Study on Acid Phosphatase Enzyme Activity in
Semen Mixed with Various Body fluids
Ajay Singh Rana1*, Priyanka Verma2, Hiren Vekariya3 and Priyanka Mittal1
1Research Scholar, University Institute of Applied and Health Sciences, Chandigarh University, India
2Assistant Proffessor, University Institute of Applied and Health Sciences, Chandigarh University, India
3Lovely Professional University, India
Submission: April 08, 2018; Published: May 03, 2019
*Corresponding author:Ajay Singh Rana, Research Scholar, University Institute of Applied and Health Sciences, Chandigarh University, Gharuan Punjab, India
How to cite this article: Ajay SR, Priyanka V, Hiren V, Priyanka M. Study on Acid Phosphatase Enzyme Activity in Semen Mixed with Various Body Fluids.
J Forensic Sci & Criminal Inves. 2019; 11(5): 555823. DOI: 10.19080/JFSCI.2018.11.555823.
In case of sexual offences the dry seminal stains are encountered in variety of forms on victims and accused clothes, on the body and on the spot where sexual offence had occurred. Acid phosphatase test is one of the most common tests used by forensic laboratories for the detection of semen. Sometimes in sexual offences the seminal fluid got mix with various other body fluids. Therefore, its detection after mixing with other body fluids is one of the most challengeable tasks for forensic laboratories by using acid phosphatase test. Various studies conducted on acid phosphatase enzyme showed that it is not specific to semen and can also be found in other body fluids. The purpose of this study is to show the activity of acid phosphatase enzyme in semen after interference with other body fluids by serial dilutions method. In this study we have collected semen samples from six donors and then serial diluted with various body fluids and then there activity was checked for acid phosphatase enzyme activity.
Keywords: Body fluids; Semen; Acid phosphatase; Menstrual blood
Human semen consists of spermatozoa, seminal plasma and epithelial cells. The seminal plasma is a mixture of secretions derived from the male accessory reproductive organs like epididymis, seminal vesicles, the prostate, vasa-deferantia, bulbourethral and urethral glands . The precise and efficient detection of semen and saliva in sexual assault case-work items is a critical step in the forensic science. Semen detection is usually based on the activity of acid phosphatase (AP), an enzyme found in high concentration in the seminal plasma [2,3]. SAP is a glycoprotein having molecular weight of 10,0000 to 120,000. It is a dimeric protein made up of two subunits of 50000 to 55000 dalton of molecular weight and a nonspecific orthophosphoric monoester phosphohydralase, which catalyses the hydrolytic removal of an ester linked phosphate group from a monophosphate substrate with an optimal pH of 4.9 . The method currently accepted by operational forensic science laboratories allows 2 min for a reaction to be obtained, and until relatively recently, this has not been challenged . Acid phosphatase enzyme is found in seminal fluid in high concentrations as compared to other body fluids and is water soluble enzyme .
The Acid Phosphatase (AP) Brentamine test for the presence of semen is detected either by direct testing or indirect testing of a questioned extractor on a surface for the presence of semen. For direct testing a drop of extract is placed onto filter/blotting paper. In case of indirect testing a visible possible semen stain is pressing with dampened filter/blotting paper on to the surface to transfer some seminal fluid onto the paper. The filter/blotting papers are then tested with a chemical reagent, which changes colour from orange to purple in the presence of acid phosphatase. This is due to hydrolysis of the α-napthylphosphate to produce α-napthol, which couples with a Brentamine Fast Black/Blue salt, resulting in a purple azo dye [7-12].
The detection of acid phosphatase can be best achieved by direct testing rather than detection of AP from the extract of swab . Sometimes exhibits seized for forensic examinations have been washed before send to forensic laboratories. Sarah Noel, et al.  in their study utilized commonly screening tests for the detection of semen on washed fabrics. They also showed that the semen stains washed once shows positivity for AP test in only 15% of cases and strongly positive in 2% of cases . Ashley et al.  in their study showed that there is persistence
of spermatozoa on cotton and terry towel even after six wash
cycles. Therefore, they emphasizes in their study that washed
clothes must be examined even they have washed multiple times
When the semen is diluted or mixed with other body fluid
then it behaves differently. Therefore, the detection of acid
phosphatase activity is critical under such circumstances for the
interpretations of sexual offences cases . Hooft and Voorde
 in their study showed that 5 out of 50 samples for whole
blood, none out of 50 urine samples and 1 out of 50 samples
showed positive results for Acid Phosphatase test. Therefore, in
their study they showed that positive acid phosphatase results
must be interpreted with great caution and this only indicated
that stain must be retained for further investigations .
Allery et al.  in their study showed that acid phosphatase
enzyme test has good sensitivity and sensitivity and has a
negative predictive value of 98%. Thus, acid phosphatase tests
used as a screening test rather than a confirmatory test. Acid
phosphatase used to semen detection is not 100% reliable as the
acid phosphatase is present in other biological material which
is a semen free like semen free vaginal material, faeces and
other food stuffs which can produce false reactions. Although
the concentrations of acid phosphatase enzyme is significantly
higher in seminal fluid as compared to other biological material
Recently various techniques have been developed for
identification of semen such as Lumatic Superlight 400, XRF
(Zinc), PRM1, PRM1/PRM2, KLK (RT-PCR), RSID-semen test &
nanotrapSg . Acid phosphatase test is not a confirmatory test
for semen detection. The confirmation of semen is done through
microscopic examinations by identification of spermatozoa. The
human sperm is a flagellated cell with its head about 4-5 μm long
and 2-3 μm wide. The sperm had a long tail of about 50-55 μm
in length .
Collection of samples: Six samples each of human seminal
fluid, urine, blood from male volunteers who were neither
connected in blood relation neither showed hereditary to each
other were collected. Six samples of female menstrual blood
were also collected from human female’s volunteers who were
also not related in blood relations with each other. The samples
were preserved in culture tubes and placed in refrigerator at -4
0C for further analysis.
0.2 gm of sodium acetate and 0.1 ml of acetic acid was taken
into a conical flask and stirred with glass rod until dissolved. Then
0.025 gm brentamine fast blue B salt was added and covered
with aluminum foil and heated in water bath for two minutes
to dissolve. Then 0.0125 gm sodium α naphthyl phosphate was
added and stirred to dissolve. Then 10 ml distilled water was
added into the solution. Then solution was placed in amber
colour bottle for further analysis .
A series of dilutions of semen, semen mixed with fresh
blood, semen mixed with menstrual blood and semen mixed
with urine (neat, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256,
1/512, 1/1028) by using distilled water were performed. These
dilutions were done in a cavity tiles by transferring the solutions
with the help of micropipette to the adjacent well. A drop of
each dilution was applied on the whatman grade 1 filter paper
and allowed to air dried at room temperature for two hours and
marked as per dilutions. The marked whatman filter papers
having seminal stains then added with a 100μl acid phosphatase
solution and change in colour and time of reaction was noted
down. Two minute cut off time was fixed for the detection of
colour change, beyond two minute the results were declared
The record of reaction occurred was noted on the basis of
the purple colour formed. The colour of reaction formed was
categorized on the basis of purple colour obtained like strong
purple, purple, faint purple, very faint purple and negative. This
process was repeated for all the six samples utilized for serial
dilutions by various body fluids.
In the present study detection of semen in six samples
mixed with various body fluids were diluted by serial dilutions
method. All the observations made were mentioned in Table-1
and 2. The results of our study showed that detection of seminal
fluid was done within 15 sec by acid phosphatase test and as the
dilutions steps increases the time of reaction is also increases.
The purple colour formed during the reaction gets fades away
as the dilution progress from strong purple, purple, faint purple,
very faint purple and negative. The results of our study showed
that semen sample can be detected maximally up to 1/512
dilutions in samples no. 1-6 and up to 1/256 in sample no. 1 &3.
Semen sample mixed with urine sample was detected for acid phosphatase activity up to 1/128 in sample no. 1,2,4 & 6 and
up to 1/256 dilutions in sample no. 3 & 5. Semen sample mixed
with blood sample was detected for acid phosphatase activity up
to 1/128 in sample no. 1-6. Semen sample mixed with menstrual
blood was detected up to 1/128 in sample no. 1 & 2 and up to
1/64 in sample no. 3-6.
Time of reaction is the time period between the addition of
acid phosphatase solution in the stain and appearance of purple
colour in the sample. In the present study no result was detected
in the samples after 120 sec. Out of the 264 samples studied
after serial dilutions with various body fluids 37 samples gave
positive reaction within 15 sec, 54 samples gave positive results
in 30 sec, 38 samples in 45 sec, 17 samples in 60 sec, 23 samples
in 75 sec, 14 samples in 90 sec, 13 samples in 105 sec, 5 samples
in 120 sec and negative in 63 sec respectively (Table-1).
Reaction time was recorded in every 15 sec on the basis of
colour change. Reactions were recorded on the basis of strength
of purple colour formed during the acid posphatase reaction.
The change in colour reaction was noted and categorized as
strong purple (+++), purple (++), faint purple (+), very faint
purple (+) and negative (-) (Table-2 & Figure-1). In our study the
change in colour was strong in neat samples but as the dilutions
progressed the color becomes faint and finally no colour change
was observed after 1/512 dilutions.
From the Table 1 it can be seen that the majority of semen
samples tested for acid phosphatase activity by serial dilutions
works up to 1/512 dilutions maximally and 1/256 minimally.
Similarly, semen mixed with urine works maximally at 1/256
and minimally at 1/128 dilutions, semen mixed with blood
works maximally at 1/128 and semen mixed with menstrual
blood works maximally at 1/128 and minimally at 1/64. Similar
study have also done Bhoopendra Singh et al.  in semen
sample who showed that maximum detection limit was 1/512
and minimum was noticed 1/64. These sets of experiments have
identified the potentiality of acid phosphatase enzyme in semen
after mixing with various body fluids. Redhead and Brown in
their study showed that acid phosphatase activity was detected
positive up to 1/1000 dilutions by direct testing and 1/400 by
using the press method. In their study no reaction was observed
after 1/400 dilutions by press method and 1/1000 dilutions by
direct method . Allard et al.  in their study showed that acid phosphatase test was detected strong to moderate up to 1
in 25 dilutions. The study made was found variable from other
two laboratories where strong positive results were detected in
1 in 200 dilutions and other obtained a weak or negative results
after 1 in 40 dilutions .
In our study time of reaction in 201 samples out of 264
samples tested for acid phosphatase test varied from 15 sec to
120 sec. 37 samples (14.01%) were tested positive with in 15
sec, 54 samples (20.45%) in 30 sec, 38 samples (14.39%) in 45
sec, 17 samples (6.43%) in 60 sec, 23 samples (8.71%) in 75
sec, 14 samples (5.30%) in 90 sec, 13 samples (4.92%) in 105
sec and 5 samples (1.89%) in 120 sec. Davidson and Jaloweicki
 in their study on 34 different types of fabrics showed that
acid phosphatase reaction time on top blot was from 1 sec
to 90 sec and on bottom blot was from 1 sec to 120 sec .
Redhead & Brown  in their study showed that time of reaction
varied from 1 min to 16 min from neat to a sample diluted up
to 1/1000. In their study they also showed that concentration
of acid phosphatase varies with in the different donors and also
the replicated samples from the same donar. Lewis et al. 
in their study showed that most brands of filter paper used to
detect semen by acid phosphatase test can detect up to 1 in 40
From the results it’s a clear that as the semen becomes more
diluted the time of a reaction is increased significantly. Moreover,
a significant difference was observed between the neat samples and when the samples are diluted which signifies the weakness
of Acid Phosphatase enzyme activity (Figures 1-7).
The identification of body fluids is very important as it is
very informative for linking the victim and suspect with crime
to aid in forensic investigation. From this study it is concluded
that there is correlation between the acid phosphatase enzyme
activities with the serial dilutions of semen samples with various
body fluids. As the dilutions progresses the time of reaction also
increases. The results of our study showed that when the semen
get mixed with other body fluids the dilutions limit to which acid
phosphatase enzyme can be detected also decreases significantly.
Acid phosphatase test is commonly used by forensic laboratories
for screening the forensic exhibits for further examinations. The
positivity of acid phosphatase test is based on the development of
purple colour within two minutes. As other biological fluids also
contained acid phosphatase enzyme and thus further testing is
required for the confirmation of seminal stains. Since other body
fluids may also contains acid phosphatase enzyme but in lower
concentrations as compared to semen. Thus, acid phosphatase
test is a presumptive test for the semen detection and further
testing is required for the confirmation of semen. Although the
limitations of acid phosphatase test are widely known but still it
is utilized by forensic laboratories for the screening of semen in
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