TECHNICAL BULLETIN: VISCOUS AND NON-LIQUEFIED SAMPLES TREATMENT WITH CHYMOTRYPSIN

BACKGROUND:

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).

OVERVIEW: LIQUEFACTION

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.

SPERM TRANSPORT

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. http://coe.ucsf.edu/ivf/conception.html.

 

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TECHNICAL BULLETIN: USING QWIKCHECK TEST STRIPS
Thursday, October 31, 2013

BACKGROUND:
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.

OVERVEIW:
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.

INSTRUCTIONS:
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.).

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

 

DOWNLOAD THE TECHNICAL BULLETIN HERE

 

Lev Rabinovitch, CTO MEDICAL ELECTRONIC SYSTEMS
Distribution: All SQA Users

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Re-Issue date/Distribution: Tuesday, October 22nd, 2013/ALL SQA-V Users

Subject: It is recommended that twice per year, the SQA-V Gold calibration be checked against the original factory calibration parameters. Although there are acceptable calibration ranges for the SQA-V, the system parameters may be close to the high or low end of the range and proactive maintenance will ensure continued uninterrupted use and optimal clinical performance from the instrument. This is a free service to all SQA-V users.

CALIBRATION CONFIRMATION INSTRUCTIONS

Twice per year, MES recommends sending us the “Service Data Report” from your SQA-V Gold for a calibration confirmation. To print your Service Data Report and return it to MES, please follow these instructions:

• NOTE: The SQA-V internal printer will need to be loaded with paper and printer ribbon. Please see user guide for paper and ribbon loading instructions.

• Navigate to the “Service” menu, once in the “Service” menu select “Print Default Settings” and click “Print Self-Test Data”.

• Tape this report to a piece of blank white paper and fax the report to MES @ 310-670-9069 or scan/e-mail it to service@mes-llc.com. Please include an e-mail address and your contact information with the Self-Test report for follow-up.

• Your SQA-V calibration parameters will be compared to the initial calibration parameters from when the instrument was manufactured and presented back to you in report format.

 

THESE INSTRUCTIONS MAY BE DOWNLOADED HERE

Also, please remember to check the Technical Bulletins section of the website often for updates HERE.

 

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BACKGROUND:

Room temperature is a general term describing common indoor temperatures. It is usually in the range of 20 °C (68 °F or 293 K) to 25 °C (77 °F or 298 K)*

*http://en.wikipedia.org/wiki/Room_temperature

 

OVERVEIW:

The WHO 5th edition manual recommends assessing sperm motility at either room temperature or at 37 °C (with a heated microscope stage). “These conditions should be standardized for each laboratory” (WHO 5th edition manual, p. 22).

 

INSTRUCTIONS:

The SQA-V GOLD is calibrated and room temperature and has been developed to operate at room temperature conditions, so there is no heating stage on the device. When performing a validation of the SQA-V based on comparing the test results to manual results, the manual results should be performed at room temperature as well.

 

MANUFACTURER’S RECOMMENDATION:

Clinical laboratories are air conditioned facilities. It is therefore recommended to maintain the room temperature of the lab in the range of 20 °C (68 °F or 293 K) to 25 °C (77 °F or 298 K).

 

DOWNLOAD THE TECHNICAL BULLETIN HERE

Lev Rabinovitch, CTO MEDICAL ELECTRONIC SYSTEMS
Distribution: All SQA Users


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Medical Electronic Systems recommends that WHO 5th Edition (2.2 Sample collection) standards be followed for sample collection and processing for all automated and manual evaluation methods:

2.2.1 Preparation

The sample should be collected in a private room near the laboratory, in order to limit the exposure of the semen to fluctuations in temperature and to control the time between collection and analysis (see Sections 2.2.5 and 2.2.6 for exceptions).

The sample should be collected after a minimum of 2 days and a maximum of 7 days of sexual abstinence. If additional samples are required, the number of days of sexual abstinence should be as constant as possible at each visit.

The man should be given clear written and spoken instructions concerning the collection of the semen sample. These should emphasize that the semen sample needs to be complete and that the man should report any loss of any fraction of the sample.

The following information should be recorded on the report form (see Appendix 6, section A6.1): the man’s name, birth date and personal code number, the period of abstinence, the date and time of collection, the completeness of the sample, any difficulties in producing the sample, and the interval between collection and the start of the semen analysis.

2.2.2 Collection of semen for diagnostic or research purposes

The sample should be obtained by masturbation and ejaculated into a clean, wide-mouthed container made of glass or plastic, from a batch that has been confirmed to be non-toxic for spermatozoa (see Box 2.1).

The specimen container should be kept at ambient temperature, between 20 °C and 37 °C, to avoid large changes in temperature that may affect the spermatozoa after they are ejaculated into it. It must be labelled with the man’s name and identification number, and the date and time of collection.

The specimen container is placed on the bench or in an incubator (37 °C) while the semen liquefies.

Note in the report if the sample is incomplete, especially if the first, sperm-rich fraction may be missing. If the sample is incomplete, a second sample should be collected, again after an abstinence period of 2–7 days.

 

 

2.2.3 Sterile collection of semen for assisted reproduction

This is performed as for diagnostic collection (see Section 2.2.2) but the specimen containers, pipette tips and pipettes for mixing must be sterile.

2.2.4 Sterile collection of semen for microbiological analysis

In this situation, microbiological contamination from non-semen sources (e.g. commensal organisms from the skin) must be avoided. The specimen containers, pipette tips and pipettes for mixing must be sterile.

The man should:
1. Pass urine.
2. Wash hands and penis with soap, to reduce the risk of contamination of the specimen with commensal organisms from the skin.
3. Rinse away the soap.
4. Dry hands and penis with a fresh disposable towel.
5. Ejaculate into a sterile container.

 

2.2.5 Collection of semen at home

A sample may be collected at home in exceptional circumstances, such as a demonstrated inability to produce a sample by masturbation in the clinic or the lack of adequate facilities near the laboratory.

The man should be given clear written and spoken instructions concerning the collection and transport of the semen sample. These should emphasize that the semen sample needs to be complete, i.e. all the ejaculate is collected, including the first, sperm-rich portion, and that the man should report any loss of any fraction of the sample. It should be noted in the report if the sample is incomplete.

The man should be given a pre-weighed container, labelled with his name and identification number.

The man should record the time of semen production and deliver the sample to the laboratory within 1 hour of collection.

During transport to the laboratory, the sample should be kept between 20 °C and 37 °C.

The report should note that the sample was collected at home or another location outside the laboratory.

2.2.6 Collection of semen by condom

A sample may be collected in a condom during sexual intercourse only in exceptional circumstances, such as a demonstrated inability to produce a sample by masturbation.

Only special non-toxic condoms designed for semen collection should be used; such condoms are available commercially.

The man should be given information from the manufacturer on how to use the condom, close it, and send or transport it to the laboratory.

The man should record the time of semen production and deliver the sample to the laboratory within 1 hour of collection.

During transport to the laboratory, the sample should be kept between 20 °C and 37 °C.

The report should note that the sample was collected by means of a special condom during sexual intercourse at home or another location outside the laboratory.

 

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TECHNICAL BULLETIN UPDATE:

 

Red Blood Cells in Semen Samples Analyzed by the SQA-V Instrument - (VERSIONS: All Human SQA-V GOLD, SQA-V and Spermalite Systems)

 

Tuesday, June 18, 2013

 

BACKGROUND:

The SQA-V utilizes frequencies of light optimized for the analysis of sperm cells only. Red blood cells (RBCs) are not ‘counted’ during the assessment of sperm cell concentration or other relevant clinical parameters. However, in extreme conditions when semen contains a gross amount of RBC’s (3.0~4.0 M/mL+) the results may be influenced slightly by this large quantity of Red Blood Cells.

 

MANUFACTURER’S RECOMMENDATION:

It is recommended in all cases where a laboratory is uncertain about the number of RBC’s in a sample, to quickly view the sample in the SQA-V visualization system. If a quantitative count is required, prepare a standard slide per the SQA-V user guide instructions, and count the number of relevant cells. Remember to use 10 microliters of sample on a 1” x 3” slide with a 22x22mm cover slip. ZOOM OUT to x300 and count each RBC in the visualization field. Each cell counted represents 1M/ml.

 

If a large amount of red blood cells are visualized – it is recommended the sample be tested manually.

 

For additional Questions or Concerns please contact MES: service@mes-llc.com

 

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For Manual and Automated Semen Analysis:

 

The nature of the liquefied ejaculate makes taking a representative sample of semen for analysis problematical. If the sample is not well mixed, analysis of two separate aliquots may show marked differences in sperm motility, vitality, concentration and morphology. To be certain of obtaining reproducible data, the sample should be thoroughly mixed before aliquots are taken for assessment.

 

Sample Mixing Instructions:

 

Before removing an aliquot of semen for assessment, mix the sample well in the original container, but not so vigorously that air bubbles are created. This can be achieved by aspirating the sample 10 times into a wide-bore (approximately 1.5 mm diameter) disposable plastic pipette (sterile when necessary). Do not mix with a vortex mixer at high speed as this will damage spermatozoa.

 

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Issue date: January 31, 2013

 

Background
QwikCheck™ beads are an in-vitro use only external quality control material for automated and manual sperm counting systems. The beads are used as a tool to assess the accuracy and precision of the laboratory’s sperm counting methods by providing a known target value and +/- range. The beads were developed for use on the SQA-V semen analyzer however, they are also labeled for manual proficiency testing and calibration on hemacytometers such as Neubauer counting chambers, Makler chambers and conventional fixed coverslip chambers. 

 

QwikCheck™ beads are supplied in a kit containing known concentrations of 4-micron latex beads suspended in an aqueous solvent and negative concentration/motility control. According to the CLIA ’88 regulations, “…for most moderately complex tests, the general requirement is to analyze two levels of QC materials on each day of testing.” It is recommended that QwikCheck™ beads be run on the SQA-V automated and visualization systems prior to each day of semen analysis testing. 

 

QwikCheck™ beads Compliance
QwikCheck™ beads comply with the WHO 3rd, 4th, 5th edition manuals and ASCP recommendations as an external quality control material. Accordingly, the WHO recommends the use of QC samples and procedures for semen analysis as follows:

 

Manufactured QC samples: Commercially available samples, manufactured and analyzed (assayed) according to manufacturing guidelines.

 

Internal quality control: Quality tests measuring the variability in a procedure that exists within a laboratory. Such tests evaluate the precision of day-to-day operations. Useful for detecting random variation (assessing precision).

 

In control: A process is in control when all values are within expected control limits.

 

Out of control: process is out of control when a measured value exceeds expected control limits, or is within control limits but shows a significant trend in values. A process that is out of control must be evaluated.

 

CLICK HERE TO DOWNLOAD THIS BULLETIN IN PDF FORMAT

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Issue date: Monday, November 26, 2012

Attn: All SQA-V /QwikCheck™GOLD (WHO 4th & 5th compliant software)

 

Background:
The WHO 5th edition manual (page 13) describes the semen liquefaction as follows:

 

“Immediately after ejaculation into the collection vessel, semen is typically a semi-solid coagulated mass. Within a few minutes at room temperature, the semen usually begins to liquefy (become thinner), at which time a heterogeneous mixture of lumps will be seen in the fluid. As liquefaction continues, the semen becomes more homogeneous and quite watery, and in the final stages only small areas of coagulation remain. The complete sample usually liquefies within 15 minutes at room temperature, although rarely it may take up to 60 minutes or more. If complete liquefaction does not occur within 60 minutes, this should be recorded.”

 

WHO 5th describes high viscosity and the effect on semen parameters (page 15):

“High viscosity can interfere with determination of sperm motility, sperm concentration, detection of antibody-coated spermatozoa and measurement of biochemical markers.”

 

Further, WHO 5th describes Delayed Liquefaction (on page 14):

 

 

Information:

 

According to the WHO 5th edition manual, highly viscous or incompletely liquefied semen samples should be treated in order to reduce their viscosity which can interfere with the accuracy of the reported semen parameters. The methods for treating high viscosity or incompletely liquefied samples differ. The most effective method is limited proteolysis by broad-specificity proteolytic enzymes like bromelain or α-chymotrypsin (QwikCheck™Liquefaction Kit).

In order to provide a complete description of the sample and any additives for the physician, the SQA-V has an entry concerning the nature of the sample LIQUEFACTION and VISCOSITY that should be assessed and entered prior to the sample treatment (see below patient / sample data entry screen).

 

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An excellent series of "Questions and Answers" that came through our Austrian Distributor regarding cutoffs and reference ranges:

 

Question:

We are presenting the SQA-V at a conference and I fear that the "reference" range cutoff values for morphology will be questioned. The technical bulletin you sent says there is high correlation between SQA and humans.....however, human semen analysis is prone to massive CV's and errors. It is hard to believe (and certainly from our data as regards motility and morphology) there is any correlation between a human and a machine value, as machines are far more accurate.

 

Answer:
WHO established reference ranges based on the results of studies conducted on a population of fertile men. What was done is that the lowest 5% of the values (5th centile) from this group was used as the lower reference limit (please see below). This is a purely clinical study involving the semen assessment of men with PROVEN FERTILITY which is quite different from the scope of the SQA-V technology. The SQA-V technology provides automated semen analysis comparable to the manual standards, based on the WHO guidelines for semen analysis. The SQA-V technology is therefore presented as comparable to the current laboratory standard (manual) with decided advantages such as: Automation, speed, ease of use, standardization, objectivity, precision, and accuracy that is not depend on the proficiency level of the operator. Because the only current globally recognized standard is manual semen analysis based on WHO guidelines, this was used as the gold standard in the SQA-V algorithm development trials. Without a standard with which to develop an automated system, the results will be far from reality and will not stand up to validation or comparison studies. Hence, in the course of algorithm development, MES strictly followed the WHO recommendations for manual semen analysis. As a result, the manual results used for establishing the SQA-V algorithms are (and have proven to be) more accurate than would be expected from any given laboratory. Fact is, this algorithm development is not a simple correlation, but a much more complex and interrelated process.

Reference ranges and reference limits. Data characterizing the semen quality of fertile men, whose partners had a time to pregnancy of 12 months or less, provided the reference ranges for this manual. Raw data from between about 400 and 1900 semen samples, from recent fathers in eight countries on three continents, were used to generate the reference ranges. Conventional statistical tradition is to take the 2.5th centile from a two-sided reference interval as the threshold below which values may be considered to come from a different population. However, a one-sided reference interval was considered to be more appropriate for semen, since high values of any parameter are unlikely to be detrimental to fertility. The 5th centile is given as the lower reference limit, and the complete distribution for each semen parameter is also given in Appendix 1. (WHO 5th ed., p. 3).
Comment 7: There may be regional differences in semen quality, or differences between laboratories; laboratories should consider preparing their own reference ranges, using the techniques described in this manual. (WHO 5th ed., p. 224).

 

Question:

If the results with consistent human counting errors were used to determine the lowest 5th-centile WHO 5th morphology value of 4% and if the SQA-V doesn’t have massive errors, what is the lowest 5th-centile range from the SQA analyzed samples? It would have to be different from the manual analyses. So if the accuracy is improved using a SQA, then the precision may be lifted (or lowered) to form a level that over a large number of samples becomes the new reference level.

Answer:
Just to reiterate a point, the WHO reference limits were established based on the test results from a population of HEALTY MEN with proven fertility. In contrast, the population of our clinical trials consists of men who applied for medical assistance due to infertility problems. Some of them are healthy as the fertility problem is the result of the female, others are infertile. So 5th centile in this population is different when compared to the healthy group WHO used to establish reference limits.

Please find below two distribution graphs: One shows manual and the other SQA-V Morphology assessed in a recent trial conducted in France (Nantes trial data) on 250 patients. You will noted that the distribution patterns of the manual and automated results are quite similar. Also, you will note that approximately 21% of the samples assessed manually fall below the 4% cutoff (WHO 5th) and 18% of the samples assessed automatically fall below 5% cutoff. Based on this extrapolation, 20th centile instead of 5th centile can be used in this group, and the morphology cutoff for the SQA-V can possibly be shifted from 4% to 5% if the lab will find it reasonable.

 

 

Question:

It’s impossible to believe that if there are minimal sampling errors, the human derived cut off of 4% (lowest 5% of all values analysed) is the same. Same will have to be determined for the lowest 5% of all measured values for sperm count and sperm motility, and see where the numerical cut off is for these lowest 5th-centile ranges.

Answer:
This question relates to the difference between accuracy and precision. Even if the precision of the manual method is low, averaging multiple results will bring a result close to the "true" value (accuracy). To establish a reference value, multiple results are used, so it is quite accurate even though precision may not be high. Where precision becomes very important is in routine practice where EVERY individual result needs to be reproducible. Concerning the 5th centile, I would like to emphasize that it is not applicable to patients with potential infertility problems. I think it is well demonstrated in the graphs above.

The questions at the end of your e-mail are addressed by the explanations above I believe. Please let us know if this answers your questions.

Best regards,

Dr. Lev Rabinovitch ~ Chief Technology Officer
 

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