2. UNIT OUTCOMES
1. Perform the pre-warming technique accurately and precisely.
2. Read and interpret the results correctly.
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3. OVERVIEW
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▪ Immune hemolytic anemia is defined as shortened RBC survival mediated
through the immune response, specifically by humoral antibody.
▪ Immune hemolysis is destruction of RBCs as a result of antibody production
and is an acquired characteristic of the RBC membrane associated with
demonstrable antibodies, as opposed to intracorpuscular defects such as
enzyme deficiencies and hemoglobinopathies, which represent intrinsic
abnormalities of the patient’s RBCs.
▪ The three broad categories of immune hemolytic anemias are:
1. Alloimmune
2. Autoimmune
3. Drug-induced
4. COLD REACTIVE AUTOANTIBODIES
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▪ Cold reactive autoantibodies are frequently encountered in serologic testing.
Most of the time, the antibodies detected are not clinically significant but
nevertheless can present challenges for the blood banker.
▪ Occasionally, cold autoantibodies are clinically significant and cause immune
hemolytic anemia. The antibody specificity may be of interest to the
technologist or may be a clue to the disease process, but the important
distinction between clinically significant and benign cold autoantibodies is
their thermal range.
▪ Any antibody that reacts at or near body temperature, regardless of specificity,
is potentially clinically significant; this includes “cold” autoantibodies.
6. Techniques useful in differentiating between cold autoantibodies and
alloantibodies are:
1. Prewarm technique
2. Cold autoadsorption technique
▪ There have been reports of clinically significant alloantibodies being unintentional
“prewarmed away,” either due to poor technique or because the antibodies were
partially IgM, yet this technique persist in practice and does have value in
investigating an identified antibody’s thermal amplitude.
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OVERVIEW
7. PRINCIPLE OF PRE-WARD TECHNIQUE
The principle of the prewarm test is that by first warming the cell suspension and
serum prior to mixing, avoiding room temperature centrifugation after 37°C
incubation, and washing with saline warmed to 37°C, any reaction between the
cold autoagglutinin and RBC antigens can be prevented, thus avoiding
complement activation.
While cold reacting antibodies should not react by this method, alloantibodies
that are reactive at 37°C (i.e., potentially clinically significant) would still bind to
the cells and be detectable at the antiglobulin phase.
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8. LABORATORY EXPERIMENTATION
The reactivity of IgM cold auto-antibodies can be reduced or eliminated by
performing pre-warmed tests.
Most problems encountered in compatibility testing, antibody detection, and
identification tests that are caused by cold agglutinins can be resolved with the
use of this technique.
By preventing the reaction between the cold agglutinin and the red cell at room
temperature (during centrifugation, and so on), one prevents complement
activation.
The anti-C3 in polyspecific antihuman globulin reagents with the remaining C3
makes the detection of significant AHG-reactive antibodies difficult. Pre-warming
of the serum, cells and additive will hinder the binding of the cold autoantibody
and the resulting complement activation.
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10. 1. Label 1 tube for each reagent or donor cell sample to be tested.
2. Add 1 drop of the appropriate 2 to 4% red cell suspension to each tube.
3. Place the tubes containing the red cells, a tube containing small amount of
patient's serum, and a tube containing the additive solution, if any, at 37 °C for
5-10 minutes.
4. Transfer 2 drops of pre-warmed serum into each tube containing pre-warmed red
cells. Mix without removing the tubes in the incubator or water bath.
5. Incubate at 37°C for at least 30 minutes with no additive, or the appropriate
time for the additive used.
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PROCEDURE
11. 6. Without removing the tubes from the water bath, fill all tubes with pre-warmed
saline (37°C). Centrifuge and wash 2-3 times with warm saline.
7. Add anti-IgG antiglobulin reagent, and record reactions.
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