Single Cell Transcriptome Analysis of Peripheral Blood Mononuclear Cells in Freshly Isolated versus Stored Blood Samples.
Author(s): Qu HQ, Kao C, Garifallou J, Wang F, Snyder J, Slater DJ, Hou C, March M, Connolly JJ, Glessner JT, Hakonarson H
Publication: Genes (Basel), 2023, Vol. 14, Page
PubMed ID: 36672883 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare single-cell transcriptomes of case-matched peripheral blood mononuclear cells (PBMC) that were isolated immediately after blood collection with those obtained after storage of blood for 72 h at 4°C.
Conclusion of Paper
The two blood specimens stored for 72 h at 4°C prior to PBMC isolation had fewer cells (significance not evaluated) and fewer numbers of five cell types (CD4+ T cells, CD8+ T cells, NK cells, monocytes, and B cells) than case-matched PBMCs that were isolated immediately. Cells from specimens stored before PBMC isolation showed much higher red blood cell contamination [amplification of hemoglobin subunit alpha 1 (HBA1) gene], but the only genes differentially expressed in specimens with elevated HBA1 levels were hemoglobin genes. Single cell sequencing revealed shared and cell-type specific changes in the transcriptomes of all five PBMC subtypes. CD4+ T cells had the greatest number of genes that were differentially expressed (≥1.5-fold change) when stored and unstored specimens from both volunteers were compared, while NK cells had the fewest. Importantly, more genes were upregulated following storage than downregulated in all five PBMC cell types examined. Gene Set Enrichment Analysis revealed upregulation of the TNFA signaling pathway in stored specimens via the NFKB gene set in all five cell types examined and upregulation of the apoptosis gene set in CD4+ T-cells and monocytes.
Studies
-
Study Purpose
The purpose of this study was to compare single-cell transcriptomes of PBMCs obtained immediately after blood collection with those isolated after storage of blood for 72 h at 4°C using case-matched blood specimens collected from two volunteers. EDTA blood was collected from one healthy female and one healthy male volunteer. PBMCs were isolated from blood by Ficoll density gradient centrifugation immediately and after storage of blood for 72 h at 4°C. Isolated PBMCs were suspended in freezing media and stored in liquid nitrogen until RNA isolation. Cells were thawed, single cells were isolated, and libraries were prepared (details not provided) and then sequenced using an Illumina Hiseq2500 SBS v4 machine. Data was processed using the Cell Ranger 7.1.0 analytical pipeline. A Benjamini Hochberg corrected P-value of <0.1 was considered to be a significant change.
Summary of Findings:
Blood specimens stored for 72 h at 4°C prior to PBMC isolation had fewer cells than those isolated immediately after blood collection (significance not evaluated), with only 12.3-91.3% of each cell type found in the stored versus fresh specimen although percentages differed between specimens collected from different volunteers. Further, the stored specimen displayed much higher red blood cell contamination [amplification of hemoglobin subunit alpha 1 (HBA1) gene] than specimens processed immediately. Single cell sequencing revealed that stored specimens had both common and cell-type specific changes in the transcriptomes of all five PBMC subtypes evaluated (CD4+ T cells, CD8+ T cells, NK cells, monocytes, and B cells). CD4+ T cells had the greatest number of genes that were differentially expressed (≥1.5-fold change) when stored and fresh specimens from both volunteers were compared (72 genes, 64 increased and 8 decreased), followed by B cells (51 genes, 26 increased and 15 decreased), monocytes (29 genes, 27 increased and 2 decreased), CD8+ T cells (25 genes, 18 increased and 7 decreased) and NK cells (6 genes, 5 increased and 1 decreased). Gene Set Enrichment Analysis revealed specimens that were stored prior to PBMC isolation had evidence of upregulation of the TNFA signaling via the NFKB gene set in all five cell types examined as well as the apoptosis gene set in CD4+ T-cells and monocytes. CD4+ cells from stored specimens also displayed significant changes in expression of genes involved in the UV response, inflammatory response, KRAS signaling, apical junction, IL2 STAT5 signaling, hypoxia, P53, MYC targets, oxidative phosphorylation, E2F targets, and epithelial mesenchymal transition pathways while monocytes displayed significant downregulation of the apical junction pathway. Importantly, the TNFA signaling (quantified via the NFKB gene set) and the apoptosis gene set were not upregulated in specimens with elevated red blood cell contamination (log2 HBA1 >1); in fact, the only three genes upregulated in specimens with elevated red blood cell contamination were hemoglobin genes.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 days
3 days