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Multicolor Flow Cytometry and Cytokine Analysis Provides Enhanced Information on Kidney Transplant Biopsies.

ABSTRACT: Introduction:Current processing of renal biopsy samples provides limited information about immune mechanisms causing kidney injury and disease activity. We used flow cytometry with transplanted kidney biopsy samples to provide more information on the immune status of the kidney. Methods:To enhance the information available from a biopsy, we developed a technique for reducing a fraction of a renal biopsy sample to single cells for multicolor flow cytometry and quantitation of secreted cytokines present within the biopsy sample. As proof of concept, we used our technique with transplant kidney biopsy samples to provide examples of clinically relevant immune information obtainable with cytometry. Results:A ratio of CD8+ to CD4+ lymphocytes greater than or equal to 1.2 in transplanted allografts is associated with rejection, even before it is apparent by microscopy. Elevated numbers of CD45 leukocytes and higher levels of interleukin (IL)-6, IL-8, and IL-10 indicate more severe injury. Antibody binding to renal microvascular endothelial cells can be measured and corresponds to antibody-mediated forms of allograft rejection. Eculizumab binding to endothelial cells suggests complement activation, which may be independent of bound antibody. We compared intrarenal leukocyte subsets and activation states to those of peripheral blood from the same donor at the time of biopsy and found significant differences; thus the need for new techniques to evaluate immune responses within the kidney. Conclusion:Assessment of leukocyte subsets, renal microvascular endothelial properties, and measurement of cytokines within a renal biopsy by flow cytometry enhance understanding of pathogenesis, indicate disease activity, and identify potential targets for therapy.

SUBMITTER: Muczynski KA

PROVIDER: S-EPMC6035131 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Multicolor Flow Cytometry and Cytokine Analysis Provides Enhanced Information on Kidney Transplant Biopsies.

Muczynski Kimberly A KA Leca Nicolae N Anderson Arthur E AE Kieran Niamh N Anderson Susan K SK

Kidney international reports 20180303 4

<h4>Introduction</h4>Current processing of renal biopsy samples provides limited information about immune mechanisms causing kidney injury and disease activity. We used flow cytometry with transplanted kidney biopsy samples to provide more information on the immune status of the kidney.<h4>Methods</h4>To enhance the information available from a biopsy, we developed a technique for reducing a fraction of a renal biopsy sample to single cells for multicolor flow cytometry and quantitation of secre ...[more]

PMID: 29989006

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Project description:BackgroundCytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).ResultsThree sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells.ConclusionICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Dataset Information

Multicolor Flow Cytometry and Cytokine Analysis Provides Enhanced Information on Kidney Transplant Biopsies.

Publications

Multicolor Flow Cytometry and Cytokine Analysis Provides Enhanced Information on Kidney Transplant Biopsies.

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