TREC and KREC Levels as a Predictors of Lymphocyte Subpopulations Measured by Flow Cytometry

Abstract

Primary immunodeficiency diseases (PID) is a heterogeneous group of disorders caused by genetic defects of the immune system, which manifests clinically as recurrent infections, autoimmune diseases, or malignancies. Early detection of other PID remains a challenge, particularly in older children due to milder and less specific symptoms, a low level of clinician PID awareness and poor provision of hospital laboratories with appropriate devices. T-cell recombination excision circles (TREC) and kappa-deleting element recombination circle (KREC) in a dried blood spot and in peripheral blood using real-time polymerase chain reaction (PCR) are used as a tool for severe combined immune deficiency but not in PID. They represent an attractive and cheap target for a more extensive use in clinical practice. This study aimed to assess TREC/KREC correspondence with lymphocyte subpopulations, measured by flow cytometry and evaluate correlations between TREC/KREC, lymphocyte subpopulations and immunoglobulins. We carried out analysis of data from children assessed by clinical immunologists at Speransky Children’s Hospital, Moscow, Russia with suspected immunodeficiencies between May 2013 and August 2016. Peripheral blood samples were sent for TREC/KREC, flow cytometry (CD3, CD4, CD8, and CD19), IgA, IgM, and IgG analysis. A total of 839 samples were analyzed for using TREC assay and flow cytometry and 931 KREC/flow cytometry. TREC demonstrated an AUC of 0.73 (95% CI 0.70–0.76) for CD3, 0.74 (95% CI 0.71–0.77) for CD4 and 0.67 (95% CI 0.63–0.70) for CD8, respectively, while KREC demonstrated an AUC of 0.72 (95% CI 0.69–0.76) for CD19. Moderate correlation was found between the levels of TREC and CD4 (r = 0.55, p < 0.01) and KREC with CD19 (r = 0.56, p < 0.01). In this study, promising prediction models were tested. We found that TREC and KREC are able to moderately detect abnormal levels of individual lymphocyte subpopulations. Future research should assess associations between TREC/KREC and other lymphocyte subpopulations and approach TREC/KREC use in PID diagnosis.

Newborn Screening for Primary Immunodeficiencies: The Gaps, Challenges, and Outlook for Developing Countries

“…In spite of the added costs by measuring both TRECs and KRECs level, it allows identification of more types of PIDs such as late onset ADA deficiency and some cases of Nijmegen breakage syndrome (20). Also, measuring them is considered less expensive than flow cytometry assay, which is not only expensive, but needs a lot of training and not available in all countries (21). Adding to the merits of NBS, the cost reduction calculated for early diagnosis was $85,882 and $55,882 for patients treated with immunoglobulin replacement therapy (3).…”

Expanding TREC and KREC Utility in Primary Immunodeficiency Diseases Diagnosis

“…Parents/guardians were informed of the procedures in lay terms. The study design has been described in detail elsewhere (18).…”

“…Sample analysis was performed as described elsewhere (18). In brief, three-four color flow cytometric immunophenotyping with directly labeled monoclonal antibodies was used to determine the following immune cell subsets: CD3-CD19+, CD3-CD(16 + 56)+, CD3+CD4+, and CD3+CD8+ following manufacturer's protocol.…”

“…TREC and KREC assays were performed using realtime PCR with fluorescent hybridization probes and reagents for TREC/KREC assays: T&B PCR kit (ABV-test, Russia) (20), in whole blood. The TREC/KREC levels were assayed in whole blood samples as described previously (16,18,20), In brief, DNA was extracted from 100 µl EDTA anticoagulated whole blood by using RIBO-prep nucleic acid extraction kit (Amplisense R , Russia). The Real-time qPCR was performed using CFX 96 Real-Time PCR System (Bio Rad, USA).…”

Modern diagnostic capabilities of neonatal screening for primary immunodeficiencies in newborns

“…Flow cytometry, one of the most widely used methods for diagnosing PID, enables assessment of the immune system: specific cell populations and subpopulations, specific cell membrane, intracellular and intranuclear proteins, biological effects associated with specific immune defects, as well as certain functional immune characteristics Thus, it is a phenotypic analysis. However, despite the fact that flow cytometry is a sensitive and important tool for assessing immune system function and diagnosing PID, this method is targeted, which is why it helps to suspect PID only in the presence of certain symptoms, while these diseases cover several hundred various conditions affecting immune system development and/or functioning [7][8][9] .…”