What is the indirect immunofluorescence test?

I hope you found yesterday’s post on the direct immunofluorescence (DIF) test on skin biopsies helpful.  Today, we will cover the indirect immunofluorescence (IIF) test.  This IIF test is a way to find whether a patient’s blood contains antibodies.  In the case of autoimmune blistering disorders such as pemphigus vulgaris, bullous pemphigoid or paraneoplastic pemphigoid, we are looking for autoimmune antibodies (autoantibodies) to one’s own skin.

For the indirect immunofluorescence test, the patient’s blood is drawn and submitted to the pathology lab (NOT the patient’s skin). In the case where autoimmune blistering diseases are suspected, the pathology lab takes the serum of the blood to see if it has antibodies that bind to certain cells or tissues.  The lab then adds an antibody which is fluorescently labeled that can bind to the suspected antibodies.  In autoimmune blistering diseases, different tissues may be used by the pathology lab, e.g. rat bladder, guinea pig esophagus, monkey esophagus, etc.  Some tissues are more sensitive or specific for a certain disease.

What is the direct immunofluorescence test on a skin biopsy?

Image by Wikipedia user Kauczuk. DIF of Henoch-Schonlein Purpura shows IgA around the vessels of the skin dermis.
DIF of Henoch-Schonlein Purpura shows IgA around the vessels of the skin dermis. Image by Wikipedia user Kauczuk.
This biopsy showed a positive "lupus band" test. Note the granular fluorescent deposits between the epidermis and dermis. Image from Wikipedia user Magnus Manske.
This biopsy showed a positive “lupus band” test. Note the granular fluorescent deposits between the epidermis and dermis. Image from Wikipedia user Magnus Manske.

Normal skin biopsies get sent for hematoxylin and eosin staining (or what we call H&E staining).  However, for certain skin conditions, we do a special test.  We take a skin or oral biopsy for direct immunofluorescence (DIF) examination.  These conditions that we are looking for may include blistering diseases such as pemphigus vulgaris, pemphigus foliaceus or bullous foliaceus, or non-blistering diseases such as lupus or Henoch-Schonlein purpura where one is looking for a specific vasculitis (inflammation around blood vessels).

So how is the direct immunofluorescence (DIF) exam done? First, a biopsy of skin or the oral mucosa is taken.  Next, it is sent to the pathology lab where the skin is cut into small slivers and mounted onto a glass slide.  Antibodies which have fluorescent tags on them are then thrown into the mix with this tissue.  These antibodies that the pathology lab adds to the tissue may recognize other antibodies (e.g. IgA, IgM, IgG) or other products such as C3 or fibrinogen.  Because the pathology lab’s antibodies have fluorescent tags, the pathologist can then see whether the fluorescence forms a specific pattern. For instance, the fluorescence may be brightest around the vessels (e.g. as in Henoch-Schonlein purpura), between the epidermis and dermis at the basement membrane (e.g. as in bullous pemphigoid), or between the cells of the epidermis (e.g. as in pemphigus vulgaris).  Even a test which is negative may be helpful, since the negative test sways us away from certain diagnoses such as the ones we had listed above.  For instance, we’d expect a disease such as a genetic epidermolysis bullosa (EB) to have a negative DIF.

Hope that helps you interpret your pathology biopsy report.  Stay tuned to future posts to learn how indirect immunofluorescence (IIF) differs from direct immunofluorescence!