For best use of immunodermatology tests, it makes a difference where the biopsy is taken from in order to effectively search for evidence of an antigen-antibody immune-mediated dermatosis. Apparently,
at least in BP, the lower extremity is often DIF false-negative3. We have seen classical BP with H&E subepidermal blister and copious superficial dermal
eosinophiles which had to be BP but was DIF negative (though IFA positive for anti-skin antibodies). And we have had a case of probable PF with DIF and anti-skin antibody
negativity. So, one must judiciously interpret immunobullous skin biopsies on the basis of clinical, H&E, DIF, and serological IFA (circulating serum anti-skin autoantibodies)
lines of evidence. "Positive" DIF is likely positive for a serum mediated immunodermatosis, but "negative" very well may not be negative.
I have yet to see a clear written exposition of the dynamics of immunobullous lesions in the sense of whether false negativity by DIF will also find negativity or
positivity for the global IFA test on rat bladder (as to paraneoplastic pemphigoid4) or monkey esophageal or pig esophageal substrate for anti-skin antibodies and if tests for specific antibodies (such as anti-pemphigus foliaceus anti-DSG1
anti-pemphigus vulgaris anti-DSG3) are more sensitive. The IgG autoantibody from the patient's serum is incubated on the above substrate so that it binds to the appropriate antigenic, morpho-specific tissue site. Serum is washed off, and fluoroscein-tagged animal anti-IgG-human-antibody (an IgM) is then incubated on the substrate slide and attaches to the patient's bound autoantibody. This is observed using a fluorescent microscope, and the amount of the autoantibody is measured by retesting at various serum dilutions for a test series that can begin with "neat" (undiluted) serum...though many start at a 1:10 dilution.
It seems reasonable that serum could be temporarily depleted of circulating auto-antibodies at the temporal apex of lesional outbreak (especially in the ill and
undernourished). And then the lesion-inducing immunodeposits degrade to the point of indetectibility fairly rapidly (hours to days).
So, non-lesional skin might harbor (be tagged by) immunodeposits which "are there" at levels below the threshold of producing lesions (for example, H-S-like vascular deposits but no H&E evidence of leukocytoclastic vasculitis) & with
circulating serum auto-antibodies still able to stay in production-consumption equilibrium and still at levels of detectibility. DIF-positive "lesions" without H&E-specific classical, corrobarative changes are likely an earlier, more sensitive...and less specific...situation of "DIF positivity of uncertain clinical significance" [S07-11392]. Perilesional skin immunodeposits may stay
detectible because, being nonlesional, they are not exposed as drastically to the dynamics of protease degradation (the degrading proteases coming to the epidermis from the
direction of the papillary dermis). At least as to vascular deposits (& probably all deposits), there is rapid (24-48 hours) degradation with IgG the first to go & fibrinogen the last.
In short, how does one rationally spend money to rule in or rule out (by lab tests and histopathology) various immunobullous dermatoses? Specimen acquistion
& handling is highly important! We can work with any type of biopsy, but a good shave biopsy seems best to me and allows maximum flexibility in doing a progressive workup.
It is important to blot or wash (with or in saline) the blood away from the DIF specimen at biopsy in order to remove endogenous immunodeposit-degrading proteases so that the
protease inhibitors in the transport fluid are not promptly depleted by just the raw-surface attached blood/serum proteases.
It is easily possible within our practice for a consulting dermatologist to do a shave biopsy, transport right away by courier in a sealed container so it won't dry, and hand deliver
to one of us pathologists. History & rule outs are communicated as the specimen changes hands. A frozen section diagnosis can be rendered as slides are cut on the cryostat
for DIF prcedures. Processed early enough in the day, it is technically possible in our situation to interpret DIF slides the same day...the very first case we ever did in 1985
was done so (an ER case). And the specimen half that was not frozen (or the frozen rapidly thawed into fixative...we prefer a mercury based fixative) can be processed overnight.
Also, we can be more specific and helpful if biopsies are also accompanied by a non-anticoagulated ("red-top tube") blood specimen so that we could add
on clarifying serological testing when needed (global histological "anti-skin antibodies" [anti-EBA, anti-BP by inference due to exact location of reaction], andor
such specific "lab test" anti-skin antibodies as the anti-pemphus antibodies, desmoglein 1 & 3).
Click here for a list of a few of our biopsy case examples [HERE]
The Preferred Sites for Direct Immunofluorescence
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