REVISED PROTOCOL
TO THE EUROPEAN AGREEMENT ON THE EXCHANGE
OF TISSUE-TYPING REAGENTS
(ETS № 84 Prot.)
(Strasbourg, 28.III.1985)
GENERAL PROVISIONS
1. Specificity
A. Tissue-typing reagents to be used in cytotoxic
techniques on lymphocytes
These reagents must, when used according to the technique recommended by the producer, react with all lymphocytes known to contain the antigen(s) corresponding to the specificity(ies) mentioned on the label. They must not react with any cell known not to contain this antigen (these antigens). If a sole reagent does not satisfy these conditions, a combination of four sera of the same specificity must be used together. In this case, at least three sera must react with each lymphocyte sample containing the corresponding antigen and, inversely, not more than one should react with cells not containing this antigen.
When these reagents are used according to the technique recommended by the producer there must be no evidence of any interfering serological phenomena such as:
a. prozone effects,
b. anticomplementarity.
B. Tissue-typing reagents for use in a complement
fixation technique on platelets
These reagents must, when used according to the technique recommended by the producer, give complement fixation with all platelets known to contain the antigen(s) corresponding to the specificity(ies) mentioned on the label. They must not give complement fixation with any platelets known not to contain this antigen (these antigens). If a sole reagent does not satisfy these conditions, a combination of four reagents of the same specificity must be used together. In this case, at least three sera must react with each platelet sample containing the corresponding antigen and, inversely, not more than one should react with cells not containing this antigen.
When these reagents are used according to the technique recommended by the producer there must be no evidence of any interfering serological phenomena such as:
a. prozone effects,
b. anticomplementarity.
2. Potency
A. Tissue-typing reagents to be used in cytotoxic
techniques on lymphocytes
The titre of such a reagent is determined by making successive twofold dilutions of the serum under study in inactivated AB serum or in another appropriate medium from a donor who is negative for the antigen(s) corresponding to the antibody (antibodies) in the reagent and who should also not have been immunised against tissue antigens by transfusion, pregnancy or other means. Each dilution is then tested with lymphocytes known to contain the corresponding antigen(s) in the reagent, using the technique recommended by the producer. The titre is the reciprocal of the figure representing the highest serum dilution in which a significantly positive reaction occurs, the dilution being calculated without the inclusion of the volume of the corpuscular suspension or any other additive in the total volume.
B. Tissue-typing reagents for use in a complement
fixation technique on platelets
The titre of such a reagent is determined by making successive twofold dilutions of the serum under study in a solution containing inactivated AB serum in Veronal (R) buffer with a volume fraction of 0.01. Each serum is then tested with platelets known to contain the antigen homologous to the antibodies in the reagent, using the technique recommended by the producer. The titre is the reciprocal of the figure representing the highest serum dilution in which a significantly positive reaction occurs, the dilution being calculated without the inclusion of the volume of the corpuscular suspension or any other additive in the total volume.
Further provisions, for tissue-typing reagents to be used in cytotoxic techniques on lymphocytes as well as for reagents to be used in a complement fixation technique on platelets:
3. Preservation
Tissue-typing reagents may be preserved in the liquid or in the dried state. Liquid reagents shall be kept at a temperature not above - 40°C and dried reagents at a temperature not above + 4°C.
Thawing and refreezing of the reagents during the period of storage must be avoided as much as possible.
Dried reagents shall be kept in an atmosphere of inert gas or in vacuo in the 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 % of its weight when tested by further drying over phosphorous 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 producer may decide that an antiseptic and/or antibiotic shall be added to the reagent. In such cases the reagent must still fulfil the requirements for specificity and potency in the presence of the added substance.
The above also applies to any other additives such as anticoagulants. Reagents, after thawing or after reconstitution, should be transparent and should not contain any sediment, gel or visible particles.
4. Stability and expiry date
Each reagent, when kept under the appropriate conditions of storage, should retain the requisite properties for at least one year.
The expiry date of a reagent in the liquid state 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.
5. Dispensing and volume
Tissue-typing 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 as described in this Protocol.
6. Records and samples
Written records shall be kept by the producing laboratory of all steps in the production and control of tissue-typing 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.
7. Shipment
Frozen reagents must be shipped in such fashion that they
remain frozen until arrival. Care must be taken to protect
reagents against inactivation by the entry of CO . Dried reagents
2
may be shipped at ambient temperatures.
8. Labels, leaflets and certificates
Two labels, one printed in English and one in French, in black on white paper, shall be affixed to each final container and shall contain the following information:
a. name and address of producer,
b. the specificity of the reagent,
c. name and amount of antiseptic and/or antibiotic, if present, or indication of absence,
d. the volume or, when the reagent is dried, the volume and composition of the fluid needed for reconstitution,
e. expiry date,
f. identification,
g. conditions of storage,
h. results of the test for HB Ag.
s
Moreover, the leaflet accompanying the containers shall include the following information:
a. full name and address of producer,
b. the recognised specificity of the reagent,
c. the volume or, when the reagent is dried, the volume and composition of the fluid needed for reconstitution,
d. date of last potency test,
e. expiry date (if any),
f. identification and (if possible) the name of the reagent,
g. adequate description of the method of use recommended by the producer including technique, volume and dilution to be used,
h. conditions of storage of unopened ampoules and precautions to be taken after opening,
i. exact composition, including antiseptic and/or antibiotic if any,
j. statement whether the product contains or does not contain material of human origin,
k. the reaction score ++, -+, +-, --, and the values of coefficient r (serum/antigen).
Each consignment shall be accompanied by a certificate as provided in Article 4 of the Agreement and the Annex to the present Protocol. Examples of label and leaflet are attached to the present Protocol.
SPECIFIC PROVISIONS*
____________________________
*To be completed under Article 4, paragraph 4 of the European Agreement on the Exchange of Tissue-typing Reagents.
EXAMPLE OF LABEL
COUNCIL OF EUROPE
EUROPEAN AGREEMENT
ON THE EXCHANGE OF TISSUE-TYPING REAGENTS
a. Laboratoire national de reference pour le ¦a. National Tissue-typing Reference Labo-
groupage tissulaire: ¦ ratory:
1 Main Street, Metropolis, Westland ¦ 1 Main Street, Metropolis, Westland
¦
b. Reactif pour groupage tissulaire: ¦b. Tissue-typing reagent:
anti-HLA-A1 ¦ anti-HLA-A1
¦
c. Une solution de № Na a 1 g/l a ete ajoutee¦c. № Na solution of 1 g/l has been added
3 ¦ 3
¦
d. Volume: 1 ml ¦d. Volume: 1 ml
¦
ou: Reconstituer avec 1 ml d'eau distillee ¦or: To be reconstituted with 1 ml of distilled
¦ water
¦
e. Date de peremption: 5 decembre 1985 ¦e. Expiry date: 5 December 1985
¦
f. Identification ¦f. Identification
¦
g. A conserver a: - 40°C ¦g. To be stored at: - 40°C
¦
h. Resultat de l'epreuve AgHB : ... ¦h. Result of the test for HB Ag: ...
s ¦ s
____________________________
This label must be affixed to each final container.
EXAMPLE OF LEAFLET
COUNCIL OF EUROPE
EUROPEAN AGREEMENT
ON THE EXCHANGE OF TISSUE-TYPING REAGENTS
____________________________----------T____________________________-----------¬
¦a. Nom et adresse complets du producteur ¦a. Full name and address of the producer ¦
¦ ¦ ¦
¦b. Reactif de groupage tissulaire: ¦b. Tissue-typing reagents: anti-HLA-A1 ¦
¦ anti-HLA-A1 ¦ ¦
¦ ¦ ¦
¦c. Volume: 1 ml ¦c. Volume: 1 ml ¦
¦ (ou: reconstituer avec 1 ml d'eau dis-¦ (or: to be reconstituted with 1 ml of ¦
¦ tillee) ¦ distilled water) ¦
¦ ¦ ¦
¦d. Date du dernier controle d'activite: ¦d. Date of last potency test: ¦
¦ ¦ ¦
¦e. Date de peremption: ¦e. Expiry date: ¦
¦ ¦ ¦
¦f. Identification et (si possible) nom du¦f. Identification and (if possible) name of¦
¦ reactif: ¦ the reagent: ¦
¦ ¦ ¦
¦g. Mode d'emploi; technique a utiliser: ¦g. Methode of use; technique to be used: ¦
¦ Lymphocytotoxicite NIH, etc. ¦ NIH Lymphocytotoxicity, etc. ¦
¦ ¦ ¦
¦h. A conserver a: (temperature, ...) ¦h. To be stored at: (temperature, ...) ¦
¦ ¦ ¦
¦i. Composition ¦i. Composition ¦
¦ ¦ ¦
¦j. Le reactif contient du serum humain ¦j. The reagent contains human serum ¦
¦ ¦ ¦
¦k. Resultats de reactions: ¦k. Reaction score: ¦
¦ ++ -+ +- -- ¦ ++ -+ +- -- ¦
¦ 30 0 1 300 ¦ 30 0 1 300 ¦
¦ Serum/antigene r = 0,90 ¦ Serum/antigen r = 0.90 ¦
L____________________________---------+____________________________------------
This leaflet must accompany a container enclosing several final containers.
Annex
to the Protocol
COUNCIL OF EUROPE
EUROPEAN AGREEMENT ON THE EXCHANGE OF
TISSUE-TYPING REAGENTS
Certificate (Article 4 of the Agreement)
NOT TO BE SEPARATED FROM THE SHIPMENT
........................ 19..
(place) (date)
Nombre de Le soussigne declare que l'envoi cite en marge ......................
colis
Number of The undersigned certifies that the shipment specified in the margin
packages .....................................................................
.............. prepare sous la responsabilite de ...................................
.............. prepared under the responsibility of ................................
Marque(s) .....................................................................
Marked .....................................................................
.............. l'un des organismes vises a l'article 6 de l'Accord, est conforme aux
.............. one of the bodies referred to in Article 6 of the Agreement, is in
Identification spуcifications de l'Article 5
Identification conformity with the specifications of Article 5
.............. du Protocole a cet Accord et doit etre delivre immediatement au
.............. of the Protocol to the Agreement and must be delivered immediately to
.............. destinataire (nom et lieu) ..........................................
.............. the consignee (name and place) .....................................
(nom et lieu) .......................................................
(name and place) ....................................................
(cachet) (signature) (qualite)
(stamp) (signature) (title)