Clinical and Genetic Spectrum of Inborn Errors of Immunity in a Tertiary Care Center in Southern India

Objectives To study the incidence, clinical manifestations, and genetic spectrum of primary immunodefciency diseases (PID)/inborn errors of immunity (IEI) in a tertiary care hospital in Southern India. Methods A retrospective analysis of all patients with a clinical suspicion of PID/IEI seen at a tertiary care hospital was performed. All patients had at least one or more warning signs of PID. Serum immunoglobulin levels and other targeted investigations were performed as warranted by the clinical presentation. All families with suspected PID were counseled and ofered genetic testing. Results A total of 225 children were evaluated for PID during the study period of 6 y. Fifty-six of them did not meet the European Society of Immunodefciencies (ESID) criteria (working defnition of clinical diagnosis) and were excluded. An IEI was found in 30/49 (61.2%) patients. The most frequent reason for referral was recurrent/unusual or serious infections (28%), or cytopenia (16%). Group IV diseases of immune dysregulation was the most common category (19%), followed by group III predominant antibody defciencies in 23/163 (14%), as per the International Union of Immunological Societies (IUIS) classifcation. Conclusions This study highlights the heterogeneity of the present cohort, the underuse of genetic tests, and eforts to provide optimal care for children with possible IEI in this center.

Rapid flow cytometry based cytotoxicity assay for evaluation of NK cell function.

Assessment of natural killer cells (NK-cell) cytotoxicity is used not only in research settings but is also important in diagnosis of various diseases. NK-cell cytotoxicity assays are based on measurement of target cells killed by cytotoxic cells analyzed either by chromium (51Cr) release assay or flow cytometry. Both these methods use peripheral blood mononuclear cells (PBMC) or pure NK-cell population and hence require large volume of blood sample which is difficult to obtain in pediatric patients and patients with cytopenia. Hence, a flow cytometric assay was designed to determine NK cell activity using whole blood, eliminating the need for isolation of PBMCs or pure NK cells. This assay is based on a dual fluorescent staining of target cells (K562 cell line). The DIOC18 dye labeled K562 cells are incubated with whole blood and then counterstained with 7-AAD enabling the measurement of dead target cell and then percent cytotoxicity is calculated. This study compared the NK cell cytotoxicity using PBMC and whole blood in clinically relevant samples. There was no significant difference between two assays in the measurement of lytic activity or in reproducibility in the repeated samplings of healthy individuals. The whole blood assay required less volume of blood and also less processing time as compared to PBMC assay. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis expected to have low NK-cell activity. This assay is rapid, sensitive and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK-cell function.

FLT3 and NPM-1 mutations in a cohort of acute promyelocytic leukemia patients from India.

Background: Acute promyelocytic leukemia (APL) with t (15;17) is a distinct category of acute myeloid leukemia (AML) and is reported to show better response to anthracyclin based chemotherapy. A favorable overall prognosis over other subtypes of AML has been reported for APL patients but still about 15% patients relapse. Methods: This study evaluated the presence of Famus like tyrosine kinase‑3 (FLT3) and nucleophosmin‑1 (NPM1) gene mutations in a cohort of 40 APL patients. Bone marrow/peripheral blood samples from patients at the time of diagnosis and follow‑up were processed for immunophenotyping, cytogenetic markers and isolation of DNA and RNA. Samples were screened for the presence of mutations in FLT3 and NPM1 genes using polymerase chain reaction followed by sequencing. Results: Frequency of FLT3/internal tandem duplication and FLT3/tyrosine kinase domain was found to be 25% and 7% respectively. We observed a high frequency of NPM1 mutation (45%) in the present population of APL patients.

Comparison of in-vitro and in-vivo response to fetal hemoglobin production and γ-mRNA expression by hydroxyurea in Hemoglobinopathies.

BACKGROUND: Hydroxyurea, which induces Fetal hemoglobin (HbF) synthesis, is the only drug widely used in different hemoglobinopathies; however, the response is very variable. We compared the efficacy of hydroxyurea in-vitro in erythroid cultures and in-vivo in the same patients with different hemoglobinopathies to induce HbF production and enhance γ-messenger RNA expression. MATERIALS AND METHODS: A total of 24-patients with different Hemoglobinopathies were given hydroxyurea and their response was studied in-vivo and in-vitro on mononuclear cells collected from them simultaneously. RESULTS: A total of 57.7% of patients (responders) showed no further crisis or transfusion requirements after hydroxyurea therapy with a mean increase in fetal cells (F-cells) of 63.8 ± 59.1% and γ-mRNA expression of 205.5 ± 120.8%. In-vitro results also showed a mean increase in F-cells of 27.2 ± 24.7% and γ-mRNA expression of 119.6% ± 65.4% among the treated cells. Nearly 19.0% of the partial-responders reduced their transfusion requirements by 50% with a mean increase in F-cells of 61.2 ± 25.0% and 28.4 ± 25.3% and γ-mRNA-expression of 21.0% ± 1.4% and 80.0% ± 14.1% in-vivo and in-vitro respectively. The non-responders (15.3%) showed no change in their clinical status and there was no significant increase in F-cells levels and γ-mRNA expression in-vivo or in-vitro. CONCLUSION: Thus, this method may help to predict the in-vivo response to hydroxyurea therapy; however, a much larger study is required.