General provisions

1. Specificity

A blood-grouping

1) At the time of approving the present version of the Protocol and its annexes, it was understood by the representatives of the Contracting Parties that when in the English text of the Agreement the expression “blood incompatibilities” was mentioned, “blood grouping incompatibilities” was implied.

It was also agreed that the expression “blood-grouping” with a hyphen in the English text of the Agreement and of the Protocol should read as “blood grouping” without a hyphen.

reagent must react with all blood samples tested which contain the antigen homologous to the antibody or other substance mentioned on the label.

When a reagent is used according to the technique recommended by the producer there must be no evidence of any of the following factors or phenomena:

(a) haemolytic properties;

(b) antibodies or other substances besides those mentioned on the label;

(c) bacterial products liable to cause false positive or false negative reactions;

(d) pseudo-agglutination through the formation of rouleaux;

(e) prozone phenomena.

2. Potency

Titre is measured by making successive two-fold dilutions of the reagent under study in an appropriate medium. To each dilution is added an equal volume of a suspension of red corpuscles. The titre is the reciprocal of the figure representing the highest serum dilution in which a reaction occurs, the dilution being calculated without the inclusion of the volume of the corpuscular suspension in the total volume.

In the case of anti-A, anti-B and other reagents intended for use on slides, avidity is expressed by means of the time required for agglutination on a slide.

3. International Standards and International Units

International Standards have been established by the World Health Organisation for anti-A and anti-B and incomplete anti-D blood-grouping reagents and are in process of being established for blood-grouping reagents of other specificities. An International Standard Preparation contains, by definition, a certain number of International Units per mg or ml and this definition is independent of the titres observed against particular red corpuscle preparations1).

1) The potency of blood-grouping reagents of most specificities is expressed as the agglutination titre observed in a dilution series, against a suspension of red-cells. The titre indicates the dilution of reagent in the last mixture of the series which shows agglutination microscopically visible.

The potency of blood-grouping reagents for which International Standard Preparations exist (at present anti-A and anti-B and incomplete anti-D) can be expressed in International Units (*) on the basis of the titration of the unknown reagent in comparison with the International Standard, or national sub-standard.

The International Standard Preparations of blood-grouping sera are dispensed in ampoules containing dried human serum. When reconstituted to the volume of 1 ml, the anti-A and anti-B sera contain by definition 256 International Units per ml. They can be obtained free of charge, from the International Laboratory for Biological Standards of WHO, Statens Seruminstitut, Copenhagen.

The following table shows an example of a comparative titration of the International Standard anti-A Serum (S) and an “unknown” anti-A reagent (U) against A1 red corpuscles and A2B red corpuscles.

Serum S | Reagent U | Serum S | Reagent U

A1 corpuscles | 1:512 | 1:128 | 256 | 64

A2B corpuscles | 1: 32 | 1: 16 | 256 | 128

titres (observed) | titres (observed) | Units (by definition) | Units (by comparison)

(*) See Bull, Wld. Hlth. Org. 1954, 10, 937, 941 - 1950, 3, 301.

4. Stability and expiry date

Each reagent, when kept under the conditions of storage recommended by the manufacturer, should retain the requisite properties for at least one year.

The expiry date of a reagent in the liquid form as given on the label shall be not more than one year from the date of the last satisfactory potency test. The expiry date can be extended for further periods of one year by repetition of potency tests.

The expiry date of reagents in the dried form as given on the label, shall be in accordance with evidence obtained from experiments on stability and shall be approved by the national authorities.

5. Preservation

Blood-grouping reagents may be preserved in the liquid or dried state. Dried reagents shall be kept in an atmosphere of an inert gas or in vacuo, in the glass container in which they were dried and which shall be closed so as to exclude moisture. A dried reagent must not lose more than 0.5 per cent of its weight when tested by further drying over phosphorus pentoxide at a pressure not exceeding 0.02 mm of mercury for 24 hours.

Reagents shall be prepared with aseptic precautions and shall be free from bacterial contamination. In order to prevent bacterial growth the competent national authority may decide that an antiseptic and/or antibiotic shall be added to the reagent (or to any solvent issued with dried reagents), provided that, in the presence of the added substance, the reagent still fulfils the requirements for specificity and potency.

Blood-grouping sera of human origin must contain at least 2.5 mg of protein nitrogen per ml of liquid or reconstituted serum.

Reagents whether in the liquid state or after reconstitution should be transparent and should not contain any sediment, gel or visible particles.

6. Coloration

Blood-grouping reagents for international exchanges should preferably not be artificially coloured at least until an international agreement is reached on a uniform system. Any added colouring matter must not interfere with the specific reaction.

7. Dispensing and volume

Blood-grouping reagents shall be dispensed in such a way and in such volumes that the reagent in one container is sufficient for the performance of tests with positive and negative control corpuscles in addition to the performance of tests with the unknown corpuscles. The volume in one container shall be such that the contents can if necessary be used for the performance of the appropriate tests for potency described in this Protocol.

8. Records and samples

Written records shall be kept by the producing laboratory of all steps in the production and control of blood-grouping reagents. Adequate samples of all reagents issued shall be retained by the laboratory until it can be reasonably assumed that the batch is no longer in use.

9. Classification of reagents

Reagents used for blood-grouping may contain substances of human, animal, vegetable (or mineral) origin, of which some constitute the active principle and others are adjuvants for enhancing the activity or maintaining the stability of the reagent.

For technical reasons these reagents have been divided into three categories according to the origin of their active principle. This does not mean that reagents of human origin contain exclusively substances of human origin or that animal or vegetable reagents cannot contain substances of human origin.

10. Labels, leaflets and certificates

A label printed in English and French, in black on white paper, shall be affixed to each final container and shall contain the following information:

1. Name and address of producer

2. Name of the reagent as it appears in the heading of the relevant specification.

3. Name and amount of antiseptic and/or antibiotic, if present, or indication of absence

4. The volume or, where the reagent is dried, the volume and composition of the fluid needed for reconstitution

5. Expiry date

6. Batch number.

Moreover, this label or the label of the carton enclosing several final containers, or the leaflet accompanying the containers, shall contain the following information:

1. Full name and address of producer

2. Name of the reagent as it appears in the heading of the relevant specification

3. The volume, or, where the reagent is dried, the volume and composition of the fluid needed for reconstitution

4. Date of last potency test