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Background: Pain is a major limiting factor in patient's recovery from major thoracic surgical procedures. Thoracic epidural analgesia (TEA), the current gold standard of perioperative management, has contraindications, can technically fail, and carries a risk of complications such as epidural abscess and spinal hematoma. The ultrasound-guided serratus anterior plane (SAP) block is a promising regional analgesia technique., Objectives: Since the anatomic space involved in the SAP block corresponds to the area exposed by the surgeon during right posterolateral thoracotomy, we investigated the feasibility of a "surgically guided" continuous SAP block as an alternative to TEA in selected esophagectomy patients., Study Design: This was a pilot case-series study., Setting: This study was carried out in a tertiary-care university hospital., Methods: The demographic and clinical data of patients in whom the continuous SAP block was performed were retrieved from a prospectively maintained database of hybrid (laparoscopy plus right thoracotomy) Ivor Lewis esophagectomy. The SAP block was performed upon closure of the thoracotomy incision using a 19-gauge catheter tunnelized subcutaneously and positioned in the deep plane between the serratus anterior muscle and the ribs. A bolus dose of 30 mL of levobupivacaine 0.25% was injected, followed by a continuous infusion of the 0.125% solution at 7 mL/h until postoperative day 4., Results: Between January 2016 and July 2016, seven (20%) out of 37 esophagectomy patients underwent a SAP block rather than TEA for the following reasons: inability to insert the epidural catheter, antiaggregation or anticoagulant therapy, or unplanned thoracotomy. The procedure was uneventful in all patients. Only two patients required rescue analgesia on day 1., Conclusion: Continuous SAP block under direct vision is feasible and safe. This novel "surgically guided" application of the SAP block may be useful in case of failure or contraindications to TEA., Competing Interests: The authors report no conflicts of interest in this work.

Thoracoscopic esophagectomy in the prone position is associated with better surgical ergonomics compared to the left lateral decubitus position due to the effects of gravity pooling blood outside the operative field and the reduced need for lung retraction. The aim of this study was to evaluate the physiological effects of prone thoracoscopic esophagectomy with single-lumen intubation on ventilation, respiratory gas exchange, and cardiovascular parameters. Thirty-two consecutive patients underwent esophagectomy either through a prone thoracoscopic approach or through a right thoracotomic approach. Samples of arterial and central venous blood, as well as ventilation and cardiovascular parameters were obtained at baseline, during induction of anesthesia, throughout the operation, and after extubation. Patients undergoing prone thoracoscopic esophagectomy showed higher oxygenation levels (p < 0.001), and a significantly lower mean pulmonary shunt fraction (p = 0.001). Perioperative hemodynamics remained stable throughout the surgical procedures. Thoracoscopic esophagectomy in the prone position with two-lung ventilation was associated with a significant improvement of global oxygen delivery and a significant reduction of the pulmonary shunt when compared to the Ivor Lewis operation.

The immunophenotypic analysis of ex vivo-expanded mesenchymal stromal cells (MSC) has so far been confined to single or dual staining analysis in normal subjects. In this study, using a four-color cytofluorimetric protocol, we demonstrated that cultured MSC derived from the bone marrow of patients with hematologic malignancies showed alterations in the expression of CD105, CD90, CD184, and HLA-DR molecules. The decrease in the percentage of CD105+ and CD90+ MSC correlated with an increased bone marrow angiogenesis. This paper provides evidence that multiparametric flow cytometry is essential for the establishment of a standardized protocol to identify various MSCs subsets and aberrant phenotypes.

In myelodysplastic syndromes (MDS), ineffective hematopoiesis and cytopenia may arise either from increased susceptibility to apoptosis of hematopoietic cells, or possibly from alterations of bone marrow stroma that contributes to defective development of marrow lineages. In order to test the significance of the endothelial growth in MDS patients, two in vitro assays are presented in this study: a long-term culture method for the detection of human bone marrow endothelial colonies (CFU-En) (77 patients) and a human primary model for the evaluation of the influence of a "bone marrow conditioned medium" on the formation of new vessels in a culture matrix ("angio-kit assay") (in 24 out 77 patients). The in vitro growth pattern of bone marrow CFU-En as well as the generation of microvessels in the angio-kit system was increased in RA, RARS and RAEB in comparison with controls. No CFU-En were detected in RAEB-T. The occurrence of large islands, formed by clusters of endothelial cells, unable to generate microcapillaries was also reported. Chromosomal abnormalities did not correlate with the angiogenetic pattern. These in vitro assays may constitute an useful approach to the analysis of angiogenesis in MDS.

The distribution and functional characteristics of in vitro bone marrow (BM) endothelial colonies (CFU-En) were studied in 70 non-Hodgkin's lymphoma (NHL) patients in different phases of the disease to explore the association between CFU-En growth and angiogenesis, and between the number of CFU-En and the presence of hematopoietic and mesenchymal progenitor cells. The mean number of CFU-En/10(6) BM mononuclear cells seen in remission patients was significantly higher than that seen in newly diagnosed patients (P=0.04), and in normal subjects (P=0.008). Patients with low-grade NHL in remission displayed a higher CFU-En value compared with high-grade NHL (P=0.04). In the autograft group (40 patients), a significant reduction of CFU-En number was detected in the first 4-6 months after transplantation. In remission patients, the CFU-En number positively correlated with the incidence of BM colony-forming unit granulocyte-macrophage (CFU-GM) (P=0.013) and CFU-multilineage (CFU-GEMM) hematopoietic colonies (P=0.044). These in vitro data show that CFU-En numbers increase following standard-dose chemotherapy, thus providing a rationale for further investigating the effects of different cytostatic drugs on BM endothelial cells growth and function.

To investigate the mechanisms of mobilization and of the factors implicated in the homing of progenitors and possibly understand the reasons for unpredicted mobilization failure, we analyzed CXCR-4 (CD184) expression on bone marrow (BM) CD34+ cells prior to peripheral blood stem cell (PBSC) mobilization in 24 patients affected by hematologic malignancies (non-Hodgkin lymphoma, multiple myeloma, and acute myeloid leukemia). We wanted to determine whether the level of CXCR-4 expressed by hematopoietic stem cells could influence mobilization process and therefore could be considered a predictive factor for mobilization adequacy. These data were also compared with stromal cell function as assessed by colony forming unit-fibroblast (CFU-F) and CFU endothelial cells (CFU-En) assays and stromal layer confluence capacity exhibited by patients' BM cells. In this study, we also compared CXCR-4 expression on CD34+ cells from different sources and at different migration stages specifically bone marrow (BM), steady state peripheral blood (SSPB), fetal cord blood (FCB), cord blood (CB), and mobilized PBSC. Seven (29%) of the 24 patients undergoing mobilization failed to achieve an adequate number of CD34+ stem cells (5 x 10(6)/kg CD34+ cells) and showed a very high expression frequency of CXCR-4 on BM CD34(+) stem cells (mean number of positive cells, 97%) investigated before the mobilization regimen. We also found that high expression intensity per cell for CXCR-4 was associated with lower amounts of mobilized CD34+ cells whereas those patients (17 out of 24 patients, 71%) with lower expression intensity per cell of CD184 on BM CD34+ cells prior to mobilization harvested at least 5 x 10(6)/kg CD34+ cells. Setting a cut off of 5 x 10(6)/kg CD34+ cells harvested, patients mobilizing less had a mean value of 97% CD34+ cells expressing CXCR-4 with a relative mean channel fluorescence of 458 whereas patients mobilizing more than 5 x 10(6)/kg CD34+ progenitors showed a mean value of 59.8% CD34+/CXCR4+ cells with a relative mean channel fluorescence value of 305. Interestingly, in the poor mobilizers group, the marrow stromal microenvironment was found to be more severely damaged in comparison with that of good mobilizers. The comparative analysis of CXCR-4 expression showed no difference in percentage values between steady-state PB (87.4%) and BM (85.1%) stem cells whereas mobilized CD34+ stem cells have a lower expression frequency of CXCR-4 (71.6%) compared to that of progenitors from other sources. Fetal blood CD34+ stem cells had the lowest mean expression frequency of CD184 antigen (36.3%), while CB cells had the highest (94.8%). In conclusion, this study provides evidence that monitoring CXCR-4 CD34 double positive cells before mobilization can be regarded as a predictive factor for mobilization outcome, giving us directional cues for the choice of the best stem cell mobilization regimens.

Background: In vitro cultures of BM cells from newly diagnosed patients with AML displayed a defective BM stromal compartment, with a reduced number of fibroblast-colony-forming unit (CFU-F: 1 +/- 1.25 SD) and a decreased proliferative ability. The purposes of our study were: 1). to select BM mesenchymal stem cells (MSC) and BM-derived stromal cells (BMDSCs) from AML patients at diagnosis and from healthy subjects, using an immunomagnetic system and either anti-CD105 or anti-fibroblast MAbs; 2). to study the immunophenotypic and functional properties of freshly isolated and cultured mesenchymal cells; 3). to test the in vitro plasticity of the selected cells to differentiate towards an endothelial phenotype., Methods: Fresh mononuclear cells obtained from BM of 20 patients newly diagnosed with AML and from eight healthy subjects were selected by using anti-fibroblast and anti-CD105 MAbs. Freshly isolated cells were analyzed, characterized by flow cytometry using a wide panel of MAbs and seeded in long-term culture medium to assess CFU-F formation. The level of confluence after 30 days and functional capacity in a long-term colony-forming cell culture (LTC-CFC) were tested. Furthermore, the cultured selected cell populations were assayed for their ability to differentiate into an endothelial-like cell phenotype with the addition of vascular endothelial growth factor (VEFG) and endothelial cell growth supplement (ECGS)., Results: In normal subjects the selection produced an increase of the CFU-F number of 2.6-fold with anti-fibroblast MAb and 2.7-fold with the anti-CD105 MAb. Anti-fibroblast and anti-CD105 MAb selection from AML BM cells resulted in a statistically significant greater count of CFU-F that was respectively 10.6-fold (P = 0.04) and 14.4-fold (P = 0.00001) higher in comparison with the unselected AML samples. Interestingly, in 80% of AML samples immunoselection was also able to restore the capacity of the CFU-F to proliferate and form confluent stromal layers. The isolation of those layers sustained the proliferation and differentiation of hematopoietic stem cells in the LTC-CFC. The phenotypic profile of cultured BMDSCs was different from that of the freshly isolated cells, and changed in relation to the culture conditions: CD105+ selected cells cultured with VEGF and ECGS expressed endothelial markers, a finding that suggests that this cell subpopulation may have the potential to differentiate toward an endothelial-like phenotype., Discussion: We report that immunomagnetic selection represents a valid tool for the selection of BM mesenchymal cells in samples obtained from both healthy subjects and patients with AML. This technique was able to rescue two functional and immunophenotypic compartments related to two different selected populations. In particular, the CD105+ cells isolated in AML displayed, after stimulation with VEGF and ECGS, the ability to change towards an endothelial-like cell phenotype, thus revealing an unexpected plasticity. Both CD105+ and fibroblast+ cells once successfully isolated might represent sources of mesenchymal cells populations useful for in vitro investigations and, above all, as therapeutic devices.

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.

Objective: The aim of this study was to evaluate which CD34(+) cell subset contained in leukapheresis products could be regarded as the most predictive of long-term hematopoietic recovery after autologous peripheral blood stem cell transplantation (auto-PBSCT)., Materials and Methods: Based on data from 34 patients with hematologic malignancies, doses of CD34(+) cells and CD34(+) cell subsets, defined by the expression of HLA-DR, CD38, CD117 (c-kit/R), CD123 (alpha subunit of IL-3/R), CD133 (AC133), and CD90 (Thy-1) antigens, were correlated with the number of short-term (i.e., colony-forming cells [CFC]) and long-term culture CFC (LTC-CFC) (generated at week 5 of culture) and with the kinetics of hematopoietic engraftment following auto-PBSCT. The capacity of autologous stroma (AS), normal human bone marrow stroma, and M2-10B4 murine cell line to sustain CD34(+) cell growth was comparatively evaluated in the LTC assay., Results: Our data demonstrated that some of the most primitive progenitor subsets (CD34(+)CD117(-)HLA-DR(-), and CD34(+)CD38(+)HLA-DR(-)) showed the strongest correlation with LTC-CFC numbers generated within the AS, whereas no significant correlation was noted using normal bone marrow stroma. Multivariate analysis showed that the only CD34 cell subset independently associated with long-term (3 to 6 months) platelet engraftment after auto-bone marrow transplantation was the CD34(+)CD117(-)HLA-DR(-) phenotype; long-term erythrocyte engraftment was correlated with CD34(+)CD38(+)HLA-DR(-) cell content. The latter further influenced platelet engraftment in the first 3 months after auto-PBSCT. The most predictive parameters for neutrophil engraftment were CD34(+)CD38(+)HLA-DR(-) cell subtype and the total LTC-CFC quantity infused., Conclusions: These data further support the hypothesis that the type of stromal feeders influences the frequency of LTC-CFC, possibly because they differ in their ability to interact with distinct subsets of hematopoietic stem cells. Furthermore, as the use of AS in LTC assay can mimic in vitro the human bone marrow microenvironment, it can be speculated that this culture system could be a useful means to study the kinetics of recovery of bone marrow stroma following chemotherapy and PBSCT. From these results, it can be concluded that some CD34(+) cell subsets appear to be more reliable predictors of long-term hematopoietic recovery rates than total CD34(+) cell quantity.

Mouse models and studies performed on fixed bone marrow (BM) specimens obtained from patients with multiple myeloma (MM) suggest that plasma cell growth is dependent on endothelial cell (EC) proliferation within the BM microenvironment. In order to assess whether EC overgrowth in MM reflects a spontaneous in vitro angiogenesis, BM mononucleated cells from 13 untreated (UT) MM, 20 treated (11 with melphalan and nine with DAV schedule) MM, eight patients with monoclonal gammopathy of uncertain significance (MGUS) and eight controls were seeded in an unselective medium to assess EC proliferation. Furthermore, the influence of IL6 on the EC growth was investigated. Endothelial colonies (CFU-En) appeared as small clusters, formed by at least 100 slightly elongated and sometimes bi-nucleated cells expressing factor VIII, CD31 and CD105 (endoglin). The CFU-En mean number/10(6) BM mononucleated cells in untreated MM samples (2.07 s.d. +/- 1.3) was significantly higher than in normal BM (0.28 +/- 0.48), while no difference was seen between normal BM and MGUS (0.28 +/- 0.54). Interestingly, the mean number of CFU-En in the DAV group (1.88 +/- 1.6) did not differ from the UT, while it was found to be lower in the melphalan group (0.31 +/- 0.63). The addition of anti-IL6 monoclonal antibody induced a reduction of both the plasma cells in the supernatant and the CFU-En number. This study describes a rapid and feasible assay providing support for the association between EC and plasma cells further suggesting that the in vitro angiogenesis process may parallel that observed in vivo.