Relationship of V617F-ET to V617F⁺ ET and comparison of proportion of V617F⁺ granulocytes to proportion of clonal granulocytes estimated by HUMARA. (A) Allele-specific PCR for V617F mutation (3-primer method) performed on peripheral-blood leukocyte samples obtained at follow-up of 12 ET patients who were V617F^- a median 77 months earlier. Lanes 1-12, follow-up ET samples; lane 13, healthy individual; lane 14, patient with V617F⁺ ET; lane 15, water. (B) Standard curve for quantitation of proportion of V617F⁺ alleles using 4-primer allele-specific PCR. Three separate experiments are shown, and the line is the nonlinear least squares fit of a 3-parameter logistic curve. The x-axis shows the logarithm of the ratio of mutant allele peak area to WT peak area and the y-axis is the known proportion of mutant allele in a dilution series of known homozygous V617F DNA into normal DNA. Expt indicates experiment. (C) Estimated proportion of clonal granulocytes by XCIPs (x-axis) and proportion of V617F⁺ granulocytes (y-axis) in 20 women with V617F⁺ MPDs (10 ET, blue; 10 PV, red). Each assay was performed in duplicate and the boxes for each patient represent the 2 values for each measurement for both HUMARAand V617F to demonstrate the interassay variability. For discussion of patients marked A, B, and C, see “Comparison of XCIPs with proportion of V617F⁺ granulocytes” in the text.