Analytical Specificity

The analytical specificity of an assay is defined as the degree by which the assay detects the specific target yet does not produce a positive result in the presence of nonspecific targets. The analytical specificity of the assay is assessed to verify that the assay only detects the target it claims to detect or analyze and does not crossreact with related targets. In infectious disease testing, one must show that the assay is able to discriminate between the target pathogen and related pathogens. Also, the assay must be able to differentiate between the target pathogen and other pathogenic or commensal flora that might be found in the specific specimen type(s) to be tested by the assay. In genetic testing, it must be documented that the assay can discriminate between the intended genetic sequence and related genes, pseudogenes, or alleles.

If multiple specimen types are to be assayed using the molecular test, specificity of the assay should be verified using all organisms that can potentially be found in the various specimen types. Table 2 gives an example of the various pathogens one would want to test when assessing the analytical specificity for an assay that is designed to detect CMV in either CSF or amniotic fluid. The list of potential pathogens causing meningitis differs from the list of potential pathogens in amniotic fluid, and both groups of pathogens must be tested to verify the specificity of the assay.

Included in the requirement for verification of analytical specificity by CLIA is the requirement for verification of potential interfering substances. A number of studies have identified potential inhibitors to the polymerase chain reaction (PCR), and specimens containing these interfering substances should be avoided (16-21). Most molecular assays will incorporate an inhibitory control that verifies the absence of amplification inhibitors in the extracted nucleic acids of the samples tested. The inhibitory control (sometimes referred to as the "internal control") can be a nucleic acid target inherent to the specimen or can be exogenously added to the specimen prior to nucleic acid extraction. The inhibitory control is extracted and amplified in parallel with the analyte nucleic acid target. Detection of the amplified internal control signifies successful nucleic acid extraction and successful amplification. Documentation of the usage of this control during the verification procedure should fulfill the requirement for verification of this assay parameter.

Table 2

Analytical Specificity of a CMV Assay

Table 2

Analytical Specificity of a CMV Assay

Specimen type

Cerebrospinal fluid

Amniotic fluid

Clinical syndrome associated with

Meningitis

Prenatal infection

infection of specimen type

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