Kastle-Meyer test

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The Kastle-Meyer test is a forensic presumptive blood test, first described in 1903, in which the chemical indicator phenolphthalein is used. It relies on the peroxidase-like activity of hemoglobin in blood to catalyze the oxidation of phenolphthalin (the colorless reduced form of phenolphthalein) into phenolphthalein, which is visible as a bright pink color.

Contents 1 Method 2 Limitations 3 Mechanism 4 References

[edit] Method

A presumed blood sample is first collected with a swab. A drop of phenolphthalin reagent is added to the sample, and after a few seconds, a drop of hydrogen peroxide is applied to the swab. If the swab turns pink rapidly, it is said to test presumptive positive for blood. Waiting for periods over 30 seconds will result in most swabs turning pink naturally as they oxidize on their own in the air.

Optionally, the swab can first be treated with a drop of ethanol in order to lyse the cells present and gain increased sensitivity and specificity. This test is nondestructive to the sample, which can be kept and used in further tests at the lab; however, few labs would use the swab used for the Kastle-Meyer test in any further testing, opting instead to use a fresh swab of the original stain.

[edit] Limitations

While the Kastle-Meyer test has been reported as being able to detect blood dilutions down to 1:10⁷, there are a number of important limitations to the test. The test will give a false positive result when in the presence of vegetable peroxidases, such as those in horseradish, broccoli, cauliflower, etc.^[1] Additionally, other oxidizing species in the sample will also cause a false positive. Chemical oxidants such as copper and nickel salts will cause the Kastle-Meyer reagent to turn pink before the addition of the hydrogen peroxide, thus it is vitally important to add the reagent first, then wait a few seconds, then add the hydrogen peroxide.

The Kastle-Meyer test has the same reaction with human blood as it does with any other hemoglobin-based blood, so a confirmatory test such as the Ouchterlony Test must be performed to definitively conclude what from which species the blood originated.

Color catalytic tests are very sensitive, but not specific. The positive color test alone should not be interpreted as positive proof of blood. However, a negative result is proof of the absence of detectable quantities of heme.

[edit] Mechanism

The phenolphthalein used in this test has been modified from its conventional form, in that it has been reduced by two electrons and is pre-dissolved in alkaline solution. This is typically achieved by boiling an alkaline solution of phenolphthalein with powdered zinc, which reduces the phenolpthalein into phenolphthalin. Upon reduction, the very intense pink color of the cationic form of phenolphthalein fades to a faint yellow color. It is this form of phenolphthalein that is present in Kastle-Meyer test kits. In order to generate the intense pink color indicative of a positive test, the reduced phenolphthalein must be oxidized back to its normal, colored form.

In the relevant reaction, hydrogen peroxide reacts with the hemoglobin in the blood. Phenolphthalein does not participate in this first process. In its reaction with hydrogen peroxide, the heme center of hemoglobin undergoes the following O-O bond homolysis reaction:

HOOH + Fe³⁺[heme] → Fe⁴⁺=O[heme] + OH· + H⁺

The products of this reaction are one equivalent each of a high-valent iron-oxo species and hydroxyl radical, either of which can oxidize the reduced phenolphthalein back to its colored form. The amount of acid produced in this reaction is insignificant in comparison to the concentration of base present in the phenolphthalein reagent solution. In addition, this reaction of heme with peroxide is catalytic, making this test very sensitive to small quantities of blood present on the test swab.

[edit] References

Culliford, B., The Examination and Typing of Bloodstains in the Crime Laboratory. U.S. Government Printing Office, 1971. Metropolitan Police Forensic Science Laboratory, Biology Methods Manual. 1978. Saferstein, R., Forensic Science Handbook. Prentice Hall, Inc., 1982. Kirk, P.L., Crime Investigation. John Wiley and Son, 1974. Gaensslen, R., Sourcebook in Forensic Serology, Immunology, and Biochemistry. U.S. Government Printing Office, 1983.