Characteristics of an Immunoassay’s Performance

Being the very initial tests for illicit-drug testing programs, immunoassays are efficiently capable to accurately differentiate between negative and non-negative test samples. NPV – Negative Predictive Value is highly important, this avoids ‘false negative’ results to appear. However, it should also be known that the slope drawn from the reaction changes is adequate so that the threshold concentration distinguishes between the change in drug concentration and the change is the reaction’s response. Therefore, when a manufacturer develops immunoassays for commercial use, it determines its performance characteristic. But, a laboratory should also verify the performance of the assay while it is still in validation period. It should evaluate the assay for its accuracy, range, precision and stability. If need be, it should also be compared with the results of other assays to determine the chances of error. The important characteristics which should be determined in an immunoassay are as follows:

Precision And Correctness

Analytical accuracy estimates how close the resultant value is to the true value. As far as workplace drug testing regimes are concerned, the immunoassay is a part of the initial testing mechanisms and should be dealt with great care. Therefore, a cutoff point is given to which readings are measured according to the cutoff level. Immunoassays are able to give precise results with the help of reagents, temperature control, analyzers and other factors that affect the predictability of the results as far as repetition is concerned. Moreover, while validating an assay, the known content is repeatedly analyzed below and above the cutoff levels so as to judge the precisiveness of the assay. For automated assays, it is highly significant that temperature stability of reagent’s surroundings and compartment, instrument sample along with reagent’s pipetting are carefully monitored. The reagent’s properties and the antibody’s affinity can deeply affect the exactness and correctness of the assay by affecting the reaction’s rate and change in absorbance over time. If proper equilibrium is maintained, then it greatly improves the precision of the assay. The parts of test specimen that might interfere with the assay also affect its accuracy.

Dynamic Change

The response and proportionality of change in analyte’s concentration is measured by sensitivity and linearity. The cutoffs points of drug testing programs are usually set at concentrations that do not really affect the sensitivity of the test’s result. Therefore, the main role of the assay is to separate the negative and non negative samples at cutoff points. Therefore, accuracy of the test is quite enhanced at that point. This limits the overall dynamic range so that the distinctive calibration curves are normally curvilinear with proportional area around the cutoff range (See Figure 2.8). Because the non negative samples are confirmed by further testing after the initial test, therefore the limitation of range is not much of a concern. But, the value of immunoassay is important as it determines the confirmation process and the dilution factors become reduced. This also impacts the comparison between initial and confirmatory tests results in order to review data properly.

The shape of the curve depends on the type of immunoassay test conducted. The test’s ability to demarcate negative and non negative results around the cutoff point is important as it infers the readings according to the slope of the curve at that point. It is now common that the workplace illicit drug testing regimes use immunoassays that easily distinguish non negative and negative samples at ± 20% around the cutoff points at 100% of the time. Therefore, performance anticipation for immunoassays which are used for alternate matrices in which cutoff and drug concentration is comparatively lower can be changed according to the validation data.

Stability And Constancy

It is important that manufacturers take great care to maintain the stability of the reagent which depends on the formulation of reagent and its storage properties. Homogenous immunoassay reagents such as DRI®, Abbott AxSYM®, Emit®  II Plus, etc. are supplied in ready to use liquid phase, with the expiry date written and instructions given on how to handle and store these reagents. The reagent CEDIA® is supplied in lyophilized form requires reconstitution and is stable while it is not opened until the expiry date. It is also stable if it is reconstituted, provided that it is stored from 2-8°C. Reagent such as Online Dat® have different expiry dates which are applicable to unopened kit (i.e. the label date) and opened kit (approx. 56 days). Many automated analyzers have reagent compartments which are refrigerated so that optimum storage conditions are provided. The laboratory should also adjust the size of the kit according to the volume of the specimen so that opened kits can be utilized before expiry date nears, as there is a difference in the expiry dates of opened and unopened kits. Generally seen, RIA and ELISA kits are stable till they reach their label dates.

Most often, it is also seen that calibration is validated as a result of the quality control material’s performance at the concentration which is below and above the cutoff range for the assay. Calibration stability therefore varies according to the type of the assay use, the drug it is used with, depending on the reagent, calibration standard and the use of the instruments. Minimally, laboratory should calibrate such that the frequency matches that which is provided by the manufacturer and according to the daily demands. If circumstances are controlled, then a calibration stability of over several weeks is achieved.

Even though, there is minor differentiation between different assays, it is evident that the correlation between initial and confirmatory screening test results is definitely good enough especially with single compound assays for Benzoylecgonine (a cocaine metabolite) and carboxy THC. However, due to problems of cross reactivity, both amphetamine and PCP assays show lesser confirmation rates for specific (amphetamines) and non specific (PCP) results. However, some cross reactions were diminished when the laboratories used a different cross reactivity immunoassay such as secondary screen (FPIA) and the rate of confirmation improved to more than 80% for KIMS & FPIA and more than 65% for CEDIA and FPIA. The lower confirmatory rate for opiates is more due to the cross reactivity with opoids which are not included in the confirmation tests and the presence of opiates below the confirmation assay cutoffs. The cutoffs for initial screening of the sample in this study were at 2000 ng/mL. Even though, commercial assays for opiates are optimized to detect morphine, they show cross reactivity to codeine to a greater extent and to hydrocodone and hydromorphone to some extent. Therefore, a positive initial test result can be obtained if a sample contains codeine and morphine together in combined phase even though their concentrations are below the cutoff i.e. 2000 ng/mL. Therefore, the confirmation test below the cutoff of 2000 ng/mL would be deemed as negative. Moreover, hydrocodone and hydromorphone’s presence is not evaluated under SAMSHA guidelines, thereby leading to lower overall confirmation rates. In workplace drug testing programs which are non regulated, a broader list of opiate substances are included for the confirmation test, thereby leading to higher confirmation rates for opiate assays. For instance, a group having 106,676 specimens for opiates using KIMS had a cutoff of 300 ng/mL and the confirmation rates increased to 71.2% when the inclusion of hydromorphone and hydrocodone was done along with codeine and morphine. Therefore, while reviewing the performance of an assay, it is quite important to realize the impact of initial test’s correctness and the design of the confirmatory tests.

Correlation

An important performance indicator for immunoassays relating to workplace drug testing programs is to compare the initial and the confirmatory screening test. This comparison should then be evaluated under two important criteria: Firstly, check if the specimens deemed as positive and negative show confirmation by GC/MS technique. Secondly, check if the initial test result when relatively compared to the cutoff can predict the quantified concentrations. The dealer also provides inserts having comparative studies of data which are performed while the assay is being developed and while submitting it to the FDA for authorization. However, this data belongs to a smaller number of samples. Therefore, assay performance is compared over the time for a larger number of data collected from multiple sources to better indicate the performance of the assay. Every laboratory performs validation tests under specified instrumentation and proper environment. In 2003, a study took place to evaluate 4 million laboratory results with 11 SAMSHA-certified laboratories to compare the rates of EIA, KIMS and CEDIA for initial tests. Amongst the 11 laboratories, 6 used EIA-DRI, 3 used KIMS-ONLINE and 2 used CEDIA tests. Results of this study are summarized in Table 2.1 below:

Even though, there is minor differentiation between different assays, it is evident that the correlation between initial and confirmatory screening test results is definitely good enough especially with single compound assays for Benzoylecgonine (a cocaine metabolite) and carboxy THC. However, due to problems of cross reactivity, both amphetamine and PCP assays show lesser confirmation rates for specific (amphetamines) and non specific (PCP) results. However, some cross reactions were diminished when the laboratories used a different cross reactivity immunoassay such as secondary screen (FPIA) and the rate of confirmation improved to more than 80% for KIMS & FPIA and more than 65% for CEDIA and FPIA. The lower confirmatory rate for opiates is more due to the cross reactivity with opoids which are not included in the confirmation tests and the presence of opiates below the confirmation assay cutoffs. The cutoffs for initial screening of the sample in this study were at 2000 ng/mL. Even though, commercial assays for opiates are optimized to detect morphine, they show cross reactivity to codeine to a greater extent and to hydrocodone and hydromorphone to some extent. Therefore, a positive initial test result can be obtained if a sample contains codeine and morphine together in combined phase even though their concentrations are below the cutoff i.e. 2000 ng/mL. Therefore, the confirmation test below the cutoff of 2000 ng/mL would be deemed as negative. Moreover, hydrocodone and hydromorphone’s presence is not evaluated under SAMSHA guidelines, thereby leading to lower overall confirmation rates. In workplace drug testing programs which are non regulated, a broader list of opiate substances are included for the confirmation test, thereby leading to higher confirmation rates for opiate assays. For instance, a group having 106,676 specimens for opiates using KIMS had a cutoff of 300 ng/mL and the confirmation rates increased to 71.2% when the inclusion of hydromorphone and hydrocodone was done along with codeine and morphine. Therefore, while reviewing the performance of an assay, it is quite important to realize the impact of initial test’s correctness and the design of the confirmatory tests.

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