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LABORATOR IUM DNA

BIDDOKPOL PUSDOKKES POLRI

0-TOLIDINE BLOOD SCREENING TEST

PROCEDURE MANUAL

Table of Contents
1. SCOPE ....................................................................................................................... 2
2. PRINCIPLES ...............................................................................• ................................. 2
3. MATERIALS .............................................................................•.......................................................................................................................................2
3 1 REAGENTS ·· ····· ...................... .......... ....... ..... ....••.... ................. •............. .. •........•• .. ....2
32 EQUIPMENT .. .•• ·••···· •••··••····•·••·· •·• • • •··• • ·•·•••••·•·••• · ............................................ •···· • •· •····•·•••·•· .• 2
4. PROCEDURE................................................................................................................... 2
5. INTERPRETING & REPORTING RESULTS ............................................................... 3
6. REFERENCES ................................................................................................................... 3

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0-TOUDINE BLOOD SCREENING MANUAL Page 1of 3
 1. SCOPE
There are a number or presumptive tests available to the rorens1c scientist to &Cteen suspected
bloodstains One of these tests 1s based on the reaction of Hydrogen Peroxide and o-Tolk1ne Nlth
the haemoglobin molecule in blood, o form a coloured product This manualdetails the procedur'!
to follow for employing the o-Tolid1ne reagent as a presumptive 1nd1cator for the presence of blood

Although this test 1s highly sensitive to minute traces of blood. it 1s not specific Any substan
capable of catalysing the oxidation of o-Tolid1ne will give a positive reaction Such substances are
common and include metal salts such as copper and nickel; chemical oxidants such as some
bleaches hypochlonte permanganates and dichromate and plant sources such as lettuce. gar11c
cucumber and tomato

Due to its rapidity this test is extremely useful1n screening large areas and large numbers of items
for blood However care should be taken in the interpretation of blood screening results and any
report should reflect the non-specific nature of this test.

2. PRINCIPLES
The o-Tolidine (3,3-dimethyl-benzidine) test is a catalytic blood-screening test which relies upon
the enzyme-like (peroxidise) activity of the haem group of haemoglobin (or haemoglobin
derivatives) found in the blood As with all catalytic tests for blood. this peroxidase-like (ability to
catalyse the oxidation of peroxide) activity of haemoglobin. catalyses the reaction between the
active reagents - o-Tolidine and hydrogen peroxide (H202) in an acid pH, producing a visible
coloured product.

o-Tolidine + H202 o-Tolidine Blue + 2H20

(n tile presence or acidic pH and Haemog
lobin)

The o-Tolidine screening test 1s an indicator for the presence of blood and does not conclus vely
prove its presence. Any report produced must clearly reflect this lim1tat1on.

3. MATERIALS
3.1. Rea gents
Refer to the Laboratory Solutions Manual for details of the O-Totid1ne solution and the reagents
used in its production

3.2. Eq u ipm ent

Brown glass eye-dropper bottles and filter paper

4. PROCEDURE
N 8. The o-Tolidine solution should always be tested on a known blood sample prior to use.

• Touch a piece of filter paper on the suspected stain

• o
Place one or two drops of o-Tol1dine solution on the filter paper and wait -1 seconds,
checking for any colour change.
• Place one or two drops of 3°k hydrogen peroxide solution on the filter paper and wait -10
seconds .
• Observe any colour change, note when It occurs and record the result.

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5. INTERPRETING & REPORTING RESULTS
All results must be recorded on the examination record

A fast blue colour change,after the addition of hydrogen peroxide indicates a posrt1ve result

A slow (greater than approximately 10 seconds) blue colour change , after the addition of hydrogen
peroxide, indicates a weak positive result This result may occur 1f there is a contaminant or if
there is a weak blood sample.

No colour change, after the addition of hydrogen peroxide, indicates a negative result.

A blue colour developing, after the addition of o-Tolldine but before the addition of hydrogen
peroxide,possibly indicates the presence of a contaminant and should be noted.

A blue colour, developing after approximately 5 minutes, is due to the oxidation of o-Tolidine by
atmospheric oxygen and does not Indicate a positive result.

N B.An area on an item. that gives a pos11tve reaction to this test, may be outlined

The o-Tohd1ne screening test is an indicator for the presence of blood and does not conclusively
prove its presence. Any report produced must clearly reflect this limitation.

6. REFERENCES
Gaensslen R E (1983), Sourcebook in Forensic Serology, Immunology and Biochemistry, USA,
Unit II, page 109.

NSW Institute of Forensic Medicine, Forensic Biology (1987), Forensic Biology Methods Manual,
"Detection of Blood Stains·

Saferstein R (1993), Forensic Science Handbook , Vol. Ill, Regents/Prentice-H all, USA,pages 272-
276.

State Forensic Science, South Australia (1988), Forensic Biology - Serology Manual, Part B
(81.4 .10).

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