(a) Sera of human origin for ABO grouping

(i) Anti-A blood-grouping serum (human)

Anti-A serum is derived from the blood of selected group B persons, who may or may not have been immunised by group A red corpuscles or group A specific substance. Anti-A serum agglutinates human red corpuscles containing A antigen, i.e. those of blood groups A and AB, including sub-groups A1, A2, A1B and A2B, and does not agglutinate human red corpuscles which do not contain A antigen, i.e. those of blood groups O and B.

POTENCY

Titration

An anti-A serum shall be titrated separately against suspensions of A1, A2, and A2B corpuscles, in parallel with the reconstituted but undiluted International Standard Preparation of anti-A blood-grouping serum or an equivalent reference preparation. The potency of the serum shall in each case be not less than 64 International Units per ml.

Determination of avidity

When anti-A serum is mixed on a slide with an equal volume of a suspension of A1, A2 and A2B cells with a volume fraction of 0.05 to 0.1, agglutination of each suspension should first appear in not more than twice the time taken when the same test is performed with the reconstituted but undiluted International Standard Preparation of anti-A blood-grouping serum or with a reference standard of equivalent avidity.

(ii) Anti-B blood-grouping serum (human)

Anti-B serum is derived from the blood of selected group A persons, who may or may not have been immunised by group B red corpuscles or group B specific substance. Anti-B serum agglutinates human red corpuscles containing B antigen, i.e. those of blood groups B and AB, and does not agglutinate human red corpuscles which do not contain B antigen, i.e. those of blood groups O and A.

POTENCY

Titration

An anti-B serum shall be titrated against a suspension of group B corpuscles in parallel with the reconstituted but undiluted International Standard Preparation of anti-B blood-grouping serum or an equivalent reference preparation. The potency of the serum shall be not less than 64 International Units per ml.

Determination of avidity

When anti-B serum is mixed on a slide with an equal volume of a suspension of B cells with a volume fraction of 0.05 to 0.1, agglutination should first appear in not more than twice the time taken when the same test is performed with the reconstituted but undiluted International Standard Preparation of anti-B blood-grouping serum or with reference standard of equivalent avidity.

(iii) Anti-A + Anti-B (group O) blood-grouping serum (human)

Anti-A + anti-B (group O) serum is derived from the blood of selected group O persons who may or may not have been immunised by group A and group B red corpuscles or group A and group B specific substances. Anti-A + anti-B (group O) serum agglutinates human red corpuscles containing A or B agglutinogens or both, i.e. those of group A including sub-groups A1 and A2, group B and group AB including sub-groups A1B and A2B, and does not agglutinate human red corpuscles which do not contain A or B agglutinogens, i.e. those of group O. It agglutinates human red corpuscles containing the Ax (Ay or A0) antigen (which are not, in general, agglutinated by anti-A serum derived from group B donors).

POTENCY

Titration

An anti-A + anti-B (group O) serum shall be titrated separately against suspensions of A1 and A2 corpuscles in parallel with the reconstituted but undiluted International Standard Preparation of anti-A blood-grouping serum or an equivalent standard preparation. It shall also be titrated against a suspension of group B corpuscles in parallel with the reconstituted but undiluted International Standard Preparation of anti-B blood-grouping serum or an equivalent standard preparation.

The potency of the serum shall in every case be not less than 64 International Units per ml.

Anti-A + anti-B (group O) blood-grouping serum used undiluted shall also give readily detectable agglutination of group Ax (Ay or A0) corpuscles.

Determination of avidity

When anti-A + anti-B (group O) serum is mixed on a slide with equal volumes of suspensions of A1 of A2 cells with a volume fraction of 0.05 to 0.1, agglutination shall first appear in not more than twice the time taken when the same tests are performed with the reconstituted but undiluted International Standard Preparation of anti-A blood-grouping serum or with a reference standard of equivalent avidity. When anti-A + anti-B (group O) serum is mixed on a slide with an equal volume of a suspension of B cells with a volume fraction of 6.05 to 0.1, agglutination shall first appear in not more than twice the time taken when the same test is performed with the reconstituted but undiluted International Standard Preparation of anti-B blood-grouping serum or a reference preparation of equivalent avidity. When anti-A + anti-B (group O) serum is mixed on a slide with an equal volume of a suspension of Ax (Ay or A0) cells with a volume fraction of 0.05 to 0.1, agglutination shall first appear in not more than 5 minutes at a temperature between 18 and 25° C.

(b) Sera of human origin for Rh grouping

Anti-Rh blood grouping sera, whatever their specificity, may be of either of two varieties differing in the conditions under which agglutination of homologous corpuscles is obtained. Certain sera commonly known as “complete” agglutinate corpuscles suspended in saline. With others, commonly known as “incomplete”, agglutination can only be obtained in the presence of certain colloids such as bovine albumin or by means of other special techniques. The sera should be used under the conditions specified by the laboratory preparing them.

Some “incomplete” sera will also agglutinate homologous red corpuscles suspended in their own serum or plasma on slides.

The following requirements of potency for Rh grouping sera may need to be revised when International Standard Preparations become available.

(i) Anti-D (anti-Rho) blood-grouping serum (human)

Anti-D serum is derived from the blood of one or more persons immunised by the D antigen of the Rh system. It reacts with human red corpuscles containing the D antigen, but not with human red corpuscles which do not contain the D antigen.

POTENCY

Titration

“Complete” anti-D sera shall have a titre not less than 32 against CcDee cells in a solution containing 9 gram sodium chloride per litre.

An “incomplete” anti-D serum shall be titrated against CcDee corpuscles in parallel with the reconstituted but undiluted International Standard Preparation of Incomplete Anti-D (anti-Rho) or an equivalent reference preparation. It shall have a potency of not less than 32 International Units. Besides reacting with all red corpuscles containing the D antigen, the serum should, as far as possible, react with corpuscles containing the Du antigen.

Determination of avidity

Anti-D sera intended for use in the slide test of Diamond and Abelson should, when mixed on a slide with an equal volume of a 40% to 50% suspension of CcDee corpuscles at approximately 40° C, show visible agglutination within 30 seconds, and agglutination should be complete within 120 seconds.

(ii) Anti-C (anti-Rh') blood-grouping serum (human)

Anti-C serum is derived from the blood of one or more persons immunised by the C agglutinogen of the Rh system. It agglutinates suspensions of human red corpuscles containing the C antigen, but not with human red corpuscles, which do not contain the C antigen. In this connection the C antigen is regarded as including the Cwantigen.

Most diagnostic anti-C sera contain “complete” anti-C together with “incomplete” anti-D. These sera are therefore specific for the C antigen only when the cells under test are suspended in a solution containing 9 gram sodium chloride per litre.

POTENCY

Titration

Anti-C sera (“complete” or “incomplete”) should have a titre not less than 8 against Ccddee corpuscles.

Determination of avidity