Kastle-Meyer test
Encyclopedia
The Kastle-Meyer test is a presumptive
blood
test, first described in 1903, in which the chemical indicator phenolphthalein
is used to detect the possible presence of hemoglobin
. 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. The Kastle-Meyer test is a form of catalytic blood test, one of the two main classes of forensic tests commonly employed by crime lab
s in the chemical identification of blood. The other class of tests used for this purpose are microcrystal tests, such as the Taichmann crystal test and the Takayama crystal test.
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.
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 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. A negative result is generally proof of the absence of detectable quantities of heme, however a false positive can be generated in the presence of a reducing agent.
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 phenolphthalein 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. Phenolphthalin does not directly participate in this process; instead, it acts as an external source of electrons. In its reaction with hydrogen peroxide, the heme
center of hemoglobin behaves as a peroxidase
, reducing the peroxide to water. This activity depletes hemoglobin of electrons that are, in turn, re-supplied by the phenolphthalin. Donating electrons to hemoglobin converts the phenolphthalin back into the intensely-colored phenolphthalein. As long as the enzyme survives, the reaction of heme with peroxide is catalytic
, making this test very sensitive to small quantities of blood present on the test swab. The hemoglobin-catalyzed reduction of peroxide that occurs is shown in the reaction below. The two electrons are supplied by phenolphthalin:
The consumption of protons during the course of the reaction has the effect of raising the pH of the solution, but the amount of base produced is negligible compared to the amount of base already present in the reagent mixture.
Presumptive test
In medical and forensic science, a presumptive test is an analysis of a sample which establishes either:#The sample is definitely not a certain substance#The sample probably is the substance....
blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....
test, first described in 1903, in which the chemical indicator phenolphthalein
Phenolphthalein
Phenolphthalein is a chemical compound with the formula C20H14O4 and is often written as "HIn" or "phph" in shorthand notation. Often used in titrations, it turns colorless in acidic solutions and pink in basic solutions...
is used to detect the possible presence of hemoglobin
Hemoglobin
Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates, with the exception of the fish family Channichthyidae, as well as the tissues of some invertebrates...
. It relies on the peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
-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. The Kastle-Meyer test is a form of catalytic blood test, one of the two main classes of forensic tests commonly employed by crime lab
Crime Lab
A crime laboratory - often shortened to crime lab - is a scientific laboratory, using primarily forensic science for the purpose of examining evidence from criminal cases.- Lab personnel :A typical crime lab has two sets of personnel:...
s in the chemical identification of blood. The other class of tests used for this purpose are microcrystal tests, such as the Taichmann crystal test and the Takayama crystal test.
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 peroxideHydrogen peroxide
Hydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent...
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.
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. 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
Ouchterlony Double Immuno Diffusion
Ouchterlony double immunodiffusion is a simple, rather dated method which is still considered to be the gold standard for detection of extractable nuclear antigens...
must be performed to definitively conclude 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. A negative result is generally proof of the absence of detectable quantities of heme, however a false positive can be generated in the presence of a reducing agent.
Mechanism
The phenolphthalein used in this test has been modified from its conventional form, in that it has been reducedRedox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
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 phenolphthalein 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
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
back to its normal, colored form.
In the relevant reaction, hydrogen peroxide reacts with the hemoglobin
Hemoglobin
Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates, with the exception of the fish family Channichthyidae, as well as the tissues of some invertebrates...
in the blood. Phenolphthalin does not directly participate in this process; instead, it acts as an external source of electrons. In its reaction with hydrogen peroxide, the heme
Heme
A heme or haem is a prosthetic group that consists of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin. Not all porphyrins contain iron, but a substantial fraction of porphyrin-containing metalloproteins have heme as their prosthetic group; these are...
center of hemoglobin behaves as a peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
, reducing the peroxide to water. This activity depletes hemoglobin of electrons that are, in turn, re-supplied by the phenolphthalin. Donating electrons to hemoglobin converts the phenolphthalin back into the intensely-colored phenolphthalein. As long as the enzyme survives, the reaction of heme with peroxide is catalytic
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
, making this test very sensitive to small quantities of blood present on the test swab. The hemoglobin-catalyzed reduction of peroxide that occurs is shown in the reaction below. The two electrons are supplied by phenolphthalin:
- HOOH + 2 e- + 2 H+ → 2 H2O
The consumption of protons during the course of the reaction has the effect of raising the pH of the solution, but the amount of base produced is negligible compared to the amount of base already present in the reagent mixture.