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How to test Blood: Confirmatory Tests in Forensic Practice


Preliminary examination of blood stains do not confirm the presence of blood. It merely indicates that the positive stain might be due to blood.
In order to confirm the identity of blood, there are certain crystal and spectroscopic tests that are regularly employed in the forensic science laboratories.

1. Takayama Test:

Reagent Preparation:

Standard Glucose Solution (100g/100ml) 3 ml
10% Sodium hydroxide (NaOH) 3 ml
Pyridine 3 ml
Distilled Water 7 ml
Reagents should be made fresh daily.


Procedure:
1. Place material to be tested on a microscopic slide and cover with a cover slip.
2. Add a drop of Takayama Reagent and allow to flow under the cover slip.
3. Warm slide gently at 65oC for 10-20 seconds
4. Allow to cool and observe under microscope at 100X magnification.
The appearance of pink needle shaped crystals of pyridine hemochromogen (Pyridine ferroprotoprophyrin) is positive reaction for heme.

2. 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.
2. Apply the Reagent so as to flow under the cover slip.
3. Warm slide gently at 65oC for 10-20 seconds.
4. Allow to cool and observe under microscope at 100X magnification.
The appearance of brown rhombohedron shaped crystals of ferroprotoprophyrin chloride is a positive reaction for heme.

3. Spectroscopic Test:

Reagent Preparation:
Solution No 1: 0.2% Sodium lauryl sulphate in water
Solution No 2: 0.2% Mercaptoethanol in 1% NH3 (Ammonia) solution
These reagents will keep approximately 4 days.

Procedure: 
  1. To a small stained thread, add 10 ml of Solution No 1.  
  2. Incubate at 37oC for 15-20 minutes.
  3. Add 10 of Solution No 2 and mix.
  4. Transfer liquid to a microcappilary cuvette.
  5. On a UV-Vis Spectrophotometer, monitor the reaction at 560 mm against a reaction blank until absorption reaches maximum. 
  6. When the reaction is complete, after 5-10 minutes, scan the sample between 600 and 500 nm. Two peaks, which are clearly defined at 558 nm and 529 nm, indicate the presence of haemoglobin derivatives.
Note: Oxyhaemoglobin exhibits absorption peak at 576 and 538 nm.

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