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Immunology Terms
Acute phase Protein that increases due to infection, injury, or
reactant trauma (e.g., C-reactive protein, alpha-1 antirypsin,
haptoglobin, fibrinogen, ceruloplasmin, alpha-1 acid
glycoprotein, complement)
Alloantibody Antibody formed in response to antigens from
individuals of the same species
Antigen A foreign substance that stimulates antibody
production. Large, complex molecules
(MW>10,000), usually protein or polysaccharide.
Antibody Immunoglobulin produced by plasma cells in
response to an antigen
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Autoantibody Antibody against self
Avidity Strength of bond between antigen and antibody
Chemotaxin Chemical messenger that causes migration of cells in
a particular direction
Clusters of Antigenic of leukocytes
differentiation
(CD)
Cytokine Chemical messenger produced by stimulated cells that
affect the function of the other cells
Epitope Determinant site on an antigen
Hapten A low molecular weight substance that can bind to an
antibody once it is formed, but that is incapable of
stimulating antibody production unless it is bound to a
larger carrier molecule
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Histamine A vasoactive amine that is released from mast cells and
basophils during an allergic reaction
Hypersensitivity A heightened state of immune responsiveness that causes
tissue damage in the host
Immunity Resistance to infection
Immunogen Any substance that is capable of inducing an immune
response
Immunoglobulin Antibody
Inflammation Cellular and humoral mechanisms involved in the reaction
of the body to injury or infection
Interferons Cytokines produced by T cells and other cells that inhibit
viral synthesis or act as immune regulators
Interleukins Cytokines produced by T cells or macrophages that
stimulate a number of cell types
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Ligand A molecule that binds to another molecule of a complementary
configuration. The substance being measured in an immunoassay.
Lymphokines Polypeptides given off by antigen-stimulated T cells, which
regulate the function of other cells
Lysozyme Enzyme found in tears and saliva that attacks cell walls of
microorganisms
MHC Major histocompatoibility complex. Genes that control the
expression of proteins found on all nucleated cells.
Monoclonal An antibody derived from a single B-cell clone
antibody
Opsonin Serum proteins that attach to a foreign substance and enhance
phagocytosis
Phagocytosis The engulfment of cells or particulate matter by neutrophils and
macrophages
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Plasma cells Transformed B cell that secretes antibody
Polyclonal antibody Antibody produced by many B-cell clones
Postzone False-negative reactions in a serological test due to antigen excess
Prozone False-negative reactions in a serological test due to antibody
excess
Seroconversion The change of a serological test from negative to positive due to
development of detectable antibody
Serum sickness A type III hypersensitivity reaction that results from the buildup of
antibodies to animal serum used in some passive immunizations
Thymus A small, flat bilobed organ found in the thorax. Site of T-
lymphocyte development.
Titer A means of expressing the concentrations of an antibody. The
reciprocal of the highest dilution in which a positive reaction
occurs.
Vaccination Injection of immunogenic material in order to induced immunity
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Branches of the Immune System
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Types of Immunity
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Adaptive Immunity
Type Explanation Example Specific? Immediate? Long-
term?
Naturally An individual Clinical or Yes No Yes
acquires infected with a subclinical
active microorganism infection
immunity produces an
antibody.
Artificially An individual DPT, MMR, Yes No Yes
acquired exposed to an polio,
active antigen through a tetanus,
immunity vaccine develops Haemophilus
immunity influenzae
without having type b
the infection. vaccine
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Adaptive Immunity Continued
Type Explanation Example Specific? Immediate? Long-
term?
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IgG IgM IgA IgD IgE
Antigen- 2 10 2 or 4 2 2
binding sites
Crosses Yes No No No No
placenta?
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IgG IgM IgA IgD IgE
Other Defense First Ig produced In tears, Function Role in allergic
against in an immune sweat, unknown reactions.
bacteria and response. Only saliva, Binds to mast
viruses. Ig at initiating respiratory cells and
Neutralizes complement and GI triggers
toxins. cascade. More mucosa. degranulation
Opsonin. efficient at First line and release of
More efficient agglutination of defense. histamine and
at precipitation than IgG. heparin.
than Destroyed by
agglutination. sulfhydryl
compounds.
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Cells of the Immune System
Cell Function Comments
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Cells of the Immune System Continued
Cell Function Comments
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Cells of the Immune System Continued
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Lymphoid Organs
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Laboratory Identification of
To Obtain Lymphocytes Lymphocytes
Density gradient centrifugation with Layers from top to bottom: plasma, mononuclear cells,
Ficoll-Hypaque Ficoll-Hypaque, RBCs and granulocytes
Flow cytometry (FACS: fluorescence Use of labeled monoclonal antibodies against specific
activated cell sorter) surface antigens. Light scattering is measured as cells
flow through a laser beam.
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Common Lymphocyte Markers
T cells B cells
CD2 CD19
CD3 CD20
CD4 CD21
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Complement
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Immunoglobulin Structure
Basic structure A four-chain polypeptide unit that consists of two heavy chains
and two light chains held together by disulfide bonds
Fab fragment Fragment antigen-binding. Consists of one light chain and one-
half of a heavy chain. The intact immunoglobulin has two Fab
fragments, each representing one antigen-binding site.
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Immunoglobulin Structure Continued
Fc fragment Fragment crystalline. The carboxy-terminal halves of the two
heavy chains. This portion of the molecule has no antigen binding
ability.
Constant region The amino-terminal end of the immunoglobulin molecule, where
the amino acid sequence is the same for all chains of that type.
Variable region The amino-terminal end of the immunoglobulin molecule, where
the amino acid sequence varies. This part of the molecule is
responsible for the specificity of a particular immunoglobulin.
Also known as the antigen-recognition unit.
Hypervariable Regions within the variable region that actually form the antigen-
region binding site. Through changes in the hypervariable region, an
immense diversity of the antigen-binding sites can be created.
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Immunoglobulin Structure Continued
Hinge region The flexible portion of the heavy chain, located between the first
and second constant regions. This allows the molecule to bend to
let the two antigen-binding sites operate independently.
Joining chain A glycoprotein that serves to link immunoglobulin monomers
together. Only found in IgM and secretory IgA molecules.
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Complement Continued
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Immune Phenomena
Explanation Application
Primary Combination of Not easily detectable. Can be measured
phenomena antibody with antigen indirectly by radioimmunoassay (RIA),
enzyme immunoassay (EIA),
immunofluorescence.
Secondary Precipitation, Measured more readily. Basis for many
phenomena agglutination, serological tests.
complement fixation
Tertiary In vivo reactions; e.g., Some are useful diagnostically, e.g., skin
phenomena inflammation, testing.
phagocytosis,
desposition of immune
complexes, immune
adherence, chemotaxis
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Hypersensitivity Reactions
Type I: TypeII: Type III: Type IV: Cell
Anaphylactic Cytotoxic Immune Mediated
Complex
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Hypersensitivity Reactions Continued
Type I: TypeII: Cytotoxic Type III: Type IV: Cell
Anaphylactic Immune Complex Mediated
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Agglutination Methods
Method Principle Examples
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Agglutination Methods Continued
Method Principle Examples
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Agglutination Methods Continued
Method Principle Examples
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Precipitation Methods Continued
Counter Antigens and antibodies are placed in Bacterial antigens
immunoelectrophoresis wells that are directly opposite one
(CIE) another in a gel. An electrophoretic
charge is applied to drive the reactants
toward each other. A precipitin band
forms where they meet.
Immunoelectrophoresis Proteins are separated by Identification of
(IEP) electrophoresis, then subjected to immunoglobulins in
double diffusion with reagent monoclonal
antibodies placed in a trough cut in the gammophaties
agar. The shape, intensity, and location
of the precipitin arcs that develop are
compared with those of a normal
control.
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Precipitation Methods Continued
Method Principle Application
Radial Antigen is measured based on the Quantitation of
immunodiffusion diameter of a precipitin ring that forms immunoglobulins and
(RID) when it diffuses out of a well nin a gel- complement
containing antibody.
Rocket An electrical charge is applied to an Immunoglobulins,
electrophoresis RID assay. The height of the rocket- complement,
shaped precipitin band obtained is
proportional to the concentration of the
antigen.
Nephelometry Light scattering by immune complexes Immunoglobulins,
is measured. complement, C-
reactive protein
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Method Other
Principle Serological Methods
Comments Example
Complement Patient serum is Patient serum must be Viral, fungal,
fixation incubated with antigen inactivated. Best for rickettsial
and complement. If the demonstration of IgM antibodies
corresponding antibody antibodies. More
is present in the serum, it sensitive than
forms a complex with agglutination and
the antigen and precipitation, but less
complement. When sensitive than labeled
sensitized RBCs are immunoassays. Not
added, there is no free widely used.
complement to lyse
them. No hemolysis is a
positive reaction.
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Other Serological Method Continued
Direct The specimen is placed on Detects antigens. Bacterial
fluorescent a glass and overlaid with Fluorescent compounds antigens
antibody fluorescein-labeled absorb energy from light
antibody. If the source and convert it
corresponding antigen is into light of a longer
present, the labeled-antiody wavelength (lower
binds and fluorescence will energy) than that
be seen with a fluorescent absorbed. Fluorescent
microscope. labels: fluorescein
isothiocyanate or
rhodamine B
isothiocyanate.
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Other Serological Method Continued
Method Principle Comments Example
Indirect Reagent antigen on a glss “Sandwich Fluorescent
fluorescent slide is overlaid with technique.” Detects antinuclear antibody
antibody patient serum. If the antibodies in serum. (FANA), fluorecent
corresponding antibody is treponemal
present in the serum, it antibody (FTA)
attaches to the antigen.
When fluorescein-labeled
antihuman globulin is
added, it attaches to the
antibody. Fluorescence
will be seen with a
fluorescent microscope.
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Labeled Immunoassay Terminology
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Labeled Immunoassay Terminology Continued
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Comparison of Radioimmunoassay and
Enzyme Immunoassay
RIA EIA
Label Radioisotope, usually 125I Enzyme
Measurement Counts per minute in Color development after
scintillation counter addition of substrate
Advantages Sensitivity Sensitivity
Specificity Specificity
Long shelf-life of reagents
No radiation hazard
No special disposal
requirements
No need for scintillation
counter
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Comparison of Radioimmunoassay and
Enzyme Immunoassay Continued
RIA EIA
Disadvantages Short shelf-life of reagents
Radiation hazard
Regulations governing disposal
of radioisotopes
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Immunoassay Labels
Radioisotopes Enzymes Fluorochromes* Chemiluminescent Molecules↑
125 I Horseradish Fluorescein Luminol
peroxidase
3H β-D- Rhodamine Acridium esters
galactosidase
14C Alkaline Dioxetane phospate
phosphatase
• *Fluorochromes absorb energy from light source and convert itinto light of a longer
wavelength and lower energy.
• ↑Chemiluminescent molecules produce light energy from a chemical reaction.
•
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Principles of Labeled Immunoassays
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Principles of Labeled Immunoassays Continued
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Principles of Labeled Immunoassays
Continued
Method Type of Assay Principle
Indirect or no- Noncompetitive Antibody in the specimen attaches to a
competitive ELISA Nonisotopic solid-phase antigen. After incubation and
Heterogeneous washing to remove unbound antibody, an
enzyme-labeled antiglobulin is added. This
second antibody reacts with the Fc portion
of the patient antibody bound to the solid
phase. Following another wash, substrate is
added. The amount of enzyme label
detected is directly proportional to the
amount of antibody in the serum. Indirect
ELISAs are more sensitive than direct
ELISAs.
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Principles of Labeled Immunoassays
Continued
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Principles of Labeled Immunoassays
Method Type of Assay
Continued
Principle
Sandwich Noncompetitive The antigen in the specimen is sandwiched
enzyme- Nonisotopic between antibody attached to a solid phase and
multiplied Heterogeneous enzyme-labeled antibody. Enzymatic activity is
immunoassay directly proportional to the amount of antigen in
the test sample.
Enzyme- Competitive The antigen in the specimen and an enzyme-
multiplied Nonisotopic labeled antigen compete for binding sites on
immunoassay Homogeneous reagent antibody. When enzyme-labeled antigen
technique binds to antibody, enzyme activity is inhibited.
(EMIT) Enzyme activity is directly proportional to the
concentration of the antigen in the specimen.
This is an automated method that is frequently
used for drug assays.
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Principles of Labeled Immunoassays Continued
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Principles of Labeled Immunoassays Continued
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Labeled Immunoassays
Isotopic Non-Isotopic
Competitive Radioimmunoassay (RIA) ELISA
Heterogeneous
Homogeneous EMIT
SLFIS
FPIA
Noncompetitive Immunoradiometric assay Indirect ELISA
Heterogeneous (IRMA) IEMA
Sandwich enzyme-multiplied
immunoassay
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Nontreponemal Tests for Syphilis
VDRL RPR
Method Flocculation Flocculation
Detects Reagin Reagin
Reagent(s) Cardiolipin Cardiolipin with charcoal
added
Positive reaction Microscopic clumps Microscopic agglutination
Specimen(s) Inactivated serum, CSF Serum (inactivation not
required), plasma
Reactivity during May be negative in primary stage. Same as VDRL
disease Titers usually peak during
secondary or early late stages.
Titers decline in late stage, eve3n
when untreated. More rapid decline
with treatment. Becomes
nonreactive following successful
treatment.
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Nontreponemal Tests for Syphilis
Continued
VDRL RPR
False- 10-30% may be biological false positive; Same as VDRL
positives e.g., infectious mononucleosis (IM),
infectious hepatitis, malaria, leprosy, lupus
erythematosus, rheumatoid arthritis,
advanced age, pregnancy. Positive in other
treponemal infections such as yaws and
pinta.
Other Replaced by RPR for serum. Still Good for screening and
performed on CSF.. treatment monitoring
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Treponemal Tests for Syphilis
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Treponemal Tests for Syphilis Continued
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Treponemal Tests for Syphilis Continued
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Test for the Diagnosis of Infectious
Mononucleosis (IM)
Antibodies Interpretation
Heterophile antibodies*
IM antibodies: Agglutinate sheep, beef, Agglutination of sheep or horse cells
ox, and horse RBCs. Absorbed by following adsorption with GPK but not with
beef RBCs but not by guinea pig kidne beef RBCs = IM.
cells (GPK). No agglutination of sheep or horse cells
Serum sickness antibodies: Agglutinate following adsorption with GPK or beef
sheep, beef, and horse RBCs. RBCs = serum sickness.
Adsorbed by beef RBCs and guinea pig
kidney cells. Aggllutination of sheep or horse cells
Forssman antibodies: Agglutinate sheep following adsorption with beef RBCs but
and horse RBCs. Adsorbed by guinea pig not with GPK = Forssman.
kidney cells but not by beef RBCs.
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Test for the Diagnosis of Infectious
Mononucleosis (IM) Continued
Antibodies Interpretation
Specific viral antibodies
IgM-VCA (viral capsid antigen): Peaks 3-4 IgM anti-VCA, anti-EA, or IgA anti-
weeks after infection. Undetectable after 12 VCA without EBNA = recent or current
weeks. infection.
IgG-VCA: Appears in late acute phase and Anti-EBNA or IgG anti-VCA without
persists for life. IgM anti-VCA = past infection.
Anti-EA (early antigen): Appoears early and
persists for years.
Anti-EBNA (EB nuclear antigen): Appears 2-
3 months after infection and persists
indefinitely.
*Nonspecific antibodies that cross-react with antigens other than those originally
responsible for their production.
•
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Weil-Felix Reaction
Disease Organism Transmission Weil-Felix Reaction
OX-2 OX-19 OX-K
Rocky Rickettsia Dog or wood tick + ++++ 0
Mountain rickettssi
spotted fever*
Epidemic R. prowazekii Body or head louse + ++++ 0
typhus
Endemic R. typhi Rat flea + ++++ 0
typhus
Scrub typhus R. tsutsugamushi Mite 0 0 ++++
Q fever Coxiella burnetii Inhalation of 0 0 0
barnyard dust or
ingestion of infected
meat or milk
• Most common rickettsial disease in U.S.
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Antinuclear Antibodies
Antibody Against Specificity Sensitivity Indirect Comments
Fluorescent
Antibody
(IFA) Pattern
Anti-ds- Double- Specific for Found in Peripheral or Considered
DNA stranded DNA SLE only 50- homogeneous diagnostic
80% of for SLE,
patients especially
with SLE when C3 is
low.
Anti-Sm Extractable Specific for Only found Speckled
nuclear SLE. Not in 30% of
antigen found in other patients
associated disorders. with SLE
with RNA
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Antinuclear Antibodies Continued
Antibody Against Specificity Sensitivity Indirect Comments
Fluorescent
Antibody
(IFA) Pattern
Anti-DNP Deoxyribonuc- Present in Present in Peripheral or LE factor
leoprotein SLE, and 70-90% of homogeneous
drug-induced patients
SLE with SLE
Anti-SS- Extractable Present in Present in Speckled Most often in
A/Ro nuclear SLE, 25-40% of patients with
antigen. RNA scleroderma SLE cutaneous
protein and patients manifestations
conjugate. Sjogren’s (photosensiti-
syndrome vity
dermatitis)
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Antibody Against Specificity Sensitivity Indirect Comments
Fluorescent
Antibody
(IFA) Pattern
Anti-SS- Ribonucl- Present in Present in 10- Speckled
B/La eoprotein SLE, 15% of SLE
scleroderma patients
and
Sjogren’s
syndrome
Anti-RNP Proteins Present in Speckled
complexed SLE, mixed
with connective
nuclear tissue disease
RNA and other
autoimmune
12/8/21 diseases 61
Antinuclear Antibodies Continued
Antibody Against Specificity Sensitivity Indirect Comments
Fluorescent
Antibody
(IFA) Pattern
Anti- Histone Present in Present in Homogeneous Diagnostic
histone (nucleoprot SLE, RA, almost all drug-induced
ein that is a primary patients with lupus. High
major biliary drug-induced levels
constituent cirrhosis lupus associated with
of more active and
chromatin) severe forms of
SLE.
– Multiple ANAs can be present in a specimen. More than on epattern will be observed.
– Fluorescence of nucleoli is due to antibody to RnA. Seen in scleroderma and polymyositis.
•
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Hepatitis Tests
Test Indication
Hepatitis A
Anti-HAV (total) Past infection and immunity
Anti-HAV (IgM) Acute hepatitis A
Hepatitis B
HbsAg Acute or chronic infection HBV (carrier), infectivity
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Hepatitis Tests Continued
Test Indication
Hepatitis C Acute, chronic, or previous hepatitis C infection
Anti-HCV
Hepatitis D Past or current hepatitis D infection
Anti-HD
Hepatitis E
None
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Hepatitis Profiles
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HIV Testing
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HIV Testing Continued
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HIV Testing Continued
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HIV Testing Continued
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HIV Testing Continued
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Viral Testing
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Other Serological Tests
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Other Serological Tests Continued
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Test Common Reagents Interpretation Comments
Method
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Other Serological Tests Continued
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Test Common Reagents Interpretation Comments
Method
Febrile Bacterial Salmonella O Positive reaction Screening test for fever of
agglutinins agglutination and H, is agglutination. unknown origin. Weil-
Brucella, Felix reaction: rickettsial
abortus, antibodies cross react
Proteus OX with OX-2, OX-19, and
19. OX-K strains of Proteus.
Widal’s test: detects
antibodies in typhoid
fever, tularemia,
brucellosis. Antibodies to
O = recent infection.
Antibodies to H = past
infection. Best indicator
of active infection is four
fold increase in titer.
Non-specific test.
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Infrequently ordered
Interpretation of Serological Tests
12/8/21 78
Serology Calculations
How would you prepare a 5% suspension of human group O red blood cells?
5% = mL per mL.
5 mL of packed RBCs + 95 mL of buffer.
How would you prepare 5 ml of a 1:10 dilution of serum?
1 =x
10 5
10x = 5
x = 0.5 mL
Mix 0.5 mL of serum with 4.5 mL of buffer.
How would you prepare 10 mL of 1:100 dilution from a 1:10 dilution?
A 1:10 dilution of a 1:10 dilution yields a 1:100 dilution (1/10 x 1/10 = 1/100).
Mix 1 mL of the 1:10 dilution with 9 mL of buffer. (1 mL + 9 mL = 1:10)
How would you prepare 10 mL of a 1:500 dilution from a 1:100 dilution?
A 1:5 dilution of a 1:100 dilution yields a 1:500 dilution (1/5 x 1/100 = 1/500).
Mix 2 mL of the 1:100 dilution with 8 mL of buffer. (2 mL + 8 mL = 2:10 = 1:5.)
What is the dilution in tube 4 of a twofold serial dilution, if is undiluted?
1 x ½ x ½ x ½ = 1/8
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The End
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