Protoblock - A biological standard for formalin fixed samples

Abstract

Background: 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. 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.

Results: To 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.

Conclusion: The 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. Video Abstract.

Keywords: Bacteria; DNA; FFPE; Microbiome; Microscopy; PCR.

Fig. 1

Making a Protoblock. a Schematic of the workflow for making a Protoblock described in methods. b Schematic of the architecture of a Protoblock, demonstrating average measurements of volume, height and radius

Fig. 2

Validation of cell architecture and numbers in a Protoblock. a Flow cytometry dot plots measuring the cell density of fixed bacterial suspensions used to make Protoblocks. Events were gated either for SYTOBC+ cells or beads. The averages of 3 reads for 4 populations per cell type are shown here and in Table 1. b Light microscopy images confirming cell architecture of Protoblock slides stained with H&E (4T1 cells) or Gram staining (bacteria). c Fluorescence microscopy images confirming cell content of Protoblocks. Slides were with α-E. coli (green), α-S. aureus (red) or DAPI (grey). Counts in Fig. 1 are the average of 20 FOV in 3 × 4 μm slides

Fig. 3

Assessing the recovery of FFPE bacterial DNA by quantitative PCR and 16S rRNA sequencing. a Evaluating PCR recovery of FFPE bacterial DNA from Protoblocks fixed for 24 h (green) or 48 h (cyan) and compared with the recovery of paired NF samples (red). (i) % of absolute PCR recovery (% shown above corresponding box). A 2-log fold decrease in recovery is observed for FFPE-treated samples, which was found to be statistically significant in all cases as per 1 sample Wilcoxon signed-rank test. In addition, longer fixation periods lead to a significantly greater reduction in recovery (p = 0.04). (ii) % deviation in recovery after compensating for 10-fold loss in recovery. Input = 0 (dotted line). % deviation shown above corresponding box. Significant deviation from input values, even after compensation for 10-fold decrease shown in all FFPE-treated samples. (In all cases, p = + < 0.1, * < 0.05, ** < 0.01 and *** < 0.001). b Sample composition bar plot of calculated input of bacterial cells added to Protoblock and 16S rRNA gene sequence analysis of Protoblocks fixed for 24 h or 48 h. c Average concentration of DNA purified from samples

Fig. 4

Evaluating bias in sample composition. a Metapolyzyme lyses FFPE bacteria. (i) Bar plot showing quantitative PCR DNA recovery after lysis (cyan)/no lysis (grey) with Metapolyzyme. Increase in recovery is shown above each test. For each bar, n = 6. Treatment with 100 μg of Metapolyzyme for 4 h markedly increased the recovery of DNA in all tests (p < 0.001) as per Wilcoxon signed-rank test. (ii) Immunofluorescence microscopy images of Protoblocks stained with DAPI (blue) for 4T1 cells, α-E. coli (green) and α-S. aureus (red). Protoblocks were fixed for 24 h. b Measuring bias introduced by host DNA. (i) Box plot comparing DNA recovery of bacteria in Protoblocks loaded with (cyan) and without 4T1 cells (orange). Quantitative PCR recovery was normalised to a sample input of 10⁶ cells. For each box, n = 6. Protoblocks without 4T1 cells had a higher recovery of all bacteria taxa. Difference of means between tests was measured using a Wilcoxon signed-rank test, for all bacterial taxa. (ii) Immunofluorescence microscopy images of Protoblocks with and without mammalian cells, stained with α-E. coli (green) and DAPI (blue) for 4T1 cells. Protoblocks were fixed for 48 h. c Testing host DNA depletion strategies. DNA recovery of 4T1 cells (orange), Escherichia (cyan) and Staphylococcus (green) after a 10-min treatment with either Triton-X (0.1%), Saponin (0.1%) or Molysis CM buffer. For each bar, n = 3. % increase or decrease in recovery from untreated is shown above each bar. Dotted lines indicate the PCR recovery of samples without host depletion. (In all cases, p = + < 0.1, * < 0.05, ** < 0.01 and *** < 0.001)

Fig. 5

DNA fragmentation in FFPE bacteria. a Evaluation of DNA integrity with fragment analyser. Electropherograms of DNA purified from Protoblocks with a mix of 5 bacterial strains (red) and Protoblocks loaded with Escherichia only (yellow) and compared with matched NF bacterial mix (blue) and Escherichia (green). NF bacterial DNA had a higher integrity (GQN > 6.6), while FFPE bacterial DNA from either sample was highly fragmented (GQN ≤ 0.1). No significant difference was observed between Protoblocks or NF samples. GQN = % of DNA above the threshold. The GQN threshold (dotted line) was set to that used for sequencing libraries (10,000). b Measuring the recovery of PCR readable DNA from FFPE bacteria in Protoblocks by qPCR. (i) Schematic of primer design for targeted fragments. Both 200 bp and 460 bp DNA fragments target the same E. coli K-12 regions. (ii) PCR recovery. Box plot of DNA recovery from 460 bp (green) and 200 bp (orange) FFPE DNA fragments (for each box, n = 9) compared with NF DNA (cyan; for each box n = 6) normalised to 10⁷, 10⁵ and 10³ genomes. Mean recovery of DNA from Protoblocks compared with input DNA significantly differed in both FFPE sample types (p < 0.001) as per one-sample Wilcoxon signed-rank test. Fragment length also significantly influenced DNA recovery of FFPE samples (p < 0.001), as per Wilcoxon signed-rank test. (iii) Gram-stained slides used for confirming bacterial content. (In all cases, p = + > 0.1, * < 0.05, ** < 0.01 and *** < 0.001)

Fig. 6

Evaluating sequence quality of bacterial FFPE DNA. a Evaluation of DNA sequence aberrations by high-resolution-melt analysis. (i) Box plots of normalised DNA quantities from Protoblock FFPE Escherichia (cyan) and NF Escherichia (orange). Significant shifts in the melting temperatures in 2 of the 3 sequences were observed as per Wilcoxon signed-rank test, with temperature shifts that were on average 0.1–0.5 °C apart from NF counterparts. (ii) Schematic of sequences used for HRM analysis: 3 DNA fragments with an average length of 100 bp were analysed. For each test and each sample type, n = 6. b Confirmation of sequence alteration by WGS. DNA from Protoblocks loaded with Escherichia and Staphylococcus and their NF paired reference was analysed by whole genome sequencing to determine chimeric reads and single-nucleotide polymorphisms (SNP) against the reference genome E. coli K12 MG1655 and S. aureus Newman. Here, the SNP are plotted on the x-axis and the rate of occurrence on the y-axis. Variant calling and level of coverage are measured using SAMTOOLS/BCFTOOLS. (i) Chimeric reads per layer of coverage. (ii) Distribution of SNPs found per bacterial strain

Fig. 7

Evaluation of sources of environmental contamination and their effect on Protoblock samples. Composition bar plot per sample showing proportional composition of bacterial taxa per negative control, with corresponding number of reads detected by 16S rRNA gene sequencing. Compared with representative Protoblock sample