Comparison of estimation of volume of fetomaternal hemorrhage using Kleihauer-Betke test and microcolumn gel method in D-negative nonisoimmunized mothers.

In this study we assessed the efficay of the microcolumn gel method in the detection and quantification of the volume of fetomaternal hemorrhage (FMH) in comparison with the Kleihauer-Betke test (KB) in nonisoimmunized D- mothers. We collected blood samples from 80 D- indirect antiglobulin test-negative mothers over a span of more than 1 year. FMH was determined by KB and microcolumn gel method, and the results were compared. FMH was recorded as less than 4 mL by KB if no fetal cells were seen after examining 25 fields using 10x objective. If fetal cells were seen, slides were examined furhter to quantify FMH. By microcolumn gel method, FMH was reported as less than 0.1 percent, 0.1 percent, 0.2 percent, and 0.4 percent or greater. None of the patients had FMH greater than 15 mL by KB . Sixty-two patients (77.5%) had FMH less than 4mL by KB. In all these cases , FMH was less than or equal 0.2 percent (approximately 4mL) by microcolumn gel method. The mean volume of FMH in the remaining 18 (22.5%) cases by KB was 8.3 ± 1.7 mL. Fifteen (83.3%) of these 18 cases had FMH of at least 0.4 percent (approximately 8 mL) by gel technology. Three cases (16.7%) that differed from KB results had FMH of 0.2 percent by microcolumn gel method with a maximal FMH of 6.4 mL by KB. FMH was significanlty increased in cesarean delivery (mean FMH 9.5 ± 0.8 mL, range 7.9-10.4 mL, p=0.001) abd abtepartum hemorrahge (mean FMH 9.5 ± 0.9 mL, range 7.9-10.4 mL, p< 0.001). Microcolumn gel method is an effective screening test . Technologies like KB and flow cytometry are better options for detecting a large volume of FMh. Antepartum hemorrhage and cesarean delivery are risk factors for FMH. the 300-µg dose of cases. We need to analyze the relative cost-effectiveness of universal administration of 300µg of Rh immune globulin vs. FMH quantitation with subsequent administration of titrated doses.

A positive feedback loop between HER2 and ADAM12 in human head and neck cancer cells increases migration and invasion.

Increased activation of epidermal growth factor receptor (EGFR) family members such as HER2/Erbb2 can result in more aggressive disease, resistance to chemotherapy and reduced survival of head and neck squamous cell carcinoma (HNSCC) patients. In order to identify mechanisms through which these receptor tyrosine kinases accelerate tumor progression, the regulation of metalloprotease expression by EGFR family members was investigated in 11 squamous cell carcinoma (SCC) cell lines. HER2 expression was significantly correlated with ADAM12 (A Disintegrin And Metalloprotease 12) expression in these cell lines and was co-expressed in human head and neck cancers. Inhibition of HER2 or EGFR decreased ADAM12 transcripts whereas HER2 transfection upregulated ADAM12 expression. To understand the molecular mechanisms underlying HER2 regulation of ADAM12, we investigated the signaling pathways directing ADAM12 production in SCC cells. Inhibition of phosphatidyl inositol-3-kinase or mammalian target of rapamycin decreased ADAM12 transcripts in HER2-expressing SCC cells, whereas transfection with AKT increased ADAM12 mRNA. Experiments utilizing ADAM12 transfection or siRNA targeting of ADAM12 revealed that the protease increased both the migration and invasiveness of oral SCC cells. Surprisingly, ADAM12 also increased HER2 message, protein levels and activity through an Ets1-dependent mechanism. Collectively, these results reveal a novel positive activation loop between ADAM12 and HER2 that may contribute to HNSCC progression.

Efficacy of autologous bone marrow-derived stem cell transplantation in patients with type 2 diabetes mellitus.

Progressive and inexorable beta-cell dysfunction is the hallmark of type 2 diabetes mellitus (T2DM) and beta-cell regeneration using stem cell therapy may prove to be an effective modality. A total of 10 patients (8 men) with T2DM for >5 years, failure of triple oral antidiabetic drugs, currently on insulin (> or = 0.7 U/kg/day) at least for 1 year, and glutamic acid decarboxylase antibody negative were included. Patients on stable doses of medications for past 3 months were recruited. Primary end points were reduction in insulin requirement by > or = 50% and improvement in glucagon-stimulated C-peptide levels at the end of 6 months of autologous bone marrow-derived stem cell transplantation (SCT), while secondary end points were a change in weight and HbA1c and lipid levels as compared to baseline. Seven patients were responders and showed a reduction in insulin requirement by 75% as compared to baseline. Mean duration to achieve the primary objective was 48 days. Three patients were able to discontinue insulin completely, although it was short-lived in one. Mean HbA1c reduction was 1% and 3 of the 7 responders had HbA1c value <7%. A significant weight loss of 5.5 kg was noted in the responders, whereas, nonresponders gained 2.2 kg of weight. However, weight loss did not correlate with reduction in insulin requirement (r = 0.68, P = 0.06). There was a significant improvement in both fasting and glucagon-stimulated C-peptide level in the group (P = 0.03) and responders (P = 0.03). HOMA-B increased significantly in the whole group (P = 0.02) and responders (P = 0.04) whereas, HOMA-IR did not change significantly (P = 0.74). Reduction in insulin doses correlated with stimulated C-peptide response at the baseline (r = 0.83, P = 0.047) and mononuclear cell count of infused stem cells (r = 0.57, P = 0.04). No serious adverse effects were noted. Our observations indicate that SCT is a safe and effective modality of treatment to improve beta-cell function in patients with T2DM. However, further large-scale studies are needed to substantiate these observations.

Multicenter evaluation of individual donor nucleic acid testing (NAT) for simultaneous detection of human immunodeficiency virus -1 & hepatitis B & C viruses in Indian blood donors.

BACKGROUND & OBJECTIVE: India has a high prevalence of HIV-1, hapatitis C and B virus (HCV and HBV) in the blood donors but has yet to implement nucleic acid testing (NAT) in blood screening. We undertook a multicentre evaluation of blood donor testing by NAT for simultaneous detection of HIV-1, HBV and HCV in a single tube and also to determine the feasibility of NAT implementation in India's low volume setting. METHODS: A total of 12,224 unlinked samples along with their serological results were obtained from representative eight blood banks in India and were individually manually tested by the Procleix Ultrio Assay (Chiron Corp. Emeryville, CA) for simultaneous detection of HIV-1, HCV, and HBV. RESULTS: Of the 12,224 samples tested, 209 (1.71%) were seroreactive. One hundred thirty three samples (1.09%) were reactive by Ultrio assay, 84 samples were seroreactive but NAT non reactive. There were eight NAT yield cases: 1 HIV, 1 HIV-HCV co-infection, and 6 HBV. INTERPRETATION & CONCLUSION: Our observed NAT yield for all three viruses was 1 in 1528 (0.065%). We estimate NAT could interdict 3272 infectious donations a year among our approximate 5 million annual donations.

Platelet audit: Assessment and utilization of this precious resource from a tertiary care hospital.

BACKGROUND: To assess the appropriate utilization of platelet transfusions [random donor platelets (RDP) and single donor platelets (SDP)]; a six-month retrospective audit was carried out in a tertiary care hospital. MATERIALS AND METHODS: A six-month retrospective platelet audit was carried out from May to October 2005 to estimate its preparation, appropriate utilization and wastage rate. Patient's demographics, transfusion triggers and episodes and ABO and Rh (D) group specific or non-group specific transfusions were also assessed. RESULTS: About 5525 units of platelets [PRP-PC, 3,813 (69%); BC-PC, 983 (17.8%); PRP, 648 (11.7%) and SDP 81 (1.5%)] were prepared and transfused to 853 patients (RDP to 814 patients and SDP to 39 patients) in 2,093 transfusion episodes. Adult and pediatric hemato-oncology were the main user specialties utilizing 39.1 and 87.6% of the RDPs and SDPs prepared. Of the patients receiving RDPs, 95% were transfused ABO and Rh (D) group specific platelets whereas 100% SDPs transfusions were of group specific platelets. 88% of prophylactic platelet transfusions were appropriate as per the recommended BCSH guidelines. However, 12% of the prophylactic platelets were transfused inappropriately in cardiopulmonary bypass (CPB) surgeries with normal platelet counts and no evidence of bleeding related to platelets. Out of 5,444 RDPs prepared 1,585 (29.11%) units were not utilized. CONCLUSIONS: Regular audit of blood and blood components is a must so that necessary remedial measures can be taken to maximize appropriate and judicious utilization of each component.

Algorithm for recall of HIV reactive Indian blood donors by sequential immunoassays enables selective donor referral for counseling.

BACKGROUND: HIV/AIDS pandemic brought into focus the importance of safe blood donor pool. AIMS: To analyze true seroprevalence of HIV infection in our blood donors and devise an algorithm for donor recall avoiding unnecessary referrals to voluntary counseling and testing centre (VCTC). MATERIALS AND METHODS: 39,784 blood units were screened for anti-HIV 1/2 using ELISA immunoassay (IA-1). Samples which were repeat reactive on IA-1 were further tested using two different immunoassays (IA-2 and IA-3) and Western blot (WB). Based on results of these sequential IAs and WB, an algorithm for recall of true HIV seroreactive blood donors is suggested for countries like India where nucleic acid testing or p24 antigen assays are not mandatory and given the limited resources may not be feasible. RESULTS: The anti-HIV seroreactivity by repeat IA-1, IA-2, IA-3 and WB were 0.16%, 0.11%, 0.098% and 0.07% respectively. Of the 44 IA-1 reactive samples, 95.2% (20/21) of the seroreactive samples by both IA-2 and IA-3 were also WB positive and 100% (6/6) of the non-reactive samples by these IAs were WB negative. IA signal/cutoff ratio was significantly low in biological false reactive donors. WB indeterminate results were largely due to non-specific reactivity to gag protein (p55). CONCLUSIONS: HIV seroreactivity by sequential immunoassays (IA-1, IA-2 and IA-3; comparable to WHO Strategy-III) prior to donor recall results in decreased referral to VCTC as compared to single IA (WHO Strategy-I) being followed currently in India. Moreover, this strategy will repose donor confidence in our blood transfusion services and strengthen voluntary blood donation program.

Change in tissue thromboplastin content of brain following trauma.

BACKGROUND: Tissue thromboplastin (TTP) is an integral membrane protein contributing to coagulopathy after trauma of brain, which is a rich source of TTP. AIMS: A study was undertaken to establish the TTP content of various areas of normal brain and estimate the changes in TTP activity of brain in response to varying degrees of trauma. MATERIALS AND METHODS: Samples from different areas of brain of ten cadavers were used as controls and they were compared with contused brain tissue obtained after surgery in 25 head injury (HI) patients of varying severity. RESULTS: In the study group, the TTP activity of the frontal, parietal, and temporal lobes after HI was significantly raised in contrast to that of the control group. The TTP activity was also significantly higher in the severe HI patients than those having moderate HI. The mode of injury and the time lapse after HI had no significant bearing on the TTP activity. Subjects above 40 years of age demonstrated a higher mean TTP activity after HI, though it was not statistically significant. CONCLUSION: The study provides quantitative data on TTP activity of normal brain and highlights the role of TTP in coagulopathy following HI through its increased activity after HI, more so in the severe HI group.

Vitamin K deficiency in diarrhoea.

The study was undertaken to determine the frequency of occurrence of vitamin K deficiency in infants with diarrhoeal illness. Infants were categorized into four groups as follows: A(acute diarrhoea), B(protracted diarrhoea) C(intractable diarrhoea) and D(healthy controls). Screening coagulation tests, PT and PTTK along with estimation of functional activity and total antigenic levels of prothrombin were performed. The ratio of functional to total prothrombin was calculated. PT was prolonged in 30% (24/75) of all infants with diarrhoea as compared to controls where the abnormality was observed in 11.1% infants (2/18). The ratio of functional to total prothrombin was significantly lower in infants with diarrhoea, the mean +/- SD values being 0.65 +/- 0.41 vs 1.1 +/- 0.26. This difference was statistically highly significant (p < 0.001). Low ratio was observed in 57.3% (43/75) infants with diarrhoea. Thus functional to total prothrombin ratio identified approximately twice as many diarrhoeal infants with vitamin K deficiency as compared to PT alone. There was no significant correlation with breast feeding as the only mode of diet, or the prior administration of antibiotics in infants with diarrhoea. The inherent malabsorptive state in diarrhoea may be a major contributory factor.

Alternative splicing of Drosophila calcium/calmodulin-dependent protein kinase II regulates substrate specificity and activation.

Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate multiple isoforms that vary only in a region between the calmodulin-binding domain and the association domain. This variation has been shown to modulate activation of the enzyme by calmodulin. In this study we examine the ability of seven of the Drosophila isoforms to phosphorylate purified protein substrates and to be inhibited by a substrate analog, and the response of six of the isoforms to a mutant form of calmodulin (V91G) that was isolated in a genetic screen. Significant variation in Kms for Eag, a potassium channel, and Adf-1, a transcription factor, were found. In the case of the a peptide inhibitor, AC3I, there were significant variations in Ki between isoforms. Kact for V91G calmodulin was increased for all of the isoforms. In addition, one isoform, RI, exhibited a lower Vmax when assayed with this mutant CaM. These results indicate that the variable domain of calcium/calmodulin-dependent protein kinase II is capable of altering the substrate specificity of the catalytic domain and the activation response to calmodulin.