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. 2023 Dec;103(12):992-1003.
doi: 10.1002/cyto.a.24793. Epub 2023 Sep 28.

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

Claire Imbratta  1 , Anele Gela  1 , Nicole Bilek  1 , Simbarashe Mabwe  1 , Yolundi Cloete  1 , Rasmus Mortensen  2 , Álvaro H Borges  2 , Pholo Maenetje  3   4 , Mandla Mlotshwa  3   4 , Gavin Churchyard  3   4 , Lwitiho Sudi  5 , Issa Sabi  5 , Peter Meewes  6 , Carole L Wallis  6   7 , Mark Hatherill  1 , Thomas J Scriba  1 , Elisa Nemes  1
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Free article

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

Claire Imbratta et al. Cytometry A. 2023 Dec.
Free article

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, 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 versus 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.

Keywords: absolute cell count; clinical trials; fixed whole blood; flow cytometry; immunophenotyping; leukocytes.

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