NDOLA TEACHING HOSPITAL PATHOLOGY LABORATORY

Document Title: : Validation Of ABO And Rhesus Blood Grouping Sera

Document No. SOP-BB-017 Effective Date: 06/09/2018
Version 1
Section: Blood bank/serology Control Copy No. 5

SOP-BB-017

VALIDATION OF ABO AND RHESUS BLOOD

GROUPING SERA PROCEDURE

Author Name Title: Signature: Date:

Evans Njovu Biomedical Technologist Njovu 11/04/2012
Approved By
Joseph Kasongo Laboratory Manager Kasongo 06/09/2018

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 NDOLA TEACHING HOSPITAL PATHOLOGY LABORATORY
Document Title: : Validation Of ABO And Rhesus Blood Grouping Sera
Document No. SOP-BB-017 Effective Date: 06/09/2018
Version 1
Section: Blood bank/serology Control Copy No. 5

1. PURPOSE
To provide the procedure for the determination of the potency, specificity and avidity of
antisera against the known red cell antigens.

2. PRINCIPLE
The principle is based on antigen/antibody reactions between the different blood groups,
the speed of their reactions and measuring the titre of these reactions.
Validation of grouping sera is performed prior to putting anti sera into use and this is a
quality check to determine antisera viability.

3. EQUIPMENT AND MATERIALS

1. Centrifuge
2. Test tubes and Racks
3. Test tube Markers
4. Normal Saline
5. Neat monoclonal anti-A
6. Neat monoclonal anti-B
7. Neat monoclonal anti-D
4. SAFETY
Use Personal Protective Equipment (PPE) and treat all Specimens as potentially
infectious.
5. STORAGE AND STABILITY
• Reagents – Store at 2-8oC
• Specimen type – Anti sera and known A, B, AB and O red blood cells
6. QUALITY CONTROL
The known RBCs to be used should be confirmed with validated antisera.
7. PROCEDURE
1. Prepare 1:40 dilution of

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 NDOLA TEACHING HOSPITAL PATHOLOGY LABORATORY
Document Title: : Validation Of ABO And Rhesus Blood Grouping Sera
Document No. SOP-BB-017 Effective Date: 06/09/2018
Version 1
Section: Blood bank/serology Control Copy No. 5

• Anti-A
• Anti-B
• Anti-D
2. Preparation: Add 1 drop of each of the above sera to 39 drops of normal saline in
clearly labeled test tubes.
3. Add 1 drop each of the 2-5% known cell suspensions to each of the diluted antisera
(refer to ID009 blood bank reagent preparation for the preparation of 2-5% cell
suspension).
4. Tap the tubes slightly to mix
5. Leave at room temperature for up to 1 hour or centrifuge at 1000 rpm for 1 minute
6. Read results

7. INTERPRETATION
Anti-A Anti-B Anti-D
Diluted 1:40 Diluted 1:40 Diluted 1:40
Known A cells ++++ O ++++/ O
Known B cells O ++++ ++++/ O
Known AB ++++ ++++ ++++/ O
cells
Known O Rh O O ++++
Positive cells
Known O Rh O O O
Negative cells

++++ = A clump of cells surrounded by a clear suspension after centrifugation at 1000rpm for 1
minute.

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 NDOLA TEACHING HOSPITAL PATHOLOGY LABORATORY
Document Title: : Validation Of ABO And Rhesus Blood Grouping Sera
Document No. SOP-BB-017 Effective Date: 06/09/2018
Version 1
Section: Blood bank/serology Control Copy No. 5

Note
Potency
(A) Diluted Anti-A and Anti-B give a ++++ reaction, against known
A cells, and B cells respectively.
(B) Diluted Anti –D gives at least a ++++ reaction against known O
Rh positive cells and a negative reaction against known O Rh
negative cells.
Specificity
(A) Diluted Anti-A and Anti-B does not react with non corresponding known
A and B Cells. Known O cells do not react with diluted Anti-A
and Anti-B.
8. LIMITATIONS OF PROCEDURE AND SOURCES OF ERROR
• Use of undiluted antisera will give inaccurate results
• Use of concentrated red cells will give inaccurate results
• Use of turbid antisera will give inaccurate results
9. DISPOSAL
Reagents: Not applicable.
Specimen: Blood specimen should be disposed of in the biohazard bag for autoclaved
and then incineration.
Waste: Used tubes, cotton swabs must be discarded in a biohazard bag and Incinerated

10. SUPPORTING DOCUMENTS

• F091Antisera Validation Record.
• F069_Antisera QC Form

11. RECORDS

RECORD TITLE RECORD ID CUSTODIAN/LOCATION

Antisera Validation Record F091 Head of Section-Blood Bank
Antisera QC Form F069 Head of Section-Blood Bank

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 NDOLA TEACHING HOSPITAL PATHOLOGY LABORATORY
Document Title: : Validation Of ABO And Rhesus Blood Grouping Sera
Document No. SOP-BB-017 Effective Date: 06/09/2018
Version 1
Section: Blood bank/serology Control Copy No. 5

12. REFERENCES
1. ZNBTS SOP no. LP/10.018/06 Version 02, Validation of ABO and RhD Blood
Grouping Sera
2. Standard Operating Procedures For District Hospital Laboratories Level 1 – SOP
no. BLTR 006/99 Validation of Grouping Sera

13. REVIEW/ AMENDMENT RECORD

NAME DATE SUMMARY OF CHANGES
Darius Chiwala 05/05/2012 Reviewed with no changes
Chileshe Musonda 15/04/2016 Reviewed with no changes
Patricia M Kafweta 04/09/2017 Changed Ndola Central Hospital to Ndola Teaching
Hospital
Patricia M Kafweta 06/09/2018 Reviewed with no changes
Patricia M Kafweta 06/09/2019 Reviewed with no changes

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 NDOLA TEACHING HOSPITAL PATHOLOGY LABORATORY
Document Title: : Validation Of ABO And Rhesus Blood Grouping Sera
Document No. SOP-BB-017 Effective Date: 06/09/2018
Version 1
Section: Blood bank/serology Control Copy No. 5

I have read, understood and agree to follow the procedure as documented:

No Name Signature Date

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 NDOLA TEACHING HOSPITAL PATHOLOGY LABORATORY
Document Title: : Validation Of ABO And Rhesus Blood Grouping Sera
Document No. SOP-BB-017 Effective Date: 06/09/2018
Version 1
Section: Blood bank/serology Control Copy No. 5

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