Principle: | Clinical Significance: | Specimen: | Materials: | Reagents: | Standardization: N.A. | Procedure: | Limitations: N.A. | Results Derivation: | Expected Results | Quality Control: | References:
The blood grouping and Rh(D) typing procedure is based on the principle of agglutination. Normal red blood cells, possessing antigens, will agglutinate in the presence of antibodies directed toward those antigens. Commercial antisera are used to test patient and donor cells. Patient plasma/serum is then tested against reagent A1 and B red cells. The ABO reaction should be reciprocal and does not require red cell stimulation.
II. Clinical Significance:
ABO compatibility testing between a donor and a recipient is the foundation of all pretransfusion testing. The antibodies of the ABO system may cause severe intravascular hemolysis as well as other manifestations of an acute hemolytic transfusion reaction. After the A and B antigens, the D (Rh) antigen is the most important red cell antigen in transfusion practice. The D antigen is highly immunogenic and will stimulate antibody production in 80% of D negative recipients receiving D positive red cells.
Refer to the Specimen section of the “General Processes, Blood Bank” protocol.
Note: The technologist performing the testing on the patient sample must confirm that all identifying information on the request form agrees with that on the specimen tube label before using the specimen for blood grouping, typing or compatibility testing. This is noted by placing a check mark by "Specimen Integrity" on the requisition for the test.
Refer to the Materials section of the “General Process; Blood Bank” protocol for vendor and product information.
2. Cellwasher (optional)
3. Illuminated optical aid
4. 12 x 75 mm test tubes
5. Graduated transfer pipettes
6. MTS incubator
7. Marking pen (water resistant)
Refer to Reagents section of the “General Process; Blood Bank” protocol for vendor and product information.
1. 0.85% Saline
2. Anti A
3. Anti B
4. Anti D
5. Anti IgG
6. A1, B Reagent cells,
7. Coombs Control Cells
8. Rh Control
VI. Standardization: N.A.
1. Perform a records check for all patients. Refer to “Patient Demographics & Orders (Electronic & Manual); Patient/Order Module: SafeTrace” or “Downtime, Computer; Blood Bank” protocols.
2. Blood grouping (forward and reverse) and Rh(D) typing:
A. Label a 12 x 75 mm test tube with proper patient or donor identification.
B. Transfer a few drops of the patient or donor red blood cells to the labeled 12 x 75 mm test tube.
Note: Interference with blood grouping and typing tests on cord blood can occur due to contamination by Wharton’s jelly. These cells must be washed before testing.
C. Prepare a 3-5% suspension of the cells, using saline as the diluent. Use the A1 or B reagent red cells as a guide in preparation of the 3-5% suspension.
D. Label five 12 x 75 mm test tubes as follows: A, B, D, a1, and b.
Note: Reverse grouping (plasma/serum testing) is not performed on cord blood, neonatal specimens or donor units.
E. Add one drop of the appropriate commercial antisera to the tubes labeled A, B and D (forward grouping).
F. Add two drops of patient plasma/serum to the tubes labeled a1 and b (reverse grouping).
G. Check each tube to make sure that they contain the appropriate antisera or plasma/serum.
H. Add one drop of the 3-5% suspension of patient or donor red blood cells to the tubes from step E.
I. Add one drop of A1 and B reagent red blood cells to the appropriately labeled tubes from step F.
J. Mix the contents of the tubes by gentle shaking. The tubes may incubate for up to 15 minutes at room temperature or may be centrifuged immediately.
K. Centrifuge the tubes at high speed. (See time requirements for centrifugation on each instrument.)
L. Gently resuspend the cells and using an illuminated optical aid, examine each tube macroscopically for agglutination. Record the results, grading the strength of reactions. (Refer to “Reading and Grading Reactions; Blood Bank” protocol.)
Note: If the patient or donor blood group is AB, an Rh control must be performed using the Rh Control reagent and patient or donor cells.
3. Indirect antiglobulin weak D testing of Rh(D) Negative typing: Indirect antiglobulin weak D testing is required only for cord blood specimens.
A. Take the patient’s negative D tube from the blood typing procedure and incorporate an Rh control tube using one of the negative reacting forward blood grouping tubes (A or B), or the Rh Control tube for the AB patient that doesn’t have a forward blood grouping tube.
B. Incubate the two tubes at 37°C for a minimum of 15 minutes to a maximum of 60 minutes.
C. Wash the cell suspensions from step B at least three times with saline, decanting thoroughly after the last wash. Refer to the Washing Cells Suspensions section of the “General Processes, Blood Bank” protocol.
D. Add two drops of Anti IgG to both tubes.
E. Mix the contents by gently shaking.
F. Centrifuge the tubes at high speed. (See time requirements for centrifugation on each instrument.)
G. Gently resuspend the cell button and using an illuminated optical aid, examine macroscopically for agglutination. Record the results, grading the strength of reactions. (Refer to “Reading and Grading Reactions: Blood Bank” protocol.)
H. Add one drop of Coombs Control Cells to each tube yielding negative results in step G.
I. Centrifuge the tubes at high speed. (See time requirements for centrifugation on each instrument.)
J. Gently resuspend the cells and observe for agglutination. Record the results, grading the strength of the reactions. (Refer to “Reading and Grading Reactions; Blood Bank” protocol”.) If no agglutination is present at this stage of the procedure, the test is invalid and the antiglobulin phase must be repeated.
VIII. Limitations: N.A.
IX. Results Derivation:
1. Group A: agglutination of patient cells with Anti-A antiserum and negative with Anti-B antiserum. Reverse grouping yields agglutination or hemolysis of only group B reagent red cells with patient plasma/serum.
2. Group B: agglutination of patient cells with Anti-B antiserum and negative with Anti-A antiserum. Reverse grouping yields agglutination or hemolysis of only group A1 reagent red cells with patient plasma/serum.
3. Group AB: agglutination of patient cells with Anti-A and Anti-B antisera. Reverse grouping yields no agglutination or hemolysis of group A1 or B reagent red cells with patient plasma/serum.
4. Group O: no agglutination of patient cells with Anti-A or Anti-B antisera. Reverse grouping yields agglutination or hemolysis of both group A1 and group B reagent red cells with patient plasma/serum.
5. Rh(D) Positive: agglutination of patient cells with Anti-D antiserum. No agglutination in the control tube. (Refer to the Quality Control section.)
6. Rh(D) Negative: no agglutination of patient cells with Anti-D antiserum or the control tube before or after weak D testing.
7. Rh (weak D) Positive: Agglutination of the patient cells with Anti-D antiserum after indirect antiglobulin (weak D) testing. No agglutination in the control tube before or after indirect antiglobulin (weak D) testing.
X. Expected Results and/or Critical Values: N.A.
1. The patient records check should match the results obtained at the present testing. If the present results and the history do not correlate, an investigation of the discrepancy must be performed before reporting. Since each case could vary significantly, it is difficult to articulate an exact procedure to follow for this investigation. A detailed report must be recorded in the incidence log and a supervisor or pathologist must be notified. An Rh negative result (immediate spin) on a previously weak D positive patient is an exception to the above. In this case, note the Rh change in the incidence log before reporting.
2. The donor unit’s group and type must match its component history in SafeTrace or be returned to the blood supplier.
3. Reverse grouping with patient plasma/serum is a quality control check of the forward grouping. If a discrepancy is found, further testing must be performed. Refer to “ABO Discrepancies” in the Technical Manual or consult a Pathologist.
4. Anti D (Bioclone) is a low protein reagent. It approximates the viscosity of the forward grouping (Bioclone) antisera; therefore, negative reactions using these antisera can be used in place of another Rh(D) control in all instances except in the case of a group AB individual, where a commercial Rh control is used. Agglutination in the Rh control tube may indicate a cold agglutinin, rouleaux or other forms of agglutination. If the Rh control tube has agglutination, notify a Pathologist.
5. When an indirect antiglobulin (weak D) test is necessary, an Rh control tube is used. If the control tube demonstrates reactivity, it is possible that the patient has a positive direct antiglobulin test and therefore the weak D status of this patient cannot be determined. A Pathologist must be notified.
6. Negative antiglobulin tests are controlled with Coombs control cells, which are group O red cells sensitized with IgG antibody. A positive test indicates the Anti-IgG was added and has not been neutralized by improper washing. Conclusion, the Anti-IgG was reactive.
7. Quality control is performed on commercial antisera daily and as new vials are opened. (Refer to the “Quality Control of Blood Bank Reagents; Testing & Recording” procedure)
1. Committee on Standards. "AABB Standards for Blood Banks and Transfusion Services." American Association of Blood Banks; Washington DC, current edition.
2. Committee on Technical Manual. "AABB Technical Manual". American Association of Blood Banks, Washington DC, current edition.
3. Package inserts for Ortho Diagnostics Systems, commercial antisera and reagent red cells, current version.
i. 8/18/91 M. English
ii. 6/10/92 N. Combs
iii. 8/96; S. Hosch (Revised III, Added VII.2.C.a-d)
iv. 2/98 B. Lucas/M. Burger (Revised: III.note; V.; VII.1. & Rh control; X.1.)
v. 2/12/99 M. Burger (Revised: Du changed to weak D; XI.1.)
vi. October 2003 S. Rodriguez (Revised: I., II., III., IV., V., VII.1-3., VIII.1., X.2.)
vii. September 2005 M. Burger, S. Hosch, S. Rodriguez (Revised: VII.2.B.Note, 3.A&C.)
viii. April 2006 S. Hosch, M. Burger, S. Rodriguez (Revised: weak D test for cord blood only)
ix. December 2006 S. Raymond, S. Rodriguez (Revised: XI.1.)
x. September 2009 M. Burger (Revised: changed to backup for new MTS Monoclonal Cards)
May 2010 L. Cooksley (no changes)
August 2011 R. Shireman (Revised: V.)