Comparison of two immunomagnetic separation technologies to deplete T cells from human blood samples

Comparison of Two ImmunomagneticSeparation Technologies to Deplete T CellsFrom Human Blood Samples

Oscar Lara,1 Xiaodong Tong,1 Maciej Zborowski,2 Sherif S. Farag,3 Jeffrey J. Chalmers1,4

1Department of Chemical and Biomolecular Engineering, The Ohio State University,120 Koffolt Laboratories, 140W. 19th Avenue, Columbus, Ohio 43210; telephone: (614)292-2727; fax: (614) 292-3769; e-mail: [email protected] Engineering Department, The Cleveland Clinic Foundation,Cleveland, Ohio3Department of Internal Medicine, Division of Hematology/Oncology and theComprehensive Cancer Center, The Ohio State University, Columbus, Ohio4Director,UniversityCell Analysis andSortingCore,Heart andLungResearch Institute,The Ohio State University, Columbus, Ohio

Received 26 June 2005; accepted 18 November 2005

Published online 3 March 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20807

Abstract: The objective of this study was to compare theperformance of two immunomagnetic separation tech-nologies to deplete T cells from buffy coats of humanblood. Specifically, two versions of the commercialMACS1 Technology: MiniMACS and SuperMACS, and aprototype, flow-through system, the QMS, were evalu-ated. Peripheral blood mononuclear leukocytes (PBL)were isolated from buffy coats and an immunomagneticseparation of CD3þ cells was conducted using companyand optimized labeling protocols. To mimic peripheralblood containing bone marrow purged hematopoieticstem cells, HSC, CD34 expressing-cells (KG1a) werespiked into PBL prior to T-cell depletion once optimizeddepletion conditions were determined. Once the labelingprotocol was optimized, the MiniMACS system per-formedwell byproducingahighlyenrichedCD3þ fraction,and a respectable level of depletionof T cells and recoveryof KG1a cells in the depleted fraction; an average log10

depletion of T cells of 2.88� 0.17 and an average recoveryof the KG1a cells of 60.8� 5.94% (n¼ 14). The perfor-manceof theSuperMACSsystemwasvery similarwith anaverage log10 depletion of T cells of 2.89� 0.22 and anaverage recovery of KG1a of 63.1� 8.55% (n¼ 10). Incontrast, the QMS system produced an average log10

depletion of T cells of 3.98� 0.33 (n¼ 16) with a corre-sponding average recovery of 57.9� 16.6% of the spikedCD34þ cells. The aforementioned QMS performancevalues were obtained using sorting speeds ranging from2.5� 104 to 1.7� 105 cells per second. It is suggested thatthe lack of a 100% recovery of the unlabeled KG1a cells isthe result of a previously reported ‘‘drafting’’ phenomenawhich pulls unlabeled cells in the direction of themagnetically labeled cells thereby resulting in loss of theunlabeled cells. � 2006 Wiley Periodicals, Inc.

Keywords: cell separation; immunomagnetic; T celldepletion; QMS; MACS; HCS transplantation

INTRODUCTION

Effective isolation and/or depletion of a target cell population

from amore complex cell population is fundamental to many

basic biological and clinical applications. With respect to

enrichment of desired cells by depletion of undesirable cells,

a large number of cell types have been magnetically targeted

for depletion, several of which include CD3, CD45, and

phosphatidyl serine expressing (apoptotic) cells (Barfield

et al., 2004; Lara et al., 2004; Leon and Roy, 2004; Tondreau

et al., 2004).

While it is always desirable to obtain a high level of

performance in a separation process, that is, a high purity and

recovery of the target cell in a positive cell selection, or

alternatively, a high level of depletion of an undesirable cell

type in a depletion cell separation, some of the highest and

most demanding applications are in the clinical environment.

For example, a significant effort has focused on the

production of cellular grafts with low or no potential to

develop Graft-versus-Host Disease (GvHD) to treat patients

after myeloablative procedures (Ho and Soffer, 2001; Kiehl

et al., 2000; Koh et al., 1999). T cells have been identified as

the general target cell population to deplete and to prevent

GvHD, and it has been suggested that up to 4–4.5 log

depletion of T cells is needed to eliminate GvHD following

allogeneic hematopoietic stem cell (HSC) (Aversa et al.,

1998; Lang et al., 1999).

While different population of T cells may be depleted,

including specific alloreactive T cells, the majority of

investigations to date have involved pan-T-cell depletion.

To achieve a high-level of depletion of T cells for

hematopoetic stem cell (HSC) transplants, three general

�2006 Wiley Periodicals, Inc.

Correspondence to: Jeffrey J. Chalmers

Contract grant sponsors: National Science Foundation; SHOT, INC;

National Cancer Institute

Contract grant numbers: BES-9731059, BES-0124897; NSF SBIR 02-056;

R01 CA62349, R01 CA97391-01A1, 5P30 CA16058, 1 R01 AI056318-01A1

approaches have been taken: (1) positive selection of the

harvested stem cell product, (2) negative depletion of the T

cells, or (3) a combination of both.

Cell isolation and or depletion systems can be classified as:

(1) batch systems (i.e., immunoaffinity or immunomagnetic

columns, rosetting combined with centrifugation), (2) con-

tinuous flow through systems (i.e., flow cytometry (FCM)-

based sorting), or (3) a hybrid of the two. With respect to

clinical cell sorting, two approaches have predominantly

been used: (a) a positive selection for HSC followed by a

negative depletion of T cells (i.e., use of Isolex 300 (Debelak

et al., 2000; Martin-Henao et al., 2001), CEPRATE System

(Martın-Hernandez et al., 1997)), or (b) a single positive

selection of CD34þ or CD56þ cells (i.e., use of CliniMACS

system fromMiltenyi Biotec GmbH, Germany) (Gaipa et al.,

2003; Lang et al., 2002). While, on average, a significant

depletion of T cells and recovery of HSC has been reported

with the CliniMACS (a 4.1 log depletion of T cells has been

reported) in 10%–20% of the procedures a second, positive

depletion of T cells was needed to obtain a sufficient depletion

for transplantation (Lang et al., 2002).

As an alternative to the current immunomagnetic cell

sorting systems, a flow-through immunomagnetic cell sorting

method has been developed in the author’s laboratories.

Figure 1 presents a schematic diagram of the system and a

picture of thewhole device (Lara et al., 2004; Sun et al., 1998).

The system is composed of a columnwith concentric inlet and

outlet splitters to separate the cell suspension injection site (a0)

Figure 1. A: Schematic drawing of the separation column of theQuadrupolemagnetic cell sorter (QMS) showing cell injection port (a0), carrier (sheath fluid)injection port (b0), and elution of non-magnetically labeled cells (a) andmagnetically labeled cells deflected by themagnetic field (b). The topview,B, shows themagnetic forces acting on the column to deflect cells. C is a photograph of the bench scale system. D: is an enlarged diagram of the magnetic separation zone

indicating the location of the inner and outer splitting cylinder.

Lara et al.: Comparison of Two Immunomagnetic Separation Technologies 67

Biotechnology and Bioengineering. DOI 10.1002/bit

from the carrier injection site (b0). The column is placedwithin

a strong quadrupolemagnet and themagnetic forces, acting on

the previously labeled cells with antibody-conjugated mag-

netic beads are deflected towards stream b or the wall. Non-

labeled cells are recovered from stream a.

In an attempt to quantify, compare, and address the limi-

tations of the current systems to separate cells, for this study

the authors have selected T-cell depletion from peripheral

blood lymphocytes as an example system to study in a

commercial immunomagnetic cell sorting system (the bench

scaleMiniMACSand the larger scale SuperMACSsystems) as

a well as our prototype, flow through, quadrupole magnetic

cell sorter, QMS. Ultimately, it is intended to apply the QMS

system to T-cell depletions to achieve a rapid, four to five log

depletion on a scale compatible with clinical applications.

With this background, the current work presents the

results of an experimental comparison of the two technolo-

gies. In addition, a discussion is given which outlines the

performance and operational conditions needed for a clinical

application.

MATERIALS AND METHODS

Cell Sources

Peripheral blood buffy coats from apparently healthy donors

were purchased from the American Red Cross, Central Ohio

Region. Isolation of mononuclear cells was carried out by

diluting buffy coat aliquots with Hank’s balanced salt solution

(HBSS, JRH Biosciences, Lenexa, KS), and laid over a Ficoll-

Hypaque (Accuprep, Accurate Chemical and Scientific Corp.

Westbury, NY) density gradient (1.077 g/mL). Tubes were

spun for 30 min at 350g with the centrifuge brake off. The

recovered mononuclear cell layer was washed twice with PBS

buffer (PBS, JRH Biosciences) supplemented with 0.5%

bovine serum albumin (BSA, Invitrogen Corporation,

Carlsbad, CA) and 2 mM EDTA (Invitrogen Corporation).

After the second washing step, the supernatant was decanted,

and the cells were resuspended in labeling buffer and

transferred to 75 cm2 T-flasks (Corning Corporation/Life

Sciences, Acton, MA) to deplete adherent cells (monocytes/

macrophages) from the suspension, as this has been shown to

prevent non-specific uptake of magnetic nanoparticles

(Comella et al., 2001). Flasks were placed in an incubator at

378C and 5% CO2 for 2 h. Non-adherent cells were removed

and centrifuged, the supernatant was decanted and cells were

resuspended in RPMI-1640 (ATCC, Manassas, VA) supple-

mented with 10% fetal bovine serum (FBS, JRH Biosciences).

Cells were next transferred to T-flasks and incubated at the

same condition as the adherent cell depletion step. Mono-

nuclear cell culturewasmaintained for amaximumof 2 days;

upon completion of such cultures, labeling, and separations

were conducted.

In order to mimic HSC present in a stimulated leukaphor-

esis product, for the final, optimized studies with the

MiniMACS, SuperMACS, and QMS, the monocyte/macro-

phage depleted peripheral blood buffy coats were spikedwith

cells from the CD34þ cell line KG1a (Cat # CCL246.1,

ATCC). KG1a cells were maintained in Iscove’s modified

Dulbecco’s medium (ATCC) supplemented with 20% FBS

(JRH Biosciences) in an incubator at 378C and 5% CO2. On

the day of the experiment, cells were harvested and washed

twice with PBS buffer, resuspended, and cell concentration

determined.

Four types of immunomagnetic separations were con-

ducted in two types of instruments: (1) separations in a

MiniMACS system (MS column with a reported capacity of

107 magnetically labeled cells) following the manufacturer’s

protocol, (2) separations in a MiniMACS system (MS

column) with an elevated magnetophoretic mobility (sug-

gested by manufacturer for some depletion separations), (3)

separations in a SuperMACS system (LD column with a

reported capacity of 108magnetically labeled cells) using the

manufacturer’s protocol, and (4) separations in the QMS

system.

Cell Labeling

Four labeling protocols were used: (1) manufacturer’s

protocol for the MiniMACS system, (2) an elevated

concentration of labeling reagents to increase the magnetic

force on the targeted cells in the MiniMACS, (3) manufac-

turer’s protocol for the SuperMACS, and the (4) optimized

labeling protocol for the QMS.

Primary Antibody Saturation Curves

It has been previously demonstrated that using both the batch

Miltenyi Biotec MACS system or the flow-through, QMS

system performance is partially based on the degree towhich

a cell is immunomagnetically labeled, quantitatively referred

to as a cell’s magnetophoretic mobility (Comella et al., 2001;

Nakamura et al., 2001;Williams et al., 1999). In addition, the

manufacturer of theMACS system states that theMS column

can be used for depletion separations if the target cells are

‘‘strongly magnetically labeled’’ (Miltenyi Biotec Catalog,

Subsection 14.3).

Mathematically, the magnetophoretic mobility of an

immunomagnetically labeled cell can be represented by:

m ¼ v

Sm¼ ABC n3 �nano

3pDc�¼ b ABC �nano ð1Þ

where m is the magnetophoretic mobility of the labeled cell,

and v is the velocity of the labeled cell in the magnetic

energy gradient, Sm. The mobility can be expressed in terms

of ABC, the antibody binding capacity (defined further

below), n3, the number of magnetic nanoparticles con-

jugated to the antibody, fnano, the field interaction parameter

which is the product of the difference in the magnetic

susceptibility of the magnetic nanoparticles and the

suspending buffer times the particle’s volume, Dc, the

diameter of the cell, and �, the viscosity of the suspending

buffer (McCloskey et al., 2003b; Zhang et al., 2005).

68 Biotechnology and Bioengineering, Vol. 94, No. 1, May 5, 2006

DOI 10.1002/bit

The ABC and be further defined by:

ABC ¼ n1�1�1 or ABC ¼ ðn1�1�1Þðn2�2�2Þ ð2Þwhere the first equation holds for a single antibody binding

step and the second equation is for a two step labeling. The

term n refers to the number of binding sites for the primary or

secondary antibody, y, is fraction of the antibody-binding sitebound with an antibody, and � is the valence of the antibody

binding. It has been demonstrated that the binding to these

sites follows a Langmuir-type saturation; consequently,

the saturation of these sites is a strong function of the

equilibrium, antibody concentration (Chosy et al., 2003).

For immunomagnetic separation, either a two step,

primary antibody followed by a secondary antibody binding

to the primary or a single primary step was used. Primary

antibodies used were anti CD3 PE (BD Biosciences, San

Jose, CA:Cat# 555333, UCHT1 clone) and saturation studies

were performed by labeling 1� 106 cells in PBS buffer, with

different amounts of anti-CD3 PE antibody, for a total cell

suspensionvolume of 1mL. The fluorescence intensity, FI, of

these samples, and appropriate isotype controls weremeasur-

ed using a FACSCalibur flow cytometer (BD Immuno-

cytometry Systems, San Jose, CA). For a given set of

experiments, all conducted sequentially with the same FCM

settings, the FI was normalized by dividing the FI of a given

sample by the highest value of FI obtained in a given set of

experiments.

Secondary (or Single Step, Antibody-MagneticBead Conjugate) Antibody Saturation Studies

Two types of antibody-magnetic particle conjugateswere used

in this study: anti-CD3DMnanobeads (BDBiosciences, Cat#

552593, HIT3a clone) and anti-PE MACS nanobeads

(Miltenyi Biotec, Auburn, CA; Cat# 130-048-801). As with

the primary antibody saturation studies, 1� 106 cells in PBS

were labeled with different amounts of the antibody-bead

conjugate in question. A total suspension volume of 1 mLwas

obtained and before mobility measurements, the cell suspen-

sion was diluted to a final concentration of 0.3� 106 cells/mL

to prevent any cell–cell interactions. Unlike the FI measure-

ments obtained when using FCM, magnetophoretic mobility

measurements in the CTV apparatus are determined on an

absolute basis; consequently, the results were not normalized.

Finally, an estimation of a combined magnetophoretic

mobility using a saturating amount of anti-CD3DMbeads, and

three different amounts of anti-CD3 PE and anti-PE nanobeads

was conducted. All experiments were based on the largest

measured magnetophoretic mobility for a given antibody

combination. Additionally, the effect of FcR blocking agent

(Miltenyi Biotec, Cat# 130-059-901) was determined.

Magnetically Activated Cells Sorting WithMiniMACS and Super MACS System Using Eitherthe Manufacturer’s or Optimized Labeling Protocol

An aliquot of isolated peripheral blood mononuclear

leukocytes (PBL) containing the number of cells required

by the experimental protocol (between 5 and 7� 106 total

cells for the MS columns and 7� 106 for the LD columns)

was transferred to 12� 75 mm tissue culture tubes (Fisher

Scientific, Hampton, NH). Following either the manufac-

turer’s or optimized protocol, cells were labeled with a

primary antibody, anti-CD3 PE (Cat # 555333, BD

Biosciences), and a secondary antibody, anti-PE MACS

(Miltenyi Biotec, Cat # 130-048-801). Cells were kept at 48Cuntil cell sorting. Consistent with the manufacturers’

operational protocol, cell suspension was poured into

the MACS columns, the negative eluent was collected, and

1.5 mL of buffer was added after the cell suspension flowed

through the column and subsequently collected in the nega-

tive eluent. The columnwas then removed from themagnetic

housing and washed twice with buffer to collect the

magnetically labeled fraction.

Magnetic Labeling of PBL for QMS Studies

For the QMS studies, the labeling approach for T-cell

depletion relied on either one antibody clone or a combina-

tion of antibodies reportedly targeting two different binding

sites on the CD3 molecule: anti-human CD3 antibodies,

clone HIT3a, conjugated to the DM, magnetic particle (BD

Biosciences, Cat# 552593), and the anti-human CD3

antibody, clone UCHT1, which is conjugated to PE (BD

Biosciences, Cat# 555333).

Cell Concentrations and Flow Cytometry,FCM, Analysis of the Eluted Fractions

Cell concentrations were determined with a Coulter Multi-

sizer II (Beckman Coulter, Miami, FL). FCM analysis was

performed using a FACSCalibur flow cytometer (Beckton

Dickinson, San Jose, CA). Two methods of analysis were

used: (1) a relatively simple gating of lymphocytes based on

forward scatter, FS, and side scatter, SS, and then subsequent

CD3þ expression, and (2) a more complex analysis based on

FS, SS, cell viability, and CD3, CD45, and CD34 expression.

Controls used included unstained cells and single-color cells

stained to perform compensation adjustments. In all cases a

minimum of 10,000 events (50,000 events in post-sort

analysis for fractions derived fromQMS samples spikedwith

KG1a cells) were acquired in the selected analysis region.

Antibodies used in the analysis prior, and subsequent, to the

actual separation could have included (depending on the

experiment): anti-CD3 PE (Cat# 555333, BD Biosciences),

anti-CD45 (Cat# 555482, BD Biosciences), and anti-CD34

APC (Cat# 340441, BD Biosciences).

To assist in distinguishing between viable and dead cells,

7-AAD was used as a viability stain in most of the studies

(Schmid et al., 1992, 2000) (Cat # 555815, BD Biosciences).

To set the gate value distinguishing viable and dead cells,

1� 106 of peripheral blood leukocytes (PBL) or CD34-

expressing cell line (KG1a) were resuspended in 50 ml oflabeling buffer, and the cell suspension was placed into a

water bath at 428C for 30 min following published protocols.



The cell suspension was then labeled with 5 ml of 7-AADstaining solution and incubated for 15 min in the dark at 48C.The heat shocked, stained cells were then used to set the

threshold FI signal, which distinguished dead cells (gate).

To increase the accuracy of the FCM analysis, for the

optimized MiniMACS, SuperMACS, and QMS experi-

ments, for an event to be considered a viable T-cell, it had

to be above a specific size on the FS axis, exclude the 7-

ADD dye, and be positive for CD45 and CD3. For an event

to be considered a KG1a cell, the same criteria held except

that it had to be positive forCD34 instead of CD3.As before,

cell concentration was determined with the Coulter

Multisizer II.

Magnetophoretic Mobility Measurements UsingCell Tracking Velocimetry, CTV

The principles and method of operation of the cell tracking

velocimetry (CTV) system have been presented previously

(Chalmers et al., 1999). Basically, significant analogies

between CTV and FCM exist, with the exception that

currently the CTV measures a single parameter, magneto-

phoretic mobility, and that it provides these values in an

absolute scale (with units).

Cell Sorting With Quadrupole MagneticCell Sorter

The description of the QMS system as well as general

operating principles have been described previously (Lara

et al., 2004; Sun et al., 1998;Williams et al., 1999). Figure 1 is

a schematic drawing presenting the general operation of

the system. For separations used in this study, the channels

were manufactured by SHOT, Inc. (prototype # 10597). The

characteristics of the QMS system include: a maximum

magnetic field strength of the quadrupole, Bo, of 1.37 Tand a

mean force field strength, Sm, of 2.382� 108 T�A/m2. The

length of the field, L, is 15.5 cm.

Two modes of operation were used in this study: the

complete, continuous flow through mode of both the cell

suspension feed,Qa0 toQa, and the sheath flow,Qb0 toQb; and

the deposition mode in which the sheath flow (Qb0 to Qb) is

turned off prior to injection of the cell suspension to a0 (flowonly from Qa0 to Qa).

Theoretical relationships to predict the performance of the

complete flow-through mode of operation have been

previously developed and reported (Williams et al., 1999).

These relationships are based on: the geometry of the system,

the magnetic field strength, knowledge of the mean, and

distributions of the magnetophoretic mobility of the

immunomagnetically labeled cells (or particles) from

experimental, CTV measurements, and the fact that the flow

in the system is laminar.

For example, the throughput, TP, through the QMS system

is given by (Williams et al., 1999):

TP ¼ 2pLB20

m0m1C

I2 �1; �ISS½ �I1 �1; �OSS½ �

1

m1=�mð3Þ

where:

m1=�m ¼ m1=ðm1 � m0Þ ð4Þ

which is also referred to as the resolving power of the system.

If a cell has a magnetophoretic mobility value of mm, then a

mathematical representation of m0 and m1 can be assumed

such that:

if mm < m0; then Fa ¼ 1 and Fb ¼ 0 ð5Þ

if mm > m1; then Fa ¼ 0 and Fb ¼ 1 ð6ÞIn addition, C refers to the cell concentration, and the term

‘‘�’’ can be interpreted as a non-dimensional radius for a

specific location with respect to the inner surface of the outer

wall of the annulus (Fig. 1). Finally, I2 and I1 are the results of

specific integrations over the boundary radii used to calculate

cells (or particle) positions (Williams et al., 1999).

A previously developed (Hoyos et al., 2002) computer

algorithm using these performance relationships was written

in Maple V (Waterloo Maple, Ont., Canada) to calculate the

fractional recoveries of the magnetically labeled cells or

particles eluting in the exit streams, Fa or Fb, or retained on

the wall of the column Fw. Inputs to this program include

paired mobility-frequency data obtain from CTV measure-

ments, QMS geometric characteristics, the total flow rate

through the system, QT, and specific flow rate ratios into and

out of the QMS, Qa0/QT and Qa/QT. Figure 2 is an exampleof a CTV analysis used as an input for this computeralgorithm: specifically, a sample of human lymphocyteslabeled with anti-CD3-PE and anti-PEMACS. The program

then predicts the fraction of magnetically labeled cells in the

exit streams, Fa and Fb, or deposited on the wall, Fw.

The program assigns a starting position and trajectory to

eachparticle.Basedon these trajectories and the total flow-rate,

the final position of each particle is calculated. Both further

details of the code and experimental evaluation of its prediction

for cell and particle suspensions have been previously

published (Hoyos et al., 2000; McCloskey et al., 2003a).

Figure 2. Representative magnetophoretic mobility distribution obtained

from the labeling of human lymphocytes with anti-CD3-PE and anti-PE

MACS.


DOI 10.1002/bit

In terms of operating the QMS in a deposition mode, the

system has yet to be modeled; nevertheless, the operation is

conceptually much simpler than the completely continuous

mode in that the flow rate of the cell suspension in equals the

flow rate of the cell suspension out. Also, qualitatively, one

can assume that the longer the residence time in the magnetic

energy gradient, the great the chance of the magnetically

labeled cells to be retained within the column.

Relationships Used to Evaluate Performance

As presented in the introduction, one of the measures the

clinical bone marrow transplant community uses to evaluate

the performance of a T-cell depletion system is the log10

depletion of the T cells. Mathematically, this is determined

by:

Log10 depletion ¼ log10Ninitial � FT; initial

Nfinal � FT; final

� �ð7Þ

where Ninitial is the initial number of cells in the sample

injected, FT,intial is the fraction of the specific cell population

which is the target cell (T cells in this case), Nfinal is the

number of cells in the final, depleted, cell sample, and FT,final

is the fraction of cells that are the target cell (T cells) after

sorting, respectively.

A second important term used to evaluate the effectiveness

of a separation process is the recovery of desirable cells, in

Figure 3. A: Secondary antibody saturation curve maintaining constant primary antibody and different amounts of secondary antibody (anti-PE MACS, no

concentration provided by the manufacturer), (B) saturation curve for anti-CD3 DM antibody, (clone HIT3a) (concentration is not provided by manufacturer),

(C) effect of FcR blocking agent onmagnetophoretic mobility whenmaintaining constant the amount of anti-CD3DMbeads. There is no noticeable difference

between measurements.



this case non-T cells, and when specifically labeled, HSCs.

Recovery is defined as:

RESULTS

Primary and Secondary AntibodySaturation Curves

To determine the optimum labeling concentration to max-

imize the magnetophoretic mobility of labeled cells,

saturation studies were conducted on the binding of primary

and secondary antibodies. A saturation curve of the binding

of the primary antibody, anti-CD3 PE (BD Biosciences, Cat

#555333) to human PBL was conduced, and it was

determined that 50 mL (or 1.25 mg) of antibody per 106 cells,in 200 mL staining solution, was sufficient to guarantee

saturation. Figure 3A is a saturation curve of the binding of the

secondary antibody anti-PEMACS, using 50 mL per 106 cells

for the primary antibody. Visual inspection indicates that a

maximal saturation is achieved with a secondary antibody

concentration of 75 mL/106 cells in 200 mL of total volume.

Introduction of a Second Antibody Targeting aDifferent Antigen Site on the CD3 Complex

As discussed previously, the anti-CD3 DM antibody-

magnetic nanobead conjugate (clone HIT3a) targets a

different site on the CD3/TCR complex in contrast to the

anti-CD3-PE UCHT1 clone. As with the other labeling

reagents, a saturation curve was created (Fig. 3B) of the

measured magnetophoretic mobility as a function of the

concentration of anti-CD3 UCHT1 clone. The highest

concentration used, 50 mL/106 cells in 200 mL of total

volume, is ten times that recommended by the manufacturer,

raising the concerns of potential non-specific binding. To

investigate this further, the effect of blocking the FcR

receptor on magnetophoretic mobility was measured using

FcR blocking reagent (Milteny Biotec, Cat# 130-059-901),

while maintaining a constant concentration (50 mL/106 cells�200 mL) of CD3-DM antibody (Fig. 3C). As can be

observed, no significant effect of the FcR blocking agent can

be detected.

Magnetophoretic Mobility UsingBoth Primary Antibodies

To attempt to further increase the magnitude of the

magnetophoretic mobility of the labeled cells, a combination

of anti-CD3 PE/anti-PE MACS and anti-CD3 DM antibody

was tested. A steric effect is a potential limitation of this

labeling scenario; therefore, CD3 PE saturation curves with

PBL labeled with and without anti-CD3DM antibodies were

conducted. Cells were labeled with increasing amounts of

anti-CD3-PE, and either no or a constant level of anti-CD3-

DM (50 mL/106 cells). Figure 4A indicates that low, but

statistically significant levels of steric hindrance was

observed at lower parts of the saturation curve (two-sided t-

test, �¼ 0.05, p-value <0.002).

Percent Recovery of target cells ¼ ðNumber of total cells recoveredÞ � ðPercentage of target cellÞðNumber of total cells addedÞ � ðPercentage of target cellÞ

� �� 100 ð8Þ

Figure 4. A: Steric effect when co-labeling with anti-CD3 DM and anti-

CD3 PE; saturation curve for anti-CD3 PE antibody, no anti-CD3DM added

(&), saturation curve maintaining constant anti-CD3 DM (50 mL/200 mL—106 cells) and anti-CD3 PE (^), (B) Magnetophoretic mobility of cells co-

labeled with anti-CD3 PE, anti-PE MACS (amount of CD3-PE/amount of

MACS) in 106 cells in 200 mL The amount of anti-CD3-DM used was

constant at 50 mL.


DOI 10.1002/bit

Figure 4B is a plot of the magnetophoretic mobilities of

PBL labeled with a constant FcR blocking agent (50 mL/106

cells), anti-CD3-DM (50 mL/106 cells), and three different,

increasing amounts of anti-PE MACS. As shown, while not

increasing with increasing concentration, the magnetophore-

tic mobility using this multiple conjugate labeling protocol

results in a total magnetophoretic mobility significantly

higher than the sum of either magnetic-colloid conjugates

individually.

CD3þ Cell Depletion Using the MiniMACSSystem (MS Columns)

Initial studies of CD3 depletion using theMiniMACS system

with MS columns used the company recommended protocol

and basic FCM analysis (FS, SS gating, CD3-PEþ positive

population). A total of four experiments were conducted

and these columns provided high purity of CD3þ cells

(99.14� 0.27%), although the depleted fraction still contains

a considerable percentage of CD3þ cells (4.54� 1.30%).

The average total cell recovery was 100%.

To determine the effect of increasing the magnetophoretic

mobility of the target cells on performance in theMiniMACS

MS columns, the concentration of the secondary antibody,

(anti-PEMACS nanobeads) was increased while keeping the

concentration of the primary antibody (anti-CD3 PE; Beck-

man Coulter) constant at 20 mL/106 cells. A significant

decrease in the proportion of CD3þ cells in the depleted

fraction was observed as the concentration of secondary

antibody was increased from 2 mL/106 cells (recommended

by the manufacturer) to 30 mL/106 cells (Fig. 5B). The purityof the enriched, CD3þ fraction remained almost constant at

greater than 99%. However, along with the decrease in the

proportion of CD3þ cells in the depleted fraction, a decrease

in the overall cell recovery (sum of the depleted and enriched

fractions) was observed with increasing secondary antibody

concentration (Fig. 5A).

With this knowledge of the impact of increased magne-

tophoretic mobility on MiniMACS performance, additional

experiments were conducted using increased labeling

concentrations (higher magnetophoretic mobility) and a

more complex FCM analysis protocol. Specifically, mono-

nuclear cells, this time spiked with KG1a cells, were labeled

with 100 mL anti-CD3-PE, 30 mL FcR blocker, 10 mL anti-

CD45 FITC, 10 mL of antiCD34 APC (total volume of

150 mL), and 150mLof anti-PEMACS (total labeling volume

of 150 mL).Figure 6 presents a representative FCM analysis imple-

mented to asses T-cell depletion as described in the

experimental methods section. Figure 6A through Figure 6C

present the initial dot plots used to discriminate between cells

anddebris (6A), anddead andviable cells (6B, 6C). Figure 6D

and E present the percentages of viable T cells and KG1a

cells prior to sorting; Figure 6F and G present the recovered

non-magnetic fraction indicating a significant depletion of T

cells. In contrast, Figure 6H and I presents the recovered

magnetic fraction, indicating a significant amount of T cells

and CD45þ cells (6H), and a low percentage of KG1a cells

(6I), respectively.

A total of 14 runs from four different buffy coats were

conducted using the modified labeling protocol and the

improved FCM analysis technique. Several significant results

merit comment. First, the average log10 depletion of CD3þ

cells was 2.88, significantly higher than the depletions using

the company protocol (1.02 log10). Second, average recovery

of the KG1a cells in the depleted fraction was 60%, which

while respectable is not ideal. Third, the overall recovery of

cells from the depleted and enriched fraction was 80%,

indicating that some of the cells remain in the MS column.

CD3þ Cell Depletion Using Super MACS System(LD Columns)

Similar to the two previous studies with the MiniMACS

system, a total of ten runs, from two buffy coats were

Figure 5. A: Percentage of CD3þ cells in the depleted and enriched

fraction after sorting using MiniMACS1MS columns maintaining constant

primary antibody (20 mL/200 mL �106 cells) concentration and different

amounts of secondary antibody (anti-PEMACS).B: Recovery of CD3þ cells

in the depleted, enriched fractions, and sum of both fractions as a function of

the secondary antibody.



conducted to depleted CD3þ cells in the SuperMACS system

following the company protocol. On average, a 2.89 average

log10 depletion and 63% recovery ofKG1a cellswas obtained

in the depleted fraction, similar to the results obtained with

the optimized MiniMACS.

CD3þ Cell Depletion Using QMS System

As presented previously, the QMS system can be operated in

two modes of operation: continuous flow through and

deposition. Figure 7 and Table I presents the results of

initial studies using the QMS in the complete flow through

mode of operation and with human blood positively labeled

for CD3. The mean magnetophoretic mobility was

1.1� 10�4 mm3/T�A�s.Several salient points are worthy of comment. First, a

measure of agreement between model predictions and

actually system performance can be observed in Figure 7A

and B. This reasonable agreement is consistent with previous

studies comparing the model to actual QMS performance

(McCloskey et al., 2003a). Unfortunately, while a somewhat

higher level of depletion of T cells was observed relative to

model predictions at higher flow rates, at lower flow rates,

where high levels of depletion should be theoretically

observed, the experimental results did not meet predictions.

Such non-ideal performance, especially at extremes of the

operating range of the system can be the result of a number of

causes, including geometric imperfections of the separation

channel as well as inaccuracies in the operation of the pumps

(Williams et al., 2003).

Simulations of the QMS performance with T cells having

highermagnetophoretic mobility is presented in Figure 7B in

terms of T-cell depletion. As can be observed, while a high

level of depletion of T cells can be theoretically obtained at

higher flow rates through the system, these flow rates are still

not high enough to process a sufficient number of cells for

clinical applications. Finally, in this mode of operation, a

reasonable level of recovery of the CD3� cells was obtained

(Table I).

These less than ideal results, as well as previously

published results of the performance of the QMS system in

a continuous mode of operation led to the studies of the

QMS operation in the deposition mode of operation for

T-cell depletion. Specifically, once the system was primed

Figure 6. Example of a flow cytometry (FCM) analysis using a four color, six-parameter analysis methodology: (A) FS vs. SS; (B) FS vs. 7-ADD, (C) SS vs.7-ADD.To be counted as a viable T-cell orKG1a cell in the feed (D andE), the non-magnetic fraction (F orG) or themagnetic fraction (H or I), an eventmust be

in gate R1, it must be 7-ADD negative (belong to R2 gate) and be stained as CD45þ/CD3þ (T-cell) or CD45þ/CD34þ (KG1a cell), respectively.


DOI 10.1002/bit

and ready for operation, flow was only conducted through

theQa stream at a number of flow rates andmagnetophoretic

mobilities. The results of these studies are presented in

Table II. Since the theoretical relationships, and the

computer model, was based on the assumption of complete

flow through of both the cell suspension and the sheath fluid,

simulations were run with flow rate ratios of Qa0/QT of 0.99and 0.999, mobilities of 3.4 and 2.3� 10�4 mm3/T�A�s,and aQTof 1.5, 3, and 10 mL/min. ForQT equal to 1.5 and 3

mL/min simulation, the model predicted no labeled cells

exiting stream a. For the case of QT equal to 10 mL/min

the model predicted up to 15% of the magnetically labeled

cells exiting in stream a when the mobility was 2.3�10�4 mm3/T�A�s; 3.0% exiting when themobility was 3.4�10�4 mm3/T�A�s. It also predicted a transport lamina thick-

ness of zero.

Since initial, preliminary studies indicated improved

performance, a total of 16 runs, from five different buffy

coats were conducted to deplete CD3þ cells fromPBL spiked

with KG1a cells. Analyses of the runs were performed with

the more complex FCM protocol. The average log10depletion of CD3þ cells for the 16 runs was 3.98 with an

average recovery of the spiked CD34þ cells of 57.9%. The

cell TP in these studies ranged from 2.5� 104 to 1.65�105 cells/s. For the first set of runs using the same cell

suspension (Runs 1 through 4), only one analysis of the feed

was performed; however, for the remaining sets of runs, the

cell suspension was analyzed before and after each separa-

tion; hence the slight, but significant variation in the

distribution of the types of cells in the feed. In addition,

no significant trends in the performance are detected as a

function of sorting speed, suggesting that depletion of T cells

is robust, with respect to sorting speed, within a range

of magnetophoretic mobilities. Finally, a summary of all

separation methods and results is given in Table III including

means and their standard deviations.

Figure 6. (Continued )



DISCUSSION

Overall, the results presented are consistent with expecta-

tions. While there are extensive reports of the MACS1

Technology systems (MiniMACS through CliniMACS) used

for positive selection of cells, including clinical scale

selections of CD34þ cells for transplantation, a different

approachwas tested here, namely, the depletion of unwanted,

CD3þ lymphocytes (i.e., T cells). Using the positive selec-

tion mode of operation, (i.e., selection for the CD34

surface marker), the MACS systems (MiniMACS through Tab

leI.

Perform

ance

ofT-celldepletionfrom

PBLspiked

intheQMSsystem

under

complete

flow

throughoperation.

Feedstock

Outleta

Outletb

Combined,aandb

Run

Cell

throughput

(cells/s)

Cells

added

%

CD3þ

Totalflow

rate

(mL/m

)

Cells

recovered

(�105)

%

CD3þ

CD3þlog

depletion

Recoveryof

CD3�from

feed

(%)

Cells

recovered

(�105)

%

CD3þ

Totalcells

recovered

(%)

Fractionof

CD3þcells

from

feed

ina

Fractionof

CD3þcell

from

feed

inb

TotalCD3�

recovered

(%)

TotalCD3þ

recovered

(%)

18.00Eþ03

2�106

57.6

12

7.00

16.9

0.99

68.6

3.0

96.9

0.10

0.25

69.7

35.5

26.70Eþ03

2�106

57.6

10

7.80

13.8

1.03

79.3

3.0

96.3

54.0

0.09

0.25

80.6

34.4

35.30Eþ03

2�106

57.6

86.20

9.40

1.30

66.2

2.5

93.2

43.5

0.05

0.20

68.2

25.3

44.00Eþ03

2�106

57.6

66.20

3.10

1.78

70.8

2.0

83.8

41.0

0.02

0.15

74.7

16.2

52.70Eþ03

2�106

57.6

44.70

1.80

2.13

54.4

2.1

31.0

34.0

0.01

0.06

71.5

6.4

61.30Eþ03

2�106

57.6

24.10

23.0

2.09

47.2

1.7

5.1

29.0

0.01

0.01

66.3

1.6

71.30Eþ04

2�106

57.6

84.70

23

1.03

42.7

5.5

49.4

51.0

0.09

0.24

75.5

33.0

Av

5.81�1.36

10.0�8.22

1.48�0.51

61.3�1.34

2.83�1.28

65.1�36.8

4.32�9.26

0.05�0.04

0.16�0.1

72.3�4.92

21.7�13.9

Mm¼1.1�10�4mm

3/A�s�T;Qa0 /Q

T¼0.2,Qa/Q

T¼0.5,transportlaminar

thickness¼486mm

.

Figure 7. Theoretical simulations using the complete flow-through mode

(continuous line) compared to actual date (open symbols); (A) fraction

recovery as a function of total flow rate; (B) simulations of Log10 depletion as

a function of total flow rate for three different magnetophoretic mobility

means.


DOI 10.1002/bit

Tab

leII.

Perform

ance

ofT-celldepletionfrom

PBLspiked

withKG1acellsin

theQMSsystem

usingthedepositionmodeofoperation.

Feedstock

concentration

Finalresults

Run

Cell

throughput

(cells/s)

Cells

added

Flowrate

(mL/m

in)

CD3þCD45þ

(%)

CD34þ

CD45þ

(%)

Magnetophoretic

mobility

mm

3/(T�A�s)

Cell

recovered

a

CD3þ

CD45þ(%

)

CD34þ

CD45þ(%

)

CD3�

(%)

Recoveryof

CD34þ45þ

(%)b

CD3þlog

depletionc

17.5�104

6�106

1.5

61

14

7.8�10�4

0.68�106

0.1

47.2

45.60

38.5

3.73

27.5�104

6�106

1.5

61

14

7.8�10�4

0.99�106

0.06

43.5

66.17

51.4

3.79

37.5�104

6�106

1.5

61

14

7.8�10�4

1.19�106

0.09

47.7

79.66

68.0

3.53

47.5�104

6�106

1.5

61

14

7.8�10�4

1.15�106

0.075

43.6

76.58

59.6

3.63

54.05�104

16.33�106

1.5

21.56

0.40

3.02�10�4

5.34�106

0.004

0.72

39.27

58.88

4.21

64.05�104

16.75�106

1.5

24.60

1.64

3.02�10�4

7.03�106

0.002

3.08

39.17

78.84

4.45

74.05�104

17.28�106

1.5

23.59

5.35

3.02�10�4

6.54�106

0.008

5.21

43.46

36.87

3.86

83.09�104

12.57�106

1.5

41.30

1.97

3.39�10�4

1.96�106

0.026

5.44

28.52

42.99

4.00

92.5�104

10.05�106

1.5

53.11

1.14

3.39�10�4

2.00�106

0.012

5.07

34.67

88.47

4.44

10

3.13�104

12.62�106

1.5

31.50

1.68

2.07�10�4

3.84�106

0.024

1.82

45.96

32.96

3.63

11

2.5�104

13.77�106

1.5

28.81

1.00

2.28�10�4

4.73�106

0.010

1.66

39.16

57.02

3.92

12

5.00�104

13.77�106

1.5

21.85

0.98

2.28�10�4

4.28�106

0.002

1.81

40.23

57.39

4.54

13

7.50�104

13.77�106

1.5

23.49

1.24

2.28�10�4

4.28�106

0.004

1.80

37.74

45.11

4.27

14

10.00�104

13.77�106

3.0

24.40

0.84

2.28�10�4

5.39�106

0.008

1.67

47.86

77.88

3.89

15

12.50�104

13.77�106

2.5

23.48

1.34

2.28�10�4

4.64�106

0.004

2.24

42.29

56.29

4.24

16

16.50�104

13.77�106

3.0

21.69

1.08

2.28�10�4

6.04�106

0.012

1.89

54.65

76.81

3.61

Average

—0.03�0.03

—47.6�14.6

57.9�16.6

3.98�0.33

aCellnumber

was

determined

byCoulter

Counter.

bRecoveryofCD3þCD45þ¼N

umber

oftotalcellsrecovered

�PercentageofCD34þCD45þcellsin

theproduct

Number

oftotalcellsadded

�PercentageofCD34�CD45þcellsin

feedstock

�100%.

cLogdepletion¼

log(Initialnumber

ofCD3þcells/Finalnumber

ofCD3þcells)CD45þcellsin

theproduct.



CliniMACS) is capable of obtaining high purity with cell

recovery values within a wide range; however, as also

reported previously, to obtain consistent, high level depletion

of T cells for transplantation at a clinical scale in 10%–20%

of the cases a second separation targeting T cells was needed.

In the depletion mode of operation, after optimization and

a more accurate FCM analysis methodology was employed,

both the MiniMACS and SuperMACS were able to produce

similar results, namely, on average, a 2.88 to 2.89 log10depletion with a 60.8%–63.1% recovery of spiked CD34þ

cells. In contrast, the QMS system achieved, on average, a

3.98 log10 depletion with an average 57.9% recovery of

spiked CD34þ cells.

Statistical analysis of the data to compare all means using

the Tukey–Kramer HSD test (�¼ 0.05, JMP v. 5.1, SAS

Institute) showed that there is no significant difference

between MiniMACS, CliniMACS, and QMS for KG1a

mean cell recovery, as shown in Table III. In contrast, when

the mean of the log10 depletion levels were compared using

the same Tukey–Kramer test, depletion levels attained by

QMS are significantly different from those obtaining using

the MiniMACS and CliniMACS. A summary of this

analysis is presented in Figure 8A and 8B demonstrating

the superiority of QMS in terms of depletion. While this

level of analysis may appear excessive, it is important to

note that in the clinical application of T-cell depletion, not

only does the mean of the performance of a system matter,

but also the distribution. If the distribution is wide, in some

cases the number of T cells remaining may be too high and

the patient will develop GvHD.

In an attempt tomore accuratelymeasure the pre- and post-

concentration of T cells, a more complex FCMmethodology

was used in this study. Also, it should be noted that 50,000 or

100,000 events were obtained to facilitate a more accurate

analysis of the rare events. While a significant improvement,

some level of ambiguity still exists in the selection of the

events to be further evaluated when FS is used to set the gate.

Staining of cell nuclei using DRAQ5 may permit more

accurate analysis. Use of DRAQ-5 would potentially allow

the complete removal of the manual selection of the cell

population of interest on the FS versus SS plot. Use of

DRAQ-5 and 7-AAD with automatic selection (after the

input of appropriate single color controls) will yield viable

events (7-AAD negative) containing nuclei (DRAQ5 posi-

tive). We believe that such a selection process of events,

combined with the need for the event to be CD45 and CD3

positive will increase the accuracy of the FCM analysis.

While the results are promising with respect to a high level

of depletion of T cells with the QMS system, a sample

between 600 and 1,000 times larger needs to be sorted for a

clinical application. At the rate of maximum rate of

1.65� 105 cell/s (Run 16 in Table II) that would take

approximately 100 min.

It is also interesting to note that all three-separation

systems presented a very similar recovery of the KG1a cells.

Since the goal of depletion separation is usually twofold: the

removal of unwanted cells and the enrichment of desired

cells, as is the case in T-cell depletion for HSC transplanta-

tions, this loss of theCD34þ cells needs to be addressed. In an

actual clinical application, such a loss results in the need for

the use of a larger initial sample, resulting in further

complexities and expenses.

Table III. Comparison of T-cell depletion using the three different methods.

MiniMACS, n¼ 4,

(company protocol)

MiniMACS, n¼ 14,

(modified protocol)

SuperMACS, n¼ 10

(company protocol) QMS, n¼ 16

Rate of cell separation (cells/s) — — — 2.5� 104–1.65� 105

Recovery of spiked KCG1a cells — 60.75� 5.94% 63.1� 8.5% 57.9� 16.6%

Percentage of cells in depletion fraction

that are CD3þ4.54� 1.30 0.06� 0.04 0.06� 0.05 0.03� 0.03

Log depletion 1.02� 0.15 2.88� 0.17 2.89� 0.22 3.98� 0.33

Figure 8. A: Tukey–Kramer HSD Test analysis of recovery of KG1a data

after sorting using MiniMACS, SuperMACS, and QMS. The confidence

interval is set at a¼ 0.05. B: Tukey–Kramer HSD Test analysis of log10depletion of T cells data after sorting using MiniMACS, SuperMACS, and

QMS. The confidence interval is set at �¼ 0.05.


DOI 10.1002/bit

At this point is not clear why these three systems have this

similar performance with respect to the recovery of KG1a

cells. It is possible that there is a low level of non-specific

binding of the magnetic reagents to the cells which results in

the removal of some of the KG1a cells. However, it is

probably more likely that a previously reported ‘‘drafting’’

phenomenon is causing this loss. We have reported that

unlabeled cells can be induced to move in the direction of the

magnetically labeled cells and that this induced movement is

a function of cell concentration (McCloskey et al., 2001;

Zhang et al., 2005). We are continuing to address this

observation. Finally, as stated previously, 2-day cultureswere

conducted to remove macrophages and/or monocytes. Such

procedures are not practical for typical separation processes

and were conducted here purely to remove potential non-

specific uptake of the magnetic labels, which potentially

masks the analysis and interpretation of the separation

performance.

To evaluate the performance of theQMS to separate T cells

on a clinical scale (on the order of 109 total cells), a number of

issues need to be addressed. First, the labeling of such a large

number of cells and insuring that the labeling concentration is

high enough to achieve the desiredmagnetophoreticmobility

is non-trivial. Other on-going research has experimentally

measured the binding constants of antibody-nanoparticle

conjugates to target antigens. With this data, theoretical

models have been developed which will guide these labeling

studies to guarantee sufficient binding in the most econom-

ical manner. Second, it needs to be demonstrated that

apheresis product from human donors can be labeled with a

minimum of processing steps. Third, an improvement in the

recovery of non-labeled, and in this case KG1a cells, needs to

be demonstrated. Fourth, functional assays are being

developed to corroborate the FCM analysis with respect to

the degree of T-cell depletion. Finally, it needs to be

demonstrated that the QMS can perform at this level of

depletion and higher recovery of the non-labeled cells when

processing a thousand fold as many cells.

This work has been supported by the National Science Foundation

(BES-9731059 andBES-0124897 to J.J.C.;NSFSBIR02-056awarded

to SHOT INC.) and the National Cancer Institute (R01 CA62349 to

M.Z., R01 CA97391-01A1 to J.J.C., 5P30 CA16058) and 1 R01

AI056318-01A1 to S.F.

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DOI 10.1002/bit

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Comparison of two immunomagnetic separation technologies to deplete T cells from human blood samples