Chapter III: The Blood (1)

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.

{140} 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 {141} 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.

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.

{142} 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.

{143} _Estimation of Hemoglobin_.--There are many methods, but none is entirely satisfactory. Those which are most widely used are here described.

* * * * *

(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 {144} 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 {145} diameter, and a pipet of 20 c.mm. capacity. The two tubes are held in a black frame with a white ground-glass back.

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 {146} 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.

(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 {147} 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 HEMOGLOBIN SPECIFIC HEMOGLOBIN
GRAVITY. PER CENT. GRAVITY. PER CENT.
1.033-1.035 25-30 1.048-1.050 55-65
1.035-1.038 30-35 1.050-1.053 65-70
1.038-1.040 35-40 1.053-1.055 70-75
1.040-1.045 40-45 1.055-1.057 75-85
1.045-1.048 45-55 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, {148} 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.

* * * * *

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 {149} 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.

{150} _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.

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. {151} The hematocrit is, however, useful in determining the relative volume of corpuscles and plasma, and seems to be gaining in favor.

* * * * *

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 {152} 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.

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 {153} 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 {154} 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

{155} _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 {156} 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 {157} 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 {158} 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.

{159} (_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.

{160} (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.

{161} (_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 {162} 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:

* * * * *

{163} 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).

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. {164} 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.

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 {165} 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 {166} 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 {167} 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 150 c.c.
(Color with methyl-violet if desired.)

{168} 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 {169} 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).

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 {170} 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).

* * * * *

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.

{171} 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.

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: {172} 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. {173} 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.

* * * * *

{174} 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.

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.]

{175} 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.