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301 Elution Techniques in Blood Bank
George H. Roberts
Heat at 560C with Heat causes thermal Rapid and easy. ABO antibodies
Temperature agitation. Spin dissociation of Eluate stained with
Landsteiner Heat and harvest eluate AgAb complex and hemoglobin. Less
denatures cell effective for warm
membrane auto/alloantibodies
Digitonin lysis of Acid alters charge Clear eluate. Most allo and
RBC. Wash yields a of proteins causing Slow process autoantibodies.
hemoglobin-free a change in requiring Decreased
Acid Elution stroma. Add structural many washes sensitivity for
glycine buffer configuration Kidd system
Digitonin-Acid (pH 3.0) to stroma.
Readjust pH with
phosphate buffer
after elution
Add cold (40C) Acid alters the Requires less time Some alloantibodies
glycine (pH 3.0) charge of proteins than digitonin. and autoantibodies.
directly to RBC. causing a change in Eluate stained with
Cold Acid Incubate one minute structural hemoglobin. Less
at 40C. Spin and configuration sensitive in
harvest eluate. eluting many
Readjust pH with alloantibodies
phosphate buffer
Mix cells and Same as ether Flammable, but does Most alloantibodies
xylene and incubate not require and autoantibodies
at 560C for 10 minutes. explosion-proof
Xylene Remove xylene and refrigerator.
stroma by vacuum Requires vacuum
aspiration. aspiration
Harvest eluate
Heat 450C with Heat causes thermal Rapid and easy. Preparing DAT-
Procedures for frequent agitation dissociation of Some antigens may positive RBC for
antigen-typing AgAb complex be damaged. RBC antigen typing
DAT-positive RBC without denaturing remain intact
Modified Heat red cell membrane
Incubate 1 volume Causes dissociation of Requires two hour Same as modified heat
Chloroquine cells and 4 volumes immune complexes incubation
diphosphate chloroquine
diphosphate
some instances, rather than using multiple absorp- the cell surface without damaging red cell integrity
tions, the scientist may choose to use selected panel or altering antigenic reactivity. The desired end prod-
cells to demonstrate specificity in cases of multiple uct is the red cell, not the antibody. As an example,
antibodies. in Rh and other forms of HDN (those due to antibod-
ies other than Rh), the DAT may be positive due to
To accurately phenotype red cells with blocking of the antigen sites by high titers of anti-
positive DAT body. For example, Rh positive red cells giving a
When cells are heavily coated with IgG antibod- strongly positive DAT may type as Rh negative due to
ies, tests with antisera of high protein content are dif- the blocking of Rh receptors. If an attempt is made to
ficult and those with antiglobulin reactive antisera are perform a weak D test, the test and the associated Rh
impossible. It is necessary to dissociate antibody from control will both be positive. This fact can be a seri-
Comments on Elution
Elution removes antibody molecules from the red
cell membrane either by disrupting the antigen or
changing conditions to favor dissociation of antibody
from antigen. Many techniques are available, and no
single method is best in all situations. If an eluate pre-
pared by one technique is unsatisfactory, it may be
helpful to prepare another eluate utilizing a different
technique.
The red cells used for any elution technique must
Together…
be thoroughly washed to remove all antibody except Making a World
that bound to the cells. Six washes with large volumes
of saline is usually sufficient. Adequacy of washing is of Difference
tested by examining saline from the last wash for the
presence of antibody by the indirect antiglobulin
(IAT) procedure. If antibody is detectable in the last
wash, there could be enough unbound antibody mol-
ecules still present so that results obtained on testing
the eluate are not valid. This assumes the possible
mixture of alloantibody and autoantibody.
As soon as the elution is completed, remove the
supernatant fluid and place it into a separate tube to
avoid reattachment of antibody to cell stroma and a
possible false negative test result.
Elution Techniques
There are several different techniques for elution
based upon the process used to cause dissociation of
the antigen/antibody complex. The table below indi-
cates the various techniques used, describes the pro-
cedure and action, lists the advantages and disadvan- Dental Assistants Recognition Week
tages of each, and gives times when each method is a tribute to the commitment
might be appropriate to use. and dedication dental assistants
exhibit throughout the year.
Suggested Readings Whether working at chairside with
Blaney, K, & Howard, P. (2000). Basic and Applied Concepts the dentist, taking X-Rays or man-
of Immunohematology. St. Louis: Mosby.
Brecher, M. (2002) American Association of Blood Banks
aging the business office, teaching
Technical Manual, 14th edition. Bethesda: AABB. or working in insurance or sales,
Harmening, D. (2005). Modern Blood Banking and Transfu- dental assistants are vital to the
sion Practices, 5th edition. Philadelphia: F. A. Davis Company. success of the dental practice.
Quinley, E. (1998). Immunohematology Principles and Prac-
tice. Philadelphia: Lippincott.
Rudmann, S. (1995). Textbook of Blood Banking and Transfu-
American Medical Technologists
sion Medicine. Philadelphia: W. B. Saunders Company. (AMT) invites you to join in the
Turgeon, M. (1995). Fundamentals of Immunohematology. celebration to recognize the skills
Baltimore: Williams and Wilkins. and vital contributions of the
dental assistant to the team effort
that enables patients to receive
the quality care they expect and
desire.
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