Prevalence of the Direct Antiglobulin Test and Its Clinical Impact on Multiply Transfused Thalassemia Patients: A Prospective Study Conducted at a Tertiary Care Center in Northern India.

OBJECTIVE: This study was conducted to estimate prevalence of direct antiglobulin test (DAT) positivity and its impact on transfusion support in patients with thalassemia. METHODS: The DAT testing was performed for patients with ß-thalassemia who received transfusion from November 2021 to March 2022. Elution was done for DAT-positive samples. RESULTS: Of 180 patients, 21 (11.6%) were DAT positive. Immunoglobulin G (IgG) was present in 4 (19%) and IgG+C3d was present in 8 (38%). Only complement was present in 9 (42.8%) patients. The IgG-reactive DATs were associated with pan-reactive eluate. Patients who were DAT-positive had significantly higher levels of serum bilirubin, ferritin, and IgG than those who were DAT-negative. CONCLUSION: Autoantibody formation in multiply transfused thalassemia patients is common and merits equal attention as alloimmunization. It is particularly important as DAT-positive red blood cells may undergo clinically significant hemolysis, which may increase the transfusion requirements with associated sequalae such as increased serum ferritin and splenomegaly.

Red cell alloimmunization and associated risk factors in multiply transfused thalassemia patients: A prospective cohort study conducted at a tertiary care center in Northern India.

BACKGROUND: One of the complications of chronic transfusions in thalassemia is the development of red cell alloimmunization. AIMS: The aim of the study was to determine the frequency, specificity of red cell alloantibodies, and factors influencing alloimmunization in multiply transfused thalassemia patients. MATERIALS AND METHODS: The study was carried out prospectively on beta-thalassemia patients over 10 months. Plasma samples were used for antibody screening and identification using the column agglutination technique. Patients' clinical, laboratory, and transfusion details were obtained from hospital information system and patient files. STATISTICAL ANALYSIS: Continuous variables were reported as median and quartile, whereas categorical variables were provided as numbers and proportions. P < 0.05 was considered statistically significant. RESULTS: Out of 255 patients, 17 (6.6%) patients developed alloantibodies. Alloimmunized patients had significantly higher median ages at their first transfusions (1 year vs. 0.5 years; P = 0.042) than nonalloimmunized patients. Alloimmunized patients had significantly higher conjugated bilirubin (P = 0.016) and serum ferritin (P = 0.007). The majority of alloantibodies had specificity toward K antigen, followed by E, C, D, JKa, and JKb antigens. Alloimmunized patients received more units per year than nonalloimmunized patients (median, 30 vs. 24 units; P < 0.001). The average transfusion interval time between two successive transfusions showed a significant difference (P < 0.001). CONCLUSIONS: The prevalence of alloimmunization in thalassemia patients in North India is relatively low. Since most of the alloantibodies belong to Rh and Kell blood group system, extended phenotype-matched blood for Rh and Kell will be helpful in further preventing or decreasing the development of alloantibodies in multiply transfused thalassemia patients.

Effect of donor parameters and cell separators on yield of apheresis platelet and their impact on corrected count increment in aplastic anemia patients.

BACKGROUND: The new cell separators make it simple to collect single donor platelets (SDP), although the platelet yield may vary depending on the cell separator used and donor-related clinical and laboratory variables. AIMS: This study aims to study the factors affecting SDP yield and corrected count increment (CCI). MATERIALS AND METHODS: This retrospective study was carried out at a tertiary care facility in northern India, over 4 years (May 2017-April 2020), data were retrieved and analyzed. STATISTICAL ANALYSIS: Categorical variables were presented as proportions, while continuous variables were presented as mean with standard deviation, P < 0.05 was considered significant. RESULTS: We found a positive correlation between predonation platelet count and yield (r = 0.243, P = 0.000). No such significant correlation was found with Hb concentration (r = 0.025, P = 0.720), age (r = 0.016, P = 0.820), sex (r = -0.038, P = 0.584), and weight (r = -0.025, P = 0.714). Maximum platelet yield and minimum time were seen with Trima. Only 39.3% (33/84) meet the 24 h CCI. The majority of patients did not meet the desired CCI could be due to the patients' clinical condition. On logistic regression, we found a significant association of 24 h CCI with product yield (odds ratio [OR] = 0.168, P = 0.015) and posttransfusion platelet count (OR = 0.454, P < 0.05). CONCLUSION: The only donor-related factor that influences yield is predonation platelet count, whereas 24 h CCI may depend on the clinical status of the patient and yield.

Molecular basis of RhD-negative phenotype in North Indian blood donor population.

Background & objectives: RHD gene typing is highly complex due to homology with RHCE genes. Molecular polymorphism of the RHCE and RHD genes have been characterized among various populations, but no studies have been undertaken among Indians. This study was undertaken to assess the genetic basis of RHD-negative phenotype in Indian blood donor population. Methods: Sample from a total of 200 phenotypically RhD-negative blood donors were analyzed for presence of RHD gene using polymerase chain reaction (PCR). RHD genotyping was done using three primer sets designed for exons 4 and 10 and one set for identification of pseudo (RHDΨ) gene between introns (int) 3 and 4. Amplified PCR products were analyzed by gel-electrophoresis (XY Loper, Uvitech, Cambridge) and confirmed by nucleotide sequencing (ABI 3730 xl 96 capillary system). Results: No PCR product was found in 195/200 (97.5%) of study samples indicating homozygous gene deletion. Of the 5/200 (2.5%) showing RHD gene polymorphisms, 4/200 (2%) were positive for presence of exon 10 only (RHD-CE-D hybrid). RHDΨ gene was not detected in any of the samples tested. One sample showed presence of all three tested regions and was negative for RHDΨ gene. Interpretation & conclusions: RHD gene deletion was found to be the most common cause of an RHD-negative phenotype while RHDΨ gene was, reported to be present in up to 39 per cent of various ethnic populations, but was not detected. RHD-CE-D hybrid gene (found in 2.5% individuals) is important for predicting the requirement of Rh prophylaxis during the antenatal period.

Red blood cell alloimmunization among recipients of blood transfusion in India: A systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: There is a varied prevalence of red cell alloimmunization being reported from different parts of India. This study aimed to estimate the overall prevalence of alloimmunization in India by performing a systematic review of the literature and to establish the most suitable antigen-matching strategy to reduce the red blood cell (RBC) alloimmunization rate among transfusion recipients. MATERIALS AND METHODS: A systematic search of all the original articles published in English on RBC alloimmunization among transfusion recipients from India in MEDLINE, SCOPUS, CINAHL and Google Scholar bibliographic databases was conducted. After screening the articles as per inclusion/exclusion criteria, data extraction was done independently by two sets of investigators. Meta-analysis was performed by the binary random-effects model using the restricted maximum likelihood method. RESULTS: A total of 44 studies on RBC alloimmunization, with a cumulative sample size of 309,986 patients, were grouped into hospital-based and multiply-transfused patients, which yielded a prevalence of 0.5 (95% confidence interval; 0.3-0.8) and 4.8 (95% confidence interval; 3.9-5.7) per 100 patients, respectively. As many as 1992 alloantibodies were identified among the 1846 alloimmunized patients. The most common antibody identified was anti-E (127; 31.99%), followed by anti-c (75; 18.89%) in multiply-transfused patients. CONCLUSION: The rate of alloimmunization was 0.5 per 100 patients tested for antibodies and 4.8 per 100 patients receiving transfusion. Considering E- and c-antigen-matched red cells along with ABO and RhD matching may significantly reduce the overall occurrence of alloimmunization among Indian population who are transfusion-dependent.

Plateletpheresis donor deferral pattern: A retrospective 4-year data analysis at tertiary care center in India.

BACKGROUND: Increasing demand of single donor platelet requires blood banks to expand the donor pool. A reassessment of donor deferral criteria for plateletpheresis is required to ensure that this increasing demand is met without compromising on product quality and donor safety. AIMS: (1) To list the various causes of SDP donor deferral. (2) To discuss various approaches to minimize it. MATERIALS AND METHODS: Data of plateletpheresis donor deferral were collected from records retrospectively over a period of 4 years from January 2017 to December 2020. STATISTICAL ANALYSIS: All statistical tests were performed using IBM SPSS software for Windows version 20. Categorical variables were presented as proportions, while continuous variables were presented as mean with standard deviation, mean calculated P < 0.05 was considered statistically significant. RESULTS: Out of the 7478 donors screened for plateletpheresis procedure, 3232 (43.2%) were deferred among which 3089 (42.5%) were male and 142 (63.1%) were female donors. Majority (96.5%) of deferral were temporary. These included low platelet count (47.4%) followed by poor venous access (22.4%) and low hemoglobin (Hb) (7.2%). Among the donors deferred for low Hb, 24.7% (58 out of 234) had Hb between 12 and 12.4 g%. Similarly, among donor deferred for low platelet count, 12% (184 out of 1532) had platelet count between 140 and 149 × 103/µl. CONCLUSION: There is potential for increasing the number of eligible plateletpheresis donors if the present donor selection criteria were relaxed to a minimum Hb of 12 g/dl and minimum platelet count of 140 × 103/µl.

Roles of Salmonella typhi and Salmonella paratyphi in Gallbladder Cancer Development.

BACKGROUND: Typhoid (Salmonella typhi and paratyphi) carriers and gall bladder cancer (GBC) are endemic in northern India. Results of previous studies about association of typhoid carriers with GBC are inconsistent. We studied antibodies against Salmonella typhi and paratyphi in serum samples of patients with GBC. METHODS: We performed modified Widal test for antibodies against Salmonella typhi (Vi and O) and Salmonella paratyphi (AO and BO) antigens in patients with GBC (n=100), xanthogranulomatous cholecystitis (XGC, n=24), chronic cholecystitis (CC, n=200) and healthy controls (HC, n=200). RESULTS: Serum antibodies against Salmonella were more frequently positive in GBC (22%) and XGC (29%), particularly in males in age ≥50 years (GBC: 47% and XGC: 50%) vs. HC (0) (p <0.01). Vi antibody was more common in GBC (13%, OR:9.8) and XGC (8%, OR:5.9) than HC (2%). O antibody was more common in GBC (8%, OR: 8.6) and XGC (8%, OR: 9.0) than HC (1%). O antibody was also more common in males with GBC (12%) than CC (1%) and HC (1%) (P=0.02 and p <0.001, respectively). AO (6%) and BO (4%) antibodies were detected in GBC, particularly in males, than HC (0), (p <0.01). Salmonella antibodies were more frequent in GBC with GS than those without GS (50% vs. 20%, OR=3.94, P=0.01). CONCLUSIONS: Salmonella carrier state was more common in GBC and XGC, particularly in elderly males than HC. The Vi antibody was more common in GBC and XGC than HC. Salmonella infection was more common in GBC with GS than those without GS.

Molecular characterisation of RhD variants in North Indian blood donor population.

OBJECTIVES: A molecular analysis of serologically RhD variant samples was conducted to find the incidence of various D variants in our blood donor population. BACKGROUND: Determining a blood donor's RhD phenotype and genotype is important as transfusion of units with a weak D or partial D phenotype can result in immunisation of the recipients. METHODS: Samples with discrepant D and weak D phenotypes identified on testing with at least five different monoclonal anti-D antisera were considered serological RhD variant and subjected to molecular testing (Massarray kit, Agena Bioscience, San Diego) for variant RHD gene. RESULTS: A total of 39 samples, including 19 RhD discrepant samples and 20 weak D samples, were identified as serological RhD variant from a total of 4386 samples. Thirteen (13/39) samples carried variants leading to weak D phenotype, and eight samples had variants leading to partial D categories. Seven samples (7) could not be characterised, whereas 11 samples were identified as Rh negative (RHD*01N.01) after molecular testing. Overall incidence of D variants in the study population was 0.48%. RHD*weak D type 1(5, 0.1%) and RHD*DFR1 (5, 1%) were the most common variants identified. CONCLUSIONS: Few samples with weak reaction on serological testing were found to be partial D variant and vice versa. Donor centres should develop a protocol for genotyping of samples with aberrant results on serological testing for assessing the actual RhD status of an individual as results of serological testing may be misleading.

Comparative evaluation of gel column agglutination and erythrocyte magnetized technology for red blood cell alloantibody titration.

Antibody titration is traditionally performed using a conventional test tube (CTT) method, which is subjected to interlaboratory variations because of a lack of standardization and reproducibility. The aim of this study is to compare newer methods such as get column technology (GCT) and erythrocyte magnetized technology (EMT) for antibody titration in terms of accuracy and precision. Patient serum samples that contained immunoglobin G (IgG) red blood cell (RBC) alloantibodies of a single specificity for Rh or K anitgens were identified during routine transfusion service testing and stored. Titration and scoring were performed separately by and stored. Titration and scoring were performed separately by different laboratory personnel on CTT, GCT, and EMT. Testing was performed a total of three times on each sample. Results were analyzed for accuracy and precision. A total of 50 samples were tested. Only 20 percent of samples tested with GCT shoed titers identical to CTT, whereas 48 percent of samples tested with EMT showed titers identical to CTT. Overall, the mean of th titer difference from CTT was higher using GCT (+0.31) compared with that using EMT (+0.13). Precision shown by CTT was 30 percent, EMT was 76 percent, and GCT was 92 percent on repeat testing. GCT showed higher titer values in comparison with CTT but was found to be the most precise. EMT titers were comparable to CTT, and its precision was intermediate. Further studies to validate this method are required.

Knowledge, attitude, and beliefs of young, college student blood donors about Human immunodeficiency virus.

INTRODUCTION: Young people, who tend to be healthy, idealistic, and motivated, are an excellent pool of potential voluntary unpaid blood donors. Recruiting and retaining young blood donors improves the long term safety and sufficiency of a country's blood supply. Knowledge, attitude, and beliefs about Human immunodeficiency virus (HIV) should play an important role in prevention of disease transmission. MATERIALS AND METHODS: This study was a questionnaire based survey, conducted to explore the levels of knowledge, attitude, and beliefs about HIV in young college student blood donors. RESULTS: The results showed that the proportion of participants with comprehensive knowledge of HIV prevention and transmission was lesser than expected. Increase in education level and male gender was found to be significantly associated with high HIV-related knowledge. The responses on the different aspects of HIV-related attitude were also varied and there is still stigma associated with Acquired Immunodeficiency Syndrome (AIDS) even in the educated groups. DISCUSSION: There was a spectrum of myths and misperceptions emphasizing the need of education that recognizes the social context of attitude towards HIV. Results from this study may contribute to the development of appropriate educational and training material for this group of donors which in turn, may assist in achieving the elusive goal of safe blood supply in future.

A case of passenger lymphocyte syndrome following minor ABO incompatible renal transplantation.

Immune hemolysis is one of the adverse effects that can occur following solid organ transplantation. Understanding the clinical settings and the various causes is necessary for prompt diagnosis and appropriate management. One such condition is passenger lymphocyte syndrome (PLS). This case report describes the case of a 27-year-old male renal allograft recipient of the B-positive blood group who received a kidney from an O-positive donor. Postoperatively, the patient showed declining hemoglobin (Hb) level and was transfused with B-group packed RBCs (PRBCs), following which there was steep fall in Hb level. A request for PRBCs was sent to the blood bank and this time cross-match with B-group PRBCs showed incompatibility. The patient's RBCs were found to be strongly DAT (direct anti-globulin test) positive and the eluate showed the presence of anti-B with a titer of 32. Thus, diagnosis of probable PLS was made. The patient was managed with methylprednisolone, plasmapheresis and O-group PRBCs. Gradually his condition improved and was discharged in stable condition.

Cross-match-compatible platelets improve corrected count increments in patients who are refractory to randomly selected platelets.

BACKGROUND: Cross-match-compatible platelets are used for the management of thrombocytopenic patients who are refractory to transfusions of randomly selected platelets. Data supporting the effectiveness of platelets that are compatible according to cross-matching with a modified antigen capture enzyme-linked immunosorbent assay (MAC-ELISA or MACE) are limited. This study aimed to determine the effectiveness of cross-match-compatible platelets in an unselected group of refractory patients. MATERIALS AND METHODS: One hundred ABO compatible single donor platelet transfusions given to 31 refractory patients were studied. Patients were defined to be refractory if their 24-hour corrected count increment (CCI) was <5×10(9)/L following two consecutive platelet transfusions. Platelets were cross-matched by MACE and the CCI was determined to monitor the effectiveness of platelet transfusions. RESULTS: The clinical sensitivity, specificity, positive predictive value and negative predictive value of the MACE-cross-matched platelets for post-transfusion CCI were 88%, 54.6%, 39.3% and 93.2%, respectively. The difference between adequate and inadequate post-transfusion 24-hour CCI for MACE cross-matched-compatible vs incompatible single donor platelet transfusions was statistically significant (p=0.000). The 24-hour CCI (mean±SD) was significantly higher for cross-match-compatible platelets (9,250±026.6) than for incompatible ones (6,757.94±2,656.5) (p<0.0001). Most of the incompatible cross-matches (73.2%) were due to anti-HLA antibodies, alone (55.3% of cases) or together with anti-platelet glycoprotein antibodies (17.9%). DISCUSSION: The clinical sensitivity and negative predictive value of platelet cross-matching by MACE were high in this study and such tests may, therefore, be used to select compatible platelets for refractory patients. A high negative predictive value demonstrates the greater chance of an adequate response with cross-matched-compatible platelets.

Removal of antibodies from red cells: Comparison of three elution methods.

BACKGROUND: Direct antiglobulin test (DAT) is the most common test done in immunohematology lab, which detects immunoglobulin and fragments of complement attached to the red blood cells. These coated red blood cells are difficult to accurately phenotype, which may be required for selection of appropriate unit of red blood cells for transfusion. AIMS: We have studied the efficacy of various elution methods in removing the antibodies coating the red cells and their impact on different blood group antigen activity. MATERIALS AND METHODS: Patient samples sent for serological evaluation of autoimmune hemolysis were included in the study. DAT and Indirect antiglobulin test (IAT) were performed using gel cards (ID system, DiaMed Switzerland). Antibody coated red cells, either by in-vivo or in-vitro sensitization, were used to assess the outcome of three elution methods. RESULTS: Out of 93 DAT positive samples already sensitized in vivo, 28 (30 %) samples became DAT negative post elution using either of three methods, while 36 (38.8%) showed reduction in strength of reaction, whereas in 29 (31.2%) there was no change in strength of reaction. Similarly, out of the 17 samples prepared by in vitro sensitization, 12 samples became completely negative after glycine-HCl/EDTA elution, 9 and 5 samples became negative after heat elution and chloroquine diphosphate elution methods, respectively. CONCLUSION: On comparative analysis glycine-HCl/EDTA elution method was better than the other two methods and can be used for eluting immunoglobulins from intact red cells.

Seroprevalence of Toxoplasma gondii antibodies in North Indian blood donors: implications for transfusion transmissible toxoplasmosis.

INTRODUCTION: Transfusion transmitted Toxoplasma gondii (T. gondii) can result in significant clinical consequences in immunocompromised and multiply transfused patients, pregnant women and fetus etc. Anti-T. gondii seroprevalence, specifically IgM antibodies reflect the risk of transfusion transmission. METHODS: Four hundred and ninety-three blood donors in a tertiary care hospital in North India were screened for IgG and IgM anti-T. gondii antibodies by enzyme linked immunosorbent assay (ELISA). RESULTS: The prevalence of IgG and IgM anti-T. gondii antibodies was 51.8% and 5% respectively. The prevalence was higher in females (M=51.6%, F=89.2%) and in replacement donors (replacement donors=63.2%, voluntary donors=33.5%). CONCLUSION: The donor population constitutes a significant risk of transfusion transmitted toxoplasmosis. Effective strategies are required to prevent transfusion transmitted toxoplasmosis.