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Buccal swab as a suitable sample for a microarray-based rapid detection assay using a warfarin genotyping kit
Clinica Chimica Acta 430 (2014) 77–78
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Letter to the editor Buccal swab as a suitable sample for a microarray-based rapid detection assay using a warfarin genotyping kit
To the Editor: Genetic testing for common variants in the CYP2C9 and VKORC1 genes may provide useful clinical information to guide dosing for patients receiving oral warfarin. Although methods for sequencing the regions of the CYP2C9 and VKORC1 genes containing the clinically significant SNPs could potentially be used in a clinical setting, most molecular diagnostic laboratories choose alternate methods for routine genotyping applications due to regulatory and quality assurance issues, as well as financial constraints and a need for rapid TATs [1]. The Verigene® Warfarin Metabolism Nucleic Acid Test (Verigene® Warfarin assay, Nanosphere, Inc., Northbrook, IL, USA) is one alternate method, which has been approved by the Food and Drug Administration (FDA) [2]. This microarray-based rapid detection assay employs signal amplification rather than DNA amplification and is suitably designed to genotype CYP2C9*2, CYP2C9*3 and VKORC1 1173CNT [3,4]. Venous blood is a commonly used specimen in the pharmacogenomic era. However, in certain situations, such as after allogeneic hematopoietic stem cell transplantation (HSCT), genotype results from blood samples do not reflect the patient's pharmacogenomic status because the leukocytes in the blood originated from the donor's hematopoietic cells. Saliva or buccal swab sampling provides a good, replicable sample source [5,6]. In this study, we evaluated the feasibility of a buccal swab specimen as an alternative to peripheral blood for use in a microarray-based, rapid detection assay, the Verigene® Warfarin assay. Fifteen buccal swab specimens were obtained using a cotton-tipped applicator in a conical
tube containing 2 mL of phosphate buffered solution, and peripheral blood samples were acquired from the same volunteers. All samples were obtained with informed consent and were in accordance with the Institutional Review Board (IRB) guidelines of Seoul St. Mary's Hospital (IRB No. KC12EISI0407). Verigene® Warfarin assay successfully obtained the genotyping results from all 15 paired peripheral blood and buccal swab specimens (Table 1). The homozygous CYP2C9*1/*1 genotype was detected in 87% of the specimens (13/15) and the remaining 13% (2/15) presented the heterozygous CYP2C9*1/*3 genotype. The VKORC1 1173TT haplotype and 1173CT haplotype were found in 80% (12/15) and 20% of the specimens (3/15), respectively. The genotype results from buccal swab specimens were 100% concordant with those from peripheral blood samples. To evaluate the accuracy of the genotype results obtained with the Verigene® Warfarin assay, we performed Sanger sequencing in parallel. We found 100% concordance in all genotype results for all three loci examined. To determine the minimum number of epithelial cells required for the Verigene® Warfarin assay, we counted the cells in buccal swab suspensions using a Neubauer chamber. The epithelial cell counts of buccal swab specimens were variable (4.4 ± 1.9 × 105/mL, minimum 2.0 × 105/mL, maximum 10.0 × 105/mL). Resampling was performed in three patients whose initial Verigene® Warfarin assay showed a “no call” result. The epithelial cell numbers in those specimens were fewer than 2.0 × 105/mL. In addition, we compared the yield of DNA extracted from peripheral blood and buccal swab specimens. The concentrations of DNA extracted from buccal swab specimens, measured by an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), were much lower than those from the peripheral blood leukocytes (34.9 ± 16.5 ng/μL vs. 216.0 ± 97.4 ng/μL, respectively). However, the A260/A280 ratios of DNA from buccal swab specimens and peripheral blood leukocytes
Table 1 DNA yields and genotype results from matched specimens and epithelial cell counts of buccal swab specimens. Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean
Cell count (/mL)
Genotype
Buccal swab
A260/280 ratio Blood
Buccal swab
DNA concentration (μg) Blood
Buccal swab
CYP2C9
VKORC1
1.78 1.82 1.81 1.69 1.78 1.87 1.85 1.87 1.93 1.61 1.69 1.71 1.77 1.87 1.90 1.80
1.82 1.86 1.92 1.90 1.84 1.85 1.84 1.94 1.92 1.99 1.98 1.98 2.00 1.98 1.98 1.90
60.4 49.0 30.4 15.0 28.4 27.0 36.8 60.2 12.4 16.8 17.8 27.2 39.6 54.4 48.6 34.9
137.1 505.2 215.9 263.2 154.1 124.5 178.6 114.2 140.8 278.1 285.8 233.7 202.4 174.3 231.5 216.0
10.0 5.8 6.3 3.0 3.3 4.3 5.0 4.8 2.0 3.7 4.0 3.4 3.5 4.5 3.0 4.4
*1/*1 *1/*1 *1/*1 *1/*3 *1/*1 *1/*1 *1/*3 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1
1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173CT 1173CT 1173TT 1173TT 1173CT
0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.12.025
78
Letter to the editor
were very close to the ideal value of 1.8 (1.80 and 1.90, respectively). Agarose gel electrophoresis showed less intense bands and smearing in some of the buccal swab specimens, but the majority of the samples showed a single, sharp, high molecular weight band with minimal degradation (Supplementary Fig. 1). Our data revealed that buccal swab specimens contained approximately five-fold lower DNA concentrations: however, the quality of the DNA was comparable with that from peripheral blood. The complete concordance of results from the Verigene® Warfarin assay also convinces us of the feasibility of using a buccal swab specimen. We concluded that buccal swab specimens were an excellent substitute sample source for the Verigene® Warfarin assay. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2013.12.025. Acknowledgements This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Korea (SN: A092258). References [1] Carlquist JF, Anderson JL. Using pharmacogenetics in real time to guide warfarin initiation: a clinician update. Circulation 2011;124:2554–9. [2] Bao YP, Huber M, Wei TF, Marla SS, Storhoff JJ, Muller UR. SNP identification in unamplified human genomic DNA with gold nanoparticle probes. Nucleic Acids Res 2005;33:e15. [3] Qin WJ, Yung LY. Nanoparticle-based detection and quantification of DNA with single nucleotide polymorphism (SNP) discrimination selectivity. Nucleic Acids Res 2007;35:e111. [4] Lefferts JA, Schwab MC, Dandamudi UB, Lee HK, Lewis LD, Tsongalis GJ. Warfarin genotyping using three different platforms. Am J Transl Res 2010;2:441–6. [5] Woo JG, Sun G, Haverbusch M, et al. Quality assessment of buccal versus blood genomic DNA using the Affymetrix 500 K GeneChip. BMC Genet 2007;8:79. [6] Abraham JE, Maranian MJ, Spiteri I, et al. Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping. BMC Med Genomics 2012;5:19.
Jiyeon Kim1 Ahlm Kwon1 Hayoung Choi Catholic Laboratory Development and Evaluation Center, College of Medicine, The Catholic University of Korea, Seoul, Korea Hyojin Chae Myungshin Kim ⁎ Yonggoo Kim Catholic Laboratory Development and Evaluation Center, College of Medicine, The Catholic University of Korea, Seoul, Korea Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea ⁎ Corresponding author at: Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-Gu, Seoul 137-701, Korea. Tel.: + 82 2 2258 1645. E-mail address: [email protected] (M. Kim). Dong-Gun Lee Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Yong-Seog Oh Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Division of Cardiology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea 3 August 2013
1
J. Kim and A. Kwon contributed equally to this study.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Letter to the editor Buccal swab as a suitable sample for a microarray-based rapid detection assay using a warfarin genotyping kit
To the Editor: Genetic testing for common variants in the CYP2C9 and VKORC1 genes may provide useful clinical information to guide dosing for patients receiving oral warfarin. Although methods for sequencing the regions of the CYP2C9 and VKORC1 genes containing the clinically significant SNPs could potentially be used in a clinical setting, most molecular diagnostic laboratories choose alternate methods for routine genotyping applications due to regulatory and quality assurance issues, as well as financial constraints and a need for rapid TATs [1]. The Verigene® Warfarin Metabolism Nucleic Acid Test (Verigene® Warfarin assay, Nanosphere, Inc., Northbrook, IL, USA) is one alternate method, which has been approved by the Food and Drug Administration (FDA) [2]. This microarray-based rapid detection assay employs signal amplification rather than DNA amplification and is suitably designed to genotype CYP2C9*2, CYP2C9*3 and VKORC1 1173CNT [3,4]. Venous blood is a commonly used specimen in the pharmacogenomic era. However, in certain situations, such as after allogeneic hematopoietic stem cell transplantation (HSCT), genotype results from blood samples do not reflect the patient's pharmacogenomic status because the leukocytes in the blood originated from the donor's hematopoietic cells. Saliva or buccal swab sampling provides a good, replicable sample source [5,6]. In this study, we evaluated the feasibility of a buccal swab specimen as an alternative to peripheral blood for use in a microarray-based, rapid detection assay, the Verigene® Warfarin assay. Fifteen buccal swab specimens were obtained using a cotton-tipped applicator in a conical
tube containing 2 mL of phosphate buffered solution, and peripheral blood samples were acquired from the same volunteers. All samples were obtained with informed consent and were in accordance with the Institutional Review Board (IRB) guidelines of Seoul St. Mary's Hospital (IRB No. KC12EISI0407). Verigene® Warfarin assay successfully obtained the genotyping results from all 15 paired peripheral blood and buccal swab specimens (Table 1). The homozygous CYP2C9*1/*1 genotype was detected in 87% of the specimens (13/15) and the remaining 13% (2/15) presented the heterozygous CYP2C9*1/*3 genotype. The VKORC1 1173TT haplotype and 1173CT haplotype were found in 80% (12/15) and 20% of the specimens (3/15), respectively. The genotype results from buccal swab specimens were 100% concordant with those from peripheral blood samples. To evaluate the accuracy of the genotype results obtained with the Verigene® Warfarin assay, we performed Sanger sequencing in parallel. We found 100% concordance in all genotype results for all three loci examined. To determine the minimum number of epithelial cells required for the Verigene® Warfarin assay, we counted the cells in buccal swab suspensions using a Neubauer chamber. The epithelial cell counts of buccal swab specimens were variable (4.4 ± 1.9 × 105/mL, minimum 2.0 × 105/mL, maximum 10.0 × 105/mL). Resampling was performed in three patients whose initial Verigene® Warfarin assay showed a “no call” result. The epithelial cell numbers in those specimens were fewer than 2.0 × 105/mL. In addition, we compared the yield of DNA extracted from peripheral blood and buccal swab specimens. The concentrations of DNA extracted from buccal swab specimens, measured by an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), were much lower than those from the peripheral blood leukocytes (34.9 ± 16.5 ng/μL vs. 216.0 ± 97.4 ng/μL, respectively). However, the A260/A280 ratios of DNA from buccal swab specimens and peripheral blood leukocytes
Table 1 DNA yields and genotype results from matched specimens and epithelial cell counts of buccal swab specimens. Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean
Cell count (/mL)
Genotype
Buccal swab
A260/280 ratio Blood
Buccal swab
DNA concentration (μg) Blood
Buccal swab
CYP2C9
VKORC1
1.78 1.82 1.81 1.69 1.78 1.87 1.85 1.87 1.93 1.61 1.69 1.71 1.77 1.87 1.90 1.80
1.82 1.86 1.92 1.90 1.84 1.85 1.84 1.94 1.92 1.99 1.98 1.98 2.00 1.98 1.98 1.90
60.4 49.0 30.4 15.0 28.4 27.0 36.8 60.2 12.4 16.8 17.8 27.2 39.6 54.4 48.6 34.9
137.1 505.2 215.9 263.2 154.1 124.5 178.6 114.2 140.8 278.1 285.8 233.7 202.4 174.3 231.5 216.0
10.0 5.8 6.3 3.0 3.3 4.3 5.0 4.8 2.0 3.7 4.0 3.4 3.5 4.5 3.0 4.4
*1/*1 *1/*1 *1/*1 *1/*3 *1/*1 *1/*1 *1/*3 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1
1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173TT 1173CT 1173CT 1173TT 1173TT 1173CT
0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.12.025
78
Letter to the editor
were very close to the ideal value of 1.8 (1.80 and 1.90, respectively). Agarose gel electrophoresis showed less intense bands and smearing in some of the buccal swab specimens, but the majority of the samples showed a single, sharp, high molecular weight band with minimal degradation (Supplementary Fig. 1). Our data revealed that buccal swab specimens contained approximately five-fold lower DNA concentrations: however, the quality of the DNA was comparable with that from peripheral blood. The complete concordance of results from the Verigene® Warfarin assay also convinces us of the feasibility of using a buccal swab specimen. We concluded that buccal swab specimens were an excellent substitute sample source for the Verigene® Warfarin assay. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2013.12.025. Acknowledgements This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Korea (SN: A092258). References [1] Carlquist JF, Anderson JL. Using pharmacogenetics in real time to guide warfarin initiation: a clinician update. Circulation 2011;124:2554–9. [2] Bao YP, Huber M, Wei TF, Marla SS, Storhoff JJ, Muller UR. SNP identification in unamplified human genomic DNA with gold nanoparticle probes. Nucleic Acids Res 2005;33:e15. [3] Qin WJ, Yung LY. Nanoparticle-based detection and quantification of DNA with single nucleotide polymorphism (SNP) discrimination selectivity. Nucleic Acids Res 2007;35:e111. [4] Lefferts JA, Schwab MC, Dandamudi UB, Lee HK, Lewis LD, Tsongalis GJ. Warfarin genotyping using three different platforms. Am J Transl Res 2010;2:441–6. [5] Woo JG, Sun G, Haverbusch M, et al. Quality assessment of buccal versus blood genomic DNA using the Affymetrix 500 K GeneChip. BMC Genet 2007;8:79. [6] Abraham JE, Maranian MJ, Spiteri I, et al. Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping. BMC Med Genomics 2012;5:19.
Jiyeon Kim1 Ahlm Kwon1 Hayoung Choi Catholic Laboratory Development and Evaluation Center, College of Medicine, The Catholic University of Korea, Seoul, Korea Hyojin Chae Myungshin Kim ⁎ Yonggoo Kim Catholic Laboratory Development and Evaluation Center, College of Medicine, The Catholic University of Korea, Seoul, Korea Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea ⁎ Corresponding author at: Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-Gu, Seoul 137-701, Korea. Tel.: + 82 2 2258 1645. E-mail address: [email protected] (M. Kim). Dong-Gun Lee Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Yong-Seog Oh Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Division of Cardiology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea 3 August 2013
1
J. Kim and A. Kwon contributed equally to this study.