clumping of cells due to the antigen-antibpody reaction
agglutination (form clumps)
- first observed in 1896
- observe hemeagglutination
heme (blood)
agglutination (clotting)
= clotting of blood due to the interactions of antigen-antibody
who first reported about the ability of an antibody to clump cells based on observations of agglutintion of bacterial cells by serum
Gruber and Durham, 1896
who developed one of the earliest diagnostic tests for the detection of antibodies occurring in typhoid fever, brucellosis and tularemia
Widal and Sicard, 1896
2 stages of agglutination
- sensitization or initial binding
- binding of antibody to epitope (antigenic site) on the surface of RBC
[Example:
If the RBC has Antigen A (Blood Group A)
Anti-A antibody attaches to it]
- reversible reaction through elution
- no visible agglutination
- non covalent bonds [weak forces like hydrogen bonds] - lattice formation
- multiple antigen–antibody bridges btwn sensitized rbc antigens and antibodies
- formation of a stable lattice
- visible agglutination
**antibodies bind with multiple antigenic sites = binding stable = lattice formation
antigenic determinant site
epitope
[Example:
If the RBC has Antigen A (Blood Group A)
Anti-A antibody attaches to it]
a manipulated procedure to break the antigen-antibody binding and release the antibody into the surrounding medium
elution
- use solutions like chemicals to break the bonds btwn the antigen-antibody
- example:
treating the sensitize rbc in a solution = break the bond and it removes the antibody and remain suspended in the illusion solution
*sensitize rbc is the product of the sensitization
what are the different tests and methods on how we can observe agglutination
tube method (most commonly used)
slide method
gel card method
agglutination grading tube method
notes
the following is present in
antigen:
anitbody:
antigen: found on the surface of red blood cell
antibody: for ex: anti-sera (anti-A, anti-B, anti-D)
antibodies against antigen A, B and D
EXAMPLE
Anti-A → reacts only with Antigen A
Anti-B → reacts only with Antigen B
Anti-D → reacts only with Antigen D (Rh factor)
explain why true agglutination has not been occurred in mixed field
there are few small clumps (isolated aggregates) present
most rbc remain free-floating
which grade in tube method where agglutination is visible with the naked eye
1+
true or false:
whenever we perform test in the blood bank, always check it under the microscope
as there might be weak reactions
why does the grade 2+ in agglutination grading tube method is clear
as it starting to clump
Interpretation: which antisera causes agglutination
Anti-A only =
Anti-B only =
Anti-A and Anti-B =
No agglutination with Anti-A or Anti-B =
Anti-A only = Group A
Anti-B only = Group B
Anti-A and Anti-B = Group AB
No agglutination with Anti-A or Anti-B = Group O
Interpretation:
Agglutination With Anti-D = Rh Type
Yes = Rh positive (+)
No = Rh negative (−)
next step red cell button is formed after centrifugation
dislodge gently to discard the supernatant
**if vigorous shaking = might break agglutination and report it as negative especially if the reaction is weak
what tests is usually done with slide method
forward blood typing
when do we check the blood under the microscope
crossmatching
coombs test
*must know how agglutination looks like (literal clumping, cant see the red cells shape)
*if iba fields na meron and yung iba ay meron = still considered as +ve in agglutination
how to differentiate rouleaux formation and agglutination
rouleaux: stacking of coins
- meron excess proteins in the serum/ plasma of the patient
how to resolve rouleaux formation
replacement method
- remove either antisera/ diluent/ solution
- replaced it with NSS
= check if the cells are still stacked
process of gel card technology
- mix the red cell and reagent
- incubate for 15 min
- centrifuge for 5 to 10 min
- grade it (notes)
GRADING
0:
clear at whole tube
weak +ve:
v. small of agglutination, very visible, near the bottom of the gel with most cells forming a pallet
1+:
almost throughout and visible in the lower half of the column
2+:
throughout the entire length of the gel column
3+:
strong agglutination is visible at the upper half of the gel column
4+:
solid pallet at the top, all blood cells are trapped as a solid bond at the top of the column
mixed field:
sometimes called as mixed population
has 2 reaction, meron pallet at the top and bottom
Hemolysis:
change in color
clear red color distributed, throughout the solution
principle of gel card technology
gel card has microtubes (columns) filled with a compact gel matrix
for example:
RCS mixed with antisera (antibodies) –> centrifuged –> RBC move downward through the gel column
POSITIVE REACTION
- antibodies bind to antigens on RBCs = lattice formation
- clumped RBCs are too big to pass through the gel = trapped at the top or within the gel column
NEGATIVE REACTION
- no agglutination = RBCs remain single and free-floating
- an pass through the gel easily
- RBCs form a pellet at the bottom of the column
[Gel = strainer
Agglutinated cells = big particles → get stuck
Non-agglutinated cells = small particles → pass through]
factors that influence agglutination reactions
- centrifugation
- high-speed centri (one of the most efficient methods used in BB for increased agglutination reaction)
- enhanced agglutination - antigen-antibody ratio
(notes)
- incorrect antigen antibody ratio, false negative result = no agglutination reaction - effects of pH
- ideal: 6.5 - 7.5 - temp
- immunoglobulin type
- enhancement media
it is one of the most efficient methods used in BB for increased agglutination reaction
centrifugation
- high speed centri
