THE SERUM-FORMALIN PROPORTION IN THE ALDEHYDE TEST FOR KALA' AZAR.

By C. R. DAS GUPTA, m.b. (Cal.), Assistant, Kala-azar Enquiry, Indian Research Fund

Association.

(From the School of Tropical Medicine, Calcutta?) A number of serum tests for the diagnose

of kala-azar have been described from tune to

time, namely, the globulin precipitation tes

(Brahmachari, 1917), the hemolytic tes

(Ray, 1921), the aldehyde test (Napier, 1921' 1922 and 1923) and the antimony test (Chopra Gupta and David, 1927), but of all these the

writer has found the last two the most reliabj?* and for practical purposes he prefers _ yi aldehyde test as it never gives a false posi^Y, result if the criteria of its originator are strict^ observed; also in all rough and field work tn

aldehyde test has the advantage of extrem6 simplicity. Simple though the test is, nevel" theless in the procedure of properly mixing

a

definite quantity of serum with a defini

quantity of formalin and in reading the resin '

of the test the rules laid down by its originate should be strictly followed. Violation of th.eS rules has led to many a fallacious result bein?

obtained with this very simple test, even W

people with some amount of laboratoO experience. Frequently we have receive

SERUM-FORMALIN PROPORTION IN ALDEHYDE TEST: DAS GUPTA. 501

samples of blood said to have given _

a

strongly positive aldehyde reaction which in our hands have given either absolutely negative or only doubtful results. Also We have been told by some that within a short period the same serum gives different results

-"-sometimes positive and sometimes doubtful ?r negative. In all these cases when informa- tion was sought as to the procedure followed

jn doing the test the answer has invariably been that,

" a little formalin was added to a

little serum."

In order to ascertain how these anomalous results have been obtained the writer carried out a few experiments varying the proportion

serum and formalin. Tests were carried out v>'ith the serum of 56 kala-azar patients from the kala-azar out-patient department of the

Calcutta School of Tropical Medicine; these ean be divided into three groups, kala-azar eases with a strongly positive aldehyde reaction, non-kala-azar cases in which there was a

doubtful or negative aldehyde reaction, and

kala-azar cases with a negative or doubtful

aldehyde reaction diagnosed by parasitological means. The experiments were carried out with the following mixtures of serum and formalin:

^-2:1, 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6. (Pre- uminary experiments with serum-formalin fixtures of 8:1, 6:1, 4:1 and 3:1 were found t? give identical results with the standard

aldehyde test so that these dilutions were

subsequently omitted.) In all cases the read- nigs recorded were taken within half an hour ?f adding the formalin to the serum; not after ^4 hours, as in the standard aldehyde test. In some instances 2 sets of readings were taken, one within half an hour and the other after 24 hours, but the difference between the two

readings was found to be negligible.

Table I.

Kala-azar cases with positive aldehyde reaction.

A.R.

10

Serum-formalin mixtures.

3:1 2:1 1:1

10

1:2 1 1:3 1:41:5* 1:6

10 10

Standard aldehyde reaction. n two instances this mixture was not included.

^ hen there were 3 parts or more of serum

as ?3ne formalin the reaction was positive,

fn.m ^le standard test, but when the serum-

rmalin proportion fell to 2:1 only 2 out of

j M'ere positive. After this, with the increase

in Proportion of formalin there was an

Unr?aSe *n ^le number of positive reactions

ji a proportion of 1:5 was reached when

1Cle was again a falling off.

Table II.

Non-kala-azar cases with negative or doubtful aldehyde reaction.

A.R.

24 17

Serum-formalin mixtures.

2:1 1:1

3 38

1 3 37

1:2 1:3

9 9

23

31 10

1:4 1:5* 1:6

37 4

31 4

27 13 1

A. R. = Standard aldehyde reaction. * In 6 instances this mixture was not included.

The above table shows an analysis of 41 non- kala-azar cases with negative or doubtful alde- hyde reaction. It will be seen that when the amount of serum was greater than that of the formalin up to a proportion of 2:1 not a single false positive result was obtained, and when the proportion was 1:1 there was only one false

positive result. As the proportion of serum to formalin fell more and more false positives were obtained up to a maximum of 37 out of 41 at a proportion of 1:4, and in the last two mixtures 1:5 and 1:6 there was a slight falling off in false positive results. Let us consider tables i and n together. It

is apparent that, provided the proportion of serum to formalin is not less than 3 to 1, no false results will be obtained; that is to say the test retains its specificity. False negatives are found with a serum-formalin mixture of 2 to 1, but it is not until the serum and formalin are

equal that any false positives will be obtained. When the serum-formalin mixture is 1 to 4 a

positive result is obtained in practically all sera; that is to say the specificity of the test is

entirely lost. As one would expect, when the proportion of serum to formalin is still further

lowered, the coagulable substance?which is in the serum?is still further diluted and posi- tive results become fewer. It is, however, rather interesting that it is among the kala- azar sera that the percentage of positive results declines more rapidly.

It seems possible that the coagulation pheno- menon on which the aldehyde test depends is associated with the globulin fraction in the

serum, and that the coagulation which occurs

when the formalin is in great excess is asso-

ciated with the albumin fraction; in an ad- vanced case of untreated kala-azar there is an excess of globulin and a marked deficiency of albumin. The results with the few aldehyde-negative

sera from kala-azar patients approximate more closely to those of the non-kala-azar cases

than to those of the advanced kala-azar cases in most of the mixtures, but it will be noted that with the serum-formalin dilutions of 1 to 6 the results were closer to those of the ad- vanced kala-azar cases; with only five cases in this group it is impossible to attach any

502 THE INDIAN MEDICAL GAZETTE. [Sept., 1931.

significance to this fact, until further observa- tions have been made.

Table III.

Kala-azar cases with doubtful or negative aldehyde reaction.

A, R.

+ ?

Serum-formalin mixtures.

2:1 1:1 1:2 1:3 | 1:4 I 1:5 1:6

A. R. = Standard aldehyde reaction.

Conclusion.?It will be seen that very diverse results are obtained by indiscriminate mixing of serum and formalin; these may or may not be in accordance with the results of the standard test. Hence in order to get the best possible result, the rule laid down by its originator, namely, of putting a drop of 30 per cent, for- malin into 1 c.cm. of clear* serum in a small test tube and then intimately mixing it by gently shaking, should be strictly adhered to.

Although in this series when the serum-formalin mixture was 3:1, or more, the results obtained were almost identical with those of the standard

aldehyde test, we do not recommend any

departure from the originally-advocated pro-

portions, because we are of opinion that this ratio of the reagent?and the ratio is after all the most important factor in all precipitation tests?is optimal. The end result, which depends entirely on

whether there is complete opacity, partial opacity (i.e., clouding or opalescence), or no

change at the end of 24 hours, should be noted and the results recorded as positive, doubtful or negative, accordingly, though in the case of a strongly positive reaction complete opacity will occur and the result can be read in about half an hour. It must be clearly understood that gel formation, which is popularly spoken of as solidification, is only an associated factor and when present by itself is mostly of nega- tive value in the diagnosis of kala-azar. My grateful thanks are due to Dr. L. E.

Napier, for his most valuable guidance in doing these tests and for his help in preparing this paper. The paper is published with the kind permission of the Secretary of the Indian Research Fund Association.

References.

Brahmachari, U. N. (1917). Fourth report on the treatment of kala-azar and some blood reactions in this disease. Indian Med. Gaz., LII, 319.

Chopra, R. N., Gupta, J. C., and David, J. C. (1927). A preliminary note on the action of antimony com- pounds on the blood serum. A new serum test for kala-azar. Indian Med. Gaz., LXII, 325.

Napier, L. E. (1921). A new serum test for kala- azar. Indian Med. Gaz., LVI, 338.

Napier, L. E. (1922). A new serum test for kala-azar. Indian J own. Med. Res., IX, 830.

Napier, L. E. (1923). Further experience with the

aldehyde test. Indian Med. Gaz., LVIII, 104.

Ray, C. B. (1921). Hajmolytic test in kala-azar. Indian Med. Gaz., LVI, 9.

* Difficulty is said to be encountered sometimes in

getting clear serum from the blood. But this can be

very easily surmounted by washing out the syringe with normal saline just before taking the blood.