In this study, a high CD34% in autologous peripheral blood stem cell (PBSC) products from 71 AML patients was associated directly with a high relapse rate (P = 0.006) and inversely with disease-free survival (P = 0.003), irrespective whether patients were transplanted or not. The relapse rate at 12 months was 67% in a group with Ͼ0.8% CD34 + cells and 34% in a group with р0.8% CD34 + cells. Although the percentage of malignant CD34 + cells in the CD34 + compartment in the relapses of the first group was not high (median 8%), the total number of malignant cells as a percentage of WBC was about 13 times higher than for the patients remaining Ͼ12 months in remission. When all patients evaluable were taken together, this frequency of malignant cells correlated strongly with disease-free survival (P Ͻ 0.001). Both this massive mobilization of normal CD34 + cells and high frequency of malignant cells in the subgroup of patients with CD34 Ͼ0.8% and relapse within 12 months indicate an insufficient in vivo purging, as well as low chemotherapeutic bone marrow toxicity. This was confirmed by an inverse correlation between hypoplasia period after the induction therapy and CD34% in PBSC products (P Ͻ 0.002). It is concluded that a subgroup of patients has been identified that might benefit from a more intensive chemotherapeutic treatment.

No abstract

Flow-cytometric detection of minimal residual disease (MRD) has proven in several single-institute studies to have an independent prognostic impact. We studied whether this relatively complex approach could be performed in a multicenter clinical setting. Five centers developed common protocols to accurately define leukemia-associated (immuno)phenotypes (LAPs) at diagnosis required to establish MRD during/after treatment. List mode data files were exchanged, and LAPs were designed by each center. One center, with extensive MRD experience, served as the reference center and coordinator. In quarterly meetings, consensus LAPs were defined, with the performance of centers compared with these. In a learning (29 patients) and a test phase (35 patients), a mean of 2.2 aberrancies/patient was detected, and only 1/63 patients (1.6%) had no consensus LAP(s). For the four centers without (extensive) MRD experience, clear improvement could be shown: in the learning phase, 39–63% of all consensus LAPs were missed, resulting in a median 30% of patients (range 21–33%) for whom no consensus LAP was reported; in the test phase, 27–40% missed consensus LAPs, resulting in a median 16% (range 7–18%) of ‘missed' patients. The quality of LAPs was extensively described. Immunophenotypic MRD assessment in its current setting needs extensive experience and should be limited to experienced centers.

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.

Background: The fluorescent probe Syto16 has been used successfully to measure P-glycoprotein (Pgp) function and, separately, early apoptosis and cell death. The present study was designed to evaluate whether the combined use of Syto16, the Pgp blocker PSC833, and 7-AAD allows simultaneous detection of all parameters, with emphasis on applications in acute myeloid leukemia (AML).Methods