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Trier, Germany

Casadonte R.,Proteopath GbR | Kriegsmann M.,University of Heidelberg | Amann K.,Friedrich - Alexander - University, Erlangen - Nuremberg | Suckau D.,Bruker | Kriegsmann J.,Proteopath GbR
Nephrologe | Year: 2015

Background: Amyloidosis is characterized by extracellular accumulation of misfolded proteins. Aim: The exact subtyping of renal amyloidosis is essential for prognostic stratification, genetic counseling and therapeutic management. Material and methods: Amyloidosis typing may be mainly achieved through two methods: immunohistochemistry and mass spectrometry (MS). Laser microdissection coupled to liquid chromatography/MS represents one possible method that is well established in the routine diagnostics in many laboratories. Additionally, matrix-assisted laser desorption/ionization imaging (MALDI) is available for diagnosis of amyloidosis in formalin-fixed paraffin embedded material. Results: The latter method (MALDI) will be introduced into the clinical practice and is a sensitive and rapid technique not only for subtyping but also for a steric correlation with Congo red stained areas. Discussion: Currently the general application in routine diagnostics is not yet feasible in most areas, as sophisticated technical equipment is needed. Nevertheless, in the future these applications may be the standard for subtyping of amyloidosis. © 2015, Springer-Verlag Berlin Heidelberg.

Gravius S.,University of Bonn | Randau T.M.,University of Bonn | Casadonte R.,Proteopath GbR | Kriegsmann M.,University of Heidelberg | And 3 more authors.
International Orthopaedics | Year: 2015

Results: Specific peaks associated with a high amount of neutrophils were detected. Of these m/z peaks, four could be assigned to predictive neutrophil molecules. These peptides include annexin A1, calgizzarin (S100A11), calgranulin C (S100A12) and histone H2A. By MALDI IMS, these peptides could be shown to be co-localised with the infiltration of neutrophils in the immediate vicinity of the periprosthetic interface, whereas more distant areas did not show neutrophil invasion or infection-related peptides.Conclusions: MALDI IMS is a new method allowing identification of neutrophil peptides in periprosthetic tissues and may be a surrogate for counting neutrophils as an objective parameter for PJI.Purpose: The accurate diagnosis of periprosthetic joint infection (PJI) relies on clinical investigation, laboratory parameters, radiological methods, sterile joint aspiration for synovial fluid leucocyte count and microbiological analysis and tissue sampling for histopathology. Due to the limits in specificity and sensitivity of these methods, molecular techniques and new biomarkers were introduced into the diagnostic procedure. Histological examination is related to the amount of neutrophils in the periprosthetic tissue in frozen sections and formalin-fixed paraffin embedded material (FFPE). However, the threshold of neutrophils per defined area of tissue among various studies is very inconsistent.Methods: We have applied matrix-assisted laser desorption ionisation time-of-flight imaging mass spectrometry (MALDI IMS) to a total of 32 periprosthetic tissue samples of patients with PJI to detect peptides associated with areas of neutrophil infiltration. © 2014, SICOT aisbl.

Longuespee R.,University of Liege | Longuespee R.,Proteopath GbR | Alberts D.,University of Liege | Pottier C.,University of Liege | And 8 more authors.
Methods | Year: 2015

Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest. © 2016.

Petzold J.,Zentrum fur Molekulare Orthopadie | Casadonte R.,Proteopath GbR | Otto M.,Proteopath GbR | Kriegsmann M.,University of Heidelberg | And 7 more authors.
Zeitschrift fur Rheumatologie | Year: 2015

Background: The classification of meniscal lesions requires correlation with clinical data. For the standardization of histopathology reports a discrimination between normal, low-grade lesions and high-grade lesions is feasible. This classification can be further specified using other methods. Material and methods: Formalin-fixed, paraffin-embedded specimens of meniscal tissue from 68 patients were analyzed by matrix-assisted laser desorption ionization (MALDI) imaging. Results: The classification of meniscal lesions and differentiation between low-grade and high-grade and acute versus non-acute degeneration is possible by determination of the differential expression of mass-to-charge ratios by statistical comparisons using the P-value from combined Wilcoxon and Kruskal-Wallis (PWKW) tests and a predefined average two-fold difference in intensity. Conclusion: The concept of a “meniscus report” is introduced for documentation of meniscus tissue specimens integrating histological, histochemical and proteomic data, thereby specifying the degree of degeneration and the assessment of acute or non-acute lesions. Mass spectrometry contributes to an objective histopathology report. An advisory opinion should always be based on close correlation of clinical and morphological evaluations. © 2014, Springer-Verlag Berlin Heidelberg.

Kriegsmann J.,Medizinisches Versorgungszentrum fur Histologie | Kriegsmann J.,Proteopath GbR | Arens N.,Molekularpathologie | Altmann C.,Molekularpathologie | And 3 more authors.
Pathologe | Year: 2014

The diagnosis of infections in patients with arthritis and/or in joint prostheses requires interdisciplinary cooperation and the application of up-to-date methods. The histological investigation of the synovial membrane allows the differentiation of acute, chronic and granulomatous synovialitis. Detection of conserved regions of the microbial genome by PCR, especially 16S rRNA for bacteria and 18S rRNA for fungi, is a broad approach for the classification of pathogens which cannot be cultured. Acute infectious arthritis and periprosthetic infections share the spectrum of pathogens with sepsis, therefore multiplex PCR-based methods for the detection of sepsis can be employed. Molecular diagnostics can detect minimal infections in periprosthetic tissues even after antibiotic therapy. The anamnesis (enteral or urogenital infection), clinical picture (oligoarthritis) and further parameters (e.g. HLA B27 status) are important for the diagnosis of reactive arthritis. In many cases of reactive arthritis, molecular methods allow the detection of bacterial DNA or RNA in synovial fluid or tissue samples. The low sensitivity of histopathological methods may be compensated by application of PCR techniques, especially in the differential diagnosis of granulomatous synovitis including mycobacterial infections. Molecular methods can be used to support the differential diagnosis of septic and reactive arthritis. MicroRNA techniques combined with PCR for detection of pathogens support the differential diagnosis of rheumatoid arthritis with severe inflammatory activity compared to infectious arthritis. Proteomic methods could expand the methodological spectrum for the diagnosis of infections. © 2014, Springer-Verlag Berlin Heidelberg.

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