STANDARD OPERATING PROCEDURE (SOP): ANTIBODY SCREENING

BLOOD CENTRE NAME

SOP Number	Effective date	Pages	Author	Authorised by
Version	Review Period	No. of copies	Reviewed by	Date

LOCATION     Red Cell Serology Laboratory	SUBJECT        Antibody Screen
FUNCTION      Detection of Unexpected Blood group Antibodies	DISTRIBUTION – Supervisor in charge of Red Cell Serology Laboratory – Master File

1. SCOPE & APPLICATION

This procedure applies to all testing that requires antibody screening, including donor units, patient’s pre-transfusion blood grouping and prenatal specimens.

2. RESPONSIBILITY

It is the responsibility of the technician/supervisor in the red cell serology laboratory to perform the antibody screen using proper cell concentrations. One technician performs all tests and another checks it. If any unexpected blood group antibody is detected, inform the staff of Advanced Red Cell Serology for further investigations.

• REFERENCES

1. Technical Manual of the American Association of Blood Banks 20^th Edition, 2020.

• Modern Blood banking & Transfusion Practices. DM Harmening, 6^th edition

• MATERIAL REQUIRED

4.1       Equipment:

• Refrigerator to store samples & reagents at 2-6⁰C.

• Deep Freezer to store enzyme papaine  cystein in frozen state.

• Tabletop centrifuge.

• Automated cell washer (for patient pre-transfusion and prenatal testing).

• Microscope.

• Dry bath/ Incubator

4.2       Specimen:

Clotted blood sample of donors/patients.

4.3       Reagents:

• Group O polled cells/Antibody-screening reagent red blood cells (two or three cells).

• Papain cystein.

• 22% Bovine albumin.

• Antihuman globulin reagent(anti-IgG+anti-C3d)

• IgG  sensitised control cells.

• 0.9% saline

• Distilled water

4.4       Glassware:

• Serum tubes.

• Coombs’ tubes(for patient pre-transfusion & prenatal testing).

• Micro tubes.

• Pasteur pipettes.

• Glass slides.

4.5       Miscellaneous:

• Rubber teats.

• Disposal box.

• 2 plastic beakers.

• Wooden blocks to hold micro tubes.

• Aluminium racks to hold serum and coombs’ tubes.

• PROCEDURE

5.1       Principle:

The antibody screen test is used in the detection of unexpected blood group antibodies. In this test, pooled O cells or the antibody-screening reagent red blood cells are combined with serum under investigation. The addition of a potentiating medium enzyme / albumin helps to promote the interaction of red cells and antibodies allowing antibody/antigen reactions to occur. Positive reactions (haemolysis or agglutination) in any tests indicate the presence of allo antibody or auto antibody in the serum.

5.2       Antibody Screen:

1. Label tubes with donor/patient and test identification.

• Add two drops of test serum to each tube.

• Add 1 drop of papain cystein to all tubes labelled ‘enzyme’ (if enzyme method is being followed).*

• To each of the tubes labelled ‘saline’ or ‘enzyme/albumin’, add 1 drop of 2% pooled O red cell suspension (or 2% suspension of the antibody-screening reagent red cells).

• Add 1 drop of 22% abovine albumin to tubes labelled ‘albumin’ (if albumin method is being followed).*

• Add 1 drop of 5% pooled O red cell suspension (or 5% suspension of antibody-screening reagent red cells) to tubes labelled ‘IAT’, followed by 2 drops of 22% bovine albumin.

• Mix the contents of the tubes gently and incubate for minimum 15 minutes.

TEST	INCUBATION	IDEAL INCUBATION
	TEMPERATURE	TIME
Saline	Room Temperature	1 hour
Enzyme	370C	45 minutes
Albumin	370C	45 minutes
IAT	370C	1 hour

Follow manufacturer’s directions when using commercial reagents.

• Either enzyme or albumin method may be followed for detection of incomplete antibodies.

5.1       Results:

1. Centrifuge saline, enzyme and albumin tests at 1000rpm for 1 minute.

• Examine for haemolysis.

• Gently resuspend the red cell button and examine for agglutination.

• Examine all visually negative tests microscopically.

• Grade and record test results immediately.

• Proceed to perform antiglobulin phase of the indirect antiglobulin test on tubes labelled ‘IAT’.
• Wash the cells 3 times with saline. Decant completely after last wash. (washing can be done manually or using automated cell washer).
• Add 2 drops antihuman globulin reagent to the dry cell button.

• Mix well and centrifuge at 1000 rpm for 1 minute.

1. Read and record results.

1. Add drop IgG sensitised cells to all negative results. This shows a positive agglutination.

5.2       Interpretation:

1. Hemolysis or agglutination in any test may indicate the presence of an unexpected antibody.
2. The absence of agglutination and hemolysis in all tests is a negative test result.

• After addition of IgG-sensitized cells to a negative test, the presence of agglutination indicates that the AHG serum added was capable of reacting and that the negative antiglobulin test is valid.

• If IgG-sensitised cells added to confirm the activity of the anti-IgG show only weak or no agglutination after centrifugation, the test is invalid and must be repeated.

5.5       Limitations:

If tests with all reagent red cells are reactive, the possibility of spontaneous agglutination should be considered. A control of cells washed three to four times added to two drops of saline must be non-reactive.

6. DOCUMENTATION

Results of donor unit antibody screen are entered in the donor grouping register and computer.

Results of patients antibody screen are entered in the patient grouping register, blood group requisition form, serial case number register and computer.

All records are initialled by the technician who has performed the test and by the technician who has checked the results.

7. END OF DOCUMENT