6.6.1 Blood
In Paper I biochemical analyses were only collected for patients with PSC. We collaborated with 15 regional and local hospitals to retrieve clinical biochemistry from the closest routine control time point from time of stool donation at home (median ~2 months difference).
Thanks to national laboratory standards efforts like Norwegian Clinical Chemistry External quality assessment Program (NNK, http://www.nkk-ekv.com/) in cooperation with Norwegian Quality Improvement of Primary Health Care Laboratories (NOKLUS, http://www.noklus.no/) sampling and subsequent measurements are comparable across the different hospital databases in Norway. Although minor differences could still exist between hospitals, they should be negligible. Matched blood and stool samples would have fewer limitations, but was not feasible at the time as participants performed sampling at home.
Serum samples in Paper II were prepared after non-fasting blood was collected, in a standardised fashion following internal biobank protocols, and stored at -70°C. Other biochemical analyses, and measurement of prothrombin time were retrieved from the databases at hospital laboratories if available within 7 days of biobank sampling. In Paper III blood from mice was drawn from the heart directly after they were sacrificed, left in room temperature, centrifuged and finally stored at -80°C awaiting further analyses.
6.6.2 Tissue sampling in mice
The weight of the mouse, liver, spleen and caecum were noted when the mice were sacrificed, together with any common bile duct dilatation (CBDD). Liver tissue was fixed in 4% formalin and subsequently embedded in paraffin using standard procedures.
6.6.3 Sampling for microbiota analyses
In Paper I, all participants were given a standardised collection devise and a simple procedure facilitating sample preparation at home.186 It was explicitly emphasised that voiding should be performed prior to sampling to avoid contamination. Sampling was then done using Stool Collection Tubes with Stool DNA Stabilizer (Stratec Molecular GmbH, Berlin, Germany), and the samples were shaken to facilitate homogenisation. The tubes were then sent by mail to the NoPSC biobank to ensure equal processing and adequate
follow-up of missing information on sampling and/or participant, and frozen at a minimum of -20°C (according to the instructions from the manufacturer) awaiting DNA extraction.
An alternative to stool in this setting would be mucosal samples.77 This is feasible in PSC due to regular screening of patients by colonoscopy, as well as for IBD controls, but is still an invasive procedure with potential complications. Samples from healthy controls are considerably more challenging as most are referred to colonoscopy for evaluation of certain symptoms, and one could therefore argue that they no longer should be considered
‘healthy’.170 An exemption to this is participants in national screening programs involving colonoscopy, but such programs are so far not implemented in Norway, and the participants would also be older than a typical PSC patient cohort. Bowel preparations have a striking effect on the mucosal microbiota.187 Bowel preparation is essential before the procedure, and there are indications that this effect is not equal between study groups, and that it could have long standing effects.187,188 Nevertheless, both stool and mucosa inhabit different and distinct niches of the microbiota,189,190 so both must be explored if we are to fully understand its role in PSC. Besides being non-invasive, the use of stool samples has several advantages as it facilitates longitudinal follow up of a larger number of participants, at a reasonable cost. Another important advantage, although not performed in Paper I, is probably the possibility to perform WGS for identification of functional contents of the microbiota. This is still not cost-effective and has several limitations when performed at DNA extracts from mucosal samples, mainly due to the low bacterial-to-human DNA ratio in these samples.190,191
The ‘gold standard’ method is still fresh stool, with DNA extraction performed on arrival at study centre, or frozen as soon as possible after sampling, all to avoid post-sampling changes to different bacteria in the samples.87,183 The method is, however, challenging to use in large scale studies involving participants over large distances.192
The Stool Collection Tubes with Stool DNA Stabilizer allowed us to greatly increase sample size, something we considered important given the large variation of the human gut microbiota. Importantly, this method has performed at par with more standard methods in comparative studies.193,194 Samples stored in room temperature for more than 72 hours were excluded according to the manufacturers recommendations. One stool sample was collected per participant. Our experience from other studies supports that double baseline samples are unnecessary in this kind of large-scale surveys, as samples from the same individual taken
at separate time points tend to harbour highly similar gut microbiota profiles,86 as illustrated by Figure 7. Samples were frozen immediately on arrival. An alternative could have been same-day DNA extraction, but this is highly laborious, and freezing per se does not appear to have minor impact on the bacterial community.195
PC2 – 6.7%
PC1 – 14.4%
Figure 7. Double baseline samples. Illustration of β-diversity based on unweighted UniFrac distance showing highly similar bacterial profiles in samples from the same individual (r2=0.92, p<0.001, 999 permutations). Samples from one individual are connected with a straight line (Cases in red, healthy family controls in blue). Samples were collected and processed using the same methods and protocols as described for Paper I.
In Paper III we sampled caecal contents and ~15 mm of caecal mucosa (whole transverse sections) from mice using disposable sterile equipment. To avoid removal of mucosal adherent bacteria we avoided liquid flushing. Caecal contents and mucosal tissue were then put in separate sterile tubes, snap-frozen in liquid nitrogen and stored at -80°C awaiting DNA extraction. The caecum was chosen as it is easily identified anatomically, thereby securing uniform sampling. Sampling was done at 10 weeks to assure sampling before development of diabetes in NOD control mice, confirmed by fasting blood glucose measurements.