Prevalence of hemoglobin variants and hemoglobinopathies using cation-exchange high-performance liquid chromatography in central reference laboratory of India: A report of 65779 cases.

CONTEXT: Hemoglobinopathies constitute the world's most common genetically inherited red blood cell disorder. Screening and accurate identification of hemoglobin (Hb) variants have become increasingly important in antenatal diagnosis and prevention of Hb disorders. AIM: The aim of this study was to screen and identify Hb fractions prevalent in the Central Reference Laboratory of India. MATERIALS AND METHODS: A total of 65,779 cases were screened for hemoglobinopathies on the bio-rad variant high-performance liquid chromatography (HPLC) system by beta-thalassemia short program. The retention times, proportion of the hemoglobin (%) and the peak characteristics for all hemoglobin fractions were recorded. Molecular analysis of the beta-globin gene was carried out by DNA sequencing on eight cases. RESULTS: Total number of abnormal Hb fractions on cation exchange-HPLC (CE-HPLC) was seen in 12,131 (18.44%) cases. Beta-thalassemia trait was the predominant genetic Hb disorder accounting for 7377 cases (11.21%) of the total cases. This was followed by sickle cell trait (2.01%), sickle cell disease (1.59%), beta-thalassemia syndrome (0.80%), HbE trait (0.79%), and borderline HbA2 (0.51%). Molecular characterization of eight rare cases of hemoglobin variants by beta-globin gene sequencing identified three cases of Hb Beth Israel, two cases of Hb Hofu trait, and one case each of Hb J Cambridge, Hb Mizunami, and Hb Sherwood Forest. CONCLUSION: Superior resolution, rapid assay time, and accurate quantification make CE-HPLC suitable for the routine investigation of hemoglobinopathies.

HLA-B27 testing: A journey from flow cytometry to molecular subtyping.

BACKGROUND: Determination of HLA-B27 status plays an important role as adjuvant in suspected cases for diagnosis of Ankylosing Spondilytis (AS). Objectives of this study were to evaluate (i) flow cytometry method in comparison with DNA microarray for HLA-B27 typing and (ii) EUROArray HLA-B27 Direct assay for HLA-B27 allele detection along with discrimination of AS/non-AS subtypes in Indian population. METHODS: A total of 7543 patients with a presumptive clinical diagnosis of AS were referred for screening of HLA-B27. All samples were initially tested by flow cytometry, and based on its findings, 1560 samples were analyzed for the presence of HLA-B27 allele by microarray technology. A subset of samples (n = 200) were further tested by DNA sequencing for identification of HLA-B27 subtypes. RESULTS: Screening of HLA-B27 by flow cytometry reported 1551 positive (20.56%) and 5556 negative (73.65%) cases. Remaining 436 (5.78%) samples were identified within equivocal zone. Of cases (n = 1560) analyzed by microarray method, 1333 (85.44%) and 227 (14.55%) were detected microarray positive and negative, respectively. DNA sequencing identified HLA-B*27:07 as the predominant subtype among cases showing ex2 positivity by microarray method. Of 200 cases, 20 cases (14 of HLA-B*07 and 6 of HLA-B*37) of HLA-B27 cross-reactive subtypes were also identified. CONCLUSION: We recommend DNA typing as a complementary tool along with flow cytometry to accomplish successful HLA-B27 phenotype determination. This is the first study among Indian population to evaluate efficacy of EUROArray to detect B27 allele and its potential to indicate the presence of nondisease-associated alleles in Indian population.

An analysis of 24 autopsied cases with supramitral rings.

The supramitral ring is a rare congenital malformation formed by presence of a ridge of connective tissue, usually attached at or above the mitral annulus. The incidence and clinical presentation is highly variable due to difficulty in diagnosis. A review of autopsied congenital heart diseases at our institute over a 17-year-period revealed 24 cases of supramitral ring. These were classified with respect to the morphology of the ridge and the presence of associated cardiac lesions. The ring was found in 1.5% of the autopsied specimens of congenitally malformed hearts, and in 37.5% of those with obstructed left-sided inflow tracts. The majority of the specimens came from children (79.2%). A clinical diagnosis had been made in only two. In one-third of the cases, the ring was associated with incomplete Shone's complex. Varied anomalies were seen in others, chiefly ventricular septal defects. An interesting association was the presence of rheumatic mitral valvar disease, found in 3 cases. There was no difference in the completeness or width of the supramitral ridge in the hearts from those with or without Shone's complex. Circumferential rings were fleshy and stenosing, while incomplete rings had variable locations and stenosis. The presence of a supramitral ring may be underestimated due to association with other cardiac anomalies, both congenital and acquired. Since the ridge need not always produce stenosis, the correct designation would be simply a supramitral ring.