Have you stress tested your assay?

Area of Science:

• Clinical Chemistry
• Laboratory Medicine
• Assay Validation

Background:

• High-volume clinical assays require rigorous validation to prevent systematic errors.
• Wellness screening often involves processing large numbers of specimens rapidly.
• Hemoglobin A1c (HgbA1c) assays are critical for diabetes monitoring and screening.

Purpose of the Study:

• To evaluate the performance of a high-throughput HgbA1c assay under high-volume screening conditions.
• To highlight the importance of stress testing during assay validation for clinical assays.
• To identify potential sources of bias in automated HgbA1c testing.

Main Methods:

• Over 15,000 whole blood specimens were analyzed for HgbA1c using an immunoturbidimetric method on a high-throughput automated line.
• HgbA1c population distribution was compared to NHANES data.
• Correlation studies were performed against a medium-throughput HPLC method.

Main Results:

• A shift in HgbA1c distribution towards higher values was observed compared to NHANES data.
• Stress testing revealed a 15-20% bias in the immunoturbidimetric assay when processing large batches.
• A residual bias of 5-7% persisted even after implementing a cuvette washing protocol.

Conclusions:

• High-volume, batched HgbA1c testing can introduce significant systematic bias.
• The observed bias may be attributed to the accumulation of interfering substances, such as blood cell fragments, in cuvettes.
• Early incorporation of stress testing into assay validation is essential for high-volume screening applications to ensure accuracy and efficiency.