LABORATORY TOOLS IN AUTOIMMUNITY

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LABORATORY TOOLS IN AUTOIMMUNITY

• By Wessam El Gendy MD
• Prof. of clinical Pathology

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• Laboratory test results help diagnose autoimmune
  diseases
• monitor disease course
• predict disease outcome
• assess the response to therapy
• and gain insight into disease etiology or
  pathogenesis.

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Screening

• With few exceptions, broad-based screening tests
  are generally not recommended in the absence of
  suggestive history and/or physical findings
  because false-positive rates may be high.

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• However, systemic lupus erythematosus (SLE) is
  rarely, if ever, present when an ANA test is
  negative.
• Test is overly sensitive 95 not diagnostic
  without clinical features

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Confirming a diagnosis

• Examples of a confirmed diagnosis are RF in RA
  (recognizing that 20-30 of RA patients are
  sero-negative, especially early in the disease
  course)
• anti-neutrophil cytoplasmic antibodies (ANCAs)
  in Wegener's granulomatosis.
• anti-dsDNA (double-stranded DNA) and anti-Sm
  antibodies in SLE.
• anti-Ro and anti-La antibodies in Sjogren's
  syndrome.

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Assessing prognosis

• Only a few serologic tests are of prognostic
  value one example is testing for Jo-1 in
  myositis.
• Anti-ribosomal P - Uncommon antibodies that may
  correlate with lupus cerebritis

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Anti-nuclear AbANA

• While the ANA is often positive in connective
  tissue diseases, it is also often positive in
  other non-autoimmune diseases such as
• Infectious disease (e.g. EBV, and viral
  hepatitis)
• Neoplastic diseases (e.g. leukemia, lymphoma,
  melanoma, and other solid tumors), andother
  diseases such as primary biliary cirrhosis.

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• A significant number of healthy individuals may
  have a positive ANA.
• However, positive ANAs in these non connective
  tissue diseases usually occur at a lower titre
• An accompanying cautionary statement in the
  reporting of low positive results (below the
  significant level) may also alleviate some of
  these unnecessary referrals.

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ANA titre

• If the patient has an ANA of 140 or less and no
  one knows why the test was done there is a good
  chance it means nothing but you cant be sure.
• If the ANA is 180 you are in no mans land.
• If the ANA is 1160 or higher, rheumatologist
  should take a look not all of them will have
  something, but some will.

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Second line Autoantibodies

• Anti-dsDNA (double-stranded DNA)
• Anti-Sm antibodies in SLE.(30-40 sensitivity)
• Anti-Ro(SS-A) and anti-La(SS-B)
• Anti-Scl-70 antibodies
• Anti-centromere
• Anti-histone

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Following patient progress

• ANA titers do not correlate with disease activity
  and the practice of ordering this test to monitor
  the course of SLE should be abandoned
• Sedimentation rate
• Anti-double stranded DNA (dsDNA) complement
  levels,
• better correlate with disease activity and serves
  to guide treatment decisions.

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Following patient progress

• Most autoantibodies (RF, ANA , ENA extractable
  nuclear antigen, anti-Jo-1, etc) do not vary
  with disease flare-ups and remissions and need be
  ordered only once during the diagnostic work-up.
  

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Anti-ds-DNA

• Testing is not recommended in patients with a
  negative ANA test.
• Anti-ssDNA antibodies are nonspecific and have
  little clinical utility.

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"Does ANA-negative lupus exist"

• "ANA-negative" person with strongly positive
  antibody to Sm would unequivocally have lupus

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Anti-phospholipid SyndromeAPS

• Lupus Anticoagulant (LA), Anticardiolipin Test
  (Acl)
• Anticardiolipin antibody is one of the few
  autoantibodies that have assays which allows the
  identification and quantification of specific
  isotypes (IgG, IgM and IgA).

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Anti-cardiolipin antibodies

• Anti-cardiolipin Ab if positive should be
  repeated after 3 6 months to diagnose
  anti-phospholipid syndrome
• Level cannot predict thrombosis as once
  suggested.
• IgG is more specific than IgM

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Relationship of the LA and aCL

• The test may be positive for one, negative for
  other, or positive for both.

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Lupus anticoagulant

• Precautions
• Not on heparin or oral anticoagulant
• Is not useful as a follow up test

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Sjögrens syndrome

• Between 40 and 70 of patients with this
  condition have a positive ANA test result. While
  this finding supports the diagnosis, a negative
  result does not rule it out.
• The doctor may want to test for two subsets of
  ANA Anti-SS-A (Ro) and Anti-SS-B (La).

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Scleroderma
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• A positive result on the ANA also may show up in
  patients with Raynauds disease, rheumatoid
  arthritis, dermatomyositis, mixed connective
  tissue disease, and other autoimmune conditions.

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Autoimmune Disease Virus Assay (ADVA )

• a human retrovirus called the Human
  Intracisternal A-type Particle, or HIAP. It is
  the first A-type retrovirus to have been found in
  humans. Research data strongly suggests that this
  virus is the cause of four well-known autoimmune
  disorders. These disorders are lupus (systemic
  lupus erythematosus), Sjögren's syndrome, Graves'
  disease, and juvenile rheumatoid arthritis.

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• The Autoimmune Disease Virus Assay (ADVA) detects
  antibodies against HIAP. These antibodies appear
  in approximately 95 of patients with one or more
  of those four disorders but in fewer than 2 of
  healthy individuals. It is believed that
  infection by this virus may produce the differing
  symptoms of the disorders in different patients
  because of genetic variations in the immune
  systems of the patients.

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Conditions Associated with a Positive Rheumatoid
Factor Test

• Rheumatoid arthritis (50 to 90)
• Systemic lupus erythematosus (15 to 35)
• Sjögren's syndrome (75 to 95)
• Systemic sclerosis (20 to 30)
• Cryoglobulinemia (40 to 100)
• Mixed connective tissue disease (50 to 60)

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Non-Rheumatic Conditions

• Aging
• Infection bacterial endocarditis, liver disease,
  tuberculosis, syphilis, viral infections
  (especially mumps, rubella and influenza),
  parasitic diseases
• Pulmonary disease sarcoidosis, interstitial
  pulmonary fibrosis, silicosis, asbestosis
• Miscellaneous diseases primary biliary
  cirrhosis, malignancy (especially leukemia and
  colon cancer)

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Rheumatoid factor in Rheumatoid Arthritis

• Negative in 30 percent of patients early in
  illness
• If initially negative, repeat 6 - 12 months
  after disease onset
• Not an accurate measure of disease progression.

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Anticyclic citrullinated peptideAnti-CCP

• Tends to correlate well with disease progression
• Increases sensitivity when used in combination
  with rheumatoid factor
• More specific than rheumatoid factor (90 versus
  80 percent)

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• If present in such a patient at a moderate to
  high level, it not only confirms the diagnosis
  but also may indicate that the patient is at
  increased risk for damage to the joints.
• Low levels of this antibody are less significant.

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• When is it appropriate to have this test?
• Does anti-CCP determine if a patient has
  rheumatoid arthritis?

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Anti-CCP

• Can detect approximately 80 of all RA patients,
  but is rarely positive in non-RA patients, giving
  it a specificity of around 98.
• In addition, ACP antibodies can be often detected
  in early stages of the disease, or even before
  disease onset.

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Serial anti-CCP as a follow up measure

• Serially determined anti-CCP antibodies as well
  as baseline determination should be considered
  from the predictable viewpoint.
• Drop of level denotes suppression of joint damage

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Flow chart
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Anti-neutrophil Cytoplasmic AbANCA

• Diagnostic tool and marker of disease activity
  for vasculitis
• The c-ANCA pattern has predominantly been
  associated with Wegener granulomatosis,
• p-ANCA has been associated with microscopic
  polyarteritis, other vasculitides, idiopathic
  necrotizing and crescentic glomerulonephritis,
  and other diseases

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Atypical ANCA

• ANCA occur in 50 to 70 of patients with
  ulcerative colitis, 10 to 30 of patients with
  Crohn disease, 90 of patients with autoimmune
  hepatitis type I ,and 90 of patients with Felty
  syndrome. ANCA also occur in up to 30 of
  patients with active rheumatoid arthritis.

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• C-ANCA is usually directed against PR3, but
  C-ANCA (atypical) often has specificity for
  bactericidal/permeabilityincreasing protein.
• The PR3-ANCA level will usually distinguish
  patients with Wegener granulomatosis from
  patients with a true atypical ANCA due to
  inflammatory bowel disease or other autoimmune
  disease.

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• PR3-ANCA and MPO-ANCA levels in chronic
  infections, inflammatory bowel disease, and
  autoimmune diseases are usually low (1) and do
  not correlate with disease activity.

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Autoantibodies and Celiac Disease

• Endomysial antibodies (EMA) testing is highly
  specific (false positives are rare) and sensitive
  for the diagnosis of celiac disease.
• Tissue transglutaminase (tTG) the most useful
  first level screening test for celiac disease.
  The levels of these antibodies decline following
  institution of a gluten-free diet.

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Celiac Disease

• IgA and IgG gliadin antibodies are less specific
  than anti-tTG and anti-endomysial antibodies and
  may occur in other intestinal diseases.

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Type I Autoimmune Hepatitis

• ANA
• ASMA more than 1/320
• Anti-actin ( more sensitive )
• Anti-soluble antigen (SLA) 10 of cases
• P-ANCA (atypical pattern)
• Anti-ds-DNA
• Anti-ss-DNA
• AMA /-

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• The presence of SLA antibodies has almost 100
  specificity for autoimmune hepatitis, although
  only 12-30 of patients have these antibodies

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Type II AIH

• Anti-LKM-1
• Liver cytosol antigen ALC-1

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ANTI-MITOCHONDRIAL Ab

• Antimitochondrial abs (AMA) have been reported in
  90-96 of patients with Primary Biliary Cirrhosis
  (PBC).
• AMA are also occasionally found in sera of
  patients with other liver conditions, including
  Chronic Active Hepatitis, Cryptogenic Cirrhosis
  and in patients with clinical, but no biochemical
  evidence of Liver Disease.

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Complement Assay

• It may be used to help diagnose and to monitor
  the activity of acute or chronic autoimmune
  diseases such as systemic lupus erythematosus
  (SLE).
• It may be tested and monitored with immune
  complex-related diseases and conditions such as
  glomerulonephritis serum sickness, rheumatoid
  arthritis, and vasculitis

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Complement Tests

• When immune complexes form, complement helps to
  clear them from the blood, making levels of
  complement low.

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Anti-Thyroid peroxidase Ab

• A positive anti-TPO Ab test provides strong
  evidence for early, subclinical autoimmune
  disease.
• Is also used to monitor response to
  immunotherapy, to identify at-risk individuals
  (with family history of thyroid disease), and as
  a predictor of postpartum thyroiditis.

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Interpretation

• The physician must be aware of the sensitivity
  and specificity of each test. Tests with low
  sensitivity but high specificity are helpful only
  if positive, whereas tests with low specificity
  should not be ordered in the absence of a high
  diagnostic suspicion

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• The clinical laboratory shouldclearly state
  the type of immunologicprocedure performed
• Define the reference range when including the
  cut-off values and indicate what percent of the
  normal population is included in the reference
  range.

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• Sensitivity the goal is no false negatives
• Specificity the goal is no false positives

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THANK YOU