IMMUNOHEMATOLOGY LABORATORY: Act. 2 – Hemolysis and Agglutination
MOGATO
Hemolysis
- Indicates a presence of strong antibodies
- Determined by red or pink coloration of saline solution after
centrifugation
- Release of hemoglobin from red blood cells due to premature
breakdown of cells
False positive results:
- Overcentrifugation
- Warming of tubes while centrifuging
- Improper method of drawing blood
- Presence of recurring in-vivo hemolysis
- Improper inversion procedure
Agglutination
1. Basic reaction in blood banking
2. Two stages:
A. Sensitization/coating
a. Binding of antibody to surface RBCs
b. Dependent of multiple factors
B. Bridge formation
a. Linkage of adjacent RBCs that are coated with antibody
b. IgM is far more capable of forming bridges between
adjacent cells due to its pentameric structure (total of 10
possible binding sites.)
c. IgG is less capable, since it has binding regions
 Antigens that extend from the surface of the RBC,
such as M, N, and ABO, make it easier for IgG to
directly bind and form bridges
 RH and other antigens that live closer to the
surface of the cells are not usually directly
agglutinated by IgG anitbodies
3. Sensitization is simple a chemical reaction
A. Antigen + Antibody ↔ Antigen-antiody
B. KO = equilibrium constant of reaction
I. Larger K means a push to the right side of the
equation, with more stable and rapid reactions
C. Affinity to RBC antigens and antibodies affected by multiple
factors
MLS 4F AY 2017-2018
J. YAP – A.
I. Cold reactive (usually IgM) vs. warm-reactive (usually
IgG)
a. Must react in appropriate temperature for the best
antibody detection
1. “Cold” antibodies are usually vs. carbohydrate
antigens (ABO, Lewis, I/I, P, M, N)
2. “Warm” usually vs. protein antigens (Rh, Kell,
Kidd, Duffy, etc.)
3. Warm antibodies are the most important with
ABO
II. Size
a. RBC is over 700 times bigger than an antibody
b. Overcoming the size difference in-vitro requires the
manipulation of environment as well as forcing
RBCs closer together (centrifugation)
III. Electrical repellence
a. RBC surfaces have a negative charge due to sialic
acid at their surface
b. RBCs are naturally repelled by the negative
charges (zeta potential) and by the positively
charge ionic cloud that forms around the cells
c. Reduce zeta potential with:
1. Low ionic strength solutions (LISS) or albumin;
fewer ions to surround RBCs
2. Water – exclusion (polyethylene glycol, “PEG”)
d. Zeta potential is the reason IgG molecules are
challenged to directly agglutinate RBCs (limited
reach of monomeric antibody)
1. RBCs usually don’t get closer 14nm apart.
IV. pH
A. Optimal pH (7.0 in vitro) encourages an
environment where
1. RBC surfaces are negatively charged
2. Antibodies are weakly positive
B. Decreasing pH leads to disassociations of antibody
from RBC surface
V. Relative amount of antibody and antigen
A. Typical: 2 drops serum, 1 drop RBCs
B. This gives mild antibody excess, to promote shift of
equation to right
1 of 1
 1. Too much antibody/prozone effect and too
much antigen/postzone effect inhibit
agglutination

False positive results:

- Contaminated test tubes

- Clots/microclots
- Presence of agglutinins in plasma/serum
- Overcentrifugation
- Improper rinsing of pipettes

False negative results:

- Incorrect techniques for the particular Ab involved

- Insufficient washing
- Poor storage of reagents
- Delays in washing cells, setting up or reading
- Failure to add reagent
- Heavy or light suspension
- Centrifugation at low speed
- Too much agitation or resuspending the cell button
- Poor anti-complement acitivty

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