A novel high throughput immunomagnetic cell sorting system for potential clinical scale depletion of T cells for allogeneic stem cell transplantation.
OBJECTIVE
To develop an immunomagnetic cell separation system for allogeneic hematopoietic stem cell (HSC) transplantations, which can achieve a high level of T-cell depletion (at least 4.0 log(10)), high level of recovery of hematopoietic stem cells (>90%), with a high throughput (>10(6) cells/second).


METHODS
Peripheral blood leukocytes (PBLs) from buffy coats were spiked with CD34-expressing cells (KG1a) to mimic a leukaphoresis product containing stimulated HSCs. T cells were labeled with anti-CD3(+) Dynabeads and separated in a quadrupole magnetic cell sorter (QMS). The performance of the system with respect to T-cell depletion and recovery of non-T cells and spiked KG1a was determined using four-color, flow cytometry analysis, with the aid of Trucount cell-concentration calibration beads. Limiting dilution assays were also performed to quantify the log(10) depletion of clonable T cells.


RESULTS
While the general performance of the QMS system is governed by proven theoretical principles, significant system variability exist, not all of which can be explained by our current understanding. Consequently, a factorial design was employed, guided by JMP software, to optimize the labeling conditions and operation of the QMS focused on maximizing the depletion of T cell, and recovery of unlabeled cells including KG1a cells. From these studies, an optimized, no wash, immunomagnetic labeling protocol and optimized QMS operating conditions were developed. For an average initial cell concentration of 1.7 x 10(8) total cells, an average 3.96 +/- 0.33 log(10) depletion (range, 3.53-4.34) of CD3(+)CD45(+) cells with a mean 99.43% +/- 4.23% recovery of CD34(+)CD45(+) cells (range, 94.38-104.90%) was achieved at a sorting speed of 10(6) cells/s (n = 6). Limiting dilution assays on the T-cell depleted fractions, which gave a log(10) depletion of 3.51 for the clonable T cells.


CONCLUSIONS
We suggest that this system will provide superior performance with respect to T-cell depletion and CD34(+) recovery for clinical allogeneic bone marrow transplants. Ongoing studies, on a clinical scale, are being conducted to demonstrate this claim.
Tong
Xiaodong
X
Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus Ohio, USA.
Xiong
Ying
Y
Zborowski
Maciej
M
Farag
Sherif S
SS
Chalmers
Jeffrey J
JJ
D018952:Antigens, CD34 / Q000096:biosynthesis; D017252:CD3 Complex / Q000096:biosynthesis; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000592:standards; D007519:Isoantigens / Q000096:biosynthesis; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D008212:Lymphocyte Depletion / Q000295:instrumentation / Q000592:standards; D036102:Peripheral Blood Stem Cell Transplantation; D012015:Reference Standards; D015203:Reproducibility of Results; D014184:Transplantation, Homologous
D018952:Antigens, CD34; D017252:CD3 Complex; D007519:Isoantigens; C060224:Kg, red cell antigen; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
Transplantation. 1985 Sep;40(3):317-22
3875918
J Immunol. 1981 Apr;126(4):1614-9
7009746
Blood. 1992 Jun 15;79(12):3380-7
1596577
Blood. 1994 Dec 1;84(11):3948-55
7524753
Bone Marrow Transplant. 1997 Jun;19(11):1069-77
9193748
Transplantation. 1997 Aug 15;64(3):472-9
9275115
Bone Marrow Transplant. 1997 Oct;20(7):599-605
9337063
N Engl J Med. 1998 Oct 22;339(17):1186-93
9780338
J Immunol Methods. 1999 May 27;225(1-2):113-24
10365788
Springer Semin Immunopathol. 2004 Nov;26(1-2):155-68
15378270
Klin Padiatr. 2005 Nov-Dec;217(6):334-8
16307419
Biotechnol Bioeng. 2006 May 5;94(1):66-80
16518837
Cytotherapy. 2006;8(5):465-72
17050251
Exp Hematol. 2007 Apr;35(4):662-72
17379076
Clin Lab Haematol. 1999 Oct;21(5):301-8
10646072
Bone Marrow Transplant. 2000 Jun;25(12):1223-8
10871725
Transfusion. 2000 Dec;40(12):1475-81
11134567
J Chromatogr A. 2000 Dec15;903(1-2):99-116
11153960
Bone Marrow Transplant. 2001 Apr;27(7):683-7
11360106
Cytotherapy. 2001;3(6):467-81
11953031
Bone Marrow Transplant. 2002 Jul;30(2):69-74
12132044
Transplantation. 2002 Aug 27;74(4):562-70
12352920
Bone Marrow Transplant. 2003 May;31(10):857-60
12748661
Biol Blood Marrow Transplant. 2003 Oct;9(10):633-42
14569559
Br J Haematol. 2004 Jan;124(1):72-9
14675410
Cytotherapy. 2004;6(1):1-6
14985161
Exp Hematol. 2004 Oct;32(10):891-904
15504544
Blood. 1990 Mar 15;75(6):1356-63
2310832