Can Trimodal Distribution of HbS Levels in Sickle Cell Traits Be Used To Predict the Associated Alpha-Thalassemia For Screening Cases in Central India?

BACKGROUND: Until now, trimodal distribution of HbS has been seen by six different studies in the world when associated with alpha-thalassemia with confirmation by corresponding alpha-genotyping studies. The RBC indices reduce as alpha-globin genes reduce in sickle cell trait (SCT) patients, which decreases the extent of intra-vascular sickling and thus betters the clinical course of the patients. This is a pioneer study conducted on Central Indian poor population to use the already proven six studies to screen associated alpha-thalassemia in SCT patients thus, circumventing the much costlier alpha-genotyping studies. Moreover, it aimed to study the haematological parameters in such cases. METHODS: The study was performed at RHDMC, IGGMC, Nagpur, India from 2003 to 2012. The sample population was suspected cases of haemolytic anaemia. CBC and RBC indices were obtained by a cell analyzer. The sickle solubility test positively screened cases were confirmed by agar-gel haemoglobin electrophoresis at pH 8.6. Finally, quantitative assessment of haemoglobin variants was performed by HPLC. RESULTS: Out of total 5819 cases over ten years, 933 cases were sickle heterozygotes. Overall, 180/933 subjects were predicted to be homozygous alpha-thalassemia and 338/933 were heterozygous alpha-thalassemia, based on trimodal distribution of HbS. CONCLUSION: Genotyping is costlier for majority of the poor non-affording patients in Indian government set-ups, so this study is suitable to screen for associated alpha-thalassemia in SCT patients.

Prenatal diagnosis of sickle cell disease by the technique of PCR.

Sickle cell disease (SCD) is prevalent in Central India and causes major morbidity and mortality. There is a lack of prenatal diagnostic facility near population affected with SCD. This is the pilot study in our region with the aim to establish prenatal diagnostic facility for the couples carrying sickle cell gene in Central India, in order to help them take an informed decision regarding fetus affected with SCD and also to calculate sensitivity of polymerase chain reaction (PCR) technique in our set up with follow up high performance liquid chromatography (HPLC) of baby's blood sample. Fetal sampling was done by chorionic villous biopsy. Extracted DNA was subjected to amplification refractory mutation system (ARMS-PCR) to detect sickle cell mutation (GAG â†’ GTG) in the sixth codon of ß globin gene. Follow-up HPLC was done to detect baby's Hb pattern. Prenatal diagnosis of sickle cell anemia was offered in total 37 cases out of which one (2.7 %) fetal sample was inadequate. Total 26 (70.27 %) fetuses had AS Hb genotype, 3 (8.11 %) had AA Hb genotype and 3 (8.11 %) had SS Hb genotype while remaining 4 (10.81 %) were given AA/AS Hb genotype. All couples with SS fetuses opted for MTP. Follow up HPLC was performed in 24 cases, out of which 18 (75 %) were correlated and 6 (25 %) were mismatched. In present study sensitivity of ARMS-PCR was 75 %. ARMS-PCR is a simple technique to be established initially for providing rapid prenatal diagnosis to the couples with known sickle cell mutation. The sensitivity of ARMS-PCR can be increased by using suitable techniques to detect maternal cell DNA contamination.

Hematological profile of sickle cell disease in central India.

Hematological profile of homozygous sickle cell disease patients attending RHDMC from Central India is presented. Central India has a huge population of sickle cell disease patients. Though predicted SS in the region is 22-44 %, 81 homozygous of sickle cell patients reported during study period of Jan 2003-Dec 2005. The clinical course of these patients is characterized in most of the cases by relatively long period without any symptoms punctuated by acute clinical events. Hematological profile of these 81 patients with age ranging from 6 month to 64 years is presented. There are 44 males and 37 females with an average age of 14.55yrs in males and 18.13 yrs females. Males out number females in pediatric age group where as females with SCD are attending hospital more in reproductive age group. Very few patients are reported after the age of 30 yrs. Average hemoglobin in males is 7.11 ± 2.13 gms/dl and in females 6.75 ± 1.85 gms/dl with parallel low RBC count.Hemoglobin rise is seen after 14 years of age in males and females. Age related rise in MCV is more noted in females after the age of 5 as compared to males. No age or sex related difference was seen in MCHC values. Hb A(2) levels for males is 2.13 ± 0.95% and for females 2.04 ± 0.91 %.Hb F in males is 19.58 + 5.86% and in females is 20.99 + 4.9%. There is no age and sex related difference in Hb F levels. Moderate to severe anemia with high Hb F dominate Central Indian sickle cell disease patient's hematological profile. The hematological profile in Central India is similar to the profile in other parts of India and Saudi Arabia but different from Jamaica and Africa.

Double heterozygous for hemoglobin S and hemoglobin E - a case report from central India.

Double heterozygosity for HbS and HbE is rare. HbS and HbE are seen in SC, ST and OBC communities from this part of country. Inter caste marriages amongst these communities have resulted into this compound heterozygous condition. Double heterozygous state for HbS and HbE is clinically silent as compared to HbS-ß Thalassaemia and HbSS cases. At Regional Hemoglobinopathy Detection and Management Center, we report a case of 15-year-old male, Teli (OBC) by caste who came for screening for sickle cell disorder. Sickling, solubility test and Hb electrophoresis on agar gel at alkaline pH was carried out. His sickling and solubility tests were positive and on hemoglobin electrophoresis it showed two bands one at Hb A(2) position and another at HbS position. For further confirmation sample was subjected for quantitation of haemoglobin on high performance liquid chromatography (HPLC), Bio-Rad. On quantitation he was having HbS 59.8%, HbE 33.5% and HbF 3.2% confirming his double heterozygous state for HbS and HbE. On family screening his father turned out to be sickle cell trait and mother as hemoglobin E trait.

Seroreactivity of 31 kDa and 41 kDa mycobacterial secretory proteins isolated from culture filtrate in extra pulmonary tuberculosis.

Despite rapid advances in molecular genetics for detection of mycobacteria, it is clear that interest in serodiagnosis remains high, especially for those situations in which a specimen may not contain the infecting agent in particular in extrapulmonary tuberculosis. Immune response to excretory-secretory (ES) proteins of Mycobacterium tuberculosis (M.tb) has been of diagnostic interest in tuberculosis. In earlier study from our laboratory, a secretory protein M.tb ES-31 has been shown to have diagnostic potential in pulmonary tuberculosis. Further, another M.tb H37Ra ES protein (ES-41) was isolated and purified by trichloroacetic acid solubilization followed by Fast Performance Liquid Chromatography (FPLC). These two protein fractions viz ES-31 and ES-41 secreted by M.tb H37 Ra bacilli were employed in stick indirect penicillinase ELISA to study seroreactivity in extra pulmonary tuberculosis namely tuberculous lymphadenopathy, tuberculous meningitis, abdominal tuberculosis and bone & joint tuberculosis. While using ES-31 antigen 88% (22/25) of tuberculous lymphadenopathy and 90% (9/10) of tuberculous meningitis cases showed positive reaction for tuberculous IgG antibody, ES-41 showed 80% positivity in both groups. In abdominal and bone & joint tuberculosis cases, ES-41 antigen showed better sensitivity of 81.5% (22/27) and 84.6% (22/26) respectively in IgG antibody detection compared to 70% (19/27) and 69.2% (18/26) shown by ES-31. This study is of interest that different antigen protein fractions of M.tb exhibit differential seroreactivity, as ES-31 protein showed good potential in detecting tuberculous IgG antibodies in tuberculous lymphadenopathy (TBLN) & tuberculous meningitis (TBM), while ES-41 in abdominal and bone & joint tuberculosis cases.

Cytodiagnosis of discharge per vaginum.

Simple, rapid, inexpensive methods such as pH determination, wet mount, KOH mount, amine test, Gram staining of vaginal discharge were undertaken prior to Pap stained smears of 158 patients of leucorrhoea. We were able to detect non-specific vaginitis (44.30%), Trichomoniasis (16.45%), Candida vaginitis (9.49%), gonococcal vaginitis (0.63%) and senile vaginitis (12 cases). No specific pathology was shown in 26 cases. 1.69% of the cases were of cervical erosion and 3.79% cases suggestive of squamous malignancy.

A simple screening test for the detection of heterozygous beta thalassemia.

Beta-Thalassemia is prevalent in a high frequency of 5-15% in selected geographical areas and ethnic communities. There is need to screen large populations for the heterozygotes of beta-Thalassemia. We have evaluated NESTROFT (Naked eye single tube red cell osmotic fragility test) with 0.36% buffered saline in this study. Of the population screened, 42 cases were positive with NESTROFT, of which 40 were positive with the confirmatory test for beta-Thalassemia. This implies a positive predictive value and specificity of 100%, negative predictive value of 83.3% and sensitivity of 95.2%.

Immunochemical studies in multiple myeloma.

The study deals with a total of 72 patients with Plasma cell dyscrasias (PCD) selected on the basis of atypical plasmacytosis in the bone marrow aspirate and radiological evidence of osteolytic lesions. Males(48) outnumbered the females (24). Pathological fracture and paraplegia was the commonest presenting symptom encountered in 38 patients. Electrophoresis of serum for 'M' band and Immunoelectrophoretic analysis of the serum revealed IgG myeloma in 40 patients followed by, IgA myeloma(13), Light chain disease (12) and other variants in remaining seven cases. The urinary Bence Jones proteins were detected in a total of 34 cases and was frequently encountered with IgA myeloma (7 out of 13) compared with IgG myeloma (13 out of 40) when analysed in Disc electrophoresis. Kappa light chain was observed in 21 cases and lambda counterpart in nine cases without any clinical significance. One case of solitary myeloma terminated in characteristic multicentric multiple myelomatosis within a span of six months in the sequential follow up study. We recommend the triangular approach to diagnosis of paraproteinemia with a special emphasis on immunoelectrophoresis for typing multiple myeloma and allied disorders along with disc electrophoresis for the demonstration of urinary Bence Jones protein in the routine set up.

Bisalbuminemia: an interesting electrophoretic abnormality--a report of two cases.

We hereby report two cases of Bisalbuminemia for its extreme rarity in Indian literature. In an attempt to look for 'M' component, polyacrylamide gel disc electrophoresis of serum displayed observation. The saline eluates of the slower (variant in our cases) and the faster component showed a reaction of complete identify in the agar gel double diffusion test when tested against monospecific antialbumin antibody. The immunoelectrophoretic analysis also confirmed the antigenic similarity between the two fractions. A wider use of disc electrophoresis perhaps would disclose additional cases of Bisalbuminemia in normals and in disease states.

Sequential study of IgG antibody response in immunized rabbit and development of immunization protocol for raising monospecific antibody.

The present study was planned to develop an immunization protocol to decide the bleeding modalities for harvesting anti IgG antibody from the immunized rabbit. A fourteen dose immunization protocol (four primary and ten boosters) of the purified human IgG spread over the one calendar year was executed. The antibody titre estimated by Reverse Single Radial Immunodiffusion displayed a six-phased pattern. The titre following the initial immunization ranged between nil to 1.02, characteristic of the primary response while the titres after 7th and 8th boosters (phase V) ranged between 0.5 to 3.87 consistent with secondary response. Phase II, III, IV had moderately elevated titres. The antibody titre amongst the six phases reached to its peak generally by the 12th day after the last dose of protocol and it took about 60 days to reach to its basal level. Administration of antigen with the higher levels of residual antibody did not produce high titre antibody and is probably ascribed to elimination of antigen through an immunecomplex mechanism. Based on the data we recommend that 15 batches. (3 per phase, phase I to phase V) with a total yield of 100 to 120 ml of serum can be procured from one immunised animal over the span of one calendar year and that should make the programme cost effective.

Reverse single radial immunodiffusion for estimation of titre of anti IgG antibody.

Reverse Single Radial Immunodiffusion (SRID) for estimating titre of anti IgG antisera is reported. Unlike the conventional radial immunodiffusion, the antigen (IgG) is held immobile in the gel while the antibody (Anti IgG) diffuses radially from the well (7 microliters) and the diameter of the resulting immuneprecipitates after immunodiffusion at 4 degrees C for 24 hr, represents a linear correlation with the antibody titre. The procedure was standardised by an extensive trial and error employing different concentrations of human IgG in the gel (60-240 micrograms) against varying dilutions of the standard antibody (titre: 3.8 mg/ml). The best results were obtained at 80 micrograms of IgG in the gel. The locally raised rabbit anti IgG antisera displayed a distinctive titre pattern under optimised conditions. Technical reproducibility, high-sensitivity threshold (0.25 mg/ml), simultaneous visual scrutiny of several antibody batches at a glance and ability to assess the shelf life of the stored antisera are its distinct assets.

Isolation and characterization of a fraction from brain that inhibits 1,4-[3H]dihydropyridine binding and L-type calcium channel current.

Bovine brain was subjected to acid extraction and several purification steps. A fraction from brain that eluted from C18 reverse-phase columns at 30-35% acetonitrile inhibited [3H]nitrendipine binding to cardiac membranes. Further purification of this fraction on a sizing column in the presence of 40% acetonitrile yielded a low molecular mass fraction (less than 1 kDa) that produced a time- and voltage-dependent inhibition of L-type (but not T-type) Ca2+-channel current in GH3 cells. The results suggest that this fraction contains an endogenous substance that binds directly to slowly-inactivating Ca2+ channels and thereby inhibits current flow.

[3H]BAY K 8644, a 1,4-dihydropyridine Ca++ channel activator: characteristics of binding to high and low affinity sites in cardiac membranes.

The binding of [3H]BAY K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] to high and low affinity sites in rabbit ventricular membranes was characterized. Binding affinities were 0.66 and 138 nM at 15 degrees C and 9.1 and 72 nM at 37 degrees C, for the high and low affinity sites, respectively, and binding site densities were 0.3 and 14 pmol/mg at 15 degrees C and 0.41 and 1.4 pmol/mg at 37 degrees C, for the respective sites. The modification of high affinity [3H]BAY K 8644 binding by verapamil, diltiazem, tiapamil, Ca++ and EDTA appeared to be the same as that for nitrendipine binding, consistent with the hypothesis that the high affinity binding site for [3H]BAY K 8644 on isolated membranes is the same as the 1,4-dihydropyridine antagonist binding site. The binding of [3H]BAY K 8644 to a low affinity binding site was modified by temperature, Ca++ and diltiazem, but the lack of stereoselectivity, lack of denaturation by heat and the large number of sites indicated that most of the low affinity binding sites were not associated with Ca++ channels. It is concluded that the high affinity binding site for BAY K 8644 is associated with Ca++ channels, and is modified by at least some of the factors that modify the binding site for Ca++ channel antagonists, whereas many or all of the low affinity binding sites detected are not related to Ca++ channels.