Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

A Blood Bank Standardized Production of Human Platelet Lysate for Mesenchymal Stromal Cell Expansion: Proteomic Characterization and Biological Effects.

Human platelet lysate (hPL) is considered a valid substitute to fetal bovine serum (FBS) in the expansion of mesenchymal stromal cells (MSC), and it is commonly produced starting from intermediate side products of whole blood donations. Through freeze-thaw cycles, hPL is highly enriched in chemokines, growth factors, and adhesion and immunologic molecules. Cell therapy protocols, using hPL instead of FBS for the expansion of cells, are approved by regulatory authorities without concerns, and its administration in patients is considered safe. However, published data are fairly difficult to compare, since the production of hPL is highly variable. This study proposes to optimize and standardize the hPL productive process by using instruments, technologies, and quality/safety standards required for blood bank activities and products. The quality and improved selection of the starting material (i.e., the whole blood), together with the improvement of the production process, guarantee a product characterized by higher content and quality of growth factors as well as a reduction in batch-to-batch variability. By increasing the number of freeze/thaw cycles from one (hPL1c) to four (hPL4c), we obtained a favorable effect on the release of growth factors from platelet α granules. Those changes have directly translated into biological effects leading to a decreasing doubling time (DT) of MSC expansion at 7 days (49.41 ± 2.62 vs. 40.61 ± 1.11 h, p < 0.001). Furthermore, mass spectrometry (MS)-based evaluation has shown that the proliferative effects of hPL4c are also combined with a lower batch-to-batch variability (10-15 vs. 21-31%) at the proteomic level. In conclusion, we have considered lot-to-lot hPL variability, and by the strict application of blood bank standards, we have obtained a standardized, reproducible, safe, cheap, and ready-to-use product.

Counting circulating endothelial cells in allo-HSCT: an ad hoc designed polychromatic flowcytometry-based panel versus the CellSearch System.

Physio-pathologic interrelationships between endothelial layer and graft-versus-host disease (GVHD) have been described leading to assess the entity "endothelial GVHD" as the early step for clinical manifestations of acute GVHD. The availability of the CellSearch system has allowed us to monitor Circulating Endothelial Cells (CEC) changes in allogeneic hematopoietic stem cell transplantation (allo-HSCT) as useful tool to help clinicians in GVHD diagnostic definition. We have compared CEC counts generated by an ad hoc designed polychromatic-flowcytometry (PFC) Lyotube with those of the CellSearch system. CEC were counted in parallel at 5 timepoints in 50 patients with malignant hematologic disorders undergoing allo-HSCT (ClinicalTrials.gov, NCT02064972). Spearman rank correlation showed significant association between CEC values at all time points (p = 0.0001). The limits of agreement was demonstrated by Bland Altman plot analysis, showing bias not significant at T1, T3, T4, while at T2 and T5 resulted not estimable. Moreover, Passing Bablok regression analysis showed not significant differences between BD Lyotube and CellSearch system. We show that CEC counts, generated with either the CellSearch system or the PFC-based panel, have a superimposable kinetic in allo-HSCT patients and that both counting procedures hold the potential to enter clinical routine as a suitable tool to assist clinicians in GVHD diagnosis.

A standardized flow cytometry network study for the assessment of circulating endothelial cell physiological ranges.

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.

Changes in circulating endothelial cells count could become a valuable tool in the diagnostic definition of acute graft-versus-host disease.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is burdened by life-threatening complications, with graft-versus-host disease (GvHD) being the major cause of morbility and mortality. Recently, clinical and physiopathologic evidences showed that vascular endothelium can be a target of GvHD in the early phase and circulating endothelial cells (CECs) represent surrogate markers of endothelial damage. METHODS: Using the CellSearch System (Veridex LLC, Raritan, NJ), CECs were counted before (T1), after conditioning regimen (T2), at engraftment (T3), at GvHD onset (T4), and after steroid treatment (T5) in 40 patients (7 Hodgkin's Disease, 13 Acute Myeloblastic Leukemia, 5 Acute Lymphoblastic Leukemia, 8 Multiple Myeloma, 3 Chronic Lymphocytic Leukemia, 1 Non-Hodgkin Lymphoma, 1 Chronic Myeloid Leukemia, 2 Severe Aplastic Anemia) undergoing allo-HSCT. RESULTS: The median CEC per milliliter at T1 was 20 (n=33, range 4-718), in comparison to a value of 2 (range, 1-14) in controls (P<0.001). At T3, CEC per milliliter were 47 (range, 16-148) in GvHD patients and 92 (range, 23-276) in patients without GvHD (P=0.006). This difference remained significant in multivariate analysis (odds ratio, 0.97; 95% confidence interval, 0.96-0.99; P=0.02). At GvHD onset, the relative increase of CEC counts from time of engraftment (T4 vs. T3) was 44% (range, -43% to 569%) in GvHD patients versus 0% (range, -49% to 2%) in patients without GvHD (P=0.003), being confirmed as significant in multivariate analysis (odds ratio, 1.04; 95% confidence interval, 1.0-1.08; P=0.04). CONCLUSION: Changes in CEC count can represent a promising marker to monitor endothelial damage in patients undergoing allo-HSCT and could become a valuable tool in the diagnostic definition of GvHD.