ASHI Governance 

Standards for Histocompatibility Testing
(4/98) Copyright © 1995 - 2002 American Society for Histocompatibility and Immunogenetics. All rights reserved.

Section Q - Flow Cytometry

These standards apply to histocompatibility testing and leukocyte phenotyping by flow cytometry.

Q1.000 Instrument Standardization/Calibration

Q1.100 An optical standard , consisting of latex beads or other uniform particles, shall be run to insure proper focusing and alignment of all lenses in the path for both the exciting light source and signal (light scatter, fluorescence, etc.) detectors.

Q1.110 The optical standard shall be run each time the instrument is turned on and any time maintenance, adjustments or sample problems likely to have altered optical alignment (obstruction of fluidics) occur during operation.

Q1.120 The results of optical focusing/alignment must be recorded in a daily quality control log.

Q1.130 A threshold value for acceptable optical standardization must be established for all relevant signals for each instrument and the focusing procedure repeated until these values are achieved or surpassed.

Q1.140 In the event a particular threshold value cannot be attained, a written protocol for instituting corrective action must be available. This protocol should include appropriate corrective actions including clear guidelines describing when a service call is warranted.

Q1.200 A fluorescent standard for each fluorochrome to be used, shall be run to insure adequate amplification of the fluorescent signal(s) on a day-to-day basis.

Q1.210 This standard may be incorporated in the beads or other particles used for optical standardization or may be a separate bead or fixed cell preparation.

Q1.220 The fluorescent standard must be run each time the instrument is turned on and any time maintenance, adjustments or sample problems likely to have altered the gain or high voltage settings (e.g.obstruction of fluidics) occur during operation.

Q1.230 The results of fluorescent standardization shall be recorded in a daily quality control log.

Q1.240 In the event that acceptable fluorescence separation cannot be attained, a written protocol for instituting corrective action must be available. This protocol should include appropriate corrective action including clear guidelines describing when a service call is warranted.

Q1.300 If performing analyses that require the simultaneous use of two or more fluorochromes, an appropriate procedure must be used to compensate for “spill over” into the other fluorescence detectors.

Q1.400 For laser based instruments, the current input (amps) and laser light output (milliwatts), at the normal operating wavelength measured after the laser is peaked and normal operating power set, must be recorded as part of a daily quality control record.

Q2.000 FLOW CYTOMETRIC CROSSMATCH TECHNIQUE

Q2.100 A multi-color technique is highly recommended. However, if a single color technique is used, the purity of the isolated cell population must be documented and should be of sufficient purity to define the population for analysis.

Q2.110 The binding of human immunoglobulin should be assessed with a fluorochrome labelled (e.g.fluorescein) F(ab’)2 anti-human IgG.

Q2.120 Binding of antibody to T cells, B cells and/or monocytes should be positively confirmed with a differently labelled (e.g. phycoerythrin) monoclonal antibody that detects the corresponding cluster designated antigen (e.g.CD3 for T cells, CD19 or CD20 for B cells and CD14 for monocytes).

Q2.130 Multicolor staining of other immunoglobulin classes and target cells may also be justified.

Q2.140 Each laboratory should establish and document the optimum serum/cell ratio i.e. a standard number of cells to a fixed volume of serum.

Q2.200 Controls

Q2.210 The normal human serum control should be from a non-alloimmunized and otherwise healthy individual and must be screened by flow cytometry to insure lack of reactivity against human lymphocytes.

Q2.220 The positive control should be human serum containing antibodies of the appropriate isotype, specific for the HLA antigens or any other alloantigens deemed to be important for detection in the crossmatch.. Positive controls should react with lymphocytes of all humans..

Q2.230 The anti-human immunoglobulin reagent should be titered to determine the dilution with optimal activity (signal to noise ratio). If a multicolor technique is employed, the reagent must not demonstrate crossreactivity with the other immunoglobulin reagents used to mark the cells..

Q2.240 Regardless of the method used for reporting raw data(mean, median, mode channel shifts or quantitative fluorescence measurements), each lab must establish its own threshold for discriminating positive reactions. Any significant change in protocol, reagents or instrumentation requires repeat determination of the positive threshold.

Q2.300 Interpretation

Q2.310 Each laboratory must define the criteria used to define positive and negative crossmatches.

Q3.000 IMMUNOPHENOTYPING BY FLOW CYTOMETRY

Q3.100 Terminology used must be defined and/or conform to nomenclature recommended/approved by the most recent International Workshop of Differentiation Antigens of Human Leucocytes or other appropriate scientific organizations.

Q3.200 Cell Preparation.

Q3.210 The method used for cell preparation should be documented to yield appropriate preparations of viable cells.

Q3.220 The viability of cell preparations should be recorded and should exceed the laboratory’s established minimum standards for each procedure used.

Q3.230 For internal labelling, the method used to allow fluorochrome labelled antibodies to penetrate the cell membrane must be documented to be effective.

Q3.300 Labelling of specimens.

Q3.310 Specificity controls, consisting of appropriate cell types known to be positive for selected standard antibodies must be run within laboratory-defined intervals sufficiently short to assure the proper performance of reagents.

Q3.320 A negative reagent control(s) shall be run for each test cell preparation. This control should consist of monoclonal antibody(ies) of the same species and subclass and should be prepared/purified in the same way as the monoclonal(s) used for phenotyping.

Q3.330 For indirect labelling, the negative control reagent should be an irrelevant primary antibody, if available, and in all cases, the same secondary antibody(ies) conjugated with the same fluorochrome(s) used in all relevant test combinations.

Q3.340 For direct labelling, the negative control reagent should be an irrelevant antibody conjugated with the same fluorochrome and at the same fluorochrome:protein ratio used in all relevant test combinations.

Q3.350 Whether analyzed directly or fixed prior to analysis, labelled cells must be analyzed within a time period demonstrated by the laboratory to avoid significant loss of any cell subpopulation or total cell numbers. Control samples must be analyzed within the same period after staining as the test samples.

Q3.360 If analysis will be based on a population of cells selected by flow cytometry “gating” on size or density parameters, or selected by depletion or enrichment techniques, control stains must be run for each test individual to detect the presence of contaminating cells in the selected population. (e.g. Monocyte contamination of ‘lymphocytes’ gated by forward angle or forward angle vs 90° light scatter must be detected with a monocyte specific marker antibody.

Q3.370 Conclusions about abnormal proportions or abnormal numbers of cells bearing particular internal or cell surface markers must only be drawn in comparison with local `control’ data obtained with the same instrument, reagents and techniques.

Q3.380 Determination of percent positives must take into consideration the results of the negative control reagent. However, when clearly defined positive and negative populations are evident in the test sample, it may be appropriate to adjust the threshold based on the test sample.

Q3.400 REAGENTS

Q3.410 The specificity of monoclonal antibodies shall be verified by published and/or manufacturer’s documentation and whenever possible verified locally through tests with appropriate control cells prepared and tested by the same method employed in the laboratory’s test sample analysis.

Q3.420 The quantities of reagents used for each test sample must be determined by the manufacturers or from published data and whenever possible should be verified locally by appropriate titration procedures.

Q3.430 Reagents must be stored according to manufacturers’ instructions or according to conditions verified to maintain stability by documented local tests.

Q3.440 Monoclonal antibodies which have been reconstituted from lyophilized powder form for storage at 4°C should be centrifuged according to the manufacturer’s instructions or locally documented procedures to remove microaggregates prior to use in preparation of working stains.

Q4.000 HLA TYPING BY FLOW CYTOMETRY (e.g. HLA B27)

Q4.100 Terminology used must be defined and/or conform to nomenclature recommended/approved by the most recent W.H.O. nomenclature committee meeting.

Q4.200 Cell Preparation.

Q4.210 The method used for cell preparation should be documented to yield appropriate preparations of viable cells.

Q4.220 The viability of cell preparations should be recorded and should exceed the laboratory’s established minimum standards for each procedure used.

Q4.2300 Labelling of specimens.

Q4.2310 A negative reagent control(s) shall be run for each test cell preparation. This control should consist of monoclonal antibody(ies) of the same species and subclass and should be prepared/purified in the same way as the monoclonal(s) used for phenotyping. Negative reagent controls should consist of:

Q4.2311 For indirect labelling, an irrelevant primary antibody, if available, and in all cases, the same secondary antibody(ies) conjugated with the same fluorochrome(s) used in all relevant test combinations.

Q4.2312 For direct labelling, an irrelevant antibody conjugated with the same fluorochrome and at the same fluorochrome: protein ratio used in all relevant test combinations.

Q4.2320 Whether analyzed directly or fixed prior to analysis, labelled cells must be analyzed within a time period demonstrated by the laboratory to avoid significant change in test results. Control samples must be analyzed within the same period after staining as the test samples.

Q4.3000 Reagents.

Q4.3100 The specificity of monoclonal antibodies shall be verified through tests with appropriate control cells prepared and tested by the same method employed in the laboratory’s test sample.

Q4.3200 Cell controls must be tested for each batch of monoclonal antibodies received.

Q4.3210 The control cells must include at least five cells known to express the specified antigen.

Q4.3220 The control cells must also include two cells for each crossreacting antigen which might be confused with the specific antigen.

Q4.3230 The control cells must also include at least two cells lacking the specific and crossreacting antigens.

Q4.3300 The quantities of reagents used for each test sample must be determined by the manufacturers or from published data and whenever possible should be verified locally by appropriate titration procedures.

Q4.3400 Reagents must be stored according to manufacturer’s instructions or according to conditions verified to maintain stability by documented local tests.

Q4.3500 Monoclonal antibodies which have been reconstituted from lyophilized powder form for storage at 4 degrees centigrade should be centrifuged according to the manufacturer’s instructions or locally documented procedures to remove microaggregates prior to use in preparation of working stains.

Q4.3600 A single monoclonal antibody may be used to define an antigen provided its monospecificity has been sufficiently verified by local testing.

Q4.3700 Minimum reactivity for assignment of a positive reaction must be established by the laboratory.

Q4.3800 If the monoclonal antibody(ies) is (are) known or found to react with antigens other than the one specified, a written protocol must explain how its presence or absence is finally determined.