Diana Campioni

Airborne microbial contamination and particulate matter in operating rooms (ORs) contribute to the risk of surgical site infections (SSIs). However, few studies have integrated culture‐based approaches with MALDI‐TOF MS identification and metagenomic NGS during real surgical activity. This study combines microbiological, molecular, particulate, and procedural data to provide a multifactorial view of intraoperative contamination dynamics. This study aimed at characterizing airborne microbiota, particulate matter, and key procedural determinants of contamination in ORs. Forty‐two surgical procedures across three specialties were monitored. Airborne microorganisms were isolated using culture‐based methods and identified with the MALDI Biotyper, whereas the OR airbiome was assessed using shotgun metagenomic sequencing. Airborne particle concentrations (≥ 0.5 and ≥5 μ m), microclimatic parameters, staff presence, door openings, electrosurgical use, and procedural order were recorded. Statistical analyses included Wilcoxon and Kruskal–Wallis tests with Dunn′s post hoc comparisons, Kendall′s τ and Spearman′s ρ correlations, and rank‐based effect sizes. Airborne microbial contamination was low in both ISO 5 and ISO 7 rooms (mean 14.02 ± 6.88 CFU/m ³ ), with a predominance of skin‐associated taxa. Culture‐based methods detected viable, fast‐growing species, whereas NGS revealed additional environmental, slow‐growing, and anaerobic taxa, including clinically significant bacteria and yeasts such as Cutibacterium acnes ( C . acnes ), Finegoldia magna ( F . magna ), and Malassezia restricta ( M. restricta ). Cross‐method concordance was weak to moderate (Spearman ^′ s ρ = 0.35; Kendall ^′ s τ = 0.24), indicating complementary detection. Median particle concentrations were significantly higher in ISO 7 than in ISO 5 ORs. In ISO 7 rooms, particle loads varied by specialty and tended to be higher during open procedures. Integrated analyses showed a decoupling of microbial and particulate contamination in ISO 5 rooms, whereas ISO 7 rooms exhibited more interconnected contamination dynamics, with significant associations between electrosurgical device use and airborne microbial contamination ( ρ = 0.45, p = 0.025), between fine (≥ 0.5 μ m) and coarse (≥ 5 μ m) particle concentrations ( ρ = 0.65, p < 0.001), and an inverse association between procedure order and staff count ( ρ = −0.66, p < 0.001). Integrating culture‐based and molecular techniques may provide a more comprehensive characterization of the airborne microbiota in ORs. The findings suggest that ISO 5 laminar‐flow systems are associated with more stable airborne contamination patterns, whereas ISO 7 rooms exhibit greater interactions between airborne contamination and procedural variables. Incorporating molecular profiling and particle metrics into OR surveillance may support environmental monitoring strategies relevant to infection prevention and control.

Background: The expression of the immunoglobulin superfamily cell membrane adhesion molecule CD146 has been reported on several normal and pathological cell types in human. The aim of this study was to investigate CD146 expression in acute leukemia using a multiparametric cytofluorimetric approach.Methods: Cytofluorimetric and cytogenetic studies were performed on peripheral blood and bone marrow samples from 162 patients with acute myeloid leukemia (AML,n = 121) and acute lymphoblastic leukemia (ALL,n = 41). ALL patients were subdivided in B‐ALL (n = 38) and T‐ALL (n = 3). Adult (n = 18) and pediatric (n = 20) B‐ALL were considered as a whole group.Results: Four out of 121 (3.3%) AML cases, 14/38 (36.8%) B‐ALL, and 2/3 (66.6%) T‐ALL expressed CD146 on 12–98% of blasts (p < 0.001). CD146 expression was not observed in 10 healthy controls. Among B‐ALL CD146‐positive cases, 78.6% were associated with a “common”/BII‐ALL and 21.4% with a pre‐B/BIII‐ALL immunophenotype while pro‐B/BI‐ALL and mature‐B/BIV‐ALL cases were CD146‐negative. Statistical analysis showed CD146 expression strongly associated with Ph+ positivity in B‐ALL with the highest percentage of CD146‐positive blasts in all Ph‐positive B‐ALL cases (84 ± 22% Ph‐positive B‐ALL SD vs. 40 ± 24% SD in Ph‐negative B‐ALL;p < 0,001).Conclusion: In our series, CD146 was expressed in all cases of Ph‐positive B‐ALL and in the vast majority of T‐ALL, whereas it was rarely expressed by AML blasts. We suggest that CD146 may be considered as an additional marker for acute lymphoblastic leukemia diagnosis and monitoring of minimal residual disease in those cases which are CD146‐positive at diagnosis. © 2015 International Clinical Cytometry Society

Human herpesvirus 6A (HHV-6A) causes ubiquitous infections and has been associated with several diseases in immunosuppressed and immune dysregulated individuals. Although considered a lymphotropic virus, HHV-6A has the potential to infect many cell types, inducing important alterations in the infected cell. In our search for additional potential targets for HHV-6A infection, we analyzed the susceptibility of human mesothelial cells to viral infection. HHV-6A infection was performed and analyzed on primary human mesothelial cells isolated from serous cavity fluid, infected in vitro with a cell-free HHV-6A inoculum. The results demonstrated that mesothelial cells are susceptible to in vitro HHV-6A infection, and more importantly, that the virus induces an alteration of HLA expression on the cell surface, inducing HLA class II and HLA-G de novo expression. Since mesothelial cells play a pivotal role in many processes, including inflammation and antigen presentation, we speculate that, in vivo, this virus-induced perturbation might be correlated to alterations in mesothelium functions.

Accumulating evidence indicates that bone marrow microenvironment plays an important role in the pathogenesis of some myeloid and lymphoid hematological malignancies (HM). Among different environmental associated parameters, those related to functional, cytogenetic and immunological integrity of mesenchymal stromal cells (MSC) are particularly relevant. Functional alterations and immunophenotypic abnormalities have been described in MSC obtained from HM patients. These data seem to confirm the defective biological pattern of MSC especially in myeloid diseases, while MSC cytogenetic profile in HM is still an open question, because it is not clear whether BM stromal cells are "culprit or bystander" displaying or not an abnormal karyotype. Contradictory findings were reported in different HM but the functional implications of altered MSC karyotype need to be further addressed also in light of a clinical use of MSC. A "pathological" in vivo supportive function of endogenous MSC, which provide important survival and drug resistance signals to leukemic cells especially in lymphoproliferative disorders, is suggested. Thus, the mechanisms underlying these protective versus cytotoxic effects exerted by MSC on leukemic cells need further investigations.

A brief review of the literature on HLA-G role in multipotent stromal cells anti-inflammatory functions

The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin. By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7–14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities. Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

The possibility that human mesenchymal stromal cells (hMSC) may derive from the malignant clone in hematological malignancies (HM) is a controversial issue. In order to clarify hMSC origin and disclose possible cytogenetic heterogeneity in hMSC belonging to different patients, bone marrow (BM)-derived hMSC samples from chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) were expanded in culture, characterized by flow cytometry, and screened by conventional cytogenetic analysis and fluorescent in situ hybridization for the presence of possible cytogenetic aberrations, related or not to the hematopoietic neoplastic clone. Our data showed that the presence of cytogenetic aberrations in successfully expanded HM-MSC stromal layers derives from the persistence of contaminating hemopoietic cells (HC), which is greatly supported by in vitro culture conditions that could mimic in vivo microenvironmental niche. Interestingly, the presence of binucleated HM-MSC maintaining a diploid numerical setting has been also detected, while aneuploidies were observed more frequently in mononucleated HM-MSC from patients with an altered karyotype than in patients with a normal karyotype and controls. In conclusion, here, we showed that in ALL and in CLL, the BM-MSC has a normal karyotype, thus supporting a distinct origin from hematopoietic cells (HC). The presence of in vitro hMSC aneuploidy is associated with lymphoid neoplasias carrying chromosome abnormalities, suggesting that hMSC should be characterized before clinical application. The adequacy of hMSC cytogenetic characterization here proposed could represent a “prerequisite” to standardize the hMSC analysis before their use in the autologous setting and cellular therapy.

Acute leukaemia is a neoplastic disorder of haematopoietic precursors characterized by a rapid clinical course and a relatively high early death rate in spite of recent therapeutic progress. Prompt and reliable diagnostic and prognostic assessment has a favourable impact on patient outcome. Diagnostic suspicion relies on signs and symptoms of bone marrow failure or quali-/quantitative abnormalities of blood cells, which are accurately detected by modern automated counters. Cytomorphological examination of a stained blood and bone marrow film plays a key role in the diagnostic process, with relevant additional information coming from immunophenotyping of blast cells. Cytogenetic and molecular genetic data are the basis of prognostic stratification. In this context, a coordinated intervention of specialized laboratories combining solid expertise in each field involved in the diagnostic work-up is essential; at the same time, the availability of all the required technologies at the same hospital structure would probably lack efficiency due to the low number of tests performed. The organization of laboratory networks, either at regional or national level, especially in the context of clinical trials, may offer a great opportunity for centralization of more sophisticated technologies in reference laboratories, each highly specialized in a particular field of investigation and all interconnected. The challenge for laboratory haematology is the reorganization of clinical and scientific activity according to this model, without loosing educational potential in favour of new generations of medical doctors, haematologists, biologists and laboratory technicians. La leucemia acuta è una malattia neoplastica dei precursori emopoietici caratterizzata da una rapida evolutività clinica, con un tasso di mortalità precoce che si mantiene elevato nonostante i continui progressi terapeutici. La tempestività di un corretto inquadramento diagnostico secondo criteri classificativi internazionali e di una precisa stratificazione prognostica ha un impatto clinico dimostrato in termini di outcome. Il sospetto diagnostico può essere formulato sulla base di sintomi o segni, in genere associati alla soppressione della normale emopoiesi, oppure sulla base di alterazioni emocromocitometriche, quantitative o qualitative, sensibilmente rilevate dai moderni contatori automatici. La valutazione morfologica dello striscio di sangue periferico e della citologia midollare rappresenta un momento fondamentale nell’iter diagnostico e può ricevere contributi di rilievo dall’indagine immunofenotipica. La stratificazione prognostica si fonda su dati citogenetici e molecolari sempre più specifici e dettagliati. In questo contesto, l’integrazione tra le diverse competenze cliniche e laboratoristiche coinvolte nel processo diagnostico acquista grande importanza; allo stesso tempo però la disponibilità presso ogni centro delle risorse strumentali necessarie e di personale altamente qualificato nei diversi settori si scontra con esigenze di efficienza economica. Lo sviluppo, a livello locale e nazionale, di reti di laboratori a elevata specializzazione in ognuno dei differenti ambiti diagnostici e di ricerca, consente una proficua ottimizzazione delle risorse. La sfida che si propone quindi all’Ematologia di laboratorio è quella di una riorganizzazione dell’attività clinica e scientifica secondo un modello che soddisfi elevati standard di accuratezza diagnostica ed efficienza economica, senza sacrificare le esigenze formative delle nuove generazioni di medici, specialisti ematologi, biologi e tecnici di laboratorio.