Collection of peripheral blood mononucleated cells for chronic graft-versus-host disease immunology research: safety and effectiveness of leukapheresis in 132 patients.

BACKGROUND: Chronic graft-versus-host disease (GVHD) is a major cause of late morbidity and non-relapse mortality in recipients of allogeneic hematopoietic cell transplantation (HCT). Its biology, however, remains poorly understood, making the studies of its biology and immunomodulatory therapies a difficult task. Such research is often hampered by lymphopenia which is common in these patients and precludes studies of critical cellular subsets across the spectrum of severity of disease. This study explores the potential of leukapheresis to safely acquire and efficiently store immune cells for immunology research in chronic GVHD. METHODS: This is a cross-sectional study in which 132 consecutively accrued patients undergo optional research leukapheresis and a one-week comprehensive outpatient evaluation. Baseline clinical and laboratory data and efficiency of the procedure were reported. RESULTS: Ninety-four of 132 patients (71%) achieved the goal collection of 2 × 10^9 PBMNCs with a mean volume processed of 4.6 L. Only mild decreases in hemoglobin, platelet, lymphocyte and monocytes were observed. All adverse events were mild (grade 1) and had resolved by the time of discharge from the apheresis unit. CONCLUSION: This study demonstrates feasibility, safety, and efficiency of research leukapheresis in a frail patient population. Results presented promote leukapheresis as a standard research practice option in studies of chronic GVHD in humans which may expedite advances in our understanding of this complex multisystem disease.

The effect of repeated stimulated granulocyte donations on hematopoietic indexes in donors: a 24-year donor center experience.

BACKGROUND: Short- and long-term effects of mobilization regimens in hematopoietic stem cell and granulocyte donors have been well characterized. In this study, we examined the longitudinal hematopoietic changes related to repeat stimulated granulocyte donation. STUDY DESIGN AND METHODS: Complete blood counts for consecutive granulocyte donors between October 1994 and May 2017 were compared to unstimulated granulocyte donors. Plateletpheresis donors served as controls. The longitudinal change in precollection white blood cell (WBC) counts for these donor groups were modeled using a linear mixed-effects model. The investigated variables were granulocyte, lymphocyte, and monocyte counts and the granulocyte collection yield. Contrasts were performed to explore the effect of donation number on precollection counts. RESULTS: For the granulocyte-colony-stimulating factor plus dexamethasone (G-CSF/Dex)-stimulated group, both the granulocyte and the lymphocyte counts decreased 6.51 × 109 /L (-23.1%, p < 0.001) and 0.21 × 109 /L (-20.4%, p < 0.001), respectively, between Donation 1 and Donation 20. This effect was still present at the 3- to 4-year interval (b = -0.0008313, SE = 0.00029, p = 0.004). For the unstimulated donor group between Donation 1 and Donation 20, the lymphocyte count decreased by 0.62 × 109 /L (-51.5%, p < 0.001). This effect was only significant up to Year 2 (b = -0.0026, SE = 0.0010, p = 0.013). CONCLUSIONS: Past granulocyte donations were found to have a statistically strong negative effect on precollection granulocyte counts and lymphocyte counts and decreased granulocyte yield both in the G-CSF/Dex-stimulated donors and the unstimulated donors. In this statistical model, for both these groups, the effect of past donations on granulocyte and WBC counts were still detectable 2 years later.

The effect of iron balance on platelet counts in blood donors.

BACKGROUND: Thrombocytosis (or, less commonly, thrombocytopenia) is associated with iron-deficiency anemia and resolves with iron therapy. Many volunteer blood donors have low iron stores, with or without anemia. Iron balance could affect platelet counts in blood donors. STUDY DESIGN AND METHODS: Whole blood donors deferred for finger-stick hemoglobin levels less than 12.5 g/dL were evaluated by complete blood count and serum iron panel before and after oral iron treatment. Group assignment for iron depletion was based on serum ferritin cutoffs of less than 20 µg/L for women and less than 30 µg/L for men or was based on changes in serum ferritin levels after iron replacement. RESULTS: Among 1273 Hb-deferred whole blood donors, 55% (619 of 1128) of the women and 70% (102 of 145) of the men were iron depleted. Iron-depleted donors had higher platelet counts compared with donors who had normal ferritin levels (women: 286 vs. 268 × 103 /µL; p < 0.0001; men: 246 vs. 222 × 103 /µL; p = 0.0454). Only 4.4% of iron-depleted donors had thrombocytosis (> 400 × 103 /µL) compared with 2.0% of donors who had normal ferritin levels (p = 0.017). Iron replacement decreased platelet counts in iron-depleted female donors (mean, -19,800/µL; interquartile range, 8000 to -45,000/µL), but not in donors who had normal or stable ferritin levels. The same trends were observed in male donors. CONCLUSION: Iron-depleted donors had higher platelet counts than donors who had adequate iron stores. Oral iron replacement decreased platelet counts on average by about 20,000/µL in iron-depleted donors but had no effect on platelet counts in donors who had normal or stable ferritin levels.

Heme-bound iron activates placenta growth factor in erythroid cells via erythroid Krüppel-like factor.

In adults with sickle cell disease (SCD), markers of iron burden are associated with excessive production of the angiogenic protein placenta growth factor (PlGF) and high estimated pulmonary artery pressure. Enforced PlGF expression in mice stimulates production of the potent vasoconstrictor endothelin-1, producing pulmonary hypertension. We now demonstrate heme-bound iron (hemin) induces PlGF mRNA >200-fold in a dose- and time-dependent fashion. In murine and human erythroid cells, expression of erythroid Krüppel-like factor (EKLF) precedes PlGF, and its enforced expression in human erythroid progenitor cells induces PlGF mRNA. Hemin-induced expression of PlGF is abolished in EKLF-deficient murine erythroid cells but rescued by conditional expression of EKLF. Chromatin immunoprecipitation reveals that EKLF binds to the PlGF promoter region. SCD patients show higher level expression of both EKLF and PlGF mRNA in circulating blood cells, and markers of iron overload are associated with high PlGF and early mortality. Finally, PlGF association with iron burden generalizes to other human diseases of iron overload. Our results demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to markers of vasculopathy and pulmonary hypertension. These trials were registered at www.clinicaltrials.gov as #NCT00007150, #NCT00023296, #NCT00081523, and #NCT00352430.

Hematopoietic progenitor cell mobilization is more robust in healthy African American compared to Caucasian donors and is not affected by the presence of sickle cell trait.

BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF)-stimulated hematopoietic progenitor cells (HPCs) collected by apheresis have become the predominant graft source for HPC transplantation in adults. Among healthy allogeneic donors, demographic characteristics (age, sex, body mass index [BMI]) and baseline hematologic counts affect HPC mobilization, leading to variability in CD34+ apheresis yields. Racial differences in HPC mobilization are less well characterized. STUDY DESIGN AND METHODS: We retrospectively analyzed data from 1096 consecutive G-CSF-stimulated leukapheresis procedures in healthy allogeneic African American (AA) or Caucasian donors. RESULTS: In a multivariate analysis, after adjusting for age, sex, BMI, baseline platelet and mononuclear cell counts, and daily G-CSF dose, peak CD34+ cell mobilization was significantly higher among AAs (n = 215) than Caucasians (n = 881; 123 ± 87 × 10(6) cells/L vs. 75 ± 47 × 10(6) cells/L; p < 0.0001). A ceiling effect was observed with increasing G-CSF dose (10 µg/kg/day vs. 16 µg/kg/day) in AAs (123 ± 88 × 10(6) cells/L vs. 123 ± 87 × 10(6) cells/L) but not in Caucasians (74 ± 46 × 10(6) cells/L vs. 93 ± 53 × 10(6) cells/L; p < 0.001). In AA donors, the presence of sickle cell trait (SCT; n = 41) did not affect CD34+ mobilization (peak CD34+ 123 ± 91 × 10(6) cells/L vs. 107 ± 72 × 10(6) cells/L, HbAS vs. HbAA; p = 0.34). Adverse events were minimal and similar across race. CONCLUSIONS: AAs demonstrated significantly better CD34 mobilization responses to G-CSF than Caucasians. This was independent of other demographic and hematologic variables. Studying race-associated pharmacogenomics in relation to G-CSF may improve dosing strategies. Adverse event profile and CD34 mobilization were similar in AA donors with and without SCT. Our findings suggest that it would be safe to include healthy AA donors with SCT in unrelated donor registries.

Initial serum ferritin predicts number of therapeutic phlebotomies to iron depletion in secondary iron overload.

BACKGROUND: Therapeutic phlebotomy is increasingly used in patients with transfusional siderosis to mitigate organ injury associated with iron overload (IO). Laboratory response variables and therapy duration are not well characterized in such patients. STUDY DESIGN AND METHODS: We retrospectively evaluated 99 consecutive patients undergoing therapeutic phlebotomy for either transfusional IO (TIO, n = 88; 76% had undergone hematopoietic transplantation) or nontransfusional indications (hyperferritinemia or erythrocytosis; n = 11). Complete blood cell count, serum ferritin (SF), transferrin saturation, and transaminases were measured serially. Phlebotomy goal was an SF level of less than 300 µg/L. RESULTS: Mean SF levels before phlebotomy among TIO and nontransfusional subjects were 3093 and 396 µg/L, respectively. Transfusion burden in the TIO group was 94 ± 108 (mean ± SD) RBC units; approximately half completed therapy with 24 ± 23 phlebotomies (range, 1-103). One-third were lost to follow-up. Overall, 15% had mild adverse effects, including headache, nausea, and dizziness, mainly during first phlebotomy. Prior transfusion burden correlated poorly with initial ferritin and total number of phlebotomies to target in the TIO group. However, number of phlebotomies to target was strongly correlated with initial SF (R(2) = 0.8; p < 0.0001) in both TIO and nontransfusional groups. ALT decreased significantly with serial phlebotomy in all groups (mean initial and final values, 61 and 39 U/L; p = 0.03). CONCLUSIONS: Initial SF but not transfusion burden predicted number of phlebotomies to target in patients with TIO. Despite good treatment tolerance, significant losses to follow-up were noted. Providing patients with an estimated phlebotomy number and follow-up duration, and thus a finite endpoint, may improve compliance. Hepatic function improved with iron offloading.

Mobilization characteristics and strategies to improve hematopoietic progenitor cell mobilization and collection in patients with chronic granulomatous disease and severe combined immunodeficiency.

BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF)-mobilized autologous hematopoietic progenitor cells (HPCs) may be collected by apheresis of patients with chronic granulomatous disease (CGD) and severe combined immunodeficiency (SCID) for use in gene therapy trials. CD34+ cell mobilization has not been well characterized in such patients. STUDY DESIGN AND METHODS: We retrospectively evaluated CD34+ cell mobilization and collection in 73 consecutive CGD and SCID patients and in 99 age-, weight-, and G-CSF dose-matched healthy allogeneic controls. RESULTS: In subjects aged not more than 20 years, Day 5 preapheresis circulating CD34+ counts were significantly lower in CGD and SCID patients than in controls; mean peak CD34+ cell counts were 58 × 10(6) , 64 × 10(6) , and 87 × 10(6) /L, respectively (p = 0.01). The SCIDs had lower CD34+ collection efficiency than CGDs and controls; mean efficiencies were 40, 63, and 57%, respectively (p = 0.003). In subjects aged more than 20 years, the CGDs had significantly lower CD34+ cell mobilization than controls; mean peak CD34+ cell counts were 41 × 10(6) and 113 × 10(6) /L, respectively (p < 0.0001). In a multivariate analysis, lower erythrocyte sedimentation rate (ESR) at mobilization was significantly correlated with better CD34+ cell mobilization (p = 0.007). In SCIDs, CD34 collection efficiency was positively correlated with higher red blood cell (RBC) indices (mean RBC volume, R(2) = 0.77; mean corpuscular hemoglobin [Hb], R(2) = 0.94; mean corpuscular Hb concentration, R(2) = 0.7; p < 0.007) but not Hb. CONCLUSIONS: CGD and SCID populations are characterized by significantly less robust CD34+ HPC mobilization than healthy controls. The presence of active inflammation or infection as suggested by an elevated ESR may negatively impact mobilization. Among SCIDs, markedly reduced CD34 collection efficiencies were related to iron deficiency, wherein decreased RBC size and density may impair apheresis cell separation mechanics.

Leukocyte integrin activation mediates transient neutropenia after G-CSF administration.

After administration of granulocyte colony-stimulating factor (G-CSF), there is a marked, albeit transient, drop in circulating neutrophils. To determine the role of leukocyte integrins in this disappearance, a dog having canine leukocyte adhesion deficiency (CLAD) or CLAD dogs who had undergone gene correction either by matched littermate allogeneic transplant or autologous gene therapy were evaluated. Shortly after G-CSF administration, a dramatic, yet transient, neutropenia was observed in the control littermates. This neutropenia was not as marked in the CLAD dogs. In all instances, it was CD18(+) neutrophils that preferentially egressed from the circulation. The association of CD18 with this rapid loss suggested leukocyte integrin activation after G-CSF administration. To determine the activation status of the integrin, a monoclonal antibody recognizing the activated α-subunit cation binding domain (mAb24) was used to evaluate human leukocytes after G-CSF administration. Mirroring the dramatic decrease in circulating neutrophil numbers, there was a dramatic and specific increase in the activation of the α-subunit after G-CSF expression on polymorphonuclear leukocytes. This activation, like the drop in neutrophil count, was transient. These results demonstrate that the leukocyte integrin on circulating neutrophils is transiently activated after G-CSF administration and mediates the transient neutropenia observed after G-CSF administration.

Ascertainment of iron deficiency and depletion in blood donors through screening questions for pica and restless legs syndrome.

BACKGROUND: Pica and restless legs syndrome (RLS) are associated with iron depletion and deficiency. The presence of pica and RLS was prospectively assessed in blood donors. STUDY DESIGN AND METHODS: During a 39-month period, 1236 donors deferred for fingerstick hemoglobin (Hb) level of less than 12.5 g/dL and 400 nondeferred "control" donors underwent health screening and laboratory testing (complete blood count, ferritin, iron, transferrin). Pica and RLS were assessed by direct questioning. Deferred donors and iron-deficient control donors were given 325 mg of ferrous sulfate daily for 60 days. Reassessments were performed and additional iron tablets dispensed at subsequent visits. RESULTS: Pica was reported in 11% of donors with iron depletion or deficiency, compared with 4% of iron-replete donors (p < 0.0001). Pagophagia (ice pica) was most common and often of extraordinary intensity. Female sex, younger age, and lower mean cell volume and transferrin saturation values were strongly associated with pica. Donors with pica given iron reported a marked reduction in the desire to consume the nonnutritive substance by Days 5 to 8 of therapy, with disappearance of symptoms by Days 10 to 14. RLS was reported in 16% of subjects with iron depletion or deficiency compared with 11% of iron-replete donors (p = 0.012). Iron replacement generally resulted in improvement of RLS symptoms; however, at least 4 to 6 weeks of iron therapy was necessary. CONCLUSION: The presence of pica is associated with a high probability of iron depletion or deficiency in blood donors; however, RLS lacks a strong correlation in this population. Screening questions for pagophagia may be useful in the ascertainment of iron deficiency in donors and may identify those who would benefit from oral iron.

Iron replacement therapy in the routine management of blood donors.

BACKGROUND: Iron depletion or deficiency in blood donors frequently results in deferrals for low hemoglobin (Hb), yet blood centers remain reluctant to dispense iron replacement therapy to donors. STUDY DESIGN AND METHODS: During a 39-month period, 1236 blood donors deferred for a Hb level of less than 12.5 g/dL and 400 nondeferred control donors underwent health history screening and laboratory testing (complete blood counts, iron studies). Iron depletion and deficiency were defined as a ferritin level of 9 to 19 and less than 9 µg/L in females and 18 to 29 and less than 18 µg/L in males. Deferred donors and iron-deficient control donors were given a 60-pack of 325-mg ferrous sulfate tablets and instructed to take one tablet daily. Another 60-pack was dispensed at all subsequent visits. RESULTS: In the low-Hb group, 30 and 23% of females and 8 and 53% of males had iron depletion or deficiency, respectively, compared with 29 and 10% of females and 18 and 21% of males in the control group. Iron-depleted or -deficient donors taking iron showed normalization of iron-related laboratory parameters, even as they continued to donate. Compliance with oral iron was 68%. Adverse gastrointestinal effects occurred in 21% of donors. The study identified 13 donors with serious medical conditions, including eight with gastrointestinal bleeding. No donors had malignancies or hemochromatosis. CONCLUSION: Iron depletion or deficiency was found in 53% of female and 61% of male low-Hb donors and in 39% of female and male control donors. Routine administration of iron replacement therapy is safe and effective and prevents the development of iron depletion and deficiency in blood donors.