Hemolytic Episodes Research Articles

PB1866 SUCCESSFUL MANAGEMENT OF A CHILD FOR PAROXYSMAL NOCTURNAL HEMOGLOBINURIA WITH LONG‐TERM ECULIZUMAB THERAPY

Background:PNH arises from a genetic mutation of hematopoietic stem cells which leads to the acquired nonmalignant clonal expansion of cells lacking glycosyl phosphatidylinositol‐anchored proteins (GPI‐APs). Lack of GPI‐APs translates into PNH's most significant clinical features: bone marrow failure, intravascular hemolysis and thrombosis. PNH rarely occurs in children and has been reported to have a distinct clinical presentation compared to the adult population. Most of the pediatric patients in the litearture have arisen in a previous setting of bone marrow failure. There are few pediatric case reports presented with reccurrent hemolytic episodes.Aims:There is a paucity of published data on outcomes in children with PNH treated with eculizumab. Therefore, we find it significant to share our experience regarding the use of eculizumab for a pediatric patient, who was diagnosed as PNH at 5.5 years of age and have been managed successful by eculizumab for more than 9 years at Kanuni Sultan Süleyman Education and Research Hospital.Methods:The case is retrospectively evaluated.Results:The patient was diagnosed in March 2008 with immunphenotyping and verified with FLAER method. She had been given blood transfusions, hyperhydration therapy, low‐dose oral prednisolone continuously initially and then during hemolytic attacks. During follow‐up, attacks had become more frequent and recurred at 2‐3 weeks intervals. With the informed consent of the family, eculizumab therapy was commenced in December 2011. She was 9 year old at this time.The dose of eculizumab was set according to the weight of the child as 600 mg/week for 4 week, then 900 mg/every other week parenterally. After introduction of eculizumab, both the frequency and the severity of the attacks had declined significantly. She has been under eculizumab therapy for more than 9 years without any considerable advers effect. Reoccurence of hemolytic attack was observed, when the patient had infections or the treatment was interrupted due to problems in obtining drugs.Summary/Conclusion:Depending on our experience, eculizumab may have a substantial role in controlling hemolytic episodes and preventing thrombotic complications and improving quality of life in children similiar to adults.

Double-Blind Clinical Trial of Arginine Supplementation in the Treatment of Adult Patients with Sickle Cell Anaemia.

Background Sickle cell anaemia (SCA) is the most prevalent monogenic disease in Brazil. In SCA, haemoglobin S (HbS) is formed, which modifies red blood cell morphology. Intravascular haemolysis occurs, in which free Hb and free radicals degrade nitric oxide (NO) and release arginase, which reduces arginine levels. Because arginine is a substrate for NO formation, this decrease leads to reduced NO (vasodilator) synthesis. SCA treatment uses hydroxyurea (HU) to maintain high foetal haemoglobin (HbF) levels and reduces HbS to avoid haemolytic episodes. Objective To analyse the efficacy of L-arginine as an adjuvant in the treatment of SCA patients. Setting The State Blood Centre of Ceará, Brazil. Methods This was a randomized double-blind clinical study of adults with SCA with continuous use of HU at the State Blood Centre of Ceará. The clinical study enrolled 25 patients receiving HU + L-arginine (500 mg) and 25 patients receiving HU + placebo. The treatment was carried out over four months. Laboratory tests were performed to determine the levels of the following: (1) complete blood count; (2) nitrite + nitrate; (3) HbF; and (4) reticulocytes. The clinical experiments were performed by a haematologist. The main outcome measures were nitrite and pain. Results Statistical analysis showed that the levels of NO were increased in the study group, and there was also a reduction in pain frequency using a pain frequency scale by day, week, and month. The levels of nitrite plus nitrate in the group receiving placebo plus HU did not change among the times evaluated (38.27 ± 17.27 mg/L, 39.49 ± 12.84 mg/L, 34.45 ± 11.25 mg/L, p >0.05), but in the patients who received supplementation with L-arginine plus HU, a significant increase in nitrite plus nitrate levels was observed between M0 and M4 (36.55 ± 20.23 mg/L versus 48.64 ± 20.63 mg/L, p =0.001) and M2 and M4 (35.71 ± 15.11 mg/L versus 48.64 ± 20.63 mg/L, p <0.001). It is important to note that the increase in nitrite plus nitrate levels occurred only in the fourth month of follow-up of patients in the treatment group, showing that at least 4 months of supplementation with L-arginine is necessary to show an increase in these metabolites in the serum. Conclusion The use of L-arginine as a coadjuvant in the treatment of sickle cell anaemia may function as a potential tool for pain relief, consequently improving the life of patients.

Prevalence of Glucose-6-Phosphate Dehydrogenase Deficiency among Steady-State Sickle Cell Disease Children at University of Ilorin Teaching Hospital, Ilorin

Background: Both glucose-6-phosphate dehydrogenase (G6PD) deficiency and sickle cell disease (SCD) are prevalent in malaria-endemic regions. Controversy however persists as to whether G6PD deficiency is commoner in SCD subjects compared with the general population. Co-existence of the enzyme-deficient state with a chronic haemolytic disorder, like SCD, could potentially predispose to fatal haemolytic episodes. There is however a dearth of paediatric studies on this subject. Aims and Objectives: To determine the prevalence and demographic determinants of G6PD deficiency in children with SCD. Methods: Red cell G6PD activity was determined in 115 steady-state sickle cell disease children aged 0.5 - 17 years, in steady state and equal number of age & gender-matched non-SCD controls using the quantitative method. A measured G6PD activity of <6.97 U/gHb defined G6PD deficiency while values ≥6.97 U/gHb were regarded as normal. Data were analysed using SPSS version 20. Statistical analyses done include chi-square, student t-test and ANOVA. For all statistical analyses, p values less than 0.05 were considered significant. Results: There were 64 (55.7%) males and 51 (44.3%) females in each of the arms of the study. The mean age of the study population was 8.4 ± 4.7 years. Seven of the subjects were G6PD-deficient giving a prevalence of 6.1% which was not significantly different from the 7.0% obtained in the controls (p = 0.789). G6PD deficiency was more frequently encountered in the older age groups although the difference was not statistically significant (p = 0.438). Similarly, mean G6PD activity was highest in the under-5 age-group compared to the older age-groups analysed (p = 0.573). The condition was also commoner in the males than females although the difference did not attain statistical significance (p = 0.897). Conclusions: The prevalence of G6PD deficiency among SCD children was 6.1%. The condition is marginally commoner in males and older children.

The enzymopathy of G6PD deficiency in Jordan: a demographic and biochemical analysis

Background: G6PD deficiency is an inherited X-linked recessive condition leading to insufficient levels of glucose-6-phosphate dehydrogenase, thus causing hemolytic anaemia under certain circumstances.&#x0D; Materials and Methods: Our study is explorative for cases admitted to Jordan University Hospital. The studied parameters include demographics, clinical manifestations, biochemical markers including Hb level, WBC count, liver enzymes, and blood grouping.&#x0D; Results: Most of the patients were admitted to the emergency unit (53.13%). Individuals who were Rh-positive represented 57.81%, while patients of AB blood group accounted for 75%. The mean values were 4.81 years (age), 29.06 hours (time-to-hospital admission), 38.10 degree Celsius (temperature), 6.11 gm/dl (Hb), 13242.19 (WBC count), 343.20 U/L (S. ALP), and 50.98 IU/L (S. ALT). There was no significant difference between males and females or between favism-induced versus drug-induced hemolytic episodes. AB and Rh positive blood groups are of a protective effect in relation to liver enzymes. Patients who were admitted to the hospital within 24 hours from having clinical manifestations had a better prognosis.&#x0D; Conclusion: This study is the first inferential research on G6PD deficiency from the Middle East to explore cases from one of the largest healthcare centres in Jordan. The role of blood grouping should be investigated prospectively.

Study of Some Predictors of Severe Hemolysis in Children with Glucose-6-Phosphate Dehydrogenase Deficiency

AbstractBackground: Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is one of the most common inherited disorders of mankind; more than 400 million people are affected worldwide, and it is a major health problem in Egypt.Aim of Work: To study some predictors of severe hemolysis in children with glucose-6-phosphate dehydrogenase deficiency and the degree of DNA damage in these patients.Patients and Methods: Seventy-two patients with G6PD deficiency admitted with acute hemolytic attacks to Diarb Negm Central Hospital-Sharkia Governorate, Egypt-over a period of 1 year from 1st May 2014 to 30th April 2015. We collected demographic data, details of signs and symptoms; special attention was given to history of fava bean ingestion, history of drug use or history of recent infection, history of neonatal jaundice, family history of G6PD deficiency disease, abdominal pain, vomiting or fever at admission and the results of tests for hemoglobin, reticulocytic count, hepatic function, G6PD enzyme activity and detection of DNA damage by DNA extraction, purification and agarose gel electrophoresis. We classified patients into mild and severe groups based on hemoglobin levels at admission. The mild cases were consid-ered as the control group.Results: 72 children with G6PD deficiency admitted with acute hemolytic attacks were included in this study, 52 (72%) patients with severe hemolysis and 20 (28%) patients with mild hemolysis (control group). There were no statistically significant differences in many factors between the two groups, but some factors were detected to have statistical significance for severe hemolysis included younger age (p<.0001), male gender (p<.0001), presence of fever at admission (p<.0001), presence of vomiting (p<.0001) and abdominal pain (p<.0001) during the attack, higher Alkaline Phosphatase (ALP) (p <.0001), Aspartate Aminotransferase (AST) (p<.0001), Lactate Dehydrogenase (LDH) (p<.0001) and Total Serum Bilirubin (TSB) (p<.05). DNA damage was significantly higher in the severe cases compared to the mild cases (p<.0001).Conclusion: Severe hemolysis can be predicted during hemolytic episodes in children with G6PD deficiency by young age, male gender, the presence of abdominal pain, vomiting and fever, high levels of ALP, AST, LDH enzymes and elevated TSB. This severe hemolysis predisposes patients to higher degree of DNA damage.

Evaluation of Phenotype-Genotype Correlation in Two Common PIEZO1 Mutations p.R2456H and p.L2495_E2495dup

Hereditary xerocytosis (HX) is a rare autosomal dominant hemolytic anemia caused by mutations in the mechanosensitive cation channel PIEZO1 or, less commonly, in the Ca2+-gated K+ channel KCNN4 (Gardos channel). It is a clinically heterogeneous condition, characterized by erythrocyte dehydration. As erythrocytes traverse narrow capillaries and sinusoids, PIEZO1 is thought to be activated by mechanical stimuli, leading to increased intracellular Ca2+ which then may activate KCNN4, causing K+ efflux and water loss. The p.R2456H and p.L2495_E2496dup PIEZO1 mutations are likely the most common of the PIEZO1 disease-causing mutations and both result in delayed channel inactivation (Glogowska et al., 2017, Blood 130:1845). The prolonged channel activity in response to mechanical stimulation is likely to contribute to erythrocyte dehydration.Ten patients with PIEZO1-associated HX (5 with p.R2456H and 5 with p.L2495_E2496dup PIEZO1 heterozygous mutations) had their diagnosis established utilizing a Next Generation sequencing panel for hereditary hemolytic and congenital dyserythropoietic anemias (HHA/CDA panel). These patients, along with family members who had similar clinical characteristics (a total of 8 patients with p.R2456H and 6 patients with p.L2495_E2496dup) ranging from 2 - 63 years of age, were enrolled in an IRB-approved study to explore the phenotype-genotype correlation of the disease. We collected patient and family histories and laboratory data and performed phenotypic testing including ektacytometry and erythrocyte cation determinations by atomic spectroscopy.Most of the patients in our cohort had compensated hemolysis without anemia at the time of evaluation and had not required any transfusions. All of the patients had elevated reticulocyte counts ranging from 5.7 to 15.7 %. Reticulocyte counts of individuals with the p.R2456H mutation were significantly higher than those with the p.L2495_E2496dup mutation (Student's t test, p=0.01). Some of the patients had splenomegaly, but it was not a universal finding. Although elevated MCHC is often considered a hallmark of HX, most of the individuals in this cohort had increased MCV and MCH but only high-normal MCHC. Varying percentages of hyper dense cells were detected in blood samples analyzed using an Advia Hematology System and stomatocytes were notable in most peripheral blood smears.Several complications have been associated with HX including hydrops fetalis, hemolytic episodes, thrombosis (especially after splenectomy), gallstones, and iron overload. One patient in our cohort had experienced ascites and a pleural effusion in the perinatal period and one was diagnosed with gallstones at age 11. Nearly all the patients had an increase in total bilirubin. Ferritin levels were increased in the majority of the patients tested after 15 years of age. The three patients older than 40 years of age had iron overload verified by T2* MRI imaging of the liver.HX presents several diagnostic challenges due to its heterogeneity. Two of the subjects in our study had been previously diagnosed with hereditary spherocytosis (HS) and one had the diagnosis of congenital dyserythropoietic anemia (CDA) prior to genetic evaluation. Misdiagnosis of individuals with HX as having HS is especially problematic since splenectomy is contraindicated in HX due to a greatly increased incidence of thromboembolic complications. Another diagnostic consideration concerns the use of osmotic fragility tests. Although osmotic fragility tests are frequently used to diagnose HX, ektacytometry provides greater accuracy. In the present study two patients who had ektacytometry results characteristic of HX had reportedly normal osmotic fragility tests in previous work-up. All individuals in our cohort with confirmed disease-causing mutations in PIEZO1 demonstrated the classic left shifted ektacytometry osmoscan indicative of erythrocyte dehydration. In addition, all individuals with these mutations demonstrated the expected decrease in erythrocyte potassium content not associated with a proportional increase in sodium. This illustrates the importance of specialized testing (ektacytometry and/or intracellular cation determinations) and the use of appropriately designed genetic panels to establish an accurate diagnosis in patients with hemolytic anemia of ambiguous phenotype, especially when splenectomy is being contemplated. DisclosuresQuinn:Amgen: Research Funding; Global Blood Therapeutics: Research Funding; Silver Lake Research Corporation: Research Funding.

WED 241 Clinical relevance of regular blood monitoring in IG treatment

BackgroundABN immunoglobulin (Ig) guidelines advise routine FBC and U and E monitoring with every treatment episode and screening for IgA deficiency. AimsWe audited compliance in inflammatory neuropathy patients on longterm treatment in two UK Neurology departments. We looked for evidence of clinically relevant haematological or AKI Ig-related events.MethodsData was collected from Nov 2015 to Nov 2017. Accepted definitions for clinically and/or biochemically significant haemolysis, neutropenia, thrombocytopenia and AKI were used.Results1919 treatment episodes in 90 patients were analysed. Mean age (SD)=57.6 (14.4)years, 69.1% male, 74% CIDP (26% MMN), 94% IVIg (6% SCIg). Mean dose=1.57 (0.74) g/kg/month or 97.1 (37.3) g/infusion. No clinically significant episodes of haemolysis, neutropenia, thrombocytopenia or AKI occurred in relation to Ig treatment. An asymptomatic drop of &gt;10 g/L Hb occurred in 68/1919 episodes in 38 individuals (3.5%); mean reduction 17.7 g/L, lowest Hb 99 g/L. Two patients with CRF (stage 3) received 28 (IV) and 104 (SC) infusions respectively without impact on eGFR. Two individuals with relative IgA deficiency (0.38 g/L, 0.4 g/L) received 16 infusions over 1.5 years without complications.ConclusionsNo clinically significant Ig-related events were identified in this representative cohort. We suggest annual screening or clinically indicated testing as safe and more appropriate in longterm IVIg use.

The clinical significance of CD(4)(+)CD(69)(+) T lymphocytes in patients with autoimmune hemolytic anemia/Evans syndrome

Objective: To investigate the significant of peripheral CD(4)(+) CD(69)(+) T lymphocytes in patients with autoimmune hemolytic anemia (AIHA)/Evans syndrome (ES). Methods: In this study peripheral blood samples from 32 patients with AIHA/ES (15 hemolytic episode patients, 17 remission patients) and 13 healthy controls were collected. Patients with AIHA/ES were recruited in Tianjin Medical University General Hospital from October 2015 to May 2016. The percentages of CD(69)(+) T lymphocytes were analyzed by flow cytometry. The expression of CD(69) mRNA in CD(4)(+) T lymphocytes which was sorted from peripheral blood by magnetic activated cell sorting (MACS) was detected using real-time PCR. Soluable CD(69) was measured by ELISA. Results: In hemolytic episode patients, the ratio of CD(3)(+)CD(69)(+)/CD(3)(+)T lymphocytes [(3.08±1.48)%] was significantly higher than that in healthy controls [(1.28±0.83)%, P<0.01] and in remission group[(1.96±1.33)%, P<0.05]. The absolute count of CD(3)(+)CD(69)(+)T lymphocytes in hemolytic episode group [(2.94±1.81)×10(7)/L] was higher than that in healthy controls [(1.48±1.42)×10(7)/L, P<0.05]. The ratio of CD(3)(+)CD(4)(+)CD(69)(+)/CD(3)(+)CD(4)(+)T cells in hemolytic episode group [(2.16±1.56)%] was significantly higher than that in remission group [(1.16±0.62)%, P<0.05] and healthy controls[(0.94±0.78)%, P<0.05]. The quantity of CD(3)(+)CD(4)(+)CD(69)(+)T lymphocytes in hemolytic episode group[(1.04±0.98)×10(7)/L] was higher than in healthy controls [(0.44±0.38)×10(7)/L, P<0.05]. The ratio of CD(3)(+)CD(8)(+)CD(69)(+)/CD(3)(+)CD(8)(+)T lymphocyte in hemolytic episode group [(4.87±2.56)%] was significantly higher than that in healthy controls[(1.83±1.27)%, P<0.01]. The quantity of CD(3)(+)CD(8)(+)CD(69)(+)T lymphocytes in three groups did not show significant difference. The ratio of CD(3)(+)CD(4)(+)CD(69)(+)/CD(3)(+)CD(4)(+) T lymphocytes in hemolytic episode group was negatively correlated with hemoglobin (Hb) (P<0.01) , positively correlated with the percentage of reticulocytes (Ret%) (P=0.01) total bilirubin(TBil), indirect bilirubin(IBil) (P<0.01) and not correlated with absolute reticulocytes count, lactic dehydrogenase (LDH), complement 3(C3), complement 4 (C4). The ratio of CD(3)(+)CD(4)(+)CD(69)(+)/CD(3)(+)CD(4)(+)T lymphocytes in remission group was negatively correlated with Hb (P<0.05). In hemolytic episode patients CD(69) mRNA (32.26±35.11) was significantly higher than that in remission group(6.05±5.87) (P<0.05) and healthy controls (1.76±1.85)(P<0.01). CD(69) mRNA in remission group was significantly higher than healthy controls (P<0.05). Serum CD(69) in hemolytic episode patients [(494.21±16.06) ng/L] was significantly higher than that in healthy controls [(441.39±104.6) ng/L, P<0.05]. Conclusion: Our findings suggest that the proportion of CD(4)(+)CD(69)(+) T lymphocytes increase in AIHA/ES patients, which is correlated with the severity of disease.

Digital epidemiology and geographic mapping of G6PD deficiency: retrospective analytic of trends database existing on the surface web

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an inherited X-linked recessive condition in which the body does not synthesise a sufficient quantity of the G6PD enzyme. Hemolytic anaemic episodes occur following exposure to some medications, foods, or even infections in G6PD-deficient individuals.Aims and Objectives: To assess the digital epidemiology as well as the geographic mapping of G6PD deficiency in the world including countries of the Mediterranean basin.Materials and Methods: This study is primarily based on a digital epidemiological analysis and geographic mapping based on data retrieved from Google Trends database, a very large database, existing on the surface web. A retrospective analysis was carried out for the period from the 22nd of March 2015 to the 24th of January 2016. Trends data will also be contrasted with a collateral data set of a cross-sectional analytic for the same period which was conducted based on the exploration of cases of G6PD deficiency admitted to Jordan University Hospital.Results: Concerning geographic mapping, countries of the Basin of the Mediterranean Sea and the Arabian Gulf accounted for one-third of the entire geographic map at 15.66% and 18.18% respectively. Countries of the Mediterranean basin included Jordan, Italy, Lebanon, Israel, Egypt, Greece, Syria, Cyprus, Tunisia, Algeria, and Morocco. Contributing countries surrounding the Arabian Gulf included Kuwait, Kingdom of Saudi Arabia, Bahrain, Iran, United Arab Emirates, Qatar, and Iraq. The Hashemite Kingdom of Jordan contributed to 2.78% of the global map which accounted for 17.74% of the entire Mediterranean basin. Concerning digital epidemiology, the prevalence was recorded as highest during May 2015 (12.10%), August 2015 (11.17%), and November 2015 (11.28%). Concerning the percentile contribution of monthly records, data were in harmony for those cases admitted to hospital and signals recorded via Google Trends. Both datasets averaged a percentile contribution of approximately 9% per month.Conclusion: This study is the first inferential research on G6PD deficiency based on data from a trends database and parallel data from Jordan University Hospital. Ambitious future research should deploy the use of machine learning for real-time and predictive analytics which will provide an excellent value for public health services and epidemiological studies.Asian Journal of Medical Sciences Vol.9(5) 2018 57-61

Induction of NLRP3 inflammasome activation by heme in human endothelial cells

Aim: Atherosclerosis, a chronic inflammatory disease is characterized by increased level of IL-1beta. Hemolytic or hemorrhagic episodes are often associated with inflammation. Heme released from RBCs is a DAMP and induce IL-1beta production through the activation of NLRP3 inflammasome in macrophages, however other cellular targets of heme-mediated inflammasome activation were not investigated.

MiR-150 regulates B lymphocyte in autoimmune hemolytic anemia/Evans syndrome by c-Myb.

The objective of the study was to study the regulation of B lymphocytes in patients with autoimmune hemolytic anemia (AIHA)/Evans syndrome. From October 2015 to May 2016, 35 patients with AIHA/Evans in the Department of Hematology, Tianjin Medical University General Hospital were enrolled into this study. c-Myb mRNA and miR-150 expression in B lymphocytes were measured using real-time PCR (RT-PCR). Correlation between c-Myb and miR-150 and clinical parameters in patients with AIHA/Evans were analyzed. c-Myb mRNA expression in hemolysis patients was significantly higher than in remission patients and CLL patients, negatively correlated with hemoglobin (Hb) level and complement 3(C3) levels, and positively correlated with total bilirubin (TBIL) concentration and indirect bilirubin (IBIL) concentration. miR-150 expression in hemolysis patients was significantly lower than in patients in remission and CLL patients. miR-150 was negatively correlated with TBIL and IBIL, and positively correlated with Hb, C3. c-Myb mRNA expression levels in hemolytic episode patients were negatively correlated with the expression levels of miR-150. The expression of c-Myb, a regulatory factor of B lymphocytes, is increased in B lymphocytes of AIHA/Evans patients, while miR-150 expression is decreased. c-Myb was negatively correlated with miR-150.

Examination of Hydroxychloroquine Use and Hemolytic Anemia in G6PDH-Deficient Patients.

Some sources urge caution when prescribing hydroxychloroquine (HCQ) to patients with G6PDH deficiency, presumably due to a risk of hemolytic anemia. There are limited published data, however, to support this risk. Additionally, not all patients with G6PDH deficiency are at similar risk for hemolysis, and people with the African variant are at particularly low risk. Through a retrospective chart review, we aimed to quantify the frequency of G6PDH-deficient patients with hemolysis attributed to HCQ. We identified Duke University Medical Center rheumatology patients with HCQ use and a measured G6PDH level. A retrospective chart review was performed, recording demographics, G6PDH levels, episodes of anemia, laboratory values consistent with hemolysis, and HCQ use. Of the 275 patients reviewed, 84% were female; 46% were African American and 48% were white. The leading diagnoses were systemic lupus erythematosus (32%), rheumatoid arthritis (29%), and inflammatory arthritis (14%). Only 4% of patients were G6PDH deficient (all African American). Two G6PDH-deficient patients had hemolysis during severe lupus flares that occurred while not taking HCQ. There were no reported episodes of hemolysis in more than 700 months of HCQ exposure among the 11 G6PDH-deficient patients. This is the largest study to date evaluating G6PDH deficiency with concurrent use of HCQ. Of 11 patients with G6PDH deficiency, 2 had episodes of hemolysis, but these did not occur during HCQ therapy. These data do not support routine measurement of G6PDH levels or withholding HCQ therapy among African American patients with G6PDH deficiency.

Pro-Inflammatory Actions of Red Blood Cell-Derived DAMPs.

Damage-associated molecular patterns (DAMPs) or alarmins are endogenous danger signals that are derived from damaged cells and extracellular matrix degradation, capable of triggering innate immune response to promote tissue damage repair. Hemolytic or hemorrhagic episodes are often associated with inflammation, even when infectious agents are absent, suggesting that damaged red blood cells (RBCs) release DAMPs.Hemoglobin (Hb) composes 96% of the dry weight of RBCs; therefore upon hemolysis, tremendous amounts of Hb are released into the extracellular milieu. Hb oxidation occurs outside the protective environment of RBCs, leading to the formation of different Hb oxidation products and heme. Heme acts as a prototypic DAMP participating in toll-like receptor as well as intracellular nucleotide-binding oligomerization domain-like receptor signaling. Oxidized Hb forms also possess some inflammatory actions independently of their heme releasing capability. Non-Hb-derived DAMPs such as ATP, interleukin-33, heat shock protein 70, as well as RBC membrane-derived microparticles might also contribute to the innate immune response triggered by hemolysis/hemorrhage.In this chapter we will discuss the inflammatory properties of RBC-derived DAMPs with a particular focus on Hb derivatives, as well as therapeutic potential of the endogenous Hb and heme-binding proteins haptoglobin and hemopexin in the prevention of hemolysis/hemorrhage-associated inflammation.

Induction of NLRP3 Inflammasome Activation by Heme in Human Endothelial Cells.

Hemolytic or hemorrhagic episodes are often associated with inflammation even when infectious agents are absent suggesting that red blood cells (RBCs) release damage-associated molecular patterns (DAMPs). DAMPs activate immune and nonimmune cells through pattern recognition receptors. Heme, released from RBCs, is a DAMP and induces IL-1β production through the activation of the nucleotide-binding domain and leucine-rich repeat-containing family and pyrin domain containing 3 (NLRP3) in macrophages; however, other cellular targets of heme-mediated inflammasome activation were not investigated. Because of their location, endothelial cells can be largely exposed to RBC-derived DAMPs; therefore, we investigated whether heme and other hemoglobin- (Hb-) derived species induce NLRP3 inflammasome activation in these cells. We found that heme upregulated NLRP3 expression and induced active IL-1β production in human umbilical vein endothelial cells (HUVECs). LPS priming largely amplified the heme-mediated production of IL-1β. Heme administration into C57BL/6 mice induced caspase-1 activation and cleavage of IL-1β which was not observed in NLRP3−/− mice. Unfettered production of reactive oxygen species played a critical role in heme-mediated NLRP3 activation. Activation of NLRP3 by heme required structural integrity of the heme molecule, as neither protoporphyrin IX nor iron-induced IL-1β production. Neither naive nor oxidized forms of Hb were able to induce IL-1β production in HUVECs. Our results identified endothelial cells as a target of heme-mediated NLRP3 activation that can contribute to the inflammation triggered by sterile hemolysis. Thus, understanding the characteristics and cellular counterparts of RBC-derived DAMPs might allow us to identify new therapeutic targets for hemolytic diseases.

Oxidized Mutant Human Hemoglobins S and E Induce Oxidative Stress and Bioenergetic Dysfunction in Human Pulmonary Endothelial Cells.

Cell free hemoglobin (Hb), becomes oxidized in the circulation during hemolytic episodes in sickle cell disease (SCD) or thalassemia and may potentially cause major complications that are damaging to the vascular system. Hemolytic anemias are commonly associated with pulmonary hypertension (PH) and often result from dysfunction of lung endothelial cells. The aim of this study was to determine the effect of different Hbs on cultured human lung endothelial function. Toward this goal, endothelial permeability, oxidative stress response parameters, glycolytic and mitochondrial bioenergetic functions were monitored in cultured human pulmonary arterial endothelial cells (HPAEC) following incubation with human adult Hb (HbA), and Hb isolated from patients with sickle cell Hb (HbS, βV6E) and HbE (βE26K) that commonly co-exist with β-thalassemia. These mutant Hbs are known for their distinct oxidative profiles. HPAEC treated with the ferrous forms of HbE, HbS for 24 h showed higher loss of endothelial monolayer integrity with concomitant rise in reactive oxygen radical production, lipid hydroperoxide formation and higher expressions of oxidative stress response proteins including heme oxygenase-1 (HO-1) accompanied by a rise in uncoupled mitochondrial respiration. Loss of membrane permeability was diminished in part by haptoglobin (Hp, protein scavenger), hemopexin (Hpx, heme scavenger) or ascorbate (reducing agent). To understand the role of Hb oxidation, HPAEC were exposed to ferric or ferryl states of the mutant Hbs. Ferryl forms of all proteins caused a significant damage to the endothelial monolayer integrity at a higher degree than their respective ferric Hbs. Ferryl forms of HbS and HbE also caused a loss of respiratory chain complex activities in isolated endothelial mitochondria and basal oxygen consumption in HPAEC. However, longer incubation with ferryl Hbs produced bioenergetic reprogramming including higher degree of uncoupled respiration and glycolytic rate. The data in this report collectively indicate that higher oxidation forms of HbS and HbE cause endothelial dysfunction through distinct damaging mechanisms involving mitochondrial bioenergetic function.

Cardiovascular complications in patients with sickle cell disease.

Sickle cell disease (SCD) is an autosomal recessive disease in which homozygosity for a single point mutation in the gene encoding the β-globin chain produces hemoglobin S molecules that polymerize within the erythrocyte during deoxygenation; the result is sustained hemolytic anemia and vaso-occlusive events. As patients live to adulthood, the chronic impact of sustained hemolytic anemia and episodic vaso-occlusive episodes leads to progressive end-organ complications. This scenario culminates in the development of 1 or more major cardiovascular complications of SCD for which there are no approved or consensus therapies. These complications include elevated pulmonary artery systolic pressure, pulmonary hypertension, left ventricular diastolic heart disease, dysrhythmia, sudden death, and chronic kidney disease with associated proteinuria, microalbuminuria, and hemoglobinuria. In patients with advancing age, cardiopulmonary organ dysfunction and chronic kidney injury have significant effects on morbidity and premature mortality. Over the last 15 years, a number of tests have been validated in multiple replicate cohort studies that identify patients with SCD at the highest risk of experiencing pulmonary and systemic vasculopathy and death, providing for screening strategies tied to targeted, more aggressive diagnostic and therapeutic interventions.

RETRACTED: Delayed Hemolytic Transfusion Reaction and Hyperhemolysis Syndrome without Detectable Alloantibodies or Autoantibodies in a Patient with Sickle Cell Disease: A Fatal Case Report and Literature Review.

L'éditeur a le regret de vous informer que cet article a déjà été publié dans: Journal of Blood Disorders & Transfusion, 2017, 8:4. DOI: 10.4172/2155-9864.1000388. Cette seconde publication faite par erreur a été retirée.

Structural analysis of clinically relevant pathogenic G6PD variants reveals the importance of tetramerization for G6PD activity.

Over 220 different amino acid variants have been identified in human glucose-6-phosphate dehydrogenase (G6PD), covering over 30% of the protein sequence. Many of these variants are pathogenic, causing varying degrees of G6PD deficiency with symptoms ranging from severe chronic anemia (class I) to milder triggered hemolytic episodes (classes II and III). The phenotypic effects of most G6PD variants have been reported, providing an opportunity to correlate phenotypic and structural information. In particular, we sought to investigate the tetramer interface of G6PD in relation to pathogenic variation, as there are conflicting reports indicating the importance of tetramerization for G6PD activity. Using a three-dimensional spatial scan statistic, hotspots of structural enrichment were identified for each class of pathogenic G6PD variants. Class I variants, the most phenotypically severe, were enriched at the dimer interface, consistent with previous evidence that dimerization is essential for G6PD activity. Class II variants were enriched near the tetramer interface, suggesting that tetramerization is also important for G6PD activity. This analysis explains why these two classes, both yielding 10% or less G6PD activity as compared to normal, lead to different clinical outcomes.

Wilson’s Disease Presenting with Severe Anaemia and Bleeding Manifestation: A Case Report

Wilson’s disease is an inherited disorder in which defective biliary excretion of copper leads to its accumulation, particularly in liver and brain. Presentation can vary, but the key features of Wilson’s disease are hepatic and neuropsychiatric disturbances, Kayser–Fleischer rings of the cornea, acute episodes of hemolysis and sometimes renal impairment. Here, we report a case of Wilson’s disease in a young female having severe anaemia without other evidence of hepatic and neuropsychiatric manifestation.Birdem Med J 2017; 7(3): 233-234

Sickle cell disease: an update on management

Abstract Sickle cell disease (SCD) is a common inherited disease affecting 12–15,000 individuals in the UK with approximately 250 new births per annum. Life expectancy has improved with the majority of those affected now surviving to adulthood, but it is associated with acute and chronic complications including haemolytic anaemia and intermittent episodes of severe bony pain, which may need hospital admission for management. Other acute complications include acute chest syndrome, stroke, priapism, splenic sequestration and red cell aplasia. Individuals with SCD also have an increased risk of infection and may develop renal dysfunction, respiratory complications and bony complications including avascular necrosis. Newborn screening will identify affected individuals and ensures early entrance into comprehensive care, which should include infection prophylaxis and primary stroke prevention by trans-cranial doppler screening. In addition annual review by a specialist team should continue throughout life. Optimal care provision comes from a strong multidisciplinary approach, with easy access to psychological services and an active community support team. With these measures, patients and their families can be educated to manage the minor complications of SCD with minimal impact to their daily lives and to recognize the more serious complications early, allowing quick and effective intervention to reverse the sickling process. Current treatments options are hydroxycarbamide (hydroxyurea), blood transfusion and haematopoietic stem cell transplant.