MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 2 - ISBB

IMMUNOLOGY & SEROLOGY

Lecturer: Sir Jayson Sanchez, RMT
By: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King

WHAT IS IMMUNE SYSTEM? TYPES OF IMMUNITY

IMMUNOLOGY INNATE IMMUNITY ADAPTIVE IMMUNITY

- Study of Immune System “Certified Pakboy” “Nagsawa na/ Committed”
o Network of Organs (if one fails, there will be an abnormality of the Characteristic Non-specific Specific, Diverse, and with
process) Memory
o Function: Recognize, Respond, React, Destroy Cells Phagocytes Lymphocytes:
o Primary Goal: IMMUNITY (the state of being resistant to infection) Basophils B cells
Macrophage T cells
Immune PRIMARY SECONDARY Mast cells NK cells (bridges innate
System Dendritic cells and adaptive immunity)
Organs Response IMMEDIATE DELAYED
Organs Bone Marrow and Thymus Spleen, Lymph Node, Time
MALT, GALT Natural Stimulated
Functions Bone Marrow - Site of T and B cells Memory Without memory With memory
- Produce Hematopoietic antigen encounter Processes Phagocytosis, T cell activation (cell lysis)
Stem Cells - Filters blood from involved Antigen presentation B cell activation (Ab
- Site of B-cell maturation pathogens and foreign production) → cell lysis
- BURSA OF FABRICIOUS: substances Specificity Non-specific Specific
functionally similar to - Responsible for Span Short-term Long-term
appendix in human trapping or preventing Pathogen Toll-like receptors (TLR) Memory Cells
entry of organisms
Recognition
Thymus
- Site of T-cell maturation and
differentiation

HISTORICAL BACKGROUND

YEAR DISCOVERER DISCOVERY

1000 AD Chinese Practice of
Insufflation (administration
of antigen through nose to
achieve immunity)
1798 Edward Jenner Smallpox vaccine;
Cross Immunity
1862 Haeckel Phagocytosis
1865-1866 Gregor Mendel Mendelian Inheritance
1880-1881 Louis Pasteur Live attenuated (weakened)
MECHANISM OF INJURY
chicken cholera and anthrax
vaccine
Type 1 – Allergic or Anaphylactic
1883-1905 Metchnikoff Cellular Theory of Immunity:
PHAGOCYTOSIS (step by Type 2 – Cytotoxic
step process) Type 3 – Immune Complex Formation
1885 Louis Pasteur Live, attenuated rabies Type 4 – Cell-mediated or Delayed
vaccine
1890 Emil von Behring, Kitasata Humoral Theory of Immunity
1891 Robert Koch Type IV Hypersensitivity
Demonstration
(Tuberculosis)
1894 Jules Bordet Complement
1900 Ehrlich Antibody Formation Theory
1902 Portier, Richet Immediate – Anaphylaxis
Hypersensitivity
1903 Arthus Arthus Reaction,
Hypersensitivity
1903 Almroth Wright Opsonins
1930 Tillet and Francis C-Reactive Protein
1938 Marrack Antigen – Antibody Binding
1949 Salk and Sabin Polio Vaccine
1951 Reed Yellow fever Vaccine
1957 Burnet Clonal Selection Theory STAGES OF INFLAMMATORY RESPONSE
1975 Georges Kohler, Monoclonal antibodies
Cesar Milstein 1. VASCULAR RESPONSE
1977 Rosalyn Yallow Radioimmunoassay - Whenever collagen is exposed, it will trigger histamine release by
1985-1987 T cell Receptor Gene the mast cells → localized the vasodilation (blood vessel lumen
1986 Monoclonal Hepatitis B dilates/widens; spaces between the endothelial cells open).
Vaccine o Localized vasodilation facilitates WBC release from Circulating
1987 Susumu Tonegawa Antibody Diversity Pool (50%) to Marginating Pool (50%) through the endothelial
2005 Frazer Human Papilloma Virus spaces
(HPV) vaccine o Vasodilation only occurs at the area where histamine was
released
o Vasodilation decreases the blood flow → this allows WBCs to
move slowly → WBCs become attracted to chemoattractants
which are signals at the site of infection
2. CELLULAR RESPONSE OXIDATIVE PATHWAY NON-OXIDATIVE PATHWAY
- Phagocytosis (Mnemonic: “I criede”) NADPH oxidase : central killing of NADP oxidase: will induce
o Initiation, Chemotaxis, Recognition, Ingestion/Engulfment, microbes; facilitate conversion of depolarization of the bacterial
Digestion, Exocytosis NADH into NAD+ to produce first membrane (holes) → cell lysis
oxygen radical SUPEROXIDE
- Physical contact between bacteria and WBCs (oxidative pathway)
- 3 Interleukins released: - SUPEROXIDE will then be converted to a more stable oxygen radical →
o IL-1: β-pyrogen; induces fever (↑ temperature prevents microbial Hydrogen Peroxide (H2O2) by superoxide dismutase
survival in the body) - Hydrogen Peroxide is then converted to Hypochlorite (HOCl-) by
INITIATION

 ↑ WBC = ↑IL-1 = fever ↑ Myeloperoxidase (MPO)

 Viral: ↑Lymphocytes = ↓IL-1 = low grade fever <40oC o Myeloperoxidase deficiency: cannot kill catalase positive organisms
 Bacterial: ↑Neutrophil = ↑IL-1 = high grade fever >40oC (Staphylococcus)
o IL-6: stimulate the liver to produce Acute Phase Reactants,
specifically C-Reactive Proteins (CPR) - Excrete antigens (tiny) outside of WBCs which can be presented by

EXOCYTOSIS
o IL-12: Stimulate the NK cells (Kiss of death; automatically lyse Antigen presenting cells
foreign organisms) - End product of phagocytosis (innate response): antigens (captured
- Recruitment of phagocytic cell towards the site of infection by APCs)
- Chemoattractants/Chemotaxins – chemicals which initiate
chemotaxis
CHEMOTAXIS

o C5a – MOST POTENT CHEMOTAXIN

- 3 Stages of Chemotaxis
o Rolling = Initiate interleukin signaling; leave interleukins along ANTIGEN PRESENTATION
the way to guide other WBCs towards the site of infection - Bridges innate and adaptive immunity
o Adhesion = attach to blood vessel wall - Dendritic cells – Most potent APC; it can capture and present antigen
o Transmigration = WBCs squeeze out towards the site of despite being far.
infection; changes shape
- To recognize only pathogenic organisms; normal flora should be not APC T-cells
recognized MHC Class 1 → CD8+/ Cytotoxic
MHC Class 2 → CD4+/ T-helper
WBC BACTERIA - MHC Class 1: Viral / Intracellular Infection
RECOGNITION

Direct Primitive Pattern Pathogen Associated - MHC Class 2: Bacterial / Extracellular Infection (B cells → Memory
Recognition Recognition Molecular Pattern Cells/Plasma cells → Antibody production → Complement → Cell lysis
Receptor (PPRR) (PAMP) - End product of adaptive response: Antibodies
Indirect PPRR + OPSONIN PAMP
Recognition (coat bacteria/ (intracellular/extracellular) 3. RESOLUTION AND REPAIR
capsule) not recognized if coated - Collagen synthesis (heal)
with capsule - Mast cell → stop histamine-release → No vasodilation
- There is edema (swelling), temporary numbness, puss (small volume of
- A.k.a. Ingestion WBCs), and abscess (large volume of WBCs) in the process of healing
ENGULFMENT

- WBC Ingests pathogen → Phagosome (Vacuole) is formed

- Macroproteins → Microproteins: for easy antigen presentation

(means faster adaptive response)

Primary Digestion Secondary Digestion

DIGESTION

Phagosome + Lysosome = Activate HMP Shunt →

Phagolysosome → bacteria produce oxygen radical
is killed (harmful oxygen) →
Organism bursts (lysis: tiny - Function of CD4+ T lymphocyte:
antigen particles) o Activation of macrophages too enhance phagocytosis
*Lysosome: suicide sac of the cell because it contains enzyme that will o Inflammation to enhance killing
destroy protein. o Activation of T and B lymphocytes

OXYGEN RADICALS FROM THE HMP SHUNT

Superoxide (O2) First oxygen radical Kill anaerobic

organisms
Hydrogen Peroxide Kill most microbes Kill aerobic organisms
(H2O2) except catalase +
organism
Hypochlorite (HOCl-); Most powerful oxygen All organisms
bleach radical
- Hexose Monophosphate Shunt → process of converting sugar into a - B lymphocyte:
functional ATP (energy) o It will become plasma cell with “cartwheel appearance” nucleus
o NADH is produced → converts to NAD+ by NADPH oxidase → to become antibodies
releasing SUPEROXIDE o Either it will bind to antigen and initiate phagocytosis by
macrophage (ADCC –phagocytosis) or
o Bind to antigen and initiate complement activation

- Function of CD8+ T lymphocyte:

o Class I MHC – intracellular infection/virally-infected cell
o Cytotoxic = killing of infected cells by cytotoxins (cytolytic toxins)
o “kills not only the virus but the virally infected cell with pathogen”
 INNATE IMMUNITY - MONOCYTE/MACROPHAGES
- Consist of the defenses against infection that are ready for immediate o Largest cell in the peripheral blood
action  Largest cell in the bone marrow: Megakaryocyte
- Considered non-adaptive or nonspecific o Monocyte: macrophage in the blood
- Composed of 2 parts: o Monocytosis is involved in Tuberculosis and Chronic infection (long
term)
A. EXTERNAL DEFENSE SYSTEM o Slow motility, microbial killing, anti-tumor, intracellular parasite
- Prevents entry and establishment of pathogen in the body (preventive eradication, phagocytosis, secretion of cell mediators
action) o 1st type of Granules: Peroxidase, ACP, arylsulfatase
- Structural Barriers: o 2nd type of Granules: β-glucuronidase, lysozyme, lipase, but no ALP
Physical and Chemical o Releases:
Unbroken/intact skin Keratin (impermeable to most infectious agent)  1L-1β: regulates temperature
 1L-6: stimulates liver to release APR (CRP, as most potent)
Psoriasin Against gram negative-like E.coli
 IL-12: during phagocytosis; activate NK cells which will destroy cells
Mucosal Membrane Mucus/Sputum traps pathogens
without prior sensitization
Tears IgA antibodies in secretions (dimeric IgA)
Sweat/Sebaceous Lactate and fatty acid maintain skin at 5.6 pH - DENDRITIC CELLS
Hair/Cilia Traps pathogen entry o Most effective antigen presenting cell (has many protrusions)
o Most potent phagocytes (can eat, kill, and present)
- Biological Barriers: o Resemble dendrites in structure
Normal Flora (Microbiota) Competitive Exclusion
Gut produce colicins Binds negatively charged surface of certain bacteria MACROPHAGES ACCORDING TO LOCATION
and penetrate to kill
Skin S. epidermidis LOCATION COMMON NAME ANOTHER NAME
Oral Cavity Viridans strep Lungs Alveolar Macrophage Dust Cells
Vagina L. acidophilus Liver Kupffer Cells
Intestine Gram negative anaerobes (Bacteroides) Brain Microglial
Enterobacteriaceae (10% only E.coli) Bone Osteoclasts
A. INTERNAL DEFENSE SYSTEM Connective Tissue Histiocytes
Placenta Placental Macrophage Hoffbauer cells
- Kills pathogen which has entered and established inside the body (corrective
action) Spleen (graveyard) Splenic macrophage Littoral cells
o Cellular Factors: Phagocytosis Blood Monocytes
o Soluble Factors: Acute Phase Reactants (APR) Kidney Mesangial Cells
 Stimulate phagocytosis of microorganisms Skin Skin Epithelial Macrophage Langerhans cells
Synovium Type A lining Cells
CELLULAR FACTORS
PHAGOCYTOSIS
1. BASOPHIL AND MAST CELLS - “CELL EATING CELL”
- BASOPHIL - Consist of 7 main steps (C.D Stevens)
o Histamine, cytokines, growth factors, and small amount of heparin - 2 types:
 Histamine: for vasodilation 1. DIRECT – via PPRR, PAMP and
 Heparin: natural anticoagulant of the blood to maintain fluidity 2. INDIRECT – via OPSONINS
inside the blood; inhibit thrombin so fibrinogen is not converted to
fibrin to form clot that will cease the blood flow → WBC cannot PHAGOCYTIC PROCESS
move freely STEP MECHANISM
 Cytokines: messenger chemicals of the body (ex. ILs) INITIATION - Physical contact between WBC and Foreign Cell
 WBC – used up = ↓ WBC = Growth factor will signal the WBCs to CHEMOTAXIS - Recruitment of immune cells to the site of infection
mature faster towards the bloodstream - Positive and Negative Chemotaxis
o Regulates T-Helper Cell response → stimulate B cells to produce IgE o Positive chemotaxis: if chemotaxins are
o Has a short life span; pulled out and destroyed by littoral cells (splenic released, the WBCs will move towards the site of
macrophages) infection
- MAST CELLS o Negative chemotaxis: if chemotaxins are
o Has a different lineage from basophil released, the WBCs will move away from the site
o Contains ACP, ALP, proteases, and many histamines unlike basophils of infection
o Involved in allergic reaction and antigen presentation - WBC movements aided by chemical messengers
o Can enhance and suppress adaptive immune response called CHEMOTAXIN/CHEMOATTRACTANTS:
 If mast cells release histamine → Enhance immune response o C5a, C3a, CRP, IL-8
 If mast cells do not release histamine → Suppress  C5a: most potent chemoattractant
o Without this process, WBC movement is
2. PHAGOCYTIC CELLS
RANDOM
- NEUTROPHIL (Segmented Neutrophils/Segs/Microphage) - Diapedesis: squeezing out of WBC from blood
o Principal Phagocyte; First WBC to go to the site of infection; increased vessel to tissue
in bacterial infection
o 2 Population of Granules: DIAPEDESIS
 Primary/Azurophilic
 Contains MYELOPEROXIDASE (MPO), Lysozyme, Elastase, 1. ROLLING
Proteinase 3, Cathepsin G, and Defensins - Leave cytokines/IL along the way to guide/signal other WBCs towards
 Secondary/Specific the site
 Lysozymes, Lactoferrin, collagenase, gelatinase, and - Neutrophil contains L-selectin (Leukocytes-selectin): will bind to the
respiratory burst components (oxygen radicals). Lewis (Sialyl-Lewis X) antigen present in endothelial cells
o Lactoferrin: temporarily binds iron to deprive bacteria of it  Lewis is not intrinsic to RBCs but the surrounding tissues (blood
 Receptors (also present in macrophages): FcyR, CR1, CRP- vessels; endothelial cells)
receptor → facilitate the binding of neutrophil to opsonins - Shedding of L-selectin
- EOSINOPHIL - Integrin of the neutrophil will bind to E-selectin (Endothelial-selectin)
o Important Role: Regulation of Immune Response (ex. Mast Cell)
o Frustrated (indirect) phagocytosis 2. ADHESION
 Eosinophils cannot directly ingest helminths since eosinophils are - WBCs will adhere to endothelial cells
smaller than them 3. TRANSMIGRATION
 IgE must coat the helminths (serves as opsonins) so that eosinophil - Squeezing of WBC out of the tissues towards the pathogens
can destroy the helminths (not eat) - Whenever WBC successfully arrived at the site of infection, WBC eats
 Eosinophil will bind to IgE (FcEpsilon Receptor; Epsilon heavy the microbes (phagocytosis)
chain), resulting to degranulation of eosinophil - Activating substances released by bacteria and damaged tissues
 Cationic Proteins (Major Basic Protein) released → (chemoattractants)
Depolarization of membrane → Helminth death.  Lipopolysaccharides, interleukin-1, and tumor necrosis factor α
o Increased in allergic reaction and parasitic infection  C3a, C5a, chemokines, histamine, prostaglandins, leukotrienes
o Granules content: Catalase, Lysozyme, Cytokines, Growth Factors,
Cationic Proteins (Eosinophilic Cationic Protein), and MAJOR BASIC
PROTEIN (MBP), Histaminase
 CHRONIC GRANULOMATOUS DISEASE

NEUTROPHIL REDUCTION TEST METHOD:

- Nitroblue tetrazolium dye (NBT) converted into a colorless complex
FORMAZAN by NADPH oxidase
- If there is NADPH oxidase deficiency/ CGD → Blue colored NBT is not
reduced to formazan, colorless crystals
- BLUE: (+) for CGD

TOLL – LIKE RECEPTORS

RECEPTOR SUBSTANCE RECOGNIZED TARGET

ORGANISM
TLR FOUND ON CELL SURFACE (EXTRACELLULAR)
TLR 1 Teichoic Acid and Peptidoglycan; Mycobacteria, Gram
Lipopeptides Positive
TLR 2 Lipoproteins Gram-negative
Bacteria
TLR 4 Lipopolysaccharide Gram-negative
Bacteria, RSV fungi
RECOGNITION DIRECT PHAGOCYTOSIS TLR 5 Flagellin Bacteria with flagella
- Pathogen recognition receptor: distinguish cell from non- TLR 6 Lipopeptides, Lipoteichoic Acid, Mycobacteria, Gram
self Zymosan positive, Yeast
Note: - PAMP (Pathogen associated molecular patterns) → TLR FOUND IN ENDOSOMAL COMPARTMENTS (INTRACELLULAR)
Receptors are present in the foreign organism TLR 3 dsRNA RNA virus
found in o Found in: TLR 7 & 8 ssRNA RNA virus
phagocytic cells  Gram-Positive: Peptidoglycan TLR 9 dsDNA DNA virus, Bacterial
 Gram-Negative: Lipoproteins, LPS DNA
 Yeast: Zymosan
TLR 10 Unknown Unknowns
 Flagella: Flagellin
- Toll-like receptors (TLR)
o Toll → protein originally discovered from fruit fly 3. NATURAL KILLER CELLS
Drosophila (antifungal immunity in adult fly) - aka “kiss of death”
o Very similar molecules found in Human WBC: - First line of defense against cells that are:
phagocytes o Virally infected
o Once bind, produce cytokines and chemokines o Cells infected with intracellular pathogen
- C-type Lectin Receptor (CLR) o Tumor cells
o Bind to Mannan and β-glucans (found in fungal cell - Appearance: LARGE GRANULAR LYMPHOCYTE
walls) - Ability to recognize damaged cells, eliminate such target cells without prior
o Once bound, produce cytokines and chemokines exposure
- Cytokine production: - CD Markers: CD16 (FcRy) and CD56 (CAM) Positive
o Retinoic acid-inducible gene-I-Like Receptor (RLR) - Stimulated by: IL-12, Interferon alpha, Interferon beta
o Nucleotide-Binding Oligomerization domain - Produces cytokines when activated: Interferon-gamma, TNF-α (help recruit
Receptor (NOD) T cells)
- Link between Innate and Adaptive Immunity
INDIRECT PHAGOCYTOSIS - NK Cell Mechanism: constantly monitors potential target cells through…
- Opsonins o Inhibitory Signal:
o Coats the foreign cell to enhance the phagocytosis  Presence of MHC Class I in cells (all cells in the body have these)
o Example: CRP, C3b, Antibodies  Any cell who do not express MHC Class I, will be recognized
 CRP: most potent opsonin as a tumor and is eliminated by NK cells
INGESTION/ - Forms PHAGOSOME (vacuole)  Receptors of NK cell which binds to MHC-I
ENGULFMENT  Killer-cell Immunoglobulin-like receptors (KIRs)
DIGESTION Primary Digestion  CD49/NKG2A receptor
- Lysosomal Granules + Phagosome = o Activating Signal:
PHAGOLYSOSOME  Cancer cells:
- CHEDIAK HIGASHI: defect in lysosomal enzyme  Decreased to lack MHC-1 aka “Missing Self”
(lysosomal granules fuse together)  Under stress releases CD16 and NKG2D (bind to NK cell)
 When either of the two met, NK cells will be activated
Secondary Digestion  Viral infection:
- Oxidative burst or NADPH oxidase  Foreign proteins recognized by and bounded to an antibody
- Digestion of Microbes by Hydrolytic Enzymes  Protein remnants of virus/organism left outside the cell
- CHRONIC GRANULOMATOUS DISEASE: defect in  ADCC: CD16 receptors of NK cells bind to the immobilized
NADPH oxidase (cytochrome oxidase deficiency) antibody (also available in Monocytes, Macrophage,
Neutrophil)
2 Different Processes: o Once activated, they release:
1. Oxygen-Dependent - Oxidative Burst via HMP shunt  PERFORIN: cause destruction of the target cell of NK cell
o Production of Oxygen Radicals  GRANZYMES
o Superoxide, H2O2, Hypochlorite
o Nitric Oxide: oxygen radical that reacts with ANTIGEN PRESENTATION
superoxide - Follows phagocytosis
- Most potent APC: DENDRITIC CELLS
2. Oxygen-Independent – NADPH oxidase
- Via Major Histocompatibility Complex (MHC)
o Depolarization of Membrane
- MHC CLASSES:
 Hydrogen and Potassium ions enter the
vacuole
 pH is altered → activate proteases MHC Class I Present antigen to CD8+ T cells Intracellular
o Examples: MHC Class II Present antigen to CD4+ T cells Extracellular
 Defensins – from lysosomal granules - CD4 T-helper: helps B cell to 1.) be activated, 2.) identify antigen
 Cleave segments of bacterial cell wall - CD80/86 and CD28: allow the binding/attachment of APC and T-cells
 Wide spectrum (g+/-, fungi, some viruses)
MAJOR HISTOCOMPATIBILITY COMPLEX
 Cathepsin G – damages cell membrane
EXOCYTOSIS/ - Release debris (fragmented antigens) outside the cell - LOCATION: Chromosome 6p (short arm)
EGRESS - Produces: Human Leukocyte Antigens (HLA)
- Basis of T cell to identify self from non-self
- Inherited haplotypes (one from each parent)
- Major Function: Aids in Antigen Presentation to T cells
- Test of Choice: MOLECULAR METHODS
 2. Mixed Lymphocyte Reaction
- PRINCIPLE: Quantify the degree of Class II MHC compatibility
between potential donors and a recipient
- For easy understanding: Donor T cell and Recipient T cell are mixed
o (+) proliferation means incompatible
o without proliferation compatible
- Steps:
o Donor lymphocytes (DL) will be x-irradiated or with Mitomycin C
serves as stimulator cells
o Recipient Lymphocytes (RL) will be responder cells
o Increase proliferation of RL means T cell be responder cells
o Measured by the uptake of Thymidine into cell DNA
o Intense proliferation of RL = poor prognosis for graft survival

NEWER DETECTION METHODS

METHOD NOTES
ELISA crossmatch Tests for: HLA ANTIGEN
Uses purified HLA antigens instead of lymphocytes
Flow Cytometric Uses T or B lymphocytes or purified HLA antigens
Antibody Screen coated onto Microparticles
Multiplex Tests for: PATIENT’S HLA ANTIBODIES
Immunoassay
- MHC Class III: C2, C4, Factor B, TNF, 21-hydroxylase (LUMINEX) (+) Mean Fluorescence Intensity (MFI)

MHC Class I MHC Class II MHC Class III HUMORAL FACTORS

Location All nucleated B cells, - aka BIOLOGIC RESPONSE MODIFIERS
Cells Macrophage, - MODULATE individual’s own immune system
Neutrophils, RBC - 4 Main Sources of BRMs secreted by Mononuclear Leukocytes:
Dendritic cells
Antigen - Present to - Present to CD4+ Not Capable B lymphocyte Secrete specific ANTIBODIES
Presentation CD8+ - Exogenous T lymphocyte Secrete soluble mediators
- Endogenous Antigen (e.g., IL-2, GM-CSF, IFN-γ, TNF-β)
Antigen - Bacterial Antigen NK lymphocyte Secrete IFN-α
- Viral or Cytosol Monocyte & Secretes IFN-α, IL-1, TNF-α, GM-CSF, M-
Antigen Macrophages CSF
Antigens HLA-A, HLA-B, DP, DQ, DR Benett - Interferon (IFN): interfere with viral replication
HLA-C Goodspeed - Interleukin (IL): communication between leukocytes
Structure α1, α2, α3 = 3α α1, α2 = 2 alpha - Small, soluble proteins secreted by WBC, and a variety of cells
(Connected by β2-m = 1 β β1, β2 = 2 beta - Chemical signals functions primarily in recruiting other immune cells to the
disulfide bonds) site of infection
o ↑ factors released = ↑ WBC response at the site of infection
DISEASE ASSOCIATION
CYTOKINES
HLA PRESENT DISEASE ASSOCIATED - Polypeptide product of activated cells that control a variety of cellular
DR2 Goodpasture’s Syndrome, Multiple Sclerosis responses and thereby regulate the immune response
DR3 SLE, Autoimmune Thyroid Diseases, Dermatitis - Non-specific in chemical structure
Herpetiformis - Migratory Inhibitory Factor (MIF) – first cytokine activity described
DR4 Rheumatoid Arthritis o T cell - derived activity
B27 (~90%) Ankylosing Spondylitis (aka Ankyloarthritis, o Immobilized macrophage migration
Bamboo Spine Disease) o Retention and accumulation of phagocytes at sites of inflammation
B8 Celiac Disease, Dermatitis Herpetiformis o Cytokine Examples:
Cw6, B17, B13 Psoriasis vulgaris  Interleukins (IL), Tumor Necrosis Factor (TNF), Interferon (IFN),
B47 Congenital Adrenal Hyperplasia Transforming Growth Factor (TGF; hasten maturation of WBCs)

POLYGENIC DISEASES WITH MHC ASSOCIATIONS INNATE IMMUNITY CYTOKINES ADOPTIVE IMMUNITY
CYTOKINES
Disease MHC Gene OMIM No. Chemokines IFN-γ
Abacavir hypersensitivity HLA-B57:01 142830 IFN Type 1 (IFN-α & IFN-β) IL-2, IL-4, IL-5, IL-13
Ankylosing sppondylitis HLA-B27 106300 IL-1, IL-6, IL-10, IL-12, IL-15, IL-18 LYMPHOTOXIN
Autoimmune thyroid disease HLA-DR3 608173 TNF TGF-B
Behcet disease HLA-BS1 109650 - Types of release:
Celiac disease HLA-DQ2, DQ8 212750 o Autocrine – affects the same cell that secreted it
Multiple sclerosis HLA-DR, DQ 126200 o Paracrine – affects target cell in close proximity (ex. IFN-γ)
Narcolepsy HLA-DQB1 *06:02 161400 - CYTOKINE STORM: massive overproduction of cytokine leading to shock,
Rheumatoid arthritis HLA-DR4 180300 multi-organ failure, death
Type 1 diabetes HLA-DR, DQ 222100 o ↑↑↑ APRs (protein – maintains oncotic pressure) → water is pulled
towards the blood vessel from tissues/organ → leading to
dehydration in tissues/organ → blood flow and oxygenation is
LABORATORY TESTS
decreased → multi-organ failure/death
1. Microlymphocytotoxicity Test INTERFERON
- aka Complement-Mediated Cytotoxicity - Interfere with VIRAL REPLICATION
- Purpose: To identify specific HLA antigen - FUNCTION:
- PRINCIPLE: Presence of antigen on the peripheral blood o Enhance specific gene expression
lymphocytes, in addition or reagent anti-HLA and complement (under o Inhibit Cell Proliferation
oil) will cause them to become porous and unable to exclude the o Augment Immune Effector Cells
added dye; (Oil – prevents evaporation) - Examples:
- Dye: Trypan Blue (penetrate dead cells)
(+): BLUE
Type IFN Name Secreted by Functions
- Reagent: Battery of reagent antisera
I IFN- Leukocyte Leukocytes, Activates NK cells
- Specimen: ACD or Phenol-Free Heparinized Blood
α IFN Null cells Inhibits Viral Replication
- AHG: Enhance sensitivity
o T-cell – detection of Class I antigens IFN- Fibroepithelial dsRNA-induced Inhibits Viral Replication
o B-cell – detection of Class II antigens β IFN Fibroblast
- Microscope used: Phase Contrast Microscope II IFN- Immune IFN T Cells, Th1, Increases expression of
γ NK cells* MHC class I & II
*NK cells will release IFN-γ which will signal neighboring cells of the viral
infection
 TUMOR NECROSIS FACTOR - Apolipoproteins synthesized in the liver
Principal mediator of Acute Inflammatory response to Gram Negative - High Affinity for HDL (HDL transports

AMYLOID A
-
SAA to site of infection)

SERUM
- Site of Production: from LPS-activated Macrophage
- Function: ~5-8 ug/mL - Function: Activate Monocytes and
1. stimulate recruitment of phagocyte to site of infection Macrophage
2. activate recruited phagocyte to kill microbes - ↑ in Chronic Inflammation,
- TNF Concentration: Atherosclerosis, and Cancer
o Low concentration: induce acute inflammation - Protein synthesized in the Liver
o Moderate Concentration: mediates systemic effect of inflammation - Function: General Plasma Inhibitor of
o Large concentration: causes clinical and pathologic abnormalities proteases released from leukocytes
 Septic Shock syndrome: complication of severe gram-negative - Inhibits ELASTASE; degrade elastin and
bacterial sepsis fiber (in chronic pulmonary inflammation,

α-1-ANTITRYPSIN
- Examples: elastase damage lung tissue)
o TNF-α – CACHETIN o Elastase normally destroys type II
145-270 mg/dL
o TNF-β – LYMPHOTOXIN pneumocytes in the lungs which
2-4 g/L may lead to premature emphysema
BETA – LYSIN - Regulates: Elastase, IL-1β, IL-6, TNF-a
- Released by platelets during coagulation, heat stable - AAT Deficiency: Premature
- Effective against gram-positive except Streptococcus Emphysema, Liver Disease (destruction
of parenchymal cells in the lungs
INTERLEUKINS develops into Emphysema or ldiopathic
- Peptides and proteins that acts as signal molecules Pulmonary Fibrosis)
- Mediate local interactions between WBCs but DO NOT BIND ANTIGEN - Function: lysis of cell

COMPLEMENT
o Each IL functions through a separate receptor system - Nine series of serum proteins normally
- Site of Production: Leukocytes present and mediates inflammation
- Nomenclature: According to order of discovery and characterization
- IL-3 → multicolony stimulating factor

- Function: ANTIOXIDANT

HAPTOGLOBIN
- Mechanism: binds irreversibly to free
hemoglobin (because free hemoglobin is
40-290 mg/dL toxic)
- Free Hemoglobin mediates Oxidative
Damage
- Complex is cleared by Kupffer Cells
- Cleaved by Thrombin to produce Fibrin
Clot
Clot increases strength of a wound
FIBRINOGEN

-
- Clot stimulates endothelial cell adhesion
200-400 mg/dL and proliferation
- Promotes aggregation of RBC and
Platelet by making blood viscous
- Risk: may develop Coronary Artery
Disease
- Principal Copper-transporting Protein
CERULOPLASMIN

- Binds Six (6) Cupric ions per molecule

- Converts toxic Fe3+ (ferric) to non-toxic
ACUTE PHASE REACTANTS Fe2+ (ferrous)
20-40mg/dL
- Depletion will lead to WILSON'S
- GLYCOPROTEINS DISEASE
- Normal serum constituents; increase rapidly during infection, injury, tissue o Copper is ↓ in serum, Copper in
trauma urine is ↑
- Function:
o Promote phagocytosis
o Limit Destruction by proteolytic enzymes (α1-antitrypsin/α2- INFLAMMATION
macroglobulin)
- Site of Production: HEPATOCYTES within 12-24 hours in response to - “Inflammare” = to set on fire
increased cytokines - Body's overall reaction to injury or invasion by an infectious agent
- Negative APR: - Brings cells and humoral factors to the injured area
- MAIN PURPOSE: attract cells to the site of infection for phagocytosis
o Albumin - Roles: INITIATE, AMPLIFY, SUSTAIN
o Transferrin (downregulated) - Primary process in inflammation: LOCALIZED VASODILATION
- Initiated and sustained by Cytokines: IL-1, IL-6, TNF-α - Final process in inflammation: RESOLUTION and REPAIR by Fibroblast
o produced by Monocyte and Macrophage at the site of inflammation - Acute Inflammation: early stage of infection; short term; begins process of
- Half-life: mostly 2-4 days (CRP = 5-7 hours) tissue repair
- CRP → most potent Acute Phase Reactant - Chronic Inflammation: prolonged infection; long term
APR
- Stages of inflammation
NORMAL DESCRIPTION o Vascular Response: Vasodilation (increased blood supply & capillary
PROTEIN CONCENTRATION permeability)
- Originally thought to be antibody to C- o Cellular Response: Margination and Migration; Phagocytosis
0.47- 1.34mg/L polysaccharide of Pneumococci o Resolution and Repair: Fibroblast proliferation resulting to
- Produced by the liver from IL-6 1. Total Repair
Risk for CVD: stimulation 2. Abscess Formation
C-REACTIVE PROTEIN

- Function: Opsonization, Complement 3. Granuloma Formation (scar)

<1mg/L = Low Risk activation
- Main Substrate: Phosphocholine CARDINAL SIGNS OF INFLAMMATION
1-3mg/L = Average - Antibody-like function
Risk - Chronic Inflammation: SIGN ENGLISH TERM CAUSES
>3mg/L = High o ↑ CRP react w/ endothelial cells Rubor Erythema/Redness ↑ blood flow and vasodilation
Risk o Stimulate vasoconstriction, Platelet ↑ blood flow, ↑ IL-1β release of
Activation, Thrombosis (clot Calor Heat
WBCs
Hs-CRP = detect formation) and vascular inflammation Vasodilation, plasma leakage in
future CVD Inactivation: Heat serum to 56oC for 30 Tumor Edema
- tissue
minutes Pressing of nerve endings
Dolor Pain
(Pacinian corpuscle)
Repeated primary 4 cardinal
Functio laesa Loss of Function
signs, death of organ
 ADAPTIVE IMMUNITY Th2: IL-4, IL-5, IL6, IL-9, IL-10, IL-13
 Help B cells produce Antibody; Regulates B-cell Activity
- Third line of defense  T-regulatory: CD4 and CD25
- Types:  5% of CD4+ Cells; Suppress Immune Response to Self-
o Humoral Immunity: Antibodies Antigens
o Cell-mediated Immunity: T cells, B cells o T cytotoxic Cells: CD8
- Characteristics:  1/3 of Population
o Specific for each pathogen
o Ability to remember a prior exposure (memory cells) 4. ACTIVATED T CELLS
o Increased response to pathogen upon repeated exposure o Produce CD25: receptor for Il-2 (secretes cytokines enhance B-cell Ab
o Select cells that will be helpful and not harmful to the host production); prevent autoimmunity

CELLS OF ADAPTIVE IMMUNITY SUB-POPULATIONS OF T-CELLS

1. Th9: produce IL-9; proinflammmatory effect
T CELLS o Wards off fungi and extracellular bacteria at epithelial surfaces
- 60-80% of Lymphocyte Fraction o Stimulate growth of hematopoietic cells (esp. Mast Cells)/ during allergic
- Site of production: bone marrow reactions
- Site of differentiation: THYMUS (Thymocytes) 2. Th17: produce IL-17 and IL-22
- FUNCTION: Cell-Mediated Immunity o ↑ Cytokines = increased inflammation and bone destruction

DEVELOPMENTAL STAGES B CELLS

- Site of differentiation: BONE MARROW (Stromal cells forming niches)
1. DOUBLE NEGATIVE - FUNCTION: Humoral Mediated Immunity (produce antibodies)
o No CD4 and CD8 markers - Phases of B cell development:
o Proliferate actively due to IL-7 (created at the outer cortex of the thymus) o Phase 1 – Antigen Independent
o Gene rearrangement for T-Cell Receptor (TCR)  Pro-B cells: rearrangement of genes coding for heavy and light
o Allelic Exclusion: screening process before double negative thymocytes chain Ab
can become double positive; selection of an allele on 1 chromosome  Pre-B cells: synthesis of heavy – chain, mu-chain
only  Immature B cells: presence of complete IgM
 Hallmark of Adaptive Immune System: pre-existing diversity of
2. DOUBLE POSITIVE receptor for Antigen
o Express both CD4 and CD8  CENTRAL TOLERANCE: eliminate B-cell bearing self-
o Positive Selection: retain thymocytes with functional TCR reactive receptors
o MHC Restriction: selects thymocytes that interact only with self MHC  Mature B cells
antigens (activates kinases; change in shape and motility; ↑ cell  Surface IgD is present: not required for B cell function;
survival) prolong B-cell life-span
o Negative Selection: apoptosis of cells with strong reactions with self-  Occurs in the Spleen: Marginal Zone B cells or Follicular B
peptides other than MHC cells
o Clonal Deletion: eliminates clone capable of autoimmunity
 Marginal Zone B cells: remain in the spleen to quickly
respond to Blood-borne pathogen
Positive selection:
 Follicular B cells: migrate to secondary lymphoid organs
(recirculate).
o Phase 2 – Antigen Dependent Phase
 B cells are activated when it meets an antigen and produce CD25
o Phase 3 – Differentiation into Plasma Cells
 Plasma Cells: most fully differentiated lymphocyte
 Function: Antibody production
 Not normally found in blood, but in the lymphoid organs or
bone marrow
 Can survive longer in the niches in the Bone Marrow
surrounded by Stromal Cells
 Stromal Cells provide chemical stimulation by cytokines
- Lymphocyte: not an end-stage cell; is a surveillance cell

Negative selection:

3. MATURE T CELLS
o Either CD4 or CD8 is present
o CD is dependent on Strength of Reaction to MHC Protein and to which
cytokines they are exposed
o T-Helper Cells- CD4 (2/3 of Population)
 Th1: production of IFN-γ, IL-2, TNF-β;
 Activate Cytotoxic Lymphocytes and Macrophages
ACTIVATORS OF LYMPHOCYTES
- Monoclonal Activators – Antigens MECHANISM OF T & B-CELL INCREASE & DECREASE
- Oligoclonal Activators – Superantigens
- Polyclonal Activators – Mitogens (induces mitosis)
o B cell Mitogen – Lipopolysaccharides
o T cell Mitogen – Concanavaline A, Phytohemagglutinin
o Both T and B mitogen – Pokeweed Mitogen

LABORATORY IDENTIFICATION OF LYMPHOCYTES

- Density Gradient Centrifugation
o Harvest lymphocytes using Ficoll-Hypaque which will serve as a
mesh/filter (SG 1.007-1.114)
o The rest of the cells will settle at the bottom after centrifugation while
lymphocytes remain and are separated
- Roswell Park Memorial Institute Medium (RPMI 1640)
o Traditional culture medium for human lymphocyte
- Flow cytometry
o GOLD STANDARD for Lymphocyte Identification

- Antigen recognition: Phagocytosis → Antigen Presentation to Naïve T

and B- cells
- T cells and B cells will increase whenever antigens are presented
- T cells will activate B cells → Plasma cells → Antibodies
o While other T cells will activate more b cells and kill more tumor/virally
infected cells; Cell mediated
o Antibodies produced will bind to antigen so that it will activate the
COMPLEMENT and kill the antigen (Humoral immunity)
- T cell and B cells → Apoptosis
- Memory Cells
o Will survive so that whenever there is another exposure to the same
antigen, the memory cell will recognize the antigen and activate T
cells and B cells and repeat the process again. Has a faster effect
upon secondary exposure.
- Antibody production: as early as 7 days
- Full blown: 14 days/2 weeks
- Effector T lymphocyte: Cytotoxic T-cell which can kill other virally infected
cells.

ANTIBODIES

- Proteins that are different in structure, bind to antigen

- Serum Protein Electrophoresis (SPE): Gamma Region at pH 8.6
- Function: When bound to antigen, complement system is activated leading
to cell lysis
- Site of Production: Plasma Cells, Spleen, Lymph nodes
o All proteins in the body are synthesized in the liver, except for
antibodies
- Produced inside the gamma globulins
- Determinant: PARATOPE
B AND T-CELL DIFFERENTIAL TESTS
PLASMA CELLS
T CELL B CELL
Test E-rosette Test Surface Ig Detection - produce and secrete antibody
- Chemical composition: Protein in nature
Principle Based on CD2 receptor on IgD and IgM are on the - Function: Binds Exogenous Antigen; Viral Neutralization
Sheep RBC surface of naive B cells - Structure:
Positive Result “ROSE” agglutination under o 2 Light Chains (Kappa or Lambda) and
the Microscope o 2 Heavy Chains (Gamma, Alpha, Mu, Epsilon, Delta)
End Product of Cytokines Antibody
Activation
Location Paracortical Region Cortical Region

FORMS OF ACQUIRED IMMUNITY

TYPE MODE OF HOST ANTIBODY
ACQUISITION PRODUCTION
ACTIVE Natural Infection Yes
(Antigen is Artificial Vaccination (Long term duration)
given)
PASSIVE Natural In-vivo transfer No
(Antibody is (IgA in colostrum) (Short term duration)
given) Artificial Infusion of
Plasma/serum - Structural Regions:
(convalescent plasma for o Variable Region: Amino Terminal End
COVID19/SARS-CoV-2) o Constant Region: Carboxy Terminal End
o Hinge Region: contains PROLINE
 Proline is an amino acid which accounts for the FLEXIBILITY of
the hinge region
 Between CH1 & CH2
o Fc Region: Fragment of Crystallization; WBC Binding Site
 Granules of WBCs will crystallize
o CH2 (IgG), CH3 (IgM): Complement Binding Site: second to the last
constant heavy chain
 IgM and IgE = has CH4
- Enzyme Digestion:
o PAPAIN
 Target: 1st disulfide bond FIVE ANTIBODY ISOTYPES
 3 Fragments = Fc + FAB + FAB ISOTYPE SUBCLASSES ACTION/FUNCTION
o PEPSIN IgG - IgG1: most efficient in - Anamnestic Response
 Target: 2nd disulfide bond crossing placenta Antibody
 2 Fragments = Fc + F(ab)2 - IgG2: cannot cross placenta; - Activates Complement
Longest may cross but slower Classical Pathway;
half-life: - IgG3: most efficient in fixing Opsonin, Viral
23 DAYS complement Neutralization, ADCC
- IgG4: cannot fix - Most Abundant in Serum
complement; antibodies of - Most Efficient in
Rh (extravascular PRECIPITATION
hemolysis)
IgA - Monomeric IgA: found in - B-cell production of IgA
the blood primarily happen in the
- Dimeric IgA: mostly found in Mucosa Associated
secretions; the only Ab with Lymphoid Tissue (MALT)
secretory components that
protects it from enzymatic
digestion while it patrols in
the mucosal surface
IgM - Pentameric IgM: present - Primary Response
in plasma Antibody
- Monomeric IgM: present - Activates Complement
on the surface of naïve B Classical Pathway
cell - Indicates Acute Infection
- w/ J CHAIN (Joining Chain)
- 10 Antigen Binding Sites
IgD - Found on the surface of naïve B cells
- Role in B-cell Activation, Maturation, and Differentiation
- Isotypes: Heavy chain Differences (IgM, IgG, IgA, IgD, IgE)
- With Extended Hinge Region
- Allotypes: Minor variation in heavy chain (IgG1, IgG2, IgG3, IgG4)
- Idiotypes: Variable part of LC and HC unique to particular Ig IgE - Formerly Reagenic Antibody, aka Homocytotropic Antibody
- SVEDBERG (S): Unit of sedimentation rate of Immunoglobulin (Ig) - Reagin: a term used to denote an Ab that is able to bind to
cardiolipin
HYBRIDOMA TECHNOLOGY - Binds to Mast Cells, Eosinophils, and Basophils
- Most Heat Labile
- Used in the commercial synthesis/production of antibodies
- Mnemonic for immunoglobulin abundance in serum: GAMDE
- HYBRIDOMA CELL: Immortally Producing Antibodies
o Hybridoma Cells = Plasma Cell + Myeloma Cell
- Surfactant: Polyethylene Glycol
o Sendai virus: formerly used as potentiator before polyethylene glycol
- Medium: Hypoxanthine-Aminopterin-Thymidine (hot medium)
o Aminopterin blocks De Novo Pathway
o Myeloma cell lacks HGPRT and Thymidine Kinase making them not
possible to create DNA

ANTIGEN-ANTIBODY INTERACTION
Affinity Association constant between antibody and univalent
antigen (epitope + paratope)
Avidity Overall binding between antigen-binding sites and
multivalent antigen

Specificity Test is negative in the absence of homologous antigen

(Negative)
Sensitivity Test is positive in the presence of homologous antigen
(Positive)
Cross Bind similar but not identical epitope; binding strength
Reactivity differs
Interactions PRIMARY:
- Van der Waal’s forces: Hydrophobic interaction
- Ionic and Hydrogen Bonds: Hydrophilic interaction
- Covalent Bonds: Not included in the interaction
 SECONDARY: ANTIBODY GENERATORS (ANTIGEN)
- Precipitation (Ab + soluble Ag)
- Agglutination (Ab + particulate Ag) - EPITOPES: Antigenic Determinants
o Stages of Agglutination - IMMUNOGEN: antigens capable of inducing an immune response
 Sensitization: Ab bind Ag - Forms of Antigen
 Lattice Formation: Ab w/ Ag bind to another o Autologous: autoantigen (hosts antigen) induces autoantibody
Ag formation
 Effect on Tissue o Homologous: induces antibody production specific to it – antigen
Factors that - Antigen-Antibody Ratio o Heterologous: “cross-reaction”; reacts with antibody it did not induce
Affect Ag and o Prozone: excess Ab; remedy is Dilution (ex. cowpox & smallpox)
Ab Binding o Zone of Equivalence: optimal - HAPTENS: small molecules, immunogenic only when paired with high MW
o Postzone: excess Ag; remedy is repeat test after carrier
2 weeks - ADJUVANTS: added to a vaccine, enhances immune response by coating
the antigen prolonging the stay of antigen to host’s body
o Squalene, MF-59, Freund’s Complete Adjuvant, Alum Precipitate
o Seronegative: exposed to an antigen but failed to produce a stable
amount of titer

TRAITS OF IMMUNOGEN

1. Macromolecular Immunogen: ≥ 10kDA – 100KDA

Size ROT: the greater the MW, the more immunogenic
2. Foreignness Non-self
3. Chemical Proteins and polysaccharides are the most
- pH – optimum pH (6.5 and 7.5) Composition immunogenic
- Length of Incubation (30 min. – 1hr.)
- Number of Antigens Mnemonics: ProPoLipiN (most immunogenic to
- Location of Antigens least immunogenic)
- Centrifugation - Proteins
- Potentiators (bring Ag-Ab closer) - Polysaccharides
o LISS: reduce zeta potential - Lipids and Lipoproteins
o 22% Bovine Albumin - Nucleic Acids (least immunogenic)
o Polyethylene glycol (PEG) 4. Ability to be processed and presented with MHC
- Rouleaux and True Agglutination 5. Route and IV and Intraperitoneal (both are effective)
- Antibody Isotype Dosage
- Temperature
o IgG: warm Temperature
o IgM: cold Temperature
o Warm serum to inactive cold agglutinins

DETECTION OF ANTIBODIES IN THE DISEASE PROCESS

1. LAG PHASE: No antibody is detectable but with phagocytosis - The strongest bonding develops when antigens and antibodies are close
2. LOG PHASE: Antibody titer increases logarithmically to each other and when the shapes of the antigenic determinants and the
3. PLATEAU PHASE: Titer is Stable antigen-binding site conform to each other.
4. DECLINE PHASE: Antibody is Catabolized - This complementary matching of determinants and binding sites is referred
to as goodness of fit.
- A good fit will create ample opportunities for the simultaneous formation of
several noncovalent bonds and few opportunities for disruption of the bond.
- If a poor fit exists, repulsive forces can overpower any small forces of
attraction. Variations from the ideal complementary shape will produce a
decrease in the total binding energy because of increased repulsive forces
and decreased attractive forces.
- Goodness of fit is important in determining the binding of an antibody
molecule for a particular antigen.
 COMPLEMENT SYSTEM LECTIN PATHWAY
- Induces cytolytic destruction by forming the Membrane Attack Complex - presence of lectin in the organism will initiate binding of MBL (Mannan-
(MAC) making a hole in the membrane of the cell Binding Lectin)
- Inactivated at 56oC for 30 mins. – use within 4 hours
- If not used within 4 hours, heat at 56oC for 10 minutes
- Complement Pathways:

CLASSICAL PATHWAY
- 1st complement system that was discovered
- Activated by: Antigen-antibody reaction
- Antibody directed: IgM, IgG1, IgG2, IgG3
- CRP, viruses, mycoplasma, some protozoa, gram-negative bacteria like
- Clear out debris (Ag + Ab)

STEPS
- IgG complement bind to CH2
- This Antigen-antibody reaction is recognized by
the recognition unit.
o C1qrs is the recognition unit that binds to the - The lectin pathway is triggered by binding of MBP to mannose on bacterial
Fc portion of two antibody molecules. cell walls. MASP-1, MASP-2, and MASP-3 bind to form an activated C1-
o To make this stable Ca2+ is needed like complex. MASP-2 cleaves C2 and C4 and proceeds like the classical
- C1s is activated and cleave C4 and C2 to form pathway.
C4b2a, also known as C3 convertase.
o C3 convertase needs Mg2+ as a cofactor to
activate C3
- C3 convertase cleaves C3 to form C4b2a3b,
known as C5 convertase.
- The combination of C4b2a3b is the activation unit.
C5 convertase cleaves C5.
o C5 becomes C5b (labile) and C5a
o In order to stabilize C5b it cleaves to C6
- C5b attracts C6, C7, C8, and C9, which bind
together, forming the membrane attack complex
(MAC).
- C9 polymerizes to cause lysis of the target cell.
o Non-nucleated = C5b678
o Nucleated = C5b6789
o Membrane Inhibitor of Reactive Lysis (MIRL) – prevents the lysis of
the cells caused by the complement system

ALTERNATIVE PATHWAY
REGULATORY PROTEINS
- Activating surfaces like LPS where opsonin C3b will bind PROTEIN MECHANISM OF ACTION
- Not activated by antigen-antibody reaction C1 inhibitor (C1 INH) - Detaches C1r and C1s from C1
- Initiated by microbial surfaces such as: - Inactivates MASP-2
o Bacterial cell wall (lipopolysaccharide), fungal cell walls, yeast, - Attacks recognition unit
viruses, virally infected cells, tumor cell lines, parasite (trypanosomes)
Factor I - Cleaves C3b and C4b from 2a
STEPS - Attacks activation unit
- Gram (-) bacteria with LPS will Factor H - Cofactor to Factor I to inactivate C3b
allow opsonin C3b to coat - Prevents binding of B to C3b
- When an opsonin is bound to a - Attacks activation unit
bacterium it will be able to C4 Binding Protein - Cofactor to Factor I to inactivate C4b
activate the alternative - Attacks activation unit
pathway through the activation S Protein/ Vitronectin - Prevents attachment of C5b67 to cell
of Factor B membrane
- C3 is hydrolyzed by water to - Attacks MAC
produce C3b, which binds
Factor B and together they DISORDERS RESULTING FROM COMPLEMENT DEFICIENCY
attach to the target cell DEFICIENT PROTEIN ASSOCIATED DISEASES
surface. C1 inhibitor Hereditary Angioneurotic Edema
- B is cleaved by Factor D into C1, C2, C4, C7 SLE-like Syndrome
the fragments Ba and Bb. C5 – C8 Neisseria Infection (Intracellular)
- Bb combines with C3b to form C3 Severe Recurrent Infection;
C3bBb, an enzyme with C3 Recall Dec. C3 Most severe deficiency
convertase activity. C2 Most common Deficiency
- More C3 is cleaved, forming C9 No known associated diseases
more C3bBb. This enzyme is stabilized by properdin, and it continues to
cleave additional C3.
- If a molecule of C3 remains attached to the C3bBbP enzyme, the convertase
now has the capability to cleave C5. The C5 convertase thus consists of
C3bBbP3b. After C5 is cleaved, the pathway is identical to the classical
pathway.
ADDITIONAL NOTES: DEFICIENCIES OF COMPLEMENT COMPONENTS
DEFICIENT ASSOCIATED DISEASE
PROTEINS OF THE COMPLEMENT SYSTEM COMPONENT
C1 (q, r, s) - Lupus-like syndrome; recurrent infections
CLASSICAL PATHWAY C2 - Most common complement fraction deficiency
C1q - Binds to Fc region of IgM and IgG - Lupus-like syndrome; recurrent infections;
atherosclerosis
C1r - Activates C1s
C3 - Most severe complement fraction deficiency
C1s - Cleaves C4 and C2 (because C3 is involved in all pathways)
C4 - Part of C3 convertase (C4b) - Severe recurrent infections; glomerulonephritis
C2 - Binds to C4b C4 - Lupus-like syndrome
- Form C3 convertase C5 - Neisseria infections/syndrome
C3 - Key intermediate in all pathways C6 - MAC deficiency
C5 - Initiates membrane attack complex C7
C6 - Binds to C5b in MAC C8
C7 - Binds to C5bC6 in MAC C9 - No known disease association
C8 - Starts pore formation on membrane C1-INH - Hereditary angioedema
C9 - Polymerizes to cause cell lysis DAF and HRF - Paroxysmal nocturnal hemoglobinuria
- CD markers of DAF: CD55 and CD59
ALTERNATIVE PATHWAY Factor H and I - Recurrent bacterial (pyogenic) infections
Factor B - Binds to C3b to form C3 convertase - Factor H: Inhibits the M protein of S. pyogenes
Factor D - Cleaves factor B
Properdin - Stabilizes C3 convertase (C3bBb) DISORDERS OF THE IMMUNE SYSTEM

1. HYPERSENSITIVITY REACTION
MBL PATHWAY
MBL - Binds to mannose - Exaggerated response to a typically harmless antigen that results in injury
to tissue, diseases, or even death
MASP-1 - Helps cleave C4 and C2
- Influenced by: ENVIRONMENT and GENE
MASP-2 - Cleaves C4 and C2 - Treatment:
o Antihistamine: during hypersensitivity reaction, the mast cells and
COMPLEMENT REGULATORY PROTEIN basophils release histamine and cause vasodilation
PROTEIN REGULATORY FUNCTION o Decongestant: lungs normally produce mucous during an allergic
C1 INH - Binds to C1, thereby preventing it from binding to the reaction and the mucous congest the respiratory tract
active sites of C1r and C1s o Corticosteroid: reduce the swelling and inflammation caused by
Properdin - Stabilizes the alternative pathway C allergic reaction; general anti-inflammatory drug
C4bp - Accelerates dissociation of C3 convertase - Omalizumab: monoclonal anti-IgE; BLOCKS the binding of IgE to mast
DAF - Accelerates dissociation of C3 of both the classical and cells and basophils (prevent histamine release)
alternative pathway - Preferred Method of Screening Type I - IN VIVO PRICK TEST
Factor I - Cleaves and inactivates C3b and C4b o Very small amount of allergen is injected under the skin
AI - Proteolytically cleaves anaphylatoxins o Observe wheal and flare reaction within 20 min.
MIRL / HRF/ - Inhibits MAC formation by inhibiting the attachment of the o Alternative test if is unable to tolerate skin testing: Noncompetitive
S protein C5b67 complex to cell membranes Solid-Phase Immunoassay for allergen-specific IgE

PLASMA COMPLEMENT REGULATION

PROTEIN FUNCTION
C1 inhibitor - Inactivate C1 by binding to the active sites of
C1r and C1s
- Inactivates MASP-2 binding to MBL-MASP
C4b Binding protein - Binds C4b
(C4bBP)
Complement - CD35
Receptor 1 (CR1) - Binds C4b and C3b receptors on platelets and
RBCs
Membrane cofactor - CD46
protein (MCP) - Binds C3b
Decay Accelerating - CD55
Factor (DAF) - Dissociates C3 convertase of both classical and
alternative pathway
Factor I - Cleaves C3b and C4b
Factor H - Cofactor with I to inactivate C3b
- Prevents binding of B to C3b
S protein - Prevents attachment of the C5b67 complex to
(Vitronectin) cell membranes

TYPES OF HYPERSENSITIVITY REACTIONS

Mnemonic: ACID (Allergic, Cytotoxic, Immune, Delayed)
TYPE I TYPE II TYPE III TYPE IV
Synonym Allergic or anaphylactic Cytotoxic Immune Complex Delayed
Immune mediators IgE IgG or IgM IgG or IgM T cells
Timing Immediate
Involve complement? No Yes Yes No
Immune mechanism Release of mediators from ADCC destruction, Cell Ag-Ab Complex activates Ag-sensitized Th1 cells release cytokines
IgE-sensitized mast cells function is inhibited or complement. Neutrophils are that recruit macrophages and induce
and basophils stimulated by antibody recruited and release lysosomal inflammation or activates cytotoxic T cells
binding enzymes that cause tissue damage to cause direct cell damage
Clinical examples - Anaphylaxis - HTR (ABO Incompatibility) - Serum sickness - Contact Dermatitis
- Allergic rhinitis - AIHA - Arthus Reaction - Tuberculin and anergy skin test
- Allergic asthmas - HDFN - SLE and RA - Hypersensitivity pneumonitis
- Food allergies - Drug Reaction - Drug reactions
(peanut & milk - 2 most - Myasthenia gravis
common allergen) - Good Pasteur
- Urticaria - Grave's
(localized wheal and flare)
Test In Vivo Skin Test DAT Skin Test and Purified Protein Derivative
(PPD) Testing (+ in 48-72 hours)
 2. AUTOIMMUNITY LABORATORY DIAGNOSIS
- If TOLERANCE fail, lymphocytes cannot distinguish self-antigens from
non-self - LE Prep
- List of Autoantibodies: o Detects neutrophil that has engulfed the antibody-coated nucleus of
another neutrophil
AUTOIMMUNE AUTOANTIBODY o Lupus antibodies target the nucleus of the neutrophils which causes
DISEASE other neutrophils to ingest such neutrophil
SLE Anti-DNA, Anti-nuclear, anti-ribosome, Anti-DNP
Primary biliary cirrhosis Anti-mitochondrial - Rheumatoid Factor
Chronic Active Hepatitis Anti-Smooth Muscle Antibody o IgM reacting with Fc portion of IgG
Hypothyroidism Anti-TPO, Anti-microsomal Anti-thyroglobulin o Not specific (present in Rheumatoid arthritis and SLE)
o Titer:
Hyperthyroidism Anti-TSH receptor, Anti-thyroglobulin
 ≥80 - Generally Positive
Goodpasture Syndrome Antiglomerular Basement Membrane
 20-40 - Weakly Positive Reaction
Wegener’s Disease c-ANCA
 No agglutination at 1:20 - Negative for RF
Churg-Strauss Syndrome p-ANCA
Diabetes Mellitus Type I Anti-Insulin, Anti-Beta cells. Anti-GAD65 - Immunochromatography for the detection of autoantibodies
Addison’s Disease Antibodies against adrenal glands - Immunofluorescent Staining for Autoantibodies (FANA or ANA)
Multiple Sclerosis Anti-myelin sheath antibody o uses hemoflagellate
(Hyperactivity)
Myasthenia Gravis Anti-acetylcholine Receptor Crithidia lucilliae
(Hypoactivity)
- circular dsDNA substrate in anti-dsDNA detection; source of DNA in
Pernicious Anemia Anti-parietal cells, Anti-Intrinsic Factor
ANA staining
Rheumatoid Arthritis Rheumatoid Factor, Anticyclic citrullinated
Peptide (more specific and sensitive than RF)
ITP Anti-platelet FLUORESCENCE PATTERN AUTOANTIBODY
Pemphigus vulgaris Anti-desmoglein (Anti-desmosome) Diffuse or Homogeneous Anti-dsDNA
Bullous Pemphigoid Anti-hemidesmosome Nucleolar Anti-RNP
Speckled Anti-RNP and anti-Smith
REGULATION OF THYROID HORMONES Peripheral Anti-DNA and anti-lamins
- Hypothalamus releases thyrotropin- Centromere (discrete speckled) Anti-centromere seen in CREST
releasing hormone (TRH) and targets - Calcinosis
the Pituitary gland NO FLUORESCENCE - Raynaud’s Phenomenon
- Pituitary gland release TSH to the - Esophageal dysmotility
Thyroid - Sclerodactyly
- Thyroid gland contains thyroglobulin - Telangiectasia
(TG)
- TSH will bind to the TSH receptor
3. TRANSPLANT IMMUNOLOGY
present in the TG
- Iodine will be able to enter the TG - Allorecognition
- Iodine will be converted into o Autograft - same individual
monoiodine or diiodine through the o Syngeneic graft - between identical twins
Thyroid peroxidase (TPO) o Allograft - two individuals of the same species
- Monoiodine + Diodine = T3 formation o Xenograft - two individuals of different species
- Diodine + Diodine = T4 formation
- If T3 or T4 increases, it will send a - GVHD (Graft Versus Host Disease)
negative feedback loop which will then o Determined by the degree of immunodeficiency in the host, rather
signal the hypothalamus and the than the number of transfused immunocompetent lymphocyte
pituitary gland to be downregulated, and the release of TSH will decrease o Immunocompetent Lymphocytes transfused to an immunodeficient or
- If T3 or T4 decreases, it will send a positive feedback loop which will then immunocompromised patients
signal the hypothalamus and pituitary gland to release more TSH o Donor lymphocytes attacks host/recipient lymphocytes
- Therefore, increasing the TSH production
- GRAFT REJECTION
HYPERTHYROIDISM o Most Immunogenic: BONE MARROW
- Antibodies bind to TSH receptors instead of TSH o Lest Immunogenic: CORNEA (low number of blood vessels)
- Unlimited iodine will just enter thus increasing both the T3 and T4
- Due to an increase T3 and T4, negative feedback loop will be used thus Cornea
the TSH will decrease - best allograft survival rate; risk for CJD transmission
- ↑ T3 & T4, ↓ TSH

HYPOTHYROIDISM CATEGORIES
- The Anti-TPO will destroy the thyroid peroxidase antibody Type Time of Tissue Predominant Cause
- Iodine will be able to enter however it will cause a decrease the production Damage Mechanism
of monoiodine and diodine and thus also decreasing T3 and T4 Hyperacute Within minutes Humoral Preformed
- Due to a decrease T3 and T4 positive feedback loop will be used thus antibodies
increasing TSH Accelerated 2-5 days Cell-mediated Previous
sensitization
MULTIPLE SCLEROSIS VS. MYASTHENIA GRAVIS
Acute 7-21 days Cell-mediated Allogeneic
- Neurotransmitters are responsible for sending signals to the brain
(ADCC) reaction to
- Acetylcholine, a neurotransmitter, transfer to another Schwann cells
donor
through the synapse (synaptic movement)
Chronic Later than 3 Cell-mediated Disturbance
- The purpose of the myelin sheath is to allow the acetylcholine to move
months of host-graft
through synapse
tolerance
- In Multiple sclerosis, there is hyperactivity since the myelin sheath is
being destroyed therefore the neurotransmitter can no longer travel through Immunopathologic Immune
the synapse Damage to the complex
- In Myasthenia Gravis, there is hypoactivity since the acetylcholine New Organ disorder
receptor have been block by the autoantibodies complex
formation with
soluble antigens
 4. TUMOR IMMUNOLOGY B CELL IMMUNODEFICIENCY
- Neoplasia: uncontrolled growth of normal cells X-linked Bruton’s - Primarily affects men
o Benign: NON-CANCEROUS Agammaglobulinemia - Markedly decreased Ig levels
o Malignant: CANCEROUS Common Variable - One or Two Ig Classes are deficient
 Carcinoma: epithelial tissue hypogammaglobulinemia - Usually compensated
 Sarcoma: connective tissue - Usually with IgG
- Deficiency develops recurrent
 Leukemia or Lymphoma: bone marrow
bacterial infection
- Metastasis: spread of cancerous cells
Selective IgA deficiency - Most common congenital
TUMOR MARKERS immunodeficiency
ONCOFETAL ANTIGENS Neonatal - Physiologic
Hypogammaglobulinemia - Normal immaturity of the immune
(increase in fetus)
system
CEA Gastrointestinal carcinoma, colorectal cancer - Liver of neonates are immature, thus it
AFP Hepatocellular carcinoma cannot synthesize enough protein
CARBOHYDRATE ANTIGENS T CELL IMMUNODEFICIENCY
CA125 Ovarian Cancer DiGeorge Syndrome - Abnormal development of Thymus
CA15-3 Breast Cancer gland during embryogenesis
CA19-9 Pancreatic, Gastric, Related to Lewis BGS (normal Nezelof Syndrome - Athymic patient (no thymus)
source is from sialyated Lea blood group antigen) COMBINED T and B CELL IMMUNODEFICIENCY
CA72-4 Gastric Carcinoma Severe Combined - Markedly decreased T and B cells
ENZYMATIC MARKERS Immunodeficiency (SCID)
PSA Prostate Cancer Wiskott-Aldrich - Triad:
ALP Bone Cancer o Thrombocytopenia
HORMONES o Immunodeficiency
Beta-HCG Testicular Carcinoma (Leydig Cells) o Eczema
Calcitonin Medullary Thyroid Carcinoma Bare Lymphocyte Syndrome - Defect in Class I or II or Both MHC
Antigen expression
Gastrin Gastric Cancer
ONCOGENES
BRCA-1 and 2 Breast Cancer BASIC SEROLOGIC TECHNIQUES
mutations - Study of fluid component of blood, especially antibodies
HER2/neu Breast Cancer
- SERUM: most frequently encountered specimen in the immunologic testing
CYFRA21-1 Lung Cancer
OTHERS
- Specimen preparation:
○ Red top sterile tube, allow to clot at RT or 4oC, spin to separate
Bombesin Oat Cell Cancer serum/plasma
IGF-1 Pituitary Cancer  Gold top can be used but it produces erroneous results
IL-2 Receptor Leukemia  Spin immediately the sample after it clots
Nuclear Matrix Protein Bladder Cancer ○ Inactivation of complement: heat to 56oC for 30 mins (4 hrs. validity)
 Do not heat whole blood because the sample will be hemolyzed
QUESTION: Which of the following is an oncogene tumor marker for breast ○ Storage:
cancer?  If not used immediately, store at 2-8oC for up to 72 hrs.
a. CA15-3  Additional delay, store at -20oC or below
b. CA125 ○ Pipettes used in serology:
c. HER2/neu  Volumetric pipette
 Graduated pipette
 Micropipette
5. IMMUNODEFICIENCY
- Depleting host immune system and resistance to infection
- DILUTION: makes less concentrated solution from a reagent
𝑆𝑜𝑙𝑢𝑡𝑒
- Classification: 𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 =
𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒
o Primary: inherited, due to developmental defects
o Secondary: caused by different factor (nutritional deficiency, aging, 𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 = 𝑆𝑜𝑙𝑢𝑡𝑒 + 𝑆𝑜𝑙𝑣𝑒𝑛𝑡
chronic disorders, drug therapy, lifestyle, viruses)
𝑆𝑜𝑙𝑢𝑡𝑒 = 𝑆𝑒𝑟𝑢𝑚; 𝑆𝑜𝑙𝑣𝑒𝑛𝑡 = 𝑁𝑆𝑆
PHAGOCYTIC CELL DEFICIENCY
Chronic Genetic defect in Cytochrome b → decreased H2O2 SIMPLE DILUTION
Granulomatous production Example #1: 2 mL of a 1:20 dilution is needed to run a specific serological
Disease (CGD) - Test: Neutrophil Reduction Test test. How much serum and how much diluent are needed to make this
- Reagent: Nitroblue Tetrazolium Dye dilution?
- Positive: Failure to Reduce NBT Dye 1 𝑥
= = 20𝑥 = 2
- Treatment: GM-CSF or G-CSF and IFN-γ 20 2 𝑚𝐿
Myeloperoxidase - Decrease conversion of H2O2 into Hypochlorite 20𝑥 2 1
Deficiency (MPO) - Review Hexose Monophosphate Shunt 20
=
20
=
10
𝑜𝑟 𝟎. 𝟏 𝒎𝑳 𝒐𝒇 𝒔𝒐𝒍𝒖𝒕𝒆 (𝒔𝒆𝒓𝒖𝒎)
G6PD Deficiency - Genetic impairment of Neutrophil’s aerobic
system 2𝑚𝐿 − 0.1𝑚𝐿 = 𝟏. 𝟗𝟗 𝒎𝑳 𝒐𝒇 𝒔𝒐𝒍𝒗𝒆𝒏𝒕
- Decreased H2O2
Chediak Higashi - Genetic abnormal fusion or primary granules in COMPOUND DILUTION
neutrophil - Composed of many sets of dilution
- Albinism and Photosensitivity
Lazy Leukocyte Job Syndrome Poor Chemotaxis, Example #2: The MTOD prepared a dilution of 1 mL serum with 3 mL diluent.
Deficiency (Hyper-IgE) abscess 2mL of this was further diluted with 2 mL diluent. What is the final dilution?
Tuftsin Deficiency Poor phagocytic motility,
engulfment, and 1 𝑚𝐿 1
1𝑠𝑡 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = = 𝑜𝑟 1: 4
oxidative metabolism 1 𝑚𝐿 + 3 𝑚𝐿 4
Actin Dysfunction Decreased Cell Motility
2 𝑚𝐿 1
and Chemotaxis 2𝑛𝑑 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = = 𝑜𝑟 1: 2
4 𝑚𝐿 2
CHEMOTAXIS RANDOM MOVEMENT 1 1 1
𝐹𝑖𝑛𝑎𝑙 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = 𝑥 = 𝑜𝑟 𝟏: 𝟖
Job’s Syndrome ✘ ✔ 4 2 8
Lazy Leukocyte Syndrome ✘ ✘
 SERIAL DILUTION QUALITY CONTROL AND QUALITY ASSURANCE IN SEROLOGY
- Used to obtain TITER: indicator of antibody’s strength; last dilution
showing the positive result - Cross-reactivity: antigenic determinants closely resemble one another;
- Most common serial dilution: doubling dilution (1/2) antibody formed against one will react with the other
- Composed of 1 set of dilution only ○ Prevention: use MONOCLONAL ANTIBODIES
- Agglutination tests are SCREENING TESTS only
Example #3: What is the dilution of tube number 5, if the undiluted sample - A negative result does not always mean absence of the antigen or the
from tube 1 is subjected into two-fold dilution? disease
○ Safest result in the laboratory = negative result
*Dilution factor = 2 - Tube testing: more sensitive (more sample is used) than slide testing; will
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 not evaporate immediately
1 1 1 1 𝟏 - Slide testing: requires rapid reading; small volume of specimen used
1 2 4 8 𝟏𝟔 - Elution: removal of bound antibodies from the RBC surfaces
- Adsorption: attachment of unbound antibodies to an adsorbing agent e.g.
Example #4: What is the titer if the first dilution of a five-fold dilution, showing charcoal
a negative result at the 5th tube, is 1:20? ○ Free floating antibodies and unnecessary proteins will attach to
adsorbing agents such as charcoal
*Dilution factor = 5
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
1 1 1 𝟏 1 GRADING AGGLUTINATION
20 100 500 𝟐𝟓𝟎𝟎 12,500 GRADE CELLS SUPERNATANT
0 - No agglutinates Dark, turbid,
Example #5: What is the final concentration of a 400 mg% stock of HCl diluted homogenous
to 1:5, then 1:10 and finally 1:20? w+ (weak positive) - Many TINY agglutinates Dark, turbid
1 - Many free cells
400 𝑚𝑔% 𝑥 = 80 𝑚𝑔%
5 - not reported in - May not be visible without
1 compatibility microscope
80 𝑚𝑔% 𝑥 = 8𝑚𝑔%
10 testing, may be
1 dirt
8𝑚𝑔% 𝑥 = 𝟎. 𝟒𝒎𝒈%
20 - repeat the test,
use clean
TEST SENSITIVITY AND SPECIFICITY apparatus
- Sensitivity: proportion of people who have the disease with a positive test 1+ (25%) - Many SMALL agglutinates Turbid
𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 - Many free cells
𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 (%) =
𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 + 𝐹𝑎𝑙𝑠𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒
𝑥 100 2+ (50%) - Many MEDIUM-sized Clear
agglutinates
○ Example: - Moderate number of free
A new laboratory assay gave the following results: cells
Number of patients tested = 100 3+ (75%) - Several LARGE Clear
Number of true positive = 54 agglutinates
Number of true negative = 42 - Few free cells
Number of false positive = 2 4+ (100%) - One large SOLID Clear
Number of false negative = 2 agglutinates
What is the sensitivity? - No free cells
54 54
𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 (%) = = 𝑥 100 = 𝟗𝟔. 𝟒𝟑%
54 + 2 56

- Specificity: proportion of people who don't have the disease with a

negative test
𝑇𝑟𝑢𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒
𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐𝑖𝑡𝑦 (%) = 𝑥 100
𝑇𝑟𝑢𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 + 𝐹𝑎𝑙𝑠𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒
○ Example (same data in sensitivity but now solve for specificity)
42 42
𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐𝑖𝑡𝑦 (%) = = 𝑥 100 = 𝟗𝟓. 𝟒𝟓%
42 + 2 44

- Positive Predictive Value: probability that a positive screening test

actually has the disease
𝑇𝑃
𝑃𝑃𝑉 =
𝐹𝑃 + 𝑇𝑃

- Negative Predictive Value: probability that a negative screening test does CAUSES OF FALSE REACTIONS IN AGGLUTINATION
have the disease
𝑇𝑁
𝑁𝑃𝑉 =
𝐹𝑁 + 𝑇𝑁 FALSE POSITIVE REACTION FALSE NEGATIVE REACTION
Overcentrifugation Undercentrifugation
A B C
Contaminated glasswares, slides, Inadequate washing of cells
Specificity 91 92 94
reagents
Sensitivity 81 96 95
Autoagglutination Delay in testing especially AHG
As the CMT, what test kit will you choose? testing (Ab eluted)
- Answer: Either B or C. Both have high specificity and sensitivity. Saline stored in glass bottles Incorrect incubation temperature
(colloidal silica may cause
agglutination)
Cross-reactivity Insufficient incubation time
Rheumatoid factor (cross-reacts with Prozone phenomenon
other antibodies), heterophile
antibody
Delay in reading a slide test Failure to add AHG reagent
- add the lightest color solution first
(clear)
 BASIC SEROLOGIC REACTIONS o AHG-mediated Agglutination
 Done to visualize agglutination of Ab-Ag that only reached
sensitization phase (visible result cannot be seen)
1. AGGLUTINATION REACTIONS  Most important step: washing
 If reagent is not added → check → add check cells
 Reagent is added → negative → check cells → agglutination
 Check cells: Type O RBCs bound to Ab
 Direct Antiglobulin Test – corrective maintenance
○ Detects IN-VIVO SENSITIZATION of RBC
○ Specimen: RBCs
○ Reagent: AHG (color indicator: Janus Green)
○ Positive: HTR, AIHA, HDFN
 Indirect Antiglobulin Test - preventive
- Antibody + Particulate Antigen ○ Detects IN-VITRO SENSITIZATION of RBC
- Types: ○ Specimen: serum
○ Direct Agglutination ○ Use: crossmatching, Ab panel, Ab detection
■ Natural Carrier of ANTIGEN (e.g. RBC, bacteria) ○ Viral hemagglutination
■ Example: ○ Hemagglutination Inhibition
● Febrile Agglutination Test
○ Widal Test: detects Salmonella Ab 2. PRECIPITATION REACTIONS
 Reagent Antigen: O, H, K, Vi in bacteria - Antibody + Soluble Antigen
o Weil Felix Test: detects Rickettsial Ab - Types:
 Reagent Antigen: Proteus OX-2 (P. vulgaris), OX- ○ Precipitation by Light Measurement
19 (P. vulgaris), OX-K (P. mirabilis)  NEPHELOMETRY
 ABO Forward Typing  Measures light scattered
o Most famous direct agglutination test
 Application: Ig quantitation
o Detects antigens
o Reagent: antibodies  Scattered light is measured by photodetector → galvanometer
will convert it to electrical energy → reflected in the computer
o Indirect Agglutination  TURBIDIMETRY
 Artificial Carrier of ANTIGEN  Measures light blocked
 Carriers: polystyrene latex, bentonite, beads, charcoal  Photodetector 180 degrees
 Example: ASO Latex Agglutination Test
 Detects ASO (Anti-Streptolysin O) ○ Passive Immunodiffusion
 Reagent: SLO coated to Latex Particle  SINGLE IMMUNODIFFUSION
 Only ANTIGEN diffuses, antibody is incorporated in the gel
o Reverse Passive Agglutination
 Artificial Carrier of ANTIBODY via Fc region SINGLE LINEAR SINGLE RADIAL
 Carriers: polystyrene latex, bentonite, beads, charcoal IMMUNODIFFUSION IMMUNODIFFUSION
 Example: CRP Latex Agglutination Test Ab in the agar tube Ab is in the agar plate
 Detects CRP (C-reactive proteins) Antigen is overlain on top Placed in wells
 Reagent: anti-CRP antibodies Example: James Oudin Test Example: C3 assay, RID
Positive Result: Precipitin Line Positive Result: Precipitin Ring
Types of RID:
- Fahey-McKelvey
○ Kinetic method
○ Measure after 18 hours
○ Diameter = log of
concentration
- Mancini
○ End-point method
○ Measure after 24 hr
(IgG) to 50-72 hr (IgM)
○ d2 = concentration

 DOUBLE IMMUNODIFFUSION
o Agglutination Inhibition  Both ANTIGEN and ANTIBODY discusses towards each other
 2-stages:  Example: Ouchterlony Double/Angular Immunodiffusion
 1st stage: add soluble reagent antibodies, neutralizes ○ Antigen: placed on the outer well
antigen ○ Antibody: placed on the inner well
 2nd stage: add antigen-coated latex particle; indicator phase ○ Result patterns:
 Example: pregnancy HCG test  Identity: “common epitope”; smooth arc
 Positive result: no agglutination = primary reagent antibody  Non-identity: “no common epitope”; crossed lines
was able to bind the true antigen  Partial identity: “common epitopes but some Ab are
not captured by antigen and travel toward the initial
precipitin line”; single spur pointing towards simpler
antigen
  Electrophoresis ENZYME IMMUNOASSAY (EIA)
 General Steps: - Cheap and readily available, safe to use
○ Separation: electrophoresis - Quantitative or qualitative; homogenous or heterogenous
○ Staining: Amido Black, Ponceau S, Coomassie Brilliant - Antigen or antibody detection use
Blue - Most commonly used labels:
○ Densitometry: quantify the bands; Densitometer (machine) ○ Alkaline phosphatase
 Source: bovine intestine
 Separation Electrophoresis
○ Horseradish peroxidase
o Goal: separate different types of proteins
 Source: horseradish (malunggay/moringa)
o Zone Electrophoresis: 5 bands (minimum); e.g. serum
○ β-galactosidase
protein electrophoresis
 Escherichia coli
o High Resolution Electrophoresis: 12 bands (maximum);
e.g. hemoglobin electrophoresis
- Forms of EIA:
 One-Stage Electrophoresis – electrophoresis only NON-COMPETITIVE COMPETITIVE IMMUNO-
EIA EIA ZYMETRIC
ROCKET ELECTROPHORESIS COUNTERIMMUNO- INDIRECT DIRECT
ELECTROPHORESIS Detects Antibodies Antigen Antigen Antigen
Rocket immunoelectrophoresis or Countercurrent immunoelectrophoresis Solid-phase Antigen Antibodies Antibodies Unattached
Voltage Facilitated Single ID or Laurel Antibodies
Electrophoresis Labels Anti- Ab Antigen Antibodies
Single ID + Electric Current Double ID + Electric Current Human against
Rocket Precipitin Precipitin Line Globulin the Ag
Ab incorporated in the gel, Ag diffuses Ag moves toward Anode (+) (AHG)
via electric current Ab moves toward Cathode (-) Concentration Direct Direct Inverse Direct
Illustration: Illustration: proportion

- Direct Non-Competitive EIA

○ Solid-phase with Abs → Ags will attach to Abs → Ab that carries
enzyme label will attach to Ag

- Indirect Non-Competitive EIA

Positive result: precipitin line ○ Solid-phase with Ag → Abs specific to the Ag will attach → a special
Gel is incorporated with antibodies. - “meet halfway”: antibody is compatible antibody, named AHG will bind to exposed Abs
Antigen diffuses via electric current with antigen ○ Note: In labeled immunoassays, all terms that have the word “indirect”
towards the antibodies. always use AHG

 Two-Stage Electrophoresis – electrophoresis + - Competitive EIA

(electrophoresis or precipitation) ○ Solid-phase with Abs → px Ag will attach to the Abs → if all px Ags are
already bound to Abs, labeled Ag will bind to free Abs → labeled Ag will
CLASSIC IMMUNO- IMMUNOFIXATION CROSSED (2-D) IMMUNO- be removed if there is a free px Ag
ELECTROPHORESIS ELECTROPHORESIS ELECTROPHORESIS ○ Inverse: the greater the label activated, the lesser the concentration and
Electrophoresis + Electrophoresis + Electrophoresis + vice versa
Immunodiffusion immunodiffusion Electrophoresis
Ab placed in trough Ab overlain on gel Ab incorporated in the 2nd - Immunozymetric:
gel ○ Same with direct but Abs are not attached in the solid phase
Precipitin Arc Precipitin Bands Precipitin Rockets ○ Free floating Abs in a tube → allow the Ag to bind to Abs → add a label
Formation of precipitin Same with classic but Distribute antigen and (antibody in nature) → Abs with the label will start to bind with the Ag
arc = antigen is has no trough antibody in gel 1 and 2 → exposed
specific for the incorporate the 2
antibody placed in the Presence of bands = FLUORESCENCE IMMUNOASSAY
trough antibody is specific for Apply electricity → antigen in - Typically used label:
that particular antigen gel 1 will migrate towards the o FITC (Fluorescein Isothiocyanate), Tetramethylrhodamine,
antibodies in gel 2 → rocket Phycoerythrin, Europium (β-naphthyl trifluoroacetone), Lucifer yellow
precipitin is only formed in gel (fluorescent dye)
2 - Labeling:
o Either primary antibody (direct) or secondary antibody (indirect)
- Read-out device: Fluorescent microscope
3. LABELED IMMUNOASSAY
- Forms of FIA:
- Uses label to quantify the amount of binding
- Labels: marker of antigen-antibody binding
DIRECT FIA INDIRECT FIA
- Example of Label: enzyme, fluorescein dye, radioisotopes
Reagent: labeled specific antibody Reagent 1: antigen
- STEPS:
Reagent 2: labeled antibody
1. Separation
○ Purpose: separate bound and free reactants
Specimen: Antigen Specimen: Antibody
○ Physical Methods:
 Decantation, centrifugation, filtration
 Wash after separation to remove unbound analyte
○ Solid-phase Vehicle
 Polystyrene test tubes, microtiter plates, glass or polystyrene
beads, magnetic beads, cellulose membranes
 Ag or Ab is attached by physical adsorption
 When binding happens, complexes remain attached to solid-
phase
2. Detection
o Depends on the label
o Label:
 Enzyme: spectrophotometer; change in absorbance
 Fluorescein dye: fluorometer, fluorescent microscope, flow
cytometer, spectrofluorometers
 Radioisotopes: gamma scintillation counter
 RADIOIMMUNOASSAY
- First type of immunoassay developed
- Pioneered by Yalow and Berson in 1950s
- Oldest immunoassay developed
o I 125 − most popular label; other labels: I131, H3
- Extremely sensitive and precise technique for
determining trace amount of analytes that are small in
size
- Chief disadvantage:
o Radiation hazard, short shelf life of reagents,
disposal problem, licensing and federal regulations
o Philippine Nuclear Research Institute: only agency in
PH that collects radioactive labels

- Calculating of half-life
o An I131 with a value of 20,000 CPM, has a half-life of
60 days. How long will it take for the value to become
1,250 CPM?
▪ 20,000 in 60 days will become 10, 000
▪ 10,000 in 60 days will become 5, 000
▪ 5,000 in 60 days will become 2,500
▪ 2,500 in 60 days will become 1,250
▪ = sum all days: 60+60+60+60 = 240 days
- Forms of RIA:
o Non-competitive (indirect and capture)
o Competitive
o Immunoradiometric 4. MOLECULAR DIAGNOSTIC TESTS
- Applications: - Basic Principles (cell gene):
o Radioimmunosorbent Test (RIST) − quantitate total IgE o Two types of nucleic acids:
o Radioallergosorbent Test (RAST) − antigen specific IgE 1. Deoxyribonucleic acid (DNA)
▪ Nucleotide contains deoxyribose sugar, with one of the
CHEMILUMINESCENT IMMUNOASSAY following base pairs: adenine, guanine, thymine, cytosine
- Emission of light caused by a chemical reaction (typically oxidation reaction) ▪ Double stranded arranged in double helix
- Labels: luminol, acridinium esters, ruthenium derivatives, nitrophenyl 2. Ribonucleic acid (RNA)
oxalate ▪ Nucleotide containing ribose sugar, with one of the base pairs
o All are luminescent substances; no need for read-out device (can be except that instead of thymine, uracil is used
observed by the naked eye) ▪ Typically, single stranded
- Excellent sensitivity, reagents are stable and non-toxic
- Application: CENTRAL DOGMA:
o Cardiac markers
o Hormones
o Vitamin D levels
o Total IgE
- New technique:
o Electro-chemiluminiscence Immunoassay
▪ Uses electrochemical compound
▪ Ruthenium undergoes electrochemical reaction with tripropylamine
(TPA) at electrode surface
▪ Light is measured by photomultiplier tube
▪ Solid phase: magnetic beads
- Hybridization Technique
Hook Effect o Southern Blot Analysis
- In case of antigen excess, majority of binding sites are filled, and the o Microarray Technology for Simultaneous Assessment of Multiple Genes
remainder of the patient’s antigen has no place to bind o Fluorescent in Situ Hybridization of Specific Genetic Regions
- Resulting to unexpected fall in the amount of measured analyte when
extremely high concentration is present - Amplification Techniques
- Example: Solid-phase with Abs attached → 6 binding site for Ag → if there o Target Amplification
are 10 antigens, excess: 4 antigens → false decrease ▪ Polymerase Chain Reaction
- Remedy: immunozymetric assay (add Ab in proportion to the specimen)  Reverse Transcriptase – PCR (RT-PCR)
 Quantitative PCR (qPCR)
▪ Transcription-Mediated Amplification – targets RNA instead of
DNA; produce cDNA copy from RNA
o Probe Amplification
▪ Ligase Chain Reaction: amplifies probe rather than the target DNA
▪ Strand Displacement Amplification (SDA): amplifies probe rather
than the target DNA
▪ Cleavase / Invader Technology
o Signal Amplification
▪ Branched DNA
▪ Hybrid Capture Assays
o Whole Genome Amplification

POLYMERASE CHAIN REACTION

- Amplifies tiny quantities of nucleic acid up to detectable levels
- Reagents: freshly prepared master mix of reagents
o Water: medium to suspend reagents
o Buffer: maintains pH
o MgCl2: cofactor to enzyme
o Nucleotide: building blocks of DNA
o Fore primer and reverse primer: starting point
o Taq polymerase: stabilize temperature (from Thermus aquaticus)
 ○ Principle of Flocculation: physical process of contact and adhesions
- Steps: wherein the aggregates form larger-size clusters called flocs or flakes
1. Denaturation: heat to 95oC; separate dsDNA into single strands
2. Annealing: cool to 52oC; primers bind to complementary sequence on - VDRL (Venereal Disease Research Laboratory):
each DNA strands ○ Principle: qualitative or quantitative slide flocculation test
3. Elongation: 72oC; heat-stable DNA polymerase binds to each primer ○ Specimen: serum and CSF
synthesize new strand of DNA ○ Read microscopically
○ Requires serum inactivation:
 Heat serum for 30 mins at 56oC
 Use serum within 4 hours
○ Reagents:
 Alcoholic solution of cardiolipin-cholesterol-lecithin
● 0.03% cardiolipin: phospholipid isolated from beef heart;
reactivity; Ag source
● 0.9% cholesterol: center for absorption of tissue lipids to
increase Ag size
● 0.21% lecithin: produce standard reactivity
 Prepare and use on the same day
 Gauge of needles of the Hamilton Syringe used to deliver Ag
suspension:
● Qualitative test: 18g = 60±2 drops of Ag suspension/mL
● Quantitative test: 19g = 75±2 drops of Ag suspension/mL
○ 23g = 100±2 drops of Ag suspension/mL
● Rotator for 4 mins at 180 rpm

- RPR (Rapid Plasma Reagin)

○ Antigen
 Cardiolipin, lecithin, cholesterol
 EDTA, Na2HPO4, thimerosal, charcoal (allow visualization of
FLOW CYTOMETRIC ANALYSIS flocculation), choline chloride (stabilizes antigen and inactivates
- Identify and enumerate various cell population complement; why heating is bypassed)
- Measures multiple properties of cells suspended in a moving fluid medium  Place serum-antigen mixture in a rotator for 8 mins @ 100 rpm
o As each cell or particle passes single file through a laser light source, it  Uses plastic-coated disposable card (10-18 mm circles) instead of
produces a characteristic light pattern that is measured by multiple glass slide
detectors for scattered light (forward, 90oC) and fluorescent emission if ○ Gauge number of needles = 20g needle = 60±2 drops of Ag
the cell is stained with a fluorochrome suspension/mL
○ Read macroscopically
INFECTIOUS DISEASES SEROLOGY
TREPONEMAL TEST FOR SYPHILIS
- detects antibodies to T. pallidum
1. SYPHILIS SEROLOGY
- T. pallidum is not recovered in blood, serum or plasma when stored at 4oC TEST SPECIMEN REAGENT INTERPRETATION
for >48 hours T. pallidum Serum Motile bacterial POS: >50%
- Species of Treponema: Immobilization suspension from live Immobilized
○ T. pallidum subsp. pertenue - Yaws Test (antibodies experimental rabbits,
○ T. pallidum subsp. endemicum - Bejel NEG: <20%
are guinea pig complement Immobilized
○ T. carateum - Pinta detected)
○ T. cuniculi - Rabbit DOUBTFUL: 20-50%
- 4 clinical stages of syphilis FTA-Abs Serum Antigen: dead T. Viewed under UV light
○ Primary: hard painless chancre; specimen: blood pallidum in slide (Nichols FITC-AHG
 In bacte: soft + painful = Haemophilus ducreyi Virulent strain)
○ Secondary: condylomata lata; specimen: blood
○ Latent: no signs and symptoms; specimen: blood, CSF Label: FITC-AHG
○ Tertiary: Gummata’s Lesion, Neurosyphilis; specimen: CSF Sorbent: Reiter strain to
remove antibody against
commensal spirochete

T. pallidum Immobilization Test

- Motile syphilis from live experimental rabbits → patient antibodies will bind
to T. pallidum → still motile → add complement → immobilized
- If T. pallidum is immobilized, the patient has a high titer of Ab against syphilis
- Causes of doubtful result: cross-reactivity, died during collection → repeat
testing

FTA-Abs
- Place the dead T. pallidum on slide → add patient serum → antibody will
bind to antigen → add AHG with label (FITC)
- FITC (fluorescein isothiocyanate)

- Laboratory Tests AGGLUTINATION TEST

○ Direct Microscopy TEST REAGENT NOTES
 Darkfield Microscopy - “corkscrew” motility TP Hemagglutination Tanned sheep RBC Confirmatory test;
 Direct Fluorescent Antibody Staining - specimen: chancre (TPHA) coated with Ag from indirect
 Levaditi’s Silver Impregnation - silver reagent; positive is black Nichol’s strain hemagglutination
○ Non-Treponemal Test Microhemagglutination Formalinized tanned Performed in
 Detects anti-cardiolipin antibodies (aka reagin or antibodies to (MHA) TP sheep RBC coated microtechnique
Wasserman antigen [cardiolipin] or anti-lipoidal antibodies) with Ag from Nichol’s
 Unheated serum reagin (USR) strain
 Toluidine Red Unheated Serum (Trust) Hemagglutination Glutaraldehyde- Automated version of
 Reagin Screening Test Treponemal Test for stabilized turkey RBCs TPHA
 Automated Reagin Test Syphilis
 Wasserman Complement Fixation Test TP-Particle Gelatin particles Indirect Agglutination
○ Oldest test for syphilis Agglutination (instead of RBC) Test
 Kahn Flocculation Test sensitized with T.
 Plasmacrit pallidum Ag
 VDRL and RPR
 ○ Specimen: EDTA
2. HIV SEROLOGY - 5 days from initial contact of infective agent: use NAT (nucleic acid testing)
- Retrovirus containing RNA and reverse transcriptase to detect
○ Reverse transcriptase will convert RNA HIV to DNA HIV - HIV strains:
- Target cell: CD4+ T cells ○ T-tropic (X4) strain: HIV infecting T-cells
○ CXCR4 and CCR5 (co-receptors) ○ M-tropic (R5) strain: HIV infecting both T-cells and macrophages
- HIV-1:
○ Formerly Human T-cell Lymphotropic Virus Type III (HTLV-III), TEST NOTES
Lymphadenopathy-associated Virus (LAV), AIDS-associated retrovirus CD4 T-Cell Gold standard: Immunophenotyping with Flow
(ARV) Enumeration Cytometry
○ Causative agent of AIDS in US and Europe HIV AIDS CD4 Count: <200 cell/mm3 (similar with µL)
- HIV-2: HIV SCREENING TEST CONFIRMATORY TEST
○ Endemic in WEST AFRICA; less pathogenic, lower rate of transmission Antibody - ELISA - Western Blot/Immunoblot
Detection - Agglutination - Immunofluorescence
- 3 major routes of transmission: Test
- Dot-Blot Testing Recommended in US:
○ Intimate sexual contact (friction) immunochromatography
○ Parenteral from body fluid/blood
○ Perinatal from infected mother rHIVda (rapid HIV diagnostic
- gp120: responsible for binding CD4 to receptor of T cells algorithm): new confirmatory test
- Anti-p24: first antibody to appear only in PH; follows 4th gen. ELISA;
can detect Ab and Ag
P24 Antigen “Window Period” before antibody is detectable
Detection P24: First antigen detected
HIV NAT Determine VIRAL LOAD (no. of alive HIV inside the body)
and development of drug resistant strain

VIRAL LOAD TEST: quantitative test for HIV nucleic acid

PCR - Most sensitive and most specific
- Preferred methods for INFANTS, CHILDREN younger
than 18 months
- HIV cannot be transmitted to the fetus (inside the
womb). It is only transmitted during delivery.

Suspected patient have s/s of HIV → pre-counselling

1. ELISA testing → at least 2 (+)
2. Confirmatory test (detect Ag, Ab) → positive
3. Visit doctor for antiretroviral therapy/drug (ARD/ART)
4. CD4 monitoring
○ if CD4 is increased = treatment is effective
5. If CD4 is decreased, → NAT (nucleic acid testing) / viral load
○ NAT/viral load (to detect if viral load is undetectable) → if viral load is
undetectable, concentration of virus inside the body is very low →
lesser chance to transmit HIV
○ After confirmatory testing (neg/pos) → post-counselling
○ Post-counselling:
 Remind to get tested regularly
 Take PREP (pre-exposure prophylaxis): antiretroviral treatment
taken regularly

GENERATION OF ELISA
GENERATION NOTES
THE HIV REPLICATION CYCLE FIRST Solid-phase indirect-assay using viral lysate antigen from
- HIV virus has gp160, as it matures, it will become gp120 and gp41 HIV-1
- CD4 will bind to gp120 → CD4 will transfer gp120 to CCR5 or CXCR4 (T- Detects Ab to HIV-1 only
cells/monocytes) SECOND Indirect binding assay using highly purified recombinant
- HIV will enter → RNAse (for breakdown) → Reverse transcriptase converts or synthetic antigens from both HIV-1 and HIV-2
RNA HIV to DNA HIV → undergoes DNA synthesis → Integrase will Detects Ab to HIV-1 and HIV-2
integrate viral DNA to host DNA THIRD Sandwich technique
- Synthesis of new viral RNA → new RNA proteins will move to the cell Detects HIV antibodies of different Ig classes including
surface → new immature HIV/virion is formed (budding) → Protease IgM
releases mature HIV FOURTH Detects HIV-1 and HIV-2 antibodies and p24 antigen

- According to CDC:
○ When ELISA yields a positive result:
 It should be RETESTED IN DUPLICATE by THE SAME ELISA
TEST
 If 2 out of 3 specimens are reactive, the results must be confirmed
by a more specific test (usually WESTERN BLOT)
 WESTERN BLOT (2 out of 3): p24, gp41, gp120/160

○ Structural
GENE PRODUCT
Gag p25: precursor protein which form core proteins
(Group Antigen Core Proteins: p6, p9, p17, p24
Gene) p24: 1st core protein detected in the patient
- Screening test: ELISA Pol - Reverse transcriptase (p66, p51): transcribes RNA to
○ 2 positive ELISA → Western Blot (Polymerase) DNA
○ Example: 1st test (+), 2nd test (-) → perform 3rd test → if 3rd test is (+) - Integrase (p31): inserts viral DNA to host
→ confirmatory test - Protease (p10): cleaves protein precursors; for
maturation of HIV
○ If 3rd test is (-) → report as negative
- RNAse
- Confirmatory: Western Blot
Env - gp160: cleaved to form gp120 and gp41; both came
○ Positive at least 2 bands to consider positive
(Envelope) from gp160
○ Bands: p24, gp41, gp120/160 - gp120: binds to CD4 on T CELLS
- Ratio of CD4:CD8 in HIV = 1:2 (0.5-1.0 in other books) - gp41: binds to transmembrane protein (CCR5/CXCR4)
○ CD4 count in AIDS is = <200 cells/µL of blood
○ Normal: 60-70% CD4
○ Normal CD4:CD8 ratio = 2:1, but CD4 dies in HIV
 remains
3. HEPATITIS SEROLOGY 5. INFECTIOUS MONONUCLEOSIS SEROLOGY
- Markers of Hepatitis B infection: - EBV infects B cells, T cells become reactive
○ HBsAg: active infection, blood donor screening - Heterophile Antibody (HA)
○ HBeAg: high degree of infectivity, active viral replication ○ Cross react with a group of similar antigens, can be found in unrelated
○ IgM Anti-HBc: current/recent infection; “core window period” animals or microorganism
○ IgG Anti-HBc: lifelong marker ○ IgM that usually appears during acute phase of infection
 Vaccinated individuals: (+) Anti-HBs, (-) IgG anti-HBc ○ Does not react with EBV-specific antigens
○ Anti-HBe: recovery marker ○ Reacts with:
○ Anti-HBs: immunity to hepatitis (protective: ≥ 10 mIU/mL of serum)
 Horse RBC
- Markers of Hepatitis A infection  Ox RBC
○ IgM anti-HAV: incubation period and early phase  Sheep RBC
○ IgG anti-HAV: immunity to Hepa A ○ Absorbed by: beef RBC
○ Total anti-HAV: immunity to Hepa A ○ Not absorbed by: guinea pig kidney cells
○ HAV RNA: detection of HAV in clinical, food and water samples
- Other names of hepatitis - Serologic Tests:
○ Hepatitis A: Infectious Hepatitis (may be transmitted by clotting factors) 1. PAUL-BUNNEL SCREENING TEST
○ Hepatitis B: Serum Hepatitis  Hemagglutination, detect heterophile antibody
 Dane Particle: complete HBV that causes infection  Reacts with sheep RBC
○ Hepatitis C: Non-A, Non-B Hepatitis, Post Transfusion Hepatitis  Heterophile antibody titer: reciprocal of the highest dilution showing
- Surrogate Test for HCV: increased ALT and positive anti-HBc agglutination
- Specific Test for HCV: positive anti-HCV  Normal titer: ≤ 56

2. DAVIDSOHN DIFFERENTIAL TEST

 Differentiates HA associated with IM, serum sickness or Forssman
antigen
 Agglutination: sheep RBC
 Absorbed by: guinea pig kidney and beef RBC

ABSORPTION PATTERNS
Type of Serum Absorbed by Absorbed by
GUINEA PIG KIDNEY BEEF RBC
IM - +
Serum Sickness + +
- Hepatitis has an incubation of 8-13 weeks Forssman + -
- Antigen is detected during acute infection (2 weeks to 3 months)
1. HBsAg: 1st detected
2. HBeAg AGGLUTINATION PATTERN AFTER ABSORPTION
○ Symptoms will appear once HBsAg peaks Type of Serum Agglutination with Sheep RBC after Agglutination
- During early recovery (3-6 months) Absorption with Guinea Pig Kidney with Sheep
1. anti-HBc total: 1st antibody that will increase IM + -
2. anti-HBc Igm Serum Sickness - -
3. anti-HBe Forssman - +
4. anti-HBs
 (+) in absorption patterns = Ab is absent → (-) in agglutination
4. STREPTOCOCCAL SEROLOGY
patterns
- Onset of clinical symptoms of rheumatic fever or glomerulonephritis typically  (-) in absorption patterns = Ab is present → (+) in agglutination
coincides with the peak of antibody response patterns
- If acute and convalescent phase sera are tested in parallel, a FOUR-FOLD
rise in titer is considered significant
3. MONOSPOT TEST (Rapid Differential Slide Test)
○ Four-fold rise in titer signifies recurring infection
- Tests:  Requires absorption of the patient’s serum
○ Anti-streptolysin O (ASO)  HORSE RBC: agglutinated by heterophile antibodies of IM
○ anti-DNAse B Test
○ Streptozyme Test 6. SALMONELLA SEROLOGY
- Carrier specimen: fluid from gallbladder
TEST NOTES
- Widal Test: detects antibody to Salmonella, Brucella, Tularemia
Streptozyme - 5 in 1 test; slide agglutination screening test
- Clinically Significant Titer: ≥160 (indicates current Salmonella infection)
- Reagent: sheep RBCs or latex coated Streptococcal
○ O antigen – LPS
extracellular products
○ H antigen – Protein
ASO - Latex Agglutination Test: based on indirect or passive
agglutination ○ K or Vi antigen – Polysaccharide
- Tube Test: based on neutralization or inhibition
- Reported either using: 7. RICKETTSIA SEROLOGY
○ Todd Units: when streptolysin reagent standard is - Causes spotted fever and typhus
used - Weil Felix Test
○ IU: when WHO international standard is used ○ Detects antibodies against rickettsia
○ Normal: <200 TODD UNIT (less or equal to 166 ○ Four-fold rise in titer or 1:160 titer is significant
TODD UNIT) ○ Antigens: OX-K, OX-2, OX-19
○ Control: SLO control – show hemolysis
 POS control: SLO shows subsurface hemolysis Epidemic typhus R. prowazekii OX-19 (+)
 NEG control: RBC control – no SLO, no
Rocky Mountain Spotted fever R. rickettsia OX-19 or OX-2 (+)
hemolysis
Anti-DNAse B - Detects Ab capable of preventing DNAse Scrub Typhus R. tsutsugamushi OX-K (+)
(streptodornase) from depolymerizing DNA
Tube Grading: ○ DNAse enzyme: reduces DNA-methyl green 8. H. pylori SEROLOGY
4+ unchanged conjugate - With endoscopy:
color ○ DNA-methyl green + DNAse → colorless ○ H. pylori culture
0 total loss of ○ If patient has antibody against DNAse enzyme → ○ Histological examination: gastric biopsy
colon color is retained ○ Urease biopsy test
- Principle: neutralization - No endoscopy:
- Reagent: DNA-methyl green substrate ○ Urea breath test
○ if there is no antibody to neutralize DNAse B, ○ Enzyme immunoassay for bacterial antigens in the stool
DNAse B will hydrolyze the DNA-methyl green ○ Molecular test
conjugate → methyl green becomes colorless
○ Serum EIA
○ if anti-DNAse B is present, it will neutralize DNAse
B, preventing it from depolymerizing DNA → color - H. pylori: causative agent of peptic ulcer
 9. SEROLOGICAL TEST FOR M. pneumoniae
- Associated with cold agglutinins
- Auto-anti-I

10. SEROLOGICAL TEST FOR MALARIA

- Definitive diagnosis: thick and thin smear
- Immunochromatography:
○ OptiMAL Assay
 Detects parasitic LDH
o Can be detected when there are 100-200 parasites/µL blood
 Detects viable (alive) parasite
 Distinguish between spp. through detection of different isoforms of
pLDH
○ MalaQUICK Standby Malaria Test
 Detects P. falciparum Histidine Rich Protein (HRP)-2 Antigen

11. SEROLOGICAL TEST FOR HISTOPLASMA

- 2 precipitin bands of diagnostic significance:
○ H line: most specific but can be found up to 2 years after recovery
○ M line: found in patient with active infection (can also be found in past
infection or patient who had a recent skin test)

12. SEROLOGICAL TEST FOR INFLUENZA VIRUS

- Tests:
○ Hemagglutinin (HA): required for entry into cells; H1 to H18
○ Neuraminidase (NA): facilitates release of virus from cells; N1 to N11
- Mutations in influenza:
○ Antigenic Shift: major antigenic change in influenza A, resulting in HA
and NA; occurs about every 10 years; sudden in onset and can cause
serious epidemics
○ Antigenic Drift: minor mutation that occur at the site of the virus as it
replicates; pre-existing antibodies cross react and can render protection

13. OTHERS
- CMV: most common cause of congenital infection; fetus; CMV has tropism
to WBC so WBC must be absent in transfusion
- Brucellosis, Tularemia: no serologic tests, both are agents of bioterrorism
- Chlamydia trachomatis: Frei Test; for Lymphogranuloma venereum
- Borrelia: diagnosed thru skin manifestation
- Toxoplasmosis: Sabin-Feldman Dye Test
- Echinococcosis: Casoni Skin Test