LEUCOSCREEN^TM

Cytochemical Stain For Detecting
Granulocytes in Semen

(about 25 - 300 determinations)

FOR  RESEARCH  USE  ONLY
NOT FOR USE IN DIAGNOSTIC PROCEDURES

Principle:

This staining method detects the  presence of the enzyme peroxidase in cells.

A semen sample is mixed with a substrate specific for the enzyme peroxidase.  If peroxidase is present, it will  reduce the substrate, hydrogen peroxide.  At the same time, diaminobenzidine (DAB) will be oxidized to form an insoluble brown product:
                       peroxidase   
DAB + H₂O₂    ----------------> oxidized DAB (brown pigment)

Using a formula, it is possible to calculate the number of peroxidase-positive cells in each semen sample by knowing the  concentration of spermatozoa.

Reagents:

Buffer:   7 ml buffer, pH 7.4.  Ready to use.
DAB:  1 ml diaminobenzidine solution.  Ready to use. Warning: Diaminobenzidine is a possible carcinogen.
Peroxide:  1 ml hydrogen peroxide solution.  Ready to use.
Fixative:  12 ml dilute ethanol.  Ready to use.
Peroxidase:  0.5 ml peroxidase suspension.  Ready to use. 

Materials Required But Not Provided:

1.  Deionized water.
2.  Sperm counting chamber.
3.  Glass slides and coverslips.
4.  Pipettors and tips.
5.  Test tubes and rack.
6.  Positive and negative controls (see  Controls).
7.  Bright-field microscope with 100X - 400X total magnification. 

Warning and Precaution:

All semen samples should be considered potentially infectious.  Handle all specimens as if capable of transmitting HIV or hepatitis. Specimens should be disposed of in accordance with OSHA guidelines.

Storage and Stability:

Store the reagents at 2^o - 8^oC.  They can be used until the expiration date shown on each reagent label.

Specimen Collection:

Semen should be collected in a clean cup.  The semen sample can be stored at room temperature until using.

Preparation:

1. Bring all reagents to room temperature.
2. Prepare fresh substrate by adding the following to a test tube:
                 1 ml water
                 250  µl Buffer
                 40  µl  DAB
                 exactly 1 drop Peroxide
3.  Mix gently.  Discard after use.

Procedure:

1.  Allow semen sample to liquify.
2.  Count spermatozoa.
3.  Pipette 20  µl semen into a test tube.
4.  Pipette 20  µl Peroxidase, as a  positive control, into a second test tube.
5.  Pipette 20 µl water, as a negative control, into a third test tube.
6.  Add exactly 1 drop Fixative to each test tube.
7.  Pipette 60  µl fresh substrate into each test tube and mix briefly.
8.  Observe the test tubes with Peroxidase and water and note any color change.  The test tube with Peroxidase should turn dark brown.  This indicates that the fresh substrate is working properly. Proceed with the next step if the fresh substrate is working properly.  
9.  Prepare specimen for viewing using Method I or Method II.
     Method I:  Pipette 10 - 20 ul onto a glass slide and place a coverslip on top of the liquid.  Examine at a total magnification of 400X using a microscope.  Count brown cells and sperm within the same viewing area.
     Method II:   Pipette about 5 ul onto a Makler Chamber or or a disposable counting chamber.  Examine at a total magnification of 100X  using a microscope.  Count brown cells within the entire grid area.

Calculation Of Granulocytes In Semen, Method I:

Granulocytes/ml =   Sperm Count     X Number of  brown cells
                                                           Number of sperm

Example: The following data were obtained for a tested semen specimen placed on a glass slide with a coverslip and viewed at 400X:
            Sperm Count = 73 X 10⁶ cells/ml
            Number of  brown cells = 4
            Number of sperm = 66
Applying the formula:  73 x 10⁶  X  4/66   =  ~4 x 10⁶ granulocytes/ml

Calculation Of Granulocytes In Semen, Method II:

Granulocytes/ml =   Number of  brown cells X 5 X counting chamber factor where 5 is the dilution factor because semen was diluted 20 ul  in a total volume of 100 ul.

Example: The following data were obtained for a tested semen specimen viewed at 100X in a Cell-VU:
           Number of  brown cells in 100 squares = 16
           Cell-VU chamber factor = 10⁵ /2  cells/ml
Applying the formula:  16 x 5  X 10⁵ /2 =   4 x 10⁶  granulocytes/ml

Addtional Optional Controls:

For a  Positive Control,  use  any human fluid that contains white  blood cells, such as whole blood.  Whole blood should be used within 10 minutes of drawing  and should contain no additives such as anticoagulants. The tested blood will contain a  variety of white blood cells (leucocytes) including some that will be stained brown.  Some will be unstained.  Thus, the same human fluid specimen preparation will have both a Positive Control, stained brown leucocytes (granulocytes)  and a Negative Control, unstained leucocytes,  on the same slide.

For a separate Negative Control, run the test but omit the Peroxide.  With this method, the tested blood will contain unstained leucocytes.

Selected Reference:

World Health Organization. 1999.  WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge University Press. Fourth Edition.

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