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

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Abstract

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

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Acknowledgements

The authors wish to thank Rita Guerrieri, Angela Zaccaria, and Marina Panealbo for their expert nursing assistance and Anna Messina for her excellent administrative support.

Financial support was obtained from the Piedmont Health Service, ARB (Associazione Ricerca Biomedica, ONLUS), and Fondazione Cavalieri Ottolenghi per le Neuroscienze.

MxA IBRIDOGEN® Sq-PCR assay was developed and provided by Amplimedical SpA which had no role in data collection, data analysis, data interpretation, or writing of this paper.

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Authors and Affiliations

  1. Regional Multiple Sclerosis Center (CReSM) & Clinical Neurobiology, Orbassano, S. Luigi Gonzaga Hospital, Torino, Italy

    Francesca Gilli, Fabiana Marnetto, Simona Malucchi, Marco Capobianco, Marzia Caldano, Arianna Sala & Antonio Bertolotto

  2. Laboratorio Ricerca e Sviluppo, Amplimedical SpA - Bioline Division, Buttigliera Alta, Torino, Italy

    Guglielmo Stefanuto, Valentina Rinaldi & Federica Farinazzo

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  1. Francesca Gilli
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  2. Fabiana Marnetto
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  3. Guglielmo Stefanuto
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  4. Valentina Rinaldi
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  5. Federica Farinazzo
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  6. Simona Malucchi
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  7. Marco Capobianco
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  8. Marzia Caldano
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  9. Arianna Sala
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  10. Antonio Bertolotto
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Correspondence to Francesca Gilli.

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Gilli, F., Marnetto, F., Stefanuto, G. et al. Comparison of three PCR assays for the evaluation of interferon-β biological activity in patients with multiple sclerosis. CNS Drugs 8, 185–194 (2004). https://doi.org/10.1007/BF03260063

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