Intradonor reproducibility and changes in hemolytic variables during red blood cell storage: results of recall phase of the REDS-III RBC-Omics study

Abstract

Background: Genetic determinants may underlie the susceptibility of red blood cells (RBCs) to hemolyze in vivo and during routine storage. This study characterized the reproducibility and dynamics of in vitro hemolysis variables from a subset of the 13,403 blood donors enrolled in the RBC-Omics study.

Study design and methods: RBC-Omics donors with either low or high hemolysis results on 4°C-stored leukoreduced (LR)-RBC samples from enrollment donations stored for 39 to 42 days were recalled 2 to 12 months later to donate LR-RBCs. Samples of stored LR-RBCs from the unit and from transfer bags were evaluated for spontaneous and stress-induced hemolysis at selected storage time points. Intradonor reproducibility of hemolysis variables was evaluated in transfer bags over two donations. Hemolysis data at serial storage time points were generated on LR-RBCs from parent bags and analyzed by site, sex, race/ethnicity, and donation frequency.

Results: A total of 664 donors were successfully recalled. Analysis of intradonor reproducibility revealed that osmotic and oxidative hemolysis demonstrated good and moderate reproducibility (Pearson's r = 0.85 and r = 0.53, respectively), while spontaneous hemolysis reproducibility was poor (r = 0.40). Longitudinal hemolysis in parent bags showed large increases over time in spontaneous (508.6%) and oxidative hemolysis (399.8%) and smaller increases in osmotic (9.4%) and mechanical fragility (3.4%; all p < 0.0001).

Conclusion: Spontaneous hemolysis is poorly reproducible in donors over time and may depend on site processing methods, while oxidative and osmotic hemolysis were reproducible in donors and hence could reflect consistent heritable phenotypes attributable to genetic traits. Spontaneous and oxidative hemolysis increased over time of storage, whereas osmotic and mechanical hemolysis remained relatively stable.

Fig. 1.

RBC-Omics subject enrollment, characteristics recall, and hemolysis variable testing. (A) Flow chart of the RBC-Omics study cohort and donor testing for hemolysis; (B) donor enrollment and recall statistics stratified by self-identified group; (C) count of number of recalled participants successfully completing each of the hemolytic tests at the three time points. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 2.

Correlation between hemolysis variables in parent versus transfer bags at 39 to 42 days using samples from recalled donors. Scatter plots shown for percent spontaneous storage hemolysis (A), percent osmotic hemolysis (B), percent oxidative hemolysis (C), and percent mechanical hemolysis (D). Percent hemolysis of blood samples derived from parent unit is shown on the x-axis and that from transfer bag on y-axis. Pearson correlation statistics, p values, and sample sizes are shown on each panel.

Fig. 3.

Intraassay correlations for screening and recall results from transfer bags.Scatter plots shown for percent spontaneous storage hemolysis (A), percent oxidative hemolysis (B), and percent osmotic hemolysis (C), with hemolysis of blood samples derived from transfer bag from the enrollment donation shown on the x-axis and that of recall donation shown on the y-axis. Pearson correlation statistics and p values are shown on each panel.

Fig. 4.

Hemolysis variables over time of storage in the recall population by site, sex, race/ethnicity, and donation frequency. Line plots showing the mean and confidence interval of percent spontaneous storage hemolysis (A) and percent stress-induced hemolysis (B) for oxidative hemolysis, osmotic hemolysis, and mechanical hemolysis, stratified by site (Row 1), by race/ethnicity (Row 2), or by sex (Row 3). Mechanical hemolysis measurements from BCP were excluded from plotting due to difference in assay performance at the laboratory testing for BCP donations. Measurements were taken at three time points (Postcollection Days 10 [range, 8-12 days], 20 [range, 18-23 days], and 42 [range, 39-42 days]).