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How to test Blood: Preliminary Tests in Forensic Practice

Blood is an important evidence in many criminal cases. A forensic scientist frequently finds blood as an evidence of a crime.
In a forensic investigation, blood as an evidence can answer a lot of questions. For example, it can establish that a crime has taken place, it can also establish the identity of a victim and suspect. Besides, blood evidence can also provide clues as to how the act of crime had taken place.
There are several tests that can be performed on a suspected sample for the identification of blood. The standard procedure of a forensic science lab is to first apply the preliminary tests on the samples to screen out non-blood samples from the actual blood. Once the preliminary tests give positive results, the more comprehensive confirmatory tests are performed in order to establish that the positive samples are indeed blood.
Preliminary Tests for Blood: Here are some of the most common preliminary tests.
1. Kastle-meyer test:
Theory: This test is based on the fact that blood can catalyze the breakdown of hydrogen peroxide (H2O2) into water and oxidizing species.
H2O2 -----> H2O + O
Blood has this property due to the presence of peroxidase enzyme. So, technically, any material which possesses this enzyme will give this test positive.
Zinc keeps the Phenolphthalein in reduced form. This reduced phenolphthalein does not show any color. But, in presence of blood and H2O2, the color changes to Pink due to the conversion of reduced phenolphthalein into its acidic form.
Reagent Preparation:
Stock Solution:
Phenolphthalein 2.0 g, Potassium Hydroxide 20.0g, Distilled Water 100 ml, Zinc Dust 20.0 g.
Mix, add a few boiling chips and boil under reflux 2-3 hours or until the solution has lost its pink color.
Structure of Phenolphthalein
Cool and decant into a bottle containing some zinc to keep in the reduced form.
Working Solution:
Solution 1: Ethanol 10 ml
Solution 2: Phenolphthalein Stock 2 ml
Distilled Water 10 ml
Ethanol 2 ml
Solution 3: 3% Hydrogen Peroxide 10 ml
1. A small cutting, swabbing or extract of the suspected bloodstain is placed on filter paper.
2. Two or three drops of Ethanol are placed on the stain.
3. Two drops of working phenolphthalein solution are added to the stain.
4. Wait to see that no color develops at this stage
5.  Two or three drops of 3% Hydrogen Peroxide (H2O2) are added.
6. An intense pink colour is a positive test for peroxidase activity, indicative of hemoglobin.
Limitations of Kastle-Meyer test: This test is not confirmatory test for blood. There are other materials which also give this test positive. For example, potato also gives this test.
This test can detect the blood in the dilutions down to 1:107.

3. Luminol Test:
Theory: In this test also, blood catalyzes the oxidation of luminol by hydrogen peroxide in a basic solution.
Structure of Luminol
Reagent Preparation:
Luminol stock solution (2 g luminol + 15 g potassium hydroxide + 250 mL water)
3% hydrogen peroxide (H2O2) in water
potassium ferricyanide or a sterile blood lancet and sterile alcohol pad
In this test, luminol solution is mixed with hydrogen peroxide and a hydroxide (e.g., KOH) in a spray bottle.
The luminol solution is sprayed where blood might be found.
The iron from the hemoglobin in the blood serves as a catalyst for the chemiluminescence reaction that causes luminol to glow, so a blue glow is produced when the solution is sprayed where there is blood.
Limitations of Luminol test: This test is not confirmatory test for blood. Several other substances also give this test positive. For example, oxidizing agents such as sodium hypoclorite (bleach), metals (such as copper), and plant peroxidases may also cause luminescence with luminol.

3. Tetramethyl Benzidine (TMB) Test:
Reagent Preparation:
Acetate Buffer: Sodium acetate 5.0g + Glacial Acetic Acid 43.0 ml + Deionized Water 50.0 ml
Working Solution: TMB 0.4g + Acetate Buffer 20.0 ml
Mix, filter and store in brown coloured bottle in refrigerator.
1. Place a cutting or swabbing of the stain on filter paper or spot test paper.
2. A drop of TMB Solution is placed on the stain, followed by a drop of 3% Hydrogen Peroxide.
3. An immediate blue-green colour is a positive test for peroxidase activity, indicative of hemoglobin.
Note: TMB is carcinogenic therefore its use is extremely limited. In some laboratories, it is no longer used.


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