A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and Molecular Analyses

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules

Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues

One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease

Comparison of proteins extracted from five different tissues with EXB and EXB Plus, respectively.

<p><b>A</b> Using EXB Plus considerably higher lysate concentrations could be obtained for each of the tissue types. <b>B</b> Western blot analysis of E-Cadherin, Erk and β-actin in five different tissues. For both buffers we obtained clear bands in the western blot, confirming that we were able to extract non-degraded, full-length, and immunoreactive proteins. In addition it is distinguishable that the extracted protein amount from all five tissues of all three analysed proteins is higher using EXB Plus.</p

Protein amounts gained by extraction from FFPE tissues from two different hospitals in three technical replicates.

<p>EXB Plus allowed high protein yields independently of the tumor sample or the hospital the tissue was processed in. Additionally only minimal variations between the replicates (mean standard deviation: 0.47 mg/ml) could be detected.</p

Protein yield from long-term stored FFPE tissue blocks.

<p>Protein yields from one pair of colon carcinoma and two pairs of gastric cancer tissues from 1990 and 2010 were analysed. The same block was extracted twice, once with EXB and once with EXB Plus. The protein amounts from the newer blocks were higher than from the older ones. But for both time periods the amount extracted with EXB Plus was higher.</p

Protein extraction from FFPE samples from 1990 in comparison to samples from 2010.

<p>For both years the usage of EXB Plus resulted in higher protein yields compared to EXB but for the twenty years old samples the difference was more striking.</p

Protein amounts and western blot analysis of lysates extracted from lymph node samples fixed for 6 h, 24 h, 48 h and 144 h, respectively.

<p>For a short fixation time no real difference could be detected between the two buffers tested. But already at the standard fixation time of 24 h EXB Plus shows 25% higher protein yield. For a fixation time of 144 h the difference increased to 30% higher protein yield compared to EXB. <b>B</b> Western blot analysis of Hsp70, Akt, Erk and β-actin in lymph node samples fixed for 6 h, 24 h, 48 h or 144 h. Protein yield decreases with extension of fixation for EXB, for all four analysed proteins. In contrast, using EXB Plus the decline is much less pronounced. All eight samples detected with one antibody were run on one gel and detected under exactly the same conditions (e.g. blocking, washing steps, exposure time).</p

Comparison of proteins extracted from four FFPE tissues and the corresponding cryo tissues with EXB Plus.

<p><b>A</b> Cryo tissue was additionally extracted with EXB Plus but without any heat and T-Per, a buffer designed for extraction of cryo material. To be able to compare protein yields from different sized tissue pieces the protein amount is calculated per mm³ of used tissue. The yields extracted from cryo material with EXB Plus or T-Per were equal, or especially for stomach tissue even higher in the EXB Plus extracts. Cryo tissue compared to FFPE material discloses a loss of protein amount in two tissues (colon and muscle) and no difference in the two others (ovarian carcinoma and stomach). <b>B</b> Western blot analysis of protein lysates from colon FFPE and cryo material. For both tissue types we obtained clear bands in the western blot, confirming that we were able to extract non-degraded, full-length, and immunoreactive proteins. In addition it is visible that the extracted protein amount from FFPE is higher than from fresh-frozen tissue, but hardly any difference could be seen for cryo material extracted using EXB Plus compared to a standard buffer for cryo extraction (T-Per).</p

Protein extraction from FFPE samples from two different xenograft mouse models.

<p>For both mouse models EXB Plus resulted in higher protein yields compared to EXB. This shows the advantage of EXB Plus independently of the organism.</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