Gino Tripodi

Multiple Target Molecular Monitoring of Bone Marrow and Peripheral Blood Samples From Patients With Localized Neuroblastoma and Healthy Donors

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.

Changes in neonatal transfusion practice after dissemination of neonatal recommendations.

OBJECTIVE: To evaluate the change in neonatal transfusion practices after the introduction of national recommendations for transfusion of blood products to neonates in 2006. METHODS: A questionnaire-based survey on neonatal transfusion practice of 79 Italian NICUs was completed in 2008. Results were compared with those obtained from a previous national Italian neonatal transfusion-practice survey performed in 2001. RESULTS: Responses were received from 62 of 79 (78.5%) neonatal units. Prophylaxis for transfusion-transmitted cytomegalovirus infection in 2001 and 2008 had been performed in 96.8% and 98.4% of NICUs, respectively. Filter leukoreduction of red blood cell donor units was preferred over cytomegalovirus antibody testing to obtain cytomegalovirus-safe blood components. Prophylaxis for graft-versus-host disease increased from being performed at 61.3% of neonatal units in 2001 to 77.4% in 2008 (P = .08, Pearson χ2), whereas usage of dedicated red blood cell donor units (paedipack system), permitting multiple transfusions from the same unit, improved from 53.2% to 82.2% (P = .001, Pearson χ2). The 2008 survey documented a continuation of wide variability in transfusion practice for fresh-frozen plasma and platelet concentrates. CONCLUSIONS: This nation-wide Italian self-report survey highlighted improvements in NICU transfusion practice after the neonatal recommendations issued in 2006. Prophylaxis for transfusion-transmitted cytomegalovirus infection continued with nearly total adherence to national recommendations, and both prophylaxis for graft-versus-host disease and paedipack-system usage suggested a trend of improvement of adherence rates. The continuing wide diversity observed among neonatal units in fresh-frozen plasma and platelet-concentrate transfusion practice may indicate a lack of acceptable criteria for the administration of these blood products.

Whole-genome sequencing as standard practice for the analysis of clonality in outbreaks of meticillin-resistant Staphylococcus aureus in a paediatric setting.

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-associated infections. This study investigated the potential use of whole-genome sequencing (WGS) for surveillance purposes by re-examining MRSA strains related to past outbreaks among hospitalized paediatric patients. WGS data ameliorated the genotypic profile previously obtained with Sanger sequencing and pulsed-field gel electrophoresis typing, and discriminated between strains that were related and unrelated to the outbreaks. This allowed strain clonality to be defined with a higher level of resolution than achieved previously. This study demonstrates the potential of WGS to trace hospital outbreaks, which may lead to WGS becoming standard practice in outbreak investigations.

Positive Selection and Expansion of Cytomegalovirus-specific CD4 and CD8 T Cells in Sealed Systems : Potential Applications for Adoptive Cellular Immunoreconstitution

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-γ 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.

Quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma by liquid chromatography/tandem mass spectrometry

Therapeutic drug monitoring is a cornerstone of antibacterial therapy, especially in an era of increasing antibacterial resistance in individualizing antimicrobial therapy. Liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was used for the simultaneous measurement of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in 50 μl plasma samples over a wide range. The overall turnaround time for the assay was 20 minutes. Intra-assay precision and accuracy for quality control samples ranged within 1·8–8·5 and 91·4–106·7%, respectively. Inter-assay precision and accuracy ranged within 1·3–14·4 and 95·8–104·6%, respectively. The lower limit of quantification was below 1·5 μg/ml for all the five antibiotics. No ion suppression due to matrix effects was found. A simple and rapid LC–MS/MS method which provides high specificity, precision and accuracy for the simultaneous quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma has been developed and validated. The present method is suitable for therapeutic drug monitoring in paediatrics.

Recommendations for transfusion therapy in neonatology.

The indications for transfusion therapy in the neonatal period are based on specific knowledge of various aspects of this particular period of life, such as the dynamic interaction of the mother-placenta-foetus/neonate, the pathophysiological changes in the perinatal and neonatal periods and the profound haematological modfications that are characteristic of the first weeks of life. Furthermore, the physiological immaturity of various organs and systems can expose neonates, in particular those with a very low birth weight (VLBW) (Appendix I), to metabolic alterations following the transfusion of various blood components and the additives in them, and to infectious and immunological risks, such as Graft-versus-Host disease (GVHD). This implies the need for close and continuous collaboration between paediatricians-neonatologists and transfusion medicine specialists in order to obtain “dedicated” blood components, both with regards to quality and quantity, able to meet the particular needs of the neonate, especially considering the now increased survival of extremely low birth weight (ELBW) babies. ELBW and “critically ill” neonates are categories of patients with high transfusion needs, even though the number of transfusions given to premature neonates has progressively decreased over the last decade. It is, however, essential to establish appropriate transfusion criteria for these subjects. The scientific contributions on transfusion medicine in the neonatal period derive predominantly from consensus of opinions rather than controlled studies and the lack of clear scientific evidence makes it difficult to formulate high-grade recommendations based on solid levels of evidence. Furthermore, it should be appreciated that neonatal transfusion medicine is, like all other scientific fields, a continuously evolving discipline. These Recommendations, which represent the opinions of the authors and include evidence-based data, when available, have been formulated to facilitate the implementation of uniform transfusion practices. They are not intended to provide absolute indications, but aim to be a “guide” which nevertheless guarantees individual healthcare professionals freedom of choice in the various different clinical situations. This document deals with pre-transfusion tests, indications for the transfusion of blood components, characteristics of the blood components and methods of their administration for neonates. Details on the levels of evidence and strengths of the recommendations are provided in Appendix II. This document does not consider the indications for the use of blood derivatives and some highly specialised, life-saving techniques used in particular emergencies, such as extracorporeal membrane oxygenation and cardiopulmonary bypass.

A validated LC–MS/MS method for the quantification of piperacillin/tazobactam on dried blood spot

Background Therapeutic drug monitoring (TDM) of antibiotics in children is of fundamental importance for effective patient management. The use of dried blood spot (DBS) offers a number of advantages over conventional blood collection. The aim of this study is to develop and validate a method to measure piperacillin/tazobactam in DBS. Results The analysis was performed by using LC-MS/MS operating in multiple reaction monitoring mode. The method has been validated by applying EMA guidelines and its suitability for TDM was evaluated by using samples from low birth weight neonates. Conclusion This paper describes a fast and cost-effective micromethod for the simultaneous determination of piperacillin/tazobactam levels on dried blood spot that is suitable for TDM in children.

The first case of drug-induced immune hemolytic anemia due to hydrocortisone

BACKGROUND: 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.

Occult hepatitis B virus infection: a case of reactivation in a patient receiving immunosuppressive treatment for allogeneic bone marrow transplantation

The presence of hepatitis B virus (HBV) genome in HBsAg-negative subjects is known as occult HBV infection1. This particular form of hepatitis, already recognised in the 1980s, has been confirmed and studied using molecular biology techniques. In fact, occult infection is usually associated with the presence of anti-HBc and anti-HBs, but given the relatively high percentage (approximately 20%) of subjects who are negative for all markers1, the introduction of a test to detect HBV DNA was fundamental. Occult infection is related in some cases to mutant viruses that are not detectable by the commonly used tests; it has also been observed that the reactivation of HBV related to variants of the viral genome often has an unfavourable clinical prognosis2,3. Much more frequently, however, an occult infection is associated with strong suppression of viral replication, which is responsible both for the negativity for HBsAg and the undetectable or very low levels of HBV DNA in the serum, although this latter can be found in liver tissue4,5. An occult infection can have a important impact in various different clinical settings, including transmission through blood transfusion or organ transplants6,7 and reactivation following immunosuppressive therapy. Indeed, it has been shown that the hosts immune response, co-infections (e.g. with hepatitis C virus) and epigenetic factors all play significant roles in occult infection8. We present the case of a Georgian child negative for HbsAg who, after receiving an allogeneic bone marrow transplant and immunosuppressive therapy, was found to be positive for HBV. We then describe the investigations conducted in order to determine whether this was due to a new infection or reactivation of an occult infection.

Validation of a miniaturized assay based on IFNg secretion for assessment of specific T cell immunity

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.