Indirect immunofluorescence

Authors: Rachel Xuan, Medical Student, University of New South Wales; Dr Anes Yang, MA, BMed, MPH, Dermatology Clinical Research Fellow, Premier Specialists, NSW, Australia; Professor Dedee F. Murrell, Head, Department of Dermatology, St George Hospital, University of New South Wales, Sydney, Australia. DermNet New Zealand Editor in Chief: A/Prof Amanda Oakley, Dermatologist, Hamilton, New Zealand. December 2017.

What is indirect immunofluorescence?

Indirect immunofluorescence, or secondary immunofluorescence, is a technique used in laboratories to detect circulating autoantibodies in patient serum. It is used to diagnose autoimmune blistering diseases.

  • Unlabelled primary antibodies from the patient serum bind to the target molecule in pre-prepared tissue samples.
  • Secondary antibodies, conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC), bind to the primary antibody.
  • When exposed to light, the fluorescent dye is excited and emits a wavelength that can be seen with a fluorescent microscope.

The tissue substrates used (monkey oesophagus or human skin) will depend on the suspected disease. Different staining patterns will help diagnose an autoimmune bullous disease. 

Which blistering skin conditions does indirect immunofluorescence test for?

Indirect immunofluorescence is used to diagnose autoimmune bullous diseases.

Describe the laboratory method 

Preparing patient serum for testing 

  1. 5–10 mL of blood is drawn and collected in a tube.
  2. The blood is allowed to clot.
  3. The blood sample is then centrifuged to separate the serum from the clot.
  4. Serum is aliquoted and frozen until testing.

Indirect immunofluorescence test  

  1. Prepare 4–6 micron thick slices of tissue substrate slides (this can be done in a laboratory or obtained commercially)
  2. Several slides are prepared with different tissue substrates.
  3. Tissue section is incubated with patient serum for 30 minutes and doubling dilution is performed if a titre is required.
  4. The slides are washed to remove any unbound primary antibodies.
  5. Slides are incubated for another 30 minutes with secondary antibodies containing fluorescent dye.
  6. The slide is mounted under a cover slip.
  7. It is examined using fluorescence microscopy.

Interpreting the results of indirect immunofluorescence

The immunofluorescent slides are examined to determine the presence of autoantibodies via the patterns of immune deposition. The results are subjective and indirect immunofluorescence cannot be used reliably to monitor disease severity and/or its treatment. 

Different staining patterns

  • Intercellular staining (ICS) pattern
  • Basement membrane zone (BMZ) pattern

Immunofluorescent patterns in skin diseases

  • Sensitivity of a diagnostic tool refers to its ability to accurately identify those with the disease (true positive).
  • Specificity is its ability to accurately identify those without the disease (true negative).

Occasionally the test can produce false results.

  • The result may be negative even if the patient has the disease (false negative).
  • The result may be positive in a patient without the disease (false positive).

Pemphigus vulgaris

  • ICS pattern IgG or C3
  • Sensitivity 81-87% (4, 7)
  • Specificity 81% (7) 

Pemphigus foliaceous

  • ICS pattern IgG or C3
  • 60-86% (4, 7) 

Paraneoplastic pemphigus

  • C3 IgG antibodies
  • Sensitivity 80% (3)
  • Specificity 83% (3)

Bullous pemphigoid

  • Linear BMZ with IgG antibodies and C3
  • Sensitivity 70% (3)

Mucous membrane pemphigoid

  • BMZ with IgG/IgA antibodies
  • Sensitivity 20% (3)

Pemphigoid gestationis

  • C3
  • Sensitivity 93% (3)

Epidermolysis bullosa acquisita

  • BMZ with IgG antibodies
  • Sensitivity 50% (3)

Dermatitis herpetiformis

  • IgA anti-endomysal antibodies          
  • Sensitivity 80% (3) 

Linear IgA bullous dermatosis and chronic bullous disease of childhood

  • BMZ with IgA antibodies
  • Sensitivity 50% (3)

Bullous lupus erythematosus

  • BMZ with IgG antibodies 


Related Information


  1. Chhabra S, Minz RW, Saikia B. Immunofluorescence in dermatology. IJDVL 2012; 78; 6; 677-692. DOI: 10.4103/0378-6323.102355. Available at;year=2012;volume=78;issue=6;spage=677;epage=691;aulast=Chhabra
  2. Fitzpatrick RE, Newcomer VD. The correlation of disease activity and antibody titers in pemphigus. Arch Dermatol 1980; 116; 3; 285-290. DOI: 10.1001/archderm.1980.01640270045011. Available at
  3. Fleming Dermatopathology. Immunodermatology Guidelines. 2010. Available at
  4. Jiao D, Bystryn JC. Sensitivity of indirect immunofluorescence, substrate specificity, and immunoblotting in the diagnosis of pemphigus. J Am Acad Dermatol 1997; 37; 2; 211–216. PubMed.
  5. Mascaro Jr JM. Histological and Immunofluorescence Diagnosis of Autoimmune Blistering Diseases. In: Murrell DF (eds). Blistering diseases. New York: Springer-Verlag, 2015:161–192.
  6. Mayo Medical Laboratories. Cutaneous Immunofluorescence Testing. August 2014. Available at (accessed 26 May 2017)
  7. Zagorodniuk I, Weltfriend S, Shtruminger L, Sprecher E, Kogan O, Pollack S, Bergman R. A comparison of anti-desmoglein antibodies and indirect immunofluorescence in the serodiagnosis of pemphigus vulgaris. Int J Dermatol, 2005; 44; 541–544. PubMed.

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