Blood donation for iron removal in individuals with HFE mutations: study of efficacy and safety and short review on hemochromatosis and blood donation.

Background: Elevated serum ferritin with/without HFE variants in asymptomatic persons leads frequently to referral for blood donation. Hemochromatosis (p.C282Y/p.C282Y) only requires treatment. We evaluated safety and feasibility of iron removal in healthy persons with elevated ferritin and HFE variants using blood donation procedures. Materials and methods: Thirty subjects with ferritin >200 ng/mL (women) or >300 ng/mL (men) with p.C282Y/p.C282Y, p.C282Y/p.H63D or p.H63D/p.H63D were randomized to weekly phlebotomy (removal of 450 mL whole blood) or erythrapheresis (removal of 360 mL red blood cells) every 14 days. The ferritin target was <100 ng/mL. A full blood count and ferritin were measured at each visit. Hemoglobin (Hb) ≥140 g/L was required at inclusion. If Hb dropped to <120 g/L (women) or <130 g/L (men), procedures were postponed (7 or 14 days). Primary endpoint was the number of procedures needed to the ferritin target; secondary objectives were duration of treatment and compliance. The treatment effect was tested with Poisson regression; number of procedures and treatment duration were compared between study arms with the Kruskal-Wallis test. Results: Twenty-five of 30 participants were men (83%); mean age was 47 years (SD 10.5), mean BMI 26.6 kg/m2 (SD 3.6); 17 had p.C282Y/p.C282Y, nine p.C282Y/p.H63D, four p.H63D/p.H63D. Median baseline Hb was 150 g/L (IQR 144, 1,559), median ferritin 504 ng/mL (IQR 406,620). Twenty-seven subjects completed the study. Treatment arm (p < 0.001) and HFE variant (p = 0.007) influenced the primary endpoint significantly. To ferritin levels <100 ng/mL, a median number of 7.5 (IQR 6.2, 9.8) phlebotomies and 4.0 (IQR 3.0, 5.8) erythraphereses (p = 0.001) was needed during a median of 66.5 days (IQR 49,103) and 78.5 days (IQR 46139), respectively (p = 0.448). Low Hb was the principal reason for protocol violation; anemia occurred in 13 participants (48%). Immediate complications were infrequent; fatigue was reported after 25% of phlebotomies and 45% of erythraphereses. Thirty-five procedures were postponed because of low Hb and 15 for non-medical reasons. The median interval was 7.0 (IQR 7.7) and 14.0 (IQR 14, 20) days between phlebotomies and erythraphereses, respectively. Conclusion: Blood donation procedures remove iron effectively in HC, but frequent treatments cause Hb decrease and fatigue that can impair feasibility.

Red blood cell transfusion-related dynamics of extracellular vesicles in intensive care patients: a prospective subanalysis.

Extracellular vesicles (EVs) accumulate during packed red blood cell (PRBC) storage. To date, the involvement of EVs in transfusion-related immunomodulation (TRIM) has not been prospectively evaluated in intensive care unit (ICU) patients. This was a prospective subanalysis of a recent observational feasibility study in postoperative ICU patients after: (1) open aortic surgery (Aorta), (2) bilateral lung transplantation (LuTx), and (3) other types of surgery (Comparison). Patient plasma was collected three times each before and after leukoreduced PRBC transfusion at 30-min intervals. The total number of EVs and EVs derived from erythrocytes (EryEVs), total platelets (total PEVs), activated platelets, granulocytes (GEVs), monocytes, and myeloid cells in PRBC samples and patient plasma were analyzed by flow cytometry. Statistical analysis was performed by Spearman's correlation test, linear mixed models and pairwise comparisons by Wilcoxon matched-pairs test. Twenty-three patients (Aorta n = 5, LuTx n = 9, Comparison n = 9) were included in the final analysis. All EV subgroups analyzed were detectable in all PRBCs samples (n = 23), but concentrations did not correlate with storage time. Moreover, all EVs analyzed were detectable in all plasma samples (n = 138), and EV counts were consistent before transfusion. Concentrations of total EVs, EryEVs, total PEVs, and GEVs increased after transfusion compared with baseline in the entire cohort but not in specific study groups. Furthermore, the change in plasma EV counts (total EVs and EryEVs) after transfusion correlated with PRBC storage time in the entire cohort. Extracellular vesicles were detectable in all PRBC and plasma samples. Individual EV subtypes increased after transfusion in the entire cohort, and in part correlated with storage duration. Future clinical studies to investigate the role of EVs in TRIM are warranted and should anticipate a larger sample size.Trial registration: Clinicaltrials.gov: NCT03782623.

Red blood cell transfusion-related eicosanoid profiles in intensive care patients-A prospective, observational feasibility study.

Introduction: Eicosanoids are bioactive lipids present in packed red blood cells (PRBCs), and might play a role in transfusion-related immunomodulation (TRIM). We tested the feasibility of analyzing eicosanoid profiles in PRBC supernatant and in plasma samples of postoperative intensive care unit (ICU) patients transfused with one unit of PRBCs. Methods: We conducted a prospective, observational feasibility study enrolling postoperative ICU patients: 1) patients treated with acetylsalicylic acid following abdominal aortic surgery (Aorta); 2) patients on immunosuppressants after bilateral lung transplantation (LuTx); and 3) patients undergoing other types of major surgery (Comparison). Abundances of arachidonic acid (AA) and seven pre-defined eicosanoids were assessed by liquid chromatography and tandem mass spectrometry. PRBC supernatant was sampled directly from the unit immediately prior to transfusion. Spearman's correlations between eicosanoid abundance in PRBCs and storage duration were assessed. Patient plasma was collected at 30-min intervals: Three times each before and after transfusion. To investigate temporal changes in eicosanoid abundances, we fitted linear mixed models. Results: Of 128 patients screened, 21 were included in the final analysis (Aorta n = 4, LuTx n = 8, Comparison n = 9). In total, 21 PRBC and 125 plasma samples were analyzed. Except for 20-hydroxyeicosatetraenoic acid (HETE), all analyzed eicosanoids were detectable in PRBCs, and their abundance positively correlated with storage duration of PRBCs. While 5-HETE, 12-HETE/8-HETE, 15-HETE, 20-HETE, and AA were detectable in virtually all plasma samples, 9-HETE and 11-HETE were detectable in only 57% and 23% of plasma samples, respectively. Conclusions: Recruitment of ICU patients into this transfusion study was challenging but feasible. Eicosanoid abundances increased in PRBC supernatants during storage. In plasma of ICU patients, eicosanoid abundances were ubiquitously detectable and showed limited fluctuations over time prior to transfusion. Taken together, larger clinical studies seem warranted and feasible to further investigate the role of PRBC-derived eicosanoids in TRIM.

Indices of iron homeostasis in asymptomatic subjects with HFE mutations and moderate ferritin elevation during iron removal treatment.

We analysed iron biomarkers and their relationships in 30 subjects with HFE mutations and moderate hyperferritinaemia undergoing iron removal at our blood donation centre. Body mass index (BMI) and liver enzymes were assessed. Serum iron (SI), ferritin, transferrin saturation (TSAT), hepcidin and non-transferrin bound iron (NTBI) were measured serially. Seventeen subjects had p.C282Y/p.C282Y, nine p.C282Y/p.H63D, four p.H63D/p.H63D. Median age (p = 0.582), BMI (p = 0.500) and ferritin (p = 0.089) were comparable. At baseline, 12/17 p.C282Y/p.C282Y and 2/9 p.C282Y/p.H63D had measurable NTBI (p = 0.003). The p.C282Y/p.C282Y had higher TSAT (p < 0.001), lower hepcidin (p = 0.031) and hepcidin/ferritin ratio (p = 0.073). After treatment, iron indices were similar among groups, except TSAT (higher in p.C282Y/p.C282Y; p = 0.06). Strong relationships were observed between ferritin and TSAT (R = 0.71), NTBI and TSAT (R = 0.61), NTBI and SI (R = 0.54) in p.C282Y/p.C282Y. Hepcidin correlated weakly with ferritin in p.C282Y/p.C282Y (R = 0.37) but strongly in p.C282Y/p.H63D (R = 0.66) and p.H63D/p.H63D (R = 0.72), while relationships with TSAT were weak (R = 0.27), moderate (R = 0.55) and strong (R = 0.61), respectively. Low penetrance p.C282Y/p.C282Y phenotype displays hepcidin dysregulation and biochemical risk for iron toxicity.

Extracorporeal Photopheresis With Low-Dose Immunosuppression in High-Risk Heart Transplant Patients-A Pilot Study.

In severely ill patients undergoing urgent heart transplant (HTX), immunosuppression carries high risks of infection, malignancy, and death. Low-dose immunosuppressive protocols have higher rejection rates. We combined extracorporeal photopheresis (ECP), an established therapy for acute rejection, with reduced-intensity immunosuppression. Twenty-eight high-risk patients (13 with high risk of infection due to infection at the time of transplant, 7 bridging to transplant via extracorporeal membrane oxygenation, 8 with high risk of malignancy) were treated, without induction therapy. Prophylactic ECP for 6 months (24 procedures) was initiated immediately postoperatively. Immunosuppression consisted of low-dose tacrolimus (8-10 ng/ml, months 1-6; 5-8 ng/ml, >6 months) with delayed start; mycophenolate mofetil (MMF); and low maintenance steroid with delayed start (POD 7) and tapering in the first year. One-year survival was 88.5%. Three patients died from infection (POD 12, 51, 351), and one from recurrence of cancer (POD 400). Incidence of severe infection was 17.9% (n = 5, respiratory tract). Within the first year, antibody-mediated rejection was detected in one patient (3.6%) and acute cellular rejection in four (14.3%). ECP with reduced-intensity immunosuppression is safe and effective in avoiding allograft rejection in HTX recipients with risk of severe infection or cancer recurrence.

Vorinostat in the acute neuroinflammatory form of X-linked adrenoleukodystrophy.

OBJECTIVE: To identify a pharmacological compound targeting macrophages, the most affected immune cells in inflammatory X-linked adrenoleukodystrophy (cerebral X-ALD) caused by ABCD1 mutations and involved in the success of hematopoietic stem cell transplantation and gene therapy. METHODS: A comparative database analysis elucidated the epigenetic repressing mechanism of the related ABCD2 gene in macrophages and identified the histone deacetylase (HDAC) inhibitor Vorinostat as a compound to induce ABCD2 in these cells to compensate for ABCD1 deficiency. In these cells, we investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ß-oxidation activity, accumulation of very long-chain fatty acids (VLCFAs) and their differentiation status. We investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ß-oxidation activity, accumulation of very long-chain fatty acids (VLCFA) and differentiation status. Three advanced cerebral X-ALD patients received Vorinostat and CSF and MRI diagnostics was carried out in one patient after 80 days of treatment. RESULTS: Vorinostat improved the metabolic defects in X-ALD macrophages by stimulating ABCD2 expression, peroxisomal ß-oxidation, and ameliorating VLCFA accumulation. Vorinostat interfered with pro-inflammatory skewing of X-ALD macrophages by correcting IL12B expression and further reducing monocyte differentiation. Vorinostat normalized the albumin and immunoglobulin CSF-serum ratios, but not gadolinium enhancement upon 80 days of treatment. INTERPRETATION: The beneficial effects of HDAC inhibitors on macrophages in X-ALD and the improvement of the blood-CSF/blood-brain barrier are encouraging for future investigations. In contrast with Vorinostat, less toxic macrophage-specific HDAC inhibitors might improve also the clinical state of X-ALD patients with advanced inflammatory demyelination.

The assessment of platelet function by thromboelastometry as a point-of-care test to guide Intercept-treated platelet support in hemato-oncological patients and hematopoietic stem cell transplantation recipients.

BACKGROUND: Pathogen inactivation (PI) techniques for platelet concentrates (PCs) are one of the latest innovations to improve blood safety and reduce the risk of transfusion-transmitted infections (TTIs). An impaired function and in vivo recovery of platelets as well as an increased PC demand are concerns regarding these techniques. The intent of this study was to evaluate the hemostatic effect of PCs treated with the Intercept™ System by thromboelastometry (TEM) and to assess the clinical validity of its results in comparison to post-transfusion increase (PTI) and corrected count increment (CCI). STUDY-DESIGN AND METHODS: This prospective-observational study included 47 patients (m:f = 25:22; median age: 54 years [21-70]) of our Bone Marrow Transplantation unit with hemato-oncological malignancies transfused with Intercept™-treated PCs. Serial TEM measurements were performed just before and 1 hour after PC transfusion and were analyzed for their correlation with PTI and CCI as well as for clinical variables. RESULTS: The majority of our patients had received a hematopoietic stem cell transplantation (HSCT) (n = 41; 87%). In median 9 (1-50) PCs were transfused. Serial TEM, PTI, and CCI measurements were available for 150 transfusion episodes. The median platelet dose transfused was 2.65 × 1011 /unit (1.8-6). The median CCI was 9.250 (0-28.000). We observed a significant improvement in TEM parameters (p < 0.05) after transfusion of PI PCs, which did not mandatory correlate with the 1-hour PTI and CCI. CONCLUSION: Serial TEM measurements indicate the hemostatic effect of Intercept™-treated PCs. The 1-hour PTI and CCI may not appropriately reflect the in vivo function of platelets after PI PC transfusion.

Transfusion of standard-issue packed red blood cells induces pulmonary vasoconstriction in critically ill patients after cardiac surgery-A randomized, double-blinded, clinical trial.

BACKGROUND: Experimental and volunteer studies have reported pulmonary vasoconstriction during transfusion of packed red blood cells (PRBCs) stored for prolonged periods. The primary aim of this study was to evaluate whether transfusion of PRBCs stored over 21 days (standard-issue, siPRBCs) increases pulmonary artery pressure (PAP) to a greater extent than transfusion of PRBCs stored for less then 14 days (fresh, fPRBCs) in critically ill patients following cardiac surgery. The key secondary aim was to assess whether the pulmonary vascular resistance index (PVRI) increases after transfusion of siPRBCs to a greater extent than after transfusion of fPRBCs. METHODS: The study was performed as a single-center, double-blinded, parallel-group, randomized clinical trial. Leukoreduced PRBCs were transfused while continuously measuring hemodynamic parameters. Systemic concentrations of syndecan-1 were measured to assess glycocalyx injury. After randomizing 19 patients between January 2014 and June 2016, the study was stopped due to protracted patient recruitment. RESULTS: Of 19 randomized patients, 11 patients were transfused and included in statistical analyses. Eight patients were excluded prior to transfusion, 6 patients received fPRBCs (10±3 storage days), whereas 5 patients received siPRBCs (33±4 storage days). The increase in PAP (7±3 vs. 2±2 mmHg, P = 0.012) was greater during transfusion of siPRBCs than during transfusion of fPRBCs. In addition, the change in PVRI (150±89 vs. -4±37 dyn·s·cm-5·m2, P = 0.018) was greater after transfusion of siPRBCs than after transfusion of fPRBCs. The increase in PAP correlated with the change of systemic syndecan-1 concentrations at the end of transfusion (R = 0.64,P = 0.034). CONCLUSION: Although this study is underpowered and results require verification in larger clinical trials, our findings suggest that transfusion of siPRBCs increases PAP and PVRI to a greater extent than transfusion of fPRBCs in critically ill patients following cardiac surgery. Glycocalyx injury might contribute to pulmonary vasoconstriction associated with transfusion of stored blood.

Microparticle detection to guide platelet management for the reduction of platelet refractoriness in children - A study proposal.

Microparticles have been shown to shed from a variety of viable cells as a consequence of inflammatory processes, activation or physical stress. Seventy to 90% of circulating microparticles are thought to be platelet-derived. The content of microparticles in blood collected from normal blood donors is highly variable and transfers into the final blood component. Elevated microparticle content (MPC) in donor blood might indicate an asymptomatic clinical condition of the donor which might affect the transfusion recipient, particularly pediatric patients. ThromboLUX is a new technology designed to routinely test biological samples for microparticle content. We compared MPC in platelet-rich plasma (PRP) of apheresis donors and the corresponding INTERCEPT-treated apheresis products (N=24). The MPCs in donor and product samples were correlated (r=0.74, P<0.001). Microparticles were significantly reduced after plasma replacement and INTERCEPT treatment. These findings are supported by phase contrast microscopy. Platelet transfusions given to patients with fever or systemic inflammation are less efficacious. In addition, transfusing heterogeneous platelets - concentrates with high MPC and activated platelets - to patients whose immune systems are activated might tip them over a threshold and cause platelet refractoriness. Restricting prophylactic platelet transfusions to homogeneous products - concentrates with resting platelets and therefore low MPC - may reduce the risk of refractoriness in cancer patients, especially children with immature immunity. To test this hypothesis we introduce an evaluation protocol for platelet management, i.e., keeping a split inventory of homogeneous and heterogeneous platelets, and using only homogeneous platelets for prophylaxis as a strategy to reduce refractoriness.

Plerixafor as preemptive strategy results in high success rates in autologous stem cell mobilization failure.

Plerixafor in combination with granulocyte-colony stimulating factor (G-CSF) is approved for autologous stem cell mobilization in poor mobilizing patients with multiple myeloma or malignant lymphoma. The purpose of this study was to evaluate efficacy and safety of plerixafor in an immediate rescue approach, administrated subsequently to G-CSF alone or chemotherapy and G-CSF in patients at risk for mobilization failure. Eighty-five patients mobilized with G-CSF alone or chemotherapy were included. Primary endpoint was the efficacy of the immediate rescue approach of plerixafor to achieve ≥2.0 × 106 CD34+ cells/kg for a single or ≥5 × 106 CD34+ cells/kg for a double transplantation and potential differences between G-CSF and chemotherapy-based mobilization. Secondary objectives included comparison of stem cell graft composition including CD34+ cell and lymphocyte subsets with regard to the mobilization regimen applied. No significant adverse events were recorded. A median 3.9-fold increase in CD34+ cells following plerixafor was observed, resulting in 97% patients achieving at least ≥2 × 106 CD34+ cells/kg. Significantly more differentiated granulocyte and monocyte forming myeloid progenitors were collected after chemomobilization whereas more CD19+ and natural killer cells were collected after G-CSF. Fifty-two patients underwent transplantation showing rapid and durable engraftment, irrespectively of the stem cell mobilization regimen used. The addition of plerixafor in an immediate rescue model is efficient and safe after both, G-CSF and chemomobilization and results in extremely high success rates. Whether the differences in graft composition have a clinical impact on engraftment kinetics, immunologic recovery, and graft durability have to be analysed in larger prospective studies.

Complete neurologic and cognitive recovery after plasmapheresis in a patient with chronic inflammatory demyelinating polyneuropathy after allogeneic hematopoietic stem cell transplantation.

UNLABELLED: Neurologic complications after allogeneic hematopoietic stem cell transplantation (HSCT) are rare but poorly understood. We present a case report of a 57-year-old-male patient who was diagnosed in 2009 with acute myeloid leukemia (AML). He received two standard induction chemotherapies, as well as a following consolidation. Six months later, an allogeneic HSCT was performed. Shortly after HSCT the patient developed progressive polyneuropathy of the lower legs and hypoesthesia. Five months later a severe dementia followed. All images of the brain and spine showed no specific pathologies. High dose corticosteroids and immunoglobulins did not improve the neurologic symptoms. Due to severe worsening of the neuropsychiatric status and the clinical presentation, chronic inflammatory demyelinating polyneuropathy (CIDP) was suspected. Therefore, the patient received ten cycles of plasmapheresis. The patient showed a significant improvement of the neuropsychiatric symptoms and cognitive status. CONCLUSIONS: Immune mediated neuropathies after allogeneic HSCT, such as CIDP, have great variability in symptoms and presentation and are challenging to diagnose and treat. Plasmapheresis is a safe and efficient treatment for patients with unclear persisting autoimmune neuropathy after HSCT.

Suitability Criteria for Adult Related Donors: A Consensus Statement from the Worldwide Network for Blood and Marrow Transplantation Standing Committee on Donor Issues.

The number of allogeneic hematopoietic stem cell (HSC) transplants performed globally each year continues to increase. Advances in HLA typing, better supportive care, and administration of reduced-intensity conditioning regimens allow treatment of older patients with older sibling donors. Pretransplant donor assessment and testing are very important processes affecting the quality and safety of donation. For unrelated HSC donors detailed recommendations for health assessment have been published, allowing donation only if they are unrestrictedly healthy. Eligibility criteria for related donors are less strict and vary significantly between centers. In situations where a family donor does not meet the suitability criteria for unrelated donors, involved physicians often struggle with the decision whether the matched relative is suitable for donation or not. On behalf of the Worldwide Network for Blood and Marrow Transplantation Standing Committee on Donor Issues, we intended to develop a consensus document with recommendations for donor workup and final clearance of family donors who would not be able to serve as unrelated donors because of their age or pre-existing diseases. This article covers different topics intending to support decision-making, with the goal of minimizing medical risk to the donor and protection of the recipient from transmissible diseases.

Granulocyte collection using a novel apheresis system eases the procedure and provides concentrates of high quality.

BACKGROUND: The latest technical innovation for granulocyte (PMN) collections is the fully automated Spectra Optia (Optia) device (TerumoBCT). In a retrospective investigation we evaluated the impact of the technical automation on the product quality in a routine working field. STUDY DESIGN AND METHODS: A total of 71 granulocyte collections (GCs) were collected from either routine random blood donors, mobilized with prednisolone (P; two females/23 males; median age, 42 years; range, 25-63 years), or family donors (three females/12 males; median age, 29 years; range, 21-59 years) who were mobilized with recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) at a dose of 5 µg/kg body weight. All collections were performed with the Optia device. Fifty-nine concentrates (GTX) produced with the Cobe Spectra (Cobe; TerumoBCT) served as a historical control. RESULTS: In total a mean of 452 ± 60 mL with a mean purity of 83 ± 9.6% PMNs was collected with the Optia. Compared with the Cobe collections (298 ± 52 mL), the product volumes in general as well as the absolute PMN yield in P-mobilized products were significantly higher with the Optia: PMN count, 1.9 × 10(10) ± 0.49 × 10(10) versus 1.5 × 10(10) ± 0.85 × 10(10) , respectively (p < 0.05), due to higher white blood cell (WBC) yields. rHuG-CSF-mobilized products showed no significant differences in the absolute WBC (7.2 ± 3.0/Optia vs. 7.0 ± 2.1/Cobe) and PMN (5.9 ± 2.6/Optia vs. 5.7 ± 1.9/Cobe) yield. The PMN purity was equal in both devices (mean, 83%) although it was slightly lower in the rHuG-CSF group (82 ± 9.2%) than in the P group (84 ± 6.2%). In none of the procedures were side effects recorded. CONCLUSION: GC with the fully automated Optia device is safe for donors, is not inferior to its forerunner Cobe Spectra, produces GTX of a high quality, and requires less manpower than the Cobe Spectra.

Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

BACKGROUND: The short-term safety profile of recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) in the allogeneic stem cell setting seems acceptable; only few data on long-term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year. STUDY DESIGN AND METHODS: In this study, 20 stem cell donors (14 male/six female; median age, 40 years; range, 22-54 years) and 20 sex- and age-matched blood component donors (controls) were included. Sampling was performed before rHuG-CSF administration; at the time of donation; and on Days (+1), 7, 30, 100, 180, and 360 in both groups. Analysis of DNMT activity in nuclear extracts was performed using a modified radionuclide assay. We performed methylation-specific polymerase chain reaction to detect the methylation status of promoter CpG islands of the genes of the retinoic acid receptor beta (RAR-B) and the Ras association domain family 1A (RASSF1A). RESULTS: DNMT activity increased significantly on the day of donation and 1 day after (p < 0.05). By Day +7 baseline values were reached. No further significant alterations of DNMT activity in the treated group compared to the controls were observed. We could not detect any differences in the gene methylation of RAR-B and RASSF1A between both groups. CONCLUSION: In our prospective study no evidence of long-lasting increased DNMT activity or enhanced DNA methylation in a limited panel of target genes after recombinant human G-CSF administration was observed in healthy stem cell donors.

Monitoring of platelet activation in platelet concentrates using transmission electron microscopy.

OBJECTIVE: The quality of platelet concentrates (PC) is important for the in vivo recovery of thrombostasis in patients suffering from bleeding disorders and in tumor patients after chemotherapy. In this respect, activated platelets (PLT) cannot display their full functionality in the recipient and even can cause adverse effects. Therefore, we developed a transmission electron microscopy (TEM) method for quality assessment of PC. METHODS: Score values taken from panorama TEM images describe the progress of PLT activation. To exemplify this method, i) 19 apheresis PC isolated with the Baxter Amicus system (BA) were compared with 14 PC obtained from pooled buffy coats (BC). ii) The score values of 33 PC derived from BA as well from BC were compared with flow-cytometric CD62P determinations by cross correlation. iii) Changes in the score value profiles during storage of a single pathogen-reduced BA PC were monitored over a period of 7 days. RESULTS: The TEM evaluation described allows for demonstrating particular PLT activation stages. i) Significant differences between the percentages of the score values 0, 1 and 2 could be demonstrated in both processing groups. No significant differences were found comparing these two groups. ii) A weak correlation could be shown when comparing the percentages of score values 2 plus 3 with the percentage of CD62P-positive PLT. iii) The pathogen reduction affected slightly the score profiles during storage due to an increase of dead PLT. CONCLUSION: Our investigations demonstrate the unique detailed quality information of PC obtained by the TEM method. This method can be performed in every routine electron microscopy laboratory.

Clinical Results of Extracorporeal Photopheresis.

Extracorporeal photopheresis (ECP) is a combination of leukapheresis and photodynamic therapy in which blood is treated with photoactivable drugs which are then activated with ultraviolet light and re-infused to the patient. It has been used successfully for more than 30 years in the treatment of erythrodermic cutaneous T-cell lymphoma (CTCL) and over 20 years for chronic graft-versus-host disease (GVHD). ECP has also shown promising results in the treatment of acute GVHD and other T-cell-mediated diseases, including systemic sclerosis, treatment and prevention of solid organ rejection, and more recently Crohn's disease. The use of ECP may allow a significant reduction or even discontinuation of corticosteroids and/or other immunosuppressants, thus leading to reduced long-term morbidity and mortality and improved overall survival. ECP is a well-tolerated therapy. No significant side effects have been reported during the last 30 years. It has been shown that ECP is not associated with an increased incidence of infections, malignancies, or recurrence of underlying malignant disease, neither during short-term nor during long-term therapy.

Processing of autologous bone marrow cells by apheresis technology for cell-based cardiovascular regeneration.

BACKGROUND AIMS: Bone marrow (BM)-derived mononuclear cell (MNC) preparations are increasingly used in experimental studies exploring the potential effect of progenitor cell-derived therapies in cardiocirculatory diseases. We analyzed the cellular BM composition, side-effects and other process-related variables of BM harvest and BM-MNC preparation in 80 patients with cardiovascular disease. METHODS: BM (median 828 mL, range 223-1038 mL) was collected from the iliac crest. After BM harvest the MNC fraction was enriched by semi-automatic apheresis to reduce the total volume of the transplant. Autologous red blood cells (RBC) were salvaged from the initial BM harvest and autotransfused to the patients. RESULTS: There were no serious side-effects related to BM collection, particularly no serious bleeding complications. Twenty- five of 80 (31%) patients developed mild pain. BM harvest resulted in the collection of a median of 2.8 × 10(9) MNC, containing a median of 66.5 × 10(6) CD34/45 cells, 39.5 × 10(6) CD133/45 cells and 50.3 × 10(6) CD34/CD133 cells. Apheresis technology-based MNC enrichment of harvested BM resulted in a progenitor cell recovery of 69-75.3% of total cells. Additional salvage of RBC from the initial BM harvest resulted in the recovery of a median of 175.0 mL autologous RBC mass. Transfusion of salvaged RBC was well tolerated and resulted in a significant increase in hemoglobin levels. CONCLUSIONS: Collection of BM of up to 1 L in combination with in vitro processing using a semi-automated apheresis device is a safe and feasible approach to increasing the number of progenitor cells necessary for cellular therapies, particularly when combined with RBC salvage.

Frequency and prognostic value of D alloantibodies after D-mismatched allogeneic hematopoietic stem cell transplantation after reduced-intensity conditioning.

BACKGROUND: Due to the fact that the ABO and D system is inherited independently from the HLA system, approximately 40% of allogeneic hematopoietic stem cell transplants (HSCT) are performed across the blood group barrier. Reports on the development of de novo anti-D in patients undergoing reduced-intensity conditioning (RIC) followed by D-mismatched allogeneic HSCT are rare. The objective of this study was to evaluate the frequency of anti-D alloimmunization after D-mismatched HSCT following RIC and its prognostic impact on transplant outcome. STUDY DESIGN AND METHODS: Forty patients with hematologic diseases who underwent D-mismatched HSCT were retrospectively analyzed: 19 D- patients with a D+ donor and 21 D+ patients with a D- donor. Routine serologic testing for blood group typing and antibody screening was performed by a column agglutination method every time when transfusion of red blood cell units was requested and in the posttransplantation course to demonstrate establishment of donor ABO type and to detect alloimmunization. RESULTS: After a median serologic follow-up of 21 (range, 0 to 73) months after HSCT, anti-D was identified in 2 of 21 (10%) D+ patients receiving a D- transplant, 23 and 34 months after HSCT. None of the 19 D- patients with a D+ donor developed an anti-D. CONCLUSION: We observed an infrequent de novo anti-D formation that is more likely in D+ recipients of D- grafts. However, the development of anti-D does not normally impair the transplant outcome and is not of clinical relevance in the posttransplant course.

Granulocyte transfusions in children and young adults: does the dose matter?

BACKGROUND: Granulocyte transfusions (GTs) may increase the absolute neutrophil count (ANC) before hematopoietic regeneration in neutropenic patients after chemotherapy or hematopoietic stem cell transplantation and support anti-infective immunity. PROCEDURE: We assessed efficacy and tolerability of 778 GTs in 70 treatment episodes of 49 children and 10 young adults [median age 6.28 y (range: 0.13 to 17.7 y) and 21 y (18.0 to 28.0), respectively] suffering from bacterial (n=55) and/or fungal (n=31) infections during neutropenia owing to conventional chemotherapy (n=14), hematopoietic stem cell transplantation conditioning (n=44), or the underlying disease (n=1). We analyzed the impact of body weight, organ dysfunction, neutrophil dose on ANC increment, infection elimination, and survival. RESULTS: The median day-5 ANC increment was 1460/microL, correlating to the administered dose. However, 28-day survival did not correlate to the neutrophil dose nor to the ANC increment, potentially owing to the high number of neutrophils transfused to all patients (median >6x10(9)/kg within 5 d). The 28-day survival probability of the total patient cohort was 0.72+/-0.06 and the 100-day survival was 0.52+/-0.07. Adverse reactions were rare including fever (< or =World Health Organization grade III, 14%), chills (3%), and mild pulmonary complications (1%). CONCLUSIONS: These data corroborate the empirical evidence that GT with sufficient cell doses and rapid availability are a feasible, well-tolerated supplemental means to fight severe infections in neutropenic patients.

Therapeutic apheresis in hematopoietic cell transplantation.

With the introduction of hematopoietic stem cell transplant (HSCT), transplant-associated problems like graft-versus-host disease (GVHD) and transplant-associated microangiopathy (TAM) have occurred. In addition, approximately 40% of allogeneic HSCTs are performed across the ABO blood group barrier, bearing the risk for immunohematological complications like severe hemolysis and pure red cell aplasia (PRCA). All these problems can potentially require therapeutic apheresis. In this review, we address recent developments in therapeutic apheresis for patients undergoing allogeneic HSCT for prevention or treatment of hemolysis in minor ABO-incompatible transplantation, treatment of PRCA after major ABO-incompatible transplantation, and treatment of TAM and GVHD.