title: Capillary electrophoresis artifact due to eosin: implications for the interpretation of molecular diagnostic assays.
abstract: Capillary electrophoresis (CE) is a commonly used tool in the analysis of fluorescently labeled PCR amplification products. We have identified a CE artifact caused by the tissue stain eosin that can complicate the interpretation of CE data. The artifact was detected during routine analysis of a DNA sample isolated from a formalin-fixed, paraffin-embedded tissue sample considered histologically suspicious for a B-cell neoplasm. A standard clinical PCR and CE assay for immunoglobulin heavy chain (IGH) gene rearrangement revealed a weak polyclonal population of rearranged IGH genes and a 71 base peak suspicious for IGH clonality. The spectral properties of the 71 base peak were unusual in that although the dominant fluorescence of the peak was blue, it also fluoresced in green and yellow (blue>green>yellow), raising the suspicion that the peak might represent an artifact. CE analysis of the genomic DNA sample without PCR amplification demonstrated the presence of the 71 base peak, suggesting that the artifact was caused by a contaminant within the DNA sample itself. We demonstrate that eosin, which was used to stain the formalin-fixed tissue during processing, yields a discrete 71 base peak of similar morphology to the contaminant peak on CE analysis. The data suggest that eosin in the fixed tissue was not completely eliminated during nucleic acid extraction, resulting in the artifact peak. We discuss the implications of this potentially common contaminant on the interpretation of CE data and demonstrate that artifacts caused by eosin can be avoided by using more stringent DNA purification steps. Histological dyes may fluoresce, and artifacts from them should be considered when primary peaks contain additional underlying peaks of other colors.
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Murphy
authors.forename: Karin D
authors.initials: KD
authors.lastname: Berg
authors.forename: Tanya
authors.initials: T
authors.lastname: Geiger
authors.forename: Michael
authors.initials: M
authors.lastname: Hafez
authors.forename: Katie A
authors.initials: KA
authors.lastname: Flickinger
authors.forename: Lisa
authors.initials: L
authors.lastname: Cooper
authors.forename: Patrick
authors.initials: P
authors.lastname: Pearson
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
mesh_terms: D000368:Aged; D016477:Artifacts; D019075:Electrophoresis, Capillary; D004801:Eosine Yellowish-(YS) / Q000737:chemistry*; D005260:Female; D005456:Fluorescent Dyes; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D016393:Lymphoma, B-Cell / Q000175:diagnosis* / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction
chemical_list: D005456:Fluorescent Dyes; D007143:Immunoglobulin Heavy Chains; D004801:Eosine Yellowish-(YS)