P1252428711rpxKL

1
Correlation of eumelanin content in human skin
determined by histological and analytical means
with a new non-invasive technique, chromophore
mapping
Matts, P.J., Dykes, P.J.? and Marks,
R.? Procter Gamble, Egham, Surrey, UK
?Cutest, Cardiff, South Glamorgan, UK
INTRODUCTION
RESULTS
OBJECTIVE
CONCLUSION
There continues to be a need for objective,
non-invasive methods to measure melanin
concentration in situ and in vivo in human skin,
independent of the confounding chromophore,
hemoglobin. Existing methods are limited by a
number of factors, including lack of specificity
and inability to resolve the spatial distribution
of these chromophores. A new measurement
capability, SIAscopy (Spectrophoto-metric
Intracutaneous Analysis)?, operates on the
principle of chromophore mapping, that is, the
in vivo measurement of the concentration and
distribution of eumelanin, oxyhemoglobin and
dermal collagen, to produce mutually-exclusive
grayscale concentration maps of these
chromophores. Here we report a study to
validate and calibrate the measurement of
eumelanin using SIAscopic techniques, relating
these with histologically and analytically-determi
ned eumelanin concentrations in non-sun exposed
skin from subjects of Fitzpatrick Skin Types I-VI.
To correlate the eumelanin content in human skin
determined by existing histological and
analytical means with a new, non-invasive
technique, chromophore mapping, based on the
principle of SIAscopy (Astron Clinica,
Cambridge, UK).
Clear, significant (plt0.0001) correlations
(r2gt0.7) were obtained between Contact and
Non-Contact SIAscope-derived eumelanin values and
actual eumelanin tissue loading (determined both
histologically and analytically), across the full
range of Fitzpatrick skin types. There was no
correlation between SIAscope-derived eumelanin
and hemoglobin values, indicating efficient
separation of the two chromophores (data not
shown). This non-invasive method for eumelanin
determination is potentially useful as a new
clinical tool, for example in the measurement,
quantification and tracking of photodamage
endpoints (e.g., lentigos / diffuse
hyper-pigmentation), skin disease states (e.g.,
pigmentation disorders such as vitiligo) and
induced melanogenesis (e.g., UV therapy),
independent of hemoglobin-based features. We
conclude that these new Contact and Non-Contact
chromophore mapping techniques, based on
SIAscopy, provide robust, rapid non-invasive
measures of the concentration and spatial
distribution of eumelanin in vivo, independent of
hemoglobin, which correspond to true tissue
values for this chromophore.
Example of full-face Non-Contact SIAscope
chromophore maps (female subject aged 35).
(3a) original cross-polarised white-light
digital photograph (3b) eumelanin concentration
map (3c) oxyhaemoglobin concentration map
1a
1b
3a
3b
3c
corresponding to eumelanin concentration maps,
were analysed by custom image analysis algorithms
to yield a mean index for eumelanin concentration
across the imaged field. Subsequently two 4mm
punch biopsies were taken from each inner upper
arm, one per arm after injection of local
anaesthesia. One biopsy was fixed in formalin
and processed for histology. Specifically,
sections were stained for melanin using a silver
staining technique (Von Kossa stain) and the
amount of melanin graded microscopically (using a
continuous visual analogue scale electronic
Vasmeter Innovaderm meter). The other biopsy
was sent to the laboratories of Professor Shosuke
Ito and Dr Kazu Wakamatsu (Fujita Health
University, School of Health Sciences, Toyoake,
Aichi, Japan) and subjected to the
micro-analytical techniques developed by these
researchers to yield precise quantitative
measures of melanin (ng eumelanin / mg wet
tissue). The correlation between the different
methods of melanin measurement was determined
using simple regression analysis.
Simple regression analysis of (4a)
Contact SIAscope eumelanin map mean grayscale and
(4b) Non-Contact SIAscope eumelanin map mean
grayscale vs mean silver stain density / field
1c
4a
4b
Simple regression analysis of (4c)
Contact SIAscope eumelanin map mean grayscale and
(4d) Non-Contact SIAscope eumelanin map mean
grayscale vs analytically-determined eumelanin
tissue concentration (ng / wet mg)
Examples of Von Kossa staining of epidermal
melanin in different Fitzpatrick skin types from
this study (x200) (1a) Type I (1b) Type IV
(1c) Type VI
4c
4d
METHODS
Means and 95 LSD Intervals for (5a) Contact
SIAscope eumelanin map mean grayscale values (256
map value) and (5b) Non-Contact SIAscope
eumelanin map mean grayscale values (256 map
value) vs Fitzpatrick Skin Type
Skin melanin content was assessed in 5 subjects
each from Fitzpatrick Skin Types I-VI (total 30
subjects) using chromophore mapping via Contact
SIAscopy (using a SIAscope II instrument Astron
Clinica, Cambridge, UK) and a custom Non-Contact
SIAscopy system (calibrated Fuji S2Pro and
cross-polarised flash illumination). Measurements
were performed on the inner (i.e., non
sun-exposed) aspect of both upper arms. The
resulting grey-scale images,
2b
2a
5a
5b
Means and 95 LSD Intervals for (5c) melanin
silver stain density and (5d) eumelanin
concentration (ng / wet mg) vs Fitzpatrick Skin
Type
References Ito S, Wakamatsu K. Quantitative
analysis of eumelanin and pheomelanin in humans,
mice, and other animals a comparative review.
Pigment Cell Res.16(5) 523-31, 2003 Moncrieff
M, Cotton SD, Claridge E, Hall PN.
Spectrophotometric intracutaneous analysis - a
new technique for imaging pigmented skin lesions.
Br J Dermatol 146(3) 448-57, 2002
2d
2c
Example of SIAscope II chromophore maps taken
from inner upper arm skin of Subject 1 (Type I
skin 12mm diameter). (2a) composite white
light image (2b) oxyhaemoglobin concentration
map (2c) eumelanin concentration map (2d)
collagen concentration map
5d
5c
This work was funded by PG Beauty