Specialized immunofluorescence testing helps uncover rare autoimmune blistering diseases, identify underlying cancers, and guide precise treatment, improving diagnostic accuracy and outcomes for patients with challenging skin disorders.
Autoimmune blistering diseases affecting the skin are often misdiagnosed as conditions like eczema or hives because they initially present with similar symptoms and thus can be treated inappropriately. Meanwhile, the condition worsens, and a delayed correct diagnosis for these rare diseases can compromise the benefits of treatment.
Mayo Clinic excels at testing for and diagnosing autoimmune skin diseases, which fall into two categories:
- Pemphigoid diseases, when the autoimmune attack is at the basement membrane zone, which is the “glue” that holds the top layer of skin to the second layer of skin.
- Pemphigus diseases, in which the autoimmune attack is against the glue that holds the skin cells together.
Leading the way in dermatologic immunology diagnostics is Julia Lehman, M.D., chair of Mayo Clinic’s Division of Dermatopathology and Cutaneous Immunopathology within the Department of Dermatology, and director of Mayo Clinic’s Immunodermatology Laboratory. As a board-certified dermatologist, she also sees patients affected by autoimmune blistering skin diseases.
“The trick is to try to catch the autoimmune attack early in the disease course before the immune system gets more brittle,” says Dr. Lehman. “Because we know, especially with pemphigus diseases, that if you don't treat early and treat aggressively, the patient can become more and more recalcitrant to treatment and much less likely to get a complete therapeutic response.”
“The trick is to try to catch the autoimmune attack early in the disease course before the immune system gets more brittle.”
Julia Lehman, M.D.
Detecting underlying cancers
Dr. Lehman designed an Autoimmune Blistering Disease Diagnostic Algorithm, which features a comprehensive test portfolio that includes the rare subepithelial autoimmune blistering disease variants (Mayo ID: RSBV).
“RSBV is a mosaic indirect immunofluorescence assay where we test for three of the most common antibodies in very rare forms of pemphigoid,” she says. “And we're the only laboratory in the country to offer a screen for antibodies against all three key antibodies—p200, collagen VII, and laminin-332. It's really important to know which one of these antibodies is involved, because they have different associations.
“For example, if a patient has anti-laminin-332 antibodies, there's a much higher risk that the skin or mucosal disease is being driven by an underlying cancer. And that triggers the clinician to make sure the patient has appropriate imaging and to do a really thorough history and physical examination. That's one way where the testing can help improve patient care, by allowing early detection and treatment of underlying malignancies.”
Such a scenario recently happened with a patient who presented with nonspecific mouth erosions. A standard biopsy and other conventional testing didn't really clinch the cause. That’s when the specialized RSBV test was ordered at Mayo.
“We found that he had anti-laminin-332 antibodies, which then triggered a full-body search for cancer,” says Dr. Lehman. “And that's when a tumor was found in his duodenum, allowing early surgical intervention. Once the cancer was removed, his mouth sores also improved.”
Pemphigoid diseases will often present with tense blisters on a hive-like base, although they can also present with itching of the skin or dermatitis-like changes of the skin.
“Sometimes patients with atypical clinical findings can go months, even years, without diagnosis, unless an astute clinician thinks to test for these diseases,” says Dr. Lehman. “Pemphigus can present with a number of different manifestations as well, including mouth sores or sores on the skin, or sometimes just rough scaly changes of the skin. So again, clinicians need to know that these diseases exist in order to be able to diagnose them properly in patients, or to refer them to someone who can.”
Direct and indirect immunofluorescence for rare skin diseases
Due to the rarity of these disorders, many primary care physicians don’t have a high familiarity with autoimmune blistering diseases of the skin. Even for general dermatologists — and associated specialists like ophthalmologists, otolaryngologists, and gynecologists — these diseases may be encountered only a handful of times in a career. Thus, determining which test(s) to order to confirm a diagnosis may be challenging.
“The standard approach to patients is to start with a regular tissue biopsy for routine microscopy,” explains Dr. Lehman. “And the changes on that biopsy are not often specific for autoimmune blistering diseases. So you can get lulled into thinking that the diagnosis of eczema or hives is correct because the biopsy isn't specific. That's where the clinician really has to think to do the specialized testing.”
Direct immunofluorescence is one of the mainstays of this test portfolio. The method is a tissue assay where the patient's biopsy is tested using various substrates. This allows clinicians to look at different components of the immune system.
“With this testing, we use fluorescent labels to visualize where exactly various components of the immune system, including immunoglobulin and complement, are depositing in the tissue,” Dr. Lehman explains. “We added IgG4 to the test because IgG4 is the subclass of immunoglobulin G that is usually involved with these processes. And the nice thing is, we don't have a lot of IgG4 circulating in our system. Therefore, there's very minimal background when we look at the tissue with IgG4. So when we see a signal with IgG4, it's almost always very specific for the diseases we're looking for.”
Mayo Clinic has led the field by being the first to incorporate IgG4 into direct immunofluorescence testing. They were also the first to publish data on how this specialized testing improves sensitivity and specificity for detecting these diseases.
Indirect immunofluorescence testing is often done at the same time, which entails incubating the patient’s serum sample with tissue substrates as well as a fluorescent tag to see if there are circulating antibodies that deposit along the basement membrane zone, or around the keratinocytes (cells in the epidermis). Though conventional indirect immunofluorescence testing was developed decades ago, Mayo Clinic upgraded the assay substantially in the last several years. IgG4 was added as a substrate to this test as well to enhance sensitivity and specificity.
Dr. Lehman continues, “When we saw how well IgG4 performed in the indirect immunofluorescence assay, we wondered, ‘Well, can we just replace our usual IgG indirect immunofluorescence with the IgG4?’ But then, with our rigorous validation testing, we found if you only do one or the other, then you lose 20% sensitivity. So testing both together gives us our best shot at identifying these diseases.”
Multiple tests for a single diagnosis
Anatilde Gonzalez Guerrico, Ph.D., is an assay developer in Mayo Clinic’s Department of Dermatology, who works closely with Dr. Lehman. She has been instrumental in helping to validate and optimize immunofluorescence assays. To further increase sensitivity, she also modifies assays by, for example, optimizing time, incubation, and temperature conditions.
Even so, there is no “perfect test.” This is why the test portfolio guides clinicians to order more than one assay to confirm a diagnosis. “Some tests are more sensitive, but they lack a little bit of specificity, and vice versa,” says Dr. Gonzalez Guerrico. “So it's good to have at least two or three tests where the combined results, along with careful clinical correlation, lead to the actual diagnosis. Just one result in isolation is not very reliable.”
NEW CONDITION, CURATED TREATMENTS
Mayo Clinic’s nationally renowned dermatopathology specialists are involved in interactive research that pushes the boundaries of what is known about rare autoimmune blistering skin conditions. These experts are available to consult on test ordering, results, and next steps to support personalized patient care.
Correct diagnosis opens the door to newer curative treatments
An accurate diagnosis is more important than ever before as newer drugs get approved by the Food and Drug Administration (FDA). Further, a correct diagnosis better ensures that insurance companies will pay for these latest treatments that are very effective and safe for patients.
“It's a really exciting time for those of us who care for patients with autoimmune diseases,” says Dr. Lehman. “Historical treatments for pemphigus — high-dose steroids or steroid-sparing immunosuppressive medications — can confer considerable risk for side effects, requiring clinicians to balance the risk with the benefit with those treatments. Then in 2018, rituximab became the first FDA-approved medication for this disease. Rituximab can lead to complete remissions in a substantial percentage of patients. And in patients with skin disease, rituximab tends to be well-tolerated and generally safe.
“And then for pemphigoid, it was the same story; it used to be that we would treat with high dose steroids, then we transitioned to using steroid-sparing immunosuppressive agents that still have their own toxicities. Then in 2025, the FDA approved the use of dupilumab, which is a biologic treatment that had previously been FDA-approved for atopic dermatitis. This breakthrough has given us a much safer and precisely targeted medication for patients with pemphigoid, which is very exciting.”
As the future of dermatologic immunologic diagnostics unfolds, Mayo Clinic will continue to lead the field by innovating and developing new tests while modifying existing commercial ones. And the race to stay ahead can, in part, mean slowing down.
“If you want to improve performance of an assay, it can take a little longer,” says Dr. Gonzalez Guerrico. “You need special instruments or testing conditions. You have to take everything into consideration if you can. There is pressure to do things super-fast in clinical labs, but sometimes you need a little longer incubation or to take other quality measures to be able to detect these very rare diseases.”
“There is pressure to do things super-fast in clinical labs, but sometimes you need a little longer incubation or to take other quality measures to be able to detect these very rare diseases.”
Anatilde Gonzalez Guerrico, Ph.D.
To learn more, watch this “Answers From the Lab” episode, where Julia Lehman, M.D., speaks about dermatologic autoimmune diagnostic advances.
