Assessment of leucocyte

in Semen

- Sushant Pokhrel
(B.Sc MLT
3rd Year)
INTRODUCTION
 Predominantly polymorphonuclear leucocyte are present in most
human ejaculates
 Sometimes be differentiated from spermatids and spermatocytes
in a semen smear stained with the Papanicolaou procedure
 Differentiation is based on differences in staining coloration, and on
nuclear size
and shape
 Polymorphonuclear leukocytes can easily be confused morphologically
with multinucleated spermatids, but stain a bluish colour, in contrast to
the more pinkish colour of spermatids
 Peroxidase-positive granulocytes are the predominant form of
leukocytes in semen, routine assay of peroxidase activity is useful as
an initial screening technique
 Can be further differentiated with more time-consuming and expensive
immunocytochemical assays against common leukocyte and sperm
antigens
Peroxidase Method using Ortho-toluidine

 Quick & inexpensive

 Useful initial screening for granulocytes
 Leukocytes in human semen are counted after a histochemical procedure that
identifies the peroxidase enzyme, which is characteristic of granulocyte
 This technique has the advantage of being relatively easy to perform,
but it does not detect:
activated polymorphs which have released their granules;
other types of leukocyte, such as lymphocytes, macrophages and monocytes,
which do not contain peroxidase.
 Useful in distinguishing polymorphonuclear leukocytes from multinucleated
spermatids, which are peroxidase-free
 Reagents
1. Phosphate buffer, 67 mmol/l, pH 6.0: dissolve 9.47 g of sodium hydrogen phosphate
(Na2HPO4) in 1000 ml of purified water and 9.08 g of potassium dihydrogen
phosphate (KH2PO4) in 1000 ml of purified water. Add one solution to the
other (approximately 12 ml of Na2HPO4 solution to 88 ml of KH2PO4 solution)
until the pH is 6.0.
2. Saturated ammonium chloride (NH4Cl) solution: add 250 g of NH4Cl to 1000 ml
of purified water.
3. Disodium ethylenediamine tetra-acetic acid (Na2EDTA) 148 mmol/l: dissolve
50 g/l in phosphate buffer (pH 6.0) prepared in step 1.
4. Substrate: dissolve 2.5 mg of o-toluidine in 10 ml of 0.9% (9 g/l) saline.
5. Hydrogen peroxide (H2O2 ) 30% (v/v): as purchased.
6. Working solution: to 9 ml o-toluidine substrate, add 1 ml of saturated NH4Cl
solution, 1 ml of 148 mmol/l Na2EDTA, and 10l of 30% (v/v) H2O2 and mix well.
This solution can be used up to 24 hours after preparation.
 Procedure
 Mix the semen sample well
 Remove a 0.1-ml aliquot of semen and mix with 0.9 ml of working solution(1:10
dilution)
 Vortex the sperm suspension gently for 10 seconds and incubate at room temperature
for 20–30 minutes.
 Remix the semen sample before removing a replicate aliquot and mixing with
working solution as above.
Assessing peroxidase-positive cell number in the haemocytometer chambers
 After 20–30 minutes, mix the sperm suspensions again and fill each side of a
haemocytometer with one of the replicate preparations.
 Store the haemocytometer horizontally for at least 4 minutes at room temperature in a humid
chamber (e.g. on water-saturated filter paper in a covered Petridish) to prevent drying out and
to allow the cells to settle.
 Examine the chamber with phase-contrast optics at ×200 or ×400 magnification.
 Count at least 200 peroxidase-positive cells in each replicate, in order to achieve an
acceptably low sampling error. Peroxidase-positive cells are stained brown, while peroxidase-
negative cells are unstained
 Make a note of the number of grids assessed to reach at least 200 peroxidasepositive cells. The
same number of grids will be counted from the other chamber of the haemocytometer.
 Tally the number of peroxidase-positive cells and grids with the aid of a laboratory counter.
 Switch to the second chamber of the haemocytometer and perform the replicate count on the
same number of grids as the first replicate, even if this yields fewer than 200 peroxidase-
positive cells.
 Calculate the sum and difference of the two numbers of peroxidase-positive cells.
 Determine the acceptability of the difference from Table
 If the difference is acceptable, calculate the concentration. If the difference is too
high, prepare two new dilutions and repeat the replicate count estimate
 Report the average concentration of peroxidase-positive cells to two significant
figures.
 Calculate the total number of peroxidase-positive cells per ejaculate
 RESULT

 Peroxidase positive cell: Brown colour

 Peroxidase negative cell: Round cell
Calculation of the concentration of peroxidase-
positive cells in semen

 The concentration of peroxidase-positive cells in semen is their number (N)

divided by the volume of the total number (n) of grids examined for the
replicates
(where the volume of a grid is 100 nl), multiplied by the dilution factor.

For a 1 + 9 (1:10) dilution, the concentration is C = (N/n) × (1/100) × 10 cells
per nl =
(N/n) × (1/10) cells per nl. Thus (N/n) is divided by 10 to obtain the
concentration in
peroxidase-positive cells per nl (106 cells per ml).
Worked examples

 Example 1. With a 1 + 9 (1:10) dilution, replicate 1 is found to contain 60

peroxidase-positive cells in nine grids, while replicate 2 contains 90
peroxidase-positive cells in nine grids. The sum of the values (60 + 90) is 150
in 18 grids and the difference (90–60) is 30. From Table 2.5 this is seen to
exceed the difference expected by chance alone (24), so the results are
discarded and new replicates are made.
Example 2. With a 1 + 9 (1:10) dilution, replicate 1 is found to contain 204 peroxidase-
positive cells in five grids, while replicate 2 contains 198 peroxidase-positive
cells in five grids. The sum of the values (204 + 198) is 402 in 10 grids and the
difference (204–198) is 6. From Table 2.5 this is seen to be less than that found by
chance alone (39), so the values are accepted.
The concentration of peroxidase-positive cells in the sample, for a 1 + 9 (1:10) dilution, is
C = (N/n) × (1/10) cells per nl or (402/10)/10 = 4.02 cells/nl, or 4.0 × 106 cells
per ml
Thank You