Chapter III: The Blood (2)

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Red corpuscles which vary from the normal in size and shape are present in most symptomatic anemias, and in the severer grades are often very numerous. Irregularities are particularly conspicuous in leukemia and pernicious anemia, where, in some instances, a normal erythrocyte {176} is the exception. In pernicious anemia there is a decided tendency to large size and oval forms, and megalocytes are rarely found in any other condition.

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

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

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

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

(_c_) _Basophilic Stippling_.--This term has been applied {177} to the finely granular appearance often seen in red corpuscles, which harbor malarial parasites (Plate VI). It is commonly classed with the degeneration just described, but is probably distinct. Not all stains will show it. With Wright's stain it can be brought out by staining longer and washing less than for the ordinary blood-stain. The minute granules stain reddish purple.

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

Normally, erythroblasts are present only in the blood of the fetus and of very young infants. Pathologically, normoblasts occur in severe symptomatic anemia, leukemia, and pernicious anemia. They are most abundant in myelogenous leukemia. While always present in pernicious anemia, they are often difficult to find. Megaloblasts are found in pernicious anemia, and with extreme rarity in any other condition. They almost invariably {178} exceed the normoblasts in number, which is one of the distinctive features of the disease. Microblasts have much the same significance as normoblasts, but are less common.

2. The Leukocytes.--An estimation of the number or percentage of each variety of leukocyte in the blood is called a _differential count_.

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

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

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

The usual classification of leukocytes is based upon their size, their nuclei, and the staining properties of the granules {179} which many of them contain. It is not altogether satisfactory, but is probably the best which our present knowledge permits.

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

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

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

(_b_) Large Mononuclear Leukocytes (Plate VI).--These cells are two or three times the diameter of the normal red corpuscle. Each contains a single round or oval nucleus, often located eccentrically. The zone of protoplasm surrounding the nucleus is relatively wide. With {180} Wright's stain the nucleus is less deeply colored than that of the lymphocyte, while the cytoplasm is very pale blue or colorless, and sometimes contains a few reddish granules. The size of the cell, the width of the zone of cytoplasm, and the depth of color of the nucleus are the points to be considered in distinguishing between large mononuclears and lymphocytes. When large forms of the lymphocyte are present, the distinction is often difficult or impossible. It is then advisable to count the two cells together as lymphocytes. Indeed, they are regarded by some hematologists as identical.

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

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

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

(_d_) Polymorphonuclear Neutrophilic Leukocytes (Plate VI).--There is usually no difficulty in recognizing these cells. Their average size is somewhat less than that of the large mononuclears. The nucleus stains rather deeply, and is extremely irregular, often assuming shapes comparable to letters of the alphabet, E, Z, S, etc. (Fig. 86). Frequently there appear to be several separate nuclei, hence the widely used name, "polynuclear {181} leukocyte." Upon careful inspection, however, delicate nuclear bands connecting the parts can usually be seen. The cytoplasm is relatively abundant, and contains great numbers of very fine neutrophilic granules. With Wright's stain the nucleus is bluish-purple, and the granules, reddish-lilac.

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

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

Normally, the cytoplasm of leukocytes stains pale yellow with iodin. Under certain pathologic conditions the cytoplasm of many of the polymorphonuclears stains {182} diffusely brown, or contains granules which stain reddish-brown with iodin. This is called _iodophilia_. Extracellular iodin-staining granules, which are present normally, are more numerous in iodophilia.

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

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

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

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

Slight eosinophilia is physiologic during menstruation. {183} Marked eosinophilia is always pathologic. It occurs in a variety of conditions, the most important of which are: infection by animal parasites; bronchial asthma; myelogenous leukemia; scarlet fever; and many skin diseases.

Eosinophilia may be a symptom of _infection by any of the worms_. It is fairly constant in trichinosis, uncinariasis, filariasis, and echinococcus disease. In this country an unexplained marked eosinophilia warrants examination of a portion of muscle for _Trichina spiralis_ (p. 255).

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

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

_Scarlet fever_ is frequently accompanied by eosinophilia, which may help to distinguish it from measles.

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

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

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

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

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

(_b_) Atypical Forms.--Leukocytes which do not fit in {185} with the above classification are not infrequently met, especially in high-grade leukocytosis, pernicious anemia, and leukemia. The nature of most of them is not clear, and their number is usually so small that they may be disregarded in making a differential count. Among them are: (_a_) Border-line forms between polymorphonuclear neutrophiles and neutrophilic myelocytes; (_b_) small neutrophilic cells with a single round, deeply staining nucleus: they probably result from division of polymorphonuclear neutrophiles; (_c_) "irritation forms"--large non-granular mononuclear cells, whose cytoplasm stains fairly deep purple with Wright's stain, and intense brown with Ehrlich's: they appear in the blood under the same conditions as myelocytes; (_d_) degenerated forms: vacuolated leukocytes, or merely palely or deeply staining homogeneous or reticulated masses of chromatin (Plate VI).

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

Blood-platelets are being much studied at present, but, aside from the facts mentioned under their enumeration (p. 165), little of clinical value has been learned. They have been variously regarded as very young red corpuscles (the "hematoblasts" of Hayem), as disintegration products of leukocytes, as remnants of extruded nuclei of erythrocytes, and as independent nucleated bodies. The most probable explanation of their origin seems to be that of {186} J. H. Wright, who, from his recent studies, regards them as detached portions of the cytoplasm of certain giant-cells of the bone-marrow and spleen.

VII. BLOOD PARASITES

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

Of the numerous animal parasites which have been {187} found in the blood, three are especially interesting clinically: _Plasmodium malariæ_, _Filaria sanguinis hominis_, and _Trypanosoma hominis_.

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

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

(_a_) _Asexual Cycle_.--The young organism enters the blood through the bite of the mosquito. It makes its way into a red corpuscle, where it appears as a small, pale "hyaline" body. This body exhibits ameboid movement and increases in size. Soon, dark-brown granules derived from the hemoglobin of the corpuscle make their appearance within it. When it has reached its full size--filling and distending the corpuscle in the case of the tertian parasite, smaller in the others--the pigment-granules gather at the center or at one side; the organism divides into a number of small hyaline bodies, the spores or merozoites; and the red corpuscle bursts, setting spores and pigment free in the blood-plasma. This is called segmentation. It coincides with, and by liberation of toxins causes, the paroxysm of the disease. A considerable number of the spores are destroyed by leukocytes or other agencies; the remainder enter other corpuscles and repeat the cycle. Many of the pigment-granules are taken up by leukocytes. In estivo-autumnal fever segmentation occurs in the {188} internal organs and the segmenting and larger pigmented forms are not seen in the peripheral blood.

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

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

(_b_) _Sexual Cycle_.--Besides the ameboid individuals which pass through the asexual cycle, there are present with them in the blood many individuals with sexual properties. These are called _gametes_. They do not undergo segmentation. Many of them are apparently extracellular, but stained preparations usually show them to be surrounded by the remains of a corpuscle. In tertian and quartan malaria they cannot easily be distinguished from the asexual individuals until a variable time after the blood leaves the body, when the male gamete sends out {189} one or more flagella. In estivo-autumnal malaria the gametes take distinctive ovoid and crescentic forms, and are not difficult to recognize. They are very resistent to quinin and often persist in the blood long after the ameboid forms have been destroyed, but are probably incapable of continuing the disease until they have passed through the cycle in the mosquito.

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

The sexual cycle can take place only within the body of one genus of mosquito, _anopheles_. Absence of this mosquito from certain districts explains the absence of {190} malaria. It is distinguished from our common house-mosquito, _culex_, by the relative lengths of proboscis and palpi (Fig. 79), which can be seen with a hand-lens, by its attitude when resting, and by its dappled wing (Fig. 80). Anopheles is strictly nocturnal in its habits; it usually flies low, and rarely travels more than a few hundred yards from its breeding-place, although it may be carried by winds. These facts explain certain peculiarities in malarial infection; thus, infection occurs practically only at night; it is most common near stagnant water, especially upon the side toward which the prevailing winds blow; and the {191} danger is greater when persons sleep upon or near the ground than in upper stories of buildings. The insects frequently hibernate in warmed houses, and may bite during the winter. A mosquito becomes dangerous in eight to fourteen days after it bites a malarious person, and remains so throughout its life.

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

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

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

Various forms of malarial parasites (Thayer and Hewetson).

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

(_b_) _In Stained Films_ (Plate VI).--Recognition of the parasite, especially the young forms, is much easier in films stained by Wright's or some similar stain than in fresh blood. When very scarce, they may sometimes be found, although their structure is not well shown, by the method of Ruge. This consists in spreading a very thick layer of blood, drying, placing for a few minutes in a fluid containing 5 per cent. formalin and 1 per cent. acetic acid, which removes the hemoglobin and fixes the smear, rinsing, drying, and finally staining. If Wright's stain be used in this method, it is recommended that the {193} preparation be subsequently stained for a half-minute with borax-methylene-blue (borax, 5; methylene-blue, 2; water, 100).

In films which are properly stained with Wright's fluid the young organisms are small, round, ring-like or irregular, sky-blue bodies, each with a very small, sharply defined, reddish-purple chromatin mass. Many structures--deposits of stain, dirt, blood-plaques lying upon red cells, etc.--may simulate them, but should not deceive one who looks carefully for both the blue cytoplasm and the reddish-purple chromatin. A plaque upon a red corpuscle is surrounded by a colorless zone rather than by a distinct blue body. Young estivo-autumnal parasites commonly take a "ring" form (the chromatin mass representing the jewel), which is infrequently assumed by the other varieties. The older tertian and quartan organisms show larger sky-blue bodies with more reticular chromatin, and contain brown granules of pigment, which, however, is less evident than in the living parasite. The chromatin is often scattered through the cytoplasm or apparently outside of it, and is sometimes difficult to see clearly. Typical "segmenters" present a ring of rounded segments or spores, each with a small, dot-like chromatin mass. With the tertian parasite, the segments more frequently form an irregular cluster. The pigment is collected near the center or scattered among the segments. In estivo-autumnal fever usually only the small "ring bodies" and the crescentic and ovoid gametes are seen in the blood. The gametes are easily recognized. Their length is somewhat greater than the diameter of a red corpuscle. Their chromatin is usually centrally placed, and they contain more or less coarse pigment. The remains of the {194} red cell often form a narrow rim around them or fill the concavity of the crescent.

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

VARIETIES OF THE MALARIAL ORGANISM

2. Filaria Sanguinis Hominis.--Of the several varieties of this worm, _Filaria nocturna_ is most common and {195} most important clinically. The adults are thread-like worms about 8 to 10 cm. long. They are rarely seen. They live in pairs in the lymphatic channels and glands, especially those of the pelvis and groin, and often occur in such numbers as to obstruct the flow of lymph. This is the most common cause of elephantiasis. Infection is very common in tropical countries, especially in Samoa, the West Indies, Central America, and the Isthmus of Panama. It is said that in Samoa 50 per cent. of the natives are infected.

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

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

3. Trypanosoma Hominis.--Various trypanosomes are common in the blood of fishes, amphibians, birds, and {196} mammals (Fig. 81). They live in the blood-plasma and do not attack the corpuscles. In some animals they are apparently harmless; in others they are an important cause of disease.

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

VIII. SERUM REACTIONS

1. Agglutination.--In the blood-serum of persons suffering from certain infectious diseases there exist soluble bodies, called agglutinins, which have the property of {197} rendering non-motile and clumping the specific micro-organism of the disease, and have little or no influence upon other bacteria. This "agglutination" takes place even when the blood is greatly diluted. Undiluted normal blood can agglutinate most bacteria, but loses this power when diluted to any considerable degree. These facts are taken advantage of in the diagnosis of several diseases.

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

Either blood-serum or the whole blood may be used. Serum is the better. To obtain it, it is convenient to use little vials, such as can be made by breaking off the lower half-inch of the tubes which have contained peptonizing powder. They must, of course, be well cleaned. One of these is filled to a depth of about one-fourth inch from a puncture in the finger, and is set aside for a few hours. When the clot has separated, it is picked out with a needle, leaving the serum. One drop of the serum is then added to nine drops of normal salt solution, making a dilution of 1:10. Distilled water may be used for dilution, but is more liable to cause error. The dilution can be more accurately made in the leukocyte pipet of the Thoma-Zeiss instrument. When the whole blood is used, it can be secured in the pipet and at once diluted with the salt solution. When it must be transported a considerable distance, dried blood is most convenient. A large drop is allowed to dry upon a clean slide or unglazed paper. It will keep for months without losing its agglutinating power. When ready to make the test, the dried stain is dissolved in ten drops of normal salt solution, care being {198} taken that the drops are about the same size as the original drop of blood.

The reaction can be detected either microscopically or macroscopically:

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

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

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

(3) Examine at intervals with a high dry lens--a one-sixth will answer very well. The light must be very subdued. At first the bacilli should be actively moving about. If the blood be from a case of typhoid, they will gradually lose their motion and gather together in clumps (Fig. 82). The clumps should be large, and the few bacilli remaining isolated should be motionless. Pseudo-reactions, in which there are a few small clumps of bacilli whose motion is not entirely lost, together with many freely moving bacilli scattered throughout the field, should not mislead. As a control, a drop {199} of the culture should always be examined before making the test.

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

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

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

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

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

To measure the amount of any particular opsonin in the blood Wright has devised a method which involves many ingenious and delicate technical procedures. Much skill, such as is attained only after considerable training in laboratory technic, is requisite, and there are many sources {201} of error. It is, therefore, beyond the province of this work to recount the method in detail. In a general way it consists in: (_a_) Preparing a mixture of equal parts of the patient's blood-serum, an emulsion of the specific micro-organism, and a suspension of washed leukocytes; (_b_) preparing a similar mixture, using serum of a normal person; (_c_) incubating both mixtures for a definite length of time; and (_d_) making smears from each, staining, and examining with a one-twelfth objective. The number of bacteria which have been taken up by a definite number of leukocytes is counted, and the average number of bacteria per leukocyte is calculated; this gives the "phagocytic index." The phagocytic index of the blood under investigation, divided by that of the normal blood, gives the _opsonic index_ of the former, the opsonic index of the normal blood being taken as 1. Simon regards the percentage of leukocytes which have ingested bacteria as a more accurate measurement of the amount of opsonins than the number of bacteria ingested, because the bacteria are apt to adhere and be taken in in clumps.

Wright and his followers regard the opsonic index as an index of the power of the body to combat bacterial invasion. They claim very great practical importance for it as an aid to diagnosis and as a guide to treatment by the vaccine method. This method of treatment consists in increasing the amount of protective substances in the blood by injections of normal-salt suspensions of dead bacteria of the same species as that which has caused and is maintaining the morbid process, these bacterial suspensions being called "vaccines." The opinion of the majority of conservative men seems to be that while vaccine therapy is undoubtedly an important addition to our {202} methods of treatment of bacterial infections, particularly of those which are strictly local, yet the value of the opsonic index in measuring resisting power or as an aid to diagnosis and guide to treatment is still _sub judice_.

IX. TESTS FOR RECOGNITION OF BLOOD

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

2. Teichmann's Test.--This depends upon the production of characteristic crystals of _hemin_. It is a sensitive test, and, when positive, is absolute proof of the presence of blood. A number of substances--lime, fine {203} sand, iron rust--interfere with production of the crystals; hence negative results are not always conclusive. Dissolve the suspected stain in a few drops of normal salt solution upon a slide. If a liquid is to be tested, evaporate some of it upon a slide and dissolve the residue in a few drops of the salt solution. Let dry, apply a cover-glass, and run glacial acetic acid underneath it. Heat _very gently_ until bubbles begin to form, replacing the acid as it evaporates. Allow to cool slowly. When cool, replace the acid with water, and examine for hemin crystals with two-thirds and one-sixth objectives. The crystals are dark-brown rhombic plates lying singly or in crosses, and are easily recognized (Fig. 83). Failure to obtain them may be due to too great heat or too rapid cooling. If not obtained at first, let the slide stand in a warm place, as upon a hot-water radiator, for an hour.

X. SPECIAL BLOOD PATHOLOGY

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

A. ANEMIA

This is a deficiency of hemoglobin, or red corpuscles, or both. It is either primary or secondary. The distinction is based chiefly upon etiology, although each type presents a more or less distinctive blood-picture. Secondary anemia is that which is symptomatic of some other {204} pathologic condition. Primary anemia is that which progresses without apparent cause.

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

(_a_) _Poor nutrition_, which usually accompanies unsanitary conditions, poor and insufficient food, etc.

(_b_) _Acute infectious diseases_, especially rheumatism and typhoid fever. The anemia is more conspicuous during convalescence.

(_c_) _Chronic infectious diseases_: tuberculosis, malaria, syphilis, leprosy.

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

(_e_) _Chronic poisoning_, as from lead, arsenic, and phosphorus.

(_f_) _Hemorrhage_--either repeated small hemorrhages, as from gastric cancer and ulcer, uterine fibroids, etc., or a single large one.

(_g_) _Malignant tumors_: these affect the blood partly through repeated small hemorrhages, partly through toxic products, and partly through interference with nutrition.

(_h_) _Animal Parasites_.--Some cause no appreciable change in the blood; others, like the _Uncinaria_ and _Bothriocephalus latus_, may produce a very severe anemia, almost identical with pernicious anemia. Anemia in these cases is probably due both to toxins and to abstraction of blood.

The blood-picture varies with the grade of anemia. {205} Diminution of hemoglobin is the most characteristic feature. In mild cases it is slight, and is the only blood change to be noted. In very severe cases hemoglobin may fall to 15 per cent. Red corpuscles are diminished in all but very mild cases, while in the severest cases the red corpuscle count is sometimes below 2,000,000. The color index is usually decreased.

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

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

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

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

Hemoglobin and red corpuscles are always greatly diminished. In none of Cabot's 139 cases did the count exceed 2,500,000, the average being about 1,200,000. In more than two-thirds of the cases hemoglobin was {206} reduced to less extent than the red corpuscles: the color-index was, therefore, high. A low color-index probably indicates a mild type of the disease.

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

Fig. 1.--Preparation from an advanced case of progressive pernicious anemia from unknown cause: _a_, Megaloblasts or gigantoblasts; the protoplasm shows marked polychromasia; _b_, stained granules in erythrocytes with normally stained protoplasm; _c_ and _d_, polychromatophilic degeneration; _e_, megalocytes; _f_, normocytes.

Fig. 2.--Preparation from the same case taken some time later while the patient was subjectively and objectively in perfect health: _a_, Punctate erythrocytes with normal and anemic degenerated protoplasm; _b_, polynuclear leukocyte; _c_, normal red blood-corpuscles; _d_, somewhat enlarged erythrocytes.

Fig. 3.--Series of cells from a case of severe progressive pernicious anemia of unknown etiology; preparation made two days antemortem: _a_, Nucleated red blood-corpuscles characterized as normoblasts by the intense staining of the nuclei; _a'_ and _a"_, karyokinetic figures in erythrocytes; the protoplasm finely punctate; _b_, beginning karyolysis in a megaloblast; _c_, erythroblasts with coarse granulation of the protoplasm; _d_, nuclear remains (?) and fine granulation of the protoplasm; _e_ and _f_, finely punctate red blood-corpuscles; _g_, megalocyte with two blue nuclei; nuclear remains (?) in the polychrome protoplasm.

(Nothnagel-Lazarus.)]

The red corpuscles show marked variation in size and shape (Plate VIII and Fig. 84). There is a decided {207} tendency to large oval forms, and despite the abundance of microcytes, the average size of the corpuscles is generally strikingly increased. Polychromatophilia and basophilic degeneration are common. Nucleated red cells are always present, although in many instances careful search is required to find them. In the great majority of cases megaloblasts exceed normoblasts in number. This ratio constitutes one of the most important points in diagnosis, since it is practically unknown in other diseases. Blood-plaques are diminished.

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

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

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

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

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

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

B. LEUKEMIA

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

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

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

Most striking is the immense increase in number of leukocytes. The count in ordinary cases varies between 100,000 and 300,000. Counts over 1,000,000 have been {209} met. During remissions, the leukocyte count may fall to normal.

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

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

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

The leukocyte count is high, but lower than in the myelogenous type. Counts of 100,000 are about the average, but in many cases are much lower. This high {211} count is referable almost wholly to increase of lymphocytes. They generally exceed 90 per cent. of the total number. In chronic cases they are chiefly of the small variety; in acute cases, of the large form. Myelocytes are rare.

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

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

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

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

{212} DIFFERENTIAL DIAGNOSIS OF BLOOD DISEASES

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A Manual of Clinical DiagnosisChapter III: The Blood (2)

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