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Flow Cytometric Analyses of Lymphocyte Markers in Immune Oncology: A Comprehensive Guidance for Validation Practice According to Laws and Standards.

ABSTRACT: Many anticancer therapies such as antibody-based therapies, cellular therapeutics (e.g., genetically modified cells, regulators of cytokine signaling, and signal transduction), and other biologically tailored interventions strongly influence the immune system and require tools for research, diagnosis, and monitoring. In flow cytometry, in vitro diagnostic (IVD) test kits that have been compiled and validated by the manufacturer are not available for all requirements. Laboratories are therefore usually dependent on modifying commercially available assays or, most often, developing them to meet clinical needs. However, both variants must then undergo full validation to fulfill the IVD regulatory requirements. Flow cytometric immunophenotyping is a multiparametric analysis of parameters, some of which have to be repeatedly adjusted; that must be considered when developing specific antibody panels. Careful adjustments of general rules are required to meet legal and regulatory requirements in the analysis of these assays. Here, we describe the relevant regulatory framework for flow cytometry-based assays and describe methods for the introduction of new antibody combinations into routine work including development of performance specifications, validation, and statistical methodology for design and analysis of the experiments. The aim is to increase reliability, efficiency, and auditability after the introduction of in-house-developed flow cytometry assays.

SUBMITTER: Lambert C

PROVIDER: S-EPMC7528430 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Flow Cytometric Analyses of Lymphocyte Markers in Immune Oncology: A Comprehensive Guidance for Validation Practice According to Laws and Standards.

Lambert Claude C Yanikkaya Demirel Gulderen G Keller Thomas T Preijers Frank F Psarra Katherina K Schiemann Matthias M Özçürümez Mustafa M Sack Ulrich U

Frontiers in immunology 20200917

Many anticancer therapies such as antibody-based therapies, cellular therapeutics (e.g., genetically modified cells, regulators of cytokine signaling, and signal transduction), and other biologically tailored interventions strongly influence the immune system and require tools for research, diagnosis, and monitoring. In flow cytometry, <i>in vitro</i> diagnostic (IVD) test kits that have been compiled and validated by the manufacturer are not available for all requirements. Laboratories are ther ...[more]

PMID: 33042129

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Project description:Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were analyzed. Using FC, BAL cells and PBMC were comprehensively characterized by cell surface markers ex vivo. An increased percentage of DH24A+ polymorphonuclear cells, and decreased percentages of CD14+ macrophages were detected in BAL from horses with sEA compared to healthy horses or horses with mEA, while lymphocyte proportions were similar between all groups. Independently of EA, macrophages in BAL were CD14+CD16+, which contrasts the majority of CD14+CD16- classical monocytes in PBMC. Percentages of CD16-expressing BAL macrophages were reduced in BAL from horses with sEA compared to healthy horses. While PBMC lymphocytes predominantly contain CD4+ T cells, B cells and few CD8+ T cells, BAL lymphocytes comprised mainly CD8+ T cells, fewer CD4+ T cells and hardly any B cells. These lymphocyte subsets' distributions were similar between all groups. After PMA/ionomycin stimulation in vitro, lymphocyte activation (CD154 and T helper cell cytokine expression) was analyzed in BAL cells of 26 of the horses and group differences were observed (p=0.01-0.11). Compared to healthy horses' BAL, CD154+ lymphocytes from horses with mEA, and CD4+IL-17A+ lymphocytes from horses with sEA were increased in frequency. Activated CD4+ T helper cells were more frequent in asthmatics' (mEA, sEA) compared to healthy horses' PBMC lymphocytes. In summary, FC analysis of BAL cells identified increased polymorphonuclear cells frequencies in sEA as established, while macrophage percentages were mildly reduced, and lymphocyte populations remained unaffected by EA. Cytokine production differences of BAL lymphocytes from horses with sEA compared to healthy horses' cells point towards a functional difference, namely increased local type 3 responses in sEA.

Project description:UNLABELLED: Claims have been made in a pilot study that a new form of short-term rehabilitation according to 'Best Practice' standards would change signs and symptoms of patients with scoliosis in the short-term. Aim of this study is to repeat the study published 2010 with a larger sample of patients using the same protocol. Both authors have undergone training in this special approach to scoliosis rehabilitation in 2010. MATERIALS AND METHODS: 34 patients with Adolescent Idiopathic Scoliosis (AIS), 32 girls and 2 boys, average age 13.7 years and an average Cobb angle of 28.7 degrees (21-43 degrees) underwent Scoliosis Short-Term Rehabilitation (SSTR) of seven days. Two days with an intensity of 3 × 90 min sessions/day, and five days with an intensity of 2 × 60 min sessions/day. Angle of trunk rotation (ATR) was measured before and after the time of treatment as well as the active correctability of the ATR after the programme as it has been done in the pilot investigation. Additionally to that, we also recorded the changes in Vital Capacity (VC) before and after the programme. RESULTS: ATR was reduced significantly from 11,5 degrees to 8,4 degrees, the active correctability as measured with the Scoliometer (TM) was also reduced significantly from the ATR after treatment 8,9 degrees to 6,5 degrees in the patients with thoracic curves. VC improved significantly (P < 0,05) from 2073 ml to 2326 ml. DISCUSSION: The results achieved in the pilot investigation published previously are repeatable. The deformity of the trunk can be reduced significantly after SSTR. During the pilot study VC was not investigated. In our study VC improved significantly. Therefore, also shorter rehabilitation times with an appropriate programme seem to be able to change signs and symptoms of a patient with scoliosis. Like the out-patient Schroth programme as described in a study from Turkey, the SSTR provides benefits leading to an improvement of the condition. CONCLUSION: Out-patient rehabilitation following the Scoliologic (TM) 'Best Practice' standards seems to provide an improvement of signs and symptoms of scoliosis patients in this study using a pre-/post prospective design. The results of the pilot study therefore seem to be repeatable.

Dataset Information

Flow Cytometric Analyses of Lymphocyte Markers in Immune Oncology: A Comprehensive Guidance for Validation Practice According to Laws and Standards.

Publications

Flow Cytometric Analyses of Lymphocyte Markers in Immune Oncology: A Comprehensive Guidance for Validation Practice According to Laws and Standards.

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