Negative Direct Antiglobulin Test Research Articles

Flow cytometry for RBC damage and complement activation

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

#2262 Renal outcomes of postpartum atypical hemolytic uremic syndrome treated with plasma exchange

Abstract Background and Aims Postpartum acute kidney injury (AKI) with features of thrombotic microangiopathy (TMA) are particularly challenging and complex cases. Causes include Preeclampsia (PE), hemolysis, elevated liver enzymes and low platelet count syndrome (HELLP) and atypical hemolytic uremic syndrome (aHUS). In a limited resource setting, it is difficult to diagnose such cases with more than standard blood tests. Therapeutic Plasma exchange (TPE) is the only available option for treatment in the absence of complement inhibitors. This study evaluates renal outcomes in patients with aHUS treated with plasma exchange. Method This is a single centre observational study conducted in a Tertiary Obstetric Intensive Care unit. The data of women who were admitted with postpartum AKI from January 2021 to October 2023 was retrospectively collected and analyzed. We used the following criteria to diagnose postpartum atypical HUS (Serum creatinine ≥1.9 mg/dL, Lactate dehydrogenase (LDH) ≥1832 U/L, or serum creatinine ≥1.9 mg/dL in combination with LDH ≥600 U/L) [1]. Inclusion criteria include women older than 18 yrs with microangiopathic hemolytic anaemia, schistocytes on peripheral blood film, negative direct antiglobulin test (DAT) and platelet count <100 /mm3.Lack of improvement of kidney functions and hemolysis 72 hours after delivery. Patients with sepsis, disseminated intravascular coagulopathy (DIC), postpartum hemorrhage (PPH) and chronic kidney disease were excluded. We identified 10 patients with postpartum aHUS. Peak creatinine, LDH, AST and Nadir platelet count and hemoglobin were recorded. Patients were followed up in the nephrology outpatient clinic. Results Fifty-two patients developed postpartum AKI in the study period. Ten patients (19%) were diagnosed with aHUS according to the above-mentioned criteria. Mean age was 24.5 ± 6.9 yrs. Median peak creatinine 5.1 (2.6-7) mg/dl, LDH 1950 (612-4000) U/L, AST 246 (150-699) U/L. Median nadir hemoglobin 7.25 (6-8.2) g/dl and platelets 57 (41-95)/mm3. All 10 patients received 3 to 6 sessions of TPE guided by platelets and LDH levels. All patients required at least 3 sessions of hemodialysis. Eight patients received TPE with plasma filter and 2 had centrifugal TPE (due to lack of plasma filters). Two patients died during ICU admission. Two months after discharge 4 (50%) patients had partial recovery of kidney functions average creatinine (3.5 ± 0.8) mg/dl and four (50%) remained dialysis dependent. Six of these patients had kidney biopsies, three biopsies showed a picture of thrombotic microangiopathy and two showed thrombotic microangiopathy with renal cortical necrosis. The other patient was lost to follow up. Conclusion It is difficult to diagnose and treat aHUS in a resource limited setting. The available treatment with therapeutic plasma exchange has poor renal outcomes.

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.

Unraveling the Clinical Spectrum: A Case Study of Classic and Aplasia-Related Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare nonmalignant clonal disorder of hematopoietic stem cells characterized by a phosphatidylinositol glycan protein A (PIGA) gene mutation. This mutation results in the deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59, on the cell surface, rendering red blood cells (RBC) susceptible to complement-mediated destruction. This leads to hemolytic anemia, thromboses, and bone marrow failure. Here we describe two cases of PNH presenting to a tertiary care center. Case 1 A 29-year-old male with a history of polysubstance use presented to the Emergency Department with a 2-day history of progressively worsening left lower quadrant abdominal pain. CT abdomen pelvis showed multiple renal infarcts and findings suggestive of pneumatosis intestinalis. A complete blood count (CBC) revealed pancytopenia with a leukocyte count (WBC) of 3 x 10 9/L, hemoglobin (Hb) of 94 g/L, and platelet count (Plt) of 48 x 10 9/L. Lactate dehydrogenase (LDH) was elevated (900 IU/L), haptoglobin level was low ( <8mg/dL), and direct antiglobulin test (DAT) was negative. Urine was positive for hemosiderin, and flow cytometry showed RBCs (6.7%), granulocytes (97.97%), and monocytes (89.02%) with PNH phenotype. Bone marrow biopsy (BMB) was negative for dysplasia and fibrosis with normal hematopoiesis but showed increased erythroid precursors. Enoxaparin was started for thromboses and levofloxacin for neutropenia. Treatment for PNH was initiated with a loading dose of ravulizumab and meningococcal vaccination. The patient was lost to follow-up after receiving the first maintenance dose. Case 2 An 81-year-old female with a medical history of hypertension and coronary artery disease was hospitalized for gradually worsening shortness of breath and fatigue ongoing for 2 months. She reported easy bruising but denied a history of obvious bleeding from any orifice. Physical exam was significant for petechial rash in her lower extremities. Labs revealed pancytopenia (WBC 1.44 x 10 9/L, Hb 68 g/L, Plt 8 x 10 9/L), low reticulocyte count, LDH of 276 IU/L, and negative DAT. BMB showed marked aplasia, and she became transfusion dependent. Subsequently, flow cytometry revealed PNH phenotype in 1.45% of RBCs, 54% granulocytes, and 54% monocytes. Eculizumab therapy was started for the diagnosis of PNH. However, her transfusion requirements did not decrease. She developed iron overload and showed positive conversion on DAT. Darbepoetin alfa and romiplostim were added. Her Hb stabilized at >70g/L in 3 years, following which she only required romiplostim to maintain Plt >20,000 ( Graph 1). After 5 years, she opted to discontinue all treatment. She is being followed up regularly and has since remained stable. Discussion The two cases above are reflective of how PNH can present in different ways, ranging from the classic triad of symptoms to those with subclinical findings associated with other underlying conditions, such as aplastic anemia or myelodysplastic syndrome. The introduction of complement inhibitors completely changed the natural history of PNH, as patients' median survival shifted from 15-20 years to align with age-matched controls. Indications for treatment include severe anemia, thrombosis, or PNH symptoms like dyspnea, fatigue, and painful paroxysms. Currently, eculizumab and ravulizumab are the only FDA-approved drugs for PNH treatment. Ravulizumab is non-inferior to eculizumab, with the added advantage of less frequent dosing (every 8 weeks versus 2 weeks). Regular monitoring for hemolysis is warranted with treatment. Many patients require long-term therapy, which continues to be a significant challenge, even with the spaced dosing of ravulizumab. Eculizumab protects from terminal complement-mediated damage preventing intravascular hemolysis. However, these cells are still deficient in CD55, making them vulnerable to opsonization by C3 and premature removal in the spleen. As a result, >50% of PNH patients receiving treatment show positive conversion on DAT, as seen in case 2. Heparin is preferred in acute thrombotic states. Most hematologists recommend anticoagulation till a few months after starting complement inhibition.

Favorable outcome without corticosteroids during post-artesunate delayed hemolysis with positive direct antiglobulin test in severe imported Plasmodium falciparum malaria, France

Objectives: Positive direct antiglobulin tests (DATs) have been reported in cases of post-artesunate delayed hemolysis (PADH), but the causal role of auto-immune hemolysis remains unclear. We aimed to analyze a cohort of patients with PADH and DAT during severe malaria.Methods: We describe PADH and DAT results in a 7-year multi-center retrospective cohort of patients receiving artesunate for severe imported malaria.Results: Of 337 patients treated with artesunate, 46 (13.6%) had at least one DAT result within 30 days of treatment initiation, and 25/46 (54.3%) had at least one positive DAT. Among 40 patients with available data, 17 (42.5%) experienced PADH. Patient characteristics were similar for patients with a positive or negative DAT, and DAT positivity was not associated with PADH occurrence (P = 0.36). Among patients, 5/13 (38.5%) with a positive DAT after day 7 experienced PADH, compared to 10/13 (76.9%) of those with a negative DAT after day 7 (P = 0.11). Overall, 41% of patients required blood transfusions, and outcome was favorable without corticosteroids, even in cases of PADH.Conclusions: DAT does not appear to be a marker of PADH, but rather an indirect marker of an immune-mediated mechanism. DAT positivity should not lead to the administration of systemic corticosteroids during PADH.

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.

Rates of phototherapy among ABO-incompatible newborns with a negative direct antiglobulin test.

We analyze phototherapy rates after implementation of a Hyperbilirubinemia Clinical Pathway (HCP), which placed full-term ABOi DAT negative newborns on the low risk phototherapy nomogram, rather than medium risk, as previously done. A chart review was performed for ABOi newborns born ≥36 weeks gestation between January 2020 and October 2021. Primary outcome measures were rates of phototherapy across pre- and post-intervention groups and among DAT negative newborns. There was an increased proportion of newborns assigned to the low risk curve after the intervention. There were no significant differences in phototherapy rates among the intervention groups, although there was a non-significant decrease in phototherapy rates among DAT negative newborns after the intervention. There were no increases in adverse outcomes. Providers adhered to the guidelines after implementation of the HCP. ABOi DAT negative newborns can be viewed as low risk for hyperbilirubinemia requiring phototherapy.

A single-center, retrospective analysis of 17 cases of hemolytic disease of the fetus and newborn caused by anti-M antibodies.

We aimed to summarize the laboratory findings and clinical features of hemolytic disease of the fetus and newborn (HDFN). We retrospectively analyzed the data for 17 infants with anti-M-induced HDFN (anti-M-HDFN) diagnosed between June 2013 and May 2019. Their maternal history, neonatal diagnosis on admission, and laboratory test results were compared with those of 15 infants with HDFN involving the ABO blood group system, 15 infants with HDFN involving the Rh system, and 15 premature infants. In the anti-M-HDFN group, 94.12% (16/17), 35.29% (6/17), and 17.65% (3/17) had free antibodies in plasma, a positive direct antiglobulin test, and a positive elution test, respectively. In 12 infants, free antibody reactions were stronger at 4°C than at 37°C, and the antibody titer at 4°C ranged from 1 to 512. All 17 infants with anti-M-HDFN developed anemia: 14 were treated with blood transfusion and 1 with neonatal exchange transfusion. Sixteen infants improved, and one died. Anti-M-HDFN had a higher rate of maternal stillbirth, lower gestational age, lower birthweight, and higher incidence of respiratory distress than other HDFN types. Anti-M may cause HDFN. It may present with varying degrees of anemia, low regenerative anemia, and low bilirubin levels. In addition, infants with anti-M-HDFN may have a negative elution test and direct antiglobulin test. These tests are helpful in examining antibody responses at a low temperature of 4°C.

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.

Minor Cross-Matching in the Diagnosis of Pneumococcal Hemolytic Uremic Syndrome in an 18-Month-Old Boy.

In developing nations, limitations in diagnostic facilities act as a barrier for differentiation of hemolytic uremic syndrome (HUS) based on the etiology. A sick-looking 18-month-old boy presented to our hospital in Bhubaneswar, India, with clinical signs and symptoms of left lobar pneumonia, abnormal hematological and renal parameters, no growth in blood culture, a negative direct antiglobulin test (DAT) result, and low complement levels. A rapid deterioration in his clinical condition necessitated intensive care support, blood transfusion, and renal replacement therapy (peritoneal dialysis and hemodialysis). Because his health care team suspected atypical HUS, therapeutic plasma exchange (TPE) was initiated as soon as possible. In the absence of a lectin panel, minor cross-matching confirmed T-antigen exposure. With a diagnosis of HUS induced by Streptococcus pneumoniae (sp-HUS), TPE was stopped immediately, and washed blood components were administered. Despite the aforementioned measures, the boy died of HUS on day 20 after presentation. This case emphasized the role of minor cross-matching in the detecting of polyagglutination in resolving the diagnostic dilemma of sp-HUS.

Atypical Hemolytic Uremic Syndrome Precipitated by Recurrent Pancreatitis

Atypical hemolytic uremic syndrome presents with negative direct antiglobulin test microangiopathic hemolytic anemia, acute kidney injury, and thrombocytopenia in the absence of diarrhea. Atypical hemolytic uremic syndrome may be triggered by drugs, infections, systemic lupus erythematosus, or, rarely, pancreatitis. Furthermore, recurrent hemolytic uremic syndrome should raise suspicion for complement-mediated atypical hemolytic uremic syndrome treated with eculizumab, an anti-C5 monoclonal antibody. Here, we report a case of complement-mediated atypical hemolytic uremic syndrome likely precipitated by recurrent alcohol-induced pancreatitis.

Rare case of self-limiting haemolysis associated with Dengue fever in a Sri Lankan female – case report

A 61-year old female with dengue haemorrhagic fever developed anaemia and rising transaminase levels on the 6th day of illness. She was found to have haemolysis with a negative direct antiglobulin test (DAT), and no red cell fragmentation. She recovered with supportive care. Haemolysis with associated Haemolytic uraemic syndrome (HUS), Disseminated intravascular coagulation (DIC) and cold Auto-immune haemolytic anaemia (AIHA) is reported previously; DAT negative haemolytic anaemia associated with dengue fever has not. This case suggests a need for further studies on other immune mechanisms that can lead to haemolysis with dengue fever.

Positive Direct Antiglobulin Test: Is It a Risk Factor for Significant Hyperbilirubinemia in Neonates with ABO Incompatibility?

ABO blood group (ABO) incompatibility is a common cause of neonatal indirect hyperbilirubinemia. The direct antiglobulin test (DAT) can identify infants developing hemolytic disease. This study aims to evaluate the significance of DAT positivity among neonates with ABO incompatibility. This retrospective study included 820 neonates with blood group A or B who were born to blood group O mothers. The study group consisted of neonates (n = 79) who had positive DAT, and the control group consisted of infants (n = 741) who had negative DAT. Demographic and clinical data of the neonates regarding jaundice were collected and compared statistically. The bilirubin level at 24 hours of life (study group: 8 ± 2.6 mg/dL, control group: 6 ± 2.2 mg/dL, p < 0.001) and the highest bilirubin level (study group: 12.7 ± 3.6 mg/dL, control group: 10.4 ± 4.2 mg/dL, p < 0.001) were higher in infants with positive DAT. A total of 37 (46.8%) infants in the study group and 83 (11.2%) infants in the control group received phototherapy (PT) in the nursery (p < 0.001). In neonates with positive DAT, direct bilirubin level, duration of hospitalization, and PT in the nursery were higher (p = 0.002, <0.001, and <0.001, respectively), whereas hemoglobin level was lower (p < 0.001). In neonates with ABO incompatibility, a positive DAT is a risk factor for developing significant hyperbilirubinemia. Close follow-up of newborn infants with ABO incompatibility is crucial for early detection and treatment of neonatal jaundice to avoid early and late complications. · The clinical spectrum of ABO incompatibility varies widely.. · The ABO incompatibility with positive DAT are at greater risk for high bilirubin levels.. · Infants with blood group incompatibilities must be monitored closely..

Bortezomib As Frontline Therapy in the Management of TTP

IntroductionThe current International Society on Thrombosis and Hemostasis (ISTH) guideline of Thrombotic Thrombocytopenic Purpura (TTP) recommends Therapeutic Plasma exchange (TPE) and corticosteroid in the management of TTP, with Rituximab and Caplasizumab as additional potential therapies [X Long Zheng et al, J Thromb Haemost. 2020;18:2496-2502]. This potentially fatal condition requires prompt diagnosis and treatment but large volume plasma treatment is not without risk and in settings with limited access with sufficient compatible plasma for apheretic exchange, this can delay this emergency therapy. We have been running the first therapeutic apheresis services with an Optia instrument in Uganda at the Joint Clinical Research Centre in Kampala city and TTP is the main indication for TPE. We present 2 recent sequential cases of TTP that were successfully managed when Bortezomib was combined with plasma therapy.Case1.A 25yr woman referred to our service on 29/09/2020, with recent onset of bruising, icterus, severe anemia Hgb5.7g/dL, Severe thrombocytopenia 8 x 10 3/uL, a high LDH 3909U with schistocytes on the peripheral blood film severe thrombocytopenia and a negative Direct Antiglobulin test. She was Blood Group B+. She was managed with 3 sessions of TPE of 2.1, 1.1 and 2.0 plasma volume exchanges with 6727mL, 3026mL and 5459mL of replacement plasma respectively and oral prednisone 60mg daily without remission. Subsequent quantities of plasma were insufficient for apheresis and were instead transfused. She also received weekly Rituximab 500mg (IV) but without significant recovery in the platelet count until she received Bortezomib 2mg (SC) when there was corresponding recovery of the platelet count and with each dose to a total of 4 doses (Figure 1).Case2.A 55yr man was previously well and shopping in a mall when he had an index episode of seizures on 19.Jun.2021 and was hospitalized that day with altered consciousness and focal motor signs. He required intubation for 3 days and at the time of hospitalization, he had Hgb 9gm/dL, PLT 17 x 10 3/uL, LDH2177U, with numerous schistocytes on the peripheral blood film and a negative Direct Antiglobulin Test. The baseline blood sample functional ADAMTS13 was 3% and had Blood Group AB+. He had received the 1 st dose of the Astra-Zeneca Covid19 vaccine on 06.May.2021. It was not possible to get any AB plasma and we considered it risky to perform large volume TPE with non-AB plasma and he was instead treated with daily transfusion with Group A+ Plasma concurrent with daily Prednisone 60mg. Because of our previous experience of poor response to Rituximab, we opted to treat him upfront with 4 doses of 2mg bortezomib (SC) given 3 days apart. He made brisk recovery to complete remission and was discharged for weekly outpatient CBC and LDH monitoring. His platelet count subsequently dropped without a corresponding drop in the Hgb and no rise in the Hgb. We initially re-hospitalized him for 4 more days of plasma transfusion but without a brisk response and since the LDH was not increasing, the isolated Thrombocytopenia was considered a side effect of Bortezomib that was expected to resolve and he receive no further treatment but continued to recover to complete remission as an out-patient (figure 2).Discussion:Our findings suggest a possible role for Bortezomib in frontline therapy for TTP with potential to reduce the plasma requirement in TPE and this approach warrants a randomized clinical trial. [Display omitted] DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:Bortezomib Indicated in the management of Multiple Myeloma and Plasma Cell Dyscrasia

Real World Use of Narsoplimab in a Case of Severe HSCT-TMA

Introduction: Hematopoietic stem cell transplantation (HSCT) offers a curative approach for hematologic malignancies. However, endothelial injury from HSCT activates the lectin pathway of complement, resulting in complications such as thrombotic microangiopathy (TMA). HSCT-TMA causes significant mortality. Incidence is reported up to 39%, although lack of consensus on diagnostic criteria results in under-recognition. Risk factors for HSCT-TMA include calcineurin inhibitors (CNI), conditioning, and infection. There is currently no approved treatment for HSCT-TMA.Narsoplimab is a monoclonal antibody that inhibits MASP-2, the effector enzyme of the lectin pathway. We report a patient with late-onset HSCT-TMA who was successfully treated with narsoplimab via a compassionate use program.Methods and Results: A 60-year-old woman with de novo acute myeloid leukemia t (9;11) (p22; q23) who achieved first complete remission (CR1) with minimal residual disease positivity confirmed by flow cytometry (0.04%) underwent matched related allogeneic HSCT from her sister. Patient was cytomegalovirus (CMV) seropositive and blood group O, whereas donor was CMV seronegative and blood group B. Patient received induction with 7+3 and HiDAC consolidation, achieving CR1. Conditioning regimen was Bu4Flu with graft versus host disease (GVHD) prophylaxis of mini methotrexate and tacrolimus.Letermovir was used for CMV reactivation prophylaxis until Day +100. Post-transplant maintenance therapy with azacitidine 36 mg/m 2 was administered by four 5-day cycles (de Lima Cancer 2010). Patient achieved completed donor myeloid CD33 chimerism at Day +30, but donor lymphoid CD3 chimerism was not complete until Day +239. Tacrolimus was discontinued at Day +180. Patient initially had red cell aplasia but achieved transfusion independence by 6 months post-transplant.Circa achieving complete donor CD3 chimerism, patient developed extensive skin and gastrointestinal-overlap GVHD requiring admission for high-dose steroid therapy and resumption of tacrolimus. Onset of GVHD precluded stopping CNI. Patient became steroid refractory and ruxolitinib was added for immunosuppression with resolution of symptoms.Four weeks later, patient developed CMV reactivation and started induction therapy with valganciclovir, during which time she became red cell-transfusion dependent. Patient required 3 weeks induction and 10 days maintenance to clear CMV viremia.Subsequently, she reported dark red urine with evidence for hemoglobinuria without viruria, bilateral leg edema unresponsive to diuretic therapy, and rising serum lactate dehydrogenase (LDH). Evaluation revealed negative direct antiglobulin test, schistocytes on peripheral blood smear (~5/hpf), elevated sC5b-9 levels of 334 ng/mL (72-244 ng/mL), and elevated random urine protein of 124 mg/dL indicative of end organ damage. LDH was modestly elevated 302 IU/L (84-246 IU/L) and haptoglobin was elevated. Based on clinical symptoms and lab results, she was diagnosed with late-onset HSCT-TMA on Day +282 (9 months) using published diagnostic criteria proposed by Cho (2010) and Jodele (2014).The patient's clinical symptoms worsened with severe leg edema - which impeded her ability to walk - and red cell and platelet transfusion dependency. With no approved therapies, compassionate use of narsoplimab was requested for this clinical course of HSCT-TMA. Treatment commenced on Day +309.Patient received a total of 21 doses of narsoplimab 370 mg administered twice weekly on an outpatient basis. All infusions were well tolerated. No adverse events were noted with narsoplimab. Peripheral counts, LDH, anemia, thrombocytopenia and sC5b-9 normalized with reduction in proteinuria, reflecting resolution of HSCT-TMA by dose 19 (10 weeks of treatment); two additional doses were administered (Figure 1). Narsoplimab was given concomitantly with tacrolimus. Patient became transfusion independent and leg edema resolved.Conclusions: This report presents a real-world case study of HSCT-TMA treated with narsoplimab via compassionate use. HSCT-TMA resolved after 10 weeks of treatment, as evidenced by peripheral counts, normalization of LDH and sC5b-9, reduction in urinary protein reflecting improvement in renal function, and improvement in clinical symptoms. Narsoplimab was effective in this complicated case despite several risk factors for poor outcomes. [Display omitted] DisclosuresPantin: Omeros Corporation: Honoraria. Billy: Omeros Corporation: Current Employment. OffLabel Disclosure:Off-label use of azacitidine for post-transplant maintenance therapy

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.

The Spectrum of Hemolytic Disease of the Newborn: Evaluating the Etiology of Unconjugated Hyperbilirubinemia Among Neonates Pertinent to Immunohematological Workup.

Background and objectiveThe exact burden of hemolytic disease of the newborn (HDN) attributed to neonatal unconjugated hyperbilirubinemia (NUH) in developing nations is still unclear. Still, anti-D is reported to be the most common cause of HDN in India. ABO incompatibility has emerged as a leading cause of exchange transfusion (ET) in many countries. But many centers in our country rely on direct antiglobulin test (DAT) as a screening tool to evaluate immunological causes, whereas advanced immunohematological workup like antibody screening, identification, and elution tests are also required. Early identification of implicated antibodies resulting in HDN can aid in the proper selection of blood units when ET is indicated, and hence also in managing the subsequent pregnancy. This study focused on determining the causes of neonatal hyperbilirubinemia (NH), especially with respect to immunohematological evaluation. This cross-sectional study was conducted on 240 neonates requiring neonatal intensive care unit (NICU) support for NUH at a tertiary care hospital.Materials and methodsDemographic data, along with detailed history pertaining to the cause of hyperbilirubinemia, was collected. Clinical and laboratory evaluation and complete immunohematological work including DAT, heat elution, antibody screening, antibody identification, and Rh Kell phenotyping were performed from neonate blood samples. Data were analyzed using SPSS Statistics version 19 (IBM Corp., Armonk, NY).ResultsPathological jaundice was more common (62.1%) than physiological jaundice (37.9%). The various pathological causes identified were HDN (42.6%), sepsis (12%), cephalohematoma (5.4%), and idiopathic (1.7%). Among HDN cases, ABO incompatibility (39.2%) was the most prevalent cause, followed by Rh HDN and G6PD deficiency (1.7% each). DAT was positive in only 14 cases out of 94 ABO-incompatible cases. Elution revealed antibodies in four DAT-negative neonates with ABO incompatibility and more specificity to the OA setting. DAT was positive with 100% sensitivity in Rh HDN cases (n=4). Elution demonstrated the presence of anti-D (n=2), anti-D + anti-C (n=1) and anti-E (n=1), confirming Rh HDN. DAT strength was found to be significantly associated with hemoglobin (Hb) level (p=0.048). The majority of cases were treated with phototherapy only (94.1%), and 10 cases received both ET and phototherapy. Four neonates' condition improved without any intervention.ConclusionThis study highlighted the shift in the trend from Rh HDN to ABO incompatibility as the cause of hemolytic jaundice in NICU neonates. Elution tests can aid in the diagnosis of DAT-negative ABO-incompatible hemolytic anemia. Early diagnosis, along with timely intervention and appropriate measures, can prevent neonatal morbidity and mortality. Negative DAT does not rule out HDN. Sensitive techniques like elution must be used in the presence of clinical suspicion.

Red cell alloimmunization in haemato‐oncology patients transfused with packed red blood cells extended phenotype matched for Rh and Kell antigens versus the standard crossmatched units

Background and ObjectivesThere is limited evidence regarding the advantage of transfusing extended phenotype‐matched (EPM) packed red blood cells (PRBCs) to prevent alloimmunization in haemato‐oncology patients. This randomized control trial aimed to determine the alloimmunization in haemato‐oncology patients transfused with PRBCs which are EPM for Rh (C, E, c, e) and Kell (K) antigens (group I) versus those receiving standard compatible PRBCs (group II).Materials and MethodsPatients having a negative antibody screen (ABS), direct antiglobulin test (DAT) and autologous control (AC) were randomly allocated to either group I or II (n = 50 each). A follow‐up (FU) testing post‐transfusion was conducted for haemoglobin (Hb), ABS and DAT at 1 week, 1 month and 3 months. ABS was conducted using fully automated immunohaematology analyser, while DAT, compatibility testing and extended phenotyping were conducted manually using column agglutination technique.ResultsThere was no significant difference between the two groups with respect to mean (±SD) Hb at initial enrolment (group I, 5·06 ± 1·29 g/dl and group II, 5·41 ± 1·37 g/dl; P = 0·179), age (40 ± 17·3 years v/s 42·2 ± 17·2 years; P = 0·523), gender (P = 0·835) and diagnosis (P = 0·06). All patients in group I and II had negative ABS, DAT and AC at all three FUs. Group II patients were transfused more PRBCs (372 v/s 187, P = 0·000), while the mean Hb increment between successive FU was significant in both the groups (P = 0·003).ConclusionIt appears that there is no added benefit of providing EPM PRBCs in haemato‐oncology patients, as none developed RBC alloantibodies.

Systematic analysis of direct antiglobulin test results in post-artesunate delayed haemolysis

BackgroundPost-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.MethodsArticles 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.ResultsTwenty-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.ConclusionsIn 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.