Preparation and microscopic visualization of multicolor luminescent immunophosphors.

The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 micron and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads.

Continuous measurement of leakage during isolated liver perfusion with a radiotracer.

Isolation and perfusion of an organ or a part of the body has been attempted to improve cancer chemotherapy results. Our study was aimed at developing a reliable method of leakage detection permitting safe clinical application of isolated liver perfusion with high dosages of cytostatics. In a pig model a method of continuous leakage monitoring was developed, which would give a fast indication of leakage using a minimum quantity of radioactive tracer. To this end we added 99mTc labelled erythrocytes (total activity: 3.7 MBq) to the isolated external circuit. Initially a scintillation phototube detector was placed upon the body above the heart (three experiments). However, this method did not meet our requirements of accuracy and reproducibility, and a measurement technique was chosen, where an arterio-venous shunt outside the body functioned as a measuring cavity. The results of 11 experiments showed that the accuracy was 98 +/- 12%, and the detection threshold was below the required sensitivity of 1% (of the perfusate volume). From this study it can be concluded that our method of leakage measurement using a minimum quantity of 99mTc is an accurate method allowing safe application of isolated liver perfusion, and is in principle able to be carried out in any isolation and perfusion technique in the clinical situation.

A successful technique of in vivo isolated liver perfusion in pigs.

A technique was developed to isolate the hepatic circulation from the general circulation using a double lumen intracaval shunt. Low flow normothermic perfusion of the liver was performed for 1 hr 25 min in pigs. All pigs survived the procedure. The isolated liver perfusion without chemotherapy (n = 11) was well tolerated as monitored by hepatic enzymes and histologic examination during and after the operation. Mild transient elevations of SGOT and LDH returned to normal values within 1 week. No significant pathological alterations were found in the liver biopsies. Twenty-two pigs were subjected to isolated liver perfusion with 20, 40, or 80 mg 5-FU/kg. Up to four times the conventional dose of the drug could safely be administered when a washout was performed. To evaluate the efficacy of the isolation a method for leakage detection was developed, using tracer quantities of 99mTc-labeled red blood cells. This method was sensitive and permitted continuous monitoring of leakage. Negligible leakage was found during 15 isolated liver perfusions. The described technique of isolated liver perfusion was a reliable and technically feasible method, and has been adapted for clinical use to evaluate its value in the treatment of hepatic metastases.