Histological assessment of paxgene tissue fixation and stabilization reagents

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities

Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues.

RNA and DNA analyses from paraffin-embedded tissues (PET) are an important diagnostic tool for characterization of a disease, exploring biomarkers and treatment options. Since nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissue are of limited use for molecular analyses due to chemical modifications of biomolecules alternate, formalin-free fixation reagents such as the PAXgene Tissue system are of evolving interest. Furthermore, biomedical research and biomarker development critically relies on using long-term stored PET from medical archives or biobanks to correlate molecular features with long-term disease outcomes. We therefore performed a comparative study to evaluate the effect of long term storage of FFPE and PAXgene Tissue-fixed and paraffin-embedded (PFPE) tissue at different temperatures on nucleic acid stability and usability in PCR. Matched FFPE and PFPE human tissues from routine clinical setting or rat tissues from a highly controlled animal model were stored at room temperature and 4°C, as well as in case of animal tissues frozen at -20°C and -80°C. RNA and DNA were extracted in intervals for up to nine years, and examined for integrity, and usability in quantitative RT-PCR (RT-qPCR) or PCR (qPCR) assays. PET storage at room temperature led to a degradation of nucleic acids which was slowed down by storage at 4°C and prevented by storage at -20°C or -80°C. Degradation was associated with an amplicon length depending decrease of RT-qPCR and qPCR efficiency. Storage at 4°C improved amplifiability in RT-qPCR and qPCR profoundly. Chemically unmodified nucleic acids from PFPE tissue performed superior compared to FFPE tissue, regardless of storage time and temperature in both human and rat tissues. In conclusion molecular analyses from PET can be greatly improved by using a non-crosslinking fixative and storage at lower temperatures such as 4°C, which should be considered in prospective clinical studies

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.

BACKGROUND:Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. METHODS:Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. RESULTS:All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. CONCLUSION:PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment

Investigated strains cover the spectrum of bacterial life conditions.

<p>Specific media were used for bacteria cultivation before and after inactivation treatment.</p

a,b,c,d,e,f,g: Results of bacterial inactivation experiments after treatment with PAXgene and formalin.

<p>Bacterial strains show variable viability after treatment with PAXgene Fix, PAXgene Fix and Stab compared to formalin and PBS (viability control) for 30 minutes (a, b, c, e, f, g) and 2 hours (d). The x-axis indicates the amount of experiments, the y-axis cfu/ml. To obtain comparable results all counted cfus were normalised to 10⁶. Columns represent the mean values (and error bars) calculated from the results of a series of different dilutions for one experiment. Dashed lines indicate the reduction limit of 10⁵.</p

Work flow of bacterial and fungal inactivation experiments.

<p>Work flow of bacterial and fungal experiments. Bacteria experiments were performed with four (<i>Pa</i>) and six (<i>Ms</i>) independent experiments, respectively, when no colony was detected after inactivation, six experiments if colonies were detected (<i>Bs</i>, <i>Sa and Mt</i>) and seven experiments with the most variable strain <i>Cs</i>. Two-hour-treatments were performed with fungi (two experiments and all strains listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151383#pone.0151383.t001" target="_blank">Table 1</a>) and additionally with <i>Cs</i> (three experiments).</p

Human relevant fungi cultivated, treated with PAXgene and formalin and investigated for viability.

<p>Human relevant fungi cultivated, treated with PAXgene and formalin and investigated for viability.</p

Reverse transcription qPCR: comparison of Cq values of PAXgene and formalin-fixed CMV samples.

<p>RT-qPCR sensitivity assay was performed to detect CMV early-immediate gene <i>TRS1</i> and reference gene <i>GAPDH</i> after fixation of CMV infected MRC-5 cells with PAXgene (<i>TRS1</i>_PF, <i>GAPDH</i>_PF), formalin (<i>TRS1</i>_FF, <i>GAPDH</i>_FF) and not fixed control samples (<i>TRS1</i>_PBS; <i>GAPDH</i>_PBS) in triple biological samples. Low Cq values indicate early detection. Statistical significance p < 0.0001 (***) or p < 0.03 (*).</p

a,b: Results of inactivation experiments of human-relevant fungi by two hours fixation with PBS as positive control, PAXgene Fix, PAXgene Fix and Stab, and formalin.

<p>At least two assays per species (1–3 experiments) were performed. a) Cfu/mL was normalised to 10⁵. The dashed line indicates the threshold for minimum of reduction of 10⁴ used for disinfectants for fungi. b) Bold printed numbers indicate minimal growth after inactivation.</p

Histological assessment of PAXgene tissue fixation and stabilization reagents.

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities