Latest advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered mutations as a major driver in these disorders. Thus, the detection of mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method. Introduction Bone marrow (BM) biopsy histology is mandatory for discriminating the different chronic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) from reactive BM lesions and from each other. This discrimination is in a proportion of cases not possible on purely histological grounds. The discovery of mutations in and genes has greatly facilitated this differential diagnosis. Polycythaemia vera is associated with mutations and exon 12 mutations) in virtually all cases. In contrast, mutations are present in essential thrombocythaemia (ET) and primary Cerovive myelofibrosis (PMF) in only 50C60%. Mutations of the gene are detectable in 3C5% of ET and 5C8% of PMF patients.1C3 and mutations were selected as the major diagnostic criteria for MPNs in the 2008 World Health Organization (WHO) classification.4 Recently, mutations of the gene were found in 50C80% of and mutation-negative ET and PMF patients.5, 6 Because of this high mutation frequency, detection of mutations is already widely included in the diagnostic programme for MPN. So far mutations are only detectable by molecular assays. These assays are complicated because of the high heterogeneity of mutations with at least 40 different types. These mutations are represented by insertions or deletions, all located in exon 9.7 All mutations cause a frameshift, which lead to a unique Cerovive alternative reading frame coding a novel protein C-terminus consisting of approximately 36 amino acids.5, 6, 8 Vannucchi mutations. However, the polyclonal antibody approach provides only a limited amount of antiserum and usually requires affinity purification of the obtained antiserum by the immobilized immunogene. These limitations can be overcome by the monoclonal antibody (mAb) technology. Here, we report about the generation of a mouse hybridoma designated as CAL2, which secrets antibodies that selectively stain cells carrying mutated proteins in routinely processed BM paraffin sections. Materials and methods Antigen peptide, immunisation and hybridisation The hybridomas were generated by a standard protocol of Synaptic Systems (G?ttingen; see also http://www.sysy.com/mabservice.html) as followed. Briefly, Cerovive we expressed the novel C-terminus peptide (-KM SPARPRTSCR EACLQGWTEA) of mutated in (BL21 D3) as immunogene. Three 8- to 10-week-old BALB/c female mice were subcutaneously immunized over a period of 75 days. Cells from the knee lymph nodes were fused with the mouse myeloma cell line P3X63Ag8.653 (ATCC CRL-1580). The clones used in this study were re-cloned two times by Smad4 limiting dilution and the immunoglobulin subclass was determined. Hybridoma screening The antibodies secreted by the hybridomas were screened for their reactivity against the immunogene by ELISA. The positive mAbs were retested by immunofluorescence on HEK 293 cells transiently transfected with a pEGFPC2-(KMSPARPRTSCREACLQGWTEA) fused to the C-terminus of enhanced green fluorescent protein (EGFP), using the Mirus TransIT package (Madison, WI, USA) based on the manufacturer’s guidelines. To check the performance from the chosen mAbs on paraffin parts of formalin-fixed HEK 293 cells transiently transfected with pEGFPC2-mutated and wt HEK 293 cells were stained with the supernatants of the obtained clones using the immunodetection method described below. The clones with the best performance were selected and Cerovive designated as CAL1, CAL2 and CAL3. Human tissue specimen One hundred and seventy-three specimens including BM samples consisting of myeloid and non-myeloid neoplasms as well as non-neoplastic samples (details in Table 1) were obtained from the archive of the Pathodiagnostik Berlin (Germany), Institute of Pathology of the University Frankfurt (Germany) and from Dr K?mpfe (Ldenscheid, Germany). Table 1 Correlation between CALR mutations.