Phenolphthalein (Kastle–Meyer) Test for Blood Detection 

Blood Testing Techniques

Forensic testing and blood analysis are crucial in crime scene investigation. Here are some key aspects:

- Bloodstain Pattern Analysis: Examines the shape, size, and distribution of bloodstains to reconstruct crime scenes and determine the sequence of events.

- Blood Testing: Various tests, such as the Kastle-Meyer test, can detect the presence of blood, even if it's been cleaned or washed away.

- DNA Analysis: Blood samples can be analyzed for DNA, which can help identify individuals or link evidence to suspects.

- Blood Typing: Determines an individual's blood type (A, B, AB, or O) and can be used to exclude suspects or confirm identity.

These techniques help investigators piece together the events surrounding a crime and build cases against suspects.

Preparation of Reagent

The Kastle-Meyer test uses a phenolphthalein solution, which is prepared as follows:

- Phenolphthalein solution:

1. Dissolve 2 grams of phenolphthalein powder in 100 mL of ethanol (ethyl alcohol).

2. Add 20 grams of potassium hydroxide (KOH) to the solution.

3. Add a small amount of zinc powder (reducing agent) to the solution.

4. Boil the solution until it becomes colorless or pale yellow.

Important: The solution is light-sensitive and should be stored in a dark bottle. The test reagent is ready to use after preparation.

The Kastle-Meyer test relies on the reaction between phenolphthalein and hemoglobin in blood, producing a pink color in the presence of hydrogen peroxide (H2O2).

Phenolphthalein (Kastle–Meyer) Test for Blood Detection

Principle

The Kastle–Meyer test is a presumptive (screening) test used to detect the possible presence of blood.
It relies on the peroxidase-like activity of the hemoglobin present in red blood cells.
Hemoglobin catalyzes the oxidation of phenolphthalin (the reduced form of phenolphthalein) by hydrogen peroxide (H₂O₂), producing a pink color if blood is present.

Chemical Reaction

Phenolphthalin (colorless) + H2O2→HemoglobinPhenolphthalein (pink)+H2O{Phenolphthalin (colorless)} + H₂O₂ {Hemoglobin} \text{Phenolphthalein (pink)} + H₂O Phenolphthalin (colorless) + H2​O2​Hemoglobin​Phenolphthalein (pink)+H2​O

Reagents Required

1. Phenolphthalein reagent (Kastle–Meyer reagent)
• Prepared by reducing phenolphthalein with zinc in an alkaline solution to form phenolphthalin.
2. Hydrogen peroxide (3%)
3. Ethanol or distilled water (for wetting the sample)

Procedure

1. Moisten a sterile cotton swab or filter paper with distilled water or ethanol.
2. Rub the suspected stain gently with the swab to collect a small portion of the sample.
3. Add 1–2 drops of the phenolphthalein reagent to the swab.
• No color change at this step (it should remain colorless).
4. Add 1–2 drops of hydrogen peroxide to the same swab.
• If blood is present, a bright pink color appears almost immediately.

 Interpretation of Results

Observation	Interpretation
Rapid pink color	Presumptive positive for blood
No color change	Negative result
Delayed or faint pink	May be weakly positive; further confirmatory tests required

Confirmatory Tests (after positive Kastle–Meyer)

• Teichmann (Hemin crystal) test
• Takayama (Hemochromogen crystal) test
• Immunochromatographic tests (ABAcard® HemaTrace) or other DNA-based confirmatory methods

Limitations

• It is not specific for human blood — other substances with peroxidase activity (e.g., certain plant materials, rust, vegetables) can give false positives.
• A very old or degraded blood stain may give false negatives due to loss of enzyme activity.

Safety and Precautions

• Use gloves and safety goggles — avoid direct contact with biological samples.
• Dispose of swabs in biohazard waste.
• Always follow up presumptive tests with confirmatory tests.