semen id and detection Flashcards
what is semen
male reproductive fluid
production starts at puberty 8-17 yrs old
spermatozoa + seminal fluid = semen
biological characteristics of semen
typical ejaculate is 2-5 mL
water soluble
normal sperm count ranges from 10^7 - 10^ 8 spermatozoa per mL
principle cell in semen is spermatozoa
pH is 7.2 - 7.8 so ~ 7.5
slightly basic
75 - 90% semen is contributed by accessory glands
20% prostate
60% seminal vesicle
Cowper’s gland
urethral galnds
seminal fluid
combined secretions of several accessory glands
seminal fluid is thick because it contains mucus and proteins
fructose, citric acid, creatine, spermine, choline, urea
also contains epithelial cells and WBCs (source of DNA)
seminal vesicle fluid
contains proteins that play a role in coagultion
contains flavin which causes semen to fluoresce under UV light
prostatic fluid, contributed by the prostate
contain high concentrations of acid phosphatase (AP) and prose-specidic antigen (PSA)
used to screen for semen
anatomy
sperm are created in the testes, mature in the epididymis, traverse the vas deferent. and ejaculatory dict to enter the urethra
accessory glands: seminal vesicle, prostate, Cowper’s gland, urethral glands provides the bulk of semne
emission - movement of semen into the urethra by contraction of the smooth muscles of the vas deferents and seminal vesicles
ejaculation - semen is propelled out of the urethra by contraction of the bulbocavernosus muscle
muscle of the perneum
acid phosphates (AP)
primary screening test, colorimetric
the prostate is the most abundant source of AP
not specific for seminal fluid
found in lower concentration in other bodily fluids
fecal stains vaginal secretions blood
seminal fluid AP conc is 20-400 times greater than other bodily fluids
elevated levels of prostatic AP are present in serum of males with prostate cancer used to diagnose
also can be found in fungi bacteria and many plants
AP levels are not affected by vasectomies because it is generated by the prostate
not testes
can use to screen for seminal fluid (semen)
prostate specific antigen (PSA)
has molecular weight of 30 kDa so also known as P30, interchangeable with PSA
present in seminal fluid in concentrations of 0.5 to 2.0 mg/mL
produced in the prostate epithelium and secreted into the semen
responsible for hydrolyzing semenogelin which mediates gel formation in semen
can also be found in the paraurethral glands, perianal glands, aprocrine sweat glands, and mammary glands
not prostate specific, not sex specific
not as specific for the prostate as once thought
detected in low levels in urine fecal material sweat and breast milk breast tumors amniotic fluid
abnormally high P30 levels in blood serum can be associated with prostate cancer as well
presumptive identification of semen stains
dried semen stains are typically off white to yellow in color
can appear crust or you can feel them
problems can occur in locating semen stains on clothing items that are thick or coarse or the color could make it difficult too
dried semen stains fluoresce bluish white on most substrates with UV excitation (ALS)
innate fluorescent compounds (flavins)
fluorescent compounds elaborated by bacteria
pseudomonas fluorescens
note taking: document document document
document color appearence whether it fluoresces what wavelength, etc
can circle stains under ALS to view in ambient light if stain is positive, document size of stain color
if visible under ambient, etc
UV fluorescence under UV or ALS
white cotton in underpants commonly fluoresces under ALS, document any observations, may need to continue with additional screening tests
light sources for semen search
using a handheld UV light, semen stains will appear bluish in color
DNA can be detrimentally affected by brief high intensity UV exposure
using an ALS (430-470 nm wavelength range) semen stains will be visible as a yellow-colored stain
crime lite
ALS is not specific for semen
other bodily fluids will fluoresce
some detergents and other chemicals have fluorescent properties
when using all you always use a filter to render the emitted light as a single wavelength colored goggles
saliva and urine fluoresce
identification process of semen stains from clothing
semen stain
presumptive tests - ALS => AP
confirmatory tests - microscopic ID of semen
stop if sperm is seeen
if not seen, P30
positive P30, possible vasectomy, possible low conc sperm
at LASD, positive P30 was semen indicated
P30 isn’t as specific as once thought
AP presumptive test for semen
semen contains high levels of acid phosphatase which is produced by prostate gland
the enzyme has optimal pH of 5.4, acidic and liberates inorganic phosphate from phosphoric esters
sodium α-Naphthyl phosphate is used as the substrate
Naphthyl liberated in the reaction couples with an ago dye (brentamin Fast Blue B) to form an insoluble colored product
colorimetric test
2-step test
time sensitive 20 sec
in presence of semen the result is a color change to purple
can test directly or indirectly
you can cut sample directly and perform in test tube (more sensitive) or swab stain and use taco method with filter paper
chemistry of AP and Fast Blue b
the enzyme has optimal ph of 5.4 and liberates inorganic phosphate from phosphoric esters
sodium α-Naphthyl phosphate is used as substrate
AP false positives may have weak reactions
fecal stains
vag secretions
plants, cauliflower sprouts
yeasts react with brentamine dye
bacteria can be present in vagina due to pregnancy or infection
contraceptive creams react with brentamine dye
combination of solutions can be delivered backwards to make more specific
fast blue B is added 1st to eliminate false positives
add dye first, color change - substrate α-Naphthyl not added yet therefore false positive
different types of colorimetric screening tests for AP
test strips were found to detect semen down to a 1/2000 dilution whereas comparative testing with two other AP tests and a zinc test showed that their limit of detection was 1/150 - 1/300
simply moisten stain, remove protective test cover and either collect swab and apply to test paper or press test paper on stain
return test cover and in approx 1 min a positive result is indicated by a deep purple color
AP mapping
blotting paper dampened with water
use mister of spray bottle
not too wet to dilute stain
place on fabric
press
remove and AP reagents are sprayed onto the blotting paper
prostate specific antigen P30 identification
immunochromatographic assays
Seratec PSA semi quant, One step ABA card PSA
used as screening test for semne
utilizes antibody-antigen relationship
3 lines indicates positive for P30 2 is negative because semi quant line
line used for detecting prostate cancer
not forensic
Enzyme linked immunoabsorbent Assay (ELISA)
antibody sandwich ELISA in which an antibody-antigen-antibody sandwich complex is formed
intensity of the signal is detected and proportional to amount of bound antigen
time consuming therefore bad for screening
Sertec PSA semiquant
cartridge technology replaced CIE (crossover immunoelectrophoresis)
more sensitive less time
specificity
shows no cross reactivity with other seminal fluids
shows no cross reactivity with neat breast milk, female urine, or female serum samples
no cross reactivity with other animal seminal fluid, except higher primates apes
sensitivity 1ng/uL
can suffer from high dose hook effect
false negative oversaturated with P30 PSA
dilute sample and run again
competitive binding
served p30 cards
free gold labeled mouse monoclonal anti-P30 antibodies just upstream of test zone, in sample well
immobilized mouse monoclonal anti-p30 antibodies in T zone
polyclonal anti-mouse antibodies present at the control region and internal standard line
when p30 present it forms fold labeled mouse anti p30 antibody - p30 antigen - mouse anti p30 antibody sandwich at test site
how Elisa works
sample contains PSA is applied to polystyrene tubes in which anti-pas Antibody is immobilized
PSA binds to immobilized anti-PSA antibody to form PSA-ant-PSA-antibody complex
a second anti-PSA antibody, specific for a different epitope of PSA, is added to form a anti-PSA-antibody(1)-PSA-antigen-anti-PSA-antibody(2) sandwich
a labeled antiblobulin then binds to the sandwich
the bound anti globulin can be detected by various reporting schemes
identification of seminal vesicle specific antigen semenogelins
these are also detected in several tissues including skeletal muscle kidney colon and trachea
purpose in semen is to coagulate the ejaculation p30 liquifies them
immunochromatographic assay
commercially available tests : RSID semen test and the nano trap Sg
similar to P30 but different target antibody
no internal standard zone, no semiquant
so 2 lines for +
ELISA
RNA based assays for identifying semen
based on expression of certain genes in certain cell or tissue types
mRNA expressed exclusively in spermatozoa and certain cells of male accessory glands
utilizes reverse transcriptase (RT-PCR)
different genes can be used
RNA-based methods have higher specificity and can be automated
limitations
RNA is unstable (degradation by endogenous ribonuclease)
microscopic examination of spermatozoa
cells from a questioned stain can be extracted and transferred to a microscopic slide
swab \ cutting
presence of sperm provides proof of a seminal stain
sperm can be on clothes laundered with clothes with seminal fluid
presence of sperm in totality with +P30 and +AP
histological staining can fascillitate microscopic examination
laser capture microdissection (LCM)
an effective technique for separating spermatozoa from non-sperm cells on a slide (but so is differential extraction and way cheaper
laser cuts spermatozoa drops in tube for extraction
spermatogenesis
formation of mature sperm cells
spermatozoa are produced in the seminiferous tubules of the testes
spermatozoa mature and acquire motility in the epididymis
spermatozoa lack various intracellular organelles such as endoplasmic reticulum, Golgi apparatus lysosomes and peroxisomes
at least 60% in normal males have normal morphology
sperm starts off as haploid cells with all organelles
mitochondria in middle piece for energy to move
flagellum to move
nucleus nuclear dna haplod
leaves only nucleus mitochondria and flagellum
review
sperm created in testes
mature in epididymis
traverse the vas deferent and ejaculatory duct into urethra
anatomy of spermatozoa
3 district regions
head
middle piece
tail
head
oval or elliptical in shape
contains the nucleus/DNA in densely packed chromosomes (haploid)
acrosomal cap
used in fertilization to enter egg
ultimately sperm genetic material is transferred to egg once penetrated
loses everything else including mitochondria middle piece
all mitochondrial dna comes from mom
Ystr on paternal line
number mitochondria > number of nucleus per cell
can get mito dna from degraded dna
more mito than nuclear
middle piece
mitochondria provide energy for the tail
tail flagellum
responsible for motility
oligospermia
condition which males have abnormally low sperm counts
temporary due to disease or virus
aspermia
complete lack of semen with ejaculation
azoospermia
term used when there are no sperm in ejaculate
may be temporary due to disease or virus
vasectomy
surgical removal of a bilateral segment of the ductus deferens
prevents spermatozoa from reaching the distal end of the reproductive tract
can still produce ejaculate containing seminal vesicle fluid and prostatic fluid
fluoresces + P30 and + AP
no DNA
no Christmas tree
visualization of sperm
histological staining provides contrast to visualize the sperm
Christmas tree H&E
christmas tree
nuclear fast red - dye used to stain nuclei (all nuclei of all cells) red
picroindigocarmine (PIC) used as counterstain (Green)
colors everything else
acrosomal cap is pink nuclei red sperm tail green mid piece blue
epithelial cells will appear blue green and have red nuclei
H&E
hematoxylin
basic dye stains nuclei purple
eosin
acidic dye used as counterstain
stains acrosmal cap pink
sperm morphology
sperm cells were first observed by Antony van Leeuwenhoek 1678
total length is 50-60 um in length
tail and mid piece make up 90% of the sperm cell length (tail >10x length of the head)
normal semen sample may contain up to 40% abnormal sperm forms and 2-3% immature forms
only need head to make identification
tails cna degrade or fall off over time thru extraction
vag cavity smear and swabs tails on smears thru staining can be telling of timing
some abnormal sperm have weird shaped or sized heads, sometime double
or odd midpieces
comparison of human spermatozoa to other animals
may be difficult to distinguish from rabbit ram boar bull and other primates
bestiality cases
sexual activity with an animal
there are animal specific primers if needed per circumstances
outsource to a specific lab
if you send forward to dna
forensic dna analysis is human specific so you see it at that point
animal - no dna
higher primate - weird dna
some animal sperm heads have a hook
phase contrast microscopy for visualization of spermatozoa
enables the viewing of transparent and colorless objects without staining
light passing through a substance is slowed so that it os out of phase with light that did not pass through the specimen
light passing through the specimen and diffracted is ocmbined with direct light that passed through a phase shifting element the result of combining the two beams (out of phase and in phase) is a darker or brighter spot in the phase of the two light beams
positive id of semen H&E
purple nucleus, pink acrosomal cap
screen at 100x, confirmation at 400x
under phase
nucleus bright yellow
cap becomes dark purple
confirmation, other cells won’t do that
positive id of semen Christmas stain
red nucleus
pink acrosomal cap
blue mitochrondrion
green tail
switching from phase to bright field helps to ID sperm
interpretation of sperm findings
sperm must be consistent in size and shape with known sperm cells
sperm may consist of just a head or a head and a tail or some portion of the tail
sperm cell must display a distinct body and acrosomal cap
phase shift must be seen when viewing under ordinary light and phase contrast at 400x
sperm density grading is performed under 400x
T for presence of tails
few less than 5
1+ hard to find
2+ some in the same fields, easy to find
3+ many or some in most fields
4+ many in every field
record the presence of tails (T) if observed 4+T or ++++T
misidentification of sperm
small lymphocytes WBCs
yeast cells
can resemble sperm heads in shape size and staining characteristics
WBC is bigger than sperm and won’t phase shift
yeast has no acrosomal cap, although orientation may lead to it looking like it does
won’t have phase shift
candida albicans
oval budding yeast
produces a pseudomycelium
elongated adding cells that resemble sperm
hyphae long filaments
part of normal flora
respiratory tract
gi tract
female genital tract
thrush
infection of oral tissues
misidentification of sperm
human stool can contain the spores of the fist parasite
henneguya slminicola (salmon)
spore body about 10 um in length (50-60um total length for sperm)
tails about 3x length of the body (10x for sperm)
no acrsomal cap
no phase shift
artifacts
not cells, common in condoms
particles
requirements for sperm id
cell must be consistent in size and shape
display distinct nucleus and acrosomal cap
color shift in head from phase to bright field
document the coordinates of sperm
fixed stage
numbers on dials
document microscope number
last requires a second opinion and documentation of confirmation with analyst initials
