STANDARD OPERATINAL PROCEDURE FOR SEMINAL ANALYSIS

PREPARED: Mumba, intern BMLS scien st.

SUPERVISOR: Mbughi, Head of Histo and Cyto sec on.
HoD: Dr. Tupokigwe, MD, Pathologist
OBJECTIVE

1. PURPOSE
2. SCOPE
3. RESPONSIBILITY
4. PRINCIPLE
5. SAMPLE REQUIREMENT
6. EQUIPMENTS
7. MATERIAL NEEDED
8. STORAGE AND STABILITY
9. SAFETY
10. CALIBRATION
11. TEST PROCEDURE
12. REFERENCE RANGE
13. INTERPRETATION OF THE RESULTS
14. REPORTING THE RESULTS
15. QUALITY CONTROL
16. LIMITATION AND SOURCE OF ERROR
17. BIOSAFETY AND WASTE DISPOSAL
18. REFERENCES
PURPOSE
A semen analysis is a screening test which is frequently requested for testing male infertility for
assessing the function of testes, accessory glands and tubule ducts also rarely for confirmation of
completely vasectomy.

SCOPE
SOP applied to all laboratory personnel at SJMHP.

RESPONSIBILITY
Hospital director/ Lab manager/ Head of cytopathology section: To ensure all technical lab
staff oriented, trained and understood S.O.P and documented.
Technical lab staff:
 To ensure all consumable materials and other reagents are meet its criteria of being used
and accessible.
 To prepare and identify the client for sample collection.
 To ensuring all necessary information of requested client are accessible when needed
include abstinence periods.

Note: any trained and oriented laboratory technician, technologist or scientist are legible to
perform the procedure.

PRINCIPLE
Semen analysis assesses the quality and quantity of spermatozoa including volume, motility,
concentration, morphology, viability, and presence of leukocytes or abnormal cells as per WHO
guidelines.

SAMPLE REQUIREMENT
Instructions to Patient:
a) Fresh semen collected within 30 minutes no more than 60 min, and processing should fall
within one hour of collection.
b) Abstain from sexual activity for 2–7 days before collection.
c) Collect sample by masturbation into a sterile wide-mouthed container provided by the lab
personnel.
d) Avoid use of lubricants or condoms unless provided by the lab.
e) Avoid any alcohol, caffeine, marijuana from 2 to 5 days
f) Avoid any hormone medication at least 3 days before collection date.
Collection Location:
 Preferably within the hospital (private semen collection room if available} otherwise
direct the client to the toilet for collection.
NOTE: If collected at home, should be delivered to the laboratory within one hour, kept at body
temperature (e.g. underarm transport) and the patients/client should instructed to withdrawal
from his partner before ejaculation.
Labeling:
 Full name, ID number, date/time of collection, abstinence period.
EQUIPMENTS AND MATERIALS NEEDED
Equipment:
1. compound Microscope with 400x and 1000x magnification
2. pH meter or any pH paper digital machine
3. Glass load or applicator
4. Improved Neubauer chamber
5. Calculator
6. Timer (stop watch)
Supplies and consumable
1. Cotton wall or gauze.
2. Pasteur pipette.
3. Personal protective equipment (PPE).
4. Labelling material includes Pencil and ball pen.
5. Microscopic glass slides, coverslips, slide tray.
6. Register book log.
7. Wide mouth transparent dry clean disposable collection sample container.
8. Applicator stick
9. Water bath at 37°C.
10. WHO semen analysis manual (latest edition).
Chemicals and reagents
1. 0.5% Sodium bicarbonate.
2. 10% Formalin.
3. 96% or 95% Ethanol.
4. Sperm staining reagents (e.g., Papanicolaou or Eosin-Nigrosin).

STORAGE AND STABILITY

 Specimen should be processed with 60 minutes after reaching to cytology laboratory.
 The temperature of specimen should be maintained to 37°C, water bath may be used to
warm the specimen.
 Below or above the maintained temperature, the motility of spermatozoa cells affected.
Note: After analysis discard the specimen.

SAFETY
Safety and bio hazard precaution must be adhered. Handle all biological specimen with care,
semen may contain harmful pathogens includes HIV and Hepatitis virus.

CALIBRATION
The machines used, must be calibrate as manufacture instructed within a specific time interval at
least twice per year include Calculators, Timer, Microscope and pH meter.
TEST PROCEDURE
1. Macroscopic Examination:
 Appearance: Normal = Greyish-white/ Tan whitish, homogenous
Abnormal= Red-brown when red blood cells are present (haemo-
spermia), or yellow in a man with jaundice or taking certain vitamins or drugs.
NOTE: Any turbidity must be reported.

 Liquefaction Time: Report the time for complete liquefaction, normally

within (30-60) min. If no changes after the specified time, report the
findings. Liquefaction is a product after coagulum reacted with Protease
enzymes, the enzyme produced by prostate gland and the coagulum is
produced by seminal vesicle.

 Volume: Measure the volume using a clean and graduated Pasteur pipette,
open the lid of specimen container, press the head of Pasteur pipette, insert
pipette into the container containing sample slowly aspirate all specimen
without allowing air bubble. Read the mark on the pipette and report your
findings.
(≥1.5 mL is normal and ≤1.5 mL is abnormal).
Alternative:
Measure the volume using weighing the sample in the vessel in which it is collected,
sample should be collected from a pre-weighed, clean, disposable container after
collection weight the vessel with semen in it, and subtract the weight of the container.
Density(g/ml) = Mass(g) / Volume (mL)
Note: Semen density varies between 1.043 and 1.102 g/ml.

pH: For assessing accessory gland secretions mainly the alkaline seminal vesicular secretion and
the acidic prostatic secretion, the pH should be measured after 30 minutes of liquefaction.
 Mix the semen sample well, dip the strip into the container containing semen, insert the
dipped strip into the pH meter for processing. Note and report the results.
 Mix the semen sample well, spread a drop of semen evenly onto the pH paper, wait for
the colour of the impregnated zone to become uniform (<30 seconds) and compare the
colour with the calibration strip to read the pH.
(normal range: 7.2–8.0)
.
Viscosity: Using Pasteur pipette draws about 50µL of liquefied and allow slowly about 20µL to
drop back in the container, note and report your findings.
(Normal sample pours drop-wise, abnormal thread of semen drawn back exceed more than 2 cm.
high viscosity may affect motility.

2. Microscopic Examination (within one hour of collection)

Make a wet preparation for assessing motility.
i. Take a clean and dry microscope glass slide, correctly labeling for the unique
cytology specimen client/ patient identification number.
ii. mix well liquified semen by slowly stir the specimen container
 iii. Open the lid of specimen container and pipette a little bit of semen, place one drop of
semen on microscope glass slide.
iv. Cover the glass slide with coverslip.
v. Observe under microscope for the presence agglutination, aggregation of the
spermatozoa, other cellular materials other than spermatozoa cells includes epithelial
cells from the genitourinary tract, as well as leukocytes and immature germ cells, the
latter two collectively referred to as “round cells”. Report your findings.

Sperm Motility: moving ability of spermatozoa cells, categorized into three Progressive motile
(PR}, Non-progressive motile (NP) and Immotile (IM).
i. Observe clearly the slide using 10X microscope objective lens to see for the presence of
spermatozoa on different fields of view and if spermatozoa not seen (azoospermia) go
for Spermatozoa viability test.
ii. After identified the cells turn into 40X objective lens for assessing spermatozoa motility.
Count a total of 100 spermatozoa cells and note out of 100, to see how many spermatozoa
are motile. Report your findings in percentage (%).
(≥ 50% motility of spermatozoa cell in the preparation is normal or ≥ 25 PR within 60
min after collection).
Note:
If the motility is ≤ 40% known as ASTHENOZOOPERMIA include (PR + NP), IM ≥60%
Spermatozoa viability test is recommended.
Rarely Seliwanoff’s test nutritive test for spermatozoa, testing for the presence of fructose
(accessory gland function test particularly seminal vesicle). Normal ≥13 µmole.

Sperm vitality/viability: estimated procedure for assessing the membrane integrity of the cells
especially if the motility is ≤ 40%, dehydration and temperature affect viability test.
 0.5% Eosin-Nigrosin method where by live spermatozoa cells exclude the eosin dye
and appear white while dead spermatozoa cells take up the dye and appear pinkish red.
Nigrosin provide a dark background for contrast. Preferred within 30 minutes.
Procedure:
i. Allow semen to liquefy (30 minutes at room temperature)
ii. mix well the sample and prepare a wet preparation by dispense one drop
of semen on the glass slide.
iii. Add one drop of 0.5% Eosin-Nigrosin mix well the mixture on the glass
slide and wait for 2 minutes before the examination.
iv. Coverslip and observe under the microscope (100X oil immersion).
v. Count at least 200 spermatozoa
 Pink/red = dead
 White/unstained = live
vi. Record and report your findings.
Result Interpretation:
 Calculate % viability:

Viability (%) = Number of live spermatozoa X 100

Total counted
NOTE: Normal > 60% live
Sperm Morphology: Evaluation of structural appearance of spermatozoa cells to determine the
percentage of a normal spermatozoa morphologically.
 Papanicolaou’s Stain Method it consists of three solutions (Hematoxylin, Orange G-6
and Eosin Azure) to provide good nuclear and cytological contrast, allowing detailed
assessment of sperm head, midpiece and tail for abnormalities.
Procedure:
i. Use Pasteur pipette, place one drop of well mixed liquefied semen on the clean
and dry microscope glass slide and make a thin smear.
ii. Fix the smear into 96% or 95% Ethanol for (15-30) minutes.
iii. Follow Papanicolaou’s’ staining technique to stain the smear.
iv. After staining mount with coverslip using DPX and allow to dry.
v. Examine under light microscope (1000X oil immersion)
vi. Evaluate at least 200 spermatozoa cells.
Criteria for Normal morphology (WHO):
 Head: smooth, oval, regular outline and its acrosome covers (40-70) % of head.
 Midpiece: slender, regular, aligned with head.
 Tail: single, uncoiled, approximately 45 µm its lengths.
Results Interpretation:
% normal spermatozoa morphology = Normal sperm X 100
Total counted
(Normal forms ≥4% by strict criteria)

Sperm Count and Concentration: To determine the concentration of spermatozoa per milliliter
of semen using manual counting with hemocytometer.
 Improved Neubauer Chamber: Diluted semen is loaded into the counting chamber;
spermatozoa cells are counted in specific squares under a microscope and the
concentration is calculated using the chamber’s known volume.
Procedure:
i. Mix well liquefied semen sample gently thoroughly
ii. Take graduated test tube or any graduated clean and dry container with
visible graduated volume range 1ml to 25 ml.
iii. Using Pasteur pipette withdrawal 1 ml of well mixed liquefied semen and
dispense into a graduated test tube.
iv. Dilute with 19 parts (19 ml) of semen diluting solution (5% Sodium
bicarbonate + Formalin) to make 1:20 ratio.
v. Mix gently avoid air bubble for at least (30-60) seconds.
vi. Using Pasteur pipette, load the diluted semen into clean and dry Neubauer
counting chamber under the coverslip by capillary action, avoid overfilling
or air bubble.
vii. Leave the filled Neubauer chamber to stand for (3-5) minutes to allow
spermatozoa cells to settle.
viii. Examine under microscope, using 400X count spermatozoa cell in the
central large square. (divided into 25 smaller square), count sperm in 5 of
the 225 squares diagonally (top left, top right, center, bottom left, bottom
right).
 ix. If count is low, count all 25 squares for accuracy.
x. Report your findings

Calculation:
 Each large square = 0.1 mm3 (1 ml X 1 ml X 0.1 ml)
 Sperm Concentration (million/ml) = Total sperm counted in 5 squares X 10 6 X Dilution
factor. 5

Sperm conc. (million/mL) = average count per squares X 20 X 10

Average sperm conc. (million/ml) = average sperm counted X 1,000,000
Normal reference range (WHO)
Sperm conc. ≥ 20 million/ml

REFERENCE RANGE (WHO)

 Normal semen volume per single ejaculation ranging between (1.5 – 7.5) ml.
 Abnormal volume of semen ≤ 1.5 ml (Hypospermia) and may be due to incomplete
collection of samples, obstruction in seminal vesicles or ejaculation ducts or partial
retrograde ejaculation.
 Normal semen viscosity ranging between (1.0 – 2) cm/ drop by drop
 Normal pH ranging between (7.2 – 7.8).
 Normal sperm count ≥ (15 – 20) million of spermatozoa cells per ml.

INTERPRETATION OF THE RESULTS

Reports your finding by using common terms used in seminal analysis
 Azoospermia: total absence of spermatozoa cells in a semen
 Necrospermia: all spermatozoa cells are non-motile and pus cells may be seen.
 Oligospermia: low sperm count.
 Polygospermia: high sperm count.

REPORTING THE RESULTS

 Patient information includes abstinence duration.
 Collection date/time
 All parameters with units and reference ranges
 Report motility of spermatozoa by considering PR regardless of the direction, NP and IM
 Reporting any abnormality of sperm morphology
 Interpretation/comments.

QUALITY CONTROL
 Use internal QC (e.g., standardized sperm samples if available).
 Participate in external quality assurance when possible.
 Maintain equipment calibration and maintenance logs.
LIMITATION AND SOURCE OF ERROR
i. Error arises during sample collection includes incomplete collection when the first
portion is lost, delayed of transportation, uses of spermicidal lubricants.
ii. Error due to biological variations includes stress or illness according to WHO single
analysis many not represent true fertility status recommended at least two tests 2 – weeks
apart.
iii. Error during technical procedure may include improper staining or improper smear
preparation may affect morphology assessment, overload or underloading of counting
chamber.
iv. Error due to environmental conditions spermatozoa cells are temperature sensitivity
outside of recommended temperature sperm cells died false positive for motility may be
obtained also exposed to light, pH changes or any toxic substance affect the results during
findings.
v. Error during interpretation.

BIOSAFETY AND WASTE DISPOSAL

i. Treat all semen samples as potentially infectious material.
ii. Always wear appropriate PPE, and if available conduct procedures within a biosafety
cabinet.
iii. Disinfect reusable items; discard waste as per biomedical waste management protocols.

REFERENCES
1. WHO Laboratory Manual for the Examination and Processing of Human Semen, 6th
Edition
2. ISO 15189:2012 Standards
3. MOHCDGEC Tanzania Laboratory Guidelines
4. Internal S.O.P for seminal analysis at SJMHPL