Presumptive Test for Blood

Presumptive blood tests (or screening tests) are spot tests that are easy, economical, and quick to perform. The
results are evaluated within a few minutes. Most of the blood presumptive tests can develop color to a dilution
level of 1:1,00,000.

Reason for Not Getting Positive Result

There are major because of:
 Blood is not present.
 Due to the presence of certain chemicals.
 The blood dilution ratio is not of considerable value.

Presumptive Color Test for Bloodstains- A color test for blood is based on the action of the peroxidase
enzyme in RBCs. Reagents are chosen based on their reaction to activate the peroxidase with a corresponding
color as the sign of blood.

The following is the general reaction for the presumptive blood color test.
H2O2 + reduced reagent (color 1) ↔ H2O + oxidized reagent (color 2)
It is based on the blood oxidizing potential which becomes apparent through a change in color.
Table of All Presumptive Blood Test With Their Color

Presumptive Test For Blood Reagent Color

Adler Test (Benzidine Test) Benzidine + H2O2 Blue

Phenolphthalein (Kastle-Meyer Test) Phenolphthalin + H2O2 Pink

Orthotolidine Test O-tolidine + H2O2 Blue

Leucomalachite Green (LMG) Test LMG + H2O2 Blue-green

Tetramethylbenzidine (TMB) Test TMB + H2O2 Blue-green

Guaiacum Test alpha-guaiaconic acid + H2O2 Deep blue

Aloin Test Barbaloin + H2O2 Red

3-amino-phthalhydrazide + sodium
Luminol Blue-white
carbonate+ sodium perborate + H2O2

Bluestar Commercial alternative of luminol Intense blue-white light

Fluorescein Fluorescin + UV Green fluoresces

The following are the most common examples of presumptive color test for bloodstains:

1. Adler Test (Benzidine Test)

2. Phenolphthalein (Kastle-Meyer Test)
3. O-Tolidine Test (Kohn test)
4. Leucomalachite green (LMG Test)
5. TetraMethylbenzidine [TMB]
6. Guaiacum Test
7. Aloin Test
8. Other common test
These are 3 types of presumptive blood tests with respective examples.

1. Color Test (visible color product)

2. Chemiluminescent Test
3. Fluorescent Test
They are either recognized as the visible color reaction or result in the release of light as
a result of the catalytic properties of blood.

1 Adler Test (Benzidine Test)

Adler test (benzidine) test is considered to be one of the oldest tests. The test was first developed by Adler (so
as on his name) in 1904. Due to carcinogenic in 1976, it was completely banned as one of the presumptive tests
for blood in the US.

Principle and Reaction

The principle is based on the reaction between the benzidine substrate as the coloring agent that flourishes the
dark blue color on reacting with blood in presence of hydrogen peroxide.

Reagent Preparation

A. Preparation of Benzidine Reagent

1. Dissolve 0·25 g of benzidine in 50 ml of glacial acetic acid
2. Add 50 ml of 2N cupric acetate solution to it.
3. Store the solution in a dark bottle.
4. Solution can be used for up to 2 weeks.
Preparation of 3% Hydrogen Peroxide
1. Take 10 ml of 30% hydrogen peroxide.
2. Dissolve it in 90 ml of distilled water and mix well.
3. Store in cool condition.
4. Solution can be used for up to 1 year.
Procedure
The following is the procedure for benzidine (Adler test):
1. Take a small amount of blood sample in a petri dish.
2. Add a few drops of benzidine reagent to the sample.
3. Add 3% hydrogen peroxide to it.
4. Wait up to 20 seconds.
Advantage of Benzidine Test
1. Develop color even with old bloodstains
2. Can develop stains that are exposed to heat or cold.
 3. Develop color reaction with a blood dilution up to 1:300,000.
Disadvantages of Benzidine Test
1. Some false-positive results
2. Benzidine fumes are highly carcinogenic.
3. Reagents such as glacial acetic acid should be handled with care, as it is highly corrosive.

#2 Phenolphthalein (Kastle-Meyer Test)

Firstly used for detection of plant peroxidases by J.H. Kastle (1901).In 1903, meyer used the test for the
detection of ythe blood and pus fluids. That’s the reason why it is called the Kastle Meyer Test.

Principle And Reaction

The principle of the Kastle Meyer test is based on the resultant reaction in which phenolphthalein—a colorless
compound, catalyzed by heme with hydrogen peroxide as the oxidant. The resultant reaction consists of reduced
phenolphthalein (phenolphthalin) in an alkaline solution to produce phenolphthalein of bright pink color.

Preparation of Kastle Meyer Test

A. Preparation of Phenolphthalein Stock Solution
1. Dissolve 1gm of Phenolphthalein (Aldrich Co.) in a little amount of distilled water in the beaker.
2. Add 10 grams of potassium hydroxide to the beaker.
3. Make the total solution up to 50 ml by adding distilled water.
4. Reflux the solution with 10 gm of powdered zinc and mix the solution for 2 hrs or until the solution
becomes colorless.
B. Preparation of Phenolphthalein Working Solution
1. Pipette out 10 ml of phenolphthalein working solution in a beaker.
2. Add 40 ml of ethanol to the beaker and swing well.
Procedure of Kastle Meyer Test
1. Take a cotton swab and moisten it with 95% of ethanol.
2. Add 2-3 drops of working solution of phenolphthalein.
3. Wait for 10 seconds, and add 2-3 drops of 3% hydrogen peroxide reagent.
4. Observe the immediate color change.
Observation
 Positive Test Result: An immediate pink color is seen.
 Negative Test: No color change is seen.
Sensitivity-The sensitivity of the phenolphthalein test can be discussed with the dilution factor of blood
samples.
Test performed as Solution medium
 Immediate Pink color: seen up to the dilution level of 1:500,000.
 Within 2 seconds: up to dilution of 1:1000,000.
 Beyond the value: no positive result.
Blood on Cotton Cloth
 Immediate Pink color: seen up to the dilution level of 1:10,000.
 Beyond 1:10,000: no positive result.
False Positive Result in Kastle Meyer Test
1. Certains vegetables and fruit juices.
2. Biological fluid: saliva, and pus.
3. Bacteria, formalin, and milk.
4. With traces of Cu.
Advantage of Phenolphthalein Test
1. Known to be one of the most specific tests (presumptive) for the detection of blood with fewer false-
positive result.
2. Reagents are known to be non-carcinogenic.
 3. Develop a positive color reaction of up to dilution level of 1:1000,000 (in solution).
Disadvantages of Phenolphthalein Test
1. Less sensitive than benzidine test.
2. Hinders the DNA analysis. It reduces the amount of recoverable DNA.
3. Less sensitivity than other comparable screening tests for blood.
3 Leucomalachite Green (LMG) Test

first developed by Adler and Adler in 1904. Leucomalachite green is a diphenylmethane dye whose base form is
colorless and produces a blue-green color in the presence of blood. Principle and Reaction

The principle of the LMG blood test uses the leuco base form of malachite green (colorless) that is made to
react with blood samples to get converted into its oxidized green form in an acidic medium.

Chemical Required for the LMG Test

1. Leucomalachite Green
2. Glacial Acetic acid
3. Zinc pellet
4. 3% of Hydrogen peroxide solution
Preparation of Leucomalachite Green (LMG) Solution
1. Add 0.1 gm of LMG in a 100 ml volumetric flask.
2. To the beaker, add 66ml of glacial acetic acid.
3. Now, make the solution up to 100ml using distilled water.
4. If the color persists, add 5 gm zinc pellets and reflux the solution (boil and condense the solution).
5. At this stage, the LMG solution should be colorless.
6. Store the solution in a glass bottle and add a small amount of zinc pellets to it.
Procedure
1. Using a moistened (with ethanol) cotton swab, graze out some of the dried bloodstains.
2. 1-2 drops of LMG solution should be added.
3. Wait for 10-20 seconds. Meanwhile, if the color develops, the test is inconclusive.
4. If no color change is seen, add 3% of hydrogen peroxide to the cotton swab.
Observation
Positive Indication: Immediate blue-green color change
Sensitivity
 Up to 1: 500 blood dilution: Immediate color change
 Up to 1: 1000 blood dilution: Color develops within 5 seconds.
 Up to 1: 5000 blood dilution: Color develops within 15 seconds
 Beyond 1: 10000 blood dilution ratio: No definite color observed.
Advantages of LMG Blood Test
 More specific than other presumptive tests.
 Highly stable LMG reagent solution can be stored for a long period of time.
Disadvantage of LMG
 Sensitivity is very low as compared to other blood screening tests.
 Obstruct the DNA recovery from the blood stains.

Tetramethylbenzidine (TMB):-
TMB is known to be one of the best alternatives to benzidine and orthotolidine tests.
Tetramethyl benzidine (TMB) is a tetramethyl derivative of benzidine that is used as a screening test for blood
by giving a characteristic color of blue-green on reacting with blood (hemoglobin).
Store it in cool condition.The shelf life of the solution is one year.
Procedure For Tetramethylbenzidine (TMB) Test
Method 1: Using Liquid Blood
 In a test tube, take 0.02 ml of diluted liquid blood (Blood:water=1:5).
 To the test tube, add 0.5 ml of TMB reagent.
 Add 0.5 ml of 3% hydrogen peroxide solution.
 Note the color change (wait up to 20 seconds)
Method 2: Using Dried Blood Stain
 Take a swab and moisten it with distilled water.
 Scab the dried blood stain with a moistened swab.
 Put 1 drop of TMB on the swab.
 Add 1 drop of hydrogen peroxide solution.
 Wait up to 20 seconds for a possible color change.
Observation:-Blue-green color as the indication of blood.

A chemiluminescent test:- is a light-producing reaction. In these reactions, light is produced as the byproduct
of the electronically excited state and their electronic transition.
Luminol Test:- Its chemical name is 3-aminophthalhydrazide.Walter Specht (in 1937) who introduced the use
of luminol at crime scenes.
Principle and Reaction:-Luminol reacts with blood and hydrogen peroxide, producing blue-white to
yellowish-green light under very low light conditions (usually dark).
Luminol Reagent Preparation
 0.1g of 3-amino-phthalhydrazide
 5g of sodium carbonate
 100 ml distilled water
 Mix all the above reagents and store.
 Before use, add 0.7g of sodium perborate
Procedure:-Luminol solution is sprayed over the suspect area.
Observation
 If blood is present, blue-white to yellow-green light is seen in darker conditions.
 It should be photographed immediately. Because the light is visible for up to 30 seconds.
Advantages of Luminol
 Highest sensitivity to blood.
 Doesn’t interfere with DNA analysis.
 Even multiple applications to the same scene results in chemiluminescence.
 Can detect drained or washed one month old blood stains
Disadvantages of Luminol
 Darker conditions needed
 Up to 30 sec visible window
 Interferes with the determination of protein and enzyme genetic markers.
Blood Confirmatory Test :- To clearly depict whether the blood sample is blood or not, a special type of test is
needed to perform called confirmatory test. These special tests are highly specific to blood i.e. nearly no false-
positive result. Confirmatory blood tests depends majorly on the detection of Hb molecules in RBCs.

1. Microscopic Examination:-Examiners first have to look for the intact RBCs. They are
readily seen in cases where blood is fresh or clotted. However, when the bloodstain
dries, intact RBCs are no longer visible.
Factors Affecting Microscopic Examination of Blood
Common factors are:
 Aged stains
 Environmental factors
 Heating
 Certain bleached chemicals
A. Microscopic Examination of Intact RBCs
Microscopic examination of intact RBCs is only used in fresh or clotted blood samples. As
in dried stains, all cellular material is broken down and becomes unrecognizable.
Reagent Required
 Vibert’s fluid: A mixture of sodium chloride, mercuric chloride, and distilled water
(or normal saline).
Procedure
1. Take the stained sample and placed it inside a petri dish.
2. With a dropper, add 2-3 drops (make sure stained cloth is fully soaked) of Vibert’s
fluid.
3. Wait for at least 6 hours, remove the stain extract, and place it on a glass slide.
4. Cover the glass slide with a coverslip and observe it under a microscope.
Note: If the blood sample is in liquid form, you directly observe under a microscope. If
stain is on cloth, soak the cloth sample in normal saline or Viberts fluid for at least 6
hours. However, the recommended time to be soaked is 12 hours.

Observation:-Intact RBCs confirm the presence of blood.

Species Identification From Intact RBCs

1. Human RBCs Features:
 Human RBCs are circular, biconcave, and non-nucleated.
 Average diameter of 7 μm.
 Exceptions: newborns have nucleated RBCs, and plumbism (lead poisoning).
2. Mammalian RBCs Features:
 Circular, biconcave and non-nucleated
 Exceptions: camels and yaks
3. Non-mammalian RBCs Features:
 oval, biconvex and nucleated

Microcrystal Assay Test For Blood:-they only react with the non-protein part of
Hemoglobin (porphyrins) i.e.only with the oxygen-carrying protein of RBCs.they only react with the non-
protein part of Hemoglobin (porphyrins) i.e.only with the oxygen-carrying protein of RBCs.These microcrystal
tests are used to detect the presence of haem (a part of haemoglobin) in the suspected blood sample, based on
the formation of characteristic coloured crystals, confirming the presence of blood.

Disadvantages of Microcrystal Assay Test

It comes with majorly two disadvantages:
 It is less sensitive than presumptive test, and
 Can’t define whether the blood is human or not

A. TAKAYAMA TEST:- The test was introduced in 1912 by a Japanese forensic pathologist, Masao
Takayama. It is based on the formation of distinctive needle-shaped pink-coloured crystals of pyridine
haemochromogen when a blood sample is treated with Takayama’s reagent. This test is also known as
haemochromogen crystal assay.

Preparation of Takayama’s Reagent:

To a beaker, add 3ml of 10% sodium hydroxide solution (NaOH), 3ml pyridine solution, 3ml saturated glucose
solution, and mix well. Finally, add 7ml of distilled water to the solution and mix well.
Principle:
Heat causes the red blood cells to rupture, releasing haemoglobin. During the process, haemoglobin gets
denatured and the ferrous form of iron (Fe2+) is converted to ferric form (Fe3+) in the presence of NaOH. This
oxidized haem is known as alkaline haematin. Haematin combines with pyridine to form insoluble pink needle-
shaped crystals of pyridine haemochromogen (also known as pyridine ferroprotoporphyrin). In the process,
saturated glucose solution functions as a reducing agent, which lowers the solubility of haemochromogen and
promotes the formation of numerous crystals.
Procedure:
Put a small drop of blood on a glass slide and spread it evenly.
Add 2-3 drops of Takayama’s reagent and cover the slide with a coverslip.
Heat the slide gently at 65°C for 10-12 secs.
Keep the slide to cool at room temperature for 2-3 mins.
Observe under the microscope at 40X magnification.
Observation: Pink needle-shaped crystals of pyridine haemochromogen confirm the presence of haemoglobin in
the sample.
B. TEICHMANN TEST:- This test was first introduced by a Polish anatomist Ludwig Karl Teichmann in
1853. It is based on the formation of distinctive rhombus-shaped crystals of haemin when a blood sample is
treated with Teichmann’s reagent.

Preparation of Teichmann’s Reagent:

Dissolve 0.1 mg each of Potassium chloride (KCl), Potassium bromide (KBr), and Potassium iodide (KI) in
100ml glacial acetic acid (CH3COOH).
Principle:
KCl, KBr, and KI act as sources of chloride, bromide, and iodide ions, respectively. The chloride ions react with
the iron present in hemoglobin, forming ferric chloride (FeCl3) and hydrochloric acid (HCl). The ferric chloride
then reacts with hematin, a breakdown product of hemoglobin, producing a green-colored complex known as
hemochromogen. Subsequently, hemochromogen reacts with HCl, resulting in the formation of a brown-colored
precipitate of haemin (ferroprotoporphyrin chloride), which appears as brown rhombus-shaped crystals under
the microscope. The bromide and iodide ions in the reagent also react in a similar manner with hematin, leading
to the formation of a brownish precipitate of haemin.
The acetic acid in the reagent acts as a solvent to dissolve the hemoglobin and helps to stabilize the reagent and
prevent the potassium salts from reacting with each other prematurely.
Procedure:
 Put a small drop of blood on a glass slide and spread it evenly.
 Add 2-3 drops of Teichmann’s reagent and cover the slide with a coverslip.
 Heat the slide gently at 65°C for 10-12 secs.
 Keep the slide to cool at room temperature for 2-3 mins.
 Observe under the microscope at 40X magnification.

Observation: Brown rhombus-shaped crystals of ferroprotoporphyrin chloride (haemin) confirm the presence
of haemoglobin in the sample.

Wagenaar Test :-It is a microcrystal test where blood detection is based on the formation of dark brown
crystals called acetone-chlor-hemin crystals. It was first developed in 1935 by Wagenaar.
3. Spectroscopic Examination of Blood:- Spectroscopic examination of blood (or precisely hemoglobin and its
derivative) is considered to be one of the most reliable confirmatory tests for both fresh and dried bloodstains.
In addition, the analysis is also non-destructive in nature.

Test-A small portion of the suspected stain (as small as 2mm) is put in0.5% potassium cyanide solution for
15min rest and then filtered. The filtered thus obtained is Potassium take in 1cm. cell and passed a U-V light in
a spectrometer, the absorption observed at from 300-600 micron. The absorption maximum at 422 milli micron
obtained that the presence of cyclohaemoglobin.
Observation- Characteristic dark absorption bands that differ as per Hemoglobin and its derivatives.

Detection of species origin of blood-The biological evidence has been identified n ified necer cessarily to
determine and confirm whether it is of human origin or not. If it is non-human origin, then to which species it
belongs to the species specific proteins in the bloodstains or other body tissues may be identified with the help
of species specific antibodies-

The species specific proteins from the bloodstain or tissue are extracted in normal saline (8.5 g of sodium
chloride in one liter of distilled water) or 5% ammonia solution. The following method are applied to detect the
species of origin-

Antibody (Ab)
An antibody is a component that the immune system produces in response to antigens. Thus, antigens result in
the production of antibodies. They act together to exhibit an immunological response. The general
characteristics of an antibody are as follows:
 An antibody is also known as an immunoglobulin (Ig)
 They are Y-shaped
 Glycoproteins
 Generated by plasma B-cells.
 Paratope is the name of the antigen binding site.
 Five types: IgG, IgA, IgM, IgE, and IgD.

Antigen (Ag)
(Anti = opposite; gen = anything that causes)
Immunogens are any foreign substances that, once entering our bodies, frequently cause a sequence of
immunological responses. While others, known as haptens, require the assistance of other molecules (carrier
proteins) to activate an immunological response. All of the immunogens and haptens are referred to as antigens.
 They could be polysaccharides, lipids, proteins, or peptides.
 An epitope is an antibody-binding location.

Precipitation reactions are based on the interaction of antibodies and antigens. They are based on two soluble
reactants that come together to make one insoluble product, the precipitate. These reactions depend on the
formation of lattices (cross-links) when antigen and antibody exist in optimal proportions. Precipitation
reactions can be used to determine the quantity and quality of both antigens and antibodies. This reaction occurs
when the antigens and antibodies are present in the right proportions and at the right temperature and pH