Presumptive and Confirmatory Tests for Blood
Presumptive Test
- A Presumptive test is a Qualitative Analysis that allows to identify, or confirm, the presence of substance in a sample.
- These determinations usually occur, after a chemical reaction, and a specific colour is produced.
- A false positive - another substance reacting the same way, producing the expected result.
- Negative result means the questioned stain is not likely Blood
- Positive Result means the Questioned Stain is likely to be Blood
1. Tetramethyl Benzidine (TMB) Test
NOTE: TMB is carcinogenic. Use of gloves is required.
Reagent preparation
Acetate Buffer
Sodium acetate anhydrous: 10 g
Glacial acetic acid: 5 mL
Distilled water: 500mL
Working Solution 1
TMB: 0.25 g
Acetate Buffer: 25 mL
Working solution 2
3% H₂O₂: 10 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.
2. A drop of TMB Solution is placed on the stain, followed by a drop of 3% Hydrogen Peroxide.
3. An immediate blue-green colour is a positive test for peroxidase activity, indicative of hemoglobin.
This is not a confirmatory test for blood.
Standards and Controls: A known bloodstain and unstained control must be tested.
Read the Beautiful Forensic Love Fiction: Mission Forensic (Click to Read)
2. Phenolphthalein Test (Kastle-Meyer Test)
Reagent Preparation
Stock Solution:
Phenolphthalein: 2 g
Potassium Hydroxide: 20 g
Distilled Water: 100 mL
Zinc Dust: 20 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: 2mL
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.
2. Two or three drops of Ethanol are placed on the stain.
3. Two drops of working phenolphthalein solution are added to the stain.
4. After waiting to insure that no colour develops at this stage, two or three drops of 3% Hydrogen Peroxide are added.
5. An intense Pink colour is a positive test for peroxidase activity, indicative of hemoglobin.
This is not a confirmatory test for blood.
Standards and Controls: A known bloodstain and unstained control must be tested.
NOTE:
Zinc powder or dust in contact with water or damp air evolves Hydrogen. The heat of the reaction is sufficient that the Hydrogen may ignite. Therefore, the Zinc should not be discarded in the wastebasket. The following procedure should be followed for less than 20 g of Zinc:
1. Follow standard laboratory procedures of wearing gloves and safety aprons. Add dilute Hydrochloric acid to the Zinc dust in a beaker. Allow the bubbles to form and slowly add more of acid till no more bubbles are seen.
2. When all the Zinc has dissolved, add Sodium Carbonate solution to the mixture. Bubbles will be formed. Keep on adding the Sodium Carbonate small quantity at a time till the Zinc precipitates as Zinc Carbonate.
3. The Zinc Carbonate can be now filtered and disposed off as it is non-toxic.
Confirmatory Tests
- Due to possibility of false positives with the presumptive tests, confirmatory tests are necessary.
- Confirmatory tests involve making crystals that detect the presence of haemoglobin.
- Crystal tests are based on the formation of heme derivative crystals such as hema-tin, hemin and hemochromogen.
1. Takayama Test
Reagent Preparation
Standard Glucose Solution (100g/100ml): 3mL
10% Sodium Hydroxide: 3mL
Pyridine: 3mL
Distilled Water (DW): 7mL
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 on a hot plate at 65°C for 10-20 seconds
4. Allow to cool and observe under microscope at 100X.
5. The appearance of pink needle shaped crystals of Pyridine Hemochromogen (Pyridine ferroprotoporphyrin) is positive reaction for heme.
2. Teichmann’s Test
Reagent Preparation
Potassium Chloride or : 0.1 g
Potassium Bromide or: 0.1 g
Potassium Iodine : 0.1 g
Glacial Acetic Acid: 100mL
Mix and store in a stoppered bottle.
Procedure
1. Place material to be tested on a microscopic slide and cover with a cover slip.
2. Let the Reagent flow under the cover slip.
3. Warm slide gently on a hot plate at 65°C for 10-20 seconds.
4. Allow to cool and observe under microscope at 100X.
5. The appearance of brown rhombohedron shaped crystals of Ferroprotoporphyrin Chloride is a positive reaction for heme.
3. Spectrophotometric Estimation
Reagent Preparation
Solution # 1: 0.2% Sodium Lauryl Sulphate in water
Solution # 2: 0.2% Mercaptoethanol in 1% NH₃ solution
These reagents will keep approximately 4 days.
Procedure
1. To a 1 cm long stained thread, add 10 ml of Solution # 1.
2. Incubate at 37 °C for 15-20 minutes.
3. Add 10 ml of Solution # 2 and mix.
4. Transfer liquid to a microcappillary cuvette.
5. On a Spectrophotometer, monitor the reaction at 560 nm 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.
Standards and Controls: Known bloodstains of various ages must be tested, Oxyhaemoglobin exhibits absorption peak at 576 and 538 nm. The apparent shift is thought to be due to the formation of reduced haemoglobin derivatives.
Source and References
1. DFSS manual; biology manual 2019 07.08.2019
2. MSc. Forensic Science (II) NFSU Class Notes.
