Effect of the technical variability of counting chambers upon the interpretation of sperm concentration results.

RESEARCH QUESTION: Does the choice of sperm-counting chamber affect the proportion of samples generating results with an erroneous interpretation? DESIGN: Laboratories in an external quality assurance programme were sent 141 semen samples over a 12-year period and asked to return the sperm concentration and whether or not the result was abnormal. Only those using 5th edition of the World Health Organization manual (WHO5) interpretation criteria were included. Submissions from specialist fertility laboratories were used to calculate assigned values for each sample. Laboratory50 values determined the sperm concentration at which the laboratories reported a majority transition from abnormal to normal interpretations, i.e. the tipping point, which should coincide with the lower reference limit. RESULTS: The median and range of bias from the assigned values of each sample were determined for the Makler (-3.3%; -88.6% to +332.8%), haemocytometer (10.6%; -93.3% to +645.5%), Kova (+65.3%; -71.7% to +581.8%) and Vetriplast (+72.4%; -100.0% to +709.1) chambers. Laboratory50 values for the Makler (17.3  ×  106/ml), haemocytometer (13.6  ×  106/ml), Kova (10.0  ×  106/ml) and Vetriplast chambers (8.8  ×  106/ml) reflected the under- and overestimation of the chambers and confirmed a shift in the adjusted lower reference limit then used. The proportion of laboratories reporting erroneous interpretations of the four chambers for oligozoospermic samples were 10.9%, 15.1.%, 40.1% and 44.0%, respectively, and rose as the adjusted lower reference limit decreased. CONCLUSIONS: The between-laboratory and within-sample variation for all the chambers was high and remains a concern. The main impact of an increasing bias of the chambers was a lowering of the laboratory50 tipping point, resulting in an under-reporting of abnormal semen samples.

Analytical variability and interpretation of results of a 3-category sperm motility assessment: 5 years' of an Australian external quality assurance programme.

RESEARCH QUESTION: How do laboratories perform when assessing sperm motility with a 3-category system and interpreting results as per the fifth edition of the World Health Organization manual (WHO5), and will the use of a 4-category system as per the sixth edition of the WHO manual improve their performance? DESIGN: Eighty video recordings of sperm samples were sent to over 200 laboratories spanning a 5-year period for the assessment of progressive motility. The results were reviewed relative to the all-laboratory trimmed mean (ALTM) in terms of the minimum and maximum values reported, the coefficient of variation and the proportion of laboratories indicating an abnormal result. A further 20 video recordings were sent over 1 year, with 6-11 laboratories per distribution adjusting to reporting rapid progressive motility using the 4-category system. RESULTS: For the 3-category system, the videos covered a mean assessed progressive motility range of 12.0-81.1%. The mean difference between the minimum and maximum values per sample was 50.3% and the coefficients of variation were negatively correlated with the ALTM (r = -0.87, P < 0.00001). Progressive motility abnormality reporting formed a sigmoid curve, and the inflection point (50% of laboratories identifying an abnormality) gave an ALTM value of 32.01%. Preliminary results for laboratories using the 4-category system showed no performance improvement but the number of laboratories was small. CONCLUSIONS: Analytical variation can result in laboratories crossing the clinical cut-off of the lower reference limit for samples whose motility is close to the WHO5 lower reference limit, but is less important for samples with extreme values. The benefits of a 4-category motility system are yet to be shown.

Human sperm morphology assessment since 2010: experience of an Australian external quality assurance programme.

RESEARCH QUESTION: Which classification criteria of sperm normality were used after the publication of the World Health Organization (WHO) 5th Edition manual (WHO5), and how did the laboratories perform? DESIGN: Semen samples were sent to enrolled laboratories over a 10-year period for the determination of the proportion of spermatozoa with normal morphology. The coefficient of variation was used to indicate the level of precision between laboratories. RESULTS: Before the publication of WHO5, at least six different classification criteria were in use. After 2010, WHO5 was quickly adopted, with 50% of laboratories using WHO5 criteria after the first 2 years, increasing to 94% after 10 years. Reported normal forms by WHO3 and WHO4 users remained consistent; however, the morphology results for each distribution declined significantly over time for WHO5 users (P < 0.001), suggesting laboratories were becoming stricter in their identification of normal spermatozoa. The precision of WHO5 users improved over time as shown by a steady decline in the coefficients of variation. CONCLUSIONS: The introduction of WHO5 resulted in the effective adoption of its morphology classification system, with laboratories showing improved between-laboratory variation over time; however, the identification of normal forms by WHO5 users over time was inconsistent, as laboratories became stricter. Given the reduction in reported normal forms by WHO5 users, it seems that increased training of laboratory personnel or the consideration of validated objective automated analysers in the assessment of sperm morphology would seem warranted.

Performance of four chambers to measure sperm concentration: results from an external quality assurance programme.

RESEARCH QUESTION: What are the changes in the use of four types of counting chambers by laboratories enrolled in an Australian-based external quality assurance programme, and what are their accuracy and precision? DESIGN: Samples of latex beads of known concentration up to 20 × 106/ml were distributed quarterly to enrolled laboratories over a 12-year period. The results of each distribution were then used to calculate a bias relative to the target value as an indicator of accuracy and a coefficient of variation to indicate the level of precision. RESULTS: The proportion of laboratories in 2007-2008 using improved Neubauer haemocytometers (44%), Makler® (9%) and Vetriplast chambers (19%) remained constant in 2018-2019, unlike Kova chamber users (20%), which reduced. The mean (range) bias of improved Neubauer haemocytometers (-2.8% [-22.5 to +32.0%]) was less than Makler® chambers (+17.0% [-2.9 to +41.2%]), Kova chambers (+33.9% [0.0 to 115.0%]) and Vetriplast chambers (+47.9% [0.0 to 170.0%]). The coefficient of variation of improved Neubauer haemocytometers (14.6% [8.7 to 25.0%]) was less than both Vetriplast (20.7% [8.8 to 36.4%]) and Makler® (24.1% [13.6 to 48.6%]) and Kova chambers (35.5% [15.9 to 123.0%]). CONCLUSIONS: The improved Neubauer haemocytometer has been shown to be superior in accuracy and precision to the Makler®, Kova and Vetriplast chambers in their estimation of concentrations up to 20 × 106/ml. Users of Makler® chambers, specifically designed for counting spermatozoa, should take care to monitor the performance of their own chambers, whereas Kova and Vetriplast chambers (designed for microscopic urinalysis) should not be used.

Within-laboratory and between-laboratory variability in the measurement of anti-müllerian hormone determined within an external quality assurance scheme.

Ten laboratories in an external quality assurance scheme used the same assay to measure anti-müllerian hormone concentration (Beckman Coulter Gen II) and received twenty serum samples distributed over a 15 month period. The mean bias for all results was only -0.089%, but there was large coefficient of repeatability of 38.8% (sample bias ranged from -37.9% to +54.7%). While each laboratory showed good reproducibility, there was a wide range of average values relative to the consensus value from -24.0% to +22.7%. This between-laboratory variability suggests clinicians should use the same laboratory to avoid problems with result interpretation.

The use of latex beads in external quality assurance and internal quality control for routine semen analysis.

The usefulness of latex beads of defined concentration was assessed as a substitute for sperm in the performance of External Quality Assurance (EQA) and Internal Quality Control (IQC) of semen analysis. Within the EQA programme, mean±SEM bias (%) was significantly reduced in 2007 compared to 2002 for both specialist (6.0%±5.4% vs. 55.0%±5.9%) and non-specialist (18.4%±5.9% vs. 90.9%±13.4%) laboratories (both p<0.0001), indicating improved accuracy over time. Within the IQC programme, the beads were used in the appraisal of two scientists, one experienced and one inexperienced, against a known standard. Beads were also used to calibrate eleven counting chambers, resulting in one old chamber being discarded due to its poor performance. The present study has shown that the use of a defined concentration of beads is an excellent adjunct to IQC and EQA programmes enabling the performance of both people and equipment to be assessed in an objective manner.