Aim: The diagnosis of infection in patients with arthritis and/or in joint prostheses requires interdisciplinary cooperation and the application of up-to-date methods. Histological evaluation of the synovial membrane is made by counting neutrophils per area of tissue, giving a highly imprecise and unsatisfactory diagnosis. The aim of this study is to establish an automated MALDI imaging method for the detection of neutrophilic aggregates. Methods: Periprosthetic formalin-fixed paraffin-embedded tissue samples containing a very high number of neutrophils from patients with proven infections were on-tissue digested with trypsin and sprayed with a matrix solution for MALDI analysis. MALDI spectra from regions with high number and lower number of neutrophils were statistically compared. In parallel, peptide digests were extracted from the tissue and analyzed by LC-MS/MS for the identification of peaks that were specifically mapped by MALDI imaging in the regions containing a high number of neutrophils. Results: MALDI imaging allowed us to specifically localize 20 m/z peaks in the region containing a high number of neutrophils. The subsequent LC-MS/MS analyses allowed to retrieve the identification of proteins containing peptides with the same monoisotopic mass analyzed in the MALDI images. Among the identified proteins, some were known to be related to neutrophil biological activities. Myeloperoxidase, endoplamins, 60S ribosomal protein, tenascin, protein disulfide isomerase, 78 kDa glucose-regulated proteins are some of the candidate proteins that we found specifically located in neutrophil areas. Conclusion: In the present study we compared joint tissue with high- and low neutrophilic counts. The different peptide patterns observed may be markers for a joint infection. In the next future, tissues from infected and non-infected patients will be analyzed by MALDI imaging and compared in order to determine the peaks that are specifically correlated to infection. A final step of the workflow will be the validation of the infection biomarkers by immunohistochemistry. MALDI imaging may then be used as a routine technique for the discrimination of infected and non-infected patients. References: 1. J. Kriegsmann et al., [Molecular pathological diagnostics of infections in orthopedic pathology]. Pathologe. 2014 Nov;35 Suppl 2:225-31. 2. M. Kriegsmann et al., [MALDI MS imaging as a powerful tool for investigating synovial tissue]. Scand J Rheumatol. 2012 Aug;41(4):305-9.
Investigation of neutrophilic proteins/peptides in periprosthetic tissue by matrix-assisted laser desorption/ionization (MALDI) imaging
Casadonte R;
2016-01-01
Abstract
Aim: The diagnosis of infection in patients with arthritis and/or in joint prostheses requires interdisciplinary cooperation and the application of up-to-date methods. Histological evaluation of the synovial membrane is made by counting neutrophils per area of tissue, giving a highly imprecise and unsatisfactory diagnosis. The aim of this study is to establish an automated MALDI imaging method for the detection of neutrophilic aggregates. Methods: Periprosthetic formalin-fixed paraffin-embedded tissue samples containing a very high number of neutrophils from patients with proven infections were on-tissue digested with trypsin and sprayed with a matrix solution for MALDI analysis. MALDI spectra from regions with high number and lower number of neutrophils were statistically compared. In parallel, peptide digests were extracted from the tissue and analyzed by LC-MS/MS for the identification of peaks that were specifically mapped by MALDI imaging in the regions containing a high number of neutrophils. Results: MALDI imaging allowed us to specifically localize 20 m/z peaks in the region containing a high number of neutrophils. The subsequent LC-MS/MS analyses allowed to retrieve the identification of proteins containing peptides with the same monoisotopic mass analyzed in the MALDI images. Among the identified proteins, some were known to be related to neutrophil biological activities. Myeloperoxidase, endoplamins, 60S ribosomal protein, tenascin, protein disulfide isomerase, 78 kDa glucose-regulated proteins are some of the candidate proteins that we found specifically located in neutrophil areas. Conclusion: In the present study we compared joint tissue with high- and low neutrophilic counts. The different peptide patterns observed may be markers for a joint infection. In the next future, tissues from infected and non-infected patients will be analyzed by MALDI imaging and compared in order to determine the peaks that are specifically correlated to infection. A final step of the workflow will be the validation of the infection biomarkers by immunohistochemistry. MALDI imaging may then be used as a routine technique for the discrimination of infected and non-infected patients. References: 1. J. Kriegsmann et al., [Molecular pathological diagnostics of infections in orthopedic pathology]. Pathologe. 2014 Nov;35 Suppl 2:225-31. 2. M. Kriegsmann et al., [MALDI MS imaging as a powerful tool for investigating synovial tissue]. Scand J Rheumatol. 2012 Aug;41(4):305-9.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
