Professional Documents
Culture Documents
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 Serology
Study of the immune system The detection/measurement of elements of humoral
immune system (antibody)
Study of body’s defense against Help in the diagnosis of infectious diseases and
specific pathogen immunologic disorders
Study of reactions of a host when Determine immune status
foreign substances are introduced
into the body
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
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
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.
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.
Normal
Protein Response time concentration Increase Function
(mg/dL)
Opsonization,
C-reactive Protein 6-10 0.5 1000x
complement activation
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
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
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
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
Enzymes Substrate
Aryl sulfatase Sulfate esters
Proteoglycans (collagen)
Cathepsin
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
Erythrocytes: RBC
o carry oxygen and carbon dioxide
Leukocytes: WBC
o defense mechanisms
o Granulocytes: Neutrophils, Eosinophils, Basophils
o Agranulocytes: Monocytes, Dendritic Cells, Lymphocytes
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
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) 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
Passive Immunity
- an immunity in which antibodies produced elsewhere are given to the individual:
Active Immunity
- product of the individuals own immune system in response to a foreign antigen
Acquired Immunity
Active Passive
Natural source: Natural Source:
clinical/subclinical disease Congenital IgG (across placenta)
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
B. Lymphatic Tissue
Diffuse
Nodular
C. Lymphatic Organs
Lymph node
Spleen
Thymus
D. Lymphatic Vessels
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
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
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
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
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
Activated T cell
- Express receptors for IL2
- Activated T cells change their migration patterns
- Activated CD4 T cells can make different types of cytokines
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
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
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
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
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:
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
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
Symbols Isotypes
IgM Mu
IgD Delta
IgG Gamma
IgA Alpha
IgE Epsilon
Pepsin Digestion
- F(ab’)2
- No Fc recovery, digested entirely
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
Hinge region
Rich in proline residues (flexible)
Proline residues are target for proteolytic digestion (papain and pepsin)
Rich in cysteine residues (disulfide bonds)
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
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
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
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)
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
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
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
Properdin
- protects C3b and stabilizes C3 convertase
Factor I
- cleaves cell-bound or fluid phase C3b and C4b-> inactivates C3b and C4b
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
Antigens bind with serum antibodies Antigen does not bind with anything
(IgM, IgG) as serum does not have antibodies
Complement binds with the complex Complement does not bind
Addition of Hemolysin-sensitized Addition of Hemolysin-sensitized
RBC (as indicator) RBC (as indicator)
RESULT: NO LYSIS (+) 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
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
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
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
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
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
Testing:
Radioimmunosorbent Test (RIST)
Measures total IgE by capturing the antibody with solid-phase anti IgE
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
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
Drug Hypersensitivity
Antibodies are produced that react with the drug
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
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
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
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
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
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
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
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
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
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
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
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
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)
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
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
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
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”
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
INVASION MECHANISM
SpiC Protein
- Produced by Salmonella typhi
- Inhibits fusion of lysosome with phagosome
- Inhibit the formation of reactive oxygen
VACCINES
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
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
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)
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
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
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
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
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. 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
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
IMMUNE RESPONSE
1. Phagocytosis
2. Complement activation
Dependent on antibody production
INVASION MECHANISM
CONTRIBUTORY FACTOR
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
Capsule
o Inhibit phagocytosis
CLINICAL MANIFESTATION
IMMUNE RESPONSE
1. Antibody production
2. Complement activation
3. Mediated phagocytosis
LABORATORY DIAGNOSIS
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
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
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
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
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
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
LABORATORY DIAGNOSIS
Screening
o Rapid Test
o ELISA
o CLE-IA
Confirmatory
o Neutralizing assay
Monitoring test
o PCR
o Branched-DNA assay
ALGORITHM
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
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
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
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
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
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
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
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
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
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 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
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)
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
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
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
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
SUMMARY
Positive Result Negative Result
Direct Agglutination Agglutination No Agglutination
Hemagglutination Agglutination No Agglutination
Passive Agglutination Agglutination No Agglutination
Reverse Passive Agglutination No Agglutination
Latex Agglutination No Agglutination Agglutination
Hemagglutination Inhibition No Agglutination Agglutination
Coagglutination Agglutination No Agglutination
Anti-Globulin 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
Causes of False Positive and False Negative Reaction in Agglutination Testing and the Corrective Action
*too low incubation temperature may result in the *check appropriate temperature for the specific test
lack of association of antigen and antibody
Undercentrifugation: Cells may not be close Calibrate centrifuge to proper speed and
enough to interact. time.
Prozone phenomenon: too much patient Dilute patient serum containing antibody,
antibody for the amount of test. and repeat the procedure.
Reagent not active: may be caused by Refrigerate antisera, but don’t freeze
improper storage because loss of activity may occur.
Delays in testing procedure: This especially Once procedure has started, follow through
pertains to antiglobulin testing. Antibody until the end without delay.
may be eluted from rbc.
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
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 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)
Applications:
Gelling Substances:
Agarose- Most commonly used
Agar
Polyacrylamide
Cellulose acetate
Gelatin, starch
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
Zone Electrophoresis
o Proteins or nucleic acids are separated and localized into separate bands/zone
o Most common medium: cellulose acetate and agarose
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
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 Examples of Fluorophores
Fluorescein isothiocyanate (FITC) → emits green color; high intensity and
good photostability
Lissamine Rhodamine B → emits red light
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
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
Radioimmunoassay (RIA)
Makes use of radioactive labels
2 types:
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
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
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:
Prerequisites
Zone Phenomenon
Concentrations of either Ag or Ab higher than the equivalence may result in a weak reaction or no
precipitate being formed
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
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
Gelling Substances
- Agarose: most commonly used
- Agar
- Polyacrylamide
- Cellulose acetate
- Gelatin
- Starch
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:
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
Procedure:
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
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)
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
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 Testing Errors Post Testing Errors
Improper patient Improper measurements Transcription error in
preparation of specimen or reagents reporting
Mislabeled/ Unlabeled Dilution and pipetting Illegible reports
Specimen errors Report sent to wrong
Improperly transported Incorrect reagents used location
specimen Country algorithm not Information system not
Inappropriately stored followed maintained
test kits Use of inappropriately Recording
Right specimen stored/ expired reagents Interpretation
Right collection Laboratory professionals Turnaround time
Right labelling Reagents Report to right user
Right quantity Equipment
Right transport Selection of test-
Right Storage SOP(Standard Operating
Procedure)
Records
Biosafety
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
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
0.046
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)
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:
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%
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