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Presumptive Tests for Blood: 11 Important Forensic Test Included

Presumptive Tests for Blood: 11 Important Forensic Test Included

Blood at the crime scene? The first question would be, “Is the suspected bloodstain is possibly the blood or not?” For that, an examiner has to perform one of the presumptive tests for blood on the field.

Table of All Presumptive Test With Their Color

Presumptive Test For BloodReagent Color
Adler Test (Benzidine Test)Benzidine + H2O2Blue
Phenolphthalein (Kastle-Meyer Test)Phenolphthalin + H2O2Pink
Orthotolidine TestO-tolidine + H2O2Blue
Leucomalachite Green (LMG) TestLMG + H2O2Blue-green
Tetramethylbenzidine (TMB) TestTMB + H2O2Blue-green
Guaiacum Test alpha-guaiaconic acid + H2O2Deep blue
Aloin Test Barbaloin + H2O2Red
Luminol3-amino-phthalhydrazide + sodium carbonate+ sodium perborate + H2O2Blue-white
BluestarCommercial alternative of luminolIntense blue-white light
FluoresceinFluorescin + UVGreen fluoresces

What is a Presumptive Test for Blood?

Presumptive tests (or screening tests) for blood are spot tests that are easy, economical, and quick to perform. The results are evaluated within a few minutes. Plus, it majorly doesn’t need too many blood samples—because of specificity.

Most of the blood presumptive tests can develop color to a dilution level of 1:1,00,000.

Though a presumptive test offers high sensitivity, one has to perform a confirmatory test (because of low specificity) to absolutely specify whether the given sample is blood or not.

So, if a screening test calls off to be positive. You must have to perform the confirmatory test.

Conversely, if the presumptive test is found to be negative, the examiner doesn’t need to do any further labor.

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.

Types of Presumptive Tests for Blood

Types of Presumptive Tests for Blood

In general, there are three broad categories on which the presumptive tests are divided to justify whether the given stain is blood or not?

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. 

These are 3 types of presumptive blood tests.

  1. Color Test (visible color product)
  2. Chemiluminescent Test
  3. Fluorescent Test

A. Presumptive Color Test for Blood Stains

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 color test of blood.

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.

The following are the most common color test for blood stains:

  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

#1 Adler Test (Benzidine Test)

Adler Test  or Benzidine Test for bloodstains

Adler test (benzidine) test is considered to be one of the oldest tests that are used for screening whether the given sample is blood or not.

The test was first developed by Adler (so as on his name) in 1904.

The test was used for half the decade, till in 1946, it opted to be carcinogenic. And 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.

The following chemical reaction is involved.

Adler Test or Benzidine Test chemical reaction

Please Note: In some cases, with the initial addition of benzidine solution only, the sample may impart dark blue color. This can happen due to chemical oxidants. And in that case, the test should be held inconclusive. 

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.

B. 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.
Observation and Sensitivity

The appearance of dark blue color is the indication that the possible sample has blood.

If the blood sample has a dilution ratio of 1:100,000, immediate dark color will appear.

However, if the blood sample is diluted with a ratio of 1:300,000, it will take up to 20 seconds to impart color.

False Positive Result in Benzidine Test
  • Certain vegetables and fruit juices.
  • Biological fluid: milk, saliva, and pus.
  • Bacteria and formalin
Advantage of Benzidine Test
  1. Develop color even with old bloodstains
  2. Can develop stains that are exposed to heat or cold.
  3. Blood treated with a cleaning agent can also be characteristically categorized.
  4. 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)

Phenolphthalein or Kastle-Meyer Test for blood

The emergence of the phenolphthalein test was from the discovery of a technique for the detection of plant peroxidases by Joseph H. Kastle and Oliver March Shedd (1901).

Later in 1903, Meyer used the test for the detection of 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.

The pink color of phenolphthalein is carried out only in alkaline conditions.

Phenolphthalein or Kastle-Meyer Test reaction
Reagent 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.
  5. You can perform a side-by-side negative control test by using distilled water.
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.

False-positive tests could also be seen when no time is given between the addition of phenolphthalein and hydrogen peroxide.

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 Orthotolidine Test

O-tolidine is known to be the derivative of benzidine and follows the same principle that benzidine has. The reaction of orthotolidine reagent in the presence of peroxide with blood leads to the production of blue color as the sign of blood. 

For, principle, reagent preparation, procedure, pros, and cons are discussed in our dedicated article Orthotolidine Test (Kohn & O’Kelly test)

#4 Leucomalachite Green (LMG) Test

Leucomalachite Green (LMG) blood Test

The use of Leucomalachite as the presumptive test was first developed by Adler and Adler in 1904. Earlier, in their study, they used LM green and violent. But later, many researchers settle on the LM green for blood screening. 

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.

The following reaction involved an LMG presumptive test for blood.

Reaction involved in Leucomalachite Green (LMG) Test
Reagent Preparation (Mc Phail’s Reagent or Hemident)

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.

The second reagent is 3% of Hydrogen peroxide, which is already stated in the benzidine test.

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.
False Positive and Specificity

LMG is considered the least sensitive towards blood as compared to other presumptive tests for blood.

But, LMG serves a remarkable advantage. It doesn’t give false-positive results to most vegetables and fruits that are seen in other peroxidase-based presumptive blood tests like the benzidine test.

False-positive result in the LMG test is seen in:

  • Hypochlorite compound: present in bathroom and kitchen cleaning agents and some washing powders.
  • Ferrous sulfate: a distinctive blue-green color (turquoise color).
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.

#5 Tetramethylbenzidine (TMB) Test

Tetramethylbenzidine (TMB) is a tetramethyl derivative of benzidine that is used as a screening test for blood by giving a characteristic color of blue-green in an acidic medium.

This color test proceeds in the presence of oxidizing agents such as hydrogen peroxide.

Moreover, TMB is known to be one of the best alternatives to benzidine and orthotolidine tests.

You can check our dedicated post on Tetramethylbenzidine (TMB) Test for the reaction, principle, procedure, false-positive result, advantages, and disadvantages.

#6 Guaiacum Test For Blood

Guaiacum Test for blood forensic

The Guaiacum test is known to be the oldest forensic blood identification test that imparts a deep blue color.

It was first proposed by Van Deen in 1862 and served for forensic purposes for more than 40 years until it was replaced by a benzidine test.

The Guaiacum test for blood is also known as Van Deen’s test or Day’s test.

For acknowledgment, Guaiacum (alpha-guaiaconic acid) is a resin isolated from Guaiacum Officinale and Guaiacum sanctum trees.

Principle and Reaction

Guaiaconic acid uses the same principle of peroxidase action on the blood. α-guaiaconic acid on reacting with blood, it imparts a deep blue color.

The following are the reactions involved in Guaiacum presumptive tests for blood.

Guaiacum Test reaction
Reagent Required
  1. Guaiacum resin (α-guaiaconic acid) strips
  2. Hydrogen peroxide
Procedure 
  1. Take guaiacum resin strips and place it on a china dish.
  2. Dilute the guaiac resin strip with a few drops of the suspected blood sample.
  3. Add the hydrogen peroxide dropwise to the resin strip.
  4. Note any immediate color change.
Observation
  • Positive detection of blood indicated by deep blue color.
Sensitivity
  • Up to 1:10,000: Immediate color change
  • Up to 1: 40,000: Develops within 15 seconds
  • Beyond 1: 40,000: No positive result.
False Positive and Specificity

There is a lot of false-positive result including:

  • Vegetable extracts
  • Milk casein 
  • Tanned sheep
  • Nasal mucus and pus,
  • Bile and gastric secretions.
Advantages
  • Easy to perform with a decent sensitivity.
Disadvantages
  • Guaiac resins are expensive.
  • Very low specificity towards blood.
  • Not currently employed for forensic blood identification.

#7 Aloin Test

Aloin test is similar to Van Deen’s test but develops a bright red product instead of deep blue.

Alion is extracted from aloes, a genus of plants of the family Liliaceae. By composition, Alion is a mixture of pentosides of aloes. Majorly, Barbaloin is the major ingredient. 

It was first employed by Klunge (1883) as a detection test for blood.

However, the Aloin blood test never got the popularity as van Deen’s test. Later, Sutherland 1907, states that it was a good confirmatory negative test.

#8 Other Forensic Presumptive Catalytic Test for Blood

I. 2,7 Diaminofluorene

In 1932, Schmidt and Eitel used Diamine derivatives of fluorene 2,7-fluorenediamine for blood identification. However, it suffers from the stability issue of the reagent.

Later, the test is used for the detection of various halides, nitrate, persulfate, and several metals which could lead to false-positive results for blood. 

This is the reason why the test is forensically terminated for possible detection of blood.

B. Eosin 

Ganassini (1910) uses eosin reagent for blood identification as a momentary yellow-to-red colored product in strong alkali with hydrogen peroxide.

Eosin reagent was prepared by heating the crystalline form of eosin in a strong base followed by washing with acid precipitate. 

Later, in 1911, Belussi questioned the sensitivity and false-positive result of Ganassini’s test. He showed the sensitivity of Belussi was far lower than benzidine.

C. Rhodamine B

Rhodamine B is of red color and was first demonstrated by Fuld in 1917. 

Preparation of Rhodamine B Reagent: Rhodamine B + Base + Zinc dust.

The sensitivity of the Rhodamine B blood test is 1: 105 to 1: 106

This test was majorly used as the blood that:

• putrefied blood, or 
• blood heated above 200° C.

However, the test doesn’t get that much popularity because Diels (from Ziemke notes, 1938) showed that chlorophyll can also give a positive response to Rhodamine B.

However, it doesn’t react with rust that most of the  other presumptive reagents do.

D. 2,7-Fluorenediamine and 2,5-fluorenediamine

A. Inagaki et.al. uses peroxidase activity for the hematological (the science of blood) uses. They use two Diamine derivatives of fluorene i.e. 2,7-fluorenediamine and 2,5-fluorenediamine. Their results show the positive detection of blood even washed by water.

Reagents:

Method 1: Saturated solution of 2,7-fluorenediamine + Hydrogen peroxide

Method 2: 0.05 per cent solution of 2,5-fluorenediamine + Hydrogen peroxide

Positive Result: Clear brown granules in cytoplasms

B. Presumptive Chemiluminescent Test for Blood Stains

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.

In this, chemicals react to form an excited unstable intermediate that breaks down, releasing energy in the form of photons of light, and finally reaches its ground state.

The most common presumptive test that is used for blood detection at the crime scene is Luminol. However, Bluestar is another major alternative to luminol.

1. Luminol Test For Blood

Luminol Test For Blood

Luminol is 3-aminophthalhydrazide, was first synthesized in A. Schmitz, Heidelberg, in 1902. However, it was first observed by W. Lommel (Germany) in 1928.

From per forensic point of view, it was 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).

Reaction involved:

mechanism for Luminol test chemical reaction
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. 
Sensitivity of Luminol
  • Can detect blood with dilution level of 1:100,000
  • Know to be the best presumptive blood test as per sensitivity
False Positive Result in Luminol
  • Household items such as bleach
  • Vegetables such as turnip, beetroot, potatoes, and horseradish
  • Metals such as copper, steel, and black iron
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

2. Bluestar

Bluestar is an alternative to luminol.

Though Bluestar is based on luminol but it is better than it.

It is better in;

  • Developing intense characteristics light,
  • Ease to make a solution,
  • Available in versatile options: tablets, liquid, and pourable powder,
  • No health risk

C. Fluorescent Presumptive Test for Blood

Chemiluminescence is different from luminescence (Fluorescence). 

Chemiluminescence is due to the release of energy while transitioning from an excited state to the ground state. While luminescence is the production of light as the release of earlier absorbed light or other electromagnetic radiation

This is the reason why, a fluorescence substance is needed to be exposed to a particular wavelength of light (majorly shorter, like UV) and emits longer wavelengths of electromagnetic radiation.

Fluorescein Test For Blood

Fluorescein blood test forensic

Most common luminescence reagent is Fluorescein. It was first proposed by Fleig in 1910. 

Fluorescein is an organic compound that is prepared from heating phthalic anhydride and resorcinol in the presence of zinc catalyst. Before the reaction, it is a deep red color. 

However, on reaction with heme in alkaline solution, it exhibits an intense green fluorescence.

Other names:

  • Solvent Yellow 94
  • Resorcinol phthalein
  • Yellow fluorescein
Principle and Reaction Involved

While preparing Fluorescein is of deep red color. But in reaction with heme, the fluorescin releases radiation that was firstly absorbed by the treatment of UV light that accelerates the oxidation to form fluorescein (a green color fluorescent light).

Reaction involved:

Fluorescin react with h202 and give fluorescein

Please Note: Fluorescin and Fluorescein (with an extra ‘e’) are different. Fluorescin is a reduced form of Fluorescein.

Fluorescein Reagent Preparation
  • 0.1 g fluorescein
  • 1 g of Sodium hydroxide pellet
  • 2 g powdered zinc
  • 20 ml distilled water
  • Heat the solution at low flame for 1 minute

Working Solution of Fluorescein: For field operational use, take a part of the fluorescein reagent and dilute it with a 1:100 ratio.

Procedure
  • Lightly spray the solution in the form of mist over the suspected area.
Observation
  • Intense green fluoresces is seen after it was exposed to an alternative light source, range of 425 nm to 485 nm
Sensitivity
  • Sensitivity of blood dilutions on white filter paper is approximately 1×104
  • Sensitivity depends upon the amount of blood dilution
  • Capable to detect blood dilution level up to 1:100,000
False Positive Result
  • Household items such as bleach and cleansers
  • Urine, with or without blood
  • Iron, copper, and hypochlorite
  • Fruits and vegetables in Kastle Meyer test (listed above)
Advantages of Fluorescein Test
  • Highly diluted blood can be detected
  • Intense green color
  • Reagent is stable and can be stored for months at 4°C
Disadvantages Fluorescein Test
  • Used extra equipment such as alternative light source

Reference

Books

  • Saferstein, Criminalistics: An Introduction to Forensic Science
  • Siegel, Forensic Science: The Basics 
  • Nordby, Forensic Science: An Introduction to Scientific and Investigative Techniques
  • Sourcebook in Forensic Serology, Immunology, and Biochemistry
  • Anil Aggrawal, Essentials of Forensic Medicine and Toxicology
  • Reddy, The Essentials of Forensic Medicine and Toxicology

Research Papers and Journals

  • Adler, 1904. Certainly more organic about behavior Connections to blood with special consideration of the Evidence of blood
  • The Kastle-Meyer Test for the Detection of Blood [NCBI.NLM.Gov]
  • Kohn, J. and T. O’Kelly, 1955. An ortho-tolidine method for the detection of occult blood in faeces. [PubMed]
  • Lee, et.al., An evaluation of fluorescin as a presumptive test reagent for blood.
  • Lucas A. Forensic chemistry and scientific criminal investigation. [Wiley]
  • Milton Cox. A Study of the Sensitivity and Specificity of Four Presumptive Tests for Blood. [PubMed]
  • Srch. M. Proof of blood in stains after the application of luminol test. [PubMed]
  • Evaluation of Six Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High Molecular-Weight DNA [ResearchGate]
  • 2,7-Fluorenediamine and 2,5-fluorenediamine as peroxidase reagents for blood smears. [Link]

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