Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Abstract

sUMMarYDevelopment and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

Detection of papillomavirus DNA in formalin-fixed paraffin-embedded equine aural plaque samples

“…Therefore, FFPE samples represent an important source for retrospective studies (Newkirk et al 2014); however, the use of an adequate fixation process to conserve tissue morphology may negatively affect nucleic acid preservation and may consequently also affect molecular diagnostic procedures (Barcelos et al, 2008). In the present study, the following measures were taken to minimize the adverse effects of the FFPE process: the samples used were approximately 6-mm thick, a 10:1 ratio of formalin to tissue was used, neutral-buffered formalin solution was used instead of unbuffered formalin solutions, and a 24-h fixation time and pure paraffin were used.…”

“…In the present study, the following measures were taken to minimize the adverse effects of the FFPE process: the samples used were approximately 6-mm thick, a 10:1 ratio of formalin to tissue was used, neutral-buffered formalin solution was used instead of unbuffered formalin solutions, and a 24-h fixation time and pure paraffin were used. These strategies are generally used to avoid overfixation and to facilitate the extraction of usable nucleic acids (Barcelos et al, 2008;Qiagen, 2010). In the present study, the DNA quality and concentration were considered adequate for PCR procedures (Qiagen, 2010).…”

Retrospective study of Bovine herpesvirus 5 meningoencephalitis in cattle from São Paulo State, Brazil

“…The variation in the results exemplified above can be a result of tissue fixation process, which ensures proper morphological conservation but may affect the preservation of nucleic acids and negatively affect the PCR results (Barcelos et al, 2008). According to Karlsen et al (1994), the fixation time causes DNA degradation and compromises the PCR results.…”

Clinical Usefulness of PCR for Differential Diagnosis of Tuberculosis and Nontuberculous Mycobacterial Infection in Paraffin-Embedded Lung Tissues

“…6,7 PCR efficiency in FFPE samples may have been influenced by several factors, such as fixation protocol, age of paraffin block, DNA quality from FFPE tissues, different kits, examiner's skill, and the presence of endogenous or exogenous inhibitors. 19 Bacterial load and distribution in the FFPE tissue block may be other factors that affect PCR efficiency. Histologically, AFB is more frequently distributed in a necrotic lesion.…”