Hemolytic Conditions Research Articles

A case report of hepatic subcapsular hematoma: Newborn with an autoimmune hemolytic condition.

We report a case of hepatic subcapsular hematoma in a newborn baby detected with an autoimmune hemolytic condition due to ABO incompatibility. Magnetic resonance imaging could be helpful in differentiating hepatic subcapsular hematoma from other abdominal masses.

B-080 Development and Validation of a GQ1b IgG ELISA as a Diagnostic Aid for Subacute Demyelinating Polyneuropathies

Abstract Background Autoimmunity targeting GQ1b is associated with group of disorders that includes Miller-Fisher Syndrome (MFS), Bickerstaff Brainstem Encephalitis (BBE) and classic Guillain Barre Syndrome (GBS) with ophthalmoplegia. Collectively these are referred to as GQ1B-IgG related syndromes. The prevalence of GQ1b-IgG in this population of patients is high and has been reported to be >80% in well-defined clinical cohorts. Clinical phenotypic presentation is diverse and requires highly accurate antibody testing. The objective of this study was to evaluate a newly developed custom GQ1b-IgG assay, using a semi-quantitative ELISA-based method. Methods A GQ1b-IgG ELISA was developed and validated. Patient sera along with assay controls and a single-point calibrator are diluted and incubated in duplicate wells in a 96-well plate coated with GQ1b antigen. The plate is washed and incubated with anti-human-IgG secondary antibody conjugated with HRP. After a colorimetric reaction, optical densities (OD) are read. The OD of patient samples are compared against the calibrator, generating a cut-off index (COI). Samples ≥1.0 COI are considered positive. During the validation phase, analytical and clinical performance characteristics were evaluated. Studies included assessment of imprecision at multiple concentrations of GQ1b-IgG, accuracy using method comparison, analytical sensitivity and specificity, reference range, common interferences, plate drift, and clinical performance in relevant disease and control cohorts. Results Intra-assay imprecision (coefficient of variation; %CV) ranged from 1.0 - 16% across all concentrations of GQ1b-IgG. Inter-assay imprecision (%CV) ranged from 12.4 - 44.5% but was <20% at or above the assay cut-off. We found perfect qualitative agreement with an independent ELISA reference method (20 positive and 20 negative samples selected based on reference method results [reference interval <1:100]). Limit of detection (LOD) was calculated at a COI of 0.22. To assess analytical specificity, 40 samples with high concentrations of immunoglobulins (hypergammaglobulinemia [N=15], IgG M-protein [N=15] and systemic lupus erythematosus [N=10] were tested and all were negative. A reference interval study was performed on 140 healthy donor serums, all were negative. Interference studies were conducted on 2 positive and 2 negative serum samples, spiked for highly hemolytic (1000 mg/dL), lipemic (2000 mg/dL) and icteric (60 mg/dL) conditions. There was no impact on the assay even with high levels of a given interferent. Plate drift on 2 distinct patient pools tested across the plate (COI range 0.7 - 1.3) had overall plate CV’s ≤10%. ROC analysis was performed on a cohort of 278 patient samples (15 MFS cases and 263 controls [suspected demyelinating neuropathy or disease mimics]). A cut-off value of 1.0 (COI) was selected based on the Youden index. At this cut-off, clinical sensitivity for MFS was 87% while clinical specificity was 99.2%. Conclusions The observed performance of the GQ1b-IgG ELISA was acceptable for clinical laboratory implementation. A COI of ≥ 1.0 was selected for clinical use.

Counteraction of unconjugated bilirubin against heme-induced toxicity in platelets

Platelets are essential for normal hemostasis and thrombosis but become hyperactive in hemolytic disorders. Cell-free heme is known to be toxic to platelets and endothelial cells, playing a significant role in the progression of pathological complications in various hemolytic conditions. The abnormal activation of circulatory platelets results in micro/macrovascular thrombosis and clot formation in the lungs, worsening the disease. This work aimed to establish the potent bioactive molecule that can regulate the hemin-induced toxicity in platelets. We found that unconjugated bilirubin (UCB), an endogenous antioxidant and a byproduct of heme degradation, exhibited a higher protective effect against hemin-induced platelet aggregation and activation. This protective effect could mainly be due to reducing ROS and lipid peroxidation-mediated ferroptosis in hemin-treated platelets. Further experiments suggested that by blocking the interaction between hemin and the CLEC-2 receptor, UCB regulates the downstream Syk phosphorylation, a key event in hemin-induced platelet toxicity. Thus, UCB is emerging as a natural regulatory molecule that mitigates hemin-induced platelet toxicity and holds promise as an adjunctive therapy for managing platelet-associated complications, particularly in hemolytic disorders.

Antioxidant, membrane stabilizing and thrombolytic potentials of the leaves of erythrina fusca lour

Erythrina fusca (Family: Fabaceae) is famous for many traditional healing. In the present study, the leaves of E. fusca were collected, dried, powdered and then extracted with methanol. It was further partitioned into n-hexane, dichloromethane and aqueous soluble fractions. All the extracts were subjected to antioxidant, membrane stabilizing and thrombolytic assays. In case of free radical scavenging assay, the aqueous soluble fraction showed highest scavenging activity. In ferric reducing anti-oxidant power assay, the dicholoromethane extract showed highest reduction of ferric ion in a dose dependent manner. In total antioxidant assay, the methanol extract and dichloromethane extract showed stronger capacity as antioxidant. During the determination of total phenolic and flavonoid contents, the methanol extract and dichloromethane soluble fraction showed higher contents of phenolics and flavonoids, respectively. The erythrocyte membrane stabilizing assay revealed the capacities of methanol extract and its aqueous fraction to stabilize the RBC membrane in both hypotonic solution- and heat- induced hemolytic conditions. However, the clot lysis ability of the E. fusca leaf extracts was not so prominent. Bangladesh J. Bot. 53(3): 439-445, 2024 (September)

Preventing kidney failure in hemolytic anemias with a novel heme and hemoglobin scavenger

Chronic hemolytic conditions, seen in genetic and acquired hemolytic diseases, result in increased levels of circulatory hemoglobin (Hb) released from erythrocytes, free heme, and iron. Cell-free Hb's toxicity arises from the Hb dissociation into dimers that can traverse tissue barriers, impacting crucial compartments like arterial smooth muscle cells and renal glomeruli. Chronic hemolysis leads to vasoconstriction, renal injury, and activates pathways via Hb oxidation and heme-ligation of TLR4 on endothelial cells, as well as leukocytes, affecting adhesion pathways. Hb and heme exposure to the immune system causes phagocyte dysfunction, inflammation suppression, and impaired antigen presentation. Kidneys are particularly susceptible to Hb toxicity due to Hb and dimer penetration into glomeruli and tubules, disrupting filtration forces, elevating the glomerular filtration rate (GFR), and causing acute kidney injury (AKI). To combat this issue, our group developed a novel Hb and heme scavenger based on apohemoglobin-haptoglobin (apoHb-Hp), functioning as both a hemopexin (Hpx) and Hp mimetic to scavenge cell-free Hb and heme during hemolytic states. Our study utilized the dorsal window chamber model in Golden Syrian hamsters, employing FITC-Sinistrin for non-invasive GFR measurement, avoiding the need for blood and urine sampling throughout time to estimate GFR. Two experiments were conducted, volumetric exchange groups with 20% of the animal’s blood volume either with Hb at different concentrations or a combination ApoHb-Hp and Hb. Results showed a significant acute decrease of 35% in half-life of the Hb group at 1.2g/dL and 5g/dL after two hours, and further decrease is seen of 45% at 24hrs. In contrast, the ApoHb-Hp group experienced a 30% decrease at 2hrs but reverted back to baseline at 24hrs, signifying Hb's predominant effect on the acute decrease rather than the volume change. Furthermore, the ApoHb-Hp group demonstrated a quicker return to baseline compared to the Hb group after the 24-hour mark, suggesting prolonged Hb exposure's lasting impact on kidney filtration rate, potentially leading to irreversible damage. Further research is required to comprehend these findings fully and explore ApoHb-Hp's protective role in mitigating Hb-induced kidney damage. Title of the project: Bioengineering a Dual Function Protein Construct to Detoxify Heme and HemoglobinProject number: R01HL159862Title of the project: Bioengineering a novel therapeutic protein complex to minimize the effects of medical device induced hemolysis.Project number: R01HL162120. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In-depth structure-function profiling of the complex formation between clotting factor VIII and heme

Background and aimsBlood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. MethodsWe herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. ResultsFVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. ConclusionOur study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.

Exploring the intersection: Peptic ulcers and hemolysis-Unraveling the complex relationship.

This paper investigates the intriguing relationship between peptic ulcers and hemolysis, 2 seemingly distinct medical conditions, aiming to unravel their potential interconnections and clinical implications. While traditionally studied in isolation, recent evidence has surfaced suggesting possible links and shared mechanisms between these conditions. This paper explores the underlying pathophysiological associations, shared risk factors, diagnostic challenges, management strategies, and implications for clinical practice and health policy. The interplay between peptic ulcers and hemolysis stems from shared inflammatory pathways, notably attributed to Helicobacter pylori infection in peptic ulcers, which might trigger systemic inflammatory responses contributing to hemolysis. Common risk factors including genetic predispositions, autoimmune disorders, and medication use (such as nonsteroidal anti-inflammatory drugs) are implicated in the development of both peptic ulcers and hemolytic conditions, suggesting a potential convergence of these disorders in affected individuals. Diagnostic considerations pose challenges, as overlapping symptoms and laboratory findings may complicate accurate differentiation between peptic ulcers and hemolysis. Recognizing the potential interplay between peptic ulcers and hemolysis holds significant implications for clinical practice and health policy. Streamlining diagnostic algorithms, fostering interdisciplinary collaborations, and developing tailored guidelines are pivotal in optimizing patient care. Continued research efforts, collaborative clinical approaches, and informed health policies are essential in advancing our understanding and enhancing patient care for individuals navigating the intersection of peptic ulcers and hemolysis.

Genetics of Gallstone Disease and Their Clinical Significance: A Narrative Review.

Gallstone (GS) disease is common and arises from a combination of genetic and environmental factors. Although genetic abnormalities specifically leading to cholesterol GSs are rare, there are clinically significant gene variants associated with cholesterol GSs. In contrast, most bilirubin GSs can be attributed to genetic defects. The pathogenesis of cholesterol and bilirubin GSs differs greatly. Cholesterol GSs are notably influenced by genetic variants within the ABC protein superfamily, including ABCG8, ABCG5, ABCB4, and ABCB11, as well as genes from the apolipoprotein family such as ApoB100 and ApoE (especially the E3/E3 and E3/E4 variants), and members of the MUC family. Conversely, bilirubin GSs are associated with genetic variants in highly expressed hepatic genes, notably UGT1A1, ABCC2 (MRP2), ABCC3 (MRP3), CFTR, and MUC, alongside genetic defects linked to hemolytic anemias and conditions impacting erythropoiesis. While genetic cases constitute a small portion of GS disease, recognizing genetic predisposition is essential for proper diagnosis, treatment, and genetic counseling.

Transcription Factor NRF2 in Shaping Myeloid Cell Differentiation and Function.

NFE2-related factor 2 (NRF2) is a master transcription factor (TF) that coordinates key cellular homeostatic processes including antioxidative responses, autophagy, proteostasis, and metabolism. The emerging evidence underscores its significant role in modulating inflammatory and immune processes. This chapter delves into the role of NRF2 in myeloid cell differentiation and function and its implication in myeloid cell-driven diseases. In macrophages, NRF2 modulates cytokine production, phagocytosis, pathogen clearance, and metabolic adaptations. In dendritic cells (DCs), it affects maturation, cytokine production, and antigen presentation capabilities, while in neutrophils, NRF2 is involved in activation, migration, cytokine production, and NETosis. The discussion extends to how NRF2's regulatory actions pertain to a wide array of diseases, such as sepsis, various infectious diseases, cancer, wound healing, atherosclerosis, hemolytic conditions, pulmonary disorders, hemorrhagic events, and autoimmune diseases. The activation of NRF2 typically reduces inflammation, thereby modifying disease outcomes. This highlights the therapeutic potential of NRF2 modulation in treating myeloid cell-driven pathologies.

Hemolysis and Free Heme Impact Therapeutic Activity of Anti-CD20 Antibody

Hemolysis is a process of destruction of erythrocytes that can occur in different hereditary or acquired pathologies. It is accompanied by release of hemoglobin and heme, which both manifest potent pro-oxidative and pro-inflammatory activities. If released in large quantities hemoglobin and heme can directly contribute to systemic inflammation and tissue injury in hemolytic diseases. Moreover, it was demonstrated that heme binds to a significant fraction (ca. 10 %) of human antibodies (Abs) and this binding can result in considerable functional changes. Thus, heme-bound human IgG acquire reactivity towards a large set of unrelated proteins, including autoantigens. In a previous study we observed that 14 % of a repertoire of 117 clinical-stage therapeutic mAbs are able to bind heme and as a result they acquired autoreactivity and polyreactivity. Among these Abs some are specific for CD20 and used in lymphoma therapy (e.g. Rituximab). Importantly, therapeutic mAbs can be administered in patients with ongoing hemolysis. Thus, it is of critical importance to decipher molecular mechanism and functional consequences of interaction of heme with therapeutic mAbs and understand how the therapy of lymphoma can be affected by underling hemolysis and heme release. The present study has the following objective: to elucidate the effect of heme on the therapeutic activity of anti-CD20 therapy in in vitro and in vivo models of B cell lymphoma. Effect of heme on anti-CD20 therapeutic Abs was assessed by i) binding assays - ELISA, western blot; protein microarrays; absorbance spectroscopy; surface plasmon resonance, and ii) molecular modeling. The impact of heme on functional and therapeutic activity of Rituximab was analyzed by i) site-directed mutagenesis; ii) flow cytometry; iv) in vitro and in vivo models of human B cell lymphoma. Our data reveled molecular and functional details about the interaction of heme with Rituximab and other anti-CD20 Abs. Thus, we characterized the interaction of Abs with heme, and identified in case of Rituximab the putative binding site for heme that overlapped with antigen binding site. By using a protein array with >9800 human proteins, we identified the target proteins of heme-bound Rituximab. A mouse model of human B cell lymphoma revealed that the therapeutic activity of Rituximab was compromised following exposure to cell free heme under condition of acute intravascular hemolysis. Our study delineates that a fraction of currently used therapeutic mAbs show high sensitivity to heme and their therapeutic activity can be compromised under hemolytic conditions. This finding suggests that therapeutic interventions that reduce free heme and /or intravascular hemolysis should precede therapy with these Abs. Moreover, we identified sequence features of Abs that determine the sensitivity to heme. This finding can help to reduce sensitivity of heme of therapeutic Abs by protein engineering.

A Case of Catastrophic Atypical Hemolytic Uremic Syndrome Unresponsive to Eculizumab and the use of Ravulizumab Off-label

“A 40-year-old woman with melanoma, under treatment with Dabrafenib and Trametinib, was evaluated in our hospital for rapidly progressive deterioration of renal function”. 8 months before the current admission, the patient had been diagnosed with melanoma, and underwent radical surgery and subsequent therapy with Dabrafenib and Trametinib. After 5 months of therapy, the patient was brought to this hospital for precordial pain, with a diagnosis of myopericarditis, therapy was started for heart failure with a good response. However, the patient developed a progressive impairment of renal function, associated with hemolytic anemia and thrombocytopenia. The peripheral smear showed the presence of schistocytes. The suspicion of atypical Hemolytic Uremic Syndrome (aHUS) was confirmed by the assay of C5B-9 induced by serum on endothelial cells, which showed a deposition of 331%, treatment with Eculizumab was initiated. After 3 administrations the patient did not improve, with further worsening of the hemolytic condition, and progression of renal damage. Due to the failure of Eculizumab, we considered the use of Ravulizumab. However, in Italy only can be administered to patients in Eculizumab stable treatment for at least three months. Nevertheless, faced with the catastrophic condition, it was decided to shift the therapy and use off-label Ravulizumab. After 10 days of the first administration, the laboratory tests showed a continuous rise in the values of haptoglobin, platelets, and hemoglobin, and a decrease in LDH. The renal function failed to return to normal values but after 20 days of therapy with Ravulizumab, there was complete resolution of the hemolytic condition.

B-069 Performance Evaluation of a Ma2 Autoantibodies ELISA

Abstract Background Ma-2 is a neuronal nucleolar protein with purported involvement in RNA transcription and apoptosis regulation. Patients with Ma2 antibodies present with autoimmune encephalitis (limbic or brainstem). Ma-2 antibodies are a high-risk marker for paraneoplastic neurologic cause for autoimmune encephalitis (>75% of patients having either testicular cancer or non-small cell lung cancer). In older patients, there is a higher frequency of small cell lung cancer or other cancers especially when Ma 1 antibodies are also detected. Identification of Ma2 antibodies is important to help diagnose autoimmune encephalitis, guide the cancer search and aid treatment selection. The objective of this study was to develop and document the performance of a qualitative enzymatically amplified sandwich-type Ma2 Antibody ELISA. Methods The Ma2 ELISA was produced in-house utilizing commercially available protein and was performed on a Hamilton Star liquid handling instrument equipped with a plate washer and plate reader. Each assay plate included patient samples along with 3 independent negative serum calibrators and 4 quality controls (Both positive and negative controls per matrix [CSF or serum]) each tested in duplicate. Mean optical densities (OD) for patient specimens were compared to the average OD of the negative serum calibrators to generate a ratio. A ratio cut-off of 8 was selected for reporting positivity based on preliminary studies and was validated during the method performance assessment. To assess assay performance we measured precision (3 positive pools at or above the cut-off ratio for each specimen type were tested 20 times in one assay and in duplicate across 10 independent assays), accuracy comparing to the current reference method at Athena® Diagnostics (N = 10 positives / 40 negatives), analytical specificity (20 samples with high titer autoantibodies directed against other proteins), and the impact of common interferences (lipids, bilirubin, hemolysate were spiked into 2 positive and 1 negative sample per interference). Clinical performance of the assay for differentiating healthy donors without disease (N = 140) and those with non-Ma2 neurological autoimmunity (N = 23) from those with clinical phenotypes consistent with Ma-2 associated encephalitis (N = 8) was measured. Results Serum intra-assay imprecision (coefficient of variation; %CV) ranged from 3.0–6.5%. Inter-assay imprecision ranged from 21–29%. CSF intra-assay imprecision ranged from 3.5–10%. Inter-assay imprecision ranged from 13–15.5%. Accuracy was assessed via qualitative comparison to a reference method [western blot or nanoliter scale immunoassay]. There was perfect agreement between methods. To assess analytical specificity, samples with high concentrations of immunoglobulins (hypergammaglobulinemia [N = 10] or Systemic Lupus Erythematosus [N = 10]) were tested in serum (and spiked into CSF); all were negative. Interference studies were conducted on 2 positive Ma2 serum and CSF samples, spiked for highly hemolytic (1000 mg/dL), lipemic (2000 mg/dL) and icteric (60 mg/dL) conditions. All samples remained positive under given interferents. A negative sample was tested for each matrix, both remained negative. Clinical specificity was 100% with all samples from patients without Ma-2 encephalitis testing negative (N = 183). All ELISA positives identified in this study had phenotypes consistent with Ma-2 autoimmune encephalitis (N = 8). Conclusion The performance of the Ma2 ELISA was acceptable for clinical laboratory implementation.

Peroxiredoxins in erythrocytes: far beyond the antioxidant role.

The red blood cells (RBCs) are essential to transport oxygen (O2) and nutrients throughout the human body. Changes in the structure or functioning of the erythrocytes can lead to several deficiencies, such as hemolytic anemias, in which an increase in reactive oxidative species generation is involved in the pathophysiological process, playing a significant role in the severity of several clinical manifestations. There are important lines of defense against the damage caused by oxidizing molecules. Among the antioxidant molecules, the enzyme peroxiredoxin (Prx) has the higher decomposition power of hydrogen peroxide, especially in RBCs, standing out because of its abundance. This review aimed to present the recent findings that broke some paradigms regarding the three isoforms of Prxs found in RBC (Prx1, Prx2, and Prx6), showing that in addition to their antioxidant activity, these enzymes may have supplementary roles in transducing peroxide signals, as molecular chaperones, protecting from membrane damage, and maintenance of iron homeostasis, thus contributing to the overall survival of human RBCs, roles that seen to be disrupted in hemolytic anemia conditions.

Patient-specific stent for hemolytic anemia due to a kinked ascending aortic graft.

Hemolytic anemia after thoracic aortic surgery is rare. A 69-year-old female patient, who underwent ascending aorta replacement for acute type A aortic dissection 7 years ago, was transferred from another hospital due to hemolytic anemia necessitating regular blood transfusions. After engaging in interdisciplinary discussions, we ruled out alternative medical diagnoses and treatments, ultimately identifying the kinked aortic graft as the primary cause of hemolysis. Due to the patient's comorbidity, it was deemed high-risk to perform a redo thoracic aorta surgery for the replacement of the kinked aortic graft. Therefore, we decided to insert a patient-specific bare-metal stent into the kinked aortic graft to alleviate the kinking and promote improvement in hemolysis. We decided to perform the stent procedure under local anesthesia. The bare-metal stent was designed to be approximately 15% larger than the size of the pre-existing kinked aortic graft. A stent measuring 32 mm × 80 mm was successfully inserted and ballooned within the patient's ascending aortic graft. As a result, the patient's hemolytic condition showed gradual improvement over time. In this way, we aim to share our experience of inserting a patient-specific bare-metal stent under local anesthesia in patients who exhibit mechanical hemolysis due to a kinked aortic graft, especially when redo surgery is considered high-risk.

Nrf2 sensitizes ferroptosis through l-2-hydroxyglutarate–mediated chromatin modifications in sickle cell disease

AbstractSickle cell disease (SCD) is a chronic hemolytic and systemic hypoxia condition with constant oxidative stress and significant metabolic alterations. However, little is known about the correlation between metabolic alterations and the pathophysiological symptoms. Here, we report that Nrf2, a master regulator of cellular antioxidant responses, regulates the production of the metabolite l-2-hydroxyglutarate (L2HG) to mediate epigenetic histone hypermethylation for gene expression involved in metabolic, oxidative, and ferroptotic stress responses in SCD. Mechanistically, Nrf2 was found to regulate the expression of L2HG dehydrogenase (L2hgdh) to mediate L2HG production under hypoxia. Gene expression profile analysis indicated that reactive oxygen species (ROS) and ferroptosis responses were the most significantly affected signaling pathways after Nrf2 ablation in SCD. Nrf2 silencing and L2HG supplementation sensitize human sickle erythroid cells to ROS and ferroptosis stress. The absence of Nrf2 and accumulation of L2HG significantly affect histone methylation for chromatin structure modification and reduce the assembly of transcription complexes on downstream target genes to regulate ROS and ferroptosis responses. Furthermore, pharmacological activation of Nrf2 was found to have protective effects against ROS and ferroptosis stress in SCD mice. Our data suggest a novel mechanism by which Nrf2 regulates L2HG levels to mediate SCD severity through ROS and ferroptosis stress responses, suggesting that targeting Nrf2 is a viable therapeutic strategy for ameliorating SCD symptoms.

Normalisation of haemoglobin and control of breakthrough haemolysis with increased frequency pegcetacoplan dosing in treated paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinura is an acquired life-threatening haemolytic condition, which is generally well controlled with terminal complement blockade with eculizumab. Whilst almost all patients treated with terminal complement inhibitors develop extravascular haemolysis, only a small proportion of these results in symptomatic anaemia limiting their activities and requiring red cell transfusion. This case highlights the potential role for the C3 inhibitor, pegcetacoplan, in controlling both intravascular and extravascular haemolysis, and is the first case to report on the use of additional doses of pegcetacoplan to control breakthrough haemolysis.

Observing temporal variation in hemolysis through photoacoustics with a low cost LASER diode based system

Patients under hemolytic condition need continuous monitoring of lysis as depletion of Red Blood Cells (RBC) and the presence of antioxidant free hemoglobin (Hb) in excess amount due to hemolysis lead to severe deterioration of their health. Out of many modalities, Photoacoustics (PA) offers real time information noninvasively from deep lying blood vessels since Hb is the strongest chromophore in mammalian blood and the PA response of blood varies with the amount of Hb present. During hemolysis, total Hb content in blood however remains unchanged, thus, questions the use of PA in hemolysis detection. In this report, a hypothesis that the amplitude of the PA signal would not change with the amount of lysis is framed and tested by applying osmotic shock to the RBCs in hypotonic environment and the PA response is recorded over time using a low cost NIR based PA system. The experimental outcome indicates that PA amplitude falls off as lysis progresses in course of time consequently rejecting the hypothesis. The decaying PA response also carries the signature of RBC swelling during the early phase of lysis. The PA measurement can detect hemolysis as low as 1.7%. These findings further advocate transforming this NIR-PA system into a portable, noninvasive patient care device to monitor hemolysis in-vivo.

A SILENT CASE OF SICKLE CELL DISEASE: A SERENDIPITOUS DISCOVERY

We present here a 4-year-old male child who came to hematology out-patient department for follow up for anemia which had required blood transfusion one and half year back. On clinical examination child was found to neither have pallor nor icterus. Blood was sent for complete hemogram. The hemogram showed a total hemoglobin of 8.4 gm%. Other RBC indices and WBC and platelet counts were within normal expectations. The reticulocyte count however was raised to 11%. Based on such a hemogram, on a high index of suspicion of a chronic hemolytic condition, despite clinical absence of any hemolytic features, a hemoglobin HPLC was performed in the Biochemistry laboratory. The HPLC revealed a percentage of HbS compatible with a HBS homozygous state. Based on this nding, the routine hemograms of both the parents were performed. These were found to be within normal limits. However, HPLC analyses for hemoglobin variants revealed HbS percentages compatible with HbS carrier i.e., heterozygous states. We thus provisionally concluded that this child was a case of Sickle cell homozygous, but atypical as because there was no history or clinical evidence of sickle cell disease symptomatology.

Purine nucleoside phosphorylase: A new pharmacological target in sickle cell disease and hemolytic vasculopathy

Under hemolytic conditions, there is a significant interaction among red blood cells (RBCs), endothelial cells (ECs), and platelets in the injured vasculature. In sickle cell disease (SCD), this interaction leads to painful vaso-occlusive crisis and end organ damage, results in poor quality of life, and unacceptably short life expectancy. We propose that these cells share two biochemical properties that are highly relevant to SCD. First, per cell volume, RBCs have the highest level of purine nucleoside phosphorylase (PNP, a key enzyme in purine metabolism), and ECs and platelets are very rich in PNP. Second, RBCs have to, ECs choose to, and platelets, when activated, switch to using glycolysis as a major pathway for intermediate phosphates and ATP production. We propose that these similarities are highly relevant to sickle cell disease (SCD) and hemolytic vascular injury. Human PNP catalyzes the phosphorolysis of guanosine/ deoxyguanosine and inosine /deoxyinosine into guanine and hypoxanthine, respectively, and generates ribose-1-phosphate and deoxyribose-1-phosphate. This results in forming a large amount of downstream pro-oxidant and vasculotoxic xanthines.Recently, we have detected that in SCD, hemolysis is associated with increased activity of extracellular PNP, accelerated guanosine and inosine metabolism, and augmented extracellular production of vasculotoxic hypoxanthine and xanthine. We identify PNP as a critical pathogenic mechanism in SCD and have proposed a link between PNP activity and the glycolytic production of intermediate phosphates and ATP. This link promotes (i) RBC sickling/hemolysis, (ii.) hemolytic angioproliferation, (iii.) oxidative vascular injury, and (iv.) platelets activation/aggregation.Furthermore, PNP accelerates guanosine/inosine metabolism and may diminish their well-documented anti-inflammatory, anti-ischemic, anti-thrombotic, and anti-nociceptive effects. We hypothesize that inhibition of PNP may lessen those four (i.to iv.) deleterious events and may potentiate the beneficial effects of guanosine/inosine, reducing the risk of vaso-occlusion and organ damage in SCD patients. Thereby, the PNP inhibitors may serve as a multipronged treatment approach for SCD.

Non-erythropoiesis stimulating agent, non-iron therapies for the management of anemia: A scoping review.

Non-erythropoiesis stimulating agent, non-iron therapies for the management of anemia: A scoping review.