Chapter 7

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 amountof 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, orleukopenia, 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 mayproduce 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 leukocytesis 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, orleukocytosis. B. Increase of leukocytes due toleukemia. The former may be classed as atransient, the latter, as apermanent, 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 increasedformation 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 ofphagocytes. 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 (seep. 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-picturemay 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).

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.

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 eighttimes 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 numberincreases 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.

(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 thefinger 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;andP=pxR/r.

rrepresents 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; andP, 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:

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 ANDSTAININGBLOOD-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 aclean 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 heldin 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.

Heat Fixation.—This may precede any of the methods which do not combine fixation with the staining process; itmustbe 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: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 calledbasophilic. Certain structures take up only acid dyes, and are calledacidophilic,oxyphilic, oreosinophilic. Certain other structures are stained only by combinations of the two, and are calledneutrophilic. 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 (seeFig. 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.

B. STUDY OFSTAINEDFILMS

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(SeePlate VIII and Fig. 84).—The cells may be abnormally small (calledmicrocytes, 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 calledpoikilocytes, 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 erythrocyteis 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(SeePlate 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 appliedto 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 (PlatesVIandVIII and Fig. 84). Nucleated red corpuscles, orerythroblasts, are classed according to their size:microblasts, 5 µ or less in diameter;normoblasts, 5 to 10 µ; andmegaloblasts, 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 invariablyexceed 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 adifferential 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 anabsolute increase;an increase in percentage only, arelative 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 granuleswhich 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 VIandFig. 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. WithWright'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, "polynuclearleukocyte." 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 stainsdiffusely brown, or contains granules which stain reddish-brown with iodin. This is callediodophilia. 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 calledeosinophilia, 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 ofinfection 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 forTrichina spiralis(p. 255).

True bronchial asthmacommonly 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.

Inmyelogenous leukemiathere 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 feveris frequently accompanied by eosinophilia, which may help to distinguish it from measles.

Eosinophilia has been observed in a large number ofskin 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 inwith 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 ofJ. 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.

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