Adjustment of the interface detector (location 71) to the absolute number of mononuclear cells in the peripheral blood: no improvement of the collection efficiency of the Fenwal CS3000 plus during progenitor cell harvests.

Improvement of the collection efficiency (CE) of the Fenwal CS3000 plus in collecting circulating progenitor cells (CPC) might diminish the number of leukapheresis procedures (LP) required to obtain the CPC required to assure engraftment. We analyzed whether adjustment of the optical setting (location 71,L71) to the number of MNC present in the peripheral blood could enhance the CE of the MNC. Thirty-five patients underwent 121 LP with an adjusted L71. We compared the results retrospectively with 26 LP performed with a fixed L71 (1:100) in 12 patients. The CPC were mobilized with chemotherapy followed by subcutaneous administration of granulocyte colony-stimulating factor (G-CSF) in both groups. Adjustment of the L71 did neither improve the CE of the MNC, the estimated CE of CD34+ cells nor diminished granulocyte contamination. For the total 121 LP with an adjusted L71 and for the total 26 LP with a fixed L71 the mean CE of MNC were, respectively, 44.6 +/- 18.3% and 46.4 +/- 14%. The mean granulocyte contamination, determined by manual white blood cell differentiation, was 1.7 +/- 2.3% for the adjusted L71 group and 2.3 +/- 3 for the fixed L71 group. There was no difference in the median number of LP required to obtain 3 x 10(6) CD34+ cells/kg between both groups. We found a weak significant correlation between WBC and pre-LP MNC count and the CE of MNC (r = 0.36, P = 0.012, resp.r = 0.33, P = 0.023), but no correlation between the CE of MNC and the estimated CE of CD34+ cells (r = 0.24, P = 0.113). In conclusion, adjustment of the L71 to the MNC did not improve the CE of MNC of the Fenwal CS3000. The lack of correlation between the CE and MNC and the estimated CE of CD34+ cells should be further explored.

Mobilisation of blood progenitor cells with ifosfamide and etoposide (VP-16) in combination with recombinant human G-CSF (Filgrastim) in patients with malignant lymphomas or solid tumours.

The mobilisation characteristics of ifosfamide and etoposide followed by Granulocyte Colony-Stimulating Factor fGCSF, filgrastim) were analysed in 17 patients with malignant lymphoma and 24 patients with solid tumours, with respect to the optimum time to harvest progenitor cells and to the yields of progenitor cells that could be achieved. In addition, we analysed patient characteristics which could influence the size of the progenitor cell harvest. Clinical parameters which were co-related with the size of the circulating progenitor cells (CPC) harvests were: the dose of G-CSF, dose of if osfamide, sex, age, diagnosis and extent of pretreatment. CPC were mobilised with 3 g/m2 (n = 11) or 4 g/m2 (n = 30) ifosfamide on day 1 and etoposide 100 mg/m2/day, on days 1-3 i.v., followed by daily s.c. injections with filgrastim 5 micrograms/kg (n = 26) or 10 micrograms/kg (n = 15) from day 4. The maximal progenitor cell harvest was achieved on either day 12 or day 13 after the start of the ifosfamide/etoposide course. The median number of leukaphereses necessary to harvest the target quantity of 3 x 10(6) CD34+ cells/kg body weight was 1 (range 1-9). Thirteen/41 (32%) of the patients did not achieve the target yield of 3 x 10(6) CD34+ cells/kg. By multivariate analysis, the dose of GCSF and prior irradiation were associated with the number of progenitor cells harvested, while all other parameters, induding the dose of if osfamide and number of previous chemotherapy courses, were not. Sixteen patients received two or more mobilisation courses. Despite the fact that the same mobilisation schedule was used, the progenitor cell yields after the first mobilisation course did not predict the results after the subsequent mobilisation courses, indicating that unknown transient factors may significantly influence the CPC yield.

Bioanalysis of the investigational anti-tumour drug 5,10-dideaza-5,6,7,8-tetrahydrofolic acid by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 278 nm is presented for the determination of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid in plasma. Sample pretreatment was achieved by using cation-exchange solid-phase extraction columns with methotrexate as internal standard. Chromatographic separation was based on ion-pair HPLC with 1-octanesulphonic acid as the ion-pairing compound. The detection limit was 10 ng/ml using an 500-microliters sample volume. The assay was linear from the detection limit up to 5000 ng/ml with good reproducibility. The applicability of the assay was demonstrated in a study in the rat.

Biodetermination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan, a metabolite of vintriptol, by high-performance liquid chromatography with fluorescence detection.

The determination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan (vintriptol acid, VtrpA), a metabolite of the investigational semi-synthetic vinca alkaloid vintriptol [N-(deacetyl-O-4-vinblastoyl-23)-L-ethyltryptophan, VtrpE], in plasma and urine samples is described. Sample pretreatment included liquid-liquid extraction of the buffered (pH 5.0) biological samples with chloroform-2-propanol (95:5, v/v). The analyses were performed by ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection. The assay was applied to the analysis of samples from cancer patients who had been treated with VtrpE in a phase I clinical study. VtrpA was found to be a principal metabolite of VtrpE with up to 1.2% of the administered dose excreted in the urine.

Bio-analysis of vinorelbine by high-performance liquid chromatography with fluorescence detection.

A simple and selective procedure for the determination of vinorelbine, a new semi-synthetic vinca alkaloid, is presented. The method is based on ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection, combined with liquid-liquid extraction using diethyl ether for sample clean-up. The absence of endogenous interferences and the excellent chromatographic behaviour of vinca alkaloids provides accurate results even at low concentrations. The limit of determination in plasma is 1.5 micrograms/l (500-microliters sample). Reproducible recoveries in urine were obtained if 10-50 microliters of sample were processed supplemented with 500 microliters of blank plasma.

High-performance liquid chromatographic determination of vinblastine, 4-O-deacetylvinblastine and the potential metabolite 4-O-deacetylvinblastine-3-oic acid in biological fluids.

Procedures for the determination of vinblastine (VBL), 4-O-deacetylvinblastine (DVBL) and 4-O-deacetylvinblastine-3-oic acid (DVBLA) in biological samples using high-performance liquid chromatography (HPLC) combined with selective sample clean-up are presented. VBL and DVBL were determined in plasma and urine using ion-exchange normal-phase HPLC with fluorescence detection. The limit of detection was 1 microgram/l for both compounds using a 500-microliter sample. Successful chromatographic analyses of DVBLA were achieved by using a glass column packed with 5-microns Hypersil ODS and acetonitrile-0.05 M phosphate buffer (pH 2.7) (23:77, v/v). Positive identification was supported by the use of diode-array detection. The limit of detection (at 270 nm) was 10 micrograms/l using 1-ml samples.

Cerebrospinal fluid tumour markers in patients treated for meningeal malignancy.

The results of cerebrospinal fluid (CSF) biochemical markers were compared with conventional CSF cytology in patients treated for leptomeningeal metastases from extra cranial malignancies. For lumbar CSF, before treatment, no statistically significant difference of the probabilities of being positive was found between CSF cytology and a classification by linear discriminant analysis, based on patient's age, of beta-glucuronidase and beta 2-microglobulin. During treatment, classification by linear discriminant analysis was found more often positive than cytology. Possible mechanisms for this difference are discussed. For ventricular CSF a correlation was found between CSF cytology and beta-glucuronidase for solid tumours, and between CSF cytology and beta 2-microglobulin for haematological malignancies. Reference values for ventricular protein, CEA beta-glucuronidase and beta 2-microglobulin were obtained for cytological negative samples.

Analytical methods for the determination of vinca alkaloids in biological specimens: a survey of the literature.

The bio-analysis and pharmacokinetics of vinca alkaloids have been the subject of many investigations. In most cases radiolabelled compounds have been used for quantification purposes. Although this method lacks selectivity, it has provided valuable information on tissue distribution of unchanged drug and metabolites in an early stage of clinical and preclinical investigations. During the last few years, methods based on high-performance liquid chromatography have been presented. This paper reviews the methods described in the literature for the bio-analysis of vinca alkaloids, supplemented with our own experience in this field. Special attention is paid to the problems that may arise during the analytical processes.

High-performance liquid chromatographic bio-analysis and preliminary pharmacokinetics of the experimental antitumour drug vintriptol.

A high-performance liquid chromatographic procedure, including sample pretreatment, is presented for the analysis of the experimental antitumour drug vintriptol in plasma. The sample pretreatment involved liquid-liquid extraction of the buffered (pH 3) sample with chloroform. Vinblastine was used as internal standard. Separation was achieved on a Hypersil ODS (5 microns) column with a mobile phase of acetonitrile-phosphate buffer. Electrochemical detection (at +0.70 V) was used, giving a detection limit of 2 micrograms/l. The applicability of the assay was demonstrated in a pharmacokinetic study with eight cancer patients who received 45 or 50 mg/m2 vintriptol in a phase I study. A three-compartment model was used to fit the plasma concentration-time curves. Pharmacokinetic parameters are presented.

High-performance liquid chromatographic method for the analysis of 10-ethyl-10-deaza-aminopterin and metabolites in plasma.

10-Ethyl-10-deaza-aminopterin (10-EdAM) is a novel folic acid antimetabolite currently being tested in phase II clinical trials. We have developed an isocratic high-performance liquid chromatographic method for the quantification of 10-EdAM and metabolites in plasma. Solid-phase extraction was used for sample clean-up. Adequate accuracy was obtained without the use of an internal standard. Fluorometric detection with excitation at 243 nm and emission at 488 nm was used for accurate quantification of samples containing small amounts of drug or metabolites (2.0-4.0 nM, depending on the compound). Ultraviolet detection at 350 nm was only applicable for the analysis of plasma concentrations of 10-EdAM exceeding 50 nM. The usefulness of the assay was demonstrated by the results obtained in a pharmacokinetic study. The assay could separate the parent compound from seven identified and two unknown products.

Serial lumbar and ventricular cerebrospinal fluid biochemical marker measurements in patients with leptomeningeal metastases from solid and hematological tumors.

This study presents results of investigations of lumbar and ventricular cerebrospinal fluid (CSF) biochemical markers (Beta-glucuronidase (B-gluc), Beta-2-microglobulin (B2-m), and carcinoembrionic antigen (CEA] in 28 patients with five different tumor types with leptomeningeal metastasis diagnosed by CSF cytology and/or autopsy. All received methotrexate and radiotherapy at some stage. Decadron or other symptomatic treatments were not used. Measurements of the concentrations of B-gluc, B2-m and CEA were evaluated with the aim of correlating the results of these measurements to site of disease, of monitoring response and early relapse of leptomeningeal disease, and of establishing the duration of survival. In almost all our patients the results of ventricular CSF B-gluc, B2-m and CEA measurements were lower than those obtained from lumbar CSF. The markers did not correlate with site of disease or CSF cytology. A clear relationship was found between pretreatment lumbar CSF B2-m and CEA levels, response to therapy and survival. The markers are also useful for monitoring response. The findings of this study indicate that B2-m and CEA levels have a prognostic value with regard to response to therapy and time of survival.

Stable and sensitive method for the simultaneous determination of N5-methyltetrahydrofolate, leucovorin, methotrexate and 7-hydroxymethotrexate in biological fluids.

A high-performance liquid chromatographic method for the determination of N5-methyltetrahydrofolic acid, leucovorin, methotrexate and 7-hydroxymethotrexate in plasma and liquor samples is presented. Gradient elution is used to increase the sensitivity. Four sample preparation methods were compared with respect to the stability of the injectable sample. Samples can be pretreated with a simple deproteinization method. For enhanced selectivity a solid-phase extraction procedure is described.

Tamoxifen, serum lipoproteins and cardiovascular risk.

The influence of tamoxifen on plasma lipids and lipoproteins was monitored in 46 postmenopausal and 8 premenopausal women treated for advanced breast cancer up till 6 months. Total cholesterol (total-C) did not significantly change. However, high density lipoprotein cholesterol (HDL-C) and the HDL-C/total-C ratio rose significantly. Low density lipoprotein cholesterol was significantly decreased. Triglycerides and free fatty acids did not change markedly. The concomitant rise of sex hormone binding globulin and thyroxine binding globulin indicates that the increase of HDL-C with prolonged use of tamoxifen is compatible with an intrinsic oestrogenic effect of tamoxifen on the liver. The increased HDL-C/total-C ratio lends no support to the concern that long-term administration of this anti-oestrogenic drug might lead to an increased cardiovascular risk.

Tumour markers in the cerebrospinal fluid of patients with central nervous system metastases from extracranial malignancies.

We have assessed the diagnostic value of the determination of cerebrospinal fluid lactate dehydrogenase, carcinoembryonic antigen, beta 2-microglobulin, beta-glucuronidase and total protein, using linear discriminant analysis, in detecting central nervous system metastases from extracranial malignancies. We conclude that, using these tests, it is impossible to differentiate between control individuals and patients with brain or epidural metastases. Leptomeningeal dissemination from either solid tumours or non-Hodgkin lymphoma could be differentiated from control individuals and patients with brain or epidural metastases. In this differentiation it is essential that bacterial, fungal or tuberculous meningitis be excluded from the differential diagnosis by other diagnostic procedures. The combination of beta-glucuronidase and beta 2-microglobulin provides almost the same diagnostic information as the combination of all parameters.

Improved method for the quantification of aminoglutethimide in urine samples.

Due to an unknown mechanism aminoglutethimide (AG) in urine samples is partly present in a form in which it is not detected. Though most evident if samples had been stored at -20 degrees C (about 60% trapped), it also occurred in fresh urine samples (about 20% trapped). Heating the urine sample for 15 min at 100 degrees C liberated all AG. This phenomenon has led to the underestimation of the excretion of unchanged AG in previous publications. This study shows that AG was excreted unchanged for 47.5 +/- 7.3% (mean +/- SD) in patients received 2 X 125 mg or 2 X 250 mg AG. Direct measurement of hydroxylaminoglutethimide (HxAG) was found difficult, due to its instability with respect to oxidation. We found azoxyG to be a stable oxidation product derived from HxAG. The concentration of azoxyG formed was proportional to the amount of HxAG converted. HxAG seemed subjected to the same phenomenon as AG. A conversion of HxAG into an unknown compound which seemed to occur very easily and which was even accelerated by heating, made it impossible to accurately measure the concentration of HxAG.

Sensitivity and specificity of single and combined tumour markers in the diagnosis of leptomeningeal metastasis from breast cancer.

The clinical efficacy of four laboratory tests in detecting leptomeningeal metastases in 57 patients with breast carcinoma was assessed. The sensitivity and specificity of beta-glucuronidase, beta 2-microglobulin, carcinoembryonic antigen and lactate dehydrogenase in cerebrospinal fluid were determined. As a single test beta-glucuronidase was the most sensitive (93%) and specific (93%) for discriminating between leptomeningeal metastases and other CNS metastases from breast cancer. Lactate dehydrogenase was the next most useful marker. Both beta 2-microglobulin and carcinoembryonic antigen had a sensitivity of 60%. More specific results were achieved by combining beta-glucuronidase and lactate dehydrogenase. CSF beta-glucuronidase may be useful by itself and in combination with lactate dehydrogenase in the detection of leptomeningeal metastases from breast carcinoma.

Cerebrospinal fluid beta 2-microglobulin: a study in controls and patients with metastatic and non-metastatic neurological diseases.

We studied cerebrospinal fluid Beta 2-microglobulin (CSF B2-m) in 197 patients with a variety of neurological diseases to evaluate the usefulness of B2-m in the detection of meningeal dissemination of malignancy. In the control group we found a relationship between CSF log B2-m and age (P less than 10(-4)). Age standardized reference values were established as 0.65-2.2 mg/l. The results show that CSF B2-m was elevated in leptomeningeal metastases from solid and haematological tumors. We observed slight elevations of CSF B2-m in epidural and parenchyma metastases from solid tumors. Our study shows that B2-m in CSF is a sensitive marker for meningeal metastases especially from hemopoietic tumors.

Cerebrospinal fluid carcinoembryonic antigen in patients with metastatic and nonmetastatic neurological diseases.

Cerebrospinal fluid (CSF) carcinoembryonic antigen (CEA) values were determined in 200 patients suffering from various neurological diseases. We found no relationship between CEA levels and age or sex. A positive test was defined as an upper limit of at least 4.0 ng/mL of CEA. We found raised CSF CEA levels in patients with leptomeningeal spread from carcinoma, but not in patients with leptomeningeal metastases from lymphoma. We also found high values of CSF CEA in three of 21 patients with epidural metastases and in two of 28 patients with cerebral metastases from solid tumors. Comparison was made with the CSF levels of total protein, glucose, and lactate dehydrogenase. The sensitivity of the CSF CEA determination in patients for the presence of leptomeningeal involvement of cancer is 31% and the specificity is 90%.