Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices.

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.

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.

Prophylactic red blood cell exchange for prevention of severe immune hemolysis in minor ABO-mismatched allogeneic peripheral blood progenitor cell transplantation after reduced-intensity conditioning.

BACKGROUND: Delayed severe immune hemolysis due to donor-derived passenger lymphocytes is observed in minor and/or bidirectional ABO-mismatched transplants, especially after reduced-intensity conditioning (RIC). The incidence is reported in up to 30 percent of patients and can result in multiorgan failure (MOF) and death. STUDY DESIGN AND METHODS: A first group of 32 patients (historical control) underwent RIC followed by allogeneic hematopoietic peripheral blood progenitor cell transplantation at our institution. In 5 of 10 patients with a minor and/or bidirectional ABO-mismatched graft, severe immune hemolysis was observed, leading to death in 3 of them. Therefore, we initiated a protocol with prophylactic red blood cell (RBC) exchange in minor and/or bidirectional ABO mismatch of a second group of patients (study group) and investigated the incidence of hemolysis, transplant-related mortality (TRM), and overall survival (OS) and compared these data with the historical control group. Twenty-two of 80 patients in the study group had a minor and/or bidirectional ABO-mismatched donor. RESULTS: In 20 patients, a prophylactic RBC exchange was performed. Three patients showed mild to moderate citrate reactions, and in 1 patient the procedure had to be stopped because of hypotension. Eighteen of 20 patients engrafted uneventfully, 1 patient rejected his graft, and another 1 showed signs of mild hemolysis. In the minor and/or bidirectional ABO-mismatched setting patients in the study group had a lower risk for TRM at 1 year compared to patients in the historical control group (16% vs. 53%, p < 0.05) and a better 1-year OS (65% vs. 40%, p < 0.05). CONCLUSION: RBC exchange is a safe procedure, reducing the incidence of delayed severe immune hemolysis and thus the risk of TRM in minor and/or bidirectional ABO-mismatched cases.

ABO-incompatible allogeneic hematopoietic stem cell transplantation following reduced-intensity conditioning: close association with transplant-associated microangiopathy.

Transplant-associated microangiopathy (TAM) is a severe complication following allogeneic hematopoietic stem cell transplantation (HSCT) even after reduced-intensity conditioning (RIC). Data on 112 patients following RIC were analyzed with respect to TAM according to the ASBMT and risk factors, response to well-defined therapy and outcome were determined. TAM occurred in 11 of 112 patients. Univariate analysis determined acute graft-versus-host disease and ABO-incompatibility as risk factors for TAM. Treatment consisted of withdrawal of calcineurin inhibitors and plasma exchange (PE). Response to PE was 64%. PE seems to be an effective therapeutic option that should be assessed in larger patient cohorts.

Autologous stem-cell transplantation in progressing amyloidosis is associated with severe transplant-related toxicity.

BACKGROUND: Amyloid light-chain (AL) amyloidosis is a disorder of plasma cells in which depositions of immunoglobulin light-chain fragments cause progressive organ failure and death with a median survival of one year, but autologous stem-cell transplantation can induce high response rates, especially in isolated renal involvement. METHODS: Six patients aged between 43 and 59 years were diagnosed with AL-amyloidosis and had stem cells mobilized with either recombinant human granulocyte colony-stimulating factor (rhG-CSF) alone (n = 2) or cyclophosphamide (2-4 g/m(2)) and rhG-CSF (n = 4). All six patients had kidney involvement and nephrotic syndrome, four had cardiac involvement and two involvement of the vascular, nervous and gastrointestinal systems. Five of the patients received high-dose melphalan (200 mg/m(2)) and autologous blood stem-cell support. RESULTS: One patient died as a result of sepsis after stem-cell mobilization. The other five patients received high-dose melphalan but experienced severe toxicity. One patient died as the result of gastrointestinal perforation on day 6, one presented with hyperfibrinolysis and spontaneous rupture of the spleen, and another experienced severe bleeding of the gastrointestine, tachyarrhythmia and hemolytic anemia. Four patients had acute renal failure: three required hemodialysis and one underwent renal transplant 21 months later. Restaging after a follow-up of 31-52 months revealed reversal of nephrotic syndrome in all three patients who regained adequate renal function. With respect to cardiac involvement (n = 2), one patient showed a decrease in NYHA class from II to I but baseline wall thickness remained stable. CONCLUSION: Treatment of selected patients with AL-amyloidosis by high-dose melphalan and stem-cell support results in reversal of amyloid-related disease in a substantial proportion of patients and improved survival.

Surface disinfection of packed red blood cells with 70% ethanol.

No data or recommendations are available on feasibility of surface disinfection of blood bags, but some circumstances can make such procedures inevitable. Impact of immersion of blood bags in 70% ethanol for 30min was investigated with respect to alcohol penetration and changes of hemolysis parameters in the product, and bag material changes influencing material stability and composition. After immersion ethanol concentration in blood bags was below detection limit. Hemolysis parameters did not differ between blood products that had been exposed to ethanol and a control group. Inner surface of the bag material was unchanged according to our infrared spectrometry results. Also endurance testing showed no altered results. We conclude that immersion of blood bags in 70% ethanol for surface disinfection is a safe procedure for the quality of the blood product and the bag material.

Lack of impact of ABO blood group or corresponding isoantibodies on the immune response after rabies vaccination.

Demand for rabies hyperimmunoglobulin has increased recently, requiring optimization of vaccination schemes for immunized plasma donors. Possible resemblance of rabies vaccine to blood group antigens and consequential association of the immune response to rabies vaccine and blood group or corresponding isoantibodies has not yet been investigated. We analyzed antirabies antibodies after rabies vaccination and ABO blood group in 142 individuals, and isoantibody titers in 92 of those individuals. We did not find any correlation of the immune response with blood group or isoantibody levels. There was also no correlation with the sex of individuals, but there was a weak correlation between age and rabies-specific antibody level. Rabies vaccination schemes for immunized donors cannot be optimized on the basis of blood groups or isoantibody titers.

Biochemical characterization of autologous fibrin sealants produced by CryoSeal and Vivostat in comparison to the homologous fibrin sealant product Tissucol/Tisseel.

Different principles for production of "autologous fibrin sealant" have been established, and commercial devices employing these methods are nowadays available and used in clinical routine. Users might anticipate for these autologous fibrin sealants features comparable to commercial homologous fibrin sealants, used in surgical routine for many years. However, only little is known about biochemical properties, formation, cross-linking and stability of fibrin sealant clots produced for autologous use with the aid of commercially available devices. We have investigated protein composition, formation and stability of clots obtained from autologuous fibrin sealants produced with commercially available devices (CryoSeal and Vivostat) and compared these parameters to those of the industrially produced homologous fibrin sealant Tissucol/Tisseel. The CryoSeal product is a mixture of many plasma proteins; the Vivostat product and Tissucol/Tisseel appear as comparatively pure plasma derivatives. The products differ in their protein composition and concentrations, including their concentration in fibrin. Significant fibrin alpha and gamma-chain cross-linking by FXIIIa occurs only in Tissucol/Tisseel clots. In test tubes CryoSeal and Vivostat (tranexamic acid-free formulation) fibrin clots liquefy within 1-2 days, but Vivostat (tranexamic acid containing formulation) clots were stable for 4 days and showed partial liquefaction after 5 days. Tissucol/Tisseel clots, containing the protease inhibitor aprotinin, appeared unchanged over the observation period of 5 days. In an in vitro model mimicking in vivo conditions (diffusion of protease inhibitors and proteolytic digestion) clot liquefaction occurs at day 1 for all autologous fibrin sealants clots, with an observable delay for the tranexamic acid containing Vivostat, and day 5 for Tissucol/Tisseel clots. Characterization of the CryoSeal and Vivostat fibrin sealants and Tissucol/Tisseel and their performance show a clear difference in biochemical properties.

Skin plugs in phlebotomy puncture for blood donation.

Contamination at the site of the donor's skin may occur despite proper disinfection, because pathogens in deeper regions (such as pores) may not be eliminated by skin disinfection. It is suspected that the cannula detaches fragments of tissue when it penetrates the skin; the tissue fragments may reach blood products and release pathogens there. In the present study we punctured piglet skin with cannulas commonly used for blood donation and performed histological as well as cytological investigations of the lavage fluid in the cannula to identify superficial skin cells and skin plugs. Histological specimens of the pierced skin showed frayed puncture sites with loosely attached tissue fragments. In the lavage fluid of the cannula, a collection of epidermal cells was found in one of 150 punctures. Our results confirm that the phlebotomy cannula may cause superficial tissue fragments to be punched out of the donor's skin during blood donation. This fact should be taken into account when devising methods to reduce bacterial contamination in blood products.

Transfusion-related exposure to the plasticizer di(2-ethylhexyl)phthalate in patients receiving plateletpheresis concentrates.

BACKGROUND: Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that can leach from medical devices including storage bags for plateletpheresis concentrates (PCs). In this study, the DEHP exposure to patients receiving PCs was determined and several variables were evaluated to reduce DEHP load to PC recipients. STUDY DESIGN AND METHODS: In 12 patients, serum DEHP was assessed before and after PC transfusion. For in vitro investigations, PCs were produced either with donor plasma or with 65 percent additive solution (AS; T-Sol) and stored for 5 days. Washing of PCs was performed according to AABB standards. DEHP levels were determined by gas chromatography-mass spectrometry. RESULTS: Transfusion of PCs led to a significant increase in serum DEHP. DEHP levels in the PCs continuously increased during storage, although the accumulation of DEHP was less in PCs stored in the AS, T-Sol, than when stored in plasma. Storage-related accumulation of DEHP contributed to a major part of the total DEHP load in PCs stored for 5 days. Washing of PCs led to a reduction of DEHP load. CONCLUSION: Recipients of PCs are exposed to DEHP, although the total amount represents only a small percentage of the defined tolerable intake. Reduction of storage time, the storage of PC in T-Sol, or the exchange of the storage medium before transfusion are practicable means to reduce the DEHP load in PC.

Potential approaches to prevent uncommon hemolytic side effects of AB0 antibodies in plasma derivatives.

Since hemolytic reactions in patients after administration of plasma derivatives like immunoglobulins or coagulation factor preparations have been described, titers of anti-A and anti-B-antibodies have to be below defined levels for batch release of these plasma-derived therapeutic products according to the European Pharmacopoeia. We have summarized clinical relevance of AB0 antibodies in plasma derivatives and related legal issues in the European Union, United States of America, and Japan. We have also discussed potential approaches for the prevention of hemolytic side effects with feasible steps in preparation of plasma derivatives, viz., (1) selection of donors, (2) exclusion of "dangerous donors", (3) optimizing ratio of the types of plasma, (4) removal of antibodies, (5) production of blood-group-specific plasma derivatives, (6) rejection of batches of plasma derivatives with high titers of antibodies, and (7) crossmatching before administration. For harmonization of standards for anti-A and anti-B in plasma-derived therapeutics the regulators and manufacturers will have to realistically deal with complex clinical, practical, and economic issues.

Quality of drainage blood: survival of red cells after re-transfusion and content of free hemoglobin and potassium.

Re-transfusion of drainage blood is widely used in orthopedic surgery, but objective evidence of the efficacy of re-transfusion of drainage blood in view of post-transfusion survival of RBC has not been given so far. With this study we wanted to evaluate the efficacy and safety of transfusion of drainage blood collected with HandyVac autotransfusion system. In 7 patients red cells in drainage blood were labeled with biotin and percentage of labeled red cells in circulation were determined immediately after re-transfusion, and during 10 days after surgery. To assess further unwanted side-effects of re-transfusion of drainage blood potassium and free hemoglobin were determined in the collected blood. Ten days after re-transfusion at mean 78.9% of drainage-blood derived RBC were found in circulation. Free hemoglobin in drainage blood ranged from 16.8 to 59.2 mg/dL; potassium in drainage blood ranged from 3.84 to 4.52 mmol/L. Our results suggest that re-transfusion of drainage blood collected with HandyVac autotransfusion system is an efficient procedure that seems to be safe in view of free hemoglobin and potassium in the product.

Impact of manufacturing, irradiation and filtration steps to bacterial contamination of autologous fibrin sealant.

BACKGROUND: Preoperative production of autologous fibrin sealant has become a routine procedure during the last years. As a certain percentage of blood products is contaminated with bacteria, contamination of plasma used for the production of fibrin sealant cannot be excluded. Especially in the orthopaedic setting, application of contaminated fibrin sealant can cause severe infections. MATERIALS AND METHODS: We contaminated plasma with Staphylococcus epidermidis, Corynebacterium striatum, Bacillus subtilis or Escherichia coli and produced fibrin sealant by cryoprecipitation and alcohol precipitation. Additionally, the products were gamma-irradiated at a dose of 30 Gy, frozen at -55 degrees C and filtered through a 0.2 microm filter after thawing. After each preparation step, samples were drawn and numbers of colony forming units were counted after incubation on agar plates. RESULTS: Cryoprecipitation, irradiation, freezing at -55 degrees C, and alcohol precipitation have only little impact on numbers of colony forming units. Filtration through a bacterial filter results in a sterile product. CONCLUSION: Bacteria in plasma as a starting material for production of fibrin sealant survive all routine steps of production, including gamma irradiation and freezing. Filtration of the product through a qualified bacterial filter is the only safe means to provide a sterile product.

Reduction of adverse citrate reactions during autologous large-volume PBPC apheresis by continuous infusion of calcium-gluconate.

BACKGROUND: Citrate-related side effects are common adverse reactions during PBPC apheresis. To reduce the incidence of citrate-related reactions, the effect of a continuous calcium-gluconate infusion on the appearance of hypocalcemic symptoms and on the subjective tolerance toward large-volume leukapheresis (LVL) was tested. STUDY DESIGN AND METHODS: A double-blinded, placebo-controlled trial was carried out in 50 patients undergoing standardized LVL at a median ACD-A ratio of 1.99 mg per kg and minute. Patients were randomly assigned to receive a continuous IV infusion of either saline or calcium-gluconate at a dose of 1.8 mmol calcium per hour. Subjective tolerance toward LVL was determined by standardized rating systems. Further, hormonal and electrolyte changes were monitored to assess the effect of continuous calcium infusion on calcium homeostasis. RESULTS: Continuous IV administration of calcium-gluconate throughout LVL reduced the incidence of citrate-related effects by 65 percent. In patients who developed signs of hypocalcemia, the symptoms were weaker, and less medical intervention was needed to resolve clinical symptoms. The subjective tolerance toward LVL was superior in patients receiving calcium support compared to control patients. Continuous calcium infusion attenuated changes in serum phosphorus compared to patients receiving saline. No differences were observed in the variation of serum potassium and serum magnesium between the control group and the treatment group. The administration of calcium was not associated with technical problems related to the apheresis procedure, neither was any effect of calcium support on the total number of CD34+ cells collected observed. CONCLUSION: These results indicate that continuous support of calcium-gluconate during LVL is an effective means of reducing the incidence of citrate-related symptoms and improving subjective tolerance toward LVL, without affecting the technical performance or the number of CD34+ cells collected.

Donor exposure to the plasticizer di(2-ethylhexyl)phthalate during plateletpheresis.

BACKGROUND: Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that is contained in most PVC devices, including apheresis disposables. Because DEHP can be extracted from apheresis disposables as the blood passes through the apheresis device, DEHP exposure was determined in healthy donors undergoing plateletpheresis performed with commercially available apheresis systems. STUDY DESIGN AND METHODS: The study population consisted of 36 healthy PLT donors undergoing plateletpheresis with either continuous or discontinuous apheresis devices. Serum concentrations of DEHP were determined from peripheral blood obtained before and after plateletpheresis, with gas chromatography-mass spectroscopy. RESULTS: Plateletpheresis performed with standard collection disposables resulted in a median increase of 232 percent of serum DEHP compared to levels before apheresis, corresponding to a total amount of DEHP exposed during a single apheresis of a median of 6.46 (range, 1.8-20.3) microg per kg of body weight. Endogenous levels of triglycerides showed a positive correlation with the amount of DEHP released. Increase in serum DEHP was short-term as serum DEHP rapidly returned to levels obtained before apheresis within 3 hours after completion of the apheresis course. Donor exposure to DEHP led to no variation in liver cell function within 48 hours after plateletpheresis. CONCLUSION: Commercial plateletpheresis disposables release considerable amounts of DEHP during the apheresis procedure, but the total dose of DEHP retained by the donor is within the normal range of DEHP exposure of the general population.

ABO mismatch increases transplant-related morbidity and mortality in patients given nonmyeloablative allogeneic HPC transplantation.

BACKGROUND: ABO mismatch has not been thought to affect the outcome of patients undergoing myeloablative conditioning and allogeneic HPC transplantation. Data on transplant-related complications after ABO-mismatched transplantation after nonmyeloablative conditioning are limited. STUDY DESIGN AND METHODS: Therefore, 40 patients were analyzed after nonmyeloablative conditioning with regard to ABO compatibility. Eleven received a minor and bidirectional and 8 a major ABO-mismatched graft. RESULTS: Four patients had evidence of hemolysis during engraftment, being lethal in one, and three developed pure RBC aplasia. Six patients in the ABO-mismatched group developed thrombotic microangiopathy, and three of them died. ABO-identical and ABO-mismatched patients had a similar incidence of GVHD. Viral infections occurred in both groups in equal shares. Patients with an ABO-mismatch had to be rehospitalized until Day 100 for a median of 19 days versus 0 days in the identical group (p < 0.05). Overall survival was 60 and 57 percent in the ABO-identical and ABO-mismatch groups, respectively. The probability of transplant-related mortality was 0 versus 28 percent in the identical group compared to patients with an ABO mismatch (p < 0.05). The probability of relapse or progression was 76 versus 25 percent in the ABO-identical group compared to the ABO-mismatched group, respectively. CONCLUSION: Significantly more patients with ABO mismatch showed transplant-associated complications and died as a result of transplant-related causes.

Severe immune hemolysis after minor ABO-mismatched allogeneic peripheral blood progenitor cell transplantation occurs more frequently after nonmyeloablative than myeloablative conditioning.

BACKGROUND: Hemolysis as a result of donor-recipient minor ABO mismatching is a complication of allogeneic peripheral blood progenitor cell (PBPC) transplantation (PBPCT). The increased B-lymphocyte content of PBPC grafts and immunosuppressive regimens without methotrexate (MTX) may increase incidence and severity of this event. STUDY DESIGN AND METHODS: A total of 93 patients were analyzed after allogeneic PBPCT. In 25 (27%) cases, minor (n=21) or bidirectional (n=4) ABO mismatching was present. Of these, 15 patients received myeloablative and 10 received nonmyeloablative conditioning regimens. For GVHD, prophylaxis cyclosporin A (CsA) alone (n=2) or CsA with MTX (n=13) was given after myeloablative conditioning and CsA with mycophenolate mofetil (MMF) after nonmyeloablative conditioning (n=10). RESULTS: Hemolysis occurred in 4 out of 25 (16%) patients with minor or bidirectional ABO mismatching only. Three patients underwent nonmyeloablative conditioning and were given CsA with MMF, and one patient underwent myeloablative conditioning and was given CsA alone for GVHD prophylaxis. Hemolysis began 7 to 10 days after transplantation, and donor type alloantibodies were detectable concomitantly with recipients type RBCs. CONCLUSION: Patients receiving minor or bidirectional ABO-mismatched PBPC grafts and GVHD prophylaxis without MTX are at risk of hemolysis. Prophylactic interventions in patients before minor ABO-mismatched PBPCT not receiving MTX should be taken into consideration. Careful monitoring of hemolysis parameters during the first 15 days after PBPCT transplantation is mandatory.