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Evaluation of the GeneXpert assay in the detection of Factor V Leiden and Prothrombin 20210 in stored, previously classified samples
Clinica Chimica Acta 413 (2012) 814–816
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Short communication
Evaluation of the GeneXpert assay in the detection of Factor V Leiden and Prothrombin 20210 in stored, previously classified samples G. Gessoni a,⁎, S. Valverde a, F. Manoni b a b
Service of Laboratory Medicine, Chioggia, Venice, Italy Service of Laboratory Medicine, Monselice, Padua, Italy
a r t i c l e
i n f o
Article history: Received 17 September 2011 Received in revised form 23 December 2011 Accepted 15 January 2012 Available online 21 January 2012 Keywords: Factor V Leiden GeneXpert Prothrombin 20210
a b s t r a c t Background: In this study, we evaluated the GeneXpert HemosIL Factor II and Factor V assay, an innovative assay for the detection of Factor V Leiden (FVL) and prothrombin G20210A mutation (GPRO). Patients and methods: We evaluated 132 patients that were previously classified (with a concordant result) using two commercial real-time PCR assays supplied, by Applied Biosystems and Roche Molecular Biochemicals. The cohort comprised 75 normal subjects, 10 FVL homozygous, 35 FVL heterozygous, 7 GPRO heterozygous, 2 GPRO homozygous and 3 double heterozygous FVL and GPRO subjects. All of the samples were evaluated using the GeneXpert HemosIL Factor II and Factor V assay. Results: All of the samples were correctly identified using the GeneXpert HemosIL Factor II and Factor V assay; therefore, in this patient series, the specificity and sensitivity of the test under evaluation was 1.00. Discussion: We have shown that the GeneXpert HemosIL Factor II and Factor V assay, a rapid fully automated assay, can accurately characterise the presence of FV G1691A and FII G20210A polymorphisms with specificity and sensitivity that are comparable to other current real-time PCR-based methods. The theoretical advantages of such an assay include improved standardisation across varying healthcare environments, more thorough sample manipulation and reduced human error. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Factor V Leiden (FVL) is an autosomal dominant condition that exhibits incomplete dominance and results in a factor V variant that cannot be as easily degraded as the wild type by activated Protein C (aPC). The gene that codes factor V is referred to as F5. FVL is characterised by a single nucleotide polymorphism in exon 10, with a G to A substitution at position 1691, G1691A, and it also predicts a single amino acid replacement of an arginine to glutamine (R506Q) at one of three aPC cleavage sites in factor Va. Because this residue 506 is normally the cleavage site for aPC, the mutation prevents the efficient inactivation of factor V. When factor V remains active, it facilitates the overproduction of thrombin, leading to excess fibrin generation and excess clotting. Clinical Laboratory evaluation for factor V Leiden can begin with a test for activated protein C resistance because aPC resistance is usually (95%) due to a factor V Leiden mutation. If resistance is present, then a test for the factor V Leiden gene mutation is Abbreviations: aPC, Activated C Protein; FVL, Factor V Leiden; Lab A, Laboratory Monselice; Lab B, Laboratory Chioggia; PCR, Polymerase Chain Reaction; GPRO, Prothrombin 20210 variant (Prothrombin G). ⁎ Corresponding author at: Service of Laboratory Medicine, Ospedale Civile, Via Madonna Marina 500, 30015 Chioggia Venice, Italy. Tel.: + 39 041 5534 400; fax: + 39 041 5534 401. E-mail addresses: [email protected], [email protected] (G. Gessoni). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.01.016
performed, both to confirm the diagnosis and to determine whether the person is heterozygous or homozygous for the mutation [1,2]. Prothrombin is the precursor to thrombin in the coagulation cascade, and thrombin is required to convert fibrinogen into fibrin, which is the primary goal of the coagulation cascade. The Prothrombin 20210 gene mutation (GPRO) is caused by a G to A transition in the 3′-untranslated region of the prothrombin (Factor II) gene at position 20210. This condition is associated with mild increases in plasma prothrombin levels and an increased risk of venous thrombosis. Diagnosis of the Prothrombin 20210 gene mutation must be made through specific genetic testing, which can also reveal whether the patient is heterozygous or homozygous for the condition. Although prothrombin levels are usually moderately elevated in subjects with this mutation and could be measured, they are not clinically useful in identifying the mutation [3,4]. In this study, the analytical sensitivity and specificity of the GeneXpert HemosIL Factor II and Factor V assay were determined using a panel of 132 subjects previously characterised using two commercial real-time PCR assays adopted for routine diagnosis in our laboratories. 2. Materials and methods 2.1. Study design We compared the results obtained using the HemosIL GeneXpert test for the detection of FVL and GPRO with those obtained from
G. Gessoni et al. / Clinica Chimica Acta 413 (2012) 814–816
two commercial tests supplied by Applied Biosystems (Aplied Biosystems and Life Technology Corporation; Carlsbad CA) and Roche (Roche Molecular Diagnostics, Plesanton CA) which are routinely used in our laboratories. After approval from the local Ethics Committees, this study was carried out according to the Principles of the Declaration of Helsinki; informed consent was obtained from all of the subjects.
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fluorescence intensity depends on the amount of specific PCR products. The amplifications per cycle can be monitored with the LightCycler instrument: at the end of the amplification process, the LightCycler instrument increases the temperature, and the fluorescence obtained is plotted against the temperature. The mutations are then identified by their characteristic curves [9–11]. 2.5. GeneXpert HemosIL Factor II and Factor V
2.2. Sample selection Samples considered in this study had been previously classified (with a concordant result), by using two commercial real-time PCR assays supplied by Applied Biosystems and Roche Molecular Biochemicals. A total of 132 stored samples were evaluated from an external shared repository: 75 normal subjects, 10 FVL homozygous, 35 FVL heterozygous, 7 GPRO heterozygous, 2 GPRO homozygous and 3 double heterozygous FVL and GPRO. Samples were selected by F.M. in Lab A and sent to G.G. and S.V. in Lab B where the GeneXpert assay was performed without any knowledge of the sample classification. The results were then sent to Lab A for comparison. For the genetic tests, the blood samples were collected into vacuum tubes (Becton Dickinson) containing K3-EDTA. The whole blood was then transferred from the vacutainers to prelabelled 1.8 mL cryovials containing 180 μL DMSO using a sterile plastic transfer pipette. The blood and cryopreservative were mixed by inverting the cryovials four to five times, and they were then placed in a tube rack and stored at approximately − 80 °C [5,6]. 2.3. Applied biosystems assay Genomic DNA was extracted from 200 μL of whole blood collected in K3-EDTA tubes. The extraction method adopted in our Laboratory was based on paramagnetic silica beads (BioSprint 15 DNA blood kit, Qiagen) performed using devoted automated equipment, King Fischer m1 (Thermo Labsystem). The presence of the G1691A or G20210A single point mutations were evaluated using real-time PCR and allelic discrimination using Taqman probes. The reference sequences for the FV and FII genes were obtained from the National Center for Biotechnology Information (NCBI) database (http://www. ncbi.nlm.gov). The primers and probes used in these tests were designed using Primers Express 2.0 software (Applied Biosystems). The analytical process was performed following the manufacturers' instructions using an automated analyser, ABI Prism 7900 HT (Applied Biosystems) [7,8]. 2.4. Roche LightCycler assay DNA samples were isolated from whole blood using a MagNA Pure LC DNA Isolation Kit I with a MagNA Pure LC Automated DNA isolation instrument (Roche Molecular Biochemicals). The DNA samples were stored at −20 °C until the mutations were investigated. RealTime PCR: The factor V Leiden and prothrombin G20210A mutations were detected with the aid of the LightCycler Factor V Leiden and LightCycler-Prothrombin 20210GA Mutation Detection Kits (Roche Molecular Biochemicals), respectively. All of the mutation-related gene regions were amplified in 20 μL PCR capillary tubes. After preparation of the master mixture, 18 μL of the reaction mixture and 2 μL (approximately 40 ng) of genomic DNA or control template were added to each LightCycler capillary tube. For the negative control, PCR-grade water was added instead of template DNA. The capillary tubes were sealed and briefly centrifuged in a microcentrifuge and then placed into the LightCycler carousel. The PCR products were detected using 3′-fluorescein (FLU)-labelled and 5′-Red 640-labelled probes. When both probes hybridise in close proximity, fluorescence resonance energy transfer (FRET) occurs, producing a specific fluorescence emission of LC-Red as a result of the FLU excitation, and the
The GeneXpert assay is a software-driven cartridge processor with an integrated thermal cycler with a fluorescence-based detection system that adopts single-use disposable cartridges to obtain nucleic acid isolation from whole blood and perform a quantitative polymerase chain reaction (PCR). The GeneXpert cartridge consists of multiple chambers for the complete automation of the nucleic acid extraction from whole blood, PCR reaction and product analysis and quantification. The cartridges contain chambers for sample introduction, the lysis buffer, the purification and elution buffers, and all of the RTPCR reagents and enzymes on two freeze-dried beads: bead one for the polymerase and nucleotides and bead two for the primers and probes. Moreover, all of the sample-processing wastes are retained within the cartridge. The cartridge also has an attached PCR tube with fluid connections to the reagents in the cartridge chambers; after introduction into the analyser, this PCR tube is surrounded by heating/cooling plates and by optical blocks that enable amplification and the real-time, fluorescence-based PCR product detection. A syringe barrel at the centre of the cartridge has a dry interface (to minimise the potential for contamination) with the GeneXpert through a syringe plunger, thereby allowing the movement of fluids within the cartridge and between the PCR tube and the cartridge [12–14]. Fluid movement within the cartridge is controlled by a rotary valve; the valve body contains a cavity containing nucleic acid purification beads. Inlet and outlet ports are contained within the cavity and permit fluid movement over the purification beads. These beads are retained within the cavity by screens that cover the two ports on the back of the valve body and a flat cap on the front of the valve body. For nucleic acid capture, a whole blood lysate solution from one reagent chamber is flowed through the beads within the valve body [14–16]. The bound nucleic acid is then washed, eluted, mixed with PCR reagents and dispensed into the PCR tube for amplification. The real-time detection is achieved with the Scorpion technique. Scorpion primers are bi-functional molecules in which a primer is covalently linked to the probe; the molecules also contain a fluorophore and a quencher. In the absence of the target, the quencher nearly absorbs the fluorescence emitted by the fluorophore. During the Scorpion PCR reaction, in the presence of the target, the fluorophore and the quencher separate, which leads to an increase in the emitted fluorescence. The fluorescence can be detected and measured in the reaction tube [17–20]. 2.6. Statistical analysis Statistical analysis was performed using dedicated software (MedCalc version 8.1.1.0, MedCalc Software, Mariakerke, Belgium). 3. Results All of the samples considered in this study were obtained from patients of Italian ancestry with ages ranging from 16 to 89 years (mean 49 years); 85 (64%) were female and 48 (36%) were male. The samples considered in this study were obtained from our frozen repository and were classified by concordant results obtained using two different commercial real-time PCR assays for the detection of FV G1691A and FII G20210A single point mutations. An aliquot of these samples was thawed and retested using the GeneXpert HemosIL Factor II and Factor V assay. As reported in Table 1, all of
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G. Gessoni et al. / Clinica Chimica Acta 413 (2012) 814–816
Table 1 Results obtained using the GeneXpert HemosIL Factor II and Factor V assay. Subject classification
Number
Correctly classified
Normal subjects FVL homozygous FVL heterozygous GPRO homozygous GPRO heterozygous GPRO + FVL double heterozygous Total
75 10 35 2 7 3 132
75 (100%) 10 (100%) 35 (100%) 7 (100%) 7 (100%) 3 (100%) 132 (100%)
the evaluated samples were correctly identified using the GeneXpert system. In this patient series, the specificity and sensitivity of the test under evaluation was 1.00. 4. Discussion In our opinion, the GeneXpert system is an important new development in the field of molecular diagnostics. This system automates all of the steps of PCR in a disposable, microfluidic cartridge. The test is moderately complex, but the assay is simple enough to be performed reliably by individuals without a background in nucleic acid diagnostics; the sample preparation reagents and sample are all that are required to be added to the cartridge by the user. The assay has independently controlled and operated analysis modules that facilitate the testing of individual samples in a random-access mode. The test incorporates an internal control that ensures that the entire test system is functioning properly, and a probe-check control step is performed before the PCR to verify the reagent rehydration, probe integrity, and PCR tube filling in the cartridge. Thus, with the GeneXpert HemosIL Factor II and Factor V assay, it is possible to perform completely automated genetic tests from the addition of the whole blood and reagents to the cartridge through the final results. With this system, there is no need for a further analytical phase, i.e., DNA extraction, or the need for a dedicated room for the molecular biology assay. The system is uniquely suited for clinical applications of molecular diagnostics when on-demand testing and rapid-result capability are needed [15]. The diagnosis of thrombophilia risk factors should be one such application. To our knowledge, this is the first study in which the GeneXpert HemosIL Factor II and Factor V assay was evaluated using stored samples classified with two commercial real-time PCR methods for the detection of FV G1691A and FII G20210A polymorphisms. The analytical performance of the GeneXpert HemosIL Factor II and Factor V assay was quite satisfactory; indeed, all of the considered samples were correctly classified, and the specificity and sensitivity of the assay under evaluation were 1.00, in good agreement with the literature [17]. An interesting characteristic of the subject series considered in this study is the presence, in addition to homozygous normal subjects, of patients who were homozygous and heterozygous for FVL, patients who were homozygous and heterozygous for GPRO and three double heterozygous patients showing the presence of FVL and GPRO. We previously reported a high prevalence of FVL carriers in Chioggia in both patients with previous thrombosis in addition to healthy individuals from the general population and blood donors [21,22]. Thus, the study of this thrombophilia risk factor is an important aspect of the routine workflow in our laboratory, and the adoption of GeneXpert HemosIL Factor II and Factor V assay has significantly improved our capability of a rapid and affordable response. In this study, we have shown that the GeneXpert HemosIL Factor II and Factor V assay, a fully automated real-time PCR assay that was completed in less than 35 min, can accurately characterise the presence of FV G1691A and FII G20210A polymorphisms. This assay was clearly
comparable in specificity and sensitivity with other current realtime PCR assay based methods. The theoretical advantages of such an assay include improved standardisation across varying healthcare environments, more thorough sample manipulation and reduced human error [23]. Conflict of interest The authors are employed by the Italian National Health Service, and no financial contribution for this study was obtained from the manufacturers of the analyser used in this study. Moreover, none of the authors has a financial relationship with the manufacturers of the analyser used in this study. References [1] De Stefano V, Rossi E, Paciaroni K, Leone G. Screening for inherited thrombophilia: indications and therapeutic implications. Haematologica 2002;87:1095–108. [2] Gessoni G, Valverde S. Clinical evaluation of a functional prothrombin time-based assay for identification of factor V Leiden carriers in a group of Italian patients with venous thrombosis. Blood Coagul Fibrinolysis 2007;18:603–10. [3] Tripodi A, Mannucci P. Laboratory investigation of thrombophilia. Clin Chem 2001;47:1597–606. [4] Somma J, Sussman I, Rand J. An evaluation of thrombophilia screening in an urban tertiary care medical center: a “real word” experience. Am J Clin Pathol 2002;126: 1–8. [5] Sayinalp N, Haznedaroğlu I, Aksu S, et al. The predictability of factor V Leiden (FV: Q506) gene mutation via clotting based diagnosis of activated protein C resistance. Clin Appl Thromb Hemost 2004;10:265–70. [6] Stevens V, Patel A, Feigelson H, et al. Cryopreservation of whole blood samples collected in the field for a large epidemiologic study. Cancer Epidemiol Biomarkers Prev 2007;16:2160–3. [7] Gessoni G, Valverde S, Canistro R, et al. Valutazione di un esame funzionale basato sul tempo di protrombina nello studio della resistenza alla proteina C attivata. Biochim Clin 2007;31:567–72. [8] Castley A, Higgins M, Ivey J, et al. Clinical applications of whole-blood PCR with real-time instrumentation. Clin Chem 2005;51:2025–30. [9] Nauck M, Marz W, Wieland H. Evaluation of the Roche doagnostic light cycler factor V Leiden mutation detection kit and the light cycler prothrombin mutation detection kit. Clin Biochem 2000;33:213–6. [10] Mammo L, Sheereen A, Saour T, et al. Comparative study between the Light Cycler and the PCR-restriction fragment length polymorphism in detecting factor V Leiden\ and factor II 20210G > A mutations. Clin Biochem 2006;39:767–9. [11] Irdem A, Devecioglu C, Batun S, et al. Prevalence of factor V Leiden and prothrombin G20210A gene mutation. Saudi Med J 2005;26:580–3. [12] Hughes S, Xi L, Raja S, et al. A rapid, fully automated molecular based assay accurately analyzes sentinel lymph nodes for the presence of metastatic breast cancer. Ann Surg 2006;243:389–98. [13] Kost C, Rogers B, Oberste M, et al. Multicentric beta trial of the GeneXpert enterovirus assay. J Clin Microbiol 2007;45:1081–6. [14] Raja S, Ching J, Xi L, et al. Technology for automated rapid and quantitative PCR or reverse transcription PCR clinical testing. Clin Chem 2005;51:882–90. [15] Miller S, Moayeri M, Wright C, et al. Comparison of GeneXpert FluA PCR to direct fluorescent antibody and respiratory viral panel PCR assays for detection of 2009 novel H1N1 influenza virus. J Clin Microbiol 2010;48:4684–5. [16] Moure R, Muñoz L, Torres M, et al. Rapid detection of Mycobacterium tuberculosis complex and rifampin resistance in smear-negative clinical samples by use of an integrated real-time PCR method. J Clin Microbiol 2011;49:1137–9. [17] Morelli B, Novelli C, Grassi C, et al. An automation experience in molecular biology: the GeneXper Dx System for FV Leiden and FH 20210A mutation detection. Poster presented at SISET Congress Florence 25–28 September 2008; 2008. [18] Mamotte C. Genotyping of single nucleotide substitutions. Clin Biochem Rev 2006;27:63–75. [19] Carters R, Ferguson J, Gaut R, Ravetto P, Thelwell N, Whitcombe D. Design and use of scorpions fluorescent signaling molecules. Methods Mol Biol 2008;429(9): 9–115. [20] Reynisson E, Josefsen MH, Krause M, Hoorfar J. Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR. J Microbiol Methods 2006;66:206–16. [21] Gessoni G, Valverde S, Canistro R, et al. Factor V Leiden in Chioggia: a prevalence study in patients with venous thrombosis, their blood relatives and the general population. Blood Transfus 2010;8:193–5. [22] Valverde S, Antico F, Trabuio E, et al. Thrombophilia risk factors evaluation in a group of Italian patients with deep venous thrombosis. Recenti Prog Med 2008;99:348–53. [23] Bianchi M, Emanuele E, Davin A, et al. Comparison of three methods for genotyping of prothrombotic polymorphisms. Clin Exp Med 2010;10:269–72.
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Short communication
Evaluation of the GeneXpert assay in the detection of Factor V Leiden and Prothrombin 20210 in stored, previously classified samples G. Gessoni a,⁎, S. Valverde a, F. Manoni b a b
Service of Laboratory Medicine, Chioggia, Venice, Italy Service of Laboratory Medicine, Monselice, Padua, Italy
a r t i c l e
i n f o
Article history: Received 17 September 2011 Received in revised form 23 December 2011 Accepted 15 January 2012 Available online 21 January 2012 Keywords: Factor V Leiden GeneXpert Prothrombin 20210
a b s t r a c t Background: In this study, we evaluated the GeneXpert HemosIL Factor II and Factor V assay, an innovative assay for the detection of Factor V Leiden (FVL) and prothrombin G20210A mutation (GPRO). Patients and methods: We evaluated 132 patients that were previously classified (with a concordant result) using two commercial real-time PCR assays supplied, by Applied Biosystems and Roche Molecular Biochemicals. The cohort comprised 75 normal subjects, 10 FVL homozygous, 35 FVL heterozygous, 7 GPRO heterozygous, 2 GPRO homozygous and 3 double heterozygous FVL and GPRO subjects. All of the samples were evaluated using the GeneXpert HemosIL Factor II and Factor V assay. Results: All of the samples were correctly identified using the GeneXpert HemosIL Factor II and Factor V assay; therefore, in this patient series, the specificity and sensitivity of the test under evaluation was 1.00. Discussion: We have shown that the GeneXpert HemosIL Factor II and Factor V assay, a rapid fully automated assay, can accurately characterise the presence of FV G1691A and FII G20210A polymorphisms with specificity and sensitivity that are comparable to other current real-time PCR-based methods. The theoretical advantages of such an assay include improved standardisation across varying healthcare environments, more thorough sample manipulation and reduced human error. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Factor V Leiden (FVL) is an autosomal dominant condition that exhibits incomplete dominance and results in a factor V variant that cannot be as easily degraded as the wild type by activated Protein C (aPC). The gene that codes factor V is referred to as F5. FVL is characterised by a single nucleotide polymorphism in exon 10, with a G to A substitution at position 1691, G1691A, and it also predicts a single amino acid replacement of an arginine to glutamine (R506Q) at one of three aPC cleavage sites in factor Va. Because this residue 506 is normally the cleavage site for aPC, the mutation prevents the efficient inactivation of factor V. When factor V remains active, it facilitates the overproduction of thrombin, leading to excess fibrin generation and excess clotting. Clinical Laboratory evaluation for factor V Leiden can begin with a test for activated protein C resistance because aPC resistance is usually (95%) due to a factor V Leiden mutation. If resistance is present, then a test for the factor V Leiden gene mutation is Abbreviations: aPC, Activated C Protein; FVL, Factor V Leiden; Lab A, Laboratory Monselice; Lab B, Laboratory Chioggia; PCR, Polymerase Chain Reaction; GPRO, Prothrombin 20210 variant (Prothrombin G). ⁎ Corresponding author at: Service of Laboratory Medicine, Ospedale Civile, Via Madonna Marina 500, 30015 Chioggia Venice, Italy. Tel.: + 39 041 5534 400; fax: + 39 041 5534 401. E-mail addresses: [email protected], [email protected] (G. Gessoni). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.01.016
performed, both to confirm the diagnosis and to determine whether the person is heterozygous or homozygous for the mutation [1,2]. Prothrombin is the precursor to thrombin in the coagulation cascade, and thrombin is required to convert fibrinogen into fibrin, which is the primary goal of the coagulation cascade. The Prothrombin 20210 gene mutation (GPRO) is caused by a G to A transition in the 3′-untranslated region of the prothrombin (Factor II) gene at position 20210. This condition is associated with mild increases in plasma prothrombin levels and an increased risk of venous thrombosis. Diagnosis of the Prothrombin 20210 gene mutation must be made through specific genetic testing, which can also reveal whether the patient is heterozygous or homozygous for the condition. Although prothrombin levels are usually moderately elevated in subjects with this mutation and could be measured, they are not clinically useful in identifying the mutation [3,4]. In this study, the analytical sensitivity and specificity of the GeneXpert HemosIL Factor II and Factor V assay were determined using a panel of 132 subjects previously characterised using two commercial real-time PCR assays adopted for routine diagnosis in our laboratories. 2. Materials and methods 2.1. Study design We compared the results obtained using the HemosIL GeneXpert test for the detection of FVL and GPRO with those obtained from
G. Gessoni et al. / Clinica Chimica Acta 413 (2012) 814–816
two commercial tests supplied by Applied Biosystems (Aplied Biosystems and Life Technology Corporation; Carlsbad CA) and Roche (Roche Molecular Diagnostics, Plesanton CA) which are routinely used in our laboratories. After approval from the local Ethics Committees, this study was carried out according to the Principles of the Declaration of Helsinki; informed consent was obtained from all of the subjects.
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fluorescence intensity depends on the amount of specific PCR products. The amplifications per cycle can be monitored with the LightCycler instrument: at the end of the amplification process, the LightCycler instrument increases the temperature, and the fluorescence obtained is plotted against the temperature. The mutations are then identified by their characteristic curves [9–11]. 2.5. GeneXpert HemosIL Factor II and Factor V
2.2. Sample selection Samples considered in this study had been previously classified (with a concordant result), by using two commercial real-time PCR assays supplied by Applied Biosystems and Roche Molecular Biochemicals. A total of 132 stored samples were evaluated from an external shared repository: 75 normal subjects, 10 FVL homozygous, 35 FVL heterozygous, 7 GPRO heterozygous, 2 GPRO homozygous and 3 double heterozygous FVL and GPRO. Samples were selected by F.M. in Lab A and sent to G.G. and S.V. in Lab B where the GeneXpert assay was performed without any knowledge of the sample classification. The results were then sent to Lab A for comparison. For the genetic tests, the blood samples were collected into vacuum tubes (Becton Dickinson) containing K3-EDTA. The whole blood was then transferred from the vacutainers to prelabelled 1.8 mL cryovials containing 180 μL DMSO using a sterile plastic transfer pipette. The blood and cryopreservative were mixed by inverting the cryovials four to five times, and they were then placed in a tube rack and stored at approximately − 80 °C [5,6]. 2.3. Applied biosystems assay Genomic DNA was extracted from 200 μL of whole blood collected in K3-EDTA tubes. The extraction method adopted in our Laboratory was based on paramagnetic silica beads (BioSprint 15 DNA blood kit, Qiagen) performed using devoted automated equipment, King Fischer m1 (Thermo Labsystem). The presence of the G1691A or G20210A single point mutations were evaluated using real-time PCR and allelic discrimination using Taqman probes. The reference sequences for the FV and FII genes were obtained from the National Center for Biotechnology Information (NCBI) database (http://www. ncbi.nlm.gov). The primers and probes used in these tests were designed using Primers Express 2.0 software (Applied Biosystems). The analytical process was performed following the manufacturers' instructions using an automated analyser, ABI Prism 7900 HT (Applied Biosystems) [7,8]. 2.4. Roche LightCycler assay DNA samples were isolated from whole blood using a MagNA Pure LC DNA Isolation Kit I with a MagNA Pure LC Automated DNA isolation instrument (Roche Molecular Biochemicals). The DNA samples were stored at −20 °C until the mutations were investigated. RealTime PCR: The factor V Leiden and prothrombin G20210A mutations were detected with the aid of the LightCycler Factor V Leiden and LightCycler-Prothrombin 20210GA Mutation Detection Kits (Roche Molecular Biochemicals), respectively. All of the mutation-related gene regions were amplified in 20 μL PCR capillary tubes. After preparation of the master mixture, 18 μL of the reaction mixture and 2 μL (approximately 40 ng) of genomic DNA or control template were added to each LightCycler capillary tube. For the negative control, PCR-grade water was added instead of template DNA. The capillary tubes were sealed and briefly centrifuged in a microcentrifuge and then placed into the LightCycler carousel. The PCR products were detected using 3′-fluorescein (FLU)-labelled and 5′-Red 640-labelled probes. When both probes hybridise in close proximity, fluorescence resonance energy transfer (FRET) occurs, producing a specific fluorescence emission of LC-Red as a result of the FLU excitation, and the
The GeneXpert assay is a software-driven cartridge processor with an integrated thermal cycler with a fluorescence-based detection system that adopts single-use disposable cartridges to obtain nucleic acid isolation from whole blood and perform a quantitative polymerase chain reaction (PCR). The GeneXpert cartridge consists of multiple chambers for the complete automation of the nucleic acid extraction from whole blood, PCR reaction and product analysis and quantification. The cartridges contain chambers for sample introduction, the lysis buffer, the purification and elution buffers, and all of the RTPCR reagents and enzymes on two freeze-dried beads: bead one for the polymerase and nucleotides and bead two for the primers and probes. Moreover, all of the sample-processing wastes are retained within the cartridge. The cartridge also has an attached PCR tube with fluid connections to the reagents in the cartridge chambers; after introduction into the analyser, this PCR tube is surrounded by heating/cooling plates and by optical blocks that enable amplification and the real-time, fluorescence-based PCR product detection. A syringe barrel at the centre of the cartridge has a dry interface (to minimise the potential for contamination) with the GeneXpert through a syringe plunger, thereby allowing the movement of fluids within the cartridge and between the PCR tube and the cartridge [12–14]. Fluid movement within the cartridge is controlled by a rotary valve; the valve body contains a cavity containing nucleic acid purification beads. Inlet and outlet ports are contained within the cavity and permit fluid movement over the purification beads. These beads are retained within the cavity by screens that cover the two ports on the back of the valve body and a flat cap on the front of the valve body. For nucleic acid capture, a whole blood lysate solution from one reagent chamber is flowed through the beads within the valve body [14–16]. The bound nucleic acid is then washed, eluted, mixed with PCR reagents and dispensed into the PCR tube for amplification. The real-time detection is achieved with the Scorpion technique. Scorpion primers are bi-functional molecules in which a primer is covalently linked to the probe; the molecules also contain a fluorophore and a quencher. In the absence of the target, the quencher nearly absorbs the fluorescence emitted by the fluorophore. During the Scorpion PCR reaction, in the presence of the target, the fluorophore and the quencher separate, which leads to an increase in the emitted fluorescence. The fluorescence can be detected and measured in the reaction tube [17–20]. 2.6. Statistical analysis Statistical analysis was performed using dedicated software (MedCalc version 8.1.1.0, MedCalc Software, Mariakerke, Belgium). 3. Results All of the samples considered in this study were obtained from patients of Italian ancestry with ages ranging from 16 to 89 years (mean 49 years); 85 (64%) were female and 48 (36%) were male. The samples considered in this study were obtained from our frozen repository and were classified by concordant results obtained using two different commercial real-time PCR assays for the detection of FV G1691A and FII G20210A single point mutations. An aliquot of these samples was thawed and retested using the GeneXpert HemosIL Factor II and Factor V assay. As reported in Table 1, all of
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G. Gessoni et al. / Clinica Chimica Acta 413 (2012) 814–816
Table 1 Results obtained using the GeneXpert HemosIL Factor II and Factor V assay. Subject classification
Number
Correctly classified
Normal subjects FVL homozygous FVL heterozygous GPRO homozygous GPRO heterozygous GPRO + FVL double heterozygous Total
75 10 35 2 7 3 132
75 (100%) 10 (100%) 35 (100%) 7 (100%) 7 (100%) 3 (100%) 132 (100%)
the evaluated samples were correctly identified using the GeneXpert system. In this patient series, the specificity and sensitivity of the test under evaluation was 1.00. 4. Discussion In our opinion, the GeneXpert system is an important new development in the field of molecular diagnostics. This system automates all of the steps of PCR in a disposable, microfluidic cartridge. The test is moderately complex, but the assay is simple enough to be performed reliably by individuals without a background in nucleic acid diagnostics; the sample preparation reagents and sample are all that are required to be added to the cartridge by the user. The assay has independently controlled and operated analysis modules that facilitate the testing of individual samples in a random-access mode. The test incorporates an internal control that ensures that the entire test system is functioning properly, and a probe-check control step is performed before the PCR to verify the reagent rehydration, probe integrity, and PCR tube filling in the cartridge. Thus, with the GeneXpert HemosIL Factor II and Factor V assay, it is possible to perform completely automated genetic tests from the addition of the whole blood and reagents to the cartridge through the final results. With this system, there is no need for a further analytical phase, i.e., DNA extraction, or the need for a dedicated room for the molecular biology assay. The system is uniquely suited for clinical applications of molecular diagnostics when on-demand testing and rapid-result capability are needed [15]. The diagnosis of thrombophilia risk factors should be one such application. To our knowledge, this is the first study in which the GeneXpert HemosIL Factor II and Factor V assay was evaluated using stored samples classified with two commercial real-time PCR methods for the detection of FV G1691A and FII G20210A polymorphisms. The analytical performance of the GeneXpert HemosIL Factor II and Factor V assay was quite satisfactory; indeed, all of the considered samples were correctly classified, and the specificity and sensitivity of the assay under evaluation were 1.00, in good agreement with the literature [17]. An interesting characteristic of the subject series considered in this study is the presence, in addition to homozygous normal subjects, of patients who were homozygous and heterozygous for FVL, patients who were homozygous and heterozygous for GPRO and three double heterozygous patients showing the presence of FVL and GPRO. We previously reported a high prevalence of FVL carriers in Chioggia in both patients with previous thrombosis in addition to healthy individuals from the general population and blood donors [21,22]. Thus, the study of this thrombophilia risk factor is an important aspect of the routine workflow in our laboratory, and the adoption of GeneXpert HemosIL Factor II and Factor V assay has significantly improved our capability of a rapid and affordable response. In this study, we have shown that the GeneXpert HemosIL Factor II and Factor V assay, a fully automated real-time PCR assay that was completed in less than 35 min, can accurately characterise the presence of FV G1691A and FII G20210A polymorphisms. This assay was clearly
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