Allele-specific PCR of BRAF^V600E in PBMCs. (A) The distribution of BRAF^V600E in PBMCs sorted into quadrants as shown according to expression of HLA-DR and lineage. Pie charts summarize the distribution of mutant alleles in HCL, LCH, and ECD. The area of each quadrant in the pie is proportional to the total number of mutant alleles in that quadrant (ie, the percentage positivity of the quadrant multiplied by the number of cells in the quadrant). All patients had active disease at the time of sampling. Two children with LCH included for comparison are indicated. Asterisk indicates the threshold of detection for a negative result where cell numbers were limiting. (B) The distribution BRAF^V600E among peripheral blood cells showing the percentage of mutated alleles detected. Gray shading indicates positive fractions. Asterisk indicates child with MS-LCH shown for comparison. All patients had active disease at the time of sampling. Italics indicate those who had received prior treatment. B, B cell; Mono, monocyte; Neuts, neutrophil; NK, natural killer; NT, not tested owing to lack of or insufficient material; pDC, plasmacytoid dendritic cell; T, T cell.