Question

Describe the identification methods of blood? Forensic significance of biological materials.

Answer 1

The Forensics of Blood 

After a homicide or an assault has been committed, police investigators usually find blood at the scene of the crime, giving them clues as to what happened. The blood’s texture and shape and how it is distributed around the victim often help investigators determine when the crime was committed, whether the crime was preceded by a fight between individuals, and which weapon was used—say, a knife, a gun, or an object used to hit a person.

Physical Examination: In natural light examination of exhibits for brown, reddish brown stains, powder or crystals of reddish brown colour, these areas should be demarcated. In case of absence of clear and visible stains, washed stains should be examined under 230-269 nm frequency UV light.

Luminol Tests: The luminol is first activated with an oxidant, usually a solution of hydrogen peroxide and a hydroxide salt in water. When luminol reacts with the hydroxide salt, a di-anion is formed. The oxygen produced from the hydrogen peroxide then reacts with the luminol di-anion. Luminol is sensitive to the presence of extremely small amounts of blood. 

Fig 1. A chemical reaction between hydrogen peroxide (H2O2) and luminol (molecule pictured to the left) in the presence of hemoglobin (Hb) produces 3-aminophthalic acid (3-APA) and blue light.

Presumptive Test: These suspected bloodstains; contaminated materials should be tested for positive for blood.

Tetramethyl Benzidine (TMB) Test:

NOTE: TMB is carcinogenic. Use of gloves is required.

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.

Procedure:

1. Place a cutting or swabbing of the stain on filter paper or spot test paper.

1. A drop of TMB Solution is placed on the stain, followed by a drop of 3% Hydrogen Peroxide.

1. An immediate blue-green colour is a positive test for peroxidase activity, indicative of hemoglobin. This is not a confirmatory test for blood.

OR

Phenolphthalein Test (Kastle-Meyer Test):

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 colour. 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

Procedure: 

1. A small cutting, swabbing or extract of the suspected bloodstain is placed on filter paper or spot test paper. 

1. Two or three drops of Ethanol are placed on the stain. 

1. Two drops of working phenolphthalein solution are added to the stain. 

1. After waiting to insure that no colour develops at this stage, two or three drops of 3% Hydrogen Peroxide are added. 

1. An intense pink colour is a positive test for peroxidase activity, indicative of hemoglobin. This is not a confirmatory test for blood.

Confirmatory Test: Stains positive for the presumptive test should be further examined by the following tests:

Takayama Test:

Reagent Preparation:

 Standard Glucose Solution (100g/100ml) 3 ml

 10% Sodium hydroxide 3 ml

 Pyridine 3 ml

 Distilled Water 7 ml

Note: Reagents should be made fresh daily.

Procedure:

1. Place material to be tested on a microscopic slide and cover with a cover slip.

1. Add a drop of Takayama Reagent and allow to flow under the cover slip.

1. Warm slide gently on a hot plate at 65oC for 10-20 seconds

1. Allow to cool and observe under microscope at 100X.

1. The appearance of pink needle shaped crystals of pyridine hemochromogen (Pyridine ferroprotoprophyrin) is positive reaction for heme.

OR

Teichmann’s Test:

Reagent Preparation:

Potassium Chloride or 0.1 g

Potassium Bromide 0.1 g

Potassium Iodine 0.1 g

Glacial Acetic Acid 100 ml

Mix and store in stoppered bottle.

Procedure:

1. Place material to be tested on a microscopic slide and cover with a cover slip.

1. Let the Reagent flow under the cover slip.

1. Warm slide gently on a hot plate at 65oC for 10-20 seconds.

 4. Allow to cool and observe under microscope at 100X.

1. The appearance of brown rhombohedron shaped crystals of ferroprotoprophyrin chloride is a positive reaction for heme.

Forensic significance of biological materials:

Biological fluids such as blood, semen, and saliva are frequently encountered as physical evidence in many types of criminal investigations such as homicides, sexual assaults, assaults, and robberies. Since blood, semen, and saliva originate as liquids, they quickly coat or penetrate surfaces they are deposited on, and can be difficult to remove when dried. Because no two humans are genetically the same (except for identical twins) these body fluids are unique to the person they originated from. By performing DNA analysis of these fluids or stains, a genetic marker profile can be obtained which can then be compared to DNA profiles obtained from reference standards or from other items of evidence.