We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.

An increased prevalence of nephrolithiasis has been reported in patients with diabetes. Because insulin resistance, characteristic of the metabolic syndrome and type 2 diabetes, results in lower urine pH through impaired kidney ammoniagenesis and because a low urine pH is the main factor of uric acid (UA) stone formation, it was hypothesized that type 2 diabetes should favor the formation of UA stones. Therefore, the distribution of the main stone components was analyzed in a series of 2464 calculi from 272 (11%) patients with type 2 diabetes and 2192 without type 2 diabetes. The proportion of UA stones was 35.7% in patients with type 2 diabetes and 11.3% in patients without type 2 diabetes (P < 0.0001). Reciprocally, the proportion of patients with type 2 diabetes was significantly higher among UA than among calcium stone formers (27.8 versus 6.9%; P < 0.0001). Stepwise regression analysis identified type 2 diabetes as the strongest factor that was independently associated with the risk for UA stones (odds ratio 6.9; 95% confidence interval 5.5 to 8.8). The proper influence of type 2 diabetes was the most apparent in women and in patients in the lowest age and body mass index classes. In conclusion, in view of the strong association between type 2 diabetes and UA stone formation, it is proposed that UA nephrolithiasis may be added to the conditions that potentially are associated with insulin resistance. Accordingly, it is suggested that patients with UA stones, especially if overweight, should be screened for the presence of type 2 diabetes or components of the metabolic syndrome. 17: 202617: -203317: , 200617: . doi: 10.1681 I ncidence of urinary stone disease rose considerably in recent decades in all industrialized countries (1,2), as did the incidence of obesity, the metabolic syndrome, and type 2 diabetes (3-6). These epidemiologic changes took place in parallel with marked modifications in dietary habits and lifestyle that occurred in all Western and westernized populations, characterized by a high calorie intake coupled with reduced physical activity (7-9). This temporal parallelism suggested that an association might exist among diabetes, obesity, and urinary stone disease. Two recent studies revealed an increased prevalence of nephrolithiasis in patients with diabetes as compared with patients without diabetes (10,11), but in these studies, the chemical type of nephrolithiasis was not identified. Therefore, it was not defined whether calcium (Ca) or uric acid (UA) stones or both contributed to the increased prevalence of urinary stone disease in patients with diabetes, as alterations in urine biochemistry associated with obesity and type 2 diabetes may favor the formation of UA as well as of Ca stones (12-15). J Am Soc NephrolInsulin resistance, which constitutes the fundamental metabolic disorder that is associated with the metabolic syndrome and type 2 diabetes (16,17), results in defective renal ammoniagenesis and low urine pH (18,19) and therefore may be expected to favor the production of UA ...

Incidence of atherosclerotic CV complications is abnormally high in predialysis CRF patients, suggesting that the uraemic state per se is associated with atherogenesis. As several of the identified clinical and metabolic risk factors for such accidents are potentially remediable by specific therapeutic interventions, prophylactic measures should be initiated long before start of renal replacement therapy.

Our results suggest that inflammatory status and duration of dialysis treatment are the most important factors relating to oxidative stress in haemodialysis patients.

The clinical impact of circulating tumor cell (CTC) detection is controversial, mainly due to drawbacks of molecular approaches applied to this field. We sought to determine if the specific identification and counting of circulating tumor cells by cytomorphologic analysis has clinical usefulness. Peripheral blood (6 mL), treated using isolation by size of epithelial tumor cells, was obtained from 44 patients with primary liver cancer (PLC) and without metastases, 30 patients with chronic active hepatitis, 39 with liver cirrhosis, and 38 healthy individuals, and followed up for a mean period of 1 year. We searched for ␤-catenin mutations in 60 single microdissected CTCs. One patient with liver cancer developed extrahepatic metastases during follow-up. CTCs and microemboli were found in 23 of the 44 patients with liver cancer and in none of the patients with chronic active hepatitis, patients with cirrhosis, or healthy subjects. Their presence was significantly associated with tumor diffusion (P ‫؍‬ .0001) and portal tumor thrombosis (P ‫؍‬ .006). Both the presence (P ‫؍‬ .01) and number (P ‫؍‬ .02) of CTCs and microemboli were significantly associated with a shorter survival. ␤-Catenin mutations were found in 3 of 60 CTCs, arguing against their impact on the initial step of tumor cell invasion. In conclusion, the highly sensitive and specific detection of CTCs and microemboli may have clinical implications for cancer staging and outcome prediction. We also show the feasibility of molecular studies of individual circulating tumor cells, aimed at identifying gene mutations involved in tumor invasion. (HEPATOLOGY 2004;39:792-797.)