Implementation of TREC/KREC detection protocol for newborn SCID screening in Bulgaria: a pilot study.

Neonatal screening for inborn errors of immunity (IEI), based on quantification of T-cell-receptor- excision circles (TRECs) and kappa-deleting recombination-excision circles (KRECs) from dried blood spots (DBS), allows early diagnosis and improved outcomes for the affected children. Determination of TREC/KREC levels from prospectively collected newborns' Guthrie cards and from DBS samples of patients with confirmed IEI was done using a commercial kit. Retrospective assessment of flow cytometry evaluation of TREC/KREC correspondence with lymphocyte subpopulations and evaluation of the correlations between TREC and KREC with immune cells, based on the data from patients with suspected or confirmed immune disorders, were conducted. 2,228 Guthrie cards were tested, 1276 for TREC only and 952 for both TREC and KREC. Eight newborns (0.36%) were TREC positive and 10 (1.05%) had KREC below the cut-off. The re-testing rate was 1.88%. Retrospective analysis demonstrated that the TREC/KREC assay identifies 100% of severe combined immune deficiencies (SCID) cases when DBS were collected at birth. Correlation analysis showed moderate significant correlations between TREC and the absolute numbers of CD4 cells (r = 0.634, p < 0.01) and total T cells (r = 0.536, p < 0.01). The ability of KREC levels to predict abnormal absolute (AUC of 0.772) and relative (AUC 0.731) levels of B cells was demonstrated.

Immune Cell Subsets Evaluation as a Predictive Tool for Hepatitis B Infection Outcome and Treatment Responsiveness.

BACKGROUND: The patient's immune response is one of the major factors influencing HBV eradication or chronification, and it is thought to be responsible for the treatment success. AIM: Our study aimed to investigate whether cellular defense mechanisms are associated with the course of HBV infection (spontaneous recovery [SR] or chronification [CHB]) and with the therapeutic approach. PATIENTS AND METHODS: A total of 139 patients (118 with CHB, 21 SR) and 29 healthy individuals (HI) were immunophenotyped by flowcytometry. Fifty-six patients were treatment-naïve, 20 were treated with interferons and 42 with nucleoside/ nucleotide analogues. RESULTS: Deficiency of T lymphocytes, helper-inducer (CD3+CD4+), suppressorcytotoxic (CD8+CD3+) and cytotoxic (CD8+CD11b-, CD8+CD28+) subsets, activated T cells (CD3+HLA-DR+, CD8+CD38+) and increased CD57+CD8- cells, elevated percentages of B lymphocytes and NKT cells were observed in CHB patients compared with HI. In SR patients, elevated CD8+CD11b+, NKT and activated T cells were found in comparison with controls. The higher values of T cells and their subsets in SR patients than in CHB patients reflect a recovery of cellular immunity in resolved HBV infection individuals. In both groups of treated patients, reduced T lymphocytes, CD3+CD4+ and CD8+CD38+ subsets were found in comparison with HI. Higher proportions of cytotoxic subsets were observed in treated patients compared with treatment-naïve CHB patients, more pronounced in the group with interferon therapy. CONCLUSION: Our data demonstrate that cellular immune profiles may be of prognostic value in predicting the clinical course of HBV infection, and the determination of the therapeutic response.

Evaluation of lymphocyte subpopulations in cord blood of Bulgarian newborns.

BACKGROUND: Umbilical cord blood (UCB) is a clinically useful source of hematopoietic stem and progenitor cells for treatment of a wide variety of malignant and non-malignant disorders. An important way to completing infor- mation on the quality and composition of units for transplantation is more extensive immunophenotyping of UCB. Moreover, phenotyping of lymphocyte subpopulations is essential for the diagnosis and follow-up of children with immunodeficiencies and other immune disorders and therefore, establishment of age-matched reference values of lymphocyte subsets is a necessity for each population. The aim of this study was to determine the normal range of T and B lymphocytes, and NK cells as well as the CD4 and CD8 subpopulations of T cells in cord blood collected from healthy term infants. METHODS: The relative and absolute number distributions (median, 5th and 95th percentile) of lymphocyte subsets in cord blood samples from 72 healthy newborns were examined by multi-colour flow cytometry with a view to obtaining reference values for Bulgarian neonates at birth. RESULTS: Mean percentages of lymphocyte subpopulations were: CD3 (62.27 ± 9.64), CD19 (17.47 ± 5.46), CD3- CD16/CD56+ (17.27 ± 8.4). Our results show the prevalence of helper-inducer CD3+CD4+ (44.88-8.21) compared to the suppressor-cytotoxic CD3+CD8+ (16.65 ± 4.54) T-cell subpopulation, which determines the positive CD4/CD8 ratio (2.86 ± 0.82; 1.4-4.8). Also, the absolute numbers of studied populations varied widely due to differences of the absolute number of lymphocytes in the samples. CONCLUSIONS: This study on distribution of lymphocyte subpopulations in UCB helps to enhance our knowledge about cell phenotypes in cord blood and improve characterization of products for cellular therapy, as well as contributes to the correct interpretation of laboratory results for infants with possible immune disorders. Our data can be used as normal intervals for lymphocyte subsets in Bulgarian neonates.