Overview of Immunology and

Serology
„ Immunology is defined as
The study of the molecules, cells, organs, and
Serology Review systems responsible for the recognition and disposal
of nonself substances
The response and interaction of body components
and related interactions
MEDT 4600 The way the immune system can be manipulated to
protect against or treat diseases

Overview of Immunology and

Types of Immunity
Serology
„ Serology is a division of immunology that „ Innate
specializes in laboratory detection and Most primitive form of immunity
measurement of specific antibodies that develop General recognition
in the blood during a response to exposure to a
„ Adaptive
disease-producing antigen.
Specific recognition of small portion of
organism or triggering antigen
Can generate memory of initiator
System should eliminate self-reacting cells

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 Autoimmunity General Types of Immunity
„ When self-reacting cells persist and are „ Innate (natural)
not destroyed, autoimmunity may result First line of defense
Abnormal immune response to the host’s own No previous exposure to agent required
cells or tissues Nonspecific
„ Pathogenic destruction of tissues or organs „ Physical and chemical mechanical barriers
„ Adaptive (acquired)
Specific response to infectious agent
Immunological memory for invader

Innate Immunity Innate Immunity

„ Physical and chemical barriers
Intact skin – effective physical barrier
„ Low pH – bactericidal for many organisms

„ Normal skin flora – helps prevent

colonization by pathogens
Loss of normal flora allows resistant organisms
or fungi to proliferate

FIGURE 2-1 Innate immune defenses located at different body

sites.

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 Innate Immunity Innate Immunity
„ Mucous membranes of respiratory, „ Natural secretions
gastrointestinal, and urogenital tracts
Chemical barriers
Ciliated epithelial cells – trap and sweep away
airborne particles and organisms „ Tears
Goblet cells – produce mucus to make epithelial „ Saliva
surface sticky
Enzymes in secretions inhibit invasion by organisms „ Mucus

„ Fatty acids

„ Bile acids

Innate Immunity Innate Immunity

„ Plasma proteins – antimicrobial and
antiviral effects
Acute phase reactants
Complement cascades and components
Tumor necrosis factor alpha (TNF)
Interferons (IFN)
Opsonins – fibronectin

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 Additional Cells of the
Cells of the Immune System
Immune System
„ Leukocyte (WBC) „ Natural killer (NK)
Phagocytic cells primary to host defense 10-15% of total lymphocytes
„ Neutrophil (PMN) Destroy virus-infected and tumor cells
„ Lymphocyte
Do not require sensitization
„ Monocyte/Macrophage
Lack antigen specificity
Auxiliary cells „ Lymphokine-activated killer (LAK)
„ Eosinophil Enhanced cytotoxicity
„ Basophil „ Eosinophils – allergy; parasitic infections

Additional Cells of the

Complement
Immune System, Cont.
„ Group of proteins that assist or
“complement” the immune response
Lysis of bacteria
Opsonization
Chemotaxis
Increased vascular permeability
„ Phagocytes released to infection site
Removal of infectious agents

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 Complement Pathways and
Complement Pathways
Nomenclature, Cont.
Components and factors act in a cascade
„ Classical pathway – antibody dependent
FIGURE 5-1 Comparison of the three
„ Alternative pathway – independent of complement pathways. This figure
compares the three pathways of
antigen/antibody reaction complement activation. Note that
although each pathway has a different
„ Mannan-binding lectin (MBL) pathway – mechanism for activation and different
C3 convertases, they all share the C5-9
triggered by MBL binding to carbohydrates membrane attack complex.

on microorganisms

Inflammation Causes of Inflammation

„ Host’s response to injury or infection „ Physical agents – burns, radiation
Localizes infection; removes agent; repairs
damage; removes debris „ Chemical agents – acids, corrosives
„ Cardinal signs of inflammatory response „ Microbial – most common
Redness, heat, swelling, pain Gram negative bacteria produce
„ 3 phases of inflammatory response: endotoxins and lipopolysaccharides that
Ï blood flow to site can cause strong inflammatory responses
Ï vascular permeability Other bacteria produce exotoxins
Migration of WBCs to site Inflammation can be beneficial or
detrimental to the host

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 Phagocytosis Phagocytosis
Ingestion and digestion of foreign particles
„ Chemotaxis – migration of PMNs to site
„ Adherence of organism to PMN
Capsule, if present, helps prevent attachment of
organism to PMN
„ Opsonins coat bacteria to enhance
phagocytosis
FIGURE 2-9 Schematic diagram of
„ Particle engulfed into cell phagosome – processes in phagocytosis (CM-
phagosome fuses with lysosome – enzymes TDM).

digest and kill organism

Phagocytosis, Cont. Adaptive Immunity

Also known as acquired immunity
„ More highly evolved than, and enhances, innate
immunity
„ Features:
Response to distinct molecules
Development of immunological memory
„ Enhanced response on repeated exposure to same agent
FIGURE 2-10 Chemotaxis. Phagocytes are directed to the site of injury or inflammation.
Migration is induced by chemotactic factors such as C5a, a complement component. Other
chemoattractants also released by phagocytes adhere to the endothelial cells lining the
capillary vessel (pavementing), then squeeze between the cells (diapedesis) to reach the
target site.

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 Immunoglobulins
Antigens and Antibodies
(Antibody)
„ Immunogen – substance capable of Proteins that bind to antigen in a “lock and
eliciting a humoral or cellular immune key” fashion
response
„ Antigen – stimulates antibody
production and binds to the produced
antibody
Epitope – specific site on the antigen to
which antibody or T cell receptors bind
„ One antigen can have many epitopes

Immunoglobulins
Antibody Structure
(Antibody)
„ Each monomer of antibody - “Y” shaped
2 heavy chains – give antibody its name
2 light chains – both kappa or both lambda
„ Each monomer has 2 Fab (antibody-binding)
regions
Each Fab contains 1 heavy and 1 light chain
„ Each monomer has 1 Fc (crystallizable)
(a) (b) containing 2 heavy chains
FIGURE 2-14 (a) Immunoglobulin structure (b) Antibody shows “lock and
Complement fixation occurs at Fc region
key” specificity for the antigen.

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 Immunoglobulin Classes Humoral Immune Response
„ IgG – major immunoglobulin in blood B lymphocyte
4 subclasses
Ó Ô
„ IgM – largest; effective in microbial killing
„ IgA – secretory; present in body fluids Plasma cell Memory B cell
2 subclasses Ó
„ IgE – seen in allergy and parasites Antibody
„ IgD – regulates activation of B cells

Humoral Immune Response, Cell Mediated Immune

Cont. Response
„ Benefits of antibody production to the host: From bone marrow stem cell – cells migrate to
Opsonization of organisms thymus – become immature T cells
„ T cell receptors (TCR) bind specific ag
Agglutination or precipitation of particles
Must be on T cell surface before cell leaves thymus
Complement activation
„ Immature T cell positive for both CD4 and CD8
„ Causes lysis of organisms

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 T Cell Maturation Antigen-Presenting Cells
„ Immature T cells undergo positive and
negative selection „ Major antigen-presenting cells:
“Bad” and “useless” T cells eliminated Macrophages
“Good” T cells mature into naïve T cells:
„ Some naïve T cells become CD8 cells
B lymphocytes
Recognize MHC Class I antigens Dendritic cells
„ Some naïve T cells become CD4 cells
Recognize MHC Class II antigens

„ T cells activated when they encounter

specific antigen

Major Histocompatibility
Primary Immune Response
Complex (MHC)
„ Also known as human leukocyte antigen
(HLA)
„ IgM antibody appears first, then IgG on
„ Must be on surface of antigen-presenting cell for
first exposure to antigen
T cell activation
„ Associated with organ and tissue rejection
„ Class I MHC – on most cells
„ Class II MHC – on antigen-presenting cells

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 Overview of Immunology and
Secondary Immune Response
Serology
„ Follows re-exposure to the same antigen
„ Shorter response time
„ Larger quantity of IgG
Persists longer due to memory cells

Immunogens Antigens and Antibodies

Immunogenicity – ability to elicit an „ Nature of Antigens

immune response Size
„ Characteristics of good immunogens: Chemical composition/complexity
Complexity – large complex molecules best Foreignness
Ability to be processed by MHC
High molecular weight
Structural stability „ Characteristics and Production of Antibodies
Classes of Immunoglobulins (Antibodies)
Foreignness of antigen
Degradability

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 Classification of
Hypersensitivity
Hypersensitivity
Hypersensitivity – exaggerated immune „ Type I – Immediate (anaphylactic)
response against normally harmless „ Type II – Antibody-Dependent Cytotoxic
antigens „ Type III – Immune-Complex-Mediated
„ Can cause inflammation and/or tissue
„ Type IV – Delayed (cell-mediated)
damage
„ Involves either humoral or cell-mediated
immunity

Major Features of
Hypersensitivity Reactions
„ Serological methods and detection of
antigens/antibodies

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 Monoclonal vs. Polyclonal
Antigen or Antibody Detection
Antibodies
„ Monoclonal antibodies – homogeneous Purpose of detecting antigen or antibody in
Originate from a single B cell clone patient sample – rapidly detect and identify
Highly specific; less cross-reactivity infectious agent without culture
„ Polyclonal antibodies – heterogeneous „ Test antigen or antibody reagent used to
Produced by isolating and purifying antibody detect its opposite in patient sample
from animals immunized with an antigen
Resulting ab/ab complex can be detected by
Lack specificity; recognize multiple antigenic
determinants another method, such as enzyme,
fluorescent, or chemiluminescent

Advantages of Antigen Diagnostic Criteria for

Detection in Specimens Evaluation of Technology
„ Rapid detection and reporting „ Criteria used to validate new methods:
The infectious agent can be detected rapidly Accuracy
in the specimen; no need to wait until Precision
antibody is produced during immune Sensitivity
response
Specificity
„ Can be used to detect non-culturable Positive predictive value (PPV)
organisms
Negative predictive value (NPV)
„ Can be used to confirm identification of an
organism isolated on culture

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Principles of Immunologic and
Precipitin Tests
Serologic Methods
„ Agglutination vs Precipitation Principle – ag and ab diffuse toward each other
Agglutination is the term used to describe the
from wells cut in agar or gel
aggregation of particulate test antigens A precipitate forms at equivalence
„ Two phases: sensitization and lattice formation „ Ouchterlony double immunodiffusion
Requires 24 hours
Precipitation is the term applied to aggregation of
soluble test antigens „ Counterimmunoelectrophoresis (CIE)
„ Zones of equivalence Similar principle to Ouchterlony but current is
Pro-zone, equivalence, post-zone applied – results available in 1 hour or less
Has been replaced by other more-rapid methods

Latex Agglutination Tests Hemagglutination Tests

Agglutination is similar in principle to „ Hemagglutination inhibition (HI) assays are
precipitation (ag/ab reaction), except in latex used to detect titer (dilution) of viral antibody
agglutination (LA), where the ag or ab is that inhibits the agglutination of RBCs
bound to a particulate carrier (latex) Dilutions usually made in microtiter plate
„ Tests must be standardized and controls „ Disadvantages of HI tests:
included: Nonspecific inhibitors may be present in serum
Positive antigen control Experience necessary for correct interpretation of
results
Negative antigen control
Control latex suspension

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Advantages and Disadvantages of
Particle Agglutination
Latex Agglutination Tests
„ Advantages:
Do not require viable organism
Reagents complete in kit form
Relatively rapid and easy to perform
Good sensitivity
„ Disadvantages:
Subjectivity in reading results
Nonspecific reactions – may be false positive or
false negative when compared to culture

Principles of Immunologic and

Immunofluorescent Assays
Serologic Methods
„ Immunofluorescent Assays Mono- or polyclonal antibodies are conjugated
Direct Immunofluorescent Assay with fluorescent dyes
Indirect Immunofluorescent Assay Fluorescein isothiocyanate is a popular
fluorochrome (green fluorescence)
Reaction observed with fluorescent microscope
„ Direct fluorescent antibody (DFA):
Antigen-specific labeled antibody applied to
specimen on slide; wash; examine for fluorescent
organism

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 Immunofluorescent Assays,
DFA and IFA Tests
Cont.
„ Indirect fluorescent antibody (IFA)
Patient serum applied to known antigen (i.e.,
cell) on slide and incubated Î wash, add FIGURE 7-7 A schematic
fluorescein-labeled antihuman globulin (AHG), representation of direct and
indirect fluorescence antibody
which will bind to human serum Î wash, tests.
examine slide for fluorescence
„ If antibody of interest is present in patient serum –
antigen on slide will fluoresce
„ No antibody – no fluorescence observed

Advantages and Disadvantages of

DFA and IFA Tests, Cont.
Immunofluorescent Assays
„ Advantages:
Rapid visualization of infection
Increased specificity since the morphology of the
organisms can be observed
„ Disadvantages:
Expensive; requires fluorescent microscope
Subjective
Not easily automated
Instability – fluorescence fades over time
FIGURE 7-8 A diagram of incident light microscope showing the light path.

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Principles of Immunologic and
Enzyme Immunoassays (EIA)
Serologic Methods
„ Enzyme Immunoassays Enzymes are conjugated to antibodies –
Enzyme immunoassay (EIA) uses enzyme molecules both antibody-binding and enzymatic
that can be conjugated to specific monoclonal or properties are preserved
polyclonal antibodies.
Common enzymes used:
Direct and Indirect Sandwich Technique
„ Alkaline phosphatase, horseradish peroxidase
Membrane-Bound Technique
„ Like fluorescent tests, can be direct or
indirect; principle is same except the
label is an enzyme, not a fluorochrome
„ Advantage – easily automated

Enzyme Immunoassays (EIA),

EIA, Cont.
Cont.
„ If antigen in the specimen has bound to the
enzyme-conjugate antibody complex, the
enzyme will act on the specific substrate added,
producing a visible-colored end product
„ If specimen does not contain antigen, enzyme-
FIGURE 7-9 antibody is removed during wash and substrate
Schematic
flow diagram
does not change color
of direct
sandwich
immunassay.

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Principles of Immunologic and
Electrochemiluminescence
Serologic Methods
„ Other Labeling Techniques „ Principle – certain chemical compounds emit
Chemiluminescence light when electrochemically stimulated
„ When current is applied, excited chemicals
serve as a detection signal for specific
antigen/antibody reactions
„ Advantages:
Highly specific; less background luminescence
compared to fluorescent methods

Complement Fixation Tests,

Complement Fixation Tests
Cont.
Laboratory tests used to detect antibodies to
viruses, fungi, and rickettsia:
„ Since complement is required for both
systems (Test and Indicator):
„ Patient complement destroyed – heating
If sheep RBCs do not lyse, patient has
„ Patient serum mixed with specific test antigen
antibody to organism (complement bound in
and standardized amount of complement test system) – positive test
(Test System)
If sheep RBCs lyse, patient does not have
„ After incubation, sheep RBCs and antibody to
antibody to organism (complement not bound
sheep RBCs added (Indicator System) in test system; bound in indicator system) –
negative test

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 Complement Fixation Test Syphilis
„ Caused by Treponema pallidum, a non-
FIGURE 5-9 Complement fixation culturable spirochaete
test. The use of the complement
fixation test in demonstrating „ Syphilis is a sexually transmitted disease
presence of antibody in a (STD) occurring in 4 stages:
patient’s serum is shown in the
figure. Primary – chancre (lesion) is first symptom
Secondary – systemic spread and skin rash
Latency – no longer contagious; may last for years
Tertiary – cardiovascular or neurological lesions
„ Congenital – fetus acquires disease from
mother who has syphilis while pregnant

Non-Treponemal Tests for

Treponemal Tests for Syphilis
Syphilis
„ Detect reagin in patient serum – an antibody- „ Confirmatory tests for syphilis
like substance „ Detect antibody to treponemes – persists
„ Useful for screening – Ï sensitivity; for life (seropositive)
Ð specificity – many false reactives „ Use treponemes as the antigen
„ Flocculation – most common type of test „ Treponemal tests:
Antigen is cardiolipid
Treponema pallidum immobilization (TPI)
VDRL – microscopic test using serum „ Patient antibody immobilizes live treponemes
RPR – macroscopic test on serum or plasma „ Rarely done except in syphilis research labs

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 Treponemal Tests for Syphilis, Treponemal Tests for Syphilis,
Cont. Cont.
Fluorescent treponemal antibody absorption Direct fluorescent antibody for T. pallidum
(FTA-ABS) (DFA-TP)
„ Directs immunofluorescent antibody test „ Fluorescent-labeled anti-T. pallidum antibody is used to stain
lesion material on slide
FTA-ABS double staining (DS)
„ Test is specific for T. pallidum and does not cross-react with
„ Adds a contrasting fluorochrome-labeled counterstain to
other treponemes
the FTA-ABS test
Microhemagglutination assay for T. pallidum NOTE: Darkfield microscopy, once used to
(MHA-TP) visualize motile spirochaetes in lesion material,
„ Qualitative hemagglutination test is now rarely performed
„ Less sensitive than FTA-ABS for primary syphilis

Viral Hepatitis Hepatitis A (HAV)

„ Non-culture methods are most common „ Transmission – oral-fecal
for laboratory diagnosis Associated with foodborne outbreaks or
contaminated water (floods, etc.)
„ Methods:
„ Testing – EIA for anti-HAV antibodies
Viral antigen detection
Anti-HAV IgM appears 5-10 days prior to
Serological response to viral antigens symptoms; decreases to undetectable levels
Performance of both antigen and antibody tests in ~6 months
important in differentiating acute from chronic Anti-HAV IgG develops later in infection and
disease confers lifelong immunity

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 Hepatitis A (HAV), Cont. Hepatitis B (HBV)
„ Transmission – parenteral (blood and
body fluids), sexual, congenital
„ 10% of infected patients become carriers
with Ï risk of liver cancer
„ Testing – EIA for ag or ab in serum
Hepatitis B early antigen (HBeAg) – early
marker of HBV infection; marker of active viral
FIGURE 7-11 Serologic response to HAV infection showing the rise and replication
decline of detectable antibodies. LFT: liver function tests.

Timeline of Antigens and

HBV, Cont.
Antibodies in HBV Infection
Hepatitis B surface antigen (HBsAg) – early
marker of HBV infection; should be undetectable
in ~3 months
Anti-HBsAg – appears 6-7 months post-exposure
„ Marker of immunity after natural infection
„ Marker of immunity in HBV-vaccinated individuals
Anti-HBc (core antibody) – remains detectable for
life
„ Only found in natural HBV infection
FIGURE 7-12 Serologic response to HBV infection showing the rise and
decline of detectable antibodies.

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 Timeline of Antigens and
Hepatitis C (HCV)
Antibodies in HBV Infection, Cont.
„ Now known to be the agent of ~90% of all
hepatitis formerly known as non-A non-B
(NANB) hepatitis
Was formerly associated with transfusion-
related hepatitis
„ Transmission – parenteral, sexual,
congenital

Human Immunodeficiency Virus

HCV, Cont.
(HIV)
„ Screening test: „ Agent of acquired immunodeficiency
ELISA for anti-HCV syndrome (AIDS)
„ Confirmatory tests: „ Markers of AIDS infection:
Recombinant immunoblot assay (RIBA) Decrease in CD4 (T helper cells)
„ Detects antibody to 4 recombinant viral antigens Altered CD4/CD8 ratio
on a solid phase
„ Normally 2:1; in AIDS 1:2
Nucleic acid test (NAT)
„ Detects HCV nucleic acid

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 HIV, Cont. HIV, Cont.
„ Antibody screening tests – various HIV EIA
tests have high sensitivity; may have false
„ Fourth generation HIV tests
positives Developed to cover window period – time
Indirect after exposure when patient is infectious
„ First-generation binding assays nonspecific but has not developed measurable
„ Second-generation recombinant technology improved
specificity
antibody titer
Antibody capture – anti-human IgG to Fc of anti- EIA assay – detects HIV-1 and HIV-2 viral
HIV captures anti-HIV IgG in serum antigen and antibody combinations
Sandwich – third generation most sensitive simultaneously
„ Detects all classes of antibody against HIV-1
„ Used in Europe, but not FDA approved in U.S.

HIV, Cont. HIV, Cont.

„ Confirmatory tests: Rapid testing methods – results in less than 10
Western blot (WB) – disrupted HIV virus is minutes
electrophoresed; blotted onto nitrocellulose „ Useful in needlestick accidents
and incubated with patient serum „ Suited for screening in remote areas without
„ Specific antibody binds to viral protein and modern laboratories
produces bands „ Easy to perform
„ Bands are detected by EIA or biotin-avidin
„ Test methods:
„ CDC interpretation for positive HIV – bands at 2
of 3 locations – p24, gp41, gp120/160 Flow through or dot blot membrane EIA

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 HIV, Cont. HIV-2
„ HIV antigen test – EIA for p24 antigen „ Low prevalence in U.S., but blood
Detects active viral replication products are screened for both HIV-1 and
Provides early detection of infection in a
HIV-2
neonate born to an HIV-positive mother „ Screening test:
Used for screening banked blood EIA – combined HIV-1/HIV-2 sandwich assay
– 99.5% sensitivity
„ Confirmatory test for HIV-2:
Recombinant immunoblot

Herpes Simplex Virus

Herpesvirus Family
(HSV-1 and HSV-2)
„ Many members of this family cause a wide „ HSV-1 most common in oral lesions
variety of illnesses from asymptomatic Î rashes „ HSV-2 most common in genital lesions
and lesions Î serious infection (meningitis, NOTE: HSV-1 can cause genital lesions and HSV-2
encephalitis) oral lesions
„ These viruses cause infection, followed by „ Clinical presentation on reactivation:
latency in neurons – may reactivate Mucocutaneous blisters rupture and heal
Can cause serious infections in the Ocular or skin lesions
immunocompromised Encephalitis or meningitis
„ Gold standard – culture from active lesion

23
 Varicella-Zoster Virus
HSV-1 and HSV-2, Cont.
(VZV)
„ Serological assays: „ Varicella (chickenpox) – easily
EIA – herpes-specific, HSV-1-specific, or diagnosed clinically by characteristic
HSV-2-specific fever and rash in young children
„ Significant serological cross-reactivity between
„ Zoster (shingles) – painful rash along
HSV-1 and HSV-2
Specific test to distinguish HSV-1 from HSV-2
sensory nerves in adults due to
– detection of antibody to glycoprotein G1 or reactivation
G2 „ Confirmation of infection:
„ Rarely done, but commercially available Viral culture
DFA of vesicular scrapings

Epstein-Barr Virus
VZV, Cont.
(EBV)
„ Serological diagnosis – rare „ Associated with infectious mononucleosis
Latex agglutination – low sensitivity (IM), Burkitt’s lymphoma, nasopharyngeal
carcinoma
Fluorescent antibody membrane antigen
(FAMA) „ Rapid serological diagnosis:
Rapid spot test for IM – detects heterophile
„ Most sensitive but time-consuming
antibody in 80-90% of IM cases
„ Requires viral replication in cell culture
Sensitized latex particle tests – less chance of
cross-reactivity
„ Beads coated with IM antigen from bovine RBCs used to
test for agglutination in serum

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 EBV, Cont. EBV, Cont.
„ Testing for EBV-specific viral proteins –
IFA or EIA methods help differentiate
acute and chronic disease
Viral capsid antigen (VCA)
„ VCA-IgM appears early in disease, followed by
VCA-IgG
Early antigen (EA) – peaks at 2-4 months
Epstein-Barr nuclear antigen (EBNA)
„ Hallmark of convalescence FIGURE 7-16 Serologic response to EBV infection showing the rise and
decline of detectable antibodies.

Cytomegalovirus
CMV, Cont.
(CMV)
„ Usually mild, often asymptomatic infection „ Serological testing for CMV IgM or IgG
in immunocompetent hosts; infection can available, but not FDA-approved for
be life-threatening in the screening blood donors
immunocompromised „ Rapid detection of CMV viremia in the
„ CMV antigenemia assay provides much immunocompromised:
faster evidence of infection than culture Nucleic acid amplification
FA staining of buffy coat of whole blood – Hybrid capture assay
number of positive PMNs are counted

25
 Measles
Measles, Cont.
(Rubeola)
„ Measles, mumps, rubella (MMR) vaccine Serological methods:
has been successful in decreasing the „ Measurement of IgG:
prevalence of measles CF
Epidemics still occur in poorly vaccinated HI
populations Plaque reduction neutralization assay (PRNA)
Rubeola in geriatric patients associated with „ EIA tests:
brain dementia – subacute spongiform pan-
IgM EIA – diagnosis of recent infection
encephalitis (SSPE)
IgG EIA – assessment of immune status

Rubella
Mumps
(German Measles)
„ Most common feature – swelling of „ Usually causes mild fever and rash in
salivary glands and mild disease children
„ EIA most common for serology „ Can cause serious congenital defects in a
fetus born to a mother who acquired rubella
EIA for IgM – indicates recent exposure
during pregnancy
EIA for IgG – immune status Congenital infection – IgM detected at birth
„ CF, HI, SN, IF methods also used „ Serological methods:
Passive hemagglutination (PPA) or HA
Solid phase capture EIA for IgG and IgM

26
 Systemic Autoimmune
Systemic Autoimmune Diseases
Diseases, Cont.
„ Systemic diseases characterized by: „ Most common systemic diseases:
Nonspecific symptoms Rheumatoid arthritis (RA)
Autoantibodies that react with antigens in Systemic lupus erythematosus (SLE)
multiple cells/organs of the body „ Linked to disease predisposition:
Damage to collagen in vascular or connective Genetic, gender, environmental factors
tissue „ Symptoms of these diseases overlap; few
„ Immune complexes, autoantibodies, and acute tests are specific for a particular disease
inflammatory response cause most damage

Laboratory Testing for Systemic Nonspecific Tests for

Diseases Inflammation
„ Initial laboratory workup for a suspected „ ESR – increased in inflammation
autoimmune disease includes: Affected by incorrect handling of specimen
CBC, metabolic panel, urinalysis, C-reactive Affected by physiological factors not related to
protein (CRP) or erythrocyte sedimentation inflammation
rate (ESR), test for rheumatoid factor, test for „ CRP – unaffected by noninflammatory
presence of antinuclear antibodies (ANA),
complement levels conditions or gender
Markedly elevates just after acute
inflammation and falls in 48 hours after
resolution

27
 Nonspecific Tests for Nonspecific Tests for
Inflammation, Cont. Inflammation, Cont.
„ Complement levels „ Rheumatoid factor (RF)
May be helpful in monitoring disease activity IgM antibody to Fc portion of IgG
and response to therapy Seen in various autoimmune conditions
CH50 (complement hemolytic activity), C3, Can be seen in the healthy
„ RF may be positive in 10-25% of people >70 years old
and C4 commonly measured
RF Ï (titer >1:80) in 2/3 of patients with RA
SLE – complement usually decreased A positive test is not diagnostic; a negative test
RA – complement usually increased does not rule out RA
Methods of detection – latex agglutination, EIA,
nephelometry

Nonspecific Tests for Nonspecific Tests for

Inflammation, Cont. Inflammation, Cont.
„ Anti-nuclear antibodies
Common screening test
„ A positive test is not diagnostic; a negative test
virtually rules out SLE
False positive results seen in aged, thyroid
conditions, chronic infection, viral infection
Methods – IFA most common; significant
titer >1:160; multiple patterns of
fluorescence can be seen
„ Peripheral pattern is highly specific for SLE

28
 Nonspecific Tests for
Rheumatoid Arthritis
Inflammation, Cont.
„ Confirmatory test for SLE „ Chronic disease with inflammation and
IFA for anti-dsDNA – examine slide for fluorescent destruction of joints
kinetoplast in hemoflagellate Crithidia luciliae
„ Probable cause – infection with virus
„ Extractable nuclear antigens (ENA)
Nuclear proteins associated with RNA
(EBV) or bacterium
Anti-SS-A/Ro and anti-SS-B/La – seen in SLE or Response to this foreign antigen attracts cells
Sjogren’s syndrome to the synovium; cytokines cause
Anti-nucleolar ab. – seen in scleroderma inflammatory response

Rheumatoid Arthritis, Cont. Rheumatoid Arthritis, Cont.

FIGURE 8-7 Normal joint vs. rheumatoid arthritis.

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 Clinical Features of Rheumatoid
Rheumatoid Arthritis, Cont.
Arthritis
Immune complexes (IgM anti-IgG bound to „ Most common rheumatic disease
IgG) in joint binds complement and attracts „ Onset of disease – 3rd-5th decade
PMNs and macrophages
„ 2-3 times more common in women
Cytokines released from macrophages and
„ Linked to disease predisposition:
lymphocytes, lysozymes, and other proteases
from neutrophils damage the cartilage in the HLA-DRB1*0101, DRB1*0104, DR4
joint „ Nonspecific symptoms include:
Weight loss, malaise, stiffness, and joint
tenderness (especially in AM)

Laboratory Testing for

Clinical Features, Cont.
Rheumatoid Arthritis
„ Treatment – non-steroidal anti- „ Nonspecific findings:
inflammatory drugs (NSAIDs) to start; as Normochromic, normocytic anemia, Ð serum iron, Ï
total protein, Ï ESR, positive CRP
disease progresses, methotrexate,
„ Laboratory marker for RA – presence of
steroids, biologic disease modifiers may
rheumatoid factor; not specific for RA, but ~80%
be used of people with RA have Ï RF
Positive – >60 U/ml by nephelometry; titer of >1:80 by
agglutination

30
Systemic Lupus Erythematosus
Clinical Features of SLE
(SLE)
Classic example of systemic autoimmune „ Onset of disease – 18-65 years of age
disease (20-40 most common)
„ Circulating immune complexes deposited „ 9 times more common in women

in various tissues cause inflammation and „ 3 times more common in African

destruction Americans than Caucasians
Joints, skin, kidney, brain, and lungs most „ Linked to disease predisposition:
commonly affected HLA-DR2, DR3; HLA-A1, B8, DR3 haplotype
UV radiation, chemical exposure

Clinical Features, Cont. Laboratory Testing for SLE

„ Symptoms range from mild to severe „ Preliminary tests:
Fatigue, fever, weight loss, joint pain, ANA screen, CBC, urinalysis
photosensitivity, butterfly rash (<40% of
patients) „ Findings in patients with SLE:
Renal complications occur in ~50% of patients Positive ANA with peripheral pattern (most
„ Treatment – NSAIDs, steroids, antimalarial common), Ð complement, Ï
drugs for joint pain, methotrexate immunoglobulins, immune complex deposits
visualized by DFA in biopsy material

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Principles of Immunologic and
Serologic Methods
„ Molecular Techniques
Polymerase chain reaction
Southern blot and Northern blot
Western blot
Microarrays

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