(a) Sera of animal origin

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

Anti-A serum is derived from the blood of animals which may or may not have been immunised by group A red corpuscles or group A specific substances. 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 red corpuscles, in parallel with the reconstituted but undiluted International Standard Preparation of anti-A bloodgrouping serum or an equivalent reference preparation

1) The International Standard Preparation is of human origin; an equivalent reference preparation, if used, may be of human or non-human origin.

. 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 shall in each case 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 (animal)

Anti-B serum is derived from the blood of animals which may or may not have been immunised by group B red corpuscles or group B specific substances. 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

1) The International Standard Preparation is of human origin; an equivalent reference preparation, if used, may be of human or non-human origin.

. 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 0.05 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 with a reference standard of equivalent avidity.

(iii) Anti-human-globulin serum (animal)

2) Coombs, R. R. A., Mourant, A. E. and Race, R. R. (1945), Lancet, ii 15

Coombs, R. R. A., Mourant, A. E. and Race, R. R. (1945), Brit. J. exp. Path, 26, 255.

Anti-human globulin serum for use in blood group serology must contain agglutinating antibodies against IgG globulin and agglutinating antibodies against complement factors. It is derived from the blood of animals immunised by the injection of human serum protein. It must agglutinate all human red corpuscles coated with human IgG and/or complement factors. Under the conditions specified by the manufacturers it does not agglutinate uncoated human red corpuscles, to whatever group they may belong.

Specificity

The specificity of an anti-human globulin serum for use in blood group serology must be tested with human red corpuscles coated with a variety of antibodies i.e. red corpuscles sensitised with human incomplete antibodies anti-D, anti-K and anti-Fya, red corpuscles sensitised with complement-binding incomplete antibodies anti-Lea in the presence of fresh human serum, and red corpuscles sensitised with so-called “incomplete cold antibodies” and with tanned red corpuscles sensitised with human IgG and, finally, with 10 different samples of non-coated human red corpuscles with and without A and B antigens.

POTENCY

Titration

An anti-human globulin serum, as supplied, or at the dilution recommended on the label, shall strongly agglutinate human red corpuscles coated with a human incomplete anti-D serum, having a titre of 4 (or less) against D-positive corpuscles, when the titration is performed by the albumin replacement method. At the same dilution it shall agglutinate K-positive human red corpuscles sensitised with selected weak anti-K antibodies and Fya positive red corpuscles sensitised with selected weak anti-Fya antibodies.

It shall also, at the same or a different dilution, as specified on the label, agglutinate human red corpuscles sensitised with weak complement-binding incomplete anti-Lea antibodies in the presence of fresh serum.

For clinical use it is desirable that the coating of all the types of incomplete antibodies above shall be detectable with a single dilution of the anti-human globulin serum.

(b) Blood-grouping reagents of vegetable origin

(i) Anti-A blood-grouping reagent (vegetable)

Anti-A reagent is prepared by extraction from the seeds or other parts of a suitable plant, followed, if necessary, by purification. Anti-A reagent agglutinates human red corpuscles containing A antigens, 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 antigens, i.e. those of blood groups O and B.

POTENCY

Titration

An anti-A reagent 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 bloodgrouping serum or an equivalent reference preparation

1) The International Standard Preparation is of human origin; an equivalent reference preparation, if used, may be of human or non-human origin.

.

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

Determination of avidity

When anti-A reagent 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 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-A blood-grouping serum or with a reference standard of equivalent avidity.

(ii) Anti-B blood-grouping reagent (vegetable)

Anti-B reagent is prepared by extraction from the appropriate part of a suitable plant, followed, if necessary, by purification. Anti-B reagent 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 reagent shall be titrated against a suspension of group B corpuscles in parallel with the reconstituted International Standard Preparation of anti-B blood-grouping serum or an equivalent reference preparation

1) The International Standard Preparation is of human origin; an equivalent reference preparation, if used, may be of human or non-human origin.

. The potency of the reagent shall not be less than 64 International Units per ml.

Determination of avidity

When anti-B reagent 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 shall first appear in not more than twice the time taken when the same test is performed with the reconstituted but undiluted Standard Preparation of anti-B blood-grouping serum or with a reference standard of equivalent avidity.