Guide notes in

Immunology and
serology

LAITAN, PAULO P.
20155879
INTRODUCTION
Immunology
- study of molecules, cells, organs and systems responsible for the recognition and disposal of foreign bodies

Serology
- Antigen-Antibody reactions
- Agglutination, Precipitation, Complement Fixation and the use of such reactions:
 To measure serum antibody titers in infectious diseases
 Clinical correlation of antibody titer (the serology of disease)
 Use of serological reaction to detect antigen

Immunology
Study of the immune system
Study of body’s defense against
specific pathogen
Study of reactions of a host when
foreign substances are introduced
into the body
Serology
The detection/measurement of elements of humoral
immune system (antibody)
Help in the diagnosis of infectious diseases and
immunologic disorders
Determine immune status

Antigen (Immunogen)
- a foreign substance that can stimulate the production of antibodies (immune response)

Antigenicity
- ability of antigen to stimulate an immune response

Immunity
- resistance to infectious disease
- process of being protected against foreign antigen

Immune Response
- Reaction of Immune System to an antigenic challenge

Complement
- series of proteins that are normally present in serum whose overall function is the mediation of inflammation
HISTORICAL DEVELOPMENT
1. Edward Jenner
 1796: Small Pox Vaccination earliest 10. Paul Erlich
disease found to induce immunity  1900: Side Chain Theory of antibody
 Sarah Nelmes: cowpox formation
 James Phipps: smallpox  Antibodies: “Magic bullets”
 1798: 1st person who studied the body’s  First effective treatment for syphilis
response  Therapeutic antiserum to combat diphtheria

2. Albert Neisser 11. Ian Hector Frazer

 1879: isolated 1st human pathogen  2005: Human Papilloma Virus vaccine
(gonococcus)
12. Albert Sabin Salk
3. Louis Pasteur  1949: Polio vaccine
 Father of Immunology
 1885: Therapeutic vaccination 13. Charles Robert Richet
 First report of live attenuated vaccine for  Anaphylaxis
rabies
 1880: Demonstrated that injection of killed 14. Frank Macfarlane Burnet
microbes provided protection  Clonal selection

4. Elie Metchnikoff 15. Dr. Eckhard Podack

 1883: cellular theory of immunity through  Cancer therapy
phagocytosis
 Nobel Prize: 1908 16. Susumu Tonegawa
 1939: cloning of immunoglobulin gene
5. Emil Adolf von Behring  1987: Nobel Prize
 1890: Humoral theory of immunity
 1901: discovered serum antitoxins which 17. Bart J. Ben
led to the development of toxoids for  Major Histocompatibility Complex
diphtheria
 1901: Nobel prize 18. Cesar Milstein and Georges J. F. Köhler
 Monoclonal Antibody
6. Jules Bordet
 1896: discovered the complement that 19. Gerald Edelman and Rodney Robert Porter
lysed bacteria  1960: chemical structure of
 The Complement System: ability to antibody
complement antibacterial properties
20. Rosalyn Yallow
7. Robert Koch  1960: Radioimmunoassay
 1891: cutaneous (delayed) hypersensitivity
21. Joseph Murray and Edward Donnall Thomas
8. Karl Landsteiner  Organ and cell transplantation in
 1902: Human blood group antigen— treatment of human disease
beginning of safe and effective transfusion  1996: Nobel prize
medicine
 isolated A,B,O 22. Haraldzur Hausen
 AB- 2 years after  Human Papilloma Virus (HPV)

9. Sir Peter Brian Medawar 23. Françoise Barré-Sinoussi and Luc Montagnier
 Father of transplantation  Human Immunodeficiency Virus (HIV)
 1944: Immunologic Tolerance
Immunity
Immunity
- is the sum of all occurring defense mechanisms that protect humans from infectious diseases
- the ability of the body to resist damage from foreign elements

Immunological experimentation
- The discovery of a remarkable relationship between exposure to cowpox and immunity to small pox

Immunity is classified into two types of Resistance mechanisms:

1. Natural immunity
a. Nonspecific immunity
b. Non adaptive immunity
c. Innate immunity
2. Adaptive Immunity
a. Specific Immunity
b. Adaptive Immunity

I. Active Immunity
-The individual has responded to an antigen and produced his own antibodies- thus lymphocytes are
activated and the memory cells formed provide long lasting resistance.

II. Passive Immunity

-the individual is given antibodies produced by someone else

Components of Natural Immune System

1. Cellular 2. Humoral
a. Mast cells a. Complement
b. Neutrophils b. Lysozyme
c. Macrophages c. Interferon

Components of Adaptive immunity

1. Cellular 2. Humoral
a. T Lymphocyte a. Antibodies
b. B Lymphocyte b. Cytokines
c. Plasma Cells
 Resistance Mechanisms
Types of Resistance Examples
Mucous membrane
Phagocytic Cells
Natural Immunity
Enzymes in secretions
Interferon
Acquired Immunity
Recovery from disease
(Active)
Naturally Acquired
Placental transfer of
antibody (Passive)
Administration of antitoxin
Artificially Acquired (Passive)
Vaccination (Active)
Innate (Non Specific) Immunity

 First Line of Defense

o Intact Skin
o Mucous membranes and their secretions
o Normal microbiota

 Second Line of Defense

o Natural killer cells and phagocytic white blood cells
o Inflammation
o Fever
o Antimicrobial substances

Adaptive (Acquired) Immunity

- Third line of defense
o Specialized lymphocytes: T cells and B cells
o Antibodies

Natural/ Nonspecific/ Nonadaptive/Innate Immunity

- The ability of the individual to resist infection by means of normally present body functions
- Characterized as a non-specific mechanism
- Eliminate or neutralize any toxic substances elaborated by infectious agent

1. External Defense System (First line of defense)

- designed to keep microorganisms from entering the body
- are composed of structural barriers that prevent most infectious agents from entering the body
- Physical and mechanical barriers
- Unbroken skin
- Mucus Membranes

A. Unbroken Skin
- outer keratinized layer serves as barrier to the entry of microorganisms
- the surface of the skin is also inhibitory to the growth of most microorganisms because of low moisture, low
pH, and the presence of several secretions discourages the growth of microorganisms
- Lactic Acid: Sweat
- Fatty Acid: sebaceous gland
- maintain the skin at an acid pH of app. 5.6
B. Mucus Membranes
- The inner surface of the body are guarded by mucous membranes that line the respiratory, digestive, urinary
and reproductive systems and protect the internal lining.

 Digestive tract: the acidity of the stomach which is due to the production of hydrochloric acid, keeps the pH
as low as 1 and serves to halt microbial growth

 Respiratory Tract: the action of coughing removes mucous that contains microorganisms

 Genitourinary tract: the flushing action of urine, plus its slight acidity, helps remove many potential
pathogens.

 Reproductive tract: lactic acid formation in the female genital tract keeps the vagina at a pH of 5.

Biochemical barriers to infection

a) Keratin
b) Hydrochloric Acid
c) Bile Salt
d) Lysozyme
e) Complement

Examples of Nonspecific Defense Mechanisms

2. Internal defense system: Second line of defense

- designed to recognize molecules that are unique to infectious organisms
- are centered on the process of phagocytosis
- the most important function of the internal defense system
- enhance by soluble factors called your acute-phase reactants
 Acute Phase Reactants

Normal
Protein Response time concentration Increase Function
(mg/dL)
Opsonization,
C-reactive Protein 6-10 0.5 1000x
complement activation

Serum Amyloid A 24 3.0 1000x Removal of cholesterol

Alpha1-antitrypsin 24 200-400 205x Protease inhibitor

Fibrinogen 24 110-400 2-5x Clot formation

Haptoglobin 24 40-200 Binds hemoglobin

Binds copper and

Ceruloplasmin 48-72 20-40
oxidizes iron

Complement C3 48-72 60-140 Opsonization lysis

Mannose-binding protein 0.15-1.0 Complement activation

Biologic activities of secretory products in Natural Immunity

1. Organic Acid
- found at low pH in sebaceous gland secretions
- many microbes are susceptible to low concentrations

2. Fatty Acids
- interfere with the functions of the cell membrane

3. Saliva
- Contain enzymes that damage the microbial cell wall and membrane and cause leakage of cytoplasm
- also contains antibodies that opsonize microbes

4. Tears
- contain lysozymes, which lyses bacteria, particularly gram positive bacteria by destroying the bacterial cell
wall

5. Lactoferrin and Transferrin

- bind iron interfering with microbial acquisition of this essential metabolite

6. Lactoperoxidase
- catalyzes the peroxidation in bacterial cell wall and membrance

7. HCL
- interfere with vital functions of the cell membrane

8. Bile Acids
- interfere with vital functions of the cell membrane
9. Trypsin
- hydrolyzes proteins of cell membrane and cell wall

10. Mucus
- entraps foreign particles

11. Spermine
- a pH dependent polyamine found in sperm and seminal fluid
- inhibits growth of gram positive bacteria

Cellular Mechanism  Lymphokine-activated killer cells

 mast cell
 neutrophil Humoral Factors
 macrophages
 monocytes  Complement
 eosinophils  Lysozyme
 NK cells  Interferon
Phagocytosis
- the engulfment of cells or particulate matter by leukocytes, macrophages, and other cells
- this process destroys most of the foreign cells that penetrate the external defense
- phagocytic cells are the major cellular components of natural immunity

 Requirements:
1. Energy generated through glucose metabolism
2. Synthesis of a new cell membrane
3. An active cytoplasmic contractile protein system

 Process:
1. Physical contact between the white cell and the foreign particle
2. Formation of phagosome
3. Fusion with cytoplasmic granules to form a phagolysosome
4. Digestion and Excretion of phagolysosome to the outside by exocytosis

Movement of Phagocytic Cells

1. Ameboid Movement
- phagocytic cells migrate in and out of blood vessels and throughout tissues (diapedesis)

 Diapedesis: movement of granulocytes from the circulating blood to the peripheral tissues

2. Chemotaxis
- the migration of cells in the direction of the chemical messenger (chemotaxin)

 Chemotaxins: a protein or other substance that acts as a chemical messenger to produce chemotaxis
 Pseudopodium: refractile process of the cytoplasm of the cell that function as an organ of locomotion
 Endocytosis: into the cell
 Exocytosis: from inside to outside the cell

Chemotactic Factors of Neutrophils

Chemotaxins Source Function
Fibrinopeptides Fibrinogen Generated via
fibrinolytic pathway
Histamines Mast cells Increase capillary
permeability
Platelet-activating Mast cells, Aggregates platelets
factor Neutrophils and causes release
of serotonin and
histamine
Interleukin 8 Macrophage Migration inhibiting
factor
 Opsonization: the coating of the organisms by molecules that speed up the process of phagocytosis.
 Opsonins: serum proteins that attach to a foreign substance and enhance phagocytosis
1. C-reactive proteins
2. Complement components
3. Antibodies
 Lysosomal enzymes: work best at pH 5 (acid)
 Functions: hydrolyze proteins, fats, polysaccharides and nucleic acids.
 Can fuse with food vacuoles to digest food.

Enzymes Substrate
Aryl sulfatase Sulfate esters
Proteoglycans (collagen)
Cathepsin

Cholesteryl esterase Lipoproteins

Glycosidase Carbohydrates
Lysozyme Peptidoglycan
Phosphatase Phosphate ester
Phospholipids Lipoprotein
INFLAMMATION
 The cellular and humoral mechanisms involve in the overall reaction of the body to injury or invasion by an
infectious agent.
 A vascular and cellular response to trauma
 The purpose is to initiate the healing of the injured tissue

Four Cardinal Signs or Clinical Symptoms

1. Redness (Rubor)
- caused by blood vessel dilation
- chemical mediators promote the vessel dilation:
 Histamine
 Serotonin
 Bradykins
 Prostaglandins

2. Swelling (Tumor)
- Edema fluid varies with the stage of inflammation
- as capillary permeability increases and plasma proteins escape, the extravascular fluid becomes
cloudy and more viscous (exudate)

3. Heat (Calor)
- Loss of function: due to the pain causing reflex guarding or muscle spasm
- spasm decreases metabolic activity and constricts blood flow which causes more pain due to
ischemia

4. Pain (Dolor)
- Results from irritation of nerve endings by physical or chemical factors
- trauma may result in cell anoxia- because of interference with blood flow due to capillary damage

Inflammation
1. Increased blood supply to the affected area (redness and heat)
2. Increased capillary permeability (swelling and pain)
3. Increased migration of WBCs, mainly PMN
4. Migration of macrophages to the injured area

Formed Elements in Blood

 Erythrocytes: RBC
o carry oxygen and carbon dioxide

 Leukocytes: WBC
o defense mechanisms
o Granulocytes: Neutrophils, Eosinophils, Basophils
o Agranulocytes: Monocytes, Dendritic Cells, Lymphocytes

 Platelets: Thrombocyte particles

o clotting factors
 Neutrophils/Polymorphonuclear leukocytes/ PMNs
o Are granulocytes that circulate in the blood and migrate quickly in response to local invasion by
microorganisms
o Mobile cells can pass through capillaries
o Engulf bacteria by phagocytosis
o Secrete a fever inducing agent called pyrogen which also helps the body fight infection

 Monocyte
o Are mononuclear cells, the largest cells in the blood peripheral blood
o Characterized by irregularly folded horseshoe-shape nucleus
 Composition of Granules:
- Peroxidase - B-glucoronidase
- Acid Phosphatase - Lysozyme
- Arylsulfatase - Lipase

o Migrate to the tissues, where they differentiate into specific macrophages

 Macrophage
o Alveolar Cells: lungs
o Kupffer Cells: Liver
o Microglial: brain
o Histiocyte: connective tissue

 Monocyte-Macrophage system
o Function:
 Microbial killing: macrophage activated
 Tumoricidal activity
 Intracellular parasite eradication
 Phagocytosis
 Secretion of cell mediators
 Antigen presentation
Secreted Products Macrophages
Products Examples
Plasminogen activator
Proteinases
Collagenase
Lysozyme, phosphatase, lipase,
Hydrolases glycosidase
Plasma Proteins Fibronectin
Tissue thromboplastin
Coagulation factors
Factors V, VII, IX, X
C1, C2, C3, C4, C5
Complement components
Properdin
Superoxide anion
Oxygen metabolites Hydrogen peroxide
Hydroxyl radical
Nucleotide metabolites cAMP, thymidine
IL-1
Cell function regulators IFN-a
-erythropoietin
  Eosinophils
o Allergic reaction and asthma
o Response to many parasitic infection
o Capable of phagocytosis but are much less efficient than neutrophils because of the smaller
number and lack of digestive enzymes
 Composition of granules
 ACP
 Arylsulfatase

 Basophils
o Inducing and maintaining immediate hypersensitivity reactions
o Some cell-mediated delayed reactions such as in skin graft
o Composition of granules:
 Histamine
 Heparin
 Eosinophil chemotactic factor A

 Mast Cells
o Connective tissue cells of mesenchymal origin
o Hypersensitivity reactions by binding IgE
o Composition of granules
 ACP
 ALP
 Protease

 Dendritic Cells
o Most potent phagocytic cell in the tissue
o Main function: phagocytose antigen and present to T lymphocyte
o Express high levels of MHC class II and co-stimulatory B7 molecules
 Classifications:

1. Langerhans Cells
 found in epidermis and mucous membranes
 contain large organelles called Birbeck granules

2. Interstitial Dendritic Cells

 populate most organs (heart, lungs, liver, kidney, GI tract)
 capacity to bind antigen and stimulate T lymphocyte responses
 represent a population of mature bone marrow-derived DC, exhibiting a strong
lymphocyte stimulating potential

3. Interdigitating Dendritic Cells

 present in T lymphocyte
 Lymphocytes
o Nucleus is usually round to slightly indented with sharply defined edges
o Cornerstone of the immune system: antibodies production and cell-mediated immunity
o Functions:
 Recognition of antigens
 Storage of immunologic memory
 Immune response to specific antigens
 Immunological functions such as cellular immunity and humoral immunity
o Can be divided into 3 classes
 1) B lymphocytes
- activation causes proliferation and differentiation into plasma cells that produce
antibodies
- lymphoyctes that play a large role in the humoral immune response
- principal functions of B cells is to make antibodies against antigens, perform the
role of antigen-presenting cell
b cells recognize the spatial arrangement of proteins, nucleic acids and
polysaccharides

2) T Lymphocytes
- mature in thymus
- belong to a group of WBC and play a central role in cell mediated immunity
- recognize a linear sequence of amino acids

 Helper T Cell
o assist other WBCs in immunologic processes, including maturation
of B cells into plasma cells and memory b cells, and activation of
cytotoxic t cells and macrophages

 Cytotoxic T cells
o destroy virally infected cells and tumor cells, and are also implicated
in transplant rejection

 Regulatory T cells
o formerly known as suppressor T cells, are crucial for the
maintenance of immunological tolerance

 Memory T cell
o are a subset of antigen-specific T cells that persist long-term after an
infection is resolved

3) Natural Killer Cells

- responsible for immune surveillance
- lack marker molecules characteristics of B and T cells
- compromise about 10-15% of the lymphocytes of circulating blood
- NK cells provide rapid response to virally infected cells and respond to tumor
formation

 Antigen recognition in B and T cells

o In B cells: the initial antigen-receptor complex is bimolecular (Ag-BCR)
o In T cells: the initial antigen-receptor is trimolecular (MHC protein-Ag-TCR)
o B cell antigens include proteins, lipids, carbohydrates, and nucleic acids
o T cell antigens are confined to protein fragments
  Platelets
- Functions:
 secrete vasoconstrictors which constrict blood vessels causing vascular spasms in broken
blood vessels
- Form temporary platelet plugs to stop bleeding
- Secrete procoagulants (clotting factors) to promote blood clotting
- Digest and destroy bacteria
- Secrete chemicals that attract neutrophils and monocytes to sites of inflammation
- Secrete growth factors to maintain the linings of blood vessels

Acquired/ Adaptive/ Specific immunity

- Specific resistance acquired by introduction of an antigen into a responsive host.

Specific immune response: 2 components

1. Primary immune response: initial exposure to a particular infectious agent
2. Secondary (anamnestic) immune response: on further contact with the same immunogen
 increased resistance develops

Characteristics of Specific Immune Response:

 The ability to recognize self from non self
 Specificity
 Immunologic memory

Specific Immune Response

 are mediated by two interrelated and interdependent defense mechanisms

1. Antibody-Mediated Immunity (Humoral)

 primarily involves Bursa or bone marrow derived B lymphocytes, or B cells
 involves activation and clonal selection of B cells, resulting in production of secreted antibodies

2. Cell Mediated Immunity (Cellular)

 T cells
 involves activation and clonal selection of cytotoxic T cells

Acquired Immunity Antibody-mediated

 allergic reactions
 blood incompatibilities (Rh, ABO)
 Graves’ disease (in goiter)
 SLE
 Immune complex kidney disease
 acute and hyperacute transplant reactions
Acquired Immunity Cell-mediated
 TB
 contact dermatitis
 tumors
 viral infections
 chronic transplant reactions (vascular damage)

Acquisition of Adaptive immunity

Active Passive
Mode of antibody Active production of Transfer of antibody
acquisition antibody from another source
Temporary/ permanent Always temporary 3-6
Duration of effect
weeks
Source May have both natural and Artificial

 Passive Immunity
- an immunity in which antibodies produced elsewhere are given to the individual:

1. Natural Acquired Passive Immunity:

- antibodies transferred from mother to fetus across the placenta and to the newborn in
colostrums and breast milk during the first few months of life

2. Artificially Acquired Passive Immunity

- introduction of antibodies that are formed by an animal or a human to an individual to
prevent or treat infection

 Active Immunity
- product of the individuals own immune system in response to a foreign antigen

a) Naturally Acquired Active Immunity:

- immunity that comes from infections encountered in daily life

b) Artificially Acquired Active Immunity

- stimulated by initial exposure to specific foreign macromolecules through the use of vaccines to
artificially establish a state of immunity

Acquired Immunity
Active Passive
Natural source: Natural Source:
clinical/subclinical disease Congenital IgG (across placenta)

Artificial Source: Artificial source:

-vaccines: inactivated (killed) -antiserum, antitoxin, gamma
-attenuated: weakened globulin
NOTE:
 Memory cells are only produce in active immunity
 Protection for active immunity is permanent whereas in passive immunity it is only temporary
 Antigens are only encountered in active immunity
 Active immunity takes place several weeks to become active but passive immunity is immediate.

Main components of Natural and Acquired Immunity that contribute to humoral and cell mediated immunity

Humoral Cell-Mediated
Complement Macrophages
Natural Neutrophils NK cells
Dendritic Cells
B cells Helper T cells
Artificial Antibodies Cytotoxic T cells
THE LYMPHATIC SYSTEM
 The immune system is a network of cells and organs that extend throughout the body and function as a
defense against infection
 The immune system has been recognized as a separate body system: “Lymphatic System”
 Lymphatic Organs: organs containing lymphatic tissues
 Lymphatic Tissue: connective tissue fibers + lymphocytes
 The precursors of lymphocytes arise from progenitor cells of the yolk sac and liver
 The bone marrow becomes the main provider of undifferentiated progenitor cells, which can further develop
into lymphoblasts
 The continued cellular development of lymphoid precursors and proliferation occurs as the cell travel to the
primary and secondary lymphoid tissues

Components of Lymphatic System

A. Cells:

 lymphocytes (B, T, NK Cells)

 Antigen Presenting Cells (Dendritic Cells, Macrophages)

B. Lymphatic Tissue
 Diffuse
 Nodular

C. Lymphatic Organs
 Lymph node
 Spleen
 Thymus

D. Lymphatic Vessels

 Carry cells and fluid

Functions of the Lymphatic System

 Filters bacteria, foreign

materials, toxins, and any
harmful materials
 Drains away excess fluid to prevent water clogging of the tissue and cells
 Transport proteins back into the blood supply
 Produces lymphocytes which protect and defend the body against infection
 React to the presence of potentially harmful antigens recognized as “nonself”
 Absorbs fat from the intestines and transport it to the liver
Lymphocytes

 The primary cell involved in the immune response

 They arise from hematopoietic stem cell
 They mature in the primary organs

Primary Lymphoid Organs

- They provide conductive microenvironments that are essential for initial production of lymphocytes from
progenitor cells.

1. Thymus
 Gland situated in front of the heart and behind the sternum
 Contains cells that mature into T lymphocytes and specifically react to viruses, parasites, fungi,
foreign substances and other antigens
 Controls cell mediated immunity
 Primary function: production of thymic lymphocytes
 Thymocytes: immature lymphocytes
 Serves as site for T lymphocyte maturation and development
 A major organ for proliferation and differentiation of T-lymphocytes in body
Node Location Function
Buccal Face, Cheek Drains the eyelids, nose and facial skin
Parotid Face, in front of ear Drains the eyelids, nose and ears
Posterior Behind ear Drains behind the ear and temple
auricular
Occipital Back of head Drains the back of the scalp and the upper neck region
Submental Under chin Drains lower lip, chin and the floor of the mouth
Submandibular Under jawline Drains the chin, lips, nose, cheeks and tongue
Superficial In the neck, below the ear Drains lower part of ear, parotid area and neck
Deep cervical neck Drains back of the scalp and neck
Axillary underarms Drains the pectoral are and the upper arm
Supratrochlear elbow Drains the fingers, thumb, hand, forearm
Intestinal Inside abdominal cavity Drains abdominal viscera
Iliac Hip Drains the pelvic are including reproductive organs and the
bladder
Inguinal Groin Drains pelvic area and legs
Popliteal Behind the knees Drains the toes, feet and lower legs
Cisterna chyli Sack like chamber in the Receives lymph from the lower abdomen, lower limbs, and
abdomen pelvis and conveys it into the thoracic duct

2. Bone Marrow
 The yellow tissue in the center of your bones that is responsible for making WBCs that are destined to
become lymphocytes
 The source of progenitor cells
 They differentiate into lymphocytes, granulocytes, erythrocytes
 Differentiation of progenitor cells into B lymphocytes and functions as the bursa equivalent in humans
 “Bursa of fabricus”
 B cell maturation
  Functions as the center for antigen-independent lymphoiesis
 In the peripheral blood, approximately: 10-20% of all lymphocytes are B cells, 61-89% are T cells, 22%
are NK cells

Secondary Lymphoid Organs

 Are tiny clusters of glands which filter out bacteria and toxins
 Primary function: generation of B cell memory
 Filter mechanisms for fluid from the tissues
 Sinuses: Lymph Fluid

1. Lymph Nodes
 Fluid and lymphocytes exit by the way of the efferent lymph vessels
 Lymphocytes are able to circulate continuously between lymph nodes and the peripheral blood
 In time of infection: proliferation of activated cells-lymphocytes accumulation in the site of infection-
causes the lymph nodes to becomes enlarged: “Lymphadenopathy”
2. Spleen

 Situated behind the stomach

 Responsible for producing antibodies and lymphocytes, and destroying old RBC
 Filtering mechanisms for antigens in the blood stream
 The largest secondary lymphoid organs
 Splenic Tissue: red pulp and white pulp

3. Tonsils
 Protective ring of encapsulated lymphatic tissue
 Are found in the mucous membrane lining of the oral and pharyngeal cavities
 To respond to pathogens entering the respiratory and alimentary tracts

4. Appendix
 Acts as filter to remove debris and antigens entering the gastrointestinal tract

5. Diffuse Lymphatic Tissue

o Mucosal Associated Lymphoid Tissue (MALT)
 Found in the gastrointestinal, respiratory and urogenital tract
 Macrophages and lymphocytes are localized
 Peyer’s Patches: represent a specialized type of MALT

o Gut-Associated Lymphoid Tissue (GALT)

 Includes lymphoid tissue in the intestines and the liver
 Involved in lymphocytes circulation

 B cells: plasma cells and memory cells (humoral immunity)

 T cells: production of sensitized lymphocytes that secrete Cytokines (Cell mediated)
  Cytokines: chemical messenger produced by stimulated cells that regulate the function of lymphocytes and
other cells involved in immune response

T Lymphocytes and B Lymphocytes

Approximate Percentages of Lymphocytes in Lymphoid Organs

Lymphoid Organ T Lymphocytes B Lymphocytes

Thymus 100 0
Blood 80 20
Lymph Nodes 60 40
Spleen 45 55
Bone Marrow 10 90

Differentiation of T cells and B Cells

T Cell B Cell
Bone Marrow Origin Bone Marrow Origin
Maturation: Thymus Bone Marrow, Bursa (birds)
Long lived Short lived/ Long lived
Highly mobile Fairly Mobile/ stationary
No complement receptor w/ complement receptor
No surface Ig w/ Surface Ig
No antibody synthesis Ab synthesis
Effector: cellular, humoral Humoral only
Located in paracortical region of lymph nodes Located in the cortical region of lymph nodes
End prod of activation: Cytokines End prod of activation: Antibody
Ag: CD2, CD3, CD4, CD8 Ag: CD19, CD20, CD21, CD40, MHC Class II
Identified by rosette formation with SRBCs Identified by surface Ig

Similar Characteristics of T and B cells

 Antigen specific
 Increased in number in secondary response
 Antigen-binding receptors on surface

T cell Development
1. Double Negative Thymocytes
- Rearrangement of the genes that code for the antigen receptor (T cell receptor)
- Lacks CD4 and CD8 markers

2. Double Positive Thymocytes

 Rearrangement of genes coding for the alpha chain

a. Positive Selection
  Permits the survival of only those T cells whose TCR’s are capable of recognizing
self-MHC molecules

b. Negative Selection
 Eliminates T cells that react too strong with self-MHC or with self MHC plus
self-polypeptides
3. Mature T cells
o CD4/Helper/Inducer T cells
 receptor for MHC class II molecules
 they activate Th cells, suppressor T cells and macrophages

o CD8/Suppressor/Cytotoxic T cells
 receptor for MHC class I molecules
 involved in the prevention of autoimmunity
 eliminates virus-infected cells, cancer cells, causes graft rejection

o Normal ratio: CD4+: CD8= 2:1

o 2 subsets:
 Helper TH1 Cell
 primary functions connected with cytotoxicity and local inflammation
 the “inducer” cell involved in delayed-type hypersensitivity
 secretions of IFN-Y and TNF-B

 Helper TH2 Cell

 direct the immune response toward production of IgE, IgA and IgG
 promotes the proliferation of eosinophils and mast cells
 secrete variety of IL: 3,4,5,6,10
 IL 4,5,6: stimulate antibody production
 IL 4: also enhances class switching to IgE

Activated T cell
- Express receptors for IL2
- Activated T cells change their migration patterns
- Activated CD4 T cells can make different types of cytokines

Principal cells that function in cell-mediated immunity

- TH1 cells: activates cells related to cell mediated immunity; macrophages, CD8, NK cells
- TH2 cells: stimulates production of eosinophils, IgM and IgE
- Cytotoxic T cells: destroys target cells on contact
- Regulatory T cells: regulates immune response and helps maintain balance
- Activated macrophage: enhanced phagocytic activity
- NK Cells: attacks and destroys target cells, participate in antibody-dependent-cell-mediated cytotoxicity

B Lymphocytes
- Progenitor cells: “antigen independent maturation process”- Bone marrow
- Bone marrow derived lymphocyte
- Surface membrane antigen-binding receptor
- Are an essential component of the adaptive immune system
- Precursor cells in antibody production: primary source for cells-responsible for humoral response
 - Represent less than 15% of the circulating lymphocytes
- B cell originate from the progenitor cells: Stromal Cells (bone marrow)
- Stromal cells produce
SCF (stem cell factor) necessary for development at early period
- Ig gene rearrangements and the appearance of surface markers identify the stage of B cell development

B cell Development
 “gene rearrangement”
 “antigen specificity”
 “Immunoglobulin class switching”

1. Pro B cell
- Rearrangement of genes on chromosome 14
- Coding for the production of heavy (H) chain
- Surface markers: CD19, CD45R, CD43, CD24
- Intracellular proteins: terminal deoxyribonucleotidetransferase (TdT) and recombination-activating
genes RAG-1 and RAG-2

2. Pre B cell
- The signal for commencement or initial transcription of L chain
- Rearrangement of genes on chromosome 2 and 22 coding for production of light (L) chain
- Distinguishing feature: u heavy (H) chains in the cytoplasm
- Only pre b cells expressing the u heavy chains survived and proceed to further development

3. Immature B cell
- Expression of immunoglobulin on the cell surface
- U chains are no longer detectable in the cytoplasm
- Monomeric IgM appears: the primary surface marker on the B cell membrance
- Surface IgM (sIgM): antigen recognition site and binds specific epitopes
- Initiates the differentiation of the B cell into plasma cells.
- CD antigens: CD10
- Cd10: expressed on the malignant cells of patients with common acute lymphocytic leukemia
“CALLA Ag”
- CD21: acts as C3 receptor
- Cd40 and MHC class II: interaction of B cells and T cells
- Immature B cells leave the bone marrow and proceed for development in the spleen and other
secondary lymphoid organs

4. Mature B Cells
- Immature B cells: marginal zone B cells
- Cells are released from the BM and seed in peripheral lymphoid organs
- Other immature cells: Follicular B cells: found in the lymph nodes and other secondary organs
- IgD: second immunoglobulin on the surface of B cell membrane
- Provide the primary activating signal to B cells
- Prolong the life span of mature B cells
- “Antigen dependent phase” of B cell development
- Transformation: memory cells and plasma cells
B cell activation and antibody synthesis
- Proliferation and maturation: B cell growth factors (IL)
- IL 1,2,6: enhance antibody production
- IL1: has a positive effect on pre B cell maturation, as well as on clonal expansion of B cells after antigen
stimulation
- IL4,5: influence immunoglobulin class switching
- IFN-Y: act as a stimulant to B cell differentiation
- Proliferation and differentiation: plasma cell
- Immunoglobulin disappears from the cell membrane
- Secretion of antibody begins
- Some B lymphocytes do not undergo terminal differentiation- “memory cells”
- Memory cells: express IgG on the cell membrane
- Antibody production: lymphatic tissues, spleen, lymph nodes, site of inflammation
- Expression of identifying markers: CD25, (receptor for IL2)

Plasma Cell
- The result of antigenic stimulation and transformation of activated B cells
- Plasma cells are the end stage of B cell differentiation
- “Terminally differentiated B cells”
- Characterized by the presence of abundant rough endoplasmic reticulum
- Secretes large amount of the antibody that had been anchored in the parent B cells membrane
- Plasma cells are not found normally in the circulating blood
- Account for <3.5% of nucleated cells in the bone marrow

Memory Cell
 Major cells responsible for the anamnestic response
 Express IgG in the cell membrane
 Secondary immune response: 3 ways
 Memory cells produce antibodies that bind with greater affinity to their antigens than the antibodies
produced in the initial response
 The response time is much faster than the primary response
 A greater number of antibodies are produced

HUMORAL IMMUNE RESPONSE

 Primary immune response
o primary antigenic stimulation
o production of small amount of antibody

 Secondary immune response

o AKA: anamnestic, booster, memory immune response
o consists of a rapid proliferation of plasma cells
o production of large amount of specific antibody
o memory cells are produced
Immunoglobulin class switching/ Isotype Switching
- Primary immune response: plasma cells (IgM)
- Subsequent exposure to antigen, plasma cell switch from producing IgM to IgG, or to another
immunoglobulin class
- There is no alteration in the L chain and variable portion of H chain
- There is no change in antigen-binding specificity of plasma cell
- The switch involves a change in the H chain constant domains
- The plasma cell loses its ability to produce that immunoglobulin class
- The cell loses its ability to produce that immunoglobulin class
- The cell that has switched to IgG will not be able to revert to IgM synthesis but could switch again to
other Igs
ANTIGENS
Antigen Recognition by the Specific Immune System:

 Recognition by Antibodies on Bone marrow derived lymphocytes (B cells) or by antibodies secreted

by B cells (Plasma Cells)
 Recognition by T cell receptor on T cells (Thymus derived Lymphocytes)

Antigen (Ag)
- binds specifically to an antibody binding site (Ab), or to a T cell receptor (TCR)
- a substance that reacts with the products of a specific immune response

Immunogen
- Binds specifically to an antibody binding site or to a TCR
- generates a humoral or cellular immune response
- a substance that induces a specific response

Characteristics B Cell T Cell

Interaction with antigen
Binding of soluble antigen
Involvement of MHC
molecules
Chemical nature of antigens
Epitope
Involves tertiary complex of
Involves binary complex of
TCR, Ag and MHC
membrane Ig and Ag
molecules
Yes No
Required to display
None required
processed antigen
Mostly proteins, but some
lipids and glycolipids
Protein, Polysaccharide, lipid
presented on MHC-like
molecules
Internal linear peptides
Accessible, hydrophilic, mobile
produced by processing of
peptides containing sequential
antigen and bound to MHC
or nonsequential amino acids
molecules

Properties of Antigens
1. Immunogenicity
- inherent ability of a substance to induce specific response resulting in the formation of immune
lymphocyte or antibodies

2. Antigenicity/ Specificity
- The ability to react specifically with the antibodies or cells that caused it to be produced.
Factors that Affect Immunogenicity

A. Foreignness
a. Autologous
- same individual
- ex: skin graft (autograft)

b. Syngenic/ Isologous
- identical twins (syngenic graft)

c. Allogeneic/ Homologous
- same species but different individual (mother-daughter) (allograft/homograft)

d. Xenogeneic/Heterologous
- found between different species (monkey-man)

e. Sequestered
- brain and corneal antigens which are not accessible to antibody

f. Tissue specific antigens

- antigens common and unique in some organs
- Thyroid: Thyroglobulin

B. Size
- the bigger the size, the more immunogenic

C. Chemical Complexity:
 Proteins: best and strongest
 Polysaccharide: endotoxin, pneumococcal capsule
 Glycoproteins: ABO, Rh
 Polypeptide: insulin
 Nucleic Acid and Lipids: least immunogenic

Parts of Antigen
1. Carrier portion
- responsible for the molecular weight

2. Epitope or Determinant
- that portion of an antigen that combines with the products of a specific immune response
- determines the specificity of antigens

A. Structure
1. Size: very Small
2. Conformation: Linear, Conformational
3. Site: Topographic epitopes, Internal epitopes
 B. Functions
- determines the specificity of antigen molecules
-
1. Paratope: area of the antibody molecule that reacts to epitopes

2. Valence of an antigen: is equal to the total number of epitopes the antigen possesses

3. Altering antigenicity: new antigens are produced by altering epitopes; denaturation or hydrolysis of
proteins

Definition of Terms

 Hapten:
- an incomplete antigen
- partial antigen
- not immunogenic by itself, but when coupled by a carrier protein can elicit and immune response

 Allergen
- a special class of immunogen that induces hypersensitivity reactions

 Adjuvants
- Are substances added to an immunogen to enhance immune response
- Actions of adjuvants
 Prolongs the retention time of the immunogen in the body
 Increases the effective size of immunogen
 Stimulates the influx of macrophage and or lymphocytes

Other Factors that Influence the Strength of Immune Response

1. Degradability
- that are not biodegradable, such as polystyrene particles or asbestos are nonimmunogenic

2. Structural Stability
- highly flexible molecules that have no fixed shape are poor immunogens
- gelatin

3. Route of Immunization
- The subcutaneous or intramuscular routes of antigen injection are best for soluble substances
- The intravenous injection is more effective for cellular immunogens: erythrocytes or bacterial
vaccines
 Hapten Molecular Weight
Penicillin 320
Aspirin 180
Methyldopa 211
Urushiols (poison ivy) 211-290
Gentamicin 700
Antigen
Oxytocin 1000
Vasopressin 1000
Gastrin 1800
Calcitonin 3600
Insulin 6000
Haptoglobin 9000

Affinity
 The strength of the attraction between an epitope and the antigen combining size of antibody
 Initial force of attraction that exist between a single Fab site on an antibody molecule and a single epitope or
determinant site on a corresponding antigen

Avidity
 Refers to the strength of interaction between complex antigen
 Sum of all attractive forces between multivalent antigen and multivalent antibody
Examples of the immunogenicity of different transplant Tissues:

 Most Immunogenic  bone

 bone marrow  xenogeneic valve replacement
 islets of Langerhans
 heart  Least Immunogenic
 kidney  cornea
 skin

MHC Antigens
- Antigen processing and recognition
- Genetic capability to regulate an immune response
- Found on all nucleated cells in the body
- Graft rejection, transfusion reactions and autoimmune diseases

Class I MHC Molecules Class II MHC Molecules

- Proteins coded for by genes at thee loci (ABC) - Proteins coded for by the DR, DP and DQ loci
- Nucleated cells - B cells, macrophages, activated T cells,
- Transplantation of tissues monocytes, dendritic cells, endothelium
ANTIBODIES
 are glycoproteins which are sensitized and secreted by plasma cells in response to specific antigenic
stimulation and it forms about 20% of plasma proteins

Complete Antibodies
 Are antibodies which are heat resistant
 When they combine with its specific antigen they will produce different immunologic reaction
 Are capable of passing the transplacental barrier

Incomplete Antibodies
 Blocking antibodies
 Are heat labile substances
 Are not able to cross the placental barrier

Antibodies Reaction with Antigens

 Antitoxin: antibodies to toxins or toxoids, which neutralize the antigen

 Agglutinin: antibodies which first immobilize motile bacteria and aggregate cells forming clumps
 Precipitins: antibodies, which form complexes with soluble antigens forming precipitates
 Lysine: antibodies which together with complement dissolve the antigenic cells
 Opsonins: antibodies which combine with surface components of microbial and other cells so that they are
more readily phagocytized

Basic Structure of an Immunoglobulin

 4 polypeptide chains
 2 identical light chains
 2 identical heavy chains

H chains
- A molecular weight of 50-70 kilodaltons (kDa)
- Contain approximately 400 amino acids
- Amino acid difference: carboxy-terminal portion H chain isotypes- five classes of Ig
- H chain determines immunoglobulin classes

L chains
- A molecular weight of about 23,000
- Are composed of approximately 200 amino acids
- Kappa, Lambda
- K:L= 65:35; 2:1
- Both L and H chains are held together by covalent disulfide bonds
- H chains are interconnected by disulfide linkages in the hinge region
2 Terminal Regions

 Carboxy Terminal
- With constant amino acid sequence
- Constant Region
- Demonstrate a much more uniform amino acid sequence

 Amino Terminal
- With varying antibody specificity
- Variable Region
- Shows a wide variation in AA sequence

 Class switching (Immune Response)

- Only the constant portion of the H chain changes and the antibody specificity remains the same

Sequencing of Heavy Chains

- 100-110 amino terminus, highly variable
- five basic sequence patterns
- Ig A,G,D,E,M
- The above classes are called isotype
- Each class can have either kappa or lambda light chains
- Minor differences lead to subclasses for IgA
- IgA1, IgA2, IgG1, IgG2, IgG3, IgG4

Symbols Isotypes
 IgM Mu
 IgD Delta
 IgG Gamma
 IgA Alpha
 IgE Epsilon

Enzymatic Digestion of Antibodies

 Digestion with papain yields

- 3 fragments
- 2 identical Fab and 1 Fc
- Fab because fragment that is Ag binding
- Fc because found to crystallize in cold storage

 Pepsin Digestion
- F(ab’)2
- No Fc recovery, digested entirely

Mercapyoethanol Reduction: eliminated disulfide bonds

Basic Structure of an immunoglobulin

 One Fc fragment (Fragment Crystallizable)

- Comprises of the carboxy-terminal portion of the H chain
- Involve in the Ig biologic function
- Involve in complement fixation, placental transfer of Ig and serve as the binding site of the cell

 Two Fab fragments (fragment-antigen binding)

- Capable of antigen binding even without the Fc
- Cannot facilitate Ag precipitation or agglutination

 Immunoglobulin structure
- Treatment with proteolytic enzymes (papain and pepsin)
- Treatment with pepsin results in digestion of Fc fragment, leaving 2 Fab fragments
- These 2 fab fragments has two antigen sites (bivalent)
- Thus capable of antigen binding, precipitation and agglutination

 Antigenic Determinants on Abs fall in 3 categories

A. Isotype
- A unique amino acid that is common to all immunoglobulin molecules in a given
species
- Constant Region of antibody
- If you inject antibody in a different species Anti-isotype is generated
- If within same species, no anti-isotype
B. Allotype
- Variation in amino acid sequence
- Same isotypes within one species
- Differences (1-4 a/a) arise in different individuals (form of polymorphism)
- If injected with such Ab you generate anti-allotype ab
- Ex. During pregnancy, Blood transfusion
C. Idiotype
- Unique Vh and Vl
- Antigen binding site, but can also behave as antigenic determinant
- If you inject a monoclonal antibody into a genetically identical recipient then anti-
idiotype antibodies are generated
- No anti-isotype and no anti-allotype Abs will be generated

 The flexible portion of the heavy chain (hinge region)

 All of the immunoglobulin classes have a hinge region except IgE

Hinge region
 Rich in proline residues (flexible)
 Proline residues are target for proteolytic digestion (papain and pepsin)
 Rich in cysteine residues (disulfide bonds)

General Functions of Immunoglobulins

 Facilitate phagocytosis and kill microbes
 Neutralize toxic substances
 Combine with antigens on cellular surfaces causing destruction of these cells
 Extravascular (outside the blood vessels)
 Intravascular (within blood vessels)
Antibodies
 Immunoglobulins are “Y” shaped proteins
 The arms of the “Y” bind to antigens
 The tail of the “Y” is responsible for biological activity
 The ability of immunoglobulins to bind antigen is determined by AA sequence in variable region
 Synthesized by plasma cells
 Constitute 25-30% of total serum proteins
 Antibodies are present in serum, tissue fluids and mucosal surfaces

Antibody Classes
Classification is based on structure and antigenic nature of H chain, the immunoglobulins are classified into 5 classes

IMMUNOGLOBULINS
Immunoglobulin G
 Most abundant class in serum
 80% of total immunoglobulin
 Present in blood, plasma and tissue fluids
 Contains less carbohydrates than other immunoglobulins
 Serum Half Life: 23 days.
 Longest of all the immunoglobulin isotypes
 Most versatile immunoglobulin
 Major immunoglobulin in serum
 Major immunoglobulin in secondary immune response
 Effective antitoxic immunity (exclusive)
 Activate complement by classical pathway
 4 Subclasses: decreasing serum concentration
 IgG1, IgG3, IgG4 can cross placenta
 IgG3: most effective complement activator
 IgG1 and IgG3: binds to Fc receptor on phagocytic cells- Opsonization (enhances phagocytosis)

 IgG1: 77%
 IgG2: 22%
 IgG3: 7%
 IgG4: 4%

 Functions
 Providing immunity for the newborn
 Fixation of the complement
 Opsonization (Coating antigen)
 Neutralizing toxins and virus
 Agglutination and Precipitation reactions
Immunoglobulin M
 One J chain is present per pentamer
 J chain served as linkage points for disulfide bonds between two adjacent monomers
 Pentamer stabilized by a J chain
 Does not contain hinge region
 5-10% of total serum proteins
 Polymer of five monomeric units
 Held together by disulfide bond and J chain
 Molecular weight: 900,000-10,000,000 (millionaire molecule)
 Half-life: 5 days
 First immunoglobulin of primary immune response
 Found mainly in the intravascular spaces
 Membrane-bound monomeric immunoglobulin
 Synthesized during fetal life
 More efficient than IgG in complement fixation
 Antibody most often formed in response to stimulus by G- bacteria
 Functions
 Agglutinates bacteria
 Activates complement by classical pathway
 Causes opsonization and immune hemolysis
 Believed to be responsible for protection against blood invasion by microorganisms
 Toxin neutralization

Immunoglobulin A
 10-15% total immunoglobulins
 present in milk, saliva, tears, mucous of respiratory tract, digestive tract and genitourinary tract
 in serum exists as a monomer
 in external secretions exist as dimer called secretory immunoglobulin
 Has J chain and secretory piece
 Half-life: 6 days
 Major class of immunoglobulins in secretions
 2nd most common serum immunoglobulins
 transport mechanism across endothelial cells
 IgA1
- Alpha 1 heavy chain
- Can be inactivated by an IgA protease produced by gonococci, meningococci,
pneumococci and Haemophilus influenza
 IgA2
- Alpha 2 heavy chains
- Important in mucosal immunity
 Serum IgA
- Accounts for about 15-20% total normal serum Ig
 IgA
- Predominance in body secretions (saliva, tears, colostrum, bronchial, genitourinary
and intestinal secretions.)
 Functions
- Provides localized defense of mucous membrane by cross-linking and neutralization
of antigens
- Presence in breast milk confers passive immunity on nursing infant
Immunoglobulin D
 Structure is similar to IgG
 Serum concentration: 30mg/mL
 0.2% of total immunoglobulins
 Half-life: 3 days
 IgD together with IgM is major membrane bound immunoglobulins on unstimulated B lymphocytes
 Acts as receptors for antigens
 Represents <1% of the total serum immunoglobulins
 Membrane immunoglobulins
 Susceptible to enzymatic degradation
 Presence of only a single interchain bond between heavy chains
 Functions
 As an antigen receptor, in B cell activation
 Antibody activity for a variety of haptens and antigens (penicillin, insulin and diphtheria toxoid)

Immunoglobulin E
 Structure is similar to IgG
 4 constant region domains
 Molecular weight: 190,000
 Half-life: 2 days
 Heat labile (inactivated at 56C in 1 hour)
 Normal serum concentration: 0.3 ug/mL
 Mostly present extracellular
 Does not cross placenta
 Present in trace amount in normal serum (0.05 mg/dL)
 A monomer with five domains in the heavy chains
 Produced by B cells in the spleen, in lymphoid tissue of tonsils and in the respiratory and gastrointestinal
mucosa
 Participate in immediate hypersensitivities
 Binds mast cells and blood basophils
 Causes degranulation- mediates the release of histamine and heparin
 Produced in the lining of respiratory and intestinal tract
 Reagin antibody
 Does not activate complement nor agglutinate antigens
 Binds to the Fc receptors on the membranes of blood basophils and tissue mast cells
 Mediates immediate hypersensitivity reactions and PK reactions
 Responsible for symptoms of anaphylactic shock, hay fever and asthma
 Play a role in immunity against helminthic parasites
THE COMPLEMENT SYSTEM
 A defensive system consisting of over 30 proteins produced by the liver and found in circulating blood serum
 Complement: a set of proteins that play a role in cytolytic destruction of cellular antigen by specific
antibody
 Synthesized mainly by the liver except C1 (intestinal epithelial cells) and Factor D (adipose tissue)
 Activation: antigen-antibody complex or endotoxin, capsule series of proteins
 Biological effects: either beneficial or harmful to host
 Opsonization: C3b and C1q: enhance phagocytosis
 Chemotaxis: C5a and C5, 6, 7 complex attract neutrophils. C5a enhances adhesiveness of
neutrophils to the endothelium
 Anaphylatoxin (C3a, C4a, C5a): cause degranulation of mast cells; binds directly to smooth
muscles of bronchioles (bronchospasm)
 Cytolysis (MAC): disrupt the membrane and the entry of water and electrolytes into the cell
 Enhancement of antibody production: binding of C3b to its receptors on the surface of activated B
cells → enhanced antibody production
 Heat labile: inactivated by heating serum at 56C for 30 minutes
 Able to augment the effects of other components of the immune system
 Specific immune system
 Catabolic rate: 1% to 3% per hour

Genetic Variants
- Most variants are specified by autosomal codominant genes
- Polymorphic complement proteins (Factor B, C2, C4)
- Genes for 6 of the complement receptors and regulatory proteins—Chromosome 1
- Congenital deficiencies (except factor B): have been observed in humans
- Complement proteins: appear in the fetal circulation, first 13 weeks of pregnancy
- Complement proteins in neonates: present in 50% of adult levels

Complement Components
- Functions:
 Increase vascular permeability
 Recruit monocytes and neutrophils to the area of antigen concentration
 Trigger secretion of immunoregulatory molecules

Three pathways of activation

a) Classical Pathway
- Triggered by antigen-antibody complex

b) Alternative/ Properdin// By-Pass pathway

- Pillemer, 1950
- Triggering factors are LPS (g-), fungal cell wall, aggregated IgA, Cobra venom factor

c) Lectin Pathway
- MBL (acute phase protein) pathway
- Binds to carbohydrates of bacteria, yeasts, viruses, and some parasites in a calcium-dependent
manner
- Identical to the classical pathway
  Most of the proteins of the complement system are inactive enzyme precursors or zymogens, that are
converted to active enzymes

Nomenclature of complement proteins

- The complement components are designated by “C” followed by an identifying number 1 to 9: C1,
C9
- The numerals indicate the order in which components are activated (Except C4)
- Cleaved peptides: fragmented peptides are denoted by lower case letters (C3a)
- If proteolysis results in loss of fragment activity an (i) is added to indicate inactivation (iC3b)
- Horizontal bar over the numerals of component complexes: activated states
- Alternative pathway: capital letters (Factor B, factor H)

Chronic activation can lead to:

 Inflammation
 Tissue Damage
 Life threatening systemic effects
 Plasma proteins act as system regulators (infectious agents and non-self-antigens)

Three Main Effects

 Opsonization: enhancement of phagocytosis
 Generation of mediators of inflammation
 Cytolysis (lysis of cells-bacteria, allografts, tumor cells)

Molecular Weight (MW), Concentration
Serum Protein Function
Kilodalton (kDa) (ug/mL)
Classical Pathway
Binds to Fc region of
C1q 410 150
IgM and IgG
C1r 85 50 Activates C1s
C1s 85 50 Cleaves C4 and C2
Part of C3 convertase
C4 205 300-600
(C4b)
Binds to C4b- forms C3
C2 102 25
convertase
Key intermediate in all
C3 190 1200
pathways
Initiates membrane
C5 190 80
attack complex
C6 110 45 Binds to C5b in MAC
Binds to C5bC6 in
C7 100 90
MAC
Starts pore formation
C8 150 55
on membrane
Polymerizes to cause
C9 70 60
cell lysis
Alternative Pathway
Binds to C3b to form
Factor B 93 200
C3 convertase
Factor D 24 2 Cleaves factor B
Stabilizes C3bBb-C3
Properdin 55 15-25
convertase

MBL Pathway
MBL 200-600 0.0002-10 Binds to mannose
MASP-1 93 1.5-12 Unknown
MASP-2 76 Unknown Cleaves C4 and C2

Plasma Complement Regulators

Dissociates C1r and
C1 inhibitor (C1 INH) 105 240
C1s from C1q
Factor I 88 35 Cleaves C3b and C4b
Cofactor with factor I
to activate C3b
Factor H 150 300-450
Prevents binding of B
to C3b
Acts as a cofactor with
C4 binding protein 520 250
factor I to activate C4b
Prevents attachment of
S protein (vitronectin) 84 500 the C5b67 complex to
cell membranes

A Cascade System
- The complement works as a cascade system
- Cascade: is when one reaction triggers another reaction which trigger others and so on. These types of
systems can grow exponentially very fast
- Complement proteins are often designated by an uppercase letter C and are inactive until they are split into
products (C1)
- When the products are split they become active
- The active products are usually designated with a lowercase a or b (example C1a and C1b)

THE CLASSICAL PATHWAY

 Immunoglobulins involved: IgM and IgG (except IgG4)
 Requires interaction of all nine major complement components
 The constant regions of antibody contain a binding site for C1q (a single molecule of IgM is enough to
initiate the pathway)
 Complement Activation:
 The Recognition Unit
- The classical pathway is considered to be part of the specific immune response because it
relies on antibodies to initiate it
- C1 becomes activated when it binds to the end of antibodies
- Stabilized by calcium
- Three polypeptides: C1q, C1r, C1s
- C1q: binds to antibody molecules, activates C1r proenzyme
- C1r and C1s: generate enzyme activity to begin the cascade
 - C1r: when activated, cleaves the proenzyme C1s
- C1q recognizes the fragment crystallizable region (Fc)
- Binding occurs at the CH2 region for IgG and CH3 region for IgM

 The Activation Unit

 The building of First activation unit (C4) and Second activation unit (C2)
- Once C1 is activated it activates 2 other complement proteins, C4 and C2 by cutting
them in half
- C4 is cleaved into C4a and C4b
- C2 is cleaved into C2a and C2b (C3 convertase)
- Both C2b and C4b bind together on the surface of the bacteria
- C2a and C4a diffuse away

 Third activation unit: C3 activation complex

- C2b and C4b bind together on the surface to form a C3 activation complex
- The function of the C3 activation complex is to activate C3 proteins
- This is done by cleaving C3 into C3a and C3b

 C3b
- Many C3b molecules are produced by the C3 activation complex
- The C3b bind to C4b2a and coat the surface of the bacteria to form C4b2a3b (C5
convertase)
- C3b is an opsonin
- Opsonins are molecules that bind both to bacteria and phagocytes
- Opsonization increases phagocytosis by 1000 fold

 The Membrane-Attack Complex

 Opsonization: enhancement of phagocytosis by coating with C3b

 Inflammation: increase of blood vessel permeability and chemotactic attraction of phagocytes
 Cytolysis: loss of cellular contents through transmembrane channel formed by membrane attack complex
C5-C9

THE ALTERNATIVE PATHWAY

 Also known as the PROPERDIN PATHWAY
 The alternative pathway is slower than the Classical pathway
 Initiated by bacterial cell walls
 Does not require complement components C1, C4, and C2 (bypasses)
 Nonspecific immunity
 Does not wait for antibody to be formed for activation
 Usually activated by products of microorganisms (endotoxin)
 Other Activators:
 Complexes containing IgA
 Some virus infected cells (EBV)
 Gram positive and Gram negative organisms
 Parasites (Trypanosomes, Leishmania)
 Erythrocytes
 Carbohydrates (agarose)
 Tumor Cell Lines
  Initiation of the Alternative pathway
- C3 contains an unstable thioester bond
- This unstable bond makes C3 subject to slow spontaneous hydrolysis to C3b and C3a
- The C3b is able to bind to foreign surface antigens

 Factor B
- Once activated, bind to factor B
- C3b on the surface of a foreign cells binds to another plasma protein called factor B

 C5 Activation complex
- When an additional C3b binds to the C3 activation complex it converts it into a C5 activation
complex
- The C5 activation complex cleaves C5 into C5a and C5b
- C5b begins the production of the MAC

THE LECTIN PATHWAY

 Also known as MANNOSE BINDING LECTIN (MBL) PATHWAY
 Activated by binding of mannose-binding lectin
 Binding causes activation of MASP (MBP-associated serine proteases cleave to C2 and C4)
 Part of the non-specific immune system
 Activator: mannose-binding lectin (MBL) a plasma protein that is similar to C1q
 MBL binds to terminal mannose (sugar) on the surface glycoproteins of microbes
 Triggered by binding of MBP to mannose on bacterial cell wall
 MASP-1, MASP-2, MASP-3 bind to form an activated C1
 MASP-2 cleaves C2 and C4 proceed as the classical

Note:
 All three pathways lead to production of C3b-> the central molecule of complement cascade
 Presence of C3b on surface of a microbe marks it as foreign and targets it for destruction
 C3b with two important functions
 Combines with other complement components to generate C5 convertase
 Opsonizes bacteria

Regulation of Complement System

 Properdin
- protects C3b and stabilizes C3 convertase

 Factor I
- cleaves cell-bound or fluid phase C3b and C4b-> inactivates C3b and C4b

 Decay accelerating factor (DAF)

- Glycoprotein on surface of human cells
- Prevents assembly of C3bBb or accelerates disassembly of performed convertase-> no formation of
MAC
- Acts on both classical and alternative
  C4b-binding protein (C4BP)
- Inhibits the action of C4b in classical pathway
- Splits C4 convertase and is a cofactor for factor I

 Complement Receptor 1 (CR- 1):

- cofactor for factor I, together with CD46

 Protectin (CD59) and Vitronectin (S protein)

- Inhibits formation of MAC by binding C5b678
- Present on self-cells to prevent complement from damaging them

Classical Alternative Lectin

Immune complex (IgM,
Activating Substance LPS, G- Mannose
IgG)
Recognition Unit C1q, C1r, C1s C3, Factor B, Factor D MBP, MASP1, MASP2
C3 Convertase C4b2a C3bBb C4b2a
C5 Convertase C4b2a3b C3bBb3b C4b2a3b
MAC C5b6789 C5b6789 C5b6789
End Result Lysis Lysis Lysis

Biologic Functions of Complement System

 Anaphylatoxins: C3a, C5a, C4a

- Substances that degranulate mast cells causing the release of pharmacologic mediators of
inflammatory response
- Anaphylatoxin: releases histamine, serotonin, and other vasoactive compounds from mast cells
increasing capillary permeability
- C3a, C5a: have the ability to contract smooth muscle and increasing capillary permeability
- C3a: function as a immunoregulatory molecule
- C5a: stimulates the production of leukotrienes

 Chemotaxins: C5a, C5b, C6, C7

- Chemotaxis: attracts phagocytic cells to sites of inflammation and increases their overall activity
- C5a:
 is the major complement derived chemotactic factor in serum
 causes neutrophils to adhere to endothelium vessels which lead to neutropenia
 activates neutrophils which triggers a bacterial oxidative burst and degranulation

 Immune Adherence: C3b

- The generation of C3b and the coating of target cells by C3b the major biologic function of
complement
- C3b- coated cells, aggregate immune adherence
- Activation of the alternative pathway
- Major factor in opsonization
- Play a role in the induction of humoral immune response

 C4a( Anaphylatoxin)
- Virus neutralization
- Anaphylatoxin activity
  C4b (opsonin)
- Involved in opsonization
- C4b receptor sites exists on several cell types (phagocytic cells, lymphocytes, erythrocytes)

 C2
- Production of kinin-like molecule that increases vascular permeability and contracts smooth
muscles
- Closely associated with the gene of factor B on chromosome 6 in MHC
- Kinin activator

 Ba and Bb
- Ba: is a chemotactic for neutrophils
- Bb: activates macrophages and causes them to adhere to and spread on surfaces

 C8 and C9
- Are responsible for the membrane damage that causes lysis of bacteria and other cells

 Factor B
o Binds to C3b to form C3 convertase enzyme

 Factor D
o Cleaves factor B

Measurements of Complement Components

 CH50 Hemolytic Assay

- Determine the hemolytic activity of the complement system
- Uses antibody-coated sheep RBCs which is incubated with patient serum
- The degree of hemolysis is proportional to the hemolytic activity of the complement
- Elevated complement components
 inflammation
- Decreases components:
 Complement has been consumed
 Presence of genetic defects

 Complement Fixation Test

- 2 systems
 Test System/ Bacteriologic System
 Composed of patient serum, measured antigen and complement
 Indicator System
 Composed of sheep RBC’s, hemolysin (antibody used in sensitizing the indicator
cells)
 Best source of complement: Guinea pig serum

- Classical CFT: Process

 Antigens bind with serum antibodies
(IgM, IgG)
 Complement binds with the complex
 Addition of Hemolysin-sensitized
RBC (as indicator)
RESULT: NO LYSIS (+)
 Antigen does not bind with anything
as serum does not have antibodies
 Complement does not bind
 Addition of Hemolysin-sensitized
RBC (as indicator)
RESULT: LYSIS (-)
 Non Lytic CFT/ Agglutinating Complement Absorption Test
- Serum from a patient contains an unknown complement fixing antibody
- Addition of antigen and complement (from horse, cattle, mice)
- Addition of indicator system: SRBC + conglutinin
- RESULT: No agglutination

 Rice Test
- Test for present antibodies but not react with complement
- Done to be certain that a negative CFT result did not miss detecting a complement fixing
antibody
- Result: (+): Lysis; (-) No Lysis

Deficiencies of Complement Components

C1q,r,s Lupus-like syndrome; recurrent infections
Hereditary Angioneurotic Edema (HANE);
C2 Lupus-like syndrome; recurrent infections;
atherosclerosis
C3 Recurrent pyogenic infections; glomerulonephritis
C4 Lupus-like syndrome
C5-C8 Neisseria infections
C8-C9 Neisseria meningitides, Neisseria gonorrhea
congenital deficiency which results in a clinical
syndrome called Hereditary Angioma results in
C1INH
excess of C4 and C2 which generate kinin-like
proteins that increase vascular permeability
DAF Paroxysmal nocturnal hemoglobinemia (PNH)
Factor H/ Factor I Recurrent pyogenic infections
MBL and MASP2 Pneumococcal infection
Properdin Neisseria infection
MAJOR HISTOCOMPATIBILITY COMPLEX
The Genetic Control of Immune Response: Major Histocompatibility Complex
 In all vertebrates there is a genetic region that has a major influence on graft survival
 This region is referred to as the Major Histocompatibility Complex (MHC)
 Individuals identical for this region can exchange grafts more successfully than MHC non identical
combinations
 Unlike minor histocompatibility antigens, the MHC products play as important role in antigen recognition by
T cells
 Cluster of genes found in all mammals
 Its products is associated with intracellular recognition and self/non self-discrimination
 Participate in both humoral and cell mediated immunity
 Act as antigen presenting structures
 Major role in determining whether transplanted tissue will be histocompatible or histoincompatible
 Group of genes associated with transplacental antigen and the immune response
 In human: MHC is found on Chromosome 6 referred to as HLA complex
 In Mice: MHC is found on Chromosome 17 referred to as H-2 complex

Immune Graft Rejection

- If unmatched MHC transplantation takes place, then T cells recognize them as foreign and generate
an immune response that can lead to graft failure
- Rejection of transplanted tissue was associated with inflammation and lymphocyte infiltration

Mouse H-2 Complex

Complex H-2
MHC Class I II III I
Region K IA IE S D
Gene Products H-2K IA  IE  C’ TNF  H-2D H-2L
proteins TNF 
*Not present in all haplotypes
Human HLA Complex
Complex HLA
MHC Class II III I
Region DP DQ DR C4, C2, BF B C A
Gene Products DP DQ DR C’ TNF  HLA HLA HLA
  proteins TNF  B C A

General Organization and Inheritance of MHC

- Gorer and Snell (1930’s) concept of rejection of foreign tissue is an immune response to cell surface
molecules
- Snell (1980): nobel prize
MHC Polymorphism and Antigen Binding
- Diversity of antigen presentation, mediated by MHC classes I and II in three ways
 The MHC’s genetic encoding is polygenic
 MHC genes are highly polymorphic and have many variants
 Several MHC genes are expressed from both inherited alleles

Molecular basis of MHC types and Variants

- Polygenism: several <HC class I and Class II genes encoding different types of MHC molecule with
a range of peptide-binding specificities
- Polymorphism: Variation >1% at a single genetic locus in a population of individuals MHC genes
are the most polymorphic known
- Allele: one of a number of alternative forms of the same gene occupying s given position on a
chromosome
- Epitope: part of a molecule (such as protein) that is the target of an immune response
- Polygenic: having an infinite number of derivatives at a point

MHC genes are polymorphic

- Highly polymorphic
- Vary considerably from person to person
- Inherited as 2 sets (one from father, one from mother)
- Haplotype: refers to set from mother or father
- Both mother and father alleles are expressed

MHC molecules are targets for immune evasion by pathogens

- Without T cells there is no effective immune response
- Ag-specific T cells are activated by peptide/MHC complexes
- The MHC has two strategies to prevent evasion by pathogens:
 More than one type of MHC molecule in each individual
 Extensive differences in MHC molecules between individuals
 Terms
 Restitope: part of the TCR that interacts with the MHC
 Desetope: part of the MHC that interacts with the antigen peptide
 Paratope: part of the TCR that specifically reacts with the epitope of an antigen
 Agretope: site of the molecule that makes contact with MHC class of molecules
 Histotope: part of the MHC that interacts with the TCR

 MHC
o Genes of MHC Organized in 3 Classes
 MHC 1
- Glycoproteins expressed on all nucleated cells
 MHC 2
- Glycoproteins expressed on M, B cells and Dendritic Cells
 MHC 3
- Products that include secreted proteins that have immune functions. EX. Complement
System, Inflammatory molecules
 Tissue MHC Class I MHC Class II
Lymphoid Tissue
 T cells +++ +
 B cells +++ +++
 Macrophages +++ ++
 Other antigen-presenting cells (Langerhans cells) +++ +++
 Epithelial Cells of the thymus + +++
Other Nucleated Cells
 Neutrophils +++ -
 Hepatocytes + -
 Kidney + -
 Brain + -
Non Nucleated Cells
 RBC - -

T cell Receptors

T cell Type Co-receptor MHC Restriction Function

B cell help
macrophage
Helper T cell CD4 Class II activation
help for CD8 T cells
Cytokine secretion
Killing virus
Cytotoxic T cells CD8 Class I infected cells
killing tumor cells

MHC Class I
 MHC class I mediates immune responses against endogenous antigens, anitgens that are already found in
the cell
 Usually, these cells that are expressing MHC class I are viral infected cells or are tumor cells
 MHC class I present peptides that are 8-10 amino acids in size, which will then be recognized by the
cytotoxic T cells
 Present in all nucleated cells and some have been found on red cells
 Process cytoplasmically derived antigens and presented to CD8+ cells
 Includes HLA A, HLA B, HLA C, B2 macroglubulin, HLA E
 Described as serologically defined antigens
 Test: Microlymphocytoxicity test
 Demonstrates that if the antigen is present on the lymphocytes, addition of complement will
cause them to become porous and unable to exclude the added dye
 Process:
 Reagent: antiserum of known HLA specificity
 Dye: Trypan Blue
 Patient’s sample (with Ag) + reagent (with Antibody) = Antigen-Antibody complex
 Antigen-Antibody complex + complement + Dye = Blue Color
 Process
 Virus infects cells
 Viral proteins synthesized in cytosol
 Peptide fragments of viral proteins bound by MHC class I in ER
 Bound peptides transported by MHC class I to the cell surface
MHC Class II
 MHC class 2 mediates immune responses against exogenous antigens, antigens that are found outside of the
cell, in the cytosol
 MHC class 2 will bind with amino acid residues that are 13-18 in size and will be recognized by T helper
cells
 The MHC class 2 protein is found on cells like the B lymphocytes, macrophages, monocytes, DC and
endothelial cells
 These cells are phagocytic and can engulf an extracellular antigen
 Present in macrophages, B cells and DC
 Process extracellular derived antigens and presented to CD4+ cells
 Important in antigen presentation and interaction between immunocompetent cells
 Includes HLA DP, HLA DQ, DLA DR
 Determined by Mixed Lymphocyte Culture
 Stimulator cells (HLA D) are used
 Are irradiated or mixed with Mitomycin C (to inactivate/ nullify them)
 Patient’s lymphocytes are then mixed with the stimulator
 Incubate
 Proliferation of patient’s lymphocytes will occur if patient does not express the HLA D found in
stimulator cells
 No proliferation will occur if patient expresses the HLA D
 Process
 Antigen bound by B cell surface receptor
 Antigen internalized and degraded to peptide fragments
 Fragments bind to MHC class II and are transported to cell surface

MHC Class III

 MHC Class III minor MHC antigen
 Involves complement components C2, C4 and Factor B
 Includes several complement components, TNF, 2 heat shock proteins
 Not membrane proteins and have no role in antigen presentation, although mostly play a role in
immune response

HLA Deficiencies
 HLA B27: Ankylosing Spondylitis associated with Reiter’s Syndrome
 HLA DR2: Goodpasteure’s Syndrome
 HLA DR3: SLE and Type I DM
 HLA DR4: Rheumatoid Arthritis
 HLA DR5: Bechet Disease (Bw51), Gold induced nephropathy, Chronic Lymphatic Leukemia,
Kaposis Sarcoma
 HLA B8: Celiac Disease, Addison’s Disease, Myasthemia gravis, dermatitis herpetiformis, Chronic
active hepatitis, Sjogrens syndrome, insulin syndrome, insulin dependent DM, thyrotoxicosis
HYPERSENSITIVITY
 Undesirable side effect of immunity manifesting discomfort such as itching of the skin to potentially fatal
disease such as bronchial asthma
 Initiated by the interaction of antigen with humoral antibody or by cell-mediated immune mechanisms
 Adaptive response which occurs in an exaggerated/ inappropriate manner causing tissue damage
 The excessive stimulation of the normal effector mechanisms of the immune system which can lead to tissue
damage
 Four Types
 TYPE I: Immediate or Anaphylactic hypersensitivity
 TYPE II: Cytotoxic Hypersensitivity
 TYPE III: Immune Complex Hypersensitivity
 TYPE IV: Cell-mediated/ Delayed Type Hypersensitivity

 TYPES I-III: Immediate hypersensitivity reactions

 TYPE IV: manifestation are not seen in 24-48 hours after exposure to antigen

TYPE I TYPE II TYPE III TYPE IV

Immune
IgE IgG IgG/IgM T cells
mediator
Ag specific IgE Autologous/
Antigen Cellular antigens Soluble antigens
mast cells Heterologous
Complement Yes (activated,
No Yes (activated) No
Involvement exocytosis)
Immune Complex Cytokine production
Immune
Histamine release Cell Lysis deposition (inflammatory
Mechanism
(tissues) reaction)
 Serum
 Anaphylaxis  Transfusion sickness  Contact
 Hay fever reactions  Arthus dermatitis
Examples  Food,  Autoimmune reaction  Tuberculin
Allergies hemolytic  SLE test
 Asthma anemia  Rheumatic  Pneumonitis
Arthritis

 Terms
 Allergy: a pathologic condition in which the body produces immunologic responses to
environmental antigens bringing about tissue inflammation and organ dysfunction
 Allergen: the antigen that causes allergic reactions
 Examples: pollen grains, house dust mites, animal hair/dander/feathers, food, drugs,
chemicals
 Atopy: inherited propensity to respond immunologically to many common naturally occurring
allergens with continual production of IgE antibodies
 Anaphylaxis: acute generalized allergic reaction with simultaneous involvement of several organ
systems: cardiovascular, respiratory, cutaneous, gastrointestinal
 Urticaria: localized cutaneous form of anaphylaxis
TYPE I: Immediate or Anaphylactic Hypersensitivity
 They are initiated by antigens reacting with cell-bound antibody IgE
 This type of hypersensitivity manifests in many ways, depending on the target organ or tissue
 Involve the release of active mediators from mast cells and basophils
 Occurs 2-30 minutes (between exposure to antigen and the onset of clinical symptoms)
 May occur as a systemic or as a local reaction
 Effector Cells: tissue mast cells and circulating basophils
 IgE
 With high affinity to mast cells and basophils (homocytotropic)
 Have a receptor for the epsilon chain (FceR) membranes
 Mast cells may be triggered by other stimuli
 Exercise
 Emotional stress
 Chemicals
 Anaphylatoxins (C4a, C3a, C5a)
 These reactions mediated by agents without IgE allergen interaction are not hypersensitivity reactions,
although they produce the same symptoms

Regulation of IgE production

 The IgE production: influence of helper T cells (Th)
 The 2 subpopulation of Th cells have opposing effects on IgE production by B cells
 Th2 cells produce IL-4 which induces class switching from IgM to IgE
 Th1 cells produce gamma interferon which blocks the IL4 effect
 A defect in suppressor T cells- involved in the increase of IgE response in the atopic individual

The release of Mediators: IgE

 Influx of Calcium: mast cells
 Intracellular phosphodiesterase: activated
 Cyclic-adenosinemonophosphate (cAMP): decreased level
 Mediator rich cytoplasmic granules migrate to cell surface
 Exocytosis occurs: granules are released

Examples:
 Anaphylaxis
 Most dramatic and life-threatening
 Affects many organs within seconds to minutes after allergen exposure
 Not common
 Fatal

 Hay Fever
 Represent the most common atopic allergic disorders resulting from exposure to inhaled
allergens

 Allergic Asthma
 S/S: Bronchoconstriction, shortness of breath (difficulty of getting air, wheezing, cough,
chest tightness)
 Long term allergen exposure can cause chronic changes of increased difficulty breathing and
chest tightness
  Food Allergies
 Symptoms limited to the gastro intestinal tract including cramping, vomiting and diarrhea
 Exposure to house dust or mice

Treatment:
 Hyposensitization

 Involves injecting the patient with gradually increasing doses of allergen.

 The main objective – immunotherapy – to stimulate the production of IgG-Blocking
antibody.
 IgG-Blocking antibody binds to the allergen and prevents complexes with IgE on the mast
cell surface.
 Avoid products containing latex:
- Surgical gloves
- Tubings
- Condoms

Testing:
 Radioimmunosorbent Test (RIST)
 Measures total IgE by capturing the antibody with solid-phase anti IgE

 Radioallergosorbent Test (RAST)

 Measures antigen-specific IgE by using solid-phase antigen to capture patient antibody

 Laboratory
 Complete Blood Count
- Increase WBC-eosinophil count
- Increased serum IgE levels (Normal Values 39 IU/mL)

 Skin Test
 Antigens are either injected intradermally or into small scratching made into the patient’s
skin → if the patient is allergic to the substance – a visible inflammation reaction will
usually develop in 30 minutes
Mast Cell and Basophil Mediators of Atopic Disease

Mediators Effects Comments

Preformed Mediators
- Smooth muscle contraction
- Probably the most
- Increase capillary
Histamine important mediator in
permeability
anaphylaxis
- Increase airway resistance
- Complexed to histamine in
Heparin - Anticoagulant
cell
- Smooth muscle contraction
- Important in anaphylaxis in
Serotonin (5-hydroxytyptamine) - Increase capillary
animals other than humans
permeability
- Chemotaxis of eosinophils
that control allergic
ECF A
reaction-arylsufatase B and
histamine
NCF A - Chemotaxis of neutrophils
- Activation of complement
Protease cascade-generate C3a and
C5a
- Platelet aggregation
Release of vasoactive - An acetyl glyceryl ether
amines phosphoryl choline,
PAF
Increased vascular inactivated by
permeability, neutrophil phospholipases
chemotaxis
- Lipoxygenase metabolite
- Smooth muscle contraction
of arachidonic acid
SRS-A - Increased capillary
- A major factor in asthmatic
permeability
broncospams
LTB 4 - Chemotaxis of basophils
LTC 4, LTD 4 - Mucus secretion in airways
- Cyclooxygenase
Prostaglandins and - Strong bronchodilation,
metabolites of arachidonic
thromboxanes disaggregation of platelets
acid
TYPE II: Cytotoxic Hypersensitivity
 Body makes special autoantibodies directed against self-cells (antigens present on the surface of cells or
other tissue components)
 Antigen
 May be intrinsic to the cell membrane
 May take the form of an exogenous antigen adsorbed on the cell surface
 The damage is localized to a particular tissue or cell type
 Reactions involved antibodies directed to antigen or surface of specific cells or tissues resulting to cytolysis
 Hypersensitivity results from the binding of antibodies to normal or altered cell-surface antigens
 Initiated by antibody (IgG of IgM) reacting with cell bound antigen which results in the activation of
complement and destruction of cells
 Lysis or inactivation of target cells by activation of complement
 Phagocytosis of target cells with or without complement activation
 Lysis or inactivation of target cells by antibody dependent cell mediated cytolysis (ADCC) – action
of NK cells

Examples:
 Transfusion reactions
 Cells from an incompatible donor react with the host’s antibody
 Occurs in the first 10-15 minutes or first 50cc of blood

1. Hemolytic reactions
 Hemoglobinuria
 Chest pain
 Low back pain
 Decreased blood pressure
 Increased respiratory rate

2. Allergic reactions
a. Allergic reaction (MILD)
 Facial flushing
 Rash
b. Allergic reaction (SEVERE)
 Anxiety
 Decreased blood pressure
 Anaphylactic shock

3. Febrile reaction
- Sensitivity of the patient’s blood to white blood cells, platelets or plasma proteins
- Clinical signs: fever, warm and flush skin, chills, headache, anxiety, muscle pain

 Management of Transfusion Reaction

 Stop the transfusion immediately
 Check and monitor vital signs
 Maintain intravenous access (NSS)
 Investigate
  Hemolytic Disease of the Newborn (Erythroblastosis fetalis)
 A medical condition where an Rh negative mother’s Abs attack the red blood cells of an Rh
positive fetus
 Prevention:
- Prophylactic treatment is passive immunization of the mother with anti Rh
agglutinins (Rhogam) shortly after delivery of the first baby
- It prevents sensitization in the Rh negative mother by neutralizing the Rh agglutinins
in the mother
- Anti-D antibody is also administered to the expectant mother starting 28-30 weeks
of gestation

 Autoimmune Hemolytic Anemia, Agranulocytosis, Thrombocytopenia

 Individuals produce antibodies to their own blood cells which are then destroyed

 Drug Hypersensitivity
 Antibodies are produced that react with the drug

TYPE III: Immune Complex Hypersensitivity

 Results from the effects of soluble antigen-antibody complexes
 Formed locally at the site of tissue damage or deposited in the tissues from the circulation
 Activation of complement – causes inflammatory damage
 This immune-complex disease may result from:
 Persistent low-grade infection, as in malaria and viral hepatitis
 Autoimmunity, as in rheumatoid arthritis and SLE
 Inhaled antigens: farmer’s lung and pigeon fanciers lung where there is repeated exposure to mold
or pigeon A
 Antigen involved maybe:
o Exogenous
 Chronic bacterial, viral or parasitic infections
o Endogenous
 Non-organ specific autoimmunity (SLE)

Examples:
 Arthus reaction
 Seen when boosters are administered to individuals who already possess high antibody titers
to vaccine molecules.
 Localized area to tissue necrosis – edema, hemorrhage, ulceration
 Develop over a few hours

 Serum Sickness
 Systemic immune complex-deposition phenomenon
 Administration of diphtheria antitoxin (prepared in horses) in humans
 Characterized by vasculitis – vascular basement membrane
 S/S: fever, rash, splenomegaly, lymphadenopathy, arthritis and glomerulonephritis
  Systemic Lupus Erythematosus (SLE)
 Antibodies present:
- Antinuclear antibodies
- Antibodies to membrane
- Cytoplasmic components
- Antibodies cytotoxic to blood cells

 Mixed type of disease = involves both Type I and Type II hypersensitivity reactions

TYPE IV: Cell-mediated/Delayed-type Hypersensitivity

 Initiated by specifically sensitized T lymphocytes which respond to an antigen by producing and releasing
certain lymphokines – activate macrophages to destroy the Ag
 Antibodies and complement are not involved
 Represents tissue damage resulting from inappropriate cell mediated immune response
 Features:
 Cytokines are released which activate macrophages and account for the events that occur in a typical
delayed hypersensitivity response
 Continuing provocation of delayed hypersensitivity by persisting antigen leads to formation of
chronic granulomas
 Th2 type cells producing IL-5 can also produce tissue damage through their ability to recruit
eosinophils
 CD8 T cells are activated by MHC Class I molecules to become directly cytotoxic to target cells
 Reaction involves sensitized T cells and release of its lymphokines of mediators and amplifiers
 Best manifested by tuberculin skin reaction/Mantoux Test

Examples:
 Contract hypersensitivity (poison ivy, reactions to metals in jewelry)
 Tuberculin-type (TST)
 Granulomatous hypersensitivity (leprosy, TB, schistosomiasis, Chron’s disease)

 Delayed type:
o 72 hours in contact and tuberculin-type
o 21-25 days in granulomatous hypersensitivity
 Self-limiting

 Tuberculin Skin Test (TST)

 Tuberculin skin test (TST) are administered to detect the presence of M. tuberculosis – TB
 Mantoux – the technique for administering the test
 Tuberculin – also called purified protein derivatives (PPD)
 the solution used to administer the test
 Reading of Tuberculin Skin Test
 The skin test reaction should be read between 48 and 72 hours after administration
 A patient who does return within 72 hours will need to be rescheduled for another skin test
 The reaction should be measured in (mm) of the induration (palpable, raised, hardened area
of swelling)
 The reader should not measure erythema (redness)
 The diameter of the indurated area should be measured across the forearm (perpendicular to
the long axis)
 Positive Tuberculin Test
 HIV infected patients
5mm  Close contact of active cases
 Persons with fibrotic chest x-ray
 Persons with factors that increase the risk of tuberculosis (TB)
10mm  Injection drug users
 Medically underserved low-income populations
15mm  Persons with no additional risk factors

 False Positive Reactions

 Infection with non-tuberculosis mycobacteria
 Previous BCG vaccination
 Incorrect method of TST administration
 Incorrect interpretation of reaction
 Incorrect bottle of antigen used

 False Negative Reactions

 Very young age (less than 6 months old)
 Recent live-virus vaccination (measles, smallpox)
 Some viral illnesses (measles, chicken pox)
 Incorrect method of TST administration
 Incorrect interpretation of reaction
DISEASES
SYPHILIS
 Causative agent of venereal syphilis: Treponema pallidum subspecie pallidum
 Other Treponemes:
 Treponema pallidum subspecie pertenue: YAWS
 Treponema pallidum subspecie endemicum: ENDEMIC SYPHILIS/ BEJEL
 Treponema carateum: PINTA

 Mode of Transmission (MOT)

 Sexual contact
 Nonsexual contact with active lesions (chancre)
 Passage thru placenta (congenital syphilis)
 Transfusion of fresh human blood
 Blood bank storage condition:
 Whole blood: 1-6˚ C for 72 hours
 Accidental direct inoculation

 Pathogenesis
 Penetrates abraded skin/intact mucus membrane
 Disseminates throughout the body via lymphatics and bloodstream
 Invade any organ of the body
 CNS and cardiocascular system
 Clinical manifestation is dependent of host’s immune response

 STAGES:
A. Primary Syphilis
 Chancre: solitary, painless papule, eroded and indurated
 Penis, anal canal, external labia, cervix, oral cavity
 Heals in 3 to 6 weeks
 Bilateral swelling of lymph nodes
 Non-treponemal antibody
o Regain titer: increase in the 1st 4 weeks and remains stationary within 6 months

B. Secondary Syphilis
 Multiplication and dissemination of spirochete
 Occurs 1-2 months after the primary stage
 Lesions contains Treponema pallidum
 Lesions: self-limiting
 Presence of lesions in different site
 Disseminated rash
o Mucocutaneous rash (90%)
 Location: trunk, extremities, palms/soles, and face
 Localized or diffuse
 Swelling of the lymph nodes
 Allofacia
 Most contagious stage
 Start of the production of anti-treponema pallidum antibody
 Higher titer of antibodies
 Nervous system: Initial damage
 Flu-like symptoms, lymphadenopathy, rashes
 Condylomata lata
 o Wart-like lesions
o Found in moist areas of the body
o Painless
o Gray-white lesions

C. Latent Syphilis
 Absence of clinical manifestation (Asymptomatic patient)
 Non-infectious except for pregnant women
 Occurs months to years
 Characterized by positive examinationS
o SCREENING: RPR, VDRL
 Component of Classical VDRL Antigen
o Cardiolipin
o Lecithin
o Cholesterol
 Need microscope to observed the result (qualitative/ quantitative)
 180 rpm for 4 minutes

 Component of Classical RPR

o Cardiolipin
o Lecithin
o Cholesterol
o CHOLINE-CHLORIDE (chemical inacticator)
 No need microscope to observed the result (qualitative/ quantitative)
 100 rpm for 8 minutes


Reagin: antibody-like substance which is form by person infected
with syphilis
 IgM: antiphospholipid Ab
o Example: alcoholic beef extract (cardiolipin) – antigen
o CONFIRMATORY:
 TPHA ( Treponema pallidum Hemagglutination)
 TPPA (Treponema pallidum Particle Agglutination)
 FTA-ABS (Fluorescent Treponema Antibody-Absorption Test)
 Most sensitive and specific test

 Early Latent: 1st two years of infection

 Late Latent: host resistance to infection and relapse
D. Late/Tertiary Syphilis
 Slowly progressive; inflammatory disease
 Occurs 2-5 years
 Subdivided into:
o Neurosyphilis
 Affects the CNS
 Common symptoms:
 Destruction of brain parenchyma
 Destruction of spinal cord and meninges

 MENINGEAL SYPHILIS
 Brain and spinal cord
 Headaches and stiff neck

 MENINGOVASCULAR
 Inflammation of the pia mater and subarachnoid space

 Parenchymatous
 General paresis
 Joint paresis
 Joint degeneration
 Tabes dorsalis

o Congenital Syphilis
 occur in all stages of pregnancy
 stillborn
 liveborn
 no clinical symptoms during the 1st week of life
 some may remain asymptomatic
 60-90% develop later symptoms if not treated at birth
 20% may also have neurosyphilis
 Manifestations:
 Necrotizing funisitis
o Inflammation of the umbilical cord
o 1st manifestation
 Hemorrhagic rhinitis or colds
 Macropapular rash
o Surrounding the mouth
o Palms of the hands
o Sole of the feet
 Generalized lymphadenopathy
 Hepatosplenomegaly
 Jaundice
 Anemia
 Painful limbs
 Bone abnormalities (saddle nose)
 Hutchinson’s Triad
o Interstitial keratits (blindness)
o Hutchinson’s Teeth
o 8th nerve deafness/ labyrinthine disease
 o Cardiovascular syphilis
 Affects the heart (aorta)
 Aortitis/aortic aneurysm
 Develops 10 years post-infection

o Gumma Lesion (Gummas)

 Affects the skin/bones
 Do not contain Treponema pallidum

 Produced clinical illness years after initial infection

 Manifest average 10-15 years after the infection

NATURE OF IMMUNE RESPONSE

- Primary body defenses- intact skin
- Once skin is penetrated, T cells and macrophages play a role in the immune response
- Primary lesions
o shows the presence of both CD4+ and CD8+ T cells
o Cytokines produced by these cells activate macrophages
o Phagocytosis- heals the primary chancre
- Antibodies produced
o Nonspecific
o Specific
- TROMPs (Treponema pallidum Rare outer Membrane Proteins)
o Important in bringing about complement activation that ultimately kills the organisms
- Chronic nature of the disease
o Indicator that the organism is able to invade the immune response
o Treponemes may persist in the host for years if antibiotic therapy is not obtained

1) Antibody Production
o 2 types:
 Specific: treponema antibody
 Specific to outer membrane and endoflagellar protein
 Non-specific: Non-treponema antibody
 Anti-cardiolipin
 Regain
 Anti-lipoidal antibody
o Phospholipid: cardiolipin
 Constituent of the treponemal lipid
 Maybe produced in other disorder
 TB
 Leprosy
 Hepatitis
 Malaria
 Measles
 Chicken pox
 Autoimmune disease
 Arthritis
 Old
 Pregnancy
 2) Opsonin production
o C3B and IgG

3) Complement Activation
o Only the classical pathway maybe activated
o Antibody: IgM production cause lag phase

4) Cytokine Production
o IF-γ
 Enhances cytotoxity of phagocyte
o IL-2
 T cell growth factor

INVADING MECHANISM OF Treponema pallidum

1. Causes an increase of soluble IL-2 receptor
2. Decrease production of T cells
3. Decrease production of cytokines IF-γ

LABORATORY DIAGNOSIS OF SYPHILIS

1. Darkfield Examination
o Spirochete visualization
o Darkfield microscopy

2. Direct Fluorescent antibody test

o Most specific test for the diagnosis of syphilis when lesions are present
o Uses fluorescein isothiocyanate
 Labeled antibody specific to pathogenic treponemes

3. Serological Test
o Screening: RPR and VDRL
 Dilution: >1:4 (monitor treatment response)
o Confirmatory: TPPA, TPHA, FTA-ABS, TPI
o 30% are serologic active after 1 week
o 90% are serologic active after 3 weeks

4. Non-standard Treponemal Tests

a. ELISA
b. EIA
c. SRTD (Syphilis Rapid Test Device)
d. Western Blot
 INTERPRETATION CRITERIA
Pattern Reading
Particles concentrated in the shape of a button in
the center of the well with a smooth round outer NEGATIVE
rim
Particle concentrated in the shape of a compact
INCONCLUSIVE
ring with a smooth round outer rim
Definite large ring with a rough multiform outer
POSITIVE
rim agglutination
Firmly agglutinated particle spread out covering
POSITIVE
the button of the well uniformly

BIOLOGICAL FALSE POSITIVE

Infectious Diseases Non-infectious Diseases
 Leptospirosis  Drug addiction
 TB  Pregnancy
 Hepatitis  Old age
 Malaria  Chronic liver disease
 Measles  Blood Transfusion
 Pnemococcal pneumonia  Connective Tissue disorder
 Mycoplasma pneumonia
 Infectious mononucleosis
 Lymphogranuloma venereum
 Early HIV infection
 Vaccination

TERMED USED:

 Screening
o Reactive
o Nonreactive
 Confirmatory
o Positive
o Negative
Serologic Tests
Sensitivities of serologic tests for syphilis in different stages of disease
STAGES OF SYPHILIS (% SENSITIVITY)
TESTS Primary Secondary Latent Late/Tertiary
VDRL 78 (74-87) 100 95 (88-100) 71 (37-94)
RPR 86 (77-100) 100 98 (95-100) 73
FTA-ABS 84 (70-100) 100 100 96
MHA-TP 76 (69-90) 100 97 (97-100) 94
* MHA-TP (Microhemagglutination Assay for Antibodies to Treponema pallidum

 Wasserman Complement Fixation Test → 1ST TEST used to detect syphilis

 Serology in Syphilis Testing
1ST Scenario
Test Result CONCLUSION

Screening RPR Nonreactive  No syphilis

 Incubating syphilis
Confirmatory TPPA Negative
 No treatment
2nd Scenario
Test Result CONCLUSION
Screening RPR Reactive  Syphilis
 Yaws/Pinta
Confirmatory TPPA Positive  Needs treatment

3rd Scenario
Test Result CONCLUSION
Screening RPR Nonreactive  Primary/ latent
syphilis
 Previously treated or
Confirmatory TPPA Positive
untreated syphilis
 Yaws/Pinta
4th Scenario
Test Result CONCLUSION
Screening RPR Reactive  Biological false
positive
Confirmatory TPPA Negative  No syphilis
 No treatment
Syphilis
 Is a sexually transmitted infectious disease caused by the spirochete bacterium Treponema pallidum
 This bacterium causes infection when it goes into broken skin or mucus membranes, usually the genitals

Organism Disease Distribution Age onset Transmission

T. pallidum Venereal syphilis Worldwide Adolescents, Sexual contact
adults
T. pertenue Yaws Tropical areas, Children Skin contact
Africa, south
America,
Caribbean,
Indonesia
T. endemicum Endemic syphilis Arid areas, Africa Children-adults Mucous
membrane
T. carateum Pinta Semiarid, warm Children Skin contact
areas, central and
south America

 Epidemiology
 Syphilis occurs worldwide, most commonly in urban areas
 The number of cases is rising fastest in men who have sex with men
 Young adults ages 15-25 are the highest risk population
 People have no natural resistance to syphilis

 Who should be tested for syphilis?

 Are pregnant
 Are members of an at-risk population
 Have HIV
 Have partner(s) who have tested positive for syphilis
 Are sexually active and live in areas with high syphilis morbidity
Spirochete
 Spiral microorganism
 Long, slender, helically coiled, spiral or corkscrew-shaped, gram-negative bacilli
 Flagella-multilayered OM (outer sheath)
 Etiologic agent: T.pallidum subsp. Pallidum
 Thin (0.2um) spirochete, 6-20 um in length with 10-13 coils
 Man is the only natural reservoir
 The causative organism, Treponema pallidum was first identified by Fritz Schaudinn and Erich Hoffmann in
1905
Antigens
 The Wasserman Antigen (cardiolipin)
 Identified as a phospholipid (diphosphatidyl glycerol)
 Normal constituent of host tissue
 Free cardiolipin is a hapten and must be bound to a suitable carrier to be antigenic
  Treponemal Antigens
 Reiter Strain: non-virulent variant of T. pallidum; present in many indigenous treponemes of the
human alimentary tract
 Nichol Strain: virulent variant

Antibodies
 Individuals infected with T.pallidum respond immunologically by producing both specific and non specific
antibodies
 Treponemal Antibodies
o Produced against the Ag of the organisms themselves
 Non treponemal antibodies (regain antibodies)
o Almost always produced by persons with syphilis but also produced in:
 Infectious diseases: leprosy, TB, Malaria, Measles, Chicken Pox, IM, Hepatitis
 Autoimmune disorders: RA
 Pregnancy
 Old age
Transmission
 Direct contact with active lesions: largely through sexual contact
 Vertical transmission: across the placenta (2nd most common mode of infection)
 Latently infected female becomes pregnant, or when a pregnant woman becomes infected
 Non sexual contact with an active lesion (rare)
 By transfusion of fresh blood products from an infected person (although organisms do not survive
>48 hours under typical bloodbank storage conditions)
 By accidental needle stick
 When infectious specimens are handled in the laboratory
Venereal Syphilis: Pathogenesis
 Penetrates an intact mucous membrane or gains access to tissue through abraded skin
 Multiplies at the inoculation site
 Enters the lymphatic and circulatory system and spread throughout the body
Congenital Syphilis: Pathogenesis
 Forms:
 An early of infantile form (<2 years of age)
- Necrotizing funisitis
- Diffuse rash with sloughing of skin
- Osteochondritis
- Periostitis
- Diffuse fibrosis of liver and lung
- Asymptomatic
  Late from following a period of latency (few years to several decades)
- TRIAD: Interstitial keratitis, Hutchinson’s Teeth, 8th nerve deafness
- Periostitis
- Saber shins
- Saddle deformity of nose

 Transplacental passage of spirochetes can occur during any trimester

 Congenital acquisition during early gestational age results in stillbirth more often than acquisition
during late gestation or parturition
 More frequent during the primary, secondary, and early latent periods of maternal infection

Stages of Syphilis

I. Primary
 Chancre
 Occurs at a median of 3 weeks post inoculation
 Primary lesion may not develop in all patients
 Painless: the lesion may go unnoticed
 Ulcerated with raised, firm edges and a smooth base
 Serum tests usually give (+) results between 1st and 3rd weeks after
appearance of the chancre
 Reagin titer increases during the first 4 weeks then remains stationary for 6
months
 Typically solitary, though multiple chancres can occurs up to 1/3 of patients
 Regional Lymphadenopathy
 Moderately enlarged
 Rubbery
 Non suppurative
II. Secondary
 Rash 90%
 Begins on the trunk and extremities (although any body surface can be involved)
 Small macules -> papules -> pustules (weeks)
 Characteristic rash on the palms and soles
 Condylomata lata
 Enlargement and condolescence of papules produce the pale plaques
 Mucous patches: 1/3
 Generalized lymphadenopathy: 90%
 CNS Symptoms 40% esp with HIV coinfection
 Headache
 Uveitis
 Sensorineural hearing loss

III. Latent Syphilis

 Patient has only serological evidence of syphilis without any clinical evidence
 Characterized by lack of symptoms
 No symptoms may appear for months or years
 Syphilis is still alive in the body
 Bacteria starts to damage internal organs
 Damage can go unnoticed until the next stage
IV. Tertiary Syphilis
 It manifests 3-10 years after the primary stage
 1/3 of untreated patients proceed to tertiary syphilis after 10-25 years

 Cardiovascular syphilis
 Results of weakening of the tunica media
 Aneurysm of the ascending aorta
 Aortic valve insufficiency
 Narrowing of the coronary artery ostia

 Syphilitic gummas
 Central area of coagulative necrosis
 Epitheloid macrophages
 Occasional giant cells
 Peripheral fibrous tissue

 Meningovascular syphilis
 to multiple small infarcts due to endarteritis in CNS
- Seizures
- Strokes
- Aphasia
 General paralysis (tabes dorsalis)
 Cardiovascular system (aortic aneurysm)

 Parenchymatous neurosyphilis
 Loss of neurons and myelinated tracts
 General paresias
 Tabes dorsalis: demyelination in dorsal columns of spinal cord
 Weakness
 Diminished reflexes
 Paresthesias/ hypothesias
 Locomotor ataxia
 Joint degeneration
Laboratory Diagnosis
Specimen:
 Lesions- mucosal, epidermal
 Tissues
 CSF
 Serum
 Plasma
Types of Serological Reactions
1. Antigen Detection Test
 Darkfield examination
 Culture/ isolation (Rabbit infectivity testing)

2. Antibody Detection Test: Serological

 Screening: (reactive, nonreactive)
 Microscopic: VDRL, USR
 Macroscopic: RPR, TRUST

 Confirmatory (positive, negative)

 TPPA/TPHA
 FTA-ABS

 Non treponemal tests

 Uses cardiolipin antigen to detect autoantibodies
 Autoantibodies are the regain-like antibodies
 Produced by tissue damage
 False positive are also present in other treponemal infections, pregnancy, acute malaria,
leprosy, TB, leptospirosis, cancer, IM, aging, LE, RA, narcotic addictions and malignancy
 Principle: flocculation
 Reporting: Reactive or nonreactive

 Darkfield Microscopy
 Definitive Method: detect T. pallidum in lesion exudate or tissue
 When direct sampling of lesions is possible (chancres, mucous patches or condyloma lata)
 The specimen must be examined immediately as visualization of motility is necessary for
definitive identification
 Can be several weeks before a positive serologic test
 Sensitivity of 80% in diagnosing syphilis

 Fluorescein Isothiocyanate (FITC)-labeled antibodies

 Definitive Method: detect T. pallidum in lesion exudate or tissue
 Can be used for direct detection of treponemes in lesions, obviating the need to observe
motility of the bacteria
 Tissue fluid that has been dried and fixed to the slide (direct fluorescent antibody DFA-
TP)
 Paraffin-embedded tissue sections (direct fluorescent antibody tissue test DFAT-TP
  When used on fluid fresh lesions, both DFA-TP and DFAT-TP have a sensitivity of
approximately 100%

 Immunohistochemical Microscopy
 Definitive Method: detect T.pallidum in lesion exudate or tissue
 Anti treponemal antibodies bound to spirochetes in tissue sections are detected after a
series of immunologic reactions
 Allows for easy detection of rare bacteria and affords the capability for histologic
localization
 Procedure reportedly offers improvement in sensitivity and specificity over silver
impregnation stains
Tests for Syphilis Infection

 Treponemal Tests
 Confirm a positive non treponemal screening test
 Confirm infection in the face of a negative non treponemal test in late or latent disease stages (up to
30% of patients with tertiary syphilis)
 Most patients who have reactive treponemal tests will have reactive test for the remainder of their
lives, regardless of treatment or disease activity
 15-25% of patients treated during the primary stage revert to being serologically nonreactive after 2-
3 years
 Treponemal test antibody titers should not be used to assess treatment response

 Congenital Syphilis Screening

 During pregnancy (at least 1x)
 Antepartum maternal serum
 In high prevalence areas or among women with potential risk factors, repeat testing may be indicated
in later trimesters

 Confirmation of congenital syphilis

 A combination of clinical and radiologic examinations
 Placental analysis
 Testing of neonatal serum
 Semiquantitatively using non treponemal tests
 Compared with maternal serologic titer to determine the source of the antibody (ie. To
exclude transplacentally derived maternal antibody)

 Neurosyphilis
 No single test can be used to diagnose neurosyphilis in all instances
 (+) serum treponemal test
 (+) VDRL on CSF: standard test; low sensitivity, high specificity
 (-) 22-69% of patients with active neurosyphilis
 10% sensitivity in inactive neurosyphilis
 CSF FTA-Abs-high sensitivity, low specificity
 CSF abnormal lab values
 Pleocytosis
 Elevated Protein
 Non Treponemal Test
 Qualitative Test
 Used to determine the presence of regain
 Quantitative Test
 Used to determine amount of regain for monitoring of treatment

 Components of the stock antigen (VDRL)

 Cardiolipin: serves as the antigen
 Lecithin: neutralizes anticomplementary properties of cardiolipin; enhances sensitivity of the
reaction
 Cholesterol: increases the effective reactive surface and complement fixing capacity of cardiolipin
with reagin

 Rapid Plasma Reagin

 Constituents of antigen
 Original VDRL antigen
 Disodium salt of EDTA: enhances the ability of the suspension by inhibiting the oxidation
or peroxidation of lipids
 Charcoal: acts as the visualizing agent
 Phosphates: buffers the suspension
 Thimerosal: acts as the preservative
 Choline Chloride: serves as chemical inactivator of inhibitors present in serum or plasma,
this eliminates the need for heating
 Procedure
 Add 1 drop of sample (50 ul)
 Add 1 drop of antigen (17 ul)
 Rotate card for 8 minutes at 100 rpm
 Reactive: characteristic clumping ranging from marked and intense (reactive) to slight but
definite (minimally to moderate) reactive
 Nonreactive: slight roughness or no clumping

 TP-HA/ TP-PA
 Principle: Agglutination. Uses Treponema pallidum as Ag and are based on the detection of Abs
directed against cellular components
 (+): Agglutination
 Gelatin particle carriers sensitized with inactivated treponemal antigens are agglutinated by the
presence Treponema pallidum of antibodies in the human serum/plasma
 Materials
 U shaped micro-well plate
 Micropipette
 Pipette tips
 Gloves
 Paper towels
 Timer
 Allow the reagents 30 minutes in room temperature before use
 Thaw the serum or plasma samples
 Vortex samples
  Wash the plate with distilled H2O
 Then tap the plate dry on a paper towel

 Sources of Error
 Use of microtitration trays other than those recommended
 Vibration during incubation
 Lint and dirt may interfere with settling pattern
 Outdated, improperly stored reagents
 Plate tapped too vigorously during mixing
 Samples containing excessive bilirubin may give erroneous results
Typhoid Fever/ Enteric Fever
 Salmonellosis
 General classification of disorder caused by Salmonella Spp.
 Intestinal pathogens
 Salmonella typhi
 Salmonella enteritidis

 Salmonella typhi
 3 antigenic molecules: serotyping
 H (Hauch) Antigen
- Protein
- Found in the cell wall and flagella
- “flagellar antigen”

 K (Kapsel) Antigen or Vi Antigen

- K (Kapsel) Antigen
 2nd somatic antigen
- Vi Antigen
 Capsular antigen
 Associated with the microorganism virulence
 Inhibit bactericidal effects

 O (Ohne) Antigen
- 1st somatic antigen
- Thermostable antigen
- Lipopolysaccharide in nature
- Endotoxin

 Pathology:
 Mode of transmission: fecal-oral route
 Commonly found in the urine or feces of infected peson
 Colonized intestinal tract
 Attaches to M cells
 Undergo endocytosis
 Migrate to lamina propia
 From lamina propia it will go to different lymph nodes to the lymphatic duct
 Enters to the circulation
  Signs and Symptoms:
 Gastroenteritis
o Nausea
o Vomiting
o Diarrhea
 Prolonged fever and headache
 Faint rush in the abdomen and chest
 Hemorrhage: severe
o From the destruction of intestinal wall in which organism is initially attached

IMMUNE RESPONSE

1. Phagocytosis with the help of opsonins

2. Activate the T cells (CD4)
 Secret cytokines (IF-γ)
 2 functions:
o Production of reactive oxygen intermediate
o Expression of nitric oxide synthase
 Enzyme
 Induced production of nitric oxide
 Toxic to Salmonella: nitric oxide

INVASION MECHANISM

 SpiC Protein
- Produced by Salmonella typhi
- Inhibits fusion of lysosome with phagosome
- Inhibit the formation of reactive oxygen

VACCINES

1. Inactivated whole cell vaccine

2. Introduction of capsular antigen (Vi Ag)
- It initiate antibody production
- Protective against the Vi Ag

DIAGNOSIS

1. CULTURE
 Standard technique
 Detect antigen

2. SEROLOGIC TESTS
 To detect antibody production
o IgG: past infection
o IgM: acute infection
 4-fold rise between the acute and convalescence phase of IgG
o Active infection
 Tests:
a) Widal Test
 Significant titer
 Vaccinated: 1:160
 Unvaccinated: 1:80
b) Typhidot (IgM, IgG)
 Typhidot IgM
c) Counter immunoelectrophoresis
d) Dipstick method
e) Other enzyme immunoassay
Streptococcal Infection
 Genus Streptococcus
 Classification:
1) Ability to lysed cell
 β- hemolytic
 α-hemolytic
 non-hemolytic

2) Landsfied classification
 C carbohydrate: cell wall (inner)
 A-H, K-V2
 Pathogenic strains:
 A, B, C, D, F, G

3) Expression of M or T protein
 Component of the cell wall (outer)

MANIFESTATIONS:

 Streptococcus pyogenes
o Virulence factor: M Protein
 Inhibit phagocytosis
 Diminished complement activation

DISEASES:

1. Pharngitis (Strepthroat)
- Slight fever
- Sore throat
- Enlargement of lymph nodes
- Untreated, it may lead to:
o Rheumatic Fever
o Upper Respiratory tract infection

2. Necrotizing fasciitis
- caused by Group A β-Hemolytic Streptococcus/ flesh eating bacteria
- associated with the production of SPE’s (Streptococcal Pyogenic Exotoxis)
- Fascia and fats of infected individual is progressively being destroyed

3. Pyoderma/ Impetigo
- Vascular lesions on the extremities
- Common among children
 4. Acute Rheumatic Fever
- Sequelae of pharyngitis/ tonsillitis
- Experience joint inflammation
- Inflammation of the heart
- Sydenham’s chorea
o Involuntary jerking movement of the arms, legs and faces due to brain tissue
inflammation
- Considered as autoimmune response
- Antibody may cross-react with the heart tissue
o M protein will have 3 similar epitope antigen in the heart
- Management: in continuous uptake of antibiotics

5. Post-Streptococcal glomerulonephritis
- Sequelae of pharyngitis/ tonsillitis
- Formation of immune complexes
- Impaired renal function

IMMUNE RESPONSE

(1) Microorganism deposited in the lamina propia and will activate the:
a. Macrophages
b. Dendritic cells
c. B and T cells

(2) Phagocytosis
 Streptococcus pyogenes has M protein and expressese hyaluronic acid → inhibit opsonin
mediated phagocytosis

(3) Inactivation of C5a

 C5a: chemotaxin; induced degranulation
 due to the presence of C5a peptidase exhibited by Streptococcus pyogenes

(4) Anti-toxin antibody production

(5) Production of secretory IgA
 Secretory IgA: prevent attachment of Streptococcus pyogenes to mucosal surfaces

Other diseases:

 Endocarditis
 Scarlet fever
 Meningitis
 Arthritis
 Streptococcus toxic shock syndrome (STSS)
LABORATORY DIAGNOSIS

I. Determination of antigen
A. Antibody or nucleic Acid Probe
B. Optical Immunoassay
 SILAS (Silicon Assay Surface Technology)
C. Antibody coated liposome
D. Genetic Probe
 GASD (Group A Streptococcal Direct)
E. EIA, Agglutination Test

II. Antibody determination (serum)

A. ASTO (Anti-DNAse)
B. Anti-hyaluronidase (Streptozyme)

III. Others:
A. Dick’s Test
 Susceptibility to scarlet fever

B. Schultz-Charlton Test.
 Diagnostic test to scarlet fever
Ricketsial Infection
 DISEASE: Rocky Mountain Spotted Fever (RMSF)

 Causative agent: Riketsia ricketsii

o Intracellular organism
o Cell wall: contain LPS and Protein
 Highly antigenic
o Cell membrane: romp A
 For cell adhesion

 MODE OF TRANSMISSION
o Tick from rodent or foxes

 MANIFESTATION
o TICK BITE
 Organism found in the saliva of the tick
 Attached to the vascular endothelial cell surface → ENDOCYTOSIS
 Escapes the vacuoles with the help of an enzyme and enter to cytosol
 Begin to multiply
 Start to adjacent cell with Filopodia → thin cell protrusion of the microorganism
 Enter the blood circulation

 Incubation Period: 1 week

IMMUNE RESPONSE

1. Cytokine Production
 IF-γ (Interferon- γ)
 Enhances phagocytosis
 Stimulate production of nitric acid synthase

2. Activation of CD
 Enhances antigen presentation to MHC

3. Antibody Production
 Only occur once the microorganism is expressed by APC
 IgG in nature
 Enhance opsonization

INVASION MECHANISM
 Capable of inhibiting phagocytosis
 Block apoptosis
LABORATORY DIAGNOSIS

 Based on history and clinical infection

o CULTURE
 Only done in reference laboratory
o SEROLOGIC TESTING
 Direct immunofluorescence with Low Sensitivity
 Enzyme immunoassay
 Agglutination
 CFT
 Well-felix test
Mycoplasma
 DISEASE: atypical pneumonia or trachobronchitis

 CAUSATIVE AGENT: Mycoplasma pneumonia

o Extracellular bacterium
o Has triple layered membrane instead of cell wall

CLINICAL MANIFESTATION
 Common cause of pneumonia of 5-35 years old
 Contagious
 Mode of transmission: Respiratory Secretions
 Signs and Symptoms:
 Fever
 Malaise
 Headache

POSSIBLE COMPLICATION
 IgA nephropathy
 Meningoencephalitis
 Anemia
 Myocarditis
 Cold Agglutinins Syndrome
o Patient have a higher titer that may lead to intravascular agglutination
o IgM antibody reacts with the glycoprotein opf RBC below 37οC (0-4 οC: Optimal reaction)

MODE OF ENTRY
 Through the mucosalmembrane
 Attached to glycoprotein of aviated bronchial epithelial cell
 Produced hydrogen peroxide → destructive to the cell and initiate inflammatory reaction

IMMUNE RESPONSE
1. Activation of B cell, CD4 and complement
2. There are more susceptible to complement activity

 Susceptibility
 Immunocompromise state
 Among AIDS patient
 Possibility of autoreactive responses
LABORATORY DIAGNOSIS
 Detection of Antigen
o Culture
o Immunoassay
o Molecular Testing (PCR)
o Counterelectrophoresis
o Immunoblotting
o Direct Immunofluorescence assay

 Does not differentiate acute and chronic infection

 Serum
o CFT
o Indirect Fluorescence Antibody Test
 Cold agglutinin antibody
 Titer: 1:3
o ELISA
Chlamydia
 DISEASE: Genital Tract Infection (STD)
 CAUSATIVE AGENT: Chlamydia trachomatis
o Intracellular bacterium
 2 morphological form:
1. Elementary Body
 Extracellular in nature
2. Reticulate
 Intracellular form
 Form of multiplication

CLIICAL MANIFESTATION

 Sexually transmitted disease (STD)

 Infected mother to infant after birth
 Asymptomatic
 Some signs and symptoms:
- Male:
o Urethritis
o Epididymitis
o Infertility
- Women:
o Cervicitis
o PID (Pelvic Inflammatory Disease)
o Urethral syndrome
- Trachoma: commonly affect the eyes

MODE OF TRANSMISSION

1. Entry of elementary body through the columnar cell with the used of its adhesion molecule
(MOMP)- Major outer membrane protein
2. Endocytosis
3. Reticulate form and multiply

IMMUNE RESPONSE

1. Production of antibody (IgG and IgM)

 IgG: facilitate phagocytosis and activate complement
 IgM: activate complement
2. Release of IF-γ
 Enhances phagocytosis
 Stimulate production of nitric acid synthase
LABORATORY DIAGNOSIS:

1. Culture
2. Direct Microscopy
3. Screening:
a. Serologic Test:
i. Optical immunoassay
ii. EIA
iii. Genetic Probe Assay
iv. Direct immunofluorescence Antibody Assay
v. Immunochromatographic Assay
vi. Nucleic Acid Amplification
vii. Hybridization Protection Assay
Meningococcal Diseases

 Causative agent: Neisseria meningitides

 With 13 serogroups
o Based on immunologic specificity of capsular polysaccharides
 A, B, C, T, W-135
 A and C: EPIDEMICS

VIRULENT FACTOR

 Lipopolysaccharide component

PATHOGENESIS

 Nasopharynx
 Form of the transitional flora and so signs and symptoms
 If enters to bloodstream, it caused bacteremia and upper respiratory tract infection
 From bacteremia it may reach to the brain and may cause meningococcemia

 “Waterhouse Friedrichsen Syndrome”

o Group of signs and symptoms
o High fever
o Hemorrhagic rash
o DIC
o Circulatory collapse
 From meningococcemia, it may lead to meningitis
o Common cause of meningococcemia
o Intense headache
o Vomiting
o Stiffness
o Coma

IMMUNE RESPONSE

1. Phagocytosis
2. Complement activation
 Dependent on antibody production

INVASION MECHANISM

 BLOCK IgA antibody production

VACCINES

 Using capsular polysacchariude of A, B,C, Y, W-135

o T cell activation

CONTRIBUTORY FACTOR

 Decrease antibody production

 Complement deficiency
o C5, C6 and C8

LABORATORY DIAGNOSIS

1. Culture
 Blood or CSF
2. Serological Testing
 Latex agglutination
 Hemagglutination tests
- Detect of antibody
Haemophilus Infection
 Causative agent:
o Haemophilus influenza
o Haemophilus ducreyi

 Responsible of meningitis in children

 Respiratory tract infection in children and adult

Haemophilus ducreyi Haemophilus influenza

 STD (Sexually Transmitted Disease)  Found in the mucous membrane of upper

 Responsible for the formation of chancroid respiratory tract
 Contain capsular polysaccharide
- Six (6) types:
o A, B, C, D, E, F
o Act as endotoxins
VIRULENCE FACTOR

 Capsule
o Inhibit phagocytosis

CLINICAL MANIFESTATION

 In the respiratory tract, enters to the bloodstream

 In meninges, it caused meningitis
 In the joint, it caused septic arthritis
 Other diseases:
o Obstructive laryngotracheitis
 Commonly seen in infants
 Epiglotis is inflamed
o Upper respiratory tract infection
 Older children and older age
o Pneumonitis
 Common disease

IMMUNE RESPONSE

1. Antibody production
2. Complement activation
3. Mediated phagocytosis

LABORATORY DIAGNOSIS

 Sample: Nasopharyngeal swab, blood or CSF

 Culture
 Serologic test: detect antigen
o Sample: CSF
 HEPATITIS VIRUS

Characteristics of Common Causative Agents of Acute Viral Hepatitis

Massive
Name of Viral Incubation Carrier Chronic
Classification Transmission Period
Prophylaxis Hepatic
Virus genome State Hepatitis
Necrosis
Hygiene,
immune
Hepatitis
Picornavirus ssRNA Fecal-Oral 15-45 days No No serum Rare
A
globulin,
vaccine
Hygiene,
Parenteral, Hep B
Hepatitis 45-160
Hepadnavirus dsDNA Sexual, Yes Yes immune Uncommon
B days
Perinatal globulin,
vaccine
Hygiene,
Parenteral,
Hepatitis 15-150 immune
Flavivirus ssRNA Sexual, Yes Yes Rare, if ever
C days serum
perinatal
globulin
Parental,
sexual, Hygiene,
Hepatitis perinatal, prevention
Deltavirus -ssRNA 30-60 days Yes Yes Yes
D **HBV of HBV
infection infection
required
Hepatitis Hygiene,
Hepeviridae ssRNA Fecal Oral 15-60 days No No No
E sanitation
Hepatitis A Virus (HAV)
 Member of the family Picornaviridae
 Mode of transmission: Fecal-Oral route
 Incubation Period: 28 days

 Hepatitis A Virus Antibodies

 IgM anti-HAV
o Marker: Acute Hepatitis
o Peak during 1st month of illness and decline to undetectable levels within 6 to 12
months
 IgG anti-HAV
o Produced as a result of natural infection or immunization
o Indication: immunity to HAV

VIRAL MARKERS
 HAV RNA
 Present in stool, plasma, from the incubation period to an average of 18 days after the clinical onset
of the disease
 Anti HAV IgM
 Indicates recent infection
 First to appear
 Develops in about 2-3 weeks after infection persisting for about 3-6 months after infection
 Anti HAV IgG
 Develops 1-2 weeks after IgM present for life (not helpful clinically)
Hepatitis B Virus (HBV)
 Virion: entire virus particle
 Dane particle: hepatitis B virion that causes infection
 Australia Antigen: original term in the HBsAg
 Hepadna: partially double stranded DNA

 DNA polymerase with a reverse transcriptase activity

o Enzyme
 Capsid + genome → Nucleocapsid
 Envelope
o Most susceptible by inactivation by environmental changes
o Example: bleach or hypochloride

 Unnaked virus
 Resistance to environmental changes
 Example: HAV
 Cause enteric disease
 MOT: Fecal-oral route
FORMS
1. Spherical
 most common form
2. Filamentous
3. Dane
 Least common form

CHARACTERISTICS
 Hepadna virus family
 Incubation: 60- 90 days
 (partially) double stranded DNA virus
 With four (4) overlapping genes that encode:
o C: nucleocapsid
o S: Envelope
o P: Polymerase with reverse transcriptase activity
o X: X proteins
 Highly viremia and infectivity
 Integrates into host genome
 Important role of host immune response in hepatocellular injury

HBV Genome
Frames Encodes Proteins
S Envelope Pre-S1, Pre-S2 and HBsAg
Pre-core precursor, HBcAg,
C Core
and HBeAg
P Polymerase HBV DNA Polymerase
X X Protein X Protein 1 and X Protein 2
MODE OF TRANSMISSION

 HBV is transmitted via contact with blood or body fluids

o 50- 100 times as infectious as HIV
o 10 times as infectious as Hepatitis C
 Other MOT:
o Perinatal/ vertical
 HBV (+) mother
 Chronic hepatitis B
 HBsAg > 6 months
o Unsafe injection and transfusion
o Sexual contact
o Organ transplantation
o Sharing of razors and toothbrushes (contaminated with blood)

OUTCOMES OF HBV INFECTION

1. Acute infection
 About 90% of healthy adults who are infected with HBV will recover and completely rid of the
virus within 6 months
 Lasts for only < 6 months

2. Chronic infection
 10% may developed
 Puts individuals at high risk of death from cirrhosis of the liver and liver cancer
 Most at risk:
o Age-dependent: young children who become infected with HBV is more likely developed
chronic infection
o 90% of infants infected during the 1st year of life
o 30-50% of children between 1-4 years
o 25% of adults who become chronically infected during childhood die from HBV-related
liver cancer and cirrhosis
 Last for more than 6 months and beyond
 Some become CARRIER

3. Fulminant
 1% may developed
 Very progressive type of infection
 Highly symptomatic
 May or may not cause cirrhosis
 Risk: age-dependent
VIRAL REPLICATION

I) Attachment
 Adsorption
 Specific-receptor
II) Penetration
 Viruses enters the host cell
III) Uncoating
 Capsid release the genome and enters to the nucleus or cytoplasm
IV) Macromolecular synthesis
 Translation and transcription occurs
V) Viral replication
 Activation of the virus
VI) Release
 To infect another host cells
 Lysis of the host cells
 Budding

HBV LIFE CYCLE

 Convalently closed circular DNA

o Serves as a stable template for transcription of viral mRNA
o Remains in the nucleus during chronic infection and may persist in the liver fro the lifetime
of the patient

i. Hepatocyte
 Envelope removed
ii. Nucleus of the hepatocyte
 Double DNA stranded it would repair to cccDNA →serves as a template for the
transcription of viral RNA
 Important in viral replication
 The virus only stay on the nucleus of the hepatocyte and multiply → carrier state
iii. HBV protein
 Expressed as transcriptional transactivator
 Mutations in the core promoter region increased viral replication and thus the risk of
hepatocellular carcinoma
 Wherein it subdivided and progresses to become packaging
SEROLOGICAL MARKERS

 HBV ANTIGEN
(1) HBsAg (Hepatitits B surface Antigen)
 Spheres and filamentous
 Australia Antigen
 Present: acute or chronic infection
 1st marker to appear in the bloodstream during acute infection
 major component of the envelope of an hepatitis B virion
 important marker for screening blood donors

(2) HBcAg (Hepatitits B core Antigen)

 Found only in the liver
 An intracellular hepatocytes
 Not detected in the bloodstream because of viral envelope that masks it
 Seen in the nucleocapsid
 Detected only through biopsy of infected liver

(3) HBeAg (Hepatitits B envelope Antigen)

 Appear in acute infection, soon after HBsAg would appear
 Highly infectious
 Correlate with high degree of therapy HBeAg to anti-HBe
 Circulatory form of HBcAg
 Present during periods of active replication of virus

 HBV ANTIBODY
(1) ANTI-HBs
 Persists for years and provide protective immunity
 Indicate recovery
 Levels of anti-HBs would decline
 10 Miu/ mL normal level
 Also produced after immunization with the Hepatitis B vaccines

(2) ANTI-HBc IgM

 1st to appear as host response to infection
 Only marker found in “core window” period

(3) ANTI-HBc Total (IgG)

 Persist with resolved infection and chronic infection
 No detectable
 Absent in vaccinated individuals

(4) HBV DNA

 Appear early in acute infection
 Most specific indication of the virus
 Indicates ctive replication
 Detect: Polymerase Chain Reaction (PCR)
 More accurate than HBeAg
 (5) ANTI-HBe
 Produced after the immune system has cleared most HBeAg
 Marker of convalescence and favorable prognosis
 Indicates that the patient is recovering from HBV infection

HBsAg Anti-HBc Anti-HBs IGM anti-HBc Interpretation

- -
+ vaccination
- -
(>10 mIU/mL)
+ +
RESULTS
+ +
- +
(+) : positive ; (-) : negative
- Susceptible
 Immunity to
 Immune caused pass
infection
 Infected
- +
 Acute infection
 Infected
- -
 Chronic infection
 False result
 HBcAg is very low
and not detected
-
 The patient is
recovering from acute
infection

LABORATORY DIAGNOSIS

 Screening
o Rapid Test
o ELISA
o CLE-IA
 Confirmatory
o Neutralizing assay
 Monitoring test
o PCR
o Branched-DNA assay

ALGORITHM

Nonreactive Inconclusive Reactive

Repeat Test
Nonreactive Inconclusive Reactive
Confirmatory Test
Report test
↓
(reactive/nonreactive) Report test
Neutralizing assay
(reactive/nonreactive)
Report test
(positive/ negative)
Hepatitis B
COURSE OF HBV INFECTION
 Following the infection with HBV the immune system attempts to clear virus
 Acute HBV infection
 HBV infection may be successfully cleared by the immune system during acute phase
 Begins with an immune tolerance phase
 Typically lasts for 45-160 days
 The next phase is immune clearance
 Between 90-95% of acute sufferers make a full recovery without medical intervention
 Acute infection progresses to chronic infection
 Immune system fails to clear HBV within 6 months
 Between 5-10% of adults, 50% of children and 90-95% of neonates develop persistent chronic
infection
 Elevation of serum ALT levels
 Between 15-40% of all individuals chronically-infected with HBV develop progressive liver disease

SEROLOGIC MARKERS
 HBsAg
 General marker of infection
 First serologic marker to appear
 Persistent for >6 months = chronic infection
 HBeAg
 Indicates active replication of virus
 Absent in precore mutant HBV infection
 Anti-HBs
 Recovery and/or immunity to HBV
 Detectable after immunity conferred by HBV vaccination
 Occasionally seen in chronic carriers
 Anti-HBe
 Indicates virus no longer replicating
 Or precore mutation
 Anti-HBc
 Evidence of a past or present HBV infection
 Anti-HBc IgM
 Usually detectable for about 6 mos
 Plus HBsAg (+) indicates acute infection
 Anti-HBc IgG
 Past infection
 Usually persists for life after infection
 Absent in vaccinated individuals
 HBcAg
 Produced by infected liver cells during replication
 Not found in bloodstream at anytime
VIROLOGIC MARKER
 HBV DNA
 Measure of the level of viral replication
 Historically measured using insensitive hybridization assays
 PCR is the current standard

BIOCHEMICAL MARKER
 Serum ALT
 An important defining point in chronic HBV
 Elevated ALT levels are an indicator of necroinflammatory activity
HISTOLOGIC MARKER
 Liver biopsy
 More sensitive and accurate than ALT as an indicator of liver disease
 Establish baseline disease prior to initiation of therapy
 Exclude other causes of liver disease
Hepatitis C Virus (HCV)
 No available vaccines against HCV
 Member of family Flaviviridae (Hepacivirus)
 Incubation period: 7 – 8 weeks

LIFE CYCLE

(i) Cytoplasmic release and uncoating

(ii) IRES (internal ribosome entry site) – mediated translation and polyprotein processing
(iii) RNA replication
(iv) Packaging and assembly
(v) Virion maturation and release

Two (2) CLASSES OF TEST

1. Detection of anti-HCV
 Screening test
2. Molecular tests
 Used to detect viral load

 Confirmatory test
o SIA (Strip Immunoblot Assay)
o NAT (Nucleic Acid test)
o HCV RNA Qualitative
 Screening
o EIA
o Rapid Test
 Monitoring
o HCV RNA Quantitative
 Other test:
o Genotyping

POST-EXPOSUSRE PROPHYLAXIS

 Hepatitits B Immunoglobulin
o Injected immediately up to 1 week exposure
o Applicable in hospital setting and expose
 Example: needle prick

Mother (Hepatitis B (+))

 Delivery: CS
 Hepatitis B Vaccine: 1st vaccine
Hepatitis C
AVAILABLE TESTS
 Screening
 EIA
 Rapid test

 Confirmatory/ Supplemental
 SIA (RIBA, LIA)
 HCV RNA (Qualitative)

 Monitoring
 HCV RNA (quantitative)

 Other tests
 Genotyping
SUPPLEMENTAL/ CONFIRMATORY ASSAYS

 To establish “true positivity” of anti-HCV EIAs

 Higher specificity; lower sensitivity
 RIBA, LIA
 Higher specificity; lower sensitivity
 Positive, negative, indeterminate
 Not a true indicator of active infection
 HCV-RNA
 To discriminate EIA +; Immunoblot -/ indeterminate
 To detect presence or absence of the virus
 Anti-HCV IgM Assay
 Acute infection (50-93%)
 Chronic (50-70%)
Hepatitis D Virus (HDV)
 Unclassified, single stranded RNA virus
 Parenterally transmitted infection that can occur in the presence of HBV
 Infection with the two viruses occur simultaneously, as a coinfection or sequentially as superinfection in
chronic HCV carriers
 Routinely indicated by the presence of anti-HDV in the patient’s serum, which is most often detected by
ELISA (IgM Anti-HDV)
 Family deltaviridae

Hepatitis E Virus (HEV)

 Member of the family Caliviridae
 Usually present as an acute
 Self-limiting hepatitis without progression to chronic carrier state
 High mortality rate: among pregnant women

HEV ANTIBODIES

 IgM anti-HEV
o Present during the acute infection
o Rapidly declines in the early recovery period
o Assays:
 ELISA
 Western Blot
 Fluorescent Antibody Blocking

HEV RNA

 Detected in the feces of the patient’s

o About 2 week after the onset of illness
 Identified by means of PCR (Polymerase Chain Reaction)
Human Immunodeficiency Virus (HIV)
Acquired Immunodeficiency Syndrome (AIDS)

 Causative agent of AIDS

 Retrovirus belonging to lentivirus
 Positively RNA virus
 Preventable, manageable BUT NOT CURABLE

TWO (2) TYPES

1. TYPE-1
 Causative agent in Europe and USA
 Formerly called
o Human Tcell lymphocyte Virus-Type III (HTLV-III)
o Lymphadenopathy-Associated Virus (LAV)
o AIDS associated retrovirus (ARV)

2. TYPE-2
 Causative agent in West Africa
 Less pathogenic and has a lower rate of transmission

STRUCTURE
(i) Outer envelope
 Contain phospholipid bilayer
(ii) Core shell protein
(iii) Inner core
 Cone-shaped
 Contain 2 identical strands of RNA

SURFACE:
 Membrane Proteins
o Serological Markers
 p24
 core protein
 large glycoprotein
 contain 2 protein:
o gp41
 found in the transverse area of phospholipid bilayer
o gp120
 extend beyond the surface to form a knob
ENZYMES
1. Reverse transcriptase
 Unables the virus to convert RA to DNA
 Has two catalytic domains:
o Ribonuclease active asite
o Polymerase active site

2. Integrase
 Insert the viral DNA to the host DNA

3. Protease
 Responsible for cleaving other enzymes and structural proteins in the formation of a new virion

DIFFERENTIATION OF HIV FROM AIDS

HIV AIDS
Successful entry of the virus in the body Terminal stage of HIV infection
NO signs and symptoms WITH signs and symptoms
Presence of opportunistic infection

MODE OF TRANSMISSION
1. Infected semen and vaginal secretion which may be contacted through sex whether it is oral, anal, or vaginal
2. Infected blood and/or blood products
a. Sharing of contaminated needles
b. Blood transfusion
c. Organ transplantation
3. Perinatal
a. Placental entry
b. During delivery
c. During breast feeding

AT RISK
 In the Philippines: through sexual contact
 Worldwide
o Risky behavior
o Multiple partners
o Unprotected sex
 Sex workers
 MSM (Man Sex with Man)
CLINICAL DISEASE
1. Primary Stage
 Generally asymptomatic
 Might also develop acute illness
o Fever
o Night sweat
o Myalgia
o Arthralgia
o Persistent Generalized Lymphadenopathy
o Anorexia
o Muscular rash
o Photophobia
 Antibody already be detected from 2 weeks to 3 months after initial infection
 Antibody: antibody against gp120

2. Intermediate Stage/ AIDS related complex

 Diagnosis: 2 of the clinical manifestations plus 2 laboratory abnormal results
 Possible manifestations:
o Lymphadenopathy for > 3 months
o Fever, > 100○F for > 3 months
o > 10% weight loss
o Persistent diarrhea
o Fatigue and night sweats
 Laboratory abnormalities:
o T4 Cell for <400/ mm2
o CD4:CD8 ratio is <1
o Patient shoul have:
 Leukopenia
 Thrombocytopenia
 Anemia
o Increase serum globulin
o Anergy to skin test
o Reduced blastogenesis
o Positive HIV antibody test

3. Final stage/ AIDS Stage

 Presence if opportunistic cells and some cancer
 Most common
o Pneumocystis
o Candidiasis
 Most frequent malignancy
o Kaposis Sarcoma
 T4 level is <200/mm2
  LATENT PERIOD
 Seroconversion
 Patient is asymptomatic but potentially infectious

 WINDOW PERIOD
 Stage from the acquisition of the infection to the appearance of antibody production
 May or may not have a positive serological test
 Occurs 3 – 6 months

IMMUNOLOGIC FEATURE

1. Progressive diffuses of CD4

2. Reversal of CD4:CD8 ratio
 Normal ratio: 2: 1
 Abnormal ratio: .5:1

OPPORTUNISTIC INFECTIONS

1. Fungal infections
 Candidiasis
 Cryptococcus

2. Protozoan infection
 Pneumocystis infection
 Toxoplasmosis
 Cryptosporidium enteritis
o Caused of persistent diarrhea

3. Viral infection
 CMB
 HIS
o Herpes Complex 1 and 2
4. Bacterial infection
 Mycobacterium tuberculosis
 Mycobacterium avium intracellulare

5. Cancer
 Kaposis sarcoma
o Cancer of the small blood vessels
6. AIDS dementia complex
SEROLOGIC DIAGNOSIS
 Demonstration of Antibody either against HIV or to its components
 Components:
o p24
 1st detectable serologic marker
o P24Ag
 Most specific indicator together with P24Ab

 Other components:
o gp41
o gp120
o gp160
o P31

 Screening Tests
a) ELISA – standard screening test
b) Agglutination test
c) Radioactive/ Fluorochrome Dye test
d) Dot blot immunobinding

 Confirmatory Tests
a) Western Blot Assay
 Most sensitive and specific
b) Line immunoassay
c) Indirect immunofluorescence assay
d) Radioimmunoprecipitation test

 Special Tests
o Eliminate biological false positives
 HIV Ag Test
 HIV gene detection
HIV/ AIDS
EPIDEMIOLOGY
 Low and Slow: 1984-2006
 Fast and Furious: 2007-2012
 Hidden and Growing: 2013 onwards
WHAT IS HIV?
 Human Immunodeficiency Virus
 A unique type of virus (retrovirus)
 Invades helper T cells (CD4 cells) in the body of the host (defense
mechanism of a person)
 Threatening a global epidemic
 Preventable, manageable but not curable
HIV SEROLOGY
 Human retrovirus
 Belongs to a group of retroviruses called “Lentivirus” (RNA)
 Lymphadenopathy associated virus (LAV)
 Human T cell lymphotropic virus III (HTLV III)
 The first indication of this new syndrome came in 1981 in homosexual drug addict males in USA
 They had two things in common
 Pneumocystis pneumonia
 Kaposi’s Sarcoma
 Recently, its origin has been traced to Kinishasa in Congo
 Size: 100-200 nm in diameter
 3 parts
 Outer envelope: derived from the host cell membrane (phospholipid layer)
 Core shell of the protein
 Cone shaped inner core that contains 2 identical strands of RNA with associated reverse
transcriptase and core polypeptides
VIRAL CHARACTERISTICS

 Icosahedral
 enveloped virus of the lentivirus
 Subfamily: retroviruses
 Retroviruses transcribe RNA to DNA

Gag Env Pol

CA
Gp160 Protease
MA
Gp120 RNAse-H
NC
Gp41 Integrase
P6, P9, P17, P24
TWO TYPES

 HIV 1
 Most common in sub-Saharan Africa, US, Europe and throughout the world
 Can be divided into group: M,N,O
 The pandemic is dominated by group M
 HIV 2
 Most often found in West Central Africa, parts of Europe and India
 Causes a slower progression of disease
 Both produce the same patterns of illness. Both are RNA viruses with single strand of genetic
material
ENZYMES

 Reverse transcriptase
 This enzyme enables the virus to convert viral RNA to DNA
 Has two catalytic domains:
o Ribonuclease active site
o Polymerase active site
 Integrase
 Inserts viral DNA into host DNA
 Protease
 Cleaves other enzymes and structural proteins from their polyproteins
LIFE CYCLE
 Attachment (binding and fusion)
 Entry (Reverse transcriptase)
 Replication (integration)
 Biosynthesis (Transcription)
 Assembly
 Release (binding and attachment)
Pathogenesis
 HIV Disease
 Profound immunodeficiency progressive quantitative & qualitative deficiency of T helper cells
 Helper T cells, Inducer T cells
 CD4 molecule
 Cell surface, serves as a main receptor for HIV with the presence of co-receptors for fusion & entry
 CCR5, CXCR4
 Window Period: 3-6 months
 HIV Asymptomatic Stage: 1-15 or more years
 HIV Symptomatic and AIDS
STAGES OF HIV

Stage 1: Primary Stage 2: Clinically Stage 3: Stage IV:

Stage Asymptomatic Symptomatic HIV Progression from
Infection HIV to AIDS
This stage of infection Lasts for an average of 10 Immune system Illness occurs leading
lasts for a few weeks years becomes severely to more severe
and is often Free of major symptoms damaged manifestations
accompanied by a Body will have produced Immune system fails,
short flu like illness. enough Ab symptoms developed
HIV test may be HIV test is positive
negative
 Asymptomatic  Unintentional weight loss  Oral candidiasis  HIV wasting
 Persistent <10% of body weight in  Oral hairy Syndrome
Generalized the absence of concurrent leukoplakia with (weight loss
Lymphadenopathy illness other systemic >10% body
 Minor mucocutaneous features weight and either
manifestations (seborrheic  Vulvo-vaginal chronic fever or
dermatitis, Prurigo, fungal candidiasis with diarrhea in the
nail infections, recurrent other systemic absence of
oral ulcerations, angular infections concurrent
chellitis)  Unintentional illness)
 Herpes Zoster within the weight loss > 10%  Pneumocystis of
last 5 years in the absence of the brain
 Recurrent upper concurrent illness  Toxoplasmosis
respiratory tract infections  Chronic Diarrhea of the brain
>1 month  Cryptosporidiosis
 Prolonged fever with diarrhea > 1
(intermittent or month
constant) > 1  Isosporiasis with
month diarrhea > 1
 Active Pulmonary month
Tuberculosis  Cryptococcosis,
 PTB within past extra pulmonary
year  CMV of an organ
 Severe bacterial other than the
infections liver, spleen or
(pneumonia, lymph node
pyomyositis)  Herpes Simplex
Infection,
mucocutaneous
for >1 month or
visceral
CD4
 A type of T cell involved in protecting against viral, fungal, and protozoal infections
 These cells normally orchestrate the immune response, signaling other cells in the immune system to
perform their special functions
 Also known as T helper cells, HIV’s preferred targets
 Lab work is done at specific intervals to measure the number of CD4 in circulation

CD4 < 500 CD4 <200 CD4 <50

 Bacterial Infections
 Tuberculosis
 Herpes Simplex
 Herpes Zoster
 Vaginal
 Candidiasis
 Hairy leukoplakia
 Kaposi’s Sarcoma
 Pneumocystic carinii
 Toxoplasmosis
 Cryptococcosis
 Coccidiomycosis
 Cryptosporiosis
 Non Hodkin’s
Lymphoma
 Dessiminated-mycobacterium
avium complex (MAC) infection
 Histoplasmosis
 CMV retinitis
 CNS Lymphoma
 Progressive multifocal
leukoencephalopathy
 HIV dimentia

MODE OF TRANSMISSION
 Through Sex
 Mother to Baby
 Injecting drugs
 Working in healthcare
 Blood Transfusion & Tissue transplant
 Myths
 Shaking hands or hugging
 Using a toilet
 Using cutlery, glasses, dishes, bed linen, clothes
 Bites from mosquitos, dogs, cats, other animals
 Tears or sweat
 Kissing or saliva
 Eating from the same plate
HIV IN BODY FLUIDS ON AVERAGE IN 1ml OF FLUID
 Blood: 18,000
 Semen: 11,000
 Vaginal Fluid: 7,000
 Amniotic Fluid 4,000
 Saliva: 1
Acquired Immunodeficiency Syndrome (AIDS)

 HIV is the virus that causes AIDS

 Disease limits the body’s ability to fight infection due to markedly reduced helper T cells
 Patients have a very weak immune system (defense mechanism)
 Patients predisposed to multiple opportunistic infections leading to death
 Predisposed to Opportunistic infection and malignancy (eg. Pneumocystis pneumonia, CNS lymphoma)
 Persons with positive HIV serology who have ever had CD4 lymphocyte count below 200 cells/mcL or a
CD4 lymphocyte percentage below 14% are considered to have AIDS

HIV TESTING
 Antibody detection assay
 Screening
 Rapid test
 Particle agglutination (heme, latex)
 Immunochrotmatography test (lateral flow assay)
 Immunodot comb assay
 Confirmatory
 Western blot
 IFA
 RIA
 LIA
 Antigen detection Assay
 P 24 antigen capture test
 PCR
 Sources of Antigen Use
 Viral Lysate Antigen
 Recombinant Antigen
 Synthetic Peptide Antigen
 Viral identification assay
 Monitoring Assay
 Carrier Format for Ab or Ag
 Microtiter plate
 Bead
 Nitrocellular paper
 Gelatin particles
 Red cells
REPUBLIC ACT 8504

 Complete Title
o An act promulgating policies and prescribing measures for the prevention and control of HIV/ADIS
in the Philippines, Instituting a Nationwide HIV/AIDS Information and Educational Program,
Establishing a Comprehensive HIV/AIDS monitoring system, strengthening the Philippine Nationals
AIDS Council and for other purposes

 Short Title: Philippine AIDS Prevention and Control Act of 1998

Autoimmune Disorders
THEORIES that explains Self-sensitization
I. Hidden Antigen Theory
 There is a release of components that does not normally circulate
 Example: antigen of Thyroglobulin (only situated in thyroid gland)

II. Neoantigen Theory

 There is mutation in the genetic material which bring about new antigenic determinants on
the cell membrane

III. Cross-Reacting Antigen Theory

 Refer to the similarity between endogenous and exogenous antigen

IV. Mutation of Immunocompetent Cell

 Mutation of immunocompetent cells
 Either T or B cells

Two (2) types of Immune Diseases

i. Organ Specific Autoimmune Diseases

 Generally there is only one antigen creating unique to an organ
 Target organ are damage through humoral or cell-mediated mechanism
 Overstimulated the organ
 Block the organ’s function

ii. Non- organ or Systemic Autoimmune Diseases

 there is a number of antigens
 target multiple organs
 common caused: defect in immune regulation resulting to hyperactive T cells
 damage may be brought about by direct action of antibody or accumulation of
immune complexes

Primary
Autoimmune Ratio HLA-
Antigen Pathology General Feature
Diseases F/M Association
Initiated by
 Systemic and multi-organ
(Non-organ specific)
 IC are formed in serum
and trapped in basement
membrane of glomeruli,
Systemic Lupus HLA-DR2 Immune skin/endothelium,
10:1 Ds-DNA
Erythematosus HLA-DR3 Complexes (IC) synovia of joints, kidney
 Anti-Ds-DNA, anti-
leukocyte, antibodies
 Anti-phospholipid
antibody (also termed,
“Lupus anti-coagulant)
  Polyclonal B-cell
activation
 Etiology Unknown
 Mixture of organ specific
and systemic symptoms
 Joint movement; lung,
cardiac, skin, and CNS
pathology
 Excessive Type- 1
cytokines
Cell-mediated  Rheumatoid Factors- IgM
Rheumatoid HLA-DR1
3:1 and Immune (or IgG/IgA) to the Fc of
Arthritis HLA-DR4
Complexes IgG. These are not
essential for disease, may
enhance IC
 Some association with
Epstein Barr Virus
(EBV), and human T
lymphocyte Virus 1
(HTLV-1)
 Organ specific – thyroid
 Anti-thyroglobulin and
anti-microsomal
antibodies are secondary
Hashimoto’s
50:1 Cell Mediated to gland destruction (use
Thyroiditis
for diagnosis)
 Manifests as goiter and/or
hypothyroidism
 Peaks at 3rd or 4th decade
 Organ specific: brain and
spinal cord
 Oligoclonal, not
polyclonal antibodies in
Cerebral spinal fluid
 Some evidence for
previous infection with
paramyxovirus
 Regions futher away
Multiple
2:1 HLA-DR2 Cell mediated frokm equator have
Sclerosis
greater incidence
 Predominantly a
Caucasian disease
 Evidence that
environmental factor
triggers disease (virus?)
 Treatment with IFNβ
(inhibits IFNγ expression
of MHC class II)
  Muti-organ: primariu=ly
in the skin/ectodermal
tissues, but can cause
multi-organ pathology as
HLA-DR3
Scleroderma 3:1 Cell mediated a result, e.g.: GI tract,
(weak)
heart, lung
 Skin fibroblasts reproduce
faster and secrete more
collagen
 Organ Specific (lung and
Kidney)
 Anti-glomerular basement
1:4 membrane antibodies
two (anti-GBM)
Basement
Goodpasture’s peaks, Antibodies to cell
membrane  Shared antigens between
Syndrome young, Surface Antigens
Antigens the lung (alveolar) and the
and
elderly glomerular basement
membrane
 Linear deposition of IgG
and complement
 Organ specific: adrenals
 Chronic hypoadrenalism
 Some evidence for
association with M.
Cytoplasmic tuberculosis infection
cortical
Addison’s  Antibodies to adrenal cell
2:1 antigens Antibodies
Disease
and/or ACTH
microsomes
receptor  Cortex involvement (not
medulla)
 Adrenocorticotrophic
Hormone (ACTH)

 Organ specific – Pancreas

 Impending diabetes
associated with antibodies
to glutamic acid
Insulin- decarboxylase (ongoing
Antibodies to
dependent HLA-DR3 Islet cells trials to treat early with
5:1 cell surface
Diabetes HLA-DR4 insulin immunosuppression
antigens
Mellitus (DI) before development of
diabetes)
 Environmental trigger
(viruses? E.g.:
Cocksackie virus B4)
Antibodies cell  Cell specific; defective
Pernicious surface antigens RBCs due to
Anemia and/or Intrinsic malabsorption of vitamin
Factor (IF) B12
 blocking  Antibodies binds cell
antibodies surface antigens and
destroy parietal gastric
cells that secrete intrinsic
factor or
 Antibodies bind to IF and
prevent binding of
vitamin B12
 Leads to megaloblastic
anemia
 Organ specific: thyroid
 Stimulatory antibodies
mimic actions to ligand
Grave’s HLA-DR3 Antibodies to (thyroxin stimulating
8:1
Disease HLA-BW35 receptor hormone)
 Get unregulated secretion
of thyroxin which leads to
hyperthyroidism
 Tissue specific: nerve and
muscle- with systemic
effects
Antibodies to  Neuromuscular
Myasthenia Complex/Age Acetylcholine receptor transmission disorder
4:1
Gravis Dependent Receptor (blocking  Antibodies are inhibitory
antibodies) and block acetylcholine
binding
 Some evidence for prior
infection with poliovirus
 Tissue specific- with
systemic effects
 Joints
Ankylosing HLA-B27  Some evidence for prior
1:9
Spondylitis (98%) infection with Klebsiella
pneumonia
 There is NO rheumatoid
Factor
 Antibodies to the insulin
receptor, with systemic
effects
 Block binding of insulin
to the insulin receptor
Acanthosis Antireceptor  Name of disease
Nigricans antibodies originates from
hyperpigmentation in the
flexer and intertriginous
areas???
  Only manifests at
temperatures below 37˚C
 Occurs only at the
extremities (nose, fingers,
Cold Antibodies to
Glycophorin toes) when the individual
Agglutinin cell surface
or li is exposed to cold
Disease receptor
temperature
 Treatment- keep patient
warm and extremities
well protected
Serology: SEROLOGIC PRINCIPLES
“Nature of Antigen-Antibody Reactions”
A. Lock and Key Concept
The combining site of an antibody is located in the Fab portion of the molecule and is constructed
from the hyper variable regions of the heavy and light chains
B. Non Covalent Bonds
 These bonds hold the antigen to the antibody combining site are all non-covalent in nature
 Hydrogen bonds, electrostatic bonds, Van der Waals forces, hydrophobic bonds
 Multiple bonding between the antigen and the antibody ensures that the antigen will be bound tightly
to the antibody
C. Reversibility
 Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature, reversible

I. Antibody Affinity
 Definitions
 Is the strength of the attraction between an antibody and an antigenic determinant
 Refers to the sum of the attractive and repulsive forces operating between the antigenic
determinant and the combining site of the antibody
 The strength of the interaction between a single antibody binding site and a single epitope
 The affinity constant describes whether the antigen-antibody complex is highly complementary,
and therefore would bind readily, or not very complementary, and therefore would not bind
readily
 Is the initial force of attraction that exists between a single Fab site on an antibody molecule and
a single epitope or determinant site on the corresponding antigen
 Antibody may react with structurally similar antigens results in cross-reactivity
 Most antibodies have a high affinity for their antigens
 Scatchard Equation: can be used to determine antibody affinity
 As epitope and binding site come to close proximity to each other, several types of non- covalent bonds
hold them together.
 Ionic Bonds/ Electrostatic Bonds: occur between oppositely charged particles.
 Hydrogen Bonds: involve an attraction between polar molecules that have a slight charge
separation and in which the positive charge resides on a hydrogen atom.
 Hydrophobic Bonds: occur between nonpolar molecules that associate with one another and
exclude molecules of water as they do so.
 Van der Waals Forces: occur because of the interaction between the electron clouds of
oscillation dipoles

II. Antibody Avidity

 Definitions
 It is a measure of the overall strength of binding of multivalent antigen and multivalent
antibodies
 Represents the sum of all attractive forces between an antigen and antibody.
 The affinity for multivalent antigens and multivalent antibodies to combine; the extent of
the binding capacity
 It is the force that keeps the molecules together
 This is greater than the cumulative affinity constants for all antigen-antibody pairs
 Is influenced by both the valence of the antibody and the valence of the antigen
 Initially, bond is easily broken, but multiple bindings at the same time the dissociation is
overcome by the number of bonds remaining
  A high avidity can compensate for a low affinity
 Stability of the antigen-antibody complex is essential to detecting the presence of an unknown,
whether it is an antigen or antibody.
 Avidity is more than the sum of the individual affinities.

III. Cross Reactivity

 Definitions
 It refers to the ability of an individual antibody combining site to react with more than one
antigenic determinant
 The ability of a population of antibody molecules to react with more than one antigen
 Specific antibody binds precise or distinct antigen
 Occurs when the antibody combines with an antigen that is structurally similar to the
immunogen that stimulated the antibody production or the antigen the antibody has the
greatest affinity for
 The closer the fit between the site and the antigen epitope, the stronger are the non-covalent, and the
higher is the affinity between the antigen and antibody
 Cross reacting antigen shares an epitope in common with the immunizing antigen
 Antibodies recognize the total configuration of antigens rather than just their chemical compositions
 The more the cross reacting antigen resembles the original antigen, the stronger the bond will be
between the antigen and the binding site.
 If the epitope and the binding site have a perfect lock-and-key relationship, as is the case with the
original antigen, the affinity will be maximal, because there is a very close fit.
Agglutination Reactions

Definitions
 Combination of particulate antigen with antibody to form aggregates
 Is the visible aggregation of particles caused by combination with specific antibody
 The immunological aggregation or clumping of insoluble particles
 Agglutinogen: participating antigen (cellular in nature)
 Erythrocytes, bacterial cells, latex particles, yeasts
 Must be exposed and able to bind with antibody
 Agglutinin: Antibodies that produce such reaction
 IgM: Best agglutinin
 Involves the interaction of antibody with a multivalent antigen (particulates) which results in the cross-
linking of various antigen particle by the antibody

Steps in Agglutination
1. Sensitization
 Antigen-antibody combination through single antigenic determinants on the particle surface
 Rapid and Reversible
 Affected by the nature of the antibody molecules themselves
 IgM: potential valence of 10 is over 700 times more efficient in agglutination
 If epitopes are sparse, or if they are obscured by other surface molecules, they are less likely to
interact with antibody
 Attachments are not stable

2. Lattice Formation
 Cross linking occurs which stabilizes complexes
 Representing
 the sum of interactions between antibody and multiple antigenic determinants on a particle is
dependent on environmental conditions and the relative concentrations of antigen and antibody
  Bordet: lattice formation is governed by physicochemical factors such as the milieu’s ionic
strength, pH, and temperature.
 Erythrocytes and bacterial cells have a slight negative surface charge, it is difficult to bring such
cells together into lattice formation
 The ability to link cells together depends in part on the nature of the antibody
 Antibodies of the IgG classes often cannot bridge the distance between particles, because their
small size and restricted flexibility at the hinge region may prohibit multivalent binding
 IgM antibodies have a diameter of about 35nm

*Enhancement of Lattice Formation

 Decreasing the buffer’s ionic strength through the use of low ionic strength saline
 Addition of albumin in concentrations of 5-30% also helps neutralize the surface charge and allows
red cells to approach
 Addition of enyzmes: reduces surface charge on RBC through cleaving of chemical groups and
decreasing hydration
 Bromelin
 Ficin: cleaves sialoglycoproteins from the RBC
 Papain
 Trypsin
 Increasing the viscosity of the solution: these agents reduce the water of hydration around cells and
allow them to come into closer proximity for antibody to join together.
 Dextran
 Polyethylene glycol (PEG)
 Polivinypyrolidine (PVP)
 Agitation and Centrifugation provide a physical means to increase cell-cell contact and thus heighten
agglutination.

Factors that affect Agglutination

1. Buffer pH  A pH of 7.0 is used (close to
physiological pH)
2. Ag-Ab Concentrations  Affects zoning phenomenon
3. Location and Concentration of  Antibodies will not readily detect
antigenic determinants on the particle determinants buried within the particle
 The more number of determinants, the
higher the likelihood of cross bridging
4. Electrostatic interactions between  Non covalent interaction required for
particles agglutination reaction
5. Electrolyte Concentration  Ionic strength reduces electrostatic
charges that interfere with lattice
formation
6. Antibody Isotype  IgM is best
7. Temperature  IgM: cold reacting: 4-22C
 IgG: warm reacting: 37C
8. Centrifugation  Promotes agglutination
9. Length of time of incubation of the  15-60 mins range
coated particles with the px serum
Types of Agglutination Reactions
1. Direct Agglutination
 Occurs when antigens are found naturally on a particle
 The process by which particulate antigens such as cells, aggregate to form large complexes when
specific antibody is present
 Insoluble particle which is in suspension is reacted with a soluble Ab thus resulting to aggregation
of the particles

Direct Immune Direct Non Immune

Reaction is due to an Ag-Ab reaction wherein the
Ag is an inherent native to the cell
Examples: ABO blood grouping, anti-A and Anti-
B reacting with native antigenic determinants of
the red cells and directly causing
hemagglutination
Aggregation of indicator red cells are NOT due to
Ag-Ab reactions
Lectins: Phytohemagglutinins are not Abs hence
the direct hemagglutination of blood group O cells
by anti-H lectin is considered a non immune
reaction
*Viral hemagglutination tests

a. Hemagglutination
 Used to detect antibodies in red cell antigen
 Grading system
Grade Interpretation
4+ one solid aggregate; clear background
3+ several large aggregates; clear background
2+ Medium sized aggregates; clear background
1+ Small agglutinates, turbid background
W+ Tiny agglutinates, turbid background
no agglutination or hemolysis
0

 Active Hemagglutination
 Ag is the RBC itself

 Passive Hemagglutination
 RBC is NOT the Ag
 RBC absorbs it and expresses it on the surface

b. Viral Hemagglutination
 Many viruses can stick to and agglutinate RBC’s
 Rubella virus, Dengue Virus, Influenza Virus, Mumps Virus
 Due to their receptors to attach to red cells called Peplomers
  Non Immune type of agglutination

2. Passive/ Indirect Agglutination

 Rxns where As has been affixed or absorbed to a carrier/ inert particle/ cells
 A reaction in which soluble antigens are bound to latex beads, bentonite or charcoal
 Antigen is attached to the carrier particle
 Agglutination occurs is patient Antibody is present
Different Passive Carriers Coupling Agents
o Human RBCs o Since many proteins adsorb
o Clay (Bentonite) poorly to cells, mild treatment
o Latex Particles with reagents may increase the
o Colloidal gold/ Colloidion amount of cell-bound Ag/Ab
o Charcoal particles o Tannic Acid
o Gel Particles o BDB Method: Bidiazotized
benzidine
o Chromic Chloride
o Glutaraldehyde
o Cyanuric Chloride
o Water soluble carbodimide
3. Reverse Passive Agglutination
 Ab is attached to the carrier particle
 Agglutination occurs if patient Ag is present
 Example
 CRP test
 Reverse passive test for Candida
 Reverse passive test for Neisseria gonorrheae
4. Agglutination Inhibition/ Latex Agglutination
 (+) No Agglutination
 HCG test
 An agglutination reaction based on the competition between particulate antigen and soluble
antigen for limited sites on a reagent antibody
 2 Stage Technique
 Patient sample incubated with Antibodies in test kit
 Complexes will form if the patient sample contains the corresponding antigen and the
Fab sites are no longer available for the Antigen coated latex particles

5. Hemagglutination Inhibition
 Red cells are used as the indicator particles
 Test for detecting antibodies to certain viruses that agglutinate RBC
 Viral Hemagglutination Inhibition (HAI)
o Competitive binding assay
o Two stage procedure
 Biological sample incubated with viral particles. The viral particles will bind to the
Fab region of anti viral Abs present in the biological sample
 Indicator Red Blood Cells added to reaction mixture
 (+): no agglutination
6. Coagglutination
 Uses bacteria as the inert particle to which antibody are attached
 Staphylococcus aureus is most frequently used because it has protein on its outer surface, called
Protein A which adsorbs the Fc portion of antibody molecules
 Protein A naturally adsorbs Fc of IgG1, IgG3
 The active sites face outward and are capable of reaching with specific Ag
 Greater stability than latex particles and are more refractory to changes in ionic strength
 Highly specific but not sensitive

7. Anti-globulin Mediated Agglutination

 Detects non-agglutinating antibody by means of coupling with a second antibody
 Detect the presence of non-agglutination antibodies on RBC
 (+): Agglutination
Direct Indirect
Detects IgG Abs bound to the Ag on the Red Detects presence of Abs in the serum that is still
Cells to be attached to an analyte
No Need for incubation Requires an incubation period
Direct Coombs Test; Anti D test for Rh HDN Indirect Coombs test
Rose Waaler test for RF detection
Sample: RBC Sample: Serum
 8. Quantitative Agglutination Reactions
 Principle: Indirect or Reverse Agglutination
Immunoassay
SPIA
(Sol Particle Immunoassay)
DIA
(Disperse Dye Immunoassay)
IMPACT
(Immunoassay by Particle
counting)
SUMMARY
Direct Agglutination
Hemagglutination
Passive Agglutination
Reverse Passive
Latex Agglutination
Hemagglutination Inhibition
Coagglutination
Anti-Globulin Agglutination
Inert Particle Detection
Gold: inorganic colloidal Qualitative: color change,
particle Quantitative: colorimetric
analysis
Dye: organic colloidal particle Qualitative: color visualized
Quantitative: color measured
optically
Latex PArticle Quantitative: automated
particle counter
*Most Sensitive
Positive Result Negative Result
Agglutination No Agglutination
Agglutination No Agglutination
Agglutination No Agglutination
Agglutination No Agglutination
No Agglutination Agglutination
No Agglutination Agglutination
Agglutination No Agglutination
Agglutination No Agglutination
 QUANTITATIVE AGGLUTINATION ASSAYS USING INERT PARTICLES
Immunoassays Technique Inert particles Detection
SPIA Indirect or reverse Gold-inorganic Qualitative, color
agglutination colloidal particle change
Quantitative,
colorimetric analysis

DIA Indirect or reverse Dye-organic colloidal Qualitative, color

agglutination particle visualized
Quantitative, color
measured optically

IMPACT Indirect or reverse Latex particle Quantitative,

agglutination automated particle
counter

Causes of False Positive and False Negative Reaction in Agglutination Testing and the Corrective Action

FALSE-POSITIVE REACTIONS CORRECTIVE ACTION

 Contaminated equipment or reagents may
cause particles to clump.
 Autoagglutination: Test cells clump without
specific antibody present; mainly a problem
with red cells
 Delay in reading slide reactions results in
drying out of mixture.
 Presence of cross-reactivity
 Presence of rheumatoid factor
 Presence of heterophil antibody: occurs
mainly when rbc are used as a carrier
particle.
 Overcentrifugation causes cells or particles to
clump too tightly. The button is packed too
tight and is difficult to resuspend
FALSE-NEGATIVE REACTIONS
 Inadequate washing of rbc in antihuman
globulin (AHG) testing may result in
unbound immunoglobulins neutralizing the
reagent.
 Store equipment and reagent in clean, dust-
free environment and handle with care.
 Use negative quality control (QC) steps.
 Use a control with saline and no antibody as
a negative control. If positive, patient’s
result is invalid.
 Follow procedural directions and read
reactions exactly as specified.
 Use purified antigen preparations and
specific monoclonal antibody whenever
possible.
 Test specifically for rheumatoid factor to
rule out its presence. If rheumatoid factor is
present, agglutination results must be
interpreted carefully
 Preabsorb serum with rbc to rule without
specific antigen, or pretreat rbc to remove
other antigen.
 Calibrate centrifuge to proper speed and
time.
CORRECTIVE ACTION
 Wash cells according to directions.
 Use positive and negative QC steps.
  Failure to add AHG reagent: occurs mainly
in direct and indirect antiglobulin testing
 Contaminated or expired reagents
 Improper incubation
*too low incubation temperature may result in the
lack of association of antigen and antibody
 Undercentrifugation: Cells may not be close
enough to interact.
 Prozone phenomenon: too much patient
antibody for the amount of test.
 Reagent not active: may be caused by
improper storage
 Delays in testing procedure: This especially
pertains to antiglobulin testing. Antibody
may be eluted from rbc.
 Use positive QC steps.
 Add check cells that are antibody-coated to
see if agglutination occurs after a negative
test. If there is still no agglutination,
disregard result and repeat.
 Use positive and negative QC steps.
 Follow procedural protocol exactly.
 Use positive and negative QC steps.
*check appropriate temperature for the specific test
 Calibrate centrifuge to proper speed and
time.
 Dilute patient serum containing antibody,
and repeat the procedure.
 Refrigerate antisera, but don’t freeze
because loss of activity may occur.
 Once procedure has started, follow through
until the end without delay.
Precipitation
 Definition of Terms
 Precipitin – antibody
 Precipitinogen – antigen
 Precipitates – insoluble complexes formed by the union of precipitin and precipitinogen
 Flocculation – natural clumping; similar to precipitation but observed as a fleecy mass
when a suspension of antigen and antibody is agitated
 Precipitation – a combination of a soluble antigen with specific antibody which leads to the
formation of an insoluble aggregation/ visible insoluble complex

 Immunoglobulins involved:
 IgG: better precipitin
 IgM: better agglutinin
 IgE: non-precipitating
 Degree of precipitating ability: IgG > IgM > IgA

 Stages of precipitation reaction:

 Rapid formation of soluble complexes (Ag-Ab complexes) that are visible
 Slow aggregation of these complexes to form a visible precipitate

 Prerequisites:
 Antigen: multivalent and soluble
 Antibody: at least 2 available antigen binding sites
 Antigen and antibody in correct proportion → Lattice formation
 Concentrations of either antigen or antibody higher than the equivalence may result in a
weak reaction or no precipitate being formed

 Factors affecting precipitation

Factors Remarks
- More rapid precipitation as temperature
Temperature rises to 40 - 45○ C
- More complete precipitation at 0-4○ C
pH - Neutral pH (6-7.5)

- High salt → increase solubility of

Ionic Strength
complexes → increases dissociation
 Major Zones of Fluid Precipitation

Zone of antibody Zone of antigen Excess/

Zone of Equivalence
Excess/ Prozone Postzone
No free antigen and no
At all stages, in this zone, At all stages, in this zone,
free antibody molecules in
free antibody is present free antigen is present
solution
Antigen is insufficient to
Represents maximal Excess antigen leads to
form large immune
amount of precipitation decrease in cross linking
complexes

I. Precipitation in a Fluid Medium

a. Dean and Webb or Alpha Procedure
b. Ramon or Beta Procedure
c. Ring Test or Interfacial Test

II. Precipitaion in a Gel Medium

a. Single diffusion Single Dimension
b. Single diffusion Double Dimension
c. Double diffusion Single Dimension
d. Double diffusion Double Dimension
Precipitation in a Fluid Medium (Semi quantitative Precipitin Tests)
C. Dean and Webb (Alpha
D. Ramon (Beta Procedure)
Procedure)
 Antigen is diluted  Antibody is diluted
 Antibody in constant amount  Antigen in constant amount
 Detects smallest amount of antigen  Detects smallest amount of antibody
visible reaction with a given amount causing precipitation with a given
of antibody amount of antigen

 Prozone: due to Antigen  Prozone: due to antibody excess

 Postzone: due to antibody excess  Postzone: due to antigen excess

E. Ring Test (Interfacial Test)

 Advantage: No zoning phenomenon
 Why? Since antibody and antigen diffuse toward each other until they reach their equivalence point
where precipitation occurs
 Antiserum is deposited at the bottom of tube, overlaid with antigen then incubated
 (+) result: hazy, cloudy, milky layer at or near junction of reactants

Applications:

 Ascoli- detection of capsular antigen of Anthrax

bacilli
 Lancefield serological typing – Streptococci
 Identification of species origin of a blood stain
 Detection of CRP
 Detection of even very small amounts of
contaminating substances in otherwise pure
preparations
 Nonclinical research purposes: determining the
purity of pharmaceutical proteins

Precipitaion in a Gel Medium

 Soluble antigen and/or antibody can diffuse through pores of gel until their concentration reaches optimum
ratio forming stable immunoprecipitate
 Terms:

Single Diffusion Only one reactant (usually antigen) is moving

Double Diffusion Both antigen and antibody are moving through medium
Single Dimension Reaction in TUBE- antigen and antibody migrate up and down
Double Dimension Reaction in PETRI DISH –antigen and antibody diffuse radially

 Four Immunodiffusion Tests:

 A. Single diffusion Single Dimension Oudin
Radioimmunodiffusion (RID) or Mancini
B. Single diffusion Double Dimension
Test
C. Double diffusion Single Dimension Oakley and Fulthrope
D. Double diffusion Double Dimension Ouchterlony and Elek

 Gelling Substances:
 Agarose- Most commonly used
 Agar
 Polyacrylamide
 Cellulose acetate
 Gelatin, starch

I. Single diffusion Single Dimension (OUDIN)

 Antibody mixed in agarose deposited at the bottom of tube
 Antigen dilution overlaid
 Antigen concentration always greater than equivalence concentration
 Mobile antigen diffuses through gel with immobilized antibody, forming insoluble antigen-
antibody complex until size of complex becomes too large to pass through pores of gel
 At equivalence concentration, antigen stop moving and stabilized band of precipitate forms
 Types of precipitation:

R type precipitation (Rabbit) H type precipitation (Horse)

 Fuzzy edge  Clean margins
 Smaller amounts of  Flocculation is complete
precipitate exists on their within equivalence zone and
side of equivalence is completely inhibited
outside that zone

II. Single diffusion Double Dimension (MANCINI TEST)

 Most common method
 Most reliable result
 Antibody mixed with hot liquid agar poured into petri dish
 Circular wells cut into gel loaded with antigen
 Ring of precipitate expands from well as antigen diffuses toward equilibrium concentration
 Diameter of disc is proportional to logarithm of concentration
 Methods for reading RID:

Kinetic (Fahey) Method Endpoint (Mancini) Method

 Measure disc while expanding  Measure when it has stopped
(at 18 hours) expanding; allow maximal
precipitation
 Result in shorter time  More reliable result
III. Double diffusion Single Dimension
(OAKLEY AND FULTHROPE)
 Overlay antibody with neutral gel agar and allow to
gel
 Overlay gel with antigen
 Both antigen and antibody diffuse through gel
forming bands of precipitates

IV. Double diffusion Double Dimension

(OUCHTERLONY and ELEK)
 Advantage: ability to detect serological
identity in 2 or more antigen-antibody
system
 Pattern of wells is cut in agarose gel (petri
dish or slide)
 Loaded with reactants
 Incubated until line of precipitate have fully developed
 Application: identify antibodies associated with autoimmune disorders
 4 possible patterns:

Single Double
Serological Non
partial Partial
Identity Identity
Identity Identity
Lines of
Spur
Fused band precipitation Double
formation
of precipitate cross each spurring
seen
other

Antigens not
Antibody is
Antigens are identical but
precipitating Rarely
serologically possess
serologically occurs
distinct common
antigens
determinants
Electrophoresis
 Principle: molecules with a net charge are separated when electric field is applied in the system
 Negatively charged particles migrate to positive pole (ANODE)
 positively charged particles migrate to negative pole (CATHODE)
 Basis: Molecular weight
 Factors that affect rate:
o Size and shape of protein
o Amount of solvation
o Viscosity of buffer
o pH of buffer
 pH > 8 usually used
o Temperature
 Room temperature

 Endo-osmosis:
o Electro-osmosis
o Net flow of hydrated ions in one direction when electric field is applied
o Flow of ions towards CATHODE → impedes movement of proteins towards ANODE
o No counteraction

 Non soluble support media

Filter paper Rarely used

Cellulose Acetae Favored because inert

Used in specialized medical

Polyacrylamide Gel (Western Blot) laboratories and research laboratories
Used in HIV confirmatory test

Useful in separation of large molecule

Agarose Gel
like DNA, IgM antibodies

 Zone Electrophoresis
o Proteins or nucleic acids are separated and localized into separate bands/zone
o Most common medium: cellulose acetate and agarose

 Categories of Immunoelectrophoresis (IE)

Single Reactant moving in a Single Dimension Rocket or Laurell Technique

Single Reactant moving in Two Dimension Crossed IE or Ressler Technique
Double Reactant moving in a Single Dimension Counter IE
Double Reactant moving in a Two Dimension Classic IE or Grabar and Williams Techniques
 IMMUNOFIXATION
 2 stages: protein (Ag) electrophoresis + immunoprecipitation
 Process:
 Specimen applied to 6 positions on
agarose plate
 Electrophorese to separate proteins
 Monospecific antisera applied to 5
patterns, the 6th for reference
 If Ag present, Ag-Ab complexes form
and precipitate; wash, stain
 Highly sensitive, easy to read
 Application: used to classify monoclonal
gammopathies
 Most commonly utilized technique

A) SINGLE REACTANT MOVING IN A SINGLE DIMENSION

 Rocket or Laurell Technique

 ONE STAGE
 Voltage needed
 Principle:
 One step:
 Antigen is pushed through the gel
containing antibody under influence
of electric field
 Antigen combines with antibody
forming cone or rocket shaped
precipitate band
 Quantitative when used with
calibration curve
 Application:
 Quantification of antigen
 Test for Factor VIII related antigens

B) SINGLE REACTANT MOVING IN TWO DIMENSION

 Double-Crossed IEP, Two-Dimensional IEP, or Ressler Technique

 TWO STAGE
 Research technique for fractionating serological active components of a complex mixture of
proteins such as whole serum
 Resulting immunoelectropherogram resembles a landscape of mountain-shaped precipitate peaks
 Two direction is perpendicular to each other
 Disadvantages:
o time-consuming
o Costly
o Difficult to perform
  Principle:
o Step 1:
 Electrophoretic separation
of proteins in biologic
samples
o Step 2:
 Separated proteins are
subjected to 2nd
electrophoresis where they
move through agarose gel
containing antibodies
leading to formation of
precipitin arc (bell shape or
mountain shape)
 Application: mainly used in the research
laboratory for identification of the
heterogeneity of certain proteins (α-1-
antitrypsin)

C) DOUBLE REACTANT MOVING IN A SINGLE DIMENSION

 Counter immunoelectrophoresis (CIE)

 Double-electroimmunodiffusion, Crossed Antigen-antibody Electrophoresis, Countercurrent
IE, Immuno-osmophoresis, Electrosyneresis, or Counter migration Electrophoresis
 ONE STAGE

 Principle:
o One Step
 Antigen and antibody are
added to separate parallel
wells cut out of agar gel
and when electric field is
applied, antigen migrates
toward anode and antibody
towards cathode
 Antigen and antibody meet
in correct proportion
forming precipitate

 Application:
o Identify antigen on bacterial
surface, fungi, or virus present in
biological fluid (e.g.: HBsAg,
Meninggococcal antigen,
Pneumococcal Antigen)
o Identify unusual protein
(Australian Antigen or Antibody,
IgM screening in newborn, alpha
fetoprotein, circulating
fibrinogen or FSP in DIC)
D. DOUBLE REACTANTS MOVING IN TWO DIMENSION
 Grabar and Williams or Immnunoelectrophoresis
 TWO STAGES
 Steps:
 Semisolid agar poured onto glass slide and antigen well and antiserum through cut out
of agar
 Antigen well filled with human serum
 Serum separated by electrophoresis
 Antiserum trough filled with antiserum to whole human serum
 Serum and antiserum diffuse into agar
 Precipitin lines forms for individual serum proteins
 Application:
 differentiates immunoglobulin classes
 typing and identification of abnormal proteins, myeloma proteins
 pharmaceutical industry: monitor purity of products for human consumption

Complement fixation test

 designed to detect complement consumption in virtually any cellular or non-cellular antigen-
antibody reaction to which complement is bound
 serum from horses, cattle, sheep and mice are essentially NONCYTOLYTIC (do not cause
lysis)
 guinea pig serum → preferred source of complement for most clinical tests involving human
serum (EXCEPT Human Histocompatibility Typing where rabbit or human complement is
preferred)
 complement is unstable: inactivation by heating at 56 degrees Celsius for 30 minutes
 nonserologic inactivation of complement: aging, vigorous agitation, treatment with:
various chemicals (acids, alkaline, alcohol, etc.), enzymes, yeast or bacterial cells, tissue
extracts, hemolysis
 antibody and antigen allowed to combine in the presence of complement
 of complement is fixed by specific Antigen-antibody reaction, complement will be unable to
combine with indicator system
 components:
 patient serum (Heat inactivation)
 reagent antigen
 exogenous complement is added
 indicator hemolytic system
 RBCs
 Hemolysin
 Hemolysin → antiserum which can activate complement and can cause immune lysis
 Heat inactivate the native complement in the patient’s serum

 LYTIC COMPLEMENT FIXATION

 Principle:
 Antigen-antibody occurs, complement is fixed → no lysis of indicator system
 Detects complement fixing antibodies (IgM and IgG)
 (+) result: non lysis of indicator RBCs
 (-) result: lysis
  Two possible reasons for a NEGATIVE LYTIC CFT
1. Absence of antibodies in the patient’s serum
2. Antibody is present but is non-complement fixing (e.g.: IgA, IgD,
IgE)
 To determine the cause of a Negative Lytic CFT, Rice test (indirect
CFT) is perfomed

LYTIC SYSTEM RICE TEST

No antibodies in
Hemolysis No Hemolysis
Patient serum
Non-complement
fixing antibody in Hemolysis Hemolysis
patient serum
 RICE TEST: NO ANTIBODY
o Introduction of a reagent antibody SPECIFIC to antigen and is
complement fixing
 Result: No antibody → No Hemolysis
o Proving that antibody is present in the patient serum but is NON-
COMPLEMENT FIXING
 Result → Hemolysis

 NON-LYTIC COMPLEMENT FIXATION TEST

o CONGLUTINATING COMPLEMENT ADSORPTION TEST (CCAT)
 Conglutinin:
 a beta globulin of cattle serum
 Protein that cause agglutinin
 Antigen-antibody complex are aggregated by conglutinin
INDICATOR LABELED IMMUNOASSAYS
 These are immunoassays where antigen-antibody complexes do not go beyond the first
phase (Ag-Ab) union

 Fluorescent Immunoassay (FIA)

 Chemiluminescent Immunoassay (CLIA)
 Radioimmunoassay (RIA)
 Enzyme Immunoassay (EIA)

Immunofluorescent (FIA)
 Fluorescent Immunoassay
o Principle:
 Uses FLUOR or FLUOROCHROME – absorb light at shorter wavelengths;
emit light at visible spectrum/longer wavelengths
 Fluor are covalently linked to IG (direct) or to antiglobulin (indirect) to detect
antigenic substance or antibodies

o Criteria of a good Fluorophores

- Stable
- High absorptivity and quantum yield
- Emit at appropriate wavelength
- NOT interfere with ligand-antibody reaction

o Examples of Fluorophores
 Fluorescein isothiocyanate (FITC) → emits green color; high intensity and
good photostability
 Lissamine Rhodamine B → emits red light

Homogenous FIA Heterogenous FIA

 No separation or washing
phase
 Competitive binding
 Direct relationship between
amount of fluorescent and
quantity of antigen in patient
sample
 Examples:
- FETI
- FPIA
 Includes separation or washing
phase to remove free form
bound fluorochrome
 Examples:
- Solid Phase FIA
- Particle concentration
FIA
 Qualitative Tests Quantitative Tests
 Direct IF  Fluorescence Quenching
 Inhibition IF
 Indirect IF
 Complement staining IF

DIRECT IMMUNOFLUORESCENCE
 Principle:
o Antigen from patient sample fixed to slide
o Fluorescent labeled antibody added
o Antibody binds to antigen fixed to slide
o Wash to remove unattached antibody
o (+) result: Fluorescence

 Example:
o Fluorescent Antibody Dark Field Technique (FADF) for Treponema pallidum
 Positive Result → immunofluorescence
 For detection of antigens

INDIRECT IMMUNOFLUORESCENCE
 Principle:
 Antigen spread on the slide and patient serum (Ab) added and incubated then washed
 Fluorescent antihuman globulin added, incubated then washed
 If patient serum contains Antibody, antibody is fixed to antigen on slide
 Tagged reagent antibody added, reacts with antibody fixed to antigen
 (+) result: Fluorescence
 Immunologic sandwich: antigen-antibody antiglobulin complex

 Example:
 FTA-ABS

 For detection of either antigen or antibody

INHIBITION IMMUNOFLUORESCENCE
 Principle:
 Antigen fixed to slide then flooded with patient serum
 Antibody in serum is fixed to antigen
 Fluorescent antibody reagent specific to antigen added but cannot bind antigen since antigen
is already fixed to antibody in patient serum
 Labeled reagent antibody washed off

 (+) result: no fluorescence

 For detection of antibodies
COMPLEMENT-STAINING IMMUNOFLUORESCENCE
 For detection of either antigen or antibody
 (+) result: Fluorescence

Chemiluminescent Immunoassay (CLIA)

 Emission of light that occurs when a substrate decays to a ground state from an excited state
produced by chemical reaction (usually oxidation)
 Oxidation of chemiluminescent label (attached to antigen or antibody) by enzyme producing light
 Most sensitive reporter system for immunoassays
o Why? Because light emission can either be detected at very low levels
 Emission is read with luminometer or captured on photographic film
 Most common chemiluminescent compounds
o Acridium Esters
o Isoluminol Derivatives (Enzyme: Peroxidase)
 Others:
o 1,2-dioxethane molecules
 Substance for ALP in commercial immunoassay
o Ruthenium-labeled Antibodies
 Detection of biological weapon agents

Radioimmunoassay (RIA)
 Makes use of radioactive labels
 2 types:

Gamma Emitters Beta Emitters

Description Emit gamma radiation Emit beta radiation
Measurement Uses solid/ crystal Uses liquid scintillation
scintillation counter counter
125 3
Examples I (commonly used) H (trithium), 14C, 32P,
and 131I and 131I

 Application:
o Monitor hormone levels (Insulin, TSH, estrogen)
o Detects vitamins
o Detects Viral antigens
o Detect therapeutic drugs (digoxin)
o Detect abused drugs (opiates, barbiturates, amphetamine)
o High sensitivity

 Advantages:
o Precision and high sensitivity
o Signal detection without optimization
 o Stability against interference from the assay environment

 Disadvantages:
o Need to protect against hazardous radioactivity
o Shorter shelf life of reagents

 Methods:
1) Indirect Radioimmunosorbent Test (RIST)
2) Direct RIST Measures the total IgE levels
3) Radioimmunoprecipitation (RIP) assay
4) Radioallergosorbent Test (RAST) → quantitate specific IgE levels

 2 main Categories:
- COMPETITIVE
 Indirectly proportional
 Patient antigen and reagent antigen compete for limited antibody in Reagent system
OR
 Patient antibody and reagent antibody competes for limited antigen in the reagent
system
- NONCOMPETITIVE
 Directly proportional
 No competition between patient antigen and reagent antigen for antibody in reagent
system OR
 No competition of patient antibody and reagent antibody for antigen in reagent
system
 Also called SANDWICH METHOD
Indirect Radioimmunosorbent Test (RIST)
 Competitive Binding Assay or Displacement/ Radioligand Inhibition
- Patient antigen and labeled antigen are incubated with known amount of antibody
- Patient antigen and labeled antigen competes for binding with antibody
- Wash to remove unbound antigen
- Radioactivity counted in gamma counter or compared to standard curve
- Result: the lower the radioactive count, the higher the concentration of patient antigen

- Example:
 Test for hepatitis antigens and antibodies
 RIST → Measures total IgE
 RAST → Measures IgE to specific allergens

- Order of addition of reagents;

 1st Reagent antibody
 2nd Patient antigen
 3rd Labeled antigen
  Why? To enhance assay sensitivity

Direct Radioimmunosorbent Test (RIST)

 Non-Competitive Binding Assay
- Antibody bound to solid phase in reagent system
- Patient antigen added
- Washing phase
- Antibody labeled with radioisotope added – directed at different determinant of patient
antigen
- Incubate and wash
- Radioactivity measured → directly proportional to patient antigen concentration
 Same principle with immunoradiometric assay (IRMA)

Radioimmunoprecipitation (RIP) Assay

 Employs a soluble 2nd antibody to precipitate the bound antigen
 DOBLE ANTIBODY TECHNIQUE

Radioallergosorbent Test (RAST)

Enzyme Immunoassay (EIA)
 Makes use of enzyme as labels
o Most commonly used:
 Horseradish peroxidase (HRPOD)
 Alkaline phosphatase
 Beta-galactosidase
 Acetylcholinesterase
 Glucose-6-phosphate dehydrogenase
 Either homogenous or heterogeneous
o Homogenous
 No separation between bound and free antigen
o Heterogeneous
 REQUIRES separation between bound and free antigen
 Can be quantitative or qualitative

 Application:
o HIV testing
o Serum hCG (Pregnancy)
o Tests for hepatitis antigens and antibodies

 EIA’s be classified as how they are measure with various detectors depending on the substrate
used:
1) Colorimetric EIA → Most commonly used HRPOD and ALP
2) Fluorescent EIA → Use Fluorescent substrate
3) Chemiluminescent EIA (CL-EIA) → Uses chemiluminescent substrate with various
enzymes employed as labels

 Advantages:
o high degree of sensitivity
o reagents are relatively cheap and can have a long shelf-life
o short run times
o multiple simultaneous assays can be developed (ease of automation)
o no radiation hazards occur during labeling or disposal of wastes
o equipment can be inexpensive and is widely available
o simplicity of sample handling

 Disadvantages:
o Measurement of enzyme activity can be more complex than measurement of the activity of
some types of radioisotopes
o Enzyme activity may be affected by plasma constituents
o Homogenous assays at the present time have the sensitivity of 10-9 M and are not as
sensitive as radioimmunoassay
o Homogenous EIAs for large protein molecules have been developed but require complex
immunochemical reagents
 Heterogeneous EIA
o Qualitative EIA
o Quantitative EIA
 Methods:
 Competitive Heterogeneous EIA
 Two-site immunometric sandwich assay (Double Antibody Sandwich/
Antigen-Sandwich EIA) → NONCOMPETITIVE
 Indirect EIA → COMPETITIVE

 Enzyme-Linked Immunosorbent Assay (ELISA)

o Patient antigens competes with Enzyme-labeled antigens for binding sits on antibodies
o Centrifuge, wash to remove unbound antigen
o Substrate added
o Colored product measured (Absorbance)
o Result:
 Inverse relationship between absorbance and patient antigen
 Low Absorbance → less enzyme-labeled antigen linked to immunosorbent → less
substrate catalyzed → lower absorbance
 Immunoenzymometric Test
o Patient antigen + enzyme-labeled antibody
o Homologous patient antigen bound to enzyme-labeled antibody
o Solid-phase antigen added, adsorb enzyme-labeled antibodies with at least one free binding
site
o Centrifuge – immunosorbent particles settle to bottom of tube
o Only antibodies with combining sites occupied by patient antigens are extracted in
supernatant
o Result:
 Direct relationship between absorbance and patient antigen
 High absorbance = high patient antigen
 More patient antigen → more enzyme-labeled antibodies in supernatant → more
substrate catalyzed → more absorbance

 Antigen- Sandwich EIA

o IMMUNOLOGICAL SANDWICH
 Solid phase antibody + patient antigen
 Enzyme-labeled antibody added
 Incubate, centrifuge, wash precipitate to removed unabsorbed antibody
 Substrate added
 Color change measured
o Result:
 More patient antigen → more enzyme-labeled antibody → more substrate catalyzed
→ greater change in color
 Increased color change = high patient antigen

Examples of Competitive vs. Non-competitive Homogeneous EIA

COMPETITIVE NON-COMPETITIVE
 Enzyme-multiplied Immunoassay
Technique (EMIT)
 Substrate-labeled Fluorescent
Immunoassay (SLFIA)  Enzyme Inhibitor Homogeneous
 Apoenzyme Reactivation Immunoassay Immunoassay (EIHIA)
(ARIS)
 Cloned Enzyme Donor Immunoassay
(CEDIA)
 Homogeneous EIA

 Enzyme-multiplied Immunoassay Technique (EMIT)

o Principle:
 Patient sample + antibody + enzyme-labeled antigen
 Unlabeled patient antigen competes with enzyme-labeled antigen for limited antibody
combining sites
 More patient antigen bound to antibody = more enzyme unbound and catalytically active
 Substrate added
 UNBOUND enzymes convert substrate to colored product

o Change in optical density:

 DIRECTLY proportional to concentration of UNBOUND enzyme-labeled antigen
 DIRECTLY proportional to concentration of patient antigen

o Result: high optical density = high concentration of patient antigen

o Characteristic of enzyme-labeled antigen
 enzyme-labeled antigen + Antibody → Enzyme CANNOT act on substrate
 Why? Because antibody sterically blocks S-E interaction
 Substrate-Fluorescent Immunoassay (SLFIA)
o Uses a characteristic fluorogenic substrate, Umbelliferyl-β-galactoside, attached to the
antigen (analyte) as a conjugate
o Umbilliferone is the fluorescent-β-galactosidase, which cannot cleave the substrate-antigen
complex when it is reacted with the specific antibody
o The free antigen (analyte) in the specimen solution competes with the antigen conjugated
with the substrate to form the immunocomplex
o The antigen concentration in the sample is proportional to the fluorescent intensity of the
cleaved fluorescent product
o Can be used to assay drugs and haptens, as well as protein ligands such as IgM and IgG
o Disadvantage: has limited sensitivity in the range of 10-9 to 10-10 molar concentration of the
analyte
 Apoenzyme Reactivation Immunoassay (ARIS)
o Uses the prosthetic group consisting flavin adenine dinucleotide (FAD)- conjugated
antigen (analyte) and glucose oxidase apoenzyme
o The antigen (analyte) and a constant amount of analyte- FAD conjugate compete for a
limited amount of specific antibody
o At equilibrium, the level of free conjugate is proportional to the amount of antigen (analyte)
in the specimen
o The apoenzyme combines with the free but not with the antibody-bound form of conjugate
to reactivate glucose oxidase activity in proportion to the amount of free conjugate in the
mixture
o APPLICATION:
 To assay the theophylline and IgG
 Measurement of high molecular weight proteins (e.g.: Thyroid binding globulin) as
well as other haptens such as phenytoin and hormones
 Cloned Enzyme Donor Immunoassay (CEDIA)
o A hapten antigen (analyte) is attached to an ED, and an analyte-specific antibody is used to
inhibit spontaneous assembly of the active enzyme
o The antigens (analytes) in patient serum compete with the analytes in the analyte-ED
conjugate for antibody, modulating the amount of active β-galactosidase formed
o The signal generated by enzyme substrate is directly proportional to the analyte
concentration in the patient serum
o Application: test for Digoxin
o The assay system is suitable for use with automated chemistry analyzers
 Enzyme Inhibitor Homogeneous Immunoassay (EIHIA)
o Consists of antibody conjugated enzyme and insoluble substrate
o Most suitable for the determination of large antigens (analytes)
o Α-amylase has been used as a labeled enzyme for the determination of ferritin and AFT
o Using this method, the measuring range of ferritin in serum is 10-800 ng/mL, and for AFT
it is 5-200 ng/mL
o Advantage: requires less incubation time to achieve a sensitive detection level
o Disadvantage: the sensitivity of the system remains inadequate for application to analytes
such as tumor markers
Precipitation Reactions
Definition of Terms:

 Precipitin: Antibody
o IgG: Best precipitin
 Precipitinogen: Antigen
 Precipitates: insoluble complexes formed by the union of precipitin and precipitinogen
 Flocculation: natural clumping; similar to precipitation but observed as a fleecy mess when a suspemsion of
Ag and Ab is agitated
 Precipitation: combination of a soluble antigen with specific antibody which leads to the formation of an
insoluble aggregation

Immunoglobulins Involved:

 Degree of precipitating ability: IgG> IgM> IgA

 IgE: non precipitating

Prerequisites

 Antigen: multivalent and soluble

 Antibody: at least 2 available Ag binding sites
 Ag and Ab in correct proportion leads to lattice formation

Zone Phenomenon
 Concentrations of either Ag or Ab higher than the equivalence may result in a weak reaction or no
precipitate being formed

Factors Affecting Precipitation

 Temperature
o More rapid precipitation as temperature rises to 40-45C
o More complete precipitation at 0-4C
 pH
o Neutral pH (6-7.5)
 Ionic Strength
o High salt: increase solubility of complexes which leads to increase dissociation
Major Zones of Fluid Precipitation

Zone of Ab Excess/ Prozone Zone of Equivalence Zone of Ag Excess/Postzone

At all stages in this zone, free No free Ab and no free Ag At all stages in this zone, free
Ab is present molecules in sol’n Ag is present
Ag is insufficient to form large Represents maximal amount of Excess Ag leads to decrease in
immune complexes precipitation cross linking

I. Precipitation in Liquid Medium (Semiquantitative Precipitin Test)

A. Dean and Webb (Alpha) B. Ramon (Beta)

Ag is diluted Ab is diluted
Ab in constant amount Ag in constant amount
Detects smallest amount of Ag giving visible Detects smallest amount of Ab causing
reaction with a given amount of Ab precipitation with a given amount of Ag
Prozone: due to Ag excess Prozone: due to Ab excess
Postzone: due to Ab excess Postzone: due to Ag excess

C. Ring test/ Interfacial test/ Fluid Precipitin Test

 Advantage: no zoning phenomenon
 Since Ab and Ag diffuse toward each other until they reach their equivalence point where
precipitation occurs
 Antiserum is deposited at the bottom tube, overlaid with Ag then incubated
 (+): hazy, cloudy, milky layer at or near junction of reactants
 No zoning occurs because both reactants diffuse into one another until sufficiently diluted to allow
precipitation to occur
 Procedure:
 Undiluted antiserum placed on the bottom of the tube
 Antigen solution layered over antiserum
 Incubated
 Result: hazy, cloudy, milky layer at or near junction of reactants

Practical Applications of Fluid Precipitin Test

 Ascoli: to detect the capsular Ag of anthrax bacilli
 Lancefield Serological Typing: Streptococci
 Forensic: identification of specie origin for a blood stain
 CRP: not an antibody; behaves as a precipitin when mixed with anti-crp
 Detecting very small amounts of contaminating substances in otherwise pure preparations
 Nonclinical research purposes: determining the purity of pharmaceutical proteins
II. Precipitation in Gel Medium
 Soluble Ag and or Ab can diffuse through pores of gel until their concentration reaches optimum ration
forming stable immunoprecipitate

Terms:

 Single Diffusion
o Only one reactant (usually Ag) is moving
 Double Diffusion
o Both Ag and Ab are moving through medium
 Single Dimension
o Reaction in tube—Ag and Ab migrate up and down
 Double Dimension
o Reaction in Petri Dish—Ag and Ab diffuse radially

Four Combinations for Immunodiffusion Tests

 Single diffusion-Single dimension: Oudin

 Single diffusion-Double dimension: Radio immunodiffusion or Manchi test
 Double diffusion-Single dimension: Oakley and Futhrope
 Double diffusion-Double dimension: Ouchterlony

Gelling Substances
- Agarose: most commonly used
- Agar
- Polyacrylamide
- Cellulose acetate
- Gelatin
- Starch

A. Single Diffusion- Single Dimension: Oudin Test

 Procedure
 Antibody mixed in agarose deposited at the bottom of the tube
 Antigen dilution overlaid
 The concentration of Ag must always be greater than the equivalence concentration
 The mobile Ag diffuses through the gel containing immobilized Ab, forming insoluble Ag-
Ab complexes until the size of the complex becomes too large to pass through the pores of
the gel
 At equivalence concentration, the Ag stops moving and a stabilized band of precipitate
forms
R type of Ppt (Rabbit) H type of Ppt (Horse)
Fuzzy Edges Clean margins
This is because smaller amounts of ppt exists on Because flocculation is complete within the
either side of the equivalence equivalence of zone and is completely inhibited
outside that zone
B. Single Diffusion- Double Dimension: Radial Immunodiffusion (RID) or Mancini Test

Uses:

 Popular method for quantitating variety of proteins normally found in serum

o Ig classes
o Complement component
o Transferring alpha 1 antitrypsin
o Alpha 2 macroglobulin
o Lysozyme
 Testing for apoproteins
 Quantitating Ab titers

Procedure:
 Ab mixed with hot liquid agar and poured into slide/ petri dish
 Circular walls cut into gel and loaded with Ag
 Ring of ppt expands from the well as Ag diffuses toward its equilibrium conc
 Diameter of the disc is proportional to the logarithm of the concentration

Technical Source of Errors:

 Overfilling or under filling the well

 Spilling sample outside of the well
 Nicking the well
 Improper incubation time or temperature

Two Methods for reading RID test:

Kinetic (Fahey) Method End Point (Mancini) Method

Measure the disc while it is expanding usually at Measures it when it essentially has stopped
18 hours expanding; allow maximal precipitation
Results obtained in a shorter period of time Produce more reliable results
C. Double Diffusion-Single Dimension: Oakley and Fulthrope

 Difference with Oudin:

 Both reactants can be diluted by diffusion so that the initial concentration of the Ag does not
need to be greater than the equivalence point with reference to the original conc of the Ab

Procedure:

 Overlay Ab with neutral agar and allow to gel

 Overlaying the gel with Ag
 Both Ag and Ab diffuse through the gel and form bands (layers) of precipitates near their zones of
equivalence concentrations

D. Double Diffusion-Double Dimension- Ouchterlony and Elek

 Chief Advantage:
 Ability to detect serological identity in two or more Ag-Ab systems

 Procedures:
 A pattern of wells is cut in an agarose gel
 Loaded with reactants
 Incubated until lines of precipitate have fully developed
Complement Fixation Test
 Detects complement consumption in any cellular or non-cellular antigen-antibody reaction to which
complement is bound

Complement Fixing Abs

 IgM
 IgG

Guinea Pig Serum

 Preferred source of complement
 An exception is in human histocompatibility typing where human or rabbit complement is preferred

Complement Mediated Cytolysis

Susceptible Resistant
- Erythrocytes - Gram (+) bacteria
- Leukocytes - Most mammalian cells
- Thrombocytes - Plant Cells
- some Gram negative bacteria - Yeast and Moulds

Precautions:
 Activated by:
 Heating serum at 56C for 30 mins
 Vigorous Agitation
 Chemicals (acids, alkali, alcohol)
 Enzymes
 Yeast, Bacterial cells
 Tissue Extracts
 Hemolysin: antiserum which can activate complement and can cause immune lysis

 Temperature:
 Rapidly loses its hemolytic activity within 24 hours at room temperature
 Loses activity over 3-4 days under refrigeration (but active up to one month if stored at -20C and 6
months at -40C)
Complement Fixation Principle
 The complement fixation test is an immunological medical test looking for evidence of infection. It tests
for the presence of either specific antibody or specific antigen in a patient's serum. It uses sheep red blood
cells (sRBC), anti-sRBC antibody and complement, plus specific antigen (if looking for antibody in serum)
or specific antibody (if looking for antigen in serum)

Lytic Complement Fixation Test

 Principle:
 Ag-Ab reaction occurs, complement is fixed
 (+): No lysis of indicator System
 (-): Lysis
 Two possible reasons for a negative lytic CFT
 Absence of Antibodies in the patient’s serum
 Antibody is present but is non-complement fixing
 To determine the cause of a negative lytic CFT, RICE test (Indirect CFT) is performed

Lytic System RICE Test

No Abs in patient serum Hemolysis No Hemolysis
Non Complement fixing Hemolysis Hemolysis
antibody in patient serum
Modifications of CFT

1. RICE Test or Indirect CFT

- Serves to detect non-complement binding Abs
- using a standard antiserum to antigen which is known to fix complement is added on one set.
- A positive result would cause hemolysis

2. Conglutinatin Complement Adsorption Test (CCAT)

- Conglutinin: a beta globulin of cattle serum
- Ag-Ab C Complex are aggregated by conglutinin
- using a horse complement which is non-hemolytic is used. The indicator system used is sensitized sheep
RBC’s mixed with bovine serum (containing a conglutinin) causing agglutination, indicating a negative
result.

3. Immune Adherence

 Occurs when bacteria combines with a specific antibody in the presence of complement,
they adhere to the erythrocytes or platelets.

4. Immobilization Test

 antigen is incubated with patient’s serum in presence of complement. Presence of antibody

would immobilize antigen.

5. Cytolytic Test

 incubation of a live bacterium with specific antibody in the presence of complement leads to
the lysis of the bacteria.
Laboratory Techniques
Pre Testing Errors
 Improper patient
preparation
 Mislabeled/ Unlabeled
Specimen
 Improperly transported
specimen
 Inappropriately stored
test kits
 Right specimen
 Right collection
 Right labelling
 Right quantity
 Right transport
 Right Storage
Testing Errors
 Improper measurements
of specimen or reagents
 Dilution and pipetting
errors
 Incorrect reagents used
 Country algorithm not
followed
 Use of inappropriately
stored/ expired reagents
 Laboratory professionals
 Reagents
 Equipment
 Selection of test-
SOP(Standard Operating
Procedure)
 Records
 Biosafety
Post Testing Errors
 Transcription error in
reporting
 Illegible reports
 Report sent to wrong
location
 Information system not
maintained
 Recording
 Interpretation
 Turnaround time
 Report to right user

Specimen Collection
 Avoid hemolysis
 Avoid hemoconcentration
 Do not force the plunger of the syringe during venipuncture
 Never transfer blood forcefully through a needle into a vacuum tube
 Wash hands
 Position the patient
 Comfortable position
 Turn the arm so that the wrist and palm face upward, and the antecubital area is accessible

Simple Steps in Venipuncture

1. Laboratory request assessment
 Read request forms VERY carefully
 Understand what is written
 Validate identification request
2. Check patient identification
3. Introduce yourself/ inform about what to do
4. Select suitable site for venipuncture

 Areas to avoid when performing venipuncture

 Edematous extremities  Scars
 Hematoma  Burn areas
 IV line  Cannula/ Fistula
 Blood transfusion line
5. Prepare materials
6. Observe infection control measures
7. Perform venipuncture
 Gently push the tube onto the needle holder so that the catheter inside the needle holder
penetrates the tube
 Blood flow should be visible at this point
 Allow tubes to fill until the vacuum is exhausted to ensure the correct blood to
anticoagulant ratio
 Blood won’t Flow
 May not yet be within the vein
 May have already passed through the vein
 May have missed the vein entirely
 May be pushed up against the inside wall of the vein

 Incomplete Collection or No Blood is Obtained

 Change the position of the needle. Move it forward ( it may not be in the
lumen) or move it backward (it may have penetrated too far)
 Adjust the angle (the bevel may be against the vein wall)
 Loosen the tourniquet. It may be obstructing blood flow
 Try another tube. There may be no vacuum in the one being used.
 Re-anchor the vein. Veins sometimes roll away from the point of the
needle and puncture site.

 Other Problems
 A hematoma forms under the skin adjacent to the puncture site- release
the tourniquet immediately and withdraw the needle. Apply firm pressure.
 The blood is bright red (arterial) rather than venous. Apply firm pressure
for more than 5 minutes.
8. Collect sample in container
 First: blood culture tube (yellow-black stopper)
 Second: Non-additive tube (red stopper/ SST)
 Third: Coagulation tube (light blue stopper)
 Last draw: additive tube in this order:
 SST (red-gray or gold): contains a gel separator and clot activator
 EDTA (lavender top)
9. Needle disposal
 Gently release the tourniquet before the last tube of blood is filled
 Remove the last tube from the needle
 Withdraw the needle in a single quick movement
 Apply pressure: quickly place clean gauze over the site, and apply pressure. You may ask the patient
to continue applying pressure until bleeding stops
  Technical Tip
 Patients often think they are helping by pumping their fists
 This is an acceptable practice when donating blood, but not in sample collection as this
can lead to hemoconcentration
 Cleansing the site
 Isopropyl alcohol swab
 Outward expanding spiral starting with the actual venipuncture site
 Allow the alcohol to dry: disinfect the site; prevent a burning sensation

Laboratory Safety
 is a careful process, with the goal of preventing injuries and diseases from occurring among
students, scientists, laboratory staff and the community
 Informs students about specific safety standards to take when doing lab procedures

I. Standard Safety Practices

 OSH Standards
 Article 162, Book IV of the labor code of the Ph
 To protect every working man against dangers of injury, sickness or death through safe
and healthful working conditions
 Scope: all working places
 Who enforces the standards?
o Regional Labor Offices and their districts

 Means of Exposure of Laboratory Personnel

 Percutaneous Inoculation
o Needle and Syringe
o Cuts and Abrasions from contaminated items
 Inhalation of Aerosols generated by work practices, work procedures, accidents
 Contact between mucous membrane and contaminated hands and surfaces
 Ingestion

II. Biosafety Levels

Basic teaching
BSL 1 Good Microbiological Technique
(bench top)
Primary health services
BSL 2 Diagnostic Teaching and Public
Health
BSL 3 Special Diagnostic
BSL 4 Dangerous pathogen units
 Prevent Contamination of  Protection of:
 Work  Worker
 Product  Co worker
 Environment  Community

III. Laboratory Accidents

 the most common injuries and diseases affect the muscular/ skeletal system, the skin and
the eyes

Muscular/ Skeletal Skin Eyes
Slips and Falls caused by Damage or burns Irritation and damage
rushing, spills and trip caused by from chemical
hazards chemicals, splashes and airborne
electricity or heat materials
Laboratory Mathematics
 Rounding off numbers

0.046

 Always place a zero before a decimal point

 In serology, 4 digits are needed
 If more than four digits, round off
 Round off to the nearest thousandths

 Preparing Dilutions
 dilution is a laboratory procedure in which the concentration of a sample, or solution is reduced by
the addition of solvent (diluent)
 In the laboratory, a dilution is commonly performed when the concentration of an unknown is
greater than the limits of linearity of a given quantitative procedure or when a working solution must
be prepared from a stock
 The technologist must be well educated in preparing solutions to yield accurate results from
preparing reagents and samples

 Simple Dilution
 Ratio between the volume of the original solution to the volume of final solution
1 𝑝𝑎𝑟𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
 2
= 𝑡𝑜𝑡𝑎𝑙 𝑝𝑎𝑟𝑡𝑠 (𝑠𝑎𝑚𝑝𝑙𝑒+𝑑𝑖𝑙𝑢𝑒𝑛𝑡)
 Example:
- 2mL of serum added to 8mL of diluent (2:10) or 1:5 dilution of 1/5
- A specimen is diluted by combining 3mL of serum with 21 mL of NSS. What is the
dilution of the serum?
 3mL (parts serum) + 2mL (parts NSS)= 24mL (total)
 Dilution= 3mL + 24 mL
3𝑚𝐿 24 𝑚𝐿 1
 + = or 1:8
3𝑚𝐿 3 𝑚𝐿 8

 Serial Dilution
 It is mixing and transferring a constant volume of serum into successive tubes containing
diluent.
 It is better to express all dilutions in fractions for easier computation.
𝟏 𝑷𝒂𝒓𝒕 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒓𝒆𝒅
 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒐𝒍𝒅
= 𝒕𝒐𝒕𝒂𝒍 𝒑𝒂𝒓𝒕𝒔 (𝑺𝒂𝒎𝒑𝒍𝒆+𝑫𝒊𝒍𝒖𝒆𝒏𝒕)

 Working and Stock Solutions

 The volume of the given solution times the concentration of that solution equals the
volume of the resulting solution time the concentration of the second solution
 C1V1= C2V2
 Where:
- C1: concentration of the stock solution
- V1: volume of the stock solution
- C2: Concentration of the working solution
- V2: volume of the working solution
 𝟏 𝑷𝒂𝒓𝒕 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆
 𝟐
= 𝒕𝒐𝒕𝒂𝒍 𝒑𝒂𝒓𝒕𝒔 (𝒔𝒂𝒎𝒑𝒍𝒆+𝒅𝒊𝒍𝒖𝒆𝒏𝒕)
 Example:
 Prepare a working solution of 500mL using a stock concentrate of 10x buffer
𝟏 𝒙
=
𝟏𝟎 𝟓𝟎𝟎𝒎𝑳
10x = 500 mL
X = 500mL / 10
X= 50 mL
(1)(10) = (50mL) (500ml)

 50 mL volume of 10x buffer

 450 mL volume of Dist H20

 ELISA Computations
 Each ELISA test kit has its own method/ formula for computing the cut-off value (COV)
 The technologist must be knowledgeable on the computations of the test kit to be used
 Basic processes like addition, subtraction, getting the mean, percent computations, more
than/ less than values, will be encountered.
 Usual Steps:
 Compute for Neg control mean
 COV
 Grayzone
 Checking assay validity
 Interpretation

 Example:

Control NC1 NC2 NC3 Ab+

0.088 0.089 0.090 1.220
Sample S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
0.045 ---- 2.891 0.010 0.270 0.298 0.520 2.697 0.060 ---- 0.300

1. Check assay validity:

 Negative control (R3)
 ODR3 < 0.170, 0.088, 0.089, 0.090 < 0.170
 Mean negative Control
o NCX < 0.150; 0.089 < 0.150
 Positive Controls (R4 & R5)
o Ab+ > 0.900; 1.220 > 0.900
o Ag+ > 0.900; 1.208 > 0.900
2. Compute for NCx:
𝑁𝐶1+𝑁𝐶2+𝑁𝐶3
 NCx =
3
0.088+0.089+0.090
 NCx = 3
0.267
 NCx = 3
 NCx = 0.089

3. Compute for COV:

 COV = NCx + 0.200
 COV+ 0.089 + 0.200
 COV+ 0.289

4. Compute for Gray Zone

 GZ = (+/-) 10% of COV
 = 10% x 0.289
 = 0.029

 Subtract  Add
 0.289 – 0.029  0.289 + 0.029
 0.260  0.318
 GZ= 0.260 -> 0.318

 Molarity
o Molarity (mol/L) is defined as the number of moles of a substance per liter of solution
𝒈𝒓𝒂𝒎𝒔
o 𝑴 = 𝑴𝑾 𝑿 𝒗𝒐𝒍𝒖𝒎𝒆
o 𝑵 = 𝑴𝒐𝒍𝒂𝒓𝒊𝒕𝒚 𝒙 𝒏
 n= number of protons exchanged in a reaction

 Conversions:
o 1000 uL = 1.0 mL
o 5 uL = 0.005 mL
o 250 uL = 0.250 mL
o 500 uL = 0.500 mL
Indicators of the Value of Diagnostic Tests
I. Sensitivity
 The ability of a test to detect very small amounts of the analyte
 The ability of a test to detect truly infected individuals. By detecting all infected individuals the
test will not produce false-negative results.
𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒
 𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 = 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒+𝐹𝑎𝑙𝑠𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑥 100
 Example:
o 100 Sera are tested; 5 Sera are from infected individuals; 95 sera are from non-infected
individuals
o Test Results: in comparison to the reference test, the test reveals only 4 positives among
the sera from the 5 infected individuals (the test produces 1 false-negative)
4
o 𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 = 𝑥100
4+1
o =80%

II. Specificity
 The specificity of an assay is the ability of the test to identify all non-infected individuals correctly
𝑻𝒓𝒖𝒆 𝑵𝒆𝒈𝒂𝒕𝒊𝒗𝒆
 𝑺𝒑𝒆𝒄𝒊𝒇𝒊𝒄𝒊𝒕𝒚 = 𝒙 𝟏𝟎𝟎
𝑻𝒓𝒖𝒆 𝑵𝒆𝒈𝒂𝒕𝒊𝒗𝒆+𝑭𝒂𝒍𝒔𝒆 𝑷𝒐𝒔𝒊𝒕𝒊𝒗𝒆

Example
o In comparison with the reference test, the test reveals 6 positives (all 5 of the infected
individuals and 1 false positive from the non-infected individuals) therefore, the test
correctly identified 94 of the non-infected individuals (produced 94 true negatives) and 1
false positive

𝟗𝟒
o 𝑺𝒑𝒆𝒄𝒊𝒇𝒊𝒄𝒊𝒕𝒚 = 𝒙 𝟏𝟎𝟎
𝟗𝟒+𝟏
o = 98.9%

III. Test Efficiency

 Test efficiency refers to the overall ability of a test to correctly identify all positives and
negatives (the absence of false-positives and false-negatives)
 It is a combination of the sensitivity and the specificity of an assay that determines the total
effectiveness of the assay.
𝑻𝒓𝒖𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆+𝑻𝒓𝒖𝒆 𝑵𝒆𝒈𝒂𝒕𝒊𝒗𝒆
 𝑻𝒆𝒔𝒕 𝒆𝒇𝒇𝒊𝒄𝒊𝒆𝒏𝒄𝒚 = 𝑻𝒓𝒖𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆+𝑭𝒂𝒍𝒔𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆+𝑻𝒓𝒖𝒆 𝑵𝒆𝒈𝒂𝒕𝒊𝒗𝒆+𝑭𝒂𝒍𝒔𝒆 𝑵𝒆𝒈𝒂𝒕𝒊𝒗𝒆 𝒙 𝟏𝟎𝟎

Example:
o 5 positives (4 from the infected group and 1 false positive from the non-infected group).
95 negatives (94 from the infected group and 1 false negative from the infected group)
𝟒+𝟗𝟒
o 𝑻𝒆𝒔𝒕 𝒆𝒇𝒇𝒊𝒄𝒊𝒆𝒏𝒄𝒚 = 𝟒+𝟏+𝟗𝟒+𝟏 𝒙 𝟏𝟎𝟎
o = 98%
 IV. Predictive Values
 Predictive values differ from the above parameters in that they describe the value of tests, taking
into account the actual prevalence of infection in the population being tested.
𝑻𝒓𝒖𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆
 𝑷𝑷𝑽 = 𝑻𝒓𝒖𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆+𝑭𝒂𝒍𝒔𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆 𝒙 𝟏𝟎𝟎
𝑻𝒓𝒖𝒆 𝑵𝒆𝒈𝒂𝒕𝒊𝒗𝒆
 𝑵𝑷𝑽 = 𝑻𝒓𝒖𝒆 𝑵𝒆𝒈𝒂𝒕𝒊𝒗𝒆+𝑭𝒂𝒍𝒔𝒆 𝑵𝒆𝒈𝒂𝒕𝒊𝒗𝒆 𝒙 𝟏𝟎𝟎

Example
o 50 positives (45 from the infected group and 5 false positives from the non-infected
group) 950 negatives (945 from the non-infected group and 5 false-negatives from the
infected group)

PPV= 90.0%
NPV=99.5%

Summary of Equations
Simple Dilution 1 𝑝𝑎𝑟𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
=
2 𝑡𝑜𝑡𝑎𝑙 𝑝𝑎𝑟𝑡𝑠 (𝑠𝑎𝑚𝑝𝑙𝑒 + 𝑑𝑖𝑙𝑢𝑒𝑛𝑡)
Serial Dilution 1 𝑃𝑎𝑟𝑡 𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟𝑟𝑒𝑑
=
𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑜𝑙𝑑 𝑡𝑜𝑡𝑎𝑙 𝑝𝑎𝑟𝑡𝑠 (𝑆𝑎𝑚𝑝𝑙𝑒 + 𝐷𝑖𝑙𝑢𝑒𝑛𝑡)
Working and C1V1= C2V2
Stock Solution 1 𝑃𝑎𝑟𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
=
2 𝑡𝑜𝑡𝑎𝑙 𝑝𝑎𝑟𝑡𝑠 (𝑠𝑎𝑚𝑝𝑙𝑒 + 𝑑𝑖𝑙𝑢𝑒𝑛𝑡)

ELISA 𝑁𝐶1+𝑁𝐶2+𝑁𝐶3
 NCx= 3
Computation
 COV=NCx + 0.200
 GZ=(+/-) 10% of COV
𝑔𝑟𝑎𝑚𝑠
Molarity o 𝑀 = 𝑀𝑊 𝑋 𝑣𝑜𝑙𝑢𝑚𝑒
o 𝑁 = 𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 𝑥 𝑛

𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒
Sensitivity  𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒+𝐹𝑎𝑙𝑠𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒
𝑥 100

𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒
Specificity  𝑥 100
𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒+𝐹𝑎𝑙𝑠𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒
Test Efficiency 𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 + 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒
= 𝑥 100
𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 + 𝐹𝑎𝑙𝑠𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 + 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 + 𝐹𝑎𝑙𝑠𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒
𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒
Predictive  𝑃𝑃𝑉 = 𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒+𝐹𝑎𝑙𝑠𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑥 100
Values 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒
 𝑁𝑃𝑉 = 𝑥 100
𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒+𝐹𝑎𝑙𝑠𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒