Chapter XLV: Blood Examinations in the Diagnosis of Tropical Diseases (2)

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

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

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

OCCULT BLOOD

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

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

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

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

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

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

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

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

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

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

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

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

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

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

ACIDOSIS

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

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

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

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

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

H_{2}CO_{3} + NaCl ⮂ HCl + NaHCO_{3}

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

Factors Operating:

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

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

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

_D. To decrease carbonic acid:_
12. Hyperpnoea.
(_a_) Voluntary.
(_b_) Due to disease processes.
(_c_) Due to low oxygen content of air.
(_d_) Emergence from warm water.
13. Low atmospheric content of CO_{2}.

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

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

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

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

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

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

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

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

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

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

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

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

CHEMICAL ANALYSIS OF BLOOD

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

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

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

RESULT OF CHEMICAL EXAMINATION OF BLOOD

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

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

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

In _comatose conditions_, nitrogen retention will indicate the
uraemic, and hyperglycaemia the diabetic cases. But _acute
nephritis_ should always be borne in mind, as it may have a
pronounced acidosis but no nitrogen retention.

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

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

LEUCOPENIA

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

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

Paratyphoid gives a similar blood picture.

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

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

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

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

EOSINOPHILIA

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

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

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

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

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

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

Scabies also gives an eosinophilia.

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

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

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

LEUCOCYTOSIS

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

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

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

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

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

According to Cabot, leucocytosis varies in infections as follows:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

LYMPHOCYTOSIS

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

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

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

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

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

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

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

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

INCREASED LARGE MONONUCLEARS

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

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

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

DISEASES IN WHICH THERE IS A NORMAL LEUCOCYTE COUNT

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

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

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

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

Coagulation Rate of Blood

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

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

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

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

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

Specific Gravity of the Blood

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

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

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

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

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

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

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

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

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

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

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

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

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

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

There is probably an increase in the percentage of lymphocytes.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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