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.

Keywords: blood plasma; clinical chemistry; glutathione; pre-analytical error; quality control; redox status.

Figure 1

Concentrations of both GSH and GSSG increase over preparation delay time in plasma most likely due to hemolysis. (A) Absolute GSH concentration (left), GSSG concentration (middle), and GSH/GSSG ratio (right) content in the plasma of 20 volunteers after their blood was incubated for 0, 60, 120, and 180 min prior to plasma preparation. Points represent mean values (n = 19 or 20) ± S.E.M on which linear regression was applied. (B) Lactate dehydrogenase activity assay (left panel, n = 14) suggests an increase in LDH activity in plasma over time (after 3 h of incubation at RT, *: p-value < 0.05). In comparison, GSH concentration increased with a steeper slope over time, and the difference of the start and endpoint was considerably larger as reflected by the lower p-value (right panel, n = 20, ****: p-value < 10⁻⁴).

Figure 2

Addition of NEM to the blood tubes does not influence common clinical blood parameters. (A) The unsupervised PCA scores plot shows the very high similarity of all 21 clinical parameters for each of the 20 volunteers (connected by the gray line) when measured from tubes spiked with PBS (circles) or with NEM (diamonds). Samples are colored according to gender (female red, male blue), showing the expected strong group separation. (B) OPLS-DA scores plot reconfirm the PCA findings that clinical parameters significantly differ between gender, while (C) the addition of NEM to PBS has no significant impact ((A,C) values in Supplementary Materials, Data 1). (D) Heatmaps with hierarchical clustering show the similarity of obtained values between tubes with PBS or NEM, always clustering both samples from the same volunteer together.

Figure 3

Direct comparison of clinical parameters measured from NEM- or PBS-spiked tubes. The linear correlation was calculated separately per gender, R² values are given in blue for males and red for females, the corresponding transparent stripes mark the 95% confidence intervals.

Figure 4

Summary of the workflow and the obtained results. Blood from 20 healthy volunteers was collected in pre-NEMylated blood collection tubes (2.5 mM final NEM concentration). At regular intervals (0, 60, 120, and 180 min), an aliquot of collected blood from each volunteer was spun down to collect the plasma, which was then used for further analysis and revealed an increase in GSH concentration in a time-dependent manner.