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PLoS One. 2019 May 22;14(5):e0217163. doi: 10.1371/journal.pone.0217163. eCollection 2019.

Standardisation of flow cytometry for whole blood immunophenotyping of islet transplant and transplant clinical trial recipients.

Author information

1
Centre for Transplant and Renal Research, Westmead Institute for Medical Research, Sydney, Australia.
2
Flow Cytometry Core Facility, Westmead Institute for Medical Research, Sydney, Australia.
3
School of Mathematics and Statistics, The University of Sydney, Sydney, Australia.
4
Westmead Clinical Schools, Faculty of Medicine and Health, The University of Sydney, Sydney, Australia.
5
Centre for Kidney Research, Children's Hospital at Westmead, Sydney, Australia.

Abstract

Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50-300 μl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation.

Conflict of interest statement

The authors have declared that no competing interests exist.

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