Q 1996 Wiley-Liss, Inc. Cytometry 24:251-259 (1996)

Immunomagnetic Isolation of Magnetoferritin-Labeled -

Cells in a Modified Ferrograph Madej Zborowski, Chwan Bor Fuh, Ralph Green, Nicholas J. Baldwin, Sridhar Reddy,

Trevor Douglas, Stephen M a n n , and Jeffrey J. Chalmers The Cleveland Clinic Foundation, Cleveland, Ohio (M.Z., C.B.F., RG., N.J.B.); Department of Chemical Engineering, Ohio

State University, Columbus, Ohio (S.R., J.J.C.); Department of Biological and Physical Sciences, Montana State University-Billings, Billings, Montana (T.D.); School of Chemistry, University of Bath, Bath, United Kingdom (S.M.)

Received for publication October 9, 1995; accepted February 19, 1 9 9 6

Pan T, helper, and cytotoxic lymphocytes were isolated from the human peripheral blood mono- nuclear cell fraction by antibody staining, ferritin labeling, and deposition on glass slides. Two dis- tinct forms of ferritin were used: one was native horse spleen ferritin, and the other was magneto- ferritin. Magnetoferritin was obtained by reconsti- tuting the horse spleen ferritin iron core with su- perparamagnetic magnetite instead of the usual paramagnetic fedhydrite crystal. The cell deposi- tion on microscopic glass slides in the magnetic field was obtained by an instrument that was adapted from an industrial magnetic deposition an- alyzer, the ferrograph. The identity of cells in the magnetic deposits was con5med by comparing the cell fractions in the feed and in the eluate with the use of flow cytometry. The hmunostaining

protocol amplitled the number of ferritin mole- cules per cell surface antigen 20-70 times. Magnet- oferritin, but not native ferritin, imparted a s&- cient magnetic moment to cells to deplete the labeled cell population between 67 and 88% of its initial concentration in a magnetic field of 1.67 Tesla (T), a field gradient of 2.57 T/mm, and a flow rate of 0.01 dmin . This study showed that the mag- netic moment of magnetoferritin was sumcient for immunomagnetic isolation of lymphocytes from mononuclear cell preparations in the modlfied ferrograph. o 1996 wuey-~iss, h c .

Keyterms: Pan T cell, CD3, helper T cell, CD4, cytotoxic T cell, CDS, immunomagnetic separa- tion, lymphocyte immunophenotyping, ferritin, magnetoferritin

Magnetic separation has found many preparative scale uses in biology and in medicine; however, it has never been developed into an accepted analytical method. Part of the reason is that the magnetic separators developed for biomedical applications operate on the principle of negative selection, that is, the removal of the magneti- cally labeled cell fraction from the cell sample ( 16,25,29). Positive selection of the magnetically labeled hc t ion in such separators, which is important for analy- sis, requires additional manipulations (recovery of the magnetic fraction in the absence of the magnetic field) that are extrinsic to the magnetic separation process itself and during which part or all of the information about the magnetic separation process is lost. Another reason has been the lack of a molecular magnetic label that would not interfere with the physical and morphological prop- erties of the cell.

In this study, we evaluated a novel magnetic separation method by using a modified industrial instrument, the ferrograph (28,34), and a novel molecular magnetic la- bel, magnetoferritin (20). The modified ferrograph pro- vided a means for positive cell selection on microscopic

glass slides and for multiple sample processing, which is important for accomplishing the immunomagnetic sepa- ration process in a realistic separation time f3ame. We dubbed the magnetic deposition process "magnetapher- esis" (34). The ferrograph has been used in the past for analysis of wear particles in arthroplastic joints and of bacteria and cells using molecular magnetic labels [lan- thanides (6,7,11,18,32) and ferritin (34)]. Whereas a number of particulate as well as colloidal magnetic labels are commercially available (8,12,16,21,29), we selected ferritin, the native iron-storage protein, as a candidate mo-

Supported by NIH grant R01 62349 and the Whitaker Foundation. Pan of this material was presented as a poster at the 23rd Annual

Meeting of the International Society for Experimental Hematology, Min- neapolis, Minnesota, August 21-25, 1994 (Abstract no. 67).

Address reprint requests to Maciej Zborowski, PhD, Department of Biomedical EngineeringNVb-3, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-5254. E-mail: zborow@ bme.ri.ccf.org.

252 ZBOROWSKI ET AL.

lecular magnetic label for immunomagnetic separation ( 32). The advantages of ferritin include its small size (8 nm diameter inner cavity containing iron core and 12 nm outer diameter protein shell), an ability to form stable physical solutions, fast reaction kinetics compared with particulate magnetic labels, a crystalline iron core that is amenable to chemical manipulation, and a well-defined protein cage that can be chemically modified (13,19,22). Unlike highly paramagnetic lanthanide ions (Er3+ and Gd’ + ), ferritin is less aggressive and, thus, is more bio- compatible in reactions with cells. However, an important limitation in the use of native ferritins as magnetic labels is the low magnetic moment of the ferrihydrite mineral core (6.94 X Am’), even at a high magnetic field [E = 1 Telsa (T)] (23,34). Considerably higher magnetic moment of 1.20 X Am’, in a field of 1 T and with a maximum gradient of 2.57 T/mm, was required for cell capture in previous experiments with a cationized native ferritin label (34). The corresponding number of native ferritin molecules per cell required to achieve this effec- tive cell magnetic moment was 1.7 X lo’, which is equiv- alent to approximately from 1.7 X 10’ to 6 X lo2 native ferritin molecules per lymphocyte surface marker (34).

In SI units, magnetic dipole moment is expressed as p = x V H (Am2), where x represents volumetric magnetic susceptibility (dimensionless), V is the volume (m’), and His the magnetic field strength (Nm). In the diamagnetic medium, such as water, W = B/po, where B represents magnetic field, and p,, = 4n . lo-’ T m/A is the perme- ability of vacuum. The term “effective” was applied in relation to the medium (water) averaged over the cell volume. The magnetostatic potential energy of the in- duced magnetic dipole in the scalar potential field is U = -f p.B (Am2 T), and the magnetic force acting on such a dipole is I; = -VU = 4 ( X / F ~ ) V VB2 (N) (2).

The reported numeric values of the magnetic suscep- tibilities of ferritin ( xfer) differ, presumably due to the differences in iron content (3,22). We chose the follow- ing numeric values for calculating the native ferritin mag- netic dipole moment effective xfer = 96.9 . lo-” (SI), corresponding to 20% (w/w) Fe content; V,, = 900 X lop9 pm3; and, for the cationized, native ferritin-labeled lymphocytes: effective xcell = 2.92 X lop6, Vcel, = 520 wm3, and E = 1 T, unless otherwise indicated.

Here, we describe studies in which we increased the efficiency and specificity of magnetic separation of hu- man lymphocytes 1) by using an immunostaining proto- col that amplified the ferritin load per labeled lympho- cyte and 2) by increasing the magnetic moment per protein molecule through chemical synthesis of a ferro- magnetic mineral core within the protein cavity. We show that the use of “magnetoferritin” (4,9,20) signifi- cantly reduces the ferritin load per lymphocyte necessary to achieve immune-specific magnetic deposition. Our re- sults demonstrate that magnetoferritin is a viable candi- date as a molecular magnetic label and that the modified ferrograph is a promising analytical instrument for inves- tigation of cell separation using weakly magnetic molec- ular labels. Additional studies are required to fully char-

acterize the analytical capabilities of such a magnetic separation system and to adapt it to practical use.

MATERIALS AND METHODS Magnetoferritin

Synthesis of magnetite (Fe,O,) within demetallated horse spleen ferritin molecules was carried out as previ- ously described (20). Iron was removed kom native horse spleen ferritin by dialysis against thioglycolic acid, under nitrogen atmosphere, at pH 4.5 followed by incre- mental addition of Fe2+ solution and slow oxidation at pH 8.5, which resulted in reconstitution of the mineral core. Transmission electron microscopy showed the pres- ence of discrete 5 nm cores of crystalline magnetite en- capsulated within the polypeptide shell. Samples, on av- erage, contained approximately 2,000 Fe atoms per protein molecule and were exposed to air after synthesis. The samples showed some aggregation on storage and were used in the magnetic separation experiments within 6 months after synthesis. Native unmodified horse spleen ferritin was used in control experiments (Sigma Chemical Co., St. Louis, MO).

The increase in magnetic susceptibility of the magnet- oferritin compared with the native ferritin was measured by using nuclear magnetic resonance (NMR) relaxome- try. The relaxation times TI and T2 were measured by using spin-lattice and spin-spin proton relaxation mea- surements (Hahn spin-echo), respectively, in the GE CSI 4.7 T Horizontal Bore Imager (GE, Fremont, CA). The sample relaxivities R1 and R2 were calculated as slopes of the 1/T1 and 1/T2 dependence on ferritin concentration, respectively, and the sample susceptibility was propor- tional to the ratio R2/R1 (10,15,24). The corresponding relaxivity ratios R2/R1 for ferritin and magnetoferritin were 6.19 5 0.91 (n = 8) and 51.5 & 2.4, (n = 10). The magnetoferritin-to-ferritin susceptibility ratio was calcu- lated as the ratio of respective RURl values, and it was equal to 8.3 2 1.3 (n = 18; P = 0.05). This finding is in agreement with results published earlier (9).

Immunofluoromagnetic Staining

A secondary antibody staining method was used to couple selectively magnetoferritin or native ferritin to human lymphocytes. Ferritin surface was modified by bi- otin conjugation in preparation for avidin-biotin binding to the antibody complex. Biotin N-hydroxysuccinimide ester (Vector Labs, Burlingame, CA) was dissolved in deionized water prior to use at a concentration of 30 mg/ml. Ferritin was dissolved in sodium bicarbonate buffer, pH 8.5, at a concentration of 1 mg/ml. An aliquot of the biotinylating reagent equal to one-tenth of the weight of the ferritin’s protein was added to 2 ml of the ferritin solution and incubated at room temperature with occasional stirring for 2 hours. Unbound biotin com- pound was removed from the solution by dialysis against 2 liters of buffer overnight. The biotinylated ferritin so- lutions were stored at 4°C following the addition of 0.02% sodium azide.

Antihuman mouse immunoglobulin (1gG)l CD3, CD4,

LYMPHOCYTE ISOLATION USING MAGNETOFERRITIN 253

Table 1 Summary of Magnetic Lymphocyte Isolation Experiments Using Modified Ferrograpb and Magnetofm.tin"

Channel no.

Labelingb seauence

1 2

Isotype + pAbb + Av + Ferb + A\CITC CD3 mAb + pAbb + Av + Ferb + A\CJTC CD4 mAb + pAbb + Av + Ferb + A?ITC CD8mAb + pAbb + Av + Fer' + A?- pAbb + Av + Fer' + A g m C Av + Ferb + A3ITC

3 4 5 Ferb +

No. experiments

6 4 1 1 4 4 4

Percent positive fraction in feed f F Y 18.3 & 8.4

74 & 6 65 27

16 f 2.3 13 & 2.7 14 f 3.5

Percent positive fraction in eluate (E) 7.8 f 4.7 32 f 12

12 3.2

7.6 ? 5.4 4.1 ? 4.1 4.0 2 4.1

Depletion of positive fraction =

57 f 28 67 f 16

81 88

52 ? 35 71 f 27 74 * 23

I(E - F)/F X

"The total cell concentration in feed was 1 X 106/ml in all five channels. The monocyte fraction in feed was (5.3 & 2.8)% (n = 5). Native ferritin showed no selective depletion in ten experiments (not shown). bAbbreviations used in the description of the sandwich label components are provided in the legend to Figure 2. Superscripts indicate conjugate compounds. 'Percent positive fraction was defined as the Eraction of high-intensity fluorescence cells in the sample relative to that in the isotype control. dRecovery of negative fraction = 100% - depletion of positive fraction in control samples (channels 1, 3, 4, and 5).

and CD8 monoclonal antibodies (mAbs) were used as the primary targeting agents (Coulter Clone Monoclonal An- tibody; Coulter Immunology, Hialeah, FL). Biotinylated rabbit antimouse lgGs were used as the secondary label- ing antibodies (Ab-biotin) (DAKO Corp., Carpinteria, CA). An avidin bridge was used to couple biotinylated ferritin or magnetoferritin to the biotinylated secondary antibody. This method was versatile and allowed ampli- fication of the number of ferritin molecules per cell sur- face marker.

Cells Peripheral blood was obtained from healthy normal

human volunteers who signed the informed consent form approved by the Institutional Review Board. The mono- nuclear cell fraction was obtained by centrifugation on a density cushion [Ficoll 400 (5.7% w/v) in diatrizoate so- dium (9% w / ~ > ; Pharmacia, Uppsala]. Monocytes were depleted from the cell sample by adhesion to the poly- carbonate surface of the 75 cm2 tissue culture flask by a 15 min incubation at 3PC. Cell sample was tested for the presence of monocytes by staining a sample aliquot with antihuman CD 14 mAb-fluorescein isothiocyanate (FITC) conjugate and by flow cytometric analysis. The cell con- centration (mostly lymphocytes) was adjusted to lo6 per ml solution, and the percent cell viability was deter- mined by using a hemacytometer and the Trypan blue dye exclusion test. All cell samples were fixed by a 10 min incubation in 1 % paraformaldehyde following sec- ondary antibody staining. Cell staining reactions were performed at 4°C. Incubation times were 15 min for both types of antibodies and 5 min for avidin and ferritin-bi- otin. Cells were washed twice in Hank's Balanced Salt Solution buffer (HBSS; GIBCO BRL, Grand Island, NY) following each staining step.

The cell sample was divided into five aliquots: one mAb test and four negative controls. The cells were stained sequentially in the following order: mAb, Ab-bi- otin, avidin, and ferritin-biotin. The mAb test aliquot was stained by using the complete staining sequence, whereas the negative control aliquots were stained by using in-

complete sequences. The staining sequences are shown in Table 1. All five cell aliquots were stained with avidin- FITC for analysis of the lymphocyte population with flu- orescent-activated cell sorter analysis (FACS). Avidin- FITC reacted only with those cells that had the biotinylated ferritin on their surface; therefore, they pro- vided a convenient means with which to identify the fer- ritin-labeled cells in the sample. Moreover, the amount of fluorescence label per cell and, thus, the cell brightness depended on the number of biotin-ferritin molecules on the cell surface. Therefore, the cell frequency distribu- tion as a function of the fluorescence intensity provided a convenient measure of the ferritin-binding distribution among the cells in the sample. The possible errors of this assumption were the likely nonspecific binding of avidin- FITC to nonstained cells and to cells that had the non- specifically attached Ab-biotin on their surface. Cell sam- ples stained with the ferritin-FITC sandwich were analyzed before (fraction F for feed) and after (fraction E for eluate) the magnetic deposition process by using a FACScan Analyzer (Becton Dickinson, San Jose, CA). Nonspecific staining was evaluated by comparing cell samples stained with the ferritin-FITC sandwich with two additional positive controls that were stained with FITC conjugated directly to either the isotype or the respec- tive primary mAb (samples X and Y, respectively).

Magnetapheresis Magnetic deposition patterns of the immunolabeled

lymphocytes by magnetoferritin were obtained by using the apparatus shown in Figure 1. The magnetic field was generated by an interpolar gap of a permanent magnet consisting of two pairs of Nd-Fe-B magnets (Dexter Mag- netics Corp., Toledo, OH) of a maximum energy product of 2.23 X lo5 T A/m arranged to provide the maximum magnetic flux density at the interpolar gap. The width- to-depth ratio of the interpolar gap, 1.4t6.0 mm, was sufficiently small to assure that the magnetic flux lines outside the magnet corresponded to those of the fringing field of a slot of infinite depth, a case that has been well

254 ZBOROWSKI ET AL.

characterized in magnetostatics (31). The length of the interpolar gap was sufficiently large to accommodate a series of five parallel-piped flow channels carrying the cell suspensions. The field measurements were per- formed by using a Gaussmeter and a Hall-effect probe (model 9200 Gaussmeter and transverse probe ATG920404; F.W. Bell, Orlando, FL) and have been de- scribed in detail elsewhere (34). The saturation magnetic field inside the interpolar gap was equal to Bo = 2.433 2 0.027 T, and the magnetic field and gradient acting on a cell placed directly over the interpolar gap, at the distal wall of the flow chamber, were B = 1.667 * 0.057 T and VB = 2.565 k 0.088 T/mm. Ten Teflon tubings with an inner diameter of 0.79 mm provided suitable means of cell sample delivery in and out of the five flow channels. The inlet tubings carrying the feed fractions were con- nected to 1 ml BD sterile disposable syringes by 19 G needles and three-way stopcocks. The outlet tubings car- rying the eluate fractions emptied to sample collection tubes. All five syringes were mounted on a single syringe pump, which provided a uniform and well-controlled flow across the magnetic barrier (Fig. 1 ). The magnet and flow chamber assembly were positioned with the flow direction parallel to gravity to avoid interference from gravitational sedimentation. The sample volume per channel was 500 pl, and the sample flow rate was 0.01 mumin. Within the magnetic barrier, the sample flow channels were formed by rectangular, rounded-edge cut- outs in a silicone rubber spacer sandwiched between a 150-km-thick glass microscope coverslip and an 2.4-mm- thick acrylic cover plate. The dimensions of the flow chamber were 0.5 X 6 X 16 mm. A microscope coverslip, which was positioned as the proximal wall of the flow channel relative to the magnet, acted as the deposition surface for cells separated in the magnetic field. Prior to cell pumping, the channels were primed with 3.7% bo- vine albumin (Ortho Diagnostic Systems Inc., Raritan, NJ) in HBSS solution to ensure that the cells entered the mag- netic deposition zone in a fully developed flow. Incuba- tion with albumin was also used to prevent nonspecific adhesion by cells, particularly monocytes, to surfaces. Af- ter each experiment, the coverslips and associated cell deposits were disassembled, stained for nucleated cells, mounted on a substrate slide, and examined by optical microscopy. The cell deposition bands forming at the magnetic barrier (following slide staining for the nucle- ated cells and mounting) were scanned directly from the slides by using the LS3510AF 35 mm Film Scanner (Ni- kon Corp., Japan). The images were digitized by using a Photoshop software package (Adobe Systems, Inc.) on a Macintosh Quadra 540 (Apple Computer, Cupertino, CA) and were reproduced on paper by using the 600 dpi La- serJet 4Si MX printer (Hewlett Packard, Boise, ID).

RESULTS Examples of kequency histograms of lymphocytes la-

beled by FITC-conjugated mAb against CD3, CD4, and CD8 cells are shown in Figures 2A, 3A, and 4A, respec- tively. These histograms served as positive controls for

FIG. 1. The modified ferrograph. Fringing field, B, of an air gap be- tween two pole pieces of the permanent magnet assembly creates a magnetic barrier to magnetically labeled cells traversing the gap in the flowing medium. The trapped cells deposited on the proximal wall of the channel are amenable to cytology. The magnified area of the inter. polar gap shows lines of constant magnetic potential energy of induced magnetic dipoles (B2 = const.; dashed lines) and the orthogonal lines of the magnetic forces acting on such dipoles (P Q VE’; solid lines), which also represent magnetic forces acting on the magnetically labeled cells (34 ) . The force lines describe quasistatic trajectories of magnetically labeled cells in a resting viscous medium. Note the concentration of trajectories at both edges of the interpolar gap. resulting in a double-bar appearance of the magnetic deposit. The movement of the carrier me- dium (not shown) displaces the cell trajectories located between the channel walls towards the channel exit, with the net effect of more cells depositing upstream than downstream, and the weakly magnetic or dis- tant magnetic cells escaping capture altogether. The channels were num- bered in correspondence to the tube numbers ( 1 through 5).

the ferritidmagnetoferritin and FITC-labeled lympho- cytes. They show a typical pattern of CD marker fre- quency distribution, with only low-intensity fluores- cence cells present in the isotype control (sample X) and with an additional high-intensity fluorescence cell frac- tion in the positive control (sample Y).

Figures 2B, 3B, and 4B summarize the magnetic depo- sition results for lymphocytes stained by anti-CD3, anti- CD4, and anti-CD8 mAbs, respectively, and labeled with the magnetoferritin-FITC sandwich. The frequency histo- grams of cells stained with the control mAb-FITC conju- gate (Figs. 2A, 3A, 4A, sample Y), as expected, are similar to those stained with the ferritin-FITC sandwich (Figs. 2B, 3B, 4B, tube 2-F). These results indicate that the ferritin was predominantly bound to the target cells stained with the mAb against respective T cell subpopulations. They also show that the distribution of cell ferritin binding in the samples stained by the ferritin-FITC sandwich was very similar to the distribution of cell FITC binding in the samples stained by mAb-FITC conjugates, suggesting that the ferritin bound to the target cells was in direct pro- portion to the CD marker surface density.

The changes in the lymphocyte population composi- tion after passing the magnetic barrier are reflected in the differences between the FACS frequency histograms in feed (fractions F) and eluate (fractions E; Figs. 2B, 3B,

LYMPHOCYTE ISOLATION USING MAGNETOFERRITIN 255

A.

CONTROLS FACS '~ llJllJ too lo1 102 lo3 lo4 loo lo1 lo2 lo3 lo4

s 0

Fluorescence

Labeling notation X: lsotypeFITC F: Feed

Y: CD3F'TC E: Eluate

1 : lsotype + pAbb + Av + Fer" + AvFITC 2: CD3 mAb + pAbb + Av + Ferb + AvFITC 3: pAbb + Av + Ferb + AvFITC 4: Av + Fer" + AvFITC 5: Ferb + AvFITc

B. FEED

1

1 -F 2-F 3-F 4-F 5-F

MAGNETIC BARRIER

1 channel 1 channel 2 channel 3 channel 4 channel 5

Fic.. 2. Magnetic deposition of CD3 lymphocytes labeled with mag- netoferritin at the magnetic barrier. A: Fluorescent- activated cell sorter analysis (FACS)-positive controls stained by direct fluorescein isothio- cyanate (FITC) conjugates [ isotype in X, anti-CD3 monoclonal antibody (mAb) in Y], to indirectly stained samples by ferritin/FITC sandwich shown in B below (samples 1-F and 2-F, respectively). The y axis in all FACS histograms shows cell count, and the x axis shows fluorescence intensity in logarithmic scale. B: Cell deposition at the magnetic barrier with the corresponding FACS results. Captured lymphocytes form a dis- tinctive deposition band at the magnetic barrier. FACS histograms dem- onstrate depletion of the captured lymphocyte fractions from the eluate,

4B). The magnetically separated fraction could be ob- served visibly on the deposition slide at the magnetic barrier as a double-bar pattern, indicating a large number of cells deposited on the slide (Figs. 2B, 3B, 4B). This was confirmed by the depletion (Fig. 2B) or the complete disappearance (Figs. 3B, 4B) of the high-fluorescence cells in the eluate (E) compared with the feed (F).

The most intense magnetic deposition was observed for samples stained with mAb (Figs. 2B, 3B, 4B, channel 2). In contrast, only light traces of magnetic deposition, which were due to nonspecific staining, were observed for the negative control samples. Typically, the highest contrast in the deposition densities between the mAb- stained fraction and the control fractions was seen for samples showing the highest depletion (Table 1). No such contrast and no capture measurable by FACS was observed for samples labeled with the native ferritin (data not shown).

E, compared with feed, F (numbers on feed and eluate samples corre- spond to the channel numbers). The histograms are numbered in accor- dance with the tube numbering shown in Figure 1. Note that the mag- netic deposition is the most intense in the mAb-stained cell sample 2-F. The double-bar appearance of the deposition is explained in the caption to Figure 1. The flow was from top to bottom. A similar pattern was seen in experiments with the anti-CD4 and anti-CD8 mAb-stained lympho- cytes shown in Figures 3 and 4. mAb, antihuman mouse monoclonal antibody; pAb, rat antimouse polyclonal antibodies; Av, avidin; Fer. mag netoferritin; b, biotin (superscripts indicate conjugated compound).

The nonspecific deposition in the control samples (Figs. 2B, 3b, 4B, channels 1 and 3-5) can be attributed to the high-fluorescence cells in the feed histograms ( 1-F and 3-F through 5-F) which disappear in the eluate his- tograms (1-E and 3-E through 5-E, respectively). These high-fluorescence cells were not seen in the direct iso- type control (Figs. 2A, 3A, 4A, sample X), suggesting an increase in nonspecific staining when using the indirect sandwich staining with magnetoferritin. The shapes of the control histograms (tubes 1 and 3-5) are all similar, even though each tube lacks a different component of the labeling sandwich. Because all tubes had the ferritin-la- beling step in common, it appears that the ferritin was responsible for the nonspecific binding, possibly to cells other than those targeted by the primary mAb having ferritin receptors, such as T cells or monocytes. The pres- ence of monocytes in feed samples was detected by using anti-CD14 mAb FITC-conjugate staining (Table 1 ) and by

256

A. FACS CONTROLS

Fluorescence

ZBOROWSKI ET AL.

Labeling notation: X:

Y:

1: 2: 3: 4: 5:

IsotypeF'Tc F: Feed

CD4F'TC E: Eluate

lsotype + pAbb + Av + Ferb + AvFITC CD4 mAb + pAbb + Av + Ferb + AvFITC pAbb + Av + Ferb + AvFITC Av + Ferb + AvFITC Ferb + AvFITC

B. ~~ 1 -F ~~ 2-F ~~ 3-F 'i!l 4-F ~~ 5-F

FEED

D 0 n

loo lo1 lo2 lo3 lo4 loo lo1 lo2 lo3 lo4 loo lo1 lo2 lo3 lo4 loo lo1 lo2 lo3 lo4 loo lo1 lo2 lo3 lo4

MAGNETIC BARRIER

1 ELUATE

channel 1 channel 2 channel 3 channel 4 channel 5

2-E 3-E 4-E 5-E

*

FIG. 3. Magnetic deposition of CD4 lymphocytes labeled with magnetoferritin at the magnetic barrier. For details, see legend to Figure 2.

the microscopic inspection of the magnetic cell de- posit (Fig. 5). Table 1 summarizes numerical values of the magnetic depletion for each experiment. The anti- body-specific cell depletion was in the range between 67 (CD3 cells) and 88% (CD8 cells) of the feed frac- tion. The nonspecific magnetoferritin binding was 14- 18% of the feed population, and the recovery of the nonspecifically stained cells in the eluate was less than 50%.

The macroscopic appearance of the magnetic deposits for anti-CD3, anti-CD4, and anti-CD8 mAb-stained sam- ples was similar (Figs. 2B, 3B, 4B, channel 2); however, the relative intensities of the mAb-stained fractions in re- lation to controls varied between the experiments. This variability appears to be related to the least understood step in the immunostaining process, the biotinylated mag- netoferritin binding. In all experiments, the large high- fluorescence cell subpopulation observed in the anti- body-stained cell sample produced dense magnetic deposition, whereas the control samples produced far weaker depositions. The highest contrast between the test mAb and controls was obtained for CD4 T cells (Fig. 3B), which may be related to the high density of the CD4 surface marker ( 1 ).

DISCUSSION Superparamagnetic magnetoferritin, but not paramag-

netic ferritin, produced immunospecific magnetic depo- sition of lymphocyte subtypes. These results were con- sistent with the observed increase in magnetoferritin magnetization when compared with native ferritin. Based on the previously published results on lymphocyte mag- netization by cationized ferritin (see above) (34) and the observed magnetoferritin susceptibility increase of 8.3- fold over that of the native ferritin, we calculated that, on average, there were 20-70 magnetoferritin molecules per surface marker. The molecular size of the magnetic label (magnetoferritin) resulted in fast reaction kinetics between its biotinylated derivative and avidin, permitting far shorter incubation times of the magnetic label (up to 5 min) compared with colloidal (up to ten min), and particulate labels (up to 90 min) (25,29).

The modified five-channel ferrograph allowed us to separate five different cell samples simultaneously, which was significantly faster than using a single-channel design. Simultaneous deposition of cell sample aliquots offered two distinct advantages: first, it produced results under identical experimental conditions (the same magnetic field and flow), which is important in comparing test

LYMPHOCYTE ISOLATION USING MAGNETOFERRITIN 257

A. FACS CONTROLS

B. FEED

1 MAGNETIC BARRIER

1 ELUATE

Fluorescence

Labeling notation: X:

Y:

1: 2: 3: 4: 5:

IsotypeFITC F: Feed

CD8F1TC E: Eluate

lsotype + pAbb + Av + FeP + AvFlTc CD8 mAb + pAbb + Av + AvFITC pAbb + Av + Ferb + AvFITC Av + Fee + AvFITC Ferb + AvFITC

channel 1 channel 2 channel 3 channel 4 channel 5

2-E 3-E 5-E

0

FIG. 4. Magnetic deposition of CD8 lymphocytes labeled with magnetoferritin at the magnetic barrier. For details, see legend to Figure 2.

results to control results; second, it made practical anal- ysis of weakly magnetic samples, which require a long time to separate. Identical experimental conditions (field and flow) were particularly important for analysis of a weakly magnetic label, such as the magnetoferritin. The simultaneous sample deposition allowed us to obtain re- sults for five samples in 50 min on average. It is possible to add channels to this system, if necessary.

The unique feature of the ferrograph is that it produces a magnetic cell deposit on the glass slide that is accessi- ble to direct optical analysis (6 ,33) . In this study, such a deposit was seen for both test (dense deposition) and control (sparse deposition) cell fractions. The specificity of lymphocyte subset deposition was confirmed by flow cytometry (antibody-specific depletion in tests and non- specific depletion in controls). The microscopic evalua- tion of the deposits confirmed the presence of mono- cytes in the feed aliquots, as measured by flow cytometry (Table 1). Monocyte contamination of the magnetic cell fraction when using colloidal magnetic labels (such as MACS microbeads) has been reported before by others (17). No attempt was made in this study to further con- firm the identity of the cells in the magnetic deposit; the identity of such cells was inferred from the depletion of the corresponding cell fraction from eluate when com-

pared with feed. The concentration of the magnetic de- posit on the glass slide provides a convenient means for analysis by immunocytochemistry and other suitable means. The molecular size of the magnetic label pre- vented its interference with cell morphology.

The modified ferrograph's design was based on high fields and gradients generated by the fringing field of an interpolar gap (34 ) . The thin deposition plate pressed against the magnet provided maximum sample exposure to the field. This design significantly increased the mag- netic force exerted on the ferritin-labeled cell in compar- ison with the original ferrograph (32). In general, the smaller the magnetic moment of the magnetic label, the higher the field and gradient necessary for successful magnetic separation (30 ,34) , as is shown above in the equation for the magnetic force I;. Large particulate mag- netic labels require a relatively small magnetic field and gradient. The best characterized particulate magnetic la- bel, Dynabead M450 (monodisperse polystyrene bead doped with 23% magnetite; Dynal AG, Oslo, Norway), has a diameter of 4.5 pm and a high magnetic suscepti- bility of 0.245 [SI] (8,26,29). It is used effectively with simple magnet systems, such as Dynal magnetic particle concentrators ( M P C series) of average field intensity on the order of 0.1 T and a gradient of 10 T/m (for the

258 ZBOROWSKI ET AL.

FIG. 5. The microscopic appearance of the cells in the magnetic de- posit following air fixing and staining by using the modified Wright stain (from the experiment shown in Fig. 3A). A Cells stained with nonspe- cific isotype antibody and magnetoferritin in channel 1. B: Cells stained with anti-CD4 mAb and magnetoferritin in channel 2. These micropho-

relationships between the magnetic and geometric quan- tities of the particle, see above). Such a field induces a magnetic dipole moment in the Dynabead M450 on the order of Am2, which is sufficient to isolate a cell attached to the Dynabead from a relatively large volume of solution (up to 50 ml for MPC 1) and in a relatively short period of time (under 1 min). The ratio of volume to the deposition area is large. Large volumes of cells can be purged by using Dynabeads in a continuous flow sys- tem (such as Isolex 300 Magnetic Cell Separator; Baxter Healthcare Corporation, Deerfield, IL). Dynabeads must be detached from cells prior to flow cytometry of the magnetic cell fraction and prior to cell culture. Dynabead sorting performance depends on the cell mixture, the type of sorting, and the quality of reagents. In a typical negative separation application, high depletion rates (98%) were achieved in purging bone marrow of CD8 cells with concomitant moderate losses of CD4, CD20, and CD34 cells (15%) (14). The CD34 cell recovery yield of 45% and purity of 92% were reported when using Dynabeads for positive selection ( 17).

Unlike the particulate labels, colloidal magnetic labels require a high magnetic field and gradient. An example of such a colloidal label is the MACS microbead (dextran microbead doped with magnetite, nominal diameter 50 nm; Miltenyi Biotec GmbH, Bergisch Gladbach, Federal Republic of Germany) (2 1 ). The small size of the MACS microbeads requires a high magnetic field and gradient to achieve sufficiently high magnetic moment. The rela- tively small magnetic force F dictates a short cell travel distance in solution to the deposition substrate necessary for cell separation in a realistic time interval, resulting in a low ratio of sample volume to the deposition area. Such fields are provided by high-gradient magnetic separators (MACS Magnetic Separator; Miltenyi Biotec GmbH) (25) . The overall performance of the MACS system is compa-

tographs show selected examples of cell appearance on the slide. Note the far less numerous and predominantly monocytic morphology in A compared with the predominantly lymphocytic morphology in B. The microscopic observations corroborated the FACS findings (see text) that monocytes are mostly responsible for the nonspecific ferritin binding.

rable to that of the Dynabead-based separation systems, with the Dynabead-based system being more efficient for negative separation and the MACS system being more efficient for positive isolation (17). High purity and cell recovery of the positive cell selection becomes im- portant in isolation of rare cells for analytical purposes, such as identification of fetal cells in the maternal blood

The depletion rate of the antibody-specific cell fraction (up to 88%; Table 1) and the loss of negative fraction (more than 50% ; Table 1 ) using magnetoferritin and the modified ferrograph are inferior to the respective values reported for the Dynabead and MACS systems described above. However, this study was successful in establishing the feasibility of magnetoferritin as a molecular label, and it identified possible improvements in relation to already established methods. Our ongoing work is directed to- ward increasing the antibody-specific depletion and de- creasing the nonspecific labeling and depletion in com- parison with other methods. This may be accomplished by increasing the iron load per magnetoferritin molecule (S), increasing the magnetic field and gradient, and re- ducing the number of components in the labeling sand- wich (preferably to one or two).

Further studies are required to fully characterize this novel analytical magnetic separation system. In particular, it would be tempting to compare the capabilities of such a system with FACS in applications to lymphocyte immu- nophenotyping. It is worth noting that, in principle, the sensitivity of the magnetic method is not limited by the intrinsic cell susceptibility (which is low for lympho- cytes) in contrast to the autofluorescence limit of the fluorescence-based sorting method (which is appreciable for lymphocytes) (27). Improvements of the labeling procedure, total and differential cell counts in the feed, the eluate, and the magnetic deposit fractions, and feasi-

(5).

LYMPHOCYTE ISOLATION

bility of the method in dealing with whole blood prepa- rations will have to be investigated before the analytic potential of lymphocyte isolation using magnetoferritin and the modified ferrograph can be compared with that of FACS. Some of these questions will be addressed in our ongoing investigations.

ACKNOWLEDGMENTS The helpful discussion of magnetostatics with V.C.

Westcott, W.W. Seifert, and R.S. Wenstrup and the tech- nical assistance with FACS analyses by T. Bendele are thankfully acknowledged.

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