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BACKGROUND: More granulocyte concentrates (GCs) could be produced for more patients from the same donor if apheresis bags were split and stored for longer periods of time. Hence, we tested the hypothesis that splitting and extension of storage of GCs do not impair granulocyte function or viability. STUDY DESIGN AND METHODS: Granulocyte apheresis concentrates were produced using modified fluid gelatin as a separation enhancer, split into two portions, and stored for 24 and 48 h. Granulocyte function, represented by cell migration, reactive oxygen species (ROS) production, and neutrophil extracellular trap formation (NETosis), was measured by live-cell imaging. ROS production, adhesive surface protein expression, and viability were measured by flow cytometry. RESULTS: Splitting had no effect on any of the tested parameters. After 24 h of storage, live-cell imaging showed no significant difference in migration, time to maximum ROS production, time to half-maximum NETosis, viability, or CD11b expression, but ROS production induced by phorbol 12-myristate 13-acetate (PMA) decreased from an initial median fluorescence intensity of 1775-590 artificial units. After 48 h, PMA-induced ROS production, viability, and migration declined, as reflected by decreases in median total distance (119 vs. 63.5 µm) and median Euclidean distance (30.75 vs. 14.3 µm). CONCLUSION: Splitting GC products has no effect on granulocyte viability or function, but extended storage >24 h does compromise granulocyte function. The findings confirm that GCs should be transfused within 24 h of collection. Longer storage cannot be recommended.
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Remoção de Componentes Sanguíneos , Granulócitos , Espécies Reativas de Oxigênio/metabolismo , Fenótipo , Movimento CelularRESUMO
OBJECTIVES: Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCD and bone marrow from an HLA-matched sibling is currently the standard of care. Haploidentical HSCT from a family donor with a TCR αß/CD19 depleted graft (T-haplo) is an increasingly successful alternative, which requires the generation of G-CSF stimulated peripheral stem cell (PBSC) from haploidentical relatives. These sickle cell trait (SCT) donors reported to develop SCD-related complications in conditions of severe stress. METHODS: In this retrospective analysis, we compared the safety and efficacy of PBSC mobilization with a G-CSF intensified mobilization regimen in SCT donors with a conventional G-CSF mobilization regimen in healthy donors. RESULTS: The reported adverse events were similar during intensified G-CSF mobilization, apheresis, and shortly after stem cell apheresis in SCT and control donors. In SCT and control donors, we were able to mobilize high yields of CD34+ stem cells and the harvested CD34+ cell count was comparable with control donors. CONCLUSIONS: Peripheral stem cell mobilization using an intensified G-CSF regimen is safe, and well tolerated among SCT donors. SCT donors are a valid alternative for collection of peripheral CD34+ stem cells for T-cell-depleted haploidentical stem cell transplantation.
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BACKGROUND: Granulocyte transfusions (GT) are used to treat progressive systemic or local infections in prolonged neutropenic patients with antibiotic or antifungal resistance. Granulocytes are most commonly collected from whole blood by apheresis using hydroxyethyl starch (HES) as the red blood cell (RBC) sedimentation agent. This is the first study on the safety and efficacy of transfusing granulocytes collected with modified fluid gelatin (MFG) instead of HES to pediatric patients. METHODS: Clinical data from 46 pediatric and adolescent patients receiving at least one MFG-based granulocyte transfusion and in total 295 granulocyte concentrates from July 2013 to August 2019 at our local university medical center were evaluated retrospectively. RESULTS: Forty-one patients (89%) survived at least 21 days after their last granulocyte transfusion. These survivors had lower CRP values and higher leukocyte counts after GT than non-survivors (mean delta of -5.34 mg/dl vs. -11.99 mg/dl and + 0.62 × 103 /µl vs. +0.18 × 103 /µl of all GT, respectively). The neutrophil corrected count increment (CCI) was 68.72 mm2 /ml in survivors versus 28.00 mm2 /ml in non-survivors. There were no major or severe adverse events. CONCLUSION: This study suggests that modified fluid gelatin is a safe and effective alternative to hydroxyethyl starch for the collection of granulocytes for transfusion to prolonged neutropenic patients with progressive systemic or local infections refractory to antibiotic or antifungal therapy.
Assuntos
Antifúngicos , Neutropenia , Adolescente , Antibacterianos , Criança , Gelatina , Granulócitos , Humanos , Transfusão de Leucócitos , Estudos Retrospectivos , AmidoRESUMO
BACKGROUND: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. METHODS: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. FINDINGS: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. INTERPRETATION: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. FUNDING: The 7th EU Framework Programme.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Rejeição de Enxerto/prevenção & controle , Terapia de Imunossupressão/métodos , Imunossupressores/uso terapêutico , Transplante de Rim , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Células Dendríticas/imunologia , Rejeição de Enxerto/imunologia , Humanos , Terapia de Imunossupressão/efeitos adversos , Macrófagos/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Extracorporeal photopheresis (ECP), an apheresis-based therapy for various immunological diseases, works mainly by inducing apoptosis in lymphocytes. Several factors influence the efficacy of ECP with the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet light A (UVA). This study aimed to optimize treatment by varying the 8-MOP starting concentration and the cell suspension medium. MATERIALS AND METHODS: All patients (n = 13) included in this study received photopheresis as medically indicated. Cells collected with a Spectra Optia apheresis system were suspended in plasma or physiological saline (NaCl) and incubated with 200 ng/ml versus 340 ng/ml photosensitizer before UVA irradiation (Macogenic G2 or UVA PIT system). Lymphocyte apoptosis and caspase activity were analyzed by flow cytometry and fluorimetry, and residual 8-methoxypsoralen concentrations by liquid chromatography-mass spectrometry. RESULTS: Raising the 8-MOP starting concentration significantly increased lymphocyte apoptosis, with values of 22% versus 35% (plasma) and 28%-46% (NaCl) at 24 h post-ECP and 37% versus 86% (plasma) and 74% versus 97% (NaCl) at 48 h for 200 ng/ml versus 340 ng/ml. Pre-transfusion residual 8-MOP levels were 168 ng/ml (plasma) and 162 ng/ml (NaCl) versus 290 ng/ml (plasma) and 266 ng/ml (NaCl) for the lower versus higher dose, respectively. DISCUSSION: Hence, 8-MOP concentration influences the efficacy of photopheresis as lymphocyte apoptosis rates were significantly higher with the higher starting concentration and with NaCl versus plasma. This indicates that increased 8-MOP starting doses and saline as additional suspension medium could help in improving ECP's efficacy.
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Apoptose/efeitos dos fármacos , Metoxaleno/uso terapêutico , Fotoferese/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Solução Salina/uso terapêutico , Adulto , Idoso , Apoptose/efeitos da radiação , Feminino , Doença Enxerto-Hospedeiro/terapia , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Raios UltravioletaRESUMO
BACKGROUND: Plateletpheresis using a leukocyte reduction system (LRS) traps donor WBCs in the LRS chamber, which may lead to lymphopenia, especially in frequent plateletpheresis donors. It seems plausible that this might cause adverse effects. However, current knowledge about potential confounders and donor health impacts is incomplete. DONORS AND METHODS: Recent platelet donors and donations collected at University Hospital Regensburg from 2016 to 2019 using the Terumo BCT Trima Accel LRS system were retrospectively analyzed and compared with historical platelet donors and donations collected mainly with Fresenius Kabi Amicus non-LRS system from 2010 to 2013. Additionally, recent donors were prospectively surveyed using a health-related topics questionnaire. RESULTS: Analysis of 819 recent donors with 11,254 blood counts and 1464 questionnaires and 1011 historical donors with 12,848 blood counts revealed that increased annual platelet donation frequencies were associated with decreased lymphocyte counts in both groups. Median lymphocyte counts in recent donors with no versus ≥24 previous annual donations declined from 2.0 to 1.2 × 103 /µL (p < 2.2 × 10-16 ), and those in historical donors with no versus ≥24 previous annual donations decreased from 2.0 to 1.5 × 103 /µL (p = 6 × 10-4 ), respectively. The questionnaire results showed that donation frequency and lymphopenia were not associated with upper respiratory tract infection (URTI) incidence or duration, but platelet donors who concomitantly donated granulocytes had significantly shorter URTI durations than those who did not (p = .008). CONCLUSION: This study confirmed that plateletpheresis-associated lymphopenia occurs in LRS and to a lesser degree in non-LRS platelet donors, but revealed no evidence of a negative impact on donor health.
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Linfopenia , Plaquetoferese , Doadores de Sangue , Humanos , Linfopenia/epidemiologia , Linfopenia/etiologia , Contagem de Plaquetas , Plaquetoferese/efeitos adversos , Plaquetoferese/métodos , Estudos RetrospectivosRESUMO
OBJECTIVES: This validation study investigated a flow cytometric apoptosis assay according to good manufacturing practice (GMP). BACKGROUND: Extracorporeal photopheresis (ECP) is a treatment for various immunological diseases and cutaneous T-cell lymphomas. It is based on the induction of apoptosis by 8-methoxypsoralene and ultraviolet A light. The quantification of apoptosis is therefore essential for ECP improvements. However, despite numerous publications on apoptosis, validated technical details are lacking. METHODS AND MATERIALS: Mononuclear cells were collected by apheresis and treated by ECP or camptothecin. Samples taken before and after ECP were cultured for 24, 48 and 72 h and analysed for apoptosis and viability of T cells and monocytes by flow cytometry with Annexin V and 7-AAD staining. Accuracy of the assay, intra- and inter-assay precision and the pre-analytical and analytical stability of the analytes were the investigated parameters. RESULTS: Our data indicate that the median intra- and inter-assay precision coefficient of variation for T cells was 3.86% and 4.80%, respectively. Pre-analytical stability of T cells and monocytes was ensured during short-term storage for up to 2 h on ice. After staining, analytical stability was limited to 30 min, likely because of ongoing apoptosis and loss of monocytes due to plastic adhesion. CONCLUSION: The results of this validation study show that the assay is GMP-compliant and that its reliability, accuracy and precision are acceptable. While pre-analytical stability of the cells was compatible with on-site procedures, our analytical stability data indicate that this assay is not suited for batch mode analysis of ECP products.
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Apoptose , Citometria de Fluxo/métodos , Monócitos/fisiologia , Fotoferese , Linfócitos T/fisiologia , Feminino , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Extracorporeal photopheresis (ECP) is an immunosuppressive treatment that involves leukocyte apheresis, psoralen and UV light treatment, and subsequent reinfusion. Patients treated with ECP are usually immunosuppressed. Bacterial contamination therefore poses a much unwanted risk, but incidence data are lacking. PATIENTS AND METHODS: We screened all 1922 consecutive ECP procedures scheduled within a roughly 3-year period for eligibility. Those with missing data on ECP method (inline or offline) or type of venous access (peripheral or central) were excluded. ECPs with complete aerobic and anaerobic microbial testing of baseline patient blood samples (n = 1637) and of ECP cell concentrates (n = 1814) were included in the analysis. RESULTS: A test for microbial contamination was positive for 1.82% of the cell concentrates, with central venous access was the most significant risk factor for the contamination (odds ratio = 19). Patient blood samples were positive in 3.85% of cases, but no patients became septic. Staphylococcus spp. were most abundant, and products with bacterial contamination did not cause side effects after reinfusion. There were no significant differences in contamination rates between inline and offline ECP. CONCLUSION: These findings stress the importance of sterile procedures and the benefits of using peripheral over central venous access for reducing the risk of bacterial contamination in ECP.
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Cateteres Venosos Centrais , Fotoferese/efeitos adversos , Infecções Estafilocócicas , Staphylococcus , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/etiologiaRESUMO
BACKGROUND: Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy for various diseases. Autologous leukocytes are collected, photoactivated with a photosensitizer (8-methoxypsoralen, 8-MOP) and UVA light, and subsequently reinfused back to the patient. Leukapheresis and UVA irradiation systems can be integrated into one device (inline) or handled by two separate devices (offline). ECP works via intercalation of 8-MOP into DNA helices and UVA-based interactions to inhibit DNA replication. 8-MOP is known to adhere to plastic materials, which might reduce its availability for intercalation. In the present study we examined the bioavailability of 8-MOP when different plastic materials and solvents are used as matrices. METHODS: Varying amounts of shredded ethylene vinyl acetate (EVA) and polyvinylchloride (PVC) from the MacoGenic irradiation bag (EVA1), UVA PIT irradiation bag (EVA2), UVA PIT recirculation bag (PVC A) and UVA PIT tubing (PVC B) by MacoPharma and PIT Medical Systems, respectively, were incubated with 200 ng mL-1 8-MOP dissolved in diisopropyl ether (DIPE) plus toluene 90/10 vol%, deionized water or plasma. After 2 h 8-MOP concentrations were determined by GC-MS. RESULTS: After incubation, 8-MOP concentrations varied depending on the amount and type of plastic (PVC > EVA) and solvent (water > plasma > DIPE/toluene). Absorption to 200 mg EVA1 or EVA2 resulted in 8-MOP concentrations of 57% or 32% in water, 91% or 80% in plasma, and 93% or 92% in DIPE/toluene, while 200 mg PVC A and PVC B yielded recovery rates of 26% and 10% in water, 76% and 75% in plasma, and 55% and 30% in DIPE/toluene, respectively. Remaining 8-MOP differed significantly between container materials (EVA vs. PVC; p < 0.022) but not manufacturers (MacoPharma vs. PIT Medical Systems). CONCLUSION: Absorption loss of 8-MOP depends on the type of plastic and solvent and is more pronounced with water than with plasma. As the DNA binding effect of 8-MOP is dose-dependent, ECP starting doses should be adjusted to ensure that a sufficient concentration of free bioavailable 8-MOP is present during UV irradiation.
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Metoxaleno/análise , Fotoferese , Fármacos Fotossensibilizantes/análise , Éteres/química , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Cloreto de Polivinila/química , Polivinil/química , Tolueno/química , Raios UltravioletaRESUMO
BACKGROUND: Patients with neutropenia or granulocyte dysfunction may require granulocyte transfusions for adequate immune restoration. High-molecular-weight hydroxyethyl starch (HES) is the most commonly used sedimentation agent to enhance granulocyte collection efficiency. However, authorities recently restricted the use of HES due to its unfavorable risk-benefit profile. As modified fluid gelatin (MFG) is already used as an alternative sedimentation agent, we tested the hypothesis that MFG is not inferior to HES in terms of the functionality and viability of granulocytes. STUDY DESIGN AND METHODS: Granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15%, or 30% MFG (Gelafundin) or HES (Hespan), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ROS) production, neutrophil extracellular trap formation (NETosis), antigen expression, and viability were subsequently investigated in vitro. RESULTS: Relative to the controls, all three concentrations of HES compared to only 15% and 30% MFG lowered migration distances, and the 15% and 30% concentrations of both sedimentation agents reduced track straightness. HES resulted in lower CD11b expression and higher CD62L expression compared to MFG and the controls, whereas the differences for CD66b did not reach statistical significance. No significant differences in the timing of ROS production or NETosis, or in neutrophil viability or respiratory burst were observed. CONCLUSION: These results indicate that MFG is not inferior to HES in terms of granulocyte function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with HES.
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Gelatina/farmacologia , Granulócitos/efeitos dos fármacos , Derivados de Hidroxietil Amido/farmacologia , Adulto , Movimento Celular/efeitos dos fármacos , Separação Celular/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Granulócitos/citologia , Granulócitos/fisiologia , Humanos , Leucaférese/métodos , Masculino , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Adulto JovemRESUMO
BACKGROUND: Granulocyte apheresis requires a sedimentation agent. Usually, hydroxyethyl starch (HES) is administered to donors for this purpose and, as granulocyte concentrate (GC) ingredient, also to patients. Authorities recently recommended suspending market authorizations for starch-containing products due to side effects. Therefore, we tested the efficacy of modified fluid gelatin (MFG, Gelafundin 4%) versus hetastarch (Hespan) for GC apheresis. STUDY DESIGN AND METHODS: This retrospective matched-pair analysis compared MFG- and hetastarch-derived GCs. Each group consisted of 15 unrelated male donors mobilized with dexamethasone and granulocyte-colony-stimulating factor for apheresis on 1 or 2 days with the COBE Spectra's PMN program. RESULTS: In each group, 24 GCs were collected from 15 male donors and analyzed. None of the HES-derived products, but two of the MFG-derived products (8.3%), had aggregates and could not be used. The HES-derived products had significantly higher neutrophil counts on the first day (7.7 × 1010 /unit vs. 4.0 × 1010 /unit; p = 0.00005) as well as second day of apheresis (4.0 × 1010 /unit vs. 1.1 × 1010 /unit; p = 0.0002). Median white blood cell collection efficacies were lower with MFG than with HES on Day 1 (24% vs. 43%) and Day 2 (15% vs. 37%). Twenty-one percent of the MFG-derived products had less than 1 × 1010 granulocytes. CONCLUSION: These results indicate that granulocyte apheresis is feasible with MFG as well as with hetastarch and that the latter is superior for GC production, if used in the same dosage. In addition, aggregates in GC from the COBE Spectra were observed in the MFG group but not in the hetastarch group.
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Doadores de Sangue , Dexametasona/administração & dosagem , Gelatina/farmacologia , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Derivados de Hidroxietil Amido/farmacologia , Leucaférese/métodos , Adulto , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
BACKGROUND: Extracorporeal photopheresis (ECP) is an efficient method to treat various autoimmune diseases, cutaneous T-cell lymphoma, and graft-versus-host disease. It is based on the ex vivo inactivation of lymphocytes by 8-methoxypsoralen (8-MOP)/UV light treatment. Despite the adhesive, lipophilic nature of 8-MOP, no quality control is established for the ECP procedure. METHODS: We developed a sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to monitor residual 8-MOP concentration after UVA irradiation in the whole blood supernatant after acetonitrile precipitation. RESULTS: The preanalytical stability of 8-MOP exceeded 7 days, allowing batch mode analysis. Linearity was determined with R2 above 0.99. The 8-MOP concentrations decreased exponentially after UV exposure, with decay constants of 0.0259 in plasma and 0.0528 in saline. The recovery of 8-MOP in photopheresates was about 68%, indicating binding to DNA as well as to plastic structures. UVA induced no 8-MOP fragmentation, but caused self-adducts under extreme conditions (10-fold UV dosage). CONCLUSIONS: Detection of 8-MOP proved to be feasible and demonstrated that the doses were in the pharmaceutically active range.
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Análise Química do Sangue/métodos , Metoxaleno/análise , Fotoferese , Fármacos Fotossensibilizantes/análise , Espectrometria de Massas em Tandem , Adolescente , Adulto , Criança , Cromatografia Líquida de Alta Pressão , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Raios Ultravioleta , Adulto JovemRESUMO
Platelet-rich plasma is a current subject of studies on chronic wound healing therapy due to possible pro-angiogenic effects. Microvascular compromise represents the major component in radiogenic wound healing complications. The effects of platelet-rich plasma on irradiated cells of the cutaneous wound healing process are poorly understood so far. In this study, the interaction of endothelial cells and adipose-derived stem cells in conjunction with treatment with platelet-rich plasma is investigated in the context of radiation effects. Therefore, the expression of surface-marker CD90 and CD31 was determined. Moreover, cell proliferation and viability after external radiation was analyzed with and without treatment by platelet-rich plasma.
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Plasma Rico em Plaquetas , Lesões por Radiação/patologia , Lesões por Radiação/terapia , Cicatrização , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Biomarcadores , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Humanos , Imunofenotipagem , Radioterapia/efeitos adversosRESUMO
BACKGROUND: Extracorporeal photopheresis (ECP) is a therapeutic technique that combines leukapheresis and ultraviolet (UV)A irradiation of the leukapheresate after 8-methoxypsoralen treatment with subsequent retransfusion. It can be achieved with a single device (online) or by combining an apheresis machine with a separate UVA light source (offline). The comparability of both established methods is unknown. STUDY DESIGN AND METHODS: In a prospective setting, four ECP systems were evaluated: one with integrated UVA irradiation for online ECP (Therakos) and three with external UVA irradiation for offline ECP (Amicus, Optia, and Cobe Spectra). Apheresis variables and cell counts were determined by methods including flow cytometry. RESULTS: The duration of apheresis ranged from 120 minutes (Amicus, Optia) to 275 minutes (Therakos). Mononuclear cell (MNC) counts in the treatment bags were comparable between offline ECP methods (median, 57 × 108 - 66 × 108 ) and lower for online ECP (14 × 108 ). CD16+ monocytes were abundant in online ECP (82%) but rarer in offline ECP (median, 14% - 19%). Hematocrit ranged from 0.1% (Therakos) to 8% (Amicus). There were no side effects in any patients. DISCUSSION: All offline ECP systems studied yielded comparable cellular compositions and highly enriched populations of MNCs. In contrast, white blood cells from online ECP displayed enrichment of nonclassical monocytes. The relevance of these findings is unknown as there is no established biomarker to predict the therapeutic efficacy of these procedures.
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Fotoferese/instrumentação , Adolescente , Adulto , Idoso , Remoção de Componentes Sanguíneos , Contagem de Células , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Sistemas On-Line/normas , Fotoferese/métodos , Estudos Prospectivos , Raios Ultravioleta , Adulto JovemRESUMO
ABO blood group antigen incompatibility (ABO mismatch) is not an obstacle to allogeneic stem cell transplantation (allo-SCT). However, the impact on clinical outcome after allo-SCT remains controversial. We analyzed 512 patients after allogeneic peripheral blood SCT (allo-PBSCT) for an association of ABO mismatch with transfusion requirements, myeloid and platelet engraftment, the incidence of GvHD, relapse, transplant-related mortality (TRM), and overall survival (OS). A total of 260 patients underwent ABO-mismatched transplantation and the control group consisted of 252 patients with ABO-matched allo-PBSCT. We found a significant association between major-0 ABO mismatch (group 0 recipient/group A, B, or AB donor) and increased red blood cell (RBC) and platelet transfusion requirements (both P<.001) as well as delayed platelet engraftment (P<.001). Minor-A (group A recipient/group 0 donor) and minor-AB (group AB recipient/group 0, A, or B donor) ABO mismatch was significantly associated with an increased TRM after allo-PBSCT (P=.001 and P=.02). In multivariate analysis performed using Cox regression, minor ABO mismatch appeared as independent risk factor for TRM after allo-PBSCT. No association was found for ABO mismatch with the incidence of GvHD, relapse, and OS. Our results suggest that ABO blood group mismatch has a significant impact on the outcome and that minor-A and minor-AB ABO mismatch represents a risk factor for increased TRM after allo-PBSCT.
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Sistema ABO de Grupos Sanguíneos/imunologia , Incompatibilidade de Grupos Sanguíneos/complicações , Transplante de Células-Tronco de Sangue Periférico , Adolescente , Adulto , Idoso , Incompatibilidade de Grupos Sanguíneos/imunologia , Transfusão de Sangue/estatística & dados numéricos , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/epidemiologia , Doença Enxerto-Hospedeiro/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Transplante de Células-Tronco de Sangue Periférico/mortalidade , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Fatores de Risco , Transplante Homólogo , Adulto JovemRESUMO
Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 10(9) particles/ml for 1 h incorporated an average of 3.39 × 10(8) Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 10(5) counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific (154)Sm-tagged anti-HLA-DR or (174)Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.
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Ouro/farmacologia , Lasers , Antígenos Comuns de Leucócito/imunologia , Pulmão , Espectrometria de Massas/instrumentação , Monócitos , Animais , Anticorpos Monoclonais Murinos , Xenoenxertos , Humanos , Antígenos Comuns de Leucócito/química , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos NOD , Monócitos/citologia , Monócitos/imunologia , Monócitos/transplanteRESUMO
Administering immunoregulatory cells as medicinal agents is a revolutionary approach to the treatment of immunologically mediated diseases. Isolating, propagating, and modifying cells before applying them to patients allows complementation of specific cellular functions, which opens astonishing new possibilities for gain-of-function antigen-specific treatments in autoimmunity, chronic inflammatory disorders, and transplantation. This critical review presents a systematic assessment of the potential clinical risks posed by cell-based immunotherapy, focusing on treatment of renal transplant recipients with regulatory macrophages as a concrete example.
Assuntos
Imunoterapia/métodos , Rejeição de Enxerto , Humanos , Imunossupressores/uso terapêutico , Transplante de RimRESUMO
BACKGROUND: Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC-EVs), and plasma. STUDY DESIGN AND METHODS: Fifty PLCs from healthy individuals were stored under standard blood banking conditions for 5 days. Changes in cholesterol, glycerophospholipid, and sphingolipid species were analyzed in PLTs, PLC-EVs, and plasma by mass spectrometry and metabolic labeling. Immunoblots were performed to compare PLT and PLC-EV protein expression. RESULTS: During 5 days, PLTs transferred glycerophospholipids, cholesterol, and sphingolipids to newly formed PLC-EVs, which increased corresponding lipids by 30%. Stored PLTs significantly increased ceramide (Cer; +53%) and decreased sphingosine-1-phosphate (-53%), shifting sphingolipid metabolism toward Cer. In contrast, plasma accumulated minor sphingolipids. Compared to PLTs, fresh PLC-EVs were enriched in lysophosphatidic acid (60-fold) and during storage showed significant increases in cholesterol, sphingomyelin, dihydrosphingomyelin, plasmalogen, and lysophosphatidylcholine species, as well as accumulation of apolipoproteins A-I, E, and J/clusterin. CONCLUSION: This is the first detailed analysis of lipid species in all PLC components during PLC storage, which might reflect mechanisms active during in vivo PLT senescence. Stored PLTs reduce minor sphingolipids and shift sphingolipid metabolism toward Cer, whereas in the plasma fraction minor sphingolipids increase. The composition of PLC-EVs resembles that of lipid rafts and confirms their role as carriers of bioactive molecules and master regulators in vascular disease.
Assuntos
Plaquetas/metabolismo , Preservação de Sangue , Glicerofosfolipídeos/metabolismo , Vesículas Secretórias/metabolismo , Esfingolipídeos/metabolismo , Adulto , Transporte Biológico , Plaquetas/fisiologia , Preservação de Sangue/métodos , Senescência Celular/fisiologia , Espaço Extracelular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plaquetoferese , Adulto JovemRESUMO
Background and Aims: Platelets are prone to activation from handling; they are therefore transported as gently as possible, most commonly by courier. Speedier methods like pneumatic tube system (PTS) transport could improve patient care but may subject platelets to mechanical stress. To test the impact of mechanical stress caused by transport, we compared a PTS with a conveyor box and courier transport on apheresis platelet function. Methods: Fourteen apheresis platelet concentrate triple donations were analyzed by light transmission aggregometry (LTA), rotational thrombelastometry (ROTEM), and flow cytometry before and after indoor transport over 800 m by PTS, conveyor, and courier, respectively, while recording shocks and vibrations with a high-frequency acceleration data logger. Shock index scores were calculated as shock intensity (g-force) times frequency. Results: The shock index was 81 for courier, 6279 for conveyor, and 9075 for PTS. Flow cytometry revealed no significant difference in platelet surface expression of CD62p before (16%) and after transport via courier (15%), conveyor (14%), or PTS (16%). LTA with adenosine phosphate and thrombin receptor-activating peptide-6 resulted in comparable platelet aggregation for courier, conveyor, and PTS. ROTEM assays showed no relevant differences in coagulation time, clot formation time, and maximum clot firmness between transport modes. Conclusion: Though the mechanical challenge was smallest with courier transport, there were no significant differences in platelet activation or aggregation between the three transport modes. These data contradict restrictions on the use of PTSs for platelet concentrate transport.
RESUMO
BACKGROUND: Orthotopic liver transplantation (OLT) is the treatment option for early-stage hepatocellular carcinoma (HCC). OLT is often associated with high blood loss, requiring blood transfusion. Retransfusion of autologous blood is a key part of blood conservation. There are, however, concerns that the retransfusion of salvaged blood might cause the spread of cancer cells and induce metastasis. Irradiation of salvaged blood before retransfusion eliminates viable cancer cells. Here, we analyzed the incidence of tumor recurrence in patients with HCC undergoing OLT who received irradiated cell-salvaged blood during transplant surgery. METHODS: We retrospectively analyzed patients undergoing OLT for HCC between 2002 and 2018 at our center. We compared the tumour recurrence in patients who received no retransfusion of autologous blood with patients who received autologous blood with or without preceding irradiation of the blood. RESULTS: Fifty-one (40 male, 11 female) patients were included in the analysis; 10 patients developed tumor recurrence within a time period of 2.45 ± 2.0 years. Statistical analysis revealed that there was no significant difference in tumor recurrence between patients who received autologous blood with or without irradiation. CONCLUSION: Intraoperative transfusion of cell-salvaged blood did not increase tumor recurrence rates. Cell salvage should be used in liver transplantation of HCC patients as part of a blood conservation strategy. The effect of blood irradiation on tumor recurrence could not be definitively evaluated.