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Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2
CLB-08998; No. of pages: 5; 4C: Clinical Biochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2 Karina Rodriguez-Capote ⁎, Trefor N. Higgins DynaLIFEDX Diagnostic Laboratory Services, Edmonton, Alberta, Canada Dept. Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
a r t i c l e
i n f o
Article history: Received 6 February 2015 Received in revised form 29 March 2015 Accepted 1 April 2015 Available online xxxx Keywords: Hemoglobinopathy screening β-Thalassemia α-Thalassemia HbF reference interval Hemoglobin A2 HbA2 reference interval Hemoglobin analysis
a b s t r a c t Objectives: The aims of this study were to identify the incidence of hemoglobinopathies and thalassemias in Northern Alberta and calculate the reference intervals (RI) for hemoglobin (Hb) HbF and HbA2. Methods: A retrospective ad-hoc analysis of the structural Hb variants and thalassemias identified on patients who had a hemoglobinopathy/thalassemia investigation performed between February 1 to December 31, 2013. Results were extracted from the Laboratory Information System. Statistical analysis was performed using MedCalc® version 11.4.2.0 for Windows software. Results: 6616 hemoglobinopathy/thalassemia investigations and HbS screens were physician requested and 602 Hb variants were fortuitously found during HbA1c analysis. 3438 were interpreted as “normal” and 532 were classified as iron deficient. 3306 individuals, with age ranging from 3 to 92 years were included in the RI calculation. HbA2 RI was 2.3% to 3.4% and HbF 0.0% to 1.8%. 524 and 423 α and β thalassemia traits respectively were identified. Additionally ten δβ thalassemia traits and twelve cases of HbH disease were identified. Regarding hemoglobinopathies, 7% were classified as α-chain variants and 93% as β-chain variants with HbS (46%), HbE (16%), HbD Punjab (8%) and HbC (7%) traits being the most prevalent. We also documented 20 homozygous hemoglobinopathies and 36 compound/double heterozygous hemoglobinopathies. Conclusion: A wide diversity of hemoglobinopathies is found in the Northern Alberta population, 80% of the hemoglobinopathies were found as a reflex to HbA1c testing. Reference intervals for HbF and HbA2 were established. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction DynaLIFEDX is the sole laboratory performing hemoglobinopathy and thalassemia investigations for a catchment area of 2 million people in Northern Alberta, Canada. Hemoglobinopathy/thalassemia investigations came from physicians based on clinical presentation, immigration medicals, red cell exchange programs for sickle cell anemia patients and as a reflex test generated by the presence of a hemoglobin (Hb) variant noted during HbA1c analysis. Diagnoses of these hemoglobin disorders are important for planning appropriate management and genetic counseling [1]. Additionally in Northern Alberta, screening for thalassemia and hemoglobinopathies is offered by the maternal care clinics if the patient and/or her partner are identified as belonging to an ethnic population whose members are at
Abbreviations: CE-HPLC, cation-exchange high performance liquid chromatography; CI, confidence intervals; Hb, hemoglobin; HPFH, Hereditary Persistence of HbF; RI, reference interval. ⁎ Corresponding author at: DynaLIFEDx, #200, 10150-102 St, Edmonton, Alberta T5J 5E2, Canada. Fax: +1 780 454 2845. E-mail address: [email protected] (K. Rodriguez-Capote).
higher risk of being carriers as recommended by the clinical practice guideline from the Genetics Committee of the Society of Obstetricians and Gynaecologists of Canada and the Prenatal Diagnosis Committee of the Canadian College of Medical Geneticists [2]. Screening during pregnancy accounts for a high percentage of the requests for hemoglobinopathy and thalassemia investigations, since in Alberta neonatal hemoglobinopathy screening is not included in the Newborn Screening Program [3]. Fetal hemoglobin (HbF, α2γ2) is elevated in newborns, usually reaching adult levels by 12 months and it commonly increases up to 5% in normal pregnancy [4]. HbF measurement is useful in the diagnosis of β-globin gene disorders and in the prognosis of patients homozygous for HbS [5]. Some acquired conditions such as aplastic anemia and myeloproliferative disorders are also associated with mild increases of HbF. Furthermore, pharmacological stimulation of HbF is used to manage conditions such as sickle cell disease and β-thalassemia major [5,6]. Thus, a reliable monitoring of HbF concentration during the treatment and follow-up of these patients is required. HbA2 (α2δ2) is recommended as the primary test for the diagnosis of β-thalassemia trait in iron replete patients in the presence of microcytosis [1,7]. The slight difference between HbA2 values in patients with and without β- thalassemia trait requires excellent precision at HbA2 values
http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001
2
K. Rodriguez-Capote, T.N. Higgins / Clinical Biochemistry xxx (2015) xxx–xxx
at the upper limit of the reference interval (RI). Thus quantification of HbA2 has to be accurate. A review of our data showed that 17% of HbF results reported in patients with a normal iron status and absence of a Hb variant exceeded the manufacturer's reference interval of b 1.0%, therefore it was necessary to reassess the RI for HbF. The aims of this study were to identify the incidence of hemoglobinopathies and thalassemias in Northern Alberta and calculate the reference intervals for HbF and HbA2.
Table 1 95% reference intervals for HbA2 and HbF. N = 3312
HbF%
HbA2%
LLRI (90% CI) ULRI (90% CI) Coefficient of skewness Coefficient of kurtosis Shapiro–Wilk test
0% 1.8% (1.78–1.90) 1.97 (P b 0.0001) 6.27 (P b 0.0001) W = 0.83, reject normality (P b 0.0001)
2.3% (2.2–2.34) 3.4% (3.4–3.5) −0.48 (P b 0.0001) 1.84 (P b 0.0001) W = 0.97, reject normality (P b 0.0001)
CI, confidence interval; LL, lower limit; UL, upper limit; RI, reference interval.
Methods Discussion This study is an ad hoc analysis of the structural Hb variants and thalassemias identified in the Northern Alberta, Canada on patients who had an investigation between February 1 to December 31, 2013. Pseudo-anonymised data containing only patient age, HbF and HbA2 concentrations and interpretative results was extracted from the Laboratory Information System. This study complied with DynaLIFEDX Institutional Ethical Board requirements. In our laboratory, EDTA-anti-coagulated blood samples for hemoglobinopathy/thalassemia investigation and those with abnormal findings during HbA1c testing, are analyzed by a high resolution cation exchange high performance liquid chromatography (CE-HPLC) using the β thalassemia program on the Bio-Rad VARIANT II. The British Committee for Standards in Haematology recommends that a second methodology based on a different analytical principle be used to make a presumptive identification of the hemoglobin variant [8–11]. Following this principle any sample with an Hb variant fraction is further investigated by electrophoresis at alkaline and acid pH (Sebia Hydrasys Electrophoresis System). Detection of common α-thalassemia mutations and HbH disease are performed by GAP-PCR analysis (Calgary Laboratory Services). A complete blood count and ferritin tests are requested as part of the hemoglobinopathy/thalassemia investigation. The interpretative report is performed by the Clinical Biochemist, who integrates the patient results from the chromatogram, electropherograms, hematology indices and calculated Mentzer Index. Total imprecision (CV%) for HbF is 2.2% and 1.2% at levels of 2.17% and 9.54% respectively. For HbA2 the total imprecision is 3.5% and 2.1% at levels of 2.8% and 5.7% respectively. Data was imported to Excel (Microsoft Office 2010). Categorical variables were expressed as frequencies and percentages. 95% reference intervals with 90% confidence intervals (CI) were calculated using the non-parametric percentile method according to CLSI EP28-A3C [12]. Statistical analysis was performed using MedCalc® version 11.4.2.0 for Windows.
Of the 6616 hemoglobinopathy/thalassemia investigations performed over a period of 11 months, 52% were considered “normal” and 8% were considered as iron deficient. The RIs obtained in our population [HbA2 2.3–3.4% and HbF 0.0–1.8%] are similar to those reported by other large reference laboratories such as ARUP Laboratories [HbA2 2.0–3.5% and HbF 0.0–2.1%] [14] and Mayo Medical Laboratories [HbA2 2.0–3.3% and HbF 0.0–0.9%] [15]. Hemoglobinopathies and thalassemias are frequent in the Mediterranean coastal region, India, Africa and in Southeast Asia, and therefore are commonly found in immigrants from these areas and their descendants [1,16]. Our laboratory serves a diverse multicultural urban population with the 2011 census results indicating that 22% of the Edmontonians were born outside Canada, from these 26% were from South Eastern Asia, 6% from the Mediterranean basin and 9% from Africa; accounting for around 14% of the total population [17,18]. Some structural Hb variant or thalassemia was found on 40% of the patients. 92% of the hemoglobin variants were β-chain variant traits with HbS (47%), HbE (16%), HbD (9%) and HbC (7%) traits being the most prevalent. Complex hemoglobinopathies such as compound or double heterozygosity due to co-inheritance of defects in α- and βglobin genes were detected in 36 patients, with the double heterozygote for sickle cell trait and α-thalassemia minor being the most prevalent. For twenty-three α-chain variants and four β-chain variants, the HPLC pattern and electrophoresis at acid and alkaline pH were not distinctive enough to make a presumptive identification of the hemoglobin variant. Eighteen of these α-chain variants and two of the β-chain variants were fortuitously detected during HbA1c analysis and the
Results Of the 6616 thalassemia and hemoglobinopathy investigation requests 3312 were on patients older than 2 years and interpreted as “normal” by a Clinical Biochemist based on their hematology indices (hemoglobin N120 g/l, mean cell volume N 80 fL), absence of a hemoglobin variant, replete iron status and calculated Mentzer Index (MCV divided by RBC N 14). These results were included in the calculation of the RIs for HbF and HbA2. Both, HbA2 and HbF failed the Shapiro–Wilk test for normal distribution (Table 1), even after logarithmic or Box– Cox transformation (data not shown). Consequently, their respective RIs were calculated by the percentile method as recommended by the CLSI C28-A3 guidelines [12]. Box-plots summarizing the data are presented in Fig. 1. A summary of the hemoglobinopathy/thalassemia investigation main findings is presented in Fig. 2, the various hemoglobin variants diagnosed in this period are tabulated as follows: Table 2, α-chain variants; Table 3, β-chain variants and Table 4, Hb homozygous and double or compound heterozygous.
Fig. 1. Box-plot, the central box represents the values from the lower to upper quartile (25 to 75 percentile). The middle line represents the median. Outlier values are displayed as X. Statistical analysis was performed using MedCalc® version 11.4.2.0 for Windows.
Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001
K. Rodriguez-Capote, T.N. Higgins / Clinical Biochemistry xxx (2015) xxx–xxx
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Fig. 2. Hemoglobin variants and thalassemias identified in Northern Alberta.
remainders were investigations requested by the Mom Care Clinics. These Hb variants were found to have minimal clinical or hematological features, and only eight of them showed retention times that interfere with the measurement of HbA1c in our HPLC.
We found twenty new Hb homozygous diseases (thirteen HbSS, five HbEE, one HbCC, and one Hb D-Iran). Even with the high incidence of HbS and HbE in the population we did not see a compound/double Table 3 Beta chain Hb variant trait.
Table 2 Alpha chain Hb variant trait.
Hb J Hb I Possible Hb G-Philadelphia Possible Stanleyville II Possible Hb J-Tongariki Possible Hb J Oxford, J Paris Or J-Mexico Possible Hb J-Toronto Possible Hb O-Indonesia Possible Hb Hopkins II Possible Hb Q-India Possible Hb Q-Iran Possible Hb St. Luke's Possible Hb Toulon Possible Hb Wayne Hb M Bostona (13) Unknown α-chain Hb Variant a
N
% with respect to alpha chain variants
% with respect to total Hb variants
10 5 5 2 1 2
18% 9% 9% 4% 2% 4%
1.4% 0.7% 0.7% 0.3% 0.1% 0.3%
1 1 1 1 1 1 1 1 1 22
2% 2% 2% 2% 2% 2% 2% 2% 2% 39%
0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 3.0%
Confirmed by molecular analysis.
HbS HbE HbD HbC Possible Hb J-Baltimore Possible Hb G-Coushatta, D-Iran or Hb Osu Christianborg Possible Hb Athens-GA, Hb Tyne or Hb Hounslow Possible Hb Hope Possible Hb Presbyterian HbJ Possible Hb O-ARAB Possible Hb J-Bangkok Possible Hb I-High Wycombe Possible Hb E-Saskatoon Possible Hb O-Arab Possible Hb Camden or Beckman Hb variant β-chain trait, unknown variant
n
% with respect to β-chain variants
% with respect to total Hb variants
346 120 63 53 18 15
54.% 19.% 10.% 8.% 3.% 2.%
47.4% 16.4% 8.6% 7.3% 2.5% 2.1%
9
1.%
1.2%
5 2 2 1 1 1 1 1 1 4
1.% 0.3% 0.3% 0.2% 0.2% 0.2% 0.2% 0.2% 0.2% 1.%
0.7% 0.3% 0.3% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.5%
Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001
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K. Rodriguez-Capote, T.N. Higgins / Clinical Biochemistry xxx (2015) xxx–xxx
Table 4 Homozygous, compound and double heterozygotes.
HbSS HbEE HbCC Hb D-Iran/Hb D-Iran HbSC HbE/Hb Hope HbS/Hb G-Philadelphia HbS trait and α-thalassemia minor HbE trait and α-thalassemia minor HbE trait and β+ thalassemia HbC trait and β+ thalassemia HbS trait and β+ thalassemia HbS trait and α-thalassemia carrier
n
% with respect to total Hb variants
13 5 1 1 5 3 1 17 5 1 1 1 2
1.7% 0.7% 0.1% 0.1% 0.7% 0.4% 0.1% 2.3% 0.7% 0.1% 0.1% 0.1% 0.3%
a high prevalence of thalassemia the identification of affected individuals forms part of pre-marital counseling. In other areas individuals with thalassemia are often referred for genetic counseling [16,19]. Alberta is one of the fast growing provinces in Canada and according to the last census this growth is mainly due to the arrival of immigrants [17,18]. Knowing the incidence of hemoglobin disorders is important for planning appropriate management and to guarantee that affected individuals receive appropriate care after the diagnosis, such as medical, psychosocial care and genetic counseling. Conclusion A wide diversity of hemoglobin variants and thalassemias are found in Northern Alberta, 80% of the Hb variants were found as a reflex to HbA1c testing. Reference intervals for HbF and HbA2 were established for our population. Acknowledgments
heterozygous HbSE, which could be explained by the less interaction and marriage between the two main ethnic groups where these hemoglobinopathies are frequent as opposed to the five double heterozygotes HbSC and three double heterozygotes for Hb Hope/HbE. HbS or HbC are the most prevalent hemoglobinopathies in African descendants and both HbE and Hb Hope are associated with Southeast Asia ethnicity [1, 19,20]. Twenty-seven patients were diagnosed with compound hemoglobinopathy (combined hemoglobinopathy and thalassemia): seventeen HbS trait and α thalassemia minor, five HbE trait and α thalassemia minor, one compound heterozygous for HbS trait and β+ thalassemia and one compound hemoglobinopathy of HbC trait and β+ thalassemia. Our current protocol uses a short HPLC as the screening method, followed by high resolution CE-HPLC. The Hb variants are identified by comparing the chromatogram with those in the Bio-Rad library and scientific literature [21–23]. We always use both, alkaline and acid electrophoresis as confirmatory tests because the extensively documented characterization of hemoglobin variants by electrophoretic techniques in the literature [10], easy accessibility to an online compilation on human hemoglobin abnormalities through a syllabus of human hemoglobin variants via the World Wide Web [24,25] and our own archive of Hb variants retention times and electrophoretic mobilities. Although with these information we can only generate a presumptive diagnosis; with our laboratory experience together with other reports on the literature we are confident that the risk of sickling variants (HbSS, HbSC, HbSE, HbS/Hb D-Punjab, HbS/Hb G-Philadelphia, HbS/Hb Hope, HbS/ Hb Lepore, HbS/HbO-Arab, and Hb S/β-thalassemia) can be identified without the need to proceed toward the most expensive molecular identification [21,26,27]. In 2013 only one Hb variant was sent for molecular diagnostic, HbM Boston [13]. The investigation of hemoglobinopathies may result from clinical presentation of hypoxia, presence of brown blood due to congenital methemoglobinemia and altered oxygen affinity particularly when using finger blood gas analyzers [13]. In addition, hemoglobinopathy investigation may form part of a pre-surgery laboratory workup. Recently a number of new hemoglobinopathies have been found as a fortuitous finding on HbA1c measurements [28,29]. We found 80% of the Hb variants during HbA1c testing. We believe that all Hb variants should be reported because they might have an effect on clinical decisions. Other laboratories, however, report the presence of a variant only if it interfered with the test [28,29]. The quantification of HbA1c provides a challenge in individuals with a homozygous hemoglobinopathy and the validity of threshold points for diagnosis and monitoring diabetes mellitus may be compromised in individuals with a hemoglobinopathy. The identification of individuals with thalassemia is important to prevent the administration of iron to individuals who are microcytic due to thalassemia rather than iron deficiency. In areas where there is
We thank DynaLIFEDx medical technologists for all their help and technical support, especially Deb Holmes and Don Zhang for their technical expertise with the Laboratory Information System. We extend our gratitude to Dr. Estey for the helpful discussions during the preparation of this manuscript. References [1] Clarke GM, Higgins TN. Laboratory investigation of hemoglobinopathies and thalassemias: review and update. Clin Chem 2000;46(8 Pt 2):1284–90. [2] Langlois S, Ford JC, Chitayat D, Desilets VA, Farrell SA, Geraghty M, et al. Carrier screening for thalassemia and hemoglobinopathies in Canada. J Obstet Gynaecol Can 2008;30(10):950–71. [3] Wilson K, Kennedy SJ, Potter BK, Geraghty MT, Chakraborty P. Developing a national newborn screening strategy for Canada. Health Law Rev 2010;18(2):9. [4] Bain BJ. Haemoglobinopathy diagnosis. Malden, Mass: Blackwell Pub; 2006. [5] Strouse JJ, Heeney MM. Hydroxyurea for the treatment of sickle cell disease: efficacy, barriers, toxicity, and management in children. Pediatr Blood Cancer 2012;59(2): 365–71. [6] Ware RE. How I use hydroxyurea to treat young patients with sickle cell anemia. Blood 2010;115(26):5300–11. [7] Mosca A, Paleari R, Ivaldi G, Galanello R, Giordano PC. The role of haemoglobin A2 testing in the diagnosis of thalassaemias and related haemoglobinopathies. J Clin Pathol 2009;62(1):13–7. [8] Ryan K, Bain BJ, Worthington D, James J, Plews D, Mason A, et al. Significant haemoglobinopathies: guidelines for screening and diagnosis. Br J Haematol 2010; 149(1):35–49. [9] Bain B, Farrar L, Henthorn J, Old J, Rees D, Roper D, et al, editors. Sickle cell and thalassaemia handbook for laboratories. Third ed. London, England: NHS Sickle Cell and Thalassaemia Screening Programme; 2012. [10] Bain BJ, Wild BJ, Stephens AN, Phelan LA. Variant haemoglobins: a guide to identification. Chichester, West Sussex: Wiley-Blackwell; 2010[vi, 254 pp.]. [11] Gallivan MVE, Giordano PC. Analysis of hemoglobinopathies, hemoglobin variants and thalassemias. Laboratory Hematology Practice: Wiley-Blackwell; 2012 562–85. [12] CLSI. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline — CLSI document EP28-A3C. Third Edition. Third ed. 2008. [Pennsylvania: Wayne, P.A.]. [13] Estey MP, Clarke G, Sia W, Toor E, Higgins T. A mother and newborn with brown blood. Clin Chem 2015;61(3). [14] ARUPLaboratories. Hemoglobin (Hb) A2 and F by Column 2015. [March 2015]. Available from: http://ltd.aruplab.com/Tests/Pub/0050613. [15] MayoMedicalLaboratories. Hemoglobin A2 and F, blood: Mayo Foundation for Medical Education and Research. [cited 2015 March 2015]. Available from: http://www. mayomedicallaboratories.com/test-catalog/Clinical+and+Interpretive/83341. [16] Giordano PC. Strategies for basic laboratory diagnostics of the hemoglobinopathies in multi-ethnic societies: interpretation of results and pitfalls. Int J Lab Hematol 2013;35(5):465–79. [17] Canada, Statistics. Immigration and ethnocultural diversity in CanadaIn: NH Survey, editor. ; 2011 [Ottawa: Canadap, Catalogue No. 99-010-X]. [18] Census, Edmonton. Municipal census resultsIn: Edmonton TCo, editor. 2014. [19] Giordano PC. Prospective and retrospective primary prevention of hemoglobinopathies in multiethnic societies. Clin Biochem 2009;42(18):1757–66. [20] Henderson S, Timbs A, McCarthy J, Gallienne A, Van Mourik M, Masters G, et al. Incidence of haemoglobinopathies in various populations — the impact of immigration. Clin Biochem 2009;42(18):1745–56. [21] Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory. Clin Chem 2004;50(10):1736–47.
Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001
K. Rodriguez-Capote, T.N. Higgins / Clinical Biochemistry xxx (2015) xxx–xxx [22] Patrinos GP, Giardine B, Riemer C, Miller W, Chui DHK, Anagnou NP, et al. Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. Database issue: D537-541; 2004. [Available from: http://globin.cse.psu.edu/hbvar/menu.html]. [23] Riou J, Godart C, Hurtrel D, Mathis M, Bimet C, Bardakdjian-Michau J, et al. Cationexchange HPLC evaluated for presumptive identification of hemoglobin variants. Clin Chem 1997;43(1):34–9. [24] Hardison RC, Chui DH, Riemer CR, Miller W, Carver MF, Molchanova TP, et al. Access to a syllabus of human hemoglobin variants (1996) via the World Wide Web. Hemoglobin 1998;22(2):113–27. [25] Huisman THJ, Carver MFH, Efremov GD. A syllabus of human hemoglobin variants augusta. GA, USA: The Sickle Cell Anemia Foundation; 1998. [26] Joutovsky A, Nardi M. Hemoglobin C and hemoglobin O-Arab variants can be diagnosed using the Bio-Rad Variant II high-performance liquid chromatography
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system without further confirmatory tests. Arch Pathol Lab Med 2004;128(4): 435–9. [27] Szuberski J, Oliveira JL, Hoyer JD. A comprehensive analysis of hemoglobin variants by high-performance liquid chromatography (HPLC). Int J Lab Hematol 2012; 34(6):594–604. [28] Behan KJ, Storey NM, Lee HK. Reporting variant hemoglobins discovered during hemoglobin A1c analysis — common practices in clinical laboratories. Clin Chim Acta 2009;406(1–2):124–8. [29] Bouzid K, Ahmed HB, Kalai E, Blibeche S, Couque N, Khiari K, et al. Prevalence of hemoglobin variants in a diabetic population at high risk of hemoglobinopathies and optimization of HbA1c monitoring by incorporating HPLC in the laboratory workup. Libyan J Med 2014;9:25768.
Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2 Karina Rodriguez-Capote ⁎, Trefor N. Higgins DynaLIFEDX Diagnostic Laboratory Services, Edmonton, Alberta, Canada Dept. Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
a r t i c l e
i n f o
Article history: Received 6 February 2015 Received in revised form 29 March 2015 Accepted 1 April 2015 Available online xxxx Keywords: Hemoglobinopathy screening β-Thalassemia α-Thalassemia HbF reference interval Hemoglobin A2 HbA2 reference interval Hemoglobin analysis
a b s t r a c t Objectives: The aims of this study were to identify the incidence of hemoglobinopathies and thalassemias in Northern Alberta and calculate the reference intervals (RI) for hemoglobin (Hb) HbF and HbA2. Methods: A retrospective ad-hoc analysis of the structural Hb variants and thalassemias identified on patients who had a hemoglobinopathy/thalassemia investigation performed between February 1 to December 31, 2013. Results were extracted from the Laboratory Information System. Statistical analysis was performed using MedCalc® version 11.4.2.0 for Windows software. Results: 6616 hemoglobinopathy/thalassemia investigations and HbS screens were physician requested and 602 Hb variants were fortuitously found during HbA1c analysis. 3438 were interpreted as “normal” and 532 were classified as iron deficient. 3306 individuals, with age ranging from 3 to 92 years were included in the RI calculation. HbA2 RI was 2.3% to 3.4% and HbF 0.0% to 1.8%. 524 and 423 α and β thalassemia traits respectively were identified. Additionally ten δβ thalassemia traits and twelve cases of HbH disease were identified. Regarding hemoglobinopathies, 7% were classified as α-chain variants and 93% as β-chain variants with HbS (46%), HbE (16%), HbD Punjab (8%) and HbC (7%) traits being the most prevalent. We also documented 20 homozygous hemoglobinopathies and 36 compound/double heterozygous hemoglobinopathies. Conclusion: A wide diversity of hemoglobinopathies is found in the Northern Alberta population, 80% of the hemoglobinopathies were found as a reflex to HbA1c testing. Reference intervals for HbF and HbA2 were established. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction DynaLIFEDX is the sole laboratory performing hemoglobinopathy and thalassemia investigations for a catchment area of 2 million people in Northern Alberta, Canada. Hemoglobinopathy/thalassemia investigations came from physicians based on clinical presentation, immigration medicals, red cell exchange programs for sickle cell anemia patients and as a reflex test generated by the presence of a hemoglobin (Hb) variant noted during HbA1c analysis. Diagnoses of these hemoglobin disorders are important for planning appropriate management and genetic counseling [1]. Additionally in Northern Alberta, screening for thalassemia and hemoglobinopathies is offered by the maternal care clinics if the patient and/or her partner are identified as belonging to an ethnic population whose members are at
Abbreviations: CE-HPLC, cation-exchange high performance liquid chromatography; CI, confidence intervals; Hb, hemoglobin; HPFH, Hereditary Persistence of HbF; RI, reference interval. ⁎ Corresponding author at: DynaLIFEDx, #200, 10150-102 St, Edmonton, Alberta T5J 5E2, Canada. Fax: +1 780 454 2845. E-mail address: [email protected] (K. Rodriguez-Capote).
higher risk of being carriers as recommended by the clinical practice guideline from the Genetics Committee of the Society of Obstetricians and Gynaecologists of Canada and the Prenatal Diagnosis Committee of the Canadian College of Medical Geneticists [2]. Screening during pregnancy accounts for a high percentage of the requests for hemoglobinopathy and thalassemia investigations, since in Alberta neonatal hemoglobinopathy screening is not included in the Newborn Screening Program [3]. Fetal hemoglobin (HbF, α2γ2) is elevated in newborns, usually reaching adult levels by 12 months and it commonly increases up to 5% in normal pregnancy [4]. HbF measurement is useful in the diagnosis of β-globin gene disorders and in the prognosis of patients homozygous for HbS [5]. Some acquired conditions such as aplastic anemia and myeloproliferative disorders are also associated with mild increases of HbF. Furthermore, pharmacological stimulation of HbF is used to manage conditions such as sickle cell disease and β-thalassemia major [5,6]. Thus, a reliable monitoring of HbF concentration during the treatment and follow-up of these patients is required. HbA2 (α2δ2) is recommended as the primary test for the diagnosis of β-thalassemia trait in iron replete patients in the presence of microcytosis [1,7]. The slight difference between HbA2 values in patients with and without β- thalassemia trait requires excellent precision at HbA2 values
http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001
2
K. Rodriguez-Capote, T.N. Higgins / Clinical Biochemistry xxx (2015) xxx–xxx
at the upper limit of the reference interval (RI). Thus quantification of HbA2 has to be accurate. A review of our data showed that 17% of HbF results reported in patients with a normal iron status and absence of a Hb variant exceeded the manufacturer's reference interval of b 1.0%, therefore it was necessary to reassess the RI for HbF. The aims of this study were to identify the incidence of hemoglobinopathies and thalassemias in Northern Alberta and calculate the reference intervals for HbF and HbA2.
Table 1 95% reference intervals for HbA2 and HbF. N = 3312
HbF%
HbA2%
LLRI (90% CI) ULRI (90% CI) Coefficient of skewness Coefficient of kurtosis Shapiro–Wilk test
0% 1.8% (1.78–1.90) 1.97 (P b 0.0001) 6.27 (P b 0.0001) W = 0.83, reject normality (P b 0.0001)
2.3% (2.2–2.34) 3.4% (3.4–3.5) −0.48 (P b 0.0001) 1.84 (P b 0.0001) W = 0.97, reject normality (P b 0.0001)
CI, confidence interval; LL, lower limit; UL, upper limit; RI, reference interval.
Methods Discussion This study is an ad hoc analysis of the structural Hb variants and thalassemias identified in the Northern Alberta, Canada on patients who had an investigation between February 1 to December 31, 2013. Pseudo-anonymised data containing only patient age, HbF and HbA2 concentrations and interpretative results was extracted from the Laboratory Information System. This study complied with DynaLIFEDX Institutional Ethical Board requirements. In our laboratory, EDTA-anti-coagulated blood samples for hemoglobinopathy/thalassemia investigation and those with abnormal findings during HbA1c testing, are analyzed by a high resolution cation exchange high performance liquid chromatography (CE-HPLC) using the β thalassemia program on the Bio-Rad VARIANT II. The British Committee for Standards in Haematology recommends that a second methodology based on a different analytical principle be used to make a presumptive identification of the hemoglobin variant [8–11]. Following this principle any sample with an Hb variant fraction is further investigated by electrophoresis at alkaline and acid pH (Sebia Hydrasys Electrophoresis System). Detection of common α-thalassemia mutations and HbH disease are performed by GAP-PCR analysis (Calgary Laboratory Services). A complete blood count and ferritin tests are requested as part of the hemoglobinopathy/thalassemia investigation. The interpretative report is performed by the Clinical Biochemist, who integrates the patient results from the chromatogram, electropherograms, hematology indices and calculated Mentzer Index. Total imprecision (CV%) for HbF is 2.2% and 1.2% at levels of 2.17% and 9.54% respectively. For HbA2 the total imprecision is 3.5% and 2.1% at levels of 2.8% and 5.7% respectively. Data was imported to Excel (Microsoft Office 2010). Categorical variables were expressed as frequencies and percentages. 95% reference intervals with 90% confidence intervals (CI) were calculated using the non-parametric percentile method according to CLSI EP28-A3C [12]. Statistical analysis was performed using MedCalc® version 11.4.2.0 for Windows.
Of the 6616 hemoglobinopathy/thalassemia investigations performed over a period of 11 months, 52% were considered “normal” and 8% were considered as iron deficient. The RIs obtained in our population [HbA2 2.3–3.4% and HbF 0.0–1.8%] are similar to those reported by other large reference laboratories such as ARUP Laboratories [HbA2 2.0–3.5% and HbF 0.0–2.1%] [14] and Mayo Medical Laboratories [HbA2 2.0–3.3% and HbF 0.0–0.9%] [15]. Hemoglobinopathies and thalassemias are frequent in the Mediterranean coastal region, India, Africa and in Southeast Asia, and therefore are commonly found in immigrants from these areas and their descendants [1,16]. Our laboratory serves a diverse multicultural urban population with the 2011 census results indicating that 22% of the Edmontonians were born outside Canada, from these 26% were from South Eastern Asia, 6% from the Mediterranean basin and 9% from Africa; accounting for around 14% of the total population [17,18]. Some structural Hb variant or thalassemia was found on 40% of the patients. 92% of the hemoglobin variants were β-chain variant traits with HbS (47%), HbE (16%), HbD (9%) and HbC (7%) traits being the most prevalent. Complex hemoglobinopathies such as compound or double heterozygosity due to co-inheritance of defects in α- and βglobin genes were detected in 36 patients, with the double heterozygote for sickle cell trait and α-thalassemia minor being the most prevalent. For twenty-three α-chain variants and four β-chain variants, the HPLC pattern and electrophoresis at acid and alkaline pH were not distinctive enough to make a presumptive identification of the hemoglobin variant. Eighteen of these α-chain variants and two of the β-chain variants were fortuitously detected during HbA1c analysis and the
Results Of the 6616 thalassemia and hemoglobinopathy investigation requests 3312 were on patients older than 2 years and interpreted as “normal” by a Clinical Biochemist based on their hematology indices (hemoglobin N120 g/l, mean cell volume N 80 fL), absence of a hemoglobin variant, replete iron status and calculated Mentzer Index (MCV divided by RBC N 14). These results were included in the calculation of the RIs for HbF and HbA2. Both, HbA2 and HbF failed the Shapiro–Wilk test for normal distribution (Table 1), even after logarithmic or Box– Cox transformation (data not shown). Consequently, their respective RIs were calculated by the percentile method as recommended by the CLSI C28-A3 guidelines [12]. Box-plots summarizing the data are presented in Fig. 1. A summary of the hemoglobinopathy/thalassemia investigation main findings is presented in Fig. 2, the various hemoglobin variants diagnosed in this period are tabulated as follows: Table 2, α-chain variants; Table 3, β-chain variants and Table 4, Hb homozygous and double or compound heterozygous.
Fig. 1. Box-plot, the central box represents the values from the lower to upper quartile (25 to 75 percentile). The middle line represents the median. Outlier values are displayed as X. Statistical analysis was performed using MedCalc® version 11.4.2.0 for Windows.
Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001
K. Rodriguez-Capote, T.N. Higgins / Clinical Biochemistry xxx (2015) xxx–xxx
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Fig. 2. Hemoglobin variants and thalassemias identified in Northern Alberta.
remainders were investigations requested by the Mom Care Clinics. These Hb variants were found to have minimal clinical or hematological features, and only eight of them showed retention times that interfere with the measurement of HbA1c in our HPLC.
We found twenty new Hb homozygous diseases (thirteen HbSS, five HbEE, one HbCC, and one Hb D-Iran). Even with the high incidence of HbS and HbE in the population we did not see a compound/double Table 3 Beta chain Hb variant trait.
Table 2 Alpha chain Hb variant trait.
Hb J Hb I Possible Hb G-Philadelphia Possible Stanleyville II Possible Hb J-Tongariki Possible Hb J Oxford, J Paris Or J-Mexico Possible Hb J-Toronto Possible Hb O-Indonesia Possible Hb Hopkins II Possible Hb Q-India Possible Hb Q-Iran Possible Hb St. Luke's Possible Hb Toulon Possible Hb Wayne Hb M Bostona (13) Unknown α-chain Hb Variant a
N
% with respect to alpha chain variants
% with respect to total Hb variants
10 5 5 2 1 2
18% 9% 9% 4% 2% 4%
1.4% 0.7% 0.7% 0.3% 0.1% 0.3%
1 1 1 1 1 1 1 1 1 22
2% 2% 2% 2% 2% 2% 2% 2% 2% 39%
0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 3.0%
Confirmed by molecular analysis.
HbS HbE HbD HbC Possible Hb J-Baltimore Possible Hb G-Coushatta, D-Iran or Hb Osu Christianborg Possible Hb Athens-GA, Hb Tyne or Hb Hounslow Possible Hb Hope Possible Hb Presbyterian HbJ Possible Hb O-ARAB Possible Hb J-Bangkok Possible Hb I-High Wycombe Possible Hb E-Saskatoon Possible Hb O-Arab Possible Hb Camden or Beckman Hb variant β-chain trait, unknown variant
n
% with respect to β-chain variants
% with respect to total Hb variants
346 120 63 53 18 15
54.% 19.% 10.% 8.% 3.% 2.%
47.4% 16.4% 8.6% 7.3% 2.5% 2.1%
9
1.%
1.2%
5 2 2 1 1 1 1 1 1 4
1.% 0.3% 0.3% 0.2% 0.2% 0.2% 0.2% 0.2% 0.2% 1.%
0.7% 0.3% 0.3% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.5%
Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001
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K. Rodriguez-Capote, T.N. Higgins / Clinical Biochemistry xxx (2015) xxx–xxx
Table 4 Homozygous, compound and double heterozygotes.
HbSS HbEE HbCC Hb D-Iran/Hb D-Iran HbSC HbE/Hb Hope HbS/Hb G-Philadelphia HbS trait and α-thalassemia minor HbE trait and α-thalassemia minor HbE trait and β+ thalassemia HbC trait and β+ thalassemia HbS trait and β+ thalassemia HbS trait and α-thalassemia carrier
n
% with respect to total Hb variants
13 5 1 1 5 3 1 17 5 1 1 1 2
1.7% 0.7% 0.1% 0.1% 0.7% 0.4% 0.1% 2.3% 0.7% 0.1% 0.1% 0.1% 0.3%
a high prevalence of thalassemia the identification of affected individuals forms part of pre-marital counseling. In other areas individuals with thalassemia are often referred for genetic counseling [16,19]. Alberta is one of the fast growing provinces in Canada and according to the last census this growth is mainly due to the arrival of immigrants [17,18]. Knowing the incidence of hemoglobin disorders is important for planning appropriate management and to guarantee that affected individuals receive appropriate care after the diagnosis, such as medical, psychosocial care and genetic counseling. Conclusion A wide diversity of hemoglobin variants and thalassemias are found in Northern Alberta, 80% of the Hb variants were found as a reflex to HbA1c testing. Reference intervals for HbF and HbA2 were established for our population. Acknowledgments
heterozygous HbSE, which could be explained by the less interaction and marriage between the two main ethnic groups where these hemoglobinopathies are frequent as opposed to the five double heterozygotes HbSC and three double heterozygotes for Hb Hope/HbE. HbS or HbC are the most prevalent hemoglobinopathies in African descendants and both HbE and Hb Hope are associated with Southeast Asia ethnicity [1, 19,20]. Twenty-seven patients were diagnosed with compound hemoglobinopathy (combined hemoglobinopathy and thalassemia): seventeen HbS trait and α thalassemia minor, five HbE trait and α thalassemia minor, one compound heterozygous for HbS trait and β+ thalassemia and one compound hemoglobinopathy of HbC trait and β+ thalassemia. Our current protocol uses a short HPLC as the screening method, followed by high resolution CE-HPLC. The Hb variants are identified by comparing the chromatogram with those in the Bio-Rad library and scientific literature [21–23]. We always use both, alkaline and acid electrophoresis as confirmatory tests because the extensively documented characterization of hemoglobin variants by electrophoretic techniques in the literature [10], easy accessibility to an online compilation on human hemoglobin abnormalities through a syllabus of human hemoglobin variants via the World Wide Web [24,25] and our own archive of Hb variants retention times and electrophoretic mobilities. Although with these information we can only generate a presumptive diagnosis; with our laboratory experience together with other reports on the literature we are confident that the risk of sickling variants (HbSS, HbSC, HbSE, HbS/Hb D-Punjab, HbS/Hb G-Philadelphia, HbS/Hb Hope, HbS/ Hb Lepore, HbS/HbO-Arab, and Hb S/β-thalassemia) can be identified without the need to proceed toward the most expensive molecular identification [21,26,27]. In 2013 only one Hb variant was sent for molecular diagnostic, HbM Boston [13]. The investigation of hemoglobinopathies may result from clinical presentation of hypoxia, presence of brown blood due to congenital methemoglobinemia and altered oxygen affinity particularly when using finger blood gas analyzers [13]. In addition, hemoglobinopathy investigation may form part of a pre-surgery laboratory workup. Recently a number of new hemoglobinopathies have been found as a fortuitous finding on HbA1c measurements [28,29]. We found 80% of the Hb variants during HbA1c testing. We believe that all Hb variants should be reported because they might have an effect on clinical decisions. Other laboratories, however, report the presence of a variant only if it interfered with the test [28,29]. The quantification of HbA1c provides a challenge in individuals with a homozygous hemoglobinopathy and the validity of threshold points for diagnosis and monitoring diabetes mellitus may be compromised in individuals with a hemoglobinopathy. The identification of individuals with thalassemia is important to prevent the administration of iron to individuals who are microcytic due to thalassemia rather than iron deficiency. In areas where there is
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Please cite this article as: Rodriguez-Capote K, Higgins TN, Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.04.001