IF - ANA Immunofluorescent Antinuclear Antibody

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IF - ANAImmunofluorescent Antinuclear Antibody
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Outline

• Introduction
• Background
• Uses
• Principle of test
• Procedure
• Interpretation of results
• QC

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Outline

• Limitations
• Accuracy
• LE test
• Alternate tests
• Confirmatory test
• Conclusion
• References

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ANAs

• Antinuclear antibodies
• autoantibodies to nuclear components
• types
• systemic
• specificity
• sensitivity

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Significance of ANAs

• ANAs are produced in a variety of autoimmune
  diseases. There is a strong correlation between
  the type of ANA produced, the titer of the ANA
  and specific diseases.

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Autoimmunity

• Represents a breakdown of the immune systems
  ability to discriminate between self and nonself
• Characterized by the persistent activation of
  immunologic effector mechanisms that alter the
  function and integrity of individual cells and
  organs
• Site of damage, organ specific or systemic,
  depends on the origination of the inappropriate
  immune response

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Factors Influencing the Development of
Autoimmunity

• Every individual, given the appropriate
  circumstances, has the potential to develop
  autoimmunity as lymphocytes that are potentially
  reactive with self antigens exist within the
  body.
• Factors effecting the control of these clones of
  lymphocytes and ultimately the antibodies
  produced are
• Genetic factors
• Age
• Exogenous factors

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Genetic Factors

• Direct genetic etiology not yet established
• Tendency for familial aggregates
• Correlation with certain HLA antigens
• More frequent in females
• Sex hormones implicated in some disorders

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Age

• Formation of autoantibodies steadily increases
  with age
• Peak seen between 60-70 years of age

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Exogenous Factors

• UV radiation
• Drugs
• Viruses
• Chronic infectious disease
• All play a role in the development of autoimmune
  disorders.

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Autoimmunity

• In theory, there are a certain number of clones
  of B cells that are not tolerant to self. These
  cells are normally suppressed by TS cells. In
  the presence of a B cell mitogen, these clones
  will proliferate in the absence of T cell
  enhancement.
• The resulting autoantibodies are
• transient
• of low affinity
• IgM primarily

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Forbidden Clone Theory

• There is a correlation between Tscell deficiency
  and the development of autoantibodies. Normally
  Tscells prevent the activation of these
  nontolerant clones of lymphocytes.

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Background

• The detection and semi-quantitation of
  autoantibodies aid in the diagnosis of autoimmune
  disease.
• Laboratories have used indirect fluorescent
  antibody (IFA) procedures to detect
  autoantibodies since 1957.
• In these procedures, a fluorescein conjugated
  AHG serves as a marker for an antigen-antibody
  binding reaction which occurs on a substrate
  surface.
• IF-ANA is commonly used as a screening test for
  certain autoimmune disorders.

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Background

• Autoantibodies Found in SLE and Other Autoimmune
  Disorders
• Anti-DNA
• Double-stranded (native) DNA (dsDNA)
• Single-stranded DNA (ssDNA)
• Antinucleoprotein (soluble nucleoprotein sNP
  DNA-histone)
• Antibasic nuclear proteins (histones)
• Antiacidic nuclear proteins (extractable nuclear
  antigens ENAs includes Smith Sm and
  ribonucleoproteinRPN)
• Nuclear glycoprotein
• Ribonucleoprotein (RNP)
• Sjogrens syndrome A and B (SS-A Ro and SS-B)
• Antinucleolas (nuclear RNA)

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Background

• This test should only be ordered for a patient
  who demonstrates the symptoms of systemic disease
  as some healthy individuals maintain low titers
  of ANAs yielding false positive results.

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Uses

• Systemic Lupus Erythematosus (SLE)
• Sjogrens syndrome
• Scleroderma
• Rheumatoid arthritis
• CREST

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Expected Results
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Kallestad? QuantafluorSanofi Diagnostic Pasteur,
IncIF-ANA test kit

• Materials provided
• Substrate slide
• Hep-2 (human epithelial cells)
• 2-8C
• Positive Control
• Pooled human sera
• Specific autoantibody activity
• Liquid form
• working dilution
• 2-8C
• Lyophilized form
• working dilution upon reconstitution
• -20C

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Kallestad? QuantafluorSanofi Diagnostic Pasteur,
IncIF-ANA test kit

• Materials provided
• Negative Control
• Pooled normal human sera
• working dilution
• 2-8C
• Universal FITC conjugate
• Fluorescein conjugated to AHG
• 2-8C
• Mounting media
• polyvinyl alcohol
• do not freeze
• ambient temp

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Kallestad? QuantafluorSanofi Diagnostic Pasteur,
IncIF-ANA test kit

• Materials provided
• Phosphate buffered saline
• pH 7.3 ? 0.10
• dibasic sodium phosphate
• monobasic sodium phosphate
• sodium chloride
• ambient temp storage for crystalline form
• prepared solution is good for 3 months at ambient
  temp
• Evans Blue counter stain
• ready to use in
• ambient temp
• Blotting strips
• ambient temp

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Materials required but not provided

• 25 ?l calibrated micropette
• Test tubes, 12 x 75 mm or 13 x 100 mm
• Test tube rack
• Humidity chamber
• Staining dish
• Magnetic stirrer (optional)
• Plastic wash bottle
• Glass cover slips, 24 x 60 mm
• Distilled or deionized water
• Fluorescent microscope

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Specimen Collection

• Serum recommended
• Avoid grossly hemolyzed or lipemic samples
• produce increased nonspecific background staining
  of the subtrate

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Specimen Storage

• Storage
• up to 2 days _at_ 2-8C
• freeze _at_ -20C for longer storage
• avoid repeated thawing and freezing
• NOTE If sample is to remain at RT (e.g. mailing)
  then add sodium azide
• 1 mg/ml to preserve.

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Procedure Step 1

• Bring all reagents, materials and patient
  specimens to ambient temperature. (Approximately
  20 min)
• Prepare a PBS wash for step 7 and a second PBS
  wash/Evans blue counter stain (if needed) for
  step 12.
• In some assay kits the counter stain and the FITC
  conjugate are combined in a ready to use form.
• Construct a humidity chamber.

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Watch point Step 1

• It is very important that the reagents are
  allowed to warm to RT.
• As warmer reacting autoantibodies may be missed
  if reagents are not warmed to RT.
• .

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Procedure Step 2

• Reconstitute controls and PBS with distilled
  water.
• Buffer is made fresh weekly. Verify pH of buffer
  before each run.

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Procedure Step 3

• Prepare a 140 dilution (0.05ml patient 1.95ml
  PBS) for each specimen.

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Procedure Step 4

• Remove slide from foil bag. Place in humidity
  chamber. Immediately add 25 ?l controls or
  diluted test serum to the appropriate wells.

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Watch point Step 4

• Carefully open slide package. Handle by outer
  edge only so that the substrate is not damaged.
• Each well must be entirely covered with control
  or diluted serum.
• This is to prevent the slide from drying and
  artifact formation.

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Procedure Step 5

• Cover humidity chamber. Incubate slides _at_
  ambient temperature for 20 minutes.

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Procedure Step 6

• Rinse each slide briefly with a stream of PBS.

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Watch point Step 6

• Apply stream above the wells.
• Wash each slide from the middle of the slide so
  that the stream is directed off of the side of
  the slide.
• Do not apply the stream directly to the wells.

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Procedure Step 7

• Wash slides for 10 minutes in a staining dish
  filled with PBS. Gently agitate staining dish
  several times or use a magnetic stirrer during
  the wash.

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Procedure Step 8

• Remove each slide from staining dish and blot
  excess PBS from around the wells using blotting
  strips.

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Watch point Step 8

• Do not touch the strip to the
• substrate wells.

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Procedure Step 9

• Place slides in humidity chamber and immediately
  dispense 25 ?l of FITC conjugate into each well.

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Procedure Step 9 (modified)

• In some kits the counter stain and the FITC
  conjugate solution are combined.

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Procedure Step 10

• Cover humidity chamber.
• Incubate for 20 minutes
• _at_ ambient temperature
• in the dark.

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Procedure Step 11

• Rinse each slide briefly with a stream of PBS.

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Procedure Step 12

• Wash slides for 10 minutes in a staining dish
  filled with fresh PBS and 5-6 drops of Evans
  blue counterstain. During wash, turn on
  microscope.

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Procedure Step 12

• NOTE This wash step should also be set up in a
  dark place. Since the fluorescent dye has be
  added any UV exposure can result in quenching of
  the dye.

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Procedure Step 12 (modified)

• If kit being used combines the counter stain and
  the FITC conjugate, then this step is modified to
  include a wash step in PBS only for 10 minutes in
  the dark.

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Procedure Step 13

• Remove slides,
• blot, and apply
• 4 ?1 drops of
• mounting media
• per slide, making
• sure to cover
• all wells.
• Add coverslip.

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Watch point Step 13

• Note If processing multiple slides, place
  slides in the humidity chamber until you are
  ready to evaluate each slide. Exposure to light
  will cause quenching of the fluorescent stain.

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Quenching

• Some fluorochromes (such as fluorescein) are
  photosensitive and are subject to photo-bleaching
  when exposed to intense excitation light.

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Procedure Step 14

• View under fluorescent
• microscope in a dark room.
• Evaluate each well for
• presence or absence of
• fluorescent staining using
• the positive and negative
• control wells as a
• reference.

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Procedure Step 15

• Record results. If positive, also record the
  staining pattern.
• All Positive results are rerun in serial
  dilutions up to
• the disappearance of the positive result.
• The final report, if the specimen is positive,
  must include the final dilution(s) and the
  staining pattern(s).

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Procedure Step 15

• Turn off the microscope.
• Log the start time and end time of use.
• As a part of quality control the bulbs are
  replaced every 200 hours or according to the
  manufacturers suggestion.

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Interpretation of Results

• Start by evaluating the positive and negative
  control wells.
• The positive control should show a 3 homogenous
  bright apple green fluoresce pattern.
• The negative control may be black with no green
  color or may show a dull green outline of some
  cells.
• NOTE The green color in the negative control
  should be in great contrast the the bright green
  of the positive control.

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Interpretation of Results

• The test for autoantibodies is NEGATIVE if no
  specific pattern of fluorescence is observed on
  the substrate.
• The test for autoantibodies is POSITVE when the
  Hep-2 substrate shows a specific pattern of
  fluorescence.

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Interpretation of Results

• The Hep-2 cells on each well are a mixture of
  cells in interphase (resting state) and in
  various stages of mitosis. Mitotic cells should
  be viewed during metaphase.
• Metaphase-stage of mitosis in which the nuclear
  envelope breaks down and the chromatids line up
  in the center of the cell.

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Interpretation of Results

• If patient specimen is POSITIVE, then determine
  titer by serially diluting the sample and
  rerunning the test with these dilutions. The
  titer of the autoantibody is the highest dilution
  demonstrating a 1 specific fluorescence of the
  substrate.
• When multiple patterns are present, determine the
  titer for each pattern separately.

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Grading of Intensity

• 4 Brilliant apple green color, clear cut
  outline,
• sharply defined cell center
• 3 Less brilliant fluorescence, clear cut cell
  outline,
• sharply defined cell center
• 2 Definite pattern but less fluorescence, cell
• outline less well defined.
• 1 Subdued fluorescence, subdued cell outline

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ANA Patterns

• HOMOGENEOUS (DIFFUSE)
• RIM (PERIPHERAL)
• SPECKLED
• fine
• coarse
• CENTROMERE (DISCRETE SPECKLED)
• NUCLEOLAR

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Homogeneous Pattern

• The nucleus stains evenly throughout. Within
  mitotic cells, the chromosomes stain as an
  irregularly shaped mass with more intensely
  stained outer edges. This pattern combination is
  suggestive of autoantobodies to nDNA, histones,
  or DNP.

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Homogeneous Pattern
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Rim (Peripheral) Pattern

• The nucleus stains predominantly at its
  periphery. Within mitotic cells, the chromosomes
  may stain as an irregularly shaped mass with more
  intensely stained outer edges. This pattern
  combination is suggestive of autoantibodies to
  nDNA or DNA-histone complexes. If the mitotic
  cell chromosomes are negative, the rim nuclear
  pattern may be suggestive of autoantibodies to
  nuclear membrane and not to nDNA (significance
  unknown).

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Speckled Patterns

• Fine speckled
• Numerous small uniform points of fluorescence
  scattered throughout the nucleus with distinct
  nuclear margins. Nucleoli appear as unstained
  patches in the speckled nucleus. Mitotic cell
  cytoplasm may retain fine speckling, while the
  mitotic cell chromosomes are negative.
• Coarse or atypical speckled
• Medium sized uniform points of fluorescence
  scattered throughout the nucleus with distinct
  nuclear margins. The mitotic cell chromosomes
  are negative.
• Fine and coarse speckled patterns are suggestive
  of autoantibodies to Sm, RNP, Scl-70, SSA, SSB,
  or other undefined antigens.

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Speckled Pattern
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Centromere Specificity (Discrete Speckled)

• Medium sized uniform points of fluorescence
  scattered throughout the nucleus with indistinct
  nuclear margins. In mitotic cells, the discrete
  speckles are clustered only in the chromosome
  mass. The specificity of the discrete speckled
  patterns is to the centromere and is highly
  correlated with the CREST syndrome of progressive
  systemic sclerosis.

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Centromere Specificity (Discrete Speckled)
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Nucleolar Pattern

• Intense homogeneous staining of the nucleoli
  often associated with dull homogeneous
  fluorescence of the rest of the nucleus. Mitotic
  cell chromosomes are usually negative however,
  some nucleolar antibodies may react with the
  chromosomes. The mitotic cells do not possess
  distinct nucleoli. This pattern is suggestive of
  autoantibodies to 4-6s RNA.

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Nucleolar Pattern
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Interpretation of ResultsExpected Values
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Expected Values
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Futher testing

• The autoantibody class can be determined by using
  class specific antisera. Fluorescein conjugated
  antisera monospecific for IgG, IgA and IgM are
  commercially available.
• Not included in most kits
• Special request by doctor

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Direction of Further Testing
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Limitations

• Diagnostic aid only
• Prozone reaction may be observed in some patients
• Sera from patients undergoing successful therapy
  for autoimmune disorders may be negative for ANA

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Limitations

• ANA specificity cannot be determined by
  morphological staining pattern alone
• Many drugs may induce ANA
• IF-ANA is not useful for therapeutic drug
  monitoring

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Specificity

• The Hep-2 cells contain nuclear antigens.
• The specificity of the Universal FITC Conjugate
  is confirmed by immunoelectrophoresis and reverse
  Radial Immunodiffusion.

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Accuracy

• The agreement between Kallestad Quntafluor test
  and Reference methods 96.4

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Quality Control

• Both a positive and negative control should be
  included with each run of the test.
• Use only the components that are provided with
  the kit being used.
• Microbially contaminated sera should not be used.

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Quality Control

• Verify pH of buffer before each run.
• Use heat inactivated sera to avoid C1q
  interference in assay.
• Replace bulb in fluorescent microscope as
  recommended by manufacturer.

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Alternate Screening Tests

• LE cell test
• Enzyme conjugated immunoassay

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LE cell test

• First reasonable good test for lupus
• Detects antibody against nuclear
  deoxyribonucleoprotein (DNP soluble
  nucleoproteinsNP, DNA-histone complex) that is
  produced in SLE.

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LE prep technique

• Uses laboratory-damaged cells, either tissue
  cells or WBCs as a source of nuclei
• Nuclei are incubated with patient serum
• Nuclear material is converted by the ANA against
  sNP into a homogeneous amorphous mass that stains
  basophilic with Wrights stain.
• The mass is then phagocytized by nondamaged PMNs
• These PMNs containing the hematoxylin
  (blue-staining) bodies in their cytoplasm are the
  so-called LE cells

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Confirmatory tests

• Ordered by Doctor
• Screening test results confirmed by assaying for
  specific antibodies (anti-Sm, anti-dsDNA, etc.)
  using Radial immunodiffusion (RID), double
  diffusion, RIA, ELISA

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Reflex or Cascade Testing

• One practice that has become more common recently
  is reflex or cascade testing. In such examples,
  panels of test for various other autoantibodies
  are performed when an ANA result is positive.
  UMMC Special Chemistry Lab commonly runs a reflex
  test upon a positive ANA screen at the doctors
  request.
• Potential problems with reflex testing include
  increased costs and erroneous diagnosis.
  Additional testing should be guided by specific
  clinical indications.

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Reflex Test Panel Used at UMMC

• Microtiter colorimetric ENA Panel
• Sm
• RNP
• SSA
• SSB
• anti-DNA

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Conclusion

• Testing for antinuclear autoantibodies should be
  done selectively and only when suspicion for
  certain autoimmune disorders is high. When
  clinical suspicion is low, most positive results
  are false-positive and may lead to unnecessary
  additional testing, needless anxiety, and
  inappropriate treatment.

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References

• Turgeon, Mary L. Immunology and Serology in
  Laboratory Medicine. St. Louis Mosby 1996.
• Kallestad? Quantafluor. Kit insert. Sanofi
  Diagnotics Pastuer, Inc. 1992
• Antinuclear antibody. Kit insert. Sigma
  Diagnostics?. 1995
• Lupus-like Disease available 4/04/01
• http//www.infotech.demon.co.uk/MCTD.htm
• Ravel. Clinical Laboratory Medicine, 6th ed.
  1995
• available http//www.home.mdconsult.com
• Postgraduate Medicine Symposium Rheumatologic
  Diseases
• available http//www.postgradmed.com
• Wiggers, Tom. Various handouts from Immunology
  and Serology