Direct immunofluorescence for autoimmune bullous diseases.
A review and update for cutaneous direct immunofluorescence (test code: CIB).
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.
I have no disclosures to report.
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.
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.
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 shows strong linear deposition of IgA along the basement
membrane zone.
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.
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.
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.
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.
Recognizing the importance of IgG 4 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.
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.
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.
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 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!