Fluorescent Staining Patterns

The detection of specific antibodies is important for the diagnosis of many autoimmune diseases. Indirect immunofluorescence (IIF) allows for the visualization of antibodies or immunoglobulins (Ig) as they adhere to certain immobilized antigens. The IIF method allows antibodies in human serum to bind to substrates that serve as proxies for human autoantigens. Unbound Ig and other serum components are washed off. A secondary antibody, specific for a class of human Ig and tagged with a fluorophore, is applied and allowed to bind. A second wash removes all unbound secondary antibodies leaving behind bound Ig that has been conjugated to a tag which will fluoresce. The specificity of the primary Ig is determined by the type of substrate used and the pattern of binding observed and informs the potential associated autoimmune disease.

Below are images, descriptions and clinical relevance of some informative IIF patterns.

Anti Mitochondrial Antibodies

show granular cytoplasmic staining of Kupffer cells and hepatocytes in the liver, renal tubular cells (distal tubules more than proximal tubules) in the kidney, and gastric cells (parietal cells more than chief cells) in the stomach.

Clinical Relevance: Serum AMAs are found at high titers in 95% of patients with primary biliary cirrhosis (PBC) which results in damage to the bile duct system of the liver. PBC is also called primary biliary cholangitis. The AMA tests are used to help diagnose and/or rule out other causes of liver disease or injury. The stated associations are only correlative and should not be the sole basis for diagnosis.

Parietal Cell Antibodies

show a bright green staining of the parietal cells as parallel lines in the gastric mucosa of stomach tissue. Kidney and liver tissues are negative. PCAs are also known as gastric parietal cell (GPC) antibodies, AGPA, and APCA.

Clinical Relevance: Serum PCAs are detected in about 90% of patients with pernicious anemia (PA) but can be found in people with other autoimmune diseases or gastritis or thyroiditis. This test in combination intrinsic factor antibody (IFA) helps evaluate patients with suspected PA. The stated associations are only correlative and should not be the sole basis for diagnosis.

Smooth Muscle Antibodies

stain the muscularis propria and the intergastric gland area in the stomach. They react with the muscle layers of arteries in all tissues. High titer antibodies may cross react with tubulin in kidney glomeruli.

Clinical Relevance: High levels of SMA and/or ANA are usually due to autoimmune hepatitis type 1. Low titers of SMA have been associated with primary biliary cholangitis (PBC), chronic viral hepatitis, alcoholic chronic hepatitis, some cancers and are found in 5% of healthy individuals. A negative SMA and an increased titer of LKM antibodies may indicate autoimmune hepatitis type 2. The stated associations are only correlative and should not be the sole basis for diagnosis.

Liver Kidney Microsomal

antibodies show a bright homogenous staining of the hepatocytes. Renal proximal tubules are also stained but not the distal tubules or any stomach tissue. Staining by both LKM and PCA can be distinguished from AMA by unstained distal renal tubules and smooth staining of the liver.

Clinical Relevance: Serum anti-LKM antibodies are detected in patients with acute and chronic liver diseases. The antibodies are most commonly associated with autoimmune hepatitis (AIH) Type 2 (80% prevalence) and typically occur in the absence of SMA and ANA. AIH Type 2 is found in Western European children but is rare in the United States. Associations are also found with chronic hepatitis C and drug-induced hepatitis. The stated associations are only correlative and should not be the sole basis for diagnosis.

Anti Endomysial Antibodies

stain a wispy network (endomysium) around smooth muscle cells mainly in the muscularis mucosa (MM). Possible staining of the submucosa (S) should be considered an AEmA negative. SMA staining of the muscularis mucosa (MM) and externa (ME) can mask an EmA positive.

Clinical Relevance: The finding of IgA-endomysial antibodies (EmA) is highly specific for celiac disease (100%) or dermatitis herpetiformis (70%). The titer generally correlates with the severity of the gluten-sensitive enteropathy. In patients with IgA deficiency, assessment of IgG-EmA is recommended. Anti-skin antibodies (ASA) can also be detected on the primate esophagus, showing patterns in the epithelium (E). The stated associations are only correlative and should not be the sole basis for diagnosis.