A Practical Technique for Differentiation of Subepidermal Bullous Diseases Localization of in Vivo–Bound Igg by Laser Scanning Confocal Microscopy

STUDY A Practical Technique for Differentiation of Subepidermal Bullous Diseases Localization of In Vivo–Bound IgG by Laser Scanning Confocal Microscopy

Katarzyna Woz´niak, MD; Takashi Kazama, MD; Cezary Kowalewski, MD

Objective: To develop a practical technique to distin- whereas basement membrane zone markers were la- guish autoimmune subepidermal bullous diseases. beled with anti–mouse Cy5-conjugated antibodies.

Design: A prospective study. Results: In patients with bullous pemphigoid, in vivo– bound IgG was localized on the epidermal side of lami- ␤ Setting: Academic referral center—the Department of nin 5 and co-localized with 4 integrin. In patients with Dermatology, Medical University of Warsaw. mucous membrane pemphigoid, IgG was in vivo bound to the dermal-epidermal junction between localization Patients: Forty-two patients fulfilling clinical, immu- of laminin 5 and type IV collagen. In patients with epi- nological, and/or immunoelectron microscopic criteria dermolysis bullosa acquisita, in vivo–bound IgG was for bullous pemphigoid (n=31), mucous membrane pem- present on the dermal side of type IV collagen. phigoid (n=6), or epidermolysis bullosa acquisita (n=5), diagnosed as having disease and treated from January 1, Conclusions: Laser scanning confocal microscopy al- 1997, to December 31, 2002. lows precise localization of in vivo–bound IgG in patients’ skin and, thus, it is a rapid method for the differ- Main Outcome Measures: We applied laser scan- entiation of mucous membrane pemphigoid from bullous ning confocal microscopy to determine the localization pemphigoid and epidermolysis bullosa acquisita. This tool of in vivo–bound IgG at the basement membrane zone is suitable for the routine diagnosis of individual pa- in biopsy specimens taken from patients’ skin to com- tients and for retrospective studies. This method is of spe- pare the localization of basement membrane zone cial value in those patients in whom circulating autoan- ␤ markers: antibody against 4 integrin, antibody against tibodies are not detectable. laminin 5, and antibody against type IV collagen. In vivo– bound IgG was visualized by labeling with fluorescein isothiocyanate–conjugated anti–human IgG antibody, Arch Dermatol. 2003;139:1007-1011

ULLOUS PEMPHIGOID (BP), cult or even impossible.4,5 The character- mucous membrane pemphi- ization of BMZ antigens on a molecular goid (MMP), and epider- level6,7 and the introduction of immuno- molysis bullosa acquisita blotting, radioimmunoprecipitation, an en- (EBA) belong to the group zyme-linked immunosorbent assay, and fu- ofB autoimmune subepidermal bullous dis- sion protein techniques for diagnosing eases (ASBDs) characterized by the devel- ASBDs allow differentiation of these en- opment of tense blisters on apparently tities in patients in whom circulating an- healthy skin. These entities clinically can tibodies are detectable. In patients with mimic each other, especially at the onset BP and MMP, BP180 antigen is a target of the disease, but they differ in course, molecule that plays a crucial role in the prognosis, and response to treatment.1-3 pathogenesis of these dermatoses.8-10 BP180 From the Departments The ASBDs are defined by the presence of antigen is a transmembrane protein con- of Dermatology, Medical circulating antibodies directed against dif- sisting of an amino terminal domain, pres- University of Warsaw, ferent basement membrane zone (BMZ) ent in the cytoplasm of basal keratino- Warsaw, Poland (Drs Woz´niak and Kowalewski), and Niigata antigens and the presence of in vivo– cytes and in the extracellular portion, that University School of Medicine, bound IgG at the dermal-epidermal junc- started with the NC16A domain just be- Niigata, Japan (Dr Kazama). tion in patients’ skin; thus, differentia- low the keratinocyte plasma membrane; The authors have no relevant tion of these dermatoses by routine it extends across the whole lamina lucida financial interest in this article. immunofluorescence (IF) may be diffi- into the upper part of the lamina densa,

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 ending by the carboxyterminal domain on the border of microscopy using a peroxidase technique was performed on the the lamina lucida and the lamina densa.11 In patients with skin originating from 2 patients whose samples were negative BP, the target epitopes for BMZ antibodies are located on for circulating anti–BMZ antibodies, and showed the presence the NC16A domain of BP180 antigen, ultrastructurally of IgG deposits within the lamina lucida. localized in the upper part of the lamina lucida, whereas Patients With MMP the serum samples of most patients with MMP recognize the carboxyterminal domain of BP180 antigen at the 12 Six patients (2 men and 4 women) fulfilled the clinical and im- lamina lucida–lamina densa border. In some patients munopathological criteria for MMP.8 All patients with MMP with MMP, autoantibodies are directed against the ␣ chain had mucous membrane and skin involvement. They had tense of laminin 5, localized in the upper part of the lamina bullae on the skin, which healed and left atrophic scars and densa. The production of autoantibodies directed against milia. Mucous membrane involvement in all patients referred type VII collagen, localized in the lower lamina densa and to scarring conjunctivitis and chronic painful erosions of oral the sub–lamina densa, leads to the development of mucosa. Of 6 patients’ serum samples, 2 were positive for cir- EBA.13,14 culating IgG anti–BMZ antibodies and reacted with the epider- In patients with ASBDs in whom circulating anti– mal and dermal side of sodium chloride salt–split skin by in- BMZ antibodies are not detectable, a final diagnosis can direct IF. The results of immunoblot studies were negative (in our studies and in the studies performed at another laboratory). be established based on direct immunoelectron micros- Direct immunoelectron microscopy using the peroxidase copy, which is time-consuming. In patients with BP, im- technique, performed in 4 patients (who were negative for 15 munoreactants are localized to the lamina lucida ;inpa- circulating anti–BMZ antibodies), showed the presence of IgG tients with MMP, immunoreactants are localized to the deposits within the lamina lucida and the lamina densa. lamina lucida and the lamina densa16; and in patients with EBA, immunodeposits are bound below the lamina Patients With EBA densa.17 In a previous study, Kazama et al18 proved that it is possible to distinguish BP from EBA based on the Three men (aged 20, 38, and 42 years at the onset of disease) comparison of the localization of target antigens for cir- and 2 children (a 3-year-old boy and a 13-year-old girl) were culating anti–BMZ antibodies and/or in vivo–bound IgG included in this study. Three patients (2 adults and 1 child) fulfilled the criteria of Roenigk et al3 for mechanobullous EBA. in patients’ skin with the localization of well-defined BMZ They had tense bullae on the skin, localized on traumatized areas markers using laser scanning confocal microscopy that were healing, and scars and milia. Two other patients had (LSCM). In the present study, we applied LSCM to in- an inflammatory type of EBA, clinically resembling BP.2 Of 5 vestigate whether it is possible to differentiate MMP from patients with EBA, 3 had oral mucous involvement. Of 5 pa- BP and EBA based on the localization of in vivo–bound tients’ serum samples, 3 were positive for circulating IgG anti– IgG within the BMZ. BMZ antibodies and revealed reactivity with the exclusively der- Therefore, this study investigates whether it is pos- mal side of sodium chloride salt–split skin by indirect IF. sible to differentiate subepidermal bullous diseases based Immunoblot studies of these serum samples showed reactivity on the localization of in vivo–bound IgG at the BMZ with with a 290-kDa molecule on the dermal extract. Serum stud- regard to the localization of lamina lucida and lamina ies using postembedding immunogold electron microscopy on Lowicryl-embedded healthy human skin demonstrated the re- densa markers using LSCM. activity of IgG anti–BMZ antibodies with the lamina densa and anchoring fibrils. Direct immunoelectron microscopy using the METHODS peroxidase technique, performed on the skin specimens of 2 patients who were negative for circulating anti–BMZ antibod- PATIENTS ies, revealed the presence of IgG deposits below the lamina densa. Data are summarized in the Table. Forty-two patients positive for in vivo–bound IgG and comple- ment C3 at the BMZ by direct IF were selected from a group of LASER SCANNING CONFOCAL MICROSCOPY 102 patients who were diagnosed as having an ASBD and treated at the Department of Dermatology, Medical University of War- Punch biopsy specimens taken from perilesional patients’ skin saw, from January 1, 1997, to December 31, 2002. Patients posi- were mounted in tissue freezing medium (Leica Instruments, tive for in vivo–bound IgA at the BMZ by direct IF as a predomi- GmbH, Nussloch, Germany) and cut into 10-µm cryosec- nant or concomitant component were not included in this study. tions. These sections were incubated with monoclonal anti– ␤ BMZ antibodies directed against 4 integrin, a marker of the Patients With BP upper part of the lamina lucida (clone 3E1; Chemicon Inter- national, Temecula, Calif); laminin 5, also called epiligrin, a Thirty-one patients (14 men and 17 women) fulfilled the clini- marker of the upper part of the lamina densa (clone P3E4; cal and immunopathological criteria for BP. Patients were aged Chemicon International); and type IV collagen, a marker of between 73 and 90 years. They developed tense blisters on the the lamina densa (clone COL-94; Sigma, Steinheim, Ger- trunk and extremities, which healed without scars or milia. Pa- many), for 30 minutes, followed by 5-minute washings with tients with BP who had bullous lesions localized on trauma- phosphate-buffered saline, performed 3 times. Then, the cryo- tized areas or had mucous membrane involvement were ex- sections were incubated with a mixture of rabbit anti–mouse cluded from this study. Of 31 patients’ serum samples, 29 were Cy5-conjugated antibody (Chemicon International) and fluo- positive for circulating IgG anti–BMZ antibodies and showed rescein isothiocyanate (FITC)–conjugated goat anti–human an- reactivity with the epidermal side of sodium chloride salt– tibodies (Kappel, Aurora, Ohio) for 30 minutes, followed by split skin by indirect IF. Immunoblot studies revealed the re- 5-minute washings with phosphate-buffered saline, per- activity of 12 patients’ serum samples with 180- and/or 230- formed 3 times; mounted in p-phenylenediamine; and viewed kDa molecules on epidermal extract. Direct immunoelectron using LSCM (Radiance 2000; BIO-RAD, Oxford, England).

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 Excitation of FITC and Cy5 was simultaneously performed with laser lines of 488 and 637 nm, respectively. To obtain a spe- Clinical and Immunopathological Characterization cific signal for FITC (green color) and Cy5 (red color) and to of Patients Included in the Study* avoid a cross-talking channel effect, we used a dichroic mirror of 560 nm and a bandpass filter of 500 to 560 nm for epifluo- Patients Patients Patients rescence of FITC and a longpass filter of 660 nm for Cy5. Green With BP With MMP With EBA and red images were overlaid by an image processing system Variable (n = 31) (n=6) (n=5) integrated with the LSCM, and photographed. Male-female ratio 14:17 2:4 3:2 Age of onset, y 73-90 60-71 3-42 Tense blisters RESULTS On the trunk and extremities 31 6 2 On traumatized areas 0 1 5 In all patients with BP, the overlay image of ␤ integrin Erosions on the oral mucosa 0 6 3 4 Scarring conjunctivitis 0 6 0 and in vivo–bound IgG showed yellow fluorescence along Scars 0 6 5 the BMZ because of an overlap of the red fluorescence Milia 0 5 5 ␤ of 4 integrin and the green fluorescence of in vivo– Reactivity by indirect IF on 29 2 3 bound IgG (Figure, A). The overlay image of laminin 5 salt-split skin and in vivo–bound IgG showed a green reaction of IgG Positive Western immunoblot 12 0 3 results on the epidermal side and red staining of laminin 5 on Positive direct immunoelectron 24 2 the dermal side, which indicates that IgG is in vivo bound microscopic results above laminin 5 (Figure, B). The overlay image of in vivo– bound IgG and type IV collagen showed green-red (from Abbreviations: BP, bullous pemphigoid; EBA, epidermolysis bullosa epidermis to dermis) staining along the BMZ, suggest- acquisita; IF, immunofluorescence; MMP, mucous membrane pemphigoid. *Data are given as number of patients in each group unless otherwise ing the localization of IgG above the localization of type indicated. IV collagen (Figure, C). In all patients with MMP, the overlay images of green on the floor of an artificial blister).20,21 In most of the pa- fluorescence dependent on in vivo–bound IgG and the red ␤ tients with MMP, in vivo–bound immunoglobulins are fluorescence of 4 integrin showed red staining on the epi- present on the roof and floor of the artificial blister mimic, dermal side and green staining on the dermal side of the BP, whereas in one fifth of the patients with MMP, immu- BMZ, suggesting that IgG is in vivo bound below the lo- ␤ nodeposits react with the floor of the blister, suggesting the calization of 4 integrin (Figure, D). The overlay images diagnosis of EBA. This discrepancy is because of the pres- of laminin 5 and in vivo–bound IgG showed red staining ence of 2 different targeted antigens recognized in the se- on the epidermal side and green fluorescence on the der- rum samples of patients with MMP: the C domain of BP180 mal side of the BMZ, suggesting that IgG is in vivo bound antigen, present in the lower part of the lamina lucida; and below the localization of laminin 5 (the epitope is recog- laminin 5 (also known as epiligrin), localized in the upper nized by the monoclonal antibody used in this study) part of the lamina densa.11,22,23 Thus, a direct salt-split pat- (Figure, E). In some areas of the BMZ, a yellow reaction tern cannot be conclusive in patients with MMP. due to the partial co-localization of in vivo–bound IgG and Later, we applied fluorescence overlay antigen map- laminin 5 was observed, but there was never green fluo- ping, using a regular epifluorescence microscope, to rescence of IgG extended above the localization of lami- investigate whether it is possible to distinguish the lo- nin 5. The overlay images of in vivo–bound IgG and type calization of in vivo–bound IgG within the BMZ in pa- IV collagen showed green-red (from epidermis to dermis) tients with BP and MMP from that in patients with EBA. staining along the BMZ, suggesting the localization of IgG The localization of in vivo–bound IgG in patients’ skin above the localization of type IV collagen (Figure, F). was compared with the localization of the carboxyter- In all patients with EBA, the overlay image of IgG ␤ minal end of type VII collagen (a marker localized 360 and 4 integrin, laminin 5, and type IV collagen showed nm below the lamina densa).13,14 We found the co- red-green (from epidermis to dermis) staining along the localization of in vivo–bound IgG in patients with EBA BMZ, indicating that IgG is in vivo bound below the lo- ␤ with the carboxyterminal end of type VII collagen and calization of 4 integrin (Figure, G) and below laminin lack of this co-localization in patients with BP and MMP. 5 (Figure, H) and type IV collagen (Figure, I). The resolution of the regular epifluorescence microscope did not allow differentiation of in vivo–bound IgG COMMENT in the lamina lucida (in patients with BP) from IgG bound in the lamina lucida and the lamina densa (in patients with MMP). In addition, an application of fluorescence Diagnosing MMP and EBA is difficult because in most overlay antigen mapping for the routine diagnosis of sub- of the patients, circulating anti–BMZ antibodies are not epidermal bullous diseases might cause many technical detectable. An alternative method to time-consuming di- problems, known as geometric error and color error.24 rect immunoelectron microscopy would be desirable. We In this study, we applied LSCM to compare the local- developed a useful option using LSCM. ization of in vivo–bound IgG with the localization of dif- In 1988, one of us (C.K.)19 was the first, to our knowl- ferent BMZ markers. In contrast to a previous study by Ka- edge, to introduce the direct IF salt-split skin technique for zama et al,18 we used a diode laser at 637 nm and a longpass the differentiation of BP (a reaction on the roof or on the filter at 660 nm for Cy5 dye, instead of a helium-neon la- roof and floor of an artificial blister) from EBA (a reaction ser at 543 nm and a longpass filter at 590 nm for tetra-

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D E F

G H I

Double immunofluorescence labeling for tissue-bound IgG (green) and basement membrane zone (red) markers in perilesional patients’ skin. In patients with

bullous pemphigoid, the in vivo–bound IgG is codistributed with ␤4 integrin (A) and localized on the epidermal side of laminin 5 (B) and type IV collagen (C). In patients with mucous membrane pemphigoid, the in vivo–bound IgG is localized on the dermal side of ␤4 integrin (D) and laminin 5 (E) and on the epidermal side of type IV collagen (F). In patients with epidermolysis bullosa acquisita, the in vivo–bound IgG is localized on the dermal side of ␤4 integrin (G), laminin 5 (H), and type IV collagen (I) (original magnification ϫ3000).

methyl rhodamine isothiocyanate, simultaneously with an MMP is more complicated because of the close distance argon laser at 488 nm and a bandpass filter at 500 to 560 between in vivo–bound IgG in both entities. nm for FITC. The system setting used in this study allows We found that in vivo–bound immunoglobulins in elimination of the cross-talking channel effect. Also, the de- patients with BP are located above laminin 5. They par- tection of FITC is increased because of the use of an emis- tially co-localized with, but did not extend below, lami- sion bandpass filter at 500 to 560 nm instead of the 500 to nin 5 (at least the epitope recognized by monoclonal an- 530 nm used in a previous study.18 tibody used in this study). In patients with MMP, in vivo– In addition to the antibodies used by Kazama et al,18 bound IgG is localized between laminin 5 and type IV we tested new markers at the lamina lucida–lamina densa collagen. It partially co-localized with laminin 5 and type border. Finally, we recommend the use of a monoclonal IV collagen, but did not extend above laminin 5 and be- antibody against the ␣ chain of laminin 5 (clone P3E4, low type IV collagen. called epiligrin by Chemicon International) in the dif- The results of our study indicate that LSCM could ferentiation of in vivo–bound IgG in patients with BP and be useful for the differentiation of MMP from BP and MMP by LSCM. The precise immunoelectron micro- EBA. This method is of special value in the diagnosis of scopic localization of the epitope recognized by this an- patients in whom circulating autoantibodies are not tibody is not known; however, by using the cryosection- detectable. ing immunogold electron microscopic method, different epitopes of laminin 5 were found in the upper part of the Accepted for publication March 28, 2003. lamina densa.11 The antibody against the ␣ chain of lami- This study was supported by grant KBN 3 PO5B 05822 nin 5 (clone P3E4) produced a stronger IF reaction on from the Polish Scientific Research Committee. the dermoepidermal junction compared with the lami- This study was presented at the World Congress of Der- nin 5 (clone D4B5) used in a previous study18; thus, it is matology; July 2, 2002; Paris, France. possible to obtain a high-quality LSCM image with this We thank Takashi Hashimoto, MD, Department of Der- particular antibody and to increase the resolution. matology, Kurume University School of Medicine, Kurume Our study has shown that EBA could be clearly dis- City, Japan, for performing some of the immunoblot stud- tinguished from BP and MMP by LSCM based on the pres- ies referenced in this article. ence of in vivo–bound IgG below the localization of type Corresponding author and reprints: Cezary Kowa- IV collagen. The differential diagnosis between BP and lewski, MD, Department of Dermatology, Medical Uni-

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 versity of Warsaw, Koszykowa 82A, 02-008 Warsaw, 13. Shimizu H, McDonald JM, Gunner DB, et al. Epidermolysis bullosa acquisita an- Poland (e-mail: [email protected]). tigen and the carboxy terminus of type VII collagen have a common immunolo- calization to anchoring fibrils and lamina densa of basement membrane. Br J Dermatol. 1990;122:577-585. 14. Shimizu H, Ishiko A, Masunaga T, et al. Most anchoring fibrils in human skin REFERENCES originate and terminate in the lamina densa. Lab Invest. 1997;76:753-763. 15. Holubar K, Wolff K, Konrad K, Beutner EH. Ultrastructural localization of immu- 1. Bean SF. Cicatricial pemphigoid. In: Beutner E, Chorzelski TP, Kumar V, eds. Im- noglobulins in bullous pemphigoid skin: employment of a new peroxidase- munopathology of the Skin. 3rd ed. New York, NY: John Wiley & Sons Inc; 1987: antiperoxidase multistep method. J Invest Dermatol. 1975;64:220-227. 355-360. 16. Fine JD, Neises GR, Katz SI. Immunofluorescence and immunoelectron micro- 2. Gammon WR, Briggaman RA, Woodley DT, et al. Epidermolysis bullosa ac- scopic studies in cicatricial pemphigoid. J Invest Dermatol. 1984;82:39-43. quisita: a pemphigoid-like disease. J Am Acad Dermatol. 1984;11:820-832. 17. Nieboer C, Boorsma DM, Woerdeman MJ, Kalsbeek GL. Epidermolysis bullosa 3. Roenigk HH, Ryan JG, Bergfeld WF. Epidermolysis bullosa acquisita: report of acquisita: immunofluorescence, electron microscopic and immunoelectron mi- three cases and review of all published cases. Arch Dermatol. 1971;103:1-10. croscopic studies in four patients. Br J Dermatol. 1980;102:383-392. 4. Jordon RE, Beutner EH, Witebsky E, et al. Basement zone antibodies in bullous 18. Kazama T, Yamamoto Y, Hashimoto T, Komai A, Ito M. Application of confocal pemphigoid. JAMA. 1967;200:751-756. laser scanning microscopy to differential diagnosis of bullous pemphigoid and 5. Kushniruk W. The immunopathology of epidermolysis bullosa acquisita. Can Med epidermolysis bullosa acquisita. Br J Dermatol. 1998;138:593-601. Assoc J. 1973;108:1143-1146. 19. Kowalewski C. New Methods in Diagnosing of Atypical Bullous Diseases [doc- 6. Bruckner-Tuderman L, Nilssen O, Zimmermann DR, et al. Immunohistochemi- toral thesis]. Warsaw, Poland: Medical University of Warsaw; 1988. cal and mutation analysis demonstrate that procollagen VII is processed to col- 20. Kowalewski C, Haftek M, Cozzani E, et al. Cicatricial pemphigoid of mucous mem- lagen VII through removal of the NC-2 domain. J Cell Biol. 1995;131:551-559. branes vs epidermolysis bullosa acquisita with exclusive IgA linear deposits in 7. Giudice GJ, Emery DJ, Diaz LA. Cloning and primary structural analysis of the the basement membrane zone. In: Ishibishi Y, Nagawa H, Suzuki H, eds. Elec- bullous pemphigoid autoantigen BP180. J Invest Dermatol. 1992;99:243-250. tron Microscopy in Dermatology. Amsterdam, the Netherlands: Elsevier Sci- 8. Chan LS, Ahmed AR, Anhalt GJ, et al. Mucous membrane pemphigoid: defini- ence Publishers; 1994:187-193. tion, diagnostic criteria, pathogenic factors, medical treatment and prognostic 21. Gammon WR, Kowalewski C, Chorzelski TP, et al. Immunofluorescence on sodium indicators. Arch Dermatol. 2002;138:370-379. chloride–separated skin in the differential diagnosis of bullous pemphigoid and epi- 9. Christophoridis S, Budinger L, Borradori L, et al. IgG, IgA and IgE autoantibod- dermolysis bullosa acquisita. J Am Acad Dermatol. 1990;22:664-670. ies against the ectodomain of BP180 in patients with bullous and cicatricial pem- 22. Lazarova Z, Hsu R, Briggaman RA, et al. Fab fragments directed against lami- phigoid and linear IgA bullous dermatosis. Br J Dermatol. 2000;143:349-355. nin-5 induce subepidermal blisters in neonatal mice. Clin Immunol. 2000;95: 10. Zillikens D. BP180 as the common autoantigen in blistering diseases with dif- 26-32. ferent clinical phenotypes. Keio J Med. 2002;51:21-28. 23. Lazarova Z, Yee C, Lazar J, et al. IgG autoantibodies in patients with anti– 11. Shimizu H. New insights into the immunoultrastructural organization of cutane- epiligrin cicatricial pemphigoid recognize the G domain of the laminin 5 alpha- ous basement membrane zone molecules. Exp Dermatol. 1998;7:303-313. subunit. Clin Immunol. 2001;101:100-105. 12. Bedane C, McMillan JR, Balding SD, et al. Bullous pemphigoid and cicatricial pem- 24. Bruins S, de Jong MC, Heeres K, Wilkinson MH, Jonkman MF, van der Meer JB. phigoid antibodies react with ultrastructurally separable epitopes on the BP180 Fluorescence overlay antigen mapping of the epidermal basement membrane zone, ectodomain. J Invest Dermatol. 1997;108:901-907. I: geometric errors. J Histochem Cytochem. 1994;42:555-560.

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