Identification and quantification of hemoglobins in whole blood: the analytical and organizational aspects of Capillarys 2 Flex Piercing compared with agarose electrophoresis and HPLC methods

Sara Altinier 1 , Mariacristina Varagnolo 1 , Martina Zaninotto 1  and Mario Plebani 1
  • 1 Department of Laboratory Medicine, University-Hospital of Padova, Padova, Italy
Sara Altinier, Mariacristina Varagnolo, Martina Zaninotto and Mario Plebani


Background: The present study was conducted to evaluate the analytical performance and the organizational aspects of Capillarys 2 Flex Piercing system (CFP) respect to agarose electrophoresis and HPLC methods in hemoglobinopathies screening.

Methods: The measurement of imprecision in HbA2 and HbF quantification was verified on HbA2 CFP control and on three samples; 74 whole blood samples were used to evaluate migration time imprecision of hemoglobin variants S, C and E (HbS, HbC, and HbE); to compare methods, 451 samples were tested on CFP and HPLC; reference values were verified as value distribution in 160 blood donors and at ROC curve analysis on 449 samples from routine analysis.

Results: Imprecision: the analytical CV%s ranged from 1.25 to 3.9 at HbA2 quantification, the CV% was 3.78 at HbF quantification; the running time imprecision for HbS and HbC and HbE ranged from 0.20 to 0.69%. Method comparison: at regression analysis findings were HbA2: CFP=1.21×HPLC–0.64, HbF: CFP=1.31×HPLC-0.75, HbS: CFP=1.10×HPLC-3.24. Reference values: the HbA2 95th percentile range was 2.5–2.8; HbF was undetectable in 154 out 160 samples tested; at ROC curve analysis the best combination of sensitivity and diagnostic efficiency was obtained using 2.2 and 3.0, as reference values, for HbA2 and 1.1 as the upper reference limit for HbF. Organizational aspects: with respect to the procedures currently implemented in our laboratory CFP requires 2 h less time and obviates the need for some manual steps.

Conclusions: The quantification, reproducibility and diagnostic efficiency provided by CFP in identification and quantification of hemoglobins appear accurate. In addition, the use of primary tubes allows improved safety, and the avoidance of some manual steps, that prolong working time and are a source of possible errors.

  • 1.

    Vichinsky EP. Changing patterns of thalassemia worldwide. Ann N Y Acad Sci 2005;1054:18–24.

  • 2.

    Pack-Mabien A, Haynes J Jr. A primary care provider’s guide to preventive and acute care management of adults and children with sickle cell disease. J Am Acad Nurse Pract 2009;21:250–7.

  • 3.

    Cogliandro T, Derchi G, Mancuso L, Mayer MC, Pannone B, Pepe A, et al. Society for the Study of Thalassemia and Hemoglobinopathies (SoSTE. Guideline recommendations for heart complications in thalassemia major. J Cardiovasc Med 2008;9:515–25.

  • 4.

    Globin gene server. Accessed on 10 January 2012.

  • 5.

    Gwendolyn MC, Higgins TN. Laboratory investigation of hemoglobinopathies and thalassemias: review and update. Clin Chem 2000;46:1284–90.

  • 6.

    Mosca A, Paleari R, Leone D, Ivaldi G. The relevance of hemoglobin F measurement in the diagnosis of thalassemias and related hemoglobinopathies. Clin Biochem 2009;42:1797–801.

  • 7.

    Kleinert P, Schmid M, Zurbriggen K, Speer O, Schmugge M, Roschitzki B, et al. Mass spectrometry: a tool for enhanced detection of hemoglobin variants. Clin Chem 2008;54:69–76.

  • 8.

    Gosselin RC, Carlin AC, Dwyre DM. Comparison of the BioRad Variant and Primus Ultra2 high-pressure liquid chromatography (HPLC) instruments for the detection of variant hemoglobins. Int J Lab Hematol 2011;33:159–67.

  • 9.

    Pornprasert S, Kasemrad C, Sukunthamala K. Diagnosis of thalassemia on dried blood spot samples by high performance liquid chromatography. Hemoglobin 2010;34:486–94.

  • 10.

    Higgins T, Mack M, Khajuria A. Comparison of two methods for the quantification and identification of hemoglobin variants. Clin Biochem 2009;42:701–5.

  • 11.

    Ryan K, Bain BJ, Worthington D, James J, Plews D, Mason A, et al. British Committee for Standards in Haematology Significant haemoglobinopathies: guidelines for screening and diagnosis Br J Haematol 2010;149:35–49.

  • 12.

    Louahabi A, Philippe M, Lali S, Wallemacq P, Maisin D. Evaluation of a new Sebia kit for analysis of hemoglobin fractions and variants on the Capillarys system. Clin Chem Lab Med 2006;44:340–5.

  • 13.

    Mosca A, Paleari R, Ivaldi G, Galanello R, Giordano PC. The role of haemoglobin A(2. testing in the diagnosis of thalassaemias and related haemoglobinopathies. J Clin Pathol 2009;62:13–7.

  • 14.

    Stephens AD, Angastiniotis M, Baysal E, Chan V, Fucharoen S, Giordano PC, et al. ICSH recommendations for the measurement of haemoglobin A2. Int J Lab Hematol 2012;34:1–13.

  • 15.

    Yang Z, Chaffin CH, Easley PL, Thigpen B, Reddy VV. Prevalence of elevated hemoglobin A2 measured by the CAPILLARYS system. Am J Clin Pathol 2009;131:42–8.

  • 16.

    Lafferty J. College of American Pathologists hemoglobinopathy survey HG–B. Chicago, IL: College of American Pathologists, 1999.

  • 17.

    Cotton F, Malaviolle X, Vertongen F, Gulbis B. Evaluation of an automated capillary electrophoresis system in the screening for hemoglobinopathies. Clin Lab 2009;55:217–21.

  • 18.

    Higgins TN, Khajuria A, Mack M. Quantification of HbA(2. in patients with and without beta-thalassemia and in the presence of HbS, HbC, HbE, and HbD Punjab hemoglobin variants: comparison of two systems. Am J Clin Pathol 2009;131: 357–62.

  • 19.

    Keren DF, Hedstrom D, Gulbranson R, Ou CN, Bak R. Comparison of Sebia Capillarys capillary electrophoresis with the Primus high-pressure liquid chromatography in the evaluation of hemoglobinopathies Am J Clin Pathol 2008;130:824–31.

  • 20.

    Anagnostopoulos K, Tentes I, Kalleas C, Margaritis D, Toli A, Pendilas D, et al. Effect of HbS in the determination of HbA2 with the Menarini HA-8160 analyzer and comparison with other instruments. Int J Lab Hematol 2009;31:665–72.

  • 21.

    Giambona A, Passarello C, Renda D, Maggio A. The significance of the hemoglobin A2 value in screening for hemoglobinopathies. Clin Biochem 2009;42:1786–96.

  • 22.

    Van Delft P, Lenters E, Bakker-Verweij M, de Korte M, Baylan U, Harteveld CL, et al. Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic populations. Int J Lab Hematol 2009;31:484–95.

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Clinical Chemistry and Laboratory Medicine ( CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. CCLM welcomes contributions on the progress in fundamental and applied research and cutting-edge clinical laboratory medicine. It is one of the leading journals in the field, with an impact factor of over three. CCLM is the official journal of nine national clinical societies and associated with EFLM.