Preliminary Examination of Blood

Preliminary Examination of Blood

The preliminary examination of blood is an essential step in forensic investigations. These tests help determine whether a suspected stain may contain blood before performing more specific confirmatory tests. Preliminary tests are widely used at crime scenes and forensic laboratories because they are quick, sensitive, and relatively inexpensive.

Objectives

• Presumptive Tests

• Principle of Preliminary Blood Tests

• Phenolphthalein Test (Kastle-Meyer Test)

• Benzidine Test (Adler’s Test)

• Leucomalachite Green (LMG) Test

• Tetramethylbenzidine (TMB) Test

• Luminol Test

Presumptive Tests

• Preliminary blood tests, also known as presumptive tests, are used to determine whether a sample might contain blood.

• These tests are conducted to eliminate non-blood materials and are generally cheaper than confirmatory tests.

• Presumptive tests are highly sensitive but not specific, meaning substances other than blood may sometimes produce a positive result.

• If a preliminary test is positive, a confirmatory test must be performed to verify the presence of blood.

Principle of Preliminary Blood Tests

• Preliminary blood tests rely on the catalytic properties of hemoglobin, which contains iron.

• The iron present in hemoglobin facilitates the oxidation of certain reagents, leading to a color change that indicates the possible presence of blood.

Phenolphthalein Test (Kastle-Meyer Test)

• Collect the suspected blood sample using a cotton swab.

• Add 1–2 drops of 95% ethanol to lyse the cells.

• Apply 1–2 drops of phenolphthalein reagent to the sample.

• After a few seconds, add 1–2 drops of 3% hydrogen peroxide (H₂O₂).

• Observe the color change. A rapid pink color change indicates the presence of blood.

Reaction:
Fe³⁺ + Phenolphthalein + H₂O₂ → Phenolphthalein (oxidized)

Benzidine Test (Adler’s Test)

• Prepare a benzidine solution by dissolving 0.4 g benzidine in 20 ml acetate buffer.

• Apply the suspected blood sample to a filter paper.

• Add 1–2 drops of benzidine reagent.

• Add 1–2 drops of H₂O₂.

• Observe the color change. A blue-green color indicates the presence of blood.

Reaction:
Benzidine + H₂O₂ → Oxidized Benzidine

Leucomalachite Green (LMG) Test

• Prepare LMG reagent by dissolving 0.1 g LMG in 10 ml ethanol.

• Apply the suspected blood sample to a cotton swab.

• Add 1–2 drops of LMG reagent.

• Add 1–2 drops of H₂O₂.

• Observe the color change. A blue-green color indicates the presence of blood.

Reaction:
LMG + H₂O₂ → Oxidized LMG

Tetramethylbenzidine (TMB) Test

• Prepare TMB reagent by dissolving 0.1 g TMB in 10 ml ethanol.

• Apply the suspected blood sample to filter paper.

• Add 1–2 drops of TMB reagent.

• Add 1–2 drops of H₂O₂.

• Observe the color change. A blue-green color indicates the presence of blood.

Reaction:
TMB + H₂O₂ → Oxidized TMB

Luminol Test

• Prepare luminol reagent by dissolving 0.1 g luminol in 10 ml sodium carbonate solution.

• Spray the luminol reagent onto the suspected blood sample.

• Observe the reaction under UV light.

• A blue-white glow indicates the presence of blood.

Reaction:
Luminol + Sodium Perborate + H₂O₂ → Oxidized Luminol

Preliminary Examination of Blood