Markers Of Intravascular Hemolysis Research Articles

Use of Carboxyhemoglobin as an Early Sign of Oxygenator Dysfunction in Patients Supported by Extracorporeal Membrane Oxygenation.

IntroductionPlasma free hemoglobin is the gold standard for monitoring hemolysis in extracorporeal membrane oxygenation (ECMO) but its routine use has some limitations. Carboxyhemoglobin (HbCO) is also a marker of intravascular hemolysis. We aimed to investigate HbCO as a marker of both hemolysis and oxygenator dysfunction in patients supported by ECMO.MethodsRetrospective analysis of patients on ECMO in an adult ICU in a tertiary hospital. HbCO was recorded every 6 h in the 48 h before and after oxygenator change in adult patients on ECMO support with an oxygenator dysfunction and replacement.ResultsThe investigation of 27 oxygenators replacements in 19 patients demonstrated that HbCO values progressively increased over time and then significantly decreased after oxygenator change. Median oxygenator lifespan was 14 days [interquartile range (IQR) 8–21] and there was no correlation between HbCO and oxygenator lifespan [Spearman coefficient 0.23 (p = 0.23)]. HbCO values at oxygenator change [HbCO median 2.7 (IQR 2.5–3.5)] were significantly higher than the HbCO values 1 week before [HbCO median 2.07 (IQR 1.86–2.8)] (p value < 0.001).ConclusionOur data highlight the potential role of HbCO as a novel marker for ECMO oxygenator dysfunction.

12-Month Analysis of a Phase 2 Study of Iptacopan (LNP023) Monotherapy for Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hematological disorder caused by somatic mutations in the phosphatidylinositol glycan A (PIGA) gene in hematopoietic stem cells, resulting in complement alternative pathway (AP)-mediated severe hemolysis, life-threatening thrombosis, and impaired bone marrow function. The current standard-of-care for PNH consists of anti-C5 blockade with either eculizumab or ravulizumab. While both monoclonal antibodies effectively control intravascular hemolysis (IVH), reduce thrombosis and improve long-term survival, a significant proportion of patients remains anemic and continues to require transfusions, largely due to persistent extravascular hemolysis (EVH). Conversely, pegcetacoplan, a recently FDA-approved anti-C3 inhibitor, prevents both IVH and EVH and showed superiority to eculizumab in improving hemoglobin (Hb) levels in PNH. Nevertheless, the need for effective oral treatment options in PNH remains unmet. Iptacopan (LNP023) is a new, oral, selective and potent first-in-class inhibitor of factor B, a key component of the AP. Recent phase 2 data showed that iptacopan effectively controls both IVH and EVH and leads to rapid, transfusion-free improvements in Hb levels in the majority of PNH patients.Methods: CLNP023X2204 (NCT03896152) is a multicenter, randomized, open-label phase 2 study in adult PNH patients with active hemolysis and no complement inhibitor treatment within 3 months prior to study entry. Patients are randomized to receive twice daily (BID) iptacopan in one of two dose-sequences, either 25 mg for 4 weeks followed by 100 mg for up to 2 years (Cohort 1) or 50 mg followed by 200 mg (Cohort 2). The key objectives are to assess the effect of iptacopan on markers of IVH/EVH (incl. lactate dehydrogenase (LDH)) and on Hb levels, as well as safety. 12-month interim results are summarized below.Results: The study has completed enrolment. A total of 13 patients (mean age 38.2 years, 7 female) were randomized to either Cohort 1 (n=7) or 2 (n=6). Mean (SD) lab values at baseline were LDH 2097.8 (911.2) U/L, reticulocytes 203.3 (82.9) x10E9/L, bilirubin 32.4 (10.1) µmol/L, and Hb 85.8 (13.2) g/L, and most patients required red blood cell transfusions in the prior 12 months (median 3.0, range 0-19). At the time of the interim analysis, 11 patients had been treated with iptacopan for at least 52 weeks, with a maximum treatment duration of 81 weeks; 2 patients discontinued treatment early, one after 2 days due to a non-serious adverse event of headache (hence not evaluable for the primary endpoint), the other after 13 weeks due to physician decision. Amongst the 12 evaluable patients, all of whom were anti-C5 naive, all reached the primary endpoint of lowering LDH by at least 60% within the first 12 weeks. The LDH response was rapid and durable, with all patients treated with ≥50 mg BID reaching this threshold after only one week of treatment and all ongoing patients except one maintaining the threshold up until the data cutoff, i.e., for at least 52 weeks. Equivalent improvements were also observed for other markers of IVH and EVH. Similarly, Hb levels improved significantly and durably in most patients, and all except one of the ongoing patients have remained transfusion-free since the start of iptacopan treatment. Moreover, no thromboembolic events occurred during the study, and the FACIT fatigue score improved significantly in most patients. Iptacopan monotherapy was safe, with no severe or serious adverse reported up to the data cutoff.Conclusion: Iptacopan is a new, well tolerated oral complement AP inhibitor that blocks both IVH and EVH in adult PNH patients with hemolytic PNH. 12-month results from this non-pivotal study demonstrate that iptacopan monotherapy leads to rapid and durable improvements in various hemolytic markers and meaningful and sustained clinical benefit as seen in improvements in Hb levels, transfusion requirement and FACIT fatigue score. These results suggest that proximal inhibition of the complement cascade parallels and further improves the hematological benefit seen with anti-C5 therapies, paving the way for the phase 3 evaluation of iptacopan as potentially new oral first-line therapy for patients with PNH. [Display omitted] DisclosuresWong: Astellas Pharma, INc.: Research Funding. Li: Novartis: Current Employment, Current equity holder in publicly-traded company. Rozenberg: Novartis: Current Employment, Current equity holder in publicly-traded company. Nidamarthy: Novartis: Current Employment, Current equity holder in publicly-traded company. Chawla: Novartis: Current Employment, Current equity holder in publicly-traded company. Junge: Novartis: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure:Iptacopan (LNP023) is a new, oral, selective and potent first-in-class inhibitor of factor B, a key component of the complement alternative pathway. It is currently being investigated in paroxysmal nocturnal hemoglobinuria (PNH) as well as in several renal indications.

Acute kidney injury is associated with impaired cognition and chronic kidney disease in a prospective cohort of children with severe malaria

BackgroundAcute kidney injury (AKI) is a recognized complication of pediatric severe malaria, but its long-term consequences are unknown.MethodsUgandan children with cerebral malaria (CM, n = 260) and severe malaria anemia (SMA, n = 219) or community children (CC, n = 173) between 1.5 and 12 years of age were enrolled in a prospective cohort study. Kidney Disease: Improving Global Outcomes (KDIGO) criteria were used to retrospectively define AKI and chronic kidney disease (CKD). Cognitive testing was conducted using the Mullen Scales of Early Learning in children < 5 and Kaufman Assessment Battery for Children (K-ABC) second edition in children ≥ 5 years of age.ResultsThe prevalence of AKI was 35.1%, ranging from 25.1% in SMA to 43.5% in CM. In-hospital mortality was 11.9% in AKI compared to 4.2% in children without AKI (p = 0.001), and post-discharge mortality was 4.7% in AKI compared to 1.3% in children without AKI (p = 0.030) corresponding to an all-cause adjusted hazard ratio of 2.30 (95% CI 1.21, 4.35). AKI was a risk factor for short- and long-term neurocognitive impairment. At 1 week post-discharge, the frequency of neurocognitive impairment was 37.3% in AKI compared to 13.5% in children without AKI (adjusted odds ratio (aOR) 2.31 [95% CI 1.32, 4.04]); at 1-year follow-up, it was 13.3% in AKI compared to 3.4% in children without AKI (aOR 2.48 [95% CI 1.01, 6.10]), and at 2-year follow-up, it was 13.0% in AKI compared to 3.4% in children without AKI (aOR 3.03 [95% CI 1.22, 7.58]). AKI was a risk factor for CKD at 1-year follow-up: 7.6% of children with severe malaria-associated AKI had CKD at follow-up compared to 2.8% of children without AKI (p = 0.038) corresponding to an OR of 2.81 (95% CI 1.02, 7.73). The presenting etiology of AKI was consistent with prerenal azotemia, and lactate dehydrogenase as a marker of intravascular hemolysis was an independent risk factor for AKI in CM and SMA (p < 0.0001). In CM, AKI was associated with the presence and severity of retinopathy (p < 0.05) and increased cerebrospinal fluid albumin suggestive of blood-brain barrier disruption.ConclusionsAKI is a risk factor for long-term neurocognitive impairment and CKD in pediatric severe malaria.

Hemoglobin S Induces Exposure of Red Blood Cell Membrane Skeleton Microdomains Bearing Mannose That Stimulate Phagocytosis By Macrophages: A Molecular Basis for Hemolysis in Sickle Cell Disease but Protection Against Plasmodium Falciparum malaria

Heterozygosity for Hemoglobin (Hb) S, sickle cell trait (SCT), affects over 40 million people and confers resistance to severe infection by Plasmodium falciparum. Homozygosity for HbS, or compound heterozygosity with certain other alleles of Hb, affects over 4 million individuals and causes sickle cell disease (SCD). Hemolytic anaemia is a prominent feature of SCD and is mainly extravascular, mediated by hepatic and splenic macrophages. No ligands for this process have been identified. As many macrophage phagocytic receptors recognise carbohydrates, we surveyed surface glycan expression by sickle cells using a panel of 8 lectins and flow cytometry. Most glycans were similar to those of healthy red blood cells (RBC), except much higher expression of terminal mannose.We investigated the structural basis for these residues using glycomic mass spectroscopy, which showed them to be N-linked high (Man5-9GlcNAc2) mannoses, a surprising conclusion as these are usually intermediates in the formation of complex glycans and not displayed on cell surfaces. High resolution microscopy revealed the mannose residues to be carried in discrete microdomains on the surfaces of sickle cells. These structures were absent on the surfaces of healthy RBC, instead being present in the membrane skeleton under the cell surface. Lectin blots and immunoprecipitation showed the mannoses to co-migrate predominantly with spectrin.We showed these mannose-bearing structures were able to stimulate phagocytosis of RBC by using a peripheral blood derived macrophage uptake assay. Sickle RBC were taken up at high rates compared to healthy RBC and this could be inhibited by congeners of mannose. We identified the importance of a cognate ligand (CD206: the mannose receptor) using blocking antibodies and knockdown of CD206 expression using siRNA.The in vivo and pathogenic relevance of mannose exposure was investigated by taking advantage of the heterogeneity of hemolysis in SCD. RBC with SCD (n=94), SCT (n=57) and healthy individuals (n=54) were assayed for mannose exposure by flow cytometry. SCT and healthy RBC showed no mannose exposure but high levels were found on HbSS RBC (p<0.0001). Co-incident inheritance of HbSS with higher HbF values and alleles encoding alpha-thalassaemia resulted in lower surface mannose values. Overall, markers of hemolysis (RBC count, haemoglobin, reticulocyte count) correlated well with mannose exposure (Spearman correlation coefficients -0.68, -0.40, 0.37; p=0.0001, 0.0032, 0.0063 respectively). Plasma LDH is a marker of intravascular hemolysis and correlated with overall hemolysis within SCD (r=-0.25, p=0.016), but not mannose exposure (r=0.14, p=0.19). Thus mannose exposure correlated only with extravascular hemolysis.Identification of a ligand pair mediating rapid clearance of sickle cells raised the possibility that they also mediate enhanced clearance of SCT RBC infected by malarial parasites. Indeed, P. falciparum cultures induced mannose expression at the pigmented trophozoite and schizont stages in infected HbAA RBCs, at levels corresponding to mild hemolysis in SCD. Mannose expression in infected HbAS RBCs was even higher, with levels corresponding to severe hemolysis in SCD.Infection with P. falciparum and selection for HbS arose only recently in human evolution, raising the question of what the physiological triggers for this mechanism are. Infection with malarial parasites causes oxidative stress. We therefore subjected healthy RBC to copper sulphate, which resulted in surface mannose exposure as well as uptake by macrophages. Oxidized SCT RBC displayed more mannose than oxidized healthy RBC.Thus, we have identified a new cell surface ‘eat me’ signalling mechanism that allows inspecting macrophages to engage with the rigid membrane skeleton and phagocytose the mannose displaying cell. The mechanism is stimulated by HbS: when present in high concentrations, the mechanism is activated constitutively, resulting in sickle cell anaemia. Heterozygosity for HbS is insufficient by itself to trigger mannose exposure. However, the mechanism is primed so that oxidative stress associated with infection by P. falciparum causes greater mannose display, increased parasitized cell clearance and protection against severe malaria. These findings should allow the design of inhibitors of sickle cell haemolysis and inducers of protection against malaria. DisclosuresCao:University of Aberdeen: Patents & Royalties. Barker:University of Aberdeen: Patents & Royalties. Vickers:University of Aberdeen: Patents & Royalties; GSK: Equity Ownership.

307: Hemolysis, a severe feature of preeclampsia

The prevalence of hemolysis in hypertensive disorders of pregnancy, independent of hemolysis, elevated liver enzymes and low platelet count (HELLP) syndrome, is unknown. We hypothesize that hemolysis is a severe feature of preeclampsia. Thus, we sought to evaluate levels of serum lactate dehydrogenase (LDH), a marker of intravascular hemolysis, in hypertensive disorders of pregnancy. This is a retrospective cohort study of subjects with a hypertensive disorder of pregnancy, that delivered ≥23 weeks gestation at a single institution (October ‘13 - May ’17). The cohort began with publication of the 2013 American College of Obstetricians and Gynecologists (ACOG) Executive Summary on Hypertension in Pregnancy. Subjects were identified by ICD-9 and ICD-10 discharge codes and charts were then individually reviewed for diagnostic accuracy. Subjects were placed into four groups by ACOG criteria: 1. chronic hypertension (CHTN); 2. gestational hypertension (GHTN); 3. preeclampsia (PE) and; 4. PE with severe features (PE-SF). LDH values were abstracted as the peak value before delivery, with 250 U/L the upper limit of normal. Clinical and laboratory data, and adverse maternal and neonatal outcomes were recorded. Statistical testing included chi-square, t-test and multivariable logistic regression. In the study period, there were 8,645 deliveries and 1,188 (13.7%) subjects with a hypertensive disorder of pregnancy, of which 812 (68.4%) had LDH measurement prior to delivery [CHTN (n=152); GHTN (n=209); PE (n=216); PE-SF (n=235)]. The median, 90th and 95th percentile (%ile) for LDH level in each group is shown in Figure 1. The 95th %ile for CHTN and GHTN is LDH <300 U/L, and for PE is <400 U/L. Those with PE-SF more often had LDH ≥400 U/L compared to other groups (9.8% vs. 2.3%, p<0.001). In those with LDH ≥400 U/L (≥1.6x normal), adverse maternal and neonatal outcomes were significantly increased (Table 1). The odds of any adverse maternal outcome (composite) with LDH ≥400 U/L persisted after adjustment for HELLP [aOR (95% CI), 3.35 (1.59-7.03), p=0.001] or ELLP (elevated liver enzymes, low platelet count only), [3.40 (1.65-7.02), p=0.001]. Our data suggest that LDH ≥1.6x normal, in our case ≥400 U/L, is a severe feature of preeclampsia. This conclusion is made because LDH values ≥400 U/L were uncommon in the absence of severe disease, and when present, increased risk for adverse outcomes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Nitric Oxide-Dependent Endothelial Dysfunction and Reduced Arginine Bioavailability in Plasmodium vivax Malaria but No Greater Increase in Intravascular Hemolysis in Severe Disease.

Pathogenesis of severe Plasmodium vivax malaria is poorly understood. Endothelial dysfunction and reduced nitric oxide (NO) bioavailability characterize severe falciparum malaria, but have not been assessed in severe vivax malaria. In patients with severe vivax malaria (n = 9), patients with nonsevere vivax malaria (n = 58), and healthy controls (n = 79), we measured NO-dependent endothelial function by using reactive hyperemia-peripheral arterial tonometry (RH-PAT) and assessed associations with arginine, asymmetric dimethylarginine (ADMA), and hemolysis. The L-arginine level and the L-arginine to ADMA ratio (a measure of L-arginine bioavailability) were reduced in patients with severe vivax malaria and those with nonsevere vivax malaria, compared with healthy controls (median L-arginine level, 65, 66, and 98 µmol/mL, respectively [P = .0001]; median L-arginine to ADMA ratio, 115, 125, and 187, respectively [P = .0001]). Endothelial function was impaired in proportion to disease severity (median RH-PAT index, 1.49, 1.73, and 1.97 in patients with severe vivax malaria, those with nonsevere vivax malaria, and healthy controls, respectively; P = .018) and was associated with the L-arginine to ADMA ratio. While the posttreatment fall in hemoglobin level was greater in severe vivax malaria as compared to nonsevere vivax malaria (2.5 vs 1 g/dL; P = .0001), markers of intravascular hemolysis were not higher in severe disease. Endothelial function is impaired in nonsevere and severe vivax malaria, is associated with reduced L-arginine bioavailability, and may contribute to microvascular pathogenesis. Severe disease appears to be more associated with extravascular hemolysis than with intravascular hemolysis.

Organ iron accumulation in chronically transfused children with sickle cell anaemia: baseline results from the TWiTCH trial.

Transcranial Doppler (TCD) With Transfusions Changing to Hydroxyurea (TWiTCH) trial is a randomized, open-label comparison of hydroxycarbamide (also termed hydroxyurea) versus continued chronic transfusion therapy for primary stroke prevention in patients with sickle cell anaemia (SCA) and abnormal TCD. Severity and location of iron overload is an important secondary outcome measure. We report the baseline findings of abdominal organ iron burden in 121 participants. At enrollment, patients were young (9·8±2·9years), predominantly female (60:40), and previously treated with transfusions (4·1±2·4years) and iron chelation (3·1±2·1years). Liver iron concentration (LIC; 9·0±6·6mg/g dry weight) and serum ferritin were moderately elevated (2696±1678μg/l), but transferrin was incompletely saturated (47·2±23·6%). Spleen R2* was 509±399Hz (splenic iron ~13·9mg/g) and correlated with LIC (r(2) =0·14, P=0·0008). Pancreas R2* was increased in 38·3% of patients but not to levels associated with endocrine toxicity. Kidney R2* was increased in 80·7% of patients; renal iron correlated with markers of intravascular haemolysis and was elevated in patients with increased urine albumin-creatinine ratios. Extra-hepatic iron deposition is common among children with SCA who receive chronic transfusions, and could potentiate oxidative stress caused by reperfusion injury and decellularized haemoglobin.

Clinical Applications of Hemolytic Markers in the Differential Diagnosis and Management of Hemolytic Anemia

Several hemolytic markers are available to guide the differential diagnosis and to monitor treatment of hemolytic conditions. They include increased reticulocytes, an indicator of marrow compensatory response, elevated lactate dehydrogenase, a marker of intravascular hemolysis, reduced haptoglobin, and unconjugated hyperbilirubinemia. The direct antiglobulin test is the cornerstone of autoimmune forms, and blood smear examination is fundamental in the diagnosis of congenital membrane defects and thrombotic microangiopathies. Marked increase of lactate dehydrogenase and hemosiderinuria are typical of intravascular hemolysis, as observed in paroxysmal nocturnal hemoglobinuria, and hyperferritinemia is associated with chronic hemolysis. Prosthetic valve replacement and stenting are also associated with intravascular and chronic hemolysis. Compensatory reticulocytosis may be inadequate/absent in case of marrow involvement, iron/vitamin deficiency, infections, or autoimmune reaction against bone marrow-precursors. Reticulocytopenia occurs in 20–40% of autoimmune hemolytic anemia cases and is a poor prognostic factor. Increased reticulocytes, lactate dehydrogenase, and bilirubin, as well as reduced haptoglobin, are observed in conditions other than hemolysis that may confound the clinical picture. Hemoglobin defines the clinical severity of hemolysis, and thrombocytopenia suggests a possible thrombotic microangiopathy or Evans' syndrome. A comprehensive clinical and laboratory evaluation is advisable for a correct diagnostic and therapeutic workup of the different hemolytic conditions.

From perpetual haemosiderinuria to possible iron overload: iron redistribution in paroxysmal nocturnal haemoglobinuria patients on eculizumab by magnetic resonance imaging

From perpetual haemosiderinuria to possible iron overload: iron redistribution in paroxysmal nocturnal haemoglobinuria patients on eculizumab by magnetic resonance imaging

Adhesion Molecules Interaction with Markers of Lipid Metabolism, Hemolysis, Endothelial Dysfunction and Medical Histories Among Steady-State Sickle Cell Anemia Patients.

Abstract 2553Poster Board II-530 Introduction:Sickle cell anemia (SCA) results from the homozygous form of hemoglobin S (Hb S). Vaso-occlusive pain episodes underlie most of the acute and chronic clinical complications of disease and it is correlated with activation of red blood cells, leukocytes, platelets and endothelial cells that express several adhesion molecules and ligand. The aim of the present study was investigates the levels of soluble ICAM-1 and VCAM-1 adhesion molecules associating with biochemical markers, expression of neutrophils adhesion molecule and medical history of sickle cell anemia patients. Patients and Methods. We studied 53 SCA patients in steady-state (25 men, 28 women, mean age: 21.17 ± 14.12 years) from northeast Brazil diagnosed with SCA in attendance of the outpatients clinic of the Foundation of Hematology and Hemotherapy of Bahia (HEMOBA). The control group was compound by 22 healthy Brazilian, with AA hemoglobin pattern matched by age, years and ethnic origin. Biochemical analyses were measured by colorimetric methods, surface adhesion molecules expressions by flow cytometry, soluble adhesion molecules by ELISA and the complete medical history was obtained by patients' record. Results:Our results show a higher serum levels of VCAM-1(s) in patients than control group (p=0.006). Moreover, in this study we found low expression of neutrophils CD18 in patients with high levels of VCAM-1(s). Total cholesterol and low density lipoprotein (LDL-C) were significantly negative associated with VCAM-1(s) (r=−0.312, p=0.023; r=−0.282, p=0.041 respectively) and the high density lipoprotein (HDL-C) was significantly negative associated with ICAM-1(s) (r=−0.348; p=0.011). Intravascular hemolysis markers such as aspartate transferase (AST) and lactate dehydrogenase (LDH) were significantly positive associated with VCAM-1(s) (r=0.459, p=0.001 and r=0.281, p=0.041, respectively). Sickle cell anemia individuals that developed necrosis and leg ulcers exhibited high serum level of VCAM-1(s) (p=0.017 and p=0.021 respectively) and patients that presented priapism had low levels of ICAM-1(s) (p=0.046). Conclusion:The presence of high levels of ICAM-1(s) and VCAM-1(s) adhesion molecules and its association with markers of intravascular hemolysis, endothelial dysfunction and lipid metabolism may indicate a differentiated mechanism of these molecules in sickle cell anemia pathogenesis, with a complex involvement of cellular, endothelial and proinflammatory interactions. The measurement of soluble adhesion molecules is of easy determination and may be an important biomarker of prognosis among sickle cell anemia patients. Additional studies should be carried out in order to explore the contribution of ICAM-1 (s) and VCAM-1 (s) in other signal pathways. Disclosures:No relevant conflicts of interest to declare.

In vivo anti-complement effect of bilirubin-IXα

The effect of the IXα isomer of unconjugated bilirubin (UB) on complement-mediated intravascular hemolysis was evaluated in rats carrying naturally occurring heteroantibodies against sheep erythrocytes. Several doses of UB were administered i.v. to these animals in order to induce different levels of hyperbilirubinemia. Intravascular hemolysis was promoted by transfusion with a sheep red cell suspension. Hemoglobin in urine was assessed as a marker of intravascular hemolysis. The urinary excretion of hemoglobin was attenuated by UB in a dose-dependent manner. To establish whether complement was involved in the hemolytic reaction, we evaluated the hemolytic activity of complement in these same animals, before and after sheep erythrocyte transfusion. The significant consumption of complement, which was partially prevented by UB, corroborated its participation in the intravascular hemolytic reaction in the current experimental conditions. The data suggest an inhibitory action of UB on complement-mediated hemolysis in vivo.