Abnormal Transcranial Doppler Research Articles

Moyamoya Disease Predicts Progression of Cerebral Vasculopathy in Patients with Sickle Cell Disease Despite Chronic Transfusion Therapy

Introduction: Patients with sickle cell disease (SCD) develop multi-organ complications due to hemolysis, inflammation, and vascular occlusion that results in small vessel obstruction throughout the body. In the brain, however, large cerebral vessels are also damaged resulting in occlusion or stenosis and subsequent development of abnormal collateral vasculature and moyamoya disease. Chronic red cell transfusion (CRCT) therapy significantly reduces the risk of stroke in children with abnormal transcranial doppler (TCD) studies and is also effective in reducing stroke recurrence in those with a history of overt or silent stroke; however, it is unclear if CRCT halts or reverses the progression of vasculopathy. The present study evaluated cerebrovascular stenosis and moya moya disease as risk factors for progression of vasculopathy over time in a cohort of patients with SCD who were started on CRCT therapy as children for stroke prevention.Methods: A retrospective cohort study (with IRB approval) was used to evaluate cerebrovascular changes in patients on CRCT.Patients were included in the study if they had received CRCT for stroke prevention for at least 12 months and had at least two serial magnetic resonance imaging and angiography (MRI/MRA) studies for review. For the imaging analysis, the patient's MRI/MRA closest to the initiation of CRCT (i.e. baseline imaging) was compared to the most recent imaging available by a neuro radiologist who was blind to the patient's clinical history. Additional demographic information included the patient's current age, gender, indication for CRCT, years on CRCT, and laboratory results for pre-transfusion % hemoglobin S (HbS).Results: Forty patients with SCD (current age: M = 16.48, SD = 5.10; 23 male, 17 female) were included. Average duration of CRCT therapy was 9.96 years (SD = 5.67) and average pre-transfusion HbS levels were 42.52% (SD = 9.88). Patients were initiated on therapy due to: overt stroke (n = 19), silent stroke (n = 2), and abnormal TCD (n = 20). Of the 20 patients initiated on therapy due to abnormal TCD, 7 were found to have abnormal MRI at baseline consistent with silent stroke. One of these patients was also found to have co-occurring moyamoya disease, despite no evidence of prior overt stroke. At baseline, 45% (n = 18) of patients had abnormal MRA and 25% (n = 10) had moyamoya disease. Progression of vasculopathy occurred in 15% (n = 6) of patients, all of whom had a history of moya moya disease at baseline (5 patients with overt stroke and 1 with silent stroke). Of the remaining 3 patients with moya moya disease at baseline, 2 remained stable with no improvement and 1 demonstrated improvement on MRA. For patients with abnormal MRA, but no history of moya moya disease (n = 9), 5 demonstrated improvement (2 patients with silent stroke and 3 with overt stroke).Conclusions: Progression of vasculopathy was common among patients with baseline moyamoya disease despite CRCT. Also notable, however, was improvement in vasculopathy (as defined by reduction of stenosis) in 6 patients, the majority of whom had not developed moyamoya prior to the initiation of CRCT suggesting that more mild vasculopathy can be reversed with early intervention. Patients with moya moya disease warrant ongoing annual assessment as they may require vascular bypass to prevent further worsening. Future large, multi-site investigations are needed to identify improved biomarkers and further understand characteristics of patients who demonstrate improvement versus progression of vasculopathy on CRCT. DisclosuresNo relevant conflicts of interest to declare.

TCD with Transfusions Changing to Hydroxyurea (TWiTCH): Hydroxyurea Therapy As an Alternative to Transfusions for Primary Stroke Prevention in Children with Sickle Cell Anemia

Transcranial Doppler (TCD) screening in children with sickle cell anemia (SCA) identifies abnormally elevated cerebral artery flow velocities that confer an elevated risk for primary stroke. Chronic transfusions offer effective stroke prophylaxis in this setting, but must be continued indefinitely and lead to transfusional iron overload. An alternative treatment strategy that offers similar effective protection against primary stroke, and provides control of iron overload, is needed. TCD With Transfusions Changing to Hydroxyurea (TWiTCH, NCT01425307) was an NHLBI-funded Phase III multicenter randomized clinical trial comparing 24-months of standard treatment (transfusions) to alternative treatment (hydroxyurea) in children with SCA and abnormal TCD velocities. All eligible children had received at least 12 months of transfusions. TWiTCH had a non-inferiority trial design; the primary study endpoint was the 24-month TCD velocity obtained from a linear mixed model, controlling for baseline (enrollment) values, with a non-inferiority margin of 15 cm/sec. The transfusion arm maintained children at HbS <30%; an elevated liver iron concentration (LIC) identified by R2 MRI FerriScan® was managed with chelation. The hydroxyurea arm included an overlap period with transfusions until a stable maximum tolerated dose (MTD) of hydroxyurea was reached; transfusions were then replaced by serial phlebotomy to reduce iron overload. In both arms, TCD velocities were obtained every 12 weeks and reviewed centrally, with local investigators masked to the results. A centralized TCD alert algorithm monitored changes from enrollment velocities. A total of 159 children were enrolled but 38 failed screening due primarily to severe vasculopathy on brain MRA or inadequate TCD exams; 121 children were randomized (61 to transfusions, 60 to hydroxyurea) with balanced characteristics including enrollment maximum TCD velocities (145 ± 21 versus 145 ± 26 cm/sec), age, duration of transfusions, serum ferritin, and LIC. Study participants randomized to transfusions maintained an average HbS <30% throughout the study, while those on hydroxyurea reached MTD after 7 ± 2 months at an average dose of 27 mg/kg/day, with expected hematological changes including HbF ~25% throughout the treatment period. After 37% of the participants exited the study, a scheduled interim analysis suggested the primary study endpoint was likely to be achieved. NHLBI allowed the study to continue until 50% of the children exited, at which time the statistical analysis was confirmed and the study was terminated; all remaining participants moved to the exit phase. The final analysis included 42 on the transfusion arm who completed all treatment, 11 with truncated treatment, and 8 withdrawn; the hydroxyurea arm included 41 who completed all treatment, 13 with truncated treatment, and 6 withdrawn. The final calculated TCD velocities (mean ± standard error) in the transfusion and hydroxyurea arms were 143 ± 1.6 and 138 ± 1.6 cm/sec, respectively; by intention-to-treat analysis, the p-value for non-inferiority = 8.82 x 10-16 and by post-hoc analysis the p-value for superiority = 0.046. Among 29 new neurological events, all centrally adjudicated by masked reviewers, there were no strokes but 6 transient ischemic attacks (3 in each arm). One child (transfusion arm) was withdrawn per the TCD alert algorithm after developing on-study TCD velocities >240 cm/sec. Exit brain MRI/MRA exams documented no new parenchymal abnormalities but one child (transfusion arm) developed new vasculopathy. Sickle cell related serious adverse events were more common in the hydroxyurea arm than the transfusion arm (23 to 15), but none was related to study treatment or study procedures. Iron overload improved more in the hydroxyurea arm than in the transfusion arm, with a greater average change in serum ferritin (-1085 compared to -38 ng/mL, p<0.001) and LIC (average -1.9 compared to +2.4 mg/g dry weight liver, p=0.001). In the multicenter Phase III TWiTCH trial, which treated children with SCA and abnormal TCD velocities but without severe MRA vasculopathy, hydroxyurea at MTD was non-inferior and possibly superior to chronic transfusions for maintaining TCD velocities. Serial phlebotomy effectively managed iron overload. Hydroxyurea may represent an effective alternative to indefinite transfusions for the prevention of primary stroke in this high risk population. DisclosuresWare:Eli Lilly: Other: DSMB membership; Bayer Pharmaceuticals: Consultancy; Bristol Myers Squibb: Research Funding; Biomedomics: Research Funding. Off Label Use: Hydroxyurea for children with SCA. Owen:Novartis: Speakers Bureau. Rogers:BioRad Labs: Consultancy; Apopharma: Consultancy; Baxter: Consultancy; Glaxo Smith Kline: Consultancy. Kwiatkowski:Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy; Sideris Pharmaceuticals: Consultancy; Novartis: Research Funding; ISIS: Membership on an entity's Board of Directors or advisory committees. Heeney:Sancillio: Consultancy; Eli Lilly: Research Funding. Nottage:Janssen Pharmaceuticals: Employment. Cohen:Novartis: Consultancy; ApoPharma: Other: DSMB member.

Genetic Modifiers Influencing the Development of Albuminuria in Children with Sickle Cell Anemia

Background: Nephropathy in sickle cell anemia (SCA) begins in childhood and portends chronic kidney disease, renal failure, and early mortality among affected adults. Individuals of African descent have disproportionately higher rates of developing non-diabetic renal disease. Several candidate genetic variants have been identified, including some specific to African Americans, which are associated with the development of albuminuria and renal disease. The influence of genetic polymorphisms on albuminuria and elevated glomerular filtration rate (GFR) in children with SCA, both early signs of sickle nephropathy, has not been investigated.Objectives: To determine the influence of selected single nucleotide polymorphisms (SNPs) on the development of albuminuria and elevated GFR in children with SCA; to identify novel genetic variants influencing albuminuria and GFR by whole exome sequencing (WES).Design/Methods: Genomic DNA was collected on children with SCA enrolled in two prospective studies with pre-hydroxyurea renal assessments (n=185): (1) Hydroxyurea Study of Long-Term Effects (HUSTLE, NCT00305175, n=79) with no prior disease-modifying therapy; and (2) Transcranial Doppler (TCD) With Transfusions Changing to Hydroxyurea (TWiTCH, NCT 01425307, n=106) on chronic transfusions for abnormal TCD velocities. Albuminuria was defined as ≥30mg albumin/gm creatinine on the pre-hydroxyurea urine specimen. GFR was measured in HUSTLE using plasma DTPA (technetium 99m-labeled diethylenetriaminepentaacetic acid) clearance, and estimated GFR (eGFR) in TWiTCH based on serum creatinine. DNA samples were genotyped for 8 candidate SNPs previously associated with renal disease, using PCR-based allelic discrimination, bidirectional Sanger sequencing, and analysis of variable number tandem repeats (VNTR). Associations between albuminuria and genetic polymorphisms were tested using an additive model and correlation trend test. Linked WES data from the same patients were analyzed to identify other variants associated with albuminuria and GFR.Results: Albuminuria was present in 13.1% of patients, including 16.3% in HUSTLE and 11.0% in TWiTCH. APOL1 genetic variants were common (G1 allele frequency = 21.9%, G2 allele = 16.0%, Table) and similar to published cohorts. Children with two APOL1 G1 alleles had an increased risk of albuminuria that approached statistical significance (p=0.053). Conversely, the presence of the DARC SNP that confers Duffy antigen expression had a protective effect (p=.038). WES analysis did not identify additional non-synonymous APOL1 variants linked with albuminuria. However, 93 non-synonymous variants were associated with DTPA GFR (p<0.001). Using patients with eGFR as a validation cohort, 7 variants in FUBP1, ZFAND4, CD163, GMFG and HLA-E maintained their association with kidney filtrative function (p<0.05). In particular, two variants in CD163, which is a macrophage scavenger receptor for hemoglobin-haptoglobin complexes, were strongly associated with increased GFR in both patient cohorts.Table 1Candidate genes associated with microalbuminuria. All SNPs were tested with either an additive or recessive genetic model. *The eNOS VNTR was analyzed by the chi-square method.GeneSNPLocationMAFAlbuminuria (Additive)Variant frequencyCasesControlsAPOL1 G1rs7388531922q1221.9%0.05332.0%20.3%APOL1 G2rs7178531322q1216.0%0.44510.0%16.0%DARCrs28147781q2313.6%0.0384.2%15.7%eNOS 4aVNTR7q3531.2%0.333*31.5%22.7%eNOSrs17999837q3512.5%0.66314.6%12.2%eNOSrs20707447q3515.6%0.29910.4%16.3%CUBNrs791897210p1216.2%0.45712.5%16.8%CUBNrs180123910p122.7%0.1990.0%3.1%Conclusion: Genetic polymorphisms associated with chronic kidney disease in African American adults may influence the development of early-onset albuminuria among children with SCA, including an increased risk among children with ≥1 APOL1 G1 alleles and a decreased risk associated with the DARC SNP. Previously published eNOS and CUBN variants had no measureable effects. WES analysis suggests novel genetic variants including CD163 SNPs may influence the development of elevated GFR in children with SCA, and provide candidate genes for future research. DisclosuresOff Label Use: Hydroxyurea is FDA approved for the treatment of sickle cell anemia in adults, but has not yet been approved in children. . Nottage:Janssen Pharmaceuticals: Employment. Ware:Biomedomics: Research Funding; Bristol Myers Squibb: Research Funding; Bayer Pharmaceuticals: Consultancy; Eli Lilly: Other: DSMB membership.

A Comparison of Hematologic Parameters in Patients with Sickle Cell Disease Undergoing Red Cell Exchange Using Terumo BCT Spectra Optia and COBE Spectra Apheresis Systems

Introduction:Therapeutic red blood cell exchange (RBCX) is a process by which diseased red cells are replaced by healthy donor red blood cells. In patients with sickle cell disease, RBCX has been used to treat acute stroke, severe acute chest syndrome, multiorgan failure, priapism and splenic sequestration. It is also being used more commonly in the prevention of vaso-occlusive pain crises and for stroke prophylaxis for patients considered to be at high risk of stroke based on abnormal transcranial Doppler flow rates. In May 2014, we transitioned from the COBE Spectra apheresis system (Terumo BCT) to the next-generation Spectra Optia apheresis system (Terumo BCT) for all red cell exchange transfusions performed on our patients with sickle cell disease. A previous small study (n = 33 RBCX transfusions) compared the two instruments (Caridian BCT) and showed no difference between exchange volumes, processing time, pre and post-exchange hematocrit and HbS levels (Perseghin et al. Transfusion and Apheresis Science, 2013). However, other clinical parameters such as changes in white blood cell count and platelet counts were not examined. In order to determine if there were any differences in hematologic parameters between the 2 apheresis instruments, we measured the differences between pre and post-exchange HbS levels, white blood cell count (WBC), hematocrit (Hct), and platelet counts for all of our adult patients who underwent RBCX transfusions on both instruments.Methods:This was a single institution, retrospective chart review of all adult patients with sickle cell disease (HbSS n=17, HbS/β0 thalassemia n = 1, HbSC n =1) who underwent routine, monthly RBCX at Children's Hospital and Research Center Oakland between November 2013 and February 2015. Indications for RBCX included a risk of stroke or a history of stroke, acute chest syndrome, or renal failure. All patients received RBCX transfusions on the COBE Spectra prior to May 2014 and were then transitioned to Spectra Optia in May 2014. Pre and post-exchange transfusion HbS levels, WBC, Hct, and platelet counts were measured for each procedure. All exchange transfusions were non-emergent and well tolerated. Statistical analyses using the student's t test and rank sum test were performed with Stata 14.0 software (College Station, Texas).Results:A total of 19 adult patients (mean average age 25 years old) underwent 153 red blood cell exchange transfusions (Spectra Optia n =87, COBE Spectra n = 66). There was a small increase in mean hematocrit percentage for both instruments (28.33 + 4.3 % to 29 + 3.06 % and 28.16 + 4.29% to 29.2 + 3.04% on the Spectra Optia and COBE Spectra, respectively). All other post-RBCX parameters decreased. The mean changes are shown in Table I. The HbS percent decreased from a combined mean of 39.86 + 12.11 % to 20.26 + 8.43 % for both instruments. The WBC decreased from a combined mean of 12.72 + 3.13 x 1000/ mm3 to 8.67 + 2.15 x 1000/ mm3. The platelet count decreased from a combined mean of 368.82 + 125.75 x 1000/ mm3 to 191.01 + 62.78 x 1000/ mm3. The mean parameter values for each patient correlated with the mean changes for each instrument based on statistical analysis using both the student's t test and rank sum test.Conclusions:The starting hematologic values were similar between the two instruments. There was no statistical difference between the raw pre and post-RBCX HbS, Hct, WBC, or platelet values or the mean changes in these parameters between the COBE Spectra and Spectra Optia instruments. There was also no statistical difference in the mean changes between the pre and post RBCX hematologic parameters amongst the 19 patients. Both instruments allowed for effective reduction in HbS percentage with comparable decreases in WBC and platelet counts while maintaining adequate hematocrit values for all 19 adult patients with sickle cell disease.Table 1Mean changes between Pre-RBCX and Post-RBCX.InstrumentChange in % HbSChange in WBC x1000/mm3Change in % HctChange in platelet count x1000/mm3Spectra OptiaN84878785Mean-20.28-4.010.67-172.44SD7.022.552.8676.98COBE SpectraN66666666Mean-19.18-4.121.09-178.02SD7.892.262.5781.02p-value0.370.780.340.67There are 3 missing values for HbS and 2 missing values for platelet count for Spectra Optia. SD = standard deviation, WBC = white blood cell count, Hct = hematocrit DisclosuresNo relevant conflicts of interest to declare.

Organ iron accumulation in chronically transfused children with sickle cell anaemia: baseline results from the TWiTCH trial.

Transcranial Doppler (TCD) With Transfusions Changing to Hydroxyurea (TWiTCH) trial is a randomized, open-label comparison of hydroxycarbamide (also termed hydroxyurea) versus continued chronic transfusion therapy for primary stroke prevention in patients with sickle cell anaemia (SCA) and abnormal TCD. Severity and location of iron overload is an important secondary outcome measure. We report the baseline findings of abdominal organ iron burden in 121 participants. At enrollment, patients were young (9·8±2·9years), predominantly female (60:40), and previously treated with transfusions (4·1±2·4years) and iron chelation (3·1±2·1years). Liver iron concentration (LIC; 9·0±6·6mg/g dry weight) and serum ferritin were moderately elevated (2696±1678μg/l), but transferrin was incompletely saturated (47·2±23·6%). Spleen R2* was 509±399Hz (splenic iron ~13·9mg/g) and correlated with LIC (r(2) =0·14, P=0·0008). Pancreas R2* was increased in 38·3% of patients but not to levels associated with endocrine toxicity. Kidney R2* was increased in 80·7% of patients; renal iron correlated with markers of intravascular haemolysis and was elevated in patients with increased urine albumin-creatinine ratios. Extra-hepatic iron deposition is common among children with SCA who receive chronic transfusions, and could potentiate oxidative stress caused by reperfusion injury and decellularized haemoglobin.

Management of Sickle Cell Disease Summary of the 2014 Evidence-Based Report by Expert Panel Members

Yawn, Barbara P.*; Buchanan, George R.†; Afenyi-Annan, Araba N.‡; Ballas, Samir K.§; Hassell, Kathryn L.∥; James, Andra H.¶; Jordan, Lanetta#; Lanzkron, Sophie**; Lottenberg, Richard††; Savage, William J.‡‡; Tanabe, Paula J.§§; Ware, Russell E.∥∥; Murad, M. Hassan¶¶; Goldsmith, Jonathan C.##; Ortiz, Eduardo##; Fulwood, Robinson##; Horton, Ann***; John-Sowah, Joylene## Author Information

G6PD deficiency and absence of α-thalassemia increase the risk for cerebral vasculopathy in children with sickle cell anemia.

The aim of this study was to test the association between hematological/genetic factors and cerebral vasculopathy in children with sickle cell anemia (SCA). A group with cerebral vasculopathy (VASC) was composed of children who had stroke (n = 6), silent infarct (n = 11), or an abnormal transcranial Doppler (n = 5). Eighty-four patients had neither positive history of stroke or silent infarct, nor abnormal transcranial Doppler (NORM group). An intermediate group (COND; n = 15) was composed of SCA children with a conditional transcranial Doppler. Biological analyses were performed on samples obtained at steady state and before the beginning of any chronic treatment. The comparisons of the three groups demonstrated a protective effect of α-thalassemia against cerebral vasculopathy through its effects on hemoglobin and reticulocyte levels. Moreover, we observed higher frequency of G6PD deficiency in the VASC group compared with the other groups. Our study confirms the key role of α-thalassemia and G6PD status in the pathophysiology of cerebral vasculopathy in SCA children.

How I manage cerebral vasculopathy in children with sickle cell disease.

Sickle cell disease induces specific brain alterations that involve both the macrocirculation and the microcirculation. The main overt neurovascular complications in children are infarctive stroke, transient ischaemic attack and cerebral haemorrhage. Silent cerebral infarction, cognitive dysfunction and recurrent headache are also common. Cerebrovascular disease selectively affects children with the HbSS or HbS-β(0) genotypes (i.e. sickle cell anaemia). The incidence of stroke peaks between 2 and 5years of age (1·02/100 patient-years) and increases with the severity of the anaemia. Most strokes can be prevented by annual transcranial Doppler screening from 2 to 16years of age and providing chronic blood transfusion when this investigation shows elevated blood-flow velocities. The role for hydroxycarbamide in children with abnormal transcranial Doppler findings is under investigation. After a stroke, chronic blood transfusion is very strongly recommended, unless haematopoietic stem cell transplantation can be performed. Routine magnetic resonance imaging shows that more than one-third of children have silent cerebral infarction, which is associated with cognitive impairments. Screening for silent infarcts seems legitimate, since their presence may lead to supportive treatments. The role for more aggressive interventions such as hydroxycarbamide or chronic blood transfusion is debated.

Chronic and acute anemia and extracranial internal carotid stenosis are risk factors for silent cerebral infarcts in sickle cell anemia

AbstractEarly transcranial Doppler (TCD) screening of the Créteil sickle cell anemia (SCA)-newborn cohort, and rapid initiation of transfusion programs, resulted in successful prevention of overt strokes, but a high cumulative risk of silent cerebral infarcts (SCI) remained, suggesting that TCD screening does not identify all patients with SCA at risk for SCI. We hypothesized that episodes of hypoperfusion/hypoxia, as observed during acute chest syndromes or acute anemic events (AAE), and extracranial internal carotid artery (eICA) stenoses, detectable via submandibular Doppler sonography and cervical magnetic resonance angiography (MRA), could also be risk factors for SCI. This study includes 189 stroke-free patients with SCA from the Créteil newborn cohort (1992-2010) followed longitudinally by magnetic resonance imaging/MRA, including cervical MRA at the last assessment. All patients with abnormal TCD and/or intracranial stenoses were placed on a transfusion program. Mean follow-up was 9.9 years (range, 2.2-19.9 years; 1844 patient-years). Annual rates of clinical events were calculated. The cumulative risk for SCI was 39.1% (95% confidence interval [CI], 23.5%-54.7%) by age 18 years, with no plateau. We confirm that baseline hemoglobin level lower than 7 g/dL before age 3 years is a highly significant predictive risk factor for SCI (hazard ratio, 2.97; 95% CI, 1.43-6.17; P = .004). Furthermore, we show that AAE rate (odds ratio, 2.64 per unit increase; 95% CI, 1.09-6.38; P = .031) and isolated eICA stenosis (odds ratio, 3.19; 95% CI, 1.18-8.70; P = .023) are significant and independent risk factors for SCI.

Dépistage de la vasculopathie cérébrale drépanocytaire par doppler transcrânien au Mali

Cerebral vasculopathy exposes patients to a high risk of stroke, a major complication of sickle cell disease (SCD) associated with a high risk of death and disability. Transcranial doppler (TCD) ultrasonography used to identify SCD patients at risk of stroke may contribute to significantly reducing morbidity and mortality in these patients by indicating appropriate treatment. From March 2008 to February 2013, we conducted systematic screening for cerebral vasculopathy using TCD in 572 SCD patients (including 375 SS, 144 SC, 26 S/β(0), and 27 S/β(+) thalassemia patients) aged 1-17 years in a comprehensive center for follow-up and research on sickle cell disease in Bamako, Mali. After exclusion of 30 inadequate results and one case of abnormal TCD observed in a multiple organ failure patient, we found an abnormal or conditional TCD in 18% of 541 children examined in a steady state. The highest prevalence of abnormal cases concerned homozygous SS patients (8.1%). No case of abnormal or conditional TCD was observed in children with S/β(+) thalassemia. Hemoglobin concentrations were significantly lower in patients with conditional or abnormal TCD (P<0.01). In a subgroup of 68 patients with conditional TCD, nine (13%) converted to abnormal TCD over 1 year. In this subgroup of 68 conditional TCD patients, a decrease or increase in baseline hemoglobin concentration was predictive of conditional or abnormal TCD at the follow-up visit. Progression towards conditional TCD was observed in four patients (0.9%) who initially had normal TCD. Children with abnormal TCD had, whenever possible, a monthly exchange transfusion program. One case of transient stroke in the context of P. falciparum malaria with low hemoglobin concentration and one death were observed. These findings highlight the need for systematic TCD in sickle cell disease monitoring and implementing regular blood transfusion programs in the context of limited access to regular and secure blood transfusions.

Association of Annexin A5 Resistance with Silent Infarct in Sickle Cell Disease

Background: In sickle cell disease (SCD) abnormally shaped RBCs interact with white blood cells and the endothelium, leading to a vasculopathy and thrombotic/ prothrombotic complications such as stroke and pulmonary hypertension. About 10 % of patients with a thrombotic event in the general population will have the presence of an antiphospholipid (aPL) antibody (Andreoli et al. 2013). One proposed mechanism for the thrombophilic nature of aPL antibodies is the disruption of annexin A5. Annexin A5 is a potent anticoagulant protein that has an affinity to phospholipids. In the presence of aPL antibodies, annexin A5 is unable to form its crystallized anticoagulant shield (annexin A5 resistance). There is a paucity of data which assesses the association of aPL antibodies with vasculopathic complications of SCD, and there have been no studies investigating the annexin A5 resistance assay (A5R). We designed a pilot study assessing aPL antibody levels and A5R in a pediatric sickle cell population.Methods: Patients with a history of stroke, abnormal transcranial doppler (TCD) and elevated TR gradient by echocardiography (>25mm of Hg) were eligible. A5R, lupus anticoagulant- DRRVT, anti β2GP1, anti phosphatidylserine and anti cardiolipin antibody (IgG, IgA, IgM) assays were performed on samples obtained prospectively when patients were at a steady state (at least 4 weeks after an acute event). A5R measures coagulation times in the presence and absence of annexin A5. Resistance to the anticoagulant effects of annexin is expressed as a reduction in this ratio (Rand et al. 2004). Statistical analysis assessed multiple variables including age, gender, hemoglobin, reticulocyte count, LDH, hydroxyurea therapy, transfusion therapy, elevated TR gradient, stroke and silent stroke. Univariate analysis and multivariate logistic regression was performed with abnormal annexin A5 as the outcome variable.Results:There were a total of 39 patients: 12 patients with a history of stroke, 6 with an elevated TCD velocity and 15 patients with an elevated TR gradient. Of the 27 patients that did not have a stroke, 25 had a screening MRI in the prior year, and 9 of these patients had silent infarcts. Only 1 of 39 patients had elevated anticardiolipin IgG antibodies and 1 had an abnormal lupus coagulant (DRVVT). In contrast, 5/39 patients (12.8%) had low or abnormal annexin A5 resistance, 7/39 (18%) were in the borderline range and 27/39 (69%) were normal. This frequency of abnormality was unexpected in the antiphospolipid antibody and lupus anticoagulant negative population. None of the patients, except the one with the positive lupus anticoagulant, developed any thrombotic events in 3.5 years of follow up. None of the patients with overt stroke or abnormal TCDs had an abnormal A5R. Multivariate logistic regression analyses showed statistically significant association of hemoglobin (p= 0.037, OR 0.25, CI 0.07-0.92)), age (p =0.047, OR 1.43, CI 1.01-2.04) and silent infarct (p =0.015, OR 28.5, CI 1.9-420.5) with abnormal annexin A5 resistance. A multivariate analysis using linear regression with annexin A5 resistance as a continuous outcome variable (Table 1), showed persistence of the significant association of silent infarcts (p =0.037).Conclusion: We report an association between annexin A5 resistance and low hemoglobin, older age and presence of silent infarct in a subgroup of SCD patients. Prevalence of abnormal aPL antibody assays and lupus anticoagulant was strikingly low in this cohort. A potential role for perturbed annexin A5 resistance in the pathophysiology of silent infarction in SCD will need to be evaluated further in carefully designed prospective studies and may be a novel therapeutic target.Table 1Hemoglobin, Age and Silent Stroke are Associated with Abnormal Annexin A5 ResistanceCharacteristicOdds RatioOdds Ratio Confidence Intervalp-valueHemoglobin*0.250.07-0.920.037Reticulocyte count0.770.54-1.080.13LDH1.000.99-1.000.51Age*1.431.01-2.040.047Monocyte count1.001.00-1.000.34Silent infarct*28.51.9-420.50.015Stroke0.470.05-4.880.53Elevated TR gradient0.340.03-3.490.36Gender0.470.05-4.880.53Allosensitization0.560.05-5.860.63HU vs no treatment0.460.03-6.930.58Transfusion vs no treatment0.180.01-4.260.29 DisclosuresNo relevant conflicts of interest to declare.

Reticulocytosis and Anemia in Early Infancy Is Associated with Abnormal and Conditional Transcranial Doppler Velocities in Pediatric Sickle Cell Anemia Patients

All sickle cell anemia patients (HbSS, SCA) have the same genetic mutation, but the clinical phenotype is highly variable and difficult to predict prior to the onset of complications. Transcranial Doppler (TCD) is the only validated predictive indicator of severe SCA currently available, identifying SCA patients age 2 years or older with the greatest stroke risk. Preliminary studies have identified that elevated absolute reticulocyte count (ARC) between 2 and 6 months of age is associated with an increased risk of hospitalization for SCA complications, stroke and death.1,2To determine if early reticulocyte levels in SCA patients are useful in classifying children at highest risk of developing an abnormal (abTCD) or conditional (cdTCD) TCD, 121 consecutive patients with SCA who had TCD screening were identified after IRB review granted a waiver of consent. Retrospective chart review was then performed to collect the steady state ARC between 2 and 6 months of age; patients were excluded if this value was not available in the medical record. Steady state was defined as a sample collected at least 30 days from an acute illness and at least 60 days since the patient received a blood transfusion. ARC and hematologic data were analyzed using Cox regression analysis to determine the relationship between ARC levels and time to cdTCD/abTCD. TCDs were considered normal (nlTCD) if the Time Average Mean Maximum Velocity (TAMMV) in the middle cerebral artery or distal internal carotid artery was <170 cm/sec, cdTCD if TAMMV was 170-199 cm/sec and abTCD if TAMMV was 200 cm/sec or greater. To evaluate the utility of ARC or hemoglobin in risk stratification, patients were divided into groups: ARC less than 200K/uL (ARC<200) and ARC greater than or equal to 200K/uL (ARC≥200), or hemoglobin less than 8.5 g/dL (Hb<8.5) and hemoglobin greater than or equal to 8.5 g/dL (Hb≥8.5).Twenty of the 121 (16.5%) patients had abTCD, while 36/121 (29.8%) had cdTCD; the remaining 65/121 (53.7%) had nlTCDs. Mean ARC between 2 and 6 months of age was highest in those children with abTCDs (264±149 K/uL) and cdTCDs (175±76 K/uL) while those who had nlTCDs had the lowest values (mean ARC 140±100K/uL, p<0.001 across groups). Mean hemoglobin was lower in the abTCD (8.1±1.2 g/dL) and cdTCD groups (8.9±1.1 g/dL) compared to the nlTCD group (9.5±1.5 g/dL, p<0.001). The three groups did not differ in age at time of ARC measurement [mean age in months: 3.6±1.2 (nlTCD) vs. 3.8±1.3 (cdTCD) vs. 3.4±1.0 (abTCD), p=0.86].Kaplan Meier analysis revealed that those children with higher ARC had an increased rate of cdTCD/abTCD (p<0.001 by the log-rank test). Lower hemoglobin was also associated with an increased rate of cdTCD/abTCD (p=0.002). Cox regression analysis revealed that those subjects with an ARC≥200 between the ages of 2 and 6 months had 2.9 times the risk of having an abTCD or cdTCD than the group with an ARC<200 (HR 2.92, 95% CI 1.68-5.08, p=0.0004). In separate analyses, patients with a hemoglobin less than 8.5 g/dL had 2.4 [95%CI 1.38-4.06 (p=0.002)] times the risk of having an abTCD/cdTCD than patients with a hemoglobin level of 8.5 g/dL or greater.These data suggest that both elevated ARC and low baseline hemoglobin during early infancy are associated with an increased risk of developing a cdTCD or abTCD later in childhood. ARC and hemoglobin at this early age should be prospectively studied as standard screening tests to assist with treatment decisions in young children with SCA. 1.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.0070794.2.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] DisclosuresNo relevant conflicts of interest to declare.

Chronic Manual Exchange Transfusions Compared with Erythrocytapheresis in Children and Teens with Sickle Cell Disease

Background:Children and young adults with sickle cell disease (SCD) are at high risk of strokes and are frequently treated with red blood cell (RBC) transfusions. RBCs may be given by simple transfusion, manual exchange transfusion (ME), or erythrocytapheresis (ECP) with a goal of suppressing hemoglobin (Hb) S while minimizing transfusion-induced iron overload. There have been no formal comparisons of these modalities, and practices for transfusion management vary among institutions. We compared transfusion therapy outcomes among patients with SCD undergoing transfusion therapy for primary or secondary stroke prevention, hypothesizing that children would be more likely to achieve Hb S suppression and ferritin goals while receiving ECP. We also compared complications of transfusion therapy across transfusion modalities.Methods:This is a single-institution retrospective cohort study of 38 patients with SCD who received chronic transfusion therapy for primary or secondary stroke prevention from 1/1/2008 through 12/31/2012. Per institutional practice, younger patients receive ME for stroke prevention; they are offered ECP when their size is adequate for a large-bore double-lumen implantable port, but may choose to continue ME. The pre-transfusion Hb S goal is <30% for at least 2 years, then may be liberalized to <50% for subjects without either abnormal transcranial Doppler ultrasound or infarct recurrence. Hb S percentage and ferritin were measured prior to each transfusion. Patients on transfusion therapy for 6 or more months were included; one child who had a stroke after brain tumor biopsy was excluded. Subjects were censored at last date of follow-up or date of hematopoietic stem cell transplant. The following factors were evaluated: duration and mode of transfusion therapy, achievement of Hb S suppression goal, ferritin levels, and catheter complications. Categorical variables were compared with Fisher’s exact test and medians with the Mann-Whitney U-test in SPSS version 21 (IBM, Armonk, NY).Results:During the study period, 38 subjects (42% male) met all inclusion criteria. Of these, 5 received exclusively ECP, 17 received exclusively ME, and 16 received both modalities during the study period. For the most recent 12-month period of data for each participant, 13 received ECP and 25 received ME. There was no association between modality of transfusion and the proportion of visits during which subjects achieved their pre-transfusion Hb S goal during the 12-month period. The median proportion of visits achieving the Hb S goal was 0.80 for ECP (IQR 0.40-1.0) versus 0.50 for ME (IQR 0.28-0.90) (p=0.27). Furthermore, there was no significant difference in ferritin concentrations between transfusion modalities: median 875 ng/ml for ECP (IQR 578-2659) versus median 1527 ng/ml for ME (IQR 731-2568) (p=0.56).Children who had ever received ECP had a significantly longer total duration of transfusion therapy (median 97 months, IQR 51.5-134) than those receiving ME only (median 28 months, IQR 12.5-47) (p<0.001). Among 21 subjects who had ever received ECP, 15 (71.4%) experienced one or more catheter complications, including infection, thrombosis, catheter leakage, or venous stenosis, compared with 1/17 subjects (5.8%) who had never received ECP (OR for catheter complications 40 for subjects who had ever received ECP, 95% CI 4.29, 372.4, p <0.001). Five subjects switched from ECP to ME due to stenosis of the great vessels that precluded double-lumen port replacement.Conclusions:Children with SCD receiving ECP and ME for stroke prevention in this cohort had similar achievement of Hb S suppression goals and iron overload management. Additional patient-specific factors may be responsible for variations in pre-transfusion Hb S and ferritin concentrations. Catheter complications were significantly more common in children and adolescents receiving ECP compared with ME, likely due to the large-bore double-lumen port utilized for ECP at our center. DisclosuresNo relevant conflicts of interest to declare.

Quantitation of Iron in Brain Structures of Children with Sickle Cell Disease and Transfusion Hemosiderosis

Introduction:Children with sickle cell disease (SCD) and abnormal cerebral blood flow often receive cyclic blood transfusions (CTx) to reduce risk of stroke. While effective for stroke prevention, CTx causes systemic iron accumulation that may damage multiple organs (Harmatz P., Blood 2000). Iron toxicity from CTx is poorly described for the brain, in comparison to the liver and heart. Quantifying iron in the subcortical structures and the choroid plexus is of renewed clinical importance, due to recent findings that iron accumulation in these regions may predict CNS injury in neurodegenerative disorders. In this study, a new imaging protocol is applied to quantify iron levels and measure structural volumes for the subcortical structures and choroid plexus of children with SCD on CTx, using healthy children for comparison.Methods:SCD patients were eligible if at least 12 consecutive months of CTx were received for abnormal transcranial doppler. Patients with history of CVA were excluded, due to potential confounding effects of prior ischemic damage.Healthysiblings and relatives were used as ethnic- and age-matched controls.MRI scans of the liver and brain, using a Siemens 3T Trio scanner, was performed without sedation for all subjects. A modified pulse sequence (R2*) for the 3T scanner was used to determine liver iron concentration. T1-weighted imaging sequence with MPRAGE was used for anatomical identification and volume measurements of brain regions (Kirk G., Cerebral Cortex 2009). A 3D multi-echo gradient-echo sequence (FOV=256x192mm, Matrix=256x192, Slice thickness = 2mm, 72 slices, TR = 50 ms, first TE = 3.7ms, 12 echoes, echo spacing = 3.8ms) was used for the quantitative susceptibility mapping (QSM), a MRI method that is sensitive to tissue iron (Shmueli K., Magn Reson Med 2009). Iron levels were generated with a post-processing protocol developed for this application (modification of Qiu D., AJNR 2014). To compare iron levels between the two groups a general linear model (GLM) was used that incorporated the ages of the subjects into the comparison. For the patients, an additional GLM analysis was performed to determine if iron level when in each region when corrected for age was associated duration of transfusions, liver R2* and average serum ferritins.Results:MRI scans were evaluable for 25 SCD patients on CTx and 23 controls. There were no significant differences between patients and healthy controls with respect to age, sex or ethnicity. The average duration of CTx was 6.0 (1.5-12.6) yrs and the average serum ferritin and liver iron R2* were 2540 ng/ml and 819 Hz (~14.8mg/g dry weight), respectively. As anticipated, iron level in the choroid plexus was significantly elevated in patients compared to controls (p=1.4x10-7), and levels correlated well with serum ferritin (p=0.0003) and liver R2* (p=0.001) (panel a). While there were no significant differences detected between the groups with respect to iron levels in the subcortical structures, there was a trend to increasing differences between the groups with age in the Substanita Nigra, Red Nucleus and Dentate Nucleus (panels b,c,d). In the GLM analysis, age was significantly associated with iron levels in the subcortical structures, not in the choroid plexus. Although iron levels in some of the subcortical structures were associated with duration of CTx, ferritin and liver R2*, the significance was predominately lost when corrected for age. These observations suggest additional studies with older subjects may reveal increased differences. Volumetric analysis was performed to assess potential injury to deep gray matter. The measured volumes of the subcortical structures were not statistically different between the groups, with a noted exception of the caudate nucleus (p=0.021), a region of known predisposition to injury in SCD (Steen R., Ann Neurol 1999).Conclusions: The MRI protocol successfully quantified iron in brain structures, associated with neurodegenerative disorders. Iron accumulation in the choroid plexus was significant in SCD patients on CTx, and correlated with other markers of iron overload. While iron was detectable in subcortical structures, the levels were not significant different from controls after adjusting for age. These finding should be helpful in planning for a longitudinal study that includes a broader age range. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Implementation of transcranial Doppler ultrasonography screening and primary stroke prevention in urban and rural sickle cell disease populations.

Transcranial Doppler (TCD) ultrasonography identifies children with sickle cell disease (SCD) at increased risk of stroke. Initiation of chronic transfusions as primary stroke prevention in children with abnormal TCD significantly reduces stroke risk. Here, we report the results describing the implementation of TCD screening and primary stroke prevention in both urban and rural clinical practices. Retrospective chart review identified children ages 2-16 years with Hgb SS or Sß0 -thalassemia and no history of stroke followed in either the local urban or rural SCD clinics at Georgia Regents University. We defined standard of care (SOC) as having one TCD performed annually between January 2010 and December 2012 starting at age 2 years. A total of 195 patients were included in the evaluation of SOC screening, overall 41% achieved SOC. There was no difference in SOC between the two clinics (35% urban and 47.4% rural). The majority of patients with abnormal TCDs are on chronic transfusions (83%), and none have experienced a stroke. Monitoring of effects of transfusion was difficult with 38% and 31% of rural patients lacking documentation of Hgb S% and ferritin levels, respectively, in the past year. We report here data describing primary stroke prophylaxis in rural patients. SOC rates are similar between the two clinical settings. While implementation of primary stroke prevention in rural patients was difficult, rural TCD screening is feasible and can achieve SOC equal to that in an urban setting. This suggests that barriers exist in provided primary stroke prevention to all patients. Pediatr Blood Cancer 2015;62:219-223. © 2014 Wiley Periodicals, Inc.

Management of sickle cell disease: summary of the 2014 evidence-based report by expert panel members.

Sickle cell disease (SCD) is a life-threatening genetic disorder affecting nearly 100,000 individuals in the United States and is associated with many acute and chronic complications requiring immediate medical attention. Two disease-modifying therapies, hydroxyurea and long-term blood transfusions, are available but underused. To support and expand the number of health professionals able and willing to provide care for persons with SCD. Databases of MEDLINE (including in-process and other nonindexed citations), EMBASE, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, CINAHL, TOXLINE, and Scopus were searched using prespecified search terms and keywords to identify randomized clinical trials, nonrandomized intervention studies, and observational studies. Literature searches of English-language publications from 1980 with updates through April 1, 2014, addressed key questions developed by the expert panel members and methodologists. Strong recommendations for preventive services include daily oral prophylactic penicillin up to the age of 5 years, annual transcranial Doppler examinations from the ages of 2 to 16 years in those with sickle cell anemia, and long-term transfusion therapy to prevent stroke in those children with abnormal transcranial Doppler velocity (≥200 cm/s). Strong recommendations addressing acute complications include rapid initiation of opioids for treatment of severe pain associated with a vasoocclusive crisis, and use of incentive spirometry in patients hospitalized for a vasoocclusive crisis. Strong recommendations for chronic complications include use of analgesics and physical therapy for treatment of avascular necrosis, and use of angiotensin-converting enzyme inhibitor therapy for microalbuminuria in adults with SCD. Strong recommendations for children and adults with proliferative sickle cell retinopathy include referral to expert specialists for consideration of laser photocoagulation and for echocardiography to evaluate signs of pulmonary hypertension. Hydroxyurea therapy is strongly recommended for adults with 3 or more severe vasoocclusive crises during any 12-month period, with SCD pain or chronic anemia interfering with daily activities, or with severe or recurrent episodes of acute chest syndrome. A recommendation of moderate strength suggests offering treatment with hydroxyurea without regard to the presence of symptoms for infants, children, and adolescents. In persons with sickle cell anemia, preoperative transfusion therapy to increase hemoglobin levels to 10 g/dL is strongly recommended with a moderate strength recommendation to maintain sickle hemoglobin levels of less than 30% prior to the next transfusion during long-term transfusion therapy. A strong recommendation to assess iron overload is accompanied by a moderate strength recommendation to begin iron chelation therapy when indicated. Hydroxyurea and transfusion therapy are strongly recommended for many individuals with SCD. Many other recommendations are based on quality of evidence that is less than high due to the paucity of clinical trials regarding screening, management, and monitoring for individuals with SCD.

Haptoglobin, alpha‐thalassaemia and glucose‐6‐phosphate dehydrogenase polymorphisms and risk of abnormal transcranial Doppler among patients with sickle cell anaemia in Tanzania

Transcranial Doppler ultrasonography measures cerebral blood flow velocity (CBFv) of basal intracranial vessels and is used clinically to detect stroke risk in children with sickle cell anaemia (SCA). Co-inheritance in SCA of alpha-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) polymorphisms is reported to associate with high CBFv and/or risk of stroke. The effect of a common functional polymorphism of haptoglobin (HP) is unknown. We investigated the effect of co-inheritance of these polymorphisms on CBFv in 601 stroke-free Tanzanian SCA patients aged <24years. Homozygosity for alpha-thalassaemia 3·7 deletion was significantly associated with reduced mean CBFv compared to wild-type (β-coefficient -16·1cm/s, P=0·002) adjusted for age and survey year. Inheritance of 1 or 2 alpha-thalassaemia deletions was associated with decreased risk of abnormally high CBFv, compared to published data from Kenyan healthy control children (Relative risk ratio [RRR]=0·53 [95% confidence interval (CI):0·35-0·8] & RRR=0·43 [95% CI:0·23-0·78]), and reduced risk of abnormally low CBFv for 1 deletion only (RRR=0·38 [95% CI:0·17-0·83]). No effects were observed for G6PD or HP polymorphisms. This is the first report of the effects of co-inheritance of common polymorphisms, including the HP polymorphism, on CBFv in SCA patients resident in Africa and confirms the importance of alpha-thalassaemia in reducing risk of abnormal CBFv.

The Cerebral Vasculopathy In Malian Sickle Cell Anemia Children: Lesson From Routine Transcranial Doppler Screening

Cerebral vasculopathy is one of the major complications of Sickle Cell Disease (SCD). 11% of the homozygous SCD patients experience stroke by age of 20 years. The use of Transcranial Doppler (TCD) allows identification of patients at risk for stroke and leads to implementation of a preventive treatment that contributes to limit the burden of SCD particularly during childhood. Using TCD screening, we evaluated the prevalence and incidence of cerebral vasculopathy in Malian SCD children. During the years 2008 to 2013, 572 children, 249 girls and 323 boys, age range (2-17yrs) were routinely screened by TCD at our Sickle Cell Disease Research and Control Center of Bamako, Mali. The overall prevalence of cerebral vasculpathy defined by conditional (1.5-1.79 cm/sec) and abnormal TCD (≥ 1.80 cm/sec) in this population is 17.1%. The highest proportion (92.9%) was observed in homozygous SCD patients while the percentage of affected patients was much lower in S/β0thalassemia (4.1%) and in SC (3.1%) patients. No cases were observed in S/β+thalassemia patients. SCD children &lt;9 years old were more susceptible to cerebral vasculopathy complications than those above this age threshold (P&lt;0.001). Low hemoglobin levels and low fetal hemoglobin were associated with an increased risk of cerebral vasculopathy. During the study, 4 of 444 children with normal TCD converted to conditional TCD, while 5 of 68 children with conditional TCD converted to abnormal TCD. This conversion from conditional to abnormal TCD was associated with a mean decrease in Hb value of 0.37g/dL (P=0.002). In conclusion this study shows high prevalence and incidence of cerebral vasculopathy in Malian SCD children. While chronic transfusion programs significantly reduces the risk of stroke in SCD patients with abnormal TCD, at present there are no well articulated strategies to prevent conversion from conditional to abnormal TCD. A more comprehensive approach would hopefully reduce the morbidity and mortality due to cerebral vasculopathy in SCD affected children. Disclosures: No relevant conflicts of interest to declare.

Liver Iron Concentration By MRI In Chronically Transfused Children With Sickle Cell Anemia In The Twitch Trial

IntroductionChronic transfusion therapy represents the standard of care for sickle cell anemia (SCA) patients with abnormal transcranial Doppler (TCD) ultrasound or prior stroke. While effective, monthly transfusions produce iron overload and toxicity if not controlled with chelation therapies. Liver iron concentration (LIC) is a powerful surrogate for total body iron stores. Unfortunately, liver biopsy is not suited for longitudinal analysis because it is invasive, expensive, and prone to sampling variability. MRI transverse relaxation rates, R2 and R2*, are highly correlated with LIC and have mostly supplanted liver biopsy for iron quantification in clinical practice and clinical trials. Since R2 and R2* have different sensitivity to the size and scale of tissue iron distribution, we compared the agreement of LIC values predicted by R2 and R2* in children with SCA and transfusional iron overload from the prospective multicenter TCD with Transfusions Changing to Hydroxyurea (TWiTCH) trial (ClinicalTrials.gov; NCT01425307). Methods133 patients underwent LIC assessment using both R2 and R2* techniques at 22 MRI sites. All sites used 1.5 Tesla magnets and torso phased array coils. Images for R2 measurements were collected on validated scanners and analyzed centrally according to the FerriScan” protocol (Resonance Health, Western Australia, see St Pierre, T.G., et al. Blood,105, 855-861, 2005). Images for R2* assessment were collected using multiple-echo gradient echo sequences (see Wood, J.C., et al. Blood,106, 1460-1465, 2005). Images were analyzed centrally at Children's Hospital Los Angeles, using an exponential-plus-constant fit to the signal decay. Bland-Altman analysis on log-transformed LIC values was used to test agreement between LICR2 and LICR2*; the residuals of this relationship were probed for association with transfusion/chelation history, markers of inflammation, and markers of hemolysis. [Display omitted] [Display omitted] ResultsFigure 1A illustrates the scattergram between LICR2* and LICR2. The variance of the disagreement between the two techniques increases with LIC, so log-transformation was performed prior to Bland Altman analysis. LICR2* was systematically higher than LICR2 below about 5 mg Fe/g dw and systematically lower above 5 mg Fe/g dw. Bland Altman comparison of the log-transformed data (Figure 1B) reveals a downward trend (r2 of 0.203, p<0.0001). After correcting for the trend, 95% limits of agreement were -0.42 to 0.42, translating to 95% limits of agreement of the ratio of the two LIC measurements of 0.66 to 1.52. After controlling for mean log LIC, differences in log LIC values were not associated with transfusion or chelation history, markers of inflammation, or markers of hemolysis. DiscussionSystematic bias is present between LICR2 and LICR2* in a cohort of children with SCA and transfusional iron overload. Even after correcting these differences, LICR2 and LICR2* also demonstrate significant intrasubject variability, comparable to the error both techniques displayed with respect to biopsy, precluding use of these metrics interchangeably. This implies that LICR2 and LICR2* have potentially clinically significant deviations from true LIC. Rather than sampling or MRI measurement errors, which are consistently < 10% in multiple studies, these disparities likely reflect calibration bias introduced by intersubject differences in tissue iron distribution. Longitudinal LIC determination should lessen their impact, however, and the changes in LIC predicted by R2 and R2* will be compared using one and two year data from the TWiTCH trial. Disclosures:Wood:Novartis: Honoraria; Apopharma: Honoraria, Patents & Royalties; Shire: Consultancy, Research Funding. Off Label Use: Hydroxyurea is FDA-approved for use in adults but not children. Kwiatkowski:Shire: Consultancy; Resonance Health: Research Funding. St. Pierre:Resonance Health Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Increased Pre-Transfusion Reticulocytosis Among Chronically Transfused Sickle Cell Disease Patients Is Associated With More Severe Cerebral Vasculopathy

Chronic red blood cell transfusion therapy is indicated for primary and secondary stroke prevention in children with sickle cell disease (SCD). The main transfusion goal is achievement of pre-transfusion sickle hemoglobin (HbS) levels of 30%. Unfortunately, there continues to be a population of patients with a history of stroke who have progressive vasculopathy and/or secondary stroke, despite chronic transfusion therapy. Predictive markers for vasculopathy and cerebral events are needed to identify patients at risk for disease progression. Increased reticulocytosis was previously associated with other sickle cell disease complications, and adherent reticulocytes may contribute to the vascular pathology. The objective of this study was to explore the hypothesis that pre-transfusion reticulocytosis may serve as a disease severity marker for cerebral vasculopathy among chronically transfused children with sickle cell disease.After obtaining consent and assent, reticulocytosis was studied in a cohort of pediatric sickle cell patients treated with chronic transfusions (n=33, ages 2-17 years). The group was stratified into three groups: group 1 with an abnormal transcranial doppler (TCD) study in the absence of magnetic resonance angiography (MRA) detected vasculopathy [TCD(+), MRA(-), n=14], group 2 with a history of a stroke in the absence of MRA-detected vasculopathy [Stroke(+), MRA(-), n=5], and group 3 with a history of abnormal TCD or stroke and more severe vasculopathy detected by magnetic resonance angiography [MRA(+), n=14]. Pre-transfusion blood samples were analyzed within 72 hours of collection. Steady-state blood samples were also examined from a control group of pediatric SCD patients (>6 years of age) with normal TCD studies who were receiving supportive care in the absence of chronic transfusions or hydroxyurea (n=7). Hematologic data, including automated complete blood counts, absolute reticulocyte counts (ARC) with reticulocyte maturity were obtained. In addition, a flow cytometric approach was developed to further examine and quantitate reticulocyte subsets based upon staining with thiazole orange combined with CD36, CD45, CD49d, CD71, and CD235.The pre-transfusion HbS levels were not statistically different among the three transfused groups ([TCD(+), MRA(-)]: 30.2 ± 11.8%; [Stroke(+), MRA(-)]: 28.4 ± 3.3%; [MRA(+)]: 33.3 ± 9%, p>0.3). The high levels of reticulocytosis in the pre-transfusion samples were similar to those measured in the control group (ARC: 451 ± 126 K/uL in the chronically transfused cohort; ARC: 369 ± 94 K/uL in the control group, p=0.11). Pre-transfusion reticulocytosis was detected in every chronically transfused subject (ARC range 151-701 K/ul). The mean ARC in the [TCD(+), MRA(-)] group was not significantly different from the [Stroke(+), MRA(-)] group (411 ± 135 K/uL and 396 ± 97 K/uL respectively, p=0.82). However, the mean ARC in the [MRA(+)] group (512 ± 107 K/uL) was significantly higher than the control group, the [TCD(+), MRA(-)] group and the [Stroke(+), MRA(-)] group (p<0.05). The increased ARC in the MRA(+) group included higher absolute numbers of circulating reticulocytes at all stages of maturation including the immature reticulocyte subset [CD36(+), CD71(+)] that was detected in every sample.These data suggest that reticulocytosis with the release of immature CD36(+), CD71(+) cells into the peripheral blood remains as a characteristic feature of pediatric SCD even among chronically transfused patients. More severe vasculopathy, detected by MRA, was associated with significantly higher levels of pre-transfusion reticulocytosis. As such, reticulocytosis should be explored further as a marker or a potential contributor to more severe vasculopathy among chronically transfused children with SCD. Disclosures:No relevant conflicts of interest to declare.