Comparison of current flow cytometry methods ... - Wiley Online Library

Cytometry Part B (Clinical Cytometry) 78B (Suppl. 1):S4–S9 (2010)

Commentary

Commentary: Comparison of Current Flow Cytometry Methods for Monoclonal B Cell Lymphocytosis Detection Wendy G. Nieto,1 Julia Almeida,1 Cristina Teodosio,1 Fatima Abbasi,2 Sallie D. Allgood,3 Fiona Connors,4 Jane M. Rachel,5 Paolo Ghia,6 Mark C. Lanasa,3 Andy C. Rawstron,4 Alberto Orfao,1 Neil E. Caporaso,7 Curt A. Hanson,8 Youn K. Shim,9 Robert F. Vogt,10 and Gerald E. Marti2* 1 Servicio General de Citometrı´a, Instituto de Biologı´a Molecular y Celular del Ca´ncer, Centro de Investigacio´n del Ca´ncer/IBMCC (CSIC-USAL) and Departamento de Medicina, Universidad de Salamanca, Spain 2 Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 3 Department of Medicine, Duke University Medical Center, Durham, North Carolina 4 Hematological Malignancy Diagnostic Service, St. James’s Institute of Oncology, Leeds Teaching Hospitals, Leeds, United Kingdom 5 Molecular Diagnostics and Flow Cytometry Laboratory, Saint Luke’s Hospital, Kansas City, Missouri 6 Laboratory of B Cell Neoplasia, Division of Molecular Oncology and Lymphoma Unit, Department of Oncology, Universita Vita-Salute San Raffaele and Istituto Scientifico San Raffaele, Milano, Italy 7 Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland 8 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 9 Division of Health Studies, Agency for Toxic Substances and Disease Registry, Atlanta, Georgia 10 Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia

Monoclonal B cell lymphocytosis (MBL) is now recognized as the B-lymphocyte analogue of a monoclonal gammopathy of unknown significance. MBL can be the precursor of chronic lymphocytic leukemia or associated with non-Hodgkin’s lymphoma. It may be associated with an autoimmune abnormality or be related to aging (immunosenescence). The combination of available new fluorochrome-conjugated monoclonal antibody reagents, multilaser instrumentation, and improved software tools have led to a new level of multicolor analysis of MBL. Presently, several centers, including the University of Salamanca (Spain), Duke University (Durham, NC), Mayo Clinic (Rochester, MN), and the National Cancer Institute (Bethesda, MD) in conjunction with the Genetics and Epidemiology of Familial chronic lymphocytic leukemia Consortium, the Food and Drug Administration (Bethesda, MD), and the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (Atlanta, GA) in collaboration with Saint Luke’s Hospital (Kansas City, MO), the Universita` Vita-Salute San Raffaele in Milan (Italy), and Leeds Teaching Hospital (UK) are all actively conducting studies on MBL. This commentary is an updated summary of the current methods used in these centers. It is important to note the diversity of use in reagents, instruments, and methods of analysis. Despite this diversity, there is a consensus in what constitutes the diagnosis of MBL and its subtypes. There is also an emerging consensus on what the next investigative steps should be. Published 2010 Wiley-Liss, Inc.† Key terms: chronic lymphocytic leukemia; immunophenotyping; lymphocyte gating

*Correspondence to: Gerald E. Marti, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD. E-mail: gemarti@mac.com.

Published 2010 Wiley-Liss, Inc. yThis article is a US government work and, as such, is in the public domain in the United States of America.

Received 12 July 2010; Accepted 14 July 2010 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cyto.b.20556

COMPARISON OF CURRENT FLOW CYTOMETRY METHODS FOR MBL DETECTION

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How to cite this article: Nieto WG, Almeida, J, Teodosio C, Abbasi F, Allgood SD, Connors F, Rachel JM, Ghia P, Lanasa MC, Rawstron AC, Orfao A, Caporaso NE, Hanson CA, Shim YK, Vogt RF, and Marti GE. Commentary: Comparison of Current Flow Cytometry Methods for Monoclonal B Cell Lymphocytosis Detection. Cytometry Part B 2010; 78B (Suppl. 1): S4–S9.

Monoclonal B cell lymphocytosis (MBL) is a benign expansion of clonal B lymphocytes that can be detected in the blood of otherwise healthy, asymptomatic individuals, and it has the potential for evolution to chronic lymphocytic leukemia (CLL) or to be associated with non-Hodgkin’s lymphoma (NHL) (1–4). MBL is often detected as part of a clinical evaluation for an elevated peripheral blood (PB) lymphocyte count or is detected during directed studies of nonaffected members of familial CLL pedigrees or during population-based studies. Recent studies suggest that the appearance of MBL predates the development of CLL and that the conversion rate of MBL associated with increased absolute B-cell counts to CLL may be as high as 1% per year, which emphasizes MBL’s role as a risk factor for CLL development (3,4); similar data for MBL conversion to NHL are not available. As such, the definition of MBL is currently based on its distinction from CLL and NHL and is based on its characteristic appearance by flow cytometry (1,2,5–9). The original detection of MBL was primarily based on two- and three-color flow cytometric analysis. These early reports have been previously reviewed (2). This report will summarize current flow cytometric methods for the detection of MBL and attempt to define area of consensus and areas in need of further investigation. Presently, this work is carried out at Duke University (Durham, NC), Mayo Clinic (Rochester, MN), Food and Drug Administration (FDA), and Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry in conjunction with Saint Luke’s Hospital (Kansas City, MO), Universita` Vita-Salute San Raffaele in Milan (Italy), University of Salamanca (Spain), and Leeds Teaching Hospital (UK). An outline of the flow cytometric procedures used at selected sites follows, including sample preparation, staining, data acquisition and analysis, standards for daily operation and compensation controls, and reagent panels. This commentary will point out the differing approaches used for the detection of MBL based on instruments, reagents, and gating strategies.

METHODS This section on methods was either taken modified from published reports with appropriate updates or by the originating laboratory. It is important to note that reagent panels, instrumentation, and software are in a continuous state of flux, and published methods are likely to have undergone revision.

Cytometry Part B: Clinical Cytometry

Salamanca A total amount of approximately 4 mL of ethylenediaminetetraacetic acid (EDTA)-anticoagulated PB per case is immunophenotyped using a direct immunofluorescence stain-and-then-lyse technique (1). A three-tube panel is used with the following antibody combinations: (1) CD20-Pacific Blue (PacB)/CD45-Pacific Orange (PO)/CD8fluorescein isothiocyanate (FITC) þ anti-sIgk-FITC/CD56phycoerythrin (PE) þ anti-sIgj-PE/CD4-peridin chlorophyll protein-cyanin 5.5 (PerCPCy5.5)/CD19-PE-cyanin 7 (PECy7)/CD3-allophycocyanin (APC)/CD38-Alexa Fluor 700 (AF700); (2) CD20-PacB/CD45-PO/cytBcl2-FITC/ CD23-PE/CD19-PerCPCy5.5/CD10-PECy7/CD5-APC/CD38AF700; and (3) CD20-PacB/anti-sIgk-FITC/anti-sIgj-PE/ CD19-PerCPCy5.5/CD10-PECy7/CD5-APC. Reagents were purchased from BD Biosciences (San Jose, CA), except for CD19-PECy7 (Beckman Coulter, Miami, FL), CD20-PacB (eBiosciences, San Diego, CA), CD38-AF700 (Exbio, Prague, Czech Republic), CD45-PO (Invitrogen, Carlsbad, CA), and Bcl2-FITC, anti-sIgk-FITC, and anti-sIgj-PE (Dako, Glostrup, Denmark). Intracytoplasmic expression of bcl2 was evaluated after staining for cell-surface antigens, using the Fix & PermTM reagent kit (Invitrogen), according to the instructions of the manufacturer. Data acquisition was performed on a FACSCanto II flow cytometer (BD Biosciences) using the BD Biosciences FACSDiva software (v. 6.1, BD Biosciences), through a double-step procedure: first, information on 1
105 events corresponding to the whole sample cellularity was stored; in a second step, information was stored on CD19þ and/or CD20þ gated events, containing a minimum of 5
106 leucocytes per tube. Instrument set-up and calibration were performed according to well-established protocols (2,3); to check for the consistent performance of the cytometer and ensure reliable and accurate results, a daily quality control program was strictly followed, based on the use of Cytometer Setup and TrackingTM beads and Cytometer Setup and Tracking module (BD Biosciences), according to the recommendations of the manufacturer. Because isotype controls were not used, those cells that did not express a certain marker were considered as negative control for positive cells. In tubes 2 and 3, in which CD3 was not present in the panel of antibodies, the possibility that coincident T-cell events were included within the CD19/CD20 gate was excluded by the pattern of forward light scatter (FSC) and side scatter (SSC), together with a phenotype incompatible with B/T lymphocyte doublets (i.e., CD5þ/ CD19þ/CD20lo vs. CD5þ/CD19þ/CD20hi). For data analysis, the Infinicyt software was used (Cytognos SL, Salamanca, Spain). Analysis of sIgk and sIgk expression was

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performed within the different B-cell compartments identified with the CD19, CD20, CD5, and/or CD10 antigens in tubes 1 and 3; further characterization of MBL populations identified in both tubes 1 and 3 was performed with markers contained in tube 2. The minimum number of cellular events considered to constitute a clonal B-cell cluster was of 50. Duke Multiparameter flow cytometry analysis is performed on either fresh or cryopreserved peripheral blood mononuclear cells (PBMC). For fresh samples, PBMC are purified by density centrifugation from approximately 10 mL of heparin-anticoagulated venous blood. Frozen samples are cryopreserved in dimethylsulfoxide-containing media in aliquots of approximately 5.0
106 PBMC. Samples are rapidly thawed and washed twice with RPMI 1640 supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin. Approximately 2.5
105 PBMC are incubated with CD23 PE, CD3 PE-Cy5, CD14 PE-Cy5, CD16 PE-Cy5, CD235a PE-Cy5, CD20 PE-Cy7, sIgk APC, CD19 APC-Cy7, sIgk APC-Cy5.5, CD5 PacB, and Vital Dye (Invitrogen). PE-Cy5-conjugated CD3, CD14, CD16, and CD235a are combined as a ‘‘dump’’ channel to exclude T cells, erythrocytes, monocytes, natural killer (NK) cells, and granulocytes. Isotype controls matched to test antibody isotype, concentration, and fluorescent conjugate are used for each test antibody. Additionally, expression of CD5 is determined using the fluorescence minus one approach. Flow cytometry is performed on a BD Bioscience LSRII flow cytometer. Instrument QC/QA using AlignFlow Plus flow cytometry alignment beads (Invitrogen) is performed by the Duke Human Vaccine Institute Flow Cytometry and Cell Sorting Facility. Compensation is performed by staining Quantum Simply Cellular antimouse IgG beads (Bangs Laboratories, Inc.) to saturation and using unstained blank beads and stained beads as negative and positive controls, respectively. Analysis of flow cytometry results is performed using FlowJo software (Tree Star Inc.; Ashland, OR). In brief, surface expression of CD5 and CD20 of gated CD19þ B cells is determined. Three B-cell subsets, CD5CD20þ, CD5þCD20þ, and CD5þCD20dim, are each analyzed for expression of CD23 and skewing of the ratio of sIgj to sIgk. CLL-like MBL are defined as CD19þ, CD5þ, and CD20dim B cells that were CD23þ. Atypical MBL are defined as CD5þ B-cell populations with skewed sIgj:sIgk ratio (3:1) not otherwise meeting criteria for CLL-like MBL, and non-CLL MBL were CD5 B-cell populations with skewed sIgj:sIgk ratio. A minimum of 50 events are required for definitive detection of CLL-like or atypical MBL. Saint Luke’s Hospital and FDA Center for Biologics Evaluation and Research At the St. Luke’s Hospital, blood donor samples from Community Blood Center (Kansas City, MO) are analyzed. Complete blood counts (CBCs) were obtained on a Sysmex X2100 instrument. EDTA-anticoagulated donor whole blood was stained with a six-color antibody cock-

tail containing CD19, CD20, CD5, CD45, and sIgj/sIgk light chains and screened on a BD Biosciences FACSCanto II flow cytometer by collecting a minimum of 500,000 events. Listmode data were analyzed using FACSDiva software (BD Biosciences) and the following gating strategy. Lymphocytes are isolated using CD45 plotted against SSC-A; these events are further isolated using FSC-H plotted against SSC-A. Singlet events were gated using FSC-A plotted against FSC-H, followed by SSC-A plotted against SSC-H. Events that fall within both singlet gates were selected for further analysis. Singlet events from Step 1 were displayed by plotting CD45 fluorescence against SSC-A. Events within the CD45-positive region with low SSC were then selected for further analysis. Lymphocyte gating was refined by displaying cells selected in Step 2 as FSC-A plotted against SSC-A and gating lymphocytes based on scatter properties. Singlet events from these sequential gates are selected for further analysis. Lymphocytes are then displayed by plotting CD19 fluorescence against CD20 fluorescence, CD19 fluorescence against CD5, and CD20 fluorescence against CD5. Dual positive events in these displays are gated, and kappa fluorescence is plotted against lambda fluorescence. CD19þ/CD5 and CD20þ/CD5 cell populations are also scanned for evidence of clonality. Finally, all discrete populations are examined for evidence of clonality. Lymphocytes from Step 3 were also displayed by plotting CD20 fluorescence against CD5 fluorescence. CD20/CD5 dual positive events were also gated, and sIgk fluorescence was plotted against sIgk fluorescence. CD20þ/CD5 cell populations were also scanned for evidence of clonality. Lymphocytes from Step 3 were also displayed by plotting CD19 fluorescence against CD20 fluorescence. All discrete populations were examined for evidence of clonality. Criteria for a donor sample to be classified as having MBL cells was based on the finding of a cluster of at least 50 B lymphocytes that were definitively clonal or expressed clonal excess, and also expressed an MBL immunophenotype that resembled classical CLL (CD5þ and CD20þ), atypical CLL (CD5þ and CD20bright), or non-CLL (CD5) subtype. Samples that screened positive in this six-color assay are sent by overnight mail to FDA for Center for Biologics Evaluation and Research confirmation. Extended immunophenotypic analysis of the donor sample includes CD23, CD38, CD69, CD27, IgM, IgD, CD11c, CD25, CD79b, and CD22. CD20, CD19, and CD45 are common to all tubes, whereas intracytoplasmic staining for ZAP-70 and bcl-2 are done separately. At FDA Center for Biologics Evaluation and Research, when possible, a CBC was obtained from an automated blood cell counter. In some cases, a single platform determination was made for the absolute lymphocyte subsets using BD TrucountTM tubes (BD Biosciences). Many of the earlier FDA studies (EDTA for fresh samples and heparin for shipped samples) were four-color using a CD20, CD5, and sIgk and sIgk panel on a FACSCalibur flow cytometer (BD Biosciences). In later studies, sixcolor analyses using a panel of CD5, CD19, CD20, and

Cytometry Part B: Clinical Cytometry

COMPARISON OF CURRENT FLOW CYTOMETRY METHODS FOR MBL DETECTION

CD45 plus sIgk and sIgk were carried out on a FACSCanto II flow cytometer. Daily calibration of photomultiplier (PMT) voltage settings were accomplished using microbead standards provided by the manufacturer and checked with a normal blood bank donor. Compensation controls were prepared by staining cells with a single fluorochrome for software-based corrections. Cell events collected varied from 100,000 to 500,000 cells. Listmode data were collected with FACS Diva software (BD Biosciences) and analyzed using FlowJo software. The lymphocyte gating strategy was identical to that used at St Luke. Biclonal MBL is subjected to further analysis and sorting. Milan EDTA-anticoagulated PB samples obtained from all individuals enrolled were processed within 24 h after blood withdrawal, incubated with the proper antibodies, followed by NH4Cl (8.6 g/L in distilled water), and washed repeatedly in phosphate-buffered saline plus 0.3% bovine serum albumin. The following antibody mixes were used: FITC-conjugated F(ab)2-anti-sIgk, PE-anti-sIgk (Dako Cytomation), PECy7-labeled anti-CD20 (BD Biosciences), PECy5-conjugated anti-CD5 (Invitrogen), and PE-Texas Red (electron coupled dye)-conjugated anti-CD19. For each sample, up to 500,000 events were acquired on a FC500 flow cytometer equipped with a 488 argon ion laser and a 635 red HeNe laser and analyzed with the CXP software system (Beckman Coulter) according to the following gating strategy: low forward and sideward light scatter (FSClo/SSClo) CD19þ cells were gated and further divided into CD5þ and CD5 subsets, and the sIgk/sIgk ratio was evaluated in both populations (indicating the presence of atypical CLL or non-CLL MBL). Concomitantly, we used a dot plot showing CD5 versus CD20 expression on gated CD19þ cells to identify CLL-like MBL (e.g., CD19þ cells, CD20dim, CD5bright). For quality control purposes, 0.4 mL Flow-Check Fluorospheres (Beckman Coulter) mixed with 0.2 mL Flow-Check 770 (Beckman Coulter PC7 (770/488) Setup Kit) were daily used to assess flow cytometer optical alignment and the performance of the fluidics system. Light scatter intensity, fluorescence intensity, and hydrodynamics were controlled using 0.4 mL Flow-Set Fluorospheres (Beckman Coulter) mixed with 0.2 mL Flow-Set 770 (Beckman Coulter PC7 (770/ 488) Setup Kit), to assess optimal conditions for quantitative analysis of human leukocytes. Leeds Teaching Hospital, United Kingdom Leukocytes were prepared using ammonium chloride as reported previously (5). To screen for MBL in samples with a normal blood count, 5
105 cells were incubated for 30 min with anti-CD19 PE-Cyanin5.5 (Cy5.5; Invitrogen, Paisley, UK), anti-CD5 allophyocyanin (APC; BD Biosciences, Oxford, UK), and FITC/PE: CD20/ CD79b (Beckman Coulter, High Wycombe, UK) and FMC7 (Chemicon Europe Ltd., Chandlers Ford, UK)/ CD23. Subjects with a clonal B-cell excess (kappa: lambda ratio 2.1:1) were assessed with the

Cytometry Part B: Clinical Cytometry

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use of an extended panel as follows. For cases with a lymphocytosis, 5
105 cells were incubated for 30 min with anti-CD19 PE-Cyanin5.5 (Cy5.5) and FITC/PE: CD3/ CD3 (control), CD20 (Beckman Coulter)/CD5, CD10/ CD38 (BD Biosciences), kappa/lambda, FMC7/CD22 (Beckman Coulter), CD11a/CD23, IgM/IgD (BD Biosciences), and IgG (BD Biosciences)/CD79b (Beckman Coulter). All other reagents were prepared in-house. Cells were washed, acquired using a FACSort cytometer and analyzed using CELLQuest software (BD Biosciences) as reported previously. B cells were purified from separated leukocytes obtained from subjects with CLLphenotype MBL, with the use of anti-CD19-coated magnetic beads and a cell separator (autoMACS, Miltenyi Biotec). In samples with more than 95% CD19 expression on selected cells, cytospin slides were prepared, airdried overnight, and stored at 20 C; if a sufficient sample was available, genomic DNA was isolated (QIAamp DNA blood mini kit, Qiagen). Mayo Clinic CBCs are obtained on all specimens using a Sysmex XE5000 or Beckman Coulter ACT-10 hematology analyzers. EDTA-anticoagulated whole blood is stained with a six-color antibody cocktail containing CD19, CD20, CD5, CD45, and sIgj/sIgk light chains and analyzed on either a FACSCanto I or II flow cytometer (BD Biosciences); a standard whole blood lysis method is used. A minimum of 300,000 events but preferably 500,000 events is collected. Daily calibration of PMT voltage settings is done using microbead standards provided by the manufacturer and checked with a normal donor. Compensation controls are prepared by staining cells with a single fluorochrome for software-based corrections. Listmode data are collected and analyzed with FACS Diva software (BD Biosciences). The lymphocyte gating strategy is based on defining singlet events using FSC-A plotted against FSC-H; CD45-positive cells with appropriate lymphocyte scatter are then identified. The next step in the analysis is to isolate the Bcell population by plotting CD19 fluorescence against CD20 fluorescence. Out of this gate, CD5þCD20þ cells are then scanned for sIgk and sIgk expression with all discrete populations being examined for evidence of clonality. Criteria for a sample to be classified as having MBL cells is based on the finding of a cluster of at least 30 B lymphocytes that are definitively clonal or express clonal excess and also express an MBL immunophenotype that resembles classical CLL (CD5þ and CD20dim), atypical CLL (CD5þ and CD20bright), or non-CLL (CD5) subtypes. Additional analysis using CD23 is done to confirm the findings and is also used if the possibility of a biclonal MBL is a consideration after the initial analysis. If CLL or clinical MBL with a clonal B-cell population of >1.0
109/L is identified, additional immunophenotypic analysis is done, including pan T-cell and NK-cell markers, CD10, CD11c, CD22, CD23, CD38, and CD103; for the prognostic markers CD49d and ZAP-70 analysis will be done separately.

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Table 1 Summary of Methods Used for MBL Detection and Analysis Site Salamancaa Duke

Mayo Clinicb St. Luke and FDAc Miland Leeds

Panels

Instrument

CD20, CD45, CD8, anti sIgk, FACSCanto II CD56, anti sIgj, CD4, CD19, CD3, CD38 CD23, CD3, CD14, CD16, LSRII CD20, sIgk, CD19, sIgk, CD5; Dump channel: CD3, CD14, CD16, CD235a CD19, CD20, CD5, FACSCanto I and CD45, sIgj/sIgk FACSCanto II CD19, CD20, CD5, FACSCanto I and CD45, sIgj/sIgk FACSCanto II F(ab)2-anti-sIgk, CD5 FC500 CD20 anti-sIgk, CD19 CD19, CD5, CD20, FACSort CD79b, FMC7, CD23

QC Cytometer Setup and TrackingTM compensation controls AlignFlow Plus Quantum Simply Cellular anti-mouse IgG beads Cytometer Setup and TrackingTM compensation controls Cytometer Setup and TrackingTM compensationcontrols Flow-check fluorospheres and flow-set fluorospheres NA

Analysis

Extended analysis

Infinicyt

IGVH and FISH

FlowJo

IGVH and FISH

FACS Diva

IGVH FISH

FlowJo

Yes

CXP

IGVH and FISH

CELLQuest

IGVH and FISH

a

Salamanca also included two other eight-color tubes, including CD23; see text. Mayo also includes other tubes including CD23 as needed; see text. The FDA also included three other seven- or eight-color tubes that included CD23; see text. d Milan also tested for CD23 expression in a separate tube. b c

General Comments Table 1 contains a brief overview of the reagents, instruments, quality control procedures, software used for analysis, and whether or not extended immunophenotyping was done. We would point to the readers that in this Issue, the article by Shim et al. has more extensive tables detailing how these differences relate to the detection of MBL prevalence. Essentially, every site, in addition to screening for MBL, does some level of extended immunophenotyping either in the same panel or with multiple panels. And all of the sites are doing Ig gene heavy chain V region mutational analysis and interphase fluorescence in situ hybridization. Multiparameter flow cytometry analysis is performed on either fresh (all sites) PB or cryopreserved PBMC (Duke, Milan, and FDA). It is important to note that there are two extremes of volume used for whole blood MBL screening. The usual 100 lL has to be expanded to over 1 mL if one wants to collect 5
106 events. At least 2.5
105 PMBC were incubated with fluorescent-conjugated CD5, CD19, CD20, j, k, and either CD45 or CD23. The fluorescent antibody conjugates were selected by the lead flow cytometry skilled investigator at each site. Of note, in recent years, the EuroFlow Consortium has developed several screening and higher-level panels (system-on-panels) for the flow cytometric analysis of hematological malignancy (van Dongen et al., www.euroflow.org) one of which included a single eightcolor tube for screening of lymphocytes, which simultaneously stains for CD5, CD19, CD20, sIgk, sIgj, CD45 plus CD38, and other additional T- and NK-cell markers. Another point to note is that even though eight colors are being detected, 10 separate reagents are being used. This implies that the same fluorochrome is used twice and is analyzed on two different lymphocyte subsets. Flow cytometry was performed on a LSRII (Duke), FACSCalibur or FACSCanto II (FDA), FACSCanto I or

FACSCanto II (Mayo and St. Luke’s), or FACSCanto II (Salamanca and St. Luke’s) instruments from BD Biosciences or an FC500 (Milan) from Beckman Coulter. Daily calibration of PMT voltage settings were accomplished using microbead standards at all sites following general good practice procedures (10) (www.euroflow.org). Compensation controls were prepared by staining cells and microbead standards with a single fluorochrome for software-based corrections. One site uses the fluorescence minus one method. The number of cell events collected at each site varied from 1
105 to 5
106 cells. Listmode data were collected and analyzed using the FACSDiva, FlowJo, Infinicyt, or CXP software programs. Gating strategies used electronic gates to eliminate cellular debris and aggregates and identified CD45positive cells with typical lymphocyte FSC and SSC features. CD19þ B cells were identified, and the surface expression of CD5 and CD20 was then determined to establish a B-cell phenotype. Three B-cell subsets (CD5CD20þ, CD5þCD20þ, and CD5þCD20dim) were each analyzed for skewing of the ratio of sIj to sIgk, and if needed for confirmation, expression of other markers, e.g., CD23. CLL-like MBL was defined as CD5þ, CD19þ, CD20dim, and sIgdim B cells that were CD23þ with a skewed sIgj:sIgk ratio (3:1 or