Single colony assays for del20q and JAK2-V617F. PBMCs were grown in methylcellulose in the presence of erythropoietin. Single erythroid colonies (BFU-E) and granulocytic colonies (CFU-G) were picked and analyzed individually for the presence of del20q and JAK2-V617F by microsatellite PCR and allele-specific PCR, respectively. Each colony is represented by a dot that is placed into one of 6 quadrangles representing the 6 possible genotypes: wild-type (wt), heterozygous (het), and homozygous (hom) for JAK2-V617F or MPL-W515L on the vertical axis, and absence (open quadrangles) or presence of del20q (gray quadrangles) on the horizontal axis. Results for BFU-Es are shown in the upper part and CFU-Gs in the lower part of the panels. The unique patient numbers, the diagnoses (PMF, ET, PV, sMF indicates secondary myelofibrosis), the allelic ratio of JAK2-V617F or MPL-W515L in purified granulocytes (%T), and the date of the sample drawing are shown above the corresponding boxes. (A) This group of patients has acquired the del20q before JAK2-V617F, as demonstrated by the presence of JAK2 wt colonies carrying del20q. For both patients shown, 2 sequential samples were analyzed. (B) In this group of patients, del20q was acquired in a cell already carrying JAK2-V617F, as indicated by the presence of JAK2-V617F-positive colonies with or without del20q. (C) Patients with a complex pattern of del20q acquisition that does not comply with a linear temporal order of events. In patient Vi346, the del20q coexists with MPL-W515L. (D) A patient with del20q that is negative for the JAK2-V617F mutation.