1. a.. ;.4-1..A.Ait.1.1,,,..,.....ac...06-ravuv.renr,....“. va...-aunowavAnactoinkvAinask.P.WAnitlAWL131.11aWiLnia212.111idaaa+effklAtaiSa,A2,51W Vascular Pharmacology ELSEVIER Vascular Pharmacology 39 (2002) 127-129 www.elsevier.com/locate/vph Internal quality control of PCR-based genotyping methods: practical experiences Else-Marie Bladbjerg a '", Jorgen Gram', Jorgen Jespersena ' b , Moniek P.M. de Maat' b' 'Department for Thrombosis Research, University of Southern Denmark, Esbjerg, Denmark bDepartment of Clinical Biochemistry, Ribe County Hospital, Ostergade 80, 6700 Esbjerg, Denmark sGaubius Laboratory TNO-PG, Leiden, The Netherlands Received 1 August 2002; accepted 1 August 2002 Abstract Internal quality control programmes for genetic analyses are needed. We have focused on quality control aspects of selected polymorphism analyses used in thrombosis research. DNA was isolated from EDTA-blood (n=500) and analysed for 18 polymorphisms by polymerase chain reaction (PCR), i.e. restriction fragment length polymorphisms, allele specific amplification, or amplification of insertion/ deletion fragments. We evaluated the following aspects in the analytical procedures: sample handling and DNA-isolation (pre-analytical factors), DNA-amplification, digestion with restriction enzymes, electrophoresis (analytical factors), result reading and entry into a database (post-analytical factors). Furthermore, we evaluated a procedure for result confirmation. Isolated DNA was of good quality,(42 . 14/m1 blood, A260/A280 ratio > 1.75, negative DNAsis tests). Occasionally, results were reanalysed because of positive reagent blanks (<1%) or because of problems with the controls (<5%). On confirmation, we observed four genotyping discrepancies. Control of data handling revealed 0.1% reading mistakes and 0.5% entry mistakes. Based on our experiences, we propose an internal quality control programme for widely used PCR-based haemostasis polymorphism analyses. 2002 Elsevier Science Inc. All rights reserved. 1. Background In recent years, analysis of genetic variation is increas- ingly integrated in the analytical repertoire of the clinical biochemistry laboratory. This development urgently in- creases the demand for quality control programmes. Quality control can be separated into external and internal quality control and recently, a few external quality control programmes for genetic methods have been introduced (Braun et al., 1998; Neumaier et al., 2000; Dequeker et al., 2001). However, there has not been much focus on internal quality control, which includes control of preanalytical factors, analytical factors, and postanalytical factors. The preanalytical phase deals with collection of the patient sample in a standardised way, verification of the patient ID, sample handling, and DNA isolation. In the analytical phase, the sample is analysed, which includes DNA amp- lification by polymerase chain reaction (PCR), digestion • Corresponding author. Department for Thrombosis Research, Depart- ment of Clinical Biochemistry, Ribe County Hospital, Ostergade 80, 6700 Esbjerg, Denmark. Tel.: +45-79-182423; fax: +45-79-182430. E-mail address: emb®ribeamidk (E.-M. Bladbjerg). with restriction enzymes, and separation of the alleles using electrophoresis. The postanalytical phase deals with process- ing and reporting of the results, and this includes reading of the genetic results and entering of the results into a database. Potential sources of errors in genetic analyses and steps that require special precautions have been described in detail (Neumaier et al., 1998; Burkardt, 2000), but to our know- ledge, there has been no reporting on experiences with internal quality control of genetic analyses. We have focused on quality control aspects of selected polymorphism analyses in haemostasis, inflammation, and blood pressure regulation genes. The methods that are used in this evalu- ation cover the most used methods of genotyping. Based on our experiences, we propose an internal laboratory quality control programme (Bladbjerg and De Maat, 2002). 2. Materials and methods 2.1. Blood samples and DNA analyses DNA from 500 blood donors was isolated from leuko- cytes by ammonium acetate precipitation, diluted to 50 ng 1537-1891/02/$ — see front matter 0 2002 Elsevier Science Inc. All rights reserved. P11: S1537-1891(02)00299-9