Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease.

Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort ("sickle") the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms.

Natural history of benign ethnic neutropenia in individuals of African ancestry.

BACKGROUND: Benign ethnic neutropenia (BEN), defined by neutrophil count <1.5 k/µL in the absence of other causes, is an asymptomatic condition more commonly observed in individuals of African ancestry. However, the natural history of this condition has been less well described. METHODS: Individuals with BEN were retrospectively identified by chart review or referral to hematology clinics. They were then invited to enroll in a prospective natural history study. Retrospective and prospective clinical and laboratory data were combined for descriptive analyses. FINDINGS: 46 participants, younger and older adults from 2 institutions, had BEN. Hypertension was reported in 30%, musculoskeletal disorders in 15%, and upper respiratory infection in 33% of these adults. Their leukopenia resulted from isolated neutropenia, ranging from 1000 and 1500 cells/µL. The severity of infections was mild and the frequency was similar to other healthy individuals in the ambulatory clinic. INTERPRETATION: In this group of BEN participants, their leukopenia was stable over time, and they had low rates of infections or common medical disorders, confirming the benign nature of this condition. The presence of BEN in children, younger adults, and older adults suggest a hereditary pattern for BEN.

Infusion of hemolyzed red blood cells within peripheral blood stem cell grafts in patients with and without sickle cell disease.

Peripheral blood stem cell (PBSC) infusions are associated with complications such as elevated blood pressure and decreased creatinine clearance. Patients with sickle cell disease experience similar manifestations, and some have postulated release of plasma-free hemoglobin with subsequent nitric oxide consumption as causative. We sought to evaluate whether the infusion of PBSC grafts containing lysed red blood cells (RBCs) leads to the toxicity observed in transplant subjects. We report a prospective cohort study of 60 subjects divided into 4 groups based on whether their infusions contained dimethyl sulfoxide (DMSO) and lysed RBCs, no DMSO and fresh RBCs, DMSO and no RBCs, or saline. Our primary end point, change in maximum blood pressure compared with baseline, was not significantly different among groups. Tricuspid regurgitant velocity and creatinine levels also did not differ significantly among groups. Our data do not support free hemoglobin as a significant contributor to toxicity associated with PBSC infusions. This study was registered at clinicaltrials.gov (NCT00631787).

Nonmyeloablative HLA-matched sibling allogeneic hematopoietic stem cell transplantation for severe sickle cell phenotype.

IMPORTANCE: Myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is curative for children with severe sickle cell disease, but toxicity may be prohibitive for adults. Nonmyeloablative transplantation has been attempted with degrees of preparative regimen intensity, but graft rejection and graft-vs-host disease remain significant. OBJECTIVE: To determine the efficacy, safety, and outcome on end-organ function with this low-intensity regimen for sickle cell phenotype with or without thalassemia. DESIGN, SETTING, AND PARTICIPANTS: From July 16, 2004, to October 25, 2013, 30 patients aged 16-65 years with severe disease enrolled in this nonmyeloablative transplant study, consisting of alemtuzumab (1 mg/kg in divided doses), total-body irradiation (300 cGy), sirolimus, and infusion of unmanipulated filgrastim mobilized peripheral blood stem cells (5.5-31.7 × 10(6) cells/kg) from human leukocyte antigen-matched siblings. MAIN OUTCOMES AND MEASURES: The primary end point was treatment success at 1 year after the transplant, defined as a full donor-type hemoglobin for patients with sickle cell disease and transfusion independence for patients with thalassemia. The secondary end points were the level of donor leukocyte chimerism; incidence of acute and chronic graft-vs-host disease; and sickle cell-thalassemia disease-free survival, immunologic recovery, and changes in organ function, assessed by annual brain imaging, pulmonary function, echocardiographic image, and laboratory testing. RESULTS: Twenty-nine patients survived a median 3.4 years (range, 1-8.6), with no nonrelapse mortality. One patient died from intracranial bleeding after relapse. As of October 25, 2013, 26 patients (87%) had long-term stable donor engraftment without acute or chronic graft-vs-host disease. The mean donor T-cell level was 48% (95% CI, 34%-62%); the myeloid chimerism levels, 86% (95% CI, 70%-100%). Fifteen engrafted patients discontinued immunosuppression medication with continued stable donor chimerism and no graft-vs-host disease. The normalized hemoglobin and resolution of hemolysis among engrafted patients were accompanied by stabilization in brain imaging, a reduction of echocardiographic estimates of pulmonary pressure, and allowed for phlebotomy to reduce hepatic iron. The mean annual hospitalization rate was 3.23 (95% CI, 1.83-4.63) the year before, 0.63 (95% CI, 0.26-1.01) the first year after, 0.19 (95% CI, 0-0.45) the second year after, and 0.11 (95% CI, 0.04-0.19) the third year after transplant. For patients taking long-term narcotics, the mean use per week was 639 mg (95% CI, 220-1058) of intravenous morphine-equivalent dose the week of their transplants and 140 mg (95% CI, 56-225) 6 months after transplant. There were 38 serious adverse events: pain and related management, infections, abdominal events, and sirolimus related toxic effects. CONCLUSIONS AND RELEVANCE: Among 30 patients with sickle cell phenotype with or without thalassemia who underwent nonmyeloablative allogeneic HSCT, the rate of stable mixed-donor chimerism was high and allowed for complete replacement with circulating donor red blood cells among engrafted participants. Further accrual and follow-up are required to assess longer-term clinical outcomes, adverse events, and transplant tolerance. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00061568.

Mobilization, collection, and processing of peripheral blood stem cells in individuals with sickle cell trait.

Mobilized peripheral blood is increasingly used as the source of hematopoietic stem cells for allogeneic transplantation, currently the only curative approach for sickle cell anemia. However, the safety and feasibility of stem cell mobilization in individuals with sickle cell trait (SCT) has not been documented. This study is a prospective controlled trial to evaluate the safety and feasibility of peripheral blood stem cell (PBSC) mobilization in 8 SCT subjects and 8 control subjects matched for age and race. Mobilization with filgrastim 10 microg/kg subcutaneous daily for 5 days was followed by 12-L apheresis on the fifth day. Filgrastim administration was accompanied by similar symptoms in all subjects; no untoward adverse events occurred in either group, including sickle cell crises. CD34+ cell mobilization response was not significantly different between SCT and control subjects. Median CD34+ cell content was also similar in PBSCs collected from SCT versus control subjects, 6.8 versus 3.9 x 10(6) CD34+ cells/70 kg, P =.165. Red cell depletion from SCT products was not possible by using hydroxyethyl starch sedimentation but was achievable with ammonium chloride lysis. There was no evidence of gelling of SCT products after thaw, and no difference in cell recovery was seen among red cell-depleted versus nondepleted products. Cryopreservation in 5% dimethyl sulfoxide/6% pentastarch was associated with superior cell recovery (both SCT and control subjects) compared with 10% dimethyl sulfoxide (P =.001). The study concluded that filgrastim mobilization, large volume apheresis, processing, and cryopreservation appears to be safe in donors with SCT, allowing PBSC use for transplantation in patients with sickle cell anemia.