Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
B2.000
Reason for Modification
v5.7
A Director/Co-Director must hold an
earned doctoral degree in a biological
science, or be a physician, or have an
equivalent qualification. In addition, the
Director/Co-Director 1) must have had four
years experience in immunology or cell
biology, two of which were devoted to full
time training in human histocompatibility
testing, or 2) five years of working
experience at full time in human
histocompatibility
testing.
Additional
qualifications required according to national
legislation also apply. The Director/CoDirector must have documentation of
professional competence in the appropriate
activities in which the laboratory is
engaged. This should be based on a sound
knowledge of the fundamentals of
immunology,
genetics
and
histocompatibility testing and reflected by
external measures such as participation in
national or international workshops or
publications in peer-reviewed journals. The
Director or Co-Director is available on site
at least 20h/week, provides adequate
supervision of technical personnel, utilises
his/her special scientific skills in developing
new procedures and is held responsible for
the proper performance, interpretation and
reporting of all laboratory procedures and
the laboratory's successful participation in
proficiency testing. The Director/CoDirector must be informed of with the
relevant national legislation.
A Director/Co-Director must hold an
earned doctoral degree in a biological
science, or be a physician, or have an
equivalent qualification. In addition, the
Director/Co-Director 1) must have had four
years experience in immunology or cell
biology, two of which were devoted to full
time training in human histocompatibility
testing, or 2) five years of working
experience at full time in human
histocompatibility
testing.
Additional
qualifications required according to national
legislation also apply. The Director/CoDirector must have documentation of
professional competence in the appropriate
activities in which the laboratory is
engaged. This should be based on a sound
knowledge of the fundamentals of
immunology,
genetics
and
histocompatibility testing and reflected by
external measures such as participation in
national or international workshops or
publications in peer-reviewed journals. The
Director or Co-Director is available on site
at least 20h/week, provides adequate
supervision of technical personnel, utilises
his/her special scientific skills in developing
new procedures and is held responsible for
the proper performance, interpretation and
reporting of all laboratory procedures and
the laboratory's successful participation in
proficiency testing. The Director/CoDirector must be informed of, and be
compliant with the relevant national
legislation.
Request
from
Commissioners
clarification of meaning.
for
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Reason for Modification
Standard v 5.6.2
v5.7
B5.000
The resources of the laboratory must be
sufficient to accommodate the workload.
Re-Introduced following a request from
the EFI Commissioners
C6.000/ B6.000
C6.100/ B6.100
Testing referred to other laboratories.
An accredited laboratory may engage another laboratory to perform testing not done by
the primary laboratory. In that event, the subcontracting laboratory must be accredited by
EFI or by ASHI, if the testing is covered by EFI Standards. If genetic systems not covered
by EFI Standards are subcontracted, the subcontracting laboratory should have
documented expertise and/or accreditation in those systems. The identity of the
subcontracting laboratory and that portion of the testing for which it bears responsibility
must be noted in the reports.
Standard moved to B6.000 from C6.000
following a request to remove H&I
specific standards out of Section C to
facilitate joint inspections for EFI and ISO
C1.6000
Laboratories performing amplification of
nucleic acids must use physical and/or
biochemical barriers to prevent DNA
contamination.
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.
The laboratory must establish and employ policies and procedures for the proper
maintenance of equipment, instruments and test systems by 1) defining its preventive
maintenance programme for each instrument and piece of equipment at least once a
year, and by 2) performing and documenting function checks on equipment with at least
the frequency specified by the manufacturer.
Incorporated into L1.1100 and L1.1200
(see below) to facilitate joint inspections
for EFI and ISO
C1.7000/ C1.600
C1.7100/ C1.700
C1.930 / L2.3310
C3.100
Assays must be performed with calibrated dispensing instruments (e.g. pipettes, etc.).
Calibration must be performed at least once a year and must be documented.
Databases of HLA sequences used for allele assignment must be accurate and updated
at least once a year with the most current version of the IMGT/HLA Database.
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
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
Standards unchanged but re-numbered
to accommodate removal of C1.600.
Standard moved to L2.3310 to facilitate
joint inspections for EFI and ISO
This introduces the option for a delegated
individual to support the Director and CoDirector
in
reviewing
procedures
annually.
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
Reason for Modification
v5.7
manual. Each procedure must be reviewed
at least annually by the Director/CoDirector and documented evidence of this
review must be available. Any changes in
procedures must be initialled and dated by
the Director/Co-Director at the time they
are initiated.
manual. Each procedure must be reviewed
at least annually by the Director/Co-Director
or a delegated individual and documented
evidence of this review must be available.
Any changes in procedures must be
initialled and dated by the Director/CoDirector/ delegated individual at the time
they are initiated.
C4.150
The Director/Co-director must evaluate the
ability 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. The laboratory must maintain
records of these evaluations for each
individual.
The Director/Co-director 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. The laboratory must
maintain records of these evaluations for
each individual.
The Standards Committee were asked to
consider the wording of this standard
after it changed in 2007 from, “The
laboratory must, at least once a year,
give each individual performing tests, and
not involved in daily routine testing in the
histocompatibility and Immunogenetics
laboratory (eg on call personnel only) a
characterised specimen as an unknown
to verify his or her ability to reproduce
test results. This standard must be
applied to all tests regularly performed by
an individual. The laboratory must
maintain records of these results for each
individual.” The Standards committee felt
that the current wording is clear and
offers more flexibility than this previous
version, but agreed to change “ability” to
“competence”.
C4.310
The laboratory must document problems
that
result
from
breakdowns
in
communication between the laboratory and
the authorised individual who orders tests
or receives results.
Standard Deleted
Comments from the Commissioners
indicated that this Standard was difficult
to understand and to inspect against.
C4.320/ C4.310
All complaints and problems reported to the laboratory must be documented. Complaints
must be investigated and corrective action taken when necessary.
Standards unchanged but re-numbered
to accommodate deletion of C4.310
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
Reason for Modification
v5.7
C4.330/ C4.320
The laboratory must, upon request, make available to clients a list of tests employed by
the laboratory.
C5.130/ E0.100
For HLA typing by complement-dependent cytotoxicity each serum-cell combination must
be recorded in a manner which indicates the approximate percent of cells killed. The
numerical scores used in the International Workshop procedure (0,1,2,4,6,8) should be
used. Other numerical codes can also be used.
Incorporated into Section E (E0.100) to
facilitate joint inspections for EFI and ISO
C5.140 / C5.130
Reports or records, as appropriate, must include a brief description of the specimen
(blood, lymph node, spleen, bone marrow, etc.) used for testing.
Standard unchanged but re-numbered to
accommodate deletion of C5.130
C5.150 / C5.140
Molecular typing: a record must be kept Molecular typing: a record must be kept
which is appropriate to the technique used, which is appropriate to the technique used,
such as a photographic record of a gel, a such as a photographic record of a gel, a
membrane,
an
autoradiograph,
an membrane,
an
autoradiograph,
an
electronic file, or the read out from a electronic file, or the read out from a
sequencer. The record must be kept sequencer. The record must be kept
according to any regulation to which the according to any regulation to which the
laboratory is obliged to abide, but for a laboratory is obliged to abide, but for a
minimum of four years.
minimum of two years.
Records may be only saved in computer files, provided that back-up files are maintained
to ensure against loss of data.
The report must contain:
The report must contain:
a. The name of the individual tested or
a. The name of the individual tested or
unique identifier of each individual tested
unique identifier of each individual tested
and relationship to the patient if applicable. and relationship to the patient if applicable.
b. The date(s) of collection of sample when b. The date(s) of collection of sample when
pertinent.
pertinent.
c. The date of the report.
c. The date of the report.
d. The test results.
d. The test results.
e. The techniques used.
e. The techniques used.
f. Appropriate interpretations
f. Appropriate interpretation of the results
g. The signature of the Laboratory
where applicable
Director/Co-Director, or in his/her
g. The signature of the Laboratory
absence, a designee who meets the
Director/Co-Director, or a designated
requirements of Technical Supervisor.
individual.
Comment from member that C5.150 was
inconsistent with C5.100 “The laboratory
must maintain records of subjects tested
for two years or longer, depending on
local regulations.” C5.150 has been
adjusted for consistency.
The standard has also been re-numbered
to accommodate the deletion of C5.130
The screening result must be predictive
Comment from Commissioners.
C5.160 / C5.150
C5.300
F6.110
The screening technique used must be at
Standard unchanged but re-numbered to
accommodate deletion of C5.130
Comment from member requested
revision.
This new standard introduces the option
for a delegated individual to sign out
reports, even if the Director and CoDirector are present in the laboratory. It
also clarifies the requirement for the
report to contain interpretive comments.
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
Reason for Modification
v5.7
of the routine crossmatch result.
least as sensitive as the routine
crossmatch technique.
Screening is now much more sensitive
than most crossmatching techniques if
using microbead technologies. The
standard has been modified to
accommodate this situation.
G3.110
A prospective crossmatch may be omitted
in recipients that have shown to be
consistently negative for the presence of
HLA-specific antibodies as relevant for the
transplant protocol. Sera must have been
collected as defined in G2.110, covering at
least one year of immunological history
and must have been tested by at least two
different techniques. Screening data must
include at least one result obtained within
the previous 3 months, using a technique of
at least equivalent sensitivity to that used
for the crossmatching.
A prospective crossmatch may be omitted
in recipients that have shown to be
consistently negative for HLA-specific
antibodies. Sera must have been collected
as defined in G2.110, and must have been
tested on at least two different samples.
Screening data must include at least one
result obtained within the previous 3
months, using a technique of at least
equivalent sensitivity to that used for the
crossmatching.
Comment from Commissioners.
This standard has been modified to allow
the omission of a pre-transplant
crossmatch if at least two samples from
the patient have been screened and
found to be HLA specific antibody
negative.
H1.000
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 should be typed for HLA-A,B
and DR antigens.
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.
H2.000
Patients should be screened
presence of HLA alloantibodies
Cardiothoracic patients must be screened
for the presence of HLA alloantibodies and
unacceptable specificities must be defined
unless a prospective crossmatch is to be
performed. For these patients, G2.100 and
G2.110 apply.
Comment from Commissioners. There
were concerns about the frequency at
which unacceptable antigens were
defined in sensitised patients awaiting
transplantation. According to our EFI
Standards, sensitised renal or
renal/pancreas patients should be
crossmatched, so attention was focussed
upon recipients of cardiothoracic organs
where crossmatching may not always be
performed pre- transplant.
H3.300
Final crossmatches performed prior to
transplantation should utilise a recipient
serum sample collected within the previous
48 hours before transplant if the recipient
for
the
Final crossmatches performed prior to
transplantation should utilise a recipient
serum sample collected within the previous
48 hours before transplant if the recipient
Comment from Standards Committee
Member.
Wording adjusted for consistency
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
I1.140
I2.320
I4.1000
Reason for Modification
v5.7
has HLA antibodies or has had a recent
sensitising event. Otherwise, a serum
collected within three months should be
used.
has HLA antibodies or has had a recent
sensitising event. Otherwise, a serum
collected within three months should be
used.
If required by the transplant protocol,
laboratories 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 at the required level of
resolution. Where the ambiguities cannot
be resolved, all the alternatives must be
reported.
If required by the transplant protocol,
laboratories 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 at the required level of
resolution.
If required by the transplant protocol, the
laboratory must be able to type the donor
and the recipient for HLA Class I by DNA
methods, to a level of resolution as defined
under D1.320. Where the ambiguities
cannot be resolved, all the alternatives
must be reported.
If required by the transplant protocol, the
laboratory must be able to type the donor
and the recipient for HLA Class I by DNA
methods, to a level of resolution as defined
under D1.320. Where the ambiguities
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 that
this information is available upon
request.
Standards in L1.0000, L2.1100, L2.1200,
L2.2000 and L2.3300 also apply.
Standards in L1.0000, L1.3400, L2.1100,
L2.1200, L2.2000 and L2.3300 also apply.
Section J – HLA and Transfusion
Complete revision of Section J to introduce Standards for HPA and HNA testing. Only new standards are listed.
J- HLA/HPA/HNA and Transfusion
J1.010
Laboratories providing diagnostic testing for all Standards covered in J must follow
documented protocols of laboratory investigations for each service (HLA/HPA/HNA
testing) provided.
Comments from members and
Commissioners.
As the number of potential ambiguities
increases, it is not reasonable to include
all the potential alternatives on a report.
However, it was felt that this data must
be retained by the laboratory, and
available upon request to users of the
service.
See also L2.3400 and L3.2560 below.
Comment from Standards Committee
member. Adjusted for consistency.
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
J1.020
J1.030
J1.000
J1.100
J1.120
J1.130
J1.140
J1.150
J1.200
J1.210
J1.220
J1.230
J1.240
J1.300
J1.310
J1.400
J1.410
J2.000
J2.100
J2.200
J2.210
J2.300
J2.310
J2.400
J2.410
Reason for Modification
v5.7
Current HPA Nomenclature (PNC Platelet Nomenclature Committee*) must be used for
recording and reporting HPA alloantigen and HPA alloantibody specificities.
Current HNA Nomenclature (ISBT Working Party **) must be used for recording and
reporting HNA alloantigen and HNA alloantibody specificities.
HLA and Transfusion
Platelet refractoriness
Platelet refractory patients who require HLA matched platelets must be typed for HLA-A
and HLA-B.
For the laboratory investigation of suspected alloimmune platelet refractoriness the patient
must be tested for HLA class I antibodies.
The specificity of detected HLA antibodies must be defined and recorded or
crossmatching must be performed to provide compatible platelets. For crossmatching
using lymphocytes standards in section F6.000 apply.
All selected plateletpheresis donors used for the provision of HLA matched platelets must
be typed for HLA-A and HLA-B.
Transfusion Related Acute Lung Injury (TRALI)
For the laboratory investigation of TRALI the sera from implicated donor(s) must be tested
for both HLA class I and class II antibodies.
The specificity of detected HLA antibodies must be defined and recorded.
If HLA specific antibodies are identified, the patient and donor must be typed at least for
the relevant loci.
For the laboratory investigation of TRALI the serum from implicated donor(s) should also
be tested for HNA alloantibodies.
Transfusion Associated Graft Versus Host Disease (TAGVHD)
The patient and donor(s) must be typed at least for HLA-A, B and DRB1.
Febrile Non Haemolytic Transfusion Reactions (FNHTR)
The patient's serum must be tested for the presence of HLA antibodies.
Human Platelet Antigens (HPA) and Transfusion
HPA Testing
HPA typing must be performed by a validated HPA typing technique. If DNA typing is
performed then relevant standards in section L also apply.
The clinically significant HPA specificities must be defined and documented.
Donor HPA Testing
If HPA alloimmunisation is identified in a patient, verification typing must be performed on
donors whose products may be used.
Composition of platelet panel for HPA alloantibody detection
Laboratories must make all reasonable efforts to include HPA antigens in their antibody
* Vox Sanguinis 2003 85, 240-245 (HPA)
** Vox Sanguinis 2008 94 277-285 (HNA)
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
J2.500
J2.510
J2.520
J2.530
J2.540
J2.600
J2.610
J2.620
J2.700
J2.710
J3.000
J3.100
J3.110
J3.200
J3.210
J3.300
J3.310
J3.400
J3.410
J3.420
J3.430
J3.440
J3.500
J3.510
J3.520
Reason for Modification
v5.7
screening protocol which will aid the identification of clinically significant HPA
alloantibodies.
Investigation of HPA antibodies
The antibody screening technique must be validated before use.
Positive and negative controls must be included in each assay.
In glycoprotein specific assays, a positive control for each glycoprotein used should be
included. For ELISA based assays section N also applies. For bead array techniques
section M 2.000 also applies.
The specificity of detected HPA alloantibodies must be defined and recorded.
Neonatal Alloimmune Thrombocytopenia (NAIT)
The maternal serum sample 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 and their serum investigated for HPA antibodies.
Human Neutrophil Antigens (HNA) and Transfusion
HNA Testing
The clinically significant HNA specificities must be defined and documented.
HNA Typing
HNA typing must be performed by a validated HNA typing technique. If DNA typing is
performed then relevant standards in section L also apply.
Composition of neutrophil /granulocyte panel for HNA alloantibody detection
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.
Investigation of HNA antibodies
The antibody screening technique must be validated before routine use.
Positive and negative controls must be included in each assay.
In glycoprotein specific assays, laboratories must make all reasonable efforts to include a
positive control for each glycoprotein used. For ELISA based assays section N also
applies. For bead array techniques section M2.000 also applies. For flow cytometry
sections M2.000 to M5.000 also apply.
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.
HNA typing of the mother, father and neonate should be performed.
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
K2.000
Reason for Modification
v5.7
Comment from member – other
techniques may also be used to test for
single antigens of allele groups.
Standards modified to be more obviously
inclusive.
Typing for a single antigen by CDC (eg
HLA-B27)
Typing for a single allele-group by
molecular techniques (eg HLA-B*27)
Typing for a single antigen by CDC
L1.1100
All pre-amplification procedures must be
performed in a dedicated work area.
Physical separation and restricted flow are
recommended (C1.600)
Laboratories performing amplification of
nucleic acids must use a dedicated work
area with restricted traffic flow and
physical barriers to prevent DNA
contamination.
L1.1200
Pre-amplification physical containment
must include use of dedicated equipment,
laboratory coats, and 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.
Pre-amplification
physical
containment must include use of dedicated
equipment,
laboratory
coats,
and
disposable supplies.
L1.3100
Accuracy of thermal cycling instruments
must be verified a) by maintenance
according to the manufacturer and b) by
locally performed functional checks at
least every six months.
Accuracy of thermal cycling instruments
must be verified by maintenance according
to the manufacturer or annual thermal
verification of the block using a
calibrated device designed specifically
for this purpose.
There has been some confusion over
exactly how thermal cyclers must be
maintained. This standard has been
modified for clarity.
L1.4200
Laboratories must have a policy for quality
control of each lot or shipment of primers.
The specificity and quantity of amplified
product must be confirmed with reference
material. For commercial kits, each lot or
shipment must be tested against at least
one DNA sample of known type. The
signal intensity must be defined,
monitored and fall within an acceptable
range.
Laboratories must have a policy for quality
control of each lot or shipment of primers.
The specificity and quantity of amplified
product must be confirmed with reference
material. For commercial kits, each lot or
shipment must be tested against at least
one DNA sample of known type.
Comment from the French Inspectors. It
is very difficult to define signal intensity in
an SOP and laboratories often write
complicated descriptions just to comply
with this standard. Many commercial
reagents use manufacturer’s
recommendations for signal intensity.
K3.100
Typing for a single allele-group by
molecular techniques
These standards have been merged with
C1.600 from v5.6.2 to enable joint
EFI/ISO inspection.
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
L2.3400
Reason for Modification
v5.7
If the typing result is ambiguous, the report
must indicate all possible combinations.
If the typing result is ambiguous, all
possible combinations must be
documented. If ambiguities are not
included on the report, a comment that
additional data are available in the
laboratory must be added.
L3.2560
If a determined sequence is ambiguous,
the report must indicate all possible
allelic combinations.
If a determined sequence is ambiguous, all
possible allelic combinations must be
documented.
If ambiguities are not
included on the report, a comment that
additional data are available in the
laboratory must be added.
L3.3280
There must be in place systems to ensure
reliable identification of the samples
throughout all stages.
Standard deleted
L3.3281/
L3.3280
Acceptable limits of signal intensity must be specified for positive and negative results. If a
test is accepted with probe signals out of the set limits, this must be documented and
justified.
L3.3282/ L3.3281
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.
Non specific and weak hybridization signals must be defined and documented.
Comments from members and
Commissioners.
As the number of potential ambiguities
increases, it is not reasonable to include
all the potential alternatives on a report.
However, it was felt that this data must
be retained by the laboratory, and
available upon request to users of the
service.
Comment from the French Inspectors Duplication of C2.130
Standards unchanged but re-numbered
to accommodate removal of L3.3280.
M - FLOW CYTOMETRY
Complete revision of Section M to update and separate standards specific for Flow Cytometry and those specific for Microbead-based technologies.
Only new standards are listed.
M1.000
M1.100
M2.000
M2.100
M2.110
Application
Section M2.100 applies to flow cytometry and flow analysis using equipment designed for
beads only (fluoroanalyzer). Sections M2.200 to M5.000 apply to flow cytometry.
Instrument Standardisation and Maintenance
General Instrument Standardisation and Maintenance
For instruments that perform an automated integrated multi-parameter standardisation
(e.g. Luminex), this function may be used instead of individual optical alignment and
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
M2.120
M2.130
M2.140
M2.150
M2.160
M2.200
M2.210
M2.220
M2.230
M2.240
M2.300
M2.310
M2.320
M2.330
M2.400
M2.410
M2.420
M2.430
M2.440
M2.500
Reason for Modification
v5.7
fluorescence standardisation described in M2.200, M2.300 and M2.400. 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 as specified in
M2.200 to M2.400 if there are no manufacturer’s instructions.
There must be a procedure for regular cleaning of the instrument which must conform to
manufacturer’s instructions. Cleaning must be documented.
The instrument must be maintained according to manufacturer’s instructions but at least
once a year.
Optical Standardisation
The optical standard must be run every day of instrument use unless otherwise specified
by the manufacturer.
The optical standard must also be run at any time when 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 and 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 and must be run any time maintenance or adjustment of the
instrument during operation is likely to have altered settings.
A fluorescence standard must be used for each fluorochrome employed in analytical
procedures.
The results of fluorescence standardisation must be recorded and fall within the defined
acceptable range.
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.
Acceptable compensation values must be defined and recorded.
Equipment Maintenance
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
M2.510
M3.000
M3.100
M3.200
M3.300
M3.400
M3.500
M3.600
M3.700
M4.000
M4.100
M4.110
M4.120
M4.130
M4.140
M4.200
M4.210
Reason for Modification
v5.7
For laser-based instruments, the laboratory must record the current input (amps) and
laser light output (milliwatts), at the normal operating wavelength. Readings must be
measured after the laser has peaked and with normal operating power set. Other
pertinent regular functional checks must be defined and documented.
General Reagents
The specificity of labelling reagents for the identification of cell subset must be verified
using a published method and/or the manufacturer's documentation and/or 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 titred to determine the dilution with optimal 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 and
thresholds for adequate intensity must be defined and documented.
The quantities 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 cross-matching
Cell based testing
For cell based antibody screening and crossmatching an individual fluorochrome per
subset should be used for the identification of each population subset (multicolour
technique).
If a single colour technique is used, the purity of the isolated cell population must be
sufficient to define the population for analysis and must be documented.
The target sub-populations must be defined and identified by appropriate labelling
antibodies (e.g. T lymphocytes: CD3, B lymphocytes: CD19) and must include a sufficient
number of events per sub-population, relevant to the test performed.
The binding of human immunoglobulin 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 using cell
and bead based antibody screening the composition of the panel must conform to the
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
M4.300
M4.310
M4.320
M4.330
M4.400
M4.410
M4.420
M4.430
M5.000
M5.110
M5.120
M5.200
M5.210
M5.220
M5.230
M5.250
M5.260
Reason for Modification
v5.7
standards in section F1.300.
Controls
A negative control must be used. This must be a serum from a non-alloimmunised human
donor(s) which has been screened and found negative by flow cytometry.
A positive control must be used. This must be human serum with antibodies of the
appropriate isotype, specific for HLA antigens.
Control sera must be tested at the same time and under the same conditions as the sera
under test.
Policies and procedures:
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 and temperatures.
Interpretation instructions must include details of the threshold for significant levels of
antibody binding (positivity) and 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, for
the test to be valid must be defined and documented.
Cell-based HLA typing by flow cytometry (e.g. HLA B27)
The specificity of each lot of labelling reagents for the identification of HLA specificities
must be determined by testing (1) at least five cells known to express the target antigen,
(2) at least two cells for each cross-reacting antigen, and (3) at least two cells which lack
the specific and cross-reacting antigens. Acceptable criteria for validation must be defined
and results must be recorded.
The specificity of each lot and shipment of labelling reagents for the identification of HLA
specificities must be shown to have comparable reactivity to the previously validated lot or
shipment.
Controls
Controls for HLA typing by flow cytometry must be run for each test cell preparation.
For direct labelling, a negative control must be conjugated with the same fluorochrome.
For indirect labelling a negative control (eg. an irrelevant primary antibody), if available,
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 under the same conditions as the specific antibodies used in
the test.
A control cell known to express the HLA specificity under test must be included in each
run.
Documentation of Changes to EFI Standards: v 5.7
Modified Standard
Standard v 5.6.2
M5.300
M5.310
M5.320
M5.330
Reason for Modification
v5.7
Policies and procedures:
There must be policies and procedures in place to address at least HLA typing technical
instructions including reagent standardisation and optimisation, reagent validation,
incubation times and temperatures.
Interpretation instructions must define the required reaction criteria in the negative and
positive control samples for the test results to be valid, as well as 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.