Proteins are involved in many biological processes. Misfolded, truncated, or mutated proteins as well as over- or underexpressed proteins have been implicated in many diseases. Therefore, detection and quantification of proteins is extremely important. Conventional techniques such as the enzyme-linked immunosorbent assay, Western Blot, and mass spectrometry have enabled discovery and study of proteins in biological samples. However, many important proteins are present at low concentrations, rendering them undetectable using conventional techniques. Furthermore, limited ability to simultaneously measure multiple proteins in a sample has constrained our ability to fully study the proteome. In this review, we comprehensively discuss approaches for protein detection. We first discuss the fundamentals of proteins and protein assays, including affinity reagents, surface functionalization, assay formats, signal detection, and multiplexing. We then discuss the challenges with these methods and review existing met...

Highly Sensitive and Multiplexed Protein Measurements

A Kwan, RS Abraham, R Currier. JAMA. 2014;312(7):729–738

To assess the results of a spectrum of state-based newborn screening programs using the T-cell receptor excision (TREC) testing approach to develop more accurate population-based incidence of severe combined immunodeficiency (SCID) and other conditions associated with T-cell lymphopenia and document the effectiveness of early intervention in those newborns found to have a significant immunologic abnormality.

Cumulative TREC newborn screening data were evaluated from 10 states plus the Navajo Nation involving ∼3 million newborns.

This was an epidemiologic and retrospective observational study based on states invited to submit SCID screening algorithms, test performance data, and deidentified clinical and laboratory information regarding infants screened and cases with nonnormal results. The data evaluated were collected from January 2008 through …

https://pediatrics.aappublications.org/content/pediatrics/136/Supplement_3/S275.full.pdf

Newborn Screening for Severe Combined Immunodeficiency in 11 Screening Programs in the United States

pdf/site-specific-gene-editing-of-human-hematopoietic-stem-cells-u9hmb4psov.pdf

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

Advancement in leishmaniasis diagnosis and therapeutics: An update.

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naive pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

/pdf/euroflow-based-flowcytometric-diagnostic-screening-and-2dtvx2tntb.pdf

EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System

Genetic defects of the immune system are referred to as primary immunodeficiencies (PIDs). These immunodeficiencies are clinically and immunologically heterogeneous and, therefore, pose a challenge not only for the clinician but also for the diagnostic immunologist. There are several methodological tools available for evaluation and monitoring of patients with PIDs, and of these tools, flow cytometry has gained prominence, both for phenotyping and functional assays. Flow cytometry allows real-time analysis of cellular composition, cell signaling, and other relevant immunological pathways, providing an accessible tool for rapid diagnostic and prognostic assessment. This minireview provides an overview of the use of flow cytometry in disease-specific diagnosis of PIDs, in addition to other broader applications, which include immune phenotyping and cellular functional measurements.

Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies.

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Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies.