A single step density gradient separation for large scale enrichment of mobilized peripheral blood progenitor cells collected for autotransplantation

Transcription

1 Bone Mrrow Trnsplnttion, (1998) 21, Stockton Press All rights reserved /98 $12.00 A single step density grdient seprtion for lrge scle enrichment of mobilized peripherl blood progenitor cells collected for utotrnsplnttion D Ferrero, C Trell, C Chersco, P Bondesn, P Omedè, R Rvgli, D Crcciolo, C Cstellino nd A Pileri Diprtimento di Medicin e Oncologi Sperimentle, Divisione di Emtologi dell Università di Torino, Aziend Ospedlier Sn Giovnni Bttist, Torino, Itly Summry: cells in leukpheresis collections. 4 7 Therefore, the use of in vitro purging techniques is now considered mjor issue Peripherl blood leukocytes re becoming the preferred in PBPC-bsed utogrfting procedures. source of hemtopoietic progenitor/stem cells for Applying in vitro purging techniques to blood-derived utologous trnsplnttion. However, in vitro purging progenitors, few specific problems hve emerged, minly procedures re complex nd expensive when pplied to relted to the lrge quntities of cells. Indeed, two to four peripherl blood progenitor cells hrvests. This is leukpheresis collections re usully required to hrvest minly due to the lrge quntities of nucleted cells enough progenitors for sfe engrftment. 2,8,9 This implies present in leukpheresis collections. Aiming to reduce two- to five-fold increse in the totl cellulrity compred totl cellulrity without significnt loss of CD34 + cells, to BM collections. The lrge mounts of cells to be processed we developed n in vitro cell seprtion procedure bsed mke ny purging procedure cumbersome nd on ficoll/metrizote grdient used t finl density of highly expensive, prticulrly those bsed on immunomgnetic g/ml. To obtin this density, stndrd Lymphoprep methods. 10 (1.077 g/ml) ws diluted with norml sline sol- In this study we describe simple procedure, bsed on ution (NCl 9 g/l). Twenty-six leukpheresis collections modified ficoll-metrizote grdient seprtion, tht ws (medin cellulrity , rnge ) from 14 employed to enrich progenitor cells leukpheresis collections. ptients with non-hodgkin s lymphom, multiple myelom The grdient seprtion llowed mrked reduction or plsm cell leukemi were processed (medin of totl cellulrity, with enrichment nd miniml loss of two leukphereses per ptient). Men ( s.d.) recovery hemtopoietic progenitors. The technique lso llowed of totl nucleted cells, CD34 + cells nd CFU-GM ws prtil removl of contminting neoplstic cells. Both %, % nd %, respectively. observtions indicte tht our pproch is useful, Cumultive per ptient progenitor cell recovery ws preliminry step prior to immunologicl purging. lwys bove 50%, nd s high s 80% in 10/14 ptients, while totl cellulrity ws reduced to medin 21.5% (10 33%) of pre-seprtion vlues. Contminting neoplstic cells, identified by immunofluorescence in Mterils nd methods five collections, were reduced by 1 2 logs. The results indicte tht our density grdient seprtion is n effective Ptients nd tretments method to reduce totl cellulrity prior to immuno- Leukpheresis products from 15 ptients, collected fter logicl purging, without significnt loss of progenitor high-dose chemotherpy regimen, were processed in vitro. cells. Eleven ptients hd B cell non-hodgkin s lymphom Keywords: leukpheresis; progenitor cells; density grdient; utotrnsplnttion (NHL), two hd multiple myelom (MM), nd two plsm cell leukemi (PL). NHL ptients were prt of pilot study imed to evlute the efficcy of high-dose chemotherpy nd utogrft with in vitro purged hemtopoietic cells in folliculr nd mntle-cell NHL (mnuscript in Mobilized peripherl blood progenitor cells (PBPC) re preprtion). All ptients gve informed consent to in vitro incresingly used for utogrft purposes. 1 3 However, mnipultion of their leukpheresis products. highly sensitive moleculr or immunologicl techniques The high-dose chemotherpy regimen ws designed hve shown the frequent presence of residul neoplstic ccording to the originl sequentil HDS scheme, 11,12 with minor modifictions. During G-CSF-stimulted (Filgrstim; Roche, Miln, Itly, 5 g/kg/dy) leukocyte recovery fter Correspondence: Dr D Ferrero, Divisione di Emtologi dell Università di Torino, Aziend Ospedlier Sn Giovnni Bttist, Vi Genov 3, Torino, Itly Received 4 June 1997; ccepted 15 September 1997 high-dose (7 g/m 2 ) cyclophosphmide, peripherl blood progenitor cells (PBPC) were collected s soon s CD34 + cells reched minimum vlue of 10 20/ l. PBPC collec-

2 Blood progenitor enrichment by density grdient 410 tion (one to four leukpheresis procedures) ws performed g/ml) ficoll/metrizote preprtion 17 (Lymphoprep; by processing two plsm volumes with Fresenius Cell Nycomed, Oslo, Norwy) with norml sline solution Seprtor AS 104 (Wlnut Creek, CA, USA). Cells (NSS) (NCl 9 g/l, clinicl grde solution) t the following underwent density grdient seprtion nd cryopreservtion. proportions: 0, 10, 15 nd 17% v/v. Two density grdients, Cells from 10 NHL ptients were submitted, A nd B, were chosen for lrge scle seprtion of leukph- prior to cryopreservtion, to in vitro purging by n immunomgnetic eresis collections on the bsis of results previously obtined method (mnuscript in preprtion). Microbio- with smll volume smples. Grdient A (density logicl tests were performed to check cell sterility fter in g/ml) ws prepred by mixing 90% of Lymphoprep with vitro mnipultion. 10% NSS, v/v; grdient B (density g/ml) ws mde with 85% Lymphoprep + 15% NSS, v/v. The whole leukpheresis Immunofluorescence nd CFU-GM ssy mteril ws centrifuged nd concentrted to finl volume of ml by removl of 80% of plsm The proportion of PBPC, identified s CD34 + leukocytes, nd pltelets. The cell suspension ws then diluted with ws evluted on whole blood smples, s previously PBS/3% utologous plsm to finl cell concentrtion of described. 13,14 Briefly, 100 l of heprinized whole blood leukocytes/ml nd poured into 50 ml plstic were incubted with the pproprite mount of phyco- tubes (Flcon; Becton Dickinson, Lincoln Prk, NJ, USA), erythrin-conjugted CD34 monoclonl ntibody (HPCA-2, ech contining mximum of leukocytes. Becton Dickinson, Sn Jose, CA, USA) for 30 min t room Eleven milliliters of Lymphoprep/NSS solution were gently temperture. Erythrocytes were then lysed by exposure to dded t the bottom of ech tube, llowing them to strtify mmonium chloride for 15 min. After extensive wshing, beneth the cell lyer. Centrifugtion ws performed t leukocytes were nlyzed by flow cytometry using FAC- 400 g, for 30 min t 20 C. Low density cells were col- Scn (Becton Dickinson). Bivrite scttergrms were gen- lected, wshed twice with PBS + 0.5% humn lbumin, erted by combining right-ngle light sctter nd red fluorescence. counted nd either processed for in vitro purging or cryopsctter CD34 + cells were identified by low right-ngle reserved fter evlution of CD34 + cell nd CFU-GM nd positive fluorescence. Three consecutive cquisitions recovery. The percent recovery of CD34 + cells nd CFU- of events were performed for ech smple GM fter density grdient seprtion ws clculted s fol- nd the result ws expressed s the men CD34 + vlue. The lows: number of circulting CD34 + cells/ l of blood ws number of recovered cells % CD34 + cells (CFU-GM) fter seprtion obtined by multiplying the percentge of CD34 + cells in the whole leukocyte popultion by the totl number of leukocytes number of cells strtified % CD34 + cells (CFU-GM) before seprtion 100 in 1 l of blood. The sme procedure ws employed to evlute the proportion of CD34 + cells mong leukocytes collected by pheresis. In two ptients with PL, CD38 (Becton Dickinson) nd CD138 MoAb (Serotech, Results Oxford, UK) were used to detect residul plsm cells in Seprtion on Lymphoprep t different dilutions: results pheresis collections. Plsm cells were identified becuse on smll volume smples of high density CD38 ntigen nd CD138 ntigen coexpression. In ptient PA, with NHL coexpressing CD19 nd Pure Lymphoprep (1.077 g/ml) llowed ner complete CD5 ntigens, CD19 (Becton Dickinson) nd CD5 MoAb recovery of CD34 + cells but removed medin 30% of (Immunotech, Mrseille, Frnce) were employed to identify cells only. Indeed, t morphologicl exmintion, unseprresidul lymphom cells (low forwrd nd right-ngle sct- ted leukphereses were mostly constituted by monocytes ter cells coexpressing CD19 nd CD5 ntigens). The bsolcytes (30 45%), immture grnulopoietic cells (blsts to myelo- ute number of contminting cells ws clculted by multilower 10 25%) nd lymphocytes (4 15%), with density plying the totl cellulrity by the percentge of cells thn g/ml. The use of grdients t progressively expressing the ntigenic phenotype described bove. lower density ws ssocited with incresing reduction of The quntity of myeloid progenitors in the mteril totl cellulrity (60 90%), while mintining stisfctory before nd fter the seprtion procedure ws estimted by recovery (50 100%) of PBPC. CFU-GM evlution, s described. 15 Briefly, All dt refer to seprtion procedures performed within cells were cultured in two 35-mm Petri dishes (Becton 1 h of cell collection; processing fter longer intervls Dickinson), ech contining 1 ml of Iscove s modified Dulreducing the efficiency of the procedure. Mintennce of llows pltelet nd leukocyte ggregte formtion, thus becco s medium (GIBCO) with 20% fetl bovine serum (Hyclone, Logn, UT, USA), 0.3% gr (Difco, Detroit, temperture t 20 1 C before nd during the centrifug- MI, USA) nd 10% superntnt of the 5637 cell line, s tion ws lso criticl for optiml enrichment in CD34 + source of colony-stimulting fctors. 16 Colonies contining cells (dt not shown). t lest 50 cells were scored fter 14 dys of incubtion. Lrge scle seprtion of leukpheresis products Density grdient seprtion Three lrge scle cell seprtions were crried out t Different density grdients were initilly tested on smll volume leukpheresis smples. Grdients of progressively lower density were prepred by mixing stndrd (1.077 g/ml density (grdient A, ie Lymphoprep solution t 90%). Cells from ptients GP (both leukphereses) nd GM (first leukpheresis) were processed nd virtully ll CD34 + cells

3 Tble 1 Blood progenitor enrichment by density grdient Differentil recovery of leukpheresis cells from lrge scle seprtions on g/ml density Leukpheresis Medin Men s.d. Rnge evluted 411 Totl cells 10 9 pre-seprtion Totl cells recovered 10 9 (% recovery) (20) ( ) (6 47) % CD34 + cells recovered % CFU-GM recovered Percent recovery of totl nucleted cells compred to pre-seprtion vlues. Contminting tumor cells could be detected in the leukpheresis products of ptients PA with low-grde NHL; CR nd FR with plsm cell leukemi. Lymphom cells were identified s CD19 + /CD5 + cells while neoplstic plsm cells were identified s CD cells (high-inten- sity fluorescence), coexpressing CD138 ntigen. A mrked reduction (1 2 logs) in the bsolute number of neoplstic cells ws recorded following ll seprtion procedures per- formed in the three tested ptients (Tble 3), while recovery of norml progenitors reched 60, 86 nd 85%, respect- ively, of pre-seprtion vlues. nd CFU-GM were recovered. However, s opposed to results obtined in smll volume experiments, removl of myelomonocytic cells ws unstisfctory, with totl cell recovery of 54, 52 nd 45% in the three seprtion pro- cedures, respectively (dt not shown). Therefore, the remining 26 leukpheresis collections were seprted on g/ml density grdient (grdient B, ie Lymphoprep solution t 85%). As illustrted in Tble 1, the elevted cellulrity in the strting popultion ws mrkedly reduced, to medin 20% of pre-seprtion vlue, with stisfctory recovery of CD34 + cells nd CFU-GM (medin vlues 85.5 nd 76.5%, respectively). Although cell removl nd progenitor cell recovery were somewht vrible, it must be emphsized tht progenitor recovery lower thn 50% ws recorded only in three seprtion procedures. Moreover, by dding the results of ll seprtion procedures performed on ech ptient, progenitor cell recovery bove 50% ws recorded in ll cses, with more thn 80% recovery in 10/14 ptients (Tble 2). Cumultive removl of totl nucleted cells remined high, with finl cellulrity vlue rnging between 10 nd 33% (medin 21.5% of the pre-seprtion vlue). Thus, three- to four-fold enrichment in hemtopoietic progenitors ws obtined in ll ptients. Reduction of neoplstic cells following density grdient seprtion Discussion The im of this study ws to develop seprtion procedure, bsed on density grdient, cpble of removing t lest 75 80% of leukpheresis collection cellulrity without significnt loss of hemtopoietic progenitors. We chose Lymphoprep ficoll/-metrizote solution s bsic regent Tble 2 Effects of density grdient seprtion t g/ml on totl leukocyte nd CD34 + cell recovery in 14 ptients Ptient Collections Pre-seprtion vlues of: Post-seprtion vlues of: performed Totl cells CD34 + cell Totl CD34 + Totl cells % Totl cell CD34 + cell Totl CD34 + % CD34 + cell 10 9 frequency cells recovery b frequency cells 10 6 recovery c CF PA B AR CR FE P G P AT C C MF VF Men percentge of CD34 + cells before nd fter density grdient seprtion. b Percentge of totl nucleted cells recovered fter cell seprtion. c Percentge of totl CD34 + cells recovered fter cell seprtion.

4 412 Tble 3 Blood progenitor enrichment by density grdient Depletion of contminting neoplstic cells by seprtion on g/ml density grdient Ptient Leuk. Totl nucleted cells 10 9 % Neoplstic cells b Totl neoplstic cells 10 6c Pre- Post- Pre- Post- Pre- Postseprtion seprtion seprtion seprtion seprtion seprtion PA PA CR CR VF Sequentil number of leukphereses performed for ech ptient. b Expressed s percentge of CD19 + /CD5 + cells for ptient PA nd CD /CD138 + for ptients CR, VF. c Vlues obtined by multiplying the number of totl nucleted cells by the % of neoplstic cells. becuse of its low cost nd stbility t room temperture PBPC enrichment chieved by our procedure is certinly s well s its lck of toxicity on hemtopoietic cells, s hs lower thn those obtined with other methods employing been proven in BM purging procedures. 18,19 However, some sequentil Lymphoprep nd Percoll grdients or monoclonl preliminry experiments showed low efficcy of the commercilly ntibodies However, our seprtion technique vilble g/ml solution in removing mture is simple nd does not require sophisticted devices or nucleted cells present in leukpheresis products. This my expensive regents. be explined by the high content of monocytes nd imm- Finlly, the g/ml density grdient proved to be n ture grnulopoietic cells in the popultion tht hd to be effective method to reduce tumor contmintion. In five removed. Therefore, the grdient density ws reduced by smples from three ptients with immunofluorescencedetectble mixing Lymphoprep with different proportions of NSS. residul tumor cells, 1 2 logs neoplstic cell A finl 85% Lymphoprep/15% NSS v/v solution gve reduction ws documented following low-density grdient the best results in terms of both cellulrity reduction nd seprtion. The precise degree of neoplstic cell removl PBPC recovery in lrge-scle seprtions. Recovery of should be further investigted in more cses, using moleculr more thn 75% of PBPC, identified either s CD34 + cells or biology techniques when residul tumor cells re too s CFU-GM, ws obtined in most seprtions. However, few to be detected by immunofluorescence. However, the low progenitor recovery, below 50%, ws occsionlly combintion of high CD34 + cell recovery nd reduction of observed with the first leukpheresis collection. This could cellulrity nd neoplstic cell contmintion mkes our procedure be scribed to vritions in cell composition mong the useful preliminry step prior to PBPC in vitro different collections. In fct, low CD34 + recovery ws purging. lmost invribly ssocited with erly recovery from cytopeni nd reltively low vlues of totl leukocytes hrvested ( ). Low-density grdients showed poor efficiency in the Acknowledgements frctiontion of BM cell hrvests: CD34 + cells were highly The present study ws suppported by Associzione Itlin per enriched but their totl recovery ws vrible nd unstis- l Ricerc sul Cncro (AIRC). fctory (dt not shown). The discrepncy between mobilized PB cells nd BM-derived cells could be explined by differences in progenitor cell density. Thus, our procedure seems specificlly suitble for leukpheresis collections, in References prticulr for those with high cell counts. Indeed, in spite 1 Kessinger A, Armitge JO. The evolving role of utologous of some vritions in the procedure efficiency, the finl pro- peripherl blood stem cell trnsplnttion following high dose genitor cell recovery per ptient ws in ny cse bove 50% therpy for mlignncies. Blood 1991; 77: nd bove 80% in 10/14. In ddition, the procedure llowed 2 Henon PR. Peripherl blood stem cell trnsplnttion: pst, three- to five-fold enrichment of CD34 + cells. present, future. Stem Cells 1993; 11: Recently, Di Nicol nd coworkers 20,21 chieved 3 Coiffier B, Philip T, Burnett AK, Symnn ML. Consensus slightly higher PBPC enrichment, with similr recovery, conference on intensive chemotherpy plus hemtopoietic by the dhesion of mture myelomonocytic cells to nylon stem cell trnsplnttion in mlignncies: Lyon, Frnce, June wool. However, in most of their experiments the procedure 4 6, J Clin Oncol 1994; 12: Ross AA, Cooper BW, Lzrus HM et l. Detection nd ws pplied to reltively smll cell mounts, ie vibility of tumor cells in peripherl blood stem cell colleccells, with only two experiments performed t cellulrity tions from brest cncer ptients using immunocytochemicl up to Since most collections contin cell counts nd clonogenic ssy techniques. Blood 1993; 82: fr bove , the fesibility of the nylon dherence 5 Moss TJ, Ciro M, Sntn VM et l. Clonogenicity of circulseprtion in the presence of high cellulrity needs to be ting neuroblstom cells: implictions regrding peripherl verified. blood stem cell trnsplnttion. Blood 1994; 83:

5 Blood progenitor enrichment by density grdient 6 Corrdini P, Voen C, Astolfi M et l. High dose sequentil tion in multiple myelom. Bone Mrrow Trnsplnt 1993; chemo-rdiotherpy in multiple myelom: residul tumor cells 11: re detectble in bone mrrow nd peripherl blood cell hrsion 15 Trell C, Ferrero D, Bregni M et l. Peripherl blood expnvests nd fter utogrfting. Blood 1995; 85: of erly progenitor cells fter high dose cyclophosphm- 7 Hrdinghm JE, Kotsek D, Sge RE et l. Moleculr detec- ide nd rhgm-csf. Eur J Cncer 1991; 27: tion of residul lymphom cells in peripherl blood stem cell 16 Welte K, Pltzer E, Lu L et l. Purifiction nd biochemicl hrvests nd following utologous trnsplnttion. Bone chrcteriztion of humn pluripotent hemtopoietic colony- Mrrow Trnsplnt 1993; 11: stimulting fctor. Proc Ntl Acd Sci USA 1985; 82: Peters W, Rosner G, Ross M et l. Comprtive effects of grnulocyte mcrophge colony-stimulting fctor (GM-CSF) 17 Boyum A. Isoltion of mononucler cells nd grnulocytes nd grnulocyte colony-stimulting fctor (G-CSF) on prim- from humn blood. Scnd J Clin Lb Invest 1968; 21 (Suppl. ing peripherl blood progenitor cells for use with utologous 97): 77. bone mrrow trnsplnttion fter high dose chemotherpy. 18 Ferrero D, De Fbritiis P, Amdori S et l. Autologous bone Blood 1993; 81: mrrow trnsplnttion in cute myeloid leukemi fter in 9 Bensinger W, Singer J, Appelbum F et l. Autologous trnsrbbit complement. Leukemi Res 1987; 11: vitro purging with nti-lcto-n-fucopentose III ntibody nd plnttion with peripherl blood mononucler cells fter dministrtion of recombinnt grnulocyte colony-stimulting 19 Gribben JG, Freedmn AS, Neuberg D et l. Immunologicl purging of mrrow ssessed by PCR before utologous bone fctor. Blood 1993; 81: mrrow trnsplnttion for B-cell lymphom. New Engl J Med 10 Gribben JG, Sporito L, Brber M et l. Bone mrrow of non- 1991; 325: Hodgkin lymphom ptients with bcl-2 trnsloction cn be 20 Di Nicol M, Sien S, Bregni M et l. Lrge scle enrichment purged of polymerse chin rection-detectble lymphom of mobilized CD34 + peripherl blood hemtopoietic progenicells using monoclonl ntibodies nd immunomgnetic bed tors by removl of nylon wool-dherent mture cells. Bone depletion. Blood 1992; 80: Mrrow Trnsplnt 1994; 14: Ginni AM, Bondonn G. High dose chemo-rdiotherpy for 21 Di Nicol M, Bregni M, Sien S et l. Combined negtive sensitive tumors: is sequentil better thn concurrent drug nd positive selection of mobilized CD34 + blood cells. Br J delivery? Eur J Cncer Clin Oncol 1989; 25: Hemtol 1996; 94: Ginni AM, Sien S, Bregni M et l. Grnulocyte mcro- 22 Mrtin H, Neumnn M, Fche I et l. Grdient seprtion of phge colony-stimulting fctor to hrvest circulting hemo- grnulocytic progenitor cells (CFUc) from humn blood poietic stem cells for utotrnsplnttion. Lncet 1989; ii: mononucler leukocytes. Exp Hemtol 1985; 13: Henschler R, Brugger W, Luft T et l. Mintennce of trns- 13 Sien S, Bregni M, Brndo B et l. Flow cytometry for clinicl plnttion potentil in ex vivo expnded CD34 + selected estimtion of circulting hemtopoietic progenitors for uto- humn peripherl blood progenitor cells. Blood 1994; 84: logous trnsplnttion in cncer ptients. Blood 1991; 77: Gbbinelli M, Srgicomo M, Pelosi E et l. Pure humn 14 Trell C, Boccdoro M, Omedè P et l. Role of chemo- hemtopoietic progenitors: permissive ction of bsic fibroblst therpy nd GM-CSF on hemopoietic progenitor cell mobiliz- growth fctor. Science 1990; 249: