Formalin fixing and paraffin embedding may lead to extra band development in PCR

The molecular biological analysis of infectious agents requires the availability of a reliable source of microorganisms to be used to recover DNA. Clinical samples can be obtained directly from infected patients or can be propagated using in vitro or in vivo systems. However, repeated sampling from patients is not always possible as the procedure may be invasive or unpleasant, or it is not possible to catch the same agent at the time of second sampling. Moreover, the techniques used may also produce false-positive and false-negative results. We therefore studied the impact of formalin-fixing and paraffin embedding on tissue sampling, and the methodologies such as DNA isolation and PCR amplification of DNAs from archival materials in the diagnosis of Mycobacterium tuberculosis. PCR analyses were done according to standard methods with some modifications. Demonstration of mycobacteria was successful both in tissue sections of the formalin-fixed lymph nodes and in stained fresh materials from patients. However, the results showed the presence of two extra bands in the gel. We accounted for extra band development due to the harshness of the methodology used to isolate nucleic acids from formalin-fixed and paraffin embedded tissue samples or the nature of the fixation procedure, or because of the time passed during storage in which alteration in the chromosomal DNA would take place. Thus, if disease- and tissue specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity are ignored, errors because of sampling and methodologies used may lead to false-positive and false-negative results.