|Associate Professor Elisa Nemes||Doctor Claire Imbratta||Doctor Anele Gela|
SATVI researchers Drs. Claire Imbratta, Dr Anele Gela, Associate Professor Elisa Nemes and others have published findings from a study into the performance of a new assay which makes it possible to quantify the absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers in fixed and cryopreserved whole blood.
The research results "Qualification of the Differential Leukocyte count and Immunophenotyping in Cryopreserved ex Vivo Whole Blood Assay" have just been published in the Cytometry Part A journal.
This flow cytometry-based assay, which was reported on earlier in (Nemes, 2014), “Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE)", allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. In this study the researchers evaluated the performance of this DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites.
In this study the researchers evaluated the performance of this DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. The study also assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants.Its accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer.
Finally, they developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB.
Overall the researchers 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.
Keywords: absolute cell count; clinical trials; fixed whole blood; flow cytometry; immunophenotyping; leukocytes.
Imbratta C, Gela A, Bilek N, Mabwe S, Cloete Y, Mortensen R, Borges ÁH, Maenetje P, Mlotshwa M, Churchyard G, Sudi L, Sabi I, Meewes P, Wallis CL, Hatherill M, Scriba TJ, Nemes E. Qualification of the Differential Leukocyte Count and Immunophenotyping in Cryopreserved Ex Vivo Whole Blood Assay. Cytometry A. 2023 Sep 7
Nemes E, Kagina BM, Smit E, Africa H, Steyn M, Hanekom WA, Scriba TJ.
Differential leukocyte counting and immunophenotyping in cryopreserved ex vivo whole blood. Cytometry A. 2015 Feb;87(2):157-65.