Other Antibodies

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ANCA: Autoantibodies in Vasculitis

B.M, Canada "I wonder if you would give me permission to use the image of p-ANCA that you have posted on your website as an illustration for our poster" 

Neutrophil Cytoplasmic Antibodies

Anti-neutrophil cytoplasm antibodies (ANCA) should be routinely tested during investigation for possible small vessel vasculitis or rapidly progressive glomerulonephritis. Two typical patterns have been described using indirect immunofluorescence (IIF) on ethanol-fixed healthy neutrophils; the cytoplasmic (cANCA) and perinuclear (pANCA) patterns. 

The table below shows that although ANCA can be detected in a number of inflammatory diseases (including bacterial endocarditis which can also be associated with a vasculitic glomerulonephritis), the best defined clinical association is with the small vessel vasculitides (Wegener’s granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome). In these diseases the pANCA pattern is usually an anti-myeloperoxidase (MPO) specific autoantibody and the cANCA pattern an anti-proteinase 3 (PR3) antibody.

ANCA
Disease  C-ANCA P-ANCA  Atypical ANCA 
Wegener's Granulomatosis 90% 10% -
Microscopic Polyangiitis 10%  90%
Churg-Strauss Syndrome  + + -
Polyarteritis nodosa   - + -
Idiopathic Crescentic glomerulonephritis + -
SLE  + +
Rheumatoid Arthritis  + +
Ulcerative Colitis - +
Crohn's Disease  - +
Primary Sclerosing cholangitis - +
Autoimmune Hepatitis  - +
Primary Biliary Cirrhosis  - +


The presence of cANCA or pANCA by IIF in patients with a high clinical index of suspicion is very sensitive for the diagnosis of vasculitis, however it is not very specific. When positive IIF is combined with a positive anti-MPO or anti-PR3 ELISA the specificity for the diagnosis increases to nearly 100% (although the sensitivity may be only 50-88%).

In patients with a diagnosis of ANCA-associated vasculitis the ANCA should be routinely monitored during follow-up. There is evidence that in a proportion of patients the ANCA titre reflects disease activity and that a rising ANCA titre may herald disease relapse.

ANCA with an atypical staining pattern that is not specific for MPO or PR3 may be detected in a number of other inflammatory diseases. In inflammatory bowel disease the presence of pANCA may help differentiate Crohn’s disease from ulcerative colitis: for the latter, it may be associated with a specific phenotype. There is no evidence that pANCA titres correlate with disease activity in inflammatory bowel disease. (Dr. M Morgan, UHB)

When should ANCA be requested?

ANCA are both specific and sensitive for the list of vasculitidies in table 1. They are performed on an urgent basis for patients with suspected vasculitis and also on a routine basis for follow up of diagnosed patients.

Initially screening is performed on ethanol-fixed neutrophils where 1:25 diluted patient serum is added followed by fluorescent anti-IgG. If staining is seen then an ELISA is performed which determines the quantitative level of myeloperoxidase (MPO; dominant P-ANCA autoantigen) and proteinase-3 (PR-3; dominant C-ANCA autoantigen).

Antineutrophil antibodies

There are a number of antigens which can be recognised by ANCA antibodies. Two most commonly occurring include; one found in the cytoplasm (proteinase 3 (PR3)), which produces a pattern referred to as cANCA, and the other is myeloperoxidase (MPO) which can be seen as perinuclear (pANCA) staining on neutrophil.

ANCA

Cytoplasmic (cANCA)


Granular cytoplasmic staining

Clinical:
 
Primary vasculitis
Wegener's granulomatosis
Microscopic polyangitis
Churg-Strauss syndrome
Polyarteritis

Perinuclear (pANCA)


Homogenous perinuclear staining 

Clinical:  
Primary vasculitis
Microscopic polyangitis (MPO)
Churg-Strauss syndrome (MPO)
Polyarteritis nodosa (MPO)

Collagenosis
Felty's syndrome (lactoferrin)
Systemic lupus erythematosus (lactoferrin)
Rheumatoid arthritis (lMPO/lactoferrin)
Sjögren's syndrome (MPO/lactoferrin)

Chronic inflammatory bowel disease
Ulcerative colitis (cathepsin G/elastase)
Crohn's disease (elastase)

Chronic liver disease
Primary sclerosing cholangitis (cathepsin G)
Antigen: PR3 (90%), MPO (5%), & BPI (4%)* Antigen: MPO (70%), lactoferrin (10), elastase (8%), cathepsin G (5%) & PR3 (2%)

* BPI = Bacterial/permeability Increasing Protein
Reference: Protein Reference Unit: Handbook of Autoimmunity, 3rd edition, 2004.

dsDNA antibodies (Crithidia Luciliae) in SLE

Crithidia luciliae consist of a giant mitochondrion (kinetoplast) containing pure DNA which is not associated with histones. This can be used for detecting double stranded DNA (dsDNA) by indirect immunofluorescence.
Positive dsDNA Normal serum
Staining of kinetoplast lying near the base of flagellum. Negative staining of both kinetoplast and the nucleus.
Ethidium bromide (red) added to show the nuclei of crithidia. Staining of basal body can be mistaken for kinetoplast.
Antigen Deoxyribonucleic acid (DNA) may be either double stranded (dsDNA) or single stranded (ssDNA)
Clinical SLE with specificity of 95% and lower sensitivity of 70%

Note: The problem with ELISAs is that they detect both low and high avidity dsDNA antibody (some ELISAs will also detect single stranded DNA). High avidity anti-dsDNA antibodies are specific for SLE associated nephritis which is successfully detected by crithidia and FARR assay. The disparity between ELISA and crithidia may reflect other clinical conditions

Endocrine antibodies

Islet cell antibodies - Diabetes Mellitus


Antigens Primate pancreas showing staining of the islet cells of Langerhans. Pancreas produces glucagon (ά-cells), insulin and GAD65 (ß-cells) and somatostatin (δ-cells). All these can be targeted by ICA.

Clinical Insulin Diabetes Mellitus (type I) and autoimmune polyglandular disease. Antibody presentation may occur years prior to clinical symptoms and may disappear after the onset of diabetes. 

Antibodies to steroid producing cells

The main target antigen in these cells is cytochrome P450 enzymes (steroid dehydrogenases) which are involved in steroid hormone production. The table below shows the distribution of antigens in the steroid producing cells of the adrenal, the testis and the ovary.  Except for P450s21 all other antigens appear in all three tissues and therefore can be dectected throughtout the compositie section.

Endocrine antigens
 
.
Adrenal   Testis  Ovary
 Glomerulosa Fasciculata     
 P450s17

 P450scc

+

+

+

+

 P450s21

+

+

+

 -

Adrenal antibodies - Addison's disease

Image: The adrenal antibody is direct to the cell cytoplasm of the granulosa and fasciculata layer.

Antigens: Cytochrome P450c17 (~57KDa), P450scc (~60KDa) and P450c21 (~56KDa). These antigens are also common to the ovary and the testis.

Disease: Rare autoimmune disease leading to adrenocorticol deficiency. Positive in 60-70% of patients with Addison's disease. Also found in cases of ovarian failure and autoimmune polyglandular disease.

Ovarian antibodies - Ovarian failure

Antigens: Primate ovary showing the location of antigens (cytochrome P450c17 (~57KDa) and P450scc (~60KDa) in the theca interna cell layer around the follicle.

Disease: Autoimmune ovarian failure

Human IgG staining primary follicles. Not commonly seen. Significance of this antibody is known. This antibody also showed specificity for both sperm head and tail.

Antibodies to testis - Autoimmune polyglandular disease

Antigens: Antibody is directed against steroid producing (Leydig) cells that are found between the seminiferous tubules and target cytochrome P450c17 (~57KDa) and P450scc (~60KDa).

Disease: Autoimmune polyglandular disease

Sperm tail Antibodies

Mature primate testis: Antibody against sperm tail. Significance unknown.

Gastric Parietal Cells - Pernicious anaemia

 Antigen: H+K+-ATPase located in the gastric parietal cells of rodent stomach.
Clinical Antibody present in more than 90% of patients with Pernicious anaemia. Autoimmune gastritis leads to pernicious anaemia which is characterised by antibodies to GPC and intrinsic factor.

J.H.L, France "While looking on the net for a good antibody against snake' H+K+-ATPase, I found a very nice picture of your H+K+-ATPase staining located in the gastric parietal cells of rodent stomach"