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.

 

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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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SQA-V GOLD Morphology Algorithm Compliance with WHO 4th and 5th (strict Kruger) Assessment Criteria

 

Issue date: October 25, 2012

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

 

Background:

 

The SQA-V GOLD system reports “Normal Morphology” in accordance with the WHO 4th or 5th edition manual criteria (based upon the selected SQA-V software version - 2.48 reports WHO 4th and version 2.60 reports WHO 5th criteria). Both WHO 4th and 5th edition guidelines and the SQA-V morphology are based on assessing sperm in compliance with strict Kruger criteria.

 

WHO 4th ed. manual, p. 19: "Strict criteria should be applied when assessing the morphological normality of the spermatozoon (Menkveld et al., 1990)". Then the classification scheme is described. "This classification scheme requires that all borderline forms be considered abnormal (Kruger et al., 1986; Menkveld et al., 1990). Using these criteria of classification, there are data to show the predictive value of sperm morphology for fertilization in vitro (Kruger et al., 1986, 1988; Kobayashi et al., 1991; Enginsu et al., 1991; Liu & Baker, 1992; Ombelet et al., 1995)".

 

As it is seen above, the WHO 4th edition manual refers to the following publications regarding morphology assessment: Kruger et al., 1986; Menkveld et al., 1990. The WHO 5th edition manual sites the following references: Kruger et al., 1986; Menkveld et al., 1990; Coetzee et al., 1998. In both manuals, it is recommended that all borderline forms should be considered abnormal. The basic criteria for Kruger’s method of assessing normal spermatozoa, is to identify as normal those spermatozoa that have the potential to successfully migrate through the cervical mucus on the way to the egg. This is different from previous methods that evaluate sperm based on shared traits among a group of presumably fertile men rather than the Kruger method for assessing the potential for functionally successful sperm to fertilize an egg.

 

The only difference between WHO 4th and 5th criteria is based on the cut-off for normal.
The SQA-V GOLD morphology algorithm was developed by assessing stained smears of semen samples under the microscope in compliance with the WHO manual guidelines for assessing normal morphology per strict criteria (Kruger). In parallel, the varying electronic signals generated by the motion patterns of sperm cells in relation to the large overall cell population were recorded (10,000+ spermatozoa analyzed in the 300 micron depth SQA-V testing capillary). The SQA-V algorithm was then developed to “read” these electronic signals and “report” them as a % normal morphology in accordance with the microscope readings performed on the same samples according to the WHO 4th and 5th edition. Thus, the % normal morphology reported by the SQA-V is in compliance with strict Kruger criteria and follows the same basic premise, which is to look at the potential for the sperm to functionally migrate through the cervical mucus on the way to fertilize an egg. For this reason, the SQA-V GOLD reports Normal vs. Abnormal morphology as opposed to a full Morphology differential of specific defects.

The SQA-V GOLD also has a 500x visualization system that allows the operator to verify the test results by microscopic examination. Based on both the automated reading and the ability to verify the results visually, the SQA-V can be used effectively to evaluate % normal morphology and provides the ability to reflux to the onboard visualization when indicated.

The Cleveland Clinic discusses the SQA-V morphology in the article published in Fertility and Sterility: "Automation is the key to standardized semen analysis using the automated SQA-V sperm quality analyzer". Agarwal A. and Sharma RK concluded that the automated SQA-V analyzer is more precise and shows the ability to accurately classify normal versus abnormal sperm morphology (Fertil Steril. 2007 Jan;87(1):156-62).

Akashi T. et al. investigated the usefulness of the sperm quality analyzer SQA-V for the assessment of sperm quality in infertile men. Significant correlations of sperm concentration (p < 0.0001), sperm motility (p < 0.0001), and normal morphology (p < 0.0001) were observed between SQA-V variables and manual semen analysis (Arch. Androl. 2005 Nov-Dec;51(6):437-42).

Shimada T et al showed that morphology reported per Kruger strict criteria by the SQA-V was predictable for fertilization and pregnancy rate outcome (Jpn J Fertil Steril 2000;45:95-100).

Additional publications and the complete Cleveland Clinic article can be found by following this link: http://www.mes-global.com/studies

 

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SQA-V users have asked MES a number of questions concerning the QwikCheck™ Liquefaction kit. We have compiled a list of those questions and our tech support answers to assist with the use of this product:

 

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Question: When should more than 1 Vial of QwikCheck™ Liquefaction kit powder be used to treat a sample?

 

Answer: For samples with a volume of 5ml or less, 1 Vial should be used. For samples over 5ml total volume, 2 Vials may be used for treatment.

Question: Does the use of the QwikCheck™ Liquefaction kit affect the pH of the sample? If so, should we perform the pH testing before and/or after the liquefaction powder has been added?

 

Answer: Use of the QwikCheck™ Liquefaction kit does not affect pH. However, it might affect the testing outcome when using Urine Test Strips or MES QwikCheck™ Test Strips (adverse chemical reaction). Therefore, if using Urine Test Strips or the QwikCheck™ Test strips for pH and WBC, use the strips before the Liquefaction kit has been implemented.

Question: How soon after treating the sample with the QwikCheck™ Liquefaction kit (time-frame) should we run Motility testing? If we use more than one vial, should we wait any additional time?

 

Answer: No, you should not wait additional time if multiple vials are used. Sample analysis should begin immediately after Liquefaction per the QwikCheck Liquefaction Kit Product Insert.

Question: If the Viscosity is normal, but gel clumps remain, should we still use a vial of Liquefaction?
Answer: Per WHO 5th manual (1) Normal, liquefied semen samples may contain jelly-like granules (gelatinous bodies) which do not liquefy. However, these gelatinous bodies do not appear to have any clinical significance. The presence of mucus strands, however, may interfere with semen analysis. So to make the semen sample more uniform and eliminate sample preparation variables, it is recommended that samples of this kind be treated with the QwikCheck™ Liquefaction kit.Question: When a highly viscous sample is treated with the QwikCheck™ Liquefaction kit, will motility increase slightly due to the drop in viscosity and, as a result will the % normal morphology also increase slightly? Question: I believe that the liquefaction kit contains an enzyme. If that is true, doesn’t it have the potential to break down the spermatozoa? If so, what may be affected? The head? The neck? The tail? Lastly, will it change the morphology?

 

Answers to both questions above: Even though “slightly" is used to describe the impact to motility, it is very difficult to characterize the exact impact to motility for all samples due to the fact that each semen sample is different in terms of viscosity level and reaction to sample treatment.  The active ingredient in the QwikCheck™ Liquefaction Kit is chymotrypsin. This is an enzyme with “proteolytic” activity (http://en.wikipedia.org/wiki/Proteolysis). Chymotrypsin performs limited proteolysis. As semen viscosity is associated with glycoproteins, limited proteolysis for the treatment of viscous semen samples is optimal. The limited proteolysis caused by chymotrypsin cannot destroy the spermatozoa, because the cells are protected by an external phosphorlipid membrane that cannot be cleaved by the protease.  There are a number of publications which address the use of limited proteolysis for the treatment of viscous and incompletely liquefied semen samples for both analytical and sperm preparation purposes. The WHO 5th edition manual recommends treating highly viscous or samples with delayed liquefaction with a proteolytic enzyme (1). Further, it was demonstrated that chymotrypsin had no effect on the detection of sperm parameters and biochemistry markers, and could be used to treat non-liquefied samples before semen analysis in the andrology laboratory (2).

Initiating limited proteolysis on high viscosity semen specimens with a-chymotrypsin was shown to be an effective treatment. Treating viscous samples with a-chymotrypsin can significantly improve the sample handling and preparation process. Furthermore, limited proteolysis of high viscosity semen samples using a-chymotrypsin resulted in the recovery of both a better quality and a higher number of spermatozoa which can be used for assisted reproduction.(3).

Other information of note: Anti-sperm antibodies in semen have been associated with a decrease in fertility potential. Treatment of these sperm samples with chymotrypsin/galactose resulted in increased pregnancy rates by IUI insemination (4). As anti-sperm antibodies are proteins, the positive effect of chymotrypsin treatment may be explained by its proteolytic activity which destroys the anti-sperm antibodies.

 

It was also shown that the outcome of a sperm penetration assay was improved by treating the semen samples with chymotrypsin (5).

The conclusions based on the findings described in the cited publications are summarized below.
Treatment of highly viscous and incompletely liquefied semen samples with chymotrypsin results in:

 

• A more homogenous spread of spermatozoa in the semen volume. This results in a decreased statistical error when assessing semen (the testing sample is more representative of the overall sample).

 

• More reliable sample handling and semen analysis

 

• No or VERY minimal effect on semen parameters (test results)

 

• Improved outcome for assisted reproductive techniques

 

• Increased pregnancy rates when used to treat semen that contains anti-sperm antibodies

References:
1. WHO, Laboratory Manual for the Examination and Processing of Human Semen. Fifth edition ed. 2010, Geneva: World Health Organization.
2. Chen F, Lu JC, Xu HR, Huang YF, Lu NQ. Chymotrypsin effects on the determination of sperm parameters and seminal biochemistry markers. Clin Chem Lab Med., 2006;44(11):1335-9.
3. P.M. Zavos, P.N. Zannakoupis-Zavos, J.K. Correa. Effect of treatment of seminal viscosity difficulties with α-chymotrypsin on the recovery of spermatozoa for assisted reproductive technologies: comparison between the SpemPrep filtration and Percoll gradient centrifugation methods. Middle East Fertility Society Journal, 1997;21(3):223-9.
4. A. Bollendorf, J.H. Check, D. Katsoff, A. Fedele. The use of chymotrypsin/galactose to treat spermatozoa bound with anti-sperm antibodies prior to intra-uterine insemination. Hum. Reprod., 1994; 9(3):484-8.
5. K.L. Honea, V.L. Houserman, D.C. Merryman, D.A. Free, S.E. Stringfellow. Effect of limited proteolysis with α-chymotrypsin on semen with an abnormal sperm penetration assay and possible application for in vitro fertilization or intrauterine insemination. Journal of Assisted Reproduction and Genetics, 1993;10(4):255-60..

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According to the SQA-V GOLD specifications, it is essential that semen samples are not heated prior to testing. The SQA-V GOLD is calibrated to conduct tests at room temperature: 22-26ºC (68-79ºF). Heating a sample prior to analysis in the SQA-V GOLD impacts the motility of the sample. When heated, the spermatozoa use the nutrients in the seminal plasma at a different metabolic rate than when the sample is not heated. The SQA-V GOLD is programmed to read non-heated samples, run within one hour of collection. The SQA-V algorithms convert electronic signals produced by non-heated spermatozoa into clinical parameters. To establish these algorithms with a very high level of accuracy and precision, samples at ROOM TEMPERATURE were tested on the SQA-V and compared to samples manually tested at ROOM TEMPERATURE following WHO testing protocols. The problem with pre-heating samples for SQA-V testing is twofold: Because there is no heating stage in the SQA-V GOLD, the temperature of a pre-heated sample will drop when running the sample in the system resulting in an unpredictable distortion of the test results (with particular impact to PROGRESSIVE motility). Also, if the sample is heated for a prolonged time, the motility may actually decrease over time as the seminal plasma nutrients are metabolized by the sperm cells at a faster rate. Therefore, for both routine SQA-V semen testing and for running comparisons of the SQA-V to manual test results, only non-heated samples should be tested or the results may differ.

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Background: On occasion, laboratories may need to perform quality control for the QwikCheck Test Strips (pH and WBC) manufactured by Medical Electronic Systems, Ltd. in order to insure they are reporting test results accurately. Please follow the instructions below to prepare the reagents and QC the test strips:

 

 

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