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A Study on Whole Blood Cryopreservation for Immunophenotyping by Flow Cytometry and Its Application on COVID-19 Research

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Title: A Study on Whole Blood Cryopreservation for Immunophenotyping by Flow Cytometry and Its Application on COVID-19 Research
Author(s): Nguyen, Ngoc Anh
Contributor: University of Helsinki, Faculty of Medicine
Degree program: Master's Programme in Translational Medicine
Specialisation: Cross-disciplinary translational medicine
Language: English
Acceptance year: 2019
Immunophenotyping by flow cytometry (FC) is an established practice to identify immune cells and their cellular changes at the single-cell level. Since preserving the structural integrity of cellular epitopes is vital for immunophenotyping, samples should be processed shortly after being collected. However, the requirements of complex facilities and trained personnel for flow cytometry make it challenging to handle samples immediately. Fixation and cryopreservation extend sample shelf life and allow analysing longitudinal samples simultaneously while minimizing technical variation. Nevertheless, usage of whole blood cryopreservation in flow cytometry is limited due to challenges in preserving epitope structures during fixation and detecting dim antigens. This thesis investigates the performances of four commercial whole blood cryopreserving kits; 1) Cytodelics, 2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), 3) Proteomic stabiliser (PROT-1), and 4) Transfix. Peripheral blood samples were processed with these stabilising buffers immediately after the collection and cryopreserved until further analysis by flow cytometry. Here, we measured the stability of major immune lineages, T cell subpopulations, and activated neutrophil profiles in samples treated with these commercial whole blood stabilisers. Our flow cytometry data showed that PROT-1, Transfix and Cytodelics maintained the distribution of major leukocyte subsets – granulocytes, T cells, natural killer cells and B cells, comparable to unpreserved samples despite the attenuation of fluorescence intensities. Moreover, these three stabilisers also preserved phenotypes of activated neutrophils upon stimulation with N-Formylmethionyl-leucyl-phenylalanine and Lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16) and granule proteins (CD66b) as well as the shedding of surface L-selectin (CD62L) on activated neutrophils was conserved most efficiently in PROT-1, followed by Cytodelics. On the other hand, none of the stabilisers provided a reliable detection of CCR7 for accurate quantification of T cell subpopulations. COVID-19 is caused by a highly transmissible and pathogenic coronavirus, so-called severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). To test the potential of whole blood cryopreservation kits for flow cytometry in COVID-19 research, we studied the detectability of major leukocyte lineages and granulocyte subsets in longitudinal patient samples processed with Cytodelics. High dimensional analysis with Uniform Manifold Approximation and Projection (UMAP) and Self-Organising Maps (FlowSOM) clustering revealed remarkable stability of CD3, CD15, and CD14 expression in samples stored with Cytodelics. It allowed the detection of lymphopenia and emergency granulopoiesis often found during the acute phase of severe SARS-COV-2 infection. Nonetheless, we could not determine signatures of granulocyte subsets, notably suppressive neutrophils, during the acute and convalescent phases of COVID-19. Variable detection of lowly expressed markers and diminished fluorescence intensities in Cytodelics - preserved samples might have hindered the analysis. In conclusion, this study demonstrates that PROT-1, Transfix, and Cytodelics enabled reliable detection of highly expressed leukocyte markers, whereas SLSS-V2 preservation resulted in the most inaccurate identification of studied markers. Notably, our results show that Cytodelics can be applied in COVID-19 studies to immunophenotype major immune lineages by flow cytometry. Nevertheless, more optimisation is needed for less abundant or fixation-sensitive epitopes to enhance the efficacy of whole blood cryopreservation for flow cytometry.
Keyword(s): Cryopreservation Immunophenotyping Flow Cytometry Immune lineages Lymphocytes Granulocytes COVID-19 suppressive neutrophils

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