SEARCH FOR A SPECIFIC CHEMICAL

TEST FOR BLOOD STAINS : ? A COM-

PARATIVE STUDY OF THE PRELIM-

INARY CHEMICAL TESTS FOR BLOOD

By S. N. CffAKRAVARTI M. B. ROY

and

S. N. ROY

(From the Chemical Examiner's Laboratory, Agra)

No specific chemical test for the detection of blood stains has yet been discovered. A sensitive conclusive chemical test would be of very great value as such a test would not only be a valuable confirmatory test, but would also enable a

medico-legal or chemical examiner to examine a larger number of exhibits in a short time. Experiments were, therefore, started in this laboratory for discovering such a specific test, and as a preliminary we thought it best first to study critically the possibilities of the existing more important colour reactions for blood. To find out the limitations of the existing colour tests the work here reported was done to study, (i) the action of substances which simulate the colour of blood, e.g. certain fruit and vegetable stains, paints, dyes, iron rust, (it) the action of substances which are likely to stain seized exhibits (clothes, etc.), e.g. saliva, urine, perspira- tion, seminal fluid, leucorrhceal discharge, albumin, human milk, cow's milk, soap solution, fseces, colostrum, secretions from throat and nose, pus, and (Hi) the action of the commoner acidic and metallic radicles and ions on the more impor- tant colour reagents for blood stains. The tests used were the benzidine test, the orthotoluidine test, the leuco-malachite green test, the phenolph- thalein test and the guaiacum test for blood. (For the preparation of these reagents and

details of the tests see Taylor's Medical Juris- prudence, Vol. I, page 463; Sydney Smith's Forensic Medicine, page 204. A 5 per cent

solution of orthotoluidine in glacial acetic acid and 6 per cent hydrogen peroxide were used for

preparing the orthotoluidine reagent. The ortho-

toluidine test was carried out in exactly the

264 THE INDIAN MEDICAL GAZETTE [June, 1944

same manner as the benzidine test.) The results of these experiments are outlined below :?

(?) The following substances gave strongly positive reactions with benzidine, orthotoluidine, leuco-malachite green and guaiacum, but not with phenolphthalein:?

(1) Potassium dichromate, (2) potassium chromate, (3) potassium ferricyanide, (4) potassium perman- ganate, (5) bromine, (6) calcium hypochlorite, (7) manganese dioxide and (8) potatoes.

(ii) Lead peroxide and iodine gave strong colours with benzidine, orthotoluidine and leuco-malachite green, but not with phenolphthalein and, guaiacum.

(in) Rye and silver nitrate gave positive reactions with benzidine, orthotoluidine and guaiacum, but not with leuco-malachite green and phenolphthalein.

(iv) Formalin and potassium cyanide gave positive reactions with benzidine and orthotoluidine, but not with the remaining reagents. Orthotoluidine gave deeper colours with potassium cyanide than benzidine and was found to be a more sensitive reagent than benzidine for detecting traces of hydrocyanic acid in the atmosphere of a factory, etc.

(v) The following substances gave positive reactions with orthotoluidine, leuco-malachite green and guaia- cum, but not with benzidine and phenolphthalein:?

(1) Aluminium chloride, (2) calcium chloride, (3) calcium fluoride, (4) ferrous sulphate, (5) manga- nese chloride, (6) potassium bromide and (7) potassium iodide.

(vi) Ammonium chloride, banana, iron oxide, lead acetate, and potassium iodate gave positive reactions with orthotoluidine and guaiacum, but not with the

remaining reagents. (vii) Bismuth nitrate, calcium carbonate and

magnesium oxide gave faint positive reactions with orthotoluidine and leuco-malachite green, but not ivitli the remaining reagents.

(viii) Calcium sulphate, gluten, pleural fluid and

potassium bromate gave faint positive reactions with orthotoluidine only.

(ix) Potassium ferrocyanide gave positive reaction with leuco-malachite green and guaiacum, but not with the remaining reagents.

(x) The following substances gave faint positive reactions with leuco-malachite green alone : barium

chloride, calomel, phenylhydrazine, potassium chlorate and stannous chloride.

(xi) Apple, betel leaf, black pepper, magnesium chloride, sodium nitrite and zinc chloride gave positive reactions with guaiacum only.

(xii) Chlorine and ammonium or potassium sulpho- cyanide gave positive reactions with all the five

reagents. (xiii) Besides chlorine and the sulphocyanides, only

copper gave strongly positive reactions with phenolph- thalein. Copper salts did not give positive results with the benzidine reagent.

(xiv) The following substances gave negative results with all the five reagents :?

Leucorrhceal stains, seminal stains, healthy human faeces (free from blood), animal faeces (free from blood), sweat, nasal discharge (free from blood), sputum (free from blood), urine (free from blood), colostrum (free from blood), human milk, cow's milk, logwood, dyes, orange juice, tomato, guava, sago, soaps, pearl powder, Tokalon poudre, Cusson's talcum powder, Colgate powder, Cuticura talcum powder, Pond's vanishing cream, Icilma vanishing cream, Tokalon vanishing skin food, methyl red, neutral red, fuchsin, cochineal, oxalic acid, lactic acid, citric acid, gallic "acid, tannic acid, pyrogallic acid, sulphanilic acid, phenol, lysol, -naphthol, jS-naphthol, aniline, a-naphthylamine, alum, aluminium phosphate, ammonium sulphide, cobalt

sulphate, cobalt nitrate, nickel nitrate, arsenic oxide, antimony sulphide, antimony sulphate, barium sulphide, cadmium sulphate, magnesium carbonate, magnesium sulphate, mercuric chloride, lithium carbonate, lead nitrate, potassium nitrate, potassium hydrogen sulphate, sodium chloride, sodium citrate, sodium acetate, sodium

arsenate, sodium nitrate, sodium phosphate, sodium sulphate, sodium bisulphite, sodium thiosulphate, strontium chloride, zinc sulphate, hydrochloric acid, nitric acid, sulphuric acid, boric acid, ammonia, calcium hydroxide, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, coal tar pitch, pure cow ghee, bazaar ghee, mustard oil, coconut oil; red pepper (chillies), prepared pan (betel), coriander, kattha (catechu), turmeric, filter paper, India rubber, rubber solution, starch, sand, gum .arabic, gum acacia, gum sandrac, gum tragacanth, resin, clay or earth, sealing wax, this office ink stains, lead pencil marks and copying pencil marks.

Conclusions.?It would be obvious from the above that none of the above tests can be

regarded as a conclusive test for blood. The

great value of these tests, specially of the benzidine test and the phenolphthalein test, lies in the fact that if these tests give negative results, blood is certainly not present, and thus a large number of stained articles may rapidly be examined and those that are negative discarded. Of all the five tests, the phenolphthalein test (for details see British Medical Journal, 1926, i, 650) is the most selective, and the benzidine test the most convenient. If a suspected stain gives positive results with both phenolphthalein and benzidine, and if the presence of chlorine and sulphocyanides (which are rarely likely to be present) is excluded, it may be concluded that in all probability the stain is due to blood, for with the phenolphthalein test a strongly positive reaction is seen only with copper salts and blood, and it gives negative results with pus, secretions from throat and nose (unless these contain blood), plant juices, commercial formalin and most of the salts and oxidizing agents which give a positive reaction with benzidine, whilst the benzidine test gives negative results with copper salts. It would, therefore, be worth while to use both these tests as a part of routine procedure for detecting blood stains. The orthotoluidine test is not so useful as the

benzidine test as a sorting test, as many more substances give positive results with this test, but it gives a deeper and more permanent blue colour with blood stains than does benzidine. It

^ is also more sensitive, and in cases of suspected very old blood stains it could be used with advantage in place of benzidine. This test deserves to be better known and should be more

widely used as a preliminary test for blood. The leuco-malachite green test) which Rhodes

describes as the most delicate known for blood, was found to be generally not so useful as the benzidine or phenolphthalein test as many more substances (besides blood stains) give a positive reaction with this test than with the latter tests- The action of these five reagents on very old

blood stains was also studied. The ortho- toluidine test was found to be the most sensitive test for old blood stains. Orthotoluidine, phenolphthalein, leuco-malachite green and benzi- dine gave positive reactions with blood stains six years old, but the guaiacum test.gave negative results.