_Climatic Fevers._—From many parts of the tropical world there

have been reported cases of fever supposed to be due to exposure to

prolonged action of tropical heat. They are often designated as

climatic or inflammatory fevers.

A careful study of the clinical manifestations tends to show that

many of them are much like dengue. Some may be due to infection

with the Gärtner group of bacteria.

_Rat Bite Disease._—Following a rather long incubation period of

from six to eight weeks, during which time the bite has healed,

we have a rather sudden invasion with high fever, 103° to 104°F.,

chill and at the same time inflammation of the site of the bite with

lymphangitis and some swelling of tributary glands.

After two or three days of high fever we have a fall by crisis

with profuse perspiration. The temperature remains normal for a

few days during which time the local swelling and inflammation

subside. The fever again comes on, frequently with an eruption, to

later on disappear and reappear. At such times the fever course is

irregular. There may be as many as 12 of these febrile accessions.

_Tsutsugamushi._—The disease sets in about a week after the bite

of the Kedani mite with headache, chills and fever of about 101°F.

There is also pain in certain lymphatic gland groups which will be

found to drain the area in which is located a small necrotic ulcer,

the site of the bite of the mite. The temperature continues to rise

during the next two or three days to 104°-105°F. and remains as a

high continuous fever for about a week, when an eruption of irregular

dusky macules appears, first on the face and later on chest,

extremities and trunk. About the tenth day the fever begins to go

down by lysis and the eruption fades. Injection of the conjunctivae

is marked.

Certain authorities have considered that there is a striking

clinical similarity and possible relation attaching to

tsutsugamushi, Rocky Mountain spotted fever, trench fever and

typhus fever. At present we believe that tabardillo or Mexican

typhus is the same as the well-known typhus of temperate climates,

hence that which describes typhus fever obtains for tabardillo.

_Spotted Fever of the Rocky Mountains._—In tabardillo the onset

and termination of the fever is rather abrupt while in spotted

fever of the Rocky Mountains it climbs up gradually for a week to

reach its maximum and falls by lysis.

All these diseases are characterized by a more or less stuporous

state.

_Oroya Fever._—It was formerly supposed that this fever was the

first stage of verruga, but it is now considered as a distinct

disease entity, caused by a protozoon of bacillary form which invades

the red cells. With pains of various joints and bones we have a

gradual rise of temperature which after a few days reaches 103° to

104°F. and tends to become remittent or continuous.

There is a remarkable and excessive destruction of the red cells

which may fall to a million or less per c.mm. The fever after about

three weeks begins to fall by lysis. Enlargement of liver, spleen

and lymphatic glands are common. Pain over the bones, especially

the sternum, is often excruciating.

_Epidemic jaundice_ shows an irregular pyrexia of from 102° to 103°F.

with jaundice about the second or third day.

_Trench Fever._—Cases of varying types of fever, some charts more or

less resembling the dengue ones, while others show repeated relapses

of short duration, have been designated _trench fever_.

In _tularaemia_ we have an irregular fever course of rather rapid

onset, extending over two or three weeks. There is very little

evidence of toxaemia. Convalescence is tedious.

_Typhoid fever_ and the _paratyphoid infections_ are far from

uncommon in the tropics and present clinical courses at variance with

those observed in temperate climates. The temperature charts in such

cases are irregular and atypical.

It must be remembered that paratyphoid infections may show marked

gastro-intestinal symptoms and that the rose rash of such cases

tends to be far more profuse than that of typhoid.

_Intestinal Parasites._—There are many conditions which seem to

be productive of febrile attacks as evidenced by the disappearance

of the fever upon removing such cause. Thus patients presenting

abdominal distress and a fever of varying type may be completely

relieved of all symptoms upon evacuating the larvae of various flies

following purgation. This condition is designated intestinal myiasis.

Abdominal pains and fever may also be caused by various helminths

usually considered nonsymptom-producing as has been noted in heavy

_Ascaris_ infections.

CHAPTER 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.

[Illustration: FIG. 147.—Neubauer’s ruling.]

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.

[Illustration: FIG. 148.—Thoma-Levy counting chamber, Bürker double

type with two Neubauer rulings.]

=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.

[Illustration: FIG. 149.—The Türck ruling. Thoma-Zeiss

Haemacytometer.]

=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.

[Illustration: FIG. 150.—1, 2, 3, 4, Making blood smears on slide.

5. Smear ready for staining—grease marks prevent Wright stain

from running over slide. 6. U-shaped glass tubing to hold slide in

staining. 7. Right hand holding two cover-glasses. One cover-glass is

being touched to drop of blood from ear. 8. Cover-glasses transferred

to left hand in preparing to place one cover-glass on another and

spread film. 9. Separating cover-glasses by sliding one from the

other.]

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.

[Illustration: FIG. 151.—Sahli’s Haemoglobinometer. (Greene.)]

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}.

[Illustration: FIG. 152.—Carbon dioxide absorption curves. (Modified

from Peters, Barr, and Rule, and Van Slyke).]

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.