Antineutrophil cytoplasmic antibody (ANCA)

What sample is needed for Antineutrophil cytoplasmic antibody (ANCA)?

1. The patient’s serum is needed.
   • How to Obtain a Good Serum Sample: Collect 3 to 5 mL of blood in a disposable syringe or a Vacutainer tube. Keep the syringe at 37 °C for 15 to 30 minutes and then centrifuge for 2 to 4 minutes to obtain a clear serum.
2. No special precaution is required.
3. Separate the serum as soon as possible and freeze it.
4. Slides from the blood are made, and the indirect immunofluorescent technique is performed for the neutrophils.
   1. Two distinct staining patterns were observed for two different antibodies.

What are the Indications for Antineutrophil cytoplasmic antibody (ANCA)?

1. For the diagnosis of Wegener’s granulomatosis.
2. This is also done to follow the course of the disease and monitor the response to treatment of Wegener’s granulomatosis.
3. This test may also be used to diagnose other autoimmune diseases.
4. This test is indicated in the case of vasculitis and inflammatory bowel disease.
5. This will help diagnose and classify various vasculitis-associated and autoimmune disorders.

What is the mechanism of damage by Antineutrophil cytoplasmic antibody (ANCA)?

1. This is also called an anti-cytoplasmic antibody. These are formed against the antigen in the cytoplasm of neutrophils and monocytes.
2. The majority of these are IgG types.
   1. ANCA activates neutrophils and is followed by the activation of the complement pathway.
   2. There is damage caused by vasculitis, accompanied by a chronic inflammatory response.
3. Regional vasculitis involves the small arteries of the kidneys, lungs, and upper respiratory tract.

4. The pathologic damage is caused by granulomatous inflammation.
5. These are autoantibodies against the myeloid-specific lysosomal enzyme.
6. ANCA is seen in autoimmune diseases with vasculitis, e.g., Necrotising vasculitis, active Wegener’s granulomatosis, Polyarteritis nodosa, and renal failure.
7. These are 85% to 100 % positive in Wegener’s granulomatosis.

What are the types of Antineutrophil Cytoplasmic Antibody (ANCA)?

• When patient sera are incubated with alcohol-fixed neutrophils, indirect immunofluorescence shows two patterns:

1. c-ANCA(cytoplasmic ANCA):
2. 1. It is highly specific, affecting 95% to 99% of Wegener’s granulomatosis, and is characterized by diffuse granular staining of the cytoplasm of neutrophils and monocytes (using a fluorescent antibody technique).
   2. Its sensitivity is greater than 90% in the systemic vasculitis phase.
   3. It is present in only about 65% of cases of respiratory disease.
   4. Not all patients with limited kidney disease in Wegner’s syndrome are positive for c-ANCA (i.e., negative c-ANCA).
   5. When Wegner’s is not active, the percentage drops to 30%.
   6. A negative result for c-ANCA does not rule out Wegener’s.
   7. False-positive results are rare.
   8. A rising titer for c-ANCA suggests relapse.
   9. When the titer is falling, it indicates successful treatment.

2. p-ANCA(perinuclear ANCA):
   1. It produces a perinuclear pattern of staining in the neutrophil’s cytoplasm.
   2. It is against various proteins like myeloperoxidase, elastase, and lysozyme.
   3. It is only seen in the alcohol fixation, and not in the formalin.
   4. A positive result is confirmed by ELIZA,
   5. This test has poor specificity and a sensitivity of 20% to 60% in autoimmune diseases.
   6. It is found in about 50% of patients with kidney disease.
   7. This is also positive (approximately 75%) in other autoimmune diseases, such as ulcerative colitis or sclerosing cholangitis.
   8. c-ANCA appears to have anti-proteinase 3 specificity, while p-ANCA has predominantly anti-myeloperoxidase activity.
   9. Result: Negative when there is very little fluorescence or no fluorescence.

Wegener’s granulomatosis (Granulomatous vasculitis)

What sample is needed for Wegener’s granulomatosis?

1. A biopsy of the affected tissue is taken, subjected to culture and special stains to exclude mycobacteria and fungal infections.
2. Advise anti-myeloperoxidase antibody.

How will you define Wegener’s Granulomatosis?

1. Wegener’s granulomatosis is a rare autoimmune systemic necrotizing or granulomatous vasculitis, mostly affecting the lungs and kidneys.
2. Wegner’s granulomatosis is defined as granulomatous inflammation containing foci of vasculitis.
3. It involves the nasopharynx, as well as a proliferative vascular lesion of the kidney.
4. A proliferative vascular lesion obliterates a focal area of the Bowman’s capsule of the kidneys (crescentic glomerulonephritis).
5. There is regional systemic vasculitis or fatal granulomatous vasculitis.
6. Small arteries in the kidneys, lungs, and upper respiratory system (including the nasopharynx) are involved.
   1. The damage is due to granulomatous inflammation.
   2. The renal lesion is focal segmental necrotizing glomerulonephritis.
7. It is a vasculitis involving small and medium-sized blood vessels in various organs.
   1. The common sites are the kidneys and the respiratory system.

What are the diagnostic criteria of Wegener’s Granulomatosis?

1. Necrotizing granulomas in the respiratory tract.
2. Generalized necrotizing arteritis.
3. Glomerulonephritis.

What are the signs and symptoms of Wegener’s Granulomatosis?

1. The patient has a fever and arthralgia.
2. Nosebleed, epistaxis.
   1. Stuffy nose, nasal ulceration, and crusty secretions.
   2. Rhinitis is the first symptom.
   3. There is sinus involvement.
3. The ear shows conductive hearing loss due to auditory dysfunction.
   1. There is otitis media.
4. The kidney shows glomerulitis.
   1. There is hypertension.
5. The oral cavity shows nonspecific mucosal ulceration.
   1. There is gingivitis.
   2. Bone destruction and loosening of the teeth.
6. The lungs show pulmonary nodules, resembling coin lesions.
   1. There is an infiltrate looking like pneumonia.
   2. There is a pulmonary hemorrhage leading to hemoptysis.
   3. Rarely is there bronchial stenosis.
7. The skin may exhibit a subcutaneous nodule, resembling a granuloma.
   1. There may be cutaneous vasculitis.
8. CNS may show sensory neuropathy.
   1. Multiple cranial nerve involvement.
9. The eye shows Inflammation like:
   1. Scleritis and conjunctivitis are common.
   2. There is proptosis or ptosis.
   3. There are orbital granulomas.
   4. There is a corneal ulcer.
   5. There may be scleritis or episcleritis.
   6. There may be uveitis.
   7. There may be retinitis.
10. Damage to the heart, lungs, and kidneys may be very severe.
    1. Usually, involvement of the heart, central nervous system (CNS), and gastrointestinal (GI) tract is rare.
11. Histologically, there are poorly formed granulomas, characterized by the presence of necrosis and numerous multinucleated giant cells.
    1. It is believed that ANCA antibodies are responsible for inflammation.

How will you diagnose Wegener’s Granulomatosis?

1. Serological method for detecting ANCA.
2. c-ANCA:
   1. Detection of c-ANCA is sensitive and specific for diagnosing Wegener’s disease.
   2. Biopsy of the affected area using the fluorescent method reveals antibody localization in the neutrophil cytoplasm, with a granular appearance throughout the cytoplasm (c-ANCA).
   3. c-ANCA is reported to be 90% to 95% positive in Wegner’s granulomatosis (range 84% to 100%).
   4. In inactive Wegner’s disease, the positivity rate is 30% (range: 13% to 41%).
   5. In cases involving only the respiratory system, the rate is 65% (range: 60% to 85%).
3. p-ANCA reacts with myeloperoxidase and is detected in 50% to 80% of cases of localized Wegener’s granulomatosis involving the kidneys.
   1. p-ANCA is seen in other diseases as well, like;
      1. Ulcerative colitis (in approximately 75% of cases).
      2. Sclerosing cholangitis also shows 75% positivity.
      3. Autoimmune hepatitis shows 50% positivity.
      4. HCV and HBV in active diseases show 5% to 7% of the cases.
      5. Crohn’s disease shows an 8% positivity rate.
4. A biopsy is more diagnostic.
5. There is hematuria, and proteinuria.
6. Urine culture is negative.
7. WBC count and ESR are raised in active disease.

What are the normal ANCA levels?

• Serological method
  • Not normally found in the serum.
  • Negative = <1:20
• ANCA by EIA =
  • Negtive    =     <21 units
  • Weak positive  =  21 to 30 units
  • Positive =>30 units.

Source 4

• Tissue biopsy
  • Negative for ANCA by IFA.
  • In positive c-ANCA, then the results are titrated.
  • In positive p-ACNA, MPO (myeloperoxidase) testing is performed by ELISA.
  • Not all p-ANCA cases are positive for MPO.

Source 2 

• Blood = Negative
  • ANCA is also found in ulcerative colitis and inflammatory bowel disease.
  • Also positive in Crohn’s disease, specifically p-ANCA.

What are the causes of increased Antineutrophil cytoplasmic antibody (ANCA)?

1. Wegener’s granulomatosis (with a positive predictive value of over 90%).
2. Polyarteritis nodosa
3. Necrotizing vasculitis.
4. Inflammatory bowel disease.
5. Systemic lupus erythematosus.
6. Rheumatoid arthritis.

What is the specificity of p-ANCA, and where are these seen?

1. Polyarteritis nodosum.
2. Primary sclerosing cholangitis.
3. Microscopic polyangiitis.
4. Ulcerative colitis.

Questions and answers: