b. Factors Affecting the First Stage (Sensitization). Red blood cell
sensitization with antibody obeys the law of mass action. Thus, the reaction between
antigen on the RBC surface and antibody is reversible and the quantity of
cell-bound antibody at equilibrium will vary depending on the reaction conditions and the
equilibrium constant of the antibody. The reaction conditions should be designed to
maximize the quantity of cell-bound antibody at equilibrium in order to facilitate
detection of either blood group antigen or antibody. Some of these reaction conditions
are described below:
(1) Temperature. Most blood group antibodies show their greatest reactivity
over a restricted temperature range, some reacting optimally at 4C, others at 37C.
Antibodies reacting optimally at 37C have been described as "warm" antibodies, and
those reacting optimally at lower temperatures as "cold" antibodies. Agglutinins
(antibodies) having maximum reactivity at one temperature may have sufficient thermal
amplitude to be active at others. Antibody activity is usually tested at room temperature
and at 37C. Antibodies active at 37C are the most clinically significant, although "cold"
antibodies cannot be ignored if they have a wide thermal amplitude (for example, above
30C). Antibodies only reacting at lower temperatures may be of importance in patients
subjected to hypothermia.
(2) pH. The pH optima for antibody reactivity in most blood-group systems
have not been investigated. For anti-RhO(D), the optimum pH lies between 6.5 and 7.
Antibodies of other blood-group specificities may have different pH optima (for example,
some examples of anti-M react best at pH 5.5).
(3) Incubation time. Time is required for the antibody RBC reaction to reach
equilibrium. The amount of time required to reach this state will depend upon other
variables. The rate of antibody binding is greatest initially, so incubation times for
routine laboratory procedures may be relatively short (for example, 15 to 30 minutes).
(a) The ionic strength of the reaction medium is one of the
physiochemical conditions that play an important role in the binding of antibody to RBC
antigens. Ionic strength is a measure of intensity of the electrical field resulting from
ions in solution. Electro-static forces (interaction of positive and negative charges) play
an important role in antibody reaction involving RBCs. Red blood cells carry a large
electronegative charge, which serves to keep them from spontaneously aggregating.
This enables them to function efficiently in oxygen transport by maintaining a maximum
surface area available for gas diffusion. When RBCs are suspended in an electrolyte
solution (0.85 percent NaCl), the cations (positive) are attracted to the negatively
charged RBCs, and the RBC becomes surrounded by a diffuse double layer ("ionic
cloud"), that travels with the RBC as if it were part of it. The outer edge of this layer is
called the surface of shear or the
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