Kanchan Mishra

Assessment of semi-automated nucleic acid testing programme in a Regional Blood Transfusion Centre

Abstract Background: Detection of human immunodeficiency virus type-1 (HIV-1), hepatitis-C (HCV) and hepatitis-B virus (HBV) in the blood donors is crucial. An efficient form of detection is nucleic acid testing (NAT) in blood screening. We assessed the suitability of commercial NAT testing in a developing country, focusing on the Altona RealStar assay and the method of Sacace Biotechnologies. Methods: We have standardised and validated commercially available NAT kits with a semi-automated system for detection of HBV, HCV and HIV-1 in blood donations. The MP-NAT (mini-pool) assay consists of pooling of sample, virus extraction, amplification and detection with commercially available NAT kits. An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification and detection process. Results: The sensitivity of the Altona RealStar assay at 10-MP for each viral target was evaluated, HBV showed amplification in all diluted positive samples of 100, 50, 25, 10 and 5 IU/ml. HIV and HCV infected samples showed amplification in all diluted positive samples of 500, 100, 50 and 30 IU/ml. For HIV, out of six diluted samples of 30 IU/ml, five were amplified. A total of 14,170 seronegative blood samples were tested by RealStar PCR kit in 10-MP and 6 (0.042%) samples/pools were positive. A total of 65,362 seronegative blood donations were also tested by kits of Sacace Biotechnologies, in 10-MP and 45 (0.075%) pools were positive. The prevalence of combined NAT yield cases among routine donors was 1 in 1559 donations tested for all the 3 viruses. Conclusion: The semi-automated combined system for NAT screening assays is robust, sensitive, reproducible, and this gives an additional layer of safety with affordable cost.

Prevalence of Macrothrombocytopenia in Healthy College Students in Western India

Macrothrombocytopenia is being increasingly described across the globe. There is paucity of data on the prevalence of this condition from different parts of India. 10,047 healthy college students from the city of Surat in western India were investigated for macrothrombocytopenia i.e. those with Mean platelet Volume of > 11 fL and platelet count of less than 150 × 109/L. ABO blood groups, complete blood counts, peripheral smear examination and haemoglobinopathy work up was also done. Siblings and parents of the macrothrombocytopenic individuals were also studied when available. Bleeding assessment tool of International society of thrombosis and haemostasis were applied to see if there were excessive bleeding in macrothrombocytropenia patients. One hundred and ninety-six students (1.95%) had asymptomatic macrothrombocytopenia. More female students (P < 0.0001) had this condition and blood group A was under represented (P = 0.019) with this condition. Prevalence of macrothrombocytopenia was not related to ethnic subgroups to which the students belonged to, nor was it linked to presence of any haemoglobinopathy gene. In 38 of the 52, 1st degree relatives studied macrothrombocytopenia was confirmed at least in one of them. Excessive bleeding in none of the individuals with macrothrombocytopenia was noted. Asymptomatic macrothrombocytopenia is rare in western parts of India and affects 1.95% of the healthy population. Females were over represented with this condition raising a suspicion of X linked dominant inheritance. Underrepresentation of blood group A in this condition requires further study.

Inherited Macrothrombocytopenia: Correlating Morphology, Epidemiology, Molecular Pathology and Clinical Features

AbstractInherited macrothrombocytopenia is increasingly being recognized as a relatively common condition. This descriptive review aims at focusing on the different areas of advancement that have taken place with this condition with particular reference to India. A pubmed search of articles between January 1990 and October 2017 with the key words-macrothrombocytopenia, asymptomatic macrothrombocytopenia, macrothrombocytopenia India, syndromic macrothrombocytopenia, molecular pathology, megakaryopoiesis and platelet formation were searched. The shortlisted articles were then read. Review articles provided additional references and the articles thus obtained were also read. Special interest and research conducted by the authors provided further sources of information. A total of 487 articles were found of which 68 articles were related to our subject of review. Review articles were read and additional articles from the reference quoted. Forty-four percent of nonsyndromic Inherited macrothrombocytopenia showed mutations of MYH9, GP1BB, GP1Ba, GPIX, ABCG5 and 8, ACTN, FLI, TUBB and RUNX1 frequently in heterozygous state. All types of inheritance pattern namely autosomal dominant, recessive and sex linked patterns have been described. Syndromic causes of this phenomenon are well known and have been described. Many asymptomatic patients do have mild or moderate bleeding history. Clinical algorithms to differentiate chronic ITP associated macrothrombocytopenia from inherited variety have been explored. Inherited macrothrombocytopenia is an emerging area of interest in platelet biology with its implication in diagnosis, prognosis, genetic counseling, management and in transfusion medicine.

Reasons for Discarding of Whole Blood/Red Cell Units in a Regional Blood Transfusion Centre in Western India

Abstract To analyze the reason for discarding whole blood and red cell concentrates in a Regional Blood Transfusion Centre in India. Retrospective analysis of electronic data on collection of blood and reason for discard of whole blood and red cell concentrate between January 2012 and December 2016. 1,70,431 units of blood were collected between January 2012 and December 2016 in various blood donation camps. On an average 6.60% whole blood or red cell units were discarded because of various reasons. Out dating was the single important cause for discarding such units leading to loss of 6.7–7 million rupees (USD 1,00,000) to the blood bank. Infective units, haemolysed units, insufficient amount collected units and leakage were other important causes for discarding the units. Using multiple approaches of donor selection, staff training rescheduling of blood camps and sharing this precious resource with other blood bank can significantly minimize the discard rate. The reasons for discard of blood units varied not only from one blood centre to other but also from one country to another.

NAT positivity in seronegative voluntary blood donors from western India

BACKGROUND AND OBJECTIVES Prevalence and composition of Hepatitis B, Hepatitis C and HIV-1, NAT positive but seronegative voluntary blood donors from western part of India is yet to be documented. MATERIAL AND METHODS Over last 2 1/2 years all the seronegative voluntary blood donors were tested using 10 minipools on a semiautomated NAT testing platform. The positively tested donors were followed up for at least five months for development of seropositivity. RESULTS 79532 seronegative donations were tested by 10 minipool (MP) NAT leading to 51 positive sample (44 Hep B, 5 HIV 1 and Hep C positive). All the HIV and Hep C NAT positive donors eventually developed seropositivity and out of 44 Hep B NAT positive donors, 31 developed seropositivity within six months of follow up, following counseling of the donors. This data translate into NAT yield of 1:1559 donors for all virus taken together. NAT yield for Hep B 1:1807 donors were much higher than HIV 1 in 1:15906 and HCV yield of 1:39761. Semiautomated minipool NAT testing system was found to be cost effective way for improving blood safety. INTERPRETATION AND CONCLUSION Seronegative NAT yield in voluntary blood donors are quiet high in western part of India and in line with rest of the country is mainly due to Hepatitis B infection. Implementation of strict donor screening, Hep B vaccination of the population and sample mutation of NAT testing should be under taken on war footing.

Nucleic acid amplification testing in Indian blood banks: A review with perspectives

Background: Nucleic acid amplification testing (NAT) is restricted to a few blood banks in India since 2008. This review was directed toward understanding NAT yield in different parts of the country and prevalence in the NAT of different types of virus. Materials and Methods: English literature was searched from 1990 to 2016 in PubMed, Scopus, Ind med, and Google database using properly constructed key words. Literature was collected and finally the data were synthesized. Results: NAT results from 11 publications and one personal communication showed that till date 389387 blood units have been NAT tested from various parts of the country. NAT yield varied from 1:476 to 1:4403 in various studies. Till date, 58/2550 (2%) blood banks of India are doing NAT testing but all of them have not published their results. Majority of the centers have used ID-NAT (Individual NAT) protocol and 21 blood banks are using minipool format of the test. One center has used in-house NAT testing system. In >70% of the time, the NAT positivity with due to hepatitis B (Hep B). For individual infection, NAT yield from the pooled data showed HIV in 1:66,000, Hep C virus 1:5484 and Hep B in 1:1761 seronegative donors. Discussion and Conclusion: In view of the very high NAT yield (1:1361), NAT in some from needs to be universally applied in Indian blood banks. However, the high Hep B occult infection suggests stricter donor selection and immunization of adults for Hep B may be way forward toward ensuring the viral safety of blood components in India.

A multiplex ARMS PCR approach to detection of common β-globin gene mutations

BACKGROUND β-thalassaemia is a group of inherited single-gene disorders worldwide. Each ethnic population has its own common mutations. Heterogeneity of β-thalassaemia mutations in multi-ethnic population of Surat, makes molecular diagnosis expensive and time consuming. METHODS Specific primers were used to differentiate four common mutations, IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G), by a simple PCR involving a multiplex amplification refractory mutation system. RESULTS Several high prevalence β-Thalassemia trait groups constituted by Muslims, Patels, Sindhis, ModhBanias, and Mahayavanshi. Four most common mutations detected in them are IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G). We identified each of these β-thalassemia mutations in multiplexed ARMS from positive control samples. Our multiplex-ARMS-PCR system was first standardized on positive DNA samples with above known four most common β-thalassemia mutations, and these positive samples had been diagnosed with β-thalassemia and also all these samples belonged to Surat ethnic groups. The system was subsequently tested on 110 blood samples from different ethnic backgrounds with unknown β-thalassemia mutations which were in all specimens. CONCLUSION The ARMS multiplex system was found reliable, cost effective, fast and most applicable for mutation screening of Thalassemia in Surat populations.