Blood Plasma Quality Control by Plasma Glutathione Status

Abstract

Timely centrifugation of blood for plasma preparation is a key step to ensure high plasma quality for analytics. Delays during preparation can significantly influence readouts of key clinical parameters. However, in a routine clinical environment, a strictly controlled timeline is often not feasible. The next best approach is to control for sample preparation delays by a marker that provides a readout of the time-dependent degradation of the sample. In this study, we explored the usefulness of glutathione status as potential marker of plasma preparation delay. As the concentration of glutathione in erythrocytes is at least two orders of magnitude higher than in plasma, even the slightest leakage of glutathione from the cells can be readily observed. Over the 3 h observation period employed in this study, we observed a linear increase of plasma concentrations of both reduced (GSH) and oxidized glutathione (GSSG). Artificial oxidation of GSH is prevented by rapid alkylation with N-ethylmaleimide directly in the blood sampling vessel as recently published. The observed relative leakage of GSH was significantly higher than that of GSSG. A direct comparison with plasma lactate dehydrogenase activity, a widely employed hemolysis marker, clearly demonstrated the superiority of our approach for quality control. Moreover, we show that the addition of the thiol alkylating reagent NEM directly to the blood tubes does not influence downstream analysis of other clinical parameters. In conclusion, we report that GSH gives an excellent readout of the duration of plasma preparation and the associated pre-analytical errors.

Changes in levels of the antioxidant glutathione in brain and blood across the age span of healthy adults: A systematic review

“…This preference for plasma may be attributed to some of its advantages over serum, such as a larger volume, no clotting time delay and a lower risk of haemolysis or the breakdown of erythrocytes ( Uges, 1988 ). In fact, contamination from degrading/leaking erythrocytes can artificially increase GSH and GSSG levels in both plasma and serum, given that these cells contain approximately two orders of magnitude higher glutathione than the liquid portion of blood ( Tomin et al, 2021 ). A linear increase in both plasma GSH and GSSG levels was demonstrated for blood left at room temperature over a three hour observation period, with significant increases becoming evident as early as one hour post-collection ( Tomin et al, 2021 ).…”

“…In fact, contamination from degrading/leaking erythrocytes can artificially increase GSH and GSSG levels in both plasma and serum, given that these cells contain approximately two orders of magnitude higher glutathione than the liquid portion of blood ( Tomin et al, 2021 ). A linear increase in both plasma GSH and GSSG levels was demonstrated for blood left at room temperature over a three hour observation period, with significant increases becoming evident as early as one hour post-collection ( Tomin et al, 2021 ). Ten of 16 (63%) blood studies in our review reported the time to processing of plasma or serum, and all performed this step within 20 min of blood draw ( Supplementary Table S3 ), thereby minimizing glutathione contamination from erythrocytes.…”

“…Ten of 16 (63%) blood studies in our review reported the time to processing of plasma or serum, and all performed this step within 20 min of blood draw ( Supplementary Table S3 ), thereby minimizing glutathione contamination from erythrocytes. Oxidation of GSH during sample preparation ( Nuhu et al, 2020 , Tomin et al, 2021 ) can also lead to an under-estimation of GSH and an over-estimation of GSSG concentrations ( Nuhu et al, 2020 ). While this error can be counteracted by the use of thiol-blocking (alkylating) agents ( Tomin et al, 2021 ), this information was not clearly stated by the majority of studies included in our review.…”

“…Oxidation of GSH during sample preparation ( Nuhu et al, 2020 , Tomin et al, 2021 ) can also lead to an under-estimation of GSH and an over-estimation of GSSG concentrations ( Nuhu et al, 2020 ). While this error can be counteracted by the use of thiol-blocking (alkylating) agents ( Tomin et al, 2021 ), this information was not clearly stated by the majority of studies included in our review. Given that accurate biochemical measures of glutathione (both reduced and oxidized forms) in blood are crucially dependent on proper blood-collection protocol and pre-analytical processing factors, we recommend that these details be provided by studies.…”

Direct Derivatization in Dried Blood Spots for Oxidized and Reduced Glutathione Quantification in Newborns

“…GSH quantification in whole blood is challenged by the complexity of the blood matrix as well as the rapid deterioration of the sample, including enzymatic- and non-enzymatic GSH oxidation [ 12 , 13 , 14 , 15 , 16 ]. Furthermore, GSH levels were proposed as a surrogate for assessing the time-dependent degradation of blood samples prior to plasma extraction, as even minor leakages of GSH from erythrocytes lead to an increase in plasmatic GSH levels [ 17 ]. Whilst GSH concentrations in blood plasma ranging between 2 and 20 μM have been reported in the literature, in whole blood, they are notably higher (mM range), as over 95% of GSH is located inside the erythrocytes [ 18 ].…”