Journal of Assisted Reproduction and Genetics

Purpose: To evaluate the drop-to-drop and pellet-to-pellet repeatability and stability of frozen sperm pellets. Methods: Ten pellets were thawed per batch (low and normal concentration) and evaluated by two investigators to establish a quality control chart. Then low and normal concentration pellets were thawed and evaluated daily for 10 days by both investigators. The values for both investigators were averaged and plotted on the chart. Results: The low sperm concentration specimen had a systematic error while the normal sperm concentration specimen had a random error as well as a systematic error. The low sperm concentration specimen violated the warning rule for motility whereas the normal concentration violated the warning rule, the random error rule, and the systematic error rule when applied to motility. Conclusions: Frozen sperm pellets are not acceptable as a daily-use quality control material for semen analysis when using a computer assisted semen analyzer. INTRODUCTION The issue of quality control of semen analyses has long been a thorny one for scientists working in clinical Andrology laboratories. One of the major stumbling blocks of this issue is the imprecision of the analytical method of semen analysis. All analytical methods require the performance of quality control procedures to verify the proper functioning of the method. These control procedures require long-term, repeated analysis of control materials followed by the application of statistical procedures that will aid in the characterization of the error of the analytical method. Imprecision of an analytical semen analysis method can be divided into four areas: 1) the instrumentation used to evaluate the semen specimen, 2) the counting chamber used, 3) the differences in techniques used by individual laboratorians, and 4) the analyte. In recent years, the development of an instrument called the computer assisted semen analyzer (CASA) has greatly improved the standardization of semen analysis ( ). The CASA is primarily computer-driven and operator intervention is required only in selecting fields for analysis and reviewing results. The algorithms used to determine sperm concentration and motility characteristics are programmed into the software and cannot be changed by the operator. Analysis parameter settings (either preset by the manufacturer or developed by the operator) do not change from analysis to analysis. In addition, no external reagents are needed and no calibration of the CASA by the operator is required. 1,2 The MicroCell disposable sperm counting chamber (Conception Technologies, San Diego, CA), with its fixed cover slip and standardized chamber depth, has greatly reduced counting chamber-related error ( ). The chamber fills by capillary action and cannot be overfilled. Under filled chambers and chambers demonstrating air bubbles are readily observable and easily discarded. The CASA is compatible with MicroCells and, when they are selected in the CASA's software as the chamber to be used, calculations of sperm concentration and motility are based on the MicroCell's chamber depth of 20 μm. 2,3 Error can be introduced into an analytical method by the specimen handling techniques of the laboratorians. Failure to adequately mix a specimen prior to sampling, dilution errors, pipetting errors, and incorrect loading of counting chambers may all contribute error to a method. Proper training and periodic review of the work habits 05.08.04 09:46 Ovid: Johnson: J Assist Reprod Genet, Volume 20(1).January 2003.38–45 Page 2 of 9 http://gateway2.ovid.com:80/ovidweb.cgi of all andrology laboratory personnel will help ensure that semen analyses are performed correctly and in the same manner by all laboratorians. This similarity in performance was confirmed when we reported no differences between two investigators collecting semen analysis data ( ). 2 Characteristics of the analyte in question also may contribute error to a method, particularly if the analyte is semen. Because semen is a nonhomogenous fluid, it is difficult to obtain an even distribution of sperm cells in seminal plasma ( ). In addition, the motility of sperm decreases over time ( ). Furthermore, over time, sperm cells clump to each other and to debris in the seminal plasma ( ). Because of the aforementioned instability of semen, its suitability as a control material for semen analysis is being brought into question. 4 5 4 Characteristics of a good control material include 1) similarity to “real” patient specimens, 2) availability in large enough quantities to allow evaluation over a long period of time, 3) availability in concentrations representative of normal values or important medical decision-making levels, 4) similarity in concentration from drop-to-drop and vial-to-vial (precision), and 5) stability over a long period of use (a year or more) ( ). 6 Semen Concentration and Motility Control (CMC) (Conception Technologies, San Diego, CA) is marketed as a control material for the CASA. Semen CMC is a pelletized frozen human semen product of known sperm concentration and motility. It is available in an abnormal or “Low” concentration and motility and a “Normal” concentration and motility to provide a two-level control material. This product meets the first three requirements of a good control material described above. Whether it meets the last two requirements needs investigation. The purpose of this study is to investigate whether, when used with a stable analytical method, Semen CMC meets the fourth and fifth requirements of a good control material, i.e., similarity in concentration from drop-todrop and pellet-to-pellet and stability over a long period of use. We also want to discover if Westgard Quality Control Rules can be used, in this instance, to determine the stability of a control material rather than the stability of an analytical method. Also at issue is whether a control material of questionable stability may still be useful in a laboratory quality control program. MATERIALS AND METHODS Parameter Settings for CASA The CASA used for this study was the Hamilton-Thorne Internal Visual Optics (IVOS), Version 10.7b (Hamilton-Thorne Research, Beverly, MA). The parameter settings used for the analysis of Semen CMC in our laboratory were as follows: frames acquired: 7; frame rate: 60 Hz; minimum contrast: 30; minimum size: 2 pixels; static head size gates: 0.53–2.99; static head intensity gates: 0.45–1.23; nonmotile head size: 5 pixels; nonmotile head intensity: 100; medium path velocity (VAP) cutoff: 25.0 μm/s; low VAP cutoff: 5.0 μm/s; slow cells motile: yes; and threshold straightness: 80. The IVOS is equipped with a built-in “Playback” feature that allows the operator to monitor how well the computer is “capturing” the sperm analyzed in a particular field. Motile sperm are labeled with green tracks indicating how far they moved in the field during the 0.67 s they were analyzed by the computer. Nonmotile sperm are labeled with red dots. This labeling allows the operator to visually assess whether the computer has failed to capture sperm in the field or if debris commonly found in semen specimens has been erroneously labeled as sperm. Adjustable gates for sperm head area (μm ), image intensity, and sperm head elongation allow the operator to change these settings to give the best labeling of sperm and screen out mislabeled debris. 2 Definition and Application of Westgard Quality Control Rules In 1981, Westgard and coworkers published a series of control rules that are now commonly referred to as “Westgard Rules” ( ). When used in combination with a Shewhart quality control chart (an versus graph 6 X Y 05.08.04 09:46 Ovid: Johnson: J Assist Reprod Genet, Volume 20(1).January 2003.38–45 Page 3 of 9 http://gateway2.ovid.com:80/ovidweb.cgi demonstrating the mean and standard deviation [SD] of repeated measurements of a control product; ), Westgard Rules provide a simple statistical procedure to determine whether observed control measurements represent the stable or unstable performance of an analytical method. When used with a control material that is analyzed repeatedly over a long period of time, Westgard Rules can tell an investigator when an analysis method is “out of control” and thus, that any subsequent patient data should not be used by the physician in making diagnostic decisions. Figs. 1 and 2