Forensic Serology: Color tests

By: Mike O'neillMazin OsmanClaudia PalmaAdam Ramirez

Color Tests

• Determination of blood are made by conducting color tests

• The Benzidine test was the most commonly used for many years.

• However it has been replaced by the Kastle-Meyer test because the benzidine has been identified as a known carcinogen (Can cause cancer)

Color Tests

• The Benzidine and Kastle-Meyer color tests are based on the observation that hemoglobin possesses peroxidase-like activity

• Hemoglobin- A red blood cell protein that transports oxygen in the bloodstream; it is responsible for the red color of blood

• Peroxidases are enzymes that accelerate the oxidation of several classes of organic compounds when combined with peroxides

Kastle-Meyer Test

• The Kastle-Meyer test relies on the iron in hemoglobin to promote the oxidation of phenolphthalin to phenolphthalein.

• Phenolphthalin is colorless, but in the presence of blood and hydrogen peroxide, it changes to phenolphthalein, which makes the solution pink.

Sensitivity and Limitations• It is extremely sensitive

• Capable of detect blood dilutions as low as 1x10^7

• If the test result is negative, heme might be absent in the sample

• The test will give a false positive result in the presence of any oxidizing agent like cauliflower or broccoli

Performing The Kastle-Meyer Test1.Collect a sample of the blood using a

cotton swab (if the blood is dry, water could be used to moisten the swab)

2.Add two drops of 70% ethanol to the swab (Helps increase the sensitivity of the test)

3.Add a drop or two of the Kastle-Meyer solution

4.Add two drops of hydrogen peroxide solution

Results

Pink Color Develops Results Interpretation

After Kastle-Meyer is applied False Positive The reaction did not occur as the result of blood’s presence

After hydrogen peroxide is applied

Positive blood is indicated

Never Negative Blood is not indicated (may not be present)

False Negative Blood may be present, but is too dilute to react

Benzidine Color Test

• The benzidine color test was, for many years, the most popular type of preliminary blood test.

• In 1988, however, benzidine was identified as a carcinogen and since then its use has mostly been discontinued.

• Benzidine + Blood Stain + Hydrogen Peroxide = Pink Color

Strengths of Benzidine

• It was a common test for blood.

• It is a presumptive test for identifying Blood

• An enzyme in blood causes the benzidine to be oxidized to a polymer

• which is blue colored. This makes it very easy to find the result

Weaknesses of Benzidine• This test is very unreliable. Although a

test may appear that it has been positive, the substance that was found due to the color presence may not be the actual substance that was involved.

• It is a carcinogen which has been used as part of a test for cyanide and also in the synthesis of dyes. It has been linked to bladder cancer and pancreatic cancer.

• It is highly sensitive.

Hemastix

• Presumptive field test for blood

• Designed as a dipstick test for blood

• Involves moistening a strip in distilled water and then placing it into contact with a suspect blood stain

• If the strip appears to turn green, it is positive for blood

Strength and WeaknessesStrengths:

• Easy to use

• Can be used on the field easily

Weaknesses:

• Not as accurate as the Kastle-Meyer or Benzidine tests

Susan May Case

• On March 12, 1992, Susan May was visiting her aunt Hilda Marchbank. She arrived at the house at approximately 9:30 am and discovered Hilda dead on her bed. She was savagely beaten on her head and face and was smothered with a pillow.

Susan May Case

• Investigators found a number of stains that appeared as blood on the walls of the scene.

• The Kastle-Meyer tests was performed to indicate whether it was blood or not

• Some of the stains contained Susan May's blood

• Susan May was arrested on March 30, 1992 in connection with the murder of Hilda Marchbank

Meredith Kercher Case

• Meredith Kercher was found dead in the bedroom of her apartment in Perugia, Italy. She had several stab wounds in her throat.

• The Kastle-Meyer test was performed on a substance that appeared to be blood and came back positive.

• However, this could have been wrong because the pink color could have been caused by reagents used in fingerprint analysis.

Review Questions

1.What are color tests used for?2.What are the three tests mentioned in

this presentation?3.What color does a positive Kastle-

Meyer tests turn?4.What is one 5.What color does the Benzidine test

turn?6.Why was the Benzidine test

discontinued? 7.What color does a hemastix strip turn

if it is positive for blood?

Sources• Saferstein, R. (2011). Forensic Science - An Introduction (Second ed., p. 293).

Upper Saddle River, NJ: Pearson Prentice Hall.

• Bloodstains. (n.d.). Retrieved April 2, 2013, from http://www.bcps.org/offices/science/secondary/forensic/Bloodstain.pdf

• Benzidine (CASRN 92-87-5). (n.d.). In EPA Integrated Risk Information System. Retrieved April 2, 2013, from http://www.epa.gov/iris/subst/0135.htm

• http://www.sciencebuddies.org/science-fair-projects/project_ideas/BioChem_p037.shtml#background

• http://chemistry.about.com/od/homeexperiments/a/Kastle-Meyer-Test-To-Detect-Blood.htm

• http://lem.ch.unito.it/didattica/infochimica/2006_Luminolo/determinazionesangue.html

• http://www.susanmay.co.uk/student-report2.htm

• http://viewfromwilmington.blogspot.com/2011/07/forensic-tests-for-presence-of-blood.html