IMMUNOLOGY

PART I. INNATE/NATURAL/NONSPECIFIC
IMMUNITY
-affords protection against many differential pathogen Basophils and Eosinophils
-present at birth Third Population Lymphocyte
-standardized response for all antigens -Null lymphocyte/Natural Killer Lymphocyte
-lacks memory -5-10% circulating lymphocytes
-lack markers found on T/B cells
1st Line of defense
-CD 16 and CD 56
Physical Skin and mucous membrane, Cilia lining of respiratory tract -kill viruses or tumor cells without prior exposure
Biochemical Lysozymes, Acidity of GIT and vagina LAK cells (Lymphocytes Activated Killer)
2nd Line of defense NK cell + IL2 = LAK cells
Cellular Components IL2: enhances NK activity,
potentiates T cell proliferation
Phagocytes
Humoral (Fluid) Components
Neutrophils Complement proteins
Monocytes: Tissue: Macrophage -Major components of Humoral
CT: Histiocyte -Alternative Pathway
Brain: Microglial Interferon A and B
Lysozyme
Kidney: Mesangial cells
Antimicrobial substances
Liver: Kupffer cells -TNF, properdin, betalysin
Bone: Osteoclast Inflammation
Lungs: Alveolar macro./Dust phagocytes
Spleen: Splenic macrophage
Skin: Langerhans
Lymph nodes: Dendritic cells
INFLAMMATION PHAGOCYTOSIS (METHCNIKOFF)
-reaction to tissue injury -engulfment of foreign material
Chronic Inflammation: INC. Gamma globulins
-kills extracellular organisms Neutrophils: 1st to migrate
CARDINAL SIGNS: Rubor (Redness),Calor (Heat),Tumor (Swelling),Dolor
(Pain),Functio laesa (Loss of function) because increase number in circulation
STAGES Mono/Macro: 2nd to migrate
1. Vascular response- primary response to inflammation STAGES
Injured cells: MAST CELLS (tissue basophils) 1. Initiation Stage
Releases: Histamine, promotes vasodilation -INC. surface receptors that allows for adherence
 Vasodilation- first response to inflammation E.g. CR3, laminin receptor, Leucyl-formyl- phenylalanine receptor
INC. blood flow to injured area (Hyperemia: Redness + heat)
-initiated by cell injury (microorganisms, trauma, small injury)
INC. Capillary permeability Plasma leakage to tissues (Swelling + pain)
*In Hema: first response is vasoconstriction 2. Chemotaxis
2. Cellular Response -migration of neutrophils and monocytes to the site of injury
Neutrophils: 1st to migrate; short lived; acute inflammation -migration to a certain direction under stimulation of chemical
Mono/Macro: 2nd to migrate; long lived; chronic inflammation substances (chemotaxin)
-Phagocytic cells -Chemotaxin: C’ Complement (C5a)
-Antigen-presenting cells
(+) Chemotaxis: towards the stimulus (E.g. WBCs)
-Secretes MONOKINES Ex. IL1
Cytokines: T cell- Lymphokine, Mono- monokine (-) Chemotaxis: away from the stimulus
 Effects of IL1: Without chemotaxin: movement is said to be RANDOM
Fever Job’s Syndrome Normal Random Activity Abnormal chemotactic activity
INC. Acute Phase Reactants-IL1 is the mediator of APR Lazy Leukocyte Syndrome BOTH RANDOM AND CHEMOTACTIC ACTIVITY
-plasma proteins that increases rapidly by at least 25% due to infection, trauma or ARE ABNORMAL
injury  Test for CHEMOTAXIS:
E.g. (CRP, Serum amyloid A, Complement proteins, α-1 antitrypsin, haptoglobin,
Boyden Chamber Assay
fibrinogen)
Stimulates T cells to produce IL2 (causes lymphocyte proliferation)
3. Resolution and Repair
-Initiated by FIBROBLAST proliferation (Stabilized wound area)
3. Engulfment INTERFERON
-enclosing the pathogen into a phagocytic vacuole/ Phagosome -are proteins produced by virally infected cells and protect the
-INHIBITED by large capsule neighboring cells
E.g. H. influenzae, S. pneumoniae, N. meningitides -exert a virus nonspecific but host specific anti-viral activity
-ENHANCED by OPSONINS (E.g.: Specific Antibody, C’ proteins C3b) -interfere with viral replication
Opsonization: coating of particle with plasma factors to speed up phagocytosis.
TYPES
4. Digestion -digestive enzymes
NATURAL IMMUNITY Type 1 Interferon: Alpha and Beta
TYPES OF PHAGOCYTOSIS
DIRECT: recognition of CHO and lipid sequence in microorganism -non immune, product of initial response to viral replication
(PRP: Primitive Pattern Recognition Receptors) Alpha IFN
INDIRECT: Via Opsonins -“Leukocyte Interferon”
HOW ANTIGENS ARE DESTROYED? -produced by virus induced leukocytes
1. Lysosome: lysozyme (destroys bacterial cell wall), defensins (permeabilize some -Major producer: Null Lymphocytes/NK cells/ Third Population
bacterial and fungal membranes), lactoferrin (competes with Iron) Beta IFN
Nitric oxide: by macrophages, NK cells -“Epithelial or Fibroepithelial IFN”
-Gamma Interferon -Secreted by double stranded RNA fibroblasts
2. NADPH oxidase -Major producer: Fibroblasts, epithelial cells and macrophages
toxic oxygen molecules will be produced, reduced nicotinamide
H2O2, superoxide anion, hydroxyl radicals Type 2 Interferon ACQUIRED IMMUNITY
CGD (Chronic Granulomatous Disease)
Gamma IFN
Impaired NADPH oxidase
-“Immune Interferon”
Inability of the phagocyte to kill ingested microorganism
Oxidative metabolism or respiratory burst -component of the specific immune response to viral and other pathogens
Associated with Kell Blood Group, MacLeod Phenotype -Inhibitory effect to ERYTHROPOIESIS
TEST FOR CGD: NBT Dye Test -Produced by immunologically stimulated lymphocytes: T CELLS
NBT dye:
Clear (Pale yellow) to Blue (Formazan)
Uses HEPARINIZED BLOOD
MODIFIED: Get BUFFY COAT and expose to bacterial suspension
NORMAL: 80-100% Formazan (+) Blue to violet granules
ABNORMAL: For CGD-<50% Formazan-Pink to red granules
TUMOR NECROSIS FACTOR (TNF) PATHWAYS
-Cytotoxic activity against tumor cells and virally infected cells CLASSICAL PATHWAY
TNF Alpha -“Cachetin” -antibody dependent
-produced by macrophages
-Activated by: Ag-Ab complex
TNF Beta (ACQUIRED IMMUNITY) -“Lymphotoxin”
-produced by CD41 and CD8 cells
(Immune complex)
-TH1 and TC Cells IgM: one molecule
Laboratory Phenomenon Schwartz Reaction (Coagulation, Necrosis) IgG: 2 molecules (IgG 3>1>2)
BETALYSIN -Recognition Unit: C1q, C1r, C1s
-heat stable cationic substances released by PLATELETS during coagulation C1: recognition unit, trimolecular complex held by calcium ions (Ca2+)
 -Bactericidal for G (+) except STREP C1q: with 6 globular structure
PROPERDIN 2 globes must attach to Fc
-serum protein with bactericidal and viricidal property CH2: IgG
-only acts in the presence of C3 and Mg++
CH3: IgM
COMPLEMENT SYSTEM (JULES BORDET)
Order of Discovery: 1 2 3 4 5 6 7 8 9 -Sequence of Activation: C4, C2, C3
Order of Activation via Classical Pathway: 1 4 2 3 5 6 7 8 9 -C3 convertase: C4b2a
Functions: -C5 convertase: C4b2a3b
Activation of Immune System ALTERNATIVE / BYPASS / PROPERDIN / ALTERNATE PATHWAY
Opsonization: C3b, C4b, C5b -Activating substance or Initiated by:
Cell lysis: end reaction of complement activity A. Aggregates of IgA, IgG4
Chemotaxin: C5a, C5b, C6, C7 B. Yeast cell wall or zymosan
Immune Adherence: C3b
C. LPS (Bacterial Capsule)
Kinin Activator: C2b
Anaphylotoxins: C3a, C4a, C5a D. Cobra Venom Factor (CVF)
Virus neutralization: C4b, C1 -Bypasses C1, C4, C2
DESTROYED AT: 56oC for 30 minutes -Recognition Unit: C3, Factor B, Factor D
COMPLEMENT PROTEINS -C3 Convertase: C3bBb
-are found on the β region of serum electrophoresis -C5 Convertase: C3bBb3b
-Major constituent: C3 -Begins with activation of C3 (activated at slow rate by water and plasma
-Produced by the LIVER except C1 (Intestinal epithelial cells), Factor D (adipose) enzymes)
LECTIN PATHWAY DEFIENCY OF COMPLEMENT COMPONENTS
Lectin: Proteins that attach to CHO C1 LE-Like Syndrome
-Initiated or activated by: microorganism with mannose or similar sugar in their C2 LE-Like Syndrome, recurrent infections, most common deficiency
cell wall or outer membrane C3 Severe recurrent infection
-MBL (Mannose binding Lectin): attaches to mannose or similar sugars in the C4 Lupus-like syndrome
cell wall or outer membrane of microorganism C5-C8 Neisseria gonorrhea, N. meningitides
*C1 is not needed. C9 Neisseria infection, No known disease (in some references)
-C3 convertase: C4b2a C1INH HANE (Hereditary Angioneurotic Edema)
-C5 convertase: C4b2a3b -Swelling of the face, extremities and GIT
-Recognition Unit: MBP, MASP-1 and Factor H or I Recurrent Bacterial Infections
MASP: MBL Associated Serine Protease, Cleaves C4 and C2 DAF and HRF for PNH
Formation of C3 convertase -Paroxysmal Nocturnal Hemoglobinuria
Proceed as in Classical -Screening: Sucrose Hemolysis Test
PLASMA COMPLEMENT REGULATION -Confirmatory: Ham’s Test
C1 inhibitor (C1INH): dissociates C1r and C1s from C1q (+) Hemolysis
Factor I: cleaves C3b and C4b, Inactivates C3b and C4b MEASUREMENT OF COMPLEMENT COMPONENTS
Factor H: competes for Factor B, Causes dissociation of C3 concentrates of CH50 Assay
Alternative and Classical Pathway, Cofactor to Factor I, Inactivates C3,Previous -total complement activity; amount of serum that can cause hemolysis of 50%
attachment of C3b to Factor B reagent RBC
C4 binding protein: acts as cofactor with Factor I to inactivate C4b RID (Radial Immuno Diffusion)
S protein (Vitronectin): prevents attachment to the C5b67 complex to cell CLINICAL SIGNIFICANCE of complement proteins
membrane Elevated complement components have little clinical importance
Decay Accelerating Factor (DAF)-Accelerates dissociation of C3 convertase Decreased complement components
INHIBIT MAC (Membrane Attack Complex) Causes:
HRF (C8bp): Homologous Restriction Factor Complement has been consumed as seen in Autoimmunity
CD59 or MIRL (Membrane Inhibitor of Reactive Lysis) Complement may be decreased or absent due to genetic defect
Complement has been excessively activated
Complement is not synthesized
 PART II.
ACQUIRED/ADAPTIVE/SPECIFIC IIMUNITY
-developed as a result of exposure to a variety of agents capable of inducing an immune response PASSIVE: Antibody production is not done by the body
-with memory Natural: develops after the placental passage of antibody from mother to fetus.
-highly evolved mechanism Example: Transfer in vivo: (IgG) Colostrum: (IgA)
-Specificity, specialization, memory Artificial: immunity obtained after injection of gamma globulin for the induction of
-starts with T cells an immune state.
3rd Line of defense -administration of serum Ig’s
Cellular components
Example: Anti-rabies
(Specialized Lymphocytes)
ADVANTAGES: Immediate Response
B cells- can further differentiate to plasma cells
DISADVANTAGES: Immunity is short-term
T cells
Gall bodies- lysosomes associated with lipid droplet LYMPHOID ORGANS
TH1: cell mediated effector mechanism 1.Primary or Central Lymphoid Organ
TH2: regulates Antibody production -Site of maturation of T and B cells -Site of growth and maturation
Humoral components a. Thymus (Mature site): T cells
From T cells: Lymphokines Cortex: Positive selection recognition
From B cells: Antibodies lymphokines Medulla: Negative selection
TYPES of ACQUIRED IMMUNITY b. Bone marrow: B cells
ACTIVE- Antigen is acquired 1.Secondary or Peripheral Lymphoid Organ
-Antibody is produced by the body -Antigen trapping site -site of proliferation and differentiation of T and B cells
a. Natural: includes the type of immunity that develops during convalescence from an infection a. Spleen: largest secondary lymphoid organ
-Antigen from the infection -Major site where antibodies are synthesized
b. Artificial: immunity is obtained from vaccination -Antigen trapping site: Via IV or intraperitoneal
-Antigen from the vaccine b. Lymph nodes
Examples: -Para cortex: T cells: thymus dependent area
Live organism: small pox -Cortex: B cells: thymus independent area
Attenuated or weakened: BCG-for MTB-Bacillus of Calemette of Guerin-Attenuated from M. bovis -Medulla: plasma cells and macrophages
Toxoid: C. tetani Modified virus: Poliovirus Dead organism: Cholera, Typhoid -Antigen trapping site: Via subcutaneous
ADVANTAGES: Immunity is Long term c. Peyer’s Patches: intestines -Antigen trapping site: Antigen ingested
DISADVANTAGES: Immune response is short term d. Adenoid; tonsils
e. Appendix
 4. T delayed hypersensitivity (<1%)
CELLS INVOLVED IN SPECIFIC IMMUNITY a.Hypersensitivity reactions
T CELLS Th : Ts Ratio
General Information: Normal 2 : 1
HIV + 0.5 : 1
-Cell mediated Immunity -Antibody regulation -Responsible for immune response
AIDS CD4+ cell count <200/uL
-Thymus: near the heart N.V.: 500-1300/uL
Cortex: Immature T cells (85%) Ontogeny of T cells
Medulla: Mature T cells (15%) I. Double Negative Thymocytes (Immature T cells)
-Lymphokines (-) CD4, CD8 (+) CD2, CD5, CD7
-60%-80% of circulating lymphocytes II. Double Positive Thymocytes (+) CD4, CD8
III. Mature T cells Single Positive
-Longer life span: 4-10 years
CD4 or CD8
-Identified by: CD3- T cell Antigen Receptor
Erythrocyte-Rosette Assay -Using sheep RBCs - (+) Rosette formation TcR- recognizes antigen on surface of T cells
- CD2: receptor for sheep RBCs a.α- similar to light chain
-CD3: Part of T cell antigen receptor complex b.β- similar to heavy chain
Subsets of T cells IV. Activated T cell
CD25 (+) : receptor for IL2
1.T helper/inducer cells (70%)
IL2: lymphocyte proliferation
a.CD4 + V. Sensitized T cells
b.Receptor for MHC class II molecule Secretes or releases Lymphokines
c.TH1: activation of T cytotoxic cells and hypersensitivity reaction; Unsensitized T cells: “Memory Cells”
produced by IFN- gamma and IL2 B CELLS
d.TH2: activation of B cells; produced by IL4 and IL5 General Information:
2.T suppressor cells (30%) -Humoral Immunity
a.CD8 + -Bone marrow
-Birds: Bursa of Fabricius
b.Suppress B cell to plasma cells for antibody production -Antibodies (Plasma cells)
c.Receptor for MHC class I molecule -Shorter Life Span:3-5 days
3.T cytotoxic (<1%) -20%-35% circulating lymphocytes
a.CD8 + -Identified by: Surface Immunoglobulins (SIgs)
b.Kills intracellular organisms with NK cells -Precursor cell for antibody production
Ontogeny of B cells MITOGEN -Substance that causes the cells to divide
I. Pro B cells (Progenitor/Immature) A. for T cells Concanavilin A (ConA) Phtyohemagglutin (PHA) *Pokeweed mitogen (PWM)
-CD19, CD45R, intracellular proteins B. for B cells Lipopolysaccharide *Pokeweed Mitogen (PWM)
-Chrom. 14: rearrangement of genes coding for the heavy chain MAJOR HISTOCOMPATIBILITY COMPLEX
-Bone marrow -molecular basis of self and non self
II. Pre B cells (Precursor B cell) -genes located on the short arm of
-mu chains in the cytoplasm Chromosome 6
-coding for light chain -set of genes that controls tissue compatibility: Organ Transplantation
Chrom. 2: Kappa -In humans, referred to as Human Leukocyte Antigen (HLA) Complex- encoded
Chrom. 22: Lambda by MHC genes
-CD19, CD45R I. MHC Class I
III. Immature B cells -Found on: all nucleated cells (Highest in Lymphocytes, T cells and B cells)
-with heavy and light chain -Genetic Loci (Antigens): HLA-A, B, C
-CD19, CD45R -Chain structure: Alpha and Beta Chain (β2 Microglobulin)
-IgM: First to appear and is monomeric on the surface of B cell -At plasma: Pentamer -Process cytoplasmically derived antigens and presented to CD8+ (Cytotoxic T cells)
-Primary cause of immune mediated platelet transfusion refractoriness
-CD21: Receptor for EBV (IM) presence of Atypical Lymphocytes II. MHC Class II
-Atypical Lymphocytes: T cells reacting on B cells attracted by EBV -Found on: B cells only, Antigen presenting cells, macrophages, and dendritic cells
IV. Mature B cells Genetic Loci (Antigens): HLA-DP, DQ, DR
-IgM and IgD -Chain structure: Alpha and Beta chains
-IgD: small amount in plasma because it is mainly found on B cells -Restricted to immunocompetent cells of immune system
V. Activated B cells -process extracellularly derived antigens and presented to CD4+ (T helper cells)
-with CD25: Receptor for IL2 III. MHC Class III
-Lymphocyte capping: surface Ig concentrated on one pole where contact -Genetic Loci (Antigens): C2, C4 and Factor B
with antigen takes place. -Minor MHC antigens
VI. Plasma cells  NOTE
-No surface immunoglobulins HLA on RBC surface:
-Cytoplasmic Antibodies Bennett –Goodspeed Antigen
-Many cytoplasmic Antibodies are released in the circulation as antibodies -BGA: HLA-B7
-mistaken as OSTEOBLAST -BGB: HLA-B17
-with perinuclear halo, cartwheel appearance -BGC: HLA-A28
IMPORTANCE OF HLA TYPING
-Tissue/ Organ Transplant
a. Kidney: most common organ to be
donated
b. Heart: If brain dead, irreversible
c. Cornea: safest organ to be transplanted –Avascular -NO to DEC. MHC Class I
d. Skin
e. Liver
f. Lungs
g. Pancreas
-Studies of racial ancestry and migration
-Paternity Testing
-For diagnostic and genetic counseling
-Risk for disease association:
(By Kuby)
a. HLA-B27: Ankylosing Spondylitis
-Disease with the highest relative risk associated with HLA For Class II- B cells
- Reactive arthritis (Yersinia, Salmonella, Gonococcus), Reiter Syndrome
b. HLA-DR3: Multiple sclerosis, Gluten-sensitive enteropathy, Myasthenia gravis
c. HLA-DR2, DR3: SLE
d. HLA DR3, DR4: Insulin-dependent diabetes mellitus
e. HLA-DR4: Rheumatoid Arthritis
f. HLA-DR2: Hashimoto’s Thyroiditis
METHODS OF DETECTING HLA ANTIGENS
1. Lymphotoxicity Testing
-identifies Class I and Class II HLA Antigen
For Class I:
-Uses polyspecific reagents to classify subsets of HLA Class I
Mixed Lymphocyte Reaction Factors affecting Immunogenecity
-Check compatibility of D related antigens of HLA Class II Foreignness
a. More foreign, more immunogenic
b. Different types of antigens
i. Autoantigen: same individual
ii. Allo/Homologous: diff individual same specie
iii. Hetero/xenogeneic: diff specie; has the greatest immune response
iv. Heterophile Ag: found in unrelated plants or animals, the cause cross
reactions
c. Grafts “transferred part or material”
i. Autograft: same individual
ii. Iso/Syngraft: identical twins
iii. Allograft: diff individual same species, Fetus
ANTIGEN and IMMUNOGEN iv. Hetero/xenograft: different specie; most immunogenic
Antigen: substance that react with specific antibody Chemical composition and complexity
Immunogen: substance that stimulates antibody production INC. complexity = More immunogenic
-complete (Hapten + protein carrier) i. Proteins: most immunogenic, most complex
-Nucleic acid + Protein ii. Polysaccharide: 2nd
-Substance becomes an Immunogen if that substance has: iii. Lipids and nucleic acid (hapten): Least
1. Immunogenecity: ability to stimulate antibody production Size
2. Antigenecity: Ability to react with its corresponding antibody The bigger the size; the more immunogenic
Hapten: incomplete or partial antigen Potent Ag: MW> 10,000 Daltons
-low molecular weight substance, has the ability to react with corresponding antibody but not Albumin: 40,000 Daltons -Good Immunogen
able to stimulate antibody production Excellent Immunogen: HEMOCYANIN -1 million Daltons
-with Antigenecity but no Immunogenecity E.g. Nucleic acid (RNA or DNA) Penicillin: penicillin group + albumin
Main parts of an Antigen Poison Ivy: Urushiol + any protein
Carrier: protein in nature; High molecular weight -Immunogenic Dosage
Hapten: non-protein in nature; low molecular weight -Antigenic INC Dosage= INC Immune response
Complete Ag: Carrier + Hapten
Incomplete Ag: Hapten only
 Adjuvant Structures of Immunoglobulins
-added to vaccines and less immunogenic substance Heavy Chain- determines Ig class
-substance that enhances immune response Chromosome 14
IgG: Gamma γ
a. Stimulates T cells -enhanced cell mediated immunity -produce lymphokines
IgA: Alpha α
b. Stimulates B cells -enhanced humoral immunity -produce antibodies IgM: Mu
c. Stimulates phagocytic cells IgD: Delta δ
Examples of Adjuvants: IgE: Epsilon ε
1. CFA (Complement Freund’s Antigen) -water in oil emulsion of Mycobacterium Light chain Kappa : Lambda
(found on the cell wall: E. muramyl dipeptide), Butyricum, B. pertussis Chromosome 2 22
-Stimulates T cells 65% 35%
2. Lipopolysaccharide (LPS) -Stimulates B cells Ratio 2 : 1
3. Alum adjuvants -Stimulates phagocytic cells  NOTE
4. Squaline -HIV vaccine (MF59) -from shark oil -Found on human sebaceous glands Bence Jones Protein (Plasma cell disorder as seen in Multiple Myeloma, proliferation of
Antibodies/ Immunoglobulins/ Gamma globulins Immunoglobulin-producing plasma cells)
 NOTE Light to Light chain: Abnormal
All antibodies are found on the gamma region of serum electrophoresis except IgA Screening Test: 40-60oC- precipitates/ coagulates 100oC- dissolves or disappears
(beta region) Beta-Gamma bridging can be seen in Biliary Cirrhosis because of an -Can also be detected in urine
-Serum electrophoresis
increase in IgA.
-Ig Light chains
General Information: Disulfide bond H-H and H-L : Normal
-Products of antigenic stimulation except ABO Blood Group Hinge region-flexible region -AA responsible for flexibility: Proline -Between CH1 and CH2
-Functions: Cell cytotoxicity Neutralization Opsonization Immobility of Pathogens Domains -regions or sections in Ig molecule
Theories of Antibody Production CH3: responsible for cytotropic reaction of macrophages and monocytes
Side Chain Theory By Paul Erlich Cells had specific receptors for antigen that were CH2: binds with complement specifically C1q
present before contact with antigen occurred First 110 amino acids are variable, the rest is constant.
Instructive or Template Theory By Felix Haurowitz The antigen serves as the mold or Immunoglobulin Light Chain 2 domains Variable light + Constant light
template and alters the protein synthesis so that antibody with a specific fit is made. Immunoglobulin Heavy Chain 4 domains 1 Variable heavy + 3 Constant heavy (IgG, IgA, IgD)
 NOTE
Clonal selection Theory By Niels Jeme and MacFarlane Burnet With extra CH4
Most acceptable theory of antibody production IgM and IgE
Clonal selection process for antibody formation. Individual lymphocytes are Heavy chain: 5 domain
genetically programmed to produce one type of immunoglobulin. = 1 Variable heavy + 4 Constant heavy
 Definitions Reduction of POLYMER
Isotype- Heavy chain that determine the Ig class -conversion of polymeric antibody into monomeric antibody
st
Allotype: Variation in the constant region of both heavy chain and light chain (1 110 Amino
Acid variable)
Idiotype: Variation in the variable region of both heavy chain and light chain
MONOMER
-2 binding sites
-Basic Ig structure
-2 heavy chains and 2 light chains
-Valence: Number of binding sites
E.g. IgG, IgD, IgE, serum IgA CLASSES OF IIMUNOGLOBULINS
A.IgG
Fragmentation of MONOMER General Information:
Papain: cut monomer exactly at the Hinge region Structure: Monomer
-Monomer is cut into 3 parts: Domains: 4
2 Fab and 1 Fc Percentage: 80%
*1 Fab= 1 light chain + ½ Heavy chain MW: 150,000 Daltons
1 Fc= 2 halves of heavy chain Sedimentation rate (Svedberg): 7s
Pepsin: cuts monomer below the hinge region Half-life: 21-23 days
-Monomer is cut into 2 parts: 23 days
*1 Fc and Fab2 Activate complement: Yes, except IgG4
Fab2= 2 light chains + 2 halves of heavy chains Functions:
DIMER Provide immunity to newborn (cross placenta) except IgG2. Most efficient is IgG1.
-consist of 2 monomer linked by a J chain Complement fixation via Classical Pathway. All except IgG4. Most efficient is IgG3
E.g. Secretory IgA (with 4 binding sites) followed by IgG1 then IgG2.
Secretory component: prevents enzyme degradation of IgA Opsonization.
POLYMER ->2 Monomers -E.g. IgM Agglutination and precipitation reactions
-valence no. is 10 IgM: Agglutination
-No. of monomers: 5 IgG: Precipitation
1. Starlike: not combined with an Ag Neutralization of toxins and viruses
2. Crablike: combined with an Ag ADCC: Antibody dependent cell mediated cytotoxicity
 4 subclasses: Differ in number and arrangement of disulfide bonds IgA
IgG1- 66% Number of disulfide bonds: 2 Structure: Monomer (serum), Dimer (secretions)
IgG2- 23% Number of disulfide bonds: 4 Domains: 4
IgG3- 7% Number of disulfide bonds: 15 Percentage: 10-15%
IgG4- 4% Number of disulfide bonds: 2 MW: 160,000-400,000 Daltons , 160,000-350,000 Daltons (In some references)
IgM Sedimentation rate (Svedberg): 7s
Structure: Pentamer, CH4 (Extra domain), J chain, starlike (free state), crablike (with Ag) Half-life: 5-6 days , 6 days (In some references)
Domains: 5 Activate complement: Alternative Pathway only
Percentage: 5-10% Functions: Major antibody on secretions Associated with anaphylaxis
MW: 900,000 Daltons 2 subclasses
Sedimentation rate (Svedberg): 19s IgA 1: serum, monomer
Half-life: 5 days IgA 2: secretions, dimer
Activate complement: Yes, most efficient -responsible for mucosal immunity -prevents attachment of pathogens to mucosal surfaces
Functions: -With secretory component, which prevents enzyme degradation of IgA. It is produced by
1.Primary response epithelial cells near IgA producing plasma cells.
2.1st Ab to be produced after antigenic stimulation IgE
3.1st to appear on B cell surface Structure: Monomer
4.1st to be produced by an infant Domains: 5, with extra CH4 domain
5.First to appear in phylogeny and last to leave in senescence Percentage: 0.002%
6.Best complement fixer MW: 190,000 Daltons
7.Opsonization Sedimentation rate (Svedberg): 8s
8.Neutralization of toxins and viruses Half-life: 2-3 days
9.Agglutination- BEST 2 days
IgD
Activate complement: No
Structure: Monomer, with tail
Functions:
Domains: 4
1.Reaginic antibody
Percentage: 0.2%
2.Has affinity to basophils and mast cells
MW: 180,000 Daltons
3.High in allergy and parasitic infections
Sedimentation rate (Svedberg): 7s
4.Attaches to eosinophils and releases Major basic protein and Cationic protein
Half-life: 2-3 days (3 days)
5.Atopy: IgE mediated allergic reaction
Activate complement: No
a.Heat labile
Functions: Immunoregulation, Second to appear on the surface of B cells, Found on
b.Defense against parasites
unstimulated but immunocompetent B cells, Sensitive to enzymatic degradation, Postulated to
ab anti-idiotypic antibody
 TYPE IV (Delayed or Cell mediated) Hypersensitivity
Time course: Delayed, 24-72 hours
HYPERSENSITIVITY
Immune mediator: T cells (T delayed type hypersensitivity cells)
-immune responses that produce tissue injury upon subsequent exposure to an
Complement involvement (Classical Pathway): No
agent that is not intrinsically harmful
Effector cells: Macrophages (Antigen presenting cells)
-heightened state of immune responses
Immune Mechanism: release lymphokines from T cells
-Classified by: Gell and Coombs
Examples Tuberculin test (Mantoux), Contact dermatitis, Poison Ivy, Leprosy
TYPE I (Anaphylactic/Immediate) Hypersensitivity
Other different categories for Hypersensitivity:
Time course: Acute, 2-30 minutes
1.Time course
Immune mediator: IgE
a.Acute: seconds to minutes
Complement involvement (Classical Pathway): No
b.Sub-acute: Hours
Effector cells: Mast cells, basophils, B cells
c.Delayed: Days
Immune Mechanism: Release of histamine and other mediators
2.Whether Antibodies or T cells are the principle immune elements
Examples Food allergy, Bee sting, Bronchial asthma, Hay fever, Urticaria (Hives), Eczema, Wheal
Special Terms
and flare, Anaphylaxis
Affinity: initial force between single Fab and single epitope (exact binding site of an
antigen)
TYPE II (Cytotoxic) Hypersensitivity
Weak bonds:
Time course: Sub acute, 5 hours-8 hours
Ionic bonds
Immune mediator: Membrane bound IgG, IgM, and IgA
Hydrogen bonds
Complement involvement (Classical Pathway): Yes
Hydrophobic bonds
Effector cells: RBC, WBC and platelets
Van der Waals Force
Immune Mechanism: Cell lysis due to antibody and complement, Cytolysis
Avidity: sum of all attractive forces between and antigen and an antibody
Examples Transfusion reactions, HDN, AIHA (Autoimmune hemolytic anemia), ITP, Good
INC. Avidity = DEC. dissociation of the complex
pasture’s Syndrome, Myasthenia Gravis
Immune response
Lag (adjustment)
TYPE III (Immune Complex) Hypersensitivity
No antibody production
Time course: Sub acute, 2 hours-8 hours
Log: Antibody production
Immune mediator: Soluble IgM or IgG
Stationary (plateau)
Complement involvement (Classical Pathway): Yes
Ab production = Ab destruction or degradation
Effector cells: Host tissue cells
*Highest titer of Ab
Immune Mechanism: deposition of immune complex to host tissues, Ag-Ab complexes
Decline
Examples Serum sickness, Arthus reaction, SLE, RA, Henoch Schonlein Purpura
Ab production < Ab destruction or degradation
SEROLOGY
SEROLOGY PART I. SECONDARY IMMUNOLOGIC TESTS
-test that involves antigen-antibody reaction in vitro I. Precipitations -antigens involved are soluble antigens, there must be optimum ratio antigen
-Exact Ag binding site (Epitope) - exact Ab binding site (Paratope) and antibody.
-Detect the presence of unknown antibodies in the serum of patient using known Antibody excess: Prozone (False negative)
commercial antigen Zone of equivalence: Ag=Ab
-Unable to culture infectious agent Antigen excess: Postzone (False negative)
-Confirmation of etiologic agent Remedy: Dilution
1. Precipitation in a fluid medium
Forward typing- Unknown Ag 1. Turbidimetry
-Known Ab (Antisera) INC. Turbidity, INC. concentration
Backward typing- Unknown Ab Principle: Measures the light that passes through
-Known Ag (2-5% red cell suspension) 2. Nephelometry
INC. Light scattered, INC. concentration
Preservation of serum: Principle: Scattered light
1.Physical: refrigerate for 72 hours at 4-6oC Advantages: simple, cheap
2.Chemical: add 0.001 g merthiolate per mL of serum or 5% phenol or tricresol in Disadvantages: time consuming
0.1 mL/mL of serum
2. Precipitation by passive immunodiffusion
Inactivation of serum -Allow reaction to take place without using enhancement (E.g. Electricity: speeds up the
-removal of complement proteins that may interfere with serological reaction)
testing -Uses supporting media (agar, agarose, gel)
1.Physical: heat serum at 56oC for 30 minutes or heat at 60-62oC for 3-4 minutes. A. Single diffusion, Single Dimension
2.Chemical: add choline chloride (Test that uses choline chloride: RPR) Oudin test
-a semi quantitative procedure
Reinactivation of serum -after storage -56oC for 10 minutes End result: Precipitin line
B. Single diffusion, Double dimension Radial Immuno Diffusion (RID)
Immunologic Reactions
-measure the diameter
1.Primary: combination of antigen-antibody; non-visible reaction
-Quantitative: diameter is directly proportional to the concentration
2.Secondary: demonstrate antigen-antibody; visible -uses plate with antibody
3.Tertiary: immunologically in vivo. Biologic reaction is detectable. End result: Precipitin ring
E.g Phagocytosis or Opsonization Fahey and McKelvey (Kinetic method) 18 hours Did not allow the reaction to complete
Mancini (Endpoint) IgG: 24 hours IgM: 5-72 hours
 II. Agglutination -antigens involved are particulate
Types of Agglutination reaction
C. Double Diffusion, Double dimension Ouchterlony Technique Direct agglutination
Qualitative procedure a. Antigens are found on the surface of the particles
-Both antigen and antibody is moving b. Examples:
-1st generation for HbsAg i. Blood typing, Hemeagglutination
ii. Kauffman and White Scheme (To serotype Salmonella)
iii. Widal (Typhoid fever)
3. Precipitation by Electrophoretic Techniques iv. Weil-Felix (Rickettsia/ Typhus Fever)
Advantages: STAT v. Cold Agglutination disease (Mycoplasma)
Disadvantage: High-cost Passive Agglutination
Rocket Electrophoresis (Laurell Technique) a. Antigen is artificially attached to a particulate carrier
RID + Electrophoresis b. Pinalaki si antigen para madetect si antibody
The total distance of antigen migration and precipitation is directly proportional c. Examples
to antigen concentration i. Cells
Immunoelectrophoresis (IEP) ii. Latex
Useful procedures for identification of monoclonal protein iii. Bentonite
iv. Celloidin
Example: Bence Jones
v. Charcoal
Precipitation: 40oC-60oC
d. (+) Agglutination
Disappears at: 100oC
Reverse Passive Agglutination
Double diffusion + Electrophoresis a. Antibodies are attached to a particulate carriers
Confirmatory for Bence Jones Protein b. Pinalaki si antibody para madetect si antigen
c. Examples
Counter Immunoelectrophoresis (CIE) i. CRP
Antigen and antibody on the well directly each other ii. Rheumatoid Factor Determination
Immunofixation Electrophoresis d. (+) Agglutination
Immunoprecipitation + electrophoresis Coagglutination: Carrier (inert particle in which the antibody is attached): Bacterium
Similar to Immunoelectrophoresis except antiserum is layered on a medium Agglutination Inhibition
a. (+) No agglutination d. Red cells are used as the indicator particles
b. (-) Agglutination e. Hemeagglutintion inhibition
c. Example: HCG Test
Antiglobulin Technique False Negative reactions in Antiglobulin Testing
-Anti-human IgG is added to bridge the gap between the cells to demonstrate incomplete a. Reagent failure
antibodies. b. Improper washing
-IgG do not cause agglutination; coating only. c. Failure to add antiglobulin reagents
a. Direct Antiglobulin Test-detects in vivo sensitization of cells d. Improper centrifugation
Examples: HDN, HTR, AIHA, Drug induced Hemolytic anemia e. Serum/cell ratio is low
Drug Absorption: penicillin f. Delayed washing (elution of weakly attached antibody)
Membrane modify: cephalosphorin Types of AHG Reagents
Formation of immune complexes: phenacetin, rifampin 1. Polyspecific AHG: Anti-C3d and anti-AHG
Autoantibody formation: mefenamic acid (Ponstel) and Methyldopa (Aldomet)- 2. Monospecific AHG: Anti-C3d or anti-HCG
Antibody to KIDD
b. Indirect Antiglobulin Test -detects in vitro sensitization Neutralization
Examples: Crossmatching, Antibody screening, weak D, confirmation of Rh (-) -antigenic activity is stopped by its specific antibody
-Target: Toxins, viral agents, antibodies to toxin or viral agents
Types:
a.Toxin Neutralization
i.ASO Titration

i. Dick Test (Scarlet fever)

ii. Schick Test (Diptheria)
a. Virus Neutralization
O Check Cells -Group O RBCs sensitized with IgG
-Added to negative Antiglobulin tests to validate the negative reaction III. Complement Fixation Test
-After addition of O check cells, agglutination indicates: -determine complement fixing antibody
1. AHG reagent was added -Components:
2. AHG reagent was not neutralized a. known target antigen reagent
False Positive reactions in Antiglobulin Testing (E.g: beef heart extract, bacterial antigen)
a.Contamination of reagents b. Complement
b.Over centrifugation Best source: Guinea pig serum
c.Direct agglutination by strong agglutinin c. Hemolysin or amboreceptor: antibody used to sensitized indicator cells
d.Over incubation with enzyme treated cells d. Indicator cells: Sensitized sheep RBC
e.Improper use of enhancement reagents
f.Saline stored in glass or metal container
 2 systems involved for complement fixation: Direct Immunofluorescent or Single Layer Technique
1. Test system or Bacteriolytic System -antigen (Tissue or cell) + fluoresceinated antibody = (+) Fluorescence
2. Indicator/Hemolytic System -Uses/application: diagnosis of viral diseases (HSV, CMV, EBV), detect cell
a. No hemolysis is the positive result. surface antigen, Chlamydial antigen, flow cell cytometry
Indirect Immunofluorescent or Double Layer Technique
-solid phase antigen + antibody (sample) + fluoresceinated anti-Immunoglobulin
-Uses: FANA, FTA-ABS
II.B Radioimmunoassay
-use radioactive as label
Examples:
131I- Gamma counter
125I- Gamma counter
3H- Beta counter
14C- Beta counter

-radioactivity is measure by Scintillation Counter

Competitive RIA -Ligand is labeled
-Antigen (sample) + antibody (reagent) + radioactive antigen
-Radioactivity in the solid phase is inversely proportional to the antigen concentration
-Most sensitive for drug assay, hormone
RIST (Radioimmunosorbent test)
PART II. PRIMARY IMMUNOLOGIC TESTS (reaction is not visible) -a competitive RIA for total serum IgE
Immunoassays -RIST activity is indirectly proportional to IgE concentration
Ligand: any substance that will complex to another substance that will be measured RAST (Radioallergosorbent test)
Analyte: substance to be measured. -Measure allergen specific IgE
Ligand assay: one reactant is labeled so that the amount of binding can be measured
II.A Fluorescent Immunoassay Noncompetitive RIA
-Uses fluorescent compounds known as fluorophores or flurochromes as labels -antigen (sample) + radioactive antibody
Examples FITC (Fluorescein isothiocyanate) - most common label- GREEN -amount of radioactivity is directly proportional to the concentration of the
Phycocyanin, Texas Red, Tetramethyl rhodamine antigen
(+) Fluorescence -IRMA (ImmunoRadioMetric Assay)
Measure using Fluorometer, Flow Cytometer, Fluorescence microscope: Qualitative
II.C Enzyme Immunoassay III.D Skin Tests
-EIA or ELISA Trichinella spiralis
-Uses enzymes as labels Xenodiagnosis: Beck’s Test (Using albino rats)
Enzyme labels are: Highly stable, Extreme specificity, Cannot be altered by inhibitors Adult: small intestine
Examples: HPO (Horseradish peroxidase), ALP, Glucose oxidase Larva: muscle
-Measured using spectrophotometry Chlamydia trachomatis -glycogen containing bodies (Halberstaedter Prowazeck bodies)
 NOTE HRP is not horseradish peroxidase Chlamydia psitacci -non glycogen containing bodies (Levinthal Lillie Cole bodies)
-Histidine Rich Protein -Found in malarial parasite (P. falciparum) DISEASE SKIN TESTS
Competitive ELISA Trichinosis Bachman Intradermal
Principle based in RIA Test
Enzyme activity is inversely proportional to the concentration of the analyte Tuberculosis Tuberculin skin test
Noncompetitive ELISA Von Pirquet’s
Indirect ELISA- captures antibody Vollmer’s patch
Solid phase antigen + antibody (sample) + enzyme labeled anti-Ig (not participate in the Mendel’s
initial antigen-antibody reaction) Mantoux
Amount of enzyme label is directly proportional to the amount of the test substance Brucellosis Brucellergin skin test
Coccidiomycosis Coccidiodin skin test
Capture Assay/ Sandwich Immunoassay-Capture Antigen
 Lymphogranuloma Frei test
Enzymatic activity is directly proportional to the amount of antigen in the sample
venereum
Other tests: Histoplasmosis Histoplasmin skin test
Raji Cell Assay  Diptheria Schick test
-measure immune complexes  Scarlet fever Dick test
Chemiluminescence (S. pyogenes)
-chemical reaction + luminal compound = luminescence Glander’s Mullein test
-measured in luminometer, no light source required Toxoplasmosis Toxoplasmin skin test
Anthrax Ascoli Test
Ascariasis Moan test
Hydatid disease Casoni Intradermal skin
test
S. pneumoniae infection Francis skin test
PART III. SEROLOGICAL TEST SEROLOGICAL TESTS FOR SYPHILIS
A. SYPHILIS/GREAT POX/ 1. NON TREPONEMAL/ NON SPECIFIC METHODS
EVIL POX/SPANISH DISEASE -use to detect REAGIN (antibody), a non-specific marker
-caused by Treponema pallidum -Screening tests only
Other Treponemes: VDRL (Venereal Disease Research Laboratory)
T. pertenue: Yaws
Uses heated 50 uL of serum, result is read microscopically
T. endemicum: non-venereal (Endemic) syphilis, Bejel
T. carateum: Pinta
Serum: 56oC for 30 minutes
Stages of Syphilitic infection CSF: No heating is needed.
Primary syphilis Principle: Flocculation
-appearance of hard chancre (painless) Reagents
Laboratory test: Dark field microscopy Cardiolipin (Main reagent) “Wasserman Ag or Diphosphatidyl glycerol”
Coiled with corkscrew motility Cholesterol (provides adsorption center and increase the reacting surface)
Specimen: From the lesions Lecithin (helps neutralize the anticomplimentary activity of Cardiolipin)
Early infection: no antibody production Rotator Serum: 180 rpm for 4 minutes CSF: 180 rpm for 8 minutes
 NOTE Soft chancre: H. ducreyi-school of fish Ring diameter Depth: 1.75 mm Serum: 14 mm CSF: 16 mm
Secondary syphilis Antigen delivery needles
-characterized by general rash and lesions Serum
Appearance of condylomata lata
Qualitative: gauge 18 without bevel that will deliver 60 drops of antigen suspension per
Laboratory test: dark field microscopy and serological test
Latent syphilis
mL of serum
-generally after 2nd year of infection Quantitative: gauge 19 that will deliver 75 drops of antigen per mL of serum or gauge
Patient is asymptomatic 23 with or without bevel that will deliver 100 drops of saline/mL
Laboratory test: Serological test CSF Gauge 21 or 22 that will deliver 100 drops per mL
Tertiary syphilis Reporting
-Characterized by destructive lesions known as Gummas Non-reactive: no clumps
Neurosyphilis: Asymptomatic Weakly reactive: small clumps
 NOTE
Specimen of choice: CSF Reactive: medium to large clumps
Chediak-Higashi Triad
Congenital syphilitic infection *reports last reactive dilution
1.Albinism
-Maternal Spirochetemia False positive:SLE, RF, IM, Malaria, Pregnancy
2.Bleeding (Thrombocytopenia)
 HUTCHINSONIAN TRIAD  Pag gumaling, nawawala ang Reagin.
3.Immunodeficiency
1.Hutchinson’s Teeth
2.Interstitial keratitis
3.Nerve deafness
RPR (Rapid Plasma Reagin)
 TPI (T. pallidum Immobilization Test)
Uses unheated serum, results are read macroscopically
Most specific test for syphilis
Principle: Flocculation
Standard Test
Reagents (Colorless alcoholic solution)
Principle: the antibody produced against T. pallidum plus complement can immobilize the live
1.Lecithin
Treponemes
2.Charcoal (for easier visualization)
Reagent (Antigen): live actively motile T. pallidum organism *extracted from the lesions of
3.Choline chloride (chemically inactivates serum)
infected rabbit
4.Thimerosal (preservative)
(+) : >50% immobilized treponemes
Rotator: 100 rpm for 8 minutes
Ring diameter: 18 mm
B. GROUP A STREPTOCOCCAL INFECTION
Antigen delivery needle: gauge 20, 60 drops/mL
-caused by Streptococcus pyogenes (β hemolytic, Lancefield group A, G (+) cocci in chains)
-Upper respiratory tract infection
2. TREPONEMAL SEROLOGIC TEST or SPECIFIC METHODS
Complication:
-uses the true treponemal antigens, detect ATA (Anti Treponemal Antibodies)
Erysipelas (skin infection) to Acute Glomerulonephritis
2 sources:
Upper respiratory tract infection to Rheumatic heart fever
1.Nonpathogenic (Reiter strain)
Cross reacts with M-protein: prevents phagocytosis
2.Pathogenic (Nichol’s Strain)
Streptolysin S
-responsible for β hemolysis
FTA-ABS (Fluorescent Treponemal Test for Syphilis)
-Oxygen stable
Principle: Indirect Fluorescent Immunoassay
-not antigenic
Reagent (Antigen): Nichol’s strain fixed on the slide
Hyaluronidase
Absorbent
-spreading factor
-Uses Reiter strain
Tissues made up of hyaluronic acid
-to remove cross reactivity with other Treponemes which shares common
Streptokinase
epitope
-dissolves clots
 Test of choice to be run at Dark Field Microscopy
Activates plasmin to dissolve clots
Specimen: Chancre
Eythrogenic toxin-causes scarlet fever
HAATS (Hemeagglutination Treponemal Test for Syphilis)
Streptolysin O
Principle: Hemeagglutintion
-Oxygen labile
Reagent (Antigen): glutaraldehyde stabilized turkey RBC coated with treponemal antigen
-Highly antigenic
-Bacterial toxin released during S. pyogenes infection
MHA-TP (Microhemeagglutination T. pallidum Test)
-hemolytically inactive in oxidized form
Principle: Hemeagglutination
-Can be neutralized by ASO
Reagent (Antigen): tanned formalin sheep RBCs coated with treponemal antigen
 C. FEBRILE AGGLUTININS
ASO TITRATION (Macrotechnique of Rantz and Randall) -Antibodies demonstrated in microbial diseases that are manifested by high fever
Interpretations of bacterial agglutination test:
Principle: Neutralization
1.Rapid slide test is only for screening. A positive result must be confirmed by tube test.
-The test estimates the amount of ASO (Antibody) that in the presence of a constant
2.A single negative test does not necessarily mean no infection.
dose of SLO completely inhibit hemolysis of a constant given number of Red Cells 3.A single positive test is of no significance unless the titer is sufficiently high
-Defines a minimal hemolytic dose of SLO as that will completely hemolyze 0.5 ml of a 4.Peak titer may occur in convalescence.
5% rabbit RBC suspension Febrile diseases
ASO Titer: reciprocal of highest dilution showing no hemolysis, expressed in Todd units Typhoid fever
Controls Typhus Fever
Red cell tube # 13: No hemolysis Brucellosis
SLO Tube #14: complete hemolysis Tularemia-To diagnose F. tularensis, use Brucella Antigen. They share common epitope
Normal values (cross reactivity).-Agglutination Test
Children: <125 Todd units Febrile Agglutinin Tests
Adults: <166 Todd units Widal test (Slide method)
-For diagnosis of typhoid fever
Positive result: No hemolysis
Principle: Direct agglutination
RAPID LATEX AGGLUTINATION TEST
Tube test (Confirmatory): highest dilution showing ++ or 50% agglutination
Principle: Passive agglutination -Significant titer: 80 and above (+) Agglutination
Significant Titer: >200 IU/mL Typhidot: detection of specific IgM (present) and IgG (past) to Salmonella typhi
Anti-DNAse B testing Salmonella typhi
Principle: Neutralization Acquired by: ingestion
-Sometimes earlier than ASO to appear in streptococcal pharyngitis. Mary Mallon- cook
-overnight incubation at 37oC is required to permit the antibodies to H antigen and K antigen (thermolabile)
inactivate the enzyme. Tubes are graded with a +4 to 0 depending on the intensity of Hauch- flagellar antigen
the color. Kapsel- capsular antigen
Streptozyme testing Vi antigen- Virulence antigen Seen in salmonella typhi and paratyphi
Principle: Agglutination O antigen (thermostable)
Ohne- somatic or body antigen
-slide agglutination screening test for detection of antibodies to several
Lab diagnosis of typhoid fever
streptococcal antigens.
Culture technique: standard
-Sheep red blood cells are coated with streptococcal antigens. Week 1 and 2: Blood
Week 3: urine
Week 4: stool
Weil-Felix Test
-a nonspecific test for the detection of antibodies against Rickettsial diseases
Principle: Direct agglutination
Leptospirosis (Weil’s disease)
Significant titer: >160
-Caused by: Leptospira interrogans
Antibody: Rickettsial antibodies in the serum
Chlamydia and Rickettsia
Infection stages:
-“Energy parasite”
1.Septicemic stage: fever, headache
-cannot make their own energy
2.Immunological stage: infectious jaundice
-not classified as virus: because they contain both DNA and RNA even if they are
Laboratory diagnosis
obligate intracellular organism
Culture method
-Gram (-) obligate intracellular organism
Microscopy
Antigen used: Proteus Ag (cross reactivity) share common epitope
Serological Test
Disease OX-19 OX-2 OX-K Primary Atypical Pneumonia
Scrub - - ++++ -Caused by Mycoplasma pneumoniae, “Eaton’s Agent”
RMSF ++++ + - -Cold agglutinins: antibodies that react best with RBC at temperature below
o
37 C
Epidemic typhus
Murine typhus Cold agglutinin Test
Q Fever - - - -Clinical samples with M. pneumoniae infection contain IgM antibodies that target the I-
Rickettsial pox - - - Antigen on RBC and induce agglutination
-Direct Agglutination
OX-19 and OX-2: Proteus vulgaris -Rapid screening test for cold agglutinins
OX-K: Proteus mirabilis -Principle: Hemeagglutination
Epidemic Typhus: R. prowazekii -Antigen: Fresh human Group O cells
Murine typhus: R. mooseri -Antibody: cold agglutinins in patient serum *Hortsman and Tatlock for cold agglutinins
RMSF: R. rickettsi -Tubes are incubated at 4oC for 18 to 24 hours
Scrub: O. tsutsugamushi Diagnosis
Q fever: Coxiella burnetti Complement fixation
Rickettsial pox: R. akari ELISA
Lyme disease -caused by Borrelia burgdorferi -Rash: bull’s eye rash or erythema Chronicum
migrans -Transmitted by Ixodes tick
 NOTE Ixodes tick: also transmits Babesia microti, mistaken as P. falciparum
Laboratory Tests: IFA-Borrelia antibodies, EIA, Western blot, PCR
 Serologic markers for HBV
Hepatitis surface antigen (HBsAg) Australian antigen
PART IV. VIRAL DISEASES Best indicator of early acute infection
IV.1 Hepatitis Indicates acute or chronic HBV infection
-Inflammation of the liver Hepatitis core antigen (HBcAg) Not detected in serum because it is found only on
Hepatitis A “Infectious hepatitis” the hepatocytes. Liver biopsy
Year of discovery: 1973 HBeAg (e: Envelope) High levels of virus and high degree of infectivity Enfectious or
Enfective
Short incubation hepatitis
Anti-HBc
Agent: hepatitis A (HAV) RNA virus
IgM: useful in detecting infection during window period, indicator of current
PicoRNAviridae: smallest RNA virus
infection
Most common type of hepatitis
Window period
MOT: fecal-oral route False negative result
Not isolated in serum because it is shed on the feces Only ANTI HBC is positive
Self-limiting disease IgG: lifelong marker
Hepatitis A markers of infection Anti-Hbe Gumagaling First serologic marker of convalescent phase
Early shredding of virus in the stool Anti-HBs Protected, immunity Viral clearance HBV ANTI hbS (Shield)
Appearance of IgM anti-HAV with the onset of symptoms (icterus and INC. liver Tests for HBV (Increasing sensitivity)
enzymes First generation: Ouchterlony
Development of anti-HAV IgG and immunity on recovery Second generation
Specimen: Serum, detects antibodies (Anti-HAV IgG and IgM) Counterelectrophoresis, Rheophoresis, Complement fixation
Laboratory Diagnosis Third generation (most sensitive)
RIA and ELISA, detects the presence of specific HAV antibodies Reverse Passive Latex Agglutination, ELISA, Reverse Passive Hemeagglutination
Radioimmunoassay
Hepatitis B “Serum hepatitis” Hepatitis C “NANB”
Year of discovery: 1963 studied by Blumberg Past: Most common cause of post transfusion hepatitis
Long incubation Agent: Flavivirus
Agent: HepaDNAviridae MOT: parenteral (blood transfusion)
Dane particle: Infectious form Complete form of Hepatitis B Tests for HCV
MOT: parenteral, vertical, sexual Surrogate test ALT and anti HBc
Tumor causing virus Serologic Tests ELISA, RIA
Leads to liver cirrhosis and Hepatoma RIBA: Detects HCV specific antibody or anti HCV CHON
Most common transfusion Hepatitis RT-PCR: HCV RNA (persistent HCV infection)
Hepatitis D “Delta hepatitis” ANTIGENS
“Defective hepatitis” VCA-Viral Capsid Antigen
Agent: Hepatitis D virus (HDV), no proper classification If positive: Primary Infection
A defective virus kasi kailangan ni Hepatitis D si Hepatitis B for replication EA- Early Antigen
MOT: parenteral, sexual EA-Diffuse
Co-infection and superinfection with HBV EA-Restricted
Often detected by ELISA (IgM-Anti HDV) If positive: Reactivation
Laboratory diagnosis NA-Nuclear Antigen
Indirect ELISA EBNA-Epstein Barr Nuclear Antigen
Anti HDV If positive: Past infection
Anti-HBc IgM differentiates co-infection (present) from superinfection (absent)
PCR TYPES OF HETEROPHILE ANTIBODIES
-Refers to Ab the body produces as part of an immune response to an infection
Hepatitis E “Water borne hepatitis” but not related to the causative agent
Agent: HEV (Caliciviridae) 1.Heterophile Ab in IM
a.Reacts with sheep cells (T lymphocytes: CD2), OX (Beef cells) and horse cells but
High mortality rate in women
Self-limiting disease not with Guinea pig cells
b.Produced response to EBV infection
HEV RNA: detected on the feces 2 weeks after the onset of the disease. Identified by means of
PCR. 2.Heterophile Ab of Forssmann
a.Reacts with sheep cells, horse cells, guinea pig cells but not with Beef cells
Laboratory diagnosis Electron microscopy Indirect ELISA RT-PCR
b.Produced by Salmonella, Shigella and other bacterial species

Hepatitis G “Blood borne hepatitis” 3.Heterophile Ab in Serum sickness

a.Reacts with sheep, OX, Horse and guinea
Agent: HGV
b.Horse products during immunization
Flavivirus
No available serological tests TESTS FOR IM
Causes syncytial giant cell hepatitis 1.Paul Bunnel
a.Screening test
IV.2 INFECTIOUS MONONUCLEOSIS b.Principle: Hemeagglutination
-“Kissing disease” or Glandular fever (RES) c.Reagent: 2% suspension of sheep RBC
-Agent: EBV d.(+): Agglutination
-Target: B lymphocytes (CD21 receptor) 2.Davidson Differential Test
-Common in adolescent and adults a.Principle: Absorption-Hemeagglutination
-Downey cells: atypical T lymphocytes b.Antigen: guinea pig kidney cells and beef RBC
c.Indicator cells: SHEEP RBCs
 ABSORPTION PATTERN Pol (Polymerase)
Heterophile Beef RBCs Guinea Pig a.Reverse transcriptase: RNA to DNA
Forssmann NO YES b.Integrase: inserts viral DNA to host DNA
c.RNAse
IM NO
d.Protease: cleaves structural protein located near the nucleic acid
Serums sickness YES YES Must marker for HIV:
P24
AGGLUTINATION PATTERN Gp41
Gp120
Heterophile After After absorption
Gp160
absorption to to GUINEA PIG
BEEF NOTE AIDS-HIV LAW 8504 FEB 13
Forssmann HIGH titer LOW titer HIV (+)
IM LOW titer High titer DONOR: TO RITM Research Institute of Tropical Medicine
Serums sickness LOW titer PATIENT: TO SACCL TD, AIDS Cooperative Central Laboratory
Monospot (Slide Method)
TESTS for HIV
Principle: Absorption-Hemeagglutination
1.Screening test
a. Indicator cells: Horse RBC (more sensitive indicator of antibodies found in IM)
a.ELISA
IV.3 HUMAN IMMUNODEFICIENCY VIRUS (HIV) b.RIA
-also known as HTLV-III (Human Lymphotropic Virus III), LAV (Lymphadenopathy Associated c.Agglutination tests
Virus), ARV (AIDS-associated Retrovirus) 2.Confirmatory tests
Types of HIV: a.Western blot
1.HIV-I: Global -positive if gp 41 band appears alone or when an envelope antibody
2.HIV-II: Africa (gp41, gp120 or gp160) appears combination with another HIV band
-Family: Retroviridae (with reverse transcriptase enzyme); subfamily Lentiviridae
(Detects Proteins)
-Has marked preference for T helper cells (CD4+) which serves as the receptor site for the virus
Main structural genes:
Testing is often done at 6 weeks, 3 months and 5 months after exposure to find
1.Envelope: gp 120, aid in the fusion and attachment of HIV to CD4 (+) cells out if a person is infected with HIV
2.Gag gene (group antigen) p15, p17, p24 (found in nucleocapsid) The US CDC defines positive test for HIV as a “repeatedly positive ELISA followed
Anti p24 (first antibody to be produced) by a positive Western Blot Test”.
IV. 4 Dengue Virus (Flaviviridae) 2. Systemic or non-organ specific- multiple organs
Clinical manifestation
Thrombocytopenia, Hemoconcentration, Positive tourniquet test
V.1 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)
Lab diagnosis Complement fixation test, Immuno assay
-disease of the connective tissue
PART V. AUTOIMMUNE DISEASES -immune complexes disease characterized by
Clinical Types overproduction of
1.Organ specific- localized autoantibodies
a.INC. gamma globulins -Arthritis: most common manifestation
b.(+) diverse antibodies
-Manifests itself by skin lesions “butterfly rash” or RED wolf
c.DEC. complement in serum
d.Immune complexes in serum *red rash across nose and cheeks
e.DEC. Ts cells Laboratory Observations
Autoimmune disease Antigens
-Most striking feature: presence of Anti-Nuclear antibodies
Addison’s disease Microsomal proteins of adrenal cells (ANA) - not diagnostic of SLE
Acute disseminated encephalomyelitis Basic protein of myelin LE cell: PMN leukocyte with ingested LE body, often in rosette formation
Hashimoto’s thyroiditis Thyroglobulin, microsomal antigen -Hypocomplementemia
IDDM Islet cells -Hypergammaglobulinemia
Goodpasture syndrome Type IV of collagen membrane SEROLOGICAL TESTS
Grave’s disease TSH receptors Antinuclear Antibody (ANA)
Myasthenia gravis Acetylcholine receptors oVisible method
Autoimmune chronic active hepatitis Smooth muscles oPrinciple: Indirect Immunoenzyme
Pernicious anemia Gastric parietal cell antigens, intrinsic factor
oHep2 cells (Nuclear antigen) + Patient serum (with ANA) +
Sjogren’s syndrome Salivary gland nucleolar antigen
AHG + Stain
Primary biliary cirrhosis Mitochondria
Autoimmune myocarditis Striated cardiac muscles
= (+) for ANA (brown cytoplasmic or nuclear stain)
Pemphigus vulgaris Epidermal antigen Indirect Fluorescent Antibody Test Detection for ANA
Bullous pemphigoid Skin basement region antigens oPrinciple: Indirect Immunofluorescent
Autoimmune rheumatic heart fever Heart and joint tissue antigen
STAINING PATTERN ANA DISEASE ASSOCIATION
V.2 RHEUMATOID ARTHRITIS
HOMOGENOUS Anti DNP Rheumatoid dse. -Autoimmune disease causing chronic inflammation of the joints
(SOLID OR DIFFUSE) Anti nDNA SLE and periarticular tissue
Anti dsDNA Sjogren’s -Rheumatoid Factor:
Anti ssDNA MCTD Group of immunoglobulins that interacts specifically
with the Fc portion of IgG molecules (Antibodies)
Mainly IgM that attacks IgG
PERIPHERAL (RING, Anti DNP Active stage of SLE Anti-cyclic citrullinated peptide
MEMBRANOUS, Anti nDNA Sjogren’s (Marker of RF)
SHAGGY) Anti dsDNA Laboratory Tests
-Sheep cells agglutination test (Rose et Al or Rose-Waaler Test)
Anti ssDNA -Latex Fixation Test (Singer and Plotz)
SPECKLED (MOTTLED, Anti ENA (extractable SLE -Sensitized Alligator Erythrocytes test (Cohen et al)
PEPPER DOTS) nuclear antigen) RA -Bentonite Flocculation test (Bloch and Bunim)
Anti-SMITH or anti-SM MCTD
VI. TUMOR MARKERS
(marker of SLE) -molecules occurring in the blood that are associated with cancer
Anti RNP
NUCLEOLAR Anti RNP Scleroderma CEA- GI cancer
AFP- hepatocellular CA
PSA- prostate CA
DISCRETE Anti-centromere CREST CA 15-3- breast CA
Calcinosis CA 19-9- pancreas, stomach, bile duct
Reynauds CA 125- ovarian CA
Esophageal dysmotility CA 72-4- gastric CA
Sclerodactyl
Telangiectasia
 READING AGGLUTINATION TUMOR MARKERS ASSOCIATED CANCERS
GRADING DESCRIPTION AFP Hepatic and testicular cancer
Cells Supernatant ALP (placental) Lung cancer
0 No agglutinate Dark, turbid, homogenous Amylase Pancreatic cancer
W+ Many tiny agglutinates Dark turbid BRCA-1 Breast or ovarian cancer
Many free cells CA 125 Ovarian cancer (treatment and recurrence)
May not be visible without microscope CA 15.3 or CA 27.29 Breast cancer (treatment and recurrence)
1+ Many small agglutinates Turbid CA 19.9 Gastric, pancreatic, colorectal cancers
Many free cells  Calcitonin Medullary thyroid cancer
2+ Many medium sized agglutinates Clear Cathepsin D or Estrogen Breast cancer
Moderate number of free cells receptor (ER)
3+ Several large agglutinates Clear CEA Colorectal, stomach, breast, lung cancer (treatment and recurrence)
Few free cells CK-1 Small cell lung cancer
4+ One large, solid agglutinate Clear GGT Hepatoma
No free cells HER-2/neu Breast CA (efficiency of trastuzumab or Herceptin therapy)
 Nuclear matric protein Urinary bladder cancer

SUMMARY OF ANTIGEN USES ASSOCIATED TUMORS

 Bence Jones protein Screening Multiple myeloma
TUMOR  AFP Screening, staging, pathologic diagnosis Hepatocellular cancer, nonseminomatous
ASSOCIATED testicular cancer
 PSA Screening, staging, pathologic diagnosis, Prostate cancer
ANTIGENS monitoring
AND THEIR  CA-125 Screening, staging, pathologic diagnosis, Ovarian adenocarcinoma
monitoring
AREAS OF  CEA Staging, pathologic diagnosis, monitoring, Tumors of GI tract, colorectal cancer
CLINICAL USE metastasis
Beta-2-microglobulin Staging Lymphoma
AND THE Lactate dehydrogenase Staging Lymphoma
ASSOCIATED
CA 19.9 Monitoring Colonic and pancreatic adenocarcinoma
TUMORS CA 15.3 Monitoring Breast adenocarcinoma
 BLOOD BANK
The FORMATION of A, B, and H Antigens

- ABO genes code not for the antigen themselves but for the production of glycosyltransferases that add
immunodominant sugars to a basic precursor substance.
Gene Glycosyltransferase Immunodominant sugar
H gene L-fucosyltransferase L-fucose
A gene N-acetylgalactosaminyl- N-acetyl-D-galactosamine
transferase
B gene D-galactosyltransferase D-galactose

***99.99% of all individuals possess the H gene.

 ABO DICREPANCIES
GROUP 1
GROUP 2
- due to weakly reacting or missing antibodies. Most commonly encountered.
- due to missing antigens and is the least frequently encountered.
It is found in the following:
It is found in:
1. newborn
1. subgroups of A or B
2. elderly patients
2. leukemia
3. leukemic patients
3. Hodgkin’s dse
4. patients with lymphoma
4. “Acquired B” in cases of GIT obstruction
5. pxs using immunosuppressive drugs
ACQUIRED B phenomenon - when bacterial enzymes (of
6. with congenital agammaglobulinemia
7. pxs with immunedeficiency Proteus vulgaris) modify N-acetylgalactosamine into D-
8. pxs who undergone BM transplant galactose)
9. CHIMERISM - a rare group 1 discrepancy. 5. Ab to low incidence Ag
- presence of two cell populations in single individual like in cases of fraternal twins 6. excess blood group specific soluble substances (BGSS)
- Artificial CHIMERA:
- Blood transfusion (e.g. O to A or B)
- BM transplant
- Exchange transfusion
- Fetal-maternal bleeding

GROUP 3
- caused by protein or abnormalities resulting to rouleaux formation. GROUP 4
It is found in: - due to miscellaneous problems seen in:
1. increased globulin (e.g. Multiple myeloma, Waldenstrom 1. polyagglutination
macroglobulinemia) 2. cold reacting Abs (allo and auto)
2. increased fibrinogen 3. unexpected ABO isoantigen
3. presence of plasma expanders (e.g. dextran) 4. warm autoantibodies
4. Wharton’s jelly 5. RBCs with cis AB phenotype
 REACTIVITY OF ANTI-H ANTISERA OR ANTI-H LECTIN WITH ABO
BLOOD GROUPS

Greatest Amount of H Ag Least Amount of H Ag

O > A2 > B > A2B > A1 > A1B

WIENER FISHER-RACE ROSENFIELD

Rh0 D Rh1
rh’ C Rh2
rh” E Rh3
hr’ c Rh4
hr” e Rh5

Inheritance:
Rh locus - located on chromosome 1 (along with the genes for elliptocytosis)

B SUBGROUPS - are infrequent LW - is the antigen closely associated phenotypically with Rh

Anti-B lectin - Bandeiraea siimplicifolia - it is formerly known as Rh25
Ne-a - is the recently discovered anti-thetical antigen to LW
BOMBAY (Oh) PHENOTYPE

The allele h is very rare and does not produce the L-fucose necessary for the
formation of H structure. The genotype hh or Hnull is also known as the Bombay phenotype
or Oh.
 SHORTHAND WIENER BLOOD FISHER LEWIS SYSTEM
FACTORS -RACE - Lewis antigens are manufactured by tissue cells and secreted into the body fluids then
R0 Rh0 Rh0, hr’, hr” Dce adsorbed onto the red cell membrane (not really an integral part of the red cell membrane
LEWIS ANTIGENS
R1 Rh1 Rho, rh’, hr” DCe - Lewis substances ( in secretions) - are glycoproteins
- Lewis antigens (cell bound Ags) - are glycosphingolipids
R2 Rh2 Rho, hr’, rh” DcE - Poorly developed at birth (not found in cord blood or newborn RBC)
-  Le(a-b-)  Le(a+b-)  Le(a+b+)  Le(a-b+), the true phenotype
Rz Rhz Rho, rh’, rh” DCE Inheritance:
Le genes:
r Rh hr’, hr” dce - located on the short arm of chromosome 19
- genes does not actually code for the production of Lewis antigens but,
r’ rh’ rh’, hr” dCe - rather, produces a specific L-fucosyltransferase to type I- precursor substances.

r” rh” hr’, rh” dcE 2. Le(a-b+): Secretors

- this phenotype is the result of the genetic interaction of Lele and Sese genes- Both Lea-
ry rhy rh’, rh” dCE soluble and Leb-soluble antigens can be found in the secretion but only Leb adsorbs onto
the red cell membrane
R1r or DCe/dce - most common in whites approx. 35%. 3 . Le(a-b-): Secretors or Nonsecretors)
R0r or Dce/dce - most common in blacks approx. 42%. - 80% ABH Secretors
- 20% ABH Nonsecretors
IMMUNOGENICITY OF COMMON Rh ANTIGENS LEWIS ANTIBODIES - are usually nsturally occurring IgM; react best at RT or lower ;
considered clinically insignificant
D > c > E > C > e - binds complement and therefore capable of triggering in vitro hemolysis
Rh ANTIBODIES - enhanced by enzyme treatment
- are usually IgG1 or IgG3 rbc-stimulated, either during transfusion of during pregnancy - readily neutralized by Lewis blood group substances
- us. do not agglutinate in saline Anti-Lea - most commonly encountered Ab of the Lewis system
react best at 37ºC and can be demonstrated by testing in high-protein media or by the indirect Biologic Significance:
antiglobulin test. 1. Leb has receptors for Helicobacter pylori.
- Reaction is enhanced by the use of enzyme-treated rbcs 2. Lex antigen is marker for Reed-Sternberg cells of Hodgkin’s dse
- Do not us. bind complement
- They cross the placenta and can cause HDN
 MNSs U BLOOD GROUP SYSTEM P BLOOD GROUP SYSTEM
- The two loci system P ANTIBODIES
MNSs U ANTIGENS ANTI-P1
- MNS are inherited as close linkage. MN is associated with - naturally occurring IgM Abs in the sera of P2 individuals; us. a weak cold
glycophorin A; Ss is associated with glycophorin B. reactive saline agglutinin
- MNS antigens are important markers in paternity testing - can be neutralized with soluble P1 substance in hydatid cyst fluid
- Are found on red cells, not in body fluids and secretions (Echinococcus granulosus infection)
U - stands for universal. RBCs with the S or s antigen also have the U antigen. ANTI-P
- naturally occurring alloantibody in the sera of all Pk individuals.
INCIDENCE OF PHENOTYPES AUTOANTI-P (Donath Landsteiner antibody)
MN = 50% M = 30% N = 20% - IgG biphasic hemolysins (attaches to rbcs in cold, lyses rbcs in
S = 55% s = 45% warm temp.) associated with PAROXYSMAL COLD HEMOGLOBINURIA
U- = common among blacks U+ = common among whites ANTI-PP1 Pk (ANTI-Tja)
MNSs ANTIBODIES - predominantly IgM; binds complement
ANTI-M and ANTI-N - associated with spontaneous abortions in early pregnancy
- Mostly IgM, naturally occurring cold reactive saline agglutinins that do not bind complement - may demonstrate in vitro hemolysis
or react with enzyme-treated cells (DESTROYED by ENZYMES!)
- Anti-N = seen in renal pxs who are dialyzed with I BLOOD GROUP SYSTEM
equipment sterilized with formaldehyde  At birth, infant red cells are rich in i antigen. (I almost
- Anti-M- reaction enhanced by acidification undetectable)
ANTI-S and ANTI-s  During the first 18 mos. of life, i slowly decreases, I increases
- most are IgG, reactive at 37ºC and the antiglobulin phase  Adult red cells, rich in I and only trace amount of I
- may bind complement and have been associated with HDN and *** CDA Type II or HEMPAS - associated with much greater i
HTRs activity on red cells than control cord cells
P BLOOD GROUP SYSTEM PHENOTYPES DETECTABLE Ags POSSIBLE Abs
P ANTIGENS P1 P, P1, -
- consists of three antigens: P,
P1 and Pk which are P2 P Anti-P1
biochemically related to the P - Anti-PP1 Pk
CHO chain that makes up the
ABH and I antigens P1K P1, Pk Anti-P
- P1 antigens are poorly P2K Pk Anti-P, Anti-P1
developed at birth
 K null McLEOD
Kx antigens Present in abundance/  Lacking/ -
Autosomal Kell antigens Lacking/ - Decreased Expression/ 
I ANTIBODIES
Benign ANTI-I - is a weak naturally occurring saline reactive IgM autoagglutinin detectable Red cell abnormality NO YES
only at 4ºC.
Pathologic ANTI-I - is a potent cold autoagglutinin that demonstrates high titer reactivity and Other Kell Antigens:
reacts over a wide thermal range (0 to 30ºC) Kell (K) Sutter (Jsa) Peltz (Ku)
***Pxs with Mycoplasma pneumoniae infections may develop strong cold agglutinins with Cellano (k) Matthew (Jsb) Penny (Kpa)
autoanti-I specificity. Class (KL) Rautenberg (Kpb) Williams (Kw)
ANTI-i - an IgM agglutinin and reacts optimally at 4ºC. **Cellano (k)- occurs in over 99% Caucasians and Negroes
- associated with INFECTIOUS MONONUCLEOSIS **Kell (K)- 9% Caucasians; 3.5% Negroes
KELL BLOOD GROUP SYSTEM DUFFY BLOOD GROUP SYSTEM
KELL ANTIGENS DUFFY ANTIGENS
- are found only on red cells, are well-developed at birth and are not destroyed by enzymes. Fya and Fyb
- The K (Kell) antigen is rated second only to D antigen in immunogenicity. - well-developed at birth
- Destroyed or inactivated by sulfhydryl reagents like AET, DTT, ZZAP (artificial Kellnull) - easily destroyed by common proteolytic enzymes
*** Fy (a-b-) - important anthropological marker for African blacks
KELL ANTIBODIES - were shown to resist infection caused by the Plasmodium vivax and Plasmodium knowlesi.
ANTI-K- outside of ABO & Rh, anti-K is the most common antibody seen in blood bank DUFFY ANTIBODIES
- us. IgG “immune” antibodies reactive in AHG phase Anti-Fya and Anti-Fyb
- can cause both HDN and HTR - usually IgG antibodies and react best at the AHG phase
PHENOTYPES - both are implicated in delayed HTR (DHTR) and HDN
KO or Knull Phenotype
- lacks Kell antigens; have no membrane abnormality KIDD BLOOD GROUP SYSTEM
McLEOD Phenotype KIDD ANTIGENS
- lacks the Kx antigen (which might be a precursor for Kell antigens) Jka and Jkb- well developed at birth, contributing to the potential for HDN
- rare phenotype with decreased Kell system antigen expression and - show in vitro hemolysis
abnormal red cell morphology - reactivity enhanced by enzyme treatment
** Jk (a-b-) - resists lysis in 2M urea and occurs mainly in Mongoloids
- McLeod Syndrome is asso. with the ff:
KIDD ANTIBODIES Anti-Jka and Anti-Jkb
- Chronic but often well-compensated hemolytic anemia -
- show dosage
reticulocytosis, acanthocytosis
- both are IgG immune antibodies(1ºly IgG3) and antiglobulin reactive
- muscular dystrophy (increased serum CK-MM)
- bind complement - common cause of delayed HTRs
- common among males suffering from Chronic Granulomatous Disease
 Lutheran
Xg SYSTEM
LUTHERAN ANTIGENS
- has only one antigen, Xga and two resulting phenotypes Xga (+) and Xga (-)
Lua and Lub
- Xga allele - located on the short arm of X chromosome;
- poorly developed at birth
more prevalent in women and associated with multiple transfusion
**Lu (a-b-) - may result from three different genetic backgrounds
- Anti- Xga - predominantly IgG reactive in IAT, binds
- Dominant In (Lu) Types - expression of Lutheran was
complement but do not cause HDN & HTR
thought to be suppressed by a rare dominant regulator
- sensitive to enzymes
gene, In(Lu) for “inhibitor of Lutheran”
Bg SYSTEM
- Recessive Lulu type - lacks all Lu Ags
- Recessive Sex-linked Inhibitor Type - X-borne inhibitor of Lu - related to Human Leukocyte Antigens (HLAs) on RBCs
Bg Antigen Related HLA Ag
LUTHERAN ANTIBODIES Bga HLA -B7
Anti-Lua Bgb HLA -B27
- naturally occurring saline agglutinins that react best at RT Bgc HLA -A28
- Characteristically show loose and mixed-fixed reactivity in vitro Anti-Bg Abs - clin. Insignificant; IgG reacting weakly in IAT- destroyed by treatment with
Anti-Lub chloroquine diphosphate or glycine-HCl/EDTA soln
- most are IgG (often IgG4) immune antibodies; reactive at AHG at 37ºC and the AHG phase
DIEGO BLOOD GROUP SYSTEM
Sd ANTIGEN- Anti-Sd Abs - demonstrates characteristic shiny, refractile,
Dia Antigen - useful tool in anthropological studies of
mixed-field agglutination
Mongolian Ancestry
SUMMARY
CHIDO/RODGERS BLOOD GROUP SYSTEM
Antibodies that causes HDN/EF
CH/RG Antigens - Antigenic determinants of human complement C4
Anti-C/Anti-AB (ABO) Anti-f (Rh)
Anti-CH/RG Abs - can be neutralized by fresh human serum
Anti-U (MNSs U) Anti-D
- HTLA Abs; weak reacting; clinically insignificant
Anti-Fya Anti-K
Abs Formerly Classified as HTLAs (High Titer, Low Avidity)
Anti-Ss Anti-Jk
Anti-Ch Chido
IgM ‘naturally occurring’ antibodies (generally) but can become IgG
Anti-Rg Rodgers
Le P
Anti-Kn Knops
I Lu P1
Anti-McC McCoy
IgG ‘immune’ antibodies
Anti-Yka York
K Fy
Anti-Csa Cost-Sterling
Jk Ss Xga
Anti-JMH John Milton Hagen
 DONOR SELECTION, BLOOD COLLECTION &
Blood groups asso. With secretor genes COMPONENT PREPARATION
Lewis Lutheran ABH
PHYSICAL EXAMINATION
Enhanced by Proteolytic Enzymes
1. General Appearance.
Kidd Rh I P1
2. Weight. 110 lbs (50 kg)
Lewis If donor is less than 110 lbs:
Inactivated/Destroyed by Proteolytic Enzymes Amount of blood to be drawn:
Duffy MNSs U ALLOWABLE AMOUNT = Donor’s weight(lb) x 450ml
Do not store well (labile antigens) 10 lb
Kidd Duffy ANTICOAGULANT NEEDED = Allowable amount x 14
Antigens that are well-developed at birth 100
MNSs U Kidd Kell Duffy ANTICOAGULANT TO REMOVE = 63ml - anticoagulant needed
Antigens that are poorly-developed at birth E.g. If WB must be drawn from a donor who weighs 70 lbs, the calculations would be:
Lewis P I Lutheran (70/110) x 450 = 286.4 ml of blood to be drawn
Antigens commonly showing dosage effect (286.4/100) x 14 = 40.1 ml of anticoagulant needed
63 - 40.1 = 22.9 ml of anticoagulant to be removed
M/N S/s K/k Jka/Jkb
Rh antigens except D 3. Temperature: Orally should not exceed 99.5ºF or 37.5ºC
Low Frequency antigens 4. Pulse: 50 to 100 beats per minute
5. Blood Pressure: Systolic no greater than 180mmHg, diastolic no greater than
Kpa Jsa Lua
100 mmHg
High Frequency antigens
6. Hematocrit and Hemoglobin: 38% Hct (12.5 g/dL Hb)
ABO Rh Kell P
Kpb U COPPER SULFATE METHOD (CuSO4)
Jsb I Lub Principle: A drop of whole blood when dropped in a solution of CuSO4 , which has a given
specific gravity, will maintan its density for approximately 15 seconds.
Specific gravity of CuSO4 is 1.053 which is equivalent to 12.5 g/dL.
 12-month deferral
1. close contact to patient with hepatitis
2. donors who received blood or blood products, an organ or tissue transplant
III. MEDICAL HISTORY 3. tattoo, ear and skin piercing
DONOR DEFERMENT: 4. those who have received HBIg bec, it is given for exposure to
Indefinitely or permanently possible infection and it may delay the onset of symptoms of disease
1. history of viral hepatitis 5. donors who have had or been treated for syphilis or gonorrhoea
- (+) HBsAg 6. (+) serologic test for syphilis
- reactive for Anti-HBc 7. donors who have traveled to areas considered endemic for malaria
- past/present evidence of Hepatitis C infection (don’t defer a donor who started antimalarial therapy in
- donor involved in post transfusion hepatitis preparation for travel to areas endemic for malaria)
2. history of jaundice of unknown cause 8. rabies vaccine
3. past/present abuse oof self-injected drugs 9. sexual contact with any person who has high risk of exposure to HIV
4. cancer Two-week deferral
5. abnormal bleeding tendencies - Vaccination: attenuated virus vaccines
6. cardiopulmonary diseases a. Mumps e. Yellow fever
7. leukemia, lymphoma b. oral polio (Sabin) f. Influenza (live virus)
c. Rubeola (measles) d. Smallpox)
8. high risk sexual behavior 6 weeks
9. high risk occupation (e.g. prostitute) - pregnant: deferred during pregnancy and 6 weeks following a
10. Chaga’s disease third trimester delivery
11. Babesiosis - 1st and 2nd trimester abortion or miscarriage need not to ne a
12. those receiving growth hormone (Creutzfeldt-Jakob dse.) cause for deferral.
13. symptoms of AIDS Related Complex (ARC), HIV/AIDS One Month (4-week deferral)
14. donor’s taking Tegison for psoriasis because its potentially teratogenic - donors taking Accutane (isotetinoin for acne therapy is also a potent teratogen)
15. Active pulmonary TB - Vaccination: Rubella (German measles), Varicella zoster (chicken pox)
Three Years Deferral 3-days
1. those with infected with malaria - tooth extraction or dental work
2. visitors, immigrants or refugees from an area considered 48-hours
endemic for malaria/ residents of area endemic for malaria - donors participating in pheresis program
Until signs and symptoms are gone
- donors who have active cold or flu symptoms must be deferred
until the symptoms are gone.
 4. Immediate preoperative hemodilution – takes place in the
operating room when 1-3 units of WB are collected and the
HEMAPHERESIS DONOR SELECTION patient’s volume is replaced with colloid or crystalloid. The blood is reinfused
ADDITIONAL DONOR GUIDELINES
during the surgical procedure.
1. At least 48 hours is the elapsed time after hemapheresis donation.
5. Post-operative salvage – an autologous donation in which a
2. A donor must not exceed more than two times in a week or 24 times in a year
drainage tube is placed in the surgical site and postoperative
unless otherwise aloowed by bloodbank physician.
bleeding is salvaged, cleaned and reinfused.
3. A donor must be tested to detect cytopenia.
4. If a donor donates whole blood, at least 8 weeks must be elapsed before he
can donate for pheresis. C O M P O N E N T P R E P A R A T I O N
5. Extracorporeal blood must not exceed 15% of the donor’s total blood volume. & I N D I C A T I O N S
6. If platelet pheresis is to be performed a donor must have PRIOR TO BLOOD COLLECTION, the intented venipuncture
above 150 x 109/L platelet count. site must be cleaned with a scrub solution containing:
7. Possible adverse reactions to HES, steroids and/or heaparin must be determined. These a. hypochlorite
substances are use in the apheresis procedures. b. isopropyl alcohol
AUTOLOGOUS DONOR SELECTION c. 10% acetone
Autologous Donor – one who is donating blood for his or her OWN future use d. PVP iodine complex
Autologous blood – the safest blood possible for transfusion
A. no risk of disease transmission; alloimmunization to red cells, platelets, wbcs,
or plasma proteins; transfusion reactions
B. phlebotomy process stimulates the BM to increase cell production
C. decreases the need for allogeneic blood and may actually increase the supply
for allogeneic blood supply.
CRITERIA
No age limit. No strict weight requirements.
Hemoglobin/hematocrit – should not be less than 11 g.dl and 34%
Frequency – Donations should not be more frequent than every 3 days and the final
donation must be completed at least 3 days prior to the scheduled surgical procedure.
Type of Autologous Donations/Transfusions:
1. Predeposit donation – refers to the blood that is drawn some
time before the anticipated transfusion and stored,, usually liquid but occasionally frozen.
2. Intraoperative autologous transfusion – occurs when blood is collected
during the surgical procedure and usually reinfused immediately.
 2. PACKED RED BLOOD CELLS
Shelflife: same with WB
Blood Components Storage Temp: 1-6ºC
Oxygen Carrying Components/Products Contents: Hematocrit should be 80 % or less
Red cell concentrates Indication: Restore oxygen carrying capacity (anemia)
Leukocyte-poor red blood cells Immediate effect of one unit: increase Hematocrit by 3% and
Frozen-thawed red cells increase hemoglobin by 1g.
Platelet Products 3. LEUKOPOOR RED BLOOD CELLS
Platelet rich plasma (PRP) Shelflife: closed system – same with Packed RBC
Platelet concentrates (PC) Open System – 24 hours
Plasma Products
Storage Temp: 1-6ºC
Fresh frozen plasma (FFP)
Frozen plasma (FP) Contents: 5 x 106 residual WBC
Cryoprecipitate Indications: Anemia with history of febrile reactions; to decrease
Stored plasma alloimmunization to WBC or HLA antigens or CMV transmission

PLASMA DERIVATIVES MEANS OF LEUKOCYTE REMOVAL

Ex. NSA ISG FACTOR VIII CONC. 1.Centrifugation
PPF Rhogam FACTOR IX CONC. 2.Washing procedures using saline or glycerol
3.Mechanical separation using leukoreduction filters
Component Transfusion Therapy
1.First generation filters-170 um
- one unit may be used for multiple transfusion
2.Second generation filters-20-10 um
- it is the effective utilization of a limited natural resource by providing therapeutic 3.Third generation filters (3-log filter)
material to several patients from a single donation
1. WHOLE BLOOD
Shelflife: CPD – 21d CPD-A1 – 35d
CPD-AS – 42d ACD – 21d
CP2D – 21 d Heparin – 2 d
Storage Temp: 1-6°C
Indications: active bleeding, hemorrhagic shock and exchange transfusion. Indicated
when both oxygen-carrying capacity and volume expansion are required.
Immediate Effect of one unit: Increase hematocrit by 1-3%.
 8. PLATELETS (SINGLE DONOR, prepared by pheresis)
4. REJUVENATED RED BLOOD CELLS Shelflife: Closed system – 5 d
Addition of Rejuvnation soln ( PIGPA-Phosphate, Inosine, Open system - 24 hours
Glucose, Pyruvate, Adenosine) to regenerate ATP and 2,3-DPG. Storage Temp: 20-24ºC with constant agitation
Shelflife: can be prepared 3 days after expiration date Contents: 3.0 X 1011 platelets in approx. 300 mL of plasma
Storage Temp: 1-6ºC Indications: Thrombocytopenia; for pxs refractory to random plts. due to
*For transfusion, wash properly and transfuse within 24 hours. platelet antibodies
REJUVESOL - the only FDA-approved rejuvenation soln Immediate effect: increase platelet count by 30,000-60,000/unit
5. WASHED RED BLOOD CELLS
9. FRESH FROZEN PLASMA (SINGLE DONOR, prepared from whole blood)
Shelflife: Open System: 24 hours
Shelflife: frozen= 1 year thawed= 24 hours
Storage Temp: 1-6ºC
Storage Temp: frozen= -18ºC thawed= 1-6ºC
QC Requirement: Plasma removal
Contents: All coagulation factors; 400mg Fibrinogen
Indications: anemia with history of febrile reactions; PNH; for pxs with
Indication: Treatment of multiple coagulation factor deficiencies
plasma proteins antibodies to reduced allergic reactions (for IgA-deficient pxs)
(caused by massive transfusion, trauma, liver dse, DIC)
6. FROZEN< THAWED< DEGLYCEROLIZED RBC Also for treatment of AntiThrombin III deficiency, TTP, HUS
Shelflife: Frozen – 10 years 10. SINGLE DONOR PLASMA (SDP) LIQUID/FROZEN
Deglycerolized – 24 hours Shelflife: Liquid – 5 days beyond whole blood expiration
Storage Temp: freezing - 65ºC (High Glycerol-40%) Frozen – 5 years
- 120ºC (Low Glycerol-20%) Storage Temp: Liquid – 1-6ºC
- 65ºC (using 79%glycerol with dextrose fructose and EDTA) Frozen= -18ºC or colder
Deglycerolizing Process - 1-6ºC Indication: Treatment of stable clotting factor deficiencies
Indications: anemia, long term storage of “rare” units and/or autologous units
11. CRYOPRECIPITATED ANTIHEMOPHILIC FACTOR
7. PLATELETS (RANDOM DONOR, prepared from whole blood)
(CRYOPRECIPITATE)
Shelflife: 3-5 days (5 days with continuous agitation)
Shelflife: Frozen – 1 year Thawed – 6 hours
Storage Temp: 20-24ºC with constant agitation
Pooled – 4 hours
Contents: 5.5 X 1010 platelets in 50-65 mL of plasma
Storage Temp: Frozen= -18ºC or colder Thawed – 20-24ºC
Indications: Thrombocytopenia, DIC, platelet disorders, bleeding
Contents: Factor VIII:C - 80-150 IU
Immediate effect: increase platelet count by 5,000-10,000 per unit
Factor VIII:vWF
Corrected Platelet Count (Plt/uL) Example: Fibrinogen – 150-250mg
= PostTransfusion Platelet – Pretransfusion Platelet X BSA Post Plt. Count= 80,000 Factor XIII
No. of Bags x 0.55 Pre-transfusion Plt. Count= 50,000 Indications: Hemophilia A, von Willebrand’s dse, Fibrinogen deficiency,
NO. of bags transfused= 4 bags Factor XIII deficiency
CCI = 23,591 ***FIBRIN GLUE -
12. GRANULOCYTE CONCENTRATE 17. PLASMA PROTEIN FRACTION
Shelflife: 24 hours Shelflife & Storage Temp:3 years at 20-24ºC
Storage Temp: 20-24ºC without agitation 5 years at 1-6ºC
Contents: 1 x 1010 wbc Contents: 80-85% Albumin and 15-20% Globulin
Indications: To correct severe neutropenia, fever unresponsive to antibiotic Indication: Plasma volume expansion
therapy and myeloid hypoplasia of the bone marrow
18. SYNTHETIC VOLUME EXPANDERS
PLASMA DERIVATIVES – are concentrates of plasma proteins that are prepared from NSS
pools (many units) of plasma. Ringer’s lactate
13. FACTOR VIII CONCENTRATE Electrolyte Solution
Shelflife: varies on expiration date on vial Dextran
Storage Temp: 1-6ºC (lyophilized) Hydroxyethyl starch (HES)
Indication: Hemophilia A 19. Rho (D) Ig (Rhogam)
14. FACTOR IX CONCENTRATE (PROTHROMBIN COMPLEX) Shelflife and Storage Temp: 3 years at 1-6ºC
Shelflife: varies on expiration date on vial Contents: Full dose- 300ug Anti-D
Storage Temp: 1-6ºC (lyophilized) Mini Dose- 50ug Anti-D
Indication: Hemophilia B Indication: Prevention of Rho (D) immunization
20. IRRADIATED BLOOD
15. IMMUNE SERUM GLOBULIN (ISG) Shelflife: 28 days or the normal dating period of the blood, which ever comes first
Shelflife: Intramuscular: 3 years (irradiation uses Cesium-137 or Cobalt-60)
Intravenous : 1 year Indications: GVH reactions, BM trnsplant, direct
Indications: prophylactic treatment to pxs exposed to hepatitis, donation from a blood relative, exchange
measles or chickenpox; treatment of congenital transfusion, IUT, transfusion for
hypogammaglobulinemia immunocompromised patients
16. NORMAL SERUM ALBUMIN (NSA)
Shelflife & Storage Temp: 3 years at 20-24ºC
5 years at 1-6ºC
Contents: 96% Albumin and 4% Globulin
Indications: Plasma volume expansion: surgery, trauma, burns
 THERAPEUTIC CYTAPHERESIS THERAPEUTIC PLASMAPHERESIS (Plasma Exchange)
1. Platetletpheresis Replacement Fluids Used: NSS, NSA, PPF, FFP
- Equivalent to 6-10 random platelet concentrates Note: FFP has the disadvantage of possible disease transmission, ABO
Contents: 3 x 1011 platelets incompatibility, citrate toxicity and sensitization to plasma proteins and
Therapeutic Indications: Used to treat patients who have cellular Ags. Therefore, FFP is now the recommended replacement fluid
abnormally elevated platelet counts (plt. ct. >1,000,000/uL) such as in the case primarily during plasma exchange for TTP and HUS.)
of Polycythemia vera
BLOOD BANKING PROCEDURES
2. Leukapheresis
HES (Hydroxyethyl starch) – sedimenting agent used for ANTIHUMAN GLOBULIN TEST (COOMB’S TEST)
granulocyte collection which causes red cells to form Principle: A technique for detecting cell-bound immunoglobulin. It is used to detect incomplete
rouleaux thus allowing wbcs to be harvested more efficiently antibodies (IgG).
Corticosteroids – administered to the donors 12-24 hours before
pheresis to increase the number of circulating IgM IgG
granulocytes by pulling them from the marginal pool Natural Immune
Therapeutic Indications: Used to treat patients with leukemia Complete Incomplete
(wbc >100,000/uL) such as Hairy cell leukemia, AML, Cutaneous T cell lymphoma
Agglutinating Coating/Sensitizing
3. Lymphocytapheresis
Therapeutic Indications: means of producing
immunosuppression in conditions like RA, SLE, Kidney Cold-Reacting Warm-reacting
transplant rejection and autoimmune and alloimmune dses. Saline-reactive Albumin/AHG-reactive
4. Neocytapheresis - transfusion of young RBCs “neocytes”
Therapeutic Indications: for young pxs with certain Ex. ABO antibody Ex. Rh antibody
hematologic disorders especially thalassemia syndromes Complement binding Complement binding
5. Erythrocytapheresis (more potent)
- considered an exchange procedure
- predetermined quantity of red cells is removed from the px AHG reagents (Commercially Prepared)
and replaced with homologous blood 1. Polyspecific AHG Reagents – consists of a pool of rabbit
anti-human IgG and mouse monoclonal anti-C3b and anti-C3d.
Therapeutic Indications: Used to treat various complications of Sickle cell
- Also referred to as Broad Spectrum Coombs Reagent.
disease, such as priapism and impending stroke
2. Monospecific AHG Reagents – contains only one antibody specificity.
- Also in pxs with severe parasitic infections from malaria and babesia
Either: a. Anti-IgG
b.Anti-C3b or C3d
Stages of Antigen-Antibody Interaction FACTORS AFFECTING THE AHG TEST
The first stage is sensitization. Sensitization occurs when antibodies react with antigens on the 1. Ratio of serum to cells.
cells and coat the cells. Minimum ratio 40:1 = 2 drops serum and 1 drop of 5%v/v cell
suspension
The second stage of the reaction is agglutination. Agglutination occurs when antibodies on 2. Temperature- Optimal: 37ºC
coated cells form cross-linkages between cells resulting in visible clumping. 3. Incubation Time – In saline suspension: 30-120 minutes
LISS suspension: 10-15 minutes
TYPES OF AHG PROCEDURES
4. Reaction medium
1. DIRECT AHG TEST (DAT)
60-minute saline test = 30-minute albumin technique
- Detects in vivo sensitization of red cells with IgG and/or complement.
22% Albumin – 2 drops 22% albumin + 2 drops serum + 1 drop 3-5% cell suspension
- Useful in the ff. situations:
- is said to reduce the zeta potential between RBCs thus increasing the rate of
- investigation of transfusion reactions (e.g. HTR) antibody uptake on the cell
- diagnosis of HDN
LISS – 2 drops 3% RBC suspension in LISS + 2 drops serum
- diagnosis of autoimmune and drug-induced hemolytic anemias
- also increases sensitivity and shortens incubation times
***Cells used for DAT should be collected into either EDTA or citrates containing
5. Washing of cells – minimum of three times
anticoagulant to minimize the possibility of in vitro attachment of complement components.
6. Saline for washing –should be fresh and buffered to a pH of 7.2-7.4
2. INDIRECT AHG TEST (DAT) 7. Addition of AHG reagents should be added to washed cells
- A two step procedure (sensitization and agglutination) that immediately after washing.
determines in vitro sensitization of red cells 8. Centrifugation- 1000 rcf for 15-20 seconds,
- Useful in the ff. situations:
- Detection of incomplete antibodies in compatibility testing or to screening cells SOURCES OF ERROR IN THE AHG TECHNIQUE
in antibody screen False-Positive Results
- Identification of antigen specificity, using a panel of red cells 1. autoaaglutinable cells
- Determination of red cell phenotype using known antisera (e.g.Du testing) 2. bacterial contamination or other contamination in cells or saline
- Titration of incomplete antibodies 3. cells with a POSITIVE DAT used for IAT
4. overcentrifugation and overreading
5. polyagglutinable cells
6. dirty glasswares
False Negative Results COMPATIBILITY TESTING PROTOCOLS
1. Inadequate or improper washing of cells (most common cause) 1. ABO GROUPING. Most critical pretransfusion serologic test.
2. AHG reagent nonreactive owing to deterioration or neutralization *If the patient’s ABO group cannot be satisfactorily determined and
3. AHG reagent not added immediate transfusion is essential, group O packed red cells should be utilized.
4. serum not added in the indirect test 2. Rh TYPING
5. serum nonreactive owing to deterioration of complement *If Rh type of the recipient cannot be determined and
6. inadequate incubation conditions transfusion is essential, Rh-negative blood should be given.
7. Postzone and Prozone (cell suspension either too weak or too heavy) *The test for Du is unnecessary when testing recipients.
8. Undercentrifugation (p.225 harmening)
9. Poor reading technique
COMPATIBILITY TESTING
COLLECTION AND PREPARATION OF SAMPLES
1. Patient Identification.
2. Collection. SERUM is the preferred specimen for compatibility
testing. Hemolysis should be avoided.
Why SERUM and NOT PLASMA?
- Plasma may cause small fibrin clots to form
which may be difficult to distinguish from true agglutination.
- Plasma may inactivate complement so that antibodies may not
be detected.
3. Age of Specimen. The freshest sample possible should be used for
compatibility testing. Specimens must be less than 3 days old if the
patient has been transfused or pregnant within the past 3 months.
4. Sample Storage. The AABB requires that patient samples
must be stored between 1-6ºC for at least 7 days after transfusion.
 ANTIBODY SCREENING Other techniques may be used to eliminate clinically
Purpose: To detect as many “clinically significant antibodies” as possible. insignificant reactions and make identification of significant antibodies easier.
“Clinically significant Abs” – refers to Abs that are reactive at 37ºC and/or in c.1 Use of AET, DTT, and ZZAP which inactivates some antigens especially Kell.
the AHG test and are known to have caused a transfusion reaction or unacceptably short survival c.2 Prewarm procedure. Clinically insignificant cold antibodies may be removed by
of the transfused red cells. this technique. Patient serum, reagent red cells and enhancement medium can be warmed
separately at 37°C for 5-10 minutes prior to mixing
Antibodies regarded as always being potentially clinically significant:
c.3 Use of sulfhydryl or thiol reagents (DTT and 2-ME) which denature IgM
ABO Duffy
antibodies by breaking disulfide bonds.
Rh Kidd
Kell SsU c.4 Use of adsorption and elution techniques to remove unwanted
a. Antibody screening is done by testing the patient/donor serum against antibodies such as cold or warm autoantibodies, or to help resolve multiple
screening cells, a panel of commonly encountered and clinically significant antigens. antibodies
Screening cells are Group O that have known antigens present. Commercially prepared ADSORPTION & ELUTION TECHNIQUES
available sets of screening cells contain D, C, E, c, e, M, N, S, s, Lea, Leb, P K, k, Fya, ADSORPTION- used to remove unwanted antibodies from SERUM
Fyb, Jka, and Jkb antigens. Testing is performed in three consecutive phases using If an autoantibody such as I, H, or IH are defined, it can be adsorbed onto the
patient serum: patient’s enzyme pretreated cells at 4ºC. Rabbit cells may also be used as adsorbents
for anti-I since they are rich in I antigen.
- Immediate Spin in saline at RT.
- 37ºC incubation with enhancement medium ELUTION - used to dissociate IgG Abs from sensitized red cells
- the recovered antibody, eluate, can be tested like serum
(e.g. albumin, LISS, PEG) to determine the antibody’s specificity
- - AHG Phase. - techniques include heat, freeze-thaw process, use of
organic solvent, acid eluates, or by using ZZAP or
Techniques to enhance antigen-antibody reactions thus facilitating antibody chloroquine diphosphate
identification include *ZZAP- mixture of DTT and papain that is used remove Ab from sensitized red
b.1 enzyme treatment with ficin, papain, trypsin, or bromelain. cells and to enzyme treat them at the same time
Enhanced: Kidd, Rh, Ii, P, Lewis *Chloroquine diphosphate- reagent used to remove IgG Abs from the surface os
Destroyed: MNS, Duffy sensitized cells; inactivates Bg antigens
b.2 Increasing the amount of serum to increase the number of available
antibody molecules
b.3 Lengthening incubation time
 c.5 Another technique for facilitating antibody identification is
NEUTRALIZATION.
Commercial substances are available to neutralize or to inhibit
reactivity of some antibodies.
Sources of Substances for Neutralization of Antibodies:
Hydatid cyst fluid – anti-P1
Plasma or serum w/ Le substances – anti-Lea & anti-Leb
Pooled seum or plasma – anti-Chido, anti-Rogers
Urine – anti-Sda
Saliva of “secretors” – anti-ABH
Human milk – anti-I
CROSSMATCHING
MAJOR X-MATCH: Donor’s cells + Recipient’s serum
MINOR X-MATCH: Donor’s serum + Recipient’s cells
Purpose:
1. Final ckeck of ABO compatibility between patient and donor to prevent
transfusion reaction.
2. Detects presence of antibody in patient’s serum that
TRANSFUSION THERAPY
will react to donor’s RBC that is not detected n antibody screen.
“LESIONS OF STORAGE”
3 Phases of Crossmatching 1. Decrease in glucose (due to cell consumption)
1. Immediate Spin in saline at RT - Detects IgM 2. Decrease pH (acidic)
2. Thermophase/37ºC incubation for 30 minutes with 3. Build up of lactic acid
enhancement medium (e.g. albumin, LISS, PEG) – Detects IgG 4. Decrease ATP levels
3. AHG Phase after washing incubated cells with saline. 5. Loss of red cell function
*Check cells/Coomb’s control cells(IgG sensitized cells) should be added to tubes that 6. Hemolysis
demonstrate no agglutination. For results to be considered valid,agglutination must occur. 7. Hyperkalemia
8. Hyponatremia
 TRANSFUSION THERAPY TRANSFUSION REACTIONS
1. Autologous Transfusion ACUTE TRANSFUSION REACTIONS
- is a donation of blood by patients for transfusion to themselves IMMUNOLOGIC
in the future 1. ACUTE/IMMEDIATE HEMOLYTIC TRANSFUSION REACTION
2. Emergency Transfusion - most severe and may be life threatening due to ABO incompatibilities
- is given to patients who are bleeding rapidly and - the associated hemolysis is Intravascular
uncontrollably. Group O negative units should be used especially - Mediators: IgM Abs(usually to ABO antigens), complement
if the patient is a woman of childbearing years. - S/S: fever, chills, hemoglobinuria, dyspnea, hypotension
3. Massive Transfusion - Most severe cases may result to DIC and renal failure
- defined as the replacement of one or more blood volume(s) 2. FEBRILE NONHEMOLYTIC TRANSFUSION
within 24 hours or about 10 units of blood in an adult. REACTION (FNHTR)
Treatment Strategy For Massive Hemorrhage - increase temperature of greater than 1ºC after transfusion
Condition Treatment - mild immunologic reactions that are caused by the interaction of recipient
Loss of blood volume Crystalloid or colloid antibodies against HLA antigens on donor’s WBC and platelets
Low O2-carrying capacity Red cells - Most common type of transfusion reactions
Loss of blood volume and Low Whole blood - Most common S/S: fever and chills
O2-carrying capacity - Management/Prevention: Use of leukocyte filters during transfusion; Antipyretic
Hemorrhage owing to
Thrombocytopenia Platelet concentrates 3. ALLERGIC TRANSFUSION REACTION
Coagulopathy Fresh Frozen Plasma - Second most common type of transfusion reactions
Adverse Conditions Associated with Massive Transfusion - IgE-mediated
a. citrate toxicity - S/S: Urticaria, Erythema, Hives, Itching, anaphylaxis
b. hypocalcemia - Management/Prevention: Administration of antihistamines before the transfusion
c. hypothermia
4. ANAPHYLACTIC TRANSFUSION REACTION
d. 2,3 DPG depletion
- Mediator: Plasma proteins, antibodies to IgA (Anaphylactic reaction)
e. depletion of coagulation factors and platelets
- Management/Prevention: Transfusion of IgA-deficient components
f. accumulation of biochemicals and microaggregates
5. NONCARDIOGENIC PULMONARY EDEMA
Aka TRALI
- Most consistent finding is Anti-leukocyte Abs in donor or patient plasma
 NON-IMMUNOLOGIC NON-IMMUNOLOGIC
1. BACTERIAL CONTAMINATION 1. TRANSFUSION-INDUCED HEMOSIDEROSIS
- caused by the endotoxins produced by Gram-negative bacteria - Iron deposition in vital organs seen in patients who are
- Mostly associated with cold growing Yersinia enterocolitica thalassemics and with chronic transfusions
Also with Pseudomonas sp. and Escherichia coli 2. TRANSMISSION OF DISEASE
a. Hepatitis B, NANB Hepatitis (HCV), HIV, HTLV-1 (oncogenic retrovirus that
2. TRANSFUSION-ASSO. CIRCULATORY OVERLOAD (TACO)
causes adult T cell leukemia), CMV, EBV
- good example of iatrogenic (physician-caused) transfusion reaction
b. Syphilis – although, transfusion of stored blood has not been shown to transmit
- common in patients with cardiac annd pulmonary disease
the disease because spirochetes do not survive at ref temp for 72 hours.
- may lead to congestive heart failure and pulmonary edema
c. Hepatitis A – occurrence is very rare. Infection by transfusion requires that the
donor has viremia (occurs briefly at the same time of onset of acute illness)
DELAYED ADVERSE EFFECTS OF TRANSFUSION
IMMUNOLOGIC
1. DELAYED HEMOLYTIC TRANSFUSION REACTION (DHTR) HEMOLYTIC DISEASE OF THE NEWBORN (HDN)
- characterized by the accelerated destruction of transfused RBCs HDN - sometimes referred to as Erythroblastosis Fetalis
- Most commonly associated with a secondary (anamnestic) response - Occurs when the mother is alloimmunized to antigen(s)
- The associated hemolysis is generally Extravascular found on the RBCs of the fetus, which results in the destruction of the fetal RBCs by the
- Mediators: IgG Abs to Rh, MNS, Kell, Kidd and Duffy antigens mother’s IgG antibodies.
- Hemolysis causes:
2. TRANSFUSION-ASSO. GRAFT VS. HOST DISEASE
- anemia in the fetus, and
(TA-GVHD)
- anemia and hyperbilirubinemia in the newborn
- these reactions occur when immunologically competent lymphocytes are
KERNICTERUS
transfused into an immunocompromised host
- Management/Prevention: Transfusion of irradiated blood components
3. POST-TRANSFUSION PURPURA
- rare transfusion reaction usually seen in older female patients who have been
sensitized to platelet antigens, either by previous pregnancy or transfusion.
- Characterized by severe thrombocytopenia one week after
transfusion due to antibody to platelet specific antigens
 Volume of FMH (mL) = # of fetal cells X maternal blood volume
# of maternal cells
*** 2000 cells are to be counted
simpler way,
% of fetal cells X 50

To determine the # of vials, you divide FMH volume by 30.

Ex. 70 mL bleed = 2.3 ; therefore, you will give 3 vials of RhIg
30 mL
Rules:
 For decimals less than 5, round down and add one dose.
Ex. 2.4 round down to 2 + 1 dose = 3 vials
 For decimals more than 5, round up and add one dose
Ex. 2.7 round up to 3 + 1 dose = 4 vials
DOSE AND ADMINISTRATION
1. 300 ug Dose RhIg- can neutralize the effects of up to 15 mL of Rh- positive
packed RBCs or 30 mL of whole blood
2. 50 ug Dose RhIg (Microdose) – is sufficient for abortion,
FETOMATERNAL BLEEDING can be assessed by: amniocentesis and ectopic rupture at up to 12 weeks gestation
1. Rosetting Test – A qualitative test that distinguishes Rh positive fetal cells from Rh
negative maternal cells

2.Kleihauer-Betke stain(Acid Elution Technique)

– A quantitative test that distinguishes hemoglobin F-containing
fetal RBCs from those adult cells that contain hemoglobin A.
Principle: Hemoglobin F resists acid elution. Therefore, Hb F-containing cells
take up the stain, and the Hb A-containing cells appear as ghost cells.
NOTES TO REMEMBER
• Anti M, anti N, anti Lea and anti Leb: cold • anti Tja was first discovered from Mrs. Jay
reacting antibodies, IgM antibodies. They who had a tumor in the stomach. It is also
can be detected in saline phase or known as anti-PP1Pk
immediate spin phase. • Anti-K: IgG antibody, severe HTR
• Cryoprecipitate: primarily used for fibrinogen replacement. • Dick test: susceptibility to scarlet fever.
• Blood component should be transfused quickly. At most is 4 hours. Reddening of the area about 10 mm (0.4
• Anti-D is not naturally occurring. A normal patient should exhibit inch) over 24 hours indicates lack of
negative anti-D. immunity to the disease. Developed in
• Direct AHG: RBC plus AHG reagent plus centrifugation 1924 by George and Gladys dick.
• Indirect AHG: addition reagent antibodies, washing, addition of Lutheran and H antigens: chromosome 19
AHG reagent, centrifugation. Example: Du testing
• Copper sulfate method: drop of blood should sink within 15
seconds. 1 cm above the surface of the solution. Specific gravity of
the solution: 1.053
• ACD, CPD and CPD2: 21 days
• CDPA-1: 35 days
• CDPDA-2: 45 days
Allowable mL of blood to donate: (donors weight in lbs x 450 mL) / 110 lbs
Amount of anticoagulant needed: (allowable blood/100) x 14
Amount of anticoagulant to be removed: 63 mL – anticoagulant needed
• Cryoprecipitate:
• FVIII:C 80-120 units
• vWF 40-70%
• FXIII 20-30%
• Fibrinogen 150 mg/dL
Minor blood group system and their molecular associations
Diego Anion exchange protein; erythrocyte band 3
Cartwright Acetylcholinesterase
Colton Aquaporin
Chido/rogers c4 complement
Knops Cr1; cd35
Indian CD44 adhesion molecule
Gerbich Glycophorin C and D; rbc membrane band 4.1
JMH (John Milton Hagen) Semaphoring; PNH III
Dombrock PNH
cromer DAF; CD55; PNH III
Effect of enzyme treatment to RBC antigens Storage lesions
Destroyed Enhanced Variably affected Not affected DEC
Duffy ABO Lutheran Kell pH
MN Rh Ss ATP
Chido rogers Ii
JMH P Glucose
Pr Lewis 2,3 DPG
Xga Kidd
INC
• Ellie Metchnikoff: phagocytosis K
• Jules Bordet: complement Lactic acid
• George kohler and cesar Milstein: monoclonal Ab Hemoglobin
production or hybridization Hydrogen ion concentration
• Susumo tonegawa: antibody diversity
• Karl Landsteiner: human blood group system
• Robert Koch: cellular immunity in TB as well as
delayed hypersensitivity
• Rosalyn yallow: radioimmunoassay
• Rodney porter and Geral Edelman: structure of
antibodies
Amino Nh2 terminal end of protein: VL
Antigen binding site: VH
Carboxyl terminal: Fc portion of the Ig molecule