Abstract
Early detection of Primary Immunodeficiencies Disorders (PIDDs) is of paramount importance for effective treatment and disease management. Many PIDDs would be strong candidates for newborn screening (NBS) if robust screening methods could identify patients from dried blood spots (DBS) during the neonatal period. As majority of congenital PIDDs result in the reduction or absence of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool. Unfortunately, detection is often limited by the extremely low protein concentrations in blood cells and limited blood volume present in DBS. We have recently developed a robust novel method for quantification of low abundance proteins in DBS for PIDDs using peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-SRM). Here, we further generated a multiplexed Immuno-SRM panel for simultaneous screening of eight signature peptides representing five PIDD-specific and two cell-type specific proteins from DBS. In samples from 28 PIDD patients including two carriers, representing X-Linked Agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), X-Linked Chronic Granulomatous Disease (XL-CGD), DOCK8 Deficiency and ADA deficiency, peptides representing each disease are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. Also included in the multiplex panel are cell specific markers for platelets (CD42) and Natural Killer Cells (CD56). In patients with WAS, CD42 levels were found to be significantly reduced consistent with characteristic thrombocytopenia. A patient with WAS analyzed before and after bone marrow transplant showed normalized WAS protein and platelet CD42 after treatment highlighting the ability of immuno-SRM to monitor the effects of PIDD treatment. The assay was readily reproduced in two separate laboratories with similar analytical performance and complete agreement in patient diagnosis demonstrating the effective standardized methods. A high-throughput Immuno-SRM method screens PIDD-specific peptides in a 2.5-min runtime meeting high volume NBS workflow requirements was also demonstrated in this report. This high-throughput method returned identical results to the standard Immuno-SRM PIDD panel. Immuno-SRM peptide analysis represents a robust potential clinical diagnostic for identifying and studying PIDD patients from easily collected and shipped DBS and supports a significant potential for early PIDD diagnosis through newborn screening.
Highlights
Primary Immunodeficiency Disorders (PIDD), referred to as inborn errors of immunity (IEI), are a group of more than 416 rare genetic disorders in which components of the immune system are missing or improperly functioning
The lower limits of detection (LLOD) values were equivalent in between the 2.5- and 20min runs while the Lower limit of quantification (LLOQ) values increased in each case except for CD56 122, Adenosine Deaminase (ADA) 93, and CYBB 509 when moving to the HT method
We have demonstrated that Immuno-SRM is a highly sensitive multiplexed assay that correctly identifies patients with five genetically defined PIDDs by directly quantifying low abundance proteins present in dried blood spots (DBS)
Summary
Primary Immunodeficiency Disorders (PIDD), referred to as inborn errors of immunity (IEI), are a group of more than 416 rare genetic disorders in which components of the immune system are missing or improperly functioning. Early detection of PIDDs is extremely important in controlling and preventing potentially life-threatening infections and chronic sequelae [12, 13]. Intervention is limited by the difficulty in diagnosing PIDDs clinically and the lack of straightforward population screening tools. Laboratory evaluations are typically elicited by evidence of recurrent and/or chronic infections. Laboratory tests that are required for a diagnostic confirmation often involve technically demanding analyses, including immune cell subset analysis, protein expression, and/or enzymatic activity in patient’s white blood cells [14]. It is not possible to perform these clinical diagnostics from dried blood spots (DBS) as all the tests require either whole blood or isolated peripheral blood mononuclear cell (PBMC) samples. Genetic sequencing is most often used as a final confirmation [14]
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