C-204. The Role of Laser Capture Microdissection in PCR Detection of Mycobacteria from Formalin-Fixed Paraffin-Embedded Tissues

S. P. Buckwalter, N. L. Wengenack, B. S. Pritt;
Mayo Clin., Rochester, MN.

Background: Molecular amplification is a promising alternative to insensitive microscopic stains for detection of acid fast bacilli (AFB) in formalin-fixed paraffin-embedded tissue. However, poor DNA quality, PCR inhibitors, and high human-to-AFB DNA ratios may compromise isolation and detection of target DNA from this source. Sampling exclusively from granulomatous foci could potentially increase sensitivity. We tested this hypothesis using laser capture microdissection (LCM) of granulomas for real time PCR detection of Mycobacteria. Methods: Fifty formalin-fixed paraffin-embedded tissue sections were selected from 34 AFB-culture positive and 16 culture negative cases. Five tissues grew Mycobacterium tuberculosis (MTB). Granulomas were dissected from a 10μm tissue section and captured onto a CapSure Macro LCM cap using the Veritas™ Microdissection instrument and processed with the Pico Pure™ DNA Extraction kit. An adjacent 50μm section was subjected to routine tissue PCR protocol, including de-paraffinization with xylene, and digestion with proteinase K. All digested tissues were extracted on the MagNA Pure Compact Instrument and subsequently assayed on LightCycler Instrument using previously described assays for the Mycobacteria Genus Screen and MTB complex. Results: Using culture as the gold standard, PCR with standard 50μm tissue sections had a sensitivities of 44% and 20% for Mycobacterium genus and MTB detection respectively. In comparison, LCM samples yielded detection sensitivities of only18% and 0% respectively. Conclusions: Our results failed to support the hypothetical advantages of LCM over whole-tissue section PCR. Dissection of additional granulomas per case could increase the yield but would also increase technical time and cost. Overall, the additional time and effort are not justified.