Pre-analytical and post-analytical evaluation in the era of molecular diagnosis of sexually transmitted diseases: cellularity control and internal control

  • Loria Bianchi | loriabianchi@tin.it U.O. Laboratorio Analisi, Sezione Microbiologia, Ospedale “San Jacopo”, Pistoia, Italy.
  • Zaleida Napoli U.O. Laboratorio Analisi, Sezione Microbiologia, Ospedale “San Jacopo”, Pistoia, Italy.
  • Simona Turrisi U.O. Laboratorio Analisi, Sezione Microbiologia, Ospedale “San Jacopo”, Pistoia, Italy.
  • Swan Donati U.O. Laboratorio Analisi, Sezione Microbiologia, Ospedale “San Jacopo”, Pistoia, Italy.
  • Riccardo Lari U.O. Laboratorio Analisi, Sezione Microbiologia, Ospedale “San Jacopo”, Pistoia, Italy.

Abstract

Background. Increase of molecular tests performed on DNA extracted from various biological materials should not be carried out without an adequate standardization of the pre-analytical and post-analytical phase.
Materials and Methods. Aim of this study was to evaluate the role of internal control (IC) to standardize pre-analytical phase and the role of cellularity control (CC) in the suitability evaluation of biological matrices, and their influence on false negative results. 120 cervical swabs (CS) were pre-treated and extracted following 3 different protocols. Extraction performance was evaluated by amplification of: IC, added in each mix extraction; human gene HPRT1 (CC) with RT-PCR to quantify sample cellularity; L1 region of HPV with SPF10 primers. 135 urine, 135 urethral swabs, 553 CS and 332 ThinPrep swabs (TP) were tested for C. trachomatis (CT) and U. parvum (UP) with RT-PCR and for HPV by endpoint-PCR. Samples were also tested for cellularity.
Results. Extraction protocol with highest average cellularity (Ac)/sample showed lowest number of samples with inhibitors; highest HPV positivity was achieved by protocol with greatest Ac/PCR. CS and TP under 300.000 cells/sample showed a significant decrease of UP (P<0.01) and HPV (P<0.005) positivity. Female urine under 40.000 cells/mL were inadequate to detect UP (P<0.05).
Conclusions. Our data show that IC and CC allow optimization of pre-analytical phase, with an increase of analytical quality. Cellularity/sample allows better sample adequacy evaluation, crucial to avoid false negative results, while cellularity/PCR allows better optimization of PCR amplification. Further data are required to define the optimal cut-off for result normalization.

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Published
2014-06-30
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Original Articles
Keywords:
microbiological molecular tests, cellularity quantification, sample suitability
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How to Cite
Bianchi, L., Napoli, Z., Turrisi, S., Donati, S., & Lari, R. (2014). Pre-analytical and post-analytical evaluation in the era of molecular diagnosis of sexually transmitted diseases: cellularity control and internal control. Microbiologia Medica, 29(1). https://doi.org/10.4081/mm.2014.4718