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MODULE
Agglutination
Microbiology
60
Notes
AGGLUTINATION
60
...
Agglutination word comes from
the Latin “agglutinare”, meaning "to glue,” it is also clumping of particles
...
The antibody binds multiple antigen particles and joins them, creating
a large lattice like complex which we can see with naked eye
...
Example of agglutination reaction using
particulate antigens is Salmonella typhi bacteria to detect specific antibody in
serum from patient suffering from typhoid fever (Widal test)
...
The soluble antigen is first made particulate by coating it on inert particles
like red cells, latex particles, gelatin particles and micro beads
...
Agglutination assays have good sensitivity, do not require sophisticated
equipment, are easy to perform, require no wash procedures and are cost
effective
...
OBJECTIVES
After reading this lesson, you will be able to:
z
522
define agglutination
MICROBIOLOGY
Agglutination
z
z
z
z
discuss the process of agglutination
describe the various methods of agglutination
read the result of agglutination reaction with naked eye and under the
microscope
describe the various applications of agglutination
60
...
This
reaction which is sensitive and specific is termed agglutination
...
Most common example of
agglutination is the testing for blood group
...
However, you can make precipitation reaction also
very sensitive by attaching/coating soluble antigens to large, inert carriers, such
as erythrocytes or latex beads so that precipitation reaction now becomes an
agglutination reaction
...
3 HISTORY
Two bacteriologists namely, Herbert Edward Durham and Max von Gruber
discovered specific agglutination in 1896
...
Later, Gruber named any substance
that caused agglutination reaction as “agglutinin” (from the Latin)
...
Widal found that blood serum from a typhoid carrier caused a
culture of typhoid bacteria to clump, whereas serum from a typhoid-free person
did not
...
Karl Landsteiner found another important practical application of the agglutination
reaction in 1900 i
...
for blood group (ABO) typing
...
60
...
We can picture a lock and Key concept
MICROBIOLOGY
523
MODULE
Microbiology
Notes
Agglutination
to understand the specificity of agglutination reaction
...
The two arms of “Y” are the Fab portion and this has the
combining site and is made of the hypervariable regions of the heavy and light
chains
...
1
...
If the fit is appropriate then agglutination will happen
...
The process of agglutination involves
two steps
...
Fig
...
1
60
...
1 Sensitization
It is attachment of specific antibody to corresponding antigen
...
IgM antibodies react best at 4 to
22 degrees C and IgG antibodies react best at 37 degrees C
...
60
...
2 Lattice formation:
Lattice is just like a “Jaal”
...
It takes more time than sensitization and we may be able to see the
result with naked eyes
...
INTEXT QUESTIONS 60
...
Agglutination means
...
Most common example of agglutination is testing for
...
Agglutination is clumping together of antigen with their specific antibodies
called
...
Attachment of specific antibody to antigen is
...
4
...
Electrokinetic potential in colloidal systems is termed as
Zeta potential
...
A high zeta potential confers so it will resist
agglutination
...
Notes
60
...
4 Grading agglutination reactions
Grading may be macroscopic or microscopic
...
Example of blood grouping used in blood
banks is given below
...
4
...
3+ Several large aggregates, clear background
...
1+ Small aggregate, turbid reddish background
...
MF Mixed Field – Any degree of agglutination in a sea of un-agglutinated cells
...
Both hemolysis and agglutination may be recorded on the
same tube
...
60
...
6 Microscopic
+ Positive - aggregates of at least 3-5 cells
...
MICROBIOLOGY
525
MODULE
Microbiology
Agglutination
60
...
These
include: rapid agglutination tests; slow agglutination tests in tubes; slow
agglutination tests in micro titration plates
...
5
...
The antigen and serum are usually mixed in fixed
proportion
...
Sometimes strong agglutination reactions need to be
confirmed by heating the sera (56 C
...
60
...
2 Slow agglutination in tubes/tube agglutination:
This involves dilution of the serum and mixing with fixed amount of unstained
antigen
...
The positive results are visualized by the
presence of a precipitate in the bottom of the tube and a clearing of the
supernatant (as compared to antigen without any serum)
...
5
...
A large number of samples/ various
dilutions can be tested at a time in one plate
...
The negative result appears as a button of un-reacted
antigen in the well
...
The
antibody is mixed with the particulate antigen and a positive test is indicated by
the agglutination of the particulate antigen
...
g
...
“Quantitative agglutination test” - agglutination tests used to quantitate the level
of antibodies to particulate antigens
...
The results are
reported as the reciprocal of the maximal dilution that gives visible agglutination
...
6 MISCELLANEOUS TYPES OF AGGLUTINATION
The agglutination of a particulate antigen by antibody raised against a different
but related antigen is termed Cross agglutination; agglutination of members of
a group of biologically related organisms (bacteria) or corpuscles by an
agglutinin specific for that group is called “Group agglutination” and clumping
of particulate elements within the blood vessels/red blood cell aggregation
within the blood vessels is called “Intravascular agglutination”
Microbiology
Notes
INTEXT QUESTIONS 60
...
Qualitative Agglutination (a) to measure the level of antibodies
2
...
Group
(c) to detect presence of antigen / antibody
4
...
Intra vascular
(e) antibody against related antigen
60
...
This
can happen if either antibody is in excess (Prozone) or when antigen is in excess
(Post zone)
...
7
...
The same sera
when tested after making dilution show a positive agglutination/precipitation
reaction
...
Excessive levels of antibody
result in false negative reaction as antibody excess results in formation of very
small complexes which do not clump to form visible agglutination
...
Prozone reaction can also
result from presence of blocking antibody or to nonspecific inhibitors in serum
...
7
...
Antigen excess is also the
probable cause of false-negative antigen-antibody agglutination/precipitation
reaction
...
3
1
...
2
...
favours agglutination
3
...
in
the serum
4
...
5
...
6
...
7
...
60
...
Let us now try to understand what the importance of
agglutination reaction in medicine is
...
The sample can be any body fluids such
as urine, blood, saliva, and cerebrospinal fluid (CSF) in which either we want to
detect antigen or antibody, the sample can also be bacterial culture which we
want to type to know whether it is pathogenic or not
...
Blood is collected by venipuncture following standard work precautions;
CSF is collected by the clinician by lumbar puncture and urine is collected by
patient using the clean catch method
...
Applications of agglutination in clinical medicine include:
60
...
1 Typing blood cells of the recipient and donor for blood transfusion
60
...
2 To identify and type bacterial cultures
60
...
3 To detect the presence of specific antibody and quantitate the amount of
antibody in patient’s serum
528
MICROBIOLOGY
Agglutination
60
...
4 Latex agglutination
MODULE
Microbiology
60
...
5 Haemagglutination
...
60
...
1 Typing blood cells of the recipient and donor for blood transfusion:
ABO and Rh blood grouping
Notes
Principle and application
Agglutination of red cells with known anti sera (antibody) indicates presence of
the corresponding antigen on the red cells
...
ABO blood groups are classified as A, B, AB, or O depending on the presence
or absence of the A or B antigens on the red cells and presence or absence of
the corresponding anti-A or anti-B antibodies
...
There are various methods of performing this test, such
as slide, test tube, microplate or Column Agglutination Technique
...
We describe here the conventional slide, test tube and Column Agglutination
Technique
...
from donor and patient in EDTA bottle;
z
Commercial blood grouping anti sera (Anti-A, -B, -D, -D blend)
z
Reagent (pooled) red cells (O-cell, A-cell, B-cell), prepared in-house
...
9 % NaCl)
z
Test tube rack
z
Pasteur pipette/Automated pipettes
z
Table top centrifuge
z
370C dry incubator
z
Incubator, Centrifuge
z
Gloves
z
Glass slides/applicator sticks
MICROBIOLOGY
529
MODULE
Microbiology
Agglutination
Procedure
Procedure for ABO cell grouping (slide method)
Notes
z
Confirm the identity of blood sample by checking registration number and
name of the patient/donor
...
z
Put 1 drop of Anti-A, 1 drop of Anti-B and 1 drop of anti D anti serum
on left, middle and right portion of slide
...
z
Mix well with a applicator stick
...
z
Record in the register/form
...
z
Record in the register/form
...
z
Prepare 2 - 5% suspension of the EDTA red cell in normal saline
z
Label three test tubes with patient name or number and tube contents (-A,
-B and –D)
...
To the tube
B, add 1 drop of Anti-B and to the tube labeled D add 1 drop of anti D
anti serum
...
z
Mix well and centrifuge the test tubes for 1 min at 1000 rpm
...
z
Remove aluminum foil from the top of microcolumn(s)
...
z
Centrifuge the cassette X 5 min in column agglutination centrifuge
z
Read and grade the reaction as per manufacturer’s instructions
...
z
It is important to follow the manufacturer’s instruction for the specific
antisera in use
...
z
Observe agglutination against a well lighted back ground
...
MODULE
Microbiology
Notes
60
...
2 To identify and type bacterial cultures
When we isolate bacteria from a sample say blood of a patient suffering from
fever, it is important to know whether the bacteria isolated are Salmonella typhi
or Acinetobacter or any other bacteria
...
However, to know the exact identity of bacteria we have to type
the bacteria using the Salmonella typhi “O antigen” specific and “H antigen
specific” sera available commercially
...
Mix the two
with nichrome loop or a wooden stick and look for macroscopic agglutination
with naked eye
...
Same way using genus specific,
species specific, strain specific commercially available antisera against many
pathogens (e
...
Pneumococci, Meningococci and others), the bacteria isolated
from patient samples can be identified
...
8
...
The agglutinins against ‘0’
(somatic) and ‘H’ (flagellar) antigens of Salmonella typhi, paratyphi A and
MICROBIOLOGY
531
MODULE
Microbiology
Agglutination
paratyphi B are estimated qualitatively (slide test) and quantitatively (Tube test)
employing killed suspension of appropriate organisms
...
Notes
Specimen: Blood sample 3-5 mL is collected in sterile dry screw capped
unbreakable tubes and transported to the lab in upright position or stored in
refrigerator (2-8°C) in case of delay
...
Material and instruments required
z
S
...
z
S
...
z
S
...
z
S
...
z
Blood sample 3-5 ml
z
Plain vacutainer/sst
z
Normal saline
z
Micropipettes
z
Glass test tubes
Reagents: Commercially available test kits are used
...
Follow the instructions given in the kit insert to perform the Widal
tube agglutination test
...
For each serum sample under test ,arrange four rows of 4
tubes each in a rack
Prepare master dilutions by taking 4 tubes in another rack
...
85% sodium chloride) in the
first tube and 3
...
ml in each of the remaining four tubes
...
5
...
Transfer 3
...
ml from the first tube to the next tube and mix
well
2
...
4
...
6
...
Continue successive transfer of 3
...
ml quantities till the last
tube is reached
...
5
...
Put
0
...
e,5th
row) to serve as controls
...
To each of the 4 tubes in the first, second, third and fourth
horizontal rows, add 0
...
ml of S
...
paratyphi B(H) antigens respectively
...
Shake the rack well to mix and incubate at 37ºC overnight
(16-20 hours)
...
Note the highest dilution in which there is evidence of
agglutination as observed by naked eye or a hand lens
...
In addition to the pattern of sedimented organisms, the
decrease in opacity of the supernatant as compared to the
saline control tube must be observed and taken into account
while judging the degree of agglutination
...
Agglutination titers of 1:120 and more are significant and rise in titers
or repetition of the test after a few days will confirm the diagnosis of enteric
fever
...
However, follow the instructions in the kit insert for
interpretation
...
Persons who have suffered from enteric infections in past or who had received
TAB vaccine may show appearance of agglutinins in moderate titer when
MICROBIOLOGY
533
MODULE
Microbiology
Notes
Agglutination
suffering from other unrelated illness
...
A moderate rise in titer of
all three ‘H’ agglutinins simultaneously against all ‘H’ antigens is suggestive of
recent TAB vaccination
...
Standard work precautions (gloves) should be used for handling and
disposal of materials during and after use
...
60
...
4 Latex agglutination
As already discussed soluble antigens are coated on inert particles like latex to
develop sensitive, specific easy to perform rapid tests termed the latex
agglutination tests
...
Always follow the instructions given in
the kit insert to perform the test
...
Cryptoccus antigen test is
a simple, qualitative or semi-quantitative, test to detect polysaccharide antigens
associated with Cryptococcus neoformans infection
...
Sample of operative procedure based on
a commercial kit “Remel Cryptococcus Antigen Latex Test” is given below
...
Principle of the test:
Cryptococcus antigen test kit incorporates the use of latex particles sensitized
with murine IgM monoclonal antibodies
...
Safety precautions:
534
z
All specimen used in this test should be considered potentially
infectious
...
z
Use soap for routine hand washing
...
MICROBIOLOGY
Agglutination
Reagents and materials
MODULE
Microbiology
Reagents
z
Test latex: Latex particles sensitized with IgM anti-CPS monoclonal
antibody suspended in a buffer and preserved in 0
...
5
ml)
...
1%
sodium azide (1 × 0
...
z
High positive control: Contains approx 50 mg/ml of C
...
1% sodium azide
...
8 ml)
z
Low positive control: Contains approx 12 mg/ml of C
...
1% sodium azide
...
8 ml)
z
Protease: 1 enzyme tablet contained in a vial
...
0% sodium
azide
...
z
z
Notes
Reaction cards: (12 × 6 circles)
Dispensing pipette- 50 ul
Equipments
z
Centrifuge
z
Micropipette for serial dilution of specimens
...
z
Timer
z
Boiling water bath
z
Graduated cylinder
z
Vortex mixer
...
Prepare a working strength solution (1x concentration) in a separate bottle by
combining the entire contents of the 10x specimen diluent bottle with 90 ml of
demineralized water
...
Use as needed or store at
2-8°C
...
Allow 30 minutes for
MICROBIOLOGY
535
MODULE
Microbiology
Notes
Agglutination
complete reconstitution, swirling the vial at least twice during this time
...
Protease solution can be stored at 2-8°C
for 1 month from the date of reconstitution
...
B
...
z
Allow contents to cool to room temperature
...
Serum:
z
If necessary dilute specimen diluent (10x) to the 1x working strength
...
z
Dispense 100-200 ul of serum in a tube and add an equal of Protease
...
Mix by vortexing
...
z
Allow contents to cool to room temp and gently mix before testing
...
Decant the
supernatant (50 μl) to avoid aspirating the pellet
...
Dispence one drop of the Test latex into a sepatate test circle for each
specimen and control to be tested
...
Use the paddle end of a separate pipette to
thoroughly mix each control and test latex
...
z
Using pipettes provided in the test kit dispense one drop of each pretreated
patient specimen (approximately 50 μl) into a test circle containing Test
latex
...
Discard the pipette
after this step
...
z
Immediately following the 5-minute rotation tilt the slide to obtain a flow
pattern and carefully examine each circle for any agglutination (see results
section below) and record the results
...
Record
the results
...
z
Obtain and mark eight tubes 1 to 8
...
1 ml of specimen diluent to
each tube
...
1 ml of heat-treated CSF, or protease-treated serum contents of tube
1 and transfer 0
...
Do not mix the contents of tube 2 with
the pipette
...
1
ml to tube 3
...
Do not mix the contents
of tube 3 with the pipette
...
The serum dilutions which have been established are from
1:4 to 1:512 for tubes 1 to 8 respectively, For CSF 1:2 to 1:256 dilutions
are made
...
z
Test each specimen dilution following the protocol as described in the
Qualitative Testing section
...
The absence of
agglutination of the test latex is considered a negative result
...
Semi-quantitative test results: When positive specimens are examined by serial
dilution the titer is the reciprocal of the last dilution which produces a positive
result (agglutination)
...
MICROBIOLOGY
537
MODULE
Microbiology
Notes
Agglutination
The high positive and low positive controls must agglutinate the test latex
differentially
...
z
The low positive control must produce weaker but clearly visible
agglutination
...
60
...
5 Haemagglutination:
As the name indicates this is agglutination of RBCs as such by RBC antigen
specific antibody (example of ABO blood grouping given under 8
...
The
antibodies/proteins which agglutinate RBCs are called Haemagglutinins
...
Adjustable multi-channel micropipette (50-300 µl)
2
...
Adjustable single-channel micropipette (5-50µl)
4
...
Reagent troughs
6
...
Micro plates (U bottom)
2
...
Control cells (uncoated)
4
...
pallidum antigen)
5
...
Approximately 30 min prior to the beginning of the test procedure; bring
kit components to room temp
...
Mix the liquid reagents gently
...
of specimens to be tested and no
...
538
MICROBIOLOGY
Agglutination
2
...
Label the plate with last three digits of donation Id on left lower side of
well, identifying the positions
...
4
...
5
...
Notes
6
...
7
...
8
...
9
...
10
...
11
...
Keep the plate
on a smooth steady surface
...
of incubation at room
temp
...
All control wells should have compact button formation
...
(Agglutination of the control cells as well as the test cells indicates the
presence of anti-cell antibody and in this event the test is not valid & should
be repeated after first performing absorption of the test serum
...
After centrifuging the sample (1000 rpm/5 min
...
Test this dilution directly, without any further dilution, using
test & control cells suspensions)
...
If there is a compact button in a test well, the result is considered non
reactive
...
If there is a characteristics ring pattern or net of the cells in the and there
is a compact button formation in the control well, specimen is considered
as reactive for T
...
MICROBIOLOGY
539
MODULE
Microbiology
Agglutination
4
...
5
...
Notes
Agglutination has been commonly used to determine whether a patient had or
has a bacterial infection
...
If an
agglutination reaction occurs, shown as clumping of the bacteria, the patient
either had or has an S
...
Since certain antibodies can persist in
a patient’s blood for years after the patent has recovered from the infection, a
positive reaction does not mean that the patient currently has the infection
...
WHAT HAVE YOU LEARNT
540
z
Agglutination word means to glue
z
Agglutination is one of the antigen and specific antibody reactions in-vitro
in laboratory in the presence of electrolytes at a suitable temperature and
pH
...
z
Agglutination involves two steps
...
z
Sensitization is the atachment of specific antibody to corresponding antigen
...
z
Reducing Zeta potential will favour agglutination
...
z
Clinically agglutination is used
Typing blood cells of the recipient and donor for blood transfusion
To identify and type bacterial cultures
MICROBIOLOGY
Agglutination
MODULE
Microbiology
TERMINAL QUESTIONS
1
...
Describe the process of agglutination
...
Explain the methods of agglutination
...
Explain prozone and postzone phenomena
...
List the clinical applicaiton of agglutination
...
Describe ABO and RH blood grouping in brief
ANSWERS TO INTEXT QUESTIONS
60
...
To glue
2
...
Agglutinins
4
...
2
1
...
(e)
3
...
(a)
5
...
3
1
...
Zeta potential
3
...
Precipitate in the bottom & clearing of supernatant
5
...
Pro-zone phenomenon
7