Laboratory detective work identifies a mishandling problem in sample aliquoting.

Area of Science:

• Biochemistry
• Clinical Chemistry
• Oncology Research

Background:

• Ovarian cancer biomarker studies rely on accurate measurements of analytes like CA125II.
• Previous studies using Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial serum aliquots showed unexpectedly low CA125II concentrations compared to prior measurements from the same subjects.
• This discrepancy raised concerns about potential compromise during the sample aliquoting process.

Purpose of the Study:

• To investigate whether serum aliquots used in a prior ovarian cancer biomarker study were compromised during the aliquoting process.
• To assess the impact of sample handling on CA125II concentration measurements.
• To determine if inadequate mixing of parent vials contributes to CA125II concentration gradients in stored serum.

Main Methods:

• CA125II concentrations were measured in "sister" vials (aliquoted during the same process as the reference study) and "cousin" vials (newly aliquoted from a different parent vial of the same blood draw) from 15 healthy controls.
• The study assessed CA125II concentration gradients among sister vials aliquoted sequentially.
• Statistical significance was determined using the Wilcoxon signed-rank test.

Main Results:

• Mean CA125II concentration was significantly higher in sister vials compared to cousin vials across all 15 subjects (p<0.001).
• The mean coefficient of variation for CA125II was higher in sister vials (0.25) than in cousin vials (0.11) (p<0.008).
• A concentration gradient was observed among sister vials, with the 5th aliquot showing a mean CA125II concentration 1.66 times higher than the 3rd aliquot (p<0.001).

Conclusions:

• The data suggest that parent serum vials were inadequately mixed before aliquoting, leading to CA125II concentration gradients.
• CA125II may partially precipitate during long-term storage, exacerbating concentration differences.
• Rigorous vortexing of serum samples after thawing and before aliquoting is critical to ensure accurate biomarker measurements in future studies.