AFCG Clinical Standards: HLA-B27

AUSTRALASIAN FLOW

CYTOMETRY GROUP

JUNE 1996



RECOMMENDED GUIDELINE STANDARDS FOR

HLA-B27 TYPING BY FLOW CYTOMETRY



1994/1995 COMMITTEE MEMBERS

Mr Arnold Kabral, Mr Peter McCardle, Mr Graeme Morrison, Mr Lyndsay Peters

1996 COMMITTEE MEMBERS

Ms Kerryn Weekes, Ms Deborah Shapira, Mr Peter Hobson



CONTENTS

Introduction

Laboratory Safety

Specimen Collection

Specimen Transport

Specimen Integrity

Specimen Processing

Flow Cytometer Quality Control

Sample Analysis

Data Storage

Data Reporting

Quality Assurance

References

Appendix 1: Determination of Reference Ranges

Appendix 2: Cell Freezing and Thawing

Appendix 3: Gating Control

Appendix 4


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.

Determination of the tissue antigen HLA-B27 is important because of the strong association with ankylosing spondylitis, a chronic inflammatory disease affecting the axial musculo-skeletal system.

Traditionally, it has been the lymphocytotoxicity assay that was used to determine HLA status1. The development of monoclonal antibodies to HLA antigens has rendered flow cytometry a proficient alternative procedure. As with the lymphocytotoxicity assay, peripheral blood lymphocytes are used as the marker population. In this respect flow cytometry procedures for HLA status will generally follow the lymphocyte subset immunophenotyping procedure.

The advent of monoclonal antibodies to the HLA-B27 antigen led the way for a flow cytometric method2. The early clones suffered because of their cross-reactivity with other HLA antigens, especially HLA-B7. False positives result when homozygous B7 expression occurs, and this allelic configuration has a population frequency of approximately 6%. HLA-B27 is expressed in about 9% of the Caucasian population.

Problems may arise, however, when antibody cross-reactivity occurs between different HLA antigens. Combating this requires the use of either monospecific antibodies or procedures which block the expression of irrelevant antigen3. The cytotoxicity assay may also be utilised as a confirmation of positive antigen expression.

HLA-B27 typing by flow cytometry is performed as a lysed whole blood technique using a single colour, directly conjugated antibody and gated peripheral blood lymphocytes as the marker population.


LABORATORY SAFETY

Each laboratory will adopt internal procedures and policies for the safe handling of biological specimens.

1. Use universal precautions4 with all specimens.

2. Develop appropriate internal procedures to cope with accidents such as spillage.

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 mins before leaving a safe biological confinement area.

5. Final cell suspension should be in a 0.5% ­ 1% available reactive 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 manufacturers' 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 precautions4 should be strictly observed when collecting blood samples.

2. Each specimen should be labelled with the patient 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 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. A total white cell count 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. Heparin or ACD anticoagulated specimens may be processed immediately or up to 48 hours after collection.

7. Any specimen more than 72 hours old, or unlabelled, or incorrectly labelled or short collection should be re-collected.

8. Frozen whole blood or gradient separated peripheral blood mononuclear cells (PBMC) may also be used.


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 18o ­ 22oC in a light proof container.

3. Temperatures below 10oC or above 37oC 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 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 report should reflect the effect that these deficiencies may have on the results.


SPECIMEN PROCESSING

1. Whole blood lysis of red cells is recommended for routine analysis. Most methods of HLA-B27 analysis5-11 employ either a single colour direct immunofluorescence or a second colour conjugated to the T-cell marker CD3 in a dual assay on a gated lymphocyte population (see Appendix 1 for lymphocyte gating).

2. Several lysing techniques are available12. These include water, ammonium chloride, and hypotonic buffer. 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. It is important to note that valid results will also be obtained from gradient density separated PBMC. These cells can be stored in frozen aliquots and either be used as controls or tested at a later date (see Appendix 2).

3. Where possible, a full blood count and differential must be performed before processing. If this is not possible, each laboratory must have a procedure to identify specimens with abnormal leucocyte counts and correct for any associated artefacts. Specimens with pronounced leucocytopaenia may have insufficient cells for flow cytometric analysis, thus requiring a larger volume of sample or a buffy coat preparation. Conversely, normal concentrations of antibody reagents may be insufficient to saturate all binding sites in specimens with leucocytosis, leading to possible false negative results. Samples with leucocytosis need to be diluted before testing.

4. For each lot of given reagent, the laboratory should verify that the manufacturer's recommended amount of antibody gives optimal positive/negative resolution and optimal positive staining intensity with the laboratory's method of sample preparation. For any deviation from the manufacturer's recommended staining conditions (e.g. time, volume, temperature, or cell number), the laboratory must determine the minimum amount (volume and/or weight) of reagent required to give optimal positive/negative resolution and optimal positive staining intensity.

This is best done by using the following HLA typed control samples: HLA-B7, HLA-B27 negative, HLA­B27 positive, and an HLA-B7 positive. Control samples need not be collected fresh on the day they can be stored as frozen whole blood aliquots from suitable donors. If liquid nitrogen is unavailable, frozen aliquots may be kept at 80oC (see Appendix 2).

Any deviation from the protocol should be documented in a laboratory protocol book to give results comparable to those obtained using the recommended procedure.

The machine protocol for typing HLA-B27 should then be checked for every new batch of antibody and after every service using these typed controls.

5. For HLA B27 typing, a putative positive result requires confirmation if the primary antibody is known to cross-react with other antigens. To do this one may:


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 performed according to the manufacturer's recommended alignment procedures. These procedures should use the recommended alignment particles, which 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 3, Optical Alignment Log).

Optical alignment can be verified by:

1.3 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.

Note: Overcompensation leads to fewer errors than undercompensation.


(A) (B)

Figure 1


Representation of application of correct compensation. Gated correlated display of anti­CD3 FITC and anti­CD19 PE. (A) Uncompensated. (B) Correctly compensated

Overall system performance can be verified by:


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. The appropriate isotype controls should be run next, followed by the subsequent test panel to investigate the provisional diagnosis.

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 non viable prior to fixation. However, this can be performed using ethidium monoazide (EMA) as described by K. Muirhead, 2nd AFCG Methods Course, 198914.



Definition of a Lymphocyte Gate


Figure 2

Representation of common ways of displaying correlated low angle versus 90o angle light scatter seen from lysed whole blood preparations. (L = predominantly lymphocytes, M = predominantly monocytes, P = predominantly polymorphonuclear leucocytes).

4. Count at least 2000 gated events in each sample. This number assures with 95% confidence that the result is < 2% 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 severely leucocytopaenic specimens.

6. Set leucocyte 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 taken are to adjust light scatter gates and re-analyse the immunofluorescent correlated two colour plot.

Typical satisfactory values for lymphocytes are 90% of all lymphocytes and 85% purity in the gate as determined by CD45-FITC/CD14-PE gating control (Appendix 3 ).

8. If levels of contamination by non-lymphocytes cannot be minimised to within acceptable limits, then test results may be suspect. If this contamination cannot be explained by reinterpretation of the data, a second specimen should be requested.

9. Most commercially available directly conjugated reagents give good resolution between low intensity negative and higher intensity positive cell populations. When simultaneous two-colour immunofluorescent correlated data are 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 cytometry 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. Ideally listmode data on all samples analysed should be retained. This allows for the complete re-analysis of the raw data. At a minimum retain correlated dual fluorescent data for each test and any interpretive comments.

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 positive/negative or equivocal.

3. Report description and results of confirmatory testing where applicable.


QUALITY ASSURANCE

1. Where possible, the laboratory should belong to and participate in a recognised external Quality Assurance program such as the RCPA Quality Assurance Programs Pty Ltd, HLA-B27 program.

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.


REFERENCES

1. Terasaki, PI, and McClelland, JD (1964): Microdroplet assay of human serum cytotoxins. Nature 204:998-1000.

2. Albrecht, J, and Muller, HAG (1987): HLA-B27 typing by use of flow cytofluorometry. Clin. Chem. 33:1619-623.

3. Trapani, JA, Vaughan, HA, Tait, BD, McKenzie, IFC (1988): Immunoradiometric assay for the rapid detection of HLA-27. Immunol. Cell Biol. 66: 215-19.

4. 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:

5. Pei, R, Ar jomand-Shamsai, M, Deng, CT, Cesbron, A, Bignon, JD, Lee, JH (1993): A monospecific HLA-B27 fluorescein isothiocyanate-conjugated monoclonal antibody for rapid, simple and accurate HLA-B27 typing. Tissue Antigens, 41:200-203.

6. Janssen, WCM, Rouwen, JACN, and Hoffman, JJML (1992): Improved flow cytometric method for HLA-B27 typing. Ann. Clin. Biochem. 29: 663-669.

7. Zuber, C, Ulrich, G, Monseaux, S, Cado, S, and Parmentier, S. (1994). Reliable flow cytometry HLA-B27 typing with B27-FITC/B7-PE combination. Cytometry Suppl. 7:77.

8. Orr, K, Thomson, GTD, and Alfa, M. (1994): Utilization of commercial antisera and flow cytometry in HLA-B27 typing. Cytometry 18:17-20.

9. Hulstaert, F, Albrecht, J, Hannet, T, Lancaster, P, Buchner, L, Kunz, A, Falkenrodt, A, Tongio, M, De Keyser, F, Veys, EM (1994): An optimized method of routine HLA-B27 screening using flow cytometry. Cytometry 18:21-29.

10. Reynolds, WM, Evans, PR, Lane, AC, Howell, WM, Wilson, PJ, Wong, R, and Smith, JL (1994): Automated HLA-B27 testing using the FACS prep/FACScan system. Cytometry 18:109-115.

11. Ward, AR, and Nikaein, A (1995): Comparison of monoclonal antibodies for flow cytometry analysis of HLA-B27 antigen. Cytometry 22:65-69.

12. Loken, MR, Meiners, H, Terstappen, LWM (1988): Comparison of sample preparation techniques for flow cytometric analysis of immunofluorescence. Cytometry Supplement 2:53.

13. Steffens-Nakken HM, Zwart, G, and van der Bergh, F (1995): Validation of allele-specific polymerase chain reaction for DNA typing of HLA-B27. Clin. Chem 41(5):687-692.

14. Muirhead, KA, Wallace, PK, Schmitt, TC, Rescatore, RL, Ranco, JA, Horan, PK. 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, NY, 1986.



APPENDIX 1: DETERMINATION OF REFERENCE RANGES

1. Definitions

Reference values: Set of values for a measured quantity.

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. Procedure for Determining Reference Ranges

Statistical methods, both parametric and nonparametric, and graphical methods are discussed in detail in references 1-3. Only a brief summary of the steps involved is presented here.

3.0 Pitfalls in Determining Flow Cytometric Reference Ranges

Each laboratory should determine its own reference range using its particular preparation method and instrumentation because significant laboratory-to-laboratory 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 intervals 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 (4-5).

One practical solution to the dilemma is to accumulate and analyse reference data in smaller sets (eg. 10-20 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 TO APPENDIX 1

1. Winkel, P, and Statlan, BE. Reference values. In Clinical Diagnosis and Management by Laboratory Methods (ed. J B Henry), Philadelphia, WB Saunders Co, 1979, pp 19-52.

2. Martin, HF, Gudzinowicz, BJ, Fanger, H. Normal Values in Clinical Chemistry. New York, Marcel Dekker, 1975, pp 102-236.

3. Henry, RJ, Cannon, DC, Winkelman, JW. Clinical Chemistry Principles and Technics. New York, Harper and Row, 1974, pp 102-236.

4. McCarthy, RC, and Fetterhoff, TJ. Issues in Quality Assurance in Clinical Flow Cytometry. Arch. Pathol. Lab. Med. 113:658-666, 1989 (in press).



APPENDIX 2: CELL FREEZING AND THAWING

REAGENTS

1. Blood cells: These may be density gradient separated peripheral blood mononuclear cells (PBMC), PBMC from buffy coats, or transformed B-cell lines. This method may not be suitable for whole blood techniques.

2. Cell culture medium: 20-50% foetal calf serum in RPMI or other balanced salt solution. This should be cooled for freezing, warmed for thawing.

3. Freeze mix: Dimethyl sulphoxide (DMSO) at 20% in cell culture mediium.

4. Cryotubes: Prelabelled and chilled if possible.


PROCEDURE

NOTE: All preparation should be carried out on ice as DMSO is toxic to cells at room temperature.

1. Count PBMC.

2. Gently centrifuge cells and resuspend in cell culture medium at 107 per ml.

3. Place cells in a small beaker on ice and slowly add to the centrifuge tube an equivalent volume of pre-cooled freeze mix, mixing well.

4. Pipette 1 mL aliquots into cryotubes.

5. As quickly as possible place tubes in an insulated container into 70oC overnight. Cells may be stored at this temperature for many months if liquid nitrogen storage is unavailable.

6. Thaw cells as quickly as possible by warming the cryotube in a 37oC water bath.

7. Pipette into a centrifuge tube containing pre-warmed cell culture medium or equivalent and centrifuge as before.

8. Reconstitute cell pellet as desired.


APPENDIX 3: GATING CONTROL

An example of a gating control for lymphocytes where contamination cell types include but are not limited to the following:



Figure 1. Lymphocyte gating utilising CD14 and CD45.

1 = nonleukocytes

2 = polymorphonuclear leukocytes

3 = lymphocytes

4 = monocytes

Lymphocyte gates should be adjusted to maximise number of cells in region 3 and minimise cells in regions 1,2, and 4.


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