Biochemically Tracked Variability of Blood Plasma Thawed-State Exposure Times in a Multisite Collection Study

Abstract

The integrity of blood plasma/serum (P/S) specimens can be impacted by preanalytical handling and storage conditions that result in thawed-state exposures (> -30°C). We recently reported a simple dilute-and-shoot, intact-protein liquid chromatography/mass spectrometry (LC/MS) assay called ΔS-Cys-Albumin that quantifies cumulative exposure of P/S to thawed conditions based on the change in relative abundance of the oxidized (S-cysteinylated) proteoform of albumin (S-Cys-Albumin) in the native sample to that of an aliquot of the sample intentionally driven to its maximum oxidation state. Herein, we evaluated the effect of prestorage delay and initial storage temperature on sample integrity by applying the ΔS-Cys-Albumin assay to a set of plasma samples (n = 413) collected under a single clinical study but from 12 different collection sites. Major differences (p < 0.0001) were observed between different groups of samples with modestly inconsistent initial handling conditions (i.e., initial processing of whole blood to plasma and placement at -80°C completed in under 3 hours, 3-13 hours, and over 17 hours). ΔS-Cys-Albumin was significantly inversely correlated with delay time at 4°C before centrifugation and total delay before final storage at -80°C (p < 0.0001). Samples from two collection sites had much lower ΔS-Cys-Albumin values relative to samples from other sites, in accordance with the fact that they were stored at -20°C for an average of 7.6 months before shipment to the central repository for final storage at -80°C. Based on the rate law for S-Cys-Albumin formation in plasma ex vivo, the average time that each plasma specimen had been exposed to the equivalent of room temperature (23°C) was back calculated from the measured ΔS-Cys-Albumin values. A survey of clinical analytes in P/S whose measured concentrations are sensitive to the initial handling/storage conditions documented in this study is provided and the ramifications of the plasma integrity findings from this multisite clinical study are discussed.

Keywords: WELCA; albumin; biospecimen integrity; plasma; thawed; ΔS-Cys-Albumin.

FIG. 1.

ΔS-Cys-Albumin concept: Half a microliter of P/S is diluted 1000-fold and injected onto a liquid chromatograph-mass spectrometer for analysis of intact albumin (brown spectrum) and determination of the percentage of albumin that is S-cysteinylated (oxidized). A 9.5-μL aliquot of the same sample is then intentionally incubated at 37°C for 18 hours to drive the percentage of albumin in the S-cysteinylated form to its maximum possible value—which is always less than 100%—and the sample is measured once again (blue spectrum). The difference between the two measurements, known as “ΔS-Cys-Albumin,” is in the range of 12%–29% for fresh plasma samples and as low as zero for samples that have been exposed to thawed conditions (> −30°C for prolonged periods of time). Since the multireaction rate law for formation of S-cysteinylated albumin at room temperature was also established, measurements of ΔS-Cys-Albumin can be related to an estimated time of exposure to the equivalent of room temperature. This figure was originally published in Jeffs et al. P/S, plasma or serum.

FIG. 2.

Effect of prestorage delay and storage temperature on ΔS-Cys-Albumin. (a) Univariate distribution of ΔS-Cys-Albumin from four sample groups with different initial handling conditions. “−80°C, <3 hours” represents the initial handling condition as samples were stored at −80°C within 3 hours from the blood draw. Similarly, “−80°C, 3–13 hours” and “−80°C, >17 hours” stand for conditions under which samples were delayed at ∼4°C before storage at −80°C for 3 to 13 hours, and longer than 17 hours, respectively. No prestorage delays of 13–17 hours occurred. Samples in the “−20°C” group were initially stored at −20°C for 42 up to 456 days before shipment to the central repository where they were stored at −80°C. Error bars represent mean ± SD, n = 124, 162, 70, and 46 for the four groups, respectively. Eleven samples were excluded from the total 413 samples due to lack of initial handling information. The yellow shaded area indicates the observed range for ΔS-Cys-Albumin in freshly collected plasma from 97 nonacute cardiac patients (12%–29%); the ΔS-Cys-Albumin reference range has not yet been established in cancer patients, but is not expected to differ (cf. Fig. 6). The different letters above the data points in panel a indicate statistically significant differences between groups (p < 0.0001 for each between-group comparison; Kruskal−Wallis test followed by the Benjamini−Hochberg false discovery correction procedure). (b) ROC curves for the three groups compared with the group with ideally handled samples (−80°C, <3 hours). Areas under the ROC curves are provided in parenthesis next to the specified stages. For all three ROC curves, p < 0.0001. ROC, receiver operating characteristic; SD, standard deviation.

FIG. 3.

Correlation between ΔS-Cys-Albumin and (a) precentrifugation delay (excluding specimens with a postcentrifugation delay longer than 1.5 hours or that were temporarily stored at −20°C), or (b) total prestorage delay (excluding specimens that were temporarily stored at −20°C). Panels include data from both case and control specimens. Spearman's rank correlation coefficients are provided above the data points. “****” next to the coefficients in (a, b) indicates that the Spearman's rank correlation was statistically significant with p < 0.0001.

FIG. 4.

Univariate distributions of ΔS-Cys-Albumin at (a) the different WELCA study collection sites, and (b) from different collection batches at site 18. The letters at the top of each set of data points within each panel demonstrate statistically significant differences between groups (p < 0.05; Kruskal−Wallis test followed by the Benjamini−Hochberg false discovery correction procedure); any overlap in letters indicates a lack of significant differences between groups (p ≥ 0.05).

FIG. 5.

Estimate of plasma sample exposure times to the equivalent of 23°C based on the rate law for formation of S-Cys-Albumin. The curved line traced out by the data points represents the rate law-predicted ΔS-Cys-Albumin value in a plasma sample with population-wide average initial concentrations of albumin, cystine, cysteine, and copper. The upper curved dashed line was calculated based on starting albumin and total copper concentrations of 2 SDs below the population mean but with a cystine concentration 2 SDs above the population mean; the lower curved dashed line was similarly calculated based on starting albumin and total copper concentrations of 2 SDs above the population mean but with a cystine concentration 2 SDs below the population mean. Thus, for each data point, the horizontal range between the two dashed black dashed lines represents the population-wide possible error for the calculated time of exposure to the equivalence of room temperature. Even though ΔS-Cys-Albumin should, theoretically, always be positive, some slightly negative ΔS-Cys-Albumin values were observed due to a modest degree of analytical error.

FIG. 6.

Univariate distribution of (a) S-Cys-Albumin and (b) ΔS-Cys-Albumin for lung cancer patients and controls. Letters above the data points indicate small but statistically significant differences (p < 0.01; Mann–Whitney tests) between case and control groups. n = 207 for controls and n = 206 for lung cancer patients.