Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells

Abstract

In centralized immune monitoring for a multi-center allergen immunotherapy trial, we observed frequent loss of CD4+ T cell integrity following staining of cultured PBMCs with our regulatory T cell flow cytometry panel. Samples were marked by a loss of total cellular events, altered scatter properties, and reduced CD3+CD4+ events. This occurred only in samples that were stained with Foxp3 and were therefore treated with Foxp3 fixation-permeabilization buffer. We identified granulocyte contamination in samples associated with a loss of integrity, and went on to test the impact of granulocyte depletion on day-old blood samples. Granulocyte depletion prevented loss of cell integrity and CD3+CD4+ events, and reduced variability in detection of Foxp3+ cells. Addition of purified neutrophils back to PBMCs altered scatter properties and detection of CD4+ T cells. Implementation of a granulocyte depletion step in our standard operating protocols has reduced assay failure due to loss of sample integrity from 31% to 0%. Routine incorporation of a granulocyte depletion step during PBMC isolation is recommended prior to downstream immune monitoring in blood with next-day processing.

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples

“…Furthermore, we observed an increase in LD neutrophil and CD3+ T cell populations at Day 2 in thawed PBMC from the blood that was stored at RT prior to processing when compared with samples stored at 4 • C prior to processing. It was previously published that aged blood has higher neutrophil contamination compared to blood freshly processed [17,33] Our findings report that CD4 T cell and CD8 T cell proportions remained unchanged after 2 days of blood storage prior to processing. Samples stored >24 h at RT had a decrease in B cells to 2.3% (95%CI: 0, 6.3) by day 2.…”

“…For example, a significant loss (36-56%) in IFN-γ T cell frequencies by enzyme-linked immune absorbent spot (ELISPOT) assay was observed when the blood was processed 24 h after venipuncture. These losses are coupled with increases in low density activated granulocytes, i.e., mainly neutrophils [17]. Therefore, almost all studies to date recommend a short timeframe (<8 h) between blood venipuncture and PBMC isolation.…”

Phenotypic characteristics of peripheral immune cells of Myalgic encephalomyelitis/chronic fatigue syndrome via transmission electron microscopy: A pilot study

“…PBMCs encompass a heterogeneous cell population comprising monocytes (which can be differentiated to macrophages and dendritic cells) and lymphocytes (T cells, natural killer cells, and B-cells). However, most PBMC purification methods contain a considerable amount of platelet contamination [ 57 ] as well as traces of erythrocytes, and low-density granulocytes (neutrophils, basophils, and eosinophils) [ 58 , 59 ]. The existence of a round nucleus in PBMCs can help easily distinguish most immune cells under TEM from erythrocytes and platelets, which have no nuclei and from granulocytes, which show a lobulated segmented nucleus [ 60 ] as well as various types of cytoplasmic granules.…”

Rho‐kinase pathway activation and apoptosis in circulating leucocytes in patients with heart failure with reduced ejection fraction

“…To isolate blood cells, we used ficoll gradient, which selects PBMCs including both lymphocytes (T and B) and monocytes but not neutrophils. Neutrophils can influence T cell activation and produce a marked loss of CD3+ and CD4+ T cells . Thus, to determine the influence of neutrophils on ROCK activity, it is necessary to isolate lymphocytes/monocytes from neutrophils.…”