Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies.

Modelling the utility of body temperature readings from primary care consults for SARS surveillance in an army medical centre.

INTRODUCTION: There is interest in surveillance systems for outbreak detection at stages where clinical presentation would still be undifferentiated. Such systems focus on detecting clusters of syndromes in excess of baseline levels, which may indicate an outbreak. We model the detection limits of a potential system based on primary care consults for the detection of an outbreak of severe acute respiratory syndrome (SARS). MATERIALS AND METHODS: Data from an averaged-sized medical centre were extracted from the Patient Care Enhancement System (PACES) [the electronic medical records system serving the Singapore Armed Forces (SAF)]. Thresholds were set to 3 or more cases presenting with particular syndromes and a temperature reading of >or=38oC (T >or=38). Monte Carlo simulation was used to insert simulated SARS outbreaks of various sizes onto the background incidence of febrile cases, accounting for distribution of SARS incubation period, delay from onset to first consult, and likelihood of presenting with T >or=38 to the SAF medical centre. RESULTS: Valid temperature data was available for 2,012 out of 2,305 eligible syndromic consults (87.2%). T >or=38 was observed in 166 consults (8.3%). Simulated outbreaks would peak 7 days after exposure, but, on average, signals at their peak would consist of 10.9% of entire outbreak size. Under baseline assumptions, the system has a higher than 90% chance of detecting an outbreak only with 20 or more cases. CONCLUSIONS: Surveillance based on clusters of cases with T >or=38 helps reduce background noise in primary care data, but the major limitation of such systems is that they are still only able to confidently detect large outbreaks.