Molecular- and Flow Cytometry-based Diagnosis of Primary Immunodeficiency Disorders

Abstract

Primary immunodeficiencies are an expanding group of genetic disorders resulting in recurrent and/or severe infections, autoimmunity, or autoinflammation. The laboratory plays a critical role in the diagnosis of these conditions given their frequently overlapping signs and symptoms. We discuss here advances in flow cytometry and molecular techniques applied to the study of primary immunodeficiencies.

Determination of lymphocyte subset reference ranges in peripheral blood of healthy adults by a dual-platform flow cytometry method

“…This method has emerged as a useful screening approach to evaluate whether specific cell populations and subpopulations are present or absent, which has been useful to clarify or assist diagnosis of leukemias, lymphomas, and immunodeficiencies [1]. For diagnosis purposes and/or treatment decisions, flow cytometry results should be compared with a reference interval [2][3][4] and, due to the characteristics of different population, it is recommended that each laboratory establish their own reference range [2,5].…”

Primary immunodeficiency

“…Other important diagnostic tools include lymphocyte proliferation assays and flow cytometry which allow for the enumeration of B-cells, T-cells, and NK, and the evaluation of lymphocyte markers, T-cell variability, and adhesion receptors that may be associated with specific immune defects. Standard flow cytometry analysis is abnormal in most cases of SCID and in many cases of CID [6,13]. …”

“…In some cases, more advanced testing involving specialized molecular methods may be required to confirm a diagnosis of PID [13]. Once the diagnosis is established, it is important that therapy be initiated as soon as possible, since delays can lead to permanent organ damage or even death from overwhelming infection [5].…”

Contribution of high‐throughput DNA sequencing to the study of primary immunodeficiencies

“…As a result, the number of genes identified as responsible for PIDs has rapidly increased . Although Sanger DNA sequencing remains the reference method for the molecular diagnosis of PIDs, physicians have recently begun using NGS for this purpose, particularly for cases in which the evaluation of multiple genes by Sanger sequencing would be required to obtain a diagnosis (reviewed in ). NGS techniques include whole‐exome sequencing (WES), which can be used to detect mutations in protein‐coding and RNA‐coding genes, and whole‐genome sequencing (WGS), which can be used for comprehensive sequencing of the entire genome, including introns.…”