Alectinib-induced Hemolytic Anemia with Positive Direct Antiglobulin Test in a Patient with Lung Adenocarcinoma: A Possible Drug-drug Interaction Effect.

Recent studies have reported that direct antiglobulin test (DAT) results were negative in cases of alectinib-induced hemolytic anemia with abnormal red blood cell (RBC) morphology. We herein report the case of a 72-year-old female patient who was diagnosed with alectinib-induced hemolytic anemia who - in contrast to previous reports - showed a positive DAT result. After discontinuing famotidine and alectinib, the DAT results turned negative; however, when alectinib was resumed, hemolysis recurred. Although alectinib-induced hemolytic anemia has been previously thought to be associated with abnormal morphological changes of the RBCs, we suggest that alectinib-induced anemia may manifest as DAT-positive immune hemolytic anemia because of a complementary effect with other drugs.

Erythrophagocytosis in autoimmune immunoglobulin A-mediated hemolysis.

INTRODUCTION: Warm antibody-mediated autoimmune hemolysis (WAIHA) is most often due to immunoglobulin G (IgG) antibodies and is detected by direct antiglobulin test (DAT). However, about 10% cases of hemolytic anemia are DAT negative. Herein, we describe a patient with DAT-negative hemolytic anemia due to an anti-IgA antibody. We investigate if isolated IgA can promote erythrophagocytosis. METHODS: We isolated patient and control IgA on Jacalin agarose. Isolated IgA was used to sensitize healthy ABO/Rh-matched donor red blood cells (RBC). Antibody binding was examined by flowcytometry. The effect of IgA on erythrophagocytosis was evaluated using Macrophage colony stimulating factor 1 (M-CSF)-differentiated autologous macrophages by Giemsa staining and immunofluorescence microscopy. RESULTS: We show that isolated IgA from the serum can bind to red cells. In addition, RBCs were phagocytosed efficiently by autologous macrophages in the presence of patient IgA. CONCLUSION: Our results show that IgA antibodies are capable of inducing erythrophagocytosis like IgG antibodies in the absence of complement fixation.

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.

Immunohematological challenges in COVID infected patients.

BACKGROUND: Several autoimmune disorders have been reported to be related with COVID infection. In continuation to these autoimmune phenomenon, autoimmune hemolytic anaemia (AIHA) also has been noted in COVID infected patients. The aim of the study was to find out the prevalence of red cell alloimmunization, ABO discrepancy and positive direct antiglobulin test (DAT) results in COVID infected patients hospitalised in a tertiary care centre in North India. METHODOLOGY: This was a retrospective observational study done from July 2020 to June 2021. All symptomatic patients admitted to ICU tested positive for SARS CoV-2 whose blood samples were received in the immunohematology laboratory of department of Transfusion Medicine for determination of blood group and issue of packed red cells, and found to have positive antibody screen, blood group discrepancy and positive DAT results, were included in the study. RESULTS: A total of 10,568 tests were run, out of which 4437 were for determination of blood group, 5842 were for antibody screen and 289 were for direct antiglobulin test. Included in this study were 146 patients who either had blood group discrepancy, or had a positive antibody screen or had a positive DAT. Out of 115 positive antibody screen, 66 patients had only alloantibodies, 44 patients had only autoantibodies while only 5 patients had both auto as well as alloantibodies. Total number of positive DAT cases was 50 (50/289 = 17.3 %). There were 26 ABO discrepancies (26/4437 =0.58 %) found. CONCLUSION: Our results also indicate that there is rise in rate of alloimmunization and DAT positivity among COVID patients.

Clinical value of combination detection of direct antiglobulin test and serum albumin globulin ratio in severe hyperbilirubinemia caused by ABO hemolytic disease of the newborn.

Background To explore of a combination of antiglobulin test(DAT) and albumin globulin ratio(AGR) could predict the severity of ABO hemolytic disease of the newborn(ABO-HDN).Methods The measurement of DAT, AGR and combination detection of DAT and AGR was done to predict severe ABO-HDN hyperbilirubinemia in 270 full-term infants based on whether the infants received transfusions of blood components. The infants were divided into three groups according to the results of DAT and ARG and compared the differences of phototherapy day and hospitalization day of the three groups.Results Of the 270 cases enrolled in this study, 69 infants were DAT positive. Peak total bilirubin, AGR, and positive DAT were independently associated with the need for blood components transfusion. ROC curve analysis for blood components transfusion showed that DAT cutoff value >± with a sensitivity of 39.4% and a specificity of 83.9%, AGR cutoff value <2.05 with a sensitivity of 54.1% and a specificity of 85.7%, and combination detection of DAT and ARG with a sensitivity of 62.1% and a specificity of 91.2%. The AUCs for DAT, AGR, and combination detection of DAT and AGR were .621, .740, and .750 respectively. The phototherapy day and hospitalization day were significantly longer in group of AGR <2.05 and DAT >± than that of a group of AGR <2.05 and group of DAT >±.Conclusions DAT and ARG could be early predictors for the severity ABO-HDN hyperbilirubinemia and combination detection of DAT and AGR could further increase its predictive value.

Direct antiglobulin test positivity in Serbian blood donors in the era of gel card technology.

BACKGROUND AND OBJECTIVES: In the era of gel cards, we aimed to determine the incidence of a positive direct antiglobulin test (DAT) in a population of blood donors in Serbia and to compare results with the period when a conventional tube technique was used. MATERIALS AND METHODS: A retrospective study was conducted on 184,171 blood donors over 10 years (2012-2021). Positive DAT results initially were detected after positive result of the compatibility test. After a DAT had been initially performed on fresh blood samples, a control DAT was performed 6 months later. RESULTS: Using gel cards, we found a low incidence (0.09%) of DAT positivity among 55,254 female and 128,917 male blood donors, with no difference found in the occurrence of a positive DAT between them. Positive DAT moderately positively correlated with increasing age of blood donors, but without statistical significance. The vast majority of blood donors (98.86%) were IgG positive. All donors were in good health, did not take medication or had anaemia or haemolysis. During the period 1980-1989, when the test tube technique was used, we found 0.05% DAT-positive blood donors. CONCLUSION: The incidence of positive DAT results in Serbian blood donors is higher compared with that found using the previous test tube technique, but the incidence is consistent with the findings of other studies.

Strategies to overcome the diagnostic challenges of autoimmune hemolytic anemias.

INTRODUCTION: The direct antiglobulin test (DAT) or Coombs test is the cornerstone of the diagnosis of autoimmune hemolytic anemia (AIHA). It can be performed by several methods with different sensitivity and specificity and enables the distinction of warm, cold, and mixed forms, which require different therapies. AREAS COVERED: The review describes the different DAT methods, including the tube test with monospecific antisera, microcolumn and solid phase methods that are routinely accessible in most laboratories. Additional investigations include the use of cold washes and low ionic salt solutions, the identification of auto-Ab specificity and thermal range, the study of the eluate, and the Donath-Landsteiner test, available in most reference laboratories. Experimental techniques are the dual-DAT, flow cytometry, ELISA, immuno-radiometric assay, and mitogen-stimulated DAT, which may help the diagnosis of DAT-negative AIHAs, a clinical challenge with delayed diagnosis and possible improper therapy. Further diagnostic challenges include the correct interpretation of hemolytic markers, the infectious and thrombotic complications, and the possible underlying conditions (lymphoproliferative disorders, immunodeficiencies, neoplasms, transplants, and drugs). EXPERT OPINION: These diagnostic challenges may be overcome by a 'hub' and 'spoke' organization among laboratories, a clinical validation of experimental techniques, and a continuous dialogue between clinicians and immune-hematologic laboratory experts.

ABO hemolytic disease of the newborn: a need for clarity and consistency in diagnosis.

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.

Evaluation of solid-phase panreactivity with negative direct antiglobulin testing.

Solid-phase red cell adherence (SPRCA) is a sensitive platform for antibody detection, but nonspecific reactions may occur. One pattern of apparent nonspecific reactivity is a panagglutinin with a negative direct antiglobulin test (DAT). The purpose of this study was to define the clinical characteristics of patients with these nonspecific reactions and their associated serologic findings. Twenty patients with panreactive SPRCA testing results were identified between November 2022 and May 2023. In addition to panagglutinins, these patients had (1) a negative polyethylene glycol (PEG) antibody detection test, (2) a negative PEG autocontrol, and (3) a negative DAT. The strength of SPRCA panreactivity and the results of eluate testing (by tube and SPRCA) were studied. Clinical characteristics of patients included age, sex, and primary diagnosis. Each patient was also assessed for evidence of hemolysis. Fourteen female and six male patients were evaluated (average age 44 years). Primary diagnoses included pregnancy (n = 10), acute bleeding (n = 4), orthopedic (n = 3), and other (n = 3). There was no clinical or laboratory evidence of hemolysis. The predominant strength of SPRCA panreactivity was evenly distributed across reaction grades (1+ to 3+). Fifty-five percent of the eluates tested in PEG showed panreactivity, consistent with warm-reactive autoantibodies, while 85 percent of eluates tested by SPRCA were panreactive. Six discrepant cases, in which PEG eluate testing was negative and solid-phase eluate testing showed panreactivity, were associated with weak solid-phase plasma panreactivity (1+). In addition, the reactivity strengths of the eluates tested by SPRCA were invariably more strongly reactive than those eluates tested in PEG. Panagglutination is a distinct SPRCA-only plasma reactivity pattern. Despite a negative PEG tube and DAT, most panagglutinins are warm-reactive autoantibodies. Fortunately, these "interfering" panagglutinins do not appear to be clinically significant and are easily managed by an alternative testing method such as PEG.

[Clinical and Laboratory Characteristics of Primary Autoimmune Hemolytic Anemia Patients with Negative Results of DAT by Tube Test But Positive Results by Microcolumn Gel Assay].

OBJECTIVE: To investigate the clinical features and laboratory characteristics of primary autoimmune hemolytic anemia (AIHA) patients with negative results of direct antiglobulin test (DAT) by tube test but positive results by microcolumn gel assay, in order to provide references for the diagnosis of these patients. METHODS: 59 patients diagnosed with primary AIHA in our hospital from January 2015 to December 2020 were retrospectively analyzed. According to the results of tube test and microcolumn gel assay, the cases were divided into 3 groups, and the clinical and laboratory characteristics of each group were compared. RESULTS: The cases were grouped as follows: Group I, cases with negative results by both methods of DAT (n=5); Group II, cases with negative results by tube test but positive results by microcolumn gel assay (n=26); Group III, cases with positive results by both methods of DAT (n=28). There was no significant difference in age and sex between Group II and other groups, whereas the positive rate of anti-IgG + anti-C3d of Group II was lower than that in Group III (P=0.015). The main clinical manifestations of Group II were chest tightness, shortness of breath, fatigue, as well as yellow skin and sclera or dark urine, but the incidence rate of these symptoms was not significantly different from other groups. Anemia related indexes in Group II such as red blood cell (RBC) count and hemoglobin (Hb) were lower than the reference intervals, but there was no significant difference compared with other groups. Hemolysis related indexes in Group II such as reticulocyte (Ret) ratio, indirect bilirubin (IBIL), lactate dehydrogenase (LDH) and free-hemoglobin (F-Hb) were higher than the reference intervals, and the latter two items were signficantly higher than those in Group I (P=0.031 and P=0.036). Serum complement C3 and C4 in Group II were higher than those in Group III (P=0.010 and P=0.037). CONCLUSION: Anemia severity of primary AIHA patients who were negative of DAT by tube test but positive by microcolumn gel assay was similar to those with negative or positive results by both DAT methods, but the mechanism and degree of complement system involved in hemolysis might be different. Results above may be helpful for laboratory diagnosis of this kind of patients.

Laser incubation for the rapid detection of red cell alloantibodies in human blood samples.

BACKGROUND AND OBJECTIVES: Pre-transfusion antibody screening requires the detection and identification of immunoglobulin G (IgG) antibodies against red blood cells (RBCs). Using the indirect antiglobulin test (IAT), plasma-RBC solutions are incubated at 37°C in gel cards, typically by heating block technology. Here, we apply the newly developed laser incubation method to detect RBC alloantibodies in the plasma from human donors. MATERIALS AND METHODS: Donated human plasma samples (N = 128) containing clinically significant IgG antibodies directed against Rh (D, C, c, Cw and E), Kell (K and Kpa ), Duffy (Fya and Fyb ), Kidd (Jka ) and MNS (S) blood group system antigens were tested by the indirect antiglobulin test (IAT). Samples were heated to 37°C by infrared laser (980 nm) for incubations of up to 5 min. Samples were also incubated in a heating block for comparison. RESULTS: When heating by laser, the presence of an alloantibody is detected after only a 1-min incubation for 96% of samples. No samples required longer than 3 min of laser incubation in order to detect the antibody. For all samples, incubation by laser gave the same or stronger result within 5 min. No samples required longer than 5 min to achieve an equivalent result to that of the 5-min heating block incubation. The laser was not found to damage cells or antibodies. CONCLUSION: Laser incubation provides comparable results in shorter time frames than the heating block. Laser incubation can rapidly detect even very weak antibodies.

The significance of antiglobulin (Coombs) test reactivity in patients with COVID-19.

Previous case reports have described patients with COVID-19-associated autoimmune hemolytic anemia (AIHA), and cold agglutinin disease (CAD) which is characterized by a positive direct antiglobulin (DAT) or "Coombs" test, yet the mechanism is not well understood. To investigate the significance of Coombs test reactivity among COVID-19 patients, we conducted a retrospective study on hospitalized COVID-19 patients treated at NMC Royal Hospital between 15 April and 30 May 2020. There were 27 (20%) patients in the Coombs-positive group and 108 (80%) in the Coombs-negative group. The cold agglutinin titer was examined in 22 patients due to symptoms suggestive of cold agglutinin disease, and all tested negative. We demonstrated a significant association with reactive Coombs test results in univariate analysis through clinical findings such as ICU admission rate, the severity of COVID-19, and several laboratory findings such as CRP, D-dimer, and hemoglobin levels lactate dehydrogenase, and RDW-CV. However, only hemoglobin levels and disease severity had a statistically significant association in multivariate analysis. A possible explanation of COVID-19-associated positive Coombs is cytokine storm-induced hyperinflammation, complement system activation, alterations of RBCs, binding of SARS-CoV-2 proteins to hemoglobin or its metabolites, and autoantibody production. Coombs-positive patients were tested for hemolysis using indirect bilirubin, consumed haptoglobin, and/or peripheral smear that ruled out any evidence of hemolysis. Understanding this etiology sheds new light on RBC involvement as a pathophysiological target for SARS-CoV-2 by interfering with their function; consequently, therapies capable of restoring RBC function, such as erythrocytapheresis, could be repurposed for the treatment of worsening severe and critical COVID-19.

DAT-Negative Autoimmune Hemolytic Anemia.

Hematologists often rely on the results of a positive direct antiglobulin test to confirm a diagnosis of autoimmune hemolytic anemia, but immune hemolytic anemia can occur when no immunoglobulin is detectable by routine methods. Negative DATs in these patients may be due to a small quantity of IgG on their red blood cells (RBCs) (below detectable levels), or when low-affinity anti-IgG is present, or when the autoantibodies are IgA or IgM in nature. A panel of tests developed to detect immunoglobulins on these patients' RBCs may be performed in a few specialized laboratories. These tests can be helpful in instances whereby the clinical picture of AIHA seems obvious, but the laboratory values are misleading.

Diagnostic algorithm for classification and characterization of direct antiglobulin test-negative autoimmune hemolytic anemia with 1-year clinical follow-up.

BACKGROUND: Approximately 5%-10% of autoimmune hemolytic anemia (AIHA) cases are negative for direct antiglobulin test (DAT). We previously reported a classification system for untreated patients with DAT-negative AIHA by quantifying levels of red blood cell (RBC)-bound IgG. This study investigated the clinical utility of a novel diagnostic algorithm with a comprehensive classification system and characterized each subgroup in DAT-negative AIHA. STUDY DESIGN AND METHODS: We assessed 637 patients with undiagnosed hemolytic anemia using our diagnostic algorithm and classification system, which was based on RBC-bound IgG levels and results of column method-DAT before and after washing RBCs. RESULTS: Patients were diagnosed with DAT-negative AIHA with 97% sensitivity and 84% specificity when the laboratory tests were performed before treatment and classified into the following six categories: tube DAT-negative, low-affinity IgG, double DAT-negative, IgA- or IgM-positive, low-affinity IgM, and s/o non-AIHA. The first three types were major conditions and accounted for 76% of DAT-negative AIHA cases. Based on multivariate analyses of idiopathic DAT-negative AIHA (n = 71), platelet count and albumin concentration were significant factors for survival at 1-year follow-up. The low-affinity IgG group showed the highest platelet count and albumin levels, better response to steroids, and higher 1-year survival rate than those in other groups. DISCUSSION: Our classification included DAT-negative, IgA-driven, and warm-IgM AIHA categories, which were atypical forms of AIHA with the severe onset and increased risk of relapse. When treating a patient with DAT-negative hemolysis, atypical AIHA should be considered and tested in reference laboratories, especially before treatment.

Review: Effects of anti-CD38 monoclonal antibodies on red blood cell transfusion and interventions.

BACKGROUND: Highly expressed in almost all myeloma cells, CD38 is an attractive treatment target. AIM: Anti-CD38 monoclonal antibodies have been approved for first-line treatment in non-transplantable multiple myeloma (MM) patients. MATERIALS AND METHODS: However, it has been found in clinical use that anti-CD38 monoclonal antibodies bind to CD38 on red blood cells (RBCs) and cause panagglutination in indirect antiglobulin test (IAT), resulting in false positives of IAT (Transfusion, 55, 2015 and 1545; Transfusion, 55, 2015 and 1555). RESULT: Thereby, interfering with blood bank testing and leading to the delay of further diagnosis and treatment. CONCLUSION: With more and more patients receiving anti-CD38 treatment, it is of great importance to recognize this problem and optimize relevant diagnosis and treatment procedures to prevent RBC transfusion delays and reduce laboratory costs.

Low hemoglobin levels are associated with direct antiglobulin test positivity in patients with acute-on-chronic liver failure.

BACKGROUND: Multiple factors contribute to anemia in patients with Hepatitis B virus (HBV)related acute-on-chronic liver failure (ACLF); however, the mechanism is unclear. The purpose of this study was to evaluate the clinical significance of the direct antiglobulin test (DAT) in patients with HBV related ACLF. METHODS: DAT was used to detect immunoglobulins and/or complement proteins on the surface of erythrocytes. RESULTS: We recruited 78 HBV-associated ACLF patients, 30 chronic hepatitis B(CHB)patients and 40 healthy people between October 2015 and May 2016. In HBV related ACLF patients, the hemoglobin concentration, number of erythrocytes, and hematocrit value were significantly lower, while the erythrocyte distribution width was significantly higher, compared to patients with CHB and healthy controls (HCs) (P < 0.001). The rates of DAT positivity in HBV related ACLF patients, CHB patients, and HCs were 62.8 %, 13.3 %, and 0%, respectively. DAT-positive ACLF patients exhibited lower Hb levels, older average age, as well as higher total bilirubin, alanine aminotransferase, and complement component 3 levels compared to DAT-negative patients. CONCLUSIONS: HBV related ACLF patients showed significant alterations in erythrocyte parameters, possibly reflecting disease development and severity. The high presence of erythrocyte autoantibodies suggested that immunologic clearance of erythrocytes contributed to multifactorial anemia in HBV related ACLF patients.

Is it necessary to add the eluate testing to the direct antiglobulin test to improve the detection of maternal erythrocyte alloantibodies?

BACKGROUND: The screening of umbilical cord blood samples by the Direct Antiglobulin Test (DAT) is the reference tool for the identification of maternal erythrocyte alloantibodies present in erythrocytes; however, its diagnostic usefulness is controversial. OBJECTIVE: To evaluate the diagnostic validity, safety, and efficiency of the eluate testing (detection of antibody in erythrocyte eluates by the Indirect Antiglobulin Test/IAT) in cord blood samples for detection of maternal erythrocyte alloantibodies in comparison with the DAT. MATERIALS AND METHODS: Evaluation study of diagnostic tests. DAT and eluate testing were performed in 306 cord blood samples from neonates born to mothers admitted at Clínica Somer in Rionegro, Colombia; then, antibodies present in the eluates were identified with erythrocyte panels. Percentage of positive results by DAT and IAT were compared with the Pearson's chi-square test and the agreement between both assays with the Cohen's kappa coefficient. The diagnostic sensitivity, specificity, safety, and efficiency of the eluate testing were calculated, taking into account the use of DAT as an imperfect reference test. RESULTS: The DAT detected alloantibodies in 6.21% of samples and the eluate testing in 14.1 %; the strength of agreement between both tests was moderate (k = 0.56) due to 25 discrepancies. The eluate testing showed sensitivity and specificity of 98.83 % and 92.31 % respectively, and a negative predictive value of 99.9 %. The diagnostic efficiency was sufficient for detection of maternal erythrocyte alloantibodies. The antibodies identified in the erythrocyte eluates were anti-A or anti-B (79.5 %), anti-D (136%), anti-C (2,3%), and anti-Fya (2,3%). CONCLUSION: The eluate testing in cord blood samples is a valid, safe, and efficient test for the diagnosis of maternal erythrocyte alloantibodies.