Screening false negatives

Zebrafish 50-200 (lateral line) AG, CP, LD, AM + HC regenerate + High-throughput screens - False-negative ototoxins identified - Only one HC type - Immature characteristics of HCs... 

Sensitive to toxins, in this case means that the assay presents no false negative results. Primary hepatocytes can elucidate hepatotoxins, and mouse neuroblastoma cells can elucidate sodium channel-blocking neurotoxins therefore these assays can be used to screen for the appropriate toxins. 

A major consideration in screening is the detection capability of the screen for both false negatives (lack of detection of an active drug) and propensity to find false positives (detection of a response to the compound not due to therapeutic activity of interest). Ostensibly, false positives might not be considered a serious problem in that secondary testing will detect these and they do not normally interfere with the drug discovery process. 

The use of animal models for depression has two main objectives. One is to provide a behavioural model that can be used to screen potential antidepressant treatments. For this, the behaviour does not have to be an animal analogue of depression all that is needed is for it to be consistently prevented by established antidepressant agents (i.e. no false negatives) but not by drugs which have no antidepressant effect in humans (i.e. no false positives). 

Assessment and definition of sensitivity are often described for quantitative analysis but are of equal importance for qualitative devices of the dip-stick type that are very popular for farm- or field-based screening assays. Because of the somewhat subjective nature of visually assessed assays, the assay s sensitivity must be validated using a number of observers to determine at what level a test is deemed positive. The number of false positives and false negatives must be carefully determined in order to balance consumer safety and potential economic loss to animal producers. 

In general the rate of false negatives are by definition difficult to ascertain. There are two general approaches to get a handle on false negatives. The first approach is based on what is known about the aqueous solubility of screening compounds since truly active compounds out of solution are the most common cause of false negatives. One can infer that perhaps 15% of true positives will be missed in an HTS. This inference comes from an analysis of the concordance or lack of concordance between nominal concentrations in DMSO stocks and nominal... 

A second approach to handling false negatives relies on a computational analysis of actives in the primary HTS. Were there analogs or similar compounds to actives that appeared inactive in the original HTS These inactives are retested perhaps in a more careful screen and some of the original inactives will now be found to be active. Most commonly these were truly active compounds that appeared inactive in the original screen because they were not in solution under the initial HTS assay conditions. 

An assay or screen is said to exhibit ROBUSTNESS when it has a high discriminatory power and produces a low number of FALSE NEGATIVE and FALSE POSITIVE results. 

Table 4.3. Scheme of test results for screening procedures tp true positive,)p false positive, tn true negative, fn false negative, n total number of tests...

The effective permeability of ionizable molecules depends on pH, and the shapes of the permeability-pH profiles can be theoretically predicted when the pKa of the molecule is known [7]. The pH dependency of ionizable molecules is illustrated in Fig. 3.3 for a series of weak acids (A. Avdeef, not published). It is clear that if the wrong pH is used in screening the permeabilities of molecules, then highly promising molecules (e.g., probenecid Fig. 3.3) may be characterized as false negatives. The ideal pH to use for in vitro screening ought to reflect the in vivo pH conditions. 

The most commonly used screening method for HIV is an enzyme-linked immunosorbent assay, which detects antibodies against HIV-1 and is both highly sensitive and specific. False positives can occur in multiparous women in recent recipients of hepatitis B, HIV, influenza, or rabies vaccine following multiple blood transfusions and in those with liver disease or renal failure, or undergoing chronic hemodialysis. False negatives may occur if the patient is newly infected and the test is performed before antibody production is adequate. The minimum time to develop antibodies is 3 to 4 weeks from initial exposure. 

The screen must be very sensitive in its detection of potential effective agents. An absolute minimum of active agents should escape detection that is, there should be very few false negatives (in other words, the type II error rate or beta level should be low). Stated yet another way, the signal gain should be way up. 

---