TMB Test for Blood Examination

-By Shivani Kumari

Introduction

The tetramethylbenzidine (TMB) preliminary test for blood is a catalytic test which is based on the peroxidase-like activity of hemoglobin. This color test proceeds in the presence of oxidizing agents such as hydrogen peroxide present in blood. This reaction proceeds in acidic conditions. Hemoglobin has the capability to split oxygen molecules from hydrogen peroxide and catalyze the reaction to convert the reduced form of 3,3,5,5’-tetramethylbenzidine to the oxidized blue-green colored salt.

Prepration of Reagents

Tetramethylbenzidine (TMB) reagent

1. 0.2 grams of TMB is weighed out

2. It is added to a 50 ml beaker

3. 10 ml glacial acetic acid is pipette out and added to the tetramethylbenzidine placed in 50 ml beaker

4. It is mixed thoroughly until the tetramethylbenzidine is completely dissolved

5. It can stored or refrigerated as its expiration date is one week

Hydrogen Peroxide 3%

1. 10 ml of 30% hydrogen peroxide is pipette out and put in a beaker

2. 90 ml of deionized water is added to it

3. It can be stored at room temperature or refrigerated as its expiration date is one year.

INSTRUMENTS USED

• Top loading balance

CONTROLS

• Positive control also known blood stain

• Negative control

Principle

When the hemeglobin present in the blood reacts with the Tetramethyl benzidine in the presence of oxidizing agent, hydrogen peroxide, it leads to oxidation of TMB to form a blue-green colored salt.

This happens as a result of splitting up of the oxygen from hydrogen peroxide by hemoglobin which in turn catalyzes the reaction to form the reduced form of the TMB and thus impart blue color.

Procedure

1.     Suspected blood stain is swabbed with clean filter paper or a swab, which may be moistened if necessary with deionized water, ethanol or saline.

2.     1-2 drops of the TMB reagent is added.

3.     Blue-green color change is noted if any. A blue-green color change at this step indicates a chemical oxidant and the test should be considered inconclusive. If there is no color change observed proceed further.

4.     1-2 drops of 3% hydrogen peroxide is added.

5.     Any instant blue-green color change is noted down.

6.     An instant blue-green color change indicates a positive result. If there is no color change it indicates a negative result. A negative result indicates that either no blood is present or if present is below the limit of detection of the test.

Method for Liquid Blood

1.     In a clean test tube, 0.02 ml of diluted liquid blood, blood to water ratio is 1:5, is taken.

2.     To the same test tube, 0.5 ml of TMB reagent is added.

3.     0.5 ml of 3% hydrogen peroxide solution is then added to the test tube.

4.     The color change observed is noted down after waiting for twenty seconds.

 

Method for Dried Blood Stain

1.     A clean swab is taken and moistened with distilled water.

2.     The dried blood stain is scabbed with a moistened swab.

3.     1 drop of TMB is added on the swab.

4.     1 drop of hydrogen peroxide solution is added.

5.     Wait up to 20 seconds to observe a possible color change.

 

Observation

The color can instantly appear after the addition of hydrogen peroxide however, if the sample is too much diluted, then it can take up to 20 seconds to show color.

Sensitivity of Tetramethylbenzidine (TMB) Test

·         Blood with the dilution up to 1:10000 gives an instant positive reaction.

·         Blood with dilution up to 1:1000000 gives 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 a time duration of 20 seconds.

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

False Positive Result in TMB Test (Specificity)

It is observed that many vegetables and fruits give false positive color test for tetramethylbenzidine due to presence of hydrogen peroxide,

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

Green grapes, cherry, banana, peach, pear, pineapple, Tangerine apricot, red apple, red plum, cantaloupe, orange and watermelon

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

They all produce false-positive results within 20 seconds to hydrogen peroxide addition while Red grapes give color within 60 seconds.

Advantage of Tetramethylbenzidine (TMB) Test

·      It is highly sensitivity of about 1: 1,000,000 blood dilution.

·      It is easy, cost effective, and less time-consuming.

·      There is no need for a confirmatory test, if the test result is negative.

·      TMB reagent can be used for up to a week.

 

Disadvantages of TMB Method

1.     It has very less specificity that is why there are a lot of false-positives result in several vegetables and fruits.

2.     Some of the reagents used are corrosive and acidic reagents.

 

SAFETY CONSIDERATIONS

1. Hydrogen Peroxide 30% is dangerous and corrosive hence needs to be handled carefully

2. Glacial Acetic Acid is dangerous and corrosive hence needs to be handled carefully

3. TMB is a possible carcinogen.