Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Robust Preanalytical Performance of Soluble PD-1, PD-L1 and PD-L2 Assessed by Sensitive ELISAs in Blood.

Author(s): Krueger K, Mayer Z, Kottmaier M, Gerckens M, Boeck S, Luppa P, Holdenrieder S

Publication: Biomedicines, 2022, Vol. 10, Page

PubMed ID: 36289796 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate potential effects of centrifugation delays (up to 7 days at 4°C or room temperature, and 48 h at 37°C), centrifugation speed (1500 g, and 6000 g versus 3000g), plasma storage (up to 24 h at 4°C or room temperature) and sample preparation (pre-analysis mixing and centrifugation) on levels of PD-1, PD-L1 and PD-L2 in heparinized plasma. Potential effects of freezing (3 h at -20 or -80°C) and up to 3 freeze-thaw cycles on levels of PD-1, PD-L1, and PD-L2 in serum, EDTA-, citrate- and heparin-plasma were also investigated.  Blood was collected from nine cardiac patients (diagnosis not specified). Unless otherwise specified, plasma was separated by centrifugation at 3000 g for 10 min within 20 min of blood draw. Plasma aliquots were then immediately stored at -80°C. PD-L1, PD-L2, and PD-1 levels were quantified in plasma by ELISA. To investigate the potential effects of a centrifugation delay, heparin blood and heparin plasma were stored at 2-8°C, room temperature, and 37°C for 0, 3, 6, 24, 48, and 168 h (2-8°C and room temperature only) and levels of PD-1, PD-L1, and PD-L2 were quantified by ELISA after plasma separation. To investigate the effects of centrifugation speed and post-separation storage, plasma was separated from blood aliquots by centrifugation at 1500 g, 3000 g, and 6000 g for 10 min and subsequently stored at room temperature for 0, 3, 6 and 24 h before freezing at -80°C. To investigate the effects of freezing, heparin, EDTA, and citrate plasma as well as serum from five cardiac patients were stored at room temperature, 2-8°C, -20°C, and -80°C for three hours before analysis. To investigate the effects of freeze-thaw cycling, heparin, EDTA, and citrate plasma as well as serum from five cardiac patients were stored at -80°C and thawed for three hours at room temperature once, twice, or three times before analysis. To investigate the effects of sample preparation, heparin plasma from two cardiac patients was stored at -80°C and then thawed and aliquots were mixed by vortex (10 sec), mixed by pipetting, or left unmixed after which aliquots were centrifuged (3000 g for 10 min) or left uncentrifuged and used directly. The acceptance criterion was defined as within 20% of the specimen processed using the standard protocol and a CV of ≤20%.

   Summary of Findings:

   Compared to plasma obtained by centrifugation at 3000 g, plasma obtained by centrifugation at 1500 or 6000 g had similar recovery of PD-L1 (97% and 97%, respectively), PD-L2 (109% and 107%, respectively) and PD-1 (97% and 102%, respectively). PD-1, PD-L1 and PD-L2 levels remained stable (within 20% of baseline) when heparinized blood was stored at 2-8°C or room temperature for ≤48 h with changes of ≤20% but more variability observed when stored for 7 days at room temperature. In contrast, when heparinized blood was stored at 37°C, PD-1 remained stable but with high variability for 24 h, and PD-L1 and PD-L2 were only stable for ≤6 h. Storage of heparin plasma at 2-8°C or room temperature had no effect on PD-1 or PD-L2 levels, but when heparinized plasma was stored at room temperature PD-L1 levels displayed a high degree of variability (>20% at all timepoints). PD-1 and PD-L2 levels were comparable when serum, heparin-, EDTA-, and citrate-plasma aliquots were stored at room temperature, -20° C, and -80° C for 3 h and were comparable to plasma specimens stored at 4°C for 3 h, but citrate plasma stored at -20 or -80°C had higher (>20%) PD-L1 levels than specimens stored at 4°C.  Generally, PD-1, PD-L1, and PD-L2 were comparable in serum -, EDTA-, and citrate-plasma subjected to ≤3 freeze-thaw cycles and serum, EDTA-, and citrate-plasma that had not undergone freeze-thaw cycling.  Comparatively, PD-1 and PD-L1 were only stable for a single freeze-thaw cycle in heparinized plasma and PD-L1 was unstable in citrated plasma. Compared to specimens that were mixed by pipetting and centrifuged before analysis, those that were in standard tubes and not centrifuged had higher levels of PD-1 and PD-L1 regardless of mixing. Acceptable levels of PD-1 were only achieved when the specimen was centrifuged and a cryotube was used. Acceptable levels of PD-L1 levels were achieved for all specimens placed in cryotubes but also for specimens in standard tubes that were centrifuged after mixing by pipette or no mixing. Acceptable levels of PD-L2 were achieved when the standard tube was mixed by vortexing and then not centrifuged or the cryotube was mixed by vortex and then centrifuged.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	ELISA

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	EDTA Citrate Heparin None
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared Centrifugation delays investigated
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Storage	Storage duration	0 h 3 h 6 h 24 h 48 h 168 h
   Storage	Storage temperature	-80°C -20°C 4°C 37°C Room temperature
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles
   Storage	Storage conditions	Standard tube Cryotube
   Analyte Extraction and Purification	Sample mixing	Pipette Vortex None
   ELISA Specific	Targeted peptide/protein	PD-1 PD-L1 PD-L2

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