Prolonged Rewarming Was Associated with Reduced Alkaline Phosphatase Bias in External Quality Assessment Materials: A Laboratory Study

Recurrent negative alkaline phosphatase (ALP) bias in external quality assessment (EQA) materials despite stable internal quality control prompted investigation of handling conditions. This study examined elapsed time after removal from frozen storage in five independent EQA materials. Materials were separately aliquoted (200 µL) and stored at -20 °C before testing. Different aliquots were analyzed on system Y at 30, 40, 50, and 60 min after removal, following routine shaking at 40 rpm for the first 20 min. Environmental records documented ambient temperatures of 20-25 °C; specimen temperatures were not measured. ALP and six comparison enzymes were measured in technical duplicate; 30-min ALP means were submitted for the first 2026 EQA round. Relative bias was calculated against method- or peer-group consensus targets, with materials as independent statistical units. Mean ALP bias (± standard deviation) changed from -6.7% ± 1.1% at 30 min to -1.7% ± 1.0% at 50 min and -1.3% ± 1.2% at 60 min. The exploratory 30-to-50-min comparison showed less negative bias in all five materials: mean paired change, 4.9 percentage points (t-based 95% confidence interval, 4.3-5.6; paired t-test: t = 21.248, df = 4, p < 0.001; two-sided exact sign test: p = 0.063). Comparison enzymes provided descriptive context without formal between-enzyme testing. Two subsequent EQA rounds yielded mean biases of 1.6% ± 1.2% and -0.2% ± 0.7%; different materials precluded paired comparisons or intervention validation. Longer elapsed time was associated with reduced negative ALP bias in these previously frozen materials. No matched refrigerated aliquots were tested, and commutability with patient specimens was not assessed. Measurement order remained confounded with elapsed time. Findings do not establish the original deviation's cause, improved analytical trueness, or an optimal rewarming interval. They support investigation of material-specific EQA handling; extrapolation to patient specimens requires separate evidence.