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Early state detection of skin inflammation by AFM
Introd
Peter Schön1, Ercüment Unsurorlu1, Cas Damen2, Christoph Riethmüller3, Martin Bennink1 1) NanoBioInterface and 2) NanoPhysics Group, Saxion University of Applied Sciences, Enschede 3) Serend-ip GmbH, CenTech Münster, Germany
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Atomic Force Microsopy (AFM)
tactile method
marker-free
ultra-resolution (1 nm! in height)
Diagnosis
by
AFM ?
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Atopic Dermatitis (AD)
Skin inflammations (in general):
230 million diagnoses in 2010
(3,5% world population)
Detection:
– Visible marks, itching, weeping
and crusting
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AD Diagnosis: patch test
series of allergens to the skin surface, evaluation 1-3 days
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Stratum corneum
Integrity of the skin barrier is a prerequisite for health !
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Corneocytes = ‘messengers’
Dynamic equilibrium= proliferating epidermis balanced by permanent abrasion of cornified epithelial cells
Corneocytes = messengers
minute changes of their morphology might hint at out-of-balance processes underneath
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Atopic Dermatitis
• Atopic dermatitis (AD) :
no clear knowledge about the exact cause
and mechanism
disturbed organization of the cytoskeleton on
desmosome disruption
immature and fragile cornified envelopes (CEs), and attachment sites of desmosomes
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Materials and methods
No labelling or sample processing
Pain free method
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Materials and methods
Nanoscale Topography
Atomic Force Microscopy
with network analysis based software
‘Biomarker’: Villus-like projections (VP)
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394
topography recording computer vision number value
Dermal
Topographical
Index
DTI
On skin samples, the assay is termed
x/y = 40 µm, z = 500 nm
The extraction method is patented (US, Western Europe)
DERMATACT
VERUM INDEX SUI ET FALSI
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Healthy vs inflamed skin
DERMATACT Index (DMI)
Risk factor: +100 corneocytes/400 µm²
Franz
et al.
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Skin depth
– 1 patient, 1 position, 6 different layers
– Decrease in VPs in depth skin
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Age and Pigmentation do not matter
Outliers reveal a hereditary background,
(elevated count hints at individual risk)
Age (years)
outliers are healthy, but have a
hereditary background in skin
disease
Skin Type
Study on healthy individuals (2013/14)
Franz
et al.
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Clinical tests
Second batch:
– 9 different patients, same layer
– P1, P2, P6, P10 are risk factors
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AFM vs clinics
Patient Risk factor Disease Match
P1 Yes Skin inflamation Yes
P2 Yes Skin inflamation Yes
P3 No Contact with skin inflamation Yes
P4 No P3 without contact (healthy) Yes
P5 No Healthy person Yes
P6 Yes Scar No
P7 No Healthy person Yes
P9 No Healthy person Yes
P10 Yes Skin inflamation in the past Yes
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Scar vs healthy tissue
20 µm2
Healthy Scar
z= 1 µm
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FLG mutation and DMI
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Genetics:
Filaggrin LOF mutations
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Immunogold labelling
targeting corneodesmosin
Dr. Marek Haftek, Uni Lyon
FLG
+/+
F
LG
-/-
F
LG
-/+
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(6 weeks with topical glucocorticoids) THERAPY Monitoring
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Conclusion
• DTI indicates (inflammatory) barrier damage
• Objective, accurate, painless method
• works at non-lesional skin areas
➤ predict an individual risk for skin diseases (subclinically)
➤ abbreviate clinical studies (reduce study population)
➤ enable effective therapy monitoring (pro-actively)
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Atomic Force Microsopy (AFM)
tactile method
marker-free
ultra-resolution (1 nm! in height)
Diagnosis
by
AFM !
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Detergent causes drastic DTI increase
20 µm2 z= 1 µm
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Sodium Lauryl sulfate
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Genetics:
Filaggrin LOF mutations
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Biochemistry:
Natural Moisturizing Factor (NMF)
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Physiology:
Barrier function as TransEpithelial Water Loss (TEWL)
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DERMATACT Index (DTI) of non-lesional sites
correlates to severity of skin lesions (SCORAD)
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Immunogold labelling
targeting corneodesmosin
Dr. Marek Haftek, Uni Lyon
FLG
+/+
F
LG
-/-
F
LG
-/+
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Method Comparison
Intensity Height
SEM AFM
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SE
M
20 µm
AF
M
80 µm 5 µm
Circular protrusions on corneocytes
kn
ow
n s
ince 1
97
9