Testing Methods and Interferences Flashcards
ABO Discrepancies: False Positives
Cold Agglutinins Warm Agglutinins Positive Direct Antiglobulin Test Using a sample post-transfusion Use of wrong antiserum Reagent contaminated with low-incidence antibody or other contaminants Polyagglutinable cells
ABO Discrepancies: False Negatives
Failure to add serum Blocking of antigen Incorrect cell suspension Incorrect antiserum-cell ratio Resuspension too vigorous Antisera does not react with a weakly expressed or variant antigen Reagent Deterioration Antisera that is directed against a compound antigen Use of wrong antiserum
False Positives: Fixing Cold Agglutinins
Washing the cells with warm saline should resolve this
False Positives: Fixing Warm Agglutinins
Washing cells or using IgM reagents or treating the cells to remove IgG
Fixing a False Positive DAT
Weak D (IAT) will be invalid with IgG coated RBCs, IgG must be removed from the cell surface
False Positives: Fixing Polyagglutinable Cells
Will agglutinate with any reagent containing human serum, monoclonal reagent should correct this
False Negative: Blocking of Antigens
In severe HDFN, the RBCs are so heavily coated that antisera cannot bind, heat elution should correct this
IAT
Crossmatch, testing Donor cells and patient plasma using an enhacement serum and a 37C incubation and includes the addition of antiglobulin (AHG in our lab)
DAT
ABO-Rh testing
Serologic test to detect in-vivo binding of Ab and Complement to RBCs, Useful for determining potential hemolytic reactions
Phenotyping
Testing a cell with known antisera (e.g. anti-E, anti-C, or anti-K) to find out if the cell carries the antigen. The principle is the same as forward typing except the term phenotyping is reserved for antigens other than A, B, or D.
Reasons to Phenotype
- To prove that the patient’s cells lack a particular antigen. If an antibody has been identified in a patient’s serum, the final step is to prove his/her cells do not carry the antigen.
- When an antibody is found in a patient, antigen negative units of blood must be found for transfusion. The way to find such units is to phenotype donor units.
- Phenotyping is sometimes helpful in genetic studies, and is used in paternity testing. The red cell antigens used in paternity testing include the Rh antigens D, C, c, E, and e. Also M, N, S, s, K, and k, and those in the Kidd and Duffy systems are used.
Positive Control in Phenotyping
Always run a positive control cell that is heterozygous for the antigen. This ensures that the serum is strong enough to react with a weak (heterozygous) antigen, and gives confidence in the results.
Negative Control in Phenotyping
Always run a negative control cell. This proves that the antiserum is not giving you a false negative reaction.
Purpose of Antibody Screen
The purpose of the antibody screen is to detect red blood cell antibodies other than “expected” anti-A and anti-B. These antibodies are called alloantibodies.
Instances where an Antibody Screen is required
- Pre-transfusion testing
- Prenatal testing
- Donor testing
Antibody Screening
The procedure is called the indirect antiglobulin test (IAT) in which the patient’s serum is incubated with screening cells. If alloantibodies are present in the patient, they sensitize the screening cells which agglutinate at some point during testing.
Enhancement Reagent Function
Promote antigen-antibody binding or agglutination by reducing the ion cloud (zeta potential) that surrounds normal red cells. Various enhancement reagents are available but the most widely used are low ionic strength saline (LISS), polyethylene glycol (PEG), and bovine albumin.
LISS
Uses: Sensitive, economical quick
Drawbacks: Enhances cold autoantibodies
Albumin
Uses: Does not enhance warm autoantibodies
Drawbacks: Needs longer incubation; not sensitive for most antibodies except Rh
Polyethylene Glycol
Uses: Shows increased sensitivity
Drawbacks: Enhances warm autoantibodies
Enzymes
Uses: Eliminate reactivity of Fya, Fyb, M, N, S
Drawbacks: Enhances cold and warm autoantibodies;
Should not be used as the only method
IgM in Ab Screen
Can agglutinate the cells at room temperature and may be detected by centrifuging the mixture before incubation, I.S. (or immediate spin) phase.
Comparison of room temperature and 4°C antibody screens can be done to confirm the presence of an IgM antibody in the patient’s plasma. The reactions should be stronger at 4°C if an IgM is causing agglutination.
Purpose of Ab ID
Panels are done to identify antibodies when the ABS (antibody screen) is positive and also in cases when a patient has a positive DAT.
Positive Reactions in Ab ID
Indicate the phase and strength of reactivity which can suggest specificity.
Negative Reactions in Ab ID
Negative reactions allow exclusion of antibodies to antigens expressed on the non-reactive cells.
Main Functions of Crossmatching
- It is a final check of ABO compatibility between donor and patient.
- It may detect the presence of an antibody in the patient’s plasma that will react with antigens on the donor red blood cells that was not detected in antibody screening.
Crossmatching Results: (+) ABS and (-) Crossmatch
If the ABS is positive and the crossmatched units are negative; you must do further testing to identify and Phenotype the unknown antibody(s). The units should be phenotyped for the antigen(s) to the corresponding antibody(s).
Crossmatching Results: (+) ABS and (+) Crossmatch
If the ABS is positive and the crossmatched units are positive (agglutinated), the antibody must be identified before crossmatching more units. The new units crossmatched should be phenotyped for the antigen(s) to the corresponding antibody(s) identified.
Crossmatching Results: (-) ABS and (+) Crossmatch
If the ABS is negative and the crossmatched units are positive, run a DAT on the donor cells. If the donor DAT is positive indicating that the cells are sensitized, the AHG phase of the crossmatch will be positive.
