Direct Antiglobulin Test Result Research Articles

A cold case of hemolytic disease of the fetus and newborn resolved by genomic sequencing and population studies to define a new antigen in the Rh system.

We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs. Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals. The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063. This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population.

The Severity of Direct Antiglobulin Test Negative ABO Hemolytic Disease of Newborn: A Retrospective Analysis at a Tertiary Children’s Hospital

This study aimed to evaluate the severity of ABO hemolytic disease of newborn (ABO-HDN) with negative direct antiglobulin test (DAT), which was identified by elution test. We retrospectively reviewed the clinical records of all neonates admitted with the diagnosis of neonatal hyperbilirubinemia requiring phototherapy or exchange transfusion. Neonates were divided into four groups according to their immunohematology test results. Then their essential laboratory results, magnetic resonance image (MRI), brainstem auditory evoked potential (BAEP) findings, and rate of exchange transfusion were compared between different groups. We found that neonates in ABO-HDN with negative DAT group developed jaundice faster and anaemia more severely than those in the non-HDN group. Although they might get less severe anaemia than neonates in ABO-HDN with positive DAT group and the Rh-HDN group, neonates in ABO HDN with negative DAT group might develop jaundice as quickly as the latter two groups. As to MRI and BAEP findings, there were no significant differences among the four groups. The rate of exchange transfusion in ABO-HDN with negative DAT group was higher than that in the non-HDN group but lower than that in ABO-HDN with positive DAT group, though without statistical significance. It suggested that in the presence of clinical suspicion of ABO-HDN with negative DAT result, the elution test should be added to rule out or confirm the diagnosis to help prevent the morbidity from hyperbilirubinemia.

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

IntroductionThe 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). MethodologyThis 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. ResultsFour 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. ConclusionEnhanced DATs generally did not impact medical decision-making in adults withhemolytic anemia.

Immunohematological challenges in COVID infected patients

BackgroundSeveral 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. MethodologyThis 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. ResultsA 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. ConclusionOur results also indicate that there is rise in rate of alloimmunization and DAT positivity among COVID patients.

Role of direct antiglobulin test in assessing the severity of extravascular hemolysis in autoimmune hemolytic anemia – analysis from a cross-sectional study

Background: Autoimmune hemolytic anemia (AIHA) is an immune hemolytic disease characterized by hemolysis and anemia which results from increased red cell destruction due to auto antibodies directed against self-antigens on red cells. Clinical characteristics and potential associated hemolysis in AIHA can greatly depend on the serological characteristics. Aims and Objectives: This study was done to find out the association of Grade of polyspecific Direct Antiglobulin test (DAT), complement involvement and immunoglobulin G (IgG) subclass with severity of hemolysis in an AIHA patients. Materials and Methods: This was a cross-sectional study conducted for a period of 18 months from March 1, 2012, to August 31, 2013. Fifty consecutive AIHA patients diagnosed by a positive DAT in EDTA anticoagulated blood samples using poly specific anti human globulin reagent were followed up and results of various immunohematological tests such as polyspecific and monospecific DAT, IgG subtyping, and thermal amplitude were performed. Hematological and biochemical parameters such as hemoglobin, percentage of reticulocyte, total serum bilirubin, and serum lactate dehydrogenase also were recorded. Data were analyzed using SPSS v17. Results: Majority of patients were belonging to 31–40 year age group and there was a remarkable female predilection. Secondary AIHA was more common (56%) than primary. SLE was identified as the most common underlying disorder in secondary AIHA. Majority (46%) were Grade 4 DAT positive. Predominant autoantibody identified was IgG (52%). Of the total IgG positive cases, IgG1 was the predominant subclass. C3d alone was detected in 18% of cases and C3d and IgG together in 30%. About 48% of our patients were of warm type, 32% mixed type, and 20% cold type. About 80% of the study population were treated with steroids alone. A small percentage of cases (16%) needed immunosuppressant in addition to steroids and still a smaller percentage (4%) needed splenectomy. Higher grades of DAT positivity were associated with more severe degree of anemia and hyperbilirubinemia. The study also demonstrated a positive correlation between DAT grade and severity of hemolysis. Conclusion: AIHA patients in this study were predominantly of Warm AIHA with more of IgG1 involvement. A significant association is found between higher grades of DAT results and severity of anemia and jaundice in this study group. A positive correlation exists between grades of DCT and hemolysis as well.

A Rare Case of Bm (Bweak) Phenotype Detected in a Healthy Blood Donor from Eastern India

A 52-year-old male repeats voluntary blood donor donated at our blood center for the first time. Historical blood group was O, Rh (D) positive. Initial blood grouping was done on the fully automated immunohematology analyzer using column agglutination technique (CAT). Discrepant results (forward as group O and reverse as group B) were observed while performing the ABO blood grouping by CAT. Repeat testing by conventional tube technique showed the same discrepancy. Antibody detection test and direct antiglobulin test results were negative. After adsorption-elution, the eluate reacted only with group B red cells. Saliva testing for secretor status shows the presence of B and H substances. Finally, it was classified as a case of Bm (now called Bweak) phenotype. As a precautionary measure, the donor was recalled and a special blood group card was issued with a clear mention of his respective donor and recipient status. This case showed the importance of ABO subgroup determination that can help blood centers to establish defined transfusion protocol and prepare an elaborate rare blood donor registry.

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

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. 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. 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). 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.

Predicting the Need for Phototherapy After Discharge: Update for 2022 Phototherapy Guidelines.

In 2021, Kuzniewicz et al1 reported that the difference (Δ-total serum bilirubin [TSB]) between last TSB before discharge and the 2004 American Academy of Pediatrics (AAP) phototherapy threshold was an excellent predictor of a postdischarge TSB above the 2004 AAP phototherapy threshold (area under the receiver operating characteristic curve = 0.93). The 2004 AAP hyperbilirubinemia guidelines have since been updated with thresholds for phototherapy that now increase with each week of gestation and are about 1 to 3 mg/dL higher than those from 2004. We sought to quantify how well the predischarge Δ-TSB predicts a postdischarge TSB exceeding the new 2022 AAP phototherapy thresholds.2This retrospective cohort study used the same cohort as our 2021 publication: 163 930 infants born at ≥35 weeks’ gestation July 1, 2012, to December 31, 2017, at 11 Kaiser Permanente Northern California hospitals employing universal TSB screening with at least 1 predischarge TSB. We excluded 9800 for prolonged hospitalization, 4 for direct hyperbilirubinemia, 476 with a predischarge TSB before 12 hours of age, and 2425 who received phototherapy or had a TSB above the 2022 AAP threshold before discharge from all analyses.For all analyses except those used to calculate inverse-probability of censoring weights, we also excluded 4546 infants who, after discharge, received phototherapy before having a TSB above the 2022 AAP threshold. Thus, the final cohort included 146 679 infants. The Kaiser Permanente Northern California institutional review board approved the study (CN-17-3051 01).Our primary predictor was Δ-TSB, defined as the difference between the 2022 AAP phototherapy threshold (for infants of the same age, gestational age and direct antiglobulin test result) and the last inpatient TSB level. (Note in our previous article, our definition was the negative of the above: the TSB minus the threshold.) We created a 5-category Δ-TSB variable to match the AAP 2022 follow-up guideline, as well as an 8-category Δ-TSB variable. We did not have data on transcutaneous bilirubin levels.Our outcomes were a TSB level above the 2022 AAP phototherapy threshold at <24 hours, <48 hours or <30 days from the predischarge TSB. We used linear interpolation to estimate the time that the TSB crossed the phototherapy thresholds.To avoid bias from censoring those who received phototherapy before exceeding the threshold (“subthreshold phototherapy”), we used inverse probability of (non)censoring weights.3 We created models for the probability P of receiving subthreshold phototherapy, based on data available at the time of discharge. We then excluded those infants who had received subthreshold phototherapy and weighted the remaining observations by 1/(1-P) after replacing weights above the 99th percentile with 99th percentile weights.Results by either in 1-mg/dL categories of Δ-TSB or in the 5 categories used in the 2022 guideline (0–<2; 2–<3.5; 3.5–<7; or ≥7 mg/dL) were consistent with those previously reported,1 showing excellent prediction and discrimination (Table 1). Eighty-four percent of the infants had Δ-TSB ≥5.5 mg/dL, with a risk of crossing the phototherapy threshold ≤0.3% (Table 1).We have documented that a single number available at the time of hospital discharge, the Δ-TSB, was a strong predictor of the probability of exceeding the AAP's 2022 phototherapy threshold. The maintenance of discrimination with the higher thresholds is not surprising: Although the slightly raised phototherapy thresholds reduced the risk of the outcome, for an infant with the same TSB level, the Δ-TSB would also be slightly increased, thereby reflecting that lower risk.The excellent discrimination we observed (area under the receiver operating characteristic curve ≥0.93) is largely a result of 84% of the infants having a Δ-TSB that correctly identified them as being at very low risk.Strengths of this study include the large sample size and population with near universal TSB screening. As previously described,1 prediction could be improved by including additional variables, including the most recent rate of rise, formula feeding, age, and gestational age. Nonetheless, our results suggest that a single number, the Δ-TSB divided into 5 categories, provides considerable prognostic information. This supports its use in the 2022 AAP guideline, which provides explicit follow-up recommendations based upon the Δ-TSB in 5 categories.

Patient ABO blood type is a major predictor of a positive DAT following a transfusion reaction.

A direct antiglobulin test (DAT) checks for antibody or complement on the surface of RBCs and is often done following a transfusion reaction. While passive anti-A and anti-B antibodies are known to cause positive DATs, the extent this occurs following transfusion is unknown. DAT results, ABO type, eluate information, and blood product information were recorded on 1097 transfusion reactions at a large academic hospital over 8 years. The effect of patient blood type, product type, and plasma compatibility of blood product transfused on DAT results were determined. Statistical significance was determined using Chi-squared testing. Patient ABO blood type was a strong predictor of a positive DAT, with type O patients having 6.7% positive rate and non-O patients having a positive rate of 20.6% (p< .0001). Plasma compatibility of the product was a strong predictor of a positive DAT, with plasma compatible transfusions having a 9.4% positive rate while plasma incompatible transfusions were positive 44% of the time (p< .0001). Elution studies found that anti-A/B antibodies were the most common antibody identified. Platelets were more likely to be associated with a positive DAT when compared with RBC transfusions (p< .05). These results demonstrate the patient ABO type and plasma incompatibility are strong predictors of positive DAT results following a transfusion reaction. Anti-A and anti-B antibodies are estimated to account for about 50% of positive DATs in this study.

A Rare Cause of Fever in a Term Neonate.

A Rare Cause of Fever in a Term Neonate.

Retrospective analysis of direct antiglobulin test positivity at tertiary academic hospital over 10 years

BackgroundHemolytic disease of the fetus and newborn (HDFN) is a clinically significant problem that may potentially affect any pregnancy. Direct antiglobulin test (DAT) is considered to be an important test in identifying newborns who are suspected to have HDN. This study aims in reviewing data regarding a positive DAT result concerning etiology and the development of HDN over a period of 10 years. Study design and methodsA retrospective study of all neonates with a positive DAT result between January 2011 and December 2020 was performed. Data were obtained from patients' electronic hospital files, transfusion medicine databases, and medical birth records. Laboratory parameters along with clinical interventions in neonates with a DAT-positive result and a comparison group of DAT-negative neonates were performed. Results36,000 deliveries were registered in this period. 176 (2.65 %) neonates had a positive DAT result. ABO-incompatibility was the most common cause with 59.1 %; Rh incompatibility 13.8 %, minor blood group incompatibility, and other RBC-related antibodies 10.1 %, and unspecified etiology in 17 % of cases. Among DAT-positive cases, 32.7 % of neonates were diagnosed with HDN. ABO-incompatibility was the major reason as well. Initial mean total bilirubin levels were higher in the DAT-positive group than the control group (p < 0.001), and these neonates also had a lower initial hemoglobin level (p < 0.001). The need for therapeutic interventions was significantly higher in DAT-positive neonates (p < 0.001) as 86.8 % underwent phototherapy, with 32.7 %, and 17.6 % receiving exchange transfusion (ET) and intravenous immunoglobulin (IVIG), respectively. ConclusionIn conclusion, ABO incompatibility was the most common cause for neonatal DAT positivity. Besides the common causes of DAT positivity, there would be rare but important conditions that may lead to a positive result, such as antibodies passively acquired from mothers in the context of alloimmunizations or using drugs. In addition, as a high rate of therapeutic intervention was identified among neonates with a DAT-positive result, there is a crucial need for increasing awareness regarding early diagnosis of the condition, careful monitoring, and the employment of prenatal alloimmunization screening tests.

A Study of Clinical and Serological Correlation of Positive Direct Antiglobulin Test in Blood Bank at a Tertiary Care Center.

Objectives Detection of red cell bound immunoglobulins and/or complement by direct antiglobulin test (DAT) is a crucial serological assay in the diagnosis of autoimmune hemolytic anemia (AIHA). However, DAT may be positive in a variety of clinical conditions with or without hemolysis. We aimed at evaluating the clinical and serological correlation of positive DAT by categorizing the clinical conditions associated with positive DAT, estimating the presence of in vivo hemolysis in case of positive DAT with polyspecific and monospecific antisera and correlating the strength of positive DAT with the presence of hemolysis. Materials and Methods The prospective observational study was performed on 200 samples that were positive for DAT with polyspecific antiglobulin reagent as the baseline investigation. These samples were further tested with anti-immunoglobulin G and anti-C3 monospecific DAT reagents to evaluate the type of protein responsible for positive DAT. The antiglobulin tests were performed by tube technique. DAT positivity was graded (1+ to 4 + ) in each patient. Autocontrol test was included. The patients with positive polyspecific DAT were categorized into different clinical conditions. The presence or absence of in vivo hemolysis was evaluated in all clinical categories and also for each grade of positivity with polyspecific and monospecific antiglobulin reagents. Statistical Analysis Binomial logistic regression and Mann–Whitney U test were applied to between the group analyses. For categorical variables, Fisher's exact test and relative risk were used. The qualitative data were expressed in numbers and percentages. Results The highest number of patients (75/200, 37.5%) belonged to the autoimmune diseases group. Tuberculosis and hepatitis C were the main infectious diseases associated with positive DAT. Out of 200 DAT-positive patients, 98 (49%) had in vivo hemolysis and 102 (51%) did not have hemolysis. AIHA (22) and systemic lupus erythematosus (18) were the commonest clinical conditions associated with in vivo hemolysis. All the 11 samples that showed positivity with only anti-C3 reagent did not show any hemolysis. There was statistically significant increase in the incidence of in vivo hemolysis with increasing grades of DAT positivity with all the three antihuman globulin reagents. Conclusion There are different disease conditions which show positive DAT with or without hemolysis. So, it is important to clinically and serologically correlate positive DAT results.

The Impact of the “Super Coombs” Test on Patient Management and Outcomes at a Large Tertiary Care Center: A Retrospective Review

Background: The direct antiglobulin test (DAT) identifies the presence of immunoglobulins and complement on the surface of red blood cells and is used to differentiate immune from non-immune hemolysis. Standard DATs done by conventional methods, using anti-IgG or anti-C3 antihuman globulin (AHG) in tube or gel, are performed routinely by laboratories associated with hospital transfusion services. However, if the standard DAT is negative but a strong clinical suspicion for immune-mediated hemolysis remains, serologic testing at an immunohematology reference lab (IRL) can be performed. Subtle differences in reagents and technique in the IRL may identify red cell-bound antibodies missed at the local hospital-based laboratory. In addition, the clinician can request an enhanced DAT, colloquially referred to as a Super Coombs, as a send-out test. This test can detect antibodies that are not detected by standard methods and that have been reported to cause autoimmune hemolysis, namely IgG bound at low levels, low affinity IgG, and IgA. We conducted a retrospective review of enhanced DATs sent by hematologists at the University of Pittsburgh Medical Center and the Allegheny Health Network to an outside IRL to determine the incidence of new autoantibodies detected by the enhanced DAT and assessed the impact of these results on patient management.Methods: We retrospectively identified patients at our center on whom enhanced DATs were sent from January 2019 to January 2021. Demographic, laboratory, and clinical data were collected and analyzed.Results: Twenty one patients were identified as having enhanced DATs sent to one outside IRL during the two year period. The majority of patients had abnormal clinical laboratory markers of hemolysis (81% with an elevated lactate dehydrogenase, 90% with an undetectable haptoglobin) and a history of immune dysregulation (79%). About 2/3 of the patients had a hemoglobin nadir above 6 g/dL. The median number of DATs performed locally prior to requesting the enhanced DAT evaluation was 2 (5 th to 95 th percentile: 1 - 3). Four out of 21 patients (19%) had previously tested positive at the local IRL by conventional serologic methods and 17 (81%) had tested negative.Among the 21 patients, 7 (33%) had at least one positive test on the enhanced DAT battery, including 5 out of the 17 (29%) who had previously tested negative locally. In 5 out of those 7 patients who tested positive by enhanced methods, the positive tests included standard serologic test done by conventional means. The only specialty serologic tests that yielded positive results was testing done at 4 °C, meant to detect low affinity IgG. The mean (±SD) overall turnaround time from sample collection to receipt of the enhanced DAT results was 3.8 (±1.5) days, the bulk of which was transit time from the local hospital to the outside IRL (2.5 (±1.5) days). Empiric high-dose steroid therapy was started in 68% of the patients prior to receipt of the enhanced DAT results. The median time between the first DAT result at the local IRL and the initiation of high dose steroid therapy was 1.5 (5 th to 95 th percentile: 0 - 12.5) days, while the median time from steroid initiation to the time the enhanced DAT was ordered was 5 (5 th to 95 th percentile: 1-256) days. Four patients were diagnosed with DAT-negative autoimmune hemolytic anemia based on response to steroids, whether complete (Hb ≥ 12 g/dL and normalization of LDH and haptoglobin) or partial (Hb improvement by 2 g/dL and no transfusion requirement), despite negative local and enhanced DAT results (table 1). Patients who had self-limited (2 patients) or subacute, non-severe anemia (5 patients) did not undergo a steroid trial and were diagnosed by non-serologic testing, specifically peripheral blood flow cytometry or bone marrow biopsy.Conclusions: The tests that most often yielded positive results on the enhanced DAT were standard serologic tests, suggesting that differences in timing of sample collection or technique may have explained result disparity between the local and outside IRL. Specialty testing done at 4 °C detects low affinity IgG antibodies bound to red cells, but the clinical significance of these antibodies is unclear. The majority of patients in our cohort underwent an empiric trial of high dose steroids prior to receipt of the enhanced DAT results. For these patients, the response to steroids was the deciding factor in AIHA diagnosis rather than the enhanced DAT results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of Polybrene DAT and Conventional DAT in Cases of Suspected DAT Negative Autoimmune Hemolytic Anemia (AIHA)

Abstract Introduction/Objective A negative Direct Antiglobulin Test (DAT) is seen in approximately 1-5% of patients presenting with signs and symptoms of immune hemolysis. A well-performed ‘standard’ DAT detects ~100-500 molecules of bound IgG per red cell. A more sensitive polybrene DAT may be helpful in order to confirm a diagnosis of AIHA in patients whose samples have tested negative via standard DAT. However, there has been little reported on utility of the polybrene DAT in evaluating such cases with modern DAT reagents. We hypothesized that the polybrene DAT would not contribute substantially to the analysis of Coombs-negative hemolytic anemia (C-NHA), based primarily on our anecdotal observation. As such, we undertook a study to assess results of polybrene DAT in cases evaluated for possible (C-NHA). Methods/Case Report Two study sites were used for data analysis (Yale-New Haven Hospital, Site A, and VA Connecticut, Site B) over a five year period (2016-2021). During this time, standard DATs were performed at both study sites by the tube method using polyspecific antiglobulin and, if positive, reflex to anti-IgG and -C3. For cases of suspected C-NHA (which are reported to Blood Bank via a consult mechanism), conventioned DAT- samples are referred to our regional immunohematology laboratory (American Red Cross, Farmington, CT) for manual hexadimethrine bromide (Polybrene) DAT. Polybrene DAT is reported as negative with two sources of polyspecific AHG. Subsets of patients also underwent acid elution studies (Gamma ELU-KITII) as part of a C-NHA algorithm developed during the study period. Results of standard DAT, acId elution, and polybrene DAT were extracted from Blood Bank electronic records at both study sites. Results (if a Case Study enter NA) Evaluation for C-NHA was performed in 32 patients/cases over the study period. Amongst these individuals, 96.8% (31/32) underwent polybrene DAT assessment and none (0%; 0/31) demonstrated a positive polybrene DAT result. Notably, acid elution studies were performed in 90.6% (29/32) of traditional DAT negative cases. Of these, 10.3% (3/29) had reactive eluates. Conclusion Performance of the polybrene DAT appeared to be of no value in the assessment of suspected C-NHA. Given that nearly 10% of individuals with a negative conventional DAT had a positive acid elution, this testing step appears to be of greater value in potentially identifying an autoantibody in suspected cases of C-NHA.

Comparative study of immunohematological tests with canine blood samples submitted for a direct antiglobulin (Coombs\u2019) test

BackgroundA 2019 ACVIM consensus statement on diagnostics for immune-mediated hemolytic anemia (IMHA) in dogs made testing recommendations. As data on the performance of immunohematological tests was lacking, we undertook a comparative analysis.Material and methodsAnticoagulated blood samples from 126 dogs suspected of having IMHA submitted to a diagnostic veterinary laboratory for a routine direct antiglobulin test (DAT) and from 28 healthy control dogs were evaluated for spherocytosis and autoagglutination before and after three saline washes. Samples were also subjected to different DATs: a gel minitube and an immunochromatographic strip kit used in clinics; neutral gel column cards, microtiter plates (at 4°, 22°, and 37°C), capillary tubes, and flow cytometry used in laboratories.ResultsSamples from healthy dogs yielded negative results with all immunodiagnostic tests. Among the 126 samples submitted for DAT 67 were positive by a DAT utilizing microtiter plates with goat anti-dog antiglobulin DAT at 22°C. Notably, DAT results were comparable and consistent across all evaluated methods regardless of antiglobulin and temperature used. DAT+ dogs were more severely anemic and more likely to have erythroid regeneration compared to DAT- dogs. Macroscopic agglutination in tubes or on slides was observed in 48 samples after 1:1 and 1:4 blood to saline dilution, but only persisted in four samples after washing. Among the DAT+ samples, 57% had agglutination, 87% had spherocytosis, and 45% had both. There was good correlation between spherocytosis and DAT results from the six DAT techniques, but the correlation with autoagglutination was only fair. Clinical follow-up was available for 42 dogs. Of the sample from 12 DAT+ dogs collected during treatment, 10 remained DAT+ when tested 1–24 weeks after initial assessment.ConclusionsBased upon this comparative prospective survey, all in-clinic and laboratory DAT techniques produced similar results when performed by trained personnel and can therefore be recommended for detection of antibody-coated erythrocytes and immunohematological diagnosis. In addition, use of these tests for monitoring response of IMHA dogs to treatment might be valuable.

Case of Hemolytic Disease of the Newborn due to a Rare Red Cell Antibody.

Case of Hemolytic Disease of the Newborn due to a Rare Red Cell Antibody.

Rash, Anemia, and Thrombocytopenia in a 6-week-old Boy.

Rash, Anemia, and Thrombocytopenia in a 6-week-old Boy.

Investigation of anemia of unknown origin

Abstract One of the most difficult concepts to explain when training immunohematology staff involves the investigation of hemolytic anemias. In the transfusion service and in the immunohematology reference laboratory setting, rapid and efficient investigation can be extremely important for patients with critical anemia requiring transfusion. The flow charts presented here provide possible patient scenarios and a logical sequence for initial and subsequent serologic testing for investigation. A clinical assessment of anemia of unknown origin or the finding of an unresolved positive antibody screen in pre-transfusion patient testing begins the investigational flow process. The testing sequence is predicated on the fact that performing a direct antiglobulin test (DAT) on all patients has a low predictive value and should be reserved for patients with unexplained anemia. The process begins with assessing the results of DATs with anti-IgG and anti-C3. Subsequent charts A, B, and C aid in investigating these results. Flow Chart A is for the investigation of warm or cold autoantibody, Chart B is for the investigation of cold-agglutinin or drug-induced immune hemolytic anemia, and Chart C is for the investigation of autoantibody in the transfused patient. While the most common approaches to the initial and subsequent test results are in these flow charts, the charts are not inclusive of all possible diagnoses or presentations. These flow charts are meant to be a guide to assist the laboratory in developing a standard approach to efficient investigation and resolution in patients with unexplained anemia.

Investigation of anemia ofunknownorigin.

One of the most difficult concepts to explain when training immunohematology staff involves the investigation of hemolytic anemias. In the transfusion service and in the immunohematology reference laboratory setting, rapid and efficient investigation can be extremely important for patients with critical anemia requiring transfusion. The flow charts presented here provide possible patient scenarios and a logical sequence for initial and subsequent serologic testing for investigation. A clinical assessment of anemia of unknown origin or the finding of an unresolved positive antibody screen in pre-transfusion patient testing begins the investigational flow process. The testing sequence is predicated on the fact that performing a direct antiglobulin test (DAT) on all patients has a low predictive value and should be reserved for patients with unexplained anemia. The process begins with assessing the results of DATs with anti-IgG and anti-C3. Subsequent charts A, B, and C aid in investigating these results. Flow Chart A is for the investigation of warm or cold autoantibody, Chart B is for the investigation of cold-agglutinin or drug-induced immune hemolytic anemia, and Chart C is for the investigation of autoantibody in the transfused patient. While the most common approaches to the initial and subsequent test results are in these flow charts, the charts are not inclusive of all possible diagnoses or presentations. These flow charts are meant to be a guide to assist the laboratory in developing a standard approach to efficient investigation and resolution in patients with unexplained anemia.

Maternal Blood Group and Routine Direct Antiglobulin Testing in Neonates: Is There a Role for Selective Neonatal Testing?

Recommendations for the screening of hemolytic disease of the newborn (HDN) advise taking a selective approach in using the direct antiglobulin test (DAT) for mothers with blood group O or RhD-negative. This study assessed the relation of DAT results to maternal and neonatal blood groups and evaluated the risk of HDN. A retrospective analysis of all healthy newborns admitted during 2018 was performed. Of 1463 newborns, 4.4% had a positive DAT. There were 541 (37%) maternal–neonatal pairs with ABO incompatibility, most commonly born to mothers with blood group O. The cohort of neonates born to mothers with blood group O was divided into three groups: the O-A and O-B groups and the O-O group as a control. The DAT was positive in 59 (8.3%) neonates; most were in the O-B group (49.2%), whereas 13.6% were in the control group (p < 0.01). While the neonates in the O-B group were more likely to require phototherapy (p = 0.03), this finding was not related to DAT results. We found that selective testing of mothers with blood group O, mothers with blood group O or RhD-negative, neonates with blood group B, and neonates with blood group B born to mothers with blood group O or RhD-negative was ineffective in detecting phototherapy requirements. Our results indicate no difference regarding the need for phototherapy in neonates born to mothers with different blood types regardless of the DAT results.