IMMUNOSEROLOGY REVIEW

Overview and Historical Background

IMMUNOLOGY

 Study of reactions of the host when foreign substances are introduced into the body
 Study of all aspects of body defenses such as antigens and antibodies, allergy, and
hypersensitivity.

IMMUNE SYSTEM

 The human immune system is designed to produce coordinated response to the

introduction of foreign substances or antigens into the body.
 Divided into two complementary arms: Innate (native or natural) immune system and the
ADAPTIVE (acquired or specific) immune system
 Once the barriers of the innate immune response have been breached, the adaptive
immune response is activated in an Antigen-specific fashion to eliminate the antigen and
provide lasting protection from future challenge.

Characteristics Innate Immunity Adaptive Immunity

Specificity For structures shared by For specific antigens of microbial

groups of microbes and nonmicrobial agents
Diversity LIMITED HIGH

Memory NO YES

Physical Barriers Epithelial tight junctions

Mucus
Humoral Factors Lysozyme Immunoglobulin Antibodies
Completement
Defensins, Acute
Phase Reactants
Interferons
Cells Neutro/Eo/Basophils, Mast Lymphocytes (except NK cell)
cells NK cells, Monocytes,
Macrophages
Key Features in Toll-like Receptors: pattern Memory cells: activated B and T
Pathogen Recognition recognition receptors that cells; subsequent exposure to a
recognize pathogen- previously encountered antigen
associated molecular results to a stronger quicker
patterns (PAMPS) immune response.
LYMPHOID ORGANS

Primary Lymphoid Organs

The site of maturation of B and T cells

A. Thymus – T cells
B. Bone Marrow – B cells

Secondary Lymphoid Organs

Site of proliferation and differentiation of T

and B cells.

A. Spleen
B. Lymph Nodes
C. MALT

THYMUS

 Site of T cell differentiation and

maturation
 Cortex is dense with immature T cells
 Medulla is pale with mature T cells and Hassal corpuscles containing epithelial reticular
cells.

LYMPH NODE

 Encapsulated, with trabeculae

 Functions are nonspecific filtration by Macrophages, storage of B and T cells, and
immune response activation.
 Cortex site for B cell localization and proliferation
 Medulla – consists of medullary cords and sinuses containing reticular cells and
macrophages
 Paracortex region between follicles and medulla; houses T cells

SPLEEN

 T cells are found in the periarteriolar lymphatic sheath within the white pulp
 B cells are found in follicles within the white pulp
 The marginal zone, in between the red pulp and white pulp, contains macrophages and
specialized B cells, and is where antigen-presenting cells capture blood-borne antigens
for recognition by lymphocytes.
HISTORICAL BACKGROUND 2%

Year Scientist Discovery

A.D 1500 Chinese Variolation
1718 Lady Montagu Injection of material from
crusts/fluids from smallpox blisters
1798 Edward Jenner (Father of Cross Immunity
Immunology)
1862 Haeckel Describe phagocytosis
1883-1905 Metchnioff Demonstrated phagocytosis
1885 Louis Pasteur Live, attenuated rabies vaccine
1890 Von Behring & Kitasata Humoral theory of immunity
1891 Robert Koch Delayed hypersensitivity
1894 Jules Bordet Complement
1897 Robert Kaus Precipitation
1900 Paul Ehrlich Antibody formation theory
1901 Von Behring Serum therapy or serum antitoxins
1902 Portier & Richet Immediate hypersensitivity
(anaphylaxis)
1903 Maurice Arthus Arthus reaction
1903 Von Pirquet & Schick Serum Sickness
1903 White & Douglas Opsonization
1905 Robert Koch Cellular immunity in TB
1920 Prausnitz & Kustner Immunologic basis of some allergy
1928 Alexander Fleming Penicillin
1944 Medawar Immunologic processes in
transplantation
1949 Salk & Sabin Polio vaccine
1951 Reed Vaccine against yellow fever
1957 Burnet Clonal selection theory
1972 Edelman and Porter Structure of antibodies
1976 Kohter Milstein First monoclonal antibodies using
hybridoma
1977 Rosalyn Yalow Radioimmunoassay
1987 Susumu Tonegawa Antibody diversity
1980 Snell, Dalisset & Benenceraf MHC
1983 Luc Montagnier HIV
1984 Robert Gallo HIV
1984 T cell receptor T cell receptor
2005 Frazer HPV vaccine

Good to Know

 Variolation is the deliberate exposure to material from smallpox lesions

 Edward Jenner used a strain of cowpox virus to protect a child from smallpox
 Vaccination was derived from Latin word “vacca” which means cow
 Radioimmunoassay was the first Immunoassay developed
 NATURAL (INNATE) IMMUNITY, Including Role of Macrophages, Monocytes and Granulocytes

INNATE IMMUNITY 5%

Components of Natural/Innate Immune System

CELLULAR Phagocytic Cells

Basophils, mast cells
Eosinophils
Natural Killer Cells
HUMORAL Complement
Fluid Components Interferon
Lysozymes
Natural antimicrobial substances

Innate Immunity Adaptive Immunity

Present intrinsically Inducible
Nonspecific Specific
No memory Memory
Limited diversity Extensive Diversity
Distinction of self vs Non-self
Self limiting

First Line of Defense Second Line of Defense Third Line of Defense

Skin Phagocytic Leukocytes Lymphocytes
Mucus Membrane Inflammation and Fever (IL1 Antibodies
and IL6 induce fever)
Lyzosome Natural Antimicrobial Memory Cells
Substances

ANTIGEN PRESENTING CELLS

 Phagocytose, process, and present the antigen to T cells through MHC molecules
 Dendritic cells are the most effective APCs for initiating primary immune responses. They
are located under the epithelium (common site of entry) and in the interstitium of tissues
(site of antigen production)

NATURAL KILLER CELLS

 A large granular lymphocyte that kills tumor cells and virally infected cells using
granzymes and perforins.
 Kill target lacking in MHC Class I
 Share common early progenitor with T cells, but do not develop in the thymus
 Identified by the presence of CD16 (FcRy) and CD56 (CAM) 611 Adhesion Molecule
 Killing by inducing apoptosis

PROPERDIN

 Bactericidal, viricidal in the presence of C3 & Mg2

BETA-LYSIN

 Heat stable cationic substance released by platelet during coagulation with bactericidal
activity found in serum of may animal species including humans

TUMOR NECROSIS FACTOR

 Cytotoxic against tumor cells and virally infected cells

 TNF – α – produced by macrophages and NK cells; aka CACHECTIN
 TNF – β – produced by CD4 CD8 T cells; aka LYMPHOTOXIN

INTERFERONS

 Family of glycoproteins produced by animal cells that exert a VIRUS-NONSPECIFIC but

HOST-SPECIFIC antiviral activity.
 FUNCTIONS AS:
 Immune regulator
 Antiviral
 Anti-neoplastic

Type IFN Name Secreted By Features

I IFN - α Leukocyte IFN Leukocytes Inhibits viral replication
IFN - β Epithelial cell IFN Fibroblasts Inhibits viral replication
II IFN - γ Immune IFN Th1, NK cells Increases expression of
MHC Class I & II

ACUTE PHASE REACTANTS

 Produced by the liver induced by IL-6

 CRP and Serum amyloid A are 1000x increased in acute inflammation
 CRP is a nonspecific but most widely used indicator of acute inflammation
 CRP is originally thought to be an antibody to the c-polysaccharide of pneumococci

Positive (Upregulated) Negative (Downregulated)

C – reactive protein Albumin
Serum amyloid A Transferrin
Ferritin, Fibrinogen
Hepcidin, Haptoglobin
Ceruloplasmin, Complement C3
Mannose binding protein

STAGES OF INFLAMMATION

1. Vascular Response
o Increased Vascular Permeability is the hallmark of acute inflammation
o Injured cells (mast cells) release histamine; promotes VASODILATION
Hyperemia – Increased Blood flow to injured area
Increased capillary permeability – plasma leakage to tissue (causing
swelling/pain)
 2. Cellular Response
o Margination – movement of leukocytes from center to periphery of blood vessel
o Rolling – transient adhesion of leukocytes to the endothelial cells
o Adhesion – firm attachment of the leukocytes to the endothelial cells
o Transmigration – migration of the leukocytes through the endothelium into the
tissues; aka Diapedesis
o Chemotaxis – unidirection or targeted movement of the leukocytes towards
antigens/bacteria in response to certain chemicals
o Opsonization – coating of the bacteria so that they are easily phagocytosed
o Phagocytosis – process by which bacteria are killed/eaten up by the white blood
cells

3. Resolution and Repair

o Final stage associated with fibroblast proliferation, which may result in:
 Complete repair and restoration of function
 Abscess formation with some loss of function
 Granuloma function

CARDINAL SIGNS OF INFLAMMATION

CARDINAL SIGNS OF INFLAMMATION

CALOR RUBOR Tumor Dolor Functio laesa
(Heat) (Redness) (Swelling) (Pain) (Loss of Function)

PHAGICYTOSIS (ICED)

Initiation

o Phagocytosis is initiated as a result of Tissue damage

Chemotaxis

o Unidirectioal or targeted movement of the leukocytes towards antigens/bacteria in

response to certain chemicals
o Without chemotaxis, cell motion is (lai) Random
o Examples of chemotoxins: C3a C5a IL-8
o Job Syndrome: impaired chemotaxis
o Lazy Leukocyte Syndrome: impaired random movement and chemotaxis

Engulfment

o Fusion of phagosome and lysosome to form phagolysosome

Digestion

o Oxygen dependent killing

 Respiratory burt – production of reactive oxygen species which are toxic to
bacteria; enzymes involved are NADPH oxidase, catalase, SOD
o Oxygen independent killing
 Performed by lysozyme, lactoferrin, major basic protein, defensins

ACQUIRED IMMUNITY – Humoral responses, immunogens, immunoglobulins, B cells; Cellular

Responses, T Cells, Cytokines, And Chemokines

ACQUIRED IMMUNITY
CELLULAR T Cells
B Cells
HUMORAL Lymphokines
Antibodies

T LYMPHOCYTES

 Represents approximately 80% of the circulating lymphocytes in peripheral blood.

 Most circulating T cells express 3 CD Markers
CELLULAR ACQUIRED IMMUNITY

T CELL

 T-cell precursors move from the bone marrow to the thymus here they are selected for
self-tolerance by exposure to MHC antigens on stromal cells
o POSITIVE SELECTION occurs in the Thymic Cortex T cells expressing TCRS capable
of binding self-MHC on cortical epithelial cells survive.
o NEGATIVE SELECTION occurs in the Thymic Medulla T cells expressing TCRs with high
affinity for self-antigens undergo apoptosis

 Double negative T cells – no CD4 and CD8

 Double positive T cells – both CD4 and CD8 positive
 Mature T cell – either CD4 or CD8 positive
o Cytotoxic T cells
 have CD8 which binds to MHC Class I
 They kill virus-infected and tumor cells by inducing apoptosis
o Helper T cells
 Have CD4 which binds to MHC Class II
 They act as the orchestrators of the effector mechanisms of the immune
response
 (antibody synthesis, macrophage activation, cytotoxic T cell killing, NK cell
killing)
o The Naïve helper T cell (Th0 cell) further differentiates into Th1, Th2, or Th17
depending on the type of antigen causing the infection
 Th1 – intracellular infections; secrete IFN- γ
 Th2 – extracellular parasites & allergens secrete IL-4, IL5
 Th17 – extracellular bacterial & fungal infections, secrete IL-17, L-22
o T regulatory cells
 Develops from the Th0 cell
 Identified by the expression of C02
 Critical for prevention o autoimmunity

ADJUVANTS

 Is a substance, distinct from antigen, that enhances T and B cell activation mainly by
promoting the accumulation and activation of APC’s at the site of antigen exposure.

MITOGEN

 Is a substance that stimulates cell division.

B LYMPHOCYTES

 Bone Marrow derived lymphocytes

 Precursor cells in antibody production

B CELL

 B cells are generated and mature in the bone marrow. Main function is Antibody
production. Each B cell produces antibody of only one specificity.
 Self-tolerance is induced by deletion of self-reactive B cells in the bone marrow (Clonal
Deletion) or inactivation of self-reactive cells in the periphery (Clonal Anergy)
  Elimination of self-reactive cells in the bone marrow is intended to minimize the number
of self-reactive B-lymphocytes released to the periphery
 Only those cells that are selectively unresponsive (tolerant) to self-antigens are allowed
to leave the bone marrow

Maturation of Stage of B Cell Bone Marrow to Periphery

IMMUNIZATIONS

 Toxoid vaccines are made from inactivated exotoxins from toxigenic bacteria (e.g DPT
vaccine)
 Live attenuated – Microorganisms loses its pathogenicity but retains capacity for transient
growth within inoculated host; e.g. BCG, Poli (Sabin)m Measles, Mumps, Rubella,
Varicella, Yellow fever
 Inactive or Killed – Pathogen is inactivated by heat-or-chemicals; e.g. “RIP Always”
Rabies, Influenza (injection), Polio (Salk), Hepatitis A

HUMORAL ACQUIRED IMMUNITY

B Cell T Cell
 IgM, IgD  TCR
 Can bind unprocessed Ag  Can only bind peptides complexed to
MHC
 Ultimately secreted as antibody  Never released from membrane
bound location

TYPE MODE OF ACQUISTION

ACTIVE NATURAL Infection (Antigen)
 Antibody production is done ARTIFICIAL Vaccination (Antigen)
by the body a. Live Organism
 Advantage: LONG-TERM b. Attenuated or weakened organism
 Disadvantage: Immune c. Dead organism
Response is SLOW d. Toxoids
e. Modified virus
PASSIVE NATURAL Transfer in vivo (IgG) Antibody
 Antibody production is not Colustrum (IgA) Antibody
done by the body ARTIFICIAL Immune serum (IgG) Antibodies
 Advantagae: IMMEDIATE
Response/PROJECTION
 Disadvantage: SHORT TERM
ANTIBODIES (Ig)

 Substances produced in response to antigenic stimulation that as capable of specific

interaction with provoking immunogen.
 Present on the surface of B616 or secreted by plasma cells
 When subjected to electrophoresis at pH 8.6, they appear in the Gamma region

ANTIBODY

 A tetrapeptide consisting of 2 heavy chairs and 2 light chairs linked by disulfide bonds
 The amino terminal end of each chain contains the variable region
 The carboxyl terminal end of each chain contains the constant region
 A hinge region joins the heavy chains and is rich in proline for flexibility. It is found between
CH1 and CH2.
 A Fab fragment consists of 1 light chain and ½ heavy chain
 The complement binding site is found in CH2 and CH3
 IgM and IgE contains CH4

Papain digestion yields 3 fragments:

 2 F(ab) fragments
 1 fc fragment

Pepsin digestion yields 2 fragments:

 1 F(ab) 2 fragments
 1 fc fragment

GENERAL FUNCTIONS OF IMMMUNOGLOBULINS

1. Neutralize toxic substances

2. Facilitate Phagocytosis
3. Combine with antigens on cellular surfaces and thereby cause the destructions of these
cells either extravascularly or intravascularly
 5 TYPES OF IMMUNOGLOBULINS

IgG

 Most abundant isotype in serum (75-80% of total serum Ig)

 Longest half-life (23-25 days)
 Most efficient in (75-80%) Precipitation
 Secondary (anamnestic) response antibody
 4 major subclasses (IgG1-IgG4) differ in number and position of disulfide bridges between
the γ chains
 IgG1 most efficient in crossing placenta
 IgG3 most efficient in complement fixation
 Major functions of IgG: providing immunity for the newborn, complement fixation,
opsonization, neutralization of toxins and viruses, agglutination and precipitation

IgM

 “Largest antibody”; aka macroglobulin

 Most efficient in Agglutination
 Monomer on B cell surface; Pentamer with J chain when secreted
 Produced in the Primary (immediate) response to an antigen
 Fixes complement but does not cross the placenta

IgA

 Monomer in circulation; Dimer with J chain when secreted

 The secretory compound facilitates transcytosis IgA across mucosal surfaces and
protects the Fc portion from enzymatic digestion.

IgD

 Present on the surface of immunocompetent but unstimulated 8 cells

 Plays a role in B-cell activation, maturation, and differentiation

IgE

 Least abundant immunoglobulin in the serum and the most heat labile
 Attaches to basophils and tissue mast-cells through high affinity FcR
 Mediates Anthelminthic and allergic responses (type I hypersensitivity)
 Also called reaginic antibody, homocytotropic antibody

IgG IgM IgA IgD IgE

Structure Monomer Pentamer Serum- Monomer Monomer
Monomer
Percent of 70-75% 5-10% 10-15% 0.2% 0.002
total Globulin
MW (Daltons) 150,000 900,000 160,000- 180,000 190,000
350,000
Sedimentation 7S 19 S 7S 7S 8S
Coefficient
Serum half-life 23 6 5 1-3 2-3
(Days)
C’ fixation YES YES ALT NO NO
 Crosses YES NO NO NO NO
placenta

COMPLEMENT SYSTEM 2%; MHC, HLA and Transplantation 3%

COMPLEMENT SYSTEM

 A humoral mechanism of nonspecific immune response consisting of 14 distinct serum

proteins that proceed in cascading sequence of activation resulting to cell lysis.
 The complement system proteins are named with a capital C followed by a number. A
small letter after the number indicates that the protein is a smaller protein resulting from
the cleavage of a larger precursor by a protease.
 Several complement proteins are cleaved during activation of the complement system;
the fragments are designated with lower case suffixes, such as C3a and C3b.
 Usually, the larger fragment is designated as “b” and the smaller fragment as “a”.
 The exception is the designation of the C2 fragments;
 The larger fragment is designated C2a and the smaller fragment is C2b.
 Proteins of the alternative activation pathway are called factors and are
symbolized by letters such as B. Control proteins include the inhibitor of C1 (C1
INH), factor I, and factor H.

ACTIVATION

 Classical pathway: Antigen-Antibody complex

 Alternative pathway: microbe surface molecule
 Lectin pathway: Mannose

FUNCTIONS OF IMPORTANT INDIVIDUAL COMPLEMENT PROTEINS

C3a, X5a – Anaphylaxis

C3b, inactive C3 – Opsonization

C5a – Neutrophil chemotaxis

C5b – 9 – Cytolysis by membrane attack complex

REGULATOR MOLECULES OF COMPLEMENT SYSTEM

 C1 inhibitor – Dissociates C1r and C1s from C1q

 Factor I – Proteolytically cleaves C3b and C4b
 Factor II – Cofactor of Factor I to inactivate C3b; Prevents binding of B to C3b
 C4 binding Protein Cofactor with Factor I to inactivate C4b
 DAF – Increases the dissociation of 3convertase
 CD59 – Membrane inhibitor of reactive lysis (MIRL); Inhibits formation of MAC
 S Protein (Vitronectin) – Prevents attachment of C5b67 to cell membrane

CLASSICAL PATHWAY ALTERNATIVE PATHWAY

C19 - Binds to Fc of IgM or IgG B – Binds 3b to form C3 convertase
C1r – Activates C1s D – Cleaves factor B
C1s – Cleaves C4 & C2 P – Stabilizes C3 convertase
C3 – Key intermediate (all pathways) LECTIN PATHWAY
C5 – Initiates MAC MBL – Binds to mannose
C6 – Starts pore formation MASP-1 – Helps cleave C4 & C2
Polymerizes to cause cell lysis MASP-2 – Cleaves C4 & C2

DEFICIENCIES OF COMPLEMENT COMPONENT AND ASSOCIATED DISEASE

 C1 esterass inhibitor – Hereditary angioneurotic edema

 C1, C2, C4 – SLE-like and collagen vascular disorders
 C2 – Most common complement deficiency
 C3 – Severe recurrent infections, Glomerulonephritis
 C5 to C8 – Recurrent Neisseria infections
 C9 – No associated disease
 DAF, CD59 – Paroxysmal nocturnal hemoglobinuria
 CD46, Factors, H factor I – Atypical or non-epidemic hemolytic uremic syndrome

MHC, HLA, & TRANSPLANT

MAJOR HISTOCOMPATIBILITY COMPLEX

 Genes that control expression of a large group of proteins

 Regulates the immune response
 Play a role in graft rejection
 MHC is a cluster of genes found in the short arm of chromosome 6
 HLA genes are inherited as haplotypes, one from each parent.

MHC CLASS I MHC CLASS II MHC CLASS III

Genetic Loci HLA-A, HLA-B, HLAC HLA-DP HLA-DO, HLA-
DR
Expression Expressed on all Expressed on APCS C2, C4
nucleated cells & (macrophages, B Properdin factor B
platelets cells, dendritic cells) TNF α and β
Not expressed on
Heat shock protein
RBCs
 Present Present exogenously Tyrosine hydroxylase
endogenously synthesized antigens
synthesized antigens to CD4+ helper
to CD8+
Function Present Present exogenously
endogenously synthesized antigens
synthesized antigens to CD4+ helper T cells
to CD8+ cytotoxic T
cells
Antigen Examples Viral cytosoloic Bacterial Proteins
proteins
Associated Proteins B2-microglobulin Invariant chain

IMMUNOGEN

 Substance that stimulate an immune response e.g antibodies

ANTIGEN

 Substance that reacts with specific antibody

EPITOPE (determinant site)

 The key portion of the immunogen against which the immune response is directed;

IMPORTANCE OF HLA TYPING

1. Tissue/Organ Transplant
 It must be HLA and ABO Matched
 Siblings: 50% chance of HLA Match
 Parents: 25% chance of HLA Match
2. Disease Association
 Refer to the table next slide
3. Paternity Testing
 DNA Definitive test for Paternity Testing

HLA SUBTYPES ASSOCIATED WITH DISEASES

HLA DISEASE
A3 Hemochromatosis
B8 Addison disease, Myasthenia gravis, Grave’s disease
B27 Psoriatic arthritis Ankylosing spondylitis, Inflammatory bowel,
Disease-associated arthritis, Reactive-arthritis (formerly Reiter
Syndrome)
B51 Behcet’s disease
CW6 Psoriasis vulgaris
DQ2/DQ3 Celiac disease
DR2 Hay fever, Goodpasture syndrome, SLE, Multiple sclerosis
DR3 DM type 1, Sjogren’s syndrome, Chronic active hepatitis, Addison
Disease, Myasthenia gravis, SLE, Grave’s disease, Hashimoto
Thyroiditis, Dermatits herpetiformis
DR4 DM type 1, Rheumatoid Arthritis, Pemphigus Vulgaris
DR5 Pernicious anemia, Hashimoto thyroiditis
TRANSPLANTATION

Most Immunogenic

 Bone Marrow
 Skin
 Islets of Langerhans
 Heart
 Kidney
 Liver
 Bone
 Xenogenic Valve replacement
 Cornea

Least Immunogenic

FACTORS AFFECTING IMMUNOGENECITY

1. FOREIGNNESS & GRAFT

 Autoantigen - from the same individual
 Alloantigen - from different individuals, but of the same species
 Heterantigen - from different species
 Heterophile antigens - antigens from unrelated plants and animals, cross-react with
antibody of another

 Autograft - same individual

 Synthetic graft - between identical twins
 Allograft - Between two individuals of the same species
 Xenograph - Between two individuals of a different species

2. SIZE
 POTENT ANTIGEN: MW>10,000 Dalton
 Albumin- 40,000 Dalton, This are good immunogen
 Hemocyanin- MW IM Dalton- Excellent immunogen

3. CHEMICAL COMPOSITION AND COMPLEXITY

 PROTEINS - MOST IMMUNOGENIC
 POLYSACCHARIDES - SECOND
 LIPIDS, NUCLEIC ACID – LEAST

4. ROUTE, DOSAGE AND TIMING

 Intravenous and Intraperitoneal routes are effective
 Intradermal route-stronger stimulus than subcutaneous or intramuscular route
 Smaller the dose, the less likely a response

5. ADJUVANTS
 Substances added to vaccines and less immunogenic substances to enhance
immune response.
A. Complete Freund's Adjuvant (CFA)- Stimulates T Cells
B. Lipopolysaccharides (LPS)- Stimulates B Cells
 C. Alum Adjuvants- stimulates phagocytic cells
D. Squalene – for HIV Vaccines; from sharks’ oil

Type of Onset Pathogenesis Features

Rejection
Hyperacute Within minutes Pre-existing receipt anti- Thrombosis =>
bodies react to donor antigen ischemia and
(Type II HS), activate necrosis
complement
Acute Weeks to months Cellular: CD8+T cells activated Parenchymal and
against donor MHCS (Type IV vascular injury
HS), activate complement

Humoral: similar to
hyperacute, except
antibodies develop after the
transplant
Chronic Months to years CD4+ T cells respond to Graft arteriosclerosis
recipient APCS, presenting
donor peptides, including
allogeneic MHC. Both cellular
and humoral components
(Type II and IV HS)
Graft-versus- Varies Grafted immunocompetent T Maculopapular rash,
host disease cells proliferate in the jaundice,
immunocompromised host hepatosplenomegaly
and reject host cells with
"foreign" proteins>severe
organ dysfunction (Type IV
HS)

Tumor Immunology (tumor marker,s oncoproteins) 3%; Hypersensitivity 1%; Autoimmune

disorders 3%

TUMOR IMMUNOLOGY

TUMOR MARKERS

 are substances present in or produced by tumors that can be used to detect the
presence of cancer based on their measurement in blood, body fluids, cells, or tissue.
 A tumor marker may be produced by the host in response to a tumor that can be used
to differentiate a tumor from normal tissue or to determine the presence of a tumor.
 Non-neoplastic conditions can also exhibit tumor marker activity.
 Some tumor markers are used to screen for cancer,
 But markers are more often used to monitor recurrence of cancer or determine the
degree of tumor burden in the patient.
 To be of any practical use, the tumor marker must be able to reveal the presence of the
tumor while it is still susceptible to destructive treatment by surgical or other means.
 Tumor markers can be measured quantitatively in tissues and body fluids using
biochemical, immunochemical, or molecular test
 TUMOR MARKER CANCER
AFP Hepatic and testicular carcinoma
ALP Bone carcinoma and Lung Carcinoma
Bence Jones Protein Multiple Myeloma
Beta-HCG Testicular carcinoma
BRCA-1 Breast or ovarian carcinoma
CA 125 Ovarian carcinoma
CA 15-3 Breast carcinoma
CA 19.9 Pancreatic carcinoma
Calcitonin Medullary thyroid carcinoma
CEA Gastrointestinal tumors, stomach, breast, and
Lung carcinoma
CYFRA Lung Carcinoma
Estrogen receptor Breast carcinoma
Gastrin Gastric carcinoma
HER-2/neu Breast carcinoma (efficiency of trastuzumab
or Herceptin therapy)
Nuclear Matrix Protein (NMP) Urinary bladder cancer
PSA Prostatic Carcinoma

HYPERSENSITIVITY

 can be defined as a normal but exaggerated or uncontrolled immune response to an

antigen that can produce inflammation, cell destruction, or tissue injury.
 It has traditionally been classified on the basis of time after exposure to an offending
antigen.
 When this criterion is used, the terms immediate hypersensitivity and delayed
hypersensitivity are appropriate.
 Immediate hypersensitivity is antibody mediated
 Delayed hypersensitivity is cell mediated

Type I Type II Type III Type IV

Name Anaphylactic/ Cell- Immune Complex Delayed/Cell-
Atopic bound/Cytotoxic Hypersensitivity mediated
Hypersensitivity Hypersensitivity Hypersensitivity
Mediator IgE IgM, IgG IgM, IgG T cell
Effector Cell Basophil, mast RBC, WBC, Host tissue cell APC,
cell Platelet macrophage, T
cell
Antigen Allergen Cell-bound Soluble antigen Sensitized
involved antigen antigen
Complement No YES YES No
involvement
Mechanism Release of Cell Lysis Deposition of Ag- Release of
inflammatory Ab complex cytokines
mediators
Examples Anaphylaxis AIHA Serum sickness Contact
 Bee sting HTR Arthus reaction dermatitis
 Food/drug HDN SLE Poison ivy
allergies ITP Poststreptococcal GVHD
Goodpasture glomerulonephritis Tuberculin test
Allergic/Atopic syndrome Rheumatic fever
  Rhinitis Rheumatic fever
 Hay fever
 Eczema
 Hives
 Asthma

AUTOIMMUNE DISEASES

 A condition in which damage to body organ results from the presence of autoantibodies
or autoreactive cells.

Autoanitbody Associated Disorder

Anti-dsDNA, anti-Smith SLE
Anti-acetylcholine receptor Myasthenia gravis
Anti-basement membrane Goodpasture syndrome
Anti-centromere CREST syndrome
Anti-desmoglein (anti-desmosome) Pemphigus vulgaris
Anti-hemidesmosome Bullous pemphigoid
Anti-histone Drug-induced lupus
Anti-insulin, anti-beta cells, Anti-glutamic Diabetes mellitus type 1
acid decarboxylase
Anti-Jo-1, anti-SRP, anti-Mi-2 Polymyositis, dermatomyositis
Anti-mitochondrial Primary biliary cirrhosis
Anti-myelin sheath Multiple sclerosis
Anti-parietal cell Pernicious anemia
Anti-ribonucleoproetin (anti-RNP) Mixed connective tissue disease
Anti-Scl-70 (anti-DNA topoisomerase 1) Scleroderma (diffuse)
Anti-smooth muscle Chronic active hepatitis
Anti-SSA, anti-SSB Sjogren’s syndrome
Anti-thyroglobulin, anti-thyroid peroxidase, Hashimoto thyroiditis
anti-microsomal
Anti-TSH receptor Grave’s disease
C-ANCA Wegener’s granulomatosis
p-ANCA Churg-Strauss syndrome
Cold/warm autoantibodies AIHA
Rheumatoid factor, anti-CCP Rheumatoid arthritis

SYSTEMIC LUPUS ERYTHEMATOSUS

 Prototype of human autoimmune diseases associated with HA DRS

 Chronic systemic inflammatory autoimmune disease characterized
by presence of antinuclear antibodies
 Joint, skin (butterfly rash), renal involvement

LABORATORY DIAGNOSIS

1. Demonstration of LE cell
 Neutrophil that engulfed the antibody-coated nucleus of another neutrophil
2. Fluorescent Antinuclear Antibody (FANA) Test
 Screening test for antinuclear antibodies; sensitive but nonspecific
3. Indirect Immunofluorescence
 Considered the benchmark in identification of autoantibodies in SLE
  Crithidialuciliae - a hemoflagellate with circular dsDNA in the kinetoplast, used as
substrate to detect anti-dsDNA

ANTINUCLEAR ANTIBODY

Patterns Autoantibody Disease Association

Homegenous or Peripheral Anti-dsDNA Diagnostic for SLE
Anti-histone Drug-induced SLE
Anti-DNP Drug-induced SLE
Coarsely speckled Anti-Sm Diagnostic for SLE
Anti-RNP SLE, RA, MCTD, Sjogren's
syndrome, Systemic
sclerosis, Dermatomyositis
Finely speckled Anti-SSA Cutanenous SLE,
Scleroderma, Sjogren’s
syndrome
Homogenous nucleolus Anti-nucleolar Scleroderma, Systemic
sclerosis
Atypical speckled Anti-Scl-70 Scleroderma, Systemic
sclerosis
Fine cytoplasmic speckling Anti-Jo-1 Polymyositis
No fluorescence Anti-centromere CREST

PATTERNS OF ANTINUCLEAR ANTIBODY

ANTINUCLEAR ANTIBODY UNDER FLOURESCENT MICROSCOPE

RHEMATOID ARTHRITIS

 Autoimmune disease that affects the synovial membrane of multiple joints

 Characterized by presence of HLA-DR4 which is an IgM that targets Fc region of IgG
 Anti-CCP is more specific for rheumatoid arthritis

LABORATORY DIAGNOSIS

 Latex test is more sensitive but sheep cell agglutination is more specific
 Based on the detection of RF in serum or synovial fluid

1. Sheep Cell Agglutination Test/Rose Waaler Test (Rose et al)

2. Latex Fixation Test (Singer and Plotz)
 Positive: Titer of 2 80
 Weak positive: Titer of 20-40
 Negative: If there is no agglutination at 1:20, even if subsequent dilution shows
agglutination
3. Sensitized Alligator Erythrocyte Test (Cohen et al)
4. Bentonite Flocculation Test (Bloch and Bunim)

IMMUNODEFICIENCY

Disease Defect Presentation Findings

Bruton’s N B-cell maturation Recurrent bacterial Absent B cells in
agammaglobulinemia and enteroviral peripheral blood ⭣Ig
infections after 6 of all classes
months (⭣maternal Absent/scanty lymph
IgG) nodes and tonsils
Selective IgA Most common Majority ⭣IgA with normal
deficiency immunodeficiency asymptomatic IgG, IgM
Common variable Defect in B-cell Can be acquired in ⭣plasma cells
Immunodeficiency differentiation 20s-30s
⭣immunoglobulins
Thymic aplasia Absent thymus and Tetany due to ⭣T cells, ⭣PTH, ⭣Ca2
(DiGeorge syndromw) parathyroids hypocalcemia
Recurrent
viral/fungal
infections
Job syndrome Deficiency of Th17 ⭡IgE, dermatologic ⭡IgE, ⭣IFN-γ
impaired chemotaxis
problems
of neutrophils to sites
of infection
 B AND T CELLS DISORDERS

Disease Defect Presentation Findings

Severe combined Most severe Failure to thrive Absence of germinal
Immunodeficiency immunodeficiency centers in lymph
Adenosine Treatment: BM node
deaminase transplant
deficiency Absence T cells in
flow cytometry
Ataxia- Failure to repair DNA Cerebellar ataxia ⭡AFP
telangiectasia double strand breaks and telangiectasia
- cell cycle arrest ⭣IgA, IgG, IgE
Hyper-IgM syndrome Class switching Severe pyogenic Normal or ⭡IgM
defect infections early in life
⭣⭣IgG, IgA, IgE
Wiskott-Aldrich Mutation in WAS Trias of ⭣or normal IgG, IgM
syndrome gene immunodeficiency,
eczema, ⭡IgE, IgA
thrombocytopenia Fewer and smaller
platelets

PHAGOCYTE DYSFUNCTION

Disease Defect Presentation Findings

Leukocyte adhesion Impaired migration Absent pus formation ⭡neutrophils
deficiency and chemotaxis
Absence of
Impaired wound
neutrophils at
healing
infection site
Chediak-Higashi Defect in LYST gene Recurrent pyogenic Giant granules in
syndrome Dsyfunction in infections by granulocytes and
phagosome- staphylococci and platelets
lysosome fusion streptococci
Pancytopenia

Mild coagulation
defects
Chronic Defect of NADPH ⭡Susceptibility to Abnormal
granulomatous Oxidase catalase (+) dihydrorhodamine
disease test
organisms
⭣Respiratory burst in NBT test: failure to
reduce dye
neutrophils
 Immunologic test for detection of antigens and antibodies – principles procedures 16%

SEROLOGY

 Study of noncellular portion of the blood.

AFFINITY

 Initial force of attraction that exists between a single Fab site on the antibody molecule
and a single epitope or determinant site on the corresponding antigen.

AVIDITY

 Sum of all attractive forces between an antigen and an antibody.

SEROLOGY TEST: PRECIPITATION

PRECIPITATION

 Involves combination of soluble antigen with soluble antibody to produce insoluble

complexes that are visible.

 Zone of equivalence - optimum precipitation occurs; number of multivalent sites of

antigen and antibody are Approx equivalence
 Prozone - antibody excess; corrected by serum dilution
 Postzone - antigen excess; corrected by Repeat test after a week
MEASUREMENT OF PRECIPITATION IN A FLUID MEDIUM

1. Turbidimetry
 Measures light blocked
 Light detection device is indirect light with the incident light

2. Nephelometry
 Measures light scattered
 Light detection device is at an angle from the incident light

MEASURMENT OF PRECIPITATION BY PASSIVE IMMUNODIFFUSION

 Passive because no electric current is used to speed up the process

 Support medium: gel, agar, agarose

1. Single Diffusion
 Antibody is incorporated into agarose in a test tube
 Antigen is layered on top, moves down the gel  precipitation
 Precipitation moves down the tube in proportion to antigen concentration

2. Radial Immunodiffusion
 Antibody is uniformly distributed in the support gel
 Antigen is placed in a well cut into the gel
 Area of the ring obtained is a measure of antigen concentration
A. Mancini/Endpoint Method
 Antigen is allowed to diffuse to completion and when equivalence is reached, there
is no further change in diameter
 Square of the diameter is proportional to antigen concentration
B. Fahey & McKelvey/Kinetic Method
 Measurements are taken before point of equivalence is reached (Stevens: 18 hours
/ Calbreath: 24 hours)
 Diameter is proportional to the log of antigen concentration

3. Ouchterlony Double Diffusion

 Both antigen and antibody diffuse independently through a semisolid medium in
two dimensions
 Antibody is placed in the central well
 Antigen is placed in the surrounding wells to determine if antigens share identical
epitopes
 Result: Precipitin lines
o Serological Identity - smooth arc
o Nonidentity - crossed lines
o Partial Identity – spur
MEASUREMENT OF PRECIPITATION BY ELECTROPHORESIS

1. Rocket Immunoelectrophoresis/One-Dimension Electroimmunodiffusion

 RID + Electrophoresis
 Antibody is uniformly distributed in the gel
 Antigen is placed in wells cut in the gel
 Result: Precipitin rocket

2. Immunoelectrophoresis
 Serum (containing antigens) is electrophoresed
 Antibody is placed on a trough
 Result: Precipitin arc

3. Immunofixation Electrophoresis
 Serum (containing antigens) is electrophoresed
 Antibody is overlaid directly to the gel surface
 Result: Preciptin band

SEROLOGY TEST: AGGLUTINATION

AGGLUTINATION

 Process by which particulate antigens such as cells aggregate to form larger complexes
when a specific antibody is present.

SENSITIZATION

 Antigen-antibody combination through single antigenic determinants on the particle

surface.

AVIDITY

 Sum of all attractive forces between an antigen and an antibody.

 READING OF AGGLUTINATION

Grade Description
Cells Supernate
0 No Agglutinates Dark, turbid, homogenous
W+ Many tiny agglutinates Dark, turbid

Many free cells

May not be visible with

microscope
1+ Many small agglutinate Turbid

Many free cells

2+ Many medium sized Clear
agglutinates

Moderate number of free

cells
3+ Several large agglutinates Clear
Few free cells
4+ One large, solid agglutinate Clear

No free cells
AGGLUTINATION REACTION

1. Direct agglutination
 Antigen is found naturally on the surface of participle
 Example: Hemagglutination, Widal test

2. Passive/Indirect agglutination
 Antigen is artificially attached to carrier particle (bentonite, charcoal, RBC, latex,
gelatin, silicates)
 Agglutination occurs if patient Antibody is present

3. Reverse passive agglutination

 Antibody is artificially attached to carrier particle
 Agglutination occurs if patient Antigen is present

4. Agglutination inhibition
 Competition between particulate and soluble antigens for limited antibody-
binding sites
 Positive result: lack of agglutination
 Example: hCG test, Hemagglutination inhibition (RBC is indicator)
5. Coagglutination
 Uses bacteria as inert particles to which antibody is attached
 S. aureus is most frequently used because of its Protein A which naturally absorbs
the Fc portionof the IgG except for IGG3.

6. Antiglobulin-mediated agglutination
 Detects non-agglutinating antibody by means of couple with a second antibody
(antihuman globulin)

A. Direct Antiglobulin Test (specimen: red cell)

 In vivo sensitization
 Investigation of HDN, HTR, AIHA, Drug induced hemolytic anemia.

B. Indirect Antiglobulin Test (specimen: serum)

 In vitro sensitization
 Cross matching
 Antibody determination and identification
 Red cell antigen phenotyping

FALSE REACTIONS IN AHG TESTING

FALSE-POSITIVE REACTION FALSE-NEGATIVE REACTION

Contamination of reagents Reagent failure
Overcentrifugation Improper washing
Direct agglutination by strong agglutinins Failure to add antiglobulin reagent
Over-incubation with enzyme treated cells Improper centrifugation
Improper use of enhancement reagents Serum/cell ratio is too low
Saline stored in glass or metal containers Delayed washing (elution of weakly
attached antibody)
QUANTITATIVE AGGLUTINATION REACTIONS

1. Sol Particle Immunoassay (SPIA)

2. Disperse Dye Immunoassay (DIA)
3. Immunoassay by Particle Counting (IMPACT)

INSTRUMENTATION

Particle Counting Immunoassay (PACIA)

 Measurement of the number of residual non-agglutinating particles in a specimen using

a laser beam in an optical particular counter similar to one that is designed to count
blood cells.

LABELLED IMMUNOASSAY

 Homogeneous assay
 no separation step; enzyme activity diminishes when antigen-antibody binding occurs
 Heterogenous assay
 requires a separation step
 Solid-phase
 medium to which an antigen or antibody may be attached

ENZYME IMMUNOASSAY

 Label: enzymes
 Horseradish peroxidase
 Glucose-6 phosphate dehydrogenase
 Alkaline phosphatase
 B-D Galactosidase
 Change in absorbance is measured using SPECTROPHOTOMETRY

1. Competitve ELISA
 Enzyme-labeled antigen competes with unlabeled patient antigen for a limited
number of antibody binding sites
 Enzyme activity is inversely proportional to the analyte concentration.
2. Noncompetitive/Indirect
 Indirect because enzyme-labeled secondary antibody does not participate in the
initial antigen-antibody binding
 Enzyme activity is DIRECTLY proportional to the analyte concentration.

3. Capture Assay/Sandwich
 Antigen captured must have multiple epitopes
 Enzyme activity is DIRECTLY proportional to antigen concentration.
 Prone to HOOK EFFECT
 4. Enzyme Multiplied Immunoassay Technique (EMIT)
 Homogeneous assay
 Antigen is labeled with an enzyme tag
 When antibody binds to specific determinant sites on the antigen, the active site
on the enzyme is blocked, resulting to LOSS OF ENZYME ACTIVITY.

FLUORESCENT IMMUNOASSAY

 Label: fluorophore or fluorochrome

 Flourescein
 Tetramethyrhodamine
 Phycoerythrin
 Europium (B-naphthyl triflouroacetone)
 Lucifer Yellow VS
 Fluorescence can be measured using fluorometer, fluorescent microscope, flow
cytometer, or spectrofluorometer.
 Disadvantages: autofluorescence, quenching

1. Direct Immunofluorescent Assay

 Unknown antigen is fixed to a microscope slide
 Fluorescent labeled antibody added to directly to unknown antigen
 Slide is read using a fluorescence microscope

2. Indirect Immunofluorescence Assay

 Same principle as indirect ELISA

3. Inhibition Immunofluorescent Assay

 Blocking Test in which antigen is first exposed to unlabeled antibody, then to
labeled antibody, and is finally washed an examined.
 If the unlabeled and labeled antibodies are both homologous to the antigen, there
should be no fluorescence.

4. Fluorescence Polarization Immunoassay (FPIA)

 Based on the change in polarization of fluorescent tag emitted from a labeled
molecule when it is bound by antibody.
 Degree of fluorescence polarization is inversely proportional to analyte
concentration.

RADIOIMMUNOSAY

 First type of immunoassay developed

 Radioactivity is measured by a SCINTILLATION COUNTER
 Crystal scintillation counter: gamma
 Liquid scintillation counter: beta
 Disadvantages: health hazard, disposal problems, short shelf life, and the need for
expensive equipment
 Applications
 Radioimmunosorbent Test (RIST)determining low serum IgE levels
 Radioallergosorbent Test (RAST) allergen-specific IgE
 1. Competitive Binding Assays
 Radiolabeled antigen competes with unlabeled patient antigen for a limited number
of antibody-binding sites
 Radioactivity is inversely proportional to the antigen concentration

2. Noncompetitive Immunoradiometric Assays (IRMA)

 Analyte measured is sandwiched between two antibodies
 Radioactivity is directly proportional to the analyte concentration.

SEROLOGY TEST: IMMUNOCHROMATOGRAPHY

IMMUNOCHROMATOGRAPHY

 Rapid diagnostic tests (RDTS) for malaria have been developed that employ
immunochromatographic methods based on the detection of malarial antigens present
in peripheral blood.
 Most RDTS use monoclonal antibodies and detect particular malarial antigens in blood
specimens including the histidine-rich protein I (HRP-11), aldolase, and parasite lactate
dehydrogenase (PLDH).
 These tests generate results within 10 to 15 minutes.
 Commercially available kits for HRP-Il detect P. falciparum HRP-Il only and therefore
diagnose only P. falciparum malaria.
SEROLOGY TEST: PCR
POLYMERASE CHAIN REACTION

 Its principle is based on the use of DNA polymerase which is an in vitro replication of
specific DNA sequences.
 This method can generate tens of billions of copies of a particular DNA fragment (the
sequence of interest, DNA of interest, or target DNA) from a DNA extract (DNA template).
 This amplification is based on the replication of a double-stranded DNA template. It is
broken down into three (3) phases: a denaturation phase, a hybridization or annealing
phase with primers, and an elongation phase. The products of each synthesis step serve
as a template for the following steps, thus exponential amplification is achieved.

 The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-
tolerant bacterium from which it was isolated (Thermus aquaticus).
 PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in
length.
 When the primers are bound to the template, they can be extended by the polymerase,
and the region that lies between them will get copied.
 The key ingredients of a PCR reaction are:
1. Taq polymerase
2. Primers - forward and reverse
3. Template DNA – from the patient's sample
4. Nucleotides (DNA building blocks)

1. Denaturation – dsDNA is heated to 95°C to separate the DNA into single strands.

2. Annealing - DNA is cooled to 52°C to allow primers to bind/anneal to complimentary

sequences on the separate DNA strands.
 3. Elongation - at 72°C, the heat-stable DNA polymerase (Taq polymerase) binds to the 3'
end of each primer and synthesizes a new strand of DNA.

VIRAL INFECTIONS, INCLUDING HEPATITIS AND HIV 5%

HEPATITIS VIRUS

Hepatitis A (Infectious Hepatitis)

 Belongs to Picornaviridae (RNA)
 Transmission by Fecal-oral route
 May be transmitted by clotting factor concentrates
 Virus has an average incubation of 28 days
 Shed in feces during incubation period and early acute infection

HAV RNA Direct detection of HAV in food and water samples

IgM anti-HAV Acute HAV
Peak during month of illness and decline to undetectable levels
within 6 to 12 months
IgG anti-HAV Evidence of previous HAV infection

Hepatitis B (Serum Hepatitis)

 Belongs to Hepadnaviridae Family (DNA)

 Transmission through Parenteral, Sexual, Perianal
 Incubation Time of 60-90 days
 Dane particle: complete HBVV that causes infection
 HB envelope Ag: InfectivitEE
 HB envelope Ab: RecoverEE

TESTS FOR HBSAG Detection:

 First Generation- Ouchterlony Double Diffusion

 Second Generation
 Counterelectrophoresis
 Rheophoresis
 Complete Fixation
 Third Generation
 Radioimmunoassay
 ELISA
 Reverse Passive hemagglutination
 Reverse Passive latex Agglutination
 HEPATITIS B

HBsAG Active Hepatitis B infection

HBeAg Active Hepatitis B infection (High Degree of
Infectivity)
Anti-HBc Current or past HBV infection
Anti-HBe Recovery phase of Hepatitis B
Anti-HBs Past-infection- evidence of immunity
HBV DNA Various manifestations of HBV

HEPATITIS C

Hepatitis C (non A- non B Hepatitis)

 Belongs to Flaviviridae Family (DNA)

 Transmission through Parenteral, Sexual, Perianal
 Incubation Time of 7-8 weeks
 Average incubation period of 7 to 8 weeks
 Surrogate Test for HCV
 >ALT
 (+) Anti- HBC
 Specific Test for HCV
 (+) Anti-HCv

Anti-HCV Current or past HCV infection

HCV RNA Current HCV Infection

HEPATITIS D

 Belongs to Deltaviridae Family (RNA)

 Transmission mostly through Parenteral, Sexual, Perianal
 HBV Infection is required!
 Requires HBV INFECTION
 Most common for IV Drug users
 o Coinfection: Simultaneously
o Superinfection: Sequentially

IgM anti-HDV Active or Chronic Hepatitis D infection

IgG anti-HDV Chronic Hepatitis D, Convalescent Hepatitis D status
HDV RNA Active HDV Infection

Hepatitis E

 Belongs to Deltaviridae Family (RNA)

 Transmission of Fecal-Oral Route
 Associated with fulminant liver failure in pregnant women.
 Incubation Time of 3-8 weeks

IgM anti-HEV Current/new hepatitis E infection

IgG anti-HEV Current/former Hepatitis E infection
HEV RNA Current Hepatitis E infeciton

HUMAN IMMUNODEFICIENCY VIRUS

 Retrovirus containing RNA and reverse transcriptase
 Targets helper CD4 T Helper Cells
 HIV-1 was formerly called human T-cell lymphotropic virus-type III (HTLV-II),
lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV)
 HIV-1 is the causative agent of AIDS in the United States and Europe
 HIV-2 is endemic in West Africa; less pathogenic with a lower rate of transmission
 3 Major routes of transmission intimate sexual contact, parenatally from infected
blood/body fluids, perinatally from infected mother to infant => vertical transmission
 ELISA have been the cornerstone of screening procedures for HIV
 Standard treatment is HAART (HIGHLY ACTIVE ANTIRETROVIRAL THERAPY) – regimen
involving a combination of at least 2 drug classes
 gp120 is responsible for binding to CD4 receptor of T cell
 Antibody tc124 is first to appear during infection

HIV LABORATORY TESTS

Test What You Need To Know

CD4 T Cell Gold standard: CD4 T cell enumeration/ immunophenotyping w/ flow
cytometry
Enumeration
HIV AIDS - inverted CD 4/CD 8 ratio (< 1:1)
HIV Antibody SCREENING CONFIRMATORY
Detection  ELISA-Best  Western
 Agglutination Tests Blot/Immunoblot*-Best
 Dot-Blot Testing  Immunofluorescence
P24 Antigen To identity infection during the Window period before antibody is
detection detectable
HIV Nucleic Acid Test To determine viral load and development of drug-resistant strains
Viral Load Tests Quantitative test for HIV nucleic acid
Polymerase Chain Preferred method for infants and children younger than 18 months
Reaction
 ELISA GENERATIONS

Generation What You Need To Know

First Solid-phase indirect-assay using viral lysate antigens from HIV-1
Detects antibodies to HIV-1 only
Second Indirect binding assay using highly purified recombinant or synthetic
antigens from both HIV-1 and HIV-2

Detects antibodies to HIV-1 and HIV-2

Third Sandwich technique
Detects HIV antibodies of different Ig classes, including IgM
Fourth - special Detects HIV-1 antibodies, HIV-2 antibodies, and p24

ELISA FALSE TESTING

FALSE POSITIVE ELISA TEST FALSE NEGATIVE ELISA TEST

 Heat inactivation of serum prior to
testing
 Repeated freezing/thawing
 Autoreactive antibodies
 Multiple pregnancies
 Severe hepatic disease
 Passive Ig administration
 Recent exposure to certain vaccines
 Malignancies
 Collection of the test serum prior to
seroconversion
 Immunosuppressive therapy
 or replacement transfusion
 Hypogammaglobulinemia
 Technical errors
 Patient harbors a genetically diverse,
recombinant strain of HIV

HIV STRUCTURAL GENES

Gene Product
Group Antigen  P55- precursor protein which forms core proteins
 Core proteins p6, p9, pl7, p24

Polymerase Gene – For replication  Reverse transcriptase (p66, p51) – transcribes

RNA to DNA
 Integrase (p31)- inserts viral DNA to host DNA
 Protease (p10)-cleaves protein precursors
 RNase

Envelope Gene  gp160 - cleaved to form gpl20 and gp41

 gpl20 - binds to CD4 to T cells
 gp44 - transmembrane protein

EPSTEIN BARR VIRUS

 Infectious mononucleosis is a self-limiting disease caused by the Epstein Barr Virus

 The disease may be confused with diphtheria, pharyngitis, Vincent's angina,
lymphadenitis, scarlet fever, hepatitis, or pertussis
 The Epstein-Barr virus is transmitted through saliva, blood transfusion, transplacental
routes, possibly by mosquitoes
 EBV infects CDI Cells. The variant lymphocytes produced in response to infection have T
cell characteristics
  Infection from blood transfusion is known as IM perfusion syndrome.
 Other diseases associated with EBV are Burkitt's lymphoma and nasopharyngeal
carcinoma
 Enlarged lymphocytes with atypical nuclei (Downey cells) are characteristic

HETEROPHILE ANTIBODY

 Heterophile is the name given to several groups of antigens that occur in the cells or fluids
of apparently unrelated animals and microorganisms yet are so closely related that they
will cross react with antibodies against any one member of the particular heterophile
group
 IgM that usually appears during acute phase of infectious mononucleosis
 Reacts with horse, ox, sheep RBC
 Absorbed by beef erythrocytes but NOT absorbed by guinea pig kidney
 Does not react with EBV-specific antigens

LABORATORY TEST FOR THE IDENTIFICATION

1. PAUL-BUNNELL SCREENING TEST

 Hemagglutination test designed to detect heterophile antibodies in patient serum
when mixed with antigen-bearing sheep RBC
 Useful screening test but cannot determine specificity
 Heterophile antibody titer is reported as the reciprocal of the highest dilution shomg
agglutination
 Normal titer: ≤ 56

2. DAVIDSOHN DIFFERENTIAL TEST

 Distinguishes between heterophile antibodies associated with infectious
mononucleosis, serum sickness, or Forssman antigen
 Absorption with guinea pig kidney and beef RBC  agglutination with sheep RBCS

ABSORPTION PATTERNS

Type of Serum Absorbed by Guinea Pig Absorbed by Beef RBC

Kidney
Forssman Absorbed (+) -
Serum sickness Absorbed Absorbed
IM (-) Absorbed

AGGLUTINATION PATTERNS WITH SHEEP RBC AFTER ABSORPTION

Type of Serum Agglutination After Agglutination After

Absorption with Guinea Pig Absorption with Beef RBC
Kidney
Forssman - Agglutination
Serum sickness - -
IM Agglutination -
 3. MONOSPOT TEST/RAPID DIFFERENTIAL SLIDE TEST
 Also differential but require absorption of the patient's serum
 Horse RBCS are agglutinated by heterophile antibodies of IM

SEROLOGIC MARKERS

Markers Clinical Significance

IgM anti-VCA Detectable early in the course of infection
IgG anti-VCA Detected after onset of signs and symptoms can be present
for life
IgG anti early antigen strongly indicates active infection
diffuse
rise in titer during reactivation latent infection

not consistent indicator of disease

IgG anti-early Antigen found in the serum of very young children but not in young
regain adults during acute stage
Epstein roar Nuclease Found in the nucleus of ALL EBV-infected cells
Antigen
does not stimulate antibody until after incubation period of IM

Bacterial infections and STD 5%

SYPHILIS

 Syphilis was called previously called 'great pox' or 'evil pox.'

 Transmitted by sexual contact, direct blood transmission, or transplacental route
 First diagnostic test developed in 1906: Wasserman test (used cardiolipin as Ag)
 Salvarsan 606 (known as arsphenamine) was used as a treatment for syphilis in the 1900s
 Penicillin-allergic: Tetracycline; Pregnant women: Erythromycin

STAGES OF SYPHILIS

1. Primary (Early) Stage – Hard Chancres (painless and firm)

2. Secondary Stage - Condylomata lata
3. Late Latent Stage - no signs or symptoms
4. Tertiary Stage - gummas, neurosyphilis
o Parenchymatous neurosyphilis may present as paresis (incomplete paralysis) or
Taberdorsalis (degeneration or the dorsal columns of the spinal cord and sensory
nerve trunks)

LABORATORY DIAGNOSIS

1. Direct Detection of Spirochetes

 Darkfield Microscopy
 luorescent Antibody Test

2. Serologic Tests
 Nontreponemal Tests -Determine the presence of REAGIN. Prone for false positive
 Detect reagin (nontreponemal ab/anticardiolipin/antilipoidal ab)
 Reagin reacts with lipid antigens from animal tissue (beef heart) in a process
known as flocculation.
 Nonspecific, only for screening
  Biologic false (+): SLE, RA, IM (Infectious mononucleosis)
 Treponemal Tests
 Detect antibodies to pallidum

NON-TREPONEMAL TESTS

VENEREAL DISEASE RESEARCH LABORATORY (VDRL) TEST

 Qualitative/quantitative slide flocculation test

 Specimen: serum/SF (heated for 30 mins for 56°C)
 Antigen: 0.03% cardiolipin, o.9% cholesterol, 0.219% lecithin
 Antigen delivery needles (Hamilton syringe)

Qualitative serum VDRL 18g 60 drops Ag suspension/mL

Quantitative serum VDRL 19g 75 drops Ag suspension/mL
23g 100 drops saline/ml
CSF VDRL 21 or 22g 100 drops/ml

 Rotator: 4 minutes 180 rpm (serum) 8 minutes 180rpm (CSF)

 Reading microscopically using LPO (100x)
 Reporting
 Nonreactive: no clumps
 Weakly reactive: small clumps
 Reactive; medium to large clumps
 All sera with reactive or weakly reactive results must be tested using the quantitative
slide test, in which twofold dilutions of serum ranging from 1:2 to 1:32 are initially used.
Sera yielding positive results at the 1:32 dilution are tittered further
 A positive VDRL test on spinal fluid is diagnostic sf neurosyphilis

2. RAPID PLASMA REAGIN (RPR) TEST

 Modified VDRL with charcoal particles (visibility)

 Specimen: serum (no heating)
 Antigen: similar to VDRL with addition of
 Charcoal – for macroscopic visibility of reaction
 EDTA
 Thimerosal - preservative
 Choline chloride - inactivate complement
 Antigen delivery needle: 20 gauge- 60 drops Ag suspension/mL
 Rotator: 8 minutes, 100 rpm
 TREPONEMAL SEROLOGICAL TESTS

1. TREPONEMA PALLIDUM IMMOBILIZATION (TPI) TEST

 Treponemal test of reference
 Treponemal antibody, in the presence of complement, inhibits the normal
movement of actively motile treponemes extracted from testicular chancre of
rabbit
 Positive: ≥ 50% immobilized
 Negative: ≤ 20% immobilized
 Doubtful: 20-50% immobilized

2. FLUORESCENT TREPONEMAL ANTIBODY ABSORPTION TEST

 Patient serum is heat inactivated and made with a sorbent consisting of
nonpathogenic treponemes (Reiter strain) which removes cross-reactivity with
treponemes other than T. pallidum
 Nichols strain of T. pallidum have been fixed to slides used for the test

3. AGGLUTINATION TESTS
1. Hemagglutination Treponemal Test for Syphilis (HATTS)
 Uses glutaraldehyde-stabilized turkey RBCS
2. T. pallidum Hemagglutination Assay (TPHA)
 Uses tanned sheep RBCS coated with antigen from Nichols strain
3. Microhemaggutination Assay for Antibodies to T. pallidum (МНАТР)
 Similar to TPHA, but performed in microtiter plates
 Uses formalinized, tanned sheep RBCS sensitized with antigen from Nichols
strain
4. T. pallidum Particle Agglutnation (TPPA)
 Uses gelatin particles instead of RBCS sensitized with T. pallidum antigens

FEBRILE ANTIBODY TESTING

Widal Test

 V For the detection of antibodies in typhoid fever, brucellosis, and tularemia

 V Clínically significant titer: ≥160

Weil-Felix Test

 Classic test that uses cross - reacting Proteus vulgaris and

 Proteus mirabilis antigens to diagnose a rickettsial infection
 Clinically significant titer: ≥ 320
 Antigens used: Ox-2, OX19
OX-K
Organism Disease Ox-2 Ox-19 OX-K
R. prowazeki Epidemic typhus + + 0
Brill-Zinsser disease
R. typhi Endemic typhus + + 0
Murine typhus
R. rickettsia Rocky mountain fever + + 0
R. akari Rickettsial pox 0 0 0
R. tsutsugamusi Scrub typus 0 0 +
 B. Quintana Trench fever 0 0 0
C, burneti Q fever 0 0 0

STREPTOCOCCAL INFECTION

GROUP A STREPTOCOCCAL INFECTION

 Onset of clinical symptoms of rheumatic fever or glomerulonephritis typically coincides
with the peak of antibody response
 If acute and convalescent phase sera are tested in parallel, fourfold rise in titer is
considered significant

LABORATORY DIAGNOSIS

1. ANTISTREPTOLYSIN O TITER
 based on neutralization of hemolytic activity of streptolysin O
 Titer is reported as the reciprocal of the highest dilution showing no hemolysis
 Expressed in Todd units or international units
 Normal titer: ≤ 166 Todd units
 Moderately elevated: at least 240 Todd units (adult)
 Moderately elevated: at least 320 Todd units (child)

2. ANTI-DNASE B TEST
 Based on neutralization of depolymerizing activity of DNase B
 DNase B hydrolyzes DNA-methyl green conjugate. Methyl green is reduced and
becomes colorless
 BUT, if anti-DNASE B is present, it will neutralize DNase B, preventing it from
depolymerizing DNA; therefore, the color remains
 Tubes are graded for color: 4+ color intensity is unchanged
0 Indicates total loss of color

3. STREPTOZYME TEST
 Slide agglutination screening test for detection of antibodies to several
streptococcal antigens
 Sheep RBCS are coated with streptolysin, streptokinase. hyaluronidase, DNase,
and NADase so that antibodies to any of the streptococcal antigens can be
detected
 Hemagglutination represents a positive test, indicating that antibodies to one or
more of these antigens are present.

PARASITIC INFECTION: MALARIA

MALARIA

OptiMAL

 Detects parasite lactate dehydrogenase which is produced by viable parasite

 Different isoforms of lactate dehydrogenase are present in different species
 PLDH can be detected when there are 100-200 parasites/ul blood

MalaQuick Standby Malaria Test

 Detects P. falciparum histidine-rich protein-2 antigen

REFERENCES
Turgeon, M.L.(1996). Immunology & Serology in Laboratory Medicine. St. Louise: Mosby

Stevens, C.D., Miller, L.E. (2017). Clinical Immunology and Serology: a Laboratory
Perspective (4th Ed.) Philadelphia, PA: F.A. Davis Compact

Acierto, M (2017). Immunology Notes

Contreras, S (2017) Lemar Notes