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Application of Flow Cytometry in Primary Immunodeficiencies: Experience From India.

ABSTRACT: Primary immunodeficiency diseases (PID) are a clinically and immunologically heterogeneous group of disorders of immune system. Diagnosis of these disorders is often challenging and requires identification of underlying genetic defects, complemented by a comprehensive evaluation of immune system. Flow cytometry, with its advances in the last few decades, has emerged as an indispensable tool for enumeration as well as characterization of immune cells. Flow cytometric evaluation of the immune system not only provides clues to underlying genetic defects in certain PIDs and helps in functional validation of novel genetic defects, but is also useful in monitoring immune responses following specific therapies. India has witnessed significant progress in the field of flow cytometry as well as PID over last one decade. Currently, there are seven Federation of Primary Immunodeficiency Diseases (FPID) recognized centers across India, including two Indian Council of Medical research (ICMR) funded centers of excellence for diagnosis, and management of PIDs. These centers offer comprehensive care for PIDs including flow cytometry based evaluation. The key question which always remains is how one selects from the wide array of flow cytometry based tests available, and whether all these tests should be performed before or after the identification of genetic defects. This becomes crucial, especially when resources are limited and patients have to pay for the investigations. In this review, we will share some of our experiences based on evaluation of a large cohort of hemophagocytic lymphohistiocytosis, severe combined immunodeficiency, and chronic granulomatous disease, and the lessons learned for optimum use of this powerful technology for diagnosis of these disorders.

SUBMITTER: Madkaikar MR

PROVIDER: S-EPMC6581000 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Application of Flow Cytometry in Primary Immunodeficiencies: Experience From India.

Madkaikar Manisha Rajan MR Shabrish Snehal S Kulkarni Manasi M Aluri Jahnavi J Dalvi Aparna A Kelkar Madhura M Gupta Maya M

Frontiers in immunology 20190611

Primary immunodeficiency diseases (PID) are a clinically and immunologically heterogeneous group of disorders of immune system. Diagnosis of these disorders is often challenging and requires identification of underlying genetic defects, complemented by a comprehensive evaluation of immune system. Flow cytometry, with its advances in the last few decades, has emerged as an indispensable tool for enumeration as well as characterization of immune cells. Flow cytometric evaluation of the immune syst ...[more]

PMID: 31244832

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Project description:PURPOSE:While there is an urgent need for diagnosis and therapeutic intervention in patients with primary immunodeficiency diseases (PIDs), current genetic tests have drawbacks. We retrospectively reviewed the usefulness of flow cytometry (FCM) as a quick tool for immunophenotyping and functional assays in patients suspected to have PIDs at a single tertiary care institute. METHODS:Between January 2001 and June 2018, patients suspected of having PIDs were subjected to FCM tests, including lymphocyte subset analysis, detection of surface- or intracellular-target proteins, and functional analysis of immune cells, at Samsung Medical Center, Seoul, Korea. The genetic diagnosis was performed using Sanger or diagnostic exome sequencing. RESULTS:Of 60 patients diagnosed with definite or probable PID according to the European Society of Immune Deficiencies criteria, 24 patients were provided with useful information about immunological dysfunction after initial FCM testing. In 10 patients, the PID diagnosis was based on abnormal findings in FCM testing without genetic tests. The FCM findings provided strong evidence for the diagnosis of severe combined immunodeficiency (n = 6), X-linked chronic granulomatous diseases (CGD) (n = 6), leukocyte adhesion deficiency type 1 (n = 3), X-linked agammaglobulinemia (n = 11), autoimmune lymphoproliferative syndrome-FASLG (n = 1), and familial hemophagocytic lymphohistiocytosis type 2 (n = 1), and probable evidence for autosomal recessive-CGD (n = 2), autosomal dominant-hyper-immunoglobulin E (IgE)-syndrome (n = 1), and STAT1 gain-of-function mutation (n = 1). In PIDs derived from PIK3CD (n = 2), LRBA (n = 2), and CTLA4 mutations (n = 3), the FCM test provided useful evidence of immune abnormalities and a tool for treatment monitoring. CONCLUSIONS:The initial application of FCM, particularly with known protein targets on immune cells, would facilitate the timely diagnosis of PIDs and thus would support clinical decisions and improve the clinical outcome.

Project description:BackgroundThere are many important clustering questions in computational biology for which no satisfactory method exists. Automated clustering algorithms, when applied to large, multidimensional datasets, such as flow cytometry data, prove unsatisfactory in terms of speed, problems with local minima or cluster shape bias. Model-based approaches are restricted by the assumptions of the fitting functions. Furthermore, model based clustering requires serial clustering for all cluster numbers within a user defined interval. The final cluster number is then selected by various criteria. These supervised serial clustering methods are time consuming and frequently different criteria result in different optimal cluster numbers. Various unsupervised heuristic approaches that have been developed such as affinity propagation are too expensive to be applied to datasets on the order of 106 points that are often generated by high throughput experiments.ResultsTo circumvent these limitations, we developed a new, unsupervised density contour clustering algorithm, called Misty Mountain, that is based on percolation theory and that efficiently analyzes large data sets. The approach can be envisioned as a progressive top-down removal of clouds covering a data histogram relief map to identify clusters by the appearance of statistically distinct peaks and ridges. This is a parallel clustering method that finds every cluster after analyzing only once the cross sections of the histogram. The overall run time for the composite steps of the algorithm increases linearly by the number of data points. The clustering of 106 data points in 2D data space takes place within about 15 seconds on a standard laptop PC. Comparison of the performance of this algorithm with other state of the art automated flow cytometry gating methods indicate that Misty Mountain provides substantial improvements in both run time and in the accuracy of cluster assignment.ConclusionsMisty Mountain is fast, unbiased for cluster shape, identifies stable clusters and is robust to noise. It provides a useful, general solution for multidimensional clustering problems. We demonstrate its suitability for automated gating of flow cytometry data.

Project description:BackgroundEosinophilia is not an uncommon clinical finding. However, diagnosis of its cause can be a dilemma once common culprits, namely infection, allergy and reactive causes are excluded. Primary immunodeficiency disorders (PID) are among known differentials of eosinophilia. However, the list of PIDs typically reported with eosinophilia is small and the literature lacks an inclusive list of PIDs which have been reported with eosinophilia. This motivated us to review the literature for all PIDs which have been described to have elevated eosinophils as this may contribute to an earlier diagnosis of PID and further the understanding of eosinophilia.MethodsA retrospective PubMed, and Google Scholar search using the terms "eosinophilia" and "every individual PID" as classified by Expert Committee of the International Union of Immunological Societies with the limit of the English language was performed. Results were assessed to capture case(s) which reported eosinophilia in the context of PID conditions. Absolute eosinophil counts (AEC) were retrieved from manuscripts whenever reported.ResultsIn addition to the typical PID conditions described with eosinophilia, we document that MHC class II deficiency, CD3γ deficiency, STAT1 deficiency (AD form), Kostmann disease, cyclic neutropenia, TCRα deficiency, Papillon-Lefevre syndrome, CD40 deficiency, CD40L deficiency, anhidrotic ectodermal dysplasia with immune deficiency, ataxia-telangiectasia, common variable immunodeficiency disorders (CVID), Blau syndrome, CARD9 deficiency, neonatal onset multisystem inflammatory disease or chronic infantile neurologic cutaneous and articular syndrome (NOMID/CINCA), chronic granulomatous disease, MALT1 deficiency and Roifman syndrome have been noted to have elevated eosinophils. Severe eosinophilia (>5.0 × 10(9)/L) was reported in Omenn syndrome, Wiskott Aldrich syndrome, ADA deficiency, autoimmune lymphoproliferative syndrome, immunodysregulation polyendocrinopathy enteropathy X-linked, STAT3 deficiency, DOCK8 deficiency, CD40 deficiency, MHC II deficiency, Kostmann disease, Papillon-Lefevre syndrome, and CVID.ConclusionsThis literature review shows that there is an extensive list of PIDs which have been reported with eosinophilia. This list helps clinicians to consider an extended differential diagnoses when tasked with exclusion of PID as a cause for eosinophilia.

Dataset Information

Application of Flow Cytometry in Primary Immunodeficiencies: Experience From India.

Publications

Application of Flow Cytometry in Primary Immunodeficiencies: Experience From India.

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