EVALUACIÓN DE IMPACTO AMBIENTAL

Results from the Affymetrix protocol were encouraging but consistency was proving a major challenge. To address this issue, an alternative kit was identified from Advanced Cell Diagnostics (ACD) that had been developed for use with a Leica Bond RX automated system – the RNAscope® 2.5 LS Automated Assay. Liver tissue samples were acquired from the Queen Elizabeth Hospital pathology department rather than the Centre for Liver Research, which would provide a more consistent fixation time, preventing over-fixation of tissue samples and standardising pre-treatment times.

The Bond RX staining protocol was supplied by ACD, and in essence was very similar to the ViewRNA protocol but all incubations and washes were performed by robot rather than by hand. As with the ViewRNA kit, a manufacturer supplied positive control kit was

used to validate the method on our equipment, and the results were in line with the manufacturer’s criteria, with the low expression control peptidyl-prolyl cis-trans isomerase B (PPIB) and high expression control RNA polymerase II subunit A (Polr2A) visualised correctly in the brightly coloured red and DAB brown respectively (Figure 3.10a).

As before, the gamma delta probes were then tested on tonsil tissue as a positive control tissue (Figure 3.10b), with positive staining observed for the CD3 and Cδ probes. Each gamma delta TCR probe, in this case Vδ1, Vδ2, Vγ9 and Cδ, was then tested in duplex with CD3 in liver tissue sections. Three cases were selected to be stained based on previous flow cytometry-based frequency analysis from the same tissue, with all three having 5-10% of their CD3+ T cell population represented by γδ T cells, of which roughly half were Vδ1+ in each case. Of the three cases tested using the Bond RX autostainer, only one case had significant positive staining for all of the gamma delta TCR probes used, and would be used as a positive control tissue henceforth (Figure 3.11).

In order to elucidate close cell-cell interactions between γδ T cells and other cells in the hepatic microenvironment, a combination of ISH to stain the γδ T cells and fluorescence IHC was employed. To label the ISH probes with a fluorescent tag, the protocol was modified to include addition of a tyramide signal amplification (TSA) step. However, since this work was carried out, ACD have released their own Multiplex Fluorescent Assay kit that allows combination of ISH and IHC analysis on the same tissue, using a similar approach.

Figure 3.10: 2-plex staining of tonsil sections using ACD RNAscope in-situ hybridisation

a) Representative images of FFPE human tonsil derived sections stained using ISH probes specific for: negative control (left) and PPIB (red) + PolR2A (brown) (right). Sections were counter-stained using haematoxylin. Magnification 20x (left) 40x (right).

b) Representative image of FFPE human tonsil derived section stained using ISH probes specific for CD3 (brown) and constant-region δ chain (red – highlighted with arrows). Sections were counter-stained using haematoxylin. Magnification 40x.

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Figure 3.11: 2-plex staining of liver sections using ACD RNAscope in-situ hybridisation

Representative images of FFPE human liver derived sections stained using ISH probes specific for: in all cases CD3 (brown) + TRCD (a), Vδ1 (b), Vδ2 (c) and Vγ9 (d) (red). Positively staining of gamma delta TCR probes (red) is identified with black arrows. Sections were counter-stained using haematoxylin. Magnification 40x.

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Firstly, to assess how good the ISH staining was for analysis of individual cells under high levels of magnification (40x), the CD3 ISH probe was stained in conjunction with a CD3 antibody on the same section (Figure 3.12). While a high level of co-staining was observed in CD3+ cells, the punctate, intracellular nature of the ISH staining was clearly not as useful for staining cell-cell interactions as the cell surface IHC staining. Nevertheless, this did highlight the possibility of using a combination of ISH and IHC on the same sections, which would potentially allow for a wide variety of conventional cell surface protein targets to be stained in multiple fluorescent colours by IHC in combination with gamma delta T cell specific ISH probes, then visualised using multi- channel imaging technology such as the Vectra 3.0 Automated Imaging Platform (PerkinElmer). Additionally, since the number of fluorescent tags for ISH probes is much greater than the colours available for bright-field visualisation, this opened the possibility of combining more than two ISH targets in the same tissue, thereby allowing localisation analysis of the three main γδ T cell subsets; Vδ1, Vδ2 and Vδ3; in the same tissue.

Next, duplex staining of two ISH probes visualised in fluorescence was performed in tonsil sections and the slides were scanned digitally using the Vectra platform and analysed using the Definiens Tissue Studio software (Figure 3.13). Using this analysis Vδ1+ cells were found to account for 2.52% of tonsil cells while Vδ2+ cells accounted for 4.85%. While this was considered a successful attempt to use digital scoring to analyse ISH results, something that would be very important for this study going forward, there was some concern over the signal to background noise ratio. According to the manufacturer, as long as the washes are performed correctly, there should be no positive signalling if there is no hybridisation of the probes to their target. With the bright-field analysis,

Figure 3.12: Dual IHC and ISH staining of liver sections

Representative images of FFPE human liver derived sections stained using IHC αCD3 antibody (upper) (red) and ISH probes specific for CD3 (centre) (green), and combined (lower). Sections were counter-stained using DAPI. Magnification 40x.

Figure 3.13: Definiens analysis of gamma delta T cell staining in human liver

Example of images generated using Definiens tissue analysis software. Top left: example field of liver section stained using RNAscope probes specific to CD3 (brown) and TRDC (red). Bottom left: the same field with staining scored using Definiens software – yellow dots are negatively stained cells, red dots are CD3+ cells, blue dots are TRDC+ and CD3+ cells. Top right: liver tissue coloured according to histology – red areas are parenchyma, blue areas are portal and green areas are fibrous septa. Bottom right: Overlay image including histology, cell identification and cellar boundaries (indicated in dark green colour). Sections were counter-stained using haematoxylin. Magnification 40x.

background did generally seem low, so differing concentrations of the TSA tags were used in an attempt to reduce fluorescence background, which essentially seemed to consist of random dots of colour in areas that were clearly not positive, such as outside cell boundaries. However, no reduction in the number of false positive background dots could be achieved. Clearly non-specific “positive” dots, even at low level, would cause over estimation of staining by the scoring software. This was exacerbated by the generally low level of staining observed for the variable-δ and –γ chain probes, which were only 7 oligonucleotides in length rather than the manufacturer’s preferred 20, which the strongly staining CD3 probe was.

To investigate whether the variable-region TCR probes were working effectively enough to raise positive staining above background noise, critical to the success of any scoring analysis, paraffin embedded tissue blocks were prepared from two T cell lines that had been transfected with either a Vδ1+ TCR clone or a Vδ2+ TCR clone. The blocks were sectioned and stained as per the RNAscope protocol. In addition to determining the level of staining, this would also provide a good control for probe specificity, since none of the assays performed thus far used tissue with precisely known frequencies of gamma delta T cells (Figure 3.14).

While there was no Vδ2+ staining on the Vδ1+ tissue block when the Vδ2 probes were used, and vice-versa, comparison of the CD3staining and both Vδ1 and Vδ2 showed that while CD3 staining was clearly evident in all cells, many cells that were known to be positive for a given variable-region were often very dimly or negatively stained. Considering the mRNA levels in these transfected cells was likely to be higher than cells in

Figure 3.14: Fluorescent ISH assessment of Vδ1- and Vδ2-specific probes using transfected cell lines

Representative images of ISH staining of sectioned paraffin-embedded Jurkat cell lines transfected with either a Vδ1+ TCR clone (left) or a Vδ2+ TCR clone (right). Top: Probes specific to the alternate TCR were used to assess potential non-specific binding. Middle: CD3+ staining using ISH probe. Lower: Vδ1+ or Vδ2+ positive staining, with image intensity increased by 300% in post- processing. Sections were counter-stained using DAPI. Magnification 20x.

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vivo, and that the fixation of these cells was very quick after harvesting, minimising RNA

degradation, it seems likely that these probes are not sensitive enough to stain enough of their targets to produce a reliable, quantifiable result. Since the CD3 probes did work consistently, it can be speculated that the reduced number of oligonucleotide sequences targeted by the variable-region probe sets, a necessity due to their short overall length, is the primary reason for this shortcoming. While this may not be an issue if the target cells were high in frequency, the relative paucity of them in liver tissue meant in the context of this study this technique would ultimately be flawed and not likely to yield useful results.