Comparison of three PCR assays for the evaluation of interferon-beta biological activity in patients with multiple sclerosis.

Background: The gene expression of the myxovirus-resistant protein A (MxA) gene is a sensitive measure of the biological response of therapeutically applied interferon-beta (IFNbeta) and of its reduced bioavailability due to inhibiting factors such as IFNbeta-induced neutralizing antibodies (NAbs).

Methods: We compared three methods for MxA mRNA quantification in 826 peripheral blood mononuclear cell (PBMC) samples obtained from patients with multiple sclerosis (MS). MxA mRNA measurements were performed using quantitative-competitive (qc)-PCR, real time-PCR, and the new semi-quantitative (sq)-PCR assay (MxA IBRIDOGEN).

Results: According to the treatment status (untreated samples versus NAb-negative treated samples), real time-PCR gave the highest specificity (93%). Slightly lower specificities were obtained with qc-PCR and sq-PCR (both 91%). qc-PCR showed the highest sensitivity (97%) compared with both real time-PCR (94%) and sq-PCR (95%). A positive correlation was found between qc-PCR and real time-PCR measurements (rspearman=0.776; p<0.0001), which also showed 90% agreement based on a statistically calculated threshold. Likewise, sq-PCR evaluations showed 84% and 79% agreement with qc-PCR and real time-PCR measurements, respectively. In addition, we showed a concordance of 89% between three sq-PCR kits.

Conclusions: All three methods displayed high specificity for MxA gene expression analysis, allowing the detection of patients in whom IFNbeta did not have any biological action. qc-PCR and real time-PCR are both useful during clinical trials demanding quantitative data of biological activity, whereas sq-PCR could prove useful for routine screening purposes because it is easy to perform and can be done in not specialized laboratories.