XLV. BLOOD EXAMINATIONS IN THE DIAGNOSIS OF TROPICAL DISEASES…

XLV. BLOOD EXAMINATIONS IN THE DIAGNOSIS OF TROPICAL DISEASES

In a short chapter on such a large subject only the more important methods and findings can be considered. As regards interpretation of blood findings in various tropical diseases one may note in the recent work of Schilling-Torgau the difficulties which at present beset the subject. Until some universal agreement as to standard methods of technique and in particular complete accord as to the characteristics of the diagnostic cells can be arrived at, conflicting reports as to findings must of necessity be obtained.

In taking up this subject it has seemed convenient to divide it into 4 heads: (1) The microscopical examination of fresh preparations or stained blood smears; (2) blood culture methods; (3) serological examinations, and (4) other practical methods of haematological study.

In the companion volume on laboratory work I have endeavored to take up rather in detail the various methods and techniques but in this chapter I shall only give single methods or point out short cuts in well-recognized ones or make suggestions as to new methods of blood study which may eventually aid us in diagnosis.

Those who work in temperate climates cannot realize the difficulties which beset the tropical laboratory worker from the lack of proper assistance, damaging effects of heat and moisture on stains and media and, of greater importance, the impairment of that driving energy so necessary for the carrying out of complicated methods. A short and simple method has a far greater value in the tropics than at home.

BLOOD PREPARATIONS

To obtain blood, except for blood cultures, use either a platino-iridium hypodermic needle which can be sterilized in the flame, a small tenotome, or a surgical needle with cutting edge.

Needles should be sterilized by boiling since flaming dulls the edge. A steel pen with one nib broken off or the glass needle of Wright may also be used. To make a glass needle, pull straight apart a piece of capillary tubing in a very small flame. Tap the fine point to break off the very delicate extremity. Scarcely any pain attends the use of such a needle. In puncturing either the tip of the finger or lobe of the ear a quick piano-touch-like stroke should be used. The ear is preferable, as it is less sensitive and there is less danger of infection. Before puncturing, the skin should be cleaned with 70% alcohol and allowed to dry. It is advisable to sterilize the needle before using it.

Note that in order to secure in the specimen a cell count that corresponds to that obtaining in the circulation as a whole it is necessary to massage the ear vigorously prior to making the puncture. Subsequently there should be no manipulation of the part, the blood examined being that which exudes freely. This procedure renders more likely the finding of blood parasites.

The first drop of blood which exudes should be taken up on the paper of the Tallquist haemoglobinometer, using subsequent ones for the blood pipettes and smears. If it is necessary to make a complete examination, it is rather difficult to draw up the blood in the pipettes, dilute it, and then get material for fresh blood preparations and films without undue squeezing, which is to be avoided. Of course, fresh punctures can be made. Ordinarily, complete blood examinations are not called for. It is only a white count or a differential count or an examination for malaria that is required.

As a practical point it is very rare that a red count is indicated. There is one point not sufficiently recognized by physicians and that is that a routine blood examination is not apt to be as carefully conducted as one calling for a specific feature. Without disparaging the necessity of routine examination of urine as well as blood it is a fact that the internist who knows what he wants gets better results from the laboratory man.

THE MICROSCOPICAL EXAMINATION OF FRESH PREPARATIONS OR STAINED BLOOD SMEARS

As regards haemocytometry it may be stated that in the tropics the counting of red cells is required more frequently in comparison to white ones than is the case in temperate climates where probably 100 white counts are necessitated as against 1 red count. This is on account of the frequency of secondary anaemias in the tropics.

The idea that time may be saved by making a white and red count from the same preparation is not borne out practically so that it is better to make white and red counts separately.

As a diluting fluid for red counts a normal salt solution, preferably about 0.9%, answers perfectly and if desired may be tinged with neutral red, methyl green or gentian violet to bring out white cells. When available, however, I prefer a 2½% aqueous solution of potassium bichromate for red cell counts.

=Rulings.=—The most desirable rulings are those of Türck, Zappert and Neubauer.

In these the entire ruled surface consists of nine large squares, each 1 mm. square. These are subdivided, and in the central large square are to be found the small squares used for averaging the red cells. These small squares are 1/20 mm. square and are arranged in nine groups of 16 small squares by bordering triple-ruled lines. As the unit in blood counting is the cubic millimeter, if one counted all the white cells lying within one of the large squares (1 mm. square), he would have only counted the cells in a layer one-tenth of the required depth, so that it would be necessary to multiply the number obtained by 10. This product, multiplied by the dilution of the blood, would give the number of white cells in a cubic millimeter of undiluted blood. The Neubauer ruling is the most satisfactory.

=Bürker Haemacytometer.=—Some workers prefer the Bürker haemacytometer. In this there are two ruled wedge-shaped pieces of glass, separated at their bases, which take the place of the ruled disc of the Thoma apparatus. Two oblong pieces of glass are on either side of the ruled wedges and are 0.1 mm. higher, thus taking the place of the shelf. Clamps fix a cover-glass on these shelves giving a space 1/10 mm. over the ruled surfaces. The blood is run in by capillarity from the mixing pipette. I gave up this type of counter because the clamps made manipulation awkward.

=Thoma-Levy Chamber.=—In the Thoma-Levy modification of the Bürker apparatus the central portion of the slide is cut away and in this depression is cemented a rectangular strip of glass, divided by a central channel. Each half of this strip of glass has a Neubauer ruling on it so that one can make a white count on one side and a red one on the other, simply touching the tip of the red pipette to the space separating the under surface of the cover-glass from the ruled rectangular slips on one side and then with the white pipette repeating the same on the other side. An advantage of the Thoma-Levy is that the original thickness of the slide makes the shelf on which the cover-glass rests instead of the support being strips on either side of the ruled surfaces and cemented to the slide. The Neubauer ruling is undoubtedly the most satisfactory of the haemacytometer rulings, its rulings being simpler than those of the Türck system. The unit square in all these haemacytometers is the small square for counting red blood cells, 1/20 mm. square.

=To Make a Red Count.=—Having a fairly large drop of blood, apply the tip of the 101 pipette to it and, holding the pipette horizontal, carefully and slowly draw up with suction on the rubber tube a column of blood to exactly 0.5. The variation of 1/25 of an inch from the mark would make a difference of almost 3%. If the column goes above 0.5, it can be gently tapped down on a piece of filter-paper until the 0.5 line is cut. Now insert the tip of the pipette into some diluting fluid, and revolving the pipette on its long axis while filling it by suction, you continue until the mark 101 is reached.

A variation of 1/25 of an inch at this mark would only give an error of about 1/30 of 1%. This gives a 1-200 dilution. After mixing thoroughly by shaking for one or two minutes, the fluid in the pipette below the bulb is expelled (this of course is only diluting fluid). A drop of the diluted blood of a size just sufficient to cover the disc when the cover-glass is adjusted, is then deposited on the disc and the cover-glass applied by a sort of sliding movement, best obtained by using forceps in one hand assisted by the thumb and index-finger of the other.

In red counts we use exclusively the small 1/20 mm. squares which are in groups of 16 bounded by triple-ruled lines.

The depth of fluid over the ruled surface is 1/10 mm., hence each of these small squares is 1/10 × 1/20 × 1/20 = 1/4000 of a c.mm., so that it takes 4000 such spaces to equal the unit for blood counting (1 c.mm.). My practice in making red counts is to count the red cells in five of the groups of 16 small squares. This in normal blood is about 100 for the 16 squares. After counting 5 groups of 16 we have counted the red cells of 80 small squares which is 1/50 of 4000 (the number in the 1 c.mm. unit). For this reason 50 × 200 (the blood dilution) = 10,000, so that it is only necessary to multiply the number of red cells found in 5 groups of 16 small squares by 10,000 in order to obtain the number of red cells per c.mm. For more accurate determination the process can be repeated with a second or third drop of the diluted blood, which would give an average from 160 or from 240 small squares.

=To Count White Cells.=—Draw up the blood in the white pipette to the 0.5 line. Then, still holding the pipette as near the horizontal as possible, because the column of blood tends to fall down in the larger bore, draw up by suction a diluting fluid which will disintegrate the red cells without injuring the whites. The best fluid is 0.5% of glacial acetic acid in water. This makes the white cells stand out as highly refractile bodies. Some prefer to tinge the fluid with neutral red or gentian violet. The 0.5 mark is preferred because it takes a very large drop of blood to fill the tube up to the 1 mark and if there is much of a leucocytosis a 1 to 10 dilution is not sufficient.

The blood having been drawn up to 0.5, we have a dilution of 1 to 20.

Making a preparation, exactly as was done in the case of the red count, we count all of the white cells in one of the large squares (1 sq. mm.). The cross ruling greatly facilitates this. Note the number. Then count a second and a third square. Strike an average of the large squares counted and multiply this by 10, as the depth of the fluid gives a content equal to only 1/10 of a c.mm. Then multiply by the dilution.

EXAMPLE.—First large square 50; second large square 70; third large square 60. Average 60. Then 60 × 10 × 20 = 12,000, the number of leucocytes in 1 c.mm. of blood. In order to save time the count is preferably made with a low power (⅔-inch objective) as the leucocytes stand out like pearls. It is more accurate, however, to use a higher power, so that pieces of foreign material may be recognized and not enumerated as white cells.

If one will accustom himself to comparing the distribution of the leucocytes in a well-made stained dried-blood film, prepared according to Ehrlich’s cover-glass method, with that in a haemacytometer preparation, he can readily acquire an experience which will enable him to determine with considerable accuracy the degree of leucocytosis by the examination of a stained, cover-glass preparation alone.

After making a blood count, the haemacytometer slide should be cleaned with soap and water and then rubbed dry, preferably with an old piece of linen. As the accuracy of the counting chamber depends upon the integrity of the cement, any reagent such as alcohol, xylol, etc., and in particular, heat, will ruin the instrument. The pipettes should be cleaned by inserting the ends into the tube from a vacuum pump, as a Chapman pump. First draw water or 1% sod. carbonate solution through the pipette, then alcohol, then ether, and finally allow air to pass through to dry the interior. If the interior is stained, used 1% HCL in alcohol. If a vacuum pump is not at hand, a bicycle pump or suction by mouth will answer.

PREPARATIONS FOR THE STUDY OF FRESH BLOOD

Many authorities prefer a fresh blood specimen to a stained dried smear in the study of parasites of the blood. In malaria in particular there is so much information as to species to be obtained from a fresh specimen that the employment of this method should never be neglected. While waiting for the film to stain one has five or six minutes which could not be better spent than in examining the fresh specimen which only requires a moment to make.

=Manson’s Method.=—Have a perfectly clean cover-glass and slide. Touch the apex of the exuding drop of blood with the cover-glass and drop it on the center of the slide. The blood flows out in a film which exhibits an “empty zone” in the center. Surrounding this we have the “zone of scattered corpuscles,” next the “single layer zone” and the “zone of rouleaux” at the periphery. It is well to ring the preparation with vaseline. When desiring to demonstrate the flagellated bodies in malaria, it is well to breathe on the cover-glass just prior to touching the drop of blood.

=The Method of Ross= is very easy of application and gives most satisfactory preparations. Take a perfectly clean slide, and make a vaseline ring or square of the size of the cover-glass. Then, having taken up the blood on the cover-glass, drop it so that its margin rests on the vaseline ring. Gently pressing down the cover-glass on the vaseline makes beautiful preparations which keep for a very long time. If it is desired to study the action of stains on living cells, this method is also applicable. A very practical way to do this is to tinge 0.85% salt solution containing 1% sodium citrate (the same as is used in opsonic work) with methylene azur, gentian violet, or methyl green. With a capillary bulb pipette, take up one part of blood, then one part of tinted salt solution. Mix them quickly on a slide and then deposit a small drop of the mixture in the center of the vaseline ring and immediately apply a cover-glass and press down the margins as before. This method will be found of great practical value.

PREPARATION AND STAINING OF DRIED FILMS

When preparations are desired for a differential count, Ehrlich’s method of making films is to be preferred, as the different types of leukocytes are more evenly distributed. In making smears by spreading, there is a tendency for the polymorphonuclears to be concentrated at the margin while lymphocytes remain in the central part of the film.

=Cover-slip Films.=—In Ehrlich’s method we have perfectly clean dry cover-slips. Take up a small drop of blood without touching the surface of the ear or finger. Drop this cover-glass immediately on a second one and as soon as the blood runs out in a film, draw the two cover-slips apart in a plane parallel to the cover-glasses. Ehrlich uses forceps to hold the cover-glasses to avoid moisture from the fingers, but I find I can work more quickly and satisfactorily with the fingers alone. The method shown in Fig. 150 is a very convenient one. In making malarial smears it is better to wash the finger or ear with soap and water to get rid of all grease and dirt. Then dry thoroughly before puncturing. Alcohol is not so efficient.

Slides and spreaders should be absolutely clean and grease-free. Scrubbing with soap and water, thorough rinsing and drying, then subjecting the slide to the flame to make it grease-free is satisfactory.

For removing dirt and grease from skin, a mixture of acetone, 40; alcohol, 60; is the best and quickest means. A bottle is kept on hand, with the puncturing needle embedded in the stopper.

For cleaning a slide, nothing equals Bon Ami. Rub up some with the wet finger, rub the slide with the lather until there is a friction squeak; let dry; polish with a clean, dry cloth. This is far better than soap and water, alcohol, ether and flaming combined. Note how a drop of water spreads on a glass so treated.

=Smears on Slides.=—Of the various methods of spreading films on slides, that described by Daniels is quite satisfactory. In this the drop of blood is drawn along and not pushed along. The films are even, can be made of any desired thickness by changing the angle of the drawing slide, and there is little liability of crushing pathological cells. Take a small drop of blood on the end of a clean slide. Touch a second slide, about ½ inch from end, with the drop and as soon as the blood runs out along the line of the slide end, slide it at an angle of 45° to the other end of the horizontal slide. The blood is pulled or drawn behind the advancing edge of the advancing slide. An angle less than 45° makes a thinner film; one greater, a thicker film.

Instead of a slide a square cover-glass may be used and if the edge be smooth it makes a more satisfactory spreader than the slide.

Instead of the Daniels method I prefer to take up the drop of blood on the slide on which the smear is to be made, about ½ inch from the end. Then apply the spreader slide and so soon as the drop runs along the end of the spreader slide proceed as above described. This method is shown in Fig. 150.

=Spreaders.=—Of the various methods of making smears by means of cigarette paper, rubber tissue, needles, etc., the best seems to be to take a piece of capillary glass tubing and use this instead of a needle in making the film. There is one advantage about the strip of cigarette paper touched to the drop of blood and drawn out along the slide or cover-glass, and that is that it is almost impossible not to make a working preparation by this method.

THICK-FILM METHODS

Such methods are of the greatest practical value in searching for malarial parasites when they are in very small numbers in the peripheral circulation, in finding trypanosomes, relapsing fever spirochaetes and filarial embryos. Ruge’s method so brings out the polymorphonuclears that such a technic can be used for opsonic index. Many workers prefer the Ross thick-film method in examining for malaria. In this about one-half of a drop of blood is smeared out over a surface about equal to that of a square cover-glass and allowed to dry. It is then flooded with 1/10 of 1% aqueous solution of eosin for about fifteen minutes. The preparation is then gently washed with water and then treated with a polychrome methylene-blue solution. After a few seconds this is carefully washed off and the preparation dried and examined.

James smears out an ordinary drop of blood so that it makes a circular smear about ¾ inch in diameter. This may be easily accomplished with a spatulate toothpick. When dry, treat the blood smear with alcohol containing HCl (Alcohol 50 cc., HCl 10 drops) until the haemoglobin is dissolved out. Then wash thoroughly in water for five or ten minutes. Allow to dry and then stain as ordinarily with the Wright or Giemsa stain.

=Ruge’s Method.=—The best thick-film method is that of Ruge. After the blood has dried well gently move the slide about in a glass containing a 2% solution of formalin to which has been added 1% of glacial acetic acid. After laking is completed, as shown by disappearance of brown color, treat the slide in the same way in a glass of tap water to remove all traces of acid. Next wash gently in distilled water and stain with dilute Giemsa (1 drop to 1 cc. of water) for twenty to thirty minutes. Wash in water and allow to dry without heat or blotting paper. Some workers prefer to stain the dried thick smear for one hour in a jar containing dilute Giemsa stain (1 to 40) without previous fixation or dehaemoglobinization. At present, I make my thick films by taking up a large loopful from the exuding drop of the puncture wound.

This is deposited at one end of the slide and from it three or four more daubs are made in succession toward the other end of the slide. These daubs are quickly smeared out before coagulation takes place in the first daub.

With all thick-film methods it is extremely important to have thorough drying of the smear before dehaemoglobinizing or staining. This ordinarily requires one or two hours in the air or twenty to thirty minutes in the incubator. It is particularly important in working with such smears, although holding for ordinary smears, to protect them from flies, ants, etc., as such insects will eat up the smear in a few minutes if left exposed.

=Fixation of Film.=—In Wright’s, Leishman’s, and other similar stains the methyl-alcohol solvent causes the fixation. In staining with Giemsa’s stain, or haematoxylin and eosin, separate fixation is necessary. For Giemsa either absolute alcohol (ten to fifteen minutes) or methyl alcohol (two to five minutes) answers well.

For haematoxylin and eosin, heat gives the best results. The best method is to place the films in an oven provided with a thermometer. Raise the temperature of the oven to 135°C. and then remove the burner. After the oven has cooled, take out the fixed slides or slips.

One of the handiest methods is to drop a few drops of 95% alcohol on the slide or cover-glass. Allow this to flow over the entire surface; then get rid of the excess of alcohol by touching the edge to a piece of filter-paper for a second or two. Then light the remaining alcohol film from the flame and allow the burning alcohol to burn itself out.

=Staining Blood-films.=—As separate staining with eosin and methylene blue rarely gives good preparations and as the modifications of the Romanowsky stain recommended are easy to make and employ, and give much greater information, the separate method of staining is not recommended.

Wright’s Method.—The stain is made by adding 1 gram of methylene blue (Grubler) to 100 cc. of a ½% solution of sodium bicarbonate in water. This mixture is heated for one hour in an Arnold sterilizer. The flask, containing the alkaline methylene-blue solution should be of such size and shape that the depth of the fluid does not exceed 2½ inches. When cool, filter the methylene blue solution, and add 500 cc. of a 1 to 1000 eosin solution (yellow eosin, water soluble). Add the eosin solution slowly, stirring constantly until the blue color is lost and the mixture becomes purple with a yellow metallic lustre on the surface, and there is formed a finely granular black precipitate. Collect this precipitate on a filter-paper and when thoroughly dry (dry in the incubator at 38°C.) dissolve 0.3 gram in 100 cc. of pure methyl alcohol (acetone-free). Wright lately has recommended using 0.1 in 60 cc. methyl alcohol. This constitutes the stock solution. For use filter off 20 cc. and add to the filtrate 5 cc. of methyl alcohol.

A modification by Balch is very satisfactory. In this method instead of polychroming the methylene blue with sodium bicarbonate and heat, the method of Borrel is used. Dissolve 1 gram of methylene blue in 100 cc. of distilled water. Next dissolve 0.5 gram of silver nitrate in 50 cc. of distilled water. To the silver solution add a 2 to 5% caustic soda solution until the silver oxide is completely precipitated. Wash the precipitated silver oxide several times with distilled water. This is best accomplished by pouring the wash-water on the heavy black precipitate in the flask, agitating, then decanting and again pouring on water. After removing all excess of alkali by repeated washings, add the methylene-blue solution to the precipitated silver oxide in the flask. Allow to stand about ten days, occasionally shaking until a purplish color develops. The process may be hastened in an incubator. When polychroming is complete, filter off and add to the filtrate the 1 to 1000 eosin solution and proceed exactly as with Wright’s stain.

In Leishman’s method the polychroming is accomplished by adding 1 gram of methylene blue to 100 cc. of a ½% solution of sodium carbonate. This is kept at 65°C. for twelve hours and allowed to stand at room temperature for ten days before the eosin solution is added. The succeeding steps are as for Wright’s stain.

In all Romanowsky methods distilled water should be used. If not obtainable, the best substitute is rain-water collected in the open and not from a roof.

Method of staining:

1. Make films and air dry.

2. Cover dry film preparation with the methyl-alcohol stain for one minute (to fix).

3. Add water to the stain on the cover-glass or slide, drop by drop, until a yellow metallic scum begins to form. It is advisable to add the drops of water rapidly in order to eliminate precipitates on the stained film. Practically, we may add 1 drop of water for every drop of stain used.

4. Wash thoroughly in water until the film has a pinkish tint.

5. Dry with filter-paper and mount.

Red cells are stained orange to pink; nuclei, shades of violet; eosinophile granules, red; neutrophile granules, yellow to lilac; blood platelets, purplish; malarial parasites, blue; chromatin, metallic-red to rose-pink.

Giemsa’s Modification of the Romanowsky Method.—This is one of the most perfect of the modifications. The objection is that greater time in staining films is required than with the Wright or Leishman method and the stain is very expensive.

Take of Azur II eosin 0.3 gram. Azur II 0.08 gram.

Dissolve this amount of dry powder in 25 cc. of glycerine at 60°C. Then add 25 cc. of methyl alcohol at the same temperature. Allow the glycerine-methyl alcohol solution to stand overnight and then filter. This is the stock stain. To use: Dilute 1 cc. with 10 to 15 cc. of distilled water. If 1 to 1000 potassium carbonate solution is used instead of water it stains more deeply. These same dyes, mixed with methylene violet, are now obtainable commercially as a powder ready for solution in methyl alcohol.

The alkaline diluent is used to obtain the coarse stippling in malignant tertian (Maurer’s clefts). Having fixed the smear with methyl alcohol for one to five minutes, pour on the diluted stain, and after fifteen to thirty minutes wash off and continue washing with distilled water until the film has a slight pink tinge. For Treponema pertenue stain from one to twelve hours.

=Haematoxylin Staining.=—While the Romanowsky methods are more satisfactory for differential counts and for the demonstration of the malarial parasites, and especially for differentiating species, yet by reason of the liability to deterioration in the tropics of methylene blue the haematoxylin methods may be preferable. Many workers in blood-work and cytodiagnosis prefer the haematoxylin.

1. Fix the film either by heat, with methyl alcohol for two minutes or with Whitney’s fixative. Heat is to be preferred.

2. Stain with Meyer’s hemalum or Delafield’s haematoxylin for from five to fifteen minutes according to the stain. Frequently three minutes will be found sufficient. To make the hemalum, dissolve 0.5 gram of haematin in 25 cc. of 95% alcohol. Next dissolve 25 grams of ammonia alum in 500 cc. of distilled water. Mix the two solutions and allow to ripen for a few days. The stain should be satisfactory in two or three days.

3. Wash for two to five minutes in tap water to develop the haematoxylin color.

4. Stain either with a 1 to 1000 aqueous solution of eosin or with a one-half of 1% eosin solution in 70% alcohol. The eosin staining only requires fifteen to thirty seconds.

5. Wash and examine.

DIFFERENTIAL COUNT

In making a differential count I would recommend the following from the directions of Schilling-Torgau. It will be remembered that considerable interest was raised a few years ago in what was termed the Arneth index. In this the more normal, more mature, better resisting polymorphonuclears were considered to have 3 or 4 lobes to the nuclear structure, even occasionally 5. The immature cells had only one or at most two lobes to the nucleus. The index was obtained by adding the percentages of cells showing 1 and 2 lobes to ½ the percentage of those with 3 lobes. As will be understood a high percentage of these immature cells was unfavorable in prognosis. These cells are graded from left to right, I, II, III, IV, V, as to separate masses in the nucleus, so that when the percentage is shoved or displaced to the left it indicates an increase in the immature cells.

Schilling-Torgau divides his polymorphonuclears into: (1) The myelocyte which is always of course a pathological cell. (2) The immature form polymorphonuclear. In this there is a close resemblance to the neutrophile myelocyte but there is a nuclear indentation instead of the round nucleus of the myelocyte. It is this cell which often puzzles us as to whether to regard it as a true myelocyte. It is the meta-myelocyte of many authorities. (3) Between the mature or segmented polymorphonuclear and the immature one or metamyelocyte we have what may be designated the band-form nucleated one. These show the type of nucleus which one is familiar with in the nucleus of the transitional. (4) The mature, multilobed or segmented nucleus of the typical polymorphonuclear.

It would seem that if all tropical workers would agree upon some single method of recording differential counts it would be advantageous.

Under the blood findings in liver abscess, in a paragraph to follow in this chapter, I give suggestive counts indicating the value of Schilling-Torgau’s method.

In the differential count he not only divided up the polymorphonuclears but makes no separation of small from large lymphocytes. Although I have always divided lymphocytes into large and small ones I believe it unnecessary and unpractical and shall henceforth group all such cells in one grouping. The statement that large mononuclears and transitionals are cells of a similar origin, type and significance has always been my view.

SCHEME OF SCHILLING-TORGAU -------------------------------------------------+-------+--------------- | Normal| Percentage Type of Cell |Percen-|Moderate Sepsis | tage |(W. C. 14,000) -------------------------------------------------+-------+--------------- 1. Mast cells | 1 | 1.0 2. Eosinophiles | 3 | 1.5 { a. myelocytes | 0 | 0.5 3. Neutro- { b. immature forms (metamyelocytes) | 0 | 5.0 philes { c. band-form (Stabkernige) | 4 | 13.5 { d. multilobed (Segmentkernige) | 63 | 64.0 4. Lymphocytes | 23 | 10.5 5. Large mononuclears and transitionals | 6 | 4.0 -------------------------------------------------+-------+---------------

BLOOD CULTURING

Among tropical diseases, only malta fever, kala-azar and plague demand this method of diagnosis, although there are met commonly in the tropics many cosmopolitan diseases in which blood culturing is a principal diagnostic procedure. There are many ways of carrying out the cultivation of organisms from the blood but the one which may be strongly recommended is the following. The blood is obtained from a vein, the overlying skin of which has been painted with tincture of iodine to insure a sterile skin surface.

A stout hypodermic needle is attached to about 6 inches of rubber tubing which in turn is pushed over a downward bent glass tube which passes through a doubly perforated rubber stopper. A second glass tube, which also passes through the stopper, is bent upward to be attached to a second piece of rubber tubing for use in suction by the mouth. The glass tubes project about ½ inch below the under surface of the rubber stopper and above are about 2½ inches including the bent arm. This system of tubing and stopper is readily sterilized by boiling in a pan or instrument sterilizer. As a receptacle for the blood we employ Erlenmeyer flasks of 100 cc. capacity, containing 25 cc. of salt solution with 1% of sodium citrate, for prevention of coagulation. Blood that contains 0.2% of sodium citrate will not coagulate so that a 0.5% solution could be used instead of the usual 1% one. These citrated salt solution flasks are plugged with cotton, sterilized and kept on hand ready for immediate use, so that we only have to sterilize the stopper and tubing by boiling and flame the neck of the flask when removing the cotton plug to insert the stopper of the system. By suction we can take any amount of blood desired. I usually count the drops of blood as they fall into the citrated salt solution allowing 16 drops to the cc. In this way we may take from 10 to 25 cc. of blood at the bedside and then later on in the laboratory, when it is convenient, inoculate various media from the flask. For plates add 2 or 3 cc. of this citrated blood to 6 or 8 cc. of melted agar at 45°C. The blood mixture can also be added to various sugar bouillons for fermentation reactions. Finally we place the receiving flask in the incubator and culture it as well as the other media.

=Clot Cultures.=—A very simple method is to take blood with a Wright U-tube. Then centrifuge and use the serum for agglutination tests and the clot, emulsified in some liquid medium, for the blood culturing. For paratyphoid culturing bile media are preferable, just as for typhoid.

=Lyon Blood Tube.=—Quite recently I have been using the blood tube recommended by Lyon. To make it, heat a 5- or 6-inch section of ¼ inch tubing in the centre and draw out as for making 2 bacteriological pipettes. Divide and seal off the large end in the flame. Next seal off the capillary end. Then apply a very small flame to a point on the large end just before it begins to taper to the capillary part. The heat causes the heated sealed-off air inside to force out a blow hole. To use: Break off the sealed capillary end and allow the capillary end to suck up blood from a drop just as with the Wright tube. I consider this tube superior to the Wright one.

=N. N. N. Medium.=—In culturing blood for protozoa the N. N. N. medium is usually employed. Novy and MacNeal originally used a 12½% meat infusion containing 2½% agar, 2% peptone, 1% normal sodium carbonate solution and ½% salt. To one part of this agar, melted and cooled to 60°C., they added twice the amount of defibrinated rabbit’s blood. In the N. N. N. medium, as modified by Nicolle, there is beside the blood only salt and agar—no peptone or meat extractives.

Citrated salt solution was the medium used by Rogers in the cultivation of splenic juice from kala-azar patients.

THE TAKING OF BLOOD FOR SEROLOGICAL TESTS

This can be done with the Wright tube, pipetting off the clear serum after centrifuging. We usually draw blood from a vein by use of the system of stopper and tubing described under blood culturing but employing an empty, sterile centrifuge tube.

Agglutination Tests

There are two methods of testing the agglutinating powers of a serum—the microscopical and the macroscopical or sedimentation method.

=For the microscopical method= draw up serum to the mark 0.5 of the white pipette. Then draw up salt solution to the mark 11. This when mixed gives a dilution of 1 to 20. One loopful of the diluted serum and one loopful of a bouillon culture or salt solution suspension of the organism to be tested gives a dilution of 1 to 40. One loopful of the 1-20 diluted serum and 3 loopfuls of the bacterial suspension give a dilution of 1-80. These two dilutions answer in ordinary diagnostic tests. The red pipette with a 1-100 or 1-200 dilution may be used where dilutions approaching 1-1000 are desired. Having mixed the diluted serum and the bacterial suspension on a cover-glass, we invert it over a vaselined concave slide and examine with a high power dry objective (⅙ inch). It is simpler to make a ring of vaseline to fit the cover-glass and make the mixture of diluted serum and culture in the centre of this ring or square. Then apply the cover-glass, press it down on the vaseline ring and examine as with the ordinary hanging drop. In making dilutions it is preferable to use salt solution, as the phenomenon of agglutination requires the presence of salts. Ordinarily, thirty minutes is a sufficient time to wait before reporting the absence of agglutination. Agglutination is more rapid at body temperature than at room temperature. In reporting agglutination, always give time and dilution. It is absolutely necessary that a control preparation be prepared in every instance; that is, one with the bacterial culture alone or with a normal serum of the same dilution as the lowest used. Some normal sera will agglutinate in 1 to 10 dilution, and group agglutinations (as paratyphoid with typhoid serum) may occur in 1 to 40 or possibly higher. It is very unusual for sera to agglutinate any other bacteria then the specific one in dilutions as high as 1-80.

=Macroscopic Agglutination.=—For the macroscopical or sedimentation test, take a series of small tubes (⅜ × 3 inches) and deposit 1 cc. of salt solution in each of the series. Now, having taken an empty test-tube, drop 4 drops of serum in it and then add 12 drops of salt solution. This approximately gives 1 cc. of a 1 to 4 dilution of the serum. It is more exact to make the 1 to 4 dilution with a graduated pipette. With a rubber-bulb capillary pipette, which has been graduated to hold 16 drops or 1 cc., draw up the contents of the tube containing the 1 to 4 serum and add it to the next tube containing 1 cc. of salt solution. This gives 2 cc. of a dilution of 1 to 8. Now mix thoroughly by drawing up and forcing out with the bulb pipette, and then withdraw 1 cc. and add to the next tube containing 1 cc. of salt solution. This gives a dilution of 1 to 16. Having mixed as before, again withdraw 1 cc. of the mixture and add it to the 1 cc. in the next tube. We now have a dilution of 1 to 32. Again withdrawing 1 cc. and adding it to the fourth tube containing 1 cc. of salt solution we have a dilution of 1 to 64. In tube 1 there is now 1 cc. of a dilution of the serum of 1 to 8; in tube 2, there is 1 cc. of a dilution of 1 to 16; in tube 3 of 1 to 32. Tube 4 contains 2 cc. of 1 to 64. The dilutions can be carried on in the same manner to any extent that may be desirable. In cholera agglutinations we may run up to 1 to 5000 or thereabouts. Of course, where such dilutions are employed, we generally start with 2 cc. of 1 to 50 in the first tube. When we have completed the series, each tube having 1 cc. of diluted serum, and the last 2 cc., we remove with the pipette 1 cc. from the last tube and discard it by ejection from the pipette leaving 1 cc. in the last tube. Now adding 1 cc. of a culture of typhoid or any other organism, we have the dilution of the serum in each tube doubled. Tube 1 now contains a serum in dilution of 1 to 16, acting on the bacteria; tube 2 of a 1 to 32; tube 3 of a 1 to 64. Now place these tubes in the incubator and, after two to five hours or overnight, we examine for the clearing up of the supernatant fluid. If the serum in a certain dilution agglutinates, the clumps gravitate to the bottom and the upper part becomes clear. If so desired, these dilutions may be carried on to 1 to several hundred in the same way. It is safer to work with dead cultures instead of living ones. To prepare, take a twenty-four-hour agar slant culture of typhoid or paratyphoid and emulsify in salt solution (about 6 cc. to a slant).

By adding 0.1 of 1% of formalin to the typhoid emulsion and placing in the ice-box the cultures will be found sterile in about three days. The emulsion should be shaken twice daily while undergoing sterilization in the ice-box. Such cultures are not easily contaminated and appear to retain their agglutinable qualities for several months. The macroscopic methods are preferable with such dead cultures. For our Dreyer emulsions we use a two-billion suspension of typhoid or para-typhoid organisms in 1 cc. of the formalinized culture.

Combination of Microscopical and Macroscopical Methods.—Microscopic: Prepare dilutions of serum as above described and take from each or several of the series, a loopful of the diluted serum. For control use a loopful of salt solution. Place on a cover-glass and add loopful of bouillon culture of the living organisms. Make hanging drop preparation, report after one hour at room temperature. Use ⅔ inch lens for examination.

Macroscopic: Add to each of the series, including the control, an equal amount of an emulsion of killed organisms.

The method of using a slide with two vaselined rings, one containing an emulsion in the specific serum and the other in salt solution, is of great practical value. This method is described under cholera.

Complement Fixation.—Complement fixation tests have been employed in the diagnosis of several tropical diseases but do not seem to be at present sufficiently reliable or practical with the exception of that for yaws and tularaemia. The chief difficulty with complement fixation tests for suspected sera is to obtain a reliable antigen. Should we later on be able to prepare bacterial antigens as satisfactory as Noguchi’s acetone-insoluble antigen is for the Wassermann test there may be a field for such tests in tropical pathology.

OTHER PRACTICAL METHODS OF HAEMATOLOGICAL STUDY

Haemoglobin Estimation

The standard method now is the estimation of the oxygen capacity of the blood, using some gas apparatus, such as Van Slyke’s. Otherwise, the most accurate instrument for this purpose is the Miescher modification of the v. Fleischl haemoglobinometer.

The apparatus is expensive, requires considerable time and care in the making of estimations, and is exclusively an instrument for a well-equipped laboratory.

=Sahli’s Haemometer.=—A simple and apparently very scientific instrument which has been recently introduced is the Sahli modification of the Gower haemoglobinometer. Instead of the tinted glass, or gelatin colored with picrocarmine to resemble a definite blood dilution, Sahli uses as a standard the same coloring matter as is present in the tube containing the blood. By acting on blood with 10 times its volume of N/10 HCl, haematin hydrochlorate is produced, which gives a brownish yellow color. In the standard tube, which is sealed, a dilution representing 1% of normal blood is used. To apply this test, pour in N/10 HCl to the mark 10 on the scale of the graduated tube. Add to this 20 cubic millimeters of the blood to be examined, drawn up by the capillary pipette provided. So soon as the mixture assumes a clear bright dark-brown color, which requires about ten minutes, add water drop by drop until the color of the tubes matches. The reading of the height of the aqueous dilution on the scale gives the Hb. reading. The tubes are encased in a vulcanite frame with rectangular apertures. This gives the same optical impression as would planoparallel glass sides.

The most accurate readings are obtained with artificial light in a dark room but almost as satisfactory comparisons can be obtained with natural light from a window. It is advisable to turn the ruled side around so that one may match colors without being influenced in his determination by the scale.

The apparatus must be kept in a dark place as strong light will change the color of the standard tube. It is recommended that the N/10 HCl be preserved with chloroform.

The Dare instrument is excellent.

Pappenheim has recently proposed an instrument in which the blood is converted into haematin hydrochloride as for the Sahli apparatus. Instead of matching a standard tube, with a dilution made drop by drop in the second tube, the new method employs a wedge-shaped glass vessel showing graduations of the brown colored blood, the treated blood being matched against the wedge-shaped container (Autenreith-Koenigsberger Haemocolorimeter).

=Tallquist’s Haemoglobin Scale.=—This is a small book of specially prepared filter-paper with a color-scale plate of ten shades of blood colors. These are so tinted as to match blood taken up on a piece of the filter-paper and are graded from 10 to 100. So soon as the blood on the filter-paper has lost its humid gloss, the comparison should be made. This is best done by shifting the blood-stained piece of filter-paper suddenly from one to the other of the holes cut in each shade—the piece of filter-paper being underneath the color plate. At least a square centimeter of the filter-paper should be stained by the blood. Daylight coming from a window to the rear or at the side should be used in making the comparison. The error with this method is probably not over 10% after a little experience. If the colored plate is not kept in the dark, the tints tend to fade.

NORMAL BLOOD

In considering what may be termed normal blood, it must be borne in mind that the normal varies for men, women, and children:

Hb. Red Cells Leucocytes

Men, 90 to 110%, 5 to 5½ million, 7500. Women, 80 to 100%, 4½ to 5 million, 7500. Children, 70 to 80%, 4½ to 5 million, 9000.

COLOR INDEX

This is obtained by dividing the percentage of the haemoglobin by the percentage of red cells, 5,000,000 red cells being considered as 100%.

To obtain the percentage of red cells it is only necessary to multiply the two extreme figures to the left by two. Thus if a count showed the presence of 1,700,000 red cells the percentage would be 34 (17 × 2 = 34). If the Hb. percentage in this case were 50, then the color index would be 50 ÷ 34, or 1.4.

In normal blood the color index is, approximately, 1.

In anaemias we have three types of color index: 1. The pernicious anaemia type which is above 1. Here we have a greater reduction in red cells than we have of the haemoglobin content of each cell. For example, in a case of pernicious anaemia we have 2,000,000 red cells (40%) and 60% of haemoglobin, 60 ÷ 40 = 1.5. 2. The normal type, when both red cells and haemoglobin are proportionally decreased, as in anaemia fallowing haemorrhage. 3. The chlorotic type. Here there is a great decrease in haemoglobin percentage, but only a moderate decrease in the number of red cells. Hence the color index is only a fraction of 1. For example, in a case of chlorosis we have 40% of haemoglobin and 4,000,000 red cells, 40 ÷ 80 = 0.5.

One can judge fairly well the approximate color index by noting the character of the staining of the red cells. This is faint in bloods of low color index and deeper than normal in cells in a case with high color index.

TESTS FOR AGGLUTINATION AND HAEMOLYSIS OF THE RED CELLS (TRANSFUSION)

Transfusion of blood has become a method of greatest value in many types of anaemia.

In the selection of a donor for blood for transfusion it is always necessary to try his red cells against the serum of the recipient as well as the patient’s red cells against the serum of the donor, in order to prove the absence of haemolyzing or agglutinating bodies.

Certain persons have isohaemolysins in their blood which dissolve the red cells of other persons and in paroxysmal haemoglobinuria autohaemolysins may be present which can destroy the patient’s own red cells. This autohaemolysin seems operative only when a low temperature is followed by a high one. When haemoglobinaemia exists the liver converts it into bile pigment, causing bilious stools and jaundice. If one-sixth of the red cells are destroyed haemoglobinuria results.

In the following tables, two groupings of blood are given. Both are quoted in text-books, and both are in common use. Although that of Moss is more generally followed in France, England and the United States, the obvious desirability of having one classification universally employed, in order to avoid confusion and the possibility of serious accidents, has led to the recommendation that, on the basis of priority the grouping of Jansky be adopted.

In 1907, Jansky described the following four groups.

Group 1, the serum of which agglutinates the corpuscles of Groups 2, 3 and 4, while the cells are not agglutinated by any serum.

Group 2, the serum of which agglutinates the corpuscles of Groups 3 and 4, but not those of Groups 1 and 2, while the corpuscles are agglutinated by the serum of Groups 1 and 3, but not by those of Groups 2 and 4.

Group 3, the serum of which agglutinates the cells of Groups 2 and 4, but not those of Groups 1 and 3, while the corpuscles are agglutinated by the serum of Groups 1 and 2, but not by those of Groups 3 and 4.

Group 4, the serum of which does not agglutinate any corpuscles, while the corpuscles are agglutinated by the serum of all other groups.

In 1910, Moss made the following classification:

Group 1, the serum of which does not agglutinate any corpuscles, while the corpuscles are agglutinated by the serum of Groups 2, 3 and 4.

Group 2, the serum of which agglutinates the corpuscles of Groups 1 and 3, while the corpuscles are agglutinated by the serum of Groups 3 and 4.

Group 3, the serum of which agglutinates the corpuscles of Groups 1 and 2, while the corpuscles are agglutinated by the serum of Groups 2 and 4.

Group 4, the serum of which agglutinates the corpuscles of Groups 1, 2 and 3 while the corpuscles are not agglutinated by any serum.

At the present time it is accepted that the four groups considered include all adult persons; i.e., that the classification is complete.

=Before transfusing= carry out the following tests:

From a vein take about 1 cc. of blood in a centrifuge tube containing 1% of sod. citrate salt solution; then shift the stopper of the blood system to a dry centrifuge tube and draw into it about 3 or 4 cc. of blood. Throw down the citrated blood, pipette off the supernatant fluid and wash the sediment with normal saline.

Again pipette off the saline after centrifuging and make a 10% emulsion of the red-cell sediment in normal saline.

Centrifuge the coagulated blood in the other tube and collect the serum which separates from the clot.

Carry out these procedures for both donor and recipient.

Tests: 1. In a small test-tube deposit 1 drop of the donor’s 10% red-cell emulsion and then add 4 drops of the recipient’s serum.

2. Treat similarly 1 drop of the recipient’s red-cell emulsion with 4 drops of the donor’s serum.

3. Treat 1 drop of donor’s red-cell emulsion with 4 drops of his serum.

4. Treat 1 drop of recipient’s red-cell emulsion with 4 drops of his serum. Finally add 1 cc. of salt solution to each of the four tubes, shake gently and place in incubator for two hours.

5. Treat one drop of donor’s red-cell emulsion with four drops of salt solution.

6. Treat one drop of recipient’s red-cell emulsion with four drops of salt solution.

Tubes 5 and 6 are controls of saline.

Tests 3 and 4 should fail to show either agglutination or haemolysis. If agglutination or haemolysis appears in tubes 1 or 2, the donor is not satisfactory; but if agglutination appears in tube 2 only, he may be used in an emergency.

Some prefer to keep the tubes overnight in ice-box after the preliminary examination following incubation.

Lee’s Technique.—For the regular carrying out of this method one should keep on hand the sera of individuals belonging to groups 2 and 3 (Moss). To carry out the tests prepare a suspension of the donor’s red cells by dropping 2 or 3 drops of his blood into 1 cc. of citrated salt solution. Deposit a platinum loopful of standard serum 2 on a slide and emulsify in it a loopful of the donor’s red-cell suspension. A concave slide with two concavities is convenient, the serum-cell emulsion being made on the cover-glasses which are to be inverted over the vaseline ringed concavities. The agglutination can be observed with a high power magnifying glass or the ⅔-inch objective. Agglutination, when it occurs, is usually complete in five to fifteen minutes. Repeat test with serum 3. If both test sera agglutinate the donor’s cells he belongs to group one. If neither agglutinate, to group four.

Agglutination by group two serum but not by three puts the donor in group three. Agglutination by group three serum but not by group two shows a group two donor. It would seem safe to use the cells of any donor of group 4, as such cells are not agglutinated by the sera of any group. It is, however, advisable to try to obtain a donor whose blood belongs to the same group as the donee. When standard sera 2 and 3 are not on hand one may use the following emergency method of Lee:

“A small amount of blood is collected from a patient (1 cc. from the ear or finger is sufficient), and allowed to clot. The serum is then obtained. One drop of this serum is placed on a slide and mixed with a drop of suspension of blood of the donor taken into 1.5% citrate solution. (A few drops of blood are taken into approximately 10 times the amount of 1.5 citrate solution and shaken. It is very important that the blood be dropped directly into the citrate, and should not be partially coagulated.) The test will appear in a few moments, and is best examined under the microscope, where, in the event of a positive test, marked agglutination will be evident. The test will also be evident macroscopically. In the event of a negative test it is a wise precaution to raise the cover-glass, and after making sure that the serum and cells are well mixed, to examine the preparation again. The only possible source of confusion is the appearance of rouleaux of the red corpuscle, indicating a too thick emulsion. If the test is negative, transfusion may be regarded as entirely safe.”

In the absence of agglutination haemolysis never occurs. Only about one-fifth of agglutinating sera prove also haemolytic. Rarely a pernicious anaemia patient’s serum may agglutinate his own red cells. This auto-agglutination is regarded as an important test in acquired haemolytic jaundice.

OCCULT BLOOD

When the presence of blood in the faeces, gastric contents, urine or body fluids, is suspected but cannot be recognized by macroscopic or microscopic methods, it is necessary to resort to spectroscopic or chemical tests. These tests are, however, individually unsatisfactory. The spectroscopic method is not delicate, the haemin-crystal method does not give uniform results and the various color tests, although very sensitive, are given by many substances other than blood. Consequently, it may be said that, with the color tests, it is negative results that are significant, and with other than the color tests it is positive findings that are informative. Serological tests are the most satisfactory medicolegally.

Haemin Crystal Test (Teichmann).—Prepare a solution (stable) of 0.1 gm. each of KI, KBr, and KCl in 100 cc. acetic acid. Mix a few drops with some of the material on a slide, apply a cover-glass, and gently warm until bubbles begin to appear. Then cool slowly, and examine for the characteristic dark-brown crystals.

Haemochromogen Crystals (Donogány).—Mix one drop each of suspected fluid, pyridin, and 20% NaOH on slide, and let dry. If positive, radiating needles will form after several hours.

Spectroscopic Tests.—These depend upon the recognition of the characteristic absorption spectra of haemoglobin or its derivatives (Fig. 24). The degree of concentration influences their appearance, and one should start with a relatively concentrated solution, diluting cautiously until the bands are typical.

The small, direct-vision (hand) spectroscope suffices. A wavelength scale is a convenient attachment. Daylight or strong artificial light (such as the “daylite” lamp) is used. Have solution in a small test tube or, preferably, a flat cell with a thickness of about 1 cm. Before use, focus Frauenhofer’s lines sharply.

Reducing agents are employed, such as ammonium sulphide, or Stokes’ solution made up as follows: Dissolve 3 gm. FeSO_{4} in cold H_{2}O; add cold, aqueous solution of 2 gm. tartaric acid; make up to 100 cc.; immediately before use, add strong NH_{4}OH until precipitate first formed is dissolved. Both solutions must be freshly prepared, and the sulphide must be warmed to about 50°C.

Material that is uncontaminated, relatively fresh and in relatively concentrated aqueous solution may give any or all of the upper three spectra, a few drops of reducer changing the first to the second.

If the material is older, dissolve the suspected stain in 1-2 cc. of 10% NaOH, heat almost to boiling, cool, and add a few drops of reducer. Examination shows Spectrum 5.

It is better, however, especially with much contamination, to prepare an ethereal, acid extract. After having ground the material thoroughly with water, if it is not already in liquid form, shake it with an equal volume of neutral ether. Reject ether extract, and, to 10 cc. of residue, add 3 to 5 cc. of glacial acetic acid. Shake thoroughly with an equal volume of ether. If the ether does not separate readily, mix gently with a few drops of alcohol. Remove ethereal extract, and evaporate it to a small bulk for use in tests. Examination will show spectrum of acid haematin, which, however, in ethereal solution, resembles Spectrum 3 more than 4.

Donogány’s Method increases the delicacy of the spectroscopic test, and is also a color test. Dissolve the pigment with 20% NaOH, add fresh pyridin and, if necessary, fresh ammonium sulphide. Filter. The filtrate will be more or less orange-red according to blood content, and will show Spectrum 5.

Color Tests.—The reliability of these may be enhanced by the use of methods which involve the removal or destruction of interfering substances. In such a method, the original aqueous solution is boiled for 15 to 20 seconds, and the acid ethereal extract is prepared as previously described. This extract is dropped on filter-paper, the reagents being applied to the moistened spot. The delicacy of these several tests is variable, being greater with blood in aqueous solution than in biological fluids, but it may be given as approximately 1-25,000 for the guaiac and aloin, and 1-250,000 for the benzidine test.

(a) Treat moist spot with a few drops of freshly prepared 2% alcoholic solution of guaiac resin, and then a few drops of hydrogen peroxide. A blue color is “positive.”

(b) Treat moist spot with a few drops of 3% aloin in 70% alcohol, and then with ozonized turpentine (turpentine that has stood for a few days in an open vessel in sunlight). A purplish-red color within 10 minutes is “positive.”

(c) Treat moist spot with 2 drops of glacial acetic acid, a few crystals of benzidine (preferably white), and finally 2 drops of hydrogen peroxide. A greenish-blue color is “positive.”

ACIDOSIS

Everyone is familiar with that form of respiratory disturbance associated with diabetic coma that is known as Kussmaul’s air hunger. Here we have hyperpnoea, a form of dyspnoea typically without cyanosis, and furnishing the best clinical evidence of acidosis. Acidosis, however, is now recognized to be but a particular phase of disturbance of the acid-base equilibrium of the body, and recent work has radically changed our conceptions of its features and its intricate relationships.

Van Slyke restricts the use of the term “acidosis” to describe a condition caused by acid retention sufficient to lower either the bicarbonate or the pH of the blood below normal limits. The pH of the blood may be considered the danger sentinel; as long as it is normal, the acid-base equilibrium is normal or compensated; otherwise, it is uncompensated, and life is seriously threatened. The normal pH of the blood may be given as 7.3 to 7.5 (a slightly alkaline reaction), each individual, however, probably having normally narrower limits of variation. That of the blood serum is about 0.2 pH higher, and that of the other body fluids (not the excretions) probably closely approximates and promptly follows any change in that of the blood plasma. Variations to the acid side may, for a short time at least, be as low as 7.0, although not much lower without fatal results; 7.0 is considered the point where coma occurs. Variations to the alkaline side (Alkalosis) beyond 7.8 are accompanied by symptoms of tetany, although one is not at present justified in assuming that all tetany is either caused, or accompanied, by alkalosis. So, the extreme range of reaction compatible with life probably lies approximately between pH of 7.0 and 7.8.

Recent, but as yet unconfirmed, work suggests that the severe reactions following intravenous medication or infusions may be due, at least in part, to the fact that the pH of the fluid introduced is decidedly more acid or alkaline than that of the blood. This applies to solutions of glucose, the salines, and possibly also to sodium citrate, arsphenamine, sera, antitoxins, etc. The question of suitably buffering such solutions, e.g., with suitable phosphate mixtures, in order to avoid disturbance of the acid-base equilibrium, is being studied, and the preliminary results are promising.

The hydrogen-ion concentration (or its derivative, pH) of the blood varies as the ratio between the concentrations of dissolved carbonic acid and bicarbonate (generally indicated by (H_{2}CO_{3})/(NaHCO_{3})), i.e., a relative increase in the H_{2}CO_{3} increases the hydrogen-ion concentration and lowers the pH, and vice versa. The stability of this ratio is preserved by body mechanisms operative in controlling its two factors,—the H_{2}CO_{3} being under respiratory control, and the NaHCO_{3}, considered as representing the alkali reserve, being normally maintained by food.

The erythrocytes control the concentration of bicarbonate by virtue of their haemoglobin and the reversible reaction.

H_{2}CO_{3} + NaCl ⮂ HCl + NaHCO_{3}

The HCl passes into the cell, and is probably held by the haemoglobin. In the lungs, the CO_{2} is excreted and NaCl reformed. This ability of haemoglobin to form bicarbonate is important inasmuch as the corpuscles can conceal 5 to 10 times as much acid as the plasma bicarbonate can ordinarily neutralize. A full appreciation of the significance of this ratio being the basis of an intelligent comprehension of acid-base equilibrium, a detailed analysis of factors that tend to influence the ratio is given.

Factors Operating:

A. To increase or protect bicarbonates: 1. Administration of bicarbonate. 2. Loss of gastric HCl induced by obstructing the pylorus, and regularly washing out the stomach for some days. 3. Processes indicated by increased excretion in the urine of ammonia compounds, the ammonia being probably diverted from urea formation, and of substances producing a titrable acidity, they including buffer acids such as acid phosphates. 4. Possibly a shift of HCl to the tissue cells from the plasma like that from the plasma to blood cells.

B. To decrease bicarbonate: 5. Acid substances, by their (a) Increased production. (b) Decreased elimination or (c) Ingestion. 6. Diuresis, with elimination via the urine. 7. Lack of Factor A (3). 8. The hyperpnoea associated with deficient oxygen.

C. To increase carbonic acid: 9. Administration of carbonic acid. 10. Impaired diffusion in the alveoli of the lungs. 11. Slowing of respiration.

D. To decrease carbonic acid: 12. Hyperpnoea. (a) Voluntary. (b) Due to disease processes. (c) Due to low oxygen content of air. (d) Emergence from warm water. 13. Low atmospheric content of CO_{2}.

Figure 152 is a graphic representation of essential facts in acid-base equilibrium. Ordinates represent total CO_{2} content, which comprises that in simple solution and that as bicarbonate of whole blood in volumes per cent, and abscissae the mm. CO_{2} tension in the blood as drawn. The line OT gives the proportion of total CO_{2} present in simple solution. pH values are shown by the lines OL, OM, etc. The extreme normals for carbon dioxide absorption curves are OP and OR. The CO_{2} tension of alveolar air may be the same or vary as much as 20 mm. below, while that of venous blood will be about 6 (0.8-10.0) mm. higher than that of arterial blood. The “CO_{2} capacity” (or “CO_{2} combining power”) of plasma may be as much as 15 vol. % more than the total CO_{2} of whole blood.

The actual state of acid-base balance, then, can only be determined by the use of any two of a number of interdependent variables, such as total CO_{2}, CO_{2} tension, pH, H_{2}CO_{3} concentration, other buffers than bicarbonate, plasma chloride, ratio of oxyhaemoglobin to haemoglobin, etc. Findings that fall within ABCD and at about 40 mm. tension indicate a normal equilibrium for the resting individual at ordinary altitudes. Or, such a normal would be a total CO_{2} of about 49 (43-56) vol. % for whole blood, and 50-65 vol. % for plasma. The normal for the individual falls within narrower limits.

If either H_{2}CO_{3} or bicarbonate varies from normal values, there is apparently an effort on the part of the body to compensate by adjusting the other so as at least to maintain a normal pH. This is accomplished by respiration, or by diverting alkali from or recalling it to the blood stream. Naturally, treatment of any such abnormal condition will do well to imitate Nature’s efforts. Haggard and Henderson have demonstrated that blood alkali may be decreased in two ways—by acids (the acidotic process) or by acapnia (the acapnial process). By the forced breathing of the acapnial process the lungs are over-ventilated and an excessive amount of carbon dioxide is washed out of the blood, thus bringing on a temporary alkalosis. In case of prolonged forced breathing, nature prevents an extreme alkalosis by causing the alkali to leave the blood, it being stored in the tissues or excreted in the urine. Blood relatively poor in carbonic acid or relatively rich in alkali acts to depress respiration, and the slowing of respiration produces an acidosis by the resultant retention of H_{2}CO_{3}, this causing alkali to be recalled from the tissues. Thus acidosis, in calling more alkali into the blood from the tissues, represents what may be regarded as a restorative effort. Hence, administration of bicarbonate is indicated in acidotic processes, and of CO_{2} in acapnial; the use of the wrong one is dangerous.

The numbered regions of the chart are associated with various clinical conditions, e.g., tetany from 1, 2, and 3; the acidosis of diabetes mellitus, nephritis, or infantile marasmus with 6 or 9; pneumonia, morphine narcosis, and breathing of air containing 3-5% CO_{2} with 7 or 8; emphysema with 4; some cardiac cases with 9; overdose of bicarbonate with 1 or 4; fever with 2; as the result of high altitudes, 2 or 3, or, when acclimated, 6; shock (handling of intestines), deep ether anesthesia, and carbon monoxide asphyxia with lowered bicarbonate. The disturbances of acid-base equilibrium in the last two are the result of acapnial processes.

Normal metabolism results in the constant formation of acids, especially H_{2}CO_{3}, and disease processes may occasion the presence of still more. A constant loss of alkali results, the neutralization products being eliminated mostly in the urine, and the H_{2}CO_{3} via the lungs; the body fluids are excellently buffered, the most important buffers being bicarbonate, proteins (especially haemoglobin), and phosphates. In the maintainance of the normal pH, the CO_{2} (or H_{2}CO_{3}) is the easily variable factor. The onslaught of invading acids is first met by the bicarbonates (acidotic process); hyperpnoea lowers the H_{2}CO_{3} and a normal pH is maintained until the bicarbonates are reduced to one-fourth (perhaps even to one-eighth) of their normal concentration. If, nevertheless, the pH falls (and only then), the other buffers are used, and, if it reaches 7.0, most of the remaining bicarbonate becomes available. The blood handles the situation, but buffers from the tissues or other body fluids also become available in extreme cases.

As noted above, measurement of one variable will be inadequate exactly to determine the state of acid-base equilibrium. As long, however, as the pH is normal, which is the usual finding in most pathological conditions, including mild acidosis, one determination will suffice. Clinical methods comprise tests for whole blood or plasma CO_{2} or bicarbonate, alveolar CO_{2} tension, bicarbonate tolerance, pH of blood or urine, Sellard’s test, NH_{3} quotient of urine, or presence of abnormal acids (particularly acetone bodies) in blood or urine. The first two methods are the ones of choice, particularly the first, as, by it, one can estimate the reserve of the very important blood buffer, bicarbonate, and its result closely indicates the total buffers.

The tolerance for bicarbonate is a very convenient and practical measure of acidosis, and means the dose of NaHCO_{3} required to produce a urine alkaline or amphoteric to litmus. A normal finding is 5-10 grams; 20 is required with a mild, 30-40 with a more severe, and more than 40 gm. with extreme degrees of acidosis. In coma, it is usually impossible to produce an alkaline urine.

Certain changes in the urine are recognized and acceptable as indirect evidence of acidosis, but these changes are not synonymous with acidosis, being dependent in part upon renal integrity and other factors. The NH_{3} quotient of urine (ammonia nitrogen; total nitrogen), as usually determined with patient on a mixed diet, is normally about 5%. Values of 10-40% occur in acidosis. It may be increased by diet, disturbances of protein metabolism, ammoniacal fermentation, etc., and there may be no increase in certain diseases with acidosis. The ammonia probably does protect the blood alkali, but its efficacy is intimately associated with renal function, inasmuch as Nash and Benedict have presented strong evidence to the effect that urinary and blood NH_{3} is the product of an active synthetic function of the kidneys themselves. Acetone bodies in the urine (ketonuria), in the blood (ketosis), or in the breath, have diagnostic value but are poor indices of severity of acidosis and may be absent in acidosis. Acetone and diacetic acid have the same significance: a progressive increase gives a grave prognosis, and it is generally considered that the presence of β-hydroxybutyric acid indicates greater severity.

Acidotic acidosis is due either to the abnormal formation or ingestion of acid substances, or to decreased elimination of normal metabolic products. Ketosis is the important example of the former, and retention of acid phosphates of the latter. In either case, the body is robbed of its bases. The acidosis of diabetes mellitus is characterized by ketosis and increased NH_{3} quotient of urine, while that of nephritis is a phosphate retention without ketosis, and, as one would expect, the NH_{3} quotient is usually not increased. Infantile diarrhoea with ileocolitis shows a marked ketosis, but, lacking the ileocolitis, the ketosis is only moderate and the acidosis is due to phosphate retention.

The appearance of an acidosis in disease constitutes a serious development demanding immediate attention. It is usually present at time of death and may be the immediate cause. We must be prepared for the appearance of acidosis in the course of numerous cosmopolitan diseases, and its presence has been recognized in a few tropical conditions. Before we generally recognized the great importance of the acidosis factor in pathology, there were two standard treatments for yellow fever and blackwater fever, the Sternberg one in the former and the Hearsey one for the latter, both of which had as a basis the administration of alkalis, which is our best means for neutralizing the deleterious action of increased acid production in the body or defective elimination of the same. It was a very important contribution to the therapeutics of cholera when Sellards, recognizing the tendency of the nephritis to produce an acidosis in this disease, made use of intravenous injections of NaHCO_{3} to combat the condition, thus counteracting the anuria, one of the chief complications leading to death. More recently, the Egyptian workers noted an acidosis in kala-azar, a finding verified and emphasized by Rogers. There is also an acidosis in heat stroke, so that intravenous or rectal injections of NaHCO_{3} are of value. It will thus be seen that acidosis is a most important condition to keep in mind in tropical conditions, and it will be well to be on the watch for other varieties of disturbance of acid-base equilibrium.

Relative to the administration of bicarbonate in treatment, there is now a decided reaction against the use of amounts that may prove injurious by reason of the danger of alkalosis. There is a tendency to employ it only in decompensated acidosis, and control it by estimations of plasma CO_{2} capacity, 0.5 gm. NaHCO_{3} per 19 kg. body weight will raise the plasma CO_{2} capacity by 1 vol %. It is distinctly contraindicated in cases whose low plasma CO_{2} is due to acapnial processes. Early administration is desirable in children, and good results are obtained, especially with the older ones. An acidosis, however, once established in infants may cause death despite alkali. In order to avoid over-dosage of bicarbonate, methyl red, which is more sensitive than litmus to early changes in the reaction of the urine, should be employed as an indicator. The appearance of a yellow color upon its addition to the urine is the sign to suspend further administration of alkali.

Glucose is indicated in conditions with ketosis due to carbohydrate deficiency, providing the organism can assimilate it.

CHEMICAL ANALYSIS OF BLOOD

The chemical analysis of the blood has attained a clinical simplicity and significance that demands recognition. It provides points of value in diagnosis, prognosis, and treatment, being especially useful in nephritis, diabetes, acidosis, comatose conditions, gout, and in questions of renal function and treatment, especially dietetic. Urine findings are always dependent upon kidney function, and, by blood chemistry, we can pass behind this barrier.

Few diseases have been as yet studied thoroughly in this respect, but our fund of knowledge is receiving constant additions. The field of tropical medicine is practically untouched, and it is quite possible that an investigation along this line might there yield facts of interest and value.

The following table (amplified from Myers), is a concise summary of normal findings and those encountered in various clinical conditions. The diagnostic significance is evident. Some of the results are based upon the analysis of many cases; others upon but few. One might include the findings mentioned elsewhere regarding acidosis in certain tropical diseases, but, except for such, we have no other data relative to them, unless one mentions that blood sugar is increased in the tropics. The values are given in milligrams per 100 cc. whole blood (the usual system), except those for diastatic activity (recorded in Winslow’s empirical units) and acidosis (expressed in terms of plasma carbon dioxide combining power—volumes %). “Inc.” and “Dec.” signify increased and decreased respectively.

RESULT OF CHEMICAL EXAMINATION OF BLOOD

CONDITION --+-------+-------+------+-------+--------+-------+-------+-------+------ | Non | | | | | | | |Plasma |protein| Urea | Uric |Creati-| Sugar |Choles-|Chlor- | Dias- |CO_{2} | nitro-|nitro- | acid | nine | | terin | ides | tase |capa- | gen | gen | | | | | | | city --+-------+-------+------+-------+--------+-------+-------+-------+------ NORMAL | 25-300| 10-15 | 2-3 | 1-2 | 90-120 |170-250|450-500| 8-64 | 53-77 | | | | | | | | | DIABETES MELLITUS, MILD | | | | |150-300 | | | Inc. | | | | | | | | | | DIABETES MELLITUS, SEVERE | | 20 | 4-10 | 2-4 |300-1200| Inc. | Dec. | Inc. | 10-50 | | | | | | | | | NEPHRITIS, ACUTE | | 40-100| 5-15 | 2-6 |120-180 | | Inc. | 20-45 | | | | | | | | | | NEPHRITIS, INTERSTITIAL, EARLY | | 15-25 | 5-12 | 2-3.5 |120-150 | | | Inc. | | | | | | | | | | NEPHRITIS, INTERSTITIAL, TERMINAL |100-300| 60-300| 5-27 | 5-28 |120-240 | Inc. | Vari- | Inc. | 12-40 | | | | | | | able | | NEPHRITIS, PARENCHYMATOUS (NEPHROSIS) | 20-50 | 2-5 | 2-4 |120-200| Inc. | Inc. | | | | | | | | | | | | NEPHRITIS, CHRONIC DIFFUSE, SEVERE | | to 230| to 10| to 16 | to 250 | | | | | | | | | | | | | URAEMIA | 90-350| 70-300| | | | | | | | | | | | | | | | KIDNEY POLYCYSTIC, DOUBLE | | to 75| to 5 | to 8 | to 200 | | | | | | | | | | | | | PROSTATIC OBSTRUCTION | Inc. | 12-40 | 3-9 |1.5-3.5|110-160 | | | | | | | | | | | | | GOUT | | | 4-10 | | | | | | | | | | | | | | | HYPERTHY- ROIDISM | | Inc. | | | Inc. | | | Inc. | | | | | | | | | | HYPOENDOCRINE CONDITIONS | | Dec. | | | 60-90 | | Dec. | Dec. | | | | | | | | | | ECLAMPSIA | 25-45 | 10-25 | 4-8 | | | | | | 43-58 | | | | | | | | | INTESTINAL OBSTRUCTION, ACUTE | 75-170| 45-120| Inc. | Inc. | | | | | | | | | | | | | | FEVER, ACUTE | Inc. | Inc. | to 4 | | Dec. | Dec. | | | | | | | | | | | | PNEUMONIA, SEVERE AND LATE | | to 53 | to 18| to 3.5| to 180 | Inc. | Dec. | | Dec. | | | | | | | | | ANAEMIA, PERNICIOUS | to 108| to 75 | to 10| to 3.1| to 300 | Dec. | Inc. | | Dec. | | | | | | | | | MALIGNANCY, LATE | Inc. | Inc. | Inc. | Inc. | | Dec. | Inc. | | Dec. | | | | | | | | | DEMENTIA PRAECOX, CATATONIC | | 6-10 | Dec. | | Inc. | | | | | | | | | | | | | SHOCK | Inc. | Inc. | | Inc. | Inc. | | | | Dec. | | | | | | | | | BICHLORIDE OF MERCURY POISONING |to 370 | to 300| to 15| to 33 |120-200 | Inc. | | | | | | | | | | | | PLUMBISM | Inc. | Inc. | Inc. | | | | | | --+-------+-------+------+-------+--------+-------+-------+-------+------

Interstitial nephritis is characterized by a nitrogen retention, while parenchymatous nephritis has relatively little nitrogen retention but does have a decided tendency towards chloride retention. Essential hypertonia with its normal blood chemistry is differentiated from arteriosclerosis with its frequent nitrogen retention. The imminence of uraemia may be judged by the extent of the nitrogen retention. We have an aid in the differentiation of the uraemia of nephritis accompanied by a flagging heart from the passive congestion of cardiac decompensation, especially as to which is the secondary condition, and thus therapeutic indications relative to mooted questions of treatment (hot packs, morphine, renal stimulants, etc.). Unsuspected cases of nephritis showing only gastric symptoms clinically have been detected by blood chemistry. The significance of albumin in traces and occasional casts in urine has been more definitely established by examination for increase of uric acid in the blood—an increase arguing for an organic lesion. Values of over 4 for creatinine do not occur without great impairment of renal function, and findings of more than 5 have practically uniformly foretold a fatal termination in less than six months, except in acute nephritis and mild bichloride of mercury poisoning. The creatinine is also the best guide to the status of renal function in terminal cases. The chloride and nitrogen content afford guides to diet.

The blood may indicate a prediabetic state, and place the practitioner upon his guard. There is a condition but recently recognized in which there is a normal blood sugar, a persistent glycuresis of usually less than 1% and independent of carbohydrate intake, occasionally polyuria, but with no other symptoms of diabetes mellitus. It is known as renal diabetes, is apparently harmless, probably not uncommon, and may represent the condition affecting most of those “diabetics” who can disregard diet with impunity. The blood sugar and plasma CO_{2} are usually considered the only safe guides in the treatment of diabetes mellitus and no extended medical treatment or surgical interference should ever be attempted without their estimation. Glycosuria is a poor guide, especially in advanced cases.

In comatose conditions, nitrogen retention will indicate the uraemic, and hyperglycaemia the diabetic cases. But acute nephritis should always be borne in mind, as it may have a pronounced acidosis but no nitrogen retention.

A high uric acid finding alone is characteristic of gout, and aids in differential diagnosis from simple rheumatic fever and other arthritides, any uric acid retention in them being usually accompanied by retention of other nitrogenous elements. It is especially useful in the diagnosis of gouty arthritis without tophi.

The efficacy of treatment will, in general, be shown by the degree of approach to normal blood findings.

LEUCOPENIA

This is a term used to designate a reduction in the normal number of leucocytes. A leucocyte count of 5000 would represent a slight leucopenia; one of 2000, a marked leucopenia. In the later stages of typhoid, and in acute miliary tuberculosis, we expect a moderate leucopenia. Glandular tuberculosis may give a very marked leucopenia. Tuberculous peritonitis will show moderate leucopenia or a normal count.

The leucopenia of typhoid is moderate and is often preceded in the first few days by a moderate neutrophile leucocytosis. Later on we have a decided increase in the lymphocytes. A marked diminution or absence of eosinophiles is so characteristic that any increase in eosinophilic percentage negatives a diagnosis of typhoid.

Paratyphoid gives a similar blood picture.

Chronic alcoholism and chronic arsenic poisoning cause a reduction in the number of the white cells. Pernicious anaemia, especially the aplastic type, shows a marked leucopenia, as is also the case with Banti’s disease. Two tropical diseases, kala-azar and dengue, show a marked leucopenia, the counts often being below 2500. During the apyrexial period of malaria we may have a white count of 5000.

It has recently been claimed that a leucopenia with a coincident marked reduction in the lymphocytes is characteristic of measles and that this occurs several days before the Koplik spots appear.

Kocher notes that in exophthalmic goiter the leucocyte count is considerably diminished and that the polymorphonuclears are not much more than one-half the usual percentage while the percentage of the lymphocytes is almost double the normal.

X-ray treatment tends to destroy leucocytes in the exposed region, especially polymorphonuclears. The small lymphocytes are least affected.

EOSINOPHILIA

Where the eosinophiles are increased to 5%, we have a moderate eosinophilia. In some cases of infection with intestinal parasites, especially hookworms, but also from other parasites, as round and whip-worms, we may have an eosinophilia of 30 to 50%. In Guam, among the natives, it is difficult to find an eosinophile count under 15%. The eosinophilia tends to disappear when the anaemia becomes very severe.

Echinococcus infection has an eosinophilia which disappears when the cyst is removed. Continuance of the eosinophilia indicates that all cysts were not gotten rid of.

The eosinophilia of trichinosis is best known, and a combination of this blood finding with fever and marked pains of muscles, would justify the excision of a piece of muscle for examination for encysted embryos.

In true asthma eosinophilia is marked, and its absence is of value in indicating other causes for the condition. Certain skin diseases, especially pemphigus, show eosinophilia. Blastomycoses are usually found to show eosinophile increase.

An increase of eosinophiles always attracts attention to the possibility of intestinal parasite infections or to skin affections. The explanation of eosinophilia is obscure although Neisser regards the increased production of eosinophiles as an expression of sympathetic system irritation.

Eczema and psoriasis are not apt to give more than 3 or 4% eosinophiles. A rather high degree of eosinophilia is found in mycosis fungoides.

Scabies also gives an eosinophilia.

The proportion of eosinophiles in the blood of children is greater than in that of adults.

Increase of both eosinophiles and mast cells is found in myelogenous leucaemia.

An eosinophilia tends to appear following splenectomy. With a Wright stain showing acid tendencies one may count polymorphonuclears as eosinophiles unless noting smaller size of granules.

LEUCOCYTOSIS

It is to an increase in the polymorphonuclears that this term is usually applied, the term lymphocytosis or eosinophilia being employed where white cells of eosinophile or lymphocyte nature are increased. We have physiological leucocytosis in the latter weeks of pregnancy, also in the new-born, and in connection with digestion.

=Pathological Leucocytosis.=—Pneumonia. In this disease we have a leucocytosis of 20,000 to 30,000 or higher. The eosinophiles are almost absent. A normal leucocyte count in pneumonia makes a prognosis unfavorable.

The leucocyte count drops about the time of the crisis, and with the reappearance of eosinophiles is a favorable sign.

Toxaemic conditions as uraemia, diabetic coma and poisoning by CO_{2} tend to show a leucocytosis.

Septic processes. The leucocyte count is of great value, especially when we obtain a leucocytosis with 80 to 90% of polymorphonuclears, as in appendicitis, cholecystitis, or other suppurative conditions. A marked leucocytosis is of diagnostic importance in acute ulcerative endocarditis provided it is not fulminant in type.

According to Cabot, leucocytosis varies in infections as follows:

1. Severe infection—good resistance; early, marked and persistent leucocytosis.

2. Slight infection—slight resistance; leucocytosis present, but not marked.

3. In fulminating infections we may have no increase in whites, but a higher percentage of polymorphonuclears.

4. Slight infection and good resistance may not be productive of leucocytosis.

It is in connection with the question of operation in appendicitis or similar conditions that the matter of a leucocyte count is of prime importance. If there be a leucocytosis but with less than 75% of polymorphonuclears it indicates an infection of little virulence or a walled-off process with an exacerbation. It is difficult to form an opinion when the polymorphonuclears are under 80%. Leucocytosis with polymorphonuclear percentage of 85 to 90 indicates immediate operation; percentages over 90 point to peritonitis and if with such percentages of polymorphonuclears there is absence of leucocytosis the prognosis is grave.

The blood of cases with malignant tumors tends to show a moderate leucocytosis except in epithelioma of the skin. When a cancer is ulcerating quite a high white count may be obtained.

Spirochaete fevers, as relapsing fever, may give a leucocytosis of from 25,000 to 50,000.

Smallpox, especially at time of pustulation, plague, scarlet fever, and liver abscess give a leucocytosis of from 12,000 to 15,000.

Smallpox often shows a very large percentage of very characteristic large mononuclears.

The leucopenia and lymphocyte increase in measles are important points in differentiating it from scarlatina.

Influenza shows a leucopenia at first, then a leucocytosis and, following the fall in fever, a second lowering. The very fatal pneumonias of the 1918 epidemic of influenza showed a marked leucopenia.

With meningitis counts of 25,000 are not unusual, in abscess of the brain the white count rarely exceeds 15,000.

Poliomyelitis and polioencephalitis give a slight leucocytosis during the febrile accession.

Erysipelas and epidemic cerebro-spinal meningitis also give a leucocytosis of from 15,000 to 20,000. In malignant diseases we sometimes have a moderate leucocytosis. Rogers states that in liver abscess, with a leucocytosis of 15,000 to 20,000 we have only about 75 to 77% of polymorphonuclears—there being also a moderate increase in the percentage of large mononuclears.

Drugs such as antipyrin may give a leucocytosis. The leucocyte increase of pilocarpine is rather a lymphocytosis. Cinnamate of soda, sodium nucleate, bacterin injections and turpentine have been used in kala-azar to increase leucocytes.

LYMPHOCYTOSIS

Of course, the disease in which we have the most marked lymphocytosis is lymphatic leucaemia.

The lymphocytosis of typhoid fever has been taken up under leucopenia.

Whooping-cough may give a lymphocytosis of 20,000 to 30,000.

Young children have normally an excessive proportion of lymphocytes even to a reversal of the polymorphonuclear-lymphocyte relation of adults. This is apt to be particularly marked in hereditary syphilis. Enlarged tonsils may give rise to lymphocytosis of 10,000 to 15,000 when more than 50% of the white cells will be lymphocytes. Rickets and scurvy give a lymphocytosis.

In pellagra there is a moderate lymphocytosis, averaging 34% in about a normal count.

Varicella and mumps may also give an increase in the percentage of lymphocytes.

Malta fever is a disease which may show quite a lymphocyte increase, this going with a reduction in polymorphonuclears.

Glandular fever (Pfeiffer, 1889) is a mild acute febrile disease, the fever coming on after a short incubation period and lasting about one week. Its main characteristics,—soft enlargement of the lymphatic glands, splenomegaly, and a leucocytosis of about 20,000 with 80% lymphocytes of the lymphoblastic type and many with bilobed Rieder nuclei,—lead often to its being mistaken for lymphatic leucaemia. Throat infections, particularly Vincent’s spirillosis, are thought by some to be concerned in its genesis.

INCREASED LARGE MONONUCLEARS

In tropical work we combine the large mononuclears and transitionals in a differential count. They are the phagocytes of animal cells or parasites. The disease in which their increase is best recognized is malaria and an increase to 15% where the blood shows moderate leucopenia is very significant. The melaniferous leucocytes of malaria are cells of this type.

Other protozoal infections, as kala-azar, trypanosomiasis and amoebiasis cause it. Filterable-virus diseases may show a mononuclear increase, thus yellow fever and dengue both give an increase about the fifth or sixth day.

In Banti’s disease there is an increase in cells of this type and a transitional increase is reported for Hodgkin’s disease.

DISEASES IN WHICH THERE IS A NORMAL LEUCOCYTE COUNT

Uncomplicated tuberculosis, influenza, Malta fever, measles, trypanosomiasis, malaria, syphilis, and chlorosis.

In malaria we have a leucocytosis at the time of the rigor, while during the apyrexial period there is a moderate leucopenia. In malaria we have a marked increase in the percentage of the large mononuclears and transitionals. These may form from 20% to 30% of the leucocytes. When bearing particles of pigment they are known as melaniferous leucocytes—macrophages which have ingested malarial material. In dengue, at the time of the terminal rash, we may have as great a percentage of large mononuclears. In this disease, however, we have a great diminution of polymorphonuclears from the start (25 to 40%). Instead of a large mononuclear we have at the onset a lymphocytic increase. There is an increase of large mononuclears in trypanosomiasis.

The white count is about normal in uncinariasis (Ashford’s average was 7800). Some have reported a leucopenia in severe cases.

While eosinophilia is the most marked feature in hookworm disease yet in very severe cases it may be absent.

Coagulation Rate of Blood

This determination is of value in connection with operations on jaundiced patients.

Wright’s coagulometer is a standard instrument but is cumbersome.

A simple method of determining the rate is to take a piece of capillary glass tubing and hold it downward from the puncture to let it fill for 3 or 4 inches. Then at intervals of thirty seconds scratch with a file the capillary tubing at short distances and break off between the fingers. When coagulation has taken place a long worm-like coagulum is obtained. Normally coagulation occurs in about three to four minutes, when the temperature is that of the hand in which the tubes are conveniently held. Rudolf recommends placing the tubes in metal tube-containers in a Thermos bottle at 20°C. He gives the normal coagulation rate for this temperature as eight minutes, while at a temperature below this the period is lengthened. Age and sex do not influence the rate. Sabrazes, the originator of this method found no appreciable variation in tubes from 0.8 to 1.2 mm diameter.

In Burker’s test you mix a drop of blood in a drop of distilled water on a slide and with a capillary tube sealed off at the end stir the mixture every half minute. So soon as fibrin threads appear you have coagulation.

For the proper testing for coagulation rate the blood should be taken from vein and not from that exuding from a needle stab of ear or finger. Our experience shows that it is not necessary to use venous blood.

Specific Gravity of the Blood

Hammerschlag has a method for the determination of the Hb. percentage based upon the specific gravity of the blood.

In this method a mixture of benzol and chloroform is made of a specific gravity of about 1050. A medium size drop of blood is then taken up with a pipette and dropped into the mixture. If it sinks add more chloroform from a dropping bottle, if it tends to rise, more benzol. The mixture in which the drop of blood tends to remain stationary, near the top of the mixed benzol and chloroform, has the same specific gravity as that of the blood. This is determined by an accurately graduated hydrometer. The normal average specific gravity for men is 1059, for women 1056. A table, giving the Hb. percentage corresponding to the specific gravity, accompanies the outfit.

To determine the necessity for intravenous infusion in cholera Rogers has recently recommended the employment of small bottles containing aqueous solution of glycerine with specific gravities varying from 1048 to 1070, increasing the specific gravity in each successive bottle by 2°.

An accurate hydrometer will suffice to determine the specific gravity. Drops of blood from the cholera patient are deposited at the center of the surface of the fluid in the bottles from a capillary pipette. If the specific gravity of the blood is 1062 at least a liter of saline or sodium bicarbonate solution is needed. If 1066 at least two liters. Formerly he estimated the indications by blood pressure considering a pressure of 80 in Europeans or of 70 in natives as indicating intravenous injections.

PRACTICAL APPLICATION OF METHODS OF BLOOD EXAMINATIONS TO THE VARIOUS TROPICAL DISEASES

In considering the value of blood examinations in the various tropical diseases we may first note those in which such examinations are of little or no value and second those in which such examinations are crucial or at any rate of prime importance.

1. IN THE FIRST GROUP WE MAY INCLUDE THE FOLLOWING:

Beriberi.—The leucocytes are about normal in number with possibly a slight increase in lymphocytes. Of course there may be anaemia present with the progress of the disease. Some think there is a slight diminution from the normal percentage of eosinophiles.

Noc found the percentage of lymphocytes in beriberi patients to be about 35 as against 32 for those unaffected.

Sprue.—There is considerable reduction in red cells which may fall below 2,000,000 in advanced cases. The whites may show a slight tendency to leucopenia with a relative increase in lymphocytes. The haemoglobin is not as much reduced as the red cells so that we obtain a color index of from 1.1 to 1.3.

Poikilocytosis and punctate basophilia are often noted, but rarely does one find nucleated reds. In a severe case the blood picture is rather that of an aplastic anaemia than a typical pernicious anaemia. The eosinophiles are rare or absent as the case advances. One often finds many (7-9) nodes in the polymorphonuclears.

Pellagra.—This disease may show a chloranaemia. Some authorities have stated that we have an increase in the percentage of large mononuclears but Hillman found a rather definite increase in the lymphocytes (34%) and a normal large mononuclear percentage.

Yaws.—This disease may show a moderate anaemia with a low color index. The leucocytes are about normal in number with a moderate increase in the percentage of large mononuclears.

Leprosy.—There is, as would be expected, with the progress of the disease, an anaemia which is of the chlorotic type. Leprosy bacilli may be found in the blood, especially during the time of the febrile accessions, but such examinations are of very little value in practical diagnosis and there are so many liabilities to error, as shown in the work with tubercle bacilli in blood, that we should be very conservative in this direction.

There is probably an increase in the percentage of lymphocytes.

Yellow Fever.—The blood findings are usually given as normal although Noc states that at first we have an increase in polymorphonuclear percentage to be followed by an increase in the large mononuclears about the fifth day. He also noted an absence or diminution of eosinophiles.

Intraperitoneal inoculation of animals with blood from patient should be practised. Should the diagnostic reliability of the procedure be established, yellow fever should then be placed in Group 2, among those diseases in which examinations of the blood are of prime importance.

Cholera.—As cyanosis develops the red count goes up even to 8,000,000 with a corresponding or greater increase in the leucocyte count. The estimation of the low blood pressure is important as indicating the necessity for intravenous injections. The determination of the degree of serum acidosis is also indicated with reference to alkaline treatment. In a convalescent from a disease suspected as cholera an agglutination test would be of value, and in the absence of the serum of immunized animals one could use that of a cholera convalescent against a spirillum isolated from the stool of a suspected case of cholera.

2. OF THE DISEASES IN WHICH AN EXAMINATION OF THE BLOOD SHOULD ALWAYS PLAY A PART IN DIAGNOSIS MAY BE NOTED THE FOLLOWING:

Malaria.—The examination of the blood is necessary not only to prove the existence of a malarial infection but, as well, to determine the species of parasite present, this latter a matter of much importance as to prognosis and intensity of treatment according as one has to deal with a benign or malignant parasite. More exact information (and with the expenditure of much less time) can be obtained from a smear stained with some Romanowsky modification than by examining a fresh preparation.

At the same time it is advisable to make a wet preparation and study it for amoeboid activity of the parasites and character of the pigment while awaiting the completion of the staining process.

In the blood of a malarial anaemia the central vacuolation of many of the red cells may give an appearance of young nonpigmented parasites. Malarial parasites tend to move about to take peripheral locations and furthermore they do not change in size upon focussing up and down as do the vacuoles.

Melaniferous leucocytes can be made out better in a fresh specimen than in a dried, stained one.

One can better differentiate species by an even thin film than by a thick-film method. There is often great doubt with a thick film as to whether the object noted is an artifact or a parasite. The Ruge thick-film method has given very good results.

There is only a moderate variation from a normal white count but in cases when the parasites are very scanty or when they have been driven from the peripheral circulation by quinine treatment we may make a tentative diagnosis of malaria on a leucocytosis during the paroxysm with a leucopenia during the afebrile interval with, at this time, an increase in the percentage of large mononuclears to 10 to 15%.

Melaniferous leucocytes are rarely noted in the benign tertian infections but in some of the very puzzling aestivo-autumnal fevers they may give the diagnostic clue.

Schüffner’s dots are yellowish dots in the infected red cells and are characteristic of benign tertian. The Maurer clefts of malignant tertian are less commonly noted. Always carefully note the pale, swollen, infected red cells of benign tertian, the shrunken degenerated cell of malignant tertian and the normal one of quartan. The fine hair-like ring of malignant tertian is often noted on the periphery of the red cell as a narrow line while the half-grown schizont of quartan is often seen as an equatorial band.

In the anaemia following malaria we may have very low red counts and haemoglobin percentages. They usually run parallel, so that the color index approximates 1.

Punctate basophilia is quite common in malarial anaemias. Up to the present time the culturing of the parasite can scarcely be considered an aid to diagnosis as it is difficult to carry the development beyond one generation so that we do not get multiplication of parasites. In cases where confusion exists as to the nature of the species of parasite present culturing would help as regards the possibility of noting the developmental stages of Plasmodium falciparum.

Blackwater Fever.—The same points which hold for malaria hold for blackwater fever.

The striking feature of blackwater, from the side of the blood, is the rapid and great reduction in red cells and haemoglobin. As a result of the pathognomonic haemoglobinuria we may have in a few days a fall of red cells from 4 or 5 million to approximately 1 million with haemoglobin down to 20%. The color index is usually about 1. The blood is thin and the serum tinged. Probably from the excessive haemolysis one does not see degenerated cells as frequently as would be expected. Tests for acidosis and coagulability of the blood are indicated as there is a reduction in titrable alkalinity of the serum and coagulation rate.

Oroya Fever.—This disease, within two or three weeks, gives the blood picture of a marked pernicious anaemia. The rod-shaped protozoon may be seen lying in the red cells singly or in V-shapes.

These rods show a chromatin granule at one extremity. Normoblasts are very numerous and megaloblasts appear later. There is both polychromatophilia and poikilocytosis. The color index is that of pernicious anaemia, above 1. The leucocytes are increased to about 20,000 with 75% of neutrophiles, among which are many immature forms or metamyelocytes. The pathological process shows its greatest activity in the bone marrow.

Malta Fever.—In this disease blood cultures offer the surest and most practical way of making the diagnosis. The blood should be taken from a vein at the time of the height of the fever rise. To prevent coagulation the blood should be forced from the syringe into about an equal amount of citrated salt solution and subsequently added to melted agar to then be poured into Petri dishes. Cultures can also be made by smearing the citrated blood over poured plates of agar.

It must be remembered that the colonies are quite small and do not develop for four or five days.

The citrated blood can also be added to bouillon. The blood culturing has rather replaced the culturing from spleen juice. As the coccus is in the blood it is eliminated in the urine and plates should be made from the urine as well as the blood.

Malta fever is one of the diseases which can be diagnosed quite early by agglutination tests, the reaction often appearing before the end of the first week and often continuing for months after recovery. There is a liability to error when low dilutions are employed so that the former use of dilutions of 1 to 20 and 1 to 40 is no longer advised. Probably a dilution of 1 to 100 would be sufficiently specific but dilutions of 1 to 500 and even higher are frequently obtained. It is now thought best to heat the patient’s serum to 56°C. for twenty minutes before applying the test so as to destroy nonspecific agglutinins. Opsonic index and complement fixation tests have been employed in diagnosis.

As the disease progresses a secondary anaemia develops. The white count is about normal but with the polymorphonuclears somewhat reduced in percentage and the mononuclears increased.

Some observers have reported a leucopenia as of some diagnostic value but others find the leucocyte count normal and Rogers considers the absence of leucopenia as differentiating kala-azar from Malta fever.

Plague.—In septicaemic plague blood cultures offer the surest method of diagnosis as clinically there may be very little to suggest plague. This is about the only disease in which one may find the causative bacterium in a blood smear. For this examination the thick-film method has been recommended. Just as with the material from a puncture of a bubo or the sputum from plague pneumonia we should employ animal inoculation as well as cultural procedures with the blood.

We usually have a marked leucocytosis due to a great increase in the polymorphonuclears. The white count may exceed 50,000. Just as septicaemic plague may so overwhelm the organism that it does not respond with fever so may the leucocytosis be absent. Bubonic and pneumonic plague tend to become septicaemic, so that in such types of the disease we may obtain results with blood cultures.

Liver Abscess.—Schilling-Torgau brings out the point that even with an absence of the usual blood findings it is possible to diagnose the disease and make a just prognosis with his method of differential counting. Ordinarily we have a leucocytosis of from twelve to twenty thousand with only about 70% of polymorphonuclears and about 12 to 15% of large mononuclears. When a bacterial infection accompanies the amoebic one of course the leucocytosis and polymorphonuclear percentage reach higher figures. The eosinophiles may entirely disappear in an uncomplicated case of amoebic abscess.

In comparing his method with the ordinary one Schilling-Torgau notes a case with a differential count showing 72% of polymorphonuclears, 17% of lymphocytes and 8% of large mononuclears with a white count of 6000—apparently a normal blood. By his method 33% of these neutrophiles were found to be of the band-form or less mature cells, thus showing that the blood really did deviate from the normal.

In other examinations he noted very unfavorable indications from the high percentage of metamyelocytes and even myelocytes when the ordinary count did not suggest the serious condition.

As stated previously this method would seem to offer many advantages over the ordinary one.

Trypanosomiasis.—While the blood, when examined in ordinary smears or with thick-film methods, does not give as good results as by examining the gland juice for trypanosomes, yet, by taking 5 or 10 cc. of blood in citrated salt solution with 2 or 3 centrifugalizations, we may obtain greater success in finding the parasites in this way than when using gland juice.

In wet preparations we may note the clumping of the red cells. This is the phenomenon of auto-agglutination thought by some to be rather characteristic of trypanosomiasis.

We may carry out the leucocyte attachment test using the inactivated serum of the suspected patient.

As the disease progresses we get a secondary anaemia. The leucocyte count is usually normal but the differential count shows an increase in the large mononuclears. Bacterial infections often supervene when a leucocytosis will be noted.

Kala-azar.—Quite recently there has been success in the diagnosis of kala-azar by culturing the blood of the suspect on N. N. N. medium. The key to success when culturing from the blood is to wait for two or three weeks before giving up the examination of the cultures. It will be remembered that almost invariably leishman bodies are present in the blood only in extremely small numbers so that there is not time by the end of a few days for sufficient development to have taken place. In probably 80% of cases the parasite of kala-azar may be found in stained smears from the peripheral blood but only after prolonged and patient search. They may be found phagocytized by large mononuclears or polymorphonuclears. Of course splenic puncture examinations show far greater abundance of parasites than blood smears but it is not without danger.

The marked anaemia of kala-azar does not appear until the earlier symptoms of fever and splenic enlargement have gone on for some time. Very characteristic and important in diagnosis, however, is the marked leucopenia of kala-azar, approximating 2000 leucocytes on the average. Again the white cells are only about in the proportion of 1 to 1000 red cells. There is an increase in the percentage of large mononuclears. Some authorities have reported an acidosis of the blood serum. Coagulation rate is delayed.

In kala-azar the coagulability of the serum is altered as shown by the formol-gel test. In this test, a drop of clear serum from the patient is placed on a slide which is then inverted over a watch glass containing a few drops of liquor formaldehyde. In cases of kala-azar the serum will solidify, appearing as an opaque, stiff jelly which adheres to the slide; while other sera will remain fluid, running off the slide when it is tilted. The reaction appears not to be specific since it has been reported for syphilis and other diseases.

Relapsing Fever.—The spirochaetes are not so numerous in the blood of the peripheral circulation in tropical relapsing fevers as in those of Europe.

The spirochaetes can best be seen in stained smears but the agitation of the red cells in a wet preparation by the motile spiral organisms is of assistance in their recognition. Dark-field illumination, India ink smears and Fontana’s silver method are used as well as Giemsa staining.

During the afebrile period the parasites disappear from the peripheral circulation.

If the disease is first seen during the afebrile stage we may try Lowenthal’s reaction, which consists in taking a drop of the blood of the suspected patient, mixing it on a vaseline ringed slide with the blood of a patient showing spirochaetes, then covering with a cover-glass and incubating for thirty minutes at 37°C. A positive reaction shows clumping and loss of motility of the spirochaetes.

Reports vary as to the white count but on the whole there would seem to be more evidence in favor of a moderate leucocytosis although some observers have noted a fall from the normal. The usual statements give a leucocytosis of from 12 to 15 thousand with a polymorphonuclear increase to between 75 and 80%. The statement is usually made that the normal percentage of large mononuclears helps in the differentiation of malaria. Kieseritzky has reported leucopenia and slight increase in lymphocytes.

The leucocyte count tends to be higher about the time of crisis.

Weil’s Disease.—This spirochaete infection is due to Leptospira icterohaemorrhagiae. The spirochaete has been found in the blood and has possibly been cultured anaerobically from the blood. The practical method is by inoculating guinea pigs with blood or urine sediment. Spirochaetes are found in the liver smears of the sick guinea pigs. In the first week of Weil’s disease we have a leucocytosis—later on a leucopenia.

Filariasis.—The sheathed embryos of Filaria bancrofti are found in the peripheral circulation at night only, hence F. nocturna, while those of F. loa are only to be found in the daytime, hence F. diurna. In the islands of the South Pacific the filarial infection is considered as of F. bancrofti but the embryos are present in the peripheral circulation both by day and by night.

Instead of being uncommon it seems rather to be the rule to fail to find embryos in the blood preparations in cases showing marked evidences of filarial disease, as in elephantiasis, calabar swellings, etc. The positive blood findings are most frequent in those who do not as yet show symptoms. There has not yet been sufficient obstruction in the lymphatics to keep the embryos from reaching the blood stream.

In some countries where a large percentage of the population may show embryos in the peripheral circulation, manifestations of the disease are very rare.

We may examine the blood either with fresh preparations, when the movements of the embryos assist in their detection, or by staining dried smears. Haematoxylin staining is better than the Romanowsky one as the break in cells and other points are better brought out.

An eosinophilia is usually considered as constantly present but this is not invariable. The leucocyte count is about normal.

Dengue and Phlebotomus Fever.—In these diseases a leucopenia, which begins to show itself by the second day, is very characteristic.

The average leucocyte count is about 3500 and along with this we have a reduction in the percentage of polymorphonuclears to about 50%. Towards the end of the terminal fever we have an increase in the percentage of large mononuclears.

Bacillary Dysentery.—The agglutination tests are of little value in diagnosing the presence of or type of an infection with dysentery bacilli, as the agglutinating power does not appear until during convalescence.

It is now customary to use a polyvalent antidysenteric serum in treatment so that it is not very essential to ascertain the strain involved in an infection. As a practical matter we make our diagnosis of the presence as well as type of dysentery bacillus involved in an infection by isolating the organism from the dysenteric stool.

During the fever we may have a moderate polymorphonuclear leucocytosis.

Enteric Group of Fevers.—In fevers of atypical course in the tropics one must always remember that typhoid and the paratyphoid fevers are anything but uncommon and blood cultures should always be made when such suspicion arises. In some tropical regions paratyphoid A infections seem most common although the usual experience is to encounter the paratyphoid B infection more frequently. In temperate climates the noting of a moderate leucopenia with an absence of eosinophiles is important in the diagnosis of typhoid, but in the tropics there are so many intestinal parasites and skin infections productive of eosinophilia that we cannot attach any importance to such a finding.

Typhus Fever.—Plotz attaches importance to the culturing of B. typhi exanthematici from the blood of typhus cases, but the relationship is now regarded as not causal. Rickettsia bodies, which can be demonstrated in the louse or in capillaries at autopsy, are now considered to be the exciting organism.

A mononuclear leucocytosis has at times been reported.

Spotted Fever of the Rocky Mountains.—Injection of the blood of the patient into guinea pigs produces the disease in the animal. Frick has reported the finding of chromatin-staining bodies in the red cells of such pigs and Wolbach has found chromatin-staining bacteria in the endothelial cells of such animals. These bodies are now classed as Rickettsia.

These findings cannot as yet be considered of diagnostic value.

Various Helminthological Infections.—In the earlier stages of ancylostomiasis and schistosomiasis we have a rather notable increase in the percentage of eosinophiles but with the advanced stages of these infections, with severe anaemia, the eosinophiles may even be absent.

One should always keep in mind the very characteristic and marked eosinophilia of trichinosis when such a blood finding is encountered. There is often a leucocytosis of 15,000 to 20,000 in this disease.

In the urticarial fever stage of Japanese schistosomiasis the marked eosinophilia is of great assistance in diagnosis. One trouble about attaching importance to eosinophilia in the tropics is the confusion which is difficult to eliminate and which arises from infections with the more common but less important group of intestinal parasites such as Ascaris, Trichuris, etc.

The eosinophilia-producing characteristics of many skin diseases must also be kept in mind.

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Chapter XLVI

THE CIRCULATORY, RESPIRATORY AND LYMPHATIC SYSTEMS TOGETHER WITH ANAEMIA, HAEMORRHAGES AND OEDEMA IN TROPICAL DISEASES

THE CIRCULATORY SYSTEM

Beriberi.—Almost as important in diagnosis as the weakness of the legs, with anaesthetic and oedematous areas, is the early palpitation of the heart upon the slightest exertion. Later on as the vagal degeneration becomes more prominent we have a loss of the normal cardiac rhythm, to even become embryocardial, together with dilatation of the right heart, pulsating jugulars and various blowing murmurs, which are propagated into the vessels of the neck. The pulse is weak and rapid and this combination of a tumultuous heart action and weak pulse is striking. Blood pressure is below normal.

Cardiac involvement is also a feature of ship beriberi as well as infantile beriberi. In the latter a marked hypertrophy of the right heart is characteristic.

In yellow fever we have at first a high blood pressure. The pulse rate, which at first corresponds with the rise of temperature, soon shows Faget’s law—a falling pulse with a constant temperature or a constant pulse with a rising temperature. It is a markedly slow pulse after the third day. The blood pressure is low in the asthenic stage.

In dengue we do not have the rise in blood pressure but the slow pulse is quite a feature of many of the dengue-like fevers.

In blackwater fever the pulse is rapid and soon becomes weak and of low tension.

Plague shows a striking toxic action on the heart muscle so that we soon get a soft, dicrotic pulse, rapid from the first and soon becoming thready. Patients with plague may die from cardiac failure upon getting up from bed.

A rapid pulse, especially in the morning, is thought to be a feature of active leprosy.

In cholera the pulse is rapid and feeble during the stage of evacuation and with the onset of the algid stage we practically have a cessation of the circulation. The systolic pressure may fall as low as 65 or 70 mm.

Hookworm anaemia shows early and marked cardiac palpitation. The pulse rate averages about 110 and the blood pressure is low. There is often some right-side dilatation of the heart.

Malaria generally gives a small, rapid, high tension pulse in the cold stage to become full and bounding in the hot stage. A cardiac type of pernicious malarial fever has been described, particularly by the French.

Both Malta fever and bacillary dysentery tend to have a toxic effect on the heart.

Typhus fever is a disease which tends markedly to affect the heart. Along with faint heart sounds we have a rapid, low tension pulse. In bacillary dysentery the tendency to an increase in pulse rate is of some value in differentiating it from amoebic dysentery.

African trypanosomiasis shows a rapid pulse rate whether the case shows temperature or not. In Brazilian trypanosomiasis the parasites may tend to invade the cells of the heart muscle thus producing manifestations of myocardial disease. The parasite (Schizotrypanum cruzi) may also affect the adrenals, causing a low blood pressure along with other signs of Addison’s disease.

THE RESPIRATORY SYSTEM

Sputum Examination.—We should make a routine of examining a fresh specimen of sputum as well as stained smears. It is in such a specimen we search for the ova of the lung fluke.

Frequently the material submitted for examination as sputum is simply buccal or pharyngeal secretion, or more probably secretion from the nasopharynx, which has been secured by hawking. It should always be insisted upon that the sputum be raised by a true pulmonary coughing act, and not expelled with the hacking cough so frequently associated with an elongated uvula. When there is an effort to deceive, some information may be obtained from the watery, stringy mucoid character of the buccopharyngeal material and also from the presence of mosaic-like groups of flat epithelial cells (often packed with bacteria).

The pulmonary secretion is either frothy mucus or mucopurulent material, and if the cells are alveolar they greatly resemble the plasma cells. At times these cells may contain blood-pigment granules (heart-disease cells).

In the microscopic examination a small, cheesy particle, the size of a pin head, should be selected. This should be flattened out in a thin layer between the slide and cover-glass and should be examined for elastic tissue, heart-disease cells, eggs of animal parasites, amoebae, and fungi. Echinococcus hooklets, Curschman spirals besprinkled with Charcot-Leyden crystals, and haematoidin and fatty acid crystals may also be observed.

Curschman spirals indicate bronchial as against cardiac or uraemic asthma. Charcot-Leyden crystals have no special significance, except in certain tropical diseases when these crystals often are present in paragonomiasis sputum and in the pus of amoebic liver abscesses discharging by way of the lungs.

It may facilitate the examination of the sputum for elastic tissue and actinomycosis and other fungi to add 10% sodium hydrate to the preparation.

To make smears for staining, the sputum should be poured on a flat surface, preferably a Petri dish, and a bit of mucopurulent material selected with forceps. A dark background facilitates picking out the particle. A toothpick is well adapted to smearing out such material on a slide. After using the toothpick it can be burned. When dry, the smear is best fixed by pouring a few drops of alcohol on the slide, allowing this to run over the surface, and then, after dashing off the excess of alcohol, to ignite that remaining on the film in the flame and allow to burn out.

In beriberi we have shortness of breath with the early cardiac palpitation. In acute pernicious beriberi the pulmonary congestion and oedema divide with the heart the terrible manifestations of such an attack. The diaphragm may become paralyzed in beriberi. Some authors refer to the dyspnoea of beriberi as the beriberic corset.

Paragonomiasis gives rise to a chronic cough attended with the expectoration of more or less bloody sputum containing ova. Haemoptysis is not infrequent. The physical signs on percussion are slight but may be more marked on auscultation.

Hirudiniasis.—In Northern Africa, as well as in many islands of the Orient, the drinking water of ponds may contain leeches and these water-leeches tend to attach themselves to the pharyngeal mucosa. They may also attach themselves to the tissues about the larynx. In these cases we not only have cough and haemoptysis but dyspnoea from laryngeal oedema. It is probable that cases of dyspnoea called halzoun, and due to the attachment in the region of the larynx of flukes (Fasciola hepatica), as the result of eating raw liver, may often be due to leeches, as the two affections occur in the same regions.

Plague pneumonia is characterized by profound prostration in a patient whose physical signs do not seem to justify such extreme illness. The rather abundant and watery sputum soon becomes sanguinolent. Herpes labialis is absent. Besides primary plague pneumonia which develops directly from contact with a former case we have a secondary pneumonia which develops in the course of a typical case of bubonic plague.

In malaria we have a slight bronchitis in the ordinary types and many recognize a pulmonary type of pernicious malaria.

Malta fever tends to show a bronchial involvement about the twelfth day of the disease. Crepitant râles, a moderate cough and slight dyspnoea may be noted. It was the presence of pulmonary signs along with the profuse sweating and anaemia of the disease that justified the designation Mediterranean phthisis.

In liver abscess the crepitation at the base of the right lung, following congestion incident to the abscess of the right lobe of the liver, is of value in diagnosis. Rupture of a liver abscess into the lung occurs in about 10% of cases.

In heat stroke we may have Cheyne-Stokes respiration and pulmonary oedema.

Japanese river fever often shows bronchial involvement and cough at the time of the height of the fever.

In ancylostomiasis cases with cough and bronchitis have been reported and it seems probable that such manifestations may be connected with the course of the larvae through the pulmonary passages to reach the intestinal tract.

The filarial embryos of F. bancrofti remain in the lung capillaries during the day and recently such embryos have been found in blood coughed up from the lungs.

Katayama disease may show a localized bronchitis early in the attack and from its rapid appearance and disappearance would seem to be a sort of patchy pulmonary oedema. This is connected with the passage of the larvae through the lungs.

Broncho-pneumonia is probably the most common complication of typhus fever.

An affection known as gangosa or rhino-pharyngitis mutilans causes great tissue loss about nasal and buccal cavity. The voice has a peculiar nasal quality. It is possibly a manifestation of tertiary yaws.

Kala-azar patients are often carried off by a terminal pneumonia probably connected with the leucopenia and marked diminution of polymorphonuclears.

In leprosy, also, the victims are frequently carried off by pulmonary tuberculosis.

Relapsing Fever.—In relapsing fever there is frequently a moderate bronchitis at the time of the first febrile accession.

Bronchial Spirochaetosis.—There is a condition which more or less resembles lobar pneumonia, even to rusty sputum, but without signs of consolidation, and with negative Roentgenograms, when we find spirochaetes in the sputum. Another type of bronchial spirochaetosis is when the clinical picture is more that of pulmonary tuberculosis. There is question whether these spirochaetes are causative or only accidental.

Cases have been reported where a phthisis-like condition was due to a mould infection (Monilia). While such a condition may be primary it is more often secondary in cachexias as may be the case with buccal Monilia infections (thrush) which occur in the victims of cachectic states.

Guha.—In Guam there is also a rather fatal capillary bronchitis affecting young children which goes under the name of epidemic asthma or, as termed by the natives, guha. This affection comes on during the rainy season and is attended with marked dyspnoea and slight elevation of temperature.

Nasal Myiasis.—In the tropical and subtropical parts of North and South America a fly, Chrysomyia macellaria, is apt to deposit its eggs about the nasal orifices of persons with an offensive discharge from the nose. The fly seems to be attracted by foul odors. The larvae which develop are called “screw-worms” on account of the segmental bands of bristles and tend to invade the various sinuses, causing great destruction of tissue.

The case sets in with signs of a very severe coryza, together with fever and marked frontal headache. The face becomes swollen, red, and tender in the region of the nose. As the larvae reach maturity they come out of the nose. A nasal douche of 15 parts chloroform in 100 parts milk is often efficacious in bringing away the larvae. At times Sacrophaga larvae may be found.

THE LYMPHATIC SYSTEM

Plague.—The buboes are the most characteristic feature of the more common form of plague, bubonic plague. There may also be slight enlargement and tenderness of the glands in septicaemic and pneumonic plague but many such cases fail to show any evidence of superficial glandular enlargement. In pestis minor the only feature suggestive of plague is the glandular enlargement.

Very characteristic of the glandular involvement in plague is the marked tenderness of such glands. The slight pressure of palpation causes some pain and a sharp punch over an affected gland, excruciating pain. So exquisitely painful are these buboes that the patient with groin or axillary buboes will flex the leg or extend the arm to relieve pressure. In about 70% of cases the bubo is located in the groin, with 15% to 20% for axillary involvement and 5% to 10% for the submaxillary or cervical region. There may be involvement of both deep and superficial glands of a region, such buboes giving a large area of induration. As a rule there is a single bubo. The bubo is formed not only by the glands but by a periglandular oedema which fuses the glands into a solid mass. The buboes tend to suppurate about the commencement of the second week, so that gland puncture with subsequent culturing for plague bacilli and animal inoculation should be carried out before this time as pyogenic organisms replace the plague bacilli upon suppuration taking place.

Trypanosomiasis.—One of the characteristics of the disease recognized as diagnostic more than 100 years ago is enlargement of the glands of the posterior cervical triangle (Winterbottom’s sign).

There may be general enlargement of the lymphatic glands which are rather hard, discrete and not bound down to the overlying skin. These glands may be somewhat tender or entirely painless. One of the most valuable methods of diagnosis of trypanosomiasis is by gland puncture, the juice obtained therefrom being examined in smear or inoculated into a monkey or guinea pig. Brazilian trypanosomiasis also shows glandular involvement.

Filariasis.—Varicose groin glands are frequently associated with lymph scrotum, chylocele or chyluria. The glandular masses are soft and doughy. The consistency is often that of a lipoma.

The overlying skin slips over the glandular mass. These glands are often mistaken for inguinal hernia. They do not give a tympanitic note and disappear slowly upon firm pressure with the patient lying down but return even with the pressure maintained upon assuming the upright position. There is no impulse on coughing. If a sterile hypodermic needle be inserted into the mass a chylous fluid slowly and persistently comes out of the needle drop by drop and this material may show filarial embryos.

The filarial worms Onchocerca volvulus obstruct the lymphatics and may give rise to swellings of considerable size along the course of the lymphatics.

Climatic Bubo.—The onset is gradual often accompanied by a low remittent type of fever. There is an absence of venereal sore.

These glands are only slightly tender and are often called fatigue glands as they produce a feeling of weariness after even moderate exercise. The inguinal glands of one or both sides are the ones involved but the overlying skin does not show the redness of a chancroidal or gonorrhoeal bubo. There is often a softening in the center of the affected glands.

Tsutsugamushi.—The glands which drain the area in which is located the ulcer at the site of the bite of the Kedani mite show swelling and tenderness.

Rat bite fever also shows glandular enlargement in the glands tributary to the healed infecting bite of the rat.

In tularaemia the lymph glands draining the site of the infecting bite become inflamed and swollen, often suppurating.

In leprosy the glands draining involved regions become enlarged but do not show a tendency to suppuration. The glands most frequently involved are the cervical and groin glands.

In kala-azar the recommendation has lately been made to excise the somewhat enlarged glands and make smears from a piece of such gland and then examine the smear for leishman bodies. Gland puncture has not given as satisfactory results.

It is often stated that the superficial cervical glands are enlarged in dengue but not in dengue-like fevers. I have not observed in the cases I have seen either constant or well marked glandular enlargements.

In yaws there may be glandular enlargement. According to Finucane the cervical glands are often involved in Fiji children. These glands do not tend to break down.

In pediculosis of the hairy scalp the scratching back of the neck may result in pus infection with enlargement of the tributary cervical glands.

American leishmaniasis.—Not only is there often enlargement of the lymphatic glands but likewise we may have lymphangitis lines leading from the ulcer to the glands. The glands may be large and painful and may remain enlarged after the recovery of the patient.

ANAEMIA

The old idea that tropical life produced an anaemia is no longer held, the view now being that such anaemic conditions are almost invariably due to some well recognized cause, the most important of which is malaria. Natives of the tropics may appear bleached out but show a normal red count and haemoglobin percentage. Chamberlain’s observations have shown that a residence in the tropics of approximately two years has no appreciable influence on the red cell count or haemoglobin content of the blood of white men and that the actinic rays do not seem to be operative in producing anaemia.

Malarial Cachexia.—Although the malignant tertian infection has the greatest tendency to produce anaemia yet any type may, when untreated, bring about the more or less profound anaemia with earthy skin, enlarged spleen, dyspnoea on slight exertion, and oedema of the ankles characteristic of malarial cachexia.

Oroya Fever.—In this disease we have what might well be termed a fulminating pernicious anaemia. The rod-shaped protozoon which attacks the red cells seems to be peculiarly active in the bone marrow, excruciating bone pains being quite a feature of the disease.

There may be a reduction in red cells to one million per c.mm. within a few days. Normoblasts are abundant and megaloblasts may be observed in the more severe cases. The anaemia is intense and 20% to 40% of cases die within two or three weeks. A severe anaemia in which the blood picture is that of pernicious anaemia may accompany infections with the Balantidium coli.

Blackwater fever may produce a fall in red cells almost as marked as in Oroya fever.

Sprue shows a slowly progressive anaemia which in the later stages of the disease may become extreme, going down to one million, with a fairly high color index.

Ancylostomiasis is along with malaria the disease to be first thought of in connection with anaemia. The splenic enlargement of malaria should be thought of, although the view has recently been advanced that the spleen may be enlarged in hookworm disease.

In advanced cases of hookworm disease, showing a picture of profound anaemia, there may be so few worms present that the method of making diagnosis by finding ova may be unsuccessful. I have seen a case of typical aplastic pernicious anaemia, confirmed by autopsy, undoubtedly following a vicious cycle set up by the hookworm infection, in which scarcely a worm was to be found in searching the intestines.

Kala-azar gives a marked anaemia with an earthy color of the skin. The leucopenia and splenic enlargement are characteristic and the finding of parasites confirmatory.

Malta fever is usually followed by a moderate anaemia.

The helminthic infections, besides hookworm disease, are always to be thought of in the presence of anaemia. Very important among these are rectal and vesical schistosomiasis as well as that from the Japanese schistosome, together with liver and lung fluke disease. Even the ordinary round-worm, Ascaris lumbricoides, is to be thought of in a tropical anaemia.

Cases of anaemia, in which no other demonstrable cause has been noted, have been thought to be due to trichocephaliasis.

Tropical dysenteries are often responsible for anaemia and in liver abscess the patient becomes quite earthy in color, provided no operation is performed. In chyluria there is a marked drain on the patient.

The anaemia in liver abscess is not so great as the muddy complexion would indicate. The emaciation is greater than the anaemia.

HAEMORRHAGES

The loss of blood through haemoglobinuria and haematuria has been taken up under the urine. The haemoglobinuria is the pathognomonic symptom of blackwater fever. There is also recognized a haemorrhagic form of pernicious malaria with epistaxis and alimentary tract haemorrhages. Moderate haemoglobinuria may be found in severe cases of malignant tertian infections.

Yellow Fever.—During the asthenic period of the disease, which sets in about the fourth day, we have, as a result of the damage to the endothelial lining of the capillaries, various haemorrhages.

Of these the best known and most dreaded is that from the stomach, black vomit. The bleeding from the gums is apt to appear before that from the stomach. Not only may bleeding occur from the intestines but from any mucosa, as that of the nose, conjunctiva or vagina.

In vesical and rectal bilharziasis the perforation of the terminal branches of the portal vein by the terminal or lateral spined eggs gives rise to haemorrhages.

In dengue we may have an epistaxis at the time of the crisis of the first febrile paroxysm.

In dysentery the blood-admixed mucous stools are of diagnostic importance.

In endemic haemoptysis the operculated eggs of Paragonimus westermanni are to be sought for in the sputum.

In leprosy epistaxis may be an early sign.

The damage to the endothelial lining of capillaries in plague gives rise to frequent haemorrhages into the skin.

There is a question whether the hookworms abstract blood from the intestines, although tests for occult blood are deemed important by some authorities in the diagnosis of this disease.

The granulomatous lesions of verruga are markedly haemorrhagic.

Some consider ship beriberi to be of the nature of scurvy in which case one should have in mind spongy, bleeding gums and the intramuscular haemorrhages of scurvy.

Typhus Fever.—The petechial rash of this disease (mulberry rash) is a distinctive feature.

OEDEMA

Oedema, especially about the ankles, is to be looked for in all the secondary anaemias of the tropics, particularly malaria and ancylostomiasis.

Beriberi.—The oedema begins at first about the feet, especially about the dorsal junction of phalanges and metatarsus. It is characteristically pretibial. It may remain confined to the shin or go up to knees, scrotum, sternal region or trunk. It is generally symmetrical but may be unilateral. It may become a general anasarca, even in forty-eight hours.

The swelling of the face is at times enormous, the eyelids being so oedematous that the patient can see only by separating them with the fingers. The oedema is more solid than that of nephritis. It not only rapidly appears but disappears as rapidly.

The oedema of beriberi may involve the glottis (oedema of glottis).

Oedema of genital regions is less marked than in nephritis or cardiac disease. We may also have localized areas of oedema 3 or 4 inches in diameter.

Ship beriberi, which has points in common with both beriberi and scurvy, shows oedema which may be limited to the lower extremities or generalized. Epidemic dropsy is a type of beriberi in which there are fever and an erythema over the dropsical areas.

Calabar Swellings.—These seem connected with infections with Filaria loa. The swellings originate suddenly and disappear in three or four days. They are hard and do not pit on pressure. These smooth swellings, often 2 to 4 inches in extent, are most often seen on arms, face or ankles.

In trypanosomiasis oedema of the face and especially of the eyelids may be striking. There may also be patches of oedema elsewhere.

In Katayama disease the urticarial areas of oedema have given it the name of urticarial fever.

A peculiar disease of North China, known as atriplicism, is caused by the eating by the very poor of a weed, Atriplex, common around Pekin. There is itching of the fingers, quickly followed by swelling. This tends to extend to the back of the hands and up the outer surface of the forearm. The face becomes so swollen that the eyelids may be closed.

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XLVII. JAUNDICE AND THE LIVER AND SPLEEN IN TROPICAL DISEASES

JAUNDICE

Although the appearance of jaundice immediately suggests a disease of the liver yet as a matter of fact those diseases of the tropics in which the liver involvement is the sole or chief feature rarely show marked jaundice.

In tropical hepatitis or congestion of the liver or, as it is often termed, tropical liver, there is rarely a distinct jaundice and if such occur it is only temporary. Such terms as earthy, muddy, sallow, sub-icteric or pale lemon tint are more often applied than jaundice.

Liver abscess rarely gives rise to a definite jaundice unless the abscess be so situated as to cause pressure on the bile ducts.

In clonorchiosis, or the liver fluke disease of man, jaundice is not a feature of the disease except in the very late stages.

In those liver cirrhoses associated with Katayama disease, malaria or kala-azar there is no typical jaundice.

The tropical diseases in which jaundice is an important diagnostic feature are yellow fever, blackwater fever, bilious remittent fever and relapsing fever.

Yellow Fever.—There are cases which succumb without having shown jaundice but immediately following death the yellowish discoloration has been noted. At autopsy the yellow fever cadaver is almost invariably deeply jaundiced.

Very important is the fact that the jaundice of yellow fever does not appear until late, about the third or fourth day. When jaundice appears earlier, as by the second day, the prognosis is almost surely a fatal one.

According to Dutroulau the designation red fever would be more appropriate for the deeply congested facies of a yellow fever case in its first days.

The icterus is more marked about the face, neck and upper parts of the trunk. The albuminuria precedes the jaundice.

Blackwater Fever.—In a typical case of this disease we have within a few hours a marked jaundice which tends to deepen. It is usually more or less marked according as the haemoglobinuria may be. It does not show the tendency to persist as does the jaundice of yellow fever.

Bilious Remittent Fever.—The jaundice appears rather earlier than that of yellow fever but is rarely seen on the first day of the paroxysm as with blackwater fever.

Of great diagnostic value is the early appearance of bile-colored urine as different from the haemoglobin-tinged urine of blackwater. The albuminous urine of yellow fever is not apt to show any bile coloring in the first three or four days of the disease.

Relapsing Fever.—There is a clinical type of relapsing fever associated with jaundice and a high death rate which was first described by Griesinger from Egypt. This icteric type is not infrequent in Asia. This jaundice is late and the disease much resembles yellow fever. The enlarged painful spleen and the finding of spirochaetes in the peripheral circulation are essential to differentiation.

Weil’s disease.—Much interest has been recently aroused in Weil’s disease, or epidemic jaundice, on account of the frequency of the disease in soldiers in the Balkan campaign. While a spirochaete has been shown by Inada and others to be the cause, yet many workers have isolated paratyphoid B organisms from the blood of such cases. Frugoni obtained cultures of this organism from the duodenal fluid of 11 cases from 48 cases investigated. The accepted cause of true Weil’s disease is Leptospira icterohaemorrhagiae. The jaundice begins about the third day of an irregular fever. Like yellow fever these cases showed injection of the conjunctivae and albuminuria. There were, however, usually a leucocytosis and enlarged spleen.

In severe cases of spotted fever of the Rocky Mountains we may have a generalized jaundice. Rarely cases of typhus fever may show jaundice.

ALTERATIONS IN SIZE OF THE LIVER

There is only a slight enlargement in the ordinary case of tropical liver but in some cases it may extend 3 or 4 fingers’ breadth below the costal cartilages or rarely to the umbilicus.

In liver abscess the enlargement is a rather late feature, and the condition should be diagnosed before we have the assistance of protruding ribs and distention of the intercostal spaces. As the abscess is usually located in the upper portion of the right lobe the enlargement is usually upwards and is best made out with the X-ray, showing the cupola-like projection.

In kala-azar the liver does not begin to enlarge until after about three months from the time of onset at which time the spleen will be quite large. Decided enlargement is generally noted by the sixth month.

The liver cirrhoses due to schistosomiasis or malaria may show slight enlargement or no particular change.

Sprue is a disease which gives a decided atrophy of the liver. Some authorities have noted a decrease of the size of the liver in cholera. The liver of yellow fever is of normal size and is not associated with splenic enlargement.

In the tropics one must always keep in mind the possibility of a liver enlargement being due to syphilis.

PAINS OF THE LIVER

In tropical liver there is more a sensation of weight than one of pain. In liver abscess, however, there are painful dragging sensations and, at times, with abscess of the upper right lobe, pain referred to the right shoulder. There is a marked tendency to splint the liver as shown by the costal breathing and moderate rigidity of the right rectus. The patient tends to lie on his back as shifting to either side, especially the left, causes pain. The legs are drawn up to relieve tension. Any jolting of the liver in palpation is exquisitely painful.

When active suppuration is going on in the liver the pain may be of an acute throbbing character. With abscess of the left lobe the pains may suggest gastric disease while with an abscess of the concave surface of the liver there may be referred pain in the appendix region.

In blackwater fever and bilious remittent fever there may be tenderness of the liver as well as the more prominent pain in the spleen.

Epidemic jaundice shows tenderness of the liver.

In plague there is a marked congestion of the liver as of other viscera and there may be some tenderness.

The liver becomes tender as well as showing enlargement in infections with Clonorchis.

SPLENIC ENLARGEMENTS AND PAINS

Splenic puncture has been carried out for diagnosis chiefly in kala-azar, malaria and Malta fever. Some authorities have reported fatalities from spleen puncture in kala-azar approximating 1% so that many advise the safer liver puncture to that of the spleen.

Spleen puncture would only exceptionally be called for in malaria as there is usually no difficulty in making the diagnosis from a blood smear. Malta fever can usually best be diagnosed by blood culture taken at the height of fever and recent work by Wenyon and others would indicate that blood cultures on N. N. N. medium might take the place of spleen puncture in kala-azar.

Kala-azar.—The splenic enlargement is the most conspicuous change in the disease, the spleen often reaching the umbilicus by the third month and later possibly filling up the entire left side of the abdomen. The coincident emaciation of the patient makes the splenic tumor more apparent. When first enlarging the spleen may be the source of considerable pain and tenderness.

Fluctuations in the size of the spleen have been noted in the course of the disease, diminution in size often attending severe diarrhoeal attacks. In spleen or liver puncture the needle must be dry so that the parasites shall not suffer distortion.

Malaria.—Splenic enlargement and tenderness are important points in diagnosis of malaria.

In acute malignant tertian infections the spleen is often diffluent so that it is liable to rupture upon slight injury. One should even exercise care not to palpate the spleen too violently and the possibility of accident should be thought of in making a spleen puncture.

The typical malaria spleen is the ague cake of malarial cachexia. Here we have a greatly enlarged spleen with a thickened capsule and firm consistence. This spleen may fill up one side of the abdomen.

Malta Fever.—The splenic enlargement in this disease usually corresponds about to that of typhoid fever. At times, however, the size may be so great as even to suggest kala-azar.

Relapsing Fever.—Splenic enlargement and tenderness are marked features in this disease, often being noted early in the course.

Blackwater Fever.—The spleen is painful and enlarged. The splenic enlargement in this disease and relapsing fever is important in differential diagnosis from yellow fever, a disease in which the spleen is unaffected.

The spleen may be enlarged in Japanese schistosomiasis as well as in rectal schistosomiasis.

Darling has recently noted that it may be difficult to differentiate the anaemia of malarial cachexia from that due to hookworm disease. As a matter of fact most authorities note a diminution in the size of the spleen in ancylostomiasis rather than an increase and splenic enlargement would certainly favor a diagnosis of malarial anaemia.

One point of distinction between spotted fever of the Rocky Mountains and typhus fever is that the spleen of the former disease is enlarged three or four times the normal, while that of typhus fever shows no increase in size. The palpable spleen of Rocky Mountain fever is firm instead of soft as with typhus fever.

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XLVIII. THE CUTANEOUS SYSTEM AND THE ORGANS OF THE SPECIAL SENSES

THE SKIN

Ringworm infections of the skin are so common in the tropics that one should always make an examination for the causative fungi when doubt as to the nature of the lesion exists. Another point is that many hyperaemias, incident to other diseases, seem to furnish a favorable soil for fungi; thus, not infrequently I have found abundant spores and mycelial structures in scrapings from the erythema of the early syphilitic secondaries. Again pruritic lesions may become infected with fungi as the result of scratching, which scratching may not only have this result but furthermore may obscure the dermatological characteristics of the primary disease.

The most expeditious way to examine for fungi is to treat the scales or hairs with a 10% solution of caustic potash or soda. Then crush between two slides; heat moderately over the flame and examine after from 10 to 30 minutes.

A very satisfactory method is to scrape the scales with a small scalpel, and smear out the material so obtained in a loopful of white of egg or blood serum on a glass slide. By scraping vigorously the serum may be obtained from the patient. After the smear has dried, treat it with alcohol and ether to get rid of the fat. It may then be stained with Wright’s stain or by Gram’s method. The ordinary Gram method may be used or the decolorizing may be done with aniline oil, observing the decolorization under the low power of the microscope.

Hanging-block cultures.—To make these, pour melted agar in a Petri dish and cut from the film so formed sections 1 cm. square. Place a section on a slide, inoculate and drop on a cover-slip. Another method is to allow a drop of melted agar, previously inoculated, to spread over a cover-slip which is then inverted on a concave slide.

Prickly heat is another condition extremely common in the tropics and the scratching to relieve the itching often leads to infection with fungi or pyogenic cocci.

Pellagra.—In no other general disease is the skin eruption of such importance in diagnosis and it is practically impossible to make a sure diagnosis of pellagra in the absence of an eruption or the history of an eruption.

The eruption tends to show itself in the spring but may first appear in the early fall. The lesions resemble a sunburn and burn instead of itch. The characteristics of the eruption are bilateral symmetry and sharp delimitation from the sound skin.

As a rule the lesions are dry and atrophic but more rarely, and usually in severe cases, the eruption may be moist and oedematous.

The backs of the hands are the most common sites for the eruption but frequently there is an extension up the forearm. The neck, the bridge and alae of the nose, the region back of the ears and the front of the chest are often involved. In children the feet and legs are frequently involved. Scrotal eruptions are early manifestations.

Leprosy.—In nodular leprosy we have the appearance of macules of greatly varying size and shape with a tendency sooner or later to symmetry. They tend to appear and recur in association with febrile accessions and, even when they have become permanent spots, they show increased redness, infiltration and tension when there is fever.

The color is rather that of a sunburn and may be uniform or the center may be pale with copper-colored periphery. These spots appear by preference on face, backs of hands, buttocks, extensor surfaces of extremities and back. They may mark the location of later developing nodules. At first they are oily rather than scaly. We soon note a disappearance of hair within the spot. These spots soon tend to become anaesthetic. The tubercles of leprosy are usually of a reddish-brown color.

In nerve leprosy the spots tend to appear on parts of the body usually covered by clothing, as scapular region, shoulders, arms, thighs or buttocks. The outline is ovoid rather than round and the spots may at first be hyperaesthetic rather than anaesthetic, as they later tend to become.

In circinate eruptions we often note a pale center with brownish-red borders. These borders may be hyperaesthetic while the centers show anesthesia. Bilateral symmetry is more common in this than in nodular leprosy.

Besides the spots nerve leprosy may show blister-like lesions on backs of hands and feet especially in the region of the knuckles. Ulceration may follow.

Malaria.—The most common cutaneous manifestation of malaria is herpes labialis. This is more common in benign types than in malignant ones. Urticaria is next in frequency. Malaria has seemed to be the cause of certain cases of purpura simplex.

In attributing skin manifestations to malaria one must always have in mind the scarlatiniform, urticarial and erythematous rashes due to quinine used in treatment.

Urticarial Fever.—In Japanese schistosomiasis the earliest symptoms are the urticarial rash and fever.

Plague.—Rarely cases of bubonic plague may show a small vesicle marking the site of the flea bite. Areas of necrosis of the skin, which are really sloughing patches, and incorrectly designated “carbuncles,” may be noted, especially over the site of the buboes.

In the later stages haemorrhages into the skin (petechiae) are common.

Trypanosomiasis.—Patchy areas of erythema are often noted in Europeans affected with this disease. These are frequently circinate with fading in the center and tend to appear on the trunk.

In natives a dry skin is more often noted.

Rat Bite Disease.—An eruption of purplish spots may accompany the fever. There is a resemblance to erythema multiforme.

Dengue.—The true eruption of dengue is the one that appears about the fourth or fifth day as a measles-like eruption, starting about wrists or ankles.

Kala-azar.—There is a darkening of the colored skin of the natives suffering from this disease and it is to this feature that the disease owes its name.

In Europeans the appearance is more that one sees in old malarial cachectics, an earthy gray color. The characteristics of cutaneous leishmaniasis are discussed under that heading.

Typhus Fever.—Gangrene is particularly a feature of spotted fever of the Rocky Mountains and typhus fever, chiefly of the scrotum and prepuce with the former and of the extremities in the latter.

The distinctions of the eruptions of these two diseases and of tsutsugamushi are given on page 445.

Tsutsugamushi.—A small necrotic ulcer with a dusky red areola, often located in the armpits or region of the genitals, marks the site of the bite of the infecting mite. From it a lymphangitis leads to the swollen glands. About the seventh day a macular eruption, which never becomes petechial, appears on face, then on trunk and extremities.

Tularaemia.—There is often a local lesion at the site of the bite of the infecting Chrysops. The tributary glands are swollen.

In ancylostomiasis the site of entrance of the infecting stage of the larvae is marked by a dermatitis—ground itch.

Tibial ulcers are also features of this disease.

In filariasis we not only have the bleb-like lesion of guinea worm infection but also the calabar swellings of Filaria loa. Elephantiasis and lymph scrotum are the best known skin manifestations of F. bancrofti, but there may also be present filarial abscesses. The tumors of O. volvulus are most often on sides of chest, are quite superficial with the skin freely movable over them.

Epidemic Dropsy.—It is a question, whether such a disease as epidemic dropsy is distinct from beriberi. An erythematous eruption about the face and a macular one of the trunk and extremities are usually stated to be features of this disease.

Juxta-articular Nodes.—This is a condition in which small tumors form under the skin especially in the region of the elbows. These bean to walnut-sized tumors of the subcutaneous tissues may also be noted about the knees. A fungus has been reported as cause but the present view is that the cause is unknown.

Oriental Sore.—This form of cutaneous leishmaniasis is especially common in Asiatic Turkey and Northern Africa. It begins as a small papule which eventually ulcerates, the sore scabbing over from time to time and again breaking down. Indolent granulations and a very protracted course are rather characteristic features.

American Leishmaniasis.—The most important point of differentiation of this form of leishmaniasis from oriental sore is the occurrence of ulcerating lesions of the mucous membranes of mouth or nose subsequent to the appearance of the oriental sore-like lesions on forearm, legs, trunk, or rarely the face. In Peru the term uta more properly belongs to the skin affections while espundia is the designation applied to the lesions of the mucous membranes. It may be stated that a form of oriental sore has been reported from Greece where mucous membrane ulcerations have been associated with the ordinary skin-type lesion.

Dermal Leishmanoid.—Brahmachari has described a form of generalized cutaneous leishmaniasis, bearing a superficial resemblance to leprosy, which may develop a variable number of months after apparent cure of kala-azar by antimony. Having found leishmania bodies in the lesions, he conjectured that some of the parasites survive the action of the drug, but with their virulence so attenuated that they can give rise only to a milder disease, a variant of cutaneous leishmaniasis, to which he gives the name “dermal leishmanoid.”

NOTE.—The special tropical diseases of the skin are discussed under their respective headings.

THE EYE

Glaucoma.—According to Elliott glaucoma is very much more common in the East than in Europe. He states that simple chronic glaucoma is extraordinarily common in India. Often the only symptoms are retraction of the field, cupping of the disc and at a later stage impairment of the central visual acuity. He notes that the advancement of the disease is often as unobtrusive as it is relentless. These patients often only present themselves at the clinic in the late stages of the disease; thus at the Madras hospital the vision was only that of hand perception, or less, in 40% of cases.

Cataract.—The general impression is that cataract is more frequent in the tropical regions than in Europe and as bearing out this view Elliott notes that cataract among those Europeans who have served in India seems more frequent than among those who have remained in England. Cataract is also more common in the southern part of China than in the northern portion.

Lachrymal Obstruction.—Elliott notes the extreme frequency of this condition in India, and states that in the Madras Ophthalmic Hospital 125 operations for excision of one or both lachrymal sacs were performed in 1907.

Trachoma.—There are certain tropical countries where trachoma is a disease of the greatest importance. Thus in China its prevalence is great, as is also true of India, Egypt and Japan.

Outside of imported cases it is very prevalent in many parts of the United States.

In this disease there is hypertrophy of the conjunctiva, granule formation and subsequent cicatricial changes. Pannus and corneal ulcerations are frequent complications.

The disease is contagious through transfer of the secretion by hands or flies. It is usually considered as caused by the so-called trachoma bodies or cell inclusions, which are best brought out by Giemsa staining. The trachoma granules are yellowish, translucent bodies set in the reddened conjunctiva.

Leprosy.—The eye is more frequently involved in nodular than in nerve leprosy. In the former we have infiltration of the conjunctiva which may extend to the cornea.

The leprous nodules invading the palpebral conjunctiva tend to ulcerate and bring about various distortions of the eyelids, producing ectropion. Iritis, irido-cyclitis and irido-choroiditis are less frequent than conjunctivitis and keratitis. The optic nerve and the retina are only rarely involved.

In nerve leprosy the eye changes are chiefly connected with the lesions of the fifth and facial nerves. Ptosis and paralytic ectropion occur with frequency.

Ophthalmia and corneal ulcerations may lead to total destruction of the eye. The cornea may be anaesthetic. Paralysis of one or more ocular muscles may cause squint or diplopia.

Malaria.—It is questionable whether the forms of conjunctivitis and keratitis at times reported as due to malarial infection are not rather of other origin.

Iritis is rarely a complication of malaria.

Retinal haemorrhages may occur in malarial cachexia and cerebral types of pernicious malaria.

Another rare malarial complication is amblyopia. In this there is an optic neuritis with grayish-red disc and the loss of vision is not complete, while in quinine amblyopia the disc is white and the vision completely lost for a time.

Filariasis.—In that filarial infection caused by Loa loa, at one time designated Filaria oculi, there seems a special tendency for the adult worms to wander to the subcutaneous tissues in the neighborhood of the eyes or under the palpebral or ocular conjunctivae. When moving under the conjunctiva the worms cause marked irritation and at times pain.

There may be considerable swelling so that the patient cannot for a time see out of the invaded eye. It has been stated that the worms may enter the anterior chamber of the eye but this is questionable.

It is believed that lesions of the cornea and iris may result from the migrations through the body of the Onchocerca volvulus.

Trypanosomiasis.—Eye lesions are quite frequent in this disease these being keratitis, irido-cyclitis or conjunctivitis.

Oedema about the eyes is of importance in diagnosis. Eye lesions seem more common in Rhodesian trypanosomiasis. The atoxyl treatment of the disease may cause optic neuritis and blindness.

Tick Fever.—In the relapsing fever of South Africa iritis has been noted as occasionally occurring.

Yellow Fever.—In the period of onset a feature of the so-called “facies” of the disease is marked injection of the conjunctivae with sensitiveness to the light.

Rush likened it to the eye of a wild animal as contrasted with the less ferocious eye of bilious remittent fever which more resembled that of a domesticated animal. About the third day the earliest manifestation of jaundice presents itself in the ocular conjunctivae.

Ancylostomiasis.—Retinal haemorrhages may occur with marked hookworm anaemia. Stiles notes a fixed stare in hookworm cases, the eye itself somewhat resembling the eye of a fish.

Among other diseases showing ocular manifestations may be mentioned one associated with fibrous nodules in the upper lid due to a larval dibothriocephalid, Sparganum mansoni.

Bacillary Dysentery.—Quite a number of cases have recently been reported where along with an arthritic complication there has been conjunctivitis. In 6 cases of dysenteric conjunctivitis, Maxwell noted that 4 cases had arthritis and 3 of the latter showed anterior uveitis. The conjunctivitis lasted about a week. Relapses seem liable to occur. In none of the cases has there been recovered from the conjunctival secretion the organism of dysentery.

Cholera.—As a result of the loss of all body fluids the lachrymal secretion is scanty or absent and we have various conjunctival and corneal troubles unless the eye is frequently irrigated with normal saline. Vitreous opacities and cataract may follow cholera.

Beriberi.—In this disease there have been reported the following eye complications: (1) retrobulbar neuritis; (2) paralyses of the muscles of the eye and (3) decreased sensibility of the cornea and conjunctiva.

Night Blindness and Xerophthalmia.—Both of these conditions are quite common in certain parts of the tropics and the view that the heat of the tropics and the tropical sunlight were potent factors had precedence until our study of vitamine requirements showed the etiology to rest in deficiency of fat soluble A. It is now known that cod-liver oil is particularly rich in this vitamine and that in this agent we have our best preventive and curative agent for these eye conditions. It is well known that rats fed on a diet deficient in fat soluble A develop xerophthalmia.

Night blindness (nyctalopia) is best known among the crews of sailing ships, especially when becalmed in tropical waters, and some influence of sunlight was considered the cause, but we now know that it is among such personnel that ship beriberi and scurvy are prone to occur by reason of deficiencies in water soluble B and the antiscorbutic vitamines. In such a dietary fat soluble A would also probably be lacking.

In those parts of the tropics where famine conditions are common both night blindness and xerophthalmia are frequent in young children, not necessarily associated but frequently combined.

In a mild case of xerophthalmia there is a dry area of triangular shape extending from either side of the cornea and covered with a fine, whitish foam. In bad cases the whole eye may be dry, wrinkled and opaque and eventually the cornea may slough away and bring about destruction of the eye. There is very little pain attending this frightful condition.

Elliot notes that from earliest times it was the custom in China to treat these conditions with extracts of liver. We now know that liver and kidneys are glandular organs rich in fat soluble A although not so rich in this vitamine as cod-liver oil.

Typhus fever.—There may be a rapid development of cataract of the soft variety, particularly in young patients. Ocular palsies, especially of the third and sixth nerves may occur during the febrile period. Corneal ulcers are not uncommon during convalescence and may be severe and very painful, often accompanied by conjunctivitis and iritis.

THE EAR

Aural Myiasis.—While the larva of Chrysomyia macellaria, known as the “screw worm,” is the one most frequently reported from the external auditory canal, yet many such cases have been connected with the larvae of Sarcophaga carnaria, Calliphora vomitoria and Anthomyia pluvialis. These larvae are usually deposited in the auditory canals of those with otorrhoea.

The symptoms are intense earache, giddiness and possibly convulsions. The larvae tend to perforate the tympanic membrane. Instillations of 10% chloroform in milk or the use of oils kill the larvae.

In the stuporous states of plague and typhus fever there often appears to be a state of deafness.

One must always keep in mind the ringing of the ears indicative of the physiological action of quinine. Permanent deafness may be produced by the long continued use of quinine.

In leprosy the lobes of the ears are special sites of preference for the nodules and I always palpate the lobes where the nodules are not distinctly visible.

THE NOSE

Nasal Myiasis.—In cases of ozaena certain flies appear to be attracted and to deposit their eggs at the nasal orifices. The larvae developing from the eggs of Chrysomyia macellaria, a fly common in tropical America, are known as “screw-worms” and cause frightful destruction of the nasal structures.

They may bore into the adjacent sinuses. Marked frontal headache and a purulent or bloody discharge are symptoms. Great swelling of the nasal structures precedes the destruction of the cartilaginous and bony tissues.

Leprosy.—The nasal mucosa is apt to be the seat of leprous nodules. Those located on the septum may ulcerate and material from these ulcers show abundant leprosy bacilli. Alternations of dryness and hypersecretion of the Schneiderian membrane are among the early manifestations of the disease.

=Epistaxis.=—This is a feature of the early stages of leprosy often associated with rhinitis, in particular the alternation of coryza-like conditions with others characterized by dryness of the nasal mucosa.

There is also a peculiar nasal tone to the voice of lepers.

In yellow fever and plague epistaxis is often the first sign of the degeneration of the endothelial linings of the capillaries.

Gangosa.—A disease of certain islands of the Pacific, especially Guam, characterized by naso-pharyngeal lesions and a nasal voice, is known as gangosa.

In goundou we have exostoses from the nasal processes of the superior maxillary bones.

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Chapter XLIX

THE URINE AND THE GENITO-URINARY APPARATUS IN THE DIAGNOSIS OF TROPICAL DISEASE

THE URINE

Of the chemical tests employed in the examination of urine that for the presence of sugar is rarely of value, as there is no tropical disease in which the presence or absence of sugar is of diagnostic importance.

The determination not only of the presence of albumin in the urine but, as well, of the variations quantitatively from day to day is, however, most necessary in many of the tropical diseases and particularly in yellow fever and blackwater fever.

=Tests for Albumin.=—The simplest and most reliable test for albumin is the heat test with the subsequent addition of a sufficient number of drops of 5% acetic acid to make the boiled urine acid and incidentally to dissolve any phosphates which may have separated out on boiling.

Ulrich’s test is a very simple one and only calls for reagents which are usually at hand. Heat a saturated solution of common salt, containing 2% of glacial acetic acid, and superimpose the urine to be tested upon the hot reagent. A ring shows the presence of albumin.

For Heller’s test, pour a small amount of nitric acid into a narrow test tube and, while holding the tube at an angle of about 45°, superimpose a layer of the urine to be tested, which is delivered drop by drop from a pipette and allowed to flow down the side of the tube.

This test can be converted into a quantitative one which is sufficiently accurate for clinical purposes. It is based on the fact that a specimen of urine containing 0.003% of albumin will give a perceptible ring at the layering of the urine and acid in two minutes. If the ring appears at once or in a few seconds the albumin content is greater. From the qualitative test an idea can be formed as to the amount of albumin which the urine contains, a heavy ring forming immediately showing a considerable albumin content. Probably the highest elimination of albumin is found in chronic parenchymatous nephritis where it may run from 1 to 3%. In an ordinary case of acute nephritis O.5% would be an average content.

Recently I have been using for both qualitative and quantitative albumin tests the following apparatus. This is simply a 5-inch piece of ¼-inch soft glass tubing heated at a point 2 inches from one end, drawn out for about 2 inches and bent to form a U-tube with one end shorter than the other. This form of tube enables one to perform two tests with the same column of nitric acid and is easily cleaned and dried. They may be kept suspended around a glass tumbler’s rim. Taking up a small amount of nitric acid with a capillary bulb pipette it is deposited in the capillary curve of the bent tube. This acid pipette should be kept attached to the acid bottle. With a second pipette the urine is deposited in the short arm of the U-tube and the presence of albumin shows by a distinct ring at the junction of urine and acid in the clear capillary tubing. The long arm will serve for the introduction of a second specimen of urine for the albumin test.

For quantitative test we dilute the filtered urine with one or more parts of normal salt solution according to the intensity of the albumin ring. A very convenient way of making the dilution is with a graduated centrifuge tube. Make a one to ten dilution of the urine, mix and draw up with a bulb pipette and deposit in the short arm of the U-tube. A distinct ring forms in two or three seconds. Pour off one-half of the diluted urine and make up with an equal amount of saline. Deposit this one to twenty dilution in the long arm. The ring forms in about a minute. With further testing it is found that a one to forty dilution shows a perceptible ring in just two minutes. This final and successful dilution multiplied by 0.0033 gives the percentage of albumin in the urine (40 × 0.0033 = 0.13%).

Should it be desired to determine the nature of the proteids present either in urine or in exudates or transudates the following method is applicable. Determine the percentage of total proteid by the method employed above. Then throw down the globulins by the addition of an equal amount of a saturated solution of ammonium sulphate, filter and estimate the proteid content of the filtrate. The difference between that and the total gives the percentage of globulin. The filtrate is now treated with 5% acetic acid until a precipitate of nucleo-proteid ceases to form; the fluid is filtered and the clear filtrate (which should not show any turbidity with a drop of 5% acetic acid) is tested for its proteid content, which represents the serum albumin. When the combined percentage of globulins and serum albumin is subtracted from the total proteid percentage we have the percentage of nucleo-proteid.

=Tests for Blood.=—Very important in tests of the urine are those for blood. With an unaided eye a smoky colored urine, more or less reddish-brown in color, is suggestive in cases of haematuria, while in haemoglobinuria we usually have a more or less porter-colored, turbid fluid which, however, shows a clear haemoglobin-tinged fluid when centrifuged to throw down the haemoglobin casts and granular débris of the disintegrated red cells. Upon shaking such urine we get a pinkish foam instead of the yellowish one of icteric urine.

A strip of white filter-paper when partially dipped into urine shows pinkish-colored waves which are more deeply colored at the summit of the waves while the paper which absorbs bile-containing urine shows the yellowish color and waves less yellow at the summits of the colored waves.

For haematuria we may use either the microscopic method for the recognition of red cells or chemical ones. The red cell is best recognized by the double contour of the 7.5 micron disk. Spores of moulds, which greatly resemble red cells, are smaller, usually not more than 5 microns.

The following technic is of the greatest value not only because it makes the red cells more distinct but because by staining the various epithelial elements it gives us more exact information as to distinction between the segmented nucleus of pus cells and the single one of renal cells. Make a streak of vaseline across a slide one inch from one end. Then deposit a drop of urinary sediment, taken up from the centrifuge tube with a pipette, about ¼ inch from the grease line. Then drop a large drop of Gram’s iodine solution on this sediment and then apply one side of a square cover-glass to the vaseline line and allow it to fall gently on the drop of sediment and stain. There is no current motion, and casts and other urinary elements remain under the cover-glass instead of floating out beyond the margins. It is well to examine the unstained sediment with a ⅔ inch objective before adding the iodine and applying the cover-glass, as one gets a better idea of casts with a low power and unstained than in any other way.

With haemoglobinuria we necessarily turn to chemical or spectroscopic tests which are also applicable to microscopically doubtful cases of haematuria.

For the detection of occult blood, the technique described on page 524 should be observed, and alkaline urines faintly acidified with acetic acid before examination.

=Indicanuria.=—In sprue and pellagra we have a rather marked increase in indican. It is probable that many cases of vague manifestations of neurasthenia with loss of physical and mental energy are connected with auto-intoxication rather than tropical heat or intestinal parasites.

=Urobilinuria.=—In conditions where there is a great destruction of red cells tests for urobilin are important. Plehn considers the presence of urobilin as of importance in the diagnosis of latent malaria, which is true, provided other causes for red blood cell destruction are excluded. Blackwater fever cases usually show an intense urobilinuria. Urobilinuria is also a sign of deficient functioning of the liver.

=Bile Pigment Tests.=—In conditions associated with the presence of bile pigments in the urine we may conveniently employ the Gmelin test in the following manner. Filter the urine several times through the filter and then touch the moist inner surface of the paper with a glass rod dipped in commercial nitric acid. A ring-like play of colors, green, blue, violet and red circle out from the spot touched. A green color must be noted for positive diagnosis.

Tests for bile acids seem to have but slight value in differential diagnosis.

A very simple and apparently quite reliable test for deficiencies in liver functioning is that known as Ehrlich’s aldehyde reaction. The reagent is a 2% solution of p. dimethylaminobenzaldehyde in equal parts of water and concentrated hydrochloric acid.

For the test treat 5 cc. urine with 5 to 10 drops of the reagent. Agitate a few minutes and a positive reaction is shown by a fine cherry-red color, thought to be due to urobilinogen.

The urine sample should be perfectly fresh and not long exposed to light.

AMOUNT OF URINE IN 24 HOURS

Normally a man passes about 1200 cc. of urine in twenty-four hours, a woman somewhat less. When the amount is under 750 cc. we have an oliguria. To consider a polyuria as present the patient should pass more than 3000 cc., as this amount may be considered the upper normal limit. In anuria we have a cessation of renal activity.

The disease in which anuria is most characteristic is cholera. During the stage of evacuation the urinary secretion becomes less and less along with the progressive failure of circulation and, during the algid stage, we have a suppression of urine.

The anuria seems to run parallel with an acidosis and intravenous injections of bicarbonate of soda solutions tend to prevent anuria. In the stage of reaction the favorable outcome is the reappearance of urine, which increases in amount to become a polyuria. In unfavorable cases the anuria continues.

In blackwater fever anuria may result from the blocking up of the renal tubules by haemoglobin casts.

Blackwater fever also shows an acidosis and alkaline treatment is here indicated. Blackwater urine is irritating so that there is vesical tenesmus with frequent urination.

The degree of renal involvement is of great prognostic value in yellow fever, and those cases where the oliguria goes on to suppression are apt to terminate fatally.

In heat stroke there is an oliguria or anuria which may be followed, during convalescence, by a polyuria. Marked irritation of the bladder, associated with suppression of sweating, may be indicative of oncoming heat stroke.

In dropsical beriberi there is an oliguria or, rarely, an anuria which with the rapid disappearance of the general body oedema may become an excessive polyuria.

Rarely one may observe a critical flow of urine in dengue at the time of the fall of the primary febrile accession.

ALBUMINURIA

The disease in which this is of peculiar diagnostic and prognostic value is yellow fever. We expect albumin about the second day with a steady increase in amount during succeeding days of the fever. The degree of oliguria or rather anuria is of greater prognostic value than the degree of albuminuria. The albuminuria is of great diagnostic value in differentiating yellow fever from dengue.

Blackwater fever shows a great abundance of albumin with the appearance of the haemoglobinuria and diminishes as the color of the urine clears up.

In malaria albumin was present in 38% of benign tertian infections and 58% of malignant ones at Johns Hopkins Hospital.

The absence of albumin in beriberic urine is important in differential diagnosis from acute nephritis.

HAEMOGLOBINURIA

Paroxysmal haemoglobinuria or haemoglobinuria resulting from potassium chlorate poisoning, severe burns, intravenous injections of foreign sera, or—most commonly—syphilis, may be noted in the tropics.

The vast majority of cases of true tropical haemoglobinuria, however, are due either to blackwater fever or to the administration of the acid salts of quinine to one predisposed to quinine haemoglobinuria. While it must be admitted that haemoglobinuria may result from quinine it is certainly so rare in subtropical countries, where great amounts of quinine are administered in treatment of malaria, as to be unimportant. It is only where the malignant tertian parasite flourishes that we have the question of the importance of quinine in producing haemoglobinuria brought up.

Certain persons have isohaemolysins in their blood which dissolve the red cells of other persons and in paroxysmal haemoglobinuria autohaemolysins may be present which can destroy the patient’s own red cells. This auto-haemolysis seems operative only when a low temperature is followed by a high one. When haemoglobinaemia exists the liver converts it into bile pigment causing bilious stools and jaundice. If one-sixth of the red cells are destroyed haemoglobinuria results.

The dark, porter-colored urine of blackwater is diagnostic even to the patient. The urinary sediment consists of granular débris with occasional haematoidin crystals. Albuminuria runs parallel with the haemoglobinuria. Pain in the loins, probably, from the plugging of the renal tubules by the detritus of red cell destruction, is a feature of blackwater fever. In blackwater fever we have the early appearance, even in a few hours, in a patient who is markedly asthenic and miserable, of jaundice, porter-colored urine and albuminuria.

HAEMATURIA

Among tropical diseases that which immediately suggests haematuria is vesical bilharziasis. The blood in the urine is in the form of red cells; it is a haematuria and not a haemoglobinuria. The passage of blood usually occurs at the end of micturition and it is either in the last few drops of urine or in the sediment obtained after centrifuging that we note the terminal spined eggs of S. haematobium which prove the diagnosis.

Red blood cells in the urine may also be noted in the haematochyluria of filarial disease.

When we have blood in the urine in yellow fever it is a haematuria and comes on about the same time as the black vomit and other haemorrhages resulting from degeneration of the endothelial linings of the blood capillaries, which only takes place about the third or fourth day of the disease.

Haematuria may also be noted in plague at the time when the haemorrhages into the skin occur.

CHYLURIA

Vesical varices from lymphatic obstruction, due to filarial disease, are the most frequent cause of the milky urine of chyluria. The urine usually has a pinkish tinge from blood admixture so that the condition is really a haematochyluria. The thoracic duct may not be the seat of obstruction which has taken place elsewhere when the condition is lymphuria instead of chyluria. Lymph and chyle differ in fat content, the former having from very little to about 3% while the latter has 5% or more of emulsified fat. Chyle has also more than twice as much proteid as does lymph.

In chyluria the morning urine is often clear while that at night is milky. On standing, chylous urine separates into an upper cream-like layer with a pinkish sediment and, between, a pinkish-white fluid in which floats a clot. Filarial embryos may or may not be found.

KIDNEY FUNCTION AND ITS DETERMINATION

The ability of the kidney to excrete substances from the blood stream is frequently affected by disease, especially such as disturbs the kidney, and is usually diminished after the age of 50 years. Disease of the kidney, however, does not necessarily imply an inability to eliminate substances—the functional need not parallel the anatomical lesion. Function may be normal especially when the changes are of focal type.

Impairment of renal function does not affect the excretion of different compounds to the same extent. It is well established that functions for chlorides and for urea are independent of each other. In any urinary examination, then, one should bear in mind the possible effect of an impaired excretory power relative only to the substance under consideration. In this connection, we speak of the kidney threshhold for a substance, i.e., the concentration required in the blood stream before the kidney will excrete it, at least in abnormal amounts. For chlorides, this is quite definite at 562 for blood plasma; for sugar, it is about 160-180 for whole blood. The threshold is not necessarily absolute, but simply indicates that with less sugar, for instance, only the normal traces will be passed. Chloride excretion begins only when their concentration passes the threshold value, and the rate of elimination depends upon their excess. Other compounds, such as urea, for example, may have no definite threshold value. Disease may affect the value, either by raising it and causing abnormal retention, or by lowering it and giving rise to depletion.

We can establish the status of the renal function in any given case, and are then in a position to intelligently prescribe dietetic and other treatment. Chemical analysis of the blood indicates the metabolic products affected and guides us in the adjustment of the diet, etc., to the excretory powers of the kidneys. One must, however, not overlook the nutritive needs of the body.

Many methods are employed for the determination of kidney function, and their relative values are still debatable. Probably those least open to criticism are chemical analysis of the blood, the phenolsulphonephthalein test, and Mosenthal’s method. Ambard, McLean, Van Slyke, and others have devised formulae to this end, based upon chloride, urea, or sugar excretion, which have many warm advocates as well as severe critics. A rough clinical comparison of the two kidneys may be obtained by determining the urea in specimens of urine simultaneously collected by the ureteral catheter.

Blood Chemistry, now that its value has been established, is generally given preference and allowed greater weight in case of disagreement with other tests. It measures excretory function for normal metabolic products, and has the additional value of an aid in diagnosis and prognosis and a guide to treatment, especially dietetic. It has the disadvantage of a possibly unfamiliar technique, and does not afford a comparison of the two kidneys.

The substances usually considered are the nitrogenous compounds (nonprotein nitrogen, urea nitrogen, uric acid, etc.), but retention of others (sugar, chlorides, cholesterin, etc.) are also of significance in this connection. Of the nitrogenous constituents, the kidney excretes creatinine most readily, urea next, and uric acid with the most difficulty. As a consequence, an impairment of function results first in the retention of uric acid, then urea, and, finally, creatinine is also retained. Owing to the relatively small amounts of uric acid and creatinine present, the nonprotein nitrogen, which includes the nitrogen in them as well as in other compounds, is not appreciably affected except by the urea increase. This is the basis of an intelligent interpretation of the findings. The urea and nonprotein nitrogen are so markedly affected by diet, especially among nephritics, that judgment must be exercised when they are employed as indices of renal function. This fact was not properly appreciated until recently, and probably accounts for much of the discredit cast upon blood chemistry in this connection. The uric acid, being less exogenous in origin, is perhaps the most delicate and the safest index; the increase appears early, and 3.5 may be considered the high normal value. On the usual restricted hospital diet, over 20 for urea nitrogen should be considered suggestive of impaired kidney function; over 75 speaks decisively for renal involvement and probably uraemia.

Phenolsulphonephthalein (Phthalein, or Red) Test.—This was developed by Rowntree and Geraghty, and its simplicity makes it very useful, especially to the isolated practitioner with limited laboratory facilities. It estimates only function for a foreign substance, is not considered quite as reliable as chemical analysis of the blood, and, of course, does not give the additional information that the latter supplies. It is, however, of much value, has no contraindications, and does compare the kidneys when combined with ureteral catheterization or use of a separator. Positive results are of more significance than negative, and it is less affected by glomerular than tubular changes. Values of more than 75% for 2 hours may be accompanied by diuresis, and Frank considers such a finding suggestive of renal disturbance with irritation if there is any corroborative evidence.

The technique comprises administration of the dye, determination of the interval before it appears in the urine, and the amount then excreted during definite periods. The dye is employed in solution, and is conveniently purchased already sterilized in ampules, each containing slightly more than 1 cc. of a solution of its monosodium salt of the strength of 6 mg. per cc. The patient drinks 200-400 cc. water, and 6 mg. of the dye are injected intramuscularly (lumbar muscles), or intravenously, 20 minutes later. The bladder is immediately emptied, and the urine discarded. The succeeding portions of urine may be collected by voiding, but it is more accurate to catheterize the bladder or ureters, and catheterization is practically a necessity for determination of the “appearance time.”

The appearance time is the interval of time elapsing between injection of dye and its appearance in the urine. It is determined by allowing the urine to drip from the catheter into a receiver containing a drop of 10% NaOH. The first traces of the dye will cause a pink color.

The time interval chosen for calculating excretion is then computed from the instant of this appearance. There is considerable diversity in practice as regards this time interval, and it would seem that shorter intervals and quicker results are gaining in preference, as well as being considered equal in value to longer periods.

The percentage excretion of the dye is now measured in each sample of urine. To do this, prepare a standard solution, made by diluting 0.5 cc. of the phenolsulphonephthalein solution mentioned above to about 200 cc. with water, adding 5-10% NaOH until no further intensification of the red color is produced (requires a few cc.), diluting to one liter, and mixing. This standard then represents 50% (3 mg.) of the amount of the dye injected. The color of the urine sample is similarly developed with alkali, and the mixture diluted to 1 liter, mixed, and compared with the standard in a colorimeter. The per cent excretion in the specimen equals the reading of the standard solution multiplied by 50 and divided by the reading of the urine mixture, when the colors are matched in the instrument.

For accurate work, it is desirable to balance in the standard the urine color of the unknown, and this is accomplished by including in the standard a volume of urine (dye-free) proportional to that in the unknown. Also, with a low excretion, it is better not to dilute the unknown to 1 liter, but to some lesser volume that will give a tint closer to that of the standard, and then allow for the variation in the calculation.

Dunning has devised a simple, inexpensive colorimetric outfit with permanent standards in ampules for this test. It is satisfactory unless the colors of the standards fade.

After intravenous injection, the normal appearance time is 4-6 minutes, and the normal elimination is 35-40% in 15 minutes, and totals of 50-65% for 30 minutes and 65-80% for 60 minutes; or, for the first 30 minutes, it may be stated as about 1% per minute from each kidney.

After intramuscular injection, the normal appearance time is nearer 10 minutes, and the normal elimination is 30-40% in 30 minutes, and totals of about 50% (40-60) for 60 minutes, and about 80% (60-85) for 2 hours, or 20-25% during the second hour. If the appearance time is not determined, it is customary to allow for it, collecting the first hour’s specimen at 70 minutes after injection of dye and the second hour’s 60 minutes later.

Impairment of kidney function, of course, increases appearance time and lessens excretion, serious cases not unusually excreting less than 1% during two hours.

When the question of the kidney involved arises, the urine must be taken by ureteral catheterization or by a separator.

=Starches and Fibres.=—In examining urinary sediments it is important to be familiar with the various textile fibres and starch grains which are so frequently present, the fibres coming from the clothing and the starch grains from dusting powders. Wool fibre fragments show bark or scale-like imbrications and are round. Cotton fibres are flattened and twisted, while linen ones show a striated flattened fibre with frayed segments as of a cane stalk. Silk shows a glass-like tube with mashed-in ends.

Corn and rice grains are the most common of the starch grains and their nature is immediately disclosed by their blue color when mounted in iodine.

AFFECTIONS OF THE GENITO-URINARY ORGANS

In blackwater fever we have marked pain in the region of the kidneys due to the plugging of the tubules with haemoglobin casts. Vesical tenesmus and pain along the ureters may also be present.

In malaria Thayer states that nephritis occurs in about 2% of malignant tertian cases.

In bilharziasis the kidneys are involved secondarily—the change being brought about by stone in the bladder and cystitis leading to hydronephrosis and pyelonephritis.

Cases of cystitis occurring in dysentery have been reported which showed amoebae in the sediment of the urine. Such cases probably were connected with recto-vesical fistulae caused by amoebic ulceration.

In cholera the kidneys are markedly affected, especially the epithelial lining of the tubules.

Malta fever may rarely be attended by an orchitis.

One of the manifestations of filarial disease is lymph scrotum in which the scrotum is covered with small blebs containing a chylous fluid which may possibly contain microfilariae. It is associated with recurring attacks of lymphangitis. There is also a filarial orchitis and we may have a lymphangitis of the lymphatics of the cord. Again filarial disease may show a chylocele in which the tunica vaginalis contains a fluid similar to that seen in the varices of lymph scrotum. This fluid may also show filarial embryos.

In endemic funiculitis there is a sudden onset with high temperature and pain in spermatic cord and epididymis. The general condition rapidly becomes grave with a hard, tender, cylindrical swelling along the cord and also pain and swelling of epididymis. It is a streptococcus infection usually engrafted on a filarial or bilharzial process and demands immediate surgical measures.

Kala-azar may be accompanied by sloughing of the scrotum at the time manifestations of cancrum oris are noted.

Cases of gangrene of the scrotum have been reported as connected with malaria.

Gangrene of the scrotum and penis is not infrequently noted in Rocky Mountain fever.

In puzzling febrile cases in the tropics one should always think of a possible pyelitis. Then too keep in mind renal tuberculosis.

If leprosy comes on before puberty the sexual organs remain in an undeveloped condition. Leprous infiltrations are noted in the testicles and ovaries. In nerve leprosy, which does not usually come on until after puberty, the women may bear healthy children and it is now thought that the view that leprosy markedly tends to produce sterility is lacking in confirmation.

In ancylostomiasis menstruation is markedly interfered with and amenorrhoea is often a prominent symptom. Young men who have been affected before puberty show lack of development of pubic hair along with infantile genital organs. The girls do not show normal breast development.

Granuloma of the pudenda is a disease which is rather frequent in British Guiana.

Dhobie itch is characteristically located in the crotch region.

BACTERIOLOGICAL EXAMINATION OF URINE

About the only tropical disease in which a bacteriological examination of the urine is of particular value is that in connection with Malta fever. It is advisable to cleanse the meatus with alcohol and then having discarded the first ounce or so of the urine to receive the remainder in a sterile salt mouth bottle. A drop of this urine can be deposited on a poured agar plate and smeared out over the surface.

As dysentery bacilli and cholera spirilla are practically absent from the blood, urine examination for the causative organisms in these diseases is fruitless.

The culturing of the urine to find paratyphoid or typhoid organisms should be carried out, as well as blood cultures, where we are dealing with puzzling fevers in the tropics. The Teague plating medium described under the chapter on Faeces is a very satisfactory one.

In culturing urine from a case of pyelitis blood agar is a most excellent differentiating medium for streptococci.

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