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agglutination methods Flashcards

1
Q

visible expression of the aggregation of antigens and antibodies

A

Precipitation and agglutination

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2
Q

aggregation of soluble test antigens

A

Precipitation

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3
Q

soluble antigen + soluble antibody =

A

visible insoluble complex

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4
Q

whereby specific antigens (e.g., red blood cells) aggregate to form larger visible clumps when the corresponding specific antibody is present in the serum

A

Agglutination

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5
Q

In Agglutination…

_______ may be needed to indicate visibly that an antigen-antibody reaction has taken place (latex particles and colloidal charcoal)

A

Artificial carrier particles

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6
Q

easy to perform; most sensitive tests currently available

A

Agglutination

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7
Q

first agglutination tests were developed

A

1960s

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8
Q

types of agglutination

A
  1. Latex Agglutination
  2. Flocculation Tests
  3. Direct Bacterial Agglutination
  4. Hemagglutination
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9
Q

(1)

bound to the surface of latex beads (C-reactive protein)
forming visible cross-linked aggregates of latex beads and antigen

A

Antibody molecules

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10
Q

(1)

  • coated the latex particles (pregnancy testing, rubella antibody testing)
  • agglutinate into large visible clumps
A

Antigen

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11
Q

Variations of latex agglutination

A

Coagglutination
Liposome-enhanced Testing

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12
Q

(1)

  • Uses antibodies bound to a particle to enhance the visibility of agglutination
  • Highly specific method
  • Not as sensitive as latex agglutination
A

Coagglutination

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13
Q

detect minute amounts of human chorionic gonadotropin (hCG)

A

LATEX AGGLUTINATION: PREGNANCY TESTING

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14
Q

a glycoprotein hormone secreted by the trophoblast of the developing embryo

A

hCG

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15
Q

first 6 to 8 weeks after conception: helps maintain the ______ and stimulate the production of ______

A

corpus luteum ; progesterone

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16
Q

hCG two noncovalently linked subunits

A

a unit and B subunit

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17
Q

identical to luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH)

A

a-unit

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18
Q

many pregnancy test kits contain ________ directed against the ______ to increase the _______ of the reaction

A

monoclonal antibody (MAb) ; β subunit ; specificity

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19
Q

level of hCG should double every ___ to ___ days

A

2 to 3

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20
Q

incubation of the patient’s specimen with anti-hCG, then addition of latex particles coated with hCG

A

Agglutination Inhibition

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21
Q

agglutination inhibition reaction results

A

(+) rxn: no agglutination (neutralizes the antibody)
(-) rxn: agglutination

If hCG is present, it neutralizes the antibody; thus,
no agglutination of latex particles is seen. If no
hCG is present, agglutination occurs between the
anti-hCG and hCG-coated latex particles.

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22
Q

latex agglutination that is rapid, direct, uses monoclonal Antibodies

A

Pregnancy Latex Slide Agglutination

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23
Q

Pregnancy Latex Slide Agglutination reaction results

A

(+) rxn: agglutination within 2 mins
(-) rxn: no agglutination

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24
Q

In pregnancy latex agglutination..

agglutination should be read within _______ to avoid erroneous results caused by _______

A

3 minutes ; evaporation

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25
Q

False-Positive Results (latex agglutination pregnancy testing)

A
  • hCG injection (Pregnyl): can remain in the patient’s system for as long as 10 days
  • Chorioepithelioma
  • hydatidiform mole
  • excessive ingestion of aspirin
  • testicular tumor
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26
Q

False-Negative Results (latex agglutination pregnancy testing)

A

Testing before reaching detectable levels of hCG

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27
Q

(2) for antibody detection

A

FLOCCULATION TESTS

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28
Q

(2) soluble antigen + antibody =

A

formation of a precipitate of fine particles

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29
Q

used in syphilis serologic testing

A

FLOCCULATION TESTS

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30
Q

tests in the flocculation tests..

A

VDRL (Venereal Disease Research Laboratories) Test
RPR (Rapid Plasma Reagin) Test

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31
Q
  • antibody-like protein, reagin, binds to the test antigen, cardiolipin-lecithin–coated cholesterol particles,
  • produces the particles that flocculate
A

Venereal Disease Research Laboratories (VDRL) test

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32
Q

antigen, cardiolipin-lecithin–coated cholesterol with choline chloride, also contains charcoal particles that allow for macroscopically visible flocculation

A

Rapid Plasma Reagin (RPR) Test

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33
Q

Used to detect antibodies directed against the
pathogens, test produced by the host to antigen
determination on the surface of bacterial agent in
response to infection within that bacterium.

A

Direct Bacterial Agglutination

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34
Q

(3) In a thick suspension of the bacteria, the binding of specific antibodies to surface antigens of the bacteria causes the bacteria to clump together in visible aggregates. This type of agglutination is called

A

Bacterial Agglutination

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35
Q

use of _______ enhances the aggregation of bacteria since most bacterial surfaces exhibit a _______ that causes them to repel each other

A

sterile physiologic saline ; negative charge

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36
Q

_______ is considered more sensitive than slide testing because it allows more time for the antigen-antibody reaction

A

tube testing

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37
Q

(4) detects antibodies to erythrocyte antigens

A

HEMAGGLUTINATION

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38
Q

HEMAGGLUTINATION specimen

A

contains Ab

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39
Q
  • detect antibodies to antigens other than those present on the cells
  • used to cross-link antigens to the cells: chromic chloride, tannic acid, and glutaraldehyde
A

Indirect hemagglutination or passive hemagglutination (PHA)

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40
Q

______ is the clumping of particles that have antigens on their surface

A

Agglutination

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41
Q

Mechanisms of Agglutination

A

Sensitization
Lattice Formation

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42
Q
  • First phase
  • physical attachment of antibody molecules to antigens on the erythrocyte membrane
  • combination of antigen and antibody is a reversible chemical reaction
  • Elution: when physical conditions are purposely manipulated to break the antigen-antibody complex, with subsequent release of the antibody into the surrounding medium
A

Sensitization

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43
Q

In sensitization, combination of antigen and antibody is a _______

A

reversible chemical reaction

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44
Q

when physical conditions are purposely manipulated to break the antigen-antibody complex, with subsequent release of the antibody into the surrounding medium

A

Elution

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45
Q

Inert particles such as latex, RBCs, and bacteria
have a NET NEGATIVE SURFACE CHARGE called the
________

A

zeta potential

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46
Q

a technique to reduce zeta potential that removes negatively charged sialic acid residues from cell surface membrane

A

Enzyme pretreatment of red blood cells

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47
Q

a technique to reduce zeta potential that increases electrical conductivity of environment

A

Addition of colloids (e.g., albumin)

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48
Q

a technique to reduce zeta potential which action is mechanical process to force red blood cells closer together.

A

Centrifugation

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49
Q

each antibody molecule must have at least 2 binding sites and that an antigen must be multivalent

A

Lattice hypothesis

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50
Q

Zone of equivalence=

A

detectable reaction

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51
Q

overcome by serially diluting the antibody-containing serum until optimum amounts of antigen and antibody are present in the test system

A

prozone phenomenon

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52
Q

small aggregates (clumps) are surrounded by excess antigen

A

postzone phenomenon

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53
Q

Repeat blood specimen should be collected _______ weeks later

A

1 or more

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54
Q

______ and _______ of antigenic determinants both affect agglutination

A

placement and number

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55
Q

If dissimilar antibodies with the same binding constant are directed against antigenic determinants located close to each other, antibodies will compete for space in reaching their specific receptor sites

A

Steric Hindrance

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56
Q

effect of antibody competition

A

antibody blocking

57
Q

(4) pH of the medium used for testing:

A

optimum pH of 6.5 to 7.5

58
Q

cold-reacting (thermal range, 4° C to 22° C)

A

IgM antibodies

59
Q

warm-reacting, with an optimum temperature of reaction at 37° C

A

IgG antibodies

60
Q

establishment of cross-links between sensitized particles RBCs) and antibodies, resulting in aggregation which is a slower process than the sensitization phase

A

Lattice Formation

61
Q

Methods of enhancing agglutination

A
  1. Centrifugation
  2. Treatment with proteolytic enzymes
  3. Use of colloids
  4. AHG Testing
62
Q

method of enhancing agglutination..

  • physically forces the cells together
  • overcome the problem of distance by subjecting sensitized cells to a high gravitational force
A

Centrifugation

63
Q

method of enhancing agglutination..

alters the zeta potential or dielectric constant

A

Treatment with proteolytic enzymes

64
Q

method of enhancing agglutination..

antigens may be so deeply embedded in the membrane surface

A

AHG testing

65
Q

Observation of agglutination: gently shaking the test tube containing the serum and cells and viewing the lower portion, the button

A

Graded Agglutination Reactions

66
Q

Because agglutination is a
reversible reaction, the test tube must be
treated delicately, and _______

A

hard shaking

67
Q

False appearance of clumping

A

Pseudoagglutination

68
Q

Pseudoagglutination rarely occur because of _____

A

Rouleaux Formation

69
Q

Rouleaux formation can be encountered in patients with …..

A

high or abnormal types of globulins in their blood (multiple myeloma or after receiving dextran as a plasma expander)

70
Q

To disperse the pseudoagglutination, a few drops of ______ can be added to the reaction tube, remixed, and reexamined.

(PS: performed after pseudoagglutination is suspected)

A

physiologic NaCl (saline)

71
Q

grading agglutination reactions…

D: no aggregates

72
Q

grading agglutination reactions…

D: A few isolated aggregates; mostly free-floating cells; supernatant appears red

A

Mixed field

73
Q

grading agglutination reactions…

D: Tiny aggregates barely visible macroscopically; many free erythrocytes; turbid and reddish supernatant.

74
Q

grading agglutination reactions…

D: A few small aggregates just visible macroscopically; many free erythrocytes turbid and reddish supernatant

75
Q

grading agglutination reactions…

D: Medium-sized aggregates; some free erythrocytes; clear supernatant.

76
Q

grading agglutination reactions…

D: Several large aggregates; some free erythrocytes; clear supernatant.

77
Q

grading agglutination reactions…

D: All erythrocytes are combined into one one solid aggregate; clear supernatant.

78
Q

In Microplate Agglutination Reactions, _____ and _____ allows for the performance of a large number of tests on a single plate

A

hemagglutination and solid-phase adherence assays

79
Q

a compact plate of rigid or flexible plastic with multiple wells

A

microplate

80
Q
  • ______ well has been used most often in immunohematology
  • volume capacity of each well: ______
A
  • U-shaped well
  • approx 0.2 mL
81
Q

In microplate agglutination reactions..

Samples and reagents are dispensed with ________ pipettes

A

small-bore Pasteur pipettes

82
Q

In microplate agglutination reactions..

After the specimens and reagents are added to the wells, they are mixed by _______ of the plates. The microplate is then _______ for an immediate ______

A

gentle agitation ; centrifuged ; reading

83
Q

After centrifugation, the cell buttons are resuspended by _________ or by using a __________ which is a shaker provides a more consistent and standard resuspension of the cells.

A

gently tapping the microplate ; flat-topped mechanical shaker

84
Q

Microplate Agglutination Reactions Results

A

(+) rxn: particles settle in a diffuse uneven button
(-) rxn: smooth compact button

85
Q

Reading agglutination..

Cells: No Agglutinates
Supernate: Dark, Turbid, Homogenous

86
Q

Reading agglutination..

Cells: Many tiny agglutinates, Many free cells, May not be visible without microscope.
Supernate: Dark, Turbid

87
Q

Reading agglutination..

Cells: Many small agglutinates, Many free cells
Supernate: Turbid

88
Q

Reading agglutination..

Cells: Many medium-sized agglutinates, Moderate number of free cells.
Supernate: Clear

89
Q

Reading agglutination..

Cells: Several large agglutinates, Few free cells
Supernate: Clear

90
Q

Reading agglutination..

Cells: One large, solid agglutinate and No free cells
Supernate: Clear

91
Q

In immunofixation electrophoresis..

______________: exhibit faintly staining bands

A

Hypogammaglobulinemia

92
Q

In immunofixation electrophoresis..

_________: show darkly staining bands in the gamma region

A

polyclonal hypergammaglobulinemia

93
Q

In immunofixation electrophoresis..

__________: dark and narrow bands in specific lanes

A

presence of monoclonal antibody

94
Q

In direct bacterial agglutination..

__________: rapid screening test useful in diagnosing typhoid fever

A

Widal test

95
Q

employs particles that are coated with antigens not normally found on their surfaces

A

Passive, or Indirect, agglutination

96
Q

the particles used in Passive, or Indirect, agglutination

A

Erythrocytes, Latex and Gelatin

97
Q

found that IgG was naturally adsorbed to the surface of polystyrene latex particles.

A

(1955) Singer and Plotz

98
Q

In Passive, or Indirect, agglutination..

______ is attached to the carrier particle

99
Q

have been used to detect rheumatoid factor, antibodies to Group A Streptococcus antigens, and antibodies to viruses such as rotavirus, cytomegalovirus, rubella, and varicella-zoster

A

Latex agglutination tests

100
Q
  • antibody is attached to a carrier particle
  • often used to detect microbial antigens
A

Reverse Passive Agglutination

101
Q

based on competition between particulate and soluble antigens for limited antibody combining sites

A

Agglutination inhibition

102
Q

(+) R in agglutination inhibition

A

no agglutination

103
Q

agglutination inhibition, involves _____ complexed to proteins; the _________ is then attached to a carrier particle

A

haptens ; hapten–protein conjugate

104
Q

use the same principle, except RBCs are the indicator particles

A

Hemagglutination inhibition

105
Q

involves measuring the number of residual nonagglutinating particles in a specimen

A

particle-counting immunoassay (PACIA)

106
Q

These particles are counted by means of a ______ in an optical particle counter similar to the one that is designed to count blood cells.

A

laser beam

107
Q

Measurements are made by looking at the rate at which the number of unagglutinated particles decrease, called a ______ or the total number of unagglutinated particles left at the end, known as an ________

A

rate assay ; end-point assay

108
Q

An _________ exists between the number of unagglutinated particles counted and the amount of unknown antigen in the patient specimen

A

inverse relationship

109
Q

physical preservation of serum

A

refrigerate for 72 hours at 4 to 6 degree celsius

110
Q

chemical preservation of serum

A

add 0.001g merthiolate powder per mL serum or 5% phenol or tricresol at 0.1 mL per mL serum

111
Q

physical inactivation of serum to remove complement

A

heat serum at 56 degree celsius for 30 mins

112
Q

physical inactivation of serum for VDRL

A

heat serum at. 60 to 62 degree celsius for 3 to 4 mins

113
Q

chemical inactivation of serum

A

choline chloride

114
Q

soluble ag/ small ag

A

Ag = antigen

115
Q

measurement of precipitation by light scattering = at fluid

A

Turbidimetry
Nephelometry

116
Q

measure turbidimetry/cloudiness

A

turbidimetry with the use of spectrophotometer

117
Q

measure light scattered in a particular angle

A

nephelometry with the use of nephelometer

118
Q
  • no current used
  • uses agar/agarose because no negative charge
A

precipitation by passive immunodiffusion at solid/semi-solid medium

119
Q

precipitation by passive immunodiffusion at solid/semi-solid medium advantage

A

cheap, simple

120
Q

precipitation by passive immunodiffusion at solid/semi-solid medium disadvantage

A

longer turn around time (TAT)

121
Q
  • single diffusion, single dimension
  • the antibody you incorporated into agarose at test tubes
  • semi-quantitative
  • end: presence of precipitin line
  • thicker line = increased concentration
A

Ouidin Test

122
Q
  • Single Diffusion, Double Dimension
  • Uses plate with antibody
  • Quantitative: diameter is directly proportional to concentration
  • (+) R = precipitin ring
A

Radial Immunodiffusion (RID)

123
Q

2 methods in RID

A

Fahey and McKelvey Method (Kinetic Diffusion)
Mancini Method (Endpoint Diffusion)

124
Q
  • Measure diameter before the completion of the reaction.
  • Diameter is proportional to log of concentration.
A

Fahey and McKelvey Method (Kinetic Diffusion)

125
Q
  • measures diameter after the completion of the reaction
  • square of diameter is proportional to concentration
  • IgG: 24 hours
  • IgM: 50 to 72 hours
A

Mancini Method (endpoint diffusion)

126
Q
  • Qualitative ; 1st generation test for HBsAg
  • both Ag and Ab is moving
A

Ouchterlony Technique (Double diffusion, Double dimension)

127
Q

Ouchterlony Technique (Double diffusion, Double dimension) pattern

A

Identity = smooth curve
Partial Identity = spur formation
Non-identity = cross/intersect

128
Q

____ separates molecules using electric current

A

Electrophoresis

129
Q

Precipitation by Electrophoretic Techniques

A
  1. Rocket Electrophoresis (Laurell Technique)
  2. Immunoelectrophoresis (IEP)
  3. Immunofixation Electrophoresis
  4. Counter Immunoelectrophoresis
130
Q
  • one dimension electroimmunodiffusion
  • RID + electrophoresis
  • total distance of antigen migration and precipitate directly proportional to antigen concentration.
  • (+)R: resembles rocket
  • increased peak = increased concentration
A

Rocket Electrophoresis (Laurell Technique)

131
Q

Rocket Electrophoresis (Laurell Technique) advantage and disadvantage

A

adv: decreased TAT
dadv: expensive, complex

132
Q
  • described by Grabar and Williams
  • useful for ID of monoclonal protein (Bence Jones)
  • double diffusion + electrophoresis
A

Immunoelectrophoresis (IEP)

133
Q

1st diffusion: separate component trough + antisera
2nd diffusion: towards the trough

A

Immunoelectrophoresis (IEP)..

134
Q

Immunoelectrophoresis (IEP) disadvantage

A

interpretation of the test result is complex

135
Q

Immunoelectrophoresis (IEP) (+)R

A

ppt toward the trough

136
Q

canal-like structure

137
Q
  • replaced IEP
  • to identify and characterize serum and urine CHON
  • antisera is directly added to the medium
  • described by Alper and Johnson
  • ex. western blot
A

Immunofixation electrophoresis

138
Q

ag and ab = placed on the well directly opposite to each other.

A

Counter electrophoresis

139
Q

antibody is towards the ___
antigen is towards the ___

A

cathode ; anode

MT2 (45 decks)

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