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Impact of delayed PBMC processing on functional and genomic assays.

Author(s): Yi PC, Zhuo L, Lin J, Chang C, Goddard A, Yoon OK

Publication: J Immunol Methods, 2023, Vol. 519, Page 113514

PubMed ID: 37353001 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared viability, cell type profile, gene expression and functionality of PBMCs isolated from sodium heparin blood that was subjected to increasing delays to separation. The effect of RosetteSep treatment on granulocyte contamination and gene expression clustering was also investigated.  Blood was collected from 15 healthy donors into sodium heparin glass vacutainer tubes and stored at 20-25°C on a shaker. PBMCs were isolated after 2 to 48 h using either a Ficoll gradient with and/or without RosetteSep (visual inspection, flow cytometry and sequencing experiments) or Leucosep tube (visual inspection and ELISpot assays) method. Isolated PBMCs were resuspended at a concentration of 20 million cells per mL in CTL-Cryo C reagent and cryopreserved at -150°C.  PBMCs were thawed in a 37°C water bath. PBMCs were counted using a Sysmex XN-450. The success of PBMC isolation was assessed macroscopically using PBMCs isolated from the blood of 3 donors that was stored for 2, 24, and 48 h before isolation using both a Ficoll gradient and a Leucosep tube.  The immunophenotype profile was investigated by flow cytometry using the 7-Color Human Immunophenotyping Kit on PBMCs isolated from the blood of two donors; blood specimens were stored for 2, 6, 24 and 48 h at room temperature.  Single cell RNA sequencing libraries were constructed using the Chromium Next GEM Single Cell 5’ Library & Gel Bead Kit v1.1 first from PBMCs isolated from blood stored for 2, 6, 24, and 48 h (2 donors) and then from PBMCs isolated from blood stored for 4, 8, 10, and 21 h (3 donors). Libraries were sequenced at 20,000 reads/cell by MedGenome Inc. Immune function of PBMCs stored 4-48h before isolation (13 donors) was assessed by measuring Production of IFN-γ, Granzyme B, and Perforin by ELISpot assays.

   Summary of Findings:

   When blood was stored for 48 h before PBMC isolation, RBC contamination increased and the authors report “increasingly deteriorating quality of density gradient separations from both the Ficoll and Leucosep methods”. Post-thaw viability of Leucosep-isolated PBMCs was not altered when isolation was delayed by ≤24 h, but was reduced in all three specimens when isolation was delayed by 48 h. Further, leukocyte count declined and granulocyte contamination increased progressively with delay to PBMC isolation in specimens from all three donors. While the observed increase in granulocyte count with delayed PBMC isolation by Leucosep was confirmed in specimens from two donors using flow cytometry, the contamination was almost eliminated when RosetteSep treatment was performed. Single-cell sequencing was performed using approximately 6000 cells per specimen, with approximately 1500 genes detected per cell. A total of 23 different gene expression clusters were identified in the initial analysis (2, 6, 24, and 48 h timepoints) and 22 of these clusters were able to be assigned to immune cell types based on gene expression. The cluster that was not assigned to a known cell type had a higher percentage of mitochondrial genome reads, which the authors suggest indicates lower cell quality.  The 23 observed gene expression clusters were not affected by the use of RosetteSep to deplete granulocytes. Most of the changes in gene clustering were observed between the 6 and 24 h delay to PBMC isolation timepoints. In the second sequencing cohort (4, 8, 10 and 21 h), a total of 21 gene clusters were identified and assigned to immune cell types. In this second cohort, the number of cells in some clusters were reduced at the 21 h timepoint, and a trend toward a reduction in the number of expressed genes was observed at the 24 h timepoint. The strength of the correlations in gene expression decreased for all major cell types (T-cells, B-Cells, natural killer cells, monocytes and dendritic cells) when longer delays were compared to the 2 h timepoint.  Correlations in gene expression between the 2 h and 6 h delay to separation timepoints were very strong (ρ>0.9) for T-cells, B-Cells, natural killer cells, and monocytes; strong for dendritic cells (ρ>0.8); but, correlations between the 2 h and 48 h were generally ≤0.70 Some of the genes affected by a processing delay were cell type-specific while others displayed altered expression in multiple cell types. Post-thaw viability remained stable as the delay to PBMC isolation increased, but there was a trend toward fewer IFN-γ secreting cells in response to both CEF and HBsAg stimulation, perforin secreting cells in response to CEF, and granzyme B secreting cells in response to PHA-M with progressive delays to PBMC isolation.

   Biospecimens

   • Cell - White Cells
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Flow cytometry
   Cell count/volume	ELISpot
   RNA	Next generation sequencing
   Cell count/volume	Hematology/ auto analyzer
   Morphology	Macroscopic observation
   Cell count/volume	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	2 h 4 h 6 h 8 h 10 h 21 h 24 h 48 h
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Analyte Extraction and Purification	Analyte purification	RosetteSep treatment performed RosetteSep treatment not used

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