Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Abstract

bCryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosisinducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-␥]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r ‫؍‬ 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P < 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3 ؉ CD8 ؉ amine-positive cells was found before cryopreservation (P ‫؍‬ 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-␥-producing CD3؉ cells after stimulation compared with healthy controls and convalescent-phase patients (P < 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho ‫؍‬ ؊0.56; P ‫؍‬ 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs. Cryopreservation is the maintenance of cells and biological tissues at low temperatures and is based on the use of various media or solutions that form hydrogen bonds with water molecules, preventing cellular damage. The low temperatures allow the cells to enter a quiescent state in which cellular functions are suspended without affecting their intrinsic characteristics (1). Peripheral blood mononuclear cells (PBMCs) are frequently cryopreserved for use in transplants or immunological studies (2, 3). However, the cryopreservation process may affect viability, phenotype, and cellular functionality due to factors such as inadequate temperatures, the freezing protocol used, the expertise of the personnel, and freezing time (4, 5). The disease of the individual from whom the PBMCs come also affects cryopreservation. For example, PBMCs from subjects infected with human immunodeficiency virus (HIV) presented reduced viability after cryopreservation, possibly due to the increased numbers of apoptotic cells circulating during the course of the disease (6). Similar findings have been found in the acute phases of diseases, such as visceral leishmaniasis (7). Particularly for HIV, great efforts have been undertaken to optimize the evaluation and comparability of immune tests...

Quantification of dengue virus specific T cell responses and correlation with viral load and clinical disease severity in acute dengue infection

“…This difference in experimental approach may have affected the cytokine production profile of the responding T cells in the different disease states. In addition, as the viability and function of T cells have been shown to be affected in those with acute dengue infection [46], we used freshly isolated PBMCs in all our experiments to limit extraneous cellular stress in contrast to previous studies [15, 21, 45, 47].…”

Cell-Based Blood Biomarkers for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

“…It would be useful to examine the viability levels of frozen lymphocytes in similar diseases to assess how specific the elevated death rate of frozen lymphocytes is to ME/CFS patients. It has been documented previously, that frozen PBMC viability is also reduced in paediatric Dengue fever [38]. However, the strength of the frozen lymphocyte viability test is its high sensitivity as a screening step to successfully detect ME/CFS individuals and correctly triage true-positive ME/CFS samples towards the subsequent confirmatory tests.…”

Cell-Based Blood Biomarkers for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

“…It would be useful to examine the viability levels of frozen lymphocytes in similar diseases to assess how specific the elevated death rate of frozen lymphocytes is to ME/CFS patients. It has been documented previously that frozen PBMC viability is also reduced in paediatric Dengue fever [44]. However, the strength of the frozen lymphocyte viability test is its high sensitivity as a screening step to successfully detect ME/CFS individuals and correctly triage true-positive ME/CFS samples towards the subsequent confirmatory tests.…”