ISBB Flashcards
- Study of our immune system
- Study of host’s reaction when foreign antigens are introduced to the body
IMMUNOLOGY
IMMUNOLOGY HISTORY
___: Chinese developed a practice of inhaling powder made from smallpox scabs
___: Developed a vaccine against Smallpox
* ___: A phenomenon that occurs when an antibody reacts with antigen that is structurally like the original antigen that induced antibody production
* Eradicated Infectious Organism: (2) __
* Stocks of Variola Virus:
1. Centers for Disease Control and Prevention in Atlanta, Georgia
2. State Research Center of Virology and Biotechnology (VECTOR Institute), Russia
___: developed the first attenuated vaccine
___: involves the use of bacteria or viruses that have been weakened through exposure to modifying conditions such as chemical treatment, elevated or cold temperatures, or repeated in vitro passage in cell culture
___: * Phagocytosis (1800)
___: * Demonstrated that diphtheria and tetanus toxins could be neutralized by the noncellular portion of the blood of animals previously exposed to the microorganisms.
___: * linked the two theories by showing that the immune response involved both cellular and humoral elements. He observed that certain humoral, or circulating, factors called opsonins acted to coat bacteria so that they became more susceptible to ingestion by phagocytic cells
___: * (1917) Immunized rabbits with haptens attached to a carrier molecule and then tested the serum to measure how the antibodies produced reacted with different haptens. He discovered that antibodies not only recognize chemical features such as polarity, hydrophobicity, and ionic charge, but the overall three-dimensional configuration is also important.
Variolation
Edward Jenner
Cross-reactivity
Smallpox and Rinderpest
Louis Pasteur
Attenuation
Elie Metchnikoff
Emil von Behring
Almroth Wright
Karl Landsteiner
BRANCHES OF IMMUNITY:
___:
Ability of the individual to resist infection by means of normally present body functions
* No prior exposure is required; Response does not change with subsequent exposures
___:
Spcificity for each individual pathogen
* Ability to remember a prior exposure
* Results in an increased immune response upon repeated exposure
NATURAL IMMUNITY
ADAPTIVE IMMUNITY
PARTS OF NATURAL IMMUNITY:
Anatomical barriers designed to keep microorganisms from entering the body
Physical: skin and mucous membrane
* cilia lining the respiratory tract
Biochemical: Lactic acid in sweatl lysozyme (tears and saliva); acidity of GIT and vagina; Normal Flora
EXTERNAL DEFENSE SYSTEM
PARTS OF NATURAL IMMUNITY:
recognize specific molecular components of pathogens
Cellular: phagocytes (extracellular) and NK cells (intacellular)
Humoral: acute phase reactants; Interferons (A & B); Defensins; Complement proteins
INTERNAL DEFENSE SYSTEM
APRs:
- marker for acute inflammation (non-specific ab)
__: * Discovered by __ and __ in 1930
* Originally thought to be an _________ to the C-polysaccharide of _________
* Structure: consists of five identical subunits held together by __
* Capable of opsonization, agglutination, precipitation, and activation of complement by the classical pathway
* Main substrate: ___________
CRP Concentration
<1 mg/dL = __
1 – 3 mg/dL = __
>3mg/dL = __
~ Risk Cardiovascular Disease is?
C-reactive Protein (4-6 hrs response time)
Tillet and Francis
antibody; pneumococci
noncovalent bonds
phosphocholine
low
average
high
- Plasma proteins that increase rapidly by at least 25% due to infection, trauma, or injury
- Produced primarily by the hepatocytes
- Cytokines involved are: __ (3)
ACUTE PHASE REACTANTS
interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)
APRs:
___:
* binds irreversibly to free hemoglobin released by intravascular hemolysis
* acts as an antioxidant to provide protection against oxidative damage mediated by free hemoglobin
____:
* clot increases the strength of a wound and stimulates endothelial cell adhesion and proliferation
* creates a barrier that helps prevent the spread of microorganisms further into the body
* makes blood more viscous and serves to promote aggregation of red blood cells (RBCs) and platelets
___:
* Principal copper-transporting protein in human plasma
* acts as an enzyme, converting the toxic ferrous ion (Fe2+) to the nontoxic ferric form (Fe3+)
haptoglobin (dec in hemolytic anemia)
fibrinogen
ceruloplasmin (response time: 48-72)
APRs:
___:
* Apolipoprotein
* Has a high affinity for HDL cholesterol and is transported by HDL to the site of infection
* Thought to contribute to localized inflammation in coronary artery disease
___:
* Series of serum proteins that are normally present and whose overall function is mediation of inflammation
* Major functions of complement are opsonization, chemotaxis, and lysis of cells.
___:
* major component of the alpha band when serum is electrophoresed
* general plasma inhibitor of proteases released from leukocytes
* acts to “mop up” or counteract the effects of neutrophil invasion during an inflammatory response
Serum amyloid A
Complement (most abundant is C3) (response time: 48-72)
Alpha-1-antitrypsin (protease inhibitor)
CELLULAR DEFENSE MECHANISM:
50-70% of the total peripheral White Blood cell
* Lobes: 2-5; Contains large number of neutral granules
- Primary granules (mnemonic)
- Secondary granules?
- Tertiary granules
Neutrophils
CLEMP:
* Cathepsin defensins
* Lysozyme
* Elstase
* Myeloperoxidase
* Proteinase
CLLR:
* collagenase
* Lactoferrin
* Lysozyme
* Reduced NADPH oxidase
GP:
* Gelatinase
* Plasminogen activator
CELLULAR DEFENSE MECHANISM:
Most important role: neutralizing basophils and mast cell products; inc in parasitic infx
- major basic protein
- Eosinophil cationic protein and ___________
Eosinophils
peroxidase
CELLULAR DEFENSE MECHANISM:
smallest of the granulocytes
what is the granule content?
Basophils
Cytokines, growth factors,
histamine and heparin
CELLULAR DEFENSE MECHANISM:
largest cell in the peripheral blood
Type 1 granules: __, ACP, arylsulfatase
Type 2 granules: ___, lysozyme, and lipase
Monocytes
peroxidase
Glucoronidase
PATHOGEN RECOGNITION RECEPTORS:
Acts as sensors for intracellular function; Detects Pathogen Associated Molecular Pattern (PAMP)
a. Peptidoglycan in Gram-positive
b. Lipoproteins in Gram-negative
c. Zymosan in Yeast
d. Flagellin in Bacteria with Flagellate
________
o Discoverer: Charles Janeway
o Recognize molecules that are commonly found in microbial pathogens but not on host cells
o Once TLRs have bound to their ligands, cell-signaling pathways are triggered that result in the production of cytokines that enhance the inflammatory response, resulting in more efficient pathogen destruction
o Highest concentration on:
▪ ____________
▪ ____________
▪ ____________
what is the minor phagocyte?
TOLL-LIKE RECEPTOR
monocyte
macrophage
neutrophil
(also major phagocytes)
minor: eosinophil
Macrophages may not be as efficient as neutrophils in phagocytosis because their motility is __ compared with that
of the neutrophils.
* Macrophages play an important role in __ and __ both innate and adaptive immune responses.
_____ - Resemble basophils but different lineage. It is larger than basophil with small round nucleus
o Granules contains: ACP, ALP, Protease, __
____ - Covered with Long Membranous extensions that make them resemble ______
* Main function: phagocytose antigen and present it to the __
slow
initiating and regulating
mast cells; Histamine
Dendritic Cells; nerve cell dendrites
T cells
___:
* Engulfment and destruction of foreign cells or particulates by leukocytes, macrophages, and other cells.
* kills extracellular organisms ONLY
Steps involved in Phagocytosis
1. Physical contact between the phagocytic cell and the microorganisms aided by opsonins
2. Outflowing of cytoplasm to surround the microorganism
3. Microorganism is surrounded by a part of the cell membrane
4. Lysosomal granules contact and fuse with the phagosome
5. Contents of the lysosome are emptied into this membrane-bound space.
6. By hydrolytic enzymes
7. contents of phagolysosome are expelled to the outside by exocytosis
PHAGOCYTOSIS
adherence
engulfment
formation of phagosome
granule contact
formation of phagolysosome
digestion of the microorganism
excretion
_______; A substance released by bacteria, injured tissue, and white blood cells that stimulates the movement of neutrophils and other white blood cells to the injured area.
_______: substances that coat particles and other organisms and make them more susceptible to phagocytosis
_______: the release of cellular substances (as secretory products) contained in cell vesicles by fusion of the vesicular membrane with the plasma membrane and subsequent release of the contents to the exterior of the cell
chemotaxins
opsonins
exocytosis
ELIMINATION OF ORGANISM VIA PHAGOCYTOSIS
1. ________
Increase in oxygen consumption, occurs as the pseudopodia enclose the particle within a vacuole
2. ________
* Defensins
* Cathepsin G
_______: the process by which degraded peptides within cells are transported to the plasma membrane where T cells can then recognize them
oxygen dependent process
oxygen independent process
antigen presentation
DISEASES ASSOCIATED TO PHAGOCYTOSIS:
* ___ - both chemotaxin and random movement is abnormal
* ___ - chemotaxin is abnormal, random movement is normal
* ___: Affects neutrophil microbicidal action; Impaired NADPH oxidase production
▪ Test: Nitro Blue Tetrazolium Test
▪ Positive result: ______________
Flow Cytometric Assay
* Neutrophils are labeled with _____
* Neutrophil activation: ____
* Patient with CGD: _________
D. NATURAL KILLER CELLS
* First Line of Defense Against: ______ (kills without exposure)
* __ is a receptor for the nonspecific end of antibodies. Because of the presence of this, NK cells can make contact with and then lyse any cell coated with antibodies. play an important role as a transitional cell _______________________ the innate and the adaptive immune response against pathogens
o Releases:
▪ ____________ - induce programmed cell death in the target cell
▪ ____________ - membrane-disrupting protein
Lazy Leukocyte Syndrome
Job’s Syndrome
Chronic Granulomatous Disease
(+) yellow –> blue ppt
CGD (+) is CLEAR (no blue ppt)
dihydrorhodamine
Phorbol Myristate Acetate
less fluorescence
intracellular organism and tumor cell;
CD16
bridging/link
granzymes
perforins
- Overall reaction of the body to tissue injury or invasion by an infectious agent
Cardinal Signs:
___: Redness; Increased Blood Flow
___: Swelling; Exudation of Fluid
___: Heat; Increased Blood Flow, Exudation of Fluid; Release of mediators
___: Pain; Stretching pain receptors and nerves by inflammatory exudates, chemical mediators
___: Loss of Function; Pain, Disruption of tissue structure - Stages
o __ : mast cells/basophils release histamine (causes vasodilation and inc WBC)
o __ : neutrophils and macrophages->(secrete IL-1 = inc fever and APRs)->(secrete IL-2: activation and proliferation of B and T cell)
o __ : initiated by fibroblast proliferation
INFLAMMATION
rubor
tumor
calor
dolor
funcio laesa
Vascular response
Cellular Response
Resolution and Repair
ADAPTIVE IMMUNITY:
Types:
___: immunization with a specific antigen by natural exposure to infection or administration of a vaccine
___: Infusion of serum or plasma containing high concentrations of antibody or lymphocytes from an actively immunized individual.
*_________________: results from the transfer of cells of the immune system, usually lymphocytes, from an immunized host
to a nonimmune individual.
life span of T cells?
- Naturally Acquired
a. _________ – includes the type of immunity that develops during convalescence from an infection
b. _________ – develops after the placental passage of antibody from mother to fetus - Artificially Acquired
a. _________ – immunity obtained from vaccination
b. _________ – immunity obtained after injection of gamma globulin for the induction of an immune state
Active (slow and long term)
Passive (immediate and short term)
Adaptive immunity
4-10 years
Active
Passive
Acitve
Passive
Vaccines
1. Based on Cross reactivity/ Weakened organism (low dose only)
2. consist of intact, killed viruses or bacteria (high dose)
3. contain a portion of the bacteria or virus.
3.1. are made using inactivated toxins produced by bacteria
3.2. are produced by chemically attaching a polysaccharide from the surface of bacteria to a protein molecule through a process called conjugation
3.3. composed of proteins from a pathogen
3.4. vaccines are produced by recombinant DNA technology.
NOTES:
* Not to administer vaccines containing live organisms to __ individuals; they may cause severe, disseminated, and potentially fatal infections.
* On rare occasions, mutations may occur in the vaccine organism, causing it to lose its attenuation and revert to the pathogenic form
* They have the potential for uncontrolled replication and may cause disseminated disease in immunodeficient persons.
live, attenuated
inactive vaccine
subunit vaccines
toxoid vaccines
polysaccharide vaccines
purified protein vaccines
recombinant vaccines
immunocompromised
___: substance administered with an immunogen that increases the immune response to provide immunity to a particular disease
◦ Stimulates ___ (3)
Types:
1. Preferentially stimulate Th2 responses; Ex. aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, or mixture
2. Stimulate the immune response by inducing release of chemokines and enhancing antigen uptake and migration of APCs; Ex. FCA, FIA, MF59, AS03, AS01B
3. capable of fusing with APCs to facilitate antigen presentation; Ex. Virosomes
*_____- indirect protection from an infectious disease that happens when a population is immune either through vaccination or immunity developed through previous infection.
Adjuvants
phagocytes, B cells and T cells
- aluminum salts
- oil-in-water emulsions
- microparticle
population/herd immunity
A. LYMPHOID ORGANS
1.Primary Lymphoid Organs/Central Lymphoid Organ:
a. __ (site of differentiation and maturation)
* Pluripotential stem hematopoietic stem cell
b. __; Small, flat, bilobed organ found in the thorax, or chest cavity, right below the thyroid gland and overlying the heart. T cell development; it decreases in size; nonfunctional in old people
bone marrow
thymus
A. LYMPHOID ORGANS
2.Secondary Lymphoid Organs
a. ___: characterized as a large discriminating filter as it removes old and damaged cells and foreign antigens from the blood
* 2 main types: __
b. ___: central collecting points for lymph fluid from adjacent tissues; Function: Filtration of __ from around cells in the tissues; Provide an ideal environment for the generation of B cell memory
Take Note:
* Lymphocytes and any foreign antigens present enter nodes via __; lymph fluid flows slowly through spaces (sinuses);
* Secondary follicles consist of antigen-stimulated proliferating B cells; The interior of a secondary follicle is known as the __ because it is here that transformation of the B cells takes place.
* Fluid and lymphocytes exit by way of the ___
c. Tonsils
d. Appendix
e. Peyer’s Patches
f. Adenoid
g. (3) ___
spleen; Red pulp and White pulp
Lymph nodes; interstitial fluid
afferent lymphatic vessels
germinal center
efferent lymph vessels.
MALT, BALT, GALT
LOCATION:
T CELLS
* ___________ and ___________of the lymph nodes
* ___________of the Lymph Nodes
* ___________ Regions of spleen
* Thoracic duct of the circulatory system
B CELLS
* ___________ and ___________ of lymph nodes
* ___________and red pulp of spleen
* ___________ region of GALT
- The __ is the largest lymphatic vessel in the body.
- Thoracic duct collects most of the body’s lymph fluid and empties it into the __.
perifollicular; paracortical regions
medullary cords
periarteriolar
follicular; medullary (germinal centers)
primary follicles
follicular
thoracic duct
left subclavian vein
LYMPHOCYTE
* ____________ of the circulating white blood cells with large, rounded nucleus, Nuclear Chromatin is dense, no granules
Major Types: (what are their markers)
1. B Cells
2. T Cells
3. NK Cells - formerly considered as T cell, it has CD2 but no CD3
*______ - Antigenic features of leukocytes that are differentiated by groups of monoclonal
antibodies expressing common activity.
~ __ is a DNA polymerase active during the process of immunoglobulin and T-cell receptor gene rearrangement early in a precursor B or T cell’s life
20-40%
- B Cells: TRAD: surface Ig (IgM and IgD); NEW: CD19, CD20, CD21 (receptor of EBV)
- T Cells: TRAD: CD2 (sheep RBC); NEW: CD3, CD4 (Th), CD8 (Tc)
- NK Cells: NEW: CD16, CD 56 [CD2]
cluster of differentiation (CD)
Terminal deoxynucleotidyl transferase (TdT)
T CELLS: (in charge of arm to arm combat)
* ____ of lymphocytes
* Product: ______; Markers: ________
* Responsible for immune response and are involved in
antibody regulation
* Subsets
1. ____________________
2. ____________________
*Subpopulation: T regulatory cell (T suppresor cell) - it prevents immune response againts self ag; supress autoimmunity)
T-Helper Types
1. Type 1
* Producers of _________________________
* protect cells against intracellular pathogens by activating cytotoxic lymphocytes and macrophages
2. Type 2
* Producers of IL-4, IL-5, IL-6, IL-9, IL-10, IL-13
* help B cells __________________ against extracellular pathogens and to generally regulate B-cell activity
3. Type 17
Subpopulation: __
* CD____ and ____; 5% all CD4 T cells; play an important role in suppressing the immune response to self-antigens
* inhibit proliferation of other T-cell populations by secreting inhibitory cytokines
60-70%
cytokines (lymphokine); CD2, CD3, CD4, CD8
T helper cell (60%)
T cytotoxic cell (30%)
IFN gamma, TNF beta, IL-2
produce antibodies
T-regulatory
CD4 and CD25
DEVELOPMENT OF T CELLS
1. Double negative thymocytes
a. Lacks _______________
b. Proliferates under the influence of _____
c. Rearrangement of the genes that code for ____
- Double positive thymocytes
a. ______ and ______ are expressed
b. Positive and Negative Selection
c. Clonal Deletion - removal of particular cell from BM circulation - __ - Exhibit only one type of marker
- Activated T cell - IL-2
- Sensitized T cell - produce ___
Memory T cell - acts as backup ; contains memory of ag
T CELL RECEPTORS
* ____ – sheep red blood cell receptor
* ____ – part of T cell antigen-receptor complex
* CD4 – receptor of MHC class __ molecule (Th)
* CD8 – receptor of MHC class __ molecule (Tc)
CD4 and CD8
IL-17
TCR
CD4 and CD8
positive - allowed to multiply
negative - not allowed
Mature T cell
cytokines
CD2
CD3
II
I
LABORATORY IDENTIFICATION OF LYMPHOCYTES:
* ___: current gold standard; ID of blood cells
* Fluorescence Microscopy
* ___: gold standard in differentiating T cell and B cell in the past
* _____: separate lymphocytes from other blood cells
o Ficoll-Hypaque - SG 1.077
o Roswell Park Memorial Institute (RPMI) - medium; also used to cultivate Plasmodium spp.
Mitogen
* T cells
✓ _________________
✓ _________________
✓ _________________
* B cells
* _________________
* _________________
Flow cytometry
Rosette assay
Density Gradient Centrifugation
Pokeweed antigen/mitogen (PWA/PWM)
Phytohemagglutinin (PHA)
Concanavalin (con-A)
Pokeweed antigen (PWA/PWM)
LPS
B CELLS; humoral immunity; arrows, missles, bullets of immune system
* ___ of lymphocytes
* Product: _____; Markers: _______
* precursor cell in antibody production
* Subsets:
* _________
* _________ - most common
10-20%
antibody production
markers:
Surface Ig: IgM and IgD (old marker); CD19, CD20, CD21 (new)
MHC Class II is found in B cells (not on T cell
B1
B2
DEVELOPMENT OF B CELLS
1. ___: Require direct contact with bone marrow
stromal cells; C-Kit interacts with a cell surface molecule called stem cell factor found on __
* First Step: rearrangement of genes that code for the heavy and light chains of an antibody molecule
* Differentiation of pro-B cells into pre-B cells occurs upon successful rearrangement of heavy-chain genes on one of the numbers ___________
- Pre-B cell: synthesis of the heavy-chain part of the antibody molecule occurs; accompanied by an unusual light chain molecule called a __
- ___: Distinguished by the appearance of complete IgM ab molecules on the cell surface
* Markers: ____________________ - Mature B cell: IgM and IgD; spleen and circulation; Marginal Zone B cell
- Activated B cell
- ___- cytoplasmic Ig
▪ Represent the most fully differentiated
lymphocyte; Main Function: antibody production
Pro-B cell
stromal cells
Chromosome 14
surrogate light chain
Immature B cell
CD21, CD40, Class II MHC
Plasma cell
FACTORS INFLUENCING IMMUNE RESPONSE:
- Macromelocules capable of trigerring an adaptive immune response by inducing the formation of antibodies or sensitizied T Cells in an immunocompetent Host
- Substance that reacts with antibody or sensitized T cells but may not be able to evoke an immune response in the first place
- Incomplete/ partial antigen; low molecular weight substance, has the ability to react with corresponding antibody but not able to stimulate antibody production
ex. nucleic acid, lipids
epitope: 2 types
___: straight amino acid sequences
___: discontinuous
ALL IMUNOGEN ARE ___
___: hapten + carrier; ability to stimulate ab production; ability to react with a corresponding ab
immunogen
antigen
hapten;
linear
folding
“All immunogen are antigen; not all antigen are immunogen”
immunogen
TRAITS OF AN IMMUNOGEN:
1. ___; The greater the molecular weight, the more potent the molecule is as an immunogen
* Usually ______ daltons to be recognized
- Chemical composition and molecular complexity:
a. Best Immunogens: ________________________
b. Less Immunogenic: carbohydrates
c. Not Immunogenic: nucleic acids and lipids - Foreignness; the more foreign. the more immunogenic
Relationship of Antigens to the Host:
_____ – antigen derived from the same individual.
____________ – antigen derived from different individual from same species.
____________ – antigen derived from different species.
____________ – found in unrelated plant and animals, cross react with other antibodies
Graft: transferred part
____________ – graft derived from same individual.
____________ – graft derived from different class but identical individual (identical twins)
____________ – found on different individual but same species.
____________ – from different species
fetus - type of antigen is ___
- Ability to be processed and presented with __
Macromolecular size
10,000
proteins and polysaccharides
autoantigen
alloantigen/homologous
heteroantigen/xenogeneic
heterophile
autograft
isograft/syngeneic graft
allograft
heterograft/xenograft
ALLO
MHC molecules
MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)
* genes located on the short arm of __
* set of genes that controls tissue compatibility
* in humans it is referred to as _______ complex – encoded by MHC genes
MHC Class I
* present in all nucleated cells
* Present endogenous antigen to _____
* Genetic Loci: _____
* Chain structure: alpha chain and B2 microglobulin
MHC Class II
* present in macrophage, B cells, dendritic cells and antigen presenting cells (APCs); restricted to immunocompetent cells of immune system
* Present exogenous antigens to _____
* Genetic Loci: HLA, DP, DQ, DR
* chain structure: ______
MHC Class III
* minor; involves complement components C2, C4a, C4b, and factor B
* HLA on RBC: ______
Take Note:
✓ Class I molecules are thus the __ of viral, tumor, and certain parasitic antigens that are synthesized within the cell
✓ Class II molecules help to mount an immune response to bacterial infections or other pathogens found outside cells
chromosome 6 at band 21
human leukocyte antigen (HLA)
T-cytotoxic cells (CD8)
HLA - A, B, C
T helper lymphocytes (CD4+)
alpha chain and beta chain
Bennett-Goodspeed
watchdogs
HLA PHENOTYPING:
Grade Cell Death (%) Interpretation
0 – 10; Negative
11 – 20; Doubtful negative
21 - 50; Weak positive
51 - 80; Positive
81 - 100; Strong Positive
–; Unreadable
HLA GENOTYPING
* Principle: ___________________
* Cells do not need to be __ in order to obtain DNA
* Typing reagents are chemically synthesized
IMPORTANCE OF HLA TYPING
* Tissue/ Organ transplant
* Paternity testing!!!
* Studies of racial ancestry and migration
* For diagnostic and genetic counselling
* Disease association (determines the risk only)
o HLA – B27 = _____ (high risk of having only)
o HLA – DR3 = Multiple sclerosis
o HLA – DR2, DR3 = SLE
o HLA - DR4 = RA
o HLA – DR3, DR4 = Type I DM
1
2
4
6
8
0
PCR-based amplification
viable
ankylosing spondylitis
HLA TYPING
* Phenotypic or genotypic identification of the HLA antigens or genes in a transplant candidate or donor
◦ ________ -HLA class I Typing
◦ ________ -HLA class II Typing
HLA PHENOTYPING:
* Principle: ______________________________________________
* Antisera + Patient Lymphocyte (Microplate wells)
* Incubation; Binding of antibody occurs only if the lymphocytes express the HLA antigen targeted by the antisera
* Complement is added; cells possess the HLA antigen defined by the antibody in that well, complement is activated and the cells are killed.
* Dye is added
* Result:
o Dead Cells : dead cells are able to take up the dye and appear colored
o Live Cells : live cells cannot take up the dye and remain colorless
T and B lymphocytes
B lymphocytes
Complement-dependent cytotoxicity
IMMUNOSUPPRESSIVE AGENTS
___: Act by blocking production and secretion of cytokines, inflammatory mediators, chemoattractant, and adhesion molecules
___: Interfere with the maturation of lymphocytes and kill proliferating cells
___: Blocks signal transduction in T lymphocytes
Monoclonal Antibodies Interfere in T Cell activation
* Basiliximab and daclizumab: – Binds to CD__ Receptor
* Alemtuzumab: – Binds to CD__
___: Deplete lymphocyte from the circulation; Thymoglobulin
Corticosteroids
Antimetabolites
Calcineurin inhibitors
CD25
CD52
Polyclonal antibodies
TYPES OF GRAFT REJECTIONS:
___: Preformed antibodies to ABO, HLA, and certain endothelial bind to donor vascular endothelium, activating complement and clotting factors.
___: Same as above;
___: Cell-mediated response to foreign MHC-expressing cells. CD4+ T cells produce cytokines and induces Delayed Type Hypersensitivity
___: Delayed type hypersensitivity response and possibly antibodies, to foreign HLA antigens on graft
___: T cells in HSC, lung, or liver transplants reacts against foreign HLA proteins in the recipient’s cells, causing massive cytokine release, inflammation, and tissue destruction in various locations throughout the body
Hyperacute (mins to hrs)
Accelerated (days)
Acute (days to months)
Chronic (year or more)
GVHD (100 days or more)
MIXED LYMPHOCYTE REACTION (direct allorecognition)
* Mitomycin C-treated or___ (prevent DNA synthesis) + live recipient lymphocytes = inc. proliferation of patient lymphocyte
* ___ is the ability of an individual organism to distinguish its own tissues from those of another.
Irradiated donor lymphocytes
Allorecognition
ANTIBODIES / IMMUNOGLOBULINS:
➢ products of antigenic stimulation
➢ Functions: (4) mnemonic
ANTIBODY STRUCTURE
✓ Gerald Edelman, working at the Rockefeller Institute in the United States
✓ Rodney Porter, working at Oxford University in England.
1. Heavy Chain – Determines immunoglobulin ____
IgG - gamma Heavy Chain
IgA - mu Heavy Chain
IgM - alpha Heavy Chain
IgD - delta Heavy Chain
IgE - epsilon Heavy Chain
2. Light Chain
* Kappa (__): Lambda (__)
3. ___:
- H-H; H-L normal; L-L abnormal - Bence Jones protein; Multiple myeloma
4. ___: flexible region
ANTIBODY VARIATIONS
* ____________ – heavy chain that determines Ig class
* ____________ – variation in the constant region of both HC and LC
* ____________– variation in the variable region of both HC and LC
CANO: Cell cytotoxicity; Agglutination; Neutralization; Opsonization
class
2; 1
Disulfide bond
Hinge Region
isotype
allotype
idiotype
__:
o major immunoglobulin in the blood
o produced during the _____
o best known function is complement activation vie the classical cascade
o the only antibodies that can pass ____
Major functions:
▪ Providing immunity for the newborn because IgG is the only antibody that can cross the placenta
▪ Fixing complement
▪ Coating antigen for enhanced phagocytosis (opsonization)
▪ Neutralizing toxins and viruses
▪ Participating in agglutination and precipitation reactions
IgG
secondary immune response
from mother to fetus
IgG subclasses:
placental transfer: +
C’ fixation: ++
Disulfide bonds: 2
%: 67%
IgG1
IgG subclasses:
placental transfer: +/-
C’ fixation: +
Disulfide bonds: 4
%: 22%
IgG2
IgG subclasses:
placental transfer: +
C’ fixation: +++
Disulfide bonds: 15
%: 7%
IgG3
IgG subclasses:
placental transfer: +
C’ fixation: -
Disulfide bonds: 2
%: 4%
IgG4
- __: largest
o Major class of antibody secreted into the blood in the early stages of a ___
o Pentameric IgM: ____
o ___ of antibody to be produced by developing B cells
Functions:
▪ Best complement fixer
▪ Agglutination
▪ Opsonization
▪ neutralization
o The pentamer form is found in ___________, whereas the monomer form occurs on the _____________ - __ - mucosal immunity
o major antibody on secretions
o associated with anaphylaxis
o Structure
▪ Serum: _________________
▪ Secretions: ______________ - facilitate transfer
- prevents enzymatic degradation
what are the 2 types and where are they found?
~ anti-inflammatory agent
IgM
primary antibody response
10 antigen binding sites
first class
serum
surface of B cells
IgA
monomer
(s IgA) secretory component
Type I: serum IgA; monomer
Type II: secretory IgA; dimer
- __:
o function: immunoregulation
o sensitive to ___
o found on the surface of __
o major membrane immunoglobulin found on the surface of a high proportion of ______
o postulated to be an ____________ antibody - __: allergen; most heat-labile; has __ domains
o has the ability to attach to human skin (hemocytotrophic antibody) and to initiate aspects of the allergic reaction (reaginic antibody)
o has affinity to ______ and _____
o Provide a striking example of the bifunctional nature of antibody molecules - Fc portion of the molecule binds to the __
- Fab portion binds the __
~ Take Note: best indication of a current infection is a ____ rise in antibody titer when comparing two serum samples collected from a patient during the beginning and later stages of the infection
______: one L chain and one-half of an H chain held together by disulfide bonding
______: - represent the carboxy-terminal halves of two H chains
IgD
enzymatic degradation
B cells
B lymphocytes
anti-idiotypic
IgE; 5
basophils and mast cells
target cells
allergen
four-fold
Fab
Fc fragment (crystallize in cold temp)
FRAGMENTATION OF MONOMER
a. ____: cut monomer exactly at the Hinge region, monomer is cut into 3 parts.
b. ____: cut monomer below the HR, monomer is cut into 2
__ - Lymphocyte are genetically preprogrammed to produce one type of Ig and that a particular cells capable of responding to it, causing them to proliferate
___ - certain cells had specific receptors for antigen that were present before contact with antigen occurred.
Key principles:
___ concept
Antigen selected cells with the built – in capacity to respond to it
Papain
Pepsin
Clonal Selection
Ehrlich ‘s side chain theory
Lock - and - key
___: Initial force of attraction between single Fab and single epitope
o Ionic bonds
o Hydrogen bonds
o Hydrophobic bonds
o Van Der Waals Forces
___: sum of all attractive forces between antigen and an antibody
IMMUNE RESPONSE (phases - 4)
___: longer lag, dec antibody, IgM shorter half-life, abrupt decrease
___: shorter lag, inc antibody, IgG longer life (23 days), slow decrease
MONOCLONAL ANTIBODIES
o __ cloned from a single cell
o Uses: analysis of cell membrane antigen
AFFINITY
AVIDITY
- Lag
- Log
- Stationary
- Decline
Primary Response
Secondary response
Purified antibodies
Monoclonal Antibodies
treatment of cancer and autoimmune diseases
Main concern: ________________
WHO nomenclature:
◦ __ – “-omob”
◦ __ – “-ximob”
◦ __ – “-zumob”
◦ __ – “umob”
common animals used for mono and poly?
Take Note:
➢ Fused cell is added with Hypoxanthine, aminopterin and thymidine (HAT medium)
➢ __ – drug that prevents myeloma cells from making their own purines and pyrimidines
highly immunogenic
Mouse
Chimeric
Humanized
Human
mono - mice; poly - rabbit
Aminopterin
C. ___ - cytotoxic activity against tumor cells and virally infected cells
TNF ALPHA – produced by ___
TNF BETA – produced by ___
D. __ -Enhances motility and promote migration of WHITE Blood CELLS
E. ___ – release by platelets during coagulation
F. ___
* Induces antiproliferative activity in a wide variety of types
* Control to help downregulate the inflammatory response when no longer needed
TUMOR NECROSIS FACTOR (TNF)
macrophages – aka cachectin
CD4 and CD 8 cells – aka lymphotoxin
Chemokines
Betalysin
TRANSFORMING GROWTH FACTOR- B
___: Generic terms for Soluble mediators; Regulates the immune system
A. ___
o Function primarily in immune cell differentiation and activation
o Examples
__ – Endogenous Pyrogen
IL – 2 – __
* Growth and differentiation of __
* Induces lytic activity of the NK cells
IL – 6
* Stimulates B-Cells to become __
* Induces CD4+ cells to produce ___
___
* Regulates Th2 immune activities
* Helps drives antibody response
___
* Inhibitory effect to the immune systems
Cytokines acts in 3 ways
___: cytokine bind to receptor on cell that secreted it
___: cytokine bind to receptors on near by cells
___: cytokine bind cells in distant parts of the body
CYTOKINES
INTERLEUKINS:
IL – 1
T Cell growth Factor; B and T cells
plasma cells
cytokines
IL – 4
IL – 10
autocrine
paracrine
endocrine
B. ___ - Proteins produced by virally infected cells and protect the neighboring cells; exert a virus-nonspecific but host-specific anti-viral activity
* Types
a. Type 1: ___
▪ non-immune, product of initial response to viral infection
o Interferon Alpha
▪ secreted by leukocytes
▪ major producer: ____
o Interferon Beta
▪ secreted by __
▪ major producers: Fibroblast and epithelial cells
b. Type 2: ___
▪ immune, component of the specific immune response to viral and other pathogens
▪ major producers: ___
INTERFERON
Alpha and Beta
Null lymphocytes/ NK cells
double stranded RNA (dsRNA) fibroblast
gamma; Th1 cells
___: a set of proteins that play a role in cytolytic destruction of cellular antigens by specific antibody
* reaction is non specific to the target cell
* functions/ examples
o ____________– C5a, C5b, C6, C7
o Immune Adherence – ___
o Kinin Activator – __
o Anaphylotoxins – C3a, C4a, C5a
o ____________ – C3b, C4b, C5b
o Virus neutralization – __
___: nature of complement proteins
___: coined the term “complement”
complement fragments either a or b - which is bigger and what is the exception
COMPLEMENT SYSTEM
chemotaxin
C3b
C2b
opsonins
C4b, C1
Jules Bordet
Paul Ehrlich
b is bigger but in C2b vs C2a, C2a is bigger than b
THREE PATHWAYS OF ACTIVATION:
1. ____ - end result lysis, triggered by ag-ab
* Initiated by: ______
* Recognition Unit: C1 (C1q, C1r, C1s)
* Activation Unit: C4, C2, C3; order of activation: ___
* MAC: C5 – C9
C5 convertase: ___
C3 convertase: ___
Classical Pathway
IgG and IgM
C412356789
C4b2a3b
C4b2a
THREE PATHWAYS OF ACTIVATION:
2. ___ - bypass pathway
Recognition unit; order of activation: ___, no ___
___ - is only stabiliaing C3 and C5 convertase
__ can trigger this pathway
Recognition unit: C3, factor B, factor D
o Triggers include:
▪ Bacterial cell walls (esp. w. LPS!!!)
▪ Fungal cell walls
▪ Yeast
▪ Viruses
▪ Virally infected cells
▪ Tumor cell lines
▪ Some parasites (esp. trypanosomes)
C5 convertase: ___
C3 convertase: ___
Alternative Pathway
C356789; C124
Properdin pathway
IgA
C3bBb3bP
C3BbP
THREE PATHWAYS OF ACTIVATION:
3. ___; Mannose Binding Protein/Lectin Associated Serine Protease
* Recognition unit: MBP,
* MASP-1,-2,-3 complex attaches to:
* ___
* Others include glycoproteins or CHO on bacteria, yeasts, viruses, some parasites
* __ dependent
* C5 convertase: __
* MAC: C5- C9
Lectin Pathway
MANNOSE/MANNAN
Calcium
C4b2a3b
PLASMA COMPLEMENT REGULATION
➢ ___________ – dissociates C1r and C1s from C1q
➢ ___________ – cleaves C3b and C4b
➢ ___________ – cofactor with I to inactivate c3b; prevents binding of B to c3b
➢ ___________– acts as a cofactor with I to inactivate C4b
➢ ___________– prevents attachment of the C5b67 complex to cell membrane
➢ ___________– accelerates dissociation of C3 convertase
INHIBITORS OF MAC
1. HRF- Homologous Restriction Factor
2. CD59-or MIRL Membrane Inhibitor of Reactive Lysis (ISBT 035 blood group associated)
C1 inhibitor
Factor I
Factor H
C4 binding protein
5 protein (vitronectin)
decay accelerating factor (DAF)
DEFICIENCIES OF COMPLEMENT COMPONENTS:
- Lupus-like Syndrome; Recurrent Infections; atherosclerosis
- Neisseria Syndrome
- Severe recurrent infections; glomerulonephritis
- No known associated disease
- Hereditary Angioedema
- Paroxysmal Nocturnal Hemoglobionuria
- Neisseria infections
C1 and C2 (arthero in only here)
C5 – C8
C3
C9
C1-INH
DAF and/or MIRL
Properdin
IMMUNOLOGICAL DISORDER
A. HYPERSENSITIVITY
* Heightened state of ________________________
* Different types of hypersensitivity reactions are distinguished by:
o Their time course
o Whether antibodies or T cells are the principle immune elements involved
immune responsiveness (exaggerated)
MEASUREMENT OF COMPLEMENT COMPONENTS
* _________ - amount of C’ serum that can cause hemolysis of the 50% of a standardized concentration of antibody–sensitized sheep RBC
* _________ - Magnesium chloride, ethylene glycol tetraacetic acid
Take Note: 50% is used because this is when the change in lytic activity per unit change in complement is at maximum
* Radial Immunodiffusion (RID) - concentration
CLINICAL SIGNIFICANCE
o Elevated complement components – have little clinical importance.
o Decreased complement components.
▪ Complement is currently being consumed
▪ Complement may be decreased or absent due to ________
COMPLEMENT FIXATION TEST
o Reporting: ________________________
o 2 Systems Involved for Complement Fixation
o Test System/ Bacteriolytic System
o Indicator System/ Hemolytic System
CH50 Assay
AH50 Assay
genetic defect
Highest diution showing no hemolysis
HYPERSENSITIVITY REACTIONS:
immune mediator: IgE
Synonym: Anaphylactic
Timing: Immediate
Antigen: Heterologous
Complement involvement: No
Ex: Anaphylaxis, allergic rhinitis, allergic asthma, food allergies, urticaria
TYPE I
HYPERSENSITIVITY REACTIONS:
immune mediator: IgG or IgM
Synonym: Antibody-mediated cytotoxic
Timing: Immediate
Antigen: Cell surface: autologous or heterologous
Complement involvement: Yes
Ex: Transfusion reactions, autoimmune hemolytic anemia hemolytic disease of the newborn, drug reactions, myasthenia gravis, Goodpasture’s syndrome, Graves disease
TYPE II
HYPERSENSITIVITY REACTIONS:
immune mediator: IgG or IgM
Synonym: Complex-mediated
Timing: Immediate
Antigen: Soluble: autologous or heterologous
Complement involvement: Yes
Ex: Serum sickness, Arthus reaction, Lupus erythematosus, Rheumatoid arthritis, drug reactions
TYPE III
HYPERSENSITIVITY REACTIONS:
immune mediator: T cells
Synonym: Cell-mediated or delayed type
Timing: Delayed
Antigen: autologous or heterologous
Complement involvement: No
Ex: Contact dermatitis, tuberculin and anergy skin tests, hypersensitivity pneumonitis
TYPE IV
TESTING FOR IMMEDIATE HYPERSENSITIVITY
✓ IN VIVO: ________________
o __ - small amt of ag, recommended (wheal and flare)
o Intradermal test - inc ag
✓ IN VITRO: ________________
o RIST: radioimmunosorbent test - total serum IgE
o RAST: radioimmunosorbent test - allergen specific IgE
MEDIATORS OF TYPE 1- HYPERSENSITIVITY
Primary Mediator (preformed) = Secondary (Newly Synthesized)
Histamine =
Heparin =
Eosinophil Chemotactic Factor for Anaphylaxis (ECF-A) =
Neutrophil Chemotactic Factor for Anaphylaxis (NCF-A) =
Skin test
Percutaneous
Prostaglandins
Leukotrienes
Platelet Activating Factor
IL-1,Il-3, IL-4
__________ Reaction:
IgG and IgM antibodies are produced against antigens found on redd cell surfaces; Ag-Ab reaction (Immunologic Mechanism)
Examples: HTR, AIHA, PCH, Cold Agglutinins
Test: ____
_________ Reaction:
Antigen is soluble (precipitation)
Deposition of ag-ab complex in the tissue
Examples: Arthus reaction, Serum Sickness, RA
Test: __________________
_________ Reaction:
Mediators: _______
Antigens Involved
◦ Intracellular Pathogens: bacteria, fungi, parasites, viruses
◦ Contact antigens: poison ivy, poison oak, metal
Examples: contact dermatitis, hypersensitivity pneumonitis
Test: _____________________________________________________
Type II Hypersensitivity
AHG (Direct and Indirect)
Type III Hypersensitivity
Labelled immunoassays
Type IV Hypersensitivity
T cells
Skin test (patch tesst, tuberculin test); interferon gamma release assays
B. AUTOIMMUNE DISEASES
✓ Conditions in which damage to organs or tissues results from the presence of ______________ or _________________
✓ _____________- Ability of the immune system to accept self-antigen and not initiate a response against them
o Types: (2)
_________:
✓ Individual _________or ___________ agents contain antigens that closely resemble self – antigen
✓ Examples: (mnemonic)
o Polio Virus VP2 –_________
o Measles Virus P3 –________
o Papilloma Virus VP2 –_____
______________ - Microorganism can induce a local inflammatory response that recruits leukocytes and stimulates APCs to release cytokines that activates T Cells
______________ -Ability to bind to both MHC Class II and TCRs, regardless of the antigen specificity
autointibody or autoreactive cells
self tolerance
Central & Peripheral
MOLECULAR MIMICRY
viral or bacterial
(PA MeMe PI)
acetylcholine
myelin basic protein
insulin receptors
bystander effect
super antigens
CLINICAL TYPES
1. SYSTEMIC OR NON-ORGAN SPECIFIC
____: Immune complex disease characterized by overproduction of autoantibodies
Manifests itself by skin lesions ___
✓ Laboratory Diagnosis
o Complement Proteins (C3)
o Presence of anti-nucear antibodies (ANA - nonspecific)
o ______ – PMN leukocyte with ingested LE body, often in rosette formation
Drugs Associated to Lupus
✓ Procainamide
✓ Hydralazine
✓ Chlorpromazine
✓ Isoniazid
✓ Quinidine
Take Note:
o Anti-Nuclear Antibodies
o autoantibodies that are directed against antigens in the nuclei of mammalian cells
o Targets: double-stranded (ds) and single-stranded (ss) DNA (deoxyribonucleic acid), histones, nucleosomes (DNA histone complexes), centromere proteins, and extractable nuclear antigens
(ENAs)
o Extractable Nuclear Antigens (ENAs): ribonucleoproteins (RNP), Sm antigen, SS-A/Ro , SS-B/La antigens, Scl-70, Jo-1, and PM-1
SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)
“butterfly rash/RED WOLF rash”
Le cell
CREST SYNDROME
METHODS OF DETECTION
o ____
✓ Principle: Indirect Immunofluorescence (ab)
✓ Most widely used and accepted test
✓ Human epithelial cell line, Hep-2, is the standard substrate.
✓ Fluorescent Staining Pattern
Calcinosis
Raynaud’s phenomenon
Esophageal dysmotility
Sclerodactyly
Telangiectasia
Fluorescent Antinuclear Antibody Test (FANA)
Fluorescent Antinuclear Antibody Test (FANA); what is the pattern?
___: Uniform staining of the entire nucleus
___: Diffuse staining is throughout the nucleus, but greater intensity around the outer circle surrounding the nucleus
___: Discrete, florescent specks throughout the nucleus
___: Prominent staining of the nucleoli
___: Numerous discrete speckles are seen
homogenous/diffusse
peripehral/outline/rim
speckled
nucleolar
centromere
___: Microtiter plate well containing a suspension of polystyrene microspheres that are coated with individual nuclear antigens or with a HEp-2 extract; Antibodies in the patient serum will bind only to the beads containing their specified antigens
✓ Phycoerythrinlabeled anti-human IgG is added
✓ Bead suspension is analyzed for fluorescence by a flow cytometer that has two lasers, one that identifies each bead and another that detects the amount of fluorescent conjugate attached
o Immunofluorescence Using _____________________
✓ used to detect antibodies to dsDNA.
✓ trypanosome has a circular organelle called a kinetoplast that is composed mainly of dsDNA
o ____: used to determine the immunologic specificity of a positive FANA test
Take Note:
o Antiphospholipid Antibodies: antibodies that bind to phospholipids alone or phospholipids complexed with protein
o ___: produces a prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT)
o Antibody to a preparation of extractable nuclear antigen was first described in a patient named Smith, hence the name anti-Sm antibody.
o Extractable nuclear antigens represent a family of small nuclear proteins that are associated with uridine-rich RNA.
o The anti-Sm antibody is specific for ___, because it is not found in other autoimmune diseases.
o Double-stranded DNA (ds-DNA) antibodies are the most specific for SLE, the presence of these antibodies is considered diagnostic for SLE
Microsphere Multiplex Immunoassay (MMIA)
Crithidia lucilliae
Ouchterlony test
Lupus Anticoagulant
lupus
___: DR4
➢ Chronic, symmetric, and erosive arthritis of the peripheral joints
➢ Women are __ as likely to be affected as men
➢ Progresses to joint ___________________
➢ ___: Generated when enzyme peptidyl arginine deaminase (PAD) modifies the amino acid arginine by replacing an NH2 group with a neutral oxygen
➢ Antibody Marker
o Rheumatoid factor
▪ Group of immunoglobulins that interacts specifically with the ___portion of ___ molecules
o _________________ = specific marker
➢ Laboratory Tests
o Sheep Cell Agglutination Test (Rose et al)
o Latex Fixation Test (Singer and Plotz)
o Sensitized Alligator Erythrocytes test (Cohen et al)
o Bentonite Flocculation Test (Bloch and Bunim)
RHEUMATOID ARTHRITIS
three times
deformity and disability
Citrulline
Fc; IgG
anti-cyclic citrullinated peptide
OTHER SYTEMIC AUTOIMMUNE RHEUMATIC DISEASE (SARDS)
▪ ___ - characterized by chronic inflammation of the exocrine glands, most notably the ocular and salivary glands
▪ ___ - rare SARD that is characterized by excessive fibrosis and vascular abnormalities that affect the skin and joints and progress over time to involve internal organs, most commonly the esophagus, lower gastrointestinal tract, lungs, heart, and kidneys.
▪ ___ - overlap syndrome of limited cutaneous SSc combined with clinical features of SLE, polymyositis, and RA.
▪ ___ - group of diseases characterized by chronic inflammation of the skeletal muscles (“myositis”) and progressive muscle weakness.
Sjögren’s syndrome
Scleroderma (SSc)
Mixed Connective Tissue Disease (MCTD)
Other Inflammatory myopathies
✓ Inflammation of blood vessels
✓ Primarily affects the upper respiratory tract, lungs, and kidneys.
✓ Laboratory Diagnosis
1. Nasal or Oral Inflammation with oral ulcers or purulent or bloody nasal discharge
2. Abnormal chest X-ray, showing presence of nodules, fixed infiltrates, or cavities
3. Urinary sediment with microhematuria or RBC Cast
4. Granulomatous inflammation or biopsy
✓ ANCA: Antineutrophil Cytoplasmic Antibodies
o Cytoplasmic - c-ANCA
o Perinuclear - p-ANCA
GRANULOMATOSIS WITH POLYANGIITIS
ORGAN SPECIFIC: Autoimmune disease?
___: -HLA - DR4 and DR5; mosst common autoimmune dse - hypothyroidism
✓ Thyroglobulin
✓ Thyroid microsome
✓ Second colloid antigen (CA2)
✓ Thyroid membrane receptors
✓ T3 and T4!!
___: Thyroid peroxidase antibody (TPO); TSH Receptor antibody (TRAb); hyperthyroidism
___: HLA – DR3, DR4, DQ2, DQ8; Insulin autoantibodies (IAA); Glutamic acid decarboxylase autoantibodies (GAD); Islet cell antigen – 2 (IA-2)
___: Environmental Trigger: Gluten!!!; HLA-DQ2 and HLA DQ8; anti – Tissue Transgulatminase; Endomysium (EmA); Deaminated Gliadin peptides
___: AIH-1: Smooth muscle antibodies; Anti-nuclear antibodies
___: AIH-2: Anti-liver kidney microsomal antibody (anti-LKM-1), Anti-liver cytosol type 1 antibody (anti-LC-1)
___: Anti-mitochondrial antibody (AMA);
___: Antibody against parietal cells; H+/K+-ATPase proton pump; Cobalamin – absorbing protein
___: Anti – IF or anti – parietal cells
___: Antibodies to myelin basic protein
___: Deoxyribonuclease – sensitive perinuclear ANCA; Anti – Saccharomyces cerevisiae antibody; Pancreatic antibody; Anti – outer membrane porin form E.coli
___: acetylcholine receptor p binding ab;
___: anti-basement membrane ab; other name?
Hashimoto’s
Grave’s
Insulin independent Diabetes Mellitus (IDDM)
Celiac Disease
Autoimmune Hepatitis
Chronic Active Hepatitis
Primary Biliary Cholangitis
Autoimmune Gastritis
Pernicious anemia
Multiple Sclerosis
Inflammatory bowel disease
Myasthenia gravis
Anti-Glomerular Basement membrane Disease; Goodpasture’s Synrome
TUMOR IMMUNOLOGY
✓ Study of relationship between the immune system and cancer cells
✓ Encompasses: Antigen associated with tumor, Host immune response to tumor, Mechanisms by which tumor are thought to escape responses, Therapeutic use of the immune system in an attempt to eradicate tumors
o Apoptosis – Physiologic cell death
o Tumor – “to swell”
o Neoplasm – “new growth”
o ___ – slowly growing cells that are well differentiated and organized, similar to the normal tissue from which they originated
o ___ - disorganized masses that are rarely encapsulated, allowing them to invade nearby organs and destroy their normal architecture
o ___ - ability of cells to break away from the original tumor mass and spread through the blood to nearby or distant sites in the body
o Cancer - named after the Latin word for “___,” derives its name from this property of invasiveness, which can resemble the legs of a crab when viewed in microscopic tissue sections
o ___ , derived from the skin or epithelial linings of internal organs or glands
o ___, derived from bone or soft tissues such as fat, muscles, tendons, cartilage, nerves, and blood vessels
o ___ - biological substances that are found in increased amounts in the blood biological substancesthat are found in increased amounts in the blood, body fluids, or tissues of patients with a specific type of cancer
Benign
Malignant
Metastasis
crab
carcinomas
sarcomas
Tumor Markers
TUMOR MARKERS:
HEPATIC AND TESTICULAR CANCER
LUNG CANCER
BREAST CANCER
OVARIAN CANCER
COLORECTAL CANCER
PROSTATE CANCER
URINARY BLADDER CANCER
PANCREATIC CANCER
MEDULLARY CANCER
HEMATOPOIETIC MALIGNANCIES
AFP
ALP
CA 15-3
CA 125
CEA
PSA
NUCLEAR MATRIX PROTEIN NMP
AMYLASE
CALCITONIN
CD-45
IMMUNOPROLIFERATIVE DISORDER:
___: characterized by the presence of very poorly differentiated precursor cells; (blast cells) in the bone marrow and peripheral blood; usually seen in children between __ and is the most
common form of leukemia in this age group
___: Group of diseases almost exclusively of B-cell origin; Includes: chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL)
___: rare, slowly progressive disease characterized by infiltration of the bone marrow and spleen by leukemic cells without the involvement of lymph nodes; Splenomegaly is striking, whereas lymphadenopathy is generally absent; malignant lymphocytes are round and often have irregular “hairy” cytoplasmic projections from their surfaces
___: most common lymphomas; often-curable disease occurs both in young adults and the elderly characterized by the presence of Hodgkin and Reed-Sternberg (RS) cells in affected lymph nodes and lymphoid organs
___: patients are greater than 60 years of age and the incidence is greater in men than women; Immunosuppression: greatest risk factor
___: Malignancy of mature plasma cell; Most serious; Bence Jones Proteins - monoclonal light chains can be found in the blood, but are rapidly excreted in the urine; excess of kappa or lambda light chains
___: malignant proliferation of IgM-producing lymphocytes; elevated serum monoclonal protein: macroprotein or IgM paraprotein; In 10 to 20 percent of patients, the IgM paraproteins behave as cryoglobulins.
Acute Lymphocytic Leukemia (ALL); 2-5 yrs old
Chronic Lymphocytic Leukemia or Lymphoma
Hairy Cell Leukemia
Hodgkin Lymphoma (HL)
Non-Hodgkin Lymphoma (NHL)
Multiple Myeloma
Waldenström’s Macroglobulinemia
IMMUNODEFICIENCY DISEASES:
Category 6: ___
o Interruption in signaling pathways of innate immune cells
Category 7: ___
o Two Classifications
o Inflammasome
o Hyper IgD syndrome and Muckle-Wells syndrome
o Noninflammasome
o Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) and early-onset inflammatory bowel disease (IBD).
o inflammasome is a protein oligomer that contains caspase enzymes and other proteins associated with apoptosis
Category 8: ___
o Complement deficiencies
Category 9: ___
o new classification of PIDs
o inherited genetic component but also include an acquired component, such as somatic mutations or autoantibody production
o Ex. Chronic mucocutaneous candidiasis
Defects in Innate Immunity
Autoinflammatory Disorders
Complement Deficiencies
Phenocopies of Primary Immunodeficiencies
__: the study of the fluid components in the blood, especially antibodies
BLOOD SPECIMEN AND MEASURING
o Serum
* complement must be inactivated because it interferes with test results; heated to 56degC for 30 mins (VDRL; non-treponemal: Syphilis) REAGIN!!!
* Add choline chloride (RPR)
Storage:
* 2°C and 8°C for up to 72 hours
* Frozen at –20°C or below
* Take note: When more than ______ has elapsed since inactivation, a specimen can be re-inactivated
by heating it to ______________ (removes intefering substances)
SEROLOGY
4hrs; 56degC for 10 mins
IMMUNOASSAYS/ SEROLOGIC TEST
o Test that involves _______________ reaction
* Types:
o ___________: Detect unknown antigen in specimen by using known or commercial anti-sera
o ___________: Detect the presence of unknown antibodies in the serum of patient by using known commercial antigen.
IMMUNOLOGIC REACTIONS:
__: combination of antigen-antibody; non-visible reaction
__: demonstrates antigen-antibody reaction
__: immunologically in vivo; biologic reaction is detectable
antigen-antibody
direct/forward
indirect/reverse
primary
secondary
tertiary
PART I. PRECIPITATION REACTION
* First noted by Kraus (1897)
* involves combining ____________ with soluble antibody to produce insoluble complexes that are visible.
* Optimum precipitation occurs in the ________________
antibody excess is ___
antigen excess is ___; both causes __
soluble antigen
zone of equivalence
prozone
postzone; false negative
MEASUREMENT OF PRECIPITATION BY LIGHT SCATTERING
* A measure of the turbidity/cloudiness of a solution; Measures the reduction in light intensity caused by reflection, absorption, or scatter; recorded in absorbance unit, a measure of the ratio of incident light to that of transmitted light.
- Measures light that is scattered in a particular angle ; The amount of light scattered is an index of the solution’s concentration
PRECIPITATION IN A SUPPORT MEDIUM
-Immunodiffusion
IMMUNODIFFUSION
1. PASSIVE IMMUNODIFFUSION
o No ___________________
o Rate of diffusion is affected by the size of the particles, the temperature, the gel viscosity, and the amount of hydration
o “Passive” because they are allowed to react to completion with no enhancements!!! such as an electrical charge applied.
ADV: cheap and simple; DIS: longer TAT
TURBIDIMETRY
NEPHELOMETRY
electrical current
PASSIVE IMMUNODIFFUSION:
1.Single Diffusion, Single Dimension (__________)
✓ End: precipitin LINE!!!
✓ semi quantitative (1+, 2+, 3+, 4+); developed by James (last name same as test)
2.Single Diffusion, Double Dimension (_______)
✓ Antibody is uniformly distributed in the support gel and antigen is applied to a well cut into the gel
✓ Result: precipitin RING!!
✓ Quantitative: diameter is directly proportional to conc.
✓ Methods
1. Fahey and McKelvey method (_______); 19hours; Diameter is proportional to log of the concentration
2. Mancini method (________); IgG=24 hr, IgM = 50-72 hr; Square of diameter is proportional to the conc.
3.Double Diffusion, Double Dimension (____)
✓ Antibody that is multispecific is placed in the central well and
different antigens are placed in the surrounding wells to determine if the antigens share identical epitopes
✓ Patterns
* _______________- serological identity or the presence of
a common epitope
* _______________- compared antigens share no common
epitopes
* _______________- Partial Identity
Oudin Test
Radial Immunodiffusion
kinetic diffusion
end-point diffusion
Ouchterlony Technique
arc formation
crossed lines
spur formation (common epitope)
- PRECIPITATION BY ELECTROPHORETIC TECHNIQUES:
o electrophoresis is a technique which separates molecules using electrical current.
o A direct current is forced through the gel, causing antigen, antibody, or both to migrate; distinct precipitin bands are formed - ___:
✓ RID + electrophoresis
✓ the total distance of antigen migration and precipitation is directly proportional to antigen concentration - ___:
✓ double diffusion + electrophoresis
✓ Serum is electrophoresed to separate out the main proteins
✓ A trough is then cut in the gel parallel to the line of separation
✓ useful procedures for the ID of monoclonal proteins - ___:
✓ immunoprecipitation + electrophoresis
✓ antiserum is applied directly to the gel’s surface rather than placed in a trough; Immunodiffusion takes place in a shorter time and results in a higher resolution
✓ Highly sensitive and specific assay that is used to identify the type of monoclonal protein present in a sample
✓ Serum samples are electrophoresed in six separate lanes on an agarose gel and specific antisera are applied directly to the lanes
✓ Take Note:
o ___ will exhibit faintly staining bands
o __ show darkly staining bands in the gamma region
o Presence of monoclonal antibody will result in ___ bands in specific lane
- ___:
✓ Ag and Ab are placed on the well directly opposite each other
✓ Ab will migrate to the __ and the Ag to the __
✓ Two parallel lines of wells are cut into agarose;
✓ Useful to detect bacterial Ags in cerebrospinal fluid and other fluids when a rapid laboratory response is needed
Rocket Electrophoresis (One Dimention Electroimmunodiffusion)
IMMUNOELECTROPHORESIS (IEP);
IMMUNOFIXATION ELECTROPHORESIS
Hypogammaglobulinemias
Polyclonal hypergammaglobulinemias
dark and narrow
COUNTER IMMUNOELECTROPHORESIS (CIE)
cathode; anode
AGGLUTINATION REACTIONS:
o In 1896, ___ and ___
o Process by which large particulate antigen such as cells aggregate to form larger complexes when a specific antibody is present
o Agglutinins (IgM) – Antibodies that cause agglutination
o 2-Step Process: Sensitization (initial attachment of ag-ab) and Lattice formation (visible reaction, clumping)
TYPES OF AGGLUTINATION REACTION:
1. ___: Antigens are found naturally on the surface of the particles
2. ___: antigen is artificially!! attached to a particulate carrier (e.g. cells, latex, bentonite, celloidin, charcoal); Detects: antibodies to viruses
3. ___: antibodies are attached to particulate carriers; active sites of antibody are facing outward; often used to detect microbial antigens.
o used to measure levels of certain therapeutic drugs, hormones, and plasma proteins such as haptoglobin and C-reactive protein
4. ___: Uses antibodies bound to a particle to enhance the visibility of agglutination; Carrier: ___;
5. ___: based on competition between particulate and soluble antigens for limited antibody combining sites
o lack of agglutination is an indicator of a positive reaction.
o Involves: hapten attached to carrier particles
Gruber and Durham
Direct Agglutination
Passive Agglutination
Reverse Passive Agglutination;
Co-agglutination; Bacterium
Agglutination-Inhibition
Take Note: Instrumentation:
✓ ___:
o specific type of precipitation that occurs over a narrow range of antigen concentrations; antigen consists of very fine particles that clump together in a positive reaction; Examples: ________
✓ ___:
o Measures the ability of the patient’s antibody to neutralize infectivity and protects cells from infection
o antigenic activity is stopped by its specific antibody
o Target: to detect toxins, viral agents or antibodies to the toxin or viral agents
o Types
▪ ___ (Schick test[diphtheria], Dick Test [Scarlet], ASO Titration Test)
▪ Virus Neutralization
✓ Precipitation and agglutination are considered ___ assays because a marker label is not needed to detect the reaction.
✓ Relatively insensitive because they rely on a high enough concentration of the unknown to visualize the reaction.
Flocculation; VDRL and RPR
Neutralization; Toxin Neutralization;
unlabelled
___:
o Designed for antigens and antibodies that may be small in size or present in very low concentration
o Determined indirectly by using a __________ to detect whether or not specific binding has taken place
LABELLED IMMUNOASSAYS; labelled reactant
LABELLED IMMUNOASSAYS;
___:
* All reactants are mixed simultaneously
* labeled antigen competes with unlabeled patient antigen for a limited number of antibody-binding sites
* concentration of the labeled analyte is in excess
* After separation, the amount of bound label is measured and used to determine the amount of patient antigen present
* the amount of bound label is inversely proportional to the concentration of the labeled antigen
Competitive
LABELLED IMMUNOASSAYS;
___:
* capture antibody, is first passively absorbed to a solid phase such as microtiter plates, nitrocellulose membranes, or plastic beads
* Excess antibody is present so that any patient antigen present can be captured
* unknown patient antigen is then allowed to react with and be captured by the solid-phase antibody
* After washing to remove unbound antigen, a second antibody with a label is added to the reaction
* the amount of label measured is directly proportional to the amount of patient antigen
Noncompetitive
LABELLED IMMUNOASSAYS;
___: more sensitive
* Require a step to physically separate free from bound analyte
* antigen or antiobody is attached by physical adsorption
* when specific binding takes place, complexes remain attached to the solid phase
* The sample is then thoroughly washed and the remaining activity is determined
Heterogeneous
LABELLED IMMUNOASSAYS;
___: less sensitive
* Do not need a separation step (no washing)
* Activity of the label attached to the antigen is diminished when binding of antibody and antigen occurs
* simpler to perform because there is no washing step
Homogeneous
I. ___; first assay to be developed
* pioneered by ___________ and _____________ in the late 1950s
* radioactive elements have nuclei that decay spontaneously, emitting matter and energy
* uses radioactive substance as label
131I – _________ counter
125I – _________ counter - most common
3H – _________ counter
14C – _________ counter
* Radioactivity is measured by a ___________________
Types:
a. ___
o Antigen (sample) + antibody (Reagent) + radioactive antigen
o Radioactivity in the solid phase is inversely proportional!! to analyte concentration; Most sensitive for drug assay, hormone
o Take Note:
➢ Analyte being detected competes with a radio-labeled analyte for a limited number of binding sites. The concentration of radioactive analyte is in excess so that all binding site in the antibody will be occupied.
b. Non-Competitive RIA
✓ Antigen (sample) + radioactive antibody
✓ Amount of radioactivity is _____ to the concentration of analyte.
✓ IRMA (immunoradiometric assay)
Advantage: sensitive and precise technique
Disadvantages: health hazard, Disposal of radioactive wastes, Requires expensive equipment
RADIOIMMUNOASSAY
Yallow and Berson
gamma
gamma
beta
beta
Scintillation counter
Competitive RIA
directly proportional
___:
* 1941, Albert Coons demonstrated that antibodies could be labeled with molecules that fluoresce; uses fluorescent compounds known as fluorophores or fluorochromes as labels
* FITC (Fluorescein Isothiocyanate)
* Tetramethyl rhodamine
* Phycocyanin
* Texas red
Types:
a. Direct Immunofluorescent
* ___ that is conjugated with a fluorescent tag is added directly to unknown ______ that is fixed to a microscope slide
* slide is read using _______________.
* Uses/ Application: diagnosis of viral disease (HSV, CMV, EBV), detect cell surface antigen, Chlamydial antigen, flow cell cytometry
b. Indirect Immunofluorescent
* 2 Step Procedure
1. patient serum is incubated with a __ attached to a solid phase
2. washed and an ________________ containing a fluorescent tag is added
* Uses: FTA-ABS, FANA
c. ___:
* based on the change in polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody
* the degree of fluorescence polarization is inversely proportional to concentration of the analyte
* polarization analyzer is used to measure the amount of polarized light.
FLUORESCENT IMMUNOASSAY
Antibody; antigen
fluorescence microscope
known antigen
anti-human immunoglobulin
Fluorescence Polarization Immunoassays
- uses enzymes as labels; Enzymes are naturally occurring molecules that catalyze certain biochemical reactions; React with suitable substrates to produce breakdown products: chromogenic, fluorogenic, or luminescent
- Types:
a. ___:
✓ enzyme labeled antigen competes with unlabeled patient antigen for a limited number of binding sites on
antibody molecules that are attached to a solid phase
✓ Enzyme activity is ___ to the concentration of the analyte
✓ Used for measuring small antigens that are relatively pure, such as drugs and hormones
b. Non-Competitive EIA
✓ ________________
▪ Solid phase antigen + antibody (sample) + enzyme labeled anti-Ig + substrate
▪ The amount of color, fluorescence, or luminescence detected is _________ to the amount of antibody in the specimen
▪ used to measure antibody production to infectious agents that are difficult to isolate in the laboratory and for autoantibody testing
c. ___:
✓ antibody is bound to the solid phase; Antigens captured in these assays must have multiple epitopes
✓ Solid phase Antibody + Antigen (sample) + enzyme labeled anti-Ig + substrate
✓ Enzymatic activity _______________ to the amount of antigen in the sample
d. ___:
✓ Rapid, Simple to perform, adapt easily to automation
✓ Example: Enzyme Multiplied Immunoassay Technique (EMIT)
✓ determination of low-molecular-weight analytes such as hormones, therapeutic drugs, and drugs of abuse in both serum and urine
ENZYME IMMUNOASSAY
Competitive EIA; inversely proportional
Indirect Immunoassay; directly proportional
Capture Assay/ Sandwich Assay; directly proportional
Homogeneous Assay
___:
➢ assays in a plastic cartridge. The membrane is usually nitrocellulose, which is able to easily immobilize proteins and nucleic acids. The rapid flow through the membrane and its large surface area enhance the speed and sensitivity of ELISA reactions.
➢ immunochromatography, combines all the previously mentioned steps into one. The analyte is applied at one end of the strip and migrates toward the distal end, where there is an absorbent pad
to maintain a constant capillary flow rate. The labeling and detection zones are set between the two ends. As the sample is loaded, it reconstitutes the labeled antigen or antibody, and the two form a
complex that migrates toward the detection zone. An antigen or antibody immobilized in the detection zone captures the immune complex and forms a colored line for a positive test
RAPID IMMUNOASSAYS
___:
✓ Emission of light caused by a chemical reaction
✓ luminal compound: luminol, acridinium esters, ruthenium derivatives
✓ measured in luminometer
✓ Advantages: excellent sensitivity, reagents are stable and relatively nontoxic, inexpensive to perform, a faster turnaround time
~ HAS NO LIGHT SOURCE
CHEMILUMINESCENT IMMUNOASSAY
A. GROUP A STREPTOCOCCAL INFECTION
✓ Caused by ___ -> pus formation
✓ Lancefield Group: _____
_____: Major Virulence Factor
▪ Inhibits phagocytosis
▪ Diminishes complement activation
▪ Attaches to Host Cell
Clinical Manifestation
* Upper Respiratory Tract Infection: ________________________
* Skin Infection: Impetigo, (streptococcal pyoderma), Erysipelas, cellulitis
* Streptococcus Toxic Shock Syndrome
* Otitis media
* Puerperal sepsis
* sinusitis.
* Septic arthritis
* acute bacterial endocarditis
* meningitis
* Scarlet fever - 2nd dse, strawberry tongue
1-6th diseases? (mnemonic)
S. pyogenes
A
M protein
Pharyngitis
(MSG stop popping heads)
1st: measles
2nd: scarlet fever
3rd: German measles
4th: Staph infections
5th: Parvorvirus B19
6th: Herpes
Damaging Sequalae
1. _____________: sequela to pharyngitis or tonsillitis.
2. _____________: Characterized by damage to the glomeruli in the kidneys.
Laboratory Diagnosis
1. Culture
a. Screening Test: susceptibility to __
b. Confirmatory Test: Testing for L-pyrrolidonyl-β-naphthylamide (PYR) activity; Lancefield typing
2. Detection of Group A Streptococcal Antigens
a. detection of bacterial, viral, fungal, and parasitic antigens in clinical samples
3. Detection of Streptococcal Antibodies
a. Diagnostically Important Antibodies (use at least 2)
a.1. ______________________
a.2. ______________________
a.3. ______________________
a.4. ______________________
Take Note:
* If acute and convalescent phase!!! sera are tested in parallel, a __ in titer is considered significant.
* The use of ___________ for antibodies to different exotoxins is recommended.
Acute rheumatic fever (heart)
Post streptococcal Glomerulonephritis (+ RBC cast)
Bacitracin
anti-Streptolysin O
anti-DNAse B
anti-NADase
anti-Hyaluronidase
four-fold rise
at least 2 test
___:
o detect antibodies to the Streptolysin O enzyme produced by Group A streptococcus
o based on the ability of antibodies in the patient’s serum to neutralize the hemolytic activity of Streptolysin O
o Titer: reciprocal of the highest dilution demonstrating no hemolysis
- Todd units
- International units -> WHO
Controls: red cell control tube & SLO control Tube
ASO titer: moderately elevated if the titer is >240 Todd units in an adult , >320 Todd units in a child
___:
o automated procedure that provides rapid, quantitative measurement of ASO titers
o antibody-positive patient serum combines with the antigen reagent, immune complexes are formed, resulting in an increased light scatter that the instrument converts to a peak rate signal
o Reporting: __________________
___:
* Highly specific for group A streptococcal sequelae
* Presence is measured by its effect on a DNA methyl-green conjugate. This complex is green in its intact form, but when hydrolyzed by DNase, the methyl green is reduced and becomes colorless.
* Tubes are graded for color, with a 4 indicating that the intensity of color is unchanged, and a 0 indicating a total loss of color. The result is reported as the reciprocal of the highest dilution demonstrating a color intensity of between 2 and 4
Anti-Streptolysin O Testing
ASO Titer Test; International units
Anti-DNase B Testing;
B. ___ INFECTION
* Agent of Gastric and Duodenal ulcers; Gastric Carcinoma
* Virulence Factor:
1. ___: dysfunction of the cells signal transduction pathway
2. ___: codes for a toxin precursor
H. pylori is able to survive and multiply in the gastric environment:
* Spiral shape and flagella
* Urease
* Flagella are coated with a flagellar sheath
✓ Diagnosis:
o Invasive Detection Methods: Endoscopy and Biopsy
o Noninvasive Detection Methods: urea breath testing, Enzyme or lateral flow immunoassays, molecular tests for H pylori DNA
▪ Serological Test: Detection of Helicobacter pylori Antibodies
* Primary Screening Method: ___
* antibody testing is not as well suited for determining eradication of infection as are other methods
Helicobacter pylori
CagA
Vacuolating Cytotoxin (VacA)
IgG, IgM, IgA
C. ____ INFECTION
▪ class Mollicutes; leading cause of upper respiratory infections worldwide; Cold Isoagglutinins
o IgM antibodies directed against the altered I antigens found on the surface of human RBCs
o agglutinate the RBCs at ____
o reaction is reversible when the samples are ______
o IM, CMV Infection
▪ Laboratory Diagnosis of Mycoplasma pneumoniae Infection
o Culture: Trypticase soy broth with 0.5% albumin, SP4 medium, or a viral transport medium; Delay: ___________
▪ Detection of Antibodies
o M pneumoniae-specific IgM
o M pneumoniae-specific IgG
▪ ___: Gold standard for the diagnosis of Mycoplasma infections
Mycoplasma pneumoniae
cold temperatures
incubated
frozen at 70degC
Molecular Diagnosis
D. ___ Infections
- energy parasite; obligate intracellular; gram neg bacteria; arthropod-borne; GENERA: Rickettsia, Orentia
✓ Weil-Felix Test
▪ A nonspecific test for the detection of antibodies against Rickettsial diseases
▪ Principle: Direct agglutination
▪ Antibody: Rickettsial antibodies in serum
Rickettsial Disease
___: ++++ for OX-K only
___: ++++ for OX-19 and + for OX-2
___: - for all (OX-19, OX-2, OX-K)
~ ___: is currently the method of choice for detecting Rickettsial infections.
Rickettsial
Scrub
RMSF (Rocky Mountain Spotted Fever)
Q Fever & Rickettsial Pox
Serodiagnosis
E. ____
✓ Agent: Salmonella enterica subsp. enterica serotype typhi
o Acquired by: ingestion
o TSI: K/A, (-) gas, (+) H2S (Salmonella, Shigella, Yersinia - NLF)
o IMViC Reaction: ___
✓ Salmonella Antigens: H ag and K ag (capsular, thermolabile); O antigen (somatic, thermostable)
✓ Culture
o Specimens for Salmonella Identification:
▪ _______-1st week of infection
▪ _______-2nd week of infection
▪ _______-3rd week of infection
✓ ___:
o For the diagnosis of typhoid fever
o Slide test: principle-direct agglutination
o Tube test: highest serum dilution showing ++ or 50% agglutination, significant titer: 80 and above
✓ Typhidot
o detection of specific IgM and IgG to Salmonella typhi
TYPHOID FEVER
-,+,-,-
blood
stool
urine
Widal Test
F. ___:
* slender, flexuous, helically shaped, unicellular; periplasmic flagella (endoflagella); gram-negative, microaerophilic bacteria
* corkcrew flexion or motility
Treponema pallidum sub speciation (mnemonic)
T. pallidum subspecies pallidum - ___
T. pallidum subspecies pertenue - ___
T. pallidum subspecies endemicum - ___
T. carateum - ___
SPIROCHETES
(PaSs or PeY? Edi Bawal Ca Pumunta)
Syphilis
Yaws
non-venereal syphilis/Bejel (non-sexually transmitted)
Pinta
___:
✓ caused by Treponema pallidum subsp. pallidum
✓ ability to cross intact mucous membranes and the placenta, disseminate throughout the body, and infect almost any organ system
✓ Treponemal Rare Outer Membrane Protein (TROMPs); VF is delayed immune response
✓ Take Note: Syphilis comes from a poem written in 1530 that described a mythical shepherd named Syphilus, who was afflicted with the disease as punishment for cursing the gods
✓ Mode of Transmission: _______________________________________
~ least likely to be transferred thru blood trans bc ___
SYPHILIS/greatpox/evilpox/Spanish disease/The Great Imitator
sexual transmission, parenteral exposure, congenital infection during pregnancy
dies at cold temp
Stages of Syphilitic Infection
1. Primary Syphilis
➢ Appearance of ___
o endothelial cell thickening occurs with aggregation of lymphocyte, plasma cells, and macrophages
o ____________, solitary lesion characterized by raised and well-defined borders; lasts from ____
➢ Laboratory test: Direct detetion; Darkfield and Fluorescence microscope
➢ Early infection: No ab production yet
- Secondary Syphilis/Systemic infection
➢ Usually observed about ___ after the primary chancre disappears
➢ generalized lymphadenopathy; malaise; fever; pharyngitis; and a rash on the skin and mucous
membranes
➢ Appearance of _______ (flat, wart-like)
➢ Laboratory Test: Darkfield microscopy, Serologic test
hard chancre!!
painless!!
1 to 6 weeks
1-2 months (2-10 weeks)
Codylomata lata
Stages of Syphilitic Infection
3. Latent Syphilis
➢ Patients are ________
➢ Generally, after 2nd year of infection
➢ characterized by a lack of clinical symptoms
➢ The only indication of infection is a ______________
➢ Types of Latency
▪ Latency within 1 year of infection is referred to as __
▪ Latency greater than 1 year is __
4.Tertiary Stage
➢ Occurs most often between ___ following the secondary stage.
➢ Three major manifestations:
▪ ______________
▪ ______________
▪ ______________
➢ ________ are localized areas of granulomatous inflammation that are most often found on bones, skin, or subcutaneous tissue
non-infectious
positive serological test
early latent syphilis
late latent syphilis
10 and 30 years
gummatous syphilis
cardiovascular disease
neurosyphilis -> CNS
Gummas
Other Clinical Manifestations of Tertiary Syphilis
* _____________________: Asymptomatic involvement of the CNS and detected only by examination of cerebrospinal fluid (CSF).
* _____________________: Caused by maternal spirochetemia and transplacental transmission of microorganism.
Hutchinsonian Triad
1. _______________________
2. _______________________
3. _______________________
Other characteristics:
Perforation of the palate and collapse of nasal bones → Saddle-nose deformity
Neurosyphilis
Congenital syphilis
Hutchinson’s teeth
interstitial keratitis
nerve deafness
Laboratory Diagnosis of Syphilis
1. ___ of spirochetes;
a. Dark-Field Microscopy; b. Fluorescent Antibody Testing
▪ uses a fluorescent-labeled antibody conjugate to T pallidum
- Serologic Test for Syphilis
1.Non Treponemal/ Non-Specific methods -> REAGIN!!!
1.1. ___; Developed by the bacteriologist August Paul Von Wasserman (1906)
▪ Antigen crude liver extract from a fetus that was infected with syphilis
▪ Principle: complement fixation test (+): no hemolysis
Direct Detection
Wasserman Test
1.Non Treponemal/ Non-Specific methods
1.2. ____: Uses heated serum; result is read macroscopically
▪ Principle: ___________________
▪ Reagent
o _________: Main Reagent
o _________: helps neutralize anticomplementary properties of cardiolipin
o _________: provides adsorption centers and increases the effective reacting surface of cardiolipins.
▪ Rotator: 180 rpm for 4 mins (serum VDRL) 180 rpm for 8 mins (CSF VDRL)
▪ Ring Diameter: 14mm (serum VDRL) 16mm (CSF VDRL) [depth:1.75mm]
▪ Antigen delivery needles
o Qualitative serum VDRL: __________ needle without bevel that will deliver 60 drops of antigen suspension per mL
o Quantitative serum VDRL: __________ needle that will deliver 75 drops of antigen suspension per mL or gauge 23 needle with or without bevel that will deliver 100 drops os
saline/mL
o CSF VDRL: __________ needle that will deliver 100 drops/mL
▪ Reporting
o Non reactive – no clumps
o Weakly reactive - small clumps
o Reactive – medium to large clumps
▪ False positive VDRL results - SLE, RA, IM, malaria, pregnancy
1.3. ___
▪ Uses unheated serum, result is read macroscopically
▪ Principle: ___
▪ Reagent: colorless alcoholic solution containing cardiolipin, lecithin, charcoal (visualize result), choline chloride (chemical inactivator of serum) and thimerosal
▪ Rotator: 100 rpm for 8 mins
▪ Ring diameter:18mm
▪ Antigen delivery needle: _________ needle , 60 drops/mL
VDRL (Venereal Disease Research Laboratory Test); flocculation
cardiolipin - 0.03%
lecithin - 0.21%
cholesterol - 0.9%
gauge 18
gauge 19
gauge 21 or 22
RPR (Rapid Plasma Reagin)
flocculation
gauge 20
2.Treponemal Serologic Test/ Specific methods
2.1 ____
▪ Principle: Indirect Fluorescent Immunoassay
▪ Reagent antigen: Nichol’s strain dried and fixed on slide
▪ Absorbent: _______ (removes cross-reactivity w other non-pathogenic treponemes
2.2. HATTS (Hemeagglutination Treponemal Test for Syphilis)
▪ Principle: hemagglutination
▪ Reagent antigen: glutaraldehyde stabilized turkey RBC coated with treponemal antigen
2.3. MHA-TP (Microhemagglutination T.pallidum Test)
▪ Principle: Hemagglutination
▪ Reagent antigen: tanned formalin sheep RBC coated with treponemal antigen
2.4. ___
▪ Most specific for syphilis, standard test
▪ Principle: the antibody produced against T.pallidum plus complement can immobilize the live treponemes
▪ Reagent antigen: live actively motile T.pallidum organisms
▪ (+): ___________________
~ 2 Treponemal Antigen sources:
1.non pathogenic __________
2.Pathogenic __________
FTA-ABS (Fluorescent Treponemal Antibody Absorption Test)
Reiter’s treponemes
TPI (T.pallidum Immobilization Test);
>50% immobilized treponemes
Reiter strain
Nichol’s strain
Borrelia (causes ___)
* All pathogenic Borrelia are arthropod-borne
* much less tightly coiled than those of the leptospires
* borreliae stain easily and can be visualized by ______________________
Lyme Disease
▪ Causative agent: ___________________________
▪ Transmitted via: ____________________________
o Ixodes scapularis
o Ixodes pacificus
o Ixodes Ricinus
o Ixodes persulcatus
▪ Stages of the Disease
1. localized rash,
2. early dissemination to multiple organ systems,
3. late disseminated stage often including arthritic symptoms
▪ Serological Test
o ____________ confirmation of ______ antibody presence includes reactivity for two of the three
following bands: 24, 39, 41 kDa.
o ____________ confirmation of ______ antibody presence includes reactivity for 5 of the 10 following
bands:18, 28, 39, 45, 66, 21, 30, 41, 58, 93kDa
Lyme disease
bright-field microscopy
Borrelia burgdoferi sensu latu complex
Ixodes spp. (tick)
Western blot; IgM
Western blot; IgG
___:
▪ spirals are very close together
▪ One or both ends of the organism have hooks
▪ they can be impregnated with silver
▪ Culture Medium
o ________________________
o Stuart liquid medium, or Ellinghausen-McCullough-Johnson-Harris (EMJH) semisolid medium.
Leptospires
Fletcher semisolid medium
___:
* Causative agent: Leptospira interrogans
Infection stages
* septicemic stage
* immunological stage
Laboratory diagnosis
* Culture method
* Microscopy
* Serological test
Leptospirosis
VIRAL SEROLOGICAL TESTS
Viral Escape Mechanism
* Acquisition of genetic mutations that result in new viral antigens
* Inhibition of immunologic components
* Suppression of the immune system
* Establishment of a latent state
I. HEPATITIS
____________________: viral RNA is treated with the enzyme reverse transcriptase to generate a complementary DNA
(cDNA) sequence
Reverse Transcriptase Polymerasse Chain Reaction (RT-PCR)
Hepatitis A:
Agent: ________________________________
o Non-enveloped; Single stranded RNA!!!
o Family ______; Genus ______
MOT: fecal-oral, blood transfusions
serological markers: Anti-HAV IgG, IgM, HAV RNA
Most common type of hepatitis; Self Limiting Disease
Hepatitis A markers of infection:
- Early shedding of virus in ___
- Appearance of IgM anti-HAV with the onset of symptoms
- Development of anti-HAV IgG and immunity on recovery
Laboratory Diagnosis: RIA and ELISA detect the presence of specific HAV antibodies
; Hepatitis A virus
Picornaviridae
Hepatovirus
stool
Hepatitis E (water-borne)
Agent: ________________________________
o Non enveloped; RNA Virus
o Family ____________; Genus Hepevirus
MOT: Fecal-Oral; Blood Transfusion
serological markers: Anti-HEV IgG, IgM, HEV RNA
4 Genotypes
- waterborne: (2)
- zoonotic: (2)
High mortality rate in ___
Self limiting disease
Laboratory diagnosis
o Electron Microscopy
o Indirect ELISA
o RT-PCR
Hepatitis E virus
Hepeviridae
HEV 1 & HEV 2
HEV 3 & HEV 4
women
Hepatitis B (long incubation); Australian aborigines
Agent: ________________________________
o _____ only one in hepatitis
o Family _________________
o Genotypes: A – H
o Infectious Form: _____________
Leads to ____; MOT: Parenteral, sexual, perinatal
serologic markers: HBsAg, HBeAg, Anti-HBc IgG, IgM, Anti-Hbe, Anti-HBs, HBV DNA
Serologic Markers for HBV:
___: best indicator of early acute infection; indicates acute or chronic HBV infection
___: Not detected in serum because it is found only in hepatocytes
___: High levels of virus and high degree of infectivity
___: IgM (useful in detecting infection during the window period, indicator of current infection); IgG (Life long marker of Hepatitis B)
___: First serologic evidence of convalescent phase
___: Bestows immunity to further HBV infection; viral clearance of HBV
Tests for HBV:
First Generation Test: Ouchterlony
Secondary Generation Test
▪ Counter electrophoresis
▪ Rheophoresis
▪ Complement fixation
Third Generation Test:
▪ Reversed Passive Latex Agglutination
▪ ELISA
▪ Reversed Passive Hemagglutination
▪ Radioimmunoassay
Hepatitis B virus
DNA Virus
Hepadnaviridae
Dane particle
liver cirrhosis and cancer
Hepatitis Surface Antigen (HBsAg)
Hepatitis Core Antigen (HBcAg)
HBeAg
Anti-HBc
Anti-HBe
Anti-HBs
Status of hepatitis if:
HBsAg: +
Anti HBs: -
Anti HBc: -
Early Infection
Status of hepatitis if:
HBsAg: -
Anti HBs: +
Anti HBc: +
Recovery
Status of hepatitis if:
HBsAg: -
Anti HBs: -
Anti HBc: +
Window Period
Hepatitis C (NANB) (___); successfully cultured
Agent: _________
o Enveloped; Single stranded RNA; MOT: Parenteral, sexual, perinatal
Serological markers: Anti-HCV, HCV RNA
o Flaviviridae; Genus ___
o Genotypes: 1-7
Tests for HCV
o Surrogate Test: ALT and anti HBc
o Serologic Tests for anti HCV
▪ ELISA
▪ RIA
o ___ = HCV specific antibody/ anti HCV CHON
o ___ = HCV RNA (persistent HCV infection)
Post Transfusion Hepatitis
Hepatitis C virus
Hepacivirus
RIBA
RT-PCR
HEPATITIS D INFECTION SEROLOGIC MARKER:
Anti-HbC IgM: +
HBsAg: +
Anti-HDV: +
Anti-HDV IgM: +
Anti-HbC IgM: -
HBsAg: +
Anti-HDV: +
Anti-HDV IgM: NA
co-infection
superinfection
Status of hepatitis if:
HBsAg: +
Anti HBs: -
Anti HBc: +
Chronic Infection
Status of hepatitis if:
HBsAg: -
Anti HBs: +
Anti HBc: -
Vaccination
II. ____:
* Large, ____; Enveloped
* latent infection with lifelong persistence in the host
* Herpesviridae
- HSV-1 (stool) & HSV-2 (genital)
- VZV, EBV, CMV
- Human Herpes Virus (HHV-6, HH-7 & HH-8)
HERPES INFECTION; Complex DNA
Hepatitis D
Agent: : _______ (defective virus); Genus: Deltavirus
MOT: parenteral, sexual, perinatal; SM: Anti-HDV IgG, IgM, HDV RNA
Co-infection and super infection with HBV
Laboratory diagnosis:
o Indirect ELISA
▪ Anti HDV
▪ Anti HBc IgM differentiates co-infection (present) from super infections (absent)
hepatitis D virus
II. HERPES INFECTION
___: result from intimate contact with salivary secretions from an infected individual
* Initial infection: oropharynx
* Receptors
* _______-Epithelial Cells
* _______-B cells (precursors in ab production)
Virus-infected B cells
* EBV-specific antibodies
* heterophile antibodies
* autoantibodies
EBV ANTIGENS:
___:
o EA-diffuse
o EA_restricted
___:
o viral capsid antigen
o membrane antigens
___:
o EBV nuclear antigen
o latent membrane proteins
Epstein-Barr Virus
B1 integrins
CD21
Early Antigens
Late Antigens
Latent Antigens
II. HERPES INFECTION
___: Kissing disease or glandular Fever (RES)
Common in adolescence and adult
Types of heterophile Antibodies
- Heterophil Antibodies in IM (most significant; EBV infx)
* reacts with sheep cells, OX (BEEF) cells and Horse cells but not with __________________ - Heterophil antibodies of Forssman (bacterial infx)
* reacts with sheep cells, Horse cells, guinea pig cells but not with __________________ - Heterophil antibodies in Serum Sickness (exposure to horse serum
* reacts with Sheep, OX, Horse and Guinea pig cells
INFECTIOUS MONONUCLEOSIS
GUINEA PIG CELL
BEEF CELLS
II. HERPES INFECTION
Tests for Heterophil Antibodies:
1. ____
* screening/general test
* principle: hemagglutination
* reagent: 2% suspension of ___
* (+) result: agglutination
2. ____ - screening test
* Principle: ______________________________
* Antigen: Guinea pig cells and Beef RBCs
* Indicator cells: _______________________
Paul Bunnel Test; sheep RBC
Davidson Differential Test; absorption-hemagglutination
sheep RBCs
Tests for Heterophil Antibodies:
Absorption Pattern:
Beef RBCs: NO
Guinea Pig Cells: YES
Beef RBCs: YES
Guinea Pig Cells: NO
Beef RBCs: YES
Guinea Pig Cells: YES
FORSSMAN
IM
SERUM SICKNES
Tests for Heterophil Antibodies:
Agglutination Pattern:
after ab w/ Beef cells: inc titer
after ab w/ Guinea Pig Cells: dec titer
after ab w/ Beef cells: dec titer
after ab w/ Guinea Pig Cells: dec titer
after ab w/ Beef cells: dec titer
after ab w/ Guinea Pig Cells: inc titer
FORSSMAN
SERUM SICKNES
IM
Tests for Heterophil Antibodies:
3. ____ (slide Method)
* Principle: absorption-Hemagglutination
* Indicator cells: horse RBC
Specific EBV Antibodies
* Anti-VCA IgM
* Anti-VCA IgG
* Anti-EA-D
* Anti-EBNA
antibodies: IFA using EBV infected cell & ELISA (gold standard)
Monospot
II. HERPES INFECTION
___:
* spread by close contact, intimate sexual contact, blood transfusions
* virus is shed in saliva, tears, urine, stool, and breast milk
* Virus persists in a latent state in monocytes, dendritic cells, myeloid progenitor cells, and peripheral blood leukocytes
* Laboratory Diagnosis: Culture, Molecular Methods, Serology
CYTOMEGALOVIRUS
II. HERPES INFECTION
1___:
* Agent of: Varicella and Zoster
* MOT: inhalation of infected respiratory secretions or aerosols
* Laboratory Diagnosis
* Identifying the characteristic vesicular lesions associated with the infection
* culture of fresh lesions (vesicles)
* fluorescent-labeled monoclonal antibodies against VZV
* PCR
VARICELLA-ZOSTER VIRUS
III. ___
* RNA Virus!!!
* Genus Rubivirus
* Family Togaviridae
* Respiratory Droplet/ transplacental infection
* Diagnosis: RT-PCR, IFA, EIA, Culture
RUBELLA
IV. ___
* Genus Morbilivirus
* Family Paramyxoviridae
* highly contagious and spreads by aerosol
* diagnosis: RT-PCR, IFA, EIA
RUBEOLA
V. ____
✓ Require an RNA-dependent DNA polymerase to synthesize DNA from the RNA genome
✓ Family _____________ Subfamily: ___________________
✓ Has marked preference for T-helper cells (CD4 (+)) which serve as a receptor site for the virus
✓ Types:
HIV-1 (pandemic)
▪ Four Groups:
* M - ___; majority of HIV infections; subtypes: A,B,C,D,E,F,G,H,J,K
* O - ___
* N - new
* P- new
o HIV-2
▪ Discovered in _________
▪ less pathogenic and has a lower rate of transmission
HUMAN IMMUNODEFICIENCY VIRUS (HIV)
Retroviridae; Lentivirinae
main group; C the most common subtype
outlier
1986
HUMAN IMMUNODEFICIENCY VIRUS (HIV):
Transmission:
o Intimate sexual contact
o Contact with blood or other body fluids
o Perinatally (from infected mother to infant)
*HIV is transmitted via: Blood, Semen, Breast Milk, Vaginal Fluid, CSF, Synovial Fluid, Pleaural Fluid
✓ Main structural genes
o Gag gene: Codes for _______________
o Env (envelope): Codes for ___________________
Pol
▪ codes for enzymes necessary for HIV
replication
* Reverse Transcriptase (___)
* Ribonuclease (RNAse H; p66)
* Integrase (p31)
* Protease (p10)
▪ located in the core of the virus in association with HIV RNA
p55
gp160, gp120, gp41
p51
HUMAN IMMUNODEFICIENCY VIRUS (HIV):
✓ Regulatory Gene
o tat - transactivator
o rev - regulator of expression of virion proteins
o nef - negative effector
o vpu - viral protein “U”
o vpr – viral protein “R”
o vif - viral infectivity factor
✓ HIV Replication
o Virus (_____) attaches to susceptible host cell (____)
o Fusion of HIV envelope with plasma cell membrane
▪ Co-receptor - _________________
o ___________________ produces complementary DNA from the
viral RNA
o Double-stranded DNA is synthesized and becomes integrated
into the host cell’s genome as a provirus
gp120; CD4
CXCR4 (t helper cell), CCR5
revere transcriptase
✓ Stages of HIV infection:
o Stage 1: _________________
▪ 2-4 weeks of infection
▪ Body’s natural response to infection
▪ large amount of virus in the blood and are very contagious
o Stage 2: _________________
▪ period can last a decade or longer
▪ people can still transmit HIV to others
▪ end of this phase, a person’s viral load starts to go up and the CD4 cell count begins to go down
o Stage 3: ________________________________
▪ most severe phase of HIV infection
▪ badly damaged immune system
▪ opportunistic illnesses
~ CD4 count <200/mm3;
~ normal: >1000/mm3
Acute HIV infection
Clinical latency
Acquired Immunodeficiency syndrome (AIDS)
✓ Testing Algorithm for HIV infection:
Initial Screening Tests:
▪ Detects antibodies to HIV-1, HIV-2 and HIV-1 p24 antigen (Combination) Confirmatory Test
Confirmation - detect type of HIV:
▪ additional testing with a rapid immunoassay that discriminates between HIV-1 and HIV-2 antibodies
▪ samples that are reactive in the initial test and nonreactive in the second test should then undergo __________________
nucleic acid testing
o Screening Test: ELISA
▪ __________ - used purified viral lysate as antigens
▪ __________ - used recombinant viral proteins
▪ __________ - relied on the double-antigen sandwich assay
▪ __________ - detected antibody and p24 antigen
▪ __________ - multiplexed screening test that detects and differentiates all three HIV analyte markers: HIV-1 antibodies, HIV-2 antibodies, and the HIV-1 p24 antigen
o Rapid Test for HIV Antibodies
▪ Detect antibodies to HIV-1 alone or to both HIV-1 and HIV-2
▪ Followed by the current recommended testing algorithm
▪ Recommended by the CDC as _________________________
o Western Blot
▪ Nitrocellulose or nylon strips containing individual ____________
* Low molecular weight antigen: ____________
* High molecular weight antigen: __________________
▪ During incubation period: HIV antibodies bind to their corresponding antigens on the test strip
▪ Anti-human immunoglobulin with an enzyme label is added
▪ Bound conjugate is detected after adding the appropriate substrate
▪ Colored bands appear
first generation screening
second generation
third generation
fourth generation
fifth generation assay
confirmatory tests
HIV proteins
bottom of test strip
top of test strip
o Take Note:
▪ CDC recommends routine HIV testing for all persons 13-64 years old and annual testing for individuals in high-risk groups
▪ Serological tests for HIV antibody are used in the initial diagnosis of HIV infection because most individuals develop antibody to the virus
within 1 to 2 months after exposure
▪ According to criteria, a result should be reported as positive if at least two of the following three bands are present: _______
gp120/160 & gp41 & p24
o Testing of Infants Younger Than 18 Months for HIV
▪ best diagnosed using molecular methods
▪ Detects proviral DNA within the infants’ peripheral blood mononuclear cells
▪ Alternative: quantitative HIV RNA assays
* Confirmatory test for infants who initially had a positive HIV DNA test.
Take note:
HIV in dried smear/blood = infective for _____ @ room temp
HBV – infective for _____
Diluted bleach inactivate HBV (_______) and HIV (______)
3 days
7 days
10 mins; 7 mins
Refers to the serologic, genetic, biochemical adn molecular study associated with membrane structures on the cellular constituents of blood, as well as the immunologic properties and reactions of blood components and constituents.
IMMUNOHEMATOLOGY
IMMUNOHEMATOLOGY HISTORY
Pope Innocent VII
___ * recommended sodium phosphate; first example of blood preservation research
___ * discovered the ABO blood groups; explained the serious reactions that occur in humans as a result of incompatible transfusion
___ * first to succeed in making devices designed for performing the transfusions; vein-to-vein transfusion of blood by using multiple syringes and a special cannula for puncturing the vein through the skin
___ * syringe-valve apparatus that transfusions from donor to patient by an unassisted physician became practical
___ * the use of sodium citrate as an anticoagulant solution for transfusions
___ * Determined the minimum amount of citrate needed for anticoagulation and demonstrated its nontoxicity in small amounts
Braxton Hicks
Karl Landsteiner
Edward E. Lindemann
Unger
Hustin
Lewisohn
BLOOD GROUP IMMUNOLOGY
Blood Group Antigens
* Refers to the genetically encoded antigen system on the erythrocytes, Leukocytes, thrombocytes and plasma
* Characteristics: ___ and ___
Incidence
* Low incidence: ___ - present in family (ex. K)
* High incidence: ___ - public, (ex. k, cellano)
immunogenicity of blood groups (most to least):
glycolipids (ABO) and glycoproteins (Lewis)
rare (family/private ag)
common
A,B>RhD>Kell(K)>Duffy(Fya)>common Rh antigens
___: produced as a result of exposure to foreign antigens; products of antigenic stimulation and that demonstrate specific antibody activity
___ : reacts with an antigen on the patient’s own cells.
___ : reacts with a foreign antigen not present on the patient’s own erythrocytes
___ : Antigenic stimulus is unknown; ex. ABO antibodies; individual was exposed to a biochemical structure similar to A or B antigens present in the environment (Bacteria, Pollen, and plants
___ : Produced as the result of immunization to foreign erythrocyte antigens by exposure through transfusion of blood components or through pregnancy
Blood group Antibodies
autoantibody
alloantibody
naturally occurring (expected/regular)
immune antibodies
___: conversion of polymeric antibody into monomeric antibody
a. 2-mercapthoethanol (2-ME)
b. dithiothreitol (DIT)
Factors That Influence Antigen-Antibody Reaction:
1. Antigen-Antibody Ratio
a. Effect of Zonal Reaction
b. ____: phenomenon in which red cells from homozygous people possess more antigens per red cell than do cells from heterozygous people
2. pH: 6.5-7.5
3. Temperature for IgM and IgG ___
4. Immunoglobulin Type
5. Centrifugation
Reduction of antibody
dosage effect
IgM - cold; IgG - warm; both- biphasic
ENHANCEMENT MEDIUM:
4. Antihuman Globulin Reagent (AHG)/___
* 1945 Coombs, Mourant and Race
* Detects antibodies and complement proteins that have attached to the RBC; Involved the injection of human serum into rabbits to produce antihuman serum
4.1. Direct AHG
* Detects ___ sensitization of RBC with IgG/ complement components
* Clinical Conditions
- Diagnosis of HDFN
- Investigation of HTR
- Diagnosis of AIHA
* Procedure
- Washed RBC + AHG reagent
- Does not require ___
~ add check cells; if truly negative, the reaction must be __
4.2. Indirect AHG
* Detects in vitro sensitization of RBC
* Used in the following:
ü Antibody detection
ü Antibody Identification
ü Antibody titration
ü Red Cell phenotyping
ü Crossmatching
* Procedure:
Coombs Reagent
in vivo
incubation
(+) agglutination; (-) agglu is invalid
___: Reagents that enhance the detection of IgG antibodies by increasing their reactivity
* Reduces the ___
* May enhance ___ or ___
1. Protein Medium - inc the dielectric constant that reduced the zeta potential of RBC
1.1. ____ – increased concentration promotes roalleux formation
1.2. ____: Removes water molecule in the environment; Hydrophilic polar heads of lipid molecules making up the outer cell membrane bilayer attract water molecules. The water thus creates a surface tension that helps to keep the cells apart
* Incubation: 10-30 mins
* More effective than albumin, LISS for detection of weak antibodies
1.3. Protamine
1.4. Polyvinylpyrrolidone (PVP)
- ____: Generally, contain 0.2% sodium chloride (+glycine); Decreases the ionic strength of a reaction medium, which reducesthe zeta potential and therefore allows antibodies to react more efficiently with RBC membrane antigens
* Incubation time: ___
* Disadvantage: prone to false positive result - Use of Enzymes
* Target the ____, protein molecule
* Enhances: ___ (mnemonic)
* Destroys: ___ (mnemonic)
Example:
* (Papaya) -
* (Fig plants) -
* (Pig stomach) -
* (pineapple) -
ENHANCEMENT MEDIUM
zeta potential
antibody uptake; promote direct agglutination
22% bovine serum albumin
Polyethylene Glycol (PEG)
Low Ionic strength Solution (LISS)
5-15 mins
sialic component
Rh, Kidd, P1, Lewis and I antigens “Rest in peace (RhIP) Lewis the Kidd.”
Fya, Fyb, M, N, S antigens (Must Not Ssmell Duff’s destroyer
Papain
Ficin
Trypsin
Bromelain
AHG Reagents:
___: contain antibody to human IgG and to the C3d component of human complement
___: Contain only one antibody specificity: either anti-IgG or antibody to specific components of complement, such as C3b or C3d
AHG Preparations:
___: Mixture of antibodies from different plasma cell clones
Recognize different antigenic determinants (epitopes), or the same portion of the antigen but with different affinities
___: Derived from one clone of plasma cells and recognize a single epitope
Polyspecific AHG
Non-specific AHG
polyclonal (Rabbits)
monoclonal (Mouse)
4.5. ___
* Red cells with attached antibody
* Added to AHG negative result
4.6. Factors Affecting AHG Test
* Cell: serum = 1:40 or 1gtt/2gtt
* Reaction medium
* Temperature: ___
* Incubation time: depends on the medium
* Washing: removes excess ab
* Centrifugation: 1000RCF for 20 sec
Take note:
4.5. Check Cells
* Red cells with attached antibody
* Added to AHG result
4.6. Factors Affecting AHG Test
* Cell: serum = 1:40 or 1gtt/2gtt
* Reaction medium
* Temperature
* Incubation time
* Washing
* Centrifugation
* 1000RCF for 20 sec
- ___: detect a level of 100 to 500 IgG molecules per RBC and 400 to 1,100 molecules of C3d per RBC
- ___: there must be between 100 and 200 IgG or C3 molecules on the cell to obtain a positive reaction
Check Cells
37degC
DAT
IAT
SEROLOGIC GRADING: grade and score?
One solid agglutinate with clear background
Several Medium to Large agglutinates with clear background
Medium-sized agglutinates with clear background
Small agglutinates with a turbid background
Tiny agglutinates with turbid background
No agglutination or hemolysis
Mixed Field - small agglutinates within predominantly unagglutinated red cells
Hemolysis
Partial Hemolysis
4+; 10
3+; 8
2+; 5
1+; 3
w+; 1
0; 0
Mf; NA
H; 10
PH; –
POSITIVE RESULT IN BLOOD BANK
___: Antibody – mediated clumping of particles that express antigen on the surface
___: Rupture of red cells with release of intracellular hemoglobin
___: Formation of an insoluble complex when soluble reacts with soluble antibody
Take Note: ____ is the single most important in vitro immunologic reaction in blood banking because it is the endpoint of almost all test systems designed to detect RBC antigens and antibodies
Agglutinaton
Hemolysis
Precipitation
Specific hemagglutination
- Unit of inheritance that encodes a particular protein
- Structures within the nucleus that contain DNA
- Illustrate the probabilities of phenotypes from known genotypes
gene
chromosome
Punnett square
- site of a gene in a chromosome
- alternate forms of a gene at a given locus
- Opposite antigens encoded at the same locus
- refers to having two or more alleles at a given locus
- equal expression of two different inherited alleles
- always expressed
- gene that in the presence of a dominant gene does not express itself
- gene that does not produce any detectable trait
- identical alleles
- Different alleles
- gene that suppresses the expression of another gene
Take Note:
Examples of Amorph: O - Blood type O; __ - Bombay Phenotype.
When writing the identity of a gene, use italics. Red cell __ phenotypes do not require italics.
MENDEL’S LAW OF INHERITANCE:
- Alternative versions of genes account for variations in inherited characters.” Alleles are different versions of genes that impart the same characteristic.
- genes for different traits are inherited separately from each other
- locus
- alleles
- Antithetical
- Polymorphic
- codominant
- dominant gene
- recessive gene
- amorph
- homozygous
- heterozygous
- Suppressor Gene
h
antigen
Law of Segregation
Law of Independent Assortment
___:
-Only blood group system in which individuals already have antibodies in their serum to antigens that are absent from their red blood cells (RBCs) without any prior exposure to RBCs through transfusion or pregnancy
- transfusion of the wrong ABO group remains a cause of death in hemolytic transfusion reaction fatalities
- only blood group system that affects ___
- ____ –donor pairs must be ABO identical or compatible to avoid this adverse outcome
Landsteiner Law
1. Antigen!! on the RBC determines the blood group
2. The corresponding antibody is never found in the individual’s serum
3. The opposite antibody is always present!! in the individual’s serum
ABO BLOOD GROUP SYSTEM
clinical transplantation
Recipient
Blood Group: O
Antigens on RBC surface: none
Naturally Occurring antibodies: ??
Percentage in Population: 45%
ISBT: ??
Blood Group: A
Antigens on RBC surface: A
Naturally Occurring antibodies: ??
Percentage in Population: 40%
ISBT: ??
Blood Group: B
Antigens on RBC surface: B
Naturally Occurring antibodies: ??
Percentage in Population: 10%
ISBT: ??
Blood Group: AB
Antigens on RBC surface: A & B
Naturally Occurring antibodies: ??
Percentage in Population: 5%
ISBT: ??
Blood Group: A1?
Note: The __ are found on the surface of red blood cells and the ___ are found in serum or plasma.
anti-A and anti-B; —
anti-B; 001001/1.1
anti-A; 001002/1.2
none; 001003/1.3
001004/1.4
antigens; antibodies
ABO ANTIBODIES/expected/regular
- It has been postulated that bacteria, pollen particles, and other substances present in nature are chemically similar to A
and B antigens.
- Reacts at room or cold temperature; __ (developed 3-6 months after birth)
- cause rapid intravascular hemolysis
ABO GROUPING:
* Most frequently performed test in the blood bank
* Both __ and __ grouping must be performed.
1. Forward Typing (aka ____,____ )
- Using known sources of commercial ___ (anti-A, anti-B) to detect antigens on an individual’s RBCs
Characteristics of a typing serum
1. __: react to its corresponding ag
2. __: dec chance of dissociation of ag-ab
3. __: zone of equivalence
IgM
forward and backward
Direct/Red Cell Typing
antisera
specificity
inc avidity
specific agglutination titer
ABO GROUPING:
2. Backward Typing (aka ___, ___)
- detecting ABO __ in the patient’s serum by using known reagent ___.
- Reagent: 4-5% red cell suspension
Blood Group; Patient’s Serum + A Cells; Patient’s Serum + B Cells
A =
B =
AB =
O =
* Not required in:
* In infant less than ___ old
* For confirmation testing of labeled previously typed donor cells
Take Note: As a general rule, always drop ___ first, then __ second to make sure you added both a source of antibody and antigen
Reverse Typing/Indirect/Serum/Plasma Typing)
antibodies; RBCs
0 = 4+
4+ = 0
0 = 0
4+ = 4+
4 months
clear solutions; RBC
ABO Lectins Other Blood Group Lectins (mnemonic)
* A –
* B -
* O –
- Arachis hypogaea (anti-T)
- Glycine max (anti T, anti-Tn)
- Vicia graminea (anti-N)
- Salvia horminum (anti-Tn/Cad)
- Salvia sclarea (anti-Tn)
Dolichos biflorus (A1 vs A2) (ADoBo)
Griffonia Simplicifolia (Bandeirea simplicifolia) (GiniSang Baboy)
Ulex europaeus (O vs Oh) (Oh, UbE)
__:
Glucosyltransferase: N-acetylgalactosaminyltransferase (A-transferase)
Immunodominant Sugar: N-acetylgalactosamine
__:
Glucosyltransferase: Galactosyltransferase (B-transferase)
Immunodominant Sugar: D-galactose
__:
Glucosyltransferase: a-2-L-Fucosyltransferase
Immunodominant Sugar: L-fucose
Take Note:
- These genes do not actually code for the production of antigens but rather produce specific glycosyltransferases that add sugars to a basic precursor substance.
- ____ -sugars that occupy the terminal positions of this precursor chain and confer blood group specificity
Blood Group A
Blood Group B
Blood Group H (precursor to A and B)
immunodominant suagrs
Precursor Substance:
___: terminal galactose on the precursor substance is attached to the N-acetylglucosamine in a beta 1 → 3 linkage; found in secretion
___: terminal galactose on the precursor substance is attached to the N-acetylglucosamine in a beta 1 → 4 linkage; found in RC
Location:
o The presence or absence of the ABH antigens on the __ membrane is controlled by the __
o The presence or absence of the ABH antigens in __ is indirectly controlled by the __
Note: ____ refers only to secretion of A, B, and H soluble antigens in body fluids
Type 1
Type 2
red blood cell; H gene
secretions; Se gene
AB secretor
Antigen Present: A1, A
Anti-A (Anti-A plus Anti-A1): +
Anti-A1Lectin: +
Antigen Present: A
Anti-A (Anti-A plus Anti-A1): +
Anti-A1Lectin: -
~ ___: show increased reactivity with anti-H lectin compared to A1 RBCs
A1 (80%)
A2 (20%)
A2 RBCs
Weak A Subgroup Characteristics:
o Decreased number of __ sites per RBC
o Varying degrees of agglutination by human anti-A,B8
o Increased variability in the detectability of H antigen, resulting in strong reactions with ___
o Presence or absence ___ of in the serum
o ____, and molecular testing can be utilized to subdivide A individuals into A3, Ax, Aend, etc
order of H ag from most to least?
A antigen
anti-H?
Anti-A1
Secretors studies, adsorption-elution tests
O>A2>B>A2B>A>A1B
(Oh ito Bhie, itoBa Ang nA1wan?)
H DEFICIENT PHENOTYPE:
* first reported by ___ in 1952 in ____
* lacks normal expression of the ___
* Typing of Bombay Phenotype __
O vs BOMBAY:
- O: 4+ in anti-H; 0 in O cell
- Oh: 0 in anti-H; 4+ in O cell
Bhende, Bombay India
ABH antigens
Oh
CLASSIFICATION OF BOMBAYS:
GENES: hh/sese
GLYCOSYLTRANSFERASE: none
RBC Ag PRESENT: none
PRESENCE OF ABH SUBSTANCE: none
ANTIBODIES PRESENT: Anti-A, anti-B, anti-H
GENES: hh/Se
GLYCOSYLTRANSFERASE: A &/or B transferase
RBC Ag PRESENT: Weak to none
PRESENCE OF ABH SUBSTANCE: yes
ANTIBODIES PRESENT: Weak anti-H, anti-A/B
GENES: Weak variant hh/se
GLYCOSYLTRANSFERASE: A &/or B transferase
RBC Ag PRESENT: weak
PRESENCE OF ABH SUBSTANCE: none
ANTIBODIES PRESENT: Anti-H, anti-A/B
____: Substances-group specific substances from other Species
a. A substance- ___
b. B substance- ___
Classic Bombay
Para Bombay
H-partially def
Witebsky
hog stomach
horse stomach
ABO DISCREPANCIES
* Discrepancy between ___ and ___
* When a discrepancy is encountered, results must be recorded, but interpretation of the ABO type must be delayed until the discrepancy is resolved
forward and reverse
GROUP 1: ABO DISCREPANCY
Unexpected ___ reactions in due to ____ weakly reacting or missing antibodies
EX:
* Newborns
* Elderly patients
* Hypogammaglobulinemia
* Agammaglobulinemia
* Patients whose existing ABO antibodies may have been diluted by
plasma transfusion or exchange transfusion
Note: Rare Group 1: ___ - Presence of two cell population in a single individual
missing; reverse grouping
chimerism
GROUP 2: ABO DISCREPANCY (least encountered)
Unexpected ____ reactions in the ____ due to weakly reacting or missing antigen
* Subgroups of A(B) may be present
* Leukemias may yield weakened A or B antigen
* “______” phenomenon will show weak reactions with anti-B antisera
* BGSS
Note: Acquired B Phenomenon
* Anti-B Clone ES4 (+) AB (-) TB
* Anti-B (pH >8.5 or <6.0) (-) AB (+) TB
* Treating RBCs with ____ re-acetylates the surface molecules,
then markedly decreases the reactivity of the cells tested with
anti-B.
missing; forward grouping
Acquired B
acetic anhydride
GROUP 3: ABO DISCREPANCY
Discrepancies between forward and reverse groupings are
caused by ____ and result in rouleaux formation or __ (add NSS!)
* Elevated levels of globulin from diseases
* Multiple myeloma
* Waldenstrom’s macroglobulinemia (WM)
* Moderately advance cases of Hodgkins lymphoma
* Elevated levels of fibrinogen
* Plasma expanders (such as dextran and polyvinylpyrrolidone)
* ___ - found in cord blood cells
protein or plasma; pseudoagglutination
Wharton’s jelly
GROUP 4: ABO DISCREPANCY
Discrepancies between forward and reverse grouping due to _____ problems
* Cold reactive autoantibodies
* More than one ABO group due to RBC transfusion or BMT
* Unexpected alloantiodies
* Unexpected ABO isoagglutinins
Note: Rare Group 4
___ -refers to the inheritance of both AB genes from one parent carried on one chromosome and an O gene inherited from the other parent
~The unexpected reaction can be due to an extra positive reaction or a weak or missing reaction in the forward and reverse grouping.
- When a discrepancy is encountered, results must be recorded, but interpretation of the ABO type must be delayed until the discrepancy is resolved ___!
miscellaneous
Cis-AB
transfuse O
RH BLOOD GROUP: (ISBT?)
HISTORY OF RH
Ø ____: described a hemolytic transfusion reaction in an obstetrical patient; Type O father donated blood to Type O mother (delivered a still born infant) → acute hemolytic transfusion reaction (AHTR).
Ø ____: Guinea pigs and rabbits + Rhesus macaque monkeys’ blood → (anti Rhesus) antibody agglutinated 85% human RBCs
most to least immunogenic DCE?
004
Levine and Stetson
Landsteiner-Weiner
D>c>E>C>e
Rh SYSTEM NOMENCLATURE:
FISHER-RACE (CDE); WIENER (Rh-Hr); ROSENFIELD (Numerical); ISBT
D; __; 004001
C; __; 004002
E; __; 004003
c; __; 004004
e; __; 004005
Take Note:
* Placing ____ around (D), (C), and (e) indicates weakened antigen expression
* ____: agglutinogen is described by a letter and symbol assigned based on the factors present
*
WIENER:ROSENFIELD
Rh0; Rh1
rh’; Rh2
rh”; Rh3
hr’; Rh4
hr”; Rh5
parenthesis
Modified Wiener Nomenclature
Modified Wiener Nomenclature:
presence of the D antigen
absence of D antigen
C antigen
c antigen
E antigen
e antigen
When both D, C and E are present
if C and E are present ONLY
Wiener; Fisher?
R1:
r’:
R0:
R
r
1 or ‘
no 1 or ‘
2 or “
no 2 or “
Rz
rz?
DCe
Ce
Dce
Rosenfield Nomenclature
o System that assigned a ___ to each antigen of the Rh system in order of its discovery or recognize relationship to the Rh system
o Demonstrates the presence or absence of the antigen on the RBC
o A ___ preceding a number designates the absence of the antigen
o If an antigen has not been phenotyped, its number will not appear in the sequence
Fisher-Race Rosenfield?
Dce/DCe:
dCE/dCe:
International Society of Blood Transfusion Committee (ISBT): Updated Numeric Terminology
o establish a ___ that is both eye- and machine-readable and is in keeping with the genetic basis of blood groups
o adopted a ___ for each authenticated antigen belonging to a blood group system
number
minus.
1, 2, -3, 4, 5
-1, 2, 3, -4, 5
uniform nomenclature
six-digit number
TIPPET GENETIC MODEL:
RH GENETICS MOLECULAR STUDIES
* Chromosome 1
* Rh Genes
Dce/ce; Ce/Ce; D- (exalted D); -/- (Rh null)
* RHD gene / x / x
* RHCE gene / / x x
___:
* gene important to ___ expression
* termed a coexpressor and must be present for successful expression of the Rh antigens
RH-ASSOCIATED GLYCOPROTEIN (RHAG)
Rh antigen
___
* Amino acid position 103 is important in determining C or c
* Position 226 differentiates E from e
RH-BIOCHEMISTRY
WEAK D: VARIATIONS OF D ANTIGEN EXPRESSION:
* variations in the quantity of D antigen or the specificity of D antigen epitopes
Three Categories:
* ___: C trans to D (Dce/dCe); The Rh antigen on the RBC is normal, but the steric arrangement of the C antigen in relationship to the D antigen appears to interfere with the ___.
* ___: inheritance of RHD genes that code for a weakened expressiong of the D antigen; antigens expressed appear to be complete but fewer in number; __ extremely low number of D antigen sites that most reagent anti-D are unable to detect
* ___: Wiener and Unger postulated that the D antigen is made of antigenic subparts, genetically determined, that could be absent in rare instances; Individual lack of ____, of the total D antigen, alloantibody can be made to the missing epitope(s) if
exposed to RBCs that possess the complete D antigen
Position Effect; expression of D antigen.
Quantitative
Del
Partial-D antigen
1 or more pieces/epitopes
___: Reacts best at 37C; Immune antibodies; do not bind complement; Shows dosage!!!!
* Clinical Considerations
- HTR
- HDFN
Laboratory Procedure: Rh Typing
A. ___: Rh View Box with a temperature of 45C
B. ___: Incubated at 37C
RH DEFICIENCY SYNDROME:
___ * fail to express any Rh antigens on the RBC surface; Genotype: Can result from either the regulator type (mutation in the RHAG gene) or the amorphic type
___ * severely reduced expression of all Rh antigens
RH ANTIBODIES
Slide test
Tube test
Rh Null/somatocytes
Rh mod
RARE/ UNUSUAL PHENOTYPES
* Cw; f (ce); rhi (Ce)
* G - antigen present on most D-positive and all C-positive RBCs
* exalted D (D-)
____:
Ø the destruction of the red blood cells (RBCs) of a fetus and neonate by antibodies produced by the mother
Ø Mother can be stimulated to form RBC antibodies naturally (ABO), by previous pregnancy, or transfusion (RBC alloimmunization).
3 IMPORTANT FACTORS FOR HDFN TO OCCUR:
1) The red cell antibody produced by the mother must be of the __
2) The fetus must possess an antigen that is ____
3) The antigen must be ___ at birth
HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN (HDFN)
IgG class
lacking in the mother
well/fully developed at birth (N is 2-4 of age is fully developed)
PATHOGENESIS
*___ : occurs when maternal IgG attaches to specific antigens of the fetal RBCs
*___ : Destruction of red cells
*___ : Increased production of red cells (liver helps but is destroyed leading to hydrops fetalis)
Take Note:
- ___ (caused by decreased hepatic production of plasma proteins) lead to the development of high-output cardiac failure with generalized edema, effusions, and ascites, a condition known as hydrops fetalis
- The antibody-coated cells are removed from the circulation by the macrophages of the ___.
- When the bone marrow fails to produce enough RBCs to keep up with the rate of RBC destruction, erythropoiesis outside the bone marrow is increased in the hematopoietic tissues of the __. These organs become enlarged (hepatosplenomegaly), resulting in portal hypertension and hepatocellular damage.
- RBC destruction releases hemoglobin, which is metabolized to bilirubin. This bilirubin is called indirect because indirect methods are required to measure the bilirubin in the laboratory. - if left untreated can cause __ or permanent damage to parts of the brain.
hemolysis
anemia
erythropoiesis
Severe anemia and hypoproteinemia
spleen
spleen and liver
kernicterus
DIAGNOSIS AND MANAGEMENT OF HDFN
Ø Serologic testing of the mother: ABO Typing, Rh Typing, Antibody Screening and Identification, Antibody Titration
____ : highly specialized prenatal test in which a sample of the baby’s blood is removed from the umbilical cord for testing.
____ : collection of amniotic fluid for testing fetal lung maturity
____ : is performed by accessing the fetal umbilical vein (cordocentesis) and injecting donor RBCs directly into the vein. (to correct anemia and maintain Hgb >10g/dL); Most centers treating HDFN use group O RBCs for intrauterine and neonatal transfusions.
~ the blood should be __ old to maximize the 2,3 DPG & prevent hyperkalemia; must be O Rh (-) blood; must be antigen negative
____ : use of whole blood or equivalent to replace the neonate’s circulating blood
____ : competes with the mother’s antibodies for the FC receptors on the macrophages in the infant’s spleen
Cordocentesis
Amniocentesis
Intrauterine transfusion; 7 days old
exchange transfusion
Intravenouc Immune Globulin
___: applicable only to Rh; prevent immunization to D antigen
o Mechanism of Action: attaches to the fetal Rh-positive RBCs in the maternal circulation
o Given in: __ week of gestation, __ after birth
o Regular-dose vial: sufficient anti-D to protect against __ of packed RBCs or __ of whole blood.
o Screening test: Rosette tesing (qualitative)
o Quantitative Test: ___
§ Ghost Cell: mother’s cell (HgA1 susceptible)
§ Intact Cell: infant cell (HgF resistant to acid)
§ Formula:
%FRC: #FRC/2000 cells x 100% (20 OIO field: 2000 cells)
FMH: %PRC x 50
§ Indicated in:
* Rh (-) without anti-D
* Rh(-) with complicated pregnancy
* Rh(-) with Rh (+) baby
Rhogam
28th; 72 hours
15mL; 30mL
Kleihauer-Betke Acid
OTHER MAJOR BLOOD GROUP SYSTEM; __ blood grps system; __ blood grp antigen
- Blood group antigens are defined by __ (sugars) attached to glycoprotein or glycolipid structures or by amino acids on a protein
45; 357
carbohydrates
Take Note:
* Serologic tests determine only RBC phenotype (not genotype)
* A genotype is composed of the actual genes that an individual has inherited and can be determined only by family or DNA studies
* International Society of Blood Transfusion (ISBT)- Working Party on Terminology for Red Cell Surface Antigens
* To facilitate computer storage and retrieval of blood group information and to help standardize blood group system and antigen names
* Six-digit Identification number
- ___ digits represent the system, collection, or series
- ___ digits identify the antigen
* ___ - consists of one or more antigens controlled at a single gene locus, or by two or more very closely linked homologous genes with little or no observable recombination between them
* ___ - are antigens that have a biochemical, serologic, or genetic relationship but do not meet the criteria for a system
* Remaining RBC antigens that are not associated with a system or a collection are catalogued into the of low prevalence antigens or the ___ of high-prevalence antigens
first three
last three
system
collections
901 series
- ___: not intrinsic to to RBCs (also Chido/Rogers)
o Type 1 glycosphingolipids – adsorbed in the RBC membrane (uptake of ag/ab by cell)
o 2 antigens: __
o Lewis gene: ___
Phenotypes:
- ____: found in cord blood, cancer and pregnant women
- ____: present in nonsecretors
- ____: present in secretors
Lewis antibodies:
- Naturally occurring __; Generally, __class; Neutralizable by the __
o Take Note:
* Leab - associated with receptor of __ and __ (causes gastroenteritis in adutls)
* Lex – marker for ___ cell - owl’s eye
* __ – increase renal graft and heart disease; increased susceptibility to infections by Candida and Uropathogenic E. coli!!
___:
* reacts best when both the Leb and the H antigens are present
* recognizes any Leb antigen regardless of the ABO type
Lewis System/Plasma Antigens
Lea and Leb
Chromosome 19
Le (a-b-)
Le (a+b-)
Le (a-b+)
[ Le(a-b-)]; IgM; plasma
H. pylori; Norwalk virus
Reed Sternberg
Le (a-b-)
- P1PK(003), GLOBOSIDE (028) and Related Antigens
1.P1PK (003, P1PK): ____
2.Globoside (028, GLOB): P, Px2
3.Globoside Collection (209): LKE
Note: ___ - poorly expressed at birth and may take up to 7 years to be fully expressed
P1, Pk, NOR
P1 antigen
Antibodies of the P Blood Group:
- naturally occurring IgM (P1-); found in patient with: (both has P1-like ag)
§ Echinococcus granulosus
§ Fasciola (cephalic cone)
- first described in the serum of Mrs. Jay, a p individual with adenocarcinoma of the stomach; Increased incidence of spontaneous abortion (SLE); IgG or IgM
- associated with habitual early abortion cold-reactive IgG autoantibody; PCH; Donath-Landsteiner Antibody Test!!!;
Tube 1: 37C
Tube 2: incubated at cold temperature, then warmed at 37C
(if cold->hot=lysis; RT->hot=no lysis)
Take Note:
o ___: Phenotypically related to the P Blood group
o a marker of ____ and ____ Stem Cells
Disease Association
o P antigen: Receptor for ___
o Pk antigen: Receptor for ___ toxin and E.coli – associated hemolytic uremic syndrome (HUS)
o provides some protection against HIV infection of peripheral blood mononuclear cells
o marker of apoptosis in germinal center B cells, Burkitt lymphoma, and lymphoblastic leukemia
Anti-P1
Anti-PP1Pk
Autoanti-P/DL ab (Donath-Landsteiner)\biphasic hemolysin (react to cold and hot)
Luke (LKE) antigen
embyonic and mesenchymal
Parvovirus B19
Shiga
I (027) System
o “Individuality”
o Normal transition: ___
o I (___, Symbol I)
o i (___, Symbol I)
o High prevalence antigens
o Antibodies
___:
- Cold Hemagglutinin Disease
___:
- Infectious Mononucleosis
Take Note:
- At birth, infant RBCs are rich in ___; I is almost undetectable. During the first ___ of life, the quantity of i slowly decreases as I increases until adult proportions are reached; adult RBCs are rich in __ and have only trace amounts of i antigen.
- Increased antigen is seen in acute leukemia, hypoplastic anemia, megaloblastic anemia, sideroblastic anemia, thalassemia, sickle cell disease, paroxysmal nocturnal hemoglobinuria (PNH), and chronic hemolytic anemia, Chronic dyserythropoietic anemia type II or hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS)
i-I
027001
207002
Anti-I
Anti-i
i; 18 months; I
- MNS (002) System
- Glycoprotein, found only on __
M: ___, ser, thr, thr, __!!
N: ___, ser, thr, thr, __!!
~ are destroyed by ___ and ___ (elution)
___: is located on GPB bet amino acids 33 and 39
___: naturally occurring, IgM (IgG in Harmening); reacts best at 6.5 (acidic)
___: “f - formaldehyde”; seen in renal patients; IgM
Take Note:
- MkMk is the null phenotype in the MNS system
- ____: serve as the receptor of pyelonephritogenic strains of E. coli
- ____: receptor of Plasmodium falciparum
RBC
SER; GLY
LEU; GLU
enzymes & ZZAP
GPA^M
M & N
Anti-N^f
U antigen
Anti-M
- Kell (006) and Kx(019) - only found in RBCs
- first blood group system discovered after the introduction of ___.
- identified in the serum of ___; _ is common, k is rare
Take Note:
- Antigens: K, k, Kpa, Kpb, Jsa, Jsb
- Kell antigen expression is dependent upon the presence of the ____.
- Kx is the only antigen in the Kx system
____ -Null Phenotype
o ____: Most common antibody found; E. coli O125:B15: bacterial organism associated with anti-K; IgG (can cause HDFN)
Note: Mcleod Phenotype!!
ü Absence of Kx, Km
ü Marked depression of ALL other Kell Antigens
ü Associated to CGD
ü Male
antiglobulin testing
Mrs. Kelleher; K; k
Kx protein
Ko
Anti-K
- ___
o Antigens: Fya and Fyb
o Destroyed by ___
o Member of the superfamily of chemokine receptors (Duffy antigen receptor for chemokines)
o ___ – resistant from Plasmodium vivax and Plasmodium knowlesi
o Function: chemokine receptor
~ Anti-Fya and Fyb - associated with __
~ IgG- demonstrates dosage
Take Note:
ü The amino acid at position __ on the Duffy glycoprotein defines the Fya and Fyb polymorphism: Fya has glycine, and Fyb has __
Duffy (008) System
enzymes
Fy null
HDFN
42; aspartic acid
- ___
o Antigens: Jka, Jkb, Jk3
o function: urea transport
o Anti-Jka and Anti-Jkb: not easily detected
Take note: ___ has been associated with severe immediate and ___ and with mild HDFN
Kidd (009) System
Anti-Jk3; delayed HTRs
- Lutheran (005) System
o found in the serum of a patient with ___
o Lutheran for the donor; the donor’s last name was Lutteran but the donor’s blood sample was incorrectly labeled
o Antigens: Lua Lub
o Affected by ___
o Antibody: Anti Lua (IgG, IgM, IgA); Anti-Lub (IgG, immune antibody)
lupus erythematosus
enzyme
- ___
* Antigens: Dia/Dib, Wra/Wrb, and Wu/DISK
* Band 3: Red Cell Anion Exchanger (AE1) and Solute Carrier Family-4. anion exchanger, member 1 (SLC4A1 SLC4A1)
* __ = marker of Mongoloid ancestry
* Diego antibodies: Usually ___
Diego (10) System
Dia
IgG!!!
- ___
* Yta :high-prevalence antigen in all populations
* Ytb : low-prevalence antigen
* Not found in Japanese
* amino acid substitution on the glycosylphosphatidylinositol (GPI)linked RBC glycoprotein acetylcholinesterase (AChE)
* Antibodies: usually IgG (cant cause HDFN)
Yt (011) System
- ___
* X chromosome and g for “Grand Rapids,” where the patient was treated
* Controlled by X – linked gene
* Antigen has a higher prevalence in females than in males
* Two antigens: Xga and CD99 (12E7 and MIC2)
Xg (012) System
- ___
* High prevalence antigen: Sm (Sc1)
* Low prevalence: Bua (Sc2)
* SC gene =chromosome 1 at 1p34.
* Product of the gene : Erythroid Membrane Associated Protein (ERMAP)
* Phenotype Sc:-1,-2
* Anti – Sc3 - Dombrock (014) System
* Dombrock antigens : carried on a mono-ADP ribosyltransferase 4 (ART4) attached to the RBC membrane by a GPI anchor.
* The Doa and Dob antigens :poor immunogens
* Gya: highly immunogenic.
Scianna (013) System
- ___
* The high- and low-prevalence antithetical antigens are Coa and Cob, respectively
* Co3
* Co(a-b-)
* The Colton antigens :carried on an integral membrane protein, aquaporin 1 (AQP1)
Colton (015) System
- ___
ü The structure that carries the LW antigens is a glycoprotein known as intracellular adhesion molecule 4 (ICAM-4), a member of the immunoglobulin superfamily.
ü Autoanti-LW is also common in serum from patients with warm autoimmune hemolytic anemia
Landsteiner-Wiener (016) System
- ___
* Not intrinsic to the RBC membrane
* C4 Component
* C4b- Ch antigens
* C4a- Rg Antignes
* Adsorbed
* Antigens: Ch1 – Ch6, Rg1-Rg2
* Antibodies: Neutralized by the plasma
Chido/Rodgers (017) System
- ___
ü was named in 1960 after Mrs. __
ü The antigens are carried on sialoglycoprotein structures GPC and GPD
ü The glycoproteins help to maintain the RBC membrane integrity through interaction with protein band 4.1,
Gerbich (020) System; Gerbich
- ___
* Antigens are carried by the DAF (CD55)
* DAF is strongly expressed on placental tissue and will adsorb Cromer antibodies
Cromer (021) System
- ___
* Antigens are located on Complement Receptor 1 (CR1)
* “Helgeson phenotype” represents the serologic null phenotype for the Knops blood group
Knops(022) System
- __
* Antigens: Ina - 4% of Indians, 11% of Iranians, and 12% of Arabs
* Located in CD44
Indian (023) System
- ___
* Named after the antibody maker, Mrs. Kobutso
* Carried in CD147 - ___
* Only antigen: MER2
* Antigen name is derived from monoclonal, and Eleanor Roosevelt - John Milton Hagen (026) System
- The Gill (029) System: ound on the glycerol transporter aquaporin 3 (AQP3)
- RH-Associated Glycoprotein (030) System
Ok (024) System
Raph (025) System
- ___
§ Forssman glycosphingolipid was first thought to be a subgroup of A called Apae
§ in honor of John Forssman
§ Antigen: FORS1
§ receptor for pathogens such as Escherichia coli
The FORS (031)
- ___§ Jra is a high-prevalence antigen in most populations; the Jr(a–) phenotype is found more commonly in Japanese
§ Associated with ABCG2
§ member of the adenosine triphosphate (ATP) binding cassette transporters
§ involved in multidrug resistance in tumor cells,
§ named for the antibody maker Rose Jacobs
The JR (032) System
- ___
§ named in 1961 after the first antigen-negative proband, Langereis
§ Associated with ABCB6
o Functions in heme synthesis with the ATP-dependent uptake of heme and porphyrins into mitochondria
The LAN (033) System
- The Vel (034) System: absence of SMIM1, a single-pass integral membrane protein
- The CD59 (035) System
§ based on a CD59-deficient child who formed an alloantibody with CD59 specificity
§ complement-regulatory glycoprotein also known as the membrane inhibitor of lysis (MIRL) - ___: first described in 1967 in the serum of a black woman named Mrs. Augustine
- ___:
§ located on the prion protein (PrPC) classically associated with Creutzfeldt-Jakob disease (CJD)
The Augustine (036) System
Kanno (037)
- SID (038)
* Sda; Found in _________
* named for Sid, who was the head of the maintenance department at the Lister Institute in London
* The soluble form of Sda is _____ glycoprotein found in urine. - CHOLINE TRANSPORTER-LIKE PROTEIN 2 (CTL2/SLC44A2) (039)
* widely expressed on human tissues, including neutrophils, lymphocytes, and platelets
* The human neutrophil antigen HNA3 is located on CTL2:
* Alloantibodies to HNA3a have been associated with several cases of transfusion-related acute lung injury (TRALI).
35.PEL (040) - MAM (041)
37.EMM (042) - ABCC1 (043)
- ER (044)
saliva, urine and meconium
Tamm-Horsfall
Miscellaneous Antigen
*__________: Receptor of Haemophilus influenza to enter RBCs.
* HLA Antigens on RBCs; HLA class I antigens that are detectable on mature RBCs; Bg (___)
* Bga: HLA-B7
* Bgb: HLA-B17
* Bgc: HLA-A28
AnWj antigen
Bennett-Goodspeed
(maling mahiwalay/nuisance RBCs)
* exhibit reactivity at high dilutions of serum, but the strength of agglutination is weak at any dilution
* Not clinically significant, but serological reactions make them look like they are
* High Titer if the antibodies are titered. The titers are usually at least 1:64 and often will be over 1:1000
* Reactions are very weak and will break apart very readily due to the weak attraction between the antigens and antibodies (low avidity).
High – Titer Low Avidity HTLAs
DETECTION AND IDENFITICATION OF ANTIBODIES:
Clinically Significant Alloantibodies
* cause decreased survival of RBCs
* typically, IgG antibodies that react at ___
Antibody screen:
* Detect any potentially clinically significant in a donor’s or recipient’s sample
* Involves the reaction between patient serum with __ phenotyped for multiple antigens.
Reagent RBC: cell with: C, c, D, E, e, Fya, Fyb, Jka, Jkb, K, k, Lea, Leb, P1, M, N, S, and s antigens
Purpose:
* To detect as many clinically significant antibodies as possible
* To detect as few clinically insignificant antibodies as possible
* To complete the procedure in a timely manner
STUDY ANTIGRAM!!!!
37C
2 or 3 reagent
Interpretations:
* _______: specificity should be suspected when all screen cells yielding a positive reaction react at the same phase(s) and strength
* _______: are most likely present when screen cells react at different phases or strengths.
* _______: should be suspected when the autologous control or DAT is positive and all screen cells tested yield a positive reaction
single antibody
multiple antibodies
autoantibodies
Note: Antibodies associated with:
_________:
ü anti-Lea
ü anti-Leb
ü anti-PP1Pk
ü anti-Vel
_________:
ü anti-Sda
hemolysis (in-vitro)
mixed field (Mf)
Antibody identification:
* ______: of an antibody to red cell antigen(s) requires testing the serum against a panel of selected red cell
samples with known antigen composition.
* Antibody Identification Panel
* collection of _______ group O reagent RBCs
* antigen expression should be diverse.
* Steps
____: Cross out panel cells with “0” reaction; However, Do not cross out heterozygous antigens that show dosage!!
____:Look for matching patter (cells with reaction)
____:Consider the antibody’s usual temperature reactivity
Note: Rule of Three The “_____ ”must be met
Patient serum MUST be:
◦ Positive with ____ cells with the antigen
◦ Negative with ____ cells without the antigen
identification
11 to 20
step 1
step 2
step 3
3 and 3
3
3
Additional Techniques for Resolving Antibody Identification
1. ___:
* The cells selected for testing should have minimal overlap in the antigens they possess
2. ___:
* modify the RBC surface by removing sialic acid residues and by denaturing or removing glycoproteins
* results in the destruction of certain antigens and the enhanced expression of others
3. ___:
* used to neutralize the corresponding RBC antibodies in serum
4. ___:
* Process of removing antibody from the serum
* Adsorbent: RBC or another antigen-bearing substance
- Human platelet concentrate - adsorb Bg-like antibodies
- Rabbit erythrocyte stroma (RESt) – Cold-reacting autoantibodies
5. ___:
* Process of quantifying the amount or concentration of antibody
* Titer level: the reciprocal of the greatest dilution in which agglutination of 1+ or greater is observed
Selected Panel
Enzymes
Neutralization
Adsoption (Absorption)
Antibody Titration
BLOOD DONATION
___: hospital based, performs compatibility testing & prepares blood components
___: screens donors, draws donors, performstesting on donor blood, & delivers appropriate components to hospital BB
___: to collect blood from donor
blood bank
blood center
Blood Collection Unit
A. DONORS
* Types
Autologous - “self”, “safer” higher cost (unused must be discarded, not used to others)
___: 5 to 6 weeks preceding a scheduled surgery; No later than 72 hours before the scheduled surgery
___: collection of whole blood with the concurrent infusion of crystalloid or colloid solutions
___: collecting shed blood from the surgical site, processing the blood, reinfusing the blood
___: collected from a drainage tube placed at the surgical site
___: - blood for others
§ Voluntary Non-Remunerated Donors
§ Directed Donors
§ Paid Donors
Take Note:
* The tag for the directed unit is a distinct color (e.g., __, __)
* If the donor is a blood relative, the unit must be ___ to prevent graft versus host disease so that viable T cells from the donor that enter the patient’s circulation do not mount an attack against patient’s cells and tissue.
Pre-operative
Acute Normovolemic Hemodilution
Intraoperative Collection
Post Operative Salvage
Allogeneic
yellow, salmon
irradiated
B. DONOR SCREENING
* Components
1. ___: Documenting information that fully identifies the donor on an individual donation
2. ___: Essential to ensure the protection of the donor and benefit to the recipient.
3. Physical Exam: Autologous Donor? Allogeneic Donor?
1. Age: no age req; at least 16 yrs old
2. Weight: no weight req; ___
3. Temperature: __
4. Pulse Rate: __
5. Blood pressure: 90-100 systolic, diastolic: ___
6. Hemoglobin: ___; male and female ____
7. Hematocrit: __; male and female __
Take Note:
Hazardous Occupations
- Shall normally entail an interval of not ____ hours between donation and returning to the occupation or hobby
~RA ____: Data Privacy Act of 2012 - protect vital information of donor
- ___:
- This implies the willingness to donate blood given by a mentally competent person who “ received the necessary information, who has adequately understood the information
Donor Registration
Medical History
110/50kg
37.5C/99.5F
50-100bpm
50-100mmHg
11.0g/dL; male 13; female: 12.5
33%; male 39%; female 38%
less than 12 hrs
10173
Informed Consent
Types of deferrals:
___: Donor is unable to donate blood for a limited period of time
___: Donor will never be eligible to donate blood for someone else
___: Donor is unable to donate blood for someone else for an unspecified period of time due to current regulatory requirement
Temporary Deferral
Permanent Deferral
Indefinite Deferral
Deferrals:
Permanent/ Indefinite Deferral (AABB)
Ø History of ___ after eleventh birthday
Ø Confirmed positive test for hepatitis B surface antigen
Ø Reactive test to antibodies to hepatitis B core on more than one occasion
Ø Present or past clinical or laboratory evidence of infection with hepatitis C virus, human T-cell lymphotropic virus, or HIV
Ø History of __
Ø Family history of ___
Ø Recipient of dura mater or human pituitary growth hormone
Ø Risk of vCJD
Ø Use of a ___ to administer nonprescription drugs
viral hepatitis
babesiosis or Chagas disease
CJD
needle
Deferral Periodsfor Potential Transfusion-Transmitted Infections (AABB):
Malaria
* History of Malaria - __
* Lived in endemic area for 5 consecutive years ___
* Travel to endemic Area ___
Others
* Babesiosis __
* Chagas Disease __
* Leishmaniasis __
3 Years
3 years from departure
1 year from departure
Indefinite deferral
Indefinite deferral
1 year from departure
Medication Deferrals (AABB):
Warfarin (Coumadin) Prevention of blood clots
Aspirin and piroxicam (Feldene) Nonsteroidal anti inflammatory
Hepatitis B immune globulin Exposure to hepatitis B
German Measles Vaccine and Varicella-Zoster Vaccine
Conclusion of Pregnancy
High-risk sexual contact, Tattoos or permanent makeup, Incarceration >72 hrs, therapy of Syphilis,Transfusion of blood components/tissue and rabies vaccine
Take Note:
- bacterial, or rickettsial vaccines such as diphtheria, hepatitis A, hepatitis B, influenza, Lyme disease, pneumococcal polysaccharide, polio injection (Salk), anthrax, cholera, pertussis, plague, paratyphoid, rabies, Rocky Mountain spotted fever, tetanus, or typhoid injection, if the donor is symptom-free and afebrile.
ü First trimester or second trimester __ or __ is not cause for deferral
ü Apheresis donation – __
ü FDA limits platelet apheresis procedures to no more than 24 in a calendar year
ü Apheresis donor may only donate __ in a period of 7 days and only once in 7 days for a double or triple apheresis donation
ü Infrequent plasma apheresis requires a 4-week deferral
ü Double red cell apheresis: 16 weeks
ü Alcohol intake: __
ü Skin lesions: __
ü Skin disorders that are not cause for deferral include poison ivy and other rashes
1 week
2 days after last dose for platelet donors
1 year
4 weeks; (other vaccines are 2 weeks)
6 Weeks Sexual contact
12 months/1 yr
abortion/miscarriage
48 hours
twice
12 hours
indefinite deferral
C. ___
a. A numeric or alphanumeric system is used to link the donor to the donor record, pilot tubes, blood container, and all components made from the original collection
b.The phlebotomist should ask the donor to state or spell his or her name
D. __
a.PVP-iodine or polymer iodine complex
b.Venipuncture site is scrubbed at ___ in all directions
c. Minimum ___
d.To prevent contamination with epidermal cell and bacteria from entering the bag
Ø Use of collection container that divert the first 10-20ml to a “__” (where contamination is present, used for blood testing purp)
DONOR IDENTIFICATION
ASEPTIQUE TECHNIQUE
least 4 cm
30 secs
diversion pouch
E. DONOR’S REACTION
§ Mild - syncope
§ Moderate - dec pulse rate
§ Severe - convulsions
- ___ - a localized collection of blood under the skin, resulting in a bluish discoloration; caused by the needle going through the vein, with subsequent leakage of blood into the tissue.
hematomas
F. __:
* Collection of a specific blood component while returning the
remaining whole blood components back to the patient
Advantages:
o Large volume of blood
o Removal of unwanted substance
Anticoagulant: ___
Methods of Centrifugation
o ___: Blood is processed in batches or cycles
o ___: Blood withdrawal, processing, reinfusion are performed simultaneously in a ongoing manner
o Types of Apheresis:
Plasma
Platelets
Leukocytes
RBC
Hematopoietic progenitor cells (HPC)
APHERESIS
citrate
Intermittent Flow Centrifugation
Continuous Flow Centrifugation
Plasmapheresis
Plateletpheresis
Leukapheresis
Erythrocytapheresis
HPC Apheresis
Take note:
Leukapheresis
◦ Hydroxyethyl Starch (HES) - ___
◦ Corticosteroids (Prednisone or dexamethasone) & Growth Factors increase ____
Double RBC Pheresis - double units of RBCs
Plateletpheresis - single donor plt conc; __ random donor platelets
Plasmapheresis:
o ____: no more than one procedure in a 4-week period
o ____: donate more frequently than 4 weeks but no more than every 48 hours and no
more than two donations in a 7-day period.
- Therapeutic Cytapheresis
1. A pathologenic substance exists in the blood that contributes to a disease process or its symptoms
2. The substance can be more effectively removed by apheresis than by the body’s own homeostatic mechanisms.
sedimenting agent
inc leukocyte harvest
6-8
Infrequent donor
Serial donor
Indication Categories for Therapeutic Apheresis
Category Description
___: Apheresis is standard and acceptable, either as primary therapy or as a first-line adjunct to other initial therapies.
___: Apheresis is generally accepted in a supportive role or as second-line therapy, rather than first-line therapy.
___: Apheresis is not clearly indicated based on insufficient evidence, conflicting results, or inability to document a favorable risk-to-benefit ratio.
___: Apheresis has been demonstrated to lack efficacy or be harmful and should be discouraged in these disorders.
I
II
III
IV
G. ___
* FDA recognizes two acceptable languages for blood component labeling:
a.ABC Codabar
b.ISBT 128
4 QUADRANTS:
upper left: ___
upper right: ___
lower left: ___
lower right: ___
Blood Label (FDA):
Blood Type A
Blood Type B
Blood Type AB
Blood Type O
Hold for further processing
For emergency use only
For Autologous use only
Not for transfusion
Irradiated
Biohazard
From therapeutic phlebotomy
Take note:
ü Once the component has been made, it must be labeled in accordance with AABB Standards, FDA regulations, and ISBT (International Society of Blood Transfusion) Code 128
Labeling
donation & collection of facility identifiers
blood type
product description
expiration and special labels
yellow
pink
white
blue
tan
orange
green
gray
purple
red
chartreuse
H. Testing of Donor Blood
⚫ (3)
1. Syphilis
2. Hepatitis B Surface antigen (HBsAg)
3. Hepatitis B Core antibody (anti-HBc)
4. Hepatitis C virus antibody (anti-HCV)
5. Human Immunodeficiency Virus antibodies (anti-HIV 1/2)
6. Human T-cell lymphotropic virus antibody (anti-HTLV I/II)
7. Human Immunodeficiency virus (HIV-1)
8. Hepatitis C virus
9. West Nile Virus
10. Trypanosoma cruzi antibody
ABO/Rh Typing
Antibody Screen
Screening test for infectious diseases
BLOOD COMPONENT PREPARATION
Transfusion Therapy:
1. Inadequate oxygen-carrying capacity because of anemia or blood loss
2. Insufficient coagulation proteins or platelets to provide adequate hemostasis
Blood Collection and Storage:
___: Collection of blood in a sterile container
___: Collection or exposure to air through an open port that would shorten the expiration because of potential bacterial contamination
Areas Crucial for Normal Erythrocyte Survival and Function:
* Normal chemical composition and structure of the RBC membrane
* Hemoglobin structure and function
* RBC metabolism
close system
open system
Blood Preservation
* Goal: provide viable and functional blood components for patients requiring blood transfusion
* FDA: requires an average 24-hour post-transfusion RBC survival of more than ____
* Types of Anticoagulant - Preservative
STORAGE TIME:
Acid-Citrate-Dextrose (ACD)
Citrate-phosphate-dextrose (CPD)
Citrate-phosphate-double dextrose (CP2D)
Citrate-Phosphate-adenine (CPDA-1 & CPDA-2)
Benefits of RBC Additive Solutions:
1. Extends the shelf-life of RBC to __ by adding nutrients
2. Allows harvesting of more plasma and platelets from the unit
3. Produces an RBC concentrate of ___ that is easier to infuse
Additive Solutions: (4)
75%
21
21
21
35
42 days
lower viscosity
- Adsol/AS-1
- Nutricel/AS-3
- Optisol/AS-5
- SOLX/AS-7
Chemicalsin Anticoagulant Solutions:
___: Chelates calcium
___: Maintains pH during storage
___: Substrate for ATP production
___: Production of ATP
___: Protects against storage-related hemolysis
Rejuvenation Solution:
* regenerate ATP and 2,3-DPG
* Red cells stored in liquid state for fewer than 3 days after their outdate are rejuvenated for 1 to 4
hours at 37C with the solution
* PIGPA: ___
* ___ – only FDA approved rejuvenation solution
Citrate
Monobasic Sodium Phosphate
Dextrose
Adenine
Mannitol
pyruvate, inosine, glucose, phosphate, adenine
Rejuvesol
RBC Storage Lesions:
- A loss of viability and function associated with certain biochemical changes that are initiated when blood is stored in vitro
Characteristics:
Percent (%) Viable cells, Glucose, ATP, pH, 2,3-DPG; change observed ___
Oxygen Dissociation Curve; change observed ___
Plasma Potassium (K), Plasma Hemoglobin, Lactic Acid; change observed ___
__: risk for TTIs is dec
- UV Irradiation and Photosensitizers
- Psoralen Treatment
dec
shift to the left
inc
Pathogen Reduction Technology
____: for patients with anti-leukocyte (FNHTR/TRALI)
* Removal of white blood cells (WBCs) from blood or blood components prior to transfusion
* ___: residual white blood cells per each whole blood, red blood cells, or apheresis platelet
* ___: residual WBCs per each platelet derived from whole blood
* ___: of the original component must be recovered after leukoreduction
Leukoreduction Categories
* ___: Performed shortly after collection
*____: at the patient’s bedside
Filters
* Multiple layers of ___ or ___ nonwoven fibers that trap leukocytes and platelets
* First generation
- Pore size: 170 um
- Removes fibrin clot
* Second generation
- Pore size: __
- Removes micro aggregates
* Third generation
- Removes ___% of WBC
Leukoreduction
<5.0x10^6
<8.3x10^5
>85%
Pre-storage
Post-storage
polyesters; cellulose acetate
20-40um
99.9
___:
* The process of removing the extracellular solution and relacing it with normal saline
* ___ are stored at 1° to 6°C for up to 24 hours
* ___ are stored at 20° to 24°C and must be transfused within 4 hours
* Main Indications for Washing
- allergic reactions
- removal of antibodies
- removal of other substances
Washing
Washed RBCs
Washed platelets
Other methods to achieve RBC leukoreduction: (5)
Quality Control:
___ : designed to count WBCs at exceptionally low levels (manual)
Flow Cytometer (automatic)
___: expiration ___
* indicated to prevent the development of transfusion-associated graft-versus-host disease
* indicated for recipients of components collected from a blood relative or HLA-matched donor
* Minimum dose of gamma irradiation: 25 Gy (central portion), 15 Gy (any part of the blood unit)
* Radioactive source: ___
* Quality Control: Radiographic film label is affixed = darkening of the film
centrifugation, washing, freezing, thawing, deglycerolizing
Nagoette chamber
Irradiation; 28 days expiration
cesium-137 or cobalt-60
___:
* Allow for multiple blood components to be transfused at a single event
* As a rule: multiple units of red blood cells are not typically pooled together, but other product types may be pooled
Reconstituted Whole Blood:
* Only example of pooling two different products together
* Produced to support neonatal exchange transfusions
* Type O rbc+ AB plasma
Pooling
RBC Freezing:
* Cryoprotective agent
§ ___: small molecules that cross the cell membrane into the cytoplasm
§ ___: large molecules that do not enter the cell but instead form a shell around it
Advantage; High Glycerol; Low Glycerol
Initial Freezing Temperature:
Type of Freezer:
Maximum storage temperature:
Shipping Requirements:
- Deglycerolization -24 hrs expiration
§ 1st: ___
§ 2nd: ___
§ 3rd: ___
Note: Donor with Sickle Cell Trait ___ is omitted - Automated Removal of Glycerol (Closed System)
§ EXPIRATION DATE: ___
Penetrating agent
Non-penetrating
-80C; 196C
mechanical; liquid nitrogen
-65C; -120C
dry ice; –
12% saline
1.6%
0.09% saline with 0.2% dextrose
1.6%
14 days at 4C suspended in AS-3
COMPONENT THERAPY:
1. RBC Products
___: Source material for blood component preparation to replace the loss of both RBC and Plasma volume; Loss of 25% of Blood Volume
___: History of reoccurring febrile reactions due to white cell antibodies; Reduction of CMV transmission and HLA alloimmunization
___: a history of plasma protein antibodies, diagnosis of PNH, history of febrile reactions owing to leukoagglutinins
___: reduced exposure to WBC platelets, plasma proteins; Long term storage of “rare” units and/or autologous units
Note:
Adult: 1unit PRBC/WB causes an increase of:
◦ __
◦ __
Pediatrics: 10 to 15ml/mg will increase the hemoglobin about __g/dL or hematocrit by __%
Whole Blood
Leukocyte Poor Red Blood Cells
Washed RBC
Frozen/Thawed/Deglycerolized RBC
1g/dL Hgb
3-5% HCT
2-3; 6-9%
Pros and Cons of RBC Freezing:
Pros
Long term storage
Maintenance of RBC viability and function
Low residual leukocytes and platelets
Removal of significant amounts of plasma proteins
Cons
A time-consuming process
Higher cost of equipment and materials
Storage requirements (-65C)
Higher cost products
COMPONENT PROCESSING FROM WB: ?
1st: WB (soft spin: 3200, 3 mins) PRP and PRBC
2nd: PRP (hard spin: 3600, 5 mins) PPP and Plt conc
PPP->FFP (when frozen)
- Platelet Products
___: To correct thrombocytopenia due to decreased platelet function, decreased platelet production, or increased platelet consumption
___: To correct thrombocytopenia in patients who demonstrates refractoriness to “random” donor platelets (platelet alloantibodies); increase plt by 20,000-60,000/uL
Note:
- Buffy Coat Method
o Whole blood - ___ (leukocyte and platelet rich buffy coat is harvested)
o buffy coat concentrates-pooled together
o Pooled product - ___ (harvesting the platelets)
- Agitation: facilitate ____ transfer into the platelet bag and consumption by the platelets
FORMULA: Corrected Count Increment =
(Absolute platelet Increment/uL x body surface (m2))/number of platelet transfused
Where:
* Absolute platelet Increment (post transfusion platelet count – pretransfusion platelet count)
* Body surface = in sq meters
* Number of platelets transfused:
* 3.0 platelet pheresis
* 0.55 x number of bags
Random Donor Platelets
Single Donor Platelets (plt alloab)
hard spin
soft spin
oxygen
- Plasma Products
___: Contain the maximum levels of both stable and labile clotting factors; Treatment of multiple coagulation factor deficiencies
___: Contains all stable proteins found in FFP normal levels of factor V, and has only slightly reduced levels of factor VIII and Protein C
___: FFP/ PF24 not transfused within the initial 24-hour period.
Treatment of stable clotting factor deficiencies
___: Patients undergoing massive transfusion; Very low levels of Coagulation factors
___: Components: factor VIII, fibrinogen, factor XIII, von Willebrand factor (vWF), cryoglobulin, and fibronectin
___: Contains: residual albumin; factors II, V, VII, IX, X, XI; and ADAMTS13; Treatment for Thrombotic Thrombocytopenia Purpura
Fresh Frozen Plasma
Plasma Frozen within 24 hours (PF24)
Thawed Plasma
Liquid Plasma
Cryo-Precipitated Antihemophilic factor
Cryo-poor plasma (Cryoprecipitate reduced plasma )
- ___
§ Correct severe neutropenia (<500 PMN/ml)
§ Fever unresponsive to antibiotic therapy
§ Myeloid hypoplasia of Bone marrow
§ A typical therapeutic dose:
Blood Derivatives:
* Recombinant DNA Technology or Monoclonal Antibody Purification
* Source Plasma VS Recovered Plasma
* Examples
1. ___
- indicated in patients who are hypovolemic and hypoproteinemic and in clinical settings for shock and burn patients.
2. Plasma Protein Fractions
3. Synthetic Volume Expanders
* Crystalloids (Ringers Lactate and NSS)
* Colloids (Dextran, HES)
Granulocyte Concentrate
Normal Serum Albumin (NSA)
PRE-TRANSFUSION COMPATIBILITY TESTING:
4. Selection of Appropriate Blood
Recipient 1st Choice, 2nd Choice, 3rd Choice, 4th Choice
AB - AB, A, B, O
A - A, O
B - B, O
O - O
- ___
§ Final Check of ABO compatibility between donor and patient
§ May detect the presence of antibodies not detected during antibody identification
§ Classifications
Serologic:- Test patient serum with Donor RBC
- Components: ___
- Possible reasons for incompatibilities: Incorrect ABO grouping of recipients or of donor unit selected; Cold-reactive allo- or autoantibody in the plasma not detected in antibody detection tests; Abnormalities in the recipient’s plasma
Crossmatching
Immediate spin (IgM); anti-human globulin (IgG)
PRE-TRANSFUSION COMPATIBILITY TESTING:
* All steps in the identification & testing of a potential transfusion recipient and donor blood before transfusion in an attempt to provide a blood product that survives in vivo
1. Specimen Collection
* ___ blood samples and donor units is absolutely essential
* The recipient’s blood specimen label should be legible and should include at least the: ____
2. Specimen Requirements
* anticoagulated or clotted specimens are acceptable for pretransfusion testing
* If serum is used, blood samples should be collected in siliconized plain tubes ___
* Unacceptable: ___
* age of the specimen: ___
* Must be retained post-transfusion for at least ___
* ___ is not usually a cause for rejection of a pretransfusion sample
3. Test Performed
* ABO Typing
* Rh Typing
* AB screening and ID
positive identification of patient
patient’s full name, hospital identification number, and specimen collection date
without serum separator gel
intravenous line (IV) contamination, hemolyzed
3 days
7 days
Lipemia
o ___
§ May be used only for patients who havs no currently detected/clinically significant antibodies or any history of alloantibodies.
§ Compares ABO serologic results and interpretation
§ Advantages:
* Increased time efficiency
* Reduced volume of sample needed on large crossmatch orders
* Greater flexibility in staffing
* Potential for a centralized transfusion service
* annual savings
* Reduced sample collection
* Reduced exposure to biohazardous sample
Computer Crossmatch
What if the Unit is Unused?
* Unit closure has to remain __
* Storage temperature must have remained in the required range (___)
* If not at correct temp, unit must be returned within ___ of issue
___:
* Less than 7 days old
* CMV negative or leukocyte reduced
* O-negative
* Hemoglobin S negative
* Dose: 10ml/Kg over 2 to 3 hours
___:
* Reduces the possibility of transfusion reaction or transmission of infectious disease
___:
* Transfusion of RBC Components prior to completion of pretransfusion testing; ABO specific blood; O Rh negative
___:
* Platelets, Thawed Plasma, Cryoprecipitate
* Compatibility Test: not required
Take Note:
* In Vivo Compatibility of Blood: Chromium (51 Cr) and Technetium (99mTc)
* Starting the IV: NSS
* Diluting RBC: NSS, 5% Albumin, ABO compatible plasma
* Speed of Infusion:
o One unit of blood should be transfused ___
o Platelet concentrates/ FFP should be transfused within __
unopened
1C to 10C for RBCs
30 mins
Neonatal Transfusion
Autologous TRANSFUSION
Emergency transfusion
Plasma Products Transfusion
within 4 hours
20 minutes
___:
* Reaction period varies from 1-2 hours
* Main cause is INCOMPATILE BLOOD
___:
* acute complication of transfusion presenting with at least a 1°C increase in body temperature
* 2 mechanisms
* Immune – mediated
* Platelet storage changes
___:
* occurs as a response of recipient antibodies to an allergen present in the blood component
* The more common, milder reactions consist of weals, hives, erythema, or pruritus. Severe reactions (anaphylactoid or
anaphylactic)
___:
* With respiratory distress and severe hypoxemia during or within 6 hours of transfusion in the absence of other causes of acute lung injury
* Mediated by Anti WBC antibodies
___:
* detection of “new” red cell antibodies after 24 hours of transfusion.
* Occurs secondarily to an amnestic response but can also occur during a primary immune response and may (delayed hemolytic) or may not (delayed serologic) be associated with shortened survival of the transfused cells
Immediate Hemolytic Transfusion Reaction
FNHTR - Febrile non-hemolytic transfusion reaction
Allergic Reaction
Transfusion Related Acute Lung Injury (TRALI)
DHTR - delayed hemolytic transfusion reactio
___:
* presents with profound thrombocytopenia, frequently accompanied by bleeding, 1 to 24 days after a blood transfusion
* The antigen most commonly implicated in this condition is the human platelet antigen (HPA)1a
___:
* due to an immunologic attack by viable donor lymphocytes contained in the transfused blood component against the transfusion recipient
___:
* with respiratory distress and hypoxemia that can be accompanied by cough, headache, chest tightness, hypertension, jugular vein distention, elevated central venous pressure, and elevated pulmonary wedge pressure during or after transfusion
* occurs when the patient’s cardiovascular system’s ability to handle additional workload is exceeded, manifesting as congestive heart failure.
Additional Testing
⚫ Clerical Check
⚫ ABO and Rh Typing
⚫ Urine test
⚫ Antibody screening
⚫ Bilirubin test
⚫ Hgb and Hct
Post-transfusion purpura (PTP)
Transfusion-Associated Graft-Versus-Host Disease
Transfusion Associated Circulatory Overload
