Evaluation of the ABAcard HemaTrace for the

Forensic Identification of Human Blood

Connie J. Swander, and Jennifer G. Stites

Michigan State Police, Forensic Laboratory
103 S. James
Grayling, MI 49738

ABSTRACT:

This study examined the ABAcard HemaTrace for it's usefulness in the forensic identification of
human blood. The goal of this study was to verify and compare the sensitivity of the HemaTrace card to
other immunohematological tests currently available. Serial dilutions of human blood were examined as
were bloodstains subjected to different conditions encountered at crime scenes, or in the laboratory. The
results have shown the ABAcard HemaTrace to be a highly sensitive, convenient, and rapid test for the
identification of human blood both in the laboratory and at the crime scene.

BACKGROUND:

The ABAcard HemaTrace is an immunochromatographic 1-step test for the detection of human
blood. If human hemoglobin is present in the questioned bloodstain, it will react with the mobile
monoclonal antihuman antibody and a mobile antigen antibody complex is formed. This complex then
migrates through the HemaTrace's absorbent membrane towards the test area 'T'. In this test area 'T', a
polyclonal antihuman hemoglobin antibody is immobilized. This immobilized antibody captures the above
complex so that an antibody-antigen-antibody sandwich is formed. When the human hemoglobin
concentration is above a certain minimum detection limit (0.05 ug/ml) the purple dye particles present in
the device will form a purple band at the 'T' area which is indicative of a positive result for the presence of
human blood.
As an internal control, human hemoglobin antibody-dye conjugates cannot bind to the antibody in
the test area 'T', but are captured by an immobilized anti immunoglobbulin antibody present in the control
area 'C forming a complex. The captured purple dye particles will thus form a band in the control area 'C
forming a complex, indicating that the test has worked properly and proper procedures have been followed.
To interpret results, the presence of two colored bands, one in the test area 'T' and one in the control area
‘C’, indicates a positive result, while a band only in the control area 'C' would indicate a negative result
(provided no "high dose hook effect"). If no band occurs at the control area 'C' the test is determined invalid
meaning either the HemaTrace card itself was defective or proper procedures were not followed.

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MATERIALS AND METHODS:

1. Known human blood.

2. One Step ABAcard HemaTrace (manufactured by Abacus Diagnostics, West Hills, CA) Used
according to manufacturer's directions.
3. Titan IV Double Diffusion plates (manufactured by Helena Laboratories, Beaumont, Texas)
4. Anti human sera (manufactured by Serological Research Institute, Richmond, CA)
5. Tetramethylbenzidine solution (TMB)
3.3,5.5 tetramethylbenzidine (Aldrich Co.) 2 g.
Glacial acetic acid 100 ml.
3% Hydrogen peroxide
6. Leucocrystal Violet solution (LCV)
5-sulfosalicylic acid 10g.
3% Hydrogen peroxide 500 ml.
1.1 gms leucocrystal violet
4.4 gms sodium acetate
7. "Hemastix" test strips (manufactured by Bayer)

Sensitivity:

Known human blood with a hemoglobin concentration of approximately 13 g/dl was used. Serial
dilution's of the known blood were made using the extraction buffer provided in the ABAcard HemaTrace
kit. The required 150 uls of each dilution were added directly to a HemaTrace sample 'S' well respectively.
The results were read and recorded at 10minutes. Ouchterlony double diffusion plates were run on the same
serial dilutions. Approximately 5 uls of dilutions 1: 16 through 1 :65,536 were applied to their respective
wells and the results were read and recorded at 24 hours.

Conditioned bloodstains:

Known human blood was subjected to various conditions and chemicals commonly encountered at
crime scenes and in the laboratory. This was done by applying the blood directly to the condition in
question or applying the condition to a bloodstained cotton swatch. A portion of each of the conditioned
bloodstains was then tested utilizing the HemaTrace and Ouchterlony double diffusion technique. The
results were read and recorded as documented.

Forensic Correlation:

Approximately 100 ul of each of the above serial dilutions was applied to respective 1 cm square
white cotton swatches. The swatches were allowed to air dry for 48 hours. The visual color of the swatch
was then graded and recorded on a red/brown, yellow/brown, tan, off-white, to white scale. A portion of
each of the stained cotton swatches was then tested with Tetramethelbenzidine, Leucocrystal Violet, and
"Hemastix" strips (presumptive tests for blood). The results were read and recorded on a 1 + to 4+ scale
with 4- being the strongest reaction. The same cotton swatches were tested for species identification
utilizing HemaTrace cards. The results were read and recorded as documented.

RESULTS AND DISCUSSIONS:

Sensitivity: Serial dilutions of known human blood: Positive results were recorded if a purple band occurred
at the 'T' and 'C' areas. Negatives were recorded if a purple band occurred only at the 'C' area, and a test was
recorded as invalid if no purple band occurred at the 'C' area. These results were all read. and recorded at 10
minutes. Comparison studies were done using the Ouchterlony double diffusion technique. These results
were read and recorded after 24 hours. (See Figure 1)
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Human Blood Dilutions HemaTrace Ouchterlony
1 :16 + +
1:32 + +
1:64 + +
1 :128 + +
1:256 + +
1:512 + +
1: 1024 + +
1 :2048 + 0
1 :4096 + 0
1:8192 + 0
1: 16384 + 0
1 :32768 + 0
1 :65536 + 0
1 :262144 +
1:1048576 +
1:16777216 +
1 :33554432 0
1 :67108864 0
Tris extraction buffer 0 0

Figure 1: Serial dilution of human blood comparison

0 =negative + =positive

Positive results were obtained with the HemaTrace test up to a dilution of 1: 16,777,216 The Ouchterlony
Double Diffusion test showed positive results up to a dilution of 1: 1,024.

Conditioned Blood: Human blood subjected to conditions commonly encountered at crime scenes, and in
the laboratory. Comparison studies of the conditioned bloods were carried out using HemaTrace, and the
Ouchterlony double diffusion technique. Results for each technique were read and recorded the same as
the above mentioned results for the comparison of sensitivity. (See Figure 2).

Human Blood Dilutions HemaTrace Ouchterlony

Treated w/ luminol (swatch) + +
Treated w/ leucocrystal violet (swatch) + +
Treated w/ tetramethylbenzidine (swatch) + +
Heat fixed (swatch) 0 0
Bleached (swatch) + 0
Soil debris + +
Off of plant material + +
Off of leather + +
From washed jeans + 0
1 yr . old frozen lysate + +
10 yr. old frozen lysate + 0
10 yr. old rm. temp. stain + 0
Rust from vehicle (no blood) 0 0
Figure 2: Human blood subjected to various conditions and chemicals
0 =negative + =positive
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Positive results were obtained with both the HemaTrace and Ouchterlony techniques for most of the
conditioned blood stains tested. Bleached bloodstains, washed (from denim) bloodstains, and the ten year
old blood stains all gave positive results with HemaTrace and negative results with Ouchterlony. It was
found that a longer extraction time (up to four hours) of older stains gave a positive HemaTrace reaction
(within the 10 minute reading time) whereas Ouchterlony results were still negative.

Forensic Correlation: An attempt to correlate the visual appearance of the stain dilution to the sensitivity
of the presumptive test and the sensitivity of the species(human) identification. (HemaTrace) This study
attempted to compare the visual appearance of the blood stain to its reaction with presumptive and species
identification testing. (See Figure 3).

Presumptive Human
Dilution Visual Hemastix TMB LCV HemaTrace
1:2 Red/brown 4+ 4+ 4+ +
1: 16 brown/yellow 4+ 4+ 4+ +
1:64 light tan 4+ 4+ 4+ +
1: 128 off white 4+ 4+ 4+ +
1 :256 off white 4+ 3+ 4+ +
1 :512 off white 3+ 2+ 3+ +
1: 1024 white 3+ 1+ 3+ +
1 :2048 white 2+ 1+ 2+ +
1 :4096 white 1+ 0 2+ +
1:8192 white 1+ 0 1+ +
1:16384 white +/0 0 0 +
1 :32768 white 0 0 0 +
1:262144 white 0 0 0 0
1: 1048576 white 0 0 0 0
1:4194304 white 0 0 0 0
1:16777216 white 0 0 0 0
1:33554432 white 0 0 0 0
1:67108864 white 0 0 0 0

Figure 3: Correlation between dilution, color, presumptive, and HemaTrace

0 =negative +=positive

Visual observation detected coloring (staining) of the cotton swatches up to a dilution of 1:512.
Presumptive tests were able to detect a positive reaction up to 1:8192 (Hemastix), 1:2048 (TMB), and
1:8192 (LCV), although visually no staining or discoloration of the swatch was noted. The HemaTrace test
gave a positive reaction for the presence of human blood up to a dilution of 1:32,768, well past any visual
or presumptive determination of the presence of blood.
It should be noted here that the swatch supporting the blood dilutions were extracted using the 2 ml of
buffer provided in the HemaTrace kit, thus adding another dilution factor and could therefore explain the
difference in sensitivity from the original serial dilutions.

Known animal bloods from deer, cow, pig, horse, dog and cat were also tested utilizing the HemaTrace
method with negative results obtained. Human saliva and urine stains also gave negative results.

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CONCLUSION:

The ABAcard HemaTrace is a convenient and rapid test for the identification of human blood. It is
far superior to the Ouchterlony Double Diffusion technique in terms of sensitivity and tolerance to different
conditions. The correlation between the visual appearance of a blood stain and the ability to determine the
presence of human blood, makes the ABAcard HemaTrace an acceptable and highly useful tool for the
forensic science community both in the laboratory and at crime scenes.

References:

(I) Hochmeister, M., Rudin O., Sparkes, R., Gehrig, C., Schmidt, L. Evaluation of an
Immunochromatographic 1-Step Occult Blood Test for the Forensic Identification of Human Blood. J. For.
Sciences. 1998. Submitted

(2) Spear, T.F. , Binkley, S.A. The HemeSelect test: a simple and sensitive forensic species test. J. For. Sci.
Soc. V34 (1), P 41-6, 1994.

(3) Fernando, S.A., Wilson, G.S. Studies of the 'hook' effect in the one-step sandwich immunoassay. J.Imm.
Methods. V151(l-2), p 47-66,1992

(4) Cox, M. A study of the sensitivity and specificity of four presumptive tests for blood. J. For. Sci.
V36(5), p 1503-11, 1991.

(5) Doherty, P.E. & Mooney, D.J. Deciphering bloody imprints through chemical enhancements. J. For.
Sci V35(2), p 457-65, 1990.