Automated Semen Analysis Validation vs Manual Microscopic Semen Analysis | SQA Sperm Quality Analyzer Comparison
The SQA Automated Sperm Quality Analyzer offers Rapid Sperm Analysis with far greater Accuracy, Precision, and Objectivity compared to Manual Methods. When Validating an Automated Semen Analysis method compared to your Manual Semen Analysis procedure it’s a good idea to brush up on your techniques to ensure a smooth validation. Below are excerpts from the WHO 5th Edition Manual for Semen Analysis which details the proper steps for counting Sperm Concentration, Motility, and Morphology. MES also publishes a Technical Bulletin that presents WHO recommended sample handling guidelines. The SQA Sperm Quality Analyzer is designed to report results based on these standards, and when followed, this will help ensure a successful Manual Semen Analysis vs SQA Automated Semen Analysis comparison. Here are some key instructions for conducting Manual Semen Analysis broken into 4 categories: SAMPLE PREPARATION, CONCENTRATION, MOTILITY, and MORPHOLOGY.
SAMPLE PREPARATION (Manual-Semen-Analysis-and-Automated-Semen-Analysis-Sperm-Preparation-and-Sample-Handling-Recommendations)
- Samples must be collected within 2-7 days of abstinence by dry masturbation without lubricants or any foreign substances, including vaginal fluid and saliva.
- Analysis should happen within one hour of collection by testing completely liquefied samples with normal viscosity.
- Liquefaction and viscosity are tested by expelling sample from the pipette. Semen should drip in individual drops or a thread less than 2cm long.
- In case of delayed liquefaction or high viscosity, the sample can be treated with the MES QwikCheck Liquefaction kit.
- Before this treatment, pH and WBC should be tested using the QwikCheck Test Strips.
- Samples should be kept at Room Temperature from collection through testing.
- Samples should be mixed thoroughly by aspirating them in and out 10x using a medium bore transfer pipette or by rotation – 10 seconds in both directions in a sample container.
- Sample should be tested right after mixing.
- The use of a 100-µm-deep improved Neubauer haemocytometer is recommended.
- Count at least 200 sperm cells per replicate (at least two replicates representing two independent dilutions is recommended).
- Prepare dilutions using at least 50 microliters of semen and count grids microscopically per WHO recommendations.
- Compare replicate counts to see if they are acceptably close (per WHO tables).
- Low Concentration (0 to 25), Medium Concentration (26 to 100), and High Concentration (100+) should be tested to demonstrate complete Linearity.
- A few negative control “Blank” samples may be included to show Lower Limit Detection.
- A “wet preparation” (standard slide, 10-microliter drop of semen, and 22mm x 22mm coverslip) should be used to create 20 µm depth for observation.
- Wait for the sample to stop drifting (within 60 seconds) before starting the count.
- Examine the slide with phase-contrast optics at ×200 or ×400 magnification.
- Assess approximately 200 spermatozoa per replicate for the percentage of different motile categories.
- Assess total Motility. Do not add up different categories, as it results in Motility overestimation.
- Compare the replicate values to check if they are acceptably close. If so, proceed with calculations; if not, prepare new samples.
- The human eye is drawn to motion, and Motility is often overestimated: “It is common to overestimate sperm motility, but this can often be avoided by reversing the order of analysis (NP and IM first), using an eyepiece reticle, and being aware of, and avoiding, to the extent possible, potential sources of bias (see Section 7.13.3 in the attached WHO Manual document)”.
- First, the Immotile Cells should be counted. Then, the sample should be immobilized (or frozen if using the SQA visualization system), and All Cells should be counted. Finally, Motility will be calculated by subtracting Immotile from the Total count.
- Motility cannot be an “estimation”; it needs to be a true count
- It is essential to count random fields and include agglutinated and aggregated fields in the count. Do not skip areas that have clumps of sperm since the SQA will analyze them.
- The SQA reports % Normal Morphology, and WHO recommendations for a “Normal Sperm” must be followed closely.
- The use of MES 1-Step Morphology Slides, Papanicolaou, Shorr, or Diff-Quik stain is recommended.
- WHO 5th Edition offers a Strict interpretation of what is considered “Normal.” Any borderline forms with even the slightest Abnormalities are to be graded as Abnormal:
- The head should be smooth, regularly contoured, and generally oval in shape.
- The midpiece should be slender, regular, and about the same length as the sperm head.
- The principal piece should have a uniform calibre along its length, be thinner than the midpiece, and be approximately 45 µm long (about ten times the head length).
- The staining quality is of high importance for correct results. In case of overstaining and non-visible acrosome, most spermatozoa will be categorized as abnormal shifting all results of tested samples towards the low end of the Normal Morphology dynamic range specified by the WHO 5th ed. manual for both fertile and infertile patients to be 0 – 30%.
- The WHO 5th Edition offers a comprehensive “Blind Challenge” to practice identifying Normal Sperm Cells.
We look forward to supporting your validation process at every turn. Please review these materials and let us know what questions, comments, or concerns you have before beginning the comparison. For an overview of Medical Electronic System’s flagship Automated Semen Analysis instrument, the SQA-Vision Automated Sperm Quality Analyzer, please check out this demonstration video:
Remember, it ALL Started with a Sperm!