Review Cards - Immunology Flashcards
Proteins that increase due to infection, injury, trauma (e.g., C-reactive protein, alpha-1 antitrypsin, haptoglobin, fibrinogen, ceruloplasmin, alpha-1 acid glycoprotein, complement)
Acute phase reactants
Antibody formed in response to antigen from individuals of same species
Alloantibody
Foreign substance that can stimulate antibody production; most often a large, complex molecule (MW = >10,000), usually protein or polysaccharide
Antigen
Immunoglobulin produced by plasma cells in response to an antigen
Antibody
Antibody against self
Autoantibody
Strength of bond between antigen & antibody
Avidity
Cytokines that attract cells to a particular site; important in the inflammatory response
Chemokines
Migration of cells toward chemokine
Chemotaxis
Antigenic features of leukocytes
Clusters of differentiation (CD)
Chemicals produced by activated immune cells that affect function of other cells; includes interferons, chemokines, tumor necrosis factors, transforming growth factors, colony stimulating factors, interleukins
Cytokines
Determinant site on antigen
Epitope
Low molecular weight substance that can bind to antibody once it’s formed, but is incapable of stimulating antibody production unless bound to a larger carrier molecule
Hapten
Heightened state of immune responsiveness that can cause tissue damage in host
Hypersensitivity
Resistance to infection
Immunity
Any substance capable of inducing an immune response
Immunogen
Antibody
Immunoglobulin
Cellular & humoral mechanisms involved in reaction to injury or infection
Inflammation
Cytokines with antiviral properties; also active against certain tumors & inflammatory processes
Interferons
Cytokines produced by leukocytes that affect inflammatory response through increase in soluble factors or cells
Interleukins
Molecule that binds to another molecule of complementary configuration; the substance being measured in an immunoassay
Ligand
Enzyme found in tears & saliva that attacks cell walls of microorganisms
Lysozyme
System of genes that control expression of MHC molecules found on all nucleated cells; originally referred to as human leukocyte antigens (HLA)
Major histocompatibility complex (MHC)
Antibody derived from single B-cell clone, frequently used in clinical laboratory assays such as enzyme-linked immunosorbent assay (ELISA)
Monoclonal antibody
Serum proteins that attach to a foreign substance & enhance phagocytosis; most often complement or antibody
Opsonin
Step-wise engulfment of cells or particulate matter by neutrophils & macrophages
Phagocytosis
Transformed B cells that secret antibody
Plasma cells
Antibody produced by many B-cell clones
Polyclonal antibody
Reduced ag/ab complexes due to antigen excess; can cause false negative in serological test for antibodies; repeat test in 1-2 weeks
Postzone
Reduced ag/ab complexes due to antibody excess; can cause false negative in serological tests for antibodies; dilute serum & retest
Prozone
Change of serological test from negative to positive due to development of detectable antibodies
Seroconversion
Small, flat bilobed organ found in thorax; site of T-lymphocyte development; one of two primary lymphoid organs (bone marrow is the other primary lymphoid organ)
Thymus
Means of expressing antibody concentration; reciprocal of highest dilution with positive reaction
Titer
Injection of immunogenic material to induce immunity
Vaccination
When the number of multivalent sites of antigen & antibody are approximately equal; results in optimal reactions
Zone of equivalence
Branches of the immune system - Cellular - definition
cell-mediated
Branches of the immune system - Cellular - defense against
-viruses
-fungi
-mycobacteria
-other intracellular pathogens
-tumor cells
Branches of the immune system - Cellular - cells involved
-T-cells
-macrophages
Branches of the immune system - Cellular - examples
-graft rejection
-hypersensitivity reactions
-elimination of tumor cells
Branches of the immune system - Humoral - definition
antibody mediated
Branches of the immune system - Humoral - defense against
bacteria (extracellular)
Branches of the immune system - Humoral - cells involved
-B cells
-plasma cells
Branches of the immune system - Humoral - examples
antibody production
Types of immunity - innate - explanation
defense mechanisms present at birth; not antigen specific
Types of immunity - innate - components
External defense system:
-intact skin
-mucous membranes
-cilia & mucus in RT
-stomach acid
-flushing of urine
-lactic acid in vagina
-lysozyme in tears & saliva
-normal flora
Internal defense system:
-neutrophils
-macrophages
-acute phase reactants
-complement
-chemokines
Types of immunity - innate - memory?
NO
Types of immunity - acquired or adaptive - explanation
defense mechanisms that are antigen specific
Types of immunity - acquired or adaptive - components
-T-cells
-B-cells
-plasma cells
-antibodies
-cytokines
Types of immunity - acquired or adaptive - memory?
YES
Adaptive immunity - naturally acquired active immunity - explanation
individual infected with microorganism produces antibodies
Adaptive immunity - naturally acquired active immunity - example
clinical or subclinical infection
Adaptive immunity - naturally acquired active immunity - specific?
yes
Adaptive immunity - naturally acquired active immunity - immediate?
no
Adaptive immunity - naturally acquired active immunity - long-term?
yes
Adaptive immunity - artificially acquired active immunity - explanation
individual exposed to antigen through vaccine develops immunity without having the infection
Adaptive immunity - artificially acquired active immunity - explanation
individual exposed to antigen through vaccine develops immunity without having the infection
Adaptive immunity - artificially acquired active immunity - example
-Diphtheria, tetanus, pertussis vaccine (DTap)
-measles, mumps, rubella vaccine (MMR)
-polio vaccine
-tetanus vaccine
-Haemophilus influenzae type b (Hib) vaccine
Adaptive immunity - artificially acquired active immunity - specific?
yes
Adaptive immunity - artificially acquired active immunity - immediate?
no
Adaptive immunity - artificially acquired active immunity - long-term?
yes
Adaptive immunity - naturally acquired passive immunity - explanation
individual protected by antibodies produced by another person
Adaptive immunity - naturally acquired passive immunity - example
maternal antibodies that cross the placenta and are present in breast milk
Adaptive immunity - naturally acquired passive immunity - specific?
yes
Adaptive immunity - naturally acquired passive immunity - immediate?
yes
Adaptive immunity - naturally acquired passive immunity - long-term?
no
Adaptive immunity - artificially acquired passive immunity - explanation
individual receives immune globulin containing antibodies produced by another person
Adaptive immunity - artificially acquired passive immunity - example
-Rh immune globulin
-convalescent plasma
-antitoxins
Adaptive immunity - artificially acquired passive immunity - specific?
yes
Adaptive immunity - artificially acquired passive immunity - immediate?
yes
Adaptive immunity - artificially acquired passive immunity - long-term?
no
Cells of the innate immune system - granulocytes - neutrophils - function
-phagocytosis
-inflammatory response
Cells of the innate immune system - granulocytes - neutrophils - respond to?
Chemotaxins
Cells of the innate immune system - granulocytes - neutrophils - granules contain?
Bactericidal enzymes
Cells of the innate immune system - granulocytes - eosinophils - function
-neutralization of basophil & mast cell products
-destruction of some helminths
-hypersensitivity reactions
Cells of the innate immune system - granulocytes - eosinophils - phagocytic ability?
Some
Cells of the innate immune system - granulocytes - basophils - function
Hypersensitivity reactions
Cells of the innate immune system - granulocytes - basophils - granules contain?
Histamine, heparin, eosinophil chemotactic factor A
Cells of the innate immune system - granulocytes - basophils - bind to what in an allergic reaction?
IgE
Cells of the innate immune system - granulocytes - basophils - when do granules release contents?
In presence of antigen
Cells of the innate immune system - mononuclears - monocytes - function
Phagocytosis
Cells of the innate immune system - Tissue cells - mast cells - function
Hypersensitivity reactions
Cells of the innate immune system - Tissue cells - mast cells - what type of cells?
Connective tissue cells
Cells of the innate immune system - Tissue cells - mast cells - similar to basophils
Yes, but they are larger and have more granules
Cells of the innate immune system - Tissue cells - mast cells - what do they bind to?
IgE
Cells of the innate immune system - mononuclears - monocytes - migration
Migrate to the tissues and become macrophages
Cells of the innate immune system - Tissue cells - mast cells - respond to?
Chemotaxins
Cells of the innate immune system - Tissue cells - macrophages - function
-phagocytosis
-elimination of bacteria, intracellular parasites, & tumor cells
-secretion of cell mediators
-antigen presentation
Cells of the innate immune system - Tissue cells - macrophages - activated by?
Contact with microorganisms or cytokines from T cells
Cells of the innate immune system - Tissue cells - dendritic cells - function
-phagocytosis
-presentation of antigens to T cells
Cells of the innate immune system - Tissue cells - dendritic cells - initiate?
Adaptive immune response
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - function
1st line of defense against tumor cells & cells infected with viruses
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - lymphocytes without?
T or B markers
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - surface antigens?
no unique surface antigens, but CD16+ and CD56+
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - bridge between?
innate and acquired immunity
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - specificity?
no
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - stimulated by?
cytokines
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - released?
early infection - provides time for T & B cells to be activated
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - % of lymphs?
<20%
Cells of the innate immune system - Lymphocytes - Natural Killer (NK) cells - key cell in what?
antibody-dependent cellular cytotoxicity (ADCC)
Cells of the acquired immune system - T-lymphocytes (T cells) - function
cell-mediated immunity
Cells of the acquired immune system - T-lymphocytes (T cells) - derived from?
cells in bone marrow
Cells of the acquired immune system - T-lymphocytes (T cells) - where do they develop T-cell-specific surface antigens?
thymus
Cells of the acquired immune system - T-lymphocytes (T cells) - % of lymphs?
60-80%
Cells of the acquired immune system - Helper T cells - function
-orchestrate cell-mediated immunity
-activate B cells, cytotoxic cells, & NK cells
Cells of the acquired immune system - Helper T cells - surface antigen
CD4+
Cells of the acquired immune system - Helper T cells - constitutes how much of peripheral T cells?
2/3
Cells of the acquired immune system - Helper T cells - normal CD4 concentration
1,000/uL
Cells of the acquired immune system - Helper T cells - CD4 concentration in AIDS
<200/uL
Cells of the acquired immune system - Cytotoxic T cells - function
-suppressor cells inhibit helper T cells
-cytotoxic cells kill other cells
Cells of the acquired immune system - Cytotoxic T cells - surface antigen
CD8+
Cells of the acquired immune system - Cytotoxic T cells - constitutes how much of peripheral T cells?
1/3
Cells of the acquired immune system - Cytotoxic T cells - normal CD4/CD8 ratio?
2:1
Cells of the acquired immune system - Cytotoxic T cells - CD4/CD8 ratio in AIDS?
<0.5:1
Cells of the acquired immune system - T regulatory cells - function
suppress immune response to self
Cells of the acquired immune system - T regulatory cells - surface antigens
CD4+ and CD25+
Cells of the acquired immune system - B lymphocytes (B cells) - function
after antigenic challenge, transform into blasts that give rise to plasma cells & memory cells
Cells of the acquired immune system - B lymphocytes (B cells) - develop where?
bone marrow
Cells of the acquired immune system - B lymphocytes (B cells) - mature cells have what on their surface?
immunoglobulins (IgM, IgD) that act as receptors for antigens
Cells of the acquired immune system - B lymphocytes (B cells) - constitute what percentage of lymphs in peripheral blood?
10-20%
Cells of the acquired immune system - plasma cells - function
antibody production
Cells of the acquired immune system - plasma cells - where are they located?
in peripheral lymphoid organs
Cells of the acquired immune system - plasma cells - do they divide?
no
Cells of the acquired immune system - memory cells - function
respond to antigens when encountered again with increased speed & intensity
Cells of the acquired immune system - memory cells - located?
in peripheral organs
Cells of the acquired immune system - memory cells - live span?
months to years
Cells of the acquired immune system - memory cells - what type of cells?
can be B or T cells
List the primary lymphoid organs.
-bone marrow
-thymus
List the secondary lymphoid organs.
-spleen
-lymph nodes
-tonsils
-appendix
-cutaneous-associated lymphoid tissue
-mucosal-associated lymphoid tissue (MALT), including Peyer patches in the lower ileum
Isolation & identification of lymphocytes - isolation
-density gradient centrifugation with separation media (e.g., Ficoll-Hypaque)
-layers from top to bottom:
–plasma
–mononuclear cells
–separation media
–RBCs
–granulocytes
Isolation & identification of lymphocytes - identification
-flow cytometry
-fluorescent-labeled monoclonal antibodies against specific surface antigens
-each antibody has different fluorescent tag
-light scattering measured as cells flow through laser beam
-common T-cell antigens tested: CD2, CD3, CD4, CD7, CD8
-common B-cell antigens tested: CD19, CD20, CD22, surface Ig
Immunoglobulin (Ig) structure - basic structure
2 heavy (H) chains + 2 light (L) chains held together by disulfide (S-S) bonds
Immunoglobulin (Ig) structure - heavy (H) chains
-determine Ig class (IgG, IgA, IgM, IgD, IgE)
-gamma, alpha, delta, mu, epsilon
Immunoglobulin (Ig) structure - light (L) chains
-kappa or lambda
-both found in all classes of Igs, but only 1 type per molecule
-free L chains = Bence Jones proteins
Immunoglobulin (Ig) structure - Fab fragment
-fragment antigen binding
-consists of 1 L chain & 1/2 H chain held together by S-S bonds
-2 per Ig
-each can bind antigen
Immunoglobulin (Ig) structure - Fc fragment
-fragment crystallizable
-carboxy-terminal halves of 2 H chains held together by S-S bonds
-site of antibody biological activity such as opsonization & complement fixation
Immunoglobulin (Ig) structure - Constant region
carboxy-terminal ends of H & L chains where amino acid sequence is same for all chains of that type
Immunoglobulin (Ig) structure - variable region
-amino-terminal ends of H & L chains where amino acid sequence varies
-also known as antigen-recognition unit
-responsible for Ig specificity
Immunoglobulin (Ig) structure - Hinge region
-flexible portion of H chain between 1st & 2nd constant regions
-allows molecule to bend so that 2 antigen-binding sites can operate independently
Immunoglobulin (Ig) structure - Joining chain
glycoprotein that links Ig monomers in IgM & secretory IgA
Immunoglobulins - IgG - form in serum
monomer
Immunoglobulins - IgG - molecular weight (daltons)
150,000
Immunoglobulins - IgG - H chain
gamma
Immunoglobulins - IgG - L chain
Kappa or lambda
Immunoglobulins - IgG - % of total Ig
70-75%
Immunoglobulins - IgG - % of total Ig
70-75%
Immunoglobulins - IgG - serum concentration (mg/dL)
800-1,600 mg/dL
Immunoglobulins - IgG - antigen binding sites
2
Immunoglobulins - IgG - complement fixation
yes
Immunoglobulins - IgM - form in serum
pentamer
Immunoglobulins - IgM - molecular weight (daltons)
900,000
Immunoglobulins - IgM - H chain
Mu
Immunoglobulins - IgM - L chain
kappa or lambda
Immunoglobulins - IgM - % of total Ig
10%
Immunoglobulins - IgM - serum concentration (mg/dL)
120-150 mg/dL
Immunoglobulins - IgM - antigen binding sites
10
Immunoglobulins - IgM - complement fixation
yes
Immunoglobulins - IgA - form in serum
monomer & dimer
Immunoglobulins - IgA - molecular weight (daltons)
160,000 or 400,000
Immunoglobulins - IgA - H chain
alpha
Immunoglobulins - IgA - L chain
kappa or lambda
Immunoglobulins - IgA - % of total Ig
10-15%
Immunoglobulins - IgA - serum concentration (mg/dL)
70-350 mg/dL
Immunoglobulins - IgA - antigen binding sites
2 or 4
Immunoglobulins - IgA - antigen binding sites
2 or 4
Immunoglobulins - IgA - complement fixation
no
Immunoglobulins - IgD - form in serum
monomer
Immunoglobulins - IgD - molecular weight (daltons)
180,000
Immunoglobulins - IgD - H chain
delta
Immunoglobulins - IgD - L chain
kappa or lambda
Immunoglobulins - IgD - % of total Ig
<1%
Immunoglobulins - IgD - serum concentration (mg/dL)
1-3 mg/dL
Immunoglobulins - IgD - antigen binding sites
2
Immunoglobulins - IgD - complement fixation
no
Immunoglobulins - IgE - form in serum
monomer
Immunoglobulins - IgE - molecular weight (daltons)
190,000
Immunoglobulins - IgE - H chain
epsilon
Immunoglobulins - IgE - L chain
kappa or lambda
Immunoglobulins - IgE - % of total Ig
0.002%
Immunoglobulins - IgE - serum concentration (mg/dL)
0.005 mg/dL
Immunoglobulins - IgE - antigen binding sites
2
Immunoglobulins - IgE - complement fixation
no
Immunoglobulins - IgG - crosses placenta?
Yes
Immunoglobulins - IgG - role(s)
-defense against bacteria & viruses
-neutralizes toxins
-opsonin
-passive immunity in newborns
Immunoglobulins - IgG - precipitation/agglutination
more efficient at precipitation than agglutination
Which immunoglobulin is the main Ig produced during memory (recall) response to antigens?
IgG
Immunoglobulins - IgM - crosses placenta?
no
Immunoglobulins - IgM - role(s)
-neutralizes toxins
-opsonin
Immunoglobulins - IgM - more efficient at what than IgG?
agglutination
Immunoglobulins - IgM - destroyed by?
sulfhydryl compounds
Which immunoglobulin is the first Ig produced in the immune response?
IgM
Which immunoglobulin is the only Ig produced by newborns?
IgM
Which immunoglobulin is the only Ig produced by newborns?
IgM
Which immunoglobulin is the most efficient Ig at initiating the complement cascade?
IgM
Immunoglobulins - IgA - crosses placenta?
no
Immunoglobulins - IgA - role(s)
-1st line of defense
-patrols mucosal surfaces
-prevents adherence of bacteria and neutralizes toxins
Immunoglobulins - IgA - located?
-tears
-sweat
-respiratory mucosa
-GI mucosa
-breast milk
Immunoglobulins - IgD - crosses placenta?
no
Immunoglobulins - IgD - role(s)
may play a role in B-cell maturation
Immunoglobulins - IgD - on surface of what cells?
B cells
Immunoglobulins - IgE - crosses placenta?
no
Immunoglobulins - IgE - role(s)
-role in allergic reactions
-binds to basophils & mast cells
-when 2 adjacent molecules on mast cells bind antigens, degranulation of cell with release of histamine & heparin
Immunoglobulins - IgE - type of hypersensitivity reaction
Type I immediate hypersensitivity reaction
Complement - definition
-group of >30 proteins involved in phagocytosis & clearance of foreign antigen
-most are inactive enzyme precursors that are converted to active enzymes in precise order (cascade)
Complement - functions
-inflammation
-opsonization
-chemotaxis
-cell lysis
Complement - classical pathway
-triggered by ag/ab reaction
-IgM is most efficient activator
-single molecule attached to 2 adjacent antigens can initiate cascade
-IgG1, 2, & 3 can activate complement but at least 2 molecules required
-recognition unit: C1 (first to bind)
-activation unit: C4, C2, C3
-membrane attack complex: C5, C6, C7, C8, C9 (cell lysis)
Complement - alternative pathway
-antibody dependent
-activated by bacteria, fungi, viruses, tumor cells, some parasites
Complement - lectin pathway
-antibody independent
-initiated by mannose-binding lectin (MBL)
-nonspecific recognition of sugars on microorganisms
-important defense mechanism in infancy
Complement - present in highest concentration in plasma
C3 (key component of all 3 pathways)
Complement - deficiencies
-increased susceptibility to infection
-accumulation of immune complexes, which can lead to glomerulonephritis
Complement - ions required
calcium and magnesium
Complement - inactivation
56*C for 30 minutes
Hypersensitivity reactions - Type I: Anaphylactic - key reactant(s)
IgE
Hypersensitivity reactions - Type I: Anaphylactic - mechanism
release of mediators from eosinophils, mast cells, & basophils
Hypersensitivity reactions - Type I: Anaphylactic - onset of symptoms
immediate
Hypersensitivity reactions - anaphylaxis - type of hypersensitivity reaction?
Type I: Anaphylactic
Hypersensitivity reactions - hay fever - type of hypersensitivity reaction?
Type I: Anaphylactic
Hypersensitivity reactions - asthma - type of hypersensitivity reaction?
Type I: Anaphylactic
Hypersensitivity reactions - food allergies - type of hypersensitivity reaction?
Type I: Anaphylactic
Hypersensitivity reactions - transfusion reactions - type of hypersensitivity reaction?
Type II: Cytotoxic
Hypersensitivity reactions - HDN - type of hypersensitivity reaction?
Type II: Cytotoxic
Hypersensitivity reactions - autoimmune hemolytic anemia - type of hypersensitivity reaction?
Type II: Cytotoxic
Hypersensitivity reactions - Arthus reaction - type of hypersensitivity reaction?
Type III: Immune complex
Hypersensitivity reactions - serum sickness - type of hypersensitivity reaction?
Type III: Immune complex
Hypersensitivity reactions - SLE - type of hypersensitivity reaction?
Type III: Immune complex
Hypersensitivity reactions - rheumatoid arthritis (RA) - type of hypersensitivity reaction?
Type III: Immune complex
Hypersensitivity reactions - contact dermatitis - type of hypersensitivity reaction?
Type IV: Cell dependent
Hypersensitivity reactions - hypersensitivity pneumonitis - type of hypersensitivity reaction?
Type IV: Cell dependent
Hypersensitivity reactions - tuberculin skin test - type of hypersensitivity reaction?
Type IV: Cell dependent
Hypersensitivity reactions - Type II: Cytotoxic - key reactant(s)
IgG, IgM, complement, cellular antigens
Hypersensitivity reactions - Type II: Cytotoxic - mechanism
cytolysis due to antibody & complement
Hypersensitivity reactions - Type II: Cytotoxic - onset of symptoms
immediate
Hypersensitivity reactions - Type II: Immune Complex - key reactant(s)
IgG, IgM, complement, soluble antigen
Hypersensitivity reactions - Type III: Immune Complex - key reactant(s)
IgG, IgM, complement, soluble antigen
Hypersensitivity reactions - Type III: Immune Complex - mechanism
deposits of antigen-antibody complexes in tissues
Hypersensitivity reactions - Type III: Immune Complex - onset of symptoms
immediate
Hypersensitivity reactions - Type IV: T-Cell Dependent - key reactant(s)
T cells, antigen-presenting cells (APCs)
Hypersensitivity reactions - Type IV: T-Cell Dependent - mechanism
release of cytokines
Hypersensitivity reactions - Type IV: T-Cell Dependent - onset of symptoms
delayed - sensitization after 1st contact with antigen, symptoms upon reexposure
Agglutination methods - direct agglutination - principle
-naturally occurring antigens on particles (e.g., bacterial antigens)
-particles agglutinate in the presence of corresponding antibody
Agglutination methods - direct agglutination - application
-Widal test for typhoid fever
-Salmonella O & H antigens used to detect antibodies in patient serum
-test used in developing countries
Agglutination methods - hemagglutination - principle
antigen-antibody reactions that results in clumping of RBCs
Agglutination methods - hemagglutination - application
ABO typing
Agglutination methods - passive (indirect) agglutination - principle
-soluble antigens bound to particles, e.g., latex
-particles agglutinate in presence of corresponding antibody
Agglutination methods - passive (indirect) agglutination - application
antibody to Group A Streptococcus (GAS) or antibody to rotavirus or cytomegalovirus (CMV)
Agglutination methods - reverse passive agglutination - principle
-antibody attached to carrier particles
-particles agglutinate in presence of corresponding antigen
Agglutination methods - reverse passive agglutination - application
-kits available for rapid ID of bacteria such as Group B Streptococcus (GBS), Staphylococcus aureus, and Cryptococcus neoformans
Agglutination methods - agglutination inhibition - principle
-competition between particulate antigen (reagent) & soluble antigen (in specimen) for sites on reagent antibody
-lack of agglutination is a positive results
Agglutination methods - agglutination inhibition - application
-detection of illicit drugs
-controls are crucial to confirm lack of agglutination
Agglutination methods - hemagglutination inhibition - principle
-detects antibodies to certain viruses that agglutinate RBCs
-in presence of antibody, virus is neutralized & hemagglutination doesn’t occur
Agglutination methods - hemagglutination inhibition - application
-Rubella & other viruses
-controls are crucial to confirm lack of hemagglutination
Precipitation methods - precipitation - principle
-soluble antigen combines with soluble antibody to produce visible complexes
-less sensitive than agglutination
Precipitation methods - Ouchterlony double diffusion - principle
antigens & antibodies diffuse from wells in gel & form precipitin lines where they meet
Precipitation methods - Ouchterlony double diffusion - application
fungal antigens
Precipitation methods - Radial immunodiffusion (RID) - principle
-antigen diffuses out of well in gel containing antibody
-precipitin ring forms
-diameter proportionate to concentration of antigen
Precipitation methods - Radial immunodiffusion (RID) - application
largely replaced by more sensitive methods such as nephelometry and enzyme-linked immunoassays
Precipitation methods - Immunofixation electrophoresis (IFE) - principle
-proteins separated by electrophoresis
-antiserum places directly in gel
-antigen-antibody complexes precipitate
Precipitation methods - Immunofixation electrophoresis (IFE) - application
-identification of Igs in monoclonal gammopathies
-Bence Jones proteins
-also useful for detection of antigen present in serum, urine, or CSF at low concentrations
Precipitation methods - Nephelometry - principle
-light scattering by antigen-antibody complexes
-amount of light scattered is proportional to concentration
Precipitation methods - Nephelometry - application
-Igs
-complement
-C-reactive protein (CRP)
-haptoglobin
-ceruloplasmin
substance being measured in immunoassay; can be antigen or antibody
ligand
immunoassay in which patient antigen & labeled reagent antigen compete for binding sites on reagent antibody
competitive
immunoassay that doesn’t involve competition for binding sites; more sensitive than competitive assays
noncompetitive
immunoassay with separation step to remove from bound analyte; more sensitive than homogeneous assays
heterogeneous
immunoassay that doesn’t require separation step; easier to automate
homogeneous
any immunoassay that uses an enzyme as a label; a substrate is added to measure enzyme activity
EIA
1st type of EIA developed; competitive; enzyme-labeled reagent is part of initial ag-ab reaction; all reactants added at same time; 1 incubation & 1 wash
Direct EIA
noncompetitive EIA; enzyme-labeled reagent isn’t involved in initial ag-ab reaction; 2 incubations & 2 washes; more sensitive than direct assays; also known as ELISA
Indirect EIA
reagent ag or ab bound to support medium, e.g., polystyrene test tubes, microtiter plates, cellulose membranes, glass beads
solid phase
EIA formats - EIA - description
heterogeneous, competitive, direct
EIA formats - EIA - principle
-enzyme-labeled ligand & unlabeled patient ligand compete for binding sites on antibody attached to solid phase
-free labeled ligand removed by washing
-substrate added
-color inversely proportional to concentration of ligand in specimen
EIA formats - EIA - used to measure?
small relatively pure antigens, e.g., insulin, estrogen
EIA formats - ELISA - description
heterogeneous, noncompetitive, indirect
EIA formats - ELISA - principle
-antigen attached to solid phase
-antibody in specimen attaches
-unbound antibody removed by washing
-enzyme-labeled antiglobulin added
-attaches to antibody on solid phase
-substrate added
-color directly proportional to antibody concentration
-more sensitive than competitive EIA
-one of the most common immunoassays
EIA formats - ELISA - used to detect?
antibodies to viruses, e.g., HIV, hepatitis A (HAV), HCV, EBV
EIA formats - Sandwich ELISA or capture assay - description
heterogeneous, noncompetitive, indirect
EIA formats - Sandwich ELISA or capture assay - principle
-antibody attached to solid phase
-antigen in specimen attaches
-enzyme-labeled antibody added, attaches to different determinant
-enzymatic activity directly proportional to amount of antigen in sample
EIA formats - Sandwich ELISA or capture assay - antigen must have?
multiple determinants
EIA formats - Sandwich ELISA or capture assay - used to measure?
-hormones
-proteins
-detect tumor markers
-viruses
-parasites
-fungi
EIA formats - Sandwich ELISA or capture assay - high concentration of antigen can cause?
Hook effect - too much antigen for binding sites so undiluted sample has lower absorbance than dilutions
EIA formats - Rapid ELISA (lateral flow) - description
membrane based
EIA formats - Rapid ELISA (lateral flow) - principle
-reagent antigen or antibody bound to membrane in single use cassette
-sample added
-presence of ag-ab complex indicated by colored reaction
EIA formats - Rapid ELISA (lateral flow) - examples
pregnancy tests, cardiac troponin, SARS-CoV 2
-usually qualitative and designed primarily for POCT or at home testing
EIA formats - Enzyme-multiplied immunoassay technique (EMIT) - description
homogeneous
EIA formats - Enzyme-multiplied immunoassay technique (EMIT) - principle
-antigen in specimens & enzyme-labeled antigen compete for binding sites on reagent antibody
-when enzyme-labeled antigen binds, enzyme activity inhibited
-enzyme activity is directly proportional to concentration of antigen in specimen
EIA formats - Enzyme-multiplied immunoassay technique (EMIT) - used for?
determination of low molecular weight analytes not readily measured by other methods, e.g., drugs and some hormones
-automated
Fluorescent immunoassays (FIA) - direct fluorescent antibody (DFA) staining - principle
-specimen on glass slide overlaid with fluorescein-labeled antibody
-if corresponding antigen is present, labeled antibody binds
-fluorescence observed with fluorescent microscope
Fluorescent immunoassays (FIA) - direct fluorescent antibody (DFA) staining - detects?
antigen
Fluorescent immunoassays (FIA) - direct fluorescent antibody (DFA) staining - fluorescent labels
Fluorescein isothiocyanate or rhodamine B isothiocyanate
Fluorescent immunoassays (FIA) - direct fluorescent antibody (DFA) staining - examples of analytes
bacteria, viral antigen
Fluorescent immunoassays (FIA) - indirect fluorescent antibody (IFA) staining - principle
-reagent antigen on glass slide overlaid with patient serum
-if corresponding antibody is present in serum, attached to the antigen
-when fluorescein-labeled antihuman globulin is added, attaches to antibody
-fluorescence observed with fluorescent microscope
Fluorescent immunoassays (FIA) - indirect fluorescent antibody (IFA) staining - detects?
antibodies in serum
“Sandwich technique”
Fluorescent immunoassays (FIA) - indirect fluorescent antibody (IFA) staining - examples of analytes
-antinuclear antibody (ANA)
-fluorescent treponemal antibody (FTA)
Fluorescent immunoassays (FIA) - fluorescence polarization immunoassay (FPIA) - principle
-labeled antigen competes with antigen in specimen for sites on reagent antibody
-free labeled antigen rotates rapidly, emits little polarized light
-bound labeled antigen rotates more slowly, emits more polarized light
-amount of polarized light is inversely proportional to concentration of antigen in specimen
Fluorescent immunoassays (FIA) - fluorescence polarization immunoassay (FPIA):
-competitive or noncompetitive?
-heterogeneous or homogenerous?
-competitive
-homogeneous
Fluorescent immunoassays (FIA) - fluorescence polarization immunoassay (FPIA) - examples of analytes
therapeutic drugs, hormones
Comparison of labeled immunoassays - EIA - labels
-alkaline phosphatase
-horseradish peroxidase
-Alpha-D-galactosidase
-glucose-6-phosphate dehydrogenase
Comparison of labeled immunoassays - EIA - detection
enzymes react with substrate to produce color change
Comparison of labeled immunoassays - EIA - type(s) of assays available
-mostly noncompetitive now
-heterogenous or homogeneous
Comparison of labeled immunoassays - EIA - advantages
-sensitivity
-specificity
-no health hazard or disposal problems
-reagents with long shelf life
-can be automated
Comparison of labeled immunoassays - EIA - disadvantages
-natural inhibitors in some specimens
-cross reactivity of some substances
Comparison of labeled immunoassays - EIA - use
common
Comparison of labeled immunoassays - FIA - labels
-fluorescein
-rhodamine
Comparison of labeled immunoassays - FIA - detection
fluorochromes absorb energy from light source, convert to longer wavelength (lower energy)
Comparison of labeled immunoassays - FIA - type(s) of assays available
-usually competitive
-heterogeneous & homogeneous
Comparison of labeled immunoassays - FIA - advantages
-sensitivity
-specificity
-no health hazard or disposal problems
-reagent with long shelf life
-automated
Comparison of labeled immunoassays - FIA - disadvantages
-autofluorescence from organic substances in serum
-nonspecific binding to substances in serum
-expensive, dedicated instrumentation
Comparison of labeled immunoassays - FIA - use
common
Comparison of labeled immunoassays - Chemiluminescent Immunoassay (CIA) - labels
-luminol
-acridinium esters
-ruthenium derivatives
-nitrophenyl oxalates
Comparison of labeled immunoassays - Chemiluminescent Immunoassay (CIA) - detection
chemiluminescent molecules produce light from chemical reaction
Comparison of labeled immunoassays - Chemiluminescent Immunoassay (CIA) - type(s) of assays available
-competitive & noncompetitive
-heterogeneous & homogeneous
Comparison of labeled immunoassays - Chemiluminescent Immunoassay (CIA) - advantages
-sensitivity
-specificity
-no health hazard or disposal problems
-reagent with long shelf life
-automated
Comparison of labeled immunoassays - Chemiluminescent Immunoassay (CIA) - disadvantages
quenching of light emission by some biological materials
Comparison of labeled immunoassays - Chemiluminescent Immunoassay (CIA) - use
common
Nontreponemal tests for syphilis - VDRL - method
flocculation
Nontreponemal tests for syphilis - VDRL - detects
reagin
(antibody against cardiolipin that is in serum of patients with syphilis)
Nontreponemal tests for syphilis - VDRL - antigen
cardiolipin
Nontreponemal tests for syphilis - VDRL - positive reaction
microscopic clumps
Nontreponemal tests for syphilis - VDRL - specimen(s)
-inactivated serum
-CSF
Nontreponemal tests for syphilis - VDRL - reactivity during disease
-Primary stage: may be negative
-Secondary or early late stages: titers usually peak
-more rapid decline with treatment
-becomes non-reactive in 1-2 years following successful treatment
Nontreponemal tests for syphilis - VDRL - false positives
Biologic false positives:
–infectious mono (IM)
–infectious hepatitis
–malaria
–leprosy
–lupus erythematosus
–RA
–advanced age
–pregnancy
Reactive in other treponemal infections such as yaws & pinta.
Nontreponemal tests for syphilis - VDRL - reactives should be confirmed by?
treponemal test
Nontreponemal tests for syphilis - VDRL - replaced by?
RPR for serum
Nontreponemal tests for syphilis - VDRL - CSF
diagnosis of neurosyphilis
Nontreponemal tests for syphilis - RPR - method
flocculation
Nontreponemal tests for syphilis - RPR - detects
reagin
Nontreponemal tests for syphilis - RPR - antigen
cardiolipin with charcoal
Nontreponemal tests for syphilis - RPR - positive reaction
macroscopic agglutination
Nontreponemal tests for syphilis - RPR - specimen(s)
-serum (inactivation not required)
-plasma
Nontreponemal tests for syphilis - RPR - reactivity during disease
-Primary stage: may be negative
-Secondary or early late stages: titers usually peak
-more rapid decline with treatment
-becomes non-reactive in 1-2 years following successful treatment
Nontreponemal tests for syphilis - RPR - reactives should be confirmed by?
treponemal test
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - detects
antibody to T. pallidum
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - reagent(s)
-sorbent (nonpathologic treponemes - Reiter strain)
-slides with Nichols strain of T. pallidum
-fluorescein-labeled antihuman globulin
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - positive reaction
fluorescence
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - specimen(s)
-serum
-CSF
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - reactivity during disease
-usually positive before non-treponemal tests
-some false negatives in primary syphilis
-usually positive for life
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - detects
antibody to T. pallidum
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - reagent(s)
colored gelatin particles coated with treponemal antigen
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - positive reaction
-agglutination of sensitized gel particles
-smooth mat over surface of well
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - specimen(s)
serum
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - reactivity during disease
-not as sensitive in primary syphilis as FTA
-sensitivity close to 100% in secondary syphilis
-usually positive in late stages
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - detects
antibody to T. pallidum
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - reagent(s)
enzyme-labeled treponemal antigen
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - positive reaction
color development following addition of substrate
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - specimen(s)
serum
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - reactivity during disease
high sensitivity
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - false positives
-fewer than nontreponemal tests
-reactive with other treponemal diseases (e.g., yaws, pinta)
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - used for?
screening & confirmation of reactive nontreponemal tests
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - used for screening
reactives should be confirmed with nontreponemal test (reverse sequence screening), followed by TP-PA or FTA-ABS if nontreponemal test is nonreactive
Treponemal tests for syphilis - Antibody capture enzyme-linked immunosorbent assay (ELISA) - Dx of congenital syphilis
IgM capture assay
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - false positives
-fewer than nontreponemal tests
-reactive with other treponemal diseases, e.g., yaws, pinta
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - what does absorbent remove?
non-specific antibodies
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - used to confirm?
reactive nontreponemal tests
Treponemal tests for syphilis - Fluorescent treponemal antibody absorption (FTA-ABS) - not good for?
treatment monitoring
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - false positives
fewer than nontreponemal tests
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - used to confirm?
reactive nontreponemal tests
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - positive result
a smooth mat will form in the base of the well of a microtitre plate, indicating a positive result
Treponemal tests for syphilis - Treponema pallidum particle agglutination (TP-PA) - not good for?
treatment monitoring
Interpretation of syphilis test results:
RPR: reactive
FTA: reactive
Positive for syphilis
Interpretation of syphilis test results:
RPR: reactive
FTA: non-reactive
Negative for syphilis
Interpretation of syphilis test results:
ELISA: reactive
RPR: reactive
Positive for syphilis
Interpretation of syphilis test results:
ELISA: reactive
RPR: non-reactive
FTA-ABS: reactive
late, latent, or previous syphilis
Serological tests for other bacterial infections - Anti-streptolysin O (ASO) - diagnosis
Sequelae of GAS infection: rheumatic fever, poststreptococcal glomerulonephritis
Serological tests for other bacterial infections - Anti-streptolysin O (ASO) - common method(s)
nephelometry
Serological tests for other bacterial infections - Anti-streptolysin O (ASO) - antigen used?
recombinant streptolysin antigen
Serological tests for other bacterial infections - Anti-streptolysin O (ASO) - positive result
if antibody present, antigen-antibody complexes form, & increased light scatter
Serological tests for other bacterial infections - Anti-DNase B - diagnosis
Sequelae of GAS infections: rheumatic fever, glomerulonephritis following skin infection, i.e., impetigo
Serological tests for other bacterial infections - Anti-DNase B - common method(s)
EIA, nephelometry
Serological tests for other bacterial infections - Anti-DNase B - advantages
-highly specific
-may be positive when ASO is negative
Serological tests for other bacterial infections - Streptozyme - diagnosis
Sequelae of GAS infection
Serological tests for other bacterial infections - Streptozyme - common method(s)
slide agglutination
Serological tests for other bacterial infections - Streptozyme - principle
uses sheep RBCs coated with several streptococcal antigens
Serological tests for other bacterial infections - Streptozyme - disadvantages
-more false positives & false negatives
Should be used in conjunction with ASO & anti-DNase
-serial titers should be performed
Serological tests for other bacterial infections - Helicobacter pylori antibody - diagnosis
gastric & duodenal ulcers caused by H. pylori
Serological tests for other bacterial infections - Helicobacter pylori antibody - common method(s)
-method of choice: ELISA
-rapid tests, PCR available
Serological tests for other bacterial infections - Helicobacter pylori antibody - detect what Ig?
IgG
Serological tests for other bacterial infections - Helicobacter pylori antibody - successful treatment
25% decrease in titer
Serological tests for other bacterial infections - Helicobacter pylori antibody - antibody lifespan
years
Serological tests for other bacterial infections - Helicobacter pylori antibody - positive rapid tests should be confirmed by?
ELISA
Serological tests for other bacterial infections - Mycoplasma pneumoniae antibody - diagnosis
primary atypical pneumonia (PAP)
Serological tests for other bacterial infections - Mycoplasma pneumoniae antibody - common method(s)
-most common: EIA
-also agglutination, IFA
-molecular methods available
Serological tests for other bacterial infections - Mycoplasma pneumoniae antibody - antibodies tested?
IgM & IgG antibodies
Serological tests for other bacterial infections - Rickettsial antibody - diagnosis
-typhus
-rocky mountain spotted fever
-other rickettsial infections
Serological tests for other bacterial infections - Rickettsial antibody - common method(s)
-Gold standard: IFA, micro-IFA
-PCR available
Serological tests for infectious mononucleosis (IM) - heterophile antibodies - specificity
-non-specific antibodies that agglutinate horse, sheep, & bovine RBCs
-heterophile antibodies are antibodies that react with similar antigens from different species
Serological tests for infectious mononucleosis (IM) - heterophile antibodies - occurrence
-90% of patients develop in 1st month of illness
-can persist for 1 year
-negative in 10% of adults & up to 50% of children with IM
-if symptomatic & heterophile negative, test for EBV-specific antibodies
Serological tests for infectious mononucleosis (IM) - heterophile antibodies - tests
-rapid latex agglutination
-solid-phase immunoassay
-antigen is purified bovine RBC extract
-screening tests
Serological tests for infectious mononucleosis (IM) - EBV-specific antibodies - specificity
-specific antibodies against EBV antigens present in different phases of infection:
–early: early antigen (EA)
–late: viral capsid antigen (VCA)
–latent: EBV nuclear antigen (EBNA)
Serological tests for infectious mononucleosis (IM) - EBV-specific antibodies - occurrence
-anti-VCA IgM: appears at onset of symptoms, disappears in 3 months
-anti-VCA IgG: appears at onset of symptoms, persists for life
-anti-EBNA: present during convalescence
Acute infection:
-anti-VCA IgM,
-anti-VCA IgG
-anti-EA
Past infection:
-anti-EBNA
-anti-VCA IgG
-negative anti-VCA IgM
Serological tests for infectious mononucleosis (IM) - EBV-specific antibodies - tests
-IFA - gold standard but time consuming & harder to interpret
-ELISA
-CIA
-molecular tests can be used for immunocompromised patients who don’t produce antibodies
Hepatitis tests - Hepatitis A - Total anti-HAV - significance
past infection & immunity
Hepatitis tests - Hepatitis A - IgM anti-HAV - significance
acute infection
Hepatitis tests - Hepatitis A - HAV RNA - significance
current infection
Hepatitis tests - Hepatitis A - HAV RNA - used to detect?
HAV in food and water
Hepatitis tests - Hepatitis B - Hepatitis B surface antigen (HBsAg) - significance
acute or chronic infection, infectivity
Hepatitis tests - Hepatitis B - Hepatitis B surface antigen (HBsAg) - confirmation of positive
repeat testing and another assay such as hepatitis B DNA PCR
What is the first serological marker to appear during early acute infection of Hepatitis B?
Hepatitis B surface antigen (HBsAg)
Hepatitis tests - Hepatitis B - Hepatitis B e antigen (HBeAg) - significance
acute or chronic infection
Hepatitis tests - Hepatitis B - Hepatitis B e antigen (HBeAg) - indicative of?
high degree of infectivity
Hepatitis tests - Hepatitis B - Total anti-Hepatitis B core (HBc) - significance
current or past infection or carrier
Hepatitis tests - Hepatitis B - Total anti-Hepatitis B core (HBc) - prominent immunoglobulin
IgG, which persists for life
Hepatitis tests - Hepatitis B - IgM anti-HBc - significance
current or recent infection
Hepatitis tests - Hepatitis B - IgM anti-HBc - useful for detecting?
HBV infection when HBsAg is no longer detectable (“window period”)
What is the first antibody to appear in hepatitis B?
IgM anti-HBc
What tests are used to screen blood donors for hepatitis B?
HBsAG and IgM anti-HBc
Hepatitis tests - Hepatitis B - Anti-HBe - significance
recovery, reduced infectivity
Hepatitis tests - Hepatitis B - Anti-HBs - significance
recovery & immunity
What antibody develops following immunization for hepatitis B?
Anti-HBs
Hepatitis tests - Hepatitis B - Hepatitis B virus (HBV) DNA - significance
current infection
What is detectable 21 days before HBsAg in hepatitis B?
Hepatitis B virus (HBV) DNA
Hepatitis tests - Hepatitis C - Anti-HCVh - significance
acute, chronic, or previous infection
Hepatitis tests - Hepatitis C - HCV RNA - significance
current infection
Hepatitis tests - Hepatitis C - HCV RNA - used for?
-viral load testing
-blood/organ donor screening
-HCV genotyping to determine optimal treatment
Hepatitis tests - Hepatitis D - Hepatitis D (HDV) (delta hepatitis) - IgM anti-HDV - significance
acute or chronic infection
Hepatitis tests - Hepatitis D - Hepatitis D (HDV) (delta hepatitis) - IgM anti-HDV - a defective virus that can only occur in the presence of?
HBV
Hepatitis tests - Hepatitis D - Hepatitis D (HDV) (delta hepatitis) - IgG anti-HDV - significance
recovery or chronic infection
Hepatitis tests - Hepatitis D - Hepatitis D (HDV) (delta hepatitis) - HDV RNA - significance
current infection
Hepatitis tests - Hepatitis D - Hepatitis D (HDV) (delta hepatitis) - HDV RNA - marker of?
viral replication
Hepatitis tests - Hepatitis D - Hepatitis D (HDV) (delta hepatitis) - HDV RNA - used to?
monitor therapy
Hepatitis tests - Hepatitis E - tests
tests are not currently approved by the FDA for use in the US
Hepatitis serological profiles - Acute Hepatitis A
IgM anti-HAV +
Hepatitis serological profiles - Recovery from Hepatitis A
Total anti-HAV +
Hepatitis serological profiles - Acute Hepatitis B
HBsAg +
Total anti-HBc +
IgM anti-HBc +
Anti-HBs -
Hepatitis serological profiles - Recovery from Hepatitis B
HBs Ag -
Total anti-HBc +
Anti-HBs +
Hepatitis serological profiles - Chronic Hepatitis B/Carrier
HBsAg +
Total anti-HBc +
IgM anti-HBc -
Anti-HBs -
Hepatitis serological profiles - Hepatitis B immunization
HBsAg -
Anti-HBc -
Anti-HBs +
Appearance of HIV markers - Viral RNA
detectable within days of infection
Appearance of HIV markers - p24 ag
-core coat for nucleic acids
-detectable in 2-3 weeks
-becomes undetectable as antibodies develop, then detectable again in late stages as immune system fails & virus replicates
Appearance of HIV markers - IgM ab
-usually detectable in 2-8 weeks
-transient
-peaks in about 1-2 weeks
-undetectable about 1-2 weeks later
Appearance of HIV markers - IgG ab
-detectable shortly after IgM
-gradual increase in titer over several months
-long lasting
HIV screening tests - ELISA/CLIA - 1st generation - detect
IgG ab to HIV-1
HIV screening tests - ELISA/CLIA - 1st generation - window period
6-12 weeks
HIV screening tests - ELISA/CLIA - 2nd generation - detect
IgG ab to HIV-1/2
HIV screening tests - ELISA/CLIA - 2nd generation - window period
6-12 weeks
HIV screening tests - ELISA/CLIA - 3rd generation - detect
IgG & IgM ab to HIV-1/2
HIV screening tests - ELISA/CLIA - 3rd generation - window period
3-4 weeks
HIV screening tests - ELISA/CLIA - 4th generation - detect
IgG & IgM ab to HIV-1/2 and pg24 ag
HIV screening tests - ELISA/CLIA - 4th generation - window period
2 weeks
HIV screening tests - ELISA/CLIA - 5th generation - detect
-IgG & IgM ab to HIV but differentiates HIV-1 from HIV-2
-also detects p24 ag
HIV screening tests - ELISA/CLIA - acute infection
P24 ag without HIV ab
HIV screening tests - ELISA/CLIA - establish infection
P24 ag & HIV ab
HIV screening tests - rapid tests - detects
IgG & IgM ab to HIV
HIV screening tests - rapid tests - window period
4-12 weeks
HIV screening tests - rapid tests - assays
immunochromatographic
HIV screening tests - rapid tests - samples
whole blood, serum, oral fluid
HIV screening tests - Nucleic acid amplification testing (NAAT) - detects
HIV RNA
HIV screening tests - Nucleic acid amplification testing (NAAT) - window period
5 days
List the causes of false positives with HIV-antibody ELISA testing.
-heat inactivation of serum
-repeated freezing/thawing of serum
-autoantibodies
-multiple pregnancies
-liver disease
-administration of Ig
-administration of certain vaccines
-some malignancies
List the causes of false negatives with HIV-antibody ELISA testing.
-blood drawn before seroconversion (window period)
-hypogammaglobulinemia
-immunosuppressive therapy
-strain of HIV not detected by assay
-technical errors
HIV supplemental tests - Western blot (WB) - detects
antibody to HIV
HIV supplemental tests - Western blot (WB) - positive
Report positive if at least 2 of the following 3 bands are present:
-p24
-gp41
-gp1220/160
HIV supplemental tests - Western blot (WB) - negative
NAAT required following negative or indeterminant results
HIV supplemental tests - Western blot (WB) - disadvantages
-time-consuming
-difficult to interpret
HIV supplemental tests - NAAT - detects
HIV RNA
HIV supplemental tests - NAAT - useful when?
serology results are inconclusive
Tests to stage and monitor HIV - CD4 T-cell count
-HIV infects CD4 cells
-Number declines as disease progresses
-<200/uL defines stage 3 infection
-also used to monitor therapy
-perform every 3-6 months
-flow cytometry is gold standard
Tests to stage and monitor HIV - HIV-1 viral load assays: PCR (RT-PCR and qPCR); branched chain DNA assay (bDNA)
-quantitative methods to determine plasma HIV RNA
-used to predict disease progression, determine when to start antiretroviral therapy, & monitor response to therapy
-qPCR most frequently performed
-bDNA assays are used in labs with high testing volumes
-test 2-8 weeks after start of therapy & then every 3-4 months
-same assay should be used in order to assess changes
Screening tests for Systemic Lupus Erythematosus (SLE) - Antinuclear antibody (ANA) - method
-indirect immunofluorescence (IIF)
-substrate is human epithelial cell line (Hep-2)
-high sensitivity (95-100%) but low specificity
-dilutions tested to eliminate low titer reaction in normal population
-cutoff dilution to report positive usually >=1:80
-endpoint titer may be reported
-generally higher in SLE
Screening tests for Systemic Lupus Erythematosus (SLE) - Antinuclear antibody (ANA) - detects?
auto-antibodies to nuclear antigen - staining patterns reported
Screening tests for Systemic Lupus Erythematosus (SLE) - Antinuclear antibody (ANA) - disadvantages
-labor intensive
-subjective
Screening tests for Systemic Lupus Erythematosus (SLE) - Complement (C3) EIA - method
EIA
Screening tests for Systemic Lupus Erythematosus (SLE) - Complement (C3) EIA - C3
C3 is turned over rapidly in SLE patients, especially during flare-ups, will see a decrease in serum C3 levels
Screening tests for Systemic Lupus Erythematosus (SLE) - urinalysis - method
dipstick
Screening tests for Systemic Lupus Erythematosus (SLE) - urinalysis - looking for?
RBCs and protein to screen for kidney damage causes by Ag-autoAb complexes
Tests for specific antinuclear antibodies (ANA) - Anti-dsDNA - sensitivity for SLE
low
Tests for specific antinuclear antibodies (ANA) - Anti-dsDNA - specificity for SLE
high
(uncommon in other diseases or normal individuals)
What antibody is the most specific antibody for SLE?
Anti-dsDNA
Tests for specific antinuclear antibodies (ANA) - Anti-dsDNA - titers
correlate with disease activity
Tests for specific antinuclear antibodies (ANA) - Anti-dsDNA - IIF patterns
peripheral or homogeneous fluorescent pattern
Tests for specific antinuclear antibodies (ANA) - Anti-Sm - sensitivity for SLE
low
Tests for specific antinuclear antibodies (ANA) - Anti-Sm - specificity for SLE
high
Tests for specific antinuclear antibodies (ANA) - Anti-Sm - IIF pattern
coarsely speckled pattern
Tests for specific antinuclear antibodies (ANA) - Anti-Sm - test methods
EIA, immunodiffusion, IIF
Tests for specific antinuclear antibodies (ANA) - Anti-dsDNA - test methods
EIA, IIF
Tests for specific antinuclear antibodies (ANA) - Antihistone, anti-DNP, anti-SS-A/R0, anti-SS-B/La, anti-nRNP - sensitivity
low
Tests for specific antinuclear antibodies (ANA) - Antihistone, anti-DNP, anti-SS-A/R0, anti-SS-B/La, anti-nRNP - specificity for SLE
low
Tests for specific antinuclear antibodies (ANA) - Antihistone, anti-DNP, anti-SS-A/R0, anti-SS-B/La, anti-nRNP - diagnosis
-generally not useful for Dx of SLE
-used to Dx other connective tissue diseases (Sjogren syndrome)
Tests for specific antinuclear antibodies (ANA) - Antihistone, anti-DNP, anti-SS-A/R0, anti-SS-B/La, anti-nRNP - method
IIF, EIA, immunodiffusion
Tests for specific antinuclear antibodies (ANA) - Extractable nuclear antigen (ENA) - sensitivity for SLE
low
Tests for specific antinuclear antibodies (ANA) - Extractable nuclear antigen (ENA) - specificity for SLE
anti-Sm is specific for SLE
Tests for specific antinuclear antibodies (ANA) - Extractable nuclear antigen (ENA) - method
immunodiffusion (Ouchterlony double diffusion) test panel that typically tests for antibodies to Smith (Sm), SS-A/R0, SS-B/La, Ribonucleoprotein (RNP)
-precipitin lines of identity/nonidentity
-new method: multiplex bead assay - immunoassay using specific antigen-coated beads & flow cytometry to detect multiple (currently 6-13) ANAs simultaneously
Serological tests for Rheumatoid Arthritis (RA) - Rheumatoid factor (RF) - common method(s)
agglutination, ELISA, nephelometry
Serological tests for Rheumatoid Arthritis (RA) - Rheumatoid factor (RF) - autoantibody
autoantibody (usually IgM) against IgG
Serological tests for Rheumatoid Arthritis (RA) - Rheumatoid factor (RF) - specificity
low
Serological tests for Rheumatoid Arthritis (RA) - Rheumatoid factor (RF) - agglutination tests
only detect IgM RF
Serological tests for Rheumatoid Arthritis (RA) - Rheumatoid factor (RF) - ELISA & nephelometry
detect IgM, IgA, & IgG classes of RF
Serological tests for Rheumatoid Arthritis (RA) - Rheumatoid factor (RF) - positive in what percentage of patients with RA?
70-80%
Serological tests for Rheumatoid Arthritis (RA) - Anti-cyclic citrullinated peptide antibody (Anti-CCP) - common method(s)
ELISA
Serological tests for Rheumatoid Arthritis (RA) - Anti-cyclic citrullinated peptide antibody (Anti-CCP) - specificity
more specific for RA than RF
Autoimmune diseases - Systemic Lupus Erythematosus (SLE) - target cells and tissues
Multiple: kidneys, joints, skin, brain, heart, lungs
Autoimmune diseases - Systemic Lupus Erythematosus (SLE) - laboratory findings
-autoantibodies to dsDNA and other nuclear components
-also see decreased serum C3
-increased CRP & ESR
-RBCs/protein in ruine
Autoimmune diseases - Systemic Lupus Erythematosus (SLE) - abnormal CBC values
-decreased HGB & HCT
-low WBC and platelet count
Autoimmune diseases - Rheumatoid Arthritis (RA) - target cells and tissues
joints, bone, and other connective tissue
Autoimmune diseases - Rheumatoid Arthritis (RA) - laboratory findings
-anti-CCP most diagnostic
-rheumatoid factor
-positive ANA titer
-elevated CRP, ESR
-decreased C3
used to monitor treatment
Autoimmune diseases - Wegener Granulomatosis - target cells and tissues
upper respiratory system, lungs, and blood vessels
Autoimmune diseases - Wegener Granulomatosis - laboratory findings
-positive antineutrophil cytoplasmic antibody (ANCAs)
-glomerulonephritis
Autoimmune diseases - Wegener Granulomatosis - severity
rare but severe due to chronic activation of neutrophils, T cells, and B cells
Autoimmune diseases - Scleroderma - target cells and tissues
skin and blood vessels - fibrosis can occur in vessels of most organs
Autoimmune diseases - Scleroderma - laboratory findings
-Scl-70 autoantibodies
-speckled/nucleolar ANA pattern
-Raynaud’s phenomenon common
Autoimmune diseases - Sjogren Syndrome - target cells and tissues
lacrimal and salivary glands
Autoimmune diseases - Sjogren Syndrome - laboratory findings
autoantibodies toward RNA complexed with cellular proteins (SS-A/Ro and SS-B/La)
Autoimmune diseases - Sjogren Syndrome - can occur with what other diseases?
SLE and RA
Autoimmune diseases - Graves’ Disease - target cells and tissues
thyroid gland (commonly TSH receptors)
Autoimmune diseases - Graves’ Disease - laboratory findings
-decreased TSH
-increased T4
-TSH receptor autoantibodies
-may see thyroglobulin & TPO autoantibodies
Autoimmune diseases - Graves’ Disease - diagnostic in 99% of patients?
TSH receptor autoantibodies
Autoimmune diseases - Hashimoto Thyroiditis - target cells and tissues
thyroid gland (eipthelial cells)
Autoimmune diseases - Hashimoto Thyroiditis - laboratory findings
-increased TSH
-decreased T4
-TPO & thyroglobulin autoantibodies in most patients
Autoimmune diseases - Hashimoto Thyroiditis - diagnostic?
microsomal autoantibodies
Immunodeficiency diseases - Severe Combined Immunodeficiency (SCID) - immune component deficient
adaptive arm, IL-2 receptor mutation
Immunodeficiency diseases - Severe Combined Immunodeficiency (SCID) - laboratory findings
decreased and/or non-functional T cells and B cells
Immunodeficiency diseases - Severe Combined Immunodeficiency (SCID) - confirmation
genetic tests
Immunodeficiency diseases - Wiskott-Aldrich Syndrome - immune component deficient
cellular arm of immune system (mainly T cells but then B cells can’t form antibodies)
Immunodeficiency diseases - Wiskott-Aldrich Syndrome - laboratory findings
-decreased platelet numbers and size
-decreased IgM (to ABO antigens, can be diagnostic)
-elevated serum alpha fetoprotein (AFP)
Immunodeficiency diseases - Wiskott-Aldrich Syndrome - patients often have?
severe eczema
Immunodeficiency diseases - DiGeorge Anomaly - immune component deficient
chromosome 22 mutation, defective thymus or lack of thymus
Immunodeficiency diseases - DiGeorge Anomaly - laboratory findings
partial or complete lack of T cells
Immunodeficiency diseases - Ataxia Telangiectasia (AT) - immune component deficient
chromosomal breaks occur, inhibiting VDJ rearrangement for T and B cells
Immunodeficiency diseases - Ataxia Telangiectasia (AT) - laboratory findings
-decreased circulating T cells
-decreased levels of IgA, IgE, and IgG2
-increased serum AFP
Immunodeficiency diseases - Ataxia Telangiectasia (AT) - physical manifestations
ataxia and capillary swelling/red blotches
Immunodeficiency diseases - X-linked Bruton Tyrosine Kinase Deficiency - immune component deficient
antibody immunodeficiency, lack of CD19+ cells and all subsequent cells (plasma and memory cells)
Immunodeficiency diseases - X-linked Bruton Tyrosine Kinase Deficiency - laboratory findings
-lack of CD19+ cells via flow cytometry
-decreased or lack of IgA, IgE, IgG, IgM
Immunodeficiency diseases - Selective IgA Deficiency - immune component deficient
IgA only is deficient
Immunodeficiency diseases - Selective IgA Deficiency - laboratory findings
lack of serum IgA
Immunodeficiency diseases - Selective IgA Deficiency - physical findings
recurrent respiratory and GI infections
Immunodeficiency diseases - Chediak Higashi Syndrome - immune component deficient
NK cells/neutrophils
Immunodeficiency diseases - Chediak Higashi Syndrome - laboratory findings
-differential shows leukocytes with enlarged granules
-increased acute phase proteins and cytokines
Immunodeficiency diseases - Chronic Granulomatous Disease - immune component deficient
neutrophil microbiocidal function
Immunodeficiency diseases - Chronic Granulomatous Disease - laboratory findings
DHR/flow cytometry (decreased fluorescence)
Immunodeficiency diseases - Chronic Granulomatous Disease - increased susceptibility to?
pyogenic infections
Immunodeficiency diseases - Leukocyte Adhesion Deficiency - immune component deficient
CD18 on phagocytic cells
Immunodeficiency diseases - Leukocyte Adhesion Deficiency - laboratory findings
decreased CD18 on dendritic cells (measured by flow cytometry)
Immunodeficiency diseases - Leukocyte Adhesion Deficiency - physical findings
-delayed wound healing
-chronic skin, intestinal, and respiratory tract infections
Interpretation of serological tests -
titer concentration
> =4-fold increase in titer from acute to convalescent specimen drawn 10-14 days later is diagnostic
Interpretation of serological tests - IgM ab
sign of recent infection
Interpretation of serological tests - IgG ab
sign of immunity
Interpretation of serological tests - IgG ab in newborn
is maternal antibody
How would you prepare a 5% suspension of human group O RBCs?
5% = 5 mL per 100 mL
Mix 5 mL of packed RBCs + 95 mL of buffer.
For a smaller amount: 0.5 mL of packed RBCs + 9.5 mL buffer
Any 1:20 dilution could be used (5:100 = 1:20)
How would you prepare 5 mL of a 1:10 dilution of serum?
(1/10) = (x/5)
10x = 5
x = 0.5 mL
0.5 mL diluted to 5 mL is a 1:10 dilution, so mix 0.5 mL serum + 4.5 mL of buffer
How would you prepare 10 mL of a 1:100 dilution from a 1:10 dilution?
A. Determine the dilution factor to make a 1:100 dilution from a 1:10 dilution:
1/10 x 1/x = 1/100
1/10x = 1/100
10x = 100
x = 10
A 1:10 dilution of a 1:10 dilution yields a 1:100 dilution (1/10 x1/10 = 1/100)
B. Determine how to make 10 mL of a 1:10 dilution:
1:10 dilution is 1 part + 9 parts
To make 10 mL, mix 1 mL of solution + 9 mL of buffer.
To make 10 mL of a 1:100 dilution from a 1:10 dilution, mix 1 mL of the 1:10 dilution + 9 mL of buffer
What is the dilution in tube 4 of a 2-fold serial dilution, if tube 1 is undiluted?
1 x 1/2 x 1/2 x 1/2 = 1/8
Which cell of the innate immune system has granules that contain bactericidal enzymes?
A. Dendritic cell
B. Neutrophil
C. T cell
D. Mast cell
B. Neutrophil
Which part of an antibody molecule is responsible for complement fixation and opsonization?
A. Fc fragment
B. Fab fragment
C. Light chain
D. Joining (J) chain
A. Fc fragment
Which hypersensitivity reaction is predominantly mediated by T cells?
A. Type I
B. Type II
C. Type III
D. Type IV
D. Type IV
From the following list, which test is most specific for RA?
A. Anti-dsDNA
B. Rheumatoid factor (RF)
C. Anticyclic citrullinated peptide (CCP)
D. Anti-SS-A/Ro
C. Anticyclic citrullinated peptide (CCP)
Which HIV marker is detectable within days of infection in patient serum?
A. Viral RNA
B. P24 antigen
C. IgM antibody
D. IgG antibody
A. Viral RNA
Which is the first antibody detected in serum after infection with hepatitis B?
A. Anti-HBe
B. Anti-HBc IgM
C. Anti-HBs
D. HBeAg
B. Anti-HBc IgM
In infectious mononucleosis, which of the following antibodies would be detected earliest during acute infection?
A. Anti-VCA IgM
B. Anti-EBNA
C. Heterophile antibodies
D. Anti-EA IgG
A. Anti-VCA IgM
Which of the following is a cause of a biological false-positive in the nontreponemal assays for syphilis?
A. Infectious mononucleosis
B. Rheumatoid arthritis
C. pregnancy
D. All of the above
D. All of the above
In which assay can a high concentration of antigen (analyte) cause hook effect?
A. EMIT
B. FPIA
C. Sandwich ELISA
D. TP-PA
C. Sandwich ELISA
Which type of immunoassay does NOT require a separation step?
A. Noncompetitive
B. Homogenous
C. Heterogenous
D. All of the above
B. Homogenous