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This review intends to survey the traditional and current technologies in the depletion and subfractionation of plasma proteins for further analyses. The value of depletion aims to enrich low-abundant proteins by removing highly abundant proteins, such as albumin or immunoglobulin G, from plasma. With this approach, one can examine both the resulting high- and low-abundant protein fractions. The depleted protein population can be further subfractionated based on their isoelectric point ranges, creating a more discrete pool of proteins for detailed post-translational modification studies by methods such as 2D gel electrophoresis and mass spectrometry. The concept of divide to conquer will greatly enhance our ability to identify and characterize low-abundant proteins and cleaved peptides from plasma as important diagnostic markers or potential drug targets. This can potentially reverse the decline in the development of new plasma diagnostic tests.

Summary: Autoradiograms obtained after middle cere bral artery occlusion (MCAO) in spontaneously hyperten sive rats show that the 99mTc complex of a 2-nitroimid azole-derivatized propylene amine oxime (BMS-181321) is selectively retained in acutely ischemic brain before disruption of the blood-brain barrier (BBB), but not in the ischemic infarct. BMS-181321 is therefore a marker of ischemic tissue at risk of infarction and its uptake, unlike that of x-ray and magnetic resonance contrast agents, does not require disruption of the BBB. In keeping with this conclusion, we have found that the single-pass cere bral extraction fraction of BMS-181321 is 0.67 at normal rat whole-brain blood flow. Sequential single-photon Delineation of viable ischemic tissue that is a tar get of various intervention strategies represents an important goal of nuclear medicine (Goldstein, 1990;Gropler and Bergman, 1991; Heiss et aI., 1992). One approach to this problem involves local Abbreviations used: BBB, blood-brain barrier; EB, Evans Blue; lAP, iodoantipyrine; MCAO, middle cerebral artery oc clusion; 2NI, 2-nitroimidazole; PnAO, propylene amine oxime; rCBF, regional CBF; ROD, relative optical density; Ro!, region of interest; SHR, spontaneously hypertensive rat; SPECT, sin gle-photon emission computed tomography. 755emission computed tomographic images obtained from cats after MCAO show that the initial distribution of BMS-181321 approximates regional CBF and that selec tive retention subsequently produces a positive image within the ischemic territory. BMS-181321 is the first Tc complex able to indicate not only ischemia, but also isch emic tissue at risk of infarction. Use of this novel Tc complex to monitor biochemical events during ischemia may contribute to the clinical management of acute stroke. Key Words: Hypoxia-Ischemia-Misonid azole-Neuronal viability-Single-photon emission com puted tomography.

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We have developed a 99mTechnetium complex for imaging of hypoxic tissue (BMS-181321). Recently, another nitroimidazole derivative, based upon a bis(amine-phenol) ligand, was described in the patent literature. To compare this compound to BMS-181321, we have synthesized the ligand, prepared its 99mTc complex, and evaluated its performance in two in vitro assays of bioefficacy: membrane permeability and uptake in normoxic and anoxic cardiocytes. In attempting to reproduce the synthesis of the ligand described in the patent application, we found that one intermediate could not be made by the method described, and alternative routes were investigated. Complexation of the bis(amine-phenol) nitroimidazole with 99mTc gave an apparent single complex; this appeared as a broad peak on HPLC analysis. Purification by a solid-phase method gave a complex with 95% radiochemical purity. This complex was not permeable to cultured bovine brain endothelial cells nor did it show preferential uptake in anoxic myocytes.

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Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.