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Indirect Immunofluorescence

Indirect Immunofluorescence is a widely used technique for the detection of autoantibodies in dilutions of patient serum. A broad range of antibodies can be identified that are relevant to a variety of clinical disciplines including rheumatology, dermatology and gastroenterology.

Although some training is required for interpretation of results the technique is quick, simple to perform, highly reproducible and often the most cost effective method of autoantibody detection for clinical laboratories. Other advantages include the ability to test for multiple antibodies simultaneously. The technique involves the incubation of a diluted serum on a tissue substrate. Substrates originate from primate and rodent organs which are cut in ultra-thin sections using cryotometry and applied to a glass microscope slide. Autoantibodies in patient serum bind to proteins on the substrate section (Fig.1). The substrate is then washed to remove un-bound antibody. A second incubation is then performed using a fluorescein labelled anti-human globulin conjugate (Fig.2). This stage is followed by a further wash. The slide is then mounted and can be examined using a fluorescence microscope (Fig.3).

Fig. 1,2,3

Positivity is indicated by the presence of fluorescence, the intensity of which can be expressed qualitatively or as a serial end point dilution. Where international standards are available such as the World Health Organisation anti-nuclear antibody standard, international units can be assigned through serial dilution.

Clinical application

Rodent tissue substrates

Immunofluorescence is the primary method for detection of anti-nuclear antibodies (ANA). IgG ANAs are observed in the connective tissue diseases. Levels of 3 iu/ml and above are seen in rheumatoid arthritis, Sjogren’s syndrome, SLE, mixed connective tissue disease, scleroderma and systemic sclerosis. The substrate of choice is usually rat or mouse liver which provide similar sensitivities.  Speckled ANAs are observed in Sjogren’s syndrome and mixed connective tissue disease. Homogenous ANA patterns are seen in SLE and DLE (Fig 4). Nucleolar ANAs are seen in scleroderma and systemic sclerosis. Perinuclear ANAs are seen in rheumatoid arthritis. ANAs have a long half life, monthly testing is therefore adequate.

 Fig 4.

The alternative substrate for ANA investigation is Hep-2 cells. Hep-2 cells have large nuclei and therefore can indicate patterns represented by the extractable nuclear antigens (ENAs). This substrate is much more expensive, requires significant training to interpret and can pick up false positives in healthy individuals.

Gastric parietal cell antibodies (GPC) are detected on rodent stomach tissue (Fig 5). Mouse and rat are both used, however mouse is preferable since it less prone to interference from heterophile antibodies. This tissue has very high sensitivity for early stage pernicious anaemia. Intrinsic factor can be used as a follow up test.

Fig 5.

Anti-mitochondria antibodies (AMAs) are observed on rat or mouse kidney sections (Fig 6). Cytoplasmic speckling can also be seen in liver. The antibodies are important in the investigation of liver disease such as chronic active hepatitis, autoimmune hepatitis and PBC. Mitochondria antibodies can be detected in patient serum years before symptoms manifest. The assay is 95% specific for the disease. In PBC the mitochondria antigen is pyruvate dehydrogenase. A simple ELISA assay can be performed to confirm the presence of M2 antibodies that are relevant to PBC.

Fig 6.

Other mitochondrial antibodies can also be viewed by immunofluorescence. The clinical significance of these antibodies is described in Table 1.

 Table 1. The mitochondrial antibodies.

Mitochondrial autoantibody

Clinical significance

M1

Syphilis

M2

PBC

M3

Lupus like disease

M5

Connective tissue diseases

M6

Drug induced hepatitis

M7

Myocarditis and cardiomyopathy

 

Type 4 hepatitis and chronic active hepatitis (CAH) patients have circulating smooth muscle antibodies (SMA). 70% of CAH patients will exhibit SMA at greater than 1:40 titre. SMA can also be used to exclude SLE since these antibodies are not prevalent in this disease. SMAs have also been found in patients with malignant disease such as melanoma and ovarian carcinoma. The muscularis mucosa on the stomach of rodent tissues shows positive fluorescence as do the F-actin filaments. On other tissues such as liver and kidney the blood vessels show positivity (Fig 7).

Fig 7.

Liver-kidney microsomal antibodies (LKM) bind to both cytochrome P450 in hepatocytes (Fig 8) and proximal renal tubes in rodent tissue (Fig 9). Three types have been characterised. LKM-1 is associated with 2a and 2b autoimmune chronic active hepatitis. LKM-2 and LKM-3 are associated with drug induced hepatitis and hepatitis-δ respectively.

Fig. 8 Fig. 9

Keratin antibodies (KAB) are detectable in the soft mucosal keratin lining of rat oesophagus (Fig 10). These antibodies are the most specific known for rheumatoid arthritis. They often appear very early in the disease process therefore allowing for early treatment to be employed.

 Fig 10.

The assays described so far are performed on rodent tissues and are therefore inexpensive. Further cost effectiveness is gained by testing samples on multiple tissue sections. A multiple tissue block allows for ANA, SMA, AMA, GPC, LKM and KABs to be investigated on each patient simultaneously.

 

Primate tissue substrates

Human and primate antigens show a high level of heterogeneity. Monkey tissues are therefore very useful substrates for autoantibody detection including both organ specific and non-organ specific autoantibodies. The following section describes some of the observed immunofluorescence patterns for primate derived substrates and the diseases associated with these patterns.

Immunofluorescence on monkey oesophagus is the principle method of IgA endomysial antibody detection for coeliac disease diagnosis. This assay is 100% sensitive and highly specific for the disease. In addition 60-70% of patients with dermatitis herpetiformis. Antibodies bind to the reticulin component of the endomysium in the oesophageal smooth muscle (Fig 11). This assay is of particular importance to the paediatrician since it negates invasive methods of coeliac diagnosis.

Fig 11.

Monkey oesophagus substrate is also used to diagnose the dermatological conditions bullous pemphigoid and pemphigus. Bullous pemphigoid is characterised by itch blisters on the limbs and trunk. IgG antibodies locate at the dermo-epidermal junction (Fig 12). In pemphigus vulgaris and foliaceus keratinocyte adhesion proteins adhere an IgG autoantibody producing a chicken wire pattern (Fig 13). End-point titres are useful for management of both clinical conditions.

Fig. 12 Fig. 13

Many other primate organs are useful for autoimmune diagnosis including pancreas islet cell for the investigation of type 1 diabetes (Fig 14), kidney for the detection of glomerular basement membrane antibodies in Goodpasture’s syndrome (Fig 15), adrenal gland for adrenal antibodies found in Addison’s disease (Fig 16), cardiac antibodies in Dressler syndrome (Fig 17) and ovarian antibodies during autoimmune ovarian failure.

Fig. 14

Fig. 15

Fig. 16

Fig. 17

 

Equipment and Protocol

 

Table 2. Equipment and reagents required for indirect immunofluorescence. 

 Material / Reagent

Details

Substrate coated glass microscope slide

Available with different well counts (4-10) and with multiple tissues.

Phosphate buffered saline

Used for dilution of samples and conjugates.

IgG and IgA F(ab)2  FITC conjugate

 

Rhodamine counterstain

Not essential but reduces background staining and allows for tissue architecture to be observed

Glass slide rack

Separates slides during washing process

Glycerol

Slide mounting buffer

Glass cover slips

Covered slides can be kept at 4°C for several months.

Tissue

Drying excess solutions from slide

Pipettes and disposable tips

200-1000ml, 30-200ml and 5-40ml are the most useful

Mixer and magnetic flea

Improves washing incubations

Fluorescence microscope and mercury bulb

 

 

Slides purchased from Immunotec are fixed and nitrogen flushed to increase the shelf life of the tissue section. Remove slides from foil pouch and either leave to dry on the bench for five minutes or dry with a hair dryer on a low heat.  Dilute patient samples and controls using phosphate buffered saline (PBS). The pH of the diluent should be checked prior to dilution and should lie between 7.3 and 7.4. Appropriate dilutions for assays are shown in Table 3.

Table 3. Assay dilutions.

Assay

Dilution (PBS)

ANA

Screen at 1:40 – Titre Strong Positives

GPC

1:10

SMA

Screen at 1:10 – Titre from 1:40

MITO

Screen at 1:10 – Titre Positives

Keratin

1:10

EMA

1:5

Skin antibodies (Basement membrane / keratinocytes)

Titre from 1:10

Islet cell antibodies

1:3

Glomerular basement membrane antibodies

1:10

Adrenal antibodies

1:3

Heart antibodies

Titre from 1:10

Ovarian antibodies

1:3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Apply each serum dilution and control to a well on the slide and incubate at room temperature for 30 minutes. Following the first incubation the slides are rinsed with PBS. Take care not to flush PBS directly on the tissue sections. The slides are then placed into a rack and immersed in PBS for 30 minutes. The washing stage is enhanced by using a mixer and magnetic flea rotating at a slow speed. The slides are then removed and shaken dry and carefully wiped with tissue to remove residual PBS from the slide. This prevents waste when adding conjugate. Care must be taken to avoid smearing the tissue on the slide. One drop of conjugate is applied to each well (50ml) and incubated at room temperature for a further 30 minutes. The conjugate solution constituents are shown in Table 4.

Table 4. Conjugate constituents

Assay

Diluent

Conjugate

Counterstain

EMA

4 ml PBS

70 ml IgA (F’ab2) FITC (DAKO F0204)

5 ml Rhodamine (Beckton Dickinson 223401)

All other tissues

4 ml PBS

70 ml IgG (F’ab2) FITC (DAKO F0315)

5 ml Rhodamine (Beckton Dickinson 223401)

 

 

 

 

 

Conjugates should be stored at 4°C and away from light. Stored conjugates can be used up to 6 months after preparation. Following the conjugate incubation the slides are washed in PBS for 30 minutes as before. The slide is then ready to mount. The mounting buffer is glycerol. 9 ml glycerol is mixed with 1 ml PBS. A 4cm smear is applied to a cover slip which is placed on tissue to absorb any excess. Slides are shaken dry to remove excess liquid and placed face down on the cover slip. Turn the slide over and carefully blot with tissue or filter paper to remove air pockets and excess mounting buffer. The slide is then ready to read or can be stored at 4°C and read at a later date.

Table 5. Immunotec Products

Product

Product Code

Monkey Oesophagus 10  well slide

APO10

Monkey Oesophagus 5  well slide

APO5

Rat Liver Kidney Stomach 10 well slide

RLKS10

Rat Liver Kidney Stomach 5 well slide

RLKS5

Mouse Liver Kidney Stomach 10 well slide

MLKS10

Mouse Liver Kidney Stomach 10 well slide

MLKS10

Rat Liver Kidney 10 well slide

RLK10

Monkey Pancreas 4 well slide

PP4

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