Improvement of tumor cell depletion by combining immunomagnetic positive selection of CD34-positive hematopoietic stem cells and negative selection (purging) of tumor cells

One possible reason for relapse after high-dose chemotherapy is retransplan tation of tumor cells contaminating autologous hematopoietic stem cell tran splants. Residual tumor cells can be diminished by various purging methods. We studied tumor cell depletion by sequentially combining immunomagnetic p ositive selection of CD34(+) hematopoietic stem cells using Isolex50 or Iso lex300SA and negative tumor cell depletion using MACS, MaxSep or Isolex50 s ystems. Using these separation systems in different selection sequences, ie positive followed by negative selection (+/- selection) or vice versa, fou r groups of double selections (Isolex50/MACS, Isolex50/MaxSep, MaxSep/Isole x50, Isolex300SA/ Isolex50) were studied. Testing these double-purging proc edures mean additional tumor cell depletion (Delta TCD) achieved by the sec ond selection step ranged from 1.1+/-0.58 log (n=5, +/- Isolex50/MACS) to 2 .0 +/- 1.1 log (n = 7, -/+ MaxSep/Isolex50). Loss of CD34+ cells during dou ble selection sometimes was extensive and mean yield of CD34(+) cells range d from 12.8 +/- 11.5% (n = 6, +/- Isolex50/MaxSep) to 43.2% (n=2, +/- Isole x300SA/Isolex50). Calculated values for mean yield-corrected Delta TCD rang ed from 0.64 +/- 0.3 log (n = 5, +/- Isolex50/MACS) to 1.4 +/- 1.3 log (n = 7, -/+ MaxSep/Isolex50). During positive selection of -/+ selection (MaxSe p/Isolex50) relative tumor cell enrichment was detectable leading to an inc rement of mean tumor cell contamination rate. Best results for total TCD we re achieved by the combination of Isolex50/MaxSep (n=6; TCD: 4.2 log; yield CD34(+): 12.8%) and Isolex300/Isolex50 (n=2; TCD: 3.8 log; yield CD34+: 43 .2%). Furthermore, we have established and tested a new simultaneous +/- se lection method by using CD34-specific releasing agent PR34+ in the Isolex30 0i. With this method we have obtained a mean total yield-corrected TCD of 4 .7 log (n = 4; range: 4.1-6.0 log) with high CD34(+) cell, yield (mean: 69. 8%) and CD34(+) cell purity (mean: 92.8%). Since this new simultaneous +/- purging procedure is safe, applicable within a closed system (GMP-like) and most effective, we recommend it for further testing in a clinical setting.