Case Report: First Case of Cefotaxime-Sulbactam-Induced Acute Intravascular Hemolysis in a Newborn With ABO Blood Type Incompatibility by the Mechanism of Non-Immunologic Protein Adsorption

BackgroundABO blood type incompatibility hemolytic disease of newborn (ABO-HDN) and drug-induced immune hemolytic anemia (DIIHA) due to non-immunologic protein adsorption (NIPA) mainly cause extravascular hemolysis. All the reported severe DIIHA were caused by drug-induced antibodies, and rare report of acute intravascular hemolysis was caused by the NIPA mechanism or ABO-HDN.Case presentationWe report the first case of acute intravascular hemolysis induced by cefotaxime sodium - sulbactam sodium (CTX - SBT) in a case of ABO-HDN which resulted in death at 55 h after birth. The mother’s blood type was O and RhD-positive, and the newborn’s blood type was B and RhD-positive. No irregular red blood cell (RBC) antibodies or drug-dependent antibodies related to CTX or SBT was detected in the mother’s plasma and the plasma or the RBC acid eluent of the newborn. Before the newborn received CTX - SBT treatment, the result of direct antiglobulin test (DAT) was negative while anti-B was positive (2 +) in both plasma and acid eluent. After the newborn received CTX - SBT treatment, the results of DAT for anti-IgG and anti-C3d were both positive, while anti-B was not detected in plasma, but stronger anti-B (3 +) was detected in acid eluent. In vitro experiments confirmed that NIPA of SBT promoted the specific binding of maternal-derived IgG anti-B to B antigen on RBCs of the newborn, thereby inducing acute intravascular hemolysis.ConclusionThe NIPA effect of SBT promoted the specific binding of mother-derived IgG anti-B in newborn’s plasma to the newborn’s RBC B antigens and formed an immune complex, and then activated complement, which led to acute intravascular hemolysis. Drugs such as SBT with NIPA effect should not be used for newborns with HDN.

An Atypical Case of Congenital Erythropoietic Porphyria

Congenital erythropoietic porphyria (CEP, OMIM #606938) is a severe autosomal recessive inborn error of heme biosynthesis. This rare panethnic disease is due to a deficiency of uroporphyrinogen III synthase (or cosynthase). Subsequently, its substrate, the hydroxymethylbilane is subsequently converted into uroporphyrinogen I in a non-enzymatic manner. Of note, uroporphyrinogen I cannot be metabolized into heme and its accumulation in red blood cells results in intramedullary and intravascular hemolysis. The related clinical symptoms occur most frequently during antenatal or neonatal periods but may also appear in late adulthood. The main antenatal clinical presentation is a non-immune hydrops fetalis. We report here two cases of antenatal CEP deficiency and a review of the reported cases in the literature.

Intravascular Hemolysis after Transcatheter Closure of a Huge Tubular Patent Ductus Arteriosus Using Cera-duct Occluder: Managed Successfully by Conservative Treatment

Isolated patent ductus arteriosus (PDA) is seen in approximately 1 in 2000 full-term life birth neonate. Transcatheter device closure is widely considered as the treatment of choice for the patient diagnosed with PDA considering the safety, efficacy and less invasiveness. Residual flow following transcatheter device closure of PDA can result in haemolysis. Our patient was a case of 9.5 years old female child weighing13 kg diagnosed as a case of large tubular PDA with severe pulmonary hypertension by echocardiography. The patient had history of recurrent chest infection, breathlessness on exertion, and growth failure. The PDA was closed by transcatheter approach using Cera-duct occluder. The patient subsequently developed hemolysis which started 18 hrs after the intervention and the resolution of hemolysis achieved by conservative management within 72 hours. JAFMC Bangladesh. Vol 16, No 2 (December) 2020: 90-92

Heme Induced Progressive Loss of Endothelial Protein C Receptor Promotes Development of Chronic Kidney Disease in Sickle Cell Disorders

Chronic kidney disease (CKD), widespread among the individuals with sickle cell disease (SCD), is a major contributor to early death in this patient population. The progressive deterioration of renal health in SCD is associated with chronic persistent intravascular hemolysis leading to anemia. We have previously reported that extracellular heme, released during acute intravascular hemolysis triggers clinically relevant acute kidney injury (AKI) in SCD mice (SS) (Blood (2020) 135 (13): 1044-1048). Although AKI is reversible, it is considered as a risk factor for CKD. The mechanistic approach elucidating the hemolysis driven pathogenesis of AKI-to-CKD transition in SCD is unknown. We found that CKD develops in the SS mice by progressive (1-10 months of age) increase in albuminuria (urinary albumin and creatinine ratio, uACR) and decrease in glomerular filtration rate (GFR) (n=5; p<0.001). Histopathology of the kidney showed age-dependent deterioration in renal peritubular vascular congestion in SS mice compared to that of the AA mice. Alongside, Evan's blue extravasation experiments showed that the SS mice are susceptible to vascular leakage that is correlated positively with age (Pearson r=0.98, p<0.001) and negatively with anemia (Pearson r= -0.46, p<0.05). We hypothesized that multiple acute hemolytic events may instigate persistent endothelial damage that ensues CKD development in SCD. To test this hypothesis, we intravenously infused vehicle or heme (14 μmoles/kg body weight; 5 times on alternate days) to 1-month old SS mice and monitored for 3 weeks following first heme injection. These mice developed severe albuminuria (n=5; p<0.01) with substantial loss of GFR (n=5; p<0.001), indicating heme induced CKD development. Next, we used ultrasound super-resolution (USR) imaging technology to determine renal microvascular changes in older SS mice (6-months) without heme challenge and in young SS mice (1-month) challenged with heme. Analysis of the USR data showed reduced renal blood volume (rBV) and substantial loss of vessel density in renal cortex as well as in corticomedullary areas of the older SS mice compared to the age-matched AA mice. Accordingly, multiple heme challenge reduced rBV and vessel density extensively in young SS mice comparable to the older SS mice without heme challenge. Since endothelial protein C receptor (EPCR) maintains vascular barrier integrity by activating protease activated receptor-1 (PAR1) signaling in the endothelium, we tested whether alterations in EPCR expression contribute to progressive endothelial damage in SS mice during CKD development. Using immunofluorescence microscopy, we determined that renal endothelium lacks expression of EPCR in older SS mice while younger SS mice retains EPCR cellular expression. In corroboration, infusion of heme in younger SS mice results in loss of renal endothelial EPCR. Shedding of EPCR from endothelium often results in a soluble form of EPCR (sEPCR). We found that SS mice had higher plasma levels of soluble EPCR (sEPCR) compared to their AA counterparts. While age dependent increase in plasma and urinary sEPCR were evident in SS mice (n=6; p<0.01), infusion of heme in younger SS mice results in significant increase in plasma sEPCR (n=6; p<0.01). In consistent with the mouse data, we discovered that the plasma sEPCR was significantly elevated in SCD patients compared to normal individuals (n=8-16; p<0.05). Moreover, the plasma sEPCR was significantly associated with the baseline albuminuria in a cohort of SCD patients (n=16; Pearson r=0.64; p<0.01). This study supports the conclusion that multiple hemolytic events may trigger CKD development in SCD by gradual loss of renal microvascular EPCR expression. Clinically, the sEPCR can be developed as risk factor for sickle CKD. Finally, our data suggest that restoration of EPCR function may protect SCD patients from CKD. Disclosures No relevant conflicts of interest to declare.

Complement Activation during Vaso-Occlusive Pain Crisis in Pediatric Sickle Cell Disease

Background: Sickle cell disease (SCD) affects millions of individuals worldwide with substantial morbidity and mortality. The sickle hemoglobin (HbS) polymerizes upon deoxygenation, causing rigid and adhesive red blood cells (RBCs), triggering vascular occlusion, greatly shortened RBC lifespan, and chronic hemolysis. Amongst acute complications in SCD, vaso-occlusive pain crisis (VOC) is the leading cause of hospitalization, with supportive care being the primary approach to management. We and others have recently demonstrated important contributions of complement to the pathophysiology of SCD. When the complement pathway (CP) is activated during SCD crises, inhibition at C5 using eculizumab, has been successful in treating various acute complications in SCD (Chonat et al, Haematologica). In this study, we prospectively analyzed the extent of CP activation among children with SCD presenting with VOC. Methods: Patients aged 0-21 years old managed at Children's Healthcare of Atlanta with homozygous sickle cell (SS) or S beta zero thalassemia genotypes were enrolled in an IRB-approved research study. Inclusion criteria included those requiring intravenous opioids for VOC, and excluded those with chronic pain, >6 VOC admission in the previous 12 months or on chronic transfusions. Blood samples were collected within 48 hours of VOC presentation, and steady-state levels were obtained at a 4-week clinic follow-up. Data was analyzed using a paired t-test, and receiver operator characteristic (ROC) curves were generated comparing intra-person complement levels during acute VOC versus respective steady-state levels. Results: Sixty-four patients have been enrolled thus far, of which 43 (67%) had steady-state samples collected. The majority of patients (90.5%) have SS genotype with a mean (SD) patient age of 14.15 (4.68) years. Fifty-three (84.1%) patients reported taking hydroxyurea (HU). Fifty-nine (93.7%) patients had at least one VOC admission in the past 12 months, with an average of 2.98 (1.67) VOC admissions. Pain Score reported on 55 patients averaged 4.93 (4.78) on a pain scale of 0 to 10. Mean values during VOC and steady-state of hemoglobin (Hb) were 8.12 and 9.01 g/dL, platelet count 431 and 511, and lactate dehydrogenase (LDH) 549 and 483 U/L, respectively. Seventeen patients had complement work-up performed during acute and steady-state, and 4 of them had additional samples collected during subsequent VOC. Complement protein levels C3, C4, C5, properdin, factor B, and complement regulatory proteins factor H and I were unremarkable during VOC and steady-state. However, complement activation markers, specifically anaphylatoxins C3a, C5a and Bb were significantly elevated during VOC compared to steady-state (see Table 1) suggesting activation of alternative CP during VOC (see Table 1 and Figure 1A-C). Terminal complement complex (C5b9) was not statistically different between VOC and steady-state (Figure 1D, red dotted lines signify normal ranges). Remarkably, patients who re-presented with acute VOC exhibited similar increases in their C3a/C5a (Figure 1E-F), substantiating the increases related to their VOC. Hemoglobin and LDH (Figure 1G-H) were similarly significant, suggestive of intravascular hemolysis. Three (7.1%) patients developed acute chest syndrome, two of whom experienced respiratory failure requiring intensive care management, and all exhibited significant CP activation. The area under the curve (AUC) of the ROC curve was analyzed to determine the ability of complement biomarkers to differentiate VOC from steady-state. Based on the AUC of these biomarkers, complement anaphylatoxins C3a and C5a exhibited the highest AUC of 0.76 and 0.87, respectively. Discussion: To our knowledge, this is the first prospective and comprehensive evaluation of CP in patients with SCD during VOC and steady-states. These preliminary findings suggest CP activation is present in a large proportion of patients during VOC, with increased activation of alternative and common CP, associated with intravascular hemolysis. Minimal increase in C5b9 could be explained by a significant proportion (> 80%) of our patients being on HU therapy, similar to prior data (Roumenina et al, AJH). Specifically, C3a/C5a, along with other biomarkers, could not only predict disease activity in patients during VOC, but provide pharmacological targets in VOC, which need further validation. Figure 1 Figure 1. Disclosures Stowell: Alexion: Consultancy; Argenx: Speakers Bureau; Grifols: Speakers Bureau. Chonat: Alexion: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Takeda: Consultancy, Research Funding.

Pozelimab, a Human Monoclonal Antibody Against Complement Factor C5, Provided Inhibition of Intravascular Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinuria

INTRODUCTION Paroxysmal Nocturnal hemoglobuinuria (PNH) is a rare, acquired, life-threatening disorder characterised by intravascular hemolysis and increased risk of thrombosis. Current available treatments represent a significant burden to patients and include anti-complement factor 5 (C5) therapies, eculizumab and ravulizumab, both intravenous infusions, or more recently, twice weekly C3 inhibitor, Pegcetacoplan, a subcutaneous (SC) infusion treatment. Pozelimab (REGN3918), a fully human monoclonal immunoglobulin G4 antibody directed against C5, has been shown to bind with high affinity to wild-type and variant (R885H/C) human C5 and block its activity. In a healthy volunteer study (NCT03115996), SC administration of pozelimab was found to be generally well tolerated while providing complete inhibition of ex vivo-assessed hemolytic activity [Weyne J et al. Blood. 2018;S1:1039]. The data suggested that pozelimab may provide control of intravascular hemolysis in patients with active PNH, and thus could provide an important new alternative for patients supporting progression to a first-in-patient study of pozelimab in patients with active PNH. OBJECTIVE To demonstrate a clinically significant reduction in intravascular hemolysis by once-weekly SC administration of pozelimab over 26 weeks of treatment in patients with active PNH. METHODS This is a phase 2, open-label, single-arm, 26-week treatment study in 24 patients with active symptomatic PNH who are naïve to complement inhibitor therapy, or who have received prior treatment with a complement inhibitor but not within 6 months prior to the start of the study (NCT03946748). The treatment regimen consists of pozelimab as a single IV loading dose of 30 mg/kg followed thereafter by weekly SC 800 mg administrations. The effect of pozelimab on intravascular hemolysis (monitored via LDH levels) and transfusion avoidance, as well as safety, is to be assessed from baseline to week 26 (study day 183; only partial data were available for some patients at the time of abstract submission). Pozelimab pharmacodynamics was assessed utilizing a sheep red blood cell (RBC) complement activity assay (CH50) and rabbit RBC complement activity assay (AH50). RESULTS All 24 patients completed the 26-week treatment period with no study drug discontinuation and have been enrolled into an ongoing open-label extension study (NCT04162470). An interim analysis on the first 17 patients was previously reported. All 17 patients had at least 71 days of treatment, with 10 patients receiving treatment for up to 183 days. All enrolled patients had baseline LDH levels ≥2 x upper limit of normal (ULN). Participants were enrolled in two cohorts: Cohort A for dose confirmation and Cohort B for further evaluation of efficacy and safety. Interim Efficacy Analysis As previously reported, treatment with pozelimab led to a rapid and sustained reduction in LDH through study week 26. All 17 patients achieved LDH reduction to below the clinically significant threshold of LDH ≤1.5 x ULN. All but one patient achieved control of intravascular hemolysis (LDH ≤1.5 x ULN) at week 2, and all but one patient achieved normalization of LDH (LDH ≤1.0 x ULN) at week 4. Importantly, one patient who is a carrier of a C5 variant known to be resistant to blockade by eculizumab/ravulizumab demonstrated rapid and sustained normalization of LDH. Interim Safety As previously reported, no serious adverse events or adverse events leading to treatment discontinuation were reported. Common treatment-emergent adverse events included headache (4 patients; 23.5%) and nausea (2 patients; 11.8%). No breakthrough hemolysis events were observed. CONCLUSIONS Pozelimab administered SC once weekly led to the inhibition of intravascular hemolysis in patients with active PNH and was generally well-tolerated. An update to the previously reported interim analysis will be provided. Upon successful completion of the 26-week treatment period, patients are enrolled into an open-label extension study. Disclosures Weyne: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Chaudhari: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Harari: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Miller: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Dain: Regeneron Pharmaceuticals, Inc.: Other: Contractor. Meagher: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Rodgers: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Perlee: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Morton: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Weinreich: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Yancopoulos: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Griffin: Alexion, AstraZeneca Rare Disease: Honoraria, Membership on an entity's Board of Directors or advisory committees; BioCryst Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sobi Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis: Other: Educational grant support.

Clinical Profile and Long-Term Outcomes of Patients with Paroxysmal Nocturnal Hemoglobinuria Treated with Eculizumab in a Real-World Setting: High Frequency of Anemia Despite Decreased Intravascular Hemolysis

Background: Eculizumab, an anti-C5 antibody, was approved for the treatment of patients (pts) with symptomatic paroxysmal nocturnal hemoglobinuria (PNH) in 2007 and has been the standard of care for over a decade. However, published data on real-world outcomes of eculizumab-treated pts with PNH are limited. The aim of this study was to describe the clinical profile of pts with PNH treated with eculizumab by characterizing their short- and long-term laboratory and clinical outcomes. Methods: This retrospective study (Versmold et al, Blood 2020) used preexisting medical records of eculizumab-treated pts with PNH (treatment duration ≥24 weeks [wks]) treated at the University Hospital Essen, Germany prior to April 2018. Anonymized data were collected via electronic case report forms. Laboratory data were extracted from the hospital computer system. Lactate dehydrogenase (LDH), hemoglobin, absolute reticulocyte count (ARC), and bilirubin profiles were assessed at baseline (12 months before treatment) and during the treatment phase (up to 13.2 years [yrs] follow-up). Breakthrough hemolysis (BTH) was defined as ≥1 new symptom or sign of intravascular hemolysis (including fatigue, hemoglobinuria, abdominal pain, dyspnea, anemia [hemoglobin <10 g/dL], major adverse vascular event [including thrombosis], dysphagia, or erectile dysfunction in the presence of elevated LDH [≥2 × the upper limit of normal (ULN)] after reduction of LDH to ≤1.5 × ULN). Extravascular hemolysis was defined as persistence of reticulocytes >100 × 10 9/L with bilirubin >1 × ULN and positive direct Coombs test or reticulocytes >100 × 10 9/L with bilirubin >1 × ULN and ≥1 positive C3c or C3d test. Complete hematologic response was zero blood transfusions with hemoglobin ≥12 g/dL and LDH ≤1.5 × ULN and major hematologic response was zero blood transfusions with hemoglobin ≥12 g/dL and LDH >1.5 × ULN within any 24-wk window (Risitano et al, Front Immunol 2019). Transfusion-dependence was ≥2 blood transfusions within any 24-wk period. Pts transferred from other centers or within 24 wks of treatment were excluded due to missing baseline data. Results: The study included 56 pts with PNH (mean age: 42.9 yrs [± 17.6]; 46.4% female) treated with eculizumab for ≥24 wks (mean follow-up: 5.24 yrs [± 3.25]) during the study period. The median duration from diagnosis to starting eculizumab was 1.57 yrs. Overall, 18 pts (32.1%) had aplastic anemia at diagnosis, 10 (17.9%) had symptoms of high disease activity, and 34 (60.7%) had a blood transfusion in the prior 12 months. The most reported disease-related symptoms at baseline were anemia (28.6%), fatigue (26.8%), thrombosis (21.4%), dyspnea (17.9%), dysphagia (10.7%), erectile dysfunction (10.0%), kidney complications (8.9%), abdominal pain (8.9%), and hemoglobinuria (7.1%). Mean hemoglobin (n=44) was 9.67 g/dL [± 2.06] and LDH in the past 12 months (n=47) was 1480 U/L [± 1010]. During the first 24-wk treatment phase, 37% (20/54) of pts had LDH >1.5 × ULN, 31% (14/45) had ARC >1.5 × ULN, and 17% (8/47) had hemoglobin ≥12 g/dL (Figure). Among pts with response data, 15% (7/47) had complete hematologic response and 2% (1/47) had major hematologic response within 24 wks. Documented BTH with symptoms occurred in 11% (6/56). Moreover, 23% (13/56) of pts were transfusion-dependent, increasing to 39% (22/56) when including pts who had ≥1 transfusion during the first 24 wks of treatment. Six pts (11%) received a higher-than-labeled dose (600 mg intravenous [IV] weekly for 4 wks, 900 mg IV 1 wk later, then 900 mg IV every 2 wks thereafter) of eculizumab. Over the long term (ie, between 25 and 246 wks), 11.1-34.7% of pts received blood transfusions and 7.0-21.7% had LDH >1.5 × ULN in any 24-wk window; whereas 36.1-72.7% had ARC >1.5 × ULN (Figure). Moreover, 65.8-77.3% of pts had hemoglobin <12 g/dL within any 24-wk period and 69.0-77.2% did not meet the criteria for major or complete hematologic response during any 24-wk period from wks 25 to 246. During the treatment phase, no meningococcal infections were reported. Conclusions: In this long-term real-world study, a considerable proportion of pts with PNH treated with eculizumab did not achieve optimal clinical outcomes with an ongoing burden of disease (ie, low hemoglobin level with high reticulocyte count due to extravascular hemolysis, BTH, etc.). Future exploration of other therapies that improve pt outcomes could help to address remaining unmet medical needs. Figure 1 Figure 1. Disclosures Alashkar: Alexion: Honoraria; Novartis: Honoraria; BMS/Celgene: Honoraria; Bluebird Bio: Honoraria. Ofori-Asenso: F. Hoffmann-La Roche Ltd: Current Employment. Xu: F. Hoffmann-La Roche AG: Current Employment. Liu: Genesis Research: Current Employment. Katz: F. Hoffman-La Roche Ltd: Current Employment. Shang: F. Hoffman-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Roeth: Apellis Pharmaceuticals: Consultancy, Honoraria; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Bioverativ, a Sanofi company: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Kira: Consultancy, Honoraria.

A Rare Case of Aluminum Phosphide Induced Thrombotic Thrombocytopenic Purpura

Aluminum phosphide (AlP) has remained a chemical cause of completed suicides in some developing countries. ALP toxicity can cause multi-system damage. As far as we know, this is the first case of ALP-induced Thrombotic Thrombocytopenic Purpura (TTP) and its successful management. A 34-year-old man, who had attempted suicide with ALP was admitted to our hospital. On the 3rd day of admission, the patient developed hematuria, hemolysis, and thrombocytopenia. Based upon available evidence, TTP was diagnosed. Following a complete patient evaluation, ALP was recognized as the probable cause of TTP. Following the treatment using prednisolone and therapeutic plasma exchange, the patient substantially improved. Finally, he was discharged on the 22nd day. Toxin-induced intravascular hemolysis should be considered for patients presenting with ALP toxicity. As reported in this patient, TTP is another manageable consequence of ALP poisoning.