Dear Editor, Calreticulin (CALR) gene mutations (CALR) have recently been discovered in about 20–35 % of patients affected by essential thrombocythemia (ET) and primarymyelofibrosis (PMF) [1, 2]. Several molecular assays have been developed to detect the most frequentCALR (type 1 consisting of a 52bp deletion, and type 2 of a 5-bp insertion) [3, 4]. All these techniques are useful for identifyingCALR at the diagnosis, but they are not suitable for minimal residual disease (MRD) monitoring, since the maximum sensitivity is 1 %. The droplet digital PCR (ddPCR) technology is a third-generation PCR method that started to be used in hematological malignancies [5–7].We describe a ddPCR assay with a sensitivity of 0.01% developed for the absolute quantification of CALR type 1 and 2 mutations and analyze a cohort of 57 JAK2V617F-negative myeloproliferative neoplasm patients. ddPCR experiments were performed using the QX-200 instrument (BioRad) and specific primers and probes were designed for both type 1 and type 2 mutations (see Supplementary Files). CALR load in each sample was expressed as fractional abundance (FA, mutant allele/mutant allele + wild-type allele). The CALR allelic burden resulted heterogeneous in both ET (min.13.8 %– max. 51%) and PMF (min. 34.5%–max. 51.3%) patients.We show that the medianCALR allelic burden at diagnosis was significantly higher in PMF patients as compared to ET case (47.9 vs 43.8 %, p = 0.008) whereas no significant difference was observed between the type 1 and 2 mutations (Fig. 1a). Moreover, no relationship between the genemutation type and the CALR amount was observed within each group of ET and PMF patients. In our ET series, there were 14 (29.7 %) patients with a very low FA, <30 %; this group was not statistically different in terms of hemoglobin, white blood cells and platelet counts, age, sex, thrombosis and/or hemorrhage, and CALR type compared to those with FA >30 %. The PMF group included ten patients, too few for any type of statistical considerations. Sequential evaluations by ddPCR experiments were performed in three patients to monitor the CALR load during treatment. CALR load at diagnosis was 15.8 and 48 % in two ET patients. The former patient was treated with interferon-α (IFN-α) and after 5 years from diagnosis the FA was 7.7 %. The latter was also treated with IFN-α and after 2 years from diagnosis, the CALR load was 14.7 %. Both patients had stable disease and a well-controlled platelet count. A 44-year-old man at PMF diagnosis showed a FA of 49.7 %; 8 years later, we observed a leukemic transformation. At the time of the AML evolution, theCALR load was 0 %; this finding was also confirmed by PCR qualitative analysis. The patient underwent induction chemotherapy, achieving complete remission, then allogeneic bone marrow transplantation (ABMT) from a HLA-matched related donor. Two months later, the FA observed by ddPCR analysis was 0.01 %; 7 months after, the ABMT and AML relapsed and at this time, the CALR load was 13.5 % (Fig. 1b, c). Although the importance of the CALR allelic burden determination has not yet been defined at the disease onset, the utility of and need for a sensitive method, like our ddPCR assay, are unquestionable for the purposes of MRD monitoring [8–10]. Electronic supplementary material The online version of this article (doi:10.1007/s00277-016-2739-2) contains supplementary material, which is available to authorized users.