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Cytometry in Clinical PracticeTranslational Applications of Flow

A. C. Harris and Edmund K. WallerDavid L. Jaye, Robert A. Bray, Howard M. Gebel, Wayne

http://www.jimmunol.org/content/188/10/4715doi: 10.4049/jimmunol.1290017

2012; 188:4715-4719; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Translational Applications of Flow Cytometry in ClinicalPracticeDavid L. Jaye,*,† Robert A. Bray,* Howard M. Gebel,* Wayne A. C. Harris,†,‡ andEdmund K. Waller†,‡

Flow cytometry has evolved over the past 30 y froma niche laboratory technique to a routine tool usedby clinical pathologists and immunologists for diagnosisand monitoring of patients with cancer and immunedeficiencies. Identification of novel patterns of ex-pressed Ags has led to the recognition of cancers withunique pathophysiologies and treatment strategies.FACS had permitted the isolation of tumor-free popu-lations of hematopoietic stem cells for cancer patientsundergoing stem cell transplantation. Adaptation offlow cytometry to the analysis of multiplex arrays offluorescent beads that selectively capture proteins andspecific DNA sequences has produced highly sensitiveand rapid methods for high through-put analysis ofcytokines, Abs, and HLA genotypes. Automated dataanalysis has contributed to the development of a “cytom-ics” field that integrates cellular physiology, genomics,and proteomics. In this article, we review the impact ofthe flow cytometer in these areas ofmedical practice. TheJournal of Immunology, 2012, 188: 4715–4719.

Flow cytometry began as “micro-fluorimetry” in the1960s as an analytic technique to measure the prop-erties of individual cells in a fluid stream following

illumination with a laser (1). Fig. 1A shows the basic opera-tion of a flow cytometer, with an example of flow cytometricanalysis of FOXP31 regulatory T cells in Fig. 1B. By breakingthe fluid stream into a series of small droplets, segregatingindividual cells into a minority of the formed droplets, andquickly interrogating the light properties of individual drop-lets following laser illumination, droplets containing singlecells could be electrostatically charged and separated from themajority of the cells in the fluid stream by deflecting thedroplets into collection tubes during their flight in air usingcharged deflector plates (2). The harnessing of mAbs thatspecifically bound to cell surface markers (3) and the discoveryof a variety of fluorescent dyes with narrow excitation andemission spectra (4) allowed the application of this technologyto basic immunology research, clinical immunology diag-nostics, and cell selection for preclinical models and clinicaltrials of transplanting phenotypically defined cell subsets. The

timeline for the technical milestones of flow cytometry isshown in Fig. 2, with the introduction of clinical applicationsof flow cytometry shown in Table I. This review highlightsthe emerging clinical applications of flow cytometry anddescribes the unique bioinformatics issues that must be ad-dressed when large list-mode files containing data on six ormore parameters from millions of analyzed cells are generatedfrom clinical samples.

Cancer diagnosis and prognosis

A growing body of literature supports the application of flowcytometry in disease prognostication and monitoring ofpatients with hematological malignancies (5, 6), as well as inclinical studies of immune status in patients with cancer,immune deficiency, and allogeneic stem cell and organ trans-plants. More than 50 y ago, when morphology was essen-tially the sole diagnostic modality, only a handful of distincthematolymphoid neoplasms, such as Hodgkin lymphoma,was recognized. A critical component of the current classifi-cation of hematolymphoid neoplasms is determination ofthe best-fit, nonneoplastic immune cell lineage (e.g., B cell,T cell) and differentiation state (e.g., precursor, mature) towhich the Ag expression profiles point. The current interna-tional classification of hematolymphoid neoplasms, with rareexceptions, requires that the immunophenotype be integratedwith morphologic, cytogenetic, and molecular genetic datato render accurate diagnoses (7). More recent advances inidentifying rare immune cell subsets, such as plasmacytoiddendritic cells, and in producing Abs to selectively detect thesesubsets have been translated to further refinements in diseaseclassification (8).Flow cytometry analysis can provide important diagnostic

and prognostic information and identify with high accuracythe distinctive immunophenotype of malignancies and cellDNA content that are used to select specific therapies. Forexample, various CD201 B cell malignancies are now com-monly treated with anti-CD20 Abs (rituximab), and a subsetof CD52-expressing T cell and B cell malignancies may betreated with anti-CD52 (alemtuzumab). Flow cytometry isused to distinguish mantle cell lymphoma, an aggressive dis-order that requires more intensive therapy, from other moreindolent mature CD201 B cell lymphomas with similarmorphology (9). Because the Ag-expression profiles of these

*Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA30322; †Winship Cancer Institute, Emory University, Atlanta, GA 30322; and‡Department of Hematology and Medical Oncology, Emory University, Atlanta, GA30322

Address correspondence and reprint requests to Dr. Edmund K. Waller, Emory Uni-versity, 1365B Clifton Road NE, Room B5119, Atlanta, GA 30322. E-mail address:ewaller@emory.edu

Copyright� 2012 by TheAmerican Association of Immunologists, Inc. 0022-1767/12/$16.00

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B cell neoplasms can be subtly different, flow cytometry canfacilitate diagnosis by identifying reproducible expressionpatterns of multiple Ags associated with specific neoplasmsand relatively quantify Ag density on malignant cells (9). Thepower of the flow cytometer to detect minute quantities ofspecific cells in heterogeneous cell mixtures is used to identifyresidual malignant cells after therapy, known as minimal re-sidual disease monitoring, in common malignancies for whichdisease persistence portends a worse prognosis (10). Lastly,flow cytometry is a rapid means of determining relative cellDNA content for acute lymphoblastic leukemia, the mostcommon childhood blood cancer, which helps to guidetreatment.

Immunologic diseases

The flow cytometer also serves as a principal clinical laboratoryplatform for assessing the immune status of patients, including

determination of CD41 T cell blood counts after HIV in-

fection (11) and classification and prognostication of various

primary immune deficiencies, such as autoimmune lympho-

proliferative syndrome, severe combined immune deficiency,

and Ab deficiencies (12). Moreover, flow cytometric quanti-

fication of specific immune cell subsets in an allogeneic stem

cell graft and of immune reconstitution following allogeneic

bone marrow transplantation serves as predictors of survival

posttransplant (13, 14). Lastly, the flow cytometric quantifi-

A B

FIGURE 1. Schematic of FACS and an example of data. (A) Cells suspended in a core stream (green) are carried in sheath fluid (light gray) to the flow cell

(yellow sphere) where they are interrogated by an excitation laser beam. Cells in the stream are detected by light scattered through the cells forward scatter (FSC)

and orthogonal to the cells side scatter (SSC); cells labeled with fluorescently labeled mAbs are detected by emitted fluorescent light (FL1). Following detection of

FSC, SSC, and FL1 signals, droplets are formed and loaded with positive or negative electrostatic charges. Droplets containing single cells are deflected to the left

or right by highly charged metal plates, and sorted cells are collected into tubes. (B) FOXP31 CD41 regulatory T cells are identified from a mixture of

lymphocytes using multiparameter flow cytometry.

FIGURE 2. Milestones in flow cytometry. Key events in

the development of flow cytometry are shown in a vertical

timeline (left panel), with the number of publications/y

identified in http://apps.webofknowledge.com listing

“microfluorimetry or flow cytometry” as key words (rightpanel).

4716 TRANSLATING IMMUNOLOGY: FLOW CYTOMETRY IN CLINICAL PRACTICE

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cation of extremely rare CD341 endothelial progenitor cellsin the blood represents a novel method to predict clinicaloutcomes in patients with peripheral arterial and cardiovas-cular disease (15, 16).

Cell therapy and transplantation

The flow cytometer has also been used as a research tool topurify specific cell populations for cell-based therapy. Theidentification of CD341 as a marker for human hematopoi-etic stem and progenitor cells (17) led to interest in using flowcytometry to isolate and purify populations of phenotypicallydefined CD341 cells for transplantation. The rationale forthis approach was that patients with cancer can be treatedwith high-intensity chemotherapy and radiation, followed byautologous stem cell rescue to achieve remission of malig-nancies that relapsed after conventional chemotherapy or forwhich conventional chemotherapy was inadequate for long-term remissions. The clinical use of cytometry-based FACSfor isolation of autologous CD341 stem cells allowed thepossibility of achieving a clinically significant depletion ofcontaminating tumor cells, reducing the risk for disease pro-gression from the reinfusion of contaminating tumor cells inthe stem cell graft. Protocols for the study of autologoustransplantation of patients with breast cancer, non-Hodgkin’slymphoma, and multiple myeloma using highly purified flowcytometry-sorted CD341 progenitors were initiated (18–22)(Table II). The results of these studies established the “proof-of-concept” that transplantation of highly purified hemato-poietic stem cells led to durable hematopoietic reconstitutionand suggest that transplantation of autologous stem cellproducts with .5-log ex vivo tumor cell depletion may leadto durable remissions in patients with relapsed cancer (16).The small size of these studies and the lack of a control groupprecludes application for U.S. Food and Drug Administra-tion approval. Although flow cytometry can identify othercellular subsets of therapeutic interest, such as FOXP31

regulatory T cell staining (Fig. 1B), techniques that usefixation preclude isolation of viable cells for therapeuticadministration by FACS (23).Another emerging use of flow cytometry is the measurement

of Abs to HLA molecules, potentially developed after exposurethrough blood transfusions, prior transplantation, or preg-nancy, as well as determination of HLA genotypes for trans-

plantation. Initially performed using cumbersome and insen-sitive serological assays, human histocompatibility testing hasmoved to the forefront in using multiplexing technology,especially in providing detailed HLAAb analysis andmolecularHLA typing. In 1983, a seminal publication (24) demon-strated that donor-directed HLA Abs detectable only by flowcytometry could pose a significant risk for early kidney allo-graft rejection and loss. Since then, numerous studies con-firmed this observation, not only for recipients of kidneyallografts but for other solid organ transplants as well (25, 26).In hematology, the ability to unambiguously identify HLAAbs directed against mismatched HLA Ags in unrelated stemcell transplantation led to the recognition that these Abs cancause engraftment failure (27, 28). In some instances, theidentification of donor-specific HLA Abs may be a contrain-dication for transplantation, whereas, in other situations, Ab-reduction protocols prior to stem cell infusion can be instituted.The clinical use of cytometry was accelerated by the devel-

opment of a new approach to detect HLA Abs using smallplastic spheres (microparticles) for interrogation by a flowcytometer. The use of microparticles displaying purified Class Ior Class II HLA Ags conjugated on their surfaces markedlyreduced false reactions due to non-HLA cell membrane pro-teins associated with using whole cells as targets. Recently, anew cytometry-based multiplexing platform (29) has emergedthat can simultaneously identify 100 unique two-color fluo-rescent microparticles displayed as a two-dimensional virtualarray. Fig. 3 shows an example of sophisticated gating strat-egies that permit the independent analysis of each individualmicroparticle within the array using a third (reporter) color.The reactivity of each bead, as determined by the intensityof the “reporter” fluorescence, determines whether a reactionis considered positive or negative. HLA Ag-coated micro-particles can be used to identify the Class I and/or Class II Absfrom the sera of sensitized patients. Compelling studies dem-onstrated a strong correlation between a positive flow cyto-metric cross-match due to microparticle proven donor-specificAb and early rejection and graft loss (30). The implementa-tion of this new technology led to significant changes in al-location of solid organs to sensitized recipients throughout theUnited States (31).Interestingly, this multiplexing flow cytometry methodology

can also be used for HLA genotyping by attaching individual

Table I. Summary of clinical applications of flow cytometry

Clinical Situation Cell/Analyte of Interest Patient Specimen Types Period of Clinical Use

Cancer Diagnosis of hematolymphoid cancers(mostly leukemias/lymphomas)

Cancer cells Blood, bone marrow,various tissues

Early 1980s

Determine cell DNA content for prognosis(e.g. childhood lymphoblastic leukemia)

Cancer cells Blood, bone marrow Late 1980s

Monitoring of hematolymphoidcancers after therapy

Residual cancer cells Blood, bone marrow Early 1990s

Immunologic diseases Diagnosis and monitoring of HIV/AIDS CD41 and CD81 T cell subsets Blood Early 1980sDiagnosis of primary immunodeficiencies B cells, T cells, and T cell subsets Blood, lymphoid tissues Late 1980s

Cell therapy andtransplantation

Determine adequacy of hematopoieticstem cell grafts (bone marrow transplantation)

to repopulate bone marrow

CD341 stem cells Stem cell graft Early 1990s

Risk assessment for graft rejection ofsolid organs and graft-versus-host disease

after hematopoietic stem cell transplantation

Abs to HLA proteins Recipient’s serum Early 2000s

HLA genotypegenomic DNA

Donor’s blood cells Middle 2000s

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DNA probes to microparticles and simultaneously testingfor ∼100 HLA allele variations (32). Thus, high-volumeHLA genotyping can now be performed easily in any HLAlaboratory with a flow cytometer. In fact, this technology issensitive and specific enough to accurately detect clinicallysignificant single amino acid sequence differences predictedbetween HLA molecules for patients undergoing stem cell orsolid organ transplantation (28, 32).

Bioinformatics: future area of growth

The use of multiplexed bead assays and DNA ploidy assaysin high-throughput diagnostic or prognostic evaluations andstudies involving detection of rare cell subsets can result inthe acquisition of exceedingly large datasets. Progress in datasharing is hampered by the lack of standardization in dataanalysis and reporting, as well as a means of efficiently sharinglarge datasets for independent evaluation and collaborationacross different sites. For example, data analysis tends to beintuitively based on serial evaluations of two-dimensionalplots, which represents a significant source of variation thatlimits standardization and reproducibility in data interpre-tation. To maximize the potential of flow cytometry forclinical use and investigation, informatics systems, such asthose applied in analyses of massive genomics and proteomicsdatasets, will need to be integrated with data-collectionefforts.The emerging field of “cytomics” represents an integrated,

whole-cell–based description of cellular physiology that in-cludes aspects of genomics (genes and regulatory processes)and proteomics (protein abundance and structure) endeavorsto develop a more comprehensive picture of the biology ofT

ableII.

Summaryofpublished

resultsfrom

clinicalstudiesof

autologousstem

celltransplantation

incancerpatientsusingFACS-purified

CD341hem

atopoieticcellprogenitors

References

No.

ofTransplantedPatients

andDisease

AgSelection/Transplanted

CellDose

Tumor

Cell

Con

tamination

Pre-/PostcellSorting

Engraftm

ent

Absolute

Neutrophil

Count.

500/ml(d)

TCellIm

muneReconstitution

Long-Term

Outcome

Bombergeret

al.(18)

9non

-Hodgkin’slymphoma

1.9

3106CD341cells/kg

Not

reported

11

300CD31cells/mlat

6mo;

restricted

VbTCRrepertoire

Not

reported

Tricotet

al.(19)

9myeloma

13

106CD341CD90low

cells/kg

16

Not

reported

Not

reported

Muller

etal.(20)

22breastcancer

1.1

3106CD341CD90low1

cells/kg

245,470-folddepletion

withmultiparam

eter

sortingversus501-fold

depletion

withCD341

selection

10

700CD31cells/mlat

12mo

23%

aliveat

10years;18%

disease

free-survival

Michalletet

al.(21)

23myeloma

0.7

3106CD341CD90low1

cells/kg

Not

reported

11

800CD31cells/mlat

6mo

Not

reported

Vose

etal.(22)

26non

-Hodgkin’s

lymphom

a0.5

3106CD341CD90low

cells/kg

3.3–5.7

logreduction

12

419CD31cells/mlat

day

100

70%

overallsurvivalat

4y;

55%

event-free

survivalat

4y

FIGURE 3. Generation of a virtual 103 10 data matrix using fluorescently

labeled beads. Data are generated by a multiplexing, bead-based flow

cytometry method. Individual beads are identified within a two-dimensional

matrix by their unique and reproducible fluorescence signature derived from

their incorporation of differing quantities of two classifier dyes (x- and y-axes).For a given combination of the classifier dyes (x, z), specific measurements of

a test analyte are assessed by a “reporter” fluorescent molecule and are mea-

sured on the vertical or z-axis. The intensity of the fluorescence from the

reporter molecule is proportional to the amount of specific binding of the

“reporter” fluorescent probe to the bead. In this example, 10 beads have re-

porter fluorescence that is above the baseline background.

4718 TRANSLATING IMMUNOLOGY: FLOW CYTOMETRY IN CLINICAL PRACTICE

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individual cells. The flow cytometer represents a logicalplatform upon which to build this field. However, a majorchallenge will be to develop standardized algorithms that al-low combining and comparing datasets from different plat-forms to foster analytical reproducibility and allow informativemeta-analyses.Automated systems for flow cytometry data analysis are

being developed that use standardized ontologies and methodsthat treat such datasets as statistical objects embedded in n-dimensional space or clustered distributions that may be com-pared with one another to foster objective data capture,evaluation, and reporting. New data standards and elementsthat incorporate variables of experimental protocols for dataacquisition and methods used for postprocessing data analysisand interpretation will be required as well. In aggregate, thesedevelopments will advance the clinical use of flow cytometryand broaden its applications in the investigation of diseasebeyond hematologic malignancies and immune monitoring intoa new age of clinical discovery and focused pathophysiology.

ConclusionsIn summary, the development and implementation of flowcytometry-based technologies has had major impacts on thediagnosis and classification of disease, monitoring, and prog-nostication of patients with cancer, as well as patients re-ceiving allogeneic hematopoietic stem cell transplants andsolid organ allografts. High-speed sorting of CD341 cells hasprovided proof-of-principle data that demonstrate the abilityof highly purified hematopoietic stem cells to provide durableand effective hematopoietic reconstitution in patients withmalignancies who are undergoing high-dose chemotherapy.The ability of flow cytometry to generate large amounts ofmultidimensional, high-complexity data that are amenable tohigh through-put, automated analysis strongly positions thistechnology as a key platform for use in clinical medicine fordecades to come.

DisclosuresThe authors have no financial conflicts of interest.

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