Abstract
BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissue is the gold standard in pathology tissue storage, representing the largest collections of patient material. Their reliable use for DNA analyses could open a trove of potential samples for research and are currently being recognised as a viable source material for bacterial analysis. There are several key features which limit bacterial-related data generation from this material: (i) DNA damage inherent to the fixing process, (ii) low bacterial biomass that increases the vulnerability to contamination and exacerbates the host DNA effects and (iii) lack of suitable DNA extraction methods, leading to data bias. The development and systematic use of reliable standards is a key priority for microbiome research. More than perhaps any other sample type, FFPE material urgently requires the development of standards to ensure the validity of results and to promote reproducibility.ResultsTo address these limitations and concerns, we have developed the Protoblock as a biological standard for FFPE tissue-based research and method optimisation. This is a novel system designed to generate bespoke mock FFPE ‘blocks’ with a cell content that is user-defined and which undergoes the same treatment conditions as clinical FFPE tissues. The ‘Protoblock’ features a mix of formalin-fixed cells, of known number, embedded in an agar matrix which is solidified to form a defined shape that is paraffin embedded.The contents of various Protoblocks populated with mammalian and bacterial cells were verified by microscopy. The quantity and condition of DNA purified from blocks was evaluated by qPCR, 16S rRNA gene amplicon sequencing and whole genome sequencing. These analyses validated the capability of the Protoblock system to determine the extent to which each of the three stated confounding features impacts on eventual analysis of cellular DNA present in FFPE samples.ConclusionThe Protoblock provides a representation of biological material after FFPE treatment. Use of this standard will greatly assist the stratification of biological variations detected into those legitimately resulting from experimental conditions, and those that are artefacts of the processed nature of the samples, thus enabling users to relate the outputs of laboratory analyses to reality.BKi5aTsaSPPC22FkaWVr1zVideo
Highlights
Formalin-fixed, paraffin-embedded (FFPE) tissue is the gold standard in pathology tissue storage, representing the largest collections of patient material
Unique factors to consider in quality control of FFPE samples are: 1. Low biomass renders samples extremely susceptible to the high burden of contaminants to which they are exposed during the non-sterile FFPE processing [31]
To achieve the desired cell numbers, formalin-fixed cell suspensions were counted with FACS or NucleoCounter (4T1) and the volume of the cell suspensions normalised to cell contents (Fig. 2a)
Summary
Formalin-fixed, paraffin-embedded (FFPE) tissue is the gold standard in pathology tissue storage, representing the largest collections of patient material. Their reliable use for DNA analyses could open a trove of potential samples for research and are currently being recognised as a viable source material for bacterial analysis. FFPE tissue is the gold standard for pathology tissue storage and represents the largest collection of available patient material [20,21,22] The availability of this material has been vital for progress in human genomics, and numerous sequencing workflows have been designed to enable use of, or are based upon, these samples [23,24,25,26,27,28]. It aggravates the influence of host DNA, rendering samples ineffective for whole genome sequencing (WGS) and introducing PCR bias to 16S rRNA gene amplicon sequencing [32]
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