Forensic Biology Screening Workshop Semen. Semen Composition Semen is a fluid of complex composition, produced by the male sex organs. There is a cellular

Forensic Biology Screening Workshop

Semen

Semen Composition

• Semen is a fluid of complex composition, produced by the male sex organs.

• There is a cellular component, spermatozoa, and a fluid component, seminal plasma.

Seminal Plasma

• Composed of salts, sugars, lipids, enzymes (such as Acid Phosphatase), nutrients, proteins, hormones, basic amines (spermine), P30 (Prostate Specific Antigen), flavins (source of fluorescence under UV light)

• The components originate from several sources, including seminal vesicles and the prostate gland

– The prostate is the source of the enzyme acid phosphatase and the protein Prostate Specific Antigen (PSA), or p30 protein

Seminal PlasmaFunction

– Lubricates sperm duct / urethra

– Nutritive and protective liquid medium for sperm to travel in

– Alkaline environment which protects the sperm against the acidic nature of the vaginal tract

Seminal Plasma

Vasectomy severs or ligates the ducts carrying sperm to the penis

– Vasectomized men will have no sperm but will have the plasma components present in their ejaculate

Sperm Cells

• Sperm are the male reproductive cells

• Each consists of a head, tail and mid-piece

– In humans, the head is a tiny disc, about 4.5 µm long and 2.5 µm wide

– The tail is about 40 µm long, and is rapidly lost in ejaculates

Sperm Cells

–The head is where the DNA is preserved

–Ape sperm are similar in size and shape

–Dogs have similarly shaped sperm but are about three times larger than human sperm

–Other animals have differently shaped sperm

Sperm Cells

• Develop in the seminiferous tubules

– Spermatogenesis has three phases:

• Spermatocytogenesis - mitotic division

• Meiosis - diploid number present in first phase becomes haploid

• Spermiogenesis – spherical sperm become elongated, acrosome forms and they are released from the seminferous tubules

• Move to the epididymis

• Upon physical stimulation:– Sperm and seminal fluid combine in vas

deferens to form semen

• Ejaculate ~ 3-4 ml of semen

• ~70-150 million sperm per ejaculate

Sperm Cells

Male Reproductive System

Effect On Spermatogensis

• Genetic abnormalities• Disease• Injury• Chemicals – chemotherapeutic agents• Drugs• Alcohol• Age

Sperm Cell Morphology

Composed of a head, midpiece and tail

• Head– Acrosome cap – aids sperm entry to egg– Houses nuclear material (DNA)– ~4.5 µm long and 2.5 µm wide

Sperm Cell Morphology

• Midpiece– Houses mitochondria – energy for sperm cell

• Tail– Used for mobility, made of protein– Long and fragile – first part to breakdown and

easily detached– ~40 µm long

Analysis of Semen

• Semen stains fluorescence under ultraviolet light

– It is common practice to visually assess items of evidence under UV light to locate possible semen stains

– The intensity of the fluorescence can be affected by the substrate, concentration of the stain, and other body fluids

Analysis of SemenPresumptive Tests

• Acid Phosphatase Detection– Human semen contains high concentrations

of acid phosphatase (AP), which can therefore be the basis of a screening test.

– While AP is detected in high concentrations in semen, it can also be detected in other body fluids

Analysis of SemenConfirmatory Tests

• P30 Identification

– Found in semen

• Microscopy

– Identification of sperm cells, usually done using a staining method

Analysis of Semen

• Acid Phosphates degrades at a much faster rate than sperm cells

• While presumptive tests for acid phosphatase can be helpful; a negative result does not necessarily mean semen is not present

• Many laboratories conduct microscopic examinations on items that have negative AP test results—based on the circumstances of the case and evaluation of the item

Survival Times• Dried Stain

– Sperm, Acid Phosphatase, Prostate Specific Antigen P30 – years if proper storage

• Vagina / Cervix– Sperm, Acid Phosphatase = ~3 days (sperm possibly

longer)– Prostate Specific Antigen P30 = ~ 1 day

• Mouth– Sperm ~ 6 hours– Acid Phosphatase / Prostate Specific Antigen P30 –

less due to water solubility• Anal cavity

– Sperm ~ 9-20 hours– Acid Phosphatase / Prostate Specific Antigen P30 -

less

Survival TimesWhy do sperm survive longer?

• Sperm heads, very strong

• Designed to last to penetrate egg

• Proteins are water soluble, breakdown quicker

• Sperm tails are lost first– Tails are fragile and break off– Made of proteins, bacteria attack first

Time Acid Phosphatase Microscopic Exam

0 hour + immediate Many sperm, easy to find, 5-10 per field, intact sperm found

1 hour + 6 seconds Many sperm, easy to find, 3-8 per field, intact sperm found

3 hours + 6 seconds Many, easy to find, 1-3 in most fields, intact sperm found

8 hours + 11 seconds 1-3 in some fields, sperm w/ broken tails, 1 intact sperm

12 hours + 14 seconds 7 sperm on slide, none intact

24 hours + 30 seconds 2 sperm on slide, none intact

37 hours - Reaction at 56 seconds Negative

48 hours - No reaction at 1 minute Negative

72 hours - No reaction at 1 minute Negative

Time Lapsed Sex Kit #1 Results Table

Time Microscopic Exam

0 hour 4+ Many sperm, intact

2 hours 1 or more in most fields, intact sperm present

12 hours Hard to find, none intact

24 hours Hard to find, none intact

48 hours 1 possible sperm on slide


Time Acid Phosphatase Microscopic Exam

0 hour + immediate < 5 seconds Neg for sperm, epi cells present

1 hour + 8 seconds

3 hours + 9 seconds

8 hours + 8 seconds

12 hours + 25 seconds



48 hours - > 30 seconds

72 hours - No reaction at 1 minute


Acid PhosphataseAcid Phosphatase

• Secreted from the prostate

• Cleaves phosphate group from molecules

phosphoryl choline choline

– Choline is important in cellular membrane composition and repair

Acid Phosphatase -BCIP TestHOW TO MAKE:

•0.5 mg 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/ml 0.01M acetate buffer, pH 5.5

Note: Dissolve the BCIP in a few drops of dimethyl sulfoxide before bringing the solution to volume with acetate buffer

HOW TO STORE:•The BCIP solution is stable for at least four weeks when stored at 4C

Acid Phosphatase -BCIP Test

HOW TO PERFORM:

•Swab question stain with a slightly moistened swab

•Place swab into tube with 200 µl BCIP substrate solution

Note: Use a known semen stain as a positive control and an unstained swab as a negative control

•Incubate at 37° for 15 minutes

Acid Phosphatase hydrolyzes BCIP and creates a blue color--Any blue color change is a POSITIVE result for phosphatase activity

Acid Phosphatase –AP Spot Test

Reaction:

– Acid Phosphatase cleaves the phosphate from α-napthyl phosphate to form napthol

– Napthol and Brentamine Fast Blue B forms a purple azo dye

Acid Phosphatase – AP Spot TestHOW TO MAKE:

• Buffer–Glacial acetic acid 5 ml–Sodium acetate, anhydrous (0.24M) 10 g–Distilled Water 500 ml

• Solution A–Buffer 250 ml–Sodium a-naphthyl phosphate, 0.25% (w/v) 0.63 g

• Solution B–Buffer 250 ml–Naphthanil diazo blue B, 0.5% (w/v) 1.25 g (Fast Blue B)

Note: Both solutions can be combined for working solution in spot test or used separately

Acid Phosphatase – AP Spot TestHOW TO STORE:

•If purchased from a commercial supplier, follow product insert regarding storage requirements

•Solution A and Solution B can be stored frozen for months

•Solution B should be stored in an amber bottle

Note: when solution A and B are combined, the reagent is not as stable as when stored separately

Acid Phosphatase – AP Spot TestHOW TO PERFORM:

•Take a small cutting of the question sample and place on filter paper/absorbent pad or swab the questioned evidence stain

Note: Use a known semen stain as a positive control and an unstained swab or filter paper as a negative control

•Add 1 drop of AP Spot Test reagent if both solutions combined or 1 drop each solution; A followed by B.

•POSITIVE if purple color within 1 min.

•NEGATIVE if no color change, pink color change or color change after 1 min.

Acid Phosphatase

False Positives

– Vaginal Acid Phosphatase– Fecal Material– Plant matter– Spermicides– Some feminine hygiene products

2 Step Acid Phosphatase Video

1 Step Acid Phosphatase Video

Acid Phosphatase (AP) Mapping

Used on large items of evidence to screen for AP activity

HOW TO MAKE:

•Follow previous formulation, place solution A and B into separate spray bottles

AP MappingHOW TO PERFORM:

• Lay out item to be mapped onto clean surface

• Staple (or otherwise attach) the appropriate size of filter paper to a piece of plastic sheeting

• Spray the paper with DI water until damp. Lay flat onto item marking the position/orientation. Plastic sheeting should be on top.

• Press for ten minutes (fifteen minutes if blood is mixed with semen). Use of a flat board or sheet of glass on top of sheeting can aid in this process


• After pressing, hang paper still attached to plastic sheeting in a fume hood.

• Evenly spray the paper with Solution A

• Evenly spray with Solution B

• Let develop for ten minutes.


• Positive stained areas appear purple. Positive stains should appear within three minutes. Weaker stains may take longer to appear (10-15 minutes).

Note: Use a known semen stain as a positive control; color reaction should occur within one minute

• DO NOT overlay paper on item and mark orientation—this should have been done in first steps

AP General SwabbingUsed on large items of evidence to screen for AP activity

•Use a slightly moistened swab to test zones or sections of a large item

– Follow the AP spot test or BCIP procedure

– The technique allow the analyst to screen large items quickly

Prostate Specific Antigen (PSA) P30

Prostate Specific Antigen (P30)

– Antigen made in the prostate gland

– Weighs 30,000 kD

– Liquefies semen and is instrumental in dissolving the cervical mucous cap for sperm entry

Prostate Specific Antigen (PSA) P30

• Early detection was based on electrophoretic or double diffusion Ouchterlony methods

• The methods relied on the formation of visible precipitation of the sample and anti-p30; oftentimes followed by staining with Coomassie Blue.

P30 ElectrophoresisMethods•The most widely used methods were crossover electrophoresis and rocket immunoelectrophoresis

– Crossover: anit-p30 is placed into a well of an agar gel opposite of the sample and allowed to electrophoreses

• The antigens and antibodies move toward each other and a precipitant formed

P30 ElectrophoresisMethods• Rocket immunoelectrophoresis

– a technique in which samples are placed in a row of wells in an agar plate containing antiserum (anti-P30) and an electric field perpendicular to the line of wells is applied; this drives the antigen through the gel, forming a spike or "rocket" precipitin pattern trailing away from each well. The length of the rocket is proportional to the amount of antigen placed in the well and is semi-quantitative

Elisa Test For SemenELISA was another method introduced to aid in semen identification

•Enzyme-linked immunosorbent assay (ELISA) crossover

ELISA typically involves a two-stage incubated immuno reaction. First the target antigen binds with a solid phase antibody. Non-bound materials are washed away and an enzyme-labeled antibody, called a conjugate, binds to form a 'sandwich' complex. Finally the antigen-antibody is introduced to a substrate where a chromogen is used to give a color change indicating the presence of the antibody

–Shown to be a very sensitive method

Commercial P30 TestsOuchterlony, electrophoresis and ELISA methods are labor intensive--this lead to the development of faster methods

• ABAcard® P30

• Seratec® PSA Semiquant–Semi-quantitative

– Slight differences between the two methods

ABAcard® p30

ABAcard® p30

Reaction:– “S” area – mobile monoclonal antihuman

Prostate Specific Antigen P30 antibodies – Prostate Specific Antigen P30 antibodies bind

to Prostate Specific Antigen P30 antigens present in sample

– Form Antibody-Antigen complex– Travel through card towards the “T” area– “T” area – stationary monocolonal antihuman

Prostate Specific Antigen P30 antibodies

ABAcard® p30

• Stationary antibodies in the “T” area catch the mobile Antigen-Antibody complex

• Forms Antigen-Antibody -Antibody complex

• Antibodies labeled with a pink dye

• When antibodies aggregate – form a pink line in the “T” area

P30 Video

Seratec® PSA Semiquant

Seratec® PSA Semiquant• “C” area – internal control ensures the test card

works properly. • Binds excess mobile antihuman prostate

specific antigen P30 antibody

• Middle line is a semi-quantitative tool (Seratec® PSA Semiquant only)– Aids in detecting semen concentration– If “T” line = middle line ~ 4ng/ml– More intense = higher concentration– Less intense = less concentration

Seratec® PSA Semiquant

High Dose Hook Effect

• Each method has noted a High Dose Hook Effect– Occurs with excess prostate specific antigen

P30 (high concentration of semen) which binds to the stationary antihuman Antibody in the “T” area

– This prevents the mobile Antibody-Antigen complex from binding

– Results in no pink line = FALSE NEGATIVE– Can be overcome by diluting the sample and

reanalysis

High Dose Hook Effect

ABAcard® p30 TEST

HOW TO MAKE:

•PBS (phosphate buffered saline) pH 7.4 •ABAcard ® P30 kit

HOW TO STORE:

•Follow product inserts for proper storage. PBS can be purchased. If made in house, follow laboratory procedures

ABAcard® p30 TESTHOW TO PERFORM:

•Place a small piece of sample material (5mm x 5mm) in a 1.5 ml microcentrifuge tube

•Add 250 µl of 1X PBS to the tube

•Incubate at room temperature for 2 hours with gentle agitation on a shaker

•Pipette 200 µl of extract to the sample well “S” of the test strip

Note: Some laboratories quality control check each kit with a 4 ng/ml and a 10 ng/ml dilution of a P30. It is common practice to include a known semen standard (positive control) and a PBS (negative control) with each run set.

ABAcard® p30HOW TO PERFORM:

• Read results at 10 minutes:

– A positive result is indicated by the presence of one pink line in the test area “T”, at or before the 10 minute reading

– A negative result is indicated by the absence of one pink line in the test area. This may result from either the absence of PSA in the sample or it may be due to “high dose hook effect”, which may give false negatives in the presence of high concentrations of PSA

• If a high concentration of PSA is suspected, a 1:100 dilution of the sample may be made to and re-run to confirm the negative result

– Results are invalid if no pink line is present in the control area “C”. In this case the test should be repeated.

Seratec® PSA SemiquantHOW TO MAKE:

• PBS (phosphate buffered saline) pH 7.4 • Seratec® PSA Semiquant kit

HOW TO STORE:

• Follow product inserts for proper storage. PBS can be purchased. If made in house, follow laboratory procedures

Seratec® PSA SemiquantHOW TO PERFORM:

•Place a small piece of sample material (5mm x 5mm) in a 1.5 ml microcentrifuge tube

•Add approximately 250 µl of 1X PBS to the tube

•Incubate at room temperature for 2 hours with gentle agitation on a shaker

•Pipette 200 µl of extract to the sample well of the test strip

Note: Some laboratories quality control check each kit with a 4 ng/ml and a 10 ng/ml dilution of a P30. It is common practice to include a known semen standard (positive control) and a PBS (negative control) with each run set.


• Read result after 10 minutes incubation at room temperature.

– There should be no remaining fluid in the sample well at this time point

– An estimate of the amount of PSA can be done by comparison with the internal standard after 10 minutes

• The intensities of the internal standard and the result may change after 10 minutes resulting in incorrect readings


• Negative result (no PSA or PSA concentration below detection limit)

– Test result line (T) is not detectable. Appearance of internal standard line and control line (C) confirm validity of the test

– If a high concentration of PSA is suspected, a 1:100 dilution of the sample may be made to and re-run to confirm the negative result


• Positive result (PSA detectable)

– Test result line (T), internal standard line, and control line (C) appear

• Invalid result – Internal standard line and/or control line (C) are not

detectable

– The assay should be repeated with a new test cassette.

– Note: If the sample contains high amounts of PSA it is possible that the color intensity the control line is only weak

Microscopic ExaminationSperm cells

– Spermatozoa-plural– Spermatozoon-singular

– Male reproductive cells

– Haploid – 1 copy of each homologous chromosome

– Confirmation of the presence of semen by microscopically identifying sperm cells

• The morphology and dimensions of the human spermatozoon are unique

Microscopic ExaminationSperm cells

• Detection is simplified by histopathological staining

– The most common stain is known as the Christmas tree stain because of the bright colors (kernechtrot picroindigocarmine stain (KPIC))

•It utilizes nuclear fast red that differentially stains the DNA containing head bright crimson, and a counter stain of picric acid - indigocarmine (PIC) that stains the tails green-blue-gray

Microscopic Analysis Using KPIC

HOW TO MAKE:

• Solution #1: – Dissolve 5g aluminum sulfate in 100ml of hot

water. Add 0.1g nuclear fast red dye. Cool and filter

• Solution #2: – Dissolve 1g of indigocarmine dye in 300ml of

saturated picric acid solution (indigocarmine dye = 5,5-indigo-sulfonic acid, disodium salt)

HOW TO STORE:

•Solutions are relatively stable when store at 4C

•If purchased commercially, follow product insert instructions


Microscopic Analysis Using KPICHOW TO PERFORM:

• Fix a smear or stain extract to the slide by gentle heating

– A small cutting can be extracted in PBS or water (stain extract) or a smear can be made directly on the slide using a small cutting

• Add Solution #1 (nuclear fast red) to the smear or stain extract on the slide and allow to stand 5 minutes

• Rinse slide with water

Microscopic Analysis Using KPICHOW TO PERFORM:

•Add Solution #2 (indigocarmine dye) to slide and allow to stand 3 seconds

•Rinse slide with ethanol or methanol

•Many procedures include the application of a cover slip using mounting media—for example: Permount

•All nuclear material will stain a red or red-purple color

•Background materials will be stained blue or green

Note: A prepared semen slide should be available for comparison purposes


•Spermatozoa will appear as differently-stained red bodies, somewhat oval in shape with a slight pink cast

•The acrosomal cap will be stained less intensely red than the nuclear portion of the sperm head

•If present, the midpiece and tail sections will be stained green or blue-green.


It is common practice to grade the abundance of spermatozoa on the smear from 1+ to 4+ at 200x magnification which aids in future DNA analysis

Note: Some procedures require that spermatozoa be verified at 1000x magnification

KIND Classification

• Method of classification originally published in 1965 by S.S. Kind

• Rare = < 10 cells on slide• 1+ = Few; difficult to locate• 2+ = Some in some fields• 3+ = Some in many fields; easy to locate• 4+ = Many in most fields

• Some laboratories have made slight modifications to this rating scale


Head of the sperm is RED

Tail is GREEN

Acrosome remains CLEAR or faintly colored

Digital Image of KPIC stained sperm cells


Yeast, bacteria, free nuclei, white blood cells, and dog sperm are stained with this method, and in some instances can interfere with the identification of sperm cells


• Always look for:

–Differential staining- acrosomal cap–Tail, if attached

–Size -larger than yeast or bacteria, but smaller than free nuclei or dog sperm

Having prepared human semen, dog semen, yeast and bacteria slides stained with KPIC should be available for comparison purposes

Christmas Tree Stain Video

Yeast

image courtesy Kimball's Biology Pages (http://biology-pages.info)

Other Stains• Hematoxylin and Eosin staining

– Hematoxylin• Affinity for nuclear material in the presence

of a metal• Stains nuclear material purple

– Eosin is a counterstain• Non nuclear material stains pink

Other Stains• Fluorescent staining

– Example: SpermPaints – fluorescent dye mixed with monoclonal antibodies which bind to the sperm head and tail antigens

– Clear, bright, selective–Will not stain other tissue types

• Giemsa staining– Nuclei stain red

Microscopes

To clean:

• Use lens paper and lens cleaner (isopropyl alcohol solution) for ocular lenses and glass surface (over the light source)

• Use cotton swabs for smaller objective lenses

Microscopes

Documents

Forensic Biology Screening Workshop Semen. Semen Composition Semen is a fluid of complex composition, produced by the male sex organs. There is a cellular