Molecular cell biologic assay of RALD. (A) Flow cytometric analysis of double-negative T cells. CD8 and CD4 double staining was performed in T-cell receptor-αβ-expressing cells. (B) Electropherogram showing KRAS G13D mutation in BM-MNCs in case 1 (left panel) and case 2 (right panel). (C) Gene dosage of mutated allele in granulocytes (Gr), T cells (T), and B cells (B). Relative gene dosage was estimated by a mutant allele-specific polymerase chain reaction method in cases 1 and 2 using albumin gene as internal control. (D) Apoptosis assay using activated T cells. Apoptosis percentage was measured by flow cytometry with annexin V staining 24 and 48 hours after IL-2 depletion. (E) Apoptosis percentage was measured 24 hours after addition of anti-FAS CH11 antibody (final 100 ng/mL). (F) Western blotting analysis of Bim expression.