Immunofluorescence   (prof. MUDr. Zdenek Vlašín, MD)

4  Skin biopsy

4.3  Immunofluorescence

prof. MUDr. Zdenek Vlašín, MD


Immunofluorescence (IF) is a commonly used laboratory method for detection of antigens of cells and tissues. In this chapter we are describing the methods commonly used in diagnostic routine. Some frequent diagnostic units are presented.

4.3.1  Direct Immunofluorescence


Direct immunofluorescence enables detecting antibodies, antigens, complement components and fibrin. Antibodies marked by a fluorochrome (eg. fluoresceinisothiocyanate, FITC) — conjugates — react with the tissue. After washing unbound antibodies out the tissue is evaluated using the fluorescent microscope. Labeled antibody-antigen complexes shine yellow-green. The contrast can further increase the staining by the Evans red (the background is reddish).

The tissue for evaluation is obtained by the 4 mm punch biopsy. Fast freezing of the tissue is extremely important. If immediate freezing is not possible, special transporting medium (maleinimide) can be used.

Direct immunofluorescence is rarely diagnostic (eg. IgG and c3 intercellulary together with the acantholytic bula in pemphigus). In most cases the combination of different immunoglobulines and complement factors together with results of classic transmitted light histologic evaluation enables specific diagnosis.

The site of biopsy is very important. In bullous or vesicular diseases its is the border of a vesicle, in lupus erythematodes and porphyria the sun exposed areas where the changes are suitable for diagnosis. In excisions of systemic disorders on the other hand the biopsy should be made from sun protected areas (volar areas of the forearm, buttocks) because of possibility of false positive results.

Uninvolved (normal) skin examination is important especially in systemic lupus erythematodes. Examination done to early can be negative. Biopsy should always follow detailed clinical and laboratory examination.  Pemphigus vulgaris and its variants (immunofluorescence)


Direct immunofluorescence contributes to the diagnosis of pemphigus vulgaris, pemphigus vegetans, pemphigus foliaceus and pemphigus erythematosus significantly by detection of autoantibodies bound to desmosomal areas (IgG, c3).


Net-like positivity of the surface of keratinocytes is common:
Pemphigus vulgaris, anti-ics, FITC 20x (10)

Pemphigus vulgaris, FITC, Evans 40x (1910)

Pemphigus vulgaris, anti-IgG, FITC, Evans 40x (1913)

Pemphigus vulgaris, anti-IgG, FITC, Evans 40x (1914)

Pemphigus vulgaris, anti-IgG, FITC, Evans 100x (1911)

Pemphigus vulgaris, anti-IgG, FITC, Evans 100x (1912)

Net-like positivity of the surface of keratinocytes, esophagus, guinea pig, pemphigus vegetans:
Pemphigus vegetans, indirect IF, FITC 10x (3676)

Pemphigus vegetans, indirect IF, FITC 20x (3677)

Pemphigus vegetans, indirect IF, FITC 20x (3678)

Pemphigus vegetans, indirect IF, FITC 40x (3679)

Pemphigus vegetans, indirect IF, FITC 40x (3680)

Pemphigus vulgaris, low titre, where positivity is limited to the lower areas of the epidermis:
Pemphigus vulgaris, low titre, anti-IgG, FITC, Evans 40x (2005)

Pemphigus vulgaris, low titre, anti-IgG, FITC, Evans 40x (2006)

Pemphigus vulgaris, low titre, anti-IgG, FITC, Evans 40x (2007)

Pemphigus vulgaris:
Pemphigus vulgaris, FITC 10x (6026)

Pemphigus vulgaris, FITC 20x (6028)

Pemphigus vulgaris, FITC 40x (6027)

Pemphigus vulgaris, FITC 40x (6029)

Pemphigus foliaceus gives positivity in the upper third of the epidermis:
Pemphigus foliaceus, anti-ics, FITC 20x (11)

Pemphigus foliaceus (another case):
Pemphigus foliaceus, anti-IgG, FITC 20x (1974)

Pemphigus foliaceus, anti-IgG, FITC 40x (1975)

Pemphigus foliaceus, anti-IgG, FITC 40x (1976)

Pemphigus foliaceus (another case):
Pemphigus foliaceus developed, anti-ig, FITC 20x (2241)

Pemphigus foliaceus developed, anti-ig, FITC 40x (2242)

Pemphigus foliaceus developed, anti-ig, FITC 40x (2243)

Pemphigus erythematosus can show the lupus band in some cases:
m. Senear-Usher, FITC (12)

ICS antibodies are not found in Hailey-Hailey disease.

Cases after therapy (high doses of steroids and cytostatics) may show negative results.  Bullous pemphigoid (immunofluorescence)


IgG and C3 positivy in the area of basement membrane of the epidermis and adnexa and partially in the roof of the bulla. Morphologically similar epidermolysis bullosa acquisita shows autoantibody positivity rather in the bottom of the vesicle. In drug induced pemphigoids is IgG and C3 often positive in the basal layer of the epidermis. Herpes gestationis is similar to the bullous pemphigoid, but IgG in basement membrane area is rare (positive is C3 only).  Pemphigoid (herpes) gestationis (immunofluorescence)


Resembles bullous pemphigoid closely.  Dermatitis herpetiformis Duhring (immunofluorescence)


Specimens from the vicinity of the vesicles show IgA granules intrapapillary.

IgA linear dermatitis will show band-like IgA positivity in basement membrane area.  Lupus erythematosus (SLE, CDE a SCLE); (immunofluorescence)


Specimen taken from sun exposed areas are positive in 90% of cases. Lupus band with IgG (and c3, IgM, IgA and fibrin too) in the area of dermoepidermal junction is crucial for the diagnosis. The band can be homogenous, granular or even fibrillar. Similar positivity is found around adnexal basement membrane as well (sweat glands, follicles). In florid SLE is the lupus band present in clinically normal skin.

Rarely the deposits of anti-Ro (SSA) and anti-La (SSB) antibodies can be demonstrated as fine granules among the cells of the basal layer. Such positivity is found in the subacuet LE, systemic LE and Sjögren's syndrome.

In addition to the lupus band the aggregates, oval formations in subepidermal and periadnexal areas are being found. Rarely (in cases with basement membrane destruction in CDE and lichen ruber) such aggregates are intraepidermally located. Usually show IgM positivity, but sometimes react with IgG and c3 as well.

In cases of CDE there is usually fluorescence of the connective tissue in the upper corium. This sign alone is not a diagnostic sign, however.


Lupus erythematodes, IF aggregates:
LE, aggregates, FITC (19)

Lupus band is formed by immune complex aggreages and can be sometimes found in other diseases as well, especially in diseases affecting the face, which are accompanied by angiectasias (rosacea, scleroderma or even tinea faciei).

In addition, in systemic lupus erythematodes cytoplasmatic fluorescence of some keratinocytes is sometimes present.  Sharp's syndrome


In Sharp's syndrome (mixed disease of connective tissue) are epidermal nuclei often positive (similarly to the cases of systemic erythematodes with high levels of antinuclear antibodies).


Sharp's syndrome, nuclear fluorescence:
Sharp s syndrome, FITC (21)  Lichen ruber planus (immunofluorescence)


Immunofluorescence helps in diagnosing not fully developed cases, especially cases affecting mucous membranes only. Characteristic are lace shaped deposits with anti-fibrin conjugates in basement area location. Aggregates (Civatte bodies) are positive as well. Deposition of fibrin is enabled by massive basement membrane destruction.


Lichen ruber, basement membrane deposits:
Lichen ruber (lace-like anti-fibrin positivity), anti-fibrin, FITC (22)  Vasculitis, immunocomplex type (immunofluorescence)


Fresh biopsies will reveal granular deposits of IgG, C3, IgM and fibrin within vascular walls. In Schönlein-Henoch purpura there is vascular positivity for IgA.  Porphyria cutanea tarda (PCT) and hepatopathies (immunofluorescence)


Skin from sun exposed areas will show characteristic changes: lupus band in dermo-epidermal junction and dilatation of blood vessels with homogenous deposits, positive for IgG and fibrin. Such changes can be found in some other diseases: systemic lupus erythematodes, hepatopathy, severe diabetes. Most common is vascular positivity for fibrin and IgG. Sweat glands are positive as well in such cases.


Porphyria cutanea tarda, fibrin deposits in vascular walls:
PCT, blood vessels, FITC (24)

Porphyria cutanea tarda, fibrin deposits in vascular walls and sweat glands:
PCT, blood vessels and sweat glands, FITC (25)

4.3.2  Indirect immunofluorescence


Indirect immunofluorescence is used especially in diagnosing autoimmune diseases. The antibody from the serum of a patient reacts with substrate (animal or human tissues, cell cultures).

One of the most common tests in dermatology is detection of antibodies against nuclear antigens (ANA), epidermal antigens in vesicular diseases, agains endomysium and against the cytoplasm of neutrophils. The results can be influenced by selection of antigenic substrate.  Antinuclear antibodies (ANA)


are detected on frozen section or imprints of rodent livers of cellular line HEp-2. Fluorescence type and antibody titre are evaluated. Clinically significant are titres at least 1:40 or 1:160 (concentration under 1:40 can be found in healthy people in higher age or some chronic diseases).

Bullous diseases are detected using antigens from monkey esophagus or guinea pig mouth.  Endomysial antibodies


are detected on monkey esophagus or human umbilical cord.  Antibodies aganist neutrophilic cytoplasm (ANCA)


Standardized spreads of neutrophils, fixed by alcohol or formaldehyde, are most common sources of antigens. This method is sometimes less sensitive.


Homogenous ANA type, rat liver imprints:
Homogenous ANA, rat liver, indirect IF (26)

Homogenous type fo fluorescecne on the substrate of cell culture HEp-2.


Homogenous (+ peripheral) type ANA, cell culture HEp-2:
Homogenous ANA, cell culture, indirect IF (27)

Peripheral type ANA:
ANA, peripheral type, indirect IF (28)

Test with the substrate from Crithidium luciliae. This protozas have their flagella attached to the kinetoplast containing native dsDNA. Positive test therefore indicates presence of anti-dsDNA antibodies. This test is highly specific but less sensitive.


Anti-dsDNA antibodies, Crithidium luciliae, indirect IF:
Anti-dsDNA antibodies, indirect IF (29)

Coarse speckled immunofluorescence indicates antibodies against U1-snRNP antigene (typical of Sharp's syndrome) or against Sm proteins (indicates systemic lupus erythematodes). Similar is large speckled type (antibodies against ribonuclear proteins of nuclear matrix), positive in some cases of Sharp's syndrome and other rheumatic diseases. Fine speckled positivity is found eg. in presence of anti Ro (SS-A) and La (SS-B).


Sharp's syndrome:
Sharp s syndrome, indirect IF (30)

Nucleolar type of fluorescence of the serum, systemic scleroderma. Further subtypes of nucleolar fluorescence are associated with systemic scleroderma.


Systemic scleroderma, nucleolar type, indirect immunofluorescence:
Systemic scleroderma, nucleolar type, indirect IF (31)

Antibodies against centromeres are characteristic for about 60% cases of CREST syndrome.


CREST syndrom, antibodies against centromeres:
CREST syndrome, anti-centromere antibodies, indirect IF (32)

Few nuclear dots type antibodies appear as about 6 discrete dots in one nucleus. These antibodies are present in primary biliary cirrhosis and chronic active hepatitis. The antigen (p80-coilin) belongs to the group of small nuclear ribonucleoproteins and is associated with fibrillarin. Its function is unknown. Similar is multiple nuclear dots fluorescence (about 10 dots/nucleus), associated with Sjögren's syndrome, less common systemic lupus erythematosus and primary biliary cirrhosis.


ANA antibodies, few nuclear dots type:
Few nuclear dots ANA antibodies, indirect IF (33)

Antimitochondrial antibodies are characteristic of primary biliary cirrhosis; sometimes is present in people with systemic scleroderma or mixed cases. This antibodies are detected using HEp-2 or rat tissues (liver, kidney, stomach).


Antimitochondrial antibodies (AMA):
Antimitochondrial antibodies (AMA), indirect IF (34)

Detection of surface antigens of keratinocytes is less sensitive than direct immunofluorescence, nevertheless positive in about 90% of developed cases of pemphigus vulgaris. Antibodies are of IgG4 and IgG1 types. Net-like deposits are characteristic of antidesmosomal antibodies (desmoglein I. in pemphigus vulgaris and desmoglein III. in pemphigus foliaceus).


Pemphigus vulgaris, net-like positivity:
Pemphigus vulgaris, ICS antibodies positivity, indirect IF (35)

Bullous pemphigoid is characterized by antibodies against the basement membrane zone. Main antigens were characterized by immunoblotting as BP 230 and BP 180 (herpes gestationis BP 180). Correlation with the disease activity is less tight. In cicatrical pemphigoid (antibodies against laminin) indirect immunofluorescence usually fails.

Antibodies (IgA) against the endomysium are characteristic of dermatitis herpetiformis Duhring and celiac sprue.


Antibodies against endomysium:
Antiendomysial antibodies, indirect IF (39)

Antibodies against cytoplasmic antigens of neutrophils (ANCA) are important in diagnosing vasculitides and monitoring on activity of some vasculitides. In C-ANCA type granular and homogenous positivity of cytoplasm with minimal fluorescence of nuclear lobes is present. Antigene is proteinase 3. Positivity is found in Wegener's granulomatosis, Churg Strauss sydrome and microscopic polyarteritis.


C-ANCA antibodies against cytoplasmic antigens of neutrophils:
C-ANCA antibodies against cytoplasmatic antigenes of neutrophils, indirect IF (40)

P-ANCA type is characterized by fluorescence of the borders of nuclear lobes. Positivity is found in polyarteritis nodosa, ulcerous colitis, sclerotising cholangitis, Crohn's disease, some forms of uveitis, microscopic polyarteritis, systemic lupus erythematodes and rheumatoid arthritis. The antigen is myeloperoxidase.


P-ANCA antibodies:
P-ANCA antibodies, indirect IF (41)

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