Cytotoxic and antigen presenting cells, and non-basement membrane zone pathology in a case of bullous pemphigoid

Abstract

Background: When performing direct and indirect skin immunofluorescence (DIF, IIF) in bullous pemphigoid (BP) utilizing single fluorophores such as fluorescein isothiocyanate (FITC), the presence of "background" fluorescence is commonly described. Aim of reporting this case: Our laboratory has noted that what is termed "background" could represent a complex immune response in BP, that may be present in addition to the classical deposits of immunoglobulins and/or complement at the dermal/epidermal basement membrane zone (BMZ). Therefore, we simultaneously used multiple colored fluorophores to further investigate this possibility. Case Report: A 68-year-old male was evaluated for the presence of rapidly appearing vesicles and bullae on the chest, with additional pruritus. Methods: Skin biopsies for hematoxylin and eosin (H&E) staining, as well as for DIF, IIF, salt split skin and immunohistochemistry (IHC) analyses were performed. Results: H&E staining demonstrated a subepidermal blistering disorder. Within the dermis, a mild, superficial and deep, perivascular infiltrate of lymphocytes, histiocytes and eosinophils was observed. A few infiltrate cells displayed positive DIF staining for CD5, CD8 and CD45 around dermal blood vessels, nerves and eccrine sweat glands. In contradistinction, CD4, CD56 and Granzyme B staining was predominantly negative in these areas. DIF, IIF and salt split skin studies revealed a strong presence of IgG, Complement/C3, IgM and fibrinogen in linear patterns at the BMZ. Around the upper dermal perivascular infiltrate, HAM56 and CD68 positive cells were also noted. Positive DIF staining for CD1a was found suprajacent to the blister in the epidermis. Utilizing identical antibodies, we repeated our staining with the IHC technique, to address the issue of autofluorescence in DIF staining. Our IHC findings correlated with DIF and IIF results. Conclusion: Using multiple DIF fluorophores in this case of BP, we observed autoantibodies to structures such dermal nerves, blood vessels and eccrine sweat glands, that were not appreciated using FITCI alone. We propose the use of this technique in autoimmune skin diseases. In addition, we observed a prominent T cytotoxic cell infiltrate, that warrants further characterization. Streszczenie Wstęp: Podczas wykonywania bezpośredniej i pośredniej immunofluorescencji skóry (DIF, IIF) w pemfigoidzie (BP) wykorzystuje się pojedyncze fluorofory takie jak izotiocyjanian fluoresceiny (FITC), a obecność "tła" fluorescencji jest powszechnie opisane. Cel niniejszego raportu: Nasze laboratorium zanotowało, że to, co jest określane jako "tło" może stanowić kompleksową odpowiedź immunologiczną w BP, która może być obecna dodatkowo w postaci klasycznych depozytów immunoglobulin i/lub dopełniacza w skórze/naskórku strefie błony podstawnej (BMZ). Dlatego równoczesne stosowaliśmy wiele kolorów fluoroforów do dalszego zbadania tej możliwości. Opis przypadku: 68-letni mężczyzna był oceniany pod kątem obecności szybko pojawiających się pęcherzyków i pęcherzy na piers...

Jam-A, Plasminogen and Fibrinogen Reactivity in a Case of a Lupus Erythematosus-Like Allergic Drug Reaction to Lisinopril

“…Our DIF was prepared and incubated with multiple fluorochromes, as previously described [5][6][7][8][9]. In brief, we transferred our biopsy from Michel's transport medium into OCT media, and froze at minus 20 degrees Celsius.…”

“…We used a cryostat to cut multiple frozen section sets, at four micron thickness. DIF was then performed utilizing antibodies directed to FITC conjugated polyclonal rabbit anti-human IgG, IgA, IgM, complement/C1q, complement/C3, albumin and fibrinogen, all from Dako (Carpinteria, California, USA) as previously described [6][7][8][9][10]. We utilized FITC conjugated monoclonal goat anti human FITCI IgE from Vector Laboratories (Burlingame, California, USA), and FITC conjugated mouse monoclonal anti-human IgG3 from Sigma (Saint Louis, Missouri, USA).…”

Immunofluorescence patterns in selected dermatoses, including blistering skin diseases utilizing multiple fluorochromes

“…The most common uses of DIF and IIF include diagnoses of vasculitides, neuropathies, rheumatologic diseases with skin compromise, autoimmune skin blistering diseases, drug eruptions; and a variety of other dermatoses including lichen planus, drug eruptions, and central centrifugal alopecia. [ 10 11 12 ]…”

“…Using DIF and IIF to confirm the presence of immune complexes in the skin biopsy at various locations such as the basement membrane zone (BMZ) of the dermal epidermal junction, dermal blood vessels, sweat glands, nerves, and other skin appendices helps in forming a diagnosis. [ 1 2 3 4 5 6 7 8 9 10 11 12 ]…”

“…The rest of the serum should be maintained at −20 C or lower, but ideally at −80 C to avoid thawing and unthawing; in our experience, this process damages the antibodies. [ 1 2 3 4 8 9 10 11 12 ] IIF testing is helpful in confirming the diagnosis of a bullous disease, and is sometimes important in the differentiation among various bullous diseases. The sensitivity and specificity of the substrates may differ among individual bullous diseases.…”