Phenotypic assay measured

Traditional methods of antibiotic susceptibility screening include phenotypic assays measuring bacterial activity in the presence of the antibiotic in consideration. Although sensitive and widely used, this process is slow and labor-intensive owing to... 

TPMT activity can be analyzed by different phenotyping assays including the radiochemical method developed by Weinshilboum and more recent non-radioactive HPLC methods using either 6-MP or 6-TG as substrates ( 145, 146, 147, 148, 149,150,151,152,153). The radiochemical and HPLC assays have been shown to lead to comparable results with 6-MP as a substrate (146,147,148,149,150). However, when using 6-TG, TPMT activity was measured at 30% higher levels (152). 

The major advantage of using the urinary S R ratio as a phenotypic trait for assessing CYP2C19 activity is that the method is fairly robust with regard to any incompleteness of urine collection or noncompliance with respect to dose administration. This is because the R-enantiomer serves as an in vivo internal standard. On the other hand, the required enantiospecific assay uses chiral capillary column gas chromatography with a nitrogen-specific detector, and such instmmentation is not commonly available. For this reason, an alternative phenotypic trait measure based on the formation and urinary elimination of 4 -hydroxymephenytoin has also been frequently used. 

Both genotypic and phenotypic drug resistance assays are valuable tools in this respect. Where genotypic tests are fast and relatively cheap to monitor the presence of known resistance-related mutations, they suffer from known and unknown synergistic and antagonizing effects of combinations of mutations. Only phenotypic assays can measure the actual inhibitory effects of the antiviral drugs on the clinical HIV-1 isolate. Specific reverse transcriptase (RT) and... 

Table 4.1 Common assay technologies and perturbation measured in a phenotypic assay [11].

Phenotypic resistance assays directly measure the ability of HlV-1 to replicate in a cell culture in the presence of different antiretroviral drug concentrations. This process is similar to that used to determine antibiotic resistance and is, therefore, more familiar to most clinicians. The recombinant virus, composed of a virus s reverse transcripfase and protease genes, is inserted into a standard reference strain of virus. The recombinant virus is then tested in vitro for fhe amount of drug needed to inhibit virus replication by 50%, relative to the amount of drug needed to inhibit a reference strain of virus. Phenotypic resistance testing is limited by the fact that it is conducted in vitro and not in vivo. 

This procedure, which complements other methods for distinguishing apoptotic and necrotic cell death, employs annexin V-PE as a marker for early apoptotic cells, and 7-AAD for late apoptotic or necrotic cells. Although other versions of this assay have used annexin V-fluorescein together with PI, that combination precludes the use of a third fluorescence color to measure an additional parameter, such as a phenotypic marker, because PI, unlike 7-AAD, has a broad emission spectrum that includes both orange and red fluorescence. 

Note that this equation applies to any in vitro assay, whether it measures direct molecular interaction or a more complex cellular phenotype. 

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