Design and test of a 434 MHz multi-channel amplifier system for targeted hyperthermia applicators.

PURPOSE: For our head-and-neck hyperthermia (HT) applicator, an amplifier system with full amplitude and phase-control to deliver the radio-frequency signals, was not available. We therefore designed and tested a 433.92 MHz multi-channel amplifier system. SYSTEM DESCRIPTION: The design consists of a direct digital synthesizer (DDS) system that generates 12 phase-controlled coherent 433.92 MHz signals, which are amplified to maximum 200 W output per channel. Directional couplers are placed at the amplifiers to couple a small portion of both forward and reflected signals to gain-and-phase detectors. The power setting is applied with a resolution of 2 W and for the phase it is 0.1 degrees . The channels are sequentially sampled at 100 Hz per channel. METHODS: We tested the performance of the designed amplifier system by measuring the RF spectrum, power and phase accuracy, and by characterising the feedback control by using highly accurate power and phase meters. RESULTS: The spurious emission is less than 60 dBc and the first two harmonic frequencies are suppressed more than 45 dB. The measurement accuracy for the power (+/-5%) is valid for at least 20 days after calibration and for the phase (+/-5 degrees ) it is valid for at least 2 months. CONCLUSIONS: The amplifier system operates according to our design criteria to support targeted HT. It can be used for both our in-house developed superficial and head-and-neck HT applicators or any other HT applicator that works on the same frequency of 433.92 MHz.

MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia.

Outgrowth of minimal residual disease (MRD) in acute myeloid leukaemia (AML) is responsible for the occurrence of relapses. MRD can be quantified by immunophenotyping on a flow cytometer using the expression of leukaemia-associated phenotypes. MRD was monitored in follow-up samples taken from bone marrow (BM) of 72 patients after three different cycles of chemotherapy and from autologous peripheral blood stem cell (PBSC) products. The MRD% in BM after the first cycle (n=51), second cycle (n=52) and third cycle (n=30), as well as in PBSC products (n=39) strongly correlated with relapse-free survival. At a cutoff level of 1% after the first cycle and median cutoff levels of 0.14% after the second, 0.11% after the third cycle and 0.13% for PBSC products, the relative risk of relapse was a factor 6.1, 3.4, 7.2 and 5.7, respectively, higher for patients in the high MRD group. Also, absolute MRD cell number/ml was highly predictive of the clinical outcome. After the treatment has ended, an increase of MRD% predicted forthcoming relapses, with MRD assessment intervals of < or =3 months. In conclusion, MRD parameter assessment at different stages of disease is highly reliable in predicting survival and forthcoming relapses in AML.

A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia.

Minimal residual disease (MRD) cells are thought to be responsible for the persistence and relapse of acute myeloid leukemia (AML). Flow cytometric MRD detection by the establishment of a leukemia-associated phenotype (LAP) at diagnosis can be used in 80% of AML patients, allowing detection and functional characterization of MRD in follow-up bone marrow. One of the mechanisms contributing to inefficient chemotherapy is apoptosis resistance. Measuring apoptosis parameters in MRD cells will help to unravel the importance of apoptosis resistance in AML. We therefore developed a four-color flow cytometry method that enables establishment of apoptosis-related protein expression such as Bcl-2, Bcl-x(L), Mcl-1 and Bax at diagnosis and in MRD. Firstly, validation of this assay using Western blot analysis in five leukemia cell lines showed a significant correlation (R=0.70: P<0.0001). Secondly, the influence of the permeabilization procedure on LAP expression was investigated in 38 AML samples at diagnosis and in 42 MRD samples. Quantification of the frequency of LAP+ cells with and without permeabilization showed no significant differences (diagnosis: P= 0.57, follow-up: P= 0.43). The flow cytometric protocol thus enables analysis of apoptosis-related proteins at different stages of the disease, which will lead to a better understanding of the role of apoptosis resistance in the emergence of MRD in AML.

Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity in acute myeloid leukemia.

Relapse is common in acute myeloid leukemia (AML) due to persistence of residual leukemia cells: minimal residual disease (MRD). In 102 out of 127 patients (80%), cells at diagnosis displayed one or more leukemia-associated phenotypes (LAP), ie combinations of cell surface markers which are absent in normal cells and can thus be used to detect MRD at follow-up. Functional characterization of MRD cells for P-glycoprotein (Pgp) and multidrug resistance protein (MRP) activity is essential to investigate the role of these drug transport proteins in multidrug resistance in AML. A fluorescent probe assay using Syto16/PSC833 and calcein-AM/probenecid as substrate/modulator of the Pgp and MRP pump, respectively, and subsequent labeling of cells with monoclonal antibodies for LAP detection allowed simultaneous detection of LAP and Pgp or MRP activity. Validation of this assay is shown for 30 newly diagnosed AML and 11 MRD situations. In addition, no significant differences were found when comparing fresh and cryopreserved de novo AML for LAP expression (n = 43), Pgp (n = 30) and MRP (n = 24) function and for MRD samples for simultaneous LAP expression and Pgp/MRP activity (n = 10). This approach enables longitudinal and multicenter studies on the detection, quantification and functional characterisation of MRD cells.

High-dose melphalan with re-infusion of unprocessed, G-CSF-primed whole blood is effective and non-toxic therapy in multiple myeloma.

In order to shorten the pancytopenic period following high-dose melphalan 140 mg/m2 (HDM) treatment of multiple myeloma patients, we studied the effects of re-infusing granulocyte colony stimulating factor (G-CSF) [Filgrastim, Neupogen]-primed unprocessed whole blood. 30 patients with multiple myeloma were treated with HDM. One litre of blood after 5 or 6 days stimulation with G-CSF (10 micrograms/kg) was drawn, kept unprocessed for 1 day and re-infused 24 h after chemotherapy. Time to granulocyte recovery (> 0.5 x 10(9)/1) and platelet recovery (> 20 x 10(9)/1) were assessed as well as length of hospital stay, number of transfusions and antibiotic use. These 30 patients were compared with 20 historical control patients who were similarly treated but without stem cell support. The response rate was 75% (21/28) including a complete remission (CR) rate of 29% (8/28). Two early deaths due to Aspergillus pneumonia were observed. The median overall survival after HDM has not been reached after a median follow-up of 14 months. 10 patients showed progression at a median of 7 months. Currently, 23 patients are alive with a median follow-up time of 14 months. Haematological recovery was significantly faster in the study group as compared to the historical control group. The neutrophil count reached 0.5 x 10(9)/1 at a median of 14 days after infusion of 1 litre of unprocessed whole blood compared with 38 days in the historical control group. A platelet count of 20 x 10(9)/1 was reached at a median of 26 days compared with 36 days in the historical control group. Length of hospital stay decreased from a median of 43 to 18.5 days. The number of days with antibiotics was reduced from a median of 21 to 6 days. HDM is effective therapy for multiple myeloma. Toxicity of the regimen is considerably reduced by the use of G-CSF-stimulated unprocessed whole blood, an easy to perform and cheap technique to mobilise and collect stem cells.

Isolation and culture of human bone marrow endothelial cells.

Bone marrow endothelial cells are likely to play an important role in the homing of hematopoietic progenitor cells. In view of analyzing the interactions between endothelial cells and hematopoietic progenitor cells, we studied several methods of isolating endothelial cells from human bone marrow, including fluorescence activated cell sorting (FACS) and separation by immunomagnetic beads. FACS sorting gave the best results as contamination with other cells did not occur. After density-gradient centrifugation of bone marrow aspirates, the mononuclear cell (MNC) fraction was depleted for T cells, B cells, and myeloid cells by immunomagnetic separation. Further enrichment of endothelial cells was achieved by FACS sorting using BNH9 or S-Endo1 monoclonal antibodies (MAbs). These MAbs, in contrast to several other endothelial-cell reactive MAbs, were found to react highly specifically with sinus endothelial cells as tested by immunohistochemistry on bone marrow tissue sections and cell culture preparations and by double-colored FACS analysis on bone marrow MNCs (BMMNC). Sorted cells, which formed 0.05% of the MNC fraction, showed strong intracytoplasmic von Willebrand factor positivity. Ultrastructural analysis revealed cells with endothelial characteristics. Cells were cultured in fibronectin-coated, 24-well culture plates in endothelial-cell culture medium or long-term bone marrow culture medium. After 1 to 3 weeks of culture, a monolayer of spindle-shaped cells developed expressing endothelial cell antigens. Cells could be kept in culture for 4 to 6 weeks. In conclusion, the method described provides highly purified preparations of human bone marrow endothelium that may permit in vitro adhesion experiments with normal and leukemic hematopoietic progenitor cells.