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ABSTRACT: BACKGROUND: Platelet-rich plasma consists of platelets concentrated in a small volume of plasma and constitutes a reservoir of bio-modulators potentially useful in tissue repair. The amounts of bio-modulators detectable in platelet-rich plasma prepared with various commercial or "in house" methods have been reported, but virtually all the analyses described have been performed on platelet-rich plasma derived from healthy donors. Since leucocyte contamination is technically unavoidable, we investigated whether platelet-rich plasma prepared from patients could contain different amounts of bio-modulators because of a possible activated status of the leucocytes. MATERIALS AND METHODS: We evaluated platelet-rich plasma prepared with three different techniques (the commercial Vivostat and Biomet recover GPS II systems and an "in house" method) starting from whole blood from healthy donors and patients. Specifically, we compared the levels of sHLA-I, sFasL, platelet-derived growth factor, transforming growth factors-beta and vascular endothelial growth factor in the platelet-rich plasma releasates according to the method of preparation and to the immune system activation status of the subjects. RESULTS: With the exception of sHLA-I levels, no differences were found in the surrogate indices of lymphocyte activation between healthy donors and patients. No significant differences were found in sHLA-I, sFasL, platelet-derived growth factor, transforming growth factors-beta and vascular endothelial growth factor levels detectable in platelet-rich plasma produced with the three different methods in either healthy donors or patients. DISCUSSION: On the whole our findings indicate that the overall content of bio-modulators in autologous platelet-rich plasma is not influenced by T-lymphocyte activation status, at least in patients with uncomplicated femoral fractures. The amounts of sFasL and sHLA-I detected in all the platelet-rich plasma releasates studied were very small, far below the amounts detectable in all clinically available blood derivatives and absolutely insufficient to induce sHLA-I and/or sFasL mediated immunomodulation.
Blood transfusion = Trasfusione del sangue 01/2013; · 2.10 Impact Factor
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ABSTRACT: BACKGROUND: The cause of transfusion-related immunomodulation (TRIM) has proved tantalisingly elusive. An ever-growing body of evidence indicates that the infusion of large amounts of soluble and cell-associated antigens into a recipient can somehow induce TRIM. One soluble molecule that has been implicated in TRIM is soluble human leucocyte antigen I (sHLA-I). However, patients infused with large amounts of sHLA-I do not always and unambiguously experience TRIM. As soluble CD8 (sCD8) molecules have been shown to capable of binding membrane and soluble HLA-I molecules, we focused on sCD8 as a possible modulator of sHLA-I-mediated TRIM. MATERIAL AND METHODS: To this aim we compared the up-regulation of circulating sCD8 in plasma from patients suffering from the same pathology, but chronically transfused with two different blood derivatives: pre- and post-storage leucodepleted red blood cells which contain low and high levels of contaminating sHLA-I, respectively. RESULTS: Significantly larger amounts of sCD8 circulating molecules were detectable in the plasma of patients transfused with post-storage leucodepleted red blood cells whose supernatants contained significantly larger amounts of sHLA-I contaminating molecules. CONCLUSION: With the limitation of indirect evidence, this report introduces a new facet of the bioactivity of sCD8 as a possible modulator of sHLA-I-mediated TRIM.
Blood transfusion = Trasfusione del sangue 12/2012; · 2.10 Impact Factor
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ABSTRACT: Multiple target molecular monitoring of minimal residual disease in neuroblastoma (NB) patients may increase sensitivity and overcome tumor heterogeneity. However, multiple target analysis is costly and time consuming, thus improvement with respect to single target monitoring needs to be achieved.
Italian patients with localized NB were evaluated at diagnosis for TH, GD2-s, DDC, DCX, ELAV-4, STX, and Phox2b mRNA expressions. Patients with metastatic NB were tested as positive controls, together with NB primary tumors and cell lines, while healthy donors were tested as negative controls.
All NB-related markers but Phox2b were expressed in healthy donors, and in a high percentage of patients with localized NB without association with clinical events. The introduction of cut-off levels increased marker specificity, although the percentage of positive results was only slightly modified. While TH positivity in PB samples significantly associated with a worse prognosis, a paradox association was found for GD2-s mRNA expression. No correlation and agreement between quantitative and qualitative results obtained with the two assays were found. In the set of samples tested for all markers, no pattern of expression was found to be associated with a specific clinical situation.
These findings suggest that positive molecular results may not reflect the presence of disease, and that correlation among different markers is small in condition of low tumor burden. Thus, to reduce cost and amount of precious samples, in addition to TH, whose prognostic value was confirmed, only Phox2b warrants further evaluation in multi-center, prospective studies for high risk patients.
Pediatric Blood & Cancer 01/2011; 58(1):43-9. · 1.89 Impact Factor
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ABSTRACT: Since there is no validated assay to monitor disease in children with neuroblastoma (NB), we tested whether NB specific cell-free RNA could be detected in their plasma samples. Moreover, with the aim of reducing patients' discomfort, we compared this assay to a recently standardized procedure that uses a larger amount of whole blood.
Using conditions that excluded RNA recovery from contaminating tumor cells, the total amount of cell-free RNA present in healthy children and patients with NB was quantified. Expression of tyrosine hydroxylase (TH) was assayed by quantitative RT-PCR.
In patients with NB the amount of cell-free RNA was higher than in healthy children. However, it was less and more degraded than in healthy adults. The median amount of cell-free RNA that was reverse transcribed, measured through the use of standard curves for reference genes, was 0.03 (range 0-30) pg of input RNA, that is, always less than 1/10,000 of that reverse transcribed from total RNA extracted from whole cells. Despite the presence of disease and the positive results obtained with RNA extracted from peripheral blood cells, few cell-free RNA samples tested positive by the TH assay. Similar results were obtained also with TH primers specifically designed to amplify 50 bp RNA fragments.
These findings suggest that for monitoring disease status detection of cell-free tumor-specific RNAs in patients with NB is not a reliable alternative to whole cell RNA.
Pediatric Blood & Cancer 07/2010; 54(7):897-903. · 1.89 Impact Factor
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ABSTRACT: Numerous mechanisms have been proposed to explain the beneficial action of intravenous immune globulin (IVIG) in autoimmune and systemic inflammatory disorders; among others, they could decrease pro-inflammatory cytokine levels and also induce anti-inflammatory cytokines.
Ex vivo analysis of cells from ten IVIG recipients showed significant increase of IL-10 mRNA and intra-cellular IL-10 molecules in both leukotypes.
In vitro comparable results were obtained incubating CD8(+) T lymphocytes and neutrophils from healthy donors with IVIG. sHLA-I and/or sFasL immunodepletion abolished IL-10 modulation. Co-culture with contaminant-free IgM or MabThera did not exert any mRNA modulation. Finally, IgM or MabThera plus purified sHLA-I molecules enhanced IL-10-mRNA in both leukotypes to levels comparable to those obtained with IVIG incubation.
As IVIG infusion involves administration of soluble contaminants, these data consent to speculate that IVIG might modulate IL-10 via the immunomodulatory activities of sHLA-I contaminant molecules inducing transcriptional and post-transcriptional modulation of IL-10 in CD8(+) T lymphocytes and neutrophils.
Journal of Clinical Immunology 05/2010; 30(3):384-92. · 3.08 Impact Factor
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ABSTRACT: A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures.
Journal of immunological methods 02/2010; 355(1-2):68-75. · 2.35 Impact Factor
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ABSTRACT: Administration of pathogen-specific T-cell lines can reconstitute the cellular immune function of immunocompromised patients. Selection and expansion of specific T cells for reinfusion pose unique challenges owing to the fact that good manufacturing procedures must be implemented. Cytokine secretion-based methods can identify and select specific T cells. We showed here that it is possible to combine this method with procedures for cell handling performed in a sealed, unbreached system from start to end. Peripheral blood mononuclear cells, obtained from blood samples and processed in a sealed system, were stimulated in Teflon bags with a library of selected CD4 and CD8 peptides derived from the immunodominant cytomegalovirus protein pp65. The stimulated T cells were labeled with reagents for interferon-gamma surface capture and selected on a magnetic column using a sealed system connected to the Teflon bags. Elution and final expansion were also performed with an unbreached protocol with preservation of sterility even if the steps were run on the bench top. Expanded cells exhibited the appropriate functions. The use of this unbreached procedure proves that safety of cellular products generated in a good manufacturing procedures facility can be further improved. Similar sealed protocols can also be applied for T-cell therapies directed against tumor antigens.
Journal of immunotherapy (Hagerstown, Md.: 1997) 10/2008; 31(8):762-70. · 3.20 Impact Factor
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ABSTRACT: Drug-induced immune hemolytic anemia (DIIHA) is a well-known complication of drug treatment. Sensitization can occur, due to interaction of the drug and/or its metabolites with cells of the immune system, after the first drug administration, while the hemolytic crisis generally occurs after repeated administration of a drug. This event occurred in the case described here of acute hemolysis due to the administration of corticosteroids.
To define the etiopathogenesis of the hemolytic crisis, immunohematologic screening and specific tests were performed to identify antibodies against a possible drug-red cell (RBC) complex and circulating drug-anti-drug antibody immune complexes. Six drugs administered to the patient were tested and results were confirmed by test repetition using other types of corticosteroids.
Indirect antiglobulin test performed with the patient's serum sample on 22 RBC samples from commercial panels was strongly positive, while it was negative on RBCs from ABO-compatible donors. The same test repeated on commercial RBCs after washing was negative. Specific tests were negative for five of the six tested drugs, while RBCs incubated with hydrocortisone strongly reacted with the patient's serum. The same tests performed using other types of corticosteroids confirmed a reaction with the same positivity score on all tested molecules.
The positive reaction observed each time the patient's serum sample was incubated with RBCs in the presence of corticosteroids suggested that the triggering cause of hemolysis was an immune-mediated mechanism and the drug responsible for DIIHA was hydrocortisone.
Transfusion 07/2008; 48(9):1925-9. · 3.22 Impact Factor
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Blood transfusion = Trasfusione del sangue 01/2008; 6(1):46-50. · 2.10 Impact Factor
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ABSTRACT: The monitoring of near miss errors, in other words events that cannot be classified as substantial errors, but whose occurrence suggests that there is probably a critical point in a working procedure, can be useful in order to prevent these 'almost errors' from occurring again or to prevent them evolving into 'relevant errors'.
The methods for picking up and studying near miss errors use widely tested systems that have recently also been applied to medicine. These systems are based on the process of identifying the risk through spontaneous notifications of events (incident reporting). In our Service of Immunohaematology and Transfusion Medicine (SIMT) these reports were assessed using root cause analysis, allowing us to introduce corrective actions to eliminate or reduce the risk.
We report the distribution, type and frequency of near miss errors, divided according to the stage of the working procedure in which they occurred, and for each of them describe the possible causes and corrective actions identified. We show how the possibility of an error, with potentially harmful consequences for the patient, is present throughout the whole transfusion chain. Near miss errors in Transfusion Medicine can be included in the wider field of 'clinical risk, a problem that concerns not only SIMT, but also numerous other sectors of health care.
The instruments identified through this study can lower the threshold of clinical risk in a Transfusion Service.
Blood transfusion = Trasfusione del sangue 12/2007; 5(4):210-6. · 2.10 Impact Factor
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ABSTRACT: Since 2002, Liguria has been part of the Interregional Agreement on Plasma Derivatives (AIP) stipulated among some Regions of north Italy with the aim of contributing to self-sufficiency of the interregional system through exchanges between the facilities lacking products and those with an excess. In Liguria , the management of plasma derivates is entrusted to the Regional Centre for Co-ordination and Compensation (CRCC) which, with strategies of compensation, tries to guarantee that the needs for plasma derivates are covered in the hospitals in its territory. The Services of Immunohaematology and Transfusion Medicine (SIMT) have a goal of increasing the production of plasma in order to participate actively in achieving regional self-sufficiency.
The SIMT of the G. Gaslini Institute introduced some strategies aimed at reaching this goal. The increase in the number of donations made with a cell separator, the introduction of multicomponent donations of plasma and platelets and the collection of high concentration platelet concentrates led to a considerable increase category A plasma sent for fractioning. Finally, the implementation of shared guidelines on the use of blood components enabled the clinical use of the plasma collected to be kept under control.
The analysis of the trends of consumption of the most widely used plasma derivatives showed an increase in the overall demands, which can be attributed to the paediatric focus of our hospital and to its highly specialised wards. ON THE BASIS OF THE INDUSTRIAL TECHNICAL YIELD, IT WAS POSSIBLE TO CALCULATE THE THEORETICAL COVERAGE OF THE REQUIREMENTS FOR PLASMA: this highlighted a better theoretical coverage for albumin but a shortfall of intravenous immunoglobulins. The amount of plasma necessary to meet the theoretical needs was calculated for each plasma derivative, revealing that the derivative requiring the greatest volume of plasma is intravenous immunoglobulins. This finding confirms the change in the "driving product": it is now the consumption of intravenous immunoglobulins that determines the amount of plasma that is sent for industrial processing.
Blood transfusion = Trasfusione del sangue 05/2007; 5(2):85-92. · 2.10 Impact Factor
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ABSTRACT: Bullous pemphigoid (BP) is an autoimmune skin disease that occurs mainly in elderly patients; onset of BP is rare in childhood. Inflammatory bowel diseases (IBD), by contrast, have a pediatric onset in 25% of presenting cases, requiring expert multidisciplinary management. Here we report a pediatric case of IBD (involving stomach, duodenum, ileum, and colon-rectum) associated with a disseminated form of drug-resistant BP successfully treated by plasma exchange (PEX), extracorporeal photochemotherapy (ECP), and corticosteroid therapy. The addition of PEX and ECP to standard treatment induced no severe side effects, prompted a rapidly achieved complete and long-term remission, and allowed dose tapering of the immunosuppressive drugs over an 18-month follow-up.
Journal of Clinical Apheresis 03/2007; 22(1):26-30. · 1.93 Impact Factor
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ABSTRACT: Recent clinical trials have demonstrated the efficacy of adoptive cellular therapy with virus-specific lymphocytes in patients with defective cellular immune responses. Immunoreconstitution has become a challenge for cellular immunology and for transfusion medicine. In fact, both expertises are required to provide effective and safe cellular products. Because of in vitro manipulation, T-lymphocyte cultures are at risk of contamination even under good manufacturing procedure (GMP) conditions.
To further improve the quality of these GMP cellular products, a procedure was designed for purification, stimulation, and expansion of antigen-specific CD4 and CD8 T-lymphocytes in a sealed, unbreached system. Leukopacks from the blood bank that fulfill the requirements of a GMP product were the starting material. Gradient separation and washing were performed in bags with sterile connecting devices on the bench-top, as well as addition of ingredients (antigen, interleukin-2) or transfer to larger bags.
The method is described in detail, and it is shown that increase in number of cytomegalovirus-specific CD4 or CD8 T-lymphocytes was similar to procedures based on open culture systems. Cell expansion after 4 weeks ranged from 800- to 2400-fold for CD4 lymphocytes and 300- to 900-fold for CD8 lymphocytes. Antigen specificity and loss of alloreactivity were demonstrated on the expanded cells with proliferation, intracytoplasmic interferon gamma-gamma staining, cytolytic activity, and pentamer binding.
This procedure can be applied to improve sterility under GMP conditions when T-cell lines are generated for adoptive immunotherapy and may increase biosafety for the staff when cell lines are generated from subjects infected with dangerous pathogens.
Transfusion 01/2007; 46(12):2053-62. · 3.22 Impact Factor
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ABSTRACT: Cellular immunity against cytomegalovirus (CMV) is essential for recovery from infection and control of viral latency. In immunocompromised hosts, this balance between CMV and cellular immunity is lost. Accordingly, restoration of the CD8 compartment specific for CMV is beneficial for immunocompromised patients. It is clear that CMV-specific CD4 cells provide helper functions facilitating long-term persistence of CD8 cells. Considering the dearth of data on CMV-specific T-helper cells, we investigated the CD4 responses to the immunodominant protein pp65 to define antigenic peptides. Such peptides were pooled and used to generate long-term T-cell lines. The lines were responsive to CMV and pp65. T cells were selected with individual peptides to produce monospecific lines for accurate definition of fine epitope specificity and to confirm human leukocyte antigen HLA-DR restriction. Furthermore, these lines lost alloreactivity, suggesting that they can be generated from the allodonor for adoptive immunoreconstitution of stem cell graft recipients.
Human Immunology 06/2004; 65(5):537-43. · 2.84 Impact Factor
