High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens

Journal of Immunological Methods, 154(1992)245-252

245

© 1992ElsevierSciencePublishersB.V. All rightsreserved0022-1759/92/$05.~2

JIM 06440

High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens Terry E. Thomas, Sara J.R. Abraham, Alan J. Otter a, Ewart W. Blackmore a and Peter M. Lansdorp Terry Fox Laboratory, British Columbia CancerAgency and Department of Medicine, Universityof British Columbia, 601 West lOth Avenue, Vancouver, B.C. VSZ IL3, Canada, and a TRIUMF, 4004 WesbrookMall, Vancouver, B.C. V6T 243, Canada

(Received16April1992,accepted4 May 1992)

The possibility of separating cells on the basis of levels of antigen expression was explored in a model system using fixed erythrocytes and high gradient magnetic separation (HGMS). Fixed human erythrocytes were labelled to varying degrees with tetrameric monoclonal antibody complexes specific for both dextrarL and glycophorin A-M. The cells were then mixed and incubated with dextran iron particles prior to magnetic separation. The small size of the dextran iron particles ( < 0.2/~m) resulted in quantitative magnetic labelling of cells as shown using fluoresceinated anti-dextran antibodies and flow cytometry. The relationships between the initial percentage of labelled cells, cell recovery, non-specific entrapment of unlabelled cells, the purity of the removed fraction, the degree of antigen expression and separation conditions (flow rate and field strength) were determined and used to establish sepa;ation conditions that allowed recovery of cells that differ only in the degree of antibody labelling. Key words: Cellseparation;Magneticseparation,highgradient;Ferrofluid

Introduction Numerous immunoadsorption techniques are currently being used to separate cells based on the expression of cell surface antigens recognized by specific monoclonal antibodies. In these procedures cells are either targeted for depletion (negative selection) or for enrichment (positive selection). In both approaches the target cells are labelled with an antibody against the selective cell surface antigen and then bound to an immunoadsorption surface which can be physically

Correspondence to: T.E. Thomas, Terry Fox Laboratory, 601 West 10th Avenue,Vancouver,B.C. VSZ IL3, Canada. Tel.: (604)877-6070;Fax:(604)877-0712.

:emoved from the cell suspension. Commonly used surfaces are columns of beads (Bensinger et al., 1990), surfaces of panning devices (Okanna, 1992) and magnetic beads (Kemshead, 1991). The ability of a given technique to effectively separate antigen-expressing cells depends on the balance between attractive forces holding the cell onto the immunoadsorption surface, and the shearing forces involved in the removal of antigen-negative cells. Cell surface antigen expression typically varies over a wide range and in most cases cannot be considered to be an "all or nothing" phenomenon. It therefore seems likely that in all immunoadsorption methods there will be a poiat where the number of available antigenic sites is insufficient to trap a cell. The level of expression at which this occurs will depend on separation

246 conditions. In order to study the effects of antigen expression in relation to the dynamics of cell separation in a batch-wise immunoadsorption technique, we have tested a number of separation variables in a model system using high gradient magnetic separation (HGMS). An HGMS device consists of a filter of fine weakly magnetic wires placed in a strong magnetic field. High gradient magnetic fields are produced around the wires, facilitating the capture of submicron magnetic particles which are too small to be retained in the magnetic field alone. The strength of the magnetic field produced by an electromagnet can be varied over a wid¢; range allowing precise control of the attractive forces acting on antigen-expressing cells. The degree of magnetic labelling can be varied using a range of antigen/antibody densities. In our model studies, fixed human erythrocytes were labelled to varying degrees with an anti-glycophorin A-M type antibody (6A7M). The anti-glycophorin antibody was non-covalently cross-linked to an anti-dextran monoclonal antibody in a tetrameric antibody complex (Lansdorp and Thomas, 1990). With this reagent cells were quantitatively labelled with submicron ( < 0.2/zm filtered) dextran iron particles (Molday and MacKenzie, 1982) as illustrated in Fig. 1. Our results show that HGMS in combination with quantitative magnetic labelling of cells can be used for batch-wise separation of cells based on quantitative differences in antigen expression.

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Antibodies The mouse IgGl anti-human glycophorin AM-type monoclonal antibody 6A7M was a gift from Dr. R. Langlois (Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA) and has been described (Langlois et al., 1990). The mouse IgG1 anti-dextran monoclonal antibody (DX1) was purified from culture supernatants produced by an IgG1 immunoglobulin switch variant of the hybridoma 341G6. The parent IgO3 producing hybridoma 341G6 (Borden and Kabat, 1988) was kindly provided by Dr. E. Kabat (Department of Microbiolog3,, Columbia University, New York). Isolation of the IgG1 producing immunoglobulin switch variant is described below. Fiuoresceinated anti-dextran antibodies were prepared by incubation of DX1 antibody (1 mg/ml in 0.1 M NaHCO 3, pH 9.5) for 2 h at room temperature with fluorescein isothiocyanate (FITC, 2 mg/ml stock in DMSO) at a final concentration of 100/~g/ml. Labelled antibody was dialysed against phosphate-buffered saline (PBS). F(ab')2 fragments of the rat monoclonal IgG1 antibody TFL-P9 specific for the Fc portion of the mouse IgO1 molecule (Lansdorp and Thomas, 1990) were obtained by pepsin digestion of purified immunoglobulin as described previously (Thomas et al., 1989). Tetramolecular antibody complexes (Lansdorp and Thomas, 1990) were prepared by mixing the anti-glycophorin A-M antibody (6A7M) with the anti-dextran antibody (DX1) and then adding the F(ab') 2 rat anti-mouse IgG1 antibody in a molar ratio of 1:4: 5 respectively O,Vognumet al., 1987). A significant fraction (32%) of the resulting tetramolecular antibody complexes recognize both glycophorin A-M and dextran.

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Fig. 1. Schematicdrawingof magneticlabellingof cells.Glycoi~horinat the cell surface is cross-linkedto magneticdextrail iron particlesusinganti-slycophorinX anti-dextrantetramericantibodycomplexes.

Magnetic particle preparation Submicron magnetic particles were prepared as described by Molday and MacKenzie (1982). Briefly, 10 g of Dextran T40 (Pharmacia, Uppsala, Sweden), 1.5 g FeCI3.6H20 and 0.64 g FeCI2.4H20 were dissolved in 20 ml H20 and heated to 40°C. The solution was continually stirred as 10 ml 4N NaOH was slowly added and the mixture heated to 700C for 5 min. The patti-

247 cle suspension was neutralized by adding acetic acid (glacial). Aggregates were removed by centrit'aging at 1000× g for 5 min, then filtering through course Watman filter paper followed by a 0.2/tm Millipore filter. Free excess dextran was removed by washing the particles in a high-gradient magnetic field (HGMF). The HGMF was produced by placing a column ~,3 ml disposable syringe) packed (17% w/v) with fine (0.025 mm) stainless steel wire (AIS1302, Goodfellow Metals Ltd., Cambridge) in a magnetic field of 1.0 Tesla generated by an electromagnet with a gap of 1 cm and a pole area of 78 cm2. Washed magnetic particles were eluted in PBS and the optical density (OD) at 450 nm recorded. Bubble formation around the stainless steel wire was prevented by initially filling the column with 70% ethanol followed by extensive washing.

Peroxidase substitution of dextran Fe particles Horseradish peroxidase (type IV-A Sigma 15 mg/ml) was oxidized with 10 mM sodium periodate in 0.1 M acetate buffer 0.15 M NaCI pH 5.5 for 15 min at room temperature. Excess periodate was removed by passage over a G25 column (PD10, Pharmacia). Dextran particles (see above) were suspended in 3 ml 0.1 M acetate buffer pH 5.5, 0.15 M NaCI. The absorbance at 450 nm was adjusted to 4.0. 10 mM sodium periodate was added and the suspension mixed for 15 rain at room temperature before being passed over a PD10 column equilibrated with acetate buffer The oxidized particles were then incubated in 1 ~ mM dihydrazide (Sigma) for 1 h at room temperature, passed over another PD10 column to remove free dihydrazide and added to an equal volume (3 ml) of oxidized horseradish peroxidase solution and mixed at room temperature for 30 min. Remaining free hydrazide an0 aldehyde groups were blocked with 0.1 M Tris pH 8.0. The suspension was rotated overnight at 4°C. Free peroxidase was separated from the magnetic particles using HGMS (see above). The magnetic particles were washed and eluted with PBS.

Isolation of an IgG1 anti-dextran producing switch variant Mouse IgG1 anti-dextral, antibodies were detected in the supernatants from confluent mi-

c:otiter well cultures of the IgG3-producing hybridoma 341G6 (10,000 cells/well at start of culture) using a mouse IgGl-specific ELISA. Probind ELISA plates (Nunc) were coated with TFLP9 antibody, specific for the Fc portion of the mouse IgG1 molecule (Lansdorp and Thomas, 1990), at 5 /~g/ml in coating buffer (200 mM sodium phosphate buffer pH 8.9). 111e plates were washed with phosphate-buffered saline containing 0.1% (v/v) Tween 20 (PBS-Tween) and incubated with 341G6 hybridoma culture supernatant for 2 h at room temperature. Positive controls consisted of 10 /~g/ml CLB-HRP-1 anti-horseradish peroxidase antibody (Lansdorp et al., 1986). The plates were washed (PBSTween) and incubated for 1 h at room temperature with peroxidase substituted dextran magnetic particles (see above) at OD450nm-0.05 in Tris 10 mM buffered saline with 0.05% thimerisol and 0.5% BSA (TBT). After washing, the wells were assayed for peroxidase activity as described (Lansdorp et al., 1986). The cells from a positive well were distributed over several 96-well plates and screened again. Cells from subsequent positive wells were serially diluted at 5.0-0.5 cells/well over four 96-well plates, screened and recloned to yield clonal populations of bybridoma cells secreting IgG1 anti-dextran (DX1).

Magnetic labelling and separation of cells Formalin fixed erythrocytes were prepared as described by Langlois et al. (1990). Briefly, 100/~1 of whole blood was added to 1 ml fixation buffer No. 1 composed of FACS flow saline solution (Becton Dickenson, CA) containing 1 mg/ml bovine serum albumin and 50 /~g/ml sodium dodecyl sulphate. Alter 1 rain 10 ml of fixation buffer No. 2 (FACS flow saline solution containing 3% formaldehyde and 10/~g/ml sodium dodecyl sulphate) was added. The cells were mixed for 90 min at room temperature. An additional 0.8 ml of formalin was added and the mixing continued overnight. The cells were then washed twice with staining buffer (phosphate-buffered saline with 0.5% BSA, 0.01% NP40 and 0.01% NaN3) and resuspended to 5 × 108 cells/ml. Fixed erythrocytes from donors which expressed glycophorin A-M were mixed with fixed erythrocytes which expressed only glycophorin A N . For

248 magnetic labelling the cells were then incubated on ice fo ~. 30 min with 6A7M × anti-dextran tetrameric antibod~ complexes at five different antibody (6A7M) concentrations (respectively at 0.01, 0.03, 0.1, 0.3, 1 . 0 / t g / m l ) and washed twice. The various cell suspensions were mixed together to produce a precolumn sample and this sample was incubated (10 s cells/ml) with magnetic dextran-Fe particles (O1)450, m = 0.01) on ice for 30 min. Labelled cells at 1.0-0.5 × l0 s cell/ml ("precolumn" sample) were passed over the HGMS filter described above. A variety of separation conditions were established by changing the flow rate, magnetic field strength and the amount of stainless steel in the 3-ml column. The column was washed with 30 ml of staining buffer. The cells which did not bind to the wire were collected in this "flow through" fraction. The magnet was then turned off and the bound cells recovered in 30 ml of buffer (" removed" fraction).

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Fig. 2. Sensitivityof lgG1 specificELISAused to isolate IgG1 anti-dextran hybridomas.The OD4s0nmof the peroxidasereaction product developed from different amounts of IgG1 anti-peroxidasemonoclonalantibody(ng/ml used for binding to anti-moaselgG1 coated wells)with either peroxidasedextran-Fe particles(o-o) or free peroxidase(o-e) as indicator. Note that at 3 ng/ml of anti-peroxidasethe maximumsignal with peroxidase-dextran-Feparticles is reached whereas 300 ng/ml of free peroxidaseis needed to achievea plateau, and that IgGl antibodiesare easilydetectablebelow 1 ng/ml with the peroxidase-dextranparticlesbut not with free peroxidase.

Precolumn, flow through and removed cell suspensions were stained with FITC conjugated F(ab') 2 fragments of sheep anti-mouse IgO (SAM-FITC, CappeU Cat-No. 1311-1744) or FITC conjugated anti-dextran DX1 for FACS analysis. 106 cells were suspended in 100 /zl of either SAM-FITC (diluted 1/100 with staining buffer)

or DX1-FITC (1 p.g/ml in staining buffer), incubated for 30 min on ice, then washed and resuspended. Stained and unstained samples were analysed using a FACScan (Becton Dickinson, San Jose, CA) flow cytometer.

TABLE I PROPORTION (%) OF POSITIVE CELLS BOUND TO THE HGMS FILTER IN RELATION TO THE LEVEL OF ANTIBODY/MAGNETIC PARTICLE LABELLING Magneticfield strength(Tesla) 0.1 •2 03 03 1.0

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O, et~ythrocytesexpressingonlyglycophorinA-N type;A, glycophorinA-M expressingcells labelledwith 0.01/zg/ml anti-dextran× anti-glycophorintetramericcomplexes;B, 0.03 p~g/ml;C, 0.1/zg/ml; D, 0.3/~g/ml; E, 1.0/~g/ml. Mixturesof cellswere labelled with dextran iron particles and separated as describedin the materials and methodssection.

249 Results A

Non-covalent cross-linking of magnetic particles to cell surface antigens In order to use the powerful cross-linking properties of tetrameric antibody complexes (Lansdorp and Thomas 1990; Lansdorp et al., 1991) for magnetic labelling of cells, an lgG1 monoclonal antibody specific for dextran iron particles was developed. This was achieved by selection and isolation of an IgG1 immunoglobu-

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Fig. 4. Relationshipbetweenthe bindingof labelled(% depletion, I1-11) and unlabelled (% non-specificbinding, zx-zx) cells to the HGMS filter, the % purity of labelled cells in the removed fraction ( • - • ), the overall recoveryof labelledcells (% recovery, e-o) in the removed fraction as a function of the flowrate. 10% of the precolumnsamplewas labelledwith 1.0 ~g/ml (A) or 0.03 Fg/ml (B) 6A7M antibody in tetrameric complexes.The fieldstrengthwas 1 Tesla.

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Fig. 3. Fluorescencehistogramsof erythrocytesamplesbefore (precolumn) and after (flowthrough) passage through a magnetic column and the cells removed from the column (removed). Cells labelled separately with 1.0, 0.3, 0.1, 0.03 and 0.0l Fg/ml 6A7M antibody in tetrameric complexeswere mixed with unlabelled cells and incubated with dextran iron particles to form the precolumnsample.Sampleswere stained with SAM FITC (A) or anti-dextran FITC (B). The separation flow rate was 6 ml/min and the field strength was 0.2 Tesla.

lin switch variant of an lgG3 anti-dextran secreting hybridoma using a sensitive ELISA technique that allowed detection of less than 1 n g / m l of IgG 1 antibodies in tissue culture supernatant (Fig. 2). With this technique the production of single IgG1 secreting clones among 104 lgG3 anti-dextran secreting hybridoma cells in the wells of microtiter plates could he detected. Such lgG1 anti-dextran secreting hybridoma cells were detected at a low frequency (4 clones per 480 wells tested) and cloned as described in the materials and methods section, lgG1 anti-dextran (DX1) was purified from hybridoma supernatant and used for the production of tetrameric antibody complexes.

250

Quantitative magnetic labelling of celts Anti-glycophorin X anti-dextran t e t r a m e r i c a n tibody complexes were u s e d at different c o n c e n trations t o g e t h e r with s u b m i c r o n d e x t r a n - F e part i d e s to achieve different d e g r e e s o f m a g n e t i c labelling o f fixed r e d blood cells. T h e p r e c o l u m n

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Fig. 6. Fluorescent histograms of erythrocyte samples befofe (A) and after HGMS (B). 25% of the precohmn cells were labelled with respectively 1.0 and 0.03 p.g/ml 6A7M antibody in tetrameric complexes. Weakly positive cells were purified via two separations: the removed sample (B) from the first (3 ml/min, 0.2 Tesla) was the pre¢olumn for the second (1.5 ml/min, l Tesla). The flow through fraction of the second passage (C)was enriched for weakly positive cells. ,

FLUORESCENCE Fig. 5. Fluorescent histograms of erythrocyte samples before (precolumn) (A) and after passage through a magnetic column. 10% of the precolumn samples were labelled with 1.0 and 0.03 p.g/ml 6A7M antibody in tetramerie complexes. Brightly positive cells were purified in the fraction which was recovered when the magnet was turned off(B). A flow rate of 9 ml/min was used with a field strength of I Tesla.

s a m p l e consisted of six distinct s u b p o p u ! a t i o n s which could be d e t e c t e d by flow c y t o m e t r y (Fig. 3). T h e s e s u b p o p u l a t i o n s were identified with a n t i - d e x t r a n F I T C staining as well as S A M - F I T C staining indicating t h a t t h e cells were labelled to varying d e g r e e s with b o t h m o n o c i o n a l antibody c o m p l e x e s a n d m a g n e t i c d e x t r a n particles (Fig. 3). T h i s cell mixture was s e p a r a t e d u s i n g H G M S at a r a n g e o f flow rates a n d field s t r e n g t h s in o r d e r to test t h e effect o f t h e s e variables o n t h e

251 number and type of cells that were depleted/ purified (Fig. 3, Table I). As expected, the proportion of positive cells bound by the magnetic column depended on their degree of antibody/ particle substitution (Table I, Fig. 3), the magnetic field strength and the flow rate (Table I). For example, lowering the field strength to 0.2 Tesla and increasing flow rates favoured the capture of strongly labelled cells and produced a removed fraction highly enriched for brightly positive cells. Doubling the flow rate had a much greater effect on the binding of weakly positive cells than reduction of the field strength by a factor of two. The capture efficiency of even highly labelled cells decreased rapidly below a magnetic field strength of 0.3 Tesla (Table I). Non-specific entrapment of negative cells was eliminated by faster flow rates and increased slightly by higher field strengths. Almost all the cells which were specifically bound to the magnetic column were recovered in the removed fraction when the magnet was turned off. The relationship between recovery of positive cells and the purity of this "removed" fraction was investigated using a precolumn cell suspension which was only 10% positive, labelled with 1.0 /zg/ml or 0.03 ~,g/mi 6A7M antibody in tetrameric complexes, respectively (Fig. 4). The recovery of positive cells in the removed fraction, the purity of the removed fraction and the non-specific entrapment of negative cells was determined at several flow rates (Fig. 4). The non-specific entrapment of negative cells decreased with increasing flow rates resulting in higher purities of the removed fraction (Fig. 4). This advantage of high flow rates was counteracted by a decrease in the recovery of positive cells (Fig. 4). Lowering the degree of antibody labelling did not affect the non-specific entrapment of negative cells but resuited in a much more iapid drop-off in recovery of positive cells at higher flow rates (Fig. 4B). The slope of the % purity curve did not appear to change with weaker magnetic labelling but the entire curve was shifted down by approximately 15-20%. The knowledge gained by testing the effects of field strength and flow rate on the purification of a mixture of cells enabled us to adjust separation conditions to purify weakly labelled cells from

strongly labelled cells. A single separation at high flow rate allowed purification of strongly labelled cells (Fig. 5). A double separation, the first to remove nnlabeUed cells (in the flow through) and the second to remove strongly labelled cells, allowed for high enrichment of weakly labelled cells in the flow through fraction of the second separation (Fig. 6).

Discussion Cell lines are often used to test immuno-separation techniques. Such cells may express an antigen of interest but often at hi~l levels compared to their normal counterparts. As a result efficient separations performed using a model cell system often cannot be reproduced with non-cultured cells. Typically, the relationship between recovery and purity will be shifted as in Fig. 4. Only with quantitative magnetic labelling and very reproducible cell suspensions have we been able to clearly dissect the relationship between purity, recovery, non-specific entrapment and antigen/label density. This relationship will determine the suitability of a given immunoadsorption technique for the separation of a subpopulation of cells at the required purity and yield. For example, with the described HGMS system highly labelled cells can be separated with > 90% purity and > 70% recovery. Such purities are unlikely to be achieved with moderate to low antibody labelling without losing most of the positive cells. In our model, 80% purity with 30-40% recovery for weakly labelled cells was achieved. Possibly the use of more magnetic wire filters which also exhibit lower non-specific entrapment could improve these results. In our studies we have used electro-magnetic HGMS for two reasons. Firstly, the submicron sized magnetic particles allow a larger range in the number of particles bound per cell than can typically be achieved with larger particles, resulting in quantitative magnetic labelling (Fig. 3). Secondly, the use of an electromagnet allows simple and accurate changes in the magnetic field strength. Several techniques for positive selection of cells with immunomagnetic particles are currently in use (Civin et al., 1990; Miltenyi et al.,

252 1990; K e m s h e a d , 1991) but n o n e fully exploit t h e advantages described above. T h e fixed erythrocyte suspension was a convenient, very reproducible model system. W e have also s e p a r a t e d h u m a n leukocytes with H G M S (manuscript in preparation). T h e efficiency o f separation d e p e n d s on t h e antigen density as predicted in this study. Highly labelled cells such as CD8 + cells from peripheral blood can be efficiently t r a p p e d in the magnetic filter in a similar m a n n e r as the " C " erythrocytes (Table I). CD34 + cells from b o n e m a r r o w which have a relatively low level o f CD34 expression, are m o r e weakly b o u n d (like " A " eryth/'ocytes in Table I). Slow flow rates and multiple passages can b e used to separate such weakly labelled cells. T h e dilemma o f purity versus yield is c o m m o n to all batch-wise purification techniques (Lansd o r p et al., 1992). T h e specific purity and recovery requirements will d e p e n d on the application o f t h e purified cells and the potential o f a given separation technique will vary with different target cells. T h e knowledge gained by this m o d e l study is currently being applied to purify rare subpopulations o f cells with m o d e r a t e levels o f antigen expression as well as to d e p l e t e specific cell types from h u m a n b o n e marrow.

Acknowledgements T h e s e studies were s u p p o r t e d by grants from the National C a n c e r Institute o f C a n a d a and G r a n t 89-AI-08 to P e t e r L a n s d o r p from the National Institutes o f Health (USA). W e thank Tannia Allnutt and K a r e n W i n d h a m for typing the manuscript. T h e electromagnet used in this study was a generous gift from T R I U M F , Vancouver, B.C., Canada.

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C.D. and Thomas, E.D. (1990) Positive selection of hematopoietic progenitors from marrow and peripheral blood for transplantation. J. Clin. Apberesis 5, 74. Borden, P. and Kabat, A. (1988) The specificities of polyclonal and monoclonal anti-idiotypes to anti-a (1-6) dextrans; possible correlations of idiotype with amino acid sequence. Mol. lmmunol. 25, 251. Civin, C.I., Strauss, L.C., Fackler, M.J., Trischmann, T.M., Wiley, J.M. and Loken, M.R. (1990) Positive stem cell selection - basic science. In: S. Gross, A.P. Gee and D.A. Worthington-White (Eds.), Bone Marrow Purging and Processing. Wiley-Liss,New York, p. 387. Kemsbead, J.T. (1991) The immunomagnetic manipulation of bone marrow. In: A.P. Gee (E"J.), Bone Marrow Processing and Purging. CRC Press, Boca Raton, FL, p. 293. Langlois, R.G., Nisbet, B.A., Bigbee, W.L., Ridinger, D.N. and Jensen, R.H. (1990) An improved flow cytometric assay for somatic mutations at the glycophorin A locus in humans. Cytometry 11, 513. Lansdorp, P.M. and Thomas, T.E. (1990) Purification and analysis of bispecific tetrameric antibody complexes. Mol. lmmunol. 27, 659. Lansdorp, P.M., Wognum, A.W. and Zeijlemaker, W.P. (1986) Stepwise amplified immunoenzyme staining techniques for the detection of monoclonal antibodies and antigens. In: J.J. Langone and H. Von Vunakis (Eds.), Methods in Enzymology. Academic Press, New York, p. 855. Lansdorp, P.M., Smith, C., Safford, M., Terstappen, L.W.M. and Thomas, T.E. (1991) Single laser three color immunofluorescence staining procedures based :~n energy transfer between phycoerythrin and cyanine :~. Cytometry 12, 723. Lansdorp, P.M., Schmitt, C., Sutheriand, H.J., Craig, W.H., Dragowska, W., Thomas, T.E. and Eaves, C.J. (1992) Hemopoietic stem cell characterization. In: S. Gross, A. Gee and D. Worthing,cn-White (Eds.), Bone Marrow Purging and Processing. Wiley-Liss, New York, in press. Miltenyi, S., Muller, W., Weichel, W. and Radbroch, A. (1990) High gradient magnetic cell separation with MACS. Cytometry 11, 231. Molday, R.S. and MacKenzie, D. (1982) Immunospecific ferromagnetic iron dextran reagents for the labelling and magnetic separation of cells. J. Immunol. Methods 52, 353. Okarma, T. (1992) A new technology for stem cell purification. In: S. Gross, A. Gee and D. Worthington-White (Fds.), Bone Marrow Purging and Processing. Wiley-Liss, New York, in press. Thomas, T.E., Sutheriand, H.J. and Lansdorp, P.M. (1989) Specific binding and release of cells from beads using cleavable tetrameric antibody complexes. J. Immunol. Methods 120, 221. Wognum, A.W., Thomas, T.E. and Lansdorp, P.M. (1987) Use of tetrameric antibody complexes to stain cells for flow cytometry. Cytometry 8, 366.