The Rheumatology Laboratory

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The Rheumatology Laboratory

• Nicole le Riche, MD, FRCPC
• Associate Professor of Medicine
• Schulich School of Medicine

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Outline

• Highlight 6 common tests utilized in Rheumatology
• RF
• ESR
• ANA
• ENA
• ds-DNA
• ANCA
• Indications for testing
• Pitfalls of testing

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Rheumatoid Factor (s) (RF)

• Auto-antibodies directed against antigenic
  determinants on the Fc portion of immunoglobulin
  (Ig) G molecules
• Can be IgM, IgG, IgA, or IgE antibodies
• IgM only one routinely tested

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Rheumatoid FactorLatex Agglutination

• Latex particles coated with human IgG - Patient
  serum added - read under a lamp 2 minutes later.
• Dilution of 1/40 generally considered as positive

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Rheumatoid FactorNephelometry

• More sensitive screening method than latex
  agglutination
• Amount of scattered light ? concentration of
  Ab-Ag complexes

Cuvette Antibody-Antigen Complexes
X
Light Source
Detector
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Rheumatoid Factor

• NOT USED AS A SCREEN
• NOT USED TO MAKE DIAGNOSIS - Not specific to
  Rheumatoid Arthritis seen in other diseases
  
• USEFUL FOR PROGNOSIS-RF ve patients may have
  more aggressive disease
• Present in 70-85 of people with RA

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Anti CCP antibodies

• Recently discovered antibodies to cyclic
  citrullinated peptide
• Not specific to patients with Rheumatoid
  arthritis but if present in a RF ve patient
  connotation is that likely to have more
  aggressive disease prompting aggressive early
  treatment

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Rheumatoid Factor

• Non- Rheumatic Diseases
• Normal Aging
• Infection
• Hepatitis B C
• SBE
• Tb
• HIV
• Sarcoidosis
• Idiopathic Pulmonary Fibrosis

• Rheumatic Diseases
• Sjogrens syndrome
• Rheumatoid Arthritis
• SLE
• MCTD
• Myositis
• Cryoglobulinemia

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Erythrocyte Sedimentation RateESR

• Initially developed as a serologic test for
  pregnancy
• Later found to be a useful but non-specific
  marker of inflammation

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Erythrocyte Sedimentation RatePrinciple

• 200 mm long X 2.5 mm diameter vertically aligned
  anticoagulated tube of blood
• Look at the distance the column of blood falls in
  one hour (mm/hr)
• Normal ESR
• Men Age/2
• Women (Age10)/2

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Erythrocyte Sedimentation Rate

• Forces affecting sedimentation of RBC
• Size of RBC
• Viscosity of Plasma
• Repellant forces between negatively charged RBC
  membrane
• The presence of asymmetric proteins (fibrinogen)
  affects quality of repellant force and allows
  formation of Rouleaux causing the RBC to settle
  more rapidly

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Erythrocyte Sedimentation Rate

• ? ESR
• Inflammatory Diseases
• Hypoalbuminemia (-)
• Hypergammaglobulinemia
• Tissue Necrosis (MI, trauma)
• Pregnancy
• Anemia
• Age
• Heparinized Blood

• ? ESR
• Plasma Viscosity
• RBC or shape (PRV, Sickle)
• Decreased plasma proteins

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Anti-Nuclear Antibodies
ds-DNA
Important ANAs
ENAs
All ANAs
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Anti-Nuclear Antibodies
In 2006 we use a Fluorescent antinuclear
antibody test (FANA)

• 1948 SLE diagnosed with LE cell
• Antibodies attack DNA complexes in nuclei
• Nuclei become damaged
• Ingested by phagocytic cells
• LE Cell - PMN with a denatured nuclei inside

PMN
Denatured Nuclei
LE Cell
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Anti-Nuclear Antibodies
Fluorescein conjugated anti-human immunoglobulin
Microscope
Patient Serum
Monolayer of Human Epithelial Cells (Hep 2)
Glass Slide
ANA in Patient Serum
Nuclei full of Antigens
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Anti-Nuclear AntibodiesDiseases Associated with
ve ANA

• Drug-Induced Lupus 100
• Systemic Lupus Erythematosus 99
• Systemic Sclerosis 95
• Mixed Connective Tissue Disease 95-99
• Polymyositis\Dermatomyositis 90
• Sjogrens Syndrome 80
• Rheumatoid Arthritis 60

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Anti-Nuclear AntibodiesDiseases Associated with
ve ANA

• Chronic Active Hepatitis 100
• Myasthenia Gravis 50
• Diabetes 25
• Normal lt 5

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Anti-Nuclear Antibodiesve ANA How do we make
a diagnosis of the specific disease?

• Clinical Features
• Symptoms
• Signs
• Other Laboratory Values
• Fluorescence Pattern of the ANA
• More specific associations
• Identify Specific Antibodies
• ENA panel
• ds-DNA titer

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Anti-Nuclear AntibodiesThe SIX (6) Fluorescence
Patterns

• Homogenous
• SLE, DILE, Overlap (PM-Scl-SLE)
• Chromatin, histone, dsDNA, Ku

Rim Enhanced SLE Lamins, Nuclear pore complex
Speckled Coarse - SLE (Sm, U1-RNP) Fine - SS,
SCLE (Ro, La)
Nucleolar SSc, PM-SSc Scl 70, RNA Polymerase 1,
PM-Scl
Centromere SSc Kinetochore
Cytoplasmic SLE, PM/DM Ribosomal - P, Jo-1
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Anti-Nuclear AntibodiesPatient with SLE -
Negative ANA?

• SSA/Ro - Sjogrens Syndrome
• Histidyl-tRNA-synthetase (Jo-1) - PM/DM
• Threonyl - tRNA-synthetase (PL-7) - PM/DM
• Alanyl-tRNA-synthetase (PL-12) - PM/DM
• Anti-Cardiolipin Antibodies/Lupus Anticoagulant -
  Antiphospholipid Ab Syndrome

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Anti-Nuclear AntibodiesSummary

• Presence of auto-immune disorder suspected
  clinically
• This suspicion was reinforced with a positive ANA
• Further testing for specific antibody detection
  (ENA, ds-DNA .)

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Extractable Nuclear AntigensENA

• Detection of SIX (6) antibodies which better
  correlate with specific disease states
• Enzyme linked Immunosorbent Assay (ELISA)

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Enzyme Linked Immunosorbent AssayELISA
Chromogen - Changes colour when cleaved by enzyme
attached to the second antibody
Anti-Human Immunoglobulin coupled to an enzyme
binds to human antibodies
Conjugate
Human Antibodies Precipitate bind to antigen form
Immune Complexes
Cuvette Coated with Antigen
Patient Serum
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Extractable Nuclear AntigensMethods

• Screen - Using plates coated with all SIX (6)
  antigens - If positive (gt20) then
• Individual ELISA with plates coated with single
  antigens - Determines specifically which
  antibodies are present in patients serum

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Extractable Nuclear AntigensDisease Associations

• Antibody
• SSA/Ro
• SSB/La
• Smith (Sm)
• RNP
• Scl 70 (Topoisomerase-I)
• Jo-1(Histidyl-tRNA-synthetase)

• Disease Association
• SS, SCLE, NLE, SLE
• SS, SCLE, NLE, SLE
• SLE
• MCTD, SLE
• Diffuse SSc
• PM/DM

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Anti-Double Stranded-DNAOverview

• Many different individual antibodies to DNA
• Isotype
• Complement fixing capabilities
• Avidity for DNA
• Antigenic specificities
• Charge

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Anti-Double Stranded-DNAOverview

• Healthy humans make antibodies to DNA
• Low titers, react with low avidity
• Typically IgM
• Humans with SLE make antibodies to DNA
• Higher titers, react with high avidity
• typically IgG

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Anti-Double Stranded-DNAWhere do these
pathogenic, high titer IgG antibodies to DNA come
from?

• Genetic Susceptibility
• Environmental Stimuli
• Initiate an immune reaction
• Formation of highly mutated, potentially
  pathogenic anti-DNA antibodies

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Anti-Double Stranded-DNAAbsClinical Correlations

• Present in 60 - 83 of patients with SLE
• Other patients with SLE have other pathogenic
  antibodies (ENA Unknown)
• Very Specific for SLE if present in high titers
  gt90
• Some correlation between increasing levels and
  disease flares

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Anti-Neutrophil Cytoplasmic Antibody (ANCA)

• A collection of antibodies directed against
  components of granules inside the neutrophil
• Detected in the laboratory by Immunofluorescence
  Assay and by ELISA methods for specific antibodies

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Anti-Neutrophil Cytoplasmic Antibody
Immunofluorescence

• Same manner as ANA
• Slide with wells coated with ethanol fixed
  neutrophils
• Add patient serum and incubate
• Add anti-human immunoglobulin with a fluorescent
  tag and incubate
• View under the microscope

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Anti-Neutrophil Cytoplasmic Antibody
Immunofluorescence

• 2 patterns possible
• Cytoplasmic
• Perinuclear - artefact of the laboratory test

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Anti-Neutrophil Cytoplasmic Antibody
Immunofluorescence - Disease Associations

• Wegeners Granulomatosis
• c-ANCA 75-80
• Microscopic Polyangiitis (MPA)
• p-ANCA 50-60

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Anti-Neutrophil Cytoplasmic Antibody
Immunofluorescence Antibodies

• c-ANCAs
• Anti-Proteinase 3 (PR3)
• p-ANCA
• Anti-Myeloperoxidase (MPO)
• Elastase
• Capthesin G
• Lactoferrin
• Lysozyme
• Azurocidin
• Histone 1

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Anti-Neutrophil Cytoplasmic Antibody ELISA

• Same manner as ds-DNA
• Wells coated with Proteinase 3 (PR3) or
  myeloperoxidase (MPO)
• Add patients serum and incubate
• Add anti-human immunoglobulin with an enzyme tag
  and incubate
• Add chromagen TURNS COLOUR!

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Anti-Neutrophil Cytoplasmic Antibody ELISA
Disease Associations

• Wegeners Granulomatosis anti-PR3
• Specificity 98
• Sensitivity 40-95 depending on disease
  activity
• Microscopic Polyangiitis (MPA)
• Anti-MPO 60
• Anti-PR3 30

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Anti-Neutrophil Cytoplasmic Antibody ELISA
Immunofluorescence

• When determining if ANCAs are present must do
• Immunofluorescence for pattern
• ELISA for MPO PR3
• Why
• Negative IF and positive ELISA
• Positive IF and negative ELISA

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Anti-Neutrophil Cytoplasmic Antibody Disease
Associations

• Strong ANCA activity is a good surrogate marker
  of small vessel vasculitides (svv)
• It must fit into good clinical context
• Diagnosis should be confirmed by histopathologic
  findings on biopsy

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The Rheumatology Laboratory

• REMEMBER
• Laboratory tests do not stand alone
• Must fit into clinical context
• IF IN DOUBT
• Consult your local neighbourhood Rheumatologist