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, ...
Background: As part of the attempt to optimize the treatment of high-grade non-Hodgkin’s lymphoma (NHL) in advanced stages, a pilot study was designed to investigate the feasibility and efficacy of the BMMP protocol (bendamustine, methotrexate, mitoxantrone, prednisolone). Material and Methods: Between October 1994 and September 1996,23 elderly patients (median age 60.1 years; range: 20-80 years) with nonresponding or relapsed high-grade NHL were treated according to the BMMP protocol. Response and toxicity were determined on the basis of the WHO criteria. Results: Complete remission was achieved in 3 patients and partial remission in 8 patients, with an overall response rate of 47.8%. Disease remained stable in 1 patient and progressed in 11 patients. Side effects mostly oberserved in a total of 72 cycles administered were leukocytopenia (WHO grade III/IV) and thrombocytopenia (WHO grade III/IV), no patient died related to treatment-associated serious adverse events. Conclusions: The BMMP protocol is able to induce a remarkable rate of remissions in elderly patients with advanced NHL, who are not eligible for high-dose chemotherapy. Further randomized studies are needed and now unter way to compare the BMMP protocol with standard salvage chemotherpy regimens.
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.
We report on a 30-year-old patient with blast crisis of a chronic myelogenous leukemia (CML) that shows immunophenotypic features similar to those of the myeloid/natural killer (NK) cell precursor leukemia previously described. Expression of CD13/CD33/CD65 as well as MPO+/LF- blasts was classified as a myelogenous blast crisis of a CML. In addition, the blasts were positive for CD7/CD56. Other lymphoid markers were not expressed. Cytogenetic and molecular cytogenetic examinations showed two Philadelphia (Ph-1) chromosomes and a trisomy 8. Similar to expression of the myeloid/NK cell precursor phenotype in acute myelogenous leukemia (AML), it is possible to exhibit this phenotype in Ph-1-positive CML. Only one case report of myeloid/NK precursor phenotype blast crisis of CML was found in the literature. Therefore, it is not clear whether this phenotype is a distinct biologic and clinical disease entity of CML, as is the case in the respective AML phenotype.
Background:The goal of this study was to evaluate a self-learning algorithm for the computer classification of information extracted from flow cytometric immunophenotype list mode files from high-grade non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). Materials and Methods: Bone marrow aspirates (BMA) were obtained from untreated NHL (n ϭ 51), HD (n ϭ 9), or MM (n ϭ 13) patients. Bone marrow aspirates were not infiltrated in NHL and HD patients as confirmed by thorough histologic and cytologic investigation; however, MM patients showed an infiltration rate Ͼ50% by malignant myeloma cells. Peripheral blood leukocyte (PBL) samples were taken from age-matched healthy volunteers (n ϭ 44) as easily available control material. A second control group of 15 healthy volunteers, from whom BMA and PBL samples were available, allowed us to differentiate whether the observed classification results on malignant samples were due to the malignant process or simply to the inherent differences between BMA and PBL. Bone marrow aspirates and PBL were analyzed by the same immunophenotyping antibody panel (CD45/14/20, CD4/ 8/3, kappa/CD19/5, lambda/CD19/5). The acquired list mode data files were analyzed and classified by the selflearning triple matrix classification algorithms CLASSIF1 following a priori separation of the data into a learning set and unknown test set. After completion of the learning phase, known patient samples were reclassified and unknown samples prospectively classified by the algorithm.
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