XCIP and V617F JAK2 mutation analysis in patients with ET. (A) Principle of method using a mismatch PCR primer and AflIII digestion of PCR products. (B) Gel showing digested products from a WT control, mutant-positive homozygote (Hm), and 3 heterozygotes (Ht) with differing levels of mutant. (C) Validation of mutant JAK2 quantification assay using mixes of DNA from homozygous WT (HL60) and mutant (HEL) cell lines. (D) Serial analysis of neutrophil XCIPs in 14 patients with polyclonal myelopoiesis. Dotted lines represent the threshold for clonality (> 75% expression of one allele) provided there was more than 20% difference from the T-cell XCIP and the patient was younger than 65 years of age. (E) Serial analysis of mutant JAK2 level in neutrophil samples from 20 mutant-positive patients. Results are expressed as the percentage difference from the first sample tested. Dotted lines represent the limits of technical variation (mean ± 1 standard deviation for percentage difference in mutant level in 46 duplicate analyses).