Abstract
Background: During sampling and processing, blood samples can be affected by hemolysis. Information is lacking regarding hemolysis for biobank samples. There is a need for a method that can easily measure hemoglobin as an indicator of hemolysis in stored samples before they are included in research projects. In this study we present a simple method for estimating hemolysis and investigate the effect of centrifugation speeds and temperatures on sample turbidity that commonly interferes with measurements. Methods: Using a variation of the Beer-Lambert law, we quantified the hemoglobin concentration in 75 long-term stored samples at a wavelength of 414 nm with a NanoDrop™ 8000 spectrophotometer. Owing to interference from turbidity, the samples underwent different treatments post-thawing: centrifugation at 10,000 and 20,000 g at two different temperatures (4°C and 19°C) for 15 minutes. In addition, freshly collected serum samples (n = 20) underwent a single freeze-thaw cycle, with hemoglobin measured prefreeze, post-thaw, and postcentrifugation. Kruskal-Wallis rank sum test groups and pairwise Wilcoxon rank test were used for statistical analysis. Results: A strong effect of centrifugation on the turbidity was shown for the long-term stored samples, however, this effect was independent of the temperature or centrifugation speeds. Centrifugation at 20,000 g for 15 minutes at 19°C reduced the turbidity up to 50%. A single freeze-thaw cycle in the fresh samples increased the optical density at 414 nm slightly, indicating a false increase of hemoglobin concentration. The following centrifugation reduced the concentration to less than the initial sample measurements, suggesting the presence of interference immediately after sampling. Conclusion: We describe here a simple and cost-effective NanoDrop-based method for measuring hemolysis levels intended for use in biobank facilities. We found that centrifugation, but not temperature, is a crucial step to reduce interference from turbidity.
Highlights
In recent decades, biobank material has become increasingly important in medical and clinical research
Blood sampling is a manual process that can introduce several preanalytical errors, including hemolysis, which can influence the measurement of serum components.[22,23]
Hemolysis is a well-recognized problem in analytical methods of serum samples, and information regarding the degree of sample hemolysis in research biobanks can be very valuable
Summary
Biobank material has become increasingly important in medical and clinical research. There is a need for a method that can measure hemoglobin as an indicator of hemolysis in stored samples before they are included in research projects. In this study we present a simple method for estimating hemolysis and investigate the effect of centrifugation speeds and temperatures on sample turbidity that commonly interferes with measurements. Owing to interference from turbidity, the samples underwent different treatments post-thawing: centrifugation at 10,000 and 20,000 g at two different temperatures (4°C and 19°C) for 15 minutes. The following centrifugation reduced the concentration to less than the initial sample measurements, suggesting the presence of interference immediately after sampling. Conclusion: We describe here a simple and cost-effective NanoDrop-based method for measuring hemolysis levels intended for use in biobank facilities. But not temperature, is a crucial step to reduce interference from turbidity
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