Analysis of abnormal clones by the fluorescent aerolysin method in paroxysmal nocturnal haemoglobinuria and other marrow disorders.

INTRODUCTION: Flow cytometry is the most sensitive and specific diagnostic modality for the assessment of clone size in paroxysmal nocturnal haemoglobinuria (PNH) and other bone marrow failure states. In this study, we attempt to distinguish PNH from aplastic anaemia (AA) and myelodysplastic syndromes (MDS) associated with PNH clones at diagnosis by clone size, clinical and laboratory features. METHODS: A total of 29 samples included 19 PNH cases and 10 AA/MDS cases with PNH clones. Flow cytometry was performed using fluorescent aerolysin (FLAER)-based assay and comparison of clinical features, laboratory parameters and PNH clone size was carried out at diagnosis. RESULTS: The PNH clone size on granulocytes varied from 0.4% to 99.2% and correlated with the clone size on monocytes (r = 0.966; P < 0.001). Paroxysmal nocturnal haemoglobinuria clone size on granulocytes (median = 34.6%) and monocytes (median = 49.9%) was always larger than erythrocytes (median = 10.9%). The median clone size in PNH (median granulocytes = 74.9%, monocytes = 71.8%) was significantly greater than in AA/MDS associated with PNH clone (median granulocytes = 2.9%, monocytes = 6%). In PNH patients, a significant negative correlation was seen between PNH clone on monocytes and the haemoglobin concentration. CONCLUSION: In our small study using the FLAER method, the clone size was >70% in majority of PNH cases. In other marrow disorders like AA/MDS, the clone size was usually <10%.

Stochastic model-based processing for detection of small targets in non-Gaussian natural imagery.

Stochastic background models incorporating spatial correlations can be used to enhance the detection of targets in natural terrain imagery, but are generally difficult to apply when the statistics are non-Gaussian. Chapple and Bertilone (see Opt. Commun., vol.150, p.71-76, 1998) proposed a simple stochastic model for images of natural backgrounds based on the pointwise nonlinear transformation of Gaussian random fields, and demonstrated its effectiveness and computational efficiency in modeling the textures found in natural terrain imagery acquired from airborne IR sensors. In this paper, we show how this model can be used to design algorithms that detect small targets (up to several pixels in size) in natural imagery by identifying anomalous regions of the image where the statistics differ significantly from the rest of the background. All of the model-based algorithms described here involve nonlinear spatial processing prior to the final decision threshold. Monte Carlo testing reveals that the model-based algorithms generally perform better than both the adaptive threshold filter and the generalized matched filter for detecting low-contrast targets, despite the fact that they do not require the target statistics needed for constructing the matched filter.

Repetitive high-dose therapy with cyclophosphamide, thiotepa and docetaxel with peripheral blood progenitor cell and filgrastim support for metastatic and locally advanced breast cancer: results of a phase I study.

This phase I study was designed to determine the optimal dosages of a novel repetitive high-dose therapy regimen for patients with metastatic breast cancer (MBC). The planned treatment was three cycles of high-dose cyclophosphamide, thiotepa and docetaxel delivered every 35 days with progressive dose-escalation in successive cohorts. Each cycle was supported by peripheral blood progenitor cells (PBPC) and filgrastim. Eighteen patients were entered into this trial. Of the planned 54 treatment cycles, 44 were delivered and 11 patients completed all three cycles. The dose-limiting toxicities were interstitial pneumonitis and mucositis with moderately severe diarrhea (n = 3) and rash (n = 3). There were no treatment-related deaths. Of the 17 patients with evaluable disease, 16 patients responded with six patients achieving a complete remission and an additional four patients achieving no detectable disease (negative restaging including PET scan) but a persistently abnormal bone scan. At a median follow-up of 12 months, median progression-free survival was 11 months with the median overall survival not reached. The recommended doses for phase II/III studies are cyclophosphamide (4 g/m2), thiotepa (300 mg/m2) and docetaxel (100 mg/m2).

Docetaxel effectively mobilizes peripheral blood CD34+ cells.

We prospectively evaluated docetaxel (100 mg/m2) with G-CSF (10 microg/kg S.C., daily) for mobilization efficiency in 26 patients with breast cancer. The minimum target yield was >4.5 x 10(6) CD34+ cells/kg (optimum = 9 x 10(6)/kg), sufficient to support the subsequent three cycles of high-dose therapy (HDT). The peak days for peripheral blood (PB) CD34+ cells were day 8 and day 9. Seven collections began on day 7, 16 on day 8 and three on day 9. The median peripheral blood progenitor cell (PBPC) CD34+ cell content ranged from 1.2 to 5.9 x 10(6)/kg per day during days 7 to 11 with a median CD34+ content of the total 72 PBPC collections of 3.4 x 10(6)/kg (0.07-15.6). Fifteen patients obtained a PBPC collection exceeding 5 x 10(6)/kg on a single day of collection. Following a median 3 days collection for each patient (range 2-4), the median total CD34+ for all individual sets of collections was 9.7 x 10(6)/kg (range 1.0-28.4). We were able to achieve the minimum CD34+ cell target yield in 22 of 26 patients with one cycle of mobilisation chemotherapy and in two of these patients a second collection yielded sufficient cells. Twenty-two patients have subsequently received repetitive HDT and PBPC transplantation with 57 cycles of HDT having been delivered. For all 57 cycles, the median time to absolute neutrophil count (ANC) >0.5 x 10(9)/l and 1.0 x 10(9)/l was 10 days (range 8-22) and 11 days (range 8-23), respectively. The median time to platelets greater than 20 x 10(9)/l, 50 x 10(9)/l and 100 x 10(9)/l was 13 days (range 11-23), 17 days (range 12-53) and 23 days (range 18-70), respectively. We conclude that docetaxel with G-CSF effectively mobilises PBPCs with apheresis needing to be commenced approximately 8 days after docetaxel administration.

Comparison of three methods of CD34+ cell enumeration in peripheral blood: dual-platform ISHAGE protocol versus single-platform, versus microvolume fluorimetry.

BACKGROUND: Quantitation of peripheral blood (PB) CD34(+) cells is now an established method for timing PBPC harvesting. Recent refinements to the dual-platform ISHAGE gating strategy for CD34(+) cells has seen the introduction of microbeads to enable absolute counting of cells on a single instrument platform. This eliminates the need for total WBCC performed on an automated hematology analyzer and potentially increases the analytical precision of the methodology. At the same time, alternative methods for CD34(+) cell enumeration have started to emerge, notably microvolume fluorimetry, which forms the basis of the fully-automated STELLer CD34 method using the Imagn 2000. METHODS: We performed a three-way evaluation of these methods. Sixty-eight samples of PB from 42 patients undergoing PBPC mobilization were analyzed by all three methods and correlations between all three calculated. The two-platform ISHAGE method was used as the reference method. RESULTS: Precision and linearity of the single-platform and STELLer CD34 assays were excellent. Correlation with the dual-platform reference method was also excellent (single-platform method slope = 1.03, intercept = -0.03 and R(2) = 0.9325, STELLer CD34 assay slope = 0.827, intercept = 4.27, R(2) =0.8215). Bias, determined by Bland-Altman analysis, was 1.16 and -1.62 for single platform and STELLer CD34 assay respectively. CONCLUSION: The three methods of CD34(+) cell enumeration gave equivalent results. The single-platform methodology negated the need for a separate white cell analyzer, while the STELLer CD34 methodology was technically the simplest.

Repetitive high-dose therapy with ifosfamide, thiotepa and paclitaxel with peripheral blood progenitor cell and filgrastim support for metastatic and locally advanced breast cancer: results of a phase I study.

BACKGROUND: This phase I study was designed to determine the optimal dosages of a novel repetitive high-dose therapy regimen for patients with metastatic breast cancer (MBC). PATIENTS AND METHODS: The planned treatment was three cycles of high-dose ifosfamide, thiotepa and conventional-dose paclitaxel delivered every 28 days with progressive dose-escalation in successive cohorts. Each cycle was supported by peripheral blood progenitor cells (PBPC) and filgrastim. RESULTS: Twenty-three patients were entered into this trial. Of the planned 69 treatment cycles, 59 were delivered and fifteen patients completed all three cycles. The dose-limiting toxicities were renal tubular acidosis, encephalopathy, mucositis and enterocolitis. There was one treatment-related hemorrhagic death. CONCLUSIONS: The recommended doses for phase II or III studies are ifosfamide (10 g/m2), thiotepa (350 mg/m2) and paclitaxel (175 mg/m2).

Comparison of COBE Spectra software version 4.7 PBSC and version 6.0 auto PBSC program.

Until recently, the collection of peripheral blood progenitor cells (PBPC) has been semi-automated by using the COBE Spectra, with the operator manually maintaining the position of the white cells being collected. The COBE Spectra Version 6.0 apheresis device offers the user an automated program for the collection of PBPC. In this study, we compared the new software Version 6.0 to that of Version 4.7. Patients (n = 46) undergoing PBPC collection were allocated to cell processing with either Version 4.7 (n = 24) or Version 6.0 (n = 22). The CD34+ cell count, mononuclear cell (MNC) count, white cell count (WCC), hemoglobin (Hb), and platelet content in the autograft product by using the two versions were compared. We divided the analysis into three subsets according to peripheral blood (PB) CD34 content: <10x10(6)/L, 10-50x10(6)/L and >50x10(6)/L. Analysis of the three subsets showed no statistical difference between results obtained when the starting PB CD34+ cell count was 10-50x10(6)/L (P=0.08) or >50x10(6)/L (P=0.4065). At lower starting PB CD34+ cell counts of <10x10(6)/L, Version 4.7 was superior (P=0.0167). However, autograft platelet contamination of the autograft was significantly higher using Version 4.7 (P=<0.0001).

Ifosfamide in combination with paclitaxel or doxorubicin: regimens which effectively mobilize peripheral blood progenitor cells while demonstrating anti-tumor activity in patients with metastatic breast cancer.

For patients with metastatic breast cancer (MBC) who undergo high-dose therapy with autologous peripheral blood progenitor cell (PBPC) transplantation, an important prerequisite is a mobilization regimen that efficiently mobilizes PBPCs while producing an effective anti-tumor effect. We prospectively evaluated ifosfamide-based chemotherapy for mobilization efficiency, toxicity and disease response in 37 patients. Patients received two cycles of the ifosfamide-based regimen; ifosfamide (5 g/m2 with conventional-dose cycle and 6 g/m2 with mobilization cycle) with either 50 mg/m2 doxorubicin (if limited prior anthracycline and/or progression more than 12 months after an anthracycline-based regimen) or 175 mg/m2 paclitaxel. For the mobilization cycle, all patients received additional G-CSF (10 microg/kg SC, daily) commencing 24 h after completion of chemotherapy. The target yield was >6x10(6) CD34+ cells/kg, sufficient to support the subsequent three cycles of high-dose therapy. The mobilization therapy was well tolerated and the peak days for peripheral blood (PB) CD34+ cells were days 10-13 with no significant differences in the PB CD34+ cells mobilization kinetics between the ifosfamide-doxorubicin vs. ifosfamide-paclitaxel regimens. The median PBPC CD34+ cell content ranged from 2.9 to 4.0x10(6)/kg per day during days 9-14. After a median of 3 (range 1-5) collection days, the median total CD34+ cell, CFU-GM and MNC for all 44 individual sets of collections was 9.2x10(6)/kg (range 0.16-54.9), 37x10(4)/kg (range 5.7-247) and 7.3x10(8)/kg (range 2.1-26.1), respectively. The PBPC target yield was achieved in 35 of the 37 patients. The overall response rate for the 31 evaluable patients was 68% with 10% having progressive disease. Thirty-three patients have subsequently received high-dose therapy consisting of three planned cycles of high-dose ifosfamide, thiotepa and paclitaxel with each cycle supported with PBPCs. Rapid neutrophil and platelet recovery has been observed. Ifosfamide with G-CSF in combination with doxorubicin or paclitaxel achieves effective mobilization of PBPC and anti-tumor activity with minimal toxicity.

Peripheral blood CD34+ cell count reliably predicts autograft yield.

A reliable measure to predict peripheral blood progenitor cell (PBPC) autograft CD34+ cell content is required to optimize the timing of PBPC collection. We prospectively examined the peripheral blood (PB) CD34+ cell count in 59 consecutive patients with various malignancies and analyzed the correlation between the PB CD34+ cell count and various parameters in the PBPC autograft. Two hundred and thirty-five collections were performed with a median of 4.0 collections per patient (range, 2-10). The median PB CD34+ cell count at the time of collection was 39 x 10(6)/1 (range, 0.0-285.6). The PBPC autograft parameters measured were the CD34+ cell, colony-forming unit granulocyte-macrophage (CFU-GM) and mononuclear cell (MNC) content. There was a strong linear correlation between PB CD34+ cells/l and autograft CD34+ cells/kg (r = 0.8477). The correlation with CFU-GM/kg (r = 0.5512) was weaker. There was no correlation between autograft CD34+ cells/kg and PB WBC (r= 0.0684), PB MNC (r = 0.1518) or PB platelet count (r = 0.2010). At our institution we aim to obtain a minimum of 0.5 x 10(6) CD34+ cells/kg with each day of collection. We demonstrate that such a collection can be reliably obtained if the PB CD34+ cell count exceeds 5.0 x 10(6)/l.

Spatial statistics of natural-terrain imagery. II. Oblique visible backgrounds and stochastic simulation.

We report on the statistics and simulation of oblique airborne imagery of natural forest in the visible wave band. Statistical correlations in the imagery are characterized by a sharp, nearly exponential, central peak and a correlation tail that falls away much more slowly than exponentially. The histogram is approximately bell shaped in appearance, but the data set fails a rigorous statistical test of the Gaussian hypothesis. Nevertheless, we find that stochastic simulations of Gaussian random fields using the experimental correlation data match the real imagery reasonably well.

Infection of cultured human type II pneumonocytes with certain respiratory viruses.

Human type II alveolar pneumonocytes were grown either as monolayers derived from a clone or as organotypic cultures of fetal lung. The type II cells in these cultures retained in their cytoplasm multilamellar bodies which were morphologically identical to similar organelles present in type II cells of intact human fetal and adult lung. A number of respiratory viruses infected the cells and produced a cytopathic effect in the cultures. Viral components were also detected in some of the infected cells. Adenovirus type 3, human coronavirus 229E, and rhinovirus types 2 and 9 produced new infectious virus, but influenza A and parainfluenza 3 virus apparently did not.

Clones of cells from a human embryo lung: their growth and susceptibility to respiratory viruses.

Colonies of cells were obtained from human fetal lung tissue and exposed to recently isolated respiratory viruses. There was a considerable variation in the number of rounded cells found in different colonies exposed to rhinovirus types 2 and 9 (RV2 and 9), human coronavirus 229E (HCV), adenovirus type 3 (Ad3) and respiratory syncytial virus (RSV). Smaller colonies had more rounded cells than larger colonies. Clones were established from 9 out of 11 colonies. They varied in their rate of growth and the pattern formed on a plastic surface. They varied also in their virus susceptibility particularly to "difficult" rhinoviruses such as RV9 and SF1340. One cell clone (HL1/77 Clone 8), was highly susceptible to all these viruses. All cultures were more sensitive to RSV when maintained in F12K medium than in MEM, whereas there was no difference for rhinoviruses. Influenza A and B and parainfluenza 3 viruses sometimes produced cytopathic effect, and always produced haemadsorption, but unlike the previous strains could not be passed serially and presumably produced little infectious virus. All clones were rather insusceptible to Ad3; but the virus could be passed, whereas coxsackie virus B3 produced no CPE. Substantial yields of coronavirus and rhinovirus were obtained in gelatin sponge cultures. Two "very difficult" respiratory viruses which had just been adapted to tissue culture; namely, HS rhinovirus and JK coronavirus grew in 7 of 9 and in 6 of 9 clones respectively.

Long-term maintenance of reaggregated hypothalamic cultures developed from embryonic rat hypothalamus: prostaglandin release during synaptogenesis in vitro.

Hypothalamic aggregate cultures were developed from hypothalami taken from rat embryos at 17-19 days gestation. The aggregate cultures exhibited a prominent morphological differentiation during 3-4 weeks in culture. The fine structure of the synapses formed in the aggregates resembled synapses in tha adult animal. During synaptogenesis the aggregates spontaneously release prostaglandin E2 (PGE2). The amount of PGE2 released in the media was reversed upon the morphological differentiation of the hypothalamic cultures. Media containing a higher PGE2 concentration increased the extracellular prolactin accumulation in monolayer cultures developed from adult rat hypophysis.

Tissue typing of cells in culture. I. Distinction between cell lines by the various patterns produced in mixed haemadsorption with selected multiparous sera.

From over 3000 sera of multiparous women about 150 were selected that reacted with some (between 1--50%) of a panel of 20--30 non-HeLa cell lines, mostly of tumour origin. In the mixed haemadsorption method the diameters of zones formed by certain sera on particular cultures were measured and scored. The haemadsorption culture patterns produced by 70--135 discriminating sera were compared in pairs with the patterns formed on other cell lines. Comparisons between replicate cultures gave 'significant' differences with 0--10% of the sera used. In these instances deviations were in one direction, whereas in every other pair comparison, deviations were in both directions. It is concluded that in cases where pair comparisons initially give only 10%, or fewer significant differences, tests using carefully selected sera or typing for HLA-antigens should be performed. When comparisons yield more than 10% significant differences and deviations occur in both directions, cultures may be considered different.

Characterization of monolayer cultures of type II alveolar pneumonocytes that produce pulmonary surfactant in vitro.

In lung, type II cells are the site of synthesis of phosphatidylcholine (PC), a major component of pulmonary surfactant. Clonal culture methods permit isolation of an epithelial cell strain (L-2) derived from type II cells of intact rat lung capable of active PC production in vitro. Isotopic labeling of these monolayer cultures show that the choline incorporation pathway is the predominant biosynthetic route for PC production. This same pathway is utilized for PC production in whole lung. Three enzymes of this pathway are readily detected in L-2 cells. Determination of specific enzyme activity in monolayers and whole lung indicate that clonally derived L-2 cells are enriched in choline kinase (10-fold), cholinephosphate cytidyl transferase (10-fold) and cholinephosphotransferase activity (5-fold). Our second approach was to develop an in vitro system in which cellular inter-relationships and cell to cell contacts present in whole lung are maintained. This organotypic system is formed by reaggregation of monodispersed fetal rat lung cells into alveolar-like structures (ALS). The ALS are comprised primarily of type II cells as evidenced by the presence of osmiophilic lamellar bodies in the cells. Biosynthesis of these lamellar bodies occurs de novo and type II cells in the ALS continue to synthesize lamellar bodies. Both systems permit study of a variety of agents of pulmonary surfactant production in vitro.

Follow-up of cases of opiate addiction from the time of notification to the Home Office.

Follow-up of 108 opiate addicts for a period of six to seven years showed that 35 were still being prescribed opiates, 25 were off drugs, 19 were dead, and for 29 it was not certain whether they were on or off drugs. Most patients who came off drugs did so in the first two years after notification to the Home Office. Clearly treatment is more likely to be successful if given early in the life history of the addict, when it is appropriate to give treatment based on withdrawal of drugs rather than substitution.