Page 242 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Chapter 9
Current confirmation practice
Laboratories participating in the largest European proficiency testing program organized by the RfB received the questionnaire by hard-copy. Laboratories in the Netherlands participating in the national proficiency testing program organized by SKML were contacted via e-mail with the questionnaire. Questionnaires were sent via RfB and SKML via their respective subscriber lists. Responding to the invitation was completely voluntary, results were collected by RfB (hard-copy) and SKML (e-mail) and were processed anonymously. Data were collected in Microsoft Excel 2010. The questionnaire contained the three following general questions;
1) Which SNPs in DPYD are currently tested in your lab?
2) Which techniques are used for these SNPs?
3) How is quality controlled? By using:
*two independent techniques, *two technicians using the same technique, *one technician using the same technique on two independent days, *using two independent samples per patient, *other; namely:, *none
The third question was used to identify which confirmation or replication methods for the current genotyping are used.
Two independent genotyping methods as confirmation practice
Two large genotyping laboratories (LUMC and Erasmus MC) performed two independent genotyping tests for over ten years.
At LUMC, both a commercially available TaqMan assay and a home-brew pyrosequencing method were employed to determine the patient’s genotype. The results of each independent method were checked by two independent technicians, and approved by a hospital pharmacist. When results between two methods were concordant, results were added to the digital system. Results and additional notes on results are then automatically available for physicians to consult. From January 2009 results were recorded electronically in a Global Laboratory Information Management System (GLIMS, version 8.10.6, MIPS©). For CYP2D6, the FDA-approved AmpliChip (Hoffmann-La Roche, Basle, Switzerland)1 was used, until discontinuation of the AmpliChip by Roche forced switching to the GenoChip CYP2D6 macroarray (PharmGenomics GmbH, Mainz, Germany) in April 2015.2 No second independent genotyping platform was used for the AmpliChip and GenoChip variants.
At Erasmus MC, commercially available TaqMan assays and Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) were used, in addition to AmpliChip, CYP2D6 kit (Luminex Corporation, Austin, TX, USA), CYP2C19 INFINITI (AutoGenomics, Carlsbad, CA, USA), DMET (Affymetrix/Thermo Fisher Scientific, Waltham, MA, USA). The results of each method were checked by two independent technicians, and approved by a clinical chemist. Results were recorded in the electronic hospital information systems EZIS and HiX (ChipSoft©) from April 2005. For CYP2D6, the FDA-approved AmpliChip was used, until discontinuation of the AmpliChip by Roche forced switching to the FDA-approved Luminex CYP2D6 Assay3 and CE-IVD marked INFINITI CYP450 2D6-BC Assay.4
The AmpliChip included 25 SNPs in CYP2D6, copy number variation in CYP2D6, and three SNPs in CYP2C19. The GenoChip is a macroarray for eleven SNPs and a deletion in
240