Direct Immunofluorescence: What’s New

Nooshin K. Brinster M.D. New York University Ronald O. Perelman Department of Dermatology [email protected] Overview

• Bullous diseases • Vasculitis • Connective tissue disease Bullous Disease Punctate Pemphigus Pemphigus

• Punctate pattern IgG (“dew-drops on a spider web”) • Pemphigus vulgaris and foliaceus – Chicken-wire: 38% – Punctate alone: 27% – Mixed: 35% – Total punctate: 62%

Ko J Cutan Pathol 2016 IgG IgG Take Home Message:

• Be aware of punctate pattern • Not false positive finding Adnexal Staining in Bullous Pemphigoid and Pemphigus Adnexal Staining in BP and Pemphigus Vulgaris • Similar sensitivity in epidermis and adnexal Zhou J Cutan Pathol 2016 structures • Rare cases where epidermis negative and adnexal positive Pemphigus Vulgaris Bullous Pemphigoid C3 IgG Adnexal Staining in PNP

2 cases PNP • Negative surface epithelial • Positive IgG, C3 intercellular follicular, eccrine, sebaceous Barnadas J Cutan Pathol 2009 Sweat Duct C3 Sebaceous IgG Pemphigus Vulgaris/Foliaceus and the Scalp October 2017 32 cases (17 PV, 15 PF)

80-88% of PV and PF had follicular acantholysis

* PV affected upper and lower segment of follicles-PF only affected upper segment

* PV and PF demonstrated sebaceous gland atrophy Take Home Message:

• Mind the adnexa • May have the answer Case Example Photos courtesy of Joyce Park MD 1/2 2/2 IgA Epidermis

1/4 IgG

2/4 IgA

3/4 IgA

4/4 IgG/IgA Pemphigus April 2016

• 13 patients • Compared to classic pemphigus (PF+PV) and IgA pemphigus • Most similar to IgG pemphigus

NYU Review IgG/IgA Pemphigus

• 43 patients – 4 NYU patients – 13 Mayo clinic – 26 case reports • Conclusions – 40% mucosal – 40% pustules, annular – Malignancy: hematologic, solid tumor – Dapsone, Immunosuppressive medications IgG/IgA Pemphigus: Conclusions

• Clinical significance: – Overlapping features of IgG Pemphigus & IgA Pemphigus – Variable mucosal involvement – Malignancy association • Unclear Pathogenesis 1. Transition from IgG to IgA pemphigus, epitope spread 2. Circulating IgA antibodies in IgG pemphigus, false negative DIF • Limitations – commercial assays for IgA to desmogleins 1 &3 not readily available – Lack of diagnostic criteria p200 Pemphigoid Anti-p200 Pemphigoid • Subepidermal autoimmune bullous disease • Overlap with EBA, BP, CP and linear IgA • Tense blisters • Pruritus • Up to 50% mucosal lesions • 30% associated with psoriasis (antigen exposure) • Favorable prognosis overall • Majority responds to conservative treatment: topical CST, dapsone Goletz et al. JAAD 2014 Anti-p200 Pemphigoid

• Antigen 200 kD dermal protein • Laminin gamma 1 – proposed antigen – lamina densa • Diagnostic assays not widely available • Under-diagnosed

Goletz et al. JAAD 2014

Anti-p200 Pemphigoid: Clinical

• Slight male predominance • Older patients: 72 yrs • Clinical heterogeneity – Tense blisters hands, feet – Urticarial – Annular – Eczematous – Scarring – Pruritus • ~50% mucosal involvement – Gingivitis – Buccal – Tongue – Anogenital – No ocular or laryngeal – No scarring or milia Meijer JAMA Derm 2016 Commin B J Dermatol 2016 p200 Pemphigoid: Pathology

• Subepidermal separation • Neutrophil rich • Eosinophils • Papillary microabscesses Anti-p200 Pemphigoid • DIF – C3, IgG in all patients – +/- IgA (58%), IgM (17%) – N serrated in 82% – Dermal on salt-split

Goletz et al. JAAD 2014 Differential Diagnosis

• Dermal C3 & IgG on salt-split: – Epidermolysis bullosa acquisita - U serrated – p200 Pemphigoid – Antiepiligrin (laminin 332, laminin 5) MMP - Fibrosis Dermatitis Herpetiformis IgA Dermatitis Herpetiformis

Fibrillar pattern • 2-4% • 36-56% in Japanese populations • Atypical clinical lesions • Psoriasiform • Urticarial • EM-like • Generalized lesions • Lack enteropathy, transglutaminase antibodies Dermatitis Herpetiformis

Vascular deposition • Up to 50% of cases • Small vessel leukocytoclastic vasculitis

Barnadas Am J Dermatopathol 2016 Dermatitis Herpetiformis Adnexal deposits • Arrector pili (30%) • Follicular (43%) • Eccrine glands/ducts (25%)

Elastic fibers (24%)

Barnadas Am J Dermatopathol 2016 Small Vessel Leukocytoclastic Vasculitis

DIF Findings in LCV

• 70-84% positive • 102 patients with LCV and DIF * – C3 80% – IgA 65% – IgM 49% – IgG 42% – Fibrinogen 50-67% – 15% negative DIF • Highest yield biopsy – early lesion (<24 hours) – biopsy edge of lesion

* Sais et al Arch Derm 1998 * Sais et al Arch Derm 1998 LCV and DIF

• 20-30% of DIF cases are for LCV • Majority of DIF cases for LCV are “rule out HSP” • We are looking for IgA Henoch Schonlein Purpura (HSP)

• 10% of all vasculitides • Children > adults • Skin + – Joint (75%): arthralgia, arthritis – GI (50-75%): pain, bleeding, perforation – Renal (40-50%): hematuria, renal insufficiency • Majority resolve without sequelae – Children: 1-3% end-stage renal disease – Adults: end-stage renal 11-14% – Adults more likely to have recurrent, chronic disease HSP and IgA

• 1973 Lancet HSP and IgA

• Pathogenesis unknown • IgA pathologic role – 88% of pediatric cases had IgA on DIF – IgA1 implicated in disease • Abnormal glycosylation leads to IgA aggregation • Increased production • Decreased clearance • Increased IgA in serum • IgA deposits in skin, kidney, GI • Aberrant IgA autoantibody production: – IgA rheumatoid factor, IgA ANCA, IgA anti-endothelial antibodies HSP and DIF

• Stats for IgA and diagnosis of HSP – Sn 81%, Sp 83% – PPV 84%, NPV 81% – 20% of HSP cases negative DIF • Diagnosis of HSP: – 5 diagnostic criteria schemes • ACR (1990), Michel (1992): no IgA • CHCC (1994, 2012), Helander (1995), EULAR (2010): IgA part of criteria – Clinical features overlap with other vasculitides Linskey et al JAAD 2012 HSP and DIF

• Chapel Hill Consensus Criteria 2012 – IgA only criteria (1994, 2012) – HSP name replaced with IgA vasculitis

• We know IgA may be seen in other conditions

Jennette et al Arthritis & Rheumatism 2013 HSP and DIF

• What this means for us • We are not to make final diagnosis of HSP or IgA vasculitis – We diagnose in clinical vacuum – Don’t know what criteria doctors use – Could IgA be secondary to other systemic condition – I use IgA as predominant immunoreactant; stronger than IgG or IgM – Say “consistent with IgA vasculitis” – Leave room for clinician to make final diagnosis and rule out other conditions Take away thoughts on HSP…

• What we call HSP may be different diseases particularly in kids vs adults – Kids: post infectious, GI, arthralgia, renal; resolves – Adults: infectious, malignancy, drug; long term sequela with kidney • Unclear pathogenesis • Nomenclature and diagnostic criteria need consensus Pitfalls for DIF in LCV

• IgA – Hepatobiliary disease • Alcoholic liver disease IgA • Primary biliary cirrhosis – Connective tissue disease • SLE, RA – Coagulopathy • Livedo vasculopathy • Cryoglobulinemia – Others • Medications • Erythema nodosum Pitfalls in DIF for LCV

• Granular, perivascular C3 C33 – Pigmented purpura – Non-specific inflammation: • Acne • Stasis dermatitis – Gravity: lower legs – Normal skin Adnexal Staining in Lupus IgM Lupus and DIF

• Lupus band test – Granular immunoreactant at dej – IgM most common – Additional reactants increases specificity • Different definitions – Lesional vs non-lesional – Sun-exposed vs sun-protected • Sensitivity 93%, Specificity 87% • Sun-exposed, lesional skin: 70-80% SLE • Non-lesional, sun-protected skin: 55% SLE – correlate with disease severity (renal, ds DNA) • DLE only positive in lesional skin • Cutaneous lupus erythematosus • 100 cases, 76 had adnexal structures • IgM in adnexal structures

Elbendary et al JAAD 2016 • DEJ: • Follicle • Eccrine Gland – IgM 89% – IgM 100% – IgM 88% – IgG 76% – IgG 75% – IgG 76% – IgA 60% – IgA 67% – IgA 66% – C3 83% – C3 82% – C3 72% Specificity and Likliehood Ratio Increase with Adnexal Staining Conclusion

• Bullous diseases • Vasculitis • Connective tissue disease

Thank you! [email protected]