Relative versus absolute quantitation

There are strong arguments for the use of standard curves and absolute quantitation; however most clinicians want to know how an individual patient is responding to treatment. There has been much discussion of the most meaningful units for expression of MRD results; copies/pL of blood (most MRD monitoring for acute leukemia is performed on bone marrow); copies/pg of RNA (this is only an indication of the RNA concentration and cDNA synthesis efficiency). Perhaps the most useful is to express the ratio of test gene to a control gene expression level (i.e. the target amount is divided by the control gene amount to obtain a normalized target value), however this is an expensive option. Most diagnostic laboratories insist on the inclusion of standard curves with each batch of test samples. Centers dealing with leukemia patient samples require to provide quick turnaround time; may have limited budgets; have access to a shared instrument only, and perform multiple assays per run; therefore multiple standard curves prove expensive for small numbers of specimens. Larger centers with high throughput use absolute quantitation; the reproducibility achieved by the best instruments is such that it is not necessary to run a standard curve with each batch of specimens. However, the principal investigator must ensure that the standard curve data are robust and not subject to individual operator variation; it is vital that the low control is included in each run if sensitivity is being reported. Figure 16.1 illustrates the use of absolute quantitation of BCRABL and ABL and Figure 16.2 illustrates the expression of the results as a percentage ratio of BCRABL/ABL of an individual patient.

Relative quantitation using the diagnostic specimen as the calibrator may be the method of choice. Results can be expressed as log reduction from the time of diagnosis. This is informative to the clinician and patient. Reports must state clearly that the transcript level is relative to the diagnostic level, not the sensitivity of the assay as AACt can result in MRD levels of <1.00E-07. Once validated, relative quantitation assays are convenient to run as multiple assays can be performed on one plate. Cell lines may be used as sources of calibrator samples; however the data obtained are relative to an arbitrary calibrator and do not reflect the percentage of malignant cells directly, this approach is useful for examining serial specimens from patients.

The final choice of method will depend on the circumstances of the individual patient and laboratory. Our laboratory uses both absolute quantitation expressing results as a ratio of test gene to control gene and relative quantitation using the individual patient's diagnostic sample as calibrator for their follow-up specimens. However the transcript ratio is established for the diagnostic sample by absolute quantitation and regular quality control is performed using standard curves.

Figure 16.1

Standard curves for absolute quantitation of BCRABL in patients undergoing treatment for CML using cABL as control gene.

Figure 16.1

Standard curves for absolute quantitation of BCRABL in patients undergoing treatment for CML using cABL as control gene.

0 0

Post a comment