Testing Methods and Interferences

Question 1

ABO Discrepancies: False Positives

Answer 1

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

Question 2

ABO Discrepancies: False Negatives

Answer 2

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

Question 3

False Positives: Fixing Cold Agglutinins

Answer 3

Washing the cells with warm saline should resolve this

Question 4

False Positives: Fixing Warm Agglutinins

Answer 4

Washing cells or using IgM reagents or treating the cells to remove IgG

Question 5

Fixing a False Positive DAT

Answer 5

Weak D (IAT) will be invalid with IgG coated RBCs, IgG must be removed from the cell surface

Question 6

False Positives: Fixing Polyagglutinable Cells

Answer 6

Will agglutinate with any reagent containing human serum, monoclonal reagent should correct this

Question 7

False Negative: Blocking of Antigens

Answer 7

In severe HDFN, the RBCs are so heavily coated that antisera cannot bind, heat elution should correct this

Question 8

IAT

Answer 8

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)

Question 9

DAT

Answer 9

ABO-Rh testing
Serologic test to detect in-vivo binding of Ab and Complement to RBCs, Useful for determining potential hemolytic reactions

Question 10

Phenotyping

Answer 10

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.

Question 11

Reasons to Phenotype

Answer 11

1. 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.
2. 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.
3. 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.

Question 12

Positive Control in Phenotyping

Answer 12

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.

Question 13

Negative Control in Phenotyping

Answer 13

Always run a negative control cell. This proves that the antiserum is not giving you a false negative reaction.

Question 14

Purpose of Antibody Screen

Answer 14

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.

Question 15

Instances where an Antibody Screen is required

Answer 15

1. Pre-transfusion testing
2. Prenatal testing
3. Donor testing

Question 16

Antibody Screening

Answer 16

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.

Question 17

Enhancement Reagent Function

Answer 17

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.

Question 18

LISS

Answer 18

Uses: Sensitive, economical quick
Drawbacks: Enhances cold autoantibodies

Question 19

Albumin

Answer 19

Uses: Does not enhance warm autoantibodies
Drawbacks: Needs longer incubation; not sensitive for most antibodies except Rh

Question 20

Polyethylene Glycol

Answer 20

Uses: Shows increased sensitivity
Drawbacks: Enhances warm autoantibodies

Question 21

Enzymes

Answer 21

Uses: Eliminate reactivity of Fya, Fyb, M, N, S
Drawbacks: Enhances cold and warm autoantibodies;
Should not be used as the only method

Question 22

IgM in Ab Screen

Answer 22

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.

Question 23

Purpose of Ab ID

Answer 23

Panels are done to identify antibodies when the ABS (antibody screen) is positive and also in cases when a patient has a positive DAT.

Question 24

Positive Reactions in Ab ID

Answer 24

Indicate the phase and strength of reactivity which can suggest specificity.

Question 25

Negative Reactions in Ab ID

Answer 25

Negative reactions allow exclusion of antibodies to antigens expressed on the non-reactive cells.

Question 26

Main Functions of Crossmatching

Answer 26

1. It is a final check of ABO compatibility between donor and patient.
2. 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.

Question 27

Crossmatching Results: (+) ABS and (-) Crossmatch

Answer 27

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).

Question 28

Crossmatching Results: (+) ABS and (+) Crossmatch

Answer 28

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.

Question 29

Crossmatching Results: (-) ABS and (+) Crossmatch

Answer 29

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.