1994/1995 COMMITTEE MEMBERS

Dr Simon Bol, Mr Greg Bryson, Dr Margaret Cooley, Ms Sue Francis

Ms Helen Hanlin, Mr Stephen Hunter, Mr Lyndsay Peters, Dr Henry Preston

Mr Steve Rockman, Mr Joseph Webster

1996 COMMITTEE MEMBERS

Dr Simon Bol, Mr Peter Hobson, Mr Arnold Kabral

Terms of reference for the AFCG leukaemia and lymphoma phenotyping standards committee

1. Committee composition: To be broadly based with a variety of experience

2. Quality control: To examine methods for inter- and intra-laboratory quality assurance for all areas of flow cytometry.

3. To report to the AFCG members on the recommendations for minimum standards for leukaemia and lymphoma phenotyping.

4. To work towards establishing a joint AFCG / RCPA Quality Assurance program directed specifically at clinical flow cytometry.

CONTENTS

Introduction

Laboratory Safety

Specimen Collection

Specimen Transport

Specimen Integrity

Specimen Processing

Controls for Immunophenotyping

Flow Cytometer Quality Control

Sample Analysis

Data Storage

Data Reporting

Quality Assurance

References

Appendix: Log Book Forms

• Sensitivity Log
• Compensation Log
• Optical Alignment Log

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 current accepted laboratory practices.

The aim of leukaemia and lymphoma phenotyping is to identify the cell type of the neoplastic process. This phenotypic identification should outline the cell lineage and level of maturation, as an aid to the classification of the leukaemia or lymphoma. Further, this phenotypic identification should assist in the determination as to whether the cell population is normal or abnormal and in the detection of a previously characterised population of cells in a sample for monitoring the disease remission, development, or recurrence.

These assays are usually performed on blood or bone marrow specimens; however, other body fluids or tissues may have to be examined.

These guidelines are written in broad terms as a gesture to indicate and not to dictate.

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% to 1% available formaldehyde-based solution, close the test tubes, and fix for a minimum of 15 mins before analysing on the flow cytometer.

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 patient samples.

2. Each specimen should be labelled with the patient's name or a unique patient identifier, and date and time of collection. 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.

3. Each specimen should be accompanied by a test requisition which should include the patient name or unique patient identifier, date, and time of collection, age, sex, pertinent medication and presumptive diagnosis of the patient, name of requesting physician, and address for return of results.

4. The request form and specimen tube(s) should carry identical patient information. Both should be checked on receipt in the laboratory; 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. A total white cell count and differential should be performed at the laboratory initiating the request. For distant laboratories and dispatch centres a white cell count and unstained blood film should accompany each specimen.

6. EDTA anticoagulated blood and bone marrow specimens are suitable if the specimen will be processed within 8 hours of collection.

7. Heparin or ACD anticoagulated bBlood and bone marrow specimens may be processed within 24 hours of collection.

8. Tissue biopsies in isotonic medium (such as phosphate buffered saline or RPMI) and CSF usually do not require anticoagulant. These specimens should be processed within 8 hours of collection.

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. Specimens should be maintained at 18^o ­ 22^o C in a light-proof container.

3. Temperatures below 10^oC or above 37^oC must be avoided.

SPECIMEN INTEGRITY

1. Visually inspect the specimen for clots, haemolysis, or container defects. Re-collect the sample if the specimen shows any visual signs of deterioration.

2. Specimens that are collected or transported outside these guidelines should be treated with caution. The viability of the cells should be tested and if <70% results must be interpreted with caution and noted in the report.

SPECIMEN PROCESSING

1. Erythrocyte lysis methods are recommended because they are less prone to differential losses of specific subpopulations. However, using this procedure assumes that all leucocyte subsets are equally tolerant to the lysis method used.

Several lysing techniques are available. These include water, tris-buffered ammonium chloride, and hypotonic buffer (4,5). Several proprietary lysing reagents are also available from instrument and monoclonal antibody manufacturers. For commercial reagents, the manufacturer's recommended protocol should always be followed unless data are available confirming that any modifications do not adversely affect results. If necessary, density gradient isolation such as ficoll hypaque can be used.

2. Where possible a total leucocyte count and differential should be performed before processing, and the cell concentration adjusted accordingly. One should aim for a cell number of 1x10⁶ per test tube. Specimens which are leucocyte-poor may have insufficient cells for flow cytometric analysis, and require a larger sample collection volume. Conversely, standard concentrations of antibody reagents may be insufficient to saturate all binding sites in specimens with leucocytosis, leading to possible false negative results.

3. Monoclonal antibody panels

Multicolour immunofluorescence is preferred, although in some cases single-colour immunofluorescence may be adequate. Because pattern recognition plays an important role in leukaemia phenotyping, it is recommended that laboratories devise panels for acute and chronic leukaemias and lymphomas. The laboratories should then become familiar with the reactivity patterns encountered with their particular reagents.

In addition to multicolour and scatter analysis, pattern recognition may also include the fluorescence intensity of antibody-labelled cells (as a measure of the level of antigen expression). In the ideal situation, cells should be labelled with excess antibody and instruments should be calibrated. In order to increase the sensitivity of the analysis, the choice of fluorochrome may be important. It is recommended that "brighter" fluorochromes (such as phycoerythrin) be used in cases of expected low antigen expression. Lower efficiency fluorochromes (e.g. FITC) can be used for expected high antigen expression. The very high efficiency "3rd colour" reagents (e.g. PECy5) cannot be used for quantitative fluorescence intensity measurements, because the antibody concentration is routinely reduced in order to bring the fluorescence into a measurable range.

Although different antibodies can be classified by cluster designation (CD) number, the different clones may show different cellular reactivity. This may be important when selecting an antibody panel. Both the CD number and the clone should be listed on the worksheet.

For the determination of lineage and maturation stage, the detection of cytoplasmic antigens may be of importance.

Any panel of antibodies must include:

(a) Isotype controls appropriate for the antibodies in the panel.

(b) A suitable large panel of antibodies for investigating the presumptive diagnosis. The selection of antibodies used in the panel should be referenced.

(c) Mechanisms for internal cross checking and lineage coexpression evaluation.

Examples of screening panels are:

4. Staining procedures

4.1 For commercially available antibodies, follow the manufacturer's instructions. Any deviations from these instructions should be verified by each individual laboratory. The results of the altered procedure should be comparable to the results obtained by the manufacturer's recommended procedure.


When no recommendations are available, antibodies can be titrated and tested on appropriate cells. Alternatively, antibodies may be used at 1­2mg per 1x10⁶ cells.

NOTE: Too low amounts of antibody per cell may result in weak staining, leading to possible false negative results. Excess reagent may cause increased nonspecific staining of negatives or reduced staining of positives and may result in decrease of positive/negative resolution.

4.2 All washes and antibody incubations should be performed in the presence of 0.1% NaN₃ to prevent shedding, capping, or internalisation of the antigen.


4.3 Generally the recommended incubation temperature and times are 4^oC for 30 minutes OR room temperature for a minimum of 10 minutes.

CONTROLS FOR LEUKAEMIA AND LYMPHOMA PHENOTYPING

1. Isotype controls are recognised as an essential part of any monoclonal antibody panel for the purpose of establishing levels of non­specific binding and autofluorescence.

In many cases the isotype control may not be optimal for determining non­specific fluorescence because of differences in fluorochrome / protein ratio and antibody concentration between the isotype control and the test reagents. This is particularly important in certain cases of leukaemia (in particular myelomonocytic) where there is a high degree of species cross-reactivity due to the presence of Fc receptors. At this time there is no solution to this problem.

2. Pooled subclass controls are not recommended. It is emphasised that dim reactivity of test antibodies cannot be interpreted with certainty in the absence of appropriate negative controls.

3. Even when appropriate negative controls show no staining, a high level of false staining with anti­immunoglobulin reagents may occur because these reagents bind to cytophilic serum immunoglobulin adsorbed onto Fc receptors. Standard washing procedures usually remove most of the serum and a proportion of cytophilic immunoglobulin. The use of multicolour fluorescence based tests utilising pan B cell antibodies in conjunction with light chain may overcome this problem. Alternatively, cells can be incubated at 37^oC for 1 hour in serum-free medium, followed by washing.

4. 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.

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. Proper 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 must 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 Appendix 1, Optical Alignment Log ).

2. Verification of instrument sensitivity and spectral overlap compensation settings should be determined and recorded using cells or fluorescent microparticles.

2.1 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.


2.2 Instrument sensitivity is verified by:

(1) Using freshly stained lymphocytes, establishing 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 (e.g. unstained versus dimly stained) in the daily log book and/or on Levy­Jennings plots (Sensitivity Log).


2.3 Two colour compensation is verified by:

(1) Freshly stained leucocytes using mutually exclusive antibodies, e.g. CD3-FITC and CD19-PE for lymphocytes.

(2) If compensation particles (e.g. FITC- and PE­labelled beads) are used, run them at test­specific settings and compensation levels established at the time of initial instrument set-up. If particles values are not within acceptable range, compensation settings should be re­evaluated using antibody­stained leucocytes.

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).

(A) (B)

Figure 1

Compensation of anti CD3-FITC and anti CD19-PE labelled peripheral blood lymphocytes.
A) Uncompensated. (B) Correctly compensated.

Note: Overcompensation leads to fewer errors than undercompensation.

2.4 Overall system performance can be verified by:
(1) Running a "normal" specimen stained with an antibody reagent such as anti CD3-FITC and CD4-PE at test­specific instrument settings.

(2) Verifying acceptable light scatter resolution of the leucocyte populations.

(3) Verifying that the percentage of antibody­positive lymphocytes falls within established ranges for the antigens selected.

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.

SAMPLE ANALYSIS

1. Sample order. Run all control specimens first and then, according to laboratory priority, run the patient samples.

2. Test order within any panel. The first tube should be a gating control to maximise the cells of interest and minimise contamination with irrelevant particles. That is, the discriminator is set to exclude debris, platelets, etc. (see Figure 2). The appropriate isotype controls should be run next, followed by the test panel to investigate the provisional diagnosis.

Figure 2

Common ways of displaying forward versus 90^o light scatter observed for lysed whole blood preparations. (L=predominantly lymphocytes, M= predominantly monocytes, P= predominantly polymorphonuclear leucocytes). Live gating should be restricted to the setting of a forward light scatter threshold. Set leucocyte gates as broadly as possible, consistent with acceptable levels of contaminating particles to avoid the exclusion of cells of interest .

3. 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 non­viable prior to fixation. However, this can be performed using ethidium monoazide (EMA) as described by K. Muirhead, 2nd AFCG Methods Course, 1989.

4. A minimum of 10,000 events per sample should be collected to allow for accurate assessment of minor cell populations. When the cells of interest are present in low frequencies, the total events collected should be increased proportionally.

5. It is recommended that correlated analysis be performed on all samples to enhance the differentiation between normal and abnormal cell populations. That is, two or more parameters are collected per cell. This can either be multicolour immunofluorescence or a combination of light scatter (forward or 90^o) and a fluorescence parameter.

6. Multiple window analysis can be performed when obvious cell populations can be discriminated by light scatter or antigen expression. However, this should be performed in conjunction with the entire population under study since it is recognised that normal elements within a population provide excellent antibody controls.

7. The reporting of percentages is not recommended for leukaemia samples due to the difficulties encountered with the expression of many antigens. Reference to percentage positivity in normal tissues such as bone marrow or peripheral blood has little relevance to leukaemic samples. Instead an estimate of the strength of expression of the antigens on a detected abnormal population should be made. This should be reported as "dim" or "weak" expression where there is some overlap with the appropriate negative control. The report can then be in the form of a qualitative description of the phenotype of the leukaemia cells.

8. It is beyond the scope of these guidelines to present a detailed analysis of different leukaemias because of the wide range of immunophenotypes that can be encountered. It is recommended to obtain familiarisation with the expression of antigens on normal cells and to use reliable reference sources such as "Report of the International Leukocyte Antigen Workshop".

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 fluorescence 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. This should also include the source, specification sheet, expiry date, etc., and the cluster designation (CD) as well as the clone number of the antibodies used.

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 designation (CD) with a short description of the main antigen recognition characteristics.

3. For unclustered antibodies, report the clone name with a short description of the main antigen recognition characteristics.

4. Report all data indicating the phenotype of the detected abnormal population. If necessary,e.g. in the case of detection of minimal residual disease, make an estimation of the percentage of abnormal cells in the total cell population.

QUALITY ASSURANCE

1. Where possible, the laboratory should belong to and participate in a recognised external Quality Assurance program.

2. Each laboratory should determine the quality of the reagents on a regular basis and at least when changing to new lot numbers. Antibodies should be tested on appropriate cell lines and freshly prepared leucocytes to determine whether they can be used to measure the level of antigen expression.

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 AS2211 - 1991, Laser safety.

(ii) Australian Standard AS 2243.3 ­ 1995, Safety in laboratories, Part 3: Microbiology.

(iii) National Committee for Clinical Laboratory Standards M29­T, Protection of laboratory workers from infectious disease transmitted by blood, body fluids and tissue.

(iv) Morbidity Mortality Weekly Report 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. National Committee for Clinical Laboratory Standards. Vol 12 No. 6. Quality Assurance and Immunophenotyping of Peripheral Blood Lymphocytes.

3. Muirhead, K.A., Wallace, P.K.,Schmitt, T.C., Rescatore, R.L., Ranco, J.A., Horan, P.K. Methodological considerations for implementation of lymphocyte subset analysis in a clinical reference laboratory. In Clinical Cytometry. M. Andreeff, ed. Ann. N.Y. Acad. Sci. Vol. 468, pp 113­127, The New York Academy of Sciences, New York, N.Y., 1986.

4. Loken, M. R., Meiners, H., Terstappen, LW. M. Comparison of sample preparation techniques for flow cytometric analysis of immunofluorescence. Cytometry Supplement 2 : 53, 1988.

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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