EFI ACCREDITATION PROGRAM
EFI NUMBER:
© EFI, October 2014
EFI ACCREDITATION PROGRAM - INSPECTION CHECKLIST
Applicant Information:
Director(s):
Institution:
City:
Inspector Information:
Name:
Institution:
Name:
Institution:
Trainee:
Institution:
Inspection Date:
1
Inspection checklist according to the Standards version 6.2.Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Required
Yes
No
EFI Standard
© EFI 2014
Recom.*
Yes No
SECTION B – PERSONNEL QUALIFICATIONS
B0
For the purposes of this document, EFI defines the Director as the person who is responsible
for the H&I laboratory.
B1
The laboratory must employ one or more individuals who meet the qualifications and fulfil
the responsibilities of:
A Director, that must:
Hold a qualification approved by EFI, such as an earned doctoral degree in a biological
science, or be a physician, and
Have minimum qualifying experience equivalent to either of the following:
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Four years’ relevant experience two of which were devoted to full time training in
human H&I testing, or
Four years of working experience at full time in human H&I testing
Additional qualifications required according to national legislation also apply.
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Have documentation of professional competence in the appropriate activities in which the
laboratory is engaged. This should be based on sound knowledge of the fundamentals of
immunology, genetics and histocompatibility testing
If a Co-Director is appointed, this person must also fulfil Standards B1.1.1 - B1.1.3
The Director and/or Co-Director must:
Be available on site to supervise the laboratory for at least 80% of the week
Provide adequate supervision of technical personnel
Utilises his/her special scientific skills in developing new procedures
Be held responsible for the proper performance, interpretation and reporting of all
laboratory procedures
Ensure the laboratory's successful participation in Proficiency testing.
Be informed of the relevant national legislation
Comply with the relevant national legislation
Demonstrate active participation in H&I related clinically relevant professional
development, such as national or international conferences or workshops
The Director or Co-Director should:
Have publications in peer-reviewed journals
A Technical Supervisor, that must:
Have minimum qualifying experience equivalent to either of the following:
Hold a bachelor's degree or equivalent and have three years’ relevant experience in
human histocompatibility and immunogenetics testing under the supervision of a
qualified Director or Co-Director
Five years of supervised experience if a bachelor's degree has not been earned
A Quality Manager, who must establish and maintain a comprehensive quality management
programme covering all aspects for the accredited facility addressed by these standards.
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B2
B3
B3.1
B1.1
B1.1.1
B1.1.2
B1.1.2.1
B1.1.2.2
B1.1.2.3
B1.1.3
B1.1.4
B1.1.5
B1.1.5.1
B1.1.5.2
B1.1.5.3
B1.1.5.4
B1.1.5.5
B1.1.5.6
B1.1.5.7
B1.1.5.8
B1.1.6
B1.1.6.1
B1.2
B1.2.1
B1.2.1.1
B1.2.1.2
B1.3
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The resources of the laboratory must be sufficient to accommodate the workload.
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Testing referred to other laboratories
An accredited laboratory may engage another laboratory to perform testing not done by the
primary laboratory.
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2
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
.....
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Required
Yes
No
EFI Standard
© EFI 2014
B3.2
B3.2.1
B3.2.2
The subcontracting laboratory:
Must be accredited by EFI or by ASHI, if the testing is covered by EFI standards.
Should have documented expertise and/or accreditation in genetic systems not
covered by EFI Standards
The identity of the subcontracting laboratory and that portion of the testing for
which it bears responsibility must be noted in the reports issued
B3.2.3
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Recom.*
Yes No
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SECTION C – QUALITY ASSURANCE
C1
C1.1
C1.1.1
C1.1.2
C1.1.3
C1.1.4
C1.1.5
C1.1.6
C1.1.6.1
C1.1.6.2
C1.1.6.3
C1.2
C1.2.1
C1.2.2
C1.2.3
C1.2.4
C1.2.5
C1.3
C1.4
C1.4.1
C1.4.2
C1.5
C1.5.1
C1.5.2
C1.5.2.1
C1.5.2.2
C1.5.2.2.1
C1.5.2.2.2
Facilities
The following facilities must be adequate and immediately available to the laboratory:
Sufficient space so that all procedures can be carried out without crowding to the extent
that errors may result, in accordance with national regulations.
Lighting
Ventilation
Refrigerators
Freezers
Storage for :
Reagents
Specimens
Records
Refrigerators and freezers:
Optimal ranges for each fridge and freezer must be documented
Must be monitored to detect unacceptable temperatures
Should be coupled to recording thermometers
Should be coupled to alarm systems with an audible alarm where it can be heard 24
hours a day
Corrective actions for when the temperature is outside the documented acceptable
range must be defined and documented
In laboratories where liquid nitrogen is utilised for storage of frozen cells, the level of liquid
nitrogen in the cell freezers must be monitored at intervals which will ensure an adequate
supply at all times
To ensure that procedures are carried out within temperature ranges specified in the
laboratory's procedure manual, the following must be monitored every working day:
Ambient temperature
Temperature of incubators in which test procedures are carried out
Laboratories performing procedures which require cell culture must have the following:
Laminar Flow Hoods or other appropriately aseptic work area
Incubators, which must be
appropriately humidified and
monitored daily in relation to:
Temperature (37°C)
CO 2 concentration (5% ± 1%)
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3
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Required
Yes
No
EFI Standard
© EFI 2014
C1.6
C1.6.1
C1.6.2
C1.6.3
C1.7
C1.7.1
C1.7.2
C1.8
C1.8.1
C1.8.2
C1.8.3
C1.8.3.1
C1.8.3.2
C1.9
C1.9.1
C1.9.2
C1.9.3
C1.9.3.1
C1.9.3.2
C2
C2.1
C2.1.1
C2.1.2
C2.1.3
C2.2
C2.2.1
C2.2.2
C3
C3.1
C3.2
C3.3
C3.3.1
Laboratories using radioactive materials must have a designated section of the laboratory for
The storage of materials
Conducting procedures
Radioactive materials must be disposed of at locations designated by local
institutions.
Laboratories performing amplification of nucleic acids must:
Use physical and/or biochemical barriers to prevent DNA contamination
Perform pre-amplification procedures in an area which excludes amplified DNA that
has the potential to serve as a template for amplification in any of the genetic
systems tested in the laboratory
The laboratory must establish and employ policies and procedures for the proper
maintenance of equipment, instruments and test systems by:
Defining its preventive maintenance programme for each instrument and piece of
equipment at least once a year
Performing and documenting function checks on equipment with at least the
frequency specified by the manufacturer.
The laboratory must use calibrated dispensing instruments (e.g. pipettes, etc) to
perform assays
Calibration of dispensing instruments must be performed at least once a year
Calibration must be documented
The laboratory must document compliance with all applicable national and local laws which
relate to:
Employee health and safety
Fire safety
Storage, handling and disposal of:
Chemical material
Biological material
Computer assisted analyses
The Laboratory Director and/or the Supervisor must
Review
Verify
Sign computer assisted analyses before issue
The computer software programme used for analyses must be:
Identified
Validated/Verified before use
Specimen submission and requisition
The laboratory must have available and follow written policies and procedures regarding
specimen collection
The laboratory must perform tests only at the written or electronic request of an authorised
person
The laboratory must assure that the requisition includes:
The patient’s or donor’s name or other method of specimen identification to assure
accurate reporting of results
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4
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
C3.3.2
C3.3.3
C3.3.4
C3.3.5
C3.4
C3.4.1
C3.4.2
C3.5
C3.6
C3.6.1
C3.6.2
C3.6.3
C3.6.4
C3.7
C3.7.1
C3.7.2
C3.7.2.1
C3.7.2.2
C3.7.3
C3.8
C3.9
C3.9.1
C3.9.2
C3.9.3
C3.9.4
C3.9.5
C4
C4.1
C4.2
C4.2.1
C4.2.2
C4.2.3
C4.3
The name and address of the authorised person or of the service who ordered the
test
Date of specimen collection
Time of specimen collection, when pertinent to testing
Source of specimen (e.g. bone marrow, spleen cells) if pertinent
Blood or tissue samples must be individually labelled with:
The name, and/or other unique identification marker of the individual
Date of collection
When multiple blood containers are collected, each container must be individually labelled
The laboratory must:
Maintain a system to ensure reliable specimen identification
Document each step in the processing and testing of patient specimens to assure
that accurate test results are recorded.
Have criteria for specimen rejection
Have mechanism to assure that specimens are not tested when they do not meet the
laboratory’s criteria for acceptability
If the laboratory provides a phlebotomy service:
Blood samples must be obtained using a location, which does not compromise
aseptic techniques
The donor's skin must be prepared by a technique, which ensures minimal possibility
of infection of the donor
of contamination of the sample
All needles and syringes must be disposable.
All biological samples must be handled and transported in accordance with the
understanding that they could transmit infectious agents
The laboratory must provide all service users with information about the requirement for
Sample labelling
Anticoagulant / preservation media
Sample packaging
Regulations relating to postal transport
The laboratory should warn users that failure to meet these requirements may
result in sample rejection.
Reagents
All reagents must be properly labelled and stored according to manufacturers’ instructions
or locally-specified conditions to maintain reactivity and specificity.
Reagents, solutions, culture media, controls, calibrators and other materials must be
labelled to indicate:
Identity and when significant, titre, strength or concentration
Recommended storage requirements
Preparation and/or expiration date and other pertinent information
For storage of larger numbers of identical samples, it might be acceptable to use short-cut
labelling of individual samples if the short-cut notation is explained on the outside of the
storage container
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5
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
.....
....
.....
....
Required
Yes
No
EFI Standard
© EFI 2014
C5
C5.1
C5.2
C5.2.1
C5.2.2
C5.2.3
C6.
C6.1
C6.1.1
C6.1.2
C6.1.3
C6.2
C6.2.1
C6.2.2
C6.2.3
C6.2.4
C6.2.5
C6.3
C6.3.1
C6.3.2
C6.3.3
C6.4
C6.4.1
C6.4.1.1
C6.4.1.2
C6.4.1.3
C6.4.2
C6.4.3
C6.4.4
C6.4.5
Laboratory Procedure Manual.
All procedures in use in the laboratory must be detailed in a procedure manual which is
immediately available where the procedures are carried out. The use of product inserts
provided by manufacturers is not acceptable in place of the procedure manual.
For each procedure:
A review by the Director/Co-Director or a delegated individual with appropriate
qualifications must be performed at least biennially.
Documented evidence of this review must be available
Any changes in procedures must be approved and documented by the Director/CoDirector/ delegated individual at the time they are initiated.
External Proficiency Testing( EPT)
The laboratory must participate in EPT programme(s) to cover
All the accredited laboratory applications (HLA typing, antibody screening and
identification, crossmatching, etc.)
All techniques used individually or in combination as routinely employed to
produce a final result
If no established scheme exists for a specific category (e.g. HNA antibody detection
and identification) the laboratory must participate in an exchange of samples based
workshop.
Procedure of EPT
The procedure for testing EPT samples including the allocation to techniques must be
documented prior to the annual commencement of the EPT cycle.
The laboratory must have a predetermined policy if they select individual shipments or
samples for EPT
The minimum number of samples as defined in C6.4 applies to all technique(s) used to
produce a final result
If the same sample is tested for more than one laboratory application, e.g. both low and high
resolution HLA typing, the results must be analysed independently
If the same sample is tested by more than one technique at low or high resolution DNA
typing, the laboratory should make only one report to the Provider, but keep results
obtained by different techniques available for inspection
EPT samples must be
tested by the same techniques as routinely employed for clinical samples, either
individually or in combination
interpreted in a manner comparable to routine clinical samples
incorporated into the laboratory’s routine workload
Minimum number of samples for EPT per year
HLA typing:
Serological typing:
10 samples
Low resolution DNA-based typing:
10 samples
High resolution DNA-based typing:
10 samples
HPA/HNA typing:
10 samples
HLA antibody detection:
10 samples
HLA antibody identification:
10 samples
HPA antibody detection and identification:
5 samples
6
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* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
....
Required
Yes
No
EFI Standard
© EFI 2014
C6.4.6
C6.4.7
C6.5
C6.5.1
C6.5.1.1
C6.5.1.2
C6.5.2
C6.5.2.1
C6.5.2.2
C6.5.2.3
C6.5.3
C6.5.3.1
C6.5.3.2
C6.5.3.3
C6.5.4
C6.5.4.1
C6.5.4.2
C6.5.5
C6.5.5.1
C6.5.5.2
C6.5.5.3
C6.6
C6.6.1
C6.6.1.1
C6.6.1.2
C6.6.2
C6.6.3
C6.6.4
C6.6.4.1
C6.6.4.2
C6.6.4.3
Crossmatching: 20 tests of different donor/recipient combinations which must include a
minimum of 10 different sera
Haematopoietic chimaerism and engraftment monitoring: 10 tests of different
donor/recipient mixtures in the range 0% - 100% excluding the reference donor and
recipient samples
Reporting of EPT results
Participants must report:
the antigen specificities and alleles identified
the method(s) used.
The following convention for reporting groups of HLA alleles is recommended:
Groups of alleles should be reported as allele x/allele y, where “/” means “or”, e.g.
DRB1*01:01/02/04 means DRB1*01:01 or DRB1*01:02 or DRB1*01:04. This shortened
presentation of alleles within a string must not be used if alleles differ within the first field,
e.g. DPB1*03:01/104:01/124:01 must not be given as DPB1*03:01/104/124.
Groups of alleles that include sequential allele numbers may be reported as allele x-allele y,
where “-” means “to”, e.g. DRB1*15:01-04 means that the allele could be any between
DRB1*15:01 to DRB1*15:04 inclusive.
Alleles should be reported in full for DRB3, DRB4, and DRB5, e.g. DRB4*01:02-01:03 and not
as DRB4*01:02-03.
For the detection of HLA class I and/or class II antibodies, participants must report
the presence or absence of HLA class I and/or class II antibodies
the antibody specificities identified
the methods used
For crossmatching, participants must report
the test results
the method(s) used
For haematopoietic chimaerism and engraftment monitoring, participants must report
the test results as a percentage of donor chimaerism
the method(s) used
details of the kit(s) and the manufacturer(s) used
Laboratory performance
If a laboratory's performance in EPT programme(s) is unsatisfactory in any category for which
EFI accreditation is sought, the laboratory must:
Participate in an additional EPT programme in that category
Document the Director’s review and any corrective action taken
Laboratories must not engage in inter-laboratory communication pertaining to EPT results
until after the reporting deadline has passed
Laboratories must not send their own EPT samples or results for analysis to another
laboratory until after the reporting deadline has passed
Participating laboratories must ensure that all the following EPT related documents are
maintained and are made available to EFI inspectors for assessment:
Submitted worksheets
EPT summary/scheme reports
Annual performance
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7
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
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....
.....
....
Required
Yes
No
EFI Standard
© EFI 2014
C6.6.4.4
C6.6.4.5
C6.6.4.6
C7
C7.1
C7.1.1
C7.1.2
C7.2
C8
C8.1
C8.1.1
C8.1.2
C8.1.3
C8.1.3.1
C8.1.3.2
C8.1.3.3
C8.2
C8.2.1
C8.2.2
C8.2.3
C8.2.4
C8.2.5
C8.2.6
C9
C9.1
C9.1.1
C9.1.2
C9.1.3
C9.2
Participation certificates
Outcomes of investigations of any unsatisfactory results
Corrective or preventive actions
Competency Evaluation and Continuous Education
The Director/Co-Director or designee must:
Evaluate the competence of each technologist to accurately perform tests. This must
be done at least yearly for each technique the technologist performs and must be
based on a defined process
Maintain records of these evaluations for each individual.
The Laboratory Director and the technical staff must participate in continuing education
relating to each category for which EFI accreditation is sought
Systems for Continuous Test Evaluation and Monitoring
The laboratory must establish and employ policies and procedures, and document actions
taken when:
Test systems do not meet the laboratory’s established criteria
Quality control results are outside of acceptable limits
Errors are detected in the reported patient results. In this instance, the laboratory
must:
Promptly notify the authorised person ordering or individual utilising the
test results of reporting errors
Issue corrected reports
Maintain copies of the original report as well as the corrected report for at
least two years
The laboratory must have mechanisms in place for continuous monitoring of all test systems
and equipment used , including:
Validation/verification, before introduction into routine use, of all new tests, by systematic
comparative evaluation of results obtained in parallel with the new and the standard system
Regular evaluation of results obtained in external and internal QC testing
Regular monitoring of test validity in routine testing, by recording observations diverging
from the expected results (e.g. cross-reactivity of probes or primer mixes, day-to-day
variations).
Comparing test results and documenting inconsistencies, if the same test is performed using
different techniques.
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Identifying and evaluating inconsistencies between test results and clinical data or diagnostic
parameters provided
Written evidence of the ongoing monitoring process must be available in the laboratory for
each method performed
Client Service Evaluation
All complaints and problems reported to the laboratory must be:
documented
investigated
followed by corrective action when necessary
The laboratory must, upon request, make available to clients a list of tests employed by the
laboratory
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8
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
C10
C10.1
C10.1.1
C10.1.2
C10.1.3
C10.1.4
C10.2
C10.3
C11
C11.1
C11.1.1
C11.1.1.1
C11.1.1.2
C11.1.1.2.1
C11.1.1.2.2
C11.1.1.2.3
C11.1.1.2.4
C11.1.1.2.5
C11.1.1.2.6
C11.1.1.3
C11.2
C11.3
C11.3.1
C11.4
C11.5
C11.5.1
C11.5.2
C11.5.3
C11.5.4
C11.5.5
Quality Assurance Evaluation
The laboratory must:
Hold quality assurance reviews
Document, assess problems identified in these reviews and discuss them with the
staff
Take corrective actions necessary to prevent recurrences
Have an ongoing mechanism to evaluate corrective action taken. Ineffective policies
and procedures must be revised based on the outcome of the evaluation
The laboratory must maintain documentation of all quality assurance activities including
problems identified and corrective actions taken, for a minimum of two years or longer,
depending on local, or national regulations.
The laboratory must maintain permanent files of all internal and external quality control
tests according to any regulation to which the laboratory is obliged to abide, but for a
minimum of four years
Records and Test Reports
The laboratory must maintain the following records:
Records of subjects tested for two years or longer, depending on local regulations.
These records must include:
Log books
Worksheets, that must clearly identify:
The sample tested
The reagents used
The methods used
The test performed
The date of the test
The person performing the test
A summary of results obtained
Records may be only saved in computer files, provided that back-up files are maintained to
ensure against loss of data.
For molecular typing, a record must be kept which is appropriate to the technique used,
such as a photographic record of a gel, a membrane, an autoradiograph, an electronic file, or
the read out from a sequencer.
The record must be kept according to any regulation to which the laboratory is obliged to
abide, but for a minimum of two years.
Reports or records, as appropriate, must include a brief description of the specimen (blood,
lymph node, spleen, bone marrow, etc.) used for testing
The report must contain:
The name of the individual tested or unique identifier of each individual tested and
relationship to the patient if applicable
The date(s) of collection of sample when pertinent
The date of the report
The test results
The techniques used
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9
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
C11.5.6
C11.5.7
C11.6
Appropriate interpretations and the signature of the Laboratory Director/CoDirector, or a designated individual.
Information regarding the condition and disposition of specimen that did not meet
the laboratory’s criteria for acceptability.
The laboratory must have adequate systems in place to report results in a timely, accurate
and reliable manner.
Laboratories must have a procedure in place for resolving any discrepancies that may occur
between laboratories.
C11.7
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SECTION D – HLA ALLELES AND ANTIGENS
D1
D1.1
D1.2
D1.3
D1.4
D1.4.1
D1.4.1.1
D1.4.1.2
D2
D2.1
D2.1.1
D2.1.2
D2.1.2.1
D2.1.2.2
D2.1.2.3
D2.1.2.4
D2.1.3
D2.1.3.1
D2.1.3.2
D2.2
D2.3
D2.3.1
Terminology
Terminology of HLA alleles and antigens must conform to the latest report of the WHO
Committee on Nomenclature.
Potential new alleles or antigens not yet approved by the WHO Committee must have a local
designation which cannot be confused with WHO terminology.
NMDP codes must only be used for recording donors or cord blood unit typings into
databases or for communication of the donor, cord blood unit or recipient typing with the
registries.
High resolution typing is defined as the identification of HLA alleles that encode the same
protein sequence within the antigen binding site:
HLA alleles must be identified at the level of resolution which defines the first and
second fields according to WHO nomenclature by at least resolving all ambiguities:
resulting from polymorphisms located within exons 2 and 3 for HLA class I
loci, and exon 2 for HLA class II loci.
That encompass a null allele, wherever the polymorphism is located, unless
it can be demonstrated that an expressed antigen is present on the cells.
Phenotypes and Genotypes
Phenotypes and genotypes must be expressed as recommended by the WHO Committee, as
in the following examples:
Single alleles: HLA-B*07. Single antigens: HLA-B7 (or B7 if HLA is obvious from context).
Phenotype
Serological assignment: HLA-A2,30; B7,44; Cw7; DR1,4; DQ5,7.
DNA assignment: HLA-A*02,*30; B*07,*44; C*07,*16; DRB1*01,*04; DQB1*05, *03:01.
If an HLA typing is performed using DNA methods, it is acceptable to report an HLA
serological assignment if required for the purposes of organ allocation.
The translation of alleles to serological equivalence must be performed according to a
documented protocol.
Genotype
Serological assignment: HLA-A2, B44, Cw-, DR1, DQ5 / A30, B7, Cw7, DR4, DQ7.
DNA assignment: HLA-A*02, B*44, C*16, DRB1*01, DQB1*05 / A*30, B*07, C*07, DRB1*04,
DQB1*03:01.
The locus designation must always be included.
Reporting Homozygosity and Heterozygosity
If no more than one single antigen or allele is found at a locus by serological typing or DNA
typing, the phenotype may include it twice only if homozygosity is proven by family studies
or if 2-digit DNA typing unequivocally demonstrates the presence of heterozygosity for two
different alleles from the same specificity.
10
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
D2.3.2
D2.3.3
D2.3.3.1
D2.3.3.2
D2.3.4
D2.4
D2.4.1
D2.4.2
D2.4.2.1
D2.4.2.2
D3
D3.1
D3.2
D3.3
D3.3.1
D3.3.2
D3.4
D3.5
D3.5.1
D3.5.2
A “blank antigen or allele” can only be assigned if proven by family studies.
If homozygosity has not been proven, the HLA type may be reported using a hyphen. For
example:
HLA-A1,3; B7,44; Cw7,- to indicate a phenotypic blank, or
HLA-A*01,*03; B*07,*44; C*07,- for DNA based typing.
If typing unequivocally demonstrates the presence of heterozygosity for two different alleles
from the same specificity (e.g. DRB1*13:01/13:59, DRB1*13:03/13:33), the report may
include it twice (e.g. DRB1*13,*13) even in the absence of family studies.
Reporting High Resolution Typing
When reporting high resolution typing, where ambiguous allele combinations cannot be
resolved, all the alternatives must be documented
If all ambiguities are not included on the report, a comment must be added stating that:
Other ambiguous HLA (define loci) results have not been excluded and
this information is available upon request.
Haplotype Assignment
Determination of haplotypes must be done by typing immediate family members including
parents, siblings and/or children of the patient.
Genotypic identity can only be proven if both parents are available or if the segregation of
the four haplotypes is clearly defined.
When appropriate, ambiguities in haplotype assignment must be resolved by:
typing for HLA-C, and/or DQ and/or DP
high resolution typing
If recombination occurs, this must be reported with the HLA haplotype assignment.
For unrelated individuals, when probable haplotypes based on population frequencies used:
Reports must clearly indicate that they were so derived
The relevant references or sources must be available.
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Recom.*
Yes No
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SECTION E – SEROLOGICAL HLA CLASS I AND CLASS II TYPING
E1
Recording Test Results
E1.1
For HLA testing by Complement Dependent Cytotoxicity, each serum-cell combination must
be recorded in a manner which indicates the percentage of cells killed. Numerical scores
used should be:
Scores used by the International Workshop (0,1,2,4,6,8), or
Other numerical codes.
Typing
For each of the following loci, the laboratory must be able to type for HLA specificities which
are officially recognized by the WHO and for those deemed relevant by EFI:
HLA A and B when applying for accreditation in the category of class I by serology
HLA DR when applying for accreditation in the category of class II by serology
Techniques used must be those, which have been established to define HLA Class I and II
specificities optimally.
Control Reagents
Each typing tray must include:
at least one positive control antibody, which reacts with cells expressing class I and
class II antigens
at least one negative control serum that should lack leukocyte reactive antibodies
E1.1.1
E1.1.2
E2
E.2.1
E2.1.1
E2.1.2
E2.2
E3
E3.1
E3.1.1
E3.1.2
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11
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Required
Yes
No
EFI Standard
© EFI 2014
E3.2
Procedures that deal with control serum failures in typing or crossmatch trays must be
described in the laboratory manual.
If the positive control fails to react as expected, there must be a procedure in place as
whether to accept or reject the test.
The minimum viability of the cells and the reactivity of control sera required for the
validation of a serological typing must be described in the laboratory manual.
Antigen Assignments
Each HLA-A and B antigen must be defined by:
At least two sera when available, if both are operationally monospecific, or
if multispecific, at least three partially non-overlapping sera
Each HLA Class II antigen should be defined by:
at least three sera, if all are operationally monospecific
at least five partially non-overlapping sera if multispecific
Criteria for antigen assignment must be described in the laboratory manual.
Ambiguity in antigen definition by serological typing must be referred for confirmation by
DNA based methods.
Typing Reagents
Cells panel of known HLA type:
must be used to prove the specificity of new antibodies
should include at least one example of each HLA antigen the laboratory is required
to define.
Each monoclonal antibody used for alloantigen assignment must be used with an
established technique at a dilution which demonstrates specificity comparable to antigen
assignment by alloantisera on a well-defined cell panel.
For reagent grade typing serum:
confirmation of specificity must be performed
supporting statistical analysis must be recorded
Specificity of individual sera received from other laboratories or commercial sources must be
confirmed to ensure that they reveal the same specificities in the receiving laboratory.
E3.3
E3.4
E4
E4.1
E4.1.1
E4.1.2
E4.2
E4.2.1
E4.2.2
E4.3
E4.4
E5
E5.1
E5.1.1
E5.1.2
E5.2
E5.3
E5.3.1
E5.3.2
E5.4
E5.5
E5.5.1
E5.5.1.1
E5.5.1.2
E5.5.2
Typing trays lots and shipments
Each lot of typing trays must be evaluated by testing, either:
at least five different cells of known phenotype representing major specificities
in parallel with previously evaluated trays with at least five cells of known phenotype.
Each new shipment of previously evaluated typing trays must be verified with at least one
cell of known phenotype.
E6
E6.1
E6.2
E6.2.1
E6.2.1.1
Complement
Complement must be kept at the recommended temperature.
Complement lot and shipment testing
Each lot and shipment of complement must be evaluated by either:
testing with at least 3 previously evaluated trays for every application for which it is
intended, or
testing a combination of at least 3 sera and 2 cells selected to include negative, weak
positive and strong positive reactions.
The test must employ multiple dilutions of complement to ensure that it is maximally active
at least one dilution beyond that intended for use.
E6.2.1.2
E6.2.2
E6.2.3
Complement must be tested separately for use with each type of target cell.
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12
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
.....
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Required
Yes
No
EFI Standard
© EFI 2014
E6.2.4
E6.2.4.1
E6.2.4.2
Evaluation of each new lot and shipment of Complement must be tested to determine that:
it mediates cytotoxicity in the presence of specific HLA antibody
it is not cytotoxic in the absence of HLA specific antibody
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SECTION F – ANTIBODY SCREENING AND CROSSMATCHING
F1
F1.1
F1.2
Sera
Serum samples stored must be retained in the frozen state.
Sera must be tested at a concentration determined to be optimal for detection of antibody
to HLA antigens. The dilution(s) must be documented.
Negative control:
Each assay must include a negative control
The negative control must be a serum from non-alloimmunised human donor(s).
Positive control:
Each assay must include a positive control
The positive control must be either a validated monoclonal antibody, or sera from highly
alloimmunised individuals and documented to react with HLA antigens
The antibodies used must be of the appropriate isotype for each assay
Techniques
For the detection of antibody to HLA antigens, the laboratory must either use:
A complement-dependent cytotoxic technique, or
Another technique performed by the laboratory with documented validation testing,
demonstrating that this technique identifies alloantibody to HLA antigens at a level
of sensitivity equivalent or superior to that of its cytotoxic technique.
To detect antibodies to HLA class II antigens, a technique must be used that
distinguishes them from antibodies to HLA class I antigens.
F1.3
F1.3.1
F1.3.2
F1.4
F1.4.1
F1.4.2
F1.4.3
F2
F2.1
F2.1.1
F2.1.2
F2.1.3
F2.2
F2.2.1
Other techniques:
Laboratories performing assays using flow cytometry and/or Luminex must also conform to
the standards in Section M.
Laboratories using micro-plate ELISA techniques for antibody screening must additionally
conform to standards in section N (ELISA).
F2.2.2
F3
F3.1
F3.1.1
F3.1.2
F3.1.3
Antibody Screening by Complement-Dependent Cytotoxicity
The following controls must be included on each tray:
Positive control
Negative control
If sera are screened after treatment with dithiothreitol, IgG and IgM positive controls must
be used
Laboratories using a CDC technique must also conform to standard E6 (Complement).
Panels
The panel of HLA antigens must include sufficient panel cell donors to ensure that they are
appropriate for the population served and the use of the test results.
For assays intended to provide information on antibody presence or antibody identification,
documentation of the HLA class I and/or class II specificities of the panel must be provided.
F3.2
F4
F4.1
F4.2
F5
F5.1
F5.1.1
Crossmatching
Crossmatching for the detection of HLA specific antibodies:
must use techniques at least as sensitive as the basic lymphocytotoxicity test.
13
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
F5.1.2
should use at least one technique documented to have increased sensitivity in
comparison with the basic microlymphocytotoxicity test, such as prolonged incubation,
antiglobulin test, ELISA, B-cell crossmatch or flow cytometry
The screening result used must be at least as sensitive as the routine crossmatch technique.
Each serum must be tested:
Undiluted
In duplicate
Crossmatches must be performed:
with unseparated lymphocytes or with T lymphocytes
With B lymphocytes if required by the relevant transplantation programmes.
The following controls must be included on each tray:
Positive control
Negative control
If sera are tested after treatment with dithiothreitol, IgG and IgM positive controls must be
used
Laboratories using a CDC technique must also conform to standard E6 (Complement)
Standards in sections M (Flow Cytometry) and N (ELISA) must be followed when applicable.
F5.2
F5.3
F5.3.1
F5.3.2
F5.4
F5.4.1
F5.4.2
F5.5
F5.5.1
F5.5.2
F5.5.3
F5.6
F5.7
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Recom.*
Yes No
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SECTION G – RENAL and/or PANCREAS TRANSPLANTATION
G1
G1.1
G1.1.1
G1.1.2
G1.1.3
G1.2
If deceased donor transplants are done:
The following personnel must be available 24 hours a day, seven days a week:
personnel for the required histocompatibility testing,
personnel for interpretation of results
personnel for advice for the clinical transplant team
Laboratories not able to perform tests 24h/day, 7d/week must arrange with an EFI or ASHI
accredited laboratory to perform tests.
Antibody Screening
Laboratories must:
have a documented policy in place to evaluate the sensitisation of each patient at the time
of their initial evaluation
G2
G2.1
G2.1.1
G2.2
have a programme to periodically screen serum samples from each patient for antibodies to
HLA antigens by:
determining and recording the specificity of detected HLA antibodies
performing testing to distinguish HLA specific antibodies from non HLA antibodies
and autoantibodies
Have a policy establishing the frequency of screening serum samples and must have data to
support this policy.
G2.2.1
G2.2.2
G2.3
Samples must be collected and tested , either:
every three months, or
as stipulated by the national and/or international organ exchange organisations.
Sensitising Events
Laboratories should maintain a record of potentially sensitising events for each patient.
Serum samples should be collected and stored after each of these events for possible
subsequent screening for antibodies to HLA and/or use in crossmatch tests.
G2.3.1
G2.3.2
G3
G3.1
G3.2
14
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Required
Yes
No
EFI Standard
© EFI 2014
G4
G4.1
G4.1.1
G4.1.2
G4.1.3
G4.2
G4.2.1
G4.2.2
G4.2.3
G4.2.4
G4.2.5
G4.2.6
G4.2.6.1
G4.2.6.2
G4.2.6.3
G4.2.6.4
G4.2.6.5
G4.3
G4.3.1
G4.3.1.1
G4.3.1.2
G4.4
G4.4.1
G4.4.2
G4.4.3
G4.4.4
G4.5
G4.5.1
G4.5.1.1
G4.5.1.2
Crossmatching
Crossmatching must be performed according to national legislation applying to the
laboratory and/or regulations from the national / international exchange organisation.
Crossmatching must be performed prospectively, or may be omitted if
Virtual Crossmatching is performed
Prospective crossmatching must be performed for all living donor transplants
If the prospective crossmatch is omitted, a retrospective crossmatch must be
performed.
Virtual Crossmatching
A transplant protocol for Virtual Crossmatching must be agreed with the clinical
transplant teams and documented.
There must be evidence that the eligibility of each patient has been evaluated when a
virtual crossmatch has been performed.
The transplant protocol must include evidence that the clinical teams are aware of the
possibility of errors in donor offer typing.
The Virtual Crossmatch result must be reported by the laboratory to the transplant
clinician before the transplant proceeds.
Evidence that the Virtual Crossmatch was reported must be documented.
Patients are only eligible for Virtual Crossmatching if
There have been no potential sensitising events since the last serum sample
screened.
Sera must have been collected as defined in standard G2.3
At least two different samples must have been tested.
At least one serum screening result obtained within the previous 3 months must
be included
Sera must be tested for antibody specificity identification by a technique of at
least equivalent sensitivity to that used for crossmatching
Virtual Crossmatching for Unsensitised Patients
A prospective crossmatch may be omitted for patients:
Who test consistently negative for the presence of HLA-specific antibodies,
as relevant for the transplant protocol.
for whom there must be documented evidence that the laboratory maintains
a record of potentially sensitising events
Virtual Crossmatching for Sensitised Patients
The prospective crossmatch may be omitted in carefully selected HLA immunised
recipients in whom acceptable and/or unacceptable mismatches have been clearly
defined and documented.
If a prospective crossmatch is omitted from an alloimmunised recipient, the method of
antibody identification must rely on single antigen technology.
The Virtual Crossmatch must include data from single antigen testing performed on a
sample collected within the last 3 months.
Patients are ineligible for Virtual Crossmatching if they have antibodies against
specificities for which the donor has not been typed.
Retrospective Crossmatching
If retrospective cross-matches are performed according to standard G4.1.3
They must be shown to be in concordance with the predicted negative result and this
must be documented
The physician in charge must be notified immediately of an unpredicted positive result.
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15
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
G4.5.1.3
G4.5.1.4
G4.6
G4.6.1
There must be a re-evaluation of this policy at least annually.
Any additional regulations either national, or of the exchange organisation must also
be applied.
Sera
The laboratory must have a policy regarding the selection of relevant sera to be used in the
final crossmatch procedure
Final crossmatches performed prior to transplantation
must use a recipient serum sample collected within the previous 48 hours before
transplant if the recipient has had a recent sensitising event.
must use the most recent available serum collected as defined in standard G2.3
should use sera obtained 14 days after a potentially sensitising event.
HLA Typing
Prospective typing of donor and recipient:
Must include typing for HLA-A, B and DR antigens.
Must include additional loci if required by national regulations.
Every effort must be made to perform verification typing for recipients prior to
transplantation.
Verification typing must be performed on living donors prior to transplantation.
G4.7
G4.7.1
G4.7.2
G4.7.3
G5
G5.1
G5.1.1
G5.1.2
G5.2
G5.3
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Recom.*
Yes No
.....
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.....
....
SECTION H – OTHER ORGAN TRANSPLANTATION
H1
H2
H2.1
H2.2
H2.3
In cases where patients are at high risk for allograft rejection (e.g. patients with histories of
allograft rejection, patients with preformed HLA antibodies), donors and recipients must be
typed for HLA-A, B and DR antigens.
Cardiothoracic patients must be screened for the presence of HLA alloantibodies, and
Unacceptable specificities must be defined, or
A prospective crossmatch must be performed
Standards G2 (Antibody Screening) also applies
H3
H3.1
H3.1.1
Crossmatching
Crossmatching must be performed according to standards in F5 (Crossmatching).
Sera from patients at high risk for allograft rejection should be prospectively crossmatched.
H3.1.2
H3.2
H3.2.1
Crossmatch results should be available prior to transplantation of a pre-sensitised patient.
Final crossmatches performed prior to transplantation should either:
utilise a recipient serum sample collected within the previous 48 hours before
transplant if the recipient has had a recent sensitising event. Or,
utilise a serum collected within three months
Sera obtained 14 days after a potential sensitising event should be used in the final crossmatch.
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Whenever possible, non-renal organs for recipients at high risk for allograft rejection should
come from cross-match negative donors as defined by the laboratory and the transplant
programme.
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H3.2.2
H3.3
H3.4
16
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Required
Yes
No
EFI Standard
© EFI 2014
SECTION I – HAEMATOPOIETIC STEM CELL TRANSPLANTATION
I1
I1.1
I1.2
I1.3
I2
I2.1
I2.2
I2.2.1
I2.2.2
I2.2.3
I2.3
I3
I3.1
I3.2
I3.3
I3.3.1
I3.3.2
I3.4
I4
I4.1
I4.1.1
I4.1.1.1
I4.1.1.2
I4.2
I4.2.1
I4.2.2
I4.2.3
HLA typing
If required by the transplant protocol, the laboratory must be able to type the donor and the
recipient for HLA Class I and HLA Class II by DNA methods, to a level of resolution as defined
in standard D1.4.
Laboratories performing transplantation using related donors who are not able to perform
high resolution Class I and/or Class II typing by DNA methods must arrange for an EFI or ASHI
accredited laboratory to perform these tests.
Laboratories performing transplantation using unrelated donors who are not able to perform
high resolution Class I typing by DNA methods must arrange for an EFI or ASHI accredited
laboratory to perform these tests.
Histocompatibility testing for related transplants
HLA-A, B or DR typing must be carried out on available members of the immediate family
Must include adequate testing
to definitively establish HLA genotype identity (D3.2 applies), or
to type at high resolution for the relevant loci defined in the transplant protocol, if only
phenotype identity has been established, or
to include high resolution typing for recipient and potential intra-familial donors who
are not HLA identical siblings
HLA-A, B and DR typing as a minimum requirement must be repeated on both the recipient
and the potential donor prior to transplantation using a new typing sample from each, so
that each individual’s typing is confirmed.
HLA typing for Donors (related cord blood unit)
The cord blood unit must be typed using DNA methods for HLA-A, B and DRB1 at a minimum
of low resolution (eg. A*02, B*44, DRB1*11)
Extended typing must be included if required by the transplant protocol (standards I2.1, I2.2
and I2.3 also apply)
Prior to transplantation, a verification typing:
Must be performed for HLA-A, B and DRB1 at a minimum of low resolution
Must be performed on a segment of the tubing integrally attached to the unit, if
available, or otherwise, on a satellite vial.
If verification typing was not performed on a segment of the tubing integrally attached, the
laboratory must recommend that an additional typing is performed on the content of the
thawed unit.
Histocompatibility Testing for Unrelated Transplants
Volunteer Bone Marrow Donor Registries
Typing of the donors must be performed
By serology or
By DNA methods at a minimum of low resolution (eg A2 or A*02, DR11 or DRB1*11)
Typing of Units for Cord Blood Banks
Typing must be performed using DNA methods for HLA-A, B and DRB1, at a minimum of low
resolution (e.g. A*02, B*44, DRB1*11).
Typing of additional loci or high resolution typing must be included if required by the policy
of the registry, or if requested.
The identity of the Cord Blood Unit must be verified by HLA typing on a separate sample to
demonstrate concordance of results.
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17
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
I4.2.4
I4.2.5
I4.3
I4.3.1
I4.3.1.1
I4.3.1.2
I4.3.1.2.1
I4.3.1.3
I4.3.1.4
I4.3.1.5
I4.3.2
I4.3.2.1
I4.3.2.2
I4.3.2.3
I4.3.3
I4.4
I4.4.1
I4.4.1.2
I4.4.1.2.1
I4.4.1.2.2
I4.4.1.2.3
I4.5
I4.5.1
I4.5.1.1
I4.5.1.2
I4.5.1.3
I4.5.2
I5
I5.1
I5.1.1
I5.1.2
I6
I6.1
I6.2
I6.3
I6.4
I6.4.1
I6.4.2
Additional typing may be performed using any stored DNA sample, provided that the
identity of the unit has previously been verified.
The verification of identity and the source of the sample tested must be reported back to the
registry.
Histocompatibility Testing for Transplants from Unrelated Donors.
HLA typing for recipient and unrelated donors must:
Be performed by DNA based methods
Include as a minimum requirement:
HLA-A/B/C and DRB1 typing at high resolution
Include additional loci if required by the transplant protocol.
Include other resolution levels if required by the transplant protocol
Be performed by the laboratory affiliated with the transplant centre
Prior to transplantation using an unrelated donor, HLA typing of the recipient and donor
must be repeated for verification:
by the laboratory affiliated with the transplant centre
using a different typing sample
for HLA-A, -B, and -DR, as a minimal requirement
For unrelated donors HLA-A,-B,-DR concordant results are required on two separate samples.
Registry typing is acceptable as one of the two required results.
Unrelated Cord Blood Unit Typing for Donor Selection
Verification typing must be performed
Including as a minimum requirement
HLA-A and -B at low resolution, and
HLA-DRB1 at high resolution
Extended typing if required by the transplant protocol
Unrelated Cord Blood Unit Typing Prior to Transplantation
Prior to the conditioning regimen of the recipient, a verification typing must be performed
at a minimum level of low resolution for HLA-A, -B, and -DRB1
upon reception of the shipped unit
on a segment of the tubing integrally attached to the unit, if available; otherwise a
satellite vial shipped with the unit may be used.
If no segment is available, this step can be performed after transplantation and must be
initiated as soon as possible after thawing the unit.
Crossmatching
Crossmatching must be performed
prior to related and unrelated transplantation if required by the local transplant
protocol
according to standards F5 (Crossmatching)
Haemopoietic Chimaerism and Engraftment (HCE) Monitoring
Standards L1, L5.1, L5.3, L6, L7.1 and L7.2 also apply.
The polymorphic gene system(s) used for HCE monitoring must be identified and
documented with regards to allelic variability
The sensitivity of the HCE assay must be validated using DNA mixtures from two individuals
at defined ratios/concentrations, before implementation into clinical use.
For locally developed PCR primers/probes the following must be documented
Sequence
Specificity
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18
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
....
Required
Yes
No
EFI Standard
© EFI 2014
I6.5
I6.5.1
I6.5.2
I6.6
I6.6.1
I6.6.2
I6.6.3
Donor and patient specific allele profiles must be
determined using appropriate reference material
Documented
Optimal ranges of DNA quantity and purity must be:
Defined
Documented
If a sample falls outside these optimal ranges, a statement must be included in the
report.
Criteria for assignment of HCE results, on a qualitative or quantitative basis, must be
defined.
When multiple PCR primers are used in the same tube (multiplex PCR), results must take into
account possible amplification bias
When HCE testing is performed on cellular subsets isolated by cell sorting, the purity of the
sorted population:
must be documented and
taken into account in the analysis of the results
If this is not possible it must be clearly stated in the report.
For quantitative HCE monitoring by quantitative PCR (Q-PCR), the following must be defined
Chemistry used
internal control gene
thresholds for positive and negative results of each reaction.
All steps of locally developed Q-PCR assays must be validated.
In addition to the requirements from standard C11.5.7, the report must contain
a description of the specimen used for testing (bone marrow, peripheral blood,
cellular subsets isolated by cell sorting etc)
the date of transplant
other information if deemed relevant for HCE interpretation (i.e. limited informative
markers or clinical condition of the patient)
I6.7
I6.8
I6.9
I6.9.1
I6.9.2
I6.9.3
I6.10
I6.10.1
I6.10.2
I6.10.3
I6.11
I6.12
I6.12.1
I6.12.2
I6.12.3
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SECTION J – HLA / HPA / HNA AND TRANSFUSION
J1
J1.1
J1.2
J1.3
J2
J2.1
J2.1.1
J2.1.1.1
J2.1.1.2
J2.1.2
J2.1.2.1
J2.1.2.2
J2.1.3
J2.1.4
Documented protocols for testing each of the following must be provided:
HLA
Human Platelet Antigens (HPA)
Human Neutrophil Antigens (HNA)
HLA and Transfusion
Platelet refractoriness
Platelet refractory patients who require HLA matched platelets
must be typed for HLA-A and HLA-B
If alloimmune refractoriness is suspected the patient must be tested for HLA class I
antibodies.
To provide compatible platelets, either:
The specificity of detected HLA antibodies must be defined and recorded, or
crossmatching must be performed
For crossmatching using lymphocytes standards F5 (Crossmatching) must be followed.
All selected plateletpheresis donors used for the provision of HLA matched platelets must be
typed for HLA-A and HLA-B.
19
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
J2.2
J2.2.1
Transfusion Related Acute Lung Injury (TRALI).
For the laboratory investigation of TRALI, the sera from implicated donors must be tested for
both HLA class I and class II antibodies.
The specificity of detected HLA antibodies must be defined and recorded.
J2.2.2
J2.2.3
J2.2.3.1
J2.3.3.2
J2.3
J2.3.1
J2.4
J2.4.1
J3
J3.1
J3.2
J3.2.1
J3.2.2
J3.3
J3.3.1
J3.3.2
J3.3.3
J3.3.4
J3.3.4.1
J3.3.4.2
J3.3.4.3
J3.3.5
J3.4
J3.4.1
J3.4.2
J3.5
J3.5.1
J3.5.2
J4
J4.1
J4.2
J4.2.1
J4.2.2
If HLA specific antibodies are identified, HLA typing must be performed at least for all
relevant loci on
The patient
The donor
Transfusion Associated Graft versus Host Disease (TAGVHD)
The patient and the donor must be typed for HLA-A, HLA-B, and HLA-DR.
Febrile Non Haemolytic Transfusion Reactions (FNHTR)
The patient’s serum must be tested for the presence of HLA antibodies
HPA and Transfusion
Current HPA Nomenclature (Platelet Nomenclature Committee; Vox Sanguinis 2003 85, 240245) must be used for recording and reporting HPA alloantigen and HPA alloantibody
specificities.
HPA Typing
HPA typing must be performed using a validated HPA typing technique. If typing is
performed using DNA based methods, standards in section L (Nucleic Acid Analysis) apply.
Clinically significant HPA specificities must be defined and documented.
Investigation of HPA antibodies
For bead array techniques, relevant standards from section M (Flow Cytometry) also apply
For ELISA based assays, standards in section N (ELISA) also apply.
Laboratories must make all reasonable efforts to include HPA antigens in their antibody
screening protocol which will aid the identification of clinically significant HPA alloantibodies.
The antibody screening technique must
be validated before use
Include positive and negative controls in each assay
In glycoprotein specific assays, a positive control for each glycoprotein used should
be included.
The specificity of detected HPA alloantibodies must be defined and recorded.
Neonatal Alloimmune Thrombocytopenia (NAIT)
The maternal serum must be investigated for the presence of HPA antibodies
HPA typing of the mother, father and neonate should be performed
Post Transfusion Purpura (PTP)
The patient must be HPA typed
The patient’s serum must be investigated for HPA antibodies
HNA and Transfusion
Current HNA Nomenclature (ISBT Working Party; Vox Sanguinis 2008 94, 277-285) must be
used for recording and reporting HNA alloantigen and HNA alloantibody specificities.
HNA Typing
HNA typing must be performed using a validated HNA typing technique. If typing is
performed using DNA based methods, standards in section L (Nucleic Acid Analysis) apply.
The clinically significant HNA specificities must be defined and documented.
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20
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
.....
....
.....
....
Required
Yes
No
EFI Standard
© EFI 2014
J4.3
J4.3.1
J4.3.5
J4.4
J4.4.1
Investigation of HNA Antibodies
For bead array and flow cytometry techniques, relevant standards from section M (Flow
Cytometry) also apply
For ELISA based assays, standards in section N (ELISA) also apply.
Laboratories must make all reasonable efforts to include HNA antigens in their antibody
screening protocol which will aid the identification of clinically significant HNA alloantibodies
The antibody screening technique must
be validated before use
Include positive and negative controls in each assay
In glycoprotein specific assays, laboratories must make all reasonable efforts to
include a positive control for each glycoprotein used.
The specificity of detected HNA alloantibodies must be defined and recorded.
Neonatal Alloimmune Neutropenia (NAIN)
The maternal serum sample must be investigated for the presence of HNA antibodies.
J4.4.2
HNA typing of the mother, father and neonate should be performed.
J4.3.2
J4.3.3
J4.3.4
J4.3.4.1
J4.3.4.2
J4.3.4.3
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Recom.*
Yes No
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SECTION K – DISEASE ASSOCIATION
K1
The following standards apply:
K1.1
If complete HLA typing is performed by serology standards in section E (Serological HLA Class
I and Class II typing) must be followed.
Typing may also be limited to all products of a single or limited number of HLA antigens,
alleles or loci.
If HLA typing is performed by DNA techniques standards in section L (Nucleic Acid Analysis)
must be followed
The clinically relevant HLA loci and alleles must be documented for each disease association
service provided.
Typing for a single antigen by CDC
Typing Reagents
Sera to define each antigen must meet requirements of section E (Serological HLA Class I
and Class II typing) as appropriate.
Cell Controls must:
Be tested on each batch
Include at least two cells known to express the specified antigen
Include at least two cells for each cross reacting antigen, which might be confused
with the specific antigen
Include at least two cells lacking the specific and cross reacting antigens
Serum Controls must
Be tested at the time of typing
include a positive and negative control
Serum controls should also include two sera for each antigen which cross reacts with the
specified antigen (if available)
Typing for a single allele-group by molecular techniques:
a positive control DNA known to encode the allele-group of interest must be
included in each test.
K1.2
K1.3
K1.4
K2
K2.1
K2.1.1
K2.2
K2.2.1
K2.2.2
K2.2.3
K2.2.4
K2.3
K2.3.1
K2.3.2
K2.3.3
K3
K3.1
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21
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
…...
Required
Yes
No
EFI Standard
© EFI 2014
K3.2
a negative control DNA known not to encode an allele belonging to the allele-group
of interest must be included in each test.
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Recom.*
Yes No
SECTION L – NUCLEIC ACID ANALYSIS
L1
General laboratory design
L1.1
Laboratories performing amplification of nucleic acids must use:
L1.1.2
L1.1.2.1
L1.1.2.2
L1.1.2.3
L1.2
Physical barriers to prevent DNA contamination, including the use of dedicated
Equipment
laboratory coats
Disposable supplies
Pre-amplification procedures must be performed in an area which excludes amplified DNA
that has the potential to serve as a template for amplification in any of the genetic systems
tested in the laboratory.
All activities occurring from and including thermal cycling must take place in the postamplification area.
For methods that use two consecutive steps of logarithmic amplification, the addition of the
template for subsequent amplifications:
must occur in an area isolated by physical or chemical barriers from both the preand post-amplification work areas
must use dedicated equipment and consumables.
Equipment
Accuracy of thermal cycling instruments:
must be verified by maintenance according to the manufacturer, or
must be verified by annual thermal verification of the block using a calibrated device
designed specifically for this purpose.
Incubators and water baths must be monitored for accurate temperature every time an
assay is performed.
Reagents
All reagents (solutions containing one or multiple components) must either be:
dispensed in aliquots for single use, or
dispensed in aliquots for multiple use if documented to be free of contamination at
each use
When reagents are combined to create a master mix, one critical component (e.g. DNA
polymerase) should be left out of the mixture.
The appropriate performance of individual products must be documented before results
using these reagents are reported for:
each shipment, and
each lot
For commercial kits, the following information must be documented:
Source
lot number
expiry date
storage conditions
L1.3
L1.4
L1.4.1
L1.4.2
L2
L2.1
L2.1.1
L2.1.2
L2.2
L3
L3.1
L3.1.1
L3.1.2
L3.2
L3.3
L3.3.1
L3.3.2
L3.4
L3.4.1
L3.4.2
L3.4.3
L3.4.4
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22
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
....
Required
Yes
No
EFI Standard
© EFI 2014
L3.5
L3.5.1
L3.5.2
L4
L4.1
L4.2
L4.2.1
L4.2.2
Reagents from different lots of commercial kit must not be mixed, unless either:
specified by the manufacturer, or
validated and documented with appropriate quality control in the laboratory
Primers
The specificity of primer combinations and the annealing positions must be defined.
Laboratories must:
have a policy for quality control of each lot or shipment of primers
Confirm the specificity and quantity of the amplified product using reference
material.
test each lot and shipment of commercial kits against at least one DNA sample of
known type.
Use primers under empirically determined conditions that achieve the defined
specificity for templates used in routine testing.
Test each lot of local primers for amplification specificity and quantity using
reference material whenever available.
Test each lot of local primers with reference DNA for appropriate sensitivity and
specificity.
Nucleic acid extraction:
The method used for nucleic acid extraction:
must be published and documented
must be validated in the laboratory
Purity and concentration of Nucleic Acids:
must be sufficient to ensure reliable test results.
should be determined for each sample, or
if not determined for each sample, the laboratory must have tested and validated
this policy.
If the DNA is not used immediately after purification, suitable methods of storage must be
available that will protect the integrity of the material.
Electrophoresis
optimal electrophoretic conditions must be documented
The laboratory must establish criteria for accepting each slab or capillary gel migration, and
each lane of a gel or capillary injection.
When the size of an amplicon is a critical factor in the analysis of data, size markers that
produce discrete electrophoretic bands spanning and flanking the entire range of expected
fragment sizes must be included in each gel.
Analysis
Signal intensity
Acceptable limits of signal intensity must be specified for positive and negative results
If these are not achieved, acceptance of the results must be justified and documented.
The method of allele assignment must be designated
The allele database must be:
Documented
Updated at least once a year with the most current version of the IMGT/HLA
database
L4.2.3
L4.2.4
L4.2.5
L4.2.6
L5
L5.1
L5.1.1
L5.1.2
L5.2
L5.2.1
L5.2.2
L5.2.3
L5.3
L6
L6.1
L6.2
L6.3
L7
L7.1
L7.1.1
L7.1.2
L7.2
L7.3
L7.3.1
L7.3.2
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23
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
....
Required
Yes
No
EFI Standard
© EFI 2014
L7.4
If a manual allele call or interpretation of positive/negative reactions is performed for SSOP
or SSP, two independent interpretations of primary data must be performed, except under
justified special emergency situations.
Databases of HLA sequences used to interpret the primary data must be:
documented
accurate
updated at least once a year with the most current version of the IMGT/HLA
database
must be archived or a record retained according any regulation the laboratory is
obliged to abide, but for a minimum of four years.
Contamination control (″wipe-test″)
Contamination must be monitored for amplification products produced in the laboratory.
Routine wipe-tests must:
include pre-amplification work areas
include pre-amplification equipment
be performed at least every two months
be performed using a method that is at least as sensitive as routine test methods.
include positive controls to assure proper performance of monitoring
include other areas of the laboratory as relevant
If amplified product is detected, there must be:
written description of how to eliminate the contamination
measures taken to prevent future contamination
evidence of elimination of the contamination
Typing using sequence-specific primers (SSP)
Each amplification reaction must include controls to detect technical failures (e.g. an internal
control such as additional primers or templates that produce a product that can be
distinguished from the typing product).
When a typing exhibits lanes with no specific amplicon or internal control amplification, the
laboratory must have a policy in place on how to accept or reject the whole typing.
The laboratory must use the following data in the interpretation phase of the typing:
information derived from the validation process
information derived from previous typings with the same lot of primers
The following must be defined and documented:
Non-specific and weak amplifications
Any tendency to form primer-dimer
Sequence-Based typing (SBT)
Sequencing Templates:
must have sufficient purity, specificity, quantity and quality to provide interpretable
sequencing data.
Should be purified after amplification to eliminate the presence of dNTPs, Taq
polymerase and amplification primers.
must not contain any inhibitors or contaminants affecting the sequencing reaction.
Validation of the methods for template preparation must ensure that the accuracy
of the final typing is not altered (e.g. mutations during cloning, preferential
amplification).
L7.5
L7.5.1
L7.5.2
L7.5.3
L7.5.4
L8
L8.1
L8.2
L8.2.1
L8.2.2
L8.2.3
L8.2.4
L8.2.5
L8.2.6
L8.3
L8.3.1
L8.3.2
L8.3.3
L9
L9.1
L9.2
L9.3
L9.3.1
L9.3.2
L9.4
L9.4.1
L9.4.2
L10
L10.1
L10.1.1
L10.1.2
L10.1.3
L10.1.4
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24
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
…...
…...
....
Required
Yes
No
EFI Standard
© EFI 2014
L10.1.5
L10.2
L10.2.1
L10.2.2
L10.2.3
L10.3
L10.3.1
L10.3.2
L10.3.3
L10.3.4
L10.4
L10.4.1
L10.4.2
L10.4.3
L10.4.4
L10.4.4.1
L10.4.4.2
L11
L11.1
L11.1.1
L11.1.2
L11.2
L11.2.1
L11.2.2
L11.2.3
L11.2.4
L11.2.5
If cloning is used as template preparation, the sequence of at least three different
clones for each allele must be determined for accurate results
Sequencing Reaction:
The specificity of the template in combination with the sequencing primer (HLA
locus and alleles) must be defined.
Quantity and quality of templates, sequencing primers and sequencing reagents
must be sufficient to provide interpretable primary sequencing data.
The conditions for the sequencing reaction must be documented and appropriate
for obtaining reliable primary sequencing data.
Nucleotide Assignment:
The following criteria for acceptance of primary data must be established (peak
intensity, baseline fluctuation, Peak shape, correct assignment for non-polymorphic
positions)
The signal to noise ratio must be sufficient to ensure reliable nucleotide
assignments
A scientific and technically sound method must be established for interpretation,
acceptance and/or rejection of sequences from regions which are difficult to resolve
(e.g. compression).
Established sequence-specific characteristics should be documented and utilized in
routine interpretation of data.
Allele assignment
Methods must ensure that sequences contributed by amplification primers are not
considered in the assignment of alleles.
Criteria for allele assignment must be established.
Established sequence-specific artefacts must be documented and the information
used in the routine interpretation of data.
Uni- and bi-directional sequencing
If allele assignments are difficult to obtain by sequencing only one strand, routine
sequencing of both strands should be performed
If a sequence suggests a novel allele or a rare combination of alleles, the sequences
of both strands must be determined
Sequence-Specific Oligonucleotide Probe (SSOP) hybridization assays
Oligonucleotide probes
The specificity of each probe and target sequence must be defined.
Probes must be stored under conditions that maintain specificity and sensitivity
Quality Control
Laboratories must have a policy in place for quality control of each lot and shipment
of probes
For home-made kits each lot must be tested with reference DNA so that each probe
is tested for specificity and signal intensity at least once.
For commercial kits each lot and shipment must be tested in parallel against at least
one DNA sample of known type.
The specificity and signal intensity for each probe must be defined and monitored.
Probes must be utilised under empirically determined conditions that achieve the
defined specificity.
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25
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
.....
....
.....
....
Required
Yes
No
EFI Standard
© EFI 2014
L11.2.6
L11.3
L11.3.1
L11.3.2
L11.3.2.1
L11.3.2.2
L11.3.2.3
L11.3.2.3.1
L11.3.2.3.2
L11.4
L11.4.1
L11.4.2
L11.4.2.1
L11.4.3
L11.4.3.1
L11.4.3.2
L11.5
L11.5.1
L11.5.1.1
L11.5.1.2
L11.5.1.3
L11.5.2
L11.5.2.1
L11.5.2.2
L11.6
L11.6.1
L11.6.2
L11.6.3
L11.6.4
L12
L12.1
L12.1.1
For commercial kits, any deviation from the manufacturer’s specifications must be
validated and documented.
Hybridization
The amplification should be monitored by gel electrophoresis before the hybridization is
performed
Each assay must include:
a probe internal to a conserved region of the amplified fragment
appropriate controls to validate the hybridization and the detection steps
a negative (no DNA) control :
that must be included in the hybridization and revelation steps of the assay
in forward SSOP assays
that must either be included in the hybridization and detection step of the
assay or monitored by gel electrophoresis in reverse SSOP assays
Equipment
Standard L2.2 must be followed for the incubators, water baths and for heated reagents.
For automated hybridization devices:
the calibration of the pumps and of the heating elements must be performed
according to the manufacturer’s specification at least once a year
For tests using an ELISA washer:
calibration must be performed at least annually according to the manufacturer’s
specifications
monthly functional checks of dispensing/aspirating must be performed.
Where a scanner is used for acquisition of the raw data, a second visual reading must be
performed to confirm data.
For automated systems for the acquisition of the primary data
all critical elements influencing the function of the instrument must be monitored at
each use
The instrument must be calibrated according to manufacturer's instructions or at
least once a year
The laboratory must define and document functional checks.
For flow cytometer-like devices, there must be evidence of:
regular cleaning
satisfactory calibration functions performed prior to use
Interpretation
Acceptable limits of signal intensity must be specified for positive and negative results.
If a test is accepted with probe signals outside the set limits, this must be documented and
justified.
The laboratory must use the data derived from the validation process and from previous
typings with the same lot of primers and probes in the interpretation phase of the typing.
Nonspecific and weak hybridization signals must be defined and documented.
Other Methods
If alternative methods (e.g. SSCP, heteroduplex, DGGE) are used for HLA typing, there must
be established procedures in place which
must be validated
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26
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
....
Required
Yes
No
EFI Standard
© EFI 2014
L12.1.2
must include sufficient controls to ensure accurate assignment of types for every
sample
must comply with all relevant standards from section L (Nucleic Acid Analysis)
L12.1.3
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Recom.*
Yes No
SECTION M – FLOW CYTOMETRY
M1
M1.1
M1.2
M2
M2.1
M2.1.1
M2.1.2
M2.1.3
M2.1.4
M2.1.5
M2.1.5.1
M2.1.5.2
M2.2
M2.2.1
M2.2.1.1
M2.2.1.2
M2.2.2
M2.2.3
M2.2.4
M2.3
M2.3.1
M2.3.1.1
M2.3.1.2
M2.3.1.3
M2.3.1.4
M2.3.1.5
Application
Standards M2 (General instrument standardisation and maintenance) applies to flow
cytometry and flow analysis using equipment designed for beads only (fluoro-analyser)
Standards M2 (General instrument standardisation and maintenance) to M5 (Cell-based
HLA typing by flow cytometry) apply to flow cytometry
General Instrument standardisation and maintenance
For instruments that perform an automated integrated multi-parameter standardisation
(eg Luminex):
This function may be used instead of the individual optical alignment and fluorescence
standardisation described in standards M2.2 and M2.3.
The reagents specified by the manufacturer to perform this test must be used.
The result of the standardisation must be recorded.
The instrument must only be used if the test has passed.
The frequency of standardisation
must conform to manufacturer’s instructions and must be performed at any time
that the temperature delta check is not correct, or
must be performed as specified in standards M2.2 o M2.2.4 if there are no
manufacturer’s instructions
Optical Standardisation
The optical standard must be run:
every day of instrument use unless otherwise specified by the manufacturer
Any time maintenance or adjustment of the instrument during operation is likely to
have altered optical alignment.
The optical standard must consist of latex beads or other uniform particles
A threshold value for acceptable optical standardisation must be established for all relevant
signals
The results of optical focusing / alignment must be recorded and fall within the defined
acceptable range.
Fluorescence standardisation
The fluorescence standard:
Must be run every day of instrument use unless otherwise specified by the
manufacturer
Must be run any time maintenance or adjustment of the instrument during operation is
likely to have altered settings.
Must be used for each fluorochrome employed in analytical procedures
The results of fluorescence standardisation must fall within the defined acceptable
range.
The results of fluorescence standardisation must be recorded
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27
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
....
Required
Yes
No
EFI Standard
© EFI 2014
M2.3.2
M2.3.2.1
M2.3.2.2
M2.3.2.3
M2.3.2.4
M2.3.2.5
M2.3.2.5.1
M2.3.2.5.2
M2.4
M2.4.1
M2.4.1.1
M2.4.1.2
M2.4.1.3
M2.4.2
M2.4.2.1
M3
M3.1
M3.1.1
M3.1.2
M3.2
M3.2.1
M3.2.2
M3.3
M3.4
M3.5
M3.6
M4
M4.1
M4.1.1
M4.1.2
Compensation
If performing analyses that require the simultaneous use of two or more
fluorochromes, an appropriate procedure to compensate for overlap in their emission
spectra must be used.
Compensation settings must be determined every day of use, and
at any time maintenance or adjustment of the instrument during operation is likely to
have altered them
Compensation must be carried out for all fluorochromes used.
Compensation values
Acceptable compensation values must be defined
The values used must be recorded
Equipment Cleaning and Maintenance
Cleaning
There must be a procedure for regular cleaning of the instrument
The frequency and protocol for cleaning must conform to manufacturer’s instructions,
if available
Cleaning must be documented
Maintenance
The instrument must be maintained according to manufacturer’s instructions, but at
least once a year.
General Reagents
Specificity of labelling reagents for identification of cell subsets:
The specificity of labelling reagents must be verified using a published method and/or
the manufacturer's documentation and/or by local documented quality control testing.
If locally defined, the specificity of labelling reagents must be verified using appropriate
control cells, prepared and tested by the same method employed in the laboratory’s
test sample analysis
Secondary labelling reagents:
must be titrated to determine the dilution with optimal activity (signal to noise ratio).
If a multicolour technique is employed, the reagent must not cross-react with the other
immunoglobulin reagents used to label the cells.
Reagents which have been reconstituted from lyophilised powder must be centrifuged
according to the manufacturer’s instructions or locally documented procedures to remove
micro aggregates prior to use.
Each lot and shipment of labelling reagents must be tested for proper performance.
Thresholds for adequate intensity must be defined and documented.
The quantity of reagents used for each test sample must be determined by the
manufacturers or from published data and verified locally by appropriate titration
procedures.
Antibody screening and crossmatching
Cell based testing
Labelling of target cells
An individual fluorochrome must be used for the identification of each population
subset (multicolour technique).
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28
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
M4.1.3
M4.1.3.1
M4.1.3.2
M4.1.4
M4.1.4.1
M4.1.4.2
M4.1.4.3
M4.1.5
M4.2
M4.2.1
M4.3
M4.3.1
M4.3.1.1
M4.3.1.2
M4.3.1.3
M4.3.2
M4.3.2.1
M4.3.2.2
M4.3.2.3
M4.3.3
M4.4
M4.4.1
M4.4.2
M4.4.2.1
M4.4.2.2
M4.4.2.3
M4.4.2.4
M4.4.3
M4.4.3.1
M4.4.3.2
M4.4.4
M4.4.4.1
M4.4.4.2
If a single colour technique is used, the purity of the isolated cell population
must be sufficient to define the population for analysis
must be documented
The target sub-populations analysed
Must be defined
Must include a sufficient number of acquired events per sub-population, relevant
to the test performed
Must be identified by appropriate labelling antibodies
The binding of human immunoglobulin on target cells must be assessed with a
fluorochrome labelled F(ab') anti-human IgG specific for the Fc region of the heavy
chain
Cell and bead based antibody screening
For the detection of anti-HLA antibodies or assignment of antibody specificity, the
composition of the panel must conform to the standards in section F4 (Panels)
Controls
Negative control
A negative control must be used, which must be
A serum from non-alloimmunised human donor(s), and
screened and found to be negative by flow cytometry.
Positive control
A positive control must be used, which must be a human serum,
specific for HLA antigens
of the appropriate isotype
Control sera must be tested at the same time and under the same conditions as the sera
under test.
Procedures and policies
There must be policies and procedures to address at least:
Antibody screening and crossmatching technical instructions, including:
Reagent standardisation and optimisation
Reagent validation
Incubation times
Incubation temperatures
Interpretation instructions must include details of:
The threshold for significant levels of antibody binding (positivity)
The mechanism for reporting positive results (mean, mode or median
channel shifts, relative mean fluorescence, or number of molecules of
fluorescent marker)
Acceptable reactivity required for negative, positive and secondary control
reagents,
in order for the test to be valid must be
defined
documented
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29
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
Required
Yes
No
EFI Standard
© EFI 2014
M5
M5.1
M5.1.1
M5.1.2
M5.1.3
M5.1.4
M5.1.5
M5.1.6
M5.2
M5.2.1
M5.2.2
M5.2.2.1
M5.2.2.2
M5.2.3
M5.2.3.1
M5.2.3.1.1
M5.2.3.1.2
M5.2.3.2
M5.3
M5.3.1
M5.3.2
M5.3.2.1
M5.3.2.2
M5.3.2.3
M5.3.2.4
M5.3.3
M5.3.3.1
M5.3.3.2
M5.3.3.3
Cell-based HLA typing by flow cytometry (eg HLA B27)
Labelling reagents for the identification of HLA specificities
The specificity of each lot and shipment must be determined by testing:
at least five cells known to express the target antigen
at least two cells for each cross-reacting antigen
at least two cells which lack the specific and cross-reacting antigens
Acceptable criteria for validation must be defined
and results must be recorded.
Each lot and shipment of labelling reagents must be shown to have comparable
reactivity to the previously validated lot and shipment
Controls
Controls for HLA typing by flow cytometry must be run for each test cell preparation
Negative Controls
For direct labelling, a negative control must be conjugated with the same
fluorochrome as the test.
For indirect labelling, a negative control should be used in conjunction with the
same secondary antibody conjugated with the same fluorochrome as used for the
specific antibody under test.
Positive Controls
Must include a pan-reacting anti-HLA monoclonal antibody, which
Must be tested against each cell
Must be tested under the same conditions as the antibodies under test
A control cell known to express the HLA specificity under test must be included in each run.
Policies and Procedures
There must be policies and procedures to address at least:
HLA typing technical instructions including:
Reagent standardisation and optimisation
Reagent validation
Incubation times
Incubation temperatures
Interpretation instructions must define:
The required reaction criteria in the negative and positive control samples for the
test results to be valid.
Criteria for positivity of the HLA antigen under test
A documented procedure must be followed for monoclonal antibodies which react with
antigens other than those expected.
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SECTION N – ENZYME-LINKED IMMUNO SORBENT ASSAY (ELISA
N1
N1.1
N1.1.1
Equipment
ELISA Reader
The light source must produce the intensity and wavelength of light required for the test
system
Periodic calibration must be performed according to the manufacturer’s instructions
The result of the calibration must be documented
N1.1.2
N1.1.3
30
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
....
Required
Yes
No
EFI Standard
© EFI 2014
N1.2
N1.2.1
N1.2.2
N1.2.2.1
N1.2.2.2
N2
N2.1
N2.1.1
N2.1.2
N2.2
N2.3
N2.4
N2.5
N3
N3.1
N3.2
N3.3
N3.4
N3.4.1
N3.4.2
N3.5
N3.5.1
N3.5.2
N3.6
N3.6.1
N4
N4.1
Microplate Washer
The performance of the microplate washer must be checked at least monthly.
The result of the performance test must be
Acceptable
Documented
ELISA Reagents
The dilution of reagents, controls and test specimens must be
Defined
Documented
Sera must be tested at a concentration determined to be optimal for the detection of
antibody to HLA antigens with the test system used.
Commercial kits must be used according to the manufacturer’s instructions, or
The laboratory must perform and document testing to support a deviation in the technique
or analysis.
Each lot of reagents must be validated and shown to have comparable reactivity to a
previously validated lot
Quality Management and Controls
Sample identity and proper plate orientation must be maintained throughout the procedure.
The lot numbers and optical density values for the reference reagents, controls and test
samples must be recorded for each assay.
The test results must meet defined criteria for reference reagents and controls in order for
the test to be valid
Negative Control
A negative control must be included in each assay, and
Must include a serum from a non-alloimmunised human donor(s)
Positive Control
A positive control must be included in each assay and
must be a human serum specific for HLA antigens and of the appropriate isotype.
Reagent Controls
A control lacking only HLA antigen must also be included in each assay.
Panels
For the detection of antibodies or the assignment of antibody specificity, the composition of
the cell panel must conform to the standards in section F4.
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31
* Recommended Standard
Inspection checklist according to the Standards version 6.2 Valid from 1st October 2014. This checklist replaces all previous versions.
Signatures:
Recom.*
Yes No
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