16.4.1: General guidelines
HLA reagents prepared from human source material should comply with the guidelines in section 22.214.171.124.
Exceptionally, reagents not tested at source as required in section 126.96.36.199, and for which no alternative exists, may be supplied for use with the expressed approval of the user and with the understanding that the reagent must be regarded as potentially infectious.
These reagents should be marked ‘Potentially infectious – not tested at source for...’, as appropriate, both on the immediate container label or multi-well tray or reservoir, and the outer packaging.
The instructions for use of these reagents should indicate that the reagent(s) has not been tested at source as required in section 188.8.131.52, and that the reagents are to be considered as potentially infectious. In addition, the package insert should give information on the safe disposal of the material and the container, multi-well tray or reservoir.
184.108.40.206: Immediate container label
HLA reagents issued separately: The label should conform to the requirements of EN ISO 18113:2009. In addition, the body of a container presented in sealed bags or foiled pouches should be marked with a unique identifier to enable identification and traceability.
Multi-component test systems: In addition to the label information required above, a test system comprising multiple components should be marked to ensure identification and traceability of all components, for example multi-well trays or reservoirs, strips and pre-prepared membranes.
The instructions for use should contain the information required by EN ISO 18113:2009, and should comply, where applicable, with the requirements of section 220.127.116.11.
16.4.2: Serological typing reagents
The following information must be provided for each individual serological HLA typing reagent or HLA typing set:
- The claimed HLA specificity(ies) of the reagent, the percentage of specific reactions giving a cytotoxicity score of 80% to 100% cell death, the values of the correlation coefficient r obtained by the pre-testing of the reagent against a well-characterised cell panel, and the reaction score.
- The manufacturer should provide information of the incidence of equivocal cytotoxicity scores within the package insert.
- HLA typing sets should include a representation of the multi-well tray or reservoir layout indicating the position, HLA specificity(ies) and batch (or sub-batch) reference of the HLA typing reagent contained in each well.
- Monoclonal antibodies should be identified as such.
- An instruction that thawing and refreezing of the HLA typing reagents should be kept to the absolute minimum from the date of manufacture to the date of use. HLA typing sera frozen in micro-well trays should be used within 1 hour of thawing. Sera supplied freeze-dried in micro-well trays should be used within 1 hour of their reconstitution; unused trays should not be refrozen for later use.
- When reagents are supplied as an HLA typing set for the detection of a single antigen, the instructions for use should indicate which controls are appropriate to demonstrate specificity and cross-reactivity.
- For HLA typing sets, a list should be provided of those specificities that cannot be adequately defined in the presence of other specified specificities.
- Each HLA typing set for Class I or Class II phenotyping should contain at least one positive control antibody preparation, previously shown to react with all target cells, and should include at least one negative control preparation, previously shown to lack antibody activity or be from a male with no history of blood transfusion.
HLA typing reagents may be preserved in the liquid or in the dried state. Reagents should be stored as recommended by the manufacturer.
HLA typing reagents, after being thawed or reconstituted, should be transparent and should not contain any sediment, gel or particles visible on microscopy (x 200).
18.104.22.168: Stability and expiry date
Manufacturers should ensure that HLA typing reagents have a shelf life of at least 1 year, when stored as recommended.
Any extension by the user of the expiry date stated by the manufacturer should be supported by documented test data.
Manufacturers should notify all primary users if an HLA typing reagent or a constituent reagent of an HLA typing set stored as recommended fails to perform satisfactorily within the expiry date allotted by the manufacturer.
16.4.3: Requirements for phenotype assignment
HLA Class I and Class II serological typing must comply with EFI Standards E1.000 to E2.740 inclusive. HLA reagents and kits to be used for phenotyping lymphocytes by cytotoxicity should comply with the following:
- HLA typing reagents, when used by all methods recommended by the producer, should react with all lymphocyte samples with the corresponding antigen(s) when tested against a panel of lymphocyte samples bearing those antigen(s) collected from at least 25 individuals. HLA typing reagents should not react with any lymphocyte samples when tested against a panel of lymphocyte samples known not to bear the corresponding antigen(s) collected from at least 100 individuals. Reagents that conform to the requirements of this paragraph are termed operationally monospecific.
- Not more than half of the HLA typing reagents used together to detect an antigen should have the same extra claimed specificity.
- None of the HLA typing reagents used together should have been shown to react with more than 5% of the separate samples of a lymphocyte panel which do not express any of the antigen(s) that the reagent is claimed to detect.
- Manufacturers should indicate in the instructions for use those specificities whose detection does not comply with the requirements of any of the above.
16.4.4: Rabbit complement for use in HLA serology
22.214.171.124: General guidelines
Rabbit complement supplied for use in HLA serology should be stored as recommended by the manufacturer.
Manufacturers should be aware that highly active complement can cause unwanted specificities to become apparent in HLA typing reagents that have been characterised on less active but adequate complement.
126.96.36.199: Immediate container label
The label of the immediate container of rabbit complement for use in HLA serology should conform to the specifications in section 188.8.131.52.
184.108.40.206: Instructions for use
The instructions for use supplied with rabbit complement for use in HLA serology should conform to the specifications in section 220.127.116.11.
The instructions for use should offer guidance on the method of thawing. In addition, they should contain an instruction that the complement, once thawed from the immediate container or reconstituted from the freeze-dried state, should not be refrozen.
The instructions for use should state whether the rabbit complement has been tested and found suitable for use with monoclonal HLA typing reagents.
18.104.22.168: Potency tests on rabbit complement for use in HLA serology
Rabbit complement should be tested prior to use, in accordance with EFI Standards E2.700–E2.740.
16.4.5: DNA typing reagents
Methods available for HLA typing of DNA samples rely on identification of polymorphic HLA gene sequence motifs. In all widely used methods, the polymerase chain reaction (PCR) is utilised, either through the use of sequence-specific primers as in PCR-SSP, or to produce a locus-specific DNA template (e.g. HLA-A) which can subsequently be typed using a panel of sequence-specific oligonucleotide probes (PCR-SSOP). The locus-specific template may also be directly sequenced using locus or allele group-specific sequencing primers.
DNA can be prepared from various tissues by a variety of methods. The laboratory should prepare DNA by a standard method that has been reported in the scientific literature and validated in the laboratory for the HLA typing method to be used.
22.214.171.124: Instructions for use
In addition to section 126.96.36.199 of these guidelines, the instructions for use must adhere to the EFI Standards for Nucleic Acid Analysis (Section L) and should include the following:
- a statement explaining the test and intended application of the kit
- the principle of the procedure
- reagents and equipment required to perform the test
- detailed instructions for all components of the test
- the gene targeted as a PCR amplification control (PCR-SSP)
- the specificity and nucleotide sequence of all primers and probes used in the HLA typing kit
- a table or diagram indicating the location of the probes and/or primers utilised in the test
- a list of ambiguous combinations of alleles defined for each test kit – this may also be given as part of interpretative software
- the HLA alleles which are claimed to be detected by the HLA typing kit, further divided into the following groups:
- those HLA alleles which have been detected in appropriately controlled validation tests
- • those HLA alleles which have not been directly detected in validation tests but where the reactivity of the allele is expected to be detected
- those HLA alleles which have not been directly detected in validation tests and whose reactivity cannot be assumed to be detected by the kit
- those HLA alleles that are known to produce weak or unreliable signals in the output systems
- the date and the source of the sequence information used in the kit design and a statement that new alleles described following the date of design may not be detected by the kit
- the control tests to be performed to check for contamination (negative control) of the test system
- the control DNA to be included to check for quality of sample DNA used
- the control test to be performed to generate a true positive signal
- acceptable limits of signal intensity should be specified for positive and negative results
- all computer software assisted interpretation of results should be validated on control DNA
- the chemical components of the kits should be listed and reference made to any toxic substances included in the kit with recommendations for their safe disposal. Reference to material safety data sheets should be given.
Manufacturers should inform all primary users of a DNA-based HLA typing kit when any changes to a kit’s ability to perform are detected. All users of DNA-based HLA typing kits should report any kit-related problems directly to the manufacturer and maintain records of such events.