Quality Control Methods for Optimal BCR-ABL1 Clinical Testing in Human Whole Blood Samples

Abstract

Reliable breakpoint cluster region (BCR)--Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 10(3) GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 10(3) GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection.

A DNA Real-Time Quantitative PCR Method Suitable for Routine Monitoring of Low Levels of Minimal Residual Disease in Chronic Myeloid Leukemia

“…4 logs above the limit for DNA qPCR, we note that recent modifications of the RT-qPCR assay for BCR-ABL1 have been claimed to provide greater sensitivity. 15,16 However, increasing the sensitivity of RT-qPCR carries the risk that false-positive results will be observed owing to the presence of rare BCR-ABL1 transcripts derived from nonleukemic cells. 8 Because rare targets have been detected using recently developed dedicated digital PCR instruments, we also investigated use of the QX100 system (Bio-Rad Laboratories) to perform digital PCR on CML samples.…”

A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

“…We recently reported that use of gene specific priming in RT increases yield of cDNA by 10–100 fold compared to oligo dT or random hexamer priming when applied to RNA from human peripheral blood leukocytes [26] . Because FFPE treatment typically reduces yield of cDNA from RNA by 100-fold, we evaluated the utility of gene specific priming relative to random hexamer priming to increase signal.…”

“…The effect of different conditions on reverse transcription (RT) efficiency, including priming with random hexamer primers (RHP) or GSP and use of 1 or 5 µg of RNA in the 30 µl RT reaction, was assessed with three FFPE samples. A previously described test using the External RNA Control Consortium (ERCC) standards was used to measure RT efficiency [26] . After completion of these studies, optimal RT conditions were selected consisting of a 30 µl RT reaction with 1 µg of RNA, gene-specific RT primer (the PCR reverse primer) (3 µ M ), and SuperScript III First Strand Synthesis System (Life Technologies, Grand Island, NY).…”

BCR-ABL1 (p210) Transcript Kinetics

“…The technique is very well described, 1 has been in use for many decades, 2 and has several advantages, including identification of preanalytical errors such as specimen misidentification and instrumentation errors. 3 Within the realm of molecular diagnostics, in contrast, most quality control in the field has taken cues from high-volume clinical chemistry laboratories, focusing on detecting preanalytical issues such as DNA integrity and input quantities 4 or analytical issues like DNA contamination or interrun variability with Levey-Jennings plots. 5,6 However, no studies that we know of have looked at the utility of intrapatient variability (delta checks) in monitoring molecular assays.…”