Confirmatory tests for Semen

Confirmatory tests for semen detection are used to provide more definitive and accurate results and reduce the chances of false positives or false negatives. In this chapter, we will discuss some of the most commonly used confirmatory tests for the detection of seminal stains:

• Kernechtrot Picoindigocarmine (KPIC) Staining or Christmas tree Staining

• Gram Staining

• Prostate-specific antigen (PSA) Test or P30 Test

1. Kernechtrot Picoindigocarmine (KPIC) Staining

Figure: Kernechtrot-Picroindigocarmine (KPIC) staining for detection of semen

Sperm are a unique and specific component of semen, and their presence is a reliable indicator of the presence of semen in a sample. Utilizing staining techniques during microscopic examination enhances the visualization of sperm cells.

Kernechtrot-Picroindigocarmine (KPIC) stain, also known as the ‘Christmas Tree stain’, is used to identify spermatozoa in forensic laboratories. The name ‘Christmas tree’ comes from the colour of the two dyes used in the process: Nuclear Fast Red (stains red) and picro-indigo carmine (stains green).

Preparation of Reagents:

Nuclear Fast Red solution: Dissolve 2.5 g aluminium sulfate in 100 ml hot water. To it, add 50 mg of Nuclear Fast Red dye.

Picro-indigo carmine solution: Dissolve 1 g of indigo carmine dye in 300 ml of saturated picric acid solution.

Procedure:

1. Take 1-2 drops of aqueous extract of the seminal stain and prepare a smear on a microscope slide. Gently heat the slide to 55-60°C for 1-2 minutes to fix the stain.

2. Add 1-2 drops of Nuclear Fast Red solution to slide and allow to stand for 5 mins.

3. Rinse the slide with deionized water.

4. Add 1-2 drops of Picro-indigo carmine solution to the slide and allow it to stand for 30 seconds.

5. Rinse the slide with absolute ethanol.

6. Observe under a microscope at 40x and 100x.

   Observation: The head of the sperm will appear red-stained with a slight pink cast. The staining intensity of the acrosomal cap will be less than the nuclear portion of the sperm head. The midpiece and tail will be stained green.

2. Gram Staining

Figure: Gram staining for the detection of semen

Gram staining was first introduced by a Danish bacteriologist, Hans Christian Gram in 1882 to differentiate and categorize different bacterial species. However, the two dyes used in gram staining i.e. crystal violet and safranin can also be used to stain sperm cells for better visualization. Crystal violet is a basic dye that stains acidic structures, such as the DNA in the sperm nucleus; and safranin is an acidic dye that stains basic structures, such as the cytoplasmic proteins. By staining sperm cells with both dyes, the morphology and structure of the sperm cells can be visualized more clearly under the microscope.

Preparation of Reagents:

Crystal violet solution: Dissolve 0.5 g of crystal violet powder in 100 mL of distilled water and mix well.

Gram’s iodine solution: Dissolve 0.5 g of iodine crystals in 10 mL of distilled water. Add 1 g of potassium iodide to the solution and mix well. Allow the solution to sit for a few minutes to ensure that the iodine and potassium iodide have fully dissolved.

Safranin: Dissolve 0.5 g of safranin powder in 100 mL of distilled water and mix well.

Procedure:

1. Take 1-2 drops of aqueous extract of the seminal stain and prepare a smear on a microscope slide. Gently heat the slide to 55-60°C for 1-2 minutes to fix the stain.

2. Flood the slide with Crystal violet and allow it to stand for 1 min.

3. Rinse the slide with water to remove the excess stain.

4. Flood the slide with Gram’s iodine solution and allow it to stand for 1 min.

5. Rinse the slide with water to remove excess iodine.

6. Decolorize the slide by flooding it with 95% ethanol for 10-15 seconds.

7. Rinse the slide with water to remove excess ethanol.

8. Flood the slide with safranin and allow it to stand for 1 minute.

9. Rinse the slide with water to remove excess safranin.

10. Air dry the slide and observe under the microscope at 40X and 100X.

    Observation: The head of the sperm cells will be stained a deep blue-purple colour, while the midpiece and tail will be stained a pinkish-red colour.

Does the absence of sperm cells mean that the sample is not semen? 🤷🏻‍♀️

The absence of sperm cells in a sample does not necessarily mean that the suspected stain is not semen. Sperm make up only a small percentage of semen, and the absence of sperm could be because of one of the following conditions:

1. Azoospermia: also known as Aspermia, is a condition in which there is no sperm present in the semen.

2. Oligospermia: is a condition characterized by a low concentration of sperm in the semen, typically under 20 million sperm per milliliter of semen.

3. Vasectomy: A vasectomy is a surgical procedure that involves cutting and sealing the vas deferens, the tubes that transport sperm from the testicles to the urethra. In such cases, the amount of sperm present in the semen drops to a very low level or undetectable level.

3. Prostate-specific antigen (PSA or P30) Test

PSA (also known as P30, because of its molecular weight of 30 kDa) is a major protein present in seminal fluid in the concentrations of 0.5–2.0 mg/mL. PSA is produced in the prostate gland and secreted into the semen. It can also be found in urine, faecal material, sweat, and milk in small quantities. Over the years, several methods have been employed to detect PSA, including immunodiffusion, immunoelectrophoresis, ELISA, and immunochromatographic assays.

📌 Read more: ELISA, Immunodiffusion Assays, Immunoelectrophoretic Assays, Immunochromatographic Assays.

Since the presence of sperm cells cannot be the only indicator to confirm the suspected stain as semen, a forensic analyst typically uses a combination of chemical tests such as the PSA test in addition to microscopic identification of sperm cells.

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