Detection of CD44 in neoplastic MCs by immunostaining and qPCR. (A) BM sections of a patient with MCL (patient 156 in supplemental Table 1) were stained by IHC with an antibody against CD44 (left) and tryptase (right). The spindle-shaped tryptase⁺ MCs coexpressed CD44. (B) CD44 was also stained in BM sections of patients with ISM (left), ASM (middle), and MCL (right). In all patients examined, CD44 was found to be expressed in the cytoplasm of neoplastic MCs. (C) CD44 expression in primary neoplastic BM MCs (top) and circulating PB MCs (bottom) was analyzed by ICC in 2 patients with MCL. (D) In addition, CD44 expression was determined in the human MC lines HMC-1, ROSA, and MCPV-1. The chronic myeloid leukemia (CML) cell line K562 served as a negative control. MCs stained positive for CD44 in all MCL patients (C) and cell lines (D) tested. Slides were examined using an Olympus DP21 camera connected to an Olympus BX50F4 microscope (Olympus Corporation; Shinjuku, Tokyo, Japan) equipped with 60×/0.90 UPlanFL (IHC) or 100×/1.35 UPlanAPO (Oil Iris; ICC) objective lenses. Images were prepared with 1000× (ICC) or 600× (IHC) magnifications and adjusted by Adobe Photoshop CS5 software version 12.0.4 (Adobe Systems, San Jose, CA). (E) CD44 mRNA expression in mononuclear cells (MNCs) obtained from BM of patients with ISM (n = 3), SM-AHN (n = 4), ASM (n = 2), or MCL (n = 3) or from normal/reactive BM (n = 9) and PB of 6 healthy controls was analyzed by qPCR. (F) CD44 mRNA expression levels were also analyzed by qPCR in various MCL-like cell lines (HMC-1, ROSA, and MCPV-1) and in the CML cell lines K562, KU812, and KCL22. Results are shown as percentage of CD44 mRNA copies relative to ABL1 mRNA levels and are expressed as mean ± standard deviation (SD) of all donors in each group (E) or as mean ± SD of 3 independent experiments (F).