Antineutrophil Cytoplasmic Antibodies (ANCA) Test

Pathophysiology

Neutrophils are the most abundant leukocyte type, comprising 50% to 75% of the white blood cell population. About 1800 to 8000 neutrophils are normally present per microliter of blood; their number can increase 10-fold under infectious conditions. Neutrophils contain 2 main types of granules: 1) peroxidase-positive (azurophilic), and 2) peroxidase-negative. Both ANCA-myeloperoxidase (MPO) and ANCA-proteinase 3 (PR3) are enzymes present in peroxidase-positive granules, and MPO is the most prevalent granular protein. PR3 is important for serving as a hematopoietic regulator and has apoptosis-inducing capabilities. Some less specific ANCA antigens include cathepsin G, lactoferrin, elastase, defensin, α-enolase, moesin, leukocidin, bactericidal-permeability-increasing protein, and lysosome-associated membrane glycoprotein 2. The pathogenicity of these antigens is thought to be low.[3]

ANCA serology is positive in about 90% of GPA and MPO cases, while it is positive in about 30% of eosinophilic granulomatosis with polyangiitis cases.[4] Anti-PR3 antibodies are about 90% sensitive for GPA.[5][6] Although sensitive and specific for GPA, ANCA positivity has also been noted in 15% to 20% of patients with systemic lupus erythematosus, especially those with lupus nephritis.[7] C-ANCA (a cytoplasmic antineutrophilic nuclear antibody with extranuclear cytoplasmic staining) is 90% PR3 reactive and 10% MPO reactive; P-ANCA (a perinuclear antineutrophilic nuclear antibody with staining around the nucleus) is 90% MPO reactive and 10% PR3 reactive.[8] Small vessel damage caused by ANCA occurs when these autoantibodies bind to granules in neutrophils. The mechanism of pathogenesis is given below:

• Neutrophils and monocytes are activated by ANCA.
• The alternative complement pathway is subsequently activated.
• Neutrophils migrate through blood vessel walls, and inflammatory mediators are released.
• Chemoattractants are released, and more inflammatory mediators are recruited.
• Inflammation, apoptosis, necrosis, and damage to small blood vessel walls occur.[9][10]

Drugs Linked to ANCA–Associated Vasculitis

Medications can induce an ANCA-associated vasculitis, usually manifesting as rapidly progressive glomerulonephritis. Specifically, a high MPO titer is common. Levamisole, which is often found in contaminated cocaine, can cause the elevation of both anti-MPO and anti-PR3 antibodies, along with other autoantibodies, skin lesions, and arthralgias. Treatment is the same as other ANCA-associated rapidly progressive glomerulonephritis cases—possibly with shorter induction. The most common offending medications include these:

• Hydralazine
• Propylthiouracil and methimazole
• Allopurinol
• Sulfasalazine
• Minocycline
• Penicillamine
• Rifampicin
• Aminoguanidine
• Sofosbuvir
• Anti-tumor necrosis factor-alpha therapy for rheumatoid arthritis and ankylosing spondylitis [11]

Specimen Requirements and Procedure

A blood sample is taken from a peripheral venous draw. A healthcare professional puts a rubber band around the arm and asks the patient to keep pumping the fist for some time, then palpates the vein for confirmation. An alcohol swab is applied to clean the area. After the alcohol has dried, a needle is introduced into the vein and a blood sample is collected in a test tube or a vial. After collecting the sample, the band is removed, the pressure is applied over the puncture site, hemostasis is secured, and a bandage is applied. Complications of the procedure include mild tenderness, bruising, oozing of blood, or infection at the puncture site.

Testing Procedures

After taking a blood sample, the specimen undergoes analysis by 2 techniques:

• Indirect immunofluorescence (IIF)
• Enzyme-linked immunosorbent assay (ELISA) [12]

The indirect immunofluorescence identifies ANCA by its staining patterns inside the cytoplasm of neutrophils.[8] In this technique, neutrophils are fixed via ethanol onto a slide; the blood sample is then mixed on the slide with the neutrophils; if present, ANCA in the sample attaches to neutrophils on the slide. The slide is then treated with fluorochrome-stained antibodies, which react with ANCA on the slide; this forms a fluorescence pattern that is visible via a microscope. As mentioned earlier, if ANCA staining is throughout the cytoplasm, it is called a C-ANCA pattern; this is usually PR3-ANCA. If staining is only around the nucleus, it is called P-ANCA (perinuclear pattern), which usually occurs with MPO-ANCA.[8] On the other hand, ELISA measures the level of MPO-ANCA or PR3-ANCA patterns in an antigen-specific pattern in a blood sample. 

Testing takes place in 2 steps. First, IIF is done, which detects the presence or absence of these autoantibodies; if the test is positive, then it is followed by ELISA.[13] The International Consensus Statement on Testing and Reporting ANCA recommends using both techniques to detect ANCA in suspected patients.[14] However, the more specific ELISA antigen testing is largely replacing the use of IIF.

Interfering Factors

ANCA testing helps diagnose and monitor small vessel vasculitis, but it has some limitations, too. There is a variant of ANCA called atypical ANCA, which is associated with drug-induced ANCA-associated vasculitis, inflammatory bowel disease, and rheumatoid arthritis.[15][16] ANCA-positive rates are also higher in type 1 diabetes mellitus than in healthy individuals.[17]

Results, Reporting, and Critical Findings

If there is a negative IIF result, ie, ANCA is not detected, the findings are normal. However, a positive IIF result occurs when ANCA is detected; therefore, in this case, the findings are abnormal (the fluorescence pattern is also described to show whether C-ANCA or P-ANCA is present).

MPO Antibody (P-ANCA) Levels

• >5.0 IU/mL = Positive
• 3.5-5 IU/mL = Equivocal
• <3.5 IU/mL = Negative

PR3 Antibody (C-ANCA) Levels

• >3.0 IU/mL = Positive
• 2-3.0 IU/mL = Equivocal
• <2.0 IU/mL = Negative

Interpretation

Positive results for C-ANCA are mostly consistent with granulomatosis with polyangiitis. Whereas positive results with P-ANCA mostly occur in microscopic polyangiitis and eosinophilic granulomatosis with polyangiitis (or Churg-Strauss syndrome). Monitoring the titers of ANCAs is useful in monitoring the clinical course of diseases.

Clinical Significance

Positive ANCA lab results with clinical suspicion aid in diagnosing some small vessel vasculitis, including pauci-immune small vessel vasculitides granulomatosis with polyangiitis, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis and anti-glomerular basement disease.[1][2] The reappearance of ANCA after the treatment of vasculitis may indicate a relapsing disease.[18][19]

Enhancing Healthcare Team Outcomes

Care coordination is pivotal in ensuring seamless and efficient patient care. Physicians, advanced clinicians, nurses, pharmacists, and other healthcare professionals must work together to streamline the patient's journey, from diagnosis through treatment and follow-up. This coordination minimizes errors, reduces delays, and enhances patient safety, ultimately leading to improved outcomes and patient-centered care.