Background information of DIF
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1 Napa Dermatopathology Meeting 2018: Immunobullous Disease Whitney A. High, MD, JD, MEng Professor of Dermatology & Pathology Vice-Chairman, Dermatology Director of Dermatopathology Director of Adult Dermatology Clinic University of Colorado School of Medicine May 17, 2018 Napa, CA Background information of DIF Clinically useful exam technique: Uses patient tissue External antibodies Can be done on typical autostainers Requires fluorescent microscope Sites of Biopsy for Bullous Disease Biopsy Site for Various Bullous Diseases Blister Routine Histology DIF Studies DIF for DH From High et al. in Bolognia 2013
2 Conclusive or tentative IF diagnosis was reached: - 92% after 24 h in saline - 83% after 48 h in saline - 68% after freezing in LN2-62% after 48 h in Michel's medium Conclusion: Use of normal saline for 24 h is an attractive method for performance of routine DIF studies. We use markedy different bottles at our lab for Michel s and 10%NBF! Results were potentially altered by 10% NBF exposure times of: Pemphigus <2 min Dermatitis herpetiformis >10 min Bullous pemphigoid >10 min
3 Bullous Pemphigoid Most common (#1) bullous condition Incidence 3x higher in last decade (4.3/100k) Chiefly elderly, often with many co-morbidities Creates tense (subepidermal) bullae (+ A-H sign) Urticarial forms can exist for months (or years) Cause of significant morbidity. Bullous Pemphigoid (Pathophysiology) Caused by antibodies to: BP230 (BPAg1) intracellular plakin-family protein BP180 (BPAg2) transmembranous NC16A region near the transmembranous portion harbors most of the antibody-reactive epitopes Bullous Pemphigoid (Clinical)
4 Bullous Pemphigoid (Histology) Shave biopsy is a beautiful way to go!!!! Subtle urticarial becoming bullous BP HAAAPPI differential ( H, triple A, PPI ) H herpes gestationis (and classic BP) A allergic contact dermatitis A arthropod bite reaction A atopic dermatitis PP pemphigus and pemphigoid (BP) I incontinentia pigmenti (not covered herein)
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6 Bullous Pemphigoid DIF Pattern Direct IF: BMZ- linear IgG +/- C 3 (85-90%) Direct IF on saline-split skin: linear IgG on roof Bullous Pemphigoid (DIF)
7 Bullous Pemphigoid- C3 recommend performance of all available tests to achieve the highest possible serologic sensitivity for BP.
8 ELISA for BP1/BP2 ELISA for BP1/BP2 (1 mo. later) Titers generally track with therapy. Feng et al Herpes Gestationis (Pemphigoid gestationis) - mid to later pregnancy - begins periumbilically - does not begin in striae
9 Herpes Gestationis Pemphigoid Gestationis Pattern Linear IgG - often focal, weak or absent Linear C3 - often strong Herpes Gestationis C3
10 Typical DIF Result Listed clinical impression: r/o BP Diagnosis: Consistent with bullous pemphigoid Comment: Bullous pemphigoid, cicatricial pemphigoid, and epidermolysis bullosa cannot be wholly distinguished by DIF examination, alone. Correlation with the clinical situation and with the results of light microscopy is necessary. Additional testing (IIF salt-split skin) can be utilized, as well, if necessary. Cicatricial Pemphigoid (Mucous Membrane Pemphigoid) Blistering disorder with tense blisters and scar Distribution oral and ocular mucosa (symblepharon) sometimes aerodigestive or anogenital area often occurs repeatedly in same areas can be a paraneoplastic condition Brunsting-Perry - variant that involves only the head and neck (can be confused with SCCIS!) Oral Cicatricial Pemphigoid
11 Ocular Cicatricial Pemphigoid Brunsting-Perry Cictricial Pemphigoid Cicatricial Pemphigoid Pattern Direct IF- linear IgG +/- C 3 along BMZ (90%) linear IgA or IgM- less common Indirect IF- linear IgG in 70%
12 Cicatricial Pemphigoid Linear IgG Cicatricial Pemphigoid- IgG Dermatitis Herpetiformis (DH) Autoimmune blistering d/o associated with gluten enteropathy Intensely pruritic condition (suicides have been reported) Classically involves skin of elbows, knees, buttocks, scalp >90% with gluten-sensitive enteropathy (perhaps subclinical) Of those with GSE ~15-25% develop DH
13 Dermatitis Herpetiformis (Pathophysiology - Simplified) Genetic predisposition for gluten sensitivity (HLA types) IgA antibodies to t-tg form in gut t-tg cross-reacts with skin based e-tg Deposition of IgA/e-TG complexes in papillary dermis activates complement & neutrophils lesions of DH DH with partial IgA deficiency described* *Samolitis et al Dermatitis Herpetiformis
14 Recognition of DH has consequences: - osteoporosis - enamel deficiencies - neurological symptoms - GI lymphomas Simple Summary 1. Do an H&E and DIF 2. Consider ELISA etc. 3. If positive, refer also to GI for scope 4. Treat patient, offer screening to family 5. Consider ongoing associations: malabsorption autoimmune dz lymphomas* *Hervonen et al. 2005, Lewis et al. 2008, Grainge et al. 2012
15 Dermatitis Herpetiformis Pattern Linear granular IgA with papillary accentuation: <90% Linear granular C 3 with papillary accentuation: 70-80% Linear granular IgM, IgG and fibrin- variably present Dermatitis Herpetiformis - IgA Dermatitis Herpetiformis- Fibrin
16 93-100%* sensitivity reported Antiga et al (159/159) Antibodies Correlate with GSE (only) anti-endomysial present in 80% of DH anti-gliadin (a portion of wheat) total IgA to screen for IgA-deficiency Circulating anti-etg may be used in dx/tx DH ELISA etg positive in only 45% commercial kit with adjusted cut-off 97% sensitive, 100% specific (Borroni et al. 2013) cost 1/6 th of DIF (without bx costs included)
17 Linear IgA Bullous Dermatosis Clinical Features Chronic bullous disease of childhood Typically before 5 years of age Tense bullae on erythematous base ( string of pearls or sausagelike arcuate lesions) Pruritic or may burn Adult variant Grouped papules, vesicles and/or bullae Clinical resembles dermatitis herpetiformis Oral lesions in 70% (adult and childhood forms) Chronic Bullous Disease of Childhood (Childhood Linear IgA Bullous Dermatosis) Vancomycin-Related LABD
18 Linear IgA Bullous Dermatosis Pattern Direct IF Linear IgA along BMZ- 100% Linear C 3 along BMZ- weak or absent Indirect IF Linear IgA along BMZ- 7-33% Linear IgA Bullous Dermatosis
19 Pemphigus Family of Disorders Caused by antibodies to DG1 & DG3 Divided into: pemphigus foliaceus (PF) and pemphigus erythematosus (PE) pemphgius vulgaris (PV) and pemphigus vegetans (PVeg) PV is especially morbid compared to PF (and to BP) Nikolsky sign - splitting of epidermis on normal skin ( direct ) or at lateral edge of lesion ( marginal ) by shear force Pemphigus Foliaceus
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22 Pemphigus Vulgaris
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24 Upper epidermis is peeling away from the lower epidermis the Alfalfa sign Eosinophils Tombstones
25 Diagnosis of Pemphigus DIF (sensitivity %*) IIF (monkey esophagusetc.) ELISA for DG1 and DG3 Sano 2008 Comparison of 82 case PV and 25 PF: ELISA = sensitivity 95% DG3, 92% DG1 IIF = sensitivity 79% DG3, 84% to DG1 Monitoring of Remissions with Hair Pluck? Epidermolysis Bullosa Acquisita Clinical Features Classic EBA : noninflammatory bullae with acral distribution with scarring, milia, scarring alopecia, loss of nails, esophageal stenosis Bullous pemphigoid-like EBA : bullae and urticarial lesions. Accounts for 25% of cases. Cicatricial pemphigoid-like EBA : Accounts for 10%. Brunsting-Perry cicatricial pemphigoid-like EBA : scarring vesiculobullous eruption of head and neck
26 Epidermolysis Bullosa Acquisita Epidermolysis Bullosa Acquista Localization of Antibodies Hemidesmosome Cell membrane Lamina lucida Lamina densa Anchoring fibrils- type VII collagen
27 Bullous SLE EBA/Bullous SLE Pattern Direct IF: linear IgG and C 3 along BMZ less commonly IgA or IgM Direct IF on split skin: linear IgG on floor Indirect IF: linear IgG anti-bmz (25-50%) Indirect IF on split skin: linear IgG on floor
28 EBA- IgG (strong and thick) EBA- C3 (< IgG) n-serration versus u-serration The n vs. u serration is a learnable criterion to differentiate pemphigoid from epidermolysis bullosa acquisita in direct immunofluorescence serration pattern analysis Terra et al. BJD 2013
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