Acid Phosphatase (AP) Semen Detection Test

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  • About Acid Phosphatase

    • Acid phosphatase is an enzyme secreted by the prostate gland that is present in large amounts in seminal fluid. It is not unique to the prostate and can be found in other biological fluids including vaginal secretions. It is therefore considered a presumptive chemical test for the presence of semen and semen must be confirmed by other means (sperm detection or PSA).

    • Prostatic Acid Phosphatase (PAP) is not a single enzyme but an array of related isoenzymes from a variety of sources. The PAP assay is a well-documented presumptive assay for the presence of semen. 

    • Acid phosphatase activity is 50-1000 times greater in human semen than in any other bodily fluid.  The use of acid phosphatase as a marker for semen is compromised because the vagina is also a source of vaginal acid phosphatase.  Since seminal and vaginal acid phosphatase can not discriminate, the only approach to differentiating semen in vaginal secretion is by quantitative analysis such as PSA or the microscopic inspection for sperm.

    • Finding a significantly elevated acid phosphatase level is consistent with the presence of semen.  For example, if semen is present the acid phosphatase assay is normally very robust.

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Frequently Asked Questions

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  • Acid Phosphatase Test Standard Crime Lab Process (Reference 11)
    • Safety Considerations
      • Glacial acetic acid - Caution! Corrosive! Flammable!
      • Sodium acetate - Caution! Irritant!
      • Sodium a-naphthyl acid phosphate - Caution! Irritant! Emits toxic fumes under fire conditions!
      • Dianisidine (Naphthanil diazo blue B) - Caution! Highly toxic! Emits toxic fumes under fire conditions!
      • Naphthanil diazo red - Caution! Avoid contact and inhalation! Emits toxic fumes under fire conditions!
    • Equipment
      • 5 ml and 500 ml Graduated cylinders
      • Balance
      • Spatula
      • Scissors
      • Tweezers
    • Materials
      • Filter paper or microtiter plate (optional)
      • Weigh boats or weigh paper
      • Cotton swabs
      • Test tubes or bottles
      • Disposable transfer pipets or droppers
    • Working Solutions
      • Acid Phosphatase (AP) Buffer
      • • 2.5 ml Glacial acetic acid • 10.0 g Sodium acetate (anhydrous) • 450.0 ml Distilled water • Mix the above ingredients until thoroughly dissolved.
        • Storage
          • The AP Buffer is stable at room temperature.
        • Labeling
          • Label the bottle as AP Buffer with a lot number (the date of preparation followed by the initials of the person preparing the stock solution). Example: AP Buffer Lot Number 100899JD was prepared by Jane Doe on October 8, 1999.
          • There is no expiration date (see 3.1.5 Minimum Standards and Controls).
      • Sodium a-Naphthyl Acid Phosphate Solution
        • Add a small amount (approximately 4 mg) of sodium a-naphthyl acid phosphate to approximately 3 ml of Acid Phosphatase buffer in an appropriately labeled 10 X 75 mm test tube or bottle.
        • Discard the solution at the end of the day.
      • Dye Solution
        • Add a small amount (approximately 4 mg) of o-dianisidine or naphthanil diazo red to approximately 3 ml of buffer in an appropriately labeled 10 X 75 mm test tube or bottle.
        • Discard the solution at the end of the day.
      • Distilled water
    • Minimum Standards and Controls
      • On the day of use a positive reagent control (known semen stain) and a negative reagent control (distilled water) must be tested to ensure that the reagents are working properly. The results of this testing must be documented in the case file.
      • If either control does not give the expected result, do not proceed with testing evidence samples until the problem has been resolved as demonstrated by testing another set of positive and negative reagent controls and achieving the expected results with both controls.
      • If the results of the test are positive, a substrate control (if available) must also be tested, unless the stain is on a cotton swab, and the results of the testing documented in the case file. It is not necessary to test submitted control swabs.
    • ACID PHOSPHATASE (AP) TEST PROCEDURE (Sodium a-Napthyl Acid Phosphate)
      • Moisten filter paper/swab with distilled water. (Do not use buffer solution, as this will contaminate the stained area.) Press the filter paper against the suspected stain or gently rub the stained area with the moistened swab. Alternatively, a small piece of the stain/swab can be placed on filter paper, in a small test tube, or in a microliter plate. Treat the substrate control in the same manner.
      • Add 1-2 drops of sodium a-naphthyl acid phosphate solution.
      • Add 1-2 drops of dye solution.
      • The development of a blue/purple color with o-dianisidine or an orange/red color with naphthanil diazo red within 10 to 15 seconds is indicative of acid phosphatase levels in the semen range.
      • The presence of semen in all samples exhibiting an inconclusive result or a positive result must be confirmed by identifying spermatozoa or, in the absence of spermatozoa, p30.
      • Interpretation
      • Positive Reaction = Blue/purple color with o-dianisidine within 10 to 15 seconds OR
      • Orange/red color with naphthanil diazo red within 10 to 15 seconds
      • Negative Reaction = No color development, slight/slow color development
      • Inconclusive Reaction = Slow moderate to strong color development


  • Ohio Bureau of Criminal Identification Laux report on Acid Phosphatase Testing
    • Conclusion  Testing for acid phosphatase remains a valuable presumptive test for the screening of swabs collected from sexual assault survivors and for the testing of stains found on clothing and bedding. The experienced forensic biologist knows that all stains that fluoresce are not necessarily semen and all semen stains do not fluoresce. In addition, semen is a heterogeneous fluid and portions of a deposited stain will contain various levels of acid phosphatase, P30 and spermatozoa. Examination of a pair of panties with an alternate light source and extraction of all the stains that fluoresce followed by psa analysis may yield semen, however, it may not, and it does not appear to this author to be the best use of time and expenses. Acid phosphatase mapping is an inexpensive and quick method for screening such stains. Years ago, forensic biologists (serologists) were taught what was termed “a systematic approach to the analysis of semen evidence” developed by Blake, Sensabaugh and Bashinski 6. The three major steps consisted of locating the stain, estimating the amount of semen found and genetic analysis of the stain. With the advent of DNA, it seems possible that one could just cut a stain from a pair of underwear, extract it and generate a DNA profile. Obtaining the subject’s DNA profile on the underwear, where it shouldn’t be, should be conclusive proof of guilt. And perhaps it is. However, this analyst, trained in the “old school” feels that a more thorough analysis is warranted. Acid phosphatase mapping in locating stains and sperm quantification of positive stains are important steps that can only aid the DNA analyst in interpreting the results. It behooves the forensic biologist to utilize all of the methods available for optimum semen detection