Should p30 Testing be Considered a Confirmatory Test for the Presence

of Semen?

Rhonda Williams, PhD, F-ABC

Kevin MacMillan, MS, F-ABC

Association of Forensic DNA Analysts and Administrators 2011 Summer Meeting

PSA

• Prostate Specific Antigen– kallikrein III

– seminin

– semenogelase

– γ-seminoprotein

– p30 antigen

• 34kD glycoprotein (Serine protease enzyme)

Concentration of PSAin human body fluids

Fluid PSA (ng/mL)

semen200,000 to5.5 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

Time after firstejaculation (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

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

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, skeneglands, 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:

Anal swabs Terminated (no DNA)

Vaginal swabs (Consistent with

complainant,no male DNA)

What to do next?

• Anal swab slide examined

• Cut another anal swab

• Send directly to DNA

• Save the PSA from extraction to test after extraction

• Look at differential slide

(No sperm observed)

(PSA positive)

(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:

• Vasectomizedand/or Azoospermic?

• Pre-pubescent(12-16 yrs-male)

• False positive P30 results

(Breast milk, urine, cancer)

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

(WebMD, 2010)

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/ulDNA, 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.html

Prostate-Specific Antigen (2009), retrieved on Oct. 18th, 2010, from

http://emedicine.medscape.com/article/457394-overview#section~CharacteristicsofProstateSpecificAntigen

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. Immunoreactiveprostate-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. ImmunoreactiveProstatic 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