Elevated Hb Research Articles

UGT1A1 promoter polymorphism associated with serum bilirubin level in Saudi patients with sickle cell disease

BACKGROUND AND OBJECTIVESPolymorphism in (TA)n of the UGT1A1 promoter influences bilirubin level and risk of gallstones in patients with sickle cell disease (SCD) of African descent. Modifiers of bilirubin level and gallstones in Saudi patients with SCD are not known.DESIGN AND SETTINGSPatients with SCD presenting to participating institutions between July 2009 and July 2012 were enrolled in our study.METHODSA total of 223 SCD patients were enrolled. Laboratory workup at steady state included complete blood count, reticulocytes, serum bilirubin, lactate dehydrogenase (LDH), G6PD level, and hemoglobin (Hb) electrophoresis. The (TA)n UGT1A1 promoter polymorphism and presence of α-thalassemia were also determined.RESULTSTA6/6 in the UGT1A1 promoter was identified in 189 patients (84.7%), TA7/7 in 26 (11.7%), TA5/5 in 6 (2.7%), and TA5/6 in 2 (0.9%). Increased (TA)n of the UGT1A1 promoter (P<.0001), male gender (P=.02), higher LDH (P=.001), and lower Hb level (P=.009) were associated with higher bilirubin level, while the co-inheritance of α-thalassemia (P=.003) was linked with lower bilirubin level. UGT1A1 (TA)n (P<.0001) and Hb level (P=.005) remained significant on multivariate analysis. Gallstones were more frequent in patients with TA7/7 (72%) compared to patients with TA6/6 (57%) and TA5/5 or 5/6 (37%); however, this difference was not statistically significance (P=.18). Older age (P=.0001) and absence of α-thalassemia (P=.03) were associated with higher risk of gallstones.CONCLUSION(TA)n in the UGT1A1 promoter and intensity of hemolysis modify steady-state serum bilirubin level in SCD. Co-inheritance of α-thalassemia reduces the risk of gallstones in Saudi patients with SCD.

KLF1 Gene Mutations in Chinese Adults with Increased Fetal Hemoglobin

We investigated the Krüppel-like factor 1 (KLF1) gene mutations in Chinese adults with increased Hb F levels (>1.5%) referred to our laboratory for thalassemia screening. Functionally effective KLF1 mutations were identified in five out of 140 samples with an elevated Hb F (1.9-11.4%). Only two different KLF1 mutations were detected. Functional KLF1 mutations were not identified in the matched cohort of 110 samples with normal Hb F values (<1.0%). The KLF1 mutations could be one of the causes of hereditary persistence of fetal hemoglobin (HPFH) in regions where thalassemias are common.

Nephroprotective activity of Cocculus hirsutus leaf extract in 5/6 nephrectomized rat model

Cocculus hirsutus, commonly known as broom creeper, belongs to the family Menispermaceae. It is widely used in folk medicine to treat leprosy, skin diseases, dyspepsia, etc. Hence, an effort has been made to investigate the nephroprotective potential of C. hirsutus. The nephroprotective activity of ethanolic C. hirsutus leaf extract (ECHE) in the 5/6 nephrectomized rat model was investigated. Different parameters like postoperative survival rate, change in body weight and levels of red blood corpuscles (RBCs), hemoglobin (Hb), triglyceride, cholesterol, creatinine, urea and uric acid were estimated in experimental rats. The findings revealed the postoperative survival rate of rats in the investigated novel method to be 100%. Change in the body weight of ECHE-treated groups I and II was found to be 32 and 30 g, respectively. These values suggested that ECHE treatment normalized the elevated body weight levels in experimental rats. Furthermore, ECHE treatment normalized the decreased RBC levels and the elevated Hb, triglyceride, cholesterol, creatinine, urea and uric acid levels in experimental rats. The results indicate that C. hirsutus has strong nephroprotective activity. However, further scrutiny is essential for isolation and characterization of the active components that can be employed to allay various human maladies.

Abstract P5-20-03: A phase 2, double-blind, randomized, placebo-controlled, dose-finding study of sotatercept for the treatment of patients with chemotherapy-induced anemia and metastatic breast cancer.

Abstract Background: Sotatercept is a recombinant activin receptor IIA (ActRIIA) ligand trap and is comprised of the extracellular domain of ActRIIA linked to the Fc domain of human IgG1 (ActRIIA-IgG1). Results of preclinical and early clinical studies provide evidence that sotatercept increases the concentration of hemoglobin (Hb) in blood and may constitute a novel treatment for chemotherapy-induced anemia (CIA), a condition that results in significant morbidity. Methods: This study evaluated the safety of, and hematopoietic response to, sotatercept in patients with CIA and metastatic breast cancer (MBC). Response was defined as increase in Hb ≥1 g/dL for ≥28 consecutive days during treatment or ≤2 months following the last dose, in the absence of red blood cell (RBC) transfusion or treatment with an erythropoiesis stimulating agent (ESA). If RBC transfusion or treatment with an ESA was required, no Hb measurements within 28 days of the RBC transfusion or administration of ESA were used to determine the Hb response. Subjects were randomized to subcutaneous injection of sotatercept (0.1, 0.3, or 0.5 mg/kg) or placebo and were treated on day 1 of each of four 28-day cycles. Results: Thirty (30) subjects were enrolled and treated (5 placebo, 25 sotatercept). Increases in mean Hb in the 0.3 and 0.5 mg/kg groups were greater than in 0.1 mg/kg and placebo groups. Increases peaked approximately 15 days following treatment and declined over the remaining interval between treatments. Among 23 subjects with confirmed CIA and administered ≥1 dose and followed for ≥57 days (per-protocol analysis), 5/18 (28%) administered sotatercept responded (0/5 [0%] at 0.1 mg/kg; 3/9 [33%] at 0.3 mg/kg; 2/4 [50%] at 0.5 mg/kg) vs 1/5 [20%] administered placebo. Among 13 nonresponders administered sotatercept, 5 (39%) experienced ≥1 dose interruption/reduction, as per protocol, due to elevated Hb. The incidence of adverse events (AEs) was consistent with that reported in patients with MBC. No dose-limiting or dose-related toxicity was observed. Conclusions: Sotatercept demonstrated dose-dependent hematopoietic activity in subjects with CIA and MBC. The safety findings were generally consistent with the known safety profile of sotatercept. These data suggest that a greater dose of sotatercept or a dose interval &amp;lt;28 days might result in a more sustained, and greater rate of, hematopoietic response. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-20-03.

Prediction of Venous Thromboembolism (VTE) in Patients with Pancreatic Cancer Using Clinical Data, Biomarkers, and VTE Risk Models

Abstract 3398 INTRODUCTION:VTE is common in patients with cancer and causes significant morbidity and mortality. Clinical risk models and biomarkers including C-reactive protein (CRP), soluble P-selectin (sPsel), and D-dimer have been used to predict VTE in diverse groups of cancer patients at varying risk for VTE. The applicability of these findings to specific high risk subtypes of cancer has not been established. Therefore, we sought to identify the value of clinical factors, plasma biomarkers, and risk models in predicting VTE in patients with pancreatic cancer, a malignancy with a high predilection for VTE. METHODS:Patients seen at the University of Michigan Comprehensive Cancer Center (UMCCC) and previously consented and enrolled in a prospective cohort study were eligible. Inclusion criteria are diagnosis of pancreatic adenocarcinoma, evaluation at UMCCC, no VTE within a month prior to cancer diagnosis, and documentation in the Electronic Medical Record (EMR) at least every 6 months until death. Primary objective was to identify factors predictive of VTE. Secondary objectives were to develop a VTE predictive model, assess the utility of published VTE risk models, and evaluate factors associated with overall survival (OS). Demographics, clinical data, and VTE (deep vein thrombosis [DVT], portal vein thrombosis [PVT], or pulmonary embolism [PE]) rate were obtained from the EMR. ELISAs were performed for CRP, D-dimer, Mac-2 binding protein, soluble E-selectin (sEsel), and sPsel using banked plasma specimens drawn at diagnosis. A retrospective cohort study was performed including univariate and multivariate regression analysis. The utility of predictive models by Khorana, et al (Blood, 2008. 111:4902–4907), which includes cancer site, body mass index (BMI), hemoglobin (Hb), platelet (plt) count, and white blood cell count, and the expanded model by the Vienna Cancer and Thrombosis Study (CATS) (Blood, 2010. 116:5377–5382), which additionally includes sPsel and D-dimer, were assessed. RESULTS:Between 2005 and 2011, 89 patients were eligible for analysis. Median follow-up was 268 (18–2433) days. Twenty (22%) cases had a VTE; 10 (50%) DVT, 2 (10%) PE, 4 (20%) PVT, and 4 (20%) multiple VTEs. Mean (SD) age was 63.4 (8.9) in cases and 65.3 (11.2) in controls. Women accounted for 55% of cases and 48% of controls.Higher BMI (median 28.8 [21.2–44.7] in cases vs. 25.4 [16.4–43.3] in controls, p=0.03) and lower plt count (median 241 [145–323] in cases vs. 289 [97–648] in controls, p=0.001) were associated with VTE on univariate analysis. On multivariate regression analysis, lower plt count (β −0.01, SE 0.004) and lower Hb (β −0.43, SE 0.20) were predictive of VTE after adjusting for BMI, tumor location, and treatment with surgery, chemotherapy or radiation (AUC 0.78). None of the biomarkers were significantly associated with VTE on univariate analysis, although there was a trend with D-dimer (p=0.09).The Khorana score was determined in 85 patients; 48 were intermediate (2 points) and 37 high risk (≥3 points) with VTE rates of 20.8% and 24.3%, respectively (p=0.70). The AUC of this model was 0.63. The risk score from CATS was calculated for 84 patients; 54 were intermediate (2 or 3 points), 17 high (4 points), and 13 highest risk (≥5 points). VTE incidence was not different among these groups and the AUC was 0.65.Factors associated with poor OS on univariate analysis were: age (per 10-year increment) (HR [95% confidence interval], p-value) (1.35 [1.07–1.71], 0.013), chronic kidney disease (5.67 [2.62–12.25], <0.0001), use of anticoagulation (3.14 [1.33–7.41], 0.009), stage III/IV vs. I/II pancreas cancer (2.05 [1.27–3.32], 0.003), and INR (1.65 [1.04–2.63], 0.035); elevated Hb (0.87 [0.76–0.99], 0.041) and sEsel (0.46 [0.29–0.72], 0.0007) were protective. CONCLUSIONS:Pancreatic cancer patients with higher BMI, lower plt count, and lower Hb were more likely to develop VTE. Other clinical variables and biomarkers did not add additional predictive information. Elevated sEsel, important for neutrophil trafficking to sites of inflammation, was found to be protective on survival analysis. The risk models developed by Khorana, et al and CATS in a diverse group of patients with cancer were not able to further differentiate VTE risk among this already high risk group. Additional work is needed to determine which patients with pancreatic cancer are at highest risk for VTE and who may benefit most from thromboprophylaxis. Disclosures:No relevant conflicts of interest to declare.

Treatment of critically ill children with kidney injury by sustained low-efficiency daily diafiltration

BackgroundContinuous renal replacement therapy (CRRT) and intermittent hemodialysis (IHD) offer diverse benefits and drawbacks for critically ill children with acute kidney injury (AKI). Sustained low-efficiency daily diafiltration (SLEDD-f) involves a conceptual and technical hybrid of CRRT and IHD. We report our SLEDD-f application to critically ill children in the pediatric intensive care unit (PICU).MethodsSLEDD-f was delivered by the new Fresenius 5008 therapy system with blood flow 5 ml/kg/min, dialysate flow 260 ml/min, hemofiltration 35 ml/kg/h for 8–10 h daily. Changes in blood pressure, blood gas, electrolyte, hemoglobulin (Hb), and hematocrit (Hct) were closely monitored.ResultsFrom February 2010 to June 2011, 14 critical patients with a total of 60 SLEDD-f sessions were studied retrospectively. Heparin was used in 46 sessions (76.6%) with no bleeding complications. Hypertension above 135 mmHg returned to normal, hypotension below 90 mmHg showed no drop. Metabolic acidosis and hyperkalemia normalized. Elevated Hb, Hct, and their ratio revealed improving hemodilution. Three episodes of intradialytic hypotension (5.0%) and one of circuit clotting (1.7%) led to premature termination. The 28-day survival rate was 71.4%.ConclusionsThis pilot investigation demonstrates that SLEDD-f provides good hemodynamic tolerance and correction of fluid overload, pH, and electrolyte imbalance for critically ill children with AKI.

Effects of photoperiod, light intensity, and their interaction on blood physiological variables of modern broilers grown to heavy weights

This study examined the effects of photoperiod, light intensity, and their interaction on blood acid‐base balance, metabolites, and electrolytes in broilers under environmentally controlled rooms in 2 trials. The study consisted of a factorial arranged in a randomized complete block design with 9 treatments that consisted of 3 photoperiod (continuous [23L:1D] from d 8 ‐ d 56; intermittent [2L:2D], and short [8L:16D] from d 8 ‐ d 48 and 23L:1D from d 49 ‐ d 56, respectively) and exposure to 3 light intensities (10, 5.0 and 0.5 lx) from day 8 through d 56 at 50% RH. A total of 540 1‐d‐old (30 male/30 female) chicks were randomly distributed into 9 chambers. Feed and water were provided ad libitum. Venous blood samples were collected on d 14, 28, 42, and 56. Short photoperiod significantly (P ≤ 0.05) reduced BW, pH level, pO2, sO2, and Ca2+, while significantly (P ≤ 0.05) elevated pCO2, Hct, Hb, and McHc concentrations. There was only an effect of light intensity on K+. Acid‐base regulation during short photoperiod and light intensity exposure had not deteriorated despite higher pCO2 that consequently decreased blood pH, due to a respiratory acidosis. Plasma corticosterone was not affected by photoperiod, light intensity or their interaction, indicating that exposure of broiler chickens to short photoperiod markedly affects some physiological blood variables without inducing stress in modern broilers grown to heavy weights.

Ten novel mutations in the erythroid transcription factor KLF1 gene associated with increased fetal hemoglobin levels in adults

We investigated whether mutations in the KLF1 gene are associated with increased Hb F levels in ethnically diverse patients referred to our laboratory for hemoglobinopathy investigation. Functionally effective KLF1 mutations were identified in 11 out of 131 adult samples with an elevated Hb F level (1.5-25.0%). Eleven different mutations were identified, 9 of which were previously unreported. KLF1 mutations were not identified in a matched cohort of 121 samples with normal Hb F levels (<1.0%). A further novel KLF1 mutation was also found in a sickle cell disease patient with a Hb F level of 20.3% who had a particularly mild phenotype. Our results indicate KLF1 mutations could make a significant contribution to Hb F variance in malarial regions where hemogobinopathies are common. All the mutations identified were heterozygous providing further in vivo evidence that a single altered KLF1 allele is sufficient to increase Hb F levels.

Stress Erythropoiesis and Genetic Regulation of Fetal Hemoglobin in Inherited Bone Marrow Failure Syndromes

Abstract 2401Patients with inherited bone marrow failure syndromes (IBMFS) frequently have manifestations of what has been called “stress erythropoiesis”. This includes macrocytosis (increased mean cell volume, MCV), increased fetal hemoglobin (Hb F) and erythropoietin (Epo) levels higher than predicted by the degree of anemia (red blood cell count, RBC). In patients with hemoglobinopathies (sickle cell disease, thalassemia, and hereditary persistence of fetal hemoglobin), Hb F levels are regulated by 3 quantitative trait loci (QTL), located at HBSIL-MYB on chromosome 6q, BCLIIA on chromosome 2p and XMN1-Gg representing HBB cluster on chromosome 11p. The role of these QTLs in the elevated Hb F levels in patients with an IBMFS has not been previously reported. Percent Hb F was measured by HPLC in blood from 97 untransplanted individuals with an IBMFS. Absolute Hb F (g/dL) was calculated by multiplication of Hb F% times total Hb in order to include data from transfused patients, and log-transformed to approximate a normal distribution. Epo levels were also log-transformed due to the wide range (8 to 1800 mU/mL). DNA was extracted from leukocytes, and candidate regions were amplified and genotyped by TaqMan. The candidate QTLs were evaluated by genotyping of tagging single nucleotide polymorphisms (SNPs): five for HBSIL-MYB, two for BCLIIA, and one for XMN1-Gg. Data were modeled using a generalized linear model (GLM), appropriate for data with a constant coefficient of variation. There were 31 patients with Diamond-Blackfan anemia (DBA), 35 with dyskeratosis congenita (DC), 25 with Fanconi anemia (FA), and 6 with Shwachman-Diamond syndrome (SDS). Hb F was elevated in 70% of the total group of patients: 48% of DBA, 83% of DC, 76% of FA, and 83% of SDS. In the pooled group of 97 IBMFS patients, 68 (70%) had Hb F >1 % (upper limit of normal), 59 (61%) were macrocytic, 55 (57%) were anemic for age, and 70 (77%) had elevated Epo. The frequencies of heterozygosity or homozygosity for the alternative alleles for the QTLs were 50% for HBSIL-MYB, >90% for BCLIIA, and 52% for XMN1-Gg. The multivariate model for Hb F in the total goup of IBMFS included the alternative allele for the XMN1-Gg SNP (p = 0.04), younger age (p<0.001), male sex (p=0.04), and increased Epo (p<0.001). In this model, the alternative allele for the XMN1-Gg QTL was associated with a 32% increase in the level of Hb F. Subset analyses indicated that the strongest association of the XMN1-Gg QTL was in FA and DC (increased Hb F by 68% and 48% respectively, p-values 0.02 and 0.09) and had no effect in DBA (decreased Hb F by 18%, p = 0.6). Data including the other QTLs were not significant. These results suggest that the alternative allele at XMN1-Gg is associated with the increased level of Hb F in FA and DC, but not DBA, after adjustment for age, sex, and Epo level. A low level of Hb F should not exclude the diagnosis of an IBMFS in a patient who has other signs of stress erythropoiesis (anemia with increased MCV and Epo), since that patient may not have the variant allele associated with increased Hb F. The degree of elevation of Hb F in FA and DC depends on the alleles at the XMN1-Gg QTL. A strength of this study is the sample size of almost 100 patients with an IBMFS who are well-characterized. A limitation is that the number within each syndrome is still small; the role of the other QTLs may be identified in future larger studies. Of major interest is that this is the first study to show regulation of Hb F by the same QTL in FA and DC as the common hemoglobinopathies, thus linking Hb F regulation across disparate hematologic disorders. Disclosures:No relevant conflicts of interest to declare.

Nine Novel Mutations in the Erythroid Transcription Factor KLF1 Gene Associated with Increased Fetal Hemoglobin Levels in Adults,

Abstract 3191Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition defined as an increased proportion of fetal hemoglobin which persists beyond infancy. HPFH has been identified in a wide range of ethnic groups but is more common in populations with a high prevalence of hemoglobinopathies and thalassemias. This is thought to be due to selective pressure resulting from the ameliorating effect which HPFH can have in some of these disorders. Consequently, although it can occur as a single entity it is often observed as a co-existing condition in a variety of hemoglobin disorders. Although several well established genetic factors have been identified that can cause the HPFH phenotype, they only account for <50% of the Hb F variance observed in adults. More recently, mutations in the erythroid transcription gene KLF1 have been found to be associated with a HPFH phenotype in a single family from Malta (Borg et.al. Nat Genet. 2010; 42(9): 801–805) and in a single family from Sardinia (Satta et.al. Haematologica 2010; 96(5):767–70).We therefore decided to investigate whether KLF1 mutations could be associated with increased Hb F levels observed in patients referred to our laboratory for hemoglobinopathy investigation. 130 adult patient samples with an elevated Hb F level above 1.5% (range 1.5 – 25%) were studied for KLF1 mutations. 55 of these samples had co-existing alpha thalassemia trait, 6 were carriers for sickle cell trait, 28 were beta thalassemia carriers and 41 had no evidence of any other hemoglobinopathy. The results showed that 8% of the cohort had a mutation in the KLF1 gene. 10 different mutations which were predicted to be deleterious (PolyPhen-2 and SIFT) were identified in 10 patients, 8 of which are previously unreported (c.159_169 del GAAGTCTGAGG, c.901C→T, c.902G→A, c.939G→T, c.1001C→A, c.913+1G→A, c.526_527Ins CGGCGCC, c.152T→G). No deleterious KLF1 mutations were identified in a matched cohort of 101 samples referred for hemoglobinopathy investigation that had normal Hb F levels (<1.0%), strongly suggesting that the KLF1 mutations are associated with a HPFH phenotype.In addition, 35 sickle cell disease patients with Hb F levels greater than 10% were investigated. One further unreported deleterious KLF1 mutation (c.914–4_914–1 del CTAG) was found in a sickle cell disease patient with a Hb F level of 20.3%. Interestingly this patient appeared to have a particularly mild phenotype and maintained a hemoglobin level of 12.7g/dl.The KLF1 mutations were predominantly found in individuals of African, Indian and Southeast Asian descent, indicating that KLF1 mutations could be a widespread cause of HPFH in malarial regions where hemogobinopathies are common, possibly making a significant contribution to Hb F variance in these populations. All the mutations identified were heterozygous, with only one case of compound heterozygosity, suggesting that haploinsufficiency for KLF1 may be sufficient to increase Hb F levels. This in turn provides further in-vivo evidence that controlled reduction of KLF1 gene expression could activate fetal hemoglobin expression and provide a potential therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Case Report of Erythroid Transcription Factor EKLF Mutation Causing a Rare Form of Congenital Dyserythropoetic Anemia in a Patient of Taiwanese Origin

Abstract 2154Congenital dyserythropoietic anemias (CDA) are a rare, inherited form of blood disorders characterized by dyserythropoiesis in the bone marrow, anemia, jaundice and splenomegaly. There are three major types of CDAs, although rarer variants have been identified. We describe a patient with an unusual type of CDA, of which only four other cases have been reported. Our patient had severe hemolytic anemia, increased fetal hemoglobin and abnormal bone marrow pathology inconsistent with previously described forms of CDA. On further study he was also found to have a mutation in KLF1, the gene encoding Erythroid-Kruppel like growth factor (EKLF). Here we describe the full clinical characteristics of our patient and define the diagnostic clinical features of this new variant of CDA by comparing with one of the previously reported patients designated as ME in the table.EKLF is an erythroid specific transcription factor that is essential for b-globin expression, the switch from fetal to adult globin and definitive erythropoiesis (Siatecka, M and Bieker, J. Blood prepublished May 2011). Various mutations in KLF1 have been identified, some causing the benign In(Lu) type of Lu blood group phenotype. Recently, a missense, dominant-negative KLF1 mutation was reported, c.973G>A, which resulted in a previously unidentified type of CDA (Singleton et al. ASH Abstract 162, 2009; Arnaud et al. Am J Hum Genet 2010,87:721-727).The G-to-A transition in exon 3 of KLF1 results in the substitution of glutamate 325 by a lysine (E325K) in the second zinc finger. The mutated area of the zinc finger was found to be essential for binding of EKLF to DNA motifs causing a profound dysregulation of globin gene expression. The mutation was found to have a dominant-negative effect on the transcriptional activity of EKLF, thus making the heterozygous patients symptomatic.Our patient, JL, is an 8 year old male Taiwanese immigrant found to have hyperbilirubinemia and anemia at birth. He is a developmentally normal child with height 10th centile, weight in the 25th centile and spleen palpable to his suprapubic area. He has chronic hemolytic anemia, with baseline hemoglobin 7–9 g/dL, MCV 83 fL and RDW 22% and reticulocyte count 16%. Peripheral blood smear shows marked anisopoikilocytosis, schistocytes, mild polychromasia and nucleated RBCs, many with double nuclei. Bone marrow aspirate revealed a hypercellular marrow with erythroid hyperplasia and dyserythropoiesis. Electron microscopy analysis of the bone marrow showed rare immature erythroid cells with marked heterochromatin. Several cells showed a peripheral double membrane of the cytoplasm and there was rare invagination of nuclear membrane with intranuclear precipitated material.JL received blood transfusions every 2 months for the first 3 years of his life while living in Taiwan. Since moving to the U.S. in 2003, he has only received 2 transfusions secondary to aplastic crisis. Osmotic fragility testing showed mildly increased increased fragility. Hemoglobin electrophoresis revealed an elevated fetal hemoglobin level of 42%. Gene analysis for alpha or beta globin mutations was negative. RBC enzyme testing revealed an ADA of 6.1 and decreased FFK. Due to the unique combination of anemia, elevated fetal hemoglobin, and bone marrow morphology suggestive of, but not fully diagnostic for CDA I, II or III, we tested his EKLF gene and identified the heterozygous E325K mutation. JL's two siblings, mother and father have normal hemoglobin levels and peripheral blood smears. They also have normal In(Lu) blood group phenotype. Since the E325K mutation is dominant-negative, his phenotypically normal family members were not tested for the mutation.Four other patients have also been identified as having an E325K mutation in their EKLF gene. The patients had severe hemolytic anemia, elevated fetal hemoglobin, and bone marrow morphology showing dyserythropoiesis. Patients' erythrocytes also had low CD44 and water channel AQP1 expression, which is known to be regulated by EKLF. One patient was described with multiple congenital anomalies: hepatomegaly, micropenis, hypospadias, enlarged fontanel and hypertelorism. Taken together, the key clinical characteristics of this rare CDA are: severe normocytic anemia, highly elevated Hb F, presence of nucleated RBCs in the peripheral blood, erythroid hyperplasia with limited dyserythropoiesis in the bone marrow, splenomegaly, and growth delay. Disclosures:No relevant conflicts of interest to declare.

Phenotypic expression of hemoglobins A 2, E and F in various hemoglobin E related disorders

Study on the phenotypic expression of hemoglobin (Hb) A 2 and Hb E in Hb E disorders has been difficult due to the co-separation of Hb A 2 and Hb E in most Hb analysis assays. Because these two Hbs are separated on capillary electrophoresis, we studied phenotypic expression of Hbs A 2, E and F in various Hb E disorders using this system. This was done on 362 subjects with several Hb E disorders including heterozygous Hb E, homozygous Hb E, β-thalassemia/Hb E, δβ-thalassemia/Hb E, and Hb Lepore/Hb E and those of these disorders with several forms of α-thalassemia. Normal controls showed Hb A 2 of 2.7 ± 0.3%. Heterozygous Hb E and homozygous Hb E had elevated Hb A 2 i.e. 3.8 ± 0.3% and 4.8 ± 0.5%, respectively. Further elevations were observed for β 0-thalassemia/Hb E (6.1 ± 1.9%) and β +-thalassemia/Hb E (7.1 ± 1.2%). Interestingly, no elevation of Hb A 2 was found in the δβ-thalassemia/Hb E, and Hb Lepore/Hb E (2.3 ± 0.3%) but higher Hb F levels were noted which could be useful diagnostic markers. The levels of Hb E were variable. Co-inheritance of these Hb E disorders with α-thalassemia were associated with lower outputs of Hb E and Hb F but the levels of Hb A 2 were not altered. Different phenotypic expression of Hb A 2, Hb E and Hb F could help in differential diagnosis of these Hb E related disorders commonly encountered in the regions where access to molecular techniques is limited.

Limitations of Hb F as a Phenotypic Modifier in Sickle Cell Disease: Study of Kuwaiti Arab Patients

Sickle cell disease is characterized by phenotypic heterogeneity and many genetic modifiers have been identified with elevated Hb F being the most recognized ameliorating factor. Kuwaiti sickle cell disease patients carry the India/Arab chromosomal haplotype, which is associated with elevated Hb F (on average ∼22%) on account of the Xmn1 site in the Gγ-globin gene promoter. Most patients had either Hb SS or Hb S-β0-thalassemia (β0-thal) and there are a few Hb SD compound heterozygotes. We have carried out longitudinal clinical studies of these patients to document the pattern of morbidity, spleen function, brain and hip magnetic resonance imaging (MRI) for prevalence of silent brain infarcts and avascular necrosis of the femoral head (AVNFH), respectively. In addition, pulmonary function, SPECT (single photon emission computerized tomography) brain cerebral blood flow and response of selected patients to hydroxyurea (HU) treatment were also studied. The Hb SS and Hb S-β-thal patients have a generally mild phenotype compared to sickle cell disease in other populations and most patients do not have their first pain crisis until about the age of 4 years. Spleen function is retained till late childhood; pneumococcemia and other severe bacterial infections are rare. Overt stroke and silent brain infarcts are uncommon in childhood (∼3% prevalence) although SPECT reveals cerebral blood flow deficits in ∼30%. Avascular necrosis of the femoral head is, however, common with a prevalence of ∼26% in children and 50% in adults. There is brisk response to HU in patients with frequent pain crises, with marked increases in Hb F levels. Patients who are compound heterozygotes for Hbs S and D-Los Angeles, have the most severe phenotype despite Hb F levels of >20% and Hb S <30%. In conclusion, although the patients have a uniformly elevated Hb F level, there are still considerable phenotypic heterogeneity and other modulating genetic factors that require further studies.

Identification of Inflammatory Biomarkers for Pediatric Malarial Anemia Severity Using Novel Statistical Methods

Areas where Plasmodium falciparum transmission is holoendemic are characterized by high rates of pediatric severe malarial anemia (SMA) and associated mortality. Although the etiology of SMA is complex and multifactorial, perturbations in inflammatory mediator production play an important role in the pathogenic process. As such, the current study focused on identification of inflammatory biomarkers in children with malarial anemia. Febrile children (3 to 30 months of age) presenting at Siaya District Hospital in western Kenya underwent a complete clinical and hematological evaluation. Children with falciparum malaria and no additional identifiable anemia-promoting coinfections were stratified into three groups: uncomplicated malaria (hemoglobin [Hb] levels of ≥11.0 g/dl; n = 31), non-SMA (Hb levels of 6.0 to 10.9 g/dl; n = 37), and SMA (Hb levels of <6.0 g/dl; n = 80). A Luminex hu25-plex array was used to determine potential biomarkers (i.e., interleukin 1β [IL-1β], IL-1 receptor antagonist [IL-1Ra], IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, tumor necrosis factor alpha [TNF-α], alpha interferon [IFN-α], IFN-γ, granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage inflammatory protein 1 alpha [MIP-1α], MIP-1β, IFN-inducible protein of 10 kDa [IP-10], monokine induced by IFN-γ [MIG], eotaxin, RANTES, and monocyte chemoattractant protein 1 [MCP-1]) in samples obtained prior to any treatment interventions. To determine the strongest biomarkers of anemia, a parsimonious set of predictor variables for Hb was generated by least-angle regression (LAR) analysis, controlling for the confounding effects of age, gender, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and sickle cell trait, followed by multiple linear regression analyses. IL-12p70 and IFN-γ emerged as positive predictors of Hb, while IL-2R, IL-13, and eotaxin were negatively associated with Hb. The results presented here demonstrate that the IL-12p70/IFN-γ pathway represents a set of biomarkers that predicts elevated Hb levels in children with falciparum malaria, while activation of the IL-13/eotaxin pathway favors more profound anemia.

De novo once-monthly darbepoetin alpha treatment for the anemia of chronic kidney disease using a computerized algorithmic approach

Anemia of chronic kidney disease (CKD) has been traditionally treated by erythropoiesis-stimulating agents (ESAs) and/or iron following manual determination of dose. We hypothesized that once-monthly (QM) algorithmically dosed darbepoetin α (DA) and iron administration would successfully treat anemia of CKD in ESA-naïve CKD subjects. QM DA and iron doses were determined via a computerized program targeting a hemoglobin (Hb) of 10.5 - 12.5 g/dl in anemic, ESA-naïve, CKD Stages 3 - 5 subjects. Six consecutive QM doses were administered. Hb, ferritin, and transferrin saturation were recorded. Data are presented as means ± standard deviation. Anemia was identified in 133 subjects, with a mean follow-up of 188 days. DA doses and Hb were significantly greater at Months 3 and 6 compared to baseline (p < 0.05); DA doses were 109 ± 68 μg and 118 ± 91, respectively, at Months 3 and 6. Hemoglobin levels were correspondingly 11.3 ± 1.1 g/dl and 11.3 ± 1.0. 78% of patients achieved the target Hb by 6 months of therapy. The elevation of Hb was greater in non-proteinuric than proteinuric subjects at 6 months of treatment (11.6 ± 0.8 g/dl vs. 11.0 ± 1.1; p < 0.05), despite lower DA dose (96 ± 76 μg vs. 139 ± 98; p < 0.05). Successful treatment of the anemia of CKD by QM DA based upon a computerized dosing program was achieved by 6 months in 78% of ESA-naïve, CKD subjects.

High blood hemoglobin concentration as risk factor of major atherosclerotic cardiovascular events in 114,159 healthy men and women in the Apolipoprotein MOrtality RISk study (AMORIS)

Background. Few studies have tested differences in relationships between hemoglobin (Hb) and long-term risk of major cardiovascular diseases according to age and gender in healthy subjects as opposed to anemia.Aims. Such relationships were examined and risk-tested in relation to Hb values in the Apolipoprotein MOrtality RISk (AMORIS) Study.Methods. Using data from AMORIS and the Swedish hospital discharge and mortality registers, a prospective cohort study of 114,159 subjects with mean follow-up of 11.8 years, the association between Hb and risk of acute myocardial infarction (AMI), ischemic stroke (IS), and congestive heart failure (CHF) by Cox regression analysis according to age and gender was studied.Results. Elevated Hb levels were associated to acute myocardial infarction (AMI) (HR 1.10 (1.06–1.13) per SD change), mostly confined to men and younger subjects but with greater sex similarity trends for CHF. Slightly increased risks were seen for the lowest Hb levels in the elderly and in females. IS risk was positively and more linearly associated to Hb.Interpretation. In AMORIS the highest AMI and CHF risks were found in the upper region of the distribution, but different shapes of relationships according to age and gender were found. IS associated positively with Hb. Key words:

Volume regulation and KCl cotransport in reticulocyte populations of sickle and normal red blood cells

The potassium chloride co-transporter (KCC) is a member of the electroneutral cation chloride family of cotransporters found in multiple tissues that are involved in transepithelial ion transport and regulation of intracellular ion content and cell volume. We have shown previously that three of the four KCC genes – KCC1, KCC3, and KCC4 – are expressed in red blood cells (RBC) (Exp. Hem. 33:624, 2005). Functionally, the KCC mediates volume reduction of reticulocytes that establishes the higher cellular hemoglobin concentration (CHC) of mature RBC. KCC activity is higher in reticulocytes and diminishes with age. KCC activity in RBC containing sickle hemoglobin (SS RBC) is elevated compared to normal (AA RBC) in part due to reticulocytosis in SS blood. However, we have demonstrated that SS reticulocytes have abnormal regulation of KCC activity leading to increased CHC upon activation of KCC compared to AA reticulocytes (Blood 104:2954, 2004; Blood 109:1734, 2007). These findings implicate KCC as a factor in the dehydration of SS RBC, which leads to elevated Hb S concentration and enhances Hb S polymerization and hemolysis. Because KCC activity correlates with cell age, standard flux measurements on blood samples with different numbers of reticulocytes or young non-reticulocytes are not comparable. The Advia automated cell counter measures cell volume (MCV) and cellular hemoglobin concentration (CHC) in reticulocytes, an age-defined population of cells, and thus circumvents the problem of variable reticulocyte counts among SS and AA blood samples. In this study, reticulocyte CHC measurements on fresh blood demonstrated a clear difference between AA and SS cells, reflecting in vivo dehydration of SS reticulocytes, although there was significant inter-individual variation, and the CHC distributions of the two groups overlapped. After KCC activation in vitro by cell swelling using the nystatin method, the initial changes in reticulocyte MCV and CHC with time were used to estimate flux rates mediated by KCC, assuming that changes were associated with isotonic KCl movements. After 20–30 min a final steady state MCV/CHC (set point) was achieved and maintained, reflecting inactivation of the transporter. CHC set points were 26.5–29 g/dl in SS reticulocytes compared to 25–26.5 g/dl in AA reticulocytes, reflecting abnormal regulation in SS cells. These results were reproducible in the same individual over time. KCC flux derived from CHC ranged from 5 to 10.3 mmolK/kgHb/min in SS reticulocytes, compared to 2.9–7.2 mmolK/kgHb/min in AA reticulocytes. Such measures of KCC activity in red cell populations controlled for cell age will facilitate further studies correlating KCC activity with phenotypic or genetic variability in sickle cell disease.

Laboratory Variables Associated with Low Near-Infrared Cerebral Oxygen Saturation in Icteric Patients Before Liver Transplantation Surgery

Although regional cerebral oxygen saturation (rSO₂) measurements can detect disturbances in cerebral oxygenation, their usefulness is limited in patients with hyperbilirubinemia. We examined the relationship between rSO₂ and other laboratory variables that may affect interpretation of low rSO₂ in awake patients with end-stage liver disease before liver transplantation surgery. Before induction of general anesthesia, rSO₂ was measured in 164 patients with liver cirrhosis (Child class A/B/C = 19/41/104) and 8 with fulminant hepatic failure. Patients with West Haven hepatic encephalopathy of grade 3 or 4 were excluded. Relationships between rSO₂ and laboratory variables were evaluated by correlation and multivariate regression, and by receiver operating characteristic curve analysis. Univariate analyses showed that rSO₂ (median 58.5%, range 15% to 82%) correlated with serum total bilirubin, hemoglobin (Hb), creatinine, sodium, and magnesium concentrations, and prothrombin time (P < 0.001 each), but not with serum concentrations of glucose, albumin, potassium, and ammonia. Multiple logistic regression analysis showed that only elevated total bilirubin (range 0.4 to 66 mg/dL; odds ratio [OR] = 1.31; 95% confidence interval [CI] = 1.18 to 1.45) and low Hb (range 5.3 to 15.7 g/dL; OR = 0.21; 95% CI = 0.11 to 0.43) were independently related to rSO₂ <50%. The optimum cutoff points for observing an rSO₂ < 50% were total bilirubin >7.2 mg/dL (sensitivity 89%, specificity 90%) and Hb <9.6 g/dL (sensitivity 70%, specificity 82%). High total bilirubin and low Hb concentrations were independently associated with rSO₂ values below 50% in end-stage liver disease patients awaiting liver transplantation. The results of this study identify patients in whom a low rSO₂ may be an artifact rather than cerebral ischemia.

Hemoglobin Lepore EF Bart’s disease: a molecular, hematological, and diagnostic aspects

We report the molecular basis and hematological phenotype associated with a hitherto undescribed interaction of hemoglobin (Hb) Lepore-Hollandia, Hb E and a deletion of three α-globin genes found in a 3-year-old Thai girl. She had mild anemia with Hb 10.9 g/dl and Hct 35.9% and had never received blood transfusion. Hb analysis revealed Hb E (22.1%) with a normal level of Hb A(2) (1.9%), unusually elevated Hb F (65.9%), Hb Lepore (4.0%), and 5.4% Hb Bart's. Globin gene analyses demonstrated that she carried the Hb Lepore-Hollandia mutation in trans to the Hb E and a compound heterozygosity for α(0)-thalassemia (SEA deletion) and α(+)-thalassemia (3.7 kb deletion), leading to the Hb Lepore EF Bart's disease. Hematological data and diagnostics using combined Hb-HPLC, capillary electrophoresis, and PCR analysis of this condition were presented and compared with those of the patients with other forms of EF Bart's disease and EE Bart's disease in our series.

Variability In Hb A2 levels among Individuals with Beta-Thalassemia Trait: Is Iron Deficiency Associated with Abnormally Low Hb A2?

AbstractAbstract 4281 Introduction:Quantification of HbA2 is a well established screening test for beta-thalassemia trait (BTT). However, there is a small subset of individuals heterozygous for beta-thalassemia mutations who have HbA2 levels less than or equal to 3.5%. Some reports have suggested that iron deficiency in BTT patients causes HbA2 to be lower than expected, while others have found no significant relationship between iron deficiency and the level of HbA2. These conflicting reports have led to confusion amongst clinicians as to the reliability of HbA2 measurement when screening for BTT in iron deficient individuals. Methods:From a database of 444 individuals with heterozygous beta-thalassemia, confirmed by molecular testing, we assessed the variability in HbA2. Individuals were classified as “iron deficient” or “non-iron deficient” based on their serum ferritin, using two definitions of iron deficiency (serum ferritin <15 ug/L or <30 ug/L), and data was analyzed independently. The association of HbA2 levels with gender, iron deficiency or beta-thalassemia mutation type was evaluated using a two-sample T-test. The relationship of HbA2 with gender, hemoglobin level, Hb F percentage, reticulocyte percentage and/or the natural logarithm of serum ferritin was evaluated using single and multiple linear regression analysis. Results:HbA2 ranged from 2.0 to 8.1%, with a mean of 5.5%. Mean HbA2 in females (5.4%, SD 0.6%) was lower than that in males (5.6%, SD 0.6%) (p=0.003). Seven individuals had a HbA2 level below 3.5% (range 2.0–3.1%), all of whom had serum ferritin ≥15 ug/L. Of these people, five had the Chinese (Aγδβ)0-thalassemia deletion, and would thus be expected to have a normal HbA2 level; one woman had a HbA2 of 3.1% and was found to be heterozygous for a novel point mutation at codon 29 of the delta-globin gene (GGC>GAC or Gly29Asp); and another woman with HbA2 of 2.0% had unexplained elevation of Hb F (8.0%). The 30 patients with serum ferritin <15 ug/L had evidence of iron-deficient hematopoiesis (significantly lower hemoglobin concentration and MCV than patients with ferritin ≥ 15), and none had HbA2 <3.5%. However, HbA2 was lower (5.3%) versus HbA2 in non-iron deficient individuals (5.5%; p=0.04). Individuals with the Chinese (Aγδβ)0-thalassemia deletion had a mean Hb A2 of 2.8%. Of the remaining beta-thalassemia genotypes, IVSII-645 and Nt -29 beta+ mutations were associated with HbA2 values significantly below the group mean, and -28 beta+ and the Codon 17, Codons 41/42 and Codon 43 beta0 mutations were associated with significantly higher HbA2 values. Multiple linear-regression analysis demonstrated a significant association of low HbA2 with low ferritin (p=0.04) and beta+-thalassemia mutation type (p<0.001). There was no significant association with gender (p=0.1), hemoglobin (p=0.2), reticulocyte count (p=0.08) or hemoglobin F level (p=1.0). Conclusions:From our population of patients with BTT, we have shown that individuals with overt iron deficiency all had HbA2 within the range expected for an individual with BTT (Hb A2 >3.5%). Nonetheless, serum ferritin < 15 ug/L was associated with a small but significant decrease in HbA2 (mean HbA2 of 5.3%, versus 5.5% in non-iron deficient individuals; p=0.04). Although a multivariate analysis confirmed that ferritin under 15 ug/L was associated with lower HbA2 levels, beta+-thalassemia mutation type explained more of the variability than did iron status in individuals with lower HbA2 values. Six out of seven patients who had HbA2 <3.5% were found to have heterozygous deletion or mutation of the delta-globin gene. Based on the results of our study, HbA2 remains a reliable test for BTT screening, even in the presence of iron deficiency. In patients known or suspected to have BTT, but in whom HbA2 is found to be <3.5%, testing for delta-globin abnormalities should be pursued. Disclosures:No relevant conflicts of interest to declare.