Feasibility of Qualitative Testing of BCR-ABL and JAK2 V617F in Suspected Myeloproliperative Neoplasm (MPN) Using RT-PCR Reversed Dot Blot Hybridization (RT-PCR RDB)

Najmiatul Masykura, Ummu Habibah, Siti Fatimah Selasih, Soegiarto Gani, Cosphiadi Irawan, Sugiyono Somoastro, Ahmad Rusdan Handoyo Utomo

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Defining the presence of BCR-ABL transcript in suspected myeloproliferative neoplasm is essential in establishing chronic myeloid leukemia. In the absence of BCR-ABL, the conventional diagnostic algorithm recommends JAK2 V617F mutation testing to support diagnosis of other MPN diseases such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis. In certain cases of thrombocythemia, simultaneous upfront testing of both BCR-ABL and JAK2 may be desirable. We wanted to test the feasibility of multiplex detection of BCR-ABL transcript variants and JAK2 V617F mutation simultaneously using the reverse transcriptase polymerase chain reaction (RT-PCR)-based reverse dot-blot hybridization (RDB) method. Material and Methods: Separate biotinylated RT-PCR primers were designed to amplify specific BCR-ABL transcripts and JAK2 V617F mutant alleles. Specific hybridization of RT-PCR products with arrays of membrane-bound probes followed by colorimetric development would allow simultaneous visualization of BCR-ABL and/or JAK2 mutant transcripts in a given specimen. To validate the RDB method, we used cDNA specimens previously referred to our laboratory for routine clinical testing of BCR-ABL and/or JAK2. Results: The limit of detection or analytical sensitivity of the RDB method using cDNA specimens was 0.5% and 6.25% in detecting BCR-ABL and JAK2 mutant transcripts, respectively. The diagnostic specificity and sensitivity to detect BCR-ABL and JAK2 were 100% and 92.3% (N = 38); and 100% and 100% (N = 27), respectively. RDB also detected BCR-ABL transcripts in 22% of JAK2 V617F mutation-positive samples (N = 14). Conclusions: RT-PCR RDB is a promising qualitative multiplex method to detect BCR-ABL and JAK2 mutant transcripts simultaneously. In this study, we described the feasibility of reverse transcriptase polymerase chain reaction (RT-PCR) reverse dot-blot hybridization for BCR-ABL and JAK2 co-testing in patients with myeloproliferative neoplasms. We have demonstrated that the sensitivity and specificity of the RT-PCR reverse dot-blot hybridization method are similar to routine methods. This method is suitable for laboratories in developing countries. We are hopeful that our results would contribute to simplify the diagnostic workup of suspected myeloproliferative neoplasms, especially in the developing countries with limited access to real time RT-PCR-based tests.

Original languageEnglish
JournalClinical Lymphoma, Myeloma and Leukemia
DOIs
Publication statusPublished - 1 Jan 2019

Keywords

  • CML
  • Diagnostic test
  • Indonesia
  • Multiplex testing
  • PV

Fingerprint

Dive into the research topics of 'Feasibility of Qualitative Testing of BCR-ABL and JAK2 V617F in Suspected Myeloproliperative Neoplasm (MPN) Using RT-PCR Reversed Dot Blot Hybridization (RT-PCR RDB)'. Together they form a unique fingerprint.

Cite this