Autoimmune

Question 1

ANCAs IIF sens/spec for vasculitis

Answer 1

85% sensitivity 96% specificity

Question 2

MPO/PR3

Answer 2

PR3- GPA 80% MPA 30% pauimmune nec GN 10% EGPA 5%, inf endocarditis, drug induced

PR3- specificity GPA 98%

MPO- MPA 60%, paucimmune GN 40%, EGPA 40%, GPA 10%, inf endocarditis, drug induced, RA, other systemic inflammatory, malignancy IBD AIH type 1

Question 3

Celiac serology tTG IgA - how is it reported in your lab

Answer 3

Bioflash tTG IgA, IgG DGP>20 CU cut off? interpretative comment

For first time new positive tTG IgA samples or those with qualitative change since previous analyses, EMA IFA on monkey esophagus also done

Question 4

Celiac serology sens/spec

Answer 4

tTG IgA sens/spec 95%, 97%
DGP IgG 65-97% 98-100% better for <2
EMA IgA 76-100%, 94-100%

Gold standard target ag is tTG - higher the titre~ greater the PPV

False neg 10-15% if on GFD, and are taking immunosuppressants

Question 5

What are the explanation for situations where coeliac disease is suspected, with no villous atrophy but with positive serology? (cite 7 causes)

In these circumstances, what is the next step in approach?

Answer 5

1. Giardia
2. CVID
3. Crohns
4. Tropical sprue
5. Autoimmune enteropathy
6. Cows milk protein intolerance
7. Olmesartan/medications

Trial GFD then re biopsy

Question 6

What are the HLA genes for coeliac disease?

Answer 6

HLA DQ2- DQ2.5 and 2.2, and DQ8 collectively 99%

Question 7

Which serology should be tested in coeliac disease?

Answer 7

1) TTG IgA and DGP IgG

DGP IgG enhances pick up, positive together results in greater predictive value

2) total IgA and tTg IgA - total IgA 3% of people w celiac dd selective IgA deficiency false neg

Question 8

What is the difference between hep 2 vs hep2000 cells

Answer 8

hep= human epitheliod carcinoma cells
hep 2 transfected SSA hyper expressing 
- malignant cells with large nuclei
-high rate of division
-more detail

transfection = introducing nucleic acids into cells utilizing means other than viral infection

technically only 60kDA Ro seen on hep2000 (52 cytoplasmic)

Question 9

ENA methods

Answer 9

• ELISA
• • Immunoblot
• nitrocellulose membrane, incub, conjugate, substrate, dry, densitometer
• • FIDIS/Lumnex/ALBIA
• microbeads coated ag, flow,
• Chemiluminescence immunoassay
• CIEP; gold standard
• radial double immunodiffusion/outcherlony
• western blotting
• unicap/FEIA

Question 10

dsDNA methods

Answer 10

• ELISA
• FIDIS
• Farr
• • crithidia
• kinetoplast tail protozoan large mitochondria high conc DNA without histone or other DNA bound ag

Question 11

Which ENA ag detection on which methods can be missed? and why

Answer 11

SSA 60 on hep2000, 52- both best by CIEP, can be denatured on IB, cant consistently detect anti52 SSA/Ro

ELISA- depends on purified or recombinant

CIEP Scl70 low negative charge pH8 so may be inadequate

cytoplasmic patterns on ANA

linear only- IB; ELISA/CIEP/DID tech conformational and linear

Question 12

Advantages/disadvantages of each method

Answer 12

Ad

• automated
• cost
• sens/spec
• random access
• direct ouput of results to reporting system/LIS interface
• range of
• ease of use
• TAT
• quantitative

Disadvantage

• labour intensive
• subjective
• cost
• reference sera may be required (DID)
• skill level
• may miss some antibodies (CIEP= 52, scl70)
• ag not well purified, differing sources false positive or over sensitivity or false neg if linear/hidden epitopes/loss conformational epitope

Question 13

ENA antigen sources?

extract antigens from nucleus of a tissue extract

Answer 13

calf thymus, rabbit thymus, human spleen

calf thymus good screen except scl70, guinea pig kidney for anti ssa 52kD only
rabbit thymus- contains scl70, can use for anti ssb , rnp, sm and jo1 but wont detect anti ssa

human spleen similar to RTE but very hard to obtain

Question 14

Interference/spurious results in immunoassays

List some antibody and non antibody mediated intereference

Answer 14

Antibody

• rheumatoid factor (anti IgG IgM) - EPG, nephel/turbidimetry, IFA
• heterophile antibodies: human ab IgM broad reactivity against ag of other species
• cryoglobuins
• paraproteins
• therapeutic mABs
• IVIG
• polyclonal hypergam

Non antibody

• hemolysis, lipemia (turb/nephel), icterus
• CRP/fibrinogen EPG

Question 15

What measures are used in place to prevent quenching?

Answer 15

• reagents kept in dark bottle/cupboard
• covered during incubation time
• minimise time between two readers
• minimise time on high power and screen at low power
• shut shutter when not looking at slides