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Extracellular vesicles in the AMC
Rienk Nieuwland
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Clinical relevance of EVs
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VOC members (AMC) • René Berckmans, PhD • Frank Coumans, PhD
(VENI) • Elmar Gool, PhD student • Aleksandra Gasecka, MD/PhD
student/Cardiologist • Chi Hau, research technician • Doortje
Horjus, PhD student • Najat Hajji, research technician • Ton van
Leeuwen, PhD • Rienk Nieuwland, PhD • Edwin van der Pol, PhD (VENI)
• Linda Rikkert, PhD student • Leonie de Rond, PhD student •
Marianne Schaap, research technician • Guus Sturk, PhD • Johannes
Thaler, MD/PhD/Haematologist • Yuanjie Yu, PhD student
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EV research lines AMC
1. Detection and isolation 2. Standardization 3. Therapy 4.
Pathology 5. Biomarker
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1. Detection and isolation
1. Signal analysis 2. Development Flow cytometry Raman
microspectroscopy Surface plasmon resonance imaging Transmission
electron microscopy Physical parameters (size, refractive
index)
3. Isolation Size exclusion chromatography
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1.1 Signal analysis
I + –
ΔI
d
D ̴L
I
J Extracell Vesicles. 2014 Dec 10;3:25922. doi:
10.3402/jev.v3.25922
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1.1 Signal analysis
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1.2 Flow cytometry
Leonie de Rond, PhD; Poster 19: generic fluorescent dyes
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1.2 Hybrid Raman microspectroscopy and resistive pulse sensing
(label-free)
Cees Otto (UT)
Hybrid AMC
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1.2 Surface Plasmon Resonance imaging
Elmar Gool, PhD
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1.2 Transmission Electron Microscopy
Linda Rikkert, PhD; poster 18: comparison of TEM protocols
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1.2 Physical parameters
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Proof of the pudding: Have developments lead to improved
detection?
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JEV 2014, 3: 23430 - http://dx.doi.org/10.3402/jev.v3.23430
1.2 Size exclusion chromatography
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1.2 Size exclusion chromatography
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EV research lines AMC
Detection and isolation 2. Standardization 3. Therapy 4.
Pathology 5. Biomarker
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2. Standardization Prerequisite for clinical application
1. Pre-analytical variables (METVES, ISTH) 2. Flow cytometry
• ISEV-ISTH-ISAC workgroup • Scatter to size • Scatter to size
and refractive index (Flow-SR) • Hollow silica beads • Exometry
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Circulation Research 2017; 120: 1632-48
2.1 Pre-analytical variables
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2.2 Flow cytometry workgroup
www.flowcytometry.org
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20
2.2 From scatter to size (requires refractive index)
Ligh
t sca
tter
ing
(a.u
.)
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2.2 From scatter to size & refractive index
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2.2 Standardization
• Aim Convert EV scatter signal to diameter • Contact
[email protected]
[email protected]
mailto:[email protected]:[email protected]
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EV research lines AMC
Detection and isolation Standardization 3. Therapy 4. Pathology
5. Biomarker
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3. Therapy
Aim: CD39/CD73 EVs for treatment of inflammatory disease
Contact: [email protected] (Arthrogen) Collaboration: VOC
mailto:[email protected]
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EV research lines AMC
Detection and isolation Standardization Therapy 4. Pathology 5.
Biomarker
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4. Department of Intensive Care
Aim Role of EVs in transfusion-associated outcome of critically
ill and injured patients Contact [email protected]
Collaboration Sanquin, VOC
mailto:[email protected]://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj3k8bJhNLWAhXBh7QKHfP3ADwQjRwIBw&url=https://depositphotos.com/14930129/stock-illustration-hospital-blood-bag-cartoon.html&psig=AOvVaw1K09h2oVL6x33g8Ad5KiM2&ust=1507037013138842https://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiagcejmNTWAhWFIlAKHQlUAFQQjRwIBw&url=https://depositphotos.com/13949806/stock-illustration-cartoon-hospital-patient.html&psig=AOvVaw0uIZ_i5VVq0dNVyg0nDuvq&ust=1507111034197845
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4. Department of Pathology
Aim Role of EVs in development of diabetic nephropathy Contact
[email protected] [email protected] Collaboration Slotervaart,
VUmc, VOC
mailto:[email protected]:[email protected]
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4. Lab of Experimental Immunology
Aim Role innate immune effector cell-derived EVs in dendritic
cell-driven immune regulation Contact [email protected]
Collaboration UU
mailto:[email protected]
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EV research lines AMC
Detection and isolation Standardization Therapy Pathology 5.
Biomarker
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5. EVs as biomarker
1. Cancer-ID 2. Parasites 3. Coagulation
• Department of Vascular medicine (AMC) • 4th PhD student
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5.1 Cancer-ID
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5.2 Biomarker
Aim Exosomes from parasites for diagnosis, drug and vaccine
development Contact [email protected] (Parasitology)
Collaboration Swiss Tropical Institute, Aarhus Univ.
Leishmania major exosomes Merlin van Loenen
mailto:[email protected]
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5.3 Coagulation
• Venous thromboembolism (VTE) is second cause of death of all
hospitalized cancer patients
• OR 6.5 when patients receive chemotherapy • Risk for
thrombosis highest in first 3 months and
in metastasized disease • Current guidelines recommend against
routine
thromboprophylaxis in ambulatory patients
Arch Intern Med 2000;160: 809-15; Crit Rev Oncol Hematol 2008;
66: 145-54; Ann Oncol 2010; 21: Suppl 5: v274-6; LoS Med 2012;
9(7): e1001275; Blood 2013; 122: 1712-23; J Thromb Haemost 2013;
11: 56-70; J Clin Oncol 2013; 31: 654-6; Cochrane Database Syst Rev
2016; 12(2): CD008500
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Study questions
• Can we identify cancer patients at high risk of developing
VTE?
• Can we prevent thrombosis in cancer
patients at risk of developing VTE?
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Clinical prediction models Khorana score
Blood 2008; 111: 4902-7
Risk score: 0 points = low risk; 1-2 points: intermediate risk;
≥ 3 points: high risk
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• 876 patients • Various types of cancer
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Conclusion
Haematologica 2017; 102(9): 1494-1502
“The present findings do not support the use of any of the
examined scores to select
patients for thromboprophylaxis”
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5.3 Coagulation (hemostasis)
41
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18,0
00 x
g
137,
000
x g
Supe
rnat
ant
18,0
00 x
g
137,
000
x g
Supe
rnat
ant
18,0
00 x
g
137,
000
x g
Supe
rnat
ant
donor 1 donor 2 donor 3
200 nm
Blood 2011; 117: 3172-3180
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Lancet 1995;346:1004-5; Br J Cancer 2007;96:290-5; J Thromb
Haemostas 2017;15:1-10
Tumors release TF+ EVs
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Can a TF-EV clotting test predict
VTE in cancer patients?
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Procoagulant activity of TF-EVs for prediction of VTE in cancer
patients
• Prospective cohort study • 648 patients • 40 patients
developed VTE (6.1%) • 6 hospitals • Various types / stages of
cancer • Primary outcome: VTE (6-months follow-up)
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Prediction of VTE
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VTE prediction in pancreatic cancer
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Statuten Fryske Ferien foar…
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Acknowledgements René Berckmans Harry Büller Frank Coumans
Frederiek van Doormaal Nick van Es Elmar Gool Aleksandra Gasecka
Chi Hau Doortje Horjus Najat Hajji Ankie Kleinjan Romaric Lacroix
(France) Ton van Leeuwen Rienk Nieuwland Edwin van der Pol Linda
Rikkert Leonie de Rond Marianne Schaap Pia Siljander (Finland) Guus
Sturk Johannes Thaler Sami Valkonen (Finland) Zoltán Varga
(Hungary) Yuanjie Yu Yuana Yuana
Extracellular vesicles� in the AMCClinical relevance of EVsVOC
members (AMC)EV research lines AMC1. Detection and isolation1.1
Signal analysis1.1 Signal analysis1.2 Flow cytometry1.2 Hybrid
Raman microspectroscopy and resistive pulse sensing (label-free)1.2
Surface Plasmon Resonance imaging1.2 Transmission Electron
Microscopy1.2 Physical parametersProof of the pudding: Have
developments lead to improved detection?1.2 Size exclusion
chromatography1.2 Size exclusion chromatographyEV research lines
AMC2. Standardization�Prerequisite for clinical application2.1
Pre-analytical variables2.2 Flow cytometry workgroup�2.2 From
scatter to size�(requires refractive index)2.2 From scatter to size
and refractive index�2.2 Label-free identification based on
refractive index differences2.2 From scatter to size &
refractive index2.2 Light scatter reference particles�Hollow silica
beads2.2 StandardizationEV research lines AMC3. TherapyEV research
lines AMC4. Department of Intensive Care4. Department of
Pathology4. Lab of Experimental ImmunologyEV research lines AMC5.
EVs as biomarker5.1 Cancer-ID5.2 Biomarker5.3 CoagulationStudy
questionsClinical prediction models Khorana scoreDianummer
39Conclusion5.3 Coagulation (hemostasis)Dianummer 42Tumors release
TF+ EVs�Can a TF-EV clotting test predict VTE in cancer
patients?Procoagulant activity of TF-EVs for prediction of VTE in
cancer patientsPrediction of VTEVTE prediction in pancreatic
cancerDianummer 48Statuten Fryske Ferien foar…Acknowledgements
