College of American
Pathologists
Sperm Count, Motility and
Forward Progression Survey
SMCD
© College of American Pathologists
Special Instructions
Note:
• The Hemocytometer images have been immobilized.
• The Hemocytometer specimens are a 1:20 dilution.
• All other specimens are not diluted.
Perform motility assessment on Makler or Disposable
Chamber images
© College of American Pathologists
Instructions
This Proficiency Testing module evaluates the ability of a laboratory to assess
motile sperm in an unknown sample and to be able to report percent motility.
You will be presented with two unknown samples at 400x magnification.
Video is best viewed in Windows Media Player Window. On the Windows
Media Player menu bar click on View and the Video Size. This should be set
to 100%. Adjust Brightness by clicking on Enhancements and then Video
Settings.
Screen resolution should be 1024 x 768 pixels. (Adjust if needed by going
to Control panel, click on Display and then Settings).
Three types of chambers are presented in this program; select the file
corresponding to the chamber type you employ in your laboratory:
1. Makler chamber
2. Disposable counting chamber (Cell-VU, MicroCell)
3. Hemocytometer
***Note: If your preferred chamber type is not listed, simply use the Makler video
clips and follow the detailed instructions and diagram included with this product
for calculation of count and motility.
© College of American Pathologists
Makler Chamber
For each sample four different fields of view will be shown.
Note: With Windows media player the clip can be paused or re-run at any time to aid in the
determination of motile and non-motile sperm by using the buttons or the space bar.
The sperm count in million/mL is determined by counting the total number of sperm in any 10
boxes in Fields 1 and 2 (5 boxes in each field) and then repeating for Fields 3 and 4 (count 5
boxes in each field). The average number of sperm calculated from the 2 counts would be
reported.
MOTILITY AND FORWARD PROGRESSION FOR LABORATORIES USING WHO 5TH
1. Four fields of 6 boxes will be presented. Count the total number of sperm exhibiting
progressive motility in 20 boxes. Then count the number of sperm exhibiting nonprogressive motility in the same 20 boxes
2. Count the total number of sperm in the same 20 boxes – regardless of progression
3. Calculate the percent progressive and percent non-progressive
4. Add these percentages together to determine percent total motility
FOR LABORATORIES USING VERSIONS PREVIOUS TO WHO 5TH
1. Four views of 6 boxes will be presented. First count motile sperm in 20 boxes
*Note: Count all moving sperm regardless of progression
2. Count non-motile sperm in same boxes
3. Determine % motility as shown below
% motile =
4.
Motile sperm
x 100 Total
sperm (Motile+non-motile)
Determine the predominant forward progression pattern of the moving cells
© College of American Pathologists
Sample of Motility Calculations on Makler
For demonstration purposes, each numbered square denotes “fields” or “squares” used to perform count
WHO 5th example
Field 1
Field 2
Progressive
Non-Progressive
Non Motile
EXAMPLE:
PROGRESSIVE
NON-PROGRESSIVE
NON MOTILE
TOTAL:
Field 3
FIELD 1
4
2
2
8
FIELD 2
5
3
2
10
FIELD 3
5
4
2
11
Field 4
FIELD 4
4
3
2
9
TOTAL
18
12
8
38
%
47.4
31.6
21.1
TOTAL MOTILITY %
78.9
Editions previous to WHO 5th example
Field 1
1
2
4
5
3
Field 2
1
2
4
5
EXAMPLE:
MOTILE
NON-MOTILE
© College of American Pathologists TOTAL:
Motile
Field 3
3
FIELD 1
6
2
8
1
2
4
5
FIELD 2
8
2
10
Field 4
3
FIELD 3
9
2
11
1
2
4
5
FIELD 4
7
2
9
3
TOTAL TOTAL MOTILITY % = 30/38 =
30
78.9%
12
38
Disposable Counting Chamber (Cell-VU, MicroCell)
For each sample four different fields of view will be shown.
Note: With Windows media player the clip can be paused or re-run at any time to aid in
the determination of motile and non-motile sperm by using the buttons or the space bar.
The sperm count in million/mL is determined by counting the total number of
sperm in any 5 boxes in one field and then counting 5 boxes in a different field
and reporting the average.
MOTILITY AND FORWARD PROGRESSION FOR LABORATORIES USING WHO 5TH
1. Four fields of 6 boxes will be presented. Count the total number of sperm
exhibiting progressive motility in 20 boxes. Then count the number of sperm
exhibiting non-progressive motility in the same 20 boxes
2. Count the total number of sperm in the same 20 boxes – regardless of progression
3. Calculate the percent progressive and percent non-progressive
4. Add these percentages together to determine percent total motility
FOR LABORATORIES USING VERSIONS PREVIOUS TO WHO 5TH
1. Four views of 6 boxes will be presented. First count motile sperm in 20 boxes
*Note: Count all moving sperm regardless of progression
2. Count non-motile sperm in same boxes
3. Determine % motility as shown below
Motile sperm
x 100
Total sperm (Motile+non-motile)
Determine the predominant forward progression pattern of the moving cells
% motile =
4.
© College of American Pathologists
Sample of Motility Calculations on Disposable Counting Chamber
For demonstration purposes, each numbered square denotes “fields” or “squares” used to perform count
WHO 5th example
Field 1
Field 2
EXAMPLE:
PROGRESSIVE
NON-PROGRESSIVE
NON MOTILE
TOTAL:
Progressive
Non Progressive
Non Motile
Field 3
FIELD 1
4
2
2
8
FIELD 2
5
3
2
10
FIELD 3
5
4
2
11
Field 4
FIELD 4
4
3
2
9
TOTAL
18
12
8
38
%
47.4
31.6
21.1
TOTAL MOTILITY %
78.9
Editions previous to WHO 5th example
Field 1
1
2
4
5
3
Motile
© College of American Pathologists
Field 2
1
2
4
5
3
EXAMPLE:
MOTILE
NON-MOTILE
TOTAL:
Field 3
1
2
4
5
FIELD 1
6
2
8
FIELD 2
8
2
10
Field 4
3
FIELD 3
9
2
11
1
2
4
5
FIELD 4
7
2
9
3
TOTAL TOTAL MOTILITY % = 30/38 =
78.9%
30
12
38
Hemocytometer – Immobilized Sperm
The center of the hemocytometer contains 25 large squares with 16 smaller squares
inside. This area represents 1 square mm and the volume above this square is 0.1 µL.
The total count in 25 squares multiplied by the dilution and 104 gives the sperm
concentration/mL. Final sperm count should be reported as million/mL.
For each sample, 5 different fields of view will be shown.
Calculate sperm concentration/mL as follows:
Sperm concentration (millions/mL) = Total number of sperm in 5 large squares x 106
Alternatively:
Sperm concentration (millions/mL) = Average sperm per large square (number of
sperm ÷5) x 25 squares x Dilution factor (20 for this exercise) x 104
Final sperm concentration should be reported in million/mL.
An example is shown on the next page.
© College of American Pathologists
Sample of Sperm Count Calculations on a
Hemocytometer
Field 1
Field 2
2
3
4
1
5
6
7
8
12
9
10
11
16
13
14
15
1
2
3
4
1
5
6
7
8
9
10
11
13
14
15
Field 3
2
3
4
5
6
7
8
12
9
10
11
12
16
13
14
15
16
Field 4
Field 5
1
2
3
4
1
2
3
4
5
6
7
8
5
6
7
8
9
10
11
12
9
10
11
12
13
14
15
16
13
14
15
16
© College of American Pathologists
Total
Field 1
9
Field 2
7
Field 3
7
Field 4
7
Field 5
8
Total
38
Specimen was a 1:20 dilution
Sperm concentration:
Total of 5 squares= 38 or
(38 ÷ 5) x 25 x 20 x104 =
38 million/mL
For demonstration purposes,
each numbered square denotes
“fields” or “squares” used to
perform count.