ISBB Flashcards by Kaori Chan

Study of our immune system
Study of host’s reaction when foreign antigens are introduced to the body

IMMUNOLOGY

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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

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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

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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

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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

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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

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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)

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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)

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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)

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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

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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

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CELLULAR DEFENSE MECHANISM:
smallest of the granulocytes
what is the granule content?

Basophils

Cytokines, growth factors,
histamine and heparin

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CELLULAR DEFENSE MECHANISM:
largest cell in the peripheral blood
Type 1 granules: __, ACP, arylsulfatase
Type 2 granules: ___, lysozyme, and lipase

Monocytes
peroxidase
Glucoronidase

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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

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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

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___:
* 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

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_______; 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

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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

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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

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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

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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

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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

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___: 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

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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

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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% 1. B Cells: TRAD: surface Ig (IgM and IgD); NEW: CD19, CD20, CD21 (receptor of EBV) 2. T Cells: TRAD: CD2 (sheep RBC); NEW: CD3, CD4 (Th), CD8 (Tc) 3. 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 ____ 2. Double positive thymocytes a. ______ and ______ are expressed b. Positive and Negative Selection c. Clonal Deletion - removal of particular cell from BM circulation 3. __ - Exhibit only one type of marker 4. Activated T cell - IL-2 5. 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 ___________ 2. Pre-B cell: synthesis of the heavy-chain part of the antibody molecule occurs; accompanied by an unusual light chain molecule called a __ 3. ___: Distinguished by the appearance of complete IgM ab molecules on the cell surface * Markers: ____________________ 4. Mature B cell: IgM and IgD; spleen and circulation; Marginal Zone B cell 5. Activated B cell 6. ___- 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 2. Chemical composition and molecular complexity: a. Best Immunogens: ________________________ b. Less Immunogenic: carbohydrates c. Not Immunogenic: nucleic acids and lipids 3. 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 ___ 4. 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

2. __: 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 _____________ 3. __ - mucosal immunity o major antibody on secretions o associated with anaphylaxis o Structure ▪ Serum: _________________ ▪ Secretions: ______________ 1. facilitate transfer 2. 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

4. __: 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 5. __: 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 1. Fc portion of the molecule binds to the __ 2. 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)

2. 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 1. ___: ✓ RID + electrophoresis ✓ the total distance of antigen migration and precipitation is directly proportional to antigen concentration 2. ___: ✓ 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 3. ___: ✓ 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 4. ___: ✓ 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 2. 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 2. 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 1. Heterophil Antibodies in IM (most significant; EBV infx) * reacts with sheep cells, OX (BEEF) cells and Horse cells but not with __________________ 2. Heterophil antibodies of Forssman (bacterial infx) * reacts with sheep cells, Horse cells, guinea pig cells but not with __________________ 3. 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) 2. ____: 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 3. 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

1. site of a gene in a chromosome 1. alternate forms of a gene at a given locus 1. Opposite antigens encoded at the same locus 1. refers to having two or more alleles at a given locus 1. equal expression of two different inherited alleles 2. always expressed 1. gene that in the presence of a dominant gene does not express itself 1. gene that does not produce any detectable trait 1. identical alleles 1. Different alleles 1. 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

1. locus 1. alleles 1. Antithetical 1. Polymorphic 1. codominant 1. dominant gene 1. recessive gene 1. amorph 1. homozygous 1. heterozygous 1. 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

1. ___: 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-)

2. 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

4. 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

5. 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

6. ___ 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

7. ___ 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

8. 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

9. ___ * 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!!!

10. ___ * 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

11. ___ * 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

12. ___ * 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 13. 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

14. ___ * 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

15. ___ 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

16. ___ * 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

17. ___ 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

18. ___ * Antigens are carried by the DAF (CD55) * DAF is strongly expressed on placental tissue and will adsorb Cromer antibodies

Cromer (021) System

19. ___ * Antigens are located on Complement Receptor 1 (CR1) * “Helgeson phenotype” represents the serologic null phenotype for the Knops blood group

Knops(022) System

20. __ * Antigens: Ina - 4% of Indians, 11% of Iranians, and 12% of Arabs * Located in CD44

Indian (023) System

21. ___ * Named after the antibody maker, Mrs. Kobutso * Carried in CD147 22. ___ * Only antigen: MER2 * Antigen name is derived from monoclonal, and Eleanor Roosevelt 23. John Milton Hagen (026) System 24. The Gill (029) System: ound on the glycerol transporter aquaporin 3 (AQP3) 25. RH-Associated Glycoprotein (030) System

Ok (024) System Raph (025) System

26. ___ § 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)

27. ___§ 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

28. ___ § 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

29. The Vel (034) System: absence of SMIM1, a single-pass integral membrane protein 30. 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) 31. ___: first described in 1967 in the serum of a black woman named Mrs. Augustine 32. ___: § located on the prion protein (PrPC) classically associated with Creutzfeldt-Jakob disease (CJD)

The Augustine (036) System Kanno (037)

33. 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. 34. 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) 36. MAM (041) 37.EMM (042) 38. ABCC1 (043) 39. 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 4. ___: - 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 1. Adsol/AS-1 2. Nutricel/AS-3 3. Optisol/AS-5 4. 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)

2. 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

3. 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 )

4. ___ § 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 5. ___ § 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

ISBB Flashcards

(258 cards)