Download Med Sch lecture Immunology Laboratory SB 2012

Clinical Immunology Overview
and use of the Laboratory
This lecture will cover
• What the immunology lab measures
• How they are measured
• Why they are measured
– (You will have other lectures expanding on
this)
Immune System
Cells
Antibodies
T-cells
B cells
Macrophages
Neutrophils
Prevent infection
Complement
Components of the immune system are measured for a variety of
reasons:
•Their amounts can vary with infections
•Abnormal components can be present with certain diseases
•Immunological components can be deficient
Questions
•Are the components of the immune system present in normal
concentrations?
•Do these components function normally?
•Are there known abnormal immunological components present?
e.g. autoantibodies, paraproteins
Immune System
Cells
Antibodies
T-cells
B cells
Macrophages
Neutrophils
Prevent infection
Complement
Immunoglobulin concentrations
in serum
Normal ranges vary with age
• IgG 6-16 g/L
• IgA 0.8-2.8 g/L
• IgM 0.5-1.9 g/L
• IgD 0.1 g/L
• IgE 0. 0001 g/L
Antibodies
Total levels
of Ig G, A,
M
autoantibodies
Allergen specific IgE
Specific
antimicrobial
antibodies
Measurement of IgG, A and M
concentrations - Nephelometry
• A fixed amount of antibody specific to the
immunoglobulin of interest is mixed with
the patient sample (serum)
• Light is directed onto the reaction chamber
• Light is scattered by the presence of
antibody-antigen complexes
• The amount of light scatter is detected
Small amount of protein.
No large complexes: little light scattering
Large amount of protein.
Large complexes: light scattering
Very large amount of protein.
Small complexes: Little light
scattering
Protein concentration
Abnormal immunoglobulins
• Monoclonal / paraproteins
• Immunoglobulin components – light chains
in serum or urine (BJP)
Protein electrophoresis
• Separating serum proteins by charge to look
for abnormalities
Autoantibodies
• Many different autoantibodies have been
found.
– Each binds to a specific self antigen
• May be found at low levels in healthy
people.
– Not always associated with disease
• Most important are of IgG class
– But IgA and IgM in some cases
Autoantibodies
• May be pathological
– i.e. the antibody causes the disease
• More often found in association with
disease
– Eg cellular attack on an organ releases
neoantigens to which antibodies develop
– Indirect measure of disease state or progression
Methods for measuring
autoantibodies
• Indirect Immunoflourescence
• ELISA
• Line blot
+
Tissue section on
microscope slide
Antibodies in
Patient Sample
Antibodies bind to
proteins on tissue section
Fluorescently
labelled antibody
to human
immunoglobulin
Fluorescence
Microscope
Indirect Immunoflourescence
Gastric
parietal cells
stained due to
presence of
autoantibody
ELISA
Line blot
Recombinant Antigens fixed on
a cellulose strip
Measurement of allergen specific
IgE
• Commonly known as RAST RadioAllergoSorbentTest
– (Misnomer as radioactivity not used)
IgE levels in serum are
very very low
•
•
•
•
•
IgG 8.0 g/L
IgA 2.0 g/L
IgM 1.0 g/L
IgD 0.1 g/L
IgE 0.0001 g/L
To detect one allergen specific
IgE requires a very sensitive
method
Immune System
Cells
Antibodies
T-cells
B cells
Macrophages
Neutrophils
Prevent infection
Complement
ALTERNATIVE
pathway (AP)
Classical
pathway (CP)
Antibody
mediated
C5 convertase (AP)
C5 convertase (CP)
Terminal pathway
Lysis of bacteria
Complement measurement
• Complement components C3 & C4
• Complement control proteins C1 esterase inhibitor
• Nephelometry as for immunoglobins
• Complement functionality
• Haemolytic assay – if all the components of the pathways
are present lysis of red blood cells occurs
Immune System
Cells
Antibodies
T-cells
B cells
Macrophages
Neutrophils
Prevent infection
Complement
Lymphocytes
Distinguished by their cell surface markers (cluster of
differentiation markers or CD)
Detected by fluorescently labelled monoclonal
antibodies to these CD markers
Lymphocytes
• T cells CD3+
– CD4 lymphocytes are CD3+ and CD4+
“T helper”
– CD8 lymphocytes are CD3+ and CD8+
“cytotoxic T cells”
• B cells CD19+
Flow Cytometry
• Cells can be differentiated by there size and
granularity
• Whole blood (or fractions thereof) can be
incubated with fluorescently labelled
monoclonal antibody to cell surface markers
• Different cell types are detected by their
different surface (CD) markers
Flow Cytometry Principle
Granularity
Granulocytes
Size
CD3
CD3
Monocytes
Lymphocytes
CD8
Functional tests
• Lymphocyte activation
– In response to mitogens
– In response to antigens
• Neutrophil activation
When are Immunology
tests useful?
Are Immunology tests ever urgent?
NO
Not in the sense of say potassium (which can
kill you)
BUT
Occasionally rapid testing is required to make
an early diagnosis so that treatment can be
instigated.
Circumstances when rapid testing can be
helpful
1. Autoimmune renal disease
– Rapidly progressive renal disease
– When Goodpasture’s syndrome (anti GBM
disease) or vasculitis is suspected
• Anti GBM and ANCA should be measured
2. Suspected primary immune deficiency
– Severe combined immune deficiency
• Lymphocytes subpopulations should be measured
Measurement of Total IgG, IgA
and IgM concentrations
Non specifically in RA, SLE
autoimmune liver disease etc
Increased production
Infection
Myeloma (monoclonal)
Primary
Decreased production
Immunodeficiency
Secondary
Allergy
Specific IgE testing
• May contribute to the diagnosis of allergy
• But only in conjunction with a careful
history
• A positive sIgE does not always mean
clinical sensitivity to an allergen
• A negative sIgE does not exclude allergy
• Allergy is a clinical diagnosis
Clinical significance of
Autoantibodies :
• Presence or absence may not rule a disease in
or out
• Key is understanding the clinical significance
of antibodies for diseases –
• We use clinical sensitivity and specificity
Clinical sensitivity = % of patients with given
disease who have positive antibody
Clinical specificity = % of healthy people who
don’t have the antibody
Anti-Nuclear Antibodies (ANA)
• Homogeneous ANA pattern consistent with
anti-double stranded DNA Ab in SLE
• Speckled ANA pattern to Anti Ro (SS-A)/
Anti La (SSB) in Sjogren’s Syndrome
• ANA pattern consistent with Anti Scl 70 in
Systemic Scleroderma
• Centromere ANA pattern in Limited
Cutaneous Scleroderma (formally CREST)
More autoantibodies
• IgM Rheumatoid Factor present in about 80% of
patients with rheumatoid arthritis and about 10% of
patients without
• Anti-CCP is highly specific for RA in patients with
clinical features of disease (not to be used as screen)
• Anti tissue transglutaminase (TTG) is highly
sensitive and specific for coeliac disease
• Anti intrinsic factor antibodies with anti gastric
parietal cell ab specific for pernicious anaemia
Anti Neutrophil Cytoplasmic Antibody
(ANCA) in renal disease
P-ANCA
C-ANCA
- Anti MPO (ELISA)
-Anti PR3 (ELISA)
- Microscopic polyangiitis> Churg
Strauss > polyarteritis nodosa
-Wegener’s
Granulmoatosis
• Anti GBM antibodies in Goodpasture’s
• Anti Mitochondrial Antibodies in Primary Biliary
Cirrhosis (kidney section) Antigen is PDH
(pyruvate dehydrogenase)
• Anti Smooth muscle Antibody in Autoimmune
Hepatitis Type 1
Use of flow cytometry
• Monitoring CD4
counts in HIV
• Looking for
lymphocyte defects in
primary
immunodeficiency
Immune System
Cells
Antibodies
Complement
T-cells
B cells
Igs
Autoantibodies
Allergen specific IgE
RAST
ANA
ANCA
Anti TTG
RF
etc
etc
Immune System
Cells
Antibodies
T-cells
B cells
Flow cytometry
HIV
Primary immunodeficiency
Complement
The Lab is open for routine service 8am-5pm
Outside these hours advice is available on an
on-call basis
Questions?