The Examination of Peroxidase-Positive Leukocytes in Semen

Male Reproductive Medicine is my primary research focus, and our protocol primarily addresses the issues of speed and cost in analyzing Peroxidase-Positive Leukocytes in semen. Semen is inherently heterogeneous, which really affects the stability of semen analysis results. Therefore, it is essential to ensure that semen is liquified and thoroughly mixed before examination.

Our protocol combines manual and automatic measures to calculate the concentration of leukocytes by utilizing sperm concentration, which saves time and is more cost effective compared to the grading counting leukocytes. To begin collect the reagents for preparing the working solution and place them at room temperature. Also, prepare an opaque bottle to store the working solution and protect it from light.

Then transfer 100 microliters of the ammonium chloride solution into the opaque reagent bottle using a 200 microliter transfer pipette. Also transfer 100 microliters of the sodium EDTA solution into the reagent bottle and mix by pipetting. Next, add 900 microliters of the ortho toluidine substrate solution into the opaque reagent bottle and gently mixed by pipetting.

Then transfer five microliters of the hydrogen peroxide solution to the reagent bottle. Using a 1000 microliter transfer pipette, gently mix the solution by pipetting. To prepare the semen samples turn on the constant temperature stand and heat it up to 37 degrees Celsius.

Also, tare the weighing scale for the weight of the polymer based receptacle. Upon receipt of the semen sample, weigh the sample and record detailed information such as identification code, collection time, abstinence duration on the outer wall of the polymer based receptacle. Next, place the semen sample on the constant temperature stand for semen liquefaction.

Draw the semen sample into a polymer based pipette and check for liquefaction every five minutes for 30 minutes until the semen sample is fully liquified. To begin, liquefy the collected human semen samples and thoroughly homogenize them using a Pasteur pipette. Then transfer 100 microliters of the semen sample into a centrifuge tube.

To this tube, add 900 microliters of the staining solution. Repeatedly mix by pipetting and thoroughly homogenize the reactant. Place the tube on a two dimensional shaker for continuous agitation at 200 RPM for 30 minutes at room temperature.

While waiting for the standing reaction, proceed for analysis of spermatozoa concentration using the CASA system. After turning on the system, double click the SCA scope icon to open the application software. Then mix the semen sample thoroughly by pipetting, and transfer up to 10 microliters of the sample on the SCA counting chamber.

Wait for 60 seconds for the semen sample to stop drifting. Next, place the SCA counting chamber onto the sample holder of the CASA system. In the application software Enter the patient's information in the dialogue box.

Click the start button to record the concentration of spermatozoa in the semen. Then stop the shaker when the 30 minute staining reaction is complete. Before analysis, mix the contents of the centrifuge tube by pipetting.

Transfer up to 10 microliters of the reactant into the sperm counting chamber and cover it with a dedicated glass cover slip. Place the sperm counting chamber onto the microscopic stage and turn on the optical microscope. Then wait for 60 seconds for the reactant sample to stop drifting.

Under 10x objective lens adjust the course and fine adjustment knobs to select a field of view. Then switch to 40x objective lens and observe the spermatozoa and peroxidase positive leukocytes. Use the electronic counter system to count out at least 200 spermatozoa and the number of peroxidase positive leukocytes in brown.

Using this technique, peroxidase positive leukocytes were stained in the semen sample. The peroxidase positive leukocytes displayed a brown hue contrasting with the unstained ordinary round cells.