A study on diagnostic performance of different immunohematological diagnostic tests in assessing the prevalence of ABO Hemolytic Disease of Newborn in the antenatal O group mothers and their neonatal outcome in a tertiary care hospital in Northern India.

BACKGROUND: Hemolytic disease of the newborn (HDN) results in the decreased lifespan of the red cells. HDN related to ABO incompatibility is mostly unnoticed because routine screening is not being done. This study was done to assess the prevalence of ABO-HDN and to compare different immunohematological tests. Methods-In this study 213 O group mothers and the 122 ABO-incompatible newborns born to them were included. Quantifying the maternal IgG anti-A/anti-B antibody titer was done by Conventional Tube Technique (CTT) using Dithiothreitol (DTT) pretreated maternal serum. Hemolysin test was performed on the mothers having titer > 256. These cases were followed up and, after delivery, were monitored for ABO HDN, along with direct antiglobulin testing and elution studies. The prevalence of ABO-HDN was calculated, and the different diagnostic parameters of the tests were calculated. Results- The prevalence of ABO-HDN in our population was estimated to be 1.7%, 6.1% & 10.6% in our population, O group mothers, and O group mothers with ABOincompatible newborns, respectively. Maternal titer≥ 512 strongly correlated with ABOHDN. DAT positivity is a good predictor of ABO-HDN, especially using sensitive techniques. Maternal IgG titers have the highest sensitivity & Negative Predictive Value, while DAT has the highest specificity & Positive Predictive Value. Conclusion - Maternal ABO antibody titration may be advocated in the centers to identify high-risk groups. It can advocate institutional delivery and dedicated follow-up of newborns with ABO-HDN. Blood grouping & DAT may be performed in all newborns born to O blood group to identify high-risk cases.

The enhanced direct antiglobulin test in current practice has a limited impact on management of adult patients.

INTRODUCTION: The direct antiglobulin test (DAT) identifies immunoglobulin IgG and/or complement onthe red blood cell surface, allowing discrimination between immune and non-immunehemolysis. When the DAT is negative but there is clinical suspicion for immunehemolysis, an enhanced DAT can be sent to an immunohematology referencelaboratory (IRL). METHODOLOGY: This retrospective study assessed the volume of enhanced DATs at a large tertiarycare center and evaluated their impact on patient care. Enhanced DATs were sent on21 adult patients (January 2019 - January 2021) at the University of Pittsburgh MedicalCenter and Allegheny Health Network. Laboratory and clinical data were collected andanalyzed. RESULTS: Four out of 21 patients had positive tests (DAT and other serologic tests) at the localIRL. Enhanced DAT testing yielded positive results in an additional 5 patients butnegative or invalid results for 2 patients. High-dose steroid therapy was started in 12patients prior to receipt of enhanced DAT results. Enhanced DAT testing was sent amedian of 5 days after initiation of steroid therapy. For the patients trialed on steroids,the enhanced DAT results impacted medical decision-making in only 3 patients, and inonly one of those patients was the enhanced DAT positive despite a negative DAT at alocal IRL. In the non-steroid treated patients, enhanced DAT results did not contributeto clinical decision-making. CONCLUSION: Enhanced DATs generally did not impact medical decision-making in adults withhemolytic anemia.

Autoantibodies against red blood cell antigens are common in a malaria endemic area.

Plasmodium falciparum malaria can cause severe anemia. Even after treatment, hematocrit can decrease. The role of autoantibodies against erythrocytes is not clearly elucidated and how common they are, or what they are directed against, is still largely unknown. We have investigated antibodies against erythrocytes in healthy adult men living in a highly malaria endemic area in Uganda. We found antibodies in more than half of the individuals, which is significantly more than in a non-endemic area (Sweden). Some of the Ugandan samples had a broad reactivity where it was not possible to determine the exact target of the autoantibodies, but we also found specific antibodies directed against erythrocyte surface antigens known to be of importance for merozoite invasion such as glycophorin A (anti-Ena, anti-M) and glycophorin B (anti-U, anti-S). In addition, several autoantibodies had partial specificities against glycophorin C and the blood group systems Rh, Diego (located on Band 3), Duffy (located on ACKR1), and Cromer (located on CD55), all of which have been described to be important for malaria and therefore of interest for understanding how autoantibodies could potentially stop parasites from entering the erythrocyte. In conclusion, specific autoantibodies against erythrocytes are common in a malaria endemic area.

Red Blood Cell Immunization and Contributing Factors in 685 Thalassemia Patients.

Background: An analysis of red blood cell alloimmunization in patients with thalassemia can help to devise specific strategies to decrease the alloimmunization rate. This study explored the frequency and specificity of alloantibodies and autoantibodies against red blood cell (RBC) antigens in patients with thalassemia referring to the Iranian Blood Transfusion Organization (IBTO) Immunohematology Reference Laboratory (IRL) in Tehran. Materials and Methods: This study first examined the laboratory records of 23,113 patients suffering from different diseases referring to IBTO's IRL for pretransfusion testing in the 2008-2015 period. ABO and Rh(D) typing and antibody screening tests were performed for all 23,113 patient records and 685 (2.97%) beta-thalassemia patients with positive pre-transfusion test results (antibody screening and/or DAT) were selected for further investigation. Results: The antibody screening test was positive in 640 out of 685 thalassemic patients (93.4%). DAT was performed for 529 patients, 226 (33%) of which showed positive results. Meanwhile, 161 out of 685 beta-thalassemia patients (23.5%) had positive auto control test results, reflecting the possible presence of allo- and/or autoantibodies. The most common antigen-specific alloantibodies were directed against K and E RBC antigens with a frequency of 25% (Anti-K) and 11.91% (Anti-E), respectively. The development of two antibodies (double antibodies) in one patient was observed in 80 individuals (11.46%). Conclusion: Age, gender, history of pregnancy, and splenectomy were not contributing factors to the antibody presence in the patient population under study. Extended red blood cell phenotyping should be considered as an essential procedure for expected multi-transfused thalassemia patients before blood transfusion. Considering the high frequency of anti-K and anti-E observed in this study, it is recommended that thalassemia patients in Iran are tested through phenotyping of RBC units for K and E antigens before transfusion.

Red Cell Antigens and Antibodies.

Autoimmune hemolytic anemia (AIHA) is caused by the production of "warm-" or "cold-" reactive autoantibodies directed against RBC antigens that may be of undefined specificity, reacting with all RBCs tested or may have an apparent specificity. Autoantibodies may be of IgG, IgM, or rarely IgA isotypes and their production can be triggered by disease, viral infection, or drugs; from breakdown in immune system tolerance to self-antigens; or from exposure to foreign antigens that induce antibodies that cross-react with self-RBC antigens. Increasingly, AIHA is being reported in patients following allogeneic hematopoietic stem cell transplantation and treatment with anti-cancer checkpoint inhibitors. Autoantibodies, whatever their etiology, interfere with pretransfusion testing of patients requiring RBCs transfusion making compatibility testing complex and labor-intensive. The availability of extended antigen typing by DNA-based assay has made transfusion of RBCs that are selected based on the patient's extended phenotype (e.g., D, C, E, e, K, Jka, Jkb, Fya, Fyb, S and s) a feasible option and this can provide a significant measure of safety as they avoid the patient being immunized to antigens absent from their RBCs.

Influencing factors of RBC products returned by hospitals due to positive DAT, from 2018 to 2020 in Shijiazhuang / 中国输血杂志

【Objective】 To analyze the RBC products returned by hospitals due to positive direct antiglobulin test (DAT), and explore measures to reduce the discarding rate of blood products and ensure the safety of clinical blood use. 【Methods】 The data of RBC products, which were returned by hospitals due to positive-DAT, in Hebei Blood Center from 2018 to 2020 were retrospectively analyzed. The donation time, hospital, gender of blood donors, donation times and DAT typing results were searched through blood donation code, input into the statistical software SPSS17.0, and analyzed by linear trend χ2 and Pearsonχ2. 【Results】 1)The discarding rate of RBC products due to positive DAT in 2018, 2019 and 2020 accounted for 0.15‰, 0.32‰ and 0.26‰, respectively, of the overall RBC collection. The total concordance rate was 89.94% by our retest. 2)The concordance rate of returned blood from secondary hospitals and tertiary hospitals was 78.26% and 91.78%, respectively (P<0.05), with the latter higher than the former. 3)No statistical significance was noticed in the DAT-positive blood by months(P>0.05). 4)The DAT-positive rate of female donors was higher than that of male donors, and that of first-time blood donors was higher than that of repeated and regular blood donors with statistical differences (P<0.05). 5)DAT-positive typing results was mainly due to IgG incomplete antibody. 【Conclusion】 In order to reduce the discarding rate of RBC products, it is suggested to strengthen the consultation before blood collection, encourage healthy males to donate blood and increase the proportion of regular blood donors. Meanwhile, the quality management of Transfusion Department in secondary hospitals should be further improved to ensure the safety of clinical blood transfusion.

Case Report: Drug-Induced Immune Haemolytic Anaemia Caused by Cefoperazone-Tazobactam/ Sulbactam Combination Therapy.

There has previously been a report of a patient developing haemolytic anaemia following exposure to cefoperazone. Another case has been reported involving the detection of cefoperazone-dependent antibodies in the absence of immune haemolytic anaemia. To date, no serological evidence has been reported to suggest that cefoperazone can lead to drug-induced immune haemolytic anaemia (DIIHA). This report aims to fill these gaps in knowledge by describing a case of DIIHA caused by cefoperazone-dependent antibodies. A 59-year-old man developed fatal haemolytic anaemia while receiving cefoperazone-tazobactam or cefoperazone-sulbactam for the treatment of a lung infection that occurred after craniocerebral surgery. This eventually led to renal function impairment. Prior to the discontinuation of cefoperazone treatment, the patient showed strong positive (4+) results for both anti-IgG and anti-C3d direct antiglobulin test (DAT), while cefoperazone-dependent IgM and IgG antibodies were detected. The patient's plasma and O-type RBCs were incubated with tazobactam or sulbactam solution at 37°C for 3 h, the results of DAT for anti-IgG and anti-C3d were both positive. Forty-three days after the discontinuation of cefoperazone, the results of DAT for anti-IgG and anti-C3d were negative. Meanwhile incubation of the patient's fresh serum and his own RBCs with cefoperazone at 37°C, gave rise to mild haemolysis, and the results of DAT for both anti-IgG and anti-C3d were positive. It is suggested that cefoperazone-dependent antibodies can activate complement, and the non-immunologic protein adsorption effect of tazobactam or sulbactam can enhance IgG and complement binding to RBCs. This may promote the formation of immunocomplexes and complement activation, thereby aggravating haemolysis.

Effect of blood component transfusion in patients with negative to positive results of direct antiglobulin test after blood transfusion / 中国输血杂志

【Objective】 To analyze the effect of blood component transfusion when the results of direct antiglobulin test (DAT) changed from negative to positive after blood transfusion. 【Methods】 The data of 215 surgical blood recipients, who were admitted in our hospital from January to October 2019 and presented negative results for both DAT and irregular antibody screening (Anti-screening), were collected via Ruimei Laboratory Management System. DAT and Anti-screening were performed again after blood transfusion, and DAT positive patients(re-test positive group) were then subject to antibody classification and polybrene cross-matching (referred to as cross-matching), and Anti-screening positive patients were tested for irregular antibodies. Patients were stratified by perioperative RBCs transfusion volume as ≤4 U (150 ± 10% mL/U), >4 to 8 U and > 8 U, and DAT-negative patients after blood transfusion were set as the controls, and the transfusion effect of DAT-positive patients after blood transfusion was compared with them. 【Results】 8.84% (19/215) of DAT-negative patients turned positive after RBCs transfusion, among which IgG type accounted for 84.21% (16/19) and IgG+ C3 15.79% ( 3/19); two patients(anti-E and-M, 10.53%) were positive in anti-screening re-test and the rest were negative (89.47%, 17/19). As for cross matching, incompatibility of both primary and secondary side, primary side and secondary side accounted for 5.26% (1/19), 5.26% (1/19) and 10.52 (2/19), respectively, while 78.95% (15/19) showed compatibility of both primary and secondary side. The Hb, RBC and Hct values of the re-test positive group, received RBC transfusion volume (U)≤4 and >4~8, were effectively elevated compared with the controls (P8 U(P>0.05). 【Conclusion】 The conversion of DAT negative results to positive after RBC transfusion indicates the patient has developed antibodies or the incidence of blood transfusion reaction, which can provide references for the clinical choice of appropriate blood components to ensure the safety and effectiveness of blood transfusion.

Warm Autoimmune Hemolytic Anemia and Direct Antiglobulin Testing With a False-Negative Result in a 53-Year-Old Man: The DAT Will Set You Free.

Warm autoimmune hemolytic anemia (WAIHA), the most common of the relatively uncommon autoimmune-mediated hemolytic anemias (AIHAs), is mediated by polyclonal immunoglobulin (Ig)G autoantibodies in most cases. Herein, we present a case of WAIHA involving a direct antiglobulin test (DAT) with an initially negative result. Using a modified DAT protocol, repeat testing of the same specimen material from a previously healthy 53-year-old man yielded positive results. This case demonstrates that investigation of an apparently negative DAT result plays a critical role in the differential diagnosis of patients with rapidly progressing hemolytic anemia and the reversal of that decline.

Importance of Direct Antiglobulin Test (DAT) in Cord Blood: Causes of DAT (+) in a Cohort Study.

BACKGROUND: The direct antiglobulin test (DAT) is the cornerstone of the diagnosis of hemolytic disease of the newborn (HDN). The aim of this study was to review the incidence and causes of positive DAT in cord blood in relation to development of HDN. METHODS: We retrospectively reviewed all results of DAT, which is routinely performed in cord blood samples, along with the laboratory and infants' medical records. RESULTS: DAT was positive in 70/2695 (2.59%) cases. In 64/70 (91.43%) cases, DAT positivity was attributed to ABO incompatibility. There were 50/218 (22.93%) DAT (+) cases in the A/O group and 13/97 (13.40%) cases in the B/O group (p = 0.0664). Two DAT (+) cases were attributed to maternal alloimmunization (anti-Fya and anti-JKb, respectively), and one to maternal IgG autoantibodies that developed after methyldopa treatment. Among the 70 DAT (+) cases, 30 (42.86%) cases required phototherapy with no difference between the A/O and B/O groups. The duration of phototherapy in the B/O group was significantly longer than in the A/O group (p = 0.024). There was a trend of correlation of increasing strength of DAT positivity with phototherapy need. No false positive DAT case was detected. CONCLUSIONS: Although ABO incompatibility remains the main reason of DAT (+), other causes (e.g., alloimmunization, drugs) should also be explored. The relevant impact of DAT (+) on HDN development should be considered.

DAT positivity in blood donors: a perplexing scenario.

A blood request was received for 70 year male patient suffering from Chronic Obstructive Pulmonary Disease with anemia. One unit was found incompatible in AHG phase. Patient's antibody screen, indirect antiglobulin test (IAT), direct antiglobulin test (DAT) and auto control was negative. DAT of donor unit was positive with anti IgG gel card and negative with C3d reagent along with positive auto control. Donor was 30 year male with no history of blood transfusion and medication and had no evidence of hemolysis. Donors with positive DAT should be deferred, notified and referred to physician but further studies are required.

Autoimmune hemolytic anemia: mixed type-a case report.

Mixed autoimmune haemolytic anemia (AIHA) is defined by the presence of both warm and cold auto antibodies. Diagnosis is based on detection of autoantibodies by monospecific direct antiglobulin test showing a pattern of IgG and complement C3d and presence of cold agglutinins. We report a rare case of primary mixed AIHA in a 12 year old girl who responded to corticosteroids.