Diagnostic tests 1. Take biopsy specimens of perilesional skin or mucosa with normal and the edge of lesional areas in Michel's transport medium for direct immunofluorescence (IF) to detect in vivo bound basement membrane zone (BMZ) antibodies. Do follow-up studies, if indicated.* 2. Take blood sample in a red top tube for serum tests for circulating BMZ autoantibodies. Test on esophagus and salt split skin sections. 3. If blisters appear, confirm with a biopsy for light microscopy.

Bullous pemphigoid (BP), other forms of pemphigoid, epidermolysis bullosa acquisita (EBA) and more rare subepidermal vesiculo-bullous eruptions with BMZ antibodies are autoimmune diseases diagnosed by the presence of in vivo bound and circulating BMZ autoantibodies. They usually produce lesions characterized by their clinical and histologic appearance. Since BP, EBA and related eruptions have an autoimmune etiology, their diagnosis depends on detecting in vivo bound BMZ antibodies by direct IF or by serum studies. These disorders can be diagnosed reliably with combined direct and indirect IF and light microscopy as first shown in 1965 (1-2).

Direct IF studies of skin or mucosal biopsies reveal BMZ deposits of IgG, IgA and/or the C3 component of complement in the BMZ of ~90% of specimens. For skin lesions, take biopsy from perilesional, non-edematous, normal area with edge of a fresh vesicle or bulla for the highest frequencies of positive BMZ findings. (Edematous areas in BP or EBA are often negative). For mucosal lesions take biopsy from normal epithelium -3 mm. from a lesion for optimal results; lesional mucosal areas are usually negative. In some cases, multiple biopsies are needed to detect BMZ immune deposits. Some experts take two biopsies, one of a normal area plus the edge of a - lesion and one of perilesional normal skin or mucosa.

Indirect IF serum studies with esophagus, unsplit skin and 1 M NaCl split skin sections reveal BMZ antibodies in ~70% to 80% of BP cases (including some that are direct IF negative), in ~50% of EBA cases and in smaller percentages of related eruptions caused by these antibodies. (See Table). These and other methods for serum studies reveal antibodies to a 180 kDa, BP2 and to a less important 230 kDa BP1 antigen in BP and related forms of pemphigoid. They reveal antibodies to type VII collagen in EBA (3) and to less common antigens (4-6) in related, subepidermal eruptions with an autoimmune etiology, for example in epiligrin autoimmunity (7).

Light microscopy reveals subepidermal or subepithelial lesions in cases with blisters. Since non-autoimmune disorders such as bullous erythema multiforme may cause similar lesions, confirmation with direct IF studies of a biopsy and/or indirect IF tests of serum is indicated. If histologic and IF studies yield discrepant results, repeat of direct IF and indirect IF studies (and/or other serum tests) are indicated.

* For information on indicated follow-up studies, see UPDATE on Differentiation of bullous pemphigoid (BP) from epidermolysis bullosa acquisita (EBA) and other forms of BMZ autoimmunity.

Clinical Relevance

Clinically, BP, other forms of pemphigoid, EBA and other conditions listed in the Table, as well as a variety of other non-autoimmune eruptions which mimic them, may present variously as bullous, vesicular or urticarial skin lesions or as ocular, oral, genital or other mucosal lesions. These disorders can have diverse clinical presentations that may resemble non-autoimmune disorders which call for other forms of treatment. If clinical findings point to the need to rule out EBA and direct or indirect IF studies yield positive findings, follow-up studies may be indicated.**

Bullous disorders	Types of epithelial lesions	Biopsies: Direct IF	Diagnostic IF methods
			Biopsy split in 1 M NaCl**	Indirect IF* for anti-BMX antibodies
Bullous pemphigoid (BP) or urticarial pemphigoid	Skin and oral mucosal blisters or unrticaria	IgG and C3 in BMZ some cases also IgA, IgM	IgG is in BMZ of roof or in floor after split**	70-80% IF* ~50% postive
Lichen planus	Lichenoid skin lesions	BP +LP type deposits*	(As for BP)	~20% positive
Gestational pemphigoid (HG)*	(As for BP)	C3 in BMZ, rarely IgG	(As for BP)	~10% CIF* pos. ("HG factor")
Cicatricial pemphigoid	Mouth, eyes, nose, skin and genital	(As for BP)	IgG is in BMZ roof & floor after split	10% to 80% pos. higher for skin + mucosa bulla
Epidermolysis bullosa acquisita, bullous LE	Skin and mucos membranes	(As for BP)	IgG in in BMZ of floor of split only.	~50% postive

* LP or lichen planus type deposits include junctional fibrin and IgM in cytoid bodies. HG = herpes gestationis; CIF = complement fixing indirect immunofluorescence
** See UPDATE on differentiation of pemphigoid from EBA or related autoimmune disorders

References:
1. Beutner EH, Lever WF, Witebsky E, Jordon RE, Chertock B. Autoantibodies in pemphigus vulgaris. Responses to an intercellular substance of epidermis. J Am Med Assoc 192:682-688,1965. (Includes direct and indirect IF BMZ findings in pemphigoid).
2. Beutner EH, Chorzelski TP, Jordon RE. Autosensitization in pemphigus pemphigoid. Charles Thomas. Springfield IL, 1970.
3. Gammon WR, Briggaman RA, Inman AO, Queen LL, Wheeler CE. Differentiating antilamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M/L sodium chloride separated skin. J Invest Dermatol 82:139144,1984.
4. Stanley JRP, Hawley-Nelson SH, Yuspa EF, Shevach EM, Katz SI. Characterization of bullous pemphigoid antigen: A unique basement membrane protein of stratified squamous epithelia. Cell 24: 897-903,1981.
5. Yancey KB. Adhesion molecules. II Interaction of keratinocytes with epidermal junction. J Invest Dermatol 104:1008-1014,1995.
6. Zillikens D. Acquired skin disease of hemidesmosomes. J Dermatol Sci 20: 134-154, 1999.
7. Kirtschig G, Marinkovich MP, Burgeson RE, Yancey KB, Anti-basement membrane autoantibodies in patients with anti-epiligrin cicatricial pemphigoid bind the alpha subunit of laminin 5. J Invest Dermatol. 105:543-548,1995.
8. Chorzelski TP, Jablonska S, Beutner EH. Bullous pemphigoid: Relationship of immunopathology to clinical findings. In 3rd ed. "Immunopathology of the skin, Ed. EH Beutner et al. 337-354,1987.
9. Gammon WR, Wilson BD, Briggaman RA. Epidermolysis bullosa acquisita. In 3rd ed. "Immunopathology of the skin, Ed. EH Beutner et al. 371-380,1987.
10. Tani M, Murata Y, Masaki H. Pemphigoid nodularis. J Am Acd Dermatol 21: 10991104,1989.

Copyright © 1999 Beutner Labs, Inc.
Published with permission.