Engraftment characteristics over serial transplantation of primary ALL cells in NSG mice. Cryopreserved diagnostic material was thawed, and 1 million cells were transplanted by intrafemoral injection into NSG mice 6 to 10 weeks of age. Mice were checked every 4 weeks by flow cytometry for human engraftment. Positive engraftment was defined as > 1% human CD19/CD45-positive and mouse CD45-negative cells in the peripheral blood. (A) Analysis of the time to engraftment for 9 VHR-ALL (black) and 13 SR-ALL (gray) patients. The time to engraftment in weeks is plotted for primary (solid lines) and secondary (dashed lines) transplantations. Statistical evaluation was performed with the log-rank test and indicates significant differences for the time to engraftment between VHR-ALL and SR-ALL (P < .05) and between primary and secondary transplantation for SR-ALL (P < .01). (B) Time to engraftment was analyzed for 7 VHR-ALL patients with up to 6 rounds of serial passages. (C) Number of cells harvested from spleens per mouse at death, when the percentage of human cells in the peripheral blood reached 70%. The mean value was 500 million for VHR-ALL and 650 million cells for SR-ALL xenografts (indicated as horizontal line).