Forensic Biology Screening Workshop

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 Acid phosphatase can be found in other bodily fluids such as vaginal fluid (vaginal acid phosphatase), which is elevated in prepubescent females and pregnant women, male urine and blood (erythrocyte acid phosphatase). Seminal acid phosphatase while found in semen can also be found in the blood of men who have prostate cancer. Most seminal fluid is produced in the prostate (~60%) and the seminal vessicles. A very small amount is produced from the bulbourethral glands (Cowpers glands) – these glands produce a small amount of viscous mucus that contributes to the overall jelly like consistency. Basic amines provide a more alkaline environments for the sperm to counteract the acidity in the vaginal tract protecting the DNA in the sperm from acidic denaturation. The consistency of semen gives sperm cells a less viscous medium to swim through and also prevents their diffusion out of the semen.

Seminal Plasma Function 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 Seminiferous tubules are housed inside the testes. At this point, sperm are immature and not capable of fertilization. They complete their maturation in the epididymis. This amount of sperm cells is what is expected in a healthy male. There are a lot of factors that can affect this.

Sperm Cells Move to the epididymis Upon physical stimulation:
Sperm and seminal fluid combine in vas deferens to form semen Ejaculate ~ 3-4 ml of semen ~ million sperm per ejaculate

Male Reproductive System
Seminal fluid from prostate, seminal vessicles and accessory glands. AP/P30 made in prostate. Pathway of sperm – Seminiferous tubules (in the testes) > epidydimis > sperm and seminal fluid combine in the vas deferens and expelled out through the urethra.

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 A micrometer is one millionth of a meter or one thousandth of a millimeter. A dime is ~ 18 mm.

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

Acrosome – aids in sperm penetration into the egg
Acrosome – aids in sperm penetration into the egg. Acrosome reaction – the membrane around the acrosome fuses with the sperm plasma membrane, exposing the acrosomal contents. The membrane at the tip fuses with the egg plasma membrane. Protamines carry a positive charge and serve to keep DNA (negative charge) packed as close as possible. The plasma membrane of the sperm cell have disulfide bonds in the outer plasma membrane. This makes the sperm resilient to a regular extraction method that would lyse open an epithelial cell. This is the basis for a differential extraction. Epithelial cells are lysed in the first stage of the extraction while the sperm cells remain intact. This epithelial DNA can be extracted and separated. Then, DTT is added for the sperm cell lysis step which breaks down the disulfide bonds and allows the sperm cell DNA to be extracted.

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 Note other body fluids also fluoresce under UV light

Analysis of Semen Presumptive 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 Semen Confirmatory Tests P30 Identification Found in semen
Microscopy Identification of sperm cells, usually done using a staining method While P30 can also be found in male blood (elevated with prostate cancer) and possibly female breast tissue and urine???, it is not found in as high a concentration as is seminal P30 and would be beyond the limit of detection of the tests.

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 The survival times are referenced in literature and vary depending on the article. These are approximate. You may see sperm on samples that were beyond 3 days once the epithelial cells are digested away during a differential extraction.

Survival Times Why 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 This is what allows for the separation of epithelial cells and sperm cells in a differential extraction

Time Lapsed Sex Kit #1 Results Table
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 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 72 hours Mention they should do this at their own lab for training purposes In my experience, a positive AP test must change before 30 seconds, so you need to follow your own lab SOP

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 48 hours 1 possible sperm on slide

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 12 hours + 25 seconds 24 hours + 30 seconds 36 hours 48 hours - > 30 seconds 72 hours - No reaction at 1 minute apparent vasectomized person

phosphoryl choline  choline
Acid Phosphatase Acid Phosphatase Secreted from the prostate Cleaves phosphate group from molecules phosphoryl choline  choline Choline is important in cellular membrane composition and repair BCIP -

Acid Phosphatase -BCIP Test
HOW 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 4C

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 Method published by the FBI. It was originally studied and implemented as a quick screening test for semen. It helps to reduce the number of samples that need to go on for further extensive testing.

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 Test
HOW 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 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 A presumptive test only indicates the presence of a substance because there are other substances that will also react with this test. Brentamine Fast Blue B – also called naphthanil diazo blue B dye = same thing. If you use the solutions separately they are more stable than in the working solution.

HOW 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

HOW 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 Again, vaginal acid phosphatase will turn pink.

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 mapping is mostly used on larger items such as panties, clothing, bed sheets.

AP Mapping HOW 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 Can also use Benchkote which is filter paper with a plastic backing

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. Good for items such as sheets, clothing that are large. Important to mark alignment of paper to the item so that you can find the stain should there be a positive reaction. Ensure your controls work – known semen stain for positive control and reagent only on the filter paper for the negative control. Also you can section the item off and take swabbings and test the swabbings using the AP spot test. This procedure is from the Illinois State Police – each lab will have its own protocol so you should follow your own SOP’s.

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 Swabbing Used 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
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. Note other markers were evaluated for the ELISA assays Note Ouchterlony method described in blood lecture

P30 Electrophoresis Methods
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 Electrophoresis Methods 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 Semen ELISA 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 Tests Ouchterlony, 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 C = control line – line must be present for the test to be valid. T= test line

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 S area is where you put the sample. The T area is where the test result is. Monoclonal means they are all clones from one type of immune cell – this means they can specifically bind a particular substance. Polyclonal are not as specific – they are similar but not identical antibodies. Antihuman antibodies are created by injecting an animal with human antigens. It then creates in response antihuman 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
Internal standard is 4ng/ml. This is the semi-quantitative tool of this card. You can approximate the intensity of the test result line compared to the internal standard line.

“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 C = control area

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 Procedure can be used for both types of cards.

ABAcard® p30 TEST HOW 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. While this protocol requires 250ul to extract the sample, please use 750ul in the workshop exercise.

ABAcard® p30 HOW 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.

HOW 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

HOW 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.

HOW TO PERFORM: 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

HOW TO PERFORM: 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

HOW TO PERFORM: 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 Examination
Sperm 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 Examination
Sperm 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) Note-these reagents can be purchased from SERI

HOW TO STORE: Solutions are relatively stable when store at 4C If purchased commercially, follow product insert instructions

HOW 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 exam is confirmatory. You can draw circles using wax crayons or there are slides you can buy with circles – analyst or lab preference If you are going to extract the sperm cells overnight you should refrigerate to prevent cell lysis.

HOW 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 Optional to do this – may be helpful for cases where a different analyst does the DNA (extraction) – during extraction the sperm:epi cell ratio can determine the number of washes you do during the extraction for a clean separation. This classification was originally published in 1964 by S.S. Kind – found in many articles.

Head of the sperm is RED Tail is GREEN Acrosome remains CLEAR or faintly colored

Digital Image of KPIC stained sperm cells
Point out tail, head and acrosomal cap

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

image courtesy Kimball's Biology Pages (http://biology-pages.info)
Yeast Most common to be confused with sperm heads. In this picture - not stained with Christmas Tree Stain, however it does absorb the Kernectrot (Red) stain. Can be differentiated from sperm by the lack of an acrosomal cap and also the size. image courtesy Kimball's Biology Pages (

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 Hematoxylin and Eosin staining is very commonly used in histology labs and can also be used to stain spermatozoa. Used for future trend in forensics – laser microdissection Mention automated microscopes for sperm ID – will get into further detail in future trends segment

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 Useful to keep covered every day with microscope cover.

Microscopes Typically binocular. Mention how to focus using coarse focus first then the fine focus. Helpful to look at slides in a grid pattern so as not to miss any. May be better to look up and down vs. side to side to avoid getting dizzy / headaches.

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