1. The Biological Crime Scene
Semen – And Others

2. Semen
Semen: Second most prominent class of biological evidence found at scenes.
Modern scene scientists/investigators locate semen using an ALS set on the CSS setting or using another appropriate wavelength that causes semen to fluoresce.
Typically, investigator wears orange goggles,
Wavelength dictates which color of goggle
For some wavelengths, e.g., 415nm, goggles may not be necessary.

3. Forensically Speaking:
What is Semen?
Semen
Complex
Connective Tissue
Plasma
Cells
Hormones
Genetic Markers
White
Cells
Sperm
Cells
Pre-Sperm
Cells
Salts
Drugs
Antigens
Genetic Markers
Enzymes
Antibodies
HLA
Antigens
Blood Group
Antigens
Blood Group
Substances
DNA
Isoenzymes
Isoenzymes
Individual
Specific
Antibodies

4. Semen Origins Contributing gland Percentage of ejaculate
Testicles and epididymus
5 per cent
Seminal vesicles
46 to 80 per cent
Prostate gland
13 to 33 per cent
Bulbourethral and urethral glands
2 to 5 per cent

5. Historically - Identifying Semen
1600’s Leuweenhook’s student first “saw” a spermatozoan in the microscope,
Identifying semen microscopically has been the forensic laboratory’s gold standard.
In the middle part of the 20th century, much of semen identification done in hospital
Received vaginal washings from rape survivor. Did quantitative AP test
Clinical laboratories performed two tests:
Sperm mobility test …
Quantitative acid phosphatase (AP or PAP) analysis (popular in the 1940’s to 1960’s) way to show that semen was present in the vaginal vault.
Acid phosphatase: high concentrations of the enzyme enzyme in Semen.
Labs establishes AP cut-off points.
Eventually shown not to be the most reliable method for identifying semen in rape cases.

6. Historically - Forensic Laboratory Testing
Received dried vaginal swabs from the police instead of a vaginal lavage,
Determining the level of AP difficult because semen stains are dried.
Forensic scientists historically employed a sequence of tests to identify semen in dried stains, culminating by identifying spermatozoa
1st: used AP reagent as a presumptive test = semen might be present by measuring time for test reagent to turn color.
If color change rapid, less than thirty seconds, the laboratory had evidence that the dried stain could contain semen.
2nd : Confirmed semen microscopically.
1970’s
Analytical sequence changed in the 1970’s with the discovery of a semen-specific protein known as p30 by forensic scientists and prostate specific antigen (PSA) by clinical scientists.
Adapted dried stains gave forensic scientists the ability to identify semen without necessarily identifying spermatozoa.

7. Finding Semen at the Scene php?option=com_content&task=view&id=593
Historically
In the 1940’s, 50’s and ‘60’s they used the crusty “feel” of dried semen as a quick check for the presence of a biological fluid, a test also used by laboratory scientists.
Fast and easily performed on evidence where semen might be expected, such as bed sheets.
Another on-scene technique employed the first true alternative light source, the black light (UV light).
Causes semen to fluoresce, and works well for semen stains on light, unwashed as well as dark fabric. It did not work on light, detergent washed fabric.
Acid Phosphatase (AP) :
Presumptive AP testing to show that semen might be present adapted for use at the scene …
Used like the chemical tests for blood.
Interpreting based on the time it takes for particular color to develop.
php?option=com_content&task=view&id=593

8. Sci Justice. 2012 Jun;52(2):106-11. Epub 2011 Sep 13.
Variations of the AP Presumptive Test
ONE – Not a best choice: not a preferred method.
Dab stain with a cotton swab moistened with the reagent and wait for the color to change on the evidence.
TWO - A preferable method
Moisten the swab with water, dissolve a small amount of the unknown stain onto the swab and then test the swab with the AP reagent.
A rapid color change from colorless to dark blue to purple is a positive test.
THREE- Not a recommended method
Moisten the unknown stain directly with the reagent by dropping micro-droplets from a micro-pipette directly onto the stain.
Again, a rapid color change is diagnostic of a positive test.
FOUR – AP screening – an older method but made obsolete by ALS
Can screen large areas to find invisible semen stains.
A piece of filter paper is moistened with water and then rubbed over larger areas of, say, a bed sheet that has been sectioned into a grid. Test the filter paper with the AP reagent.
A positive result tells investigator … semen might be present in area swabbed.
The AP screening method is not as popular, if it is used at all, because the ALS highlights those areas more easily and faster without the use of chemicals.
Sci Justice Jun;52(2): Epub 2011 Sep 13.

9. Semen – Presumptive tests
Acid phosphatase (AP) test
An older test
a-naphthyl phosphate
Sodium a-naphthyl phosphate
broken down by AP
frees naphthyl group
Fast Blue
o-dianisidine
combines with naphthyl group
produces scarlet red color
o-dianisidine
Acid Phosphatase
Scarlet Color

10. Confirming Semen @ the Scene
Confirmatory tests were not available for on-scene dried semen testing until recently.
Immunochromatographic cards available for on-scene testing to identify the presence of semen proteins,
p30 also known as Prostate Specific Antigen (PSA by Abacus Diagnostics)
Seminogelin manufactured by RSID.
Immuno-cards can be used to identify human semen at the scene.
Immuno-cards expensive AND slower than AP test

11. Rationale or Decision Tree for Locating
Biological Evidence.
Logic:
Searching for semen should not be a one-step, check-the-bedroom-only endeavor.
Knowing where semen might be is the first step. Obviously sexually motivated scenes lead investigators to search for semen in logical places:
Beds, sofas, car seats, etc.
Non-sexually motivated scenes
Perpetrator masturbates, urinates or defecates
Burglary scenes have a higher than reasonably expected amount of semen present.
For whatever reason, perhaps after fondling a female’s undergarments or jewelry, burglars masturbate, leaving their biological signature.

12. Finding Semen at the Scene
Alternative Approaches
Need written procedure for deciding which ALS positive stains to test further
How and which to collect for the laboratory.
ALS is first line of attack
Can also locate other biological evidence: saliva, urine and trace evidence.
ONE:
Use ALS to locate possible semen, saliva or urine stains and
Laboratory decides which to test further.
Safest approach …
leaves choice of DNA analysis to the laboratory.
TWO:
Use ALS to locate possible probative stains
Then use the acid phosphatase test to determine which stain most likely contains semen. …
Laboratory would re-test stain using its ALS and might re-do AP test before DNA analysis.
Semen stain highlighted by ALS
@ 450nm under orange goggles

13. Alternative Approaches – Continued
THREE: ALS & Immunocard
Use ALS followed by an immunochromatographic test.
AP test is avoided and the more definitive/confirmatory immunological test used.
Positive on-scene test identifies semen … alerts laboratory that subsequent analysis is warranted.
Negative immunocard test suggests ALS highlighted stain not semen.
In this instance, investigator might retest using AP test.
OR laboratory will re-test stain with AP.
NOTE: Can occur with old stains sitting in non-air conditioned evidence vaults for extended periods of time.
Negative test may mean stain too old (insoluble) so that dissolution is slow & insufficient material not in solution for the test to be positive.
Expect AP and the immunocard tests can be negative on older stains, especially those exposed to excessive environmental insults.

14. Biological Fluids on Garments
Using an ALS to Locate
Biological Fluids on Garments
Examine the evidence using the 450nm wavelength of the ALS
CSS (crime scene search setting) and other wavelength settings on the MiniScopeTM 400 can highlight semen or other fluorescing biological substances – some without using goggles –
Preferred method is to use the 450nm setting PLUS barrier filters or goggles - Orange).

15. With Matter (Evidence)
Interaction of Light
With Matter (Evidence)
Light is absorbed
Light is transmitted
Light is reflected
Light is given off in the form of fluorescence

16. Subsequent DNA testing identified the murderer forty plus years later.
40+ Year Old Case
Slip taken from the body of a young girl who had been attacked, raped and murdered 40 plus years before the ALS analysis.
The areas in yellow and marked with letters and numbers (A1, A2, etc) are fluorescent areas highlighted by the ALS.
The AP test, evaluated using most established standards was negative –
Long time for the reagent to show a very weak purple color;
Expected because the stains had been stored improperly for 40 years.
The only tests giving positive results were the ALS and an immunological test for prostate specific antigen (p30).
Subsequent DNA testing using LCN (Low Copy Number) techniques identified the murderer forty plus years later.
Subsequent DNA testing identified
the murderer forty plus years later.

17. Marking Evidence at the Scene
When ALS highlights something that may have a biological origin,
Mark area by circling it with a marker or by marking the location with an arrow sticker.
Shows the laboratory scientist where the scene investigator obtained a positive presumptive test result.
Means investigator observed something using an ALS
AND it might require further analysis.
Investigator’s notes should reflect “possible semen evidence.”
The scene investigator should
Clearly delineate where the positive result was obtained
Initials and date should also be marked clearly on the evidence.
Photograph what ALS highlights
Tells lab scientist what the investigator saw
Confirms location of highlighted stain.

18. Collecting and Packaging Semen Evidence
The invariant rules discussed for blood evidence hold equally for semen evidence.
Packaging guidelines are also the same.
Generally semen evidence at the scene is dry,
Tissue paper, clothing, towels, bed sheets, pillows, blankets, furniture,
Wet or dry droplets on the floor
Skin of a deceased or survivor.
If wet --- DRY IT
Collecting
Collecting the entire, dried stain
Moistened sterile cotton-tipped swab designed for semen evidence.
Dry swabs at scene using a drying device or by placing the swab into a cardboard holder also designed for that purpose.

19. Saliva Saliva is another biological fluid that occurs at crime scenes.
Saliva is a biological tissue having two parts: liquid and cells.
The cells in saliva are epithelial cells and contain DNA. The liquid part is comprised of salts, drugs, hormones as well as dissolved proteins and enzymes that differ from those
Present in semen and blood.

21. Salivary Amylase
Most important constituent of saliva, other than DNA, is enzyme amylase, important for ascertaining whether an unknown stain might be saliva.
Historically - Before ALS used to locate biological evidence
Finding at crime scenes was mostly luck because likely associated with semen in a sexual assault or associated with a bite mark.
Historically, identifying a dried saliva stain meant identifying the salivary component in the largest concentration - the enzyme alpha amylase.
The problem is that, like AP, amylase occurs in most forensically important tissues.
Body Fluid Avg. [AMY] 3 3
Saliva ………………………………………………… 94x10
Semen (vasectomized) ………………………. 10
Semen (aspermatic) …………………………

22. 40 + Year Old Case

23. Finding/Collecting Saliva Evidence
Logic
Presumptive tests
Amylase testing
ALS
Confirmation Testing
RSID – Salivary Immunochromatographic Card
Collecting & Packaging
Same as for semen evidence

24. Urine

26. Finding Urine Evidence
Logic
Odor
Presumptive tests
Urea testing = only presumptive test – not used often
ALS (weak yellow fluorescence of dried stains
Confirmatory testing
RSID – Immunochromatographic card

27. Collecting and Packaging Urine Evidence
Toilet: Substantial volume to collect and package …
Transport immediately to forensic laboratory … often impossible …
urethral epithelial cells should not remain in wet, acidic environment for extended periods … deleterious effect on the DNA.
If the urine contents cannot be transported immediately, …
Freeze as soon as possible or put on ice. Finding ice at convenience stores is not difficult or expensive.
Wet in Puddle: Collected into a container …
Put on ice/frozen
Absorb onto an absorbent material (sponge, towel or filter or bench paper) and dried.
Once dry, the DNA will be more stable than if the urine remains wet.
Clothing: Either wet or already dry.
If wet or damp dry ASAP, preferably at the scene.
Once dry, the DNA should be relatively stable, certainly for several hours until it can be taken to the forensic laboratory.

28. Collecting and Packaging Feces Evidence
Human metabolism … waste product … found at scenes … often in piles.
Perpetrators defecate at scenes, so searching for their feces at the scene is an endeavor with important identifying implications.
Locating it is not usually difficult, as Characteristic odor and/or characteristic appearance diagnostic and overwhelming … obvious.
Dried brown swipes may not have the characteristic fecal odor,
Drying mutes odor.
The easiest way to determine whether a dried, brown stain is feces is to swab a small amount onto a wet cotton swab and then smell it.
Once feces are re-dissolved, the unmistakable, offensive odor will be unmistakable.
Collecting and Packaging Feces Evidence
Forensic Value: Potential investigative information PLUS DNA.
Other forensically important considerations.
Product of digestion, it has foodstuffs that might help to identify a meal the perpetrator ate or ingested drugs.
Collecting and packaging wet feces … scooping it up … into a casserole-like dish … seal.
If cannot be transported to the laboratory immediately …
Frozen or put on ice.
Keeping the feces cold is important because it has destructive enzymes that destroy cellular DNA

29. Vomit

30. Collecting and Packaging Vomit Evidence
Not a common type of biological evidence … certainly found at scenes.
Can be important … contains cellular material … DNA … foodstuffs can tell something about the immediate eating habits of the person.
Finding: … not difficult because of distinctive odor. Dried vomit stains have an odor … dissolving small amount in water & boiling it releases odor.
Enzymatic tests for rennin … enzyme present in vomit ... odor test is sufficient for investigative purposes. The laboratory will do the appropriate tests … extract the DNA.
Collecting and Packaging Vomit Evidence
Same as for feces and urine evidence.
Like urine and feces, the acidic environment of vomit can be detrimental to the integrity of DNA, so collecting it and either drying it or freezing it as soon as possible can be critical.

31. Case Example YOKNAPATAWPHA COUNTY SHERIFF'S DEPARTMENT
Investigating Officer(s): Det. S. Murphy. Det. T. Armstrong Incident No.: I-2011 Case Description: Kimberly Pace Homicide
Investigators prepared the following summary of results obtained thus far from forensic analysis of various items taken into evidence from the crime scene at 1592 Jackson Avenue by Yoknapatawpha County CSU technicians.
Samples of vomitus found on ground, approximately 36 inches north of back door of crime scene
Analysis of content found:
Beef, texture consistent with ground beef
Plant matter
Hairs
Hairs were determined to be canine.
That result plus the presence of non-human antibodies suggest the vomit was from a dog.
Toxicological tests found ~1250 mg of acetaminophen in the vomit

32. Hair

33. Hair
Finding probative hair evidence can be crucial in the final adjudication of a case. National Academy of Sciences determined that hair comparisons should be done in conjunction with mtDNA analysis Hair has forensic value. Is biological and a pipeline to what we eat.
Hair grows from hair follicles, which means DNA is present. Although the hair has both nuclear and mtDNA, the former is found mostly in the hair root and only tiny amounts are present in the shaft.
The shaft however, is an ample source of mtDNA. Unfortunately,
mtDNA is not unique enough to provide a unique identification.
Understand the meaning of hair found at the scene.
Hair clutched in the hand of a dead victim suggests hair pulled from an assailant during a struggle.
While collecting this hair might be the responsibility of the M.E., it is critical to ensure that the hair is collected and preserved for DNA analysis. This is the responsibility of the crime scene team leader, who must capture the hair clump photographically.

34. Hair
Finding probative hair evidence requires logic and the appropriate use of technology and collection techniques. Flashlight is appropriate as is ALS.
The technique is to highlight the hair by holding the light source at an oblique angle.
Search locations that make sense.
Homicide scene
Depending on how the homicide occurred, the deceased’s body and/or its immediate vicinity should have the hair of the assailant present, if a struggle took place in that area.
If combatants fell onto furniture, that too, would be a logical place to search for probative hair evidence.

35. Collecting and Packaging Hair Evidence
Three common methods used to collect hair & other trace evidence.
Discussed in trace evidence section
Generally, the methods employed are listed below.
The hunt and peck (Picking) method using tweezers to lift hair and placing it into an appropriate container (envelope),
Taping and to lift the hair onto the sticky side of tape.
Vacuuming.

36. Fingerprint Residue Time since last washing.
Not usually categorized as a type of biological evidence.
Commonly thought of in terms of friction ridge detail.
Fingerprints contain biological material in two forms:
Emulsion and the cellular content of the finger’s epithelial cells.
When print left on a surface, cellular material remains with the print residue. The number of epithelial cells deposited depends on,
Time since last washing.
Tendency of the individual to shed cells, such as whether the individual is a shedder.
Mechanism by which the print was deposited. That is, was it rubbed onto the surface or was it gently placed.
Genomic DNA: A cell has approximately 6pg of nuclear DNA in its nucleus. While it is possible to obtain a DNA profile from a single cell, a form of forensic DNA testing called Low Copy Number (LCN) testing usually requires approximately 22-25pg (approximately 4 cells) of template DNA (useable) per test
Mitochondrial DNA (mtDNA): Present in cells in fingerprint emulsion, and even a single cell has a better chance of giving a mtDNA profile because each cell has multiple copies of mtDNA,
Consider the burglar who is looking into a prospective residence and places his head against the window in order to better see.
Forehead is good source of cells - residue on window should have DNA.
Understanding elements of crime (LOGIC) crucial for determining whether skin residue will be an important source of biological evidence.

37. Collecting and Packaging Skin Residue
Ideally - procedures for collecting fingerprints should be amenable to collecting its cellular content.
Quantitative amounts of DNA are not collected using any of the traditional fingerprint collection methods:
Print lifting tape,
Casting with silicone casting material such as MikrosilTM or with
Rubber-backed gel lifters.
Secondarily swabbing can lift the cells
DNA profiles can be obtained from the swabs.
Smudged fingerprints and skin prints … scene is first issue.
Generally, powder dusting or locating using ALS or reflected UV light.
Once located, the print can be swabbed and preserved by putting the swab into a swab collection box of the type used for collecting vaginal swabs in rape cases.
Place boxed swab into a larger paper container.
Do not use plastic: As for any biological evidence

38. Collecting and Packaging Soughed-off Cell Evidence
Sloughed-off Cells
Refers to our human penchant to continually shed
Everyone sheds cells, and in the heat of a struggle, more cells should be shed than normal. If located and collected, these cells could be an important source of DNA.
Elusive category of biological evidence for which no on-scene technology yet exists, Likely the most prevalent category of physical evidence, bar none, because it is ubiquitous where there are people.
Collecting and Packaging Soughed-off Cell Evidence
Finding sloughed-off cells at the scene. Possible choices.
Taping technique for collecting trace evidence.
It will “lift” sloughed-off cells from most any surface.
Impractical to tape entire surface of the crime scene … use logic to lead to appropriate areas to find.
Use fingerprint lift tape.
Preliminary work suggests that this is an inefficient method for collecting DNA from fingerprint residue.
Swabbing the print residue remaining after lifting will capture the print residue and the cellular material left behind.
Use a gellifter to lift the fingerprint. Preliminary work suggests that gellifters are not an efficient way to lift cellular material (DNA) from fingerprints.
As with fingerprint lift tape, swabbing after gellifting capture the DNA left behind.
For smudged prints, swabbing is the preferred method.

39. Bacteria/Spores/Viruses
Uncommon Biological Evidence
Bacteria/Spores/Viruses
The team leader and the investigator decide how to handle the situation based on the scene.
A triple homicide with lines of white powder on a dining room table suggests cocaine.
A scene with strange looking laboratory equipment may suggest something more sinister.
After the 2001 attacks on the World Trade Center and the Pentagon, packages containing Anthrax spores were sent in the U.S. mail to Florida, New Jersey and New York.
The possibility of finding a suspicious white powder containing biological material (spores) at crime scenes presents is apparently real, which presents in insidious threat.
The danger exists not only for those conducting the investigation but also the outside world if the material is not handled properly.
Do not touch suspicious powders
Seal the scene
Quarantine anyone at the scene
Contact a Hazmat crew
All white (or colored) powders are not suspicious or have a biological origin.
A murder scene where obvious drug activity is present may have powders that are clandestinely prepared drugs … these are dangerous … they can be tested scene for drug content.

40. Plant Material
Common among the four categories of not so commonly occurring biological evidence : bacteria, plant material, pollen and viruses. Utilizing for forensic applications is not so common.
Plant material: DNA, so a plant DNA profile can be used much like human DNA.
Collecting and packaging plant material like any biological evidence.
Finding probative plant material is the problem.
Sometimes plant material will be found associated with tire tracks or shoeprints.
Collecting
As carefully as possible
Lifted with gel lifter.
Use tweezers and put it into an appropriate container – a coin or glassing envelope – and labeled appropriately.

41. Plant Material Pollen: Plant material on all surfaces.
Small and hard to detect. In some seasons, it is easy to see, as the pollen grains are seemingly everywhere.
Useful when found associated with something at the scene that had been collected for another purpose –
Shirt from deceased where pollen grains were transferred during the commission of a crime, the sole of a shoe from someone who came from another country and brought the pollen with her.
Forensic Palynologists: Someone who studies pollen.
Pollen reveals where a person or object has been because regions of the world specific locations have a distinctive collection of pollen species. It can even reveal the season when a particular object picked up the pollen.
No standardized methods for collecting pollen at the scene.
Techniques in the Anthrax investigation should be considered
Taping techniques should be successful.

42. Evidence of Condom Use

43. Condom Evidence – From Vaginal Swabs
Assailants wear them, probably not from the fear of contracting a disease but to avoid leaving DNA evidence. Value of the semen evidence and the victim’s vaginal secretions or saliva on the assailant’s penis are lost to the investigation.
No possibility of getting DNA profile of assailant,
Substantiate a victim’s allegation that a sexual assault took
Evidence of penetration
Can help identify a serial rapist by linking common condom types.
Condom construction
Variety of natural and synthetic materials, each manufacturer having its propriety formulas, even among its own brands: lamb membranes, polyurethane plastic and latex rubber – the largest percent of condoms sold.

44. Condoms and trace evidence: Particulates, lubricants, and spermicide.
Classified as wet or dry and have an oily consistency – easily recovered and analyzed
Dry lubricants: water insoluble and wet ones are either water-based or water soluble.
Particulates are finely powdered corn or potato starch, the powdered lycopodium, silica and talc.
Differences because of varying viscosity grades.
Wet lubricants
Contain polyethylene glycol or a gel similar to vaginal lubricants.
Associate specific formulations with particular condom brands.
Lubricated condoms may also contain the spermicide nonoxynol-9, which can be recovered.
Can construct a profile of a condom by identifying the particulates, the lubricant and spermicide present from other evidence found at the scene or at the hospital.

45. Condom Evidence at the Scene
Finding a condom at a sexual assault scene can be important evidence that can link a suspect to the scene and thus the crime.
The DNA profile from the material collected from the condom not only should provide a DNA profile of the suspect but also that of the victim (vaginal DNA) which places the suspect in direct contact with the victim.
Collecting and Packaging Scene Condom Evidence
Locate used condom and foil packaging.
DNA from the victim on the outside proves that the condom was used with her and seminal fluid from the assailant on the inside will be the best evidence against a suspect.
Wear powder-free gloves to protect against blood borne pathogen infection and to avoid leaving particulates similar to those used by some condom brands
Package condom evidence in a breathable box or envelope.
If possible, dry wet semen before packaging.
Package investigator material (collecting gloves) separately and
Submit them with the evidence so laboratory can verify that the gloves did not leave behind any particulates.
Save empty condom packets for fingerprint analysis.
Wipe the inside of the condom package with a clean cotton swab that will become the standard for comparison with traces recovered from the victim and the suspect.
The inside of an empty condom package should not contain fingerprints but might contain lubricant, a spermicide, and particulate residues.

46. Accreditation, Certification & Quality Assurance
Accreditation: Refers to agencies
Quality assurance:
A program of quality – part of a comprehensive quality assurance program
An integral part of accreditation
Certification: Refers to people

47. Quality Assurance at the Scene
Establish a quality assurance program
Essential for ensuring reliability of scene investigation
Accreditation: Requires quality assurance program
Gives confidence that work meets minimum quality standards.
Critical component any quality assurance programs is quality testing .. referred to as quality control testing. This refers to the testing of reagents used routinely to ensure that they produce consistent and reliable results.
All testing reagents must be quality checked regularly against established quality guidelines, which means all reagents used in the field must meet established quality standards.
Related topics:
Sensitivity and Specificity of particular reagent compared with other reagents that test for the same analyte. means that if one reagent detects blood
All testing reagents have a sensitivity threshold, which t the 1in 10,000 level and the other at 1 in 100,000 it should come as no surprise that the second reagent is more sensitive and will give a positive test long after the first stops giving results.
The characteristics of reagents should be known before using them at scenes. If necessary, specificity of reagent must be tested.