Comparison of methods for polycythemia rubra vera-1 mRNA quantification in whole-blood leukocytes and purified granulocytes

In the absence of pathognomonic markers, the diagnosis of the two chronic myeloproliferative disorders polycythemia vera (PV) and essential thrombocythemia (ET) has relied on a set of clinical and laboratory criteria (1)(2)(3)(4)(5). The cloning of the cell surface receptor polycythemia rubra vera-1 (PRV-1) has recently been described (6), and the consistent overexpression of PRV-1 mRNA observed in PV patients indicates that this might constitute a new diagnostic marker for the disease. In the initial cohort examined by Northern blot analysis, PRV-1 expression was increased in all PV patients examined as well as in some ET patients, but not in healthy controls (6). These results have also been verified and extended using a quantitative reverse transcription-PCR (RT-PCR) assay. All PV as well as 50% of ET patients displayed increased PRV-1 expression (7)(8). Patients with secondary erythrocytosis and healthy controls tested showed PRV-1 concentrations within the reference interval. Interestingly, the observed increase in PRV-1 mRNA expression does not lead to a corresponding increase in protein expression on the cell surface (9). Erythropoietin-independent colony growth and PRV-1 overexpression seem to go hand in hand in both PV and ET patients (7), raising the hope that RT-PCR for PRV-1 could replace the need for the technically demanding erythropoietin-independent colony assay, although a recent report suggests that the erythropoietin-independent colony growth assay is a more reliable method (10). The aim of the present work was to develop a quantitative RT-PCR method to measure PRV-1 transcripts in whole-blood leukocytes and to determine the potential usefulness of the method in the differential diagnosis of polycythemias and thrombocytosis. The assay was compared with a method using isolated granulocytes (7)(9) to assess whether granulocyte purification is necessary before RNA extraction. Granulocyte fractionation is cumbersome to standardize, and …