ANCA Complete Kit

c-ANCA

c-ANCA and ANA

p-ANCA

p-ANCA and ANA

Negative

Atypical ANCA

ANA Positive

Ethanol Fixation

In ethanol fixed slides, MPO (p-ANCA) antigens migrate from the azurophilic granules and attach to the surface of the nucleus, whereas the PR3 (c-ANCA) antigens remain in the granules. ANCA positive samples will demonstrate either nuclear (p-ANCA) or cytoplasmic (c-ANCA) staining, dependent on antibody specificity.

Formalin Fixation

In formalin fixed slides, all ANCA antigens remain associated with the azurophilic granules, causing both c-ANCA and p-ANCA to produce cytoplasmic staining. Because antinuclear antibody positive samples may demonstrate a positive p-ANCA result, formalin fixed slides are typically used to confirm p-ANCA patterns.

Step 1. Bring all reagents to room temperature (18–26°C).

Step 2. Make 1:20 working solutions by diluting 50 µL of patient's serum in 1.0 mL diluent.

Step 3. Let pouch equilibrate to room temperature, then remove slide(s) from pouch.

Step 4. Remove slides from pouch. Place slide(s) into moisture chamber and add 50 µL of samples and controls. Cover and incubate 30 minutes.

Step 5. Rinse slide(s) by dipping into beaker with PBS. Transfer slide(s) into Coplin jar and wash 10 minutes.

Step 6. Blot edge of slide(s) on absorbent paper. Proceed immediately with next step.

Step 7. Apply ~50 µL conjugate to each well. Cover and incubate 30 minutes.

Step 8. Rinse slide(s) by dipping into beaker with PBS. Transfer slide(s) into Coplin jar and wash 10 minutes.

Step 9. Blot edge of slide(s) on absorbent paper. Proceed immediately with next step.

Step 10. Mount coverslip and read under fluorescent microscope.