In the 8 April 2010 issue of Blood, we reported characterization of IDH1 mutation in a large cohort of acute myeloid leukemia (AML) patients. 1 Very recently, a series of reports about this mutation in AML have been published in a cluster of manuscripts. 1-7 As IDH1 mutation brings prognostic information in glioma and possibly AML, 4, 8-9 identification of IDH1 R132 mutations will bring increasing clinical relevance. Moreover, the mutation seems quite stable and may serve as a marker for monitoring minimal residual disease. 1 Hence, a sensitive and simple method for detecting this mutation will be highly desirable. We here report a very sensitive, single-tube, multiplex polymerase chain reaction (PCR)–based method, which has been verified by direct sequencing method. With this method, we then determined the stability of the R132 mutation in sequential samples of AML and measured the incidence of this mutation in a cohort of patients with myelodysplastic syndrome (MDS). This study has been approved by the Institutional Review Board of the National Taiwan University Hospital. We designed 6 forward mutation-specific primers to cover all possible types of R132 mutations (Figure 1A), although only 5 of them had been reported until now. 1-7 Another upstream forward primer was used to generate a product as an internal control. Combination of these 7 forward primers and a common reverse primer (Figure 1A) would generate a shorter mutant and a longer control product in any genomic DNA containing any type of R132 mutation, whereas only the longer product would be seen in samples without this mutation (Figure 1B). PCR was performed by heating at 95 C for 10 minutes, followed by 45 cycles of 95 C, 62 C, and 72 C for 30 seconds each in a total of 25 μL containing 200nM each dNTP, 1.5 mM MgSO4, 50ng of genomic DNA, 100nM upstream forward primer, 500nM each of the other 7 primers, and 1.5 units of PlatinumTaq polymerase (Invitrogen). A single tube combining 8 primers would be easy, economic, and fast in screening any possible type of IDH1 R132 mutation. We verified the utility of this multiplex method in 103 AML marrow samples previously studied by PCR and direct sequencing. 1 Such selection is based solely on availability of samples, without consideration of any other factors. Comparison of this multiplex PCR with direct sequencing revealed perfect concordance except for 1 patient whose IDH1 mutation was detected only by multiplex PCR but not by direct sequencing (FL91 in Figure 1C). Furthermore, in 9 patients with IDH1 mutation at diagnosis the same mutation could be detected by multiplex method in all 11 samples obtained at disease relapse, including the 4 in which mutant signals were no longer seen by sequencing (Figure 1D and data not shown). The sensitivity of this method was approximately 0.5%, obviously higher than sequencing (Figure 1E). We also