Introduction: Flow cytometry analysis of lymphocyte subsets in peripheral blood is a common technique in diagnostic laboratories. Abnormal values have been identified in prevalent infections, autoimmune disorders and neoplastic diseases. Reference ranges for lymphocyte subsets of a healthy population from Spain are scarce. Methods: The study was performed on 319 healthy subjects, aged 4–88 years, from 709 individuals enrolled in the GAIT-2 Project (Genetic Analysis of Idiopathic Thrombophilia). Health status, age, sex, fertility, BMI and lifestyle (physical activity, cigarette smoking and ethanol intake) were assessed using standardized criteria. The percentage of lymphocyte subsets was determined using flow cytometry (Lymphogram™). Percentages of CD3+, CD4+, CD8+, CD19+, CD3–CD56+, CD3+CD4–CD8– double-negative (DN) T cells, CD3+CD4+CD8+ double-positive T cells and the CD4+/CD8+ ratio were recorded for each case. Results: Children had a significantly higher percentage of CD19+ and DN cells than adults. Women had a significantly higher percentage of CD3+ and CD4+ and a lower percentage of natural killer cells than men. Increases in BMI were inversely associated with the percentage of DN cells. Physical activity increased the percentage of lymphocytes and DN cells. Alcohol consumers had a lower percentage of CD19+ and DN cells, and a higher percentage of CD4+. Conclusion: This study provides reference ranges for lymphocyte subsets of healthy children and adults in a Mediterranean population (Spain) and determines the influence of lifestyle factors on these values.

To cite this article: Pabinger I, Vossen CY, Lang J, Conard J, García-Dabrio MC, Miesbach W, Legnani C, Svensson P, Kaider A, Rosendaal FR.Mortality and inherited thrombophilia: results from the European Prospective Cohort on Thrombophilia. J Thromb Haemost 2012; 10: 217-22.Summary. Background: Data on the survival of individuals with hereditary thrombophilia are rare and only come from retrospective studies. Objective: The aim of the present study was to assess mortality in individuals with known thrombophilia with and without a history of thrombosis in comparison to a control group. Patients/methods: The European Prospective Cohort on Thrombophilia (EPCOT) study is a prospective, multi-center observational study performed to assess the risk of thrombosis in persons with inherited thrombophilia. In an extension of the present study, the vital status was assessed in 1240 individuals with thrombophilia (mean age 40.9 years, 59% women, 196 with antithrombin, 341 with protein C, 276 with protein S-deficiency, 330 with factor (F)V Leiden and 97 with combined defects, and 62% with a history of venous thrombosis [VT]) and 875 controls (mean age 42.5 years, 48% women, 7% with a history of VT). Results: Seventy-two individuals with thrombophilia and 45 controls died during follow-up. The risk of death, adjusted for gender, thrombosis history and center, was not associated with thrombophilia (hazard ratio [HR] thrombophilia individuals vs. controls: 1.09, 95% confidence interval [CI] 0.66-1.78). When individuals with thrombophilia were evaluated separately, a history of thrombosis was not associated with mortality: the risk of death after adjustment for gender, anticoagulation and center was HR 0.79 (95% CI, 0.41-1.54). Conclusions: No increased risk of death in individuals with thrombophilia, not even in those with a history of thrombosis, was observed.

No abstract

CD34 positivity threshold with prognostic relevance is low (3% positive cells). Immunophenotypic findings in AML probably could only be fully exploited after a complex analysis that takes into account unconventional thresholds and the MFI.

1452 Introduction: Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by the presence of acquired genetic alterations in hematopoietic progenitor and stem cells. The prognostic impact of immunophenotypic markers has long been controversial. Despite this, only a very limited number of studies have investigated the prognostic impact of the levels of cell surface membrane (Sm) proteins commonly expressed on AML blasts. In this study we analyzed the prognostic impact of the levels of expression of several markers associated with AML blasts and normal hematopoietic precursors, as asessed by Multiparameter Flow Cytometry (MFC) in de novo AML patients (pts). Methods: Overall, 122 adult de novo AML pts diagnosed between 12/2003 and 08/2011 were studied, after excluding acute promyelocytic leukemia. All cases were diagnosed and classified according to the WHO criteria and they were immunophenotyped using an extensive 4-color panel of monoclonal antibodies by MFC, as previously described. Patients were treated according to the multicenter CETLAM AML-03 trial. Blast cells were gated after excluding all other cell populations, as well-delineated clusters of SSC low/int/CD45dimevents (“blast gate”) using the merge function of the Infinicyt software (Cytognos SL, Salamanca, Spain). For each case, the reactivity for the following markers was evaluated in terms of mean fluorescence intensity (MFI; arbitrary units) in bone marrow samples: CD123, CD117, CD34 and CD7. Normal residual bone marrow lymphocytes were used as reference for internal quality control purposes. Cytogenetic and molecular risk stratifications were based on the European LeukemiaNet (ELN) recommendations and the molecular lesions were investigated using well established protocols. Overall survival (OS), disease-free survival (DFS) and relapse rate (RR) were measured by the Kaplan–Meier method and curves were compared with the log-rank test. Multivariate analysis was performed using the Cox regression model. P-values ≤0.05 were considered to be statistically significant. Results: Median AML patient age was of 54 years (range: 20–70 y) with a M/F ratio of 63/59. Twelve pts (10%) had good cytogenetic/molecular risk, 83 (68%) intermediate, 17 (14%) high risk and 10 (8%) pts were unknown. Fifteen cases (12%) had NPM1mut, 33 (27%) showed FLT3-ITDmut, 5 (4%) pts carried CEBPAmut, 41 (33.6%) pts with no mutations and 28 (23%) pts were unknown. The median follow-up of pts alive was 19 months (range 0–97 months) and the 5-year OS of all pts was 42±5%. Univariate analysis of prognostic factors showed an association between higher CD45 (MFI >232; p=.01), CD117 (MFI>259; p=.008), CD34 (MFI >242; p=.007) and CD7 (MFI> 19; p=.03) expression and both a shorter OS and DFS. In addition, high CD123 expression (MFI >248; p=.04) was associated with a shorter DFS. In multivariate analysis only a high CD45 (p=.03) and a high CD34 (p=.03) expression were identified as independent predictors for a shorter OS. In turn, higher levels of CD123 (p=.03) and of CD34 (p=.02) were independent predictors for DFS and relapse, even when age, gender and WBC were taken in account. Conclusion: In this study, we show that higher levels of expression of immunophenotypic markers associate with immature myeloid precursors (CD45, CD117, CD34 and CD7) as assessed by MFC, have a significantly adverse prognostic impact in AML, both as regards OS and DFS. Accordingly, higher levels of CD45 and CD34 were independent predictors for both a shorter OS whereas, greater CD123 and CD34 values were associated with an increased risk of relapse and a shorter DFS, supporting the adverse prognostic impact of a more immature blast cell phenotype in de novo AML. Disclosures: No relevant conflicts of interest to declare.