IMMUNOLOGY AND

SEROLOGY
REVIEW
PREPARED BY:

DANIELLE ALEXANDRA D. CARVERO, RMT, MLS(ASCPi)

IMMUNOLOGY
• study of the reactions of a host when foreign substances are introduced
into the body

ANTIGEN
• any foreign substance that stimulates an immune response
• NOTE: All immunogens are antigens; not all antigens are immunogens.

ANTIBODY
• serum proteins that are produced by certain lymphocytes in response to
an exposure to a foreign substance (antigen)
IMMUNE SYSTEM
i. BRANCHES OF THE IMMUNE
SYSTEM
ii. TYPES OF IMMUNITY
iii. CELLS OF THE IMMUNE SYSTEM
iv. PRIMARY AND SECONDARY
LYMPHOID ORGANS
 BRANCHES OF THE IMMUNE SYSTEM
CELLULAR
• Cell-mediated
• Involves T-lymphocytes (T-helper
cells, NK cells, Cytotoxic T-cells)
• Protections against intracellular
pathogens such as viruses, fungi,
mycobacteria and tumor cells
HUMORAL
• Antibody-mediated
• Involves B-lymphocytes, Plasma
cells and Memory cells
• Protections against extracellular
pathogens such as bacteria
• Responsible for antibody
production
 TYPES OF IMMUNITY
Natural or
• Innate
present at birth; no prior exposure is required
• involves physical barriers (intact skin & mucous membrane), stomach acidity,
complement, interferons, lysozymes, IgA, phagocytes, inflammations (APRs)
• response lacks memory and specificity, but there is immediate effect

Adaptive or
• Acquired
involves T-cells, B-cells, plasma cells, antibodies & cytokines
• results to increased immune response due to specificity immunologic
memory (prior exposure to foreign pathogen)
 ADAPTIVE IMMUNITY

TYPE PROCESS EXAMPLE EFFECT

An individual is
exposed to foreign
Naturally-acquired Long term immunity;
immunogen which Clinical Infection
active immunity not immediate
results to Ab
production.

An individual is
Vaccinations (Ex.
Artificially-acquired exposed to foreign Ag Long term immunity;
Tetanus, Influenza
active immunity without having an not immediate
Type B, MMR, DPT etc.)
infection.
 ADAPTIVE IMMUNITY

TYPE PROCESS EXAMPLE EFFECT

An individual is Maternal antibodies Immediate response

Naturally-acquired protected by Abs (breast milk) crosses but short-term
passive immunity produced by another the placenta to protect immunity (no
individual. infant memory cells)

An individual receives Rhogam (Rh Immune Immediate response

Artificially-acquired an immune product globulin), Anti-toxins, but short-term
passive immunity from another animal Plasma from recovered immunity only (no
injected into the host. COVID-19 px memory cells)
CELLS OF THE IMMUNE SYSTEM
01 T-lymphocytes

02 Helper/Inducer T cells

03 Suppressor/cytotoxic T cells

04 B-lymphocytes

05 Natural Killer (NK) cells

06 Lymphokine-activated killer cells (LAK)

CELLS OF THE IMMUNE SYSTEM
07 Eosinophils

08 Basophils

09 Neutrophils

10 Monocytes
 CELLS OF THE IMMUNE SYSTEM
CELLS FUNCTION

T-lymphocytes Cell-mediated immunity

Orchestrates cell-mediated immunity. Activates B cells,

Helper/Inducer T cells
cytotoxic cells, and natural killer cells

Suppressor/cytotoxic T cells Suppressor cells inhibit Helper T cells. Cytotoxic cells

kills other cells

B-lymphocytes (differentiate
Humoral-mediated immunity. Transforms into plasma
into plasma cells & memory
cell after antigenic challenge and synthesize and
cells)
secrete immunoglobulins (Ig)

Natural Killer (NK) cells Recognize and destroy virally-infected cells & tumor
cells
 CELLS OF THE IMMUNE SYSTEM
CELLS FUNCTION

Lymphokine-activated killer
Use IL-2 to help lyse tumor cells
cells (LAK)

Eosinophils Suppress inflammatory reaction. Kill some parasites

Basophils Granules contain histamine and heparin. Role in

hypersensitivity reactions

Neutrophils Principal leukocyte associated with phagocytosis and

localized inflammatory

Monocytes Respond to chemotaxins. Process antigens and present

to lymphocytes. Produce IL-1 & other cytokines
PRIMARY LYMPHOID ORGANS
• Bone Marrow - maturation of B cells and NK cells; produces HSCs
• Thymus - maturation of T cells

SECONDARY LYMPHOID ORGANS

• Spleen - filters blood
• Lymph Nodes - filter lymphatic fluid
• Mucosal-associated lymphoid tissue (MALT) - protects mucosal surfaces
• Cutaneous-associated lymphoid tissue (CALT) - prevents antigens from
penetrating the skin
• Peyer's Patches (in the intestines)
• Tonsils
• Appendix
IMMUNOGLOBULIN STRUCTURE
 IMMUNOGLOBULIN STRUCTURE

A four-chain polypeptide units that consists of 2 heavy chains and 3

Basic Structure
lights chains held together by disulfide bonds

The 2 heavy chains are always of the same type (gamma, alpha, mu,
Heavy chains
delta, epsilon) and determine the Ig class

Either kappa or lambda are found in all classes of Igs, but only one type
Light Chains
is present in a given molecule

"Fragment antigen-binding"; consists of 1 light chain and one-half of a

Fab Fragment heavy chain. The intact Ig has 2 Fab fragments, each representing 1
antigen-binding site

"Fragment crystalline"; the carboxy-terminal halves of the 2 heavy

Fc Fragment
chains. This molecular portions has no antigen-binding site
 IMMUNOGLOBULINS
IgG IgM IgA IgD IgE

Molecular 160,000
150,000 900,000 180,000 190,000
Weight (daltons) (monomer)

Monomer &
Form Monomer Pentamer Monomer Monomer
Dimer

Heavy chain Gamma (γ) Mu (μ) Alpha (α) Delta (δ) Epsilon (ε)

Antigen
2 10 2 or 4 2 2
binding sites

Serum 800-1,600 120-150 70-350

1-3 mg/dL 0.005 mg/dL
Concentration mg/dL mg/dL mg/dL
 IMMUNOGLOBULINS
IgG IgM IgA IgD IgE

Percent of total
70-75% 10% 10-15% <1% 0.002%
immunoglobulin

Crosses
YES No No No No
placenta

Complement
YES YES No No No
fixation

IgG1, IgG2,
Subclasses None α1, α2 None None
IgG3, IgG4

Kappa (κ) or Kappa (κ) or Kappa (κ) or Kappa (κ) or Kappa (κ) or
Light Chain
lambda (λ) lambda (λ) lambda (λ) lambda (λ) lambda (λ)
 IMMUNOGLOBULINS

Ig
G• predominant serum antibody
• defense against bacteria and viruses; neutralizes toxins
• important mediator of opsonization and antibody-dependent cellular
cytotoxicity (ADCC)
• produced in secondary (anamnestic) antibody response
• participates in precipitation (more efficient) and agglutination in vitro
 IMMUNOGLOBULINS

Ig
M• largest; a.k.a "macroglobulin"

• 1st antibody to be produced after exposure to an antigen

• "primary response antibody"

• most efficient in activating the classical complement pathway

• only Ig produced by newborn

• neutralizes toxins

• more efficient at agglutination than IgG

 IMMUNOGLOBULINS

Ig
A• can be found in tears, sweat, saliva, respiratory, and GI mucosa
• occurs mainly in secretory form (IgA1)
• also found in breast milk - passive transfer of immunity from mother to infant
• an anti-inflammatory agent - 1st line of defense
 IMMUNOGLOBULINS

Ig
D• role in B-cell maturation
• found on the surface of B lymphs
• present as an antigen receptor on B-cell membrane
 IMMUNOGLOBULINS

Ig
E• responsible for type I hypersensitivity or allergic reactions
• binds to specific Fc receptors on mast cells and basophils - when two
adjacent molecules bind ag, degranulation results and releases histamine and
heparin are released
• role in response to parasites
HYPERSENSITIVITY
REACTIONS
Type I: Anaphylactic
Type II: Cytotoxic
Type III: Immune Complex-Mediated
Type IV: Cell-Mediated
 KEY
TYPE MECHANISM RESULT SYMPTOMS EXAMPLE
REACTANTS

• Anaphylaxis
• Hay fever
Release of
Cell-bound • Asthma
ANAPHYLACTIC mediators from
I IgE reacts Immediate • Food allergies
or IMMEDIATE mast cells &
with antigen (peanuts, wheat,
basophils
seafood)
• Urticaria (hives)

IgG, IgM,
complement, • HDN
Cytolysis of
CYTOTOXIC free Ab • AIHA
target cell due to
II (Antibody- reacts with Immediate • ITP
Ab and
dependent) mostly self- • Transfusion
complement
antigen on reactions
cell surface
 KEY
TYPE MECHANISM RESULT SYMPTOMS EXAMPLE
REACTANTS

IgG, IgM,
• Arthus reaction
complement Deposits of Ag-
IMMUNE • Serum sickness
III Ab reacts Ab complexes in Immediate
COMPLEX • SLE
with soluble tissues
• RA
Ag

Release of
lymphokines Delayed (max • Tuberculin skin
CELL-
from Ag- 48-72 hours test
IV MEDIATED T-cells
stimulated T cells due to cell • Contact
(Delayed Type)
induce migration) dermatitis
inflammation
IMMUNOLOGICAL
PROCEDURES
I. Agglutination Methods
II. Precipitation Methods
III. Other Serological Methods
AGGLUTINATION
• soluble Ab reacts or aggregates with insoluble Ag or soluble Ag reacts
insoluble Ab to form larger complexes
• SENSITIZATION - initial binding of antigen & antibody through
single antigenic determinants on the particle
• LATTICE FORMATION - involves the development of cross-links
that form visible aggregates
 AGGLUTINATION METHODS

Method Principle Results Examples

Patient serum is • Febrile agglutinins

Agglutination indicates
DIRECT reacted with antigen • Salmonella (Widal
the presence of patient
AGGLUTINATION that is naturally found test) & Shigella
Ab to a natural Ag
on a particle serotyping

Patient sample is
Agglutination indicates
PASSIVE reacted with particles
the presence of patient Rheumatoid factor
(INDIRECT) coated with antigens
Ab to an artificially (anti-IgG)
AGGLUTINATION NOT normally found on
attached Ag
their surfaces
 AGGLUTINATION METHODS
Method Principle Results Examples

Ab is attached to carrier Agglutination

REVERSE PASSIVE particles which indicates the • Rapid ID tests
AGGLUTINATION agglutinate in the presence of specific for bacteria
presence of Ag Ag

Competition between
antigen-coated particles Lack of agglutination
AGGLUTINATION • Detection of
and soluble patient Ag is a positive result =
INHIBITION illicit drugs
for limited no. of presence of Ag
antibody sites

A test for detecting Abs Lack of agglutination

HEMAGGLUTINATION • Rubella
to certain viruses that is a positive result =
INHIBITION Antibody
agglutinate RBCs. presence of Antibody
 AGGLUTINATION METHODS
Method Principle Results Examples

A reaction in which
bacteria are used as Agglutination indicates
• Rapid tests for ID
COAGGLUTINATION the carrier for the the presence of
of bacteria
antibody (S. aureus specific Ag
mostly used)

Used to detect Abs Ag-Ab reaction results

HEMAGGLUTINATION • ABO slide typing
to RBC antigens in clumping of RBCs
PRECIPITATION
• involves combining soluble Ag with soluble antibody to form larger and
visible complexes
• less sensitive than agglutination
⚬ Affinity - initial attraction that exists between a single Fab site on an
Ab molecule and a single epitope on the corresponding Ag
⚬ Avidity - represents the sum and strength of all attractive forces
between an Ag and Ab
PRECIPITATION
• 3 zones of the precipitation reaction:

• PROZONE - concentration of Ab molecules is greater than the

concentration of Ag molecules which results to little or no precipitation

• ZONE OF EQUIVALENCE - no. of multivalent sites of Ag and Ab are

approximately equal forming a stable lattice

• POSTZONE - concentration of Ag molecules is greater than the

concentration of Ab molecules which results to little or no precipitation

 PRECIPITATION METHODS
Method Principle Examples

soluble Ags react with specific Ab to • VDRL

Flocculation
form a precipitate of fine particles • RPR

Ags and Abs diffuse out from wells cut

Double Immunodiffusion • Fungal Ags
in agar gel to form precipitin lines where
(Ouchterlony method) • Extractable nuclear Ags
they meet

Ag is measured based on the diameter

Radial Immunodiffusion of a precipitin ring that forms when it • Quantitation of Igs and
(RID) diffuses out of a well in a gel-containing complement (C3/C4)
Ab.
 IDENTITY - single smooth arc forms
between the Ags and Abs which indicates
that the Abs are precipitating identical
Ag specificities (common epitope)

NONIDENTITY - two PARTIAL IDENTITY -

separate lines cross two lines meet forming
each other which a spur which indicates
indicates that Ags and that the Ags share
Abs have no common common epitopes but
epitopes & don't one of them has a
precipitate together unique epitope
 PRECIPITATION METHODS
Method Principle Examples

Electrophoresis of serum followed by • ID of Igs in monoclonal

Immunofixation
direct application of reagent antisera to gammopathies (Ex.
electrophoresis (IFE)
the gel Multiple Myeloma pxs)

• Igs
Nephelometry Light scattering by Ag-Ab complexes • Complement
• C-reactive protein

Ags are electrophoresed in an agar-

• Igs
Rocket containing Ab with selected pH;
• Complement
Immunoelectrophoresis combine to form precipitates (rocket
• Alpha-fetoprotein
height = Ag concentration)
 SYPHILIS
• causative agent: Treponema pallidum
• can be transmitted either through sexual, transplacental (congenital
infections) or parenteral exposure (contaminated needles or blood)
• Stages of the disease:
• PRIMARY (Early)
• SECONDARY
• LATENT
• TERTIARY
 SYPHILIS
• PRIMARY STAGE
⚬ characterized by inflammatory lesions called chancres at the contact site
⚬ predominant Abs are IgM; positive serum tests
⚬ lasts from 1-6 weeks
• SECONDARY STAGE
⚬ characterized by a generalized rash, enlarged lymph nodes & secondary
lesions possibly in the eyes, joints or CNS
⚬ observed after 1-2 months after primary chancre disappear
SYPHILIS
 SYPHILIS
3. LATENT STAGE ("Hidden")
⚬ characterized by a lack of visible clinical symptoms
⚬ still positive in serologic tests but not infectious (except pregnant
women who can still transmit the disease on to the fetus)
4. TERTIARY STAGE

⚬ characterized by granulomatous lesions called gummas or gummata,

neurosyphilis and cardiovascular disease
⚬ often occurs 10-30 years after the secondary stage
 NONTREPONEMAL TESTS FOR SYPHILIS

Venereal Disease Research

RPR (Rapid Plasma Reagin)
Laboratory (VDRL)

Method Flocculation Flocculation

Detects Reagin (anti-cardiolipin) Reagin (anti-cardiolipin)

Ag Reagent Cardiolipin Cardiolipin (charcoal-bound)

Positive rx Microscopic clumps Microscopic agglutinations

Specimen Inactivated serum, CSF Serum

Slide rotation 180 rpm for 4 minutes 100 rpm for 8 minutes
 NONTREPONEMAL TESTS FOR SYPHILIS

Venereal Disease Research

RPR (Rapid Plasma Reagin)
Laboratory (VDRL)

10-30% may be biologic false positives (e.g.,

Infectious mononucleosis, Infectious
Hepatitis, Malaria, Leprosy, SLE, RA,
False positives Same as VDRL
advanced age, pregnancy. May also be
positive in other treponemal infections
such as yaws and pinta)

Replaced by RPR for serum. Good for screening and

Other
Still performed on CSF treatment monitoring
 TREPONEMAL TESTS FOR SYPHILIS (CONFIRMATORY)
Fluorescent Treponema pallidum
Treponemal Antibody Particle Agglutination EIA, CLIA, MFIs
Absorption (FTA-ABS) (TP-PA)

Detects Antibody to T. pallidum Antibody to T. pallidum Antibody to T. pallidum

Nichols strain of T. Gel particles sensitized Enzyme-labeled

Reagent
pallidum with T. pallidum sonicate treponemal Ag

Positive
Fluorescence (green color) Agglutination Color development
rxn

Specimen Serum, CSF Serum Serum

TREPONEMAL TESTS FOR SYPHILIS (CONFIRMATORY)

MHATTS (Microhemagglutination Treponemal Test

for Syphilis) Assay

Method Hemagglutination

Detects Ab to T. pallidum

Sheep RBCs coated with antigens from Nichols strain of T.

Reagent(s)
pallidum, sorbent

Positive reaction Rough or jagged patterns of cells in microtiter plates

Specimen(s) Serum
 NONTREPONEMAL TESTS FOR SYPHILIS
FTA-ABS MHATTS

Not as sensitive in primary syphilis as

Usually positive before regain tests.
Reactivity FTA. However, sensitivity is close to
Some false-negatives in primary
during disease 100% in secondary syphilis. Usually
syphilis. Usually positive for life
positive in late stages

Fewer than nontreponemal tests.

False-positives Reactivity is seen with other Fewer than nontreponemal tests
treponemal diseases (yaws and pinta)

Sorbent removes nonspecific Abs. Sorbent removes nonspecific Abs.

Used to confirm reactive Used to confirm reactive
Other
nontreponemal tests. NOT GOOD for nontreponemal tests. NOT GOOD for
treatment monitoring treatment monitoring
SYPHILIS TESTING
 Other Serological Methods
Method Principle
Patient serum is incubated with
Ag * complement. If the
corresponding Ab is present in
the serum, it forms a complex
Complement Fixation with the Ag & complement.
When sensitized RBCs are
added, there is no free
complement to lyse them. No
hemolysis is a positive reaction.
Function Examples
Best for detection of IgM
antibodies. More sensitive
than agglutination and
Viral, fungal, rickettsial
precipitation, but less
Abs
sensitive than labeled
immunoassays. Not widely
used
 Other Serological Methods
Method Principle
The specimen is placed on a
glass slide and overlaid with
fluorescein-labeled Ab. If the
Direct Fluorescent Ab corresponding Ag is present, the
labeled Ab binds and
fluorescence will be seen with a
fluorescent microscope.
Function Examples
Detects antigens.
Fluorescent compounds
absorb energy from light
source and convert it into Bacterial Ags
light of a longer wavelength
(lower energy) than that
absorbed.
 Other Serological Methods
Method Principle Function Examples

Reagent Ag on a glass slide is

FANA (Fluorescent
overlaid with a patient serum. If
antinuclear antibody),
the corresponding Ab is present
Indirect Fluorescent Detect Abs in the serum. FTA (Fluorescent
in the serum, it attaches to the
Ab A.k.a “Sandwich technique”. Treponemal Ab, IFA-
Ag. When fluorescein-labeled
Malaria, IFA-Giardia
antihuman globulin is added, it
lamblia
attaches to the Ab.
 Labeled Immunoassay (Terminology)
A molecule that binds to another molecule of a complementary
Ligand
configuration.

Isotopic An immunoassay that uses a radioisotope as the label.

An immunoassay that uses something other than a radioisotope as the

Nonisotopic
label (e.g., enzyme, fluorochrome, chemiluminescent molecule)

An immunoassay in which the patient ligand and the labeled reagent

Competitive
ligand compete for a limited number of binding sites on a reagent Ab.

An immunoassay in which the reaction does not involve competition

Noncompetitive
for binding sites (more sensitive)
Competitive Immunoassay
Noncompetitive Immunoassay
 Immunoassay Labels

CHEMILUMINESCENT
RADIOISOTOPES ENZYMES FLUOROCHROMES
MOLECULES

125I
Horseradish
(Iodine) Fluorescein Luminol
peroxidase

3H (Tritium) � -ᴅ-galactosidase Rhodamine Acridium esters

14C (Carbon) Alkaline phosphatase Dioxetane phosphate

 Principles of Labeled Immunoassays
Method Type of Assay Principle

Radiolabeled ligand and unlabeled ligand in the

specimen compete for binding sites on reagent
Competitive
RIA Ab. The more unlabeled ligand in the specimen,
Isotopic
(Radioimmunoassay) the less labeled ligand binds. CPM are inversely
Heterogeneous proportional to the concentration of the ligand
in the specimen.

The specimen is incubated with a labeled Ab.

Solid-phase ligand is added to remove
Noncompetitive
Immuno-Radio Metric unbound labeled Ab. After centrifugation, the
Isotopic
Assay (IRMA) radioactivity in the supernatant is determined.
Heterogeneous
CPM are directly proportional to the
concentration of ligand in the specimen.
RIA
 Principles of Labeled Immunoassays
Method Type of Assay Principle

Enzyme-labeled ligand and unlabeled patient

ligand compete for binding sites on Ab molecules
Enzyme-linked Competitive
attached to a solid phase. Free labeled ligand is
Immunosorbent Assay Nonisotopic
removed by washing and a substrate is added.
(ELISA) Heterogeneous The enzyme activity is inversely proportional to
the concentration of the ligand in the specimen.

Ab in the specimen attaches to a solid-phase Ag.

After incubation and washing to remove unbound
Noncompetitive Ab, an enzyme-labeled antiglobulin is added. This
Indirect or
Nonisotopic 2nd Ab reacts with the Fc portion of the patient Ab
noncompetitive ELISA
Heterogeneous bound to the solid phase. Following another wash,
substrate is added. The amount of enzyme label
detected is directly proportional to the amount
of Ab in the serum.
ELISA
 RIA VS EIA
Radioimmunoassay (RIA) Enzyme Immunoassay (EIA)

Radioisotope (usually 125I

Label Enzyme
(Iodine)

Counts per minute in

Measurement Color development after addition of substrate
scintillation counter

Sensitivity
Specificity
Specificity Long shelf-life of reagents
Advantages
Sensitivity No radiation hazard
No special disposal requirements
No need for scintillation counter

Short shelf-life of reagents

Radiation hazard
Disadvantages
Regulations governing
disposal of radioisotopes
 Principles of Labeled Immunoassays
Method Type of Assay Principle

The specimen is incubated with an enzyme-

labeled Ab. A solid phase ligand (usually on glass
Noncompetitive beads) is added, and the tubes are centrifuged to
Immunoenzymometric
Nonisotopic remove unbound labeled Ab. The amount of
assay (IEMA)
Heterogeneous labeled Ab in the supernatant is directly
proportional to the concentration of the ligand in
the specimen.

The Ag in the specimen is sandwiched between

Noncompetitive the Ab attached to a solid phase and enzyme-
Sandwich enzyme
Nonisotopic labeled Ab. Enzymatic activity is directly
multiplied immunoassay
Homogeneous proportional to the amount of Ag in the test
sample.
 Principles of Labeled Immunoassays
Method Type of Assay Principle

The Ag in the specimen and an enzyme-labeled

Ag compete for binding sites on reagent Ab.
Enzyme-Multiplied Competitive
When enzyme-labeled Ag binds to Ab, enzyme
Immunoassay Technique Nonisotopic
activity is inhibited. Enzyme activity is directly
(EMIT) Homogeneous proportional to the concentration of the Ag in the
specimen.

Unlabeled ligand in the specimen and fluorogenic

Substrate-labeled Competitive
ligand compete for sites on reagent Ab.
Fluorescent Nonisotopic
Fluorescence is directly proportional to the
Immunoassay (SLFIA) Homogeneous
concentration of the ligand in the specimen.
 Principles of Labeled Immunoassays
Method Type of Assay Principle

Unlabeled ligand in the specimen and fluorogenic

ligand compete for sites on reagent AB. The
Competitive higher the concentration of bound labeled ligand,
Fluorescence Polarization
Nonisotopic the more polarized fluorescence. The amount of
Immunoassay (FPIA)
Homogeneous polarized fluorescence is inversely proportional
to the concentration of the ligand in the
specimen.
 COMPLEMENT SYSTEM
A series of >25 serum proteins that interact to enhance host defense
Definition reactions. Most are inactive enzyme precursors that are converted to
active enzymes in a precise order (cascade)

Functions Opsonization, Chemotaxis, cell lysis

Triggered by ag-ab reactions. IgM is the most efficient activator.

Recognition unit: C1 (1st to bind)
Classical Pathway Activation unit: C4, C2, C3
Membrane Attack Complex (MAC): C5, C6, C7, C8, C9 (drills holes in cell
membrane  cell lysis)

Alternative Pathway Activated by bacteria, fungi, viruses, tumor cells, some parasites

Activated by direct recognition of surface moieties found on

Lectin Pathway pathogens; plays an important role as a defense mechanism in
infancy
Classical Complement Cascade
Alternative Complement Cascade
 Immune Phenomena
Explanation Application

Not easily detectable. Can be

Primary
Combination of Ab with Ag measured indirectly by RIA, EIA,
phenomena
immunofluorescence (IF)

Secondary Precipitation, agglutination, Measured more readily. Basis for

phenomena complement fixation many serological tests

In vivo reaction (e.g., Inflammation,

Tertiary Phagocytosis, deposition of Some are useful diagnostically (e.g.,
phenomena immune complexes, immune skin testing)
adherence, chemotaxis)
 HEPATITIS
PROGRESS
HEPATITIS TYPE AND SEROLOGICAL &
TRANSMISSION TO CHRONIC
VIRUS FAMILY MOLECULAR MARKERS
STATE

Fecal-oral, direct
single-stranded • IgM anti-HAV
Hepatitis A contact with
RNA No • Total anti-HAV
(HAV) infectious
Picornaviridae • HAV RNA
individual

single-stranded Fecal-oral, blood Yes • IgM anti-HEV

Hepatitis E
RNA transfusion, vertical (immunocom • IgG anti-HEV
(HEV)
Hepeviridae transmission promised) • HEV RNA
 HEPATITIS
PROGRESS
HEPATITIS TYPE AND TO SEROLOGICAL &
TRANSMISSION
VIRUS FAMILY CHRONIC MOLECULAR MARKERS
STATE

Hepatitis C Parenteral, sexual, • Anti-HCV

RNA Flaviviridae Yes
(HCV) perinatal • HCV RNA

Parenteral, sexual • IgM anti-HDV

Hepatitis D
RNA Deltavirus (along with HBV Yes • IgG anti-HDV
(HDV)
required • HDV RNA
 HEPATITIS
PROGRESS
HEPATITIS TYPE AND TO SEROLOGICAL &
TRANSMISSION
VIRUS FAMILY CHRONIC MOLECULAR MARKERS
STATE

• HBsAg
• HBeAg
• IgM anti-HBc
Hepatitis B DNA Parenteral, sexual,
Yes • Total anti-HBc
(HBV) Hepadnaviridae perinatal
• Anti-HBe
• Anti-HBS
• HBV DNA
 HEPATITIS
Test Indication

Hepatitis C
• Acute, chronic, or previous Hepatitis C infection
• Anti-HCV

• Past or current Hepatitis D infection (Hepatitis D can survive in the

Hepatitis D
presence of a Hepatitis B infection)
• Anti-HDV

Hepatitis E None
 HEPATITIS PROFILE
Acute Recovery from Recovery from Chronic Hepatitis B
Acute Hepatitis B
Hepatitis A Hepatitis A Hepatitis B Carrier

• HBsAg +
• HBsAg + • HBsAg -
• HBeAg +
Anti-HAV Anti-HAV • HBeAg + • HBeAg -
• Anti-HBc (Total) +
(IgM) + (Total) + • Anti-HBc (IgM) + • Anti-HBs +
• Anti-HBs -
• Anti-Hbe + • Anti-Hbe +
• Anti-Hbe -
 SYSTEMIC AUTOIMMUNE DISEASES

DISEASE TARGET CELLS AND TISSUES ASSOCIATED AUTOANTIBODIES

• Anti-nuclear antibodies (ANAs)

• Phospholipid antibodies
Systemic Lupus Multiple cells and organs (skin, joints,
• Antibody to RBCs, platelets,
Erythematosus (SLE) kidneys, brain, heart and lungs)
lymphocytes, endothelium etc.
• Rheumatoid factor

• Anti-CCP (Cyclic Citrullinated

Rheumatoid Protein)
Joints, bone, other tissues
Arthritis (RA) • Rheumatoid factor
• ANAs

• ANAs, RF, Anti-salivary duct abs,

Sjogren’s syndrome Eyes, mouth
Anti-lacrimal gland abs
 Pattern Characteristic/s Associated disease/s

SLE, drug-induced SLE,

Homogeneou uniform staining of the
chronic AIH, juvenile
s nucleus
arthritis

discrete, fluorescent
SLE, Sjogren’s
Speckled specks throughout the
Syndrome, SSc
nuclei

CREST syndrome
numerous discrete
Centromere (a.k.a limited
speckles in the nuclei
cutaneous SSc)

prominent staining of the SSc, patients with

Nucleolar
nucleoli within the nuclei SARDs
 ANTI-NUCLEAR ANTIBODIES (Common)
Ab IFA Pattern Specificity Sensitivity Comments

Peripheral or Most specific for Found in only 50- Considered dx for SLE
Anti-dsDNA
Homogeneous SLE 80% SLE patients (especially if C3 is low)

Only found 30% of Not found in other

Anti-Sm Speckled Specific for SLE
SLE patients disorders

Present in SLE
Peripheral or Present in 70-90%
Anti-DNP and drug-induced LE factor
homogeneous of SLE patients
SLE

Diagnostic of drug-
Present in SLE, RA, Present in almost all induced SLE. High
Anti-histone Homogeneous primary biliary drug-induced SLE levels assoc. with
cirrhosis patients severe and more
active SLE
 ANTI-NUCLEAR ANTIBODIES (Common)
Ab IFA Pattern Specificity Sensitivity Comments

Present in SLE, Scleroderma Present in 10-1% of

Anti-RNP Speckled -
& Sjogren’s syndrome SLE patients

Can cross the

Anti-SS- Present in SLE, Scleroderma Present in 25-40%
Speckled placenta; assoc.
A/Ro & Sjogren’s syndrome of SLE patients
with neonatal lupus

Most often in
patients with
Anti-SS- Present in SLE, Scleroderma Present in 10-15% mucocutaneous
Speckled
B/La & Sjogren’s syndrome of SLE patients manifestation (Ex.
photosensitivity
dermatitis)
ANTI-NUCLEAR ANTIBODIES (Common)

ANTIBODY IMMUNOFLUORESCENT PATTERN DISEASE ASSOCIATION

Prominent staining of nucleoli (smooth,

Anti-nucleolar SLE, systemic sclerosis
clumpy, or speckled)

Anti-Scl-70 Compound pattern with speckling Systemic sclerosis, scleroderma

Anti-Jo-1 Fine cytoplasmic speckling Polymyositis

Anti-
Discrete speckled CREST syndrome
centromere

Usually indicates absence of

DFS70/LEDGF Dense fine speckled
SARDs
 SYSTEMIC AUTOIMMUNE DISEASES (Continuation)

DISEASE TARGET CELLS AND TISSUES ASSOCIATED AUTOANTIBODIES

• ANAs
Systemic Sclerosis Connective tissue • Anti-Scl-70
• Anti-centromere antibody

Polymyositis or
Muscles, skin • ANAs (Anti-Jo-1)
Dermatomyositis

Granulomatosis with • Anti-neutrophil cytoplasmic

polyangiitis Upper respiratory system, lungs, antibodies (ANCA)
(”Wegener’s blood vessels • Rheumatoid factor
granulomatosis) • ANAs
 ORGAN-SPECIFIC AUTOIMMUNE DISEASES
TARGET CELLS AND
DISEASE ASSOCIATED AUTOANTIBODIES
TISSUES

Addison’s Disease Adrenal glands • Antibody to adrenal glands

Autoimmune Hemolytic Anemia Red blood cells • Antibody to RBCs

• AIH-1 – smooth muscle Abs

Autoimmune Hepatitis (AIH) Liver
• AIH-2 – anti-LKM-1, anti-LC-1

Autoimmune thrombocytopenic
Platelets • Anti-platelet Ab
purpura

Small intestine and • Anti-transglutaminase (tTG)

Celiac Disease
other organs • Ab to DGPS

• Ab to an ab in the renal and

Goodpasture’s Syndrome Kidneys, Lungs
pulmonary basement membranes
 ORGAN-SPECIFIC AUTOIMMUNE DISEASES
TARGET CELLS
DISEASE ASSOCIATED AUTOANTIBODIES
AND TISSUES

• Anti-thyroglobulin
• Anti-thyroid peroxidase (TPO)
Graves’ Disease Thyroid gland
• Thyroid-stimulating hormone receptor Abs
(TRAbs)

• Anti-thyroglobulin
Hashimoto’s Thyroiditis Thyroid gland
• Anti-thyroid peroxidase (TPO)

Myelin sheath of • AIH-1 – smooth muscle Abs

Multiple Sclerosis
nerves • AIH-2 – anti-LKM-1, anti-LC-1

• Abs to acetylcholine receptors (AChR)

Nerve-muscle
Myasthenia Gravis • Anti-muscle-specific kinase (MuSK)
synapses
• Antibody to lipoprotein LRP4
 ORGAN-SPECIFIC AUTOIMMUNE DISEASES
TARGET CELLS
DISEASE ASSOCIATED AUTOANTIBODIES
AND TISSUES

Pernicious Anemia Stomach • Parietal cell antibody, intrinsic factor Ab

Poststreptococcal
Kidneys • Streptococcal Abs that cross-react with kidney tissue
glomerulonephritis

Primary biliary Intrahepatic

• Anti-mitochondrial antibodies (AMA)
cholangitis bile ducts

Rheumatic fever Heart • Streptococcal Abs that cross-react with cardiac tissue

• Anti-insulin
Type 1 Diabetes
Pancreas • Islet cell antibodies
Mellitus
• Ab to glutamic acid decarboxylase (GAD-65)
 Tests for the Diagnosis of Infectious Mononucleosis
(IM)
Antibodies Interpretation

• Heterophile Abs
- IM Abs: agglutinate sheep, beef, ox, and - agglutination of sheep or horse cells following
horse RBCs. Adsorbed by beef RBCs but adsorption with GPK but not with beef RBCs –
not by guinea pig kidney cells (GPK) INFECTIOUS MONONUCLEOSIS

- Serum sickness Abs: agglutinate sheep, - No agglutination of sheep or horse cells

beef, and horse RBCS. Adsorbed by beef following adsorption with GPK or beef RBCs =
RBCs and GPK cells Serum sickness

- Forsmann Abs: agglutinate sheep and - Agglutination of sheep or horse cells following
horse RBCS. Adsorbed by GPk cells but not adsorption with beef RBCs but NOT with GPK =
by beef RBCs Forssmann
 Tests for the Diagnosis of Infectious Mononucleosis
(IM)
Antibodies Interpretation

Specific Viral Abs

• IgM-VCA (Viral Capsid Ag): peaks 3 to 4 • With IgM anti-VCA, anti-EA, or IgG anti-VCA, but
weeks after infection. Undetectable WITHOUT Anti-EBNA: recent or current infection
after 12 weeks
• IgG-VCA: appears in late acute phase • With Anti-EBNAor IgG anti-VCA, but WITHOUT
and persists for life IgM anti-VCA: past infection
• Anti-EA (Early Ag): appears early and
persists for years
• Anti-EBNA (Ebstein-Barr nuclear Ag):
appears 2 to 3 months after infection
and persists indefinitely
 Weil-Felix Reaction
Weil-Felix Rx
Disease Organism Transmission
OX-2 OX-19 OX-K

Rocky Mountain Rickettsia Dog or wood tick + ++++ 0

Spotted Fever rickettsii

Epidemic Rickettsia Body or head louse + ++++ 0

Typhus prowazekii

Endemic Typhus Rickettsia typhi Rat flea + ++++ 0

Scrub Typhus Rickettsia Mite 0 0

tsutsugamushi ++++
Q fever Coxiella burnetti Inhalation of 0 0 0
barnyard dust or
ingestion of infected
meat/milk
HIV TESTING
 HIV TESTING
Type of
Method Detects Principle Comments
Assay

Viral Ag is coated on a solid

Most widely used screening
support. Patient serum is
procedure. Positive results must
Ab to added. After incubation &
ELISA Screening be confirmed. Anti-HIV-1 and
HIV washing, enzyme-labeled
HIV-2 (or combined) are required
antihuman globulin (AHG) is
on blood donors.
added followed by substrate.

HIV Ag is adsorbed onto

Slide
Ab to carrier particles. The Used when instrumentation is
Agglutinat Screening
HIV particles agglutinate in the not available
ion Test
presence of Ab.
 HIV TESTING
Method Detects Type of Assay Principle Comments

A lysate of HIV antigen

is separated into
components by
Most commonly used
electrophoresis and
confirmatory test. HIV infection
Western Ab to transferred to
Confirmatory is indicated by reactivity in 2 of
Blot HIV nitrocellulose paper.
the following bands: p24, gp41,
The resulting blot is cut
or gp120/160.
into strips and reacted
with serum. Labeled
AHG is added.
 HIV TESTING
Method Detects Type of Assay Principle Comments

An antibody test. Serum

is incubated with virally
infected cells on a glass
Indirect
slide. Fluorescein-labeled
antihuman globulin is Sensitivity and specificity
Immuno- Virus or
Confirmatory added. comparable to Western blot.
fluorescence Ab
Antigen test: Simple and rapid to perform.
Assay Patient cells are fixed to
a slide and incubated
with HIV specific
antiserum.
 HIV TESTING
Method Detects Type of Assay Principle Comments

A nti-H I V - 1 b o u n d t o a
solid support is P24 Ag precedes Ab by
P24 Antigen incubated with serum. several weeks. Positives
test (HIV-1- HIV Ag Screening After washing, enzyme- must be confirmed by a
Ag) labeled anti-HIV-1 is neutralization assay.
added followed by Required on blood donors
substrate.
 HIV TESTING
Method Detects Type of Assay Principle Comments

Extremely sensitive
Viral DNA from infected
Polymerase Adjunct to technique. Will detect
Viral cells is amplified, then
Chain standard infections during “window
genome identified using labeled
Reaction testing period”. Not suitable for
probes.
routine screening.

Adjunct to Expensive, time-consuming,

Virus grown on cell
Viral Culture Virus standard and hazardous. Not
culture
testing routinely performed
 Other Serological Tests
Common
Test Reagents Interpretation Comments
Method

Rheumatoid factor is an
autoantibody (usually IgM) against
IgG attached Positive in 70-
RHEUMATOID Agglutinati IgG in the joint synovium. Not
to latex or 80% of patients
ARTHRITIS on specific for RA. Also found in SLE,
RBCs with RA.
Scleroderma, and Sjogren’s
Syndrome.

PAP is caused by Mycoplasma

pneumoniae. Increased titer of Anti-
Positive in I. Test is incubated in the ref.
Patient or
COLD Hemaggluti Primary Atypical Specimen must be kept warm prior
Group O
AGGLUTININS nation Pneumonia to testing to prevent false-negs or
RBCs
(PAP) decreased titers. Positives must be
confirmed by reversal of
agglutination at 37C.
 Other Serological Tests
Common
Test Reagents Interpretation Comments
Method

Streptolysin O lyses RBCs. Anti-

Titer is the streptolysin O in pxs serum
highest dilution neutralizes streptolysin O reagent so
showing no RBCs are not lysed. Streptolysin O is
hemolysis. High oxygen labile. RBC control (RBCs +
ANTI Streptolysin
Neutralizat titer with strep buffer) should show no hemolysis.
STREPTOLYSIN O, Group O
ion infections, Streptolysin control (streptolysin O +
O RBCs
rheumatic fever, buffer + RBCs) should show
and hemolysis. Result is reported in Todd
glomerulonephri units (reciprocal of highest dilution
tis showing no hemolysis)
Normal result: <166
 Other Serological Tests
Common
Test Reagents Interpretation Comments
Method

Sensitive, but nonspecific

C- Latex
indicator of inflammation.
REACTIVE Latex particles Agglutination =
Originally named because it was
PROTEIN Agglutination coated with inflammation
thought to be an antibody to C
(CRP) anti-CRP
polysaccharide of S. pneumoniae

Rubella: German Measles or “3-

Titer is highest
day measles”. In-utero infections
dilution showing
Viral cause fetal death or birth defects.
Hemagglutination no agglutination.
RUBELLA preparation Greatest risk is first month of
Inhibition A titer of 8 or
& RBCs pregnancy. IgM abs in a neonate
more = immunity
are diagnostic of congenital
to Rubella
Rubella.
 Other Serological Tests
Common
Test Reagents Interpretation Comments
Method

Screening test for fever for unknown

region.
Weil-Felix reaction: rickettsial Abs
cross react with OX-2, OX-19, and
Salmonella O
OX-K strains of Proteus.
and H,
Widal’s Test: detects Abs in Typhoid
Febrile Bacterial Brucella Positive reaction
fever, Tularemia, Brucellosis.
Agglutinins Agglutination abortus, is agglutination
Abs to O = recent infection
Proteus OX-
Abs to H = past infection
19
Best indicator of active infection is
four-fold increase in titer.
Nonspecific test, infrequently
ordered today.
 Interpretation of Serological Tests
• A single titer is not usually meaningful because it can’t differentiate current

infection from past infection or immunity due to immunization.

• The acute specimen should be frozen and run with the convalescent specimen to

eliminate differences in titer due to technical variations.

• A convalescent specimen should be drawn 10-14 days after the acute specimen.

• A fourfold increase in titer from acute to convalescent specimen is diagnostic.

• IgM Ab is a sign of recent infection.

 Serology Calculations
• How would you prepare a 5% suspension of human group O red blood cells?

5% = 5 mL per 100 mL

5 mL of packed RBCs + 95 mL of buffer

• How would you prepare 5 mL of a 1:10 dilution of serum?

1 / 10 = x / 5

10x = 5

x = 0.5 = Mix 0.5 mL of serum with 4.5 mL of buffer

 Serology Calculations
• How would you prepare 10 mL of a 1:100 dilution from a 1:10 dilution?

A 1:10 dilution of a 1:10 dilution yields a 1:100 dilution (1/10 x 1/10 = 1/100)

Mix 1 mL of the 1:10 dilution with 9 mL of buffer (1 mL + 9 mL = 1:10)

• How would you prepare 10 mL of a 1:500 dilution from a 1:100 dilution?

A 1:5 dilution of a 1:100 yields a 1:500 dilution (1/5 x 1/100 = 1/500)

Mix 2 mL of the 1:100 dilution with 8 mL of buffer (2 mL + 8 mL = 2:10 = 1:5)

 Serology Calculations
• What is the dilution in tube 4 of a twofold serial dilution, if tube 1 is undiluted?

T1 T2 T3 T4 1 x ½ x ½ x ½ = 1/8

Titer 1 2 4 8
 REFERENCES:
• Clinical Immunology & Serology 5th edition. A Laboratory Perspective
Linda E. Miller & Christine Dorresteyn Stevens

• Quick Review Cards for Medical Laboratory Science 2nd edition

Valerie Dietz Polanzky, Med, MLS(ASCPi)

• Shirley F. Cruzada, EdD, MS, MT (AMT) – Professor - Clinical Laboratory Services

Prepared by: DANIELLE ALEXANDRA D. CARVERO, RMT, MLS(ASCPi)