Tissue processing constitutes a major challenge in molecular profiling efforts. Pre-analytical factors such as fixative temperature and infiltration rate, dehydration and embedding media affect downstream analysis, generating data associated with FFPE processing rather than disease condition. Most diagnostic laboratories are equipped with automated tissue processing machines employing raised temperatures, effective reagent circulation and incorporating vacuum/pressure cycles to enhance processing and reduce processing times. We have investigated three different tissue-processing apparatuses and in parallel compared two different sample preparation protocols for in-situ proteomic analysis of clinical tissues by MALDI IMS. Methods Tumor and non-tumor liver tissues were collected from one sample and processed to create FFPE specimens with three different automatic tissue-processors (TP); Leica-ASP300-II (TP1), Leica-Peloris-II (TP2), Sakura-Tissue-TEK-Xpress-X120 (TP3). Tissues were treated with two different sample preparation protocols (A,B) for peptide imaging and investigated with respect to deparaffinization, antigen retrieval and digestion temperature. For both protocols, trypsin and CHCA matrix were deposited on tissues using an automatic sprayer. MS measurements were performed using a rapifleX MALDI Tissuetyper (Bruker Daltonik) at 50μm spatial resolution. All experiments were done in triplicates and data were analyzed by SCiLS Lab and R software. Results The median total peak count and signal-to-noise ratios were used as quality parameters to compare all experiments. The highest variance occurred in tissues processed with TP1 with both protocols A and B. Highest peak counts and lowest variance were instead achieved in all tissues processed with TP2 and TP3 combined with protocol A. As a result, TP2 and TP3 tissue processors, working in common conditions of xylene-free agents, vacuum, high temperature and short reagent incubation time led to consistent high quality and reproducible results compared to the TP1 system. Protocol A using lower antigen retrieval temperature (95 °C), alkaline buffer (Tris pH=9) and lower digestion temperature (37 °C) produced the highest number of peaks compared to protocol B for both normal and tumor tissues. Conclusions Different tissue processor systems were evaluated in combination with two different sample preparation protocols for in-situ proteomic analysis using MALDI IMS of FFPE tissues, to achieve high quality and reproducibility results.
A comparison of different sample preparation protocols for IMS analysis of clinical tissues
Casadonte R
2018-01-01
Abstract
Tissue processing constitutes a major challenge in molecular profiling efforts. Pre-analytical factors such as fixative temperature and infiltration rate, dehydration and embedding media affect downstream analysis, generating data associated with FFPE processing rather than disease condition. Most diagnostic laboratories are equipped with automated tissue processing machines employing raised temperatures, effective reagent circulation and incorporating vacuum/pressure cycles to enhance processing and reduce processing times. We have investigated three different tissue-processing apparatuses and in parallel compared two different sample preparation protocols for in-situ proteomic analysis of clinical tissues by MALDI IMS. Methods Tumor and non-tumor liver tissues were collected from one sample and processed to create FFPE specimens with three different automatic tissue-processors (TP); Leica-ASP300-II (TP1), Leica-Peloris-II (TP2), Sakura-Tissue-TEK-Xpress-X120 (TP3). Tissues were treated with two different sample preparation protocols (A,B) for peptide imaging and investigated with respect to deparaffinization, antigen retrieval and digestion temperature. For both protocols, trypsin and CHCA matrix were deposited on tissues using an automatic sprayer. MS measurements were performed using a rapifleX MALDI Tissuetyper (Bruker Daltonik) at 50μm spatial resolution. All experiments were done in triplicates and data were analyzed by SCiLS Lab and R software. Results The median total peak count and signal-to-noise ratios were used as quality parameters to compare all experiments. The highest variance occurred in tissues processed with TP1 with both protocols A and B. Highest peak counts and lowest variance were instead achieved in all tissues processed with TP2 and TP3 combined with protocol A. As a result, TP2 and TP3 tissue processors, working in common conditions of xylene-free agents, vacuum, high temperature and short reagent incubation time led to consistent high quality and reproducible results compared to the TP1 system. Protocol A using lower antigen retrieval temperature (95 °C), alkaline buffer (Tris pH=9) and lower digestion temperature (37 °C) produced the highest number of peaks compared to protocol B for both normal and tumor tissues. Conclusions Different tissue processor systems were evaluated in combination with two different sample preparation protocols for in-situ proteomic analysis using MALDI IMS of FFPE tissues, to achieve high quality and reproducibility results.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
