Transfused Sickle Cell Disease Patients Research Articles

Tissue Iron Distribution Assessed by MRI in Patients with Iron Loading Anemias.

Bone marrow, spleen, liver and kidney proton transverse relaxation rates (R2), together with cardiac R2* from patients with sickle cell disease (SCD), paroxysmal nocturnal hemoglobinuria (PNH) and non-transfusion dependent thalassemia (NTDT) have been compared with a control group. Increased liver and bone marrow R2 values for the three groups of patients in comparison with the controls have been found. SCD and PNH patients also present an increased spleen R2 in comparison with the controls. The simultaneous measurement of R2 values for several tissue types by magnetic resonance imaging (MRI) has allowed the identification of iron distribution patterns in diseases associated with iron imbalance. Preferential liver iron loading is found in the highly transfused SCD patients, while the low transfused ones present a preferential iron loading of the spleen. Similar to the highly transfused SCD group, PNH patients preferentially accumulate iron in the liver. A reduced spleen iron accumulation in comparison with the liver and bone marrow loading has been found in NTDT patients, presumably related to the differential increased intestinal iron absorption. The correlation between serum ferritin and tissue R2 is moderate to good for the liver, spleen and bone marrow in SCD and PNH patients. However, serum ferritin does not correlate with NTDT liver R2, spleen R2 or heart R2*. As opposed to serum ferritin measurements, tissue R2 values are a more direct measurement of each tissue’s iron loading. This kind of determination will allow a better understanding of the different patterns of tissue iron biodistribution in diseases predisposed to tissue iron accumulation.

Lifetime risk and characterization of red blood cell alloimmunization in chronically transfused patients with sickle cell disease

Aims: Patients with sickle cell disease (SCD) are often exposed to multiple units of blood transfusions predisposing them to the development of alloantibodies. Alloantibodies make subsequent transfusion difficult, costly and could also result in life-threatening hemolysis. The aim of this study is to estimate and characterize alloantibodies developing over a life-time in patients with sickle cell disease who are on chronic red cell transfusion. Methods: Retrospective data were obtained from the electronic medical record and sunquest transfusion record of the DMC for a total of 121 patients with SCD aged 18 years and older who were on chronic red cell exchange transfusion. Data on demographics, blood group, genotype, total number of units transfused over lifetime, presence and type of alloantibodies identified was collected and analyzed. Results: The median age of the studied patients was 33 years with a male to female ratio of 0.73. Almost all (>95%) patients had homozygous SCD. A total of 67,586 units were transfused for the 121 patients over a lifetime approximating an average of 559 units transfused per patient. Alloantibodies were identified in 68 of the 121 patients (56.2%). The most common antibody identified was E (27%) followed by K (23%) and C (12%). Overall, antibodies against the Rh system (E, C, D and e) accounted for 39% of the identified antibodies. Conclusion: These data demonstrate a high lifetime rate of alloimmunization for patients with SCD on chronic red cell exchange transfusion. Understanding the nature of alloantibody development in chronically transfused SCD patients can help foster appropriate blood utilization paradigms, and develop preventive strategies.

Elevated Cerebral Blood Oxygen Extraction in Non-Transfused Sickle Cell Disease Patients

Introduction Chronic Transfusion Therapy (CTT) has been successful in decreasing stroke frequency in patients with sickle cell disease (SCD). Despite this, indication for CTT is largely based on empirical evidence and the mechanisms by which CTT protects the brain remain unclear. CTT improves oxygen carrying capacity and lowers hemoglobin S%, but the corresponding impact on cerebral blood flow(CBF), cerebral metabolic rate (CMRO2), and oxygen extraction fraction (OEF) is unknown. Understanding the impact of these competing influences in non-transfused (NT) and chronically transfused (CT) SCD patients will inform stroke prevention. Thus, we measured CBF, CMRO2, and OEF, in NT and CT patients with SCD using magnetic resonance imaging (MRI).Methods All patients were recruited with informed consent or assent and this study was approved by the CHLA IRB. Fourteen (6 NT, 8 CT) patients with SCD and 12 healthy ethnicity matched controls (CTL) were studied. Exclusion criteria included pregnancy, previous stroke, acute chest or pain crisis hospitalization within one month. Complete blood count and hemoglobin electrophoresis were performed. Arterial oxygen saturation (SaO2) was measured via peripheral pulse oximetery. CaO2 was calculated as the product of hemoglobin, SaO2 and the oxygen density of hemoglobin (1.36 ml/g). Phase contrast imaging of the carotid and vertebral arteries was used to measure global CBF. T2 Relaxation Under Spin Tagging (TRUST) was used to measured T2 relaxation of blood within the sagittal sinus. T2 relaxation was converted to SvO2 via previously validated calibration curves. OEF represented the difference of SaO2 andSvO2 divided bySaO2. CMRO2 was calculated as the product of CBF and OEF. High resolution, 3D, T1 weighted images were used for brain volume calculation using BrainSuiteñ software.Results Table 1 summarizes the results. Hemoglobin and oxygen content were well matched between transfused and non transfused SCD patients. Cerebral metabolic rate was also nearly identical in the two groups. However, CT patients exhibited 25% higher CBF than NT SCD patients, allowing them to have a normal oxygen extraction fraction ~30%. In contrast, OEF in NT SCD patients was abnormally high (37.8%), suggesting a decreased extraction reserve. Total oxygenation index (TOI) by NIRS also trended lower in NT SCD patients, consistent with the greater oxygen extraction and lower cerebral venous saturations observed. Abstract 1387. TableCTL (reference)NTCTp value (NT vs CT)Hemoglobin (g/dl)13.5 ± 1.229.7 ± 1.259.7 ± 1.05nsCaO2 (umol O2/ml)9.85 ± .996.84 ± 1.176.95 ±.71nsCMRO2 (umol O2/100g/min)193.1 ± 44.9239.7 ± 35.3238.6 ± 38.3nsCBF (ml/100g/min)70.0 ± 12.8101.5 ± 16.6127.1 ± 23.5< 0.05OEF (%)30.0 ± 7.137.8. ± 3.0629.7 ± 7.53< 0.05NIRS TOI56.0 ± 4.0948.5 ± 4.2153.5 ± 8.760.076SvO2 (%)65.6 ± 6.856.2 ± 5.267.1 ± 6.7< 0.05Discussion: Chronically transfused SCD patients achieve normal brain oxygenation metrics (SvO2, OEF, and NIRS) but require very high CBF to achieve this balance (lowering flow reserve). In contrast, NT SCD patients have smaller increases in CBF but require greater oxygen extraction to meet cerebrovascular demands (lowering extraction reserve). Hemoglobin S mediate changes in oxygen dissociation, blood viscosity, red cell deformability and microvascular damage potentially mediate these differences but their interplay is complicated and requires further study. DisclosuresCoates:novartis: Consultancy, Honoraria, Speakers Bureau; shire: Consultancy, Honoraria; apo pharma: Consultancy, Honoraria; acceleron: Consultancy, Honoraria.

Cardiac iron overload in sickle‐cell disease

Chronically transfused sickle cell disease (SCD) patients have lower risk of myocardial iron overload (MIO) than comparably transfused thalassemia major (TM) patients. However, cardioprotection is incomplete. We present the clinical characteristics of six patients who have prospectively developed MIO, to identify potential risk factors for cardiac iron accumulation. From 2002 to 2011, cardiac, hepatic, and pancreatic iron overload were assessed by R2 and R2 * magnetic resonance imaging techniques in 201 chronic transfused SCD patients as part of their clinical care. At the time, they developed MIO, five of six patients had been on chronic transfusion for more than 11 years; only one was on exchange transfusion. The time to MIO was correlated with reticulocyte and hemoglobin S percentages. All patients had qualitatively poor chelation compliance (<50%). All patients had serum ferritin levels >4600 ng/ml and liver iron concentration >22 mg/g. Pancreatic R2 * was >100 Hz in every patient studied (5/6). Cardiac iron rose proportionally to pancreas R2 *, with all patients having pancreas R2 *>100 Hz when cardiac iron was present. MIO had a threshold relationship with liver iron that was higher than observed in TM patients. In conclusion, MIO occurs in a small percentage of chronically transfused SCD patients and is only associated with exceptionally poor control of total body iron stores. Duration of chronic transfusion is clearly important but other factors, such as levels of effective erythropoiesis, appear to contribute to cardiac risk. Pancreas R2 * can serve as a valuable screening tool for cardiac iron in SCD patients.

Partial dysfunction of Treg activation in sickle cell disease.

Sickle cell disease (SCD) is a chronic inflammatory disease associated with multiple organ damage, chronic anemia, and infections. SCD patients have a high rate of alloimmunization against red blood cells (RBCs) following transfusion and may develop autoimmune diseases. Studies in mouse models have suggested that regulatory T cells (Treg) play a role in alloimmunization against RBC antigens. We characterized the phenotype and function of the Treg cell population in a homogeneous cohort of transfused SCD patients. We found that the distribution of Treg subpopulations differed significantly between SCD patients and healthy blood donors. SCD patients have a particular Treg phenotype, with strong CTLA-4 and CD39 expression and weak HLA-DR and CCR7 expression. Finally, we show that this particular phenotype is related to SCD rather than alloimmunization status. Indeed, we observed no difference in Treg phenotype or function in vitro using autologous feeder cells between strong and weak responders to alloimmunization.

Cardiac Iron Overload In Sickle-Cell Disease

IntroductionChronically transfused sickle cell disease (SCD) patients have lower risk of endocrine and cardiac iron overload load than comparably transfused thalassemia major patients. The mechanisms for this protection remain controversial but likely reflects lower transferrin saturation and circulating labile iron pools because of chronic inflammation and regeneration of apotransferrin through erythropoiesis. However, cardioprotection is incomplete; we have identified 6 patients out of the 201 patients (3%) followed at our Institution who have prospectively developed cardiac iron. We present the clinical characteristics of these patients to identify potential risk factors for cardiac iron accumulation. MethodsCardiac, hepatic, and pancreatic iron overload were assessed by R2* Magnetic Resonance Imaging (MRI) techniques as extensively described by our laboratory. The medical records of the selected patients were reviewed for demographic data, for transfusion and chelation history and for hematologic and biochemical parameters. ResultsTable 1 describes clinical characteristics of the six patients at the time they developed detectable cardiac iron (R2* ≥ 50 ms). Patient 6 was included because he showed a R2* of 49 Hz that was increasing rapidly.Five of the six patients were managed on simple transfusions. Five patients had been on chronic transfusion for more than 11 years. The three patients who developed cardiac iron the earliest (3.7 – 14 years of transfusions) had more efficient suppression of endogenous red cell production (HbS levels 2-5%) compared with patients who required longer transfusional exposure (HbS levels 13.3 – 41%). All patients had qualitatively poor chelation compliance (<50%), based upon their prescription refill rate. All patients had serum ferritin levels exceeding 4600 and liver iron concentration (LIC) greater than 22 mg/g. Pancreatic R2* was greater than 100 Hz in every patient studied (5/6).Figure 1 shows the longitudinal relationship between iron overload in the heart and in the other organs for each patient; initial iron levels are shown in black. Cardiac R2* appears increase dramatically once a critical LIC “threshold” is reached, qualitatively similar to the 18 mg/g threshold observed in thalassemia major patients. Cardiac R2* rose proportionally to pancreas R2*, similar to thalassemia major patients, with all of the patients having pancreas R2* > 100 Hz at the time cardiac iron was detected. [Display omitted] ConclusionsCardiac iron overload occurs in a small percentage of chronically transfused SCD patients and is only associated with exceptionally poor control of total body iron stores. Duration of chronic transfusion is clearly important but other factors, such as levels of effective erythropoiesis, may also contribute to cardiac risk. The relationship between cardiac iron and pancreas R2* suggests that pancreas R2* can serve as a valuable screening tool for cardiac iron in SCD patients. Disclosures:Berdoukas:ApoPharma inc: Consultancy. Coates:ApoPharma inc, Novartis, Shire: Consultancy. Wood:Novartis: Consultancy, Honoraria; Shire: Consultancy, Research Funding; ApoPharma: Consultancy, Honoraria, Use of deferiprone in myocardial infarction, Use of deferiprone in myocardial infarction Patents & Royalties.

Alloantibody Formation In Sickle Cell Disease

BackgroundTransfusion of red blood cells (RBC) is a common intervention to treat and prevent complications in sickle cell disease (SCD). However, frequent transfusions may lead to erythrocyte alloimmunization, thereby complicating donor matching procedures and posing patients at risk for hemolytic transfusion reactions. Little information is available about the risk of alloimmunization of sickle cell patients living in European countries. In the Netherlands extensive matching procedures to prevent alloimmunization were introduced a decade ago, but the effect on alloimmunization has not been evaluated yet. AimsThe primary aim of this study is to evaluate the cumulative incidence of first alloantibody formation in a Dutch cohort of transfused SCD patients, and to compare this with a general Dutch RBC-transfused population. In addition, the effect of extended RBC matching protocols on the incidence of alloantibody formation in SCD and potential clinical determinants of alloimmunization will be assessed. MethodsWe conducted a retrospective cohort study and collected data on SCD patients (genotypes HbSS, HbSC, HbSβ0 and HbSβ+ thalassemia), diagnosed in three Dutch Sickle Cell Treatment Centers that received non-extended matched (ABO, RhD) RBC transfusions between 1984-2004 and extended matched (at least ABO, Rhesus phenotype, Kell) RBC transfusions between 2004-2011.In addition, we compared this population with a general population of 3 042 patients that received non-extended matched (ABO, RhD) RBC transfusions between 2005-2009 in the Leiden University Medical Center (Zalpuri et al. 2012). Cohorts were not matched for ethnicity. Alloimmunization risk was calculated as Kaplan-Meier incidence with cumulative number of transfusions as time variable. The association with the clinical determinants gender, SCD-phenotype and ethnicity was analyzed with Cox-regression analysis. ResultsA total of 291 SCD patients received 7 957 RBC units. Alloantibody formation occurred in 52 (17.9%) patients. The cumulative incidence of alloimmunization was 9% after 5 RBC units, 15% after 10, 24% after 20 and 34% after 40 RBC units. Multivariate analysis, correcting for the cumulative number of transfusions, demonstrated a significantly increased risk of alloantibody formation in our SCD cohort when compared to a general population of transfused patients (HR 7.5 (95% CI: 5.06-11.14), where the cumulative incidence of alloimmunization was 1.1% after 5, 2.4% after 10, 3.4% after 20 and 6.5% after 40 RBC units. No association could be demonstrated between alloantibody formation and clinical determinants such as gender, SCD-phenotype or ethnicity. However, a significant reduction in alloimmunization was observed in SCD patients that received their first transfusion from the year 2004 onwards, after preventive matching for Rhesus phenotype and Kell was introduced for SCD patients (HR 0.48 (95% CI: 0.24-0.97)). ConclusionThe overall rate of first RBC alloantibody formation in our cohort was 17.9% and the risk of alloimmunization increased substantially with an increasing number of RBC transfusions. A unique comparison with a general cohort of Dutch transfused patients demonstrates a significantly higher risk of alloantibody formation in SCD, acknowledging earlier findings. This may partially be explained by differences in RBC antigens between patients of African descent and the predominantly Caucasian donors. Besides the number of RBC units, no other clinical risk factors for allo-immunization in SCD could be identified. The effectiveness of extended RBC matching protocols in the prevention of alloimmunization for chronically transfused patients in the participating centers was confirmed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Comments on: Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

Comments on: Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

Transfusion practices for patients with sickle cell disease at major academic medical centers participating in the Atlanta Sickle Cell Consortium

Abstract The Atlanta Sickle Cell Consortium represents more than 2600 pediatric and adult patients with sickle cell disease (SCD) in the metropolitan Atlanta, Georgia, area receiving care at four major locations, each providing comprehensive care 24 hours a day, 7 days a week. Both transfusion services that support these sites use two levels of prospective phenotype matching to decrease the rates of alloimmunization. Although exact rates are unknown and are currently under investigation, alloimmunization occurs infrequently with the exception of chronically transfused SCD patients, who represent the minority of active SCD patients. With increasing availability, red blood cell genotyping will be used in the near future both for determination of predicted patient phenotypes and for provision of genotypically matched donor units. Immunohematology 2012;28:24–6.

Potential Benefits of Extended Genotype Matching to Chronically Transfused Patients with Sickle Cell Disease

Abstract▪2144▪This icon denotes a clinically relevant abstract Background:Management of RBC alloimmunization in Sickle Cell Disease (SCD) patients has been the subject of much debate, and currently there is no standard approach. Many programs transfuse SCD patients with RBCs that are phenotype-matched for D, C, c, E, e and K. Although these approaches reduce the incidence of alloantibody production, patients still become alloimmunized. Based on this we aimed to identify the rates of alloimmunization in chronically transfused SCD patients and compare the phenotyping with genotyping methods to find a better way to match RBC units to those patients. Methods: We selected 45 SCD patients (homozygous for hemoglobin S) with multiple transfusions, previously phenotyped for ABO, Rh (D, C, c, E, e) and K1. Phenotypes were determined by hemagglutination using gel cards (Diamed® ). Genotypes were determined by a DNA array using the Human Erythrocyte Antigen BeadChip (“HEA”) from Bioarray Solutions. All SCD patients included in this study were in chronical transfusion program; receiving multiple transfusions. The median age was 24y; there were 28(62%) females and 17(37.8%) males. The median of transfusions were 53 (5–78) and 40 (88.9%) patients received more than 20 phenotype-matched units for Rh (D, C, c, E, e) and K1. Results: Of the 45 SCD patients selected, 11 (24.4%) had alloantibodies. The antibody specificities found in these patients were anti-D, -C, -CW, -E, -Jka, -Jkb, -Fya, -Dia, -s. Although the patients were receiving Rh and K phenotype-matched units 8 (17%) of them became alloimmunized to Rh antigens and on those patients we found discrepancies between the previous phenotype and genotype-derived phenotype. Our results showed that the risk of immunization increases in patients over 40 years old (p= 0.05) and with the number of transfusion events. Patients with more than 20 RBC transfusions have a tendency for alloimmunization (p=0.65). We also observed that genotyping was more effective than hemagglutination in determining patient’s correct phenotype. Conclusion: Our data show that even with the implementation of Rh and K phenotype-matching in chronically transfused patients with SCD, they still become alloimmunized to other antigens with high immunization risk and also to Rh antigens due to the limitations of the hemagglutination. The relevance of genotype determination of blood groups for the management of multiple transfused patients with SCD has been demonstrated by allowing the determination of the true blood group genotype, by assisting in the identification of suspected alloantibodies and in the selection of antigen-negative. As donor genotyping for the most clinically relevant blood group antigens by automated DNA techniques are becoming available, extended genotype matching should be considered in this group of patients. Disclosures:No relevant conflicts of interest to declare.

Low Ascorbate Levels in Transfused Patients Are Associated with Correlates of Vascular Damage in Sickle Cell Disease.

Abstract 2570Poster Board II-547 Introduction:Ascorbate is vital for endothelial homeostasis because it is responsible for regeneration of tetrahydrobiopterin (BH4) from dihydrobiopterin (BH2). BH4 is essential for proper electron transport from oxygen to arginine in the synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). When BH4 is deficient, eNOS becomes uncoupled from arginine and synthesizes superoxide, producing a vasoconstrictive rather than a vasodilatory phenotype. Low BH4/BH2 ratios are correlated with decreased flow-mediated dilation, a known marker of nitric oxide-dependent endothelial function. Dietary supplementation with ascorbate has been show to acutely improve NO-mediated vasodilation in smokers and patients with coronary artery disease. Since patients with sickle cell disease (SCD) are known to have low levels of NO and impaired vascular reactivity, we examined the correlates of ascorbate deficiency in our chronically transfused SCD patients. Methods:We recently studied micronutrient levels in convenience sample of our chronically transfused SCD patients. In that study, 56.7% of SCD patients had ascorbate levels below the lower limit of normal (0.2–1.9 mg/dl). We screened the following parameters as predictors of abnormal ascorbate: LDH, cell-free Hb, total bilirubin, liver iron concentration (LIC), pancreas iron, cardiac iron, ferritin, transferrin saturation, insulin, glucose, brain natriuretic peptide (BNP) and high sensitivity C reactive peptide( hs-CRP). A total of 28 patients (21 females, 7 males), ranging in age from 1.4 to 31.4 years old, who had low ascorbate levels at the time of micronutrient measurements were studied. All patients had chelating drugs held for 24 hours prior to being evaluated. Results:None of the above predictors demonstrated significant linear relationships with ascorbate levels. However, low or undetectable ascorbate levels were observed with extremes of a number of parameters including LIC > 30 mg/g, BNP > 40 pg/ml, LDH > 1500 U/Lcell-free hemoglobin > 30 mg/dl,hs-CRP > 7mg/L,and homocysteine > 10 μM/L. LDH, cell-free hemoglobin and hs-CRP exhibited the best specificity for prediction of ascorbate levels and were the only variables to achieve statistical significance by Fischer's Exact test (Table 1). More importantly, there was sufficient independence among these predictors that having and extreme value for one of the above parameters identified 14/15 patients with low ascorbate, with only 1/13 false positives.Table 1Correlates of low ascorbate levels in SCDAscorbate levelFischer'sLowNormalP valueFree Hb > 30 mg/dl91Free Hb < 30 mg/dl690.02hs-CRP > 7 mg/L60hs-CRP < 7 mg/L9100.05Homocysteine > 10 μM/L50Homocysteine < 10 μM/L8100.05Any Abnormal141All Normal112<0.0001 Discussion:These observations suggest that low ascorbate levels in SCD patients result from a combination of chronic inflammation, hemolysis, and hyperhomocysteinemia. Since ascorbate is vital for proper endothelial function, these observations are consistent with known associations of inflammation and hemolysis with sickle vasculopathy. Although hyperhomocysteinemia is a known vascular stressor in other diseases, its contribution in SCD is less clear because treatment with pyridoxine and folate, which reduce homocysteine levels, do not affect other markers of SCD vascular dysfunction. Taken together, these data suggest that comprehensive treatment of sickle vasculopathy may require multiple interventions, including decreasing hemolysis and inflammation as well as B vitamin supplementation. Ascorbate replacement alone is unlikely to be effective without correction of upstream stressors, similar to its use in other complex vascular disorders. Disclosures:No relevant conflicts of interest to declare.

Altered Regulation of Immune Responses in Alloimmunized Chronically Transfused Patients with Sickle Cell Disease.

Abstract 637Transfusion therapy is hampered by the development of alloantibodies to transfused red blood cells (RBCs). A recent mathematical model for RBC alloimmunization predicted that approximately 13% of the general population are antibody producers (responders) (Higgins and Sloan, 2008). However, immune molecular markers which may predict these transfusion responders have not been clearly demonstrated. We recently reported in a mouse model that key immune response regulators, CD4+ regulatory T cells (Tregs) have reduced activity in alloantibody responders compared to non-responders (Bao, 2009). (Bao, 2009). Here, we designed a study to determine whether alloimmunized humans have an altered immune response to red cell transfusions, similar to that seen in the mouse model. Peripheral Treg frequency and activity was studied in a cohort of 18 chronically transfused patients with sickle cell disease (SS), receiving either exchange transfusions (n=7) or simple transfusions (n=11). All had been receiving leukoreduced units, matched for Kell and Rh antigens on a roughly 4 weekly intervals for at least 2 years prior to the study. Nine patients were identified as having had a positive history of alloimmunization (responders), 6 on simple transfusions and 3 on exchange transfusions responders. We found no statistically significant differences in the Treg frequencies (Foxp3+CD25hi in the CD4+ population) between alloimmunized and non-alloimmunized SS patients (p>0.1). However, Treg activity as measured by suppression of proliferation of autologous CD4+CD25–cells at 1:4 and 1:16 ratios of Tregs: CD4+CD25–cells was significantly lower in responders compared to non-responders (at 1:4 ratio, 38±5% alloimmunized versus 54±3% non-alloimmunized, p=0.02 and at 1:16 ratio, 10±6% versus 28±4%, p=0.03). To determine if there was a skewing of the T helper (Th) responses as a result of reduced Treg activity in responders, we analyzed the functional phenotype of peripheral CD4+ T cells by single-cell measurement of intracellular cytokines using flow cytometry. CD4+ T cells were classified as Th1, Th2 or Th17 by assessing their intracellular cytokine profile of IFN- γ, IL-4 and IL-17, respectively. Expression of secreted cytokines was also measured in stimulated cultured supernatants by ELISA. Following stimulation with PMA and ionomycin, we found higher proportion of IL-4 single positive Th2 cells as well as higher levels of secreted IL-4 in sorted populations of CD4+CD25−T cells from responders compared to non-responders receiving either simple or exchange transfusions (p=0.01), indicating that responder status is skewed towards a Th2 response. Although we did not find statistically significant differences in either secreted IL-17 or IFN- γ or in IL-17- or IFN- γ -expressing Th cells between alloimmunized and non-alloimmunized patients, our transfused SS cohort had significantly higher levels of secreted proinflammatory IL-17 and IFN- γ compared with normal healthy race-matched controls (n=7, p=0.03). These latter data are consistent with an underlying Th cytokine imbalance in chronically transfused SS patients, although it remains to be determined if the perturbations in cytokine balance is the result of chronic transfusions, or specific to SS patients. In summary, our data indicate that in chronically transfused SS patients, responders have compromised Treg activity. This may be responsible for the observed increase in Th2 responses, known to be associated with induction of antibody production. The mechanisms responsible for these differences in responders are under further study. Our findings suggest that Treg associated molecular markers may be used to predict in advance antibody producers to reduce alloimmunization-associated morbidity and mortality. Disclosures:No relevant conflicts of interest to declare.

Virus de l'immunodéficience humaine et virus des hépatites virales C et B chez les patients drépanocytaires au CHU Campus de Lomé (Togo)

Context. – The clinical features of sickle cell disease (SCD) are vaso-occlusive and/or hemolytic crises which treatment may require blood transfusions. Objective. – This study aimed to determine the prevalence of HIV, Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV) infections in a population of SCD patients. Methods. – All the samples were analyzed by Elisa technique. We studied 119 sera for HIV using Elisa and a confirmation test in case of positive Elisa. We screened 91 sera for HCV and 119 sera for HBV. Results. – The prevalence was 5.04% for HIV, 6.5% for HCV and 20.2% for HBs Ag. Homozygous (SS) patients were more infected than compound heterozygous patients SC, (p < 0.02). 24.6% of the children (0 to 15 years of age) were infected as well as adults (35.5%). The Relative Risk to be contaminated was 7.14 for HIV, 4.29 for HCV and 5.43 for HBV in transfused compared to non transfused SCD patients. Conclusions. – This high infectious risk in SCD patients should lead us to elaborate a better strategy to increase the safety of blood transfusion in Togo.

Clinical and Economic Issues in Multiply Transfused Sickle Cell Disease (SCD) Patients.

Aim: To study alloantibody (alloAB)/autoAB formation and transfusion reactions in SCD patients before and after instituting the practice of transfusing C, E, K blood type negative (CEKneg) packed red blood cell (pRBC) units.Material and Methods: We retrospectively reviewed blood bank records of all SCD patients transfused pRBCs. Statistical analysis was performed using the Chi square test and Fischer's exact test.Results: During 1990–2004, 500 SCD patients received 16,617 pRBCs in our hospital. 387 received pRBCs crossmatched for ABO and Rh only; 121 (31.3%) developed alloantibodies (alloABs).Table 1:Transfusion characteristics of patients forming alloABsData on pts forming alloABsCEK matched patientsCEK unmatched pRBC Transfusion (Tn) patients (387)≥ 1 ABs≥ 2 ABs≥ 3 ABs≥ 4 ABs≥ 5 ABsNumber of patients (% of total)6/113 (5%)121/387(31%)57/121 (47%)29/57 (51%)16/29 (55%)11/29 (69%)# of Tn before AB-Median (range)9.5 (0-106)7(0-270)2 (0-106)0 (0-89)0 (0-180)0 (0-16)# of pts with > 5 Tn before AB4/6 (67%)68/121(56%)21/57 (37%)5/29 (17%)4/16 (25%)1/11 (9%)# of pts with >30 Tn before AB1124210There were 37 transfusion reactions: 9 febrile, 24 allergic and 4 dHTRs, none was life-threatening. 21 pts developed multiple alloABs simultaneously after a single transfusion; 15 additionally developed warm autoABs. >13% patients transfused CEK unmatched units developed ABs to C, E, K and other antigens. Once allosensitized, there was an ↑ chance of subsequent development of multiple alloABs with fewer transfusions.Table 2:Incidence of antibody formation in patient groupsMajor patient groupsTotal # of ptsTotal # of transfusionsNumber of allo ABs (%of pts) [Incidence/1000 pRBCs]Number of autoABs (%of pts) [Incidence/1000 pRBCs]AllNon-CEKTotalWarmP value (CEK vs Regular) - # of pts<0.0010.030.0050.02CEKmatched11323546 (5%)[2.55]5(4%)[2.12]1 (0.88%)[0.425]1 (1%)[0.46]Regular (ABORh)38714263240(31%)[16.8]76 (13%)[5.33]39 (10%)[2.73]30 (8%)[2.1]alloAB forming pts1217338240[32.71]76[10.36]34 (28%)[4.63]30 (25%)[4.1]No alloAB pts2666925N/AN/A5 (2%)[0.7]0P value(alloAB vs non-alloAB) - # of ptsN/AN/A<0.001<0.001Of 266 patients who did not develop any alloABs, 5 had cold and no warm autoABs. 113 patients received 2354 CEKneg pRBCs only (from 1997), 6 (p<0.01) had one alloAB each, 1 warm autoAB and no reactions. Technologists required 30 more minutes and $153 extra in reagent costs for extended CEK match. 90% of our donors are Caucasian.Conclusions: Utilizing CEK negative pRBCs ↓↓ alloAB(p<0.01), autoAB (p<0.001) formation and eliminated transfusion reactions in our multiply transfused SCD patients. Universal availability of leukopoor pRBCs (from 1997) may have eliminated febrile reactions. There was ↓ alloAB formation for C, E, K and other blood group antigens. Though unlikely, Rh neg and CEKneg pRBCs may also be negative for other minor antigens. AutoAB formation (and allergic reactions)especially ↑ in patients with multiple and simultaneous alloAB formation. CEK matching made it easier to find pRBCs due to less formation of alloABs. However, it resulted in marked overuse of Rh neg pRBCs and extra cost and effort to find CEKneg pRBCs for every transfusion. For cost efficacy, one might consider CEKneg pRBCs after first alloAB.

Allosensitization in Patients Receiving Multiple Blood Transfusions

Extended antigen (C, E, K) matching decreased the incidence of alloantibody (alloAB) and autoantibody (autoAB) formation, in addition to eliminating transfusion reactions in the multiply transfused sickle cell disease patients. AlloAB formation possibly transforms the immune system into a hyperactive state leading to further and earlier alloAB and autoAB formation. However, additional CEK matching results in marked overuse of Rh-negative packed red blood cell (pRBC) units, 30 minutes' extra time of a skilled technologist, and 153 dollars extra CEK reagent cost per unit to find CEK-matched pRBCs for every transfusion for these multiply transfused patients.

Alloimmunization to Platelets in Heavily Transfused Patients With Sickle Cell Disease

Bone marrow transplantation (BMT) is now an option for some patients with sickle cell disease (SCD). Many SCD patients are multiply transfused with red blood cells (RBCs), and may be immunized to alloantigens other than erythrocyte antigens. Because platelet refractoriness is a significant complication during BMT, we wished to determine the prevalence of alloimmunization to platelets in transfused SCD patients. Sera collected from 47 transfused and 14 untransfused SCD patients were screened for HLA and platelet-specific antibodies. Transfusion and RBC antibody histories were reviewed. A subset of the patients were rescreened 1 year later. Eighty-five percent of patients with at least 50 RBC transfusions (22 of 26), 48% of patients with less than 50 transfusions (10 of 21), and none of 14 untransfused patients demonstrated platelet alloimmunization (P < .05). Platelet alloimmunization was more prevalent than RBC alloimmunization (20% to 30%). Half of the platelet reactivity was chloroquine-elutable. Eighteen of 22 patients (82%) on chronic RBC transfusion remained platelet-alloimmunized 11 to 22 months after initial testing. In summary, 85% of heavily transfused SCD patients are alloimmunized to HLA and/or platelet-specific antigens. These patients may be refractory to platelet transfusion, a condition that would increase their risk during BMT. Leukodepletion in the transfusion support of SCD patients should be considered to prevent platelet alloimmunization.

Alloimmunization in two multitransfused patient populations.

A retrospective study was undertaken to determine the frequency of alloimmunization in two multitransfused patient populations. Three hundred eighteen unselected patients whose transfusion records were available were studied. One hundred fifty-seven patients had sickle cell disease (SCD) and 161 had thalassemia. Forty-four antibodies were detected in 23 of the 99 transfused SCD patients. The predominate antibodies were in the Rh group: seventeen were anti-rh"(E), eight were anti-rh'(C), and four were anti-rh'w(Cw). Of the remaining antibodies, five were anti-Kell, three were anti-Jka, two were anti-Jkb, three were anti-Fya, one was anti-Bga, and one was anti-S. In the thalassemic patients, six antibodies were detected in 4 of the 39 transfused patients. Of these, there were two anti-Kell and one anti-Rho (D), anti-rh'(C), anti-rh'w(Cw), and anti-Fya each. Although the SCD patients were more likely to have been transfused at the parent institution than were the thalassemic patients (69% vs. 24%, p less than 0.05), the frequencies of alloimmunized patients, the total number of antibodies, and the number of Rh antibodies did not differ significantly (p greater than 0.05) for transfused patients in the two populations.