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Effects of processing conditions on stability of immune analytes in human blood.

Author(s): Gottfried-Blackmore A, Rubin SJS, Bai L, Aluko S, Yang Y, Park W, Habtezion A

Publication: Sci Rep, 2020, Vol. 10, Page 17328

PubMed ID: 33060628 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare PBMC viability, RNA yields, RNA integrity, RNA expression profiles, and cytokine levels when blood was subjected to processing delays of up to 5 h at room temperature and 15 h at 4°C. Blood was collected between 8 and 9 AM from ten healthy subjects into Sodium Heparin and Serum BD Vacutainer tubes. Matched specimens were stored for 10 min, 2 h, or 5 h at 25°C and 10 min, 5 h, or 15 h at 4°C before PBMC, plasma, and serum separation and RNA isolation. Plasma and serum were isolated by centrifugation for 10 min at 800 and 1200 rcf, respectively, and frozen at -80°C. Cytokine levels were measured in plasma and serum using the Human 62-Multiplex Array on the Luminex 200 IS System. For RNA isolation, blood was transferred from the sodium heparin tube to PAXgene tubes, refrigerated, and then stored frozen (no details provided) before extraction using the PAXgene Blood RNA Kit. The RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer. RNA expression was investigated using the Clariom D Human Assay Microarray. PBMCs were isolated by Ficoll gradient, viability was assessed by trypan blue staining, and PBMCs in Recovery Cell Culture Freezing Medium were frozen in liquid nitrogen. PBMC viability and surface and intracellular cytokine levels were quantified by surface and intracellular cytokine mass cytometry (CyTOF) in thawed, unstimulated PBMCs or after stimulation for 4 h with PMA (10 ng/ml), ionomycin (1 µg/ml), and LPS (1 µg/ml) in the presence of brefeldin A (5 µg/ml) and monensin (5 µg/ml).

   Summary of Findings:

   The number of viable PBMCs after Ficoll separation declined significantly when blood was stored for 15 h at 4°C but was not significantly different when blood was subjected to a processing delay of 5 h at room temperature or 4°C. Although there was no change in the percentage of live singlet PBMCs counted by CyTOF after cryopreservation and thawing, regardless of stimulation, more variability in the percentage was observed in specimens subjected to a processing delay at 4°C. RNA yields and integrity (RINs) were not significantly different when blood was subjected to a processing delay but RIN variability was higher after a processing delay of ≥2 h at room temperature or 15 h at 4°C. PCA showed no effect of processing delay on variance in gene expression with very strong correlations in expression observed between specimens processed after 10 min and those processed after processing delays at 25°C (2 h and 5 h) and 4°C (5 h and 15 h) (R²=0.988-0.992). Further, PCA showed no clustering based on patients age, gender, or race. There was a trend toward lower paired correlations between control specimens (processed after 10 min) and those stored for 5 h at room temperature than other processing delays. The expression profile of housekeeping genes, cell death genes, and surface marker genes were comparable between timepoints but variance was found in levels of cytokines. Importantly, no significant changes in gene expression were found across the delay timepoints. Similarly, markers measured by CyTOF were generally unaffected by processing delay.

   Cytokine levels were poorly correlated between plasma and serum, regardless of processing delay. Less variability was observed in cytokine levels in serum than plasma but, after correcting for interindividual variability, there were more cytokines affected by processing delays in serum than plasma (18 versus 13 of 62). The changes in cytokine levels tended to be small in both serum (median − 0.4-3.1%, range − 10.8 to 43.5%) and plasma (median 0.1 to 1.7%, range − 8.4 to 19.6%). Generally, cytokines increased with pre-processing storage at room temperature but refrigeration reduced cytokines in serum and caused both increases and decreases in plasma cytokines. When the effects of processing delay were not normalized by individual, the effects of processing delay were larger (–77 to 414% in serum and −38 to 159% in plasma), but while plasma showed a trend toward increases in cytokine levels, serum showed a trend toward a decrease. The authors conclude that the majority of cytokines are stable after processing delays of up to 5 h at room temperature or 15 h at 4°C and that stability is higher at 4°C than room temperature.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen
   • None (Fresh)
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	Automated electrophoresis/Bioanalyzer
   Cell count/volume	Light microscopy
   Protein	MS/MS
   Protein	Flow cytometry
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Storage duration	10 min 2 h 5 h 15 h
   Light microscopy Specific	Technology platform	cyTOF
   Storage	Storage temperature	4°C Room temperature

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