CE U P D A T E — F O R E N S I C S I
Kim A. Collins, MD
The Laboratory's Role in
Detecting Sexual Assault
Sexual assault is a crime of violence. Unfortu-
nately, this crime continues to prevail in the
United States and other countries. The victims
usually are children and women, and the
assailants are most often men. Most cases of sex-
ual assault, especially in adult victims, involve
penile penetration into the vagina, rectum, or
mouth. Vaginal penetration occurs in approxi-
mately 90% of cases, and oral penetration in
approximately 20%.1>2
After an alleged assault, an experienced exam-
iner should conduct a physical examination. This
is the prime opportunity for documentation of
injuries and collection of laboratory evidence.
One must keep in mind that the lack of physical
injuries does not rule out an assault. In previous
reports, only 20% to 45% of victims ever show
evidence of bodily injuries.3'4 Furthermore, the
act of sexual assault usually is unwitnessed; there-
fore, the victim's claim of assault needs the sup-
port of corroborating laboratory evidence.
Because this evidence is often lacking or misinter-
preted, the rate of prosecution of assailants is low.5
The other scenario of which we must be
equally aware is that of an innocent individual
accused of sexual assault. Our laboratory evi-
dence also may be crucial in exonerating falsely
accused individuals. As pathologists, medical
technologists, and medical laboratory techni-
cians, we enter the realm of sexual assault with a
definite challenge to examine four areas:
1. Can we prove sexual contact occurred?
2. When did the assault occur?
3. How can we best process and document our
evidence?
4. Who is the assailant?
In order to address these areas, a knowledge of
semen, spermatozoa, and the physical examina-
tion and collection of laboratory evidence is
needed [see Table for a list of laboratory tests and
specimens used].
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ABSTRACT Sexual assault, a crime of violence and control,
is prevalent in the United States. Most of the victims are
female and the assailants male. The crime usually is
unwitnessed and, unfortunately, the majority of the assailants
are never prosecuted. The number one reason for lack of
prosecution is lack of demonstrative evidence. The most
crucial evidence in court is that of the laboratory, used to
prove or disprove the presence of semen. As clinical laboratory
professionals, we bear great responsibility in the handling and
interpretation of this evidence. With the discovery of better
staining techniques, chemical analyses, and molecular
pathology, we are now able to bring the laboratory into the
courtroom and use our knowledge to bring positive proof
against the assailant. We are obligated to the victim and theIDC0
community to properly use this knowledge to document, "M
0
process, and interpret the evidence to the best of our ability. ac
3
This is the first in a three-part continuing education series on forensic pathology. At £
the end of the series, the reader will be able to describe the traditional as well as E
o
new molecular pathology techniques used in detecting sexual assault; describe 0
u
techniques used to recover, examine, and identify skeletal remains; and describe
DNA techniques used in the forensic pathology laboratory.
From the
Semen Department of
Semen, or ejaculate, is the crucial evidence sought Pathology and
Laboratory
in cases of sexual assault. Normal semen is light
Medicine, Forensic
gray to white and has an odor similar to sodium Section, Medical !
hypochlorite (bleach). Semen actually is com- University of South
posed of various constituents. Sixty percent of its Carolina,
volume originates from the seminal vesicles and Charleston.
20% from the prostate. The spermatozoa com- Reprint requests to
prise 5% of the volume.6 The remaining 15% is Dr Collins,
Department of
composed of secretions from glands such as the
Pathology and
bulbourethral and urethral glands. Because vari- Laboratory
ous glands produce the ejaculate and it passes Medicine, Forensic
through ducts and moves along its course Section, Medical
University of South
through the urethra during ejaculation, it also
Carolina, 171 Ashley
Ave, Charleston, SC
29425.
JUNE 1998 VOLUME 29, NUMBER 6 LABORATORY MEDICINE
 Specimens and Laboratory Procedures Used in Obtaining
Evidence of Sexual Assault
Specimens*
Anorectal swab/smear
Cervicovaginal swab/smear
Control specimen of blood in EDTA (from the victim)
Control specimen of saliva (from the victim)
Extragenital semen residue
Hair and fibers
Nail scrapings and clippings
Oral swab/smear
Swab/smears from locations that fluoresce to light from a
Wood's lamp
Procedures*
"Christmas tree" (nuclear fast red-picroindigocarmine) stain
DNA fingerprinting using Southern blot analysis
Extragenital semen residue analysis
Fluorescence in situ hybridization (FISH) studies for nonsperm
male cells, eg, epithelial cells or inflammatory cells
Hair and clothing fiber analysis
Papanicolaou stain
Polymerase chain reaction to amplify DNA
Prostatic acid phosphatase analysis
p30 glycoprotein analysis
Saline wet mount (no stain) in the emergency department (to
detect motile spermatozoa)
*ln alphabetical order.
contains epithelial and possible inflammatory
cells. The normal volume of a single ejaculation is
1.5 to 5.0 mL and contains 60 to 200 million sper-
matozoa per mL.6 Semen also is rich in fructose, a
sugar that provides the energy for the spermato-
zoa. The normal pH of semen is 7.0 to 8.3. When
semen mixes with the acid environment of the
vagina, the pH decreases toward the pH for opti-
mum sperm motility, 6 to 6.5.
The first fraction of the ejaculate is rich in
sperm. As mentioned, the prostate provides a sig-
nificant volume to the ejaculate. The prostatic
emission contains acid phosphatase and spermine
phosphate. Since 1945, prostatic acid phosphatase
has proven extremely useful in the evaluation of
alleged sexual assault. An elevated level is a specific
_ . ' indication of recent intercourse and ejaculation
;
spermatozoon.
Head
Nucleus
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(semen). Even in an individual who has undergone
a vasectomy, spermatozoa will be absent, but the
acid phosphatase level will be elevated in the
semen specimen from the victim's vagina, mouth,
or anorectal area. Acid phosphatase levels in the
vagina and cervicovaginal area have been shown to
decrease rapidly over time following coitus, how-
ever.3'7'8 The enzyme level will remain elevated for
approximately 8 hours and then begin to decrease
with time. Most investigators agree that the level of
acid phosphatase will not be elevated 36 to 48
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hours after coitus.7 The level of acid phosphatase
remains elevated longer in dried secretions on the
extragenital areas, bedding, or clothing. As investi-
gators, we must be accurate with the analysis of
this enzyme level because the level will aid in con-
firming the victim's statement regarding the time
of the assault.
Another substance whose analysis has recently
become more sensitive is the p30 glycoprotein.9'10
This glycoprotein is a prostatic antigen found in
the semen, even in vasectomized men.
Spermatozoa
The spermatozoa are positive proof of sexual con-
tact, and their presence is strong evidence for the
prosecution of any sexual assault case. Dutch
microscopist Anton van Leeuwenhoek first
described spermatozoa, a free-swimming cell, in
the 17th century. The basic structure is composed
of a head, neck, midpiece, and tail. The head is
approximately 4 to 5 u-m (length) X 2 to 3 u,m
(width) and has an anterior acrosome that con-
tains enzymes needed to penetrate the ovum dur-
ing fertilization (Fig 1). The head is packed with
DNA, which determines its staining characteris-
tics.11 The head is connected by a neck to a mid-
piece (7-8 u-m in length) and a tail (50-55 u,m in
length). Spermatogenesis occurs in the testes, sper-
matozoa are stored in the epididymis, and then
carried via the vas deferens to the urethra during
ejaculation.
Spermatozoa are extremely motile and this
motility can be observed if the specimen is col-
lected soon after ejaculation. In reported studies,
only 50% of the smears will have motile sperm
after 3 hours.7'12 Generally, motile spermatozoa
are not found in cervicovaginal smears after 12
hours.
Spermatozoa are sensitive to the environment
and begin to degenerate within hours after ejacu-
lation. The first obvious sign of degeneration is
loss of the tail. This loss occurs after approximately
16 hours. Nonmotile spermatozoa and spermato-
zoan heads can be detected in cervicovaginal
 smears days after intercourse; with time, however,
the positive recovery rate is low. After 72 hours,
only 50% of cervicovaginal smears of sexual
assault victims will have spermatozoa present.7
Spermatozoa rarely are found in cervicovaginal
smears after 10 days.13,14 In the rectum, spermato-
zoa seem to lose their tails more quickly, after 6
hours.15 Successful recovery of spermatozoa in the
rectum can occur up to 24 hours after ejaculation
and has been reported up to 65 hours.16 These
positive findings are usually only the spermato-
zoan heads, however. Recovery from the oral cavity
requires an even shorter interval of approximately
6 hours.17'18 This decreased survival is due to the
action of saliva and bacteria in the mouth. Of
course, the positive identification of spermatozoa
in the vagina, anorectum, or mouth may be detri-
mentally affected by douching, urinating, ingesting
oral contraceptive pills, defecating, mouth wash-
ing, and drinking. Oral contraceptives can change
the vaginal environment, ie, pH, and thus alter
sperm survival time (decrease survival). Prepuber-
tal young girls also have decreased spermatozoal
survival in cervicovaginal specimens due to a
decreased cervical mucus.19
Physical Examination and Collection
of Evidence
After a sexual assault has occurred, an experienced
examiner should conduct a physical examination
as soon as possible. If the victim is dead, an exam-
ination should be conducted before the autopsy is
performed. Even if the performance of the autopsy
is delayed a day or two, the sexual assault workup
should not be delayed. During the sexual assault
examination, injuries should be documented and
photographed, and any evidence properly
obtained.
One must keep in mind, not only are we look-
ing for injuries to the anogenital region, but any
injuries over the body including those of defense.
Defense injuries may include scratches on the
hands and forearms, broken nails, and injuries to
the soles of the feet. A lack of physical injuries does
not rule out an assault. In one report, only 20% to
45% of victims ever show evidence of bodily
injuries.3'4 All physical and medical evidence
obtained at the time of the examination must be
complete and properly handled so that the legal
case is not dismissed because of insufficient and
mishandled evidence.1
Laboratory evidence in sexual assault cases
includes vaginal, anorectal, or oral swabs; extra-
genital semen residue; foreign material such as hair
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andfibers;and clothing from the victim or bedding
that could prove as evidence. Although some labo-
ratories favor aspirated specimens, we prefer sterile
cotton swabs to collect the specimens. Swabs taken
from the vaginal, anorectal, or oral areas are trans-
ferred (smeared) onto separate glass slides. All
smears should be spread as thinly as possible, espe-
cially anorectal smears. Simultaneous swabs
should be taken and air-dried for prostatic acid
phosphatase analysis and DNA fingerprinting if
needed. Swabs should be kept out of direct sun-
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light because ultraviolet light irrevocably damages
DNA.20 Specimens on the air-dried swabs can later
be resuspended in 1 to 2 drops of sterile water or
saline. Extragenital sites also can be examined for
the presence of semen. Semen will fluoresce blue-
green when exposed to the ultraviolet light of a
Wood's lamp. If a fluorescent area is detected, a
sterile cotton swab moistened with sterile water or
saline can be used to swab the fluorescing area. As
above, smears can be prepared and swabs air-dried Test Your
for further analysis. Knowledge
The victim's clothing also can be examined with Look for the CE
a Wood's lamp. Spermatozoa adhere tenaciously to Update exam on
Forensics (805) in the
cotton fabric.11 Analysis using extraction can be August issue of
performed on the clothing at a forensic laboratory. Laboratory Medicine.
Nail scrapings and, if possible, nail clippings Participants will earn
should be taken as evidence, placed in a small 3 CMLE credit hours.
envelope, sealed, and secured. All evidence e
o
obtained during the examination must be labeled
n
using permanent markers with the victim's name, u
e
sample, site, and if possible an identification num- 3
ber for reference.1 The evidence must pass directly E
£
from the examiner to the persons who will process o
0
the specimens with signatures using a chain-of- u
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custody procedure. Each person who handles the 5
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specimens should be documented.1 Before and a
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after processing, specimens should be kept secure w
in a locked area.
Because evidentiary specimens often are a mix-
©
c
ture of fluids from the victim and the assailant, .2
control specimens of blood and saliva from the
victim also are needed.21-24 Blood is best collected
in a tube that contains EDTA (usually the tube
with a purple top). EDTA samples give higher
yields of DNA, are easier to process, and are less
susceptible to bacterial contamination. Saliva can
be collected in a plain sterile container/tube. These
also must be properly labeled and the chain of cus-
tody well documented.
A saline wet mount may be prepared during the
initial examination of the victim, in an effort to
detect motile spermatozoa. Often, because the
search for spermatozoa can prove challenging, a
clinical laboratory professional is asked to assist in
JUNE 1998 VOLUME 29, NUMBER 6 LABORATORY MEOICINE 363
 '**ffl? -j. , i ' . ^

> ' • * ' . ' .

any positive finding should be documented clearly.

The use of borderline terminology such as "suspi-
cious for" or "could be" or "possibly" should be
avoided at all times.
• . v
• * *

Molecular Pathology and Sexual

Assault
In alleged sexual assault cases, the absence of the
aforementioned sexual assault evidence does not
disprove the victim's claim, but does leave the vic-
tim without laboratory support for the allegation.

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«
A lack of laboratory evidence—that is, spermato-
zoa are not detected and acid phosphatase level is
not elevated—does not mean the assault never
took place and is not necessarily due to an error in
the collection, processing, or interpretation of the
•

specimens. The assailant may be oligospermic,

Fig 2. Cervical smear
from a sexual
the emergency department examination. A drop of azospermic, have had a previous vasectomy, have
assault victim. sterile saline is placed in the center of a clean slide. been sexually dysfunctional during the assault, or
Spermatozoa exhibit A specimen swab is mixed with the saline to create withdrew before ejaculation. New advances in
intact tails, a suspension on the slide. A coverslip is placed over pathology and laboratory medicine have opened
indicating that
the sample. Some observers prefer to "rim" the doors for the alternative demonstration of positive
ejaculation occurred
within 16 hours of edges of the coverslip with petroleum jelly to keep evidence indicative of sexual assault. Two recent
the microscopic the suspension in place. Some investigators first techniques are fluorescence in situ hybridization
21 24 26 31
examination. smear the specimen on the slide before adding a (FISH) and DNA fingerprinting. - ' - These
Bacteria, squamous drop of sterile saline to the slide. I have found no two areas have taken us into a realm of science and
cells, and
inflammatory cells
reference as to which method yields the best technology that we never dreamed possible.
are seen in the results. No stain is used with the wet mount; only FISH is a highly sensitive and highly specific
background (X600, the refraction of the light at the proper plane of procedure that uses a DNA probe specific for a
Papanicolaou). focus is necessary for the trained observer. Most chromosome or chromosome region. The probe
observers begin on low power, find the proper is labeled with a nonradioactive fluorescent mol-
plane, and advance to X400 magnification. Turn- ecule and hybridized to the homologous region
ing the light source down often helps define the in cell DNA.26'27 Studies using FISH and
motile and nonmotile spermatozoa. Y-chromosome-specific DNA probes have iden-
The glass slides from various body sites then are tified nonsperm male cells even when acid phos-
stained in the pathology laboratory. Various stain- phatase levels are not elevated and no sperm are
ing methods have been employed, however, the present.26'27 The assailant's epithelial cells and/or
most widely used is the Papanicolaou stain. The inflammatory cells originating from the penis
Pap stain was first used to identify spermatozoa in and genital tract, cells from the hand, or cells
1964 and since this time has been successfully used from the mouth may be identified in smears from
to detect intact spermatozoa and spermatozoan the victim. This is especially important in cases of
heads in smears from sexual assault cases.11 With child sexual assault when the assault is digital or
the Pap stain, the head is blue-purple and the tail is oral in nature. Even without the presence of sper-
blue-black (Fig 2). One can easily scan a slide at matozoa and an elevated acid phosphatase level,
X100 and confirm positive findings at X400. we can prove that contact has occurred.
Many crime laboratories use the "Christmas tree" In the 1980s, Alec Jeffreys opened the doors of
stain, or nuclear fast red-picroindigocarmine DNA applications in law enforcement.21,24'28'29 The
stain.9,25 With this stain, the head is red and the tail DNA double-helix ladder can be cut with restric-
is yellow-green.9,25 Again, spermatozoa can be tion enzymes, thefragmentsanalyzed by the South-
detected at X100 and confirmed at X400. The ern blot using electrophoresis, and the ladder
"Christmas tree" stain is especially helpful when fragments "unzipped" by denaturation.24 The frag-
analyzing anorectal smears due to the dense fecal ments are blotted onto a membrane and single-
material. Regardless of the staining method used, strand DNA probes are added. The complementary
sites bind, or hybridize, and the membrane is
exposed to X-rayfilm.When the film is developed,
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 we see dark bands showing the location of the
radioactive probes. The appearance is like a bar-
code, and this is our fingerprint. Next, we look for
bands from the known sample to match with bands
from the evidence. For example, we can compare
the bands from the suspect assailant's DNA with
the bands from a vaginal swab (evidence). We take
into account that the female victim's DNA also will
appear as bands from the vaginal swab. Because
interpretation can be difficult, the entire process is
repeated; in most cases, three to five different
probes are eventually used.24
Problems can arise if not enough evidentiary
DNA is present for the performance of a Southern
blot. The technique of polymerase chain reaction,
or PCR, was developed and perfected to amplify the
DNA.22,24 PCR amplifies the DNA by replication.
The enzyme used to replicate the DNA is a poly-
merase. PCR can make millions of copies of a sin-
gle-cell DNA, enough DNA with which to perform
a Southern blot. We therefore can match a single
hair root, a small drop of blood or semen, or skin
cells found under a victim'sfingernailsto a suspect
assailant. The potential that molecular pathology
has in the world of forensics is almost limitless.
Now, not only can we prove that male cells are pre-
sent, but also we can examine the DNA in these
cells and match them to an individual. No longer
do we rely only on the presence of spermatozoa and
elevated acid phosphatase levels.
Conclusion
Sexual assault is a crime of violence and control. It
is a crime that is prevalent not only in the United
States, but in many other countries as well. Sexual
assault affects both sexes, all races, all ages, and all
socioeconomic groups. Because the crime usually is
unwitnessed, the case often becomes one of hearsay
and testimonies, without demonstrative evidence
supporting either side. The pathology laboratory,
with its new techniques and skilled observers, is at
the forefront of the sexual assault investigation. We
gather, document, process, and interpret the evi-
dence. The responsibility of sexual assault prosecu-
tion is great and lies in our laboratories. We have
become the ones best qualified, and law enforce-
ment relies on our expertise. Now we can critically
examine laboratory evidence using both sensitive
and specific techniques and answer questions con-
cerning a sexual assault case.®
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