Antineutrophil cytoplasmic antibody (ANCA), Wegener’s Granulomatosis

Sample

1. The patient serum is needed.
   • How to get good serum: Take 3 to 5 ml of blood in the disposable syringe or a vacutainer. Keep the syringe for 15 to 30 minutes at 37 °C and then centrifuge for 2 to 4 minutes to get the clear serum.
   • No special precaution is required.
   • Separate serum as soon as possible and freeze it.
2. Slides from the blood are made and perform the indirect immunofluorescent technique for the neutrophils.
   1. Two types of staining patterns were seen for two different antibodies.

Purpose of the test (Indications)

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 be done for other autoimmune diseases.
4. This test is indicated in the case of vasculitis and inflammatory bowel disease.
5. This will help in the diagnosis and classification of various vasculitis-associated and autoimmune disorders.

Pathophysiology

1. This is also called an anti-cytoplasmic antibody. These are formed against the antigen in the cytoplasm of the neutrophils and also of the 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 by vasculitis and accompanied by a chronic inflammatory response.
3. There is regional vasculitis involving the small arteries of kidneys, lungs, and upper respiratory tract.
   

   ANCA mechanism for damage

4. The pathologic damage is 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.

Types of ANCA

1. When patient sera are incubated with alcohol-fixed neutrophils, indirect immunofluorescence shows two patterns:
2. c-ANCA (cytoplasmic ANCA) is highly specific 95 to 99% of Wegener’s granulomatosis, and there is diffuse granular staining of the cytoplasm of neutrophils and monocytes (by fluorescent antibody technique ).
   1. In respiratory disease, it is present in only about 65 % of the cases.
   2. All Wegner’s patients with limited disease in the kidney are not positive for c-ANCA.
   3. When Wegner’s is not active, then % drops to 30%.
   4. A negative result for c-ANCA does not rule out Wegener’s.
   5. False-positive results are rare.
   6. A rising titer for c-ANCA suggests relapse.
      1. When titer is falling indicates successful treatment.
         

         ANCA (autoantibodies) formation

3. p-ANCA (perinuclear ANCA)  produces a perinuclear pattern of staining in the neutrophil’s cytoplasm.
   1. It is found in about 50% of the patient with kidney disease.
   2. This is also positive (about 75%) in other autoimmune diseases like Ulcerative colitis or sclerosing cholangitis.
4. c-ANCA appears to have anti-proteinase 3 specificity, while p-ANCA has predominantly anti-myeloperoxidase activity.
   1. Result: Negative when there is very little fluorescence or no fluorescence.
      

      ANCA-C and ANCA-P type of antibodies

Wegener’s granulomatosis

1. Definition:
   1. Wegner’s granulomatosis is defined as granulomatous inflammation containing foci of vasculitis. involving the nasopharynx plus proliferative vascular lesion of the kidney.
   2. There is a proliferative vascular lesion obliterating a focal area of the Bowman’s capsule of kidneys (crescentic glomerulonephritis).
2. There is regional systemic vasculitis or fatal granulomatous vasculitis.
3. Small arteries of the kidneys, lungs and upper respiratory system (nasopharynx) are involved.
   1. The damage is due to granulomatous inflammation.
   2. The renal lesion is focal segmental necrotizing glomerulonephritis.
4. It is a form of vasculitis which involves small and medium-sized blood vessels in various organs.
   1. The common sites are the kidneys and respiratory system.
5. Diagnostic criteria:
   1. Necrotizing granulomas in the respiratory tract.
   2. Generalized necrotizing arteritis.
   3. Glomerulonephritis.
6. Signs and symptoms are:
   1. The patient has fever and arthralgia.
   2. Nose bleed, epistaxis.
      1. Stuffy nose, nasal ulceration, and crusty secretions.
      2. Rhinitis is the first symptom.
      3. There is sinus involvement.
   3. Ear shows conductive hear loss due to auditory dysfunction.
      1. There are 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. Lung shows pulmonary nodules like coin lesions.
      1. There is infiltrate looking like pneumonia.
      2. There is pulmonary hemorrhage leading to hemoptysis.
      3. Rarely there is bronchial stenosis.
   7. The skin may show a subcutaneous nodule which is like 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, heart, CNS, and GI tract involvement are rare.
7. Histologically there are poorly formed granulomas, with the presence of necrosis and many multinucleated giant cells.
   1. It is believed that ANCA antibodies are responsible for inflammation.

Lab diagnosis

1. Serological method for the detection of ANCA.
2. Detection of c-ANCA is sensitive and specific for the diagnosis of Wegener’s disease.
   1. Biopsy of the affected area with fluorescent method show antibodies localization in the neutrophil cytoplasm, having a granular appearance throughout the cytoplasm (c-ANCA).
   2. c-ANCA are reported 90% to 95% positivity in the Wegner’s granulomatosis (range 84% to 100%).
   3. In inactive Wegner’s disease 30% positivity (range is 13% to 41%).
   4. In the case of only involvement of the respiratory system is 65% (range is 60% to 85%).
3. p-ANCA reacts to myeloperoxidase and is detected in 50% to 80% of the localized Wegner’s granulomatosis involving the kidneys.
   1. p-ANCA is seen in other diseases as well like;
      1. Ulcerative colitis (roughly in 75% of the cases).
      2. Sclerosing cholangitis also shows 75% positivity.
      3. Autoimmune hepatitis shows 50% positivity.
      4. HCV and HBV in active disease show 5% to 7% of the cases.
      5. Crohn’s disease shows 8% positivity.
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.

Normal

• Serological method
  • Not found normally 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 ELIZA.
    • 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.

Increased in:

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

p-ANCA is not specific, but these are seen in:

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

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