Association of Forensic DNA Analysts and Administrators 2011 Summer Meeting

Should p30 Testing be Considered a Confirmatory Test for the Presence of Semen? Rhonda Williams, PhD, F-ABC Kevin MacMillan, MS, F-ABC

PSA • Prostate Specific Antigen – – – – –

kallikrein III seminin semenogelase γ-seminoprotein p30 antigen

• 34kD glycoprotein (Serine protease enzyme)

Concentration of PSA in human body fluids Fluid

PSA (ng/mL)

semen

200,000 to 5.5 million

amniotic fluid

0.60-8.98

breast milk

0.47-100

saliva

0

female urine

0.12-3.72

female serum

0.01-0.53

http://mafs.net/pdf/forensicdetectionsemen3.pdf

Functions of PSA 1. Liquefies semen, allowing sperm to swim freely –

The gel formed by proteins (semenogelin) from the seminal vesicles is broken up and semen becomes more liquid Normally takes less than 20 minutes Abnormally long liquefaction (more than 30 minutes) may indicate an infection/infertility

– –

2. Helps in dissolving cervical mucous cap (allowing entry of sperm to achieve fertilization) –

Cervical caps used as contraceptive devices •

Prevents sperm entry to uterus

Semen development during puberty The first ejaculation in males often occurs about 12 months after the onset of puberty Janczewski, Z. and Bablok, L. (1985). "Semen Characteristics in Pubertal Boys". Archives of Andrology 15 (2-3): 199–205.

Time after first ejaculation (months)

Average volume (mL)

Liquefaction

Average sperm concentration (million sperm/mL)

0

0.5

No

0

6

1.0

No

20

12

2.5

No/Yes

50

18

3.0

Yes

70

24

3.5

Yes

300 http://en.wikipedia.org/wiki/Ejaculation

Where does female PSA come from? • The Skene's glands (also known as the lesser vestibular glands, periurethral glands, skene glands, U-spot, or female prostate) are glands located on the anterior wall of the vagina, around the lower end of the urethra • Thought to have similar function to male prostate -Biochemical properties (Wimpissinger, 2007) – Helps dissolve cervical mucous cap (promotes pregnancy)

Case Synopsis • Sexual Assault • 9 year old female • Pt. stated: “My brother touched my butt”

Evidence submitted: Vaginal swabs (PSA -, slide -) Oral swabs (PSA -, slide -) Anal swabs (PSA +)

DNA Results: Vaginal swabs (Consistent with complainant, no male DNA)

Anal swabs Terminated (no DNA)

What to do next? • • • •

Anal swab slide examined (No sperm observed) Cut another anal swab Send directly to DNA Save the PSA from extraction to test after extraction (PSA positive) • Look at differential slide (No sperm observed)

Next step:

• Send both cuttings for autosomal STR DNA typing • Send both cuttings for Y-STR DNA typing

(No male DNA detected)

(No male DNA detected)

Possible Explanations: • Vasectomized and/or Azoospermic? • Pre-pubescent (12-16 yrs-male)

Not likely (14-15 yrs old)

(Ding, Ding – maybe) (Medline, 2010) -no studies that assess PSA levels in tissues and secretions of pre-pubescent children

• False positive P30 results (WebMD, 2010) (Breast milk, urine, cancer)

Similar cases: • 14 yr old boy suspect, sister is complainant – Vaginal/anal swabs and panties PSA+ – No sperm detected – Y-STR gave partial profile ( maybe epithelial cells?)

• 3 male suspects, 27 yr old female – Vaginal and anal swabs PSA+ and sperm + – Oral swabs PSA+, no sperm or male DNA detected (re-cut and detected 0.009ng/ul of male DNA)

Other cases observed

Swab type/# cases

+ PSA results

Sperm observed

Male DNA

Vaginal (5)

NEG

No male DNA detected

Anal (9)

NEG

1 case with 0.008ng/ul DNA, sample terminated

Panties (1)

NEG

Case had 0.0138ng/ul DNA, partial profile

Similar cases: • Forens-dna has cases mentioned • No study/determination to date • Some labs require 1/100 dilution PSA to be positive • Some labs would report “seminal fluid detected” not “semen detected”

Interesting note • PSA levels show an increase with prostate cancer patients

Post-mortem study • A post-mortem study was performed on cadavers to analyze p30 levels in the rectum – 39 male/10 female cadavers tested with no history of sexual assault – 64% of males tested positive for p30, all females tested negative for p30 – Y-STR analysis on showed results consistent with cadaver (Lunetta, 2009)

Our lab policy • Due to the issue of false positive p30 results, our laboratory has taken the conservative approach by considering the p30 test as a presumptive test – The statement used is: “Presumptive test(s) for semen was/were positive for item___; however, the presence of semen could not be confirmed.”

References: Puberty, (2010) , retrieved on Oct. 18th, 2010 from, http://www.nlm.nih.gov/medlineplus/puberty.ht ml Prostate-Specific Antigen (2009), retrieved on Oct. 18th, 2010, from http://emedicine.medscape.com/article/457394overview#section~CharacteristicsofProstateSpecif icAntigen

References: PSA • Steven P. Balk, Yoo-Joung Ko, Glenn J. Bubley (2003). "Biology of Prostate-Specific Antigen”. Journal of Clinical Oncology 28 (2): 383-91. PSA in postmortem cadavers • Lunetta, P, and Sippel, H. (2009), “Positive prostate-specific antigen (PSA) reaction in post-mortem rectal swabs: a cautionary note”. Journal Forensic Leg. Med. 16(7):397-9.

References: Breast Tissue and Tumors • Papotti M, Paties C, Peveri V, Moscuzza L, Bussolati G. Immunocytochemical detection of prostate-specific antigen (PSA) in skin adnexal and breast tissues and tumors. Basic Appl Histochem 1989, 33(1): 25-9. • Yu H, Diamandis EP, Sutherland DJA. Immunoreactive prostate-specific antigen levels in female and male breast tumors and it’s association with steroid hormone receptors and patient age. Clin Biochem 1994, 27:75-79.

References: Periurethral glands • Frazer HA, Humphrey PA, Burchette JL, Paulson DF. Immunoreactive Prostatic specific antigen in male periurethral glands. J Urol 1992, 147: 246-248. • Iwakiri J, Grandbois K, Wehner N, Graves HC, Stamey T. An analysis of urinary prostate specific antigen before and after radical prostatectomy: evidence for secretion of prostate specific antigen by the periurethral glands. J Urol 1993 Apr; 149(4): 783-6. • Pollen JJ, Dreilinger A. Immunohistochemical identification of prostatic acid phosphatase and prostate specific antigen in female periurethral glands. Urology 1984 Mar; 23(3): 303-4.

References: Breast Milk •

Yu H, Diamandis EP. Protease prostate specific antigen in milk of lactating women. Clin Chem 1995 41:54-60.

Amniotic Fluid •

Yu H, Diamandis EP. Prostate specific antigen immunoreactivity in amniotic fluid. Clin Chem 1995 41:204-210.

Female Urine

•

Breul J, Pickl U, Hartung R. Prostate-specific antigen in urine. Eur Urol 1994, 26(1): 18-21.

Prostate Cancer •

Kim, HW, Ko, YH, Kang, SH, and Lee, JG. Predictive Factors for Prostate Cancer in Biopsy of Patients with Prostate-Specific Antigen Levels Equal to or Less Than 4 ng/ml. Korean Urol. 2011 Mar;52(3):166-71.

Acknowledgements • Dr. Roger Kahn, F-ABC • Michael Donley, MS, F-ABC • Lisa Gefrides, MS, F-ABC