INTRODUCTION

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 bodily fluids.

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.

LABORATORY SAFETY

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 precautions.

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.

SPECIMEN COLLECTION

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:

2.1 Patient's surname, given name(s), AND hospital record number, or date of birth where no hospital number is available.


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:

3.1 Patient's surname, given name(s) and hospital record number or date of birth.


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.

SPECIMEN TRANSPORT

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 18­22^oC during transit. Temperature extremes (below 10^oC or above 37^oC) 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 2­8^oC.

SPECIMEN INTEGRITY

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 are apparent.

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:

patient cells + patient serum (indirect auto)
patient cells + no serum (direct test)
patient cells + negative control serum
patient cells + positive control serum.

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 following tests:

panel cell + buffer (autofluorescence/non­specific binding control)
panel cell + negative control serum
panel cell + positive control serum
panel cell + patient serum (test)

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:

a serum with reactivity which is affected by the additive
a serum with reactivity which is unaffected by the additive

4. It is recommended that residual serum be accurately labelled and stored frozen at ­20^oC 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.

CONTROLS

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, is initiated.

2. Controls:

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 by:


(1) Using freshly stained cells of interest to establish that positive­negative separations are acceptable.


(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).



2.2 Two colour compensation is verified by:

(1) Freshly stained cells of interest brightly stained with mutually exclusive antibodies.


(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 instrument set-up.


(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.

(A) (B)

Representation of application of correct compensation. Gated correlated display of brightly staining FITC-conjugated and PE-conjugated antibodies. (A) Uncompensated. (B) Correctly compensated.

3. Overall system performance can be verified by:

(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 instrument settings.


(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.

SAMPLE ANALYSIS

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.

DATA STORAGE

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.

DATA REPORTING

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 recognition characteristics.

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.

QUALITY ASSURANCE

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 are introduced.

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.

REFERENCES

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, 1993.

APPENDIX 1: DETERMINATION OF REFERENCE RANGES

1.0 Definitions

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 Ranges

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.

2.1 Steps for parametric methods


(1) Collect data on randomly chosen set of representative individuals (e.g. 50 healthy individuals).


(2) Inspect frequency distribution of values obtained.


(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 Reference Ranges

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 10:443­441, 1989.

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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(765) 494-0757; FAX (765) 494-0517; Web http://www.cyto.purdue.edu, EMAIL cdrom3@flowcyt.cyto.purdue.edu