Transfusion Medicine Quality Manual Standard … · 1.4 Tests shall be read immediately after...

Transfusion Medicine Quality Manual Standard Operation Procedure for

Direct Antiglobulin Test

Provincial Blood

Coordinating Program

_______________________________________________________________________

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

NL09-005-TMQ

Version: 4.0

Effective Date: 2016-02-15

of 13

Standard Operation Procedure for


1.0 Policy Statements

1.1 A direct antigloulin test (DAT) shall be performed for investigation of:

1.1.1 Hemolytic disease of the newborn;

1.1.2 Autoimmune hemolytic anemia;

1.1.3 Hemolytic transfusion reactions;

1.1.4 Sensitization caused by drugs; and

1.1.5 Complications during pretransfusion and/or compatibility testing.

1.2 A DAT is required if an auto control is not completed in the antibody screen

and:

1.2.1 Antibody identification is required and an auto control cannot be

completed (e.g., limited volume of plasma);

1.2.2 Antigen typing of the patient cells is required.

1.3 A control reagent (e.g., Saline or 6% albumin) is required when

agglutination is observed with all antisera tested.

1.4 Tests shall be read immediately after centrifugation. Delay may cause bound

IgG to dissociate from the red cells causing false negative results.

1.5 The antiglobulin reagent shall contain antibodies to IgG and C3d component

of complement.

Exception: Cord blood testing that may be performed with a

monospecific anti-IgG reagent.

1.6 If a DAT performed on a clotted specimen identifies complement on the red

cell surface, the result shall be verified using an EDTA sample.

1.7 An elution shall be performed on red cells from patients who have a positive

DAT and:

1.7.1 Have received a transfusion or red cells or platelets in the past three

(3) months;

1.7.2 Have been pregnant in the past three (3) months; or

1.7.3 The patients history is questionable or unavailable.

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



2.0 Linkages

Determining Specimen Suitability. Available at:

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

df

Guidelines for Investigation of Adverse Transfusion Reactions. Available at:

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

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

3.0 Scope

3.1 All Transfusion Medicine Laboratory Technologists

4.0 General Information

4.1 The DAT can determine if red cells have been coated in vivo with

immunoglobulin, complement, or both.

4.2 The interpretation of a positive DAT result should take into consideration:

4.2.1 The patient’s recent drug, pregnancy and transfusion history;

4.2.2 Clinical data; and

4.2.3 Results of other laboratory tests.

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

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



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

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

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

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

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

http://www.health.gov.nl.ca/health/bloodservices/pdf/quality_control_of_reagents_and_antisera_ver1.pdf

http://www.health.gov.nl.ca/health/bloodservices/pdf/quality_control_of_reagents_and_antisera_ver1.pdf

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



4.3 A positive control may be due to strong cold agglutinin in the recipient’s

serum/plasma. Washing of the recipient red cells with 37°C saline and

repeating DAT may correct this issue.

4.4 Mixed field reaction may indicate a transfusion reaction.

4.5 Weak anti-C3d reactions may be enhanced if tubes containing polyspecific

AHG/red cells or anti-C3d/red cells are incubated for five (5) minutes at

room temperature (RT) after initial reading of the test.

4.6 A DAT should be performed on every patient in whom the presence of

hemolysis has been established to distinguish immune from nonimmune

hemolytic anemia.

4.7 False positive test results due to in vitro coating of red cells with

complement may occur if testing is performed on a clotted specimen.

5.0 Process

5.1 Quality Control

5.1.1 All reagents shall be used and controlled according to the

manufacturer’s written instructions.

5.1.2 The expiry date shall be checked on each reagent used. Do not use

reagents beyond expiry date.

5.1.3 The addition of IgG control cells should demonstrate agglutination

or the test is invalid and shall be repeated.

5.1.4 The addition of complement control cells should demonstrate

agglutination or the test is invalid and shall be repeated.

5.2 Procedure

5.2.1 Procedure for Testing with Polyspecific Antiglobulin Reagent

(AHG)

5.2.1.1 Determine specimen suitability.

5.2.1.2 Centrifuge specimen (speed and time as recommended by

manufacturer’s directions).

5.2.1.3 Check patient history.

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



5.2.1.4 Wash an aliquot of red blood cells to be tested at least once

with isotonic saline. Prepare a 2%–5% suspension of red

cells in isotonic saline.

5.2.1.5 Label test tube with patient identifier and ‘AHG.’

5.2.1.6 Dispense 1–2 drops of red cells into labelled tube.

5.2.1.7 Wash three (3) times with isotonic saline. Completely

decant saline after each wash and re-suspend cells prior to

the addition of saline for subsequent washes.

5.2.1.8 Completely decant saline after final wash, blot to remove

any residual saline in order to obtain a ‘dry’ red cell button.

5.2.1.9 Add two (2) drops (or volume indicated as per

manufacturer’s directions) of polyspecific AHG to labelled

tube.

5.2.1.10 Mix and centrifuge (speed and time as per manufacturer’s

directions).

5.2.1.11 Immediately after centrifugation re-suspend red blood

cells and examine macroscopically for agglutination. If

negative macroscopically, read microscopically.

5.2.1.12 Grade and record the results.

5.2.1.13 If test is negative, incubate test at room temperature for 5–

10 minutes. If test is positive, perform DAT with

monospecific reagents, anti-IgG and anti-C3d, to determine

which molecules are present.

5.2.1.14 After incubation mix and centrifuge, re-suspend the red

cells. Read macroscopically and microscopically.

5.2.1.15 Grade and record results:

If test is positive, perform DAT with monospecific

reagents, anti-IgG and anti-C3d, to determine which

globulins are present.

If test is negative add one (1) drop (or volume indicated as

per manufacturer’s directions) of IgG coated red cells to

both tubes and centrifuge.

5.2.1.16 Examine the tube for macroscopic agglutination.

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



5.2.1.17 Grade and record results:

If positive, test is valid. DAT is negative.

If negative, test is not valid. Procedure must be repeated.

5.2.2 Procedure for Testing with Monospecific Anti-IgG and Anti-

C3d

5.2.2.1 Perform steps 5.2.1.1 to 5.2.1.4 as in procedure 5.2.1.

5.2.2.2 Label two (2) test tubes: one with patient identifier and

‘IgG’ and one with patient identifier and ‘C3d.’

5.2.2.3 Dispense 1–2 drops of red cell suspension into each

labelled tube.

5.2.2.4 Wash three (3) times with isotonic saline. Completely

decant saline after each wash and re-suspend cells prior to

the addition of saline for subsequent washes.

5.2.2.5 Completely decant saline after final wash, blot to remove

any residual saline in order to obtain a ‘dry’ red cell button.

5.2.2.6 Add two (2) drops (or volume as indicated as per

manufacturer’s directions) of Anti-IgG to tube labelled

‘IgG’ and add two (2) drops of anti- C3d to the tube

labelled ‘C3d.’

5.2.2.7 Mix and centrifuge (speed and time as per manufacturer’s

directions).

5.2.2.8 Immediately after centrifugation re-suspend red blood cells

and examine macroscopically for agglutination. If negative

macroscopically, read microscopically.

5.2.2.9 IgG

5.2.2.9.1 If negative:

Add one (1) drop (or volume indicated in

manufacturer’s directions) of IgG coated red cells.

Centrifuge, examine macroscopically, grade and

record results.

5.2.2.9.2 If positive:

Report as positive, IgG coating red cells.

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



5.2.2.10 C3d

5.2.2.10.1 If negative:

Incubate at room temperature for five (5)

minutes, and follow steps 5.2.1.14 to 5.2.1.15. If

test is then negative add one (1) drop (or volume

indicated in manufacturer’s directions) of

complement coated red cells. Incubate test at

room temperature for 5–10 minutes. Centrifuge,

read and record results.

5.2.2.10.2 If positive:

Report as positive, complement coating red cells.

5.2.2.11 If C3d and IgG are both positive, perform control if not

previously completed.

5.3 Guidelines (NA)

5.4 Materials

5.4.1 Reagents:

5.4.1.1 Polyspecific Anti-Human Globulin (AHG)

5.4.1.2 Anti-IgG (Monospecific)

5.4.1.3 IgG sensitized cells

5.4.1.4 Anti-C3d (Monospecific)

5.4.1.5 C3d coated cells

5.4.1.6 Isotonic saline

5.4.2 Supplies:

5.4.2.1 Test tubes (10x75mm)

5.4.2.2 Transfer pipettes

5.4.2.3 Test tube rack

5.4.2.4 Microscope slides

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



5.4.3 Equipment:

5.4.3.1 Serological centrifuge

5.4.3.2 Cell washer

5.4.3.3 Interval timer

5.4.3.4 Microscope

5.4.4 Specimen:

5.4.4.1 Blood sample collected in EDTA anticoagulant.

5.4.4.2 Cord blood sample (clotted), if test is positive with anti-

C3d, the test must be repeated with an EDTA specimen.

6.0 Acronyms

DAT Direct anti-globulin test

EDTA Ethylenediaminetetraacetic acid

RBC Red blood cells

RT Room temperature

7.0 Definitions

Elution A technique that dissociates antibodies from sensitized red cells

In vitro Outside the living body

In vivo In living body of plant or animal

Macroscopically Large enough to be observed by the naked eye

Microscopically Observed with the use of a microscope

8.0 Records Management

8.1 The recipient transfusion data file in the transfusion service laboratory shall

be retained for 50 years.

8.2 All transfusion records in the recipient’s medical chart shall be retained in

accordance with health care facility’s retention policy for medical records.

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



8.3 Quality control of blood components, blood products, reagents and

equipment shall be retained for five (5) years.

8.4 Date and time of specimen collection and phlebotomist’s identification shall

be retained for one (1) year.

8.5 Request form for serologic tests shall be retained for one (1) month.

8.6 Documentation of staff training and competency must be kept for a

minimum of 10 years.

9.0 Key Words

Direct anti-globulin test, Coombs test, complement

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



10.0 Supporting Documents

10.1 Process Flow/Algorithm

Process Flow – Testing with Polyspecific AHG

Determine specimen suitability

Centrifuge specimen

Check patient history

Wash an aliquot of RBC once and prepare RBC suspension

Label test tube with ‘AHG’ and patient identifier

Dispense 1– 2 drops of RBC suspension into labelled tubes

Wash 3 times with saline

Add 1– 2 drops of AHG to labelled tube

Mix and centrifuge

Immediately re-suspend red cells and examine macroscopically and

microscopically

Preform DAT with monospecific reagents

Incubate at room temperature for 5 minutes

Mix and centrifugeRe-suspend red cells and

examine macroscopically and microscopically

Add one drop of IgG coated red cells to the tube

Examine macroscopically

Report test as Negative

Test invalid. Repeat

POS

POS

POS

NEG

NEGNEG

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



Process Flow– Testing with Monospecific Anti-IgG and Anti-

C3d

Determine specimen suitability

Centrifuge specimen

Check patient history

Wash an aliquot of RBC once and prepare RBC suspension

Label test tubes ‘C3d’ and ‘IgG’ with

patient identifier

Dispense1– 2 drops of RBC suspension into labelled tubes

Wash 3 times with saline

Add 2 drops of Anti-IgG to ‘IgG’ tube and 2 drops of Anti-C3d

to ‘C3d’ tube.

Mix and centrifuge

Immediately re-suspend and examine macroscopically and

microscopically

IgG Results C3d Results

Add IgG coated red cells (Check Cells)

Incubate at RT for 5 minutesReport as Positive

for IgG

Centrifuge and re-suspend

Report as Positive for C3d

Read macroscopically


Test Invalid. Repeat



and microscopically

Report as Positive for C3d

Add C3d coated red cells (complement

control cells)




NEG POS NEG POS

NEG

POS

NEG

POS

POS

NEG

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



10.2 Tables/Charts

Interpretation using Polyspecific AHG reagents:

Polyspecific

AHG

Control Test Result Interpretation

Negative Negative Negative

Positive Negative Positive. Test with monospecific reagents.

Positive Positive Test invalid.

Interpretation using Monospecific antiglobulin reagents:

Anti-IgG Anti-C3d Control Test Result Interpretation

Negative Negative Negative Negative

Positive Positive Negative Positive – IgG and Complement coating

cells

Positive Negative Negative Positive – IgG coating cells

Negative Positive Negative Positive – Complement coating cells

Positive Positive Positive Test invalid.

Provincial Blood


_______________________________________________________________________


NL09-005-TMQ

Version: 4.0


of 13



References

AABB Standards for Blood Banks and Transfusion Services, 29th

edition pg 88.

Canadian Standards Association (2015). Blood and blood components, Z902- 15.

Toronto (ON): Author.

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

Transfusion Service. Version 3. Ottawa: Author.

Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-C3d:

Instructions for use. Dartmouth, NS: Author.

Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-IgG, -C3d

Polyspecific: Instructions for use. Dartmouth, NS: Author.

Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-IgG:

Instructions for use. Dartmouth, NS: Author.

Immucor. (2011). CHECKCELL®: Instructions for use. Norcross, GA: Author.

Immucor. (2010). Complement Control Cells. Instructions for use. Norcross, GA: Author.

Transfusion Medicine Quality Manual Standard … · 1.4 Tests shall be read immediately after...

Documents

Transcript of Transfusion Medicine Quality Manual Standard … · 1.4 Tests shall be read immediately after...