Confirmatory Tests of Blood

Objectives

• Confirmatory Blood Tests

• Takayama Test (Haemochromogen Test)

• Teichmann Test (Haemin Crystal Test)

• Spectroscopic Examination

• Immunological Tests (Antigen–Antibody Reaction)

Confirmatory Blood Tests

Confirmatory blood tests are highly specific tests used to definitively confirm the presence of blood.

Unlike preliminary tests, which may sometimes produce false positive results, confirmatory tests detect hemoglobin or its derivatives using microscopic, chemical, or spectroscopic methods.

Common confirmatory tests for blood include:

• Takayama Test (Haemochromogen Test)

• Teichmann Test (Haemin Crystal Test)

• Spectroscopic Examination

• Immunological Tests

Takayama Test (Haemochromogen Test)

Principle

This test detects hemoglobin by forming pink needle-shaped crystals of pyridine haemochromogen.

Takayama Reagent Composition

Standard glucose solution (100 g/100 ml)3 ml

10% Sodium hydroxide - 3 ml

Pyridine - 3 ml

Distilled water - 7 ml

Reference: Manual, Directorate of Forensic Science, MHA, Government of India.

Procedure

1. Place a small drop of the suspected blood sample on a clean glass slide.

2. Cover it with a cover slip.

3. Add 2–3 drops of Takayama reagent and allow it to flow under the cover slip.

4. Heat the slide gently at about 65°C for 10–12 seconds.

5. Allow the slide to cool for 2–3 minutes.

6. Observe under a microscope at 100× magnification.

Result

The presence of pink needle-shaped crystals of pyridine haemochromogen (pyridine ferroprotoporphyrin) confirms the presence of blood.

Teichmann Test (Haemin Crystal Test)

Principle

This test detects hemoglobin by forming brown rhombus-shaped crystals of haemin.

Reagent Composition

Potassium chloride / Potassium bromide / Potassium iodide - 0.1 g

Glacial acetic acid - 100 ml

Procedure

1. Place a small drop of the suspected blood sample on a glass slide.

2. Cover it with a cover slip.

3. Add 1–2 drops of the reagent and allow it to flow under the cover slip.

4. Heat the slide gently at about 65°C for 10–20 seconds.

5. Allow the slide to cool.

6. Observe under a microscope at 400× magnification.

Result

The presence of brown rhombus-shaped crystals of ferroprotoporphyrin (haemin crystals) confirms the presence of hemoglobin.

Spectroscopic Examination

Principle

Hemoglobin absorbs light at specific wavelengths, which allows identification through spectroscopic analysis.

Solutions Used

1. 0.2% Sodium lauryl sulphate in water

2. 0.2% Mercaptoethanol in 1% NH3 solution

Procedure

1. Prepare a solution of the suspected blood sample.

2. Extract the sample using the above solutions.

3. Place the extracted sample in a spectrophotometer.

4. Measure absorbance at:

   • 540 nm (oxyhemoglobin)

   • 575 nm (deoxyhemoglobin)

Result

Characteristic absorption peaks at these wavelengths confirm the presence of hemoglobin.

Immunological Tests (Antigen–Antibody Reaction)

Principle

These tests detect human-specific hemoglobin using antigen–antibody reactions.

Procedure

1. Mix the suspected blood sample with anti-human hemoglobin antibodies.

2. Observe for agglutination or color change.

Result

A positive reaction confirms the presence of human blood.