Topics: Analysis of Blood

Background: Blood, most common body fluid is commonly found at the crime scene. Various
numbers of other stains, such as ketchup or tomato juice, can make a stain that might be confused
with bloodstains. Preliminary and confirmatory tests are conducted to confirm that a sample is in
fact blood. Preliminary test indicates that the blood stain may be a blood possibility. In contrast, a
negative result suggests that blood is not present. On the basis of preliminary results, these tests
should not be considered to be conclusive. Further, confirmatory tests should be performed later.
Most of the blood tests can be categorized in following category
1. Chemical test: Chemical tests are based on the color change or chemiluminescence after
the addition of a reagent when it comes into contact with a blood stain.
2. Catalytic test: In catalytic tests, heme serves as the catalyst for the oxidation–reduction
reaction and oxidizing species formed in this reaction. In this reaction, colorless substrate
is oxidized to a produce visible color changes.
3. Immunological test: Immunological tests are based on specific binding between an
antigen and an antibody.

Presumptive Test:
Luminol Test:
Background and Principle: Luminol is a chemical that produces chemiluminescence, when react
with the iron of hemoglobin. Iron of the hemoglobin is work as a catalyst in this chemical reaction.
In this reaction, hydrogen peroxide reacts with the iron in blood to form oxygen and water in which
the luminol gains oxygen atoms while losing nitrogen and hydrogen. Further, 3-aminophthalate
compound is formed. Blue light is emitted when the electron of this excited molecules return to
the ground state.
Equipment’s and materials:
 Graduated cylinder
 Test tubes
 Luminol stock solution (2 g luminol + 15 g potassium hydroxide + 250 mL water): Luminol
solution is unstable and must be mixed before use in the experiment.
 3% hydrogen peroxide in water.
Procedure:
1. Mix the 5 ml of luminal solution and 5 ml of hydrogen peroxide solution in a clear test
tube.
2. Make the area around the test site as dark as possible.
3. Spray a thin film of the on dried blood stain on non-porous surface and watch for a faint
glow.
4. The light emitted from the surface can only be observed in a dark condition.
5. Experiment with taking pictures of the light emitted surface in dim light.
Result and Analysis:
1. It is nondestructive test both to blood and crime scene. It does not interfere with the DNA
analysis.
2. It is also react with various plant peroxides (potatoes, horseradish, red onions), metals and
cleaning products such as bleach and produces false results.

Schematic representation of the reactions between luminol and hydrogen

peroxide catalyzed by horseradish peroxidase in alkaline condition. The
catalytic mechanisms is proceed with iron of hemoglobin. The enzyme
facilitates the initial reaction of oxidation by hydrogen peroxides through
complex I and II.
Phenolphthalein (Kastle-Meyer) Test:
Background and Principle: Phenolphthalein colorless compound is catalyzed by
iron of hemoglobin with hydrogen peroxide as the oxidant. It oxidized in to phenolphthalein, which
appears the pink solution.
Equipment’s and materials:
 A sample of dried blood
 70% ethanol
 Distilled or deionized water
 Cotton swabs
 3% hydrogen peroxide
 Dropper or pipette
 Kastle-Meyer solution
Reagent Preparation: In a test tube, add 0.1 g phenolphthalein in 10.0 ml of 25% sodium
hydroxide solution and 0.1 g zinc powder to the tube. Gently heat the solution until it becomes
colorless or pale yellow. If necessary, add the distilled water to maintain the volume during
heating. Allow the solution to cool and decant the solution in to 250 ml graduated cylinder and
dilute with the 100 ml of 70% ethanol. Pour the solution in to brown bottle and store in a
refrigerator.
Procedure:
1. The dried blood stain on the surface is scraped onto filter paper.
2. A dried blood stain should be moistened by swab with water.
3. A drop of alcohol (70% ethanol) is added to the swab. The alcohol does not react with
either any chemical or blood. It does serve to expose hemoglobin in blood so that it can
increase the sensitivity of the test.
4. Add a drop or two of the phenolphthalin reagent is added to the sample, and after few
seconds, a drop or two of hydrogen peroxide solution is added to the swab.
5. Waiting for a period of time over 30 seconds swab turns pink,this is a positive test for blood
Result and Analysis:
1. The speed of this test is a major advantage, however certain chemicals and biological
tissues (vegetables such as potatoes and horseradishes) that do not contain blood can also
cause the false positive results.
2. Luminol test is more sensitive than Kastle – Meyer test. Kastle – Meyer test never perform
directly on the stains since it interferes with subsequent extraction of DNA.