.......

 

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This is to certify that the

thesis entitled
THE IDENTIFICATION OF SEMINAL FLUID AND THE DETERMINATION OF

PHOSPHOGLUCOMUTASE SUBTYPE INFORMATION BY ELECTROPHORETIC

 

 

 

SEPARATION
presented by
Toby Leigh Wolson
has been accepted towards fulfillment
of the requirements for

M.S. degree in Criminal Justice

\

(It ,
/ I Major professor I
Date /0 6

0-7639 MS U is an Aflinnaa've Action/Equal Opportunity [urination

 

THE IDENTIFICATION OF SEMINAL FLUID AND THE
DETERMINATION OF PHOSPHOGLUCOMUTASE SUBTYPE INFORMATION
BY ELECTROPHORETIC SEPARATION

BY

Toby Leigh Wolson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

School of Criminal Justice

1986

ABSTRACT

THE IDENTIFICATION OF SEMINAL FLUID AND THE
DETERMINATION OF PHOSPHOGLUCOMUTASE SUBTYPE INFORMATION
BY ELECTROPHORETIC SEPARATION

BY

Toby Leigh Wolson

Seminal fluid samples were examined by electrophoretic
separations on polyacrylamide disc gels and cellulose
acetate to study the usefulness of the polyamines spermine
and spermidine as confirmatory markers in the absence of
spermatozoa. These separations indicated that spermine and
spermidine were poor confirmatory markers for the presence
of seminal fluid. They also revealed the presence of a
protein band of unknown origin and identity which may be
unique to seminal fluid and usable as a confirmatory marker.

The second portion of this study was concerned with the
development and testing of a conventional electrophoresis
procedure for the determination of phosphoglucomutase
subtypes (PGM). The procedure uses an Isogel (FMC) support
media with a pH 5.3 phosphate-citrate buffer system and a
voltage of 21 v/cm for 4 hours. This procedure provides
excellent separation and resolution of the PGM subtype bands
as well as information on the erythrocyte acid phosphatase,

adenosine deaminase, and adenylate kinase enzyme phenotypes.

Dedicated to:
Anne, Rachel, and Max

iii

ACKNOWLEDGMENTS

Thanks to Jay Siegel, Ph.D.: Joseph Melvin, Ph.D.;

Ieva Marks, Edward W. Sparrow Hospital; and the School of
Criminal Justice, Michigan State University for giving me
the guidance and assistance which enabled me to complete
this research study.

Thanks to my co-workers in the Serology Section of the
Metro-Dade Police Department Crime Laboratory for allowing
me time off casework to devote my full energy toward

development and testing of the PGM Subtyping procedure.

iv

TABLE OF

CONTENTS

List of Figures. . . . . . . . . . . . . . . . . . . . . vii

Key to Symbols, Abbreviations, and Nomenclature. . . . .viii

I. Introduction. . . . . . . . . . . . . . . . . . . . 1

Background: the crime of rape. . . . . . . . . . 3
Collection and preservation of the physical

evidence. . . . . . . . . . . . . . . . . . . . 8

Analysis of the evidence. . . . . . . . . . . . .13

II. Spermine and Spermidine as Confirmatory Markers for

Seminal fluid . . . . . . . . . . . . . . . . . .26

Literature review . . . . . . . . . . . . . . . .27

III. Disc Electrophoresis on Polyacrylamide Gels . . . .30

Introduction. . . . . . . . . . . . . . . . .30

Methods and materials . . . . . . . . . . . . . .33

Gel preparation . . . . . . . . . . . . . . . .33

Separation gel. . . . . . . . . . . . . . . .33

Stacking gel. . . . . . . . . . . . . . . . .34

Buffer preparation. . . . . . . . . . . . . . .35

Stock solution. . . . . . . . . . . . . . . .36

Working solution. . . . . . . . . . . . . . .36

Electrophoresis procedure . . . . . . . . . . .36

Staining and destaining . . . . . . . . . . . .38

Stain stock solution. . . . . . . . . . . . .39

Staining solution . . . . . . . . . . . . . .39

Results . . . . . . . . . . . . . . . . . . . . .41

Gel preparation . . . . . . . . . . . . . . . .46

Conclusions . . . . . . . . . . . . . . . . . . .48

IV. Electrophoresis on Cellulose Acetate Membranes. . .50

Introduction. . . . . . . . . . . . . . . . . . .50

Methods and materials . . . . . . . . . . . . . .51

Procedures. . . . . . . . . . . . . . . . . . .51

Results . . . . . . . . . . . . . . . . . . . . .55

Extraction in 0.85% saline. . . . . . . . . . .58
Extraction in 0.85% saline and concentration
of the sample . . . . . . . . . . . . . . . .59

Extraction in 0.85% saline at 2°C for variable
time lengths. O O O O O O O O O O I O O O I O 59
Extraction in pH 7.5 phosphate buffered saline.60

Extraction in pH 7.3 phosphate buffered saline.60

Conclusions . . . . . . . . . .

V. Determination of Phosphoglucomutase

IntrOduCtionO O O O O O O O O 0
Methods and materials . . . .

Buffers . . . . . . . . . . .
Tank buffer . . . . . . . .
Gel buffer. . . . . . . . .

Gel and plate specifications.

Samples and electrOphoresis .

Development . . . . . . . . .
EAP . . . . . . . . . . . .

Reaction buffer . . . . .
Reaction mixture. . . . .
AK. . . . . . . . . . . . .
Reaction buffer . . . . .
Reaction mixture. . . . .
ADA. . . . . . . . . . . .
Reaction buffer . . . . .
Reaction mixture. . . . .
Development procedures. . .
PGM subtypes development. .
Reaction buffer . . . . .
Reaction mixture. . . . .
Results and discussion. . . . .

PGM subtypes. . . . . . . . .

EAP . . . . . . . . . . . . .

ADA and AK. . . . . . . . . .

Conclusion. . . . . . . . . . .

VI. Summary . . . . . . . . . . . . .

List of References . . . . . . . . . . .

General References . . . . . . . . . . .

vi

Subtypes.

O O O O O O O O O O O O O O O O O O O O O O O O O

.61

.63
.63
.69
.69
.69
.70
.70
.70
.71
.71
.71
.71
.72
.72
.72
.73
.73
.73
.73
.74
.74
.75
.75
.75
.77
.78
.78

.81

.83

.89

LIST OF FIGURES

Figure
l. The morphology of spermatozoa. . . . . . . . . . .
2. The identification and analysis of seminal fluid
evidence . . . . . . . . . . . . . . . . . . . .
3. PAGE composition menu. . . . . . . . . . . . . . .
4. PGM phenotypes . . . . . . . . . . . . . . . . . .
5. PGM subtype phenotypes and p0pulation frequencies.
6. The effects of PGM subtyping on the analysis of
sexual assault evidence. . . . . . . . . . . . .
7. PGM subtype band patterns. . . . . . . . . . . . .
8. ADA, AK, and EAP band patterns . . . . . . . . . .

vii

.17

.22
.47
.64
.66

.68
.76
.80

KEY TO SYMBOLS AND ABBREVIATIONS

ADA: Adenosine Deaminase

AK: Adenylate Kinase

Bis: N,N'-Methylene-Bis-Acrylamide

C: Centigrade

cm: Centimeter

Cleland's Reagent: DL-Dithiothreitol

C: Crosslink

°: Degree

DNA: Deoxyribonucleic Acid

EAP: Erythrocyte Acid Phosphatase

EDTA: Ethylenediamine Tetraacetic Acid

G PD: Glucose—G-Phosphate Dehydrogenase
6

g: Gram

3: Greater than or equal to

JMH/RTC: Jackson Memorial Hospital/Rape Treatment Center

L: Liter

MUP: 4-Methylumbelliferyl Phosphate

ul: Microliter

mg: Milligram

m1: Milliliter

mm: Millimeter

M: Molar

NADP: fi-Nicotinamide Adenine Dinucleotide Phosphate

pH: Indicates measure of acidity

%: Percent

PGM: Phosphoglucomutase

PAGE: Polyacrylamide Gel Electrophoresis

PMR: Premixed Reaction Mixture

rpm: Revolutions per minute

RNA: Ribonucleic Acid

SERI: Serological Research Institute

T: Acrylamide

TEMED: Tetramethylenediamine

TCA: Trichloroacetic Acid

UV: Ultraviolet

V: Volts

v/cm: Volts per centimeter

viii

INTRODUCTION

One of the goals of this research was to examine the
usefulness of the polyamines spermine and spermidine as
confirmatory markers for the presence of seminal fluid when
the samples are spermatozoa free. The positive
identification of a sample as seminal fluid is a critical
determination in helping to solve sexual batteries. Because
of this, some background information concerning the crime of
rape will be presented prior to discussing the research
findings.

The second area of study in this research was the
electrophoretic determination of phosphoglucomutase (PGM)
subtypes. Several of the electrophoretic procedures which
have appeared in the literature were tested to assess their
usefulness for determining PGM subtype information. After
experiencing difficulty in obtaining usable results and
information, these procedures were modified and adapted to
create a new procedure.

The determination that a sample does or does not
contain seminal fluid will affect the forensic serologist's

analysis. Samples determined to be seminal fluid free will

not be analyzed because they will not provide any
information of value concerning the seminal fluid donor.
Samples determined to be seminal fluid positive can be
analyzed to provide additional information such as the PGM
subtypes. This information concerning the nature of the
samples and any additional information obtained from the
analysis will affect the interpretation of the information
provided by the analysis of the evidence.

This may then affect the prosecution and defense of
the subject by weakening or strengthening their cases and
how they will present them to the court during a trial. The
additional information may also affect the willingness of
the prosecution or defense to go to trial, thus affecting

the ultimate disposition of the criminal charges.

BACKGROUND; The Crime of Rape

Rape is a very serious violent crime which has been

committed at a greater frequency during the last two

decades. Between the years 1972 and 1976, the Federal
Bureau of Investigation reported a 21% increase in the
number of forcible rapes which were reported to the
police.(1) This trend toward an increase in the numbers of
reported forcible rapes has continued into the present.
During 1985, the number of forcible rapes which were
reported to police increased by 7% over those reported
during l984.(2) Furthermore, these statistics are not an
accurate representation of the real problem because most
rapes are never reported to the authorities.(3) The true
forcible rape rate is unknown but some estimates place it
between five and ten times higher than the number
reported.(4)

Determining the true frequency of occurrence for
forcible rape is impossible because of the reluctance of
many rape victims to report or discuss the incident. There
are a number of reasons for this. There may be a feeling of
guilt, shame or embarrassment. The victim may fear that the
rapist will retaliate should they report the crime. They
may fear the reactions of their friends and family, or they
may be totally subdued and frustrated by the legal system.

There are undoubtedly other reasons as well.

In recent years, many states have enacted new statutes
concerning rape. These new laws are designed to better
protect the victims and provide them with more privacy
concerning the details of the actual crime and their past
personal history. In the past, both of these areas were
open to public scrutiny during prosecution of the accused
rapist, thus creating a situation in which the victim seemed
to be on trial and not the defendant. The new rape laws
also have defined the crime in a more realistic way by
broadening the scope of what constitutes rape, who may be a
victim and who may be a rapist. This has, in turn, made it
easier for the state to prosecute this crime when it is
reported and a subject is identified.

The Florida statute which proscribes rape provides an
example of the new rape laws. This statute removes the term
"rape" from the law and renames the crime as "sexual
battery“. It then defines the term sexual battery and
specifically excludes bona fide medical procedures from this
definition. The law widens the definition of victim by
defining the victim as any individual alleging to have been
sexually assaulted. Under many of the old rape laws, men
could not be the victim of rape, while under the new law,
anyone - male or female - can be a rape victim. The new law
also broadens the definition of who can commit a sexual
battery. Many of the old laws excluded women from this
category. A husband could not be prosecuted for raping his

legal wife and males under the age of 14 were considered

incapable of committing this crime. The new law removes
these restrictions when describing a possible perpetrator.
A very important aspect of this law concerns

corroborative evidence. Under the old rape laws,

corroborative evidence or physical evidence of a rape was
required to prosecute the crime. If this corroborative
evidence could not be demonstrated, then no rape could be
proven. The new law doesn't require corroborative evidence
to prove that a rape occurred. It recognizes that there may
not be any physical evidence present in some sexual
batteries. If evidence is present, it is not required that
the prosecution use it during the trial even though it may
help prove the charges. It is a portion of this
corroborative physical evidence, namely seminal fluid, which
may be present in a sexual battery with which this
scientific study is concerned. The collection,
preservation, analysis and interpretation of this physical
evidence can help verify that the alleged victim toOk part
in sexual activity. This physical evidence can also provide
information about the alleged rapist (subject) and help
connect the subject to the alleged victim.

In a sexual battery committed by a post-pubescent
male, one type of physical evidence left behind may be
seminal fluid. Seminal fluid is the male reproductive
fluid. In the normal male, seminal fluid is a turbid white
fluid with a volume ranging between 1.5 ml and 5 ml.(5) The

fluid is a combination of several secretions and cellular

components which are mixed during the process of ejaculation
(ejection of seminal fluid from the male reproductive
organ). The ejaculate can be divided into three fractions
which correspond to the emptying of the different glands of
the genital tract.(6,7,8) The first fraction is the product
of the Cowper's gland, Littres gland and prostate gland: the
second fraction is contributed to by the ampulla and
epididymus and the third fraction by the seminal

vesicle.(9) The early portion of the ejaculate (fractions
one and two) contain the bulk of the spermatozoa (10) or
approximately 75% of the total sperm count.(ll) In the
normal male the spermatozoa constitute approximately .1% of
the total volume of the seminal fluid.(12) This translates
to 40-250 million sperm per milliliter in the normal healthy
male.(l3)

The bulk of the seminal fluid is composed of the
secretions from the seminal vesicle (45-80%) and the
prostate gland (15-30%).(14) It is because seminal fluid is
composed of multiple secretions that its characterization
and analysis make it a very valuable tool in the prosecution
of rape.

The positive identification of seminal fluid is
conclusive evidence that the alleged victim has recently
participated in sexual activity in which the partner was a
post-pubescent male capable of ejaculating seminal fluid.
This helps verify a portion of the alleged victim's

allegation of rape. It does not prove in any way that the

sexual activity was rape. There is no physical evidence
which can prove rape. The physical evidence may only help
indicate that there was sexual activity which may have been

forcible.

The other reason the identification of seminal fluid
is important is that it contains genetic materials which can
be isolated and identified by the forensic serologist. This
information is then used to help demonstrate that the
alleged rapist could have contributed the seminal fluid
sample which was identified. Because
seminal fluid can be valuable physical evidence, its

collection, preservation and analysis is very important.

COLLECTION AND PRESERVATION OF THE PHYSICAL EVIDENCE

In many large cities there exist rape crisis centers
which the victim of a rape may contact for assistance. The
purpose of these centers is to provide rape victims with
medical treatment and counseling to help them through the
rape crisis. The pe0ple who work in these centers are
specially trained in dealing with the victim so they can
determine the circumstances of the assault and properly
collect and preserve physical evidence for forensic
analysis. One of the first rape crisis centers in the
United States was Opened in the early 1970's at Jackson
Memorial Hospital in Miami, Florida. The Jackson Memorial
Hospital Rape Treatment Center (JMH/RTC) provides free,
private treatment for all rape victims.

The primary concern of the rape treatment center is
the victim's well being. The victim is not required to
report the crime to the police in order to receive medical
care. This policy recognizes that because many rape victims
never report the assault, they never receive medical aid.
By not requiring that the assault be reported to the police
as a prerequisite for receiving treatment, victims have the
opportunity to receive the pr0per medical care and
counseling they will need.

As part of the medical treatment, the victim of a
sexual battery is given a complete physical examination.

During this examination, notes are made by the doctor

concerning the presence of any physical trauma such as
bruises or lacerations. The doctor also collects physical
evidence which will be submitted to the crime laboratory for

analysis. To aid in the collection of the physical

evidence, the JMH/RTC uses a kit which was designed for them
by the Serology Section of the Metro Dade Police Department,
Crime Laboratory Bureau. The purpose of the kit is to
insure that collection and preservation of the physical
evidence is done in a manner which will help maintain its

maximum forensic value.

The kit is composed of the following items:

A) One red top vacutainer blood collection tube

B) One piece of clean cotton cloth about 1 inch x
1 inch contained in a small cardboard box

C) Sealed paper packages, each containing two sterile
swabs

D) Glass microscope slides

E) A ten ml screw top glass tube containing two to
three ml of physiological saline

A sample of the victim's blood is collected in a 10 m1
red top vacutainer blood collection tube. The red top
indicates that the tube contains no preservatives or
anticoagulants which may interfere with the analysis of the
blood sample. The purpose of this sample is to provide the
forensic serologist with information about the victim's
blood factors. This information is used for comparison to
the information which is provided by analysis of the other

physical evidence.

10

The victim provides a saliva sample by removing the
piece of clean cotton cloth from the cardboard box and
placing it in her mouth until it is saturated with saliva.

After saturating the piece of cloth, it is returned to the

cardboard box and allowed to dry. The saliva sample is used
to provide information about the victim's other body

fluids. The information is then used to help interpret the

information revealed by the analysis of the remainder of the
evidence.

The packages of sterile swabs are used to collect
samples from any of the body areas which are alleged to have
been involved in the assault. Since most sexual batteries
are vaginal, the doctor would use one package of the sterile
swabs to swab the vagina for seminal fluid. A second
package of swabs would be used to swab the cervix (the upper
limits of the vagina) for seminal fluid. The sample is
collected by rolling the cotton tip of the swab over the
area being sampled. This is done so that the entire surface
of the swab absorbs the fluid being collected, and provides
a large sample for analysis. After the sample is taken, the
wet swabs are used to make smears for microscopic
examinations of the sample. This is done by rubbing the wet
swab across the surface of a clean glass microscope slide.
After the smear is made, the swabs are returned to their
original paper package and allowed to air dry away from
light and heat. The smears are air dried and then wrapped

in gauze for protection.

11

After the affected areas have been swabbed, the saline
contained in the screw top glass vial is used to rinse the
vaginal vault. The liquid is then collected and placed back

in the glass vial. This vaginal washing is called the

vaginal aspirate.

The evidence is then properly labeled and sealed in a
paper bag for transport to the crime laboratory. All the
samples, excluding the blood standard and the vaginal
aspirate, are placed in packages which allow air to flow in
and out. This allows the samples to dry quickly, preserving
the information which has been collected. The faster the
samples dry, the greater their potential for providing
information during analysis.

Other evidence which may be collected at the rape
treatment center are swabbings and smears of any other area
involved in the assault, pubic hair combings, pubic hair
standards and the victim's clothing. The clothing which may
be the most valuable is the victim's panties. Seminal fluid
may drain out of the victim's vagina while traveling to the
rape treatment center. The panties may, in some cases,
contain more seminal fluid than the vagina, making them a
very important source of information about the individual
who deposited the seminal fluid sample. As with the rape
evidence collection kit, all these items are sealed in paper
bags or cardboard boxes and submitted as fast as possible to
the crime laboratory. Upon submission to the laboratory,

the samples are removed by the serologist and preserved in a

12

freezer while awaiting analysis. This helps preserve the
seminal fluid which may suffer rapid decomposition over
time. The faster the samples are preserved and frozen, the

more information they may provide during analysis.(15)

The final physical evidence which may be collected and
submitted to the laboratory is evidence collected at the
crime scene. This evidence is collected by crime scene
technicians who are trained in the proper collection and
preservation of physical evidence. The evidence which will
be collected depends on the details of the crime and varies

from crime to crime.

13

ANALYSIS OF THE EVIDENCE

The rape evidence collection kit and any items

impounded at the crime scene are submitted to the crime

laboratory where the forensic serologist preserves the
samples and does any analysis which may be necessary.

The initial analysis of the evidence is to determine
if seminal fluid is present on the samples which were
collected at the rape treatment center. If the samples are
seminal fluid positive, then further analysis can be
performed. This additional analysis may provide information
concerning the blood group and enzyme profile of the seminal
fluid contributor. Evidence which is seminal fluid negative
is of little or no value because it cannot provide any
information about the alleged rapist.

The first step in the analysis procedure is to locate
and identify any samples which may contain seminal fluid.

In the rape evidence collection kits, these items would be
the swabbings and the vaginal aspirate. The evidence
collected at the crime scene and any of the victim's
clothing must be examined visually to locate any stained
areas. Seminal fluid is a milky white color when in its
liquid form. This liquid, when it dries, leaves a white
stain which may be very difficult to visualize on items
which have light colorations or are white. Since the stain
may be difficult to see on clothing, it is very important

for the serologist to note areas which feel stiff in

14

comparison to the rest of the item. It is also important to
watch for areas in which the material appears more wrinkled
than the surrounding areas. These variations may indicate

that the area may contain a sample which should be tested

for the presence of seminal fluid.

A second method which may be used to locate suspected
seminal fluid stains is to examine the evidence under long
wave ultraviolet light. When a seminal fluid stain is
exposed to UV light, it will fluoresce and emit a very pale
glow. The area can then be tested by the serologist to
determine if the fluorescent activity is the result of
seminal fluid being present. A difficulty is that many
substances and materials will fluoresce under UV light, so
that many stained areas which are located will not contain
seminal fluid. Furthermore, the substrate may fluoresce,
thus preventing any stains from being visible.

After the suspect stains are located, it must be
determined if seminal fluid is present. To demonstrate that
a stain is seminal fluid, or contains seminal fluid, several
tests are used. The first test is the acid phosphatase
test, of which there are many variations, but all rely on
the detection of an enzyme called acid phosphatase. Acid
phosphatase is an extremely good presumptive marker for the
presence of seminal fluid. The enzyme is a product of the
prostate gland and is very abundant in seminal fluid
(16,17,18), and very persistent in the length of time during

which it can be detected.(19,20,2l) The detection of acid

15

phosphatase is a presumptive test for seminal fluid because
it is also present in many other biological materials
including saliva and vaginal secretions.(22,23) Even though
acid phosphatase is not specific for seminal fluid, it is a
very good marker for its possible presence. This is because
of the high levels of acid phosphatase which occur in the
seminal fluid of men with proPerly functioning prostate
glands. The levels of acid phosphatase in seminal fluid are
approximately 400 times the levels which can be found in
other biological materials.

The majority of tests and procedures for the detection
of acid phosphatase are chemical tests which produce a color
reaction which may be visualized by the unaided eye. An
example of one procedure is the use of sodium
thymolphthalein monophosphate as described by Seiden and
Duncan.(24) When a sample is being screened for the
presence of seminal fluid, a small cutting is removed from
the suspected seminal fluid sample. The cuttings, along
with a known seminal fluid sample (positive control) and an
unstained cutting of the item being examined (negative
control), are saturated with a few drops of the sodium
thymolphthalein solution. The samples are allowed to stand
for five minutes at room temperature, after which time
several drops of the color developer are added. As the
color developer is added, the sample is Observed for the
development of a blue coloration. This blue color is a

positive test for the presence of acid phosphatase. This

16

test result is then compared to the positive and negative
controls to verify that the test worked pr0perly and that
the results were not false-positive for the presence of acid
phosphatase.

The samples which are acid phosphatase positive are
then analyzed further to conclusively determine if they
contain seminal fluid. The best way to demonstrate this is
to microscopically examine the sample for the presence of
spermatozoa.(Figure l)

Spermatozoa are the male gametes (25) and are produced
in the testes of the normal adult through the process of
spermatogenesis.(26) The mature spermatozoan in man has a
filoform structure (27), due to the presence of the
flagellum or tail which is used for motility. As exhibited
in Figure l, spermatozoa consist of a head, neck, midneck
and tail. These cells are unique in their appearance and
may be easily identified by microscopic examination of the
samples. The presence of spermatozoa is conclusive proof
that seminal fluid is present, because spermatozoa does not
occur naturally in any other biological fluid. The
identification of spermatozoa demonstrates that the sample
being examined is either pure seminal fluid or a mixture of
seminal fluid with other materials such as the vaginal
secretions of the victim.

There are many ways to microsc0pically examine the
smears for the presence of spermatozoa. One of the

preferred methods is phase contrast microscopy.(28)

17

NICK

MID-PIECE

 

TAIL“ __

 

 

 

 

 

Figure l: The morphology of spermatozoa.(29)

18

When biological materials such as seminal fluid are examined
using bright field microsc0py, there is very little contrast
between the cellular matter and the background. This lack
of contrast makes it very difficult to visualize and
identify what is present in the sample. Phase contrast
microsc0py bends the light waves being projected through the
sample, creating a much better contrast between the
background and the cellular materials. Using phase contrast
makes visualization of the spermatozoa easier, and reduces
the chance that a misidentification may occur. The problem
with phase contrast microsc0py is that it will not work on
samples which are opaque, such as clothing or bedding.

These samples can be examined by extracting a small portion
of the stain in saline. The eluate may then be placed on a
clean glass microscope slide and microscopically examined
for spermatozoa using phase contrast microscopy.

In the rape evidence collection kit, the smears which
were made from the swabs at the time of the victim's
physical exam are examined for spermatozoa. The vaginal
aspirate is examined by placing one or two drops of the
liquid on a microscope slide and covering the sample with a
glass cover slip. The sample may then be examined
microscopically for spermatozoa.

What happens when the microscopic examination of the
samples which were positive for acid phosphatase fail to
reveal the presence of spermatozoa? Until recently, the

only way to conclusively identify seminal fluid was to

19

observe the presence of spermatozoa. This is not always
possible, because not all adult men produce spermatozoa.
There are many factors which can cause this situation, such
as fertility problems, cancer of the testes or blockage of
the ducts. This situation is called azoospermia and is an
abnormal trait. Azoospermia can be temporary or permanent
depending on its cause. Azoospermia can be the result Of a
vasectomy, which is a surgical procedure designed as a form
of birth control. When a vasectomy is performed, the tubes
which lead from the vas deferens are severed. The vas
deferens is the storage area for the mature spermatozoa. By
severing its connection to the ejaculatory duct and the
urethra, it prevents the spermatozoa from being mixed with
the seminal plasma and creates an azoospermic condition.
Oligospermia or low spermatozoa count may also be the
reason that no spermatozoa are seen. Spermatozoa are very
fragile after ejaculation and degrade rapidly with time. In
the vagina, the major portion of the spermatozoa will be
gone within 12 hours, while the acid phosphatase levels of
the vagina will be back to normal within 48 hours Of sexual
intercourse.(30) Positive acid phosphatase tests may be
Obtained for almost four times as long as positive
microscopic examinations for spermatozoa. The rapid decay
of spermatozoa, along with a low spermatozoa count may make
it nearly impossible to microscopically find spermatozoa on

the samples.

20

Oligospermia is usually the result of fertility
problems, high fever, or frequency of ejaculation although
there may be other causes. Frequency of ejaculation will
affect the spermatozoa count and volume of ejaculate. The
more frequently an individual ejaculates, the lower the
spermatozoa count and volume of the seminal fluid. This is
because the body does not have enough time to regenerate its
reserves between ejaculations, thus lowering the amount of
spermatozoa and seminal fluid available for ejaculation.

It is not unusual to have samples which are acid phosphatase
positive and spermatozoa negative. In a study conducted at
the MetrOpOlitan Police Forensic Science Laboratory in Great
Britain, 1.9% of the seminal stains on swabs examined were
azoospermic.(3l) When this occurs, other confirmatory tests
for seminal fluid must be used. The most frequently used
confirmatory marker besides spermatozoa is a protein called
p30.

The use of p30 as a seminal fluid specific marker was
first reported in the literature in 1978 by G.F.
Sensabaugh.(32) The value of p30 is that it appears to be
specific for seminal fluid and, to date, it has not been
shown to occur naturally in any other biological fluids
including vaginal secretions.(33,34,35) The p30 protein is
very stable and persists for long periods of time in both
the vaginal environment and in dried samples.(36) The
protein is detectable in vaginal fluids for a mean period of

27 hours after coitus compared with a mean period Of 14

21

hours for acid phosphatase.(37) Because p30 protein is a
glycoprotein secretion Of the prostate gland (38) and since
the prostate gland is a major contributor to the seminal

fluid mixture, p30 is usually present in high levels making

its detection and identification easier. Furthermore, p30
is even present in the seminal fluid Of vasectomized
men.(39)

There are several procedures which can be used to
identify the p30 protein. The procedure which is used most
frequently is crossover electrophoresis. The unknown
samples are extracted in physiologic saline while a 1%
agarose gel is poured. Two parallel rows of wells (holes)
are punched in the gel and their cores removed. TO one row
Of wells, the unknown samples are added and to the other row
is added anti-p30. Anti-p30 is an antiserum which contains
antibodies specific for the p30 protein. Electrophoresis is
then performed in a tris buffer system. After completion of
the run, the gel is removed and placed in a 1M solution of
sodium chloride overnight. The next day, the plate is
soaked in distilled water and then dried in an incubator.
After the gel has dried, it is stained in Coomassie blue and
destained. The gel is then examined for precipitin bands
which may have formed between the unknown sample wells and
the anti-p30 wells. The band is a positive test result for
the presence Of the p30 protein, which indicates the sample
contains seminal fluid. Figure 2 shows the entire seminal

fluid identification and analysis procedure.

22

The victim reports the sexual battery
to the police

victim examined crime scene tech-
at JMH/RTC and rape nician collects
evidence collection any scene evidence

evidence is submitted
to crime laboratory

samples removed visual and/or UV exam
from rape kit ‘--for possible seminal
fluid stains

 

 

ll _

AP testing of all samples -—-€> stop analysis

+
spermatozoa searches -—€>~p30 -—E> stop analysis

+ +

further analysis Of samples to
determine ABO blood group infor-
mation and PGM enzyme information

Figure 2: The identification and analysis Of seminal fluid
evidence.

23

After all the evidence is examined for samples
containing seminal fluid, a determination is made concerning
the need for additional analysis. Any samples which are

negative for the presence Of seminal fluid are of no further

value.

The samples which are seminal fluid positive have the
potential to provide information about the alleged rapist.
These samples can be tested for ABO blood group
information. If the seminal fluid contributor is a
secretor, analysis of his seminal fluid will provide
information about his blood group. A secretor is an
individual who secretes blood group information into his
other body fluids. These fluids, such as saliva, seminal
fluid or vaginal fluid, can then be analyzed to determine
his ABO blood group. Analysis Of a nonsecretor's body
fluids (excluding blood) will not provide information about
their ABO blood group. One must keep in mind that
approximately 80% Of the population are ABO secretors, and
that the seminal fluid samples being tested may be mixed
with the victim's body fluids. Therefore, interpretation Of
the blood group information must be compared to blood and
saliva standards Of the victim and subject to determine who
may have contributed the blood group information.

The seminal fluid positive samples are also tested to
determine their PGM enzyme information. PGM is an.enzyme
which is present in the blood of all people, every male's

seminal fluid, every woman's vaginal secretions, and is not

24

affected by an individual's secretor classification. Like
the ABO blood group, it is a genetic marker which occurs in
several forms, and each individual may only have one of
these forms. Testing Of the samples may reveal PGM
information which can then be compared to the PGM types Of
the victim and subject for interpretation. To further aid
in the interpretation of this information, the three major
PGM types can be broken down into 10 subtypes. The
subtyping information can be used in several ways, one of
which is helping identify samples which may be mixtures of
the victim's fluids with the subject's seminal fluid.

The genetic marker information is then used to create
a population profile. The profile illustrates who, in the
general population, could have contributed their seminal
fluid to create the genetic information which was
determined. This profile takes into account the victim's
genetic markers and any possible mixtures of the victim's
fluids with the seminal fluid sample. The profile is then
used to generate a population frequency to illustrate the
size of the pOpulation which could have provided this
genetic information. This profile of genetic markers and
the poPulation frequency is compared to the subject's
genetic markers to determine if the subject is within the

contributing population or excluded.

25

The identification Of seminal fluid is very important
because it verifies sexual activity and it may provide
genetic marker information which can implicate the subject.

It is for these reasons that this scientific study on the

electrophoretic examination of seminal fluid was done.

26

SPERMINE AND SPERMIDINE AS CONFIRMATORY MARKERS FOR SEMINAL

FLUID

A possible solution to the identification Of seminal
fluid in the absence of spermatozoa, is to employ
electrophoretic analysis of seminal fluid to establish the
presence of the polyamines spermine and spermidine, both Of
which are usually present in seminal fluid.

Seminal fluid is composed Of many potentially
electrically charged constituents.(40) Therefore, they can
be separated by electrophoresis. Two components Of seminal

fluid are the polyamines spermine (C H N ) and spermidine
26 26 4

(C H N ). These two substances give seminal fluid its
7 l9 2

characteristic Odor.(4l) They occur in variable
concentrations within seminal fluid and have been identified
as a secretion Of the prostate gland in men.(42) An
important feature Of spermine and spermidine is that they do
not occur naturally in vaginal fluid, feces, urine or
saliva.(43,44) Another important feature of these
polyamines is that they are very stable and can be used for
identification of seminal fluid even after long periods of
time have elapsed.(45)

Spermine and spermidine can be easily and rapidly
separated from the other constituents Of seminal fluid and,
thus, identify it even in the absence Of spermatozoa. The
main prOblem with employing spermine and spermidine to

identify seminal fluid occurs in cases in which the

27

subject's prostate gland is not functioning properly. These
polyamines are secreted by the prostate gland, its
malfunction may result in no spermine or spermidine being
present. Studies also indicate, however, that certain types
Of prostatic pathology may increase the seminal fluid

concentration Of these polyamines.(46)

Literature Review:

The following is a survey of the relevant literature
concerning the analytical methodology employed in the
analysis of polyamines such as spermine and spermidine.

Lewin, Beer and Glezerman (47), while studying the
prostatic and vesicular contributions of seminal fluid,
developed an electrophoretic technique for the separation of
seminal fluid into its constituents. This technique used
cellulose acetate as a support medium and provided good
separation Of the constituent bands. Following
electrophoresis of the seminal fluid on the cellulose
acetate, the electrophoretic patterns were Observed,
measured and identified. A series Of bands was Observed,
one Of which they labeled the PB band. The PB band was
determined to be a product Of the early portion Of the
seminal fluid, and was secreted by the prostate gland. An
analysis Of the band showed it to contain spermine and

spermidine.

28

While doing a study Of urinary polyamine levels for
the diagnosis of prostatic cancer, Fair, Wehner and Brorsson
(48) found that the prostate gland contained the highest
concentrations of polyamines within the human body. It was
also found that cancer of the prostate was accompanied by
increased levels Of these polyamines, especially
spermidine. They proposed that the electrOphoretic analysis
of urine for increased levels of spermidine could be used to
diagnose cancer Of the prostate. The method which they used
for analysis was paper electrophoresis in a citric acid
buffer. The procedure was complicated and required an
extended period Of time and therefore is not deemed suitable
for forensic analysis Of seminal fluid.

Chen, Forsyth, Buchanan and Holmes (49) determined the
amine content of vaginal fluid from women with treated and
untreated nonspecific vaginitis. The analysis of the
vaginal fluid samples was done using high voltage
electrophoresis on a Whatman paper support medium. They
also did a second analysis in which thin-layer
chromatography was used. ElectrOphoresis Of the amines
showed the presence Of cadaverine, histamine, putrescine and
methylamine in the vaginal fluid of most the untreated
cases. In several cases, it was also determined that
spermine and spermidine were present. Further analysis Of
these cases indicated that the presence of spermine and
spermidine was due to the presence Of seminal fluid in the

test samples. It was also determined that spermine and

29

spermidine are not naturally occurring constituents of
vaginal fluid.
Suzuki, Oya, Katsumata, Matsumoto and Yada (50)

develOped an enzymatic method for the determination Of

spermine in human seminal fluid. The method depends on an
enzymatic reaction between the spermine and the reagent to
produce a green stain. The analysis method is easy to do
and it yields good results. They proposed using this test
instead of the acid phosphatase test because spermine is
more stable than acid phosphatase and can be identified in
very Old seminal stains.

Clavert, Montagnon and Brun (51) did a study Of the
proteins present in seminal plasma. Their method of
analysis was electrophoresis on a cellulose acetate support
medium. The method and their results were similar to those
Of the Lewin, Beer and Glezerman study. Their research
method differed in that the whole seminal fluid was
analyzed. Then an analysis was done of the seminal fluid
excluding the prostatic secretions, seminal fluid minus the
vesicular secretions, and seminal fluid minus both prostatic
and vesicular secretions. They determined that when the
prostatic secretions were excluded, the seminal fluid sample
lacked the spermine and spermidine constituents, therefore
confirming that spermine and spermidine are secretions of

the prostate gland.

30

DISC ELECTROPHORESIS ON POLYACRYLAMIDE GELS

Introduction

Disc electrophoresis on polyacrylamide gels (PAGE)
refers to the discoid shape Of the protein bands, due to the
tubular shape Of the electrOphoresis cell and the
discontinuities in the electrOphoretic matrix resulting from
the layering of a stacking gel on tOp Of the separation
ge1.(52,53)

Charged particles and solutes will migrate through a
support medium in the presence of an electric fluid. This
process is known as electrOphoresis. Some molecules have an
electric charge because Of proton ionization.(54) The
particle will have a positive charge if it has less
electrons than it has protons and will move toward the
cathode (negative pole) Of the electrophoresis system,
whereas a negatively charged particle will have a surplus of
electrons and will move toward the anode (positive pole).
The rate Of migration of these particles during
electrophoresis is variable and depends on several factors
such as the net electric charge of the molecule, the
temperature Of Operation, the strength of the electric
field, the nature Of the support medium, and the size and
shape Of the molecule.(55) Because all substances are

composed Of components which may have differing electric

31

charges, under various conditions, electrophoresis can be
used to separate them into their individual components.

In PAGE electrophoresis the gels are composed Of
acrylamide and the cross-linking agent N,N'—methylene-bis-
acrylamide (Bis). The gel is prepared by polymerizing the
gel monomer (acrylamide) with the cross-linking agent (Bis)
and the appropriate catalyst to form a solid medium. There
are two methods Of polymerization (gelation) which may be
used.(56) Polymerization may be done chemically by the
addition Of ammonium persulfate and tetramethylenediamine
(TEMED) to the liquid gel solution. In the presence Of a
small amount Of oxygen, the addition Of these two substances
will cause the acrylamide-Bis mixture to solidify. This
method of polymerization is usually used when making the
separation gel which is the majority of the gel through
which the samples will be separated. The other manner Of
polymerization is photochemical. Photochemical
polymerization is achieved by the addition Of riboflavin to
the acrylamide-Bis mixture and exposure Of the mixture to
ultraviolet light. This method of polymerization is usually
used when adding the stacking gel on top Of the separation
gel. The purpose of using multiple gels is that the
stacking gels, which have a large pore size, will line up
the molecules of the sample being separated and the
separation gel will then separate the sample's constituents
based upon their molecular size. The smaller molecules will

move through the pores of the gels more rapidly than the

32

larger molecules, revealing many well defined protein bands
when the gel is stained. The pore size of polyacrylamide
gels is inversely related to the acrylamide-Bis
concentration. As the Bis concentration increases, the pore
size Of the gel decreases. The gels can be made from about
3% (large pores) to about 30% (small pores) with the usual
range being between 5% and 10%.(57)

An advantage of electrophoresis on polyacrylamide is
that the gels are easy to make and they may be stored in
refrigerated, airtight packages for extended periods Of
time. The gels are desirable because they are thermostable,
transparent, strong, provide very good sample separation,
are relatively chemically inert, and can be made over a wide
range of pore sizes.(58) The ability to vary the pore size
Of the gel lends itself to the separation of compounds which
have a small molecular size such as spermine and spermidine.

The disadvantage of electrophoresis using
polyacrylamide gels is that the acrylamide and Bis are
neurotoxins which are readily absorbed through the skin.

The chemical accumulates in the body where it remains
permanently to build up in concentration with continued
exposure. Because of this, the acrylamides must be handled
very carefully to minimize exposure prior to

polymerization. After polymerization the gels are no longer

hazardous.

33

The other disadvantage associated with the use of
polyacrylamide gels is that the staining and destaining
process used to visualize the protein bands is a very long

process due to the thickness Of the gel. This procedure
must be done over several days so the stains and destaining

reagents may permeate the gel.
Methods and Materials
1. Gel Preparation
A) Separation Gel (7.184% T, 2.56% C) (59)

Solution A
Dissolve 14.0 g Acrylamide (Bio-Rad)
0.36 g Bis (Bio-Rad)
in 35 ml Of distilled water. After all
the solids are dissolved, add enough dis-
tilled water to bring the solution up to
a final volume of 50 ml.

Solution B

Dissolve 9.05 g Trizma Base (Sigma)
12.00 ml 1M Hydrochloric Acid (Ricca)
0.06 ml TEMED (Bio-Rad)
in 25 ml of distilled water. After all
the chemicals are in solution, add enough
distilled water to bring the solution up
to a final volume of 50 ml.

Solution C
Dissolve 0.14 g Ammonium Persulfate (Bio-Rad)
in 90 ml of distilled water. After the
solids are dissolved, add enough dis-
tilled water to bring the solution up to
a final volume of 100 m1.

34

To prepare the separation gel, mix one part Solution
A, one part Solution B, and two parts Solution C. Pour the
mixture into borosilicate disc gel tubes which have been

sealed at the bottom with parafilm and placed in a level

tube rack. The tubes should be filled approximately 3/4 of
the way full. Carefully (to avoid mixing) deposit a thin
layer of distilled water on top of the mixture. Allow the
tubes to stand undisturbed until the acrylamide solution is
completely polymerized (1 hour). After polymerization is

complete, the stacking gel may then be added.

B) Stacking Gel (3.125% T, 20% C) (60)

Solution A
Dissolve 2.99 g Trizma Base

0 . 23 ml TEMED
in 20 ml of distilled water. After all
the chemicals are in solution, adjust the
mixture to pH 6.7 by adding 1M hydro-
chloric acid (approximately 24 ml). Add
enough distilled water to bring the
mixture up to a final volume of 50 ml.

Solution B
Dissolve 10.9 g Acrylamide
2.5 g Bis
in 35 ml Of distilled water. After all
of the solids are dissolved, add enough
distilled water to bring the solution up
to a final volume of 50 ml.

Solution C
Dissolve 2.0 mg Riboflavin (Bio-Rad)
in 50 m1 of distilled water

35

Solution D
Dissolve 20.0 g of Sucrose (Sigma)
in 35 ml of distilled water. After the
sucrose is completely dissolved, add
enough distilled water to bring the
solution up to a final volume of 50 ml.

TO prepare the stacking gel, mix one part Solution A,
one part Solution B, one part Solution C, four parts
Solution D, and one part distilled water. Pour the mixture
into the disc electrophoresis tubes which contain the
polymerized separation gel. This layer should be
approximately 1/4 inch thick. Carefully (to avoid mixing)
deposit a thin layer of distilled water on top of the
mixture. The stacking gel must be exposed to ultraviolet
light to polymerize. Polymerization takes approximately one
hour. The completed gels can be sealed with parafilm and
stored in a refrigerator for several weeks prior to being

used.

11. Buffer Preparation

The tank buffer is a Tris/Glycine buffer. This buffer
can be made in a concentrated form and stored under
refrigeration for long periods of time. When buffer is
needed, the concentrate can be diluted, cooled down to 4°C
and used. The tank buffer should be made fresh for every

use.

36

A) Stock Solution (61)

Dissolve 6.0 g Trizma Base
28.8 g Glycine (Sigma)
in 750 m1 of distilled water. After the
chemicals are dissolved, add enough dis-

tilled water to bring the solution up to
a final volume Of 1 L.

B) Working Solution

The working buffer solution is a 10% dilu-
tion of the stock solution. This is made
by mixing one part of the buffer stock so-
lution with nine parts distilled water.
The diluted buffer must then be cooled to
4°C prior to use. The diluted buffer
should be made up fresh for each electro-
phoresis run and discarded after each

use.

III. Electrophoresis Procedure

Electrophoresis of the samples is done in a Bio-Rad
electrophoresis cell designed for vertical electrophoresis
using polyacrylamide disc gels. The power source is a Bio-
Rad model 1420A. All runs are done at room temperature
using precooled buffers. The length of the runs is
variable, and based on the movement Of a bromophenol blue
(Eastman) tracking dye which is added to the buffer of the
upper electrOphoresis cell. The tracking dye is a low
molecular weight dye which moves through the gel at a faster
rate than most Of the proteins which are present in the
samples. The dye is used as an indicator Of the rate and

distance the samples constituents have migrated in the gel.

37

The dye will not alter or affect the constituents of the

sample being separated.

The following procedure is used for the electro-

phoretic separation of the samples being studied:

1)

2)

3)

4)

5)

6)

7)

The polyacrylamide disc gels are placed in the
electrophoresis chamber gel holders, with the
stacking gel being positioned at the tOp. The
upper buffer chamber in this procedure is the
anode (positive pole).

The lower buffer chamber (cathode) is filled with
cold 10% buffer. The buffer should rise at least
1 cm above the base of the disc gels.

The small air space at the base of each disc gel
is filled with buffer. This will insure a good
electrical contact.

The gel holder is gently lowered into the
electrophoresis chamber.

The large air space in the t0p of each gel is
filled with tank buffer. The gels are now ready
for the introduction Of the samples.

The samples must be mixed with four parts sucrose
solution (stacking gel preparation, Solution D).
This adds weight to the sample so it will not
float out of the disc gel tube. The addition of
the sucrose will not alter or affect the electro—
phoresis results. After the sample is diluted
with the sucrose solution, 5 ul are then gently
introduced into the upper end of the disc gel.

The electrodes are inserted into the electro-
phoresis chamber. The lower buffer chamber
should contain enough buffer so that the end of
the electrode is submerged in a minimum of 1 cm
of buffer.

8)

9)

10)

ll)

12)

13)

14)

38

The upper chamber is gently filled with buffer
until its level is 1/4 inch above the top of the
disc gels.

Four to five dr0ps of bromophenol blue tracking
dye are added to the upper buffer chamber. The

buffer is gently stirred to mix the tracking dye
with the buffer.

The lid is placed on the electrophoresis chamber
and the power cables are connected to the power
supply. The cables should be connected to the
chamber so the upper buffer chamber is the anode
(positive pole) and the lower buffer chamber is
the cathode (negative pole).

The power supply is turned on and allowed to warm
up for several minutes.

The power supply is set to constant voltage and
the voltage is adjusted to the desired level. In
this study the voltage was set at 240 V.

Upon completion of the run, which is either deter-
mined by the migration of the tracking dye or the
elapsed time, the power supply is turned down to
zero and then switched Off. Disconnect the power
cables and Open the electrOphoresis chamber.

The disc gel tubes are removed from the electro-
phoresis chamber. The electrophoresis procedure
is now complete and the gels must be stained to
view the band patterns of the separated sample.

IV. Staining and Destaining

The staining and destaining procedure for poly-

acrylamide disc gels is very lengthy in comparison to that

of other electrophoresis separation media. This is due to

the increased thickness of the gel. During staining, the

39

gel must be totally permeated by the stain, after which the

unfixed stain (that which did not bind to any protein) must

be removed. Removal of the excess stain, reveals the areas

of stained proteins. These areas appear as very thin, well

defined discs running the length of the gel.

Three stains were tested during this study. The

stains were Coomassie-Brilliant Blue R-250 (Bio-Rad),

Crowle's Double Stain for immunodiffusion (Polysciences,

Inc.) and Ponceau S (Gelman). The Ponceau S and the

Crowle's Double Stain were purchased premade while the

Coomassie stain must be made fresh as needed.

A) Stain Stock Solution (62)

Solution A
Dissolve

Solution B
Dissolve

0.5 g of Coomassie Brilliant Blue R-250
in 35.0 ml of distilled water. After the
Coomassie is dissolved, add distilled
water until a final volume of 40 ml is
reached.

12.5 g of Trichloroacetic Acid (TCA) in
80.0 ml of distilled water. After the
TCA is dissolved, add distilled water
until a final volume of 100 ml is
reached.

B) Staining Solution

Mix 2 ml of the stain stock solution with 40 ml TCA

solution.

40

All three Of the protein stains contain

trichloroacetic acid (TCA). The purpose of the TCA is to

fix the proteins which are present in the gel. If the

proteins are not fixed, they will diffuse out Of the gel

during the staining process.

The following staining and destaining procedure is

used:

1)

2)

3)

The polyacrylamide gels are carefully removed
from the tube. This is done by forcing water
between the edge of the glass tube and the gel
with a syringe. The gel will slide out Of the
tube. When handling the gel be very careful not
to damage it.

The gel is placed in the stain for a minimum of
24 hours or until it is totally permeated by the
stain.

The gel is placed in the appropriate destaining
solution to remove the excess stain. As the
destaining solution becomes dark with the excess
stain, it should be replaced by fresh destaining
solution. This is continued until all the excess
stain has been removed from the gel. The gel may
then be stored in distilled water for an indef-
inite period of time.

The Coomassie Blue and Crowle's stains are removed

from the gels by soaking them in a mixture which was 50%

methanol (Mallinkrodt), 40% distilled water, and 10% glacial

acetic acid (Mallinkrodt). After the majority of stain is

removed, the gels are transferred to a mixture which is 88%

distilled water, 7% glacial acetic acid, and 5% methanol for

removal Of the remaining stain.

41

The Ponceau S stain is removed by soaking the gels in
a 5% solution of glacial acetic acid, which was replaced as

necessary until the destaining process was completed.

V. Results

The initial sample separations are done at a constant
voltage of 250 volts for valuable lengths of time, depending
on the migration Of the tracking dye. The samples which
were run were human serum, human hemoglobin, and human
seminal fluid. The human serum samples and the human
hemoglobin samples were obtained from the blood bank at
Edward W. Sparrow Hospital, Lansing, Michigan. The human
seminal fluid samples were provided by the Laboratory of
Clinical Medicine which Obtained the samples from male
donors being tested for fertility problems. Since the
seminal fluid samples were submitted by men with suspected
fertility problems, the following criteria was used to

select the seminal fluid of "normal" fertile males.

1) Seminal fluid is a white-grey Opaque coagulum
which is viscous.

2) pH 7.2 to pH 7.8

3) The seminal plasma should initially coagulate and
subsequently liquefy within 10 to 30 minutes.

4) A total volume of 1.5 ml to 5.0 ml.

42

5) Normal motility Of the spermatozoa:

a) The majority (370%) of the normal spermatozoa
show fast movement.

b) The majority (370%) of the normal spermatozoa
show forward movement.

c) The majority (370%) of the normal spermatozoa
show some type of movement.

d) The majority (>70%) of the normal spermatozoa
should be motiIe after two hours.

6) A spermatozoa count between 40-250 million sperma-
tozoa/ml.

7) The majority (390%) of the spermatozoa should not
be agglutinated initially or after two hours.

8) Normal spermatozoa morphology:

a) The majority (370%) of the spermatozoa should
be normal forms.

b) Less than 4% of the spermatozoa should be ab—
normal or immature forms.

c) The seminal plasma should not contain large
numbers of foreign cells as leukocytes, bac-
teria and/or fungi.

9) The viability of the spermatozoa must be 365%.

The criteria used were the same as the standards which
were set by the Laboratory of Clinical Medicine, Lansing,
Michigan to assess the seminal fluid samples of its
patients. The information concerning each of these samples
was provided with the samples for assessment by this
researcher.

The seminal fluid samples which were selected for this
study were centrifuged for 10 minutes at 3000 rpm to remove
the cellular components. The seminal plasma was divided up
into portions and stored at -70°C until needed.

The initial electrophoretic separations were done to
determine the best electrophoretic separation parameters for

this study and to determine what sample band patterns would

43

appear on 7% polyacrylamide gels. The final initial area of
comparison was to compare the banding patterns of the
seminal fluid samples to those of the plasma and the

hemoglobin so that bands which may be unique to the seminal

fluid could be identified.

After Obtaining an understanding of the sample band
patterns and a feel for the basic electrOphoresis procedure,
the samples were run against laboratory prepared mixtures Of
spermine and spermidine. This was done to help identify
which bands in the gels were spermine and spermidine. The
laboratory prepared spermine and spermidine standards were
run simultaneously on cellulose acetate to aid in the
determination of their correct concentrations. All the
standards which were developed contained too high a
concentration of spermine and spermidine for the cellulose
acetate study of seminal fluid, but these increased levels
were useful in the polyacrylamide disc gel studies.

A comparison of the gels used to separate the seminal
fluid samples with the gels used to separate the spermine
and spermidine standards failed to reveal the presence of
any spermine or spermidine bands. Since the length of time
during which the electrOphoretic separation was done was
being determined by the movement Of the tracking dye, it is
possible that the samples were being electrOphoresed for too
long. If the time of electrophoresis is tOO long, portions
of the sample being separated can be run Off the end of the

gel and lost. In the case of spermine and spermidine, this

44

is possible because their molecular weight is lower than
that of the tracking dye and they would move through the gel
faster than, and in front of, the tracking dye.

To test for this possibility, the length of
electrOphoresis was timed and decreased to determine at
which point the spermine and spermidine bands could be
identified on the disc gels. The point at which the bands
could be identified on the disc gels would be used to set
the length of time during which the electrOphoretic
separations of the seminal fluid samples could be done.
Decreasing the length of time during which the samples were
electrophoretically separated failed to reveal the presence
of the spermine and spermidine bands on the disc gels.

Since shortening the run time of the electrophoretic
separation failed to reveal which bands were the spermine
and spermidine, different protein stains were tested. The
use of the different stains could possibly identify
different band patterns, because of the differences in their
staining sensitivity. It was possible that the spermine and
spermidine were not being stained by the Coomassie-Brilliant
Blue, which was being used. The additional stains which
were tested were Ponceau S, which was used to stain the
seminal fluid samples being electrophoretically separated on
cellulose acetate, and Crowle's Double Stain for
immunodiffusion. The gels stained with these dyes failed to

reveal the presence Of the spermine and spermidine bands.

45

Spermine and spermidine are very small molecules. The
smaller the molecule, the faster it will migrate through the
polyacrylamide disc gels. The gels being used were 7%

acrylamide which indicates that the pore size of the gel is

large. In this particular study it is possible that the
pore size of the gel was too large to trap the spermine and
spermidine within the gel matrix, thus explaining the
failure to identify these bands on all the previous
electrophoretic runs.

The pore size of acrylamide gels can be decreased by
increasing the acrylamide concentration of the gel. The
acrylamide concentration may be increased as high as 30%,
creating gels which have very small pore Openings in the gel
matrix. These gels are used to study substances with very
small molecular sizes such as DNA and RNA. TO test for the
possibility that a smaller pore size in the gel matrix would
help in the identification of the spermine and spermidine,
new gels were made. The new gels had a higher Bis to
acrylamide concentration, which decreased the pore size Of
the gel matrix. The solutions and procedures used to make

these gels are as follows:

46

Gel Preparation (63)

Solution A
Dissolve

Solution B
Dissolve

Solution C
Dissolve

Solution D
Dissolve

Solution E
Mix

11.47 g of Trizma Base in

50.0 ml of distilled water plus

28.92 ml of 1M Hydrochloric Acid

add enough distilled water to bring the
solution up to a final volume of 100 ml.

1.92 g of Trizma Base in

50.0 ml of distilled water plus

25.6 ml Of 1M Phosphoric Acid

add enough distilled water to bring the
solution to a final volume of 100 ml.

38.0 g Acrylamide and

2.0 g Bis in

70.0 ml of distilled water. After the
chemicals are dissolved, add enough dis-
tilled water to reach a final volume of
100 ml.

5.0 g Acrylamide and

1.25 g Bis in

70.0 ml of distilled water. After the
chemicals are dissolved, add enough dis-
tilled water to reach a final volume of
100 ml.

0.6 m1 of a 10% solution of Ammonium
Persulfate

10.0 ml of a 0.02% solution of Riboflavin
Phosphate

89.0 ml of distilled water

Mix the above stock solutions as prescribed in Figure

3 to produce the desired gel. The procedure for making the

gels and polymerization is the same as was described

earlier.

47

 

 

 

 

 

 

 

 

SOLUTION SEPARATING STACKING
GEL GEL
5%T 5%C 10%T 5%C 15%T 5%C
Monomer 1.25 ml C 2.5 ml C 3.75 ml C 10 ml D
C or D
Buffer 2.5 ml A 2.5 ml A 2.5 m1 A 5 m1 B
A or B
Distilled 5.0 ml 3.75 ml 2.5 ml None
Water
Catalyst 1.25 ml 1.25 ml 1.25 ml 5 ml
E
TEMED 20 ul 20 ul 20 ul 10 ul

 

 

 

 

 

Figure 3: PAGE

composition menu.(64)

 

48

The study using the disc gels with smaller pore sizes
was identical to the one which was done on the 7% acrylamide
disc gels. Electrophoresis was carried out over variable
times based upon the movement of the bromophenol tracking
dye. The gels were stained with the same three protein dyes
and all the known seminal fluid samples were run versus the
laboratory standards of spermine and spermidine.

The study which was done on the disc gels with the
smaller pore sizes resulted in the same findings as the
study using the 7% acrylamide gels. The spermine and

spermidine bands could not be isolated and identified.

IV. Conclusions

The electrophoretic separation and analysis of the
seminal fluid samples on polyacrylamide disc gels failed to
provide any information concerning the location or
identification of spermine and spermidine. The failure to
located and identify the two polyamines is probably the
result of their small molecular size. The small molecular
size of these substances allows them to migrate rapidly
through the pore matrix of the gel, making it impossible to
confine them within the gel and create identifiable bands
patterns upon staining. If this is the case, then

separation of seminal fluid on polyacrylamide disc gels is

49

not suitable for the isolation and identification of
spermine and spermidine.
The other possibility is that the spermine and

spermidine may have been negatively charged. The molecular

charge of spermine and spermidine is determined by the tank
buffer. If these polyamines have a negative charge in this
buffer system, they will migrate toward the anode which has
a positive charge. If these compounds migrate toward the
anode, they will not migrate through the disc gel and the
gel will not exhibit any band patterns when stained, because
the spermine and spermidine will not be present in the disc
gel. To determine if this was the reason for which no
results were obtained, it would be necessary to reverse the
polarity (switch the direction) of the electrophoresis run.
This possibility was not thought of at the time the research

was being done and was never examined.

50
ELECTROPHORESIS ON CELLULOSE ACETATE MEMBRANES
Introduction

Cellulose acetate membranes may be used as a support
medium for the electrophoretic separation and examination of
biological fluids such as seminal fluid. These membranes
are composed of cellulose, which is a complex
polysaccride.(65) The cellulose is acetylated with acetic
anhydride to produce a membrane with a three-dimensional
network of interlocking pores.(66) These membranes can be
purchased as a film or with an inert support backing such as
mylar. The purpose of the backing is that cellulose acetate
is very brittle when it is dry; the backing helps prevent
the film from cracking and breaking.

Cellulose acetate has many desirable properties such
as pore size, density and thickness which can be controlled
during production of the membrane. Each of these properties
is determined by the degree of acetylation (67), which is
easily controlled. This results in many different types of
cellulose acetate membranes for electrophoresis which
exhibit a good degree of uniformity from batch to batch and
even between those of different manufacturers.

An additional advantage of cellulose acetate is that
electrOphoresis is faster on cellulose acetate than on most
other mediums. The generation of heat during

electrOphoresis is usually not a problem, manipulations are

51

easier than on other mediums and they can be rendered clear
for scanning of the band patterns by a densitometer.
Electrophoresis on cellulose acetate is also fairly

inexpensive compared to electrOphoresis on other support

media. The equipment is inexpensive, the buffers are easy
to make or they may be bought premeasured or mixed and the
applications are very broad. The disadvantage of
electrophoresis on cellulose acetate is that it lacks
sensitivity in comparison to electrOphoresis on other
mediums. This means that larger sample sizes are required
to achieve the desired results. Because the advantages
outweigh the problem of lack of sensitivity, the second

phase of this research was done on cellulose acetate.

Methods and Materials

1. Procedures

All sample separations were performed on Titan III
cellulose acetate (Helena) which has an inert mylar backing
for support. Prior to use, the cellulose acetate plates
were soaked in the electrophoresis tank buffer for 15
minutes. To do this, a beaker is filled with approximately
250 m1 of tank buffer. The cellulose acetate is slowly
lowered into the beaker allowing the buffer to migrate along
the cellulose acetate by wick action. Care should be taken

to avoid the trapping of air in the pore matrix of the

52

cellulose acetate. If air becomes trapped (small white
areas will be visible), the cellulose acetate should be
discarded. After the cellulose acetate has been soaked in
the tank buffer, it is ready for application Of the samples.

The samples analyzed on cellulose acetate were a serum
control (Dade), liquid seminal fluid, used neat and prepared
as described in the previous section, reconstituted seminal
fluid stains which were made by drying the liquid seminal
fluid samples on clean pieces of cotton cloth and a
laboratory produced standard of spermine and spermidine.

The samples were applied to the cellulose acetate with a
Super-z applicator and Cellulose Acetate Template (Helena)
using the following procedure:

The Super-Z electrOphoresis tank (Helena) is filled
with cold buffer. The buffer used for this procedure is HR
buffer (Helena) which is a tris—sodium barbital buffer with
a pH range of 8.6 to 9.0. Two wicks (2" x 5”) made from
filter paper are wetted by submerging them in the tank
buffer. One wick is folded over each support bridge, being
sure they make good contact with the buffer and that there
are no air bubbles trapped under the wick. The inner
chambers of the electrophoresis tank are filled with ice,
which provides cooling during electrOphoresis. The electro-
phoresis tank is ready for use.

The wells of the sample application plate are filled
with approximately 10 ul of each sample using a Drummond

Microdispensor. The Super-Z applicator is lowered into the

53

wells on the sample application plate several times. The
first sample application using the Super-Z applicator is
applied to a blotter to determine if the samples are being

applied evenly, if not, the last two steps are repeated.

The cellulose acetate is removed from the beaker of tank
buffer and gently blotted between two pieces of filter paper
to remove any excess buffer (do not allow the cellulose
acetate to dry). The cellulose acetate plate is placed in
the aligning base, cellulose acetate side facing up,
aligning the edge of the plate with the black line labeled
”cathodic application". The samples are applied to the
plate by lowering the Super—Z applicator into the sample
wells three times and then transferring the applicator to
the aligning base. The Super-Z applicator is lowered down
to make contact with the cellulose acetate and held for five
seconds, prior to release. The application of the samples
is repeated two more times in order to deposit a sufficient
quantity of sample for analysis. The more concentrated
samples may not need multiple applications. If this occurs,
these samples can be removed from their wells on the
applicator plate prior to reapplication of the other
samples.

The cellulose acetate is placed in the electrophoresis
tank face down and a quarter is placed on it as a weight to
insure contact with the wicks. The chamber is covered with
its lid and the power cables are attached to the power

supply. Electrophoresis is anodic (towards the anode) at

54

180 V for periods of 5 minutes and 15 minutes. This was
done to examine which length of time provided the best
results. As will be explained, both lengths of time may be

suitable for examining different constituents of seminal

fluid.

After completion of electrOphoresis the cellulose
acetate is stained in Ponceau S (Gelman) stain for serum
proteins and hemoglobin using the following procedure:

The cellulose acetate is placed into the dye solution
by quickly sliding it under the liquid to submerge it
totally. The cellulose acetate should remain in the dye
solution for three minutes. The cellulose acetate is
removed from the dye and drained well to minimize the
carryover of dye to the 5% acetic acid destain bath. The
bath should be gently agitated while the cellulose acetate
is in it. After two minutes, the cellulose acetate is
removed and drained. This step is repeated in two more
acetic acid baths. After the third acetic acid bath, the
cellulose acetate is removed and drained. The cellulose
acetate is placed in a bath of absolute methanol for two
minutes and gently agitated. The methanol bath is repeated
one time prior to removal and draining of the cellulose
acetate. The unbound stain should be totally removed from
the plate. The cellulose acetate is placed in Clearing Aid
(Helena) for five minutes. This reagent transforms the
Opaque white background of the cellulose acetate to clear.

The cellulose acetate is removed from the clearing solution

55

and placed on a hot tray in a drying oven for five minutes.

The plate is then ready to read in a densitometer.

I. Results

The initial electrophoresis runs on cellulose acetate
were done to determine the best length of time for the run
and to see what the seminal fluid band patterns looked like
in comparison to a serum sample which was run as a control.
The initial runs were done for lengths of 5 and 15 minutes.
The 15 minute runs provided the best separation of the
negatively charged portions of the seminal fluid and serum
samples. The negatively charged materials migrate toward
the anode. These negatively charged constituents appeared
to comprise the majority of the materials in the samples as
indicated by the near absence of any band patterns between
the origin and the cathode. Based upon the better
separation and the anodic migration of most of the
constituents making up the samples, a 15 minute run time was
determined to be best.

An area of interest on the cellulose acetate plates
which were electrOphoresed for 15 minutes was a band which
migrated slightly Off the origin toward the anode. This
band, when stained with Ponceau S dye, would only partially
accept the stain. The partial acceptance of the stain
produced a band which was visible to the unaided eye by

virtue of it having a different coloration than the other

56

materials stained with Ponceau S. The other unique aspect
of this band was it did not occur in the serum samples which
were being run on the plates as a control. The serum

samples stained with Ponceau S did not exhibit any band

patterns in the position at which this band occurred. Since
this band does not occur in serum, it is possible that this
material may be seminal fluid specific. Because of the
possible unique nature of this band, the research which was
conducted on the dried seminal fluid stains was oriented
toward demonstrating the presence of this material and its
potential as an indicator of seminal fluid.

The second phase of this study of seminal fluid
samples on cellulose acetate was the identification of the
bands which corresponded to spermine and spermidine. To aid
in this, a standard was created which was a mixture of 1 ml
of human serum, 4 mg of spermine tetrahydrochloride (Sigma)
and 4 mg of spermidine trihydrochloride (Sigma). This
sample was electrOphoresed along with known seminal fluid
samples and a serum control for 15 minutes and stained with
Ponceau S. A comparison of the band patterns produced by
the three samples failed to reveal any bands between the
origin and the anode which may be spermine and spermidine.

A review of the literature revealed why. The polyamines
spermine and spermidine are basic and have a positive charge
in barbitol buffers (68) such as the Helena HR buffer. The
polyamines, being positively charged, will migrate toward

the cathode. The samples are applied 2.5 cm from the

57

cathode and 5 cm from the anode, leaving very little room
for cathodic migration before hitting the wick and running
off the edge of the plate. With this in mind, several

plates were electrOphoresed for 5 minutes after which the

area between the origin and the cathode was examined. The
spermine and spermidine were found to be present cathodic to
the origin. The 15 minute electrophoresis time is too long
for the identification of spermine and spermidine using this
procedure. The resolution to this problem is a run time of
5 minutes or a move of the origin to the anodic end of the
plate, both of which could result in the loss of the unique
band which was previously discussed.

The remainder of the research on cellulose acetate was
oriented toward this unique band and demonstrating that it
is present in dried seminal fluid as well as fresh liquid
seminal fluid samples. When biological fluids dry, some of
their constituents may be lost forever. This is because the
fluid changes chemically upon drying causing the loss or
destruction of some of its constituents. In forensic
serology, the majority of the biological fluids which are
examined have been dried. It is for this reason that the
only information which is of value is that which survives
the drying process unchanged.

For this portion of the study, the seminal fluid
samples were split into two equal portions. One portion was
applied to clean cotton cloth and allowed to air dry at room

temperature. The other portion was retained in liquid form

58

and frozen at -80°C until used. The purpose of this was so
that a comparison could be made of the band patterns from
liquid and dried seminal fluid samples of the same source.
Prior to electrophoresis, the dried seminal fluid
stains must be eluted from the cloth in a manner which will
provide the largest quantity of the sample. The following
elution techniques were examined to determine which would

recapture the largest quantity of sample.

A) Extraction in 0.85% saline

A 2 cm sample of the stain was cut from the cotton
cloth and placed in 5 m1 of 0.85% saline for one hour at
room temperature. At the end of one hour the cloth was
removed from the saline and discarded. The liquid extract
was then used as a sample and electrOphoresed as in the
procedure which was previously discussed. Samples extracted
by this method failed to show any band patterns on the
cellulose acetate. This extraction procedure was then
repeated with incubation at 2°C and 37°C, both of which
provided the same results. Failure to achieve any patterns
after electrophoresis on cellulose acetate may indicate that
the seminal fluid samples did not elute Off the cotton cloth
or that the quantity which did elute off the cotton cloth

was too dilute for detection.

59

B) Extraction in 0.85% saline and concentration of the

sample

The previous elution procedure was repeated at

temperatures of 2°C, room temperature and 37°C. Prior to
discarding the piece of cloth, an effort was made to wring
it out to retain as much of the eluate as possible. The
eluate was then concentrated in a Minicon B-15 clinical
sample concentrator (Amicon). The sample, after filtration,
was approximately 50 times more concentrated than it was
prior to being concentrated. ElectrOphoresis of these
samples provided very weak band patterns which were

unreadable.

C) Extraction in 0.85% saline at 2°C for variable time

lengths

The previous extraction technique was repeated except
all the extractions were done at 2°C and the time length of
the extraction was changed. Extractions were done for 2, 4,
8 and 16 hours, after which the samples were concentrated to
50 times their original concentration. These samples
produced band patterns which were detectable with a

densitometer, but they were still very weak.

60

D) Extraction in pH 7.5 phosphate buffered saline

The samples were extracted in 5 ml of phosphate
buffered saline with a pH of 7.5 at 2°C, room temperature
and 37°C for four hours. After the extraction was complete,
the samples were concentrated to 100 times their original
concentration. Electrophoresis of these samples provided

very weak band patterns on cellulose acetate.

E) Extraction in pH 7.3 phosphate buffered saline

The samples were extracted in 5 ml of phosphate
buffered saline with a pH of 7.3 at 2°C, room temperature
and 37°C for four hours. After the extraction was
completed, the samples were concentrated to 100 times their
original concentration. Electrophoresis of these samples
provided weak but readable patterns in cellulose acetate.
The extracts which were done at 37°C provided the best
results.

A comparison was then done of the densitometer charts
of the samples extracted at 37°C in pH 7.3 phosphate
buffered saline to the charts of the corresponding liquid
seminal fluid samples. These comparisons indicated that
most of the materials present in liquid seminal fluid and
detectable on cellulose acetate survived the drying process
and were extractable. The comparison also indicated that

only a small portion of the dry seminal fluid was being

61

reclaimed during the extraction process. One substance
which did not extract well was the unique band which was
identified in the liquid samples because of its failure to

accept the Ponceau S dye. Since this substance could not be

clearly identified after extraction of the seminal fluid
from the cloth, one must wonder if it survives the drying
process and, if it does survive the drying process, what is

the best extraction method.

III. Conclusions

The electrophoretic analysis of seminal fluid on
polyacrylamide disc gels and cellulose acetate to identify
spermine and spermidine is not feasible. The polyamines
spermine and spermidine have such small molecules with a
high electric charge that they are not easily captured by
electrOphoresis on polyacrylamide disc gels.
ElectrOphoresis on cellulose acetate lacks the sensitivity
necessary to identify any band patterns from dilute
samples. This can be a major problem since neat seminal
fluid is a rarity in the analysis of rape evidence. The
seminal fluid is almost always mixed with the vaginal
secretions of the victim. These secretions dilute the
seminal fluid sample and this will create prOblems for the
electrophoretic examination of the sample on cellulose

acetate.

62

The unique band which was identified, is the only
finding of potential value which was identified by the study
of seminal fluid on cellulose acetate. This substance may
be seminal fluid specific, which means it could be used to
conclusively identify the presence of seminal fluid when no
spermatozoa is identified. To determine if this band is
seminal fluid specific, several avenues of research should
be explored.

First, the band must be identified as to its chemical
composition and properties. After the band is identified,
its site of origin within the male reproductive tract must
be determined. Determination of these facts will further
indicate if the substance is seminal fluid specific or not.
If the substance appears to be seminal fluid specific, a
procedure must be developed for the identification of this
substance in seminal fluid stains. After development of a
viable identification procedure, samples of other biological
fluids must be analyzed to demonstrate that this band is not

naturally occurring in anything other than seminal fluid.

63

DETERMINATION OF PHOSPHOGLUCOMUTASE SUBTYPES

Introduction

Phosphoglucomutase is an isoenzyme which is routinely
used in determining information about the enzyme profile of
an individual. Isoenzymes are proteins which are the same
but occur in multiple molecular forms. As in the red blood
cell groups, each individual will only exhibit one of these
forms and that form of the enzyme will be the same
regardless of the tissue or body fluid which is examined.
These multiple enzyme forms may be determined and identified
by electrOphoretic separation of the sample. The best
source of isoenzyme information is blood. Blood contains
dozens of isoenzymes which can be electrophoretically
determined. Some of these enzymes are present in other body
fluids such as seminal fluid and vaginal fluid. These
fluids may be electrophoretically separated to determine
their isoenzyme information. Once such enzyme is
phosphoglucomutase (PGM). PGM information is present in
several Of the body's fluids, such as the blood, seminal
fluid and vaginal fluid. PGM can be separated into three
major phenotypes (Figure 4) by low voltage electrophoresis
on 10% starch gels. The three major PGM phenotypes and
their population frequencies for whites in Miami, Florida
are: PGM 1: 60.1%, PGM 2-1: 33.1% and PGM 2: 6.8%.(69)

It has been demonstrated that each of these PGM phenotypes

64

 

Figure 4: PGM phenotypes.(70)

65

may be further divided into subgroups by low voltage
electrophoresis on agarose (71,72) or by isoelectric
focusing.(73,74) PGM subtyping is valuable because it

divides the three major phenotypes into ten phenotypes, each

of which has a lower population frequency than the three
major forms. The PGM subgroup group phenotypes and their
population frequencies for whites in California are shown in
Figure 5.

The most important benefit of PGM subtype information
is the higher potential for identifying possible mixtures of
body fluids contributed by several individuals, such as
seminal fluid with vaginal fluid. When a man ejaculates
into the vagina of a woman, there is a mixing of their body
fluids. The PGM information present in the woman's fluids
may mask the seminal fluid donor's PGM information,
especially if the woman is a PGM 2-1. Prior to the
development Of the PGM subtyping technology, seminal fluid
samples from the vagina of 2-1 women were of little or no
value unless the victim and seminal fluid donor were
different ABO blood groups. The ability to determine PGM
subtypes increases the chances of identifying mixtures
because the victim and seminal fluid donor may be different
PGM subtypes. When this occurs, the PGM subtype of the
seminal/vaginalfluid mixture may then be compared to the
victim's PGM subtype (Obtained from analysis of the victim
blood standard), to predict the possible subtypes of the

seminal fluid donor. This information is then compared to

66

 

 

 

 

 

 

 

 

 

 

 

 

PGM Phenotype Subtype Population Frequency(75)
1+ 39.6%
PGM 1 1+1- 16.7%
1- 2.7%
1+2+ 23.1%
PGM 2-1 1+2- 6.8%
1-2+ 3.7%
1-2- 1.8%
2+ 2.9%
PGM 2 2+2- 2.2%
2- 0.5%

 

 

 

Figure 5: PGM subtype phenotypes and population frequencies.

 

67

the PGM subtype information of the subject (obtained from
analysis of the subject's blood standard) to determine if he
is in the group of possible seminal fluid contributors.

Because there are ten PGM subtypes, this situation occurs

much more frequently than when determining the PGM major
phenotypes. Figure 6 illustrates the improved analysis
benefits of PGM subtyping technology.

At present, several problems exist with the
conventional methods for determining PGM subtypes. In tests
done using a phosphate-citric acid buffer system (76) at pH
5.5 and tests of an acetic acid buffer system (77) at pH
5.3, there were problems in Obtaining readable band
patterns. Tests of a phosphate-citric acid buffer system
which used a 1% isoelectric focusing gel (78) provided good
results, but were difficult to reproduce. This system also
exhibited poor separation between the PGM 2+ band and the
more anodic PGM bands and poor separation between the PGM 2-
and 1+ bands.

Another area of consideration in the analysis of blood
and body fluids is sample quantity. Limited sample size
restricts the amount of information which may be determined
about it. This is because each test expends a portion of
the sample, continually decreasing the sample quantity
available for the next test. Electrophoresis systems which
provide information on multiple enzyme groups are preferable
to systems which provide information on only one enzyme

group because large sample quantities are not necessary.

68

 

Victim
PGM Phenotype

Seminal/Vaginal Fluid
Mixture
PGM Information

Possible PGM
Phenotype of
Semen Donor

 

PGM 2-1

PGM 2-1

PGM 1

PGM 2-1

PGM 2

 

 

PGM Subtype l—2+

 

PGM Subtype 1+l-2+

 

PGM 1+
PGM 1+1-

PGM 1+2+

 

Figure 6: The effects Of PGM subtyping on the analysis of
sexual assault evidence.

 

69

It has been demonstrated that modification of the
phosphate-citric acid buffer system by lowering the pH and
using isoelectric focusing agarose provides PGM subtype
information that has a good band pattern separation and

reproducible results. It has also been demonstrated that
the same plate can be developed to determine the Adenosine
Deaminase (ADA), Erythrocyte Acid Phosphatase (EAP) and
Adenylate Kinase (AK) enzyme information. These three
enzymes also occur in multiple forms and are also useful in
the analysis of blood samples. They are not useful in the
analysis of semen and other body fluids because of very low
concentration in these fluids or because they only occur in

the blood.

Methods and Materials
Buffers

Tank Buffer:

The tank buffer is a phosphate-citric acid buffer
composed of 19.21 g of citric acid, anhydrous (Sigma)
dissolved in 800 ml of distilled water. The buffer is then
adjusted to pH 5.3 at 23°C by the addition of approximately
35 g of sodium phosphate, dibasic (Sigma) and brought up to

a final volume of one liter.

70

Gel Buffer:

The gel buffer is a phosphate—citric acid buffer
composed Of 0.48 g citric acid, anhydrous dissolved in 800
ml of distilled water. The buffer is then adjusted to pH
5.3 at 23°C by the addition of approximately 0.75 g of
sodium phosphate dibasic and brought up to a final volume of

one liter.

Gel and Plate Specifications:

The gel is composed of l g of Isogel (FMC Corp.)
dissolved in 100 m1 of gel buffer and poured onto a glass
plate which is 24 cm long and 25 cm wide. The plate should
have borders which are 1 cm wide and 1 mm thick. The gel
should be allowed to solidify completely before being placed

on a cooling plate.

Samples and Electrophoresis:

The blood samples and body fluid samples were obtained
from regular case evidence which were in the laboratory for
routine analysis. The samples were placed in slots which
were cut into the gel 7 cm from the cathode and then wetted

with 2 ul of 0.01M Cleland's Reagent (Sigma).

71

Electrophoresis is performed at a constant temperature
of 5°C and a voltage of 21 v/cm for four hours with the

samples run anodically.

Development:

The reaction buffers and reaction mixtures used for
development of the EAP, ADA and AK enzymes are the same as

those specified in Culliford (79) and Wraxa11.(80)

Reaction Buffer:

Dissolve 0.96 g of citric acid, anhydrous and 0.4 g of
sodium hydroxide (Sigma) in 80 ml of distilled water. After
the solids are totally dissolved, bring the buffer up to a
final volume of 100 m1. This buffer has a pH of 5.0 at

23°C.

Reaction Mixture:

Mix 4 mg of MUP with 10 m1 of the reaction mixture.

Saturate a piece of 1 mm Whatman filter paper which measures

25 cm long and 8.5 cm wide with the reaction mixture.

72

51‘.

Reaction Buffer:

Dissolve 1.20 g trizma base and 0.4 g of magnesium
chloride (Sigma) in 80 ml of distilled water. The buffer is
then adjusted to pH 8.0 at 23°C by the addition of 1:1
hydrochloric acid (Fisher) and brought up to a final volume

of 100 ml.

Reaction Mixture:

Dissolve 55 mg of AK PMR (SERI) and 1 mg of

G PD hexokinase (Sigma) in 15 ml of reaction buffer. This
6

mixture is light sensitive, it must be kept in a dark place
until ready for use.

Dissolve 0.3 g of agarose Type I (Sigma) in 15 ml of
distilled water. After the agarose is totally dissolved,
cool the solution to 60°C. Pour the reaction mixture into

the agarose and apply it to the enzyme plate immediately.

73

Reaction Buffer:

Dissolve 0.355 g of sodium phosphate, monobasic
(Sigma) and 0.19 g of sodium phosphate, dibasic in 80 m1 of
distilled water. After the solids are dissolved, bring the
buffer up to a final volume of 100 ml. This buffer has a pH

of 7.0 at 23°C.

Reaction Mixture:

Dissolve 30 mg Of ADA PMR (SERI), 35 ul of xanthine
oxidase and 35 ul Of nucleoside phosphorylase in 15 ml of
reaction buffer. This mixture is light sensitive and must
be kept in a dark place until ready for use.

Dissolve 0.3 g of agarose Type I in 15 m1 of distilled
water. After the agarose is totally dissolved, cool the
solution to 60°C. Pour the reaction mixture into the

agarose and apply it to the enzyme plate immediately.

Development Procedures:

The EAP is developed first in the area covering from
the origin, 8.5 cm toward the anode by covering this area
with the Whatman filter paper soaked in the reaction

mixture. The AK overlay is positioned at the origin running

74

4 cm toward the cathode. This is done by placing plastic
borders around the area, pouring the reaction/agarose
mixture into it and allowing it to solidify. The ADA
overlay is positioned from the edge of the anodic wick, 5 cm
back toward the origin. The same procedure is followed as
in the AK overlay. The plate is then placed in an incubator
adjusted to 37°C for 20 to 30 minutes, after which the EAP
and AK results may be read.

The EAP is visualized by exposing the developed area
of the plate to ultraviolet light. During this procedure,
the AK and ADA enzyme areas should be covered to shield them
from the ultraviolet light. The AK results are visible
under white light.

After recording the EAP and AK results, the PGM
subtypes can be developed. The procedure for preparation of
the PGM subtype reaction buffer and mixture is described by

Sutton and Burgess.(8l)

PGM Subtypes DevelOpment

 

Reaction Buffer:

Dissolve 18.0 g trizma base, 2.0 9 magnesium chloride
and 1.0 g histidine mono hydrochloride (Eastman) in 400 ml
of distilled water. The buffer is then adjusted to pH 8.0
with 1:1 hydrochloric acid and brought up to a final volume

Of 500 ml.

75

Reaction Mixture:

Dissolve 90 mg PGM PMR (SERI), 2 mg NADP, 25 mg EDTA

trisodium (Sigma) and 1 mg G PD in 20 m1 of reaction
6

buffer. This mixture is light sensitive and must be kept in
a dark place until ready for use.

Dissolve 0.4 9 agarose Type I in 20 ml of distilled
water. After the agarose is dissolved, cool the solution to
60°C. Pour the reaction mixture into the agarose and apply

to the enzyme plate immediately.

The position of the PGM subtype overlay is the same
area which was developed for EAP. The overlay pouring
procedure is the same as that for the AK and ADA enzymes.
After pouring the overlay, the plate should be placed in a
37°C incubator for 60 to 90 minutes or until the band

patterns have developed to a sufficient density.

Results and Discussion

PGM Subtypes (Figure 7):

The pH 5.3 phosphate-citric acid buffer system
provides very good resolution and separation of the PGM
bands. The separation which occurs between the PGM 2+ band
and the more anodic bands is excellent, but diminishes with

each usage of the tank buffer. Due to this effect, the tank

SECONDARY
P 2+
G
M
2-
B
A 1+
N
D 1-

Figure 7:

76

PGM SUBTYPE

 

+ 2+ 2+ 2+ 2- 2- 1+

2+ 2- 2- 1+ 1- 1+ 1- 1+ 1- 1-

BANDS ___ ___ ___ ___ ___ __. ___ ___ ___ ___
ORIGIN

PGM subtype band patterns.

77

buffer should be used a maximum of three times and then
discarded.
When determining the PGM subtypes of samples it is

recommended that the major PGM type be determined in

advance. This aids in the reading of the subtype bands and
prevents samples from being misread. This is very important
in samples from individuals who are PGM 1+ or PGM 1- because
these samples may exhibit an additional band which occurs in
the PGM 2 region and may be interpreted as a PGM 2+ band.
The band is located midway between the positions of the 2+
and 2- bands and can be identified by the use of proper
standards and knowledge of the sample's major PGM type. The
nature of this band is unknown at this time. It may be a
degradation product which is evidenced by the failure of the

band to occur in fresh blood samples.

EAP (Figure 8):

The resolution and separation of the EAP band pattern
is extremely good. The band patterns are very sharp with a
strong intensity which provides for good visualization under
ultraviolet lighting. Due to the intensity of the band
patterns, the degradation band which occurs anodic to the
primary B band is more easily visualized and may be misread
as an R band. This problem is minimized when 0.01M
Cleland's Reagent is applied to the samples prior to

electrophoresis. It is essential to use minimal amounts of

78

the Cleland's Reagent, because it migrates to the position
of the AK 1 band and, when used in excess, will obscure this

band.

ADA and AK (Figure 8):

The resolution and intensity of the band patterns are
comparable to that which occurs when analysis is done using
the pH 5.5 phosphate-citric acid buffers and a starch

gel.(82)

Conclusion

The pH 5.3 phosphate-citric acid buffer system and use
of a 1% isoelectric focusing support gel provides
information on four enzyme groups simultaneously. This
minimizes the quantity of sample expended during analysis,
while maximizing the enzyme information Obtained. Care
should be taken in determining the PGM subtype and EAP
enzyme information when using this system. If the enzyme
character of the sample is in question, the sample should be
examined by isoelectric focusing and, if this methodology is
unavailable, it should be reported out as inconclusive. It
should also be taken into consideration that multisystems
such as this may provide information concerning several
enzymes, but it may not be the best or clearest way to

determine each of the individual enzyme types being

79

identified. This may lead to errors in its identity of the
enzyme types, so great care should be taken when reading

these enzyme plates.

8O

 

EAP

 

 

 

    

Figure 8: ADA, AK, and EAP band patterns.(83)

81

SUMMARY

Seminal fluid samples were examined by electrophoretic

separations on polyacrylamide disc gels and cellulose

acetate to assess the usefulness of the polyamines spermine
and spermidine as confirmatory markers. The electrOphoretic
separations indicate that spermine and spermidine are poor
confirmatory markers for seminal fluid. ElectrOphoretic
separation of samples on cellulose acetate revealed the
presence of a protein which may be specific for seminal
fluid. At present, the identity and origin of this protein
are unknown making it a potential area of future research.

The second portion of this research study involved the
modification and development of an electrophoresis procedure
for the determination of PGM subtypes. The procedure which
was developed is done on 1% Isogel in a pH 5.3 phosphate-
citrate buffer system at a voltage of 21 v/cm for 4 hours.
This electrophoresis methodology provides clear reproducible
results as well as information on the EAP, ADA, and AK
enzyme phenotypes of the samples.

The additional information which is obtained by the
determination of the PGM subtypes may affect the
interpretation of the evidence and how it relates to the
victim and the alleged rapist. This more in-depth
interpretation may, in turn, affect the prosecution and
defense of the subject by strengthening or weakening their

cases. It can also affect the planning concerning the

82

presentation of the case to the court and what should be
emphasized during that presentation. It also may cause
either side to be unwilling to go to court by providing
information which is contradictory to the position being
taken, thus affecting the ultimate disposition of the case.
If the subject is convicted of the criminal charges,
the methodology which was used, as well as the information
which was determined, may affect the appeals process.
Scientific evidence and the expert Opinions arrived at as a
result of the analysis are subject to judicial review. If
the courts decide that the methodology and/or Opinions are
flawed, the conviction may be overturned resulting in a new
trial and exclusion of either the methodology or expert
opinions from future criminal cases, thus affecting the

future prosecution or defense of all cases.

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