Primary immunodeficiency diseases (PID) are a heterogeneous group of disorders caused by genetic defects of the immune system, which manifest clinically as recurrent infections, autoimmune diseases or malignancies. The most severe forms of PID, severe combined immune deficiency (SCID) is intensively studied and has been found to be associated with fatal consequences in the first 2 years of life (1, 2).

Most forms of SCID can be detected by measuring the levels of T-cell recombination excision circles (TREC) in dried blood spots using real-time polymerase chain reaction (PCR) (3), while kappa-deleting recombination excision circles (KREC) are used to screen for agammaglobulinemia (4). TREC is a by-product of the T-cell receptor gene recombination, and KREC is a by-product of the B-cell receptor recombination. Low levels of these molecules in T- and B-cells in peripheral blood were shown to be associated with T- and/or B-cell lymphopenia (4). The best possible outcome for patients with SCID can be achieved by timely hematopoietic stem cell transplantation or gene therapy before the development of infectious complications (5, 6), while early diagnosis is associated with a significant increase in treatment effectiveness (5). In patients with agammaglobunemia, the best outcome is achieved by initiating replacement therapy using intravenous or subcutaneous immunoglobulins.

PID influence child health-related quality of life, limiting physical, emotional, social and school functioning (7). Therefore, early detection of not only SCID but all PID patients is vital to improve the chances of appropriate management, in order to significantly reduce potential complications and improve life quality (6, 8).

At present, early detection of PID remains a challenge. This is particularly true in older children and in adults, potentially due to milder and less specific symptoms, a low level of awareness of PID amongst clinicians, as well as unavailability of necessary diagnostic devices such as flow cytometry in hospital laboratories (9, 10). In some countries flow cytometry is not readily available and TREC/KREC analysis may represent a feasible alternative. It is therefore imperative to not only allow diagnosis of patients in low-resource facilities but also develop more cost-effective alternatives. Many studies showed that dried blood spots are robust (11) and useful as a potential alternative sample source for clinical purposes, epidemiological studies, and biobanking (12).

Flow cytometry is a more commonly used but more expensive diagnostic technique for PID detection, when compared with PCR (13). It requires a significant amount of training in highly specialized tertiary centers and therefore cannot be used as a screening tool.

Potential applications of TREC/KREC analysis were highlighted in the reviews by van Zelm and co-authors (14, 15). These included support therapy monitoring, patient classification and newborn screening for PID. Apart from apparent clinical benefits, assessment of B- and T- cell neogenesis in PID patients following stem cell transplantation (16) and KREC assessment in patients presenting with abnormalities in B-cell subsets to explain B-cell compartment aberrancies (17) may improve current state of knowledge.

This pilot study aims to assess diagnostic accuracy of TREC and KREC in children from 0 to 18 years of age with suspected PID.

In this study, we tested the accuracy of TREC and KREC in PID diagnosis. The models showed decent utility of TREC in the diagnosis of “Combined PID” and “Syndromic PID.” To our knowledge, this is the first attempt to use TREC/KREC not in SCID screening but in PID diagnostics.

PID is a large group of disorders encompassing more than 400 conditions affecting development and/or functioning of the immune system (23). Flow cytometry is a sensitive and important tool in evaluating the immune system function and in PID diagnosis (24). However, it is expensive, not easily available in developing countries and requires appropriate training and equipment. TREC and KREC may represent cheaper alternatives and/or add value to PID diagnosis and screening. Low cost methodology can be used in small laboratories and rural settings, where complex and expensive tools are unavailable, to provide access to primary PID evaluation.

Prior studies have noted the need in screening tool for early SCID diagnosis, to reduce the risk of infections and organ damage (6, 25). Early diagnosis is particularly important as lack of early treatment is associated with severe complications and increased mortality rates (26). TREC is a common screening approach used for early SCID detection around the globe, providing a good combination of sensitivity and specificity (27, 28) with high cost-effectiveness (29). KREC's role in early screening is still debatable but some data suggest that it may add value in certain cases (30). While TREC's indispensability in SCID screening is obvious, very little can be found in the literature on the question of TREC/KREC use in other PID diagnosis or their use as a screening tool (31–33). Previous attempts to this end have predominantly focused on Common Variable Immunodeficiency (CVID).

We hypothesized, that TREC and KREC may be adopted as a surrogate method of PID diagnosis. Our models demonstrated good AUCs indicating the potential of TREC to be used as an additional tool in PID diagnosis. Our data show that when TREC is used for differentiation between “Combined PID” and “Syndromic PID” patients and healthy individuals, the cut-off point probability of 0.4 provides high specificity (92%) with acceptable sensitivity (65%), further supporting our hypothesis that use of TREC may have its place in PID diagnosis. TREC may serve as an addition to existing tests and may be used as a prerequisite to flow cytometry. We did not use any clinical questionnaires in this study but expect that laboratory findings combined with additional clinical data using standardized instruments may facilitate development of a stronger diagnostic model, improving utility.

TREC and KREC whether individually or combined, did not show good diagnostic ability in diagnosing “Antibody PID.” However, this result was expected, with immunoglobulin measurement having the primary role in aiding the diagnosis. Our further analysis highlights potential for TREC level assessment in discrimination between “Combined PID,” “Syndromic PID,” and “Antibody PID.” When a cut-off of 0.4-point probability was used, TREC reached specificity of 84%. At present, the wide variety and lack of specificity of PID clinical manifestations, do not allow physicians to determine the exact area of immune defect during initial clinical examination. Thus, the extensiveness of the primary laboratory examination in a patient with suspected PID is often determined by subjective clinical criteria, based on physician expertise. Laboratory tests normally used vary from immunoglobulin level assessment to assessment of a wide range of lymphocyte subpopulations. There are no predefined universal guidelines on how detailed the laboratory analysis should be, while selection of diagnostic tests depends on the given clinical settings and clinical immunologist. Normally, if initial tests reveal deviations in humoral immunity the next logical step is cellular immunity assessment to exclude combined immunodeficiency and diagnose “Antibody PID.” Our data suggests that TREC appears to be a useful additional tool to aid in the differentiation between combined immunodeficiency and antibody deficiencies, particularly in the settings with limited access to flow cytometry. The qualitative method of TREC/KREC assessment is easy and can be applied equally well to both whole blood and Guthrie cards, as DNA extraction can be performed on either of these samples.

A few diagnostic algorithms/approaches to children with suspected PID were proposed. Among them excellent algorithm from Dutch immunologist Esther DeVries', based on a pattern recognition approach and decision trees (34), and the Jeffrey Modell Foundation's 4 steps (35). We do not propose changes to existing clinical approaches in routine PID diagnosis and fully acknowledge that TREC/KREC should not be used as a replacement of flow cytometry. Addition of TREC/KREC measurements to aforementioned clinical approaches, nevertheless, may improve predictive capacity of the tools, which is worth further investigation.

The main limitation of this study is related to the use of ICD-10 classification for PID diagnosis. The same ICD-10 code may sometimes include heterogenous group of immunodeficiencies. Genetic testing would be a preferable option; however, this was not available for most of the patients due to economical restrictions. Another limitation is the lack of Guthrie card use in our study with all samples analyzed using whole blood, which does not allow for a result extrapolation. No difference is expected, however, between DNA extraction from the whole blood sample and dried blood spot. Recruitment of patients of three PID groups only, may also be considered a limitation, even though it is unlikely to have influenced the outcomes of this study. We acknowledge that participants defined as “immunologically healthy” in this study can be considered as “healthy” to a high degree of certainty, but no information on naive cells and memory cells were collected. It is clear, however, that “immunologically healthy” participants in this study do not belong to Group I (“Combined PID”). Our study would also have benefited from assessment of TREC/KREC diagnostic accuracy in patients with a particular subtype of PID [e.g., X-linked agammaglobulinemia (XLA)], but it was not possible due to restricted number of patients. This should be addressed in future research.

In conclusion, we found evidence that TREC may have a place in aiding PID diagnosis. The models showed decent diagnostic accuracy measures for TREC in diagnosing “Combined PID” and “Syndromic PID”. Further investigations in a larger cohort in combination with addition of genetic diagnoses and/or questionnaires focused on clinical symptoms are needed to improve diagnostic performance and to further evaluate TREC potential on a wider scale. It is too premature to draw definitive conclusions, but with a few diagnostic algorithms available (e.g., Esther DeVries' and Jeffrey Modell Foundation's 4 steps), addition of TREC to such algorithms may allow for an improved predictive ability. We would like to stress that TREC-based PID diagnosis may be particularly important in the recourse-limited countries and further research will benefit children in these settings.

The datasets generated for this study are available on request to the corresponding author.

The studies involving human participants were reviewed and approved by Speransky Children's Hospital Ethics Committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

IK, MF, AP, AK, and DM conceived and designed the experiments and study analysis. MG and ND performed the experiments. IK, NZ, SZ, and AS collected, extracted, and sorted the data. OB, RM, and AZ analyzed the data. AE reviewed additional available evidence on the matter. IK, PH, and DM wrote the manuscript.