STANDARD OPERATING PROCEDURE:
ABO blood groups are determined by testing red cells for the presence of A and B antigens (‘Forward’ grouping). Blood grouping reagents anti-A and anti-B are used in blood typing tests to detect these antigens. Normal human red cells possessing the A and/or B antigen will agglutinate in the presence of specific antibody. The absence of agglutination is a negative test indicating the antigen is not present.
Reverse grouping using plasma or serum tested against known A1 and B cells must be performed in conjunction with red cell grouping tests. Most individuals naturally produce antibodies directed against the A and/or B antigen absent from their own red cells. The results of the forward and reverse grouping must agree before a valid interpretation of the ABO group can be made.
Agglutination of red cells with anti-D reagent indicates the presence of the D antigen. No agglutination indicates the cells are D negative.
Optimal detection of weakened forms of the D antigen may require use of the indirect antiglobulin test. However, application of this test for routine pre-transfusion testing is not necessary.
Specimen Requirements:
Clotted specimens or specimens collected into anticoagulant may be used. [Refer to specific manufacturer’s direction inserts for approved anticoagulants.] The sample should be tested as soon as possible after collection or stored at 2-8°C.
Equipment, Reagents and Supplies
10 X 75 mm test tubes blood grouping reagent anti-A
Transfer pippets blood grouping reagent anti-B
0.9% normal saline blood grouping reagent anti-A,B (donor confirmation testing))
Test tube rack reagent A1 red cells 3% suspension
Viewing light and mirror reagent B red cells 3% suspension
Heat block or water bath blood typing reagent anti-D(monoclonal blend, or monoclonal/poly serologic centrifuge clonally blend)
Rh-Hr Control
Anti-IgG reagent
Coombs Control (Check) Cells
Process:
NOTES:
1. Do not separate the patient’s plasma from the red cells for pretransfusion testing. All testing must be performed from the original sample tube.
2. Testing for ABO and Rh may be set up at the same time.
3. Antisera should be added to the tube prior to the cell suspension for easier resuspension of the cell button.
ABO/RH Testing
1. Prepare a 3% suspension of patient red cells in isotonic saline. Refer to Preparation of a 3% Red Cell Suspension for instructions.
2. Label two test tubes “A” and “B” for the forward type and two tubes “a” and “b” for the reverse type.
3. Label all tubes with patient’s last name, or donor number if testing donor segment.
4. Add two drops of patient’s serum or plasma into both tubes. Add 1 drop of A1 reagent red cells into tube labeled “a” and one drop of B reagent red cells into tube labeled “b”.
5. Add one drop of reagent anti-A into tube labeled “A” and one drop of reagent anti-B into tube labeled “B”. Add one drop of the patient’s cell suspension to both tubes.
6. Mix well and centrifuge all tubes according to the time designated on each centrifuge for saline reactions.
7. Completely re-suspend the cells by gentle agitation and examine for agglutination and/or hemolysis. Use the viewing mirror/agglutination viewer to observe reactions.
8. Grade and record reactions immediately. Refer to Grading and Interpretation of Results (Tube) for additional information.
NOTE: For AB positive patients refer to step 12 in the Rh section of this procedure.
9. Forward and reverse groupings must agree. Discrepancies between the two must be resolved prior to transfusion, or group O blood must be issued.
Procedural Notes:
ABO confirmatory tests for labeled donor red cell units will only include forward grouping. Group O donors may be tested using anti-A, B in lieu of Anti-A and anti-B. Group O red cells will be negative with anti-A, B. Red cells reactive with anti-A, B may be group A, B or AB and must be tested with anti-A and anti-B to determine ABO group.
INTERPRETATION OF RESULTS:
Forward Reverse Interpretation Approximate
Frequency (%)
Anti-A Anti-B A1 cells B cells
3-4+ 0 0 2-4+ Group A 27-40
0 3-4+ 2-4+ 0 Group B 10-20
0 0 3-4+ 3-4+ Group O 45-50
3-4+ 3-4+ 0 0 Group AB 4
If the results obtained do not agree with those listed above, refer to procedure for ABO Discrepancy Resolution. Forward and reverse grouping discrepancies must be resolved. Refer to ABO Discrepancy Resolution for additional information.
Group O red cells and group AB plasma products must be issued if transfusion is needed before the ABO group of the patient can be accurately determined.
LIMITATIONS OF THE PROCEDURE:
A. In some patients (e.g. newborns, elderly or immunocompromised patients) the expected ABO antibodies may be weak or missing.
B. Some subgroups of A or B antigens may not be detected using this method.
C. ABO tests should not be routinely performed at temperatures below 15ºC or higher than 30ºC.
D. Anti-A and –B reagents are produced from cell cultures of murine hybridomas. These reagents contain single or blended monoclonal antibodies from multiple cell lines, which may differ by manufacturer. Uncommon examples of A and B antigens may be non-reactive or will show variable reactivity dependent on the source of the antisera.
Refer to manufacturer’s inserts for specific performance characteristics of the antisera in use.
Rh Testing
1. Prepare a 3% suspension of patient red cells in isotonic saline. Refer to Preparation of a 3% Red Cell Suspension for instructions.
2. Label the test tube “D” and the patient’s last name.
3. Add one drop of reagent anti-D into tube and add one drop of patient’s cell suspension.
4. Mix well and centrifuge the test according to the time designated on the centrifuge for saline testing.
5. Completely re-suspend the cells by gentle agitation and examine for agglutination. Use the viewing (agglutination) mirror to observe reactions.
6. Grade and record reactions. Refer to Grading and Interpretation of Results (Tube) for additional information.
7. Weak D testing (DUTEST) must be ordered by Technologist and performed in the following situations:
• Newborns with Rh negative mothers,
• Patients with a historical Rh positive type due to previous weak D testing,
• Donors of bone marrow, apheresis stem cells or transplant organs
8. Weak D testing is performed as follows:
a. Label an additional tube with “D Cont” (and patient’s last name).
b. Add one drop the Rh-hr control reagent and one drop of the patient’s red cell suspension to the control tube.
c. Incubate the control with the “D” tube at 37C for 15 minutes.
d. Wash the cells 3 – 4 times with isotonic saline after incubation, decanting completely after the last wash.
e. Add two drops of anti-IgG to the tubes, or amount as specified in manufacturer’s instructions.
f. Mix well and centrifuge at the speed and time designated on the centrifuge for the AHG phase of testing.
g. Completely re-suspend the cells by gentle agitation and examine for macroscopic agglutination. Use the viewing mirror to observe reactions.
h. Grade and record reactions.
i. Add AHG Control cells (Coombs Control Cells/Check Cells) to all negative tests.
j. Mix well and centrifuge at speed and time designated for the AHG test.
k. Completely resuspend the cells by gentle agitation and examine for agglutination. Use the viewing mirror to observe reactions.
l. Weak D positive test results are valid only if the “D Cont” is negative.
m. Mixed field agglutination in a weak D test on a woman who recently delivered may indicate a mixture of maternal Rh negative and fetal Rh positive blood. Maternal Rh type must be confirmed using pre delivery sample if there is any question about maternal candidacy for Rh immune globulin. (Refer to Rh Immune Globulin)
9. If the patient types as Group AB Rh positive, an Rh control must be tested at room temperature to validate the immediate spin test as follows:
a. Add one drop of Rh-hr control reagent in a 10 x 75 mm test tube labeled D cont (and patient’s last name).
b. Add one drop of the patient’s 3% red cell suspension.
c. Centrifuge the mixture at the speed and time designated on the centrifuge for saline phase.
d. Gently dislodge the red cell button and examine for agglutination using a viewing mirror.
e. Grade and record reactions.
f. The control must be negative in order for the D typing to be valid.
g. If the control is positive, the patient must be transfused with Rh negative products
INTERPRETATION OF RESULTS:
Positive test: Agglutination of red cells at the immediate spin, 37ºC or antiglobulin phases, with appropriate controls. (See Limitations.)
Negative test: No agglutination of red cells with anti-D.
Mixed field: Mixed-field results must be investigated. In cases where the patient has been recently transfused, the patient’s historical Rh type and the Rh type of transfused blood products should be documented. Interpretation of test results should be made with extreme caution when reliable information cannot be obtained (i.e. patients transferred from other hospitals).
Document results of ABO/Rh interpretation.
Interpretation Chart:
Anti-D Control Weak D test (AHG) Control/DAT Interpretation
+ 0 NT NT D positive
0 0 NT NT D negative
+ + NT NT Invalid
0 0 ³ 2+ * 0 D positive
0 0 0 0 D negative
0 0 + + Invalid
* see below (D)
LIMITATIONS:
A. On rare occasions, red cells coated in vivo with IgG or IgM molecules may spontaneously agglutinate and lead to falsely positive results. In these cases, aggregation or spontaneous agglutination will most likely be observed in other saline tests, such as ABO grouping. It is not essential to test a control in parallel with this reagent unless the cells are reactive with anti-A, anti-B and anti-D. If the cells test as group AB, D-positive, an additional control is required for valid interpretation of results (i.e. Monoclonal Control).
B. Positive tests for weak D antigen (AHG reactions) are valid only when the red cells tested have been shown to be negative by the Direct Antiglobulin Test (DAT), or if a patient red cell control (in Monoclonal Control) was tested in parallel and found to be negative at the AHG phase.
C. Red cells that express variant Rh antigens may demonstrate weaker than expected results with some sources of anti-D.
D. Tests for weak D antigen with results of less than 2+ should be evaluated by a Technical Specialist or supervisor before valid conclusions can be made.
A. Red cells rendered DAT negative by chemical treatment (e.g. glycine-EDTA) may be tested for weak D antigen by the antiglobulin test. A test control consisting of the treated cells with Monoclonal Control must be tested in parallel.
Discrepancy Resolution
Discrepancies in ABO Group Testing
Missing or weak antibodies in reverse testing Elderly persons
Newborns
Hypogammaglobulinemia
Chimeras
Unexpected reactions in reverse testing Cold reacting agglutinin
Missing or weak antigen in forward testing Subgroup
Disease change
Recent transfusion of different ABO group
Unexpected reactions in forward testing Spontaneous aggregation
Rouleaux
Cold reacting agglutinin
Disease change
Missing or weak antigen(s) in forward testing, and or unexpected reactions in reverse grouping Bone Marrow Transplant may be the cause.
Resolution of Typing Discrepancies
Missing or weak antibodies in reverse testing Incubate reagent red cell and patient serum or plasma mixture at room temperature for 15 minutes.
Unexpected reactions in reverse testing Warm the reagent red cells and serum to 37C prior to mixing. Read serum tests at 37C.
Remove cold reactive auto-agglutinin from serum using cold autoabsorption method.
Unexpected reactions in reverse testing (continued) Identify the cold reactive alloantibody and test the patient serum or plasma using A1 and B cells that lack the corresponding antigen
Missing or weak antigen in forward testing Incubate patient red cells with reagent antisera at room temperature for 30 minutes.
