Establishment and validation of freeze dried cell lines as reference materials for the standardisation of quantitative bcr-abl1 measurements on the international scale

BIANCHINI M; RUIZ MS; FERRI C; ICARDI G; MEDINA MS; TAPIA I; BARRIO MM; MORDOH J; LARRIPA I

Estoril

Congreso; iCMLf International Conference on CML - Biology and Therapy; 2013

ESH

BACKGROUND: Current guidelines for managing chronic myeloid leukemia (CML) include molecular monitoring of BCR-ABL1 by quantitative reverse-transcription PCR (RT-qPCR). Despite the proven prognostic significance of molecular response, it is not widely appreciated that RT-qPCR potentially produces highly variable quantitative data, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve this is the use of an international reporting scale (IS). Conversion to the IS is achieved by the application of laboratory specific conversion factors (CF). AIM: Our goal was to establish and validate secondary reference panels to mitigate the inter-laboratory imprecision of quantitative BCR-ABL1 measurements, and to facilitate local laboratory standardization on the IS. METHODS: we employed 2 different methods for RT-qPCR that varied in the primers and probes used: i) Molecular MD protocol (MD) or ii) adapted EAC protocol (aEAC) for ABL1 as control gene. Both methods are based on one-step RT-qPCR approach and TaqMan chemistry. PCR platforms used were Rotor-Gene (Qiagen) or ABI7500 (Applied Biosystem). To obtain the conversion factor for MD protocol on Rotor-Gene, we sent 60 RNA patient samples to the Australian reference laboratory in Adelaide. The secondary reference material panel, comprising 4 different dilution levels of K562 cells in HL60 cells, was lyophilized by an automated freeze-drying process with AdVantage-Plus freeze dryer (VIRTIS). RESULTS: The antilog of the estimated mean bias (-0.348) was designated as the conversion factor (CF=0.45) for our method (MDIS). Our pilot analysis indicated us that a 1:10 mixture of K562:HL60 (K/HL01) cells yielded a BCR-ABL/ABLIS value of approximately 10%. Three further 1:10 dilutions (K/HL02, K/HL03 and K/HL04) were made into HL60 cells and 60 ampoules filled with 1x106 cells were prepared. Stability studies of the freeze-dried cells, showed no significant reduction in the copy number or changes in the BCR-ABL/ABL ratio. Next, fixed %BCR-ABL/ABLIS values to reference materials were assigned according to the MDIS method (12.45%, 1.71%, 0.132% and 0.006% for K/HL01, K/HL02, K/HL03, K/HL04 respectively). To evaluate the utility of reference panel we used it with the aEAC protocol in 2 laboratories, obtaining 2 specific CFs: 2.17 (95% LOA; 3.21-1.46) on Rotor-Gene and 1.34 (95% LOA; 2.49-0.73) on ABI7500. After CF-correction of the individual raw ratios, the average difference was <1.2 in both laboratories. For the evaluation of the agreement between methods we determined MMR concordance; we observed 90.5% concordance for aEACIS method on Rotor-Gene and 92% concordance for aEACIS on ABI7500. Importantly, the aEAC methods showed a higher sensitivity when compared to MD; 2 undetectable cases (MR5.0) by MD method were quantifiable by aEAC (resulting in MR4.5). CONCLUSIONS: cellular calibrator panels provide robust reagents for standardization to the IS, well-suited for any control gene, allowing harmonization of all steps involved. The availability of secondary reference reagents will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs.