Blood Stain Tests Overview: Presumptive and Confirmatory Methods

1. TESTS FOR BLOOD STAINS By Dr. Melody Joy V. Mique

2. Examination of Blood Stain  1. PRESUMPTIVE TEST a. Benzedrine Test b. Phenolphthalein Test c. Guaiacum Test d. Leucomalachite Green Test  2. CONFIRMATORY TEST  a. Microscopic Test b. Micro chemical Test  3. BLOOD TYPING a. ABO Direct Typing c. Rh Typing b. ABO Indirect Typing

3. Tests for Blood Stains  When a blood presumptive test is done at a crime or accident scene, an investigator must include the use of controls to ensure the accuracy of the result. This is because a blood presumptive test can be subject to what is known as a false positive result. This is when the characteristic color reaction is produced by a sample that is in fact not blood. As well, a false negative reaction is possible, where for some reason a blood sample does not produce the characteristic color change in the indicator chemical.

4. 1. Presumptive Test for Blood Stain a. Benzidine Test b. Phenolphthalein Test c. Leucomalachite Green Test d. Guaiacum Test

5. a. Benzidine Test = a test for blood based on its production of a blue color in a solution of Benzidine, hydrogen peroxide = A Test for blood, the suspected fluid is treated with benzidine reagent, and then treated with hydrogen peroxide ; the presence of blood is indicated by a bluish color turning to purple. Principle involved: It makes use of the peroxidase-like action of haeme group of Hemoglobin. In the presence of an oxidant like Hydrogen Peroxide, haeme group oxidizes colorless Benzedrine to produce BLUE colored products

6. a. Benzedrine Test Limitation of the Test: 1. Insoluble to blood 2. can cause false positive reaction *on substances with heme *on substances with peroxides *on substances with oxidizing agent Advantage: The test never fail to detect blood

7. b. Phenolphthalein Test = a solution of phenolphthalein, which is colorless, will turn an intense pink when added to a blood stain in the presence of hydrogen peroxide. The formation of a pink color indicates that the fluid could be, and indeed, likely may be, blood. However, confirmation requires the more detailed lab analyses. Principle involved: It makes use of the peroxidase-like action of haeme group of Hemoglobin. In the presence of an oxidant like Hydrogen Peroxide, haeme group oxidizes colorless phenolphthalein to produce pink colored products

8. b. Phenolphthalein Test Limitation: 1. can cause false positive reaction *on substances with heme *on substances with peroxides (Plants and animals), *on substances with oxidizing agent 2. Its viability decreases/Easily destroyed when stored in light transparent bottles.

9. b. Phenolphthalein Test Advantage =more sensitive than Benzidine Test( can readily detect heme component) = soluble with blood. Disadvantage: Viability of the prepared reagent, it must be kept in a dark bottle.

10. c. Leucomalachite Green Test The reagent is the dye Leucomalachite green dissolved in water along with sodium per borate (NaBO3). Principle: A blue-green color indicates a positive result. Ionic iron in the blood forms chelate (ring) structures with many organic compounds to form iron chelate ( iron chelate forms catalytic activity in oxidation rxn. Ex Peroxidase, when decomposed with H2O2 free hydroxyl radicals) LMG (colorless) + Hb --> Oxidized MG (blue green ) H2O2 + Peroxidase --> (OH-)

11. b. Leucomalachite Green Test the LMG Test is more commonly used by governmental agencies such as the FBI because it gives a faster result (catalytic action)

12. c. Guaiacum Test = one of the first developed but seldom used test. It relied on guaiacum (a resin isolated from trees) in combination with hydrogen peroxide. If a stain turned blue or bluish green when treated with these reagents, it was considered a positive result indicative of blood. Principle: The reaction of nitric to ferrous substance and oxygen of the blood

13. c. Guaiacum Test Limitation of the Test: 1. Insoluble to blood 2. It should be refrigerated and discarded after 1 month 3. can cause false positive reaction *on substances with heme *on substances with peroxides *on substances with oxidizing agent Advantage: 1. The test never fail to detect blood 2. More available than Benzedrine reagents

14. Factors that Affect Presumptive Blood Screening Forensics 1. Age 2. Temperature 3. Materials the Age of the blood that's being tested. When blood is fresh it appears red to the human eye, but over time it will darken and become brown. While the color of blood changes with age, so does its chemical composition. Hemoglobin present in the blood will change to met hemoglobin when it's exposed to oxygen over a long period of time. This chemical change in the blood can cause the test to turn out differently than with fresher blood.

15. Factors Affect Presumptive Blood Screening Forensics  2. Temperature Cold preserves blood for longer, and it can make it possible to have an accurate presumptive test much later. Heat, on the other hand, acts as a catalyst and speeds up chemical reactions in spilled blood. Regardless of which extreme is used, though, a presumptive blood screening test can be affected. The effects are positive in the case of cold since blood is preserved, and negative in the case of heat because blood is more rapidly decomposing.

16. Factors Affect Presumptive Blood Screening Forensics  3. Materials  material that the blood is found on or in. Flat surfaces that won't absorb blood are best because then blood is held in an easy- to-reach pool. Wood and concrete, though, have grains and pores, respectively, and these can prevent a blood screening test from functioning properly. The same can be said of materials that are absorbent such as cloth. Cloth provides an additional difficulty because it may be washed with bleach, which will destroy blood as far as a test is concerned.

17. 2. Confirmatory Test a. Microscopic Test Wet Preparation Streak Method b. Micro chemical Test Teichman Test Takayama Test

18. 2. Confirmatory Test A. Microscopic Test of Blood 1. Demonstrate distinction between human blood from animal blood 2. Demonstrates what kind of blood discharge it is (menstrual, wound etc. ) 3. Demonstrate blood abnormalities 2 Types of Preparation 1. Wet preparation 2. Streak preparation ( Blood Streaked)

19. 2. Confirmatory Test A. Microscopic Test of Blood 1. Wet preparation (diluting agent+ Blood) Diluting agents: Vibert’s solution ( NaCl and HgCl) Saline Solution ( salt and distilled water) 2. Streak Preparation Direct streaking of blood in a glass slide

20. 2. Confirmatory Test b. Micro chemical Test 1. Teichman Test / Heimin Test potassium bromide, potassium chloride and potassium iodide in glacial acetic acid, and is heated to react with hemoglobin Principle: The reaction first converts the hemoglobin to heamin, and then the halides react with the heamin to form characteristic brownish-yellow rhomboid crystals

21. 2. Confirmatory Test 2. Takayama Test / hemochromogen test The oldest chemical confirmation of the presence of hemoglobin or its derivatives by the formation of specific crystals. Principle: ferrous iron from hemoglobin reacts with pyridine to produce red feathery crystals of pyridine ferroprotoporphyrin.

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23. Quiz 1. discuss how do you determine the age of blood in a collected blood sample. 2. discuss what does the confirmatory test for blood demonstrate 2 Differentiate the Two Micro chemical test for blood as to  A. Their Principle  B. Positive result for blood