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Transfusion Medicine Quality Manual Standard Operating Procedure for Rh Typing Tube

Method

Provincial Blood

Coordinating Program

_______________________________________________________________________ This document may be incorporated into each Regional Policy/Procedure Manual.

NL2010.-009–TMQ

Version: 3.0

Effective Date: 2015-04-03

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Standard Operating Procedure for

Rh Typing Tube Method

1.0 Policy Statements

1.1 Each recipient blood sample for compatibility testing shall be tested for Rh (D).

1.2 Rh (D) type shall be determined by phenotyping the recipient’s red cells for the presence or absence of the D antigen.

1.3 Red cells shall be tested with anti-D reagent. An Rh control system, appropriate to the anti-D reagent in use must be included.

1.4 The Transfusion Medicine Laboratory Technologist shall check the prenatal status of the patient being tested.

1.5 Adult and pediatric specimens are tested with anti-D by direct agglutination only. The test for weak D is not required.

1.6 Weak D testing shall be performed in the case of:

1.6.1 An Rh negative neonate of an Rh negative mother; and

1.6.2 To resolve discrepancies between current and previous results.

1.7 All adult and pediatric samples that test Rh negative on initial testing shall be confirmed Rh negative using a broad reactivity anti-D reagent

(Novaclone anti-D, Bioclone anti-D).

1.8 Current and previous results should be compared to identify any discrepancy. All discrepancies shall be resolved and the resolution

documented before reporting.

1.9 If an Rh typing discrepancy is detected or in an emergency situation:

1.9.1 Rh negative recipients should be transfused with Rh negative red cells. Follow facility established policy for the transfusion of Rh

positive red cells to an Rh negative recipient when Rh negative red

cells are in diminished supply.

1.9.2 Rh negative women of child bearing age and children shall receive Rh negative red cells, except for a life-threatening situation and

when Rh negative red cells are not available.

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1.9.3 Rh positive recipients may receive red blood cells that are either Rh positive or Rh negative.

2.0 Linkages

Canadian Blood Services request for genotyping form. Available at:

https://www.blood.ca/sites/default/files/requisition-for-blood-group-genotyping-

patient.pdf

Determining Specimen Suitability. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/determining_specimen_suitability.p

df

Patient History Check. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/patient_history_check.pdf

Patient Identification and Specimen Labeling. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/patient_id_and_specimen_labeling.

pdf

Preparation of Red Cell Suspensions. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/preparation_of_red_cell_suspensio

ns.pdf

Quality Control of Reagents and Antisera. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/quality_control_of_reagents_and_a

ntisera_ver1.pdf

Weak D Typing: Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/sop_for_weak_d_typing.pdf

3.0 Scope 3.1 All Transfusion Medicine Laboratory Technologists.

https://www.blood.ca/sites/default/files/requisition-for-blood-group-genotyping-patient.pdfhttps://www.blood.ca/sites/default/files/requisition-for-blood-group-genotyping-patient.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-determining-specimen-suitability.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-determining-specimen-suitability.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-patient-history-check.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-patient-id-and-specimen-labeling.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-patient-id-and-specimen-labeling.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-preparation-of-red-cell-suspensions.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-preparation-of-red-cell-suspensions.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-quality-control-of-reagents-and-antisera-ver1.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-quality-control-of-reagents-and-antisera-ver1.pdfhttps://www.gov.nl.ca/hcs/files/bloodservices-pdf-sop-for-weak-d-typing.pdf

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4.0 General Information

4.1 Rh typing is a part of the forward blood group.

4.2 Pre-transfusion Rh testing is second only to ABO testing in importance.

4.3 Due to changes in the Rh reporting process, there may be occasions when patients were previously reported as Rh positive and must now be reported

as Rh negative. A second sample may not be required to confirm this change

because it is a result of an update in policy. The attached physician memo

may be sent to the attending physician to assist in explaining the change in

patients’ blood group.

4.4 Pre-washing the red cells is not necessary, however, if a discrepancy is detected, the cells should be washed and the tests repeated.

4.5 Agglutination less than grade 2 with anti-D requires further investigation as the reactions may not be due to interaction between reagents and test cells.

4.6 If two limited specificity anti-D reagents are used for initial testing, (example Immucor series 4 and series 5), the reaction strength should agree

within 2 grades or the patient should be investigated.

4.7 If indicated in the manufacturer’s insert, weak reactions may be enhanced by incubating at room temperature for up to15 minutes.

4.8 False positive or negatives can be caused by variables such as:

4.8.1 Improper technique;

4.8.2 Contaminated materials;

4.8.3 Omission of reagents or antisera;

4.8.4 Delays in reading tests;

4.8.5 Inadequate incubation time and temperature;

4.8.6 Inappropriate centrifugation;

4.8.7 Inappropriate or prolonged storage of red cells;

4.8.8 Rouleaux (may be eliminated by washing the red cells prior to testing);

4.8.9 Polyagglutinable red cells; and

4.8.10 Autoantibodies.

4.9 Very weak reactions may indicate the presence of quantitatively weak D or partial D antigen.

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4.10 Anti-D reagents may not detect all examples of partial D. Reagents may react with weak D cells and rare examples of partial D cells that may have

previously been tested and interpreted as Rh negative using other sources

of Anti-D.

4.11 It is recommended to note in the patients transfusion history if testing using anti-D reagent is positive but

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negative red cells. Positive control cells should be selected to

represent weak expression of the specific antigen.

5.2 Procedure

5.2.1 Determine specimen suitability.

5.2.2 Centrifuge specimen.

5.2.3 Check specimen for abnormal appearance after centrifuging (e.g. hemolysis).

5.2.4 Perform a patient history check.

5.2.5 Ensure patient information on the sample corresponds with the patient information on the worksheet.

5.2.6 Label 2 tubes, one with the recipient’s identifier and anti-sera reagent (A/D) and the other with the recipient’s identifier and

control.

5.2.7 Add 1 drop of anti-D reagent and 1drop of control to the appropriate labelled tubes.

5.2.8 Prepare a 3-5% recipient red cell suspension.

5.2.9 Add 1 drop of the recipient’s red cell suspension to each tube.

5.2.10 Mix the contents of the tubes.

5.2.11 Centrifuge the tubes (speed and time as recommended by manufacturer’s instructions).

5.2.12 Remove the tubes from the centrifuge. Verify that the recipient’s identification on the tubes, specimens and worksheet correspond.

If more than one recipient is being tested, read and record results

on one recipient at a time.

5.2.13 Re-suspend the cell button.

5.2.14 Read macroscopically.

5.2.15 Grade and record results.

5.2.16 Reaction strength of 2+, 3+ and 4+ report as Rh positive.

NOTE: If the result is negative or

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5.2.17 Add 1 drop of Novaclone anti-D reagent and 1drop of control to the appropriate labelled tubes.

5.2.18 Add 1 drop of the recipient’s red cell suspension to each tube.

5.2.19 Mix the contents of the tubes.

5.2.20 Centrifuge the tubes (speed and time as recommended by manufacturer’s instructions).

5.2.21 Remove the tubes from the centrifuge. Verify that the recipient’s identification on the tubes, specimens and worksheet correspond.

If more than one recipient is being tested, read and record results

on one recipient at a time.

5.2.22 Re-suspend the cell button.

5.2.23 Read macroscopically.

5.2.24 Grade and record results. If positive 2+, 3+ or 4+ report as Rh positive. If negative or < 2+ reaction:

5.2.24.1 Incubate tube at room temperature for 5 (five) minutes. 5.2.24.2 Centrifuge the tubes (speed and time as recommended

by manufacturer’s instructions).

5.2.24.3 Grade and record results. If positive 2+, 3+ or 4+ report as Rh positive. If negative report as Rh negative.

5.2.24.4 If the result is weak or 1+ refer to the table provided in section 10 (ten) for Rh determination and reporting.

5.2.24.5 If the control tube is positive at any time during testing, the results are invalid and must not be reported.

5.3 Guidelines (N/A)

5.4 Materials

5.4.1 Sample: 5.4.1.1 Blood sample collected in EDTA anticoagulant. 5.4.1.2 Red cells from clotted sample. (SST tubes with gel

separator should not be used).

5.4.1.3 Venous or capillary blood sample can be used for neonates. Note: Cord blood must not be used for pre-transfusion

testing.

5.4.2 Reagents: 5.4.2.1 Anti-D reagent 5.4.2.2 Broad reactivity Anti-D reagent (Novaclone D)

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5.4.2.3 Diluent control or 6-8% bovine albumin 5.4.2.4 Isotonic Saline

5.4.3 Supplies: 5.4.3.1 Test tubes (10x75mm)

5.4.3.2 Transfer pipettes

5.4.3.3 Test tube rack

5.4.4 Equipment:

5.4.4.1 Serological centrifuge

5.4.4.2 Timer

6.0 Acronyms

EDTA Ethylenediamine tetracetic acid anticoagulant

RBCs Red blood cells

SST Serum-separating tubes

RT Room temperature

7.0 Definitions

Rouleaux The stacking up of red blood cells caused by extra or abnormal

proteins in the blood that decrease the normal distance red cells

maintain between each other.

Compatibility testing Testing to determine if the donor's blood is compatible with the

blood of an intended recipient.

8.0 Records Management

8.1 The recipient transfusion data file in the Transfusion Medicine Laboratory shall be retained indefinitely.

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8.2 All transfusion records in the recipient’s medical chart shall be retained in accordance with health care facility policy.

8.3 Quality control of blood components, blood products, reagents and equipment shall be retained for 5 years.

8.4 Date and time of specimen collection and phlebotomist’s identification shall be retained for 1 year.

8.5 Request form for serologic tests shall be retained for one month or as per facility policy.

8.6 Documentation of staff training and competency must be kept for a minimum of ten years.

9.0 Key Words

Forward blood group, Rh typing

10.0 Supporting Documents

10.1 Tables/Charts

Note: Neonate and cord samples require IAT weak D testing

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10.1 Interpreting Rh Test Results: Rh control tube must be negative for valid Rh type

LIMITED

SPECIFICITY

ANTI-D (routine

testing i.e. provue,

immucor series 4 and

series 5)

BROAD

REACTIVITY

ANTI-D

(NOVACLONE

D)

RESULT/INTERPRETATION

4+ Not required Rh positive



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10.2 Process Flow/Algorithm

Process Flow for Rh Typing

Label Test Tubes

Add A/D reagent

to tube labeled

A/D

Prepare RBC

suspension

NL2012-033

Add RBC

suspension to

tubes

Mix contents of

tubes

Centrifuge tubes

Remove tubes

from centrfuge

Verify recipient’s

identification on

tubes, specimens

and worksheet match

Re-suspend cell

button

Read

Macroscopically

Grade and record

Interpret results

and compare to

previous reports

Rh

Discrepancy

Resolve

Discrepancy

before reporting

No

Yes

Yes

No

Add Rh control to

tube labeled

Control

Report Rh typing

as outlined in

table 10.1

If Rh neg or

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References

American Association of Blood Banks, (2011). Standards for Blood Banks and

Transfusion Services, (27th

ed.). Bethesda, MD: Author.

British Columbia Provincial Blood Coordinating Program. (2009, January).ABO/D

Examination by tube Procedure. (Ver. 1.0). British Columbia: Author.

Canadian Society for Transfusion Medicine, (2011). Standards for Hospital

Transfusion Services, Version 3.0. Ottawa, ON: Author.

Canadian Standards Association, (2010). CAN/CSA-Z902-10, Blood and Blood

Components. Mississauga, ON: Author.

Cooling L. ABO, H, and Lewis blood groups and structurally related antigens. In: Fung

M, Grossman BJ, Hillyer CD, Westhoff CM, eds. Technical manual. 18th edition.

Bethesda, MD: AABB, 2014:291-315.

Immucor Gamma. (2012) Blood Grouping Reagent: Package Insert.

Immucor Gamma. (2012) Reagent Red Cells; Package Insert.

Nova Scotia Provincial Blood Coordinating Program.Guidelines for Rh Testing in Nova

Scotia (Ver. 1.0.DRAFT).). Halifax, NS: Author.