I. Acid Phosphatase Test

Acid phosphatase (AP) is found in high levels in semen and originates from the epithelial cells where it is secreted into the prostate gland. The level of AP activity is 500 to 1000 times higher in human semen than in any other normal body fluids or secretions (1). It has been amply demonstrated that elevated levels of AP activity persist in the vaginal pool after sexual intercourse and in semen stains. Thus the detection of strong AP activity is considered a fairly reliable indicator of the presence of semen (2 – 20).

AP reagent is an enzymatic presumptive test which is used to indicate the possible presence of semen. AP cleaves the phosphate from the alpha-napthyl phosphate and the phosphate binds with Brentamine Fast Blue B to yield a purple azo-dye.

Alternative Light Sources (ALS) may be used to screen large items of evidence which may contain biological fluids. The shorter wavelengths induce molecular excitations in body fluids (excluding blood) and the subsequent emission of photons (fluorescence). Filter goggles (frequency selective) may be necessary to block extraneous emitted wavelengths (21).   

Fluorescence is the emission of radiation (esp. visible light) by a substance, typically during exposure to external radiation (lasers, X-rays, UV light). The substance absorbs light and re-emits the light in wavelengths longer (lower energy) than that of the illuminating source. The mechanisms of absorption and emission are based on transitions of quantum electron energy levels. The energy difference between the absorbed and emitted photons ends up as molecular vibrations or heat.

Various naturally occurring chemicals present in body fluids such as semen, as well as latent fingerprints, may fluoresce under appropriate lighting conditions. Fluorescent body secretions include semen, saliva, and sweat (perspiration). Constituents of significance to the forensic scientist include various fluorescent enzymes and proteins contained in body fluids.

Original references to fluorescence in cells of human spermatazoa resulted from tests exhibiting visible fluorescence from the Y chromosome when stained or treated with quinacryne (22). Thus, semen contains a chemical in the Y chromosome that will fluoresce when stained with quinacryne. Normal semen also contains small amounts of a fluorescent enzyme called choline (a nutrient, essential for cardiovascular and brain function, and cellular membrane composition and repair).

Moreover, as evidenced by the Woods Lamp, or black light technique (23), semen responds to illumination by longer wavelength frequencies of UV light (~ 350 nm) which is invisible to the human eye. When the substance is illuminated, it absorbs the energy and exhibits luminescence at a lower energy (longer wavelength) frequency of visible blue light. The advantage of this is that you can make invisible semen stains appear visible to the human eye.

In addition, excitation and emission spectra of untreated dry semen (24) indicate clearly that there is an alternative to using UV radiation when searching for semen stains. The primary features of these data are the following:

1) Under standard conditions of visible light (500 nm) illumination, untreated dry semen has a broad band of emission from 350 – 400 nm, just below the range of visibility to the naked eye.

2) Long wavelength UV (350 nm) illumination of untreated dry semen produces a more narrow band of emissions centered near the blue visible region.  

3) Illuminating dried semen with a band of visible (450 nm) light produces strong visible fluorescence in a broad region with a maximum around 520 nm (orange).   

Using a UV light source, one can readily see the fluorescent emissions. Using visible light sources (e.g. blue and green), a filter is needed to block the light from the source so that the fluorescent emissions may be viewed selectively. Viewers typically wear barrier goggles (orange, yellow or red) and the lens of any CCD (charge-coupled device) camera used to record the evidence must be similarly equipped with appropriate filters.

Acid phosphatase is an enzyme that is secreted by the prostate gland into seminal fluid. Its concentrations in seminal fluid are up to 400 times greater than those found in any other body fluid. 4-methyl umbelliferyl phosphate (MUP) will fluoresce under UV light when it comes in contact with acid phosphatase (25).

The above figure clearly illustrates the advantages of using 6,8-difluoro-methyl umbelliferyl phosphate (DiFMUP) over the standard MUP substrate (26).

II. Alternative Light Sources: LED’s

It is generally held that the shape of the emission spectrum is independent of the color used to excite it. What does vary with excitation color is the brightness (or intensity) of the fluorescence. A plot of the fluorescence intensity versus excitation color is called a Fluorescence Excitation Spectrum.

Variations in fluorescence intensity across the excitation spectrum are mainly caused by differences in absorption. These are characteristic of the fluorescing molecule and can be used to identify unknowns. Thus, the two principle design parameters are:

1) Varying excitation wavelength

2) Resolving emission wavelength.

The objective is to tailor the excitation and emission spectra of an ALS optimized toward a very specific function: detecting semen stains on white cotton fiber (or fabric). In this case, the goal is not the brightest fluorescence of the target -- but rather but the greatest contrast between target (semen) and background fabric. (27)

Most cotton fabrics are bleached with fluorescent brighteners. These fluorescent compounds cause the fabric to effectively emit more visible light than is incident by converting some of the invisible incoming ultraviolet into visible outgoing white fluorescence. Unfortunately, these compounds fluoresce so brightly that they can mask the fluorescence of any stains on the fabric. Similar fluorescent compounds are also used to brighten paper.

We can improve the contrast with which semen stains are seen on white fabric by tailoring both the excitation and emission spectra to the job. First, we note that the faint yellow color in dried semen is due to a significant blue absorption. Bleached cotton fabric on the other hand exhibits a decrease in the fluorescence intensity when excited in the visible blue compared to the UV. So we can preferentially excite the semen fluorescence over the cotton by exciting with blue light rather than UV. This improves the contrast.

A further improvement can be achieved by noting the different emission spectra of fabric and semen (27). The blue excited fabric has a wide emission spectrum extending from the blue-green through the red, while the blue excited semen stain emits mostly yellow fluorescence. 

Stoilivic (24) describes the use of the 'Polilight', a light source based on a xenon arc lamp, to exploit the fluorescent properties of semen as an aid to searching fabrics for stains in sexual assault cases. The broad excitation spectrum of semen allows the fluorescence to be generated at a range of wavelengths. This method permits the excitation and emission conditions to be selected that minimize interference from background fluorescence of the fabric and thereby optimizes the contrast between the fabric and the stain.

Thus, if the fabric has a four times broader emission spectrum than the semen, we can get a contrast enhancement of four by viewing the fluorescence though a filter that passes only yellow light. For both natural and synthetic fibers, a balance

between efficiently exciting fluorescence in the fiber while minimizing the excitation of background fluorescence usually leads to the blue. The most power efficient and cost effective means of generating blue light is by use of Light Emitting Diodes (LEDs), which (unlike lasers) are non-resonant and can be made to emit at any color in the spectrum except brown. (28)

The typical emission spectral bandwidth of LEDs is ~ 25 nm. Because this is still considerably less than the width of the typical absorption peaks seen in room temperature liquids and solids, the fluorescing molecule does not see any difference between light from a monochromatic laser and that from a LED. One watt of green LED light excites just as much fluorescence as a one watt green laser.                    

III. PSA (p30) Test

Prostate-Specific Antigen (PSA or p30), a glycoprotein that is produced in the prostate gland, is a valid marker for detecting semen in evidence form criminal cases including samples deposited by vasectomized or azospermic individuals (29-30). It appears in the semen and urine of males, as well as the urine of females. Its detection is based upon a two-step reaction scenario:  

1) The joining of a mobile monoclonal antibody MAB/dye molecule that is specific for PSA with a PSA molecule that may be in the sample.   

2)  The joining of the MAB – PSA antigen complex to a polyclonal antibody molecule that is already attached to the test area. 

A pink colored band develops as the antigen complex molecules adhere to the polyclonal antibodies and accumulate in the test area.

Methods for the detection of PSA include Ouchterlony double diffusion, crossover electrophoresis, rocket immunoelctrophoresis, radial immunodiffusion and enzyme-linked immunosorbent assay, or ELISA (31–38). However, some of these procedures have low sensitivities and/or are cumbersome and time consuming to perform in forensic laboratories (especially when only a few samples are analyzed weekly).  

For clinical screening of elevated levels of PSA in serum that may indicate the presence of prostatic cancer, several sensitive membrane0based PSA tests have been developed and are commercially available. These tests are simple, relatively rapid to perform, require minimal equipment, and produce results that are easy to interpret. Furthermore, these tests offer the same sensitivity as ELISA-based tests. Thus PSA membrane tests may also prove useful as confirmatory tests for the presence of seminal fluids in forensic casework analysis.

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References & Reading

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1) Gutman, A.B. and Gutman, E.B.

“Quantitative Relations of a Prostatic Component (Acid Phosphatase) of Human Seminal Fluid”  

Endocrinology, Vol. 28, p.115 (1941)

2)  Riisfeldt, O.

“Acid Phosphatase Employed as a New Method of Demonstrating Seminal Spots in Forensic Medicine”

Acta Pathalogica st Microbiologica Scadinivia Supplementum, Vol. 58, p.1 (1946)

3) Kaye, S.

“Identification of Seminal Stains”

J. Crim. Law, Criminology & Pol. Sci., Vol. 38, p.79 (1947)

4)  Fisher, R.S.

“The Acid Phosphatase Test as Evidence of Rape”

New England Journal of Medicine, Vol. 240, p.783 (1949)

5)  Kind, S.

“The Use of the Acid Phosphatase Test in Searching for Seminal Stains”

J. Crim. Law, Criminology & Pol. Sci., Vol. 47, p. 597 (1957)

6)  Brakett, J.W.

“The Acid Phosphatase Test for Seminal Stains”

J. Crim. Law, Criminology & Pol. Sci., Vol. 47, p. 717 (1957)

7)  Pinto, F.C.

“Rape for the Defense of Presumptive Seminal Testing for Acid Phosphatase”

J. For. Med., Vol. 6, p.147 (1959)

8)  Enos, W.F., et al.

“A Laboratory Procedure for the Identification of Semen”

Amer. J. Clin. Pathology, Vol. 39, p.316 (1963)

9)  Kind, S.S.

“The Acid Phosphatase Test”

Methods in Forensic Science, p.267, John Wiley & Sons, NY (1964) 

10) Rupp, J.C.

“Sperm Survival and Prostatic Acid Phosphatase Activity in Victims of Sexual Assault”  

J. For. Sci., Vol. 14, p.177 (1969)

11)  Schiff, A.F.

“Modification of the Berg Acid Phosphatase Test”

J. For. Sci., Vol. 14, p.538 (1969)

12)  Godwin, I.D. and Seitz, G.K.

“Vaginal Acid Phosphatase”

Medical Annals of D.C., Vol. 39, p. 147 (1970)

13)  Walther, G.

“Acid Phosphatase: Significance in the Determination of Seminal Traces”

 J. For. Med., Vol. 18, p.15 (1971)

14)  Willott, G.M.

“L-Tartrate Inhibitable Acid Phosphatase in Semen and Vaginal Secretions”

J. For. Sci. Soc., Vol. 12, p.363 (1972)

15)  Wraxall, B. and Adams, E.

“Phosphatases in Body Fluids: The Differentiation of Seminal and Vaginal Secretion” 

Forensic Sci., Vol. 3, p.57 (1974) 

16) Davies, A. and Wilson, E.

“The Persistence of Seminal Constituents in the Human Vagina”

Forensic Science, Vol.3, p.45 (1974)

17)  Gomez, R.R., et al.

“Determination of Acid Phosphatase Activity in Vaginal Washings”

Amer. J. Clin. Pathology, Vol. 64, p.423 (1975)

18)  McCloskey, K.L., et al.

“Prostatic Acid Phosphatase Activity in the Postcoital Vagina”

J. For. Sci., Vol. 20, p.630 (1975)

19)  Findley, T.P.

“Quantitation of Vaginal Acid Phosphatase and Its Relationship to Time of Coitus”

Amer. J. Clin. Pathology, Vol. 68, p.238 (1977)

20)  Sensabaugh, G.F.

“The Quantitative Acid Phosphatase Test: A Statistical Analysis of Endogenous and Postcoital AP Levels in the Vagina.”

J. For. Sci., Vol.24  (1979)

21)  Auvdel. M.J.

“Comparison of Laser and UV Technology used in the Detection of Body Secretions”

J. For. Sci., Vol.32, p.362 (1987)

22)  Pearson, P.L., Bobrow, M., Vosa, C.G. & Barlow, P.W.

"Quinacrine Fluorescence in Mammalian Chromosomes"

Nature (London) Vol. 231, p. 326 (1971)

23)  Woods, R.W.

“Communications Secretes au Moyen de Rayons Lumineux”

Journal de Physique (Theoretical and Applied) Vol. 9 p. 77 (1919)

24)  Stoilivic, M.

“Detection of Semen and Blood Stains Using Polilight as a Light Source”

Forensic Sci. Int.  Vol. 51, p.289 (1991)

25)  Robinson, D., Willcox, P.

“4-Methylumbelliferyl phosphate as a substrate for lysosomal acid phosphatase” 

Biochim. Phys. Acta., Vol.191, p.183 (1969)

26)  Gee, K.R., et al.

“Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases”

Anal. Biochem., Vol. 73, p.41 (August, 1999)

27)  H.J. Kobus, J. Scharnberg, E. Sileneiks 

“Improving the Effectiveness of Fluorescence for the Detection of Semen Stains on Fabrics” 

Journal of Forensic Science, Vol. 47, p.823 (2002)

28)  McGraw, D.

"New LEDs Enable Innovations in Forensic Alternative Light Sources"
Forensics Magazine, June/July (2005)

29)  Hara, M., et al.

“Some Physico-Chemical Characteristics of Gamma-Seminoprotein – An Antigenic Component Specific for Human Seminal Plasma” 

Jap. J. Leg. Med. Vol. 25 p. 322 (1971)

30)  Blake, E.T. and Sensabaugh, G.F.

“Genetic Markers in Human Semen: A Review”

J. For. Sci., Vol. 21, p.784 (1976)

31)  Blake, E.T. and Sensabaugh, G.F.

“Genetic Markers in Human Semen II: Quantitation of Polymorphic Proteins”

J. For. Sci., Vol. 23, p.717 (1978)

32)  Sensabaugh, G.F.

“Isolation and Characterization of a Semen-Specific Protein from Human Seminal Plasma: A Potential New Marker for Semen Identification”

J. For. Sci., Vol. 23, p.106 (1978)

33)  Poyntz, F.M., et al.

“Comparison of PSA and AP levels in Postcoital Vaginal Swabs from Donor and Casework Studies”

For. Sci. Int., Vol. 24, p.17 (1984)

34)  Graves, H.C., et al.

“Postcoital Detection of a Male Specific Semen Protein: Application to the Investigation of Rape”

New England J. Med., Vol. 312, p. 338 (1985)

35)  Stubbings, N.A. and Newall, P.J.

“An Evaluation of Gamma-Glutamyl Transpeptide (GGT) and PSA Determinations for the Identification of Semen on Postcoital Vaginal Swabs”  J. For. Sci., Vol. 30, p.604 (1985)

36) Kamenev, L., et al.

“An Enzyme Immunoassay for Prostate Specific PSA Antigen Detection in the Postcoital Vaginal Tract”

J. For. Sci., Vol. 29, p. 233 (1989)

37)  Stowell, L.I., et al.

“An Enzyme-Linked Immunosorbent Assay (ELISA) for Prostate-Specific Antigen”

For. Sci. Int., Vol. 50, p. 125 (1991)

38)  Hochmeister, M.N., et al.

“Evaluation of Prostate-Specific Antigen (PSA) Membrane Tests for the Forensic Identification of Semen”

J. For. Sci., Vol.44, p.1057 (1999)