IMMUNOLOGY & SEROLOGY Research on Immunity?

______________

Definition of terms in Immunology Roles of the Immune System

1. Immunology - study of the 1. Defending the body against

molecules, cells, organs, and infections
systems responsible for the 2. Recognizing and responding to
recognition and disposal of foreign foreign antigens
(non-self ) material; how body 3. Defending the body against the
components respond and interact; development of tumors
the desirable and undesirable 4. The immune system can injure cells
consequences of immune and induce pathologic inflammation
interactions; and the ways in which
the immune system can be Protective or Destructive?______________
advantageously manipulated to
protect against or treat disease; The first written records of immunological
Study of host immune system and its experimentation date back to the 1500s,
responses to microbial pathogens when the Chinese developed a practice of
and damaged tissues inhaling powder made from smallpox
2. Immune system - collection of cells, scabs in order to produce protection
tissues, and molecules that mediate against this dreaded disease. This practice
resistance to infection of deliberately exposing an individual to
3. Immune response - coordinated material from smallpox lesions was known
reaction of the immune system and as variolation. The theory was that if a
molecules to infectious microbes healthy individual was exposed as a child or
4. Immunity - state of resistance to young adult, the effects of the disease
disease would be minimized. However, this was not
5. Antigen (Ag) - any molecule that always the case.
can bind to the components of the
specific immune response Edward Jenner discovered a remarkable
6. Antibody (Ab) - family of defensive relationship between exposure to cowpox
proteins the body makes when and immunity to smallpox. After observing
stimulated by an antigen the fact that milkmaids who were exposed
7. When secreted by plasma cells, they to cowpox had apparent immunity to
are termed immunoglobulins smallpox, he deliberately injected
8. Anamnestic/ Secondary response individuals with material from a cowpox
- pertains to the immunologic lesion and then exposed them to smallpox.
memory; Rapid rise in the immune He thus proved that immunity to cowpox, a
reaction after re-exposure to an very mild disease, provided protection
antigen against smallpox. This procedure of
9. Cytokines - substances, mostly injecting cellular material became known as
proteins, secreted by cells affecting vaccination, from vacca, the Latin word for
the behavior of nearby cells; those “cow.”
cells with cytokine receptors

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The phenomenon in which exposure to one Natural and Acquired Immunity
agent provides protection against another
agent is known as cross-immunity. Natural, or innate or native, immunity is
the ability of the individual to resist infection
In working with the bacteria that caused by means of normally present body
chicken cholera, Louis Pasteur, a key functions.
figure in the development of both
microbiology and immunology, accidentally Acquired, or adaptive or specific,
found that old cultures would not cause immunity is a type of resistance that is
disease in chickens. 2 Subsequent characterized by specificity for each
injections of more virulent organisms had no individual pathogen, or microbial agent, and
effect on the birds that had been previously the ability to remember a prior exposure,
exposed to the older cultures. In this which results in an increased response
manner, the first attenuated vaccine was upon repeated exposure.
discovered.
Second and Third line of Defense
Attenuation, or change, may occur through
heat, aging, or chemical means, and it
Natural Immunity Adaptive
remains the basis for many of the Immunity
immunizations that are used today. He also
employed this principle to rabies. INnate resistance Acquired resistance

First line of defense Non-specific Clonally distributed

receptors
Normal body
1. Unbroken skin functions Specific

Keratinization - keratinocytes, Langerhans No memory Memory/ Recall

cells, melanocytes
Constant epithelial cell renewal - labile cells N response against Diverse response
antigens
Normal microbiota - microbial antagonism
Uses the cells and
Acute Inflammation molecules of innate
2. Intact mucosa system to eliminate
Fast
Mucosal secretions - sebum, nasal mucus, Slow
cerumen, lactic acid in sweat, stomach acid,
intestinal secretions, acidity of the vagina,
saliva, tears
Lysozyme - attacks and destroy the cell wall
particularly the _____________ of gram
positive organisms
Immunoglobulin A

3. Ciliary function

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 APRs are mainly produced in the?________
Phagocytes
Neutrophils -
bacterial and acute Acute Phase Reactants
inflammation
Monocytes Examples:
Eosinophils 1. C-reactive protein
Basophils/ Mast 2. Serum amyloid A
cells?
Lymphocytes - NK 3. Alpha1-antitrypsin
cells 4. Fibrinogen
5. Haptoglobin
6. Ceruloplasmin
Macrophages 7. Complement C3
Connective tissue - 8. Mannose-binding protein
Histiocytes
Lungs - Alveolar,
Dust cells Positive APRs
Brain - Microglia
Kidney - Mesangial
cells
Liver - Kupffer cells
Placenta -
Hoffbauer cells

Negative APRs

Cytokines are mainly produced by________

Lack of MBP_________________________

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Dendritic cells

Named because they are covered with long

membranous extensions that make them
resemble nerve cell dendrites.

Their main function is to phagocytose

antigen and present it to helper T
lymphocytes. While their actual
developmental lineage is not known, they
are believed to be descendents of the
myeloid line. They are classified according
to their tissue location, in a similar manner
to macrophages.

1. Langerhans cells are found on skin

and mucous membranes;
2. Interstitial dendritic cells populate
the major organs such as the heart,
lungs, liver, kidney, and the
gastrointestinal tract; and
3. Interdigitating dendritic cells are
present in the T lymphocyte areas of
secondary lymphoid tissue and the
thymus.

They are the most potent phagocytic cell

in the tissue.

Toll-like Receptors

Toll is a protein originally discovered in the

fruit fly Drosophila, where it plays an Cellular theory of immunity through
important role in antifungal immunity in the phagocytosis________________________
adult fly highest concentration of these
receptors occurs on monocytes,
macrophages, and neutrophils.

TLR2 recognizes teichoic acid and

peptidoglycan found in gram-positive
bacteria, while TLR4 recognizes
lipopolysaccharide found in gram-negative
bacteria

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Phagocytosis

Complement component which is a

chemotaxin__________________________

The principal factor in determining whether

phagocytosis can
occur______________________________
___________________________________

Neutrophil Extracellular Traps NETs

NETs function in innate immunity. They are

composed of chromatin components,
including histones, and neutrophil
antimicrobial proteins. Microbes are trapped
in NETs, where they encounter high
concentrations of antimicrobial proteins.

Mononuclear phagocyte system

Common origin, similar morphology, and

shared functions, including rapid
phagocytosis mediated by receptors for IgG
and the major fragment of C3.
1. Fixed
2. Wandering

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Interferon-gamma (IFN-γ) and Terminal stage of development
granulocyte colony-stimulating factor
(G-CSF) activates macrophage Multinucleated giant cell, which
characterizes granulomatous inflammatory
Macrophage secretes tumor necrosis diseases such as tuberculosis.
factor α (TNF-α, cachectin), which can
activate macrophages itself under certain in Both monocytes and macrophages can be
vitro conditions. shown in the lesions in these diseases
before the formation of giant cells, thought
to be precursors of the multinucleated cells.

ANTIGEN - substance that reacts with EPITOPE/ DETERMINANT SITE - key

antibody or sensitized T cells but may not portion of the immunogen, molecular
be able to evoke an immune response in shapes or configurations that are
the first place. recognized by B or T cells capable of
triggering specific antibody production or a
IMMUNOGEN - macromolecules capable of T-cell response
triggering an adaptive immune response by
inducing the formation of antibodies or PARATOPE - antigen-binding site
sensitized T cells in an immunocompetent
host. HAPTEN - non-immunogenic non-protein
materials that, when combined with a
ALL IMMUNOGENS are antigens, but carrier, create new antigenic determinants;
NOT ALL ANTIGENS are immunogens. low MW

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CARRIER MOLECULE - protein part and is
immunogenic; high MW

AUTOANTIGENS AUTOGRAFT
are those antigens that belong to the host. From same individual

ALLOANTIGENS ALLOGRAFT
Are from other members of the host’s From different individual
species, and these are capable of eliciting
an immune response. They are important to HETEROGRAFT/ XENOGRAFT
consider in tissue transplantation and in From different species
blood transfusions.
ISOGRAFT
HETEROANTIGENS From identical twins
are from other species, such as other
animals, plants, or microorganisms ADJUVANT - is a substance administered
with an immunogen that increases the
HETEROPHILE immune response. It acts by producing a
are heteroantigens that exist in local inflammatory response that attracts a
unrelated plants or animals but are either large number of immune system cells to the
identical or closely related in structure so injection site. e.g. Aluminum salts, Freund’s
that antibody to one will cross-react with complete adjuvant
antigen of the other. Ie. human blood group
A and B antigens, which are related to Freund’s complete adjuvant
bacterial polysaccharides 1. Mineral oil
2. Emulsifier
3. Killed mycobacteria (0.5 mg/mL)

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Antibody Structure and Function

Affinity and Avidity

_____________Individual antibody-antigen
interaction

_____________Cumulative binding strength

of all antibody-antigen interactions in a
multivalent molecule

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Antibody diversity_____________________

IgG Main antibody in secondary

response to an antigen
Most abundant in serum
Crosses the placenta
Can fix, opsonize and neutralize

IgM Produced in the primary Notes:

(immediate) response to an
antigen
First antibody produced in the
fetus; most primitive; also known
as MACROGLOBULIN Antigen type
Can fix, agglutinate, opsonize
and neutralize 1. Thymus-independent antigens
Monomer in B cell, Pentamer in
secretions Antigens lacking a peptide component
lipopolysaccharides
IgA Most produced antibody overall, Cannot be presented by MHC to T cells
low in serum; Monomer in Weakly immunogenic
circulation, dimer in secretions; Vaccines often require boosters and
GI secretions, tears, saliva, adjuvants. e.g. PPSV23 vaccine
mucus, breast milk
Picks up secretory component in 2. Thymus-dependent antigens
epithelial cells → protects the Fc
portion from luminal proteases Antigens containing a protein component
Class switching and immunologic memory
IgD Unclear biological function occur as a result of direct contact of B cells
On the surface of many mature with Th cells. e.g PCV13
naive B cells and in serum

IgE Reaginic antibody

Type I hypersensitivity reaction
Binds mast cells and basophils
and cross-linking
Parasitism and allergy, heat
labile

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Antigen-Antibody Complex Cross reactivity

non covalent, reversible, intermolecular ability of an individual antibody combining

forces. site to react with more than one antigenic
determinant

Affinity Zonal reactions - False negative results

strength of the reaction between a single

antigenic determinant and binding site
sum of the attractive and repulsive forces
the equilibrium constant that describes the
antigen-antibody reaction

Avidity

a measure of the overall strength of binding

of an antigen with many antigenic
determinants and multivalent antibodies.
Avidity is influenced by both the valence of
the antibody and the valence of the antigen.

Law of Mass Action

Phases of Antigen - Antibody Interaction

1. Primary Phase – initial or physical

Specificity attachment
2. Secondary Phase – visible
ability of an individual antibody combining manifestations
site to react with only one antigenic 3. Tertiary Phase – biologic
determinant consequences

Sensitivity

defined by the notion that antibodies may or

may not react to certain antigens under
specific conditions.

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Primary phase Secondary phase

Notes:

Notes:

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Precipitation Ouchterlony

Antigen: precipitinogen
Antibody: precipitin
Soluble nature converted to an insoluble
complex

Occurs when an Ag-Ab reaction results to

the cross-linking of the Ab until it forms a
lattice structure

Requires at least two antigenic

determinants per molecule and a bivalent Radial Immunodiffusion
antibody

In Solutions In Gel medium

Four Methods: Semisolid support

1. Tube
2. Capillary Agarose – most
3. Slide common and best
4. Ring/ medium because
Interfacial of its relative
negative charge
Detection of that enhances the
bacterial Ags in Ag-Ab reaction
tissues
Oudin - simple Electrophoresis
Identification and diffusion
typing of bacteria

Flocculation Test
for Syphilis

C reactive protein
test

Identification of
blood stains

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 Precipitation Measurement

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Agglutination

Antigen: agglutinogen
Antibody: agglutinin

Aggregation of particulate matter due to

combination with specific antibody

Requires at least two antigenic

determinants per molecule and a bivalent
antibody

Sensitization and Lattice formation

1. Direct - uses antigens found

naturally on the surface of cells

2. Passive - employs particles that are

coated with antigens not normally
found on their surfaces (carriers
used: RBC, polystyrene latex,
bentonite, charcoal)

3. Reverse passive - antibodies rather

than antigens are attached to carrier
particle

4. Agglutination Inhibition - Based on

competition between particulate and
soluble antigen for limited antibody
combining sites, and a lack of
agglutination is an indicator of
positive reaction

5. Coagglutination - uses bacteria as

the inert particles to which the
antibody is attached

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Neutralization Complement fixation

Antigenic activity is stopped (Neutralized) by

its specific antibody

Antibody renders the antigenic

microorganisms non-infective

Example:

1. ASO (toxin neutralization)

2. Viral neutralization
3. In vivo test
4. In ovo test
5. Pock reduction test
6. In tissue culture
7. Plaque reduction test

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Labeled Immunoassays

Labeling techniques
1. Enzymes - horseradish peroxidase,
ALP

2. Radioactive substances - 131I,

125I (the label of choice), tritriated
hydrogen

3. Fluorescence - FITC (Fluorescein Notes:

isothiocyanate)= green fluorescence,
TRITC (Tetra methyl rhodamine
isothiocyanate) = red fluorescence,
Phycobiliproteins

4. Chemiluminescence - Luminol,
Acridium esters, Dioxetane
phosphate, Peroxyoxalate,
Ruthenium derivatives

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COMPLEMENT SYSTEM Complement Activation Pathways

Who coined the term 1. CLASSIC - Antigen-antibody

“complement”?_______________________ complex; IgG or IgM mediated
2. ALTERNATIVE - Microbe surface
Nobel Prize for elucidating the nature of molecules; Amplifying C3 production
complement_________________________ 3. LECTIN - Mannose or other sugars
on microbial surface
a. IgA aggregates
b. Yeast cell wall/zymosan
c. CVF

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Complement Regulators

Opsonins___________________________

Anaphylatoxins_______________________

Membrane Attack Complex_____________

Complement regulatory proteins

___________________________________

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Complement Deficiencies NK cells (Third Population)

1. Inhibitory receptors - MHC I

Early complement Increased risk of
deficiencies (C1-C4) severe, recurrent recognition
pyogenic sinus and
respiratory tract KIRs (killer cell immunoglobulin-like
infections. Increased
receptors)
risk of SLE.
ILT/LIR
Terminal complement Increased susceptibility and CD94/NKG2A receptors
deficiencies (C5–C9) to recurrent Neisseria
bacteremia. 2. Activatory signals → perforins and
granzymes
PIGA gene defect Paroxysmal nocturnal
preventing the hemoglobinuria MICA and MICB
formation of GPI Complement-mediated
anchors for CD94/NKG2C and CD94/NKG2D
intravascular hemolysis
complement inhibitors
→ haptoglobin, dark
Perforins are pore-forming proteins that
urine
polymerize in the presence of Ca2  and
C1 esterase inhibitor Hereditary form channels in the target cell membrane.
deficiency angioedema
Unregulated activation Granzymes are packets of serine esterase
of kallikrein → enzymes that may enter through the
bradykinin → channels and mediate cell lysis.
inflammation
Decreased C4 levels

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Adaptive Immunity The spleen is the largest secondary
lymphoid organ, having a length of
Lymphoid structures approximately 12 cm and weighing 150 g in
1. Primary/ Central/ Generative the adult.
a. Bone Marrow
b. Thymus It is located in the upper-left quadrant of the
abdomen, just below the diaphragm and
surrounded by a thin connective tissue
capsule. The organ can be characterized as
a large discriminating filter, as it removes
old and damaged cells and foreign antigens
from the blood.

Periarteriolar lymphoid Sheath (PALS)

Where B and T cells become mature and

become competent to respond to antigens
___________________________________

2. Secondary/ Peripheral
a. Spleen
b. Lymph nodes
c. Tonsils
d. Peyer’s patches

The spleen has important functions to this

Where adaptive immune responses are kind of pathogens?____________________
initiated_____________________________

B and T cells are produced where?_______

Direction of maturity of T cells in the

thymus?____________________________

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Lymph nodes are located along lymphatic Enter via afferent vessels and HEV
ducts and serve as central collecting points
for lymph fluid from adjacent tissues. Lymph Cortex - B cells
fluid arises from passage of fluids and low
molecular-weight solutes out of blood vessel Paracortex - T cells
walls and into the interstitial spaces
between cells. Medulla - mixed cells

MALT - GI, GU, respiratory

Tonsils

Appendix

The epidermis contains a number of

intraepidermal lymphocytes.

Most of these are T cells, which are

uniquely positioned to combat any antigens
that enter through the skin. This association
of lymphocytes is known as the
cutaneous-associated lymphoid tissue.

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Surface Markers on T and B cells

Membrane glycoprotein found in all

hematopoietic cells____________________

Membrane glycoprotein found in all

hematopoietic cells except mature
erythrocytes_________________________

B cell Differentiation

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Positive and Negative Selection

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 T-cell Activation

T-cell and B-cell Activation

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Types of Acquired Immunity

Serum antitoxin______________________

Passive immunization_________________ Vaccination

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Autoimmunity
Principal factors for development
Inappropriate immune attack against the 1. Inheritance of susceptibility
body’s own proteins or antigens, mediated genes
by autoantibodies and self-reactive T cells. 2. Environmental factors
It is characterized by breakdown of immune
tolerance.

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Rheumatoid Arthritis

Rapid Agglutination
Principle
The principle of serologic testing for
rheumatoid arthritis is based on the
detection of macroglobulins collectively
called RF. RF behaves like antibodies
against human gamma globulin (IgG).

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Hypersensitivity

Type I Hypersensitivity
‘

Type II Hypersensitivity

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Type III Hypersensitivity

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Immune mediators
Type I - IgE Classical Complement mediated
Type II - IgM, IgG ___________________________________
Type III - IgM, IgG
Type IV - T cells

RIST vs RAST

MITCH ROY B. MARISTELA, MD

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Immunoproliferative disorders

Bence Jones Protein Screening

Procedure

Heat solubility is used to detect Bence

Jones (BJ) protein, the urinary protein
characteristic of multiple myeloma (MM).
BJ protein is soluble in urine at room and
body temperatures.

When the urine is heated, BJ protein forms

a precipitate around 60  C to 70  C, and
then dissolves at 100  C, but reappears on
cooling. The minimal detectable
concentration of BJ protein is about 30
mg/dL. Excessive amounts of acid or salt
will prevent the appearance of the
precipitate.

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Human immunodeficiency virus (HIV)

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Transplantation Genes coding for the MHC molecules in
humans are found on the short arm (p arm)
Major Histocompatibility Complex (MHC) of chromosome 6 at band 21 and are
divided into three categories or classes.
Group of genes encoding for HLA antigens
Third class are not HLA antigens, but
represent complement, TNF, etc.

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 thyroiditis, Addison disease

DR4 Rheumatoid arthritis, DM type

1, Addison
disease

DR5 Hashimoto thyroiditis

Blood group HLA type

Bennett-Goodspee
d

Bga HLA-B7

Bgb HLA-B17

HLA ASSOCIATED DISEASE/

CONDITION Bgc HLA-A28

A3 Hemochromatosis Immunologic tolerance

B8 Addison disease, Myasthenia Lack of response to antigens upon

gravis, Graves exposure of lymphocytes to antigens. The
disease lymphocytes are functionally inactivated or
killed resulting in tolerance.
B27 Psoriatic arthritis, Ankylosing
spondylitis, May be induced during encounter of
IBD-associated arthritis, self-antigen with developing lymphocytes in
Reactive arthritis the primary or secondary lymphoid tissues.

C Psoriasis Immunogenic response - activation,

proliferation and differentiation of
DQ2/DQ Celiac disease
lymphocytes into effector cells
8

DR2 Multiple sclerosis, hay fever, Tolerogenic response - lymphocytes are

SLE, either killed or inactivated
Goodpasture syndrome
Immunologic ignorance - antigen-specific
DR3 DM type 1, SLE, Graves lymphocytes will not produce any reactions
disease, Hashimoto

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Central T cell tolerance
Cell death (negative selection) and
generation of regulatory T cells

Peripheral T cell tolerance

Functional inactivation of T cells (anergy) or
death (activation induced cell death) or
suppression by regulatory T cells

Central B cell tolerance

Changes in receptor specificity (receptor
editing) or killing (negative selection)

Peripheral B cell tolerance

Anergy

What is the most sensitive

method for crossmatching and antibody
identification_________________________

What are the indications?

How long a patient waits for a transplant

depends on the
following factors:
1. Blood type (some rarer than others)
2. Tissue type
3. Height and weight of transplant
candidate
4. Size of donated organ
5. Medical urgency Most efficient and probably the most
6. Time on the waiting list economical method available for the
7. Distance between donor’s hospital prevention of posttransfusion GVHD
and potential donor organ ___________________________________
8. Number of donors in local area over
time
9. Transplantation center’s criteria for
accepting organ offers
10. Depending on the type of organ
needed, some factors are more
important than others.

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Graft Rejection

Minimum matching levels

For adults at least 6 out of 8
For cord blood units at least 4 out of 6

The donor and recipient may be

incompatible.

HLA matching is the primary

consideration in assessing whether a
donor is acceptable for a given patient and
overshadows any other non-HLA factors,
including ABO incompatibility.

1. Improves the chances for a

successful transplantation

2. Promotes engraftment, the process

of donated cells beginning to grow
and produce new blood cells in the
host.

3. Reduces the risk of

post-transplantation
graft-versus-host disease (GVHD).

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Tumor Immunology

Tumor antigens

1. Tumor-specific antigens (TSAs) on chemically induced tumors

2. Tumor-associated antigens (TAAs) on virally induced tumors

3. Carcinofetal antigens

4. Spontaneous tumor antigens

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Marker for neuroendocrine tumors
___________________________________

Antigen for Multiple Myeloma

___________________________________

Guardian of the genome

___________________________________

Governor of the cell

___________________________________

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Infectious Diseases
Bacterial infections

Clinically significant result for

Widal’s test 1:160
Weil-felix test 1:320
Cold agglutinin 1:128
ASO, Anti-DNAse 1:240

Weil-Felix test
OX2, OX19 P. vulgaris
OX-K P. mirabilis

OX-K (+) Scrub typhus

OX2 and OX19 (+) Epidemic, murine
and RMSF

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Parasitic infections

Toxoplasma

Sabin-Feldman Dye Test The DT is a sensitive and specific

IgG antibodies are primarily measured by neutralization test in which live organisms
the Sabin-Feldman dye test (DT), are lysed in the presence of complement
considered the gold standard. and the patient’s IgG T. gondii–specific
antibody.

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Rapid TORCH Procedure Cytomegalovirus (CMV)
The ImmunoDOT TORCH test uses an
enzyme immunoassay (EIA) dot technique
for the detection of antibodies. The antigens
are dispensed as discrete dots onto a solid
membrane. After adding specimen to a
reaction vessel, an assay strip is inserted,
allowing patient antibodies reactive with the
test antigen to bind to the strip’s solid
support membrane.

In the second stage, the reaction is The CMVscan Card Test* is a passive latex
enhanced by the removal of nonspecifically agglutination test for the detection of IgM
bound materials. During the third stage, and IgG CMV antibodies. It can be used as
alkaline phosphatase–conjugated a diagnostic tool or to screen donor
antihuman antibodies are allowed to react specimens for antibodies to CMV in human
serum and plasma. This assay can be
with bound patient antibodies. Finally, the performed qualitatively on undiluted serum
strip is transferred to an enzyme substrate to identify antibodies to CMV and
reagent that reacts with bound alkaline quantitatively using serial twofold dilutions
phosphatase to produce an easily seen to determine the titer of CMV antibody.
distinct dot.
The absence of CMV antibodies suggests
Positive Results no viral exposure, whereas the presence
This is a dot with an easily seen distinct of CMV antibodies indicates previous
border that is visible in the center of the exposure to the virus. Recurrent infection,
window. The outer perimeter of the window if it occurs, may not be as severe as primary
must be white to pale gray. A positive result infection. Because CMV is a bloodborne
for each antigen demonstrates the presence pathogen, infection is of greatest concern
of that antibody. The presence of T. gondii with newborn infants requiring transfusion
indicates previous or current infection and and immunosuppressed allograft recipients.
immunity to future primary infection.
The serologic detection of IgM and/or IgG
antibodies to CMV is a clinically useful aid in
the diagnosis of CMV infection.

The IgG assay is used with serum for

Most common of the TORCH infections diagnostic assessment of prior infections
___________________________________ with CMV.

The presence of IgM antibodies to CMV

is, in general, indicative of primary CMV
infection. Specific IgM antibody, however,
has been reported in reactivations and
reinfections. IgM antibody may persist for
MITCH ROY B. MARISTELA, MD
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as long as 9 months in The Davidsohn differential procedure is
immunocompetent individuals and longer performed only if the preliminary
in immunosuppressed patients. Paul-Bunnell test is positive in a titer of
1:56 or greater. Serum sickness occurs as
The immunoglobulin M (IgM) type of the result of sensitization to animal serum,
heterophile antibody usually appears during usually horse serum.
the acute phase of infectious
mononucleosis, but the antigen that
stimulates its production remains unknown.
IgM heterophile antibody is characterized by
the following features:
1. Reacts with horse, ox, and sheep
erythrocytes
2. Absorbed by beef erythrocyte
3. Not absorbed by guinea pig
kidney cells
4. Does not react with EBV-specific
antigens

Epstein-Barr Virus (EBV)

Abs to Forssman Ag Positive (+) Negative (-)

Paul and Bunnell first associated infectious

mononucleosis with sheep cell agglutination
and developed a test for the infectious Rubeola
mononucleosis heterophile.

Davidsohn modified the original

Paul-Bunnell test, introducing a differential
adsorption aspect to remove the
cross-reacting Forssman and serum
sickness heterophile antibodies. Rapid
agglutination slide tests are now available.

MITCH ROY B. MARISTELA, MD

54
Fungal infections

Spirochete infections

MITCH ROY B. MARISTELA, MD

55
Syphilis

Direct Detection of Spirochetes

Direct examination of the treponemes is

most often performed with darkfield
microscopy.

Pathogenic treponemes appear as fine,

spiral (8 to 24 coils) organisms
approximately 6 to 15 μm long. They have a
trilaminar outer membrane similar to that of
gram-negative bacteria.

Serologic tests

1. Nontreponemal

2. Treponemal

Wasserman test - based on Complement

fixation The RPR test, a charcoal agglutination test,
can be performed on heated or unheated
serum or plasma using a modified VDRL
antigen suspension of choline chloride with
ethylenediaminetetraacetic acid (EDTA).
The RPR card test antigen also contains
charcoal particles to which cardiolipin
At each stage, what samples do we utilize? containing antigen is bound for macroscopic
reading.

MITCH ROY B. MARISTELA, MD

56
The RPR test is more sensitive than the Reagin can also be exhibited by patients
VDRL test for the detection of primary with noninfectious conditions such as
syphilis. autoimmune disorders, drug addiction,
old age, pregnancy, and recent
The VDRL test, a flocculation test, is both a immunization.
qualitative and quantitative screening
procedure.

Serum for testing must be heated to 56  C

(133  F) for 30 minutes to inactivate
complement. The test serum should be
used promptly after inactivation. The
antigen suspension is composed of
cardiolipin, cholesterol, and lecithin. The
VDRL test measures IgM and IgG Treponemes may remain viable for up to 5
antibodies. days in tissue specimens removed from
diseased animals and from frozen
Specific antitreponemal antibodies in early cryoprotected specimens.
or untreated early latent syphilis are
predominantly immunoglobulin M (IgM) Refrigerated blood storage decreases
antibodies. accidental transmission of the
microorganism because T. pallidum has a
The greatest elevation in IgG short survival period in stored blood.
concentration is seen in secondary
syphilis. Spirochetes do not appear to survive in
units of citrated blood at 4  C (39  F)
Nontreponemal antibodies, often called for longer than 72 hours.
reagin antibodies, are produced by infected
patients against components of their own or VDRL Reporting
other mammalian cells. Non-reactive - no clumps
Weakly reactive - small clumps
Infectious diseases in which reagin can be Reactive - medium to large clumps
demonstrate include measles, chickenpox,
hepatitis, infectious mononucleosis, The FTA-ABS test is a direct method of
leprosy, tuberculosis, leptospirosis, observation. Although not recommended for
malaria, rickettsial diseases, screening, it is the most sensitive
trypanosomiasis, and lymphogranuloma serologic procedure in the detection of
venereum. primary syphilis.

MITCH ROY B. MARISTELA, MD

57
MITCH ROY B. MARISTELA, MD
58
Lyme Disease

MITCH ROY B. MARISTELA, MD

59
Viral Hepatitis

Vaccinated

HBsAg HBeAg Anti Anti-

-Hb HBs
c

Incubation
period

Acute
infection

Window
period

Complete
recovery

Chronic
carrier

Chronic
active

MITCH ROY B. MARISTELA, MD

60
The initial detectable marker found in
serum during the incubation period of HBV
infection is HBsAg.

This procedure screens for the presence of

the major coat-protein of the virus (HBsAg)
in serum and is considered to be the most
reliable method of choice for preventing
the transmission of HBV via blood. The
presence of HBsAg indicates active HBV
infection, acute or chronic.

HBeAg appears to be a reliable marker for

the presence of high levels of virus and a
high degree of infectivity. HBeAg is a
serum marker of active viral replication.

The development of anti-HBe in a case of

acute hepatitis is the first serologic
evidence of the convalescent phase.

Anti-HBs is probably the major protective

antibody in this disease.

The time between the disappearance of

detectable HBsAg and the appearance of
detectable antibody to HBsAg (anti-HBs) is
called the anticore window or hidden
antigen phase of HBV infection.

MITCH ROY B. MARISTELA, MD

61
Cryptococcus neoformans Dengue, Malaria, Leptospirosis, Typhoid
Notes
Latex-Cryptococcus Antigen Detection
System

Babesiosis

Babesia spp. are visualized as

intraerythrocytic organisms in thick
peripheral (rapid Field’s test) or thin
blood films.

Acute and convalescent antibody titers may

be useful; a titer higher than 1:256 is
diagnostic of acute infection.

Only IgG antibody determinations are

performed. PCR amplification can be used
for diagnosis.

MITCH ROY B. MARISTELA, MD

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Tuberculosis Infection Additional Notes on Thyroid Diseases
Notes

PPD vs IGRA

MITCH ROY B. MARISTELA, MD

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PERTINENT SEROLOGIC TECHNIQUES primers, typically 16 to 20 bp, are
used. The oligonucleotides (small
Target Amplification portions of a single DNA strand) act
as a template for the new DNA.
Amplicon - piece of genetic material, such These primer sequences are
as DNA, that can be formed as the product complementary to the 3′ ends of the
of a natural event or artificial amplification sequence to be amplified.
technique
- This enzymatic process is carried
A molecular diagnostic laboratory that out in cycles. Each repeated cycle
performs in vitro amplification reactions consists of the following:
needs to practice techniques to control a. DNA denaturation.
contamination. This is especially true if a Separation of the double
high number of thermal cycles is used for DNA strands into two single
the PCR. strands through the use of
heat.
PCR is highly sensitive but a disadvantage b. Primer annealing.
to the use of this assay is that it is prone to Recombination of the
producing false-positive results. In oligonucle- otide primers with
laboratories in which PCR is performed the single-stranded original
frequently, any false- positives are DNA.
generally caused by amplicon c. Extension of primed DNA
contamination. A broken capillary tube or a sequence. The enzyme DNA
PCR plate left carelessly at the edge of a polymerase synthesizes new
table can aerosolize those amplicons, which complementary strands by
can then adhere to lab coats and objects in the extension of primers.
the room.
- Each cycle theoretically doubles the
Spray equipment, workstations, and amount of specific DNA sequence
pipettes with 10% bleach and then let it sit present and results in an exponential
for 15 to 30 minutes. accumu- lation of the DNA fragment
being amplified (amplicons). In
Polymerase Chain Reaction general, this process is repeated
approximately 30 times. At the end
an in vitro method that amplifies low levels of 30 cycles, the reaction mixture
of specific DNA sequences in a sample to should contain about 2^30
higher levels suitable for further analysis. molecules of the desired product.

- To use this technology, the target - After cycling is complete, the

sequence to be amplified must be amplification products can be
known. Typically, a target sequence analyzed in various ways. Typically,
ranges from 100 to 1000 base pairs the contents of the reaction vessel
(bp) in length. Two short DNA are subjected to gel electrophoresis.

MITCH ROY B. MARISTELA, MD

64
 This allows visualization of the
amplified gene segments (e.g., PCR
prod- ucts, bands) and determination
of their specificity. Additional product
analysis by probe hybridization or
direct DNA sequencing is often
performed to verify the authenticity
of the amplicon further.

Applications of PCR (RT) in the initial steps.

RT-PCR is useful in the
1. Amplification of DNA identification of RNA viral
2. Identification of a target sequence agents, such as human
3. Synthesis of a labeled antisense immunodeficiency virus (HIV)
probe and hepatitis C virus (HCV).

Modified Polymerase Chain Reaction 2. Multiplex Polymerase Chain

Techniques Reaction
- uses numerous primers in a
1. Reverse Transcriptase single reaction tube to
Polymerase Chain Reaction amplify nucleic acid
- If the nucleic acid of interest fragments from different
is RNA rather than DNA, the targets. Specific nucleic acid
PCR procedures can be amplification should occur if
modified to include the the appropriate target DNA is
conversion of RNA to DNA present in the sample tests.
using reverse transcriptase Detection may be

MITCH ROY B. MARISTELA, MD

65
 accomplished by the 2. Transcription-Mediated
traditional Southern transfer Amplification
method and subsequent - Transcription-mediated
nucleic acid probe, by amplification (TMA) is
enzyme immunoassay (EIA) another isothermal assay
methods, or by gene chip that targets DNA or RNA, but
analysis. This technology is generates RNA as its
limited by the following: (1) amplified product. TMA is
the number of primers that used to detect
can be included in a single microorganisms (e.g.,
reaction; (2) primer-primer Mycobacterium tuberculosis).
interference; and (3)
nonspecific nucleic acid 3. Nucleic Acid Sequence–Based
amplification. Amplification
- Nucleic acid
3. Real-Time Polymerase Chain sequence–based
Reaction amplification (NASBA) is
- uses fluorescence-resonance similar to TMA, but only RNA
energy transfer to quantitate is targeted for amplification.
specific DNA sequences of Its applications include the
interest and identify point detection and quantitation of
mutations. Real-time PCR is HIV and detection of
particularly appealing cytomegalovirus (CMV).
because the procedure is
less susceptible to amplicon 4. Ligase Chain Reaction Nucleic
contamination and is more Acid Amplification
accurate in quantifying the - Oligonucleotide pairs
initial copy number. hybridize to target sequences
within the gene or the cryptic
Other amplification techniques plasmid. The bound
oligonucleotides are
1. Strand Displacement separated by a small gap at
Amplification the target site. The enzyme
- Strand displacement DNA polymerase uses
amplification (SDA) is a fully nucleotides in the ligase
automated method that chain reaction (LCR)–nucleic
amplifies target nucleic acid acid amplification reaction
without the use of a mixture to fill in this gap,
thermocycler. A creating a ligatable junction.
double-stranded DNA Once the gap is filled, DNA
fragment is created and ligase joins the
becomes the target for oligonucleotide pairs to form
exponential amplification. a short, single-stranded
product that is

MITCH ROY B. MARISTELA, MD

66
 complementary to the Analysis of DNA products
original target sequence.
This product can itself serve 1. Conventional Analysis - Detection
as a target for hybridization of DNA products by PCR assay can
and ligation of a second pair be convention- ally analyzed using
of oligonucleotides present in agarose gel electrophoresis after
the LCR reaction mixture. ethidium bromide staining. This
- Subsequent rounds of technique is simply an extra step
denaturation and ligation after a PCR assay has been run.
lead to the geometric DNA and other biomolecules can be
accumulation of amplification separated based on charge, size,
product. The amplified and shape. DNA has a net negative
products can be detected in charge and will migrate toward the
an LCx analyzer (Abbott anode (positive pole). PCR products
Laboratories, Abbott Park, Ill) are loaded into an agarose gel and
by microparticle EIA. electrophoresed. Ethidium bromide
is a dye that intercalates into nucleic
5. Target Enrichment Strategies acids and will fluoresce with an
- This approach overcomes orange color under ultraviolet (UV)
the limitations of traditional irradiation. An image analyzer uses
Sanger sequencing by UV light to capture computer images
providing highly parallel of the PCR products.
sequencing with a separate
sequence result for every 2. Other Techniques - include the
sequence of interest. This hybridization protection assay,
has positioned NGS as the DNA EIA, automated DNA
method of choice for targeted sequencing technology,
re-sequencing of regions of single-strand conformational
the human genome. Two polymorphism, and restriction
main technologies have been fragment length polymorphism
used to enable target (RFLP) analysis. The selection of
enrichment: PCR and one technique over another is often
hybridization. based on factors such as sensitivity
and specificity pro- files, cost,
Advantages of Molecular testing turnaround time, and local
1. Faster turnaround time experience. Other techniques are
2. Smaller required sample volumes used to enhance the sensitivity
3. Increased specificity and sensitivity and specificity of amplification
techniques.

MITCH ROY B. MARISTELA, MD

67
DNA Sequencing Branched DNA

DNA sequencing is considered to be the Branched DNA (bDNA) is another

gold standard to which other molecular quantitative test that uses signal
methods are compared. DNA sequencing amplification instead of target
displays the exact nucleotide or base amplification. Target DNA or RNA is
sequence of a fragment of DNA that is hybridized at different sites by two types of
targeted. The Sanger method, which uses probes. Branched DNA assays are used to
a series of enzymatic reactions to produce measure the viral load of hepatitis B virus
segments of DNA complementary to the (HBV), HCV, HIV-1, CMV, and microbial
DNA being sequenced, is the most organisms (e.g., Trypanosoma brucei).
frequently used method for DNA
sequencing. Automated sequencing The Versant HIV-1 RNA 3.0 assay (bDNA;
techniques use primers with four different Bayer, Berkeley, Calif ), uses bDNA
fluorescent labels. technology. It is the only viral load assay
specifically designed to target multiple
Steps in sequencing: sequences of the HIV-1 genome with more
1. The first step in sequencing a target than 80 nucleic acid probes.
is usually to amplify it by cloning
or in vitro amplification, usually Hybridization Techniques
PCR. Once the amplified DNA is
purified from the clinical specimen Many forms of probe hybridization assays
(the target DNA), it is involve the complementary pairing of a
heat-denatured to separate the probe with a DNA or RNA strand derived
double- stranded DNA (dsDNA) into from the patient’s specimen. The common
single strands (ssDNA). feature of probe hybridization assays is the
2. The second step involves adding use of a labeled nucleic acid probe to
primers to the ssDNA. Primers are examine a specimen for a specific,
short synthetic segments of ssDNA homologous DNA or RNA sequence.
that contain a nucleotide sequence Clinical probes are usually labeled with
complementary to a short strand of nonradioisotopic molecules such as
target DNA. The patient’s DNA digoxigenin, alkaline phosphatase, bio- tin,
serves as a template to copy. DNA or a fluorescent compound. The detection
polymerase catalyzes the addition of systems are conjugate-dependent and
the appropriate nucleotides to the include chemiluminescent, fluorescent, and
preexisting primer. DNA synthesis is calorimetric methodologies.
terminated when the
deoxynucleotide is incorporated 1. Liquid-Phase Hybridization
into a growing DNA chain. - the target nucleic acid and
labeled probe interact in
solution. Specific
homologous hybrids are
subsequently separated from
the remaining nucleic acid

MITCH ROY B. MARISTELA, MD

68
 component and the hybrids
are identified by an
appropriate detection
system.

2. Dot Blot and Reverse Dot Blot

- These hybridization methods
are used in the clinical
laboratory for the detection of
disorders in which the DNA
sequence of the mutated
region has been identified
(e.g., sickle cell anemia,
cystic fibrosis). These
techniques are capable of
distinguishing the
homozygous or
heterozygous state of a
mutation.
a. Dot Blot

2. Northern blot

b. Reverse Dot Blot

Blotting Protocols 3. Western blot

1. Southern blot

MITCH ROY B. MARISTELA, MD

69
Microarrays - Microarray (DNA chip) hematopoiesis. New advances have shown
technology has helped accelerate genetic that there may be wide implications for
analysis, just as microprocessors cytokines in the diagnosis and treatment of
accelerated computation. Microarrays are various disorders. Cytokines have been
basically the product of bonding or direct implicated in cancer, autoimmune
synthesis of numerous specific DNA probes disorders, and septic shock, among
on a stationary, often silicon-based support. other disorders. Cytokine detection and
The chip may be tailored to particular quantification play an important role in
disease processes. The technique is easily clinical practice.
performed and readily automated.
Assessment of cytokines

Defects in cytokine production can lead to

autoimmunity (Table 5-10). Traditional
methods for assessment of cytokines
include the following:
1. Bioassays
2. Enzyme-linked immunosorbent
assay (ELISA)
3. Intracellular staining
4. Ribonuclease protection assay
5. Polymerase chain reaction (PCR)
Newer methods of measurement include the
following:
Cytokines - are important mediators of 6. Multiplexed assay using the
cell-to-cell communication within the innate FlowMetrix; quantifies multiple
and adaptive immune systems. The cytokines simultaneously
expression of most cytokines is highly 7. Intracellular staining using flow
regulated and generally occurs in response cytometry
to foreign or self-antigenic stimulation. The 8. Cord blood mononuclear cells
functions of cytokines are extremely varied, stimulated by allergens (celELISA)
with many cytokines also displaying 9. Real-time PCR for lymph nodes or
pleiotropic effects, depending on their spleen
cellular target. Some cytokines, such as 10. Enzyme-linked immunosorbent spot
tumor necrosis factor (TNF), interleukin (ELISPOT) assays • Enhanced
(IL)-1 beta, IL-6, interferon (IFN)-alpha and immunoassays for cytokines
beta, IL-10, and IL-18 are particularly 11. Biotrak assay—high-sensitivity
important in the innate immune response. ELISA

Cytokines are soluble proteins that act as

chemical messengers in the immune
response, and play an important role for
communication with cells in other systems
and in cell growth, differentiation, and

MITCH ROY B. MARISTELA, MD

70
4 types of measurements are to be Sample reference values:
discussed below:
Tumor necrosis factor: <10.0 pg/mL
TESTS ADVANTA DISADVAN Interleukin (IL)-6: <5.0 pg/mL
GES TAGES Interferon (IFN)-beta: <20.0 pg/mL
IL-10: <7.0 pg/mL
1. Bioassays good low Monocyte chemoattractant protein-1: < or
sensitivity; specificity; =198 pg/mL
capability poor IL-1 beta: <20.0 pg/mL
of precision; IFN-gamma: <60.0 pg/mL
measuring longer Macrophage inflammatory protein-1
biologically assay alpha: <220 pg/mL
active times Granulocyte-monocyte colony
molecules stimulating factor: <15.0 pg/mL
IL-2 receptor alpha soluble: < or =959
2.Immuno- excellent measures pg/mL
assays sensitivity; total IFN-alpha: <20.0 pg/mL
high cytokine IL-18: < or =468 pg/mL
specificity; concentrati
shorter on; Elevated cytokine concentrations could
assay cross-reacti be consistent with the presence of
times; vity with infection or other inflammatory
ability for precursor processes.
automation or
degradatio Results from cytokine testing should not be
n products used to establish or exclude a specific
diagnosis.
3.Flow rapid complexity
Cytometry analysis of testing
Cytokine testing should only be used in
4. high conjunction with clinical information and
Nanoparticle- sensitivity; other laboratory testing as part of a
modified high patient’s overall assessment.
aptamers specificity;
ability for Normal concentrations of cytokines do not
automation exclude the possibility of infection or
other inflammatory conditions.

Cytokine concentrations could be affected

by immunomodulatory agents.

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71