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Developing a reference system for the IFCC standardization of HbA2
CCA-14380; No of Pages 6 Clinica Chimica Acta xxx (2016) xxx–xxx
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Developing a reference system for the IFCC standardization of HbA2 Renata Paleari a, Donatella Caruso b, Patricia Kaiser c, Cristian Gabriel Arsene d, Christine Schaeffer-Reiss e,f, Alain Van Dorsselaer e,f, Emmanuel Bissé g, Maria Ospina h, Víctor R. De Jesús h, Barbara Wild i, Andrea Mosca a,⁎, on behalf of the IFCC Working Group on Standardisation of Hemoglobin A2 (WG-HbA2): a
Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Milano, Italy Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milano, Italy Instand e.V., Düsseldorf, Germany d Physikalisch-Technische Bundesanstalt (PTB), Braunschweig, Germany e BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France f IPHC, CNRS, UMR7178, Strasbourg, France g Department of Clinical Chemistry, University Medical Center, Freiburg, Germany h Division of Laboratory Sciences, National Center for Environmental Health, US Centers for Disease Control and Prevention, Atlanta, GA, USA i UKNEQAS(H), Watford, UK b c
a r t i c l e
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Article history: Received 18 December 2015 Received in revised form 25 May 2016 Accepted 26 May 2016 Available online xxxx Keywords: Analytical goals Beta-thalassemia Quality control Reference materials Reference measurement procedure Standardization
a b s t r a c t The importance of hemoglobin A2 (HbA2) as an indicator of the presence of β-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing. © 2016 Elsevier B.V. All rights reserved.
1. Introduction
2. Clinical needs for the accurate determination of HbA2
In December 2004, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working group for the standardization of HbA2 (IFCC WG-HbA2) was established. At that time, the IFCC working group for the standardization of HbA1c, (IFCC WG-HbA1c) was almost at the end of its tasks. It seemed appropriate to commence work on another minor hemoglobin, where standardization was urgently needed but no apparent work was being yet undertaken. The terms of reference for the new WG were directed to promote the standardization of hemoglobin A2 measurement through the definition of an international reference system, including a reference measurement procedure primary and secondary certified reference materials. This brief report summarizes the achievements so far and outlines the work remaining.
The inherited disorders of hemoglobin are among the most common monogenic diseases in the world. Recent surveys indicate that after sickle-cell disease, β-thalassemia has the highest incidence, with more than 22,000 annual births and with a mean global prevalence of 4.9% [1]. In order to prevent the occurrence of severe thalassemia-major phenotypes, accurate prenatal screening has to be performed. The measurement of HbA2 has an important role in achieving the correct diagnosis, together with other laboratory investigations and clinical information. Indeed, an increase in this hemoglobin fraction is one of the most widely used indications of β-thalassemia heterozygotes. However, some technical difficulties can arise in the identification of β-thalassemia carriers with only slightly increased HbA2 values. The occurrence of such borderline subjects is not rare, since there is a great heterogeneity of mutations and other conditions such as iron deficiency or the presence of δ-thalassemia may complicate the typical phenotype [2,3]. Therefore, because of the small differences between normal and pathological HbA2 values, strict quality assurance of HbA2 measurement is crucial for accurate diagnosis, particularly concerning genetic counselling of couples at-risk.
⁎ Corresponding author at: Università degli Studi di Milano, Dip. di Fisiopatologia Medico-Chirurgica e dei Trapianti, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy. E-mail address: [email protected] (A. Mosca).
http://dx.doi.org/10.1016/j.cca.2016.05.023 0009-8981/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023
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How well HbA2 needs to be measured is a topic which was discussed some time ago by this working group in terms of the analytical performance goals [4], and also by recommendations issued by the International Committee for the Standardization in Haematology [5]. Presently, there is no consensus on these goals, however a precision of ±0.1% amount of substance fraction HbA2/total Hb in the final report for the analysis, equivalent to a CV of 2%, should be obtainable using modern automated high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) equipment. In terms of trueness, ideally a zero bias should be pursued but in the absence of a reference measurement procedure for comparison, it is difficult to assess compliance with this goal. Concerning the relative total error, an acceptable limit of 7.0% has been recently proposed [6], to correctly classify a subject with a true HbA2 value of 3.6% besides other variables, such as mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). The total error (TE) goal based on biological variability is however even more stringent (4.5%) [4]. 3. State-of-the-art of current automated methods for HbA2 The state-of-the-art of the most commonly used methods for measuring HbA2 can be assessed by means of dedicated inter-comparisons, external quality assessment schemes (EQAS) or validation studies, as well as by using information directly provided by the manufacturers. There is limited data on the comparability of the different methods used for HbA2 quantitation, especially on the commonly used HPLC and CE techniques. Recent work prepared by Sangkitporn et al. [7] evaluated the analytical performance characteristics, including precision and accuracy, of the automated capillary electrophoresis method (Capillarys 2, Sebia, France) with existing validated HPLC and LPLC methods. The coefficients of variation of HbA2 quantitation were 1.80– 2.86%, 1.26–5.13% and 1.08–6.66% for within run, between run and inter-laboratory comparison, respectively. The results of HbA2 quantified by the CE method correlate well with those of the HPLC and LPLC methods (r = 0.98–0.99). Similar findings were obtained in a more recent study performed with different HPLC (Bio-Rad Variant I, Bio-Rad Variant II, Menarini HA-8160, Tosoh G7, Tosoh G8) and CE (Beckman Coulter MDQ, ProteomeLab PA800, Sebia Capillarys 2) methods. These
studies proved that the results of the different methods were highly correlated (r between 0.974 and 0.997), despite the bias between methods, with a within-run imprecision of HbA2 measurement (expressed as CV) between 0.5% and 4.4% (HPLC) and between 1.2% and 4.4% (CE) [8]. As it can be seen, the recommended CV goal for precision of 2.0% is not achieved by many routine methods. Some years ago an EQAS pilot exercise in Italy, using fresh blood samples and involving 48 Italian laboratories routinely measuring HbA2 [9], was performed to further assess the state-of-the-art. The overall inter-laboratory CVs were 8.0%, 6.0% and 7.9% for samples with low, high and intermediate HbA2 levels, respectively. Moreover, it was found that the fraction of laboratories reporting unacceptable results, assuming an allowable total error of 7.8%, ranged from 17.0% to 31.9%. In addition, some recent data were collected either in Italy from a mandatory EQAS exercise provided by the Region of Tuscany or from an EQAS exercise provided by a manufacturer (Bio-Rad Laboratories) covering many laboratories around the world, not only using methods from the same company. The data covered the 2014 year for the Italian EQAS (the data from the 2015 exercise is not available yet), and the 2015 exercise from Bio-Rad (in the 2014 exercise no samples with high HbA2 level were distributed), and are presented in Fig. 1. Results from three different control materials, with similar HbA2 concentration were compared and the consensus means established by the EQAS providers were chosen as the target HbA2 values. It is evident from these plots that the inter-laboratory dispersions (represented by the error bars) are often within the method performance goal for the total error, but there is certainly a significant bias between the methods. For instance, the mean value obtained by the users of the Tosoh G7 is clearly above the consensus mean in the 2014 Italian exercise, as well as for the results of users of the Bio-Rad Variant II dual kit in the 2015 EQAS. The comparison between the two exercises have to be done with some caution, because the materials analyzed were not the same, both were in the lyophilized form, but produced by different manufacturers. Nevertheless, the data show clearly that the methods are not completely aligned. From the data reported above, it can be concluded that the state-ofthe-art is not optimal, and that more standardization work is needed to achieve the desired method performance targets.
Fig. 1. Inter-laboratories results obtained from two different EQAS providers, at three different levels of HbA2/total hemoglobin amount of substance fraction, grouped according to the methods used by the participants. The left panel reports the data obtained in 2014 from the Italian Inter-Regional Program. The right panel refers to the data obtained from the international EQAS provided by Bio-Rad Laboratories. The numbers of participants per method are reported in brackets, from the lower to the higher HbA2 level. Points and error bars represent means and SD, respectively. The dotted lines represent the limits of the allowable total error of ±7%.
Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023
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4. Definition of a reference measurement procedure A major task of the working group was the development of a reference measurement procedure (RMP). Much effort was put into this aspect whereby different approaches based on mass spectrometry, which potentially could deliver more accurate results for HbA2, were applied, and briefly summarized below. Two different measurement principles were then followed. In the first approach, intact δ- and α-globin chains are separated and detected using LC-ESI/MS. The HbA2-fraction is determined by calibrating the mass spectrometer for the direct measurement of δ/α globin chain ratios. As an alternative approach, the amount of substances of HbA2 and total hemoglobin in blood are determined separately from each other in a first step. For this purpose, isotope dilution mass spectrometry (ID-MS) is used. The HbA2-fraction is then calculated in a second step as a ratio from the results for HbA2 and for total hemoglobin. Unlike the measurement of intact globin chains, tryptic peptides derived from and representing HbA2 and total hemoglobin, respectively, are being quantified. The HbA2 fraction is calculated as the ratio between the amount of substance determined for the δ and α chain derived peptides. These two MS procedures, based on different analytical measurement principles, were developed in parallel within the working group. Both procedures were established and optimized in different cooperating expert laboratories. Particularly, in respect to the main objective of the working group to certify reference materials for recalibration of the routine systems, the establishment of a reference laboratory
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network operating two different and independent measurement procedures has been considered to be of major advantage. Finally, with regards to the quantity measured and the units, the working group agrees that the measurand is the molecule of native HbA2, expressed as HbA2/total hemoglobin amount of substance, and reported in %. 4.1. Based on the quantification of intact globin chains A method based on the quantification of intact globin chains by LCESI/MS, which eliminates the need for a digestion step prior to MS analysis, was developed. The two major advantages of the measurement of intact globin chains, compared to the often used proteomic approach based on the measurement of targeted proteolytic peptides, are to avoid the potential measurement bias introduced by the trypsin digestion step and to reduce the efforts going into the sample preparation. For the calibration of the method, purified HbA2 and HbA0 solutions were mixed in well-defined ratios to form a set of calibrators with known HbA2 concentrations spanning across the expected range of measurement. The purified HbA2 and HbA0 were prepared by a twostep preparative chromatographic procedure consisting of consecutive ion exchange cellulose separations. The sample preparation consisted of red blood cell lysis followed by dilution with acetonitrile and desalting [10]. The measurements were performed using LC-MS equipped with an ESI source and a linear ion trap analyzer, in full scan mode. Due to the low content of the δ globin chains relative to the others, an ion chromatogram extraction of 10 masses corresponding to the charge states with the highest intensities for δ and α chains,
Fig. 2. Ion chromatograms obtained from the separation of a hemolysate prepared from the blood of a β-thalassemia carrier. Each trace was obtained by adding the 10 most intense multicharged ions.
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4.2. Based on the quantification of tryptic peptides
Fig. 3. Comparison between the results obtained by the LC-ESI/MS method on intact globin chains (y-axis) and a routine HPLC method (Bio-Rad Variant II, dual kit) (x-axis) for the measurement of HbA2 in fresh blood samples. Each point is the mean of three replicates.
was performed. In this way, satisfactory results were obtained in terms of peaks shape, method robustness and performance. A representative chromatogram obtained for a patient sample is shown in Fig. 2. The optimized LC-ESI/MS method was then applied to the analysis of samples with HbA2/total hemoglobin ratio values within the normal and pathological ranges. The results of one exercise performed in one of the MS laboratories of our WG are presented in Fig. 3, indicating a close correlation to results obtained with an HPLC routine analyzer, calibrated according to the manufacturers' instructions.
As a promising alternative to routine methods, mass spectrometry without chromatographic separation was proposed in the past for the determination of the HbA2 fraction in blood [11]. At that time, the relative amount of the δ- and β-globin chains was used to estimate the HbA2/total hemoglobin fraction. For this purpose, δ to β peptide ratios were determined after tryptic digestion of the sample. In the approach we have developed within our working group, we focussed however our attention to the relative amount of the δ- and α-globin chains to estimate the HbA2/total hemoglobin fraction, because α-globin chains are indeed more representative of total hemoglobin, respect to β chains [12]. Isotope dilution mass spectrometry (ID-MS) has the potential to deliver highly reliable results if isotopic equilibrium can be achieved and if implemented properly. Such a measurement procedure combined with HPLC separation was developed within the activities of our WG, by employing recombinant human hemoglobins and native and 15N-labeled. Calibration solutions of the native forms of HbA2 and HbA0 were value assigned by amino acid analysis using an ID-MS based measurement procedure [13]. For calibration pure substance amino acid reference materials certified for their purity have been used. After tryptic digestion, signature peptides of δ- and α-globins are measured, thus providing values for HbA2 and total hemoglobin, respectively. The work flow of the measurement procedure is illustrated in Fig. 4, while the complete procedure will be described elsewhere [manuscript in preparation]. Briefly, a known amount of recombinant 15N-labeled hemoglobins HbA0 and HbA2 is added to the sample. Next, a calibration blend is prepared containing a known amount of recombinant hemoglobins HbA0 and HbA2 and the same amount of 15N-labeled hemoglobins HbA0 and HbA2, as those added to the sample. Both,
Fig. 4. Workflow for the ID-MS method.
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sample and calibration blends are subjected to tryptic digest and HPLCMS/MS-analysis. The peptides TYFPHFDLSHGSAQVK (α-T6) derived from the α-globin chain and TAVNALWGK (δ-T2) derived from the δglobin chain have been selected for the analysis. Additional peptides derived from both globin chains can be analyzed to increase confidence in the results. The HbA2 fraction is calculated as the δ-T2/α-T6 amount of substance ratio. Preliminary experiments performed on a few lyophilized samples used in the German EQAS scheme, showed promising results [14] and further work is in progress to propose this approach as a reference measurement procedure (RMP). 5. Preparation of a certified reference material In addition to the RMP, the certified reference materials (CRMs) represent an integral element of a complete reference measurement system. CRMs are essential tools to establish metrological traceability, being used as calibrators to establish and assure an unbroken traceability chain for values assigned to in vitro diagnostic calibrators and obtained for clinical samples traceable to RMPs and/or reference materials of higher metrological order, and in the best case to the International System of Units (SI) [15,16]. European legislation [17] requires the application of RMPs and use of reference materials of higher metrological order and has as final aim equivalence of measurement results obtained with different diagnostic methods. There are some requirements a CRM has to fulfill to be successfully used and fit-for-purpose. It should carry a property value in the relevant range. It should also have adequate homogeneity, long-term stability and commutability with clinical samples in order to minimize calibration bias. The certified value should preferably be assigned by methods based on different measurement principles but providing equivalent results or, if the property value is procedurally defined, by application of an internationally recognized RMP. To date, the only recognized material for HbA2 is the “WHO International Reference Reagent”, held at the UK National Institute for Biological Standards & Control (89/666, NIBSC, UK) [18]. This material has an assigned HbA2 value of 5.3 ± 0.07%, obtained by an international collaborative study using electrophoresis with elution of HbA2 band, microcolumn chromatography and HPLC. The preparation of the WHO material dates back more than 25 years and its commutability characteristics with current methods used for HbA2 determination is under review. Moreover, in the near future, it is expected to become exhausted, and consequently a suitable replacement material has to be planned for. For these reasons, a part of the working group activities was directed towards the development of a CRM in collaboration with the Institute of Reference Materials and Measurements (IRMM). The candidate reference material consists of a stabilized hemolysate in the lyophilized form. It is prepared from human red blood cells, following the protocol previously reported [19]. A first pilot batch at normal HbA2 fraction was prepared and extensively tested. After reconstitution, the material showed a total hemoglobin concentration and methemoglobin (MetHb) content similar to that of fresh blood (129 g/L and 1%, respectively). The material was analyzed with the most widely used methods for HbA2 determination, including HPLC, capillary and gel electrophoresis. The separation profiles compared with those obtained with fresh blood samples did not show any abnormality which may have been due to preparation or lyophilization process. The stability was evaluated for both, the reconstituted and lyophilized material, at different storage temperatures. The reconstituted materials can be stored for at least 2 weeks at + 4 °C with no change in HbA2 fraction. With regards to the lyophilized material, the stability study performed over a four years period (study is still in progress) showed that the material is sufficiently stable at −20 °C with respect to the HbA2 concentration, and that no oxidation or degradation products were detectable under these storage conditions (Fig. 5). At + 4 °C, the HbA2 fraction was
Fig. 5. Stability of the candidate reference lyophilized material stored at −20 °C. Each point is the mean of three replicate measurements. The error bars represent the SD. The dashed lines define the acceptability range equivalent to the allowable TE calculated from the measurements performed at the basal level, before the storage.
equally stable over a four years period, however a slight increase in the formation of MetHb (nearly 6%) was detected, thus indicating that for long term storage these materials have to be kept at − 20 °C, or below. Preliminary data proved that this material was commutable for an HPLC and a capillary electrophoresis method used for the determination of HbA2 in human blood [19].
6. Possible impact of standardization on the diagnosis of thalassemic syndromes More and more often, clinicians and laboratory scientists are called to discuss this topic. A useful approach in our opinion, is to look at the case of glycated hemoglobin. As it is well known, in 2007 the American Diabetes Association introduced the use of HbA1c for the diagnosis of diabetes, with a decision limit of 6.5% (48 mmol/mol) [20]. Besides discussions about possible pitfalls in such an approach, there were also some discussions on how the lack of standardization could have potentially impacted patient outcomes. An interesting point-of-view was presented by Selvin et al. [21] stating that the prevalence of HbA1c 6.5% in individuals without a history of diabetes was of 1.6% (1.4–1.8), corresponding to 3.0 million (2.6–3.3) adults in the U.S. population. It was also then speculated that a shift in the cut point of only − 0.1% units could result in an increase in diagnosis of diabetes in 1.1 million more people, thus demonstrating how important the trueness of the methods could be. In the case of HbA2, we have previously shown that the occurrence of subjects with borderline HbA2 fraction values in the range 3.3–3.7% is approximately 2%, corresponding to approximately 7 million people [22]. With the elimination of the bias between routine methods due to the achievement of a perfect global standardization of HbA2 methods, we could attain improved diagnostic sensitivity and specificity for the diagnosis of β-thalassemia trait, consequently in the ideal case with considerable savings to the health care systems.
7. Conclusions One should keep in mind that the optimization of the analytical phase does not resolve the pre-analytical and the post-analytical errors. In the case of HbA2, the test adds important information to the diagnosis of the thalassemic syndromes, together with other exams and clinical findings, including the family history. Within such a scenario, the measurement of HbA2 needs to be performed with the best possible attention, as already discussed in the first part of this document.
Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023
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Looking now to the activities of our WG, it is evident that still some points have to be addressed before a complete reference system can be put in place, particularly: a) The candidate reference measurement procedure will have to be validated and implemented in at least three independent laboratories. b) The certified reference materials will have to be produced at minimum two levels of HbA2 fraction and in large enough batches to serve for at least 5–10 years. c) The manufacturers will have to use the CRM to calibrate their methods. d) Robust EQAS exercises, using possibly commutable control materials with target values assigned by the PRMP, will have to be implemented to monitor the analytical quality among the laboratorians. Moreover, corrective actions will have to be taken to ensure the measurement of HbA2 fraction with an acceptable total error. We suggest that a TE of 7%, based on clinical needs [4], should be taken as a realistic goal, at least at the beginning. We strongly hope that all these issues could be settled in the next 2 years, and we hope that all the stakeholders (governments and international organizations, scientific societies, manufacturers and specialists in laboratory medicine) will act in a coordinated way to take corrective action every time that EQAS will clearly indicate suboptimal situations. Finally, our report may tell also that standardization of measurements is not an easy topic, and that time and coordinated efforts may be needed for a long time before coming to the end. Scientists should be aware of this aspect before starting any new standardization project. Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Author contributions AM, BW and EB worked on the clinical needs; AM, RP collected and elaborated the EQAS data; DC, CSR, AVD, MO and VRDJ worked on the development of the LC-ESI/MS method based on the measurement of intact globin chains; PK and CA worked on the development of the IDMS method based on the measurement of target peptides; RP worked on the processing of samples and calibrators for the LC-ESI/MS method, and for all the analyses by the routine methods; RP and AM worked on the preparation and evaluation of the reference material; all authors contributed to the intellectual content of this manuscript; AM and RP coordinated the activities of the whole work. Acknowledgments We thank Dr. Amalia Munoz, Dr. Ingrid Zegers and Dr. Heinz Schimmel (Institute of Reference Materials and Measurements, Geel, Belgium) for having taken active part to this project since the beginning. We acknowledge Dr. Massimo Quercioli (Azienda Ospedaliera Careggi, Firenze, Italy) for having provided the data on the 2014 EQAS in Italy and Dr. Ferruccio Ceriotti and Dr. Gabriella Passerini (San Raffaele
Hospital, Milano, Italy) for having provided fresh blood samples useful for the various methods we have developed and evaluated with our activities. Finally we wish to express our gratitude to the Scientific Division of the International Federation of Clinical Chemistry for having supported this project for more than ten years.
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Developing a reference system for the IFCC standardization of HbA2 Renata Paleari a, Donatella Caruso b, Patricia Kaiser c, Cristian Gabriel Arsene d, Christine Schaeffer-Reiss e,f, Alain Van Dorsselaer e,f, Emmanuel Bissé g, Maria Ospina h, Víctor R. De Jesús h, Barbara Wild i, Andrea Mosca a,⁎, on behalf of the IFCC Working Group on Standardisation of Hemoglobin A2 (WG-HbA2): a
Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Milano, Italy Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milano, Italy Instand e.V., Düsseldorf, Germany d Physikalisch-Technische Bundesanstalt (PTB), Braunschweig, Germany e BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France f IPHC, CNRS, UMR7178, Strasbourg, France g Department of Clinical Chemistry, University Medical Center, Freiburg, Germany h Division of Laboratory Sciences, National Center for Environmental Health, US Centers for Disease Control and Prevention, Atlanta, GA, USA i UKNEQAS(H), Watford, UK b c
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Article history: Received 18 December 2015 Received in revised form 25 May 2016 Accepted 26 May 2016 Available online xxxx Keywords: Analytical goals Beta-thalassemia Quality control Reference materials Reference measurement procedure Standardization
a b s t r a c t The importance of hemoglobin A2 (HbA2) as an indicator of the presence of β-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing. © 2016 Elsevier B.V. All rights reserved.
1. Introduction
2. Clinical needs for the accurate determination of HbA2
In December 2004, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working group for the standardization of HbA2 (IFCC WG-HbA2) was established. At that time, the IFCC working group for the standardization of HbA1c, (IFCC WG-HbA1c) was almost at the end of its tasks. It seemed appropriate to commence work on another minor hemoglobin, where standardization was urgently needed but no apparent work was being yet undertaken. The terms of reference for the new WG were directed to promote the standardization of hemoglobin A2 measurement through the definition of an international reference system, including a reference measurement procedure primary and secondary certified reference materials. This brief report summarizes the achievements so far and outlines the work remaining.
The inherited disorders of hemoglobin are among the most common monogenic diseases in the world. Recent surveys indicate that after sickle-cell disease, β-thalassemia has the highest incidence, with more than 22,000 annual births and with a mean global prevalence of 4.9% [1]. In order to prevent the occurrence of severe thalassemia-major phenotypes, accurate prenatal screening has to be performed. The measurement of HbA2 has an important role in achieving the correct diagnosis, together with other laboratory investigations and clinical information. Indeed, an increase in this hemoglobin fraction is one of the most widely used indications of β-thalassemia heterozygotes. However, some technical difficulties can arise in the identification of β-thalassemia carriers with only slightly increased HbA2 values. The occurrence of such borderline subjects is not rare, since there is a great heterogeneity of mutations and other conditions such as iron deficiency or the presence of δ-thalassemia may complicate the typical phenotype [2,3]. Therefore, because of the small differences between normal and pathological HbA2 values, strict quality assurance of HbA2 measurement is crucial for accurate diagnosis, particularly concerning genetic counselling of couples at-risk.
⁎ Corresponding author at: Università degli Studi di Milano, Dip. di Fisiopatologia Medico-Chirurgica e dei Trapianti, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy. E-mail address: [email protected] (A. Mosca).
http://dx.doi.org/10.1016/j.cca.2016.05.023 0009-8981/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023
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How well HbA2 needs to be measured is a topic which was discussed some time ago by this working group in terms of the analytical performance goals [4], and also by recommendations issued by the International Committee for the Standardization in Haematology [5]. Presently, there is no consensus on these goals, however a precision of ±0.1% amount of substance fraction HbA2/total Hb in the final report for the analysis, equivalent to a CV of 2%, should be obtainable using modern automated high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) equipment. In terms of trueness, ideally a zero bias should be pursued but in the absence of a reference measurement procedure for comparison, it is difficult to assess compliance with this goal. Concerning the relative total error, an acceptable limit of 7.0% has been recently proposed [6], to correctly classify a subject with a true HbA2 value of 3.6% besides other variables, such as mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). The total error (TE) goal based on biological variability is however even more stringent (4.5%) [4]. 3. State-of-the-art of current automated methods for HbA2 The state-of-the-art of the most commonly used methods for measuring HbA2 can be assessed by means of dedicated inter-comparisons, external quality assessment schemes (EQAS) or validation studies, as well as by using information directly provided by the manufacturers. There is limited data on the comparability of the different methods used for HbA2 quantitation, especially on the commonly used HPLC and CE techniques. Recent work prepared by Sangkitporn et al. [7] evaluated the analytical performance characteristics, including precision and accuracy, of the automated capillary electrophoresis method (Capillarys 2, Sebia, France) with existing validated HPLC and LPLC methods. The coefficients of variation of HbA2 quantitation were 1.80– 2.86%, 1.26–5.13% and 1.08–6.66% for within run, between run and inter-laboratory comparison, respectively. The results of HbA2 quantified by the CE method correlate well with those of the HPLC and LPLC methods (r = 0.98–0.99). Similar findings were obtained in a more recent study performed with different HPLC (Bio-Rad Variant I, Bio-Rad Variant II, Menarini HA-8160, Tosoh G7, Tosoh G8) and CE (Beckman Coulter MDQ, ProteomeLab PA800, Sebia Capillarys 2) methods. These
studies proved that the results of the different methods were highly correlated (r between 0.974 and 0.997), despite the bias between methods, with a within-run imprecision of HbA2 measurement (expressed as CV) between 0.5% and 4.4% (HPLC) and between 1.2% and 4.4% (CE) [8]. As it can be seen, the recommended CV goal for precision of 2.0% is not achieved by many routine methods. Some years ago an EQAS pilot exercise in Italy, using fresh blood samples and involving 48 Italian laboratories routinely measuring HbA2 [9], was performed to further assess the state-of-the-art. The overall inter-laboratory CVs were 8.0%, 6.0% and 7.9% for samples with low, high and intermediate HbA2 levels, respectively. Moreover, it was found that the fraction of laboratories reporting unacceptable results, assuming an allowable total error of 7.8%, ranged from 17.0% to 31.9%. In addition, some recent data were collected either in Italy from a mandatory EQAS exercise provided by the Region of Tuscany or from an EQAS exercise provided by a manufacturer (Bio-Rad Laboratories) covering many laboratories around the world, not only using methods from the same company. The data covered the 2014 year for the Italian EQAS (the data from the 2015 exercise is not available yet), and the 2015 exercise from Bio-Rad (in the 2014 exercise no samples with high HbA2 level were distributed), and are presented in Fig. 1. Results from three different control materials, with similar HbA2 concentration were compared and the consensus means established by the EQAS providers were chosen as the target HbA2 values. It is evident from these plots that the inter-laboratory dispersions (represented by the error bars) are often within the method performance goal for the total error, but there is certainly a significant bias between the methods. For instance, the mean value obtained by the users of the Tosoh G7 is clearly above the consensus mean in the 2014 Italian exercise, as well as for the results of users of the Bio-Rad Variant II dual kit in the 2015 EQAS. The comparison between the two exercises have to be done with some caution, because the materials analyzed were not the same, both were in the lyophilized form, but produced by different manufacturers. Nevertheless, the data show clearly that the methods are not completely aligned. From the data reported above, it can be concluded that the state-ofthe-art is not optimal, and that more standardization work is needed to achieve the desired method performance targets.
Fig. 1. Inter-laboratories results obtained from two different EQAS providers, at three different levels of HbA2/total hemoglobin amount of substance fraction, grouped according to the methods used by the participants. The left panel reports the data obtained in 2014 from the Italian Inter-Regional Program. The right panel refers to the data obtained from the international EQAS provided by Bio-Rad Laboratories. The numbers of participants per method are reported in brackets, from the lower to the higher HbA2 level. Points and error bars represent means and SD, respectively. The dotted lines represent the limits of the allowable total error of ±7%.
Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023
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4. Definition of a reference measurement procedure A major task of the working group was the development of a reference measurement procedure (RMP). Much effort was put into this aspect whereby different approaches based on mass spectrometry, which potentially could deliver more accurate results for HbA2, were applied, and briefly summarized below. Two different measurement principles were then followed. In the first approach, intact δ- and α-globin chains are separated and detected using LC-ESI/MS. The HbA2-fraction is determined by calibrating the mass spectrometer for the direct measurement of δ/α globin chain ratios. As an alternative approach, the amount of substances of HbA2 and total hemoglobin in blood are determined separately from each other in a first step. For this purpose, isotope dilution mass spectrometry (ID-MS) is used. The HbA2-fraction is then calculated in a second step as a ratio from the results for HbA2 and for total hemoglobin. Unlike the measurement of intact globin chains, tryptic peptides derived from and representing HbA2 and total hemoglobin, respectively, are being quantified. The HbA2 fraction is calculated as the ratio between the amount of substance determined for the δ and α chain derived peptides. These two MS procedures, based on different analytical measurement principles, were developed in parallel within the working group. Both procedures were established and optimized in different cooperating expert laboratories. Particularly, in respect to the main objective of the working group to certify reference materials for recalibration of the routine systems, the establishment of a reference laboratory
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network operating two different and independent measurement procedures has been considered to be of major advantage. Finally, with regards to the quantity measured and the units, the working group agrees that the measurand is the molecule of native HbA2, expressed as HbA2/total hemoglobin amount of substance, and reported in %. 4.1. Based on the quantification of intact globin chains A method based on the quantification of intact globin chains by LCESI/MS, which eliminates the need for a digestion step prior to MS analysis, was developed. The two major advantages of the measurement of intact globin chains, compared to the often used proteomic approach based on the measurement of targeted proteolytic peptides, are to avoid the potential measurement bias introduced by the trypsin digestion step and to reduce the efforts going into the sample preparation. For the calibration of the method, purified HbA2 and HbA0 solutions were mixed in well-defined ratios to form a set of calibrators with known HbA2 concentrations spanning across the expected range of measurement. The purified HbA2 and HbA0 were prepared by a twostep preparative chromatographic procedure consisting of consecutive ion exchange cellulose separations. The sample preparation consisted of red blood cell lysis followed by dilution with acetonitrile and desalting [10]. The measurements were performed using LC-MS equipped with an ESI source and a linear ion trap analyzer, in full scan mode. Due to the low content of the δ globin chains relative to the others, an ion chromatogram extraction of 10 masses corresponding to the charge states with the highest intensities for δ and α chains,
Fig. 2. Ion chromatograms obtained from the separation of a hemolysate prepared from the blood of a β-thalassemia carrier. Each trace was obtained by adding the 10 most intense multicharged ions.
Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023
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4.2. Based on the quantification of tryptic peptides
Fig. 3. Comparison between the results obtained by the LC-ESI/MS method on intact globin chains (y-axis) and a routine HPLC method (Bio-Rad Variant II, dual kit) (x-axis) for the measurement of HbA2 in fresh blood samples. Each point is the mean of three replicates.
was performed. In this way, satisfactory results were obtained in terms of peaks shape, method robustness and performance. A representative chromatogram obtained for a patient sample is shown in Fig. 2. The optimized LC-ESI/MS method was then applied to the analysis of samples with HbA2/total hemoglobin ratio values within the normal and pathological ranges. The results of one exercise performed in one of the MS laboratories of our WG are presented in Fig. 3, indicating a close correlation to results obtained with an HPLC routine analyzer, calibrated according to the manufacturers' instructions.
As a promising alternative to routine methods, mass spectrometry without chromatographic separation was proposed in the past for the determination of the HbA2 fraction in blood [11]. At that time, the relative amount of the δ- and β-globin chains was used to estimate the HbA2/total hemoglobin fraction. For this purpose, δ to β peptide ratios were determined after tryptic digestion of the sample. In the approach we have developed within our working group, we focussed however our attention to the relative amount of the δ- and α-globin chains to estimate the HbA2/total hemoglobin fraction, because α-globin chains are indeed more representative of total hemoglobin, respect to β chains [12]. Isotope dilution mass spectrometry (ID-MS) has the potential to deliver highly reliable results if isotopic equilibrium can be achieved and if implemented properly. Such a measurement procedure combined with HPLC separation was developed within the activities of our WG, by employing recombinant human hemoglobins and native and 15N-labeled. Calibration solutions of the native forms of HbA2 and HbA0 were value assigned by amino acid analysis using an ID-MS based measurement procedure [13]. For calibration pure substance amino acid reference materials certified for their purity have been used. After tryptic digestion, signature peptides of δ- and α-globins are measured, thus providing values for HbA2 and total hemoglobin, respectively. The work flow of the measurement procedure is illustrated in Fig. 4, while the complete procedure will be described elsewhere [manuscript in preparation]. Briefly, a known amount of recombinant 15N-labeled hemoglobins HbA0 and HbA2 is added to the sample. Next, a calibration blend is prepared containing a known amount of recombinant hemoglobins HbA0 and HbA2 and the same amount of 15N-labeled hemoglobins HbA0 and HbA2, as those added to the sample. Both,
Fig. 4. Workflow for the ID-MS method.
Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023
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sample and calibration blends are subjected to tryptic digest and HPLCMS/MS-analysis. The peptides TYFPHFDLSHGSAQVK (α-T6) derived from the α-globin chain and TAVNALWGK (δ-T2) derived from the δglobin chain have been selected for the analysis. Additional peptides derived from both globin chains can be analyzed to increase confidence in the results. The HbA2 fraction is calculated as the δ-T2/α-T6 amount of substance ratio. Preliminary experiments performed on a few lyophilized samples used in the German EQAS scheme, showed promising results [14] and further work is in progress to propose this approach as a reference measurement procedure (RMP). 5. Preparation of a certified reference material In addition to the RMP, the certified reference materials (CRMs) represent an integral element of a complete reference measurement system. CRMs are essential tools to establish metrological traceability, being used as calibrators to establish and assure an unbroken traceability chain for values assigned to in vitro diagnostic calibrators and obtained for clinical samples traceable to RMPs and/or reference materials of higher metrological order, and in the best case to the International System of Units (SI) [15,16]. European legislation [17] requires the application of RMPs and use of reference materials of higher metrological order and has as final aim equivalence of measurement results obtained with different diagnostic methods. There are some requirements a CRM has to fulfill to be successfully used and fit-for-purpose. It should carry a property value in the relevant range. It should also have adequate homogeneity, long-term stability and commutability with clinical samples in order to minimize calibration bias. The certified value should preferably be assigned by methods based on different measurement principles but providing equivalent results or, if the property value is procedurally defined, by application of an internationally recognized RMP. To date, the only recognized material for HbA2 is the “WHO International Reference Reagent”, held at the UK National Institute for Biological Standards & Control (89/666, NIBSC, UK) [18]. This material has an assigned HbA2 value of 5.3 ± 0.07%, obtained by an international collaborative study using electrophoresis with elution of HbA2 band, microcolumn chromatography and HPLC. The preparation of the WHO material dates back more than 25 years and its commutability characteristics with current methods used for HbA2 determination is under review. Moreover, in the near future, it is expected to become exhausted, and consequently a suitable replacement material has to be planned for. For these reasons, a part of the working group activities was directed towards the development of a CRM in collaboration with the Institute of Reference Materials and Measurements (IRMM). The candidate reference material consists of a stabilized hemolysate in the lyophilized form. It is prepared from human red blood cells, following the protocol previously reported [19]. A first pilot batch at normal HbA2 fraction was prepared and extensively tested. After reconstitution, the material showed a total hemoglobin concentration and methemoglobin (MetHb) content similar to that of fresh blood (129 g/L and 1%, respectively). The material was analyzed with the most widely used methods for HbA2 determination, including HPLC, capillary and gel electrophoresis. The separation profiles compared with those obtained with fresh blood samples did not show any abnormality which may have been due to preparation or lyophilization process. The stability was evaluated for both, the reconstituted and lyophilized material, at different storage temperatures. The reconstituted materials can be stored for at least 2 weeks at + 4 °C with no change in HbA2 fraction. With regards to the lyophilized material, the stability study performed over a four years period (study is still in progress) showed that the material is sufficiently stable at −20 °C with respect to the HbA2 concentration, and that no oxidation or degradation products were detectable under these storage conditions (Fig. 5). At + 4 °C, the HbA2 fraction was
Fig. 5. Stability of the candidate reference lyophilized material stored at −20 °C. Each point is the mean of three replicate measurements. The error bars represent the SD. The dashed lines define the acceptability range equivalent to the allowable TE calculated from the measurements performed at the basal level, before the storage.
equally stable over a four years period, however a slight increase in the formation of MetHb (nearly 6%) was detected, thus indicating that for long term storage these materials have to be kept at − 20 °C, or below. Preliminary data proved that this material was commutable for an HPLC and a capillary electrophoresis method used for the determination of HbA2 in human blood [19].
6. Possible impact of standardization on the diagnosis of thalassemic syndromes More and more often, clinicians and laboratory scientists are called to discuss this topic. A useful approach in our opinion, is to look at the case of glycated hemoglobin. As it is well known, in 2007 the American Diabetes Association introduced the use of HbA1c for the diagnosis of diabetes, with a decision limit of 6.5% (48 mmol/mol) [20]. Besides discussions about possible pitfalls in such an approach, there were also some discussions on how the lack of standardization could have potentially impacted patient outcomes. An interesting point-of-view was presented by Selvin et al. [21] stating that the prevalence of HbA1c 6.5% in individuals without a history of diabetes was of 1.6% (1.4–1.8), corresponding to 3.0 million (2.6–3.3) adults in the U.S. population. It was also then speculated that a shift in the cut point of only − 0.1% units could result in an increase in diagnosis of diabetes in 1.1 million more people, thus demonstrating how important the trueness of the methods could be. In the case of HbA2, we have previously shown that the occurrence of subjects with borderline HbA2 fraction values in the range 3.3–3.7% is approximately 2%, corresponding to approximately 7 million people [22]. With the elimination of the bias between routine methods due to the achievement of a perfect global standardization of HbA2 methods, we could attain improved diagnostic sensitivity and specificity for the diagnosis of β-thalassemia trait, consequently in the ideal case with considerable savings to the health care systems.
7. Conclusions One should keep in mind that the optimization of the analytical phase does not resolve the pre-analytical and the post-analytical errors. In the case of HbA2, the test adds important information to the diagnosis of the thalassemic syndromes, together with other exams and clinical findings, including the family history. Within such a scenario, the measurement of HbA2 needs to be performed with the best possible attention, as already discussed in the first part of this document.
Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023
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Looking now to the activities of our WG, it is evident that still some points have to be addressed before a complete reference system can be put in place, particularly: a) The candidate reference measurement procedure will have to be validated and implemented in at least three independent laboratories. b) The certified reference materials will have to be produced at minimum two levels of HbA2 fraction and in large enough batches to serve for at least 5–10 years. c) The manufacturers will have to use the CRM to calibrate their methods. d) Robust EQAS exercises, using possibly commutable control materials with target values assigned by the PRMP, will have to be implemented to monitor the analytical quality among the laboratorians. Moreover, corrective actions will have to be taken to ensure the measurement of HbA2 fraction with an acceptable total error. We suggest that a TE of 7%, based on clinical needs [4], should be taken as a realistic goal, at least at the beginning. We strongly hope that all these issues could be settled in the next 2 years, and we hope that all the stakeholders (governments and international organizations, scientific societies, manufacturers and specialists in laboratory medicine) will act in a coordinated way to take corrective action every time that EQAS will clearly indicate suboptimal situations. Finally, our report may tell also that standardization of measurements is not an easy topic, and that time and coordinated efforts may be needed for a long time before coming to the end. Scientists should be aware of this aspect before starting any new standardization project. Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Author contributions AM, BW and EB worked on the clinical needs; AM, RP collected and elaborated the EQAS data; DC, CSR, AVD, MO and VRDJ worked on the development of the LC-ESI/MS method based on the measurement of intact globin chains; PK and CA worked on the development of the IDMS method based on the measurement of target peptides; RP worked on the processing of samples and calibrators for the LC-ESI/MS method, and for all the analyses by the routine methods; RP and AM worked on the preparation and evaluation of the reference material; all authors contributed to the intellectual content of this manuscript; AM and RP coordinated the activities of the whole work. Acknowledgments We thank Dr. Amalia Munoz, Dr. Ingrid Zegers and Dr. Heinz Schimmel (Institute of Reference Materials and Measurements, Geel, Belgium) for having taken active part to this project since the beginning. We acknowledge Dr. Massimo Quercioli (Azienda Ospedaliera Careggi, Firenze, Italy) for having provided the data on the 2014 EQAS in Italy and Dr. Ferruccio Ceriotti and Dr. Gabriella Passerini (San Raffaele
Hospital, Milano, Italy) for having provided fresh blood samples useful for the various methods we have developed and evaluated with our activities. Finally we wish to express our gratitude to the Scientific Division of the International Federation of Clinical Chemistry for having supported this project for more than ten years.
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Please cite this article as: R. Paleari, et al., Developing a reference system for the IFCC standardization of HbA2, Clin Chim Acta (2016), http:// dx.doi.org/10.1016/j.cca.2016.05.023