A Manual of Clinical Diagnosis

by James Campbell Todd

CHAPTER III

THE BLOOD

Preliminary Considerations.—The blood consists of a fluid of complicated and variable composition, the plasma, in which are suspended great numbers of microscopic structures: viz., red corpuscles, white corpuscles, blood-platelets, and blood-dust.

Red corpuscles, or erythrocytes, are biconcave discs, red when viewed by reflected light or in thick layer, and straw-colored when viewed by transmitted light or in thin layer. They give the blood its red color. They are cells which have been highly differentiated for the purpose of carrying oxygen from the lungs to the tissues. This is accomplished by means of an iron-bearing proteid, hemoglobin, which they contain. In the lungs hemoglobin forms a loose combination with oxygen, which it readily gives up when it reaches the tissues. Normal erythrocytes do not contain nuclei. They are formed from preëxisting nucleated cells in the bone-marrow.

White corpuscles, or leukocytes, are less highly differentiated cells. There are several varieties. They all contain nuclei, and most of them contain granules which vary in size and staining properties. They are formed in the bone-marrow and lymphoid tissues.

Blood-platelets, or blood-plaques, are colorless or slightly bluish, spheric or ovoid bodies, about one-third or one-half the diameter of an erythrocyte. Their structure, nature, and origin have not been definitely determined.

The blood-dust of Müller consists of fine granules which have vibratory motion. Little is known of them. It has been suggested that they are granules from disintegrated leukocytes.

Coagulation consists essentially in the transformation of fibrinogen, one of the proteins of the blood, into fibrin by means of a ferment derived from disintegration of the leukocytes. The resulting coagulum is made up of a meshwork of fibrin fibrils with entangled corpuscles and plaques. The clear, straw-colored fluid which is left after separation of the coagulum is called blood-serum. Normally, coagulation takes place in two to eight minutes after the blood leaves the vessels. It is frequently desirable to determine the coagulation time. The simplest method is to place a drop of blood upon a perfectly clean slide, and to draw a needle through it at half-minute intervals. When the clot is dragged along by the needle, coagulation has taken place. This method is probably sufficient for ordinary clinical work. For very accurate results the method of Russell and Brodie, for which the reader is referred to the larger text-books, is recommended. Coagulation is notably delayed in hemophilia and icterus and after administration of citric acid. It is hastened by administration of calcium chlorid.

For most clinical examinations only one drop of blood is required. This may be obtained from the lobe of the ear, the palmar surface of the tip of the finger, or, in the case of infants, the plantar surface of the great toe. With nervous children the lobe of the ear is preferable, as it prevents their seeing what is being done. An edematous or congested part should be avoided. The site should be well rubbed with alcohol to remove dirt and epithelial débris and to increase the amount of blood in the part. After allowing sufficient time for the circulation to equalize, the skin is punctured with a blood lancet (of which there are several patterns upon the market) or some substitute, as a Hagedorn needle, aspirating needle, trocar, or a pen with one of its nibs broken off. Nothing is more unsatisfactory than an ordinary sewing-needle. The lancet should be cleaned with alcohol before and after using, but need not be sterilized. If the puncture be made with a firm, quick stroke, it is practically painless. The first drop of blood which appears should be wiped away, and the second used for examination. The blood should not be pressed out, since this dilutes it with serum from the tissues; but moderate pressure some distance above the puncture is allowable.

FIG. 64.—Daland's blood-lancet.

When a larger amount of blood is required, it may be obtained with a sterile hypodermic syringe from one of the veins at the elbow.

Clinical study of the blood may be discussed under the following heads: I. Hemoglobin. II. Enumeration of erythrocytes. III. Color index. IV. Enumeration of leukocytes. V. Enumeration of plaques. VI. Study of stained blood. VII. Blood parasites. VIII. Serum reactions. IX. Tests for recognition of blood. X. Special blood pathology.

I. HEMOGLOBIN

Hemoglobin is an iron-bearing proteid. It is found only within the red corpuscles, and constitutes about 90 per cent. of their weight. The actual amount of hemoglobin is never estimated clinically: it is the relation which the amount present bears to the normal which is determined. Thus the expression, "50 per cent. hemoglobin," when used clinically, means that the blood contains 50 per cent. of the normal. Theoretically, the normal would be 100 per cent., but with the methods of estimation in general use the blood of healthy persons ranges from 85 to 105 per cent.; these figures may, therefore, be taken as normal.

Increase of hemoglobin, or hyperchromemia, is uncommon, and is probably more apparent than real. It accompanies an increase in number of erythrocytes, and may be noted in change of residence from a lower to a higher altitude; in poorly compensated heart disease with cyanosis; in concentration of the blood from any cause, as the severe diarrhea of cholera; and in "idiopathic polycythemia."

Decrease of hemoglobin, or oligochromemia, is very common and important. It is the most striking feature of the secondary anemias ([p. 204]). Here the hemoglobin loss may be slight or very great. In mild cases a slight decrease of hemoglobin is the only blood change noted. In very severe cases, especially in repeated hemorrhages, malignant disease, and infection by the worms uncinaria and bothriocephalus latus, hemoglobin may fall to 15 per cent. Hemoglobin is always diminished, and usually very greatly, in chlorosis, pernicious anemia, and leukemia.

Estimation of Hemoglobin.—There are many methods, but none is entirely satisfactory. Those which are most widely used are here described.

FIG. 65.—Von Fleischl's hemoglobinometer: a, Stand; b, narrow wedge-shaped piece of colored glass fitted into a frame (c), which passes under the chamber; d, hollow metal cylinder, divided into two compartments, which holds the blood and water; e, plaster-of-Paris plate from which the light is reflected through the chamber; f, screw by which the frame containing the graduated colored glass is moved; g, capillary tube to collect the blood; h, pipet for adding the water; i, opening through which may be seen the scale indicating percentage of hemoglobin.

(1) Von Fleischl Method.—The apparatus consists of a stand somewhat like the base and stage of a microscope (Fig. 65). Under the stage is a movable bar of colored glass, shading from pale pink at one end to deep red at the other. The frame in which this bar is held is marked with a scale of hemoglobin percentages corresponding to the different shades of red. By means of a rack and pinion, the color-bar can be moved from end to end beneath a round opening in the center of the stage. A small metal cylinder, which has a glass bottom and which is divided vertically into two equal compartments, can be placed over the opening in the stage so that one of its compartments lies directly over the color-bar. Accompanying the instrument are a number of short capillary tubes in metal handles.

Having punctured the finger-tip or lobe of the ear as already described, wipe off the first drop of blood, and from the second fill one of the capillary tubes. Hold the tube horizontally, and touch its tip to the drop of blood, which will readily flow into it if it be clean and dry. Avoid getting any blood upon its outer surface. With a medicine-dropper, rinse the blood from the tube into one of the compartments of the cylinder, using distilled water, and mix well. Fill both compartments level full with distilled water, and place the cylinder over the opening in the stage so that the compartment which contains only water lies directly over the bar of colored glass.

In a dark room, with the light from a candle reflected up through the cylinder, move the color-bar along with a jerking motion until both compartments have the same depth of color. The number upon the scale corresponding to the portion of the color-bar which is now under the cylinder gives the percentage of hemoglobin. While comparing the two colors, place the instrument so that they will fall upon the right and left halves of the retina, rather than upon the upper and lower halves; and protect the eye from the light with a cylinder of paper or pasteboard. After use, clean the metal cylinder with water, and wash the capillary tube with water, alcohol, and ether, successively. Results with this instrument are accurate to within about 5 per cent.

A recent modification of the von Fleischl apparatus by Miescher gives an error which need not exceed 1 per cent. It is, however, better adapted to laboratory use than to the needs of the practitioner.

(2) The Sahli hemoglobinometer (Fig. 66) is an improved form of the well known Gowers instrument. It consists of a hermetically sealed comparison tube containing a 1 per cent. solution of acid hematin, a graduated test-tube of the same diameter, and a pipet of 20 c.mm. capacity. The two tubes are held in a black frame with a white ground-glass back.

FIG. 66.—Sahli's hemoglobinometer.

Place a few drops of decinormal hydrochloric acid solution in the graduated tube. Obtain a drop of blood and draw it into the pipet to the 20 c.mm. mark. Wipe off the tip of the pipet, blow its contents into the hydrochloric acid solution in the tube, and rinse well. In a few minutes the hemoglobin is changed to acid hematin. Place the two tubes in the compartments of the frame, and dilute the fluid with water drop by drop, mixing after each addition, until it has exactly the same color as the comparison tube. The graduation corresponding to the surface of the fluid then indicates the percentage of hemoglobin. Decinormal hydrochloric acid solution may be prepared with sufficient accuracy for this purpose by adding 15 c.c. of the concentrated acid to 985 c.c. distilled water. A little chloroform should be added as a preservative.

This method is very satisfactory in practice, and is accurate to within 5 per cent. The comparison tube is said to keep its color indefinitely, but, unfortunately, not all the instruments upon the market are well standardized.

(3) Dare's hemoglobinometer (Fig. 67) differs from the others in using undiluted blood. The blood is allowed to flow by capillarity into the slit between two small plates of glass. It is then placed in the instrument and compared with different portions of a circular disc of colored glass. The reading must be made quickly, before clotting takes place. This instrument is easy to use, and is one of the most accurate.

FIG. 67.—Dare's hemoglobinometer.

(4) Hammerschlag Method.—This is an indirect method which depends upon the fact that the percentage of hemoglobin varies directly with the specific gravity of the blood. It yields fairly accurate results except in leukemia, where the large number of leukocytes disturbs the relation.

Mix chloroform and benzol in a urinometer tube so that the specific gravity of the mixture is near the probable specific gravity of the blood. Add a drop of blood by means of a pipet of small caliber. If the drop floats near the surface, add a little benzol; if it sinks to the bottom, add a little chloroform. When it remains stationary near the middle, the mixture has the same specific gravity as the blood. Take the specific gravity with a urinometer, and obtain the corresponding percentage of hemoglobin from the following table:

SPECIFIC GRAVITY.	HEMOGLOBIN PER CENT.
1.033-1.035	25-30
1.035-1.038	30-35
1.038-1.040	35-40
1.040-1.045	40-45
1.045-1.048	45-55
1.048-1.050	55-65
1.050-1.053	65-70
1.053-1.055	70-75
1.055-1.057	75-85
1.057-1.060	85-95

For accurate results with this method, care and patience are demanded. The following precautions must be observed:

(a) The two fluids must be well mixed after each addition of chloroform or benzol. Close the tube with the thumb and invert several times. Should this cause the drop of blood to break up into very small ones, adjust the specific gravity as accurately as possible with these, and test it with a fresh drop.

(b) The drop of blood must not be too large; it must not contain an air-bubble, it must not adhere to the side of the tube, and it must not remain long in the fluid.

(c) The urinometer must be standardized for the chloroform-benzol mixture. Most urinometers give a reading two or three degrees too high, owing to the low surface tension. Make a mixture such that a drop of distilled water will remain suspended in it (i.e., with a specific gravity of 1.000) and correct the urinometer by this.

(5) Tallquist Method.—The popular Tallquist hemoglobinometer consists simply of a book of small sheets of absorbent paper and a carefully printed scale of colors (Fig. 68).

Take up a large drop of blood with the absorbent paper, and when the humid gloss is leaving, compare the stain with the color scale. The color which it matches gives the percentage of hemoglobin. Except in practised hands, this method is accurate only to within 10 or 20 per cent.

FIG. 68.—Tallquist's hemoglobin scale.

Of the methods given, the physician should select the one which best meets his needs. With any method, practice is essential to accuracy. The von Fleischl has long been the standard instrument, but has lately fallen into some disfavor. For accurate work the best instruments are the von Fleischl-Miescher and the Dare. They are, however, expensive, and it is doubtful whether they are enough more accurate than the Sahli instrument to justify the difference in cost. The latter is probably the most satisfactory for the practitioner, provided a well-standardized color-tube is obtained. The specific gravity method is very useful when special instruments are not at hand. The Tallquist scale is so inexpensive and so convenient that it should be used by every physician at the bedside and in hurried office work; but it should not supersede the more accurate methods.

II. ENUMERATION OF ERYTHROCYTES

In health there are about 5,000,000 red corpuscles per cubic millimeter of blood. Normal variations are slight. The number is generally a little less—about 4,500,000—in women.

Increase of red corpuscles, or polycythemia, is unimportant. There is a decided increase following change of residence from a lower to a higher altitude, averaging about 50,000 corpuscles for each 1000 feet, but frequently much greater. The increase, however, is not permanent. In a few months the erythrocytes return to nearly their original number. Three views are offered in explanation: (a) Concentration of the blood, owing to increased evaporation from the skin; (b) stagnation of corpuscles in the peripheral vessels, because of lowered blood-pressure; (c) new-formation of corpuscles, this giving a compensatory increase of aëration surface.

Pathologically, polycythemia is uncommon. It may occur in: (a) concentration of the blood from severe watery diarrhea; (b) chronic heart disease, especially the congenital variety, with poor compensation and cyanosis; and (c) idiopathic polycythemia, which is considered to be an independent disease, and is characterized by cyanosis, blood counts of 7,000,000 to 10,000,000, hemoglobin 120 to 150 per cent., and a normal number of leukocytes.

Decrease of red corpuscles, or oligocythemia. Red corpuscles and hemoglobin are commonly decreased together, although usually not to the same extent.

Oligocythemia occurs in all but the mildest symptomatic anemias. The blood count varies from near the normal in moderate cases down to 1,500,000 in very severe cases. There is always a decrease of red cells in chlorosis, but it is often slight, and is relatively less than the decrease of hemoglobin. Leukemia gives a decided oligocythemia, the average count being about 3,000,000. The greatest loss of red cells occurs in pernicious anemia, where counts below 1,000,000 are not uncommon.

FIG. 69.—Thoma-Zeiss hemocytometer: a, Slide used in counting; b, sectional view; d, red pipet; e, white pipet.

The most widely used and most satisfactory instrument for counting the corpuscles is that of Thoma-Zeiss. The hematocrit is not to be recommended for accuracy, since in anemia, where blood counts are most important, the red cells vary greatly in size and probably also in elasticity. The hematocrit is, however, useful in determining the relative volume of corpuscles and plasma, and seems to be gaining in favor.

FIG. 70.—Ordinary ruling of counting chamber, showing red corpuscles in left upper corner.

The Thoma-Zeiss instrument consists of two pipets for diluting the blood and a counting chamber (Fig. 69). The counting chamber is a glass slide with a square platform in the middle. In the center of the platform is a circular opening, in which is set a small circular disc in such a manner that it is surrounded by a "ditch," and that its surface is exactly one-tenth of a millimeter below the surface of the square platform. Upon this disc is ruled a square millimeter, subdivided into 400 small squares. Each fifth row of small squares has double ruling for convenience in counting (Fig. 70). A thick cover-glass, ground perfectly plane, accompanies the counting chamber. Ordinary cover-glasses are of uneven surface, and should not be used with this instrument.

It is evident that, when the cover-glass is in place upon the platform, there is a space exactly one-tenth of a millimeter thick between it and the disc; and that, therefore, the square millimeter ruled upon the disc forms the base of a space holding exactly one-tenth of a cubic millimeter.

FIG. 71.—Method of drawing blood into the pipet (Boston).

Technic.—To count the red corpuscles, use the pipet with 101 engraved above the bulb. It must be clean and dry. Obtain a drop of blood as already described. Suck blood into the pipet to the mark 0.5 or 1. Should the blood go beyond the mark, draw it back by touching the tip of the pipet to a moistened handkerchief. Quickly wipe off the blood adhering to the tip, plunge it into the diluting fluid, and suck the fluid up to the mark 101, slightly rotating the pipet meanwhile. This dilutes the blood 1:200 or 1:100, according to the amount of blood taken. Except in cases of severe anemia, a dilution of 1:200 is preferable. Close the ends of the pipet with the fingers, and shake vigorously until the blood and diluting fluid are well mixed.

When it is not convenient to count the corpuscles at once, place a heavy rubber band around the pipet so as to close the ends, inserting a small piece of rubber-cloth or other tough, non-absorbent material if necessary to prevent the tip from punching through the rubber. It may be kept thus for twenty-four hours or longer.

When ready to make the count, mix again thoroughly by shaking; blow two or three drops of the fluid from the pipet, wipe off its tip, and then place a small drop (the proper size can be learned only by experience) upon the disc of the counting chamber. Adjust the cover immediately. Hold it by diagonal corners above the drop of fluid so that a third corner touches the slide and rests upon the edge of the platform. Place a finger upon this corner, and, by raising the finger, allow the cover to fall quickly into place. If the cover be properly adjusted, faint concentric lines of the prismatic colors—Newton's rings—can be seen between it and the platform when the slide is viewed obliquely. They indicate that the two surfaces are in close apposition. If they do not appear at once, slight pressure upon the cover may bring them out. Failure to obtain them is usually due to dirty slide or cover—both must be perfectly clean and free from dust. The drop placed upon the disc must be of such size that, when the cover is adjusted, it nearly or quite covers the disc, and that none of it runs over into the "ditch." There should be no bubbles upon the ruled area.

Allow the corpuscles to settle for a few minutes, and then examine with a low power to see that they are evenly distributed. If they are not evenly distributed over the whole disc, the counting chamber must be cleaned and a new drop placed in it.

Probably the most satisfactory objective for counting is the special one-sixth for blood work already mentioned. To understand the principle of counting, it is necessary to remember that the large square (400 small squares) represents a capacity of one-tenth of a cubic millimeter. Find the number of corpuscles in the large square, multiply by 10 to find the number in 1 c.mm. of the diluted blood, and finally, by the dilution, to find the number in 1 c.mm. of undiluted blood. Instead of actually counting all the corpuscles, it is customary to count those in only a limited number of small squares, and from this to calculate the number in the large square.

In practice a convenient procedure is as follows: With a dilution of 1:200, count the cells in 80 small squares, and to the sum add 4 ciphers; with dilution of 1:100, count 40 small squares and add 4 ciphers. Thus, if with 1:200 dilution, 450 corpuscles were counted, the total count would be 4,500,000 per c.mm. This method is sufficiently accurate for all clinical purposes, provided the corpuscles are evenly distributed and two drops from the pipet be counted. It is convenient to count a block of 20 small squares in each corner of the large square. It distribution be even, the difference between the number of cells in any two such blocks should not exceed twenty. Corpuscles which touch the upper and left sides should be counted as if within the squares, those touching the lower and right sides, as outside, and vice versâ.

Diluting Fluids.—The most widely used are Hayem's and Toisson's. Both of these have high specific gravities, so that, when well mixed, the corpuscles do not separate quickly. Toisson's fluid is probably the better for beginners, because it is colored and can be easily seen as it is drawn into the pipet. It stains the nuclei of leukocytes blue, but this is no real advantage. It must be filtered frequently.

HAYEM'S FLUID.			TOISSON'S FLUID.
Mercuric chlorid	0.5		Methyl-violet, 5 B	0.025
Sodium sulphate	5.0		Sodium chlorid	1.000
Sodium chlorid	1.0		Sodium sulphate	8.000
Distilled water	200.0		Glycerin	30.000
			Distilled water	160.000

Sources of Error.—The most common sources of error in making a blood count are:

(a) Inaccurate dilution, either from faulty technic or inaccurately graduated pipets. The instruments made by Zeiss can be relied upon.

(b) Too slow manipulation, allowing a little of the blood to coagulate and remain in the capillary portion of the pipet.

(c) Inaccuracy in depth of counting chamber, which sometimes results from softening of the cement by alcohol or heat. The slide should not be cleaned with alcohol nor left to lie in the warm sunshine.

(d) Uneven distribution of the corpuscles. This results when the blood is not thoroughly mixed with the diluting fluid, or when the cover-glass is not applied soon enough after the drop is placed upon the disc.

Cleaning the Instrument.—The instrument should be cleaned immediately after using, and the counting chamber and cover must be cleaned again just before use.

Draw through the pipet, successively, water, alcohol, ether, and air. This can be done with the mouth, but it is much better to use a rubber bulb. When the mouth is used, the moisture of the breath will condense upon the interior of the pipet unless the fluids be shaken and not blown out. If blood has coagulated in the pipet—which happens when the work is done too slowly—dislodge the clot with a horse-hair, and clean with strong sulphuric acid, or let the pipet stand over night in a test-tube of the acid. Even if the pipet does not become clogged, it should be occasionally cleaned in this way.

III. COLOR INDEX

This is an expression which indicates the amount of hemoglobin in each red corpuscle compared with the normal amount. For example, a color index of 1.0 indicates that each corpuscle contains the normal amount of hemoglobin; of 0.5, that each contains one-half the normal.

The color index is most significant in chlorosis and pernicious anemia. In the former it is usually much decreased; in the latter, generally much increased. In symptomatic anemia it is generally moderately diminished.

To obtain the color index, divide the percentage of hemoglobin by the percentage of corpuscles. The percentage of corpuscles is found by multiplying the first two figures of the red corpuscle count by two. This simple method holds good for all counts of 1,000,000 or more. Thus, a count of 2,500,000 is 50 per cent. of the normal. If, then, the hemoglobin has been estimated at 40 per cent., divide 40 (the percentage of hemoglobin) by 50 (the percentage of corpuscles). This gives 4/5, or 0.8, as the color index.

IV. ENUMERATION OF LEUKOCYTES

The normal number of leukocytes varies from 5000 to 10,000 per cubic millimeter of blood. The number is larger in robust individuals than in poorly nourished ones, and if disease be excluded, may be taken as an index of the individual's nutrition. Since it is well to have a definite standard, 7500 is generally adopted as the normal for the adult. With children the number is somewhat greater, counts of 12,000 and 15,000 being common in healthy children under twelve years of age.

Decrease in number of leukocytes, or leukopenia, is not important. It is common in persons who are poorly nourished, although not actually sick. The infectious diseases in which leukocytosis is absent ([p. 160]) often cause a slight decrease of leukocytes. Chlorosis may produce leukopenia, as also pernicious anemia, which usually gives it in contrast to the secondary anemias, which are frequently accompanied by leukocytosis.

Increase in number of leukocytes is common and of great importance. It may be considered under two heads: A. Increase of leukocytes due to chemotaxis and stimulation of the blood-making organs, or leukocytosis. B. Increase of leukocytes due to leukemia. The former may be classed as a transient, the latter, as a permanent, increase.

A. LEUKOCYTOSIS

This term has not acquired a definite meaning. By some it is applied to any increase in number of leukocytes; by others, it is restricted to increase of the polymorphonuclear neutrophilic variety. As has been indicated, it is here taken to mean any increase in number of leukocytes caused by chemotaxis and stimulation of the blood-producing structures; and includes every increase of leukocytes except that due to leukemia.

By chemotaxis is meant that property of certain agents by which they attract or repel leukocytes—positive chemotaxis and negative chemotaxis respectively. An excellent illustration is the accumulation of leukocytes at the site of inflammation, owing to the positively chemotactic influence of bacteria and their products. A great many agents possess the power of attracting leukocytes into the general circulation. Among these are bacteria and many organic and inorganic poisons.

Chemotaxis alone will not explain the continuance of leukocytosis for more than a short time. It is probable that substances which are positively chemotactic also stimulate the blood-producing organs to increased formation of leukocytes; and in at least one form of leukocytosis such stimulation probably plays the chief part.

In general, the response of the leukocytes to chemotaxis is a conservative process. It is the gathering of soldiers to destroy an invader. This is accomplished partly by means of phagocytosis—actual ingestion of the enemy—and partly by means of chemic substances which the leukocytes produce.

In those diseases in which leukocytosis is the rule the degree of leukocytosis depends upon two factors: the severity of the infection and the resistance of the individual. A well-marked leukocytosis usually indicates good resistance. A mild degree means that the body is not reacting well, or else that the infection is too slight to call forth much resistance. Leukocytosis may be absent altogether when the infection is extremely mild, or when it is so severe as to overwhelm the organism before it can react. These facts are especially true of pneumonia, diphtheria, and abdominal inflammations, in which conditions the degree of leukocytosis is of considerable prognostic value.

As will be seen later, there are several varieties of leukocytes in normal blood, and many chemotactic agents attract only one variety and either repel or do not influence the others. These varieties may be divided into two general classes:

(a) Those having active independent ameboid motion. They are able to migrate readily from place to place and to ingest small bodies, as bacteria. From this latter property they derive their name of phagocytes. This group includes all varieties except the lymphocytes. The polymorphonuclear leukocytes are taken as the type of the group, because they are by far the most numerous.

(b) Those having very little or no independent motion—non-phagocytic leukocytes. Only the lymphocytes belong to this class.

By this classification we may distinguish two types of leukocytosis, according to the type of cell chiefly affected: a phagocytic and a non-phagocytic type.

1. Phagocytic Leukocytosis.—Theoretically, there should be a subdivision of phagocytic leukocytosis for each variety of phagocyte, e.g., polymorphonuclear leukocytosis, eosinophilic leukocytosis, large mononuclear leukocytosis, etc. Practically, however, only one of these, polymorphonuclear leukocytosis, need be considered under the head of leukocytosis. Increase in number of the other phagocytes will be considered at another place. They are present in the blood in such small numbers normally that even a marked increase scarcely affects the total leukocyte count; and, besides, substances which attract them into the circulation frequently repel the polymorphonuclears, so that the total number of leukocytes may actually be decreased.

Polymorphonuclear leukocytosis may be either physiologic or pathologic. A count of 20,000 would be considered a marked leukocytosis; of 30,000, high; above 50,000, extremely high.

(1) Physiologic Polymorphonuclear Leukocytosis.—This is never very marked, the count rarely exceeding 15,000 per cubic millimeter. It occurs in the new-born, in pregnancy, during digestion, and after cold baths. There is moderate leukocytosis in the moribund state; this is commonly classed as physiologic, but is probably due mainly to terminal infection.

(2) Pathologic Polymorphonuclear Leukocytosis.—The classification here given follows Cabot:

(a) Infectious and Inflammatory.—The majority of infectious diseases produce leukocytosis. The most notable exceptions are influenza, malaria, measles, tuberculosis, except when invading the serous cavities or when complicated by mixed infection, and typhoid fever, in which leukocytosis indicates an inflammatory complication.

All inflammatory and suppurative diseases cause leukocytosis, except when slight or well walled off. Appendicitis has been studied with especial care in this connection, and the conclusions now generally accepted probably hold good for most acute intra-abdominal inflammations. A marked leukocytosis (20,000 or more) nearly always indicates abscess, peritonitis, or gangrene, even though the clinical signs be slight. Absence of or mild leukocytosis indicates a mild process, or else an overwhelmingly severe one; and operation may safely be postponed unless the abdominal signs are very marked. On the other hand, no matter how low the count, an increasing leukocytosis—counts being made hourly—indicates a spreading process and demands operation, regardless of other symptoms.

Leukocyte counts alone are often disappointing, but are of much more value when considered in connection with a differential count of polymorphonuclears (see [p. 181]).

(b) Malignant Disease.—Leukocytosis occurs in about one-half of the cases of malignant disease. In many instances it is probably independent of any secondary infection, since it occurs in both ulcerative and non-ulcerative cases. It seems to be more common in sarcoma than in carcinoma. Very large counts are rarely noted.

(c) Post-hemorrhagic.—Moderate leukocytosis follows hemorrhage and disappears in a few days.

(d) Toxic.—This is a rather obscure class, which includes gout, chronic nephritis, acute yellow atrophy of the liver, ptomain poisoning, prolonged chloroform narcosis, and quinin poisoning. Leukocytosis may or may not occur in these conditions, and is not important.

(e) Drugs.—This also is an unimportant class. Most tonics and stomachics and many other drugs produce a slight leukocytosis.

2. Non-phagocytic or Lymphocyte Leukocytosis.—This is characterized by an increase in the total leukocyte count, accompanied by an increase in the percentage of lymphocytes. The word "lymphocytosis" is often used in the same sense. It is better, however, to use the latter as referring to any increase in number of lymphocytes, without regard to the total count, since an actual increase in number of lymphocytes is frequently accompanied by a normal or subnormal leukocyte count, owing to loss of polymorphonuclears.

Non-phagocytic leukocytosis is probably due more to stimulation of blood-making organs than to chemotaxis. It is less common, and is rarely so marked as a polymorphonuclear leukocytosis. When marked, the blood cannot be distinguished from that of lymphatic leukemia.

A marked lymphocyte leukocytosis occurs in pertussis, and is of value in diagnosis. It appears early in the catarrhal stage, and persists until after convalescence. The average leukocyte count is about 17,000, lymphocytes predominating. There is moderate lymphocyte leukocytosis in other diseases of childhood, as rickets, scurvy, and especially hereditary syphilis, where the blood-picture may approach that of pertussis. It must be borne in mind in this connection that lymphocytes are normally more abundant in the blood of children than in that of adults.

Slight lymphocyte leukocytosis occurs in many other pathologic conditions, but is of little significance.

B. LEUKEMIA

This is an idiopathic disease of the blood-making organs, which is accompanied by an enormous increase in number of leukocytes. The leukocyte count sometimes exceeds 1,000,000 per cubic millimeter, and leukemia is always to be suspected when it exceeds 50,000. Lower counts do not, however, exclude it. The subject is more fully discussed later ([p. 208]).

The leukocytes are counted with the Thoma-Zeiss instrument, already described. Recently, several new rulings of the disc have been introduced, notably the Zappert and the Türk (Fig. 72), which give a ruled area of nine square millimeters. They were devised for counting the leukocytes in the same specimen with the red corpuscles. The red cells are counted in the usual manner, after which all the leukocytes in the whole area of nine square millimeters are counted; and the number in a cubic millimeter of undiluted blood is then easily calculated. Leukocytes are easily distinguished from red cells, especially when Toisson's diluting fluid is used. This method may be used with the ordinary ruling by adjusting the microscopic field to a definite size, and counting a sufficient number of fields, as described later. Although less convenient, it is more accurate to count the leukocytes separately, with less dilution of the blood, as follows:

Technic.—A larger drop of blood is required than for counting the erythrocytes, and more care in filling the pipet. Use the pipet with 11 engraved above the bulb. Suck the blood to the mark 0.5 or 1.0, and the diluting fluid to the mark 11. This gives a dilution of 1:20 or 1:10, respectively. The dilution of 1:20 is easier to make. Mix well by shaking in all directions except in the long axis of the pipet; blow out two or three drops, place a drop in the counting chamber, and adjust the cover as already described ([p. 153]).

FIG. 72.—Türk ruling of counting chamber.

Examine with a low power to see that the cells are evenly distributed. Count with the two-thirds objective and a high eye-piece, or with the long-focus one-sixth and a low eye-piece. A one-fourth objective will be found very satisfactory for this purpose.

With the ordinary ruling of the disc, count all the leukocytes in the large square, multiply by 10 to find the number in 1 c.mm. of diluted blood, and by the dilution to find the number per c.mm. of undiluted blood. In every case at least 100 leukocytes must be counted as a basis for calculation, and it is much better to count 500. This will necessitate examination of several drops from the pipet. With the Zappert and Türk rulings a sufficient number can usually be counted in one drop, but the opportunity for error is very much greater when only one drop is examined.

FIG. 73.—Size of field required in counting leukocytes as described in the text.

In routine work the author's modification of the "circle" method is very satisfactory: Draw out the tube of the microscope until the field of vision has a diameter equal to eight times the side of a small square (Fig. 73). The area of this field closely approximates one-tenth of a square millimeter. With a dilution of 1:20, count the leukocytes in 20 such fields upon different parts of the disc without regard to the ruled lines, and to their sum add two ciphers. With dilution of 1:10, count 10 such fields, and add two ciphers. Thus, with 1:10 dilution, if 150 leukocytes were counted in 10 fields, the leukocyte count would be 15,000 per c.mm. To compensate for possible unevenness of distribution, it is best to count a row of fields horizontally and a row vertically across the disc. This method is applicable to any degree of dilution of the blood, and is simple to remember: one always counts a number of fields equal to the number of times the blood has been diluted, and adds two ciphers.

It is frequently impossible to obtain the proper size of field with the objectives and eye-pieces at hand. In such case, place a cardboard disc with a circular opening upon the diaphragm of the eye-piece, and adjust the size of the field by drawing out the tube. The circular opening can be cut with a cork-borer.

Diluting Fluids.—The diluting fluid should dissolve the red corpuscles so that they will not obscure the leukocytes. The simplest fluid is a 0.5 per cent. solution of acetic acid. More satisfactory is the following: glacial acetic acid, 1 c.c.; 1 per cent. aqueous solution of gentian-violet, 1 c.c.; distilled water, 100 c.c. These solutions must be filtered frequently.

V. ENUMERATION OF BLOOD-PLAQUES

The average normal number of plaques is variously given as 200,000 to 700,000 per c.mm. of blood. The latter figure probably more nearly represents the true normal average, since the lower counts were obtained for the most part by workers who used unreliable methods. Physiologic variations are marked; thus, the number increases as one ascends to a higher altitude, and is higher in winter than in summer. There are unexplained variations from day to day; hence a single abnormal count should not be taken to indicate a pathologic condition.

Pathologic variations are often very great. Owing to lack of knowledge as to the origin of the platelets and to the earlier imperfect methods of counting, the clinical significance of these variations is uncertain. The following facts seem, however, to be established:

(a) In acute infectious diseases the number is subnormal or normal. If the fever ends by crisis, the crisis is accompanied by a rapid and striking increase.

(b) In secondary anemia plaques are generally increased, although sometimes decreased. In pernicious anemia they are always greatly diminished, and an increase should exclude the diagnosis of this disease.

(c) They are decreased in chronic lymphatic leukemia, and greatly increased in the myelogenous form.

(d) In purpura hæmorrhagica the number is enormously diminished.

Blood-plaques are difficult to count owing to the rapidity with which they disintegrate and to their great tendency to adhere to any foreign body and to each other. The method of Kemp, Calhoun, and Harris is practical and is to be recommended:

Wash the finger well and allow a few minutes to elapse for the circulation to become normal. Prick the finger lightly with a blood-lancet, regulating the depth of the puncture so that the blood will not flow without gentle pressure. Quickly dip a clean glass rod into a vessel containing diluting and fixing fluid, and place two or three good-sized drops upon the finger over the puncture. Then exert gentle pressure above the puncture so that a small drop of blood will exude into the fluid. Mix the two by passing the rod lightly several times over the surface of the blended drop. (Some workers first place a drop of the fluid upon the finger and then make the puncture through it, this necessitating less care as to depth of the puncture.) Now transfer a drop of the diluted blood from the finger to a watch-glass which contains two or three drops of the fluid, and mix well. From this, transfer a drop to the counting slide of the Thoma-Zeiss hemocytometer, and cover. An ordinary thin cover will answer for this purpose, and is preferable because it allows the use of a higher power objective. Allow the slide to stand for at least five minutes, and then with a one-sixth or higher objective count the plaques and the red corpuscles in a definite number of squares. At least 100 plaques must be counted. The number of red corpuscles per cubic millimeter of blood having been previously ascertained in the usual manner ([p. 152]), the number of plaques can easily be calculated by the following equation:

r:p :: R:P; and P = p x R/r.

r represents the number of red corpuscles in any given number of squares; p, the number of plaques in the same squares; R, the total number of red corpuscles per c.mm. of blood; and P, the number of plaques per c.mm.

Beginners are apt to take too much blood and not to dilute it enough. Best results are attained when there are only one or two plaques in a small square. With insufficient dilution, the platelets are more or less obscured by the red cells.

The following diluting and fixing fluid is recommended:

Formalin	10 c.c.
1 per cent. aqueous solution sodium chlorid (Color with methyl-violet if desired.)	150 c.c.

This fluid is cheap and easily prepared, and keeps indefinitely. It fixes the plaques quickly without clumping, and does not clump nor decolorize the reds. It has a low specific gravity, and hence allows the plaques to settle upon the ruled area along with the reds. Fluids of high specific gravity cause the plaques to float so that they do not appear in the same plane with the reds and the ruled lines.

VI. STUDY OF STAINED BLOOD

A. MAKING AND STAINING BLOOD-FILMS

1. Spreading the Film.—Thin, even films are essential to accurate and pleasant work. They more than compensate for the time spent in learning to make them. There are certain requisites for success with any method: (a) The slides and covers must be perfectly clean; thorough washing with soap and water and rubbing with alcohol will usually suffice; (b) the drop of blood must not be too large; (c) the work must be done quickly, before coagulation begins.

The blood is obtained from the finger-tip or the lobe of the ear, as for a blood count; only a very small drop is required.

Ehrlich's Two-cover-glass Method.—This method is very widely used, but considerable practice is required to get good results. Touch a cover-glass to the top of a small drop of blood, and place it, blood side down, upon another cover-glass. If the drop be not too large, and the covers be perfectly clean, the blood will spread out in a very thin layer. Just as it stops spreading, before it begins to coagulate, pull the covers quickly but firmly apart on a line parallel to their plane (Fig. 74). It is best to handle the covers with forceps, since the moisture of the fingers may produce artifacts.

Two-slide Method.—Place a small drop of blood upon a clean slide and push it along with the edge of a second slide held at an angle of 45 degrees to the surface of the first (Fig. 75).

FIG. 74.—Spreading the film: two-cover-glass method.

FIG. 75.—Spreading the film: two-slide method.

Cigarette-paper Method.—This gives better results in the hands of the inexperienced than any of the methods in general use, and may be used with either slides or covers. A very thin paper, such as the "Zig-zag" brand, is best. Ordinary cigarette paper and thin tissue-paper will answer, but do not give nearly so good results.

Cut the paper into strips about ¾ inch wide, across the ribs. Pick up one of the strips by the gummed edge, and touch its opposite end to the drop of blood. Quickly place the end which has the blood against a slide or a large cover-glass held in a forceps. The blood will spread along the edge of the paper. Now draw the paper evenly across the slide or cover. A thin film of blood will be left behind (Fig. 76).

FIG. 76.—Spreading the film. Cigarette-paper method applied to cover-glasses.

The films may be allowed to dry in the air, or may be dried by gently heating high above a flame (where one can comfortably hold the hand). Such films will keep for years, but for some stains they must not be more than a few weeks old. They must be kept away from flies—a fly can work havoc with a film in a few minutes.

2. Fixing the Film.—In general, films must be "fixed" before they are stained. Fixation may be accomplished by chemicals or by heat. Those stains which are dissolved in methyl-alcohol combine fixation with the staining process.

Chemic Fixation.—Soak the film five to fifteen minutes in pure methyl-alcohol, or one-half hour or longer in equal parts of absolute alcohol and ether. One minute in 1 per cent. formalin in alcohol is preferred by some. Chemic fixation may precede eosin-methylene-blue and other simple stains.

Heat Fixation.—This may precede any of the methods which do not combine fixation with the staining process; it must be used with Ehrlich's triple stain. The best method is to place the film in an oven, raise the temperature to 150° C., and allow to cool slowly. Without an oven, the proper degree of fixation is difficult to attain. Kowarsky has devised a small plate of two layers of copper (Fig. 77), upon which the films are placed together with a crystal of urea. The plate is heated over a flame until the urea melts, and is then set aside to cool. This is said to give the proper degree of fixation, but in the writer's experience the films have always been underheated. He obtains better results by use of tartaric acid crystals (melting-point, 168°-170° C.). The plate, upon which have been placed the cover-glasses, film side down, and a crystal of the acid, is heated over a low flame until the crystal has completely melted. It should be held sufficiently high above the flame that the heating will require five to seven minutes. The covers are then removed. Freshly made films of normal blood should be allowed to remain upon the plate for a minute or two after heating has ceased. Fresh films require more heat than old ones, and normal blood more than the blood of pernicious anemia and leukemia.

FIG. 77.—Kowarsky's plate for fixing blood (Klopstock and Kowarsky).

Fixation by passing the film quickly through a flame about twenty times, as is often done in routine work, is not recommended for beginners.

3. Staining the Film.—The anilin dyes, which are extensively used in blood work, are of two general classes: basic dyes, of which methylene-blue is the type; and acid dyes, of which eosin is the type. Nuclei and certain other structures in the blood are stained by the basic dyes, and are hence called basophilic. Certain structures take up only acid dyes, and are called acidophilic, oxyphilic, or eosinophilic. Certain other structures are stained only by combinations of the two, and are called neutrophilic. Recognition of these staining properties marked the beginning of modern hematology.

(1) Eosin and Methylene-blue.—In many instances this stain will give all the information desired. It is especially useful in studying the red corpuscles. Nuclei, basophilic granules, and all blood parasites are blue; erythrocytes are red or pink; eosinophilic granules, bright red. Neutrophilic granules and blood-plaques are not stained.

(1) Fix the film by heat or chemicals.

(2) Stain about five minutes with 0.5 per cent. alcoholic solution of eosin, diluted one-half with water.

(3) Rinse in water, and dry between filter-papers.

(4) Stain one-half to one minute with saturated aqueous solution of methylene-blue.

(5) Rinse well, dry, and mount. Films upon slides may be examined with an oil-immersion objective without a cover-glass.

(2) Ehrlich's Triple Stain.—This has been the standard blood-stain for many years, and is still widely used. It is probably the best for neutrophilic granules. It is difficult to make, and should be purchased ready prepared from a reliable dealer. Nuclei are stained blue or greenish-blue; erythrocytes, orange; neutrophilic granules, violet; and eosinophilic granules, copper red. Basophilic granules and blood-plaques are not stained (see [Fig. 85]).

Success in staining depends largely upon proper fixation. The film must be carefully fixed by heat: underheating causes the erythrocytes to stain red; overheating, pale yellow. The staining fluid is applied for five to fifteen minutes, and the preparation is rinsed quickly in water, dried, and mounted. Subsequent application of Löffler's methylene-blue for one-half to one second will bring out the basophilic granules, and improve the nuclear staining, but there is considerable danger of overstaining.

(3) Wright's Stain.—Recently the polychrome methylene-blue-eosin stains, dissolved in methyl-alcohol, have largely displaced other blood-stains for clinical purposes. They combine the fixing with the staining process, and stain differentially every normal and abnormal structure in the blood. Numerous methods of preparing and applying these stains have been devised. Wright's stain is one of the best, and is the most widely used in this country. Directions for preparing it are given in most of the newer large text-books upon clinical diagnosis. The practitioner will find it best to purchase the stain ready prepared. Most microscopic supply houses keep it in stock. The method of application is as follows:

(1) Without previous fixation, cover the blood film with the stain, and let stand one minute.

(2) Add water, drop by drop, until a delicate metallic scum forms upon the surface. Let this mixture remain on the preparation for two or three minutes.

(3) Wash in water until the better spread portions of the film have a pinkish tint.

(4) Dry between filter-papers and mount.

The stain is more conveniently applied upon cover-glasses than upon slides. Films much more than a month old do not stain well. In some localities ordinary tap-water will answer both for diluting the stain and for washing the film; in others, distilled water must be used. Different lots of Wright's fluid vary, and a few preliminary stains should be made with each lot to learn its peculiarities. The principal variation is in the amount of water which must be added to obtain the iridescent scum. Sometimes eight or more drops must be added after the scum appears.

When properly applied, Wright's stain gives the following picture (Plate VI): erythrocytes, yellow or pink; nuclei, various shades of bluish-purple; neutrophilic granules, reddish-lilac; eosinophilic granules, bright red; basophilic granules of leukocytes and degenerated red corpuscles, very dark bluish-purple; blood-plaques, dark lilac; bacteria, blue. The cytoplasm of lymphocytes is generally robin's-egg blue; that of the large mononuclears may have a faint bluish tinge. Malarial parasites stain characteristically: the cytoplasm, sky-blue; the chromatin, reddish-purple.

Jenner's stain, which gives a somewhat similar picture, is preferred by many for differential counting of leukocytes. It brings out neutrophilic granules rather more clearly, but does not compare with Wright's fluid as a stain for the malarial parasite. Unfixed films are stained about three minutes, rinsed quickly, dried, and mounted.

For the physician who wishes to use only one blood-stain, Wright's fluid is undoubtedly the best of those mentioned.

PLATE VI

EXPLANATION OF PLATE VI

Stained with Wright's stain. All drawn to same scale. 1, Normal red corpuscle for comparison; 2, normoblasts, one with lobulated nucleus; 3, megaloblast and microblast. The megaloblast shows a considerable degree of polychromatophilia; 4, blood-plaques, one lying upon a red corpuscle; 5, lymphocytes, large and small; 6, large mononuclear leukocyte; 7, transitional leukocyte; 8, polymorphonuclear neutrophilic leukocytes; 9, eosinophilic leukocytes, one ruptured; 10, basophilic leukocyte; 11, neutrophilic myelocyte. The granules are sometimes less numerous and less distinct than here shown; 12, eosinophilic myelocytes; 13, basophilic myelocyte; 14, "irritation" or "stimulation" form, with small vacuoles; 15, degenerated leukocytes: two polymorphonuclear neutrophiles, one ruptured, one swollen and vacuolated; and a "basket cell" composed of an irregular meshwork of nuclear material; 16, large mononuclear leukocyte containing pigment-granules: from a case tertian malaria; 17, four stages in the asexual cycle of the tertian malarial parasite: the second and fourth were drawn from the same slide taken from a case of double tertian; 18, red corpuscle containing tertian parasite and showing malarial stippling; 19, estivo-autumnal malarial parasites: two small ring forms within the same red cell, and a crescent with remains of the red corpuscle in its concavity.

B. STUDY OF STAINED FILMS

Much can be learned from stained blood-films. They furnish the best means of studying the morphology of the blood and blood parasites, and, to the experienced, they give a fair idea of the amount of hemoglobin and the number of red and white corpuscles. A one-twelfth-inch objective is required.

1. Erythrocytes.—Normally, the red corpuscles are acidophilic. The colors which they take with different stains have been described. When not crowded together, they appear as circular, homogeneous discs, of nearly uniform size, averaging 7.5 µ in diameter ([Fig. 84]). The center of each is somewhat paler than the periphery. The degree of pallor furnishes a rough index to the amount of hemoglobin in the corpuscle. They are apt to be crenated when the film has dried too slowly.

Pathologically, red corpuscles vary in size and shape, staining properties, and structure.

(1) Variations in Size and Shape (See [Plate VIII and Fig. 84]).—The cells may be abnormally small (called microcytes, 5 µ or less in diameter); abnormally large (macrocytes, 10 to 12 µ); or extremely large (megalocytes, 12 to 20 µ).

Variation in shape is often very marked. Oval, pyriform, caudate, saddle-shaped, and club-shaped corpuscles are common. They are called poikilocytes, and their presence is spoken of as poikilocytosis.

Red corpuscles which vary from the normal in size and shape are present in most symptomatic anemias, and in the severer grades are often very numerous. Irregularities are particularly conspicuous in leukemia and pernicious anemia, where, in some instances, a normal erythrocyte is the exception. In pernicious anemia there is a decided tendency to large size and oval forms, and megalocytes are rarely found in any other condition.

(2) Variations in Staining Properties (See [Plate VIII]).—These include polychromatophilia, basophilic degeneration, and basophilic stippling. They are probably degenerative changes, although polychromatophilia is thought by many to be evidence of youth in a cell, and hence to indicate an attempt at blood regeneration.

(a) Polychromatophilia.—Some of the corpuscles partially lose their normal affinity for acid stains, and take the basic stain to greater or less degree. Wright's stain gives such cells a faint bluish tinge when the condition is mild, and a rather deep blue when severe. Sometimes only part of a cell is affected. A few polychromatophilic corpuscles can be found in marked symptomatic anemias. They occur most abundantly in malaria, leukemia, and pernicious anemia.

(b) Basophilic Granular Degeneration (Degeneration of Grawitz).—This is characterized by the presence, within the corpuscle, of small basophilic granules. They stain deep blue with Wright's stain; not at all, with Ehrlich's triple stain. The cell containing them may stain normally in other respects, or it may exhibit polychromatophilia.

Numerous cells showing this degeneration are commonly found in chronic lead-poisoning, of which they were at one time thought to be pathognomonic. Except in this disease, the degeneration indicates a serious blood condition. It occurs in well-marked cases of pernicious anemia and leukemia, and, much less commonly, in very severe symptomatic anemias.

(c) Basophilic Stippling.—This term has been applied to the finely granular appearance often seen in red corpuscles, which harbor malarial parasites ([Plate VI]). It is commonly classed with the degeneration just described, but is probably distinct. Not all stains will show it. With Wright's stain it can be brought out by staining longer and washing less than for the ordinary blood-stain. The minute granules stain reddish purple.

(3) Variations in Structure.—The most important is the presence of a nucleus (Plates [VI] and [VIII and Fig. 84]). Nucleated red corpuscles, or erythroblasts, are classed according to their size: microblasts, 5 µ or less in diameter; normoblasts, 5 to 10 µ; and megaloblasts, above 10 µ. Microblasts and normoblasts contain one, rarely two, small, round, sharply defined, deeply staining nuclei, often located eccentrically. Occasionally the nucleus is irregular in shape, "clover-leaf" forms being not infrequent. The megaloblast is probably a distinct cell, not merely a larger size of the normoblast. Its nucleus is large, stains rather palely, has a delicate chromatin network, and often shows evidences of degeneration (karyorrhexis, etc.). In ordinary work, however, it is safer to base the distinction upon size than upon structure. Any nucleated red cell, but especially the megaloblast, may exhibit polychromatophilia.

Normally, erythroblasts are present only in the blood of the fetus and of very young infants. Pathologically, normoblasts occur in severe symptomatic anemia, leukemia, and pernicious anemia. They are most abundant in myelogenous leukemia. While always present in pernicious anemia, they are often difficult to find. Megaloblasts are found in pernicious anemia, and with extreme rarity in any other condition. They almost invariably exceed the normoblasts in number, which is one of the distinctive features of the disease. Microblasts have much the same significance as normoblasts, but are less common.

2. The Leukocytes.—An estimation of the number or percentage of each variety of leukocyte in the blood is called a differential count.

The differential count is best made upon a film stained with Wright's, Jenner's, or Ehrlich's stain. Go carefully over the film with an oil-immersion lens, using a mechanical stage if available. Classify each leukocyte seen, and calculate what percentage each variety is of the whole number classified. For accuracy, 500 to 1000 leukocytes must be classified; for approximate results, 200 are sufficient. Track of the count may be kept by placing a mark for each leukocyte in its appropriate column, ruled upon paper. Some workers divide a slide-box into compartments with slides, one for each variety of leukocyte, and drop a coffee-bean into the appropriate compartment when a cell is classified. When a convenient number of coffee-beans is used (any multiple of 100), the percentage calculation is extremely easy.

The actual number of each variety in a cubic millimeter of blood is easily calculated from these percentages and the total leukocyte count. An increase in actual number is an absolute increase; an increase in percentage only, a relative increase. It is evident that an absolute increase of any variety may be accompanied by a relative decrease.

A record is generally kept of the number of nucleated red cells seen during a differential count of leukocytes.

The usual classification of leukocytes is based upon their size, their nuclei, and the staining properties of the granules which many of them contain. It is not altogether satisfactory, but is probably the best which our present knowledge permits.

(1) Normal Varieties.—(a) Lymphocytes.—These are small mononuclear cells without granules ([Plate VI] and [Fig. 86]). They are about the size of a red corpuscle or slightly larger, and consist of a single, sharply defined, deeply staining nucleus, surrounded by a narrow rim of protoplasm. The nucleus is generally round, but is sometimes indented at one side. Wright's stain gives the nucleus a deep purple color and the cytoplasm a pale robin's-egg blue in typical cells. Larger forms of lymphocytes are frequently found, especially in the blood of children, and are difficult to distinguish from the large mononuclear leukocytes.

Lymphocytes are formed in the lymphoid tissues, including that of the bone-marrow. They constitute, normally, 20 to 30 per cent. of all leukocytes, or about 1000 to 3000 per c.mm. of blood. They are more abundant in the blood of children.

The percentage of lymphocytes is usually moderately increased in those conditions which give leukopenia, especially typhoid fever, chlorosis, pernicious anemia, and many debilitated conditions. A marked increase, accompanied by an increase in the total leukocyte count, is seen in pertussis and lymphatic leukemia. In the latter, the lymphocytes sometimes exceed 98 per cent.

(b) Large Mononuclear Leukocytes ([Plate VI]).—These cells are two or three times the diameter of the normal red corpuscle. Each contains a single round or oval nucleus, often located eccentrically. The zone of protoplasm surrounding the nucleus is relatively wide. With Wright's stain the nucleus is less deeply colored than that of the lymphocyte, while the cytoplasm is very pale blue or colorless, and sometimes contains a few reddish granules. The size of the cell, the width of the zone of cytoplasm, and the depth of color of the nucleus are the points to be considered in distinguishing between large mononuclears and lymphocytes. When large forms of the lymphocyte are present, the distinction is often difficult or impossible. It is then advisable to count the two cells together as lymphocytes. Indeed, they are regarded by some hematologists as identical.

Large mononuclear leukocytes probably originate in the bone-marrow or spleen. They constitute 2 to 4 per cent. of the total number of leukocytes: 100 to 400 per c.mm. of blood. An increase is unusual except in malaria, where it is quite constantly observed, and where many of the cells contain ingulfed pigment.

(c) Transitional Leukocytes ([Plate VI]).—These are essentially large mononuclears with deeply indented or horseshoe-shaped nuclei. A few fine neutrophilic granules are sometimes present in their cytoplasm.

They are probably formed from the large mononuclears, and occur in the blood in about the same numbers. The two cells are usually counted together.

(d) Polymorphonuclear Neutrophilic Leukocytes ([Plate VI]).—There is usually no difficulty in recognizing these cells. Their average size is somewhat less than that of the large mononuclears. The nucleus stains rather deeply, and is extremely irregular, often assuming shapes comparable to letters of the alphabet, E, Z, S, etc. ([Fig. 86]). Frequently there appear to be several separate nuclei, hence the widely used name, "polynuclear leukocyte." Upon careful inspection, however, delicate nuclear bands connecting the parts can usually be seen. The cytoplasm is relatively abundant, and contains great numbers of very fine neutrophilic granules. With Wright's stain the nucleus is bluish-purple, and the granules, reddish-lilac.

Polymorphonuclear leukocytes are formed in the bone-marrow from neutrophilic myelocytes. They constitute 60 to 75 per cent. of all the leukocytes: 3000 to 7500 per c.mm. of blood. Increase in their number practically always produces an increase in the total leukocyte count, and has already been discussed under "phagocytic leukocytosis." The leukocytes of pus, pus-corpuscles, belong almost wholly to this variety.

A comparison of the percentage of polymorphonuclear cells with the total leukocyte count yields more information than a consideration of either alone. With moderate infection and good resisting powers the leukocyte count and the percentage of polymorphonuclears are increased proportionately. When the polymorphonuclear percentage is increased to a notably greater extent than is the total number of leukocytes, no matter how low the count, either very poor resistance or a very severe infection may be inferred. In the absence of acute infectious disease a polymorphonuclear percentage of 85 or over points very strongly to gangrene or pus-formation. On the other hand, except in children, where the percentage is normally low, pus is uncommon with less than 80 per cent. of polymorphonuclears.

Normally, the cytoplasm of leukocytes stains pale yellow with iodin. Under certain pathologic conditions the cytoplasm of many of the polymorphonuclears stains diffusely brown, or contains granules which stain reddish-brown with iodin. This is called iodophilia. Extracellular iodin-staining granules, which are present normally, are more numerous in iodophilia.

This iodin reaction occurs in all purulent conditions except abscesses which are thoroughly walled off or purely tuberculous abscesses. It is of some value in diagnosis between serous effusions and purulent exudates, between catarrhal and suppurative processes in the appendix and Fallopian tube, etc. Its importance, however, as a diagnostic sign of suppuration has been much exaggerated, since it may occur in any general toxemia, such as pneumonia, influenza, malignant disease, and puerperal sepsis.

To demonstrate iodophilia, place the air-dried films in a stoppered bottle containing a few crystals of iodin until they become yellow. Mount in syrup of levulose and examine with a one-twelfth objective.

(e) Eosinophilic Leukocytes, or "Eosinophiles" ([Plate VI]).—The structure of these cells is similar to that of the polymorphonuclear neutrophiles, with the striking difference that, instead of fine neutrophilic granules, their cytoplasm contains coarse granules having a strong affinity for acid stains. They are easily recognized by the size and color of the granules, which stain bright red with Wright's stain.

Eosinophiles are formed in the bone-marrow from eosinophilic myelocytes. Their normal number varies from 50 to 400 per c.mm. of blood, or 1 to 4 per cent. of the leukocytes. An increase is called eosinophilia, and is better determined by the actual number than by the percentage.

Slight eosinophilia is physiologic during menstruation. Marked eosinophilia is always pathologic. It occurs in a variety of conditions, the most important of which are: infection by animal parasites; bronchial asthma; myelogenous leukemia; scarlet fever; and many skin diseases.

Eosinophilia may be a symptom of infection by any of the worms. It is fairly constant in trichinosis, uncinariasis, filariasis, and echinococcus disease. In this country an unexplained marked eosinophilia warrants examination of a portion of muscle for Trichina spiralis ([p. 255]).

True bronchial asthma commonly gives a marked eosinophilia during and following the paroxysms. This is helpful in excluding asthma of other origin. Eosinophiles also appear in the sputum in large numbers.

In myelogenous leukemia there is almost invariably an absolute increase of eosinophiles, although, owing to the great increase of other leukocytes, the percentage is usually diminished. Dwarf and giant forms are often numerous.

Scarlet fever is frequently accompanied by eosinophilia, which may help to distinguish it from measles.

Eosinophilia has been observed in a large number of skin diseases, notably pemphigus, prurigo, psoriasis, and urticaria. It probably depends less upon the variety of the disease than upon its extent.

(f) Basophilic Leukocytes or "Mast-cells" ([Plate VI]).—In general, these resemble polymorphonuclear neutrophiles except that the nucleus is less irregular and that the granules are larger and have a strong affinity for basic stains. They are easily recognized. With Wright's stain the granules are deep purple, while the nucleus is pale blue and is nearly or quite hidden by the granules, so that its form is difficult to make out. These granules are not colored by Ehrlich's stain.

The nature of mast-cells is undetermined. They probably originate in the bone-marrow. They are least numerous of the leukocytes in normal blood, rarely exceeding 0.5 per cent., or 25 to 50 per c.mm. A notable increase is limited almost exclusively to myelogenous leukemia, where they are sometimes very numerous.

(2) Abnormal Varieties.—(a) Myelocytes ([Plate VI]).—These are large mononuclear cells whose cytoplasm is filled with granules. Typically, the nucleus occupies about one-half of the cell, and is round or oval. It is sometimes indented, with its convex side in contact with the periphery of the cell. It stains rather feebly. The average diameter of this cell (about 15.75 µ) is greater than that of any other leukocyte, but there is much variation in size among individual cells. Myelocytes are named according to the character of their granules—neutrophilic, eosinophilic, and basophilic myelocytes. These granules are identical with the corresponding granules in the leukocytes just described. The occurrence of two kinds of granules in the same cell is rare.

Myelocytes are the bone-marrow cells from which the corresponding granular leukocytes are developed. Their presence in the blood in considerable numbers is diagnostic of myelogenous leukemia. The neutrophilic form is the less significant. A few of these may be present in very marked leukocytosis or any severe blood condition, as pernicious anemia. Eosinophilic myelocytes are found only in myelogenous leukemia, where they are often very numerous. The basophilic variety is less common, and is confined to long-standing severe myelogenous leukemia.

(b) Atypical Forms.—Leukocytes which do not fit in with the above classification are not infrequently met, especially in high-grade leukocytosis, pernicious anemia, and leukemia. The nature of most of them is not clear, and their number is usually so small that they may be disregarded in making a differential count. Among them are: (a) Border-line forms between polymorphonuclear neutrophiles and neutrophilic myelocytes; (b) small neutrophilic cells with a single round, deeply staining nucleus: they probably result from division of polymorphonuclear neutrophiles; (c) "irritation forms"—large non-granular mononuclear cells, whose cytoplasm stains fairly deep purple with Wright's stain, and intense brown with Ehrlich's: they appear in the blood under the same conditions as myelocytes; (d) degenerated forms: vacuolated leukocytes, or merely palely or deeply staining homogeneous or reticulated masses of chromatin ([Plate VI]).

3. Blood-plaques.—These are not colored by Ehrlich's stain, nor by eosin and methylene-blue. With Wright's stain they appear as spheric or ovoid, reddish to violet, granular bodies, 2 to 4 µ in diameter. When well stained, a delicate hyaline peripheral zone can be distinguished. In ordinary blood-smears they are usually clumped in masses. A single platelet lying upon a red corpuscle may easily be mistaken for a malarial parasite ([Plate VI]).

Blood-platelets are being much studied at present, but, aside from the facts mentioned under their enumeration ([p. 165]), little of clinical value has been learned. They have been variously regarded as very young red corpuscles (the "hematoblasts" of Hayem), as disintegration products of leukocytes, as remnants of extruded nuclei of erythrocytes, and as independent nucleated bodies. The most probable explanation of their origin seems to be that of J. H. Wright, who, from his recent studies, regards them as detached portions of the cytoplasm of certain giant-cells of the bone-marrow and spleen.

VII. BLOOD PARASITES

A study of blood bacteriology is useful, but is hardly practicable for the practitioner. Most bacteria can be detected only by culture methods. The spirillum of relapsing fever can be identified by the method for the malarial parasite in fresh blood. The blood must be taken during a paroxysm. The organism is an actively motile spiral thread, about four times the diameter of a red corpuscle in length. The movements which its active motion causes among the corpuscles render it conspicuous. It can also be seen in stained preparations (Fig. 78). The disease has rarely been seen in the United States.

FIG. 78.—Spirillum of relapsing fever (Karg and Schmorl).

Of the numerous animal parasites which have been found in the blood, three are especially interesting clinically: Plasmodium malariæ, Filaria sanguinis hominis, and Trypanosoma hominis.

1. Plasmodium Malariæ.—This organism is one of a large group, the hemosporidia ([p. 247]), many of which live within and destroy the red corpuscles of various animals. Three varieties are associated with malarial fever in man—the tertian, quartan, and estivo-autumnal malarial parasites.

(1) Life Histories.—There are two cycles of development: one, the asexual, in the blood of man; and the other, the sexual, in the intestinal tract of a particular variety of mosquito.

(a) Asexual Cycle.—The young organism enters the blood through the bite of the mosquito. It makes its way into a red corpuscle, where it appears as a small, pale "hyaline" body. This body exhibits ameboid movement and increases in size. Soon, dark-brown granules derived from the hemoglobin of the corpuscle make their appearance within it. When it has reached its full size—filling and distending the corpuscle in the case of the tertian parasite, smaller in the others—the pigment-granules gather at the center or at one side; the organism divides into a number of small hyaline bodies, the spores or merozoites; and the red corpuscle bursts, setting spores and pigment free in the blood-plasma. This is called segmentation. It coincides with, and by liberation of toxins causes, the paroxysm of the disease. A considerable number of the spores are destroyed by leukocytes or other agencies; the remainder enter other corpuscles and repeat the cycle. Many of the pigment-granules are taken up by leukocytes. In estivo-autumnal fever segmentation occurs in the internal organs and the segmenting and larger pigmented forms are not seen in the peripheral blood.

The asexual cycle of the tertian organism occupies forty-eight hours; of the quartan, seventy-two hours; of the estivo-autumnal, an indefinite time—usually twenty-four to forty-eight hours.

The parasites are thus present in the blood in great groups, all the individuals of which reach maturity and segment at approximately the same time. This explains the regular recurrence of the paroxysms at intervals corresponding to the time occupied by the asexual cycle of the parasite. Not infrequently there is multiple infection, one group reaching maturity while the others are still young; but the presence of two groups which segment upon the same day is extremely rare. Fevers of longer intervals—six, eight, ten days—are probably due to the ability of the body, sometimes of itself, sometimes by aid of quinin, to resist the parasites so that a number sufficient to cause a paroxysm do not accumulate in the blood until after several repetitions of the asexual cycle. In estivo-autumnal fever the regular grouping, while usually present at first, is soon lost, thus causing "irregular malaria."

(b) Sexual Cycle.—Besides the ameboid individuals which pass through the asexual cycle, there are present with them in the blood many individuals with sexual properties. These are called gametes. They do not undergo segmentation. Many of them are apparently extracellular, but stained preparations usually show them to be surrounded by the remains of a corpuscle. In tertian and quartan malaria they cannot easily be distinguished from the asexual individuals until a variable time after the blood leaves the body, when the male gamete sends out one or more flagella. In estivo-autumnal malaria the gametes take distinctive ovoid and crescentic forms, and are not difficult to recognize. They are very resistent to quinin and often persist in the blood long after the ameboid forms have been destroyed, but are probably incapable of continuing the disease until they have passed through the cycle in the mosquito.

When a malarious person is bitten by a mosquito, the gametes are taken with the blood into its stomach. Here a flagellum from the male unites with the female, which soon thereafter becomes encysted in the wall of the intestine. After a time it ruptures, liberating many minute rods, or sporozoites, which have formed within it. These migrate to the salivary glands, and are carried into the blood of the person whom the mosquito bites. Here they enter red corpuscles as young malarial parasites, and the majority pass through the asexual cycle just described.

FIG. 79.—Mosquitos—Culex (1) and anopheles (2) (Bergey).

FIG. 80.—Showing, on the left, anopheles in resting position, its dappled wing, and the position of its larvæ in water; on the right, culex in resting position, its plain wing, and the position of its larvæ in water. The arrows indicate the directions taken by the larvæ when the water is disturbed (Abbott).

The sexual cycle can take place only within the body of one genus of mosquito, anopheles. Absence of this mosquito from certain districts explains the absence of malaria. It is distinguished from our common house-mosquito, culex, by the relative lengths of proboscis and palpi (Fig. 79), which can be seen with a hand-lens, by its attitude when resting, and by its dappled wing (Fig. 80). Anopheles is strictly nocturnal in its habits; it usually flies low, and rarely travels more than a few hundred yards from its breeding-place, although it may be carried by winds. These facts explain certain peculiarities in malarial infection; thus, infection occurs practically only at night; it is most common near stagnant water, especially upon the side toward which the prevailing winds blow; and the danger is greater when persons sleep upon or near the ground than in upper stories of buildings. The insects frequently hibernate in warmed houses, and may bite during the winter. A mosquito becomes dangerous in eight to fourteen days after it bites a malarious person, and remains so throughout its life.

(2) Detection.—Search for the malarial parasite may be made in either fresh blood or stained films. If possible, the blood should be obtained a few hours before the chill—never during it nor within a few hours afterward, since at that time (in single infections) only the very young, unpigmented forms are present, and these are the most difficult to find and recognize. Sometimes many parasites are found in a microscopic field; sometimes, especially in estivo-autumnal infection, owing to accumulation in internal organs, careful search is required to find any, despite very severe symptoms. Quinin causes them rapidly to disappear from the peripheral blood, and few or none may be found after its administration. In the absence of organisms, the presence of pigment granules within leukocytes—polymorphonuclears and large mononuclears—may be taken as presumptive evidence of malaria. Pigmented leukocytes ([Plate VI]) are most numerous after a paroxysm.

(a) In Fresh Unstained Blood (Plate VII).—Obtain a small drop of blood from the finger or lobe of the ear. Touch the center of a cover-glass to the top of the drop and quickly place it, blood side down, upon a slide. If the slide and cover be perfectly clean and the drop not too large, the blood will spread out so as to present only one layer of corpuscles. Search with a one-twelfth-inch objective, using very subdued light.

The young organisms appear as small, round, ring-like or irregular, colorless bodies within red corpuscles. The light spots caused by crenation and other changes in the corpuscles are frequently mistaken for them, but are generally more refractive or have more sharply defined edges. The older forms are larger colorless bodies containing granules of brown pigment. In the case of the tertian parasite, these granules have active vibratory motion, which renders them conspicuous; and as the parasite itself is very pale, one may see only a large pale corpuscle in which fine pigment-granules are dancing. Segmenting organisms, when typical, appear as rosets, often compared to daisies, the petals of which represent the segments, while the central brown portion represents the pigment. Tertian segmenting forms are less frequently typical than quartan. Flagellated forms are not seen until ten to twenty minutes after the blood has left the vessels. As Cabot suggests, one should, while searching, keep a sharp lookout for unusually large or pale corpuscles, and for anything which is brown or black, or in motion.

PLATE VII

EXPLANATION OF PLATE VII

Various forms of malarial parasites (Thayer and Hewetson). 1 to 10, inclusive, Tertian organisms; 11 to 17, inclusive, quartan organisms; 18 to 27, inclusive, estivo-autumnal organisms; 1, young hyaline form; 2, hyaline form with beginning pigmentation; 3, pigmented form; 4, full-grown pigmented form; 5, 6, 7, 8, segmenting forms; 9, extracellular pigmented form; 10, flagellate form; 11, young hyaline form; 12, 13, pigmented forms; 14, fully developed pigmented form; 15, 16, segmenting forms; 17, flagellate form; 18, 19, 20, ring-like and cross-like hyaline forms; 21, 22, pigmented forms; 23, 24, segmenting forms; 25, 26, 27, crescents.

(b) In Stained Films ([Plate VI]).—Recognition of the parasite, especially the young forms, is much easier in films stained by Wright's or some similar stain than in fresh blood. When very scarce, they may sometimes be found, although their structure is not well shown, by the method of Ruge. This consists in spreading a very thick layer of blood, drying, placing for a few minutes in a fluid containing 5 per cent. formalin and 1 per cent. acetic acid, which removes the hemoglobin and fixes the smear, rinsing, drying, and finally staining. If Wright's stain be used in this method, it is recommended that the preparation be subsequently stained for a half-minute with borax-methylene-blue (borax, 5; methylene-blue, 2; water, 100).

In films which are properly stained with Wright's fluid the young organisms are small, round, ring-like or irregular, sky-blue bodies, each with a very small, sharply defined, reddish-purple chromatin mass. Many structures—deposits of stain, dirt, blood-plaques lying upon red cells, etc.—may simulate them, but should not deceive one who looks carefully for both the blue cytoplasm and the reddish-purple chromatin. A plaque upon a red corpuscle is surrounded by a colorless zone rather than by a distinct blue body. Young estivo-autumnal parasites commonly take a "ring" form (the chromatin mass representing the jewel), which is infrequently assumed by the other varieties. The older tertian and quartan organisms show larger sky-blue bodies with more reticular chromatin, and contain brown granules of pigment, which, however, is less evident than in the living parasite. The chromatin is often scattered through the cytoplasm or apparently outside of it, and is sometimes difficult to see clearly. Typical "segmenters" present a ring of rounded segments or spores, each with a small, dot-like chromatin mass. With the tertian parasite, the segments more frequently form an irregular cluster. The pigment is collected near the center or scattered among the segments. In estivo-autumnal fever usually only the small "ring bodies" and the crescentic and ovoid gametes are seen in the blood. The gametes are easily recognized. Their length is somewhat greater than the diameter of a red corpuscle. Their chromatin is usually centrally placed, and they contain more or less coarse pigment. The remains of the red cell often form a narrow rim around them or fill the concavity of the crescent.

While the parasites are more easily found in stained preparations, the varieties are more easily differentiated in fresh blood. The chief distinguishing points are included in the following table:

VARIETIES OF THE MALARIAL ORGANISM
TERTIAN.	QUARTAN.	ESTIVO-AUTUMNAL.
Asexual cycle, forty-eight hours.	Seventy-two hours.	Usually twenty-four to forty-eight hours.
Substance pale, transparent, comparable to hyaline tube-cast.	Highly refractive, comparable to waxy tube-cast.	Highly refractive.
Outline indistinct.	Distinct.	Distinct.
Ameboid motion active.	Sluggish.	Active.
Mature asexual form large; fills and often distends corpuscle.	Smaller.	Young forms, only, in peripheral blood.
Pigment-granules fine, brown, scattered throughout. Very active dancing motion.	Much coarser, darker in color, peripherally arranged. Motion slight.	Very few, minute, inactive. Distinctly pigmented forms seldom seen.
Segmenting body rarely assumes typical "daisy" form. 15 to 20 segments.	Usually typical "daisy." 6 to 12 segments.	Not seen in peripheral blood.
Gametes resemble asexual forms.	Same as tertian.	Appear in blood as distinctive ovoids and crescents.
Red corpuscles pale and swollen.	Generally darker than normal.	Dark, often bronzed.

2. Filaria Sanguinis Hominis.—Of the several varieties of this worm, Filaria nocturna is most common and most important clinically. The adults are thread-like worms about 8 to 10 cm. long. They are rarely seen. They live in pairs in the lymphatic channels and glands, especially those of the pelvis and groin, and often occur in such numbers as to obstruct the flow of lymph. This is the most common cause of elephantiasis. Infection is very common in tropical countries, especially in Samoa, the West Indies, Central America, and the Isthmus of Panama. It is said that in Samoa 50 per cent. of the natives are infected.

The female is viviparous, and produces vast numbers of embryos, which appear in the circulating blood. These embryos are very actively motile, worm-like structures, about as wide as a red corpuscle and 0.2 to 0.4 mm. long ([Fig. 107]). They are found in the peripheral blood only at night, appearing about 8 P.M. and reaching their maximum number—which is sometimes enormous—about midnight. If the patient change his time of sleeping, they will appear during the day. Infection is carried by a variety of mosquito, which acts as intermediate host.

Diagnosis rests upon detection of embryos in the blood. They can be seen in stained preparations, but are best found in fresh unstained blood. A rather large drop is taken upon a slide, covered, and examined with a low power. The embryo can be located by the commotion which its active motion produces among the corpuscles. This motion consists almost wholly in apparently purposeless lashing and coiling movements, and continues for many hours.

3. Trypanosoma Hominis.—Various trypanosomes are common in the blood of fishes, amphibians, birds, and mammals (Fig. 81). They live in the blood-plasma and do not attack the corpuscles. In some animals they are apparently harmless; in others they are an important cause of disease.

FIG. 81.—Trypanosomes from blood of gray rat (Boston).

Trypanosoma hominis is an actively motile, spindle-shaped organism, two or three times the diameter of a red corpuscle in length, with one end terminating in a long flagellum. It can be seen with medium power objectives in either fresh or stained blood. Human trypanosomiasis is common in Africa. As a rule, it is a very chronic disease. "Sleeping sickness" is a late stage when the organisms have invaded the cerebrospinal fluid. Infection is carried by a biting fly, Glossina palpalis.

VIII. SERUM REACTIONS

1. Agglutination.—In the blood-serum of persons suffering from certain infectious diseases there exist soluble bodies, called agglutinins, which have the property of rendering non-motile and clumping the specific micro-organism of the disease, and have little or no influence upon other bacteria. This "agglutination" takes place even when the blood is greatly diluted. Undiluted normal blood can agglutinate most bacteria, but loses this power when diluted to any considerable degree. These facts are taken advantage of in the diagnosis of several diseases.

When applied to the diagnosis of typhoid fever, the phenomenon is known as the Widal reaction. As yet, it is the only agglutination reaction which has any practical value for the practitioner.

Either blood-serum or the whole blood may be used. Serum is the better. To obtain it, it is convenient to use little vials, such as can be made by breaking off the lower half-inch of the tubes which have contained peptonizing powder. They must, of course, be well cleaned. One of these is filled to a depth of about one-fourth inch from a puncture in the finger, and is set aside for a few hours. When the clot has separated, it is picked out with a needle, leaving the serum. One drop of the serum is then added to nine drops of normal salt solution, making a dilution of 1:10. Distilled water may be used for dilution, but is more liable to cause error. The dilution can be more accurately made in the leukocyte pipet of the Thoma-Zeiss instrument. When the whole blood is used, it can be secured in the pipet and at once diluted with the salt solution. When it must be transported a considerable distance, dried blood is most convenient. A large drop is allowed to dry upon a clean slide or unglazed paper. It will keep for months without losing its agglutinating power. When ready to make the test, the dried stain is dissolved in ten drops of normal salt solution, care being taken that the drops are about the same size as the original drop of blood.

The reaction can be detected either microscopically or macroscopically:

Microscopic Method.—(1) The blood or serum having been obtained and diluted 1:10 as just described, mix it with a bouillon culture of the typhoid bacillus to any desired dilution. One drop of each makes a blood-dilution of 1:20, etc. The culture should be between eighteen and twenty-four hours old, and the bacilli must be actively motile. A stock agar culture should be kept at room temperature, and bouillon tubes inoculated the day before the examination is to be made. Agar cultures can be purchased from dealers in biologic products. They must be renewed monthly.

Instead of the bouillon culture, McFarland recommends the use of a suspension made by removing some of the growth from the surface of a fresh agar culture and mixing it well with a little sterile water. It is then necessary to examine the suspension microscopically to make sure that there are no natural clumps.

(2) Place a few drops of the mixture of blood and culture upon a perfectly clean slide and apply a cover-glass. The cover may be ringed with vaselin to prevent evaporation, but this is not usually necessary.

(3) Examine at intervals with a high dry lens—a one-sixth will answer very well. The light must be very subdued. At first the bacilli should be actively moving about. If the blood be from a case of typhoid, they will gradually lose their motion and gather together in clumps (Fig. 82). The clumps should be large, and the few bacilli remaining isolated should be motionless. Pseudo-reactions, in which there are a few small clumps of bacilli whose motion is not entirely lost, together with many freely moving bacilli scattered throughout the field, should not mislead. As a control, a drop of the culture should always be examined before making the test.

FIG. 82.—Showing clumping of typhoid bacilli in the Widal reaction. At one point a crenated red blood-corpuscle is seen (Wright and Brown).

Normal blood may produce clumping if time enough be allowed. The diagnostic value of a positive reaction is, therefore, impaired unless clumping takes place within a limited time. With dilution of 1:20 the time limit should not exceed one-half hour; with 1:40, one hour. Tests based upon lower dilution than 1:20 are probably not reliable.

Macroscopic Method.—The principle is the same as that of the microscopic method. Clumping of the bacilli causes a flocculent precipitate, which can be seen with the naked eye. A dead culture gives the same results as a living one. This method is as reliable as the microscopic and is more convenient for the practitioner, although it requires more time.

Dead cultures, together with apparatus for diluting the blood, are put up at slight cost by various firms under the names of typhoid diagnosticum, typhoid agglutometer, etc. Full directions accompany these outfits.

The Widal reaction is positive in over 95 per cent. of all cases of typhoid fever. It may, rarely, be positive in other conditions, owing, sometimes at least, to faulty technic. It appears often as early as the sixth or seventh day; usually during the second week. It remains during the whole course of the disease, and frequently persists for years.

2. Opsonins.—That phagocytosis plays an important part in the body's resistance to bacterial invasion has long been recognized. According to Metchnikoff, this property of leukocytes resides entirely within themselves, depending upon their own vital activity. The recent studies of Wright and Douglas, upon the contrary, indicate that the leukocytes are impotent in themselves, and can ingest bacteria only in the presence of certain substances which exist in the blood-plasma. These substances have been named opsonins. Their nature is undetermined. They probably act by uniting with the bacteria, thus preparing them for ingestion by the leukocytes; but they do not cause death of the bacteria, nor produce any appreciable morphologic change. They appear to be more or less specific, a separate opsonin being necessary for phagocytosis of each species of bacteria. There are, moreover, opsonins for other formed elements—red blood-corpuscles, for example. It has been shown that the quantity of opsonins in the blood can be greatly increased by inoculation with dead bacteria.

To measure the amount of any particular opsonin in the blood Wright has devised a method which involves many ingenious and delicate technical procedures. Much skill, such as is attained only after considerable training in laboratory technic, is requisite, and there are many sources of error. It is, therefore, beyond the province of this work to recount the method in detail. In a general way it consists in: (a) Preparing a mixture of equal parts of the patient's blood-serum, an emulsion of the specific micro-organism, and a suspension of washed leukocytes; (b) preparing a similar mixture, using serum of a normal person; (c) incubating both mixtures for a definite length of time; and (d) making smears from each, staining, and examining with a one-twelfth objective. The number of bacteria which have been taken up by a definite number of leukocytes is counted, and the average number of bacteria per leukocyte is calculated; this gives the "phagocytic index." The phagocytic index of the blood under investigation, divided by that of the normal blood, gives the opsonic index of the former, the opsonic index of the normal blood being taken as 1. Simon regards the percentage of leukocytes which have ingested bacteria as a more accurate measurement of the amount of opsonins than the number of bacteria ingested, because the bacteria are apt to adhere and be taken in in clumps.

Wright and his followers regard the opsonic index as an index of the power of the body to combat bacterial invasion. They claim very great practical importance for it as an aid to diagnosis and as a guide to treatment by the vaccine method. This method of treatment consists in increasing the amount of protective substances in the blood by injections of normal-salt suspensions of dead bacteria of the same species as that which has caused and is maintaining the morbid process, these bacterial suspensions being called "vaccines." The opinion of the majority of conservative men seems to be that while vaccine therapy is undoubtedly an important addition to our methods of treatment of bacterial infections, particularly of those which are strictly local, yet the value of the opsonic index in measuring resisting power or as an aid to diagnosis and guide to treatment is still sub judice.

IX. TESTS FOR RECOGNITION OF BLOOD

1. Guaiac Test.—The technic of this test has been given ([p. 89]). It may be applied directly to a suspected fluid, or, better, to the ethereal extract. Add a few cubic centimeters of glacial acetic acid to about 10 c.c. of the fluid; shake thoroughly with an equal volume of ether; decant, and apply the test to the ether. In case of dried stains upon cloth, wood, etc., dissolve the stain in distilled water and test the water, or press a piece of moist blotting-paper against the stain, and touch the paper with drops of the guaiac and the turpentine successively.

FIG. 83.—Teichmann's hemin crystals (Jakob).

2. Teichmann's Test.—This depends upon the production of characteristic crystals of hemin. It is a sensitive test, and, when positive, is absolute proof of the presence of blood. A number of substances—lime, fine sand, iron rust—interfere with production of the crystals; hence negative results are not always conclusive. Dissolve the suspected stain in a few drops of normal salt solution upon a slide. If a liquid is to be tested, evaporate some of it upon a slide and dissolve the residue in a few drops of the salt solution. Let dry, apply a cover-glass, and run glacial acetic acid underneath it. Heat very gently until bubbles begin to form, replacing the acid as it evaporates. Allow to cool slowly. When cool, replace the acid with water, and examine for hemin crystals with two-thirds and one-sixth objectives. The crystals are dark-brown rhombic plates lying singly or in crosses, and are easily recognized (Fig. 83). Failure to obtain them may be due to too great heat or too rapid cooling. If not obtained at first, let the slide stand in a warm place, as upon a hot-water radiator, for an hour.

X. SPECIAL BLOOD PATHOLOGY

The more conspicuous characteristics of the blood in various diseases have been mentioned in previous sections. Although the great majority of blood changes are secondary, there are a few blood conditions in which the changes are so prominent, or the etiology so obscure, that they are commonly regarded as blood diseases. These will receive brief consideration here.

A. ANEMIA

This is a deficiency of hemoglobin, or red corpuscles, or both. It is either primary or secondary. The distinction is based chiefly upon etiology, although each type presents a more or less distinctive blood-picture. Secondary anemia is that which is symptomatic of some other pathologic condition. Primary anemia is that which progresses without apparent cause.

1. Secondary Anemia.—The more important conditions which produce secondary or symptomatic anemia are:

(a) Poor nutrition, which usually accompanies unsanitary conditions, poor and insufficient food, etc.

(b) Acute infectious diseases, especially rheumatism and typhoid fever. The anemia is more conspicuous during convalescence.

(c) Chronic infectious diseases: tuberculosis, malaria, syphilis, leprosy.

(d) Chronic exhausting diseases, as heart disease, chronic nephritis, cirrhosis of the liver, and gastro-intestinal diseases, especially when associated with atrophy of gastric and duodenal glands. The last may give an extreme anemia, indistinguishable from pernicious anemia.

(e) Chronic poisoning, as from lead, arsenic, and phosphorus.

(f) Hemorrhage—either repeated small hemorrhages, as from gastric cancer and ulcer, uterine fibroids, etc., or a single large one.

(g) Malignant tumors: these affect the blood partly through repeated small hemorrhages, partly through toxic products, and partly through interference with nutrition.

(h) Animal Parasites.—Some cause no appreciable change in the blood; others, like the Uncinaria and Bothriocephalus latus, may produce a very severe anemia, almost identical with pernicious anemia. Anemia in these cases is probably due both to toxins and to abstraction of blood.

The blood-picture varies with the grade of anemia. Diminution of hemoglobin is the most characteristic feature. In mild cases it is slight, and is the only blood change to be noted. In very severe cases hemoglobin may fall to 15 per cent. Red corpuscles are diminished in all but very mild cases, while in the severest cases the red corpuscle count is sometimes below 2,000,000. The color index is usually decreased.

Although the number of leukocytes bears no relation to the anemia, leukocytosis is common, being due to the same cause.

Stained films show no changes in very mild cases. In moderate cases variations in size and shape of the red cells and polychromatophilia occur. Very severe cases show the same changes to greater degree, with addition of basophilic degeneration and the presence of normoblasts in small or moderate numbers. Megaloblasts in very small numbers have been encountered in extremely severe cases. Blood-plaques are usually increased.

2. Primary Anemia.—The commonly described varieties of primary anemia are pernicious anemia and chlorosis, but splenic anemia may also be mentioned under this head.

(1) Progressive Pernicious Anemia.—It is frequently impossible to diagnose this disease from the blood examination alone. Severe secondary anemia sometimes gives an identical picture. Remissions, in which the blood approaches the normal, are common. All the clinical data must, therefore, be considered.

Hemoglobin and red corpuscles are always greatly diminished. In none of Cabot's 139 cases did the count exceed 2,500,000, the average being about 1,200,000. In more than two-thirds of the cases hemoglobin was reduced to less extent than the red corpuscles: the color-index was, therefore, high. A low color-index probably indicates a mild type of the disease.

The leukocyte count may be normal, but is commonly diminished to about 3000. The decrease affects chiefly the polymorphonuclear cells, so that the lymphocytes are relatively increased. In some cases a decided absolute increase of lymphocytes occurs. Polymorphonuclear leukocytosis, when present, is due to some complication.

FIG. 84.—A, Normal blood; B, chlorosis; C, pernicious anemia. The plate shows the sharp contrast between cells rich in hemoglobin and the pale cell of chlorosis, and also the poikilocytosis and marked variations in size noted in pernicious anemia. A normoblast and megaloblast also appear. Stained smears (from Greene's "Medical Diagnosis").

PLATE VIII

EXPLANATION OF PLATE VIII.

Fig. 1.—Preparation from an advanced case of progressive pernicious anemia from unknown cause: a, Megaloblasts or gigantoblasts; the protoplasm shows marked polychromasia; b, stained granules in erythrocytes with normally stained protoplasm; c and d, polychromatophilic degeneration; e, megalocytes; f, normocytes. Fig. 2.—Preparation from the same case taken some time later while the patient was subjectively and objectively in perfect health: a, Punctate erythrocytes with normal and anemic degenerated protoplasm; b, polynuclear leukocyte; c, normal red blood-corpuscles; d, somewhat enlarged erythrocytes. Fig. 3.—Series of cells from a case of severe progressive pernicious anemia of unknown etiology; preparation made two days antemortem: a, Nucleated red blood-corpuscles characterized as normoblasts by the intense staining of the nuclei; a' and a", karyokinetic figures in erythrocytes; the protoplasm finely punctate; b, beginning karyolysis in a megaloblast; c, erythroblasts with coarse granulation of the protoplasm; d, nuclear remains (?) and fine granulation of the protoplasm; e and f, finely punctate red blood-corpuscles; g, megalocyte with two blue nuclei; nuclear remains (?) in the polychrome protoplasm.

(Nothnagel-Lazarus.)

The red corpuscles show marked variation in size and shape (Plate VIII and Fig. 84). There is a decided tendency to large oval forms, and despite the abundance of microcytes, the average size of the corpuscles is generally strikingly increased. Polychromatophilia and basophilic degeneration are common. Nucleated red cells are always present, although in many instances careful search is required to find them. In the great majority of cases megaloblasts exceed normoblasts in number. This ratio constitutes one of the most important points in diagnosis, since it is practically unknown in other diseases. Blood-plaques are diminished.

The rare and rapidly fatal anemia which has been described under the name of aplastic anemia is probably a variety of pernicious anemia. Absence of any attempt at blood regeneration explains the marked difference in the blood picture. Red corpuscles and hemoglobin are rapidly diminished to an extreme degree. The color index is normal or low. The leukocyte count is normal or low, with relative increase of lymphocytes. Stained smears show only slight variations in size, shape, and staining properties of the red cells. There are no megaloblasts, and few or no normoblasts.

(2) Chlorosis.—The clinical symptoms furnish the most important data for diagnosis. The blood resembles that of secondary anemia in many respects.

The most conspicuous feature is a decided decrease of hemoglobin (down to 30 or 40 per cent. in marked cases), accompanied by a slight decrease in number of red corpuscles. The color-index is thus almost invariably low, the average being about 0.5.

As in pernicious anemia, the leukocytes are normal or decreased in number, with a relative increase of lymphocytes.

In contrast to pernicious anemia (and in some degree also to secondary anemia) the red cells are of nearly uniform size, are uniformly pale (Fig. 84), and their average diameter is somewhat less than normal. Changes in size, shape, and staining reactions occur only in severe cases. Erythroblasts are rarely present. The number of plaques is generally decreased.

(3) Splenic Anemia.—This is an obscure form of anemia associated with great enlargement of the spleen. It is probably a distinct entity. There is decided decrease of hemoglobin and red corpuscles, with moderate leukopenia and relative lymphocytosis. Osler's fifteen cases averaged 47 per cent. hemoglobin and 3,336,357 red cells. Stained films show notable irregularities in size, shape, and staining properties only in advanced cases. Erythroblasts are uncommon.

B. LEUKEMIA

Except in rare instances, diagnosis is easily made from the blood alone. Two types of the disease are commonly distinguished: the myelogenous and the lymphatic. Atypical and intermediate forms are not uncommon. Pseudoleukemia, because of its clinical similarity to lymphatic leukemia, is generally described along with leukemia.

1. Myelogenous Leukemia.—This is usually a chronic disease, although acute cases have been described.

Hemoglobin and red corpuscles show decided decrease. The color-index is moderately low.

Most striking is the immense increase in number of leukocytes. The count in ordinary cases varies between 100,000 and 300,000. Counts over 1,000,000 have been met. During remissions, the leukocyte count may fall to normal.

While these enormous leukocyte counts are equaled in no other disease, and approached only in lymphatic leukemia and extremely high-grade leukocytosis, the diagnosis, particularly during remissions, depends more upon qualitative than quantitative changes. Although all varieties are increased, the characteristic and conspicuous cell is the myelocyte. This cell never appears in normal blood; extremely rarely in leukocytosis; and never abundantly in lymphatic leukemia. In myelogenous leukemia myelocytes usually constitute more than 20 per cent. of all leukocytes. Da Costa's lowest case gave 7 per cent. The neutrophilic form is generally much more abundant than the eosinophilic. Both show considerable variations in size. Very constant also is a marked absolute, and often a relative, increase of eosinophiles and basophiles. Polymorphonuclear neutrophiles and lymphocytes are relatively decreased.

FIG. 85.—Blood from a case of splenomedullary leukemia, stained with Ehrlich's triple stain (Jakob).

The red cells show the changes characteristic of a severe secondary anemia, except that nucleated reds are commonly abundant; in fact, no other disease gives so many. They are chiefly of the normoblastic type. Megaloblasts are uncommon. Blood-plaques are generally increased.

FIG. 86.—Blood: Lymphatic leukemia (lower section); marked leukocytosis (upper section). Stained smears. Compare types of leukocytes with Fig. 85 (from Greene's "Medical Diagnosis").

2. Lymphatic Leukemia.—This form may be either acute or chronic. There is marked loss of hemoglobin and red corpuscles. The color-index is usually moderately low.

The leukocyte count is high, but lower than in the myelogenous type. Counts of 100,000 are about the average, but in many cases are much lower. This high count is referable almost wholly to increase of lymphocytes. They generally exceed 90 per cent. of the total number. In chronic cases they are chiefly of the small variety; in acute cases, of the large form. Myelocytes are rare.

The red corpuscles show the changes usual in severe secondary anemia. Erythroblasts are seldom abundant. Blood-plaques are decreased.

3. Pseudoleukemia (Hodgkin's disease) resembles lymphatic leukemia in that there is marked and progressive enlargement of the lymph-nodes. There is, however, no distinctive blood-picture. The changes in hemoglobin and red cells resemble those of a moderate symptomatic anemia, with rather low color-index. The leukocytes are commonly normal in number and relative proportions.

4. Anæmia Infantum Pseudoleukæmica.—Under this name von Jaksch described a rare disease of infancy, the proper classification of which is uncertain. There is enlargement of liver and spleen, and sometimes of lymph-nodes, together with the following blood-changes: grave anemia with deformed and degenerated red cells and many erythroblasts of both normoblastic and megaloblastic types; great increase in number of leukocytes (20,000 to 100,000), and great variations in size, shape, and staining of leukocytes, with many atypical forms and a few myelocytes.

The table on the following page contrasts the distinctive blood-changes in the more common conditions.

DIFFERENTIAL DIAGNOSIS OF BLOOD DISEASES
	SECONDARY ANEMIA.	PERNICIOUS ANEMIA.	CHLOROSIS.	MYELOGENOUS LEUKEMIA.	LYMPHATIC LEUKEMIA.
Hemoglobin.	Diminished according to degree of anemia.	Diminished.	Greatly diminished.	Decidedly diminished.	Markedly diminished.
Red Corpuscle Count.	Normal in mild cases; diminished in all others.	Greatly diminished.	Slightly diminished.	Decidedly diminished.	Markedly diminished.
Color-index.	Normal or slightly diminished.	High.	Low.	Usually slightly diminished.	Usually slightly diminished.
Leukocyte Count.	Not necessarily affected; leukocytosis common.	Normal or diminished.	Normal or diminished.	Extremely high.	Very high.
Red Corpuscles on Stained Films.	Variations in size and shape in moderate cases; variations in staining reactions and normoblasts in severe cases.	Marked variations in size, shape, and staining reactions. Average size increased. Tendency to large oval forms. Erythroblasts always present; megaloblasts exceed normoblasts.	Nearly uniform size and shape; average size decreased; pale centers. Erythroblasts very rare.	Similar to secondary anemia, except normoblasts generally very numerous.	Similar to secondary anemia. Erythroblasts not numerous.
Leukocytes on Stained Films.	Normal proportions or increase of polynuclears.	Lymphocytes relatively, sometimes absolutely, increased.	Lymphocytes apt to be relatively increased.	Large numbers of myelocytes (average, 20 per cent.). Absolute increase of eosinophiles and basophiles. Relative decrease of polynuclears and lymphocytes.	Lymphocytes exceed 90 per cent. Other varieties relatively decreased.