Validation and Quality Control of Immunophenotyping in Clinical Flow Cytometry

Journal of Immunological Methods 243 (2000) 33±50 www.elsevier.nl/locate/jim

Validation and quality control of immunophenotyping in clinical ¯ow cytometry Marilyn A. Owensa,* , Horacio G. Vall a , Anne A. Hurley b , Susan B. Wormsley c aIMPATH, Inc., 5230 Paci®c Concourse Drive, Los Angeles, CA 90045, USA bComprehensive Cytometric Consulting,62Bayberry Lane, Hanover, MA 02339, USA cPharMingen (BD Biosciences), 10975 Torreyana Rd, San Diego, CA 92121, USA

Abstract

Clinical ¯ow cytometry has evolved from two-parameter quantitative assessment of peripheral blood lymphocytes to six-parameter qualitative evaluation of bone marrow for hematopathology. Leukemia and lymphoma immunophenotyping represent an extremely important complement to morphology in the diagnosis and monitoring of hematopoietic malignancies. The complexity of ®ve- and six-parameter analyses and the interpretation of the data rely on standardization and validation of the instrument, the reagents and the procedure. In addition, ¯ow cytometry laboratories in the U.S. are required to document pro®ciency testing, sample preparation, method accuracy, speci®city, sensitivity and precision. NCCLS and the U.S.±Canadian Consensus Conference have provided recommendations, but each laboratory is ultimately responsible for validating its own qualitative and quantitative procedures. This paper reviews procedures for validation and quality control of all aspects of the operation of a clinical ¯ow cytometry service.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Immunophenotyping; Flow cytometry; Phenotypic analysis; Leukemia diagnosis; Lymphoma diagnosis

1. Introduction unusual in the clinical laboratory, allowing the simultaneous measurement of ®ve or six different Flow cytometry is a dynamic technology which parameters, respectively (Nicholson et al., 1996). A has allowed the multi-parameter analysis of recent consensus conference recommended ®ve-pa- heterogeneous cell populations to develop as a rameter immunophenotyping to be the minimal clinical service (Owens and Loken, 1995). Complex standard for hematological malignancies, forward analyses are able to combine immunophenotyping of and right angle light scatter and three colors of both surface and cytoplasmic antigens, DNA analysis ¯uorescence (Stelzer et al., 1997). With so many and functional evaluations. Subsets of cells can be variables in these analyses, standardization and vali- identi®ed and characterized by patterns of maturation dation of instrumentation and methodology is essen- antigens and staining intensity which can assist in tial to ensure the technical quality of the results diagnostic and prognostic interpretations as well as (Brando and Sommaruga, 1993; Carter et al., 1992; the detection of minimal residual disease. Cavelli et al., 1993; National Committee for Clinical Three- and four-color immunophenotyping are not Laboratory Standards, 1992; Hurley, 1988, 1997a,c). Reagents must be well characterized for speci®city *Corresponding author. and performance with different ¯uorochromes. All

0022-1759/00/$ ± see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-1759(00)00226-X 34 M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 monoclonal-¯uorochrome combinations must be 2. Responsibilities of the clinical service critically evaluated for staining intensity, spectral overlap, and instrument compensation (Hurley, Flow cytometry falls under the Centers for Dis- 1997b; McCoy et al., 1990; Muirhead, 1993). Fur- ease Control and Prevention (CDC) category of thermore, ¯uorescence patterns must be character- high complexity laboratory testing. Documentation ized for diagnostic combinations of antigens and of staff quali®cations and training as well as ana- diagnostic interpretation (McCoy and Overton, lytical accuracy, sensitivity, precision and QC are 1996). required. In some states, such as California, per- In attempts to assist with standardization, the sonnel responsible for generating ¯ow cytometric U.S.±Canadian Consensus Conference in 1997 and results must be licensed medical technologists who National Committee for Clinical Laboratory Stan- are required to have 12 h of continuing education dards (NCCLS) guidelines in 1998 provided recom- yearly. Other states may not have the same person- mendations for clinical ¯ow cytometry in hemato- nel quali®cations. At the very least, training and pathology (Borowitz et al., 1997; Davis et al., 1997; pro®ciency in the technology must be documented. National Committee for Clinical Laboratory Stan- Laboratories should develop SOPs for training, dards, 1997a; Stelzer et al., 1997; Stewart et al., with supervisor veri®cation of pro®ciencies in in- 1997; Braylan et al., 1997a,b). On the regulatory cremental responsibilities for each staff member. side, the Clinical Laboratory Improvement Act Instrument manufacturers and scienti®c societies (CLIA 88) (Department of Health and Human also conduct training courses and provide certi®- Services, 1992) has in¯uenced laboratory staf®ng, cates of training. Additionally, the American Socie- training, validation and documentation. As we enter ty of Clinical Pathology (ASCP) offers a specialty the 21st century, the laboratory's responsibilities exam for national certi®cation in ¯ow cytometry continue to increase. A recent publication by Hurley (QCYM). and Zito (1998b) offers approaches for CLIA compliance in the clinical ¯ow cytometry laboratory and includes templates for appropriate forms to satisfy 2.1. Pro®ciency testing documentation. The goal of this article is to discuss how regula- All high-complexity laboratories must enroll in a tory oversight in¯uences the laboratory's validation pro®ciency testing (PT) program that meets CLIA and quality control documentation, particularly in regulations (Subpart I) (Department of Health and hematopathology. Although this article covers reg- Human Services, 1992) and is approved by Health ulatory issues as they apply in the U.S., all lab- and Human Services (HHS). Regulations require that oratories world-wide are responsible for maintaining the laboratory inform HHS which programs it will performance standards. Good Laboratory Practices use, list the tests performed for each program, include Standard Operating Procedures (SOPs), participate in a program for at least a year before Quality Control (QC) and Quality Assurance (QA) choosing a different program, and must notify HHS as integral to providing good patient care (Hurley before changing. The laboratory must also authorize and Zito, 1998a). Regardless of requirements placed the PT program to release data to HHS. on manufacturers to provide analyte-speci®c reagents Pro®ciency Testing samples must be treated the (ASR) or in vitro diagnostic (IVD) reagents, each same as patient specimens, using the same personnel laboratory should validate their own panels for and work processes (Dorsey, 1975). Results must not sensitivity, speci®city, and correlation with morphol- be discussed with other laboratories and PT samples ogy and clinical ®ndings. The following sections will may not be outsourced under any circumstances. consider laboratory responsibilities, reagent and in- Documentation for handling, preparation, processing, strument validations, QC, QA, and troubleshooting analysis and interpretation of PT samples must be as a model for the integration of new technologies kept (Grannis et al., 1972). Final reports must be into diagnostic hematopathology. retained for a minimum of 2 years. M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 35

2.2. Sample handling that all clinical testing be characterized for accuracy, speci®city, sensitivity and precision (National Com- All laboratories must establish specimen require- mittee for Clinical Laboratory Standards, 1997b). ments, recommended transport conditions and However, for leukemia and lymphoma immuno- criteria for acceptability. Procedures for handling, phenotyping by ¯ow cytometry, there are no consen- packaging, labeling and transporting potentially in- sus standards or recommendations for these assess- fectious biological specimens have been published ments; thus each laboratory must establish its own (Nicholson et al., 1994; National Committee for performance criteria (National Committee for Clini- Clinical Laboratory Standards, 1990, 1997a). The cal Laboratory Standards, 1997a). U.S.±Canadian Consensus strongly recommended that every attempt be made to derive useful information from any specimen submitted for leukemia 2.4. Accuracy or lymphoma immunophenotyping analysis (Stelzer et al., 1997). Requesting physicians should always be Analytical accuracy compares the test result to a informed about sample quality issues; any compro- reference, or `gold' standard. In hematology, normal mised specimen, whether evaluated or not, must be cell populations can be counted microscopically with described in the test interpretation. a hemocytometer or with automated equipment and Clinical specimens must be appropriately iden- results compared for accuracy (Wooten and King, ti®ed Ð the minimum information is a unique 1953). In abnormal populations, especially when the patient identi®er (for cytogenetic analyses, two iden- characterization is based on multi-parameter data, the ti®ers may be required), test ordered and date of comparison becomes complex. For hematopathology, sampling. Other information helpful for interpreta- the gold standard is morphology. The ¯ow cyto- tion include presumptive diagnosis, age, gender, date metric assessment of accuracy must therefore com- and time of specimen collection, source of specimen, pare to morphology. With rare events, however, name of physician, and recent treatment or medica- morphologic diagnosis is dif®cult. Flow cytometry tions. Con®dentiality must be assured and docu- allows characterization of cell populations with mentation tracking the specimen's handling is essen- complex phenotypes and should also assess accuracy tial. Tests may only be performed if requested by an by comparison to previously characterized cells authorized person. Verbal requests must be followed (Stewart and Stewart, 1997b). Sources of well-char- by written authorization. All requests must be re- acterized cell populations are cryopreserved pedi- tained at least 2 years. greed specimens from another validated laboratory or relapse specimens from previously characterized cases. Another source of specimens for assessing 2.3. Sample preparation accuracy can be cases diagnosed by cytogenetics and molecular biology. Documentation comparing at Sample preparation for ¯ow cytometric analysis least 20 leukemia and lymphoma cases with com- must consider the type of specimen submitted and plete histopathology and clinical ®ndings should be the number of cells available for analysis (Stewart on ®le to support analytical accuracy. and Stewart, 1994). Peripheral blood, bone marrow, Another aspect of accuracy that should not be or tissue specimens should be processed to contain a ignored is the qualitative staining pattern used to suspension of the cells of interest, eliminating eryth- identify cell lineages. Descriptions of dim, moderate, rocytes, at a concentration optimal for monoclonal and bright staining patterns can be diagnostic or staining (0.5±13107 /ml; 0.1 ml per tube). All prognostic and should be well characterized and processing procedures must be written, approved and documented. For example, when multiple antibodies daily records maintained. Test records must be conjugated to ¯uorochromes are used in combina- retained for at least 2 years. tion, accuracy should be assessed for each antibody Good laboratory practices and CLIA 88 require separately and the frequency and intensity results 36 M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 compared to those obtained when using the com- pedigreed malignant specimens or well-characterized bined reagents. transfected cell lines used as positive controls.

2.6. Sensitivity 2.5. Speci®city Reagent sensitivity describes the ability to detect Speci®city of monoclonal reagents is de®ned by the minimum staining intensity above non-speci®c or how well the antibody recognizes the correct an- negative staining (Steen, 1991). The sensitivity of tigenic target. Manufacturers are responsible for detection is dependent on the titration of monoclonal reagents having the correct speci®city listed on their reagents (Stewart and Stewart, 1997a), the proper labels. However, speci®city in a `home-brew' diag- instrument setup and calibration, the number of cells nostic test has a broader meaning. For leukemia and counted and the ¯ow rate of the instrument (Wittrup lymphoma testing, the interpretation of the ¯ow et al., 1994; Zucker et al., 1991). Documentation of panel should be compared with morphology and reagent titrations and parallel testing of each new lot clinical presentation to assess the `speci®city' of the of antibody is required. Instrument calibration and testing (Zagursky et al., 1995). Each laboratory documentation is also required. should establish its own expected rate of discrepancy between ¯ow and morphology, most likely ,5%. 2.7. Precision The laboratory should then assess, on a case-by-case basis, the reason for a discrepancy, document the Precision is a standard analytical parameter which discrepancy as a QA assessment, and monitor trends measures the reproducibility of a single sample quarterly. stained and analyzed in duplicate at least 10 times. Speci®city of ¯ow cytometric reagents can be NCCLS Guidelines recommend 20 replicates. Nor- assessed by consensus workshops, publications or by mal peripheral blood, cell lines, blood standards or in-house validation. Most notable are the publica- CD Chex may be used. CD Chex are preserved white tions from the various Human Leukocyte Differentia- cell controls manufactured by Streck Laboratories. tion Antigen Workshops, the latest being Leucocyte Quantitative mean and standard deviation for each Typing VI (Kishimoto et al., 1997), which aptly and monoclonal antibody should be documented and a 2 succinctly summarize the vast body of testing results S.D. range developed. Determination of instrument on most if not all of the monoclonal antibodies precision is accomplished by running the same commonly used in leukemia and lymphoma immuno- stained sample at least three times, with results phenotyping. Helpful data summaries, reviews, and within 2 S.D. citations pertaining to these reagents can be found on the HLDA Web page at http://www.mh-han- 2.8. Analyte-speci®c reagents nover.de/projekte/hlda7/hldabase/select.htm. Also refer to the PROW database (Protein Reviews on the In the United States, clinical laboratories, reg- Web). Other sources of speci®city data are clinical ulated under CLIA 88 and performing physician- texts and journal articles. ordered ¯ow cytometric testing for leukemia and However, ¯ow cytometry monoclonal reagents are lymphoma immunophenotyping, are required to be considered `home-brew' reagents and each labora- quali®ed to perform high-complexity testing and tory must document staining performance, or `diag- must use reagents that are manufactured and labeled nostic speci®city' on both normal and abnormal cell as Analyte Speci®c Reagents (ASRs). populations, identifying both positive and negative Manufacturers of ASR must register their facility, staining (Stewart and Stewart, 1995). Clinical hema- list their products, follow current Good Manufactur- topoietic specimens will almost always contain nor- ing Practices (cGMP) under the new Quality System mal along with any abnormal populations and thus Regulations (QSRs), and comply with medical de- will allow both evaluations in one specimen. Diag- vice reporting requirements. Manufacturers are re- nostic immunophenotyping must be validated with stricted from providing any statement regarding M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 37 analytical or clinical performance, number of tests ance (Belk and Sunderman, 1947; Sax et al., 1967; provided, or instructions for use. Advertising and Shuey and Cebel, 1949; Paxton et al., 1989). promotional material for an ASR product must include the identity and purity (including source and 3.1. Instrument validation method of acquisition) of the analyte-speci®c reagent and the identity of the analyte [Code of Federal Validation of instrument performance falls into Regulations, CFR 809.30(d), Federal Register, two areas: (a) instrument setup and daily quali®ca- 1997]. tion of both light scatter and ¯uorescence measure- The identity of the analyte for monoclonal anti- ments, and (b) cross-instrument performance using body reagents is provided by the antigen distribution relevant clinical specimens. and supporting references. The HLDA references for when the antibody clone was ®rst clustered are 3.2. Instrument setup and daily quali®cation provided for the identity of the ASR. The labeling for analyte-speci®c reagents must include the reagent NCCLS recommends that the setup of a ¯ow name, concentration, purity and quality, statement of cytometer is comprehensive enough to assure proper warnings or precautions for users, date of manufac- optical alignment for adequate light scattering and ture, lot number, expiration date, storage instructions, ¯uorescence sensitivity and resolution as well as the net quantity of contents, name and place of business, proper degree of compensation to correct for spectral and the following statement: ``Analyte Speci®c overlap when multiple ¯uorochromes are used. While Reagent. Analytical and performance characteristics hardware con®guration may not allow the laboratory are not established'' [CFR 809.10(e)]. to align optics, a check of performance must be It is the responsibility of the testing laboratory made and documented. In addition, since the hard- using these reagents to validate their performance in ware and optics are different for ¯ow cytometers `home-brew' clinical assays. In addition, any labora- made by different manufacturers (the two major ones tory reports using Class I ASRs must contain the being Beckman-Coulter and Becton Dickinson, now following disclaimer: ``This test was developed and known as BD Biosciences), the recommended pro- its performance characteristics determined by hlab- cedures for instrument setup and performance qual- oratory namej. It has not been cleared or approved i®cation are instrument-speci®c. by the U.S. Food & Drug Administration'' [CFR 809.30(e)]. Further clarifying comments may also be 3.3. Light scatter sensitivity and resolution included. Research institutions and other non-clinical lab- Optical alignment for optimal sensitivity and oratories that use ASRs to make tests for purposes resolution of both forward (FSc) and side (SSc) other than providing diagnostic information to pa- scatter can be assessed by running uniformly sized tients and practitioner are not restricted under the beads that fall within the light scatter ranges ob- ASR regulation [CFR 809.30(g)]. served with most clinical samples, at a constant PMT voltage on a daily basis. The mean FSc and SSc channel numbers and percent coef®cient of variation (% C.V.) should be recorded. The acceptable ranges 3. Validation of Immunophenotyping for each parameter can be established by ®rst running the beads 20 times over a 5 day time period How does a laboratory validate ¯ow cytometric at the same PMT setting. Levy±Jennings graphs are immunophenotyping? The basic components are the then used to plot the values obtained daily and an validation of the instrument, the individual reagents, action plan is established for what to do when any and the staining protocols used to create a ®nal parameter falls outside of the expected range (Allen interpretation (Whitehurst et al., 1975). Each step in et al., 1969; Henry, 1959; Levy and Jennings, 1950). the testing process must be speci®ed and quality However, since beads and cells do not always control measurements included to monitor perform- behave similarly on a ¯ow cytometer, it is also 38 M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 recommended that the instrument be set up for ticular staining protocol, the compensation between running clinical samples using biological material ¯uorescence signals must be properly set to prevent and that the proper resolution of different cell types spill-over of one ¯uorescence signal into another be determined in daily instrument quali®cation pro- (Bagwell and Adams, 1993). This is usually done cedures (Henry and Segalove, 1952; Ladenson, automatically using hard dyed beads as seen in Fig. 1 1975). Normal peripheral blood leukocytes pro- for four-color compensation of FITC, PE, PerCP and cessed in a similar manner as the clinical samples APC on a FACSCalibur (BD Biosciences). can be used for this purpose. For example, the However, the use of a biological compensation lymphocytes can be set to fall within a speci®ed control is also recommended. When performing four- range based on FSc. The PMT for SSc can then be color immunophenotyping using a single laser at 488 adjusted so that the granulocytes fall within a nm on a Coulter XL (Beckman-Coulter), and a speci®ed range, with the ®nal FSc vs. SSc cytogram combination of monoclonal antibodies directly required to demonstrate good separation of lympho- conjugated to FITC, PE, PE-Cy5 and ECD, an cytes, monocytes and granulocytes. appropriate biological compensation control would consist of normal donor peripheral blood lympho- 3.4. Fluorescence sensitivity and resolution cytes stained with CD4±FITC, CD8±PE, CD2±PE/ Cy5 and CD45±ECD, as seen in Table 1. Acceptance values for monitoring ¯uorescence Hypothetically, in this example, there are 12 sensitivity and resolution can be established by two different combinations of ¯uorescence signals that methods: (a) recording the channel number and C.V. need to be checked for compensation but, due to of calibration beads with a pre-determined, ®xed instrument design, the optical ®lters used, and the laser power, ®lters, PMT voltages and gains, or (b) inherent spectral emissions of each ¯uorochrome, not recording the high voltage and gain to position the all combinations can or need be considered. Using beads in the same channel each time. For either the Cytosetting Matrix Display on System II 3.0 approach, acceptance ranges can be established by Expo software in the Coulter XL, each of the four using at least 20 replicate data sets collected over at ¯uorescence PMTs are ®rst adjusted to the proper least a 5 day period; new ranges must be developed settings based on the single color ¯uorescence each time a new lot of beads is phased into service. histogram and compensated with the compensation Regardless of the method used, procedures for what matrix. For a complete discussion of four-color to do if any of the measured and plotted parameters compensation, see Stewart and Stewart (1999). do not fall within the acceptable range must also be established, followed and documented by the clinical laboratory.Values for the acceptable ranges displayed 4. Monitoring instrument performance on the Levy±Jennings graphs for each parameter are typically updated based on each successive 20 day In the setup and quali®cation of a ¯ow cytometer cycle of data collection. However, QC speci®cations for daily use, NCCLS recommends a check of for instrument performance must be established to optical alignment, ¯uorescence resolution and in- allow for detection of signi®cant trends or drifts over tensity. Using the Coulter XL, a calibrator suspen- time which require corrective action. sion of latex beads with a known concentration of ¯uorescence (DNA Check) can be used to verify an 3.5. Fluorescence compensation appropriate cell-wide laminar ¯ow, a proper alignment between the laser beam and the cell at the Compensation should also be evaluated at the time interrogation point and a proper adjustment of laser of initial instrument setup and then monitored daily, power and/or PMT voltage. The ranges for the preferably using a biological compensation control in particles must be established, running 20 times addition to hard dyed beads provided by the instru- during 5 days and keeping the PMT voltage constant. ment manufacturer. Depending on the number, type The placement of the beads must meet the estab- and combination of ¯uorochromes used in a par- lished ranges and be recorded in the QC log. Any M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 39

Fig. 1. Column A: ¯uorescent beads run without compensation on FACSCalibur (four color) FITC, PE, PerCP, APC. Column B: beads showing compensation for FL12%FL251.2 and FL22%FL1523.3, FL22%FL350, FL32%FL452.5, FL42%FL355.0. 40 M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50

Table 1 Signal volts and gain showing matrix compensation for four-color phenotyping on a single laser Coulter XL MCL instrument Signal Compensation Signal Volts Gain Total Signal out5Y2%X gain YX→ FL1 FL2 FL3 FL4 FS 25 5.0 5.38 FL1 0.9 0.0 0.0 SS 402 20.0 44.12 FL1 687 1.0 FL2 11.2 12.8 4.7 FL2 873 1.0 FL3 718 1.0 FL3 0.0 31.1 2.1 FL4 909 1.0 AUX 69 5.0 6.04 FL4 0.0 0.0 19.1 values out of range must be investigated, corrective unstable. Monitoring linearity is a check of PMT action taken and documented. voltages. Using the same PMT settings used for For the BD FacsCalibur, the ¯uidics and cali- clinical specimens, a set mixture of four to ®ve bration check is performed by running CaliBrite multi-level ¯uorescence beads, each with a known Beads and allowing the instrument software to make relative ¯uorescence intensity level, should be run. adjustments in setup based on lot-speci®c perform- Products are available from several manufacturers, ance expectations. Documentation of acceptable for example Flow Cytometry Standards, or calibration is provided for record keeping. These Spherotech. Acceptable mean ¯uorescence intensity records can be used for trend analysis of the laser (MFI) ranges for each bead in the mixture should be power and voltage settings to indicate potential established by 20 replicate runs over a 5 day time technical problems. period. These same bead mixtures should then be run NCCLS recommends daily performance monitor- once a month and the linearity, or the relative ing of ¯uorescence intensity, color compensation and difference in MFI between each of the beads, should veri®cation of system performance using a QC remain constant for each ¯uorescence PMT (Fig. 2). (normal) specimen. Each laboratory must have QC Graphs of linearity should be drawn following procedures to monitor instrument performance. For manufacturers' recommendations to accommodate hematopoietic immunophenotyping, color compensa- Log 0. tion and veri®cation of performance can be evaluated with each specimen panel. Although every patient is different, the CD4/CD8/CD45/CD2 combination 4.2. Cross-instrument performance can be used to check compensation. Settings will rarely need to be changed. Laboratories that are performing the same clinical Optics should not be changed by a clinical labora- immunophenotyping protocols on more than one tory without speci®c validated procedures. It is more instrument should include a semi-yearly cross-instru- appropriate to have the manufacturer check and ment comparison of at least ®ve different and adjust optics during routine maintenance and to representative clinical samples stained with each of provide documentation for the laboratory. the standard protocols used in the laboratory. The results obtained for each sample should not differ 4.1. Fluorescence linearity between instruments by more than a pre-de®ned acceptable variance (Vogt et al., 1991, 1994). This Linearity of ¯uorescence detection should be cross-instrument sample testing process should be checked on a monthly basis or as recommended by documented and corrective action plans must be the instrument manufacturer (Vogt et al., 1989). established and followed when cross-comparison Linearity should be constant unless the laser is results fail to meet performance speci®cations. M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 41

Fig. 2. Beads with ®ve different ¯uorescence intensities for each ¯uorochrome. Setup regions A to E will provide the log intensity channel value. A plot of log intensity vs. bead number should provide a linear distribution.

5. Preanalytical QC be documented before transfer to the testing laboratory. Laboratories must not only establish acceptance criteria for immunophenotyping specimens, but must have procedures to assess acceptability and documentation to assist in troubleshooting and the inter- 6. Reagent and method validation pretation of results. NCCLS recommends a visual analysis on receipt to con®rm specimen quality. An immunophenotyping procedure ®rst requires Hemolysis, partial draw (especially in ACD tubes), the selection of monoclonal reagents and ¯uoro- temperature extremes and improper labeling should chromes which are to be used in the multi-parameter 42 M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 analysis and interpretation of results. The hemato- reagents are combined in testing panels is left up to pathology testing panel must be designed to clearly the testing laboratory, but should be based on a distinguish normal and abnormal immunoreactivity target-oriented strategy (Table 2). patterns based on differences in light scatter and/or All the combinations have to be validated in each ¯uorescence intensity. These pattern comparisons are laboratory and the analysts must be familiar with the integral to the interpretation of results. Therefore, the patterns associated with each combination. Different panel must include all relevant markers and re¯ect clones (under the same CD) may perform differently. the visual expectations of the diagnostic interpreters. Similarly, the same CD labeled with different ¯uoro- The U.S.±Canadian Consensus Conference chromes will show a different level of intensity and stopped short of recommending a diagnostic panel possible color overlap. Fluorochrome intensity and for leukemia and lymphoma immunophenotyping, antigen expression are both important in establishing however there was agreement on 42 determinants the best antibody combination to evaluate antigen which can provide diagnostic information in these density (Schwartz et al., 1990, 1996a,b). Some procedures (Stewart et al., 1997). Each specimen tandem dye combinations can help to minimize these must be considered a unique case and fully evaluated problems. to minimize missing any abnormality. The selection Different combinations of antibodies can be used of panel reagents needs to balance the economic to provide the same clinical interpretation and no need for a minimal number of monoclonal antibodies single panel will accommodate all leukemia or to identify abnormal populations with the scienti®c lymphoma phenotypes. To compound the dif®culty, need to detect abnormal antigen expression. The new markers and reagents, when determined to be committee concluded that it is more important that clinically valuable, must be validated by each labora- each laboratory have adequate experience with the tory. Any panel of monoclonal reagents used by a binding characteristics of their testing reagents than laboratory must be chosen not only for their techni- specifying what those reagents should be. How cal performance, but also to satisfy the experience

Table 2 Some common tube combinationsa Population Three-color Four-color to identify combination combination B cells Kappa/Lambda/CD20 K/L/CD45/CD20 CD10/CD5/CD19 CD10/CD5/CD45/CD19 CD5/CD23/CD20 CD5/CD23/CD20/CD19 Plasma cells CD38/cytoK/CD45 CD38/cytoL/CD45 T cells CD7/CD3/CD45 CD7/CD3/CD45/CD56 CD4/CD8/CD3 CD4/CD8/CD45/CD2 Myeloid CD33/CD13/CD45 CD13/CD14/CD45/CD33 CD34/CD117/CD45 CD15/CD117/CD45/CD34 CD11b/CD16/HLA-DR CD11b/CD16/CD45/HLA-DR Myeloid w monocytic diff CD64/CD13/CD45 CD64/CD14/CD45/CD33 Myeloid w CD34/CD41/CD61 CD41/CD61/CD45/CD34 megakariocytic diff CD42b/CD61/CD45 CD42b/CD61/CD41/CD34 Red cell precursors CD71/Glycophorin A/CD45 a These combinations are for examples only and do not represent guidelines or consensus recommendations. M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 43 and expectations of the technical and medical per- proach is to set the PMT with the single reagent and sonnel who must differentiate abnormal populations then check compensation with the ®nal cocktail. from normal cells. The proper combination of monoclonal antibodies within a cocktail must consider antigen expression 7. Sample preparation on normal and abnormal cells as well as the ¯uorochrome combinations to minimize interference be- Integral to the analytical procedure, the laboratory tween reagents. One example of potential interfering must decide on and validate a sample preparation markers is bright CD15/CD16/CD45/CD13 on procedure. Erythrocyte lysis is the most commonly granulocytes. For multiple ¯uorochrome combina- used method for preparing peripheral blood or bone tions, the laboratory must not only validate that each marrow specimens for clinical immunophenotyping. monoclonal performs as expected in the procedure, Lysis can be performed either before staining with but must also prove that the performance is not monoclonal antibodies or after. Whichever method affected by co-blocking of epitopes, ¯uorescence for erythrocyte lysis is used must be validated prior quenching, or energy transfer (Pollice et al., 1992). to reporting clinical results. Lysis is optically moni- Antibody cocktails may be purchased from the tored by observing a cloudy red suspension of cells manufacturer or prepared by the laboratory. Whether change into a clear, translucent solution in 5 to 10 reagents are mixed immediately before each use or min. If red cells are still present after centrifugation stored as a premix solution, performance of each and resuspension, the lysing process should be cocktail must be validated and expiration dates repeated. documented. Tissue specimens should be disaggregated and Each monoclonal antibody in each cocktail must ®ltered to create a single cell suspension prior to be titrated individually for optimal signal-to-noise staining. This laboratory procedure must also be separation. However, potential interference between written and validated. Cytological slides should be certain clones and/or ¯uorochromes necessitates also prepared and reviewed with the H&E slides of the performing checkerboard titrations to select the ®nal tissue to assess the loss of fragile cells, such as optimal working dilutions once the reagents are used Reed±Sternberg cells. Some large cells in ®brotic in combination. For each new multi-color combina- tissues may be dif®cult to remove and cell loss will tion added to the panel, it is necessary to ®rst occur during the processing. Other sampling errors validate that the performance (mean intensity ¯uores- are due to focal lymphoma in the lymph node, not cence and percent positive) of each antibody when seen in the isolated cells or in the ®xed tissue. Cell used in combination is comparable (within 2 S.D.) to fragility may be minimized by gentle tissue process- the performance of each antibody used alone at the ing and teasing the tissue apart with a needle. same concentration on the same target cell popula- Reproducible monoclonal antibody staining relies tion. If reagents are stored in a pre-mixed cocktail on the proper proportion of antibodies to cells. For format, it is also important to institute suf®cient QC hematopoietic immunophenotyping, cell counts procedures, using appropriate control cells, to verify should be performed on all specimens to ensure stability of performance of the cocktailed reagents correct proportions of cells to monoclonal antibodies over time. Stable cocktails will provide results and thus accurate staining intensity patterns. NCCLS wherein the mean channel and percent of gated cells recommends the following adjustments based on will not differ more than 2 S.D. between cocktailed WBC count of peripheral blood. (There are no and single reagents. Documentation must be summa- similar recommendations for bone marrow and each rized and maintained in the laboratory and should laboratory should establish and validate its own include instrument photomultiplier tube (PMT) set- procedures.) tings and compensation. No more than one combination of PMT and compensation may be used within a (a) WBC ,1000 cells/ml: use 200 ml whole blood panel (Stewart and Stewart, 1999). The best ap- and adjust amount of lysing reagent appropriately; 44 M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50

(b) WBC 1000±10,000 cells/ml: use 100 ml 30%, a gating discrimination strategy should be used whole blood and standard volume of lysing to limit the analysis to live cells, minimizing non- reagent; speci®c binding from dead cells which could lead to (c) WBC 10,000±20,000 cells/ml: use 50 ml misdiagnoses. Several different methodologies have whole blood and adjust amount of lysing reagent been used to report viability, trypan blue, 7-amino- appropriately; actinomycin D (7-AAD), or propidium iodide (PI). (d) WBC .20,000: dilute whole blood with PBS The most common is trypan blue dye exclusion to achieve WBC concentration 1000±10,000, then visualized microscopically with a hemocytometer. use 100 ml and standard volume of lysing reagent. Dead cells stain blue because their membranes are broken, allowing the dye to enter the cell.Viability is Light scatter patterns must be appropriate after reported as a percentage of the total cell population. lysing and staining. The exclusion of dead cells is now being commonly performed on the ¯ow cytometer using dyes such 7.1. Viability as 7-AAD (Schmid et al., 1992) or PI (Sasaki et al., 1987) (see Fig. 3). Several others have been de- Assessment of viability is crucial for evaluating scribed and, for most common clinical instruments, a leukemia and lymphoma specimens by ¯ow cytom- relatively simple dead cell discrimination can be etry because cell membrane integrity is required for performed simultaneously with immunophenotyping. antigen expression.Viability at the time of collection, For example, using 7-AAD, the most common the in¯uence of transportation environment and the three-color application in single laser instruments is time before testing can all affect sample viability and FITC in FL1, PE in FL2 and 7-AAD in FL3. In this compromise tumor cell detection. setting, 7-AAD (Emax 5 655 nm) is preferred over PI By recent consensus recommendations, ¯ow cy- (Emax 5 625 nm) because it has less spectral overlap tometry performed on leukemias and lymphomas with PE (Emax 5 578 nm) In our experience with should be held to less stringent viability restrictions four-color immunophenotyping with a dual laser than quantitative immunophenotyping (Stelzer et al., system (488 nm/635 nm on the FACSCalibur) the 1997). When viability is low, for example less than most powerful combination for excluding dead cells

Fig. 3. Dead cell discrimination on two lymphoma tissues. (A) 7-AAD vs. Forward Scatter with 18.4% dead cells. (B) CD451PI vs. FSC with 29.1% dead cells. M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 45 while evaluating four surface markers is FITC in be in¯uenced by the donor, the target cell type, and FL1, PE in FL2, PerCP1PI in FL3, and APC in the particular staining method used. For cytoplasmic FL4. staining, the use of isotype controls is recommended due to the signi®cant in¯uence of cell size on the 7.2. Staining process QC degree of non-speci®c staining (Jacobberger and Bauer, 2000). It is important that each CD mono- Once the instrument setup and performance vali- clonal antibody has the corresponding ¯uorochrome dation is complete, a normal blood is run to check and isotype reagent to monitor non-speci®c binding the staining process. For this purpose, there are some performance. commercially available preserved cell preparations The presence of non-speci®c binding of an isotype [e.g., CD Chex (Streck Labs), CD Chex Plus (Streck can affect the interpretation of the ¯uorescence Labs), Immunotrol (Beckman-Coulter)] which con- intensity patterns and, as such, is critically important tain most of the cellular markers evaluated in in the analysis of leukemia and lymphoma immuno- leukemia and lymphoma immunophenotyping. Stain- phenotyping. ing each new lot of stabilized cells in triplicate will provide a range to be used for quality control. Most 7.4. Procedure control importantly, each leukemia or lymphoma specimen will contain a normal cell population which can be Fluorescence staining intensity patterns are more used to document appropriate staining patterns. easily interpreted if there is a common factor in each tube. For example, CD45 can be used as the `anchor' 7.3. Isotype controls in each tube of a multi-color cocktail, providing a reference population for comparisons to other an- The appropriate use of isotype controls is still tigenic expression patterns. The most important controversial in the clinical ¯ow cytometry com- parameter, however, is experience-based. The analyst munity (Borowitz et al., 1997; National Committee must understand the markers and the patterns used to for Clinical Laboratory Standards, 1997a). For quan- identify an abnormal population. Running a normal titative immunophenotyping, the isotype control was control will provide a baseline for all the markers. included to provide a negative cell reference and to Any deviation from the normal pattern should be set up the axes for assessing the numbers of cells in reported and con®rmed with other markers or tech- each quadrant. However, immunophenotyping of nologies, such as cytogenetic or molecular tech- leukemias and lymphomas is a qualitative assessment niques. and does not usually require quadrant analysis. Furthermore, since all leukemias and lymphomas contain a mixture of cells, those cells that do not 8. Data analysis and interpretation express the antigen will be the negative control for the ones that do. Immunophenotyping by ¯ow cytometry can be Most of the commercially available antibodies used to distinguish abnormal cell populations from have been selected by screening for the IgG class of normal cells and, in the process, identify the pro®le immunoglobulin. For IgG class antibodies, however, of antigens in the abnormal population (Almasri et it is important to recognize that some IgG subclasses al., 1992; Borowitz et al., 1993a; Braylan et al., will be more problematic due to their increased 1993; Cheson et al., 1996; Hertler et al., 1988; binding to Fc receptors present on various cell types. Jamieson et al., 1993; Roth and Schmitz, 1996; In general, the order of `stickiness' is IgG2b. Tomer and Harker, 1989). Various patterns of im- IgG2a.IgG1. As a result of their carbohydrate munoreactivity with monoclonal antibodies are com- structure, some antigenic targets (e.g., CD15 and bined with light scatter to characterize populations of CD57) will invariably result in generation of only interest (Davis et al., 1994; Kilo and Dorfman, 1996; IgM class antibodies. High non-speci®c binding of Loef¯er et al., 1992; Segal et al., 1991; Wormsley et IgM class antibodies is commonly observed and can al., 1990; Davis and Bigelow, 1990; Shimenti et al., 46 M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50

1992). The speci®c reagent combinations can be populations should only be performed if all cells of chosen to identify abnormal antigen expression and interest are contained within the analysis gate. Strate- to characterize cells at various stages of maturation gies for speci®c diseases may include gating on B (Civin and Loken, 1992; Ford et al., 1995; Hurwitz cells to determine clonality or identi®cation of et al., 1988; Macedo et al., 1995; Seivers et al., leukemic blasts in a CD45 and right angle light 1996). scatter display. The complement of monoclonal antibodies neces- In Fig. 4, gating by FSc vs. SSc is shown in the sary to identify hematopoietic malignancies is not left panel and gating by CD45 vs. SSc for the same standardized. In fact, strategies for immuno- case is shown on the right. The better gating strategy phenotyping must consider antibody speci®city, uses CD45 to differentiate three populations (imma- ¯uorochrome, laboratory workload and, most im- ture myeloid, immature monocytes and normal lym- portantly, appropriate multi-parameter combinations. phocytes) (Borowitz et al., 1993b). Most laboratories use a comprehensive screening Data analysis of abnormal populations requires panel and add con®rmatory monoclonal antibodies if that ¯uorescence intensity be measured. The calcula- needed (Hassett and Parker, 1995). Reimbursement tion of percent positive cells does not aid in the for laboratory testing is a major factor, thus lab- interpretation, while presence of inappropriate deter- oratories strive to make diagnoses with a minimal minants or inappropriate intensity expression of number of antibodies. The U.S.±Canadian Consen- antigens can be diagnostic (Bene et al., 1995; Press sus Conference estimated that the average North et al., 1989). Resolving dimly positive populations American laboratory uses 19 antibodies to diagnose a from negative populations is crucial in the accurate leukemia and 16 for a lymphoma. European diag- assessment of ¯uorescence intensity (Givan et al., noses often involve a larger number of monoclonal 1991). There are no hard and fast rules for de®ning antibodies (Stewart et al., 1997). these populations; in fact, reagent combinations are Gating is one of the most important parameters in generally optimized to provide discrimination be- multi-parameter data analysis. In a diagnostic case, tween these populations based on the subjective all cells should be evaluated. The U.S.±Canadian review of the pathologist (Campana and Pui, 1993; consensus recommended that the initial analysis Rameshwar et al., 1994). For critical markers, such include all viable cells. Further analyses on gated as immunoglobulin light chains, in the diagnosis of

Fig. 4. Comparison of gating strategies: FSc vs. SSc on left; CD45 vs. SSc on right. M.A. Owens et al. / Journal of Immunological Methods 243 (2000) 33 ±50 47 non-Hodgkin's lymphoma, the redundant use of Borowitz, M.J., Guenther, K.L., Shults, K.E., Stelzer, G.T., 1993b. antibodies to light chains using alternative ¯uoro- Immunophenotyping of acute leukemia by ¯ow cytometric chromes, antibody pairs from different vendors, or analysis: use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis. Am. J. Clin. Pathol. monoclonal versus polyclonal reagents, can signi®- 100, 534±540. cantly enhance con®dence in interpretation of mono- Borowitz, M.J., Bray, R., Gascoyne, R., Melnick, S., Parker, J.W., clonality in the face of very low level expression. Picker, L., Stetler-Stevenson, M., 1997. 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