1. ACID PHOSPHATASE REACTIONS OBTAINED FROM
SECONDARY TRANSFER SEMEN STAINS.
Laura Fairley, School of Science, Engineering and Technology, Abertay University, Bell Street, Dundee,
DD1 1HG.
Bedroom
setting
simulation
Indirect test 1
Item being
tested
AP reaction Confirmatory microscopy
Underwear Instant. Strong pink to
purple.
4+ (TTT)
Bed sheet Instant. Moderate pink to
purple.
4+ (TTT)
Pillowcase Before 2 minutes. Moderate
purple.
2+ (T)
Pillow 15 minutes. Weak purple. 1+ (T)
Bedroom
setting
simulation
Indirect test 2
Underwear Instant. Strong pink to
purple.
4+ (T)
Bed sheet Instant. Moderate pink to
purple. More diffusion that
indirect test 1.
4+ (T)
Pillowcase Before 2 minutes. Moderate
pink to purple. More diffusion
than indirect test 1.
3+ (T)
Pillow After 2 minutes but before 5
minutes. Weak purple.
2+ (T)
Walking
simulation
Indirect test 1
Underwear Instant. Strong pink to
purple.
4+ (T)
Linoleum Instant. Strong pink to
purple.
2+ (T)
Sock Instant. Moderate purple. 2+
Walking
simulation
Indirect test 2
Underwear Instant. Strong purple. 4+ (T)
Linoleum Instant. Strong purple. 2+
Sock Before 2 minutes. Moderate
purple.
1+
Table 1. AP results for scenario one and two from both indirect test 1 and indirect test 2.
Introduction
Along with blood, fingermarks and footwear impressions, semen
is consistently recovered from crime scenes on a regular basis.
Crimes involving semen are typically sexually motivated,
therefore determining the presence of seminal fluid is key as it
can confirm or refute a victim’s account of the series of events.
There has been limited research presented on Acid Phosphatase
(AP) reactions obtained from secondary transfer semen stains).
The most commonly used presumptive test for the location of
seminal stains is the Acid Phosphatase, or Brentamine, test. The
AP reagent detects the presence of AP enzyme due to a
chemical reaction in which there is a colour change from orange
to purple. This reaction occurs when ∝-naphthyl phosphate is
hydrolysed, forming ∝-naphthol. The ∝-naphthol subsequently
combines with the Fast Black K salt, creating an azo dye that
exhibits a purple colour in the presence of Prostatic Acid
Phosphatase (PAP) (Figure 1) (Redhead and Brown 2013).
Figure 1. Acid Phosphatase Reaction.
The aim of this project was to discover the variety of AP
reactions obtained from secondary transfer semen stains. Two
possible scenarios that could arise in a sexual assault were
simulated, and involved the transfer of semen from one area to
another. The materials used were underwear, socks, bed sheets,
pillowcases, a pillow and linoleum flooring.
Figure 4. Negative (left) and positive (right) controls.
Confirmatory microscopy was carried out to determine the
quantity of spermatozoa present at each transfer. A portion of the
stain was excised from the item and subject to an extraction
process. The extract was then heat fixed to a microscope slide
and stained used Haematoxylin and Eosin (H&E). The slides
were then viewed under the microscope at 400x magnification.
The scale used to grade the quantity of spermatozoa was:
Negative: no spermatozoa present.
Trace: Ten or fewer spermatozoa present.
1+: Spermatozoa hard to find.
2+: Some spermatozoa present, easy to find.
3+: Many or some spermatozoa present in most fields.
4+: Many spermatozoa in every field.
The presence of tails were also noted: (T) few intact tails, (TT)
some intact tails and (TTT) many intact tails.
Figure 5. Confirmatory microscopy – positive control.
Figure 6. Strong, moderate and weak AP results.
Depletion series
A positive AP reaction was obtained for all depletions. The AP
reactions for depletions one and five were strong purple, while
ten was moderate purple. Confirmatory microscopy results
followed the same pattern as both simulations; the quantity of
spermatozoa decreased as the AP reaction became weaker.
Conclusion
This study aimed to investigate the assortment of AP reactions
obtained from secondary transfer semen stains. It also sought to
explore whether wetting the filter paper and the item being tested
would provide any distinct results compared to the standard
method. The results indicate varying strengths of AP reaction are
obtained for different levels of seminal transfer. The less transfer
a stain is subject to, the stronger the AP reaction will be, and a
higher quantity of spermatozoa will be present.
Acknowledgements
I would like to thank my supervisor, Darren Phillips, for the
continued support, encouragement and assistance he provided
me with throughout the duration of my project.
References
Davidson, G. & Jalowiecki, T.B. 2012. Acid Phosphatase screening – wetting test paper or wetting fabric and test paper? Science & Justice, 52(2),
pp.106-111.
Lewis, J. et al. 2012. The fallacy of the two minute acid phosphatase cut off. Science & Justice, 52(2). Pp.76-80.
Redhead, P. & Brown, M.K., 2013. The acid phosphatase two minute cut off: An insufficient time to detect some semen stains. Science & Justice,
53(2), pp.187-191.
Results and discussion (continued).Methodology (continued).
Methodology
Two simulations were carried out in which semen could
potentially be transferred:
Simulation 1 (Figure 2) related to a bedroom setting. Semen
transfer was: underwear > bed sheet > pillowcase > pillow.
Simulation 2 (Figure 3) related to a situation in which someone
walked through a seminal stain. Semen transfer was: underwear
> linoleum > sock.
A depletion series of 10 marks was carried out on a bed sheet
segment and linoleum tile.
The standard method of the test involves dampening a piece of
filter paper with distilled water and placing it on an area of the
item being tested that is suspected of containing a seminal stain.
Pressure is then applied and the filter paper is subsequently
placed inside a fume hood. The AP reagent is applied either by a
spray or aerosol bottle, and the filter paper is observed for purple
colouration which indicates a positive reaction and therefore the
presence of semen.
Two versions of the AP test were subsequently carried out:
• Indirect test 1: Standard method.
• Indirect test 2: Wetting the filter paper and the item.
The item and the filter paper was dampened with distilled water.
Positive and negative controls were carried out prior to the initial
AP test (Figure 4).
After application of the AP reagent, the filter papers were
observed and photographed at 0 min, 2 min, 5 min, 10 min, 15
min, 1 hour, 2 hours and 4 hours.
Results and Discussion
There was a positive AP reaction obtained in every situation
which occurred before two minutes. Table 1 indicates the
average AP reactions and microscopy results obtained in each
simulation.
Figure 2. Simulation 1 (bedroom setting).
Figure 3. Simulation 2 (walking scenario).
The main difference between indirect test 1 and indirect test 2
was an increase in diffusion of the colour in indirect test 2. These
findings agree with results produced by Davidson & Jalowiecki
(2012). There were no differences in the time taken for a reaction
to occur between the two tests.
At low levels of transfer the AP reaction was strong and there
was a high quantity of spermatozoa present. Conversely, the
higher the level of transfer, the weaker the AP reaction was, and
the lower the quantity of spermatozoa present. The strongest AP
reaction occurred during primary transfer (underwear), while the
weakest AP reaction was found during quaternary transfer
(pillow).