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Tetramethylbenzidine (TMB) Test: Principle, Reagent and Procedure

Tetramethylbenzidine (TMB) Test: Principle, Reagent and Procedure

Tetramethylbenzidine [TMB] test is a presumptive test, generally employed for the detection of blood based on reagent action of peroxide with heme to impart blue-green color.

That’s the one-line answer. Let’s know about the TMB test.

What is Tetramethylbenzidine (TMB) Test?

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).

This color test proceeds in the presence of oxidizing agents such as hydrogen peroxide. To impart blue blue-green color, the whole process needed to be in acidic condition.

Tetramethylbenzidine is known to be one of the best alternatives to benzidine and orthotolidine tests.

That’s the reason why it is widely accepted in forensic labs.

History and Development of TMB Test

In 1974, Holland et al synthesized the TMB and reported a possible way to detect blood from samples. And in the same year, the Occupational Safety and Health Administration banned the use and manufacturing of benzidine in the United States.

This creates a necessity of developing a highly sensitive test for blood.

A year later, in 1976, Garner, et al, reported that TMB is quite good as benzidine for the detection of blood. Their findings and comparable results in terms of sensitivity and specificity lead to the emergence of tetramethylbenzidine as their alternative to the benzidine test.

Moreover, Orthotolidine was also found to be carcinogenic in rats, and in 1992, the test was also replaced by TMB.

Principle of Tetramethylbenzidine (TMB) Test

When the heme compound in the blood reacts with the Tetramethyl benzidine in the presence of oxidizing agent leads to oxidation of TMB to form a blue-green colored product.

This happens because hemoglobin cleaves down the oxygen from hydrogen peroxide and catalyzes the reaction to form the reduced form of the TMB that imparts blue color.

Principle of Tetramethylbenzidine (TMB) Test

Two-Step Reaction Involved

A. Without Addition of Hydrogen Peroxide

In some cases, the blue-green color appears right after adding the TMB reagent. This happens because of chemical oxidants in the blood sample.

Though it states the sign of blood, but from a forensic point of view, the result is inconclusive.

In general scenarios, there will be no color changes seen at this stage.

B. Addition of Hydrogen peroxide:

The deep blue-green color is seen as soon as (or within 20 secs) hydrogen peroxide is added.

Reagent Preparation for TMB Test

A. Tetramethylbenzidine (TMB) Reagent Solution

  1. Weigh 0.2g of 3,3′,5,5′-Tetramethylbenzidine and put it in the 50ml beaker.
  2. To the beaker, 10 ml of glacial acetic acid should be added.
  3. Mix till the solution becomes homogeneous.
  4. The shelf life of the solution is one week.

B. Preparation of Hydrogen Peroxide 

  1. 10 ml of 30% hydrogen peroxide.
  2. To the beaker, add 90 ml of distilled water.
  3. Mix well using a glass rod. 
  4. Store it in cool condition.
  5. The shelf life of the solution is one year.

Procedure For Tetramethylbenzidine (TMB) Test

Method 1: Using Liquid Blood

  1. In a test tube, take 0.02 ml of diluted liquid blood (Blood:water=1:5).
  2. To the test tube, add 0.5 ml of TMB reagent.
  3. Add 0.5 ml of 3% hydrogen peroxide solution.
  4. Note the color change (wait up to 20 seconds)

Method 2: Using Dried Blood Stain

  1. Take a swab and moisten it with distilled water.
  2. Scab the dried blood stain with a moistened swab.
  3. Put 1 drop of TMB on the swab.
  4. Add 1 drop of hydrogen peroxide solution.
  5. Wait up to 20 seconds for a possible color change.

Observation

Blue-green color as the indication of blood.

The color may appear immediately after the addition of hydrogen peroxide. However, if the sample is too much diluted, then it may take up to 20 seconds to develop color.

Sensitivity of Tetramethylbenzidine (TMB) Test

  • Blood with the dilution up to 1:10 000 gave an immediate positive reaction.
  • Blood with dilution up to 1:1000,000 gave positive results within 20 seconds.

The TMB reagent with peroxide is able to produce positive results up to 1:1,000,000 dilution of blood within 20 seconds.

The following is the sensitivity table of TMB against dilution of blood.

Blood DilutionResultTime Taken
1:50PositiveImmediate
1:100PositiveImmediate
1:500PositiveImmediate
1:1000PositiveImmediate
1:10,000PositiveImmediate
1:100,000PositiveUp to 20 secs
1:500,000PositiveUp to 20 secs
1:1,000,000PositiveUp to 20 secs
1:5,000,000NegativeNA

False Positive Result in TMB Test (Specificity)

Most of the vegetables and fruits produce false-positive color tests for Tetramethylbenzidine.

False Positive Result in Vegetables

Some of the vegetables that produce false-positive color with the tetramethylbenzidine test are:

  • Asparagus, Avocado, green bean, broccoli, cabbage, Capsicum, carrot, cauliflower, celery, corn, cucumber, eggplant, garlic, lettuce, mushroom, sweet potato, radish, spinach, tomato, turnip root, and white onion.

They all produce false-positive results within 20 seconds to hydrogen peroxide addition.

False Positive Result in Fruits

Fruits that produce false-positive color tests in the TMB method of blood detection are:

  • Within 20 seconds: Green grapes, apricot, red apple, red plum, cantaloupe, cherry, banana, peach, pear, pineapple, Tangerine orange, watermelon.
  • Within 60 seconds: Red grapes

Advantage of Tetramethylbenzidine (TMB) Test

  1. Highly sensitivity of about 1: 1,000,000 blood dilution.
  2. All reagents are non-carcinogenic.
  3. Easy, economical, and less time-consuming.
  4. No need for a confirmatory test, if the test result is negative.
  5. TMB reagent can be used for up to one week.

Disadvantages of TMB Method

  1. Very less specificity. That is why a lot of false-positives result in vegetables and fruits.
  2. Corrosive and acidic reagents are used.

Other Uses of TMB

  • TMB is used as the colorimetric substrate for forensic DNA testing. It is used to form an insoluble precipitate of intense blue color in the presence of peroxidase in an acidic medium. 
  • TMB also used as the colored substrate for the quantitation in the slot blot assay of DNA.
  • Blood detection spot kits such as Hemastix® assay kit is based on the TMB-based assay. The kit has strips that need to be moistened with a sample, and later it produces a green to blue-green color as the indication of blood.

References:

  • Grodsky et al, Simplified Preliminary Blood Testing: An Improved Technique and a Comparative Study of Methods, Journal of criminal law, Criminology and Police Science, Vol. 42, 1951.
  • Corby et al, The Identification of Human Stains: A Critical Survey, Journal of Forensic Medicine, Vol.7.
  • Cox, M., A Study of the Sensitivity and Specificity of Four Presumptive Tests for Blood, Journal of Forensic Sciences, JFSCA [NIH.Gov]
  • Cano et al, An Evaluation of Tetramethylbenzidine as a Presumptive Test for Blood [NIH.Gov]
  • Forensic Biology by Richard Li [Book]

Further Read:

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