1. Evidence Collection in Acute Sexual Abuse
Marilyn Kaufhold, MD, FAAP

2. Learning Objectives The participant will be able to:
List the usual forensic specimens to be collected from an acute sexual abuse/assault victim.
Explain how sexual assault evidence may be contaminated or lost before collection.
Define the concept of “chain of custody.”
Justify the importance of establishing a collaborative relationship between medical providers and the forensic laboratory.

3. Evidence Only collected in acute cases
May corroborate victim’s history by…
Linking AP to victim
Linking victim to the scene
Linking the AP to the scene
Identifying the perpetrator by DNA recovery
Lack of evidence may also corroborate the victim’s history
With the advent of DNA analysis, semen evidence is fairly robust, provided there was sufficient sample present in the first place
Sperm can be found inside the vagina for up to several days. Sperm might be found inside the cervix for up to several weeks.
Semen deposited inside the rectum is generally gone within 24 hours and inside the mouth within 12 hours.
Of course if a condom was used or ejaculation didn’t occur, it’s likely that semen won’t be found even if the exam is done just a few hours after the assault.
Local practice may include collection of oral and rectal swabs even if the victim does not provide history of oral or anal contact

4. DNA Evidence
Powerful investigative tool that has replaced former serology techniques
Trace evidence (foreign hairs, debris) still collected - less frequently processed
DNA evidence may be left of the victim’s body or at the scene
Semen, saliva, blood left by suspect on victim’s body, clothing, bedding or other items from the scene
Skin cells (fingernail scrapings) if the victim scratched the suspect

5. Three Categories of Evidence
1. Standard items
Collect in all exams – typical sources of evidence in SA case
2. Evidence per patient history
Patient tells of other contact that might yield evidence
Examiner finds debris, etc.
3. Reference samples
Informs lab to differentiate foreign material from patient’s own type of blood, hair, DNA, etc.

6. 1. Standard Items All Prepubertal Adolescent Girls Boys Clothing MouthX
Mouth
Fingernails
Anal canal
Pubic hair
Vulva/ vestibule
Vagina/ Cervix
Penis/ scrotum
Collection of standard items depends on pubertal status and gender of the victim.
Items are listed because they are the most typical locations for useful evidence to be deposited n sexual assault/abuse cases.

7. 2. Evidence per Patient History/Exam
Possible saliva – any site with oral contact (usual sites: perioral, neck, suction injuries, bite marks, breasts, abdomen)
Possible semen (usual sites: face, abdomen, hands, external genitalia, legs)
Rectum - Swabs per anoscope
DFSA cases (Collect from perioral area, neck, breasts, abdomen)
Debris (Vegetation, ligatures, intra-vaginal items [condoms, retained tampons], soil or sand, fibers, paint, hair [human,animal], other
DFSA (drug-facilitated sexual assault): when the victim has no recollection of acts, perioral area, neck, breasts, abdomen are frequent sites of semen or saliva left by the perpetrator
Anoscopy is indicated in cases in which the victim reports anal penetration or the medical provides sees anal injury or there is unexplained rectal bleeding. It may be done in adolescents who consent to the procedure. If there is concern about anal penetration in prepubertal children and the medical provider assesses the child needs a rectal exam, it should be done under anesthesia by a pediatric specialist.

8. 3. Patient Reference Samples
Head hair
Pubic hair
DNA sample: blood, buccal swabs
These are required to differentiate the victim’s DNA from any other DNA collected from the body of the victim or the scene

9. Working with the Forensic Lab
There are a number of different evidence collection protocols and kits
Important to work with local/regional crime lab to establish consistent practice
Best practice has not been determined in many cases
Meet with local crime lab regularly
Regular meetings with the crime lab promote
Better evidence collection by medical providers
Better appreciation of the medical provider’s task by forensic technologists

10. Prior to the exam, biological forensic evidence may be lost by…
Rapid biochemical degradation
Loss of sperm motility and morphology
Gravity drainage from the vagina
Mechanical loss: washing, brushing teeth, urinating, defecating, wiping, douching, etc.
Pre-hospital providers should make every effort to get patient to the exam site before evidence is lost

11. Remember to obtain relevant history
Time interval since assault
Sexual acts
Condom or other contraception
Prior or subsequent sexual activity
Post assault hygiene: urination, defecation, bathing/showering/washing, douching, eating/drinking, brushing teeth/gargling, changing clothes

12. Before starting… Make sure victim is medically stable
Set up evidence kit items
Pre-label where possible
Sufficient supply of patient labels
Supply of paper bags, evidence sealing tape
Extra tubes, envelopes
Supplies to collect foreign materials
Post-its, tweezers, swabs, bindle paper
Urine containers
Supplies for drawing blood

13. If alcohol/drugs have been used to overcome the victim…
Collect toxicology specimens
If patient appears under the influence
If there is a recent history of drug or alcohol use
If patient has history of loss of consciousness
Collect at the outset of the exam as specimens will more accurately reflect patient’s level of intoxication at the time of the assault

14. Avoid Evidence Contamination
Contamination of evidence can lead to errors in interpretation of the crime circumstances
Process only one patient at a time
Clean exam environment after each patient
Avoid coughing or sneezing or chewing gum - May deposit examiner DNA on patient and/or evidence
Always wear gloves. Change gloves as needed during exam
Routine cleaning of the exam room and stationary equipment is mandatory after or just before the forensic exam
Disposable supplies and equipment should never be retained or re-used

15. Clothing Collection Collect, dry, and store correctly
Body fluid stains on clothing are stable for years
May yield more evidence than body
Collect, dry, and store correctly
Worn during assault or put on immediately after
Underwear put on even after bathing - may contain semen from gravity drainage
Collect clothes when…
Bedding/linens collected by police at scene
Christian (2000) Medical record review of 273 <10 yr old children who had ED exams and forensic evidence collected and processed between in Philadelphia
Findings
Majority of forensic evidence (64%) found on clothing and linens
Beyond 24 hr, all forensic evidence was found on clothing and linens
Only 35% of children had clothing collected
Recommendation: Remember to collect clothing

16. Step 1: Clothing Collection
Wear gloves throughout clothing collection
Use gloves throughout physical exam
Prevents contamination of evidence with examiner’s DNA

17. Step 2: Clothing Collection
Place 2 large pieces of paper on the floor for the patient to stand on while undressing
Can use exam table paper
Top paper will catch any debris or fibers that fall from patient while undressing undressing

18. Step 3: Clothing Collection
Patient removes shoes
…and steps onto paper

19. Step 4: Clothing Collection
Examiner inspects clothing while still on patient for…
Stains
Debris
Damage (tears, missing buttons, etc.
Examiner asks if due to assault or not, and records

20. Step 5: Clothing Collection
Patient removes clothing item by item
Patient hands to examiner who places in labeled paper bag

21. Step 6: Clothing Collection
Patient continues to remove clothes over the paper
Patient puts on gown and steps off paper

22. Step 7: Clothing Collection
Over the paper, examiner inspects for dry or wet stains
Should avoid excessive handling
Never use plastic bags
Biological evidence will degrade

23. If Clothing is Damp…
Fold item loosely to avoid transferring damp area to another area of item
Don’t fold clothing across stains
Damp areas will dry in paper bags

24. If Clothing is Wet… Wrap wet evidence in paper and place in bag
Even in plastic bag
Mark bag clearly
Notify law enforcement if there are “wet evidence” bags
Law enforcement will dry wet evidence before impounding it
If left wet, biological degradation occurs
The examiner may choose to use a plastic bag to place very wet evidence in so it will not leak through a paper bag and contaminate other evidence.
If a plastic bag is used, the transporting officer should be advised that the contents need to be dried as soon as possible and then stored in a paper bag. Criminalists will know this but transporting officers do not always.

25. Step 8: Clothing Collection
Seal bag: Fold top over.
Tape should go over the fold and around the other side.
Label outside of bag with…
patient’s name
examiner’s name
date/time
contents
Write initials across seal
This procedure is used because after sealing, it is not possible to tamper with the evidence without notice.

26. Step 9: Clothing Collection
Carefully fold the top sheet of paper into a “bindle”
Seal and label the bindle.
It may contain debris
from the clothes.

27. Step 10. Clothing Collection
Discard the bottom sheet of paper
It will be contaminated with debris from the floor

28. Clothing Collection: Finished!
Place the bindle and the individually-bagged garments into a large paper bag
The large paper bag usually does not need to be sealed. It is just used to carry all the smaller bags.

29. Other Collection Techniques

30. Head Hair and Fingernail Scrapings
Head hair reference sample: Pluck/cut hairs close to the scalp showing variations in length and color
Fingernail scrapings: Suspect skin cells, body fluids, semen
Use clean toothpicks
Separate bindles, R or L
If no fingernails, swab fingers down to knuckles
Head hair is collected from the victim as a reference sample. Most forensic labs allow the hair to be cut close to the scalp rather than plucked.
Fingernail scrapings are inportant if the victim scratched the suspect and may have skin cells under the nails.
Specimens may be placed in prelabeled envelopes or bindles. Instructions for bindle on next slide.

31. Instructions for Making a Bindle
7. Fold in half. Seal the open end of the bindle. Initial over the tape.
Lable contents.
A bindle is a simple method of folding paper to create a collection packet for evidence (debris, vegetation, other)
Training activity: pass out pieces of paper so participants can practice making a bindle.

32. Instructions for Collecting Swabs
For wet stains, use dry swabs. For dry stains, use swabs moistened with de-ionized water
Use 2 swabs. Rotate them to coat evenly with the available material
Use at least two swabs for each site
(Later, defense can request half the kit for tests by independent lab)
Pairs of cotton tipped swabs are the usual tools for collecting samples of possible DNS.

33. Instructions for Evidence Handling
Dry all wet swabs in a stream of ambient air for at least 60 minutes
Make bindles to hold debris of various kinds
Label all swabs and evidence collected:
As swabs are collected, they should be placed, tips up, in a prelabeled rack to ensure they do not become mixed-up as to site source or contaminated.
Patient name, dob
Date and time of collection
Contents
Examiner initials

34. Oral Cavity Evidence Swab areas in red
Purpose: Recovery of sperm, foreign material
Procedure
Collect 2 oral cavity swabs up to 12 hr post rape
Wet or dry secretions/stains around mouth
Any foreign material in or around mouth
Optional use of dental floss may recover sperm from between teeth
If an oral act occurred, the swab collection should proceed as soon as that history is obtained. Normal movement of tongue and lips (swallowing, speaking, etc) causes loss of evidence.
Patient may be thirsty or hungry by time of arrival at exam site; collect oral cavity specimens first. Then patient is permitted to eat, drink.

35. Alternate Light Source: Locating Body Fluids Dried on the Skin
Scan entire body with alternate light source. Identify areas of fluorescence
Remove with water-moistened swab
Remember: Substances other than semen and saliva fluoresce
An ultraviolet light may be used to scan the body. The room needs to be darkened in order for potential semen dried on the skin will show up. The fluorescent material is then removed with moistened cotton-tipped swabs.

36. Removing Fluorescent Material2 3 1
Arm - dried secretion not visible
Arm – dried secretion fluoresces under UV light
As fluorescent area is removed with moistened swab,
fluorescence transfers to swab

37. Swabbing for Saliva
Collect areas of possible saliva using swabs moistened with deionized water
Bite marks
Suction marks (hickeys)
Per patient history
DFSA

38. Genital Exam Sequence for Teen Girls
Scan ext. genitalia w/alt. light source
Cut matted pubic hair
Pubic hair combing
Inspect vulva and vestibule: Collect external specimens, debris
Apply TB dye
Speculum exam of vagina. Collect specimens.
Collect endocervical specimens
Document injuries in all areas as you proceed specimens
Examination of adolescent girls is the most extensive and has more steps than prepubertal girls or boys or adolescent boys. This slide lists all the steps in the order they are best accomplished.
Subsequent slides describe evidence collection for each in more detail.

39. Pubic Hair Collection: 3 Parts
Cut out matted hair or crusted material
Put in a bindle
Brush/comb hair downward over clean paper under victim’s buttocks
Fold paper with comb inside to make a bindle
Reference standard
20-30 hairs are required from random areas - Pluck/Cut as close to skin as possible
Label per general instructions
#2 will capture any loose hairs, fibers, debris, etc., that have been transferred from the crime scene or from the assailant as previously discussed
If pubic lice are noted on the victim, collect and submit. They may be able to be linked to the suspect by DNA.
Criminalists prefer plucked hair. Cut hair can be submitted. If plucked hair is critical, it can be obtained later.

40. Collect Debris, if present from Vulva, Vestibule
Inspect carefully
Photograph in place
Collect debris, vegetation, hairs, fibers using post-its, tweezers, swabs
Place in a bindle
Label
Debris can sometimes be used to corroborate the location of the sexual assault.

41. Collect 2 swabs each from the vulva and vestibule
Swab widely – include perineum

42. Collection from Vagina
Insert speculum into vagina
If no speculum, can collect blind
Collect foreign material, if present
Tampon, condom, hair, vegetation
Photograph, document
Collect 4 swabs from the vaginal pool
Preferred: All swabs at same time. Rotate to coat evenly. If not all at one time, note how done
Label swabs - code by number. Noting code number, prepare one dry mount and one wet mount slide
Only water should be used to moisten the speculum since lubricant may affect the evidence
When examining for injury, inspect for the presence of visible evidence
The vaginal pool is in the posterior fornix – red X marks the spot.

43. Wet Mount Slide for Motile Sperm Detection
Alternate methods
When the speculum is removed from the patient’s vagina, add a drop or two of normal saline, agitate slightly to mix, dump the liquid onto the slide and cover with a cover slip
- or -
When the swabs are removed from the vagina, take
one and roll it on the slide. Add a drop or two of
normal saline to the slide and place a cover slip over
the specimen.
Examine immediately for motile/nonmotile sperm. Submit with other evidence.

44. Specimens from Cervix Obtain two swabs from endocervix
Make sure to label “cervix”
Not possible unless
cervix is visualized

45. Looking for Sperm Sperm recovery confirms sexual contact
Stained slides (crime lab) will detect sperm more often than the wet mount slide
Wet mount only opportunity to see motile sperm
Sperm survive longer in cervical mucus than in vaginal pool

46. Sperm Recovery in Living Victims
Body cavity
Motile Sperm
Non-motile
Vagina
6-28 h
14 h – 10 d
Cervix
3-7.5 d
7.5 – 19 d
Mouth -
2 – 31 h
Rectum
4 – 113 h
Anus
2 – 44 h
Data refers to adolescents and adults, not prepubertal children. Data referencing other anatomic sites (mouth, rectum, anus) are from adult/adolescent studies, but may be applicable to children.

47. Microscopic Examination
Focus, using a low power, 10-x
Using a phase contrast microscope, change to 40+x
Sperm motility decreases quickly with time and removal from vagina
Without removing the cover slip, submit slide with evidence kit
Phase contrast microscope

48. Sperm visualized with the aid of Christmas tree stain
This slide is included just for another opportunity to see the morphology of sperm. The color is due to the stain used by the forensic lab. It is typical that the forensic will be able to detect sperm about three times more often than the medical provider because the stain makes the sperm more visible.
The wet mount exam by the medical provider is, however, the only opportunity to see motile sperm.
Sperm visualized with the aid of Christmas tree stain
Prepared by the forensic laboratory

49. Prepubertal Female Genital Exam Sequence
Alternate light source
No pubic hair collection
Swabs
Vulva – swab widely
Vestibule, if child cooperative
Be careful not to touch the hymen
Intravaginal ? – No need. If child goes to the OR for repair, intravaginal specimens can be collected under anesthesia
Rest – same as teen
This describes the usual sequence of collection for the prepubertal girl.
Intravaginal swabs are not generally attempted in prepubertal girls
If there is no hymen injury, it is not likely that, even if ejaculation occurred, sperm would be exclusively intravaginal. The hymenal opening is so small c/w the size of the penis that semen is more likely to be external and will be recovered from the vulva and vestibule swabs.
Attempting intravaginal swabs is likely to cause the child pain because it is difficult not to touch the hymen when inserting swabs.

50. Male Genital Exam Sequence
Prepubertal and adolescent same except for pubic hair collection.
Alternate light source exam
Collect 2 swabs, moistened with deionized water from...
Penis, both glans and shaft
Include coronal sulcus and base of penis
Include foreskin if it retracts
No swab from urethral meatus
Scrotum
Remind participants not to retract the foreskin if it doesn’t retract. Often the victim can be asked to retract the foreskin so the examiner can do the swabbing.
Observe that there is no requirement or recommendation to take urethral swabs.

51. Anorectal Evidence Collection
Same procedure for male and female
Inspect and collect, if present...
Moist/dry secretions, foreign material
Clean the perianal area with 3 water-moistened 2x2s, back to front, prior to next step. Discard.
Use buttock traction to expose anus to avoid contamination.
Routinely collect 2 anal canal swabs by inserting the swabs cleanly into the anal canal.
If anoscopy indicated (teens only), collect 2 swabs from rectal mucosa through anoscope
Contamination of anal specimens may occur if vaginal secretions run down the perineum onto the anal folds. If this occurs, positive anal specimens cannot support a sodomy charge. That’s the reason for cleaning the anus prior to collecting the swabs.
Collect even if history of assault does not include anal contact. Victims are often reluctant to report anal contact.
When anoscopy is performed, obtain swabs by touching the swabs to the rectal mucosa above tip of instrument

52. Collect perianal debris using Post-its or
a swab to brush debris into a bindle

53. Record all evidence collected…
Documentation
On diagrams and report
Clothing
Foreign materials, secretions on body
Oral, genital, and anal samples
Record results of vaginal wet mount
Toxicology
Reference samples
Record all evidence collected…

54. Recommendations for Storage
Storage of Collected Evidence
Biological evidence once dried should be stored room temperature, refrigerated or frozen – follow crime lab recommendations
Swabs and slides
Trace evidence
Reference samples
Blood and urine should be refrigerated
Do not freeze liquid blood samples
Clothing should be stored in a dry area
Storage recommendations will be influenced by local humidity and location of storage. It is critical to discuss with the forensic lab.

55. Chain of Custody
Definition: chronological documentation showing the collection, custody, control, transfer, analysis, and disposition of physical evidence
Medical provider collects, labels, and packages evidence specimens from the victim
Law enforcement signs for and receives the evidence packet
Crime lab signs for and receives the evidence packet from law enforcement

56. Questions?

57. References
De Jong, A. R. (2011). Tests used to analyze forensic evidence in cases of child sexual abuse and assault. Child Abuse and Neglect: diagnosis, Treatment and Evidence. C. Jenny, Saunders: Elsevier:
(2001). California Medical Protocol for Examination of Sexual Assault and Child Sexual Abuse Victims. Sacramento, CA, California Emergency Management Agency