li-S CORNELL UNIVERSITY, THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEQE. X897 Cornell University Library RB 145.C69 The blood; how to examine ="^,j'i^9|1!|^?^ ' 3 1924 000 274 856 Cornell University Library The original of tiiis bool< is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000274856 THE BLOOD: HOW TO EXAMINE AND DIAGNOSE ITS DISEASES. THE BLOOD: HOW TO EXAMINE AND DIAGNOSE ITS DISEASES. ^ ALFRED C. COLES, M.D. B.Sc. OF Public Health, Edinburgh ; Fellow of the Obstetrical Society, London ; Formerly Senior House-Surgeon at the County Hospital, York. WITH SIX COLOURED PLATES. LONDON: & A. CHURCHILL, 7 GREAT MARLBOROUGH STREET. 1898. PREFACE. During a somewhat prolonged investigation into the microscopic appearance of the blood in certain patho- logical conditions, I was at an early period struck with the difficulty of finding any exhaustive account of the methods employed in, and the help to be derived from, the examination of the blood. Various papers written by Drs. Gulland, Kanthack, and Muir have appeared in different journals, but these only deal with one department ; and a full and critical account of the methods, especially those dealing with the fixing and staining of films, was incomplete or absent. Most of the literature relating to the microscopic examination of the blood was in German, some in French, but very little in English, excepting on Malaria. Hay em's Du Sang, a stupendous work, treats more particularly of the red corpuscles, but the account of the occurrence of nucleated red blood corpuscles and the development of the cellular elements, is either out of date or not generally accepted as correct, whilst very little mention is made of the variety of leucocytes, classified according to Ehrlich's investigation on the staining reaction of the granules contained in their protoplasm. THE BLOOD Von Limbeck's Grundriss einer Klinischen Pathologie des Blutes gives a much fuller account of the latter, but the language in which it is written — German — prevents some taking advantage of the information it contains. Having regard to the importance and value of a careful examination of stained preparations of the blood, I have collected what information I could from the German, French, and scattered articles in the English medical journals. When near the conclusion of my work, an excellent book was brought out in English by Dr. Cabot, of America, on The Clinical Examination of the Blood. To my mind this work is deficient in the matter of technique, particularly in the. staining of dry films, and no mention is made of a subject which has quite recently attracted some attention in England, viz.. Splenic anaemia. This small work has no claim to originality, being more or less a compilation from other literature. I have followed most of the observations mentioned, and after repeated failures, wish to draw attention to an efficient and reliable method of blood examination which answers all the requirements of the physician. Certain parasites of the blood — e.g., filaria sanguinis hominis, the spirochaetae of relapsing fever — as well as the symptoms of the diseases described, except splenic anaemia, have been omitted, as these are dealt with at length in all text-books on medicine. No mention is made of the question of the chemical examination of the blood, partly because it is work that PREFACE can only be done in a laboratory, and partly that up to the present the results so obtained have not proved to be of that clinical value which they may ultimately reach. All the illustrations, except Plate VI., are taken from films v/hich I have made during the last five years. I take this opportunity of expressing my thanks to Dr. Gulland and Dr. Kanthack for kind suggestions as to methods, especially in the earlier days. I have given a list of books and articles from which I have extracted freely, and would particularly mention my indebtedness to the following: Hayem, von Limbeck, and an article by Stengel in the Twentieth Century of Medicine. Bournemouth. COLOURED PLATES Plate I. The Red Blood Corpuscles and their alterations To face page 4? II. The Leucocytes . . . „ 64 III. The Leucocytes variously stained . „ 72 IV. Chlorosis and Pernicious Anaemia . „ 100 V. Spleno-medullary Leucocythaemia and Normal Marrow To face page 132 VI. Lymphatic Leucocythaemia and Malarial Parasites To face page 146 CONTENTS. Methods of Examining the Blood- Enumeration of the Cellular Elements PAGE I Estimation of the Haemoglobin 12 Examination of Fresh Blood 17 Examination with Preserving Fluids 19 Stained dry Film Method 20 Spreading Films . . . . 20 Fixing Films . . . . 23 Staining Films . . . . 26 General Morphology of the Blood — The Red Blood Corpuscles — Effects of Physiological Conditions on Pathological Alterations in Nucleated Red Blood Corpuscles The Development of the Blood The Structure of the Bone Marrow The White Blood Corpuscles — Varieties of Leucocytes The Lymphocytes Multinucleated Leucocytes Eosinophile Cells Marrow Cells Mastzellen The Number and Proportion of the Leucocytes Influence of Physiological Conditions on Pathological Alterations in Inflammatory Leucocytosis and Phagocytosis The Blood Plates 37 39 41 47 55 58 62 63 64 67 71 76 77 79 80 81 83 88 THE BLOOD Pathology of the Blood- Anaemia considered generally The Degrees of Anaemia Primary or Idiopathic Anspmia — Chlorosis — .... The Red Blood Corpuscles and Haemoglobin in Nucleated Red Corpuscles in . The Nature of Chlorosis The Diagnosis of Chlorosis . . ■ Simple Primary Anemia Progressive Pernicious An/emia — Definition and Restriction of the Term . The Red Blood Corpuscles and Haemoglobin in Nucleated Red Corpuscles in . Nature of the Disease Diagnosis and Differential Diagnosis Leucocythaemia — Number of the Leucocytes found in The Leucocytes in Spleno-medullary Leucocythaemia Marrow Cells or Myelocytes Eosinophile Cells Other varieties of Leucocytes Mitotic Division of Nuclei The Red Blood Corpuscles in Lymphatic Leucocythaemia — The Nature of the Leucocytes Pathology of Leucocythaemia The Nature of Spleno-medullary Leucocythaemia The Nature of Lymphatic Leucocythaemia Acute Leukaemia Hodgkin's Disease or Pseudo-leukaemia — General Characters . Condition of the Blood in PAGE • 92 94 . . 98 • 99 lOI • 103 104 i°5 109 109 112 117 120 121 127 130 smia 132 133 135 138 141 142 145 146 148 149 15° 160 . 162 . 163 CONTENTS XI Primary Anaemia (contd.) — Splenic Anemia — Condition of the Blood in Diagnosis and Differential Diagnosis Differential Chart of the most important forms OF ANjEMIA . . . ... PAGE i68 170 172- Secondary or Symptomatic Anaemia — General Condition of the Blood in Diagnosis and Differential Diagnosis Secondary Ancemia due to Malignant Disease . The Condition of the Blood in Carcinoma The Condition of the Blood in Sarcoma . Post-Haemorrhagic Anemia Haemorrhagic Diseases — Purpura .... Scorbutus .... Haemophilia Haemoglobinaemia or Haemocytolysis Malarial Parasites — Method of Examining the Blood Staining Films of Malaria Blood Double Staining with Eosine-Haematoxylin The Tertian Parasite . The Quartan Parasite . The Parasite of Malignant Fevers Diagnosis and Recognition of the Malarial Parasite Tabular Chart of the Characteristic Signs of the various Parasites . . ■ ... The Blood in Acute Diseases — Pneumonia . The Serum diagnosis of Typhoid Fever The Blood in Typhoid Fever The Exanthemata Diphtheria Erysipelas . 174 177 179 179. 181 i8s 185 187 188 188 191 192 194 197 199 200 202 206 211 21S 216 218 2 20- 221 xu THE BLOOD Secondary or Symptomatic Anaemia (contd.) — PAGE Acute Rheumatism . . . , . 222 Inflammation of Serous Membranes . • .223 Pleurisy, Peritonitis, Meningitis . . ■ .224 Septicaemia and Pyaemia . . ■ .226 Suppuration and Abscess formation . . .227 Secondary Anaemia in Chronic Infectious Diseases — Syphilis . . . . . . 229 Tuberculosis . . . . . 231 Condition of the Blood in Various General Diseases — Diabetes . . . . . . 234 Gout . . . ... 236 Myxoedema . . . . . 237 Diseases of the Alimentary System . . -238 „ „ Respiratory „ . . .240 „ „ Circulatory „ ... 241 „ Kidney . ... 242 „ „ Nervous System . . . 242 The Anemias occurring in Infants — Characters of the Blood peculiar to early life . . 243 Splenic Anemia of Pseudo-leukaemica- Infants r An^mi A Infa NTUM 245 Diagnosis Inherited Syphilis in Infants Rickets in Early Childhood 248 250 251 Literature Index 253 255 THE BLOOD: JIOIV TO EXAMINE AND DIAGNOSE ITS DISEASES. METHODS OF EXAMINATION OF THE BLOOD. A COMPLETE and systematic examination of the blood would include the enumeration of the corpuscular elements, the estimation of the amount of haemoglobin, and the examination of films microscopically. The first two' procedures have been generally recog- nized for a considerable period, but the last, especially the use of differentially-stained preparations, has received an attention disproportionate to its value. THE ENUMERATION OF THE CELLULAR ELEMENTS OF THE BLOOD. Two instruments are used in England for counting the corpuscles, viz., the Thoma-Zeiss and Gower's Haemocytometer. The latter is recommended in almost all the English textbooks, but it is not as convenient or accurate as the Thoma-Zeiss apparatus. The Thoma-Zeiss Haemocytometer. This consists of a small pipette for counting the red corpuscles, the stem of which is divided into tenths, and THE BLOOD marked o'5 and ro. Just above the mark i' the tube expands into a bulb containing a small glass bead, at the end of which the number loi is marked. The second pipette (when supplied with the instrument) for counting the white corpuscles, is very similar, but slightly larger. The lobe of the ear or tip of the finger is cleaned, dried, and then pricked with a needle. When a drop of blood appears, the point of the pipette is placed into it, and gentle suction made till the blood reaches the point marked I'O at the com- mencement of the bulb. It is well not to commence to draw up the blood till the drop is sufficiently large, or otherwise the mark may not be reached, and this would render a fresh attempt necessary. The point of the pipette should not be pressed on the surface of the skin, or the flow may in that way be stopped ; and lastly, the point of the pipette should never be allowed to escape from the drop of blood till the desired mark is reached, or an air bubble will be drawn in and the result vitiated. Some writers advise that the blood be only drawn up to the point o"5, so that the dilution being i in 200, instead of i in 100, shall facilitate the counting of the corpuscles. According to these observers, if the mark be overstepped, it is possible to lower the column of blood to the point 0-5 by gently tapping the pipette on a towel. I invariably draw the blood up to the mark i', as the leucocytes can then be counted on the same film as the red corpuscles ; and further, that although with a dilution of i in 100 about I2|- corpuscles in health are seen in each square of the counting chamber, yet in ENUMERATION OF CELLULAR ELEMENTS 3 most anaemias, and it is with these that we are chiefly dealing, half, frequently less than half, that number are present. Another point in favour of the latter method of procedure is, that the less the multiplier the less the error in the calculation. As soon as the blood has reached the mark the outside of the point of the pipette is quickly wiped, to remove any blood adhering to it, and immediately im- mersed in the diluting fluid, and this is drawn up to the mark loi. Any of the diluting solutions here- after given may be used. In doing this it is advisable to roll or shake the pipette whilst suction is being applied, in order to prevent the blood floating on the top of the diluting fluid. Care should also be taken that the point of the pipette is kept under the fluid, or a bubble of air may be drawn in. It is extremely easy to overstep the mark 10 1, as the bulb seems to fill suddenly. So far the whole process should be performed as quickly as the precautions necessary to be observed will allow. As everything depends on the accuracy of the preceding technique, I will briefly mention the possible or probable errors that may at first occur. 1. The blood column should not be allowed to coagu- late before the diluting fluid is drawn up. 2. An air bubble or break in the column of the blood, usually due to starting before the drop is large enough or allowing the point of the- pipette to slip out of the drop of blood or neutral solution whilst suction is being made, may occur, which would render the preparation useless. If the puncture be deep enough a sufficient quantity of blood will quicklj^ appear. THE BLOOD 3. The blood column may suddenly stop, probably either because the top of the pipette is touching the skin, or because the capillary bore of the pipette is not dry, and coagulation has taken place. The pipette now contains one part blood and 100 parts of the neutral solution, and may safely be left for some time before counting. There is no need to carry the microscope to the bedside. All that is necessary is the pipette, which can be carried in a small case a little larger than that containing a clinical thermometer, and a small bottle holding about thirty drops of the diluting fluid. In order to carry about the filled pipette Cabot suggests that a rubber band be placed longitudinally round it, in order to close the ends. At first I tried to obviate any danger of escape of the contents by placing india-rubber caps on both ends, but found that this had the disadvantage of driving the fluid in the capillary bore into the bulb, making the dilution one in 10 1, instead of one in 100. I find now that the best method is to carefully detach the india-rubber tubing, after the pipette is filled, without compressing it — any pressure at this end forces a small drop of the liquid out at the point end — and then the tube is placed in its case, and kept horizontally till used. The Counting. The slide containing the counting chamber consists of a disc of glass, on which parallel lines are engraved, which by their intersection form 400 squares, the length of a side of each of which is -^ of a millimetre. The squares are further divided by double lines into groups of 16. When the coverglass is applied each cell has a depth of j\j- of a millimetre. ENUMERATION OF CELLULAR ELEMENTS s The charged pipette is thoroughly shaken, previously closing both ends with the thumb and ring-finger — the india-rubber tubing having been removed — and then a drop is blown out, which consists only of the diluting fluid. It is well to blow out three or four large drops before charging the counting cell. After this a small drop is placed on the centre of the ruled disc and covered with the coverglass. The drop should only cover the ruled area, and should not run into the little channel around it. Care should be taken to avoid air bubbles appearing in the film, and lastly, the coverglass should fit so closely to the outer table of glass that Newton's rings can be seen. The slide may then be placed under the microscope and kept in a horizontal position. In a few minutes the corpuscles have sunk to the bottom and may be counted. A magnification of about 200 {i.e., \ or \ inch objective) is very suitable. The light should be so arranged that the lines as well as the corpuscles are seen. It sometimes happens — I have, however, only noticed it in the Gower's ruled chamber — that the parallel lines are invisible ; and Hawkesley, in a private communication, stated that this is easily remedied by rubbing a lead pencil over the ruled area, and then wiping gently with a soft handkerchief before filling the cell. The number of red corpuscles found in several groups of 16 squares should be counted and then the slide washed,, cleaned, and thoroughly dried, and another drop counted. The greater the number of corpuscles counted the less the error. Some corpuscles will be seen on the lines and not in the squares, and in such cases it is usual to count in all corpuscles touching the top and right lines, and to THE BLOOD leave out of the count those touching the lower and left lines. Calculation. — Each square has a cubic capacity of ^ X TJT X TiT = Tinnr cubic millimetre, therefore the formula for calculation will be : — No. of corpuscles counted x dilution (ioo)x4ooo • ,. !,„ „f E- = — ^ — = per cubic mm. oi No. of squares counted. blood. Example. — Supposing 2000 corpuscles were counted in 10 groups of 16 squares, and the dilution was i in 100, then, 2000 X 100 X 4000 5,000,000 red cells in i cubic 10x16 millimetre of blood. Counting the White Corpuscles. There are two methods of counting the leucocytes : I St, with the special pipette ; and 2nd, with the ordinary pipette and at the same time as the red blood corpuscles. With the Special Pipette. — Here the process is similar to that just described for counting the red corpuscles. The diluting solution used is generally a ^ % solution of glacial acetic acid in water, which renders the white cells evident, but the red cells somewhat transparent. It requires a much larger quantity of blood than the ordinary pipette. With the Ordinary Pipette. — I find the red corpuscle pipette sufficiently accurate for clinical purposes, and it further necessitates much less discomfort to the patient, as the puncture required for filling the ordinary small pipette, the capillary tubes of Fleischl's haemo- meter, and the preparation of half a dozen films on the glass slides, need not be very deep. ENUMERATION OF CELLULAR ELEMENTS 7 Ewing, who has made numerous observations on the occurrence of leucocytosis, especially in pneumonia, commends the use of the ordinary pipette for counting the white as well as the red corpuscles. He states that the employment of the large pipette or leucocytometer has several disadvantages. " The amount of blood required is inconveniently large. Acetic acid obscures the outline of the leucocytes, and it is often difficult to distinguish them from dSbris left by the red blood cells. The leucocytes become very cohesive, and appear in the counting-chamber in considerable masses, in spite of persistent efforts to secure the even distribution neces- sary for an accurate count." [New York Med. Journal, Dec. i6, 1893.) It is very advantageous to use a tinted diluting solu- tion, as the white corpuscles are then sharply defined. I find Toisson's solution answers excellently, provided it is always filtered before use. With a dilution of i in 100 only about J^ leucocytes will be found in normal blood in the whole of the 400 squares, and it is obviously necessary to somewhat modify the process. Under the microscope it will be seen that the area of ruled squares only occupies a small part of the whole of the cell, and it is advisable to include the whole area under observation. The method which I use is the following, suggested by Stengel. The area of the field of vision of the micro- scope is first determined. The ruled area on the slide is focussed with, say, ^ inch objective, and the tube of the microscope drawn out till "one of the parallel lines of the ruled slide accurately coincides with either side of the field of vision." In my own microscope with THE BLOOD \ inch objective, eyepiece 2, and the tube drawn out to 162 mm., the diameter of the field of vision corresponds to 10 squares, each of which is ^V "^"^•' ^o that the diameter =5- mm. and the radius \ mm. From the formula RV, where '7r = 3-1416 and R = radius, in this case \ mm., then ixix 3-1416 = 0-19635 square mm. But the depth of the cell is 3^ mm., therefore the cubic capacity of a field of vision will be : — t^t^tV^ 3-1416 = 0-019635 cubic mm. Then the formula for calculation will be the following : — No. of leucocytes counted in field of vision x dilution (100) area (0-019635) x No. of fields counted. As two of these factors are constant — viz., the dilution and the cubic area of the field of vision — we can simplify it thus : — 100 Then the simplified formula will in this instance be the following : — Working with \ inch objective, eyepiece 2, tube length 162 mm., and the dilution of i in 100 : — No. of leucocytes counted x 5092-946 No. of leucocytes per No. of fields in which they were counted" i cmm. of blood. Example. — Suppose that 40 microscopic fields were examined and 64 leucocytes seen : — 64x5092.946 8148 leucocytes per cubic millimetre 4° of blood. In the same way I find that my \ inch objective when used with eyepiece No. 4, and tube length of 173 mm,, includes 8 cells of -^ mm. ENUMERATION OF CELLULAR ELEMENTS, 9 Therefore the diameter = ^®^ mfti. and the radius = j®^ or \ mm. •'• tVx¥XtX3"I4i6 = -oi2s664, the dilution being i in 100: — 100 _ 77- = 7957728. ■0125664 '^^' ' The formula in this case would therefore be : — ■ No. of leucocytes counted x 7957728 _ No. of leucocytes in cubic No. of fields counted millimetre of blood. When counting the leucocytes in the field of vision, instead of in the ruled areas, care must be taken to go regularly from one circular area to another, and to include an entirely fresh field at each count. The areas counted must not be selected, but should be taken just as they come. When the leucocytes are very numerous, as in leucocythemia, in which 45 to 50 white cells may be seen in one field with the \ inch objective and No. 2 eyepiece, it is much easier to estimate their number over some part of the ruled area, not necessarily over the squares. Cleaning the Blood Pipette. It is most important that the capillary pipette should be cleaned immediately after it has been used. The directions generally given are that, first some of the diluting fluid, then water, then alcohol, and lastly, ether should be sucked into the pipette, and after shaking, blown out. On account of the fineness of the capillary bore this is a very tedious proceeding, and involves considerable labour. I have found the following method of cleaning invalu- able, and it involves no apparatus other than that which every physician possesses, viz., an aspirator. I attach lo THE BLOOD the blood pipette by a small piece of india-rubber tubing to the inlet tube of a Potain's aspirator, and then, after exhausting the air in the bottle, allow a large quantity of water (or at first, normal saline solution), then alcohol, and finally ether, to be drawn through it. If the instru- ment is wanted at once, warm air from a spirit lamp may be drawn through, so as to rapidly dry every part of the tube. Before use the pipette must be absolutely clean and dry. Gower's Haemocytometer. This instrument is in more general use in England than the above mentioned, but after considerable experience with it, I am convinced that Gower's instru- ment cannot be compared with the one just described. It has no advantages over the Thoma-Zeiss apparatus, and is much more complicated and costly. In using this form of haemocytometer 995 cubic millimetres of the diluting solution, measured in the larger pipette, are placed in the small mixing jar supplied with the instrument. Into the capillary pipette 5 cubic millimetres of blood are drawn, and then blown into the diluting fluid. The two liquids are well mixed with the glass rod stirrer, and then a drop of the diluted blood is placed in the counting- cell and covered with a coverglass, which is held in position by two clips. The corpuscles are allowed to settle and then counted. Each square measures -g^ cmm., and the dilution is I in 200 ; therefore the formula for the calculation would be— No. of corpuscles counted x dilution (200) x 500 No. of corpuscles No. of squares counted ~ in i cmm. of blood. ENUMERATION OF CELLULAR ELEMENTS ii Neutral Diluting Fluids. For diluting the blood for the purpose of enumeration various preserving or neutral solutions may be used, the simplest being a 2 or 3 % solution of salt in water. Amongst others the following may be mentioned : — Gower's Solution consists of a solution of sodium sulphate in distilled water, having a specific gravity of 1025, or any of the following formulae may be used : — Sodium Sulphate Acetic Acid .... Distilled water .... Hayems Solution consists of — Hydrarg. Perchlor. Sodium Sulphate Sodium Chloride Distilled water Pacini's Solution — Hydrarg. Perchlor. Sodium Chloride Glycerine Distilled water The last mentioned is allowed to stand for a consider- able time (two months), and when about to be used is diluted with three times its volume of water, and filtered. The indifferent solutions may also be tinted with some of the aniline dyes, in order to render the white blood corpuscles more apparent. The 2 or 3 % aqueous salt solution may be coloured with gentian violet, about i % of the saturated alcoholic solution being added. grs. : E04. 5j. Siv. o"5 grms. 5-° >J i-o J) 200'0 )> I'O grms. 2'0 J) 130 )) 113-0 )j THE BLOOD Toissons Diluting Solution is probably one of the most convenient for clinical purposes. It consists of the following : — Aqua, distill. i6o cm. Glycerine (neutral) 30 cm. Sodium Sulphate 8 grm Sodium Chloride I grm Methyl. Violet . 0-025 grm All these indifferent solutions require very thorough filtration before being used. Toisson's fluid is particularly adapted for diluting, as it stains the leucocytes very clearly, and thus facilitates their enumeration. A Differential Count of the Leucocytes can only be satisfactorily made by means of stained films. In such specimens 500 to 1000 leucocytes are counted and classified according to their character into small uninucleated, large uninucleated, multinucleated, eosino- phile leucocytes and in pathological conditions, marrow cells or myelocytes. ESTIMATION OF THE AMOUNT OF HAEMOGLOBIN. Various instruments have been devised for estimat- ing the amount of haemoglobin, but probably the most satisfactory for clinical purposes are Von Fleischl's haemometer and Gower's haemoglobinometer. They are both colorimetric, the blood being in each case diluted, and the resulting colour compared in the one case with a tinted-glass wedge, in the other with a standard solution of some pigment. THE AMOUNT OF HAEMOGLOBIN 13 Von Fleischl's Method. The apparatus consists of the following : — 1. A metal cylinder, closed at one end by glass and divided into two equal compartments by a vertical septum. 2. A stage somewhat resembling that of a micro- scope, which carries a wedge of red glass. The latter is graduated and can be moved horizontally along beneath the stage. 3. Small capillary tubes, which fill automatically and hold a definite quantity of blood. 4. A small glass pipette for filling the compartments with water. Full instructions of the method of using are supplied with the instrument, and I shall only briefly describe the most important points. The cylinder is partially (about one-fourth) filled with water by means of the glass pipette, and as soon as a fairly large drop of blood has appeared at the point punctured, the capillary tube, held by the stem in which it is fixed, is brought in contact with it, and the blood immediately fills it by capillarity, provided that it is held in a position other than the vertical. The tube containing the exactly-measured quantity of blood is quickly cleared of any blood which may be adhering to its outer surface and immediately placed in the water in one compartment of the cylinder. It is then moved about in order that the water may dissolve out the colouring matter of the corpuscles, and then, whilst it is held vertically, drops of water from the pipette are allowed to fall on and run through it until the capillary tube is quite clean. With the handle end of the capillary 14 THE BLOOD tube the blood and water are thoroughly mixed, and then water added till the compartment is quite full. The other side is filled with water only, so that on one side we have a mixture of water and blood and on the other water alone. The cylinder is then placed on the circular opening of the stage, so that the coloured part is directed away from the finger screw, whilst the side containing the water is over the wedge of coloured glass. Artificial light, such as that afforded by a lamp or gas (daylight, electric, or incandescent light cannot be used), is reflected upwards from the plaster of Paris reflector, and it is generally better to use as little light as possible. The coloured glass is then moved along, preferably in jerks, and not too gradually, till the red tint in both compartments appears equal and the percentage noted. Daylight must be excluded as much as possible, and it is easier to estimate the intensity of the two colours when the axis of the eyes is at right angles to the septum, which divides the cylinder into two equal compartments, the lateral portions of the retina being more sensitive to colour impressions. One eye only should be used, and that at the ordinary reading distance from the stage. Sometimes a piece of paper rolled into a tube may be looked through, as this, by cutting off any light in the room, renders a sharper definition possible. The whole process only occupies one or two minutes, and the results are very fairly accurate. It renders the greatest assistance in watching the progress of cases, especially chlorosis, and sometimes obviates the necessity of a long count of the red corpuscles. THE AMOUNT OF HAEMOGLOBIN 15 The instrument should be used soon after the blood is measured and diluted, but it is possible when the cylinder is carefully covered with the small circular glass disc supplied with the instrument, to delay a short time. I generally use an elastic ring to keep on the glass disc whilst I am carrying it. Certain fallacies occur in using Von Fleischl's haemometer. Firstly, the readings are rather low, as many healthy individuals only show 85 to 90 % of haemoglobin with this instrument. Secondly, when the haemoglobin is very low it is extremely difficult to determine exactly at what point the two colours are quite equal. I have found this particularly notice- able in pernicious aneemia. Under such conditions I use two capillary tubes of blood instead of one, and therefore take one -half the percentage as the approximate measure of the haemoglobin. I find that Stengel also advises this procedure. Thirdly, in cases of leucocythemia the enormous increase in the white cells renders the mixture of blood and water turbid. Leichtenstern recommends the addition of a very small quantity of alkali to the water in these cases. Gower's Haemoglobinometer. This instrument is much used in England, and gives approximately good results. It is, however, not as convenient as Von Fleischl's apparatus, but is some- what less bulky, and costs only about one-third the price of the latter. It consists of two glass cylinders of equal size placed vertically side by side on a small stand. One con- tains a tinted solution equal to a i % watery solution 1 6 THE BLOOD of blood. The other, open at the top, is graduated in tenths up to 120. In addition there is a capillary pipette, measuring 20 cmm., and a small drop -bottle for diluting the blood. A few drops of water are first placed in the graduated cylinder and 20 cmm. of blood are drawn up into the capillary pipette and quickly discharged into the cylinder. The pipette is then filled with water in order to dissolve any blood which may be adhering to the surface of the tube, and this is added to the mixture of blood and water. The cylinder is immediately shaken to mix the blood and water before the former coagulates, and water is added drop by drop with the dropper, till the tint in both tubes is equal. This is easily ascertained by placing a piece of white paper behind the tubes, or by holding them up to the light from a window. The percentage of haemoglobin is then seen by the scale on the gradu- ated cylinder. The amount of haemoglobin as determined by either of these methods is usually expressed as so much per cent, 100 being taken as the normal, and it is advisable to add the name Von Fleischl or.jGower after it, in order to show which apparatus was used. Hayem, however, expresses the amount of haemo- globin in terms of normal red corpuscles. For example, in describing the results of an exami- nation he states that the number of corpuscles was three million per cubic millimetre, and the amount of haemoglobin was equal to that of two million normal corpuscles, i.e., 40 %. The amount of haemoglobin that each corpuscle contains — the so-called colour index or corpuscular THE AMOUNT OF HAEMOGLOBIN 17 richness in Hb — Hayem indicates this by the letter G — may be estimated thus : — The number of healthy corpuscles to which the haemoglobin was equivalent divided by the number of corpuscles counted. Supposing the number of red corpuscles = three million, and the Hb = two million of normal corpuscles, I.e. 40 %, then the amount of Hb each corpuscle contains is equal to f or o"6 — the normal being i. Or the percentage of Hb divided by the percentage of cor- puscles present will give the same result. In this case the number of red corpuscles = 60 % of the normal and the Hb = 40%, therefore the colour index per cor- puscle =^§=f or 0.6. The Examination of the Corpuscular Elements of Blood may be made in three ways : — 1. The examination of fresh undiluted blood. 2. The examination by means of diluting or in- different solutions. 3. The examination by means of dry films prepared and stained according to Ehrlich's method. All these can be used, but for clinical purposes the examination of the blood in the fresh and dry films is especially advised. The Examination of Fresh Blood. This is the ordinary procedure adopted by almost all physicians, and if certain precautions are taken is very reliable as far as it goes. The tip of the finger or lobe of the ear, thoroughly cleansed and as thoroughly dried, is pricked with a needle, preferably triangularly pointed. Puncture of the lobe of the ear is less painful, but the manipulation c 1 8 THE BLOOD of the blood is somewhat more difficult in this position than when the finger is pricked. Slight hyperaemia may be produced before puncturing by gentle friction over the part, but the blood must not be squeezed out. The drop thus obtained is allowed at once to fall on the slide and the coverglass put over it, avoiding undue pressure ; or we may touch the exuding drop with the coverglass and then immediately place it upon the slide. The blood should be in as thin and uniform a layer as possible, the elements protected against any moisture or foreign matter in the form of dirt, and the process carried out as quickly as possible. If the specimen be rung round with oil or vaseline it will be protected from evaporation, and therefore remains longer unaltered. When the blood is to be examined for micro-orranisms the place of puncture must be well washed with soap, then with i in looo Hg. perchloride, and, lastly, with alcohol and ether. The needle should be sterilized by heat, and the slides and coverglasses scrupulously clean. Some observers maintain that the pressure of the coverglass is injurious, as it tends to unduly spread out the corpuscles, and adopt means by the intervention of a layer of paper, or drop of wax at the corners of the coverglass, to prevent this. This is, however, seldom necessary if ordinary care be taken. The thinnest films are essential to the study of the nuclear structure of the leucocytes. Hayem uses a very thin ringed cell in order to follow any modifications of coagulation in inflammatory con- ditions. Specimens obtained in this simple manner show the size, shape, and colour of the red blood corpuscles, THE AMOUNT OF HAEMOGLOBIN 19 the blood plates of Hayem, and the approximate number of leucocytes present, with a slight insight into the condition of their nuclei and granules. The parasites of the blood, e.g., those of malaria and recurrent fever, are also well seen in these preparations, in fact many writers prefer fresh unstained blood for the examination of the haematozoa. The measurement of the size of the corpuscular elements is only reliable when made in this fresh condition. The Examination with Preserving Fluids. These neutral or indifferent solutions — stained or unstained — are used more particularly in pure histo- logical work. Amongst them the following may be mentioned : — Hayem's and Pacini's solutions, formulse already given above. Bizzozero's Fluid — physiological salt solution (|- %) — stained with methyl, violet (1:5000) or with gentian violet (1:3000). Arnold uses salt solution o'6 %, stained with methyl, green, whilst Mosso uses a i % methyl, green in physio- logical salt solution. A solution of osmic acid, i % or 2 %, tinted with methyl, green is very suitable for the examination of the blood plates. These solutions may be used in any of the following ways. A drop of the diluting fluid is laid on the cleansed finger and a prick made through it, so that the blood at once mixes with the neutral solution ; or some of the blood may be allowed to flow into the preserving fluid in the proportion of about i to 100, THE BLOOD and then, after letting the preparation stand, the super- natant fluid is poured off and the elements are then mounted. They may be washed after being exposed to the preserving liquid, stained, and then mounted permanently. The Examination of Stained Films. In this method, introduced by Ehrlich, the blood is spread out in a very thin layer on the coverglass or slide, the film is then fixed and subsequently stained, so that permanent preparations are thus obtained. The success of the preparation depends here, as in other histological work, very largely on the quality of the film. Making Films. The usual procedure is to prepare such films on coverglasses. The drop of blood is allowed to fall on a perfectly clean and dry coverglass, and this is covered by another. These are then separated by gently sliding one over the other, avoiding pressure. In order to obviate the presence of any moisture from the fingers, Erhlich uses a pair of forceps to hold the coverglasses whilst spreading. Other writers use a thin glass rod or platinum needle to spread the blood, or with a rapid shaking movement attain similar results. I have always used slides in preference to coverglasses for the purpose of preparing films of sputum, etc., and have found this method particularly adapted to blood films. The slides used should be the best, as the better the surfaces and edges the more uniform the film will be. These should be thoroughly cleaned by THE EXAMINATION OF FILMS means of strong sulphuric acid, then with water, and lastly with ether. The slides should be perfectly dry, but not warmed before using. Immediately the drop of blood appears at the spot punctured, it is allowed to touch one end of the surface of a slide held between the finger and thumb of the left hand. Another slide, held in the right hand, is used to spread this drop over the surface of the glass by passing it along the slide — holding the glass at an angle of about 45° — in a quick Fig. I. Author's Method of Spreading Blood Films on Slides. but steady movement from left to right. (See Fig. i.) If the drop be large enough and the glass slide perfectly true a thin uniform layer of blood is thus spread over the lower slide. The slide used as the spreader should only pass over the surface of the blood once. The following points should be attended to : — 1. The films should be as thin and uniform as possible. Very thin layers of blood dry in one or two seconds, before any alteration in the blood takes place. 2. The time that elapses from the moment that the blood issues from the puncture, to that of a completely spread film, should be as short as possible, otherwise the corpuscles tend to undergo alterations, particularly crenation. If several films are required the drop from the puncture should be wiped away and a fresh drop obtained. THE BLOOD 3. Undue pressure should be avoided, as this tends to alter the shape and size of the elements. 4. One should also very carefully prevent the access of any moisture to the films. The method of spreading films on slides meets these requirements much better than the use of coverglasses. The film so obtained is about i^ inches long and generally of the breadth of the slide, and thus presents a larger area from which a workable field can be selected. One can generally say that a film that does not dry at the ordinary temperature of the room in two or three seconds after it is spread, will be rather too thick. In cases in which the corpuscles are very considerably reduced in number, e.g., pernicious anaemia, it is some- times advisable to spread films somewhat thicker than those ordinarily required, and these can be quickly dried by swaying them in the air. If the blood films be examined without a coverglass under the microscope, the red corpuscles should appear uniformly separated one from another, otherwise if in contact, no true idea of their form is obtained. The leucocytes also suffer by thickness of the films, as they seem reduced in size, and when stained have a shrunken appearance. In manipulating the slide with its unfixed film on the stage of the microscope, it is important that the fingers should not approach the film part, or other- wise the heat and moisture from the hand or even the breath, tend to disintegrate it. When the film is successfully made, the red corpuscles are fixed in their normal shape, but the white corpuscles are flattened, lamelliform, and are generally larger than in the moist condition. (Hayem.) THE EXAMINATION OF FILMS 23 The slide method commends itself particularly on account of its convenience for clinical purposes, the only requisites being a box containing half a dozen clean slides and a needle. A number of such films can be prepared and will keep indefinitely — provided that moisture and dust are excluded — without any further fixing. I have stained such films three years after they were made, and have found that their reaction to stains, and their appearance under the microscope, were all that could be desired. Films prepared on slides, as opposed to coverglass preparations, have a further advantage in the fact that they are easily trans- mitted by post. Method of Fixing Blood Films. In order to study the effects of various stains on the elements of the blood, these air-dried films must be fixed. If any aqueous or glycerine solution of a stain be applied before they are fixed, the whole film either washes off or is converted into a granular debris. On the other hand, air-dried preparations unfixed can be stained at once, provided that the stain used be in alcoholic solution. Solution of iodine with potassium iodide does not dissolve out the haemoglobin, and is, according to Hayem, one of the most useful methods of examining for nucleated red corpuscles in blood. If films be exposed to the vapour of a i % solution of osmic acid the haemoglobin is rendered insoluble ; but this somewhat impairs their reaction to certain of the aniline stains. 24 THE BLOOD According to Hayem, when films have been prepared for a month or more the haemoglobin adheres more firmly to the corpuscles than at the time of preparation. For practical purposes it is necessary to use one of the following methods in order to fix the haemoglobin of the red cells of the film. By means of Heat. — Ehrlich, to whom we are in- debted for the film method of examination of blood, subjected the air- dried preparation, placed on a copper plate, to a temperature of iio°-i20° C. for two hours or more. Lowit placed the films — which had first been allowed to dry very thoroughly in the air — in an oven at a temperature of iio°-ii5° C. for one to two hours, allowing a gradual increase and decrease in the heat. He pointed out that too great heat, or heat extending over too long a time, destroys the structure of the nuclei. This method of treatment takes too much time for clinical purposes, and many of the recent observers agree that much of the finer detail of structure is altered by it. No doubt passing the coverglass film two or three times through the flame of a spirit lamp or bunsen burner, as commonly advised, answers the purpose fairly well. After trying the latter method of heating for some years I found that for certain stains, particularly Ehrlich's acid haematoxylin solution, very thorough fixation was necessary ; and moderate application of heat was usually followed by disintegration or total disappear- ance of the film when treated with such stains as the above, whilst too great heat spoils some of the nuclear structure. THE EXAMINATION OF FILMS 25 Muirs Method of Fixing is probably the best for all fine histological preparations, but is somewhat tedious for clinical work. " Films are placed at once, before any drying can occur, in a saturated solution of corrosive sublimate with f % of sodium chloride added, preferably heated to a temperature of 50° C, though this is not essential, and are allowed to remain for about half an hour. They are then thoroughly washed in f % common salt solution, taken through successive strengths of alcohol, and then stained in the same way as sections. I also add salt in the same proportion to the weaker strengths of alcohol." {Journal of Anat. and Phys., vol. xxvi,, 1892, P- 393-) GuUand has recently suggested a modification of this process, which I shall describe later. Fixing by Alcohol and Ether. — Nikiforoff's Method gives the same results as the application of heat, and for clinical use is much more convenient. Films dried iri the air are placed for a short time in a mixture of equal parts of absolute alcohol and ether. Von Limbeck and others advise an immersion of fifteen to thirty minutes, but I find three to five minutes quite sufficient for the staining with eosin and haema- toxylin. If preparations are slightly heated after the film has dried in air, and when cold placed in the above solution, a few minutes' immersion seems sufficient, but this is not usually necessary. It is convenient for clinical work and gives sufficiently accurate results, but for detailed structure it is not as good as that recommended by Muir or Gulland. Absolute alcohol alone may be used instead. 26 THE BLOOD Formal promises to be very useful, and as its action is rapid, most convenient for clinical examinations. [Formol is a 40 % solution of formic aldehyde in water.] Benario, for fixing blood preparations, uses a 10% watery solution of formol, and of this stock solution he dilutes one, part with ten of alcohol when it is required for use. The preparations remain in it for one minute, and can then be stained without drying. The mixture — ^formol one part; water nine, alcohol ninety parts — could be used if well stoppered to prevent evaporation. For detailed study of nuclear structure, and for the demonstration of karyokinesis, fixation by Flemming's solution or by Muir's method may be used. I find the following very satisfactory : — Mullers Method. — The films are heated, and when cold placed in a saturated aqueous solution of picric acid for twenty -four hours. They are then washed for several (twelve to twenty -four) hours, to remove as much as possible of the acid, and subsequently stained for several hours in very diluted Bohmer's haematoxylin solution. They are finally washed, and any overstaining with haematoxylin removed by weak hydrochloric acid alcohol, under frequent examination with the microscope. They should be cleared with xylol or cedar oil, and mounted in Canada balsam. I have obtained very fine specimens by this means. They may, however, be stained for a short time in undiluted borax carmine, instead of haematoxylin. Method of Staining Films. Ehrlich has divided aniline stains into three classes, viz., acid, basic, and neutral dyes. THE EXAMINATION OF FIIMS 27 The acid stains are, as Kanthack has pointed out, "chemically neutral salts, yet tinctorially they react like free acids." They include eosine, aurantia, indulin, picrate of ammonium, orange G, etc. Among the basic stains are found fuchsin and its derivatives, saffranin, methyl-green, methylene-blue, etc. Neutral stains are formed by the junction of an acid with a basic stain, e.g., a mixture of acid fuchsin with methylene-blue, or methylene-green, etc. The same observer has classified leucocytes, according to the manner in which the granules in their protoplasm react to aniline dyes, into the following : — i. Eosinophile — leucocytes containing a granules, which stain deeply with eosine or other acid aniline stains. ii. Mastzellen — cells which contain large coarse granules basophile in nature = 7 granules. iii. Basophile — cells with fine ^ granules, which stain with basic aniline dyes, e.g., methylene-blue. iv. Neutrophile, or e granules, those which stain with a mixture of a basic and acid stain. The Eosinophile leucocytes may be easily seen by staining fixed films in a concentrated solution of eosine in glycerine, or better in 5 % carbolic-glycerine. The preparation is exposed to the stain for several hours, washed, dried, and mounted in Canada balsam. Glycerine solutions of eosine have a selective, and carbolic acid increases this eosinophilous action. The eosinophile granules are stained a deep red colour. Glycerine solutions of eosine give beautiful results, but they are not as generally useful or as easily manipu- lated as the aqueous or alcoholic solutions. Aqueous or weak alcoholic solutions of eosine, or aqueous solutions of orange G may also be used. 28 THE BLOOD The Mastzellen, or leucocytes containing y granules are stained by Ehrlich's acid dahlia solution, which consists of: — Absolute Alcohol . . . . 50 cc. Distilled water . . . . 100 „ Glacial Acetic Acid . . . . 1 2 -5 „ Dahlia is added to saturation. Miiller recommends that preparations before staining should be heated for several hours, and then further fixed in absolute alcohol and ether ; but this is not essential. Stain films for several hours, wash in water, remove the excess of stain by acid alcohol, and finally wash, dry, and mount in balsam. The 7 granules appear stained of a reddish violet. They may be much more conveniently demonstrated by staining for a minute or so in an aqueous solution of methylene-blue, when they have a purplish tint, whilst all the other elements are stained blue. The Basophile cells, or leucocytes with 5 granules, are best stained by saturated aqueous solutions of methylene - blue. Loffler's methylene - blue solution, consisting of concentrated alcoholic solution of methylene - blue 30 cc, distilled water 30 cc. and 100 cc. of i(,\q(, caustic potash solution, answers very well. Preparations are stained for about ohe minute. The so-called Neutropkile, or e granules, may be stained by Aronson and Phillips' mixture, Ehrlich's triple stain (formula given below), or Ehrlich's neutral mixture. The last is made by adding one volume of strong watery solution of methylene-blue gradually with constant stirring, to five volumes of a saturated watery solution of acid fuchsin, and then five volumes of dis- THE EXAMINATION OF FIIMS 29 tilled water are added. After allowing the mixture to stand for a few days it is filtered. Preparations are stained for two to five minutes, rapidly rinsed with water, and quickly dried and mounted. The red corpuscles are stained red, the € granules violet, whilst the a granules appear of a purple colour. For clinical purposes it is desirable to use two or more stains, either separately or combined, which will show all these elements on one and the same film. Kanthack advises the following procedure : — " Films allowed to dry in the air are passed three times through the flame, and stained in a solution of eosine (o"5 gram of eosine, alcohol 70 cc, water 30 cc.) for half a minute or less. They are then thoroughly washed in distilled water, dried between blotting paper, and then passed again 3-5 times through the flame, and then counter- stained in Lofifler's methylene-blue solution for a few seconds, again washed, dried, and mounted in Canada balsam." {Medical Chronicle^ Such films should show the eosinophile granules stained deep red, the basophile granules blue, the e, or so-called neutrophile granules^which Kanthack and Hardy have shown are really fine eosinophile or oxyphile granules — light red, the nuclei of the leucocytes a brilliant blue colour, and the red corpuscles a pale red colour. Notwithstanding many very kind and suggestive hints from Dr. Kanthack, I have rarely succeeded in obtain- ing, or rather constantly obtaining, what I should consider satisfactory results. I prefer to use Kuhne's carbolic methylene-blue in preference to Lofifler's solution. 30 THE BLOOD After trying all the usually-described methods of double staining with eosine and methylene-blue, either separately or combined in mixture, e.g., Plehn's* or Canon's, t I have never succeeded sufficiently to re- commend these stains for clinical use. No doubt the results depend very largely on the time the film is subjected to the action of the methylene-blue solution, and particularly on the rapidity with which the films are dried after being stained. EhrlicKs new Triple Stain seems to be very generally used. It consists of the following formula. Saturated watery solutions of orange G, acid fuchsin, and methyl- green are first prepared. Saturated watery solution of Orange G Acid fuchsin . Methyl-green . Then add — Water Absolute alcohol Glycerine 24-27 cc. 16-33 ,. 25 » 60 „ 40 ,, 20 ,, The mixture should stand for one or two weeks before being used. Films fixed by heating or prolonged (twelve hours) action of the alcohol and ether mixture are said to give the best results. Stengel found that as a rule a rather larger quantity of methyl-green was required. * Plehn's, or Czenzinsky's Mixture, has the following formula : — Methylene-blue concentrated aqueous solution . 40 parts Eosine, \ % solution in 70 % alcohol . . . 20 „ Distilled water . . . . . . 40 t Canon's solution, very closely resembling this, is composed of : — Methylene-blue concentrated aqueous solution . 40 parts Eosine, J % solution in 70 % alcohol . . . 20 „ Distilled water . . . . . . 40 Film preparations fixed by alcohol are stained in either of these mixtures for several hours (3-6 according to MuUer) rinsed in water, dried, and mounted in Canada balsam. THE EXAMINATION OF FILMS 31 After staining for from two to eight minutes the preparation is rapidly rinsed in water, dried, and mounted. The nuclei of the leucocytes are stained a greenish blue, the eosinophile granules red or brownish red, and the neutrophile granules violet or lilac colour. The red blood corpuscles are coloured yellow, and their nucleus when present green. Gulland speaks very highly of the Ehrlich Biondi mixture, and prefers it to other triple stains. It is best to obtain Grubler's solution ready-made, or the powder for making it from one of the London agents (e.g., Baker or Kanthack). Preparations are stained in a few minutes (from ten to thirty) in the concentrated solution. Cabot recommends the following modification of the triple stain : — * Ehrlich Biondi (or Grubler's Biondi- Heidenhain) Powder . . ... grs. xv. Absolute alcohol . . . . i cc. Distilled water . . 6 cc. Preparations fixed by heat are stained for about one to five minutes. He points out that the time required for staining depends on the length of time which the specimens have been heated. If the preparations be over-heated the red blood corpuscles have a faint colour, if under-heated they are of a brownish grey tint, instead of a brilliant yellow. Personally I cannot recommend the use of any of the triple stains for clinical use. The colours are more or less blurred, and as the green colour of the nuclei is * This formula is taken from Cabot's book, but I doubt whether gr. xv. of Grubler's powder, which contains little or no fuchsin, is soluble in 7 cc. of dilute alcohol. 32 THE BLOOD very indistinct, there is considerable difficulty in obtain- ing a good insight into the nuclear structure.* Dr. Gulland, in a private communication on the subject, advised staining preparations for thirty minutes in the undiluted stain, washing with distilled water very rapidly, and dehydrating with absolute alcohol as quickly as possible. The preparations are then cleared with xylol and mounted in balsam. To overcome the faint colouring of the nucleus, he suggests staining very lightly with haematoxylin first, then with the Ehrlich Biondi solution. Dr. Gulland, in British Medical Journal, March 13th, 1897, has suggested a method of fixing, and, if neces- sary, partially staining, blood films at the same time. Fresh films before becoming dry in the air are placed in the following solution : — Absolute alcohol, saturated with eosine 25 ccm. Pure ether . . . . 25 ccm. Sublimate, in absolute alcohol (2 grms. to 10 ccm.) . . • • 5 drops (more or less). Films are fixed in three or four minutes. They are then thoroughly washed and stained for one minute in a saturated watery solution of methylene-blue, washed in water, quickly dehydrated in absolute alcohol, cleared in xylol, and mounted in balsam. * Even such an authority as BoUes Lee makes the following statement in regard to the Ehrlich Biondi mixture as a stain for general work : — " After a very careful study of this admired stain, I find I cannot commend it for any but the most special objects. It must in the first place, I think, be acknowledged that a reagent that can neither be prepared nor preserved without the minute precautions detailed above, is something much too great and good for human nature's daily food. The stain is a very fine one when successful, but the most minute precautions will not ensure its being successful. It is very capricious, it seldom gives the same result twice running." {Microtomisf s Vade-mecum, 4th edition, 1896.) THE EXAMINATION OF FILMS 33 He suggests that any acid stain which is soluble in alcohol, and not precipitated by sublimate, may be used instead of eosine, or the stain may be omitted altogether from the fixing mixture, and films subse- quently stained in any of the aniline dyes. I have not generally used this method, chiefly because the absolute alcohol and ether mixture answers very well for practical purposes. To my mind this method has one disadvantage if haematoxylin is to be the subsequent stain, that is, all the sublimate must be very thoroughly washed out from the preparation, otherwise a precipitate will be formed when brought into contact with haema- toxylin. It fixes the elements much better than the alcohol and ether solution, and for detailed study of blood and film preparations generally, it answers ex- cellently. Muller, in a very extensive paper (in Centralblatt fur Allgemeine Pathol, u. Pathol. Anatomie, 1892) discussing methods of blood examination, repeatedly draws attention to haematoxylin as being undoubtedly the best nuclear stain, and particularly recommends eosin-haematoxylin. This can be used either as Ehrlich's eosin-haematoxylin solution, or by first staining in eosine and then in haematoxylin. [As haematoxylin solutions require to be kept some time to mature, it is better to obtain them ready prepared from Grubler's agents.] Preparations require to remain in the solution some hours. Formerly I stained fixed films for several hours in Ehrlich's acid haematoxylin solution, then washed, and, if necessary, decolourised in weak acid alcohol, and then stained in glycerine or aqueous solution of eosine. The great disadvantage of this method, independently of the time required for the stains to act, was that the D 34 THE BLOOD eosinophile granules never would stain as well after immersion in the acid haematoxylin as before, and hence it is advisable to stain first with eosine and then with haematoxylin. I have modified this process, and now alwdys use the following method. Eosine and Haematoxylin. Films of blood on slides dried in the air are fixed in the alcohol and ether mixture for a minute or so. They are then stained in a dilute alcoholic solution of eosine, viz., eosine one part, water one hundred, alcohol one hundred [the eosine soluble in water is used in preference to that soluble in alcohol], for about half a minute or longer. They are then washed in water and stained with Bohmer's or Delafield's haematoxylin solution (which should always be filtered before use), for about ten to fifteen seconds, washed in water, and examined whilst wet to see if the blue of the' haema- toxylin in the nuclei of the leucocytes is sufficiently deep ; if not, they are again exposed to haematoxylin. The films are then washed — tap water being sufficiently alkaline to develop the blue colour of the haematoxylin — dried, cleared with xylol, or cedar oil, and mounted in Canada balsam. The staining with eosine requires no comment. The exact time matters little, the longer films are stained the deeper red will the corpuscles appear. As to the haematoxylin solution, the old formula of Bohmer, or Mayer's Haemalum, answers perfectly well. It is advisable to procure it ready-made. The time it requires to stain depends very largely on its maturation. THE EXAMINATION OF FILMS 35 Until one knows the staining power of the haematoxylin it is better to be on the safe side, and under rather than over - stain. We aim at having the nuclei of the leucocytes stained clearly and distinctly without dis- placing the eosine in the protoplasm of the multi- nucleated leucocytes. The time required for staining with haematoxylin may be anything from a few seconds to ten minutes. I find a half- minute's immersion gives perhaps the best results. By this means films can be made, fixed, stained, and mounted in about ten to fifteen minutes. Of all methods it is, as far as my experience goes, the most constant and reliable. The red blood corpuscles appear of a yellow-pink, or red colour, according to the time that the film is exposed to the eosine. The nuclei of the multi-nucleated leucocytes stain distinctly and sharply of a dark blue colour, the threads of chromatin connecting the nuclei being clearly seen. The nuclei of the small uninucleated leucocytes are of a somewhat deeper blue. The proto- plasm of the leucocytes is of a pink colour, whilst the granules of the eosinophile cells are stained deep red. The eosin-haematoxylin method of staining is par- ticularly adapted for the examination of the nucleated red blood corpuscles. The nuclei and, when present, the mitotic figures are very conspicuous, as they are stained of a dark blue, almost black colour. Degener- ative changes in the red blood corpuscles, " polychroma- tophilia," are well seen. This stain is also particularly suited for the demon- stration of the malaria organism, as I have pointed out when dealing with this subject. 36 THE BLOOD Coverglass films are usually stained by placing them film downwards in a watch glass containing the stain, or, in cases in which the stain acts very quickly, by simply pouring on the solution of the dye for a few minutes and then washing. In order to fix and stain slide films I use wide-mouthed, glass- stoppered bottles, about i-^ inches in diameter, 2)\ inches in height, holding two ounces. (See Fig. 2. These have been specially made for me by the York Glass Co.) They are large enough to hold two slides (or even four) back to back. This is a very convenient, economical, and clean method of using fixing and staining so- lutions. Such bottles contain alcohol and ether solutions, eosine, triple stain, etc. They are not particularly adapted for haema- toxylin solutions, as these always need filtering before using. Fig. 2. Bottle in which Blood Films made on Slides can be con- veniently fixed and stained. All the aniline stains used should be Grubler's, and these, as well as the instruments for counting the cor- puscles and estimating the haemoglobin, can be obtained of Baker, 244, High Holborn, W.C. With regard to the microscopic magnification necessary for clinical work, practically everything can be seen with a -^ or -|- inch objective ; but a ^ inch oil immersion lens is almost essential for the study of the malarial organism, ' and is in all cases of the greatest value. General Morphology of the Blood. Blood consists of a fluid, the plasma, or liquor san- guinis, containing cellular bodies, the corpuscles. Three forms of elements may be recognized — the red blood corpuscles, by far the most numerous, giving to blood its characteristic colour ; the white blood corpuscles, or leucocytes ; and the blood plaques, or blood plates. THE RED BLOOD CORPUSCLES, OR ERYTHROCYTES. In man these are biconcave, non-nucleated, flattened discs, having a circular outline. (See Plate I., Fig. i.) Their surface is smooth, permitting them to glide easily past each other, and although very extensible they are equally elastic. When seen in a thin layer they are of a yellow rather than red colour, and in consequence of their shape appear darker in the centre than at the margins when seen at a distant focus, but lighter in the middle and dark at the periphery when viewed with a near focus. The colour of the corpuscle is due to the presence of haemoglobin, and in health practically all the red cells contain exactly the same amount of colouring matter. As regards their size it is possible, according to Hayem, to recognize, firstly, large elements measuring 8 to 9 Mj constituting about 12^%; secondly, medium- sized corpuscles, having an average diameter of 7*5 Mj constituting about 75 % ; and thirdly, small corpuscles measuring 6 to 6 '6 ix, which constitute about i2|-% of 38 THE BLOOD all the erythrocytes. It is thus seen that by far the greater number of red blood corpuscles have a mean diameter of 7*5 m- (The Greek letter m is equal to xtnrtr part of a millimetre, and is usually known as a micro- millimetre.) In addition to these, the same observer speaks of dwarf corpuscles, "globules nains," having a diameter between 3 "5 and 6 M) and giant corpuscles, "globules grants," above 9*5 m- These are both occasionally found in health, but the latter only in the newly-born. Alter- ations in size are, however, very conspicuous ip several pathological conditions, which will be noted later. The erythrocytes are of an extremely delicate struc- ture, and undergo considerable alterations when exppsed to moisture, foreign material, or injury. Alterations in their form, due to artificial causes, must not be confounded with changes resulting from pathological causes. One of the earliest and most marked artificial deform- ation is that in the presence of moisture the disc-like corpuscle assumes a spherical form, or under similar conditions loses part of its colouring matter, becoming much paler, constituting the chlorocytes of Hayem ; whilst others, losing all their haemoglobin, become com- pletely decolourised, and are termed achromacytes by the same author; and it is probable that the so-called invisible corpuscles, which N orris considered as the early form of the red corpuscles, are none other than these altered by external agencies. Further, the corpuscles may become spinous, undergo fragmentation, or become crenated or vacuolated, and hence the desirability of paying considerable attention to the method of examination before regarding these changes as of pathological importance. THE RED BLOOD CORPUSCLES 39 Hayem considers that the coloured elements consist of a part insoluble in water, constituting the stroma of Rollett, having no apparent structure, the surface of which becoming somewhat hardened constitutes a sort of membranous envelope; and a part soluble in water, an albuminoid material containing haemoglobin. The corpuscles have no cell- membrane, no contractile protoplasm, and do not, therefore, undergo amoeboid movement, such as is seen in some of the leucocytes. They are by far the most numerous of the elements found in the blood, and the general consensus of opinion is, that there are five million red corpuscles per cubic millimetre in the blood of a healthy man, and 4|- million in that of a woman. Physiological Alterations of the Red Blood Corpuscles. The Effects of Age. — The number of red corpuscles shortly after the birth of a child is relatively high, being greater than that of the mother. Hayem found a maximum of 6,262,000 and a minimum number of 4,340,000. In a child 10 hours old Otto found 6,912,000, and in another 25 hours old 6,496,000. After the first day there is a gradual decrease in their number, whilst the weight of the body under- goes an increase. It appears very doubtful whether the time at which the umbilical cord is tied, influences the number of corpuscles. Hayem, however, found in six . children in whom the cord was tied imme- diately, an average of 5,087,000, whilst in eight children in whom the cord was not tied until the umbilical artery had ceased, an average of 5,576,000 per cmm., and this difference persisted for forty-eight hours. 40 THE BLOOD The colour of the capillary blood for the first few- days after birth is darker than that of the adult con- dition, and more closely resembles the colour of venous blood. The size of the corpuscles appears much more un- equal in infancy, the maximum being 9-10/*, the smaller measuring only 5 '5 ii-, and there is a marked alteration in their diameter from day to day. They more readily undergo changes in form under the influence of mois- ture, the small corpuscles readily becoming spherical. The amount of haemoglobin contained in the indi- vidual corpuscles at this early age is greater than in the adult — the colour index on an average being I'l. These alterations are probably due to developmental conditions. "In spite of the presence of large corpuscular ele- ments in the blood, nucleated red blood corpuscles are never found in the newly-born." (Hayem.) They disappear at about the 6th or 6^ month of intra-uterine life. Influence of Sex. — There are fewer coloured cor- puscles in the adult female than in the male. This may possibly be due to menstruation, as Sterlin found that in children from 9 months to 15 years the red corpuscles were slightly more numerous in the female. Constitution. — The robust have, as would be ex- pected, a larger number of coloured elements than the feeble. Effects of Food. — The taking of food causes a slight decrease in the number of red corpuscles, as opposed to an increase in the number of leucocytes — probably THE RED BLOOD CORPUSCLES 41 due to the dilution of the blood during digestion. A mixed diet would seem to be the most suited for the renovation of the blood. A fast of 24 hours' duration produces a very distinct increase in the number of the corpuscles, and a prolonged fast has much the same effect. Hayem quotes a case in which a dog was totally deprived of food until its death, which took place after 25 days, the red corpuscles steadily increas- ing till the end. This apparent increase is evidently the result of thickening of the blood when the cor- puscles become concentrated. . Exactly the same thing occurs in diarrhoea. Prolonged fatigue causes a diminution in their num- ber. Menstruation produces a slight anaemic condition, which quickly returns to the normal. Pregnancy is followed by some degree of diminution of the red cor- puscles and haemoglobin, but the alteration varies very much in different individuals. Young women and primipara show the greatest diminution in the cor- puscular elements, especially in the latter two months. Lactation does not cause any appreciable anaemia. The effects of parturition depend very largely on the quantity of blood lost, and on the resistance and vigour of the individual. Generally there is some diminution in the red cells, but reparative process slowly sets in. Hayem states that the number of red corpuscles and the quantity of haemoglobin are the same in all parts of the vascular system. Pathological Alterations in the Red Blood Corpuscles. Alterations in Number. — Polycythemia is relatively infrequent, and practically only occurs — in addition to fasting and want of nourishment — in conditions in which 42 THE BLOOD concentration of blood occurs as the result of consider- able loss of liquids, particularly from the intestinal tract, e.g., cholera and diarrhoea. Hayem points out that 6*2 to 6'5 millions of red corpuscles may be counted during the algide stage of cholera. He does not believe that the plethoric condition could be attributed to an actual increase in the number of red corpuscles in the blood. Diminution in the number of the erythrocytes, oligocythemia, is of very frequent occurrence. When produced rapidly, as by repeated haemorrhages, anaemia cannot pass certain limits without proving fatal, and patients are in a very serious condition when the number of red cells approaches one million. (Hayem.) " When the anaemia is progressive the body seems to adapt itself to the impoverished condition of the blood, and not infre- quently patients are met with, active and working for their living, in whom the blood may only show two million corpuscles per cmm., or even less. This fact is still inore remarkable when one realizes that often the haemoglobin in these corpuscles is at the same time much less than in normal elements." (Hayem.) Hayem, as a result of his extended experience, says that an anaemia may be curable when the number of erythrocytes is not lower than half a million, but does not think that life could continue when they fall lower than 3 to 400,000. In fatal cases of anaemia some authors have found less than 300,000 corpuscles per cmm. Alterations in Diameter. — Not only may the red cells undergo alterations in number, but alterations in size are often seen in pathological conditions. There may be Plate I. Red Blood Corpuscles and their Alterations. hFilm preparations stained with eosine and haematoxylin. Examined with eyepiece 2. Objective -h" oil immersion and tube length of i6omm. Fig. 1. — Normal red blood corpuscles showing ;the slight variation in size found in health. Fig. 2. — Blood plates, some isolated, some in clusters. They £^re usually coloured with a mixture of eosine and haematoxylin. Fig. 3. — Small red corpuscles or Microcytes, of, which two are' deeply stained — the so-called Eichhorst's corpuscles. Fig. 4. — Large red corpuscles or Megalocytesj two of which show some degree of vacuolation. (a) Normal corpuscles. Fig. 5. — Poikilocytes of varying size, shape, and colour. Fig. 6.-^Other degenerative changes in the corpuscles, (a) Cracked or vacuolated elements.' (b) Pale or shadow corpuscles, [c) Polychromatophile corpuscles. Fig. 7. — Nucleated red corpuscles of about the size of the normal elements — Normoblasts — from blood. In some the nucleus is single, in others double, and in a few treble. In some the nucleus is partially extruded, (a) Normoblast dividing. (b) Nucleus partially free, with only a slight amount of protoplasm surrounding it. {c) Nucleus of normoblast free, (rf) Microblast. Fig. 8. — Large nucleated red corpuscles or Megaloblasts, generally showing a somewhat indistinct large nucleus. The central figure also illustrates polychromato- phile degeneration. ■iFig. 9. — Nucleated red corpuscles from the marrow of a kitten two days old. Two karyokinetic figures [a), are seen. P L AT E ! X,.. Fi g 4. F-ii 2 Fig,, ,3 & # f» 1 'C> C-^ Exg,5. v>.lf' ' 1 e- e , Fig 7, n-' h^- ' --^'■:i>--'--'^! <- % Fig 8. Fi g, 9 # # Wes-t.lS'^jrQar. Clir THE RED BLOOD CORPUSCLES 43 a diminution in the diameter, e.g., the dwarf corpuscles or microcytes (see Plate I., Fig. 3), measuring 3-5 to 6 M, or an increase in size when numerous giant cells or megalocytes, generally 9-5 to 12 m, or even 14 m in diameter, maybe found. (See Plate' I., Fig. 4.) Eichhorsi' s corpuscles are small deeply-stained bodies, about 3 Ai in diameter, generally spherical in form. They were considered by their discoverer to be characteristic of pernicious anaemia ; but this is generally regarded as incorrect, and the opposite, viz., the occurrence of megalocytes in large numbers, is more important, though not characteristic of pernicious anaemia. The term macrocythemia has been applied to the condition in which the large elements prevail, microcy- themia when the small corpuscles are in excess. Hayem states that megalocytes rather than microcytes point to a severe anaemia. Alteration in size is usually associated with changes in the form of the corpuscles. The elements losing their disc-like form may show irregular thickenings or pro- jections from their border, giving rise to oval, pear- shaped or peculiarly elongated bodies, which have been termed poikilocytes by Quincke, or schistocytes by Ehrlich. (See Plate I., Fig. 5.) These are met with in nearly all forms of anaemia, and are by no means characteristic of pernicious anaemia, as Quincke originally supposed. Fragments of corpuscles are certainly seen in some severe conditions, although, as Hayem very strongly points out, these may occur in normal blood if due care has not been taken in the avoidance of moisture, pressure, etc. The same observer has noticed the occurrence in 44 THE BLOOD some cases of anaemia of very small rod-shaped, red corpuscles, to which he has given the name pseudo- bacilli. They have been mistaken for bacteria on account of their small size, their shape, and the very active movements which they undergo, probably partly molecular and partly amoeboid in nature. Whether these latter changes are due to artificial conditions, or whether the corpuscular elements in disease are more susceptible to injury, seems a little doubtful. Normally the amount of haemoglobin contained in a corpuscle is in direct proportion to the size of the element ; but when pathological alterations in size and shape occur, there is frequently no relation between these two factors. Diminution in the amount of haemoglobin is usually more pronounced in the small than in the large or giant corpuscles, and not infrequently the amount of colouring matter in the latter is increased, and this accounts for the high colour-index frequently found in pernicious anaemia. Sometimes, in spite of all care used in the method of examination, partially decolourised chlorocytes, or totally colourless corpuscles, achromacytes or " shadow corpuscles," may be seen, showing a diminished resisting power of the coloured elements. These are said to occur particularly in infectious diseases, adynamic typhoid, haemorrhagic small pox, haemoglobin-aneemia, etc. Gabritschewski found that the red blood corpuscles have an elective staining reaction with eosine, rather than with other aniline dyes. In the circulating blood the living red corpuscles do not stain, achromatophile ; but as they die — e.g., when fixed — they stain only with THE RED BLOOD CORPUSCLES 45 one of the dyes — the acid stain — of a mixture of aniline colours — e.g., eosine and haematoxylin ; this is termed monochromatophile. In pathological conditions, however, especially in severe anaemia, measles, scarlet fever, small pox, typhus, and purpura, a few red blood corpuscles lose their elective staining property, and from a mixture they stain with two or three colours, either diffusely or in streaks or spots, the last especially in their central part, polychromatophile. (See Plate I., Fig. 8.) Thus when eosine and haematoxylin are used the diseased erythrocytes appear of a violet or purple, instead of the normal pink or red colour. These alterations Ehrlich considered as signs of the death of the corpuscle. Maragliano and Castellino divide these changes, showing the death of a corpuscle — necrobiotic alter- ations — into endoglobular and total transformations. The endoglobular changes commence by diminution of the haemoglobin in the centre, with an increase in that at the periphery, causing an apparent enlargement ■of the central depression of the corpuscle, so that eventually only the margin of the red corpuscles is coloured. This gives the cell an appearance of a figure of 8 when seen on its side. Sometimes the decolourisation does not proceed so uniformly, and the blood corpuscle may show variously- shaped white patches, with fairly definite margins. This condition corresponds to vacuolation of a corpuscle, as described by Hayem and others. (Plate I., Figs. 4 and 6.) The decolourised areas not infrequently show amoeboid changes, and have been mistaken for un- pigmented malaria parasites. As the process advances, movement of the particles 46 THE BLOOD within the decolourised zones becomes apparent, and increases in rapidity, and finally the whole of the corpuscle degenerates into a granular mass. The changes in the corpuscle as a whole — total necrosis — appear later than the endoglobular alterations. The corpuscles show little projections as points or round knobs, which, increasing in size, constitute the condition described as poikilocytosis. Some of these projections become detached and float in the plasma. At the same time decolourisation occurs, and though at first starting from the centre, ultimately affects the whole corpuscle. Limbeck, in confirming this account, remarks that reagents of various kinds will produce similar results, especially the endoglobular changes, yet they may be seen in the blood drawn freshly under certain patho- logical conditions, proving that these diseases may be associated with necrosis of the red corpuscles. In addition to pathological alterations in the blood plasma — e.g., that resulting from cancer, severe tuber- culosis of the lung, parenchymatous nephritis, etc. — various poisons, chloroform, antifebrin, dilute acids and alkalies, and organic acids may produce similar results. Ehrlich drew attention to the fact that in anaemic conditions, the appearances of degeneration were very closely connected with those of regeneration of the blood corpuscles. He considers poikilocytosis as essen- tially a sign of degeneration in anaemias. Quincke, who first introduced the term, regarded it as characteristic of pernicious anaemia, but as it occurs in the blood in many other conditions much of its significance is lost. Hayem mentions the occurrence of increased viscosity and diminished elasticity of the red corpuscles in hyper- THE RED BLOOD CORPUSCLES 47 trophic cirrhosis of the Hver with jaundice, and in certain cachectic conditions. Under such circumstances the cor- puscles run together, fuse into a mass, arid lose their individual outline. Other important alterations of the corpuscles, which occur in malarial fevers, will be fully described when we speak of this disease. Nucleated Red Blood Corpuscles. The occurrence of nucleated red blood corpuscles in the blood is the most interesting and important of the numerous alterations which the red cells may undergo in disease. Although present during intra-uterine life, it has been generally accepted that they are never seen in the normal blood of man at birth. Associated with signs of degeneration of the blood in certain patho- logical conditions, are the signs of regeneration, and amongst these the appearance of nucleated red cells. These elements cannot be detected in fresh, unstained blood, but are best seen in dry films stained with a solution of methylene-blue, or better, with eosin-haema- toxylin, and even then careful examinations of many films may be necessary before they are met with. They have been termed, according to their size, normoblasts, microblasts, and megaloblasts. The Normoblasts (Plate I., Fig. 7) are nucleated red cells of about the same size as the ordinary red corpuscle, varying from 7-5 to 8 m in diameter. The nucleus is usually single, round or oval, and shows an intra-nuclear network. It stains very deeply with most nuclear stains, so that in films coloured with eosin-haematoxylin, even under a low magnification. 48 THE BLOOD they are seen to be the darkest objects in the prepara- tion. It is smaller than the nucleus of a lymphocyte, which, although it stains deeply, does not stain as intensely as the nucleus of these elements. (Plate III., Fig. I.) Its size bears no constant proportion to that of the whole corpuscle, but it seldom exceeds one-half the diameter of the red cell. The nucleus is usually ec- centrically situated, but is nearly always completely surrounded by protoplasm. Sometimes, however, appearances are met with, which suggest the extrusion of the nucleus. Occasionally, instead of a single nucleus, two or three apparently separate nuclei may be seen lying very near each other, or connected by delicate strands of nuclear structure, so that a figure of a dumb-bell or trefoil results. Mitotic figures are sometimes, though very rarely, seen in the nuclei of normoblasts. The protoplasm surrounding the nucleus is homogeneous, and contains haemoglobin ; it stains uniformly with the acid aniline dyes, but is somewhat paler in colour than the ordinary red corpuscle. Sometimes it shows polychromatophilia, and appears of a purplish tint when stained with eosin- haematoxylin. The normoblasts are not infrequently crenated, and in my experience in most cases of secondary anaemia they seldom show a perfectly regular outline. Microblasts (Plate I., Fig. 7, d) are nucleated red corpuscles, smaller than the ordinary coloured corpuscle. They are less frequently met with than either of the other varieties. The Megaloblasts (Plate I., Fig. 8) are considerably THE RED BLOOD CORPUSCLES 49 larger than the normoblasts, being nearly always over 10 M. not infrequently 14 to 16 /« in diameter. The nucleus is often extremely large, occupying f of the cell. It stains much less deeply than that of the normoblast, presenting at all times a very indistinct appearance. The protoplasm contains more haemo- globin than the small variety and stains deeply with eosine, and the whole cell has a swollen or distended appearance. It sometimes shows signs of degeneration, and poly- chromatophilia, vacuolation, etc., may occasionally be seen. Megaloblasts and normoblasts may undergo poikilocy- tosis, constituting poikiloblasts. I can confirm the statement that the normoblasts are less resistant to, or more easily deformed by, pressure, and have noticed that in some films three or four small free nuclei, with litrie or no surrounding protoplasm, may be met with (Plate I., Fig. 7, b and c); but whether they existed as such in the blood, or whether this was the result of the slight pressure they were subjected to in spreading the film, remains uncertain. Limbeck and Ehrlich state that the normoblasts differ from the megaloblasts in that the nucleus of the latter is not extruded, but undergoes degeneration and disinte- grates by degrees in the cell. Normoblasts are found in the bone marrow of a healthy adult (Plate I., Fig 9), whilst megaloblasts never occur in the adult, only in the marrow and blood-forming organs of the embryo. Ehrlich, therefore, regards the appearance of the latter in pathological conditions in an adult as a return to the embryonic type. The conditions under which nucleated red cells may 50 THE BLOOD be met with, and still more the importance from a clinical aspect of their occurrence, still seem somewhat uncertain. Hayem {du Sang, 1889) expresses the following opinions regarding them : — " This interesting lesion appears to me rare, notwith- standing the assertion of certain authors. I have only met with it in extreme anaemias and in leucocythemia." (p. 386.) " Nucleated red cells are constantly found in leucocythemia at some time in its course" (p. 857), "when the haemopoietic organs, especially the spleen and marrow, are full of embryonic elements, proving that in those cases in which numerous nucleated red corpuscles are formed in the haemopoietic organs, these elements can enter into the blood without losing their nuclei, and that they persist in the general circulation with their own characteristics, just as in the embryo." (p. 609.) " Excluding leucocythemia, the appearance of nucleated red corpuscles is a rare condition in human blood. I have only seen it in patients suffering from extreme anaemia, almost at the point of death, and their number has never beeh conspicuous. These elements have always been infrequent, and their appearance has coincided with an aggravation in the condition of the patient ; they have never acquired the significance of a reparative process on the part of the blood. When nucleated red corpuscles, however scanty, are found in a patient suffering from spontaneous or symptomatic anaemia the most serious prognosis ought to be given, and a near fatal termination expected. Would this be so if these elements were destined to regenerate the blood ? " (p. 609.) "They only appear in the blood of intense and THE RED BLOOD CORPUSCLES 51 extreme anaemia, and seem to take only a very limited part in its regeneration. It is a final and insufficient attempt at the restoration of the blood." (p. 610.) Speaking of chlorosis Hayem says, " Whatever be the intensity of the anaemia in this disease, I have never been able to find a single nucleated red corpuscle. For some years past a certain number of foreign pathologists have asserted that nucleated red cells are met with frequently enough in chlorosis. I have re-examined with reference to this statement my collection of pre- parations, taking care to stain the elements, and the result of this inquiry was absolutely negative." (p. 623.) "Towards the end of pernicious anaemia nucleated red corpuscles may be seen, but they are not found in all cases." (p. 794.) As to their occurrence in post-haemorrhagic anaemia, he states " that exceptionally nucleated corpuscles may be seen in anaemia, the result of haemorrhage ; firstly, in cases in which repeated haemorrhages have reduced the patient to a condition threatening existence, but they are extremely scanty and require very careful examination before they are found ; secondly, when repeated haemorrhages, or even a single large loss of blood, occur in a patient already suffering from an organic disease, and their presence under these con- ditions is of very grave import." (p. 826.) Muir, in xh^ Journal of Anat. and Physiology, 1891- 92, states that up to that time he had found these elements in twelve cases, viz., four cases of pernicious anaemia, six of leucocythemia, one of traumatic anaemia, and in one case of secondary anaemia complicated with haemorrhages. In half of these they were numerous, whilst in the 52 THE BLOOD remainder they were scanty, and required careful search. In traumatic anaemia they were generally normoblasts ; in pernicious anaemia, megaloblasts or poikiloblasts. As to their importance he mentions that in one case of post-haemorrhagic anaemia, and two of pernicious anaemia in which they were most numerous, the patients ultimately recovered, whilst in cases of most extreme anaemia they were absent immediately before death. He states that Ehrlich records their absence before death in a case in which the red blood corpuscles fell to 213,360 per cmm., and "there is therefore reason in Ehrlich's supposition that their presence indicates that the bone marrow is still producing red blood cor- puscles." Von Limbeck considers the occurrence of normoblasts as a sign of the existence of new formation of the blood. They are usually only found for any length of time in variable numbers in severe anaemias, and decrease in number with continued improvement in the blood. Very rarely they occur in groups in the blood, a condition Von Norden has termed "blood crisis." The megaloblasts are, according to Ehrlich, a sign that the formation of blood is not following a normal manner. Megaloblasts and megalocytes are, according to this observer, the criteria of genuine pernicious anaemia, whilst he regards poikiloblasts as decided signs of severe alterations in the blood. Some recent writers, contrary to Hayem, state that nucleated red corpuscles, especially and probably only normoblasts, may occur in severe chlorosis, and many record their presence in severe anaemias. Stengel expresses the opinion that " the smaller forms usually present themselves first, and in the more THE RED BLOOD CORPUSCLES 53 moderate grades of aneemia," whilst megaloblasts may be conspicuous in the severe cases. " Nucleated corpuscles are undoubtedly the expres- sion of active haemogenesis, the smaller forms being particularly significant. Occasionally they appear in separate crops (blood crisis, Von Norden), followed by increase in the number of red corpuscles in the blood. The larger forms, according to Ehrlich, are expressions of imperfectly conducted haemogenesis ; but this view, it seems to me, cannot be well maintained. On the contrary, they would appear to be the expression of unusual demands upon the haemogenetic function, in which the large as well as the small forms escape from the bone marrow before their development has been completed." I have quoted the opinions of Hayem, Muir, Von Limbeck, and Stengel as to the importance and fre- quency of nucleated red corpuscles, in order to emphasize the importance of these elements in the blood. My experience is, however, quite at variance with Hayem's as regards these two points, and I consider that the two forms — the normoblasts and megaloblasts — differ entirely in their importance. I have frequently found the former in the most varied forms of primary and secondary aneemias when the oligocythemia is pronounced, and should expect to find them, or rather should make prolonged search for them, in cases in which the red corpuscles were less than two and a half million per cmm. I quite agree with Hayem that they are constantly found, usually in large numbers, in spleno-medullary leukaemia, but are frequently absent from the lymphatic form of this disease. 54 THE BLOOD With regard to their importance I am of the same opinion as many other observers, that normoblasts originate from the bone marrow, and their presence in the blood indicates a severe anaemia, but one in which an effort is being made by the marrow to improve the blood. Megaloblasts are entirely absent from healthy adult marrow, although found in foetal red medulla, and their presence in the blood must point to a profound alteration, possibly of a degenerative nature, in the bone marrow. It would seem that normoblasts either represent a call on the blood-forming organs — the marrow — in which premature nucleated corpuscles are precipitated into the circulation, and which, in readily losing their nucleus, increase the number of corpuscles, or that their presence indicates the degree of the increased activity on the part of the blood-forming function of the marrow. On the other hand, megaloblasts are signs of per- version of the haemogenetic action of the marrow, and in that they apparently do not readily lose their nucleus, and do not form mature corpuscles, are in- dicative of retrogression rather than progression. When megaloblasts and normoblasts are present in the blood the anaemia is always severe, and when the former are more numerous than the latter, and continue to remain so, the condition is probably one of pernicious anaemia. The diagnostic importance of the presence of nucle- ated red corpuscles, especially megaloblasts, in the blood, necessitates a very careful examinatio»> of several films before their absence is determined. Sometimes THE RED BLOOD CORPUSCLES 55 they are very numerous and may be easily found, whilst at other times their discovery occasions much laborious searching. The Development of the Blood. I. In the Embryo. — In the vascular area of the embryo a mass of cells, probably formed from the mesoblast, are found, to which the term "blood islands" has been given. From these cells, which contain nuclei, the earliest blood vessels and blood are formed. They multiply by indirect division and assume a red colour. White blood corpuscles appear later, being formed in the venous capillaries of the liver, and, as in the adult, in the marrow of the bones, the spleen, and lymphatic glands. The red bone marrow consists of the following elements : — Marrow cells, nucleated red blood cor- puscles containing haemoglobin, eosinophile cells, giant cells, and mastzellen, or cells containing y granules. In the adult the bone marrow is the most important seat of blood formation. The spleen is not generally regarded as an important factor in the formation, but rather as the seat of destruc- tion of the red blood corpuscles. The Malpighian corpuscles are, however, said to be one of the chief centres of the formation of leucocytes, which pass into the splenic pulp, and from thence into the general circulation. The lymphatic glands are probably concerned in the formation of lymphocytes, as small uninucleated leucocytes often showing division by mitosis are seen there. ~. As the age of the foetus increases the number of 56 THE BLOOD nucleated red corpuscles in the blood decreases, whilst the non-nucleated increase, probably due to the trans- formation of the former into the latter. This change may, according to some observers, take place in the liver, according to others in the circulating blood or spleen, and writers differ in their view of the method by which this is brought about. Some assume that the nucleus simply disappears (Bizzozero and Neumann) ; others, that there is an actual separation of the nucleus from the cell. (Rindfleich and Howell.) Hayem states that the nucleated and non- nucleated red blood corpuscles have a distinctly separate origin. Ehrlich believes that some of the nucleated red corpuscles, those known as normoblasts, lose their nuclei by a process of extrusion, whilst others, the megaloblasts, lose them by a method of endocellular disintegration. 2. Formation of the Red Corpuscles in the Adult. — The nucleated red corpuscles of the bone marrow are generally considered as the precursors of the ordinary non-nucleated erythrocytes of the blood. In support of this view Neumann and Bizzozero adduce the following facts : — That a great number of mitotic forms of these cells are seen under normal conditions in the marrow, that they disappear with rapidity, and that in all periods of life division by mitosis or karyokinesis is seen. On the other hand, to what extent the spleen and lymphatic glands take part in the formation of red blood corpuscles opinions differ. After loss of blood in a previously healthy individual, THE RED BLOOD CORPUSCLES 57 signs of rapid regeneration appear, and when the haemorrhage has been excessive, nucleated red cells are seen in the circulating blood. The time necessary to replace the corpuscles is naturally in proportion to the amount of the haemorrhage. Lowit believes that the spleen, lymphatic glands, and marrow take an equal share in the formation of both the red and white corpuscles, and states that in these organs two different forms of cells, the erythroblasts and leucoblasts, both characterized by an absence of haemoglobin, arise. These differ in that the leucoblasts possess amoeboid movement, and multiply by direct or amitotic division, whilst the erythroblasts possess no amoeboid movement, and increase by indirect or mitotic division. Latterly, however, he thinks the leucoblasts divide by indirect division by granules. The nucleated red cor- puscles arise from the erythroblasts containing no haemoglobin, by endocellular means in the blood. Miiller considers that the red and white corpuscles have a common origin, and a common mode of division. Denys assumes, as Lowit does, two separate sources of development of white and red corpuscles, which multiply by mitosis. Hayem thinks that the red corpuscles arise directly from the blood plates, or as he terms them the haemato- blasts, which increase in size and acquire haemoglobin. In favour of his theory he adduces the facts that normally a number of erythrocytes are found in the blood, smaller than the ordinary corpuscles, and these are increased in number when there has been a loss of blood, and assumes that they are the early stages of new corpuscles. He has noticed all transitional forms between these haematoblasts and mature red corpuscles. S8 THE BLOOD He points out that there are two varieties of erythrocytes in the embryo and adult. The ordinary non-nucleated which occur in normal blood, and which increase in cases of renovation of the blood of haemato- blastic origin ; and secondly, the nucleated red corpuscles, which occur in the embryo and in many of the lower animals, but are not normally present in man after the sixth month of intra-uterine life. They are never present in the healthy newly-born child, biit appear in extreme anaemias, and seem to take only a limited part in the regeneration of the blood. These he considers are formed by the haemopoietic organs. Hayem's theory of the haematoblastic origin of the red blood corpuscles is not generally received as correct. Engel considers nucleated red cells have originated from embryonic cells containing haemoglobin : — metro- cytes. These divide by mitotic division into two, each containing a nucleus. Each of these again divides into a nucleated and non-nucleated cell, forming nucleated and non-nucleated red blood corpuscles. The non- nucleated corpuscles occur in mammals, whilst the nucleated forms decrease in number till the end of intra-uterine life, when they are entirely absent. They lose their nuclei with a little surrounding protoplasm containing haemoglobin, or the latter may be dissolved by the serum of the blood. The cell, having lost part of its contents, by pressure in the vascular system becomes concave. THE BONE MARROW. Having regard to the importance which nucleated red corpuscles and marrow cells play in pathological THE BONE MARROW 59 conditions of the blood, it may not be out of place to give a short description of the red bone marrow. I have examined the bone marrow of the mouse and newly-born kitten. Films were made on the slides by gently spreading out the fresh marrow in as thin layers as possible. They were then fixed in the usual way, and stained with eosine (preferably glycerine solutions) and haematoxylin. Films heated and then fixed in picric acid solution, as described under " methods," gave beautiful results as regards the karyokinetic figures, but the eosinophile cells stained imperfectly. The best description in English of the marrow is that given by Muir and Drummond in the Journal of Physiology, 1892, and I have somewhat followed their description. The Marrow Cells (Plate V., Fig. 2). — Large uni- nucleated cells, measuring 12-14/w in diameter, are the most conspicuous elements present. Their protoplasm contains very fine granules, neutrophile, or finely oxyphile in nature, which are well seen in preparations stained with the triple stain. (Plate III., Fig. 5.) When films are stained with eosine and haematoxylin the protoplasm of the marrow cells, as in the blood of leucocythemia, colours a bluish colour, and not pink, as is the case with the multinucleated leucocytes, and little or no sign of granules can be seen. Their nucleus is of large size, generally round or oval in shape, and frequently has an indented or lobed appearance. It is poor in chromatin, stains somewhat faintly, and some- times shows nucleoli in its interior. All stages in their division by the formation of karyokinetic, or mitotic figures, are beautifully seen, especially in the marrow of 6o THE BLOOD young animals. They are stained somewhat deeper than the nuclei of other leucocytes. (Plate III., Fig. 9.) The eosinophile, or coarsely granular marrow cells, possess a single or lobed nucleus, and sometimes show mitotic figures. Their protoplasm contains coarse eosinophile granules. They differ from those of normal blood, not only by their size, but also by the fact that they are mononuclear, not polynuclear. (Plate II., Fig. 7 ; Plate III., Fig. 6.) Nucleated red corpuscles, termed haematoblasts by Neumann, nucleated haematoblasts by Hayem, and erythroblasts by other writers, are seen in the red marrow of all mammals. (Plate I., Fig. 9.) They vary considerably in size, some being about the same as that of the ordinary red corpuscle, whilst many are nearly twice as large. Between these extremes all intermediate sizes are seen. Their protoplasm is coloured with haemoglobin, but less deeply than the non-nucleated variety. It is homogeneous, stains with eosine and other acid aniline dyes, and is usually of spherical form, but tends to undergo alterations in shape more readily than the ordinary erythrocyte. The larger nucleated cells, the youngest elements, are only faintly coloured with haemoglobin. Their nucleus is large, and often shows a well-marked intra-nuclear network, or a somewhat dotted appearance. The smaller forms are older, i.e., nearer the non-nucleated condition, and contain a larger amount of colouring matter. Their nucleus is smaller, and stains very deeply with haematoxylin, so that little beyond a granular appearance can be made out. In both forms the nucleus is usually situated in the centre or towards the periphery, but in some of the smaller varieties the nucleus may appear almost entirely THE BONE MARROW 6i pushed out of the cell, and occasionally small free nuclei may be seen without, or with extremely little, protoplasm around them. In the larger elements karyo- kinetic figures are often apparent, whilst in the smaller forms two nuclei, or a budding nucleus, may be frequently met with. In addition to these red nucleated corpuscles there are colourless nucleated corpuscles, containing no haemoglobin. Although all intermediate forms between the larger erythroblasts and the marrow cells proper are seen, Muir states that they are probably two distinct elements, and that the marrow cells belong to the leucocyte class. Giant cells are very large, conspicuous elements, many measuring over 60 m in diameter. (Plate V., Fig. 2.) According to Muir they belong properly to the haemopoietic tissue, and have no relation to the osteoblasts. They are generally of somewhat irregular, or lobed appearance, containing one or many nuclei. The nucleus, which is very lobulated, is arranged some- what in the form of a circle, having one or more apertures in the centre. The nuclear portion of the cell stains a pale blue with haematoxylin, and not infrequently red blood corpuscles are seen in its interior. According to Hayem these cells are concerned with the destruction rather than the formation of the red blood corpuscles. In addition to the marrow cells proper and the giant cells, multinucleated and other leucocytes may be seen in red marrow, and are probably the ordinary elements of the blood, which have become mixed with the marrow in preparing films. There is still another class of cell which I have 62 THE BLOOD frequently seen, both in marrow and in the blood of leucocythemia. These are larger than the ordinary marrow, but do not reach the size of the giant cells. They are stained very faintly with haematoxylin, and seem to consist of a nucleus without any surrounding protoplasm. They appear to be made up of a very loose, open network, and contain several vacuoles or unstained parts, which appear quite homogeneous. In the marrow they are very plentiful, but are not nearly as numerous in leucocythemic blood. THE WHITE BLOOD CORPUSCLES, OR LEUCOCYTES. When blood is examined in the fresh state, in addition to the red corpuscles, pale, white, or greyish bodies will be seen — the leucocytes, or white blood cells. These are small spherical masses of protoplasm, containing no haemoglobin, and although larger in size, are much less numerous than the coloured elements. Some appear almost homogeneous, others slightly granular, whilst a few contain large, highly refractile granules. If the slide be maintained at the temperature of the body it will be noticed that the smaller elements remain at rest, retaining their spherical shape, whilst the larger undergo various alterations in their form and outline, due to amoeboid movement, which they, unlike the erythrocytes, possess. The white blood corpuscles have been variously classified. Wharton Jones, in 1846, recognized three varieties, viz., leucocytes containing fine granules, leucocytes with coarse granules, and nucleated leuco- cytes without granules. Max Schultze later, 1865, pointed out the occurrence of four forms : — THE WHITE BLOOD CORPUSCLES 63 1st. Small round cells with nucleus surrounded by- little, clear protoplasm, non-amoeboid ; 2nd. Larger cells with nucleus and clear protoplasm, amoeboid ; 3rd. Cells containing many nuclei with finely granular protoplasm, amoeboid ; and 4th. Leucocytes containing coarse granules in their protoplasm, also amoeboid. , Hayem describes the leucocytes under the following heads : — [a) Those of the ist variety — the smallest cells generally spherical, finely granular, possessing a very large nucleus, which occupies nearly the whole of the cell. He includes in this class the larger or intermediate forms with an indented nucleus. (b) Those of the 2nd variety — larger spherical cells with finely granulated protoplasm, and lobed or divided nucleus. These are the most numerous in normal blood. (f) Those of the 3rd variety — including cells conspic- uous by their very granular appearance ; the granules replacing two-thirds or three-fourths of the element. Ehrlich further elaborated this structural classification, by pointing out that the white cells react peculiarly to various aniline dyes according to the nature of the granules which they contain ; and according to their staining reaction, he divides the leucocytes into : — 1. Cells with a or eosinophile granules, which stain deeply with acid aniline dyes, e.g., eosine. (Plate II., Fig. 5-) 2. Cells with /3 or amphophile fine granules, staining both with acid or basic dyes. We are not concerned with these in blood. 3. Cells with y or coarse basophile granules, the 64 THE BLOOD so-called " Mastzellen." These stain with basic dyes. (Plate III., Fig. 8.) 4. Cells with ^ or fine basophile granules, staining with basic dyes, such as methylene -blue. (Plate III., Fig. 7-) 5. Cells with e or so-called fine neutrophile granules, staining with a mixture of acid and basic dyes, (Plate III., Fig. 3.) Kanthack and Hardy, in an excellent article in the Journal of Physiology, 1894, have satisfactorily proved that the so-called neutrophile granules are really faintly acidophile, or oxyphile in their reaction. For clinical purposes the different varieties of leucocytes may be conveniently considered under the following heads : — The small uninucleated leucocytes, large uninucleated leucocytes, multinucleated leucocytes, eosinophile cells, with the additional abnormal elements, marrow cells, and mastzellen. The first four varieties always occur in normal, the two last only in pathological blood. The Small Uninucleated Leucocytes or Lymphocytes. Synonyms. — Small hyaline cell, Sherrington ; leuco- cytes of the first variety, Hayem ; small round cell I. of Max Schultze. These are the smallest of all the leucocytes, measur- ing in the fresh condition only about 6 to 7*5 m, therefore somewhat less in diameter than the red blood corpuscle. (Plates II. and III., Fig. i.) They possess a single, large round nucleus, occupying nearly the whole of the cell, which stains deeply with the usual nuclear dyes, e.g., methyl-blue, haematoxylin, etc. When very dilute solutions of stains are used it will be found that the Plate II. The Leucocytes. Blood films stained with eosine and haematoxylin. Examined with eyepiece 2. Objective iV oil immersion and tube length of i6omm. Fig. 1. — Lymphocytes or small uninucleated elements. The nucleus is deeply stained--the protoplasm small in amount, or apparently absent. (a) Two large lymphocytes approaching in size the large uninucleated elements. Fig. 2. — Large uninucleated Leucocytes or Hyaline cells. The nucleus is somewhat indistinct, the protoplasm slightly coloured with haematoxylin. Fig. 3. — Transitional forms intermediate between the large uninucleated and multinucleated elements. Fig. 4. — Multinucleated or Neutrophile leucocytes. The nuclei numerous, showing distinct intranuclear network. The protoplasm with this stain (eosine and haema- toxylin) shows no granules. Fig. 5. — Normal Eosinophile cells having usually two or more nuclei. The protoplasm contains large granules deeply stained with eosine. Fig. 6. — Marrow cells or Myelocytes from leukaemic blood. Large uninucleated elements. Fig. 7. — Eosinophile Myelocytes or Marrow cells containing eosinophile granules, showing single large nucleus surrounded by and imbedded in coarse granules. One is partially ruptured. Fig. 8. — Marrow cells from leukaemic blood containing fine oxyphile granules, and therefore intermediate between the ordinary myelocytes and the eosinophile myelocytes. Fig. 9. — Marrow cells from leukaemic blood, showing karyokinetic figures. D L A-r £ % 9 ^■"^Ss W 4 ^ "~^,.: ?is 6. #^'- ■rf^^ ^^t ^■'* <•>.•• ■%?^''' .".•••.■•, '^^'^v /^•/,y,_ • .•.■:.;'..;,3!/. • ■ -. . , ft ;v.v •* *. v- ;«!'■ < "AV as? -:# '^' THE WHITE BLOOD CORPUSCLES 65 nuclei of the lymphocytes are coloured before those of any other leucocytes, and this staining characteristic, together with their size and shape, render these cells easily recognizable. The protoplasm of the smaller lymphocytes is in- significant, the nucleus frequently appearing bare and utterly destitute of a surrounding body. When visible the protoplasm rarely exceeds the appearance of a slender ring, one point of which may be thickened. Occasionally the cell body of the larger forms of this variety contains very fine granules, basophile in nature, which stain with methyl-blue. (Plate III., Fig. 7.) These leucocytes appear to be more resistant, i.e., undergo less deformation in shape than the other varieties, and unlike these they are neither amoeboid nor phagocytic. Their number is very variable, being increased after food, and diminished during starvation. They generally constitute about 15 to 20 % of the leucocytes present in the blood. They are considerably increased in lymphatic leucocythemia, a disease affecting the lymphatic glands, and are then associated with an increase in the number of the large uninucleated elements. They are usually diminished in diseases in which the activity of the lymphatic glands is impaired. The lymphocytes, as their name implies, originate from the lymphatic tissue, and are probably immature forms of the large uninucleated leucocytes, as all intermediate forms between the small and large cell are seen. Gulland considers them to be the early form of all the other leucocytes. They are sometimes spoken of as the young white corpuscles. Lymphocytes are met with considerably larger than F 66 THE BLOOD the typical form just described. These are often indistinguishable from, and are probably intermediate forms of, the hyaline cell. Large Uninucleated Leucocytes. Synonyms. — Hyaline cell, Kanthack ; large hyaline cell, Sherrington ; large lymphocytes ; larger types of the first variety, Hayem ; macrophagocytes, according to Metschnikoff. These are considerably larger than the small variety or lymphocytes, being sometimes the largest of all forms of leucocytes met with in normal blood. They measure generally about 8^ to 12 m in diameter. (Plate II., Fig. 2 ; Plate III., Fig. 2.) The cell is usually oval or spherical in outline, but this is by no means constantly the case in dry prepara- tions, and frequently they have the most irregular form, possibly due to their arrested amoeboid movement, or possibly from the fact of their being less resistant to pressure than the other leucocytes. Their protoplasm, which is usually abundant, contains no granules, although it has a somewhat ground -glass appearance. It stains slightly with the basic aniline dyes, so that in films stained with eosine and haematoxy- lin, or methyl-blue, the protoplasm surrounding the nucleus has a faint bluish tinge instead of pink, as is the case with most of the other leucocytes. The nucleus, which is relatively large, has a round or oval shape, and not infrequently shows a deep notch, giving it a kidney-shaped appearance. The intra- nuclear network is very diffuse and the nucleus as a whole stains faintly with the usual dyes, and I have frequently noticed that haematoxylin colours it a peculiar greyish-blue tint. THE WHITE BLOOD CORPUSCLES 67 These leucocytes undergo active amoeboid movement, and are phagocytic. The large size of these cells and their often indented nucleus, their somewhat irregular outline, as well as the faint blue colour which nucleus and protoplasm alike stain with eosine and haematoxylin, make the typical forms easily recognizable ; but I quite agree with Sherrington's statement that it is often extremely difficult to decide whether some leucocytes should be classified as small or large uninucleated forms. (Plate II., Fig. I, a.) Large uninucleated leucocytes, in which the nucleus is indented or distinctly horse-shoe shaped, and which show a well-marked intranuclear network, approach in appearance the multinucleated elements, and are often classified separately under the term transitional forms. (Plate II., Fig. 3.) The number of the large uninucleated elements is estimated at about 6-8 % of all the leucocytes. Sherrington has noticed that in the blood of dogs and cats an increase in the number of both the large and small forms is associated with a diminution in the haemoglobin and red corpuscles. Hayem estimates the number of the leucocytes of "the I St variety," which includes the large and small uninucleated forms, as 23 %, . and remarks that in chronic cases, in which the lesion is due to an advanced cachectic state, there is an increase in these forms. Multinucleated Leucocytes. Synonyms. — Neutrophil cells or leucocytes with e granules, Ehrlich ; finely granular oxyphile or acido- 68 THE BLOOD phile cells, Kanthack ; the oxyphile cells, Gulland ; microphagocytes, Metschnikoff; cells with finely granu- lar protoplasm. Max Schultze ; leucocytes of the 2nd variety, Hayem. These are by far the most numerous of all leucocytes present in normal blood. They are considerably larger than the red corpuscles and lymphocytes, but not quite as large as the large uni- nucleated and eosinophile cells. They vary in size. Hayem gives their measurement as from 7*5 to 9*5 or 10, and other writers as from 8 to 10 m in diameter. (Plates II. and III., Fig. 3.) The protoplasm contains numerous very fine granules, which Ehrlich found to be e or neutrophile in man, but amphophile (/3 granules) in the rabbit. Kanthack and Hardy consider them to be faintly oxyphile or acido- phile, i.e., staining with the acid aniline dyes, and this view is generally accepted at the present time. The neutral mixture of Ehrlich, as well as the triple stains, give these granules a lilac or violet colour. Ac- cording to Kanthack, when film preparations are stained with eosine and methyl-blue, these granules take on the eosine, and are therefore eosinophilous in nature. In the method of staining films previously recom- mended, viz., with eosine first and then with haema- toxylin, the protoplasm of these cells appears uniformly stained pink, and no evidence of granules can be made out with ]3^ inch oil immersion objective. If, however, films be exposed to the fumes of osmic acid before they dry, as Kanthack has suggested, their granular appearance may sometimes, though according to my experience rarely, be seen. Their nuclei, which are not very easily seen in the spherical condition of the THE WHITE BLOOD CORPUSCLES 69 living unstained cell, become very evident when the leucocyte is flattened in the film and stained. They usually consist of several round or oval nuclei, connected by thin threads of chromatin. They vary in number, but usually three, sometimes five or six, may be seen in a single leucocyte. In some the nuclei are so closely in Contact that they appear almost fused into one irregular nucleus, and are therefore spoken of as multi- partite nuclei, or as leucocytes with polymorphous nuclei. These transitional forms often show a deeply indented nucleus. The nuclei show a very marked intranuclear network, the chromatin threads and nodes of which appear clearly defined with haematoxylin, methyl-blue, etc. Together they form variously-shaped figures, such as 3 or 5, or resembling the letters s, z, u, etc. The irregularity of the nucleus, considered by Lowit as evidence of degeneration, and by other early writers as a sign of multiplication, was shown by Arnold to be due to amoeboid movement of the cell. According to Sherrington, "if the cell is allowed to quiet down before it is killed, the nucleus often returns to the spherical form." Many writers have objected to the term "multi- nucleated " applied to these cells, and Gulland in particular has raised strong arguments against the classification of leucocytes according to the nature of their nuclei; in fact, he states that "no one shape of nucleus is associated invariably with any one type of cell body, or with granules of a special kind." As this term, however, represents a condition of things usually seen in dry films, and as these leucocytes are so very generally spoken of by it, we prefer for purposes 70 THE BLOOD of clinical enumeration to retain it, qualifying its use by the names "neutrophile or finely oxyphile." These leucocytes are actively amoeboid, and possess the power of ingesting particles, whether these be of the nature of bacteria — " phagocytosis " — or products of the degeneration of the red blood corpuscles, e.g., the malarial granules in cases of malaria, or the black pigment granules of melanotic tumours. Pus consists for the most part of neutrophile cells, but in order to study their characteristics it is essential that the pus should be as fresh as possible, otherwise they appear as degenerated cells, and their granules may show Brownian movement. This latter condition, according to Sherrington, is a sign that the cell is nearly dead, and although present in some of the pus corpuscles, is always absent from the leucocytes of healthy blood. The number of multinucleated leucocytes in the blood is generally about 70-75 % of all the leucocytes. Hayem gives the proportion as 70 %, Von Limbeck 70-80 %. They are, however, subject to consider- able variation in number. Generally speaking when leucocytosis, i.e., an increase in leucocytes, is present the multinucleated elements are those which are chiefly concerned. As to the origin of these leucocytes authorities differ. Some, including Ehrlich, maintain that they are developed from the lymphocytes, which are formed in lymphatic tissue, and the large uninucleated cells. Gulland has shown that in the early life of the embryo there are no leucocytes present in the blood, that the lymphocytes are the earliest stage of all the varieties of leucocytes, and that multiplication and THE WHITE BLOOD CORPUSCLES 71 division of the white cells by mitosis takes place in adenoid tissue. Kanthack maintains that karyokinesis or mitosis occurs in the neutrophile cells, and adduces this as an argument against their being merely transitional stages of the uninucleated cells. He considers that they appear in the blood as distinct elements, and have an individual existence. These leucocytes are considered by many to be the most mature form of the colourless elements, and are therefore referred to as the " adult " cells. Eosinophile Cells. Synonyms. — Coarsely granular oxyphile or acidophile cells, Kanthack ; leucocytes with a granules, Ehrlich ; coarsely granular cells, Max Schultze and Wharton Jones ; leucocytes of the 3rd variety, Hayem. When normal blood is examined in the fresh con- dition, or by means of dry films, a few leucocytes may be seen conspicuous by their very granular appearance. They are seen as a mass of highly refractile granules, which occupy nearly the whole of the cell, and under a high magnification the granules appear to be spherical, brighter in their centre than at the periphery. When stained with one of the acid aniline dyes, particularly eosine, they are very prominent features in the film, and on this account received from Ehrlich the name of eosinophile cells. (Plate II., Fig. 5 ; Plate III., Fig. 4.) They are generally spherical in shape, but on account of the granules contained within their protoplasm, their margin is frequently irregular. In a badly-prepared film, in which much pressure has been used, occasionally the cells are seen to have 72 THE BLOOD ruptured and the granules extruded. I do not con- sider that they resist external pressure so well as the lymphocytes and multinucleated, but probably better than the hyaline cells. Their size varies, usually they are larger than the lymphocytes and multinucleated, but in dry films at least are not as large as the large uninucleated leucocytes. Von Limbeck states that they are intermediate in size between a large and small uninucleated cell. Hayem gives their average diameter as 8-9 "5 m, Kanthack as lo-ii m. The nucleus may be single, double, or deeply in- dented. It is generally somewhat horse-shoe shaped, the transverse part being thinner than the extremities, and is usually directed towards the periphery of the cell. At other times it is irregularly lobed, consisting of two egg-shaped nuclei, connected by a thin bridge of nuclear structure, which may be almost invisible. The nucleus appears to have been pushed to one side of the cell, as it is scarcely ever central in posi- tion. It does not stain as deeply as that of the multi- nucleated leucocyte, and the intranuclear network is not as evident. The protoplasm containing the granules occupies about three-fourths of the whole of the cell. The number and size of the granules vary in different leucocytes. For the most part they are relatively large spherical bodies, highly refractile, often showing in fresh preparations a greenish-yellow tint. In some the granules are very large, and the clear and frequently unstained matrix in which they are embedded allows of their being clearly defined. In Plate III. The Leucocytes — continued. Figs. 1 to 6. — Blood films stained with Ehrlich-Biondi's stain. Fig. 1. — Four Lymphocytes contrasted with {b) nucleated red and {a) normal corpuscle. Protoplasm contains no granules. Fig. 2. — Large uninucleated or Hyaline cell. Protoplasm contains no granules. Fig. 3. — Multinucleated or Neutrophile Leucocytes showing the indistinct outline of several nuclei. The protoplasm contains fine neutrophile granules stained a violet colour. Fig. 4. — Normal eosinophile cells containing two or more nuclei surrounded by large eosinophile granules. Fig. 5. — Marrow cells or Myelocytes from leukaemic blood containing a single large nucleus surrounded by fine neutrophile granules stained a violet colour. The granules in Figs. 3 and 5 are the same ; the elements differ, however, in their size, and the shape of their nucleus. Fig. 6. — Marrow cells containing eosinophile granules, or Eosinophile Myelocytes, from leukaemic blood. Figs. 4 and 5 contain the same kind of granules, but differ in their size and the shape of their nucleus. Fig. 7. — Marrow cells and two small uninucleated leucocytes from leukaemic blood, showing fine basophile or 5 granules stained with methylene blue. Fig. 8. — Mastzellen or cells containing coarse basophile or 7 granules. Film preparation from the peritoneum of a mouse stained with methylene blue. Fig. 9.— Marrow cells or Myelocytes, from the marrow of a kitten two days old, showing all stages of karyokinetic division. Film preparation fixed in picric, stained with dilute haematoxylin and then with eosine according to Muller's method. Fig.l Plate, ni. Fig 2 7i e,3. .,>•;««■->,. h & ,—,3..-.. Fig 4. ..,...,.._ «;*-:'• i;'^,,-^ Fi g. 6 V;. .-.'.i y. Fi tf.7. i'i li. 9. Fig.e m fk m P.L. Coles del. West, Newmaxi C,\ir . THE WHITE BLOOD CORPUSCLES 73 Others they are small and more numerous, so that when stained with eosine the protoplasm takes on a very- deep red colour, which renders the individual granules indistinct. Their oxyphilous reaction is much greater than that •of the finely granular cells, and, according to Kanthack, this is increased by corrosive sublimate or the applica- tion of heat. Sherrington has shown that the granules, which he considers are situated in a cortical layer of the cell, never exhibit Brownian movement in the normal leucocyte, this only occurring in abnormal conditions. The nature of these granules is still undetermined. Hayem states that although they have a particular affinity for eosine they are not haemoglobin, for they are more highly refractile than that substance, and are not dissolved by liquids in which haemoglobin is soluble ; but he thinks that the black granules, which he states are often associated with them, are possibly altered haemoglobin or its derivatives. They are not of the nature of fat, as osmic acid does not blacken them. Ehrlich considers them to be an albuminoid substance. It is stated that Barker has recently found that they contained iron. Sherrington mentions that they give the reaction for phosphorus faintly, and suggests that they are of the nature of nucleo-albumin. The eosinophile cells are amoeboid, but not phagocytic. Ehrlich considers that they are derived from the bone marrow; Kanthack and Hardy that they originate in the connective tissue and serous spaces. An argument against their origin from the bone marrow is that one 74 THE BLOOD observer, according to Adami, has found numerous eosinophile cells in the ray, which possesses no bone marrow. As Kanthack points out, marrow after all consists only of specially developed connective tissue cells. Some writers assume that they are the mature forms of leucocytes, and consider the comparatively small size of the nucleus, and the small amount of chromatin that it contains, evidence in favour of this view. The eosinophile cells are comparatively rare in adult human blood, although abundant in areolar tissues and bone marrow. Hayem states that they constitute 7 % of all the leucocytes, Stengel 2-4 %, and Von Limbeck, quoting Zappert, gives their number in healthy adult man as varying from 0"67 to 1 1 %. Their absolute number, according to Zappert, varies from 55 to 784 per cmm. They are relatively numerous in healthy children up to 13 or 14 years of age, whilst in old age as well as middle life they are subject to great variations. Von Limbeck remarks that the eosinophile cells have taken an important place in recent literature relating to the blood. Many writers have attached diagnostic and prognostic value to the increase or decrease of their number. Present experience, however, hardly justifies the unqualified acceptance of this view. Amongst conditions attended with numerical altera- tions Von Limbeck mentions the following : — Pregnancy and menstruation have no effect on their number. In leukaemia they vary greatly, but usually constitute about 2-6 % of all the leucocytes present. Their absolute number is generally increased, but THE WHITE BLOOD CORPUSCLES 75 relatively they may be normal or even less than normal. In chlorosis and severe anaemia they are frequently increased, but sometimes during the later stages are diminished, but do not afford any diagnostic or pro- gnostic value. Cardiac failure is usually attended with a normal number. During the course of non-febrile tuberculosis of the lung a diminution may sometimes be seen. Bronchial asthma and emphysema of the lungs are often associated with an increase in their number, as is frequently the case in affections of the liver and inflammation of the kidney. Functional neuroses, as well as skin diseases, may also show an increase, whilst malignant tumours, excepting sarcomata, frequently present a diminution. Ehrlich considered that an increase in the number of these cells indicated a chronic disturbance of haemogenesis, and particularly an implication of the bone marrow, as in spleno-medullary leukaemia. This view is no longer entertained, for in leukaemia there may be a relative diminution in the number of eosinophile cells. Many infectious diseases attended with a high temperature also show a diminution. Croupous pneu- monia is especially noticeable by the great diminution, or even entire disappearance, of these cells from the blood. Injection of tuberculin was followed by a diminution of the eosinophile cells, which again increased on the disappearance of the leucocytosis. Von Limbeck, in commenting on the immense amount of literature on the subject, states that from his experi- 76 THE BLOOD ence, only two facts are constant, viz., an increase of the eosinophile cells in asthma, and a decrease, or even entire disappearance of them, in croupous pneumonia ; and adds that the reappearance of them in the blood affords a means of foreseeing the approach of the crisis with great probability. Eosinophile myelocytes, or marrow cells containing eosinophile granules, which are almost pathognomonic of spleno-medullary leukaemia, will be fully described under this heading. (Plate II., Fig. 7; Plate III., Fig. 6.) Marrow Cells, or Myelocytes. Synonyms. — Markzellen, of German writers ; globules blancs hypertrophies, Hayem ; large uninucleated cells with neutrophile or e granules. These cells are never present in normal blood, although they occur in large numbers in the red marrow of bones. They are very characteristic of spleno-medullary and mixed leukaemia, sometimes constituting the greater number of leucocytes found in that disease. They are very large cells, attaining a diameter, ac- cording to Hayem, of even 20 m- (Plate II., Fig. 6; Plate III., Fig. 5.) Their protoplasm contains fine granules, which Ehrlich considered were neutrophile, Kanthack finely oxyphile. In preparations stained with eosine and haematoxylin the protoplasm almost invariably stains a faint blue colour with the haematoxylin, rather than pink with the eosine, but does not show any granules. Stengel makes the following allusion to them : " Ac- cording to Ehrlich the myelocytes are indistinguishable from the large uninucleated or transitional form of THE WHITE BLOOD CORPUSCLES 77 leucocytes, except for the presence of neutrophile granules. In some instances, undoubtedly, Ehrlich's description applies, but the typical myelocyte differs from the ordinary large uninucleated leucocyte in the much greater size of the corpuscle, and in the unusual pallor of the nucleus. These leucocytes may be dis- tinguished by their morphology alone in many instances, but the small forms approach so nearly the ordinary uninucleated elements, that differential stains are neces- sary to distinguish the nature of the individual cor- puscles, and even after this test, doubt may exist as to the proper classification." The nucleus is usually single, often indented or some- what lobed. It is very poor in chromatin, stains only faintly with nuclear stains, and shows very little intra- nuclear structure. They are said to be identical with Cornil's "cellules meduUaires." Marrow cells are the chief diagnostic feature of spleno-medullary leucocythemia, where they are almost as numerous as the multinucleated leucocytes. Till recently it was thought that they were only to be found in this disease, but it is now known that they occur also in severe anemias, particularly pernicious, myxoedema, syphilis, and other chronic cachectic con- ditions ; but their percentage in all diseases except leukaemia is extremely small. We shall speak more fully of their characters and relationship to marrow cells proper, when we describe the condition of the blood in leucocythemia. Mastzellen, or cells containing y granules, or coarsely granular basophile cells. (Kanthack.) Sherrington states' that he has never met with them 78 THE BLOOD in normal mammalian blood, but has seen a few in the reaction stage of Asiatic cholera. According to Limbeck they sometimes occur in leukaemia ; according to Miiller they are very numerous in this disease. , They occur in health, however, in the coelomic fluid and connective tissue of the body, and in the marrow of bones. They can readily be seen in preparations made from the peritoneal fluid of the mouse, and stained with Ehrlich's acid dahlia solution, or with methylene-blue. (Plate III., Fig. 8.) The nucleus stains very faintly, and with either of these stains appears rather as a cavity, which is situated somewhat out of the centre of the cell. It is poor in chromatin, no intranuclear structure being visible by the ordinary methods of procedure. The cell protoplasm is filled with very numerous dense granules, which obscure the nucleus, and at the periphery give the outline of the cell an irregular margin. They stain very deeply with dahlia and methylene-blue, and appear of a purplish-blue colour, contrasting with the surrounding blue cells. They are not very refractile, and in unstained prepara- tions are indistinguishable from other cells except by their size. They are very large cells, often exceeding 20 m in diameter. According to Kanthack they are non-amoeboid, but Gulland states that they possess this power. They are not phagocytic. These cells originate in the connective tissue, but " owing to their large size cannot get further in the blood stream than the first capillaries." (Gulland.) Some writers speak of the finely and coarsely granular THE WHITE BLOOD CORPUSCLES 79 basophile leucocytes, i.e., cells with <5 and y granules respectively, under the general term mastzellen, and state that they may be found in small numbers in normal blood. Von Limbeck expresses doubt whether even the small finely granular basophile cells occur in the blood of a healthy adult. Canon points out that they are of constant occurrence, especially in healthy and sick children. I have frequently found the ^, or finely granular basophile cells, which are especially numerous in leu- cocythemia, but in my whole experience have never met with true mastzellen cells in the blood of this or any other condition. The Number of Leucocytes. The number of all forms of white corpuscles found in normal blood, depends on various conditions, and writers differ as to what should be considered the average. Hayem gives their number as 6000 per cmm. Limbeck as 8-9000, allowing an increase or decrease . of a thousand, and the proportion of white to red corpuscles as about i to 5 or 600. Afanassieu gives the proportion as i to 600, and Muir states that the proportion of i to 500 is rarely exceeded without some pathological condition being present. Under many physiological as well as pathological conditions they may undergo an increase in number, constituting leucocytosis or hyperleucocytosis, or a diminution in number, known as hypoleucocytosis, leu- copenia, or leucolysis. The last expression is used by Lowit to indicate a destruction of the leucocytes. 8o THE BLOOD When only the lymphocytes are increased it is some- times called lymphocytosis. Leucocytosis is usually, however, attended with an increase of the multinucle- ated or neutrophile cells. The proportion of the various forms of leucocytes present in normal adult blood is as follows : — According According to Rotch. to Stengel. Multinucleated or neutrophile cells 60-7 5% ... 6s-7o7„ Small uninucleated cells or lymphocytes 24-3°% 257„ Large uninucleated or hyaline cells 3- 6% - 3- 67o Eosinophile cells I- 2% not above 37„ Other writers give very similar figures. The Influence of Physiological Conditions on the Leucocytes. Digestive Leucocytosis.— Ki\.&c a meal the number of leucocytes increases, reaching a maximum at the time that intestinal digestion is taking place. An in- crease of 18 to 20% may be found about this time. This increase depends largely on the nature of the diet ; thus proteid food causes a much greater in- crease than a mixed diet. The lymphocytes and multinucleated leucocytes are chiefly concerned in this form of leucocytosis, the eosinophile and large uninucleated forms either remain unaffected or tend to diminish. During fasting there may be a slight diminu- tion in the number of white cells, but this is seldom pronounced. Von Limbeck records the result of Luciani's examination of the blood of Succi, the fasting- man, in whom the leucocytes sank from 14,530 to 861 per cmm. in seven days, but on the eighth day increased THE WHITE BLOOD CORPUSCLES 8i to 1530, and remained at about that figure till the end of the fast. There is said to be a relative increase in the eosinophile, but a diminution in the lymphocytes and multinucleated elements, during starvation. During pregnancy there is usually, though not in- variably, an increase in the leucocytes ; at delivery there is very little alteration ; whilst during lactation there may also be a slight rise in their number. In the early days of childhood there is a condition of leucocytosis. During the first two or three days of life, the white cells are more numerous than in the adult, but when the weight of the body is at its minimum, about the third day, the leucocytes decrease considerably, whilst the red cor- puscles increase. The leucocytosis observed in the first few days of life, is occasioned by an increase in the number of lymphocytes, and not the multinucleated elements. The eosinophile cells are also more numerous at this period. The condition of the blood during infancy and early childhood, will be more fully dealt with when the diseases of children are described. Other physiological conditions, such as fatigue, differ- ences in sex, etc., apparently have very little influence on the number of leucocytes. Hayem noticed an in- crease in their number in the Esquimaux, and attributes it to their fatty diet, milk producing similar results. Pathological Alterations in the Leucocytes. Increase in the number of the leucocytes is of very frequent occurrence in pathological conditions. It is found in acute inflammatory diseases, in which the 82 THE BLOOD leucocytes may attain the number of 36,000, but more usually 15 to 20,000 per cmm., as well as in chronic diseases. Of the latter the most striking illustration is leukaemia. Usually the multinucleated leucocytes are the form that are increased ; but sometimes, especially in advanced cachectic states, e.g., rickets, syphilis, etc., the lymphocytes are chiefly involved. Occasionally in cases of lymphosarcoma, malignant disease, and in terminal leucocytosis, as well as in the post haemorrhagic forms, the uninucleated leucocytes are increased, but this is not generally the case. It is rather difficult to say what number of leucocytes should be considered pathological. Hayem and Stengel state that when the leucocytes exceed 10,000 per cmm., i.e., a proportion of i to 500, the condition is one of leucocytosis, and the latter very rightly points out that the proportion of white to red corpuscles is not nearly as reliable a test of the existence of this condition, as the actual number of leucocytes. Decrease in the number of leucocytes, a much rarer condition, is found in many long-continued fevers, not complicated by inflammatory conditions, such as typhoid fever. It is also seen in extreme anaemias. In fact, those diseases in which the lymphatic organs are involved, are usually attended with a decrease in the number of the white corpuscles. Alterations of the leucocytes other than numerical may occur in various pathological processes. In extreme anaemia some of the multinucleated elements are found to contain haemoglobin in their protoplasm, which gives them a yellow tint, especially at their margins. Leucocytes containing haemoglobin, are said to have been mistaken for giant nucleated THE WHITE BLOOD CORPUSCLES 83 red corpuscles ; but the granular condition of their pro- toplasm, and the multipartite character of their nuclei, should in stained preparations prevent any mistake. Under certain conditions, particularly leucocythemia, some of the leucocytes may lose their amoeboid power of movement. In cases in which marrow cells appear in large numbers in the blood, e.g., splenic and mixed leukaemia, remarkably pale and indistinct leucocytes may be found, containing vacuoles of very varying size. Mention of these has already been made in connection with the bone marrow. In most forms of malarial fevers the white blood corpuscles often contain pigment granules — the so-called " melaniferous leucocytes " — and a similar condition has been noticed in general melanosis. These are generally regarded as the product of the destruction of the red blood corpuscles. Inflammatory Leucocytosis and Phagocytosis. Excellent accounts have been given by Kanthack and Hardy, Sherrington and Adami, on the part the leucocytes take in inflammation and against bacteria. All that is stated here is largely culled from the writings of these authors, as well as those of Metschnikoff "The process of inflammation is essentially the endeavour on the part of the organism to prornote the migration of leucocytes, and to aid in the inclusion and destruction of the irritant." When inflammation attacks, or poisons of the nature of bacteria are inoculated into the body, two conditions result — a temporary diminution, quickly followed by 84 THE BLOOD an increase in the number of the white cells of the blood. The initial decrease of the leucocytes, termed " leucolysis " or " leucopenia " by Lowit, or " hypoleu- cocytosis," sets in immediately after the irritant has been applied. The variety of leucocytes which are chiefly diminished is, according to Sherrington and others, the multinucleated or neutrophile cells. Although this stage is of brief duration, the diminution is sometimes very marked. The cause of this phenomenon has received many explanations. Lowit considers that it is owing to a destruction of these cells. Some assume that the leucocytes are drawn away to other parts of the circulation, whilst others think that the colourless elements accumulate in the vessels of the liver, spleen, and marrow. Sherrington has not found that the leucocytopehia bears any constant relation to the extent of the fever. This primary stage is quickly followed by leucocytosis, and Kanthack considers it a rule " that previous to recovery from bacterial products, an increase in the number of leucocytes takes place." Lowit considers this is due to an increase of the lymphocytes ; but many other observers, including Kanthack and Sherrington, attribute it to the multinucleated leucocytes, the granules of which, according to the former, become more eosinophilous, but not larger. The eosinophile cells are said to be greatly . diminished, or to actually dis- appear from the blood in this as in the former stage. It has been suggested that they may be drawn to some particular part of the circulation, or that they pass to the seat of injury. THE WHITE BLOOD CORPUSCLES 85 The increase in the multinucleated cells is, according to Romer, due to an actual reproduction of these cells in the blood. Very similar results are to be found in many infectious diseases. In pneumonia all writers recognize the occurrence of leucocytosis in favourable cases, and in pleurisy, erysipelas, scarlet fever, measles, etc., the same condition is observed. In pneumonia, and most acute inflammatory affections, leucocytosis is regarded as a favourable, and the absence of it an unfavourable sign. Kanthack states " that in very acute diseases where the process runs a rapid course, where we have resolution by crisis — in fact, processes which are comparable with artificial infections or intoxications — a well-marked leucocytosis always ushers in the crisis or recovery, or at any rate is a most favourable sign of great prognostic value." {^Medical Chronicle, 1894.) Phagocytosis. — For a considerable time it has been known that the leucocytes can take up foreign matter into their interior, and more recently it was found that they could attack and incorporate bacteria. All the leucocytes do not, however, possess this phagocytic power, the multinucleated or neutrophile, and the large uninucleated or hyaline cells are the most' important elements concerned in this process. Although a leucocyte may take up bacteria, it does not necessarily follow that it digests all. Thus in subacute and chronic diseases, such as gonorrhoea, tubercle, etc., organisms are found in their protoplasm in a living condition. In many cases the microbes are not only seized and incorporated in the substance of the leucocyte, but they are subjected to a process analogous to digestion. If the 86 THE BLOOD organisms are very virulent, exceedingly few are taken up by the leucocytes, and it is a long time before phagocytosis comes into action. Chemiotaxis, a term used to denote the attraction by which the leucocytes are drawn to the point of injury, explains why the wandering cells of the body accumulate at the part which is attacked by the micro-organisms. When the latter are not very virulent, the leucocytes are attracted in large numbers, "positive chemiotaxis," but when very virulent, the leucocytes are not drawn to the point of infection, " negative chemiotaxis," but a negative may eventually become a positive chemiotaxis. Positive chemiotaxis is very variable in amount, and many writers doubt the occurrence of negative chemio- taxis, assuming the absence of the leucocytes to be due to their destruction by the microbes. " The cure of zymotic or mycotic disease, whether localised or general, and the immunity also, are mainly brought ' about by the activity of special cells — the phagocytes — and are primarily dependent on the attraction existing between these cells and the products of bacterial metabolism." {Allbutfs Medicine, vol. i. p. 87, 1896.) Kanthack and Hardy have found an increased number of eosinophile cells at the seat of injury, as, for instance, in blisters, etc., and as these cells are very numerous in fresh pus from acute gonorrhoea, purulent sputum of tubercular disease, and possibly in pneumonic sputum, they consider that these cells migrate when- ever noxious influences — inflammatory in nature — are to be counteracted. They have suggested the theory that although the eosinophile cells are not phagocytic, yet they assist in the process by possibly preparing the THE WHITE BLOOD CORPUSCLES 87 particles for digestion, and recognize a stage of "eosinophile attack and that of phagocytic ingestion." The Leucocytosis of Malignant Disease. Many maUgnant growths, particularly sarcomata — osteo- and lympho-sarcoma — as well as carcinomata — scirrhus and encephaloid — are attended with an in- crease in the number of leucocytes. It is usually more marked in the sarcomata than the carcinomata, varying in number from normal to 50,000 or more. In cases of sarcoma, involving the lymphatic structure, the appearance of the blood may closely resemble that of lymphatic leukaemia, in that the same variety of white cells, viz., the small uninucleated leucocytes, may be increased in the former as well as the latter. Hayem states that epitheliomata are the form of malignant neoplasms, which may show no increase in the number of the leucocytes, and notes that cancers of the stomach may be divided into those attended with and those without leucocytosis, and suggests that the latter are probably epitheliomatous in nature. Hayem lays down the following rule, "that all tumours — independent of inflammatory or suppurative complica- tions — accompanied with an increase of leucocytes are of a malignant nature." Cachectic leucocytosis may occur in a variety of chronic affections, particularly in syphilis and rickets. As the spleen and glands may be enlarged in these diseases, and as the increase in leucocytes may affect chiefly the uninucleated forms, the differential diagnosis between it and lymphatic leukaemia, may present considerable difficulty. 88 THE BLOOD Agonal, or terminal leucocytosis, noticed towards the end of life in many chronic cachectic diseases, may sometimes be very pronounced. It has been noted in pernicious anaemia, and usually involves the small uninucleated leucocytes. It is said to be due to a reduction in the blood pressure, and the discharge of leucocytes from the lymphatic organs into the circula- tion. (Stengel.) Post-haemorrhagic leucocytosis occurs soon after loss of blood, and persists some few days according to the quantity of blood lost. Sometimes the uninucleated, but more usually the multinucleated, elements are increased. THE BLOOD PLATES. Synonyms. — Blood plaques, or haematoblasts of Hayem. The third corpuscular elements of the normal blood were first discovered by Hayem, and termed by him " the haematoblasts, or the germs of the red blood corpuscles," and were later described by Bizzozero as the blood plates. In fresh blood preparations made and examined as quickly as possible, very small round or oval bodies, measuring about 2 to 3|- /^ in diameter are seen. These are of a faint yellow colour, and are slightly granular in appearance. In a short time — a few seconds — they run together into grape-like clusters, and, losing their rounded form, become spinous. At the same time they appear more granular and. highly refractile, and ultimately very thin filaments of fibrin are seen starting from their angular projections. They are remarkable for their adhesiveness, in virtue THE BLOOD PLATES 89 of which they adhere to red corpuscles or adjoining blood plates. In order to examine them in detail it is necessary to use Hayem's solution, or the i % osmic acid solution mentioned before. They may also be well seen in dry film preparations, provided that the film has been quickly made and dried. (Plate I., Fig. 2.) On account of their adhesiveness, the blood plates will be most numerous in that part of the film where the blood first came in con- tact with the slide. An exact enumeration of them is not easy, as they tend to run together into little masses. They stain faintly with aniline dyes, having an ampho- phile reaction, so that in dry preparations stained with eosine and methyl-blue, or eosine and haematoxylin, they generally appear pale violet colour. This colour is, however, variable. Lowit considered them at first to be artificial products of the blood plasma, or of the degeneration of the white blood corpuscles ; but Bizzozero proved that they could be seen in the circulating blood of the bat. The majority of writers now consider that they exist as such in the blood. In the circulating blood in the web of the frog, blood plates, which are here very large and easily mis- taken for leucocytes, can be readily seen. Their high specific gravity, together with their great adhesiveness, explain their most important function — the formation of the white blood clot. Whether they have any other use is still undetermined. Hayem's theory that they are the early forms of red corpuscles is not generally accepted. According to Hayem, in man the blood plates number 250,000 per cmm. Afanassieu gives their number as 200,000 to 300,000, and Fusari from 180,000 to 250,000. 90 THE BLOOD The first-named observer considers that a diminution in their number is a serious sign. Among the causes which may lead to this, he mentions prolonged fast, long- continued typhoid fever, progressive anaemia, and ad- vanced cachexia, especially cancerous cachexia. Hayem's statement that "there is only one condition in which the decrease in the number of blood plates can be con- sidered favourable, and that is where it is temporary and associated with an increase in the number of the red corpuscles," is evidently based on his peculiar theory of blood formation. Diminution in their number may also occur after certain poisons — pyrogallic acid, glycerine, etc., in high fevers, e.g., typhus and erysipelas, but not in tuberculosis, nor in pneumonia. Pizzini points out that in all infectious fevers there is a diminution in the number of the blood plates, which is inversely proportional to the height of the temperature — the higher the fever the more marked is the diminu- tion of these elements. After the cessation of fever they often increase, reaching a maximum six to seven hours after the crisis. They are sometimes said to dis- appear entirely from the blood in malarial fever. Increase in the number of haematoblasts is of frequent occurrence. It may be noted in all anaemias unattended with fever, especially at the time that regeneration of the blood occurs, and is said to be a favourable sign. It also occurs after loss of blood, and in leucocythemia. According to Hayem a temporary increase in their number is concerned in a renovation of the blood, and augmentation of their transverse diameter is said by Fusari to occur in anaemia as soon as improvement takes place. THE BLOOD PLATES 91 In inflammatory conditions in which the film is increased very viscid masses occur, having a granular appearance and containing many blood plates, as well as red and white corpuscles. These have been termed " plaques phlegmasique " by Hayem. In certain cachectic con- ditions, somewhat similar granular masses are found in the blood, differing from the former in the following particulars : — Their outline is more defined, they contain few but large plates, and include a very limited number of coloured and colourless corpuscles. These he speaks of as "plaques cachectique." The same observer also mentions the formation of crystals in very severe anaemias, due to modifications, probably of a chemical nature, of the haematoblasts. Pathology of the Blood. Anaemia. The term anaemia is used to imply a diminution in the quantity of blood — "oligaemia," in its corpuscles — "oligocythaemia," or in its haemoglobin or colouring matter — " oligochromaemia," or a combination of these conditions. The pallor noticeable in anaemic conditions is due to a decrease in the amount of haemoglobin, whether that be from a diminution of the actual amount of blood, of the corpuscles, or of the colouring matter of the red ■cells. It must be noted that the degree of pallor is not necessarily a sign of the extent of the anaemia, and that in some individuals pallor, due to alterations of the capillary supply to the skin, or to deficient pigment, may exist without any departure of the blood from the normal. This has been termed pseudo-anaemia. From this it is apparent that the red blood corpuscles and their contained haemoglobin are only concerned in this condition, and that, although alterations in the number of leucocytes may and do occur, yet they can- not be regarded as factors in the process. Absolute diminution in the quantity of blood, "oligaemia," occurs after haemorrhage, but except in those cases in which the loss has been so great as to cause an immediately fatal result, the mass of blood ANEMIA 95, is maintained at nearly the same bulk, owing to the relationship existing between the liquid of the various- tissues and the vascular system. Decrease in the number of red corpuscles — oligocy- thaemia — is an exceedingly common condition, but diminution of the haemoglobin — oligochromaemia — may occur without any numerical alteration of the coloured elements, as is seen in some of the slighter forms of anaemia, and particularly in chlorosis. Otto found that after haemorrhage the number of corpuscles increased before the haemoglobin, and some authors consider mere diminution in the haemoglobin a sign of regeneration of the blood. Diminution of the red corpuscles may be due either to lessened blood formation, " haemogenesis," as in conditions of starvation, unfavourable hygienic sur- roundings, new growths, etc., or to excessive destruction of the red cells, "haemolysis," as in fevers, etc., or ta a combination of these factors. Admitting that in health the red corpuscles number about five million per cmm., and the amount of haemo- globin present is represented by five million normal corpuscles, or 100 %, and taking into consideration the individual variations of the blood in health, what condition constitutes anaemia? Hayem considers patients to be suffering from anaemia when they com- plain of functional troubles evidently connected with the state of the blood, and when the amount of haemo- globin is only equivalent to four instead of five million normal corpuscles, i.e., 80 %. Stephen Mackenzie states that anaemia commences when the corpuscles constitute 80 %, or four million per cmm. ■94 THE BLOOD Hayem classifies anaemias generally according to their severity into four groups : — 1. Ancemia of the ist degree, or slight ancsmta. — The haemoglobin of the blood is equal to three or four million red corpuscles, i.e., 60-80 %. The red corpuscles are in some cases normal or slightly altered, and their number is greater than is expressed by the value of the haemoglobin. In other cases their num- ber is proportional to the amount of haemoglobin, and both show slight changes in the form of the red cells. If the amount of haemoglobin each corpuscle con- tains — i.e., the colour index — be represented in health as I, then in this degree of anaemia it will most frequently be represented as from o"65 to o'g. This, according to Hayem, is not the form which is most improved by iron. 2. Ancsmias of the 2nd degree, or of average intensity. — This is the most common type. The haemoglobin is equal to 2-3 million corpuscles, i.e., 40-60 %. The number of red corpuscles varies from 3-5 million, and the small corpuscles, "globules nains," are frequent. Therefore, although their number is not reduced, their average size is diminished, and the individual value in haemoglobin or their colour index, is notably lower than the normal, and varies from 0*3 to 0'8, generally 0*5. Large corpuscles may be found, but they are not numerous enough to raise the average diameter of the red cells to the normal. It is very rarely that giant cells are seen. Chlorosis belongs to this type of anaemia, which is essentially characterized by the disproportion between the amount of haemoglobin and the number of cor- puscles. ANEMIA 95 3. Ancemias of the ■^rd degree, or intense ancemia. — This includes cases in which the ancemia, although severe, does not per se threaten life. The haemoglobin is equivalent to from 2 million to 800,000 red cells, i.e., 40 to 16%. The red corpuscles number between 800,000 and 4 million, and the amount of haemoglobin which each corpuscle contains varies considerably, sometimes being as low as 0*4, but often equals i "o or even above. Whilst small corpuscles are especially characteristic of anaemia of the 2nd degree, large and giant corpuscles (megalocytes) are essentially features of the more intense anaemic conditions. In some cases the red cells are small, but relatively numerous, whilst in other and severer types they are, ori an average, larger than in normal blood, but their number is considerably reduced, whilst in both, large and even giant cells are frequently seen. 4. Ancemias of the /^th degree, or extreme ancemia. — This includes cases in which the anaemia may pei' se cause the death of a patient. In it the amount of haemoglobin is equal to about 800,000 normal corpuscles, i.e., 16%, and the red corpuscles fall to 800,000 and below. Although death is not inevitable when the number of red cells is only equal to 800,000, yet Hayem from his experience expresses the opinion that anything below this, places the life of the patient in extreme danger, on account of the condition of the blood alone. As the average diameter of the corpuscles is greater than, and their colour almost as great as the normal, the amount of haemoglobin that each contains is often above 96 THE BLOOD I, the figure expressing the amount in a healthy cor- puscle, and frequently varies from 0"88 to \"]. Large corpuscles are the prevailing feature of this form of anaemia, and giant cells^megalocytes — reaching even i6yu in diameter, are frequent, constituting 3 to 12% of the red cells present. Sometimes dwarf corpuscles, " globules nains, or petits " — microcytes — are present, and the irregularity in the size, form, and colour of the elements is a striking feature. According to Hayem, nucleated red corpuscles only occur in some cases of extreme anaemia, but as we have previously mentioned, our experience regarding the occurrence of these elements is totally opposed to this view. It is in this type of anaemia especially that vacuoles in the corpuscles, as well as crystals, are seen. The leucocytes play a very secondary part in anaemia, and undergo very little alteration in the first two degrees, but are not infrequently modified in the severer forms. Their actual number is usually diminished, and some may be found to contain haemoglobin. Sometimes the small uninucleated leucocytes are increased, at other times — these are less numerous — the other forms of white corpuscles are increased in number, and occasionally marrow cells may occur in severe anaemia. The blood plates usually remain as numerous as in the normal condition, and are only diminished in extreme anaemia. (Hayem). Some observers, ^.j^., Muirand Eichhorst, do not agree with Hayem's statement, that the presence of large corpuscles indicates a severe, and small forms a mild, anaemia. It was the last-named writer who assumed ANEMIA 97 that very small deeply-coloured corpuscles were charac- teristic of pernicious anaemia. Hayem expresses the opinion that contrary to the statements of Eichhorst, Quincke, and others, anaemia associated with small red corpuscles, instead of being characteristic of the most serious of all anaemias — progressive pernicious anaemia — indicates rather the most benign of the severer anaemia — - and from my experience I regard this as the correct view. Stephen Mackenzie, in the Lettsomian Lectures i^B. M. Journal, January 24th, 1891), makes the following scale of anemia : — Anaemia commences at 80 %, i.e., 4 million red cor- puscles ; it is decided between 65 and 50%, i.e., ■^^ to 2\ million ; grave at 50 %, i.e., 2^ million ; very grave at 35 %> oi" if million ; and fatal at 7"5 %, i.e., when the red corpuscles number only 375,000 per cmm. It is usual for clinical purposes to divide anaemia into two classes — the primary or idiopathic, and the secondary or symptomatic ; but it must be borne in mind that many conditions which cause symptomatic anaemia may affect the blood-forming organs, and so give rise to a condition of the blood closely resembling that of primary anaemia. This classification is convenient, but not necessarily scientific, and some cases of anaemias, which are at present termed primary, may eventually be proved to be really secondary to some organic condition, which so far remains undetermined. Primary, idiopathic or essential ancemias are those in which the condition of the blood or blood-forming organs is the main characteristic of the disease, and include chlorosis, simple primary anaemia, pernicious anaemia, leucocythemia, Hodgkin's disease, and splenic anaemia. H 98 THE BLOOD Secondary or symptomatic ancsmias include those con- ditions in which the state of the blood is not the essential characteristic of the disease, but is due to other causes outside the haemopoietic system, such as those resulting from haemorrhage, malaria, malignant growths, and organic diseases generally. Primary Anaemia. Chlorosis. Synonyms. — Chloraemia, or chloranaemia ; greensick- ness ; cachexia virginum ; morbus virgineus ; bleichsucht of German writers. Chlorosis is a form of primary anaemia affecting chiefly, though not exclusively, the female sex during the time of puberty or early womanhood. It is usually character- ized by a peculiar pale, sallow complexion, having a somewhat greenish tint, and by a diminution in the amount of haemoglobin greater than that of the number of the red corpuscles. This definition requires to be somewhat modified, as this form of anaemia occurs also in boys in a somewhat modified form, and the so-called "chlorotic condition of the blood " — the greater reduction of haemoglobin over that of the corpuscles — is by no means confined to chlorosis proper, but occurs in many other forms of anaemia. Character of the Blood. The blood as it proceeds from a puncture is usually pale in colour and remarkably fluid. It coagulates normally, and there is no evidence that the amount of fibrin is other than normal. CHLOROSIS 99 The total amount of blood is probably not reduced ; its specific gravity, however, is somewhat less than that of healthy blood. According to Duncan, who was the first to investigate both the number of the corpuscles and the amount of haemoglobin, chlorosis consists in a diminution in the amount of the latter — oligochromaemia — whether the number of corpuscles are normal or less than normal ; and Graeber even went further, and stated that diminu- tion in the number of red cells was a complication of chlorosis with aneemia. In favour of this view he mentions that during treatment, the number of cor- puscles increases before any increase in the amount of haemoglobin takes place. Laache divides chlorosis into two classes, one in which there is no diminution in the number of the erythrocytes — "pseudo chlorosis" — and the other in which the cor- puscles as well as the haemoglobin are deficient, which he terms "true chlorosis." Most writers consider that there is a diminution in the amount of haemoglobin usually associated with a decrease in the number of the red corpuscles, but that the former is more conspicuous and characteristic than the latter. Thus, out of 247 cases of chlorosis examined by various writers, in 40 % the corpuscles numbered four million and upwards, whilst in the remaining 60 % they were less than that number. Hayem found that. in 60 cases of chlorosis 20 cases showed anaemia of 2nd degree, red b. corp. 4 mill., Hb = 2-7 mill, or 54%, = 0-65. 39 cases showed ansemia of 3rd degree, red b. corp. 2*9 mill., Hb= i'5 mill, or 30%, G = o'52. I case showed anaemia of 4th degree, red b. corp. 937,360, Hb= 796,756 or 16%, = 0-85. THE BLOOD G = individual corpuscular value in haemoglobin or the so-called colour index. In all these cases the amount of haemoglobin was always less than the number of corpuscles, and the individual corpuscular value in haemoglobin was re- duced as a rule to 0"5 or o"3, the normal being = i. The so-called chlorotic condition of the blood occurs in many chronic secondary anaemias, in cases recovering from haemorrhage, and in the most varied pathological conditions ; but it is seldom as constant or as pronounced as in chlorosis. Regeneration of the blood, which occurs in chlorosis during treatment with iron and arsenic, as in anaemias the result of haemorrhage, does so, as Otto and Graeber pointed out, by a primary increase in the number of red corpuscles, and then by an increase in the amount of haemoglobin. Alterations in the size, form, and colour of the cor- puscles, as well as mere numerical change, are seen in the blood of chlorotics, and for clinical examinations Hayem recommends the use of dry films, which can be compared with those of normal blood. (See Plate IV., Fig. I.) In shape the corpuscles may be more or less altered, according to the degree of oligocythemia. Poikilocytes are frequently present in all forms of chlorosis, and are generally more pronounced in cases in which the cor- puscles are considerably reduced in number. Muir, however, does not consider that the poiki- locytosis always varies proportionately to the diminution of the number of the red corpuscles, and states that it may be very well marked when the number is very little reduced. Hayem points out that alterations in Plate IV. Film preparations stained with eosine and haemaloxylin. Both examined with eyepiece 2. Objective i". Fig. 1.— 'Chlorosis. The red blood corpuscles show moderate poikilocytosis. They are, on the average, somewhat below the size of normal corpuscles. Microcytes are numerous. The cells are stained faintly and the central depression very marked. At the lower part of the film a multinucleated leucocyte is seen. Fig. 2. — Pernicious Ansemia. The red blood corpuscles have an average diameter considerably above the normal, and Megalocytes are numerous. Poikilo- cytosis is pronounced. The corpuscles are deeply stained, and the central depression almost, if not completely, absent. Two large nucleated red blood corpuscles, Megaloblasts, are seen. / o PLATt IV. Fig ,1. '^^^^ ^~^"~^?\ /-%) r- ^ * ^ X, / w \ o N ^^ ^ ' a a ^ o o ^ - - ^ -^ -£? o ^,. o '^tf ft •' '■ / Cliloi-osis. Fig. 2 T.L^Colea del. Pernicio-us Anaemia West, Newman Chr. CHLOROSIS loi form occur most frequently in the smaller corpuscles present. Alterations in the size of the corpuscles are seen in many forms of chlorosis, and generally speaking the smaller corpuscles — microcytes — are more conspicuous than the larger elements, so that the average diameter of the red cells may fall from the normal 7*5 i». to 7 or 6*5 M- (Hayem.) Some writers consider this very characteristic of chlorosis. When the chlorotic condition is very severe large corpuscles — megalocytes — may be comparatively numerous. The colour of many individual corpuscles is per- ceptibly lessened, and the pallor of the red corpuscles, seen either in fresh preparations or in dry films, justifies Hayem's description of the disease as a chlorosis of the individual corpuscles. In stained films some corpuscles are much less deeply stained than others, whilst in some, signs of degeneration described by Maragliano and Castillino are also apparent. Normal blood corpuscles when fixed, stain pink with the eosine out of a mixture of eosine and methyl-blue, or eosine and haematoxylin — " monochromatophile " — whilst those corpuscles which are undergoing necrobiotic changes are coloured with both the eosine and haematoxylin, or methyl -blue, a purple colour, " polychromatophile." Examples of this may occasionally be seen in chlorosis. (Plate I., Fig. 6.) As to the occurrence of nucleated red blood corpuscles in cases of chlorosis, opinions differ considerably. Hayem, notwithstanding the opinion of other writers, has carefully looked for, but never discovered them, even in the most severe types of chlorosis, although THE BLOOD he mentions a case in which the corpuscles numbered only 937,360 per cmm. M.uir, in his earlier writings, states that he met with no nucleated red corpuscles in a long series of cases of chlorosis, in which all the elements were carefully enumerated. Kanthack observes that nucleated erythrocytes are rare in chlorosis, and are a sign of severe anaemia. According to Von Limbeck nucleated corpuscles occur in severe chlorosis, as they do in any other form of severe anaemia, occasionally in large masses, con- stituting, according to Neudorfer, a blood crisis, and their presence is often followed by an increase in the red cells. These are for the most part normoblasts, i.e., nucleated red cells of the same size as the healthy corpuscle. (Plate I., Fig. 7.) Stengel mentions the occurrence of normoblasts, which are said by some to indicate a favourable prognosis. He, however, admits that they are rarely conspicuous in cases of moderate severity, and are never marked except in the severest forms of the disease. Large nucleated red cells — megaloblasts or giganto- blasts- — have also been met with in this disease, but are never as numerous as normoblasts. In one case of chlorosis in a girl of 16, in whom all the typical symptoms of this disease were present, I found numerous nucleated red corpuscles, although the red cells present numbered 3*5 million per cmm. These were apparently absent from time to time, but when present were fairly numerous, so that in a thin film with f inch coverglass as many as 8 to 11 would be found. They were mostly normoblasts, and nearly all showed ex- tensive crenation and appeared less resistant to external CHLOROSIS 103 injury, as some free nuclei were also seen. In many the nucleus was pushed to one side, and in some almost extruded ; the protoplasm in a few stained purple with eosin and haematoxylin — polychromatophile. It is only right to mention that the patient was subject to slight attacks of bronchial asthma. We must conclude from the later writers, as well as from our own experience, that nucleated red blood corpuscles do occur in chlorosis, but probably only in the severer forms of that disease, and when present are usually of the normoblast variety. Their presence may indicate a severe type, but also one in which a reparative effort on the part of the haemopoietic organs, probably the bone marrow, is being made. I cannot but regard the megaloblasts, should they occur, as a sign of much more serious disturbance of the blood and blood-forming organs. The blood plates are generally somewhat increased in number, although in the very severe forms they may, according to Hayem, be diminished. The leucocytes are very little affected in uncomplicated cases of this disease, their number and variety being usually normal. In severe cases Hammerschlag has, however, found a few marrow cells. The exact nature of chlorosis is still undetermined. It is almost universally admitted that it is a primary anaemia, depending upon some disturbance of- the blood- forming organs, and not merely a symptom of some general disease. " The character of the blood, its poverty in haemoglobin, comparative richness in albu- minates, the marked renovating influence of iron, all indicate a defective haemogenesis." (Stephen Mac- kenzie.) That it is not due to an increased blood 104 THE BLOOD destruction — "haemolysis" — seems to be established by the absence of signs of blood destruction taking place in the body ; and hydrobilirubin or urobilin pigments, (resulting from the disintegration of red corpuscles), in urine and faeces, and marked degeneration of the corpuscles, as well as the presence of jaundice, are all wanting in chlorosis, although present in pernicious ansemia. What conditions give rise to the defective haemo- genesis, whether menstrual, nervous, or vascular derangements, or constipation attended with absorp- tion of toxic products from the intestines, are still unknown. Von Noorden and others maintain that want of nutrition from abnormal gastric digestion or diminished amount of nourishment cannot — -per se — account for the defective regeneration of the blood, since fasting causes neither oligocythemia nor oligochromaemia. Diagnosis. — The diagnosis of chlorosis must not depend on the examination of the blood alone, as the so-called chlorotic condition of the blood may occur in other forms of anaemia, and may occasionally be absent from chlorosis. Anaemia occurring in young females, attended with yellowish-green pallor, cardiac, digestive and menstrual disorders, which rapidly improves under suitable dietetic and ferruginous treatment, and in which there is marked reduction in the amount of haemoglobin, usually with, more rarely without, diminution in the number of red blood corpuscles, renders the diagnosis of chlorosis usually very easy. Sometimes secondary anaemias, resulting from malig- SIMPLE PRIMARY ANEMIA 105 nant disease, syphilis, loss of blood, etc., very closely resemble chlorosis, and here we have to rely on a careful consideration of the history, symptoms, and physical signs. The chlorotic type of the blood is not usually as marked in these conditions as in chlorosis, but we must insist on the fact that the description of the blood given above is not absolutely charac- teristic of, or peculiar to chlorosis. Leucocytosis generally occurs in secondary anaemia, but is absent from chlorosis, except when complica- tions are present. A very important point is the diagnosis of chlorosis from simple primary, and particularly pernicious anaemia ; but this will be considered later, when these diseases have been described. Simple Primary Ansemia. Many cases of anaemia originating without any dis- coverable pathological cause, and therefore primary, are met with, in which the essential features of chlorosis are absent, and which, under suitable hygienic and medical treatment, rapidly improve, and therefore can- not be included under the definition of progressive pernicious anaemia. Many writers refuse to consider this as a special type of anaemia, and we must admit the difficulty of distinguishing some cases, which are not typical, from the anaemia just described, and pernicious anaemia. This form of anaemia includes many cases which are due to unfavourable conditions, but it is somewhat difficult to say exactly what element, whether want of fresh air or light, is most concerned in its production. io6 THE BLOOD In the milder type of cases there is no evidence of blood destruction, and hydrobilirubin and jaundice are absent ; but some of the most severe forms, which border very closely on pernicious, show indications of disintegration of the red blood corpuscles, jaundice, etc. The number of red cells and the amount of haemo- globin usually diminish in the same proportion, but whilst this is true of the less severe forms, exceptions are found in the more aggravated types. Laache pointed out that sometimes the amount of haemoglobin \s lower than the number of corpuscles, whilst in cases of severe aneemia it is higher, and the individual cor- puscular value in haemoglobin, i.e., the colour index, may approach or even exceed the normal. Von Limbeck describes a case of severe simple primary ansemia occurring in a woman aged 40, who was living in the greatest misery, subject to hard work, and who had suffered for a long time from want of food. On admission the number of red corpuscles was 306,000 and the haemoglobin 18 %, whilst under treatment in hospital at the end of three months the corpuscles, reached 2,690,000 and the haemoglobin 60 %, when she left the hospital. She was readmitted a month later, and during this short time the corpuscles had fallen to 700,000, haemoglobin 26 % ; but at the end of six weeks' residence in hospital they numbered 4,280,000, and the haemoglobin 65 %. Jaundice and other signs of blood destruction were noticed during part of her illness. In this case the reduction in the number of the erythro- cytes was not always proportionate to the amount of haemoglobin. The number of red cells vary considerably from slight diminution to those cases, as the above, in which SIMPLE PRIMARY ANEMIA 107 there is an enormous reduction. The corpuscles may undergo alterations in size and shape, microcytes, megalocytes, and poikilocytes being often present. Hayem considers, as we have previously mentioned, that the predominance of small corpuscles occurs in slighter, whilst megalocytes belong to the severer forms of this anaemia ; and he divides cases into those in which the red corpuscles are small, but relatively numerous, (these are the least serious), and those in which they are on the average larger in size, but very reduced in number ; in other words, the average size of the corpuscles is inversely proportional to their number. The corpuscles and haemoglobin have probably a development independent of each other, and the in- crease in the number of corpuscles may be suspended whilst renovation of the haemoglobin is taking place, or vice versa. (Von Limbeck.) As signs of degeneration- are associated with those of regeneration, and as the degree of the ansemia fluctuates, so the microscopic condition of the blood varies from time to time. During regeneration nucleated red cor- puscles of the normoblast type appear. Megaloblasts, except in severe cases, are not numerous in this form of anaemia. According to Von Limbeck all these con- ditions may appear in any severe anaemia, and are therefore not characteristic of any one particular form. The blood plates are, especially in severe cases of primary anaemia, frequently increased in number. (Von Limbeck.) The leucocytes usually show no important or characteristic alteration, either in number or in quality. Commonly, according to Hayem, their number is some- what diminished, probably owing to the impaired io8 THE BLOOD function of the haemopoietic organs. Sometimes the number of lymphocytes, at other times the multinu- cleated leucocytes, are relatively increased, and the latter may occasionally contain a small quantity of haemoglobin in their protoplasm. Any conspicuous change in the number or nature of the leucocytes usually indicates the presence of some complication. The diagnosis of this form of ansemia is in some cases a simple matter. The fact that simple primary anaemia may occur at almost any age in either sex, that in typical cases the chlorotic condition of the blood is absent, and the haemoglobin and corpuscles are usually diminished in the same proportion, is sufficiently characteristic to distinguish it from chlorosis. The condition of the blood, and the improvement which takes place under efficient hygienic and medicinal treatment, generally aid in the diagnosis of simple primary from pernicious anaemia. Cases, however, may occur, in which it is impossible at once to say, even after a careful examination of the blood, as well as a clinical history of the patient, which of these forms we have to deal with. Though evidence of blood destruction is always present in true cases of pernicious, it is absent from any but exceptionally severe forms of simple primary anaemia. The prevalence of megalocytes, and particularly the presence of a large number of megaloblasts, are in favour of pernicious anaemia, especially when associated with increasing oligocythemia and a general downward tendency of the case. Improvement under treatment, especially if per- manent, indicates simple primary anaemia. PROGRESSIVE PERNICIOUS ANEMIA 109 Progressive Pernicious Anaemia, Synonyms. — Primary, essential or idiopathic anaemia ; Addison's anaemia ; Biermer's disease ; anaematosis. (Pepper.) Although this disease had been previously recognized by Andral and others, it was not till 1843 that Addison accurately described cases of anaemia occurring without any discoverable cause, and resulting almost invariably in death, under the term idiopathic anaemia. Later, Biermer in 1868 drew attention to the existence of severe anaemia, and more fully, in 1872, to some of its pathological characters. He named it progressive per- nicious anaemia, and included severe primary anaemia as well as those secondary to pregnancy, lactation, and haemorrhage. Nearly all the earlier writers recognized its fatality, but differed considerably as to its being primary, i.e., without a recognizable cause, or secondary to other minor pathological conditions. Thus Eichhorst, who collected all the literature relating to it up to 1877,^ divided cases into those which are essential, primary, or idiopathic, (of these he counted seventeen), and those which were secondary to other causes. In the latter group he included twenty-nine cases in which the anaemia followed pregnancy, ten after confinement, twenty-four from affections of the alimentary canal,, seven from loss of blood, and seven were attributed to bad hygienic surroundings. He defined pernicious anaemia as a condition of severe anaemia, which defies all treatment, proceeds without being checked, and usually ends in death. no THE BLOOD Hayem describes it as a morbid condition, charac- terised by extreme anaemia, which most frequently ends fatally. " Thus the element of fatality seems to be regarded as the most important feature, and the absence of cause became of secondary importance. Now, however, there seems to be a tendency to attach more weight to the absence of apparent cause, and to consider those cases only to be pernicious anaemia in which no such cause can be recognized. But even in such cases of fatal anaemia as are preceded by some definite lesion which may rank as a cause, there may remain much to explain if the supposed cause, as sometimes happens, ceases to operate long before death, and only leaves the patient in a condition which never improves, but gradually — it may be in the course of months — deteriorates until death ensues. Thus, it seems to me, it will be too great restriction of the term to apply it only to ap- parently quite idiopathic cases, and it ought to include, or at any rate we must still recognize as very obscure, those cases in which a temporary cause is followed by a persistent anaemia with a fatal tendency." (Taylor, Brit. Med. Journal, 1896, Sept. 19, 719.) Modern English writers are still at variance as to what constitutes the nature of pernicious anaemia. Dr. Stockman maintains that pernicious anaemia is not a disease in itself, but a symptom of several ex- hausting conditions, which induce an initial anaemia followed by degenerative changes in the vessels, result- ing in capillary haemorrhages, and these cause the excessive anaemia. Dr. William Hunter holds that pernicious anaemia is a specific, definite disease, in which the blood is PROGRESSIVE PERNICIOUS ANEMIA iii destroyed in the portal circulation, and excess of iron is deposited in the liver as a result of this haemolysis. From the condition of the blood and clinical aspects of the disease we shall consider it as a disease arising in most cases without any determinable cause, some- times from insignificant causes, and characterised by extreme anaemia and marked changes in the blood, which almost invariably terminates in death. Condition of the Blood. The blood usually flows freely from a puncture, but sometimes, especially in emaciated subjects, in whom the tissues are dry and the peripheral circulation diminished, it is somewhat difficult to obtain. It appears remarkably thin and watery, and is generally very pale, sometimes yellowish in colour. It has been described as of a dirty pinkish-red colour, resembling the washings of a vessel which had contained blood, or the water used in washing meat, an appearance said to be very characteristic. Exceptionally it has been noted of a reddish-brown or coffee colour. So extremely thin and fluid is the blood that I have frequently noticed a difficulty in obtaining a drop large enough to suck up into the Thoma-Zeiss pipette, or to fill the automatic capillary tube of Von Fleischl's haemometer, as the blood from the puncture tends to run or spread out, and seldom forms a large globule. Films of blood from a case of pernicious anaemia are frequently very thin, as the cellular elements are so scanty, and it is well, particularly with a view to the differential count of the leucocytes and the examina- tion for nucleated red cells, to spread the films rather 112 THE BLOOD thicker than usual, and to hasten their drying by moving' them rapidly about in the air, or as some suggest by slightly heating them. On standing, the corpuscles in a drop of blood soon separate and fall to the bottom, leaving clear serum above. The blood has apparently lost its cohesiveness, rouleaux formation is absent, or when present defective, and coagulation is slow — a condition resembling, accord- ing to Hayem, a certain degree of haemophilia, and is found in all severe anaemias. The specific gravity is usually lower than in the normal condition. If a drop of blood be examined in the fresh condition, taking care that the film is not too thin, characteristic alterations are seen. The corpuscles group themselves into little masses composed of two to six elements, which, owing to their great dissimilarity in size, give the rouleaux a very irregular appearance, or they lie isolated in the field. The red blood corpuscles appear unequal in size and are deformed. Many giant cells — megalo- cytes — are conspicuous in each field, often associated with the presence of microcytes. The numerical decrease of the red cells is extra- ordinary, in fact usually under no other conditions is this alteration known to be so excessive. Even when the disease is first apparent the corpuscles number only 2\ million, more frequently \\ million, and usually when the disease is pronounced they frequently fall considerably below one million, and towards the end they may sink below 500,000 per cmm. Lepine records a case in which the corpuscles fell to 378,000, Muller one in which they numbered 360,000, and Quincke mentions a case in which they were only PROGRESSIVE PERNICIOUS ANMMIA 115 found to be 143,000 per cmm. This is certainly the lowest on record. Temporary increase in the number of red cells may occur several times in the course of the disease, but relapses as frequently follow. During periods of temporary improvement, the corpuscles may even reach the normal number. Thus one case is mentioned by Dr. Gibson in which at the commencement, the cor- puscles numbered 500,000 to 600,000 ; under treatment they rose to over five million, and the patient was dis- charged from the hospital in apparent health, only to return in a few weeks feeling unwell. He died within a week after readmission. The haemoglobin is always greatly reduced in amount, though usually not to the same extent as the corpuscles, so that the individual corpuscular value in haemoglobin is usually greater than normal, varying, according to Hayem, from o"88 to 17, the normal being I'o. This is due to the fact that the corpuscles, though enormously reduced in number, are well coloured, and their average size considerably increased. It is said that Von Fleischl's instrument is inaccurate for determining the small amount of haemoglobin present in this disease. In one case I found the haemoglobin= 15 %, and in order to satisfy myself, after cleaning the instrument, I filled two instead of one of the capillary tubes with blood, and found the reading for the two exactly equal to 30 %. This apparently proved that error, if present, is unimportant. Laache and Hayem concluded that the characteristic feature of the disease, consisted in an increase in the size of, and the relative excess of haemoglobin in, the cor- puscles. Nearly all writers agree that this condition is I 114 THE BLOOD very characteristic of pernicious anaemia, although von Limbeck remarks that the apparent richness of the red corpuscles in haemoglobin, advanced by Laache as pathognomonic of this disease — a statement which he says is unjustly found in text-books — is met with in nearly all cases of severe oligocythemia. The alterations in the size of the blood corpuscles is a conspicuous feature in pernicious anaemia, the average diameter of the elements being considerably increased. (Plate IV., Fig. 2.) It was previously mentioned that in health large cor- puscles, measuring 8 "5 m in diameter, are found, consti- tuting about 1 2^ % of all the red cells ; but in this disease giant cells — megalocytes — measuring upwards of 8 '5 /x, occur. These are never found in health, with the exception of the newly-born, and belong exclusively to pathological conditions. (Plate I., Fig. 4.) Laache found the average measurement of the mega- locytes about 11-13 M, but has seen some as large as 15 M- Hayem's results closely correspond with those of Laache. He states that the giant cells usually measure about 9"5 to 12 yu. The most numerous are about 10 m ; those of 12 yu are rarer, but in exceptionall cases he found them as large as' 14 m and even 16 /x in diameter. Megalocytes are not, however, numerous, seldom con- stituting more than 3 to 1 2 % of the red corpuscles ; so that increase in the average diameter of the elements is due to the prevalence of large-sized cells, not exceeding 10 /«. They are frequently oval or pear-shaped. Small or dwarf corpuscles — microcytes — occur in this disease, but they are by no means as conspicuous or as characteristic as the megalocytes. They vary in size from that of a blood plate to an erythrocyte, but are PROGRESSIVE PERNICIOUS ANEMIA 115 usually about 3 to 4 /« in diameter, and generally have about the same colour as the ordinary red corpuscles. Eichhorst thought that the occurrence of small round corpuscles, measuring about 3 or 4 />i, conspicuous on account of their deep colour and high refractive index, peculiar to idiopathic anaemia. " Eichhorst's corpuscles," as they are called, are, however, met with in other forms, and are frequently absent from pernicious ansemia. (Plate I., Fig. 3.) They are of no diagnostic value whatever. Muir noticed their absence from nine cases of pernicious anaemia which he examined. He mentions that in one case of this disease in which the corpuscles were much below one million, the red cells were practically of the same size, and considerably larger than the normal, having a diameter of 9 /i. Changes in the form of the coloured elements are commonly seen in this form of anaemia. Poikilocytes are very frequent, and Quincke, who first drew attention to them, thought characteristic of pernicious anaemia. Many of these are oval or fusiform, as if one end had been drawn out into a point ; others are lanceolate, with one end broad and round, the other tapering. (Plate I., Fig. 5.) In some the outline is irregularly indented or broken ; others are stellar or fragmented ; whilst many are so irregular as to defy description. They vary in size from a blood plate to a figure measuring 15 m or more in its greatest diameter. These changes in the outline of the erythrocytes are probably degenerative signs, constituting that variety described by Maragliano and Castellino as " total necrosis." The colour of the individual corpuscles varies con- siderably. The megalocytes are often pale in colour. ii6 THE BLOOD with an absence of the central depression or concavity, although the central portion may appear somewhat faintly stained with eosine, owing to the diminution of the haemoglobin in this part of the cell. Frequently, however, the giant corpuscles are somewhat deeply coloured, with no diminution in the colour of their centre. (Plate IV., Fig, 2.) The smaller cells often appear rich in colouring matter, and although the smallest poikilocytes are pale, yet the larger forms are frequently deeply coloured, except in their projections. Many writers point out that the cell depression or concavity is either entirely wanting, or only partially present, in many corpuscles. Very faint, almost colourless red cells, termed "shadow corpuscles," are met with in pernicious anaemia, but are not usually abundant. (Plate I., Fig. 6a.) They are sometimes only recognized by a pale circle of colour. According to Stengel, faint granular matter may occasionally be seen within them, or near their periphery. In films stained with eosine and haematoxylin, most of the corpuscles, some with, many without a central concavity, are stained a pink colour of varying intensity according to the amount of haemoglobin contained ; a few of them, however, especially the larger megalocytes, are coloured partly with the eosine, partly with the haematoxylin, a purple colour, i.e., Polychromatophile — a sign, as was previously mentioned, of degenerative or necrotic alterations in the corpuscles. This is often seen in the protoplasm of the nucleated red corpuscles, especially in that of the megaloblasts. (Plate I., Figs. 6 and 8.) Other signs of degeneration — the endoglobular, necrotic changes of Maragliano, may be met with, in PROGRESSIVE PERNICIOUS ANEMIA 117 which decolorization occurs in the centre of, or in patches in the cell, forming vacuoles and finally general granular disintegration of the interior of the red cells. Amoeboid movements of some of the poikilocytes, and other red corpuscles, have been observed by several authorities, and Hayem has drawn attention to the occurrence of rapid movements on the part of some of the very small microcytes, which have assumed the shape of little rods, closely resembling, and are said to have been mistaken for, bacteria. Nticleated red blood corpuscles or erythroblasts are usually, though not constantly, present in the blood of cases of pernicious anaemia. Stengel speaks of them as a constant feature in this disease, although varying considerably in number. Hayem mentions their frequent occurrence towards the end of the disease, but remarks that they are not present in every case, and notes their absence in one out of three fatal cases which he has fully described. Muir found them in four out of eleven cases, and remarks that " the two patients in whom they were most numerous, ultimately repovered, whilst in cases of most extreme ansemia, they were absent before death." He mentions the fact that " Ehrlich found them absent before death, in a case in which the cor- puscles had fallen to 213,360 per cmm. Limbeck states that when, at least partial, regenera- tion occurs, nucleated red cells appear. These may occur as normoblasts, i.e., nucleated red cells about the size of an ordinary erythrocyte, or smaller as microblasts, or larger as megaloblasts. When a poikilo- cyte contains a nucleus, it is spoken of as a "poikiloblast." The normoblast usually measures 7*5 to 8 ^, but it never 11 8 THE BLOOD exceeds 9*5 jx. The nucleus which is of small size is always stained deeply with haematoxylin. It is generally situated towards the periphery of the cell. It frequently shows division into two or three smaller parts, and occasionally, though extremely rarely, karyokinetic figures may be found. (Plate I., Fig. 7.) I would add, a point not usually emphasized by most writers, that frequently free nuclei, easily distinguished from other elements by their small size and deep-staining reaction, are met with in blood films of cases of per- nicious anaemia. These are no doubt derived from the normoblasts. Von Noorden describes the occurrence of blood crisis in this disease, i.e., the occasional appear- ance of a large number of normoblasts, followed in a few days by a marked increase in the red blood corpuscles. Microblasts, very small nucleated red cells, may be found in this form of ansemia, but they are very in- frequent. (Plate I., Fig. 7d.) Megaloblasts are considerably larger than an ordinary erythrocyte, being nearly always over 10 i*., and often 14-16 M in diameter. The nucleus, which is usually more centrally placed, is larger, but not nearly so deeply stained as that of the normoblast, and when found in a cell, whose protoplasm shows polychroma- tophilic degeneration, is somewhat indistinct. (Plate I., Fig. 8 ; and Plate IV., Fig. 2.) It is impracticable to estimate the number of nucleated red cells present in the blood. In some cases they appear extremely numerous, in others they require considerable looking for. Approximately their number per cubic millimetre may be determined by, first, ascertaining the number of white PROGRESSIVE PERNICIOUS ANEMIA 119 cells per cmm. by the Thoma-Zeiss pipette ; and, second, by finding how many nucleated red cells are present in a large number of leucocytes counted on the dry-stained preparations. The formula for the calculation will be : — No. of leucocytes per cmm. x number of nucleated red corpuscles counted in the dry film. No. of leucocytes counted in dry film. For example, in one of my cases the leucocytes numbered 1944 per cmm. In films stained with eosine and haematoxylin I found 2 nucleated red cells whilst counting 1000 white cells. Therefore "^^ooo^ = 3'8 nucleated red cells per cmm. I agree with many observers that the megaloblasts are more characteristic, and more important as a prog- nostic sign, than the normoblasts. The Blood Plates are, according to Hayem, Muir, etc., usually diminished in number. Other writers. Limbeck, Stengel, etc., maintain that they are not diminished, but rather increased, and are found grouped together in thick masses. I can confirm the latter statement. The Leucocytes, like the blood plates, undergo un- important alterations in this disease. Von Limbeck considers that the white corpuscles in this, as in the greater number of severe anaemias, are not increased in number. Hayem indicates that they are usually diminished, that the uninucleated forms are most numerous, and that some of the leucocytes show vacuolation of their protoplasm, whilst others contain a small amount of haemoglobin. THE BLOOD Stengel remarks that in the earlier stages they may- be diminished, although they are more frequently normal, or even increased ; and points out that before death there may be a marked terminal leucocytosis. Most observers are of the opinion that the number is somewhat diminished, and that the small uninucleated elements or lymphocytes are usually relatively greatly increased. Many recent writers mention the presence of a small number of myelocytes or marrow cells, and I have found them occasionally in severe cases of pernicious anaemia. Nature of the Disease. The majority of observers have agreed that pernicious anaemia depends on excessive blood destruction, rather than defective blood formation ; and that this haemolytic nature of the disease, so ably emphasized by Dr. Hunter, is supported by the occurrence of ferruginous blood pig- ments in various organs, the presence of jaundice, an excess of urobilin in the urine, and the occasional occur- rence of haemoglobinaemia. Where this destruction takes place, whether in the portal circulation, liver or spleen, or what exact condition gives rise to it, is not yet ascertained. Many consider it to be due to intestinal auto- intoxication, and bring forward the fact that cases of severe anaemia have been benefited by intestinal antiseptics, and that indican has been found in excess in the urine, and occasionally peptonuria, cadaverin, and other products of intestinal putrefaction. The changes which occur in the bone marrow in pernicious anaemia are essentially a return from the PROGRESSIVE PERNICIOUS ANEMIA 121 adult condition to that of the embryo ; the fat being displaced by red marrow, containing a large number of nucleated red cells, and also megalo- or giganto- blasts, which are not normally present. The marrow changes, probably secondary and compensatory in nature, help to somewhat explain the condition of the blood in this disease.* The Diagnosis. — In some cases the diagnosis may be comparatively easy, frequently, however, it is extremely difficult, and often impossible without an examination of the blood by means of dry-stained films. The clinical history, and the results of a careful physical examination, must in all cases be taken into consideration. The following clinical features are very suggestive of the presence of this disease : — The absence of any evidence of organic disease, the severe progressive anaemia, the inefficiency of ferruginous treatment, the peculiar, flabby condition of the subcutaneous tissue, and the absence, in most cases at least, of emaciation, especially if associated with extreme weakness, lemon- yellow complexion, and the occasional occurrence of slight pyrexia and haemorrhages, particularly retinal. * In a post-inortem examination of a case of pernicious ansmia in a man aged sixty-three years, I found all the characteristic alterations in the bone marrow described by Muir. One inch of the middle third of the shaft of the femur was removed, and it was seen that the yellow fat of normal marrow was entirely replaced by a deep blood-red marrow having the appearance of recently coagulated blood. The medullary canal of the bone was enlarged. Microscopically — in films as well as sections — this red marrow consisted in a great increase in the number of nucleated red corpuscles, many of which were of enormous size. The nuclei of these megaloblasts were single, double, or fragmented. The non-nucleated red corpuscles present showed all the alterations found in the blood of pernicious anaemia, viz., megalocytes, poikilocytes, and various forms of cells undergoing degeneration. THE BLOOD The blood should be carefully examined, and will in many cases clear up the diagnosis. There is, however, in the blood, no one absolutely pathogno- monic sign, although the occurrence of several altera- tions just described are together very significant. The enormous decrease in the number of red corpuscles, seldom occurs as constantly in any other condition as in progressive idiopathic anaemia. After severe haemorrhage, and during the course of malignant diseases, the corpuscles may, however, sink below one million, but this reduction is exceptional. The relative increase in the amount of haemoglobin, combined with marked oligocythaemia, is often a striking feature, but it is not pathognomonic, as some undoubted cases of pernicious ansemia have been described, in which the haemoglobin had undergone a greater re- duction than the number of corpuscles ; whilst in other severe cases of anaemia — not pernicious in nature^the high individual corpuscular value in haemoglobin has been noticed. Poikilocytosis is of very frequent occurrence in all varieties of anaemia, and I have noticed it, certainly in a slight form, in very moderate degrees of oligocythaemia. The very large irregular poikilocytes are of more significance, and the presence of these, with comparatively large numbers of megalocytes, megaloblasts, and particularly poikiloblasts, is very strong evidence of this disease. Generally speaking, nucleated red corpuscles are more frequent in this than in any form of ansemia, with the exception only of leucocythaemia ; and of the three varieties the megalo- or giganto-blasts are of the most diagnostic importance. PROGRESSIVE PERNICIOUS ANEMIA 123 Megaloblasts, associated with the presence of normo- blasts, do occur in other diseases, but in practically no other than pernicious anaemia are the former more numerous than the latter. The peculiar staining reaction — polychromatophile — of some of the red blood corpuscles is of little diagnostic value. Von Limbeck regards the occurrence of signs of rapid destruction of the blood cells, which is shown in the marked degeneration of the corpuscles, the excess of hydrobilin or urobilin in the urine, and the evidence of slight jaundice, as the only condition which distinguishes pernicious from any other severe anaemia. From Chlorosis. — Pernicious anaemia can usually be easily distinguished from chlorosis, in that the former generally shows great diminution in the number of the corpuscles, and a relative increase in the amount of haemoglobin, i.e., the individual corpuscular value is above the normal, whilst the latter is characterized by relatively moderate oligocythaemia, but great reduc- tion in the haemoglobin, so that the corpuscular value is below the normal. The corpuscles in pernicious anaemia are generally larger than in health, megalocytes and large poikilocytes being frequently seen, and nucleated red corpuscles are almost always present. The megaloblasts are more numerous than the normoblasts. (Compare Fig. i and Fig. 2, Plate IV.) In chlorosis the corpuscles are, on the whole, some- what smaller and paler than in the normal ; they do not show such marked alteration in their form ; poikilocytes, though commonly present, are not as irregular; and nucleated red blood corpuscles only 124 THE BLOOD occur in severe forms, and then are usually of the normoblast type. Megaloblasts may occur in ex- ceptionally severe cases of chlorosis, but are never more numerous than the normoblasts. Marrow cells are more frequently met with in per- nicious anaemia than in chlorosis, but in neither disease are they ever numerous. From Secondary Ancemia, especially that resulting from malignant disease and haemorrhage. Cases of severe anaemia, particularly those resulting from malignant disease, e.g., carcinoma of the stomach, may frequently present clinical features closely re- sembling pernicious anaemia, but the examination of the blood will usually render their distinction possible. In secondary anaemia the red blood corpuscles are seldom below one million, and oligochromaemia is generally more pronounced than oligocythaemia, so that a chlorotic condition of the blood is found. The eryth- rocytes are usually smaller in size and paler in colour. Nucleated red cells are not infrequently present, es- pecially in the severer forms, but they are principally of the normoblastic type. In those exceptional cases in which the corpuscles are very considerably reduced, leucocytosis, usually involving an increase in the multinucleated cells, is almost invariably present. In pernicious anaemia, on the other hand, the reduc- tion of the red cells is always extreme, and oligocy- themia is in excess of oligochromaemia. The corpuscles exceed the average in size, and are usually well coloured. Megaloblasts are present, and generally form the greater number of nucleated red cells. The leucocytes are diminished, and there is usually a relative increase of the lymphocytes. PJiOGRESSIVE PERNICIOUS ANEMIA 125 The following case illustrates the improvement, too often only temporary, which may take place in the course of Pernicious Anaemia under suitable treatment : Woman, aged 47, presented all the clinical features. of the disease, viz. : lemon-tint complexion, slight jaundice, bile pigments in the urine, progressive weak- ness without emaciation, and irregular pyrexia. She complained of loss of strength, shortness of breath, and buzzing or swimming in the head. Physical examination revealed no cause for the condition. The Blood appeared of a dirty colour. The red cor- puscles numbered 914,583 per cmm. ; their average size was increased, and megalocytes were very numerous. Poikilocytosis and other degenerative alterations in the red cells, e.g., vacuolation, crenation, and polychro- matophilia were extremely frequent. Nucleated red corpuscles were fairly numerous, and at first the megaloblasts were considerably in excess of the normoblasts. The haemoglobin was about 15 %. The leucocytes were diminished, numbering only about 19 14 per cmm. The lymphocytes were rela- tively greatly increased, whilst the multinucleated and eosinophile cells diminished. A few myelocytes, or marrow cells, were seen. The following table shows the progress which took place under treatment with bone marrow, etc. The increase in the corpuscles was attended with a diminution and ultimate disappearance of the megalo- blasts, and latterly all forms of nucleated corpuscles were absent. At the last examination the red cells numbered 4^ million, but their average diameter was greater than the normal. 126 THE BLOOD < CO o u w p-l fa o w < u O _>^ "3 H :z; w IN s OJ H 3 < H-, w pi! ,cl H « O ^ 1 — 1 psi CI O VO o 6^ ° 3-S VD ft vo O ^ -T "? O O "^ m CO bJ t-i > in li-l u CO r^ o u t3 w K-l m >j^ w % o Tf w « ffi H - fe n ^ M H ■^ ^ Z C3 O U , 1 m < ro m ■^ H 2; : : w p^ M m in t^ [n ^ m ^ o lx< CO W u c/2 t3 Pi o u o o o p; Q W PJ Q W H f 5 fe *> • "■---^ ,^a •» ^ w ^ v,.i.„- y Normal Marrow. Weat,M3virir.a,n Chr SPLENO-MED ULLAR Y LE UCOCYTHMMIA 1 33 seen in the marrow of a kitten, prepared by the method previously mentioned ; and if with this some of the blood of the animal be mixed, a condition resembling the spleno-medullary form of the disease may be seen. (Compare Figs, i and 2, Plate V.) Myelocytes, or Marrow Cells ; synonym : Leucocytes hypertrophies (Hayem). Large uninucleated leucocytes, having distinctive staining reaction, are the most characteristic elements in this form of leukaemia. They are very large cells, measuring sometimes as much as 16 m. or even 20 m in diameter, and generally having a more or less circular or oval outline. (Plate II., Fig. 6.) Some of the marrow cells are very small, not much larger than a red corpuscle, and indistinguishable from the large or small lymphocytes, except that they are the only form of white cell present in the blood, which has a single nucleus, and whose protoplasm contains fine granules which stain with Ehrlich's neutral mixtures. (Plate III., Fig. 5.) All forms of the myelocytes possess no power of amoeboid movement. The nucleus is of large size, and although lobed, it is nearly always single, and does not show the deep indentations of the polymorphous leucocytes. It is usually round or oval, sometimes indented at one side, or horse-shoe or kidney shaped, and stains faintly with nuclear stains. The nucleus is poor in chromatin, but a fine irregular network may in some cases be seen. It occupies a somewhat eccentric position, although it may sometimes be in the centre of the cell. The surrounding protoplasm is abundant, and con- 134 THE BLOOD tains very fine granules, which were formerly said to be only neutrophile, as they stain with Ehrlich's neutral stain, but are now generally considered to be also finely oxyphilous or eosinophilous. Myelocytes may in the same film contain neutrophile and fine oxyphile granules, and the latter may show all gradations, up to coarsely granular oxyphile or eosinophile cells. (Plate II., Figs. 6, 7, and 8.) In specimens stained with eosine and haematoxylin, the protoplasm is almost invariably tinted blue with haematoxylin, and the granules are not discernible even with the high power of the microscope, but these are clearly demonstrated by using one of the triple stains of Ehrlich. (Plate III., Fig. 5.) Marrow cells may be easily distinguished from lymphocytes and multinucleated leucocytes, by their large size, faintly stained large nucleus, and absence of amoeboid movement ; and typical examples are easily separated from the large uninucleated or hyaline cells ; but sometimes even when stained with the triple stain, it is extremely difficult and to me occasionally impossible to say whether the leucocyte is a large hyaline or a marrow cell. Some of these elements may show evidence of karyokinetic division, especially when the blood film has been previously fixed with Flemming's solution. (Plate II., Fig. 9.) Marrow cells were till recently thought to occur only in leucocythaemia, and von Limbeck states " that their presence in other diseases, though frequently asserted, is even now doubtful." It is now known that they are occasionally present in very small numbers, as we have mentioned before, in SPLENO-MED ULLAR Y LE UCO C YTH^MIA 1 35 pernicious and some chronic ansemias and wasting diseases, and I have found elements closely resembling them in the blood of two cases of myxoedema in 1892. Zappert is said to have met with them in some cases of skin diseases, and Neusser after an attack of uraemia. These statements do not diminish their importance and diagnostic value in leukaemia, as in no other do they ever reach the relatively large number that they attain in this disease. In spleno- medullary leucocythaemia they generally constitute about 30 to 50 % of all the leucocytes present, whilst in other diseases, in which they sometimes occur, they never, according to Cabot, exceed 9 % and usually not2%. _ In three typical cases of this form of leukaemia, I found that the marrow cells constituted 37 "2, 54*5, and 46 % respectively of all forms of leucocytes present. Mosler, who first discovered this form of leucocyte, found them in the marrow obtained from a puncture of the sternum, and their origin from, and identity with, the marrow cells of the bone, have been universally con- firmed. The cells described by Cornil and Ranvier as "cellules mddullaires" and " medulocelles," were proved by Miiller to be identical with these leucocytes. Eosinophile or Coarsely Granular Oxyphile Cells. Three forms of these leucocytes may be recognized in leucocythaemia. ist. The ordinary eosinophile cells of normal blood (Plate II., Fig. 5, Plate III., Fig. 4), spherical cells with usually two nuclei connected by a band of chromatin, placed somewhat eccentrically in the protoplasm, and surrounded by, and embedded in a 136 THE BLOOD mass of coarse, highly refractile granules, which stain deeply with the acid aniline dyes, e.g., eosine aurantia, etc. Their nuclei stain fairly deeply with haematoxylin. These cells have an average diameter of about 10 to 1 1 M- In three cases, I found that the ordinary form of eosinophile cells, constituted 43, 13, and 38% respect- ively, of all the eosinophile cells present. 2nd. Small eosinophile cells deeply stained with eosine, which do not occur in the normal blood are extremely rare, if they ever occur, in any other disease. They are about the size of a red blood corpuscle or even smaller. In the same three cases I noticed that the relative number of small eosinophile cells varied considerably. In one they counted 2 %, in another 7 %, , and in the third case 6 % of all forms of eosinophile cells. 3rd. Marrow cells containing eosinophile granules or eosinophile myelocytes. These never occur in health and probably in no other disease, although it is stated that they are occasionally seen in very small numbers in pernicious anaemia. (Plate II., Fig. 7, and Plate III., Fig. 6.) They exactly resemble the ordinary marrow cell, but have coarse eosinophile instead of fine neutrophile (or oxyphile) granules. All stages from the finely granular oxyphile to the coarsely granular eosinophile myelocytes may be seen. (Plate II., Figs. 6, 7, and 8.) Their large nucleus is always single, often indented at one side, and usually in contact with the wall of the cell at one part. It stains faintly with haematoxylin, and is poor in chromatin. Karyokinetic division of the nucleus can occasionally be seen. These elements are of very large size, measuring often 13 m and SPLENO-MED ULLAR Y LE UCO C YTH^MIA 1 3 7 sometimes 16 or 18 /u in diameter. I have frequently noticed the presence of large vacuoles in the leucocytes, especially in the marrow cells or eosinophile myelocytes. The eosinophile myelocytes, which are identical with the form found in normal marrow, are the most numerous of all the eosinophile leucocytes, and together with the ordinary myelocyte, are the most characteristic of all the elements present in the blood of leucocythaemia. I found that in three cases of this form of the disease, they numbered respectively 55, 80, and 56 %, i.e. an average of 63! % of all the eosinophile cells. The nature of the granules present in these three forms of eosinophile cells is still a matter of uncertainty. They were formerly regarded as fat granules, and Litten and Miiller are stated to have stained them black with osmic acid, but it has not yet been proved that every- thing blackened with osmic is necessarily of the nature of fat. The majority of observers regard them as more of the nature of an albuminoid material. The absolute number of eosinophile cells is always increased, but the relative number is sometimes above, sometimes below the normal of 2-4 %. Von Limbeck states that their relative number compared with the other forms of leucocytes is not raised, and mentions that Zappert found in ten cases of leukaemia not more than 8 % of eosinophile cells, a proportion which he says is often exceeded in health. [Von Limbeck and Zappert give a higher percentage for the eosinophile cells in health than other observers, their average being from o"6 to 1 1 %.J I counted the relative number of all forms of eosinophile cells in three cases of this disease, and found that in one patient — a child of 1 1 (and it is well known that these cells are easily increased in child- 138 THE BLOOD hood even in health) they were equal to i\\%, but ultimately fell to 7 %. In another, that of an adult, the average was 5 %, whilst in a third they only reached 3 % of all the white cells present. Many, particularly the earlier writers, considered that a relative increase in these cells was a characteristic sign of leukaemia, but this is certainly not the case, as frequently they are diminished, whilst in asthma, skin diseases, and a number of other pathological conditions they may be markedly increased. Mere numerical increase in the proportion of eosino- phile cells is of no assistance whatever in the diagnosis of the disease, but qualitative alterations ■ — more especially the appearance of eosinophile myelocytes — are of the highest importance. Multinucleated or Neutrophile Leucocytes. The ordinary multinucleated cells are seen in leu- kaemia, but are not such conspicuous features on account of their size, as the marrow cells. (Plate II., Fig. 4, and Plate III., Fig. 3.) They are increased in absolute number, but are relatively less numerous than in health, constituting about 50% of all the white cells present. In some cases they only number about 20 or 10%, sometimes even less. In appearance they differ from each other in size, staining characters and the shape of their nuclei, and are not so regular as in health, or even in leucocytosis. In most the protoplasm is faintly but uniformly stained with eosine, whilst in a few it is quite colourless, taking on neither the eosine nor haematoxylin, and the latter frequently show very irregular nuclei, suggestive in some SPLENO-MEDULLARY LEUCOCYTH^MIA 139 cases of mitotic division. Not infrequently very small multinucleated leucocytes are seen, not much larger than an ordinary red blood corpuscle, with the protoplasm darkly stained. In many of these cells the amount of chromatin in the nucleus is diminished and the intra- nuclear network is not nearly as well seen, as in the ordinary multinucleated elements, and in some, the distinction from marrow cells is almost impossible. Sometimes the fine granules appear to be distinctly more like fine eosinophile than neutrophile, and usually they are less pronounced than in normal blood. The multinucleated leucocytes in three of my cases constituted 43*3, 35*5 and 48"3 %, an average of about 42 % of the leucocytes present. A marked increase in the relative number of multi- nucleated elements occurring in the course of the disease,, would suggest the presence of some inflammatory complication. Small Uninucleated Leucocytes or Lymphocytes. These forms — small elements about the size or slightly larger than the red blood corpuscles, with very deeply stained nucleus, surrounded by a small border of non- granular protoplasm, are greatly reduced in their relative number. In three cases I found that the lymphocytes constituted only i^\, 5 and 2f % respectively of all forms of white cells, instead of the normal 15 to 25%: In appearance these elements have practically the same characters as those occurring in health, and also occa- sionally contain fine basophile granules. (Plate II., Fig. I, Plate III., Fig. i and Fig. 7.) Fraenkel drew attention to larger forms of lympho- 140 THE BLOOD cytes, having a large, sometimes deeply indented, or even polymorphous nucleus, poor in chromatin, which he ■considered were of lymphatic origin. Mitosis is seldom seen in the small uninucleated elements. Large Uninucleated Leucocytes — Hyaline Cells. These are probably mature forms of the small lym- phocytes, and in health contain no granule in their protoplasm. (Plates II. and III., Fig. 2.) The nucleus is large, single, or indented, poor in chromatin, and stains faintly with nuclear stains, and shows indistinct signs of an intranuclear network. The protoplasm stains very faintly with eosine, and usually takes on some of the stain of the nucleus, appearing of a very faint bluish pink with eosine and haematoxylin. In the spleno-medullary form of the disease they are of very little importance, being usually relatively diminished. Transitional Leucocytes. These are intermediate between the large lympho- cytes and the multinucleated leucocytes, differing from the former by the irregularity of the nucleus, which tends to become multipartite, by the presence of a larger quantity of chromatin and a more distinct intranuclear network. (Plate II., Fig. 3.) As we have previously mentioned, it is difficult, frequently impossible, to dis- tinguish between these different forms of cells, as all intermediate types are met with. Basophile Cells. In films stained with methylene blue alone, some of the small leucocytes, as in health, contain fine basophile SPLENO-MEDULLARY LEUCOCYTH^MIA 141 or 5 granules, but in addition to these many large marrow cells have these granules. Atypical basophile cells are very commonly met with in spleno-medullary leucocythaemia. (Plate III., Fig. 7.) Buchanan has pointed out the occurrence of mixed granules — basophile and finely granular oxyphile — in the same cell, and this, he says, is particularly notice- able in the large uninucleated or marrow cells. It seems that myelocytes may be rr^et with containing neutrophile, basophile, fine oxyphile, and coarse oxyphile or eosinophile granules, and all of these forms I have frequently seen, but so far have been unable to confirm Buchanan's interesting statement of the occurrence of mixed granules. " Mastzellen," or cells containing coarse basophile or y granules which stain deeply with basic stains, are said to occur in leukaemia. (Plate III., Fig. 8.) They are, however, as von Limbeck states, seldom found in the spleno-medullary form of the disease, and their presence is by no means characteristic, as they have been seen in the blood of patients suffering with hereditary syphilis. Mitotic or Karyokinetic Division of the Nuclei. In films of blood quickly prepared as we have de- scribed, i.e., by fixing for a short time in a mixture of ether and alcohol, and staining in eosine and haema- toxylin, occasionally all stages in the division of a leucocyte may be seen. This is more clearly demon- strated in films specially prepared by Flemming's, Muir's, or Muller's method as previously described. Indirect division of the nucleus — mitosis — is by no means frequently met with, and Flemming, who first. 142 THE BLOOD drew attention to them, found but one among several thousands of white cells. They are said to be seen in any of the leucocytes, but are much more frequently met with in the marrow cells or myelocytes. I have occasionally seen these karyokinetic figures, either in the stage of the stellar bodies with radiating, fairly deeply stained filaments, or in that of two rosette figures at either end of the cell, which in further stages show constriction of the protoplasm. (Plate II., Fig. 9.) Some of these are quite as distinct as the karyokinetic figures found in the bone marrow of young animals. (Plate III., Fig. 9.) In other cells, especially those previously mentioned, in which the protoplasm Is almost colourless, the nucleus is irregularly arranged in the form, and very suggestive of, the earlier stages of division, but I am not convinced that they are actually mitotic figures. Lowit, in accordance with his theory of blood forma- tion, does not regard these as white cells, but as erythroblasts, i.e., the first step in the formation of erythrocytes. The majority of observers, however, consider them to be undoubtedly leucocytes, undergoing multiplication by indirect nuclear division. The Red Blood Corpuscles. The red corpuscles are usually decreased in number, and it is rarely that cases of leucocythaemia are met with, in which there is no oligocythaemia present, but it is in very few cases excessive. It is stated (Quincke and Litten) that they may be normal, or even above the normal number, but this is SPLENO-MED ULLAR Y LE UCO C YTH^MIA 143 an unusual condition. Most frequently they constitute about 2 to 3 million per cmm., but may reach as high as \\ or even 5 million, and as low as, according to Eichhorst, 316,000. The amount of haemoglobin is usually diminished in proportion to the reduction of the red elements, but not infrequently it is lower than the number of the latter. It is not easy to determine exactly the percentage with von Fleischl's instrument, on account of the tur- bidity produced by the number of white cells present. This may be somewhat overcome by adding, as pre- viously mentioned, a little caustic soda or potash to the water, as suggested by Leichtenstern. It is a very remarkable fact that patients suffering with leucocythaemia frequently show little pallor; in fact, as Wilks pointed out, they have often a good colour on the lips and face, and when seen in bed do not appear to be very ill. Alterations in the size, shape, and staining reaction of the red corpuscles occur in this as in other forms of anaemia, and are more marked in those cases in which the oligocythaemia is pronounced. In cases in which the anaemia is slight, according to Hayem, small corpuscles prevail, whilst in severer types of the disease large red cells, and even megalocytes may be often seen. In one of my cases, in which the corpuscles numbered 3|- million, megalocytes were frequent, but in another, having about 3 million red cells per cmm., they were much less numerous. Often the cells show very little alteration in their shape, but when the anaemia is intense, extreme degrees of poikilocytosis may be observed. I have occasionally met with corpuscles showing 144 THE BLOOD Maragliano's degenerative signs, such as vacuoles, within the cells, and the peculiar staining reaction — Polychromatophile— due to necrobiotic changes. Amoeboid movements on the part of the red cells have been observed by Friederich, similar to those sometimes found in pernicious aneemia. The most conspicuous feature on the part of the red corpuscles is the presence of nucleated red cells, and although they may be seen in severe anaemic conditions, this is the only disease in which they are present where there is very slight oligocythaemia. (Plate V., Fig. I.) Hayem, who so emphatically denies the existence of nucleated red corpuscles in chlorosis, etc., admits that they are a constant feature in some stage or another of this form of leukaemia. In cases in which the red corpuscles number four million or more, they may be found in almost every field of the microscope. They are mostly normoblasts, about the same size as the ordinary erythrocytes, sometimes megaloblasts, and occasionally I have met with the most perfect examples of microblasts. Signs of direct — amitosis — and in- direct — mitosis — division are frequently seen in the nuclei of the normoblasts. (Plate I., Figs. 7 and 8.) The Blood Plates. These elements are sometimes increased in number, and Pruss found that in four cases they reached two million, the normal being about 200,000 to 300,000 per cmm. In some cases they are however diminished, and Hayem gives their figures in one case as 128,650, and in another as 271,800 per cmm. LYMPHATIC LEUCOCYTH^MIA 145 Charcot- Ley den Crystals. Many observers, amongst others Neumann, Westphal, and Eberth, have frequently found these crystals in the blood of patients suffering from leukaemia. They are probably identical with the crystals found in the sputum during an attack of as^ma. Charcot and Vulpian described them as octahedral, whilst Cohn thought them hexagonal pyramids. They are obtained by allowing a small quantity of blood to stand for some time in order to partially dry. Their origin seems as yet undetermined. Westphal considers that they are formed in the spleen, and obtained them by puncture of that organ in leukaemic patients, whilst other writers think that these are a crystallized product from the blood plasma or leucocytes. According to Schreiner they are chemi- cally a compound of phosphoric acid with an organic base. They are said to be found in the normal bone marrow. They are by no means constant in the blood of leucocythaemia, and von Limbeck and von Jaksch have repeatedly failed to find them, and in dry films I have never met with any crystalline forms. Lymphatic Leucocythaemia, or Lymphaemia. This form of leukaemia is of much less frequent occurrence, runs a more acute course, and usually terminates fatally in shorter time than the previously described type of the disease. It is attended with enlargement of the lymphatic glands, and occasionally, though not generally, with some slight increase in the L 146 THE BLOOD size of the spleen, but the latter never reaches, in un- mixed cases of the disease, the enormous size that is met with in the spleno-medullary forms. (Plate VI., Fig. I.) The Leucocytes. The white cells are greatly increased in number, but not to the extent that they are in the spleno-myelogenous form. The average proportion of white to red cor- puscles is usually about i to 15, or i to 45, instead of I to 2, or I to 10, as is frequently the case in the latter variety of leukaemia. The actual number of leucocytes varies considerably, but is usually about 40,000 to 250,000 per cubic mm. Hayem records one case in which they numbered 476,000 per cmm., and in four cases described by Muir they averaged about 441,600. The prevailing form of leucocyte is almost entirely that of the large and small uninucleated cells or lymphocytes, and they may constitute 95 % of all forms of white corpuscles present. In some cases the elements are almost all of the small lymphocytic variety — cells about the size of the red blood corpuscles containing a relatively large nucleus, which stains deeply with nuclear stains, and shows signs of a dense intranuclear network. (Plates II. and III., Fig. I.) The nucleus may in some cases be deeply indented. The surrounding protoplasm is very small in amount, and is usually devoid of any granules. In other cases the leucocytes are nearly exclusively, large uninucleated lymphocytes with large nuclei, which stain faintly, and their protoplasm, which is abundant, gener- ally also contains no granules, but is frequently somewhat Plate VI. Film preparations stained with eosine and haematoxylin. Fig. 1. — Lymphatic Leucocythaemia. The leucocytes are increased, but not to the same extent as in Fig. 1, Plate V. They are all — with the exception of one multinucleated leucocyte — of the lymphocyte variety. No eosinophile cells or nucleated red corpuscles are seen. Examined with eyepiece 2. Objective i". Fig. 2.— Malarial Parasites. A. Parasites of Quartan Fever, showing stages in their development, from a small intra-corpuscular pigmented body to that of segmentation. The red blood corpuscles are of about the normal size. In the segmenting stage the pigment is situated in the centre, and around it are eight spores. B. Parasites of Tertian Fever, showing stages in development. The red blood corpuscles are somewhat larger than the normal, and the number of spores found in the segmenting forms is more numerous than in that of quartan fever. C. Parasites of Irregular or Aestivo-Autumnal Fever. The amoeba is seen to assume the form of a ring. The red corpuscles are smaller and more deeply stained. Two Laveran corpuscles or crescentic bodies are shown. Examined under rather higher magnification than previous plates. Eyepiece 4. Objective iV' oil immersion. Tube length 160 mm. Fig. 1- 0^ O 0U ® o /■■ ^ o -LiynipTiatic Leucooytliffimia.. >-^^^ ^•> Fig. 2. * i-| @ ^ Malarial Parasites. F.L.ColeK dd.. ■ 't , Pifj-ivmaYi Ctur LYMPHATIC LEUCOCYTH^MIA 147 coloured with the nuclear stain. (Plates II. and III., Fig. 2.) Not infrequently cases are met with in which both the small and large hyaline cells or lymphocytes are seen, as well as intermediate sizes. When stained with methyl-blue alone some of the small and large elements show fine basophile granules, arranged in a ring round the nucleus. (Plate III., Fig. 8.) Signs of mitotic division of the nuclei are very rarely seen in lymphatic leukaemia, and this is remarkable, as these uninucleated leucocytes multiply by indirect mitotic division in the lymphatic glands. The multinucleated or neutrophile leucocytes are relatively and absolutely diminished in number, and the eosinophile cells are extremely infrequently seen. Marrow cells or myelocytes may or may not be present in the lymphatic form of the disease. Muir saw them in one out of four cases, and von Limbeck found them wanting in his case of lymphaemia. Stengel remarks that large uninucleated cells, corresponding to marrow cells, are rarely seen, although he quotes Fraenkel's statement as to the occurrence of uninu- cleated hyaline cells larger than the ordinary large lymphocyte. Generally speaking, marrow cells when present are so in very small numbers. The Red Blood Corpuscles. The number of the erythrocytes is almost invariably diminished, and usually to a greater extent than in the spleno-medullary form of the disease. Hayem records a case, in which they numbered 372 million;' MuUer, 2-55 million; and von Limbeck two cases, in which they reached 3-8 and 3-4 million per cmm. In Muir's 148 THE BLOOD four cases they did not average i'5 million per cmm. Notwithstanding the fact that the oligocythaemia is generally more pronounced in lymphatic leukaemia, yet nucleated red corpuscles are extremely rare in this form, although abundant in the spleno-myelogenic type of the disease. Miiller, Limbeck, and Wertheim have met with a few isolated forms, and Muir found them in one case, in which the red corpuscles were below one million per cmm. The latter states that he has only found them when the anaemia has been severe. Most observers agree that nucleated red cells are frequently absent, and when present are never found in large numbers. The Pathology of Leucocythaemia. Virchow and many other observers regard leucocy- thaemia as a primary disease of the blood - forming organs, and consider that the blood is only affected secondarily, whilst Lowit and some others maintain that the condition of the blood is primary, and that the haemopoietic organs are secondarily affected. The former is most generally accepted as the correct view of the process, and Is based upon the fact that organic changes of the nature of proliferation have been found in the blood-forming organs. Those who maintain that the blood Is the primary seat of the disease, consider that the proliferative changes found in the haemopoietic organs are due to elements in process of multiplication, being carried there by the general circulation, and not due to an actual proliferation taking place in the organs them- selves. PATHOLOGY OF LEUCOCYTH^MIA 149 In favour of the former and in opposition to the latter theory, is the fact that signs of multiplication of the cellular elements, i.e., karyokinetic figures, are much more numerous in the organs concerned in blood formation than in the blood itself. The earlier writers, especially Hughes Bennett, looked upon the condition as one of "suppuration of the blood"; whilst latterly some observers, Kottmann and Bard, consider the process as one of " new growth or cancer of the blood." Lowit maintains that the excessive number of leucocytes found in both forms of the disease is not due to increased proliferation, but rather to diminished destruction of the leucocytes, or an arrest in the development of the uninucleated, into more mature white corpuscles. This view is not generally enter- tained, because although uninucleated elements may prevail in both types of the disease, yet there is no evidence that diminished destruction of the leucocytes actually occurs, some even maintaining that leucolysis is increased. (a) The spleno - medullary Leukaemia is essentially characterized by the occurrence of certain elements which are not found in the normal blood, viz., myelocytes, eosinophile myelocytes, and nucleated red corpuscles, and these- in health are only found in the bone marrow. (Cornpare Figs, i and 2, Plate V.) In this disease the marrow of the spongy and long bones is found to have undergone considerable alteration, and the fat is largely replaced by purulent, or, as Neumann terms it, pyoid, material. This is of a yellowish colour, either distributed uniformly or in patches throughout the marrow, and which, though 150 THE BLOOD at first firm, becomes softer and more liquid, and eventually closely resembles pus. Microscopically it is seen to consist very largely of marrow cells of both varieties, and proliferation is evident from the large number of cells in process of division, and all stages of karyokinesis may be seen. It seems very probable that all the elements found in the blood in this form of the disease may be explained by excessive proliferation of the marrow cells and their overflow into the blood. The nucleated red corpuscles which, as Muir points out, lie for the most part at the periphery of the marrow near the vessels, might easily escape into the blood stream. Flemming and others maintain that the proliferation occurs in the marrow, and that the elements in process of division found in that situation are far too numerous to be accounted for by the assumption that they are carried there by the blood, and in the blood, mitotic figures, though occasionally found, are far too few to be regarded as the primary factors. What is the primary cause of the proliferation of the cellular elements in the marrow seems still a matter of the greatest obscurity. At present there is no evidence that bacteria are the exciting cause, although in acute cases it seems possible that infection may play an important part. (/8) Lymphatic leukaemia. — Partly owing to the greater rarity of this form of leukaemia, and partly to the fact that the elements present are not abnormal leucocytes, it is extremely difficult to determine the primary cause of the disease. In this type of leukaemia the bone marrow proper is largely displaced by lymphoid tissue, consisting of small PATHOLOGY OF LEUCOCYTH^MIA 151 uninucleated leucocytes, and a few nucleated red cells. The spleen is usually somewhat enlarged owing to the presence of an enormous number of lymphocytic elements. The lymphatic glands are frequently, but not always, enlarged in the lymphatic form of the disease. They are softer than the normal, owing to a proliferation of the cellular rather than the connective tissue elements. Mitotic figures are frequently seen, pointing to rapid multiplication of the cells. The lymphatic glands are occasionally not enlarged, and Leube, according to Muir, records a case in which neither the lymphatic glands nor the spleen were hypertrophied, but in which a lymphoid condition of the bone marrow was present. Muir further states that there may be enormous enlargement of the spleen, and yet the blood may contain an excessive proportion of small uninucleated cells. He considers that the term lymphatic, applied to this type of leucocythaemia, should refer to the variety of leucocytes present, rather than to their origin. The condition seems to depend on proliferation of the lymphoid elements in the lymphatic tissue throughout the body. Wertheim is of the opinion that the two forms of leukaemia are quite different, whilst Mliller, on the other hand, considers the process in them analogous, but that in the lymphatic form the lymphatic system is chiefly involved. Von Limbeck points out the close resemblance that exists between lymphatic leukaemia and lymphosarco- mata, and considers the temporary improvement that occasionally takes place in a leukaemic patient attacked 152 THE BLOOD with an infectious disease as analogous to the influence ot erysipelas on some forms of malignant disease. What cause gives rise to the cell proliferation in both forms of the disease is at present entirely unknown. Diagnosis of Leucocythaemia. The diagnosis of this disease is in some cases an extremely easy matter, but in others, especially in the lymphatic form, it may be exceedingly difficult to say whether we have to deal with leucocythaemia or lympho- sarcoma, and in either type of the disease stained film preparations are, in my opinion, of much greater value than mere enumeration and proportion of the red and white cells. I. Diagnosis of the Spleno-medullary Form. This, by far the commoner type of the disease, is characterized by a large increase in the number of leucocytes, a slight variable diminution of the red corpuscles, the constant appearance of nucleated red cells not accounted for by the degree of anaemia ; and, above all, by the presence of abnormal elements, namely, the marrow cells and atypical eosinophile cells or eosin- ophile marrow cells. All observers agree that the leucocytes may in certain cases be so enormously in excess of the normal as to equal, an'd occasionally to exceed, the number of red blood corpuscles present ; but at times the number of white cells, though increased, are not sufficiently so to justify the diagnosis of leucocythaemia. As previously mentioned, there is no absolute number of leucocytes, nor is there any figure representing the DIAGNOSIS OF LEUCOCYTH^MIA 153 ratio of white to red which entitles us to distinguish between leucocytosis, from whatever cause, and leucocy- thaemia. Some recent writers — Gilbert and Hayem — have placed the limit of leucocytosis at 70,000 white cells per cmm., others have asserted that when the propor- tion of white to red corpuscles exceeds i to 20 the condition is one of leukaemia rather than leucocytosis. This is, however, by no means reliable. Mere relative increase in the number of eosinophile cells, although mentioned as an early diagnostic sign by von Jaksch, is not characteristic, as frequently these elements are relatively diminished. The only diagnostic features of this form of leucocy- thaemia, are the occurrence of marrow cells and eosino- phile myelocytes, and although the former may be found occasionally in pernicious anaemia, and in a few other pathological conditions, yet they are present in extremely small numbers, whilst in leucocythaemia they constitute a very large proportion of all the elements present. It must be mentioned that during the course of this disease the splenic enlargement may to a great extent subside, and the number of leucocytes undergo a marked diminution ; yet if such a case be seen for the first time in this stage, myelocytes are present in sufficient numbers to warrant a diagnosis of the condition. Nucleated red blood corpuscles are present in large numbers, quite out of proportion to the degree of the oligocythaemia, and these, and the occasional presence of mitotic figures, help to clinch the diagnosis. As an example of the importance of stained film preparations of blood, and the comparative insignificance of the enumeration of the cellular elements and the 154 THE BLOOD examination of blood in the fresh unstained condition, I will describe one of the last cases of spleno-medullary leukaemia, which quite recently I saw in consultation. A girl, aged ii. Spleen very much enlarged, occupy- ing the greater part of the left side of the abdomen, extending anteriorly to the linea alba, and at the back as far as the posterior axillary line, and reaching within one and a half inches of the symphysis pubis. The notch was felt just below the umbilicus. The organ measured about lo inches in length, and 2>\ inches in its greatest breadth. The lymphatic glands in the groin and axilla were just palpable. There was no tenderness on deep per- cussion of the bones. The blood had been carefully examined in the fresh condition, and an enumeration of the corpuscles made by her medical attendant, but the comparatively small number of leucocytes apparently excluded the diagnosis of leukaemia, and the case appeared rather one of splenic ansemia. I found on my first examination that the red corpuscles = 3,458,333 ; the haemoglobin 37 % (von Fleischl) ; the white blood corpuscles, 44,315 per cmm., or a proportion of white to red as i to 78. From my enumeration I could make no diagnosis, nor did the examination of fresh blood films render this possible ; but in dry films stained with eosine and haematoxylin I found that the leucocytes present very largely consisted of typical marrow cells, and large eosinophile myelocytes were frequent. Nucleated red blood corpuscles, especially of the normoblastic type, were also present in large numbers, although the red cells were fairly numerous and well shaped. DIAGNOSIS OF LEUCOCYTH^MIA 155 A dififerential enumeration of the leucocytes showed that the myelocytes were 37*2 %, and a certain diagnosis of spleno-medullary leukaemia was given. The proportions of the other elements will be seen in the table below. An examination of the blood made a week later showed that the leucocytes had slightly increased, reaching 55,385 per cmm. After a lapse of five weeks the leucocytes reached 555,795, whilst the red numbered 2,143,333, or a proportion of white to red cells as i to 3 "8, or practically i to 4. The following table shows the remarkable increase in the number of white cells, and the proportion of the various forms of leucocytes found at intervals in the course of the disease : — CASE OF SPLENO-MEDULLARY LEUCOCYTHAEMIA. April 18, '97. April 25. May 29. June 5th. Red Blood Corpuscles . 3,458,333 •• 3,576,562 . • 2,143,333 • . 3,029,166 White Blood Corpuscles . 44,3i5 ■■ 55,385 ■ ■ 555,795 ■ 696,960 Haemoglobin (von Fleischl) . 37 % 44% ■ ■ 40% . .. 30%? Proportion of White to Red Corpuscles . . . 1:78 1:644 . . 1:3-8 . • 1:44 DIFFERENTIAL ENUMERATION OF THE LEUCOCYTES. Multinucleated . . 43'3% ••• 36 •■• 49-% •■• 52 Marrow Cells . 37-2 .. 33 ■■ 43 35 Lymphocytes 47 II ... I 2 Eosinophile I4'6 20 ... 7 II VARIETIES OF EOSINOPHILE CELLS. Myelocyte Eosinophile . 43% - 45 -• 70% .. 66 Ordinary Pojynuclear Eosino- phile 55 55 ... 28 .. 34 Small Eosinophile 2 ... 2 Nucleated Red Corpuscles to Leucocytes. . . 41 in 2000 ... 50 in 2000 ... 1 5 in 2000... 14 in 2000 iS6 THE BLOOD It is possible that at the time the first examination was made, and previous to it, some complication might have caused a temporary diminution in the number of the leucocytes, and the subsequent increase may have been merely a reversion to the former state of the blood. The large size of the spleen would fully permit this opinion. Against it must be mentioned that no signs of recent inflammatory complications existed, and the temperature chart for some weeks previously only showed the usual irregular rise so frequently found in this disease. On the other hand the condition first seen might have been merely an incipient form of the disease. In this case the qualitative rather than the quantita- tive alteration in the leucocytes as seen in stained films, rendered the diagnosis of spleno-medullary leukaemia certain from the first examination. (a) Diagnosis from Leucocytosis. As a general rule, the number of white corpuscles in leucocytosis does not exceed 60,000, but it may in exceptional cases reach over 100,000 per cmm., whilst commonly in leukaemia these cells are over 200,000 per cmm. In leucocytosis usually the multinucleated leucocytes are the prevailing type, although in some cases the uninucleated lymphocytes may predominate, but these are chiefly of the small variety. In leukaemia the abnormal elements so frequently alluded to, are present in large proportions, whilst the multinucleated forms are relatively diminished in number. (/3) From Hodgkins Disease and Splenic Anczmia. — These diseases are attended with, in the one case, enlargement of the lymphatic glands, and in the other, usually slight enlargement of the spleen, but the latter DIAGNOSIS OF LEUCOCYTH^MIA 157 may be greatly increased in size, and the condition may somewhat resemble this form of leukaemia. The blood, however, in these two diseases is usually more or less normal, and the leucocytes, if increased, are not very numerous, and the multinucleated forms generally predominate, and very few, if any, of the abnormal leucocytes, so characteristic of the latter disease, are seen. (y) From Malarial enlargement of the Spleen. — The condition of the blood, the absence of marked leucocy- tosis, the presence of malarial organisms, or of pigment granules in the leucocytes, as well as the entire absence of marrow cells, render the distinction from spleno- medullary leukaemia very easy. (<5) Ttimottrs in the region of the Spleen or Hydrone- phrosis, or malignant disease of the kidney, may clinically resemble leukaemic enlargement of the spleen, but the absence of the notch, and the condition of the blood, which may in malignant disease of the kidney show marked leucocytosis, involving however almost entirely the multinucleated leucocytes, render the diagnosis certain. 2. Diagnosis of Lymphatic Leukaemia. The diagnosis of this, fortunately the less common form of leukaemia, presents greater difficulty than the former. In an article in the British Medical Journal, Dec. 5th and 12th, 1896, on the "Relation between Leukaemia and Pseudo-leukaemia," Martin and Mathew- son take a very pessimistic view of the value of the examination of the blood as a means of distinguishing these two diseases. I must mention, however, that they iS8 THE BLOOD refer throughout their paper to the lymphatic form of leukaemia. The essential diagnostic feature of this form of leucocythaemia is the occurrence of permanent leucocy- tosis, which varies considerably in degree, but usually there are about 130,000 white cells per cmm., involving almost exclusively the large and small uninucleated elements or lymphocytes, whilst marrow and eosino- phile cells are extremely few. The red blood corpuscles are diminished usually to about half the normal number, but nucleated red cells are exceedingly rare. (a) Diagnosis of Lymphatic from Spleno-medullary Leukaemia. — In the lymphatic type of the disease, the leucocytes are not increased to the same extent as in the spleno-medullary form. In the former 130,000 white cells, with a ratio of white to red corpuscles of i to 30, are usually seen, whilst in the latter the leucocytes frequently number 400,000 or 500,000 per cmm., with a ratio of I to 6. In lymphatic leukaemia the leucocytes are almost entirely normal elements — lymphocytes ; and the other white cells — the multinucleated and eosinophile cells, are considerably reduced in number. Marrow cells may occasionally be present in some few cases, but even then constitute only a small percentage, not more than 0-5 %. (Compare Fig. i, Plate V., with Fig. i, Plate VI.) In the spleno-medullary form, the leucocytes present are largely abnormal elements, large uninucleated neutrophil cells or myelocytes, constituting 30 to 50 % of all forms of white cells seen. The multinucleated elements are relatively reduced,, but may constitute 50 %, whilst the eosinophile cells, though not usually relatively increased, are present in fair numbers, and many of them DIAGNOSIS OF LEUCOCYTH^MIA 159 are eosinophile myelocytes. The lymphocytes are considerably diminished in number. As regards the red corpuscles, they are usually some- what less numerous in the lymphatic than in the spleno-medullary form, but nucleated corpuscles though extremely numerous and constant in the latter, are absent or very infrequent in the former. (/3) From Hodgkins Disease, or Pseudo-leukaemia and Splenic Ancemia. — In both Hodgkin's disease and lymphaemia, enlargement of the lymphatic glands is the prominent clinical feature, but the blood in the former, as well as in splenic anatmia, is usually normal ; and although there may be an increase in the number of the leucocytes, yet this very rarely attains the high degree seen in typical lymphatic leukaemia. The leucocytosis in pseudo-leukaemia, when present, involves chiefly the multinucleated cells, hut sometimes the lymphocytes are the elements concerned. Osier has stated that occasionally the leucocytes in Hodgkin's disease are greatly increased, and the character of the blood becomes that of lymphatic leukaemia. (y) From Sarcomata and other Malignant Diseases. ■ — As a general rule leucocytosis, when it results from carcinomata, is usually attended with an increase in the number of multinucleated elements, but this is not always so in sarcomata, and cases have been recorded by Palma and others in which lymphocytosis occurred. When multinucleated leucocytes are present in excess, lymphatic leukaemia is easily excluded ; and in other cases, in which the lymphocytes are the prevailing type, the number of the white cells seldom reaches that found in lymphaemia. Yet many authorities, particularly von Limbeck, i6o THE BLOOD admit that some cases of lymphosarcomata may occur, in which the leucocytes are very considerably increased, and are mainly of the lymphocytic type, a condition which it is practically impossible to distinguish from true lymphatic leukaemia. We shall reserve the question of the diagnosis of both forms of leukaemia occurring in children, from what has been termed "anaemia infantum pseudo -leukaemica," till we have described this disease. Acute Leukaemia. A form of leucocythaemia, characterized by its rapid course, has been described under this name, and Ebstein collected an account in 1889 of seventeen such cases. Since then additional cases have been recorded, so that at the present time their number has reached to thirty-five or forty. The duration of the disease varies from six days to nine weeks. Most observers, however, limit it to six weeks, whilst Fraenkel, in a recent account (1895) of ten cases, includes those which lasted four months. In all cases the blood and blood-forming organs showed the characteristic appearances of leucocythaemia, but unfortunately many of the descriptions give merely the number and not the variety of leucocytes present. According to Leyden, Greiwe, and Kossler, all forms — the splenic, spleno- medullary, and lymphatic — may occur in acute as in chronic leukaemia. Thus Eich- horst's case was pure splenic, Leyden and Kossler's spleno-meduUary, whilst two other cases described by Kossler were the lymphatic variety ; and the last-named author noticed that in one of his cases — a woman aeed ACUTE LEUKEMIA i6i twenty -seven years, who died within seven days — myelocytes were the prevailing elements, whilst the multinucleated leucocytes were very infrequent, and the lymphocytes and nucleated red corpuscles entirely absent. The proportion of white to red corpuscles was i:6o. In Greiwe's and in two other cases of Kossler, the increase in the white cells took place at the expense of the small lymphocytes. According to Fraenkel, the characteristic condition of the blood is that of lymphaemia, i.e., the small uninucleated are enormously increased, whilst the multi- nucleated leucocytes are considerably diminished in number. He pointed out that the nuclei of the lymphocytes were somewhat poorer in chromatin than the normal, and their protoplasm frequently con- tained basophile, never neutrophile, granules. Mitotic figures were occasionally seen, as well as a few nucleated red corpuscles. The erythrocytes are usually diminished in number, and in some cases the anaemia is pronounced. Von Limbeck considers that the anaemia, which is sometimes at the commencement a prominent feature of the disease, and which later is followed by leucocytosis, may depend on the haemorrhagic tendency — a marked feature in acute leukaemia — and this might also account for a leucocytosis of 30,000 per cmm., but would not explain the occurrence of 300,000 or more leucocytes. It seems extremely difficult to explain the nature of this form of the disease. Possibly some of the cases described by the earlier writers, especially where no exact investigations of the blood were made, were of the nature of a high degree of leucocytosis, whilst in others it is probable that acute M i62 THE BLOOD symptoms might have supervened in a patient affected with chronic, latent, or unrecognized leukaemia. There are many clinical features present, e.g., sudden onset with rigors, irregular fever, enlargement of the lymphatic glands, and occasionally of the spleen, the frequent haemorrhagic tendency, and the acute course of the illness, which all point to this as a separate form of leucocythaemia, in which infection may play an important part. Hodgkin's Disease. Synonyms : Pseudo-leukaemia (Cohnheim), Lymphatic anaemia (Wilks), Addnie (Trousseau), Lymphosarcoma (Virchow), Malignes Lymphom (Billroth), Desmoid- carcinom (Schulz), Ansemia splenica (Greisinger). Hodgkin's disease is defined by Osier as "an affection characterized by progressive hyperplasia of the lymphatic glands with aneemia, and occasionally the development of secondary lymphoid growths in the liver, spleen, and other organs." The disease was first recognized by" Hodgkin in 1832, and Wilks in 1865 described a number of cases under the name of "Hodgkin's disease." Cohnheim in 1857 described cases under the term Pseudo-leukaemia, which closely resembled leucocythaemia, only lacking the characteristic condition of the blood in that disease. French writers included Hodgkin's disease and leukaemia under the general term "Addnie." A splenic form of the disease was recognized by the earlier writers, and recently described by Banti and Bruhl under the names anaemia splenica, spleno-megalie primitive, and splenic anaemia. We shall describe this later. HODGKIN'S DISEASE 163 There is no doubt that tubercular, syphilitic, inflam- matory, and malignant enlargement of the lymphatic glands have been included under the term Hodgkin's disease. Clinically, tubercular enlargement of the lymphatic glands may usually be distinguished from pseudo- leukaemia by the fact that the former is more common in the young, the disease is more or less localized, it involves the submaxillary rather than the glands in the anterior and posterior cervical triangles, and the glands often fuse together and tend to suppurate. Syphilitic enlargement of the glands is usually dis- tihguished by the history of the disease and the moderate size, though often universal distribution of the glandular hyperplasia. Malignant diseases of the lymphatics when secondary, are easily distinguished if the primary growth is found, but when the glands are primarily affected, the distinction is often impossible, many considering Hodgkin's disease as a primary malignant lymphoma. Anatomically and pathologically, pseudo-leukaemia closely resembles leucocythaemia, and some observers maintain that the former may develop into the latter. A few writers have recorded such cases, but while recognizing that transformations may undoubtedly occur, yet some of the cases published may probably be in- stances of terminal leucocytosis rather than true leukaemia. The Condition of the Blood. The examination of the blood is of the greatest im- portance, although characteristic alterations in the number and nature of the elements are conspicuously i64 THE BLOOD absent. At the commencement of the disease the blood is practically normal, but when the condition is advanced, and the patient has become cachectic, then all the signs of the anaemia associated with cachexia are present. Generally the number of red cells is somewhat below five million, but in severe cases they may be reduced to two million per cmm. The haemoglobin usually undergoes a greater diminu- tion, so that a chlorotic condition of the blood is fre- quently seen. In cases in which the anaemia is pronounced, all the alterations in size and shape of the red corpuscles may occur as in any other form of severe anaemia. Nucleated red corpuscles may occa- sionally be seen when the oligocythaemia is a marked feature of the disease, but these are very few in number and nearly always of the normoblast type. The Leucocytes are present either in normal numbers or sometimes below, more usually above, the number found in health. Von Limbeck states that the con- dition of the blood depends upon the nature of the lymphoid growths ; thus he found in two cases of splenic anaemia (it is evident that he includes splenic anaemia under the term Hodgkin's disease) that a chlorotic condition of the blood was associated with a normal number of leucocytes, whilst cases in which the lymphatic glands are enlarged are almost always attended with an increase, if only a moderate one, of he white cells of the blood. The presence of inflammation in the lymphatic glands' usually results in an increase of the leucocytes. Most observers are agreed that, generally speaking, the variety of the white cells present does not materially HODGKIN'S DISEASE 165 differ from that seen in health, and any marked leuco- cytosis is owing to an increase in the multinucleated or neutrophile elements ; occasionally, however, it is stated that the small uninucleated lymphocytes may predomi- nate. Von Limbeck considers that the nature of the leucocytosis is variable, and states that if the multi- nucleated cells are increased this would be in favour of the inflammatory character of the lymphatic tumours, whilst increase of the small lymphocytes gives the con- dition an appearance similar to that found in sarcoma of the lymphatic glands. Occasionally, as in some other forms of chronic anaemia, a few myelocytes may be seen, but these are always infrequent, and in most cases absent. Acute Pseudo- leukaemia, analogous to acute leukaemia, has been described, in which the disease takes a very acute course. These cases are attended with all the usual characters of the chronic form, but are usually associated with a marked haemorrhagic tendency and moderate pyrexia. Lannois and Courmont, in their cases, found, in ad- dition to enlargement of the lymphatic glands, certain organisms, viz., staphylococcus pyogenes aureus, and streptococcus pyogenes, in the blood and lymphatics. Von Limbeck states that it is extremely doubtful whether this is a separate disease, as all the symptoms — oligocythaemia, reduction in the amount of haemoglo- bin, slight leucocytosis, haemorrhagic tendency, enlarge- ment of the spleen and lymphatic -glands, occasionally jaundice, and usually slight fever, may be occasioned by various pathological conditions, especially acute sar- comatosis (Fagge) as well as chronic pyaemia. i66 THE BLOOD Diagnosis of Flodgkins Disease. The blood shows very little alteration, and is at first practically normal, although later all the signs of aneemia may appear. Leucocytosis, if present, is usually due to an increase in the multinucleated elements. The negative character of the blood is of the greatest assistance in distinguishing this disease from both forms of leukaemia, as was previously mentioned. Splenic Anaemia. Synonyms : Spleno - megalie primitive ; Anaemia splenica (Banti) ; Pseudo leukaemia splenica. We shall speak of this as Splenic Anaemia of adults (or children), in contradistinction to that described later as " Anaemia pseudo - leucaemia infantum," or the splenic anaemia of infants. Attention has recently been drawn to this disease in England by West, and more particularly by Frederick Taylor at the British Medical Association in London in 1896. Banti, an Italian observer, in 1882, and Bruhl, of Paris, in 1891, have both written a short account of the disease, and it is more especially from the article by Bruhl in Archives gSndrales de MSdecine, 1891, that I have gathered the following information. Splenic anaemia is characterized by a marked in- crease in the size of the spleen, progressive anaemia, an absence of leucocytosis, and a similar absence of enlargement of the lymphatic glands. Such a definition excludes leucocythaemia, and the ordinary forms of Hodgkin's disease or pseudo-leukaemia. SPLENIC ANAEMIA 167 No separate account of this disease is given by Limbeck and Stengel, who regard it as merely a variety of Hodgkin's disease, whilst Hay em and Cabot make no mention at all of its occurrence. Some writers have divided Pseudo - leukaemia into a splenic and a lymphatic form, according as the spleen or the lymphatic glands are involved, and consider this disease belongs to the former group. The Symptoms and Signs of the disease vary. Some- times anaemia is the first symptom which causes the patient to seek medical advice, whilst at other times the presence of a splenic tumour, partly on account of its size, partly also from the pain resulting from an attack of perisplenitis, calls attention to the disease. The symptoms of anaemia present no peculiarity. Pallor, progressive feebleness without emaciation, and palpitation and breathlessness on exertion are present, as in all other forms of ansemia. At other times attacks of colic -like pain in the left hypochondrium, nausea, vomiting, and diarrhoea occur. These occur in paroxysms, and" are generally followed by an interval of varying duration, in which the patient feels comparatively well. The spleen on examination is always found enlarged, sometimes reaching as far as the iliac crests. It is uni- formly increased in size, and its shape is unaltered. The liver is either normal or slightly enlarged, and in the latter case often associated with the presence of jaundice. Haemorrhages are not frequent, but occasionally epistaxis may be an early and prominent symptom. The entire absence of any enlargement of the lymphatic glands is the point of distinction between splenic ansemia and Hodgkin's disease. i68 THE BLOOD The Condition of the Blood. The examination of the blood alone will not dis- tinguish the disease from pseudo- leukaemia, as there is nothing pathognomonic of the condition to be found. On the other hand, it is impossible to differentiate between leucocythaemia and splenic aneemia without a blood examination. The red corpuscles are nearly always diminished in number, frequently falling to two and a half million, and occasionally to one million per cmm. or even less. Generally speaking, the anaemia corresponds to the second or third degree of anaemia according to Hayem's classification, which has been previously mentioned. Bruhl states that the corpuscles undergo little or no alteration in shape, and poikilocytes are absent. Banti, however, describes the red cells as being deformed, fusiform or oval. They are paler than the normal corpuscles, owing to the marked diminution in the amount of haemoglobin. The size of the erythrocytes is generally diminished, and microcytes measuring 3 to 4 m are not infrequently seen. Bruhl states that the red corpuscles are never increased in size in this disease. The amount of haemoglobin undergoes a much greater diminution than the number of corpuscles, giving the blood a very pronounced chlorotic character. It often falls to 50%, and may even reach only 13 %. The individual corpuscular value in haemoglobin — the colour index — is considerably below the normal, in this way resembling chlorosis, but in marked contrast to that found in pernicious anaemia, and Bruhl regards this pronounced oligochromaemia as very suggestive of the disease. SPLENIC ANEMIA 169 The leucocytes in the majority of cases are normal, and any marked increase points to some compUcation. In none of the cases described by Banti and Bruhl is a differential enumeration of the variety of leucocytes given. The former, however, mentions that the multinucleated elements were, as in health, the most numerous, and in one case found the eosinophile were increased, and a few leucocytes were seen containing granules which stained deeply with methylene-blue, these probably being basophile in nature. Speaking generally, no marked alteration in the number or quality of the leucocytes has been found, and they may be regarded as practically normal. The disease, in the majority of cases, progresses till a fatal termination occurs. The duration of the illness varies from six months to four years, but more usually from half to two years. Some cases have a very rapid course, analogous to that some- times seen in acute leukaemia and pseudo-leukaemia. The following table shows the number of the red and white corpuscles, and the amount of haemoglobin in a few cases that have been recorded of this disease. Name of Observer Bruhl. CASES OF SPLENIC ANEMIA. Banti. Banti. Banti. Potain. Age and Sex . Man, 54 .. Man, 18 .. Girl, 16 . .Woman, 72.. Man, 50 Red Corpuscle 2,851,350, .. 3,999,000, .. 3,948,000 .. 3,720,000, 2,000,000 Girl, 13 2,700,000 "White Corpuscle 10,757 .. 4704 Haemoglobin . 1,246,500, .. 2,400,000, or 43 % or 48 % ■Colour Index , 0*43 . . 0*632 6876 .. 19,805 . . no leucocytosis . . no leucocytosis 68 % . . 2,594,000, . . 33 % . . chlorotic diminution or 52% o*86 .. 0*697 0*825 .. diminished In the case recorded by Dr. West, British Medical Journal, June 13, 1896, the patient was a man aged 36 ; the red corpuscles and haemoglobin were diminished, and the leucocytes slightly increased, but not more than 170 THE BLOOD the fever would account for. Dr. Taylor described another case in a boy, aged 19, in which red corpuscles and haemoglobin were 55 % of the normal, but no leucocytosis at first. The Diagnosis. A careful examination of the blood, not merely the enumeration of the elements, is of the greatest value in distinguishing splenic anaemia from other pathological conditions associated with an enlargement of the spleen. A correct diagnosis is of the utmost importance, as there is some hope that the disease may be cured by splenectomy. Whilst this operation has been almost invariably attended with a fatal result in leucocythaemia, at least three cases of splenic aneemia have been recorded in which a cure followed removal of the spleen. The essential characteristics of the disease are the marked enlargement of the spleen, the absence of en- largement of the lymphatic glands, and the condition of the blood, viz., a diminution in the red corpuscles, associated with a greater diminution in the amount of haemoglobin, the absence of leucocytosis and abnormal white blood corpuscles. From Leucocythaemia. In both forms of leukaemia the leucocytes are usually, though not invariably, enormously increased. In the spleno-medullary type, marrow cells and eosinophil myelocytes are numerous and characteristic, whilst in the lymphatic form the small and large uninucleated elements predominate. I must again draw attention to the fact that an absence of a great increase in the leucocytes does not exclude leucocythaemia. In a case SPLENIC ANEMIA 171 previously mentioned of a girl of 1 1 with very enlarged spleen, I found the leucocytes only numbered 44,313, the red cells about 3^ million per cmm., and without stained films leucocythaemia would have been excluded, and a diagnosis of splenic anaemia given. From Hodgkin's Disease, or Pseudo- leukaemia. The condition of the blood will not distingfuish these two diseases. The presence of enlarged lymphatic glands without, or with only slight enlargement of the spleen, points to Hodgkin's disease. From other conditions in which an enlarged spleen is present, the fact that the spleen in splenic anaemia is always considerably enlarged uniformly, without losing its form and direction, and that its surface presents no irregularities, enables us to exclude hydatid cysts and cancer of the spleen. Waxy disease of the spleen, which might somewhat resemble it, is distinguished by a history of suppura- tion or syphilis, and by similar disease in other organs, especially the intestine and kidney, giving rise in the one instance to diarrhoea, and in the other albuminuria. Malarial enlargement of the spleen is recognized by the history of residence abroad, or of previous attacks of ague. Before considering the various forms of secondary anaemia which occur in different pathological conditions, it will be well to glance at the following table which I have compiled. It will show the main features found in the blood of the most important forms of anaemia, and includes secondary anaemia, considered generally, in order to accentuate the differences which exist between it and all forms of primary or idiopathic anaemia. DIFFERENTIAL CHART OF THE MOST 1.— PRIMARY Red Blood Corpuscles — Chlorosis. Simple Pritnary Anaemia. Pernicious Anaemia. Number Usually diminished, rarely below 2 million. Always diminished, but extent variable. Generally enormously diminished. Size and Colour Diminished in size, micro- cytes frequent. Paler in colour. Diminished in size in slight, but may be larger in severe cases. Colour variable. Increased in size. Me- galocytes frequent. Colour usually not diminished. Shape Poikilocytosis seldom excessive. Poikilocytosis pro- nounced in severe cases. Poikilocytosis always ex- tremely pronounced. Haemoglobin Relatively greater dimi- nution than number of corpuscles. Diminished proportion- ate to number of cor- puscles. Generally relatively high, in excess of the cor- puscles. Colour Index Always low. Variable, generally about normal. Frequently high. Normoblasts Present in severe cases, generally in small numbers. Present in severe cases. Almost always present. Megaloblasts Absent or extremely rare. Only found in severe cases, and not as numerous as the Nor- moblasts. Nearly always present and more numerous than the Normoblasts. Leucocytes — Number Generally normal. Generally normal, some- times diminished. Usually diminished. Lymphocytes . Sometimes increased re- latively. Sometimes increased re- latively. Usually relatively in- creased. Multinucleated or Neutrophile . Usually normal, some- times diminished. Sometimes increased, sometimes diminished. Usually diminished. Myelocytes or Marrow Cells . Absent or extremely rare. Absent or extremely rare. A very small percentage frequently present. IMPORTANT FORMS OF ANEMIA. ANEMIA. 2.— SECONDARY AN. to 1 s ■s Pigmented Quotidian Parasite. 24 hours. The unpigmented immature forms very actively amoe- boid, less active when pigment ac- cumulates. Very fine, later coa- lesce in one or two lumps, does not swarm. < Unpigmented Quotidian Parasite. 24 hours or less. Very active amoeboid movement. None. Malignant Tertian Parasite. 48 hours. Active, the move- ment remains pre- sent in the pig- mented bodies. Moderately fine, often shows oscillatory movements. THE PARASITES OF MALARIAL FEVER 207 SIGNS OF THE VARIOUS PARASITES. Maximum Size. Form of Spore Formation. Number of Spores. Crescent Bodies, Alterations in the Infested Blood Corpuscles. The size of the red blood corpuscle. Daisy form, the sin- gle spores round, with distinct nu- cleolus. 6-12. None. Red blood corpuscles are little discoloured and do not alter their size. Size of the red blood corpuscle, some- times even larger. Sunflower or grape- like, single spores small, round, nu- cleolus rarely seen.* iS-20, often less. None. Red blood corpuscles are often hyper- trophied and lose their colour quickly and completely. J-| the size of a red blood corpuscle. Irregularly formed heaps. 6-8, even more. Present. Red blood corpuscles shrink often, and are then either darker stained, cop- per coloured, or may be completely de- colorised (Schleier). \-\ size of a. red blood corpuscle. Star-shaped, or in irregular heaps. 6-8. Present. Red blood corpuscles shrink frequently, and are darkly stained. J-l the -size of a red blood corpuscle. Irregular heaps. 10-12, rarely 15-16. Present. Red blood corpuscles shrink frequently, they are darkly stained or may be perfectly colourless. * N.B, — In a fresh condition ; it is always seen in a stained preparation. 2o8 THE BLOOD The same remarks apply to the malignant fevers, but here greater difficulty will be experienced. The severity of the paroxysm is generally, accord- ing to Golgi — -and this is more or less confirmed by Mannaberg — proportionate to the number of organisms present. It must be mentioned that occasionally no parasites can be found, although malaria is present. This occurred three times in 130 cases which Mannaberg examined, and he says it is most likely to occur during the first days of the illness, and advises waiting "until one or more paroxysms have occurred, and thereafter to repeat the examination of the blood at a specially favourable time (before the paroxysm)." Secondary Anaemia resulting from Malaria or Alterations in the Blood other than the presence of Haematozoa. Anaemia, due to the destruction of the red blood cor- puscles by the malarial parasite, and also possibly to the parasitic poison in the blood serum, is present in varying degrees, and is generally proportionate to the duration and intensity of the disease. Kelsch, in one case, found the oligocythaemia so pro- nounced that the red corpuscles numbered only half a million, whilst in another patient he determined that two million corpuscles were destroyed in the course of four days. Hayem and Halla saw the red cells reduced to 1,182,760 and 2,800,000 per cmm. These figures represent severe cases. There is apparently a greater destruction of coloured elements after the earlier paroxysms, and Thayer states that when they have been reduced to two or even one THE PARASITES OF MALARIAL FEVER 209 million, they often remain stationary. The haemoglo- bin is usually reduced proportionately to the red cor- puscles. Haemoglobinuria sometimes occurs in severe cases of malaria, and also in post-malarial anaemia. Kelsch affirms that during convalescence the cor- puscles increase more rapidly than the haemoglobin, and Mannaberg found that sometimes the haemoglobin continued to decrease for several days after the cessation of the fever, whilst the corpuscles began to increase. Dionsi has stated that the anaemia following malignant fever is more persistent, and resists treatment longer, than that following the simple forms of malaria. The leucocytes are usually diminished in number, and this is most marked just after a paroxysm. In the apyrexial period they may increase in number, seldom however reaching the normal, till the next paroxysm. The decrease involves chiefly the multinucleated ele- ments ; the large uninucleated cells as well as the tran- sitional forms are relatively increased. This is said to be particularly noticeable in advanced and severe cases. The smaller lymphocytes are usually also increased, whilst the eosinophile cells are somewhat diminished, but have occasionally been found more numerous in post-malarial anaemia. The large uninucleated, and to a less extent the multinucleated elements, according to Marchiafava and Bignami, are the leucocytes which assume a phagocytic action in malaria, and these, especially the former, are often seen containing pigment in little masses, or diffusely in the form of fine granules. These observers state that the multinucleated are not so often pigmented as the large uninucleated elements, while the lymphocytes and eosinophile cells never contain pigment. p THE BLOOD Melaniferous leucocytes are found in the blood during the attack and for twelve to twenty-four hours after, and when the organisms cannot be discovered are very strongly suggestive, though not absolutely diagnostic of malarial fever. Diagnosis. The presence of the amoeba in any of its various phases, and to some extent leucocytes containing granules, are the characteristic features which distin- guish malaria from many pathological conditions clini- cally resembling it, particularly forms of pyaemia, septicaemia, and miliary tuberculosis, in which rigors, profuse perspiration, etc., occur in paroxysms, with a temperature chart not unlike that of ague. Chronic malarial cachexia, with enlarged spleen, may be differentiated from diseases presenting somewhat similar signs, such as leukaemia, Hodgkin's disease, and the so-called splenic ansemia of adults and infants, by the examination of the blood, and particularly the absence of leucocytosis. The Blood in Acute Diseases. Nearly all the infectious fevers are associated with a more or less marked increase in the number of the leucocytes. The following are, however, the most notable excep- tions to this general statement : — Typhoid fever, malaria, influenza, tuberculosis, leprosy, and sometimes measles. Fever alone may cause some alterations in the blood. According to Maragliano it may first produce contrac- tion of the peripheral vessels, hence concentration of THE BLOOD IN PNEUMONIA the blood, aided also by the increased loss of water during febrile conditions by perspiration, etc., so that the corpuscles may be actually more numerous per cubic millimetre. After the fever has disappeared, dilatation of the vessels occurs, and partly from this cause, but also from the destruction of the cellular elements due to the increased metabolism, the number of the red cells shows marked diminution. The Blood in Fneumonia. The red blood corpuscles suffer very little numerical alteration during the febrile period, and even after the crisis, and whilst convalescence is being established, the anaemia is usually not pronounced. Hayem considers that an attack of ordinary pneumonia is responsible for the destruction of about one million red cells. The blood when examined, particularly in the fresh condition, shows an extremely well-marked increase in the amount of fibrin, which is probably only equalled by that seen in acute rheumatism. Hayem divides pneumonias according to the presence or absence of this condition of the blood, into those attended with a pronounced increase in the fibrin, which usually have a favourable prognosis, and those serious typhoid types, in which the " phlegmasique " (i.e., inflammatory) characters are completely absent, the latter almost in- variably ending fatally. The Leucocytes. Numerous observations have been made regarding the number of leucocytes in this disease. Most writers agree that, at least in favourable cases, there is a THE BLOOD marked leucocytosis, which is sometimes proportionate to the temperature — more usually, however, the constant parallelism between the degree of leucocytosis and the temperature is not seen. When the crisis occurs, the leucocytes generally diminish in number ; but when the temperature falls, but again rises — false crisis — they are somewhat diminished, but are still above the normal, according to von Limbeck. Some cases show no leucocytosis, and these are generally regarded as unfavourable. Ewing has given an excellent account of the subject in the New York Medical Journal {De-c^mb^r i6, 1893). He examined the blood in one hundred cases of lobar pneumonia, and as a result he expresses the opinion that {a) leucocytosis may be absent in very mild cases, or in very severe cases in which the reaction of the system is absent ; {b) that it is to some extent pro- portional to the extent of the lung involved, but usually depends on the degree of reaction of the system against the poison; and {c) that a "well-marked leucocytosis in lobar pneumonia, while in itself a favourable sign, does not assure that the disease will pursue a favour- able course, but indicates usually a severe infection. A moderately low degree of leucocytosis in severe cases is an extremely unfavourable sign, whilst in some cases its absence indicates, with rare exceptions, that the disease will prove fatal." It has been suggested by von Jaksch that leucocytosis should be artificially induced by the injection of irritants, e.g., pilocarpin, turpentine, etc., but this has not been found to be of any real service. The following state- ment by Cabot seems to me well worth noting, viz., TYPHOID FEVER 213 that " leucocytosis is checked by anti-pyretics (Hare), but not by cold bathing, which speaks in favour of the latter method of reducing temperature." During the febrile leucocytosis the multinucleated neutrophile elements are increased, whilst the lympho- cytes, and particularly the eosinophile cells — the latter may even be absent — are decreased, but after the crisis the multinucleated cells are relatively diminished, whilst the eosinophile forms are increased. The Diagnosis. The presence of leucocytosis will distinguish pneumonia from typhoid, malaria, and sometimes influenza. Ewing has also pointed out that in acute tubercular inflamma- tions of the lung, and in acute apical lesions, the presence of leucocytosis is in favour of simple pneu- monia, its absence pointing to the tubercular form of the disease. As regards the prognosis, the absence of leucocytosis, except in the very mild cases, is admitted by all to be a bad omen. Typhoid Fever. Quite recently the diagnosis of enterica has been rendered comparatively certain by Widal's method of examining the blood serum. Various modifications of the process are constantly being introduced, but the following by Sheridan Del^pine (in Allbutt's Medicine, vol. ii., p. 1 145, and British Medical Journal, April 17, 1897) seems to be very suitable. The Quick, or Microscopic Method. I. The blood is obtained in a small pipette of such diameter that a platinum loop, having a diameter of 214 THE BLOOD about one millimetre, may be introduced into it when it is broken transversely. 2. One drop of the blood, or serum, is diluted with nine parts of neutral bouillon, and they are thoroughly mixed. 3. One drop of this -^ dilution of blood and bouillon is mixed on a glass slide, with one or more drops of a young (twenty-four hours old) culture of the typhoid bacillus. The latter may either be a growth on bouillon, or an emulsion of the bacillus on agar with a small quantity of bouillon or normal saline solution. 4. Cover with a cover-glass, and examine under a magnification of 200 to 300 diameter. Delepine does not consider it necessary or advisable to separate the serum entirely from the corpuscles, and regards the presence of a few of the latter as ad- vantageous, as they support the cover-glass, and cause the films to be of uniform thickness. If the blood be from a patient suffering from typhoid, particularly after the first week, the bacilli rapidly, or slowly, according to the potency of the serum, become sluggish, and then frequently motionless. They are soon drawn together in little masses of varying sizes, the so-called "agglomeration" phenomenon. In most cases this occurs within five to thirty minutes, but in some rare cases it may take from one to two hours. If the blood be from a non-typhoid patient the bacilli remain diffused throughout the film, moving freely, and showing no tendency to the formation of clumps or clusters. Grtinbaum [British Medical Journal, May i, 1897) recommends a greater dilution, viz., i to 32. He first dilutes the blood serum i to 16, and then mixes one drop of this with one drop of the typhoid culture. TYPHOID FEVER 215 This writer further considers that a time limit is im- portant. He prefers an emulsion of an attenuated culture of the typhoid bacillus from agar rather than a bouillon culture. For collecting the blood he thinks capillary tubes, of not too fine a bore, are better than capillary pipettes. Johnston and McTaggart {British Medical Journal, Dec. 5, 1896), instead of fresh blood or serum, use dried blood. A large drop of blood is caught on a piece of sterilized paper, and this is afterwards moistened with sterilized water. Then the solution of blood so obtained is mixed with a drop of broth culture of the typhoid bacillus. By this mearts the blood may be transmitted a distance for examination. Wright and Semple {British Medical Journal, May 15, 1897), on the principle that blood serum of a typhoid patient produces agglomeration, even when brought into contact with a culture of the bacillus which has been killed by heat, have advised the use of dead bacilli in- stead of a fresh culture. They use an emulsion of fresh agar culture, in which the bacilli have been killed by exposure to a temperature of 60° C. for five to ten minutes. They consider that this method will obviate all necessity for culture apparatus, and the physician's " equipment in connection with the serum diagnosis may in fact be narrowed down to a supply of these capsules of dead bacteria and a small supply of glass tubing for pipettes." The value of the serum reaction in typhoid fever cannot be over-estimated, but as to which method is the most convenient and reliable I am not in a position to state, as typhoid fever is very infrequent in and about Bournemouth. 2i6 THE BLOOD The Blood in Typhoid Fever. The red blood corpuscles show very little alteration in the earlier stages of enterica, in fact, owing to the fever as well as the diarrhoea and perspiration, they may be apparently increased, but generally vary frotn 4|- to 5 million per cmm. During, or towards the end of the third week a marked diminution may take place, and not infrequently they fall to 3j- million, and towards the end of defervescence may number only 2^ million. During the course of this disease there is, according to Hayem, a loss of 2\ million of corpuscles even without the occurrence of haemorrhage. Intestinal haemorrhage may occasion very considerable diminution in the cellular elements, so that aneemia may be a conspicuous and dangerous feature. The duration of the disease is not the only cause of the oligocythaemia, the increased temperature playing an important part, and Hayem has mentioned that in cases in which the temperature has been kept low by anti- pyretics, cold baths, etc., the loss of corpuscular elements has been less than in those in which less active treatment was adopted. The haemoglobin is reduced in amount corresponding to, or greater than, that of the corpuscles. The leucocytes, which are never increased in number in uncomplicated cases, usually undergo a marked dimi- nution. Hayem states that they frequently fall below 2000, sometimes numbering only 1000 per cmm. The diminution is said to be most noticeable towards the end — Thayer states about the fifth week — of the fever. TYPHOID FEVER 217 According to most observers, if leucocytosis occur during typhoid, it is due to some complication, such as perforation, suppuration or abscess formation, pneumonia, haemorrhage, etc. Von Limbeck confirms this state- ment, but notices the fact that sometimes pneumonia or inflammatory complications may occur during or towards the end of typhoid fever without producing any leucocy- tosis. He records such a case in which the formation of abscesses did not raise the number of leucocytes above 5000 per cmm. The multinucleated elements diminish, whilst the uni- nucleated leucocytes, particularly the larger forms, are relatively increased, especially towards the end of the illness. After the fever has disappeared the former begin gradually to increase, but do not become normal for some time. The eosinophile cells are present, but not numerous. The blood plates are usually reduced in number, and according to Hayem they reach their minimum about the time when the high temperature begins to decline. The fibrin is apparently normal, and never shows in uncomplicated cases the marked increase which is found in many inflammatory conditions. The coagulation of the blood is unaltered. Diagnosis. The entire absence of leucocytosis, and more usually the diminution in the number of white corpuscles, enable us to distinguish uncomplicated cases of typhoid fever from many inflammatory conditions, which may at one or other time closely resemble it clinically. Perityphlitis, inflammatory and suppurative conditions, -and most of the acute infectious diseases, are at once 2i8 THE BLOOD eliminated, as leucocytosis is as constantly present in these as it is absent in enterica. Malaria and tuberculosis are associated with a diminu- tion in the leucocytes, and therefore indistinguishable by a mere count from typhoid fever. The presence of the malarial parasite, or pigment bearing leucocytes in the blood, points conclusively to the presence of ague. Scarlet Fever. There is usually a slight diminution in the number of the red corpuscles, which according to Hayem is most marked in mild cases on the first day of defervescence, in severe cases — in which the fall in temperature is delayed— about twenty-four hours after cessation of the fever. The same observer considers that, as in pneumonia, about one million of red corpuscles are lost during scarlatina. The haemoglobin decreases as the disease proceeds. The Leucocytes are usually increased, and von Limbeck and others notice that the leucocytosis may persist for some days after the temperature has fallen to and remained at the normal. He records a case in which the white cells numbered 14,000 thirteen days after the thermometer showed a normal temperature. The multinucleated are the form of leucocytes which are increased, and Kanthack and others state that the eosinophile cells are also more numerous, and may reach 8-15 % during the second and third weeks of the disease. Some writers — Kotschetkoff, Kanthack, and others — consider the eosinophile cells, except at the earliest stage of the fever, to be an unfavourable. SMALL-POX 219 sign. According to Neusser an increase in the eosino- phile cells in scarlatinal nephritis is a favourable, and their absence an unfavourable sign. The lymphocytes, Cabot states, are decreased pro- portionately to the severity of the case. The presence of leucocytosis distinguishes scarlet fever from measles in some, but not in all cases. Measles. Mild cases scarcely show any alteration in the blood, and seldom more than half a million red corpuscles are destroyed during its course. (Hayem.) Most observers — von Limbeck, Rieder, P6e, etc. — agree that there is no leucocytosis in uncomplicated cases, but when there is marked catarrhal affection of the mucous membranes, or bronchitis, the leucocytes as well as the fibrin are increased. The multinucleated elements, Felsenthal states, are relatively increased, the eosinophile cells, unlike those in scarlatina, diminished in number. The absence of leucocytosis helps to distinguish measles from scarlet fever, and from the roseola of syphilis. Small-Pox or Variola. Hayem considers that of all the eruptive fevers small- pox is attended with the greatest destruction of the red corpuscles, causing a loss of about two million during its entire course. The corpuscles are normal during the eruptive stage, increased during suppuration, possibly owing to thicken- ing of the blood, and rapidly decrease in number — from dilution of the serum, as well as actual destruction of the elements — after the temperature has fallen. Hayem THE BLOOD further remarks that in the haemorrhagic forms of small- pox the diminution of the corpuscles occurs immediately after, and is proportional to the amount of the haemor- rhage. During the occurrence of suppuration the inflamma- tory character of the blood — increase in fibrin and white cells — becomes most pronounced. The leucocytes in slight cases occurring in vaccinated individuals may, according to von Limbeck, show no increase, but in ordinary or severe cases, in the discrete forms during the third and fourth day, and in confluent small-pox towards the end of suppuration, the leucocytes are increased, and may reach 28,000 or 30,000 per cmm. in the latter form of the disease. In haemorrhagic variola the leucocytes may show a great increase. Hayem considers that the increase in leucocytes, when not due to suppuration, indicates a severe form of infection. Diphtheria. The red blood corpuscles are in the earlier stages usually normal, or even above the normal ; later there may be slight ansemia. It is stated that antitoxin injections tend to diminish the amount of oligocythae- mia, and to somewhat increase the rapidity of blood regeneration. The haemoglobin, according to Billings, suffers a slight reduction — about 10% — which is, however, less pronounced when antitoxin has been used. The leucocytes are, according to the majority of ob- servers, usually increased, and generally the greater the severity of the disease, the greater the leucocytosis. Its absence may indicate a mild attack or a very severe con- dition. Antitoxin injections only cause a temporary fall INFLUENZA 221 in the number of white cells shortly after they have been administered. Usually the multinucleated leucocytes, though sometimes the lymphocytes, are increased. Ewing states that the staining reaction of the white cells is a measure of the degree of infection. Myelocytes are said by Engel to be present in very severe cases, and he considers that when they occur in large percentages they constitute a bad sign. Erysipelas. Erysipelas produces much the same changes in the blood that pneumonia does, and the red corpuscles decrease, till they reach their minimum a few hours before the defervescence, and usually suffer a loss of from |- to I million or more in severe cases (Hayem). The haemoglobin is reduced about 10-20%. Leucocytosis is usually present, and, according to von Limbeck, is proportionate to the temperature and the degree of severity of the case. " The inflammatory character of the blood is a measure of the extent and intensity of the erysipelatous inflammation." (Hayem.) In some cases occurring in scrofulous individuals this author found the leucocytes numbered only 7 to 8 thousand, whilst in ordinary severe cases they varied from 12 to 20 thousand per cmm. Influenza. Canon, who examined the blood in twenty cases of influenza, found a number of short bacilli about the thickness, but half the length, of the bacillus of mouse septicaemia. These bacilli are not easily found in the blood, but they are usually numerous in the THE BLOOD yellow masses seen in the sputum. He made culti- vations from the blood on glycerine, or sugar agar, at 30° C. For staining the films he uses a mixture of eosine and methyl blue. The majority of writers consider that the leucocytes are usually normal, although Hayem states that " la grippe" is attended with an increase in the fibrin and leucocytes. Typhus Fever. Leucocytosis is constantly absent, according to von Limbeck, whilst it is stated that Everard and Demoor have found it present. Follicular Tonsillitis. This generally shows slight leucocytosis, and Halla has noticed the same in the febrile period of angina tonsillaris. Acute Eheumatism. In all forms of acute rheumatism, whether the synovial membranes are involved or not, the blood shows a remarkable increase in the amount of fibrin, and this disease, and lobar pneumonia are attended with the most marked inflammatory condition. This " phlegmasique " character of the blood, as Hayem terms it, is best seen in blood examined in the fresh condition and in not too thin films. It is recognized by the delay in the coagulation, the enormous increase in the fibrin, which forms a dense fibrillar network, and by an increase in the leucocytes. When these conditions are very pronounced Hayem states that in all probability either pneumonia or acute rheumatism is present, and considers the examination of the blood in the fresh state of the greatest assistance in diagnosis. INFLAMMATION OF SEROUS MEMBRANES 223 The red blood corpuscles are diminished, and Hayem considers this disease one of those which cause the most marked destruction of the corpuscles, and notices that the sooner the attack is stopped by salicylates, the less the anaemia. Frequently one or two million corpuscles are lost during the fever. The haemoglobin undergoes a diminution somewhat greater than that of the coloured elements, and not infrequently Hayem has noticed that when the cor- puscles increase in number during convalescence, the haemoglobin may for a time diminish. The leucocytes are almost invariably increased, and generally propor- tionally to the severity of the attack. In cases of moderate intensity they number 17 thousand to 18 thousand, in some cases as much as 25 thousand, whilst in subacute attacks they are just above the normal. There is apparently no leucocytosis in chronic and muscular rheumatism. Inflammation of Serous Membranes. N on -tubercular inflammations of the serous mem- branes are usually associated with leucocytosis, and von Limbeck states that there is a relation between the cellular richness of the exudation and the number of the leucocytes in the blood. Leucocytosis is always present in inflammations of these structures when they are due to streptococci, staphylococci, or pneumococci, but is usually absent in tubercular cases. He says that Rieder's observations are in agreement with his, in regard to the question of the differential diagnosis of tubercular from non- tubercular cases. 224 THE BLOOD Pleurisy. In simple acute pleurisy the fibrin and leucocytes are generally increased, but not to the same extent as in pneumonia. Hayem found that they varied from 7500 to 12,000, whilst von Limbeck records a non-tubercular case in which at first they numbered 13 to 15 thousand, but reached 22,000 per cmm. The leucocytosis is generally more pronounced during the fever, and may disappear when the temperature falls. According to Hayem the inflammatory character of the blood, particularly the increase in the amount of fibrin, is less marked in tubercular and cancerous pleurisy than in simple or rheumatic forms, and most observers consider that leucocytosis is absent in tuber- cular cases. When there is a large and continued increase in the number of the leucocytes, it suggests that a serous pleurisy has become purulent. The red corpuscles and haemoglobin are but little altered, except in severe cases. Peritonitis. Serous or purulent, non-tubercular, and particularly septic forms of peritonitis, generally show a well- marked leucocytosis associated with a great increase in the amount of fibrin ; but occasionally in very severe cases, as we saw was the case in grave forms of pneumonia, there may be no increase in the white cells. According to Cabot, intestinal obstruction or malignant disease may cause an increase in the leucocytes, but rarely the fibrin ; whilst tubercular peritonitis generally shows an entire absence of leucocytosis. MENINGITIS 22 s Pericarditis. This follows the general rule, and shows leucocytosis in simple and septic cases, but it is frequently absent in tubercular forms of the disease. Meningitis. Hayem states that one always finds an increase in the leucocytes if not in the fibrin, and von Limbeck that leucocytosis is present in non-tubercular but absent in tubercular cases. Osier, Zappert, Cabot, etc., found that leucocytosis occurs in some cases of tubercular meningitis, and the last-named observer considers that purulent meningitis shows a marked increase in the white cells, whilst the absence of leucocytosis would point to a tubercular form. Rotch, in one case, an infant suffering from tubercular meningitis, found the leucocytes numbered 38,000, and considers that this increase pointed to some complica- tion. In another case, also an infant, in which the white cells reached 37,500 per cmm., at the post-mortem ex- amination, in addition to tubercular meningitis, an ap- pendicitis was discovered, which accounted for the large number of leucocytes. Hayem considers that the presence of leucocytosis would help to distinguish simple meningitis from hysteria, but lead "encephalopathy" and uraemic coma may be indistinguishable, as interstitial nephritis can cause similar alterations in the blood. Cabot is of the opinion that cerebral tumours, hysteria, diabetic coma, sunstroke, etc., do not cause leucocytosis, whilst cerebral abscesses and apoplexy may. Q 226 THE BLOOD Septicaemia and Pyaemia. Numerous observers have found streptococci and staphylococci in cultures of blood taken, with the greatest antiseptic precautions, from patients suffering from many forms of septic infection. In some obscure cases, as for example septic endocarditis, very valuable assistance may be obtained by this method of research, but as the whole subject is somewhat outside the range of the blood proper, I must refer readers to those books dealing with bacteriology. The red blood corpuscles are usually diminished, and in severe cases the oligocythaemia may be very pronounced. The haemoglobin generally shows a diminution pro- portionate to that of the red cells. In some cases haemoglobinaemia, recognized by the reddish coloura- tion of the blood serum, is present. The Leucocytes. Apparently contradictory accounts are given as to the number of white cells in all forms of septicaemia. Thus von Limbeck states that leucocytosis may be entirely absent in some cases, whilst in others it may reach a very high degree. As illustrating this he quotes a case of septic endometritis, ending fatally, in which the leucocytes were never higher than 5000, and on the other hand, a case of septic endocarditis, in which they reached 21,000. Rieder always found leucocytosis, and doubts the correctness of von Limbeck's observations. The latter, however, explains this difference on the ground that the whole question of leucocytosis, depends on the intensity of the infection and the power of SUPPURATION OR ABSCESS FORMATION 227 resistance of the body. Thus in dogs, in which in- oculation of very virulent cultures of organisms caused no leucocytosis, death always resulted. He thinks that leucocytosis is particularly apt to occur in those cases in which exudation takes place, that high degree of leucocytosis is associated with exudation rich in cellular elements — or pus, and only slight in serous exudations, and that an absence of leucocytosis renders the presence of exudation formation improbable. The occurrence and degree of leucocytosis in septi- caemia is probably analogous to that seen in pneu- monia, etc., that is, it is marked in most cases, but absent in very mild or in very severe cases which end fatally. Its presence would in surgical cases, in which the temperature has gone up, point to sepsis, and would exclude fever resulting from hysteria or mental worry. Suppuration or Abscess Formation. The leucocytes generally increase proportionately to the intensity of the inflammatory condition, and reach their maximum when suppuration takes place. They suddenly fall when the pus escapes, but increase again when a new focus of suppuration occurs. As illustrating this point, Hayem mentions a case of suppurative pelvic peritonitis, in which the white cells numbered 21,000, and twenty-four hours after the evacuation of the abscess they fell to 9300. Similarly in an abscess of the forearm, in which they numbered 25,000, the next day, after incision and drainage, they were only 5000 per cmm., but subsequently became somewhat more numerous. 228 THE BLOOD When the inflammation is disappearing, the leuco- cytes gradually diminish, and at the beginning of convalescence they may sink below the normal before definitely attaining the number found in health. The increase of the white cells is almost invariably on the part of the multinucleated cells. Associated with this leucocytosis there is usually an increase in the amount of the fibrin. Hayem considers that the former is more essential, and is never completely absent from inflammatory con- ditions. The examination of the blood in cases of obscure suppuration may be of the greatest diagnostic value, particularly in abdominal diseases. Leucocytosis in such conditions would point to some inflammatory condition, e.g., typhlitis, perityphlitis, peri- tonitis, pelvic suppuration, etc. Malignant diseases usually show leucocytosis, but seldom an increase in the amount of fibrin. Speaking generally, absence of leucocytosis in uncomplicated cases would suggest typhoid, tubercular peritonitis, simple biliary or renal colic, obstruction from non- malignant disease, or fcecal accumulation, ovarian and pelvic neuralgias. A perinephritic abscess would show an increase, whilst a cyst of the kidney ' usually presents no increase in the white cells. Cabot, who has made extensive observations in cases of appendicitis, has stated that mild and very severe cases may show no leucocytosis, that an in- creasing leucocytosis may be the only evidence of extension of the process, whilst a persistently high increase of the white cells indicates a large abscess. SYPHILIS 229 Secondary Anaemia in Chronic Infectious Diseases. Syphilis. Some cases of syphilis, especially in the earlier stages, show little or no alteration in the blood, but generally during the secondary period, more or less anaemia is present. The red. blood corpuscles vary considerably, being frequently somewhat diminished, but rarely in simple cases do they fall according to Hayem below ^h •million. Usually the diminution in the haemoglobin is much more pronounced than that of the coloured elements, and, according to most writers, a chlorotic type of anaemia is frequently present. Sometimes a very severe form of anaemia, termed by the early writers the " pernicious anaemia of syphilis," occurs. Miiller describes a case of this kind, in which the haemoglobin was greater than the number of corpuscles, a condition frequently occurring in, and formerly considered characteristic of, pernicious anaemia. In this case the erythrocytes fell to 720,000, the haemoglobin being 18%. On the other hand, Lezius found that in twenty cases the red corpuscles were normal or nearly so, but the haemoglobin diminished. Most observers state that syphilis produces much greater anaemia in women than in men. Microscopically the corpuscles show little alteration except in the rare and exceptionally severe cases of this disease, in which all the signs of pronounced anaemia may be present. Thus Muller found in the severest form of syphilis various alterations in the elements, e.g., megalocytes, microcytes, poikilocytes, 230 THE BLOOD and nucleated red cells, usually normoblasts, but some- times megaloblasts. As von Limbeck states, the condition of the blood is most variable, and no one type characteristic of, or peculiar to the disease. The corpuscles may be normal, or may show a chlorotic condition, particularly in the earlier stages, whilst exceptionally in the severest forms of the disease, especially towards the end, the blood may resemble that seen in pernicious anaemia. The leucocytes, according to von Limbeck, are generally normal in number in slight as well as severe cases, and Lezius found the uninucleated and multi- nucleated forms were present in the proportion seen in health. Rille noticed leucocytosis, in which the lympho- cytes and large uninucleated cells were increased, as well as the eosinophile leucocytes, especially when the specific roseola was present. He also found a few marrow cells. Bieganski observed an increase in the leucocytes, and whilst the multinucleated were diminished the uninucleated forms were increased. The administration of mercury, however, caused an increase of the former and a diminution of the latter. It seems to me that there is no doubt that when the roseola appears, leucocytosis is generally present, and the uninucleated and eosinophile cells increased; whilst in the tertiary stage severe anaemia with pronounced lymphocytosis may occur. According to Cabot the existence in adults of leucocytosis, with an increase in the proportion of the uninucleated and eosinophile cells, is in favour of syphilis, and opposed to the presence of tuberculosis, typhoid, or malignant disease. TUBERCULOSIS 231 Tuberculosis. The blood shows exceedingly varied alterations in the different forms and stages of tubercle. In the commencement, there may be no appreciable anaemia, notwithstanding the pallor, and Hay em remarks, that in cases in which the patients appear very anaemic, absence of any notable alteration of the red corpuscles is presumptive evidence of the presence of tubercle. In phthisis it is not uncommon to find a normal number of red corpuscles associated with a diminution in the amount of haemoglobin ; and these cases of tubercular pseudo-chlorosis are often, according to von Limbeck, indistinguishable from pure chlorosis. In other cases oligocythaemia as well as oligo- chromaemia are present, whilst in exceptionally severe cases an extreme anaemia may accompany the disease. Thus von Limbeck records a fatal case of acute miliary tuberculosis, closely resembling pernicious anaemia, in which the red corpuscles numbered 730,000, the white 4300, and the haemoglobin 25%. The red cells frequently show some degree of pallor, and poikilocytes may be present. Cabot states that nucleated red blood corpuscles are very rarely seen, except after haemorrhage, whilst in malignant disease they are sometimes present in large numbers. The leucocytes are as a general rule not increased in pure uncomplicated cases of tuberculosis, and, according to Limbeck, tubercular meningitis and miliary peritonitis always show, even during the febrile period, a normal number of white cells. Most observers agree as to the absence of leucocytosis ^32 THE BLOOD in pure tubercular diseases dependent on the presence of tubercle bacilli only, but when other organisms are present and secondary complications occur, then the blood presents all the signs of septicaemia, and leucocy- tosis may even be pronounced. We shall briefly consider the effects of special forms of tubercular disease on the blood. Phthisis. In the early stages of apical catarrh, when fever is absent, the red corpuscles are normal, or somewhat below the normal, and the haemoglobin diminished. The leucocytes, when no complications attend the disease, show no increase. When cavities are present, but fever absent, or present in only a slight degree, the red blood cor- puscles are normal, or nearly so, and the white cells increased, often showing a leucocytosis of from 12 to 15 thousand. When the fever is of the hectic type, the red cor- puscles and haemoglobin are almost always diminished, the leucocytes variable. In cases of mixed infection, with high temperature, and of a generally septic character, leucocytosis is almost always found, whilst according to von Limbeck, in acute miliary or subacute miliary forms of the disease, it is usually absent. Von Limbeck expresses the opinion that acute miliary or subacute granular forms can be distinguished from septic, and infiltrating or caseous pneumonia. Accord- ing to this author, in cases of chronic septic phthisis, leucocytosis is usually moderate ; in the infiltrating forms (tubercular pneumonias) it is often very pronounced, occasionally reaching that found in croupous pneumonia, PHTHISIS 233 whilst in miliary tuberculosis of the lung leucocytosis is absent. When leucocytosis is present, the multinucleated forms are increased, whilst the eosinophile cells are diminished. When it is absent the various forms of white cells may be present in their normal proportion, or there may be a relative increase in the small and large uninucleated elements. Marrow cells have been found in very small numbers in advanced cases. Neusser notes, that in certain cases of phthisis, some of the large uninucleated, and multinucleated leucocytes contain a number of basophile granules, which he con- siders as a sign of the presence of a uric acid diathesis, and maintains that they justify a good prognosis. These cells, and their staining reaction, will be described under gout. Acute miliary tuberculosis, tubercular forms of peri- tonitis, meningitis, pericarditis, and pleurisy, when un- complicated, generally follow the rule of tubercular diseases, and show no increase — sometimes a diminution in ,the number of the leucocytes. The Condition of the Blood in various General Diseases. Constitutional Diseases. Diabetes. This disease is not attended with any constant or characteristic changes in the cellular elements of the blood. The red corpuscles are sometimes normal, sometimes diminished, but may show an increase towards the end. The white corpuscles, though generally normal, may occasionally show a very pronounced digestive leucocy- tosis, even when there has been no prolonged fast previously, (von Limbeck.) Recently two methods have been recommended for enabling a distinction to be made between normal and diabetic cases, by an examination of the blood alone. I St. Bremer s Film Method. [New York Medical Journal, March 7th, 1896.) {a) Film preparations of the blood are made and fixed by immersion for four minutes in a mixture of equal parts of alcohol and ether — which should be boiling. {b) They are then stained for four minutes in the following solution : — Saturated watery solutions of eosine and methyl-blue are mixed in equal proportions. The precipitate which forms is collected on a filter paper, washed and dried. When reduced to a powder, -^-^ part of eosine and \ of methyl-blue are added. DIABETES 235 Of this dry powder '025 to "05 grms. are dissolved in 10 grms. of a 33 % solution of alcohol. This staining fluid does not keep well, and must be freshly prepared before use. (c) After staining the films are washed in water and. mounted. The red blood corpuscles of diabetic blood are stained a green or greenish-blue ; those of normal blood reddish violet. Bremer has quite recently (British Medical Journal, July 3, 1897, Epitome) suggested the following modi- fications in the process, by which the alteration in the staining reaction can be seen with the naked eye. Films of blood taken from a diabetic patient are made on the slide, and should not be too thin. They are fixed by exposure to a temperature of 135° C. for six to ten minutes in a hot-air chamber. The temperature and time mentioned must not be exceeded. They can then be stained in a i % watery solution of methyl-blue for one to two minutes, after which they are washed and dried. Diabetic blood is not stained, or only imperfectly, whilst non-diabetic blood stains of a bluish-green colour. If the preparation be stained in Ehrlich-Biondi's solution for two to three minutes, the diabetic blood is stained orange, whilst normal blood is coloured violet. If films are first stained in i % watery solution of methyl-green for one and a half to two minutes, then washed and stained in ^ % of watery solution of eosine for ten seconds, according to this authority, the diabetic blood film remains green, whilst the non-diabetic blood takes on the eosine. 236 THE BLOOD Methyl-blue and eosine give similar results. 2nd. Williamson's Method {British Medical Journal, Sept. 19, 1896) : — i. Into a test tube 40 cmm. of water are placed. ii. After puncturing, 20 cmm. of blood are added. iii. Then i cmm. of a i in 6000 aqueous solution of methylene-blue are added. iv. To this mixture 40 cmm. of liquor potassae are added, and the tube is thoroughly shaken in order to mix these solutions. V. It is then allowed to boil for about four minutes. This writer uses the capillary tube of Gower's haemo- globinometer, which holds 20 cmm., and the i cmm. pipette which accompanies Southall's ureometer, to measure these quantities. The tube containing diabetic blood changes from a blue to dirty yellow or straw-colour, whilst that contain- ing normal blood remains of a blue or bluish-green, but never decolorizes. In both of these methods control experiments with normal blood should be made at the same time. Gout. The red corpuscles may show no alteration, whilst the leucocytes are usually normal according to Limbeck, although Neusser states that they are increased. Uric acid crystals may be obtained by acidulating serum, obtained from a blister, with acetic acid (about six drops of 25 % acetic acid are added to one drachm of the serum). Crystals will form on a fibre of linen placed in the watch-glass in about twenty-four to forty- eight hours. Neusser has drawn attention to the occurrence of MYXCEDEMA 237 basophile granules in the large uninucleated and multi- nucleated leucocytes in cases of gout and uric acid diathesis. Film preparations of blood fixed in the usual way are stained in the following solution, which is a slight modification of Ehrlich's triple stain : — Saturated Watery Solution of Methyl-green . 80 cmm )) >j >j Acid Fuchsin • 5° .. )) )> )) Orange-green • 7° n Distilled Water . 150 n Absolute Alcohol ■ 80 „ Glycerine ■ 20 „ Mix. These leucocytes are found in tubercular disease, gout, uric acid condition, and occasionally in diabetes and leucocythaemia. Von Limbeck does not consider that they represent the existence of a uratic diathesis. Janowski has found similar granules in pus corpuscles, and regards them as the result of degeneration of the leucocytes. Myxoedema. In a child suffering from myxoedema, von Lebreton found that before treatment the red corpuscles numbered if million, the haemoglobin 65 %, and the leucocytes 4500 ; after treatment with thyroid gland for forty days, the red cells increased to 2,450,000, the haemoglobin 68 %, and the white cells 9600 per cmm. He found nucleated red cells before treatment, but these disappeared as the ansmic condition improved. In a somewhat extensive examination of the blood of two cases of this disease, I found the red cells somewhat diminished, and noticed the prevalence of large uni- 238 THE BLOOD nucleated leucocytes, a few of which were probably marrow cells. Since that time Thomas has also found the latter in myxoedema, but only in very small numbers. Addison's Disease. The aneemia seldom reaches a high degree, and dry preparations of the blood show little or no alteration. Diseases of the Alimentary System. In Ulcer of the Stomach the anaemia may, in some instances, be extremely pronounced, whilst in other cases only moderate oligocythaemia is present. The haemoglobin is generally diminished to a greater extent than the number of corpuscles would suggest, but this is not always the case. Osterspey found that whilst in one case the red cells numbered 2,640,000, and the haemoglobin 30%, in another they had fallen to 1,900,000, with 3 1 % of haemoglobin. The leucocytes are not usually increased except after haemorrhage, or a meal of albuminous food. In Cancer of the Stomach severe anaemia, associated with the presence of extreme poikilocytosis and numerous microcytes, may be found, whilst nucleated red cells, especially normoblasts, are not infrequently seen. The leucocytes are generally increased in number — the leucocytosis of malignant disease — particularly the multinucleated variety, and the number of the latter, according to Hartung, is directly proportional to the cachexia. The eosinophile cells are usually diminished. The occurrence of digestive leucocytosis shortly after food is usually found in gastric ulcer, whilst its absence is, according to Limbeck, Hartung, and others, a strong indication of cancer of the stomach. DISEASES OF THE LIVER 239 Intestinal parasites, particularly the bothriocephalus latus, may produce very severe anaemia, in some cases closely resembling that found in pernicious ansemia. Not only normoblasts, but even megaloblasts, have been seen in comparatively large numbers in a few cases. Acute gastritis and gastroenteritis are usually asso- ciated with leucocytosis, but this is absent in typhoid fever. In chronic gastritis there is usually no increase in the white cells, and, according to Cabot, digestive leucocytosis is sometimes absent. Diseases of the Liver. The red corpuscles vary in number considerably in different forms of hepatic diseases. In some cases, particularly towards the end of atrophic cirrhosis of the liver, very marked oligocythaemia may be found. Hayem has noticed that in many forms of cirrhosis, even when no jaundice is present, the red cells are more adhesive than in the normal condition, and vacuoles may be seen in their interior. In catarrhal jaundice von Limbeck found that the red corpuscles were larger, and the haemoglobin more firmly attached to them than in health. The leucocytes are also extremely variable in number. According to von Limbeck, in acute hepatitis and abscess of the liver they are always increased, in uncomplicated catarrhal jaundice they are never increased, whilst in cancer of the liver moderate leucocytosis is nearly always present. The leucocytes, in different forms of cirrhosis of the liver, vary considerably according to the nature and progress of the case. 240 THE BLOOD Diseases of the Respiratory System. We have previously described the occurrence of well- marked leucocytosis in pneumonia, and its absence in the early stages of phthisis, miliary tuberculosis, and tubercular pleurisy. Acute bronchitis is, at least during the febrile period, usually associated with an increase in the white cells, which generally reaches, according to Limbeck, 15,000 per cmm. Pneumonia, empyema, and cancer of the lung are often attended with a greater increase in the number of white cells, than is generally found in serous pleurisy, whilst in tubercular pleurisy leucocytosis is absent in uncomplicated cases. Malignant disease of the lung, although causing leucocytosis, is not usually associated with the marked increase in the amount of fibrin, which is found in inflammatory diseases of these organs. In pulmonary emphysema and asthma the red corpuscles may show an apparent increase, due to cyanosis. The eosinophile cells are, according to many observers, more numerous in these two con- ditions, particularly just before, during, and immediately after a paroxysm of asthma. Increase of these cells is peculiar to pure bronchial asthma, and is absent from those cases of dyspnoea, or so-called asthma of renal or cardiac origin. I can confirm the state- ment, that an increase of eosinophile cells in the blood is associated with a similar increase of these cells in the sputum, during an attack of this disease. DISEASES OF THE CIRCULATORY SYSTEM 241 Diseases of the Circulatory System. Pericarditis and endocarditis, especially the septic forms, are attended with leucocytosis. According to Hayem, endo- and pericarditis, when of rheumatic origin, are the forms which are associated with the most pronounced inflammatory character of the blood, i.e., an increase in the leucocytes and fibrin, and he is of the opinion that, even in cases in which articular manifestations of the disease are absent, the occurrence of a dense network of fibrin points to rheumatism as the primary cause. In infectious and septic forms of endo-pericarditis, although the leucocytes are frequently markedly in- creased, Hayem states it is rare to find an excessive proportion of fibrin. In obscure cases of septic endocarditis, bacteriological cultures of the blood may give invaluable help in the diagnosis. Valvular diseases of the heart, when compensation is present, usually show no alteration in the blood, but when this is imperfect or absent, and the effects of backward pressure occur, a diminution, or more frequently an increase of the red corpuscles, is found, owing to concentration of the blood serum, which results from oedema, etc. In the cyanosis of con- genital heart disease, polycythaemia may be very pronounced. The leucocytes are normal in organic valvular disease, except when complications are present. 242 THE BLOOD Diseases of the Kidney. In most forms of nephritis the red corpuscles are usually found to be normal, or somewhat diminished, but in some cases, according to von Limbeck, par- ticularly those which are of a haemorrhagic type, very pronounced anaemia, in which the red corpuscles may fall to i^ or even i million per cmm., is sometimes found. The haemoglobin undergoes a diminution propor- tional to, or somewhat greater than, that of the red cells. The majority of observers state that the leucocytes are moderately increased in the greater number of cases of acute or chronic nephritis. According to Hayem the interstitial form is attended with the greatest increase in the amount of fibrin. Von Limbeck states that there is frequently an increase in the eosinophile cells in nephritis. In uraemic conditions the blood shows practically no alteration other than existed before in the number of the red or white blood corpuscles. Malignant disease of the kidney is often attended with very pronounced leucocytosis. In floating kidney the blood is normal, whilst in renal calculus there may be some degree of leucocytosis, especially when ulceration is present. Nervous System. We have previously mentioned the occurrence of leucocytosis in simple meningitis, and its frequent, although not constant absence, in the tubercular form of the disease. THE ANEMIAS OCCURRING IN lI^FANTS 243 In many functional diseases of the nervous system — hysteria and neurasthenia — a slight reduction in the amount of the haemoglobin is not infrequently seen, whilst in chronic organic nervous diseases one often finds a slight anaemic condition. The eosinophile cells are occasionally increased in number in chorea, hysteria, and neurasthenia generally. The Anaemias occurring in Infants. The blood of infants, and to a less extent young children, presents certain differences from that of the adult. The red corpuscles are more numerous at birth, constituting, according to Hayem 5,350,000, according to Schiff 5 "8 million per cmm., and the former observer has noted greater variations in their size at this period of life. The haemoglobin, though somewhat increased in amount, is less firmly attached to the corpuscles than in the adult. Nucleated red cells, notwithstanding their presence in the earlier periods of intra-uterine life, are absent during the later months, and are rarely found in the blood of a healthy mature child at birth. Normoblasts, and even megaloblasts, may occasionally be seen in the blood of premature infants for a few days after their birth. The leucocytes are also more numerous during the first week of life, constituting "the leucocytosis of the newly-born." Hayem places their number at birth at 21,000 per cmm. The uninucleated forms, as well as the eosinophile cells, are relatively numerous, whilst 244 THE BLOOD the multinucleated, or neutrophile leucocytes, are diminished. The number and proportion of the corpuscular- elements during the early periods of life are shown in the following tables, taken from an excellent article on the blood of infants and children in Rotch's Pediatric. At birth red blood corpuscles 5,900,000, leucocytes 2 1,000. Seventh day ., „ „ 5,000,000 „ 15,000. First year „ „ „ „ ., 10,000. Sixth year and upwards ,, „ „ „ „ 7,500. The Proportion of Leucocytes in Adults and Infants. Adults. Infants. Small uninucleated lymphocytes . 24-30% ... 50-75% Large uninucleated leucocytes . . 3-6 % ... 6-14% Multinucleated, or neutrophile . 60-75% ••■ 28-40% Eosinophile cells . . . 1-2 % ... i-io% Grundobin found that at the third year the multi- nucleated were equal in number to the uninucleated leucocytes, and after the eighth to tenth year there was little difference between the white cells of children and adults. Under pathological conditions the blood of infants may show changes which do not ordinarily occur in the adult. In young children severe anemia, associated with pronounced alteration in the size and shape of the red corpuscles, may result from very trifling causes. Nucleated red corpuscles are not infrequently found in cases of slight oligocythaemia, and are generally more numerous in all forms of anemia at this age. The leucocytes are much more readily increased in number, and leucocytosis is often associated with many forms of anaemia. Whilst in adults leucocytosis is mainly SPLENIC ANEMIA OF INFANTS 245 due to an increase in the multinucleated elements, in infants it involves an increase in the large or small uninucleated leucocytes, being in fact a lymphocytosis. Marrow cells are much more frequently found in the severe anaemias of infants than in adults. Anaemias, during the early periods of childhood, are often associated with an enlargement of the spleen. Rickets, hereditary syphilis, and to a certain extent tuberculosis, are responsible for some of the severest forms of anaemia in infancy. Anaemia Infantum Pseudo-leukaemica, or the Splenic Anaemia of Infants. Synonym : "Anaemia splenica infettiva dei bambini," according to Italian writers. In 1889 von Jaksch described under this term a form of anaemia occurring in children, which was characterized by marked enlargement of the spleen and at times slight enlargements of the lymphatic glands and liver. The blood showed considerable oligocythaemia, associated with pronounced and persistent leucocytosis. He found that some of these cases recovered. Hayem and Luzet described cases resembling those of von Jaksch. Luzet was, however, of the opinion that the disease usually ended fatally. I will give a very brief outline of the clinical features of the disease, as it is not even mentioned in most of the text-books on medicine. This form of splenic anaemia occurs most frequently in children under four years of age, but particularly between the ages of seven and twelve months. 246 THE BLOOD It is not infrequently associated with the presence of rickets, and to a less extent inherited syphilis. The disease begins insidiously, the anaemia gradually increasing till the waxy pallor of the skin is conspicuous. Sometimes the infants are emaciated, whilst at other times they are well nourished, but in all there is pro- gressive weakness. The spleen may be enlarged to such an extent as to occupy nearly the entire left side of the abdomen. More frequently, however, it is only moderately in- creased in size. The organ is firm and resistant to the touch. Post-mortem examination shows hyperplasia of the splenic elements. The liver may be slightly enlarged, but frequently is of the normal size. . The lymphatic glands are in some cases enlarged. The Condition of the Blood. The red corpuscles are always considerably reduced in number, and are usually below three million. In one case von Jaksch found that the red corpuscles fell to 820,000, whilst the white cells numbered 54,666 per cmm. The coloured cells frequently show signs of Mara- gliano's degeneration and polychromatophilia. The corpuscles are also of unequal size and shape, and all degrees of poikilocytosis may be seen. Nucleated red cells are very numerous, and according to many observers characteristic. They are for the most part normoblasts, and many show karyokinetic nuclear division, which Luzet con- siders very significant of the disease. Large nucleated SPLENIC ANEMIA OF INFANTS 247 red corpuscles — megaloblasts — are sometimes found. The haemoglobin undergoes a marked diminution. The leucocytes are always increased, generally ranging from 40,000 to 114,150 per cmm. Von Jaksch found the proportion of white to red corpuscles in three cases was as 1:12, 1:17, and 1:20. Usually the multi- nucleated elements predominate, but sometimes the uninucleated are in excess. The eosinophile cells are, according to von Jaksch, diminished, but Luzet, Zappert, and others found them increased. Marrow cells have been found in small numbers in some cases. Von Jaksch considered this a special disease of infants, from the occurrence of marked enlargement of the spleen, the absence of a leukaemic character of the blood, as well as the usually favourable prognosis. Von Limbeck and many other observers regard it as not a special form of ansemia, much less a special disease of early life. Stengel considers that it is not an independent disease, but probably a form of secondary ansemia, which owes its characteristics — enlargement of the spleen, leucocy- tosis, and the numerous nucleated red cells — chiefly to the age of the patient. In favour of its being of secondary nature is the fact that Fischl found that in 18 cases of this disease 16 were affected with rickets, whilst two presented all the signs of inherited syphilis. In the latter two cases oligocythaemia and leucocytosis were very pronounced. 248 THE BLOOD Diagnosis. It would appear that sometimes so-called anaemia infantum pseudo-leukaemica shows all the signs of a primary anaemia — either pernicious anaemia, leukaemia, or Hodgkin's disease ; and at other times it occupies a position intermediate between primary and secondary anaemia. The presence of enlargement of the spleen does not- exclude it from being pernicious anaemia, as although this disease in adults is usually associated with no en- largement of that organ, and no leucocytosis, yet any severe anaemia in infancy may be attended with both these conditions. The increase in the number of leucocytes may not be considered sufficiently pronounced to suggest leukaemia, but a case of the spleno-medullary form of this disease — which we have previously described — an enlarged spleen, numerous nucleated red corpuscles, and only 44,000 leu- cocytes, would apparently be considered by many as one of splenic anaemia of infants. Here, however, the pre- valence of marrow cells, to my mind, absolutely settled the diagnosis of leukaemia. In this particular case,, within five or six weeks, the leucocytes increased from 44,000, or a proportion of white to red of i : 78, to 555-795; and a week later to 696,960, or a proportion of I : 4, and justified the diagnosis. Given a case of anaemia in a young child, in which the spleen is considerably enlarged, the following diseases in addition to anaemia infantum pseudo-leukaemica suggest themselves : leucocythaemia, — spleno-medullary or lym- phatic variety, — pernicious anaemia, or severe secondary anaemia associated with rickets and syphilis. SPLENIC ANJEMIA OF INFANTS 249 If blood films show that the marrow cells are numer- ous, constituting 30 to 50 % of all the leucocytes found, I should consider the case one of spleno-medullary leu- kaemia. As we have previously pointed out, qualitative alterations of the leucocytes are more important and characteristic of leukaemia, than mere numerical increase of the white cells. If, however, the leucocytosis were very pronounced, but consisted in an almost exclusive increase in the lymphocytes, I should regard it as probably one of lym- phatic leucocythaemia, although the diagnosis might be somewhat difficult. Prevalence of large nucleated red corpuscles — megalo- blasts — as well as megalocytes associated with very severe and progressive oligocythaemia, and to a less extent oligochromaemia, would suggest the presence of pernicious anaemia. From Hodgkin's disease, especially when this is associated with leucocytosis, the diagnosis would present the greatest difficulty. In both the spleen and lym- phatic glands may be enlarged, and in either, especially in the splenic ansemia of adults, the former may alone show enlargement. I cannot help regarding some forms of anaemia in- fantum pseudo-leukaemica as similar to the so-called splenic anaemia of adults, and am of the opinion that both may be mere varieties of Hodgkin's disease, or pseudo-leukaemia. 250 THE BLOOD Inherited Syphilis in Children. The red corpuscles may be considerably reduced in number. The haemoglobin is sometimes diminished proportionately, sometimes less than, whilst not infre- quently the reduction is greater than that of the red cells, constituting in the last case the so-called chlorotic condition of the blood. Loos and Fischl have noticed that microcytes, megalocytes, poikilocytes, and poly- chromatophilia are sometimes found, whilst in the severe forms of anaemia normoblasts and even megalo- blasts may be seen. The leucocytes vary in number, but are usually some- what increased, ranging from 12,000 to 58,000. Loos found the following percentage of the different forms of white cells : lymphocytes, 15 '9 to 337 ; multi- nucleated elements,- 26'4 to 587 ; and a marked in- crease in the transitional forms, which numbered 22 to 52-9 %• Fischl noticed that the eosinophile cells were not increased, and Loos stated that they varied consider- ably, but were usually increased when the specific ex- anthemata appeared, and this is confirmed by Neusser and Zappert. Marrow cells and leucocytes containing basophile granules were found by Loos. Rotch points out that there is nothing characteristic of congenital syphilis in infants, the condition being that of severe secondary anaemia, frequently associated with the presence of an enlarged spleen. In an infant of three months he found the red corpuscles numbered 3,387,000, the leucocytes 20,000, and the haemoglobin 47%. ANEMIA OF RICKETS IN CHILDREN 251 The Anaemia of Rickets in Children. Severe forms of rickets are nearly always associated with a marked diminution in the red corpuscles. Vort Jaksch records a case in which they fell from 1,600,000 to 750,000 in three months. The corpuscles present all the signs of anaemia, and nucleated red cells, especially normoblasts, are frequently seen. Slight forms of rickets may, however, present no signs of anaemia. The haemoglobin generally suffers a reduction pro- portional to that of the red cells. The leucocytes are almost invariably increased, and may reach as many as 45,000 per cmm. According to von Limbeck the multinucleated forms predominate, whilst Rotch states that the lymphocytes were greatly increased. These apparently conflicting statements can be explained by the age of the child. The younger the child, the more numerous are the small uninucleated leucocytes, whilst in older children the leucocytosis consists chiefly of the ordinary multi- nucleated cells. Marrow cells and karyokinetic signs of division of the nucleus of the leucocytes are sometimes seen. Rotch mentions that in some cases the spleen is enlarged. Inflammatory conditions of the alimentary tract, and tuberculosis, are usually attended with considerable anaemia in young children. Leucocytosis is almost invariably present, even if no oligocythaemia is found, and the lymphocytes are usually increased, whilst the multinucleated forms diminished. .252 THE BLOOD In the various forms of tuberculosis affecting children the leucocytes, contrary to that found in the adult, are usually more numerous. Monti and Berggrun found that, in a child of nearly seven suffering from tubercular disease of the lymphatic glands, the red corpuscles were 3 "64 million, and the white 14,000; and in another child of about the same age, with chronic tuberculosis of the lung and peri- toneum, the red numbered 3 "2 3 million, and the white corpuscles 1 7, 200 per cmm. LITERATURE TO WHICH REFERENCE HAS BEEN MADE. Hayem, "Du Sang," Paris, 1889. Von Limbeck, "Grundriss einer Klinischen Pathologie des Blutes," Jena, 1896. Muir, " Contribution to the Physiology and Pathology of the Blood," Jour, of Anat. and Phys., vols, xxv., xxvi., and xxvii., 1891. Muir, " Method of Examining Blood, Bone Marrow," etc.. Journal of Anatomy and Physiology, 1892, page 394. Muir, " Observations on Leucocythaemia," Journal of Pathology and Bacteriology, vol. i. No. 2, Oct., 1892. Muir and Drummond, "On the Structure of the Bone Marrow in Relation to Blood Yorxasixon^' Jour, of Anat. and Phys., Oct., 1893. Muir, "On Changes in the Bone Marrow in Pernicious Anaemia," Jour, of Path, and Bacter., vol. ii. No. 3, Feb., 1894. Kanthack, "The Histological Changes of Blood in Diseased Con- ditions," Medical Chronicle, July, Aug., and Oct., 1894. Kanthack and Hardy, " The Morphology and Distribution of the Wandering Cells of Mammalia," Jour, of Phys., vol. xvii., 1894. Kanthack and Hardy, "On the Characters and Behaviour of the Wandering Cells of the Frog, especially in relation to Micro- organisms," Proceedings of Royal Society, vol. lii., Jan., 1893. Sherrington, "Note on some Changes in the Blood of the General Circulation consequent upon certain Inflammations of an Acute Local Character," Proc. of the Royal Society., vol. Iv. No. 332, 1894. Gulland, "Nature and Variety of Leucocytes," The Report of the Laboratory of Royal College Physicians, Edinburgh, iii., 1891. Gulland, "On the Granular Leucocytes," ybz^r. of Phys., vol. xix., 1896. Gulland, "A Rapid Method of Fixing and Staining Blood Films," British Medical Journal, March 13, 1897. Miiller, H. F., "Die Methoden der Blutuntersuchung," Centralblatt fur Allgemeine Pathologie u. Pathologische Anatoinie, No. 19 and 20, 1892. Rieder, "Atlas der Klinischen Microscopie des Blutes," Leipzig, 1893. Mackenzie, "The Lettsomian Lectures on Anaemia," The Lancet, Jan. loth, 24th, Feb. 7th, 1 89 1. 254 THE BLOOD Ewing, "A Study of the Leucocytosis of Lobar Pneumonia," New York Med. Journal, Dec. i6th, 1893. Bruhl, " De la Splenom^galie Primitive," Archives Generales de Midecine, 1891. Laveran, "Paludism," New Sydenham Society, 1893. Marchiafava and Bignami ; Mannaberg, " Malaria and the Parasite of Malaria Fevers," New Sydenham Society, 1894. Stengel, article on "The Diseases of the Blood" in the Twentieth Century of the Practice of Medicine, 1896. Rotch, article on "The Blood in Infancy and Childhood" in Hatch's Pediatrics, 1896. Buchanan, "Leukaemia," Journal of Pathology and Bacteriology, vol. iv. No. 2, 1896. Williamson, "Method of Distinguishing Diabetic from Non-Diabetic Blood," British Medical Journal, Sept. 19, 1896. Stockman, "The Nature and Treatment of Pernicious Anaemia,'' British Medical Journal, May 4, 11, 18, 1895. Hunter, "The Pathology of Pernicious Anaemia," British Medical Journal, Feb. 8, 1896. West, "Splenic Anemia," British Medical Journal, June 13, 1896. Oliver, " The Croonian Lectures : A contribution to the Study of the Blood and Circulation,'' June, 1896, British Medical Journal. Manson, various contributions on " Malaria " in the Brit. Med. Jour. Johnston and McTaggart, "Observations on the Serum Reaction in Typhoid," British Medical Journal, Dec. 5, 1896. Taylor, "Discussion on Anaemia at the British Medical Conference," British Medical Journal, Sept. 19, 1896. Martin and Mathewson, "Observations upon the Relation between Leukaemia and Pseudo-leukaemia," Brit. Med. Jour., Dec. 5 and 12, 1896. Von Jaksch, Clinical Diagnosis, 1893. Delepine, " Technique of Serum Diagnosis, with special reference to Typhoid Fever," British Medical Journal, April 17, 1897. Bremer, " The Examination of the Blood in Diabetes," British Medical Journal: Epitome, 1896, July 3, 1897, and Aug. 28, 1897. Wright and Semple, " The Serum Diagnosis of Typhoid by means of Dead Typhoid Bacilli," British Medical Journal, May 15, 1897. Cabot, " The Clinical Examination of the Blood." Longmans, Green, and Co., 1897. AUbutt's System of Medicine, vols. i. and ii., 1896 and 1897. Osier's, Fagge's and Reynolds' Medicine. INDEX. Abscess formation Acetic acid Achromacytes Achromatophile . Acute diseases, Blood in . Addison's Disease Alimentary system, Diseases of 238 Alcohol and Ether Mixture Anaemia Classification . Definition Degrees of In Infants PAGE 6 38 44 210 238 25 92 97 93 94 243 Infantum pseudo-leuk^mica 245 Malarial . . . 208 Pernicious . . .109 Post-hsemorrhagic . -183 Primary or idiopathic . . 97 Pseudo-leukaemica infantum 245 Red Corpuscles in . -94 Secondary or symptom- atic . . 98, 174 Splenic, in adults . .166 Splenic, in infants . . 245 White corpuscles in . -96 Appendicitis . . . 228 Asthma . . -75, 240 Basophile cells — in Leuksemia . Staining Neusser's Blood- Counting Corpuscles, red Corpuscles, white Crises . • 77 . 140 . 28 • 237 I • 37 . 62 S3, 102, 118 Blood {contd.) — Destruction of . . .188 Examination of fresh . -17 Examination of stained . 20 Examination with preserv- ing liquids . . -19 in Acute diseases . .210 Methods of examining i Morphology of. 37 -Plates ... 88 -Plates, distinguished from Malarial parasites . . 203 Bone marrow . . . 58 „ „ in Pernicious Ansmia . . . 121 Bottle method of staining . 36 Bremer's method of staining Diabetic blood . . 234 Bronchitis . . . 240 Cancer or, carcinoma . .179 Canon's staining mixture . 30 Cerebral disease . . . 225 Charcot- Ley den crystals . 145 Chart of the Malarial parasites 206 Chart of the various Anaemias 172 Chemiotaxis . . . 86 Chloranasmia, see Chlorosis . 98 Chlorocytes . . . 38 Chlorosis — . . . 98 Diagnosis of . . . 104 Hsemoglobin in ■ • 99 Leucocytes in . . . 103 Nucleated red corpuscles in loi Red blood corpuscles in . 99 Chlorotic condition of the blood . . . 98 2s6 THE BLOOD PAGE Chronic infectious diseases . 229 Cleaning the blood pipette . 9 Colour index . . 16, 94 Constitutional diseases . . 234 ■Cornil and Ranviers' cells . 135 Corpuscles — Alterations in . . -38 Pathological changes in . 41 Physiological „ „ . 39 Red . . ■ ■ 37 White, see leucocytes . . 62 Counting the red corpuscles . I „ „ white „ 6 Cover-glass films . 20 Crenation . . . 38 Crescents . . 201 Crystals . . . 145 Degenerative changes in the red corpuscles . . 45 Destruction of blood cor- puscles . . 93, 188 Development of the blood . 55 Diabetes — . . . 234 Bremer's Method . 234 WiUiamson's Method . . 236 Differential chart of Anamias . . .172 Differential chart of Malarial parasites . . 206 Differential count of the leucocytes . . .12 Digestive leucocytosis — 80, 181 In cancer and ulcer of the stomach . .181, 238 Diluting fluids — Hayem's . . . u Pacini's . .11 Toisson's . 12 Diphtheria . . 220 Dried film preparations — Fixing . . . . 23 On slides . . . 20 Making . . 20 Staining . . . 26 Dwarf corpuscles • 38 Ehrlich-Biondi stain . -31 Ehrlich's Acid dahlia solution 28 Ehrlich's Triple stain . . 30 Eichhorst's corpuscles . 43, 1 1 5 Emphysema . . . 240 Empyema . . 224, 240 Endocarditis . . . 241 Endoglobular degeneration of the red corpuscles . . 45 Enumeration of the cellular elements . . . i Eosine-hsematoxylin . 34 Eosinophile cells . .71 „ „ staining . 27 Eosinophile marrow cells 76, 136 Erysipelas . . .221 Erythroblasts = nucleated red corpuscles . . . 60 Erythrocytes = red blood cor- puscles . . 37 Film preparations Fixing films Flagellated malarial parasites. Fleischl's haemometer 20 23 202 13 Floating kidney . Food- 242 Influence on red corpuscles „ „ white „ Formol . . . . 40 80 26 Gastric cancer 238 „ ulcer Gastritis . . . . 238 239 General Diseases, the blood in . . . . Giant cells of the Marrow 234 61 Giant corpuscles = megalo- cytes . . .43 Gout . . . . Gower's H^mocytometer „ Hsemoglobinometer . „ Solution 114 236 10 IS II GuUand's method of staining . 32 INDEX 257 H sematoblasts = Blood Plates . 88 Hasmatoxylin . • • 33 Haemocytolysis . . 93, 188 Hsemocytometer, Gower's . 10 „ Thoma-Zeiss i Hsemoglobin — . . . 44 Estimation of . . 12 in Chlorosis . • ■ 99 in Leucocytes . . 82, 96 in Leukaemia . . -143 in Pernicious anaemia . • 113 in Secondary ansemia . .174 Haemoglobinaemia — . . 188 Haemoglobinuria . .188 Haemolysis . 93, 188 Hsemometer . . -13 Haemophilia . 188 Haemorrhagic anaemia . 183 Hemorrhagic diseases . 185 Hayem's solution . 1 1 Heart diseases . . ■ 241 Heat for fixing films . . 24 Hodgkin's Disease, or pseudo- leukaemia . . .162 Hodgkin's Disease, or acute pseudo-leukaemia . . 165 Hyaline cells = large uninu- cleated leucocytes — . 66 Hydrobilin . . .123 Hypoleucocytosis 79; 84 Hysteria . . .243 Infancy — The blood in . . ■ 243 Rickets in . . .251 Splenic anaemia of . . 245 Syphilis . . . 250 Inflammation of Serous Mem- branes . • • 223 Influenza . • .221 Jaundice . .120, 239 Kanthack's method of staining 29 Karyokinesis Kidney — Cancer of Diseases of Floating Inflammation of Stone in PAGE 59. 141 181, 242 . 242 . 242 . 242 . 242. Lactation, influence on the red corpuscles . . -41 Laveran's corpuscles . 201 Leucocytes — . . . 62 Eosinophile . . -71 Influence of physiological conditions on . . 80 Large uninucleated or Hyaline . . . 66- Multinucleated. . . 67 Neutrophile . . 67 Number of . . . 79 Pathological alterations in . 81 Pigmented . . 83, 210^ Proportion of the various forms . . . 80 Small uninucleated or lym- phocytes . . 64 Leucocythaemia — 127 Acute . ■ ■ 160- Bloodin . . . 128 Crystals in blood of . . 145 Diagnosis of lymphatic . 157 „ spleno -medul- lary . . . 152 Effects of other diseases on. 131 Eosinophile cells in . 135 Haemoglobin in . i43 Lymphatic . -145 Lymphocytes in . 139, 146 Marrow cells or myelocytes in . . . 133, 147 Mitotic figures in . . 141 Nucleated red cells in. 144, 148 Pathology of . . 148 Red corpuscles in 142, 147 Spleno-meduUary . .132 2S8 THE BLOOD 8i Leucocythasmia {contd.) White corpuscles in Varieties of Leucocytosis — Absence of Agonal Cachectic Diagnosis from leuksemia Inflammatory . of digestion of malignant disease of the newly-born of pregnancy . Leucolysis Leucopenia Leukaemia, see leucocythsemia Liver, diseases of Lceffler's Methylene-blue Lung, diseases of Lymphaemia = lymphatic leu- cocythsemia . Lymphocytes Lymphocytosis — in infancy . .81, Lymphatic leukeemia . Malignant disease Pernicious anaemia . 129 . 127 82, 210 87 156 83 80 87 243 . 81 79,84 79.84 127 239 28 240 I4S 64 81, 24s . 146 87, 182 ■ 119 Malaria — Aestivo-autumnal Blood the, in . Crescents in Diagnosis of . Hsemoglobinuria in Malignant . 191 . 200 . 208 . 201 . 202 . 209 . 200 Methods of examining the blood in . . . 191 Parasites of . .196 Pigment . -197, 204 Quartan parasite . -199 Quotidian parasite . . 200 Staining with eosine-hama- toxylin . . -194 Tertian parasite . .197 Time of examination of the blood. . . . 19s 133 76: Malignant disease — Carcinoma of the lung Sarcoma Markzellen = marrow cells Marrow cells — in Blood in Leuksemia . in Bone-marrow Marrow eosinophile cells Marrow of bones Mastzellen — Staining Measles . Megaloblasts — . in Pernicious anemia in Secondary anaemia Megalocytes Melaniferous Leucocytes, Meningitis Methaemoglobinuria Methods of examination blood Methylene blue . Microblasts Microcytes Mitosis M onochromatophile Morphology of the Blood Muir's method of fixing films MuUer's method of fixing films Multinucleated leucocytes Myelocytes = marrow cells Myxoedema Necrobiosis Nephritis Nervous System . Neurasthenia Neusser's granules in gout „ „ in phthisis Neutrophile cells „ „ staining Nikiforoff's solution 179 179 240 181 76 76 H7 59 136 • 58 • 77 . 28 . 219 . 48 . 118 . 176 43> 114 83, 210 225 188 I 192 48 43 59 45 37 25 26 67 76 237 45 242 242 243 236 233 67 28 25 of 28 INDEX 259 Normoblasts . . . 47 Nucleated red Corpuscles . 47 bone marrow . . 60 01igsemia= diminution in the blood . . . 92 Oligochromsemia, diminution in Hb of blood . . 92 Oligocythsemia, diminution in corpuscles . . 42, 92 Osmic acid . . . 23 Osteo-sarcoma . . .181 Pacini's solution . . .11 Parasites of the blood, see Malaria . . . .191 Paroxysmal Hsemoglobinuria . igo Pathology of the blood . . 92 Pericarditis . . 225, 241 Perinephritic abscess . . 228 Peritonitis . . • 224 Pernicious anaemia — . .109 Diagnosis of . ■ 121 Heemoglobin in . .113 Leucocytes in . . .119 Nucleated red cells in . .117 Red-blood corpuscles in . 112 Phagocytosis . • • 83 Phthisis . . . . 232 Plasmodia, see Malaria . .191 Plehn's staining mixture . . 30 Pleurisy . . . . 224 Pneumonia . ■ .211 Poikiloblasts . ■ • 49 Poikilocytes . . 43, 4> Polychromatophile . . 45 Polycythaemia= increase in the number of corpuscles . 41 Post-hsemorrhagic anaemia . 183 Pregnancy, influence on leu- cocytes . . .81 Pregnancy, influence on red corpuscles . . . 41 Primary Anaemia . • 97 Pseudo-anaemia . . . 92 Pseudo-bacilli . . 44 Pseudo-chlorosis . . . 99 Pseudo-leuksemia = Hodgkin's Disease . . . 162 Pseudo-leukaemia, acute . .165 Purpura . . . .185 Pus formation . . . 227 Pyaemia . . . . 226 Ratio of white to red cor- puscles in leukffimia . 130 Ratio of white to red cor- puscles in normal blood. 79 Regeneration of the blood 47, 100 Respiratory system . . 240 Rheumatism . . .222 Rickets in infants . 251 Sarcoma . . . . 181 Scarlet fever . . .218 Schistocyte=poikilocyte. . 43 Scorbutus or scurvy . .187 Secondary ansemia — . 98, 174 Diagnosis . . -177 in malignant disease . -179 Segmenting forms of malaria parasites . . .198 Septicemia . . . 226 Serous membranes, inflamma- tion of . . . 223 Serum diagnosis of typhoid fever . . . . 213 Sex, influence on the red cor- puscles . . . 40 Shadow corpuscles = achro- macytes . . . 44 Simple primary anemia . 105 Small-pox . . . 2ig Spleen- Enlargement in infants . 245 in leucocythaemia . .128 in lymphatic leucocythaemia 1 46 in splenic ansemia . .167 in splenic anaemia of infants 246 Splenic anaemia — . .166 Blood in . . .168 26o THE BLOOD Splenic anaemia {contd.) — Diagnosis of . . . 170 Symptoms of . . .167 Splenic anaemia in infants — Blood in . . . 246 Diagnosis of . . 248 Symptoms of . . . 245 Staining Films — . 26 Ehrlich's triple stain . . 30 Eosine and Hsematoxylin . 34 Methylene-blue . .28 Neusser's solution . 237 Neutral staining mixture . 28 Starvation, effect on cor- puscles . . 41, 80 Stomach — cancer of . . 238 ulcer of. 238 Suppuration . 227 Syphilis — in adults . . 229 in children . . 250 Thionin-blue . .193 Thoma-Zeiss hffimocytometer i Toisson's solution . .12 Tonsillitis . 222 Transitional leucocytes . . 67 Transitional leucocytes in Leu- kaemia . . .140 Tubercular meningitis 225 Tuberculosis . 231 Typhoid fever — Blbod in . . 216 Serum, diagnosis of . - 213 Typhus fever . .222 Ulcer of the stomach . 238 Uninucleated Leucocytes, 66 large neutrophile = marrow cells . . . . 76 Uninucleated Leucocytes, small = lymphocytes . . 64 Uraemia . . . . 242 Uric acid diathesis . 237 Urobilin . . . 123 Variola . . . . 219 Vacuoles in red corpuscles dis- tinguished from malaria parasite . . . 203 White blood corpuscles, see Leucocytes . . . 62 Williamson's method of dis- tinguishing diabetic blood 236 vv, , * "^:?V A SELECTION FROM J. & A. 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Chauveau's Comparative Anat- omy of the Domesticated Ani- mals. Revised and Enlarged, with the Co-operation of S. Arloing, Director of the Lyons Veterinary School, and Edited by George Fleming, C.B., LL.D., F.R.C.V.S., late Principal Veterinary Surgeon of the British Army. Second English Edition. 8vo, with 585 Engrav- ings, 31S. 6d. Human Nature, its Principles and the Principles of Physio- gnomy. By Physicist. Part I., Imp. i6mo, 2s. LONDON: 7, GREAT MARLBOROUGH STREET. INDEX TO J. & A. CHURCHILL'S LIST. Allen's Chemistry ofUrine, 12 Commercial Organic Analysis, 13 Anderson's Deformities of Fingers and Toes, g Armatage's Veterinary Pocket Remembrancer, 14 Barnes (R.) on Obstetric Operations, 3 on Diseases of Women, 3 Beale (L. S.) on^ Liver, 6 Microscope in Medicine, 6 Slight Ailments, 6 ■ Urmary and Renal Derangements, 12 Beale (P. T. 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Asphyxia, 6 — ■ Medical Lectures and Essays, 6 Cholera Controversy, 6 (A. E.) Analyst's Companion, 13 Journal of Mental Science. 3 Kellogg on Mental Diseases, 3 Keyes' Genito-Urinary Organs and Syphilis, 12 Kohlrausch's Physical Measurements, 14 Lancereaux's Atlas of Pathological Anatomy, 2 Lane's Rheumatic Diseases, 7 Langdon-Down's Mental Affections of Childhood, 3 Lazarus-Barlow's General Pathology, i Lee's Microtomists' Vade Mecum, 14 Lescher's Recent Materia Medica, 4 Lewis (Bevan) on the Human Brain, 2 Liebreich's Atlas of Ophthalmoscopy, 10 MacMunn's Clinical Chemistry of Urine, 12 Macnamara's Diseases and Refraction of the Eye, 9 ; ; ; of Bones and Joints, 8 McNeill's Epidemics and Isolation Hospitals, z {Continued on the next Page. LONDON: 7, GREAT MARLBOROUGH STREET. Inijex to J. & A. Churchill's List — cdntintted. Malcolm's Physiology of Death, g Marcet on Respiration, z Martin's Ambulance Lectures, 8 Maxwell's Terminologia Medica Polyglotta, 12 Maylard's Surgery of Alimentary Canal, 9 Mayne's Medical Vocabulary, 12 Mercier's Lunacy Law, 3 Microscopical Journal, 14 Mills and Rowan's Fuel and its Applications, 14 Moore's (N.) Pathological Anatomy of Diseases, x Moore's (Sir W.J.) Family Medicine for India, 5 Manual of the Diseases of India, s Morris's Human Anatomy, i Moullin's (Mansell) Surgery, 8 Nettleship's Diseases of the Eye, 9 ' Notter and Firth's Hygiene, 2 Ogle on Tympanites, 8 Oliver's Abdominal Tumours, 3 Diseases of Women, 3 Ophthalmic (Royal London) Hospital Reports, 9 Ophthalmolo^ical Society's Transactions, 9 Ormerod's Diseases of the Nervous System, 7 Parkes' (E.A.) Practical Hygiene, 2 Parkes' (L.C.) 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Dental Metallurgy, 10 Smith's (Eustace) Clinical Studies, 4 Disease in Children, 4 WastingDiseases of Infants and Children, 4 Smith's (J. Greig) Abdominal Surgery, 8 Smith's (Priestley) Glaucoma, ic Snow's Cancer and the Cancer Process, 11 Snow's Palliative Treatment of Cancer, 11 Reappearance of Cancer, 11 Solly's Medical Climatology, 8 Southall's Materia Medica, 5 Squire's (P.) Companion to the Pharmacopoeia, 4 London Hospitals Pharmacopoeias, 4 Methods and Formulsej 14 Starling's Elements of Human Physiolog^y, -^ Sternberg's Bacteriology, 6 Stevenson and Murphy's Hygiene, 2 Sutton's (J. B.), General Pathology, i Sutton's (F.) Volumetric Analysis, 13 Swain's Surreal Emergencies, 8 Swayne's Obstetric Aphorisms, 3 Taylor's (A. S.) Medical Jurisprudence, z Taylor's (F.) Practice of Medicine, 6 Taylor's (J. C), Canary Islands, 8 Thin*s Cancerous Affections of the Skin, 11 Pathology and Treatment of Ringworm, ir on Psilosis or " Sprue," 5 Thomas's Diseases of Women, 3 Thompson's (Sir H.) Calculous Disease, 11 Diseases of the Prostate, n Diseases oftheUrinaryOrgans, 1 1 Lithotomy and Lithotrity, n Stricture of the Urethra, 11 Suprapubic Operation, 11 Surgery of theUrinaryOrgans,!! ^_ Tumours of the Bladder, 11 Thome's Diseases of the Heart, 7 Thresh's Water Analysis, 2 Tilden's Manual of Chemistry, 12 Tirard's Prescriber's Pharmacopceia, 5 Tomes' (C. S.) Dental Anatomy, 10 Tomes' (J. and C. S.) Dental Surgery, 10 Tooth's Spinal Cord, 7 Treves and Lang's German-English Dictionary, 12 Tuke's Dictionary of Psychological Medicine, 3 Tuson's Veterinary Pharmacopceia, X4 Valentin and Hodgkinson's Qualitative Analysis, 13 Vintras on the Mineral Waters, &c,, of France, 8 Wagner's Chemical Technology, 14 Walsham's Surgery : its Theory and Practice, 8 Waring's Indian Bazaar Medicines, 5 Practical Therapeutics, 5 Watts' Organic Chemistry, 12 West's (S.) How to Examine the Chest, 6 Westminster Hospital Reports, 7 White's (Hale) Materia Medica, Pharmacy, &c., 4 Wilks' Diseases of the Nervous System, 7 Wilson's (Sir E.) Anatomists' Vade-Mecum, 4. Wilson's (G.) Handbook of Hygiene, 2 Wolfe's Diseases and Injuries of the Eye, 9 Wynter and Wethered's Practical Pathology, i Year-Book of Pharmacy, 5 Yeo's (G. F.) Manual of Physiology, 2 N,B.—J. ^ A. ChurchilVs larger Catalogue of about 600 works on AnatomyA Physiology^ Hygiene^ Midwifery^ Materia Medica^ Medicine^ Surgery^ Chemistryk Botany^ ^c. Sfc, with a complete Index to their Subjects^ for easy reference \ will be forwarded post free on application. America. — -/. «^ A. Churchill being in constant communication with various publishing houses in America are able to conduct negotiations favourable to English Authors, LONDON i 7, GREAT MARLBOROUGH STREET.