scholarly journals Semi-Automatic Enrichment with High Resolution/Selected Reaction Monitoring (HR/SRM) Scan for the Detection of Urinary Hepcidin in Patients with Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3418-3418
Author(s):  
Xionghao Lin ◽  
Elena Afia Adjei ◽  
Namita Kumari ◽  
Sharmin Diaz ◽  
Marina Jerebtsova ◽  
...  
Keyword(s):  
High Resolution ◽  
Sickle Cell Disease ◽  
Sample Preparation ◽  
Sickle Cell ◽  
Kidney Injury ◽  
Urinary Albumin ◽  
Selected Reaction Monitoring ◽  
Reaction Monitoring ◽  
Cell Disease ◽  
Urine Albumin

Abstract Background Urinary hepcidin is a potential biomarker of renal inflammation and acute kidney injury (AKI) which is elevated in sickle cell disease (SCD). Hepcidin in circulation is filtered through glomeruli filtration barrier and reabsorbed by the renal tubules. Hepcidin can also be synthesized by the kidney tubular cells. Thus, increased urinary levels of hepcidin may reflect either a reduction in tubular uptake or an increase in renal production. Recent studies suggested that urinary hepcidin may protect against AKI by attenuating heme-mediated injury. Thus decreased hepcidin levels in SCD patients may contribute to AKI and serve as potentially informative marker of SCD-associated kidney injury. Previously, hepcidin was measured by ELISA and mass spectrometry. Immunoassays are limited due to the cross-reactivity of antibodies to prohepcidin and truncated hepcidin-20, -22, and -24 isoforms of active hepcidin-25. Mass spectrometric assays are specific for hepcidin-25 but sample preparation remains a challenge. Objective To develop a sensitive, reliable and reproducible nanoLC/FT-MS method with simplified sample preparation for measuring of hepcidin in urine samples. Also to correlate urinary hepcidin with urinary albumin and urinary protein to access the degree of kidney dysfunction. Methods Samples were enriched and purified semi-automaticaly on 10-uL ZipTip and online trap column. Stable isotope-labeled hepcidin was used as internal standard. The standard concentration range was 1.56-800 nM and quality control samples were 5 nM, 20 nM, 80 nM and 400 nM. Samples were subjected to an LC-20AD nano HPLC system coupled to an LTQ XL™ Orbitrap mass spectrometer with an in-house made nano-HPLC column. High resolution/selected reaction monitoring (HR/SRM) scan was carried out and the narrow mass range ([M+H]+ ±0.01 Da) was used to extract ion chromatograms (EICs) for quantification. Urinary samples were collected from 20 SCD patients and 13 controls. Urinary albumin, protein and creatinine were detected by ELISA. The urine hepcidin concentrations were normalized to urine creatinine (Cr) values. Results Semi-automatic approach simplified sample preparation and accelerated the analysis. At least 24 samples could be prepared and processed at the same time. Online column trapping further purified and enriched hepcidin and improved the sensitivity and specificity of this method by eliminating interferences from urine. Hepcidin showed a good linearity within the concentration range of 1.56-800 nM with an r2 value of 0.9994. The precision intraday (n = 5) and interday (n = 5) and the repeatability (n=5) of the method were good with relative standard deviations (RSDs) lower than 5%. The analyzed samples were stable for 3 days at +4°C (RSDs<5%). The percent mean recoveries of hepcidin was within the acceptable range of 89.65-104.79%. We found that SCD patients had significantly lower (about 2-fold) urinary hepcidin levels compared to controls, and urinary hepcidin levels in 2 SCD patients were below the lower limit of detection (<0.5 nM). We found that there was no difference in urine albumin between SCD and control subjects, total urine protein was significantly increased in SCD patients. There was no positive correlation between urine hepcidin and urine albumin or total protein. Conclusion We developed an LC-MS based method for measuring levels of urinary hepcidin. This method is promising in terms of recovery, sensitivity, selectivity, repeatability and simplicity of sample preparation. SCD patients showed significantly decreased hepcidin levels in urine suggesting a potentially novel mechanism of AKI in SCD. Acknowledgments This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005 and 5G12MD007597). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hemopexin deficiency promotes acute kidney injury in sickle cell disease

Blood ◽  
2020 ◽  
Author(s):  
Solomon Ofori-Acquah ◽  
Rimi Hazra ◽  
Oluwaseun O Orikogbo ◽  
Danielle Crosby ◽  
Bethany Flage ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Kidney Injury ◽  
Risk Factor ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Inflammatory Process ◽  
Clinical Evidence ◽  
Kidney Injury ◽  
Red Cell ◽  
Cell Disease

Acute kidney injury (AKI) is a major clinical concern in sickle cell disease (SCD). Clinical evidence suggests that red cell alarmins may cause AKI in SCD however the sterile inflammatory process involved has hitherto not been defined. We discovered that hemopexin deficiency in SCD is associated with a compensatory increase in alpha-1-microglobulin (A1M) resulting in up to 10-fold higher A1M/hemopexin ratio in SCD compared to health controls. The A1M/hemopexin ratio is associated with markers of hemolysis and AKI in both humans and mice with SCD. Studies in mice showed that excess heme is directed to the kidneys in SCD in a process involving A1M causing AKI while excess heme in controls is transported to the liver as expected. Using genetic and bone marrow chimeric tools, we confirmed that hemopexin deficiency promotes AKI in sickle mice under hemolytic stress. However, AKI was blocked when hemopexin deficiency in sickle mice was corrected with infusions of purified hemopexin prior to the induction of hemolytic stress. This study identifies acquired hemopexin deficiency as a risk factor of AKI in SCD and hemopexin replacement as a potential therapy.

scholarly journals Relationship between Vaso-Occlusive Crises and Important Complications in Sickle Cell Disease Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2167-2167 ◽  
Author(s):  
Miranda Bailey ◽  
Ajibola Abioye ◽  
George Morgan ◽  
Tom Burke ◽  
Tim Disher ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Lower Bound ◽  
Sickle Cell ◽  
Research Group ◽  
Kidney Injury ◽  
Leg Ulcers ◽  
Cell Disease ◽  
Unmeasured Confounding ◽  
The Uk ◽  
The Relationship

Background: Sickle Cell Disease (SCD) describes a group of inherited hemolytic disorders caused by structurally abnormal variants of hemoglobin, which result in the sickle-shaped red blood cells (RBCs) that are characteristic of the disease. In patients with SCD, overexpression of adhesion molecules such as P-selectin bind sickled RBCs to endothelial cells; this contributes to hemolytic anemia and vaso-occlusive crises (VOCs), which are associated with severe acute and chronic pain. Patients with sickle cell disease often experience disease-related complications, affecting a diverse range of organs, thought to be due to the systemic impact of chronically inflamed vasculature, ongoing hemolysis and ischemic damage as a result of vaso-occlusive events. Many of these SCD-related complications are associated with significant morbidity and poor quality of life. The relationship between VOC frequency and the incidence of these complications is still being assessed. This study aimed to assess the relationship between the number of VOC experienced in the previous year and the occurrence of complications using real world evidence from the UK, specifically the Hospital Episode Statistics (HES) database. OBJECTIVE: To examine the relationship between the number of VOCs reported in the previous 12 months and the presence of SCD-related complications using a mixed modelling approach. METHODS: All patients reported with a diagnosis of SCD between 2008 and 2017 in the NHS England's HES database were identified. Detailed follow-up data on the number of vaso-occlusive crisis events and occurrence of complications was evaluated using ICD-10 diagnosis codes. Assuming no unmeasured confounding, the causal effect of VOCs, categorized into 3 groups (0, 1-2, 3+), was estimated using marginal structural models (MSM) for the complications reported in the dataset. To obtain inverse probability of treatment and censoring weights (IPTW and IPCW), the probability of being in each VOC category was estimated with a multinomial logistic model, and subsequently, the probability of being censored was estimated with a binary logistic model. The two models were adjusted for age, gender, ethnicity, and the occurrence in the previous 12 months of the 20 most common SCD complications and comorbidities in the dataset. Pooled logistic regressions were used to approximate the IPW-MSM Cox model. E-values were used to assess the minimum strength of association that an unmeasured confounder would have to have with both exposure (VOC) and outcome in order to fully explain away the observed relationship. Uncertainty in the magnitude of the E-value required to explain observed associations was explored by calculating values for both the point estimate and the lower bound of the confidence interval. RESULTS: A total of 15,076 patients were identified with a diagnosis of SCD in the HES database for this analysis. Patients had a median age of 30 and a female-male ratio of 1.7:1. A broad range of SCD related-complications were experienced by patients in the UK as shown in Table 1. Rates of some complications were observed less frequently than expected, in particular, leg ulcers, pulmonary hypertension, osteomyelitis, priapism and acute kidney injury, reported at <5% (Table 1). The hazard ratio associated with experiencing 3+VOCs versus 0 VOC in the previous year was calculated for all identified complications, resulting in a HR ≥5, for: priapism, osteomyelitis and acute chest syndrome; HR ≥2 to <5 for: gall stones, avascular necrosis, sepsis, cardiomegaly, pulmonary hypertension, CNS complications, leg ulcers, cellulitis, hyposplenism, liver complications and acute kidney injury. E-values (Table 1) suggest that most outcomes are robust to considerable unmeasured confounding, although large confidence intervals resulted in small lower-bound E-values for some outcomes (e.g. leg ulcers: 3.62 lower-bound: 1.00). Large E-values (>= 3 based on similar research in SCD) suggest results are robust to considerable unmeasured confounding, while small values imply greater fragility. CONCLUSIONS: This analysis shows that vaso-occlusive crises are related to the occurrence of important complications of sickle cell disease. Reducing the annual incidence of VOC may significantly lessen the ongoing organ damage and morbidity but may also improve the patient's quality of life with respect to these conditions. Disclosures Bailey: Novartis: Employment. Abioye:Novartis: Employment. Morgan:HCD Economics: Employment. Burke:HCD Economics: Employment. Disher:Cornerstone Research Group: Employment. Brown:Cornerstone Research Group: Employment. Bonner:Cornerstone Research Group: Employment. Herquelot:HEVA: Employment. Lamarsalle:HEVA: Employment. Raguideau:HEVA: Employment.

scholarly journals UGT1A1 (TA)n genotyping in sickle-cell disease: High resolution melting (HRM) curve analysis or direct sequencing, what is the best way?

2013 ◽  
Vol 424 ◽  
pp. 258-260 ◽  
Author(s):  
Vincent Thomas ◽  
Blandine Mazard ◽  
Caroline Garcia ◽  
Philippe Lacan ◽  
Marie-Claude Gagnieu ◽  
...  
Keyword(s):  
High Resolution ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Curve Analysis ◽  
Cell Disease

scholarly journals Acute kidney injury during parvovirus B19-induced transient aplastic crisis in sickle cell disease

2018 ◽  
Vol 93 (8) ◽  
pp. E198-E200 ◽  
Author(s):  
Jamie Oakley ◽  
Rima Zahr ◽  
Inmaculada Aban ◽  
Varsha Kulkarni ◽  
Rakesh P. Patel ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Parvovirus B19 ◽  
Kidney Injury ◽  
Cell Disease ◽  
Aplastic Crisis ◽  
Transient Aplastic Crisis

Myocardial Ischemia in Patients with Sickle Cell Disease: A Retrospective Review

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2189-2189
Author(s):  
Payal C. Desai ◽  
Nicole Kendel ◽  
Spero R Cataland ◽  
Eric H. Kraut ◽  
Ying Huang ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Chest Pain ◽  
Sickle Cell Disease ◽  
Myocardial Ischemia ◽  
Sickle Cell ◽  
Myocardial Injury ◽  
Kidney Injury ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Troponin Elevation

Abstract Introduction: On an autopsy study of 306 patients with sickle cell disease (SCD), cardiovascular disease is observed in 58% of patients and myocardial microinfarcts were noted in 20% of patients. Previously data from our center indicates that approximately 13% (3/22) patients demonstrated cardiac microvascular disease in steady state. However, the incidence of myocardial ischemic injury in patients with SCD presenting with chest pain remains largely undefined. Methods: We conducted a single institution retrospective chart review from September 2009 through September 2014 to evaluate the incidence of elevated troponin-I (normal < 0.11ng/ml), which is a well-established biomarker of myocardial injury, in patients with SCD and chest pain. We further characterize each of these episodes with cardiac magnetic resonance imaging, if available, and clinical and laboratory findings at the time of the event. Kruskal-Wallis test was used to compare troponin measurement values among different groups of patients. Results: A total of 25 (10 female, 15 male) of the 352 (7%) of patients followed at the Ohio State Comprehensive Sickle Cell Center had troponin elevation over a 5 year period. They had a total of thirty-eight individual encounters with troponin elevations (range: 0.11-12.17) [72% patients with 1 elevation, 28% patients multiple troponin elevations) (range: 1-4 incidences)]. The median age at the time of troponin elevation was 36 (Range: 20.5-66 yrs). Troponin elevation was observed in patients of all genotypes (76% SS; 4% SBeta+; 20% SC). 6/25 (25%) of patients with troponin elevation in the past five years are now deceased. Thirteen patients (52%) had acute chest syndrome and ten patients (40%) had acute kidney injury at the time of troponin elevation. The degree of troponin elevation was not associated with concurrent acute chest syndrome, concurrent acute kidney injury or mortality. At the encounter level, median troponin at diagnosis was 0.3 (range: 0.11-12.2) and the median peak troponin was 0.4 (range: 0.11-38.1),. The median value of TR jet velocity at baseline was 2.8 (range: 1.1-3.7) (n=23) and the median TR jet velocity at the time of elevation was 3.0 (range: 1.7-4.6) (n=28). Median baseline hemoglobin was 8 (range: 4.5-12.6) and median hemoglobin at encounter was 7.5 (range: 4.3-12.5), resulting in a median change in hemoglobin of -0.4 (range: -6.2 - 2.5). Four of 10 MRI obtained at the time of troponin elevation showed myocardial ischemia and 3/10 patients showed late gadolinium enhancement indicating myocardial injury. Conclusion: Patients with SCD presenting with chest pain have myocardial ischemia and infarctions as demonstrated in our population by both troponin elevation and cardiac imaging. While, the long term implications of this finding are currently being studied in a multi-centered prospective study, further cardiac evaluations should be considered in patients with SCD presenting with chest pain. Disclosures Desai: Pfizer: Consultancy. Cataland:Ablynx: Consultancy. Raman:Siemens: Consultancy.

scholarly journals Acute kidney injury in children with sickle cell disease—compounding a chronic problem

2017 ◽  
Vol 32 (8) ◽  
pp. 1287-1291 ◽  
Author(s):  
Cherry Mammen ◽  
Mei Lin Bissonnette ◽  
Douglas G. Matsell
Keyword(s):  
Acute Kidney Injury ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Kidney Injury ◽  
Cell Disease ◽  
Chronic Problem

scholarly journals PERIOPERATIVE MANAGEMENT OF SICKLE CELL DISEASE: A NARRATIVE REVIEW

2018 ◽  
Vol 10 (1) ◽  
pp. 2018032 ◽  
Author(s):  
Kwame Ofori Adjepong ◽  
Folashade Otegbeye ◽  
Yaw Amoateng ADJEPONG
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Kidney Injury ◽  
Operative Management ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Volume Depletion ◽  
Transfusion Requirements ◽  
Intravascular Volume Depletion ◽  
Operative Assessment

An estimated 30 million people worldwide have sickle cell disease (SCD).  Emergent and non-emergent surgical procedures in SCD have been associated with relatively increased risks of peri-operative mortality, vaso-occlussive (painful) crisis, acute chest syndrome, post-operative infections, congestive heart failure, cerebrovascular accident and acute kidney injury.  Pre-operative assessment must include careful review of the patient’s known crisis triggers, baseline hematologic profile, usual transfusion requirements, pre-existing organ dysfunction and narcotic use. Use of preoperative blood transfusions should be selective and decisions individualized based on the baseline hemoglobin, surgical procedure and anticipated volume of blood loss.  Intra- and post-operative management should focus on minimizing hypoxia, hypothermia, acidosis, and intravascular volume depletion. Pre- and post-operative incentive spirometry use should be encouraged. 

scholarly journals Hemopexin Replacement Therapy Protects Sickle Cell Disease Mice from Acute Kidney Injury

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 78-78
Author(s):  
Samit Ghosh ◽  
Oluwaseun Orikogbo ◽  
Rimi Hazra ◽  
Bethany Flage ◽  
Danielle Crosby ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Kidney Injury ◽  
Sickle Cell Disease ◽  
Replacement Therapy ◽  
Sickle Cell ◽  
Bone Marrow Cells ◽  
Kidney Injury ◽  
Renal Tissue ◽  
Acute Chest Syndrome ◽  
Cell Disease

Acute kidney injury (AKI) is a major clinical concern during episodes of acute chest syndrome and vaso-occlusive crisis in sickle cell disease (SCD). AKI increases the risk of chronic kidney disease (CKD) and end stage renal disease (ESRD). We previously showed that alpha-1-microglobulin (A1M), a low affinity heme-binding protein that carries heme for renal clearance is elevated in patients and mice with homozygous SCD (SS). Hemopexin (Hx), which primarily scavenges circulating heme to the liver, is exhausted in SCD. In this study, we explored the idea that acquired Hx deficiency in SCD is a risk factor for AKI development in SCD. We studied mice with a global knockout of Hx with no detectable plasma Hx at baseline. Plasma A1M was significantly elevated in the Hx-/-mice compared to control (Hx+/+) mice (n=5; p&lt;0.01). We then transplanted whole bone marrow cells from SS mice into Hx+/+and Hx-/-mice to create bone marrow chimeric SSHx+/+and SSHx-/-mice with SCD phenotype. The chimeras had elevated plasma A1M compared to recipient littermates (Hx+/+and Hx-/-) and low baseline glomerular filtration rate (GFR). Modest elevation of circulating heme with infusion of hemin (20 μmoles/kg bw) worsened GFR and caused severe AKI. Next, to determine whether hemin induced AKI is attenuated by elevation of circulating Hx, we infused SS mice with purified Hx, and control SS mice with either purified A1M or vehicle immediately prior to the hemin challenge. We observed improved GFR in the Hx-treated SS mice, while vehicle and A1M-treated mice suffered 23% and 39% loss of GFR respectively compared to their baseline. In agreement with the GFR data, the levels of several AKI diagnostic markers, plasma creatinine (plasma Cr), urinary albumin-creatinine ratio (uACR) and kidney injury molecule (uKIM-1) were elevated significantly in vehicle and A1M treated mice following hemin challenge. In Hx-treated mice, these biomarkers remained unaltered. Importantly, Hx infusions re-directed excess heme in SS mice to the liver, while A1M infusion significantly increased total heme content in the kidneys. Two-way ANOVA analysis of GFR (p&lt;0.01) and plasma Cr (p&lt;0.001) revealed significant exacerbation of kidney injury in A1M treated SS mice compared to vehicle treated mice. Histopathology of renal tissue showed considerable tissue damage in vehicle and A1M infused SS mice, while Hx treated SS mice kidneys appeared relatively normal. This study provides genetic evidence that hemopexin deficiency promotes AKI development in SCD, and we provide proof-of-principle for hemopexin replacement therapy to treat AKI in SCD. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship.

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