Annals of Hematology

, Volume 98, Issue 10, pp 2339–2346 | Cite as

CAL2 monoclonal antibody is a rapid and sensitive assay for the detection of calreticulin mutations in essential thrombocythemia patients

  • Massimiliano BonifacioEmail author
  • Rachele Montemezzi
  • Alice Parisi
  • Giovanna De Matteis
  • Roberta Bertorelle
  • Luigi Scaffidi
  • Cinzia Candiotto
  • Giuseppe Lippi
  • Alberto Zamò
  • Marco Chilosi
  • Giovanni Pizzolo
  • Aldo Scarpa
  • Mauro Krampera
Original Article


Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.


Essential thrombocythemia Calreticulin CAL2 Driver mutations Diagnostic tools 



M.B. and M.C. designed the study; M.B., R.M. and L.S. treated patients, analysed the data and wrote the manuscript; A.P. and A.Z. performed histopathological tests and analysed the data; G.D.M., R.B. and C.C. performed molecular biology tests; G.L., G.P., A.S. and M.K. critically revised the manuscript. All authors have read and approved the final version.

Compliance with ethical standards

Conflict of interest

M. Bonifacio declares research funding from Novartis and received speaker bureau and advisory board honorarium from Amgen, Bristol-Myers Squibb, Incyte, Novartis and Pfizer, outside the present study. All the other authors declare no competing financial interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

277_2019_3741_MOESM1_ESM.docx (95 kb)
ESM 1 (DOCX 94 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Massimiliano Bonifacio
    • 1
    Email author
  • Rachele Montemezzi
    • 1
  • Alice Parisi
    • 2
  • Giovanna De Matteis
    • 3
  • Roberta Bertorelle
    • 4
  • Luigi Scaffidi
    • 1
  • Cinzia Candiotto
    • 4
  • Giuseppe Lippi
    • 3
  • Alberto Zamò
    • 5
  • Marco Chilosi
    • 6
  • Giovanni Pizzolo
    • 1
  • Aldo Scarpa
    • 2
  • Mauro Krampera
    • 1
  1. 1.Department of Medicine, Section of HematologyUniversity of VeronaVeronaItaly
  2. 2.Department of Diagnostics and Public Health, Section of Pathological AnatomyUniversity of VeronaVeronaItaly
  3. 3.Department of Diagnostics and Public Health, Section of Clinical BiochemistryUniversity of VeronaVeronaItaly
  4. 4.Immunology and Molecular OncologyVeneto Institute of Oncology IOV-IRCCSPaduaItaly
  5. 5.Department of Oncology, Section of Pathological AnatomyUniversity of TurinTurinItaly
  6. 6.Department of PathologyPederzoli Hospital, Peschiera del GardaVeronaItaly

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