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The direct antiglobulin test (DAT; sometimes referred to as the “Coombs” test) continues to be one of the most widely used assays in laboratory medicine. First described about 70 years ago, it is elegantly simple in design, yet it is widely complex in its applications and interpretations, and it is prone to false-positive and false-negative results. The overall objective of our review is to provide practicing pathologists with a guide to identify situations when the DAT is useful and to highlight disease-specific shortcomings as well as general pitfalls of the test. To accomplish these goals, this review will discuss the following: (1) the history of the DAT, (2) how the test is performed in the clinical laboratory, (3) clinical situations for its use, (4) its interpretation, and (5) the pitfalls associated with DAT assays, including causes of false positivity.

Abstract Objectives Identifying antibodies to red blood cell antigens is one of transfusion medicine’s critical responsibilities. The International Society of Blood Transfusion recognizes 354 red blood cell antigens. Accurate identification of clinically significant alloantibodies is imperative for preventing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. We compared the performance of the tube (polyethylene glycol–indirect antiglobulin test [PEG-IAT]) and solid-phase red cell adherence assay techniques. Methods We performed a retrospective study on all antibody screens performed between 2007 and 2021 at Stanford Transfusion Services. Initially, 631,535 antibody screens were performed using a solid-phase technique. Subsequent antibody identifications were performed using a combination of tube testing and solid-phase techniques. Results Antibody screening resulted in 28,316 (4.5%) positive samples. Antibody identification performed on both platforms identified 50 discordant samples. The anti-E antibody had the lowest sensitivity (98.99%) in the automated solid-phase technique, while anti-Jkb had the lowest sensitivity (98.78%) with the PEG-IAT method. Conclusions To our knowledge, this is the first robust, 15-year study comparing methodologic sensitivity to detect clinically significant alloantibodies. The incidence of discordant results between PEG-IAT and the solid-phase technique was low. Among discordant samples, anti-Jka was commonly detected using the solid-phase method but not with the PEG-IAT. In contrast, anti-E was commonly detected by PEG-IAT but not by the solid-phase method.

The diagnosis of ABO hemolytic disease of the newborn (ABO HDN) has been the subject of considerable debate and clinical confusion. Its use as an overarching default diagnosis for hyperbilirubinemia in all ABO incompatible neonates regardless of serological findings is problematic and lacks diagnostic precision. Data on hemolysis indexed by carbon monoxide (CO) levels in expired air (ETCOc) and blood (COHbc) support an essential role for a positive direct antiglobulin test (DAT) in making a more precise diagnosis of ABO HDN. A working definition that includes ABO incompatibility, significant neonatal hyperbilirubinemia, and a positive DAT is needed to gain clarity and consistency in the diagnosis of ABO HDN. Absent a positive DAT, the diagnosis of ABO HDN is suspect. Instead, a negative DAT in a severely hyperbilirubinemic ABO incompatible neonate should trigger an exhaustive search for an alternative cause, a search that may require the use of targeted gene panels.

Background Post-artesunate delayed haemolysis (PADH) is common after severe malaria episodes. PADH is related to the “pitting” phenomenon and the synchronous delayed clearance of once-infected erythrocytes, initially spared during treatment. However, direct antiglobulin test (DAT) positivity has been reported in several PADH cases, suggesting a contribution of immune-mediated erythrocyte clearance. The aim of the present study was to compare clinical features of cases presenting a positive or negative DAT. Methods Articles reporting clinical data of patients diagnosed with PADH, for whom DAT had been performed, were collected from PubMed database. Data retrieved from single patients were extracted and univariate analysis was performed in order to identify features potentially related to DAT results and steroids use. Results Twenty-two studies reporting 39 PADH cases were included: median baseline parasitaemia was 20.8% (IQR: 11.2–30) and DAT was positive in 17 cases (45.5%). Compared to DAT-negative individuals, DAT-positive patients were older (49.5 vs 31; p = 0.01), had a higher baseline parasitaemia (27% vs 17%; p = 0.03) and were more commonly treated with systemic steroids (11 vs 3 patients, p = 0.002). Depth and kinetics of delayed anaemia were not associated with DAT positivity. Conclusions In this case series, almost half of the patients affected by PADH had a positive DAT. An obvious difference between the clinical courses of patients presenting with a positive or negative DAT was lacking. This observation suggests that DAT result may not be indicative of a pathogenic role of anti-erythrocytes antibodies in patients affected by PADH, but it may be rather a marker of immune activation.

Warm autoimmune hemolytic anemia is a chronic, relapsing disease characterized by anemia, reticulocytosis, other evidence of hemolysis, and a positive direct antiglobulin test. First-line therapy (after prompt transfusion of ABO- and RhD-matched blood in patients with severe anemia) involves glucocorticoids and rituximab.

Autoimmune Hemolytic anemia (AIHA) a relatively uncommon form of hemolytic anemia, which is characterized by the presence of autoantibodies directed against erythrocyte surface antigens, most frequently of the IgG isotype. A positive Direct Antiglobulin Test (DAT) is a key diagnostic criterion for AIHA. However, when hemolysis involves multiple autoantibodies, the standard DAT (polyspecific, anti-IgG + C3) may fail to detect certain antibodies, potentially delaying appropriate treatment. We reported one patient with severe AIHA mediated by IgG and IgA autoantibodies was successfully treated with Multi-drug combination regimens. A 58-year-old female was admitted to the hospital presenting with a history of fatigue, jaundice and soy sauce-colored urine for one day. Upon admission, a complete blood count revealed a critically low hemoglobin level of 41 g/L and a life-threatening condition. Initially diagnosed with IgG-mediated AIHA via standard DAT, the patient showed suboptimal response to glucocorticoids, intravenous immunoglobulin (IVIG), and transfusion support. Subsequently, through the extended DAT (monospecific, anti-IgA, anti-IgG, anti-IgM, and anti-C3) test results, the patient was diagnosed as severe AIHA mediated by IgG and IgA. Based on extended DAT results, the treatment plan was modified to include combination therapy with dexamethasone, rituximab, cyclosporine, and bortezomib, alongside intensified plasma exchange. The extended DAT testing is recommended for all patients with clinical and laboratory evidence of acute hemolysis. Early detection helps in avoiding further investigations and provide efficient management. Severe AIHA mediated by multiple autoantibodies requires early intensive combination therapy, including immunosuppressive agents, IVIG and plasma exchange.

Abstract Introduction/Objective Identifying antibodies to red blood cell (RBC) antigens is one of transfusion medicine’s most critical and challenging issues. There are 354 RBC antigens recognized by the International Society of Blood Transfusion. Accurate identification of clinically significant alloantibodies is imperative for identifying and preventing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. We compared the performance of the tube (polyethylene glycol–indirect antiglobulin test [PEG-IAT]) and solid-phase techniques for antibody identification. Methods/Case Report We performed a retrospective study on all antibody screens and identifications performed between 2007–2021 at Stanford Hospital. Over this period, 631,535 antibody screens were performed predominantly using an automated solid-phase technique. Subsequent antibody identification studies were performed using manual tube testing (PEG-IAT) and automated solid-phase techniques. Results (if a Case Study enter NA) Antibody screening resulted in 28,316 (4.5%) positive samples with at least one antibody. Antibody identification performed on both platforms identified 50 discordant [DMH1] samples. 8 anti-Jka, 2 anti-Jkb, 1 anti-S, and 1 anti-M were detected by automatic solid-phase technique but were not detected by PEG-IAT. 20 anti-E, 6 anti-K, 2 anti-Fya, 2 anti-c , 2 anti-C, 2 anti-Fyb, 1 anti-cE[DMH2] , 1 anti-e,1 anti-M, and 1 anti-S were detected by PEG-IAT but were negative by automated solid-phase technique. Anti-E had the least sensitivity (98.99%) in the automated solid-phase technique, while anti-Jkb had the least sensitivity (98.78%) in PEG-IAT. Conclusion This is the first robust 15-year study comparing methodologic sensitivity to detect clinically significant alloantibodies. The incidence of discordant results between the PEG-IAT and solid-phase technique was low. Among discordant samples, anti-Jka was commonly detected by solid-phase but not by PEG-IAT. In contrast, anti-E was commonly detected by PEG-IAT but not by the solid phase.

Objective 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. Results 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%). Conclusions RRBCs used in antibody detection and identification vary in diagnostic performance and should therefore be carefully considered before being implemented in routine patient testing.

Abstract Background Saline agglutination tests (SATs) are widely recommended for diagnosis of immune-mediated hemolytic anemia in dogs, but there are frequent false-positive results. Objectives Specificity of SATs will improve at higher saline-to-blood ratios. Animals One hundred fifty dogs treated at a veterinary referral hospital with hematocrits ≤30%. Methods Prospective diagnostic accuracy study. Immune-mediated hemolysis (IMH) was considered present if a gel direct antiglobulin test (DAT) was positive and there was clinical evidence of hemolysis (n = 9), absent if another mechanism for anemia was identified and the DAT was negative or there was no hemolysis (n = 138), and if IMH status was unclear, dogs were excluded (n = 3). Saline agglutination tests were prepared at 1 : 1, 4 : 1, 9 : 1, and 49 : 1 saline-to-blood ratios, and microscopic agglutination was considered a positive result. Results Specificity for IMH increased from 29% (95% confidence interval 20-38) at a 1 : 1 dilution to 97% (93-99) at a 49 : 1 dilution. Sensitivity was 88% (47-100) at 1 : 1 and 4 : 1 dilutions and 67% (30-93%) at 9 : 1 and 49 : 1 dilutions. Diagnostic accuracy increased from 33% (24–42) at 1 : 1 dilution to 95% (90-98) at 49 : 1 dilution. Conclusions and Clinical Importance If performed using a 49 : 1 saline-to-blood ratio, SATs achieve high specificity for IMH. Based on a gold standard of positive DAT and evidence of hemolysis, lower saline-to-blood ratio results should not be used because false-positive results are common.

Background It is advised to pretreat the reagent erythrocytes with Dithiothreitol (DTT) to denature the surface CD38 to prevent anti‐CD38 monoclonal antibodies (MoAb) from interfering with the blood compatibility test. Anti‐CD38 has little impact on the Polybrene test, but it is still unknown how sensitive it is to detect irregular antibodies and how effective it is when compared to the standard DTT‐based method. Methods Twenty‐one patients receiving daratumumab (N = 13) and isatuximab (N = 8) had their serum collected. Standard anti‐sera (anti‐c, D, E, Fyb, Jka, M, Mia) with serial dilution were added to patients' serum. Antibody screening tests were performed simultaneously using the manual polybrene method (MP) and DTT‐pretreated, automatic indirect antiglobulin test (DTT‐IAT) to compare the detection sensitivity. These two methods' operating times and costs were also analyzed. Results Both MP and DTT‐IAT can overcome the interference caused by anti‐CD38 MoAb. However, MP is more sensitive in detecting anti‐M and anti‐Mia and is comparable to DTT‐IAT in detecting other antibodies. In terms of cost and operating time, MP is also far superior to DTT‐IAT. Conclusion MP is a cost‐effective alternative to DTT‐IAT in resolving anti‐CD38 interference and is especially suitable for populations with a high prevalence of anti‐M and anti‐Mia. However, both methods have a well‐known drawback of low detection sensitivity for anti‐K, and K‐units should be provided to patients to prevent hemolytic transfusion reactions. Anti‐CD38 MoAbs interfere with IAT but not MP. In terms of operating cost and time, MP is also far superior to DTT‐IAT.