Keywords
Factor V Leiden
Prothrombin
PCR-RFLP
Molecular Diagnosis
How to Cite
Abstract
Background: Hereditary thrombophilia is a predisposing factor for venous thromboembolic disease, frequently associated with Factor V Leiden (G1691A) and Prothrombin G20210A polymorphisms. Molecular identification of these variants is essential for risk stratification and the implementation of prophylactic strategies. Aim: The aim of this study was to optimize and validate an in-house method based on polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP) for the detection of both polymorphisms. Methods: An in silico polymerase chain reaction was performed to assess primer specificity, followed by optimization of amplification conditions by using a thermal gradient. Amplified products were subjected to enzymatic digestion with MnlI and HindIII and subsequently analyzed by agarose gel electrophoresis. Validation included analytical sensitivity and specificity, accuracy (by Sanger sequencing), trueness, interference testing, robustness, and precision. Results: Specific amplicons of 267 bp for Factor V and 506 bp for Prothrombin were obtained, with optimal annealing temperatures of 58 °C and 59 °C, respectively. The method demonstrated 100% sensitivity and specificity, with complete concordance compared to an external laboratory and sequencing results. No interferences were detected, and the assays showed high repeatability and reproducibility. Conclusion: The optimized technique represents a rapid, accurate, robust, and cost-effective tool for the detection of these polymorphisms, facilitating the identification of individuals with genetic predisposition to thrombotic events.
References
1. Noroña Calvachi CD. Trombofilias hereditarias. Rev Cient Cienc Med. 2015;18(1):43-9. Disponible en: http://www.redalyc.org/articulo.oa?id=426041256009
2. Gallo MC, Maneyro A, Altuna D, Ceresetto J, Fassi D, Ferro H, et al. Trombofilias. En: Sociedad Argentina de Hematología, editor. Guías de diagnóstico y tratamiento: hemostasia y trombosis [Internet]. Buenos Aires: Sociedad Argentina de Hematología; 2023 [citado 2026 Jun 9]. p. 275-83. Disponible en: https://www.sah.org.ar/docs/guias/2023/Hemostasia_y_Trombosis-Guia_2023-Libro.pdf
3. Ríos M, Fernández-Zenoff V, Hayward C, Suárez A, Rossi E. Study of hereditary thrombophilia (factor V Leiden and prothrombin 20210) in women of fertile age with thromboembolic events studied in the Tucumán public health laboratory. J Cardiol Curr Res. 2020;13(2):35-9, https://doi.org/10.15406/jccr.2020.13.00471
4. Grande Ratti MF, Posadas-Martínez ML, Vicens J, González Bernaldo de Quirós F, Vázquez FJ, Giunta DH. Incidence of hospital-acquired venous thromboembolic disease. Rev Fac Cien Med Univ Nac Cordoba. 2018;75(2):82-7, https://doi.org/10.31053/1853.0605.v75.n2.17243
5. Palomo GI, Pereira GJ, Alarcón LM, Pinochet PC, Vélez SM, Hidalgo PP, et al. Factor V Leiden y mutación de protrombina G20210A en pacientes con trombosis venosa y arterial. Rev Med Chil. 2005;133(12):1425-33, https://doi.org/10.4067/S0034-98872005001200003
6. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994;369(6475):64-7, https://doi.org/10.1038/369064a0
7. Vargas Ruíz AG. Trombofilias hereditarias: el perfil de pruebas necesarias. Hematol Mex. 2019;20(2):79-85, https://doi.org/10.24245/rhematol.v20i2.3096
8. Spector EB, Grody WW, Matteson CJ, Palomaki GE, Bellissimo DB, Wolff DJ, et al. Technical standards and guidelines: venous thromboembolism (factor V Leiden and prothrombin 20210G>A testing). Genet Med. 2005;7(6):444-53, https://doi.org/10.1097/01.GIM.0000172641.57755.3A
9. Kindermann EHS, Rodrigues Campos K, Caterino-de-Araujo A. Performance
of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene. Rev Inst Adolfo Lutz. 2023;82:e39195. Disponible en: https://periodicos.saude.sp.gov.br/RIAL/article/view/39195
10. Leotta GA, Chinen I, Epszteyn S, Miliwebsky E, Melamed IC, Motter M, et al. Validación de una técnica de PCR múltiple para la detección de Escherichia coli productora de toxina Shiga. Rev Argent Microbiol. 2005;37(1):1-10.
11. Jennings L, Van Deerlin VM, Gulley ML, College of American Pathologists Molecular Pathology Resource Committee. Recommended principles and practices for validating clinical molecular pathology tests. Arch Pathol Lab Med. 2009;133(5):743-55, https://doi.org/10.5858/133.5.743
12. Burd EM. Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev. 2010;23(3):550-76, https://doi.org/10.1128/CMR.00074-09
13. Yepes-Pérez M, Carrero K, Vásquez N, Correa E. Validación de PCR convencional para detectar Escherichia coli O157. Inf Tecnol. 2022;33(2):3- 12, https://doi.org/10.4067/S0718-07642022000200003
14. Danneberg J, Abbes AP, Bruggeman BJ, Engel H, Gerrits J, Martens A. Reliable genotyping of the G-20210-A mutation of coagulation factor II (prothrombin). Clin Chem. 1998;44(2):349-51, https://doi.org/10.1093/clinchem/44.2.349
15. Wallace RB, Shaffer J, Murphy RF, Bonner J, Hirose T, Itakura K. Hybridization of synthetic oligodeoxyribonucleotides to ΦX174 DNA: the effect of single base pair mismatch. Nucleic Acids Res. 1979;6(11):3543-57, https://doi.org/10.1093/nar/6.11.3543
16. Cardona-Ramírez C, Enríquez-Valencia CE, Méndez-Callejas G, Meza Barreto G, Tafur-Gómez GA, Sanjuanelo-Corredor DW. An integrated methodological framework for the validation and verification of clinical testing by qRT-PCR. Heliyon. 2025;11(1):e41088, https://doi.org/10.1016/j.heliyon.2024.e41088
17. Yusuf E, Schijffelen MJ, Leeflang MMG. How to verify and validate a clinical microbiology test before it can be used in routine diagnostics: a practical guide. Clin Microbiol Infect. 2024;30(10):1261-9, https://doi.org/10.1016/j.cmi.2024.06.028