Sensitivity

The sensitivity that can be obtained by real-time PCR analysis depends on several variables; some target independent and specimen dependent, i.e. specimen sensitivity; others dependent on the nature of the target, i.e. assay sensitivity. The assay sensitivity can be established by performing dilution experiments, these can be dilutions of patient material, cell lines or plasmids. The potential presence of fusion genes in healthy individuals means that MRD levels of less than 10-6 should be interpreted with caution. Sensitivity of allele-specific oligonucleotide (ASO) assays for rearranged antigen receptor genes are usually established by performing dilution of the patient's leukemic cell DNA in mononuclear cell DNA from several donors.

However, the values obtained do not reflect the sensitivity obtained for the actual test sample. This is dependent on the quality and quantity of the template used. If absolute quantitation has been used, specimen sensitivity can be estimated by dividing the maximal reproducible sensitivity of the test gene by the copy number of the control gene, e.g. in an assay where the 10 copy/l standard is the limit of reproducibility and the ABL gene copy number is 2.3 x 104/^l; the sensitivity is 10/2.34 x 104or 4.27 x 10-4.

For DNA based ASO approaches the test DNA is usually quantified by amplification of a control gene such as B2M or ALB in an absolute quanti-tation assay using genomic DNA of known concentration to prepare standards.

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