Practical Blood Bank

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Practical Blood Bank
Adsorption
Lab
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Adsorption

• Definition The removal of antibody from serum by
  combining a serum sample with appropriate RBCs
  under optimal conditions.
• After the antibody attaches to the membrane-bound
  antigens and the serum and cells are separated,
  the specific antibody remains attached to the red
  cells.
• It may be possible to harvest the bound antibody
  by elution.

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Mix serum with antibody with red cells with
corresponding antigen. Incubate at appropriate
temperature. Anti-e antibodies have been adsorbed
out of the serum and onto the RBCs.
Anti-S
Anti-S
Anti-e
Anti-e
Anti-S
Anti-e
Anti-e
Anti-e
Anti-S
Anti-e
Anti-e
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Adsorption techniques are useful in such
situations as

• Separating multiple antibodies present in a
  single serum.
• Removing autoantibody activity to permit
  detection of coexisting alloantibodies.
• Removing unwanted antibody (often anti-A and/or
  anti-B) from serum that contains an antibody
  suitable for reagent use.
• Confirming the presence of specific antigens on
  red cells through their ability to remove
  antibody of corresponding specificity from
  previously characterized serum.
• Confirming the specificity of an antibody by
  showing that it can be adsorbed only to red cells
  of a particular blood group phenotype.

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Adsorption Separating Multiple Antibodies

• Patient red cell phenotype is very helpful
  Phenotyping the patient can help in choosing a
  red cell phenotype that will give the most
  information.
• If None of the antibodies have been identified
  then a weak reacting cell can be used assuming
  that only one antibody may be reacting with it.

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Adsorption Separating Multiple Antibodies

• If One or More of the antibodies have been
  identified, then red cells lacking those antigens
  are usually chosen so that only one antibody is
  removed.
• Large volumes of red cells are required so
  Reagent vials are insufficient. Donor blood or
  staff members end up being the most convenient.

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Adsorption Positive DAT

• Auto adsorption is simply mixing patient serum
  with patient red cells to remove the auto
  antibody remaining in the serum under optimal
  conditions. If it is a warm auto antibody then
  adsorb at warm temps, if it is a cold auto
  antibody then adsorb at cold temps.
• Retest serum to determine degree of removal of
  auto antibody. May require second adsorption.
• Test panel with adsorbed serum to check for
  presence of allo antibody that was masked by the
  auto antibody.

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• Specimen
• Serum or plasma containing antibody to be
  adsorbed.
• Reagents
• Red cells (eg, autologous or allogeneic) that
  carry the antigen corresponding to the antibody
  specificity to be adsorbed.

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Procedure

• Wash the selected red cells at least three times
  with saline.
• After the last wash, centrifuge the red cells at
  800 to 1000 g for at least 5 minutes and remove
  as much of the supernatant saline as possible.
  Additional saline may be removed by touching the
  red cell mass with a narrow piece of filter
  paper.
• Mix appropriate volumes of the packed red cells
  and serum and incubate at the desired temperature
  for 30 to 60 minutes.
• Mix the serum/cell mixture periodically
  throughout the incubation phase.

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1. Centrifuge the red cells at 800 to 1000 g for 5
   minutes to pack cells tightly. Centrifuge at the
   incubation temperature, if possible, to avoid
   dissociation of antibody from the red cell
   membranes.
2. Transfer the supernatant fluid, which is the
   adsorbed serum, to a clean test tube. If an
   eluate is to be prepared, save the red cells.
3. Test an aliquot of the adsorbed serum, preferably
   against an additional aliquot of the cells used
   for adsorption, to see if all antibody has been
   removed.

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Interpretation

• If reactivity remains, the antibody has not been
  completely removed. No reactivity signifies that
  antibody has been completely adsorbed.

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Notes

• Adsorption is more effective if the area of
  contact between the red cells and serum is large
  use of a large bore test tube (13 mm or larger)
  is recommended.
• Multiple adsorptions may be necessary to
  completely remove an antibody, but each
  successive adsorption increases the likelihood
  that the serum will be diluted and unadsorbed
  antibodies weakened.
• Repeat adsorptions should use a fresh aliquot of
  cells and not the cells from the prior
  adsorption.
• Enzyme pretreatment of adsorbing cells can be
  performed to increase antibody uptake for
  enzyme-resistant antigens.