Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 49: 782-791, 2003; 10.1373/49.5.782
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Raggi, C. C.
Right arrow Articles by Orlando, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Raggi, C. C.
Right arrow Articles by Orlando, C.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Laboratory Management
(Clinical Chemistry. 2003;49:782-791.)
© 2003 American Association for Clinical Chemistry, Inc.

External Quality Assurance Program for PCR Amplification of Genomic DNA: An Italian Experience

Claudia Casini Raggi1, Pamela Pinzani1, Angelo Paradiso2, Mario Pazzagli1 and Claudio Orlando1,a

1 Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, viale Pieraccini 6, 50139 Florence, Italy.

2 Clinical Experimental Oncology Laboratory, National Cancer Institute, 70126 Bari, Italy.

aAuthor for correspondence. Fax 39-055-4271-413; e-mail c.orlando{at}dfc.unifi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: External quality assurance (EQA) programs for diagnostic tests based on nucleic acid amplification have not been widely implemented in clinical laboratories and remain limited to few tests. Development of specific EQA programs based on application-based proficiency testing for any diagnostic molecular target is challenging. Development of EQA trials based on methodologic proficiency testing and directed to the evaluation of analytical aspects common to the majority of PCR-based tests may be valuable.

Methods: We developed an EQA program for evaluation of DNA extraction and amplification and analysis of products after PCR. Participants received a package containing primers and reference materials to evaluate three specific controls for, respectively, DNA extraction (quality and quantity), PCR performance (specificity and efficiency), and interpretation of results after electrophoresis. Each participant was asked to return to the organizers a form with their numerical results and an aliquot of all amplified samples for joint evaluation.

Results: Results varied in all phases of the experimental procedure: preamplification, amplification, and post-PCR interpretation. To give a general estimation on the quality of performances for each laboratory, we designed a score scheme in which the results of any specific action were evaluated on the basis of the distribution around the median consensus values. The maximum possible score was 84. On the basis of total score obtained by each laboratory, we created a qualitative ranking list that provided the final interpretation of results as excellent (>63 points; n = 4 laboratories), good (53–63 points; n = 13), sufficient (42–52 points; n = 15), poor (31–41 points; n = 3), and not acceptable (<31 points; n = 4).

Conclusions: This survey demonstrates the importance of EQA trials based on methodologic proficiency testing directed to evaluation of analytical aspects common to the majority of PCR-based tests.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The role and importance of appropriate schemes for external quality assurance (EQA) in medical laboratories were reviewed recently (1). Most EQA schemes developed to verify the quality of laboratory performance are now evolving toward a more general concept of quality assurance, including the evaluation of method performance, postmarket surveillance, training, and technical support (2).

In recent years, molecular biology-based methods have become widely used in clinical laboratories, primarily because of the great versatility of PCR-based methods. The high sensitivity of amplification techniques and the complexity of the correlated molecular methods have produced an obvious demand for assay standardization and quality assurance. The IFCC has included participation in EQA schemes as a key tool for quality assessment of molecular amplification methods applied to clinical diagnostics (3). In a recent report, this requirement was considered to be a cornerstone of molecular testing to ensure a correct diagnosis (4).

The importance of proficiency testing in molecular genetics was recently emphasized in the US (5) and in Europe (6)(7)(8). Quality assessment programs were recently developed for Huntington disease (9), cystic fibrosis (10)(11), familial thrombophilia (12), factor V Leiden mutations (13), and Y-chromosome microdeletions (14). Proficiency testing programs were also developed for molecular diagnostic testing of infectious diseases such as the hepatitis B (15), hepatitis C (16), and hepatitis G viruses (17); enterovirus (18); JC virus (19); herpes simplex virus (20); cytomegalovirus (21); HIV-1 (22)(23)(24); toxoplasmas (25); and Mycobacterium tuberculosis (26). Finally, in molecular cancer diagnosis, only a few multicenter studies have been performed for quality-control evaluation of PCR-based tests in leukemia diagnosis (27)(28)(29)(30) and in the evaluation of multidrug resistance gene 1 expression detected by reverse transcription-PCR (RT-PCR) or Northern blot analysis (31).

In spite of these rare examples, EQA programs for many diagnostic tests based on nucleic acid amplification have not been implemented on a regular basis. This limitation is particularly evident for rare diseases and for diagnostic tests in an experimental phase. In addition, considering the large variability of methodologic variants and diagnostic approaches, the development of specific EQA programs based on application-based proficiency testing for any diagnostic molecular tool remains challenging (3). A valuable alternative to this limitation is the development of EQA trials based on methodologic proficiency testing and directed to the evaluation of analytical aspects common to the majority of PCR-based tests.

This alternative procedure was first proposed by the Central Reference Institution of the German Society of Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM), which demonstrated the value of this approach and its applicability to all diagnostic laboratories, independent from their specific field of specialization (32)(33). The main innovation of this program, compared with conventional EQA programs, is that the evaluation of results from each participating laboratory is based on two distinct analyses. The first analysis is based on the data reported by participants (i.e., quantity and quality of DNA as obtained by spectrophotometric measurement and number and size of amplicons after gel electrophoresis). The second analysis, performed in the center responsible for the EQA program, involves simultaneous densitometric analysis of all post-PCR products to evaluate the efficiency of amplification (34).

We have developed an EQA program for the evaluation of DNA extraction and amplification and the analysis of products after PCR. Our program involves three specific control levels: (a) DNA extraction (quality and quantity); (b) PCR performance (specificity and efficiency); and (c) interpretation of the results after electrophoresis. The scheme is being carried out under the auspices of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) and was included in the "National Concerted Action for Quality Control in Oncological Laboratories" of the Italian Ministry of Health. At present, two rounds of studies have been performed. The first involved 16 laboratories (35), and the second involved 39 Italian laboratories. Here we present the results of this second pilot EQA study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
experimental design
A total of 39 laboratories joined the EQA program, including 26 public hospital laboratories, 11 academic laboratories, 1 private laboratory, and 1 industrial laboratory. Each laboratory received a package containing 11 vials (3 containing primer pairs, 1 containing reference DNA, 3 containing DNA calibrators, and 1 containing a blood sample). Each participating laboratory was asked to return to the organizers a preprinted information form completed with all the requested numerical results (see below). In addition, the reference DNA and the DNA extracted from the blood sample were assessed by separate PCRs using the three pairs of primers. For each experiment, PCR was run in duplicate. For the routine evaluation of PCR performances, each participant used one vial of each duplicate for gel electrophoresis, whereas the second vial was returned to the organizers by express courier for the overall evaluation of PCR amplification products.

primers
Each laboratory received six vials containing three pairs of primers, identified as primer pairs 1A-1B, 2A-2B, and 3A-3B. Each vial contained 20 µL of 20x primer solution, ready to use. Primers were designed to amplify the following: primer pair 1A-1B, region 3901–4010 of the epidermal growth factor receptor gene (EGFR; GenBank accession no. NM_005228); primer pair 2A-2B, region 82627–82803 (exon 14) of the type 2 neurofibromatosis gene (accession no. Y18000); and primer pair 3A-3B, region 99198–99372 (exon 17) of the type 2 neurofibromatosis gene (accession no. Y18000). The primer sequences were as follows:

    Primer 1A: 5'-AGC CAT GCC CGC ATT AGC TC-3'

    Primer 1B: 5'-AAA GGA ATG CAA CTT CCC AA-3'

    Primer 2A: 5'-CTG TGC TTG TAT GAC CCA AG-3'

    Primer 2B: 5'-AGG GCA CAG GGG GCT ACA-3'

    Primer 3A: 5'-CTC TCA GCT TCT TCT CTG CT-3'

    Primer 3B: 5'-CCA GCC AGC TCC TAT GGA TG-3'

reference DNA
A DNA sample, designated "Reference DNA", was prepared from a pool of human leukocytes, collected from healthy volunteers, by a standard phenol–chloroform method and resuspended to a concentration of 0.04 µg/µL with an A260 nm/A280 nm ratio of 1.75 ± 0.05 (n = 10). To this sample we added DNA containing a synthetic target for the EGFR gene, prepared with the PCR overlap technique as described previously (36), to generate an additional PCR fragment that was 26 bp longer than the corresponding natural counterpart. For this reason, amplification of Reference DNA with primers 1A-1B produced two PCR products of different sizes (110 and 136 bp, respectively). Competitor target was added to Reference DNA in a pretested concentration to amplify the natural target and the synthetic competitor with comparable amplification efficiency and to give a densitometric ratio, after gel electrophoresis, close to unity (0.97 ± 0.09; n = 10). The remaining primer sets generated a single band each. Participants received 40 µL of Reference DNA solution, ready to use.

blood sample
Each participant received a sample vial (hereafter referred to as "Blood Sample") containing 2 mL of blood from an EDTA pool, tested to exclude the presence of HIV and hepatitis C virus.

standard DNAs
Participants received three different samples of DNA (hereafter referred to as "Standard DNA 1, 2, and 3") extracted from a blood pool collected from 10 healthy male and female volunteers. All three reconstituted samples contained 4 µg of DNA/tube and variable amounts (0, 10, and 20 µL) of a solution containing 50 ng/µL bovine serum albumin. These variable DNA/protein ratio samples were prepared to produce different readings at 260 nm and different A260 nm/A280 nm ratios. These samples (100 µL) were ready to use and were used for spectrophotometric measurements only.

instructions
Participants received detailed instructions for actions to be performed on reference materials, and they were asked to perform six PCR amplifications, using 100 ng of Blood Sample DNA and 3 µL of Reference DNA solution with the three sets of primers. Participants were requested to use the Taq polymerase, deoxynucleotide triphosphates, and other reagents commonly used in their own laboratories. The number of cycles was always 40 for PCR amplifications. For amplification with primer pairs 1A-1B and 3A-3B, PCR cycling conditions were 30 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C, and a final extension of 7 min at 72 °C. For primer pair 2A-2B, the annealing temperature was 58 °C.

actions
Participants used the Standard DNAs only to determine the quality and quantity of DNA, using their own spectrophotometric procedure. Results had to be reported on the result form.

For extraction of DNA from the Blood Sample, participants were requested to use 1 mL of blood and their own protocols. After extraction, DNA was suspended in 50 µL of Tris-EDTA or H2O and quantified spectrophotometrically (A260 nm and A260 nm/A280 nm ratio). The participants were requested to report the DNA concentration and A260 nm/A280 nm ratio in the result form.

PCR amplification of Blood Sample DNA and Reference DNA was performed in duplicate with the provided three sets of primers. One of the duplicates was used by participants to visualize their results and to provide a subjective evaluation of PCR results in terms of the number and sizes of PCR products after conventional gel electrophoresis (2% agarose). These results were reported on the result form. In addition, the participants were asked to ship the remaining duplicate samples (six tubes containing the post-PCR targets obtained from Control DNA and Sample DNA with the three pairs of primers) to the program organizers for an all-inclusive and simultaneous evaluation of all amplified samples.

evaluation
The completed EQA information forms and samples were received back from all 39 laboratories. The laboratories had completed all the procedures, with the exception of one laboratory that did not provide the results of DNA measurements. The numerical results provided by each laboratory were compared with the consensus median of all laboratories. The variability for each result was expressed as the CV (%).

The six post-PCR samples from each laboratory were checked simultaneously by gel electrophoresis (2% agarose), using 12 µL of PCR amplification products, to verify the number and sizes of the bands corresponding to the respective amplicons. An image of each gel was acquired by Kodak Image Station 440 CF (NEN) and measured by fluorescence analysis with relative densitometry measurements after background subtraction, as described previously (34). To compare the results of different gel runs, the intensity of each band corresponding to PCR products was normalized against the fluorescence produced by the 501-bp band in 3.6 µL of DNA molecular weight marker VIII (34.5 ng of double-stranded DNA/µL, corresponding to 124.2 ng of DNA; Roche Diagnostics GmbH).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
phase 1: DNA quantification in standard DNA samples
To test the ability of each laboratory to measure the exact quantity of DNA and its quality, participants were requested to measure the DNA concentration (absorbance at 260 nm) in Standard DNAs 1, 2, and 3 and the A260 nm/A280 nm ratio in the same samples. Shown in Fig. 1 are the data distributions for both values in corresponding samples. The results of the DNA quantification reported by participants revealed a consistent dispersion of data (CV, 128%, 72%, and 211%, respectively, for Standards 1, 2, and 3). This variability was dramatically reduced for the A260 nm/A280 nm ratio (CV, 51%, 15%, and 30%, respectively).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 1. Measurement of DNA quality and concentration in Standard DNAs.

Distribution in classes of results for measurement of DNA concentration (A; µg/µL) and quality (B; A260 nm/A280 nm ratio) in Standard DNAs 1, 2, and 3.

phase 2: DNA extraction from blood sample and its quantification
DNA extraction from the Blood Sample showed substantial variation of results among laboratories. The DNA concentration ranged from 0.012 to 0.54 (median, 0.1) µg/µL with a CV of 82%. The A260 nm/A280 nm ratios of the same extracts ranged from 0.8 to 2.5 (median, 1.69) with a CV of 21% (see Fig. 2 ). The results reported by the participants on the quality and quantity of DNA in this and the other three Standard DNA samples were used for assigning scores, as indicated below. The score for these sets of results ranged from 0 to 4 for each of the four determinations related to DNA quantity, with a global attainable score of 16. The same approach was used for DNA quality results.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 2. Measurement of quality and concentration of DNA extracted from the Blood Sample.

Individual results for concentration (top; µg/µL) and quality (bottom; A260 nm/A280 nm ratio) of DNA extracted from the Blood Sample by each participating laboratory. Laboratory 21 did not provide these results because of technical problems. Laboratory 39 reported a value >5. The horizontal lines indicate the consensus medians.

phase 3: PCR amplification and interpretation
After PCR amplification of the Reference DNA and DNA extracted from the Blood Sample, participants provided their interpretation of results after electrophoretic separation of PCR products. In particular, they had to indicate the number of detectable specific bands and the approximate sizes in base pairs. Shown in Table 1 are the single results as provided by each participant. In addition, these results, as related to the interpretation of the PCR results, were used for assigning the score. The score for these sets of results ranged from 0 to 4 for each of the six PCR interpretations, with a maximum attainable score of 24.


View this table:
[in this window]
[in a new window]
  		Table 1. Interpretation of results after gel electrophoresis of PCR products of Reference DNA and DNA extracted from Blood Sample as reported by each participating laboratory.

phase 4: evaluation of PCR efficiency
The simultaneous evaluation by the organizers of all post-PCR samples after gel electrophoresis and ethidium bromide staining revealed large variability in the yield and quality of PCR performance for the different participants. Examples of these heterogeneous results are shown in Fig. 3 .



View larger version (106K):
[in this window]
[in a new window]
  		Figure 3. Examples of gel results.

Four examples of PCR amplification products for the Reference DNA (lanes a–c) and DNA extracted from the Blood Sample (lanes d–f) amplified with primer pairs 1A-1B (lanes a and d), 2A-2B (lanes b and e), and 3A-3B (lanes c and f). Gel electrophoresis (2% agarose) was performed using the duplicate that each participating laboratory returned to the organizers of the program. Results from laboratory 39 (top left) show a complete and efficient amplification of all expected targets, including the two products in lane a. The sample from laboratory 2 (top right) has an acceptable amplification, but only one target is present in lane a. Samples from laboratories 4 (bottom left) and 16 (bottom right) show amplification of the expected targets, but several unspecific products were also detectable. DNA molecular weight markers: left, marker VIII (Roche GmbH); right, GeneRulerTM 100-bp DNA ladder (Genenco).

The densitometric analysis of PCR bands confirmed this high variability. The densitometric analysis of PCR products obtained by the 39 laboratories using the DNA extracted from the Blood Sample with the three pairs of primers gave comparable results with quite constant CVs: 48% for primer set 1A-1B, 39% for primer set 2A-2B; and 40% for primer set 3A-3B. One laboratory (number 5) failed to amplify DNA from the Blood Sample with any of the three primer sets, whereas laboratories 17 and 23 obtained bands only for two primer pairs and one primer pair, respectively.

The densitometric analyses of PCR products of the Reference DNA samples were comparable to those obtained with the DNA extracted from the Blood Sample. The CVs were, respectively, 45% and 44% for primer pair 1A-1B, 48% for primer pair 2A-2B, and 46% for primer pair 3A-3B. Four laboratories failed to get amplification with primer pair 2A-2B, and two laboratories failed to get amplification with primer pair 3A-3B. With primer pair 1A-1B, 31 laboratories obtained two detectable bands after gel electrophoresis corresponding to the expected products, with a good correlation with their theoretical ratio (median, 1.03; range, 0.91–1.24) and a very low CV (7.7%). However, seven laboratories were not able to detect the second PCR product and one failed to obtain any bands. All densitometric results for each laboratory were summed to provide a mean value of PCR efficiency. These results are reported in Fig. 4 .



View larger version (56K):
[in this window]
[in a new window]
  		Figure 4. Mean (SE; error bars) results of individual densitometric analyses of all products of PCR amplification.

Laboratories are ordered on the basis of increasing efficiency of amplification. Laboratory 5 did not obtain any amplification. The horizontal line indicates the consensus median of the results.

The results of the densitometric analysis were used for assigning the score. The score for these sets of results ranged from 0 to 4 for each of the seven densitometric measurements, with a maximum obtainable score of 28.

score
To give a general estimation on the quality of performances for each laboratory, we designed a score scheme in which the results of any specific action were evaluated on the basis of the distribution around the median consensus values. In practice, for each phase of this EQA program, we attributed a different score. As an example, Table 2 reports the score assigned to the results of the A260 nm/A280 nm ratio for the four DNA samples measured by the participants in phases 1 and 2. Similar tables were constructed for the other three sets of results evaluated in this study, i.e., measurement of the quantity of DNA in the four samples (phases 1 and 2); interpretation of electrophoretic results obtained for PCR products in six gels (phase 3); and the PCR efficiency as measured by densitometric analysis of seven amplifications (phase 4; data not shown). The maximum obtainable score was 84. After the evaluation of total score obtained by each laboratory, we created a qualitative ranking list that provided the final interpretation of results as excellent (>63 points; n = 4 laboratories), good (53–63 points; n = 13), sufficient (42–52 points; n = 15), poor (31–41 points; n = 3), and not acceptable (<31 points; n = 4). These results are reported in Fig. 5 .


View this table:
[in this window]
[in a new window]
  		Table 2. Example of score assignment for DNA quality: data for the A260 nm/A280 nm ratio (EQA phases 1 and 2).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 5. Ranking distribution of the laboratories as a function of the total score obtained by each laboratory participating at the EQA program.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The advent of molecular diagnostic methodologies, mainly based on PCR or PCR-derived approaches, introduced some problems of standardization because of the relative complexity of molecular methods. Amplification techniques are based on a cyclic enzymatic reaction highly sensitive to any source of possible contamination, which could generate false-positive results. PCR is particularly sensitive to predictable and unpredictable variables that can negatively influence amplification. Most of these negative aspects are represented by inappropriate amplification protocols, poor preparation and quantification of nucleic acid targets, low recovery, the presence of PCR inhibitors, use of inefficient reagents and/or thermal cyclers, and misinterpretation of results.

We partially modified, implemented, and tested in Italian laboratories an EQA scheme approach directed to the evaluation of single analytical procedures connected to PCR protocols (32), which is advantageous because it is suitable for any laboratory.

The results of this study showed the high variability of efficiency and precision among the different laboratories in all aspects that were tested: efficiency in DNA extraction, estimation of its quality and quantity, PCR performance, and interpretation of results.

In particular it was evident that different DNA extraction techniques and/or expertise generated a wide distribution of apparent DNA recoveries from the blood sample that we provided. This large spread of data was probably attributable to the incorrect estimation of DNA concentrations based on absorbance values obtained with a conventional spectrophotometer. This was confirmed by the wide distribution of results obtained for the Reference DNAs, for which participants had only to measure the DNA concentration. We had indirect confirmation of this hypothesis from our evaluation of the results for the A260 nm/A280 nm ratio because the absolute absorbance measurement should be less affected by human and/or instrument errors. As expected, the CVs for these measurements in the Blood Sample and Standard DNAs were lower than those for the absorbance readings at 260 nm.

The highest variability was in the efficiency of PCR amplification, as we detected with the simultaneous evaluation of PCR products. The densitometric analysis of each gel demonstrated the high variability in performance, which is probably attributable to differences in the efficiencies of the reagents and thermal cyclers used by individual laboratories.

Finally, we experimentally tested the use of a score ranking to provide a final and comprehensive interpretation of all data obtained by each participating laboratory. According to this type of result classification, 18% of all laboratories (7 of 39) provided unsatisfactory results, whereas 44% (17 of 39) obtained good results.

In conclusion, the approach of this study was limited to some of the analytical steps in PCR-based methods, and even in the presence of a high score, diagnostic errors could still be made. It is known that DNA extraction is a key influencing factor on PCR-based methods and that the types and calibration status of spectrophotometers and thermal cyclers can influence PCR variability (37); we therefore decided that these aspects should be taken most into consideration. The results demonstrate that there is high variability among laboratories carrying out an identical assay. This finding has implications for any assay that relies on PCR-based methods; it also demonstrates the importance and validity of EQA trials based on methodologic proficiency testing directed toward the evaluation of analytical aspects of PCR-based tests. It is possible that the recent development of real-time PCR chemistries, which use more standardized reagents and instrumentation, might be able to increase the reliability of PCR-based assays.


   Acknowledgments
 
This program was partially supported by a grant from the Italian Society of Clinical Chemistry and Clinical Molecular Biology (SIBioC) and by a special project (141/01) of the Ministry of Health, Italian Government. We thank Dr. S. Bustin for fruitful suggestions. We also thank Polymed s.r.l. (Florence, Italy) for its contribution to the project.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Libeer JC. Role of external quality assurance schemes in assessing and improving quality in medical laboratories. Clin Chim Acta 2001;309:173-177.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  2. Olafsdottir E, Hellsing K, Steensland H, Tenhunen R, Uldall A. Adding new scopes to traditional EQA schemes emphasizing quality improvement. Hyltoft Petersen P Blaabjerg O Irjala K eds. Assessing quality in measurements of plasma proteins: the Nordic Protein Project and related projects, Nordkem Clinical Chemistry Project 1994:187-193 Lasse Halle Ltd. Print Helsinki. .
  3. Neumaier M, Braun A, Wagener C. Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques. Clin Chem 1998;44:12-26.[Abstract/Free Full Text]
  4. Burkardt HJ. Standardization and quality control of PCR analyses. Clin Chem Lab Med 2000;38:87-91.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  5. Schwartz MK. Genetic testing and the clinical laboratory improvement amendments of 1988: present and future. Clin Chem 1999;45:739-745.[Abstract/Free Full Text]
  6. Muller CR. Quality control in mutation analysis: the European Molecular Genetics Quality Network (EMQN). Eur J Pediatr 2001;160:464-467.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  7. Dequeker E, Cassiman JJ. Genetic testing and quality control in diagnostic laboratories. Nat Genet 2000;25:259-260.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  8. Dequeker E, Ramsden S, Grody WW, Stenzel TT, Barton DE. Quality control in molecular genetic testing. Nat Rev Genet 2001;2:717-723.[Web of Science][Medline] [Order article via Infotrieve]
  9. Losekoot M, Bakker B, Laccone F, Stenhouse S, Elles R. A European pilot quality assessment scheme for molecular diagnosis of Huntington’s disease. Eur J Hum Genet 1999;7:217-222.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  10. Dequeker E, Cassiman JJ. Evaluation of CFTR gene mutation testing methods in 136 diagnostic laboratories: report of a large European external quality assessment. Eur J Hum Genet 1998;6:165-175.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  11. Dequeker E, Cuppens H, Dodge J, Estivill X, Goossens M, Pignatti PF, et al. Recommendations for quality improvement in genetic testing for cystic fibrosis. European Concerted Action on Cystic Fibrosis. Eur J Hum Genet 2000;8(Suppl 2):S2-S24.
  12. Preston FE, Kitchen S, Jennings I, Woods TA. A UK National External Quality Assessment scheme (UK NEQAS) for molecular genetic testing for the diagnosis of familial thrombophilia. Thromb Haemost 1999;82:1556-1557.[Web of Science][Medline] [Order article via Infotrieve]
  13. Lutz CT, Foster PA, Noll WW, Voelkerding KV, Press RD, McGlennen RC, et al. Multicenter evaluation of PCR methods for the detection of factor V Leiden (R506Q) genotypes. Clin Chem 1998;44:1356-1358.[Free Full Text]
  14. Simoni M. Molecular diagnosis of Y chromosome microdeletions in Europe: state-of-the-art and quality control. Hum Reprod 2001;16:402-409.[Abstract/Free Full Text]
  15. Valentine-Thon E, van Loon AM, Schirm J, Reid J, Klapper PE, Cleator GM. European proficiency testing program for molecular detection and quantitation of hepatitis B virus DNA. J Clin Microbiol 2001;39:4407-4412.[Abstract/Free Full Text]
  16. Robertson JS. International standardization of gene amplification technology. Biologicals 1998;26:111-113.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  17. Lefrere JJ, Lerable J, Mariotti M, Bogard M, Thibault V, Frangeul L, et al. Lessons from a multicentre study of the detectability of viral genomes based on a two-round quality control of GB virus C (GBV-C)/hepatitis G virus (HGV) polymerase chain reaction assay. J Virol Methods 2000;85:117-124.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  18. van Loon AM, Cleator GC, Ras A. External quality assessment of enterovirus detection and typing. European Union Concerted Action on Virus Meningitis and Encephalitis. Bull World Health Organ 1999;77:217-223.[Web of Science][Medline] [Order article via Infotrieve]
  19. Weber T, Klapper PE, Cleator GM, Bodemer M, Luke W, Knowles W, et al. Polymerase chain reaction for detection of JC virus DNA in cerebrospinal fluid: a quality control study. European Union Concerted Action on Viral Meningitis and Encephalitis. J Virol Methods 1997;69:231-237.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  20. Hirsch HH, Bossart W. Two-centre study comparing DNA preparation and PCR amplification protocols for herpes simplex virus detection in cerebrospinal fluids of patients with suspected herpes simplex encephalitis. J Med Virol 1999;57:31-35.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  21. Grundy JE, Ehrnst A, Einsele H, Emery VC, Hebart H, Prentice HG, et al. A three-center European external quality control study of PCR for detection of cytomegalovirus DNA in blood. J Clin Microbiol 1996;34:1166-1170.[Abstract/Free Full Text]
  22. Yen-Lieberman B, Brambilla D, Jackson B, Bremer J, Coombs R, Cronin M, et al. Evaluation of a quality assurance program for quantitation of human immunodeficiency virus type 1 RNA in plasma by the AIDS Clinical Trials Group virology laboratories. J Clin Microbiol 1996;34:2695-2701.[Abstract/Free Full Text]
  23. Schweiger B, Pauli G, Zeichhardt H, Kucherer C. A multicentre quality assessment study to monitor the performance of HIV-1 PCR. J Virol Methods 1997;67:45-55.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  24. Nowicki MJ, Benning L, Bremer JW, Meyer WA, III, Hanson C, Brambilla D, et al. Longitudinal variability of human immunodeficiency virus type 1 RNA viral load measurements by nucleic acid sequence-based amplification and NucliSens assays in a large multicenter study. J Clin Microbiol 2001;39:3760-3763.[Abstract/Free Full Text]
  25. Pelloux H, Guy E, Angelici MC, Aspock H, Bessieres MH, Blatz R, et al. A second European collaborative study on polymerase chain reaction for Toxoplasma gondii, involving 15 teams. FEMS Microbiol Lett 1998;165:231-237.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  26. Noordhoek GT, van Embden JD, Kolk AH. Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories. J Clin Microbiol 1996;34:2522-2525.[Abstract/Free Full Text]
  27. Bolufer P, Barragan E, Sanz MA, Martin G, Bornstein R, Colomer D, et al. Preliminary experience in external quality control of RT-PCR PML-RAR {alpha} detection in promyelocytic leukemia. Leukemia 1998;2:2024-2028.
  28. van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999;13:1901-1928.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  29. Morschhauser F, Cayuela JM, Martini S, Baruchel A, Rousselot P, Socie G, et al. Evaluation of minimal residual disease using reverse-transcription polymerase chain reaction in t(8;21) acute myeloid leukemia: a multicenter study of 51 patients. J Clin Oncol 2000;18:788-794.[Abstract/Free Full Text]
  30. Bolufer P, Lo Coco F, Grimwade D, Barragan E, Diverio D, Cassinat B, et al. Variability in the levels of PML-RAR {alpha} fusion transcripts detected by the laboratories participating in an external quality control program using several reverse transcription polymerase chain reaction protocols. Haematologica 2001;86:570-576.[Abstract/Free Full Text]
  31. Chevillard S, Vielh P, Validire P, Marie JP, Faussat AM, Barbu V, et al. French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hopitaux de Paris. Leukemia 1997;11:1095-1106.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  32. Braun A, Deufel T, Geilenkeuser WJ, Neumaier M, Rohle G, Roscher A, et al. External quality assessment of molecular biology-based methods used in laboratories of clinical chemistry and human genetics. Clin Chem Lab Med 1998;36:231-234.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  33. Neumaier M, Braun A, Gessner R, Funke H. Experiences with external quality assessment (EQA) in molecular diagnostics in clinical laboratories in Germany. Working Group of the German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM). Clin Chem Lab Med 2000;38:161-163.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  34. Orlando C, Sestini R, Zentilin L, Gelmini S, Pinzani P, Giacca M, et al. Image analysis in quantitative PCR. An application for the measurement of c-erbB-2 oncogene amplification in DNA from human tumours. J Biolumin Chemilumin 1994;9:223-228.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  35. Orlando C, Casini Raggi C, Pazzagli M. Molecular diagnosis and quality control: a program for DNA analysis with PCR amplification [Abstract]. Clin Chem Lab Med 2000;7:A10.
  36. Sestini R, Orlando C, Zentilin L, Lami D, Gelmini S, Pinzani P, et al. Gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2, and N-myc measured by quantitative PCR with a multiple competitor template. Clin Chem 1995;41:826-832.[Abstract/Free Full Text]
  37. Saunders GC, Dukes J, Parkes HC, Cornett JH. Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA analyses. Clin Chem 2001;47:47-55.[Abstract/Free Full Text]



The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
C. Orlando, P. Verderio, R. Maatman, J. Danneberg, S. Ramsden, M. Neumaier, D. Taruscio, V. Falbo, R. Jansen, C. Casini-Raggi, et al.
EQUAL-qual: A European Program for External Quality Assessment of Genomic DNA Extraction and PCR Amplification
Clin. Chem., July 1, 2007; 53(7): 1349 - 1357.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
S. J. Patton, A. J. Wallace, and R. Elles
Benchmark for Evaluating the Quality of DNA Sequencing: Proposal from an International External Quality Assessment Scheme
Clin. Chem., April 1, 2006; 52(4): 728 - 736.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
S. Raja, J. Ching, L. Xi, S. J. Hughes, R. Chang, W. Wong, W. McMillan, W. E. Gooding, K. S. McCarty Jr, M. Chestney, et al.
Technology for Automated, Rapid, and Quantitative PCR or Reverse Transcription-PCR Clinical Testing
Clin. Chem., May 1, 2005; 51(5): 882 - 890.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
V. Cappelletti, L. Celio, E. Bajetta, A. Allevi, R. Longarini, P. Miodini, R. Villa, A. Fabbri, L. Mariani, R. Giovanazzi, et al.
Prospective evaluation of estrogen receptor-{beta} in predicting response to neoadjuvant antiestrogen therapy in elderly breast cancer patients
Endocr. Relat. Cancer, December 1, 2004; 11(4): 761 - 770.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
L. Birch, C. A. English, M. Burns, and J. T. Keer
Generic Scheme for Independent Performance Assessment in the Molecular Biology Laboratory
Clin. Chem., September 1, 2004; 50(9): 1553 - 1559.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
C. S. Richards and W. W. Grody
Alternative Approaches to Proficiency Testing in Molecular Genetics
Clin. Chem., May 1, 2003; 49(5): 717 - 718.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Raggi, C. C.
Right arrow Articles by Orlando, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Raggi, C. C.
Right arrow Articles by Orlando, C.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Laboratory Management

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS