Laboratory Issues

The variability of results of PCR testing for IGH rearrangements and IGH/BCL2 translocations among clinical molecular laboratories has been documented multiple times over the past several years by the proficiency testing surveys of the College of American Pathologists,47 as well as by other independent interlaboratory surveys.48 It is of particular concern to clinicians, and to those setting up and running clinical treatment trials, that multiple inter-laboratory surveys have documented a high level of false-negative results for these PCR assays, as well as a lower but still significant number of false-positive results. The reasons for the lack of reproducibility among laboratories are multiple and not easily overcome. Some strategies for PCR testing and interpretation that have been shown to improve testing performance are discussed below.

Use of Appropriate Controls

Appropriate controls include positive, negative, sensitivity, and no DNA reactions. Many laboratories do not know or overestimate the diagnostic sensitivity of their IGH and IGH/BCL2 PCR assays, possibly because the sensitivity of the assay is established at the time of test validation, but sensitivity controls are not included in every run. Interlab-oratory surveys also indicate that some laboratories do not use adequate controls to allow accurate interpretation of results. An additional important PCR control, particularly with paraffin-embedded tissue, is the amplification of a nonrearranging gene to document the presence of adequate DNA in the tested sample and the absence of inhibitors of the Taq enzyme, to prevent reporting of a false-negative result.

Amplification of Duplicate Aliquots of DNA

In small samples or samples with few B cells, there are not always sufficient B cells to produce a polyclonal background. In this setting, PCR can amplify rare B cells, producing a misleading monoclonal or oligoclonal pattern of amplification for IGH PCR assays. Likewise, a sensitive IGH/BCL2 assay capable of detecting MRD will also amplify a rare benign translocation-carrying cell, as previously discussed. Overinterpretation of a single band on IGH and IGH/BCL2 PCR assays by laboratories not performing the assays in duplicate likely represents the most common cause of false-positive results for these assays. With amplification of duplicate aliquots side by side, only clearly visible bands of identical size in each duplicate should be called positive, not nonreproducible or weak bands. Bands seen in analyses of specimens containing rare B cells, or rare normal translocation-carrying cells, will not be reproducible.

Adherence to Strict Criteria for Interpretation and Reporting

The interpretation of assays for IGH rearrangements, the IGH/BCL2 translocation, and other BCL-associated translocations must be performed with caution. It is apparent that even the most commonly performed PCR assays for IGH and IGH/BCL2 have an unacceptably high level of erroneous results. Since no universal standards for interpretation of these assays have been delineated thus far, PCR results should be very carefully interpreted and reported, preferentially in conjunction with clinical history, mor phology, and immunophenotyping data. Reports should clearly indicate that a false-negative result is a possibility. Use of SBA as a backup for negative or equivocal IGH PCR results is helpful in eliminating false-negative and false-positive IGH PCR results, but it may also substantially delay obtaining a final result. Clinical molecular laboratories must develop mechanisms to avoid reporting irrelevant IGH/BCL2 translocations as MRD. Labor-intensive methods such as sequencing of PCR products and fluorescence-based PCR-single-strand conformation polymorphism (SSCP) analysis are not practical for routine clinical practice,but most false-positive IGH/BCL2 PCR results can be avoided by performing the assay in duplicate. Parallel analysis of the original BCL with a posttreatment sample being evaluated for MRD will confirm that the rearrangement is detectable by the specific PCR assay and will allow comparison of PCR amplification product sizes, which is very helpful in ruling out false-negative and false-positive results (Figure 32-3). False-negative results for MRD detection occur most frequently with inadequate test sensitivity. Laboratories performing testing for MRD must offer a sensitivity level of 1 in 104 to 106.

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