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WHO 5th Edition Manual STRICT Morphology Training and Testing

Tuesday, April 22, 2014


This document includes pages 67 through 95 of the WHO Laboratory Manual for the Examination and Processing of Human Semen FIFTH EDITION.  This updated version of the WHO manual does a very good job of showcasing normal vs. abnormal forms including all relevant defects.  Dr. Thinus Kruger assessed the slides presented personally and in addition to grading them provides comments to further explain his judgements.  It is highly recommended that all SQA-V users, and facilities conducting manual semen analysis become familiar with this new criteria as it is fast becoming the world standard.  The document also provides 12 plates worth of numbered cells which can be assessed by the operator for training and proficiency purposes (don't look at the answers ahead of time!)  The document can be downloaded by clicking the image above or by clicking HERE


Remember, it ALL Started with a Sperm.

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WHO Recommendations for Manual Sperm Concentration Analysis

Tuesday, April 22, 2014

Maintaining an accurate backup method for any automated analyzer in the laboratory is important.  When it comes to semen analysis, there are many different counting chamber options available.  Correlating manual methods to automated methods can be challenging, especially when it comes to semen analysis.  The first step is picking the correct tool for the job.  Below are the WHO 5th Edition Manual's recommendations for sperm concentration counting chambers.  To achieve the best possible correlation to the SQA-V Gold Automated Sperm Quality Analyzer, and maintain the most accurate backup method possible these recommendations should be followed.  In particular please note that disposable plastic counting chambers can lead to increased variability.  


2.7.1 Types of counting chambers (From Page 34 of the WHO 5th Edition Manual for Semen Analysis)

The use of 100-m-deep haemocytometer chambers is recommended. Dilution factors for the improved Neubauer haemocytometer chamber are given here. Other deep haemocytometer chambers may be used, but they will have different volumes and grid patterns and will require different factors for calculation. Disposable chambers are available for determining sperm concentration (Seaman et al., 1996; Mahmoud et al., 1997; Brazil et al., 2004b), but they may produce different results from those of the improved Neubauer haemocytometer. Shallow chambers that fill by capillary action may not have a uniform distribution of spermatozoa because of streaming (Douglas-Hamilton et al., 2005a, 2005b). It may be possible to correct for this (Douglas-Hamilton et al., 2005a) but it is not advised (Björndahl & Barratt, 2005). The validity of these alternative counting chambers must be established by checking chamber dimensions (see Appendix 7, section A7.8), comparing results with the improved Neubauer haemocytometer method, and obtaining satisfactory performance as shown by an external quality-control programme. For accurate assessment of low sperm concentrations, large-volume counting chambers may be necessary (see Section 2.11.2).

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Reason for Assessing WBC Levels on the SQA-V

Thursday, February 06, 2014

Thursday, February 6, 2014


The SQA-V technology for assessing sperm concentration is based on the principle of spectrophotometry coupled with software filters that eliminate ‘background’ factors that can influence the SQA-V results.


It is well known that a semen ejaculate contains cells other than spermatozoa (1). These include epithelial cells from the genitourinary tract, as well as leukocytes (WBC) and immature germ cells (2). The presence of these non-sperm cells in semen may be indicative of testicular damage (immature germ cells), pathology of the efferent ducts (ciliary tufts) or inflammation of the accessory glands (leukocytes) (1).

These non-sperm cells or along with seminal plasma are all considered ‘background’ factors that interfere with the accurate assessment of sperm concentration using spectrophotometry technology. When the SQA-V assesses sperm concentration, the ‘background’ is subtracted by the software in order to eliminate its impact on the final and accurate assessment of sperm concentration. The normal SQA algorithm compensates for ‘normal’ levels of ‘background’ which are seen in most semen, however, abnormal levels of ‘background’ require a different algorithm that compensates for this. Hence, the operator is asked to assess for abnormal levels of WBC (the most contributing background factor) and select ≥ 1 M/ml in order to activate the appropriate algorithm required to measure the sample’s concentration.

The normal value for semen WBC is < 1 M/ml. In cases of inflammation, the leukocyte level is ≥ 1 M/ml and the semen will also contain seminal plasma compounds associated with inflammation. This increases the level of ‘background’ to subtract from the algorithm. This is why it is important to test if WBC’s are NORMAL or ABNORMAL and to select the proper input on the PATIENT DATA ENTRY SCREEN in order to activate the appropriate algorithm for sample testing.


WBC levels in semen can be tested using one of the following procedures:
• Using QwikCheck™Test Strips - recommended (please refer to the SQA-V User Guide Version 2.60 I-Button, Appendix 7: Measuring WBC's in Semen).
• Using the SQA-V visualization screen or the V-Sperm and visually/manually assessing the sample for WBC’s (please refer to the SQA-V User Guide Version 2.60 I-Button, Appendix 4: Counting Cells using the SQA-V Visualization System and Appendix 7: Measuring WBC's in Semen).


WBC assessment should be conducted on FRESH and WASHED semen samples and the results entered in the SQA-V PATIENT/SAMPLE DATA ENTRY screen PRIOR to running automated semen analysis on the SQA-V and before any sample treatment with chymotrypsin or any other agent (enrichment, washing, dilution, etc.).


1. WHO laboratory manual for the examination and processing of human semen - 5th ed., World Health Organization 2010.
2. Johanisson E et al. (2000). Evaluation of “round cells” in semen analysis: a comparative study. Human Reproduction Update, 6:404-412.


Distribution: All SQA Users





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SQA-V Visualization Counting Instructions - WHO 5th Edition

Sunday, January 26, 2014

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QwikCheck Test Strip Color Interpretation

Thursday, January 23, 2014
Thursday, January 22, 2014

QwikCheck™ Test Strips are used to determine the pH and leukocyte level (WBCs) in semen based on comparing the color of the test strip pads to the color chart provided on the bottle label. They are for in vitro diagnostic use only.


- pH: Normal values for semen pH are generally ≥ 7.2; the pH pad reports a range of 5.0 to 8.5. 

- Leukocyte Level: Normal values for semen Leukocytes are < 1 M/ml; the Leulocyte pad reports semi-quantitative results: NEG is <1M/mL and POSITIVE is ≥1 M/mL. If the Leulocyte pad color is equal to or exceeds the darkest lavender color of the chart within 60 seconds, the result is ≥1 M/mL, otherwise it is <1M/mL.


Please see the package insert for complete testing instruction details.


Test results can be impacted by drugs or other chemical use. QwikCheck™Test Strip pH
and WBC testing should be conducted PRIOR to any sample treatment with chymotrypsin or in any other way (enrichment, washing, dilution, etc.).





Distribution: All SQA Users

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Post Vasectomy Testing on the SQA-V - Work Flow Recommendation

Thursday, January 23, 2014



The WHO 5th edition manual provides recommendations for testing post-vasectomy semen samples.



Per the WHO 5th edition manual, it is not recommended to centrifuge the post-vasectomy sample if the technician is searching for motile spermatozoa, as centrifugation impacts sperm motility. If no spermatozoa are found in the ejaculate it is recommended to re-assess the sample after centrifuging and re-suspending the pellet in a small volume of seminal plasma.




Fast scan:
o Prepare a standard slide per the user guide instructions (10 μl liquefied ejaculate, 22mmx22mm coverslip). Search for spermatozoa using the SQA-V visualization system, set at “Zoom Out”.

If 1-2 MOTILE spermatozoa are found in each field of view:
o Run the sample in the SQA-V Post-vasectomy mode.

If >=2 MOTILE spermatozoa are found in each field of view:
o Run the sample in the SQA-V FRESH mode.

If only IMMOTILE spermatozoa are found:
o Count the sperm cells manually using the SQA-V visualization system set at Zoom Out. Count in duplicate using the same slide. Compute the total number of sperm divided by the number of assessed fields of view. This will roughly represent the sperm concentration in millions per ml.

If NO spermatozoa are found:
o Centrifuge the semen sample at 3000g for 15 minutes.
o Decant most of the supernatant.
o Re-suspend the sperm pellet in approximately 50μl of seminal plasma.
o Test the sample manually in duplicate using a standard slide (please see above).
o The presence of spermatozoa indicates cryptozoospermia; the absence of spermatozoa indicates azoospermia.
o This method cannot be used to determine sperm concentration or total sperm number. 



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Comparison of WHO 4th and 5th Edition Morphology Criteria

Thursday, January 23, 2014

Please reference the the following comparison table of WHO 4th and 5th Morphology criteria and Dr. Menkveld's (the author of publications used by the WHO) presentation at ESHRE SIG-Andrology Campus Meeting, 2009. 


Comparison of WHO 4th and 5th Morphology Criteria


The New 5th WHO Manual Semen Parameter Reference Values - Menkveld


Based on this comparison and Dr. Menkveld’s presentation, the following conclusions can be drawn:


• In the Dr. Menkveld presentation, no WHO 5th vs. WHO 4th Morphology criteria differences are mentioned, only a lower reference limit. Strict criteria is used for morphology assessment and grading in both manuals.


• The concept of normal spermatozoon by WHO 4th and 5th manuals are the same.


• Most of WHO 4th and 5th Morphology criteria are the same.


• There are the slight but not significant differences in some dimensional details that were updated in the WHO 5th manual.


• The WHO 4th manual Morphology reference value was not final and was updated in the WHO 5th edition.
The overall conclusion is that the WHO 4th and 5th Morphology criteria are similar, as they are based on the same strict principles, similar guidelines and reference publications. As the WHO 4th and 5th criteria are similar, we did not conduct any additional study for the WHO 5th morphology algorithm and stayed with the same developed for the WHO 4th. Only the reference value was changed in the V-Sperm.

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Troubleshooting Blank Operation Screen

Wednesday, January 22, 2014
Click Image to Download full instructions:

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Understanding Liquefaction in Semen Samples

Thursday, November 21, 2013


A semen sample that presents with delayed liquefaction or high viscosity should be reported during the testing process. These are abnormal parameters and are clinically significant because they may indicate a male accessory gland malfunction (Please see two abstracts below). Nevertheless, delayed liquefaction does not necessarily indicate infertility. A post-coital test (PCT) can answer this question (Please see below). There is no direct evidence that the female tract contains enzymes that promote semen liquefaction, but in some cases, motile sperm in non-liquefied specimens can be found when examining cervical mucus after sexual intercourse (Please see below).


What Is It? When semen is ejaculated, it is thick and gelatinous. This is to help it adhere to the cervix. The semen eventually liquefies to enable the sperm to swim better.

What Is Considered Normal? Semen should liquefy within 20 to 30 minutes of ejaculation.

What Might Be Wrong if Results Are Abnormal? Delayed liquefaction may indicate a problem with the prostate, the seminal vesicles, or the bulbourethral glands, which are also known as the male accessory glands. If delayed liquefaction occurs, your doctor may perform a post-coital test (PCT). This fertility test evaluates the woman's cervical mucus after sexual intercourse. If sperm are found and moving normally, then delayed liquefaction is not considered a problem.


The transport of sperm is dependent upon several factors. The sperm must be capable of propelling themselves through the environment of the female vagina and cervix. This environment, which is under cyclic hormonal control, must be favorable to admit the sperm without destroying them. Finally, the sperm must possess the capability of converting to a form that can penetrate the cell membrane of the egg (capacitation).

Following ejaculation, the semen forms a gel which provides protection for the sperm from the acidic environment of the vagina. The gel is liquefied within 20-30 minutes by enzymes from the prostate gland. This liquefaction is important to free the sperm so transportation may occur. The seminal plasma is left in the vagina. The protected sperm with the greatest motility travel through the layers of cervical mucus that guard the entrance to the uterus. During ovulation, this barrier becomes thinner and changes its acidity creating a friendlier environment for the sperm. The cervical mucus acts as a reservoir for extended sperm survival. Once the sperm have entered the uterus, contractions propel the sperm upward into the fallopian tubes. The first sperm enter the tubes minutes after ejaculation. The first sperm, however, are likely not the fertilizing sperm. Motile sperm can survive in the female reproductive tract for up to 5 days.



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Testing PH and WBCs Before Sample Treatment or Dilution

Tuesday, November 05, 2013
Thursday, October 31, 2013

QwikCheck™ Test Strips are for in vitro diagnostic use for the determination of pH and
leukocytes (WBCs) in semen. Test results are determined by comparing the color of the
test patches to the color chart provided on the bottle label.

Normal values for semen pH are generally ≥ 7.2; the pH pad reports a range of 5.0 to
8.5. Normal values for semen Leukocytes are < 1 M/ml; the Leulocyte pad reports semiquantitative results: NEG is <1M/mL and POSITIVE is ≥1 M/mL.

Follow the instructions provided in the package insert for testing and for determining the
results. Because test results can be impacted by drugs or other chemical use, run the pH
and WBC tests on untreated semen and PRIOR to any sample treatment (Chymotrypsin,
enrichment) or dilution (washing, dilution, etc.).


Test the sample PRIOR to any sample treatments or dilutions (chymotrypsin,
enrichment, washing, etc.) as test results may be impacted due to erroneous color




Distribution: All SQA Users

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