Immunofluorescence (Direct and Indirect)

Immunofluorescence uses antibodies labelled with a fluorescent dye to detect immune deposits in tissue (direct immunofluorescence) or autoantibodies in a patient's serum (indirect immunofluorescence). The labelled antibody binds its target, and the dye glows under ultraviolet or blue light in a fluorescence microscope.

Direct immunofluorescence (DIF) looks for immunoglobulins (IgG, IgA, IgM), complement (C3, C1q) and fibrinogen deposited in tissue in vivo. It is used for skin blistering disease, vasculitis and connective tissue disease, and for renal biopsy. The pattern and the site of the deposits are often diagnostic.

Indirect immunofluorescence (IIF) mixes the patient's serum with a tissue or cell substrate, and then adds a labelled anti-human antibody. It detects and titres circulating autoantibodies, such as antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA), anti-basement membrane and anti-intercellular antibodies.

DIF takes 1 to 2 days and needs tissue that has not been fixed in formalin. IIF takes 1 day. Sensitivity of DIF for pemphigus and pemphigoid is over 90% when the sample is taken correctly.

Indications.

• Direct: suspected autoimmune blistering disease (pemphigus, pemphigoid, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita).
• Direct: cutaneous lupus erythematosus, vasculitis and lichen planus when histology is not enough.
• Direct: renal biopsy for glomerulonephritis (IgA nephropathy, membranous nephropathy, lupus nephritis, anti-GBM disease, C3 glomerulopathy), and for transplant rejection.
• Direct: some neuromuscular and other tissue biopsies.
• Indirect: screening for connective tissue disease (ANA) in a patient with suggestive features.
• Indirect: ANCA for suspected vasculitis, anti-dsDNA on Crithidia luciliae, anti-endomysial antibodies in coeliac disease, anti-mitochondrial and anti-smooth muscle antibodies in liver disease, and antibodies in blistering disease (on salt split skin).

Not indicated for ANA screening in people without features of an autoimmune disease, since low titre positive results are common in healthy people.

Specimen requirements.

• DIF skin: a 3 to 4 mm punch biopsy from perilesional skin, within 1 cm of a fresh blister or lesion, in Michel's medium or fresh on saline moistened gauze or frozen. Not fixed in formalin. A lesional biopsy is used for vasculitis (lesion under 24 hours) and lupus, with a sample of sun protected skin.
• DIF kidney: a part of the renal core, fresh or in transport medium, set aside before formalin fixation.
• IIF: serum (clotted blood), 2 to 5 mL, kept at 4°C and sent promptly. Samples are not heat treated.
• Clinical details: the suspected diagnosis, current treatment and drug history.

Normal appearances.

Normal skin shows no or only faint deposits. Kidney shows no or trace staining. Sun exposed skin and older skin can show non-specific C3 and IgM. IIF on normal serum shows no or low titre staining of the substrate.

Interpretation.

• Pemphigus: IgG and C3 in a "fishnet" pattern between keratinocytes (intercellular). Indirect IF on monkey oesophagus shows the same pattern.
• Bullous pemphigoid: linear IgG and C3 along the basement membrane zone. Salt split skin shows deposits on the roof (epidermal side).
• Epidermolysis bullosa acquisita: linear deposits on the floor (dermal side) of salt split skin.
• Dermatitis herpetiformis: granular IgA deposits in the dermal papillae of uninvolved skin.
• Linear IgA disease: linear IgA at the basement membrane zone.
• Lupus erythematosus: granular IgG, IgM and C3 at the basement membrane zone (lupus band), more reliably in lesional skin.
• Vasculitis: C3, IgM, IgA and fibrinogen in vessel walls. IgA predominant deposits suggest IgA vasculitis.
• Lichen planus: shaggy fibrinogen at the basement membrane, with cytoid bodies.
• IgA nephropathy: dominant mesangial IgA with C3.
• Membranous nephropathy: granular IgG along the capillary walls.
• Anti-GBM disease: smooth linear IgG along the glomerular basement membrane.
• Lupus nephritis: "full house" staining (IgG, IgA, IgM, C3 and C1q).
• C3 glomerulopathy: dominant C3 with little or no immunoglobulin.
• ANA patterns: homogeneous, speckled, nucleolar, centromere and cytoplasmic. Titres of 1:80 and 1:160 or more are more significant than 1:40, though low titres occur in 20 to 30% of healthy people.
• ANCA: cytoplasmic pattern (c-ANCA, mostly PR3) and perinuclear (p-ANCA, mostly MPO), confirmed by specific immunoassays.

Critical values and findings needing immediate action.

• Positive anti-GBM or ANCA with rapidly progressive renal impairment or lung haemorrhage: urgent treatment and specialist care.
• Pemphigus or pemphigoid with extensive skin or mucosal involvement: urgent specialist treatment.
• High titre anti-dsDNA with nephritis or neuropsychiatric lupus: urgent rheumatology and renal review.

Limitations and artefacts.

• A sample from a blister roof, an ulcer or an old lesion gives false negative results. A perilesional sample is best.
• Wrong transport (formalin) destroys antigens, and delay reduces staining.
• Fading (photobleaching) of the fluorescent signal limits time to read and re-read.
• Non-specific staining: sun damaged skin, leg ulcers and the area around the follicles show C3 and IgM without disease.
• Immunosuppressive treatment can reduce the deposits and titres.
• Dilution and substrate differences between laboratories change the ANA titre, so interpretation uses titre and pattern, not the result alone.
• A positive ANA in a healthy person is common and does not indicate disease.
• Subjective reading needs experienced staff, with clinical and histological correlation.
• Anti-dsDNA and specific autoantibody assays are more specific than IIF for several diseases.

Special groups.

• Children: linear IgA disease and juvenile dermatomyositis, with specialist interpretation.
• Older adults: bullous pemphigoid is the commonest blistering disease, and drug induced forms occur.
• Pregnant women: pemphigoid gestationis shows linear C3 at the basement membrane, and ANA, anti-Ro and anti-La have foetal implications.
• Patients on immunosuppressants or steroids: repeat testing if the first result is negative with a high clinical suspicion.
• Patients with coeliac disease or dermatitis herpetiformis: DIF and anti-endomysial antibodies.
• Regions: immunofluorescence microscopes and trained staff are scarce, so skin samples are sent to reference centres in Michel's medium. Where DIF is unavailable, histology and clinical features direct treatment of blistering disease. ELISA for desmoglein and BP180 helps where available.

Follow up tests.

• Routine histology of the lesional sample.
• ELISA for desmoglein 1 and 3, BP180 and BP230, and collagen VII antibodies.
• Specific autoantibody panels (anti-dsDNA, ENA, anti-PR3, anti-MPO, anti-GBM).
• Complement levels, urinalysis, renal and liver function.
• Renal biopsy light microscopy and electron microscopy.
• Tissue transglutaminase and endomysial antibodies, and coeliac workup.
• Repeat sampling of fresh lesions when the first sample is negative.
• Specialist rheumatology, nephrology or dermatology review.