Antiglobulin Test Research Articles

Cumulative rabbit anti-human thymocyte globulin dose to recipient weight during the peri-operative period is an independent risk factor for early postoperative urinary tract infection after kidney transplantation

Anti-human thymocyte globulin-Fresenius (ATG-F) is frequently utilized to achieve successful induction for kidney transplantation recipients. This study aimed to examine the association between the ATG-F dose-to-recipient-weight ratio (ADR) and the risk of developing urinary tract infections (UTIs) following kidney transplantation. Data of kidney transplant recipients who underwent ATG-F-induction peri-operatively in a medical center were retrospectively collected, and the incidence of UTIs during the first postoperative year was also recorded. The risk of UTI associated with ADR was analyzed, and receiver operating characteristic curves were drawn to determine the optimal ADR, followed by Cox regression models. In total, 131 recipients were included, with an UTI incidence of 19.08% and a mean interval of 3.08 months. The optimal ADR was 6.34, involving 41 and 90 patients in the low ADR and high ADR groups, respectively. The UTI-free rate in the low ADR group was significantly higher than that in the high ADR group (p = 0.007). Cox regression analysis indicated that a high ADR independently increased the risk of UTI following kidney transplantation (hazard ratio: 5.306, 95% confidence interval: 1.243–22.660, p = 0.024). There was no significant difference in rejection rate between the high ADR and low ADR groups. In conclusion, a high ADR increased the incidence of early postoperative UTI among kidney transplant recipients.

Nocturnal continuous subcutaneous infusion of apomorphine in advanced Parkinson's disease: a series of 37 cases

Multiple factors can cause sleep disturbances in Parkinson's disease. The quality of sleep and therefore of life is usually improved with continuous dopaminergic stimulation therapies, such as continuous subcutaneous infusion of apomorphine. We present an observational retrospective study of patients at our centre with advanced Parkinson's disease, treated with continuous infusion of apomorphine, with treatment extended to nights, between 2011 and 2022. We collected data from 37 patients, and evaluated the indication for nocturnal treatment, efficacy, safety and reasons for withdrawal. Fifty percent of patients began nocturnal treatment for motor complications, 19% for non-motor complications and 31% for both. The most common non-motor symptoms were sleep fragmentation and disturbances, neuropathic pain, psychiatric symptoms and intense nocturia. Twenty of the 37 patients (54%) were continuing treatment at the end of the study follow-up, 16 (43%) discontinued the infusion, and one (3%) was lost to follow-up. The adverse reactions that led to termination of the infusion were severe nodules (two), dopaminergic psychosis (one) and a positive Coombs test with/without anaemia (one). Four patients terminated the nocturnal infusions while continuing the daytime infusions due to suboptimal adaptation to the device. Patients whose symptoms improved without any significant adverse effects continued the treatment. Continuous infusion of apomorphine during the night was an effective and safe treatment in our series.

Genotyping for Weak and Partial D Rh Status to Optimize Usage of Limited Blood Supplies

Abstract Introduction The Rh antigen group, expressed on erythrocytes and encoded by highly polymorphic RHD and RHCE genes on chromosome 1 (1p36-p34), is notable in transfusion medicine due to its high polymorphism, strong immunogenicity and ability to cause hemolytic transfusion reactions in patients and hemolytic disease of the fetus and newborn (HDFN). The D antigen specifically contains multiple epitopes with variations that include decreased expression (weak D) or loss of certain epitopes (partial D) which expose patients to risk of alloimmunization depending on the subtype of the Rh(D) antigen. Hence, serologic weak D testing followed by molecular testing is not performed except for donors testing negative for Rh(D) antigen and newborns typing negative for Rh(D) and born to Rh(D) negative mothers. Given the current shortages of blood supplies, understanding patients’ genotypes in the context of weak or partial D-typing and risk of alloimmunization can be essential in the management of increasingly limited supplies of red blood cell (RBC) units. Objective To further characterize patients that initially type negative for Rh(D) but positive for weak D with molecular testing to investigate risk of alloimmunization, retype the Rh(D) and avoid unnecessary use of Rh(D) negative RBC units during critical shortages of RBC units. Methods We collected the results of ABO/Rh(D) typing for patients with negative Rh(D) testing but positive weak D test over a period of four weeks in December 2023 and had D-variant molecular testing performed by a reference laboratory. Initial serologic ABO/Rh(D) was performed in tube followed by weak D testing by indirect Coombs test in tube. Results The total number of patients who initially tested negative for Rh(D) but positive (weak to +4) for weak-D was three patients. Two patients showed a probable genotype of RHD*01W.1 (RHD* weak type 1, 809T>G (V270G)) homozygous which indicated the presence of weak D+ type 1. Patients with weak D+ type 1 can receive Rh(D) positive red cell units, and they are not at risk of alloimmunization and developing anti-D. The third patient showed a probable genotype RHD*DVI type 2 homozygous (lack of Rh(D) exons 4 through 6) and indicated the presence of partial D antigen. Patients with partial D antigen should receive RH(D) negative red cell units as they are at risk of alloimmunization and developing anti-D. Conclusions Although the vast majority of Transfusion Medicine services do not perform the serologic weak D test with reflex to molecular testing, characterizing patients who test negative with initial serologic Rh(D) testing with serologic and molecular D-tests may aid in retyping the Rh(D) of patients during critical shortages, particularly for patients with ABO blood group O.

Nonimmune Hemolytic Anemia in Myelodysplastic Neoplasms (MDS) with U2AF1 Gene Mutation at S34 Hotspot: A Rare and Challenging Case Presentation

Abstract Introduction/Objective Hemolysis is recognized but not well understood in a subset of patients with myelodysplastic Neoplasms (MDS), largely through non-immune-mediated mechanisms. Lower survival rates are observed in MDS patients experiencing hemolysis. It is important to investigate the underlying mechanism including the molecular features of these cases. We hereby present a rare and challenging case of nonimmune hemolytic anemia in MDS with U2AF1 gene mutation at S34 hotspot. Methods/Case Report The patient is a 75-year-old male with past medical history of hypertension that presented with two weeks of 20 pounds weight loss, dyspnea on exertion and a syncopal event complicated with a parietal subarachnoid hemorrhage. Patient was found to have pancytopenia with severe anemia (hemoglobin 3.6 g/dl), reticulocytosis (reticulocyte 13.29%), decreased haptoglobin, while increased indirect bilirubin and lactate dehydrogenase. Spherocytes were detected in peripheral blood smear. With Coombs test being negative, all above are consistent with nonimmune hemolytic anemia. There is no evidence of paroxysmal nocturnal hemoglobinuria. Bone marrow biopsy revealed trilineage hematopoiesis, many hypo-or mono-lobated dysplastic megakaryocytes and less than 5% blasts. Cytogenetics/FISH unveiled 5q deletion, 20q deletion and trisomy 8. Mutation in the U2AF1 gene at S34 hotpot is detected by next-generation sequencing. The overall findings are consistent with MDS with nonimmune mediated hemolysis. Results (if a Case Study enter NA) NA Conclusion Hemolysis, although uncommon in MDS, has a substantial impact on prognosis, treatment approaches and response. U2AF1 gene mutation is associated with poor outcomes in MDS patients. U2AF1 gene mutation at S34 hotpot is observed in 30% MDS patients with hemolysis in one recent report. Responses to erythropoiesis-stimulating agents and hypomethylating agents vary depending on different mutations, including U2AF1, SF3B1 and EZH2, with discrepancies across studies. Further investigation is warranted to elucidate the relationship between the molecular abnormalities and treatment response as well as prognosis in MDS patients with hemolysis.

Molecular testing in a patient with multiple alloantibodies and warm autoimmune hemolytic anemia: A case study

Abstract Introduction/Objective We present a case of RBC alloimmunization and warm autoimmune hemolytic anemia (WAIHA) in a patient with a recent RBC transfusion. Serological testing followed by molecular testing revealed a rare D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/- predicted phenotype. Methods/Case Report A 75-year-old black male with secondary myelofibrosis presented with a sudden onset of chest pain, dyspnea and weakness during an RBC transfusion. The patient was subsequently admitted for further workup. Labs showed low hemoglobin, low haptoglobin, elevated LDH and a positive direct antiglobulin (DAT) test. The patient was started on intravenous immunoglobulin and steroids for suspected WAIHA. On immunohematology serologic testing, blood typed as B-positive and polyspecific DAT positive with anti-IgG positive and anti-C3 negative. Serum plasma studies showed probable warm autoimmune hemolytic anemia and allo anti-S, -E, and -C by previous history. Eluate studies showed panagglutinin and antibody with D-like specificity. Immucor PreciseTypeTM Human Erythrocyte Antigen (HEA) Molecular BeadChip test predicted a partial e antigen and potential hrB-. Genotyping for RHD variants was performed and revealed RHD*01 (homozygous or hemizygous). Targeted genomic testing did not detect variants associated with common partial or weak RhD antigens. Based on this testing, the patient was not predicted to be at risk of allo-anti-D. An RHCE genotype for C, c, E and e variants including hrB and hrS showed RHCE*ce48C,733G/RHCE*ce733G with predicated phenotype of D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/-. Based on the RHCE testing, the patient was predicted to be at risk for allo-anti-c, -e, -f (ce) and possibly anti-hrB. Results (if a Case Study enter NA) NA Conclusion In this case, serologic testing alone did not reveal a precise diagnosis. Molecular testing aided in the diagnosis of WAIHA and will help guide future transfusions. Pathologists must consider molecular testing when faced with a challenging serologic diagnosis.

Flow cytometry for RBC damage and complement activation

Abstract Introduction Red blood cells (RBCs) express anionic phospholipids such as phosphatidylserine (PS) in their inner cell membrane layer. As RBCs age, physiochemical changes in their cell membranes, particularly exposure of PS on the outer membrane layer, lead to phagocytosis and clearance from circulation. PS in RBCs induces activation of the complement system, increasing binding of C3 to RBCs. Thus, quantifying PS-expressing (PS+) RBCs and complement deposition on RBCs (C3+) is a potential measure of RBC injury and may improve transfusion quality when used for predicting post-transfusion RBC recovery. Our goal was to develop a flow cytometry assay that quantifies PS+ and C3+RBCs and to assess how this assay correlates with the direct antiglobulin test for C3 (C3-DAT). Methods Using the Cytoflex S flow cytometer, we measured PS+ and C3+ RBCs using anti-annexin-PE (PS+RBC) and biotin-labeled anti-C3 with streptravidin-PE-Cy5 (C3+RBC). RBCs from healthy donors (NL-RBCs) with negative direct antiglobulin tests for C3 (C3-DAT) (n = 4) were analyzed. To assess the sensitivity of the assay and correlation with C3-DATs, complement control cells (CCC; Immucor) were used as a positive control for both PS+RBCs and C3+RBCs and diluted to different concentrations by mixing CCCs with NL-RBCs. Aliquots of diluted RBCs were analyzed blinded for C3-DAT. Differences between means for PS+RBC and C3+RBC were compared using two-sample t-test, and one-way ANOVA and Tukey’s HSD test were used to determine differences in C3+RBCs among a C3-DAT range of reactivities (0+ to 3+) in diluted CCC samples. P values less than 0.05 were statistically significant. Results In healthy, DAT-negative individuals, the mean positivity for PS+RBCs was 0.28% and for C3+RBCs was 0.34%, indicating that healthy individuals have very low percentages of RBCs with PS and C3 on their cell membrane surface. In CCC diluted samples, the mean C3+RBC percentage was 1.4% in negative C3-DAT samples, 5.9% in weak+ C3-DAT samples, 28.1% in 1+ C3-DAT samples, 67.2% in 2+ C3-DAT samples, and 99.0% in 3+ C3-DAT samples. One-way ANOVA revealed a statistically significant difference in C3+RBCs amongst all C3-DAT reactivities in diluted CCC samples (P<0.001), and Tukey’s HSD test found a mean difference in C3+RBCs percentage of 26.7% between 0+ and 1+ samples (P<0.001), 39.2% between 1+ and 2+ samples (P<0.001), and 31.8% between 2+ and 3+ samples (P<0.01). Undiluted CCC samples had a mean PS+RBC percentage of 7.0% and was statistically different from the PS+RBC percentage in NL-RBCs (P<0.01). Conclusion We describe a feasible assay to measure both PS+RBCs and C3+RBCs using flow cytometry in which CCC can be used as a positive control for PS+RBCs. This assay could serve to evaluate the quality of transfused RBC units and provide insights into the role of phosphatidylserine expression and complement deposition for RBC clearance.

Blended Phenotypes From a SERPINA 11 Pathogenic Variant Over Underlying Immune Fetal Hydrops: A Rare Case Report and Literature Review

Abstract Fetal hydrops can stem from immune or nonimmune causes. Immune causes often involve red cell alloimmunization, whereas nonimmune causes encompass structural malformations, aneuploidy, infections, lymphatic system disorders, genetic syndromes, and more. In a rare and complex case, we encountered a fetal hydrops presentation characterized by blended phenotypes, indicating both a genetic and an underlying immune etiology. The mother, Rhesus negative, presented with a history of adverse obstetric events. At 21 weeks, the current fetus was diagnosed with hydrops. Maternal blood tests unveiled Rhesus alloimmunization, featuring a positive indirect Coombs test at a 1:512 dilution and the presence of anti-D, anti-C, and anti-E antibodies. Fetal blood sampling revealed an O-positive blood group with a hemoglobin level of 10 gm/dL. Despite administering intrauterine transfusion to the fetus, there was no improvement; instead, the fetal hydrops worsened, accompanied by the emergence of nuchal and axillary masses. Exome sequencing of fetal DNA revealed the fetus was homozygous for a pathogenic variant in the SERPINA11 gene and compound heterozygous for a pathogenic variant in the PIEZO1 gene. Furthermore, the combination of pathogenic variants in SERPINA11 and PIEZO1 genes has not been described in cases of fetal hydrops before. This case posed significant challenges in management due to the concurrent presence of both immune and nonimmune hydrops. We describe some of the diagnostic challenges faced in clinical management of this case.

Evaluation of the analytical performance of four different manufacturer's reagent red blood cells in antibody detection and identification.

The detection/identification of clinically significant antibodies to red cell antigens form the foundation for safe transfusion practices. This study aimed to evaluate the diagnostic performance of commercially available 0.8% reagent red blood cells (RRBCs) in Australia. 166 patient-derived plasma samples with a positive indirect antiglobulin test (IAT) were tested using column agglutination technology (CAT) with Immulab, Bio-Rad, Grifols and QuidelOrtho screening and identification RRBCs with the respective manufacturer's proprietary CAT system. False-negative antibody screening and identification results were obtained with Bio-Rad (3/61), Grifols (14/68) and Quidel-Ortho (3/59) RRBCs when tested with the respective manufacturer's proprietary CAT system. Zero false-negative results were observed with Immulab RRBCs when tested with samples across all platforms. The sensitivity of the RRBCs used in this study were calculated to be 95.83% (95%CI 88.30-99.13%) for Bio-Rad RRBCs, 82.50% (95%CI 72.38-90.09%) for Grifols RRBCs and 95.65% (95%CI 87.82-99.09%) for QuidelOrtho RRBCs. The sensitivity of Immulab RRBCs were stratified based on performance in the 3 CAT platforms: Bio-Rad CAT (100%, 95%CI 95.01-100%), Grifols CAT (100%, 95%CI 95.49-100%) and QuidelOrtho CAT (100%, 95%CI 94.79-100%). RRBCs used in antibody detection and identification vary in diagnostic performance and should therefore be carefully considered before being implemented in routine patient testing.

Perioperative selection of blood and components for substitution: Immunohaematological assessment of immunization risk and methods for selection of optimally compatible blood

The aim of this study is to assess the risk for immune and hematologic adverse events in perioperative transfusion of blood components. Our second goal is to propose methods for the selection of an optimally compatible donor in order to limit the negative impact of complications following blood component transfusion on the hospital outcome of patients admitted to clinics of general, gastrointestinal, thoracic, and vascular surgery. The largest group of patients included in this study were patients with gastrointestinal disorders (614 patients; 31%) and patients with diseases of the organs of the blood and lymphatic system (589 patients; 30%). This was followed by patients with diseases of the skin and subcutaneous tissue (368 patients; 18%) and patients with benign and malignant neoplasms (365 patients; 18%). The remaining 65 patients (3%) were diagnosed with diseases of the endocrine system, trauma patients, and patients with genitourinary disorders. The methods used for optimal selection of compatible blood components in this study are enzyme tests, Coombs tests, and tests in agglutinating medium. The study shows that clinically significant antibodies, which could provoke a post-transfusion hemolytic reaction, were detected in 4.3% of the screened patients. In the majority of patients, the specificity of antierythrocyte antibodies cannot be established. For those patients where the specificity of anti-erythrocyte antibodies could be established, the type of antibody and the antigen system to which it belonged were as follows (in descending order): 1. Anti-erythrocyte antibodies belonging to the Rh system: 37% (n = 17) of all screened patients (1.6% of all patients), including anti-E type (5 patients), anti-D (8 patients), anti-C-1, anti-C (2 patients), and anti-Cw (1 patient). 2. In 3 (6% or anti-erythrocyte antibodies belonging to other erythrocyte antigen systems—Kidd-1 patients, Lewis-2 patients—6% of the screened patients (0.3% of all patients). 3. In 57% (n = 26) of those screened (2.4% of all patients), alloantibodies without any known specificity were identified. The largest number of identified antibodies were directed against the D antigen belonging to the Rh system. Most of the anti-D alloantibodies found in Rhesus D (-) negative patients with no history of prior transfusion of D (+) positive red blood cell (RBC) concentrate possibly have resulted from prior Rh isoimmunization during pregnancy, provided that anti-D antibodies persist for a long time after birth—in some of the studied patients, 15–20 or more years. A greater number of alloimmunizations were found in women—28 (61%), compared to men—18 (39%), due to previous sensitization with different blood group antigens during pregnancy. This study shows that timely diagnosis is essential for the selection of appropriate RBC concentrate, for the avoidance of adverse reactions, and for the improvement of hematological parameters after transfusion of blood components. Graphical abstract:

Alloimmunization and consequential delayed hemolytic transfusion reactions in sickle cell disease patients: A case series

Abstract: Alloimmunization is a significant complication of blood transfusion, especially in sickle cell patients, and may lead to a delayed hemolytic transfusion reaction (DHTR). DHTR is defined as evident hemolysis and a positive direct antiglobulin test (DAT) 24 h to 28 days posttransfusion with either a positive eluate or a newly identified alloantibody in the plasma. Hyperhemolysis syndrome (HS) is a fatal form of DHTR in which the posttransfusion hemoglobin (Hb) level is less than the pretransfusion Hb level. In this case series, we have reported three cases of alloimmunized sickle cell disease patients with clinically significant DHTR. The second case is typical of HS. All three cases were DAT positive, alloimmunized with multiple alloantibodies, and had substantial hemolysis posttransfusion. In this series, we have provided an algorithmic approach to resolve such complex immunohematological problems and have highlighted some of the limitations of serology methods. High-dose corticosteroid and intravenous immunoglobulin prove to be an effective treatment for DHTR.

Can computer crossmatch really replace antihuman globulin crossmatch? An intriguing case of missing anti-C on routine antibody screen from a multispecialty center in India

Can computer crossmatch really replace antihuman globulin crossmatch? An intriguing case of missing anti-C on routine antibody screen from a multispecialty center in India

Cold autoantibody interference in pretransfusion testing and its resolution: a case report

We report a case of cold autoantibody detected in a 69-year-old gentleman diagnosed with chronic lymphocytic leukemia and cold autoimmune hemolytic anaemia. Pretransfusion testing showed panagglutination in ABO grouping, antibody screening and identification, with positive direct antiglobulin test (DAT) and positive control cells. A cold autoantibody was suspected, however, the test using a prewarm method and washed RBC with warm saline failed to resolve it. Further tests using DTT-treated cells resolved the grouping as AB RhD positive and DAT as positive with anti-C3d only (control negative). Cold autoadsorption was done and repeated antibody screening using cold-autoadsorped plasma was negative, excluding the presence of alloantibody. The cold-agglutinin titer was >1028 having a wide thermal range. Crossmatching using cold autoadsorped plasma with the AB-positive donor blood was compatible and the patient was transfused safely. For such cold autoantibody cases, an extensive immunohematological workup is required to exclude underlying alloantibody for transfusion safety and efficacy. Bangladesh Journal of Medical Science Vol. 23 No. 04 October’24 Page : 1238-1242

Utility of Jaundice Surveillance and Bilirubin Screening in Identifying Neonates Who Qualify for Phototherapy ≤ 24 Hours After Birth

To assess the utility of jaundice surveillance and routine 24 hour bilirubin screening in identifying neonates who qualify for phototherapy (PT) at ≤ 24 hours after birth. In this retrospective, single-center observational study, records of neonates ≥ 350/7 weeks gestation born to O+, antibody negative mothers (n=6098) were screened to identify who received PT at ≤ 24 hours after birth. The hour specific TSB at which neonates qualified for PT, blood type, direct antiglobulin test (DAT), and whether treatment was triggered by jaundice detection at <24 hours or the 24-hour bilirubin screen were determined. 59 neonates (1.0%) qualified for PT ≤ 24 hours after birth; 10 (17%) were identified by jaundice detection at < 24 hours, whereas 49 (83%) were identified on 24-hour bilirubin screening. Forty-eight of the 59 (81%) were ABO incompatible and DAT+; 11 were DAT negative, one of whom had glucose-6-phosphate dehydrogenase deficiency. Among the ≤ 24 hour PT group, 17 had a PT qualifying TSB within 3 mg/dL of exchange transfusion (ET); 14 of whom were only identified first on 24-hour bilirubin screening. Six exceeded ET thresholds, 4 of whom were identified on 24-hour bilirubin screening. Neonates who qualified for PT at ≤ 24 hours were identified mostly by 24-hour bilirubin screening, a fraction of whom had a TSB that approached or exceeded ET thresholds. Our findings support routine birth hospitalization bilirubin screening and suggest screening no later than 24 hours after birth may be beneficial.

DIAGNÓSTICO E MONITORAMENTO DA ERITROBLASTOSE FETAL: UMA REVISÃO DA LITERATURA

The text addresses fetal erythroblastosis, a serious condition resulting from blood incompatibility between mother and fetus, generally due to the Rh factor. Initially, the discovery of the ABO and Rh blood systems is discussed, highlighting the importance of the latter for understanding the disease. Objective: to understand the pathophysiological mechanisms of fetal erythroblastosis, focusing on Rh incompatibility, thereby analyzing the impacts of fetal erythroblastosis on health of the fetus and newborn.Methods: The work is a bibliographical review where topics such as pathophysiology, diagnosis and prevention of the disease studied were researched.Conclusion: Diagnosis is essential, being done through prenatal exams, such as the Coombs test, which detects the presence of maternal antibodies against fetal antigens. Treatment includes administration of anti-Rh immunoglobulin to prevent the mother’s sensitization to the baby’s Rh factor. Prevention also involves the administration of this immunoglobulin during pregnancy and after birth, if necessary, to avoid future complications and ensure the baby’s safety.

Walter Brendel and the dawn of transplantation research in Germany.

Walter Brendel was a physiologist who headed the Institut of Experimental Surgery at the University of Munich (LMU) from 1961 until 1989. His legendary career began with the development of an anti-human lymphocyte globulin (ALG) at his Institute during the late 1960s. The initial successful treatment of a small number of patients culminated in the co-treatment of the first successfully heart-transplanted patient in Capetown, South Africa (successful reversal with ALG of an acute allograft rejection). Walter Brendel was a pioneering personality whose work has laid a wide platform for the promotion of interdisciplinarily conducted innovative research programs in various domains of translational science and medicine. Among the many innovative achievements, the most notable are: discovery of involvement of the alternative pathway of complement activation in hyperacute xenograft rejection; induction of immunological tolerance to horse IgG as a means to prevent anaphylactic reactions during ALG therapy; development and clinical implementation of the extracorporeal shock wave lithotripsy for extracorporeal destruction of renal and ureteral calculi. The legacy of Brendel continues with the foundation of the Walter-Brendel Kolleg für Transplantationsmedizin (i.e., the German Transplant School for Transplantation Medicine), which has been held annually since 1994.

Impact of Type and Screen Method on Turnaround Time and Man-Hour Utilization Compared to Conventional Coomb's Crossmatch: A Cross-Sectional Analytical Study.

Background Blood component transfusion is vital for various medical conditions, requiring thorough pretransfusion testing to ensure safety and compatibility. The Type and Screen (T and S) method allows for efficient detection of clinically significant antibodies while reducing unnecessary crossmatching. This study evaluates T and S's impact on turnaround time and man-hour utilization compared to conventional crossmatching. Methodology The study included 835 elective crossmatch requests. Blood grouping, antibody screening, and antihuman globulin (AHG) crossmatching were performed using column agglutination technology, while immediate saline crossmatching was done by tube technique. Turnaround time (TAT), man-hour utilization, and saved rates were calculated for both the T and S protocol and routine AHG crossmatch. Results In this study, 835 elective blood samples underwent antibody screening and immediate saline phase crossmatching, with validation by AHG phase crossmatch. The T and S protocol significantly reduced TAT for both first (51.24 vs. 71.56 minutes) and subsequent transfusions (17.47 vs. 39.67 minutes) compared to AHG crossmatching, with reductions being statistically significant (p < 0.0001). T and S also saved 279.28 man-hours (11.6 man-days), equating to 0.33 man-hours saved per sample. Conclusion Our study shows that the T and S protocol significantly enhances blood bank efficiency by reducing TAT and man-hour utilization compared to conventional AHG crossmatching. This improvement not only optimizes manpower but also makes the process more cost-effective.

Hattrick of H-deficient phenotypes at a tertiary care health center in India

Abstract: H-deficient phenotypes, commonly known as Bombay blood type, have more inclination to be found in the Indian subcontinent, and conventional gold standard tube techniques are still the recommended method to identify them, provided a strict adherence to testing protocol. There is more to these phenotypes than just the bombay blood type. This brief communication highlights the identification of three H-deficient phenotypes from a single center that adhered to standard operating procedures and basic testing methodologies. It emphasizes the requirement of indirect Coombs test with pooled “O” cells with blood grouping of every sample irrespective of the testing platform and also the H antigen typing on encountering any suspicion. These H-deficient individuals may not be as rare as we assume them to be provided there are reframed testing policies. Although international collaborations and databases like the International Society of Blood Transfusion database have made it easier to record and share knowledge on blood types, a National network and registries of these rare phenotypes are essential to make them available to the general population who actually benefit from them.

One Haemolytic Anaemia May Hide Another: Paroxysmal Nocturnal Haemoglobinuria Masquerading As Plasmodium Falciparum Infection.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, genetic and acquired haematologic disease that causes complement-mediated intravascular haemolytic anaemia, thrombosis and bone marrow failure. A 27-year-old migrant patient attended the emergency department in a context of fever and chills over the previous few days as well as chronic fatigue, dyspnoea and chest pain. His medical history included chronic anaemia and erectile dysfunction. Initial biology showed a haemoglobin of 6.3 g/dl, platelets of 25,000/μl, total leucocytes of 3,500/μl with 1,500 neutrophils. B12 vitamin, folic acid, ferritin and thyroid stimulating hormone were normal. Lactate dehydrogenase levels were high and haptoglobin was non-measurable. C-reactive protein was 46.1 mg/l. A thick blood smear revealed Plasmodium falciparum infection with 0.1% parasitaemia. The patient was treated with an oral combination of artemether and lumefantrine. Three weeks later, the patient consulted the infectious disease department given the lack of clinical improvement. The cytopenias worsened, and lactate dehydrogenase (LDH) and reticulocytes increased. Tests for schistocytes, a thick blood smear for malaria and a direct Coombs test were negative; a myelogram was reassuring. An abdominal, pelvic and thoracic CT scan showed a mild hepatomegaly with no focal lesion and no splenomegaly or adenomegaly. A 12-colour flow cytometry unveiled a PNH clone on 90.9545% of neutrophils and 80.7371% of monocytes. PNH patients can be vulnerable to parasites infection (such as P. falciparum) as it may trigger breakthrough haemolysis through uncontrolled resurgence of activity of the complement system. In our patient, P. falciparum infection was a confounding factor, as it commonly causes haemolytic anaemia and thrombocytopenia, and patients living in malaria-endemic regions can carry low parasitaemia while being slightly symptomatic or asymptomatic. Plasmodium falciparum infection can cause breakthrough haemolysis in patients with paroxysmal nocturnal haemoglobinuria.Low P. falciparum parasitemia in patients living in malaria-endemic regions is not always significant as these patients often carry acquired immunity.Patients from malaria-endemic regions presenting with severe sickness and low P. falciparum parasitemia must be assessed for other diseases, as it cannot explain heavy illness.Patients presenting with haemolytic anaemia, no schistocytes, a negative direct Coombs test and other unexplained cytopenia such as thrombocytopenia/neutropenia and other unexplained clinical manifestations such as dyspnoea, chest pain or erectile dysfunction should be assessed for paroxysmal nocturnal haemoglobinuria.

Role of dithiothreitol in the resolution of pretransfusion immunohematology testing in patients receiving the anti-CD38 monoclonal antibody daratumumab for the treatment of multiple myeloma

Abstract: INTRODUCTION: Daratumumab (Darzalex; Janssen-Cilag Pty Ltd), an anti-CD38 monoclonal antibody (MoAb), has demonstrated high efficacy in treating relapsed and refractory multiple myeloma (MM). It stands as the pioneering anti-CD38 MoAb approved by the US FDA (2015) and TGA Australia (2017). While targeting CD38 on myeloma cells, daratumumab poses a challenge in pretransfusion testing by inducing panreactivity in indirect antiglobulin tests (IATs) due to CD38 expression on red cells. This study investigates the role of dithiothreitol (DTT) in resolving pretransfusion immunohematology testing in patients treated with daratumumab for MM. MATERIALS AND METHODS: Pretransfusion samples from three hematological patients diagnosed with MM and receiving anti-CD38 drugs necessitating red cell transfusion were subjected to immunohematology serological testing. Clinicians provided patient information, including gender, age, transfusion history, and time between anti-CD38 MoAb infusion and sample collection. Tests included ABO/RhD typing, IAT, both polyspecific and monospecific direct antiglobulin test (DAT), antibody (Ab) screening, Ab identification, eluate preparation, and red cell crossmatch. Control tests were conducted by incubating patient samples at 37°C for 1 h. RESULTS: ABO/RhD typing remained unaffected, yet all samples exhibited panreactivity in IATs. Two samples showed positive DAT results but negative eluates. Treatment of reagent red blood cells (RBCs) with 0.2 M DTT effectively mitigated interference by anti-CD38 monoclonal antibodies, resulting in compatible red cell crossmatches. Notably, incubation of patient samples at 37°C for 1 h yielded identical results. CONCLUSION: Pretreatment of erythrocytes with DTT deactivated the CD38 antigen, eliminating reactivity with patient serum treated with daratumumab. DTT treatment of reagent RBCs emerges as an efficient and accessible strategy to counteract interference from anti-CD38 drugs in pretransfusion tests.

Red cell serological characterization of autoimmune hemolytic anemia in systemic lupus erythematosus patients

Abstract: INTRODUCTION: Autoimmune hemolytic anemia (AIHA) is a clinical condition with increased red cell destruction or decreased red cell survival due to autoantibodies against self-antigens on red blood cells. AIHA may be primary or secondary to other underlying illness. Systemic lupus erythematosus (SLE) is the most common secondary cause of AIHA. Although the incidence of AIHA in SLE patients is low (5%–10%), sometimes, it causes severe anemia which requires blood transfusion. Characterization of these autoantibodies in AIHA is very important, because the clinical condition and treatment of the patient depend on the optimal temperature reactivity, class, and subclass of immunoglobulin responsible. AIM AND OBJECTIVES: The aim and objective of this study are to perform detailed characterization of red cell autoantibodies for accurate diagnosis of AIHA in SLE patients, to find out specific class (immunoglobulin G [IgG]/IgM/IgA) of immunoglobulin and/or complement (C3c/C3d) responsible for AIHA, and their association with severity of hemolysis. MATERIALS AND METHODS: This was a prospective analytical study, done at a tertiary care hospital in Hyderabad, conducted between February 2021 and May 2022. SLE patients with AIHA were included in the study. Monospecific direct antiglobulin test (DAT) was performed to identify the specificity of autoantibody or complement. Based on hematological laboratory values, the study population was divided into three groups, i.e., mild, moderate, and severe hemolysis. DAT strength, hemolytic parameters, and severity of hemolysis were compared between different types of AIHA in SLE patients. Correlation statistics were used to know the association of severe hemolysis with DAT strength and different combinations of autoantibodies. RESULTS: Among 150 patients enrolled, 16 (10.6%) patients had severe hemolysis, 51 (34%) patients had moderate hemolysis, and 83 (55.3%) patients had mild hemolysis. Only IgG was positive in 109 (72.6%) patients, both IgG and C3d were positive in 37 (24.6%) patients, C3d was positive in 4 patients, and IgM was positive in 19 patients. High DAT reaction strength (3+–4+) and presence of C3d or IgM along with IgG were moderately correlated with severe hemolysis. CONCLUSION: Warm AIHA is the most common type of AIHA in SLE patients mediated by IgG. Mild and moderate hemolysis was seen in most cases. Severe hemolysis was moderately correlated with the presence of C3d or IgM. Further studies are warranted to find the predisposing factors leading to severe hemolysis.