An In-House Method for Molecular Monitoring of BCR-ABL.

"DISCUSSION: RQ-PCR is a technically demanding, yet very powerfultool. The exquisite sensitivity of the assay is also its weakness.It is essential that extreme care be taken to avoidfalse-positive results due to cross-contamination andcarry-over of RNA/DNA from a previous amplification.The calibrators themselves are the greatest threat of contamination,as they are cloned DNA containing DNA targetregions. Their preparation, storage, and handling shouldnot be performed in the same laboratory in which patientsamples are processed. All equipment, including pipettesets, kits, reagents, paper, pens, workbooks, and lab coatsshould be dedicated for use only in that particular laboratory.It is of great importance that the rules of good laboratory practice be followed when setting up PCR assaysand during post-PCR processing of samples. Though notaddressed herein, there are excellent reviews of recommendedpre-PCR procedures in the literature, includinghandling of blood samples, RNA extraction, cDNA synthesis,and reverse transcription [8,9,10,17]. A dH2O-negativecontrol (BCR-ABL negative cells), along with low and highpositive controls should be included in each RQ-PCR runto monitor assay performance. Any changes in technique,protocol, or instrument should be accompanied by a thoroughevaluation. Standard curves should be generatedafter opening a new real-time kit or when a new batch ofprimer/probe is diluted. : Over the past five years significant advances leadingto more wide-spread adaptation of RQ-PCR for BCR-ABLhave occurred. Implementation of the IS has improved thecomparability of results between laboratories, and recentlyaccredited reference reagents for BCR-ABL quantificationhave been developed [7]. It is essential that more diagnosticcenters in Turkey qualify to report BCR-ABL resultsaccording to the IS. In essence, the procedure involvesassignment of a laboratory-specific conversion factor (CF)to convert BCR-ABL measurements to the IS [15]. LogBCR-ABL values of the same sample set are compared toreference and local laboratories via linear regression [17].The results are considered linear when the correlationcoefficient of any 2 laboratories is >0.98 [17]. The prerequisitefor the procedure is that all in-house validationtests be performed and confirmed prior to application. Inthis context, the present study aimed to describe a costeffective,in house method for BCR-ABL quantificationand to illustrate an example for RQ-PCR validation testing,as well as to provide a description of DNA plasmidsthat may be implemented into any RQ-PCR methodologyto quantify BCR-ABL transcripts. The primary advantageof the presented methodology over widely used commercialkits—in addition to being cost effective—is that onceoptimized and validated, both absolute and relative quantificationcan be performed, whereas most commercial kitsare restricted to providing relative quantification results.In conclusion, the methodology described herein is suitablefor implementation into any RQ-PCR instrument and/or kit for quantifying BCR-ABL transcripts. : Conflict of Interest Statement: The authors of this paper have no conflicts of interest,including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materialsincluded."