Assay - General Steps of Any Assay

General Steps of Any Assay

An assay (analysis) is never an isolated process and needs to be preceded by certain necessary procedures which are the preanalytic steps and must be followed by certain necessary post analytic steps. The information communication (e.g. request to perform an assay and further information processing) or specimen handling (e.g. collection transport and processing) done before and till the point of beginning of an assay are the preanalytic steps. Similarly after the actual assay is done the result may be documented, verified and transmitted/communicated in steps which are called post-analytic steps related to an assay. Like any multistep information handling and transmission systems, variation and errors in the communicated final results of an assay involves corresponding parts in every such step i.e. not only analytic variations and errors intrinsic to the assay itself but also variations and errors involved in preanalytic and post analytic steps. Since the assay itself (the analytic step) gets a lot of attention, steps that get less attention by the chain of users i.e. the preanalytic and the post analytic steps are often less stringently regulated and generally more prone to errors- e.g. preanalytic steps in medical laboratory assays may contribute to 32-75% of all lab errors.

Assays can be very diverse, but generally involve the following general steps:

1. Sample processing/manipulation in order to selectively present that target in a discernible/measurable form to a discrimination/identification/detection system. It might involve a simple centrifugal separation or washing or filtration or capture by some form of selective binding or it may even involve modifying the target e.g. epitope retrieval in immunological assays or cutting down the target into pieces e.g. in Mass Spectrometry. Generally there are multiple separate steps done before an assay and are called preanalytic processing. But some of the manipulations may be inseparable part of the assay itself and will not thus be considered pre-analytic.
2. Target specific DISCRIMINATION/IDENTIFICATION principle: to discriminate from background (noise) of similar components and specifically identify a particular target component ("analyte") in a biological material by its specific attributes. (e.g. in a PCR assay a specific oligonucleotide primer identifies the target by base pairing based on the specific nucleotide sequence unique to the target).
3. Signal (or target) AMPLIFICATION System: The presence and quantity of that analyte is converted into a detectable signal generally involving some method of signal amplification, so that it can be easily discriminated from noise and measured - e.g. in a PCR assay among a mixture of DNA sequences only the specific target is amplified into millions of copies by a DNA polymerase enzyme so that it can be discerned as a more prominent component compared to any other potential components. Sometimes the concentration of the analyte is too large and in that case the assay may involve sample dilution or some sort of signal diminution system which is a negative amplification.
4. Signal DETECTION (and interpretation) system: A system of deciphering the amplified signal into an interpretable output that can be quantitative or qualitative. It can be visual or manual very crude methods or can be vary sophisticaed electronic digital or analog detectors.
5. Signal enhancement and noise filtering: may be done at any/all of the steps above. Since the more downstream a step/process during an assay, the higher the chance of carrying over noise from the previous process and amplifying it, multiple steps in a sophisticated assay might involve various means of signal-specific sharpening/enhancement arrangements and noise reduction or filtering arrangements. These may simply be in the form of a narrow band-pass optical filer, or a blocking reagent in a binding reaction that prevents nonspecific binding or a quenching reagent in a fluorescence detection system that prevents "autofluorescence" of background objects.