Assessment of Differentiation States of Hematopoietic Stem Cells Following in Vitro Culture Using Side and Forward Scatter of Flow Cytometry

Hematopoietic stem cells (HSC) are defined by the International Society of Hematotherapy and Graft Engineering (ISHAGE) as those at low side scatter, positive for CD34 and CD45dim for their numeration with flow cytometry. However, we found that these CD34+ cells increase their granularity and size following in vitro culture, which was exhibited in flow cytometry as more events at higher side scatter and forward scatter. To further determine whether such a change in the cell event distribution is related to HSC differentiation, HSC markers and differentiation markers of in vitro-cultured HSC were detected by flow cytometry at different side scatter and forward scatter levels using modified ISHAGE gating strategies.nbsp The results revealed that cultured HSC with higher side scatter have a lower percentage of cells positive for HSC markers and a higher percentage of differentiation makers, while those with higher forward scatter have a higher percentage of differentiation makers but a slightly higher percentage of stem cell markers, suggesting that side scatter and forward scatter levels of cultured HSC correlate with the differentiation level of these cells

Increased serum BAFF (B-cell activating factor of the TNF family) level is a peculiar feature associated with familial chronic lymphocytic leukemia.

In a series of 84 chronic lymphocytic leukemia (CLL) patients we sought to establish whether BAFF (B-cell activating factor of the TNF family) circulating levels correlated with clinical characteristics of disease. BAFF serum levels were significantly higher in 20 healthy controls (i.e., median 695 ng/mL, range 389-1040) in comparison to the whole population of CLL patients (median 376, range 93-8914:, P < 0.0001). After setting a cut-off at the median value observed in healthy controls (i.e., 695 ng/mL) we found that 6 out of 15 (40%) patients with familial CLL had increased BAFF levels while the same occurred only in 5 out of 64 (7.2%) patients with sporadic CLL (P = 0.0007). No significant difference in age (P = 0.82), sex (P = 0.97), Binet clinical stage (P = 0.20), incidence of autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP) (P = 0.47), mutational status of IgVH (P = 1.00), CD38 (P = 0.34) and ZAP-70 expression (P = 0.16) could be detected between patients with sporadic and familial CLL, respectively. The only feature characterizing familial CLL patients was a higher serum BAFF level (sporadic CLL 336 ng/mL, range 93-925; familial CLL 601 ng/mL, range 138-8914; P = 0.002). Our data suggest that BAFF levels are elevated in patients with familial CLL. The small cohort of patients used implies that a larger study is needed to reinforce the observation