Diagnostics of Celiac Flashcards
Celiac’s disease = immunology explanation + steps
Gluten sensitive enteropathy mediated in small intestine proinflammatory immune response resulting in malabsorption + enterocyte destruction.
Since it’s not well digested, gluten stays at large peptides and can go to lamina propria passing through the epithelia layer of gut (purple), and there’s tissue transglutaminase there. Transglutaminase is involved in tissue repair, but it also can interact with gluten peptides and deamidates them.
Due to the modification done by transglutaminase (deamidation), Glutamine is turned into Glutamate with negative charge.
Now they can fit perfectly to the HLA DQ2-8.
Then these peptides can be presented to T cells, T cells get activated and you get cytokine secretion that can directly kill the intestine cells, or you can activate the intraepithelial lymphocytes (IEL), they become activated and can kill the enterocytes too.
CD4 + T cells also secrete cytokines to provide B cells = Abs are produced.
Abs in disease progression
Gluten binds to transglutaminase, and modified gluten and transglutaminase is taken up by APCs, and modified gluten fits well to HLA-DQ 2-8 (MHC Class II) presented to T cells, T cells produce cytokines + goes and helps B cells that both produce Abs for gluten and transglutaminase.
Transglutaminase is usually recognized with a low affinity BcR, but when it’s complex with the gluten the complex is taken up, and B cell presents gluten on its HLA-DQ2 as well. = APC.
Abs in diagnosis - how to detect:
1) Take esophagus tissue, in this case monkeys
2) Cut thin slices, put them on slides, incubate with patient serum that has fluorescently labeled IgA, and in Celiac patients this typical chicken wire pattern is observed.
Below: Positive scoring of these Abs, from strongly positive to negative. In first diagnosis, this test was done in old times.
= Endomysium Ab test
Later: The antigen that causes the disease is identified, the Abs were recognizing transglutaminase! ELISA is done, first in animal cells, then in human erythrocytes for transglutaminase (then recombinant versions are used).
More automated tests are also developed for diagnosis, patient samples are placed and quantitative results are provided, rather than just checking the positivity with eye.
Establishing level of positivity/negativity in diagnostics
Compare levels of Celiac patients to healthy controls (kids/adults) = Look at the cut off. Upper limit of normal can be understood, %99 or %95 of level of normal patients. (two dotted lines)
Upper normal value: The point where it’s going to be still validated healthy but very close to be sick.
Sensitivity and specificity is important.
Sensitivity: percentage of positive tests in disease group (ability to catch positive individuals)
Specificity: percentage negative in healthy group
(ability to separate positive and negative patients)
If you have a %99 cut off in upper normal value, you will lose individuals that are between %95 and 100, and some of them might be sick. You will lose more people that aren’t sick (more specific) but you will also lose the patients (less sensitive).
10times upper limit of normal is used for diagnosing kids.
Disadvantages of tests: -When you can see false negative or positive:
You can see false negative:
1) If the mucosal damage is low, you can’t see the Abs in serology.
2) Celiac patients have IgA deficiency X10 more than normal individuals, IgA is normally tested since they show up the best, but if they are deficient of IgA, those patients might be missed. IgG tests are available for that. = If you see a negative IgA test, if you check their IgG, it might show disease = chicken wire pattern.
3) If you take low gluten, Ab levels might be low.
4) Test systems should be adequate, so quality controls are important.
You can see false positive:
1) quality controls
2) Some kind of positivity can be seen in susceptible people that aren’t necessarily Celiac, the tests need to be repeated then
Patients with normal total IgA- tests
transglutaminase IgA = %85-100 sensitivity + %90-100 specificity
Endomysium IgA = %90-100 sensitivity, specificity = %99
transglutaminase IgG = %30 sensitivity - %99 specificity
* not sensitive at all.
DGP (deaminated gliadine peptide Abs) IgG = Abs against actual gluten.
%80-95 sensitivity %98 specificity
In normal tests, always IgA is done at first, and if there’s deficiency, DGP IgG is checked. *You can also use Ttg IgG in IgA deficient patients, because surprisingly it’s as sensitive as DGP IgG in deficient people.
How to test patients genomically for their HLA types:
Sequence specific oligonucleotides (SSO)= Amplify HLA- DQ region, use different probes that recognize different HLA types, and identify which one is the one you are looking for.
Is it positive or negative for HLA DQ 2/8? = Sequence specific primers (PCR-SSP) = Different primers for HLA DQ2 and 8 are used, multiplex PCR= if its negative, patient is negative and won’t get celiac ever.
Sequence based typing (PCR-SBT) = Not done for really checking if it’s positive or negative, used for eg. transplantation purposes to make sure if really HLAs match or not. More detailed.
Example studies: relation with gluten challenge and sickness
A patient is checked over time, suspected patient. Was found to be strongly positive in 1997, and biopsy showed strong Celiac too. Patient started gluten free diet (GFD), Abs were high, then dropped to negative. Biopsy was taken at that point, patient started to question whether if she’s really Celiac’s, and biopsy showed completely normal intestine. = completely cured. She only decided to take 1 m of gluten = Abs back again. They should be really using a gluten free diet for rest of their lives.
It also shows that if you start a gluten free diet before coming to doctor = they might not see that you have the disease. Abs might be so low, and intestine can be normal.
Gluten intake only between day 0-14.
A= transglutaminase Ab levels upon gluten challenge = until day 14 its fine, then it increased very high
B = deaminated gliadin peptide Abs already go slightly up in day 14, after that increased drastically
In both cases, patients already stopped after 14 day using gluten but it still increased.
So even if you eat it for 2 weeks you are doomed. = Except %25 of people, they don’t increase it even in 6-12 weeks = lucky af. Ab titers don’t increase but they might still get complaints/tissue damage though.
B cells disappear with gluten free diet
How to detect T cells to study them:
Tetramers/dextramers: Tetramers are made of MHC molecules, in our case HLA DQ 2-8, peptides are alpha gliadin peptides. They are connected with streptavidin, marked with florophore that can be detectable with flow.
They took blood, isolated the WBCs, stained with tetramer + HLA specific + alpha gliadin = mAb florescent detection is done, mAb also has magnetic bead. Any cells labeled with 2nd Ab = get stuck inside magnetic column, rest is washed. Selected cells are eluted. Since their concentration is low, you can amplify them, then detect them with data analysis.
Does T cell disappear with gluten free diet?
Treated patients (TCDs) even though they are treated up to 10 years, T cells still can be detected! T cells don’t go away with diet
Refractory celiac disease
Occurs in %2-5 adult onset CD patients
Persisting villous atrophy + crypt hyperplasia + intraepithelial lymphocytes even when you do gluten free diet for 12 months
Diagnosis should be done after other similar diseases are excluded.
RCD type I: no response to GFD %96 survival
RCD type 2: No response + Aberrant IEL formation %60 survival, with lymphoma %6.
RCDII: huge T cell lymphoma + death risk
Even though transglutaminase Ab/ deaminated glidin, any Abs levels go 0 = disease continues. = serology negative
Aberrant IELs - how to identify them
1) TcR clonality analysis
TcRs vary with VDJ arrangements. Check VDJ = understand whether T cells you look for are clonal or not. If they are polyclonal, you have tiny reads everywhere. Oligoclonal = some larger peaks here and there, monoclonal = only 1 peak.
2) IHC
Problem: Can’t distinguish between surface expression or intracellular expression.
Problem: Majority of gamma delta T cells are also both negative for CD4 and CD8.
Might be mistaken for aberrant T cells.
CD3 positive but CD8 negative.
3) Flow cyt
No CD3, but no CD8 = aberrant T cells.
Strategy: Simple Flow cyt panel.
1) Stain with anti-CD3 first, (Ab + flourochrome)
2) Fix and permeabilize
3) Abs can go inside too.
RCD1, as expected almost all cells are double positive both in cyt. and surface CD3.
RCD2, a good percentage of cells are surface CD3 negative.
Cut off for aberrant cells are %20 for RCDII diagnosis.
They are mostly at %50, some of them are even around %90 in IEL percentage in duodenum.
Aberrant T cells: CD3 negative on surface, intracellular CD3 positive - no CD8
