Serology Review
2011
BY:
JOANNA ELLIS, (MLS)ASCP
Overview
 Study your notes and labs from the spring.
 Make sure you know the information related to the
objectives.
 The certification exam will test over knowledge of
immunology and serology, so need to know
immunological theories as well as testing specific for
conditions.
 The Serology final will also test over this.
 This review is BRIEF and covers the high points only.
Overview
 Only a few select serological tests are performed in
clinical laboratories. Due to this fact serology is
performed on campus to give everyone equal
experience.
 A kit test is a kit test. As an MLT you must be able to:

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Read and accurately follow directions of package insert.
State principle, sample requirements, limitations of the
procedure and causes of false positives and negatives.
Accurately perform test according to package insert or written
instructions.
Report out results in required format.
Traits of Immunogens
 Foreignness
 Ex. Plant protein more immungenic than animal protein
 Size
 >10,000 daltons
 Complexity
 Heterogeneity in building blocks that comprise the antigen
 Proteins>polysaccharides>>lipids and nucleic acids
Haptens
 Small molecule that, by
themselves, are NOT
immungenic.
 When coupled to a high
molecular weight
protein, the hapten
becomes the antigenic
determinant (epitope)
for the antigen .
Definitions
 Chemotaxis
 the migration of cells to an inflammatory site following
interaction with chemoattractants or chemokines.

Ex. C5a of the complement system
 Opsonization
 The process by which plasma proteins are attached to a
foreign substance and prepare it for phagocytosis

Ex. IgG
 Epitope (antigenic determinant)
 Small part of the immunogen that is recognized by B or T cells.

Can be as few as 6 amino acids
More Definitions
 Antibody

Please tell me you can define antibody by now.
 Heterophile antigen

Antigens that appear on the surface of tissues of several different species
 Heterophile antibody

Antibodies that are generated in response to an antigen in one species,
but are cross reactive with an antigen in another species.
Humoral Immunity
 Exposure to antigen causes production of specific
antibodies by B cells.
 Life long
 Two types of responses – must know length of
time for antibody production and antibody class
produced

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Primary – first exposure, takes time, IgM the IgG, slow
Secondary – second exposure to same antigen, IgG, fast
Parts of the Antibody
Another view of IgG Antibody Structure
Classes of Antibodies – Know Structure and Function
 IgG, IgD, and IgE: all
monomers

IgG: Gamma heavy chains

IgD: Delta (Δ or δ) heavy chains

IgE: Epsilon (Ε or ε) heavy chains
 IgA: Alpha -monomer or dimer
 IgM: Mu - pentamer
Comparison of Antibodies
Innate versus Acquired (Adaptive) Immunity
Innate versus Adaptive Mechanisms
Factors that determine Immune Response
 Dose
 High dose: Tolerance or immunological paralysis
 Intermediate: Best response
 Low dose: little stimulus OR induces tolerance
 Route
 Enteric administration : preferred route for intestinal parasite
immongens—frequently induces B cell tolerance rather than an immune
response
 Parenteral administration


Intradermal and subcutaneous routes stimulate the immune cells of
lymph nodes , faster and more pronounced immune response.
IV administration stimulates immune cells in spleen first, slower and less
effective
 Adjuvants enhance the immune response
 Often oil and water mixture
 Aluminum potassium sulfate is the only one approved for human use
Cellular versus Humoral Immunity
Primary versus Secondary Response
Complement
 Purpose


Promote the inflammatory response by opsonization which enhances
susceptibility of coated cells to phagocytosis.
Alter biological membranes to cause direct cell lysis.
 Three pathways:
 Classical – activation caused by IgM (1 molecule minimum) or IgG
(2 molecules minimum in close proximity) - know recognition,
activation and membrane attack units.
 Alternative (properdin) - occurs independently of antibody, must
know triggers
 Lectin - begins when mannan-binding protein (MBP) binds to the
mannose groups of microbial carbohydrates.
 Must know the activation sequence of each
Classical Pathway of Complement Activation
Specificity versus Sensitivity
 Specificity: How true is the test result?
 Ability of a test to correctly EXCLUDE individuals who do
NOT have the given disease/condition
 How likely is the test to detect the absence of a characteristic
in someone without the characteristic?
 Sensitivity: How small of an amount can be
detected?

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Ability of a test to correctly identify individuals who HAVE a
given disease/condition
How likely is the test to detect the presence of a characteristic
in someone with the characteristic?
Principle of Serological Tests
 Cause a reaction between antigen and antibody to
produce a DETECTABLE reaction.
 One must be UNKNOWN.


Look for antigen OR antibody.
An antibody MAY be the antigen in the test system, i.e., RF is
an IgM class antibody directed against IgG. In this test latex
particles coated with IgG antibody, looking for antigen which
in this case is the IgM RF present.
 MANY different test systems developed.
Factors affecting Ag/Ab Reactivity
Basic Immunologic Procedures
 Measurement by Light
 Precipitation
 Electrophoretic Techniques
 Agglutination Reactions – still popular
 Labeled reactions – very popular
 Molecular techniques – gaining in popularity
especially for pathogens.
Nephelometry
 Measures turbidity of
sample by passing light
thru it, amount of light
scatter is measured.
 Two types:


Endpoint – reaction goes
to completion
Kinetic – light scatter
measured at specific time.
Reaction occurs at a
steady rate and the timing
of measurement can be
done.
Precipitation
 Involves combining soluble antigen with soluble
antibody to produce visible, INSOLUBLE complexes.
 Relative concentrations of antigen and antibody
must be equal
Liquid Precipitation
Gel Precipitation: Radial Immunodiffusion (RID)
Double Gel Diffusion : Ouchterlony
Electrophoresis
 Process of separating
proteins in a mixture
utilizing their different
net electrical charges
 Size and shape can cause
frictional drag
 Types
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Moving Boundary
Disc
Capillary zone
Immunoelectrophoresis
Capsular Precipitation
 Swelling of the capsule surrounding a bacterium as a
result of interaction with anticapsular antibody,
consequently the capsule becomes more refractile
and conspicuous.
Agglutination
 Occurs in two stages:
 Sensitization – cannot be seen
 Lattice formation – visible
 Antigen or antibody can be
coated onto or an integral
part of a carrier particle:
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Latex particles
Red blood cells
Charcoal
Bacteria
 Agglutination indicates
presence of substance being
tested for.
Labeled Reactions
 One of the reactants labeled with a tag:
 I125 – measure radioactivity
 Enzyme – color or intensity of color measured.
 Measure intensity of light emitted as a result of reaction.
 Fluorescence
 Review lecture guide for specifics.
Molecular
 Rapidly exploding field.
 Polymerase Chain Reaction (PCR) allows replication
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of genetic material specific to an infectious agent or
malignancy.
Probe is prepared which has target sequence.
Use thermocycler to cause DNA to denature
(separate) then cool to cause annealing to probe.
Amplify the specific target.
Becoming EXTREMELY popular.
Review lecture notes for specifics.
C-Reactive Protein
 Non-specific protein that
appears in serum as a
response to inflammatory
conditions
 Involved in opsonization and
complement fixations
 Latex agglutination available

Coated with Anti-CRP
 Conditions elevated
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Bacterial infectiosn
Viral infectiosn
Active rheutmatic fever
Active rheumatoid arthritis
TB infections
Malignancies
Following surgeries
C-Reactive Protein
 ESR also used to gauge inflammation.
 C-RP has the following advantages over the ESR:
 Rises quickly DURING inflammation.
 Decreases quickly once inflammation resolved.
 Not affected by anemia or abnormal serum proteins.
Syphilis
 Caused by spirochete


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Treponema pallidum
Early: chancre: 90%
seropositive within 3
weeks
Secondary: 6-8 weeks
100% seropositive
Latent: seropositive,
symptoms absent
Tertiary: Years later, new
lesions any body part
Syphilis Lab Tests
 Direct Microscopic


Darkfield
Fluorescent Antibody
 Nontreponemal: detect antibody to cardiolipin called
reagin

VDRL: flocculation, prone to false positives

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Antigen composed of cardiolipin, cholesterol, lecithin
RPR: modified VDRL with charcoal particles, more sensitive
Trust: similar to RPR
 Treponemal: detect antibody to the spirochete or
spirochete itself
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FTA-ABS: confirmatory test that uses slides fixed with Nichols strain
of T. pallidum that will react with patient antibody
EIA: not as sensitive as FTA
DNA probe
Syphilis Testing
 REMEMBER if a non-treponemal antibody screening
test is positive MUST do specific treponemal
antibody test.
 RPR CANNOT be performed on CSF or cord blood.
 VDRL can be performed on CSF.
 Review lecture notes for causes of false positive and
false negative reagin tests.
Syphilis Testing
 VDRL
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Must heat serum to 56C for 30
minutes prior to testing to
inactivate complement which
can cause a false positive.
Antigen must be prepared
daily.
Test read microscopically.
 RPR
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Modified commercially
prepared antigen attached to
charcoal.
Serum does not have to be
heated.
Plasma can be used.
Read macroscopically.
Lyme Disease
 Causative agent: Borrelia
burgdorferi

Transmitted by Ixodes
scapularis
 Bull’s eye rash
Lyme Disease Laboratory Testing
 Ab may not be detectable
until 3-6 weeks after bite
 Acute cases (first 2
weeks) serological testing
too insensitive.
 Immunofluorescence
assay (IFA)

B. burgdorferi antigen on
slide

Cross-reactivity possible
 Enzyme immunoassay
(EIA)

Sonicated B. burgdorferi
 Western Blot most
common confirmatory
test
 PCR
Group A Streptococcus
Streptococcal Infections
 Causative agent Streptococcus pyogenes.
 Organism found only in man.
 Leading cause of oropharyngitis which may lead to
serious complications (sequelae)


Rheumatic fever
Acute glomerulonephritis
 Culture and rapid screening tests detect early
infection.
 Two major sites of infection


Upper respiratory tract - oropharyngitis
Skin - Pyoderma or Impetigo
Group A Lab Tests
 Culture
 Rapid Test
 Detects antibodies to carbohydrate specific to Group A
 PCR – DNA probes becoming popular in place of
serology and culture
 ASO

Antistreptolysin O detected suggests recent infection
 Anti-Dnase B
 Streptyozyme
 Reagent RBCs with streptokinase, hyaluronidase, Dnase, and
NADase
 Hemagglutination indicates positive result
Cold Agglutinins
 Transient Ab that appears



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in patients with
Mycoplasma pneumoniae
Anti-I specificity
Reacts preferentially at
4C, agglutination reversed
if heated at 37C.
Must run RBC control
Fourfold or more rise in
titer is significant
Herpes
 Many types of Herpes viruses, only need to know
most common
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Epstein-Barr virus (EBC)
Cytomegalovirus (CMV)
Herpes simplex virus (HSV) type I and II
Varicella-zoster virus
Infectious Mononucleosis
 Causative agent Epstein-
Barr Virus (EBV)
 Forssman
antigen=heterophile
antigen
 Paul-Bunnell Test


Sheep RBCs are added to
dilutions of patient serum
Agg indicates presences
Serum sickness Ab
 Forssman
 IM antibody

Davidson Differential Test
 If Paul Bunnell is positive
Type of Heterophile
Antibody
Absorption by Guinea
Pig Kidney Tissue
Absorption by Beef
RBCs
IM
NO
YES
Forssman
YES
NO
Serum sickness
YES
YES
CMV
 Symptoms resemble IM
 Serious for patients with defective immune systems
 In babies may cause life threatening illness
 Patients with deficient immune systems
 AIDS patients
 Transplant patients
 Detection of CMV antigen in cells using IFA
 ELISA to detect antibody to CMV
 Other
 fluorescence assays,
 indirect hemagglutination, and
 latex agglutination
 False positives can occur due to RA and Epstein-Barr
antibodies
Herpes Simplex Virus (HSV)
 Possesses viral latency – hibernation
 Two types:
 HSV-1 causes lesions above the waist, cold sores
 HSV-2 causes lesions below the waist, genital area
 Lab tests
 Recovery of virus from culture
 Direct examination of cells from lesion using IF or
immunoperoxidase stain
 DNA probes
 ELISA
 Latex agglutination
 RIA
 Indirect IF
Varicella - Zoster
 Varicella causes chicken pox.
 Zoster causes Shingles
 Important to distinguish VZV from other infections
 PCR
 Direct Fluorescent Antibody staining
 Viral culture
 IgG and IgM antibody test by ELISA
Rubella
 Single-stranded, enveloped RNA virus of the genus
Rubrivirus, belonging to the family Togaviridae
 Causes German measles
 Vaccine (MMR) given at 15 months
 Testing


ELISA most common
Hemagglutination inhibition (HI)


Ab against the viral protein responsible for haemagglutination can
prevent haemagglutination
Serology tests
IgM and IgG appear as rash of German measles begins to fad
 IgM decline by 4-5 weeks, but may persist for a year
 IgG provide immunity for life

Pregnancy Test
 Human Chorionic Gonadotropin (HCG): glycoprotein
hormone produced in increased amounts in pregnant
women and in some tumors
 Radioimmunoassay testing

Quantitative


Can determine gestational age
Serum
 ELISA

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Qualitative
Anti-HCG bound to membrane

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Can detect HCG 7-10 days after conception
Serum
Urine
Hepatitis
Type
Route of transmission
Acute/Chronic
Vaccine
A
Fecal-oral
Acute
2 doses, first at
12 mo, then 6
mo later
B
Contact with infected blood,
seminal fuid, vaginal
secretions
Chronic, can lead to
cirrhosis and cancer
3 doses anytime
with 6 mo
between
C
Infected blood
Chronic, can lead to
cirrhosis and cancer
No
D
Infected blood, sexual contact
Only infects people
with HBV
HBV vaccine
E
Fecal-oral
Acute
none
Hepatitis
 Panel done to determine whether infection is current
or past.
 Presence of IgM indicates acute infection.
 Presence of IgG indicates previous exposure, cannot
determine when.
 Hepatitis B Surface Antigen (HBsAg) first marker to
appear in HBV infection,
HIV
 HIV belongs to the genus
Lentivirinae of the virus
family Retroviridae
 HIV is a spherical
particle with an inner
core with 2 copies of
single stranded RNA,
surrounded by a protein
capsid and an outer
envelope of glycoproteins
embedded in a lipid
bilayer.
Testing for HIV
 ELISA screening test.
 Western blot is confirmatory test.
 Molecular testing used to follow HIV load and
determine success of treatment.
HIV Testing
HIV Time Line
ASCP Content Outline Serology
V. IMMUNOLOGY (7% of total exam)
 1. Immunity
 A. Autoimmunity



1) ANA, anti-DNA
2) Thyroid antibodies
B. Pre-Analytical, Test Principles
 2. Infectious Diseases
 A. Viral

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
1) EBV/infectious mononucleosis
2) Hepatitis
3) HIV/HTLV/CMV
4) Rubella/measles
5) Other viruses
B. Microbial



1) Cold agglutinins
2) Syphilis
3) Other microorganisms
End of Review
 It is important to review the course materials from
the Fall semester.
 The exam will cover BOTH immunology and
serology.