Longitudinal Analysis of Hydroxyurea and Fetal Hemoglobin in Adult Patients with Sickle Cell Anemia

Abstract Introduction: Hydroxyurea (HU) was the first FDA-approved therapy for sickle cell anemia (SCA) and remains the most well-established disease-modifying therapy, reducing painful crisis and improving morbidity and mortality. HU improves outcomes through the upregulation of fetal hemoglobin (HbF). Dosing is highly variable with doses ranging from <10-35 mg/kg/day. Most dosing strategies escalate with a goal of mild myelosuppression, commonly defined using an absolute neutrophil count (ANC) between 2,000-4,000 and platelets >80,000. Macrocytosis is often used as a surrogate marker for compliance and effect. Despite its well-established effectiveness, clinical use remains limited with only 12% of adults with SCA taking HU. The reasons for this are multifactorial but include skepticism by both providers and patients regarding its effectiveness in the adult population. Some experts suggest up to 30% of adults are non-responders with no significant HbF change, and many believe that the effect of HU fades with time. Dosing strategies are usually quite conservative to prevent excessive myelosuppression, though dose is highly correlated with clinical effect and ultimate %HbF. There is very limited real-world data regarding long term effectiveness of HU in adults with SCA over time. Methods: We retrospectively analyzed the records of the 109 adults with SCA currently treated in our multidisciplinary sickle cell clinic. Data from 1/1/2011-6/30/2021 was collected, including genotype, HU prescription status, current and maximum laboratory values (HbF, MCV, ANC), and number of admissions for painful crisis. We performed Wilcoxon rank sum testing between pts prescribed and not prescribed HU and measures of HbF (peak, average and current) and number of admissions for painful crisis over the study period. Results: Among 106 pts (3 excluded from analysis, 2 for lack of data and 1 for post-transplant status), 79 are currently prescribed HU (75%). Among our 63 pts with HbSS genotype 58 are prescribed HU (92%). Average HbF over time for all pts prescribed HU was 11.9%. Average peak HbF was 18.1% and average current HbF is 12.4%. In the pts not prescribed HU HbF average, peak and current levels were 5.5%, 6.7%, and 5.0% respectively. HU prescription was significantly associated with increased HbF at peak, average, and current levels (p-value <0.001, Figure A). HU prescription was also significantly associated with decreased number of admissions for painful crisis (p-value 0.001). Among pts prescribed HU, MCV levels >100 at time of peak HbF were associated with higher peak levels than pts with MCV <100 (p-value <0.001, Figure B). Larger peak HU doses were also correlated with higher MCVs at the time of peak HbF levels (Figure C). Larger current HU doses were also significantly associated with higher current HbF levels (Figure, D). Doses >9.9m/kg were associated with significantly higher HbF levels however there was no significant difference between dose levels 10-19.9mg/kg and 20-29.9 or 20-29.9 and 30-39.9mg/kg (p-values 0.02, 0.68 and 0.84 respectively). Among pts prescribed HU, 34 obtained ANC levels <4000 at the times of peak HbF and 24 at the current dosing level (43% and 30% respectively). 24 pts obtained both MCV>100 and ANC <4000 at time of peak HbF and only 11 achieved both at current dosing levels (30% and 14% respectively). Conclusion: In our adult multidisciplinary sickle cell clinic prescribing rates are well above current HU usage for adults with SCA. Our data notes that elevated HbF levels can be maintained over long periods of time. HU prescription was significantly associated with increased HbF levels and reduced admissions for painful crisis. This was despite a majority of pts not meeting target prescribing levels of ANC <4000 and MCV >100. While higher peak HbF levels were significantly associated with higher HU doses, this was only true for doses above 9.9 mg/kg. Further investigation is needed to explore the factors contributing to suboptimal HbF and MCV response, including careful assessment with medication adherence and dose selection. Overall, these data illustrate the importance of dosing and suggest that one size dosing of HU for adults with SCA does not fit all. We hypothesize that there may be a role individualized dosing strategies in adults with SCA, incorporating factors such as pharmacokinetics and renal function to achieve the optimal dose for each patient. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Fetal Hemoglobin in Sickle Cell Anemia: A Genome-Wide Association Study of the Response to Hydroxyurea

Blood ◽

10.1182/blood.v112.11.2471.2471 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2471-2471 ◽

Cited By ~ 1

Author(s):

Nadia Timofeev ◽

Paola Sebastiani ◽

Steven H. Hartley ◽

Clinton T. Baldwin ◽

Martin H. Steinberg

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Association Studies ◽

Fetal Hemoglobin ◽

Significant Snps ◽

Genome Wide Association ◽

P Value ◽

Genome Wide ◽

Genome Wide Significance

Abstract Fetal hemoglobin (HbF) is the major genetic modulator of sickle cell anemia. Candidate gene-based and genome-wide association studies (GWAS) have provided strong evidence that single nucleotide polymorphisms (SNPs) linked to genes on chromosome 6q (HBS1L-MYB) and 2p (BCL11A), along with elements in cis to HBG help determine HbF concentration in untreated patients with sickle cell anemia and β thalassemia, and in normal individuals. The HbF response to hydroxyurea (HU) varies considerably among treated patients, even when compliance with treatment is good and patients are treated under controlled conditions. This suggests that genetic factors might affect the response to treatment with this agent. In the Multicenter Study of Hydroxyurea, 299 patients were randomized to receive either HU titrated to maximum tolerated doses, or a placebo, and HbF levels were measured before and at the completion of the randomized phase of the study. In 123 HU-treated patients, we completed GWAS using Illumina 370K chips that include approximately 350,000 haplotype tagging SNPs, and studied the association of SNPs with the change in HbF from baseline levels to levels measured at the end of the active treatment portion of the study. We conducted a GWAS using the analytical program PLINK, of approximately 273K SNPs with minor allele frequency >0.05, using linear regression and an additive model of inheritance. We selected for further investigation those SNPs with association that reached 0.05 significance, after we adjusted for sex. Because of the limited sample size that results in relatively large p-values, no single SNP reached so-called genome-wide significance after correcting for multiple comparisons using a Bonferroni correction (p-value <10-7) or 5% false discovery rate. Two SNPs had an association with p value <10–6 and 27 SNPs reached at least 10–5 significance. Noticeably, the SNP rs6899351 in FABP7 in 6q22.31 was associated with the largest increment in HbF after treatment with HU (6.9% change per copy of allele G, p-value 4 ×10–5). We also identified 2 SNPs in PDE7B (6q23.3) that were significantly associated with positive changes of HbF and 3 SNPs in MAP7 (6q23.3) that were significantly associated with a reduction of HbF after treatment. Using candidate gene association studies, we had previously shown that PDE7B and MAP7 were significantly associated with differential expression of HbF in sickle cell anemia. These new GWAS results suggest a regulatory role for these genes, or this region of chromosome 6q, in the HbF response to HU in sickle cell anemia. Analysis of the distribution of significant SNPs per chromosome also showed that chromosome 20 had a larger number of significant SNPs than expected at random, especially in CST9, one of a family of protease inhibitors. CST9 is tagged by 3 SNPs in the 370K array (rs2983639, rs2983640, rs10485646), 2 of which were associated with significant positive changes in HbF after treatment with HU and one with significant negative changes of HbF. Specifically, the average increase in HbF was 1.5% for each copy of allele G for SNP rs2983639 (p = 0.025), and 1.6% for each copy of allele A for SNP rs2983640 (p = 0.047), while the level of HbF decreased by approximately 1.5% for each copy of allele A for SNP rs10485646 (p = 0.035). The SNP rs2983640 is an exon variant that produces the amino acid change F-L. Although these SNPs do not individually reach genome-wide significance, cumulatively they provide strong evidence of association, as the probability that they are all simultaneously associated by chance is 10-4. Furthermore, we identified significant variants in other genes that belong to the same family of type 2 cysteine protease inhibitors, specifically 2 SNPs in CTS3 and 1 SNP in CTS5. Although the small sample size and the large number of SNPs tested suggest caution until these results are replicated in independent patient treatment groups, these preliminary findings suggest that type 2 cystatin genes and pseudogenes are associated with the HbF response to HU. If confirmed, it might be possible to use results like these to build a prognostic model of the HbF response to HU in sickle cell anemia.

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Serum Transferrin: An Independent Predictor of Mortality in Sickle Cell Anemia

Blood ◽

10.1182/blood.v118.21.2126.2126 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2126-2126

Author(s):

Zahra Pakbaz ◽

Mariana E Hildesheim ◽

Shoaib Alam ◽

Darlene Allen ◽

Caterina Minniti ◽

...

Keyword(s):

Endothelial Dysfunction ◽

Sickle Cell ◽

Serum Ferritin ◽

Sickle Cell Anemia ◽

Conflicts Of Interest ◽

Independent Predictor ◽

Stable Condition ◽

Acute Chest Syndrome ◽

Serum Transferrin ◽

Significant Difference

Abstract Abstract 2126 Introduction: Serum ferritin (SF), the most convenient marker of iron burden in sickle cell anemia (SCA), is potentially confounded by effects of inflammation in SCA. Serum transferrin (STF) has been described as one of the independent predictors of elevated tricuspid regurgitant velocity (TRV) in SCA. Therefore in this report we investigate the potential predictive role of STF in morbidity and mortality of individuals with SCA. Methods: Patients with sickle cell disease documented by high-pressure liquid chromatography were eligible for the study. Four hundred and sixty SCA patients were recruited in this study from the community through multimedia advertisements, community outreach, and regional clinics. All evaluated patients were screened by history taking, physical examination, laboratory studies, and transthoracic echocardiography. All patients provided written informed consent. The advertisements and protocol were approved by the institutional review boards of the National Heart, Lung, and Blood Institute and Howard University. Only outpatients in stable condition were included; patients who had had a vaso-occlusive crisis within the previous two weeks or an episode of acute chest syndrome within the previous four weeks were excluded. Results: Two hundred and sixty two participants with HbSS were included in the data analysis. Forty-seven percent were male. Median age was 32 years old. Median TRV in this cohort was 2.4 m/s. Forty nine percent of participants had TRV≥2.5 m/s and 19% had TRV≥3 m/s. Patients with lower STF (<164 mg/dl) were older (p=0.01), had more blood transfusions in the past (p<0.0001) and did not have more complications of sickle disease (priapism, acute chest syndrome, leg ulcers or emergency room visits, all p>0.05), but all-cause death rate was higher (22% vs. 6%, p=0.0001). Hemoglobin, CRP, alkaline phosphatase, uric acid, placenta growth factor (<0.001), SF and iron saturation were found to be higher and kidney function was worse. Patients with lower STF level were also more likely to have TRV≥3.0 m/s (32% vs. 13%, p<0. 001) but there was no significant difference in BNP and ejection fraction. Patients with lower STF had endothelial dysfunction, as indicated by a blunted forearm blood flow (FBF) response to infusion of acetylcholine into the brachial artery (p<0.01). Among age, gender, BNP, TRV,GFR,WBC, STF, systolic blood pressure and fetal hemoglobin, the Cox proportional analysis of mortality found TRV, GFR and STF the independent significant predictors of mortality in this cohort. Kaplan-Meier survival curve showed that patients with transferrin <164 mg/dl had significantly lower survival (p<0.001). Conclusion: In this cohort of adults with sickle cells anemia, STF is found to be an independent predictor of endothelial dysfunction, high TRV and mortality. It may be a more sensitive predictor than serum ferritin. We propose that iron overload may induce a state of endothelial dysfunction that is a risk factor for clinical vasculopathy and death. Disclosures: No relevant conflicts of interest to declare.

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Prediction of Fetal Hemoglobin in Sickle Cell Anemia Using a Genetic Risk Score

Blood ◽

10.1182/blood.v120.21.3216.3216 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3216-3216

Author(s):

Jacqueline N Milton ◽

Paola Sebastiani ◽

Clinton T. Baldwin ◽

Efthymia Melista ◽

Victor R. Gordeuk ◽

...

Keyword(s):

Linear Regression ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Genetic Risk ◽

Genetic Risk Score ◽

Fetal Hemoglobin ◽

P Value ◽

Data Sets ◽

Genome Wide ◽

Hbf Level

Abstract Abstract 3216 Fetal hemoglobin (HbF) is the major genetic modifier of clinical course of sickle cell anemia (homozygosity for HBB glu6val). HbF level is also an important predictor of mortality. If it were possible to know at birth the HbF level likely to be present after stabilization of this measurement at about age 5 years, then an improved prognosis might be given and HbF-inducing treatments better informed. Levels of HbF in adults are highly heritable and the production of HbF is genetically regulated by several quantitative trait loci and by genetic elements linked to the HBB gene cluster. One of the most popular approaches to genetic risk prediction uses a summary of the risk alleles in the form of a genetic risk score (GRS) that is used as a covariate of the genetic prediction model. We present the development of a GRS for HbF in 841 patients from the Cooperative Study of Sickle Cell Disease (CSSCD) cohort patients and assessed its ability to predict HbF values in three independent cohorts that included PUSH (N=77), Walk-PHaSST (N=181), and C-Data from the Comprehensive Sickle Cell Centers program (N= 127). We used the results of a genome-wide association study (GWAS) of HbF in sickle cell anemia, in which patients were genotyped using the 610K Illumina array, and association of each of the ∼550K SNPs with HbF was tested using a linear regression model with gender adjusted additive genetic effects. To build the GRS, we sorted SNPs by increasing p-value, starting from the most significant SNP associated with HbF (rs766432, p-value=2.61×10−21), and pruned the list by removing SNPs in high LD (r2 > 0.8). We then used this list of SNPs to generate a sequence of nested GRS. We started with the GRS that included only the most significant SNP and generated the second GRS by adding the second SNP from the list of SNPs. The third GRS was generated by adding the 3rd SNP from the list of SNPs to the second GRS, and so on. We repeated this analysis including up to 10,000 SNPs (p-value< .02185) and hence generated 10,000 GRS, for each of the subjects in the CSSCD. Each of these GRS was included as covariate in a linear regression model and the regression coefficients of the resultant 10,000 linear regression models were estimated using Least Squares methods in the CSSCD data. The predictive value of these GRS models was then evaluated in three independent cohorts. In this evaluation, we computed the 10,000 GRS for each subjects in each data sets, and then used the 10,000 regression models estimated in the CSSCD data set to compute the expected HbF value of patients, given their GRS. We then assessed the predictive accuracy by computing the correlation between the observed and predicted values of HbF. To produce more stable predictions, we also created ensembles of predictive models. An ensemble of the first 14 GRS models including 14 SNPs had the best predictive value in all 3 data sets and explains 23.4% of the variability in HbF; the correlation between the predicted HbF and observed HbF was 0.44, 0.28 and 0.39 in the three different cohorts. Of these 14 SNPs, 6 were located in BCL11A; other SNPs were located in the olfactory receptor region and the in chromosome 11p15 and the site of the HBB gene cluster and were found previously to be associated with HbF. We next compared these results to predictive models in which we included gender, coincident alpha thalassemia, and HBB haplotypes for prediction. The model including gender and alpha thalassemia explained only 2.6% of the variability of HbF in the discovery cohort and the model including HBB haplotypes explained 2.35% of the variability of HbF in the discovery cohort and neither model showed a significant correlation between the predicted and observed HbF in the three other cohorts. In addition, combining the non-genetic information with the GRS did not help to explain more of the variability in HbF. With as few as 14 SNPs we can explain more of the variability in HbF and do a better job of prediction in comparison to using other non-genetic risk factors or genome-wide significant SNPs; however, we still cannot explain all of the variability in HbF that is due to heritability. These results suggest that knowing the genotype of a few SNPs can help to predict HbF that after they have stabilized. Prediction of HbF at an early age has the potential to help foretell some features of the severity of the clinical course of the disease and aid to optimize the clinical management of patients. Disclosures: No relevant conflicts of interest to declare.

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Clinical Complications in Adult Patients with Sickle Cell Anemia and β-Thalassemia-Sickle Cell Disease

Blood ◽

10.1182/blood.v120.21.4767.4767 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4767-4767

Author(s):

Giovanna Graziadei ◽

Alessia Marcon ◽

Martina Soldarini ◽

Ilaria Gandolfi ◽

Luisa Ronzoni ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Acute Chest Syndrome ◽

P Value ◽

Cell Disease ◽

Transfusion Therapy ◽

Clinical Complications ◽

Significant Difference ◽

The Mean

Abstract Abstract 4767 Background. Sickle-Cell Disease (SCD) is one of the most common severe monogenic inherited disorders worldwide, due to hemoglobin S (HbS), with reduced affinity for the oxygen. HbS polymerization, leading to erythrocyte rigidity, vaso-occlusion and hemolytic anemia, is central in the pathophysiology and crucial for the clinical outcome. The term SCD refers to Sickle Cell Anemia (SCA) due to homozygosis for βS allele, HbS/β-thalassemia (T-SCD) due to compound of β-thal and βS allele, and HbSC disease, owing to the coinheritance of βS and βcalleles. SCD is a multiorgan disease characterized by recurrent acute events and progressive organ damage, worsening during the life. Aims. This is a retrospective monocentric study aimed to assess and compare the clinical complications among 59 adult SCD patients, followed at the Hereditary Anemia Centre of the Foundation IRCCS “Ca Granda” Ospedale Maggiore Policlinico, in Milan, Italy. Methods. Mutation analysis of the b globin gene was established by direct DNA sequencing on the ABI Prism 310 genetic analyzer. Clinical and hematological features were evaluated by routine tests and physical examination, with special attention to the erythropoiesis stress parameters as LDH values and extramedullary erythropoietic (EE) masses. Results. Fifty-nine adult SCD patients, 16 SCA and 43 T-SCD, were evaluated. In T-SCD patients detected b-mutations were severe (b°) in 69.8%, and moderate or mild (b+-b++) in 30.2%. The mean age of SCA patients was 36±9 and 41±11 years for T-SCD patients. For both groups the mean follow-up was 20±6 years, while the mean age at the presentation in our Centre was 32±8 years in SCA patients and 31±10 years in T-SCD ones. Five out of 16 (31.2%) SCA patients and 16/43 (37.2%) T-SCD patients were male. HbF mean levels were 6.9±5.1% and 10.1±7.2%, respectively in SCA and T-SCD group; surprisingly Hb mean levels were lower in SCA (9.3±1.3 g/dl) than in T-SCD (9.9±1.4 g/dl) patients. Comparing SCA and T-SCD, there was statistically significant difference in splenic features: splenectomy was performed in 2/16 (12.5%) SCA patients vs 21/43 (48.8%) T-SCD patients (p-value < 0.01). Splenomegaly was absent in SCA, while was detected in 11/22 (50%) T-SCD (p-value < 0.0001); all SCA patients had functional asplenia, not observed in T-SCD patients; splenic infarctions were absent in SCA patients and were detected in 7/22 (31.8%) T-SCD patients, of whom 5 had splenomegaly and 2 had normal spleen size (pvalue <0.001). On the other side, there was not statistically significant difference in the prevalence of stroke, acute chest syndrome (ACS), bone pain crisis, sepsis, leg ulcers and priapism. However, we observed some clinical differences, even if not statistically significant. Cholecistectomy was performed in 4/16 (25%) SCA patients vs 17/43 (39.5%) T-SCD patients, and gallstones were detected respectively in 5/12 (41.7%) and in 14/26 (53.8%) of SCA and T-SCD patients. Thrombotic events were absent in SCA patients, compared to 4/43 (9.3%) T-SCD patients. Furthermore, we detected EE in 3/16 (18.6%) SCA and in 3/43 (7%) T-SCD, all carrying b° thal mutations. We underlie that Hb levels and LDH values were higher in SCA than in T-SCD patients (823±295 vs 689±209 U/L). About the treatment, 14/16 (87.5%) SCA and 31/43 (72%) T-SCD underwent to top-up transfusion; 5/43 (11.6%) T-SCD were regularly transfused. Seven out of 16 (43.8%) SCA and 18/43 (41.8%) T-SCD patients were treated with Hydroxycarbamide (HU). Criteria for transfusion therapy were: painful crisis not responsive to HU, major clinical complications, such as stroke or ACS, extramedullary erythropoietic masses associated with high LDH levels and low Hb values. Conclusions. These data suggest that SCA and T-SCD patients have similar clinical course. Splenomegaly is present only in T-SCD patients, probably due to the increased amount of extravascular hemolysis. Surprisingly, SCA patients showed EE and lower Hb levels with higher LDH values compared to T-SCD ones. This could be related to the prevalence of intravascular hemolysis, that can lead to erythropoietic stress in SCA, even if tissues are better oxygenated in these patients because of biochemical characteristic of HbS in terms of decreased oxygen affinity. These observations could be important to evaluate transfusion and HU treatment. Disclosures: Cappellini: Novartis: Research Funding.

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Revisiting the Role of the Stat Laboratory: Is It Safe to Perform Activated Clotting Time Determinations (ACTs) Out of the Operating Room

Blood ◽

10.1182/blood.v120.21.4256.4256 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4256-4256

Author(s):

Michael Plietz ◽

Aaron Leifer ◽

Vilma Padilla ◽

Carole Pineda ◽

Khrishan Naraine ◽

...

Keyword(s):

Half Life ◽

Conflicts Of Interest ◽

P Value ◽

Clotting Time ◽

Heparin Group ◽

Platelet Factor ◽

Activated Clotting Time ◽

Significant Difference ◽

Stat Laboratory ◽

Over Time

Abstract Abstract 4256 Background: Activated clotting time (ACT) is determined during cardiac procedures in order to assess the level of anti-coagulation. There are three main anticoagulants used during cardiac procedures:heparin, angiomax and Plavix. Heparin is often used in the cardiac OR for major procedures and is neutralized, over time, by platelet factor 4(PF4). Angiomax, bivalirudin, has a quick onset of action, but a much shorter half-life when compared to Heparin. Plavix, clopidogrel, irreversibly inhibits platelets from clotting and is used on patients in need of anticoagulation for an extended period of time. The purpose of this study was to evaluate the stability of ACT results over time utilizing different coagulation regimens. If the ACT value remains stable over time, this would enable us to remove the device from the OR and place it in a stat laboratory. Methods: ACTs were determined by the ISTAT (Abbott), a hand held point of care device, using kaolin activated cartridges. A single blood sample was obtained and was evaluated at intervals of 0, 5, 10, 15, and 20 minutes. In addition, temperature, age, body surface area and platelet count of patient were recorded. The time 0 specimen was used to determine the patients ACT and interval testing was performed on the remnant. Results: 36 samples representing 22 patients had ACT tests performed using the ISTAT. There was no significant change from 0 to 20 minutes over the entire dada set. However, when divided into different anti-clotting agents there were significant changes. Patients who had only taken Angiomax had a significant difference within the first 5 minutes (p value=.0094). Patients taking Angiomax and Plavix together had no change in ACT values at 20 minutes. Patients on Heparin alone demonstrated a loss of ACT stability at 10 minutes with values both increasing and decreasing. Conclusions: Patients on Angiomax alone demonstrated a significant difference in ACT value within the first 5 minutes and all intervals thereafter. Based on Angiomax short half-life the ACT should be performed as soon as possible. The ACT values in patients taking both Angiomax and Plavix achieved steady state throughout the 20 minutes and had little if any decrease in value. The results for the Heparin group were unreliable at 10 minutes. As a result of these findings, performing an ACT outside the OR is feasible assuming the test can be performed by 10 minutes. Disclosures: No relevant conflicts of interest to declare.

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Monocyte Shift to a Non-Classical CD14dim/CD16+ Phenotype Correlates with Fetal Hemoglobin Levels in Sickle Cell Anemia Patients Treated with Hydroxyurea

Blood ◽

10.1182/blood.v120.21.817.817 ◽

2012 ◽

Vol 120 (21) ◽

pp. 817-817 ◽

Cited By ~ 1

Author(s):

Kleber Yotsumoto Fertrin ◽

Dulcinéia Martins Albuquerque ◽

Carolina Lanaro ◽

Carla Fernanda Franco-Penteado ◽

Flavia Rubia Pallis ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Cell Activation ◽

Conflicts Of Interest ◽

Fetal Hemoglobin ◽

Pleiotropic Effect ◽

Monocyte Subset ◽

Classical Monocytes ◽

Intermediate Monocytes

Abstract Abstract 817 Vaso-occlusion in sickle cell anemia (SCA) involves inflammation and cell activation; fetal hemoglobin (HbF) elevation by hydroxyurea (HU) remains the mainstay of SCA treatment. Monocytes are activated in SCA, and their contribution to the chronic inflammatory state includes the production of cytokines and reactive oxygen species (ROS). Monocytes are a heterogeneous group of leukocytes subdivided into distinct subsets: classical monocytes comprise over 80% of circulating monocytes, are highly positive for CD14 (CD14bright) and typically CD16-negative, while CD16-positive monocytes have been further subdivided into intermediate CD14bright/CD16+, and non-classical CD14dim/CD16+ monocytes. Intermediate monocytes are recognized as the main monocytic producers of ROS and are increased in inflammatory conditions such as atherosclerosis and sepsis. The less characterized non-classical subset is believed to have a patrolling behavior in blood vessels, does not produce ROS and constitutively produces IL-1 receptor antagonist (IL1-RA). Another relevant subgroup of monocytes expresses angiopoietin-2 receptor TIE2, and the role of TIE2-expressing monocytes (TEMs) has been investigated in angiogenesis in neoplastic diseases. TEMs usually correspond to intermediate monocytes, but their importance in inflammation is still unclear. We hypothesized that monocyte subsets in SCA patients would differ from controls, and that treatment with HU might also influence monocyte phenotypes, thus shedding light on the possible role of these subsets in an inflammatory condition not previously studied. EDTA-anticoagulated peripheral blood samples were collected upon written informed consent from 21 healthy controls (CON, ages 21–63 years) and 34 SCA patients (18 on HU, ages 16–58 years) in steady state, with no transfusion or acute sickling episode in the previous three months. Monocytes were immunophenotyped by flow cytometry on a multicolor FACSCalibur cytometer. Medical history of SCA-associated complications, HbF levels and dosage of HU in mg/kg/day were obtained from medical charts. Statistical analysis was performed on GraphPad Prism 5.0 software. As expected, we found that relative percentage and absolute count of CD16-positive monocytes were higher in SCA patients than in controls. Surprisingly, a significantly higher percentage of non-classical CD14dim/CD16+ monocytes, rather than intermediate cells, was found in SCA patients on HU (SCA-HU) treatment (mean±SEM: CON 2.06±0.43%, SCA 2.91±0.50%, SCA-HU 6.42±0.80%, P<0.0001). TEMs were also increased in SCA patients compared to controls (CON 2.64±0.72%, SCA 20.48±5.40%, SCA-HU 32.97±5.92%, P<0.0001), but HU treatment did not significantly influence TEM counts. Mean TIE2 expression did not vary among the groups, and there was no correlation between TEMs and presence of SCA complications pathophysiologically associated with disturbed angiogenesis, such as pulmonary hypertension, osteonecrosis, leg ulcers and retinopathy. Higher percentages of non-classical monocytes in HU-treated patients were initially interpreted as a possible toxic effect of HU on monocytopoiesis, but the lack of correlation of monocytes subsets with the degree of relative monocytopenia made this hypothesis unlikely. Moreover, we found a significant positive correlation between percentages of non-classical monocytes and HbF levels (rS=0.4763, P=0.0068, see figure). This suggests that successful HU treatment with higher HbF could correlate with the expansion of this particular monocyte subset. During the study period, only one patient was available for comparison before and after HU, but the increase in HbF from 4.2% to 11.6% and in non-classical monocytes from 1.82% to 9.48%, in this case, corroborates that HU therapy may explain this phenotype shift in monocytes. Whether non-classical monocytes expansion represents yet another pleiotropic effect of HU, if these cells are less likely to take part in the vaso-occlusive process and have an antiinflammatory role or, furthermore, if a bone marrow counterpart of this monocyte subset could be involved in increasing HbF production, remains to be investigated. The correlation of the expansion of non-classical monocytes with HbF levels could prove to be an interesting biomarker of response to HU, and future studies may address its clinical usefulness. Disclosures: No relevant conflicts of interest to declare.

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Absolute Reticulocyte Count Is Negatively Correlated with Fetal Hemoglobin during Infancy and Early Childhood in Patients with Sickle Cell Anemia

Blood ◽

10.1182/blood.v124.21.4081.4081 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4081-4081

Author(s):

Emily R. Meier ◽

Colleen Byrnes ◽

Y. Terry Lee ◽

Maxine Weissman ◽

Jeffery L. Miller

Keyword(s):

Steady State ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Conflicts Of Interest ◽

Fetal Hemoglobin ◽

Convenience Sample ◽

Risk Of Death ◽

Increased Risk ◽

F Cells ◽

Analytical Approaches

Abstract Hemoglobin switching is largely complete in healthy infants by 6 months of age. In infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Previous studies demonstrated that patients with an ARC greater than 200 K/uL during early infancy (60-196 days of age) were at the highest risk for SCA-associated events. 1,2 The objective of this study was to determine if ARC is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study [42 (39.6%) less than 1 year of age (28-362 days old), 46 (43.4%) between the ages of 1 and 10 years, and 18 (17.0%) between 10 and 20 years old]. After consent and assent were provided, discarded peripheral blood was obtained during routine clinic visits at steady state and analyzed within 48 hours of collection and storage at 40C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated by flow cytometry following intracellular staining with a fluorescent antibody directed against HbF. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t tests were used to compare means. Initial studies compared groups based upon ARC greater than or equal to 200 K/uL (ARC≥200) during infancy because of the previously reported utility of this threshold as a predictive marker for SCA severity.1 Over one third of the infants less than 1 year of age (n=16) had an ARC≥200. Mean HbF and F-cell levels were significantly lower in the ARC≥200 group when compared to the ARC<200 group (HbF: 29.9±10.9% vs. 53.5±17.6%, respectively, p=2.2E-05; F-cells: 83.5±13.2% vs. 96.6±5.7%, p=6.2E-05). Mean hemoglobin levels were also lower in the ARC≥200 group [8.1±1.4 g/dL vs. 9.5±1.6 g/dL (ARC<200), p=0.005]. Of the 22 (52.4%) infants who had a HbF level greater than 40%, only 2 (9.1%) had an ARC greater than 200K/uL. Enrolled patients were also grouped according to age and comparisons were made between ARC and HbF or F-cell levels. HbF and F-cell levels were negatively correlated to ARC in the infant subgroup (r=-0.696, p=3.1E-07 and r=-0.795, p=0.000, respectively). HbF and F-cell levels from children between the ages of 1 and 10 years were inversely related to the ARC, but the correlation was less significant (r=-0.626, p=3.3E-06 and r=-0.538, p=1.2E-04, respectively). The inverse relationship was no longer present in the oldest group of patients (HbF vs. ARC r=-0.203, p=0.420 and F-cells vs. ARC, r=-0.258, p=0.302). According to both analytical approaches described here, increased ARC is associated with decreased HbF and F-cell levels in infants with SCA. Less robust negative correlations are maintained through age 10 years, but no significant correlation was identified in adolescence and young adulthood. Overall, the data suggest that increased ARC levels may identify SCA infants who manifest a more rapid or greater loss of fetal hemoglobin during the later stages of the HbF-to-HbS switching phenomenon. Meier ER, Byrnes C, Lee YT, et al. Increased reticulocytosis during infancy is associated with increased hospitalizations in sickle cell anemia patients during the first three years of life. PLoS One 2013; 8(8):e70794. doi: 10.1371/journal.pone.0070794.Meier ER, Wright EC, Miller JL. Reticulocytosis and anemia are associated with an increased risk of death and stroke in the newborn cohort of the Cooperative Study of Sickle Cell Disease. Am J Hematol 2014 May 31; doi: 10.1002/ajh.23777. [Epub ahead of print] Disclosures No relevant conflicts of interest to declare.

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Clinical and Hematological Evaluation of Patients with Sickle Cell Anemia Before and After Four Years of Using Hydroxyurea

International Archives of Medicine ◽

10.3823/2469 ◽

2017 ◽

Vol 10 ◽

Author(s):

Ieda Maria Gonçalves Pacce Bispo ◽

Maria Lúcia Ivo ◽

Valter Aragão do Nascimento ◽

Alexandra Maria Almeida Carvalho de Pinto ◽

Olinda Maria Rodrigues de Araújo ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Hematological Parameters ◽

Fetal Hemoglobin ◽

Hemoglobin Concentration ◽

Teaching Hospitals ◽

Acute Chest Syndrome ◽

Mean Values ◽

Significant Difference ◽

Before And After

Objective: Evaluating clinical and hematological-clinical parameters of patients with sickle cell anemia (SCA) before and after four years of using hydroxyurea (HU). Method: A retrospective cohort study implementing a quantitative, descriptive and analytical approach developed in two public teaching hospitals located in the Central-West region of Brazil, from November 2010 to October 2011. Data collection was performed through medical records of 32 patients with SCA to assess clinical and hematological parameters before and after HU treatment. The study was approved by the UFMS Ethics Committee under protocol number 1890/2010. Results: All of the 32 patients were homozygous with a mean age in the prescription of hydroxyurea of 19.72±7.58 years, an initial dose of 15.59±4.27 mg/kg/day, and 22.48±5.35 mg/kg/day in the fourth year of treatment. Regarding the use of HU, average values of some hematological parameters presented a significant difference in the fourth year compared to the mean values prior to HU use, such as fetal hemoglobin (14.49±7.52%), red blood cells (2.54±0.38x1012/L), hematocrit (25.30±4.03%) and hemoglobin (9.22±3.34g/dL). Conclusion: Treatment with hydroxyurea showed a significant increase in fetal hemoglobin levels, increased hemoglobin, hematocrit and average corpuscular hemoglobin concentration, with reduced episodes of pain, infection and acute chest syndrome in such a way as to reaffirm its efficiency in treating these patients. Keywords: Hemoglobin; Sickle Cell Anemia; Hydroxyurea.

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European Chromosome 6 Haplotypes Significantly Augment Fetal Hemoglobin Levels in Brazilian Sickle Cell Anemia Patients: Influence of Four HBS1L-MYB Intergenic Region SNPs

Blood ◽

10.1182/blood.v120.21.1002.1002 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1002-1002

Author(s):

Flávia Costa Leonardo ◽

Stephan Menzel ◽

Ana Flavia Brugnerotto ◽

Kleber Yotsumoto Fertrin ◽

Marcos André Cavalcanti Bezerra ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Intergenic Region ◽

Conflicts Of Interest ◽

Strong Association ◽

Fetal Hemoglobin ◽

Brazilian Population ◽

Minor Allele ◽

Chromosome 6 ◽

Major Allele

Abstract Abstract 1002 Fetal hemoglobin (HbF) levels significantly modulate the severity of the 2 major β-hemoglobin disorders - sickle cell anemia (SCA) and β-thalassemia. Three major quantitative trait loci (QTLs; Xmn1-HBG2, the HBS1L-MYB [HMIP] intergenic region on chromosome 6q23, and BCL11A on chromosome 2p16) account for 20–50% of the common variation in HbF levels in SCA and β - thalassemia patients, and in healthy adults (Thein et al., Hum Mol Genet (2009) 18:R216). Lettre et al. (PNAS (2008) 105:11869) confirmed the influence of SNPs at the BCL11A and HBB loci in an African American cohort and a Brazilian cohort of SCA patients; as well as a significant influence of the HMIP region SNPs (rs7776054, rs9389268 and rs4895441) on HbF expression in the Brazilian SCA cohort. A strong association between HMIP polymorphisms that have a high frequency in the European population and modulation of F cell numbers has been reported (Creary et al., PLoS One (2009) 4:e4218). Given the unusually high admixture of the Brazilian population, the current study aimed to look at the influence of such HMIP markers on HbF production in SCA patients from this population (two regions, in the Northeast and Southeast of Brazil). We studied the influence and frequencies of the HMIP allele marker rs9376090 (that specifically tracks European chromosomes), as well as the rs9399137 marker (that has a much higher frequency in European descendents than in African descendents), as well as two HMIP markers (rs9389269 and rs9402686) that are also common in African descendents. Patients (220 HbSS, aged 12–68 years) were recruited at the Hematology Center, UNICAMP and at Fundação Hemope. The study was approved by the local Ethics Committees and informed consent was provided by all participants. Patients presenting the XmnI Gγ polymorphism (N = 2) were identified and excluded from further analysis, as this polymorphism has a known influence on HbF. The HMIP markers were genotyped by Taqman assays. Percentage HbF levels were determined by HPLC, using the Variant™ Bio-Rad kit, and were log transformed to normalize distribution for regression analyses. For those patients on hydroxyurea (HU) therapy, pre-HU HbF levels were used for analyses. Tests for associations between SNPs and HbF levels were conducted using linear regression models (SPSS v.15), including age and sex as covariates. High minor allele frequencies (MAF) for all four HMIP markers were observed in the population of patients studied (MAF; 0.09, 0.10, 0.12 and 0.12 for the rs9376090, rs9399137, rs9389269 and rs9402686 markers, respectively). For all four SNPs studied, higher levels of HbF were observed for the SCA individuals that were homozygotes for the minor allele, with strikingly higher levels of HbF presented by those individuals that were homozygotes for the rs9376090 and rs9399137 polymorphisms (see Table). The clinical courses of these patients were consistent with the higher levels of HbF observed (data not shown). Significantly higher HbF was also found in heterozygotes for the HMIP SNPs, compared to the major allele homozygotes. The variance in HbF levels due to rs9376090 was 7.1% (β= 0.270; p = 6.36 10−5), due to rs9399137 was 7.1% (β= 0.270; p = 9.59 10−5), due to rs9389269 was 8.3% (β= 0.287; p = 2.31 x10−5) and to rs9402686 was 8.3% (β=0.291; p = 2.18 x10−5). Our results confirm the HBS1L-MYB intergenic region as a key determinant of HbF levels in Brazilian SCA patients. The admixture of the Brazilian population has apparently led to a much higher incidence of European haplotypes at chromosome 6 in this population studied, when compared to the British and Tanzanian SCA populations. Importantly, the presence of these SNPs at the HMIP appears to have a very significant effect on HbF levels in the Brazilian SCA population, with probable clinical benefits. Table. HbF Levels in SCA Individuals, according to HMIP genotypes SNP (HBS1L-MYB locus) % HbF (Median ± S.D.) TT TC CC rs9376090 6.35 ± 4.24 8.45 ± 3.63 17.25 ± 1.63 N = 182 N = 32 N=2 p<0.01 p<0.05 rs9399137 6.47 ± 4.38 8.68 ± 3.63 17.25 ± 1.63 N = 167 N=37 N = 2 p<0.05 p< 0.05 rs9389269 6.40 ±4.29 8.59 ± 3.70 12.68 ± 2.45 N = 162 N = 42 N = 5 p< 0.01 p< 0.05 rs9402686 GG AG AA 6.26 ± 4.28 8.36 ± 4.67 12.68 ± 2.45 N =166 N = 41 N = 5 p<0.01 p<0.05 Significant differences for heterozygotes and homozygotes for the minor allele, compared to the homozygote major allele group are indicated by P values. Kruskal-Wallis test, Dunn's multiple comparison post test. Disclosures: No relevant conflicts of interest to declare.

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Elevated Plasma Levels and Platelet-Associated Expression of the Pro-Thrombotic and Pro-Inflammatory Protein, LIGHT (TNFSF14), In Sickle Cell Anemia (SCA)

Blood ◽

10.1182/blood.v116.21.2651.2651 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2651-2651

Author(s):

Vanessa T. Garrido ◽

Venina M. Dominical ◽

Renata Proença-Ferreira ◽

Marcos André Cavalcanti Bezerra ◽

Mariana R. B. Mello ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Therapeutic Target ◽

Plasma Levels ◽

Conflicts Of Interest ◽

Fetal Hemoglobin ◽

Endothelial Activation ◽

Potential Therapeutic Target ◽

Activation Markers ◽

Inflammatory Protein

Abstract Abstract 2651 Background: LIGHT (TNFSF14; CD258), a recently-identified member of the TNF superfamily, is found associated with and produced from platelets, amongst other immune cells. Increased circulating levels of this protein have been observed in patients with myocardial infarction and acute atherothrombotic stroke and LIGHT has been proposed as a potential therapeutic target in atherosclerosis, due to its reported pro-thrombotic and pro-inflammatory effects upon human endothelial cells. This study evaluated whether production of LIGHT is altered in patients with sickle cell anemia (SCA), and the participation that the platelets (PLTs) may have in this production, since SCA is characterized by a significant chronic inflammation and endothelial activation that may initiate the vaso-occlusive process. Patients and Methods: Soluble LIGHT (sLIGHT) was determined in PLT-free plasma (obtained by sequential centrifugations and ultrafiltration) from healthy control individuals (CON), SCA patients in steady state (SCA) and SCA patients on hydroxyurea therapy (SCAHU; 20–30 mg/kg/day HU) by ELISA. Expressions of PLT-membrane LIGHT and PLT activation markers were evaluated by flow cytometry using anti-CD258-PE, anti-CD62P-FITC or anti-PAC1-FITC. Subjects had not taken ASA during the previous 14 days. Results: sLIGHT was significantly elevated in the plasma of SCA and SCAHU individuals, compared to CONs (SCA; 35.4 ± 9.4 pg/ml; SCAHU, 37.3 ± 7.5 pg/ml; CON, 9.7 ± 1.5 pg/ml, n=27, 27, 19, resp.; P<0.01 for SCA/SCAHU, compared to CON; Kruskal-Wallis/Dunn's). Plasma sLIGHT in SCA/SCAHU individuals presented no correlation with hematological variables, such as PLT counts (rs=-0.079, P=0.65) and fetal hemoglobin (rs=0.107, P=0.51). In contrast, plasma sLIGHT levels in SCA patients presented an impressive correlation with plasma levels of CD40L, another important PLT-derived inflammatory protein (rs=0.817, P<0.0001 for SCA group and rs=0.651, P<0.0001 for SCA+SCAHU) and correlated with IL-8, an endothelium-derived inflammatory mediator (rs=0.900, P<0.05 for SCA and rs=0.455, P=0.06 for SCA+ SCAHU). Expression of LIGHT protein (CD258) was significantly higher on the membrane of PLTs from SCA and SCAHU individuals, compared to CON PLTs (SCA, 27.1 ± 4.5 %; SCAHU, 33.7 ± 5.8 %; CON, 5.5 ± 1.6 % positive PLTs, n=15, 20, 15, resp.; P<0.001 for SCA/SCAHU comp. CON). Notably, when PLTs were activated by incubation with ADP (20 μM, 30 min), PLTs from SCA/SCAHU individuals still expressed significantly more surface LIGHT than CON PLT (SCA, 41.8 ± 4.5 %; SCAHU, 41.3 ± 5.4 %; CON, 11.1 ± 2.3 % positive PLTs; n=15, 20, 14, resp. P<0.001 for SCA/SCAHU comp. CON); successful activation of PLTs from all groups was confirmed by increased PAC-1 (anti-activated αIIbß3 integrin) binding and increased P-selectin (CD62P, a PLT activation marker) expression (data not shown); furthermore LIGHT expression on ADP-activated cells correlated significantly with both PAC-1 binding (rs=0.483, P=0.03) and P-selectin expression (rs=0.502, P=0.02, n=20) on SCA PLTs. sLIGHT release from SCA PLTs during 90 min (37°C, 5% CO2 in Krebs) was evaluated and demonstrated that PLTs of SCA individuals may be an important source of circulating sLIGHT, releasing 22.2 ± 4.7 pg LIGHT/108 PLTs (n=10); release of sLIGHT from SCA PLT was significantly augmented by incubation with collagen (P<0.01), but not ADP (P>0.05); basal and collagen-stimulated sLIGHT release correlated significantly with CD40L release (rp=0.654; rp=0.764, resp. P<0.05). Conclusion: The pro-inflammatory and atherogenic protein, LIGHT, is found significantly elevated in the plasma of SCA patients. The correlation of plasma LIGHT with IL-8 indicates that LIGHT may participate in, or reflect, endothelial activation, whilst the correlation of circulating LIGHT and PLT release of LIGHT with CD40L indicates that the production of these two proteins may be tightly coupled. LIGHT protein is highly expressed on the PLT surface in SCA and this expression appears to be associated with PLT activation, rather than PLT number. Interestingly, HU therapy was not associated with any significant alteration in circulating LIGHT, nor PLT surface LIGHT. Future investigations will determine the extent of the contribution of LIGHT to endothelial activation, inflammation and vaso-occlusion in SCA and whether this protein holds promise as a potential therapeutic target for the disease. Disclosures: No relevant conflicts of interest to declare.

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