Direct immunofluorescence

Author: George Zhang, Medical Student, University of Auckland. DermNet New Zealand Editor in Chief: Hon A/Prof Amanda Oakley, Dermatologist, Hamilton, New Zealand. Copy editor: Maria McGivern. April 2017.

What is direct immunofluorescence?

Direct immunofluorescence (DIF) is a technique used in the laboratory to diagnose diseases of the skin, kidney, and other organ systems. It is also called the direct immune fluorescent test or primary immunofluorescence.

DIF involves the application of antibodyfluorophore conjugate molecules to samples of patient tissue obtained from biopsies. These antibodyfluorophore conjugates target abnormal depositions of proteins in the patient’s tissue. When exposed to light, the fluorophore emits its own frequency of light, seen with a microscope. The particular staining pattern and type of abnormal protein deposition seen in the tissue sample help diagnose the disease.

Direct immunofluorescence for the diagnosis of skin diseases

DIF is useful in the diagnosis of suspected autoimmune bullous diseases, connective tissue diseases and vasculitis. The staining patterns seen in tissue samples may be specific to a disease entity or they may need to be interpreted with the clinical and histological findings.

Autoimmune bullous diseases

DIF is useful in diagnosing the following autoimmune bullous diseases:

Connective tissue diseases

DIF is useful in diagnosing the following connective tissue diseases:

Vasculitis and other conditions

DIF is useful in diagnosing cutaneous vasculitis and some other types of inflammatory skin disease:

Site of biopsy

The optimal site for taking a biopsy of the skin depends on the suspected disease.

Another biopsy is usually taken for routine haematoxylin–eosin (H&E) histology. Note that the optimal biopsy site and timeframe required differs for these examples.

Transport and storage of the biopsy sample

Specimens for DIF should not be placed in formalin, as this alters proteins and significantly diminishes the accuracy of results. Transport the specimen to the laboratory as soon as possible. Options for transporting the sample include:

Each laboratory has its own protocols. Specimens in saline may yield superior sensitivity over other media if processed within 24–48 hours.

What happens to the specimen in the laboratory?

DIF may be carried out in an automated machine or manually. The process involves first making frozen sections then carrying out immunofluorescence.

Preparing frozen sections  

  1. A punch biopsy is transported to the lab on saline-soaked gauze.
  2. The specimen is placed in a gel.
  3. Liquid nitrogen is used to freeze the specimen and gel.
  4. The frozen specimen is contained within the frozen gel.
  5. The specimen can be stored in liquid nitrogen for about one week.
  6. 4–6 micron thick slices are cut.

Carrying out direct immunofluorescence

  1. Five or six slides are made; each for a different reagent. One slide will be used for a normal H&E stain.
  2. A special pen is used to draw a perimeter to keep reagents within the slides.
  3. The slides are washed.
  4. The reagents are made up.
  5. The reagents (antibodyfluorophore conjugates to IgG, IgM, IgA, complement protein C3, and when required fibrinogen) are dropped onto the slides and the slides are left for some time in the dark.
  6. The slides are washed again in solution.
  7. Glass covers are placed over the slides.

Interpretation of direct immunofluorescence

The prepared immunofluorescence slides are examined by a pathologist to determine the primary sites of immune deposition (if any), the classes of immunoglobulin or other immune deposits, and the patterns of deposition. Staining patterns can be classed into five groups:

Examples of staining patterns 

Staining patterns of specific skin diseases

Pemphigus vulgaris

Pemphigus foliaceus

Paraneoplastic pemphigus

IgA pemphigus

Bullous pemphigoid

Mucous membrane pemphigoid (cicatricial pemphigoid)

Epidermolysis bullosa acquisita

Dermatitis herpetiformis

Lupus erythematosus

Systemic scleroderma

Mixed connective tissue disease

Dermatomyositis

Lichenoid reactions (lichen planus, drug reactions and photodermatoses)

Porphyria cutanea tarda

Vasculitis

Henoch–Schönlein purpura

How reliable are direct immunofluorescence results?

DIF testing is moderately sensitive in detecting a range of diseases. The sensitivity for DIF approaches 100% for the pemphigus group of diseases, and sensitivity has been reported to be 55–96% for bullous pemphigoid. A study of ten patients with Henoch–Schönlein purpura and nine with lupus erythematosus demonstrated positive DIF testing in all of the patients.

The number of false-negative results with DIF depends on the quality of the sample, laboratory processing, and whether or not the patient has been on treatment. Reasons for false-negative results include:

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