Immunofluorescence Patterns in Selected Dermatoses, Including Blistering Skin Diseases Utilizing Multiple Fluorochromes

Abstract

Background: Autoimmune vesiculobullous disorders represent a heterogeneous group of dermatoses whose diagnosis is made based on clinical history, histologic features, and immunopathologic features. The most commonly used techniques for the diagnosis of these diseases are direct and indirect immunofluorescence (DIF and IIF), including salt-split processing. NaCl split skin is used to determine the level of blister formation, and the localization of autoantibodies relative to the split. Classically, immunofluorescence has been performed with one fluorochrome in the diagnosis of autoimmune bullous skin diseases.

Aims: To compare DIF and IIF of the skin, using a single fluorochrome versus multiple fluorochromes.

Materials and methods: We studied 20 autoimmune skin disease cases using fluorescein isothiocyanate (FITC) alone, in comparison to multiple fluorochromes (with or without DNA counterstaining).

Results: The use of multiple fluorochromes helped to simultaneously visualize reactivity in multiple skin areas, in contrast to using FITC alone.

Conclusions: Using multiple fluorochromes allows simultaneous labeling of two or more antigens within the same cell/or tissue section, assists in colocalization of unknown antigens with known molecules, and helps in ruling out "background" staining.

Keywords: Autoimmune blistering diseases; lupus; multiple fluorochromes immunofluorescence; skin.

Figure 1

(a) An indirect immunofluorescence showing an intercellular stain between the queratinocytes (ICS) (white arrow) using monkey esophagus as substrate and fluorescein isothiocyanate conjugated anti-human immunoglobulin G antibodies. In (b), similar but using 4’,6-diamidino-2-phenylindole to counterstain the nuclei of the cells (light blue) in red, antibody against anti-intercellular adhesion molecule 1/CD54 (vascular endothelial marker) conjugated with Alexa 555. In (c), an example of direct immunofluorescence using the patient skin from the same patient as in a and b; in d, same as c but using 4’,6-diamidino-2-phenylindole to counterstain the nuclei

Figure 2

Indirect immunofluorescence using monkey esophagus. (a) A representative case of paraneoplastic pemphigus, stained with fluorescein isothiocyanate conjugated immunoglobulin G (green staining) shows positive staining in the intercellular spaces pattern (yellow arrow) and at the basement membrane zone (white arrow). The combination of intercellular spaces and basement membrane zone deposition may be seen in Senear-Usher syndrome, paraneoplastic pemphigus and in El Bagre- endemic pemphigus foliaceus. (b) In paraneoplastic pemphigus, fluorescein isothiocyanate conjugated immunoglobulin G staining is again present (green staining; yellow arrow). Texas red conjugated collagen IV antibody is positive at the basement membrane zone (red staining). Keratinocyte nuclear staining is demonstrated via 4’,6-diamidino-2-phenylindole (whitish staining; white arrow). In (b) antibodies to the sweat glands may also be seen. In (c), fluorescein isothiocyanate conjugated complement/C3 is positive in a case of paraneoplastic pemphigus (yellow staining; white arrow) and in (d) sweat gland structures in the same case as c are further highlighted utilizing Ulex (positivity in sweat gland vessels (fuchsia staining; white arrow); sweat gland nuclei are counterstained with 4’,6-diamidino-2-phenylindole (light blue staining; white arrow)

Figure 3

(a and b) Direct immunofluorescence positive staining in a case of Diffie-Hellman, utilizing fluorescein isothiocyanate conjugated anti-human immunoglobulin A (green staining; white arrow); a dermatitis herpetiformis body is also indicated (red arrow). In (b), same as (a) with colocalization of Texas red conjugated armadillo repeat gene deleted in velocardiofacial syndrome on the dermatitis herpetifomis body (red staining; red arrow). (c and d) On indirect immunofluorescence using monkey esophagus anti-human immunoglobulin G with the serum of a patient with celiac disease (green staining; white arrow). (d) Similar to (c) but, in this case, we use 4’,6-diamidino-2-phenylindole (light blue) to counterstain keratinocyte nuclei

Figure 4

(a and b) Direct immunofluorescence. These show a representative case of discoid lupus, with a serrated deposit of complement/C3 along the basement membrane zone (green staining; white arrow); in (b) The nuclei of the cells are counterstained with 4’,6-diamidino-2-phenylindole (light blue). In (c and d), a case of bullous pemphigoid, positive with fluorescein isothiocyanate conjugated immunoglobulin G (green staining at the basement membrane zone in a continuous pattern; yellow arrow); in (d) We utilize NaCl split skin with Ulex in red and 4’,6-diamidino-2- phenylindole in blue. The stain, in this case, is present on both sides of the split, but primarily on the epidermal side (yellow arrow)

Figure 5

(a) Direct immunofluorescence using fluorescein isothiocyanate conjugated anti-human immunoglobulin M antibodies, showing positive staining in a case of bullous lichen planus; the arrow indicates cytoid bodies in the upper dermis (green staining; white arrows). In (b), The nuclei of keratinocytes are stained with To-pro 3 (in red); the cytoid bodies are also present (green staining; white arrow). In (c) We show direct immunofluorescence in case of epidermolysis bullosa acquisita, with fluorescein isothiocyanate conjugated immunoglobulin G autoantibodies on dermal side of the basement membrane zone (green staining; white arrow). In (b), the nuclei of the cells were stained with To-pro 3 (in red). Please notice that in (c and d), some staining is also seen on the upper dermal vessels (yellow arrows)