Abstract
Background: Tissue microarray (TMA) technology allows rapid visualization of molecular markers by immunohistochemistry and in situ hybridization. In addition, TMA instrumentation has the potential to assist in other applications: punches taken from donor blocks can be placed directly into tubes and used for nucleic acid analysis by PCR approaches. However, the question of possible cross-contamination between samples punched with the same device has frequently been raised but never addressed. Methods: Two experiments were performed. (1) A block from mycobacterium tuberculosis (TB) positivetissue and a second from an uninfected patient were aligned side-by-side in an automated tissue microarrayer. Four 0.6 mm punches were cored from each sample and placed inside their corresponding tube. Between coring of each donor block, a mechanical cleaning step was performed by insertion of the puncher into a paraffin block. This sequence of coring and cleaning was repeated three times, alternating between positive and negative blocks. A fragment from the 6110 insertion sequence specific for mycobacterium tuberculosis was analyzed; (2) Four 0.6 mm punches were cored from three KRAS mutated colorectal cancer blocks, alternating with three different wild-type tissues using the same TMA instrument (sequence of coring: G12D, WT, G12V, WT, G13D and WT). Mechanical cleaning of the device between each donor block was made. Mutation analysis by pyrosequencing was carried out. This sequence of coring was repeated manually without any cleaning step between blocks. Results/Discussion: In both analyses, all alternating samples showed the expected result (samples 1, 3 and 5: positive or mutated, samples 2, 4 and 6: negative or wild-type). Similar results were obtained without cleaning step. These findings suggest that no cross-contamination of tissue samples occurs when donor blocks are punched using the same device, however a cleaning step is nonetheless recommended. Our result supports the use of TMA technology as an accessory to PCR applications.
Highlights
Tissue microarrays (TMAs) play an important role in translational and clinical studies [1]
Recent advances in TMA technology rely on digital pathology and automated tissue microarraying
Three patients were wild-type (WT); one patient had a colorectal cancer with G12D mutation, the second a G12V mutation and the third was mutated in codon 13 (G13D)
Summary
Tissue microarrays (TMAs) play an important role in translational and clinical studies [1]. One approach is to use a slide scanner to digitally visualize stained tissue sections, which can be annotated using a TMA tool of various sizes (Figure 1). These annotated slides are matched to their corresponding donor blocks, which are precisely punched out at the desired locations and transferred into recipient blocks, automatically. The question of possible cross-contamination across tissue samples using the same punching device, especially one embedded in an automated tissue microarrayer, has frequently been posed but never addressed This may be of particular concern if nucleic acids are amplified by PCR. In this study, we determine the level of contamination transferred between samples after automated punching using a sensitive assay for mycobacteria and a pyrosequencing assay for KRAS mutation analysis
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