A comprehensive assessment of immunophenotyping performed in cryopreserved peripheral whole blood

Verschoor, Chris P.; Kohli, Vikas; Balion, Cynthia

doi:10.1002/cyto.b.21526

Cited by 34 publications

(56 citation statements)

References 30 publications

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“…Cryopreservation also provides further benefit in that biospecimens can be analyzed in large batches, minimizing overall analytical variability; this is especially important for studies in which collection occurs over a long period of time, or a large number of samples are obtained. Recently, we and others showed that cryopreserved peripheral blood, a biospecimen that is simple and inexpensive to prepare, is comparable to fresh blood for the enumeration of a number of leukocyte types, including monocytes, neutrophils, B‐lymphocytes, T‐cells, natural killer, and (NK)‐cells; our study also showed that these measures were also comparable to that obtained from cryopreserved peripheral blood mononuclear cells (PBMCs) or a hematology analyzer. However, many of these cell types, especially monocytes, T‐cells, and NK‐cells, are known to represent distinct groups of subsets that have specific roles in maintaining health and preventing disease.…”

supporting

confidence: 72%

“…Hence, absolute estimates of that leukocyte subset (calculated as the relative proportion multiplied by the total leukocyte count) would also be inflated. Proportional estimates relative to PBMCs would be expected to be more accurate given the reduced cellular loss in the PBMC compartment (as we have shown ), which would relate to better accuracy of absolute estimates. To enumerate total PBMCs, cryopreserved blood was thawed on ice for 20 minutes and 100 μl was transferred to a 2.0 ml snap‐top tube.…”

Section: Methodsmentioning

confidence: 79%

“…A complete gating strategy can be found in Figure and a comparison of gating in cryopreserved and fresh blood in Supporting Information Figure S2. NK‐cell subsets were identified using our previously published comprehensive stain , but replacing CD123‐AlexaFluor647 with CD16‐APC (Biolegend, CA, USA). NK‐cells were CD45 + CD19 ‐ HLA‐DR ‐ CD3 – , and further divided as CD56 Bright and Dim subsets: CD56/NKp46 ++ CD16 lo/– and CD56/NKp46 + CD16 + , respectively.…”

Section: Methodsmentioning

confidence: 99%

“…To estimate the absolute count of leukocyte subsets (as opposed to the relative proportions of each subset), we first measured the absolute number of PBMCs in each blood sample using a slightly different cellular staining procedure that does not include centrifugation steps; this procedure and example calculations are provided in Supporting Information. Not including centrifugation steps is important since a significant proportion of granulocytic (neutrophils) cells in cryopreserved blood are lost during centrifugation leading to artificially reduced estimates. The rationale for basing absolute counts on PBMCs, not total leukocytes, is for that very same reason.…”

Section: Methodsmentioning

confidence: 99%

“…Cellular staining to identify monocyte, T-cell, and NKcell subsets was performed as in our previous study (5) and is described in detail in the Supporting Information. Cryopreserved blood was moved to ice from 2808C storage, left for up to 20 minutes until completely thawed and gently inverted three times to mix.…”

Section: Leukocyte Immunophenotyping Proceduresmentioning

confidence: 99%

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Cryopreserved whole blood for the quantification of monocyte, T‐cell and NK‐cell subsets, and monocyte receptor expression by multi‐color flow cytometry: A methodological study based on participants from the canadian longitudinal study on aging

Kohli

2018

Cytometry Pt A

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Immunophenotyping by multi-color flow cytometry is arguably the best tool to identify and quantify distinct cell lineages from the peripheral blood and other biological fluids/tissues. Effective in both clinical and research settings, it can be used to estimate the frequency of a given cell type or measure its phenotypic or functional properties. Normally, immunophenotyping is performed in fresh or fractionated blood (i.e., PBMCs) the same day, or within 24 hours of collection; however, this may not be feasible for all study designs. We have previously shown that cryopreserved blood, a biospecimen that is simple and inexpensive to prepare, is comparable to fresh blood for the enumeration of major leukocyte cell types. For the following study, we sought to extend these observations to distinct subsets of: monocytes (classical, intermediate, and non-classical), T-cells (CD4/CD8 naïve, central and effector memory, senescent, and terminally differentiated, and regulatory T-cells), and NK-cells (CD56 bright and dim); we also examined the expression of monocyte cell-surface receptors CX3CR1, CCR2, TLR2, and TLR4. Our results indicate that cryopreserved blood is comparable to fresh blood; with exception to relatively rare subsets and lowly expressed receptors, the absolute or relative frequency of cell subsets generally correlated >0.80 between blood types, while monocyte receptor expressed was mostly >0.70. Furthermore, the day-to-day coefficient of variation for most cell subsets and parameters was below 20%. Given these findings, we suggest that cryopreserved peripheral blood be given greater consideration for studies in which the quantification of distinct leukocyte subsets is required. © 2018 International Society for Advancement of Cytometry.

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supporting

confidence: 72%

Section: Methodsmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Leukocyte Immunophenotyping Proceduresmentioning

confidence: 99%

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Cryopreserved whole blood for the quantification of monocyte, T‐cell and NK‐cell subsets, and monocyte receptor expression by multi‐color flow cytometry: A methodological study based on participants from the canadian longitudinal study on aging

Kohli

2018

Cytometry Pt A

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A Practical Cryopreservation and Staining Protocol for Immunophenotyping in Population Studies

et al. 2018

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Large population-based cohort studies, through their prospective collection of a broad range of health information, represent an invaluable resource for novel insights into the pathogenesis of human diseases. Collection and cryopreservation of viable cells from blood samples is becoming increasingly common in large cohorts as these cells are a valuable resource for immunophenotyping and functional studies. The cryopreservation of peripheral blood mononuclear cells (PBMCs), thawing, and immunophenotyping protocols used to immunophenotype 9938 participants in the Health and Retirement Study (HRS) are described. The extensive quality control involved in a large-scale immunophenotyping epidemiological study is also outlined. The existing literature on the effect of cryopreservation on various immune cell subsets including T, B, NK cells, monocytes, and dendritic cells is provided. © 2018 by John Wiley & Sons, Inc.

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Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay

Imbratta,

Gela,

Bilek

et al. 2023

Cytometry Pt A

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We developed a flow cytometry‐based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC‐ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers.We evaluated the performance of the DLC‐ICE assay by determining inter‐operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter‐operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC‐ICE measurements to real‐time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27‐colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB.Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti‐coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti‐coagulants using the DLC‐ICE method exhibited excellent correlation with the reference method i.e complete blood count (CBC) with differential, measured using a hematology analyzer (r2>0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB vs DLC‐ICE processed blood yielded equivalent and linear results over a wide dynamic range (r2=0.94 over 10‐104 cells/μL).These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC‐ICE assay for large cohort studies involving multiple clinical research sites.This article is protected by copyright. All rights reserved.

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A comprehensive assessment of immunophenotyping performed in cryopreserved peripheral whole blood

Abstract: Given this, cryopreserved blood should be considered a feasible biospecimen in clinical and epidemiological studies requiring leukocyte immunophenotyping. © 2017 International Clinical Cytometry Society.

Cited by 34 publications

References 30 publications

Cryopreserved whole blood for the quantification of monocyte, T‐cell and NK‐cell subsets, and monocyte receptor expression by multi‐color flow cytometry: A methodological study based on participants from the canadian longitudinal study on aging

Cryopreserved whole blood for the quantification of monocyte, T‐cell and NK‐cell subsets, and monocyte receptor expression by multi‐color flow cytometry: A methodological study based on participants from the canadian longitudinal study on aging

A Practical Cryopreservation and Staining Protocol for Immunophenotyping in Population Studies

Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay

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