Research Papers:

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories

Margherita Perricone _, Francesca Palandri, Emanuela Ottaviani, Mario Angelini, Laura Bagli, Enrica Bellesia, Meris Donati, Donato Gemmati, Patrizia Zucchini, Stefania Mancini, Valentina Marchica, Serena Trubini, Giovanna De Matteis, Silvia Di Zacomo, Mosè Favarato, Annamaria Fioroni, Caterina Bolzonella, Giorgia Maccari, Filippo Navaglia, Daniela Gatti, Luisa Toffolatti, Linda Orlandi, Vèronique Laloux, Marco Manfrini, Piero Galieni, Barbara Giannini, Alessia Tieghi, Sara Barulli, Maria Luisa Serino, Monica Maccaferri, Anna Rita Scortechini, Nicola Giuliani, Daniele Vallisa, Massimiliano Bonifacio, Patrizia Accorsi, Cristina Salbe, Vinicio Fazio, Milena Gusella, Eleonora Toffoletti, Marzia Salvucci, Mirija Svaldi, Filippo Gherlinzoni, Francesca Cassavia, Francesco Orsini and Giovanni Martinelli

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Oncotarget. 2017; 8:32608-32617. https://doi.org/10.18632/oncotarget.15940

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Margherita Perricone1, Francesca Palandri1, Emanuela Ottaviani1, Mario Angelini2, Laura Bagli3, Enrica Bellesia4, Meris Donati5, Donato Gemmati6, Patrizia Zucchini7, Stefania Mancini8, Valentina Marchica9, Serena Trubini10, Giovanna De Matteis11, Silvia Di Zacomo12, Mosè Favarato13, Annamaria Fioroni14, Caterina Bolzonella15, Giorgia Maccari16, Filippo Navaglia17, Daniela Gatti18, Luisa Toffolatti19, Linda Orlandi20, Vèronique Laloux21, Marco Manfrini1, Piero Galieni2, Barbara Giannini3, Alessia Tieghi4, Sara Barulli5, Maria Luisa Serino6, Monica Maccaferri7, Anna Rita Scortechini8, Nicola Giuliani9, Daniele Vallisa10, Massimiliano Bonifacio11, Patrizia Accorsi12, Cristina Salbe13, Vinicio Fazio14, Milena Gusella15, Eleonora Toffoletti16, Marzia Salvucci3, Mirija Svaldi18, Filippo Gherlinzoni19, Francesca Cassavia20, Francesco Orsini20, Giovanni Martinelli1

1Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology ‘L. and A. Seràgnoli’, University of Bologna, S. Orsola-Malpighi Hospital, Bologna, Italy

2Molecular Hematology Laboratory U.O.C of Hematology Hospital Mazzoni, Ascoli Piceno, Italy

3Medical Genetics Unit- Hub Laboratory AUSL Romagna, Pievesestina di Cesena, Italy

4Imaging and Laboratory Diagnostic Department, Clinical Chemistry and Endocrinology Laboratory, Hematology Unit, Oncology and Technology Department, Hospital S. Maria Nuova, IRCCS, Reggio Emilia, Italy

5Clinical Pathology Laboratory, A.O. Ospedali Riuniti Marche Nord, Pesaro, Italy

6Center Hemostasis and Thrombosis, Section of Medical Biochemistry, Molecular Biology and Genetics, Department of Biomedical and Specialty Surgical Sciences, University of Ferrara, Ferrara, Italy

7Department of Medical and Surgical Sciences, Division of Hematology, University of Modena and Reggio Emilia, Modena, Italy

8Clinical Hematology Laboratory, Department of Molecular and Clinical Sciences, Polytechnic University of Marche, Ancona, Italy

9Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy

10Clinical Pathology, Molecular Biology Laboratory, and Hematology/Bone Marrow Transplantation Unit, AUSL Piacenza, Piacenza, Italy

11Section of Clinical Biochemistry and Section of Hematology, Azienda Ospedaliera Universitaria Integrata di Verona, Verona, Italy

12Department of Hematology, Blood Bank and Biotechnology, Ospedale Civile Pescara, Pescara, Italy

13UOS Molecular Diagnostics, Department of Clinical Pathology, ULSS12 Venetian, Venice, Italy

14UOC laboratory medicine, P.O. San Salvatore, Sulmona, L’Aquila, Italy

15Department of Oncology, Laboratory of Pharmacology and Molecular Biology, ULSS 18, Rovigo, Italy

16Clinical Hematology, Department of Experimental and Clinical Medical Sciences, University of Udine, Udine, Italy

17Department of Laboratory Medicine, University-Hospital of Padova, Padova, Italy

18Department of Haematology and BMT, Healthcare Company of South Tyrol, District of Bolzano, Bolzano, Italy

19Department of Pathology and Haematology, Treviso General Hospital, Treviso, Italy

20Werfen, Milano, Italy

21QIAGEN GmbH, Hilden, Germany, member of the European LeukemiaNet (ELN) Foundation Circle

Correspondence to:

Margherita Perricone, email: [email protected]

Keywords: JAK2 V617F mutation, myeloproliferative neoplasms, qPCR standardization, molecular diagnosis

Received: November 01, 2016     Accepted: February 22, 2017     Published: March 06, 2017


To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.

A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.

The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.

In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

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