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 Robyn Minchinton
10. AUTO- AND ALLO- ANTIBODIES
CONTENTS
10.1 Introduction
10.2 Specimen Collection, Transport and Integrity
10.3 Specimen Processing and Storage
10.4 Controls
10.5 Sample Analysis
10.6 Data Reporting
10.7 Quality Assurance
10.8 References
10.1 INTRODUCTION
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.
10.2 SPECIMEN COLLECTION, TRANSPORT and INTEGRITY
10.2.1 Collection
10.2.1.1 Universal precautions should be strictly observed when collecting blood samples (see 1.1 Laboratory Safety).
10.2.1.2 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.
10.2.1.3 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.
10.2.1.4 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.
10.2.2 Specimen Transport
10.2.2.1 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.
10.2.2.2 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.
10.2.3 Specimen Integrity
10.2.3.1 Visually inspect the specimen for clots, haemolysis or container defects. Recollect the sample if the specimen shows any visual signs of deterioration.
10.2.3.2 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.
10.3 PROCESSING AND STORAGE
10.3.1 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 10.3.2 and 10.3.3).
10.3.2 Autoantibody investigations using patients own cells should consist of at least the following tests:
10.3.5 Repeated freeze thawing of patient and/or control sera should be avoided to minimise immunoglobulin aggregate formation and deterioration of the antibody(ies).
10.3.6 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.
10.4 CONTROLS
10.4.1 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.
10.4.2 Controls:
10.4.2.1 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.
10.4.2.2 Positive control sera should be confirmed by an established reference laboratory.
10.4.2.3 Each laboratory should establish reference ranges for the antigens being tested (see 5.11 Determination of Reference Ranges).
10.5 SAMPLE ANALYSIS
10.5.1 Sample order - all control specimens should be run first and then, according to laboratory priority, run the patient samples.
10.5.2 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)1.
10.5.3 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.
10.5.4 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.
10.5.5 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.
10.5.6 Set gates as broadly as possible consistent with acceptable levels of contamination to minimise the exclusion of cells of interest.
10.5.7 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.
10.5.8 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.
10.6 DATA REPORTING
10.6.1 Report all unique patient identifiers.
10.6.2 Report all data in terms of cluster of differentiation (CD) with a short description of the main antigen recognition characteristics.
10.6.3 For unclustered antibodies report the clone name or the systematic name for human antibodies with a short description of the main antigen binding characteristics.
10.6.4 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.
10.6.5 Reference limits for test results should be determined by each laboratory and these should be quoted in the report.
10.7 QUALITY ASSURANCE
10.7.1 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.
10.7.2 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. e.g. tube to tube variation can be monitored by the inclusion of the same antibody in separate tubes within the one patient test series.
10.8 REFERENCES
1. 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. Ann NY Acad Sci 1986;468:113127.