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THE WASSERMANN TEST 
 
THE WASSERMANN TEST 
 
 BY 
 
 CHAELES F. CRAIG, A.M. (Hon.), M.D. (Yale) 
 
 LIEUTENANT CJOLONEL, MEDICAL COEPS, UNITED STATES ARMY; FELLOW OP THE 
 
 AMERICAN COLLEGE OF SURGEONS; PORMERLT ASSISTANT PROFESSOR OP 
 
 BACTERIOLOGY AND PATHOLOGY, ARMY MEDICAL SCHOOL, AND GEORGE 
 
 WASHINGTON UNIVERSITY; COMMANDING OFFICER, DEPARTMENT 
 
 LABORATORY, CENTRAL DEPARTMENT, UNITED STATES ARMY, 
 
 POET LEAVENWORTH, KANSAS. 
 
 ''J^<«i" 
 
 » 
 
 Published with authority of the Surgeon General, 
 United States Army 
 
 ILLUSTRATED WITH COLORED PLATES, HALFTONE 
 PLATES, AND FIFTY-SEVEN TABLES 
 
 ST. LOUIS ■M^ '■ I U ) ' 
 
 » • 
 
 C. V. MOSBY COMPANY 
 
 \, '■ 
 
 1918 
 
No.52?4 
 
 Copyright, 1918, By C. V. Mosby Company 
 
 
PREFACE 
 
 The following work upon the Wassermann test has been 
 written at the solicitation of friends who have urged me 
 to place upon record the experience gained from nearly ten 
 years of personal use of this test in the diagnosis and as a 
 control of the treatment of syphilis. I have tried to include 
 in the work all of the most important data that have keen 
 published regarding this very valuable diagnostic measure, 
 and the conclusions that I feel may rightly be drawn from 
 its use in the diaignosis, control of treatment, and as an in- 
 dex of the prevalence of this disease. 
 
 The work has been largely prepared since the outbreak 
 of the present war and, for this reason, is not as exhaus- 
 tive as I had originally intended it to be, as owing to official 
 duties it has been impossible to spend as much time in its 
 preparation as would have been necessary to make it an ex- 
 haustive treatise, and it has also been impossible for me 
 to consult much of the very extensive literature that has 
 accumulated during recent years in regard to the test. 
 However, it is believed that the work contains all of the 
 essential and really valuable facts regarding the test which 
 have been reported in the literature, and if there have been 
 any omissions I would deem it a favor to have them called 
 to my attention. 
 
 I have quoted quite liberally from some of the more re- 
 cent investigators, as Noguchi, Nichols, Vedder, and Kol- 
 mer, and have also used much data previously published by 
 myself in various medical journals, and it is a pleasure to 
 tender my thanks to the editors of the Journal of the Amer- 
 ican Medical Association, the Journal of Experimental 
 Medicine, the Journal of Infectious Diseases, and the Amer- 
 ican Journal of Syphilis, for permission to avail myself of 
 the data previously published in these journals. 
 
 11 
 
12 PREFACE 
 
 From personal experience, I believe that there is still 
 a great deal of misunderstanding and confusion among the 
 membei^s of the medical profession regarding the exact 
 value and limitations of the Wassermann test, both in the 
 diagnosis of syphilis, and when used as a control of the 
 treatment of the disease, and if this work -will help in clear- 
 ing up this confusion it will be a source of great gratifica- 
 tion. Much of this misunderstanding rests upon the shoul- 
 ders of laboratory workers, for it must be admitted that 
 too often the performance of the Wassermann test has been 
 delegated to poorly trained or careless assistants, and the 
 results obtained with the test have thus bfeen erroneous and 
 unsatisfactory. I can not urge too strongly upon the pro- 
 fession the, necessity for submitting material for this test 
 to well-qualified serologists if reliable results are to be ob- 
 tained. A standard technic for the test is much to be de- 
 sired but all efforts in this direction have failed, owing 
 largely to the difficulty of securing a standard antigen, so 
 that at the present time several methods of performing the 
 test are in use, all of which are reliable in the hands of 
 experienced serologists. The method recommended in this 
 work has stood the test of time and has been used by many 
 different workers in thousands of syphilitic infections, and 
 it is believed that it is as simple in technic and as accurate 
 in results as any method of performing the Wassermann 
 test that has been devised. 
 
 My thanks are due the publishers for their courtesy 
 and assistance in many ways and for the very excellent 
 manner in which they have reproduced the color plates pub- 
 lished in the wofk. 
 
 Chahles F. Ceaig. 
 
 Department Laboratory, Central Department, XJ. 8. Army, 
 Tort Leavenworth, Kansas. 
 June 1, 1918. 
 
CONTENTS 
 
 CHAPTEE I 
 
 PAGE 
 
 The Discovery of the Wassermann Test 17 
 
 CHAPTER II 
 
 Geneeial Description or the Wassermann Test. Nature of the 
 
 EEAt3TI0N 30 
 
 CHAPTEE III 
 
 Tactors Which Ineluencb thu Eesult op the Test 40 
 
 CHAPTER IV 
 
 Preparation and Titration op the Reagents Used in the Wasser- 
 mann Test , 60 
 
 CHAPTER V 
 
 The Technic op the Writer's Modifioation op the Wassermann 
 Test. The Test Upon the Cerebrospinal Fluid. The Origi- 
 nal Wassermann Technic. Other Modifications op the 
 Wassermann Test 89 
 
 CHAPTEE VI 
 
 Complement Fixation in Syphilis with Antigens Prepared prom 
 
 Pure Cultures op Treponema Pallidum 116 
 
 CHAPTER VII 
 
 The Results op the Wassermann Test in the Various Stages op 
 Syphilis. Time of Appearance op the Reaction. The Spec- 
 ificity OF THE Wassermann Test 127 
 
 CHAPTER VIII 
 
 The Interpretation op the Results of the Wassermann Test. The 
 Wassermann Test as an Index of the Prevalence op Syph- 
 ilis IN Communities 152 
 
 13 
 
14 CONTENTS 
 
 CHAPTEE IX 
 
 The Effect of Treatment upon the Wassermann Eeaction. The 
 Wassekmann Test as a Control of the Treatment of Syph- 
 HjIS. The Provocative Wassermann Eeaction 171 
 
 CHAPTEE X 
 
 The Wassermann Test upon the Cerebrospinal Fluid. Interpreta- 
 tion of Eesults. The Colloidal Gold Test. The Cell 
 Count and Tests for Increase in Globulins in the Cerebro- 
 spinal Fluid 205 
 
 BIBLIOGEAPHY 
 Bibliography 231 
 
ILLUSTRATIONS 
 
 PAGE 
 
 Plate I. (1) Titration of complement. (2) Titration of amboceptor pa- 
 pers. (3) Titration of antigen 78 
 
 Plate II. (1) Beading of results of Wassermann test. (2) Positive reac- 
 tion. (3) Negative reaction 98 
 
 Plate III. Paretic reaction. Tabetic reaction 218 
 
 FIG. 
 
 1. Showing hemolytic and bacteriolytic systems 24 
 
 2. Schematic representation of complement fixation in syphilis or the 
 
 Wassermann test 32 
 
 3. Luer syringes 62 
 
 4. Electric centrifuge 66 
 
 5. Electric centrifuge with shaker head attached 70 
 
 6. Small incubator 72 
 
 7. Water-bath suitable for Wassermann test incubations 74 
 
 8. Amboceptor cutter. Front view, showing gauge 80 
 
 9. Amboceptor cutter. Rear view, showing scale and set screw ... 82 
 10. Amboceptor markers 84 
 
 15 
 
THE WASSERMANN TEST 
 
 CHAPTER I 
 THE DISCOVERY OF THE WASSERMANN TEST 
 
 - The discovery of the Wassermann test for the diagnosis 
 of syphilis was the direct result of the study of immunity 
 and especially of the discovery of certain facts of basic 
 importance in that process ; i. e., bacteriolysis, hemolysis, 
 and complement fixation. 
 
 Bacteriolysis 
 
 In 1888, NuttalP demonstrated that freshly drawn blood 
 serum and defibrinated blood had a marked destructive 
 effect upon bacteria, an effect that could be definitely esti- 
 mated, and which was undoubtedly due to some substance 
 or substances in the blood serum and defibrinated blood. 
 This effect consisted in the dissolving of the bacteria and 
 the process was called bacteriolysis, the substance causing 
 the destruction of the bacteria being known as a bacter- 
 iolyein. The bacteriolytic effect of the blood serum or de- 
 fibrinated blood could be demonstrated in the following 
 manner : 
 
 Definite amounts of the blood serum or defibrinated blood 
 were mixed with definite amounts of the bacterium experi- 
 mented with, and incubated at 37° C. for several hours, 
 after which plates were poured and the number of colonies 
 of bacteria developing compared with the number upon 
 plates containing the same bacterium but to which no blood 
 serum or defibrinated blood had been added. It was ob- 
 
 17 
 
18 THE WASSEEMAK-N TEST 
 
 served that the plates prepared from the mixtures of bac- 
 teria and blood serum and defibrinated blood showed very 
 many less colonies of the bacterium than those to which no 
 blood serum or defibrinated blood had been added, and, in 
 some of the experiments, the plates were almost sterile. 
 
 Nuttall's experiments were confirmed by von Fodor^ and 
 Buchner,^ and it was the study and discussion of this sub- 
 ject that introduced into the science of medicine the "hu- 
 moral" theories regarding the nature of immunity. 
 
 Nuttall had shown that the bactericidal power of blood 
 serum was very unstable and that it was only present in 
 fresh blood or blood serum, and that heating the serum at 
 55 to 56° C, or allowing it to stand for any considerable 
 time destroyed its capacity for dissolving the bacteria. 
 Buchner,' in explaining the phenomenon of bacteriolysis, 
 evolved what is now known as the humoral explanation, 
 i. e., that the capacity of the blood or serum for dissolving 
 the bacteria resided in a constituent of the fresh blood 
 serum which he termed "alexin;" which he believed to be 
 active against all bacteria; and which he compared to the 
 action of a ferment. Buchner further stated that this 
 "alexin" might be of cellular origin, either a product of the 
 tissue cells or leucocytes. Alexin is identical with the 
 "complement" of Ehrlich and his followers, and in this 
 work the term "complement" will be used in preference 
 to "alexin." 
 
 In 1894, Pfeiffer,^ by his classical experiment upon chol- 
 ' era immunity in the guinea pig, demonstrated that the 
 process of bacteriolysis is a specific one and that the injec- 
 tion of a specific bacterium into an animal is followed by 
 the production of a specific bacteriolysin in the blood serum 
 of that animal for the bacterium injected. His experiment, 
 now a common classroom demonstration of bacteriolysis, 
 was briefly as follows : 
 
 If a guinea pig, which has recovered from cholera, be in- 
 jected in the peritoneal cavity with a suspension containing 
 cholera spirilla, it mil be found, upon examination of the 
 
DISCOVERY 19 
 
 exudate from the peritoneal cavity of the animal, that the 
 spirilla undergo rapid destruction, all stages of the process 
 being visible in samples of the exudate taken at graduated 
 intervals of time after the injection. The spirilla will be 
 observed to become swollen, distorted in shape and gran- 
 ular in appearance, and soon entirely disappear, being dis- 
 solved in the peritoneal exudate. That this process is 'pro- 
 tective in nature is shown by the fact that such an animal 
 will survive doses of the spirilla that are lethal to animals 
 that have not been protected by a previous attack of chol- 
 era, and that it is specific by the fact that other bacteria, 
 when injected into the peritoneal cavity of the cholera pig, 
 are unaffected. 
 
 In other words, the experiment proved that the pig, after 
 recovery from cholera, had present in the peritoneal ex- 
 udate some protective substance, specific in nature, which 
 was capable of dissolving the cholera spirilla and incapable 
 of acting upon other bacteria. This substance was the 
 same as that demonstrated by Nuttall in fresh blood serum 
 and defibrinated blood and to which the name ''bacterio- 
 lysin" had been given. 
 
 A very significant phenomenon observed by Pfeiff er was 
 that if the peritoneal cavity of a normal guinea pig was 
 injected with a mixture of cholera spirilla and some blood 
 serum from the immunized pig, the normal pig also escaped 
 infection. That is, it was possible thus to transfer the bac- 
 teriolytic power possessed by the peritoneal exudate of the 
 immunized pig to the peritoneal cavity of an unprotected 
 pig by mixing the cholera spirilla with the blood serum of 
 the protected animal, thus proving that the bacteriolysin 
 also existed ia the blood serum. It was also found that 
 while the bactericidal power of the blood serum of the im- 
 munized animal was destroyed, in vitro, by heating, the 
 heated blood serum is just as effectual in producing im- 
 munity in a normal guinea pig when injected with the chol- 
 era spiriUa as the unheated. This fact Pfeiffer thought 
 to be due to the necessity of some living tissue taking part 
 
20 THE WASSERMANN TEST 
 
 in the reaction and stated that the endothelial cells of the 
 peritoneum were probably the cells concerned in this par- 
 ticular experiment. His conclusions, in this respect, lead 
 him to believe that bacteriolysis, so far as it occurred with 
 the cholera spirillum, could only take place in the living 
 body. 
 
 The researches of Pfeiffer stimulated a great deal of re- 
 search upon the subject and to Bordet we ow;e the true ex- 
 planation of the process of bacteriolysis. Pfei tier's con- 
 ception that it could only occur in the living animal was 
 disproved by Bordet^ whp produced bacteriolysis of the 
 cholera spirillum in hanging drop preparations, and 
 showed, further, that while the bacteriolytic action of im- 
 mune serum is destroyed by heating the serum at 56° C, 
 the addition to this so-called "inactivated" serum of a 
 small amount of fresh normal blood serum, will "reac- 
 tivate" or restore the bacteriolytic property of the serum. 
 
 Thus, while normal blood serum may contain no bacte- 
 riolytic substances against the organism experimented 
 with, when added to an immune serum which has been 
 heated and its bacteriolytic activity destroyed, it has the 
 power of again bringing the bacteriolytic properties of 
 the immune serum into play and bacteriolysis results. 
 From this experiment it is evident that in the unheated 
 immune serum the dissolving of the bacterium must de- 
 pend upon two substances, one rendered powerless by heat, 
 while the other is unaffected. The substance rendered in- 
 active by heat is said to be thermolabile, while the unaf- 
 fected substance is said to be thermostable. It is also evi- 
 dent that the one can not act without the other upon the 
 bacteriiun, and that the thermolabile body is present in 
 normal, unheated serum, while the thermostable body is 
 only present ia the immune serum. Bordet 's researches 
 also proved the specific nature of the bacteriolysins and 
 that an animal immunized to several specific bacteria will 
 produce in its blood specific bacteriolysins against the va- 
 rious bacteria injected during the process of immunization. 
 
DISCOVEEY 21 
 
 From the researches mentioned it is evident that the 
 process of bacteriolysis depends upon the production in 
 the hlood of an animal of a specific protective substance 
 which, in the presence of fresh normal blood serum, is ca- 
 pable of dissolving the specific bacterium against which 
 the animal has been immunised. For bacteriolysis to occur 
 there must be present three factors; one, the bacterium 
 used in immunisation; the second, the blood serum contain- 
 ing the protective substance produced by immunisation; 
 and third, the substance which is present in fresh normal 
 blood serum. 
 
 In immunological literature certain terms have been 
 given the factors mentioned and in this work those used by 
 EhrUch and his followers will be used. Thus, the bac- 
 terium injected in order to produce the bacteriolytic sub- 
 stance is called an antigen, and this name may be applied to 
 any substance, which when injected into an animal, results 
 in the formation of an antibody. The bacteriolytic sub- 
 stance, itself, is called an antibody or amboceptor; while 
 the substance which is present in fresh normal blood serum 
 and which, when brought into contact with the antigen and 
 amboceptor, causes bacteriolysis is called complement. 
 This substance is identical with Buchner's "alexin." 
 
 Definitions. — As these terms are used, throughout this 
 work, in describing the Wassermann reaction, the follow- 
 ing definitions of them are inserted for convenience, as it 
 is essential that they be understood if one desires to have 
 a clear conception of the test : 
 
 Antigen. — Any substance which, when injected into suit- 
 able animals, will result in the formation of specific anti- 
 bodies. Antigens may be bacterial, cellular, or chemical in 
 nature. 
 
 Amboceptor. — The specific antibody produced by the in- 
 jection of an antigen. Amboceptors may be normally pres- 
 ent in certain blood sera but can be greatly increased by 
 the injection of the suitable antigen. Amboceptors are 
 thermostable, retaining their activity after heating at 
 
22 THE WASSEEMANN TEST 
 
 56° C, for one half hour. A hacteriolysin is a bacterio- 
 lytic amboceptor. A hemolysin is a hemolytic amboceptor. 
 
 CoMPLBMEBTT. — The substance present in all fresh nor- 
 mal sera, which when added to a mixture containing anti- 
 gen and amboceptor, results in the production of bacterio- 
 lysis or hemolysis. Complement is rendered inactive by 
 heating for one half hour at 56° C. and is, therefore, ther- 
 molabile. It also loses strength rapidly on standing at 
 room temperature. 
 
 Bacteriolytic System. — The combination of antigen 
 (bacterium), amboceptor (immune serum), and comple- 
 ment (normal serum) is called a bacteriolytic system. 
 
 Hemolysis 
 
 During his researches upon the nature of bacteriolysis 
 Bordet^ discovered that if an animal be injected with the 
 red blood corpuscles of another animal a substance is pro- 
 duced in the blood serum of the injected animal capable of 
 laking the red corpuscles injected. To this substance the 
 name "hemolysin" is generally applied and the process of 
 laking is kno■\^^l as hemolysis. Bordet's discovery of hem- 
 olysis was announced in 1898 and at the same time Belfanti 
 and Carbone* also described their experiments demonstrat- 
 ing the same phenomenon, so that to these investigators 
 belongs equally the credit for the discovery of one of the 
 most useful and important phenomena of" immunity. Al- 
 though foreshadowed by the work of Landois, who, in 1875, 
 observed that the blood serum of certain animals produced 
 laking of the blood of other species when the two were 
 added together, the real explanation of the process was 
 made clear by the work of Bordet, who proved that, while 
 hemolysins are sometimes present in the freshly drawn 
 blood of certain species against the cells of other species 
 of animals, they can be produced in large amount by the 
 injection of the washed erj'throcytes of one species into 
 another, and that the hemolysins thus produced are abso- 
 lutely specific in nature. 
 
DISCOVEEY 23 
 
 Thus, if a rabbit be injected with the washed red blood 
 corpuscles of man, the hemolysin thus produced in the 
 rabbit's blood serum will cause lakltig of human erythro- 
 cytes but will be powerless against erythrocytes of other 
 species; if injected with the erythrocytes of a sheep, the 
 hemolysin produced will only act upon sheep's erythro- 
 cytes; if injected with the erythrocytes of an ox, the rab- 
 bit's blood serum will only hemolyse ox corpuscles. In 
 other words, hemolysins are absolutely specific against the 
 kind of erythrocytes injected in immunizing an animal and 
 are powerless against other erythrocytes. 
 
 As will be seen, the hemolysins are strictly analogous to 
 the baeteriolysins, being produced by the injection of an 
 antigen, the erythrocytes, the only difference being that 
 in the one case bacteria are dissolved while in the other the 
 hemoglobin is dissolved from the red blood corpuscles em- 
 ployed during immunization. In both instances the reac- 
 tion will not occur except in the presence of complement, 
 which is furnished by the addition of fresh normal blood 
 serum, and in both instances heating for one half hour at 
 56° C. destroys the activity of the immune serum, as it de- 
 stroys the complement present in the serum. Natural hem- 
 olysins occur in the blood of one species of animal against 
 the erythrocytes of another, to a limited extent, just as nat- 
 ural baeteriolysins against certain bacteria occur in the 
 blood of certain animals, but these may be greatly in- 
 creased in amount by the injection of suitable erythrocytes. 
 
 To all baeteriolysins and hemolysins the group term 
 "cytolysins" has been applied, owing to the fact that their 
 injurious action is upon the cells injected as antigen in 
 immunization. In the case of the baeteriolysins the bacte- 
 ' rium injected constitutes the cell used as antigen, and the 
 injection is followed by the production in the blood serum 
 of a bacteriolysin capable of destroying the bacterium in- 
 jected; while in the case of the hemolysins, the erythrocyte 
 injected cons'titutes the antigen and is followed by the pro- 
 duction in the animal's blood serum of hemolysins capable 
 
24 
 
 THE WASSEEMANN TEST 
 
 of laking the erythrocytes of the species of animal from 
 which the injected erythrocytes were obtained. 
 
 Hemolytic System. — The combination of antigen (ery- 
 throcytes), amboceptor (immune serum) and complement 
 (normal serum) is called a hemolytic system. 
 
 The various theories that have been advanced in expla- 
 nation of. the cause of bacteriolysis and hemolysis and the 
 relative part played in the process by antigen, amboceptor, 
 and complement, will not be discussed here, as it is unnec- 
 essary in the consideration of the Wassermann test. 
 
 Complement Fixation 
 
 In. 1906, Bordet and Gengou^ published their paper de- 
 scribing complement fixation and as the Wassermann test 
 
 fT\'Sr3<iX A ftgr3<i3 
 
 Hemolytic system. 
 
 Bacteriolytic system 
 
 B 
 
 B 
 
 I II 
 
 Fig. 1. 
 I. — A. Hemolytic system; a, red blood corpusclei, or antigen; b, immune rabbit serum, or 
 amboceptor; c, guinea pig serum, or complement. 
 B, Showing union of antigen, a; amboceptor, b; and complement, c, with resulting 
 hemolysis. 
 II. — A. Bacteriolytic system; a, bacterium or antigen; b, immune rabbit serum,' or ambo- 
 ceptor; c, guinea pig serum, or complement. 
 •B. Showing union of antigen, a; amboceptor, b; and complement, c, with resulting 
 bacteriolysis. 
 
 is based upon this reaction it is essential that it be thor- 
 oughly understood before the technic of the test can be 
 considered. 
 
 In experiments upon bacteriolysis and hemolysis it will 
 be remembered that neither process can occur" without the 
 presence of three factors ; i. e., antigen, amboceptor, and 
 
DISCOVBEY 25 
 
 complement. For instance, if the red corpuscles of 
 a sheep be added to the blood serum of a rabbit which has 
 been immunized to these corpuscles, nothing, occurs, but if 
 to this mixture a little fresh normal blood serum be added, 
 the hemoglobin of the red cells is dissolved from them and 
 hemolysis occurs ; in the same way, if the erythrocytes and 
 complement, or fresh normal blood serum, be placed to- 
 gether, nothing will occur until the immune serum, or am- 
 boceptor be added, when hemolysis results. Furthermore, 
 in neither case is the complement in the normal blood se- 
 rum fixed by either antigen or amboceptor alone, but can 
 only be fixed, or used up, when both antigen and ambocep- 
 tor are combined with it.. In the reaction the amboceptor, 
 contained in the immune serum, apparently acts as a con- 
 necting link, uniting the antigen and the complement and 
 making hemolysis possible. 
 
 This fact Ifed Bordet and Gengou to the discovery of com- 
 plement fixation as a specific test for the differentiation of 
 bacteria, for they found that by using a bacteriolytic and 
 hemolytic system together it is possihle to discover the 
 presence of a specific antigen or specific amboceptor in a 
 mixture. In complement-fixation tests the hemolytic sys- 
 tem is used as an indicator, as hemolysis is visible to the 
 unaided eye, while bacteriolysis is not. 
 
 In order that complement fixation, as applied to the dem- 
 onstration of specific immune bodies in the blood serum 
 may be understood by the reader, the following hypothet- 
 ical experiment will be described. The complement-fixa- 
 tion test is not used in the diagnosis of typhoid fever prac- 
 tically, because it is complicated and the agglutination test 
 for the disease is more practical and easier of application, 
 but the experiment described can be carried out in the prac- 
 tice if desired. 
 
 FroUem: A patient is supposed to be suffering from typhoid fever and it 
 is desired to determine the diagnosis, if possible, by means of complement 
 fixation. ^ 
 
 Pkooeduke. — ^In a test tube containing the necessary amount of normal salt 
 
26 THE WASSBBMANN TEST 
 
 solution there is placed a certain amount of the patient 's serum, which has been 
 inactivated by heating at 56° C. for one half hour; some normal guinea pig 
 serum or complement, and a certain amount of a suspension of typhoid baeilU, 
 or antigen. This mixture is allowed to stand in a water-bath at 37° C. for 
 one-half hour, after which there are added a certain amount of a suspension 
 of human red blood corpuscles and some serum from a rabbit which has been 
 immunized against these erythrocytes. This mixture is allowed to stand in 
 the water-bath at 37° C. for one hour, placed in the ice box for one hour, 
 and the results then noted. 
 
 Result. — If it is remembered that complement is absolutely necessary 
 in order that hemolysis occur and that complement can not be fixed or used 
 up unless both antigen and amboceptor be present with it, the result of 
 this experiment can be easily understood. If the patient is really suffering 
 from typhoid fever and immune substances or amboceptor be present in the 
 blood serum, the complement in the fresh guinea pig serum which is added 
 to the typhoid bacillus suspension^ or antigen, and the patient's serum which 
 contains amboceptor against the typhoid bacillus, will be used up or fixed 
 and bacteriolysis will occur. If now the erythrocytes and the rabbit's serum 
 containing amboceptor against them be added to the mixture, hemolysis will 
 not occur, because the complement has already been used up or fixed by the 
 bacteriolytic system and is no longer available for union with the hemolytic 
 antigen and amboceptor. The test would, therefore, be positive for typhoid, 
 as it would demonstrate that the patient's blood serum contained amboceptor 
 or immune bodies for the typhoid bacillus. On the other hand, if the patient 
 did not have typhoid the complement would not be fixed, so that when the 
 erythrocytes and immune rabbit serum were added, hemolysis would occur, 
 and the test would be called a negative one. 
 
 From this experiment it will b6 seen that the principle of 
 complement fixation can be utilized in the diagnosis of dis- 
 eases due to a specific microorganism and this fact was 
 quickly recognized and investigators endeavored to work 
 out coniplement-fixation tests for many of the acute spe- 
 cific infections. Since the discovery of complement fixation 
 the test has been applied to pertussis, gonorrheal infec- 
 tions, tuberculosis, glanders, certain helminthic infections, 
 and several other conditions, but its greatest practical im- 
 portance was reached in the discovery by Wassermann, 
 Neisser and Bruck of the complement-fixation test for 
 syphilis, or, as it is commonly known, the Wassermann re- 
 action. 
 
 Discovery of the Wassermann Complement-Fixation Test 
 for Syphilis.— Many students of the subject, after the dis- 
 
DISCOVERY 27 
 
 covery of complement fixation by Bordet and Gengon, were 
 able to show that it was not necessary to employ the whole 
 bacteria as antigen in making tests but that watery extracts 
 of the bacteria, when used as antigen, gave equally good 
 results. This fact caused Wassermann and Bruck to con- 
 clude that tuberculins should act well as antigen in a com- 
 plement-fixation test for tuberculosis, and it was while 
 working upon this problem that Wassermann conceived 
 the idea that complement fixation could be applied to the 
 diagnosis of syphilis. The discovery of Treponema palli- 
 dum, by Schaudinn, had shown that the disease was due to 
 an organism that is present in large numbers in the tissues 
 of certain organs, and to these organs Wassermann turned 
 for his antigenic extract, as Treponema pallidum had not 
 then been grown; in pure culture. 
 
 Wassermann, working in conjunction with Neisser and 
 Bruck, ^'' attempted to produce complement fixation in syph- 
 ilis by using the blood serum of syphilitic monkeys as am- 
 boceptor and watery extracts of syphilitic organs as anti- 
 gen, and, as expected, it was found that such a combination 
 resulted in complement fixation. It was but a step from 
 this experiment to the application of the test to man, and 
 in 1906, Wassermann, Neisser, Bruck, and Schucht" pub- 
 lished a paper giving their results in the diagnosis of syph- 
 ilis in man by the complement-fixation test. Their paper 
 was antedated a short time by that of Detre,^^ but as Was- 
 sermann, Neisser and Bruck, in their previous publication 
 describing the test upon syphilitic monkeys antedated 
 Detre's paper, the credit for the discovery remains with 
 Wassermann and his colleagues. 
 
 Wassermann considered the complement-fixation reac- 
 tion in syphilis as a true antigen-antibody reaction, the 
 fixation being due to specific antibodies or amboceptors 
 present in the patient's blood, produced by Treponema pal- 
 lidum, the etiological agent of the disease. This conception 
 of the reaction was held by all observers until 1907, when 
 several investigators demonstrated that complement fixa- 
 
28 THE WASSEEMANN TEST 
 
 tion in syphilis occurred not only when an extract of tissues 
 containing Treponema pallidum was used as antigen, but 
 also when nonspecific substances were used, as extracts of 
 normal tissues and even certain chemical Teagents. In 
 1907, Weygandt" obtained a positive reaction in tabes by 
 using an antigen obtained by extracting a normal spleen, 
 and Marie and Levaditi" obtained positive reactions in 
 paresis with an aqueous extract of normal liver. Later, 
 Plant," Landsteiner," and others obtained positive results 
 in complement fixation in syphilis by using aqueous ex- 
 tracts of normal organs, both of man and animals, as anti- 
 gen, and finally, the publication of the paper by Land- 
 steiner, Miiller and Potzl," in which they demonstrated 
 that as good results could be obtained in the diagnosis of , 
 syphilis by the complement-fixation test with alcoholic ex- 
 tracts of guinea pig heart as antigen as when aqueous ex- 
 tracts of foetal syphilitic liver were employed, demon- 
 strated that the reaction could no longer be considered spe- 
 cific in the sense that it is a true antigen-antibody reaction. 
 The work of the last named investigators was soon con- 
 firmed by others and it was soon shown that alcoholic ex- 
 tracts of tissues were as valuable as antigens in the Was- 
 sermann test as watery extracts, and that normal organs 
 of man or animals were apparently as rich in complement- 
 fixing substances as were organs containing Treponema 
 pallidum. 
 
 Noguchi" added greatly to our knowledge of the subject 
 by proving that the substances which fix complement in the 
 Wassermann reaction are acetone insoluble, thus indicating 
 that they consisted largely of lipoids, and a further anal- 
 ysis showed that such antigenic extracts contained lecithin, 
 cholesterin, and sodium oleate. The fact that the addition 
 of a certain amount of cholesterin to antigens used in the 
 diagnosis of syphilis greatly increased their antigenic 
 value was first noted by Browning and Cruickshank" and 
 was soon confirmed by other workers, notably, in this coun- 
 
DISCOVERY 29 
 
 try, by Walker and Swift,^" and at the present time such 
 cholesterinized antigens are very generally used and, in 
 the experience of the writer and most authorities who have 
 used them, are perfectly reliable when properly controlled 
 and are more valuable in the specific diagnosis of syphilis 
 by the Wassermann test than are antigens which have not 
 been thus fortified. 
 
 The 'discovery that the antigens used in the Wassermann 
 test need not be prepared from tissues containing Trepo- 
 nema pallidum but that alcoholic extracts of normal tissues 
 are just as effective, and that certain chemicals, as choles- 
 terin, when added to antigens prepared from normal tis- 
 sues, increase their antigenic value in the diagnosis of the 
 disease, demonstrated, beyond question, that the early con- 
 ception of Wassermann that complement fixation in syph- 
 ilis is a specific antigen-antibody reaction, is erroneous, and 
 that this reaction can no longer be considered a specific 
 one on the strict sense in which this term is enaployed in 
 immunology. Although this is undoubtedly true, the prac- 
 tical application of the test in many hundreds of thousands 
 of cases of syphilis has proved that, except in a very few 
 easily recognized conditions, the test is specific for syphilis 
 and that, clinically, it is probably even more valuable than 
 it would be if it were a true antigen-antibody reaction, for 
 it is doubtful if as large a percentage of infections would 
 react positively to the test if this were true, judging from 
 the results of complement fixation in other diseases which 
 are true antigen-antibody reactions. 
 
 Although, as generally performed, the Wassermann test 
 is not a true specific reaction, the work of Noguchi" and of 
 Nichols and the writer^^ has proven that, with antigens 
 prepared from pure cultures of Treponema pallidum com- 
 plement fixation can be obtained with syphilitic sera, and 
 that in such instances the reaction is really a specific one, 
 due to antibodies in the patient's blood serum against the 
 treponema. This subject will be discussed in a later chap- 
 ter. (Chapter VI.) 
 
CHAPTER II 
 
 GENERAL DESCRIPTION OF THE WASSERMANN 
 TEST. NATURE OF THE REACTION 
 
 As stated in the preceding chapter, Wassermann and 
 his colleagues considered the complement-fixation test for 
 syphilis as a true antigen-antibody reaction, the aqueous 
 extract of tissue containing Treponema pallidum furnish- 
 ing the specific antigen, while the patient's blood serum 
 contained the specific antibody or amboceptor. This con- 
 ception of the reaction had to be abandoned when it was 
 demonstrated that aqueous or alcoholic extracts of normal 
 tissues could serve equally well as an antigen in the test. 
 Therefore, it follows that the terms "antigen" and "ambo- 
 ceptor" when used in reference to this test, except Avhen 
 applied to the hemolytic system employed, are not strictly 
 accurate, inasmuch as the antigenic extracts are not true 
 antigens, their injection into animals not being foHowed by 
 the development of antibodies ; while the substance or sub- 
 stances in the syphilitic patient's serum which, in the pres- 
 ence of complement and the antigenic extract, fix the for- 
 mer, are not true amboceptors, for they are not produced 
 by injection of the antigenic extracts employed in the test. 
 However, as the reaction agrees in all essentials with really 
 specific complement-fixation reactions, and as the antigenic 
 extracts and the syphilitic patient's serum act in the same 
 manner as do true antigens and antibodies, it is convenient 
 to retain these terms in speaking of the reaction. 
 
 As already stated the discovery of complement fixation 
 by Bordet and Gengou was quickly utilized in the diagnosis 
 of infectious diseases and the Wassermann test is simply 
 an adaptation of the principle to the diagnosis of syphilis. 
 In performing the test a hemolytic system is used as an 
 
 30 
 
GBNEEAL DESCRIPTION 31 
 
 indicator and what corresponds with a bacteriolytic system 
 is used as the diagnostic system. The hemolytic system 
 consists of a suspension of erythrocytes, the antigen; an 
 immune serum containing antibodies to the erythrocytes 
 used as antigen, the amboceptor; and fresh normal blood 
 serum, the complement. The bacteriolytic system of true 
 specific complement-fixation reactions is replaced by the 
 syphilitic system, as it may be called, consisting of an ex- 
 tract of syphilitic or normal tissues, the antigen ; the syph- 
 ilitic patient's serum, corresponding to the amboceptor; 
 and complement, or fresh normal blood serum. 
 
 These various reagents are employed in exactly the same 
 manner as are the hemolytic and bacteriolytic systems in 
 specific complement-fixation reactions, as will be apparent 
 from the general description of the test which follows : 
 
 It will be remembered that in neither a hemolytic nor bac- 
 teriolytic experiment will either hemolysis or bacteriolysis 
 be brought about unless all three factors necessary for this 
 to occur are brought together ; i. e., antigen, amboceptor 
 and complement must be placed in the same mixture before 
 either hemolysis or bacteriolysis can occur. The immune 
 serum contains the amboceptor and this body acts as an 
 intermediary or connecting link between the antigqp and 
 complement, and when this union occurs either hemolysis 
 or bacteriolysis results, as the case may be. In the Was- 
 sermann test the patient's blood serum, if he is syphilitic, 
 acts as the amboceptor, the serum containing some sub- 
 stance or substances capable of uniting with antigen (the 
 extract employed) and complement and binding the latter. 
 In order to demonstrate the presence of this complement 
 binding substance in the patient's serum the following, pro- 
 cedure is employed, and it is this procedure that is known 
 as the complement-fixation test for syphilis or the Wasser- 
 mann test : 
 
 In a test tube containing a suitable amount of normal 
 salt solution there is placed a certain amount of the blood 
 serum from the patient to be tested; a certain amount of 
 
32 
 
 THE WASSERMANN TEST 
 
 fresh normal blood serum, generally from a guinea pig, 
 which contains complement ; and a certain amount of an ex- 
 tract of syphilitic or normal tissues, the ailtigen. This mix- 
 ture is allowed to stand in a water-bath at 37° C. for one 
 half hour, at the end of which time there are added to the 
 
 Schematic representation of complement fixation in syphilis or the Wassermann test. 
 
 A. The reagents entering into the reaction; o, human red blood corpuscles or hemolytic 
 antigen; b^ hemolytic amboceptor, or immune rabbit serum; c, complement, or guinea pig 
 serum; d, patient's blood serum, which may, or may not contain syphilitic antibody; 
 e, syphilitic antigen or antigenic extract. 
 
 B. Representing a positive reaction. The patient's blood serum, d, containing syph- 
 ilitic amboceptor, has caused the union of complement, c, and syphilitic antigen, f, so 
 that when the hemolytic antigen, a, and the hemolytic amboceptor, b, are added to the 
 mixture, no hemolysis results, as the complement has already been fixed or absorbed, as 
 shown in the figui'e. 
 
 C. Representing a negative reaction. The patient's blood serum, d, containing no 
 syphilitic amboceptor, does not fix the complement with the syphilitic antigen, e, so that 
 when the hemolytic antigen, a, and the hemolytic amboceptor, b, are added, union occurs 
 between them and the complement, and hemolysis results. 
 
 mixture a certain amount of a suspension qf human or 
 other red blood corpuscles and a certain amount of the 
 blood serum of a rabbit immunized to the blood corpuscles 
 
GENERAL DESCBIPTION 33 
 
 employed in the suspension. The mixture is again incu- 
 bated in a water-bath at 37° C, this time for one hour, and 
 then placed in an ice box, and the result noted at the end of 
 an hour or two. 
 
 If the patient is suffering from syphilis, the result will 
 be that no hemolysis occurs, and the test is called positive, 
 while, if syphilis is not present, hemolysis will occur, and 
 the test is said to be negative. The positive result is due 
 to the fact that during the first incubation the substance in 
 the patient's blood serum, corresponding to a specific am- 
 boceptor, unites the antigen, or extract used in the test 
 and the complement, thus binding the latter, so that when 
 later the erythrocytes and immune serum or amboceptor 
 against them is added, there is no complement left to unite 
 with them and therefore, hemolysis can not occur. In 
 case that the result is negative, this is due to the fact that 
 the patient's blood serum contained no substance capable 
 of uniting the antigenic extract and complement, so that 
 when later the erythrocytes and the immune serum to them 
 were added, the amboceptor in the immune serum caused 
 the union of the erythrocytes and the complement and 
 hemolysis resulted, the complement having been left free 
 in the mixture, owing to nonunion during the first incuba- 
 tion. 
 
 From this brief description of the Wassermann test it 
 will be noted that it corresponds in every way as regards 
 technic and results with specific complement-fixation tests, 
 but that instead of a specific bacteriolytic antigen there is 
 substituted a nonspecific tissue extract which serves as an 
 antigen and which acts in the same manner as does the 
 specific antigen employed in bacteriolytic experiments, 
 while the complement-fixing substance or substances in the 
 syphilitic patient's blood serum act in the same manner as 
 does the amboceptor in the immune serum in bacteriolytic 
 experiments. When, however, an extract of a pure cul- 
 ture of Treponema pallidum is used as an antigen there is 
 no difference between the "Wassermann test and other com- 
 
34 THE WASSEBMANN TEST 
 
 plement fixation reactions, as in this case the complement 
 fixation is the result of a true antigen-antibody reaction. 
 Unfortunately, the antigens prepared from pure cultures 
 of Treponema pallidum are very unreliable in practice, so 
 that they can not be used for this purpose, and the nonspe- 
 cific antigens have to be depended upon in the test. 
 
 Nature of the Wassermann Reaction 
 
 The exact nature of the "Wassermann reaction is still a 
 mystery, despite an immense amount of work that has been 
 done in the endeavor to solve the problem. The fact that ex- 
 tracts of tissues which do not contain Treponema pallidum 
 serve equally well as antigens as those that do, and even 
 better than antigens prepared from pure cultures of the 
 organism, proves that the reaction is not a true antigen- 
 antibody reaction. The experiments of Noguchi^* demon- 
 strating that the active antigenic principles in the normal 
 tissue extracts were lipoids and his conclusion that the re- 
 action depends upon lipotropic substances in the patient's 
 blood serum, showed that the cause of the reaction is 
 closely connected with the chemistry of the lipoids, and 
 this is about all that we can claim to know regarding its 
 exact nature to date, although many ingenious and inter- 
 esting theories have been advanced in explanation of the 
 phenomenon. 
 
 Citron^* adopted the hypothesis that the antibody pro- 
 ducing antigen is a toxolipoid and has proposed the name 
 "Luesargine" for the syphilitic antibodies. In conjimc- 
 tion with Miinck he claims to have demonstrated that the 
 true syphilitic antigen is contained only in aqueous ex- 
 tracts of syphilitic tissues and that only rabbits inoculated 
 with such extracts produce antibodies that react positively 
 with alcoholic extracts of normal organs, but his work has 
 not been confirmed. 
 
 Schmidt^^ considers that the reaction is colloidal in na- 
 ture, depending upon the union of the colloids in the anti- 
 
NATURE OF THE REACTION 35 
 
 genie extracts and the serum globulins in the patient's 
 blood serum, which are increased in amount and changed 
 in nature in syphilitic patients. 
 
 Levaditi and Yamanouehi^' believe that the reaction de- 
 pends upon the union of two colloidal factors, one present 
 in the patient's blood serum and nonproteid in character, 
 the other consisting of lipoidal substances in the extracts 
 used as antigen. 
 
 "Weil and Braun" conclude that during the progress of 
 the disease certain tissue products, lipoidal in nature, are 
 absorbed, and give rise to antibodies which will react in 
 vitro with the lipoids present in the antigenic extracts, the 
 so-called autoantibody theory. This theory is somewhat 
 similar to Citron's, both resting upon the belief that in the 
 tissues of the patient there are produced antibodies to the 
 lipoids in the antigenic extracts, these antibodies being pro- 
 duced by the absorption in the body of albumin-lipoid sub- 
 stances produced by the growth of Treponema 'pallidum, 
 and the subsequent production of antibodies against these 
 substances. 
 
 BergeP* considers that the Wassermann reaction may be 
 due to the action of a specific lipase which is produced in 
 the syphilitic patient against "lues-lipoids." This theory 
 can not hold in the light of the facts demonstrating that 
 the reaction occurs with extracts prepared from normal or- 
 gans, when used as antigen. 
 
 Manwaring^® thinks that in guinea pig blood serum there 
 is a proteolytic ferment which may weaken or destroy com- 
 plement and that the fixation reaction is due to substances 
 present both in the antigenic extracts and syphilitic pa- 
 tient's serum which increase this effect. Bruck and Stern^^ 
 believe that the reaction is due to the interaction of the 
 lipoid bodies in the antigenic extracts and similar bodies 
 in the patient's blood serum; while Peritz^^ concludes that 
 it is due to some substance in the blood serum that has an 
 affinity for lecithin, which is always contained in the 
 extracts; 
 
36 THE WASSEEMANN TEST 
 
 None of the various theories advanced to account for 
 this reaction have been accepted as the true explanation of 
 the process of complement fixation in syphilis and none of 
 them rest upon sufficient experimental evidence to be ac- 
 cepted as the explanation. The most that can be said re- 
 garding the reaction is that it is due to an interaction be- 
 tween lipotropic bodies present in the blood serum of syph- 
 ilitics and lipoids in the antigenic extracts. A mixture of 
 a lipoid extract and syphilitic blood Avill absorb a large 
 amount of complement, so that it follows that the substance 
 or substances in the serum must be lipotropic in nature. 
 This lipotropic substance is more constantly present in the 
 blood of syphilitics than in other diseases, and is prac- 
 tically specific for that disease. Eesearches have conclu- 
 sively proved that the lipotropic substance or substances 
 are due to unknown changes in the tissues or blood serum 
 brought about by the actual presence in the tissues of liv- 
 ing Treponema pallidum, and that the reaction disappears 
 with the cure of the infection, so that its presence is proof 
 of the existence of syphilis even though months or years 
 may have passed without definite clinical symptoms having 
 been observed. 
 
 Although the Wassermann reaction, as the test is usually 
 performed, is not a true antigen-antibody reaction, there 
 is proof that in the blood of syphilitics true antibodies 
 against Treponema pallidum are formed which will fix 
 complement in the presence of antigens prepared from 
 pure cultures of the organism. 
 
 The first experiments along this line were initiated by 
 Schereschewsky,^^ who obtained positive reactions in syph- 
 ilitics by using antigens made from an impure culture of 
 what he regarded as Treponema pallidum. Owing to the 
 doubt attending the impurity of the culture and as to the 
 exact species of treponema with which he worked, Scheres- 
 chewsky's results were not conclusive, and it was not until 
 Noguchi^^ cultivated in pure culture the organism of syph- 
 ilis and employed antigens prepared from these pure cul- 
 
NATURE OF THE REACTION 37 
 
 tures, that it was proved that true complement fixation did 
 occur in syphilis. Noguchi^* demonstrated, that by using 
 aqueous extracts of pure cultures of Treponema pallidum 
 as antigen, it was possible to secure complement fixation in 
 certain cases of the disease, but that the reaction did not 
 occur as consistently as when nonspecific antigens were 
 employed nor did it occur at all stages of the disease. 
 Noguchi immunized rabbits with the pure cultures, using 
 the aqueous pallidum extracts for injection, and found that 
 their blood bound complement when the pallidum extract 
 was used as antigen but that negative results were ob- 
 tained when extracts of normal tissues were used as anti- 
 gens. These results proved that the blood serum of the 
 immunized rabbits contained a real antibody against the 
 syphilitic toxin contained in the pallidum extract used in 
 immunization. He found, however, that while the blood 
 serum of rabbits "with an active syphilitic orchitis, gave 
 positive complement fixation with the extracts of normal 
 tissue, rich in lipoids, it did not bind complement when the 
 pallidum extract was used. He explained this result as 
 follows :'^ 
 
 The findings show that the immune serums contain the specific antibodies 
 for the pallidum but not for the lipoids, and that in the serums of syphilitic 
 rabbits (active stage) there is an abundance of the lipotropic substance 
 (probably an enzyme) capable of rendering the complement inactive (so- 
 called fixation) in the presence of certain lipoids, but too little specific anti- 
 bodies to bind complement with the pallidum antigen. 
 
 When pure pallidum antigens were used with human 
 syphilitic blood serum, Noguchi obtained similar results, 
 the early stages of the disease, primary and secondary, 
 seldom reacting with the specific antigen, while in the ter- 
 tiary, latent, and late congenital cases the blood serum fre- 
 quently gave a positive result. Because of these diverse 
 results in the various stages of the disease, when the pure 
 pallidum antigen was used, Noguchi concluded that a posi- 
 tive result with the pallidum antigen indicated the forma- 
 tion of true antibodies in the blood of the patient, and was. 
 
33 THE WASSEEMANlir TEST 
 
 therefore, of favorable omen, while a negative reaction in- 
 dicated their absence. 
 
 In 1912, the writer and Nichols,^' using antigens made by 
 extracting pure cultures of Treponema pallidum with alco- 
 hol, obtained complement fixation in all stages of syphilis, 
 but positive reactions were also obtained with syphilitic 
 blood serum with antigens made from pure cultures of 
 Treponema pertenue, Treponema microdentium, and Spi- 
 rocheta refringens, while antigens made from pure cul- 
 tures of organisms like Bacillus typhosus and Spirillum 
 cholercB gave negative results. "We concluded that the pos- 
 itive reactions with the antigens prepared from the organ- 
 isms mentioned was in the nature of a group reaction, and 
 that the practical value of the test when the pallidum anti- 
 gen was used was small, owing to the fact that many cases 
 of the disease gave a negative reaction, even during the 
 late stages of the infection. Our results have since been 
 confirmed by Kolmer, Williams and Laubaugh^'' and other 
 investigators. i 
 
 While it may be considered as proved that antigens pre- 
 pared from pure cultures of Treponema pallidum are unre- 
 liable and of little value in the practical application of the 
 Wassermann test, their use has demonstrated that a spe- 
 cific fixation of complement may be obtained in syphilis 
 when such antigens are used in the complement-fixation 
 test. This fact renders it very probable that antigens pre- 
 pared from foetal syphilitic liver, rich in treponemas, may 
 give positive results in rare instances where extracts of 
 normal tissues will fail, and the use of the two kinds of 
 antigen in practice has shown that this is the case. The 
 writer is, therefore, of the opinion that the Wassermann 
 reaction, when an extract of foetal syphilitic liver is used 
 as antigen, is of a dual nature, in some instances, consist- 
 ing of a true antigen-antibody reaction between specific 
 antibodies in the patient's blood serum and specific sub- 
 stances in the antigen ; and a nonspecific reaction between 
 the lipotropic substance in the patient's serum and lipoids 
 
NATUKE OF THE EEACTION 39 
 
 in the antigen. When an extract of normal tissues is used 
 as antigen the reaction is due to the lipoids in the antigen 
 and lipotropic substances in the patient's blood serum, and, 
 in such instances, the possible reaction between true anti- 
 bodies and the specific substance in the antigen is lost, and 
 the reaction may be negative instead of positive. While, 
 in practice, in the writer's experience, this result very 
 rarely occurs, it is considered best, if possible, to make use 
 of both varieties of antigen when performing the Wasser- 
 mann test. 
 
CHAPTER III 
 
 FACTOES WHICH INFLUENCE THE RESULT 
 OF THE TEST 
 
 The Wassermann test is quantitative in nature and un- 
 less all of the reagents are carefully titrated at frequent 
 intervals and accurately used, erroneous results are sure 
 to occur. The quantity of blood serum to be tested; its 
 proper inactivation ; the dose of complement and its 
 source; and the amount of amboceptor and antigen to be 
 used; are all important factors in the test and variations 
 from the proper amount of each will be followed by faulty 
 results, as will be shown in the chapter devoted to technic. 
 
 However, aside from the reagents employed in the test 
 there are certain factors which have a very marked influ- 
 ence upon its result and which should be Tinderstood by 
 physicians using this valuable diagnostic measure. These 
 factors are: The influence of the ingestion of alcohol; the 
 influence of certain bacteria in the blood serum ; and varia- 
 tions in the amount of complement inhibiting substance in 
 the blood serum to be tested. 
 
 The Influence of the Ingestion of Alcohol. — ^In 1911, 
 Major Nichols, of the Medical Corps of the Army, and 
 the writer^^ called attention to the effect that the inges-. 
 tion of considerable amounts of alcohol has upon the re- 
 sults of the Wassermann test. It was found that the in- 
 gestion of alcohol in the foi-m of beer or whiskey, and in 
 considerable amounts, might convert a strongly positive 
 serum into a negative one, if the test were made within 
 twenty-four hours after the ingestion of the alcohol, and 
 that the blood might remain negative for as long as three 
 days, although it generally became positive again within 
 twenty-four hours. This fact was discovered by the writer 
 
 40 
 
FACTORS INFLUENCING EESTJLT OF TEST ' 41 
 
 while making routine examinations of the blood of a cer- 
 tain individual who was being treated for syphilis by- 
 Major Nichols, and who had presented several strong 
 positive reactions before the first negative one which was 
 due to the ingestion of alcohol, and further research by 
 Nichols and myself proved that this drug has a very po- 
 tent influence in weakening or destroying the lipotropic 
 substances in the patient's blood serum which apparently 
 cause the Wassermann reaction. 
 
 The case in which this phenomenon was first observed is 
 of special interest in this connection and the clinical his- 
 tory is here given : 
 
 Male, 41 years of age. Infected with syphilis in 1904. Now has palmar 
 lesions. February 25, 1911, 0.5 gm. of salvarsan was administered intra- 
 venously, the Wassermann test before administration giving a plus reaction. 
 Between this date and April 7th, 1911, his blood was tested Ave times, the 
 reaction four times being double-plus, and th« last test, on April 7th, being 
 plus. (Double-plus here equals four-plus of most authorities.) April 8th 
 his blood was again tested and was found to be negative. The result was 
 unusual and on inquiry ^it was learned that on the night of April 7th he 
 drank about 700 c.c. of Munich beer. His blood was tested again on April 
 12th, and gave a double-plus (four-plus of some authors) reaction. Be- 
 lieving that the beer might have had something to do with the sudden 
 disappearance of the positive reaction in this ease, we requested that he 
 repeat the beer on the night of April 14th, which he did, and on the morn- 
 ing of April 15th the reaction was again negative. On the 17th of April it 
 had become plus and on the 22nd again was double-plus. 
 
 As it was evident that the negative result of the Wasser- 
 mann test was due, in this case, to the ingestion of the beer, 
 we decided to repeat the experiments on other cases. We 
 selected nine syphilitic patients, who before the adminis- 
 tration of alcohol gave a double-plus reaction, and Table I 
 gives the results obtained in these patients. It will be re- 
 membered that in this table, the double-plus reaction means 
 absolute inhibition of hemolysis, and is equivalent to the 
 four-plus reaction of most writers. 
 
 Of the 9 cases included in this table, 3 were in the sec- 
 ondary stage of the disease and 6 were in the tertiary 
 stage. The quantity of alcohol administered varies some- 
 
42 
 
 THE WASSERMANN TEST 
 
 t-t 2 
 
 W 
 
 
 
 
 ' +++ 
 
 ' +++ 
 
 'i^ 
 
 s a a a a a a s a a a a a a a a a a a a a a a a a a a a a a S' a a- a a a a a a a 
 
 
 
 ■3a 
 
 ■^ >«. 
 
 
 oa 
 
 •sa 
 
 .o 
 
 Oil 
 
 ci 
 
 osa 
 
 
 5o 
 
 
 Ja 
 i< 
 
 tS 
 
 o C a 
 
 ^ <U Pi 
 
 :ia 
 
 ci 
 
 O C £* 
 
 5t^ 
 
 
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 X 
 s. 
 
 ofia 
 
 CM " * 
 
 2SS 
 
 <! <! 
 
 & 
 S 
 
 < < 
 
 ^ 
 ^ 
 
 4J "O, 
 
 » B 
 w CM 
 
 + J3 
 
 B 
 
 ■o 
 
 c 
 
 B 
 
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 B 
 
 "o 
 
 
 =•3 
 
 °o 
 
 =7? 
 
 °-s 
 
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FACTOES INFLUENCING RESULT OF TEST 43 
 
 what but in every case the positive reaction disappeared 
 after its administration. In most of the cases the blood 
 was tested one hour before the administration of the al- 
 cqhol was begun as well as afterward. A. study of the ta- 
 ble shows that in 5 of the cases the reaction was found to 
 be negative one hour after the last dose of the alcoholic 
 liquor had been administered, and that in all the cases the 
 reaction remained negative for several hours. In cases 4, 
 5, and 6 the reaction remained negative for over 24 hours 
 while in case 9 it remained negative for three days and did 
 not become double-plus again until over four days had 
 elapsed from the time the alcohol was administered. Two 
 of the patients had received salvarsan before the (admin- 
 istration of the alcohol, but in one the drug had been given 
 so long before that it could not have had" any effect, upon 
 the reaction, while in the other the fact that the reaction 
 became double-plus again after becoming negative from 
 the alcohol, proves that the salvarsan had nothing to do 
 with its disappearance. 
 
 That the disappearance of the reaction after the inges- 
 tion of alcohol is not due to the alcohol itself, which may 
 be present in the blood serum, is proved by the fact that 
 a much larger quantity of alcohol is necessary to produce 
 hemolysis, as proved by experiment, than could possibly 
 be present in the blood serum of the patients tested. Thus, 
 we found that it requires at least 0.15 c.c. of absolute alco- 
 hol to produce complete hemolysis of 1 c.c. of a 1 per cent 
 suspension of human red blood corpuscles within the time 
 used in the Wassermann test for incubation of the hem- 
 olytic system, whereas only 0.08 c.c. of the patient's blood 
 serum was used in each test. It is thus evident that if the 
 amount of blood serum used had been actually absolute al- 
 cohol the quantity would have been insufficient to have pro- 
 duced hemolysis. 
 
 From these experiments, which have been confirmed 
 by many investigators since our original publication, 
 it is evident that alcohol may render inert the sub- 
 
44 THE WASSEEMANN TEST 
 
 stance or substances in the blood serum of syphilitics 
 which react with lipoids in the antigenic extracts, and thus 
 a strongly positive serum may give a negative result. For 
 this to occur, the alcohol must be tajjen in considerable 
 quantity and probably within twenty-four hours, or at 
 most three days before the test is made, but it should 
 be remembered that smaller amounts of alcohol may ren- 
 der weak reactions negative, so that cases which should 
 present a single plus reaction will often react nega- 
 tively after even moderate amounts of alcoholic liquors 
 have been ingested. It is thus evident that very careful 
 inquiry should be made regarding the use of alcoholic liq- 
 uors before taking blood for a Wassermann test from any 
 patient, and if there is a history of indulgence in such liq- 
 uors within from twenty-four to thirty-six hours previ- 
 ously, the taking of the specimen should be postponed and 
 alcoholics forbidden until at least two days have passed. 
 The neglect of this precaution has often resulted in errone- 
 ous results and cases that should have been strongly posi- 
 tive have been reported as negative or doubtful. When 
 the test is used as a control of treatment alcohol, even in 
 small amounts, should be forbidden for some days before 
 the blood is collected, or the weak reactions often observed 
 in treated cases will be entirely missed, thus obscuring the 
 real condition of the patient as regards his serological 
 status. 
 
 From what has been said, it follows that a negative Was- 
 sermann reaction, after the ingestion of alcoholic liquors, 
 possesses no value whatever, and an immense amount of 
 harm has doubtless been done by lack of care in ascertain- 
 ing whether patients have indulged in such liquors within 
 a short time before the blood was collected. Our observa- 
 tions, as well as those of others, both in this country and 
 abroad, have proved that the effect of alcohol upon the 
 Wassermann reaction is very important from a practical 
 standpoint, and one which should not be forgotten when 
 
FACTORS INTLUENCING RESULT OF TEST 45 
 
 patients come to the physician for the collection of blood 
 for the test. 
 
 The Influence of Certain Bacteria, when Developing in 
 the Blood Serum, upon the Results of the Test.— It is a well- 
 known fact that, upon standing for varying lengths of time, 
 some human blood sera develop anticomplementary sub- 
 stances, which cause inhibition of hemolysis in both the 
 tubes containing antigen and the control tubes used in the 
 Wassermann test. Most of these bodies are destroyed by 
 heating the blood serum for one-half hour at 56° C., but, 
 in some instances, they still persist after heating, and in 
 such sera one is unable to read the result of the test owing 
 to inhibition occurring in both the antigen and control 
 tubes. From these observations it is evident that these 
 anticomplementary bodies are of two kinds, thermolabile 
 and thermostable, and that the inactivation of the blood 
 serum by heating enables us to destroy only the thermo- 
 labile group. 
 
 While the exact nature of these anticomplementary 
 bodies is still doubtful, the writer-has shown'* that some 
 of them are produced by the growth of bacteria in the blood 
 serum to be tested, if the serum is kept for some time. Not 
 only will certain bacteria produce anticomplementary 
 bodies that cause inhibition of hemolysis in both the anti- 
 gen and control tubes of the test, but certain species also 
 possess the power of causing inhibition of hemolysis in the 
 antigen tube ialone, thus giving rise to a nonspecific reac- 
 tion in the sera containing them. It is these species that 
 are of special importance in the application of the Wasser- 
 mann test and which will here be discussed. 
 
 While carrying on some comparative tests of blood se- 
 rum with another laboratory it was noted that in one in- 
 stance which gave a positive reaction in the writer's lab- 
 oratory and a negative one at another laboratory situated 
 five days' journey distant, the blood serum when received 
 was cloudy in appearance and had a bad odor. As it was 
 known to be infected and as it was thought possible that 
 
46 
 
 THE WASSEEMANN TEST 
 
 bodies developed in the serum by the growth of the bac- 
 teria might be the cause of the difference in results, this 
 serum was plated out upon agar-agar and cultures of the 
 following organisms were isolated : Staphylococcus aureus; 
 a large Staphylococcus albus; a small Staphylococcus albus, 
 a large spore-bearing bacillus of the subtilis type; and a 
 very minute diplobacillus. 
 
 In order to determine whether any of these bacteria 
 were responsible for the reaction obtained with this blood 
 serum the following experiments were undertaken to- 
 gether with experiments with a stock culture of Staphy- 
 lococcus aureus, Staphylococcus citreus, and Streptococcus 
 pyogenes. The same technic was followed in these exper- 
 iments as was used in performing the Wassermann test, 
 the mixtures of serum and bacteria being substituted for 
 the patient's blood serum. 
 
 Experiment 1.— An emulsion in normal salt solution was made from a 
 pure culture of each organism isolated from the blood serum, and 0.08 c.c. of 
 this emulsion was placed in 1 c.c. of a 1 per cent suspension of human red 
 blood corpuscles, together with 0.08 c.c. of an inactivated normal serum. 
 Table II gives the result of this experiment. 
 
 Table II 
 Results op Complement-Fixation Tests in Ezpeeiment No. 1 
 
 
 Besults 
 
 Controls 
 
 
 NAME OP BACTERIUM 
 
 ANTIGEN 
 TUiBE 
 
 CONTEOL 
 
 TUBE 
 
 BACTERIA 
 ALONE 
 
 SEEUM 
 ALONE 
 
 ORIGIN OP 
 BACTERIUM 
 
 8. albus, large 
 S. albus, small 
 S. aureus. 
 Bacillus (Spore) 
 Diplobacillus 
 Combined bacteria 
 
 - 
 
 - 
 
 — 
 
 - 
 
 Isolated 
 from 
 serum in 
 all cases 
 
 From these results it is evident that merely adding an 
 emulsion of the cultures of these bacteria to normal blood 
 serum is without effect upon the result of the complement- 
 fixation test, as all of the mixtures gave a negative reaction. 
 
 Experiment 2. — This experiment was identical with Experiment 1, except 
 that the mixtures, of serum and bacteria were inactivated at 56° C. for one-half 
 
FACTORS INFLUENCING RESULT OF TEST 
 
 47 
 
 hour before being tested. The mixtures of bacteria and serum in this experi- 
 ment all gave a negative reaction with the exception of the mixture of blood 
 serum and the diplobacillus, which resulted in a plus reaction, the heating 
 resulting in the liberation of enough inhibitory substances to cause this type 
 of reaction in both the antigen and control tubes. Of course this reaction 
 would have caused no confusion so far as the diagnosis of syphilis is concerned, 
 as the fact that the inhibition occurred in the control tube as well as in the 
 antigen tube would have thrown the test out entirely and another specimen 
 would have been requested. 
 
 Iixperiment 3. — Specimens of normal serum were inoculated with each of 
 the bacteria isolated from the serum in question as well as with the stock cul- 
 tures and allowed to stand at room temperature for one week before being 
 tested. Table III gives the results of this experiment: 
 
 Table III 
 Results of Complement-Fixation Tests in Experiment No. 3 
 
 NAME OF 
 BACTE^TCM 
 
 Besult 
 
 CONTROL, 
 SEKUM 
 ALONE 
 
 ORIGIN OF 
 BACTERIUM 
 
 antigen 
 
 TUBE 
 
 CONTROL 
 TUBE 
 
 S. alius, large 
 S. albus, small 
 S. aureus. 
 Bacillus (spore) 
 Diplobacillus 
 S. aiureus 
 S. citreus 
 S. pyogenes 
 
 + - 
 
 -1- 
 -I- + 
 + + 
 
 + + 
 -I- + 
 
 - 
 
 Isolated from serum 
 It tt it 
 
 (( t ( It 
 
 It tt ti 
 
 tt It tt 
 
 Stock culture 
 (< (( 
 
 (( It 
 
 It is evident, by consulting the table, that after incuba- 
 tion in human blood serum for a week at room temperature 
 the large 8. albus isolated from the blood serum under 
 discussion gave a plus-minus reaction in the antigen tube, 
 while the diplobacillus isolated from the same serum gave 
 a plus reaction in the antigen tube and a double-plus re- 
 action in the control tube. The stock 8. aureus gave a 
 double-plus reaction in the antigen tube, while the stock 
 culture of 8. citreus caused total inhibition of hemolysis in 
 both antigen and control tubes. 
 
 From this experiment it is evident that the stock 8. 
 aureus, growing in normal blood serum at room tempera- 
 ture foF one week produced some substance or substances 
 in the serum which gave a false positive reaction. 
 
 Experiment 4. — This experiment was identical with Experiment 3, except 
 that the mixtures of bacteria and serum were incubated at 37° C. for 24 hours 
 
48 
 
 THE WASSEEMAHN TEST 
 
 and then allowed to stand at room temperature for one week, before being 
 tested. Table IV gives the results of this experiment: 
 
 Table IV 
 
 EESDLTS of COMPLEMENT-FtXATION TESTS IN EXPERIMENT NO. 4 
 
 
 Result 
 
 CONTROL 
 
 SERUM 
 ALONE 
 
 ORIGIN OF 
 BACTERIUM 
 
 BACTEEIUM 
 
 ANTIGEN 
 TUBE 
 
 CONTROL 
 TUBE 
 
 8. albus, large 
 S. albus, small 
 S. cmreus 
 Bacillus (spore) 
 Diplobacillus 
 8. am-eus 
 S. eitreus 
 8. pyogenes 
 
 + + 
 
 + + 
 
 + + 
 + + 
 + + 
 + + 
 
 + + 
 + + 
 
 - 
 
 Isolated from serum 
 << << It 
 
 Cl It It 
 
 <( <( It 
 it ti It 
 
 Stock culture 
 II II 
 
 ti ti 
 
 From this experimental record it will be hoted that no 
 less than four of the organisms tested gave a double-plus 
 (four-plus) positive reaction in the antigen tube alone, 
 while two inhibited hemolysis completely in both the anti- 
 gen and control tubes. It would appear that the 24 hours 
 at incubator temperature lead to an increase in the inten- 
 sity of the reaction, due to a richer development of the 
 bacteria in the sera. 
 
 Considering the experiments as a whole it is perfectly 
 evident that a positive complement-fixation reaction may 
 be obtained in a normal serum if certain species of bacteria 
 are enabled to develop, within it. That every strain of a 
 bacterial species will not produce this result is shown by 
 the fact that while a double-plus reaction was obtained in 
 the serum infected with the stock 8. aureus and with the 
 8. aureus isolated from the serum under discussion, a nega- 
 tive result was later obtained with an 8. aureus isolated 
 from a blood serum that gave a negative reaction. 
 
 It is also evident that temperature has much to do with 
 the ability of the bacteria to produce the positive reaction, 
 for in the mixtures of serum and bacteria kept for 24 
 hours at incubator temperature, the reactions were most 
 marked, while two of the organisms which did not give 
 a reaction when growing in the blood serum at room tem- 
 
PACTOES INFLUENCING EESULT OF TEST 49 
 
 perature, gave a strongly positive reaction when incnbated. 
 Inactivation also appears to favor the production of the 
 false positive reaction in sera which are contaminated with 
 certain bacteria. 
 
 As regards the serum under discussion, it is evident that 
 the positive result could have been caused by either the 
 large 8. alhus or the 8. aureus, or both, according as the 
 conditions at the time of examination were favorable to the 
 production of inhibitory substances by one or both of these 
 organisms. 
 
 The fact that, under certain conditions, such common 
 bacteria as staphylococci and streptococci, when growing 
 in normal blood serum, may give rise to a positive Was- 
 sermann reaction, is of great practical importance, and 
 one might conclude from these experiments that only fresh 
 blood serum should be used in making complement-fixation 
 tests. However, if proper precautions are taken in the 
 collection of specimens, there is no danger of bacterial 
 contamination, and it is our experience that, unless such 
 contamination occurs, a positive result will not be obtained 
 in normal serum, even when kept for a considerable period 
 of time. Personal experiments have demonstrated that 
 blood sera may be kept "at room temperature for as long 
 as one month without danger of a negative serum becom- 
 ing positive, provided there is no bacterial contamination 
 in' the serum. While, in such specimens, anticomplemen- 
 tary bodies frequently develop, after they have been kept 
 for a week or more, these bodies cause inhibition of hem- 
 olysis in both the antigen and control tubes of the serum, 
 and thus while destroying the value of the test, this type 
 of reaction does not cause any confusion in the diagnosis 
 of the disease.' However, these observations emphasize the 
 importance of collecting specimens for the "Wassermann 
 test under aseptic precautions if the sera can not be ex- 
 amined within 24 hours after collection. 
 
50 THE WASSERMANN TEST 
 
 The Influence of Variations in the Strength of the Re- 
 action in Untreated Cages of Syphilis upon the Results of 
 the Test. — During the routine examinations of blood sera 
 for the Wassermann test, the writer has frequently ob- 
 served cases in which the reaction varied from a strongly 
 suspicious one to a plus-minus or negative one within 
 periods of one or two weeks, while, in rare instance^, a 
 positive reaction would be found negative upon a second 
 examination within the same period of time, no treatment 
 having been administered at any time. In all such in- 
 stances subsequent tests resulted in a positive reaction, so 
 that it was evident that the variations in results, provided 
 the technic of the test was the same, must have depended 
 upon the reduction or absence of the body or bodies in 
 the patient's serum which produce the reaction. 
 
 The fact that the blood serum of an undoubted syphilitic 
 patient may, during certain intervals of time, give a nega- 
 tive reaction in the absence of specific treatment, and when 
 previous and subsequent tests are positive, is of practical 
 importance. While in the writer's experience such anom- 
 alous results have not been very numerous, their occur- 
 rence, together with the criticisms of the test by some 
 authors who have had a similar experience, rendered an 
 experimental investigation of the phenomenon of value, 
 and in 1914, the writey*" published certain observations 
 upon the subject which will be briefly discussed at this 
 time. The observations proved that marked daily varia- 
 tions occur in the strength of the complement-fixation re- 
 action in the blood of syphilitics. While, a priori, there 
 would appear to be no reason why the strength of the re- 
 action should not vary, we have grown to consider the 
 Wassermann reaction, if once obtained, as a stable quan- 
 tity, the discordant results sometimes observed being im- 
 puted to the serologist or considered as proof of the unre- 
 liability of the test. 
 
 Ten patients suffering from undoubted syphilis were 
 selected for the observations. Most of these men were 
 
PACTOKS INFLUENCING EESULT OF TEST 51 
 
 prisoners at the U. S. Military Prison, at Fort Leaven- 
 worth, and thus all chance of the reaction being influenced 
 by the use of alcohol was obviated, the men being under a 
 rigid discipline and on a plain, wholesome diet. Of the 
 ten patients, two were in the primary stage of the disease, 
 four in the secondary stage, and four in the latent stage. 
 No treatment had been administered for over a year in 
 any case, and then only to those who were in the latent 
 stage. The patients in the primary stage had typical 
 chancres and have since developed secondary eruptions; 
 the secondary cases all presented eruptions and inucous 
 patches; while the four in the latent stage were free from 
 obvious symptoms. All gave double-plus (four-plus) Was- 
 sermann reactions at the time of the experiment. 
 
 The blood was collected every day and tested upon the 
 same day it was collected, daily titrations of the strength 
 of the complement fixation being made. The technic used 
 was that employed by the writer for several years and 
 which is described in this work. 
 
 Each patient's blood serum was titrated every day for 
 one week, the quantities of serum employed being 0.02 c.e. ; 
 0.04 e.c. ; 0.06 c.c. ; 0.08 c.c. ; and 0.1 c.c. The quantity of 
 blood serum used with the technic for diagnostic purposes 
 is 0.1 c.c. so that this quantity may be taken as the diag- 
 nostic amount in the tables that follow. 'A control of 0.1 
 c.c. of the serum titrated was always employed. 
 
 In the tables it will be understood that the sign, + +, in- 
 dicates absolute inhibition of hemolysis, or a positive re- 
 action; the sign, +, anything between absolute inhibition 
 and 50 per cent of inhibition of hemolysis; the sign, +-, 
 anything between 50 per cent of inhibition and total hem- 
 olysis, or practically a negative reaction; and the sign, -, 
 total hemolysis or a negative result. 
 
 Eesults in the Pkimaey Stage or Syphii-is. — Owing to 
 lack of material, only two cases were tested in the primary 
 stage of the disease. Both presented typical chancres and 
 both gave a double-plus, or positive, Wassermann reaction 
 
52 
 
 THE WASSBEMANN TEST 
 
 when first tested. Tables V and VI give the results of the 
 daily titrations of the blood serum in these two cases. 
 
 Table V 
 
 Eestjlts of Titration or Blood Serum for Complement-fixing Strength in 
 Case 1 — Primart Syphilis 
 
 
 AMaUNT OF BLOOD SERUM IN C.C. 'S 
 
 CONTROL. 
 
 
 0.02 
 
 0.04 
 
 0.06 
 
 0.08 
 
 0.1 
 
 0.1 C.C. 
 
 Nov. 19 
 Nov. 20 
 Nov. 21 
 Nov. 22 
 Nov. 23 
 Nov. 24 
 Nov. 25 
 
 + - 
 + - 
 + - 
 + - 
 + 
 
 + - 
 + + 
 
 + 
 + - 
 
 + 
 
 + - 
 + + 
 
 + 
 + + 
 
 + 
 
 + 
 + + 
 
 + 
 + + 
 
 + 
 + + 
 
 + 
 
 + + 
 + + 
 
 + 
 + + 
 
 + 
 + + 
 
 + 
 
 ++ 
 
 ++ 
 
 + 
 
 ++ 
 
 + 
 
 ++ 
 
 - 
 
 Table "VI 
 
 Results of Titration of Blood Serum for Complement-fixing Strength in 
 Case 2 — Primary Syphilis 
 
 DATE OP TEST 
 
 AMOUNT OF BLOOD SERUM. C.C. 
 
 CONTROL. 
 
 0.02 
 
 0.04 
 
 0.06 
 
 0.08 
 
 0.1 
 
 0.1 C.C. 
 
 Nov. 19 
 Nov. 20 
 Nov. 21 
 Nov. 22 
 Nov. 23 
 Nov. 24 
 Nov. 25 
 
 + 
 + - 
 
 + 
 + 
 + 
 
 + - 
 + - 
 
 + + 
 -f + 
 
 + + 
 
 + 
 
 + - 
 -I- + 
 -H- 
 
 -n- 
 
 + ■^ 
 -I- + 
 
 -1- 
 
 + 
 
 ++ 
 + + 
 
 -n- 
 + + 
 
 -1- 
 
 + -I- 
 
 - 
 
 A study of these tables will show that considerable varia- 
 tions occurred in the complement-fixing power of the blood 
 serum in both these cases of primary syphilis, especially 
 in the smaller amounts employed, but that in neither case 
 did the reaction become negative with the diagnostic 
 amount of serum; i.e., 0.1 c.c. However, in one case a 
 plus or doubtful reaction was obtained upon three of seven 
 days, while in the other the same result was obtained upon 
 two of the seven days. These results are very significant 
 when it is remembered that in this stage of syphilis the 
 Wassermann test is so often doubtful, or negative, and in- 
 dicates that frequent examinations would give a much 
 higher percentage of positive results than are usually re- 
 
FACTORS INFLUENCING RESULT OF TEST 
 
 53 
 
 corded. Both of these cases, if tested upon certain days 
 of the week, would have been reported serologically doubt- 
 ful, but, fortunately, in most primary infections Trepo- 
 nema pallidum may be demonstrated with the dark-field, 
 thus avoiding the confusion that might be caused by varia- 
 tions in the strength of the Wassermann reaction. 
 
 Eesults in the Secondary Stage of Syphilis. — The 
 blood serum of four typical cases of secondary syphUis, 
 
 Table VII 
 
 Results of Titration of Sbkum pok Complement-fixing Strength in 
 Case 3 — Secondary Syphilis 
 
 date of test 
 
 amount of blood serum, c.c. 
 
 control c.c. 
 
 0.02 
 
 0.04 
 
 0.06 
 
 0.08 
 
 0.1 
 
 0.1 
 
 Nov. 26 
 Nov. 27 
 Nov. 28 
 Nov. 29 
 Nov. 30 
 Dee. 1 
 Dee. 2 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 ++ 
 ++ 
 ++ 
 + + 
 ++ 
 ++ 
 ++ 
 
 ; 
 
 Table VIII 
 
 Results of Titration op Serum for Complement-fixing Strength in 
 Case 4 — Secondary Syphilis 
 
 
 AMOUNT OF BLOOD SERUM. C.C. 
 
 CONTROL C.C. 
 
 
 0.02 
 
 0.04 
 
 0.06 
 
 0.08 
 
 0.1 
 
 0.1 
 
 Jan. 3 
 Jan. 4 
 Jan; 5 
 Jan. 6 
 Jan. 7 
 Jan. 8 
 Jan. 9 
 
 -l--h 
 
 + 
 ++ 
 
 + -t- 
 -H- 
 
 -n- 
 ++ 
 
 + -H 
 
 + + 
 
 -n- 
 -f-l- 
 
 H- + 
 
 -1- -1- -1- + -1- -1- -4- 
 -H- -t- -H- -(- -1- 
 
 ++ 
 ++ 
 
 -F-l- 
 
 -f-i- 
 
 - 
 
 Table IX 
 
 Results op Titration op Serum for Complement-fixing Strength in 
 Case 5 — Secondary Syphilis 
 
 
 AMOUNT OP BLOOD SERUM. C.C. 
 
 CONTROL C.C. 
 
 DATE OF TEST 
 
 0.02 
 
 0.04 
 
 .0.06 
 
 0.08 
 
 0.1 
 
 0.1 
 
 Dec. 24 
 Dec. 25 
 Dee. 26 
 Dec. 27 
 Dee. 28 
 Dee. 29 
 Dec. 30 
 
 -t-- 
 
 +- 
 + - 
 
 -(- 
 -F- 
 
 -^ 
 + - 
 
 ^■ 
 +- 
 
 + 
 + 
 
 ■f- 
 -t- 
 + 
 
 ■^ + 
 -t•-^, 
 
 - 
 
54 
 
 THE WASSEEMANN" TEST 
 
 Table^X 
 
 Eesults of Titration of Serum for Complement-fixing Strength in 
 Case 6 — Secondary Syphilis 
 
 DATE OF TEST 
 
 AMOUNT OP BLOOD SERUM. CO. 
 
 CONTROL C.C. 
 
 0.02 
 
 0.04 
 
 0.06 
 
 0.08 
 
 0.1 
 
 0.1 
 
 Dec. 5 
 Dec. 6 
 Dec. 7 
 Dec. 8 
 Dee. 9 
 Dec. 10 
 Dec. 11 
 
 + - 
 + + 
 + + 
 + + 
 + + 
 + - 
 + - 
 
 + - 
 + + 
 + + 
 + + 
 + + 
 + 
 + - 
 
 + 
 
 + + 
 + + 
 + + 
 + + 
 
 + 
 
 + 
 
 + 
 
 + + 
 + + 
 + + 
 + + 
 
 + 
 
 + 
 
 ++ 
 
 ++ 
 
 ++ 
 
 ++ 
 
 ++ 
 
 + 
 
 + 
 
 1 1 1 1 1 1 1 
 
 all showing lesions of the disease, was titrated in the same 
 manner as described and Tables VII, VIII, IX, and X give 
 the results obtained. 
 
 In Case 3 the strength of the blood serum did not vary 
 at all during the time it was titrated, a double-plus (four-' 
 plus) reaction being obtained, even with- the smallest 
 amount of serum used. The case is of interest as showing 
 how strong the blood serum is in complement-fixing power 
 in some instances and how little it varies in strength even 
 when very small amounts of serum are used. The patient 
 presented clinically a very marked secondary eruption over 
 the chest, abdomen, and back. 
 
 In Case 4, likewise, there was practically no variation in 
 the strength of the reaction. In this case the patient pre- 
 sented mucous patches in the mouth and a weU marked 
 secondary eruption. 
 
 In Case 5, the variations in the strength of the comple- 
 ment-fixing power of the blood serum varied markedly, 
 especially when amounts of serum of less than 0.1 c.c. were 
 used. With the diagnostic amount of serum ; i. e., 0.1 c.c, 
 the serum gave a plus-minus, or practically negative result 
 upon one day and a plus, or doubtful reaction upon two of 
 the seven days during which it was tested. With 0.08 c.c. 
 of the serum, an amount which was formerly used in the 
 writer's laboratory as the diagnostic amount, a negative 
 reaction was obtained upon one day and plus-minus upon 
 one day and a plus, or doubtful reaction, upon two days. 
 
FACTORS USTFLTJENCING RESULT OF TEST 
 
 55 
 
 In this case the Wassermann reaction was practically nega- 
 tive on one day and doubtful upon two days, although very 
 marked secondary symptoms were present. 
 
 In Case 6 the variation in the strength of the reaction 
 was not very marked, when the diagnostic amount of blood 
 serum was used, the serum giving absolute inhibition of 
 hemolysis upon all but two days, when it gave a plus or 
 doubtful reaction. 
 
 The results of the titration of the blood serum in second- 
 ary cases demonstrate that variations do occur daily in 
 cases showing well marked symptoms of the disease and 
 that a case giving absolute inhibition of hemolysis upon 
 one day may be negative or doubtful upon a succeeding 
 day. When small amounts of blood serum are used the 
 variations in the strength of the reaction are very marked, 
 in some instances, as in'Case 5, while in others the strength 
 of the reaction remains unchanged. (Case 3.) 
 
 Eesults ih- the Latent Stage of Syphilis. — Pour pa- 
 tients in the latent stage of syphilis were tested, all of 
 whom gave a history of definite symptoms of the disease, 
 but in none had symptoms been present within one year. 
 All of the men had received one or two injections of sal- 
 varsan aiid all gave a double-plus (four-plus) Wassermann 
 reaction shortly before the titrations of the blood serum 
 were commenced. Tables XI, XII, XIII, and XIV give the 
 results of the titrations. 
 
 Table XI 
 
 Results of Titkation of Serum foe Complement-fixing- Strength in 
 Case 7 — Latent Syphilis 
 
 
 amount of blood serum. CO. 
 
 control CO. 
 
 
 0.02 
 
 0.04 
 
 0.06 
 
 0.08 
 
 0.1 
 
 0.1 
 
 Dec. 19 
 Dec. 20 
 Dec. 21 
 Deo. 22 
 Dee. 23 
 Dee. 24 
 Dec, 25 
 
 + - 
 
 ++ 
 ++ 
 +- 
 
 + 
 + - 
 
 + 
 
 + - 
 + + 
 + + 
 
 + 
 
 + 
 
 + 
 + + 
 
 + 
 
 + + 
 + + 
 
 + + 
 
 + 
 + + 
 
 + 
 
 + + 
 + + 
 
 + 
 
 ++ 
 
 ,+ 
 
 ++ 
 
 + 
 
 ++ 
 ++ 
 
 + 
 
 -■ 
 
56 
 
 THE WASSEEMANN TEST 
 
 Table XII 
 
 Besults of Titration or Serum for Complement-fixing Strength in 
 Case 8 — ^Latent Syphilis 
 
 DATE OF *BST 
 
 AMOUNT OF BLOOD SERUM. C.C. 
 
 CONTROL C.C. 
 
 0.02 
 
 0.04 
 
 0.06 
 
 0.08 
 
 0.1 
 
 0.1 
 
 Jan. 12 
 Jan. 13 
 Jan. 14 
 Jan. 15 
 Jan. 16 
 Jan. 17 
 Jan. 18 
 
 + 
 
 + 
 
 + 
 + - 
 + - 
 + - 
 + - 
 
 , + + 
 + + 
 + + 
 
 + 
 + 
 + 
 + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + 
 
 + + 
 ++ 
 ++ 
 ++ 
 ++ 
 ++ 
 ++ 
 
 - 
 
 Table XIII 
 
 Results of Titration of Serum for Complement-fixing Strength in 
 Case 9 — Latent Syphilis ' 
 
 
 AMOUNT of blood SERUM. C.C. 
 
 control C.C. 
 
 
 0.02 
 
 0.04 
 
 .0.06 
 
 0.08 
 
 0.1 
 
 0.1 
 
 Jan. 15 
 Jan. 16 
 Jan. 17 
 Jan. 18 
 Jan. 19 
 Jan. 20 
 Jan. 21 
 
 +- 
 
 + 
 
 -1- 
 -1- 
 
 + - 
 
 + 
 
 + + 
 -t- 
 
 + 
 + - 
 
 ++ 
 
 + 
 
 -t-t- 
 
 ++ 
 
 +- 
 
 ^ 
 
 Table XIV 
 
 Ebsults of Titration of Serum for Complement-fixing Strength in 
 Case 10 — ^Latent Syphilis 
 
 DATE OF TEST 
 
 amount of blood serum. C.C. 
 
 CONTROL C.C. 
 
 0.02 
 
 0.04 
 
 0.06 
 
 0.08 
 
 0.1 
 
 0.1 
 
 Jan. 15 
 Jan. 16 
 Jan. 17 
 Jan. 18 
 Jan. 19 
 Jan. 20 
 Jan. 21 
 
 + - 
 
 + - 
 + - 
 
 + - 
 + - 
 + + 
 
 -1- 
 
 + 
 
 + - 
 + - 
 + - 
 + -1- 
 
 + 
 
 -1- 
 
 + 
 + - 
 
 + 
 + + 
 
 -1- 
 
 -n- 
 -1- 
 + 
 
 +- 
 ++ 
 ■n- 
 
 - 
 
 In Case 7 the strength of the complement-fixation re- 
 action, using the diagnostic amount of serum, i. e., 0.1 c.c, 
 was only sufficient upon three of the seven days that the 
 serum was tested to give a plus or doubtful reaction, while 
 with smaller amounts the variations in the strength were 
 very marked. Upon these three days the test would have 
 been reported as doubtful and if further tests had not been 
 
FACTORS INFLUENCING RESULT OF TEST 57 
 
 made, the diagnosis, owing to the absence of symptoms, 
 would have been missed. 
 
 In Case 8 there was no variation from a positive reaction 
 with the diagnostic amount of serum during the seven days 
 the titrations were made, although some variation occurred 
 with smaller amounts of the serum. 
 
 In Case 9 there were very marked variations in the 
 strength of the reaction with the diagnostic amount of se- 
 rum, a negative reaction being obtained upon one day, a 
 plus-minus upon another, and a plus or doubtful reaction 
 upon a third. A double-plus, or positive reaction, was ob- 
 tained upon only four of the seven days that the serum 
 was tested. With amounts of serum smaller than the diag- 
 nostic amount of 0.1 c.c. the variations were very great. 
 
 In Case 10 there was also great variation, from day to 
 day, in the strength of the complement-fixation reaction, the 
 diagnostic amount of serum giving a plus-minus reaction 
 upon one day, a plus reaction upon three days, and a double- 
 plus or positive reaction upon three days of the week. With 
 smaller amounts of serum the variations were still more 
 marked. 
 
 General Discussion. — The most important practical point 
 brought out by these observations on the daily titration of 
 the blood serum of untreated syphilitics is that great varia- 
 tions may occur in the complement-binding power of the se- 
 rum of patients uninfluenced by any kind of treatment, and 
 that these variations occur from day to day, so that one neg- 
 ative examination, or even more, in a suspected case, is of 
 absolutely no value in excluding the disease. In several 
 of the cases mentioned the blood serum, if tested upon cer- 
 tain days only, would have given a negative, or practically 
 negative, result, although serum from the same case had 
 been previously positive, and again became positive within 
 a day or two. If this be true of untreated cases, some of 
 them showing severe lesions of the disease, it will certainly 
 be found true of a greater number of latent and treated in- 
 fections, the class of cases in which a negative Wassermaim 
 
58 THE WASSERMANN TEST 
 
 reaction is so often considered decisive as to the absence 
 of the disease. The results of these tests indicate the great 
 value of repeated examinations, when a negative Wasser- 
 mann is reported, before a patient is considered as cured, 
 or before he is assured that he is not suffering from a 
 syphilitic iafection. 
 
 As to the cause of the variations in the strength of the 
 reaction observed from day to day, it is evident that the 
 antibodies and lipotropic substances present in the pa- 
 tient's serum must vary from day to day, or such results 
 would not be obtained. It may be accepted as proved that 
 there exist in the blood of syphilitics antibodies and lipo- 
 tropic substances that are capable of fixing complement 
 in the presence of a proper antigenic extract, and that these 
 bodies depend for their origin on the presence in the body 
 of living 'treponemas, for when the latter disappear from 
 the lesions in experimental animals, as the result of treat- 
 ment, the complement-fixation reaction also disappears. 
 Whether these substances are produced by the reaction of 
 the tissues to endotoxins liberated by the breaking down 
 of dead treponemas ; to toxins produced by the living trep- 
 onemas ; or to the presence in the blood of the toxins them- 
 selves; are all mooted questions, but these experiments 
 prove that there must be a certain amount of them in the 
 blood serum before a positive Wassermann reaction can 
 be obtained, and that this amount varies considerably from 
 day to day. 
 
 It is but just to call attention to the importance of these 
 results in explaining, possibly, the discrepancies between 
 Wassermann reports from various laboratories where spec- 
 imens of blood from the same individual were examined at 
 different times, for it is evident that unless the same spec- 
 imen of blood be examined no reliable conclusions can be 
 drawn regarding the reports of different laboratories, a 
 fact ihat should be borne in mind when it is desired to 
 obtain a report from more than one laboratory upon a 
 suspected individual. 
 
FACTORS INFLUENCING EESULX OF TEST 59 
 
 The factors which have been mentioned in this chapter 
 as markedly influencing the results of the Wassermann 
 test ; i. e., the effect of alcohol ; of bacteria growing in the 
 blood serum to be tested; and normal variations in the 
 power of the blood serum to bind complement, are all of 
 practical importance and should all be considered in per- 
 forming the test and in judging of its results in any case 
 where there is a question of the accuracy of the reaction^ 
 If this were done, there would be much less criticism of 
 the Wassermann test than there is, and a much better un- 
 dersljanding of its reliability and its limitations. 
 
 '■r: 
 
CHAPTER IV 
 
 PEEPARATION AND TITRATION OF THE 
 
 REAGENTS USED IN THE 
 
 WASSERMANN TEST 
 
 The following list of apparatus and reagents needed in 
 performing the Wassermann test is here inserted and it 
 will be found to include all that is really essential for this 
 work: 
 
 List of Apparatus 
 
 1 Luer syringe, capacity 1 c.c. 
 1 Luer syringe, capacity 5 c.c. 
 1 Luer syringe, capacity 20 c.c. 
 6 needles, for collecting blood, (special needles for this purpose 
 
 can be obtained of any good surgical supply house). 
 1 electric centrifuge. International Instrument Co., Size 1, Type 
 A, with one four 15 c.c. tube head and metal holders, and 
 one four 50 c.c. tube head and metal holders. Type of 
 current and voltage must be specified. 
 20 centrifuge tubes, plain, 15 c.c. 
 10 centrifuge tubes, graduated, 15 c.c. 
 10 centrifuge tubes, 50 c.c, plain. 
 1 incubator, either electric, gas, or oil, with proper thermoregu- 
 
 lators, to run at 56° C. 
 1 water-bath, with thermometer, burner and thermoregulator, 
 large enough to accommodate the required number of 
 Wassermann racks, to run at 37° C. 
 1 set of 20 copper test tube racks, made by Topham, Wash- 
 ington, D. C. 
 1 amboceptor cutter, made by Topham, Washington, D. 0. 
 1 set of 3 amboceptor markers, 4 mm., 5 mm., and 6 mm., made 
 
 by Topham, Washington, D. C. 
 1 paraffin bath to fit incubator mentioned above, to hold tubes 
 during inactivation of blood serum. 
 100 ov more test tubes, without lip, 100 by 12.5 millimeters. 
 4 test tube baskets, 6 inches high by 7.5 inches in diameter. 
 10 Mohr pipettes, 10 c.c. capacity, graduated in tenths. 
 10 Mohr pipettes, 5 c.c. capacity, graduated in tenths. 
 
 60 
 
PKKPARATION AND TITRATION OF REAGENTS 61 
 
 20 pipettes, serologic, 1 c.o. capacity, graduated in hundredths to 
 tip. These pipettes should be of small bore and the 
 graduations far enough apart to render accurate read- 
 ings possible for quantities as small as one-one-hundredth 
 of a c.e. 
 100 or more pipettes, serologic, capacity 1 e.c, graduated in tenths. 
 These are used for measuring the sera to be tested. 
 12 Erlenmeyer flasks, capacity 250 c.e. each. 
 
 6 animal cages. 
 20 pounds soft paraffin, melting point 43° 0. 
 1 bottle, 500 gm. sodium chloride, C. P. 
 1 bottle, 30 gm. cholesterin, Merck's. 
 1 bottle, 500 gm. absolute alcohol. 
 6 triangular files, 3 inches long. 
 
 Guinea pigs and rabbits must be kept for complement 
 and amboceptor production and a small and suitable room 
 or animal house should be set aside to shelter them and to 
 allow for breeding. 
 
 Preparation of Reagents 
 
 The proper preparation of the reagents used in the Was- 
 sermann test is of great importance and explicit directions 
 are given in the following pages regarding this subject, 
 which, if carefully adhered to, will result in the prepara- 
 tion of reagents that will give accurate results. 
 
 The Wassermann test as performed in the writer's lab- 
 oratory and in most of the laboratories connected with the 
 Medical Department of the Army, is a modification of both 
 the test as originally recommended by Wassermann, and 
 the modification of that test recommended by Noguchi. The 
 technic was originally worked out by the writer and has 
 since been standardized, so far as the apparatus is con- 
 cerned, by Lieut. Colonel Vedder, of the Medical Corps. 
 A human hemolytic system is employed instead of the 
 sheep system recommended by Wassermann; alcoholic ex- 
 tracts of both foetal syphilitic liver, and cholesterinized 
 alcoholic extracts of normal human heart muscle, are used 
 as antigen ; and the blood serum is inactivated at 56° C. for 
 
62 THE WASSEBMANN TEST 
 
 one-half hour before it is tested. The following reagents 
 are employed in the test : 
 
 1. Complement. The fresh blood serum of guinea pigs. 
 
 2. Hemolytic amboceptor. The blood serum of rabbits 
 immunized to human red blood corpuscles. 
 
 3. Hemolytic antigen. A suspension of human red blood 
 corpuscles. 
 
 4. Syphilitic antigen. An alcoholic extract of foetal 
 syphilitic liver and a cholesterinized alcoholic extract of 
 normal human heart muscle. 
 
 5. Syphilitic amboceptor. The patient's blood serum. 
 This may or may not contain the lipotropic substance or 
 substances necessary for complement fixation with the 
 antigens mentioned. 
 
 Preparation of Complement. — Complement is a constit- 
 uent of the fresh blood serum of all animals but certain an- 
 
 f 
 
 imals furnish better complement for complement-fixation 
 tests and for the Wassermann test than do others, and it 
 has been demonstrated that for the WasSermann test the 
 blood serum of-the guinea pig supplies a better complement 
 than does that of other animals. Noguchi and Bronfen- 
 brenner tested the blood serum of the sheep, dog, ox, guinea 
 pig, hog, and rabbit, and found that the serum of the guinea 
 pig gave the best results when used as complement in the 
 Wassermann test, so that the blood serum of this animal 
 has been generally adopted for this purpose. 
 
 The blood serum of different guinea pigs varies consid- 
 erably in complementary activity, even in health, and in 
 unhealthy animals, and when the temperature is either 
 very hot or cold, marked variations may occur. For this 
 reason it is essential that the blood serum be titrated foi^ 
 its complementary power each time that it is used, and it 
 is always best, in practice, to employ the serum from two 
 or three guinea pigs, mixed, rather than to depend upon 
 that of a single pig, which might be found to be so weak as 
 to be inconvenient for use in the test. 
 
I,ne,- syringes. Se, of seven, suital.Ie fn,- J'e ,„ ,l,e Wasser.nann test, a„,l for serolog,. 
 
 work in general. ^ 
 
PREPARATION AND TITRATION OV REAGENTS 63 
 
 The guinea pig may be either bled from the heart or its 
 throat cut. It is the practice, in the writer's laboratory, 
 to bleed the pigs from the heart, as by this method the 
 animal is not sacrificed and can be used over and over again 
 for bleeding, provided the bleedings be from three to four 
 weeks apart. Several lots of pigs can be rotated and thus 
 considerable expense saved, as the animals usually stand 
 the bleeding well and it is seldom' that any are lost if the 
 bleeding is properly done. The operation should be per- 
 formed under ether and consists simply in entering the 
 heart with a hypodermic needle attached to a syringe hold- 
 ing 10 c.e. and nearly this amount of blood can be aspi- 
 rated from the heart without danger to the animal. It 
 is well to connect the needle of the syringe to the syringe 
 with a flexible rubber tube, as it is much easier to make the 
 puncture and handle the syringe afterward if this is done. 
 The operation requires considerable practice but when th,e 
 technic is once mastered it is by far the most satisfactory 
 way of obtaining complement for the Wassermann test, 
 especially if only a small number of tests are to be made 
 at one time. 
 
 After bleeding, the blood should be ejected from the 
 syringe into a sterile Petrie dish, allowed to stand at room 
 temperature for two hours and then placed in the ice box 
 overnight. In the morning the clear serum is pipetted off 
 and if it is to be used at once, diluted with an equal amount 
 of normal salt solution — (0.85%). If it is not to be used 
 for several hours it should be placed at once in the ice box, 
 as the complementary power of blood serum is weakened 
 by exposure to, room temperature for any great length of 
 time. The serum should be free from blood corpuscles 
 and almost colorless or but very slightly blood stained. 
 
 If a very large number of tests are to be made it may 
 be better to sacrifice the pigs by cutting their throats, each 
 pig being held over a Petrie dish, the throat cut quickly 
 with a sharp scalpel or razor, after shaving the neck, and 
 the blood allowed to run into the dish. Great care should 
 
64 THE WASSERMANIT TEST 
 
 be taken not to cut too deeply, so as to penetrate the esoph- 
 agus or trachea, as the secretions from the stomach have 
 a marked inhibitory effect upon complement and this is 
 also true to a lesser degree of the secretions of the larynx 
 or trachea. After collecting, the blood should be treated 
 in the same manner as after puncture of the heart. 
 
 The syringe, needles, Petrie dishes, and all apparatus 
 used in collecting the blood for complement should be ster- 
 ile and perfectly dry, with the exception of the syringe, 
 which should be washed out with normal salt solution be- 
 fore the puncture is made. If the blood serum, after be- 
 ing pipetted off, is much blood stained, it should not be 
 used for complement. If it contains blood corpuscles these 
 may be removed by centrifuging and pipetting off the clear 
 serum above the layer of corpuscles at the bottom of the 
 centrifuge tube. 
 
 The complement, even after being diluted one-half with 
 normal salt solution, may be kept in the ice box without 
 much loss of strength for about three days, but usually 
 upon the third day the titration of it will show such loss 
 of complementary power that it will be useless for the 
 test. Complement appears to be at the height of its com- 
 plementary power about thirty-six hours after removal 
 from the pig, but is much weaker immediately after with- 
 drawal of the blood and grows gradiially weaker from day 
 to day after this period. 
 
 Preservation of Complement. — ^Many attempts have been 
 made to preserve complement by the addition of various 
 substances, but none of the methods so far advocated have 
 been generally adopted. Noguchi*^ attempted to preserve 
 it by impregnating filter paper with it, a n^ethod very suc- 
 cessful with amboceptor serum, but without satisfactory 
 results. Kolmer*^ recommends chemically pure sodium 
 chloride as the best preservative, adding 0.425 gram so- 
 dium chloride to each 10 c.c. of sera obtained from several 
 guinea pigs. He states that complement preserved in this 
 
PEBPAEATION AND TITRATION OF REAGENTS 65 
 
 manner will maintain its hemolytic and fixing properties 
 for several weeks. 
 
 Preparation of Hemolytic Amboceptor.— The hemolytic 
 serum or amboceptor used in the Wassermann test as per- 
 formed by the writer is prepared by immnnizing rabbits 
 to human red blood corpuscles. Noguchi*^ was the first to 
 call to the attention of serologists the advantage of a hu- 
 man hemolytic system in Wassermann work and the writer 
 believes that it is the system best suited for complement- 
 fixation reactions upon the blood of man. Its adoption ob- 
 viates the danger that is present in human blood serum, of 
 the action of the natural antisheep amboceptor normally 
 present in such blood serum, and which, when the sheep 
 hemolytic system is employed, should be removed from 
 such serum before the test is made. It has not been prac- 
 ticable, owing to the immense number of Wassermann tests 
 made in army laboratories, to use any method which re- 
 moves this natural antisheep amboceptor from blood se- 
 rum, and, therefore, aside from the superiority of the 
 human hemolytic system for complement-fixation work, it 
 was necessary to adopt it from practical considerations. 
 The only objection that can be urged' against the human 
 system is that it is more difficult to immunize rabbits 
 against human erythrocytes than against sheep corpuscles 
 and that it requires a more careful technic both in collect- 
 ing the blood and injecting it into the animals, but this 
 objection is of no weight when the great advantages of the 
 human hemolytic system are considered. 
 
 Before considering the method of preparing the hemo- 
 lytic amboceptor serum there are certain general principles 
 regarding amboceptors, either bacteriolytic or hemolytic, 
 that will be mentioned. 
 
 It will be remembered that amboceptors are produced in 
 the blood serum of an animal in response to the injection 
 of an antigen, and that these amboceptors are specific for 
 the antigen injected. If the blood serum of the animal con- 
 taining them is added to a mixture of the antigen and of 
 
6Q THE WASSERMANN TEST 
 
 fresh blood serum, or complement, the amboceptor will act 
 as an intermediary body, allowing the complement to act 
 upon the antigen, and bacteriolysis or hemolysis wUl result. 
 
 Amboceptors are thermostable ; i. e., their activity is not 
 destroyed by heating the serum containing them for one 
 half hour at 56° G, They are also resistant to a consider- 
 able degree to acids and alkalies, and to drying. A good 
 hemolytic serum can be preserved upon filter paper, in a 
 dry condition, for many months with very little loss of 
 strength and this fact is made use of in the Wassermann 
 test, as will be noted later. 
 
 Amboceptors appear to act as a chemical linking body, 
 connecting the antigen with the complement, as the comple- 
 ment is unable to act upon an antigen except through the 
 agency of the amboceptor. Hemolytic amboceptors occur 
 in very small amounts in the blood of normal animals and 
 such amboceptors are sometimes present in an amount suf- 
 ficient to cause trouble in complement-fixation reactions, as 
 in the case of the normal antisheep amboceptor present 
 in human blood serum. 
 
 In producing hemolytic amboceptor for human red blood 
 corpuscles in rabbits several methods have been recom- 
 mended which will be described. The writer has obtained 
 the best results with the method first described but the 
 other methods are given in order that a choice may be 
 made if desired, according to circumstances. 
 
 Well grown, perfectly healthy rabbits are selected, pref- 
 erably pure white ones, and are given repeated injections 
 of washed human red blood corpuscles. The blood corpus- 
 cles are prepared for the injections in the following man- 
 ner : The requisite amount of blood, allowing at least twice 
 the amount of blood as the amount of corpuscles to be in- 
 jected, is withdrawn from one of the large veins of the 
 arm with a glass syringe, which has been sterilized and 
 washed out with sodium citrate solution. After drawing 
 the blood, it is at once ejected into a flask containing from 
 200 to 250 c.c. of normal salt solution (0.85%), and dis- 
 
,_fe===i«s^^l 
 
 Fig. 4. 
 Klectric centrifuge. 
 
PBBPAEATION AND TITRATION OP REAGENTS 67 
 
 tributed into large centrifuge tubes (50 e.c.) and centri- 
 fuged until the erythrocytes are deposited and the super- 
 natant fluid is practically colorless. The supernatant fluid 
 is now poured or pipetted off and the tubes again filled 
 with the salt solution, well shaken so as to distribute the 
 red cells throughout the solution, and again centrifuged. 
 This process is repeated four times, when the supernatant 
 salt solution is tested for albumin; if the merest trace is 
 demonstrated, the washing is again repeated and continued 
 until no reaction for albumin is obtained. Almost invari- 
 ably four washings will be found sufficient. Care should 
 be taken not to centrifuge for so long that the corpuscles 
 become packed at the bottom of the tubes, for the force 
 necessary to dislodge them will break up a certain per- 
 centage and render them unsuitable for injection. Care 
 should also be taken to have each centrifuge tube weigh 
 the same as all others, and this can be done by weighing 
 each in a balance ^t the time they are filled. Unless this 
 is done the centrifuge will run unevenly and is liable to 
 breakage or accident. 
 
 After the corpuscles are washed in the manner described 
 they are mixed with as little normal salt solution (0.85) 
 which must be sterile, as is necessary to secure a suspen- 
 sion that will pass through the needle of the injection syr- 
 inge, and are at once injected, either intravenously or 
 subcutaneously, with a sterilized syringe. 
 
 Methods of Securing a Good Amboceptor Serum. — The 
 methods of securing a good amboceptor serum val-y some- 
 what but the writer has found that the most rapid method 
 of immunizing the rabbits and securing a good hemolytic 
 serum is to give an intravenous injection, in the ear vein, 
 of 1 c.c. of washed erythrocytes every other day until five 
 or six injections have been given, when the rabbit's blood 
 serum should be titrated and will generally be found strong 
 enough in hemolytic properties to be used for the test. 
 If it is not, two or three more intravenous injections of the 
 same amount of red corpuscles should be given. Earely, 
 
68 THE WASSEEMANN'«TEST 
 
 animals are found that will not develop a sufficiently strong 
 serum, no matter how many injections may be given, but 
 usually from five to six injections will be found sufficient. 
 By this method it is very seldom that an animal is lost, 
 as the frequent injections prevent the development of ana- 
 phylaxis, and a good serum for use as amboceptor in the 
 Wassermann test can be secured within a comparatively 
 short period of time. 
 
 Another good method is to give one subcutaneous 
 injection of 5 c.c. of washed human erythrocytes in the 
 abdomen, followed by from three to four intravenous in- 
 jections in the marginal vein of the ear of from 3 to 4 c.c. 
 of washed erythrocytes, at intervals of from five to six 
 days. This method is much slower than the preceding, and 
 not infrequently an animal is lost through anaphylactic' 
 phenomena. 
 
 Noguchi** recommends the following method of produc- 
 ing hemolytic amboceptor for the Wassermann test: Five 
 injections of washed human erythrocytes are given subcu- 
 taneously in the abdomen, the first of 5 c.c. ; the second, 8 
 c.c. ; the third, 12 c.c. ; the fourth, 15 c.c. ; and the fifth, 20 
 c.c; at five day intervals. Nine days after the last injec- 
 tion the rabbit is bled for a small amount of blood from the 
 ear vein and the serum titrated. This method is an excel- 
 lent one but consumes much more time than the method of 
 intravenous injections made every other day, recommended 
 above. 
 
 "Whatever method is used for unmunizing the rabbits, the 
 blood serum should not be tested for its hemolytic proper- 
 ties until from 7 to 9 days have elapsed from the date of 
 the last injection of red corpuscles. 
 
 Preservation of the Amboceptor Serum.— The ambocep- 
 tor serum may be preserved in ampules holding from 1 
 to 5 c.e. in the ice box, and used as required, but the writer 
 has employed for nearly ten years the method of preserv- 
 ing amboceptor serum for the "Wassermann test recom- 
 mended by Noguchi with the most satisfactory results and 
 
PEEPAKATION AHD TITEATION OF BBAGBNTS 
 
 69 
 
 this method is used in most of the army laboratories. It 
 consists in impregnating suitable filter paper with the 
 blood serum, drying, and storing in a glass container in 
 a dark closet or drawer. The method has entirely replaced 
 the use of liquid amboceptor in our laboratories and pre- 
 served in this manner the serum keeps much better, and 
 preserves its strength for a much longer time, than when 
 it is kept iu liquid form, and the danger of bacterial con- 
 tamination, always present with the liquid serum, is 
 avoided. 
 
 Before impregnating the filter paper, a preliminary ti- 
 tration of the rabbit's blood serum should be made in order 
 to determine whether it is strong enough in hemolytic 
 amboceptor to use in the test. For this purpose a small 
 amount of the rabbit's blood is collected from the ear vein 
 in a capillary pipette or Wright tube and the serum allowed 
 to separate. It is then separated from the clot and inac- 
 tivated by heating it for one-half hour at 56° C. in the 
 paraffin bath or water-bath, and one drop of it, from a 
 capillary pipette, is mixed with 39 drops of sterile normal 
 salt solution- (0.85) and titrated for hemolytic strength. 
 The method of titration is illustrated in Table XV. 
 
 Table XV 
 Pbeliminaky Titration op Amboceptor Serum for Wassermann Test* 
 
 TUBE 
 NO. 
 
 amount op 
 salt solu- 
 tion. 0.85%, 
 
 CO. 
 
 0.9 
 0.9 
 0.9 
 0.9 
 0.9 
 0.9 
 
 number op 
 complement 
 
 UNITS 
 
 1 
 1 
 1 
 1 
 1 
 1 
 
 AMOUNT OP 
 BLOOD SUS- 
 PENSION. 
 5%. 
 CO. 
 
 0.1 
 0.1 
 0.1 
 0.1 
 0.1 
 ■0.1 
 
 AMOUNT OP 
 AMBOCEPTOR 
 SEBUM, DI- 
 LUTED 1-40. 
 DROPS 
 
 1 
 
 2 
 3 
 4 
 5 
 
 None 
 
 Incubate i n 
 water-bath at 
 37° C. for one 
 hour or in incu- 
 bator at same 
 temperature for 
 two hours and 
 read results. 
 
 *If the unit of complement has not been determined by a previous titration, use 0.05 
 c.c. of a 1 ;2 dilution in salt solution. 
 
 After incubating in the water-bath at 37° C. for one 
 hour, or in the incubator for two hours, the titration is 
 
70 THE WASSEBMANSr TEST 
 
 read, and if either of the tubes containing one or two drops 
 of the rabbit's serum shows complete hemolysis, the sertnn 
 is strong enough in hemolytic amboceptor for use in the 
 test and the rabbit should be bled at once. The cdiitrol 
 tube. No. 6, should show complete inhibition of hemolysis. 
 
 Bleeding is accomplished most quickly by cutting the 
 carotid vessels while holding the rabbit over a large glass 
 dish, into which the blood is allowed to flow. The neck of 
 the animal, where the incision is to be made, should be 
 shaved and washed carefully but no disinfectant should be 
 used. The glass dish to contain the blood should be ster- 
 ilized before use and after the blood has been collected it 
 should be kept at room temperature for about two hours 
 and then placed in the ice box overnight. In the morning 
 the clear serum should be pipetted off into small test tubes, 
 inactivated by heating it for one-half hour at 56° C. in the 
 paraffin or water-bath, and then kept in the ice box until 
 one is ready to impregnate the filter paper. It does no 
 harm if the blood serum is slightly tinged with hemoglobin, 
 as the amount used in the test is so small that it wiU not 
 interfere with the reading of the reaction. 
 
 Impregnating the Filter Paper. — The filter paper used 
 for this purpose is Schleich and SchuU's No. 597, but any 
 other make of paper of approximately the same texture 
 will serve equally as well. The paper is cut into squares 
 measuring 10 by 10 centimeters, and actual experience has 
 shown that it will take about 1.5 c.c. of the rabbit serum 
 to saturate one of these squares of paper, so that it is an 
 easy matter to calculate how many squares will be required 
 for any given amount of the serum. This number is placed, 
 one by one, in a suitable Petrie dish or other glass dish 
 containing the serum, and the process continued until all 
 the serum is absorbed by the paper. The squares of sat- 
 urated paper are then carefully lifted with a pair of for- 
 ceps, thoroughly drained, and drawn across the edge of 
 the dish a few times to remove any excess of serum, and 
 then placed upon a piece of unbleached muslin, which 
 
Same ce' 
 
 „tr,tu8C illustrated in Vi, 4, with shaker hea.l .ttach.,1. fnr 
 
 iic;c in making 
 
 aiitigfiiic extracts. 
 
PREPAEATION AND TITRATION OF REAGENTS 71 
 
 should be large enough to contain all the slips of paper. 
 The muslin containing the paper is now placed under an 
 electric fan and the paper rapidly dried, after which it 
 should be placed in air-tight glass containers and kept at 
 room temperature in a dark and dry place. Before use, 
 it should be titrated as will be described. 
 
 The amboceptor paper thus prepared will retain its 
 working strength for many months, but it should be ti- 
 trated at least once a month in order to avoid any sudden 
 loss of strength, although the writer has never observed 
 such losses in the many years that he has used the ambo- 
 ceptor ^aper. Paper over a year old has been repeatedly 
 used and in practically the same dose as when it was first 
 prepared and paper two years old was found to have lost 
 only one-eighth of its original strength at the end of that 
 time. 
 
 Preparation of the Hemolytic Antigen. — The hemolytic 
 antigen used in the test consists of a 5 per cent suspension 
 of human red blood corpuscles, prepared in the following 
 manner : 
 
 In the Wassermann test, as recommended by the writer, 
 each tube contains 0.1 c.c. of a 5 per cent suspension of 
 human red blood corpuscles in 0.85 per cent salt solution. 
 The quantity of blood required for a series of tests will 
 depend, of course, upon their number. One cubic centi- 
 meter of blood will furnish enough suspension for nearly 
 one hundred tests, and unless one is doing a very large 
 number of tests, enough blood may be easily secured by 
 constricting the middle finger, after forcibly swinging the 
 arm about for a few times, with a rubber tube, and prick- 
 ing the finger just above the root of the nail with a sterile 
 needle or glass point. The blood is allowed to drop into 
 sterile graduated centrifuge tubes, each tube being filled to 
 the 9 c.c. mark with sterile normal salt solution (0.85%), 
 and enough blood is added to fill the tube to the 10 c.c. mark. 
 The tubes are then shaken well and centrifuged until all 
 the blood corpuscles are collected at the bottom. The su- 
 
72 THE WASSBKMANK TEST 
 
 pernatant liquid is then carefully poured, or pipetted off, 
 the tubes again filled with salt solution, and again centri- 
 fuged until the cells are at the bottom of the tube. This 
 process of washing is repeated until the corpuscles have 
 been washed four times, which will be found sufficient. 
 
 The supernatant fluid is poured or pipetted off for the 
 last time, and the amount of corpuscles at the bottom of 
 the tube noted, and the tube filled mth enough salt solution 
 to make a 5 per cent suspension. Thus, if there are 0.5 c.c. 
 of red corpuscles at the bottom of the tube, it would require 
 9.5 c.c. of salt solution to make a 5 per cent suspension. 
 If only a small amount of blood is needed for a few tests, 
 one centrifuge tube wilt' be all that is required for the 
 washing but if many tests are to be performed, the wash- 
 ing should be done with a greater number of tubes. 
 
 In the test, 0.1 c.c. of this suspension is added to each 
 tube, which contains 0.9 c.c. of normal salt solution, thus 
 making a half of one per cent suspension. This makes a 
 suspension sufficiently strong to render the reading of the 
 test easy and is much more saving of complement and am- 
 boceptor than the use of a stronger suspension. 
 
 Preparation of Antigens for Wassermann Test. — The 
 syphilis antigen, as it may be called, as already stated, is 
 not a true antigen, in the strict use of the term, as it is an 
 extract containing lipoids which react mth lipotropic sub- 
 stances in the blood serum of the syphilitic patient, but as 
 it acts like a true antigen in the Wassermann test, it is 
 convenient to retain the name, but it should be understood 
 that it is not a real antigen, as are the red blood corpuscles 
 used in the hemolytic system just described. 
 
 It is certainly true that in all alcoholic extracts of nor- 
 mal tissues used as antigens in the Wassermann test, the 
 active principle is contained within lipoids in the extracts 
 and that there is nothing specific as regards Treponema 
 pallidum, in these extracts. When alcoholic extracts of 
 foetal syphilitic liver, and especially watery extracts of 
 such livers, are used as antigens, the extracts, besides eon- 
 
Small incubalnr, tn run at 5i>" C , fui inactivating!; sura. 
 
PREPAEATION AND TITRATION OP REAGENTS 73 
 
 taining the lipoids present in the normal extracts, may also 
 contain specific antigenic substances due to Treponema 
 pallidum, as has already been shoMTi. However, even in 
 such extracts, the greater part of the reaction is undoubt- 
 edly due to the lipoids present in them, although it can not 
 be denied that in some instances, where the extracts of 
 normal tissue give a negative or doubtful result, the ex- 
 tracts of foetal syphilitic liver and of cultures of Trepo- 
 nema pallidum give a positive one. Accordingly, the 
 writer advises the use of both a cholesterinized alcoholic 
 extract of normal human heart muscle and an alcoholic 
 extract of foetal syphilitic liver in performing the test. If 
 both can not be obtained the best results will be secured 
 by using cholesterinized alcoholic extracts of normal tis- 
 sues, preferably human heart muscle. The extracts of pure 
 cultures of Treponema pallidum, while giving interesting 
 results from a scientific standpoint, are useless as antigen 
 in routine Wassermann work because of their unreliability. 
 In practical work the writer recommends the antigens al- 
 ready mentioned, and, in addition, Noguchi's acetone- 
 insoluble lipoid antigen, where the other antigens fail in 
 apparently true syphilitic infections, but only in such in- 
 fections. 
 
 Alcoholic Extract of Foetal Syphilitic Liver. — A foetal 
 liver, rich in Treponema pallidum is selected, washed free 
 from blood, and any fat present removed. One hundred 
 grams is weighed out, all blood removed by washing, and 
 cut into very small pieces. The material is then placed in 
 a suitable bottle, or other container, and 1000 grams of 
 absolute alcohol, or 95% alcohol, added, and the whole 
 placed in an incubator at 37° C. and allowed to remain for 
 10 days, being thoroughly shaken three times a day during 
 that time, when it will be ready to titrate. If a shaking 
 machine is available, the material, after adding the alco- 
 hol, is placed in a suitable bottle or bottles, and shaken for 
 24 hours at a good rate of speed, at the end of which time 
 it will be found that the tissue is thoroughly extracted. 
 
74 THE WASSEKMANK" TEST 
 
 After extraction by either method is completed, the mate- 
 rial is filtered through filter paper and the filtrate titrated. 
 If the first titration is not satisfactory, the filtrate should 
 be evaporated to two-thirds its original volume and again 
 titrated. The antigenic extract should always be kept in 
 the ice box except when in use. 
 
 Cholesterinized Human Heart Extract.— The same 
 method is used in preparing the alcoholic extract of heart 
 muscle, the material being washed free from blood, all fat 
 removed, and 100 grams of the heart muscle being cut into 
 small pieces and extracted with alcohol in the same pro- 
 portions (1000 grams of alcohol to 100 grams of muscle). 
 After extraction one-half of the material is filtered through 
 paper, and 0.4% of cholesterin added, and titrated. If the 
 titration does not result satisfactorily as regards the anti- 
 genic strength of the extract, the remaining half is evapo- 
 rated to two-thirds its original volume, again filtered 
 through paper, and the cholesterin added, after which it 
 is titrated. The amount of cholesterin added gives prac- 
 tically a saturated solution and there is generally an ex- 
 cess of cholesterin left after standing, and in using the 
 antigen care should be taken not to get any of this excess 
 into the pipette used in the work and thus in the diluted 
 solution of the antigen. After the addition of the choles- 
 terin the antigenic extract is placed in glass-stoppered bot- 
 tles' and kept in an ice box, except when in use, 
 
 Noguchi's Acetone Insoluble Lipoid Antig^en, — This type 
 of antigen was recommended by Noguchi when he evolved 
 his modification of the Wassermann test and it is an excel- 
 lent antigen, especially for use in cases in which the 
 ordinary antigens described give doubtful results, as it 
 appears to be more delicate than the extracts already de- 
 scribed. However, it is true that sometimes this antigen 
 appears to give positive reactions in cases that are nega- 
 tive with other antigens and in which syphilis can not be 
 stated to exist upon clinical grounds, so that, in the writ- 
 er's opinion, the use of this antigen should be restricted to 
 
FiK. 7. 
 Watfr-balh. siiilal.lt for Wassermann lest incuhatinns. 
 
PEEPAEATION AND TITRATION OF KEAGENTS 75 
 
 the class of cases mentioned and as a control of other anti- 
 gens, when doubtful results are obtained. It is prepared 
 in the following manner: 
 
 Normal organs are used and may be from man or the 
 lower animals. Extracts made from heart muscle, liver, 
 or kidney of man or the same organs of the guinea pig or 
 rabbit are satisfactory and the extraction is done in the 
 same manner as for the antigens already described, 10 
 parts of absolute alcohol being used for each part of tis- 
 sue. After extraction, the material is filtered, and the 
 filtrate evaporated to dryness by the aid of an electric fan. 
 The residue is taken up with ether and the solution thus 
 obtained is allowed to stand over night in a cool place. In 
 the morning the supernatant ether will be found perfectly 
 clear while the insoluble portion of the residue has settled 
 to the bottom of the receptacle. The clear etheral portion 
 is decanted off and evaporated to a small quantity and 
 mixed with ten volumes of pure acetone. The precipitate 
 which forms is allowed to settle and the supernatant liquid 
 poured off. The precipitate is light brown in color and 
 forms the material from which the antigen suspension is 
 made, 0.3 gram of this acetone insoluble fraction being dis- 
 solved in 1 c.c. of ether and then mixed with 9 c.c. of methyl 
 alcohol. This mixture may be kept for a long time and 
 the antigen suspension used in making the test is prepared 
 by adding 1 c.c. of this stock solution to 9 c.c. of normal 
 salt solution, or if less is needed at the time of making the 
 tests, the same proportion of antigen and salt is preserved. 
 
 Wassermann's Aqueous Extract Antigen. — The antigen 
 employed by Wassermann in his method of performing the 
 complement-fixation test for syphilis was prepared as 
 follows : 
 
 A foetal syphilitic liver, rich in treponemas, is cut into 
 very small pieces and weighed. Four times its weight of 
 0.5 per cent phenol in physiologic salt solution is added 
 and the mixture placed in a brown bottle and shaken in a 
 shaking machine at room temperature for twenty-four 
 
76 THE WASSBRMANN TEST 
 
 hours. It is then filtered through gauze and placed in a 
 brown bottle in the ice box for several days, after which it 
 is titrated to determine its antigenic strength. A sediment 
 forms in the bottle after standing in the ice box and one 
 should be careful not to disturb this in using the antigen. 
 
 This antigen is not satisfactory, as it varies very greatly 
 in strength when made from different livers, and is very 
 prone to lose strength rapidly, especially if it is left for 
 any length of time at room temperature. It is not nearly 
 as satisfactory as the antigens heretofore described. 
 
 Various other methods of preparing antigenic extracts 
 for the Wassermann test have been described by Marie 
 and Levaditi,*^ Landsteiner, Miiller, and Potzl,*^ Porges 
 and Meier,*'' Browning, Cruickshank, and McKenzie,** and 
 others, but none of them are better in practical use than 
 those which have been treated of in the foregoing para- 
 graphs. 
 
 Cholesterinized Antigens.— Before concluding this por- 
 tion of our subject it may be well to discuss briefly the sub- 
 ject of the use of cholesterinized antigens in the Wasser- 
 maim test. The writer advocates their use, or, at least, the 
 use of an extract of human heart muscle, cholesterinized, be- 
 cause he has found by long experience that such an extract 
 is perfectly reliable and does not give false positive reac- 
 tions, as shown by the results in the many thousands of 
 tests in which he has used such an extract. Some author- 
 ities claim that, in their experience, cholesterinized ex- 
 tracts are so very sensitive that they may give false posi- 
 tive results but this has not been the writer's experience. 
 Very rarely there has been observed a plus or plus-minus 
 reaction in apparently normal serum, but the writer has 
 never observed a distinctly positive reaction with the 
 cholesterinized heart extracts used in his method in a se- 
 rum which could be proved to be normal, but, on the other 
 hand, numerous instances have been observed in which 
 ordinary extracts gave doubtful or negative results but in 
 which the cholesterinized heart extract gave positive re- 
 
PREPARATION AND TITRATION OF REAGENTS 77 
 
 actions. This is very apt to be the case in cases of syphilis 
 that have been treated and, in the writer's opinion, the 
 cholesterinized extract is far superior, as a control of 
 treatment, than are extracts of normal tissues or of foetal 
 syphilitic liver, to which cholesterin has not been added. 
 
 Titration of Reagents Used in the Wassermann Test. 
 
 The accurate titration of the various reagents used in 
 the Wassermann test is of fundamental importance, as the 
 test is essentially a quantitative one and the smallest vari- 
 ation in the amount of the reagents used may cause very 
 great variations in the result of the test. If the amount 
 of complement or amboceptor be excessive the strongest 
 positive serum may give a negative result, while if the 
 amount of these reagents is too small, normal sera will 
 give positive results. The same is true of the amount of 
 antigen, for an excess of antigen will result in a positive 
 result in normal sera or in the blood sera from other dis- 
 eases than syphilis, while too little will result in negative 
 reactions in what should be found to be strongly positive 
 sera. 
 
 It is easily seen, therefore, that the accurate titration of 
 complement, amboceptor, and antigen is of the greatest 
 practical importance, and the neglect of such titrations will 
 be sure to lead to erroneous results and to great injustice 
 being done the patient who has entrusted the testing of his 
 blood to the laboratory. 
 
 Titration of the Complement. — The amount of comple- 
 ment to be used in the Wassermann test is of vital impor- 
 tance and is determined by a titration of the g-uinea pig 
 serum each day before the tests are made. Some author- 
 ities employ exactly one unit of complement Avith one unit 
 of amboceptor for each test but this allows of no inhibitpry 
 effect by either the blood serum or antigen, and, is there- 
 fore, a dangerous proceeding unless very carefully con- 
 trolled. If the titrations are made with the amount of 
 patient's serum added to the complement and amboceptor 
 the fallacy residing in 'the possible anticomplementary 
 
PLATE I 
 
 Fig. 1. — Titration of Complement. Each of the tubes contains the amount 
 of complement marked above the tube. In the titration, as illustrated, hemo- 
 lysis is comgiete in the fourth tube, containing 0.05 c.e. of complement. Henee 
 0.05 e.e. is the complement unit and twice this amount is used in the actual 
 test, or 0.1 c.c. 
 
 The tenth tube is a control tube containing no complement and it should 
 show complete inhibition of hemolysis, as illustrated. 
 
 Fig. 2. — Titration of Amboceptor Papers. Each of the first five tubes con- 
 tains a piece of amboceptor paper of the size indicated above the tubes. In the 
 titration illustrated the first tube showing complete hemolysis is the third tube, 
 containing a piece of paper measuring 5 by 3 mm. Hence the unit of ambo- 
 ceptor paper is a piece measuring 5 by 3 mm., and twice this amount is used 
 in the test, or a piece measuring 5 by 6 mm. 
 
 The sixth tube is a control tube containing no amboceptor paper and should 
 show complete inhibition of hemolysis, as illustrated. 
 
 Fig. 3. — Titration of Antigen. Tubes 1, 2, 3, and 4 contain respectively 
 0.05, 0.1, 0.15, and 0.2 c.c. of a 1 in 10 dilution of the antigenic extract, with 
 0.1 e.c. of a known syphilitic serum. Tube 5 contains 0.1 c.c. of the syphilitic 
 serum without antigen. Tube 6 contains 0.1 c.e. of a normal blood serum with 
 0.2 e.c. of the diluted antigen. Tube 7 contains 0.1 c.c. of the normal serum 
 without antigen. Tube 8 contains complement without amboceptor, while 
 Tube 9 contains the hemolytic system, acting as a control of the latter. 
 
 A good antigen should give the results illustrated in this titration, causing 
 complete inhibition of hemolysis in the presence of 0.05 e.c. of a syphilitic 
 serum, as shown in Tube 1, and no inhibition of hemolysis with a normal serum 
 in amounts as large as 0.2 c.c, as shown in Tube 6. Complete inhibition of 
 hemolysis shoiild occur in Tube 8 and complete hemolysis in 5, 6, 7, and 9. 
 
0-o2cc. o-o3c£. 0-o^c.c. o-o5cc oo6c.c oorcc oo8croo9cc o/cc Control 
 
 'p fr"-"» 
 
 ^ 
 
 Sb^ W^ ^^ v^ 
 
 Fig. I. Tifrafion of Complement. 
 
 5x1mm. 5x2mm. 5x3/nm, 5xH-mm. 5x5mm. Control 
 
 y^^ Y" "1 
 
 Vi&^ 
 
 wife yy.aj..i w w/ 
 
 >»„-■-;* 
 
 Fig.E. Titration of Amboceptor Papers. 
 
 T 
 
 »! ■<< 
 
 •^^m, 
 
 V't'- ' '^Hf 
 
 "^ 
 
 '^e.is' 
 
 W"-""*"^ ' 
 
 '|S38 
 
 Fig. 3. Titration of Antigen 
 
PREPAKATION AND TITRATION OF EEAGENTS 79 
 
 action of the blood serum would be overcome, but this is 
 a cumbersome procedure, especially when a large number 
 of tests are to be made, and, in addition, there is still the 
 factor of the natural inhibition of hemolysis which is pres- 
 ent in almost all antigens. It is, therefore, much better 
 to use an excess of complement in making the tests and it 
 has been found perfectly safe in practice to double the 
 unit of complement found by titration, when actually do- 
 ing the test. In other words, two units of complement are 
 used for each tube employed in the test. 
 
 In order to titrate the complement it is necessary that a 
 standard amount of amboceptor and a standard suspension 
 of human red blood corpuscles be adopted and strictly ad- 
 hered to, although the strength of the blood suspension 
 may be varied, provided the complement be titrated with 
 it before each series of tests. Standard doses of ambo- 
 ceptor are adopted because this reagent changes little in 
 strength during long periods of time, while if the ambo- 
 ceptor were to be titrated before each series of tests, a 
 standard dose of complement would have to be adopted 
 and the latter varies so greatly in strength that it is pref- 
 erable to titrate the complement with a standard dose of 
 a*nboceptor. 
 
 In the titration of the complement two units of ambo- 
 ceptor, as determined by titration, are employed, or the 
 amount of amboceptor that is actually used in the test. 
 The complement, amboceptor, and blood cells should be all 
 added at once, for if the amboceptor and the blood cells 
 are left in contact, without the complement, for any length 
 of time, the cells will become "sensitized" and a smaller 
 amount of complement will produce hemolysis. 
 
 In making the titration of the complement, either a 5 or 
 10 per cent suspension of human erythrocytes, in normal 
 salt solution, may be used, but the writer prefers the 5 per 
 cent suspension as it is more economic of amboceptor and 
 is of sufficient strength to render the readings of the reac- 
 tion easy. It should be remembered that the stronger the 
 
80 
 
 THE WASSERMAISTN' TEST 
 
 blood suspension is fhe greater will be the amount of am- 
 bpceptor and complejnent necessary to produce hemolysis, 
 and the more extravagant the expenditure of these re- 
 agents, so that it is foolish to employ a stronger blood sus- 
 pension for the test than is, necessary to enable one to 
 readily read the reactions, as the hemolytic system is 
 merely used as an indicator of the presence in the patient's 
 blood of the substances that absorb or bind complement. 
 In titrating the complement, test tabes measuring 100 
 by 12.5 millimeters are used. These tubes should be per- 
 fectly clean and, whUe they do not need to be sterile, the 
 best technic is to have them sterilized. The complement 
 is diluted with an equal amount of normal salt solution 
 (0.85%) and graduated amounts are added to tubes con- 
 taining the blood suspension and amboceptor, as illustrated 
 in Table XVI. 
 
 Table XVI 
 Titration of the Complement 
 
 
 AMOUNT OF 
 
 AMOUNT OF 
 
 
 
 1- 
 
 
 
 
 NUMBER OF 
 
 AMOUNT OF 
 
 
 TUBE 
 
 SALT SOLU- 
 
 BLOOD SUS- 
 
 AMBOCEPTOR 
 
 COMPLEMENT 
 
 Incubate i » 
 
 NO. 
 
 TION 
 
 PENSION, 5% 
 
 UNITS 
 
 CO. 
 
 water-bath a t 
 
 
 CO. 
 
 CO. 
 
 
 
 37° 0. for one 
 
 1 
 
 0.9 
 
 0.1 
 
 2 
 
 0.02 
 
 hour and read." 
 
 2 
 
 0.9 
 
 0.1 
 
 2 
 
 0.03 
 
 The tube show- 
 
 3 
 
 0.9 
 
 0.1 
 
 2 
 
 0.04 ' 
 
 i n g complete 
 
 4 
 
 0.9 
 
 0.1 
 
 2 
 
 0.05 
 
 hemolysis con- 
 
 5 
 
 0.9 
 
 0.1 
 
 2 
 
 0.06 
 
 tains one unit 
 
 6 
 
 0.9 
 
 0.1 
 
 2 
 
 0.07 
 
 of complement. 
 
 7 
 
 0.9 
 
 0.1 
 
 2 
 
 0.08 
 
 Two units are 
 
 8 
 
 0.9 
 
 0.1 
 
 2 
 
 0.09 
 
 used in making 
 
 9 
 
 0.9 
 
 0.1 
 
 2 
 
 0.10 
 
 the test. 
 
 10 
 
 0.9 
 
 0.1 
 
 
 
 0.10 
 
 
 After incubating in the water-bath at 37° C. for one 
 hour, or in the ordinary bacteriological incubator, for two 
 hours at the same temperature, the titration is read, and 
 the tube that contains the smallest amount of complement 
 which shows complete hemolysis is noted and this amount 
 is called one unit of complement. For instance, if it is 
 found that Tube No. 4, containing 0.05 c.c. of complement, 
 
 / 
 
i'l 
 
 y. 
 
 Amboceptor cutter. Fr 
 Wdder, Medical Corps, I'. 
 War IV-iiartTiK-nt, Wrshingr 
 
 111 view showing gauge. 1 )fi 
 . Army, for cutting amboceiito 
 ,1. I). C, Office of the Surgen 
 
 std ii\ Ijenl. Colunc 
 
 1 E 
 
 |ia|icr. (From I!n]]cti 
 
 , Xm 
 
 GtTitTal, V. S. Arm> 
 
 , 191 
 
PREPARA.TI01i5" AND TITRATION OF REAGENTS 81 
 
 is the first of the tubes to show complete hemolysis, the 
 tubes preceding it showing only partial hemolysis, then 
 0.05 c.c. of complement constitutes one unit of complement, 
 and in performing the actual test twice this amount or 
 0.1 c.c. will be used for the complement dose for each tube, 
 or two units of complement. The amount of complement 
 is doubled in order to provide for the anticomplementary 
 action of the patient's blood serum and of the antigen, and 
 has been found in practice to be sufficient to answer this 
 purpose and at the same time not to cause fallacies in 
 the test. 
 
 In reading the titration if there is the slightest cloud- 
 iness in any of the tubes hemolysis can not be said to be 
 complete and in selecting the tube which represents one 
 complement unit, the first that shows an absolutely crystal 
 clear solution should be noted. The control tube. Tube No. 
 10, which contains no amboceptor, should show complete 
 inhibition of hemolysis. If it does not, there is somethiag 
 wrong with the complement or the salt solution used in 
 making the titration, and the titration should be repeated 
 with new reagents. 
 
 This Titration of the Complement Must be Made Before 
 Each Series of Tests or Very Serious Errors Will De- 
 velop. — The blood serum of guinea pigs varies greatly in 
 complementary strength, so that it is excessively danger- 
 ous to use a stated amount of the serum for complement. 
 A common observation, upon titrating different samples of 
 guinea pig serum, is to find differenced in complementary 
 strength all the way from a unit equalling 0X)3 c.c. to one 
 of 0.08 c.c. and sometimes a serum will be found much 
 stronger or weaker than these limits, so that the danger 
 of using any- definite amount is apparent. The complement 
 must he carefully titrated before each series of tests. 
 
 Titration of the Hemolytic Amboceptor. — As already 
 stated, the hemolytic serum or amboceptor used in the 
 Wassermann test as recommended by the writer is pre- 
 pared by immunizing rabbits to human red blood corpus- 
 
82 
 
 THE WASSEEMANN TEST 
 
 eles. The human hemolytic system, as it is called, was 
 adopted in order to obviate the danger arising from the 
 natural antisheep amboceptor present in human blood se- 
 rum, and which is frequently present in quantities suffi- 
 cient to render a positive syphilitic serum negative unless 
 special precautions are taken to guard against such an 
 occurrence. While this result is not frequent in practice, 
 when the sheep hemolytic system is used, the fact that it 
 may occur, and that, in order to be perfectly safe, it is 
 necessary to remove from each patient's blood the anti- 
 sheep amboceptor that may be present, renders the use 
 of the human hemolytic system preferable, both from a 
 scientific and practical point of view, and it is the system 
 the writer has always urged for complement-fixation tests 
 upon human blood serum. 
 
 The hemolytic amboceptor, having been prepared l)y 
 impregnating suitable filter paper Avith it, as already de- 
 scribed, must be titrated in order to determine the ambo- 
 ceptor unit. After the paper is thoroughly dry a strip is 
 cut from one of the sheets measuring 5 millimeters in 
 width and varying lengths of this strip are used in the 
 titration, as shown in Table XVII. The outer edge of the 
 saturated sheet of filter paper should be trimmed off be- 
 fore cutting the strip referred to, in order to avoid any 
 concentration of the serum which might occur at that por- 
 tion of the paper. 
 
 Table XVII 
 Titration of Amboceptor Paper* 
 
 TUBE 
 NO. 
 
 AMOUNT OF 
 SALT SOLU- 
 TION 
 CO. 
 
 0.9 
 0.9 
 0.9 
 0.9 
 0.9 
 0.9 
 
 AMOUNT OF 
 
 COMPLEMENT 
 
 UNITS 
 
 AMOUNT OF 
 BLOOD SUS- 
 PENSION 
 C.C. 
 
 0.1 
 0.1 
 0.1 
 0.1 
 
 0.1 
 0.1 
 
 AMOUNT OF 
 
 AMBOCEPTOR 
 
 PAPER 
 
 MM. 
 
 5 by 1 
 5 by 2 
 5 by 3 
 5 by 4 
 5 by 5 
 None 
 
 Incubate i n 
 water-bath a t 
 37° 0. for one 
 hour or in incu- 
 bator for two 
 hours at same 
 t e Iff p e rature, 
 and read re- 
 sults. 
 
 *If the unit of complement has not been determined against a known amboceptor by 
 titration, use 0.05 c.c. of a 1:1 dilution of the complement as the unit. It may be neces- 
 sary to carry the titration on further by using larger amounts of the amboceptor paper, 
 but a good paper should give hemolysis in a piece 5 by 5 millimeters. 
 
Ambnccptor cutter. Rear vuw, showing scale and set screw. Devised liy Lieut. 
 Cnlnnel Iv U. N'edder, JMcdical Cori.s, U. S. Army. (Frnin lUdletin No. S, War L)e- 
 jjartment. Office nf llie Surueon Cencral, U. S. Army, Washington. D. C.) 
 
PEBPARATION AND TITRATION OF REAGENTS 83 
 
 After incubating in the water-bath at 37° C. for one 
 hour, or in the incubator, at the same temperature, for two 
 hours, the tubes being shaken every fifteen minutes in or- 
 der to facilitate the liberation of the amboceptor serum, 
 the titration is read and the first tube to show complete 
 hemolysis is noted and the amount of the paper contained 
 in that tube is called one amboceptor unit. A good ambo- 
 ceptor paper should show complete hemolysis in tubes 
 Nos. 2 or 3, or with pieces of paper measuring 5 by 2 or 
 3 millimeters, but paper measuring 5 by 5 millimeters can 
 be used in the test. In many instances, the first tube will 
 show complete hemolysis and, when this occurs, the paper 
 may be reduced in width, or the strength of the blood sus- 
 pension may be increased, a ten per cent suspension being 
 used instead of a five per cent suspension. The control 
 tube. No. 6, should not show any hemolysis and if it does 
 it indicates that something is wrong with the blood sus- 
 pension or the complement, and the titration will have to 
 be repeated with new reagents. 
 
 In making the actual Wassermann test two units of 
 amboceptor paper should be used in order to allow for the 
 anticomplementary qualities of human blood serum and of 
 the antigen. Thus, if one unit of the paper was found to 
 be a piece measuring 5 by 2 mm. a piece 5 by 4 mm. should 
 be used in making the test. 
 
 Amboceptor paper, being very stable, does not need to 
 be titrated very frequently, once a month being amply suf- 
 ficient, and such paper will keep, with little loss of strength, 
 for several months. 
 
 Titration of the Syphilitic Antigen. — The antigens used 
 in the Wassermann test must be carefully titrated and 
 such titrations should be made at frequent intervals, as all 
 antigens used in the test lose strength with more or less 
 rapidity. To be on the safe side, the antigen should be 
 titrated at least once in two weeks unless it be found that 
 the particular antigens that are employed are very stable, 
 when monthly titrations may be sufficient. A really good 
 
84 
 
 THE WASSERMANN TEST 
 
 antigen loses strength slowly and the writer has often 
 worked with extracts that were practically as strong in 
 antigenic qualities after several months as when first pre- 
 pared, but the reverse is often true, and an antigen will 
 be found that rapidly loses strength, thus making frequent 
 titrations advisable. If possible, it is best to titrate the 
 antigen before each series of tests. 
 
 An antigen must be titrated with a known syphilitic se- 
 rum in order to determine its antigenic qualities; with a 
 normal serum in order to demonstrate that it will not in- 
 hibit hemolysis when used with such a serum; and in the 
 absence of either a syphilitic or normal serum, in order to 
 determine whether or not it is anticomplementary or hemo- 
 lytic of itself. Before titrating, the antigen should be 
 properly diluted, which is accomplished by adding one part 
 of the extract to nine parts of normal salt solution (0.85%). 
 Any antigenic extract prepared in the manner recom- 
 mended by the writer and diluted so as to produce an 
 emulsion containing one part of the extract to nine parts 
 of salt solution, that is neither anticomplementary nor 
 hemolytic of itself or in the presence of a normal serum 
 in a dose of 0.2 c.c. and that gives inhibition of hemolysis 
 in the presence of a syphilitic serum in a dose of 0.05 or 
 0.1 c.c, will be found satisfactory in practice. 
 
 Table XVni 
 Titration of Antigen pok Hemolytic Properties 
 
 
 AMOUNT OF 
 
 
 AMOUNT OP 
 
 AMOUNT OP 
 
 
 TUBE 
 
 SALT SOLU- 
 
 UNITS OF 
 
 ANTIGEN 
 
 BLOOD SUS- 
 
 Incubate i n 
 
 NO. 
 
 TION 
 
 COMPLEMENT 
 
 EMULSION 
 
 PENSION. 5% 
 
 water-bath a t 
 
 
 CO. 
 
 
 0.0. 
 
 CO. 
 
 37° C. for one 
 
 1 
 
 0.9 
 
 2 
 
 0.05 
 
 0.1 
 
 hour or in in- 
 
 2 
 
 0.9 
 
 2 
 
 0.10 
 
 0.1 
 
 cubator for two 
 
 3 
 
 0.9 
 
 2 
 
 0.15 
 
 0.1 
 
 hours, and read 
 
 4 
 
 0.9 
 
 2 
 
 0.20 
 
 0.1 
 
 results. 
 
 5 
 
 0.9 
 
 2 
 
 None 
 
 0.1 
 
 
 Table XVIII illustrates the titration of antigenic ex- 
 tracts for the purpose of determining their hemolytic 
 properties. The qu,antities of antigenic solution tested 
 
Fig. 10. 
 Ambocc|itor markers. Devi.sfd liy Lieut. Culontl I'". 11. Xeddrr, Mt-dic-il Cor 
 Arniv, for :icciiialily nieasurini; units of aniiiocciitnr iiaper. I Fn'iii Ihilletin N(j 
 Dciiartiiient, Office of the Sur.L;oon f.cntral, U. S. Army, Wnshmntoii, I). C.) 
 
 Ih s. 
 
 1, War 
 
PRBPAEATIOIT AND TITRATION OF REAGENTS 
 
 85 
 
 have been determined by a large number of experimental 
 tests to yield satisfactory results. 
 
 In this titration none of the tubes should show hemolysis. 
 If hemolysis occurs in all, including Tube 5, the control 
 tube without antigen, it would demonstrate that hemolytic 
 substances were present either in the complement or blood 
 suspension, or both; but if hemolysis should occur in one 
 or more of the tubes containing the antigen, the control 
 tube remaining unhemolyzed, it would demonstrate that 
 the antigenic extract is hemolytic; and, in this event, it 
 should be discarded unless, upon being further diluted with 
 salt solution, it is found to give satisfactory results. If 
 none of the tubes containing antigen show hemolysis, the 
 antigen is not hemolytic, and therefore, so far as hemo- 
 lytic properties are concerned, it is safe to use in making 
 the Wassermann test. 
 
 In order to, determine whether antigens are anticomple- 
 mentary or not, they are titrated as shown in Table XIX. 
 
 Table XIX 
 TiTEATiON or Antigen fok Anticomplementary Peopeeties 
 
 TUBE 
 NO. 
 
 AMOUNT OJ 
 SALT SO- 
 LUTION 
 C.C. 
 
 UNITS OF 
 COMPLE- 
 MENT 
 
 AMOUNT OP 
 
 ANTIGENIC 
 
 EMULSION 
 
 CO. 
 
 AMOUNT OF 
 BLOOD SUS- 
 PENSION, 
 
 5% 
 CO. 
 
 UNITS OF 
 AMBOCEP- 
 TOR PAPEE 
 
 Incub ate 
 in water- 
 bath at 37° 
 C. for one 
 hour or in 
 inou b a t r 
 
 1 
 
 2 
 3 
 4 
 5 
 
 0.9 
 0.9 
 0.9 
 0.9 
 0.9 
 
 2 
 2 
 2 
 
 2 
 2 
 
 0.05 
 0.10 
 0.15 
 0.20 
 None 
 
 0.1 
 0.1 
 0.1 
 0.1 
 0.1 
 
 2 
 2 
 2 
 2 
 2 
 
 for two 
 hours at 
 same t e m- 
 perature and 
 read results. 
 
 As the result of this titration there should be Complete 
 hemolysis in all of the tubes. If Tube 5, the control tube, 
 should show inhibition of hemolysisi, it would prove that 
 either the complement, amboceptor, or blood suspension, 
 was inhibitory, but if any of the tubes containing antigen 
 show inhibition, the control tube being hemolyzed, it would 
 demonstrate that the antigen is anticomplementary, and, 
 unless further dilution will remove this property without 
 
86 THE WASSBBMANIS- TES* 
 
 * 
 
 injuring its antigenic value, the extract should not be used 
 for the test. 
 
 Having tested an antigen for its hemolytic and anticom- 
 plementary properties, it is now necessary to titrate it 
 against a knoAvn syphilitic blood serum to determine its 
 antigenic value, and with a known normal blood serum to 
 demonstrate that it will not give a positive reaction 
 with nonsyphilitic sera. This titration is illustrated in 
 Table XX. 
 
 As the result of this titration all of the tubes containing 
 antigen should show complete inhibition of hemolysis in 
 which syphilitic blood serum is also present and .complete 
 hemolysis in the tubes iu which normal blood serum is 
 present. Thus Tubes 1, 2, 3, and 4 should show absolute 
 inhibition of hemolysis, while Tube 6 should show complete 
 hemolysis. If Tube 6 shows any trace of inhibition, the 
 antigen is not suitable and should be discarded; and the 
 same is true if there is less than complete inhibition of 
 hemolysis in Tube 2. There may be a slight trace of hem- 
 olysis in Tube 1 containing only 0.05 c.c. of the antigenic 
 emulsion, without harm ; but it will always be found better 
 in practice to use only those antigenic extracts that give 
 complete inhibition of hemolysis with 0.05 of the diluted 
 antigen. Tube 7, which is a control of the normal serum 
 used, should show complete hemolysis, and if it does not, 
 another serum should be tested. Tube 8, the control of the 
 complement, should show complete inhibition of hemolysis ; 
 and Tube 9, the control of the hemolytic system, should 
 show complete hemolysis. 
 
 If these results are obtained, they demonstrate that the 
 antigen, as diluted, is suitable for use in the Wassermann 
 test as recommended by the writer, in that it produces 
 inhibition of hemolysis in the presence of syphilitic blood 
 serum in a dose as small as 0.05 c.c, and that it does not 
 inhibit hemolysis in the presence of a normal serum in four 
 times that dose, or 0.2 c.c. Such an antigen wiU give excel- 
 lent results and even one that gives inhibition of hemolysis 
 
PREPARATION AND TITRATION OF REAGENTS 
 
 87 
 
 «o 00 *q OS en rfk. w to w 
 
 
 p> p> o opoooo 
 
 ?0 ?0 ?D CO ^ CO CD (O CD 
 
 AMOUNT OF 
 SALT SOLU- 
 TION 
 C.C. 
 
 |l|!oS g oS 2 ooooo 
 PBMSBi-'SBf-'f-^Mi-'!-' 
 
 CD CD g pa p p 
 
 AMOUNT OF 
 LUETIC SE- 
 RUM INACTI- 
 VATED 
 C.C. 
 
 MK) to KltStOtObSK) 
 
 UNITS OF 
 COMPLE- 
 MENT 
 
 ^ !^ fe! o !z! o o o o 
 29 2 iogtoMMO 
 gg g ogooiooi 
 
 AMOUNT OP 
 
 ANTIGENIC 
 
 EMULSION 
 
 C.C. 
 
 Incubate in wa- 
 ter-bath at 37° C. 
 for one-half hour 
 or in incubator for 
 one hour. 
 
 oo p ^ <^ ^ c> <:> <s 
 
 MM M MMMMMM 
 
 AMOUNT OF 
 BLOOD SUS- 
 PENSION, 5% 
 C.C. 
 
 bag l^s t3 N> cs ts t9 to 
 
 UNITS OF 
 
 AMBOCEPTOE 
 
 PAPEK 
 
 Incubate in wa-" 
 ter-bath at 37° 0. 
 for one hour or in 
 incubator for two 
 hours at same tem- 
 perature and read 
 results. 
 
 
 01 
 
 M 
 X 
 
88 THE WASSBEMANN TEST 
 
 with a syphilitic blood serum in a dose of 0.1 will be found 
 satisfactory in practice, provided it is otherwise all right. 
 The amount of antigenic emulsion necessary to produce 
 complete inhibition of hemolysis in the presence of a syph- 
 ilitic blood serum is known as an antigen wnit. The anti- 
 gen unit is not doubled in making the test as isi the unit 
 of complement and amboceptor. 
 
 ■ All blood sera used in titrating the antigen should be 
 inactivated by heating for one-half hour at 56° C. and a 
 neglect of this will produce most erroneous results. 
 
 Antigens should be kept in the ice box when not in use 
 as exposure to room temperature for any length of time 
 will result in their rapidly losing strength. 
 
CHAPTER V 
 
 THE TECHNIC OF THE WRITER'S MODIFICATION 
 OF THE WASSERMANN TEST. THE TEST 
 UPON THE CEREBROSPINAL FLUID. 
 THE ORIGINAL WASSERMANN 
 TECHNIC. OTHER MODIFI- 
 CATIONS OF THE WAS- 
 SERMANN TEST. 
 
 The technic of the writer's modification of the Wasser- 
 raann test was first fully published in 1913, in Bulletin 
 No. 3, War Department, Office of the Surgeon General, 
 entitled Studies in Syphilis, by Charles F. Craig, Captain, 
 Medical Corps, U. S. Army and Henry J. Nichols, Cap- 
 tain, Medical Corps, U. S. A. The method has been used 
 by the writer since 1910 with success and since 1913 it has 
 been the standard method adopted in nearly all of the large 
 army laboratories, and has been recommended for use in 
 all army laboratories by Lieut. Col. Vedder, of the Medical 
 Corps, who has devised special apparatus for use with the 
 method. It is a modification of both the original Wasser- 
 mann method and of that of Noguchi, following Wasser- 
 mann in using an extract of foetal syphilitic liver as one 
 antigen and in inactivating the patient's blood serum, and 
 Noguchi in using a human hemolytic system instead of the 
 sheep system. 
 
 This modification of the Wassermann test has been so 
 thoroughly tried iii the army, and so many thousands of 
 tests have been made with it, that there is no question of 
 its accuracy and value in the diagnosis of syphilis. Many 
 different laboratory workers have used it and their re- 
 sults have all been in agreement as to its accuracy and 
 
 89 
 
90 THE WASSBEMANN TEST 
 
 as, in army practice, we make many repeated examina- 
 tions of the same case in order to control the treatment, 
 any irregularities in the results of the test would have been 
 soon discovered. In the hands of different workers the re- 
 sults have been almost identical with the same character of 
 cases, as is instanced by Vedder," and it is believed that it 
 is as well adapted to civilian laboratories as to those of the 
 army and will be found at once simple and accurate. 
 
 The method of preparing the reagents used in the test 
 has already been described, as well as their titration, and 
 in this chapter will be considered the exact technic of the 
 test using the reagents as prepared and titrated. 
 
 The glassware used in preparing the various reagents 
 for the test and in the test should be perfectly clean. No 
 chemicals should be used in cleaning the glassware but 
 only hot water and soap, followed by a thorough rinsiag 
 with hot water. All glassware, except the test tubes used 
 in the test, should be sterilized by dry heat and one should 
 be careful that a temperature of 180° C. is not exceeded, 
 as a higher temperature than this is apt to so heat the 
 ends of the small 1 c. c. pipettes as to close them. The 
 test tubes do not need to be sterilized, and after washing, 
 should not be plugged, but placed open end downward in 
 a test tube basket and covered with a clean towel when not 
 in use. 
 
 The pipettes used for measuring the patient's serum 
 should be graduated in tenths and do not need to be grad- 
 uated in hundredths, thus saving considerable expense 
 where many tests are being done, as an individual pipette 
 should be used with each patient's blood serum. At least 
 a dozen 1 c.c. pipettes, graduated plainly in hundredths, 
 the graduations being at such a distance apart that 0.02 
 c.c. can be accurately measured, should be in stock, for 
 titration work and in making dilutions. In using any 
 pipette graduated to the tip the last unit of graduation 
 should not be used as it is very apt to be inaccurate. Great 
 care should be exercised in using the pipettes to avoid 
 
TECHNIC OF THE WRITER'S MODIFICATION 91 
 
 breakage, especially of the tip, and they should not be al- 
 lowed to remain unwashed after using but should be im- 
 mediately cleaned, as otherwise the blood serum will dry 
 in them and will be found very difficult to remove owing 
 to the small caliber of the pipette. 
 
 Collection and Preparation of the Patient's Blood Se- 
 rum. — ^When specimens of blood for the Wassermann test 
 are to be sent through the mail or kept for more than two 
 days before they are tested, the blood should be collected 
 with aseptic precautions and placed in sterile glass con- 
 tainers. If the test is to be made at once or within a day 
 or two such precautions are not necessary but it is always 
 essential that an aseptic technic be employed in collecting 
 the blood, in order to avoid any danger of infection to the 
 patient. The reason for observing these precautions has 
 been noted in the discussion of the effect of contaminating 
 bacteria upon the result of the "Wassermann test. All 
 specimens of blood serum for the test should be kept in the 
 ice box until used, as exposure to room temperature may 
 cause the serum to become anticomplementary. 
 _ Before taking the blood from the patient he should be 
 questioned regarding the use of alcohol and if he states 
 that he has used alcoholics within thirty-six hours the 
 blood should not be taken but the patient told to return 
 upon the following day, meanwhile abstaining from the 
 use of alcohol in any form. This precaution is necessary 
 owing to the fact that alcohol is capable of rendering a 
 positive reaction negative, as shown by the writer and 
 Nichols and already discussed. 
 
 Patients undergoing treatment for syphilis frequently 
 give a negative Wassermann reaction during the time treat- 
 ment is being administered, so that it is better to test the 
 blood serum after treatment has been stopped for a period 
 of two or three weeks. Blood for the Wassermann test 
 should not be taken during a malarial paroxysm, as some- 
 times a positive reaction is given in such instances, nor 
 should it be collected immediately after anesthesia or after 
 
92 THE WASSBEMANN TEST 
 
 eating. Bile stained blood sera may inhibit hemolysis and 
 should not be tested if it is possible to secure a specimen 
 from the patient that is not bile stained. If a positive re- 
 action occurs with a bile stained serum it should be re- 
 miembered that it may be a false positive and the diagnosis 
 of syphilis should only be made provisionally. 
 
 Blood serum should not be over five or six days old, at 
 the most, when tested, as older sera frequently are anti- 
 complementary. "If a serum inhibits hemolysis in both the 
 antigen and control tube another specimen should be re- 
 quested. Inactivation often removes the anticomplemen- 
 tary properties of old sera but it is best not to employ 
 them in the test if it is possible to avoid doing so. 
 
 Collection of the Blood. — The amount of blood serum 
 required in the writer's modification of the Wassermann 
 test does not exceed 0.2 c.c. so that 2 c.c. of blood is amply 
 sufficient for the test. This amount may be easily collected 
 from a puncture with a needle or glass needle just above 
 the finger nail upon the dorsal surface of the middle finger 
 in the adult, allowing the blood to drop into a small, sterile 
 and perfectly dry glass vial until from one and one-half to 
 two cubic centimeters are collected, or the blood may be 
 collected from a puncture in the lobe of the ear with a 
 Wright tube. Any form of special apparatus that has been 
 devised for collecting blood for the "Wassermann test is 
 unnecessary and if venipuncture is objected to by the pa- 
 tient enough blood can easily be obtained as described 
 above. 
 
 In the writer's opinion, the simplest and best method 
 of obtaining blood for the test is to remove from 2 to 5 
 c.c. of blood from one of the large veins in the forearm or 
 at the bend of th$ elbow with a sterilized glass syringe. 
 The arm should be cleaned, the site of the puncture of the 
 vein brushed with iodine, and the needle, held almost par- 
 allel with the surface of the arm, pushed through the skin 
 and directly into the vein. The blood should be withdrawn 
 slowly or the syringe can be dispensed with entirdy and 
 
TBOHKIC OP THE WRITER 's MODIFICATIOISr ' 93 
 
 the blood allowed to flow directly into a container from 
 the needle. The operation is practically painless and with 
 a little practice anyone can become very expert in the pro- 
 cedure. The advantage of the method is that more blood 
 can be collected, thus insuring an abundant supply of the 
 serum to be tested. 
 
 After collection, the blood is placed in a suitable glass 
 vial, the serum allowed to separate, after which it is 
 pipetted into a sterile glass vial or tube if it is to be mailed, 
 or used at once in the test. The serum separates best if 
 the blood be kept at room temperature for an hour and 
 then placed in the ice box. All blood serum should be kept 
 in the ice box until used, and when specimens are received 
 by mail, they should be immediately placed in the ice box. 
 A positive and negative blood serum should always be 
 kept in stock for control sera and these should be kept in 
 the ice box when not in use. AU blood sera used for the 
 Wassermann test should be clear in appearance and with 
 no disagreeable odor. Milky or cloudy sera are often due 
 to chyle but it is best, if possible, not to use such sera, 
 although they may not cause inhibition of hemolysis. 
 
 Inactivation of the Blood Serum. — All blood sera used 
 in the Wassermann test as recommended hy the writer 
 must he inactivated hy heating at 56° G. for one-half hour. 
 This is done for the purpose of removing the native com- 
 plement in the blood serum and also for removing any 
 thermolabile anticomplementary substances that may have 
 developed through age or bacterial contamination. "While 
 the inactivation of the serum also tends to make it weaker 
 in substances that give a positive reaction A^dth syphilitic 
 serum, as so well shown by Noguchi,^" it is absolutely nec- 
 essary that inactivation be employed in any modification 
 of the Wassermann test that employs either aqueous or 
 alcoholic extracts of tissues which contain proteins and 
 this is true of the original Wassermann test and most of 
 its modifications. "When Noguchi's acetone insoluble anti- 
 genic extract is used the blood serum does not require in- 
 
94 THE WASSEKMAlTlir TEST 
 
 activation and this is one of the advantages that its author 
 claims for it. However, in practice, the amount of harm 
 done by inactivation, in weakening the reacting power of 
 tlje blood serum, is small when compared with the harm 
 which would result in using active serum, and it is only 
 in very early infections or in those which have received 
 treatment that inactivation might so weaken the serum as 
 to render it negative. 
 
 In inactivating the blood serum extreme care should be 
 taken that 56° C. is not exceeded, for exposure of the 
 serum to a temperature of 60° C. will practically destroy 
 its power of reacting with syphilitic serum. The temper- 
 ature may be allowed to stand at 55° C. with as good 
 results as at 56° C, but the latter figure should never be 
 exceeded. 
 
 At the same time that the sera to be tested are inac- 
 tivated, a known syphilitic serum and a known normal 
 serum are also inactivated, to serve as controls of the 
 test. In addition, there is always provided a tube for 
 the serum to be tested, in which no antigen is placed, the 
 control tube, and this is chiefly for the purpose of deter- 
 mining that the serum tested is not anticomplementary of 
 itself. This control tube of each serum tested should never 
 he omitted for if it is no dependence can he placed upon 
 the results of the test. 
 
 The Writer's Technic. — ^Having inactivated the sera 
 to be tested and the positive and normal control, and being 
 provided with the proper blood suspension, hemolytic am- 
 boceptor, and syphilitic antigens, the test can be proceeded 
 with. 
 
 Before doing anything else, however, ihe complement 
 must he titrated as already described and the exact comple- 
 ment unit determi/ned, and this is doubled for each tube in 
 making the test. This procedure should never be omitted 
 under any consideration. Some authorities prefer to ti- 
 trate the amboceptor instead of the complement, but as the 
 
TECHNIC OP THE WRITER 's MODIFICATION 95 
 
 complement is the substance in the test that is most apt to 
 vary in strength, it should be titrated in preference to the 
 amboceptor. As a matter of fact, the titration of the com- 
 plement with a fixed dose of amboceptor, also determined 
 by previous titration, can not result in any fallacies in the 
 test, as the amount of complement necessary to produce 
 hemolysis with the dose of amboceptor used, when doubled, 
 is always sufficient to give accurate results when used in 
 the test. 
 
 It is recommended that two antigens be used in testing 
 each serum, one, the alcoholic extract of foetal syphilitic 
 liver; the other, the alcoholic extract of normal human 
 heart muscle, to which 0.4 per cent of cholesterin has been 
 added. If both antigens can not be obtained, good prac- 
 tical results can be obtained by using one, as it is seldom 
 that a positive result is obtained with one antigen and not 
 with the other. 
 
 If two antigens are used three test tubes are needed for 
 each serum to be tested, while if only one antigen is used, 
 two tubes will be sufficient. The test tubes should be placed 
 in racks having a double row, an anterior and a posterior. 
 When three tubes are used, two should be placed in the an- 
 terior row of the rack and one in the posterior, while if 
 two tubes are used, one should be placed anteriorly and 
 one posteriorly. The anterior tubes contain the patient's 
 blood serum together with the syphilitic antigen, while the 
 posterior contains the patient's blood serum without the 
 antigen, this tube acting as a control of the serum tested. 
 Besides the tubes for the patient's blood serum, there 
 must be an equal number of tubes for a knoAvn syphilitic 
 serum, the positive control ; and for a known normal serum, 
 the normal serum control. 
 
 In making the test proceed as follows: In all of the 
 tubes enumerated place 0.9 c.c. of normal salt solution 
 (0.85%). In Tube 1 and Tube 2, anterior, if two antigens 
 are used, or in Tube 1, if only one antigen is used, place 
 0.1 c.c. of the inactivated blood serum of the patient to be 
 
"7 
 
 96 THE WASSEEMANK" TEST 
 
 tested, and the same amount in Tube 1, posterior. The 
 same amount of syphilitic and normal blood serum is 
 placed in the control tubes mentioned, and in the same man- 
 ner. Thus, if two antigens are used, in Tubes 3 and 4 
 0.1 C.C of the known syphilitic serum is placed, and the 
 same amount in Tube 3, posterior, while if only one anti- 
 gen is used, 0.1 e.e. of the syphilitic serum is placed in 
 Tube 2, anterior, and in Tube 2, posterior. There is now 
 added to every tube used in the test two units of comple- 
 ment and to each anterior tube one unit of the antigen used. 
 If two antigens are used, one unit of each should be placed 
 in the anterior tubes, alternately. 
 
 The tubes are now placed in a water-bath at 37° C. for 
 one half hour, or in an ordinary bacteriological incubator, 
 at the same temperature, for one hour, and at the expira- 
 tion of this time there is added to each tube 0.1 c.c. of the 
 5 per cent suspension of human red 'blood corpuscles and 
 two units of the amboceptor paper. The tubes are again in- 
 cubated in the water-bath at 37° C, this time for one hour, 
 or in the incubator for two hours, being shaken every fif- 
 teen minutes in order to facilitate the liberation of the 
 amboceptor serum from the filter paper. After this incu- 
 bation the tubes are placed in an ice box for one or two 
 hours and the results then read. Neglect of shahing the 
 tubes, as recommended, will oftentimes result in partial 
 positive reactions in sera that should react negatively. 
 
 If desired a hemolytic and antigen control tube may be 
 included but this is not necessary in practice as the titra- 
 tions of the complement will show if anything is wrong 
 with the hemolytic system, and an antigen control is unnec- 
 essary unless it has been some time since the antigen was 
 titrated. 
 
 Results of the Test.— If all of the reagents used in the 
 test are working properly the resiilts, after the time al- 
 lowed in the ice box, should be as follows : 
 
 The anterior tube or tubes, containing the patient's blood 
 
TEST UPON THE CEEEBEOSPINAL FLUID 97 
 
 serum, and the antigen or antigens, should- show complete 
 inhibition of hemolysis (positive reaction), if syphilis is 
 present; "v^hile the posterior tube, containing the patient's 
 serum without antigen, should show complete hemolysis. 
 Of course, various degrees of inhibition will be shown in 
 the anterior tube or tubes if the case is an early one, treat- 
 ment has been given, or if the reaction is weak. If the 
 patient is not syphilitic there should be complete hemolysis 
 in the anterior tube or tubes as well as in the posterior 
 tube. The posterior, or control tube of the patient's se- 
 rum, should always show complete hemolysis, and if it does 
 notj another specimen should be requested. 
 
 The anterior tube, or tubes, containing the known syph- 
 ilitic blood serum, or the positive control, should show 
 complete inhibition of hemolysis (positive reaction) while 
 the posterior tube of the same serum without antigen, 
 should show complete hemolysis. If inhibition occurs in 
 this tube it demonstrates that the control positive serum is 
 worthless. 
 
 The anterior tube, or tubes, containing the known nor- 
 mal serum, or the normal control, with antigen, should 
 show complete hemolysis, as should the posterior tube of 
 this set. 
 
 If a control tube for the antigen and for the hemolytic 
 system be used, both should show complete hemolysis. 
 
 The Test Upon the Cerebrospinal Fluid 
 
 The great value of the Wassermann test when per- 
 formed with the cerebrospinal fluid in demonstrating the 
 existence of syphilitic infection of the central nervous sys- 
 tem renders it almost imperative that, before assuring a 
 patient that he is free from infection, the spinal fluid be 
 tested. Not only is this true, but the fluid should be tested 
 in all cases early in the infection, if possible, in order to 
 ascertain whether the nervous system is involved, and if 
 this were more generally done, it is probable that many of 
 
PLATE II 
 
 Fig. 1. — Reading of Results of Wassermann Test. The first tube shows com- 
 plete inhibition of hemolysis and may be read four-plus or double-plus, accord- 
 ing to the nomenclature used in the individual laboratory. 
 
 Tubes 2 and 3 show three-plus and two-plus reactions, or both are included 
 and reported as a plus reaction. 
 
 Tubes 4 and 5 show single-plus and plus-minus reactions, or may both be 
 included and reported as a plus-minus reaction. 
 
 Tube 6 shows a minus or negative reaction. 
 
 Fig. 2. — ^Wassermann Test. Positive Reaction. Tube 1 contains the pa- 
 tient's blood serum and syphilitic antigen and shows complete inhibition of 
 hemolysis or a positive reaction. Tube 2 is a control of the patient's serum 
 without antigen, and shows complete hemolysis. Tube 3 is a known syphilitic 
 control serum. Tube 4 is a control of the positive serum, without antigen. 
 Tube 5 is a known normal serum and Tube 6 a control of the normal serum 
 without antigen. 
 
 Fig. 3. — ^Wassermann Test. Negative Reaction. Tube 1 contains the pa- 
 tient's blood serum and syphilitic antigen and shows complete hemolysis, or a 
 negative reaction. Tube 2 is a control of this serum without antigen. Tube 3 
 is a known positive serum and shows complete inhibition of hemolysis. Tube 4 
 is a control of the known positive serum without antigen. Tube 5 is a known 
 normal serum with the antigen and Tube 6 a control of the normal serum 
 without antigen. 
 
Fig. I Reading of Results of Wassermann Tesf 
 12 5^ 5 6 
 
 fe-4l 
 
 iE^ 
 
 v_y V_y \^/ v_y 
 Fig.;^ WassermannTest PosiFive ReacFion 
 
 12 3^56 
 
 ■asi^ ?■ 't " .''^ \ -^^ * -"^ *-'"^ 
 
 Fig.3 Wassermann Test Negative Reaction 
 
 Plate II. 
 
TEST UPON THE CEEEBEOSPIIirAL FLUID 99 
 
 the severe complications characteristic of involvement of 
 the central nervous system could be avoided. 
 
 Collection of the Cerebrospinal Fluid. — The cerebrospinal 
 fluid is collected by lumbar puncture and when properly 
 performed, this operation is devoid of danger. The pa- 
 tient should be in bed and should remain in bed for twenty- 
 four hours after the puncture. The writer is well aware of 
 the fact that scores of punctures of the spinal canal are 
 made in the physician's office, the patient afterward being 
 allowed to walk home or to attend to his business, but, in 
 his opinion, this practice is unjustifiable and should be dis- 
 couraged, for while the danger of complications may be 
 small, deaths have occurred during or shortly after the 
 procedure when thus performed, and severe symptoms are 
 not uncommon. 
 
 The lumbar region is washed carefully with soap and 
 water, alcohol and ether, and the region over the lumbar 
 vertebrae brushed with iodine solution. The site of punc- 
 ture is most easily ascertained by running the finger along 
 the spines of the vertebrae until the so-called ''soft spot" 
 is reached, which is indicated by a distinct feeling of soft- 
 ness to the finger and is situated between the third and 
 fourth lumbar vertebrae. The patient may sit upon a 
 stool with his back bent so as to bring the spinous proc- 
 esses of the vertebrae close to the surface, or lie upon the 
 left side upon the edge of the bed. The latter posture is 
 necessary in the case of the sick but the sitting posture 
 is suitable for ordinary ambulant patients. The needle 
 used in the puncture should be of flexible steel measuring 
 10 cm. long, with a bore of 1 to 1.5 mm., while for a child 
 the needle should be shorter but of the same bore. The 
 needle should be sterilized before being used, as veil as 
 the stylet which should accompany it. 
 
 The space between the third and fourth lumbar vertebra 
 haiving beeu ascertained the puncture is made directly in 
 the median line, which I have found much more satisfac- 
 tory than the lateral puncture recommended by many writ- 
 
100 THE WASSBKMAWN TEST 
 
 ers. The needle is held firmly and the puncture made 
 quickly, the direction being straight forward. Enough 
 force should be used to push the needle quickly through the 
 skin and muscles but as soon as the spinal ligaments are 
 reached it may be pushed forward more slowly until there 
 is a sudden sensation of loss of resistance when the needle 
 is in the spinal canal. The stylet is now withdrawn and 
 the spinal fluid, if the puncture is successful, will flow from 
 the needle. If it does not flow, the needle may be shifted 
 very, gently or the patient told to take a deep breath, or the 
 stylet may be replaced in the needle and gently pushed 
 forward to clear the tube of any materials that have been 
 carried into it by the influx of the spinal fluid. If none of 
 these procedures are successful the puncture is said to be 
 a "dry" one and there is little use in repeating it. 
 
 Not over 5 c.c. of fluid should be withdrawn, as that is 
 amply sufficient for the test and the less fluid withdrawn 
 the slighter will be the symptoms following the puncture. 
 The fluid often comes from the needle under considerable 
 pressure and this should be noted upon the slip sent to the 
 laboratory with the fluid. It should be free from blood but 
 can be used even if blood corpuscles are present, by centri- 
 fuging the fluid until they are deposited at the bottom of 
 the tube and pipetting off the clear fluid. 
 
 The spinal fluid is kept in the ice box until used and 
 should not be inactivated, as is tJie blood serum, before it 
 is tested. 
 
 The same procedure is followed in testing spinal fluid 
 as in testing blood serum, but different quantities of the 
 fluid are tested, as in some infections of the central nerv- 
 ous system, as paresis, a very small amount of the fluid will 
 give a positive reaction, while the same amount in cere- 
 brospinal syphilis would be negative. Therefore it has 
 always been the writer's practice to test 0.2, 0.5, 0.7 and 
 1 c.e. of the spinal fluid, thus making four tubes for the 
 antigen tubes and four for the control amounts of fluid. 
 
TEST UPON THE CEREBROSPINAL FLUID 101 
 
 if only one antigen is used, and double this number if two 
 antigens are used in the test. 
 
 Precautions to be Observed in Lumbar Puncture.— Pa- 
 tients who are very weak or debilitated should not have a 
 lumbar puncture performed unless it is absolutely neces- 
 sary. Those suffering from paresis and tabes bear punc- 
 ture exceedingly well and seldom complain of pain, but 
 nervous normal adults frequently speak of the pain of 
 the puncture and of disagreeable symptoms following it. 
 Headache may be severe if the fluid is withdrawn rapidly 
 and if the bone is punctured or bruised by the needle dur- 
 ing the procedure pain may last for several hours after- 
 wards. Nausea is sometimes observed, especially if the 
 fluid is rapidly withdrawn, so that it is always best to allow 
 the fluid to flow as slowly as possible, thus relieving 
 pressure gradually. Nonne" warns against lumbar punc- 
 ture in cases of cerebral tumor, several deaths having fol- 
 lowed the operation in. this condition, and states that he 
 has himself observed four cases of sudden death in such 
 patients following the puncture. 
 
 The patient should remain in bed for twenty-four hours 
 after the puncture, if possible, and neglect of this precau- 
 tion often leads to disagreeable symptoms. In a case which 
 was observed in the writer's practice, very severe head- 
 aches and attacks of dizziness occurred for several days 
 after puncture because the patient insisted upon immedi- 
 ately returning to work. Diarrhea is a symptom some- 
 times observed, especially if the fluid has been withdrawn 
 rapidly. 
 
 The Reading of the Reaction and Nomenclature Em- 
 ployed. — The results of the Wassermann test are variously 
 reported from different laboratories and unless one is ac- 
 quainted with the meaning of the various terms used, 
 grave errors may result in the interpretation of the results 
 of the test. 
 
 In perhaps the large majority of Wassermann labora- 
 
102 THE WASSERMAITN TEST 
 
 tories the results of the test are recorded as four-plus 
 (+ + + +), indicating complete inhibition of hemolysis, and, 
 therefore, a strongly positive reaction; three-plus (+ + +), 
 indicating about 75 per cent inhibition of hemolysis, or a 
 moderately positive reaction; two-plus (++), indicating 50 
 per cent inhibition of hemolysis, or a weakly positive re- 
 action; plus (+), indicating from 25 to 50 per cent inhibition 
 of hemolysis, or a doubtful reaction; plus-minus (+-), in- 
 dicating less than 25 per cent inhibition of hemolysis or a 
 doubtful reaction; and minus (-), indicating complete hem- 
 olysis, or a negative reaction. 
 
 In the army laboratories a more simple nomenclature 
 has been employed for the reason that only aJbsolute inhi- 
 bition of hemolysis is considered a positive reaction, all 
 other degrees of inhibition being considered doubtful. 
 This standard has been adopted principally that the soldier 
 may be given the benefit of any doubt that may exist re- 
 garding syphilitic infection. Thus four designations are 
 used in the army laboratories, as follows: Double-plus 
 (++), indicating complete inhibition of hemolysis, and, 
 therefore, a positive reaction; plus (+), indicating anything 
 between complete inhibition and 50 per cent of hemolysis ; 
 plus-minus (+-), indicating anything between 50 per cent 
 of hemolysis and complete hemolysis; minus (-), indicat- 
 ing complete hemolysis, or a negative reaction. 
 
 ' Comparing this nomenclature with the foregoing, the 
 three-plus and two-plus reactions would correspond to our 
 plus reaction, the one-plus with our plus-minus reaction, 
 and the four-plus with our double-plus reaction. 
 
 The writer believes that for use in civilian life the no- 
 menclature of results first mentioned, i. e., four-plus, three- 
 plus, two-plus, plus, plus-minus and negative, is the most 
 useful; but anything below a three- plus reaction, in his 
 opinion, should be considered doubtful, unless there is very 
 good evidence of syphilitic infection, or the patient has 
 received antisyphilitic treatment. 
 
OEIGINAX, WASSBKMANI3". TECHNIC 103 
 
 The Original Wassermann Technic 
 
 In the original Wassermann technic an aqneons extract 
 of syphilitic liver was employed as the antigen; the blood 
 sera were inactivated by heating them at 55° C. for one- 
 half hour before testing; and a sheep hemolytic system was 
 used. The complement was fresh guinea pig serum diluted 
 1:10 with normal salt solution, the dose being arbitrarily 
 fixed at 1 c.c. of the diluted serum. The blood suspension 
 was a 5 per cent suspension of sheep erythrocytes in nor- 
 mal salt solution, and the dose for each tube was 1 c.c. of 
 the suspension. The hemolytic amboceptor was the blood 
 serum of a rabbit immunized with sheep's erythrocytes 
 and this was titrated carefully before each series of tests. 
 The antigen, as stated, was an aqueous extract of foetal 
 syphilitic liver, prepared in the manner already described 
 and carefully titrated before using. Two tubes were used 
 for each of the blood sera tested ; two for the positive con- 
 trol; two for the normal control; and three tubes for con- 
 trol of the hemolytic system, antigen, and blood suspension. 
 
 The tubes are arranged in double rows except the three 
 control tubes. For each serum tested there should be an 
 anterior and . posterior tube and the same for the known 
 positive serum and for the known normal serum. In each 
 of these tubes there is placed 1 c.c. of the complement, di- 
 luted 1 :10. In the anterior tube of the test set of two there 
 should be placed 0,2 c.c. of the patient's inactivated blood 
 serum, and the same amount in the posterior tube of this 
 set. In the anterior tube and posterior tube of the positive 
 control set there should be placed 0.2 c.c. of the known pos- 
 itive serum, and in the two tubes of the normal serum con- 
 trol there should be placed the same amount of a known 
 negative blood serum. In the anterior tubes the proper _^ 
 dose of antigen is placed and enough normal salt solution 
 to bring the total amount in the tubes up to 3 c.c. Of the 
 three control tubes, the first is the antigen control and 
 should contain the dose of antigen used, 1 c.c. of the com- 
 
104 THE WASSBRMANN TEST 
 
 plement, aM enough salt solution to equal 3 c.c. ; the sec- 
 ond is the hemolytic system control, and should contain 
 1 c.c. of the complement and 2 c.c. of the normal salt solu- 
 tion; while the third is the blood suspension control, and 
 contains 1 c.c. of the corpuscle suspension and 2 c.c. of 
 salt solution. 
 
 The tubes are shaken and incubated for one hour in the 
 incubator and to each tube is then added two units of am- 
 boceptor serum, as determined by the titration and 1 c.c. of 
 corpuscle suspension. The blood suspension control does 
 not, of course, receive this suspension. The tubes are 
 shaken and incubated until the blood serum controls (the 
 posterior tubes) are hemolyzed, and the results read at 
 once or after several hours in the ice box. 
 
 If the patient's serum is positive, the anterior tube will 
 show complete or almost complete inhibition of hemolysis, 
 while the posterior tube, containing the serum without an- 
 tigen, should show complete hemolysis; if the patient's 
 serum is negative, the anterior and posterior tubes should 
 both show complete hemolysis. The anterior tube of the 
 positive control set should show complete inhibition of 
 hemolysis, while the posterior tube should show complete 
 hemolysis; the anterior tube of the normal serum control 
 and also the posterior should show complete hemolysis. 
 The antigen control tube should show complete hemolysis, 
 as should the hemolytic system control, while the blood 
 suspension control should show complete inhibition of 
 hemolysis. 
 
 The nomencla,ture used in reporting the test is that al- 
 ready described, but in reporting results to physicians it 
 is better to report them simply as positive, very suspicious, 
 suspicious, doubtful or negative. 
 
 Other Modifications of the Wassermann Test 
 
 There have been many modifications of the original Was- 
 sermann technic for the complement-fixation test in syph- 
 ilis and the most important wUl be here considered. 
 
MODIFICATIOKS OP THE WASSEBMABTN TEST 105 
 
 NogncM's Modification.— In NogucM's modification a 
 human hemolytic system is used, the serum to be tested is 
 not inactivated, and the acetone insoluble lipoid antigen is 
 employed, the preparation of which has already been de- 
 scribed. If the blood serum can not be tested at once, in- 
 activation should be employed but better results are ob- 
 tained with active serum. Capillary pipettes are used in 
 measuring the patient's serum, one drop of the active se- 
 rum being employed, or practically 0.02 c.c. and four drops 
 of the inactivated serum, or practically 0.08 c.c. 
 
 Noguchi thus describes the technic of the test:"^ A rack 
 containing two rows of holes should be used, an. anterior 
 and a posterior roAV. "For each test two tubes are re- 
 quired, one in the front row and its control in the rear 
 row. There will also be two pairs of tubes to serve as 
 positive and negative controls. 
 
 "Put Into each of two small test tubes front and rear one 
 drop (0.02 c,c.) of the serum to be tested from a capillary 
 pipette. When using inactivated serum put 4 drops (0.08 
 c.c.) into each tu,be. Use 0.2 c.c. of cerebrospinal fluid not 
 inactivated. Add to each tube 0.1 c.c. of 40 per cent fresh 
 guinea pig serum made by adding 1 part of complement to 
 1^ parts of 0.9 per cent salt solution. To the front tube 
 add the unit of antigen determined by titration. Then to 
 both tubes add 1 c.c. of the one per cent suspension of 
 washed human corpuscles (the first procedure). In case 
 of using the ten per cent corpuscle suspension add 0.9 c.c. 
 of salt solution and two units of amboceptor (the second 
 procedure). Shake the tubes thoroughly from time to 
 time to distribute the reagents throughout the mixture. 
 
 "With every series of tests it is necessary to carry two 
 sets of controls and for this purpose four additional tubes 
 are necessary. To each of the first pair of these, one in the 
 front and one in the rear row, one capillary drop of a syph- 
 ilitic serum known to give a positive reaction is added. 
 This will serve as a positive control. To the second pair 
 
106 THE WASSEBMANW TEST 
 
 one drop of normal serum known to give a negative re- 
 action should be added. This tube will serve as a negative 
 control. Now put into each tube complement and into the 
 tubes of the front row antigen, adding finally 1 c.c. of the 
 one per cent corpuscle suspension (first procedure), or 0.9 
 c.c. of salt solution and 2 units of amboceptor (second pro- 
 cedure) to each tube.- 
 
 "Place the rack holding these pairs of tubes in a water- 
 bath, thermostat, or warm place not over 37° C. Allow an 
 hour from the time the mixture is made for the antibody to 
 combine with the antigen and for complement to Ipe fixed. 
 If a water-bath is used 30 minutes is a sufiicient length of 
 time. The contents of the tubes in the first procedure 
 (first method) are as follows : 
 
 ''Bear. Test serum plus complement (2 units) plus 
 1 c.c, 1 per cent corpuscle suspension. 
 
 "Front. Test serum plus complement (2 units) plus 
 antigen plus 1 per cent corpuscle suspension. 
 
 ' * The contents of the tubes in the second procedure are as 
 follows : 
 
 "Eear. Test serum plus 2 units complement plus 2 
 units amboceptor paper. 
 
 "Front. Test serum plus complement (2 units) plus 
 antigen plus 2 units amboceptor. 
 
 "First incubation at 37° C. for 1 hour, then add to each 
 tube of the first procedure a slip bearing two units of am- 
 boceptor, as follows : 
 
 "Rear. Above, plus amboceptor (2 units). 
 
 "Front. Above, plus amboceptor (2 units). 
 
 "To each tube of the second procedure 0.1 c.c. of the ten 
 per cent corpuscle suspension, as follows : 
 
 "Eear. Above, plus 10% corpuscle suspension (.1 c.c). 
 
 "Front. Above, plus 10% corpuscle suspension (0.1 c.c). 
 
 "Allow two hours in the thermostat or one hour in the 
 water-bath. After final incubation the tubes are kept at 
 room temperature for a few hours before the results are 
 recorded." 
 
MODIFICATIOIfS OF THE WASSERMANN TEST 
 
 107 
 
 It will be noted that NogucM gives two procedures or 
 methods of making the test but the first procedure is to 
 be preferred, in the opinion of the writer. 
 
 Table XII illustrates the method of performing Noguchi's 
 modification of the Wassermann test by the first proce- 
 dure recommended: 
 
 Table XXI 
 
 Noguchi's Method of Perpoeming the Wassermann Test with His 
 
 Modification 
 
 SET roE diagnosis 
 
 Posterior Sow 
 
 TJnknown serum, 1 
 
 drop (active) 
 Complement, 2 units 
 1 c.e. 1% corpuscle 
 suspension 
 
 Anterior Sow 
 
 Unknown serum, 
 drop (active) 
 
 Complement, 2 units 
 
 1 e.c. 1% corpuscle 
 suspension 
 
 Antigen, 1 unit 
 
 POSITIVE CONTEOL 
 SET 
 
 Posterior Sow 
 
 Positive serum, 
 
 drop 
 
 Complement, 2 units 
 1 e.c. 1% corpuscle 
 
 suspension 
 
 Anterior Sow 
 
 Positive serum, 
 
 drop 
 
 Complement, 2 units 
 1 CO. 1% corpuscle 
 
 suspension 
 Antigen, 1 unit 
 
 negative CONTEOL 
 SET 
 
 Posterior Sow 
 
 Normal serum, 
 
 drop 
 
 Complement, 2 units 
 1 e.c. 1% corpuscle 
 
 suspension 
 
 Anterior Sow 
 
 Normal serum, 
 
 drop 
 
 Complement, 2 units 
 1 e.c. 1% corpuscle 
 
 suspension 
 Antigen, 1 unit 
 
 a 
 
 J 
 
 
 < 
 
 u 
 
 I i, 
 
 I «■ O 
 
 f i 
 
 > (D 4^ 
 
 ^ ^ S) 9^ 
 3 2 SB'S 
 
 'in 
 
 Hecht-Weinberg's Modification.^' — ^In this modification 
 the natural amboceptor present in human blood serum to 
 sheep's corpuscles is used for the amboceptor, and for 
 complement, that present in the patient's blood serum is 
 utilized. In other words, the serum to be tested furnishes 
 both amboceptor and complement. Therefore, the blood 
 serum must not be inactivated before use and should be 
 used as soon as possible after the specimen is taken. The 
 modification is claimed to give more delicate results than 
 the usual Wassermann test but the writei; does notib^lijie^e 
 that it should be used generally, owing to the errors that 
 may occur through false positive reactions occurring with 
 the usual antigens employed and becaiis'e the bin'oUnt of 
 natural amboceptor to sheep erythrocytes is such a vari- 
 
108 THE WASSBBMANlir TEST 
 
 able quantity in human blood, being excessive in some in- 
 stances and practically absent in others. The acetone in- 
 soluble antigen of Noguchi should be used with this modi- 
 fication. 
 
 Gradwohl's Modification of the Hecht- Weinberg Modifi- 
 cation. — Gradwohl" has greatly improved the teehnic of 
 the Hecht- Weinberg modification of the Wassermann test 
 by ascertaining the hemolytic strength of each serum to 
 be tested to sheep corpuscles ; i. e., the hemolytic index of 
 each serum to sheep corpuscles is worked out before the 
 serum is finally tested. GradwohP^ thus describes his 
 teehnic : 
 
 "Place in a rack fourteen smaU test tubes. The first 
 ten of these tubes are used to determine the hemolytic in- 
 dex of the suspected blood. By this I mean the exact 
 amount of hemolytic amboceptor present in the given 
 blood serum. The last four tubes are used in the actual 
 test. Add 0.1 c.c. of fresh, unheated, patient's blood serum 
 to each of the first ten tubes. Then add decreasing 
 amounts of normal salt solution to these tubes, beginning 
 with 1 cc, then 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 and 0.1 c.c. 
 to the succeeding nine tubes. Next add increasing amounts 
 of fresh 5 per cent suspension of sheep's corpuscles, start- 
 ing with 0.1 c.c. and ending with 1 c.c. Place the rack in 
 the water-bath for one-half hour. The tube which last 
 shows complete hemolysis constitutes the 'hemolytic in- 
 dex;' if it is tube 4, the index is 4, because this tube had 
 received 0.4 c.c. of sheep's corpuscles and therefore we 
 have obtained an idea as to how much sheep's blood is to 
 be added to the last four tubes. The first three of these 
 tubes , (11, 12, and 13) constitute the tubes for the actual 
 test, while the last tube in the rack (14) serves as the se- 
 rum /control tube. Tubes 11, 12, and 13 receive, therefore, 
 the patient's serum, the proper amount of sheep's corpus- 
 cles, dependent on hemolytic index, rising strengths of an- 
 tigen, but' no c(im^lement and no amboceptor. Tube 14 re- 
 ceives only shee^p corpuscles, but no antigen." 
 
MODIFICATIONS Or THE WASSERMANN TEST 109 
 
 *'In my technic I use 0.1 c.e. of a diluted antigen, deter- 
 mined by titration, in tube 11, 0.15 c.e. antigen in tube 12, 
 and 0.2 c.c. in tube 13. In order to equalize the volume of 
 fluid in these tubes, I add 0.2 c.e. normal saline to tube 11, 
 0.15 e.e. to tube 12, 0.1 c.c. to tube 13, and 0.3 c.c. to tube 14. 
 The tubes are then agitated and placed in the water-bath 
 for half an hour. These last four tubes are filled at the 
 time I make the additions to the first ten and are left with 
 them in the water-bath for one-half hour for fixation of 
 complement, the rack is then taken out and the hemolytic 
 index computed. If the index is low, say from 1 : 4, I add 
 0.1 c.c. of sheep's blood to the last four tubes. If the in- 
 dex is between 5 and 7, I use 0.15 c.c. sheep's blood to the 
 last four tubes ; if between 8 and 10, I add 0.2 c.c. ; if the 
 index is over 10, I rack up ten mo^e tubes and repeat the 
 titration of the natural complement and amboceptor, then 
 I estimate that; if between 11 and 15, I use 0.25 c.c; if 
 between 15 and 18, I use 0.3 c.c; and if between 18 and 
 20, 1 use 0.35 c.c. If the patient's serum has an index be- 
 low 2, I regard the reaction of doubtful value. If it is 
 above 2, I regard it as absolute. The reaction is read off 
 exactly as in the Wassermann, that is, inhibition or non- 
 inhibition of hemolysis. If the amount of complement or 
 amboceptor is very low, we can add the proper amount of 
 guinea pig's serum or rabbit's immune serum, ascertained 
 by preliminary titration." 
 
 Gradwohl claims that this modification, in his hand.s, is 
 superior to the Wassermann reaction, giving 15 to 20 per 
 cent more positive reactions than the latter, but the writer 
 believes that it is so cumbersome that it could not be used 
 in laboratories where scores and, sometimes, hundreds of 
 tests are being made each day, as in army practice, and 
 that the fact that false positives have been repeatedly re- 
 ported by different observers who have used the test mil- 
 itates greatly against its value. It is an excellent control 
 
110 THE WASSBEMAWN TEST 
 
 of the Wassermann test but should not replace it in gen- 
 eral laboratory practice. 
 
 Kolmer's Modifications. — Four different methods for 
 performing the Wassermann reaction are recommended by 
 Kolmer.^° The first method is exactly similar in technic to 
 the original Wassermann, except that an alcoholic extract 
 of syphilitic liver is used instead of an aqueous extract. 
 IiTthe second method the technic is the same except that 
 three different antigens are used with each blood serum 
 tested and the amounts of each reagent are reduced one- 
 half. Kolmer strongly recommends this method as being 
 "simple, accurate, and reliable." The three antigens used 
 are : 1. A cholesterinized alcoholic extract of human heart ; 
 2, alcoholic extract of syphilitic liver; 3, acetone insoluble 
 lipoids. It will thus be noted that four test tubes are re- 
 quired for each serum tested, one as control, and three 
 for the antigen tubes. As regards the results, Kolmer 
 
 "With strongly positive serums there is complete inhi- 
 bition of hemolysis with all three antigens. * * * In from 
 15 to 20 per cent of cases the cholesterinized extract shows 
 a 50 per cent or more inhibition of hemolysis, whereas with 
 the other two antigens the reactions are negative. In our 
 experience the majority of such serums were taken from 
 patients giving a frank history of syphilis of many years' 
 standing, and from known cases undergoing treatment, fur- 
 ther therapy being indicated until the reaction finally be- 
 comes negative when cholesterinized extracts are used. In 
 a small proportion of cases a feebly positive reaction of 
 25 per cent or even less of inhibition of hemolysis may be 
 found with the cholesterinized extract alone. Many of these 
 reactions occur with serums of treated cases of syphilis ; on 
 the other hand a similar reaction may occur with about 5 
 per cfent of normal serums, so that if the histoi^y and clin- 
 ical conditions are clearly negative, a slight degree of in^ 
 hibition of hemolysis (5 to 10 per cent) with the choles- 
 
MODIFICATIONS OF THE WASSERMANN TEST 111 
 
 terinized extract and marked hemolysis with the other two 
 antigens may be interpreted as a negative reaction." 
 
 The writer has quoted Kolmer regarding his results with 
 cholesterinized extracts for antigens because his experi- 
 ence has been exactly similar and for this reason the writer 
 firmly believes in the value of such extracts in the diagno- 
 sis of syphilis with the Wassermann test and in the control 
 of the treatment of the disease. Properly titrated and con- 
 trolled, and with experience gained by using them, so that 
 a proper interpretation may be placed upon the results ob- 
 tained, cholesterinized antigenic extracts are perfectly safe 
 and furnish a much greater amount of information regard- 
 ing the existence of a syphilitic infection than do other 
 antigenic extracts. 
 
 Kolmer 's third method is devised in order to measure 
 the amount of syphilitic antibody in the patient's blood se- 
 rum. He employs only one antigen, either an alcoholic 
 extract of syphilitic liver or the acetone insoluble lipoid 
 antigen. The technic is about the same as in the original 
 Wassermann but eight tubes are used for each patient's 
 serum, increasing amounts of the serum, diluted 1 :10 being 
 placed in them, together with the unit of antigen deter- 
 mined by titration, only one antigen being employed. The 
 fourth method is somewhat similar, but in this the amount 
 of syphilitic antibody in the patient's blood serum is meas- 
 ured by the number of hemolytic doses of complement fixed 
 by a constant amount of antigen. 
 
 As routine methods, where many tests are being per- 
 formed, the third and fourth methods of Kolmer are not 
 practical, as they consume too much time and material. 
 The second method is an excellent one, but it appears to 
 the writer that practically the same results are obtained by 
 using two antigens, instead of three, as recommended in 
 his modification. The writer also believes, as already 
 stated, that a human hemolytic system is preferable to th^ 
 sheep system, which is used in all of Kolmer 's methods, 
 owing to the possibility of the natural antisheep ambocep- 
 
112 THE WASSEBMANN TEST 
 
 tor present in nearly all human blood serum interfering 
 with' the delicacy of the test. 
 
 Despite all the claims that have been made that the pres- 
 ence of antisheep amboceptor in human serum is a negligi- 
 ble factor in the Wassermann test, the fact remains that 
 in some blood sera there is so much of this amboceptor 
 present that no dependence can be placed upon the results 
 of a Wassermann test with them unless it is first removed, 
 and it is certainly logical to believe that, in some cases, 
 this amboceptor when combined with the' amboceptor 
 added, would render a reaction negative that, with the hu- 
 man hemolytic system, would be positive. This factor 
 would frequently be of very great importance in any 
 method of performing the test in which titrations of the 
 amount of syphilitic antibody in the patient's serum were 
 used. 
 
 The modifications of Tschemogubow'' which utilize the 
 natural complement and amboceptor against guinea pig 
 corpuscles present in human serum; of Detre and Bre- 
 zovsky,^* in which an antihorse hemolytic system is em- 
 ployed with rabbit complement ; of Margarette Stern^' who 
 used fresh, active blood serum and the complement present 
 in the blood serum tested; of Bauer,*" who makes use of 
 the antisheep amboceptor present in human blood serum; 
 of Browning and McKenzie," who use an antiox hemolytic 
 system and a quantitative technic ; and of Thompson,*^ who 
 uses the complement present in the fresh blood serum of 
 the patient, an antihuman hemolytic system, and the 
 acetone insoluble lipoid antigen of Noguchi have all been 
 recommended, from time to time, but none of them appear 
 to be" as reliable as the original Wassermann technic, the 
 modifications recommended by Noguchi and Kolmer, or 
 the writer's modification. 
 
 The Effect of Low Temperatures upon the Results of 
 the Wassermann Test. — Quite recently several observers 
 have called attention to the effect of incubating the com- 
 
MODIFICATIONS OF THE WASSERMANN TEST 113 
 
 plemeiit, antigen, and patient's blood serum at ice box tem- 
 perature, claiming that more delicate results are obtained 
 than when this incubation is made in the water-bath at 
 37° C. Zinsser"' states that in his laboratory two series of 
 tests were run, one with the first incubation at 37.5° C. for 
 30 minutes, the other with the first incubation in the ice 
 box at 8 to 10° C, for three hours, the same blood sera 
 being used in each series. The results showed 15 more 
 positive reactions in the series incubated at ice box tem- 
 perature, than in that incubated in the water-bath, and of 
 these cases 7 were unquestionably syphilitic, two were 
 treated syphilitics, and four were probably syphilitic. * 
 
 In a recent valuable contribution, Smith and MacNea?* 
 compared the results obtained with different antigens and 
 different temperatures of incubation in the Wassermann 
 test. They experimented with three antigens as follows: 
 1. A cholesterinized alcoholic extract of beef heart. 2., A 
 plain alcoholic extract of beef heart. 3. An acetone insolu- 
 ble extract of beef heart. Two series of tests were made, 
 with the same blood sera, one in which the first incubation, 
 with each antigen, was at 37° C. and one in which the first 
 incubation was for 4 hours in the ice box at 8° C, with each 
 antigen. Over 500 different sera were tested and their con- 
 clusions are important. They found that the cholesterin- 
 ized antigen, with incubation at 8° C. for 4 hours proved a 
 more sensitive test for syphilis than any of the other 
 methods tried but that it was apt to give nonspecific com- 
 plement fixation, and, therefore, if used alone it could not 
 be depended upon in diagnosis. The simple extract antigen 
 (alcoholic extract of beef heart), with the first incubation 
 for 4 hours at 8° C. in the ice box^ gave more sensitive re- 
 sults than the cholesterinized extract at 37° C. and in these 
 experiments did not give any false positive results. The 
 acetone insoluble extract was found less sensitive either 
 at 37° C. Or 8° C. than the cholesterinized extract and also 
 less sensitive than the simple alcoholic extract at 8° C. 
 
114 THE WASSBEMANN TEST 
 
 It was more sensitive than the simple extract at 37° C, 
 and gave no false positive results in this series of tests. 
 From their results it is evident that the eholesterinized ex- 
 tract at 8° C. gave the highest percentage of positive re- 
 sults, but sometimes gave a false positive reaction, whUe 
 the simple alcoholic extract gave the best practical results, 
 when the first incubation v/as made at 8° C, as it gave 
 better results than the eholesterinized extract at 37° C, 
 and did not give any false reactions. 
 
 The subject of the ice box temperature for the first in- 
 cubation in the Wassermann test deserves further study, 
 and, if these observations are confirmed, this temperature 
 should be adopted in practice. If it is found reliable it 
 will probably be .best to use simple alcoholic extracts for 
 antigen instead of eholesterinized extracts, but more work 
 should be done upon the subject before the method is gen- 
 erally adopted. 
 
 The Titration of the Strength of the Wassermann Reac- 
 tion. — ^In using the Wassermann test as a control of the 
 efficiency of treatment, it is often advisable to test the 
 strength of the reaction, from time to time, especially if 
 sera show a double-plus (four-plus reaction) repeatedly, 
 although intensive treatment is being administered. In 
 such cases, the physician can not be sure that the treat- 
 ment is having any effect unless the blood serum is titrated 
 as to its fixing or absorbing power of complement in the 
 presence of suitable antigen. This is done by diluting the 
 blood serum in the following manner, as recommended by 
 Vedder :°= Six tubes are used in addition to the usual num- 
 ber used in the Wassermann test as recommended by the 
 writer. In the first tube is placed 1.9 c.c. of normal salt 
 solution, and in each of the other tubes 1 c.c. of normal 
 salt solution. In the first tube is placed 0.1 c.c. of the 
 serum to be titrated and the tube is thoroughly shaken. 
 One cubic centimeter is taken from this tube and placed 
 in the second tube, which is shaken, and the same amount 
 is carried to the third tube, and the operation repeated 
 
MODiriCATIOlSrS Oli" THE WASSEEMANN TEST 
 
 115 
 
 until the sixth tube is reached, from which, after it is 
 shaken, 1 c.e. is discarded. After the dilutions are thus 
 made each tube -will contain 1 c.c. of fluid and the dilutions 
 of the blood serum -will be as follows: Tube 1, 0.05 c.c; 
 Tube 2, 0.025 c.c; Tube 3, 0.012 c.c; Tube 4, 0.006 cc; 
 Tube 5, 0.003 c.c. ; and Tube 6, 0.0015 cc. 
 
 The test is then made in the usual manner with all of 
 the dilutions and the results recorded as in Table XXII. 
 
 Table XXII 
 Results of Titration of Blood Serum of 
 
 
 DILUTIONS 
 
 
 0.1 e.e. 
 
 0.05 
 
 0.025 
 
 0.012 
 
 0.006 
 
 0.003 
 
 0.0015 
 
 Previous Eesult. 
 
 + + 
 
 with 0.05 cc. 
 
 + + 
 
 + 
 
 + - 
 
 - 
 
 - 
 
 - 
 
 / 
 
 This method of titrating the blood serum of patients 
 under treatment is recommended in all cases in which per- 
 sistent double-plus (four-plus) reactions are reported, as 
 it is the only way in which the physician can be sure that 
 the particular method of treatment being pursued is prov^ 
 ing efficient. In many instances the titration wiU demon- 
 strate that the smaller amounts of serum become progres- 
 sively negative, thus proving the efficiency of the treatment, 
 although the usual amount of serum tested, i. e., 0.1 c.c, 
 may give a positive reaction. 
 
CHAPTEE VI 
 
 COMPLEMENT FIXATION IN SYPHILIS WITH 
 
 ANTIGENS PREPAEED FEOM PURE 
 
 CULTURES OF TREPONEMA 
 
 PALLIDUM 
 
 The success attending Noguchi's efforts to obtain Tre- 
 ponema pallidum in pure culture lead to the hope that an- 
 tigens prepared from such cultures would be effective in 
 the Wassermann test and that in this manner a truly spe- 
 cific antigen would be obtained and the reaction become 
 a true antigen-antibody reaction. It is ob\dous that if 
 such a specific antigen could be obtained the value of the 
 Wassermann test, great as it is at present, would become 
 still greater, as the positive reactions frequently observed 
 in yaws, tubercular leprosy, and with less frequency, in 
 malaria and trypanosomiasis, would be eliminated. The 
 subject has already been briefly discussed but it is thought 
 of enough importance to consider somewhat more exten- 
 sively in this chapter. 
 
 Schereschewsky, in 1909,^* was the first to attempt to 
 secure an antigen from cultures of Treponema pallidum. 
 Unfortunately, his culture was an impure one and for this 
 reason his results were not conclusive. He was able to 
 obtain complement fixation with syphilitic serum, with his 
 antigen, but also obtained positive results with some of the 
 control antigens with which he worked. It is now gen- 
 erally admitted that he was not working with cultures of 
 Treponema pallidum at all but with Treponema microden- 
 tium, and, as will be shown later, his positive results were 
 in the nature of a group reaction, as the writer and Nichols 
 have shown that this organism will react positively with 
 syphilitic serum, if used as an antigen. In addition, 
 
 116 
 
COMPLEMENT FIXATION IN SYPHILIS 117 
 
 Schereschewsky's cultures were contaminated with bac- 
 teria and it is possible that some of his positive results 
 were due to this fact, for, as already noted, certain bacteria, 
 when growing in normal blood serum, produce substances 
 which give a positive reaction with the Wassermann test, 
 and it is not improbable that bacteria growing in the horse 
 serum medium, which he used for his cultures, may have 
 produced lipoidal substances which were capable of giving 
 positive results. However, his work was very suggestive 
 and, without doubt, stimulated research along these lines. 
 
 In 1912, Noguchi,"' who had succeeded in isolating and 
 growing Treponema pallidum in pure culture, published 
 the results of his work upon complement fixation in syph- 
 ilis with an antigen prepared from such cultures. His an- 
 tigen was an aqueous extract of Treponema pallidum, and 
 he also worked with an emulsion of the organisms obtained 
 from the testicles of infected rabbits. His researches dem- 
 onstrated beyond doubt that, in certain cases of syphilitic 
 infection, it is possible to obtain complement fixation with 
 antigens prepared from a pure culture of Treponema palli- 
 dum and that, in such instances, the reaction is indeed a 
 true antigen-antibody reaction. 
 
 Noguchi found that, in cases of syphilis that had been 
 treated or in which the disease was latent, he often ob- 
 tained a positive reaction with the culture antigen when 
 the ordinary antigens used in the Wassermann test gave 
 a negative result, and that the culture antigen very rarely 
 gave a positive result in primary and secondary cases of 
 the disease. Therefore, he concludes that the reaction with 
 the pallidiun antigen may be regarded as an index of the 
 resistance of the patient to the infection, stating ''We have 
 in the Wassermann reaction a fair measure of the ac- 
 tivity of the infecting agent, and now we will have in the 
 pallida fixation reaction a gauge for the defensive activity 
 of the infected host." 
 
 Noguchi 's conclusions regarding his work with the pal- 
 lidum antigen are as follows: 
 
118 THE WASSBEMAiriir TEST 
 
 1. The Wassermann reaction is caused by the lipotropic 
 substances in sera but not by the antibodies which combine 
 specifically with the pallida antigen. 
 
 2. The fixation produced by the culture pallida antigen 
 with certain syphilitic sera is caused by the specific anti- 
 bodies contained in the latter and may constitute a specific 
 diagnostic method for syphilis. 
 
 3. The fixation caused by testicular extracts behaves like 
 the culture pallida antigen in the majority of cases, but 
 when the sera (syphilitic or leprous) contains abundant 
 lipotropic substances, they may give a Wassermann re- 
 action as well, which is not the case with the culture pallida 
 antigen. 
 
 4. In the serum of rabbits with active syphilitic orchitis 
 there is no indication of the presence of a sufficient amount 
 of antibodies for the pallida antigen, although it gives a 
 strong Wassermann reaction. 
 
 In 1912, the writer and Major Nichols published the 
 results of some work upon specific antigens in comJ)lement. 
 fixation with syphilitic serum,** which differed somewhat 
 from those of Noguchi, chiefly because of the nature of 
 the antigen employed. In his work Noguchi used aqueous 
 extracts of Treponema pallidum, while in our work alco- 
 holic extracts were employed. Noguchi stated that it was 
 impossible to extract the antigenic properties of the or- 
 ganism by alcohol and that only aqueous extracts contained 
 them, but our results showed that alcoholic extracts were 
 efficient, although they were not as efficient as aqueous ex- 
 tracts, and our work has since been confirmed by other au- 
 thorities. It is true that alcoholic extracts owe a part of 
 their activity to other substances than the specific com- 
 plement-fixing substance of Treponema pallidum which is 
 best extracted in water, but it is also true that the alco- 
 holic extracts do contain this specific substance, as the re- 
 sults obtained can be explained in no other way, and dif- 
 fer so greatly from those obtained with ordinary alcoholic 
 antigens used in the Wassermann test. 
 
compijEment fixation in syphilis 119 
 
 In our experiments we tested 51 syphilitic sera and 54 
 nonsyphilitic sera. Of the latter, 38 were from patients 
 suffering from diseases other than syphilis and 16 were 
 from normal individuals. Eleven rabbit sera were also 
 tested. The sera were all inactivated by heating them at 
 55-56° C. for half an hour before testing. 
 
 The stock antigen used was an alcoholic extract of foetal 
 syphilitic liver and the culture antigens were prepared 
 from cultures kindly sent us by Dr. Noguchi. Alcoholic 
 extracts were made of the cultures, filtered, the filtrate 
 evaporated to one-third its original volume and titrated in 
 a dilution of 1-10 with normal salt solution. Antigens 
 were thus prepared from cultures of Treponema pallidum, 
 Treponema pertenue, and Treponema microdentium. The 
 test was performed in the same manner as that recom- 
 mended in this work by the writer for the Wassermann 
 test. 
 
 As a control of the culture antigens used an alcoholic ex- 
 tract of the medium employed in growing the treponemas 
 was prepared in the same manner as the culture antigens. 
 
 Results with Normal Sera. — The blood serum of 16 nor- 
 mal individuals was tested with the stock antigen and with 
 pallidum, pertenue, and microdentium antigen, and all gave 
 a negative result. Not a trace of a reaction occurred in 
 any of these tests so that it is evident that none of the an- 
 tigens employed reacted with normal blood serum. 
 
 Results with Serum from Other Diseases than Syph- 
 ilis. — The blood serum of 38 patients suffering from dis- 
 eases other than syphilis was tested with the same an- 
 tigens and was negative in every instance, except that a 
 plus reaction was obtained with the microdentium antigen 
 in a case diagnpsed as arthritis. Syphilis could not be 
 eliminated in this case but the stock syphilitic antigen gave 
 a negative result with the serum. 
 
 Results with Serum from Syphilitic Patients. — The blood 
 sera of 51 patients suffering from syphUis were tested and 
 Table XXIII shows the results obtained. In recording 
 
120 
 
 THE WASSEEMAKN TEST 
 
 these tests the sign, + +, means absolute inhibition of hem- 
 olysis; the sign, +, at least 50 per cent of inhibition; and 
 the sign, +-, weaker degrees of inhibition. The sign, -, 
 indicates total lack of inhibition, while the sign indicates 
 that no test was made. 
 
 Table XXIII 
 
 Complement Fixation in Syphilis with Stock Antigen and Specific 
 
 Antigens in Forty Infections* 
 
 
 
 T. PAL- 
 
 T. PEK- 
 
 T. MICKO- 
 
 CONTROL 
 
 
 SEKUM 
 
 STOCK 
 
 LIDUM 
 
 TENUE 
 
 DENTIUM 
 
 ANTIGEN 
 
 REMARKS 
 
 NO. 
 
 ANTIGEN 
 
 ANTIGEN 
 
 ANTIGEN 
 
 ANTIGEN 
 
 
 
 1 
 
 + + 
 
 + + 
 
 
 
 
 
 _ 
 
 Secondary, early 
 
 2 
 
 + + 
 
 + 
 
 + - 
 
 
 
 _ 
 
 Primary 
 
 3 
 
 + + 
 
 + - 
 
 + - 
 
 
 
 -, 
 
 Secondary 
 
 4 
 
 + + 
 
 ^ 
 
 
 
 
 
 
 
 Latent, 18 years 
 
 5 
 
 + + 
 
 + 
 
 + 
 
 + 
 
 _ 
 
 Latent, 4 years 
 
 6 
 
 + + 
 
 _ 
 
 + - 
 
 _ 
 
 _ 
 
 Latent, 3 years 
 
 7 
 
 + + 
 
 ■_ 
 
 - 
 
 
 
 _ 
 
 Tertiary, 10 years 
 
 8 
 
 + + 
 
 - 
 
 
 
 
 
 _ 
 
 Secondary, 9 months 
 
 9 
 
 + 
 
 + 
 
 
 
 
 
 
 
 Secondary, early 
 
 10 
 
 + 
 
 
 
 
 
 
 
 _ 
 
 Primary 
 
 11 
 
 + 
 
 + + 
 
 
 
 
 
 _ 
 
 Primary, 2 weeks 
 
 12 
 
 + 
 
 + 
 
 1 
 
 
 
 
 
 Latent, 2 years 
 
 13 
 
 + 
 
 - 
 
 ' 
 
 + - 
 
 
 
 Primary 
 
 14 
 
 + 
 
 - 
 
 
 
 + 
 
 - 
 
 Primary 
 
 15 
 
 + 
 
 + 
 
 
 
 + 
 
 - 
 
 Secondary, early 
 
 16 
 
 + 
 
 + 
 
 - 
 
 - 
 
 - 
 
 Secondary, 1 year 
 
 17 
 
 + 
 
 - 
 
 
 
 + 
 
 - 
 
 Tertiary 
 
 18 
 
 + 
 
 + 
 
 + - 
 
 + 
 
 - 
 
 Secondary 
 
 19 
 
 + 
 
 + + 
 
 
 
 + 
 
 - 
 
 Tertiary 
 
 20 
 
 + 
 
 +- 
 
 + - 
 
 + - 
 
 - 
 
 Latent, duration un- 
 known 
 
 21 
 
 + - 
 
 +- 
 
 
 
 
 
 _ 
 
 Latent, 3 years 
 
 22 
 
 + - 
 
 + 
 
 
 
 
 
 - 
 
 Primary, 3 weeks 
 
 23 
 
 + - 
 
 + - 
 
 
 
 
 
 
 
 Secondary, early 
 
 24 
 
 + - 
 
 - 
 
 - 
 
 - 
 
 -■" 
 
 Primary, 2 weeks 
 
 25 
 
 + - 
 
 - 
 
 - 
 
 
 
 - 
 
 Epilepsy 
 
 26 
 
 + - 
 
 + - 
 
 
 
 +- 
 
 - 
 
 Arthritis, syphilitic 
 
 27 
 
 + - 
 
 + - 
 
 
 
 +- 
 
 
 
 Latent, 2 years 
 
 28 
 
 +.- 
 
 + - 
 
 
 
 +- 
 
 _ 
 
 Latent, 2 years 
 
 29 
 
 + - 
 
 + 
 
 
 
 + 
 
 _ 
 
 Latent, 1 year 
 
 30 
 
 + - 
 
 +- 
 
 - 
 
 + - 
 
 --" 
 
 Secondary, 1 year 
 
 31 
 
 + - 
 
 + + 
 
 + + 
 
 + 
 
 — 
 
 Latent, duration un- 
 known 
 
 32 
 
 + - 
 
 + - 
 
 
 
 + 
 
 - 
 
 Latent, 3 years 
 
 33 
 
 + - 
 
 +- 
 
 +'- 
 
 — 
 
 - 
 
 Secondary, 3 months 
 
 34 
 
 + - 
 
 + 
 
 + - 
 
 + - 
 
 - 
 
 Latent, 4 years 
 
 35 
 
 + - 
 
 + - 
 
 - 
 
 +- 
 
 _ 
 
 Latent, 6 years 
 
 36 
 
 + - 
 
 +- 
 
 +- 
 
 - 
 
 - 
 
 Secondary, early 
 
 37 
 
 - 
 
 + - 
 
 - 
 
 + - 
 
 - 
 
 Latent, 2 years 
 
 38 
 
 - 
 
 + 
 
 + 
 
 + - 
 
 - 
 
 Latent, 1% years 
 
 39 
 
 - 
 
 +- 
 
 - 
 
 - 
 
 - 
 
 Latent, 6 years 
 
 40 
 
 - 
 
 +- 
 
 - 
 
 
 
 
 
 Primary, 2 weeks 
 
 *Each 
 
 serum test 
 
 ed.was pro] 
 
 jerly Contn 
 
 )Iled as is i 
 
 isual in the 
 
 Wassermann *ps+ 
 
COMPLEMENT FIXATION IN SYPHILIS 121 
 
 It will be noted that no case reacted with the control 
 culture medium antigen and that of the 36 cases giving 
 any reaction at all with the stock extract antigen, no less 
 than twenty-seven, or 75 per cent reacted also with the 
 pallidum antigen, but the reactions were, as a rule, much 
 weaker with the pallidum antigen than with the stock an- 
 tigen. However, there were instances in which the pal- 
 lidum antigen gave a stronger reaction than the stock an- 
 tigen, as illustrated in serums 11 and 19, while in serum 
 38 the stock antigen gave a negative result and the pal- 
 lidum antigen a plus reaction. 
 
 As regards the results given with the antigen prepared 
 from the pure cultures of Treponema pallidum, 17 of the 
 sera gave the same result as with the stock antigen, four 
 gave weaker reactions, six gave stronger reactions, and 
 four gave positive results in sera in which the stock an- 
 tigen gave a negative result. However, the pallidum an- 
 tigen gave seven negative reactions in twenty sera that 
 gave absolute inhibition of hemolysis with the stock an- 
 tigen, (not shown in table) thus proving that the pallidum 
 antigen is untrustworthy in the diagnosis of syphilis. 
 
 Of the various syphilitic sera tested, fifteen were tested 
 with all three antigens, and the results are valuable be- 
 cause they indicate the, group nature of the complement 
 fixation reaction obtained with these alcoholic extracts of 
 Treponema pallidum, Treponema pertenue, and Treponema 
 microdentium. Table XXIV gives the results obtained : 
 
 In four of the cases, the results were the same with all 
 of the culture antigens but in the remaining cases they 
 varied, the pallidum antigen giving results more like those 
 of the stock antigen, while the pertenue and microdentium 
 antigens gave quite similar results, although those of the 
 microdentium approached nearer to those of the pallidum 
 than did the pertenue. 
 
 Unlike Noguchi, we obtained positive results not only in 
 latent syphilitic infections but also in primary and second- 
 
122 
 
 THE WASSBBMANIT TEST 
 
 Table XXIV 
 Complement Fixation in Syphilitic Serum with Culture Antigens 
 
 SEEUM 
 NO. 
 
 STOCK 
 ANTIGEN 
 
 T. PAL- 
 LIDUM 
 ANTIGEN 
 
 T. PEK- 
 
 TBNUE 
 ANTIGEN 
 
 T. MICRO- 
 DENTIUM 
 ANTIGEN 
 
 CONTROL 
 ANTIGEN 
 
 EEMABES 
 
 Latent, 4 years 
 Latent, 3 years 
 Latent, duration? 
 Primary, 2 weeks 
 Secondary, 1 year 
 Secondary, duration? 
 Latent, duration? 
 Secondary, 3 months 
 Latent, 4 years 
 Latent, 6 years 
 Latent, 2 years 
 Latent, 1% years 
 Latent, 6 years 
 Primary, 2 weeks 
 Secondary, 1 year 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 18 
 
 14 
 
 15 
 
 + + 
 + + 
 
 + 
 + - 
 
 + 
 
 + 
 
 + - 
 
 + - 
 + - 
 + - 
 + - 
 
 + - 
 
 + 
 
 + 
 
 + - 
 + + 
 + - 
 
 + 
 
 + - 
 + - 
 
 + 
 
 + - 
 + - 
 
 + 
 + - 
 
 + + 
 
 + - 
 
 ary infections with the culture antigens. It should be re- 
 membered, in this connection, that Noguchi worked with 
 an aqueous extract of Treponema pallidum while we used 
 alcoholic extracts, and thus the variation may be explained, 
 although the writer does not agree with Noguchi that it is 
 impossible to extract from this organism specific comple- 
 ment-fixing substances with alcohol. Our results with these 
 alcoholic extracts of treponemas are too much at variance 
 with those obtained with the ordinary alcoholic extract of 
 foetal syphilitic liver to make it at all probable that the 
 entire complement-fixing or absorbing power of these cul- 
 ture antigens was entirely nonspecific in character, and 
 our experiments with rabbit blood serum, which gave 
 identical results with those of Noguchi, are evidence of this 
 fact. 
 
 Results With Rabbit Blood Serum.— We tested the 
 blood serum of two rabbits presenting acute symptoms of 
 syphilitic infection; of three that had recovered from a 
 syphilitic orchitis; of two that had recovered from yaws; 
 and of four normal rabbits, and the results are given in 
 Table XXV. 
 
 The important fact shown by these experiments is tha;t 
 
COMPLEMENT FIXATION IN SYPHILIS 
 
 123 
 
 Table XXV 
 Complement Fixation with Specific Timponema Antigens in Rabbit Sera 
 
 
 
 T.PAL- 
 
 T. PER- 
 
 T. MICRO- 
 
 
 
 RABiBIT 
 
 stock 
 
 LIDirM 
 
 TENTJE 
 
 DENTIUM 
 
 CONTROL 
 
 REMARKS 
 
 NO. 
 
 antigen 
 
 ANTIGEN 
 
 ANTIGEN 
 
 ANTIGEN 
 
 ANTIGEN 
 
 
 1 
 
 + + 
 
 _ 
 
 _ 
 
 _ 
 
 _ 
 
 Active lesions 
 
 2 
 
 + + 
 
 - 
 
 _ 
 
 _ 
 
 „ 
 
 Active lesions 
 
 3 
 
 - 
 
 - 
 
 - 
 
 - 
 
 
 
 Recovered from syphilis 
 
 4 
 
 - 
 
 - 
 
 - 
 
 - 
 
 
 
 Recovered from syphilis 
 
 5 
 
 - 
 
 - 
 
 - 
 
 - 
 
 - 
 
 Recovered from syphilis 
 
 6 
 
 - 
 
 - 
 
 1. 
 
 
 
 - 
 
 Recovered from yaws 
 
 7. 
 
 - 
 
 ^ 
 
 _ 
 
 _ 
 
 „ 
 
 Recovered from yaws 
 
 8 
 
 - 
 
 _ 
 
 _ 
 
 
 
 _ 
 
 Normal rabbit 
 
 9 
 
 _ 
 
 _ 
 
 _ 
 
 
 
 _ 
 
 Normal rabbit 
 
 10 
 
 - 
 
 _ 
 
 _ 
 
 
 
 _ 
 
 Normal rabbit 
 
 11 
 
 - 
 
 - 
 
 - 
 
 
 
 
 
 Normal rabbit 
 
 no reaction with the culture antigens occurred in any of the 
 rabbit sera, although the twd animals showing active le- 
 sions of syphilis gave absolute inhibition of hemolysis 
 with the stock antigen. These results are in entire agree- 
 ment with those of Noguchi, who found that his pallida an- 
 tigen did not react with rabbit serum even when the ani- 
 mals presented active lesions of the iafection. On the 
 other hand, antigens prepared by extracting with alcohol 
 the testicles of rabbits presenting a syphilitic orchitis will 
 give positive results with syphilitic sera and also with the 
 blood of rabbits injected with syphilis, thus proving that 
 the alcoholic extract of cultures of Treponema pallidum 
 is an entirely different antigen from that obliained from 
 the infected testicle of rabbits and that the reactions ob- 
 tained with such an extract are, at least, partly specific in 
 character. In 1911, Nichols published some experiments of 
 ours upon complement fixation with alcoholic antigens 
 made from the testicles of rabbits suffering from experi- 
 mental syphilis and yaws, and we found that while such 
 antigens gave positive results with certain syphilitic sera, 
 they also reacted with certain normal sera, as well as sera 
 from other diseases, and that an antigen prepared from 
 the testicles of rabbits inoculated with syphilis give posi- 
 tive results with the sera of rabbits inoculated with yaws 
 
124 THE WASSBEMANN TEST 
 
 and vice versa. In addition, we found that alcoholic an- 
 tigens prepared from the testicles of normal rabbits also 
 gave positive results, so that we did not consider that this 
 type of antigen was at all specific in character. 
 
 From our experiments with antigens prepared from 
 pure cultures of the treponemas named we reached the 
 following conclusions: 
 
 1. Alcoholic extracts of pure cultures of Treponema pal- 
 lidum can not be depended upon in the diagnosis of syph- 
 ilitic infection by -the complement-fixation test, for many 
 cases of undoubted syphilis, giving absolute iohibition of 
 hemolysis with the stock antigen, an alcoholic extract of 
 foetal syphilitic liver, gave a negative reaction with the 
 culture antigen, and the reactions obtained with the pal- 
 lidum antigen were generally weaker and less conclusive 
 than with the stock antigen. 
 
 2. The results appear to prove that there are specific 
 group reactions, in the way of complement fixation, or 
 absorption, between Treponema pallidum, Treponema per- 
 tenue and Treponema microdentium, as antigens prepared 
 from pure cultures of these organisms may all give comple- 
 ment fixation in the presence of syphilitic serum. 
 
 Later experiments®^ with antigens prepared from pure 
 cultures of Spirochceta refringens, Spirillum cholerce, and 
 Bacillus typhosus demonstrated that, while SpirochcBta re- 
 fringens sometimes reacted positively with the sera tested, 
 the antigens prepared from the cultures of Spirillum chol- 
 erce and Bacillus typhosus never give a positive reaction, 
 thus proving that the theory that positive reactions with 
 these pure culture antigens might be due to lipoidal sub- 
 stances produced in the media itself by the growth of the 
 organisms is practically untenable, and that there are 
 group reactions among this class of organisms and that 
 alcoholic extracts of these organisms contain some specific 
 substance common to all, to a greater or lesser extent. 
 
 Since our work a considerable number of investigators 
 have studied the immunological phenomena of cultivated 
 
COMPLEMENT FIXATION" IN SYPHILIS 125 
 
 treponemas and spirochetes, and our results have heen 
 confirmed. Thus, Kolmer, "Williams, and Laubaugh,'^" 
 working with aqueous and alcoholic extracts of Treponema 
 pallidum obtained positive reactions in secondary, tertiary, 
 and congenital syphilis. They found that the aqueous ex- 
 tracts gave better results than the alcoholic but that, with 
 both, the reactions were generally weaker than those ob- 
 tained with the ordinary antigenic extracts used in the 
 Wassermann test. They also obtained weak reactions with 
 control antigens made from uninoculated media and other 
 organisms. Zinsser, Hopkins, and McBurney" in an ex- 
 tended series of studies upon Treponema pallidum and 
 syphilis obtained specific agglutination and complement 
 fixation with culture treponemas but conclude that their 
 work lends no support to the belief that either agglutina- 
 tion or complement fixation with cultures of Treponema 
 pallidum will prove of any practical value in the diagnosis 
 of syphilis. Finally, the work of Noguchi and Akatsu^^ 
 proves that group reactions occur between certain tre- 
 ponemas and that specific immune bodies capable of pro- 
 ducing agglutination, complement fixation, and opsoniza- 
 tion could be produced in the blood serum of rabbits by 
 injection of these organisms. They worked with Tre- 
 poneTna pallidum, Treponema calligyrum, Spirochceta re- 
 fringens, Treponema microdentium, and Treponema mu- 
 cosum, and found that the pallidum produced higher de- 
 grees of agglutination and complement fixation, than the 
 others, but that group reactions with complement fixation 
 occurred between Treponema pallidum and Treponema cal- 
 ligyrum, between calligyrum and Spirochceta refringens, and 
 between Treponema microdentium and Treponema muco- 
 sum, while there was slight group reaction between the lat- 
 ter and Treponema pallidum. 
 
 The present status of this subject is, that while specific 
 complement fixation, at least within the limits of a group, 
 is possible with either aqueous or alcoholic extracts of pure 
 cultures of Treponema pallidum, and that aqueous extracts 
 
126 THE WASSBEMANlSr TEST 
 
 give the best results there is no evidence sufficient to 
 prove that such antigens are of real practical value in the 
 diagnosis of syphilis by the Wassermann test. The re- 
 actions obtained with them are generally weak and doubt- 
 ful and they are so often negative where the ordinary anti- 
 genic extracts are positive that their use in diagnosis is 
 not justifiable. Further research may result in giving us 
 a specific antigen for the Wassermann test but at present 
 the outlook is very doubtful that such an antigen can be 
 evolved. 
 
CHAPTEE VII 
 
 THE EESULTS OF THE WASSERMANN TEST IN 
 
 THE VAEIOUS STAGES OF SYPHILIS. TIME 
 
 OF APPEAEANCE OF THE REACTION. 
 
 THE SPECIFICITY OF THE WAS- 
 
 SEEMANN TEST 
 
 The results of the Wassermann test vary with the dif- 
 ferent stages of syphilitic infection, the highest percentage 
 of positive results being obtained in the secondary stage 
 of the disease and the lowest in latent infections. The 
 results also vary with the methods employed by different 
 observers, although, in the hands of experienced serol- 
 ogists, the results obtained with one method are practically 
 the same as those with another, and even in the hands of the 
 inexperienced the results often are almost as good as when 
 the test is done by the experienced investigator. It is one 
 of the most fortunate things connected with this test, that 
 it has given such accurate results with so many different 
 modifications of the original test, and in the hands of such 
 a great number of workers, many of whom were, and are, 
 far from being experts in serology. As Vedder well says :'^ 
 "One is impressed by the fact that the Wassermann re- 
 action must be a test of most surprising merit to have sur- 
 vived aU the clumsy technic that has been perpetrated in 
 its name." 
 
 The figures as to the results of this test in the various 
 stages of syphilis which are here given, aside from those 
 given by authorities quoted, are based upon more than 
 fifty thousand "Wassermann tests personally made by the 
 writer, using the method which has been recommended in 
 Chapter V. A great many thousands of these tests were 
 made upon conditions due to other causes than syphilis 
 
 127 
 
128 THE WASSERMANlir TEST 
 
 while a still greater number were reexaminations made 
 upon the same case, sometimes as many as twenty or more 
 tests having been made upon a single patient, in the course 
 of months or years. In the army it has been customary to 
 control the treatment of syphilis by repeated Wassermann 
 tests and this practice has not only enabled army surgeons 
 to have scientific control of the treatment of their syphi- 
 litic patients but has also enabled us to have a very ac- 
 curate check upon the efficiency and reliability of the modi- 
 fication of the Wassermann test which is recommended in 
 this work. The writer has tried to keep himself informed 
 as to its success in the hands of other laboratory workers 
 and it is gratifying to be able to report that the modification 
 has proved satisfactory in every laboratory in the army 
 in which it has been adopted, and this includes nearly all, 
 and that it has been recommended as a standard method 
 for all army laboratories by Lieut. Col. Vedder,^* who has 
 devised special apparatus Avhich makes such standardiza- 
 tion possible. 
 
 Before considering the results of the "Wassermann test 
 in each stage of syphilis, it is well to call attention to the 
 fact that many of the statistics which are to be foxmd in 
 the literature are based upon observations made during the 
 first few years after the test was discovered and are, there- 
 fore, not representative of the results that are really being 
 obtained today. A great amount of research has been ex- 
 pended upon the various reagents used in the Wassermann 
 test and a great many factors have been discovered that 
 influence the results of the test, in one way or another, 
 so that at the present time much better results are being 
 obtained with the test than formerly and many inaccura- 
 cies in technic have been corrected. This fact must be 
 borne in mind in considering the percentages of positive 
 results recorded by the earlier observers in the various 
 stages of syphilis and especially the numerous reports of 
 positive results in diseases other than syphilis. In fact, 
 the results now being obtained with this test are hardly at 
 
RESULTS IN VARIOUS STAGES Or SYPHILIS 
 
 129 
 
 all comparable with those which were obtained with it dur- 
 ing the first year or two after Wassermann's publication 
 of his technic. 
 
 As an illustration of the results obtained with the au- 
 thor's method in the various stages of syphilis Table 
 XXVI is inserted, giving the results of the test in 5,600 
 syphilitic infections, personally tested by him: 
 
 Table XXVI 
 The Results of the Wasseemann Test in 5,600 Cases op Syphilis 
 
 STAGE 
 
 TOTAL CASES 
 
 POSITIVE 
 
 % 
 
 NEGATIVE 
 
 % 
 
 Primary 
 
 1080 
 
 970 
 
 89.8 
 
 110 
 
 10.1 
 
 Secondary 
 
 2217 
 
 2132 
 
 96.1 
 
 85 
 
 4.9 
 
 Tertiary 
 
 728 
 
 633 
 
 87.4 
 
 95 
 
 13.5 
 
 Latent 
 
 1525 
 
 1039 
 
 68.1 
 
 486 
 
 31.8 
 
 Congenital 
 
 28 
 
 25 
 
 82.2 
 
 3 
 
 10.7 
 
 Parasyphilis 
 
 22 
 
 15 
 
 68.1 
 
 7 
 
 3L8 
 
 . totals 
 
 5600 
 
 4814 
 
 85.9 
 
 786 
 
 14 
 
 In the above cases no test was considered positive un- 
 less there was complete inhibition > of hemolysis (a four- 
 plus reaction) except in the very early primary cases when 
 a three-plus reaction was considered positive in a few in- 
 stances. By insisting upon absolution inhibition of hem- 
 olysis as indicating a positive reaction in these cases it is 
 undoubtedly true that some cases of infection, which gave 
 reactions almost absolute, have been erroneously consid- 
 ered as negative, but in most of the army laboratorijps we 
 have not felt justified in basing a positive diagnosis of 
 syphilis upon anything less than absolute inhibition of 
 hemolysis. In considering this table it should also be re- 
 membered that in at least 95 per cent of the cases tabulated 
 only one Wassermann test was performed and that had 
 repeated tests been made it is undoubtedly true that a 
 higher percentage of positive results would have been ob- 
 tained in all of i the stages of syphilis tested- 
 
 Our results are in practical agreement with those of 
 most recent observers, so that it may be stated that about 
 86 per cent of infections with syphilis will give a positive 
 
130 THE WASSBBMASTBT TEST 
 
 reaction with the Wassermann test, if they are distributed 
 among the various stages as in the table given; that 10 
 per cent of primary cases will give a negative result ; about 
 5 per cent of secondary cases; about 13 per cent in the 
 tertiary stage; and about 30 per cent in the latent stage 
 of the disease. With good technic-and efficient antigenic 
 extracts one should obtain 90 per cent of positive results in 
 the primary stage of syphilis; 95 per cent of positive re- 
 sults in the secondary stage ; 87 per cent of po_sitive results 
 in the tertiary stage; and 70 per cent of positive results 
 •in the latent stages. 
 
 Results in the Primary Stage of Syphilis. — It is now a 
 generally accepted fact that the earlier a syphilitic infec- 
 tion is diagnosed the better will be the results of any 
 method of treatment that may be instituted. Thus the 
 early diagnosis of the disease becomes of paramount im- 
 portance and with the dark-field apparatus and the Was- 
 sermann test we are supplied with agencies that, if prop- 
 erly used, should enable us to make the diagnosis during 
 the early portion of the primary stage of the disease in 
 the vast majority of infections. 
 
 If possible ever^^ patient exhibiting a sore upon the 
 penis should be given the benefit of an examination of the 
 serum from the sore by the dark-field apparatus. If this 
 were done, the Treponema pallidum would be found in 
 most instances of true syphilitic infection, and thus the dis- 
 ease would be diagnosed at a very early stage. However, 
 the dark-field apparatus is often unobtainable and the ex- 
 amination can not be made, so that the question arises as to 
 the diagnostic value of the Wassermann test in the primary 
 stage of syphilis. If one were to believe some writers it 
 would be concluded that during this stage of syphilis the 
 Wassermann test is of very little value, as so many cases 
 give a negative reaction, but this has not been the writer's 
 experience with his modification of the test, for, as already 
 mentioned, practically 90 per cent of the primary cases 
 he has tested have given a positive result before the de- 
 
RESULTS IK VAKIOUS STAGES OF SYPHILIS 131 
 
 velopment of secondary symptoms. Therefore, if a dark- 
 field examination can liot be made, or is negative, it is 
 recommended that every patient presenting a suspicious 
 lesion upon the penis be given a "Wassermann test, for if 
 modern research upon syphilis has proved anything, it is 
 that the morphology of a sore upon the penis is of much 
 less value than formerly supposed, and that the typical 
 Hunterian chancre is almost as much the exception as the 
 rule. Mixed infections of chancre and chancroid, with the 
 resultant confusion in clinical pictures, have been quite 
 common in the writer's experience, and with the admis- 
 sions by the best authorities upon syphilis that it is often 
 impossible to diagnose syphilis from the appearance of the 
 initial lesion, the use of the Wassermann test and the dark- 
 field apparatus in every suspicious case is imperative. 
 
 From experience and experimental observations it is 
 certain that a syphilitic infection must exist for a certain 
 length of time in man before the substance or substances 
 which give rise to a positive Wassermann reaction appear 
 in the blood. Just how long this is will vary with the viru- 
 lence of the infection and the resisting power of the pa- 
 tient, but it is beyond controversy that sometimes the test 
 proves positive a very few days after the appearance of the 
 initial lesion, although this occurs but rarely. The writer 
 has observed and reported" one case in which the reaction 
 occurred only five days after the appearance of the chancre 
 and two cases in which it occurred after eight days of the 
 appearance of the initial lesion. As will appear in Table 
 XXIX, no less than 27 of 77 cases, or 36 per cent of 
 primary cases tested by the writer, gave a positive reaction 
 during the first week after the chancre appeared and al- 
 most 60 per cent during the second week after the appear- 
 ance of the chancre. It is thus evident, that even very 
 early in the primary stage of syphilis a positive Wasser- 
 mann reaction may occur, and thus the test will prove of 
 value in diagnosis. 
 
 As would be expected, the statistics given by different au- 
 
132 
 
 THE WASSBEMANlSr TEST 
 
 thorities regarding the percentage of positive results in 
 the primary stage of syphilis vary greatly, especially those 
 published shortly after Wassermann's original paper upon 
 the subject. Kolmer" states that in general, in primary 
 syphilis positive results will be found in from 80 to 90 
 per cent of the cases tested, and the writer agrees with 
 this statement, as it has been his experience that when 
 properly performed the Wassermann test should give at 
 least this nuniber of positive results. Table XXVII gives 
 the results obtained by different observers in the primary 
 stage of syphilis : 
 
 Table XXVII 
 
 KESTJLTS of the COMPLEMENT-mXATION TEST JOE SYPHILIS IN THE PeIMART 
 
 Stage op the Disease 
 
 NAME OF OBSERVEK 
 
 NO. OF CASES ' 
 
 POSITIVE 
 
 % POSITIVE 
 
 Arning 
 Bering 
 Boas 
 
 48 
 
 56 
 
 50 
 
 25 
 
 47 
 30 
 
 60 
 84 
 60 
 
 Bruek-Stern 
 
 27 
 
 13 
 
 48.5 
 
 Fischer-Meier 
 
 8 
 
 6 
 
 75 
 
 Grosser 
 
 20 
 
 19 
 
 95 
 
 Hancken 
 
 17 
 
 15 
 
 88 
 
 Craig 
 Kaplan 
 
 1080 
 138 
 
 970 
 125 
 
 89.8 
 90 
 
 Noguehi 
 Swift 
 
 70 
 16 
 
 65 
 13 
 
 92.8 
 81 
 
 Vedder 
 
 242 
 
 178 
 
 73.5 
 
 From this table it is evident that the percentage of posi- 
 tive results with the Wassermann test in the primary stage 
 of syphilis has varied greatly in the hands of different ob- 
 servers, the percentage being as low as 48 per cent and as 
 high as 95 per cent. However, as already stated, from 80 
 to 90 per cent of the usual run of primary infections com- 
 ing to the physician should give a positive reaction. Of 
 course, if tests were made upon a very large number of 
 very early infections in a series reported, the percentage 
 would be mu6h lower than if cases were tested from two 
 to three weeks after the appearance of the initial lesion, 
 but the writer's experience has been that at least 80 per 
 cent of cases of primary syphilis should give a positive 
 
RESULTS IN VARIOUS STAGES 01* SYPHILIS 133 
 
 Wassermaim test and it is believeji that, in the army, the 
 initial lesion is reported to the surgeon at least as soon 
 as it is, in the vast majority of cases, in civil life. 
 
 It is true that the percentage of positive results with this 
 test varies more in the primary stage of syphilis than in 
 other stages, with the possible exception of the latent stage, 
 due to many reasons, among the most important being 
 that the test is made within a very few days of the ap- 
 pearance of the initial lesion or early treatment often ob- 
 scures the reaction at this stage of the disease. The 
 writer has often observed negative cases become positive 
 with the cessation of treatment, during the primary stage, 
 and hundreds of instances in which, in untreated cases, 
 a negative reaction became positive after an interval of 
 days or weeks, and before the occurrence of secondary 
 symptoms. It should be remembered, however, that a cer- 
 tain proportion of negative results must be expected in the 
 primary stage of syphilis and that a negative result is no 
 proof that a suspicious lesion is not syphilitic. 
 
 Time of Appearance of the Reaction 
 
 The date after infection with syphilis that one may ex- 
 pect the Wasseitmann reaction to be positive is a point of 
 great practical interest, for the earlier treatment is com- 
 menced in syphilis the better are the therapeutic results, 
 and unless the Wassermann test can assist in diagnosis 
 early in the disease, it loses much of its value as a diag- 
 nostic agent. While the ideal method of diagnosing syph- 
 ilis in the primary stage is by the demonstration of Tre- 
 ponema pallidum with the dark-field apparatus, this meas- 
 ure is not infrequently unsuccessful, especially if local 
 treatment has been given the initial lesion, and, in addi- 
 tion, comparatively few practitioners possess the appara- 
 tus. The demonstration of the treponema in stained prep- 
 arations or by the India ink method should always be at- 
 tempted but these methods are also often unsuccessful, so 
 
134 
 
 THE WASSBRMANN TEST 
 
 that one must depend upon the Wassermann test fre- 
 quently for an early diagnosis. 
 
 Contrary to the opinion of some observers, the writer 
 believes that this test is a most valuable aid to the diag- 
 nosis of syphilis even thus early in the disease, for, in his 
 experience, nearly 90 per cent of primary cases tested by 
 him have shown a positive result before the appearance of 
 secondary symptoms, and while a single test performed at 
 a very early date after the appearance of the chancre will 
 be negative in a very large proportion of cases, repeated 
 tests will prove that the reaction becomes positive before 
 the appearance of secondary s^Tnptoms in most patients. 
 
 In Table XXVIII Vedder" shows the results of the ex- 
 amination of cases of syphilis in the primary stage by both 
 the dark-field apparatus and the Wassermann test, and it 
 is of interest as demonstrating that both methods fail, at 
 times, to aid in the diagnosis of the disease. 
 
 Table XXVIII 
 Result of Examination of Pkimaky Cases of Syphilis by Dark-Field and 
 
 THE WASSEEMANN TEST* 
 
 ddeation of 
 
 DAKK- 
 
 WASSER- 
 
 DURATION OF 
 
 DARK- 
 
 WASSER- 
 
 sore 
 
 FIELD 
 
 MANN 
 
 SORB 
 
 FIELD 
 
 MANN 
 
 2 days 
 
 + 
 
 _ 
 
 1 month 
 
 _ 
 
 + + 
 
 3 days 
 
 + 
 
 _ 
 
 7 days 
 
 + 
 
 + + 
 
 7 days 
 
 + 
 
 _ 
 
 9 days 
 
 + 
 
 + 
 
 7 days 
 
 + 
 
 - 
 
 14 days 
 
 -1- 
 
 -1- 
 
 10 days 
 
 + 
 
 _ 
 
 15 days 
 
 + 
 
 -1- 
 
 2 weeks 
 
 » + 
 
 _ 
 
 1 month 
 
 + 
 
 + + 
 
 2 weeks 
 
 + 
 
 _ 
 
 1 month 
 
 + » 
 
 + 
 
 3 weeks 
 
 + 
 
 + - 
 
 1 month 
 
 -1- 
 
 4-4- 
 
 3 weeks 
 
 + 
 
 _ 
 
 1 month 
 
 -1- 
 
 4-4- 
 
 3 weeks 
 
 + 
 
 _ 
 
 1 mouth 
 
 + 
 
 4-4- 
 
 1 month 
 
 + 
 
 _ 
 
 1 month 
 
 + 
 
 4-4- 
 
 1 week 
 
 — 
 
 + + 
 
 1% months 
 
 + 
 
 4-4- 
 
 1 week 
 
 _ 
 
 + 
 
 2 months 
 
 + 
 
 4-4- 
 
 2 weeks 
 
 _ 
 
 + + 
 
 1 month 
 
 _ 
 
 _ 
 
 3 weeks 
 
 _ 
 
 + 
 
 1 month 
 
 _ 
 
 _ 
 
 1 month 
 
 - 
 
 + + 
 
 
 
 
 *The sign + -f equals absolute inhibition of hemolysis, the four-plus reaction of many 
 laboratories. 
 
 Table XXVTII is conclusive evidence of the value of the 
 Wassermann test as an aid in the diagnosis of syphilis in 
 
RESULTS IN VARIOUS STAGES OF SYPHILIS 
 
 135 
 
 the primary stage of syphilis. It will be noted that in no 
 less than three cases the Wassermann test was positive 
 one week after the appearance of the chancre and that in 
 two of these cases the dark-field examination Avas negative. 
 The table also demonstrates that in every case of primary 
 syphilis both a dark-field examination and a Wassermann 
 test should be made, if possible. 
 
 Table XXIX, compiled from personal tests, gives the 
 date of appearance of the positive reaction in periods of 
 weeks after the appearance of the initial lesion. Six him- 
 dred cases of primary syphilis are here considered and the 
 Wassermann tests were performed by the writer in the 
 ordinary course of routine examinations in the army. A 
 great many more cases of primary infection were really 
 tested but are not included for the reason that we could 
 not be sure of the data regarding the date of appearance of 
 the initial lesion. 
 
 Table XXIX 
 
 Date of Appearance of Wassermann Reaction in Weeks in 600 Cases of 
 
 Primary Syphilis 
 
 ■WEEK AFTER 
 
 
 
 
 
 
 appearance 
 
 total cases 
 
 positive 
 
 % 
 
 negative 
 
 % 
 
 of chancre 
 
 
 
 
 
 
 First week 
 
 77 
 
 27 
 
 86.3 
 
 50 
 
 64.9 
 
 Second week 
 
 155 
 
 92 
 
 59.3 
 
 63 
 
 40.3 
 
 Third week 
 
 158 
 
 109 
 
 68.9 
 
 49 
 
 31. 
 
 Fourth week 
 
 167 
 
 129 
 
 77.2 
 
 38 
 
 22.7 
 
 Fifth week 
 
 43 
 
 35 
 
 81.3 
 
 8 
 
 18.6 
 
 From this table it is evident that, in the writer's ex- 
 perience, 36 per cent of cases of primary syphilis have 
 given a positive Wassermann reaction by the end of the 
 first week after the appearance of the initial lesion ; almost 
 60 per cent by the end of the second week; almost 70 per 
 cent by the end of the third week ;' over 77 per cent by the 
 end of the fourth week ; and over 80 per cent by the end of 
 the fifth week after the appearance of the chancre. 
 
 It is also evident that this test is of very distinct value 
 in the diagnosis of syphilis during the primary stage of 
 
136 THE WASSEEMANN TEST 
 
 the disease, and should always be resorted to if a dark- 
 field examination is impossible or gives a negative result. 
 A single negative test, at this stage especially, should not 
 be considered as proving that a suspected lesion is non- 
 syphilitic, but repeated tests should be made before a pa- 
 tient is assured that the condition present is not due to 
 syphilis. In the army, owing to the situation of the va- 
 rious Department Laboratories, the Wassermann test is al- 
 ways available and suspected cases can be examined at 
 weekly intervals, a practice which enables the army sur- 
 geon to establish the diagnosis very early in the disease, 
 but in civil practice this is generally impossible, owing to 
 the cost of the test. However, whenever possible, repeated 
 Wassermann tests should be made upon patients suspected 
 of presenting the primary lesion of syphilis. 
 
 As regards the question as to how early the positive 
 Wassermann may appear in the blood after the primary 
 lesion appears, there are a considerable number of obser- 
 vations upon record showing that positive reactions have 
 been obtained as early as the third and fourth days after 
 the appearance of the chancre, and a few in which a posi- 
 tive reaction has been reported even before the appearance 
 of the initial lesion. In view of our knowledge of the na- 
 ture of the syphilitic poison it may be stated that one must 
 regard with suspicion the reports of a positive Wasser- 
 mann before a primary lesion was observed, for it is prac- 
 tically impossible to rule out previous syphilitic disease 
 in most patients and unless this can be done or it can be 
 definitely proved that the lesion supposed to be primary 
 in such cases is really so, by the demonstration of Tre- 
 ponema pallidum, the evidence can not be considered as con- 
 clusive. / 
 
 However, it may be accepted as proved beyond a doubt 
 that the Wassermann test is not so very infrequently posi- 
 tive by the end of the first week after the appearance of 
 the chancre, and the writer has observed two cases in which 
 it was positive five days after the sore was first noticed, 
 
EESULTS IIS" VARIOUS STAGES OF SYPHILIS 137 
 
 and in both cases Treponema pallidum was demonstrated 
 in sernm from the lesions. Well authenticated cases are 
 on record in which the test became positive two or three 
 days after the initial lesion was noted, so that the possi- 
 bility of a positive Wassermann test before the appearance 
 of such a lesion can not be denied, although it must be ex- 
 ceedingly rare. 
 
 It is now generally conceded that Avhen the primary le- 
 sion of syphilis appears the disease has already become a 
 generalized infection, so that it is not difficult to under- 
 stand why a positive Wassermann reaction may occur very 
 soon after the chancre appears, and the variation that oc- 
 curs as to the time of the appearance of the positive re- 
 action in different patients must depend upon the virulence 
 of the treponema and the reaction of the tissues of the 
 patient to the organism. The writer has observed many 
 cases in which repeated Wassermann tests always proved 
 negative, though there was no question of the presence of 
 syphilis and marked lesions were present at the time that 
 the tests were made, so that in a certain percentage of 
 cases of syphilis it is probable that a positive reaction will 
 never be obtained. The writer has also observed two pa- 
 tients in whom the primary lesion of syphilis was present, 
 as demonstrated by the finding of Treponema pallidum by 
 the dark-field, and whose blood serum gave a positive re- 
 action prior to the development of secondary symptoms, but 
 in whom no secondary synaptoms ever developed, although 
 they were under observation for nearly one year, and in 
 whom the Wassermann reaction became negative, although 
 no specific treatment Avas ever administered. In his opin- 
 ion, these cases demonstrate that syphilis is sometimes 
 spontaneously cured, and it is not improbable that this 
 occurs much more frequently than is generally supposed. 
 
 In Ariew of the reports of positive Wassermann reactions 
 occurring before the initial lesion appears, the recent work 
 of Woods and Morris" upon complement fixation in 
 trypanosomiasis is very suggestive. They found that with 
 
138 THE WASSEEMANN TEST 
 
 an antigen prepared from the spleen of a rat heavily in- 
 fected with Trypanosoma equiperdum, complement fixa- 
 tion usually appeared in an infected animal's blood serum 
 within eight days after inoculation, usually at the time 
 that the trypanosomes appeared in the blood of the infected 
 animal, but sometimes even before this occurred. In every 
 instance, however, complement fixation preceded the ap- 
 pearance of sjTuptoms of the infection. 
 
 Results in the Secondary Stage of Syphilis. — ^It is in the 
 secondary stage of syphilis that the highest percentage of 
 positive results are obtained with the Wassermann re- 
 action. The per cent of positive reactions reported by va- 
 rious observers varies somewhat, but it may be stated that 
 in this stage of the disease practically 95 per cent of the 
 patients should give a positive result, provided, of course, 
 that treatment has not been administered, but even in such 
 cases one would expect from 90 to 95 per cent of positive 
 results. Kolmer" states that in untreated secondary syph- 
 ilis he has never obtained a negative result and Boas*° in 
 437 untreated secondary cases obtained 100 per cent of 
 positive reactions. The writer, in 2,217 cases of secondary 
 syphilis, many of whom had received some specific treat- 
 ment, obtained 2,132 positive results, or 96.1 per cent, and 
 Vedder,^^ in 310 secondary cases of similar nature, obtained 
 91 per cent of positive results. 
 
 Table XXX gives the results obtained in the secondary 
 stage of the disease by various observers : 
 
 From this table it will be noted that the percentage of 
 positive results in the secondary stage of syphilis varies, 
 according to different observers, from 79 per cent to 100 
 per cent, but it should be remembered that many of the 
 cases tested by one observer may have had specific treat- 
 ment, while those tested by another had had no treatment 
 whatever, so that the low percentages reported can not be 
 considered as giving any indication of the real value of the 
 test in the absence of data regarding specific treatment. 
 
RESULTS IN VARIOUS STAGES OF SYPHILIS 
 
 139 
 
 Table XXX 
 
 The Results op the Complement-fixation Test for Syphilis in the 
 Secondaky Stage of the Disease 
 
 OBSEEVEE 
 
 no. op cases 
 
 positive 
 
 % positive 
 
 Ai-ning 
 
 107 
 
 99 
 
 93 
 
 Bering 
 
 113 
 
 111 
 
 98 
 
 Bruck-Stern 
 
 115 
 
 101 
 
 87.1 
 
 Blumenthal-Koscher 
 
 131 
 
 130 
 
 99 
 
 Craig 
 
 2217 
 
 2132 
 
 96.1 
 
 Hoene 
 
 376 
 
 260 
 
 79.1 
 
 Lesser 
 
 204 
 
 186 
 
 91 
 
 Lederman 
 
 110 
 
 108 
 
 98 
 
 Merz 
 
 377 
 
 366 
 
 97 
 
 Noguehi 
 
 197 
 
 190 
 
 96 
 
 Schonnefeld 
 
 112 
 
 112 
 
 100 
 
 Swift 
 
 76 
 
 70 
 
 92 
 
 Vedder 
 
 310 
 
 285 
 
 91.9 
 
 As already stated, the percentage of positive results in 
 untreated cases of secondary syphilis should be something 
 over 95 per cent, and unless as high a percentage as this 
 is obtained the complement-fixation system used should be 
 carefully studied for the occurrence of possible errors, as 
 the Wassermann teat, as at present performed, should not 
 be given a lower percentage of positive results in the 
 secondary stage of syphilis than that mentioned. 
 
 A negative reaction in this stage of syphilis, when there 
 is a clear history of infection and lesions are present, is 
 of no value whatever in excluding the disease ; and as nega- 
 tive reactions may occur, even when the test is repeated 
 many times during this stage of the disease, the diagnosis 
 of syphilis should be made, in such instances, without ref- 
 erence to the result of the test. It should also be remem- 
 bered that the severity of the lesions present has nothing 
 to do with the type of reaction obtained, for cases with 
 very mild symptoms will frequently react as strongly as 
 those showing severe lesions, and not infrequently a pa- 
 tient presenting the most pronounced secondary eruption 
 and mucous patches will give a weak Wassermann reaction, 
 which, unless repeated, might be interpreted as doubtful. 
 However, in these cases repeated Wassermann tests almost 
 
140 
 
 THE WASSBRMANN TEST 
 
 invariably result in the appearance of a strong positive re- 
 action. 
 
 Results in the Tertiary Stage of the Disease.— Perhaps 
 in no other stage of syphilis has the Wassermann test 
 proved as valuable as in the tertiary stage, and especially 
 in those cases in which there is involvement of the central 
 nervous system. The percentage of positive results ob- 
 tained will vary, of course, with the amount of treatment 
 that has been given the patient, for in a large majority 
 of the cases of tertiary syphilis, observed in practice, a 
 considerable amount of specific treatment has been admin- 
 istered prior to the time that the blood was collected for 
 a Wassermann test. If cases are tested that have received 
 no specific treatment and in which active tertiary lesions 
 are present, the percentage of positive results should be 
 above 95 per cent, but in the usual run of cases examined 
 the Wassermann test, if properly applied, should give posi- 
 tive results in from 80 to 85 per cent of cases examined. 
 The writer, in 728 cases of tertiary syphilis obtained 633 
 positive results, or 87.4 per cent, using the method recom- 
 mended in this work, and Vedder,*^ using the same method, 
 obtained 227 positive reactions in 263 cases tested, or 86.3 
 per cent. 
 
 Table XXXI gives the results reported by various ob- 
 
 Table XXXI 
 
 Kestjlts or THE Complement-fixation Test for Syphilis in the Tertiary 
 Stage of the Disease 
 
 OBSERVER 
 
 NO. OF CASES TESTED 
 
 POSITIVE 
 
 % POSITIVE 
 
 Arning 
 
 30 
 
 27 
 
 90 
 
 Bering 
 
 45 
 
 37 
 
 82 
 
 Bruck-Stem 
 
 47 
 
 27 
 
 57 
 
 Fleishman 
 
 41 
 
 40 
 
 98 
 
 Hohne 
 
 33 
 
 21 
 
 63 
 
 Craig 
 
 728 
 
 633 
 
 87.4 
 
 Lederman 
 
 78 
 
 75 
 
 96 
 
 Lesser 
 
 131 
 
 119 
 
 90 
 
 Merz 
 
 158 
 
 127 
 
 80.3 
 
 Noguehi 
 
 177 
 
 159 
 
 89.9 
 
 Swift 
 
 45 
 
 37 
 
 80 
 
 Vedder 
 
 263 
 
 227 
 
 86.3 
 
RESULTS IN VARIOUS STAGES OF SYPHILIS 141 
 
 servers of the complement-fixation test for syphilis dur- 
 ing the tertiary stage of the disease. 
 
 From this table it is evident that the results of the "Was- 
 sermann test in the tertiary stage of syphilis varied from 
 57 per cent to 96 per cent, according to various observers. 
 This great variation in the results may probably be ex- 
 plained by certain bf the observers including under the 
 tertiary stage cases tested, 1, cases without definite symp- 
 toms; 2, cases that had received specific treatment within 
 a recent date or were receiving treatment; and 3, cases in 
 which tertiary symptoms had never appeared. It is evi- 
 dent that, until statistics are based upon similar cases a 
 great deal of variation must be expected in the results of 
 this test in the hands of different observers. 
 
 In syphilitic disease of the blood vessels, as aortitis, 
 aortic aneurism, and aortic insufficiency, the Wassermann 
 test has proved of the very greatest diagnostic value and 
 in syphilitic disease of the central nervous system the test 
 is invaluable. This subject will be discussed more fully 
 in Chapter X. - 
 
 Results in the Latent Stages of the Disease. — The Was- 
 sermann test in the latent stages of syphilis has been of 
 the greatest use in indicating that treatment was neces- 
 sary in patients thought to be cured of the infection be- 
 cause of the absence of symptoms for long periods of time. 
 There is probably no disease occurring commonly in the 
 practice of the physician which so often exhibits intervals 
 during which clinical symptoms are absent as does syph- 
 ilitic' infection and often a positive Wassermann reaction 
 is the only tangible evidence of the disease. The patho- 
 logical investigations of careful workers, as Warthin, have 
 shown that the human body may be extensively invaded by 
 Treponema pallidum without clinical symptoms being evi- 
 dent and with a positive Wassermann reaction as the only 
 evidence of such infection, and one of the most valuable of 
 assets of the Wassermann test is its capacity for detecting 
 such infections. Of course, the test is frequently negative 
 
142 
 
 THE WASSEEMANN TEST 
 
 in this class of patients, as would be expected, for, as the 
 writer has shown, a certain amount of the substance or 
 substances causing complement fixation in syphilis must 
 be present in order to accomplish this result, and in the 
 latent cases of syphilis a sufficient amount is not present 
 in a considerable percentage of cases, as shown by the re- 
 sults of the test. 
 
 The writer has tested 1,525 cases of latent syphilis, with 
 a positive reaction in 1,039, or 68 per cent, using the method 
 of performing the test recommended in this work. Ved- 
 der,®^ using the same method, tested 114 cases Avith a posi- 
 tive result in 92, or 80.7 per cent. Boas,** in 363 cases of 
 early latent syphilis obtained positive results in only 40 
 per cent of the cases, while in older cases he obtained posi- 
 tive results in but 22 per cent of treated cases but in cases 
 treated carelessly the positive results equaled 74 per cent. 
 
 Table XXXII gives the results of the Wassermann test 
 obtained by the observers mentioned in the latent stage of 
 syphilitic infection. 
 
 Table XXXII 
 
 KESTJLT of the COMPLEMENT-riXATION TEST FOR SYPHILIS IN THE LATENT 
 
 Stages of the Disease 
 
 OBSERVER 
 
 NO. OF CASES tested 
 
 POSITIVE 
 
 % POSITIVE 
 
 Bering 
 
 147 
 
 75 
 
 48 
 
 Grosser 
 
 35 
 
 12 
 
 33.3 
 
 Craig 
 
 1525 
 
 1039 
 
 68.1 
 
 Fox 
 
 54 
 
 25 
 
 46 
 
 Lederman 
 
 78 
 
 36 
 
 46 
 
 Noguchi 
 
 265 
 
 206 
 
 77.7 
 
 Swift 
 
 39 
 
 25 
 
 64 
 
 Vedder 
 
 114 
 
 92 
 
 80.7 
 
 As would be expected, there is a great variation in the 
 number of positive reactions obtained with the test in the 
 latent stages of syphilis, as shown in this table. When it 
 is remembered that in most of the cases tested specific 
 treatment had been given for a longer or shorter period 
 of time ; that there were no sjTnptoms of the disease pres- 
 ent at the time the blood was tested ; and that in some the 
 
RESULTS IN VARIOUS STAGES OF SYPHILIS 143 
 
 only history of a syphilitic infection was a doubtful one; 
 the low percentage of positive results sometimes obtained 
 in this class of cases can be understood. In the writer's 
 cases there was always a clear history of infection but in 
 the majority of the patients some specific treatment had 
 been taken, so that the percentage of positive results ob- 
 tained ; i. e., 68 per cent, may be considered the average 
 percentage obtained in latent infections in such cases. 
 
 The value of the Wassermann test in the diagnosis of 
 syphilis in the latent periods of the disease can not be 
 overestimated either from the sociological or therapeutic 
 standpoint. The sociological aspect of latent syphilitic in- 
 fection will be treated of more fully in the discussion of the 
 subject of Wassermann surveys, but it can not be too 
 strongly emphasized that there is a very large amount of 
 syphilitic infection present in all countries that is never 
 recognized and it has only been within very recent years 
 that, because of researches upon various classes of the 
 population by means of the Wassermann test, it has been 
 realized to what a great extent this insidious disease has 
 invaded society. 
 
 From the therapeutic standpoint, the Wassermann test 
 is most valuable in the latent stage of syphilitic infection 
 because a positive reaction is the only index which we pos- 
 sess as to the existence of the infection and the necessity for 
 specific treatment. The test is often positive even after 
 specific treatment has been administered for months and 
 years, and the vast majority of patients treated with mer- 
 cury, before the days of salvarsan, and supposed to have 
 been cured, will give a positive reaction with the Wasser- 
 mann test. Some authorities have gone so far as to state 
 that they do not believe that any syphilitic infection was 
 ever cured by mercury alone, because of the revelations of 
 the Wassermann test in latent infections, but the writer 
 believes this to be too strong a statement, although it must 
 be admitted that the cases proving the contrary are com- 
 paratively few in number. 
 
144 
 
 THE WASSEEMANK" TEST 
 
 Results in Congenital Syphilis. — The results of the Was- 
 sermann test in congenital syphilis vary with the character 
 of the case tested. In children showing the lesions of 
 syphilis at birth the test gives 100 per cent of positive 
 results, in the writer's experience, but in children exhib- 
 iting tho lesions of late congenital syphilis the percentage 
 of positive results is lower, averaging from 80 to 85 per 
 cent. In still older individuals in whom the disease is 
 supposed to be congenital the percentage of positive re- 
 sults will not exceed 70 to 75 per cent. 
 
 Table XXXIII gives the results obtained by several ob- 
 servers with the Wassermann test in congenital syphilis: 
 
 Table XXXIII 
 
 Results of the Complement-fixation Test foe Syphilis in Congenital 
 
 Syphilis 
 
 observer 
 
 NO. or cases tested 
 
 POSITIVE 
 
 % POSITIVE 
 
 Arning 
 
 5 
 
 3 , 
 
 60 
 
 Craig 
 Jesionek-Meirosky 
 
 28 
 
 25 ■ 
 
 82.2 
 
 18 
 
 16 
 
 88.8 
 
 Noguehi 
 
 17 
 
 17 
 
 100 
 
 Vedder 
 
 128 
 
 123 
 
 96 
 
 The variation obtained by different observers in the re- 
 sults of the Wassermann test in congenital syphilis are 
 well sho^^Ti in the table and depend upon the causes already 
 mentioned. The recent work of Veeder®' upon the subject 
 is of interest. He studied 100 syphilitic families with the 
 following results: In the 100 families, 331 pregnancies 
 occurred resulting as follows: abortions, 100, or 30.2 per 
 cent; stillbirths, 31, or 9.3 per cent; living births, 200, or 
 60.5 per cent. Of the 200 living births, 39 of the children 
 had died at the time of the investigation and of the 161 
 living children, 12 died during the course of the investiga- 
 tion. Of the 161 tested, 107 had both clinical symptoms 
 of syphilis and positive Wassermann reactions; 5 were 
 clinically , positive but gave negative Wassermann tests; 
 16 were clinically negative but gave positive reactions, 
 leaving 33 children who were both clinically negative and 
 
RESULTS IN VARIOUS STAGES OE SYPHILIS 145 
 
 who gave a negative Wassermann test. In this series of 
 eases of congenital syphilis in young children born of 
 syphilitic parents Veeder obtained 96 per cent of positive 
 results, and his work well illustrates the value of the test 
 in the diagnosis of congenital infection. 
 
 The Wassermann test has changed our conception of 
 CoUes' and Prof eta's laws and has explained these appar- 
 ent contradictions in the etiology of syphilis. CoUes' law, 
 that an apparently healthy mother of a syphilitic child 
 may suckle the child with impunity, even though it pre- 
 sents the most infectious lesions of the disease, is ex- 
 plained by the fact that the mother, although showing none 
 of the lesions of syphilis is really in the latent phase of 
 the disease, as demonstrated by the positive Wassermann 
 reaction that is almost invariably obtained with the blood 
 of such mothers. Prof eta's law, that the child born of a 
 syphilitic mother, but presenting no evidence of the dis- 
 ease, may suckle its mother with impunity, has also been 
 shown by the Wassermann test to be due to latent syphilis 
 in the child, as in these children the test is generally pos- 
 itive although no symptoms of syphilis are present and 
 none may appear for long periods of time. In other words, 
 children born of syphilitic parents possess no immunity 
 to syphilis as was long believed, nor does a mother who 
 with impunity suckles her syphilitic child, possess an im- 
 munity, but is herself syphilitic. 
 
 Just what part late hereditary syphilis plays in the nu- 
 merous instances in which a positive Wassermann reaction 
 is found in patients who deny any syphilitic infection and 
 who may, or may not, present evidences of the disease, is 
 still a mooted question. In the writer's experience it has 
 not infrequently occurred that patients who are absolutely 
 truthful and who have given a positive Wassermann re- 
 action, have denied even any exposure to infection or any 
 lesions that could be considered syphilitic, and there has 
 been no reason to doubt the truth of their statements. In 
 
146 THE WASSERMANN ^EST 
 
 such cases, especially if there are no lesions of the dis- 
 ease present, one should be very careful to eliminate any 
 condition that has been proved to give a positive Wasser- 
 mann reaction, but after this has been done the explanation 
 of the reaction is often very difficult and even impossible. 
 That hereditary syphilis, latent in character, is the cause 
 of some of these reactions can not be doubted but the 
 writer is loth to believe that most of them are due to such 
 a cause. It is more probable that an accidental infection 
 may have occurred in infancy or childhood, the initial le- 
 sion of which went unrecognized, than that the positive 
 test is due to a hereditary infection that has never caused 
 symptoms or lesions of syphilis. 
 
 The Specificity of the Wassermann Reaction 
 
 While it must be admitted that the Wassermann reac- 
 tion is not absolutely specific for syphilis, for positive 
 results have sometimes been observed with other diseases, 
 the fact remains that a positive reaction with this test 
 indicates syphilis in so immense a proportion of individ- 
 uals giving it that the practical value of the reaction in 
 the diagnosis of syphilis is hardly at all decreased by the 
 comparatively few instances in which such a result is ob- 
 tained in other conditions. 
 
 If one reads the works of writers published shortly after 
 the discovery of the Wassermann test, the attention is at 
 once called to the large number of conditions other than 
 syphilis in which positive reactions are reported; but if 
 these writings be compared with those published by in- 
 vestigators during the past five years, it is evident that 
 most of the so-called positive reactions must have been 
 due to poor technic, for as our knowledge of the technic 
 has increased, these nonspecific reactions have decreased 
 in number, until today a positive reaction with the, test is 
 reported in but few conditions that are not due to syphilis. 
 Any serologist who reports any considerable number of 
 
EESTJLTS IN VARIOUS STAGES 0¥ SYPHILIS 147 
 
 positive reactions with the Wassermann test in diseases 
 other than syphilis proclaims that his technic is poor, for, 
 if properly performed, this test will give positive results 
 in very few nonsyphilitic conditions. 
 
 The earlier writers upon the suh.i'ect reported positive 
 results in such diseases as scarlet fever, septicemia, diph- 
 theria, carcinoma, and most of the acute infections; but 
 it is now well known that these conditions do not give a 
 positive reaction if the test is properly performed. There 
 is still an opinion prevalent among the profession that 
 the test is frequently positive in scarlet fever, but the work 
 of recent investigators proves conclusively that the test is 
 not positive in this disease but that when a positive reac- 
 tion does occur the child is syphilitic or syphilis can not 
 be excluded. Hecht, Lateiner, and Wilenko*" tested 105 
 cases of scarlet fever and obtained but one positive result, 
 and Kolmer*'' in 250 cases which he tested with both the 
 original "Wassermann method and the Noguehi modifica- 
 tion obtained positive reactions in only 5 cases, or 2 per 
 cent, and in these cases syphilis could not be excluded. 
 Browning and McKenzie^* examined 37 cases of scarlet 
 fever and did not obtain a positive reaction in one of them. 
 These results prove that the Wassermann test is not pos- 
 itive in scarlet fever and that the large proportion of pos- 
 itive results reported in the early literature of the test 
 must have been due to improper technic. 
 
 Another disease in which a considerable proportion of 
 positive reactions have been reported is tuberculosis. 
 Some authorities have reported as high as 30 to 40 per 
 cent of positive results in this disease but such reports are 
 absolutely unreliable and prove that the method of per- 
 forming the test must have been erroneous. The writer 
 has tested hundreds of cases of tuberculosis and while a 
 few have shown a positive reaction, syphilis could not be 
 excluded in any case, and the majority of the patients ad- 
 mitted infectioii. There are many instances on record of 
 syphilis of the lung existing along with tuberculosis of 
 
148 THE WASSERMAITN" TEST 
 
 the same organ and many in which syphilis of the lung was 
 diagnosed as tuberculosis, and it is these cases that have 
 caused the impression that tuberculosis frequently gives 
 a positive reaction with this test, together with the large 
 number of latent syphilitic infections which are found in 
 patients suffering from other diseases. There is also con- 
 siderable evidence to prove that tuberculosis renders an 
 individual much less resistant to infection with Treponema 
 pallidum and this probably has much to do with the prev- 
 alence of the disease in the tubercular. However, that 
 tuberculosis is a disease which causes a positive Wasser- 
 mann reaction can not be longer maintained in view of 
 the negative results of the best observers as reported in 
 the most recent literature. 
 
 There are, however, a few diseases in which a positive 
 Wassermann reaction is sometimes obtained and where 
 the most careful examination fails to disclose any evi- 
 dences of syphilitic infection and the most careful in- 
 quiries any history of the disease. These diseases are 
 yaws or frambesia; the tubercular type of leprosy; some 
 cases of relapsing fever ; some malarial infections, during 
 the febrile stage; and some instances of experimental try- 
 panosomiasis in animals. Of these, yaws is the most im- 
 portant because some of the lesions of this disease re- 
 semble those of syphilis and in the lesions the causative 
 organism, Treponema pertenue, may be found either by a 
 dark-field examination of the secretions from the lesion 
 or by staining methods. As this treponema is almost 
 indistinguishable morphologically from Treponema pal- 
 lidum, and the blood often gives a positive Wassermann 
 reaction, some confusion in diagnosis may result, but a 
 careful study of the case should enable the diagnosis of 
 yaws to be made from the clinical history and symptoms 
 alone. 
 
 The writer has observed positive reactions in five cases 
 of tertian malarial infection, during the febrile stage of 
 the paroxysm, the blood becoming negative during the 
 
EBSULTS IN VAEIOUS STAGES OF SYPHILIS 149 
 
 afebrile periods. Thompson*^ states that he has observed 
 positive results several times in malarial disease during 
 the stage of fever which became negative with the disap- 
 pearance of the fever and plasmodia, and our results have 
 been confirmed by many observers. On the other hand, 
 the occurrence of a positive reaction in malaria, even dur- 
 ing the paroxysm of fever, is comparatively rare, for the 
 writer has examined many cases in which the test was neg- 
 ative and other investigators report similar results. How- 
 ever, the microscopical examination of the blood should 
 suffice to diagnose malarial infections, so that the occur- 
 rence of a positive Wassermann reaction should cause no 
 trouble in the diagnosis of the disease. If the reaction 
 persists after the disappearance of the fever and Plas- 
 modia the diagnosis of syphilis should be made, for all 
 investigators report that the positive reaction in malaria 
 is only temporary in character. 
 
 In the tubercular forms of leprosy numerous positive 
 reactions have been reported but it is impossible to say 
 just how many of these were true false reactions and how 
 many were due to a complicating syphilitic infection. If 
 it be remembered that most of the cases of leprosy that 
 have been tested occurred in native races which were thor- 
 oughly syphilized the difficulty of ruling out that infection 
 can be appreciated. The writer believes that a positive re- 
 action does occur in tubjercular leprosy in certain stages 
 of the disease but he also believes that the majority of 
 the positive reactions reported in leprosy have been due 
 to a concurrent syphilitic infection. In the anesthetic form 
 of leprosy the "Wassermann test is generally negative, so 
 far as reports indicate. As tubercular leprosy can be 
 easily, diagnosed by the finding of the bacillus in the le- 
 sions a positive Wassermann reaction should occasion no 
 confusion in the diagnosis. 
 
 Cases of relapsing fever have been found to give a posi- 
 tive reaction and also some experimental infections with 
 trypanosomiasis but it is yet unsettled whether or not the 
 
150 THE WASSEEMANN TEST 
 
 blood of patients suffering from infection with Trypano- 
 soma gambiensi or other human trypanosomes gives a pos- 
 itive reaction. 
 
 Positive reactions have been reported in diabetes mel- 
 litus, in cases suffering from acidosis, and in pellagra, but 
 so far as the latter disease is concerned it is now definitely 
 proved that the Wassermann test gives only negative 
 results. 
 
 As regards the number of positive reactions occurring 
 in diseases other than syphilis it may be stated that, with 
 a properly performed and controlled method of making 
 the test, it will be so small as to be of practically no im- 
 portance from a diagnostic standpoint. The writer has ob- 
 tained 12 positive reactions among 4,000 individuals suffer- 
 ing from diseases other than syphilis or 0.3 of 1 per cent. 
 Of these patients five were suffering from tertian malarial 
 infection and gave a positive result during the febrile 
 stage, which became negative when the temperature re- 
 turned to normal; three were diagnosed as tuberculosis; 
 three as pityriasis rosea; and in one the diagnosis was 
 undetermined. In the pityriasis rosea cases the reactions 
 were almost double-plus (four-plus), but would not have 
 been reported as positive mthout a qualifying statement, 
 while in two of the tuberculosis cases a history of syphi- 
 litic infection was afterwards obtained, and the disease 
 could not be excluded in the remaining case. But, even 
 admitting that all of these cases gave a false positive re- 
 action, the real value of the test is but very little affected 
 as a diagnostic measure. Vedder®" has obtained results 
 almost identical with those of the writer, using the writ- 
 er's modification of the Wassermann test, as he obtained 
 only four positive reactions among 1,049 individuals suf- 
 fering from diseases other than syphilis. 
 
 When it is remembered how difficult it is to absolutely 
 exclude a syphilitic infection in any individual and the 
 comparatively few instances in which the Wassermann test 
 gives a positive result in diseases other than syphilis, one 
 
EESULTS IN VARIOUS STAGES OF SYPHILIS 151 
 
 should accept with great caution the reports of positive 
 reactions in such diseases, for the experience of those who 
 have worked with this test for years and in hundreds and 
 thousands of cases has been that a positive reaction, in the 
 overwhelming majority of instances, demonstrates the 
 presence of syphilis. There are a very few conditions in 
 which a positive reaction is obtained with this test and in 
 which syphilis can be eliminated, but as niost of them can 
 be easily diagnosed by laboratory and clinical methods, 
 there should be no confusion caused by the result of the 
 Wasserma^n test, but even in these cases a very careful 
 inquiry and examination should be made before a possible 
 complication of syphilis is excluded. 
 
 In surgical practice weak positive reactions are some- 
 times noted in blood sera collected shortly after ether and 
 chloroform anesthesia, so that the test should not be made 
 upon patients who have been anesthetized until at least 
 two days have elapsed since the anesthetic was admin- 
 istered. 
 
 In conclusion, it may be stated that the substance or 
 substances that cause a positive Wassermann reaction ap- 
 pear to be practically peculiar to the blood serum of pa- 
 tients suffering from syphilitic infection, and while it can 
 not be claimed that the test, when positive, is absolutely 
 specific of syphilis, from a practical standpoint it is doubt- 
 ful if a more specific test is employed in medicine, the 
 margin of error appearing to be less than five-tenths of 
 one per cent. 
 
CHAPTER VIII 
 
 THE INTERPRETATION OF THE RESULTS OF THE 
 WASSERMANN TEST. THE WASSERMANN- 
 TEST AS AN INDEX OF THE' PREVA- 
 LENCE OF SYPHILIS IN COMMUNITIES 
 
 The writer has often been impressed with the evident 
 misconceptions of manj practitioners of medicine regard- 
 ing the nature of the Wassermann reaction, the percentage 
 of positive results that may be expected in the various 
 stages of the disease, but, especially the exact significance 
 that may be attached to a positive or negative result. In 
 his experience, many patients have been told by their med- 
 ical attendant that they were free frdm syphilis, the state- 
 ment being based upon a single negative result with this 
 test, while others have been told that they were suffering 
 from the disease upon the strength of a plus or plus-minus 
 reaction, in the absence of either a history of infection or 
 of any symptoms of the disease. 
 
 Such interpretations of the results of the Wassermann 
 test are unwarranted and have brought the test into dis- 
 repute in certain localities. In addition, innocent individ- 
 uals have suffered great mental anguish from being un- 
 justly stigmatized as afflicted with syphilis, and others 
 have been infected by those who have been told that they 
 were free from the disease by some careless or ignorant 
 practitioner. Even today, almost ten years since the Was- 
 sermann test has been in general use by the profession, 
 there is a surprising amount of ignorance regarding the 
 interpretation of the various grades of the reaction, as re- 
 ported by different laboratories, and much of the fault 
 lies with the laboratory reports, for the terms strongly 
 positive, positive, and weakly positive, so commonly used, 
 
 152 
 
INTEKPRBTATION OF RESULTS 153 
 
 are misleading, unless they are carefully explained in ref- 
 erence to each case. In fact, the writer has always believed 
 that only three terms should be used in reporting the re- 
 sults of the Wassermann test ; i. e., positive, doubtful, and 
 negative, leaving it to the clinician to decide how much 
 weight should be attached to any grad§ of the reaction 
 which is reported as doubtful, and which, in the army, 
 would mean any degree of the reaction less than complete 
 inhibition of hemolysis. While there is no doubt that many 
 cases of syphilis react wealdy consistently, especially in the 
 early and late latent stages, never presenting complete in- 
 hibition of hemolysis, the writer believes that all cases show- 
 ing less than complete inhibition should be reported as 
 doubtful, and the interpretation of the reaction left with the 
 clinician. Of course, if there is a clear history of infection 
 and. typical clinical signs are present, the doubtful reaction 
 would at once, in the opinion of the clinician, be considered 
 as positive and as supporting the diagnosis of syphilis, but 
 if, on the other hand, there was no history of infection and 
 no symptoms are present, a doubtful Wassermann reac- 
 tion is not sufficient proof upon which to base a diagnosis 
 of syphilitic disease. 
 
 The statements here made regarding the interpretation 
 of the results of the Wassermann test are based upon over 
 50,000 tests personally performed by the writer, the major- 
 ity of them being reexaminations made as a control of the 
 treatment of the disease, the remainder being upon patients 
 presenting the symptoms common to the various stages of 
 syphilis. The percentage of positive and negative results 
 obtained in these cases has already been given (Chapter 
 VII), as weU as the terminology used in reporting the re- 
 sults, but it is well here to repeat the terminology used 
 in the army laboratories. 
 
 Four degrees of the reaction are noted in reports from 
 the army laboratories. A positive reaction is reported as 
 double-plus (+ +) and means that there was absolute inhibi- 
 tion of hemolysis. A doubtful reaction is reported as 
 
154 THE WASSERMANN TEST 
 
 plus (+) or plus-minus (+-), the former term indicating 
 that there was over 50 per cent inhibition of hemolysis, the 
 latter that there was less than 50 per cent inhibition of hem- 
 olysis. A negative reaction is reported as minus (-). In 
 most civilian laboratories the results of the Wassermann 
 test are reported as four-plus (+ + + +), three-plus (+ + +), 
 two-plus (++), plus (+), plus-minus (+-), and negative (-). 
 The four-plus reaction corresponds to the army double- 
 plus; the three-plus and two-plus to the army plits; and the 
 plus and plus-minus to the army plus-minus. 
 . These distinctions between the terminology used should 
 be borne in mind in the following discussion of the inter- 
 pretation of the results of the test. 
 
 The writer has already discussed the specificity of the 
 Wassermann reaction (Chapter VII) and there remains the 
 question as to the significance of the positive reaction. 
 Some authors have denied that a positive reaction with this 
 test indicates the presence of syphilis, claiming that after 
 the disease disappears the reaction still persists, and that 
 the positive yesult is simply an indication that the patient 
 has had syphilis sometime in the past. In the light of mod- 
 ern research upon the disease this theory can no longer be 
 considered as worthy of credence, for it is the unanimous 
 experience of those who have had the most to do with the 
 treatment of syphilis, as controlled by the Wassermann 
 test, that with the disappearance of the treponema and of 
 symptoms the reaction also disappears and that, in cases 
 that are reaUy cured, the reaction remains permanently 
 absent. That this is true has been proved experimentally 
 upon infected animals, and the disappearance of the reac- 
 tion in these animals after the administration of salvarsan 
 which has caused the disappearance of the treponemas, is 
 conclusive evidence that the positive reaction does not per- 
 sist after the cure of the syphilitic infection. 
 
 There are still others who maintain that, in the absence 
 of symptoms, a positive Wassermann reaction does not in- 
 dicate syphilis. To those who still hold to this opinion, the 
 
INTEKPEETATION 0¥ BESULTS 155 
 
 writer has only to say that all experience has shown that, 
 if the few conditions in which a positive result sometimes 
 occurs can be eliminated, a positive reaction means the 
 presence somewhere in the hody of living treponemas and 
 that the question of history or symptoms should have no 
 influence in deciding the advisability of treatment. It is 
 just this class of cases that later furnish the great bulk of 
 our patients suffering from paresis, tabes, aortitis, aneu- 
 rism, and syphilitic disease of the viscera, the latter often 
 only discovered at autopsy. In cases in which there is 
 good reason to question the possibility of syphilis the pa- 
 tient's blood serum should be titrated, for, as Vedder" 
 well says: 
 
 "One-tenth of a cubic centimeter of some sera is re- 
 quired to give a double-plus reaction, while one-thousandth 
 of a cubic centimeter of another serum may be sufficient 
 to give a double-plus reaction. There would be little hes- 
 itation in making a diagnosis of syphilis in the latter case 
 whether clinical symptoms were present or not. There 
 might be doubt in the former case." 
 
 As the interpretation of the results of the test varies in 
 the different stages of the disease, each stage will be con- 
 sidered separately. 
 
 The Interpretation of the Results of the Wassermann 
 Test in the Primary Stage of Syphilis. — ^It is in this stage 
 of the disease that the weaker grades of reaction are of 
 most value from a diagnostic standpoint. As would be ex- 
 pected, the earlier in the primary stage the blood is tested, 
 the larger will be the percentage of negative results and 
 also the larger the percentage of doubtful reactions. As 
 a certain amount of complement-binding substance must 
 be present to give a positive result, the longer the infection 
 has lasted the greater the chance that this amount is pres- 
 ent and that the test will be positive. This fact has al- 
 ready been shown in the table giving the date of the ap- 
 pearance of the positive Wassermann reaction in 600 cases 
 of syphilis, biit, with a clear iiistory of infection and a 
 
156 THE WASSBBMANN TEST 
 
 suspicious lesion, it is proper to consider as positive, in 
 this stage of the disease, reactions that would be always 
 doubtful at other stages. . , 
 
 A plus-minus , reaction (plus of most laboratories) 
 should be considered as a negative reaction, in most in- 
 stances, but if such a reaction is obtained within a week 
 or two after the appearance of the initial lesion it may be 
 considered as of some confirmatory value, but never as 
 diagnostic of syphilis. 
 
 A pltis reaction (+), the double-plus (++), and three-plus 
 (+ + +) reaction of other laboratories, when obtained in the 
 primary stage of syphilis, may be interpreted in practice 
 as a positive reaction, provided there is a history of ex- 
 posure and a suspicious lesion is present. The writer 
 often obtains this type of reaction in cases in which the 
 initial lesion has lasted for from one to three or four weeks, 
 and he has never seen a plus reaction in such cases become 
 negative, except as the result of treatment, and if un- 
 treated, the patients invariably developed secondary symp- 
 toms. In the absence of a history of infection or the initial 
 lesion a phis reaction should not be considered as diag- 
 nostic of syphilis. 
 
 A douhle-plus (+ +) reaction, the four-plus (+ + + +) re- 
 action of some laboratories, is always diagnostic of syph- 
 ilis in this, as in every other stage of the disease, provided 
 the other conditions that sometimes give such a reaction, 
 can be eliminated. This type of reaction, which means ab- 
 solute inhibition of hemolysis, in the writer's experience 
 has occurred in 14 per cent of the cases of primary syph- 
 ilis tested during the first week after the appearance of 
 the initial lesion; in 22 per cent tested during the second 
 week; in 41 per cent tested during the third week; in 53 
 per cent tested during the fourth week ; and in 61 per cent 
 of cases tested during the fifth week after the appearance 
 of the initial lesion. Eeactions so strong as to almost equal 
 complete inhibition of hemolysis occur in a large number of 
 
INTEEPRETATION OF EESULTS 157 
 
 cases of primary syphilis and these should be reported as 
 double-plus or four-plus reactions. In these, the super- 
 natant salt solution, after the undissolved corpuscles have 
 settled to the bottom of the test tube, shows a slight red- 
 dish yellow tinge, demonstrating that a small amount of 
 hemoglobin has been dissolved from the erythrocytes, but 
 practically such reactions are positive and should be re- 
 ported as such. 
 
 A negative reaction, in the primary stage of syphilis, is 
 of no value whatever in eliminating syphilis. Even though 
 it persists until the initial lesion disappears and during 
 the latent stage before the occurrence of secondary symp- 
 toms, it can not be relied upon as evidence that syphilis 
 does not exist. As the writer has already shown, at least 
 10 per cent of patients suffering from primary syphilis 
 will give a negative Wassermann reaction, so that, in the 
 presence of a suspicious lesion, the negative ^reaction is 
 of no value, and the patient should never be assured, upon 
 the strength of such a reaction, that his symptoms are not 
 due to syphilis. 
 
 As already emphasized, every suspicious lesion follow- 
 ing exposure, "Wj^hether situated upon the penis or else- 
 where, should, if possible, be examined with the dark-field 
 apparatus for Treponema pallidum. This should never be 
 neglected and a Wassermann test made in preference, for 
 the dark-field examination is the diagnostic method of 
 choice during this stage of the disease, but where such an 
 examination can not be made, or where it gives negative 
 results, a Wassermann test should always be performed. 
 The writer has observed many instances in which it was 
 impossible to demonstrate Treponema pallidum, either by 
 the dark-field apparatus or in stained preparations made 
 from the suspicious lesion, but in which the Wassermann 
 test was positive and thus the diagnosis was established. 
 In extraurethral chancres the test has been of the greatest 
 value in enabling us to make an early diagnosis and in the 
 latent period, after the healing of the initial lesion and be- 
 
158 THE WASSEEMANN TEST 
 
 fore the appearance of secondary symptoms, the Wasser- 
 mann test is the only method available for making a diag- 
 nosis. It is in this latter class of cases, where there is 
 only a history of a healed lesion to assist one, that the test 
 is of the very greatest clinical value in diagnosis and every 
 such case should be tested repeatedly, if possible, as treat- 
 ment applied before the appearance of secondary symp- 
 toms is much more efficient than when it is administered 
 afterward. 
 
 The Interpretation of the Results of the Wassermann 
 Test in the Secondary Stage of Syphilis. — ^In the secondary 
 stage of syphilis a positive result is obtained in from 95 
 to 96 per cent of all cases, so that in this stage of syphilis 
 the Wassermann test may be stated to be almost always 
 confirmatory of the clinical findings. However, from 4 to 
 5 per cent of secondary cases, in the writer's experience, 
 will give a negative reaction, even when repeatedly tested, 
 and when marked secondary lesions are present, so that 
 the mere absence of a positive reaction is no proof that 
 the patient is not suffering from the disease. 
 
 A double-plus {four-plus) reaction in patients present- 
 ing the lesions of secondary syphilis or lesions that are 
 suspicious of this stage of the disease, is conclusive evi- 
 dence of the presence of syphilis under the limitations al- 
 ready mentioned in discussing this same type of reaction 
 in the primary stage ; i. e., if the conditions which some- 
 times give a positive reaction can be eliminated in the 
 particular case examined. 
 
 A plus {two- or three-plus) reaction is not of as much 
 value in the secondary stage of syphilis as in the primary, 
 but even in this stage this type of reaction occurs in about 
 10 per cent of cases, in the writer's experience, and when 
 accompanied by suspicious symptoms, should be inter- 
 preted as a positive reaction. In the majority of such in- 
 stances the reaction is equal to the three-plus reaction 
 reported by most laboratories, inhibition of hemolysis be- 
 ing almost complete, but even if the reaction is less than 
 
INTERPEETATIOISr OP RESULTS 159 
 
 this it should be regarded as confirmatory of the presence 
 of the disease in patients presenting clinical symptoms or 
 a clear history of a primary lesion. 
 
 A plus-minus {plus or plus-minus) reaction in the sec- 
 ondary stage of syphilis is valueless from a diagnostic 
 standpoint. In these cases the diagnosis will have to rest 
 upon the clinical symptoms present or upon the history of 
 the case. On the other hand, the presence of this type of 
 reaction should not lead to any hope that the disease is 
 not present, as some secondary infections never give more 
 than a plus-minus reaction. 
 
 A negative reaction when suspicious symptoms of sec- 
 ondary syphilis are present is of greater value than at any 
 other stage of the disease, but it should be remembered 
 that practically five per cent of cases of secondary syph- 
 ilis give a negative reaction and that many of these cases 
 remain negative for weeks and months. It is only when 
 the negative reaction persists for several months and no 
 symptoms of syphilis are present during this time that it 
 may, with justice, be regarded as conclusive evidence of 
 the absence of syphilis. On the other hand, if the case 
 examined gives no history of a primary lesion and the 
 symptoms present are only slightly suspicious, a negative 
 reaction which persists for two or three months may be 
 regarded as proving the absence of syphilis, provided 
 treatment has not been administered in the meanwhile. 
 
 The Interpretation of the Results of the Wassermann 
 Test in the Tertiary Stage of Syphilis. — In the tertiary 
 stage of syphilis, when definite tertiary lesions are present, 
 the Wassermann test gives as high a percentage of posi- 
 tive results as in the secondary stage, but there are many 
 cases tested in this stage in which the symptoms are atyp- 
 ical or only slightly suspicious, or in which the nervous 
 system alone is involVed, which give weak or negative re- 
 actions, so that the total percentage of positive results in 
 this stage of the disease is slightly below that encountered 
 in the secondary stage. In many of these cases, also, spe- 
 
160 THE WASSBBMANN TEST 
 
 cific treatment has been administered and although not 
 successful in preventing the development of lesions, it may 
 have reduced markedly the strength of the Wassermann 
 reaction. 
 
 A double-plus (four-plus) reaction with the test in the 
 tertiary stage of syphilis is diagnostic of the disease under 
 the limitations already noted in discussing the interpreta- 
 tion of this type of reaction in the primary and secondary 
 stages. 
 
 A plus [two-plus or three-plus) occurs more frequently 
 in this stage of syphilis than in the secondary stage and is, 
 therefore, of greater diagnostic value. In patients pre- 
 senting suspicious lesions of this stage, especially in those 
 in which there is apparent involvement ©f the central nerv- 
 ous system, the writer belieives that this type of reaction 
 should be given the same diagnostic value as the double- 
 plus (four-plus) reaction, with the same limitations. 
 
 A plus-minus (two-plus and plus-minus) reaction, unless 
 permanent, or treatment has been administered, is of no 
 value in the diagnosis of syphilis during this stage of the 
 disease. In those instances in which there is no history of 
 infection and the symptoms present are merely slightly 
 suspicious, this type of reaction should be considered as 
 negative, provided it persists over two or three months, but 
 in the presence of a clear history of infection, especially if 
 treatment has been administered, a plus-minus reaction 
 should not lead one to consider the case as nonsyphilitic. 
 
 A negative reaction in patients suspected of tertiary 
 syphilis is of no value in excluding the disease unless it 
 persists for several months and symptoms have not de- 
 veloped meanwhile. In the absence of a history of infec- 
 tion and of symptoms which are typical, a negative re- 
 action may be interpreted as excluding syphilis, provided 
 no specific treatment has been administered and it remains 
 negative for a period of three months. In all suspected 
 tertiary cases, when the Wassermann test is negative, a 
 test should be made upon the cerebrospinal fluid. 
 
INTERPRETATION OF RESULTS 161 
 
 The Interpretation of the Results of the Wassermann 
 Test in the Latent Stages of Syphilis.— The results of the 
 Wassermann test differ in the early and late latent cases 
 but the diiference is not sufficient to justify our classify- 
 ing the cases into early and late cases, so far as the inter- 
 pretation of the results is concerned. In the early latent 
 cases, that is, the cases tested during the latent period be- 
 tween the healing of the initial lesion and the occurrence 
 of secondary symptoms, a higher percentage of positive 
 results is obtained than in latent periods occurring later 
 in the disease, and the lowest percentage of positive re- 
 sults is obtained in latent cases in which the period of 
 latency occurs after the development of secondary or ter- 
 tiary lesions. The percentage of positive results obtained 
 by the writer in latent infection, including both early and 
 late cases, has been 68 per cent, so that in this class of 
 infections one must expect about 30 per cent of negative 
 results. One of the most important reasons for the lower 
 percentage of positive results in latent cases is that the 
 vast majority of these patients have received more or less 
 specific treatment and in most of the cases the test was 
 made in order to determine the result of this treatment. 
 Therefore, it follows that the plus reaction will have more 
 diagnostic value in these treated cases than in other stages 
 of syphilis when treatment has not been given, and that 
 a negative reaction will have less value in excluding the 
 disease. 
 
 A double-plus (four-plus) reaction occurs less frequently 
 in latent cases than in other stages of syphilis, but when it 
 is obtained it is absolutely diagnostic of the disease under 
 the limitations already mentioned. 
 
 A plus (two-plus or three-plus) reaction, in latent cases, 
 where there is a clear history of infection, should be given 
 the same interpretation as the double-plus (four-plus) re- 
 action, especially if specific treatment has been admin- 
 istered. It is the writer's belief that this type of reaction 
 
162 THE WASSEKMANN TEST 
 
 in latent cases, under the conditions named, is diagnostic 
 of syphilitic infection. 
 
 A plus-minus (plus or plus-minus) reaction in latent in- 
 fections, when there is a clear history of infection, symp- 
 toms have been present in the past, or specific treatment 
 has been given, should arouse the suspicion that the disease 
 is still present, but the test should be repeated two or 
 three times, with a similar result, before a diagnosis of 
 syphilis should be entertained. In such eases a plus-minus 
 reaction usually means insufficient treatment, and indicates 
 that further treatment is required. In the absence of a his- 
 tory of infection, or of previous symptoms, a diagnosis of 
 syphilis should never be made upon a plus-minus reaction. 
 
 A negative reaction in cases suspected of being in the 
 latent stage of syphilis has no value as excluding the dis- 
 ease unless it is consistently negative over several months, 
 in the absence of specific treatment and of symptoms of 
 the disease. A negative result is obtained in at least 30 
 per cent of latent syphilitics so that it has little value in 
 excluding the disease. 
 
 Interpretation of the Results of the Wassermann Test in 
 Syphilitic Disease of the Central Nervous System. — The in- 
 terpretation of the results of the Wassermann test in syph- 
 ilitic disease of the central nervous system is considered 
 in Chapter X of this work. 
 
 Inteo-pretation of the Results of the Wassermann Test 
 after Specific Treatment.— The interpretation of the results 
 obtained in patients who have received specific treatment is 
 considered in Chapter IX of this work. 
 
 General Summary .—From the writer's experience he be- 
 lieves that the following general conclusions regarding the 
 interpretation of the results of the Wassermann test are 
 justified : 
 
 1. If the diseases, other than syphilis, that sometimes 
 give a positive result with the Wassermann test, can be 
 excluded, a double-plus (four-plus of many writers) re- 
 action is absolutely diagnostic of syphilis. It is believed 
 
INTEEPEETATIOlir OF EESULTS 163 
 
 that, under such conditions, this type of reaction is specific 
 of the disease, whether symptoms are present or not, or 
 whether there is or is not a history of infection. 
 
 2. Under the same conditions, a plus reaction (three- 
 plus or two-plus of many. writers) may, in primary, ter- 
 tiary, and latent infections he regarded as diagnostic, pro- 
 vided there is a clear history of infection, or suspicious 
 clinical symptoms are present. In secondary eases this 
 type of reaction is not diagnostic unless typical secondary 
 symptoms are present. In the absence of either history 
 or symptoms this type of reaction should not be regarded 
 as diagnostic of syphilis. 
 
 3. A diagnosis of syphilis should never be made upon a 
 plus-minus reaction alone. Many perfectly normal individ- 
 uals give this type of reaction at times in their blood serum. 
 In latent infections a plus-minus reaction, if persistent, 
 should be followed by further specific treatment. 
 
 4. A single negative reaction, where there is no history 
 of infection and where symptoms are not present, is of con- 
 siderable value as a corroborative sign that syphilis is not 
 present, but where there is any suspicion that the disease 
 may be present it has very little value in excluding syphilis. 
 The experiments already mentioned upon the variation in 
 the complement-binding power of the blood serum of known 
 syphilitics illustrate the truth of this statement, and it is 
 only when a negative reaction is repeatedly obtained over 
 a long period of time that it can be considered as good 
 evidence of the absence of the disease, and even then the 
 spinal fluid should be lested and,- if possible, a provocative 
 Wassermann test should be made upon the blood. The 
 same remarks apply to cases that have been given specific 
 treatment, a negative reaction being of no value as indi- 
 cating a cure of the infection, unless the reaction remains 
 persistently negative over a period of at least one year. 
 In such cases a provocative Wassermann test' should be 
 made, and the spinal fluid should be tested. A luetin test 
 
164 THE WASSERMANIsr TEST 
 
 should also be made in certain cases before the patient is 
 discharged as cured. 
 
 The interpretation of the results of th'e Wassermann 
 test must rest very largely with the clinician, for the clin- 
 ical picture present is often more decisive than is the re- 
 sult of the test, and it is the clinician's place to reconcile 
 the result of the test with the clinical picture, rather than 
 the serologist's. The latter should simply report what 
 actually occurs when the patient's blood serum is tested, 
 without any reference whatever to the clinical history or 
 the symptoms which may be present, and it remains with 
 the clinician to interpret the report in the light of his 
 patient's condition. The writer believes that much harm 
 has been done by the serologist allowing the history of a 
 case, or the symptoms present, to influence his reading of 
 the Wassermann test, and it is always better to have the 
 test read without the serologist knowing anything what- 
 ever about the history or symptoms of the patients from 
 whom the specimens were collected. 
 
 The Wassermann Test as an Index of the Prevalence 
 
 of Syphilis 
 
 Since the discovery of the Wassermann test it has been 
 largely used in determining the prevalence of syphilis in 
 communities, hospitals, and among various classes of peo- 
 ple, and has been of the greatest service in demonstrating 
 how widespread this disease really is, and the compara- 
 tively large number of people who suffer from it in nearly 
 all countries and among nearly all classes of society. In 
 fact, it may be stated, that before the days of the Wasser- 
 mann reaction, although it was generally recognized that 
 syphilis was a very prevalent disease in many countries, 
 we had a very poor conception of its real prevalence, espe- 
 cially in the United States, so that from a sociological 
 standpoint, the test has been of such value as to almost 
 revolutionize our ideas regarding the subject. 
 
INDEX OF THE PREVALENCE OF SYPHILIS 165 
 
 It is now well known, and has been demonstrated time 
 and again by Wassermann surveys of the class mentioned, 
 that between 80 and 90 per cent of prostitutes who have 
 pursued their calling for as long as five years or more, 
 are syphilitic, although it should be remembered, in con- 
 sidering this high percentage, that only a comparatively 
 small percentage of the women were actually capable of 
 transmitting the infection at the time the tests were made, 
 so far as clinical evidence was obtainable. However, in a 
 disease like syphilis that has its periods of infectivity and 
 quiescence, this high percentage of syphilitics among pros- 
 titutes furnishes evidence enough of the danger of illicit 
 sexual intercourse, and one often wonders why the disease 
 is not much more common than it appears to be in the 
 usual professional experience. As a matter of fact, syph- 
 ilis is much more common, as shown by the results of the 
 Wassermann test, than usually supposed and, in this coim- 
 try as well as in others, is pretty well distributed through 
 ""all classes. 
 
 As a result of the application of the Wassermann test 
 in Wassermann surveys, as they are called, a great mass 
 of evidence has been accumulated regarding the preva- 
 lence of syphilis in different communities and among dif- 
 ferent classes of the population. Thus, Southard®^ per- 
 formed 6,000 Wassermann tests in the Harvard Neuro- 
 pathologic Laboratory and found 23 per cent positive; 
 Hammond,*^ who applied the test to both patients and 
 others at the New Jersey State Hospital, found 6.3 per 
 cent positive ; Thompson^* made 1,000 tests at the Arkansas 
 State Hospital for Nervous Diseases and found 33 per 
 cent positive ; Eosenberger®^ made 5,106 Wassermann tests 
 upon the patients at the Philadelphia General Hospital, 
 during 1916, and found 27.4 per cent positive; and John- 
 son,^*" who examined by means of the Wassermann test 
 224 children showing anemia and malnutrition, found that 
 over 33 per cent gave a positive Wassermann reaction. 
 
 There is no better place to study the prevalence of syph- 
 
166 
 
 THE WASSBBMANN TEST 
 
 ills than among the personnel of the army, for recruits 
 are drawn from all classes of society for the service and 
 from all parts of the United States. The most important 
 and conclusive contribution to our knowledge of the preva- 
 lence of syphilis in the army, and, consequently, in civil 
 life, has been made by Lieut. Colonel Vedder, who pub- 
 lished his results in 1915,*" and from whose work I shall 
 quote largely?- in considering this subject. 
 
 In his work, Vedder considered that all cases giving a 
 double-plus (four-plus) reaction were without doubt syph- 
 ilitic, while those giving a plus (three-plus reaction) were 
 probably syphilitic. Summarized, his results are presented 
 in Table XXXIV: 
 
 Table XXXIV 
 Occurrence of Syphilis in Army, from Vedder 
 
 
 
 
 
 
 
 ESTIMATED 
 
 
 
 
 WAS- 
 
 UNDOUBT- 
 
 WAS- 
 
 TOTAL 
 
 
 
 KNOWN 
 
 
 
 
 NUMBER OP 
 
 
 TOTAL 
 
 
 SER- 
 
 ED SYPH- 
 
 SER- 
 
 
 CLASSES 
 
 
 CASES 
 
 
 
 
 PROBABLE 
 
 
 TESTED 
 
 
 MANN 
 
 ILITICS 
 
 MANN 
 
 
 
 
 % 
 
 % 
 
 % 
 
 % 
 
 SYPH- 
 
 ILITICS 
 
 % 
 
 Recruits 
 
 1,019 
 
 
 
 7.75 
 
 7.75 
 
 9.02 
 
 16.77 
 
 Cadets 
 
 621 
 
 
 
 2.57 
 
 2.57 
 
 2.89 
 
 5.46 
 
 White enlisted 
 
 1,577 
 
 3.44 
 
 4.77 
 
 8.21 
 
 7.87 
 
 16.08 
 
 Colored enlisted 
 
 1,472 
 
 1.08 
 
 21.80 
 
 22.21 
 
 13.11 
 
 36.00 
 
 Porto Rico Regt. 
 
 531 
 
 13.55 
 
 28.58 
 
 42.37 
 
 13.55 
 
 55.93 
 
 Military convicts 
 
 1,145 
 
 6.48 
 
 9.50 
 
 15.98 
 
 5.67 
 
 21.65 
 
 Insane soldiers 
 
 567 
 
 3.51 
 
 8.29 
 
 11.80 
 
 7.41 
 
 19.21 
 
 Tuberculous soldiers 
 
 229 
 
 7.56 
 
 15.72 
 
 23.28 
 
 15.72 
 
 39.00 
 
 Soldiers home 
 
 1,171 
 
 11;62 
 
 13.40 
 
 25.02 
 
 9.73 
 
 34.75 
 
 There may be some question as to whether the cases 
 giving a plus reaction examined by Vedder should be 
 called probably positive, but, even if these cases are ex- 
 cluded, the figures given in the table are very striking 
 and show that syphilis is a far more prevalent disease than 
 is generally supposed by the average practitioner. 
 
 Among the recruits examined by Vedder, numbering 
 1,019, a double-plus (four-plus) reaction was obtained in 
 7.75 per cent, or practically 8 per cent. These men had 
 
INDEX OF THE PKBVALENCE OP SYPHILIS 167 
 
 passed a most thorough physical exaimnation for admis- 
 sion to the army and presumably presented no evidences 
 of syphilis when accepted. If to this number be added 
 the 9.02 per cent of cases giving a plus (three-plus) re- 
 action, the total percentage of syphilitics would amount 
 to 16.77 per cent. These recruits were distributed through 
 the areas of the United State& covered by the recruiting 
 stations at Fort Slocum, New York, and Columbus Bar- 
 racks, Ohio, and represented numerous occupations before 
 enlistment. Vedder concludes that his observations, to- 
 gether with the known figures for rejection for syphilis of 
 applicants for admission, show that practically "among 
 young men in civil life between the ages of 20 and 30, and 
 of the general class belonging to the occupations mentioned, 
 the percentage of syphilis may be estimated at at least 
 16.77 per cent, and there is good reason for believing that 
 it is fully 20 per cent." 
 
 The writer's own observations upon the percentage of 
 positive Wassermann reactions obtained in recruits for 
 the army, numbering several thousands of cases, is prac- 
 tically the same as Vedder 's, as regards the occurrence of 
 double-plus reactions, the percentage obtained by the 
 writer being 8.64 per cent instead of 7.75. It is not be- 
 lieved that the cases giving a plus reaction should be con- 
 sidered as positive unless there is a history of infection 
 or of suspicious symptoms in the past; but in the writer's 
 experience such a history may be obtained upon careful 
 ■ questioning in enough of the cases to raise the percentage 
 of syphilis among recruits of the army to 10 per cent when 
 they are admitted to the service. If to these are added 
 the percentage of rejections for syphilis, based upon actual 
 symptoms of the disease, the writer believes that at least 
 15 per cent of all applicants for enlistment for the army, 
 prior to the present war, were infected with syphilis, and 
 this means that practically 15 per cent of the average male 
 civilian population of this country between the ages of 
 20 and 30 are infected. This appears to be an enormous 
 
168 THE WASSBEMANN TEST 
 
 percentage but the figures are borne out in "VVassermann 
 surveys upon a similar class of men, as regards age, in 
 other countries, and in surveys made in our large hospitals 
 upon all classes of cases admitted to them. • 
 
 Vedder's results upon the class of young men who have 
 entered West Point are important, for these young men 
 come from the average middle class fanaily of this coun- 
 try, have been well educated, and must be above the aver- 
 age in intelligence in order to pass the rigid examinations 
 for entrance to the military academy. In this class of 
 young men Vedder found 2.57 per cent of double-plus 
 (four-plus) reactions among the 621 cadets tested, and 
 2.89 per cent of plus reactions. If these be added together, 
 there is a total percentage of over 5 per cent which he con- 
 siders as probably syphilitic. The writer believes that 
 probably three per cent would be about the amount of 
 syphilis 'in this class of cases, and, applied to a similar 
 class of young men in civilian life, (the class attending our 
 colleges) it would mean that about this percentage of col- 
 lege men are syphilitic before entering college. 
 
 As regards the amount of syphilis in white and colored 
 enlisted men in the army, Vedder found that among the 
 white enlisted men 8.21 per cent gave a double-plus (four- 
 plue) reaction while among the colored enlisted men no 
 less than 22 per cent gave this type of reaction. His fig- 
 ures as regards the greater percentage of syphilitic infec- 
 tion among the colored men of the army agree with those of 
 many investigators who have made Wassermann surveys 
 upon the colored race in this country. 
 
 Vedder's results as to the prevalence of syphilis among 
 the patients at the army hospital for tuberculosis, at Fort 
 Bayard, N. M., are of interest, as they demonstrate that 
 a much larger proportion of tuberculous patients give a 
 positive reaction than patients suffering from other dis- 
 eases. He found that no less than 23.28 per cent of the 
 229 patients examined gave a positive reaction and he 
 states that his results are almost identical with those of 
 
INDEX OF THE PEEVALENCE OF SYPHILIS 169 
 
 LetuUe, Bergeron, and Lepine in Paris, who found that 
 one out of every five tuberculous patients was also syph- 
 ilitic. These results explain 'fully the large number of 
 positive reactions which some writers have claimed are due 
 to tuberculosis, but the most careful investigations have 
 shown that tuberculosis per se does not give a positive 
 reaction with the Wassermann test, so that those patients 
 who do react positively are undoubtedly suffering from a 
 coincident syphilitic infection. 
 
 The writer, while working upon his method of comple- 
 ment fixation in the diagnosis of tuberculosis, had the 
 opportunity of testing 166 patients in the General Hospital 
 at Fort Bayard and found that nearly 10 per cent of them 
 gave a positive reaction with the Wassermann test, while 
 about 6 per cent were known to be syphilitic, giving a total 
 percentage of 16 per cent of syphilitics in the number ex- 
 amined. While these figures are not as large as those 
 recorded by Vedder they amply demonstrate the. fact that 
 syphilis and tuberculosis are more often associated than 
 are other diseases, and that one may expect a large per- 
 centage of syphilitic infection among the tuberculous. 
 
 Irvine,®^ as the result of various Wassermann surveys, 
 concludes that from 10 to 15 per cent of the population of 
 the State of Minnesota are syphilitic, or from 200,000 to 
 300,000 people. He states that Wassermann surveys 
 made in the asylum at Fergus Falls showed that 30 per 
 cent of the inmates were syphilitic and that the average 
 of many similar surveys is 20 per cent. Peterson®^ found 
 that among 477 obstetric patients, no less than 5.8 per 
 cent were syphilitic, and of 606 gynecologic patients the 
 test was positive in 8.2 per cent. Regarding the value of 
 the Wassermann test in obstetric and gynecologic prac- 
 tice he says: "In many of the cases, both where there was 
 a history of infection and when this was lacking, physical 
 examination in the gynecologic clinic failed to arose sus- 
 picion of the presence of syphilis. The point to be empha- 
 sized is that the same careful physical examinations have 
 
170 THE WASSEEMANN TEST 
 
 been made in this particular clinic for years with only 
 an occasional patient suspected of being luetic. "When, 
 however, such examinations are reinforced by routine Was- 
 sermann examinations, nearly 10 per cent of the patients 
 are found syphilitic." Ladd"" found that 25.5 per cent of 
 patients applying for treatment at the dispensaries of the 
 Washington, D. C, Casualty and Asylum Hospitals gave 
 a double-plus (four-plus) Wassermann reaction, and that 
 of these 14.6 per cent were colored, and 10.9 per cent white 
 patients. 
 
 An entire volume could be written covering the statis- 
 tics of the almost innumerable Wassermann surveys that 
 have been made in different localities but the illustrations 
 here given demonstrate the value of the test in the study of 
 the prevalence of syphilis. In fact, no longer is any data 
 of scientific value regarding this subject unless it is based 
 upon the results of carefully performed Wassermann re- 
 actions, for the day has passed when clinical evidence alone 
 is to be considered as sufficient upon which to base figures 
 regarding the prevalence of syphilis in a community or 
 country. The cases of syphilitic infection that are appar- 
 ently without clinical symptoms far outnumber those that 
 present typical symptoms and the only way that we at pres- 
 ent possess of making a diagnosis in these latent infections 
 is by means of the Wassermann test. Aside from the value 
 of this test in the diagnosis of the disease and in the scien- 
 tific control of its treatment, perhaps the greatest value 
 of the test is in the demonstration of the extent to which 
 this insidious disease has permeated society, an extent al- 
 together unsuspected before the use of the reaction in Was- 
 sermann surveys. 
 
CHAPTER IX 
 
 THE EFFECT OF TREATMENT UPON THE WAS- 
 SERMANN REACTION. THE WASSERMANN 
 TEST AS A CONTROL OF THE TREAT- 
 MENT OF SYPHILIS. THE PRO- 
 VOCATIVE WASSERMANN 
 REACTION 
 
 The treatment of syphilis, either with mercury or with 
 salvarsan or heosalvarsan often has a marked effect upon 
 the results of the Wassermann test, rendering strongly 
 positive reactions negative or greatly lessening the strength 
 of the reaction in those cases in which a positive result is 
 not rendered negative. This fact has rendered the use of 
 the test invaluable as a guide to the efficiency of the treat- 
 ment of the disease with various drugs and has enabled 
 the profession to discard many drugs vaunted as specifics 
 for the disease but which have been proved by the Was- 
 sermann test to be absolutely worthless. One of the most 
 striking examples of this fact is the instance of sodium 
 cacodylate, which at one time was announced as almost the 
 equal of salvarsan in the treatment of syphilis, but' which 
 was soon proved by the Wassermann test to be practically 
 worthless, so far as curing the infection,^ and which is not 
 used at the present time in the treatment of the disease 
 except by those ignorant of the work which has been done 
 proving its worthlessness or those willfully blind to the re- 
 sults of that work. 
 
 The Wassermann reaction is generally conceded to be 
 the most reliable and delicate of all of the symptoms of 
 syphilis and for this reason the effect of treatment upon 
 the reaction is of the utmost practical importance. A posi- 
 
 171 
 
172 THE WASSBEMABTlSr TEST 
 
 tive reaction, as has been noted, means the presence in the 
 body of living treponemas, and as long as the reaction 
 remains positive, just so long is the patient a victim of 
 syphilitic infection, regardless of the occurrence of other 
 symptoms of the disease. The effect of treatment with 
 various drugs can be easily followed in the case of syph- 
 ilis by frequently repeated Wassermann tests, and in this 
 way one can secure a reliable picture of the results of any 
 particular form of treatment. In view of the importance 
 of this subject the following chapter contains the personal 
 observations of the writer upon the effect upon the "Was- 
 sermann reaction of treatment with salvarsan and mer- 
 cury, which have been previously published,^"^ together 
 with remarks upon the test as a control of the treatment 
 of syphilis, and upon the provocative Wassermann reaction. 
 
 The Effect of Treatment upon the 
 Wassermann Reaction 
 
 Prior to 1911 there was little in the literature upon the 
 effect of treatment on the Wassermann reaction but since 
 that time a great deal of data has been published show- 
 ing that the reaction is markedly influenced by treatment 
 with certain drugs, and that positive reactions often be- 
 come negative and remain so for considerable periods of 
 time after the cessation of treatment. Unfortunately, in- 
 vestigation has also shown that the early treatment with 
 mercury and salvarsan was insufficient and that most of 
 the cases so treated relapsed within a year after treat- 
 ment was stopped, so that at the present time no intelligent 
 practitioner believes that a few injections of mercury or of 
 salvarsan or neosalvarsan will cure syphilis. 
 
 The Effect upon the Reaction of Treatment with Salvar- 
 san. — The data given in regard to the effect upon the Was- 
 sermann test of treatment with salvarsan w^as collected by 
 the writer between 1910 and 1913 and is based upon a com- 
 paratively few injections of the drug, as Ehrlich's idea 
 
THE EFFECT OF TREATMENT 173 
 
 that one or two injections of this drug would sterilize the 
 body, so far as Treponema pallidum was concerned, was 
 still the belief and hope of the profession. However, the 
 facts deduced from these experiments and observations 
 have since been fully confirmed and the data demonstrate 
 the effect upon a positive Wassermann reaction of treat- 
 ment with this drug. 
 
 Among those who had contributed valuable data regard- 
 ing the effect of salvarsan upon the Wassermann reaction 
 prior to the writer's publications may be mentioned the 
 following : 
 
 Nichols and Fordyce^"^ treated 11 patients with salvarsan 
 with the following results upon the Wassermann reaction : 
 In 8 the reaction became negative, 1 in 19 days; 2 in 21 
 days ; 1 in 23 days ; 1 in 25 days ; 1 in 39 days ; 1 in 3 months ; 
 and 1 in 3 months and 11 days. Miehaelis"^ treated 110 
 patients and states that the reaction may become negative 
 within a period from ,2 to 10 weeks after the injection. 
 Fordyce"* states that the results of the Wassermann test 
 after salvarsan have varied within wide limits, in his expe- 
 rience, but in general a change occurred in from 4 to 5 
 weeks. The earliest change from a positive to a negative 
 reaction observed by him was in 6 days after the injec- 
 tion. He states that ** several cases showed alternating 
 negative and positive phases." Noguchi"^ analyzed very 
 carefully 102 cases treated with salvarsan, more than half 
 of them being under observation for over three months 
 while the remainder had been injected four weeks pre- 
 viously. He made quantitative examinations of their blood 
 serum for complement-binding power, the tests being made 
 before the injection of salvarsan, and at intervals of one 
 day, three days, one week, two weeks, three weeks, four 
 weeks, six weeks, eight weeks, etc., after the injection. He 
 found that 30 cases became negative, 24 reduced to less 
 than 1 antibody unit, while ,the remaining 48 cases still 
 contain more than 1 antibody unit, giving strong positive 
 reactions. In 40 per cent of the primary cases, in 37 per 
 
174 
 
 THE WASSERMANN TEST 
 
 cent of the secondary cases, in 35 per cent of tertiary, in 
 33 per cent of latent, in 14 per cent of hereditary, and in 
 50 per cent of incipient tabes the reaction became nega- 
 tive. The average of the negative reactions equalled 33.7 
 per cent of the total 102 cases. Of the negative eases re- 
 ported by Noguchi, 34 in number, 3 became negative in 
 two weeks, 10 in three weeks, 11 in four weeks, 5 in five 
 weeks, 4 in six weeks, and 1 in seven weeks. 
 
 Aiiimal Experiments. — The effect upon the Wassermann 
 reaction of treatment with salvarsan can be shown in rab- 
 bits experimentally infected with syphilis. Through the 
 kindness of Major H. J. Nichols, the writer had the oppor- 
 tunity of repeatedly testing the blood serum of rabbits ex- 
 perimentally infected with syphilis and yaws, and Tables 
 XXXV and XXXVI illusfrate the gradual increase in the 
 strength of the "VVassermann test in these animals after in- 
 fection, and the disappearance of the positive reaction after 
 
 Table XXXV 
 
 Eesults or Wassermann Test befoee and after Treatment with Salvarsan 
 
 IN Babbit Infected with Syphilis* 
 
 AMOUNT OF 
 BLOOD 
 
 DATE OF TESTS 
 
 SERUM, C.C. 
 
 Jan. 23 
 
 Jan. 27 
 
 Jan. 30 
 
 Feb. 6 
 
 Feb. 8 
 
 Feb. 10 
 
 Feb. 13 
 
 Feb. 18 
 
 0.05 
 
 _ 
 
 + 
 
 + 
 
 + + 
 
 _ 
 
 + 
 
 
 
 _ 
 
 0.10 
 
 _ 
 
 + + 
 
 + + 
 
 + + 
 
 + - 
 
 + + 
 
 _ 
 
 _ 
 
 0.15 
 
 
 
 + + 
 
 + + 
 
 + + 
 
 + - 
 
 + + 
 
 
 
 _ 
 
 0.20 
 
 + + 
 
 
 
 + + 
 
 + + 
 
 + - 
 
 + + 
 
 - 
 
 - 
 
 *Treponema. pallidum found in testicular lesion Jan. 19. 0.02 gram salvarsan per kilo- 
 administered intravenously Feb. 6. 
 
 Table XXXVI 
 
 Eesults of Wassermann Test before and after Treatment with Salvarsan 
 
 IN Babbit Infected with Taws* 
 
 amount of 
 blood 
 
 DATE OF TESTS 
 
 serum, C.C. 
 
 Jan. 23 
 
 Jan. 30 
 
 Feb. 2 
 
 Feb. 6 
 
 Feb.8 
 
 Feb. 10 
 
 Feb. 13 
 
 Feb. 17 
 
 0.05 
 
 _ 
 
 _ 
 
 + + 
 
 ■¥ + 
 
 _ 
 
 „ 
 
 _ 
 
 _ 
 
 0.10 
 
 _ 
 
 + - 
 
 + + 
 
 + + 
 
 _ 
 
 + - 
 
 _ 
 
 _ 
 
 0.15 
 
 _ 
 
 + 
 
 + + 
 
 + + 
 
 + - 
 
 + 
 
 _ 
 
 — 
 
 0.20 
 
 - 
 
 + + 
 
 + + 
 
 ^+ + 
 
 + - 
 
 + + 
 
 - 
 
 - 
 
 *Treponema pertenue found in lesion Jan. 21. 
 tered intravenously Feb. 6. 
 
 0.02 gram salvarsan per kilo adminis- 
 
THE EFFECT OF TREATMENT 175 
 
 treatment with salvarsan. The sign (+ +) indicates com- 
 plete inhibition of hemolysis. 
 
 In the rabbit infected with syphilis the treponema was 
 found in the lesion on January 19, but the Wassermann 
 test did not become positive, even with 0.2 c.c. of the serum, 
 until February 6. One week after the injection of salvar- 
 san the reaction had become negative and the table shows 
 that it had begun to diminish in strength two days after 
 the injection. 
 
 The results of treatment with salvarsan upon the rab- 
 bit infected with yaws were quite similar, the reaction be- 
 coming negative one week after the intravenous injection 
 of the drug. 
 
 The following summary of the writer's observation upon 
 the effect of treatment with salvarsan on the Wassermann 
 reaction embraces 500 cases whi^h were observed and 
 tested for at least four months after treatment. Of these 
 500 cases only 288 became negative, or 57.6 per cent, but 
 these results were due to the prevalence, at that time, of giv- 
 ing but one or two intravenous injections of salvarsan in- 
 stead of several, the intramuscular method of using the drug 
 giving altogether the best results as regards the disappear- 
 ance of the positive reaction. 
 
 The real effect of treatment with salvarsan upon the 
 Wassermann reaction can only be accurately understood 
 if several factors are taken into consideration, the most 
 important being the stage of syphilis during which the 
 drug is administered; the intensity of the positive Was- 
 sermaim reaction before treatment; the method of admin- 
 istration of the drug and the dose administered; as well 
 as the amount and kind of previous mercurial treatment. 
 
 The Stage of Syphilis in Relation to the Disappearance 
 of the Refwtion. — The results of the writer have shown 
 that regardless of the method of administration or the 
 dose, the stage of the disease has much to do with the 
 effect of treatment with salvarsan upon the Wassermann 
 reaction. This is shown in Table XXXVII. 
 
176 
 
 THE WASSERMAITN TEST 
 
 Table XXXVII 
 
 The Results of Treatment with Salvarsan upon the Wassermann Test in 
 THE Various Stages of Syphilis 
 
 
 TOTAL CASES 
 
 became negative 
 
 REMAINED POSITIVE 
 
 stage of disease 
 
 NUMBER 
 
 % 
 
 NUMBER 
 
 % 
 
 Primary 
 .Secondary 
 Tertiary 
 Latent 
 
 95 
 
 285 
 52 
 68 
 
 63 
 
 169 
 
 17 
 
 39 
 
 66.3 
 59.3 
 32.6 
 57.3 
 
 32 
 
 116 
 
 35 
 
 29 
 
 33.6 
 40.7 
 67.3 
 42.6 
 
 totals 
 
 500 
 
 288 
 
 57.6 
 
 212 
 
 42.4 
 
 An analysis of this table is interesting. In 95 primary 
 cases 63 became negative, or 66.3 per cent;, in 285 second- 
 ary cases, 169, or 59.3 per cent became negative; in 52 
 tertiary cases 17 or 32.6 per cent became negative; and in 
 68 latent cases, including both early and late latent infec- 
 tions, 35, or 57.3 per cent became negative. 
 
 From the above it is evident that the effect upon the 
 Wassermann reaction of treatment with salvarsan is .most 
 marked during the primary stage of the disease, as would 
 be expected and least marked during the tertiary stage, 
 and that the longer the infection has lasted the less is the 
 effect upon the reaction of the treatment. 
 
 Time of Disappearance of the Reaction in Relation to the 
 Stage of Syphilis 
 
 The rapidity with which the positive Wassermann. re- 
 action disappears after treatment with salvarsan depends 
 largely upon the stage of the disease as is shown in Table 
 XXXVIII. 
 
 Table XXXVIII 
 
 The Relation of the Stage of Syphilis to the Time of Disappearance of 
 the Wassermann Reaction, after Treatment with Salvarsan 
 
 STAGE OF 
 DISEASE 
 
 TOTAL 
 CASES 
 
 1 
 
 WEEK 
 
 2 
 
 WEEKS 
 
 3 
 
 WEEKS 
 
 4 
 
 WEEKS 
 
 5 
 
 WEEKS 
 
 6 
 
 WEEKS 
 
 7 
 
 WEEKS 
 
 8 
 
 WEEKS 
 
 Primary 
 Secondary 
 Tertiary 
 Latent 
 
 63 
 
 169 
 
 17 
 
 39 
 
 6 
 
 13 
 
 3 
 
 2 
 
 25 
 
 44 
 
 6 
 
 9 
 
 20 
 
 39 
 2 
 
 7 
 
 8 
 
 33 
 
 5 
 
 10 
 
 2 
 
 17 
 
 
 
 6 
 
 2 
 
 13 
 
 1 
 
 5 
 
 
 5 
 
 
 
 
 
 5 
 
 
 
 TOTALS 
 
 288 ■ 
 
 24 
 
 84 
 
 68 
 
 56 
 
 25 
 
 21 
 
 5 
 
 5 
 
THE EFFECT OF TREATMENT 177 
 
 Regarding the time of disappearance of the reaction 
 after treatment with salvarsan it will be noted that the 
 best results were obtained in the primary stage of the dis- 
 ease, no less than 51 of the 63 primary cases that became 
 negative becoming so within three weeks and the longest 
 time after treatment in which the reaction became negative 
 in this stage was six weeks. In the secdndary stage the 
 reaction disappeared generally during the second, third, 
 or fourth weeks, the longest period being eight weeks. In 
 the tertiary stage the results appear more favorable than 
 in the primary, 1 of the 17 cases becoining negative within 
 three weeks but the number of cases is too small to base 
 such an assertion upon, and the writer believes that the 
 evidence is sufficient to prove that the best results are 
 obtained in the primary stage of syphilis. 
 
 As will be noted, none of the reactions, in any stage of 
 the disease, became negative after eight weeks, so that it 
 would appear that a positive reaction which remains so 
 after a period of eight weeks has^ elapsed since the last 
 salvarsan treatment, should always be an indication for 
 further treatment. If the drug is given intravenously, as 
 it generally is today, the period may be shortened to less 
 than this, but it is believed that eight weeks is a practical 
 and safe period to wait in order to ascertain if the Was- 
 sermann reaction will become negative. 
 
 The Relation of the Intensity of the Reaction to Its Dis- 
 appearance after Salvarsan. — The stronger the Wasser- 
 mann reaction the less is it affected by treatment with 
 salvarsan, as is shown in Table XXXIX. In this table 
 and those that follow, it should be remembered that a 
 double-plus (+-^) reaction means absolute inhibition of 
 hemolysis, or the four-plus reaction of most writers ; a plus 
 (+) reaction at least 50 per cent of inhibition, or the three- 
 and double-plus reaction of some laboratories ; and a plus- 
 minus, less than 50 per cent of inhibition. 
 
 An analysis of this table shows that of 295 cases in which 
 the Wassermann reaction was double-plus (four-plus). 
 
178 
 
 THE WASSEEMAKN TEST 
 
 Table XXXIX 
 
 The Eelation op the Intensity of the Wasseemann Eeactionito its 
 Disappearance aptek Treatment with Salvaksan 
 
 CHARACTER 
 OP 
 
 TOTAL 
 CASES 
 
 BECAME 
 
 NEGATIVE 
 
 REMAINED POSITIVE 
 
 REACTION 
 
 NUMBER 
 
 % 
 
 NUMBER 
 
 % 
 
 + + 
 + 
 
 + - 
 
 295 
 
 169 
 36 
 
 145 
 
 110 
 
 33 
 
 41.9 
 65.0 
 91.6 
 
 150 
 
 59 
 
 3 
 
 50.8 
 
 34.9 
 
 8.3 
 
 
 500 
 
 288 
 
 57.6 
 
 212 
 
 42.4 
 
 41.9 per cent became negative ; of 169 giving a plus (three 
 or two plus) reaction, 65 per cent became negative, and of 
 36 cases showing a plus-minus reaction, 91 per cent be- 
 came negative. As regards the latter cases it may be stated 
 that they were either very early primary cases or cases that 
 had previously received specific treatment to a greater or 
 lesser extent. It will thus be seen that the poorest results 
 as regards the effect of treatment upon the Wassermann 
 test were obtained in cases shoAving complete inhibition of 
 hemolysis and the results gradually grew better with the 
 loss in the strength of the reaction. 
 
 The Relation of the Stage of Syphilis and the Intensity 
 of the Wassermann Reaction to its Disappearance after 
 Treatment with Salvarsan. — The percentage of negative 
 results obtained by treatment with salvarsan in each stage 
 of syphilis is considerably influenced by the intensity of 
 the reaction. Table XL illustrates the effect of the inten- 
 sity of the reaction upon its disappearance in each stage 
 of the disease. 
 
 From this table it is evident that the effect of treatment 
 with salvarsan upon the Wassermann test, in every stage 
 of syphilis, varies with the intensity of the reaction, being 
 most pronounced in the cases showing the weaker reactions 
 and least in those giving complete inhibition of hemolysis. 
 From these data it is evident that the earlier treatment is 
 begun in syphilis the better will be the results, for the 
 cases of tertiary syphilis, for instance, which gave a double- 
 plus reaction, gave a very low percentage of negative re- 
 
THE EFFECT OF TREATMENT 
 
 179 
 
 Table XL 
 
 Thb Relation of the Stage op Syphilis and the Intensity of the Wasser- 
 
 MANN Reaction to Its Disappearance after Treatment with 
 
 Salvarsan 
 
 STAGE OF 
 
 CHARACTER 
 
 OP reaction 
 
 TOTAL 
 
 CASES 
 
 BECAME NEGATIVE 
 
 REMAINED 
 
 posiTivii; 
 
 syphilis 
 
 NUMBER 
 
 % 
 
 NUMBER 
 
 % 
 
 Primary 
 (95 cases) 
 
 ++ 
 
 + 
 
 + - 
 
 44 
 41 
 10 
 
 24 
 
 31 
 
 8 
 
 54.5 
 75.6 
 80.0 
 
 20 
 
 10 
 
 2 
 
 45.4 
 24.3 
 20.0 
 
 Secondary 
 (285 cases) 
 
 ++ 
 + 
 
 + - 
 
 185 
 86 
 14 
 
 98 
 58 
 13 
 
 52.9 
 67.4 
 92.8 
 
 87 
 
 28 
 
 1 
 
 47.0 
 
 32.5 
 
 7.1 
 
 Tertiary 
 (52 eases) 
 
 + + 
 
 + 
 
 + - 
 
 33 
 
 16 
 
 3 
 
 5 
 
 I 
 
 15.1 
 
 56.2 
 
 100.0 
 
 28 
 7 
 
 
 84.8 
 
 43.7 
 
 0.0 
 
 Latent 
 
 (68 cases) 
 
 ++ 
 
 + 
 + - 
 
 33 
 
 26 
 
 9 
 
 18 
 
 12 
 
 9 
 
 54.5 
 
 46.1 
 
 100.0 
 
 15 
 
 14 
 
 
 
 45.4 
 
 53.8 
 
 0.0 
 
 suits after treatment; i. e., only 15 per cent and even in 
 the cases giving a plus reaction the percentage of negative 
 reactions was only a little over 50 per cent. It will be ob- 
 served that in the latent stage a higher percentage of cases 
 showing a double-plus reaction became negative than of 
 those showing a plus reaction, but this was due to previous 
 mercurial treatment in a larger proportion of the cases 
 giving a double-plus reaction. 
 
 From these data it is also evident that the intensity of 
 the Wassermann reaction in the various stages of syphilis 
 is of considerable prognostic importance. While in all of 
 the stages of syphilis, with the exception of the latent stage, 
 the results were better in cases giving a plus reaction than 
 in those giving a double-plus, the difference is most marked 
 in the tertiary stage of the disease and least in the pri- 
 mary. As regards the double-plus cases, the best results 
 were obtained in the primary and latent stages of syphilis 
 and the poorest results in the tertiary stage. The good re- 
 sults in the latent stage may be explained by the fact that 
 many of these cases were early latent cases, for the late la- 
 tent cases are much more resistant to treatment. Of the 
 cases giving a plus reaction the best results were obtained in 
 
180 
 
 THE WASSEBMANN TEST 
 
 the primary stage, the poorest in the latent stage, while 
 the results in the secondary stage were better than those 
 in the tertiary. The prognosis, as regards the disappear- 
 ance of the reaction, is most unfavorable in the tertiary 
 cases giving a double-plus (four-plus) reaction. 
 
 The Relation of the Method of Administration of Salvar- 
 san to the Disappearance of the Wassermann Reaction in 
 Treated Cases. — The method of administration of salvar- 
 san has a very marked influence upon the effect of the drug 
 on the Wassermann reaction. Of the 500 calses studied, 
 209 were treated by the intramuscular injection of the alka- 
 line solution, 249 by the intravenous injection of the drug, 
 and 42 by combined intramuscular and intravenous injec- 
 tions. Table XLI illustrates the effect of the method of 
 administration upon the 'V\''assermann reaction. 
 
 Table XLI 
 
 The Methods of Administration of Salvaesan in Relation to the 
 Disappearance of the Wassekmann Reaction in Treated Cases 
 
 METHOD of 
 
 TOTAL 
 CASES . 
 
 BECAME 
 
 NEGATIVE 
 
 remained POSITIVE 
 
 administration 
 
 NUMBER 
 
 % 
 
 NUMBER 
 
 % 
 
 Intramuscular 
 
 Intravenous 
 
 Combined 
 
 209 
 
 249 
 42 
 
 159 
 
 101 
 
 28 
 
 76.0 
 44.5 
 66.6 
 
 50 
 
 148 
 
 14 
 
 24 
 
 55.5 
 
 33.3 
 
 totals 
 
 500 
 
 288 
 
 57.6 
 
 212 
 
 42.4 
 
 It will be observed, from a study of the table, that the 
 effect of intramuscular injections of salvarsan upon the 
 reaction is much more pronounced than other methods of 
 administration. Unfortunately, this method of adminis- 
 tration has been almost abandoned by the profession and in 
 the army it has been entirely replaced by the intravenous 
 method, owing to the pain and complications that followed 
 the injections and the time lost in hospital. However, 
 there is no question that the intramuscular method is in- 
 finitely more efficient in treatment than the intravenous, as 
 shown by the results upon the Wassermann test, for of the 
 209 cases so treated, most of them receiving but one injec- 
 tion of 0.6 gram of salvarsan, 159, or 76 per cent became 
 
THE EFFECT OF TREATMENT 181 
 
 negative, and, as will be noted later, in discussing the test 
 as a control of treatment, fewer cases treated by the intra- 
 muscular method relapsed than by the intravenous. 
 
 The figures given for the intravenous method are below 
 what they should be for the reason that the majority of 
 the cases studied only received one intravenous injection. 
 Further observations upon this method of administration 
 and its effect upon the Wassermann reaction have shown 
 that with from three to five intravenous injections *the re- 
 sults are as good as those obtained with the intramuscular 
 injections, but the data here given are sufficient to show 
 that the Wassermann reaction can be rendered negative by 
 only one intravenous injection in at least 40 per cent of the 
 cases. 
 
 The effect upon the reaction of the combined intra- 
 muscular and intravenous method of using salvarsan was 
 determined in 42 cases, of which 66 per cent became nega- 
 tive, thus indicating that the results were not as good with 
 the combined method as with the intramuscular method 
 alone. However, this result was due to the fact that a num- 
 ber of the intramuscular cases had two injections of salvar- 
 san and some three, and it is considered that one intra- 
 muscular injection of the drug is equal to at least three in- 
 travenous injections so far as the effect upon the Wasser- 
 mann test is concerned. 
 
 As would be expected the effect of treatment with sal- 
 varsan upon the reaction increases with the number of 
 doses of the drug that are administered, regardless of the 
 method of administration. Of 200 cases receiving one 
 intramuscular injection of salvarsan of 0.6 gm., 152, or 
 76 per cent became negative, while of 9 cases receiving 
 two intramuscular injections, 7, or 77.7 per cent became 
 negative. There is little difference between these two 
 groups of cases so far as the apparent effect upon the 
 Wassermann test is concerned, but the number of cases 
 receiving two intramuscular injections is too small to allow 
 of our basing any accurate statistics upon, as a larger 
 
182 
 
 THE WASSEKMANH TEST 
 
 number of cases would undoubtedly give a higher per- 
 centage in this class of cases. 
 
 Of the cases treated by the intravenous method, 177 
 were given one intravenous injection of 0.6 gm. of salvar- 
 san, and 72, or 40.6 per cent became negative; 52 were 
 given two intravenous injections of the same dose, of which 
 22, or 42.3 per cent became negative; while 10 cases were 
 given three intravenous injections, of which 7, or 70 per 
 cent became negative. There was but little difference in 
 the percentage of negative results obtained in those given . 
 one and two intravenous injections, and it will be noted 
 that two intravenous injections did not have as much effect 
 upon the Wassermann reaction, as one intramuscular in- 
 jection. The percentage of negative results obtained with 
 three intravenous injections, however, approaches closely 
 to that obtained with one intramuscular injection, and jus- 
 tifies the assertion that one intramuscular injection is equal, 
 in its effect upon the Wassermann reaction, to three intra- 
 venous injections of salvarsan. 
 
 The time of the disappearance of a positive reaction 
 after treatment with salvarsan varies with the method of 
 administration as shown in Table XLII. 
 
 Table XLII 
 
 The Relation op the Method of Administration op Salvarsan to the 
 Time or Disappearance op the Wassermann Eeaotion in Cases 
 - Treated with Salvarsan 
 
 method op 
 administration 
 
 total 
 
 OASES 
 
 1 
 
 WEEK 
 
 2 
 
 WEEKS 
 
 3 
 
 WEEKS 
 
 4 
 
 WEEKS 
 
 5 
 
 WEEKS 
 
 6 
 
 WEEKS 
 
 7 
 
 WEEKS 
 
 8 
 
 WEEKS 
 
 Intramusoular 
 
 Intravenous 
 
 Combined 
 
 159 
 101 
 
 28 
 
 9 
 
 13 
 
 2 
 
 36 
 
 44 
 4 
 
 39 
 
 24 
 
 5 
 
 31 
 
 16 
 
 9 
 
 18 
 4 
 3 
 
 18 
 
 
 3 
 
 4 
 
 1 
 
 4 
 
 1 
 
 From the table it will be noted that the positive re- 
 action disappeared more slowly after intramuscular injec- 
 tions of the drug than after the intravenous injections. 
 After intravenous injections there were only four cases 
 that became negative during the fifth week after injec- 
 tion, and none after that period, while no less than 26 cases 
 
THE EFFECT OF TREATMENT 183 
 
 treated by the intramuscular method became negative after 
 the fifth week. The cases treated by the combined method 
 were also slower in becoming negative than those treated 
 by the intravenous method alone. 
 
 If the Wassermann reaction is due to the presence in 
 the tissues of living treponemas, as is now generally ac- 
 cepted, one would expect that a positive reaction would 
 disappear more rapidly after an intravenous injection of 
 salvarsan than after an intramuscular injection. The drug, 
 when administered intravenously, is quickly brought into 
 contact with large numbers of the organisms and they are 
 killed, while those not immediately affected and which are 
 accessible to the drug perish later, but as the drug is elim- 
 inated within a few days it is obvious that what, action it has 
 occurs comparatively quickly and if the reaction is depend- 
 ent upon the treponemas and they are destroyed rapidly, 
 the reaction also should disappear within a comparatively 
 short period of time. On the other hand, when salvar- 
 san is administered intramuscularly it is absorbed very 
 slowly and exerts its destructive action upon the trepo- 
 nemas for days and weeks, a smaller number are killed in 
 a given period of time, and therefore, the reaction persists , 
 for a longer period than after intravenous administration. 
 Whether the positive Wassermann reaction is due to sub- 
 stances liberated from the treponema during life; pro- 
 duced by the tissties in answer to the irritation caused by 
 the treponema multiplying, within them ; or to substances 
 liberated from the organism after its destruction or to the 
 reaction of the tissues to such substances, is immaterial, 
 so far as the disappearance of the reaction after the admin- 
 istration of salvarsan is concerned, for, however produced, 
 it is evident thai the reaction theoretically should dis- 
 appear more rapidly after intravenous injection than after 
 intramuscular, and this is what actually occurs in practice. 
 
 The Effect upon the Reaction of Treatment with Mer- 
 cury. — The Wassermann test demonstrated the true value 
 of mercury in the treatment of syphilis by the fact that 
 
184 
 
 THE WASSEKMANN ^EST 
 
 this drug is capable of causing the disappearance of a 
 positive reaction, but it has also demonstrated the fact that 
 it is of comparatively small value when compared with 
 salvarsan. For one case that becomes negative after treat- 
 ment with mercury there are hundreds that become nega- 
 tive after treatment with salvarsan, and although the 
 majority of both classes of cases relapse in time and again 
 present a positive Wassermann reaction, the relapses are 
 much less frequent among patients treated with salvarsan 
 than with mercury. 
 
 The relative efficiency of salvarsan and mercury in caus- 
 ing a disappearance of the Wassermann reaction is well 
 illustrated in patients who had previously been treated 
 with mercury before receiving salvarsan. Of 90 patients 
 who were treated with mercury by the mouth for nine 
 months or more before the administration of salvarsan 
 and who gave a positive Wassermann reaction, 68, or 70.5 
 per cent became negative within eight weeks after the ad- 
 ministration of the drug. < The intensity of the reaction 
 at the time of receiving salvarsan and the length of time 
 the patients had been taking mercury is given in Table 
 XLin. 
 
 Table XLIII 
 
 The Wassermann Test in Patients Who Had Taken Meecuet foe 
 Vaeious Periods op Time 
 
 METHOD or 
 
 TIME OP 
 TREATMENT 
 
 NTTMBEE 
 OP CASES 
 
 CHARACTER OP THE 
 
 REACTION 
 
 TREATMENT 
 
 + + 
 
 + 
 
 + - 
 
 Mercury by mouth 
 tt tt tt 
 
 {t it t i 
 
 (< tt It 
 
 9 months 
 
 1 year 
 
 2 years 
 
 3 years 
 
 17 
 26 
 17 
 
 8 
 
 8 
 
 16 
 
 7 
 
 3 
 
 7 
 8 
 9 
 4 
 
 2 
 2 
 1 
 1 
 
 TOTALS 
 
 
 68 
 
 34 
 
 28 
 
 6 
 
 The table shows that 26 of these patients had taken mer- 
 cury by mouth for a period of one year and that 16 of 
 them still gave a double-plus (four-plus) reaction; that 17 
 had received the same treatment for two years, of whom 
 7 still gave a double-plus reaction ; and that 8 had received 
 
THE EFFECT OF TREATMENT 
 
 185 
 
 treatment for three years, of whom 3 still gave a double- 
 plus reaction. Of the 68 cases not one had become nega- 
 tive as the result of mercury administered by the mouth, 
 but after the administration of salvarsan every one became 
 negative within 8 weeks. This is certainly very decisive 
 proof of the greater specific action of salvarsan and could 
 only be ascertained by the effect of the drugs in question 
 upon the Wassermann reaction. 
 
 Objection may be raised to the results recorded above by 
 calling attention to the fact that the administration of 
 mercury by the mouth is now well recognized to be the 
 very poorest of all ways of giving this drug, although it 
 is not so long ago that three years ' treatment by mouth with 
 mecury was considered by the best authorities upon the 
 disease as adequate for the cure of syphilis. In answer to 
 this objection Table XLIV is given, covering 18 patients 
 treated with hypodermic injections of mercury, a plan of 
 treatment generally acknowledged to be the most efficient 
 of the many ways of administering the drug. For pur- 
 poses of comparison the effect upon the Wasserman of 
 treatment with salvarsan is also included. 
 
 Table XLIV 
 
 The Comeaeative Eesults of Tkeatment with Mercury and Salvarsan 
 UPON THE Wassermann Eeaction 
 
 NUMBER OF 
 
 Mercmrial Treatment 
 
 Salvarsan 
 
 Treatment 
 
 INJECTIONS OF 
 
 CHARACTER OF 
 
 DOSE 0.6 GM. 
 
 CHARACTER OF 
 
 
 
 
 
 
 GRET OIL 
 
 REACTION 
 
 
 REACTION 
 
 8 
 
 7 
 
 + + 
 
 Intramuscular 
 
 ^ 
 
 1 
 
 8 
 
 + 
 
 te ti 
 
 _ 
 
 1 
 
 9 
 
 + + 
 
 11 tt 
 
 _ 
 
 1 
 
 11 
 
 + + 
 
 ti tt 
 
 _ 
 
 3 
 
 15 
 
 + + 
 
 Intravenous 
 
 _ 
 
 1 , 
 
 15 
 
 + 
 
 Intramuscular 
 
 „ 
 
 4 
 
 18 
 
 + + 
 
 tt tt 
 
 _ 
 
 2 
 
 19 
 
 + + 
 
 Intravenous 
 
 _ 
 
 1 
 
 20 
 
 + + 
 
 It It 
 
 _ 
 
 1 
 
 25 
 
 + 
 
 Intramuscular 
 
 _ 
 
 1 
 
 30 
 
 + 
 
 tt tt 
 
 - 
 
 From a consideration of this table it is evident that 
 cases having had as many as 18 to 20 injections of grey oil 
 
186 THE WASSEEMANN TEST 
 
 still gave a double-plus Wassermann reaction and that 
 eases having as high as 25 and 30 injections still gave a plus 
 (three-plus) reaction. After the administration of salvar- 
 san all became negative within 8 weeks, and in 12 of the 
 cases only one intramuscular injection of 0.6 gm. of sal- 
 varsan was administered. While there is reason to believe 
 that previous treatment with mercury may have had some- 
 thing to do with the good effects produced by salvarsan 
 upon the reaction, it must be admitted that the results ob- 
 tained in these cases demonstrate beyond all question the 
 superior specific value of salvarsan upon Treponema pal- 
 lidum. 
 
 Patients who have been previously well treated with mer- 
 cury show a higher percentage of negative results after 
 treatment with salvarsan than those who have received no 
 mercurial treatment. Thus of 75 patients who had re- 
 ceived mercurial treatment before receiving salvarsan, 84 
 per cent became negative, while of 110 patients who had 
 received no treatment before the administration of salvar- 
 san, only 74.5 per cent became negative. 
 
 In order to illustrate the persistence of the positive 
 Wassermann reaction after treatment with mercurials Ta- 
 ble XLY has been prepared- giving the method of treat- 
 ment, the length of time the patient was under treatment, 
 the number of cases observedj and the intensity of the 
 reaction. 
 
 The table illustrates very well the effect of treatment 
 with mercury upon the Wassermann reaction, as it occurs 
 in the usual routine of serological work in a Wassermann 
 laboratory. It shows that continued treatment with the 
 drug for long periods of time has but little effect upon the 
 reaction in the majority of instances and that interrupted 
 treatment continued for many years does not produce a 
 negative reaction in many cases. It should not be thought, 
 however, from the data given here, that the Wassermann 
 reaction never becomes negative after treatment with mer- 
 cury, for in a considerable proportion of cases it does, and 
 
THE EFFECT OF TEEATMENT 
 
 187 
 
 Table XLV 
 
 Illustrating the Persistence of the Wassermann Eeaction after 
 Treatment with Mercury 
 
 METHOD OF 
 
 LENGTH OF 
 TREATMENT 
 
 NUMBER OF 
 CASES 
 
 INTENSITY OP THE REACTION 
 
 TREATMENT 
 
 + + 
 
 + 
 
 + - 
 
 By mouth 
 
 1 month 
 
 2 
 
 
 2 
 
 
 '( " 
 
 2 months 
 
 3 
 
 1 
 
 2 
 
 
 t( it 
 
 3 " 
 
 4 
 
 2 
 
 2 
 
 
 1 1 it 
 
 5 " 
 
 6 
 
 3 
 
 3 
 
 
 1 1 ti 
 
 6 " 
 
 10 
 
 7 
 
 1 
 
 2 
 
 1 1 ti 
 
 7 " 
 
 4 
 
 3 
 
 1 
 
 
 1 1 it 
 
 8 " 
 
 5 
 
 4 
 
 1 
 
 
 t i it 
 
 9 " 
 
 10 
 
 7 
 
 1 
 
 2 
 
 It it 
 
 10 " 
 
 3 
 
 2 
 
 1 
 
 
 1 1 ti 
 
 11 " 
 
 3 
 
 3 
 
 
 
 it tt 
 
 1 year 
 
 24 
 
 13 
 
 6 
 
 5 
 
 it it 
 
 14 months 
 
 2. 
 
 . 2 
 
 
 
 it tt 
 
 15 " s 
 
 2 
 
 1 
 
 1 
 
 
 It it 
 
 16 " 
 
 4 
 
 3 
 
 
 1 
 
 ti it 
 
 17 " 
 
 1 
 
 1 
 
 
 
 it it 
 
 18 " 
 
 7 
 
 1 
 
 4 
 
 2 
 
 it it 
 
 19 " 
 
 4 
 
 3 
 
 1 
 
 
 it it 
 
 20 " 
 
 2 
 
 1 
 
 1 
 
 
 it tt 
 
 2 years 
 
 15 
 
 8 
 
 5 
 
 2 
 
 a tt 
 
 2.5 " 
 
 3 
 
 
 2 
 
 1 
 
 a tt 
 
 3 " 
 
 , 5 
 
 1 
 
 2 
 
 2 
 
 it tt 
 
 4 " 
 
 1 
 
 1 
 
 
 
 By mouth* 
 
 7 " 
 
 1 
 
 
 1 
 
 
 By mouth* 
 
 10 " 
 
 1 
 
 1 
 
 
 
 By mouth* 
 
 12 " 
 
 1 
 
 
 1 
 
 
 Inunctions 
 
 2 months 
 
 2 
 
 2 
 
 
 
 it 
 
 5 " 
 
 1 
 
 
 1 
 
 
 it 
 
 6 " 
 
 2 
 
 2 
 
 
 
 a 
 
 1 year 
 
 1 
 
 1 
 
 
 
 'Interrupted treatment during this time. + + indicates complete inhibition of hemolysis. 
 
 the writer has observed many cases in which a negative 
 Wassermann reaction was obtained after from one to two 
 years proper treatment with this drug but it has been his 
 experience that most cases becoming negative after mer- 
 curial treatment relapse in the course of months or a year 
 or two. While he would not go so far as to state that the 
 Wassermann test never becomes permanently negative 
 after treatment with mercury, it has been his experience 
 that it is only in very rare instances that a permanently 
 negative result is obtained from treatment with this drug 
 alone, no matter how it is administered or in what 
 dosage. In fact, it has been proved in rabbits experimen- 
 
188 THE WASSEBMANH TEST 
 
 tally infected with syphilis, that before the animals can be 
 rendered sterile as regards Treponema pallidum so much 
 mercury has to be administered as to cause either the 
 death of the animal or very serious pathological lesions 
 due to the drug. In the writer's rather large experience he 
 has never personally followed a case treated with mercury 
 alone, in which the Wassermann test was positive before 
 treatment, that became permanently negative, although 
 many cases have lost the positive reaction for a while but 
 have invariably become positive again upon the cessation 
 of treatment. However, that mercury does cure syphilis 
 in some instances, is proved by the fact that individuals 
 are encountered who undoubtedly had the disease but who 
 have been without symptoms for many years and who give 
 a negative Wassermann reaction whenever tested. 
 
 If the patient's blood serum be carefully titrated after 
 the administration of either salvarsan or mercury, it will 
 be found that in almost every instance the Wassermann 
 reaction is influenced to some extent, although it may never 
 become negative. Thus, many cases showing a double-plus 
 reaction, or absolute inhibition of hemolysis, with quanti- 
 ties of blood serum as small as 0.02 c.c. will become nega- 
 tive for this amount of serum, although giving a positive 
 reaction with the usual quantity of serum used in the rou- 
 tine tests ; i. e., 0.1 c.c. By titrating the inhibitory strength 
 of each patient's serum it will be found that, although the 
 Wassermann test may apparently be uninfluenced when 
 the diagnostic amount of serum is used, that is, 0.1 c.c, 
 some diminution in the strength of the reaction will be 
 observed in amounts less than 0.1 c.c. In using the Was- 
 sermann test as a control of treatment the titration of the 
 patient's serum is of great importance, as will be noted. 
 
 Not only will treatment with salvarsan or mercury mark- 
 edly influence the strength of the reaction but if treatment 
 is coimnenced early in the primary stage of the disease it 
 will sometimes prevent the development of a positive re- 
 action, although clinical symptoms may occur. The writer 
 
WASSEEMANN RKACTION AS CONTKOL OF TREATMENT 189 
 
 has observed several such instances, in which secondary 
 symptoms developed in spite of treatment, but in which a 
 positive WassQrmann reaction was never obtained. 
 
 The Wassermarm Reaction as a Control of Treatment 
 
 Aside from its value as a diagnostic agent, the great- 
 est value of the Wassermann test for syphilis is found in 
 its use as a guide to the treatment of the disease. It will 
 be evident, from what has been said of the effect of treat- 
 ment upon the positive rea'ction that, if scientifically used, 
 the efficacy of any form of treatment advocated for syph- 
 ilis may be absolutely determined by employing the test 
 as a control of that treatment. In the army the test has 
 been of the greatest practical value in ascertaining the rel- 
 ative efficiency of the various methods of treating syph- 
 ilis and it has been very largely used for this purpose. As 
 an illustration of the extent of the use of the reactioif' in 
 the army in the control of the treatment of syphilis, the 
 writer may say that he has personally made almost twenty 
 thousand Wassermann tests upon treated patients, all of 
 these tests being for the purpose of controlling the treat- 
 ment of the disease, and his experience has been that of all 
 other medical officers who have had charge of the army 
 laboratories where the Wassermann test is performed. 
 
 By means of the Wassermann test, repeated at frequent 
 intervals, one is able to diagnose a relapse of syphilitic 
 infection long before the appearance of clinical symptoms, 
 and in this way enabled to treat the infection before it has 
 resulted in such symptoms, thus saving the patient much 
 anxiety and discomfort, and obviously at a stage when it is 
 much more amenable to treatment than after gross clinical 
 symptoms of relapse have occurred. The writer believes 
 that the first symptom of a relapse of a syphilitic infection, 
 in the vast majority of cases, is the recurrence of a posi- 
 tive Wassermann reaction, and that this recurrence often 
 happens weeks and even months before marked clinical 
 
190 THE WASSBEMANW TEST 
 
 symptoms are noted. In other words, the positive Wasser- 
 mann reaction is the most delicate symptom of relapse in 
 syphilis that is known, a symptom that occurs long before 
 definite clinical symptoms, and thus it is of the greatest 
 value as an indication of the need of further treatment in 
 cases where it has once disappeared and then recurs. For 
 this reason all cases of syphilis that have been given specific 
 treatment should be tested at frequent intervals in order' 
 to ascertain the effect of the treatment upon the reaction, 
 and after a negative reaction is once obtained a test should 
 be made at least every three months for a period of at least 
 one year, when, if the reaction is still negative, a provoca- 
 tive Wassermann test, a Wassermann test upon the spinal 
 fluid, and a luetin test should be made, before the patient is 
 pronounced cured. 
 
 The writer has already mentioned the great importance 
 of titrating the complement-binding power of the syphilitic 
 patient's blood serum in judging of the effect of any treat- 
 ment upon the Wassermann reaction and in using the test 
 as a control of treatment, and the method of making such 
 titrations. The titration of the patient 's blood serum should 
 be done whenever it is possible^ but especially in those cases 
 that show a continued double-plus (four-plus) reaction 
 after specific treatment has been administered for some 
 time. In such cases, also, the spinal fluid should be tested 
 if the Wassermann on the blood shows no diminution when 
 the serum is titrated, for it is in this class of cases that early 
 involvement of the central nervous system is most apt to 
 have occurred. 
 
 The data derived from the use of the Wassermann test 
 as a control of treatment has enabled us to use both mer- 
 cury and salvarsan intelligently and has added greatly 
 to our knowledge of the action of other drugs that have 
 been advocated from time to time in the treatment of the 
 disease. It has demonstrated beyond all doubt that the 
 absence of clinical symptoms is no proof of cure in this 
 
WASSEEMANN REACTION AS CONTROL OP TREATMENT 191 
 
 disease, even though symptoms may be absent for months 
 or years, and that a single negative Wassermann reaction 
 is of absolutely no value in determining either the question 
 of the existence of syphilis or of its cure. ■ 
 
 Perhaps the most striking proof of the value of the Was- 
 sermann test in the control of the treatment of sypfiilis is 
 the results that have been obtained by numerous workers 
 upon the occurrence of relapse after treatment with sal- 
 varsan, as shown by the Wasserraann test. In 1913, the 
 writer published"" certain observations upon relapse in 
 syphilis after treatment with salvarsan and has continued 
 collecting data upon this subject which have confirmed the 
 observations published. They were undertaken for the 
 purpose of determining the relation of relapse to the dose 
 and method of administering the drug but serve equally 
 well to illustrate the value of the Wassermann test in con- 
 trolling the treatment of syphilis and in ascertaining the 
 therapeutic results following treatment with a specific 
 drug. Used in this manner the Wassermann test has fur- 
 nished the following information regarding the occurrence 
 of relapses after treatment with salvarsan: 
 
 Four hundred cases were selected for study, and were 
 observed for a period of eight months or more. Of the 400 
 cases, 202, or 50.5 per cent remained positive after the 
 administration of the drug, while 198, or 49.5 per cent be- 
 came negative to the Wassermann test after salvarsan was 
 given. Of the 198 cases that became negative, 172, or 86.8 
 per cent have relapsed ; i. e., the Wassermann test has 
 again become positive. 
 
 The Stage of Syphilis in Relation to Relapse. — The re- 
 lation of the stage of syphilis to relapse following treat- 
 ment with salvarsan is shown in Table XL VI. 
 
 A consideration of the table shows that 88.5 per cent of 
 the primary cases relapsed, as shown by the Wassermann 
 test ; 84.3 per cent of the secondary cases ; 88.8 per cent of 
 the latent ; and only 69.5 of the tertiary cases. The latter 
 had all received considerable treatment with mercury prior 
 
192 
 
 THE WASSERMANN TEST 
 
 Table XL VI 
 
 The Stage of Syphilis in Relation to Relapse after Treatment 
 WITH Salvaesan 
 
 stage of disease 
 
 number of cases 
 
 number of 
 relapses 
 
 % OF RELAPSES 
 
 Primary 
 Secondary 
 Tertiary 
 Latent 
 
 35 
 
 115 
 
 23 
 
 25 
 
 31 
 97 
 16 
 22 
 
 88.5 
 
 '84.3 
 69.5 
 
 88.8 
 
 to the administration of salvarsan, so that the compara- 
 tively low percentage of relapses in this stage undoubtedly 
 was due to this fact, for the writer believes that in un- 
 treated cases the tertiary stage would show as large, and 
 probably a larger, percentage of relapses than either the 
 primary or secondary. The latent stage showed the 
 largest percentage of relapses and this was expected, as 
 it is this class of cases that most often prove resistant to 
 treatment, so far as the disappearance of the Wassermann 
 test is concerned, but the difference is only slight, as shown 
 by the table. 
 
 The Relation of the Intensity of the Wassermann Reac- 
 tion to Relapse. — The cases giving the strongest Wasser- 
 mann reaction furnished the largest percentage , of re- 
 lapses, as is shown in Table XLVII. Here, as in all tables 
 in this work, the sign, + +, indicates complete inhibition of 
 hemolysis, the four-plus reaction of many authorities : 
 
 Table XLVII 
 
 The Relation of the Intensity of the Wassermann Reaction to Relapses 
 AFTER Treatment with Salvaesan 
 
 chakactee op 
 reaction 
 
 number of cases 
 
 number of 
 relapses 
 
 % of relapses 
 
 + + 
 
 + 
 
 110 
 88 
 
 104 
 68 
 
 94.5 
 
 77.2 
 
 
 198 
 
 172 
 
 86.8 
 
 This table demonstrates that of the 110 cases giving a 
 double-plus reaction, no less than 104, or 94.5 per cent 
 relapsed, while of the 88 cases giving a plus (three-plus 
 
WASSEEMANN EBACTIOlT AS CONTROL OF TREATMENT 193 
 
 or two-plus)' reaction, only 68 or 77 pter cent relapsed. 
 Therefore it is evident that the stronger the Wassermann 
 reaction the greater the number of relapses following 
 treatnaent with salvarsan. 
 
 Time of Relapse after Treatment with Salvarsan.— By 
 controlling the treatment of syphilis with salvarsan it has 
 been possible for us to obtain accurate data regarding the 
 time of relapse after treatment with salvarsan; i. e., the 
 period elapsing from the time the Wassermann test be- 
 came negative to the time in which the test again became 
 positive. The data accumulated show that, in the major- 
 ity of instances, a Wassermann relapse, as it may be called, 
 occurs within eight months after cessation of treatment. 
 Thus, of the 172 cases that relapsed after treatment with 
 this drug, no less than ' 159 cases relapsed within eight 
 months after treatment. Table XL VIII gives the time of 
 relapse after treatment observed in these cases. 
 
 Table XLVIII 
 
 The Time or Eelapse after Treatment with Salvarsan 
 
 as Shown ry the 
 
 Wassermann Test 
 
 
 TIME OF relapse 
 
 number or CASES 
 
 time of relapse. 
 
 number of cases 
 
 1 month 
 
 8 
 
 5 months 
 
 17 
 
 ' 5 weeks 
 
 7 
 
 6 " 
 
 12 
 
 6 " 
 
 5 
 
 7 " 
 
 5 
 
 7 " 
 
 6 
 
 8 " 
 
 5 
 
 2 months 
 
 38 
 
 9 " 
 
 2 
 
 9 weeks 
 
 1 
 
 10 " 
 
 2 
 
 10 " 
 
 7 
 
 11 " 
 
 1 
 
 11 " 
 
 2 
 
 12 " 
 
 2 
 
 3 months 
 
 26 
 
 13 " 
 
 1 
 
 14 weeks 
 
 1 
 
 15 " 
 
 1 
 
 4 months 
 
 19 
 
 22 " 
 
 1 
 
 
 
 24 " 
 
 1 
 
 From this table it will be noted that no less than 38 cases 
 relapsed' at the end of two months and 26 at the end of 
 three months, so that it would appear that these periods 
 are of special significance as regards the time of relapse. 
 It is also interesting to note that no less than 100 of the 
 172 cases that relapsed did so by the end of three months 
 and no less than 149 of the 172 cases by the end of six 
 
194 THE WASSEBMANN TEST 
 
 months after cessation of treatment. From this it would 
 appear that the vast majority of cases of syphilis treated 
 as these were, with salvarsan, will relapse within six 
 months so far as the Wassermann reaction is concerned, 
 and that it is during this period that repeated Wassermann 
 tests are most essential. 
 
 The statement that has already been made regarding the 
 worthlessness of a single negative Wassermann reaction 
 is well illustrated by this table, and even in those cases in 
 which the reaction remains negative for a long period of 
 time, no assurance can be given that it will not again be- 
 come positive, for it will be observed that some of these 
 cases had a nega!tive Wassermann reaction for as long as 
 oine and two years and became positive. 
 
 As regards the time of relapse in the various stages of 
 syphilis it may be stated that the Wassermann tests 
 showed that the average time of relapse in primary cases 
 was 4% months; in the secondary cases, 4 months; in the 
 tertiary cases, 3 months; and in the latent eases, 3^^ 
 months. The latent cases included both early and late 
 latent cases, which accounts for the longer period of re- 
 lapse in this class. As will be noted, relapses occur earlier 
 the longer the syphilitic infection has lasted. 
 
 The Relation of the Method of Administration of Salvar- 
 san to Relapses. — The Wassermann test has shown that the 
 percentage of relapses after salvarsan varies considerably 
 with the method of administration of the drug. The three 
 methods that have been tested in this way have been the 
 intramuscular injection of the drug, the intravenous in- 
 jection, and the combined intramuscular and intravenous 
 injection. Table XLIX gives the number of cases treated 
 which have been thus tested and the number of relapses, 
 together with the percentage of relapse in each class of 
 cases. 
 
 The number of cases tested that were treated by the com- 
 bined intramuscular and intravenous method was so small 
 that the percentage of relapses ; i. e., 66.6 per cent, can not 
 
WASSERMANN REACTION AS CONTROL OF TREATMENT 195 
 
 Table XLIX . 
 
 The Relation or the Method op Administration of Salvaksan to Belapse, 
 AS Shown by the Wassermann Test 
 
 method op 
 
 total number 
 
 OF CASES 
 
 RELAPSES 
 
 administration 
 
 NUMBER 
 
 % 
 
 Intramuscular 
 Intravenous 
 
 Combined intramuscular 
 and intravenous 
 
 74 
 115 ' 
 
 9 
 
 54 
 106 
 
 6 
 
 72.9 
 92.1 
 
 66.6 
 
 be considered as a fair estimate of the percentage that 
 would relapse were a greater number of cases tested. 
 However, the number of cases tested that were given sal- 
 varsan intramuscularly and intravenously is large enough 
 to base fair estimates upon, and it is very evident that a 
 much smaller number of relapses follow the intramuscular 
 method of administering the drug than the intravenous 
 method. In justice to the intravenous method it should be 
 stated that fully 50 per cent of the cases tested had only 
 received one dose of the drug, but even after two injections 
 the results were not as good as after one intramuscular 
 injection. Of 80 cases receiving one intravenous injection 
 of 0.06 gm. of salvarsan 78 relapsed, or 97.5 per cent, while 
 of 67 cases receiving the same dose intramuscularly, 51 
 relapsed, or 76 per cent* 
 
 From what has been stated regarding the study of re- 
 lapse in syphilis after the administration of salvarsan 
 some idea may be gained regarding the great value of the 
 Wassermann test when it is used as a control of treatment 
 or as a test of the efficiency of any drug recommended in 
 the treatment of the disease. The results that have fol- 
 lowed the modern method of treatment; i.e., numerous 
 intravenous injections of salvarsan combined with mercu- 
 rials, given in the proper manner, are far superior to those 
 which have been noted in this chapter with either drug 
 alone. There is but one way of determining what these 
 results are and that is by the use of the Wassermann test 
 as a control of the treatment of the disease. Without it, 
 the physician is at as great a disadvantage in judging of 
 
196 THE WASSBEMAKN TEST 
 
 the results of his treatment of syphilis as the mariner! 
 would be without a compass, and this test may well be 
 called the compass of the physician in the treatment of 
 this disease. 
 
 The Interpretation of the Reaction when the Test is used 
 in the Control of Treatment. — ^A Wassermann test should 
 not be performed for the purpose of controlling the treat- 
 ment of syphilis until treatment with salvarsan, mercu- 
 rials, or other agents has been omitted for at least a month 
 and the test should be repeated at monthly intervals re- 
 gardless of the reaction if treatment is not again instituted. 
 If the reaction is negative monthly tests should be made 
 in all cases possible. If it remains persistently negative 
 treatment should not be persisted in, but if there is any 
 trace of inhibition beyond a weak plus-minus reaction, the 
 treatment should be continued. It should always he re- 
 membered that a single negative reaction is ahsolutely val- 
 uless as indicating the cure of the infection and repeated 
 tests must be made with a similar result before any opin- 
 ion favoring cure is warranted. Too many patients have 
 been assured that they were cured of syphilis on the 
 strength of one or two negative Wassermann reactions, 
 with disastrous results to innocent individuals, as well as 
 to the patient, and the utmost caution should be exercised 
 in expressing any opinion regarding ultimate cure in syph- 
 ilis. It should be remembered that the "Wassermann test 
 has shown that the vast majority of treated syphilitic in- 
 fections eventually relapse, even though the treatment has 
 been scientific and thorough, so that the prognosis in every 
 case should be very guarded. 
 
 In primary infections which have been treated and in 
 which the Wassermann test has become negative, a return 
 of the reaction even to the extent of a plus-minus reaction, 
 when it is persistent, should indicate further treatment. 
 In the secondary cases a plus-minus reaction is not of as 
 much importance, as most of these cases will give a 
 stronger reaction if they relapse, but even in such cases 
 
PROVOCATIVE WASSEEMANN EBACTION 197 
 
 if the plus-minus reaction persists for several weeks, treat- 
 ment should be instituted. In tertiary and latent cases the 
 occurrence of a plus (two- or three-plus of some authors) 
 reaction after treatment has been stopped should be fol- 
 lowed by the resumption of the treatment. 
 
 Treatment in syphilis should never be discontinued until 
 the "VVasserraann test has become negative unless it is 
 found that the case is "Wassermann fast"; i. e., that no 
 amount of treatment will cause the reaction to become neg- 
 ative. Wassermann fast cases are frequently met with in 
 practice and occur during every stage of the disease but 
 such cases occur more frequently in tertiary and latent 
 infections than in primary or secondary. In the "Was- 
 sermann fast" cases the clinical symptoms, aside from the 
 positive Wassermann reaction, may, and generally do, dis- 
 appear xmder treatment, but the reaction never becomes 
 negative. 
 
 The Provocative Wassermann Reaction 
 
 In 1910, Gennerich^"^ called attention to the fact that 
 cases presenting a negative Wassermann reaction often 
 showed a positive reaction after the administration of a 
 dose of salvarsan, this reaction occurring either within a 
 few hours after the injection or within two weeks follow- 
 ing it. He called attention to the value of this phenomenon 
 in the diagnosis of obscure syphilitic infections, and as a 
 method of determining whether a patient is really cured 
 of the infection. He suggested that this so-called "pro- 
 vocative Wassermann" be used in treated cases that had 
 presented negative reactions for some time, and were pre- 
 sumably cured, as, in such instances he had found the 
 provocative test frequently positive, thus demonstrating 
 that the disease was still present. Milian,"* of Paris, 
 about the same time, called attention to the value of the 
 procedure in the study of the disease, as well as Herx- 
 heimer,"^ and Michaelis,"" but to Gennerich belongs the 
 
198 THE WASSBKMANN TEST 
 
 credit of first actually applying the test in the diagnosis 
 of syphilis and as a control of the treatment of the disease. 
 
 Method of Making a Provocative Wassermann Test. — 
 The writer recommends the following procedure in making 
 a provocative Wassermann test: The suspected patient 
 is given from 0.3 to 0.6 gm. of salvarsan intravenously and 
 the blood is collected and tested 12 hours after the admin- 
 istration of the drug; 24 hours after administration; and 
 every day for at least ten days. If the negative reaction 
 is to become positive, it generally does so within from a 
 few hours to 5 or 6 days after the salvarsan has been 
 given, but delayed reactions have occurred not infre- 
 quently up to 10 days after administration, and it is good 
 practice to make a test upon the 12th and 14th days after 
 administration, in addition to those recommended. Dur- 
 ing the first two days after salvarsan has been given, if 
 it is possible, the blood should be collected at intervals of 
 three or four hours and tested, as sometimes the reaction 
 is very evanescent in character and quickly disappears. 
 However, owing to the labor involved in maldng so many 
 tests it is seldom practicable to do this, and the procedure 
 first mentioned will generally prove sufficient to detect the 
 reaction, if it occurs. Stokes and O'Leary^^^ recommend 
 that the blood be dra"WTi at the time of injection and daily 
 thereafter for one week, and that all blood collected be 
 tested with several antigens at one sitting and with the 
 same reagents, in order to avoid any difference in the re- 
 sults that might occur from variations in the reagents used 
 in the test from day to day. 
 
 Results of the Test. — ^In a recent contribution, King,"** as 
 the result of his investigation of the provocative test upon 
 a very small number of patients, came to the conclusion 
 that the provocative Wassermann reaction does not exist, 
 but that the positive result that sometimes follows an in- 
 jection of salvarsan is due either to normal variations in 
 the complement-binding power of syphilitic serum, as 
 shown by the writer, or to variations in the technic or 
 
PBOVOCATIVB WASSEEMANN" EBACTIOST 
 
 199 
 
 reagents used in the test when made upon different days. 
 His work is very far from convincing and the united ex- 
 perience of hundreds of observers who have used the pro- 
 vocative test in their laboratories and practice is unan- 
 imous as regards the actual occurrence of this phenom- 
 enon and as to the value of the test in the diagnosis of 
 syphilis and the control of its treatment. 
 
 Nichols,"^ after stating that it is a well-established fact 
 that in a syphilitic a negative Wassermann reaction can be 
 converted into a positive one by an injection of salvarsan, 
 gives the following table illustrative of the effect of the 
 provocative test in ten patients in whom a luetin test was 
 also performed at the same time. Table L gives the re- 
 sults that he obtained in these cases : 
 
 Table L 
 
 Results of the Provocative Wassermann Test and the Luetin Test. 
 (After Nichols.) 
 
 
 time ordinary wassermann 
 
 
 PROVOCATrVE 
 
 case no. 
 
 REACTION HAD BEEN 
 
 LUETIN TEST 
 
 WASSERMANN 
 
 
 NEGATIVE 
 
 
 TEST 
 
 1 
 
 16 months 
 
 _ 
 
 _ 
 
 2 
 
 21 " 
 
 
 
 3 
 
 24 " 
 
 
 
 4 
 
 24 " 
 
 _ 
 
 
 5 
 
 24 " 
 
 _ 
 
 _ 
 
 6 
 
 15 " 
 
 + 
 
 + + 
 
 r 
 
 17 " 
 
 + 
 
 + + 
 
 8 
 
 18 " 
 
 + 
 
 + + 
 
 9 
 
 19 " 
 
 + 
 
 + + 
 
 10 
 
 ? 
 
 + 
 
 + + 
 
 This table is conclusive proof of the occurrence of the 
 provocative reaction, as it shows complete agreement be- 
 tween the results of this test 'and the luetin test, for it is 
 hardly probable that, if the positive reaction in these cases 
 was due to variation in the complement-binding power of 
 the blood serum or inaccuracies in technic, the results of 
 the provocative test would be identical with the results of 
 the Kietin test in every case. 
 
 Nichols calls attention to Case 10, in the above table, be- 
 
200 THE WASSBEMANN TEST 
 
 cause of the interesting results obtained as regards a 
 delayed provocative reaction. The patient was infected 
 in 1906 and had two years of treatment by the mouth with 
 mercury and potassium iodide. No symptoms had oc- 
 curred since 1908 and in March, 1913, he gave a negative 
 reaction. On April 18th he was given an intravenous injec- 
 tion of 0.6 gm. of salvarsan and his blood was tested every 
 day with the following results : April 18th, negative ; April 
 19th, negative; April 20th, negative; April 21st, negative; 
 April 22nd, negative; April 23rd, double-plus (four-plus). 
 
 This case well illustrates the value of the provocative / 
 reaction in patients who have had no symptoms of syphilis 
 for several years and who give a negative Wassermann test, 
 
 Stokes and O'Leary,"* in a very excellent study of th 
 provocative Wassermann reaction, obtained very signifi^ 
 cant results. They made 103 provocative tests, of whidi 
 19, or 18.4 per cent resulted in a reversal of the ordinal^ 
 Wassermann from negative to positive. This small per- 
 centage of positive results with the test is not unusual aid 
 it has been the writer's experience that it is seldom that 
 a higher percentage than 20 per cent is obtained with the 
 provocative Wassermann test upon limited groups of 
 cases. If thousands of tests were made it is probable that 
 the positive percentage would be somewhat higher but 
 from 18 to 20 per cent of positive results is about all that 
 can be expected from the reaction. 
 
 As to the character of the cases showing a positive re- 
 action with the test, Stokes and O'Leary state that 26.3 
 per cent presented no clinical symptoms of syphilis, but 
 that the remainder show suspicious symptoms although 
 the Wassermann was negative. However, they state that 
 "In no one of the positive cases were the signs so indu- 
 bitable that the provocative could be regarded as unneces- 
 sary or merely confirmatory." They say further: "we 
 believe the test has distinct value in determining the status 
 of a patent under treatment." 
 
 Stokes and O'Leary give Table LI showing the results 
 
PROVOCATIVE WASSEKMANN REACTION 
 
 201 
 
 obtained with the provocative Wassermann test in differ- 
 ent types of syj)hilis. 
 
 Table LI 
 Epficienot of the Peovocativb Test in Various Types op Syphilis 
 
 TYPE 
 
 NUMBER OP CASES 
 
 % positive 
 
 Heredosyphilis 
 
 2 
 
 
 
 Osseous 
 
 5 
 
 20 
 
 Central nervous system 
 
 12 
 
 25 
 
 Vascular 
 
 3 
 
 33 
 
 Latent 
 
 10 
 
 40 
 
 Late cutaneous 
 
 5 
 
 60 
 
 Late mucous membrane 
 
 5 
 
 80 
 
 While they admit that the number of cases treated is. 
 not large enough to base accurate conclusions upon, they 
 state that, from their series, it appears as though the pro- 
 vocative test is least useful in the class of cases where it is 
 most needed, but, even if this proves to be true, and we 
 can only expect 20 per cent of positive results in osseous 
 syphilis, 25 per cent in syphUis of the central nervous 
 system, and 33 per cent in vascular syphilis, the writer 
 believes that the test is well worth while, as often it will 
 be the only method available for mailing a diagnosis in 
 this class of cases. 
 
 The indications for a provocative Wassermann reaction 
 are given by Stokes and O'Leary in their paper and are 
 so admirable that this section of the paper is here quoted : 
 
 "1. A definite history of primary or secondary lesions 
 or a suspicious genital sore of any description. (With a 
 negative Wassermann.)' 
 
 *'2. Syphilis in husband or wife or a history of a sore 
 in either. 
 
 "3. Treated cases to determine the fact of cure or need 
 for further treatment. One-third of the cases thus tested 
 by us, gave a positive provocative effect. 
 
 "4." Obscure bone or joint lesions. 
 
 "5. Histories of miscarriages unless the anatomical 
 cause is glaringly obvious. 
 
202 
 
 THE WASSEEMANN" TEST 
 
 "6. Mothers of syphilitic children without clinical signs 
 of the disease. 
 
 "7. Cases with a history of a positive Wassermann else- 
 where, negative on present examination. 
 
 "8. Mental deviates and constitutionally inferior indi- 
 viduals with suspicious histories. 
 
 "9. Certain signs elicited by special examinations, such 
 as decreased bone conduction with normal hearing, chorio- 
 retinitis and retinitis pigmentosa, bilateral dacryocystitis in 
 childhood," etc. 
 
 In all the above conditions the writer agrees with the 
 authors that a provocative Wassermann test should be 
 made, if possible, for in nearly all cases in which the indi- 
 cations for the test cited have occurred, the writer has 
 found the test of service. 
 
 As an illustration of the results obtained with the test 
 in cases of syphilis that have not shown symptoms for a 
 long period of time and in which the ordinary Wassermann 
 test has been negative for months and even years, Table 
 LII is given : 
 
 Table LII 
 Results op Provocative Wassermann Test in Syphilis 
 
 
 TIME SINCE 
 TREATMENT 
 
 TIME SINCE WAS- 
 
 RESULT OP 
 
 RESULT OP 
 
 CASE NO. 
 
 SERMANN BECAME 
 
 WASSERMANN 
 
 PEOVOCATIVB 
 
 
 NEGATIVE 
 
 REACTION 
 
 REACTION 
 
 1 
 
 12 months 
 
 . 12 months 
 
 _ 
 
 + + 
 
 B 
 
 13 " 
 
 13 " 
 
 __ 
 
 _ 
 
 3 
 
 14 " 
 
 14 " 
 
 _ 
 
 + + 
 
 4i 
 
 15 " 
 
 15 " 
 
 _ 
 
 + + 
 
 5 
 
 16 " 
 
 16 " 
 
 _ 
 
 _ 
 
 6 
 
 17 " 
 
 17 " 
 
 _ 
 
 + + 
 
 7 
 
 18 " 
 
 18 " 
 
 _ 
 
 + + 
 
 8 
 
 19 " 
 
 19 " 
 
 _ 
 
 + + 
 
 g 
 
 20 " 
 
 20 " 
 
 _ 
 
 + + 
 
 10 
 
 21 " 
 
 21 " 
 
 _ 
 
 _ 
 
 11 
 
 24 " 
 
 24 " 
 
 _ 
 
 _ 
 
 12 
 
 24 " 
 
 24 " 
 
 _ 
 
 + + 
 
 13 
 
 24 " 
 
 24 " 
 
 _ 
 
 _ 
 
 14 
 
 25 " 
 
 25 " 
 
 - 
 
 + + 
 
 The cases given in the above table are selected from a 
 large number because they show the results of the provoe- 
 
PROVOCATIVE WASSEEMANN REACTION 203 
 
 ative reaction month after month, as regards time, and 
 because the data relating to them is of such a nature as to 
 make the test definite and conclusive. It will be seen that 
 a positive reaction was obtained as long as 25 months after 
 the cessation of treatment, during which time no symptoms 
 of syphilis had been noted and the Wassermann reaction 
 was always negative, when tested. 
 
 The writer is firmly convinced, from personal experience, 
 that a dose of salvarsan or neosalvarsan will frequently 
 convert a negative Wassermann reaction into a positive 
 one and that this phenomenon is not due to any variation 
 occurring normally in the patient's blood serum or to dif- 
 ferences in technic or in the reagents used in making the 
 test. When one has tested cases week after week, always 
 with a negative result, and when these same cases after a 
 dose of salvarsan or neosalvarsan become positive 'within 
 a few hours or days after the administration of the drug, 
 there can be only one conclusion, and that is that the posi- 
 tive result has been produced _by the drug, and the writer 
 has had this experience too many times to allow of the 
 least doubt in his mind of the existence of a provocative 
 reaction following the administration of these drugs. It 
 is true that the test often fails in cases in which one would 
 expect it to be positive, but this is no proof of the non- 
 existence of the phenomenon. 
 
 Cause of the Provocative Reaction. — The exact nature of 
 the provocative reaction is still undetermined. There are 
 two theories that have been discussed in this connection, 
 the first being that the reaction is due to the stimulation 
 of the treponemas by the drug and the consequent pro- 
 duction of more of the substance or substances which give 
 rise to a positive Wassermann reaction; while the second 
 is that the drug kills sufficient of the treponemas to increase 
 the amount of toxin and thus the syphilitic antibody, as 
 it may be called, is increased. The first theory depends 
 upon the belief that Treponema pallidum, during its life 
 and multiplication in the tissues causes the production of 
 
204 THE WASSBEMANK' TEST 
 
 some substance which produces the Wassermann reaction, 
 and that a small dose of salvarsan stimulates the few re- 
 maining organisms in the negative Wassermann cases to 
 overproduction of this substance for a short time, and thus 
 the negative Wassermann reaction becomes positive. The 
 second theory rests upon the belief that the organisms 
 killed by the drug either liberate the substance producing 
 the positive reaction, or a toxin which stimulates the pro- 
 duction of this substance in the tissues of the body. Neither 
 theory has been proved correct as we know nothing cer- 
 tain as to the actual cause of the Wassermann reaction. 
 
 Some authorities have recommended intramuscular in- 
 jection of mercury for the production of the provocative 
 Wassermann reaction but the reports concerning the effi- 
 cacy of this method are so conflicting that it is not recom- 
 mended. 
 
 The provocative Wassermann test is a useful adjunct 
 totour diagnostic methods for syphilis and should be re- 
 sorted to whenever it is possible, both in the diagnosis of 
 the disease and in determining the question of the cure of 
 the infection. The large amount of work involved in mak- 
 ing the numerous tests that are necessary in correctly us- 
 ing the provocative reaction will ahvays operate to limit 
 its use to comparatively few cases but whenever it is pos- 
 sible to use the test it should be employed. 
 
 From the evidence that has accumulated it may be stated 
 that in patients who have presented a negative Wasser- 
 mann reaction for long periods of time and in whom symp- 
 toms of syphilis have been absent during this period, the 
 provocative Wassermann test often results positively. The 
 same result is sometimes obtained in cases presenting sus- 
 picious symptoms but in which the ordinary Wassermann 
 test is negative, or in cases vjhich give a history of infec- 
 tion but with no symptoms and a negative Wassermann re- 
 action. 
 
CHAPTEE X 
 
 THE WASSERMANN TEST UPON THE CEREBRO- 
 SPINAL FLUID. INTERPRETATION OF RE- 
 SULTS. THE COLLOIDAL GOLD TEST. 
 THE CELL COUNT AND TESTS FOR 
 INCREASE IN GLOBULINS IN 
 THE CEREBROSPINAL 
 FLUID 
 
 Recent observations have shown the great importance 
 of the Wassermann test upon the cerebrospinal fluid and 
 it may be stated that no case of syphilis can be considered 
 as having had the best treatment unless this fluid has been 
 tested, even though the Wassermann test upon the blood 
 is negative and the patient has been free from symptoms 
 for long periods of time. It is now demonstrated beyond 
 doubt that involvement of the central nervous system fre- 
 quently occurs very early in this disease and this demon- 
 stration has been rendered possible by the results of the 
 "Wassermann test upon the cerebrospinal fluid during the 
 early latent and secondary stages of the infection. 
 
 The Wassermann test upon the cerebrospinal fluid often 
 shows involvement of the central nervous system before any 
 clinical symptoms are observed and the use of the test 
 has rendered intensive treatment possible and has resulted 
 in either preventing the development of such symptoms or 
 in greatly delaying their appearance. Formerly it was be- 
 lieved that if the test gave a negative reaction in the cere- 
 brospinal fluid and a positive one in the blood, the diag- 
 nosis of cerebrospinal syphilis should be made, the result 
 serving to distinguish this condition from paresis, in which 
 both the blood and cerebrospinal fluid usually gave a posi- 
 
 205 ' 
 
206 THE WASSEEMANSr TEST 
 
 tive reaction. However, it has since been shown that the 
 negative result in the cerebrospinal fluid depended en- 
 tirely upon the amount of the fluid used in the test 
 and that if a large enough amount was used a positive 
 result could be obtained in the fluid in almost 100 per cent 
 of cases of cerebrospinal syphilis. Therefore, it follows, 
 that the Wassermann test upon the cerebrospinal fluid, so 
 far as its value in differentiating various syphilitic con- 
 ditions of the central nervous system is concerned, depends 
 upon quantitative factors entirely, so that it is necessary 
 in testing this fluid to test different quantities if one is to 
 secure the best results from the test. The importance of 
 the Wassermann test upon the cerebrospinal fluid is shown 
 by the statement of Fordyce^^^ that "From 25 to 35 per 
 cent of syphilitic individuals have positive findings in the 
 spinal fluid during the first year of their infection" and 
 during this time symptoms referable to the central nervous 
 system are frequently present. This being so, the impor- 
 tance of ascertaining in every case of syphilis whether 
 the central nervous system is involved at as early a date 
 as possible, can hardly be overestimated, and for this rea- 
 son the cerebrospinal fluid should be tested as well as the 
 blood serum, although the necessity of the test upon the 
 spinal fluid has not, as yet, been fully realized by the medi- 
 cal profession. 
 
 The writer has already described the method of obtain- 
 ing the cerebrospinal fluid and the technic required (Chap- 
 ter V) in making the test. As the differentiation of some 
 of the syphilitic diseases of the central nervous system 
 depends upon the quantity of fluid tested, the writer rec- 
 ommends that 0.2, 0.5, 0.7 and 1 c.c. quantities be tested 
 in each case, with his method of performing the reaction, 
 the fluid, of course, not being inactivated before use. The 
 quantity of spinal fluid tested, with any method, should 
 always be at least four times the quantity of blood serum 
 tested with the particular method used, and it is always 
 best to use both smaller and larger amounts than this in 
 
CKREBBOSPINAL FLUID 207 
 
 order to determine the strength of the positive reaction 
 upon which depends the differential diagnosis of the con- 
 dition present. 
 
 Hauptmann and Hossli"* were the first to insist upon 
 the use of large quantities of the cerebrospinal fluid in ap- 
 plying the Wassermann reaction and their work changed 
 entirely the prevalent conception of the comparative rarity 
 of a positive reaction in diseases like tabes and demon- 
 strated that almost any syphilitic disease of the central 
 nervous system would give a positive Wassermann re- 
 action with these larger amounts of fluid. Thus, the find- 
 ing of a positive reaction in the cerebrospinal fluid, once 
 looked upon as almost pathognomonic of paresis, has 
 ceased to point to this disease unless it is obtained with 
 small amounts of the fluid, for with large amounts other 
 syphilitic diseases of the central nervous system also give 
 a positive reaction. 
 
 Because of the use of too small amounts of the cerebro- 
 spinal fluid in making the Wassermann test much of our 
 statistical data regarding the occurrence of a positive re- 
 action in the various sj'philitic infections of the central 
 nervous system are worthless and a great many divergent 
 results are reported in the literature owing to this fact. 
 Mueller^^' obtained 75 per cent of positive reactions in 
 tabes, and 98 per cent in paresis, while Boas found that 
 aU of his untreated cases of tabes gave a positive reaction. 
 This is a very good illustration of the confusion in results 
 due to the use of different amounts of the spinal fluid, for 
 Boas used larger amounts of the fluid in his tabetic cases 
 and obtained a much higher percentage of positive results. 
 The writer's experience may be summed up as follows: 
 In paresis the Wassermann test is positive in the blood 
 in nearly 100 per cent of the cases, and if 1 c.c. of the 
 cerebrospinal fluid be used, it is positive in practically 
 100 per cent of the cases. If smaller amounts of the spinal 
 fluid be tested the percentage of positive reactions will be 
 
208 THE WASSERMANH TEST 
 
 found to decline, until when but 0.2 c.c. of the fluid is tested 
 only from 75 to 80 per cent of the cases will give a positive 
 reaction. When 0.5 c.c. of the fluid is used practically 98 
 per cent of cases of paresis will give a positive reaction in 
 the spinal fluid, a result nef er found in any other syphilitic 
 condition of the central nervous system. 
 
 In cerebrospinal syphilis, if 1 c.c. of the spinal fluid be 
 tested almost 100 per cent of the cases will prove positive, 
 but if only 0.2 c.c. of the fluid be tested only about 10 per 
 cent will prove positive. The Wassermann on the blood 
 in these cases gives from 60 to 75 per cent of positive re- 
 sults, as reported by different investigators. 
 
 In tabes, if 1 c.c. of the cerebrospinal fluid be tested, the 
 Wassermann will be positive in about 95 per cent of the 
 cases, but if only 0.2 c.c. of the fluid be tested the positive 
 results will only amount to from 5 to 10 per cent. In this 
 disease the Wassermann test upon the blood gives about 
 70 per cent of 'positive reactions. 
 
 From the above it is apparent that in differentiating be- 
 tween paresis, cerebrospinal syphilis and tabes, the ac- 
 curate titration of the complement-binding power of the 
 cerebrospinal fluid is most important. If 1 c.c. of the fluid 
 is used for the test, the results will be positive in prac- 
 tically all of the cases of either disease, but if smaller 
 amounts of the fluid be used it will be found that results 
 are obtained, which, if taken in conjunction with the clin- 
 ical symptoms present, will generally enable the correct 
 diagnosis to be made. Thus, if a positive reaction is ob- 
 tained with 0.2 c.c. of the fluid, or less, the diagnosis is al- 
 most certainly paresis, for only from 5 to 10 per cent of 
 cases of cerebrospinal syphilis or tabes give a positive 
 result with this amount of fluid. If a positive reaction 
 is obtained with 0.1 c.c. of the fluid the diagnosis of paresis 
 will be foimd correct in 99 per cent of the cases examined. 
 
 The Test upon the Cerebrospinal Fluid in the Various 
 Stages of Syphilis. — As stated, recent researches have 
 
CEREBROSPINAX, FLUID 209 
 
 shown that involvement of the central nervous system in 
 syphilis occurs early in the disease in a considerable pro- 
 portion of the cases, as has been shown by the results of 
 the Wassermann test upon the cerebrospinal fluid. In fact, 
 so good an authority as Fordyce holds that paresis, tabes, 
 and the majority of other manifestations of syphilis on 
 the part of the nervous system originate "at the time of 
 the general infection in the first year" and there is much 
 to support this theory in the biological and clinical char- 
 acteristics of syphilitic infection. 
 
 Results in Primary Stage. — The writer has no knowledge 
 of a well authenticated positive result of the Wassermann 
 test upon the cerebrospinal fluid in the primary stage of 
 syphilis. 
 
 Results in the Secondary Stage. — The older writers upon 
 this subject all agreed in stating that cases showing no 
 symptoms of involvement of the central nervous system, in 
 the secondary stage of syphilis, were invariably negative 
 to the Wassermann test upon the spinal fluid, or to other 
 tests upon this fluid, while, if symptoms of involvement were 
 present the Wassermann was generally positive. One ex- 
 ception may be made to this statement in the case of 
 Eavaut"^ who, in 1903, reported that among 116 cases in 
 which the spinal fluid was tested, no less than 67 per cent 
 showed some abnormahty of the fluid, either in an in- 
 creased cell count or an increase in globuhns. The more 
 recent investigators have reported numerous instances in 
 which the Wassermann test upon the cerebrospinal fluid 
 was positive early in the secondary stage, even when there 
 were no symptoms of involvement of the central nervous 
 system. Thus Wile"" found 30 per cent of the eases of 
 secondary syphilis that he tested gave a positive result with 
 the Wassermann test upon the spinal fluid, and Craig and 
 CoUins'^^ found that a considerable proportion of their 
 early secondary cases gave a positive Wassermann reac- 
 tion in the spinal fluid. Ellis and Swift'"^ examined 22 
 
210 THE WASSERMANN TEST 
 
 cases of untreated early secondary syphilis and found the 
 spinal fluid negative in all, but in 56 cases of late secondary 
 syphilis, tested by Altman and Dreyfus,"^ no less than 
 eleven gave a positive Wassermann reaction in the spinal 
 fluid. 
 
 Results in the Tertiary Stage. — The results of the Was- 
 sermann test upon the cerebrospinal fluid in paresis, cere- 
 brospinal syphilis, and tabes have already been discussed, 
 but it should be remembered that the examination of the 
 fluid in this stage of the disease from many cases in which 
 there are no discoverable lesions in the central nervous 
 system will result positively, as stated by Kolmer.^^* The 
 writer has repeatedly observed cases of tertiary syphilis 
 that gave a positive Wassermann reaction with the cere- 
 brospinal fluid but in which there was absolutely no evi- 
 dence of involvement of the central nervous system, but 
 he would be loath to conclude from this that no lesions due 
 to Treponema pallidum were present. At any rate such 
 a finding is the signal for a very careful scrutiny of the 
 patient and a very guarded prognosis as regards future 
 involvement of the central nervous syslJejn. 
 
 Results in the Latent Stage. — The results of the test 
 upon the cerebrospinal fluid in the latent stage of syphilis 
 will vary, of course, with the involvement of the central 
 nervous system, but the percentage reported by most ob- 
 servers does not exceed 15 per cent, including both early 
 and late latent infections. 
 
 The Interpretation of the Results of the Test 
 
 The interpretation of the results of the Wassermann 
 test upon the cerebrospinal fluid, as regards the intensity 
 of the reaction, are the same as already noted in the dis- 
 cussion of the interpretation of the results upon the blood, 
 with this exception; that a positive Wassermann reaction 
 upon the cerebrospinal fluid is specific of syphilis, in the 
 opinion of practically all observers. The writer has never 
 
CEREBROSPINAL FLUID 211 
 
 seen a positive reaction upon the spinal fluid in any other 
 condition than syphilis and his experience is that of all who 
 have studied this phase of the subject. 
 
 It is also true that more double-plus (four-plus) reac- 
 tions are observed in the spinal fluid than in the blood 
 although occasionally weak reactions are noted with the 
 maximum amount of fluid tested. Of course, in cases of 
 tabes and cerebrospinal syphilis, the smaller amounts of 
 fluid show gradations in the strength of the reaction, but a 
 diagnosis of syphilis of the central nervous system should 
 never be made upon anything less than total inhibition of 
 hemolysis; i.e., a double-plus (four-plus) reaction, when 
 as large an amount of the serum as 1 c.c. is tested. 
 
 The writer has already given the significance of the posi- 
 tive reaction when obtained with the different amounts of 
 the cerebrospinal fluid tested but will here summarize the 
 interpretation to be given the -reaction in this respect : 
 
 If amounts of the spinal fluid as small as 0.05 or' 0.1 c.c. 
 give total inhibition of hemolysis the diagnosis is almost 
 certainly paresis; if 0.2 c.c. of the fluid give the same re- 
 sult, the diagnosis is, in all probability, paresis; if larger 
 amounts up to 0.5 c.c. of the fluid are used, the diagnosis 
 is probably paresis ; while if still larger amounts are used 
 the diagnosis must rest largely upon the clinical symptoms 
 present, as cerebrospinal syphilis, tabes, and paresis all 
 give positive results with the larger quantities of fluid. 
 
 In order to illustrate the difference in results obtained 
 by using different amounts of the cerebrospinal fluid. Table 
 LIII is inserted, showing the results of the "Wassermann 
 test upon cases of cerebrospinal syphilis, tabes and paresis. 
 
 A consideration of this table will show the importance 
 of testing different amounts of the cerebrospinal fluid and 
 how useful such titrations are in the differentiation of the 
 conditions mentioned, especially the differentiation of pare- 
 sis from cerebrospinal syphilis and tabes. 
 
 It will be understood that the interpretation of the re- 
 sults of the Wassermann test upon the cerebrospinal fluid 
 
212 
 
 THE WASSBEMANN TEST 
 
 Table LIII 
 
 The Results of the Wassermann Test upon the Cerebrospinal Fluid in 
 Cerebrospinal Syphilis, Tabes, and Paresis 
 
 name of 
 
 WASSERMANN 
 IN BLOOD 
 
 AMOUNT OP CEREBROSPINAL FLUID C.C. 
 
 INFECTION 
 CASE NO. 
 
 0.05 
 
 0.1 
 
 0.2 
 
 0.5 
 
 0.7 
 
 1 
 
 Cerebrospinal 
 syphilis 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 8 
 
 + + 
 
 + + 
 
 + 
 + - 
 + + 
 
 + 
 
 - 
 
 - 
 
 + 
 + - 
 
 + + 
 
 + 
 + - 
 
 + - 
 + + 
 
 + 
 
 + + 
 + + 
 + + 
 + 
 . + + 
 + + 
 + + 
 + + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 Tabes 
 1 
 2 
 3 
 4 
 5 
 6 
 
 + + 
 
 + + 
 
 + + 
 
 + 
 
 + + 
 
 - 
 
 - 
 
 + 
 
 + 
 + + 
 
 + + 
 
 + 
 
 + + 
 + + 
 
 + 
 + - 
 
 + + 
 + + 
 + + 
 + + 
 
 + 
 
 + 
 
 + + 
 + + 
 + + 
 + + 
 
 + 
 + + 
 
 Paresis 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 8 
 
 + + 
 + + 
 + + 
 
 ++ 
 
 + 
 ++ 
 
 + + 
 + + 
 
 + 
 + + 
 + + 
 + + 
 
 + 
 + + 
 
 + + 
 + + 
 ++ 
 + + 
 + + 
 ++ 
 ++ 
 + + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 + + 
 + + ■ 
 + + 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 should be based not only upon this test but also upon the 
 result of the cell count, the colloidal gold reaction and the 
 tests for the increase in globulins, as all should be taken 
 into consideration in arriving at a just conclusion regard- 
 ing the condition present in any case, but the Wassermann 
 reaction alone is sufficient to base a diagnosis of syphilitic 
 infection of the central nervous systena upon, if positive. 
 
 In using the Wassermann test upon the cerebrospinal 
 fluid in the determination of the cure of syphilis the re- 
 action should be negative with 1 c.c. of the fluid. If this 
 quantity of the fluid gives even a partial positive result 
 the case should not be considered as cured but treatment 
 should be continued. 
 
 The early occurrence of involvement of the central nerv- 
 
CEEEBEOSPINAIi FLUID 213 
 
 ous system, as shown by the results of the Wassermann 
 test, renders this procedure upon the cerebrospinal fluid as 
 essential as upon the blood in syphilitic infections, and the 
 test should be employed much more frequently than it now 
 is as a routine measure. No patient should be assured that 
 he is free from syphilitic infection unless the cerebrospinal 
 fluid has been found to give a negative reaction with quan- 
 tities as large as 1 c.c, but, on the other hand, a diagnosis 
 of involvement of the central nervous system should not be 
 made unless complete inhibition of hemolysis occurs with 
 this quantity of spinal fluid. 
 
 The Wassermann test upon the blood, in cases shoAving 
 some nerve condition, if positive, does not prove that the 
 condition is syphilitic in nature, but if the cerebrospinal 
 fluid is also positive, the condition is almost certainly syph- 
 ilitic. While the blood serum is often negative in certain 
 syphilitic conditions of the central nervous System, it is 
 but rarely that the cerebrospinal fluid is negative when 
 quantities as large as 0.7 to 1 c.c. are employed in the test, 
 so that the examination of the spinal fluid will detect many 
 syphilitic infections that would be overlooked if one de- 
 pended entirely upon the result of the Wassermann test 
 upon the blood. For this reason the writer urgently rec- 
 ommends the testing of the cerebrospinal fluid in every case 
 of syphilis in which it is possible to do so and this test 
 should never be omitted in the determination of the cure 
 of a syphilitic infection. The operation of lumbar punc- 
 ture, when properly performed, is not dangerous or very 
 painful, and the amount of information that is often se- 
 cured by the examination of the cerebrospinal fluid more 
 than compensates for any discomfort that may be caused 
 by the collection of the fluid. 
 
 The Colloidal Gold Test 
 
 The colloidal gold test was recommended by Lange,"^ in 
 1913, as a method of distinguishing between certain syph- 
 
214 THE WASSEEMANN TEST 
 
 ilitic and nonsyphilitic diseases of the central nervous sys- 
 tem, and today this test stands as one of the most useful 
 confirmatory methods that we possess in the diagnosis of 
 syphilitic disease of this system. 
 
 The exact nature of the reaction in the colloidal gold test 
 is unknown. Lange believes that it is a method which en- 
 ables us to measure the protein content of the cerebro- 
 spinal fluid, for he found that this fluid under conditions 
 in which the protein content was increased caused the pre- 
 cipitation of the gold solution and that, within certain 
 dilutions, this precipitation was characteristic of syphilitic 
 conditions of the central nervous system. Previously, Zsig- 
 mondy^^' had demonstrated that the clear red color of 
 solutions of colloidal gold, prepared first by him, changed 
 to a blue with the precipitation of the gold upon the ad- 
 dition of electrolytes, and that solutions of protein in the 
 presence of an electrolyte inhibited the precipitate and that 
 it was possible in this way to determine for various pro- 
 teins the inhibitive point and thus differentiate them. 
 Accordingly, Jaeger and Groldstein^^'^ regard the reaction 
 obtained in the colloidal gold test as a purely physical 
 reaction of an electrolytic nature, but others, especially 
 Zaloziecki,^^* believe it to be an immunity reaction. It is 
 true that the reaction is obtained when the protein content 
 of the spinal fluid is increased but it is yet too early to in- 
 sist that it is wholly due to protein increase in the fluid. 
 
 The literature concerning this test is now quite volumi- 
 nous and all investigators are unanimous in their state- 
 ments regarding the value of the test in syphilis of the 
 central nervous system, but there is considerable disagree- 
 ment as to the weight to be placed upon the various findings 
 with the test and their exact significance. However, all 
 agree that the test is practically specific for paresis and 
 some of those having a large experience with it insist that 
 a diagnosis of this condition may be made from the result 
 of this test alone. The writer can not agree with this opin- 
 
CEBEBROSPINAL FLUID 215 
 
 ion of the value of the test in paresis, although it is un- 
 doubtedly true that in at least 96 to 98 per cent of cases 
 the typical paretic curve is obtained, but the same type of 
 reaction has been observed in other conditions, although 
 very rarely. However, the fact that a paretic curve does 
 occur in other conditions than paresis renders it impos- 
 sible for the test to be considered as absolutely specific of 
 this condition, although it must be regarded as the most 
 valuable of all confirmatory evidences of the presence of 
 the disease. 
 
 Technic of the Test.— The technic of the colloidal gold 
 reaction is very simple, but the preparation of the solu- 
 tion of colloidal gold is extremely difficult and the utmost 
 care must be used at every stage of the process or results 
 will not be satisfactory. In fact, it may be ,said that the 
 entire technic of the test really consists in the preparation 
 of the solution of colloidal gold, for if a good solution is ob- 
 tained there is little difficulty in performing the test. 
 
 Preparation of the Colloidal Gold Solution. — ^It is abso- 
 lutely requisite that all chemicals used in the preparation of 
 the solution be chemically pure and the products of Merck 
 have been found most satisfactory by numerous workers. 
 The following chemicals are used in the preparation of the 
 colloidal gold: 
 »- 1. Merck's Gold Chloride, Acid. 
 
 2. Merck's Blue Label Oxalic Acid. Crystals. 
 
 3. Merck's Blue Label Potassium Carbonate. 
 
 4. Merck's Formaldehyde. Highest Purity, 40%. 
 
 All glassware used in making the colloidal gold solution 
 should be thoroughly washed in hot water with ivory soap, 
 rinsed in tap water for several minutes; then with hot 
 bichromate solution, allowing the beakers, pipettes, and 
 test tubes to remain in the bichromate solution for fifteen 
 or twenty minutes ; after which they are rinsed in distilled 
 water and finally in triple distilled water. The glassware 
 should be used as promptly as possible after cleaning, the 
 
216 THE WASSEKMANK" TEST 
 
 pipettes and test tubes being dried in a hot air oven before 
 use. Thermometers used in preparing the solution should 
 be cleaned in the same manner as the glassware. 
 
 The distilled water used should be distilled in an appa- 
 ratus in which there are no rubber connections and the 
 water should be distilled three times before it is used. In 
 certain regions, where the water is very heavily impreg- 
 nated with mineral salts, it may have to be distilled four, 
 or even five times, and this is also true if the water used 
 has been heavily impregnated with chlorine in purifying 
 it for domestic use. 
 
 The following method of preparing the colloid of gold 
 solution is recommended by Kolmer^^' who states that it 
 is adapted from the method of Miller, Brush, Hammers, 
 and Felton."" ^ 
 
 1. Heat 1000 c.c. of triply distilled water over a proper burner in a properly 
 washed beaker containing a thermometer. 
 
 2. When the temperature reaches 60° 0. add 10 c.c. of a 1 per cent solution 
 of Merck's gold chloride crystals in triply distilled water and 7 c.c. of a 2 
 per cent solution of Merck's blue label potassium carbonate in triply distilled 
 water. 
 
 3. At 80° C, while stirring briskly, add 10 drops of a 1 per cent solution 
 of Merck's blue label oxalic acid crystals, in ^iply distilled water. 
 
 4. At 90° C. remove tha burner and, while stirring, add 5 c.c. of a solution 
 of 1 CO. of Merck's highest purity formaldehyde in 40 c.c. of triply distilled 
 water, or enough to produce an initial pink color. 
 
 5. The solution must be neutral in reaction when used and to determine 
 the reaction it is titrated with a 1 per cent solution of alizarin; red, in 50 per 
 cent alcohol. With this indicator the neutral point is shown by a brownish 
 red tint; an acid solution gives a lemon-yellow, and an alkaline solution a 
 purple-red, color. 
 
 In determining the reaction the following procedure is 
 carried out: 
 
 In a properly cleaned beaker is placed 10 c.c. of the colloidal gold solu- 
 tion and two drops of the indicator are added. If a brownish red tint appears 
 the solution is satisfactory; if not, the color is noted, and according to 
 
 whether it is acid or alkaline it is titrated with either -=77 NaOH or -^ HCl. 
 
 50 50 
 
 If acid the NaOH is used and if alkaline the HCl. When the neutral point 
 is reached; i.e., a brownish red tint appears, the amount of reagent nfices- 
 
CEREBROSPINAL FLUID 217 
 
 sary to neutralize the 10 o.e. of the gold solution is noted and the amount 
 necessary to neutralize 1000 c.c. is readily calculated. Neutralization should 
 be made with normal or decinormal solutions of the acid or alkali, as the 
 case may be. 
 
 Kolmer gives the following standards for an efficient col- 
 loidal gold solution: 
 
 1. It must be absolutely transparent and of a brilliant red orange or sal- 
 mon red color. (The writer has found that if there is the least trace of 
 cloudiness in the solution, it will be found unserviceable.) 
 
 2. Five cubic centimeters of the solution should be completely precipitated 
 in one hourby 1.7 c.c. of a 1 per cent solution of sodium chloride in distilled 
 water. 
 
 3. The solution must be neutral in reaction when used. 
 
 4. The solution must not produce a reaction greater than a red blue dis- 
 coloration (No. 1 reaction) with normal cerebrospinal fluid, and should give 
 
 'a typical curve with a known paretic fluid. 
 
 To the above the writer would add that the colloidal gold 
 solution should show no evidence of having been deposited 
 on the glass flask in which it is stored or any evidence of 
 a 'precipitate within the flask. It has been noted by the 
 writer that some glassware is entirely unsuitable for use 
 with colloidal gold as it apparently is attacked by it with 
 a resulting precipitate in the solution or deposited upon 
 the sides and bottom of the storage flask. Jena glassware 
 is the best but Pyrex has been found satisfactory. The 
 colloidal gold should be kept in the flask in the dark and 
 will keep for months unchanged if properly handled. 
 
 The fact that the colloidal gold solution is perfectly clear 
 does not indicate that it will prove effective in actual prac- 
 tice for it has been found that perfectly clear solutions are 
 sometimes worthless. Such solutions are called "pro- 
 tected solutions" because no precipitation will take place 
 after the addition of any electrolyte, while ''unprotected 
 solutions," as they are called, act efficiently in the test, 
 and are those that can be completely precipitated by 1.7 
 c.c. of a 1 per cent sodium chloride solution in one hour. 
 This test of the efficiency of a colloidal gold solution is very 
 important and should always be carried out before any 
 
218 THE WASSEEMANN TEST 
 
 such solution is depended upon for use in the colloidal 
 gold test. It has also been determined that alkaline col- 
 loidal gold solutions are practically inert when used with 
 a positive cerebrospinal fluid, while acid solutions give 
 little reaction with a positive fluid, and an abnormal reac- 
 tion with a normal spinal fluid. If the colloidal gold so- 
 lution is only slightly acid it will react well with paretic 
 fluids, but will give a reaction with normal fluids similar to 
 that obtained in the luetic zone with fluid from some syph- 
 ilitic conditions. In order to secure reliable results the 
 colloidal gold solution must be neutral in reaction. 
 
 Method of Performing the Test. — The technic of the col- 
 loidal gold test is very simple but to secure proper results 
 each step must be carefully perfornied. All glassware 
 must be prepared as already described and pipettes must 
 be perfectly dry before being used. 
 
 Eleven test tubes, cleaned as described and perfectly dry, 
 are arranged in a rack and in the first tube is placed 1.8 
 c.c. of freshly prepared, sterile 0.4 per cent sodium chlo- 
 ride solution prepared from chemically pure sodium chlo- 
 ride. Into each of the ten remaining test tubes place 
 1 c.c. of the salt solution. With a dry pipette add 0.2 c.c. 
 of the cerebrospinal fluid to be tested, which must be free 
 from blood, to the first tube and mix thoroughly. After 
 mixing, 1 c.c. of the mixture is transferred to the second 
 tube, mixed, and 1 c.c. of this mixture transferred to the 
 third tube, and this process repeated until the tenth tube 
 is filled, when from that tube 1 c.c. is discarded. The elev- 
 enth tube acts as a control and will contain no cerebro- 
 spinal fluid. The dilutions which have thus been made are 
 as follows : 1-10, 1-20, 1-40, 1-80, 1-160, 1-320, 1-640, 1-1280, 
 1-2560, and 1-5120. 
 
 After the dilutions of the cerebrospinal fluid have been 
 made, as indicated, there is added to each tube 5 c.c. of the 
 colloidal gold solution. This is thoroughly mixed with the 
 fluid in the tubes and the rack kept at room temperature 
 
1 = 10 |i20 t-.HO |;80 I.-|60 |:3E0 1 = 640 I: I2B0 1=2560 |:51Z0 Control 
 
 Parefic Reaction. 
 
 I-IQ U20 1-^0 1 = 80 I-I60 1 = 380 1 = 6^0 1:1280 \-Z560 1 = 5120 Confrol 
 
 Tabetic Reaction. 
 
 Plate III. 
 
 Paretic! Reaction. — The upper row of tubes illustrates the reaction, or curve, obtained 
 with the colloidal gold test in the cerebrospinal fluid of paresis and is known as the 
 paretic curve or reaction. 
 
 Tabetic Reaction. — The lower row of tubes illustrates the reaction, or curve, obtained 
 with the colloidal gold test in the cerebrospinal fluid of tabes and is known as the tabetic 
 reaction. (Luetic-zone curve.) 
 
OBKBBBOSPIHAL FLUID 219 
 
 overnight, the test being read upon the next day and re- 
 corded by the numbers corresponding to the changes in the 
 color of the mixture observed, as follows : 
 
 5. Complete precipitation. The supernatant liquid is 
 colorless. 
 
 4. Pale blue. 
 
 3. Blue. 
 
 2. Lilac or purple. 
 
 1. Eed-blue. 
 
 0. No change. 
 
 Reading the Results.— The results of the test are read by 
 the numbers corresponding to the color changes, as noted 
 in the preceding table. Thus, if the first four tubes show 
 complete precipitation, with a colorless supernatant fluid, 
 the fifth and sixth tubes a pale blue color, the seventh tube 
 lilac or purple, and the remaining tubes no change, the 
 reaction would be read as follows : 5555442000. 
 
 The Results of the Test in Various Syphilitic Condi- 
 tions. — The change in color in the various tubes will differ 
 with different syphilitic conditions of the central nervous 
 system, the color reaction with paresis being the most char- 
 acteristic and diagnostic of all, and the changes observed 
 are called * ' curves. ' ' Thus, we have a ' ' paretic curve ' ' and 
 a "luetic zone curve," as well as a "meningitic zone curve." 
 
 The "Paretic Curve." — The reaction observed in the 
 cerebrospinal fluid in paresis is so constant and typical 
 that it may be considered as the most valuable of all the 
 confirmatory signs that we possess of this type of involve- 
 ment of the central nervous system. It is to this type of 
 the reaction that Miller and Levy"^ gave the name "pa- 
 retic curve" and it is this type of reaction that is met with 
 in practically 100 per cent of cases of paresis. In report- 
 ing the colloidal gold test it is usually customary to record 
 the reaction upon a chart and when this is done, the pa- 
 retic curve will appear substantially as in Table LIV. 
 
220 
 
 THE WASSERMANBT TEST 
 
 Table LIV 
 
 Illustrating Type op Reaction Obtained with the Colloidal Gold Test 
 IN Paresis (Paretic Curve) 
 
 COLOR 
 
 color 
 
 NO. 
 
 DILUTIONS OP CEREBROSPINAL FLUID WITH OA PER CENT NACL 
 
 REACTIONS 
 
 1-10 1-20 1-40|1-80 1-160 1-320 
 
 1-640 1-1280 1-2560 1-5120 
 
 Colorless 
 
 5 
 
 
 
 Pale blue 
 
 4 
 
 
 • 
 
 Blue 
 
 3 
 
 
 s 
 
 Mac or purple 
 
 2 
 
 
 % 
 
 Red-blue 
 
 1 
 
 1 1 
 
 -•^ 
 
 No change 
 
 
 
 1 1 
 
 •% 
 
 Eecorded in numbers this reaction would be thus re- 
 ported: 5555554210. Often the first three, four, or five 
 tubes are decolorized, while the remaining ones show some 
 changes, as would be found in the following combinations : 
 5554432200; 5555422100, 5555542210, or as many as eight 
 of the tubes may be completely decolorized. Curves of this 
 type are characteristic of paresis and are not found in 
 other syphilitic diseases of the central nervous system, 
 although a few reports are on record of cases of menin- 
 gitis and multiple sclerosis giving similar curves Tvith this 
 test. A paretic curve is also observed in taboparesis. 
 
 The Luetic-Zone Curve. — The luetic-zone curve is ob- 
 tained in cases of cerebrospinal syphilis and tabes, as well 
 as in the fluid in the tertiary stage of syphilis. In tabes 
 the colloidal gold test usually gives a luetic-zone curve of 
 No. 4 intensity, the precipitation being only partial in the 
 first two or three tubes, complete in the next two or more 
 tubes, and gradually shading off into normal. However, it 
 should be distinctly understood that there is nothing char- 
 acteristic in the curves noted in tabetic patients and there 
 is no constancy in the type of curve observed, except that 
 the "paretic curve" is never noted. Table LV illustrates 
 the "luetic-zone curve" obtained in a case of tabes. 
 
 Eecorded by numbers the above reaction would be thus 
 reported: 4445554200. In cerebrospinal syphilis the fluid 
 usually shows weaker reactions than that for tabes but al- 
 
CEBEBBOSPINAL FLUID 
 Table LV 
 
 221 
 
 Illustrating Type op Reaction Obtained with the Colloidal Gold Test 
 IN Tabes (Luetic Zone Curve) 
 
 COLOR 
 
 COLOR [dilutions OP CEREBROSPINAL FLUID WITH 0.4 PER CENT NACL 
 
 REACTIONS 
 
 NO. |1-10 1-20 1-40 1-80 1-160 1-320 1 
 
 ■640|l-1280|l-2360 
 
 1-5120 
 
 Colorless 
 
 5 ^ , 
 
 
 Pale blue 
 
 4 ' =c;7 
 
 
 Blue 
 
 z 
 
 \, 
 
 
 Lilac or purple 
 
 2 
 
 %. 
 
 
 Ked-blue 
 
 1 
 
 
 
 No change 
 
 
 
 1 
 
 ••• 
 
 ways in the luetic-zone, while the reactions obtained in 
 the fluid during the tertiary stage of syphilis when symp- 
 toms of involvement of the central nervous system are 
 absent are still weaker, only slight changes being observed, 
 as a rule, in the luetic zone of the test. 
 
 In nonsyphilitic disease of the central nervous system, 
 as in meningitis, acute poliomyelitis, multiple sclerosis, 
 etc., the findings with the colloidal gold test are often con- 
 fusing and of little practical value. In meningitis the so- 
 called "meningitic zone" curve is encountered, in which 
 the color changes occur in the highest dilutions, complete 
 precipitation occurring at the opposite end of the chart 
 from paresis. Table LVI illustrates the type of curve ob- 
 tained in tuberculous meningitis or purulent meningitis. 
 
 Table LVI 
 
 Illustrating Type op Reaction Obtained with the Colloidal Gold Test 
 IN Meningitis (Meningitic Curve) 
 
 COLOR 
 
 C0L0R|DILUTI0NS op cerebrospinal fluid with 0.4 PER CENT NACL 
 
 REACTIONS 
 
 NO. |1-1011-20|1-40|1-80|1-160 1-320 1-640 1-1280 1-2560 1-5120 
 
 Colorless 
 
 5 1 1 1 ^._.— 
 
 Pale blue 
 
 4 1 1 1 ,- \ 
 
 Blue 
 
 3 1 1 ,•' \ 
 
 Lilac or purple 
 
 2 1 1 ^.--.'- ^ 
 
 Bed-blue 
 
 1 1 ..' \ 
 
 No change 
 
 ,__..^|_.' ', 
 
 Eecorded by numbers the above reaction would be thus 
 reported: 0001223550. 
 
222 THE WASSERMANN TEST 
 
 Interpretation of the Results of the Colloidal Gold 
 Test. — There is only one condition in which, th^ colloidal 
 gold test may be practically relied upon for diagnosis and 
 that is'paresis. In the overwhelming number of instances, 
 if a typical paretic curve is obtained with this test it is 
 safe to make a diagnosis of this condition and the Wasser- 
 mann test upon the blood and cerebrospinal fluid will gen- 
 erally confirm the diagnosis. However, the writer is still 
 doubtful if we should rely upon the colloidal gold reaction 
 alone in making a diagnosis of paresis, especially if the 
 other diagnostic aids that we possess do not confirm the 
 reaction. It must be admitted, however, that the first evi- 
 dence we have in many cases of incipient paresis is a pos- 
 itive result with this test and whenever such a result is 
 obtained it should be given great weight and the most in- 
 tensive specific treatment at once instituted. 
 
 In cerebrospinal syphilis and tabes the colloidal gold re- 
 action is of no diagnostic importance and the results 
 obtained "should never be considered as more than confir- 
 matory of the diagnosis in cases which are very suspicious 
 in other respects. With the exception of paresis and tabo- 
 paresis the colloidal gold test is of value only as an aid in 
 the diagnosis of syphilis of the central nervous system. In 
 congenital syphilis, cerebrospinal syphilis, tabes, and sec- 
 ondary and tertiary syphilis the results are inconstant 
 and only of confirmatory value, but its appearance in pare- 
 sis, in many cases, before symptoms of the condition de- 
 velop, renders the test of great value in early diagnosis 
 and as this complication may occur in any case of syph- 
 ilitic infection the routine testing of the cerebrospinal fluid 
 with the colloidal gold test is to be encouraged. 
 
 The Cell Count 
 
 It is well known that in most inflammatory conditions 
 of the central nervous system there is an increase in the 
 number of cells found in the cerebrospinal fluid and this 
 
CEREBBOSPINAL FLUID 223 
 
 increase occurs to such an extent in some conditions as to 
 be of considerable service in their differential diagnosis. 
 The cell count, however, so far as syphilitic disease of the 
 central nervous system is concerned, is seldom of more 
 than confirmatory value of results obtained by other meth- 
 ods of laboratory diagnosis. In no syphilitic condition of 
 the central nervous system is the cell count specific nor 
 can one differentiate syphilitic disease from other diseases 
 of this system by its means. 
 
 An increase in the number of cells in the cerebrospinal 
 fluid, called pleocytosis, occurs in practically 95 per cent 
 of syphilitic affections of, the central nervous system but 
 also occurs in other diseases of this system, and in acute 
 infections, as cerebrospinal meningitis, although in non- 
 syphiUtic conditions the cell count is seldom as high as in 
 conditions lilse tabes, cerebrospinal syphilis, paresis, and 
 syphilitic meningitis. According to Nonne^^'' the cell count 
 in the cerebrospinal fluid may be appreciably increased in 
 from 30 to 40 per cent of cases of syphilis even in the ab- 
 sence of symptoms of organic nervous disease. In old 
 cases of tabes, or cases in which the symptoms are not pro- 
 gressive, there may be an absence of pleocytosis and even 
 in progressive cases this may sometimes occur. 
 
 Efforts have been made to differentiate the kinds of cells 
 increased in the pleocytosis of syphilitic disease of the cen- 
 tral nervous system, and Alzheimer has rendered it pos- 
 sible to recognize the different types of cells that occur. 
 However, from a practical standpoint, the differentiation 
 of the various cells found is of very little, if any, assistance 
 in differentiating the various syphilitic diseases of the 
 nervous system, a simple count of the cells present being 
 as useful as any differential count. As Nonne well says :^'^ 
 *'The attempt to recognize in the spinal fluid a character- 
 istic cell-picture for the development of a paresis, or to 
 foretell the time of progression of a syphilogenetic affec- 
 tion, has not as yet been crowned with success." 
 
224 THE WASSBEMANN- TEST 
 
 The cells which are increased in syphilitic disease of the 
 central nervous system comprise both lymphocytes and 
 leucocytes, but their origin is not absolutely determined. 
 Whether they originate from the blood or from the connec- 
 tive tissue of the leptomeninges is unsettled but the pleo- 
 cytosis is the direct result, undoubtedly, of an inflamma- 
 tory process in the leptomeninges. This is the conclusion 
 reached by Nonne and it is believed that it is the one gen- 
 erally held by the best students of the subject. 
 
 Technic of Counting the Cells. — ^In counting the cells in 
 the cerebrospinal fluid the most generally used method is 
 "with the Fuchs-Eosenthal counting chamber. The method 
 of making the dilution of spinal fluid and counting is as 
 follows: The staining fluid consists of 0.1 gram gentian 
 violet; 2.0 grams glacial acetic acid; and 50 e.c. distilled 
 water. This fluid is drawn up into the ordinary pipette 
 used for counting the white cells of the blood, to the point 
 marked I and then the cerebrospinal fluid drawn up to the 
 point marked II. The pipette is now shaken very thor- 
 oughly for at least two minutes, when a drop of the mix- 
 ture is placed on the counting chamber, a few drops being 
 expelled from the pipette before doing so. All of the 
 lymphocytes and leucocytes are counted in all the squares 
 and the total number divided by three. Nonne regards 
 from to 5 cells as normal ; 6 to 10 as borderland cases ; 
 and more than 10 cells as pathological. 
 
 The Fuchs-Eosenthal counting chamber is not necessary 
 in making a count of the cells in the spinal fluid, as the 
 Zappert, Turk, or Levy counting chamber may be used 
 just as well, the spinal fluid being undiluted or, if the cells 
 are very numerous, diluted as for making a white blood 
 cell count. 
 
 The staining of the cells is not necessary if the fluid be 
 diluted with a 0.5 per cent solution in distilled water of 
 glacial acetic acid, when the Fuchs-Rosenthal chamber for 
 counting is used, while if either the Turk or Zappert count- 
 
CEKEBKOSPINAL. FLUID 
 
 225 
 
 ing chambers are used the fluid should be used undiluted, 
 unless the fluid is purulent or contains an excessive num- 
 ber of cells. 
 
 The number of cells found in the cerebrospinal fluid in 
 the various syphilitic diseases of the nervous system varies 
 very greatly and no diagnostic data can be based upon the 
 number of the cells alone. The increase in the number of 
 cells is the important diagnostic feature, but this increase 
 is not of such a nature that one can say that paresis exists 
 in this case, and tabes in that.' In illustration of this, 
 Table LVII shows the counts observed in paresis, cerebro- 
 spinal syphilis, and tabes. 
 
 Table LVII 
 
 Cells Counts in the Cbkebeospinal Fluid Obsekved in Pakesis, Cerebro- 
 spinal Syphilis, and Tabes 
 
 paresis 
 
 CEREBROSPINAL SYPHILIS 
 
 TABES 
 
 CASE NO. 
 
 COUNT 
 
 CASE NO. 
 
 COUNT 
 
 CASE NO. 
 
 COUNT 
 
 1 
 
 18 
 
 1 
 
 13 
 
 1 
 
 9 
 
 2 
 
 45 
 
 2 
 
 64 
 
 2 
 
 8 
 
 3 
 
 67 
 
 3 
 
 243 
 
 3 
 
 43 
 
 4 
 
 16 
 
 4 
 
 11 
 
 4 
 
 44 
 
 5 
 
 29 
 
 5 
 
 86 
 
 5 
 
 55 
 
 6 
 
 88 
 
 6 
 
 65 
 
 6 
 
 69 
 
 7 
 
 12 
 
 7 
 
 50 
 
 7 
 
 186 
 
 8 
 
 36 
 
 8 
 
 590 
 
 8 
 
 237 
 
 9 
 
 168 
 
 9 
 
 344 
 
 9 
 
 36 
 
 10 
 
 243 
 
 10 
 
 156 
 
 10 
 
 110 
 
 11 
 
 507 
 
 11 
 
 6 
 
 11 
 
 124 
 
 12 
 
 323 
 
 12 
 
 402 
 
 12 
 
 116 
 
 It will be seen from the above table that there is nothing 
 characteristic in the number of cells found in the cerebro- 
 spinal fluid in the three conditions, the characteristic fea- 
 ture being that there is an increase above normal in all the 
 cases cited. 
 
 Interpretation of Results of Count. — The increase in the" 
 number of cells in the cerebrospinal fluid, as has been 
 stated, is due to inflammatory conditions of the leptomen- 
 inges, but an increase does not always indicate that the 
 condition causing it is syphilitic. Of the syphilitic con- 
 
226 THE WASSBEMANN TEST 
 
 ditions of the central nervous system the highest counts 
 are generally observed in syphilitic meningitis, the count 
 frequently exceeding one thousand cells; in tabes the 
 counts are apt to be high, running into the hundreds; in 
 paresis, the average count is not above 100, generally run- 
 ning between 20 and 60, but counts above 100 are often ob- 
 tained, and the same statement is true of cases of cerebro- 
 spinal syphilis. However,_the mere number of cells found 
 is no criterion of the condition present, and the cell count 
 has to be interpreted in conjunction with the clinical symp- 
 toms present and the results of the other methods of lab- 
 oratory diagnosis in syphilis ; i. e., the Wassermann test, 
 the colloidal gold test, and the test for increase in proteins 
 in the cerebrospinal fluid. 
 
 Tests for Increase in Globulins in the Cerebrospinal 
 Fluid. — ^In syphilitic disease of the central nervous system, 
 as well as in other organic diseases of this system, the pro- 
 tein content of the cerebrospinal fluid is generally increased, 
 and this increase can be detected by tests which have been 
 perfected for demonstrating an increased globulin content 
 in this fluid. Three methods are in general use, known as 
 the Nonne and Apelt method, the Noguchi method, and the 
 Pandy method. 
 
 The Nonne-Apelt Globulin Test.— This test is thus de- 
 scribed by Nonne :^^* "The simplest method of testing the 
 cerebrospinal fluid for albuminous bodies is to search for 
 an increase of globulia — ^the so-called 'Phase I' reaction, 
 introduced by Apelt and myself. For this purpose there is 
 added to a hot saturated solution of sulphate of ammo- 
 nium, which has been permitted to cool, an equal amount 
 of cerebrospinal fluid ; 1 c.c. of each is quite sufficient. It 
 is advisable to pour one liquid gently on top of the other; 
 if the globulins are increased, there occurs a more or less 
 distinct gray ring at the plane of contact. After this pre- 
 liminary observation the mixture is well shaken and the 
 result may be read off within three minutes. If it is dis- 
 
CEEBBEOSPIETAL FLUID 227 
 
 tinetly opalescent or cloudy, we call it a positive 'Phase I' 
 reaction." 
 
 In making the above test it is best to add the cerebro- 
 spinal fluid from a 1 c.c. pipette very slowly to the ammo- 
 nium sulphate solution, as in this way the gray ring indi- 
 cating increased globulin is rendered much more distinct 
 than when one fluid is poured into the other. This method 
 is still in general use but has been very largely displaced 
 by the Noguchi and Pandy methods. 
 
 The Noguchi Butyric Acid Test for Globulin.— This 
 method was devised by Noguchi,^^* is simple, and accu- 
 rate, and the one that the writer has generally used in his 
 laboratories. The method of making the test is thus de- 
 scribed by Noguchi :"° 
 
 "Two parts of the cerebrospinal fluid to be examined 
 are mixed with 5 parts of a 10 per cent butyric acid solu- 
 tion in physiological salt solution, and are heated over a 
 flame and boiled for a brief period. One part of a normal 
 solution of NaOH is then added quickly to the heated mix- 
 ture, and the whole boiled once more for a few seconds. 
 The actual quantities of these three reagents that I pre- 
 fer are 0.1 or 0.2 c.c. of the spinal fluid, 0.5 c.c. of the 
 butyric acid solution, and 0.1 c.c. of the normal sodium 
 hydrate. It is necessary to take the precaution to employ 
 for this test only cerebrospinal fluid entirely free from 
 blood-" 
 
 A positive reaction with this test is indicated by the ap- 
 pearance of a granular or flocculent white or gray precip- 
 itate which settles slowly to the botto^i of the test tube. 
 The amount of this precipitate and the rapidity with which 
 it is formed will vary, as Noguchi observed, with the quan- 
 tity of protein in the fluid examined, the greater the 
 amount of protein the quicker the reaction will appear and 
 the greater the amount of precipitate, in the one case the 
 reaction appearing within a few minutes, while, if the 
 amount of protein increase is small the reaction may take 
 
228 THE WASSBEMANN TEST 
 
 an hour or more to appear. Two hours should be the time 
 limit for the reaction. 
 
 The Pandy Method for Globulin Increase. — A method 
 which is spoken very highly of by those who have used it 
 is the Pandy method. It is the most simple of all methods 
 that have so far been devised for the purpose and is un- 
 doubtedly reliable. Miller,"'" who has had a large experi- 
 ence Avith this method thus describes its use: 
 
 " Pandy 's test has not received the attention it deserves. 
 None of the other reactions used to reveal an excess of 
 globulin is so simple in execution or so quickly decisive in 
 its results. The reagent consists of a saturated aqueous 
 solution of carbolic acid; ten parts of pure crystals are 
 added to 100 parts of hot distilled water; the mixture is 
 kept at room temperature for three or four days, during 
 which time it should be frequently shaken. At the end of 
 this time the clear supernatant fluid is draAvn off into an- 
 other bottle. To approximately 1 c.c. of the reagent is 
 added one. drop of the spinal fluid. Normally no change 
 occurs or at most an extremely faint opalescence; with a 
 fluid abnormal in its protein content there develops in- 
 stantly at the point of contact a bluish white cloud, often 
 resembling a ring of smoke, which gradually settles to the 
 bottom of the tube." 
 
 The writer has had very little experience with this test 
 but it has proved accurate in those cases in which it has 
 been used. 
 
 Interpretation of the Results of the Globulin Tests. — ^In 
 organic disease of the central nervous system an increase 
 in the protein content of the cerebrospinal fluid conamonly 
 occurs and this increase is present in the vast majority of 
 cases of syphilitic disease of this system. However, as 
 this protein increase, indicated by the increase in globu- 
 lins, is not confined to syphilitic disease, a positive reac- 
 tion with any of the tests mentioned does not prove that 
 the condition present is syphilitic nor is it possible to dif- 
 
CEREBKOSPINAL FLUID 
 
 229 
 
 ferentiate the various syphilitic diseases of the central 
 nervous system by their use. The tests are often positive 
 in infections of the meninges due to the pneumococcus, tu- 
 bercle bacillus, influenza bacillus, meningococcus, and in 
 acute poliomyelitis, as well as in syphilitic disease, and 
 are also present in chronic organic disease of the central 
 nervous system, so that a positive reaction can only be re- 
 garded as confirmatory of the results of other laboratory 
 methods of diagnosis of syphilitic involvement of this sys- 
 tem and of the nature of the clinical symptoms which may 
 be present. 
 
 Siunmary of the "Four Tests."— In diagnosing and in 
 differentiating syphilitic disease of the central nervous 
 system, it is necessary to carefully consider the results of 
 the "Four tests" as they may be called, upon the cerebro- 
 spinal fluid. These are 1, the Wassermann test upon the 
 fluid ;. 2, the colloidal gold test ; 3, the cell count ; 4, the test 
 for increase in globulins. As an illustration of the results 
 that are commonly obtained with these four tests. Table 
 LVIII, adapted from Miller^^' is inserted: 
 
 Table LVIII 
 
 Showing Average Fkequency of the Various Eeactions in Syphilis of the 
 Central Nervous System 
 
 TEST 
 
 paresis 
 % 
 
 tabes 
 % 
 
 cerebrospinal 
 syphilis 
 
 % 
 
 Spinal fluid Wassermann 
 Globulin test 
 Cell count 
 Colloidal gold test 
 
 97 
 
 100 
 
 98 
 
 98-100 
 
 60-89 
 90-95 
 85-90 
 85-90 
 
 85-90 
 90-95 
 85-90 ■ 
 85-90 
 
 
 Paretic 
 curve 
 
 Luetic 
 curve 
 
 Luetic 
 curve 
 
 The blood Wassermann in these diseases gives approx- 
 imately the following results: Paresis, 98-100 per cent; 
 tabes, 70 per cent; and cerebrospinal syphilis, 70-80 per 
 cent. A careful study of the cerebrospinal fluid by means 
 of these four reactions will often result in enabling one to 
 differentiate the various syphilitic diseases of the central 
 
230 THE WASSEEMANN TEST 
 
 nervous system and, in the vast majority of our cases, if 
 the tests are negative, to exclude syphilitic disease in a 
 suspected individual. If the globulin test is negative in a 
 patient suffering from disease of the central nervous sys- 
 tem it is almost certain that the condition is not syphilitic, 
 although, if positive, it can not be said that it is syphilitic. 
 A fluid showing a positive Wassermann reaction in 
 amounts as small as 0.2 c.c, together with a paretic curve 
 with the colloidal gold test, is sufficient upon which to 
 base a diagnosis of paresis, while a high cell count, a 
 luetic curve with the colloidal gold test, and a negative 
 Wassermann, or one only positive in large amounts of the 
 spinal fluid, would point to tabes. By a careful compar- 
 ison of the results of these various tests with the clinical 
 symptoms present it should be possible, in the vast ma- 
 jority of instances, to make a diagnosis of the form of 
 syphilitic disease of the central nervous system which may 
 be present, and the vahie of these tests in diagnosing very 
 early involvement of this system has already been empha- 
 sized. 
 
 In no case can it be stated that the syphilitic patient has 
 been given the best that it is possible for medical science 
 to give him, either in the way of diagnosis or treatment, 
 if the thorough examination of the cerebrospinal fluid has 
 been omitted and this examination should become as much 
 of a routine in the diagnosis and treatment of syphilis as 
 is the Wassermann test upon the blood serum. 
 
BIBLIOGRAPHY 
 
 The_ references given below do not pretend to cover the literature of the 
 subject, which is now immense, but only cover the papers actually referred to 
 in the text. 
 
 iNuttall: Ztsehr. f. Hyg., 1888, iv, 353. 
 
 2Fodor, von: Deutsch. med. Wchnschr., 1888, vi, 460. 
 
 sBuchner: Centralbl. f. Bakt., 1889, v, 252. 
 
 iBuchner : Arch, f . Hyg., 1891, x, 84. 
 
 sPfeiffer: Ztsehr. f. Hyg., 1894, xvii, 355. 
 
 BBordet: Ann. d. I'Inst. Pasteur, 1898, xii, 688; Ibid., 1899, xiii, 682. 
 
 'Bordet: Studies in Immunity, Trans., Gay, New York, 1909. 
 
 sBelfanti and Carbone: Gior. d. r. Accad. di. med. di Torino, July, 1898. 
 
 »Bordet and Gengou: Ann. d. I'Inst. Pasteur, 1901, xv, 289. 
 loWassermann, Neisser, and Bruck: Deutsch. med. Wchnschr., 1906, xxxii, 
 
 745. 
 "Wassermann, Neisser, Bruck, and Sohucht: Ztsehr. f. Hyg. u. Infections- 
 
 krankh., 1906, Iv, 451. 
 i2Detre: Wien. klin. Wchnschr., 1906, xix, 619. 
 laWeygandt: Miinchen. med. Wchnschr., 1907, liv, 1557. 
 i^Marie and Levaditi: Ann. d. I'Inst. Pasteur, 1907, xxi, 138. 
 ispiaut: Centralbl. f. Nervenh. u. Psychiat., 1908, xxxi, 289. 
 leLandsteiner : Centralbl. f. Bakt., orig. abstr. 1, 1906, p. 42. 
 I'^Landsteiner, Midler ajid Potzl: Wien. klin. Wchnschr., xx, 1421, 1565. 
 isNoguchi: Jour. Exper. Med., 1909, xi, 84. 
 
 iSBrowning, Oruickshank, and McKenzie: Biochem. Ztsehr., 1910, xxv, 85. 
 aoWalker and Swift: Jour. Exper. Med., 1913, xviii, 566. 
 ziNoguchi: Serum Diagnosis of Syphilis, ed. 3, Philadelphia, 1912, p. 159. 
 22Craig and Nichols: Jour. Exper. Med., 1912, xvi, 336. 
 asNoguchi: Jour. Exper. Med., 1909, xi, 84. 
 
 2*Citron: Immunity, Trans., Garbat, ed. 2, Philadelphia, 1914, p. 166. 
 ^Schmidt: Ztsehr. f. Hyg. u. Infectionskrankh., 1911, Ixix, 186. 
 26Levaditi and Yamanouchi: C. E. Soc, Biol., 1907, Ixiii, 740. 
 !l7WeiL and Braun: Wien. klin. Wchnschr., 1908, xxi, 151. 
 zsBergel: Ztsehr. f. Immun., 1913, xvii, 234. 
 asManwaring: Ztsehr. f. Immun., 1909, iii, 309. 
 soBruok and Stern: Ztsehr. f. Immun., 1910, xxiii, 534. 
 3iPeritz : Ztsehr. f . Path. u. Ther., 1908-1909, v, 607. 
 32Scheresehewsky : Deutsch. med. Wchnschr., 1909, xxxv, 1652. 
 ssNoguchi: Jour. Am. Med. Assn., 1912, Iviii, 1163. 
 34Noguchi : Ibid. 
 
 ssNoguchi: Serum Diagnosis of Syphilis, ed. 2, Philadelphia, 1912, p. 169. 
 ssCraig and Nichols : Jour. Exper. Med., 1912, xvi, 336. 
 s^Kolmer, Williams, and Laubaugh: Jour. Med. Research, 1913, xxviii, 345. 
 saCraig and Nichols: Jour. Am. Med. Assn., 1911, Ivii, 474. 
 3»Craig: Jour. Exper. Med., 1911, xiii, 521. 
 ^oCraig: Jour. Am. Med. Assn., 1914,. Ixii, 1232. 
 
 iiNoguchi: Serumi Diagnosis of Syphilis, ed. 3, Philadelphia, 1912, p. 72; 
 *2Kolmer: Infection, Immunity, and Serum Therapy, ed. 2, Philadelphia, 
 
 1917, p. 437. 
 *3Noguchi: Jour. Exper. Med., 1909, xi, 392. 
 
 231 
 
232 THE WASSEEMANN TEST 
 
 «Nogucki: Serum Diagnosis of Syphilis, ed. 3, Philadelphia, 1912, p. 75. 
 
 *5Marie and Levaditi: Am. de I'Inst. Pasteur, 1907, xxi, 138. 
 
 isLandsteiner, Muller and Potzl: Wien. klin. Wchnsehr., 1907, xx, 1421-1565. 
 
 47Porges and Meier : Berl. klin. Wchnsehr., 1908, xlv, 731. 
 
 48Browning, Cruickshank, and McKenzie : Biochem. Ztschr., 1910, xxv, 85. 
 
 *9"Vedder: Bull. No. 8, War Dept., Office Surgeon-General, U. S. A., June, 
 
 1915. 
 soNoguchi: Serum Diagnosis of Syphilis, ed. 3, Philadelphia, 1912, p. 95. 
 siNonne : Syphilis and the Nervous System, Trans., Ball, Philadelphia, 1913. 
 52Noguehi: Serum Diagnosis of Syphilis, ed. 3, Philadelphia, 1912, p. 59. 
 53Hecht and Weinberg: Ztsehr. f. Immun., 1910, xxxvi, 317. 
 siGradwohl: Am. Jour. Syph., 1917, i, 450. 
 ssGradwohl: Ibid., 461. 
 ssKolmer: Infection, Immunity, and Serum Therapy, ed. 2, Philadelphia, 
 
 1917, p. 459. 
 s7Tsohernogubow: Berl. klin. Wchnsehr., 1908, xlv, 2107. 
 ssDetre and Brezovsky: Wien. klin. Wchnsehr., 1908, xxi, 1700. 
 eeStern, Margarette: Berl. klin. Wchnsehr., 1908, xlv, 1489. 
 eoBauer: Deutsch. med. Wchnsehr., 1908, xxxv, 698. 
 siBrowning and McKenzie: Ztsehr. f. Immun. Orig., 1909, ii, 459. 
 saThompson: Am. Jour. Syph., 1917, i, 555. 
 63Zinsser: Infection and Resistance, New York, 1917, 208. 
 6*Smith and MacNeal : Jour. Infect. Dis., 1917, xxi, 233. 
 65Vedder: Bull. No. 8, War Dept., Office Surg. General, U. S. A., 1915, p. 109. 
 ssSchereschewsky : Deutsch. med. Wchnsehr., 1909, xxxv, 1652. 
 67Noguchi: Jour. Am. Med. Assn., 1912, Iviii, 1163. 
 ssOraig and Nichols : Jour. Exp. Med., 1912, xvi, 336. 
 BflOraig and Nichols : Bull. No. 3, War Dept., Office Surg. Genl., 1913, 66. 
 ToKolmer, Williams, and Laubaugh: Joiir. Med. Research, 1913, xxyiii, 345. 
 T^Zinsser, Hopkins, and McBoimey: Jour. Exper. Med., 1916, xxiv, 561. 
 TzNoguohi and Akatsu: Jour. Exper. Med., 1917, xxv, 765. 
 "Vedder: Bulletin No. 8, War Dept., Office Surg. Genl., TJ. S. A., 1915. 
 T4Vedder: Ibid., 96. 
 
 TBCraig: Jour. Exp. Med., 1910, xii, 726. 
 TOKolmer: Infection, Immunity, and Specific Therapy, ed. 2, Philadelphia, 
 
 1917, p. 486. 
 TT-Vedder: BuU. No. 8, War Dept., Office Surgeon General, TJ. S. A., 1915, 
 
 p. 9. 
 raWoods and Morris: Jour. Infect. Dis., 1918, xxii, 43. 
 raKolmer: Infection, Immunity, and Serum Therapy, ed. 2, Philadelphia, 
 
 1917, p. 486. 
 soBoas: The Wassermann Reaction, Berlin, 1911. 
 siVedder; Bull. No. 8, War Dept., Office Surgeon General, U. S. A., 1915, 
 
 p. 9. 
 saVedder: Ibid. 9. 
 83Vedder: Ibid. 9. 
 
 8*Boas : The Wassermann Reaction, Berlin, 1911. 
 ssVeeder: Am. Jour. Med. So., 1916, clii, 522. 
 
 seHecht, Lateiner, and Wilenko : Wien. klin. Wchnsehr., 1909, xxii, 523. 
 87Kolmer: Infection, Immunity, and Serum Therapy, ed. 2, Philadelphia, 
 
 1917, 491. 
 ssBrowning and McKenzie: Diagnosis and Treatment of Syphilis, Phila- 
 delphia, 1912, 100. 
 SBThompson: Syphilis, Diagnosis and Treatment, Philadelphia, 1916, 157. 
 "oVedder: Bull. No. 8, War Dept., Office Surgeon General, U. S. A., 1915, 
 
 p. 10. 
 siVedder: Ibid., 109. 
 
BIBLIOGRAPHY 233 
 
 92Southara : Boston Med. and Surg. Jour., 1914, olxx, 25. 
 
 ssHammond : Am. Jour. Insan., 1913, Ixx, 107. 
 
 »4Thompson: Syphilis, Diagnosis and Treatment, Philadelphia, 1916, p. 27. 
 
 BSRosenberger : New York Med. Jour., 1917, ev, 1233. 
 
 96 Johnson: Am. Jour. Syph., 1917, i, 606. 
 
 »7Vedder: Bull. No. 8, War Dept., Office Surgeon General, U. S. A., 1915. 
 
 ssirvine: Journal-Laneet, April 1, 1917. 
 
 soPeterson: Am. Jour. Syph., 1917, i, 211. 
 looLadd: New York Med. Jour., 1917, cvi, 1211. 
 loiCraig: Jour. Am. Med. Assn., 1913, Ix, 565; Bull. No. 3, "War Dept., Office 
 
 Surgeon General, V. S. A., 1913, 80. 
 io2Nichols and Fordyce: Jour. Am. Med. Assn., 1910, Iv, 1171. 
 loaMichaelis: Berl. klin. Wchnschr., 1910, xliii, 183. 
 i04Fordyce: New York Med. Jour., 1911, xeii, 897. 
 losNoguchi : Serum Diagnosis of Syphilis, ed. 3, 1912. 
 loecraig: Bull. No. 3, War Dept., Surgeon General Office, U. S. A., 1913, 
 
 p. 99. 
 lo^Gennerich: Berl. klin. Wchnschr., 1910, Sept. 19, No. 38. 
 losMilian: Paris, Dei-matological Gesellsehaft, Dec. 1, 1910. 
 io9Herxh"eimer : Deutseh. med. Wchnschr^ 1910, xxxvii, 1517. 
 iioMichaelis: Berl. klin. Wchnschr., 1910, xlv, 1195. 
 iiiStokes and O'Leary: Am. Jour. Syph., 1917, i, 629. 
 iizKing: Jour. Am. Med. Assn., 1916, Ixvii, 1669. 
 iisNichols: Bull. No. 3, War Dept., Office Surgeon General, U. S. A., 1913, 
 
 p, 28. 
 ii*Stokes and O'Leary: Am. Jour. Syph., 1917, i, 629. 
 iisFordyce: Med. Eecord, New York, 191T, xd, 927. 
 iisHauptmann and Hossli: Miinchen med. Wchnschr., 1910, Ivii, 1581. 
 "''Mueller: Die Serodiagnose der Syphilis, Berlin, 1913. 
 iisFordyce: Med. Eecord, New York, 1917, xd, 927. 
 iisEavaut: Ann. de dermat. et de syph., 1903, iv, 1. 
 laoWile: Quoted by Kolmer. 
 
 liiOraig and Collins : Jour. Am. Med. Assn., 1914, Ixii, 1955. 
 i22Ellis and Swift: Jour. Exper. Med., 1913, xviii, 162. 
 i23Altman and Dreyfus: Miinchen med. Wchnschr., 1913, Ix, 464. 
 i24Kolmer: Infection, Immunity, and Serum Therapy, ed. 2, Philadelphia, 
 
 1917, p. 488. 
 i25Lange: Ztschr. f. Chemotherap., 1912-1913; i, 44; Berl. klin. Wchnschr., 
 
 1912, xlix, 897. 
 i28Zsigmondy: Ztschr. f. anal. Chem., 1901, xl, 697. 
 
 127 Jaeger and Goldstein: Ztschr. f. d. ges. Naur. u. Psych., 1913, xvi, 391. 
 i28.Zaloziecki: Deutseh. ztschr. f. Nervenh., 1913, xlvii, 783. 
 izoKolmer: Infection, Immunity, and Serum Therapy, ed. 2, Philadelphia, 
 
 1917, p. 556. 
 isoMiller, Brush, Hammers, and Felton: Bull. Johns Hopkins Hosp., 1915, 
 
 xxvi, 391. 
 isiMiller and Levy: Bull. Johns Hopkins Hosp., 1914, xxv, 123. 
 i32Nonne: Syphilis and the Nervous System, Trans. Ball, Philadelphia, 1913, 
 
 p. 345. 
 i33Nonne: Ibid., 346. 
 i34Nonne: 341. 
 
 lasNoguchi: Proe. Soc. Exper. Biol, and Med., 1909, vi, 51. 
 i36Noguohi: Serum Diagnosis of Syphilis, ed. 3, Philadelphia, 1912, p. 223. 
 iwMiller: Southern Med. Jour., 1915, viii, 940. 
 lasMiller: Ibid. 
 
INDEX 
 
 Alcohol, influence of, 40 
 Amboceptor : 
 
 character of, 65 
 
 definition of, 21 
 
 hemolytic, 23 
 
 preparation of, 65 
 
 preservation of, 68 
 
 titration of, 69, 81 
 Anesthesia, Wassermann reaction in, 
 
 151 
 Antigen, acetone insoluble lipoids as, 
 28, 74 
 
 alcoholic extracts as, 28, 73 
 
 aqueous extracts as, 28, 75 
 
 character of, 21 
 
 chemicals as, 28 
 
 cholesterinized, 76 
 
 cultures of T. pallidum as, 29, 37, 
 116 
 
 definition of, 21 
 
 preparation of, 71 
 
 titrations of, 77 
 Apparatus, for Wassermann test, 60 
 Asepsis, importance of, 49 
 
 Bacillus typhosus, 25, 124 
 
 extracts of, 38, 124 
 as antigen, 38 
 Bacteria, action of, 45 
 Bacteriolysin, 22 
 Bacteriolysis, 17 
 
 definition of, 21 
 
 discovery of, 17 
 
 nature of, 20 
 
 production of, 21 
 
 specific nature of, 20 
 Bacteriolytic system, 22 
 Blood corpuscles, washing of, 67 
 Blood serum: 
 
 bacteriolytic substances in, 17 
 
 collection of, 91 
 
 colloids in, 35 
 
 hemolytic substances in, 22, 65 
 
 inactivation of, 20, 93 
 
 lipotropic substances in, 34, 36 
 
 Blood serum — Cont'd. 
 
 thermolabile substances in, 20 
 
 thermostable substances in, 20 
 
 titration of, 59 
 
 variations in strength of, 51 
 Bordet-Gengou phenomenon, 24 
 Butyric acid test, 227 
 
 C 
 
 Carcinoma, Wassermann test in, 147 
 Cell count, 222 
 
 results of, 225 
 
 technic of, 225 
 Cerebrospinal fluid, 97, 205 
 
 collection of, 99 
 
 in latent stages of syphilis, 210 
 
 in primary stage of syphilis, 209 
 
 in secondary stage of syphilis, 209 
 
 in tertiary stage of syphilis, 210 
 
 interpretation of results of test, 210 
 
 Wassermann test on, 210 
 Cerebrospinal syphilis, 225 
 
 cell count in, 225 
 
 colloidal gold test in, 220 
 
 globulin tests in, 229 
 
 Wassermann test in, 208 
 Cholera spirillum, bacteriolysis of, 18 
 
 extracts of, as antigen, 124 
 CoUes' law, 145 
 Colloidal gold test, 213 
 
 in cerebrospinal syphilis, 220 
 
 in paresis, 219 
 
 in tabes, 219 
 
 interpretation of results of, 222 
 
 results of, 2l9 
 
 technic of, 215, 218 
 Colloids, 35 
 Complement, 22 
 
 character of, 22 
 
 definition of, 22 
 
 preparation of, 61 
 
 preservation of, 64 
 
 titration of, 77 
 Complement fixation, 25 
 
 alcohol and, 40 
 
 discovery of, 24 
 
 illustration of, 25 ; 
 
 235 
 
236 
 
 INDEX 
 
 Complement fixation — Cont 'd. 
 
 in man, 27 
 
 in monkeys, 27 
 
 nature of, 25, 34, 36 
 
 Wassermann test and, 26 
 Cultures, as antigens, 116 
 Cytolysins, 23 
 
 D 
 
 Dementia paralytica, Wassermann test 
 
 in, 207 
 Diabetes mellitus, Wassermann test in, 
 
 150 
 
 E 
 
 Extracts, as antigens, 28 
 alcoholic, 28, 73 
 aqueous, 28, 75 
 cholesterinized, 76 
 culture, 116 
 human heart, 74 
 
 Eilter paper, amboceptor, 70 
 
 impregnation of, 70 
 
 preservation of, 70 
 
 titration of, 81 
 Frambesia, Wassermann test in, 148 
 
 G 
 
 Globulin tests, 226 
 Nonne-Apelt, 226 
 Noguchi, 227 
 Pandy, 228 
 
 Hemolysin, definition of, 22 
 Hemolysis, 22 
 
 discovery of, 22 
 
 nature of, 22 
 Hemolytic amboceptor, 23 
 
 preparation of, 65 
 
 preservation of, 70 
 
 titration of, 69, 81 
 Hemolytic antigen, 71 
 Hemolytic system, 24 
 
 Inaetivation of blood serum, 20, 93 
 L 
 
 Leprosy, Wassermann test in, 149 
 Luetic zone curve, 220 
 Lumbar puncture, 99 
 precautions in, 101 
 
 M 
 
 Malaria, Wassermann test in, 148 
 Materials used in Wassermann test, 
 
 60 
 Mercury, effect of, on Wassermann 
 
 test, 183 
 Modifications of Wassermann test, 
 
 104, 112 
 
 N 
 
 Noguchi 's butyric acid test, 227 
 Noguchi 's modification, 105 
 Nomenclature of results of Wasser- 
 mann test, 101 
 
 Pandy 's test, 228 
 Paresis, cell count in, 225 
 
 colloidal gold test in, 219 
 
 globulin tests in, 229 
 
 Wassermann test in, 208 
 Paretic curve, 219 
 Preparation of antigen, 71 
 
 of amboceptor, 65 
 
 of blood corpuscles, 67 
 
 of blood serum, 91 
 
 of complement, 61 
 Prof eta's law, 145 
 Provocative Wassermann test, 197 
 
 cause of, 203 
 
 results of, 198 
 
 technic of, 198 
 Pulmonary tuberculosis, Wassermann 
 test in, 147 
 
 K 
 
 Reaction, time of appearance of, 133 
 time of disappearance of, 175 
 
 Reagents for Wassermann test, 60 
 preparation of, 61 
 titration of, 61 
 
 Relapse in syphilis, 191 
 
 Relapse in Wassermann reaction, 191 
 
 Relapsing fever, Wassermann test in, 
 149 
 
 Salvarsan, effect of, on Wassermann 
 test, 172 
 method of administration of, 180 
 Scarlet fever, Wassermann test in, 
 
 147 
 Septicemia, Wassermann test in, 147 
 Spirillum eholerse, bacteriolysis of, 18 
 cultures of, as antigens, 38, 124 
 
INDEX 
 
 237 
 
 Spirochseta refringens, 38, 125 
 Staphylococcus albus, 46 
 aureus, 46 
 citreus, 46 
 Streptococcus pyogenes, 46 
 Syphilis, cell count in, 222 
 colloidal gol4 test in, 213 
 globulin test in, 226 
 prevalence of, 164 
 relapse in, 191 
 
 variations in blood serum in latent 
 stage of, 55 
 in blood serum in primary stage 
 
 of, 52 
 in blood serum in secondary stage 
 of, 53 
 Wassermann test in congenital; 129, 
 144 
 in latent stage of, 129, 141 
 in primary stage of, 129 
 in secondary stage of, 129, 138 
 in tertiary stage of, 129, 140 
 
 T 
 
 Tabes, Wassermann test in, 207 
 
 cell count in, 225 
 
 globulins in, 229 
 
 colloidal gold test in, 220 
 Temperature, relation of, 48, 112 
 Titration of reagents, 77 
 
 of blood serum, 51, 57 
 Toxolipoids, 34 
 Treponema ealligyrum, 125 
 
 microdentium, 38, 119 
 
 mucosum, 125 
 
 pallidum, 27 
 
 antigens prepared from, 37, 116 
 cultures of, 37 
 discovery of, 27 
 
 pertenue, 38, 119 
 Tuberculosis, Wassermann test in, 147 
 
 W 
 
 Wassermann test: 
 alcohol, and, 40 
 
 Wassermann test — Cont 'd. 
 
 amboceptor for, 67, 81 
 
 antigens for, 27, 28, 71, 116 
 
 apparatus used in, 60 
 
 cerebrospinal fluid and, 97, 205 
 
 discovery of, 26 
 
 factors influencing results of, 40 
 
 general description of, 31 
 
 influence of bacteria on, 45 
 
 influence of variation in bloX)d se- 
 rum on, 50 
 
 interpretation of results of, 44, 152 
 after specific treatment, 162 
 in cerebrospinal fluid, 210 
 in control of treatment, 193 
 in latent stage, 161 
 in nervous diseases, 162 
 in primary stage, 155 
 in secondary stage, 158 
 in tertiary stage, 159 
 
 modifications of, 104, 112 
 Gradwohl's, 108 
 Hecht-Weinberg 's, 107 
 Kolmer's, 110 
 Noguchi's, 105 
 
 nature of, 27, 30, 33, 36, 38 
 
 original teohnio of, 103 
 
 prevalence of syphilis and, 164 
 
 provocative, 197 
 
 results of, 96 
 in congenital syphilis, 144 
 in latent syphilis, 129, 141 
 in primary syphilis, 129, 130 
 in secondary syphilis, 129, 138 
 in tertiary syphilis, 129, 140 
 
 specific antigens in, 28, 33, 36, 116 
 
 specificity of, 146 
 
 technic of, 89 
 
 titration of reagents used in, 77 
 
 treatment and, 171 
 
 Yaws, Wassermann test in, 148 
 
INDEX OF AUTHOES 
 
 Akatsu, 125 
 Altman, 210 
 
 AtZHBIMEE, 223 
 
 Apelt, 226 
 
 Aening, 132, 139, 140, 144 
 
 Bauee, 112 
 
 Belfanti, 22 
 
 Beegenon, 169 
 
 Beegel, 35 
 
 Beeing, 132, 139, 140, 144 
 
 Blumenthal, 139 
 
 Boas, 132, 138, 142, 207 
 
 BOEDET, 20, 22, 24, 25, 26, 30 
 
 Bkaun, 35 
 
 Beezovsky, 112 
 
 Bronpenbeennee, 62 
 
 Beuok, 26, 27, 35, 132, 139, 140 
 
 Beush, 216 
 
 Bkowning, 28, 76, 112, 147 
 
 Caebone, 22 
 CiTEON, 34, 35 
 OOLLIN, 209 
 
 Craig, 29, 38, 40, 41, 45, 50, 89, 91, 
 113, 118, 132, 139, 140, 142, 144 
 Cruickshank, 28, 76 
 
 Detee, 27, 112 
 Deeypus, 210 
 
 Ehelich, 18, 21 
 Ellis, 209 
 
 Felton, 216 
 Fischer, 132 
 fodoe, von, 18 
 Fokdyce, 173, 206 
 Fox, 142 
 
 D 
 
 a 
 
 Gengou, 24, 25, 26, 30 
 Genneeich, 197 
 Goldstein, 214 
 Geadwohl, 108, 109 
 Geossee, 132, 142 
 
 H 
 
 Hammers, 216 
 Hammond, 165 
 Hanchen, 132 
 Hauptmann, 207 
 Hecht, 107, 147 
 Hekxheimee, 197 
 Hohne, 139, 140 
 Hopkins, 125 
 HossLi, 207 
 
 Jaeger, 214 
 Jesionek, 144 
 Johnson, 165 
 
 K 
 
 Kaplan, 132 
 King, 198 
 KoLMER, 38, 42, 
 
 110, 111, 112, 125, 
 
 138, 147, 210, 216, 217 
 
 Ladd, 219 
 Landois, 22 
 Landsteinee, 28, 76 
 Lange, 213 
 Lateinee, 147 
 Laubaugh, 88, 125 
 Lederman, 139, 140, 142 
 Lepine, 169 
 Lesser, 139, 140 
 Letulle, 169 
 Levaditi, 28, 35, 76 
 Levy, 219 
 
 238 
 
INDEX OP AUTHORS 
 
 239 
 
 M 
 
 McBuENEY, 125 
 
 McKenzie, 76, 112, 147 
 MacNeal, 113 
 Manwaring, 35 
 Maeie, 28, 76 
 Meier, 76, 132 
 Meiekosky, 144 
 Merz, 139, 140 
 MiCHAELis, 173, 197 
 MiLIAN, 197 
 
 Miller, 216, 219, 228 
 MOKEIS, 137 
 MiJLLER, 28, 76, 207 
 MiJNCK, 34 
 
 N 
 
 Neisser, 26, 37 
 
 Nichols, 29, 38, 40, 41, 89, 91, 113, 
 118, 123, 173, 174, 199 
 
 NoGUCHi, 28, 29, 34, 36, 37, 62, 64, 
 65, 68, 74, 89, 93, 105, 107, 112, 
 116, 117, 118, 119, 121, 122, 
 123, 125, 132, 139, 140, 144, 
 147, 173, 226, 227 
 
 O 
 
 O'Leary, 198, 200, 201 
 P 
 
 Pandy, 226, 228 
 Peritz, 35 
 Peterson, 169 
 Pfeipfee, 18, 19 
 Platjt, 28 
 
 PORGES, 76 
 
 POTZL, 28, 76 
 
 B 
 
 Eavaut, 209 
 
 EOSCHER, 139 
 EOSENBEEGER, 165 
 
 s 
 
 Soiiaudinn, 27 
 
 Schmidt, 34 
 
 Scheeeschewsky, 36, 116 
 
 Schonnepeld, 139 
 
 Schtjcht, 27 
 
 Smith, 113 
 
 Southard, 165 
 
 Stern, 35, 112, 132, 139, 140 
 
 Stokes, 198, 200, 201 
 
 Swift, 29, 132, 139, 140, 142, 209 
 
 Thompson, 112, 149, 165 
 Tscheenogubow, 112 
 
 V 
 
 Vedder, 61, 89, 90, 114, 127, 128, 132, 
 134, 138, 139. 140, 142, 144, 
 150, 155, 166, 167, 168 
 
 Veeder, 144, 145 
 
 W 
 
 Walkee, 29 
 
 Wassermann, 26, 27, 29, 30, 75 
 
 Weil, 35 
 
 Weinberg, 107 
 
 Weygandt, 28 
 
 Wile, 209 
 
 WiLENKO, 147 
 
 Williams, 38, 125 
 Woods, 137 
 
 Yamanouchi, 35 
 
 Zaloziecki, 214 
 Zinsser, 113, 125 
 Zsigmondy, 214