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

// Margherita Perricone 1 , Francesca Palandri 1 , Emanuela Ottaviani 1 , Mario Angelini 2 , Laura Bagli 3 , Enrica Bellesia 4 , Meris Donati 5 , Donato Gemmati 6 , Patrizia Zucchini 7 , Stefania Mancini 8 , Valentina Marchica 9 , Serena Trubini 10 , Giovanna De Matteis 11 , Silvia Di Zacomo 12 , Mose Favarato 13 , Annamaria Fioroni 14 , Caterina Bolzonella 15 , Giorgia Maccari 16 , Filippo Navaglia 17 , Daniela Gatti 18 , Luisa Toffolatti 19 , Linda Orlandi 20 , Veronique Laloux 21 , Marco Manfrini 1 , Piero Galieni 2 , Barbara Giannini 3 , Alessia Tieghi 4 , Sara Barulli 5 , Maria Luisa Serino 6 , Monica Maccaferri 7 , Anna Rita Scortechini 8 , Nicola Giuliani 9 , Daniele Vallisa 10 , Massimiliano Bonifacio 11 , Patrizia Accorsi 12 , Cristina Salbe 13 , Vinicio Fazio 14 , Milena Gusella 15 , Eleonora Toffoletti 16 , Marzia Salvucci 3 , Mirija Svaldi 18 , Filippo Gherlinzoni 19 , Francesca Cassavia 20 , Francesco Orsini 20 , Giovanni Martinelli 1 1 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology ‘L. and A. Seragnoli’, University of Bologna, S. Orsola-Malpighi Hospital, Bologna, Italy 2 Molecular Hematology Laboratory U.O.C of Hematology Hospital Mazzoni, Ascoli Piceno, Italy 3 Medical Genetics Unit- Hub Laboratory AUSL Romagna, Pievesestina di Cesena, Italy 4 Imaging and Laboratory Diagnostic Department, Clinical Chemistry and Endocrinology Laboratory, Hematology Unit, Oncology and Technology Department, Hospital S. Maria Nuova, IRCCS, Reggio Emilia, Italy 5 Clinical Pathology Laboratory, A.O. Ospedali Riuniti Marche Nord, Pesaro, Italy 6 Center Hemostasis and Thrombosis, Section of Medical Biochemistry, Molecular Biology and Genetics, Department of Biomedical and Specialty Surgical Sciences, University of Ferrara, Ferrara, Italy 7 Department of Medical and Surgical Sciences, Division of Hematology, University of Modena and Reggio Emilia, Modena, Italy 8 Clinical Hematology Laboratory, Department of Molecular and Clinical Sciences, Polytechnic University of Marche, Ancona, Italy 9 Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy 10 Clinical Pathology, Molecular Biology Laboratory, and Hematology/Bone Marrow Transplantation Unit, AUSL Piacenza, Piacenza, Italy 11 Section of Clinical Biochemistry and Section of Hematology, Azienda Ospedaliera Universitaria Integrata di Verona, Verona, Italy 12 Department of Hematology, Blood Bank and Biotechnology, Ospedale Civile Pescara, Pescara, Italy 13 UOS Molecular Diagnostics, Department of Clinical Pathology, ULSS12 Venetian, Venice, Italy 14 UOC laboratory medicine, P.O. San Salvatore, Sulmona, L’Aquila, Italy 15 Department of Oncology, Laboratory of Pharmacology and Molecular Biology, ULSS 18, Rovigo, Italy 16 Clinical Hematology, Department of Experimental and Clinical Medical Sciences, University of Udine, Udine, Italy 17 Department of Laboratory Medicine, University-Hospital of Padova, Padova, Italy 18 Department of Haematology and BMT, Healthcare Company of South Tyrol, District of Bolzano, Bolzano, Italy 19 Department of Pathology and Haematology, Treviso General Hospital, Treviso, Italy 20 Werfen, Milano, Italy 21 QIAGEN GmbH, Hilden, Germany, member of the European LeukemiaNet (ELN) Foundation Circle Correspondence to: Margherita Perricone, email: margherita.perricon2@unibo.it Keywords: JAK2 V617F mutation, myeloproliferative neoplasms, qPCR standardization, molecular diagnosis Received: November 01, 2016      Accepted: February 22, 2017      Published: March 06, 2017 ABSTRACT To date, a plenty of techniques for the detection of JAK2 V617F 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 JAK2 V617F 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 JAK2 V617F 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 JAK2 V617F 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 JAK2 V617F 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 JAK2 V617F 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 JAK2 V617F .