ELSEVIER Four-Color Flow Cytometric Analysis of Peripheral Blood Donor Cell Chimerism Diana Metes, Alison Logar, William A. Rudert, Adriana Zeevi, J ennifer Woodward, Anthony J. Demetris, Kareem Abu-Elmagd, Bijan Eghtesad, Ron Shapiro, John J. Fung, Massimo Trucco, Thomas E. Starzl, and Noriko Murase ABSTRACT: Passenger leukocytes have been demon­ 0.2%. When the fresh peripheral blood mononuclear cells strated to play significant roles in initiating and also were separated by conventional Ficoll gradienr purifica­ regulating immune reactions after organ transplantation. tion, similar, but slightly lower levels of donor cells were Reliable techniques to detect donor leukocytes in recipi­ detected. Blood samples obtained I ~ 5 months after liver, ents after organ transplantation are essential to analyze the kidney, and intestine transplants revealed that the kind of role, function, and behavior of these leukocytes. In this organ allograft influenced levels and lineage pattern of the report we describe a simple, reliable method to detect circulating donor cells. This procedure provided a simple donor cells with low frequencies using peripheral blood and reliable method in determining early chimerism in samples. Detection of small numbers of major histocom­ transplant recipients. However, the detection of MHC­ patibility complex (MHC) mismatched cells was first mismatched leukocytes of all lineages was much lower studied using four-color flow cytometry in artificially when frozen peripheral blood mononuclear cells were created cell mixtures. By selecting the CD45 + population used. Human Immunology 64, 787~795 (2003), © and simultaneous staining with several leukocyte lineage American Society for Histocompatibility and Immunoge­ markers (CD3, CD4, CDS, CD56, and CD19), MHC­ netics, 20D3. Published by Elsevier Inc. mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood KEYWORDS: flow cytometry; whole blood; chimerism; samples with a threshold sensitivity as low as D.I %~ solid organ transplantation ABBREVIATIONS mAb monoclonal antibody FISH fluorescent in situ hybridization PBMC peripheral blood mononuclear cells SS side scatter WB whole blood FS forward scatter INTRODUCTION Passenger leukocytes in solid organ allografts have been However, the persistence of small numbers of donor conventionally considered to be the main initiators of leukocytes in the recipient for decades after transplanta­ immune reactions leading to allograft rejection (1, 2). tion implies the possible function of these cells as active modulators for graft acceptance (3, 4). The persistence of mobile donor alloantigens, with sustained immune reac­ From the Depc/rtlllents lifSurgery (D.M., AL., W.AR.,]. W,., K.A-E .. tions between donor and recipient leukocytes, and con­ B.E., R,S.. JIF., AI. T., T.E.S., N.M.), Immunology (D.M,), Pathology sequent mutual immunologic regulation, are considered (A,Z .. AI!),), and Department of Pediatrics, Division of ImmunogeneticJ to be the principal factors for allograft acceptance (5, 6}. (W.A R., ;\1. T.), Thomas E. Starzl Tranplantation Institllte, U niz'enity (if Piwb!£rgh Medical Center, Pittsburgh, PA, USA The analysis of the role, function, and behavior of Address reprint reqmstJ to: Dr. N. Murase, University (if Pittsburgh, donor leukocytes after transplantation requires sensitive Thollias E. Starz! TranplantationInstitute, 200 Lothrop Street, Piwimrgh, PA 15261; Tel: (412) 648- 2155; Fax: (412) 624-6666. techniques to distinguish donor and recipient phenotype Received MelY 1, JO()): retised May 14, 2003; accepted ilia) 14, 2003. cells. The detection of small numbers of donor cells long Human Immunology 64, 787-795 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 0198-8859/03/$-see front matter Published by Elsevier Inc. doi:l0.1016/S0198-8859(03)00113-7 788 D. Metes et al. after ttansplantation has only recently become possible. Grand Island, NY, USA), in liquid nitrogen, and thawed Currently, donor cells are analyzed by polymerase chain rapidly for assay in a 37 DC water bath. reaction (PCR), immunohistochemistry, in-situ hybrid­ ization, and flow cytometry by targeting disparities at HLA Typing the human leukocyte antigen (HLA) loci or sex-deter­ HLA typing was performed at the Tissue Typing Labo­ mining region Y [4, 7-9}' Some of these techniques are ratory (Clinical Immunopathology, Central Laboratory either laborious or require particular tissue samples, Services, Inc., University of Pittsburgh Medical Center, thereby precluding their widespread use. Flow cytomet­ Pittsburgh, PA, USA) using genomic DNA extracted ric analysis is technically simple and the procedure can be from PBMC isolated from all patients in the study. completed in a short time. Although this method has been known to have limitations in detecting low fre­ Artificial Mixtures quency events, it could provide valuable information on Whole blood or isolated PBMC from healthy volunteers surface or intracellular expression of various molecules on were mixed in vitro to generate the artificial samples donor cells. Therefore, it was our goal to establish a containing known levels of HLA-mismatched and sex­ standardized flow cytometric technique that can provide mismatched cells (5%,1%,0.3%, and 0.1%). Aliquots reliable results of donor leukocytes after organ transplan­ of PBMC mixtures were cryopreserved and used in par­ tation in a timely manner. allel with WB mixtures or fresh PBMC mixtures for In this report we describe a four-color flow cytometry comparison analysis. method to detect levels and lineages of major histocom­ patibility complex (MHC) mismatched (donor) cells using Antibodies and Reagents variable types of samples, including whole blood, and fresh The panel of FITC-conjugated monoclonal antibodies and frozen peripheral blood mononuclear cells (PBMC) (mAb) specific for HLA-A or -B loci were either pur­ from normal volunteers, artificial mixtures, and samples chased from One Lambda, Inc. (Canoga Park, CA, USA) from organ transplant recipients. The method is quick and or prepared from hybridomas (A TCC, Manassas, VA, reliable, with a detection limit of 0.1 %-0.2% when used USA) and FITC-conjugated at the University of Pitts­ in combination with lineage-specific antibodies. burgh Cancer Institute Hybridoma Facility, directed by Dr. Albert Deleo. Other lineage-specific mAbs included CD3-PE, CD4-PE, CD56-PE, CD19-PC5, CD8-PC5, MATERIALS AND METHODS and CD45-ECD (Beckman Coulter Co., Miami, FL, Blood Collection and Processing USA, and Becton-Dickinson, San Jose, CA, USA). Flu­ Heparinized whole blood (WB) was obtained from orochrome-conjugated isotype-matched non-specific healthy volunteers and transplant recipients in accor­ mAbs were used as negative controls for each assay. dance with the guidelines of the Institutional Review Board at the University of Pittsburgh. Three types of Flow Cytometric Analysis blood samples were used in this study: WB, freshly Whole blood (100,....1) was incubated with relevant mAbs isolated PBMC, and cryopreserved PBMC. PBMC were or isotype controls for 20 minutes in dark at 4 DC, and obtained by Ficoll-Hypaque (Amersham Pharmacia Bio­ then mixed vigorously with FACS lysing solution (Bec­ tech, Piscataway, NJ, USA) standard density gradient ton Dickinson) for 10 minutes at room temperature for centrifugation method [l0}. Aliquots of PBMC were erythrolysis and fixation. Cells were washed twice and cryopreserved in freezing medium containing 10% fixed in 1% paraformaldehyde in PBS. Alternatively, DMSO and 30% FCS in RPMI 1640 (Life Technologies, freshly isolated or frozen PBMC (1 X 105 cells) were TABLE 1 Monoclonal antibodies used in the study Specificity Isotype Dilution Supplier A2/28 mouse IgG2a 1:300 One Lambda, Inc. A3 mouse IgG2a 1:20* ATCC (HB122)/UPCI Hybridoma Facility A9 mouse IgG2b 1:20 One Lambda, Inc. B7 mouse IgG1 1:30* ATCC (HB56)/UPCI Hybridoma Facility B8 mouse IgG2b 1:20 One Lambda, Inc. B12 mouse IgG2b 1:20 One Lambda, Inc. B27 mouse IgG1 1:20 One Lambda, Inc. * Based on the mAb concentrations of 1 mg/ml. Four-Color Flow Cytometry Analysis of Chimerism 789 0.3% 0.10/0 o o 0.01 0.02 0.18 0.16 "., "I '" CD45ECD HLA-A2FITC 0.11 05 6.62 0.04 0. 1 0.80 0.26 0.06 '''''I ,,,' ,;;' «- ..r. HLA-A2FITC FIGURE 1 Detection of major histocompatibility complex scatter (SS) versus forward-scatter (FS) cytograms, 50,000 (MHC) mismatched cells in different artificial mixtures. CD45 + events were collected per sample and analyzed Whole blood from normal volunteers who were mismatched at with EXP032 software (Applied Cytometry System, human leukocyte antigen A (HLA-A) were mixed to generate 1%, 0 . .3%, and 0.1% artificial mixtures. Level and lineage Sheffield, United Kingdom). Daily calibration of the specificity of MHC-mismatched (HLA-A2) cells were analyzed instrument was performed using flow check fluoro­ on gated CD45 (ECD) positive cells with monocloml antibod­ spheres (Beckman Coulter Co.). Fluorescent compensa­ ies for HLA-A2 (FITC), together with CD.3 (PE) and CD19 tion was performed using a normal control and staining (PE). with each fluorochrome separately. Compensation values were set to eliminate spectral overlap. preincubated with 10% goat serum for 20 minutes for the blocking of nonspecific binding, washed three times, Cytospin and Fluorescent In Situ Hybridization and incubated for 20 minutes in dark at 4 °C with for XY relevant mAbs or isotype controls, followed by washing Aliquots of 1 X 105 cells from WB or PBMC were and fixation. A typical four-color flow cytometry analysis cytospun for 5 minutes at 500 rpm using a centrifuge was performed with mAb combinations of CD45-ECD, (Thermo Shandon, Pittsburgh, P A, USA). The slidES anti-HLA-FITC, and two leukocyte lineage markers con­ were air dried and fixed in 95% ethanol for 10 minutes. jugated with PE and PE-Cy5. Data acquisition and anal­ FISH analysis was carried out using directly labeled ysis was performed on a Coulter EPICS XL flow cytom­ probes for X (red) and Y (greC'i1) chromosome alfa-satel­ eter (Beckman Coulter Co.) mounted with four lite regions, according to the manufacturer's recom­ photomultiplier tubes and a single argon laser.