BLOODSTAIN ANALYSIS AND MEDICO LEGAL ASPECTS

Blood stains are an important forensic tool. This presentation describes bloodstain splatter analysis. They are an important diagnostic tool which afford the forensic science investigators an opportunity to find out whether it is actually blood or not

1. BLOODSTAIN ANALYSIS Dr. BAISHWANAR BANERJEE 1

2. Whenever two objects come into contact with each other, there is a mutual transfer of material from one object to the other—Edmund Locard (1877-1966) French forensic scientist INTRODUCTION 2

3. Different stains having Medico legal importance Blood Seminal fluid Vaginal discharge Saliva Fecal stain Urine

4. Forensic serology: Involves the examination and analysis of a variety of body fluids which includes blood, saliva, semen and urine. Blood is a complex fluid with ph 7.4, cells about 45% and plasma about 55%. Legal requirements dictate that identification of the stain should be established to a scientific certainty, before it can be presented in court. Visual observation of an untested stain, coupled with positive chemical presumptive and confirmatory tests will provide sound data to support identification. 4 FORENSIC SEROLOGY

5. BLOODSTAIN PATTERN ANALYSIS Interpreting bloodstain patterns can yield information about manner in which a bloodstain was deposited and helps in crime scene reconstruction Distance from impact origin, object that may have been responsible for impact, direction of impact, no of impacts/ movement of individual after injury can be determined by studying blood deposition. Bloodstain shapes determined by angle of impact. When drop of blood strikes a horizontal surface at angle of 90 degrees, resulting bloodstain is round with spiked edges- crown appearance

6. Contd As angle increases bloodstain becomes longer and narrower-tapering/ tear drop stain is formed Sometimes a small separate spot may be present in front of the sharp end of the stain resembling exclamation marks-lance shaped Smearing indicates the movement of the bloodstained object across the surface. Sometimes a pattern may be left which helps to indicate shape of weapon or fingerprints in blood may help in identification

7. May establish a link between offence, offended and offender May establish link between offence and offensive agent Fictitious charges by presenting an animal stain as human In poisoning, poison chemically detected Time since death may be estimated by chemical and enzymatic study 7 MLI

8. Examination Whether the stain is actually a blood stain Physical Chemical Microscopic Spectroscopic Micro chemical Dr. Shiuli, Forensic Medicine & Toxicology, KGMU

9. Whether the blood stain is of human or other than human Precipitin test If human, which blood group Sex, source Indication as to cause of death Whether belong to victim or accused Whether bleeding ante mortem or postmortem Any disease process

10. Substances resembling blood stain Vegetable stains ammonia greenish yellow color Bleached by chlorine water M/E vegetable cells and fibers Rust stains Does not fall off in scale on being heated Positive reaction for iron Dissolved in dil. HCL

11. Presumptive/ screening test- when positive leads to the conclusion that blood is present. Further tests undertaken to confirm presence of blood If negative, stains need no further consideration Exceedingly sensitive, specificity not satisfactory May be recognized as those that produce a visible color reaction or result in release of light. Both types rely on catalytic properties of blood to drive the reaction. 11 PRESUMPTIVE TESTS FOR BLOOD

12. Employ chemical oxidation of a chromogenic substance by an oxidizing agent (H202) Heme group of haemoglobin exhibit peroxidase activity which catalyses breakdown of hydrogen peroxide H2O2+REDUCED AGENT (COLOR 1) = H2O + OXIDISED REAGENT (COLOR 2) False positive may be due to other materials catalyzing the peroxidase reaction-pus, saliva, mucus, infected CSF, formalin, plant juices 12 CATALYTIC COLOR TESTS

13. Stain in question is sampled with a clean, moistened cotton swab. 1 drop of the color reagent is added to the stain followed by equal amount of hydrogen peroxide Immediate development of color, typical of reagent used, indicates presence of blood in the sample 13 METHOD

14. Chemical examination Highly sensitive Positive reaction even with faint traces of blood Principal factor – peroxidase activity liberating nascent oxygen which leads to colour change

15. Reaction is carried out in ethanol/acetic acid solution and results in a characteristic blue color Test given by blood of any age or even blood subjected to heat/ cold Seldom used nowadays due to carcinogenic effect of benzidine 15 BENZIDINE (ADLER) TEST

16. BENZIDINE TEST stain extract ┼ benzidine ┼ 10 vol in hydogen peroxide glacial acetic acid Blue colour

17. PHENOLPHTHALEIN (KASTLE MEYER) TEST Test commonly used Reagent consists of reduced phenolphthalein in alkaline solution that is oxidized by peroxide in presence of hemoglobin in blood Reaction shows phenolphthalein being oxidized and turning a pink color More specific than benzidine but less sensitive

18. O TOLIDINE (KOHN/ O KELLY) TEST Reaction similar to that of benzidine is conducted under acidic conditions & produces a green blue color reaction

19. Color change is from green to blue green 19 TETRAMETHYLBENZIDINE TEST

20. LMG produces a green color 20 LEUCOMALACHITE GREEN (LMG) TEST

21. KASTLE MAYER TEST - Phenolphthalein in alkaline medium Pink color ORTHOTOLIDINE TEST (4% orthotolidine in ethyl alcohol)+ glacial acetic acid + H 2 O 2 Green color LEUCHOMALACHITE GREEN TEST Bluish green color

22. Washed drag/ spatter pattern in large areas is tested with luminol & fluorescein tests Involves spraying a chemical mixture on a suspected bloodstained rea and observing the result, either in darkness or in reduced light Luminol (3 aminophthal hydrazide) gives blue white to yellowish green glow which indicates presence of blood 22 CHEMILUMINESCENCE & FLUORESCENCE

23. Delicate and reliable test for detection of presence of blood in both recent and old stains Blood is dissolved in water or normal saline and is placed in a small test tube which is then kept between the spectroscope and source of light. Solution has property of absorbing some of the rays from the spectrum producing a characteristic dark absorption bands which vary with the type of blood pigment present 23 SPECTROSCOPIC EXAMINATION

24. Thin layer of silica gel is prepared on a suitable glass plate Appropriate quantity of sample extract, standard hematin chloride solution and control sample of blood are placed on prepared gel Plate is placed in a chamber having a convenient solvent system When plate is dry, benzidine & H202 are sprayed on it If stain contains blood, sample extract gives a blue spot at the same height 24 THIN LAYER CHROMATOGRAPHY

25. Stained piece is cut and dipped and teased in a watch glass with 2-3 drops of Vibert’s Fluid (sodium chloride, mercuric chloride, distilled water/ normal saline) for half an hour and then examined under the microscope Non mammalian RBCs– Oval, biconvex, nucleated Mammalian RBCs– Circular, biconcave, non nucleated except camal & llama Primates- Nucleated RBCs may be found 25 MICROSCOPIC EXAMINATION

26. Confirmatory tests for blood Dr. Shiuli, Forensic Medicine & Toxicology, KGMU

27. Crystal tests are regarded as confirmatory tests. These tests involve non protein heme group of hemoglobin called porphyrins Include Teichmann test, Takayama Test, Wagenaar Test 27 CRYSTAL TESTS

28. A sample of suspected blood is placed on a glass slide. A few crystals of sodium chloride and few drops of glacial acetic acid are added from side of cover slip and heated to form hematin derivative Crystals are brownish rhombic shaped, arranged singly/ in clusters Reaction is positive if stain is old, washed or treated with chemicals, too much salt is added, moisture in acid or overheating 28 TEICHMANN/ HEMIN CRYSTAL TEST

29. A small stain sample placed under the coverslip and allow the TAKAYAMA REAGENT (sodium hydroxide, pyridine, glucose) to flow under and saturate the sample After brief heating crystals are viewed microscopically Pink feathery crystals of reduced alkaline arranged in clusters are seen Can be carried out on small stain quantity, is effective on aged stains and is more dependable 29 TAKAYAMA TEST/HEMOCHROMOGEN CRYSTAL TEST

30. Few drops of acetone are added to suspected bloodstain, followed by a drop of dilute mineral acid Characteristic are formed which can be seen under the microscope Crystals formed even when stains are old or blood partially putrefied 30 WAGENAAR TEST

31. Microchemical test HAEMIN CRYSTAL TEST/ TEICHMAN’S TEST Sod. Chloride + strain extract + glacial acetic acid Yellowish brown rhombic crystals of haemin HAEMOCHROMOGEN CRYSTAL/ TAKAYAMA TEST takayama reagent + stain extract Salmon pink hemochromogen crystals

32. A wide variety of tests are available for the determination of species orig i n of an identified bloodstain. Most tests use immunoprecipitation to effect a result 32 SPECIES IDENTIFICATION

33. Species specification test PRECIPITIN TEST When human serum is injected in an experimental animal, antibodies develop. When any human serum is brought in contact with this animal serum, the antibodies and protein will react and a visible precipitate will develop

34. Employs simple diffusion between 2 liquids in contact inside a test tube The 2 liquids are: the antiserum and an extract of the bloodstain in question T he antiserum is placed in a small tube and a portion of human bloodstain extract is carefully layered over the denser antiserum Dissolved antigens and antibodies will begin to diffuse into the other layer A fine line of precipitate at the interface of the 2 solutions 34 RING PRECIPITIN TEST

35. Also known as Haemagglutination inhibition test When human globulin is mixed with antihuman globulin serum, the latter is absorbed and is no longer capable of agglutinating Rh + ve red cells sensitized with incomplete anti D. Test detects globulins 35 ANTIGLOBULIN CONSUMPTION TEST

36. Involves the use of agar gel plates with wells for both antibodies and antigens The 2 reactants diffuse into the gel, where the soluble antigens and antibodies form an insoluble complex- a precipitate 36 OUCHTERLONY METHOD

37. Involves both quantitative and qualitative determination of blood sample Variant of the Ouchterlony method Under influence of an electric field, the antigen and antibody migrate towards each other and a precipitate is formed at the point of their interaction 37 CROSSED OVER ELECTROPHORESIS

38. A saline extract of bloodstain is mixed with dilute suspension of latex particles sensitized with antiserum A positive reaction is shown by agglutination of the particles into clumps 38 LATEX TEST

39. 39 TAKAYAMA TEST

40. 40

41. 41 THANK YOU !