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In vivo real-time confocal imaging of non-pigmented cutaneous neoplasms
ESDR I JSID I SID Abstracts
0667 REAL-TIME CONFOCAL IMAGIN,; OF NON-PIGh;lENTED CUTANEOUS NEOPLASMS. ‘Salvador Mathew White. &na& ‘.‘M’,i”d R h .sJamesZavislan.‘Jessica’Rand Rubinstcin. I
IN VW0
Rex Wellman~Labbratories of Photomedicine, ZDermatology Surgery Dcpartm& oi Dermatology, Massachusetts General Hospital, Harvard Mcdicai School, Boston, MA, 3Lucid Technologies Inc., Henrietta. NY, USA. The Confocal scanning laser microscope (CSLM) provides real-time images of thm sections of living tissue with high resolution and contrast. Typically, the lateral resolution is OS-l.0 pm and axial resolution (virtual optical section thickness) is 3-5 pm. Imaging is possible to a maximal depth of 460 pm. This study was aimed to stablish the microscopic characteristics of non-pigmented cutaneous neoplasms using the CSLM in viva Lesions and margins of each lesion were viewed in viva using the CSLM end later correlated with routine histology. Included in the patient study were subjects with actinic kcratoses, squamous cell carcinoma, and basal cell carcinoma. Differences in nuclear and cellular morphology as well as tissue architecture between lesions and surrounding normal skin were analyzed. Microscopic characteristics from virtual sections recorded with the non-invasive, in viva CSLM correlated well with ex viva horizontal and vertical sections obtained from biopsies. In a significant number of patients, the margin of their cutaneous neoplasm was imaged and clearly distinguished from the surrounding normal skm. Our study shows that the in viva CSLM may be a powerful imaging tool in the diagnosis of cutaneous malignancies and could be helpful as adjuvant to Mohs micrographic surgery in the assessment of tumor margins.
0668 AN LJLTRASlRUCIURAL
ulo”&t to play a pathogenic role in the develop~t ot ulc.rarion. On. hypoth..is ha. .ugg..t.d that parloapillary fibrin d.po.ite *ot (I. * barrier to th. diffusion of 0xyg.n and oth.r nutriant.. Boe.v.r, th. aettlal ep*ti*l configuration of pericapillarg fibrin is “nknovn, md it r.m*ins oria**r *.th.r ft truly repr.*.*t* II barrier that Carl ilpoti+r obvsiolooic*l .xch~o.e bet”..” th. blood and dad.. Wina
horiconklly, with confoch nicro.copy. ‘kh. dilrriaution-of Fibrin d.po*ition “** hlghal “arlabl. and *t&y, with *r.a. of gr.*t int.n*ity n.xt M 0 TK. r* OF wargina P intauity. V.rtioal * ti**u. section. .hoW.d bQh.r eonc.ntr*tion of f111or..o.,,t .W.r 1 ill -Yaof tb. region next to the loL d.-l capi1l.ri.s. Borizo*tal scanning oi th. e.otion. indicata that Mxi65l fluor.w.no. “** di.tt,b”t.d *t radon, and not n.o..**rily ti th. top ..ctlons, suggeeting that thie distribution i* not the r..at Of (LII artifact Of antibody p.n.tr*tiOn. *hr.. di..o*ion*l r.oon.truot~oll of H1.Ct.d are** OF fibrin d.po.ition v.rxi.d the irregular fibrin ill the We.81 wall, oonfirsdng that it I. di*oontti”ol*. In .mry, our findings indicata that fibrin d.posit. in ‘,.nou* ulo.r* *re patch and di.COntiuou* around de-1 r.*s.la and e.nnot *ot *(I M alar. od[ o barri.=, *. orio,.nallv Dmw..d. Th. an.stion nnain..
dintribm.ionOf
0671
AND CY’I0MORPHOME’IRICAL STUDY OF MELANOCY7F.S IN PHOTODAMAGED SKIN. mrohashi Masahiko Toyoda, Department of Dermatology, ToyaaLl Medical and Pharmaceutical University, Toyama, Japan. lhe photoaging process produces some distinctive morphological skin alterations that are not present in intrinsically aged skin. To examine pathological changes of melanocytes in photodamaged skin, we performed a compamtive cytomorphometrical analysis as well as a qualitative observation of melanocytes from sun-exposed and from sun-protected facial ski at the electron microscopic level. The characteristic ultrastructural features of mzlanocytes in photodamaged skin were as follows: 1) a statistically significant increase in their number per unit area, 2) a marked nuclear heterogeneity, 3) signs of cell activation characterized by increased melanogenesis, abundant cytoplasmic organelles and elongation of dendritic processes, 4) close apposition to photodamaged degenerate keratinocytes, 5) degenerative changes represented by large intracytoplasmic vacuoles, condensed nuclear chromatin and higher electron density of cytoplasm wth disrupted mitochondria, 6) frequent direct contact with Langerhans cells, suggesting some interaction between these two types of ce!ls. Cytonxxphometrical analysis revealed significant decreases in cell and nuclear sizes, increases in cell and nuclear perimeters accompanied by irregular contours, and higher degrees of nuclear ellipticity in sun-exposed melanocytes. ‘lhis study denxmstrates that there are remarkable differences in the morphologics of melanocytes between photoaged and intrinsically aged facial ski, and supports the concept that photoaging processes contribute to cytological alterations in melanocytes.
ALTERATIONS OF DERMO-EPIDERMAL JUNCTION IN HYALINOSIS CUTIS ET MUCOSAE (HCM). I.Hausser. Denetdo~y. Universityof Heidelberg. German Cancar ResearchCenter (DB. RB), Heidelberg, Germany. Hyalinoeis cutis et rnucosee (HCM) is a rere, autosomal recessively inherited diswder effectingskin, mucosaaand Ieryrx Histdogicly, accumulationsof hyaline material are found in the connectivetissue. especially et the junctionalzones of epidermis. appendand upper dermal vessels. Since the Mrsshudwe of HCM-skin had indicated a messive deposition of basement membrane material WB investigated (i) if there wss a dysbelance of BM and associated compon&s in skin by indirect immunoQuwescenoe(IIF) and (Ii) possible consequencesfor lhe overal tissue orQenisationepplyinQepide”nal diffsrentlatwnmarkers. In general. there was e tremendous increase of BM material in the skin 8-0 induding, besides the epidermaljunctionalzone. skin-appendagesand bloodvessels. Thus. collagen IV. BM-lamini” end nidogenwere “at largely resbicted to a distinctnarrow zone as in u”Mecfed healthy control tissue but also formed huge plaques in the dermis mostly around vessels. Comparable to wound tissue Rbmnectin and tenascin were strongly expressed In upper denis. Only minor aberraticns from “o”?Isl were seer! for laminind which wss virtually restricted to junctions associated with epithelial tissue. On the contrary, epidermal compartment&&on and differentiationappeared largely unchanged.Thus. the basal layer was dearly defined by staining for the integrin chains a3 and !31, and keretin 14. As early differentiationmarkers Kerstins 1 and 10 were expressedsuprabesaliy,while the late marker kereti” 2e revealed a” bmgular reaction in the lower suprabesel layers with increasing intensity towards the epidermal surfece. Some focal staining for (he ‘hypexpmliferetlve’tyyp~ Keratins 6 end 16 wes &sewed which, however, wes also occeslonaltysee” in h!Moiog~ normal skin. Pnchorergeof epidermisappeared normal es judged by largely colocelizedlinear stainingfor the integri” chains a6 and !34.Taken together. the ImmunohUochemicel analysis suggests that in Sk,” from HCM patients the major defects ere localized in the dermal compartment.wile the epklermlsis only mildlyaffected.
0669
0672
ARM AND LEG SKlN LYMPHATIC DENSITY STUDIED IN DIFFERENf RAClAL TYPES Uslffi QUANmATlVE FLUORESCENCEMNXOLYMPHOGRAPHY. A.W.B. H.S. pa M. eDSherman. Division of Physiological Medicine (Dermatology) and lDepwtment of PhysMgy. St George’s Hespkal Meedical School, London, UK. We considered the possibility that skin lymphatic density might be higher in dependent regions of the body, providng greater capacity for fluid drainage and compensating for higher capillary filtration rate. Using fluorescence microiymphor graphy (Bollinger et. al., Circulation 1961; 64: 1195-200) and stereological analysis (Stanton et. al., Miirovasc. Res. 1997; 54: 156-63) we capered forearn, and lower leg dennal lymphatic networks. We also canpred lymphatic density in white and Asipn (In&n) subjects. Local Ethics Committee approval wss obtained. The right arms and legs of 6 white and 6 Asian me”, 19-24yr, ware studied 1Opl of 25% fluwescein isothiocysnstedenran, M, 150k, wss injected intrsdermally (366 needle). Filling of lymph vessels with dye was observed using a fluoresce”ce microscope. Lymphangiograms were constructed front mosaics of pints from videotape recordings. Dye4lled lymph vessel networks were analysed by the line intersection method to give lymphatic density at increasing radii from the depot (Lo,, an-l) and total vessel length filled (LL, sum of LD,*annular area, cm). Peak wr u. Peak L@ LL Arm (n-16) lB.Of1.4 11&1.4 White (leg, “-8) 27.3i1.7 40.0i10.6 Leg (n-16) 25.1f1.4 40.2t6.9 Asia” (leg, n-8) 23.Ot2.1 40.3t9.2 P 0.002 0.001 P 0.13 0.9 L& a”dLL were higher in the leg than in the arm, implying an anatwnical difference. Higher lymphatic vessel density in leg dermis would provide greater surface area fa interstitial fluid absorption. No differences were detected between white and Asian subjects.
MICROSCOPY CORNEUM:
OF LIPID STRUCl’URE THE
EFFECTS
OF AGE,
IN THE OUTER STRATUM SKIN
GRADE
AND
R.R. Warner. Y.L. Boissv and S.A. Lane The Procter & Gamble Conmanv. Cincinnati. OH. The effect of age, skin co&i& and exten&l soap use on the lipid structure of the outer stratum comeum (SC) in humans was investigated using transmission electron microscopy of ruthenium-te~oxide-stained tape strips. Young individuals with good skin condition had a normal lamellar lipid structure (Laodmann units) even in the outermost SC. Young individuals with poor skin condition did not have Landmann units in the outer SC; instead, the intercellular spaces were filled with disrupted lamellae, amorphous material, and fibrous material. Individuals over the age of 40, even with good ski condition, typically did not have well-formed Landmann units in the outer SC. Individuals over 40 with poor skii condition had diverse abnormal lipid structures with infrequent lamellae. Frequent soap use resulted in poor skin condition and a distinctive lipid structure consisting of extracted regions, regions with an exaggerated number of disorganized lamellae without a Landmann pattern, and regions containing darkly staining deposits. We conclude tha.t the lipids of the outer SC have a variable but predictable structure. This structure is influenced by age, skin condition, and environmental treatment. FREQUENT
SOAP
USE.
0667 REAL-TIME CONFOCAL IMAGIN,; OF NON-PIGh;lENTED CUTANEOUS NEOPLASMS. ‘Salvador Mathew White. &na& ‘.‘M’,i”d R h .sJamesZavislan.‘Jessica’Rand Rubinstcin. I
IN VW0
Rex Wellman~Labbratories of Photomedicine, ZDermatology Surgery Dcpartm& oi Dermatology, Massachusetts General Hospital, Harvard Mcdicai School, Boston, MA, 3Lucid Technologies Inc., Henrietta. NY, USA. The Confocal scanning laser microscope (CSLM) provides real-time images of thm sections of living tissue with high resolution and contrast. Typically, the lateral resolution is OS-l.0 pm and axial resolution (virtual optical section thickness) is 3-5 pm. Imaging is possible to a maximal depth of 460 pm. This study was aimed to stablish the microscopic characteristics of non-pigmented cutaneous neoplasms using the CSLM in viva Lesions and margins of each lesion were viewed in viva using the CSLM end later correlated with routine histology. Included in the patient study were subjects with actinic kcratoses, squamous cell carcinoma, and basal cell carcinoma. Differences in nuclear and cellular morphology as well as tissue architecture between lesions and surrounding normal skin were analyzed. Microscopic characteristics from virtual sections recorded with the non-invasive, in viva CSLM correlated well with ex viva horizontal and vertical sections obtained from biopsies. In a significant number of patients, the margin of their cutaneous neoplasm was imaged and clearly distinguished from the surrounding normal skm. Our study shows that the in viva CSLM may be a powerful imaging tool in the diagnosis of cutaneous malignancies and could be helpful as adjuvant to Mohs micrographic surgery in the assessment of tumor margins.
0668 AN LJLTRASlRUCIURAL
ulo”&t to play a pathogenic role in the develop~t ot ulc.rarion. On. hypoth..is ha. .ugg..t.d that parloapillary fibrin d.po.ite *ot (I. * barrier to th. diffusion of 0xyg.n and oth.r nutriant.. Boe.v.r, th. aettlal ep*ti*l configuration of pericapillarg fibrin is “nknovn, md it r.m*ins oria**r *.th.r ft truly repr.*.*t* II barrier that Carl ilpoti+r obvsiolooic*l .xch~o.e bet”..” th. blood and dad.. Wina
horiconklly, with confoch nicro.copy. ‘kh. dilrriaution-of Fibrin d.po*ition “** hlghal “arlabl. and *t&y, with *r.a. of gr.*t int.n*ity n.xt M 0 TK. r* OF wargina P intauity. V.rtioal * ti**u. section. .hoW.d bQh.r eonc.ntr*tion of f111or..o.,,t .W.r 1 ill -Yaof tb. region next to the loL d.-l capi1l.ri.s. Borizo*tal scanning oi th. e.otion. indicata that Mxi65l fluor.w.no. “** di.tt,b”t.d *t radon, and not n.o..**rily ti th. top ..ctlons, suggeeting that thie distribution i* not the r..at Of (LII artifact Of antibody p.n.tr*tiOn. *hr.. di..o*ion*l r.oon.truot~oll of H1.Ct.d are** OF fibrin d.po.ition v.rxi.d the irregular fibrin ill the We.81 wall, oonfirsdng that it I. di*oontti”ol*. In .mry, our findings indicata that fibrin d.posit. in ‘,.nou* ulo.r* *re patch and di.COntiuou* around de-1 r.*s.la and e.nnot *ot *(I M alar. od[ o barri.=, *. orio,.nallv Dmw..d. Th. an.stion nnain..
dintribm.ionOf
0671
AND CY’I0MORPHOME’IRICAL STUDY OF MELANOCY7F.S IN PHOTODAMAGED SKIN. mrohashi Masahiko Toyoda, Department of Dermatology, ToyaaLl Medical and Pharmaceutical University, Toyama, Japan. lhe photoaging process produces some distinctive morphological skin alterations that are not present in intrinsically aged skin. To examine pathological changes of melanocytes in photodamaged skin, we performed a compamtive cytomorphometrical analysis as well as a qualitative observation of melanocytes from sun-exposed and from sun-protected facial ski at the electron microscopic level. The characteristic ultrastructural features of mzlanocytes in photodamaged skin were as follows: 1) a statistically significant increase in their number per unit area, 2) a marked nuclear heterogeneity, 3) signs of cell activation characterized by increased melanogenesis, abundant cytoplasmic organelles and elongation of dendritic processes, 4) close apposition to photodamaged degenerate keratinocytes, 5) degenerative changes represented by large intracytoplasmic vacuoles, condensed nuclear chromatin and higher electron density of cytoplasm wth disrupted mitochondria, 6) frequent direct contact with Langerhans cells, suggesting some interaction between these two types of ce!ls. Cytonxxphometrical analysis revealed significant decreases in cell and nuclear sizes, increases in cell and nuclear perimeters accompanied by irregular contours, and higher degrees of nuclear ellipticity in sun-exposed melanocytes. ‘lhis study denxmstrates that there are remarkable differences in the morphologics of melanocytes between photoaged and intrinsically aged facial ski, and supports the concept that photoaging processes contribute to cytological alterations in melanocytes.
ALTERATIONS OF DERMO-EPIDERMAL JUNCTION IN HYALINOSIS CUTIS ET MUCOSAE (HCM). I.Hausser. Denetdo~y. Universityof Heidelberg. German Cancar ResearchCenter (DB. RB), Heidelberg, Germany. Hyalinoeis cutis et rnucosee (HCM) is a rere, autosomal recessively inherited diswder effectingskin, mucosaaand Ieryrx Histdogicly, accumulationsof hyaline material are found in the connectivetissue. especially et the junctionalzones of epidermis. appendand upper dermal vessels. Since the Mrsshudwe of HCM-skin had indicated a messive deposition of basement membrane material WB investigated (i) if there wss a dysbelance of BM and associated compon&s in skin by indirect immunoQuwescenoe(IIF) and (Ii) possible consequencesfor lhe overal tissue orQenisationepplyinQepide”nal diffsrentlatwnmarkers. In general. there was e tremendous increase of BM material in the skin 8-0 induding, besides the epidermaljunctionalzone. skin-appendagesand bloodvessels. Thus. collagen IV. BM-lamini” end nidogenwere “at largely resbicted to a distinctnarrow zone as in u”Mecfed healthy control tissue but also formed huge plaques in the dermis mostly around vessels. Comparable to wound tissue Rbmnectin and tenascin were strongly expressed In upper denis. Only minor aberraticns from “o”?Isl were seer! for laminind which wss virtually restricted to junctions associated with epithelial tissue. On the contrary, epidermal compartment&&on and differentiationappeared largely unchanged.Thus. the basal layer was dearly defined by staining for the integrin chains a3 and !31, and keretin 14. As early differentiationmarkers Kerstins 1 and 10 were expressedsuprabesaliy,while the late marker kereti” 2e revealed a” bmgular reaction in the lower suprabesel layers with increasing intensity towards the epidermal surfece. Some focal staining for (he ‘hypexpmliferetlve’tyyp~ Keratins 6 end 16 wes &sewed which, however, wes also occeslonaltysee” in h!Moiog~ normal skin. Pnchorergeof epidermisappeared normal es judged by largely colocelizedlinear stainingfor the integri” chains a6 and !34.Taken together. the ImmunohUochemicel analysis suggests that in Sk,” from HCM patients the major defects ere localized in the dermal compartment.wile the epklermlsis only mildlyaffected.
0669
0672
ARM AND LEG SKlN LYMPHATIC DENSITY STUDIED IN DIFFERENf RAClAL TYPES Uslffi QUANmATlVE FLUORESCENCEMNXOLYMPHOGRAPHY. A.W.B. H.S. pa M. eDSherman. Division of Physiological Medicine (Dermatology) and lDepwtment of PhysMgy. St George’s Hespkal Meedical School, London, UK. We considered the possibility that skin lymphatic density might be higher in dependent regions of the body, providng greater capacity for fluid drainage and compensating for higher capillary filtration rate. Using fluorescence microiymphor graphy (Bollinger et. al., Circulation 1961; 64: 1195-200) and stereological analysis (Stanton et. al., Miirovasc. Res. 1997; 54: 156-63) we capered forearn, and lower leg dennal lymphatic networks. We also canpred lymphatic density in white and Asipn (In&n) subjects. Local Ethics Committee approval wss obtained. The right arms and legs of 6 white and 6 Asian me”, 19-24yr, ware studied 1Opl of 25% fluwescein isothiocysnstedenran, M, 150k, wss injected intrsdermally (366 needle). Filling of lymph vessels with dye was observed using a fluoresce”ce microscope. Lymphangiograms were constructed front mosaics of pints from videotape recordings. Dye4lled lymph vessel networks were analysed by the line intersection method to give lymphatic density at increasing radii from the depot (Lo,, an-l) and total vessel length filled (LL, sum of LD,*annular area, cm). Peak wr u. Peak L@ LL Arm (n-16) lB.Of1.4 11&1.4 White (leg, “-8) 27.3i1.7 40.0i10.6 Leg (n-16) 25.1f1.4 40.2t6.9 Asia” (leg, n-8) 23.Ot2.1 40.3t9.2 P 0.002 0.001 P 0.13 0.9 L& a”dLL were higher in the leg than in the arm, implying an anatwnical difference. Higher lymphatic vessel density in leg dermis would provide greater surface area fa interstitial fluid absorption. No differences were detected between white and Asian subjects.
MICROSCOPY CORNEUM:
OF LIPID STRUCl’URE THE
EFFECTS
OF AGE,
IN THE OUTER STRATUM SKIN
GRADE
AND
R.R. Warner. Y.L. Boissv and S.A. Lane The Procter & Gamble Conmanv. Cincinnati. OH. The effect of age, skin co&i& and exten&l soap use on the lipid structure of the outer stratum comeum (SC) in humans was investigated using transmission electron microscopy of ruthenium-te~oxide-stained tape strips. Young individuals with good skin condition had a normal lamellar lipid structure (Laodmann units) even in the outermost SC. Young individuals with poor skin condition did not have Landmann units in the outer SC; instead, the intercellular spaces were filled with disrupted lamellae, amorphous material, and fibrous material. Individuals over the age of 40, even with good ski condition, typically did not have well-formed Landmann units in the outer SC. Individuals over 40 with poor skii condition had diverse abnormal lipid structures with infrequent lamellae. Frequent soap use resulted in poor skin condition and a distinctive lipid structure consisting of extracted regions, regions with an exaggerated number of disorganized lamellae without a Landmann pattern, and regions containing darkly staining deposits. We conclude tha.t the lipids of the outer SC have a variable but predictable structure. This structure is influenced by age, skin condition, and environmental treatment. FREQUENT
SOAP
USE.