Lenticular nuclei to thalamic ratio on PET is useful for diagnosis of GLUT1 deficiency syndrome.

PURPOSE: To establish an objective method of [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) that can assist in the diagnosis of glucose transporter 1 deficiency syndrome (GLUT1-DS). METHODS: FDG-PET was performed in 8 patients with a mean age of 12.5 years (range, 2-22 years) with GLUT1-DS. Their PET findings were compared with those of 45 controls with a mean age of 11.2 years (range, 2-21 years) by statistical parametric mapping (SPM12, Welcome Neurological Institute). The controls had epilepsy of unknown etiology and normal MRI findings. The age-adjusted ratios of mean radioactivities in regions of interest (ROIs) of bilateral lenticular nuclei, thalami, and the whole cerebral cortex were also measured. The sensitivities and specificities of the ratios for the differential diagnosis of GLUT1-DS were also determined. RESULTS: SPM showed significantly decreased uptake in bilateral thalami and increased uptake in bilateral lenticular nuclei in patients with GLUT1-DS. There were no areas in the cerebral cortex with significant differences between patients and controls. On ROI analysis, by setting the cut-off value of the age-adjusted lenticular nuclei/thalami radioactivity ratio to 1.54, patients with GLUT1-DS were differentiated from controls with sensitivity of 1.00 and specificity of 0.98. CONCLUSION: The age-adjusted lenticular nuclei/thalami radioactivity ratio on PET can distinguish patients with GLUT1-DS from patients with epilepsy of unknown etiology with high sensitivity and specificity. It is important to pay attention to the metabolism of the lenticular nuclei and thalami on PET for the diagnosis of GLUT1-DS.

Primary Preclinical and Clinical Evaluation of 68Ga-DOTA-TMVP1 as a Novel VEGFR-3 PET Imaging Radiotracer in Gynecological Cancer.

PURPOSE: Tumor periphery and lymph nodes of tumor-induced lymphangiogenesis often abundantly express VEGFR-3. In our previous study, we identified a 5-amino acid peptide named TMVP1, which binds specifically to VEGFR-3. The objective of this study was to develop a novel 68Ga-labeled TMVP1 for VEGFR-3 PET imaging and to investigate its safety, biodistribution, and tumor-localizing efficacy in xenograft tumor models and a small cohort of patients with recurrent ovarian and cervical cancer. EXPERIMENTAL DESIGN: The DOTA-conjugated TMVP1 peptide was labeled with radionuclide 68Ga. SPR and saturation binding assays were used for the receptor-binding studies. Gynecologic xenograft tumors were employed for small-animal PET imaging and biodistribution of 68Ga-DOTA-TMVP1 in vivo. In the clinical study, 5 healthy volunteers and 8 patients with gynecologic cancer underwent whole-body PET/CT after being injected with 68Ga-DOTA-TMVP1. RESULTS: DOTA-TMVP1 was successfully labeled with 68Ga. LECs showed higher binding capacity with 68Ga-DOTA-TMVP1 than LEC(shVEGFR-3) and human umbilical vein endothelial cells. In mice with subcutaneous C33-A and SKOV-3 xenografts, the tracer was rapidly eliminated through the kidney to the bladder, and the small-animal PET/CT helped to clearly visualize the tumors. In patients with recurrent ovarian cancer and cervical cancer, tracer accumulation well above the background level was demonstrated in most identified sites of disease; especially with recurrent endodermal sinus tumors, the diagnostic value of 68Ga-DOTA-TMVP1 was comparable with that of 18F-FDG PET/CT. CONCLUSIONS: 68Ga-DOTA-TMVP1 is a potential PET tracer for imaging VEGFR-3 with favorable pharmacokinetics.

Metabolic Evaluation of MYCN-Amplified Neuroblastoma by 4-[18F]FGln PET Imaging.

PURPOSE: This study aims to explore whether 4-(2S,4R)-[18F]fluoroglutamine (4-[18F]FGln) positron emission tomography (PET) imaging is helpful in identifying and monitoring MYCN-amplified neuroblastoma by enhanced glutamine metabolism. PROCEDURES: Cell uptake studies and dynamic small-animal PET studies of 4-[18F]FGln and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) were conducted in human MYCN-amplified (IMR-32 and SK-N-BE (2) cells) and non-MYCN-amplified (SH-SY5Y cell) neuroblastoma cells and animal models. Subsequently, short hairpin RNA (shRNA) knockdown of alanine-serine-cysteine transporter 2 (ASCT2/SLC1A5) in IMR-32 cells and xenografts were investigated in vitro and in vivo. Western blot (WB), real-time polymerase chain reaction (RT-PCR), and immunofluorescence (IF) assays were used to measure the prevalence of ASCT2, Ki-67, and c-Caspase 3, respectively. RESULTS: IMR-32 and SK-N-BE (2) cells showed high glutamine uptake in vitro (31.6 ± 1.7 and 21.6 ± 6.6 %ID/100 µg). In the in vivo study, 4-[18F]FGln was localized in IMR-32, SK-N-BE (2), and SH-SY5Y tumors with a high uptake (6.6 ± 0.3, 5.6 ± 0.2, and 3.7 ± 0.1 %ID/g). The maximum uptake (tumor-to-muscle, T/M) of the IMR-32 and SK-N-BE (2) tumors (3.71 and 2.63) was significantly higher than that of SH-SY5Y (1.54) tumors (P < 0.001, P < 0.001). The maximum uptake of 4-[18F]FGln in IMR-32 and SK-N-BE (2) tumors was 2.3-fold and 2.1-fold higher than that of [18F]FDG, respectively. Furthermore, in the in vitro and in vivo studies, the maximum uptake of 4-[18F]FGln in shASCT2-IMR-32 cells and tumors was 2.1-fold and 2.5-fold lower than that of the shControl-IMR-32. No significant difference in [18F]FDG uptake was found between shASCT2-IMR-32 and shControl-IMR-32 cells and tumors. CONCLUSION: 4-[18F]FGln PET can provide a valuable clinical tool in the assessment of metabolic glutamine uptake in MYCN-amplified neuroblastoma. ASCT2-targeted therapy may provide a supplementary method in MYCN-amplified neuroblastoma treatment.

18F-FEDAC as a Targeting Agent for Activated Macrophages in DBA/1 Mice with Collagen-Induced Arthritis: Comparison with 18F-FDG.

Activated macrophages have been known to play pivotal roles in the pathogenesis of rheumatoid arthritis (RA). 18F-FEDAC (N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-18F-fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide) is a radiolabeled ligand for the 18-kDa translocator protein (TSPO), which is abundant in activated macrophages. We evaluated the feasibility of using 18F-FEDAC in a murine RA model. Methods: RAW 264.7 mouse macrophages were activated by lipopolysaccharide. TSPO expression levels in activated and inactivated macrophages were measured by quantitative polymerase chain reaction and Western blotting. The cellular uptake and specific binding of 18F-FEDAC were measured using a γ-counter. For the in vivo study, collagen-induced arthritis (CIA) was developed in DBA/1 mice, and the clinical score for arthritis was measured regularly. 18F-FEDAC and 18F-FDG PET images were acquired on days 23 and 37 after the first immunization. Histologic examinations were performed to evaluate macrophages and TSPO expression. Results: We found increased TSPO messenger RNA and protein expression in activated macrophages. Uptake of 18F-FEDAC in activated macrophages was higher than that in nonactivated cells and was successfully blocked by the competitor, PK11195. In CIA mice, joint swelling was apparent on day 26 after the first immunization, and the condition worsened by day 37. 18F-FEDAC uptake by arthritic joints increased early on (day 23), whereas 18F-FDG uptake did not. However, 18F-FDG uptake by arthritic joints markedly increased at later stages (day 37) to a higher level than 18F-FEDAC uptake. The 18F-FEDAC uptake correlated weakly with summed severity score (P = 0.019, r = 0.313), whereas the 18F-FDG uptake correlated strongly with summed severity score (P < 0.001, r = 0.897). Histologic sections of arthritic joints demonstrated an influx of macrophages compared with that in normal joints. Conclusion:18F-FEDAC enabled the visualization of active inflammation sites in arthritic joints in a CIA model by targeting TSPO expression in activated macrophages. The results suggest the potential usefulness of 18F-FEDAC imaging in the early phase of RA.

PET Imaging on Dynamic Metabolic Changes after Combination Therapy of Paclitaxel and the Traditional Chinese Medicine in Breast Cancer-Bearing Mice.

PURPOSE: The aim of the study was to non-invasively evaluate the anticancer activity of a traditional Chinese medicine-Huaier, combined with paclitaxel (PTX) in breast cancer bearing mice by detecting dynamic metabolic changes with positron emission tomography (PET). PROCEDURES: Balb/c nude mice were randomly divided into one of the four groups: Huaier, PTX, PTX + Huaier, or the control. PET imaging with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) was performed to monitor the metabolic changes in BT474 (luminal B) and MDA-MB-231 (triple-negative) breast cancer xenografts. Immunohistochemistry (IHC) study was performed immediately after the final PET scan to assess the expressions of phosphatidylinositol 3-kinase (PI3K), phospho-AKT (p-AKT), caspase-3, and vascular endothelial growth factor (VEGF). RESULTS: Compared to the control group, [18F]FDG accumulation demonstrated a significant decrease in PTX + Huaier (p < 0.01) or Huaier group (p < 0.05), which was consistent to the decreased expression of PI3K (p < 0.05) and p-AKT (p < 0.05) in the breast cancer xenografts. CONCLUSION: The therapeutic effect of Huaier combined with PTX was superior than the PTX alone in BT474 and MDA-MB-231 breast cancer-bearing mice. [18F]FDG PET imaging could be a potential non-invasive approach to assess the metabolic changes after chemotherapy combined with traditional Chinese medicine in the breast cancer.

18F-FDG-Avid Thyroid Incidentalomas: The Importance of Contextual Interpretation.

18F-FDG-avid thyroid incidentaloma (TI) is seen in approximately 2.5% of patients imaged for staging or response assessment of malignancy and represents thyroid cancer in approximately 35% of cases. Consequently, the 2015 American Thyroid Association guidelines strongly recommend investigation of all 18F-FDG-avid nodules 1 cm or larger with ultrasound and fine-needle aspiration cytology (FNA). This study aimed to assess the overall and thyroid cancer-specific survival in a large cohort of patients with 18F-FDG-avid TI with long-term follow-up to assess the validity of this approach. Methods: Retrospective review of 45,680 PET/CT scans performed at a comprehensive cancer center from January 2007 to January 2015 identified 2,588 18F-FDG PET/CT reports referring to the thyroid. After exclusion of nonavid thyroid nodules, diffuse 18F-FDG uptake, known thyroid cancer, abnormalities adjacent to the thyroid, and repeat studies, 500 patients (1.1%) with TI were identified, of whom 362 had confirmed death or more than 12 mo of clinical follow-up. Variables including age, sex, primary malignancy, overall survival, thyroid cancer-specific survival, FNA, and histopathology were collected until January 2016. Multivariate logistic regression and survival analysis were performed. Results: The 362 analyzed patients (65% female) had a median age of 65 y (range, 19-96 y) and follow-up of 24 mo (range, 1-103 mo). Lymphoid, lung, and colorectal malignancy were the most common staging indications. Median overall survival was 20 mo (interquartile range, 9.5-39 mo). Most of the 180 observed deaths were due to the primary malignancy under investigation (92.2%) or to causes not related to cancer (7.2%); one patient (0.6%) died from incidentally detected medullary thyroid cancer. 18F-FDG avidity in the index malignancy, an advanced stage for that malignancy, and a clinician decision not to investigate 18F-FDG-avid TI were all predictors of mortality, with hazard ratios of 8.5, 3.0, and 3.3, respectively, and 95% confidence intervals of 4.6-15.8, 2.3-3.9, and 2.0-5.0, respectively (P < 0.001). Of 131 patients suitable for cytologic or histopathologic evaluation, 47 (36%) had incidental thyroid cancer (24 papillary, 11 malignant FNA, 5 oncocytic/Hürthle cell, 2 medullary, 1 follicular, and 4 metastases from underlying malignancy). Conclusion: Overall survival with 18F-FDG-avid TI was poor because of the prognosis associated with underlying malignancy, which must be considered before investigation of 18F-FDG-avid TI and certainly before aggressive treatment. Active surveillance should be considered in this group of patients.

A Pilot Study Into the Use of FDG-mNP as an Alternative Approach in Neuroblastoma Cell Hyperthermia.

Herein, we present a pilot study concerning the use of fluorodeoxy glucose conjugated magnetite nanoparticles (FDG-mNP) as a potential agent in magnetic nanoparticle mediated neuroblastoma cancer cell hyperthermia. This approach makes use of the 'Warburg effect', utilizing the fact that cancer cells have a higher metabolic rate than normal cells. FDG-mNP were synthesized, then applied to the SH-SY5Y neuroblastoma cancer cell line and exposed to an ac magnetic field. 3D Calorimetry was performed on the FDG-mNP compound. Simulations were performed using SEMCAD X software using Thelonious, (an anatomically correct male child model) in order to understand more about the end requirements with respect to cancer cell destruction. We investigated FDG-mNP mediated neuroblastoma cytotoxicity in conjunction with ac magnetic field exposure. Results are presented for 3D FDG-mNP SAR mnp (10.86 ± 0.99 W/g of particles) using a therapeutic dose of 0.83 mg/ mL. Human model simulations suggest that 43 W/kg SAR Theo would be required to obtain 42 °C within the centre of a liver tumor (Tumor size, bounding box x = 64, y = 61, z = 65 [mm]), and that the temperature distribution is inhomogeneous within the tumor. Our study suggests that this approach could potentially be used to increase the temperature within cells that would result in cancer cell death due to hyperthermia. Further development of this research will also involve using whole tumors removed from living organisms in conjunction with magnetic resonance imaging and positron emission tomography.

(31)P CSI of the human brain in healthy subjects and tumor patients at 9.4 T with a three-layered multi-nuclear coil: initial results.

OBJECTIVE: Investigation of the feasibility and performance of phosphorus ((31)P) magnetic resonance spectroscopic imaging (MRSI) at 9.4 T with a three-layered phosphorus/proton coil in human normal brain tissue and tumor. MATERIALS AND METHODS: A multi-channel (31)P coil was designed to enable MRSI of the entire human brain. The performance of the coil was evaluated by means of electromagnetic field simulations and actual measurements. A 3D chemical shift imaging approach with a variable repetition time and flip angle was used to increase the achievable signal-to-noise ratio of the acquired (31)P spectra. The impact of the resulting k-space modulation was investigated by simulations. Three tumor patients and three healthy volunteers were scanned and differences between spectra from healthy and cancerous tissue were evaluated qualitatively. RESULTS: The high sensitivity provided by the 27-channel (31)P coil allowed acquiring CSI data in 22 min with a nominal voxel size of 15 × 15 × 15 mm(3). Shimming and anatomical localization could be performed with the integrated four-channel proton dipole array. The amplitudes of the phosphodiesters and phosphoethanolamine appeared reduced in tumorous tissue for all three patients. A neutral or slightly alkaline pH was measured within the brain lesions. CONCLUSION: These initial results demonstrate that (31)P 3D CSI is feasible at 9.4 T and could be performed successfully in healthy subjects and tumor patients in under 30 min.

[18F-Berberine Derivatives: a Potential Molecular Imaging Agent for Tumor Targeting by PET/CT Tumor].

Cancer is one of the main causes of death for human beings. Clinical oncologists increasingly rely upon imaging for diagnosis, stage, response assessment, and follow-up in cancer patient. However, 18F-FDG is not a tumor specific agent, inflammation and infection also have intensive uptake of 18F-FDG, resulting in false positive diagnosis, and some tumors have low uptake of 18F-FDG or even do not uptake 18F-FDG, leading to false negative diagnosis. So it is urgent to develop non-18F-FDG novel tumor targeting agent. Recently, a large number of researches in vitro have demonstrated that berberine has anti-tumor activity against a variety of tumor cells by inducing tumor cell apoptosis through inhibition of mitochondrial respiratory chain etc. So far, there is no credible evidence of berberine targeting in tumor in vivo. We proposed a hypothesis that berberine has the characteristics of tumor targeting biodistribution in vivo, and verified the proposal by 18F-berberine PET/CT imaging in VX2 muscle tumor-bearing rabbit model. In this review, we intend to give an overview of the progress of berberine anticancer, the structural bases of berberine anticancer and the uderlying molecular mechanisms of berberine anticancer indentified so far. We also introduce the first visualization of 18F labeled berberine derivatives targeting tumor in VX2 muscle tumor-bearing rabbit model by PET/CT. These breakthrough findings suggest that 18F-berberine derivatives as a potential PET/CT tumor targeted molecular imaging agent may have important implications for cancer targeting therapy, molecular imaging and modernization of Traditional Chinese Medicine.

Can complementary 68Ga-DOTATATE and 18F-FDG PET/CT establish the missing link between histopathology and therapeutic approach in gastroenteropancreatic neuroendocrine tumors?

UNLABELLED: Gastroenteropancreatic neuroendocrine tumors (GEPNETs) are indolent neoplasms presenting unpredictable and unusual biologic behavior that causes many clinical challenges. Tumor size, existence of metastasis, and histopathologic classification remain incapable in terms of treatment decision and prognosis estimation. This study aimed to compare (68)Ga-DOTATATE and (18)F-FDG PET/CT in GEPNETs and to investigate the relation between the complementary PET/CT results and histopathologic findings in the management of therapy, particularly in intermediate-grade patients. METHODS: The relation between complementary (68)Ga-DOTATATE and (18)F-FDG PET/CT results of 27 GEPNET patients (mean age, 56 y; age range, 33-79 y) and histopathologic findings was evaluated according to grade and localization using standardized maximum uptake values and Ki67 indices. Grade 2 (G2) patients were further evaluated in 2 groups as G2a (3%-9%) and G2b (10%-20%) according to Ki67 indices. RESULTS: The sensitivity of (68)Ga-DOTATATE and (18)F-FDG PET/CT was 95% and 37%, respectively, and the positive predictive values were 93.8% and 36.2%, respectively. The sensitivity in detecting liver metastasis, lymph nodes, bone metastasis, and primary lesion was 95%, 95%, 90%, and 93% for (68)Ga-DOTATATE and 40%, 28%, 28%, and 75% for (18)F-FDG, respectively. Statistically significant differences were found between grades 1-2, 2a-2b, and 1-2b with respect to (68)Ga-DOTATATE PET/CT as well as between 1-2a and 1-2b with respect to (18)F-FDG PET/CT. However, no statistical differences were found between 1 and 2a (P > 0.05) for (68)Ga-DOTATATE and 2a and 2b (P = 0.484) for (18)F-FDG. The impact of the combined (18)F-FDG and (68)Ga-DOTATATE PET/CT on the therapeutic decision was 59%. CONCLUSION: Combined (68)Ga-DOTATATE and (18)F-FDG PET/CT is helpful in the individual therapeutic approach of GEPNETs and can overcome the shortcomings of histopathologic grading especially in intermediate-grade GEPNETs.

Small molecule PET-radiopharmaceuticals.

This review describes several aspects required for the development of small molecule PET-tracers. Design and selection criteria are important to consider before starting to develop novel PET-tracers. Principles and latest trends in (11)C and (18)F-radiochemistry are summarized. In addition an update of some new developments in regulatory aspects is supplied.

Efficacy of oregonin investigated by non-invasive evaluation in a B16 mouse melanoma model.

Oregonin has been reported to act as a mediator of antibiosis, a liver-protective agent, an antioxidant, an anti-inflammatory agent, and to prevent cancer outbreaks. B16 melanoma cells were separated with trypsin-ethylenediaminetetraacetic acid, resuspended in 50 µl of phosphate-buffered saline and transplanted into the backs of 6- to 8-week-old male Balb/c nude mice through subcutaneous injection. Treatment doses of oregonin were administered three times weekly, for 30 days from the 11th day after transplantation of the melanoma cells, in each group. The study consisted of a control group, a dacarbazine group, an oregonin group and a dacarbazine + oregonin group. Measurements were taken before treatment and on the 5th, 7th, 10th and 15th days after treatment for each group. Based on survival rates after transplantation, the control group showed less than 50% survival after 20 days, while the treatment groups showed at least 50% survival up to the 41st day.

Using microPET imaging in quantitative verification of the acupuncture effect in ischemia stroke treatment.

Acupuncture has been indispensable in Chinese medicine. However, its function still remains elusive. This paper studies the effect of acupuncture in ischemia stroke treatment using the Sprague Dawley rat animal model. We induced focal cerebral ischemia in rats using the middle cerebral artery occlusion (MCAO) procedure. For each rat in the real acupuncture group (n = 63), the sham acupoint treatment group (n = 62), and the blank control group (n = 30), we acquired 3-D fluorodeoxyglucose-microPET images at baseline, after MCAO, and after treatment, respectively. Then, we measured the changes of the injury-volume in the right hemisphere of these rats. The measurements showed that real acupuncture slightly reduced the injury-volume, sham acupoint treatment increased the injury-volume, and blank control had no obvious effect in reducing the injury-volume. Statistical tests also confirmed that acupuncture was more effective than random stimulus in improving the metabolic recovery after stroke.

Functional imaging of brown fat in mice with 18F-FDG micro-PET/CT.

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults (1). BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via ß adrenergic receptor (2, 3). Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters (4-6). BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance (6-8). While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT (9-13), there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of (18)F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of ß-adrenoceptor antagonist propranolol (14, 15), or the enhanced BAT activation by ß3 agonist BRL37344 (16). The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module.

We developed a new fully automated method for the synthesis of [18F]fluoromisonidazole ([18F]FMISO) by modifying a commercial FDG synthesizer and its disposable fluid pathway. A three-step procedure was used to prepare the tosylate precursor, 1-(2'-nitro-1'-imidazolyl)-2-O-tetrahydrofuranyl-3-O-toluenesulfonylpropanediol. Using glycerol as the starting material, the precursor was synthesized with a yield of 21%. The optimal labeling conditions for the automated synthesis of [18F]FMISO was 10 mg of precursor in acetonitrile (2 ml heated at 105 degrees C for 360 s, followed by heating at 75 degrees C for 280 s and hydrolysis with 1 N HCl at 105 degrees C for 300 s. Using 3.7 GBq of [18F]F- as a starting activity, [18F]FMISO was obtained with high end-of-synthesis (EOS) radiochemical yields of 58.5+/-3.5% for 60.0+/-5.2 min with high-performance liquid chromatography (HPLC) purification. When solid-phase purification steps were added, the EOS radiochemical yields were 54.5+/-2.8% (337+/-25 GBq/micromol) for 70.0+/-3.8 min (n=10 for each group, decay-corrected). With a high starting radioactivity of 37.0 GBq, we obtained radiochemical yields of 54.4+/-2.9% and 52.8+/-4.2%, respectively (n=3). The solid-phase purification removed unreacted [18F]fluoride and polar impurities before the HPLC procedure. Long-term tests showed a good stability of 98.2+/-1.5%. This new automated synthesis procedure combines high and reproducible yields with the advantage of using a disposable cassette system.