produce a signal in the measuring system.”
Assay interference can be “analyte dependent or analyte
independent” and can increase or decrease the measured
Increase (positive interference) is due to lack of specificity.
Decrease (negative interference) is due to lack of
Assay interference can be of different types:
¾ Postanalytical errors (Fig. 22.18).
All factors associated with the procedures before the actual
performance of the test are known as preanalytical errors.
Such as incorrect sampling times and environmental
factors such as smoking, etc. may change analyte
concentration and consequently interpretation.
There are many factors that constitute this.
Blood collection procedure and time of collection.
Certain hormones are affected by the time of collection.
For all immunoassays, serum is the matrix of choice.
Samples collected in to tubes containing sodium fluoride
may be unsuitable for some enzymatic immunoassay
methods; preservation with sodium fluoride may affect
results. Impurities in tracers interfere with direct dialysis
FIG. 22.17: Analyzers (Courtesy: Lilac Medicare)
Lipemia may cause interference with assays for fat-soluble
Stability and storage of reagents and samples are as
Certain procedures before the test is performed like:
• Bringing the kit to the room temperature
• Checking the incubator temperature
• Checking the room temperature
• Formatting and arranging the workbench
• Planning for assays to be performed
• Maintaining the fridge temperature
• Dilution of samples with distilled waterinstead of zero
These form the major part of all errors that affects the
results in immunoassays. Mostly, these are overlooked and
so attention is not paid to rectify them. These will lead to
erroneous results. They are mainly the procedural errors.
They are mostly confined to the different steps—addition,
washing, incubation, dilution, pipetting, reading the
protocol suggested by the manufacturer is not followed.
Some of the errors are as follows:
¾ Non-removal of all wash solution from wells or tubes
¾ Not following soak time (if present) during washing
¾ Leaving bubbles in the well after washing
¾ Not tapping the well after washing
¾ Use of contaminated wash buffer
¾ Wells/tubes falling off while washing.
FIG. 22.18: Factors affecting EIA
¾ Pipette/dispenser not primed
¾ Using same tips to break bubbles.
¾ Incubators not maintained at right temperature
¾ Heating not uniform throughout
¾ Washer probes blocked, contaminated
¾ Refrigerator not maintaining right temperature
¾ Defrost water falling on kits
¾ Use of dry incubators instead of water bath
¾ Instrument filters not checked periodically.
¾ Wells not covered during incubation
¾ Not running all calibrators to plot a graph
¾ Use of contaminated or uncleaned tubes to prepare
¾ Using kits/reagents beyond expiry
¾ Not mixing after adding stop solution.
The errors that occur after the performance of the test are
called as postanalytical errors. These are like:
¾ Choosing a wrong graph scale
¾ Comparison of result with inappropriate reference
¾ Not correlating the results with clinical history
¾ Transcription error when report is prepared.
The reference ranges (normal ranges) of various parameters
are different. Manufacturers specify the reference ranges.
The units for reference ranges may differ. One should only
compare with reference intervals given in the pack insert
or should establish their own reference interval.
Comparison with results of different reference intervals
will create confusion. Many laboratories make the mistake
of comparing results with other labs without knowing the
assay conditions and other factors that affect ELISA.
The units given by manufacturers may be different. For
example, T3 ng/dL is different from ng/mL. A kit having
a sensitivity of 0.4 ng/dL is more sensitive than 0.4 ng/mL
One should always make note of the units. Reports from
different laboratories may differ in this aspect and create
The errors may be of any kind, but the outcome is that
the result is incorrect and hence, a wrong report is given.
To overcome this, one should follow all the steps and
adhere to the protocol strictly.
ELISA is a technique of multiple steps. The steps must
be followed strictly to achieve good results. Errors at any
levels will affect the final result. Complete understanding
of the process is necessary for troubleshooting.
Factors affecting EIA are shown in Figure 22.18.
In washing plate manually, the most important factor is
that each well receives the washing solution so that, no air
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