AGGLU RESET Flashcards
1
Q
Visible clumping of particulate antigens (agglutinogens) when combined with agglutinating antibodies (agglutinins) in the correct proportion
A
Agglutination
2
Q
Antibodies that produce agglutination by binding to specific antigens (e.g.+on RBCs)
A
Agglutinins
3
Q
Foreign particles triggering immune responses when recognized by agglutinins
A
Agglutinogens (particulate antigens)
4
Q
Initial force of attraction between antibody Fab sites and antigen epitopes
A
Affinity
5
Q
Overall strength+stability of antigen-antibody binding
A
Avidity
6
Q
Artificial carriers used in agglutination tests
A
Artificial carriers
(latex+charcoal+gelatin+plastic+silicate)
Inert particles
7
Q
Requirement for agglutination: multivalency of agglutinin+agglutinogen
A
Multivalency
8
Q
Common agglutination test types:
A
Slide agglutination test+Tube testing+Plate agglutination test+Passive agglutination test+Microscopic agglutination+Heme agglutination
9
Q
Major agglutination reaction categories
A
Direct immune agglutination+Direct non-immune agglutination+Indirect/passive agglutination
10
Q
Advantage of agglutination
A
detects antigens+antibodies with simple instruments+kits
macroscopic results+less zoning+qualitative/semiquantitative outpu
11
Q
Most effective agglutinating antibody due to pentameric structure
A
IgM
12
Q
Pathogen clumping via antibody binding for immune elimination
A
Agglutination (immune defense mechanism)
13
Q
Diagnostic tests using agglutination:
A
Latex agglutination+Blood typing+Widal test+CRP+Rapid Plasma Reagin (RPR)+COVID test kit
14
Q
Examples of inert carriers:
A
latex (rheumatoid factor+CRP)+charcoal (RPR)+gelatin+plastic+silicate
15
Q
Direct clumping via antibody-antigen binding
A
Direct immune agglutination
16
Q
Non-antibody-mediated clumping (e.g.+viruses)
A
Direct non-immune agglutination
17
Q
Antigen/antibody-coated particle tests
A
Indirect/passive agglutination
18
Q
Typhoid fever test with Salmonella antigens
A
Widal test
19
Q
Acute inflammation marker test using CRP
A
CRP agglutination test
20
Q
Syphilis screening with charcoal particles
A
Rapid Plasma Reagin (RPR) test
21
Q
Rapid antigen detection via agglutination principles
A
COVID test kit
22
Q
What causes false positives due to cold-reacting antibodies in samples?
A
Autoagglutination
23
Q
How to resolve autoagglutination in blood samples?
A
Warm sample to 37°C
24
Q
Why does saline stored in glass bottles cause false positives?
A
Colloidal silica induces clumping
25
What mimics antigens and causes cross-reactivity in tests?
Antigenic determinant resemblance
26
How to prevent cross-reactivity in agglutination tests?
Use monoclonal antibodies
27
What non-specific antibodies cause false positives?
Rheumatoid factor+Heterophile antibody
28
Why does delayed slide reading cause false positives?
Evaporation concentrates reagents
29
What centrifugation error causes false negatives?
Under centrifugation
30
What leads to unbound antibodies neutralizing reagents?
Inadequate washing of cells
31
Why do expired reagents produce false negatives?
Loss of reagent activity
32
What delay causes antibody detachment from RBCs?
Delayed AHG testing
33
What phenomenon occurs with excess antibodies blocking agglutination?
Prozone effect
34
How to resolve the prozone effect?
Perform serial dilution
35
What is the initial reversible antigen-antibody binding stage?
Sensitization
36
What factors affect sensitization? (list 5)
Antigen/antibody nature+Temperature+Incubation time+pH+Isotonic strength
37
What temperature is required for IgG reactions?
37°C
38
What temperature is required for IgM reactions?
4°C
39
What pH range optimizes agglutination?
6.5-7.5
40
What solution reduces ionic interference?
Low Ionic Strength Solution (LISS)
41
What crosslinking stage forms visible clumps?
Lattice formation
42
What force repels RBCs and inhibits agglutination?
Zeta potential
43
Which antibody type bypasses zeta potential better?
IgM
44
What concentration balance is needed for lattice formation?
Equal antigen/antibody amounts
45
Chemical potentiators enhancing lattice formation
Low Ionic Strength Solution (LISS)+Bovine Albumin+Dextran+Polyethylene Glycol (PEG)
46
LISS mechanism
Lowers zeta potential+Increases antibody uptake+Decreases ionic strength+Improves sensitivity
47
LISS composition
NaCl+Glycine+Albumin
48
LISS storage temperature
2-8°C (36-46°F)
49
LISS discard condition
Turbidity (indicates contamination/deterioration)
50
LISS pre-use incubation
37°C
51
Delayed testing protocol for LISS samples
Store at 20°C+Test within 48 hours
52
Bovine albumin mechanism
Reduces zeta potential+Disperses cations+Increases dielectric constant
53
Bovine albumin concentration range
5-30% (22% most common)
54
Bovine albumin stability
Stable at 2-8°C for 24 months
55
Rh typing control use
Bovine albumin (detects false positives from autoagglutinins/abnormal proteins)
56
Bovine albumin validation test
Agglutination with sensitized check cells+No agglutination with unsensitized Rh-negative cells
57
Dextran/PEG mechanism
Removes water+Concentrates antibodies+PEG causes nonspecific aggregation
58
PEG centrifugation restriction
No centrifugation post-37°C incubation
59
PEG washing requirement
Minimum 4 washes
60
Normal Saline Solution (NSS) composition
0.85-0.9% NaCl+Distilled water
61
NSS osmolality/pH
308 mOsm/L+pH 5.5-7
62
Proteolytic enzymes used
Bromelain+Papain+Physin+Pepsin+Trypsin
63
Enzyme mechanism
Cleave chemical bonds+Reduce RBC surface charge+Decrease hydration
64
Physical potentiators
Centrifugation+Agitation+Temperature
65
Centrifugation effect
Forces antigen-antibody proximity+Removes repulsion+Forms larger aggregates
66
Agitation effect
Increases kinetic energy+Distributes reactants evenly+Prevents settling
67
IgG optimal temperature
37°C
68
IgM optimal temperature
4°C
69
LISS binding acceleration mechanism
Magnetizes particles via low NaCl
70
PEG immediate testing requirement
Proceed to washing phase without delay
71
Bovine albumin reactivity check
Use sensitized/unsensitized cells post-IgG incubation
72
NSS clinical uses
RBC washing+Reagent+Bedside testing mimic
73
Proteolytic enzyme storage
Not explicitly stated (assume standard enzyme storage protocols)
74
Agitation examples
Latex agglutination rotators+Bacterial test shakers
75
What are the three phases of agglutination testing?
Immediate Spin Phase+Incubation Phase+Anti-Human Globulin Phase
76
Which antibody is primarily detected in the Immediate Spin Phase and at what temperature?
IgM+Room temperature
77
What is the procedure for the Immediate Spin Phase?
Combine antigen and antibody+centrifuge+grade agglutination
78
Which antibody is primarily detected in the Incubation Phase and at what temperature?
IgG+37°C
79
What is the procedure for the Incubation Phase?
Combine antigen+antibody+chemical potentiator+incubate at 37°C+centrifuge+grade
80
What is the sequence for the Anti-Human Globulin Phase?
Combine antigen+antibody+incubate+wash+add AHG+incubate+centrifuge+grade
81
What is the purpose of agitation after centrifugation in agglutination reading?
To observe agglutination by resuspending the red cell button
82
What does a 4+ agglutination grade indicate?
One solid clump+no free cells+clear supernatant+strong agglutination
83
What does a 3+ agglutination grade indicate?
Several large clumps+few free cells+clear supernatant
84
What does a 2+ agglutination grade indicate?
Medium-sized agglutinates+moderate free cells+clear supernatant
85
What does a 1+ agglutination grade indicate?
Many small agglutinates+many free cells+turbid supernatant+weak agglutination
86
What does a 0 (negative) agglutination grade indicate?
No agglutinate+dark and turbid supernatant
87
What are the types of agglutination reactions?
Direct Agglutination
88
What is the principle of direct agglutination?
Antigen is naturally found on a particle+visible clumping occurs when antibody binds
89
What are two examples of direct agglutination tests?
ABO blood typing+Widal test
90
What is the principle of ABO blood typing?
Direct heme agglutination where RBCs clump due to antibody binding to surface antigen
91
What dyes are used for Anti-A antisera?
Bromphenol blue+Thymol blue+Patent blue+Alcian blue+Methylene blue
92
What dyes are used for Anti-B antisera?
Tartrazine yellow+Acriflavine
93
What is the minimum titer for ABO blood typing?
Greater than 256
94
What is the principle of the Widal test?
Direct bacterial agglutination to detect antibodies against O and H antigens of Salmonella typhi
95
What antigens are tested in the Widal test?
O (somatic)+H (flagellar)+AH (Paratyphi A)+BH (Paratyphi B)+NC (Negative control)+PC (Positive control)
96
What result indicates a positive Widal test?
Agglutination with O and/or H antigens
97
How are Widal test results graded?
1/20 heavy clumping+1/40 visible clumping+1/80 weak positive+1/640 negative
98
What is the Weil Felix test used for?
Detection of rickettsial infection by direct hemagglutination
99
What Proteus strains are used in the Weil Felix test?
P. vulgaris OX19+OX2+P. mirabilis OX-K
100
What does agglutination with OX19 indicate in Weil Felix test?
Rickettsia typhi infection (epidemic/endemic typhus)
101
What does agglutination with OX2 indicate in Weil Felix test?
Spotted Fever Group (Rocky Mountain Spotted Fever+Indian Typhus)
102
What does agglutination with OX-K indicate in Weil Felix test?
Scrub Typhus (Rickettsia tsutsugamushi)
103
What does no agglutination in Weil Felix test indicate?
No rickettsial infection
104
At what temperature does IgM agglutinate best?
4-22°C+Room temperature
105
At what temperature does IgG agglutinate best?
37°C
106
What particles are coated with antigens in passive agglutination?
RBCs+latex beads+synthetic beads
107
What is the main risk of using RBCs as carriers?
Cross-reactivity with heterophile antibodies
108
Who pioneered latex bead use in agglutination tests?
Singer and Plotz
109
Why are latex beads preferred over RBCs?
Inexpensive+stable+reduced cross-reactivity
110
List three tests using latex agglutination
CRP Test+Rheumatoid Factor Test+Streptococcus Antigen Test
111
What advantage do synthetic beads offer over latex?
Precise size/surface control
112
What is the key drawback of synthetic beads?
Non-specific IgM agglutination+requires controls
113
How does heme agglutination inhibition work?
Viruses clump RBCs unless antibodies block binding
114
What does no agglutination in heme inhibition indicate?
Antibodies neutralized the pathogen
115
How are heme inhibition results reported?
Red pellet=negative (no virus/antibodies)+Dispersed RBCs=positive (virus present)
116
What reacts with CRP in latex agglutination tests?
Anti-CRP coated latex particles
117
What CRP level is reported as positive?
>6 mg/L
118
What CRP level is reported as negative?
<6 mg/L
119
What detects rheumatoid factor in agglutination tests?
IgG-coated latex particles
120
What is the RF test's sensitivity cutoff?
>8 IU/ml
121
How quickly must RF results be read?
Within 2 minutes
122
What diseases link to high rheumatoid factor?
Rheumatoid arthritis+mononucleosis+sarcoidosis+lupus
123
How does reverse passive agglutination differ from standard passive?
Antibodies (not antigens) coat carrier particles
124
What principle governs reverse passive agglutination?
Antibody-coated particles clump if antigen is present
125
What carriers are used in reverse passive agglutination?
Latex beads+RBCs
126
What causes false positives in reverse passive tests?
Rheumatoid factor binding to IgG
127
How to prevent false positives in reverse passive tests?
Heat samples+use EDTA+use plasma+add enzymes (e.g.
128
Why are controls critical in agglutination tests?
Distinguish true clumping from non-specific binding
129
What is CRP's role in inflammation?
Produced by liver during infection/inflammation
130
What are normal/moderate/severe CRP levels?
Normal: 0.9 mg/dL+Moderate: 1-10 mg/dL+Severe: >50 mg/dL
131
How is CRP testing performed?
Mix serum with anti-CRP latex+rotate at 80-100 rpm for 2 minutes+compare to controls
132
What is the storage requirement for RF reagents?
2-8°C
133
What happens if RF results are read after 2 minutes?
Invalid due to potential false negatives
134
How does reverse passive agglutination handle antigen detection?
Antibody-coated carriers bind patient antigens to form clumps
135
What happens if no agglutination occurs in reverse passive tests?
Suspension remains clear (antigen absent)
136
What Proteus strains are used in Weil Felix test?
P. vulgaris OX19/OX2+P. mirabilis OX-K
137
What rickettsial infections correlate with Weil Felix results?
OX19=epidemic/endemic typhus+OX2=spotted fever+OX-K=scrub typhus
138
What is the principle of agglutination inhibition?
Competition between particulate and soluble antigens for limited antibody binding sites
139
How is a positive result in agglutination inhibition interpreted?
No agglutination
140
How is a negative result in agglutination inhibition interpreted?
Agglutination
141
Why is agglutination inhibition considered highly sensitive?
It can detect even the smallest amount of antigen
142
Name two clinical uses of agglutination inhibition tests
Urine drug screening+Urine beta HCG pregnancy test
143
What is the most common principle for beta HCG detection?
ELISA
144
What viral antibodies can be detected by hemagglutination inhibition?
Rubella+mumps+influenza+RSV+adenovirus
145
What happens when a patient sample contains antigen in agglutination inhibition?
Antibody binds to free antigen+immune complexes form+antibody cannot cross-link with antigen-coated particle+no agglutination
146
What happens when a patient sample does not contain antigen in agglutination inhibition?
No immune complexes form+antibody is free+cross-links with antigen-coated particle+agglutination occurs
147
What is coagglutination?
Agglutination test using bacteria (commonly S. aureus Cowan I) as inert particles to which antibody is attached
148
What bacterial protein is responsible for binding antibodies in coagglutination?
Protein A on S. aureus (binds Fc region of IgG except IgG2)
149
How does coagglutination work?
Bacteria coated with specific antibody bind antigen in sample+cross-linking forms visible agglutination
150
Why is coagglutination more stable than latex agglutination?
Bacteria provide greater reagent stability
151
What is a drawback of coagglutination?
Bacteria are not colored so results are harder to read
152
What are common uses for coagglutination?
Rapid bacterial antigen detection (Streptococcus pneumoniae+Neisseria meningitidis+Haemophilus influenzae+E. coli)+Toxin detection (Staphylococcal enterotoxin+Clostridium difficile)
153
What is antiglobulin-mediated agglutination also known as?
Coomb’s test
154
What reagent is used in antiglobulin-mediated agglutination?
Anti-human globulin (AHG)
155
How are AHG reagents produced?
Inject purified human globulin (IgG+C3b+C3d) into animals (rabbit or mouse) to produce anti-IgG+anti-C3b+anti-C3d
156
What does anti-IgG in AHG reagent detect?
IgG on the surface of RBCs
157
What does anti-C3b or anti-C3d in AHG reagent detect?
C3b or C3d on the surface of RBCs
158
What are the two types of AHG reagents?
Monospecific (anti-IgG or anti-C3b/anti-C3d)+Polyspecific (both anti-IgG and anti-C3b/anti-C3d)
159
What are the two main types of antiglobulin-mediated agglutination tests?
Direct Antiglobulin Test (DAT)+Indirect Antiglobulin Test (IAT)
160
What is another name for the Direct Antiglobulin Test?
Direct Coombs test+One-step AGT test
161
What does the Direct Antiglobulin Test detect?
In-vivo sensitization of RBCs with IgG or complement components (C3b/C3d)
162
What clinical conditions are investigated with DAT?
Hemolytic transfusion reactions+Hemolytic disease of the fetus and newborn
163
What is the basic procedure for DAT?
Wash patient RBCs 3 times+add AHG reagent+observe for agglutination
164
What does a positive DAT indicate?
RBCs are coated in vivo with IgG and/or complement (C3b/C3d)
165
What is another name for the Indirect Antiglobulin Test?
Indirect Coombs test+Two-step AGT test
166
What does the Indirect Antiglobulin Test detect?
In-vitro sensitization of RBCs by IgG or complement components
167
What are the main uses of IAT?
Antibody screening+Antibody identification+Crossmatching
168
What is the basic procedure for IAT?
Incubate patient serum with RBCs+wash to remove unbound antibodies+add AHG reagent+observe for agglutination
169
What special cells are used to validate negative IAT results?
Check cells or IgG reagent check cells
170
What are check cells?
Rh positive RBCs sensitized with IgG anti-D
171
Why are check cells used in AHG testing?
To confirm validity of negative AHG test and ensure AHG reagent was added and active
172
What solution is used to prolong check cell lifespan?
Alsever’s solution
173
How long can check cells be stored in Alsever’s solution at 1-6°C?
Up to 7 days
174
What is the storage condition for check cells if not in Alsever’s solution?
25°C for 24 hours only
175
What does it mean if check cells fail to agglutinate after a negative AHG test?
AHG reagent was neutralized
176
What is the purpose of screening tests like the antiglobulin test?
Initial detection of antibodies or antigens (not confirmatory)
177
Why is a negative result in the antiglobulin test not definitive?
Low antigen/antibody levels or insufficient test sensitivity can cause false negatives
178
List materials needed to prepare check cells
Cell washer+centrifuge+test tubes+water bath at 37°C+pipettes+dropper bottle+saline+IgG anti-D+Rh positive red cell segment+Alsever’s solution
179
What is the purpose of Alsever’s solution in check cell preparation?
Extends check cell lifespan by preserving RBCs
180
How are check cells prepared step-by-step?
Label tubes+add Rh-positive blood+add IgG anti-D+incubate 15 mins+wash 4x+preserve in Alsever’s solution+store at 1-6°C
181
What happens if check cells are not washed properly during preparation?
Residual Alsever’s solution may interfere with agglutination
182
Why is a 5% cell suspension used for check cells?
Ensures optimal concentration for visible agglutination
183
What is the role of IgG anti-D in check cell preparation?
Sensitizes Rh-positive RBCs to create antibody-coated control cells
184
What are clinical applications of direct agglutination?
Direct hemagglutination+Widal Test+HAV+HBV+HIV-I+HIV-II
185
What are clinical applications of passive agglutination?
Rheumatoid Factor+Antinuclear antibodies+Antibodies
186
What are clinical applications of reverse passive agglutination?
Group A (pyogenes)+Group B (agalactiae)+Streptococci+Neonatal meningitis+Staphylococcus aureus+Neisseria meningitidis+Haemophilus+Rotavirus+Cryptococcus neoformans+Vibrio cholerae+Leptospira+Identification of soluble antigens in urine+CSF+serum+Latex agglutination
187
What are clinical applications of agglutination inhibition?
Detection of illicit drugs (cocaine+heroin)+Detection of B-HCG in serum/urine (pregnancy testing)
188
What are clinical applications of coagglutination?
Identification of Streptococci+Neisseria meningitidis+Neisseria gonorrhoeae+Vibrio cholerae 0139+Haemophilus influenzae
189
What are clinical applications of antiglobulin-mediated agglutination?
Hemolytic transfusion reactions (HTRs)+Hemolytic Disease of the Fetus and the Newborn (HDFN)+Autoimmune Hemolytic Anemia (AIHA)+Drug-induced Hemolytic Anemia (DIHA)+Crossmatching (Major+Minor)+Antibody screening/detection+Antibody identification+RBC phenotyping+Antibody titration
190
What are clinical applications of quantitative agglutination reactions?
Sol Particle Immunoassay (SPIA)+Disperse Dye Immunoassay (DIA)+Immunoassay by Particle Counting (IMPACT/PACIA)
191
What is the principle of Sol Particle Immunoassay (SPIA)?
Antibody-coated gold sol particles bind specific analytes+measured by optical density or turbidity+can use colorimetric methods (CRAAS)
192
What is the principle of Disperse Dye Immunoassay (DIA)?
Dye-labeled antibodies detect antigens or antibodies+solid phase (well/dipstick)+color change visualizes presence
193
What is the principle of Immunoassay by Particle Counting (IMPACT/PACIA)?
Latex particles detect antibodies+antigens+haptens+measures residual non-agglutinating particles by laser beams (nephelometry)+amount of unknown antigen is indirectly proportional to unagglutinated particles
194
What are clinical uses of PACIA?
Therapeutic drug monitoring+Tumor markers+Viral antigen tests
