Capillary electrophoresis for the screening and diagnosis of inherited hemoglobin disorders. Ready for prime time?

Giuseppe Lippi; Mario Plebani

doi:10.1515/cclm-2015-0545

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Capillary electrophoresis for the screening and diagnosis of inherited hemoglobin disorders. Ready for prime time?

Giuseppe Lippi and Mario Plebani

Published/Copyright: August 1, 2015

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 54 Issue 1

Inherited hemoglobin (Hb) disorders, also known as hemoglobinopathies, are conventionally classified into two main categories, which entail structural Hb variants (i.e. qualitative or functional disorders) and thalassemias (i.e. quantitative disorders, characterized by defective globin production). According to recent statistics, approximately 7% of the worldwide population carries an inherited Hb disorder, and up to 500,000 babies are born each year with severe forms of these conditions [1]. Inherited Hb disorders are characteristic of tropics and subtropics, but their prevalence is now constantly increasing around the globe due to migration [2]. It is hence predictable that the diagnosis and management of inherited Hb disorders will grow exponentially around the globe, especially in some countries of the south Mediterranean area (e.g. Italy, Greece and Spain), due to the fact that civil wars and oppressive regimes in the Middle East and Africa have forced an ever increasing number of people on a perilous journey to Europe.

In agreement with the guidelines of the British Committee for Standards in Haematology, the choice of methodology and equipment for the diagnostics of inherited Hb disorders should be based on a balanced combination of workload, biological material (i.e. blood or dried blood spots), analytical performance, local availability of instrumentation and personnel, expertise and costs [3]. Although the current armamentarium for the screening and diagnosis of hemoglobinopathies includes many techniques, such as cellulose acetate electrophoresis (CAE), isoelectric focusing (IEF), low-pressure liquid chromatography (LPLC), high-performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS), capillary zone electrophoresis (CZE) and even genetic analysis, the Committee concluded that MS/MS and CE may be suitable alternative to HPLC provided that acceptable analytical and clinical performances can be fully demonstrated. Five years after the publication of these universally adopted recommendations, reliable evidence has accumulated that CZE may be an accurate tool for the screening and diagnosis of Hb disorders. In this issue of the journal we publish two studies that substantially support this assumption.

In the former article, You-Qiong et al. analyzed 15 samples (13 adult blood samples and 2 cord blood samples) of patients heterozygous for Hb New York by using both CZE and HPLC [4]. Interestingly, all cases could be diagnosed with CZE, whereas none of them could be detected by HPLC, as the variant could not be separated from HbA using the local equipment. Although the optimal performance of CZE for screening and diagnosis of Hb disorders has been recently confirmed in other studies [5], the potential advantages of this technique extend beyond the boundaries of the analytical domain. One of the major drawbacks of most commercial HPLC analyzers is represented by the challenge of accurately measuring HbA_1c in patients with hemoglobinopathies, especially in the presence of common Hb variants, such as HbC, HbS or HbF values >10–15% [6]. Nevertheless, two recent studies published in an earlier issue of this journal convincingly showed that this shortcoming may be at least in part overcome by CZE. Ji et al. measured HbA_1c in blood samples of patients with a variety of Hb disorders, and concluded that thalassemia, HbE, HbG Coushatta, HbG Taipei and Hb Kaohsiung complicated HbA_1c detection, whereas glycated Hb could be accurately measured using CZE [7]. As regards other common Hb variants, in another study Weykamp et al. evaluated the analytical interference of HbS, HbC, HbD, HbE, HbJ and HbG on HbA_1c accuracy [8], and also concluded that glycated Hb can be reliably measured with CZE.

In the second article published in this issue of the journal, Pornprasert et al developed specific quality control materials for the analysis of some forms of thalassemia and Hb variants that are commonly observed in South-East Asia [9]. Interestingly, the Hb typing control materials could be stored and then accurately analyzed by many commercially available techniques, including HPLC and CZE, thus representing a valuable resource for internal and external quality assurance in the diagnostics of hemoglobinopathies.

The use of CZE is now commonplace in clinical laboratories, due to the versatility of this technique for performing protein electrophoresis [10, 11] and immunotyping [12], as well as for the measurement of HbA_1c [13] or carbohydrate-deficient transferrin (CDT) [14]. Despite the use of CZE has several practical advantages, such as positive sample identification, cap piercing and on-board sample mixing capabilities, avoidance of some manual steps and reduced turn-around time, as well as high throughput and reproducibility [15], some technical issues still plague its accuracy, which typically include falsely elevated HbA₂ values in patients with HbC or overestimation of HbF values in HbS patients [16], as well as the different migration pattern of most Hb variants, which will force laboratory professional to revise and update their usual practice in the interpretation of chromatograms and electropherograms. Notwithstanding these limitations, it seems now reasonable to suggest that CZE may be ready for prime time for the screening and diagnosis of Hb disorders.

Corresponding author: Prof. Giuseppe Lippi, Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Via Gramsci 14, 43126 Parma, Italy, E-mail: glippi@ao.pr.it; ulippi@tin.it.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Weatherall D, Akinyanju O, Fucharoen S, Olivieri N, Musgrove P. Inherited disorders of hemoglobin. In: Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, et al., editors. Disease control priorities in developing countries, 2nd ed. Washington (DC): World Bank, 2006. Chapter 34.Search in Google Scholar

2. Lippi G, Montagnana M, Danese E, Salvagno GL, Bellorio F, Franchini M, et al. Frequency and type of newly diagnosed haemoglobin variants in Northern Italy. Blood Transfus 2010;8:307–8.Search in Google Scholar

3. 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;149:35–49.10.1111/j.1365-2141.2009.08054.xSearch in Google Scholar PubMed

4. You-Qiong L, Hui-Ping H, Zhi-Zhong C, Lin Z, Liang L, Gui-Fang Q, et al. Comparison of capillary electrophoresis and high performance liquid chromatography for detection and quantification of hemoglobin New York. Clin Chem Lab Med 2016;54:91–5.10.1515/cclm-2015-0238Search in Google Scholar PubMed

5. Oyaert M, Van Laer C, Claerhout H, Vermeersch P, Desmet K, Pauwels S, et al. Evaluation of the Sebia Minicap flex piercing capillary electrophoresis for hemoglobinopathy testing. Int J Lab Hematol 2015;37:420–5.10.1111/ijlh.12305Search in Google Scholar PubMed

6. National Glycohemoglobin Standardization Program (NGSP). Factors that interfere with HbA1c test results. Available from: http://www.ngsp.org/factors.asp. Accessed 7 June, 2015.Search in Google Scholar

7. Ji L, Yu J, Zhou Y, Xia Y, Xu A, Li W, et al. Erroneous HbA1c measurements in the presence of β-thalassemia and common Chinese hemoglobin variants. Clin Chem Lab Med 2015;53:1451–8.10.1515/cclm-2014-0598Search in Google Scholar PubMed

8. Weykamp C, Kemna E, Leppink S, Siebelder C. Glycation rate of haemoglobins S, C, D, E, J and G, and analytical interference on the measurement of HbA1c with affinity chromatography and capillary electrophoresis. Clin Chem Lab Med 2015;53:e207–10.10.1515/cclm-2014-1134Search in Google Scholar PubMed

9. Pornprasert S, Tookjai M, Punyamung M, Pongpunyayuen P, Jaiping K. Development of hemoglobin typing control materials for laboratory investigation of thalassemia and hemoglobinopathies. Clin Chem Lab Med 2016;54:81–9.10.1515/cclm-2015-0113Search in Google Scholar PubMed

10. Bossuyt X, Lissoir B, Mariën G, Maisin D, Vunckx J, Blanckaert N, et al. Automated serum protein electrophoresis by capillarys. Clin Chem Lab Med 2003;41:704–10.10.1515/CCLM.2003.107Search in Google Scholar PubMed

11. Lippi G, Battistelli L, Vernocchi A, Mussap M. Analytical evaluation of the novel Helena V8 capillary electrophoresis system. J Med Biochem 2013;32:245–9.10.2478/jomb-2013-0020Search in Google Scholar

12. Barlow IM, Kemp ML. Evaluation of the Sebia Capillarys zone electrophoresis system for monoclonal paraprotein analysis. Br J Biomed Sci 2010;67:150–3.10.1080/09674845.2010.11978217Search in Google Scholar PubMed

13. Marinova M, Altinier S, Caldini A, Passerini G, Pizzagalli G, Brogi M, et al. Multicenter evaluation of hemoglobin A1c assay on capillary electrophoresis. Clin Chim Acta 2013;424:207–11.10.1016/j.cca.2013.06.014Search in Google Scholar PubMed

14. Daves M, Cemin R, Floreani M, Pusceddu I, Cosio G, Lippi G. Comparative evaluation of capillary zone electrophoresis and HPLC in the determination of carbohydrate-deficient transferrin. Clin Chem Lab Med 2011;49:1677–80.10.1515/CCLM.2011.630Search in Google Scholar PubMed

15. Altinier S, Varagnolo M, Zaninotto M, Plebani M. 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. Clin Chem Lab Med 2013;51:791–7.10.1515/cclm-2012-0061Search in Google Scholar PubMed

16. Greene DN, Vaughn CP, Crews BO, Agarwal AM. Advances in detection of hemoglobinopathies. Clin Chim Acta 2015;439:50–7.10.1016/j.cca.2014.10.006Search in Google Scholar PubMed

Published Online: 2015-8-1

Published in Print: 2016-1-1

Articles in the same Issue

https://doi.org/10.1515/cclm-2015-0545