RT.003 Weak D Typing

Weak D Typing

1.0Principle

To test for a weak expression of the D antigen.

Red cells that react weakly or not at all in direct agglutination tests with anti-D may react with anti-D by the indirect antiglobulin test (IAT).

Red cells that fail to react a grade 2 in direct agglutination tests with anti-D are incubated with anti-D at 37° C and examined for agglutination. The red cells are washed to remove unbound antibody (IgG anti-D), then tested with anti-IgG.

2.0Scope and Related Policies

2.1When testing potential blood component recipients, the test for weak D is unnecessary.9.1

2.2Testing for weak D should be performed on infants who type as Rh negative if their mother is Rh negative and has no evidence of Rh alloimmunization.9.1 Also for infants whose mother’s blood type is unknown.

2.3Obstetrical patients that type as Rh positive or weak D positive should be designated as Rh positive. Patients whose red cells type as Rh negative should be designated as Rh negative.

2.4For apparent Rh negative obstetrical patients testing for weak D may be performed as per facility policy.

2.5A control system, appropriate to the anti-D reagent in use, shall be included. If this control is positive, the Rh typing must be repeated with an appropriate anti-D reagent and control.9.1

2.6If an Rh typing problem is detected and transfusion is necessary before resolution, for pre-menopausal females and children Rh negative blood products must be issued until the problem is resolved. Other patients, in the absence of a known anti-D, may receive Rh positive blood in emergency situations when there is a shortage of Rh negative blood.

3.0Specimens

EDTA anticoagulated whole blood

4.0Materials

Equipment:Cell washer

Serologic centrifuge

Block for test tubes

Waterbath/Heating block at 37°C

Microscope

Supplies:Test tubes – 10 x 75mm

Serologic pipettes

Reagents:Anti-D reagent

Control for Rh typing (suitable to anti-D reagent used)

Anti-IgG

IgG coated control cells

Normal Saline

5.0Quality Control

5.1See QCA.001 – Quality Control of Reagent Red Cells and Antisera.

5.2Before performing and reporting a “weak D typing” an accurate transfusion and pregnancy history should be obtained.

5.3Test for weak D should not be performed in the following situations:

5.3.1If the direct antiglobulin test (DAT) is positive on the red cells tested.

5.3.2If the patient has been transfused within the last three months with cellular donor unit(s) of different Rh type.

5.4Test for the weak D antigen should be done in the following situations:

5.4.1When weak or grade 1 reactions are found. Microscopic readings should only be done if mixed field agglutination is suspected.

5.4.2When Rh typing discrepancies are found between current and previous results.

5.4.3On Rh negative neonates born to Rh negative mothers. If the weak D testing is positive, the neonatal Rh type would be reported as “D positive” and the mother would be a candidate for Rh Immune Globulin (RhIG).

5.5If the test for weak D is performed on an obstetrical patient and result is weakly positive and/or mixed field, there must be an investigation to determine if the result is due to a large feto-maternal hemorrhage. If a feto-maternal hemorrhage had occurred the test for weak D cannot be interpreted. The patient’s physician must be notified.

5.6For other causes of mixed field refer to NRT.004 – Rh Type Problem Solving.

6.0Procedure

6.1Check the suitability of the specimen(s) to ensure that the specimen label information matches the request form. PA.002 – Determining Specimen Suitability steps 6.1 – 6.4.

6.2Perform a patient history check. See PA.003 – Patient History Check.

6.3Label tubes as per established procedure. See PA.004 – Labelling of Test Tubes and Block Set Up for Compatibility Testing.

6.4Add the appropriate reagent to the labelled test tubes according to the manufacturer’s insert.

6.4.1Add 1 drop of anti-D to tube labelled D.

6.4.2Add 1 drop of control reagent to tube labelled DC.

6.5Check the specimens for abnormal appearance. See PA.002 – Determining Specimen Suitability step 6.5.

6.6Compare the patient name or identification number on each tube with the corresponding information on the request form (or computer screen) to ensure they are the same.

6.7Prepare a 3% patient red cell suspension. It is not necessary to pre-wash the red cells, however, if a discrepancy is found the cells should be washed and the tests repeated.

6.7.1Label a test tube with the first 3 letters of the patient’s family name; transcribe this from the specimen tube, not from the request form. A pre-printed label may be used (ensure the information coincides exactly with the information on the specimen label).

6.8Add 1 drop of the 3% patient red cell suspension to the corresponding tubes labelled D and DC.

6.9Mix and centrifuge tubes at 3400 rpm for 10-15 seconds.

6.10Grade and record results. See PA.006 – Reading and Recording Hemagglutination Reactions. If the cells are strongly agglutinated (grade 2 or greater) in the anti-D tube and negative in the control tube, record the interpretations as “D positive” and do not proceed.

6.11Mix and incubate the tubes at 37°C for 15 minutes.

6.12Check and record the temperature of the waterbath or heating block on form QCA.006F.

6.13After 15 minutes of incubation, if a 37° C reading is desired:

6.13.1 Centrifuge tubes in a serological centrifuge at 3400 rpm for

10 – 15 seconds (according to the manufacturer’s directions).

6.13.2 Compare the patient name on each tube with the

corresponding information on the request form (or computer

screen) to ensure they are the same.

6.13.3 Read macroscopically. See PA.006 – Reading and

Recording Hemagglutination Reactions.

6.13.4 Grade and record results. See PA.006 – Reading and

Recording Hemagglutination Reactions. If the cells are

strongly agglutinated (grade 2 or greater) in the anti-D tube

but negative in the control tube, record the interpretation as “D positive” and do not proceed with the antiglobulinphase of the test. If there is no reaction in either tube, proceed to step 6.14.

6.14Proceed to the antiglobulin phase. See PA.005 – Cell Washing Automated and Manual.

6.15Interpret the Rh type.

6.15.1 If patient cells react as described in 7.0 – Reporting, record

the Rh typing on the request form or in the computer as per

established procedure.

6.15.2 If the patient cells do not react as described in 7.0 –

Reporting, a discrepancy exists and must be resolved

before reporting the Rh type. See NRT.004 – Rh Typing

Problem Solving and Procedural Notes 8.2.

6.16If a previous Rh typing result was found, compare it with the current Rh result. If results are discrepant, see NRT.004 – Rh Typing Problem Solving.

6.17When the procedure is complete, perform a clerical check.

For each Rh type, check that:

• The patient name and identification number are identical on all specimens and the request form or computer screen

• The patient name is the same on all the test tubes and the request form or computer screen

• The test results have been interpreted correctly

6.18Initial or sign and record the completion time and date on the request form or in the computer.

6.19Verification of results must be recorded. See 7.0 Reporting.

7.0Reporting

7.1The control must be negative. Interpret results as follows:

IAT

Interpretation

NT: Not Tested

7.1.1Reactions that are a grade 2 or stronger after 15 minutes incubation at 37°C indicate the presence of the D antigen. Report as Rh positive.

7.1.2Reactions that are a grade 1 or stronger by IAT indicate the presence of the D antigen. Report as Rh positive.

7.1.3No reaction (negative) by IAT indicates the absence of the D antigen. Report as Rh negative.

7.2If the control is positive or if the positive reaction with anti-D is weaker than grade 1 by IAT:

7.2.1Do not report the Rh type result.

7.2.2Additional investigation is required. See NRT.004 – Rh Typing Problem Solving.

8.0Procedural Notes

8.1Tests should be read immediately after centrifugation. Delay may cause bound IgG to dissociate from red cells and either leave too little IgG to detect or neutralize AHG reagent causing false negative results.

8.2In cases where the result of the weak D typing cannot be reported or is unresolved:

8.2.1If cellular blood components are required STAT, before the discrepancy is resolved, for pre-menopausal females and children Rh negative blood products must be issued. Other patients, in the absence of a known anti-D, may receive Rh positive blood in emergency situations when there is a shortage of Rh negative blood.

8.2.2If unable to determine the Rh type and the patient is a neonate delivered by a mother who is Rh negative, a Kleihauer-Betke test should be performed on the post delivery maternal specimen to determine the amount of Rh Immune Globulin (RhIG) required.

9.0References

9.1Standards for Hospital Transfusion Services, Version 2 – September 2007, Ottawa, ON: Canadian Society for Transfusion Medicine, 2007: 5.3.3.2, 5.3.3.3.

9.2Roback JD, ed. American Association of Blood Banks Technical Manual, 16th ed. Bethesda, MD: American Association of Blood Banks, 2008: 447-448, 889-890.

9.3Judd WJ, Johnson ST, Storry JR. Judd’s Methods in Immunohematology, 3rd ed. Bethesda, MD: American Association of Blood Banks, 2008:15-20.