RECOMMENDED GUIDELINES FOR
THE DETECTION OF AUTO & ALLO ANTIBODIES
1994/1995 COMMITTEE MEMBERS
Ms Jennifer Bryant, Mr Greg Bryson, Dr
Margaret Cooley, Dr Gerard Hale
Mr Stephen Hunter, Dr Robyn Minchinton,
Mr Lyndsay Peters, Mr Gideon Sinclair
1996 COMMITTEE MEMBERS
Ms Jennifer Bryant, Mr Peter Hobson, Dr
Terms of reference for the AFCG standards committee.
1. Committee composition: To be broadly based with a variety of
2. Quality control: To examine methods for inter and intralaboratory
3. To report to the AFCG members on
the recommendations for minimum standards for the detection of
allo and autoantibodies.
4. To work towards establishing a Quality
5. To report recommendations and progress
to the AFCG AGM.
Processing and Storage
Flow Cytometer Quality Control
Appendix 1: Determination of Reference Ranges
Appendix 2: Log Book Forms
These recommendations are presented
with a view to being a minimum standard. These recommendations
should not be seen to restrict the ability of any individual.
This document will be reviewed regularly to ensure that these
recommendations embrace currently accepted laboratory practices.
Although correlated multicolour
immunofluorescent techniques are preferred to ascertain the purity
of the population of interest, it is recognised that in certain
situations this may not be possible. Thus, immunofluorescent techniques
using a single colour are acceptable in the typing of isolated
or relatively homogeneous populations provided the correct controls
are included in the analysis.
This committee recognises that its expertise
is mainly in the area of detecting allo and autoantibodies in
human blood. We are unable to evaluate the suitability of these
recommendations for the detection of antibodies present in other
Laboratory Safety is an issue more fully
covered in the publications by the Australian Standards2
and NHMRC/ANCA3. These publications should be held
by the Quality Control Manager or Occupational Health and Safety
Committee of each organisation.
The use of formaldehyde in uncontrolled
areas is not recommended, as formaldehyde is a noxious chemical.
Each laboratory will adopt internal
procedures and policies for the safe handling of biological specimens.
1. Use universal precautions1
with all specimens.
2. Develop appropriate internal procedures
to cope with accidents such as spillages.
3. Handle and manipulate specimens in
a safe biological confinement area wherever possible.
4. Fix cell samples with a 0.5% available
formaldehyde-based solution for 15 min before leaving the safe
biological confinement area.
5. Final cell suspension should be in
a 0.5% 1% available formaldehyde based solution.
6. Unfixed samples outside the safe
handling area should be capped.
7. Appropriate safety devices such as
gloves, gowns, goggles, centrifuge carriers, automatic pipetting
are recommended whenever handling and processing specimens. Use
disposable plastic equipment wherever possible.
8. Wash hands with medicated soap after
working with specimens, removing gloves, or when leaving the laboratory,
and as in accordance with usual local laboratory policy and universal
9. For decontamination of flow cytometers
refer to the instrument manufacturer's recommended procedures.
10. Liquid waste should be treated with
sodium hypochlorite. Solid waste should be handled carefully in
appropriate robust containers.
11. Laser safety: Most benchtop flow
cytometers use visible lasers which pose very little risk of injury
to the operator. Operators should be aware of the potential dangers
of lasers and the need for safety devices such as shields and
goggles in given circumstances. The operator is referred to the
manufacturer of the instrument and to AS 22111 with
regard to safety of lasers.
1. Universal precautions1
should be strictly observed when collecting and processing blood
samples (Management guidelines for the control of infectious disease
hazards in health care establishments).
2. A written request form should accompany
the blood samples and should include in legible script:
2.2 Clinical diagnosis including pertinent
medication. Additional information including previous and recent
transfusion history, pregnancies, and gender is desirable.
2.3 Time and date of collection and
signature of collector.
2.4 Name of requesting physician and
address for return of results.
3. Following collection and before leaving
the patient, the specimen tube(s) should be legibly labelled with:
3.2 Date and time of collection.
3.3 If a preprinted label is used, the
signature or initials of the collector should appear on the label
to verify that the information relates to the patient from whom
the blood was collected.
4. The request form and specimen tube(s)
should carry identical patient information. Both should be checked
on receipt in the laboratory and in case of discrepancy or doubt,
a clear, documented protocol approved by the Director/Scientist
in Charge of the laboratory should be followed. Unlabelled samples
and forms should be discarded.
5. EDTA or ACD anticoagulated blood
samples are suitable as a cell source for the investigation of
alloantibodies and autoantibodies to red cells, platelets, lymphocytes,
monocytes, and neutrophils. Clotted blood, collected in a plain
tube, is required as an antibody source.
6. For cell preparations, the volume
of whole blood to collect will depend on the absolute count of
the cell type under investigation. Insufficient volume coupled
with a delay in transport and/or processing may preclude testing.
Close liaison with the testing laboratory is recommended to ascertain
acceptable parameters for volumes and transit times.
7. Cellular elements in blood for autoantibody
studies are extremely labile and a policy of early morning collections
with rapid transport the same day is recommended. Neutrophils
are the most labile cellular elements and meaningful results can
be reported only if the collection to arrival time in the laboratory
is less than 8 hours (less than 6 is preferable). Careful planning
and close liaison with the laboratory is essential, in particular
for patients in remote areas.
1. Packaging, labelling, and transport
of specimens should comply with all current local, state, national,
and international regulations for the regions through which the
specimens will pass.
2. Anticoagulated samples for the investigation
of alloantibodies and autoantibodies to platelets, lymphocytes,
monocytes, and neutrophils should be maintained at 1822oC
during transit. Temperature extremes (below 10oC or
above 37oC) will compromise the quality of the sample
and should be avoided. Samples for the investigation of erythrocyte
antibodies and clotted blood as an antibody source are best transported
and stored at 28oC.
1. Upon receipt in the laboratory, patient
details, collection and referral information on the tube(s) and
form should be checked. An inspection of the specimen for clots,
haemolysis, and/or container defects should be carried out and
an approved, documented local policy followed if any problems
2. Specimens which have been collected
inappropriately may be processed by the laboratory according to
a local approved, documented policy. The deficiencies in the sample
should be noted and the final report should reflect the effect
that these deficiencies may have had on the results.
PROCESSING AND STORAGE
1. Preparation of peripheral blood cells
(red cells, platelets, neutrophils, lymphocytes, monocytes) for
the demonstration of autoantibodies or alloantibodies by flow
cytometry requires rigorous standards of handling. If single cell
populations are to be isolated from whole blood, established,
referenced, locally validated methods should be used. Excessive
manipulation of cellular material should be avoided where possible
and manual or automated treatment of cells with lysing agents,
fixative or "stripping" agents to remove HLA antigens
should be carefully validated and controlled (see below).
2. Autoantibody investigations using
patient's own cells should consist of at least the following tests:
3. Detection of serum antibodies reactive
with normal donor cells should be performed using a typed cell
panel where possible. If phenotyped cells are unavailable, a random
panel should be selected based on phenotypic frequencies to ensure
adequate representation of all common antigens of relevance for
a given cell type. The use of large pools of donor cells in antibody
screening should be avoided as weakly expressed antigens may not
be detected. Detection of serum antibodies should include the
The shelflife of panel cells will vary considerably according to the cell type, storage conditions and fixation status; therefore, storage and extended use of panel cells should be strictly validated by the laboratory to ensure that antigens are preserved and background levels of activity are within acceptable limits. If additives (such as drugs or antigen stripping agents) are to be added to the system, the controls should include:
4. It is recommended that residual serum
be accurately labelled and stored frozen at 20oC
or lower, for a period determined appropriate to the history and
origin of the specimen.
5. Repeated freeze thawing of patient
and/or control sera should be avoided to minimise immunoglobulin
aggregate formation and deterioration of the antibody(ies).
6. For each lot or batch of secondary
antibody, the laboratory should determine the optimal concentration
and volume to use for each cell type. A selection of positive
and negative sera for each cell type should be used as primary
antibodies. The volumes and dilutions recommended by the manufacturers
for immunofluorescence are guidelines only. Documentation of this
validation should be maintained according to local statutory requirements.
1. Ideally, a method control is prepared
and run on a daily basis in parallel with patient samples. At
a minimum the method control should be prepared and run whenever
a new batch of any reagent, used in cell preparation and staining,
2.1 Commercial "isotypic"
controls are irrelevant for human alloantibody and autoantibody
investigations. Ideally a pooled sera negative control should
be prepared in house from untransfused, group AB- or ABO-compatible
males whose sera have been found to be negative for alloantibodies
and autoantibodies to the relevant peripheral blood cell antigens.
2.2 Positive control sera should be
confirmed by an established reference laboratory.
3. Each laboratory should establish
reference ranges for the antigens being tested. See appendix 1.
FLOW CYTOMETER QUALITY CONTROL
These procedures should be carried out
when the flow cytometer is first received, or when major maintenance
or repair is performed.
1. Alignment of the optical components
of the flow cytometer (laser, focusing lenses, collecting lenses,
photodetectors, etc.) should be established using the manufacturer's
recommended alignment materials and procedures. Alignment particles
are typically uniform plastic particles incorporating a fluorescent
dye. (Other materials may be recommended by the manufacturer.)
The laboratory should determine optimum settings for their own
instrument/alignment particle combination and establish their
own expected values. The expected range along with relevant instrument
settings should be recorded in an instrument log book for subsequent
use and daily monitoring (See optical alignment log).
Optical alignment can be verified by:
(1) Running alignment particles at instrument
settings determined at the time of initial instrument set-up.
(2) Recording the mean channel number
and CV for all parameters that will be analysed for test specimens
in the daily log book and/or on LevyJennings plots optical alignment log.
If particle values are not within acceptable
range, alignment should be optimised before proceeding.
2. Verification of instrument sensitivity
and spectral overlap compensation settings should be determined
and recorded using cells or fluorescent microparticles.
2.1 Instrument sensitivity is verified
(2) If sensitivity particles (e.g. fluorochromelabelled
beads or nuclei) are used, run them at testspecific settings
established at the time of initial setup.
(3) Record mean fluorescence channel
and CV for all populations of interest (eg. unstained versus
dimly-stained) in the daily log book and/or on LevyJennings
plots (sensitivity log).
(2) If compensation particles (e.g. FITC-
and PElabelled beads) are used, run them at testspecific
settings and compensation levels established at time of initial
(3) Record mean channel fluorescence
intensity for each population of interest (red only, green only,
and negative for both) in the daily log book and/or on LevyJennings
plots (compensation log).
Note: Overcompensation leads to fewer errors than undercompensation.
Representation of application of correct
compensation. Gated correlated display of brightly staining FITC-conjugated
and PE-conjugated antibodies. (A) Uncompensated. (B) Correctly
3. Overall system performance can be
(1) Running a "normal" specimen
stained with a combination of antibodies which stain and divide
the cells to be studied into definite populations at testspecific
(2) Verifying acceptable light scatter
resolution of the population(s) of interest.
(3) Verifying that the percentage of
antibodypositive cells falls within laboratory established
ranges for the antigens selected (Appendix 1).
If this positive control does not meet
laboratory criteria, remedial action should be taken. Instrument
performance and/or staining procedure should be checked to determine
the source of the problem. Any problems identified using this
sample must be rectified prior to analysis of test specimens.
4. Instruments should be calibrated
using a recognised fluorescent standard before a valid comparison
of results from different instruments can be made. The calibration
curve should be checked at regular intervals based upon instrument
performance characteristics. A calibration protocol should be
established by each laboratory and results should fall within
the limits established by each laboratory. A suitable protocol
should be established and followed in the event of unsatisfactory
results. Changes in instrument performance characteristics, e.g.
optical alignment, laser, or PMT's will necessitate recalibration.
1. Sample order - Run all control specimens
first and then, according to laboratory priority,
run the patient samples.
2. Assessment of specimen viability
is desirable; however, because of biohazard concerns, it is recommended
that all samples be appropriately fixed prior to analysis on the
flow cytometer. It is not presently possible, on a routine large-scale
basis, to distinguish those cells which were non-viable
prior to fixation. For nucleated cells, this can be performed
using ethidium monoazide (EMA) as described by K. Muirhead, 2nd
AFCG Methods Course, 1989.
3. Maintenance of specimen viability
is desirable prior to incubation with antibodies; however, because
of biohazard concerns, it is recommended that where appropriate
all samples be fixed prior to analysis on the flow cytometer.
4. Where possible, count at least 2000 gated events in each sample. This number assures with 95% confidence that the result is < 2% SD (standard deviation of the "true" value - binomial sampling).
NB: This sample mode assumes that the
variability of determining replicates is < 2% SD.
5. The counting of 2000 gated events
to ensure reasonable statistical confidence may not be achievable
in specimens where the cell of interest is severely depleted.
6. Set gates as broadly as possible
consistent with acceptable levels of contamination to minimise
the exclusion of cells of interest.
7. Each laboratory should establish
limits of contaminating cells and debris, based on documentation
that their inclusion does not significantly affect the measurement
of interest. If levels of contamination exceed established laboratory
limits, the corrective actions recommended are to adjust light
scatter gates and reanalyse. If levels of contamination cannot
be restricted to acceptable limits, test results are suspect and
a second specimen should be requested.
8. When simultaneous two colour immunofluorescent
correlated data is analysed, boundaries must be set to define four
distinct regions: cells labelled with neither antibody, cells
labelled with antibody #1 but not antibody #2, cells labelled
with antibody #2 but not #1, and cells labelled with both antibodies.
1. The possibility of patients' contesting
the diagnostic implications derived in part from flow cytometric
testing makes it incumbent upon the laboratory to be able to demonstrate
and verify the process used in arriving at the reported test results.
2. Where possible all listmode data on all samples analysed should be retained.
At a minimum retain correlated dual
fluorescent data for each test and any interpretive comments on
samples where a significant diagnosis is made.
3. Retain all primary files, worksheets,and report forms.
4. Minimum duration of data storage depends on state and federal regulations.
These regulations may vary and each
laboratory will need to remain informed of the current requirements.
1. Report all unique patient identifiers.
2. Report all data in terms of cluster
of differentiation (CD) with a short description of the main antigen
3. For unclustered antibodies report
the clone name or the systematic name for human antibodies with
a short description of the main antigen binding characteristics.
4. The testing laboratory should indicate
whether a phenotyped or random panel was used in the investigation.
Where feasible, those antigens represented in the screening panel
should be included in the report.
5. Reference limits for test results
should be determined by each laboratory and these should be quoted
in the report.
1. Where possible, the laboratory should
belong to and participate in a recognised external Quality Assurance
program. It is recognised that such programs may not be available
when pioneering a new application of flow cytometry in the study
of auto and alloantibodies. In such cases, another
QA program (e.g. lymphocyte immunophenotyping) could be used to
assure the quality of flow cytometric results from your laboratory
until a more suitable program becomes available.
2. Each laboratory should determine
the level of test variability by preparing and analysing at least
six replicates. This will provide a basis when methodologic changes
For example, tube-to-tube variation can be monitored
by the inclusion of the same antibody in separate tubes within
the one patient test series.
1. Universal precautions: There appears to be no single document that addresses the specific needs of flow cytometry. Readers are advised to refer to the following documents:
(I) Australian Standard AS 2211 - 1991, Laser Safety.
(ii) Australian Standard AS 2243.3 1991, Safety in laboratories, Part 3: Microbiology.
(iii) NCCLS M29T, Protection of laboratory workers from infectious disease transmitted by blood, body fluids and tissue.
(iv) MMWR 1988;37(24):37782, 3878.
CDC Update: Universal Precautions for the prevention of transmission
of human immunodeficiency virus, hepatitis B virus, and other
bloodborne pathogens in health care settings
2. Australian Standards AS 1386.171989
and AS 1807.021989
3. Report of the joint NHMRC/ANCA working
party on management guidelines for the control of infectious disease
hazards in health care establishments. National Health and Medical
Research Council/Australian National Council on AIDS joint report
published by Australian Government Publishing Service, Canberra,
APPENDIX 1: DETERMINATION OF REFERENCE RANGES
Reference values: Set of values for
a measured quantity defined state of health.
Reference range: Classically, the range
of values found in 95% of a reference population of healthy individuals
without overt clinical disease.
NOTE: Age, sex, and race are factors
known to influence reference intervals.
2.0 Procedure for Determining Reference
Statistical methods, both parametric
and nonparametric, and graphical methods are discussed in detail
in references 13. Only a brief summary of the steps involved
is presented here.
(1) Collect data on randomly chosen
set of representative individuals (e.g. 50 healthy individuals).
(2) Inspect frequency distribution of
(3) If frequency distribution is Gaussian,
use appropriate statistical techniques to estimate 95% confidence
interval and use endpoints of interval as the reference range.
(4) If frequency distribution is nongaussain,
back transform endpoints of 95% confidence interval to obtain
reference range, (e.g. log X, of (X + C), square root X, arcsin
X) and proceed as in step 3.
(5) If no satisfactory transformation
can be identified, use nonparametric methods which do not depend
on the exact distribution of the data.
2.2 Steps for nonparametric methods
(1) Collect data on randomly chosen
set of representative individuals.
(2) Arrange data in ascending
or descending order.
(3) Use appropriate nonparametric
techniques to identify desired limiting percentiles (e.g. 2.5
and 97.5) to desired confidence level.
Nonparametric methods are most appropriate
when data does not show a Gaussian distribution and cannot be
so transformed. However, they are very sensitive to outliers,
and final ranges chosen may be highly dependent on methods used
for removing outliers (13).
3. Pitfalls in Determining Flow Cytometric
Each laboratory should determine its
own reference range using its particular preparation method and
instrumentation because significant laboratorytolaboratory
differences related to these variables have been reported.
However, quite large data sets are technically
required to carry the above described methods for reference range
determination, typically >300 for parametric methods and >120
for establishing nonparametric interval with 90% confidence. Until
more standardised methodology allows pooling of data among laboratories
(hence this document), this is clearly an unrealistic expectation.
Other confounding variables besides
sample size have been described (45).
One practical solution to the dilemma
is to accumulate and analyse reference data in smaller sets (eg. 1020
individuals), which can then also be pooled and analysed. If the
last two sets of pooled data are found to give the same reference
range within experimental error, this gives increased confidence
that the reference range selected is not unduly affected by the
small sample size.
REFERENCES FOR APPENDIX 1
1. Winkel, P., and Statlan, B.E. Reference
values. In Clinical Diagnosis and Management by Laboratory
Methods (ed. J.B. Henry), Philadelphia, W.B. Saunders Co.,
1979, pp. 2952.
2. Martin, H.F. Gudzinowicz, B.J. Fanger,
H. Normal Values in Clinical Chemistry, New York, Marcel
Dekker, 1975, pp. 102236.
3. Henry, R.J., Cannon, D.C., Winkelman,
J.W. Clinical Chemistry. Principles and Technics, New York,
Harper and Row, 1974, pp. 343371.
4. Edward, B.S., Altobelli, K.K., Nolla,
H.A., et al. A comprehensive quality assessment approach for flow
cytometric immunophenotyping of human lymphocytes. Cytometry
5. McCarthy, R.C., and Fetterhoff, T.J.
Issues in quality assurance in clinical flow cytometry. Arch.
Pathol. Lab. Med.113: 658666, 1989 (in press).
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