ANA TestingANA Testing

Carrie MarshallCarrie Marshall

1/18/081/18/08

HistoryHistory

This is often-mentioned, never-seen LE cell. These dead nuclei are being engulfed by PMNs.

ANA TestingANA Testing

Guideline #1Guideline #1 Test for autoantibodies Test for autoantibodies only when a consistent clinical suspicion of only when a consistent clinical suspicion of rheumatic disease is present.rheumatic disease is present.

Not a good screening test for patients with Not a good screening test for patients with vague symptomsvague symptoms

ANA can be positive in sick people (non-ANA can be positive in sick people (non-rheumatic) and healthy peoplerheumatic) and healthy people

ANA TestingANA Testing

Anti-Nuclear Antibodies, but they can also Anti-Nuclear Antibodies, but they can also be anti-cytoplasmicbe anti-cytoplasmic

Immunofluorescence is commonly usedImmunofluorescence is commonly used

In the past patients serum was placed on In the past patients serum was placed on to slides with rodent (or other animal) cells to slides with rodent (or other animal) cells and IF was performed to look for and IF was performed to look for antibodies binding to cellular componentsantibodies binding to cellular components

What problems does this cause?What problems does this cause?

Human and rodent cells differ (slightly), Human and rodent cells differ (slightly), and so some people with obvious and so some people with obvious rheumatic disease would be negative on rheumatic disease would be negative on this test. “ANA-negative lupus”this test. “ANA-negative lupus”

Now there are human tumor cell lines that Now there are human tumor cell lines that are used (HEp-2 are preferred)are used (HEp-2 are preferred)

Another source of false negatives includes Another source of false negatives includes how the tissues are fixed onto the slideshow the tissues are fixed onto the slides

Ethanol and methanol fixation may remove Ethanol and methanol fixation may remove Ro/SSA antigens from cells, so the cells Ro/SSA antigens from cells, so the cells are fixed with acetoneare fixed with acetone

How is the test done?How is the test done?

Patient serum is diluted and dropped onto Patient serum is diluted and dropped onto HEp-2 slides (cells fixed into separate dots HEp-2 slides (cells fixed into separate dots on the slide)on the slide)

Incubated, washed, secondary antibody Incubated, washed, secondary antibody addedadded

Read by a tech using an IF scope (takes Read by a tech using an IF scope (takes specialized training and there is inherent specialized training and there is inherent variability between individuals)variability between individuals)

ResultsResults

Results typically include positive/negative, Results typically include positive/negative, titer and pattern of stainingtiter and pattern of staining

Titers less than 1:40 should be considered Titers less than 1:40 should be considered negative (20-30% of healthy people)negative (20-30% of healthy people)

Titers of 1:40 to 1:160 should be Titers of 1:40 to 1:160 should be considered positive at low titer (further considered positive at low titer (further workup is not recommended in the workup is not recommended in the absence of specific symptoms)absence of specific symptoms)

ResultsResults

Titers equal to or greater than 1:160 Titers equal to or greater than 1:160 should be considered positive and further should be considered positive and further workup should be done (only 5% of workup should be done (only 5% of healthy people). Prevalence of SLE is 40-healthy people). Prevalence of SLE is 40-50 in 100,000 (but 5,000 will have + ANA) 50 in 100,000 (but 5,000 will have + ANA)

Each hospital can change these cutoffs Each hospital can change these cutoffs based on their patient populationbased on their patient population

What Follow-up Testing?What Follow-up Testing?

Ideally this would depend on clinical symptoms, Ideally this would depend on clinical symptoms, but often:but often:dsDNAdsDNASmSmnRNPnRNPRo/SSARo/SSALa/SSBLa/SSBScl-70Scl-70Jo-1Jo-1

PatternsPatterns

The IF pattern is still reported, but does The IF pattern is still reported, but does not correlate well with what the antibody’s not correlate well with what the antibody’s specificity is.specificity is.

It was the most you could do “back in the It was the most you could do “back in the day”day”

Now with ELISA testing for specific Now with ELISA testing for specific antigens possible, the ANA pattern has a antigens possible, the ANA pattern has a low relevancelow relevance

PatternsPatterns

Peripheral or rim = dsDNAPeripheral or rim = dsDNA

Homogenous = dsDNA, histonesHomogenous = dsDNA, histones

Speckled = many antigensSpeckled = many antigens

Nucleoli = associated with sclerodermaNucleoli = associated with scleroderma

Centromeric = CREST syndromeCentromeric = CREST syndrome

Cytoplasmic = myositis, mitochondrialCytoplasmic = myositis, mitochondrial

PatternsPatterns

PatternsPatterns

PatternsPatterns

To summarize…To summarize…

You screen for ANAs using IF on slides You screen for ANAs using IF on slides with HEp-2 cellswith HEp-2 cellsIf it’s positive you look for the specific If it’s positive you look for the specific antigen that the antibody is reacting to antigen that the antibody is reacting to using ELISA (the antigen is stuck to the using ELISA (the antigen is stuck to the well) or other methodswell) or other methodsWe don’t screen for ANAs using ELISA We don’t screen for ANAs using ELISA because it’s hard to get all the various because it’s hard to get all the various antigens (40+) onto the well wallsantigens (40+) onto the well walls

dsDNAdsDNA

Crithidia luciliae has a large mitochondrion with dsDNA (and no histones)

dsDNAdsDNA

Guidelines suggest checking for anti-dsDNA Guidelines suggest checking for anti-dsDNA antibodies only when the symptoms are antibodies only when the symptoms are suspicious of SLE suspicious of SLE ANDAND the ANA is the ANA is positivepositive

The “ANA-negative lupus” patients are The “ANA-negative lupus” patients are REALLY rare now that we test with HEp-2 REALLY rare now that we test with HEp-2 cells rather than animal cellscells rather than animal cells

Guidelines suggest that the only Guidelines suggest that the only antibodies that need to be quantified are antibodies that need to be quantified are dsDNA (to predict a flare, and nephritis dsDNA (to predict a flare, and nephritis risk)risk) Active disease (q 6-12 weeks)Active disease (q 6-12 weeks) Less active disease (q 6-12 months)Less active disease (q 6-12 months)

Report quantitative results on isolated U-RNP Report quantitative results on isolated U-RNP antibodies (part of criteria for MCTD)antibodies (part of criteria for MCTD)

Anti-CCPAnti-CCP

IgG against Cyclic Citrullinated Peptide (CCP)IgG against Cyclic Citrullinated Peptide (CCP)Is a very specific marker, 98%, (very low rate of Is a very specific marker, 98%, (very low rate of false negatives) for Rheumatoid Arthritisfalse negatives) for Rheumatoid ArthritisWill be + in 70% of RA patients in early dzWill be + in 70% of RA patients in early dzNot found in other diseases (contrast to RF)Not found in other diseases (contrast to RF)Should be a one time test, does not need to be Should be a one time test, does not need to be repeated or followedrepeated or followedIndicates pts at high risk of progressive erosive Indicates pts at high risk of progressive erosive disease, should be treated aggressivelydisease, should be treated aggressively

QuestionQuestion

In what 2-3 diseases should you continue In what 2-3 diseases should you continue a work-up even if the ANA is negative?a work-up even if the ANA is negative?

AnswerAnswer

Sjogren’s syndromeSjogren’s syndrome

DermatomyositisDermatomyositis

PolymyositisPolymyositis

(ANA can be negative in more than 50%)(ANA can be negative in more than 50%)

QuestionQuestion

Besides a rising anti-dsDNA titer, what Besides a rising anti-dsDNA titer, what other lab test can help predict an other lab test can help predict an upcoming SLE flare?upcoming SLE flare?

AnswerAnswer

Falling C3 and C4 levelsFalling C3 and C4 levels

QuestionQuestion

What is the single greatest risk factor for What is the single greatest risk factor for SLE?SLE?

What 2 antibodies are the most specific for What 2 antibodies are the most specific for SLE (not ANA)?SLE (not ANA)?

AnswerAnswer

Female genderFemale gender

Anti-dsDNA and anti-SmithAnti-dsDNA and anti-Smith

QuestionQuestion

Why do SLE patients test falsely positive Why do SLE patients test falsely positive on VDRL tests?on VDRL tests?

AnswerAnswer

This tests uses particles coated with This tests uses particles coated with phospholipids, SLE patients who make phospholipids, SLE patients who make anti-phospholipid antibodies will make the anti-phospholipid antibodies will make the test look like it’s positive.test look like it’s positive.

QuestionQuestion

What specific autoantibody is What specific autoantibody is characteristic of drug-induced lupus?characteristic of drug-induced lupus?

AnswerAnswer

Anti-histone (H2A-H2B dimer)Anti-histone (H2A-H2B dimer)

QuestionQuestion

What is the major autoantibody in diffuse What is the major autoantibody in diffuse scleroderma?scleroderma?

In CREST syndrome?In CREST syndrome?

AnswerAnswer

Diffuse scleroderma = Scl-70Diffuse scleroderma = Scl-70

CREST = anti-centromereCREST = anti-centromere

QuestionQuestion

What enzyme class is the target of What enzyme class is the target of autoantibodies in polymyositis?autoantibodies in polymyositis?

AnswerAnswer

Transfer-RNA synthetasesTransfer-RNA synthetases

QuestionQuestion

Patients with MCTD typically have a high Patients with MCTD typically have a high titer of what autoantibody?titer of what autoantibody?

AnswerAnswer

Antiribonucleoproteins (either U1-RNP or Antiribonucleoproteins (either U1-RNP or nRNP)nRNP)