Mohit Chowdhry

Hepatitis B core antibody testing in Indian blood donors: A double-edged sword!

Background: Until lately, anti-HBc antibodies were considered an effective marker for occult Hepatitis B virus (HBV) infection and have served their role in improving blood safety. But, with the development of advanced tests for HBV DNA detection, the role of anti-HBc in this regard stands uncertain. Materials and Methods: Anti-HBc and HBsAg ELISA and ID-NAT tests were run in parallel on donor blood samples between April 1, 2006 and December 31, 2010 at the Department of Transfusion Medicine, Indraprastha Apollo Hospitals, New Delhi. A positive ID-NAT was followed by Discriminatory NAT assay. Results: A total of 94 247 samples were tested with a total core positivity rate of 10.22%. We identified nearly 9.17% of donors who were reactive for anti-HBc and negative for HBsAg and HBV DNA. These are the donors who are potentially non-infectious and may be returned to the donor pool. Conclusion: Although anti HBc testing has a definite role in improving blood safety, centers that have incorporated NAT testing may not derive any additional benefit by performing anti-HBc testing, especially in resource-limited countries like ours.

Red cell alloimmunization and infectious marker status (human immunodeficiency virus, hepatitis B virus and hepatitis C virus) in multiply transfused thalassemia patients of North India

BACKGROUND Patients with thalassemia major are largely transfusion dependent and are thus exposed to a variety of risks such as transmission of infectious diseases, iron overload and alloimmunization. This study was performed to determine the prevalence of human immune deficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and red cell antibodies among multiple-transfused thalassemic patients in and around the national capital region. MATERIALS AND METHODS The Department of Transfusion Medicine, Indraprastha Apollo Hospitals, conducted this study in collaboration with the National Thalassemia Welfare Society over a period of 1 year starting February2011. Blood samples from the patients were tested for blood group, red cell alloantibody/ies, anti-HIV, anti-HCV and hepatitis B surface antigen (HBsAg) by ELISA and for the respective viral ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) by nucleic acid testing (NAT). RESULTS A total of 462 thalassemics which consists of 290 males and 172 females were tested. The overall alloimmunization rate was 4.1% and anti-Kell was the most common antibody identified. Thirteen cases (2.8%) were positive for HBsAg by ELISA, 107 (23.1%) were reactive for anti HCV and 11 (2.38%) for anti HIV antibodies. Further screening and discriminatory assays by NAT confirmed the presence of HBV DNA in 11 cases, HIV RNA in 7 cases and HCV RNA in 48 cases. CONCLUSION In spite of advances in Immunohematology and infectious marker testing in recent years, the rates of alloimmunization and infectious marker positivity remains high among multiply transfused patients like thalassemics. Provision of safe and adequate blood supply to these patients is a key to improving their quality-of-life and longevity.

Prevalence of HIV among blood donors in a tertiary care centre of north India.

Background & objectives: India has the second highest HIV population in the world with about 2.5-3.0 million cases. HIV-2 cases among general and blood donor population have also been reported mostly from west and south India. This single centre study was carried out to observe the HIV-1 and HIV-2 prevalence among blood donors from north India. Methods: A total of 2,04,677 people were screened for the presence of HIV infection over the 11 year period (1999 to 2009). Till 2004, a third generation ELISA kit was used. From 2005 till January 2009 all tests were done using the fourth generation ELISA kit which detected the presence of HIV-1 P24 antigen and anti-HIV antibodies. From February 2009 onwards, the kits used were Genscreen ULTRA HIV Ag-Ab Assay. Results: A total of 506 (0.247%) donors were found to be repeat reactive for HIV. Of these, 486 (96%) donors tested using the Western blot were found positive for HIV-1 infection. Twenty (4%) donors showed a negative Western blot result, none of the donors were found reactive for HIV-2 infection. Interpretation & conclusions: The prevalence of HIV was 0.249 per cent among blood donors of north India. No HIV-2 case was found among the studied blood donor population indicating that it is not a threat currently.

Preoperative predictors of blood component transfusion in living donor liver transplantation

Context: Extensive bleeding associated with liver transplantation is a major challenge faced by transplant surgeons, worldwide. Aims: To evaluate the blood component consumption and determine preoperative factors that predict the same in living donor liver transplantation (LDLT). Settings and Design: This prospective study was performed for a 1 year period, from March 2010 to February 2011. Materials and Methods: Intra- and postoperative utilization of blood components in 152 patients undergoing LDLT was evaluated and preoperative patient parameters like age, gender, height, weight, disease etiology, hemoglobin (Hb), hematocrit (Hct), platelet count (Plt), total leukocyte count (TLC), activated partial thromboplastin time (aPTT), international normalized ratio (INR), serum bilirubin (T. bilirubin), total proteins (T. proteins), albumin to globulin ratio (A/G ratio), serum creatinine (S. creatinine), blood urea (B. urea), and serum electrolytes were assessed to determine their predictive values. Univariate and stepwise discriminant analysis identified those factors, which could predict the consumption of each blood component. Results: The average utilization of packed red cells (PRCs), cryoprecipitates (cryo), apheresis platelets, and fresh frozen plasma was 8.48 units, 2.19 units, 0.93 units, and 2,025 ml, respectively. Disease etiology and blood component consumption were significantly correlated. Separate prediction models which could predict consumption of each blood component in intra and postoperative phase of LDLT were derived from among the preoperative Hb, Hct, model for end-stage liver disease (MELD) score, body surface area (BSA), Plt, T. proteins, S. creatinine, B. urea, INR, and serum sodium and chloride. Conclusions: Preoperative variables can effectively predict the blood component requirements during liver transplantation, thereby allowing blood transfusion services in being better prepared for surgical procedure.

Seroprevalence of infectious markers & their trends in blood donors in a hospital based blood bank in north india

Background & objectives: Hepatitis B virus (HBV), human immunodeficiency virus (HIV), hepatitis C virus (HCV) and syphilis infections pose a great threat to blood safety. This study was undertaken to investigate the seroprevalence of serologic markers for transfusion transmitted infections (TTIs) among blood donors at a hospital based blood centre in north India over a period of nine years. Methods: The results of serologic markers for TTIs (HBsAg, anti-HCV, anti-HIV and syphilis) of all blood donations (both voluntary and replacement) at our hospital from January 2005 to December 2013 were screened. Additional analysis was conducted to examine the prevalence trends associated with each of the positive marker. Results: The data of 180,477 donors [173,019 (95.86%) males and 7,458 (4.13%) females] were analyzed. Replacement donations [174,939 (96.93%)] represented the majority whereas, only 5,538 (3.06%) donations were from the voluntary donors. The risk of blood being reactive was three times higher in male donors when compared with the female donors. The risk of blood being reactive for one or more infectious markers was 2.1 times higher in replacement donors when compared with the voluntary donors. Seropositivity of HIV, HBsAg, HBcAb, syphilis showed a significant decreasing trend (P<0.05) while there was an increasing trend in HCV infection which was insignificant. Interpretation & conclusions: This study reflects that the risk of TTIs has been decreased over time with respect to HIV, HBV and syphilis, but the trends for HCV remains almost the same in blood donors. Blood transfusion remains a risk factor for the spread of blood-borne infections. Therefore, improvements are needed to strengthen both safety and availability of blood.

Clinical significance of antibody specificities to M, N and Lewis blood group system.

Context: The clinically significant antibodies are those active at 37°C and/or by the indirect antiglobulin test. Most of the published literature refers to antibodies of Lewis blood group system to be insignificant, whereas antibodies to M and N blood groups are associated with variable clinical significance. Aims: The aim of this study is to find the frequency and clinical significance of antibodies to M, N and Lewis blood group systems. Settings and Design: The study was carried out retrospectively from January 2009 to December 2012. Materials and Methods: Antibody screening was performed by solid phase red cell adherence (SPRCA) technique using four cell screening panel on a fully automated platform GALILEO (Immucor Inc. USA). In case of a positive antibody screen, antibody identification was performed using SPRCA (GALILEO, Immucor Inc. USA). Results: A total of 49,077 red cell antibody screens were performed and a total of 427 identifications of red cell antibodies were carried out. A total of 304 specific antibodies were detected: 8.22% of antibodies were of anti-M specificity and 2.96% were of anti-N specificity. Majority (84%) of anti-M and 77.78% of anti-N were of Immunoglobulin G (IgG) class reacting at 37°C. 1.31% of the antibodies were directed against Lewis system antigens of which 0.65% were anti-Lea and 0.65% were anti-Leb. Half of the Lewis system antibodies, i.e., 1 each of anti-Lea and anti-Leb were of IgG class. Conclusion: Our study highlights the importance of detecting the thermal amplitude of antibodies with variable clinical significance especially if both IgG and IgM types of antibodies are associated with it so as to establish their clinical significance.

A multivariate analysis to assess the effect of packed red cell transfusion and the unit age of transfused red cells on postoperative complications in patients undergoing cardiac surgeries.

Background: Transfusion of blood components and age of transfused packed red cells (PRCs) are independent risk factors for morbidity and mortality in cardiac surgeries. Materials and Methods: We retrospectively examined data of patients undergoing cardiac surgery at our institute from January 1, 2012 to September 30, 2012. Details of transfusion (autologous and allogenic), postoperative length of stay (PLOS), postoperative complications were recorded along with other relevant details. The analysis was done in two stages, in the first both transfused and nontransfused individuals and in the second only transfused individuals were considered. Age of transfused red cells as a cause of morbidity was analyzed only in the second stage. Results: Of the 762 patients included in the study, 613 (80.4%) were males and 149 (19.6%) were females. Multivariate analysis revealed that factors like the number and age of transfused PRCs and age of the patient had significant bearing upon the morbidity. Morbidity was significantly higher in the patients transfused with allogenic PRCs when compared with the patients not receiving any transfusion irrespective of the age of transfused PRCs. Transfusion of PRC of over 21 days was associated with higher postoperative complications, but not with in-hospital mortality. Conclusion: In patients undergoing cardiac surgery, allogenic blood transfusion increases morbidity. The age of PRCs transfused has a significant bearing on morbidity, but not on in-hospital mortality. Blood transfusion services will therefore have to weigh the risks and benefits of providing blood older than 21 days in cardiac surgeries.

Weak D prevalence among Indian blood donors

Sir, The field of medicine has since infinity been associated with consistent discoveries thus leading to ever increasing list of advancing technologies. When talking about transfusion medicine, the discovery of blood group antigens marked the revolutionising step in the success of blood transfusions. Following the discovery of ABO antigens by Landsteiner in 1901,[1] the next most important discovery was that of Rh antigens in 1939 by Landsteiner and Weiner further leading to description of haemolytic disease of new born by Levine and Stetson.[2] The Rh antigen itself has been studied extensively and its alleles recognised. The Rh gene lies on chromosome number 1 and is carried in groups of three.[3] The Rh locus is composed of two highly homologous genes: the RHD gene, which encodes the D protein, and the RHCE gene, which encodes the C, c, E, and e proteins.[4] 6 alleles have been identified (c, C, e, E, d, D) as against only 5 antigens (c, C, e, E, D), d being an amorph gene.[5] Among these, D is the most immunogenic. Consequently, D often is called the Rh antigen, and the terms Rh+ and Rh– refer, respectively, to the presence or absence of D antigen. The Rh complex is critical to the structure of the RBC membrane. Rh null erythrocytes, which lack Rh proteins, are stomatocytic and spherocytic, and affected individuals have haemolytic anemia.[6] The RhD antigen has been reported to consist of a mosaic of at least 9 D epitopes (epD1-epD9).[7] Recent work involving testing a large number of monoclonal anti-D (MAb-D) reagents has suggested the presence of a minimum of 30 different epitope structures[8] distributed along the extracellular portions of the RhD protein. Thus a change, or changes, in the amino acid sequence of RhD may not ablate the entire D antigen but can cause epitope loss, giving rise to variant forms of D antigen. “Weak D” RBCs demonstrate reduced quantities of the D antigen. As a result weak or no agglutination reaction is demonstrated by these RBCs with the anti D reagents at the immediate spin phase. About 0.1 to 2 percent of white Caucasians have this Rh phenotype.[9] Missense mutations observed in the alleles of all weak D types have been demonstrated to be the probable cause for the reduced antigen D expression in these cases.[10] In “Partial D” RBCs, the RHD protein is mutated in an exofacial loop, eliminating at least one D-specific epitope. However, the numbers of RhD antigens on the RBC surface are normal.[10] The “DEL” phenotype is a third group of D variants. DEL cannot be detected by routine serological testing. It is, however, easily detected by genetic analysis.[11] DEL RBCs contain an extraordinarily low number of D antigens but, despite this paucity, can cause primary[12] and secondary[13] immune responses against the D antigen in D negative recipients. Fortunately its incidence is very low. It is found mainly amongst Asian populations where a recent study found a DEL allele in approximately 13% to 16% of serologically D negative Chinese and Japanese individuals.[14] A myriad of different serological techniques and reagents, each with different sensitivities, and a rapidly expanding understanding of the genetics of the RHD gene have greatly complicated D typing. In this study we tried to estimate the prevalence of weak D in our population based on the serological techniques. As ours is a prominent tertiary care hospital of the country, the donors here come from various parts of the country and are representative of the whole Indian population. This study was conducted in the Department of Transfusion Medicine, Indraprastha Apollo Hospitals from 01 January 1999 to 31st December 2008. During this period, a total of 1, 84,072 donors came to our blood bank. As a routine protocol, Rh typing was done for all these donors. Those who tested negative for Rh D antigen were further subjected to weak D testing. Routine Rh typing was done using the immediate spin tube technique with two anti-D reagents (RHOFINAL Anti D IgM + IgG by TULIP and RHESOLVE Anti D IgM monoclonal by ORTHO). Blood samples, which were negative for agglutination by immediate spin tube method, were further tested for weak D. A 5% suspension of the cells to be tested was made. Three tubes were taken and labelled as test, negative and positive control. To the tube labelled as test 2 drops of Anti D serum (RHOFINAL Anti D IgM + IgG by TULIP) was added. 2 drops of 22% bovine albumin were added to the negative tube and to the positive control 2 drops of weak anti D (i.e. 1 drop of Anti D IgM monoclonal by ORTHO + 19 drops of saline). 2 drops of 5% cell suspension was added to all the test tubes. They were mixed and incubated at 37°C in a water bath for 45-60 minutes (as per manufacturer’s instructions). The tube was gently re-suspended and the cell button observed for agglutination. If the test red cells were agglutinated (but not in the negative control tube) the test was recorded as positive. If the test cells were not agglutinated or the results were doubtful, the cells were washed three to four times with normal saline. After the final wash, the saline was decanted and one to two drops of antiglobulin serum was added according to manufacturer’s instructions. Following this, the contents of the test tube were mixed and the tube was centrifuged at 1000 rpm for 30 seconds. The cell button was then gently re-suspended and examined for agglutination. All negative results were confirmed under the microscope. Samples showing agglutination after incubation or after addition of AHG serum (Bioclone Anti IgG, C3d by Ortho) were considered to be weak D positive. The data was analysed to provide the following results. A total of 1, 84, 072 donors were studied. Among these 13, 253 (7.2%) were tested to be Rh negative. All the Rh negative samples were subjected to weak D testing. Of the 13, 253 samples that tested to be Rh negative, 16 (0.01% of total donors and 0.12% of Rh negative donors) were found to be weak D positive. [Table 1, Figure ​Figure1a,1a, and ​andbb] Figure 1 a-b Data label (value) missing in diagram b, 0.12% (pink region) Table 1 Weak D prevalence in blood donors In our study 7.19% of the donors were Rh negative and the weak D variant was detected in about 0.01%. This extensive study was done by us over a period of 10 years. To our knowledge, this study is the largest of its kind that we came across in the published literature. Our hospital, being a major tertiary care centre of the country, caters to a population covering large geographical areas and therefore our results can be considered as representative of the whole country. Rh system is the most complex of all 29 blood group systems. New discoveries relating to the RHD gene, and an appreciation of its variant phenotypes have challenged the way that D status is assigned to both blood donors and blood product recipients. The weak D, previously called Du, has been the subject of many studies ever since it was identified. Controversy as to whether the weak D is a quantitative or qualitative D variant and whether or not it stimulates anti-D immunization in RhD-negative persons still persists. Historically, RBCs that react with anti-D only after extended testing in the AHG phase are called weak D. However, the number of samples classified as weak D depends on the characteristics of the typing reagent.[15] The improved sensitivities of the anti D sera have decreased the prevalence of weak D phenotypes. The prevalence also varies from region to region. We reported Weak D in 0.01% of our donor population. Slightly lower values have been reported in a Korean study and higher values have been reported in the Caucasians.[16,17] The main concern about this Rh phenotype arises due to the risk of alloimmunisation among the recipients. Since, the “D” antigen is highly immunogenic, individuals with the Weak D phenotype are designated D pos. The patients with weak D are considered D negative and must be transfused with D negative blood. Mothers with weak D fetus must receive Rh immunoprophylaxis[18] as passage of Weak D red cells from fetus to mother may cause sensitization. However, there have been studies suggesting that the most common weak D types (1, 2, 3, 4.0, and 4.1), encompassing more than 90% of all European weak D individuals, do not appear to be susceptible to immunization to the D antigen[7] These individuals could safely receive D positive blood and do not need Rh immune globulin prophylaxis during pregnancy. However, serological tests cannot discriminate between these weak D types and those that are susceptible to alloimmunization; only a molecular analysis of the RHD gene can distinguish between weak D types. The prevalence of Rh negativity in our donor population has been estimated to be 7.19% and that of weak D antigen 0.01% in our study. However, a study analysing repeat antibody screens of serologically D negative patients with weak D alleles who have been exposed to D+ RBCs is needed to quantify the absolute risk of sensitization.

Clinicohematological correlation and chromosomal breakage analysis in suspected Fanconi anemia patients of India

Introduction: The management of patients with aplastic anemia, to an extent, depends on the etiology i.e., inherited or acquired. The classical Chromosomal breakage study involves detection of chromosomal breakage or aberrations (breaks, gaps, rearrangements, radials, exchanges, endoreduplications) in peripheral blood cells after culture with a T-cell mitogen and a DNA clastogenic (cross-linking) agent, such as diepoxybutane (DEB) or mitomycin C (MMC). The testing needs to be performed in laboratory with appropriate expertise in Fanconi Anemia testing. The present study was undertaken to find out the frequency of inherited aplastic anemia in North India. Materials and Methods: This study was carried out at the Department of Molecular Biology and Transplant Immunology, Indraprastha Apollo hospital, New Delhi. The study includes retrospective analysis of 528 aplastic anemia patients whose samples were tested at our department for Chromosomal breakage study during the period 2007 to 2011. Respective age and sex matched healthy controls were also processed for chromosomal breakage study. Patients habitat, clinical symptoms, differential blood count and history of drug exposure were documented for all patients referred to us, whereever available. Relative risk was estimated by odds ratio (OR) with 95% confidence interval (CI) in matched cases and controls. Conclusion: A significant increase in chromosomal breakages was seen in 13.1% patients. The survival data documented for 100 patients suggested 60% mortality.

Frequency of Mia antigen: A pilot study among blood donors

The Miltenberger (Mi) classes represent a group of phenotypes for red cells that carry low frequency antigens associated with the MNSs blood group system. This pilot study was aimed at determining the Mia antigen positivity in the blood donor population in a tertiary care hospital in New Delhi, India. The study was performed between June to August 2014 on eligible blood donors willing to participate. Antigen typing was performed using monoclonal anti-Mia antiserum by tube technique. Only one of the 1000 blood donors (0.1%) tested was found to be Mia antigen positive. The Mia antigen can, therefore, be considered as being rare in the Indian blood donor population.