AIEOP-BFM consensus guidelines 2016 for flow cytometric immunophenotyping of Pediatric acute lymphoblastic leukemia.

Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.

Prospective comparison of two flow cytometry methodologies for monitoring minimal residual disease in a multicenter treatment protocol of childhood acute lymphoblastic leukemia.

BACKGROUND: Minimal residual disease (MRD) is a powerful prognostic indicator in childhood acute lymphoblastic leukemia (ALL). Multiparametric flow cytometry (FC) is a rapid and sensitive methodology for detection of MRD, applicable for most patients and is being incorporated in multicenter treatment protocols. The influence of different techniques and of individual interpretation of data on the interlaboratory variability in FC-MRD determinations has not been described. METHODS: We compared FC-MRD of identical bone marrow samples processed as either Ficoll separated mononuclear cells or lyse and wash nucleated cells (NC) in two central laboratories of a national multicenter childhood ALL study. A total of 290 samples at diagnosis and 494 follow-up samples (Day-15 n = 261; Day-33 n = 233) were analyzed. A group of 52 paired list mode data (LMD) of D-15 and D-33 samples was blindly reanalyzed by both laboratories. RESULTS: Pearson correlations for all samples of D-15 (n = 261) and D-33 (n = 233) were 0.875 and 0.82, respectively (P < 0.001), being lower for T-ALL 0.716 and 0.719, respectively. Quantitative concordance defined as less than 0.5 log difference in MRD measured by the two methodologies was 80.8% at D-15 but only in 57.9% at D-33. Reanalysis of LMD revealed that data interpretation explained half of the discordance. CONCLUSIONS: FC-MRD analysis of childhood ALL is a robust method during the earliest phases of induction therapy in a multicentric setting. Standardization of data analysis could improve about half of the discordance between different technical approaches.

The effects of external counter pulsation therapy on circulating endothelial progenitor cells in patients with angina pectoris.

OBJECTIVES: External counter pulsation therapy (ECPT) offers symptomatic relief and improves ischemia in patients with refractory angina pectoris. We aimed to determine the effects of ECPT on circulating endothelial progenitor cells (EPCs). METHODS: We prospectively studied 25 patients with angina pectoris treated with ECPT (n = 15) or receiving standard care (n = 10). The number of EPCs positive for CD34 and kinase insert domain receptor (KDR) was determined by flow cytometry and the number of colony-forming units (CFUs) was assessed in a 7-day culture, before ECPT and after 9 weeks. RESULTS: ECPT improved anginal score from a median of 3.0 to 2.0 (p < 0.001). Concomitantly, ECPT increased EPC number from a median of 10.2 to 17.8/10(5) mononuclear cells (p < 0.05), and CFUs from 3.5 to 11.0 (p = 0.01). Flow-mediated dilatation was improved by ECPT from 7.4 to 12.2% (p < 0.001) and correlated with EPC-CFUs (r = 0.461, p = 0.027). The levels of asymmetric dimethylarginine were reduced by ECPT from 0.70 to 0.60 micromol/l (p < 0.01). In contrast, the same parameters did not change in the control group, before and after follow-up. CONCLUSIONS: The present pilot study shows, for the first time, that ECPT is associated with increased number and colony-forming capacity of circulating EPCs.