Summary:In order to optimize peripheral blood stem cell (PBSC) collection for transplantation, absolute CD34 counts are necessary to determine the exact time-point for sufficient leukapheresis. In an effort to establish and to validate a rapid and reproducible assay for PBSC enumeration, different recommendations for selection of monoclonal antibodies, lysis techniques, analysis parameters and gating strategies were developed. In this methodical study, two gating strategies for PBSC enumeration were compared, in order to validate the accuracy of PBSC counting in peripheral blood and apheresis products. Gating strategy I was performed using volumetric flow cytometry and reference beads whereas gating strategy II was done according to the ISHAGE guidelines. The highly standardized volumetric assay seems to be superior to the more 'expert-reliant' ISH-AGE procedure requiring more 'manual work' by the cytometrist. Keywords: progenitor cells; CD34; flow cytometry; stem cell transplantation High-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) is increasingly used for treatment of different hematological malignancies because remission induction seems to be superior in comparison to conventional treatment. 1,2 PBSC have to a large extent replaced bone marrow as source of marrow repopulating cells and are able to reconstitute hematopoiesis in a shorter time after myeloablative chemotherapy. 3,4 A number of methods are available for mobilization of PBSC from bone marrow into peripheral blood. 5,6 Since the identification and characterization of the CD34 antigen as a marker for self-replicating PBSC, flow cytometric analysis has been used for PBSC enumeration in peripheral blood and apheresis products. 7,8 Today, the decision to commence leukapheresis after PBSC mobilization depends on flow cytometric enumeration of PBSC in peripheral blood and the total number of PBSC in leukaph- eresis products often determines the time-point of a future PBSC collection. Exact quantification of PBSC in apheresis products is required to assess the quality of harvest for engraftment after PBSCT. 9 Therefore, it is essential to establish a reliable flow cytometric assay for the enumeration of PBSC.Although CFU-GM numbers correlate with the absolute number of PBSC in apheresis products, culture assays have several disadvantages as compared to flow cytometry. [10][11][12] Cell growth is subjected to the numerous inconsistencies of culture assays and results are not available until 1 to 3 weeks after collection. 13 Flow cytometric assays can be performed in less than 3 h and results are available directly after analysis. Therefore, flow cytometric procedures appear to be a more sensitive, reliable and rapid alternative to colony assays. 10,14 Different flow cytometric methods to enumerate PBSC in peripheral blood and apheresis products have been described. However, the lack of a standardized method has led to widely inconsistent data. 15,16 To validate the precision of different gating strategies in PBSC enumeration, ...

The detection of dysplastic features of hematopoiesis in de novo acute myeloid leukemia (AML) by light microscopy is defined as AML with trilineage myelodysplasia (AML/TLMD). The prognostic relevance of these dysplastic features for patients with de novo AML remains unclear. In order to evaluate the role of dysplasia in de novo AML, bone marrow aspirates from 69 patients were analyzed prospectively and investigated separately for erythropoiesis, granulopoiesis and megakaryopoiesis by three independent investigators. The overall complete remission (CR) rate was 48.8% and partial remission (PR) or nonresponders constituted 52.2% of the patients investigated. The median overall survival time was 5 months with a disease-free interval of 3.5 months for all patients. Dysgranulopoiesis (DysG) was observed in 30.4%, dysmegakaryopoiesis (DysM) in 50.7%, and dyserythropoiesis (DysE) in 43.5%. Of all patients, 26.0% showed trilineage dysplastic features and were thus classified as AML/TLMD. A significantly worse prognosis (Kaplan-Meyer plot, Student's t-test) was calculated for those patients with detection of only DysG (p = 0.002), DysM (p = 0.02), DysE (p = 0.04) as compared with patients without any dysplastic signs. An unfavorable karyotype was correlated with patients showing DysG (P = 0.02) and DysM (P = 0.04). For these patients with an unfavorable karyotype, the occurrence of any dysplastic features had no additional prognostic impact. Dysplastic features (DysG, DysM, DysE) seem to be an important prognostic factor in de novo AML correlating with short overall survival. DysG and DysM correlated well with the appearance of unfavorable chromosomal abnormalities. It may be reasonable to assume that patients with dysplastic features should be considered for more aggressive treatment schedules at the time of diagnosis.

In general, the mobilization of peripheral blood progenitor cells (PBPC) in multiple myeloma (MM) patients is poor and is achieved in most cases by combined cyclophosphamide and G-CSF. This study was performed to examine the efficacy of combined ifosfamide/epirubicine and G-CSF for PBPC mobilization and purging. Sixteen patients suffering from multiple myeloma in stage II/A and III/A according to Durie and Salmon underwent chemotherapy consisting of a total of three cycles of ifosfamide (3 g/m(2) on days 1 and 2 and epirubicine 80 mg/m(2) on day 1) and G-CSF (10 or 20 microg/kg body weight (BW) daily until harvesting). PBPC harvesting was performed after the first and third cycle of chemotherapy. The median number of PBPC after the first cycle of chemotherapy was 7.79 x 10(6) CD34+ cells/kg BW (ranging from 0.94-26.36 x 10(6)) and 6.38 x 10(6) CD34+ cells/kg BW (ranging from 0.79-29.31 x 10(6)) after the third cycle of chemotherapy. Clinical re-evaluation after three cycles of chemotherapy showed 13 (81 per cent) patients in partial remission (PR), two (12 per cent) in complete remission (CR) and one (6.25 per cent) in stable disease (SD). No major side-effects were observed, six patients developed hematological toxicity stage IV WHO for a median of 3.9 days but no serious infection episodes occurred. Combined ifosfamide/epirubicin and standard G-CSF is able to mobilize sufficient PBPC without serious side-effects for patients with MM and for purging procedures resulting in a high proportion of complete remissions after tandem high-dose melphalan chemotherapy.

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