Potential Factors for Poor Reproducibility of In Vitro Hemolysis Testing

Abstract

In vitro testing of hemolysis is essential for the validation and development of ventricular assist devices. However, as many factors influence hemolysis, such tests’ inter- and intralaboratory reproducibility is poor. In this work, CentriMag blood pumps were used to conduct a hemolysis study according to ASTM F1841 with blood from 23 bovine donors. Complementary blood analysis, including cell count, plasma composition, and viscosity, was performed to identify factors relevant to the variability of hemolysis testing results. Three strategies were tested to improve reproducibility: albumin supplementation, maintaining glucose concentration, and replacement of plasma with plasma-like buffer. Differences in red blood cell stability among donors were responsible for the largest portion of the total variance. Hematocrit varied widely among donors, and its adjustment to a standard value led to the artificial introduction of between-donor differences, especially in viscosity. It seems likely, that a more careful selection of donors with similar characteristics or repeated blood collection from the same donor could improve reproducibility. However, no direct correlations were found between the susceptibility to hemolysis and individual donor or blood characteristics in this study. The addition of albumin and glucose had a negligible effect while washing blood samples with artificial plasma significantly reduced mean hemolysis, although not its variation. The findings contribute to the understanding of variability in hemolysis experiments and give reason to question the common standard operating procedures, such as hemodilution or maintaining glucose concentration. To confirm the factors identified here, additional studies isolating the effects of individual factors are necessary.

Mechanical stress‐induced hemolysis of bovine blood is donor‐dependent

“…It was hypothesized recently that a more careful selection of donors with similar characteristics or repeated blood collection from the same donor could improve the assay reproducibility. 12 However, no related result has been published to support the hypothesis so far. Based on our study and the available data, the bovine donor age, body weight, blood gas, and glucose values once within the wide range, and native hematocrit upon adjusting to around 31% to 32%, these variables do not seem to be the decisive factors on blood hemolysis behavior.…”

“…To our knowledge, there has been no prior study or data to clearly demonstrate that the blood drawn repeatedly from the same donor on different days exhibited consistent hemolysis behavior in dynamic in vitro hemolysis testing. It was hypothesized recently that a more careful selection of donors with similar characteristics or repeated blood collection from the same donor could improve the assay reproducibility 12 . However, no related result has been published to support the hypothesis so far.…”

“…The heterogeneity of blood, even from same species, significantly contributes to the poor comparability of hemolysis test results obtained from a laboratory on different days 5,12 . A large difference in hemolysis using bovine blood from 23 donors tested on 12 different days was recently reported 12 . The results suggested that inherent differences between animals contributed more to the overall variability.…”

“…5,12 A large difference in hemolysis using bovine blood from 23 donors tested on 12 different days was recently reported. 12 The results suggested that inherent differences between animals contributed more to the overall variability. It was thought that red blood cell stability was responsible for the largest portion of the total variance.…”

Effectiveness of Erythrocyte Morphology Observation as an Indicator for the Selection and Qualification of Blood in a Mechanically Induced Hemolysis Test

“…Application of human blood is a potential strategy for overcoming interspecies differences during testing; however, securing the volume required is difficult, and regulatory challenges, such as Institutional Review Board (IRB) approval, may hamper product safety evaluation processes. Therefore, some studies have compared blood characteristics in vitro using various animal blood samples as alternatives to human blood [10][11][12][13][14][15][16].…”