Quality control methods for optimal BCR-ABL1 clinical testing in human whole blood samples

Area of Science:

• Molecular Biology
• Oncology
• Clinical Chemistry

Background:

• Accurate breakpoint cluster region (BCR)--Abelson (ABL) 1 quantification is vital for managing chronic myelogenous leukemia.
• Current methods require optimization for quality control, sensitivity, and reliability in detecting molecular response or treatment failure.

Purpose of the Study:

• To assess the impact of storage time, primer type, and RNA input on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield.
• To optimize reverse transcription (RT) conditions for reliable BCR-ABL1 measurement.

Main Methods:

• Whole blood samples spiked with K562 cells were used to evaluate room temperature storage effects (up to 48 hours).
• Reverse transcription (RT) reactions were performed using either gene-specific primers or random hexamers with varying RNA input.
• BCR-ABL1 levels were normalized to GUSB for sample loading control and to internal standards for reagent and thermal cycler variation control.

Main Results:

• BCR-ABL1/GUSB values significantly decreased (P=0.004) after 48-hour storage.
• Gene-specific primers yielded substantially more BCR-ABL1 cDNA compared to random hexamers across all RNA inputs (up to 158-fold increase).
• Increasing RNA input inhibited RT with random hexamers but not with gene-specific primers.

Conclusions:

• Optimal BCR-ABL1 measurement in whole blood requires gene-specific primers for RT.
• Sample analysis should be performed within 24 hours of blood collection to ensure reliable results.
• The optimized method demonstrates concordance with other laboratories for clinical samples and reference materials.