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Direct immunofluorescence for autoimmune bullous diseases

A review and update for cutaneous direct immunofluorescence (test code: CIB).

Introduction

Hello, I’m Dr. Julia Lehman, director of Mayo Clinic Immunodermatology Lab, and I’m a dermatopathologist and dermatologist at Mayo Clinic. I’d like to talk to you today about a recent change to our direct immunofluorescence assay, specifically the addition of the IgG4 conjugate.

Disclosures

I have no disclosures to report.

Autoimmune bullous dermatoses

Autoimmune bullous dermatoses comprise a rare category of blistering skin diseases that is caused by the development of autoantibodies against various constitutive parts of the epidermis or the dermal-epidermal junction. For example, pemphigus develops when autoantibodies are directed against desmogleins, the proteins that glue keratinocytes of the epidermis or the mucosal epithelium to one another, whereas, pemphigoid develops when autoantibodies are directed against bullous pemphigoid antigens, which help to hold the basement membrane zone together. In either case, autoantibodies can lead to loss of adhesion and cause erosions or ulcerations clinically.

Accurate diagnosis of diseases in this category of disease is essential to assure appropriate therapeutic decisions, as well as to recognize systemic diseases known to be associated with the autoimmune bullous dermatoses.

In general, the diagnosis of autoimmune blistering diseases requires careful correlation of clinical features with histopathologic findings, direct immunofluorescence features, and results of serum studies.

Historical perspective

Direct immunofluorescence was first applied to immunobullous diseases in Dermatology in 1967, when it was recognized that bullous pemphigoid was associated with the linear deposition of IgG along the basement membrane zone.

 A few years later, it was recognized that the addition of complement, specifically C3, increased sensitivity for a diagnosis of bullous pemphigoid with direct immunofluorescence.

Over time, the standard panel of conjugates tested with direct immunofluorescence for skin expanded to include IgG, IgM, IgA, C3, and fibrinogen. This panel of conjugates has been essentially unchanged for several decades at most institutions.

Pemphigoid/EBA/bullous lupus

Classically, pemphigoid, epidermolysis bullosa acquisita, and bullous lupus all show IgG with or without C3 in a linear distribution along the basement membrane zone.

Linear IgA bullous dermatosis

Linear IgA bullous dermatosis shows strong linear deposition of IgA along the basement membrane zone.

Pemphigus

Pemphigus typically shows cell surface deposition of IgG and/or C3. Sometimes IgA may also show cell surface deposition. Very rarely, IgA is the only conjugate involved.

Dermatitis herpetiformis

The direct immunofluorescence changes of dermatitis herpetiformis are very characteristic, with granular deposition along the basement membrane zone, accompanied by stippling in the dermal papillae with IgA.

Pemphigus as an IgG4-mediated disease

It is long been known the pathogenesis of pemphigus involves the development of autoantibodies directed against desmoglein 1 and desmoglein 3. These autoantibodies are typically of the IgG4 subclass.

IgG4-predominant pemphigoid

Conventional wisdom is that the pathophysiology of pemphigoid involves deposition of IgG antibodies along the basement membrane zone, which then induces complement, which ultimately leads to blister formation. Because the IgG4 subclass of IgG antibodies does not fix complement, it was generally regarded as having no substantial role in the pathogenesis of bullous pemphigoid. However, it has since been recognized that there may be a complement-independent mechanism of blister formation that is mediated by IgG4, and there have been several patients reported with IgG4 predominant pemphigoid. Interestingly, these patients often present with pruritus or urticaria, without blisters being a prominent component in early lesions.

Effect of adding IgG4 conjugate to the direct immunofluorescence panel (test code: CIB)

Recognizing the importance of IgG4 in the pathogenesis of pemphigus, as well as the increasing awareness of IgG4-predominant pemphigoid, we decided to evaluate the potential benefit to diagnosis of adding IgG4 conjugate to our direct immunofluorescence panel. 

Specifically, we aimed to assess how often the addition of the IgG4 conjugate helped to refine diagnosis.  As a secondary outcome, we documented the degree of background staining with IgG, C3, and IgG4, to see whether decreased background staining with the IgG conjugate may contribute to improved sensitivity and specificity of findings.

Effect of adding IgG4 conjugate to DIF

To assess these outcomes, we collected all cases over a one-month period in which there was definite or equivocal linear or cell surface deposition with IgG, C3, and/or IgG4.

Here is an example in the series in which there was very focal linear IgG deposition along the basement membrane zone. Due to considerable background staining, it would be very difficult to call this finding. C3 also shows some focal and discontinuous linear deposition, which would not alone be diagnostic for subepidermal autoimmune blistering disease.

In the same case, IgG4 demonstrated strong, continuous linear deposition along the basement membrane zone. With this finding, the interpretation of IgG and C3 changes were increased in diagnostic confidence.

This is another case which shows some potential cell surface deposition with IgG that is obscured by background. C3 was negative.

However, IgG4 showed strong cell surface and punctate deposition in a patent that has been described in pemphigus previously. This finding increased confidence that the changes observed in IgG were likely clinically relevant for a diagnosis of pemphigus.

Finally, in this case, there was equivocal linear deposition of IgG along the basement membrane zone. As you may be able to tell from the photograph on the left, the epidermis was slightly out of focus from the dermis, and so in this case, I would suspect that the linear deposition is actually edge artifact rather than true deposition. C3 showed nonspecific granular deposition.

IgG4 in this case was unequivocally negative. This finding increased confidence that the changes seen with IgG testing were indeed artifact.

Effect of adding IgG4 conjugate to the direct immunofluorescence panel (test code: CIB)

So, in summary, we carefully evaluated 70 cases meeting inclusion criteria. And in almost a third of the cases, the diagnosis was refined due to the addition of IgG4 conjugate. We also observed the background staining to be considerably less in IgG4 staining compared to IgG staining. Importantly, no case showed nonspecific deposition of IgG4.

Upon making these observations, our group made the careful decision to add IgG4 as part of our standard panel for the direct immunofluorescence assay.  As experience with this test is gained, we may find that the presence or absence of IgG4 staining, in pemphigus, pemphigoid, and potentially other immune mediated dermatoses may have additional diagnostic and/or therapeutic implications that are as yet unstudied.

Therefore, going forward, you will notice that the IgG4 conjugate will be reported in our standard direct immunofluorescence test report.  If you have any questions about this or how to interpret the results, please do not hesitate to contact the Immunodermatology Laboratory, and one of our cutaneous immunopathologists would be happy to discuss your case with you.

Test update

If you would like to order direct immunofluorescence at Mayo Clinic for the skin, please continue to use test code CIB. There is no need to order IgG4 conjugate testing separately.

Although we believe that the addition of the IgG4 conjugate to our direct immunofluorescence test will increase sensitivity and specificity of this component of diagnosis, it should be reiterated that the final clinical diagnosis should be based on a combination of clinical, histopathologic, immunofluorescence, and serum studies.  

Thank you

Thank you so much for your attention. If you have any questions or requests relating to this talk, please send an email to mcleducation@mayo.edu, or for more information, visit our website at www.mayocliniclabs.com. Thank you!

Questions?

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