Autoimmunity: Luminex and Flow Cytometry

Question 1

Purpose of FIDIS vasculitis

Answer 1

Help is diagnosis of small vessel vasculitis

Question 2

Principle

Answer 2

Detects 3 autoantibodies; anti-MPO, anti-PR3 and anti-GBM

Use of microspheres that are internally labelled with two fluroscent dyes (red and infra red). Each antigen needed for a panel is convantely bonded to an indiviual microsphere set. These are mixed together to form a microsphere reagent.

Firstly, sample and microsphere reagent is mixed together and incubated. Ag and Ab complexes will form. Microsphere ares washed by filtration and removes any unbound Ab. Captured Ab are tagged with phycoerythrin labelled IgG anti human. Incubate and wash. The reaction is measured by flow cytometry which indentifies:
1. the microsphere (red laser)
2. the average fluroscence emitted by conjugate (green laser)

Question 3

Does this use a calibrator

Answer 3

Yes and uses diluent, POS, NEG, 2x calibrator and external QC

Question 4

Why spin +, - and cal before use

Answer 4

bring all the solution to the bottom so the pribe doesnt get contaminated or have a short sample alert

Question 5

what is the time cut off to read 100 beads

Answer 5

Ideally 10-30 seconds but MUST be 54 seconds or less

Question 6

Purpose of Sheath fluid

Answer 6

To being all the microparticles to the laser one by one

Question 7

why use calibrator

Answer 7

allows the titre of the sample for each antigenic specificity to be determined

Question 8

What does FIDIS vasculitis test for?

Answer 8

Detects 3 autoantibodies; anti-MPO, anti-PR3 and anti-GBM

Question 9

Relationship between FIDIS vasculitis and ANCA pattern readings

Answer 9

Interferences such as lactoferrin, elastase and lysosymem cause false P-ANCA patterns and therefore the use of MPO-ANCA and PR3-ANCA Elisa’s in conjugation with with the classical ANCA IFA is for greater discrimination when considering the clinical implications of ANCA results. Ensure the pattern is a true pattern as MPO and PR3 are reliable markers for the diagnosis of vasculitides

Question 10

If you had a P-ANCA pattern on ANCA reading what would you do and the expected results

Answer 10

Perform FIDIS vasculitis and expect a positive reaction for antibodies directed against MPO