Quality Control in Clinical Flow Cytometry
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1 Clin Lab Med 27 (2007) Quality Control in Clinical Flow Cytometry Teri A. Oldaker, CLS (NCA), ASCP (QCYM) Genzyme Genetics, 5300 McConnell Avenue, Los Angeles, CA 90066, USA Flow cytometry assays cover a wide subspecialty of testing. One category of flow cytometric evaluation is the enumeration of well-defined CD4-positive or CD34-positive cell populations. For this type of assay, well-established procedures have been standardized and proved robust and reproducible in many laboratories [1,2]. The more complex leukemia/lymphoma immunophenotyping assays use the same flow cytometry instrumentation and techniques; however, they involve the recognition of many different and, many times, subtle hematologic neoplasms. Such interpretation requires knowledge of the maturation and expression patterns of normal and abnormal populations [3]. A comprehensive quality assurance/quality management program designed for these complex assays is necessary to ensure that the results obtained are accurate, consistent, and reproducible over time regardless of laboratory variables, such as staff, reagents, sample preparation, acquisition, and analysis [4]. Components of quality management system The goal of an effective clinical laboratory quality management system (QMS) is to maintain those established assay specifications consistently over time regardless of variables that are introduced into the system. Assay specifications include, but are not limited to, precision, accuracy, sensitivity, specificity, reference range, and stability. Many factors existing in clinical flow cytometry laboratories are responsible for variation. First are factors inherent to the biologic sample; second is the technical complexity of the instrumentation; and third are the immunologic and other reagent factors. Laboratories use numerous staff, use multiple instruments, and make or purchase new lots of reagents and controls during the laboratory process. In a flow cytometry laboratory, the instrumentation is technically complex; address: teri.oldaker@genzyme.com /07/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi: /j.cll labmed.theclinics.com
2 672 OLDAKER specimen collection, transport, and integrity are critical; and the reagents (especially monoclonal and polyclonal antibodies) must be optimized and verified on use [5,6]. All these variables must be controlled strictly to maintain standardization and accuracy in reported results. The following are critical components of an effective laboratory QMS [7]: 1. Validation/change control 2. Quality control (QC) 3. Proficiency testing 4. Document control of standard operating procedures 5. Training/competency assessment 6. Quality improvement This article focuses on QC; other aspects of a complete QMS are addressed in an article by Carey and Oldaker elsewhere in this issue. Quality control QC is defined as a continuous system that monitors all operational techniques and activities during the analytic (testing) phase of a process, with the goal of ensuring valid and reproducible results. In flow cytometry, many QC assessments are used: instrument QC, reagent/antibody QC, and process (assay) QC. In addition, other verifications can be made to ensure appropriate specimen integrity is maintained throughout the testing process [8]. A written QC policy is required for each assay, which includes the number, source, and frequency of control material and the tolerance limits for control values. QC must be documented with each assay, approved by the laboratory director or designee, and retained in the laboratory for at least 2 years [9]. Instrument quality control In clinical flow cytometry, it is critical to maintain optimized instrument settings to ensure laser alignment, function of photomultiplier tubes (PMT), and optimal fluidics. This is critical especially in the assessment of leukemia/ lymphoma immunophenotyping, where subtle changes in light scatter or fluorescence allow for the identification of aberrant populations. Flow cytometer instrument QC consists of the initial instrument setup and daily monitoring of performance of the established settings. The initial setup is performed at initial installation and after a major repair to determine optimum settings for the analysis of patient samples and to establish tolerance limits on these settings for daily monitoring [10 12]. Initial instrument setup The initial instrument setup includes establishing optimal laser, fluidics, optical filter and lenses, PMT, log and linear amplification, and the laser
3 QUALITY CONTROL 673 alignment. Laser alignment is the process of focusing the laser to the cell stream and optimizing the emitted light signals with the detectors and subsequent filters, resulting in the brightest and tightest signal. The goal of optical alignment is to produce the brightest fluorescent and light scatter signals (as measured by mean fluorescent channel [MFC]) and the least variation (as measured by percent coefficient of variation). Suboptimal alignment can lead to decreased fluorescence and light scatter signals, high signal variation, and increased noise, thereby decreasing sensitivity [10]. In clinical flow cytometers, the laser alignment is performed by a qualified service engineer and the alignment is fixed. Some clinical laboratories use research flow cytometers that have sorting capabilities. These instruments need to be aligned daily by an operator because of increased variability in the stream in air configuration. Daily monitoring of performance Once an initial instrument setup is performed, the laboratory must establish tolerance limits to be used for the daily instrument monitoring. Commercial type IIA latex beads are reanalyzed to determine instrumentspecific tolerance limits. This is performed by obtaining at least 20 datasets to create an adequate database of values for each scatter and fluorescent parameter to be assayed. Daily target ranges are established by calculating the geometric mean of the dataset and a SD range. Once the instrument-specific tolerance ranges are established, the clinical flow cytometry laboratory staff is responsible for the daily monitoring of performance for all components of the instrumentation on a daily basis or after each cold start. This ensures that standardization and reproducibility of these settings (and thereby results) can be maintained over time. Performance can be monitored in two ways: PMT settings or MFCs. The first approach is to place the target channel in a predefined position by adjusting the PMT settings. This placement ensures that the beads and, thereby, specific assay cell populations fall in the same position at each operation (Fig. 1). PMT settings then are recorded and compared to the tolerance limits. PMT settings can be displayed longitudinally using Levy-Jennings plots to assess trends and variations (Fig. 2). Evidence of shifts or trends in PMT settings may indicate a potential instrument problem and should be investigated. Most clinical flow cytometry vendors use this method and supply beads and respective automated software systems to perform this monitoring task easily (eg, CaliBRITE beads with FACSComp software by BD Biosciences [San Jose, California] and Flow-Set beads with Cyto-Comp or ADC software by Beckman Coulter [Miami, Florida]). The second approach is to keep the instrument PMT settings constant and monitor and document the MFC for each fluorochrome and light scatter parameters. This method is advantageous when a variety of fluorochromes and multiple protocols with specific instrument settings are used. In both situations, data should be collected, compared to established
4 674 OLDAKER Fig. 1. Example of optimizing beads where the MFI for FL1 is placed into a target range with a tight coefficient of variation (%CV). This maximizes the signal-to-noise ratio and minimizes the variation. instrument-specific tolerance limits, and displayed on Levy-Jennings plots for longitudinal assessment of data reproducibility [13]. New lot numbers of beads must be evaluated prior to implementation of daily instrument monitoring QC [2,12,14]. An optimized flow cytometer optical alignment, sensitivity, resolution, and appropriate color compensation (described later) are critical in the assessment of samples for hematologic neoplasms. It is essential that instruments be able to discriminate two closely related fluorescent intensities and distinguish dimly fluorescent populations from background fluorescence, auto fluorescence, and instrument noise. Because hematologic neoplasms are characterized by aberrant antigen expression (over- and underexpression), the ability to distinguish the neoplastic cells from the normal counterpart is critical and highly dependent on optimal instrument settings [15 19].
5 QUALITY CONTROL 675 Fig. 2. Example of a Levy-Jennings plot that monitors the PMT settings of each parameter that places the beads into a set target range. Longitudinal display of PMT settings can identify potential instrument problems. Color compensation In flow cytometry, when more than one fluorochrome are used concurrently that have overlapping emission spectrums; this spectral overlap needs to be eliminated or subtracted by a process called color compensation. Band pass filters can be used to subtract out some of the spectral overlap, but in most cases one portion of a detectors signal needs to be subtracted from another. This subtraction can be accomplished by using instrument settings or, more recently, with software manipulation. Fluorescent compensation settings are linked to other instrument parameters; therefore, if changes to PMT, voltage or gain settings, laser power, or optical filters are made, compensation settings must be revalidated. The more fluorochromes used, the more complex the compensation process. In addition to the number of fluorochromes, the use of tandem dyes can complicate compensation further. Tandem dyes
6 676 OLDAKER (example PE-Cy7) use fluorescent resonance energy transfer such that a donor fluorochrome is excited by the laser and the released energy is transferred to an acceptor molecule, which emits at a higher wavelength. Some tandem leakage can occur by the donor molecular; therefore, it is critical when designing panels to minimize the impact on the leakage. To assess compensation, unstained and singly stained control cells are stained with each fluorochrome used in the specific assay. The median value of the fluorescent signal should be equivalent for negative, dim, and bright populations. If beads are used to assess compensation, the settings must be verified with stained cells because of minor differences in antibody bound to beads versus bound to cells [20 22]. Traditionally, compensation was performed using the instrument hardware. With the complexity related to increasing numbers of fluorochromes, however, it is recommended that if more than four fluorochromes are used in the test system that compensation be performed using software-assisted compensation programs (such as FCS Express [DeNovo Software, Thornhill, Ontario, Canada], WinList [Verity Software House, Topsham, Maine], or FloJo [Tree Star Inc., Ashland, Oregon]) [14]. Appropriate compensation is essential when performing leukemia and lymphoma immunophenotyping. If samples are undercompensated, two discrete populations may fuse into one population, possibly causing a misdiagnosis or misidentification of hematologic neoplasm (Figs. 3 and 4). Linearity and sensitivity A linear response of the flow cytometer to each of the emitted fluorescent signals is critical in measuring antigen density on the surface of cells. Although only a requirement for laboratories performing DNA content analysis and quantitative flow cytometry, the assessment of the instrument linearity is recommended on a periodic basis to verify the instrument linear performance. Signal measurements at the low and high end of the fluorescent scale should be related to the signal intensity, especially when comparing one instrument or laboratory to another. Linearity assessment can be performed by running a series of type III microbeads with at least four defined levels of fluorescent intensity and a nonfluorescent bead. Fluorescent intensity is plotted against the known molecules of equivalent soluble fluorochrome. A linear regression equation between the predefined fluorescent values of the beads and the instruments response is computed and evaluated against established tolerance limits (Fig. 5) [12]. Instrument correlation When multiple flow cytometers are used in the same laboratory, it is essential that equivalency of instrument output is established and monitored at least twice per year [23,24]. This can be performed by running the same stained tubes on multiple instruments and comparing the results for equivalency. In assays that are reported quantitatively (for example, CD4 and CD34), the correlated assay results must fall within the established interassay
7 QUALITY CONTROL 677 Fig. 3. Example of (A) uncompensated, (B) overcompensated, and (C) properly compensated data. precision established at validation. For qualitative assays, such as leukemia/ lymphoma immunophenotyping, both categoric concordance for each of the markers run and equivalency in mean fluorescent intensity (MFI) also is required; for example instrument #1 CD19 ¼ dim, and instrument #2 CD19 ¼ dim. Such comparisons confirm whether or not the fluorescent intensities and light scatter properties are reproducible from instrument to instrument, thereby yielding clinically identical results. These semiannual instrument correlations must be documented along with the laboratories acceptability criteria (Table 1). Preventative maintenance Like any complex equipment, following manufacturers periodic preventative maintenance is essential to maintaining optimal instrument operation and ensuring the longevity of the equipment. In the case of high-volume clinical laboratories, it may be prudent to increase the frequency of maintenance in order to minimize down time. Documentation of the maintenance procedures is Fig. 4. Both histograms display the same data, CD45 versus CD4. The histogram on the left is compensated improperly and the CD45 and CD4 blend together, where the histogram on the right is compensated properly, allowing appropriate separation from these two populations, CD45 and CD4 dim.
8 678 OLDAKER GENZYME LOS ANGELES FLOW CYTOMETRY LAB PMT LINEARITY QC RECORD PEAK # CH # MEFL MEFL LOG CALC. RESIDUAL CALC. MEFL % % % % % % % Slope: Intercept : Rsq: SPHERO CALIBRATION GRAPH (FITC Channel) MEFL R 2 = RELATIVE CHANNEL NUMBER Rainbow Calibration Particles (RCP-30-5A) Lot No.: W02 File # FC7 Acceptable: YES By: GT Date: 1/30/07 Action taken if not linear: Fig. 5. This is an example of a linearity/sensitivity assessment performed on a fluorescein-5-isothiocyanate FITC channel. required as is documentation of problems encountered and service rendered. This documentation can assist in instrument troubleshooting and is required to remain in the laboratory for the life of the instrument. Reagent/antibody quality control In flow cytometry, monoclonal and polyclonal antibodies are the most critical and active assay reagent. An optimized ratio of antibody to antigen
9 QUALITY CONTROL 679 is required for accurate results. For in-vitro diagnostic (IVD) use flow cytometry assays (such as CD4 enumeration), manufacturers titered and established the amount of antibody and amount of sample to be used in the assay. For leukemia/lymphoma immunophenotyping assays, the antibodies used are considered analyte-specific reagents by the United States Food and Drug Administration and, therefore, clinical laboratories are responsible for determining the optimum titration for the specific assay during the validation procedure. This is critical in the assessment of leukemia/lymphoma immunophenotyping where too little antibody present in a dim fluorescent antigen can be interpreted incorrectly and erroneous result may occur. This optimization is performed by a titration experiment using a 5-point, 2-fold serial dilution of the manufacturers recommendation [10,25]. The highest signal-to-noise ratio determines the best titration to use. In addition to the initial titration, each new lot number of antibody must be tested for equivalency against the in-use lot number prior to putting the reagent in use [24]. On receipt of new lot of antibody, a known positive sample for this antigen should be identified. In many instances, previous cases or normal blood samples contain cells that express the target antigen; for instance, CD3 is expressed on normal T lymphocytes or CD33 is expressed on normal myeloid cells. In this case, a normal sample is run with the old and the new lot numbers of antibody, and the specific population that represents the positive population is gated and analyzed. The categoric concordance and MFI of the expressed antigen are compared for equivalency. Acceptability criteria must be established, met, and documented prior to placing the new antibody lot number in use. An example of acceptability criteria for antibody phase-in may be: dim ¼ dim, bright ¼ bright, moderate ¼ moderate. In the case of an antibody where it may be difficult to find a positive sample, cryopreserved cells, commercial controls, or cell lines can be used. Some laboratories archive frozen cells of rare antigenicity for parallel testing new lots of reagents. Documentation of antibody phase-in is required by all regulatory agencies [23,24]. In addition to antibody verification, all new lots of reagents must be tested for equivalency prior to putting into laboratory use. This includes manufacturer- or laboratory-prepared buffers, lysing reagent, fix and perm reagents, and so forth. Equivalency must be defined for each reagent and objective acceptability criteria defined and documented. These criteria depend on the use of each of the reagents. All reagent phase in tests must be documented. Negative and positive controls For immunophenotyping of leukemia/lymphoma, it is critical to determine negative and positive expression for the population of interest in order to identify neoplastic population and determine the phenotype appropriately. Therefore, each assay must include negative and positive controls as a verification of the staining [5].
10 Table 1 Example of an inter-instrument correlation quality control document using a stained sample run on seven instruments correlating the mean fluorescent intensity (MFI) of all markers. Categoric concordance of each marker mean required and fluorescent intensity concordance is required for acceptance Inter-instrument correlationdleukemia/lymphoma Date: Instrument # FC1 FC2 FC3 FC4 FC5 FC6 FC7 Concordance Antibody MFI MFI MFI MFI MFI MFI MFI P ¼ pass, F ¼ fail Kappa N N N N N N N P Lambda N N N N N N N P HLA-DR N N N N N N N P CD2 N N N N N N N P CD3 N N N N N N N P CD4 N N N N N N N P CD5 N N N N N N N P CD7 N N N N N N N P CD8 N N N N N N N P CD10 N N N N N N N P CD11B N N N N N N N P CD11C MOD MOD MOD MOD MOD MOD MOD P CD13 DIM DIM DIM DIM DIM DIM DIM P CD14 N N N N N N N P 680 OLDAKER
11 CD16 N N N N N N N P CD19 N N N N N N N P CD20 N N N N N N N P CD22 N N N N N N N P CD23 N N N N N N N P CD33 MOD MOD MOD MOD MOD MOD MOD P CD34 N N N N N N N P CD38 DIM DIM DIM DIM DIM DIM DIM P CD45 DIM DIM DIM DIM DIM DIM DIM P CD56 N N N N N N N P CD64 N N N N N N N P CD117 DIM DIM DIM DIM DIM DIM DIM P Acceptability criteria Legend Categoric Concordance BRI ¼ BRI BRI, bright DIM ¼ DIM DIM, dim MOD ¼ MOD MOD, moderate NEG ¼ NEG NEG, negative Normal sample is non malignant case Technologist Supervisor review QUALITY CONTROL 681
12 682 OLDAKER Negative controls are run to establish the level of background (nonspecific or autofluorescent staining) to set threshold analysis quadrants. This can be done one of two ways: set a negative threshold quadrant by using a known negative population in the same analysis tube and comparing that threshold to the unknown populationdfor example, gate on T lymphocytes to set a negative threshold for CD33 expression; alternatively, use a separate tube with matched subtype isotype controls run with each marker to establish the negative quadrant analysis threshold. The first option is easiest and more cost effective and allows for internal positive and negative populations to serve also as positive negative and process controls (discussed later). In addition, it provides opportunities in each tube for positive and negative reagent controls. The patient samples also often contain normal cells that assess specific staining (antigen positive) and can serve as positive controls in the sample. Many times, fluorochrome-matched negative and positive controls can be found with in the same tube or with the patient sample (Fig. 6) [14]. Isotype controls may be indicated for dim antigen expression, especially when using limited panels, and are recommended when intracellular staining is performed. Process quality control In order to monitor the entire assay process, control material with expected values must be run with each test or batch of testing. There are commercially available controls for many IVD flow cytometry assays, such as CD4 and CD34. For quantitative assays when a numeric result is derived and reported from the instrument, it is strongly recommended that commercial controls be used. For leukemia/lymphoma immunophenotyping, commercial controls are not available, and the previous practice of running a normal patient control with each batch is redundant. Because each sample tested contains some component of normal leukocytes, the optimal process control for this type of testing is using internal controls as a process QC [14,26]. This can be done by designing panels, such that positive and negative populations are present in each tube that is analyzed. This allows gating on multiple populations (the unknown population of interest and known populations inherent in the sample, such as lymphocytes, monocytes, and granulocytes). Normal lymphocytes expect to have heterogeneous marking for B- and T-cell markers, such as CD20, CD19, CD3, and CD2 (positive control). Normal lymphocytes do not express myeloid and monocytic markers, such as CD13, CD33, and CD14 (negative control). In addition, known normal cell populations express the specific antigen with a consistent fluorescent intensity and light scatter pattern. It is critical that staff performing the analysis of leukemia/lymphoma immunophenotyping be familiar with the staining patterns of abnormal and normal components of samples in order to assess the internal control acceptability [27]. Internal controls can be verified within each sample and each tube
13 QUALITY CONTROL 683 Fig. 6. Compares an isotype control (IgG1) used to set the negative threshold for CD13 compared to an internal negative population CD2. of each sample and used to verify that the expected marking for each population is expressed. Documentation of internal control results is required as is noting the objective acceptability criteria for each population and each marker. Out-of-range values must be investigated and corrective action documented, as with any QC material [24]. References [1] Centers for Disease Control and Prevention. Guidelines for performing single-platform absolute CD4 þ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. MMWR Morb Mortal Wkly Rep 2003;52(RR-2):1 13.
14 684 OLDAKER [2] Clinical and Laboratory Standards Institute. Enumeration of immunologically defined cell populations by flow cytometry; approved guidelinedsecond edition H24-A2. CLSI, Villanova, PA. [3] Nguyen D, Diamond L, Braylan R. Flow cytometry in hematopathology. A visual approach to data analysis and interpretation Totowa, NJ: Humana Press; [4] McCoy JP Jr, Keren DF. Current practices in clinical flow cytometry: a practice survey by the American Society of Clinical Pathologists. Am J Clin Pathol 1999;111(2): [5] US Centers for Medicare & Medicaid Services (CMS). Medicare, Medicaid and CLIA programs; laboratory requirements relating to quality systems and certain personnel qualifications. Final rule. Fed Regist 2003;16. [6] U.S. Department of Health and Human Services. Medicare, Medicaid and CLIA Programs: regulations implementing the clinical laboratory improvement amendments of 1988 (CLIA). Final rule. Fed Regist 1992;57. [7] Clinical and Laboratory Standards Institute. A quality system model for health care; approved guideline GP26-A. CLSI, Villanova, PA. 1999;17(18). [8] McCoy JP Jr, Carey JL, Krause JR. Quality control in flow cytometry for diagnostic pathology, I: cell surface phenotyping and general laboratory procedures. Am J Clin Pathol 1990; 93(Suppl 1):S [9] CAP laboratory accreditation manual. Northfield (IL): College of American Pathologists; [10] Purvis N, Stelzer G. Multi-platform, multi-site instrumentation and reagent standardization. Cytometry 1998;33: [11] Poncelet P, Besson-Faure I, Lavabre-Bertrand T. Clinical applications of quantitative immunophenotyping in immunophenotyping. In: Stewart CC, Nicholson JKA, editors. Cytometric cellular analysis: immunophenotyping. New York: Wiley-Liss, Inc; p. 34. [12] Kraan J, Gratama JW, Keeney M, D Hautcourt JL. Setting up and calibration of a flow cytometer for multicolor immunophenotyping. J Biol Regul Homeost Agents 2003;17(3): [13] Barger J. Levey and jennings revisited. Arch Pathol Lab Med 1992;116(7): [14] Clinical and Laboratory Standards Institute. Clinical flow cytometric analysis of neoplastic hematolymphoid cells; approved guideline-second edition H43-A2 CLSI, Villanova, PA [15] Tatsumi N, Kitahashi S, Kubota H, et al. Standardization in flow cytometry. Rinsho Byori 1993;41(9): [16] Wood JC, Hoffman RA. Evaluating fluorescence sensitivity on flow cytometers: an overview. Cytometry 1998;33(2): [17] Wood JC. Fundamental flow cytometer properties governing sensitivity and resolution. Cytometry 1998;33(2): [18] Chase ES, Hoffman RA. Resolution of dimly fluorescent particles: a practical measure of fluorescence sensitivity. Cytometry 1998;33(2): [19] Gaigalas AK, Li Li O, Henderson R, Vogt J, Barr G, Marti J, Weaver, Schwartz A. The development of fluorescence intensity standards. J Res Natl Inst Stand Technol 2001;106: [20] Bagwell CB, Adams EG. Fluorescence spectral overlap compensation for any number of flow cytometry parameters. Ann N Y Acad Sci 1993;677: [21] Roederer M. Compensation is not dependent on signal intensity or on number of parameters. Cytometry 2001;46(6): [22] Roederer M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 2001;45(3): [Erratum in: Cytometry 2002 Jun 1;48(2):113]. [23] College of American Pathologists, Commission of Laboratory Accreditation. General inspection checklist. Northfield (IL): College of American Pathologists; [24] College of American Pathologists, Commission of Laboratory Accreditation. Flow cytometry inspection checklist. Northfield (IL): College of American Pathologists; 2005.
15 QUALITY CONTROL 685 [25] Stelzer GT, Marti G, Hurley A, McCoy P Jr, Lovett EJ, Schwartz A. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: standardization and validation of laboratory procedures. Cytometry 1997;30: [26] National Committee for Clinical Laboratory Standards. Method comparison and bias estimation using patient samples. Wayne (PA): NCCLS; no. EP9-A.2. [27] Braylan RC. Applications in clinical oncology: lymphomas. In: Bauer KD, Duque RE, Shankey TV, editors. Clinical flow cytometry: principles and application. Baltimore (MD): Williams & Wilkins; p
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