(P-QU-1) An International Reference Protocol for Hemolysis Measurement: A BEST Collaborative Study

Red blood cell (RBC) hemolysis, a measure of RBC membrane breakdown, is a critical quality indicator for red cell concentrates (RCCs). However, despite its significance in the field of transfusion medicine, there is currently no internationally recognized reference method for hemolysis measurement. While various methods exist, the interlaboratory variability among them is high, making comparison of results between labs impractical. The objective of this study is to introduce and validate a reference method for hemolysis measurement that will allow for comparison of results between blood centers, quality testing laboratories, and industry testing facilities.

Study

Design/Methods:

Twelve international laboratories evaluated a recommended reference method alongside current in-house methods for hemolysis measurement. Reference method hemolysis was determined from measurement of (1) hematocrit by capillary tube centrifugation, (2) total hemoglobin (Hb) by a hematology analyzer, and (3) supernatant Hb prepared by centrifugation of RCC samples (2200xg, 10 min, 4°C) and measured with a HemoCue photometer. In-house methods varied among sites (ex. Drabkin’s, Harboe). Linearity of Hb measurement was determined from serially diluted samples prepared in-house (0.01 to 40 g/L Hb). Repeatability, precision (coefficient of variation, CV), and method comparison (bias) were conducted on fresh (≤8 days) and expired (centre specific) RCCs evaluated with the reference and in-house methods by 2 technicians and 10 replicates.

Results/Findings:

Linearity of HemoCue was R² ≥ 0.995 for all sites and R² ≥ 0.916 for in-house methods. Reportable range of supernatant Hb values varied between sites for the HemoCue, but all successfully obtained results in the range of 0.35 to 22.5 g/L Hb. Mean hemolysis, determined by the reference method, for fresh RCCs was 0.06% ± 0.03% and 0.17% ± 0.04% (day 35±1), 0.31% ± 0.13% (day 42±2), and 0.32% ± 0.02% (day 49) for expired RCCs. Average precision of the reference method was 26.7% CV and 8.4% CV for fresh and expired RCCs, respectively. Precision of the in-house methods ranged from 0.0 to 233.8% CV and 1.8 to 18.9% CV for fresh and expired RCCs, respectively. Bias between the reference method and in-house methods ranged from -0.13 to 0.06 and -0.13 to 0.13 for hemolysis (%) in fresh and expired RCCs, respectively.

Conclusions:

Reference method precision at low Hb concentrations (fresh RCCs) was low due to inability to read Hb values below limit of detection. However, overall analytical performance was acceptable. Further evaluations, including a send-around study between participants, are needed to establish variability of the reference method between labs. This work will establish an important reference method for international labs to use in understanding hemolysis data reported by the numerous groups performing this important RCC quality test.