Fluorescence overlay antigen mapping of the epidermal basement membrane zone: II. Color fidelity.

Abstract

In this third study on the fluorescence overlay antigen mapping (FOAM) technique, we have addressed the question of which differences of antigen distributions close to the resolving power of the light microscope can be distinguished. An answer to this question should provide clues to future applications of the technique aiming at the topographic differentiation of IgG deposits displayed at the epidermal basement membrane zone (EBMZ) in certain bullous skin disorders. For the present purpose we have developed a topographicStainingmodel~human~,uSingsauctutalEBMz antigens as topographic reference markers. The distribution of these markers relative to one another is visualized in FOAM images obtained by selective double immunofluorescence tracing and videomicroscopic overlay imaging. The theoretical resolution limit of the technique is discussed and

Fluorescence overlay antigen mapping of the epidermal basement membrane zone: III. Topographic staining and effective resolution.

“…Even the use of multiband filter sets for concomitant observation of the red and green fluorescence signals would make little difference, because shades of red, green, and yellow-orange are very difficult to define under the conditions specified (personal observation, SB and MCJMdeJ). Our present technique appears to be more promising, provided that due consideration is given to the geometric fidelity (3) and the color fidelity (2). The strict requirements of staining specificity may give rise to hopeless problems in devising the desired staining protocol.…”

“…We are still refining the technique to identify relative antigen distributions close to the resolving power of the light microscope, particularly in tissue sections (2,3). One of our ultimate goals is to identlfy topographic differences of immune deposits across the epidermal basement membrane zone (EBMZ) as found in certain autoimmune bullous skin disorders (1,12).…”

“…Ultrastructural detail of the EBMZ in normal human skin (groin), showing the distribution relative to one another of the structural molecules used to study the topographic staining efficacy of the FOAM technique: (1) laminin-1; (2) laminin-5; (3) a6D4 integrin; (4) BP180; (5) BP230 (6) HDl; (7) collagen Type VI1 (for corresponding antibodies see Table 1). The areas of interest are the hemidesmosome complex (HD).…”

Application of confocal laser scanning microscopy to differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita

“…1,4,7,8 Computer-aided fluorescence overlay antigen mapping (FOAM) has recently been developed to identify the relative antigen distributions in tissue sections. [9][10][11] This method has the potential for differentiation of tissue-bound IgG above and below the lamina densa, and has also been demonstrated to be useful for the differential diagnosis. 12 Conventional immunofluorescence microscopy was used in the original method.…”

Bullous Pemphigoid and Epidermolysis Bullosa Acquisita

“…23 Immunoblotting techniques have proved to be a major tool to identify the antigen that is recognized by the autoantibodies,3,4 but in these cases the diagnosis has to be made by some form of microscopy. 18,19 In view of these problems, we have developed the fluorescence overlay antigen mapping technique (FOAM), whereby con¬ tiguous antigenic sites at the EBMZ may be reliably local¬ ized. These include double IF microscopy identifying the site of linear IgG deposits at the EBMZ in perilesional skin,10 indirect IF mapping of serum anti-BMZ antibodies on sa¬ line-split skin substrate,11 and direct IF mapping of linear IgG deposits in perilesional saline-split skin.12 See also pages 145 and 220 Of these, the saline-split skin techniques have proved to be a major tool for the differential diagnosis of BP and EBA.…”