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

Abstract

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.

OMIP‐101: 27‐color flow cytometry panel for immunophenotyping of major leukocyte populations in fixed whole blood

“…Use of cryopreserved fixed whole blood in this assay offers multiple benefits compared to other flow cytometry-based methods, including reduction of procedural complexity and workload at the point of sample collection, cost-effectiveness, improvement, and reduction of data variability, especially in extensive studies involving multiple sites in resource-limited settings. In a previous report, we demonstrated accuracy, robustness, low inter-operator variability of this method and high concordance of readouts including a wide range of absolute counts and frequencies when fresh and cryopreserved fixed WB samples from the same donors were stained with a similar 27-color panel [3]. These results suggest that fixation/ permeabilization and cryopreservation steps do not affect the detection and resolution of the selected antibody-marker combinations.…”