Whole blood assay system

Inhibition of COX can be quantified in recombinant or natural enzyme preparations, cellular systems, isolated human cell populations such as platelets (COX-1) and white blood cells (COX-2), or in ex vivo stimulated whole blood samples The closer the experimental system is to the physiological state, the lower the selectivity for most COX-2-inhibitors. The standard test for comparison is considered to be a whole blood assay which mimics in vivo conditions like plasma binding (e.g. Patrignani et al., 1996). It is commonly accepted that reasonable variations occur between different laboratories (see data for Piroxicam). Therefore, whenever possible, data of several compounds generated with a given test system should be compared with each other. 

Piroxicam shows up to 12-fold selectivity for COX-1 compared to COX-2 in several assay systems. The variations of the results in whole blood assays from different laboratories (see also Warner et al., 1999 ratio COX-1/COX-2 0.3 and Young et al., 1996 ratio COX-1/COX-2 0.4) stress the importance of careful interpretation of COX selectivity ratios. 

The first step in the clinical development of rofecoxib was to determine the degree of selectivity when the drug was administered to humans. As outlined above, the assessment of selectivity of Cox inhibitors for the two isoforms is dependent on the systems used and in vitro systems may not reflect the degree of selectivity of the enzyme inhibitors in vivo. Two clinical models were used to assess the selectivity of rofecoxib in humans. For both tests, tissues were obtained from patients or volunteers receiving either rofecoxib or NSAIDs, and the ability of these tissues to synthesize prostanoids ex vivo was determined. The simplest of these models is the whole blood assay, which was mentioned earlier in this chapter as one of the tests than can be used for evaluation of Cox selectivity (Patrignani et al, 1994). To use this test in the clinical setting, two samples of blood are drawn from individuals receiving a Cox inhibitor. One sample is allowed... 

Commercial application of the dendrimer-based reagent technology has been demonstrated by the successful development of The Stratus CS STAT fluorometric analyzer [5] marketed by Dade Behring Inc. This rapid automated point of care immunoassay system provides quantitative analysis of whole blood or preprocessed plasma samples via unit use assay test packs. Up to four test packs can be introduced for each sample. All reagents [5-9] required for specimen analyses are contained within the test packs. 

Fig. 39 (a) Design and principle of operation of the BL-QD system and (b) spectral manifestation of the assay, (c) Two assays performed under identical conditions in mouse serum green) and mouse whole blood red). (Reprinted with permission from [222]. Copyright 2006 Macmillan Publishers Ltd)... 

Zhang S, Sun W-l, Xian Y-z, Zhang W, Jin L, Yamamoto K, Tao S, Jin, J (1999) Multichannel amperometric detection system for liquid chromatography to assay the thiols in human whole blood using the platinum microelectrodes chemically modified by copper tetra aminophthalocyanine. Anal Chim Acta 399(3) 213-221... 

Serum is the specimen of choice for many assay systems. Sometimes considerable differences may be observed between the concentrations of analytes in serum and plasma as shown in Table 2-3. However, some assay systems require a whole blood or plasma specunen. If so an anticoagulant must be added to the specimen during the collection procedure. A number of anticoagulants are used including heparin, EDTA, sodium fluoride, citrate, oxalate, and iodo acetate. 

When an assay system has been designed to analyze whole blood samples, specimen preparation time is essentially eliminated. Automated or semiautomated ion-selective electrodes, which measure ion activity in whole blood rather than ion concentration, have been incorporated into automated systems to provide certain test results within minutes of the drawing of a specimen. This approach is now commonly used for assaying electrolytes and some other common analytes. Another approach involves either manual or automated application of whole blood to dry reagent films and visual or instrumental observation of a quantitative change. This approach is exemplified by the Reflotron Plus. 

Third, assays need to describe methods used for obtaining minimal detection limits (e.g., mean plus 3 SD of 20 replicates of a zero calibrator) and total imprecision, describing at what concentration a 10% CV is attained. Preanalytical factors that should be described include the effects of storage time and temperature, glass versus plastic tubes and gel separator tubes, and the influence of anticoagulants and whole blood measurements. As more assay systems are devised for POCT, the same rigors applied to the central laboratory methodologies need to be adhered to by the POCT systems. 

We emphasize that, in terms of systemic safety for children, tacrolimus whole-blood concentrations were consistent with minimal absorption of tacrolimus through the affected skin in more than 90% of blood samples collected, the level of tacrolimus was below the limit of detection of the assay used. 

The selectivity of drugs is generally assessed with the aid of in vitro or ex-vivo assay systems, and also in human whole blood as appropriate. Occasionally, however, the data show considerable variability. This appears to apply in particular to meloxicam, where, depending on the experimental set-up, almost any selectivity factor may be obtained. [188] COX- inhibitors show in general affinity to both isozymes, cyclooxygenase-1 and -2. In many cases the selectivity factor ranges from 1 to 100 (Fig. 5.90). [189]... 

Since blood serves as the primary metabolic transport system between the body organs, its composition is the preferred indicator with regard to the pathophysiological condition of the patient. This is the reason why most determinations are concerned with whole blood or biofluids derived from it. Glucose in urine is also an index component of metabolic disorders in patients. Other biological fluids have been considered as an alternative assay for blood glucose. 

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