Hidden Sources of Variability in Semen Analysis and How Laboratories Can Reduce Them

Every source of variability is an opportunity to improve consistency and accuracy throughout the analysis process.

Start before the lab:

Many people may assume the process of semen analysis begins when the sample is being evaluated, but it actually begins with the patient. In reality, several factors that influence test quality are independent of the testing method and include both patient collection and laboratory sample handling prior to analysis.

Initial sample collection

• Patients should avoid lubricants, saliva, condoms, partner assistance, etc.
• These can introduce foreign substances and bacteria into the sample.

Temperature control

• For offsite collection, transportation is critical.
• Containers should be sealed tightly and protected from sunlight.
• Patients should avoid excessive heat or cold during transport.

Chain of custody

• Ensure there are at least two identifiers on the specimen cup to verify the sample is received appropriately.

 *Even if everything is done perfectly when collecting and transporting a sample, there are still many sources of variability within the semen analysis process regardless of method. 

Sample liquefaction and proper mixing

• Common mistakes occur during sample preparation.
• Samples may be mixed upon arrival or preparation but not mixed again immediately before testing.
• Regardless of method, samples should be swirled for 30 seconds immediately before testing to improve accuracy and precision.

Test time

• Per the WHO manual for semen analysis, samples should ideally be tested within one hour of collection and should not exceed two hours before testing.

Manual method variables:      

Once a sample has been properly collected and prepared, there are still several sources of variability that are specific to manual semen analysis.

Sperm counting errors

• Statistical variability increases as fewer sperm cells are counted.
• From a statistical perspective, counting more cells improves reliability.

Chamber variability

• Chambers (Makler, hemocytometer, MicroCell, fixed coverslips, etc.) can introduce variability.
• For example, Makler chambers wear over time and are not intended to last indefinitely.

Field selection bias

• Laboratory personnel may select fields of view instead of choosing them randomly.
• It can be tempting to choose fields that are easier to count (e.g., more sperm or less agglutination).
• Fields of view should be selected randomly to avoid bias.

Motility classification

• Sperm motility should be counted, not estimated.
• Count immotile cells, immobilize the sample, count total cells, then subtract immotile cells from the total to determine motility.

 Morphology interpretation variability

• Morphology is fundamentally subjective.
• Multiple evaluation methods and criteria make morphology one of the most subjective and challenging parameters to standardize.

 Human factors

• Training, skill level, fatigue, and adherence to SOPs can all affect results.

Clerical Challenges Addressed Through Automation:

In addition to reducing analytical variability, automation helps minimize many of the clerical and administrative challenges associated with manual semen analysis, improving workflow efficiency while reducing opportunities for avoidable errors.

Dilution rates

• The need for dilutions and duplicate counts is eliminated across many parameters.

Data recording

• LIS and EMR interface capabilities can reduce data recording errors.
• MES systems offer a bidirectional API interface and a unidirectional ASTM interface for local connections.

Unit conversion

• Reduces simple errors such as confusing microliters and milliliters or count versus concentration.

 Patient results, QC, and proficiency storage

• Automated electronic record retention eliminates manual data archiving and retrieval.

 Decimal point errors

• Reduces incorrect decimal point entry.

 Routine math

• Eliminates many manual calculations that create opportunities for avoidable errors.

 Reference range interpretation

• Abnormal results based on established reference ranges can be automatically flagged.
• The SQA system highlights out-of-range parameters for laboratory personnel.

 Handwriting

• Eliminates issues caused by illegible handwritten results or notes.

 Version control and copy-and-paste errors

• Helps reduce clerical variability caused by outdated templates or copying values between documents.

 *Automated testing, particularly with LIS/EMR integration, helps address many clerical challenges that can be overlooked during manual semen analysis.

Sources of Variability in Automated Systems:

While automation helps reduce many sources of variability associated with manual semen analysis, automated systems are not without their own challenges. Understanding these factors and following established best practices can help laboratories achieve the most accurate and consistent results.

*MES makes a point to address these sources of variability during customer onboarding.

Sample handling

• Sample handling remains the highest priority during implementation.
• Reinforcing proper sample handling helps establish a level playing field before testing begins.

 Viscosity

• Viscosity can affect the SQA-iO system.
• On the waived system, all samples are treated with the liquefaction kit, which largely reduces viscosity-related issues.
• Laboratories using the moderate complexity systems must evaluate viscosity and ensure proper liquefaction.

 Debris

• Debris can affect the instrument.
• The system includes a debris scanner that uses image processing to help correct for non-cellular debris and round cells.

 Agglutination/Aggregation

• Extreme agglutination and aggregation can affect results.
• Proper liquefaction and sample mixing generally address these issues.
• Some samples may require additional mixing before analysis.

 Unidentified contamination

• Unknown material in a sample may affect an automated system more than a manual system.
• Laboratory personnel may recognize unusual contamination during manual microscopy.
• This reinforces the importance of pre-analytical sample handling recommendations (avoiding lubricants, condoms, saliva, contaminants, excessive time delays, excessive heat exposure, improperly sealed containers, etc.).

 Manufacturer’s instructions

• Regardless of method, following the manufacturer’s instructions is essential for successful implementation and operation.

 Documentation

• Pictures, videos, and comments are valuable when something unusual is observed.
• The MES system provides unlimited cloud storage for images, videos, comments, and associated sample results.
• Links can be included in the test report, allowing providers to log in remotely and review the supporting information.