A novel syphilis Treponema pallidum lipoprotein peptide antigen diagnostic assay using red cell kodecytes in routine blood centre column agglutination testing platforms.

BACKGROUND AND OBJECTIVES: The detection of treponemal antibodies, which are used to make a diagnosis of syphilis, is important both for diagnostic purposes and as a mandatory blood donor test in most countries. We evaluated the feasibility of using Kode Technology to make syphilis peptide red cell kodecytes for use in column agglutination serologic platforms. MATERIALS AND METHODS: Candidate Kode Technology function-spacer-lipid (FSL) constructs were made for the Treponema pallidum lipoprotein (TmpA) of T. pallidum, using the peptide and FSL selection algorithms, and then used to make kodecytes. Developmental kodecytes were evaluated against a large range of syphilis antibody reactive and non-reactive samples in column agglutination platforms and compared against established methodologies. Overall, 150 reactive and 2072 non-reactive Syphicheck assay (a modified T. pallidum particle agglutination) blood donor samples were used to evaluate the agreement rate of the developed kodecyte assay. RESULTS: From three FSL-peptide candidate constructs, one was found to be the most suitable for diagnostics. Of 150 Syphicheck assay reactive samples, 146 were TmpA-kodecyte reactive (97.3% agreement), compared with 58.0% with the rapid plasmin reagin (RPR) assay for the same samples. Against the 2072 expected syphilis non-reactive samples the agreement rate for TmpA-kodecytes was 98.8%. CONCLUSION: TmpA-kodecytes are viable for use as cost-effective serologic reagent red cells for the detection of treponemal antibodies to diagnose syphilis with a high level of specificity in blood centres. This kodecyte methodology also potentially allows for introduction of the reverse-algorithm testing into low-volume laboratories, by utilizing existing transfusion laboratory infrastructure.

Molecular basis of DEL phenotype in the Indian population: Insights from next-generation sequencing analysis of two cases.

The DEL phenotype represents an intriguing and challenging aspect of blood group serology. This condition is characterized by an extremely weak expression of the D antigen on red blood cells, to the extent that it often eludes detection through routine serological methods. Identifying the DEL phenotype necessitates more specialized techniques, such as adsorption and elution tests, to reveal the presence of the D antigen. This distinctive phenotype underscores the complexity and subtlety of blood group genetics and highlights the importance of using advanced methods to accurately classify individuals with this condition, as their ability to form anti-D antibodies can have clinical implications during transfusion and pregnancy scenarios. There is a paucity of data for the DEL phenotype in the Indian population, and the molecular basis has not been elucidated yet. Our investigation delves into the genetic underpinnings of two distinct DEL phenotype cases that pose challenges for resolution through conventional serological techniques. We employ next-generation amplicon sequencing to unravel the intricate genetic landscape underlying these cases. In the D-negative donor, the DEL phenotype was first identified serologically, which was subsequently confirmed by molecular analysis. In the second case, it was associated with an anti-D antibody in a D-positive patient. Initial data analysis unveiled a substantial reduction in coverage across the exonic segments of the RHD gene in both samples, suggesting the potential presence of RHD exon deletions. On both occasions, we identified a homozygous intronic RHD polymorphism that is well established to be linked to the RHD* 01EL.32/RHD*DEL32 variant.

A national survey of current immunohematologic testing practices for the diagnosis of autoimmune hemolytic anemia in India.

Autoimmune hemolytic anemia (AIHA) is a common term for several disorders that differ from one another in terms of etiology, pathogenesis, clinical features, and treatment. Management of patients with AIHA has become increasingly evidence-based in recent years. While this development has resulted in therapeutic improvements, it also carries increased requirements for optimal diagnosis using more advanced laboratory tests. Unfortunately, limited data are available from developing countries regarding the testing and transfusion management of patients with AIHA. The main objective of this survey was to explore the current immunohematologic testing practices for the diagnosis of AIHA in India. This online survey consisted of 30 questions, covering the place of work, the number of AIHA cases encountered in the 3 preceding years, testing method(s), transfusion management, and so forth. Individuals representing 89 laboratories completed the survey; only 78 of which responded that AIHA testing was performed in their facility's laboratory. The majority of respondents agreed that the most commonly affected age-group comprised individuals of older than 20 years, with a female preponderance. Regarding transfusion management, respondents indicated that transfusion with "best-match" red blood cell units remains the most common practice. Column-agglutination technology is used by 92 percent of respondents as the primary testing method. Although a monospecific direct antiglobulin test is available at 73 percent of the sites, most of them have limited access to other resources that could diagnose cold or mixed AIHA. Merely 49 percent of responding laboratories have the resources to perform adsorption studies for the detection of alloantibodies. Furthermore, three-cell antibody screening reagents are unavailable at 32 percent of laboratories. In 72 percent of centers, clinical hematologists would prefer to consult a transfusion medicine specialist before administering treatment to AIHA patients. There is unanimous agreement regarding the need for a national registry. The survey data indicate wide variability in testing practices for patients with AIHA in India. Future studies are needed to focus on the feasibility and cost-effectiveness of different testing strategies for developing countries.

Are we missing reverse TRALI? - A real world experience from a tertiary care oncology centre in India.

Transfusion related acute lung injury (TRALI) is a rare but potentially fatal pulmonary complication of transfusion that presents as acute hypoxemia and non-cardiogenic pulmonary oedema, developing during or within six hours of transfusion. Majority of the cases reported are due to transfusion of plasma rich blood components containing antibodies to human leukocyte antigen (anti-HLA) or human neutrophil antigen (anti-HNA). Rarely, anti-HLA or anti-HNA in recipients against transfused donor leukocyte antigens, cause TRALI by a reverse mechanism. Herein, we report three cases of suspected TRALI following transfusions of buffy coat derived granulocytes and peripheral blood stem cells. Three patients with hematological malignancies developed pulmonary symptoms after transfusions of leukocyte rich blood components. All cases showed findings of bilateral pulmonary infiltrates at chest radiography and patients were managed accordingly; however, all three expired within seven days of transfusion due to progressive respiratory deterioration. The patients were transfusion dependent for a long time and had received multiple non-leukoreduced blood components in the past. Clinical findings in all three cases indicate the possibility of reverse TRALI. Although, patients' anti-HLA or anti-HNA antibodies concordance with donors' cognate antigens (HLA and HNA) was not confirmed; yet these three cases suggest that reverse pathogenesis of TRALI is not as infrequent as reported in the literature. However, reverse TRALI has not been confirmed as the presence and nature of antibodies in the transfused recipient were not investigated due to the non availability of immunodiagnostic tests in India.

An anatomically comprehensive atlas of the adult human brain transcriptome.

Neuroanatomically precise, genome-wide maps of transcript distributions are critical resources to complement genomic sequence data and to correlate functional and genetic brain architecture. Here we describe the generation and analysis of a transcriptional atlas of the adult human brain, comprising extensive histological analysis and comprehensive microarray profiling of ∼900 neuroanatomically precise subdivisions in two individuals. Transcriptional regulation varies enormously by anatomical location, with different regions and their constituent cell types displaying robust molecular signatures that are highly conserved between individuals. Analysis of differential gene expression and gene co-expression relationships demonstrates that brain-wide variation strongly reflects the distributions of major cell classes such as neurons, oligodendrocytes, astrocytes and microglia. Local neighbourhood relationships between fine anatomical subdivisions are associated with discrete neuronal subtypes and genes involved with synaptic transmission. The neocortex displays a relatively homogeneous transcriptional pattern, but with distinct features associated selectively with primary sensorimotor cortices and with enriched frontal lobe expression. Notably, the spatial topography of the neocortex is strongly reflected in its molecular topography-the closer two cortical regions, the more similar their transcriptomes. This freely accessible online data resource forms a high-resolution transcriptional baseline for neurogenetic studies of normal and abnormal human brain function.

ABO, Rhesus, and Kell Antigens, Alleles, and Haplotypes in West Bengal, India.

BACKGROUND: Few studies have documented the blood group antigens in the population of eastern India. Frequencies of some common alleles and haplotypes were unknown. We describe phenotype, allele, and haplotype frequencies in the state of West Bengal, India. METHODS: We tested 1,528 blood donors at the Medical College Hospital, Kolkata. The common antigens of the ABO, Rhesus, and Kell blood group systems were determined by standard serologic methods in tubes. Allele and haplotype frequencies were calculated with an iterative method that yielded maximum-likelihood estimates under the assumption of a Hardy-Weinberg equilibrium. RESULTS: The prevalence of ABO antigens were B (34%), O (32%), A (25%), and AB (9%) with ABO allele frequencies for O = 0.567, A = 0.189, and B = 0.244. The D antigen (RH1) was observed in 96.6% of the blood donors with RH haplotype frequencies, such as for CDe = 0.688809, cde = 0.16983 and CdE = 0.000654. The K antigen (K1) was observed in 12 donors (0.79%) with KEL allele frequencies for K = 0.004 and k = 0.996. Conclusions: For the Bengali population living in the south of West Bengal, we established the frequencies of the major clinically relevant antigens in the ABO, Rhesus, and Kell blood group systems and derived estimates for the underlying ABO and KEL alleles and RH haplotypes. Such blood donor screening will improve the availability of compatible red cell units for transfusion. Our approach using widely available routine methods can readily be applied in other regions, where the sufficient supply of blood typed for the Rh and K antigens is lacking.

Role of thromboelastography in the management of snake bite: A case report from India.

In the absence of a specific laboratory test of envenomation, there is a need for an alternative mechanism for the early recognition of envenomation following hematotoxic snake-bite in tropical countries. Abnormalities of clotting are commonly associated with hematotoxic snake bite either due to systemic envenomation or due to the release of an inappropriate tourniquet applied as 'first-aid' often by the rural people before presentation to the hospital. Thromboelastography (TEG) has been used to monitor the coagulation abnormalities in various clinical scenarios. Here we narrate our experience where regular monitoring of hemostasis by TEG had helped us to successfully manage a case of hematotoxic snake-bite in a 45 year old male patient from rural India.

An analysis of transfusion support in haematopoietic stem cell transplantation--report from a centre in India.

BACKGROUND: Transfusion support in haematopoietic stem cell transplantation (HSCT) can be very demanding and challenging. The conditioning regimen, stem cell dose, donor type, presence of GvHD, infection all influence transfusion therapy in haematopoietic stem cell transplantation (HSCT). We retrospectively analysed the first 100 days transfusion requirements among HSCT recipients with haematological as well as non-haematological malignancies in our centre. MATERIALS AND METHODS: Transfusion data were retrieved for 100 patients who had undergone HSCT over a period of two years. The HSCT recipients were divided into three groups: autologous, allogenic and haplo-identical. Allogenic group was subdivided into matched related donor (MRD) and matched unrelated donor (MUD). The allo and haplo groups were then classified on the basis of the ABO compatibility as major, minor, bi-directional and compatible. We analysed the mean requirement of blood components (RBC, RDP, SDP and FFP) within the first 100 days of HSCT in each category. RESULTS AND DISCUSSION: Haematologic malignancies constituted 97% of the indications for HSCT. Allo-HSCT constituted 50% of the HSCT, of which 92% were MRD. Auto and haplo-HSCT constituted 40% and 10% respectively. Mean requirement for all products--RBC, SDP, RDP and FFP--was highest in the haplo category, followed by the allo category and then the auto HSCT category. The mean product requirement in the MUD category was significantly higher than in the MRD category (p < 0.05). The mean product requirement in the major and bidirectional ABO incompatible group was significantly higher as compared to the minor and ABO compatible group (p < 0.05). Hence our data may help transfusion medicine specialists to understand the transfusion requirement in stem cell transplant settings from developing countries like India. The average number of blood donors required for each group of stem cell transplant patients can also be roughly predicted from this study.

Warm autoimmune hemolytic anemia with mimicking anti-e specificity causing intravascular hemolysis in a chronic ITP patient.

A 12-year-old male child presented to the emergency room with three days history of cola-colored urine, mild icterus, dyspnea, palpitation and fatigue. He had a history of chronic ITP two years ago and had since been on steroid for maintenance of platelet count. He was subsequently diagnosed as a case of warm autoimmune hemolytic anemia. Laboratory investigations were suggestive of intravascular hemolysis, and on immuno-hematological evaluation it was diagnosed that the patient had autoantibody with mimicking anti-e specificity. The specificity of autoantibody was further confirmed by adsorption study. The patient was successfully managed by transfusion of Rh(e)-negative red cells,steroid and rituximab therapy. So an autoantibody with mimicking anti-e specificity was identified in this case, which was significant in clinical point of view.

Current challenges of blood transfusions in patients with thalassemia in India and future perspectives.

The introduction of regular red blood cell transfusions transformed thalassemia major from a fatal childhood disease into a chronic disorder. Thalassemia is highly prevalent in South Asia, including the Indian subcontinent, and blood transfusion remains the cornerstone of management for these patients. But safe blood transfusions still remain a major problem in India. Difficulties in maintaining adequate blood inventory, a lack of a national blood act, and fragmented blood transfusion services are some of the major contributing factors for the delay in blood supply. In most of the blood centers, alloantibody detection facilities and extended red cell antigen typing are unavailable. Awareness is the key to reducing alloimmunization, which limits the effectiveness of transfusions and the potential availability of blood. Patients with thalassemia are also at high risk of transfusion-transmitted infections unless appropriate blood screening is in place. Hence, many patients remain under-transfused, resulting in decreased health and quality-of-life outcomes. Facilities such as leucoreduction and immunohematological monitoring following a blood transfusion are often lacking in India, especially at the sub-district level. Continuous efforts to raise community awareness, regular training of health-care workers, and proper utilization of available resources are essential to ensuring safe blood transfusions for patients with thalassemia. Access to the new treatments at an affordable cost may reduce the blood transfusion burden for thalassemia patients in India.

Management of autoimmune haemolytic anaemia in low-to-middle income countries: current challenges and the way forward.

Autoimmune haemolytic anaemia (AIHA) is a common term for several disorders that differ from one another in terms of aetiology, pathogenesis, clinical features, and treatment. Therapy is becoming increasingly differentiated and evidence-based, and several new established and investigational therapeutic approaches have appeared during recent years. While this development has resulted in therapeutic improvements, it also carries increased medical and financial requirements for optimal diagnosis, subgrouping, and individualization of therapy, including the use of more advanced laboratory tests and expensive drugs. In this brief Viewpoint review, we first summarize the diagnostic workup of AIHA subgroups and the respective therapies that are currently considered optimal. We then compare these principles with real-world data from India, the world's largest nation by population and a typical low-to-middle income country. We identify major deficiencies and limitations in general and laboratory resources, real-life diagnostic procedures, and therapeutic practices. Incomplete diagnostic workup, overuse of corticosteroids, lack of access to more specific treatments, and poor follow-up of patients are the rule more than exceptions. Although it may not seem realistic to resolve all challenges, we try to outline some ways towards an improved management of patients with AIHA.

Molecular analysis and transfusion management in a rare case of cis-AB blood group: A report from India.

Molecular characterization of a rare cis-AB blood group has not been done in the Indian subcontinent. Herein, we report a case of A2B3 blood group in an Indian patient which was subsequently confirmed to be a case of cis-AB phenotype. Blood grouping was performed by the column agglutination technique (CAT), conventional tube technique (CTT) and subsequently, whole exome sequencing for molecular analysis. The patient was initially typed as AB, RhD positive in forward grouping. However, serum grouping showed agglutination (2+) with the B red cells in CAT. In CTT, an extra reaction was observed with A1 red cells and a strong agglutination was seen with Anti-H lectin. Thus, the blood group was identified serologically as A2B3. During the next-generation sequencing, a total of 10 exonic variants in the ABO gene were filtered, of which 2 (rs8176747 and rs7853989) were found to be non-synonymous and occurring on the same allele. The other allele was found to be ABO*A1.01. The sample analyzed in the study was found to carry two previously reported nucleotide changes of cis-AB (c.803G > C and c.526C > G) on the same allele which had not been reported before. Transfusion requirement was managed with type O red cells and type AB plasma.

Genome-wide atlas of gene expression in the adult mouse brain.

Molecular approaches to understanding the functional circuitry of the nervous system promise new insights into the relationship between genes, brain and behaviour. The cellular diversity of the brain necessitates a cellular resolution approach towards understanding the functional genomics of the nervous system. We describe here an anatomically comprehensive digital atlas containing the expression patterns of approximately 20,000 genes in the adult mouse brain. Data were generated using automated high-throughput procedures for in situ hybridization and data acquisition, and are publicly accessible online. Newly developed image-based informatics tools allow global genome-scale structural analysis and cross-correlation, as well as identification of regionally enriched genes. Unbiased fine-resolution analysis has identified highly specific cellular markers as well as extensive evidence of cellular heterogeneity not evident in classical neuroanatomical atlases. This highly standardized atlas provides an open, primary data resource for a wide variety of further studies concerning brain organization and function.

Positive direct antiglobulin tests in cancer patients on immune checkpoint inhibitors: A case series from India.

Tumour cells activate immune checkpoints such as programmed death receptor-1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) signalling pathways to inhibit T lymphocyte activation and thus escape from immune surveillance. Immune checkpoint inhibitors (ICPis) reactivate T lymphocytes to recognize cancer cells by blocking CTLA-4 or PD-1. Autoimmune haemolytic anaemia is a rare, but often severe, complication of ICPis. Therefore, we performed a retrospective clinical case review, including serologic, haematology, and biochemistry laboratory results, of three patients who developed autoantibodies to erythrocytes following treatment with pembrolizumab, an anti-PD-1 inhibitor. Serologic testing of blood samples from these patients showed their red cells were positive for direct antiglobulin test (DAT + for IgG in two cases and IgG with C3d in one case). Antibody detection test was negative. No patient had clinical and laboratory features of haemolysis. There were no additional immune-related adverse events. IgG antibodies coating red cells were neither IgG1 nor IgG3 in class and elution was found negative in all. In conclusion, immunohaematology laboratories should be aware of the possibility of erythroid autoantibodies and their nature in cancer patients receiving ICPis. The result of a positive DAT should be interpreted carefully in these patients to exclude other possible causes of anaemia.

Rare blood group registry in India-current challenges and future perspectives.

Patients who require blood from rare blood group donors present great challenges even to the most advanced healthcare delivery system. It is most challenging to supply blood for a patient with an antibody to an antigen of high prevalence. The blood donor lacking the corresponding antigen would have an occurrence rate of less than one in 1,000. The International Rare Donor Panel was established in 1965, but since then there has been gross underrepresentation of South Asian countries, including India. There are several challenges to starting a rare blood group donor program in India that include technical, logistical, and administrative limitations. But the main limiting factors are poor availability of trained resources, lack of awareness, absence of antibody screening, inadequate number of laboratories with blood group genotyping facilities, and the decentralized nature of blood transfusion services. Despite that, there were several rare blood groups identified by Indian immunohematologists in the recent past. Recently, a transfusion genomic group has been established in collaboration with the clinical transfusion medicine specialists in India under the GUaRDIAN (Genomics for Understanding Rare Disease in India Alliance Network) initiative to address the domain of rare blood group genomics. Similarly, the National Institute of Immunohematology, Mumbai under the directive of the ICMR (Indian Council of Medical Research) has taken a step to start the RDRI (Rare Donor Registry of India). In this context, we explore the current challenges of setting-up a rare blood group registry in India and future goals from a developing nation's perspective.

Real-world data from India on clinical practices in the management of autoimmune haemolytic anaemia: A survey-based cross-sectional assessment.

BACKGROUND: Autoimmune haemolytic anaemia (AIHA) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. The aim of this national survey is to capture real-world data of clinical practices in AIHA by collecting responses from clinical haematologists across India. METHODOLOGY: In this cross-sectional study, a structured, 26-question online survey was conducted in India by few members of the special interest group in immunohaematology between January and March, 2022. The final survey consisted of questions covering place of work, amount of AIHA cases being evaluated by the haematologist over preceding years, basic demographic, clinical and laboratory features of the patients being treated under them etc. Descriptive statistical analysis was performed during the assessment. RESULTS: The survey response rate was 48.2% (53/110), 69.8% (37/53) have diagnosed and managed more than 10 AIHA cases in the last 3 years with a female preponderance. There was considerable variability in response. While 56.6% (30/53) of respondents do have the access to the facilities to subtype AIHA cases; 32.1% (17/53) of clinicians would prefer administering high dose steroids for 6 weeks or more in non-responding patients, and only 45.3% (24/53) would assess the risks of thrombosis in AIHA. There is unanimous agreement among the participants that health-related quality of life should be taken into consideration in patients and the need for a national registry of patients with AIHA in India. CONCLUSION: The current national survey showed that some aspects of AIHA management were consistent; others were less so, but also significant variations were observed in certain clinical practices, where the evidence base is limited. A joint effort is needed to establish a national patient registry by including both clinical haematologists and transfusion medicine specialists which could potentially standardise AIHA management and future research in India.

Should we adopt an automated de-centralized model of chimeric antigen receptor- T cells manufacturing for low-and middle-income countries? A real world perspective.

Autologous chimeric antigen receptor-T (CAR-T) cell therapy has proven itself as an effective therapeutic modality for cancers, especially hematological malignancies and is emerging as a potential candidate for solid organ cancers as well. However, the accessibility to treatment has been limited due to complexities and costs associated with manufacturing a genetically modified autologous product. The centralized model of CAR-T manufacturing which has emerged as the dominant model in developed nations does not seem well-suited to the needs and realities of the developing economies. In this context, we explore the relative advantages and disadvantages of the two models from a developing nation's perspective.