Diagnostic des maladies du foie par spectroscopie et imagerie multimodale
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1 Diagnostic des maladies du foie par spectroscopie et imagerie multimodale François Le Naour Inserm U1193, Centre HépatoBiliaire, Villejuif Médecine moléculaire et modèles animaux: pathologie hépatique Institut Pasteur 18 mai 2017
2 Function of the liver Glycogen synthesis & catabolism Lipid synthesis & catabolism (production of triglyceride) Lipoprotein synthesis Destruction of old red blood cells & leucocytes Production of coagulation factors Metabolism of toxins and drugs Urea synthesis Storage of vitamins Storage of elements (Fe, Cu) The liver is the organ with the most important activity in metabolism
3 Chronic liver diseases and cancer Normal liver Cirrhosis Hepatocellular Carcinoma Alcohol Dysmetabolic syndrome (diabete, obesity) Viral Hepatitis 6 th cause of cancer worldwide 2 nd cause of death by cancer Poor prognosis (survival < 1 year)
4 Chronic liver diseases and cancer Normal liver Cirrhosis Hepatocellular Carcinoma Alcohol Dysmetabolic syndrome (diabete, obesity) Viral Hepatitis 6 th cause of cancer worldwide 2 nd cause of death by cancer Poor prognosis (survival < 1 year) Need of markers for diagnostic & prognostic
5 Hypothèse Le développement des pathologies induit des variations de composition. La composition globale reflète l état du tissu.
6 A multimodal spectroscopy-based approach at synchrotron SOLEIL Liver Cryomicrotome Tissue section The synchrotron SOLEIL Brillance Accordability IR UV X-ray Microspectroscopy Spectral markers
7 The synchrotron SOLEIL Electrons Light Characteristics - Brightness IR UV X - Accordability
8 Light Electrons
9 X-ray fluorescence spectroscopy Jean-Philippe Braly/LookatSciences
10 Infrared
11 Infrared microspectroscopy at SMIS beamline Infrared
12 Multimodal spectroscopy 10 µm Elemental composition Autofluorescence Biochemical composition
13 Analyse qualitative de la stéatose hépatique
14 Chronic liver diseases Normal liver Steatosis Cirrhosis Alcohol Dysmetabolic syndrome (diabete, obesity) Viral Hepatitis
15 Liver steatosis Steatosis: excess of fat in hepatocytes (TG) Lipid droplets in at least 5% of cells
16 The synchrotron SOLEIL Electrons Light InfraRed
17 Infrared spectroscopy IR Polyethyleneoxide Absorbance Energy IR IR IR H C O O H H C O C N cm cm / 1540 cm-1
18 Infrared spectroscopy on tissues IR Proteins Absorbance Lipids ADN/sugars Tissue section Wave number (cm-1) The spectrum is resulting of the global biochemical composition
19 Infrared microspectroscopy on steatosis Min cm -1 Proteins cm-1 Lipids cm-1 Esters cm-1 Unsaturated Max Absorbance Absorbance Steatotic Unsaturated C=C 3500 non steatotic 3500 Lipids CH2 & CH Esters C=O 2000 Wavenumber (cm-1) Proteins Amides I & II Esters C=O
20 Analysis of various stages of steatosis Infrared spectroscopy & Principal component analysis (PCA) Healthy Low Intermediate High Lipids: cm -1
21 Analysis of various stages of steatosis Healthy Low Intermediate High CH3 Molecular changes in the composition CH2 C=C
22 Time of flight-secondary ion mass spectrometry TOF-SIMS Aggregates Bi3+ Secondary ions Desorption ionisation Molecules Tissue section
23 Time of flight-secondary ion mass spectrometry TOF-SIMS Bi kev Without any treatment of the sample Lateral resolution : 1-2µm Mass <1500 Da Lipids
24 Lipidomic in situ of steatosis using ToF-SIMS Centrilobular vein Intensity per unit surface (counts x 10 3 ) b MAG Chol DAG PC TAG Portal tract m/z d
25 Lipidomic in situ of steatosis using ToF-SIMS DAG : diacyl glycerol C36:4 C36:3 C36:2 C36:1 C36:0
26 Lipidomic in situ of steatosis using ToF-SIMS DAG C36 unsaturated DAG C36 saturated Red = Unsaturated Green = Saturated
27 Multimodal spectroscopy on steatosis Infrared & ToF-SIMS Gradation of steatosis Molecular changes in the composition steatotic vesicles Enrichment in cholesterol Lipids with longer acyl chains Enrichment in unsaturated lipids Steatotic vesicles potentially highly reactive to peroxydation
28 Stéatose & transplantation hépatique
29 Chronic liver diseases and cancer Normal liver Cirrhosis Hepatocellular Carcinoma Alcohol Dysmetabolic syndrome (diabete, obesity) Viral Hepatitis 6 th cause of cancer worldwide 2 nd cause of death by cancer Poor prognosis (survival < 1 year) TRANSPLANTATION
30 Liver transplantation : a good treatment for liver diseases Agence de la Biomédecine
31 Limitation of liver transplantation Shortage of grafts Year Transplantation Waiting patients nc 1104 Agence de la Biomédecine
32 Limitation of liver transplantation Shortage of grafts Additional grafts: living donors, graft splitting, domino program, use of marginal grafts, non-heart-beating donors Additional grafts But increased risk Quality of the graft: Steatosis
33 Graft steatosis Primary non-function 80% with steatosis > 60% 15% with steatosis 30-60% Delayed function 35% with steatosis 30-60% Long term poorer evolution of the graft: fibrosis, survival Ploeg Transplantation 1993
34 Steatosis sensibilizes liver to ischemia-reperfusion lesions through altered blood flow in liver microcirculation and enhanced peroxydation of membrane lipids
35 Ischemia-Reperfusion lesions in liver graft
36 20, 30 or 40%? Estimation of steatosis
37 Assessment of Hepatic Steatosis by Expert Pathologists: The End of a Gold Standard El-Badry, et al Annals of Surgery. 2009; 250(5): Cong Poor inter-observer reproducibility Par
38 Estimation of steatosis in the context of emergency Paraffin Frozen > 24h < 15 min A liver transplantation has to be performed in less than 8h
39 Histological estimation of steatosis is not correlated to the lipid content % steatosis Triglycerides (TG) (nmol/mg)
40 Histological estimation of steatosis - Poor inter-observer reproducibility - Poor conservation of the morphology on frozen tissue sections - Not correlated to the true lipid content
41 Analysis of the non-steatotic tissue Normal liver + Absorbance Steatosis + Absorbance Wave number (cm-1) The non-steatotic part of the tissue is NOT normal The whole tissue reflects the grade of steatosis
42 Implementation to the hospital Easy to use method No need of liquid nitrogen Regular glass slide Automatic calculation using the standard curve Only 1 spectrum Only 1 pixel 2 mm x 2 mm Only 1 minute! ATR infrared spectroscopy
43
44 Quantification of steatosis ATR-InfraRed spectroscopy Pixel 2 mm x 2 mm 500µm Absorbance Lipids Proteins Ratio: lipids / proteins Wavenumbers (cm -1 ) Estimation of lipids
45 Quantification of steatosis InfraRed microspectroscopy 25 tissue samples from liver surgical specimens - Steatosis : 5 to 90% Infrared spectroscopy allows the quantification of lipids on tissue section
46 Quantification of steatosis ATR-InfraRed spectroscopy
47 Lipid evaluation by IR microspectroscopy Estimated Measured [TG] nmol/mg Liver grafts Validation on additional 21 liver graft biopsies
48 Quantification of liver steatosis Reproducible Rapid method (1 min) Non expensive procedure Assessment of liver graft quality
49 P Dumas C Sandt I Youssef L Gadea M Réfrégiers F Jamme N Trcera S Kascakova M Danulot C Peng A Brunelle D Touboul D Debois O Laprévote C Guettier M Wavelet B Trousselle MP Bralet Denis Castaing Eric Vibert Thanks for fundings Centre Hépatobiliaire Hôpital Paul Brousse Université Paris-Sud
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