Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research

Diks, Annieck M; Bonroy, Carolien; Teodósio, Cristina; Groenland, Rick J; Mooij, Bas de; Maertelaere, Emilie De; Neirynck, J.; Philippé, Jan; Órfão, Alberto; Dongen, Jacques J.M. van; Berkowska, Magdalena A.

doi:10.1016/j.jim.2019.06.007

Cited by 72 publications

(65 citation statements)

References 45 publications

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“…For many plasma analytes, refrigeration at 4 °C is preferable for stability. However, there are conflicting reports in the literature as to whether blood sample refrigeration is beneficial or detrimental for flow cytometry analysis of different PBMC subsets 17 . We found that frequencies of 3 of 26 canonical PBMC subsets surveyed by CyTOF following a 5 h processing delay at 4 °C were significantly decreased compared to 5 h RT, including live singlets, monocytes, and Treg.…”

Section: Discussionmentioning

confidence: 99%

Effects of processing conditions on stability of immune analytes in human blood

Gottfried-Blackmore

Rubin

Bai

et al. 2020

Sci Rep

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Minimizing variability in collection and processing of human blood samples for research remains a challenge. Delaying plasma or serum isolation after phlebotomy (processing delay) can cause perturbations of numerous analytes. Thus, a comprehensive understanding of how processing delay affects major endpoints used in human immunology research is necessary. Therefore, we studied how processing delay affects commonly measured cytokines and immune cell populations. We hypothesized that short-term time delays inherent to human research in serum and plasma processing impact commonly studied immunological analytes. Blood from healthy donors was subjected to processing delays commonly encountered in sample collection, and then assayed by 62-plex Luminex panel, 40-parameter mass cytometry panel, and 540,000 transcript expression microarray. Variance for immunological analytes was estimated using each individual’s baseline as a control. In general, short-term processing delay led to small changes in plasma and serum cytokines (range − 10.8 to 43.5%), markers and frequencies of peripheral blood mononuclear cell phenotypes (range 0.19 to 3.54 fold), and whole blood gene expression (stable for > 20 K genes)—with several exceptions described herein. Importantly, we built an open-access web application allowing investigators to estimate the degree of variance expected from processing delay for measurements of interest based on the data reported here.

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Section: Discussionmentioning

confidence: 99%

Effects of processing conditions on stability of immune analytes in human blood

Gottfried-Blackmore

Rubin

Bai

et al. 2020

Sci Rep

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“…Indeed, cell surface markers show varying sensitivity to cryopreservation, with some markers, such as CD62L, clearly decreasing due to protein shedding ( 31 ). Main lineage markers may also be susceptible to cryopreservation, mainly for NK cells ( 32 , 33 ) however, the analysis of cryopreserved PBMCs has been standardized for several diseases, in particular HIV infection ( 34 , 35 ). In contrast, less information is available on other markers, in particular those with a low expression, which may increase their signal due to nonspecific binding to damaged cells.…”

Section: Discussionmentioning

confidence: 99%

“…Some authors report that even the time-lapse between cell thawing and surface marker assays by cytometry can determine marker level outcomes and hamper biomarker discovery ( 33 ). Even anticoagulant use may impact downstream features of cryopreserved blood cells ( 32 – 35 ). Although the sensitivity to cryopreservation and freeze/thaw under identical conditions might be expected to impact PBMCs from any individual to the same extent, this is in fact not the case: the individual disease state itself may in principle also influence lymphocyte cryosensitivity, particularly in diseases related to immunometabolic imbalance such as ME/CFS.…”

Section: Discussionmentioning

confidence: 99%

Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Implications for Biomarker Discovery

et al. 2020

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Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmune disorder characterized by numerous symptoms of unknown etiology. The ME/CFS immune markers reported so far have failed to generate a clinical consensus, perhaps partly due to the limitations of biospecimen biobanking. To address this issue, we performed a comparative analysis of the impact of long-term biobanking on previously identified immune markers and also explored additional potential immune markers linked to infection in ME/CFS. A correlation analysis of marker cryostability across immune cell subsets based on flow cytometry immunophenotyping of fresh blood and frozen PBMC samples collected from individuals with ME/CFS (n = 18) and matched healthy controls (n = 18) was performed. The functionality of biobanked samples was assessed on the basis of cytokine production assay after stimulation of frozen PBMCs. T cell markers defining Treg subsets and the expression of surface glycoprotein CD56 in T cells and the frequency of the effector CD8 T cells, together with CD57 expression in NK cells, appeared unaltered by biobanking. By contrast, NK cell markers CD25 and CD69 were notably increased, and NKp46 expression markedly reduced, by long-term cryopreservation and thawing. Further exploration of Treg and NK cell subsets failed to identify significant differences between ME/CFS patients and healthy controls in terms of biobanked PBMCs. Our findings show that some of the previously identified immune markers in T and NK cell subsets become unstable after cell biobanking, thus limiting their use in further immunophenotyping studies for ME/CFS. These data are potentially relevant for future multisite intervention studies and cooperative projects for biomarker discovery using ME/CFS biobanked samples. Further studies are needed to develop novel tools for the assessment of biomarker stability in cryopreserved immune cells from people with ME/CFS.

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“…To gain detailed insight into the composition of the lymphocyte compartment, including robust identification of small cell populations such as plasmablasts subsets, EuroFlow developed SOPs for acquisition of high cell numbers (≥1–5 × 10 6 total nucleated cells) and/or large sample volumes (up to 2 mL per tube) (56, 63, 64). These procedures can be used for fresh (<36 h, preferably <24 h) blood and BM samples (69).…”

Section: Methodsmentioning

confidence: 99%

EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System

et al. 2019

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Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

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Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research

Cited by 72 publications

References 45 publications

Effects of processing conditions on stability of immune analytes in human blood

Effects of processing conditions on stability of immune analytes in human blood

Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Implications for Biomarker Discovery

EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System

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