Available online at www.sciencedirect.com

Clinical Biochemistry 42 (2009) 1667–1676

Rapid identification of HBB gene mutations by high-resolutionmelting analysis

Hung-Chang Shih a,b,c,d, Tze-Kiong Er b,e, Tien-Jye Chang c, Ya-Sian Chang b,Ta-Chih Liu a,b,e, Jan-Gowth Chang a,b,e,f,⁎

a Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwanb Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

c Department of Veterinary Medicine, National Chung Hsiung University, Taichung, Taiwand Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan

e Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwanf Center for Excellence in Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

Received 24 April 2009; received in revised form 3 July 2009; accepted 14 July 2009Available online 23 July 2009

Abstract

Objective: This study was undertaken to identify HBB gene mutation.Design and methods: Herein we evaluated high-resolution melting analysis in the identification of HBB mutations.Results: We have successfully established a diagnostic strategy for identifying HBB gene mutations including c.−78ANG, c.−79ANG,

c.2TNG, c.79_80insT, c.84_85insC, c.123_124insT, c.125_128delTCTT, c.130 GNT, c.170GNA, c.216_217ins A and c.316–197 CNT fromwild-type DNA using HRM analysis. The results of HRM analysis were confirmed by direct DNA sequencing.

Conclusions: In summary, we report that HRM analysis is an appealing technique for the identification of HBB mutations. We also believe thatHRM can be used as a method for prenatal diagnosis of β-thalassemia.© 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Keywords: HBB gene; Mutation; β-thalassemia; High-resolution melting analysis; Single nucleotide polymorphism; Melting curve

Introduction

Hemoglobinopathies resulting from mutations in the α- or β-like globin gene clusters are the most common inheriteddisorders in humans, with around 7% of the world populationbeing carriers of a globin gene mutation [1]. Molecular defectsin either regulatory or coding regions of the human α-, β- or δ-globin genes can minimally or drastically reduce theirexpression, leading to α-, β- or δ-thalassemia, respectively.

Abbreviations: HBB, Hemoglobin, beta; Hb, Hemoglobin; PCR, Polymer-ase Chain Reaction; PCR-RFLP, PCR-Restriction Fragment Length Polymorph-ism; HRM, High-Resolution Melting; SNP, Single Nucleotide Polymorphism;High performance liquid chromatography, HPLC; Capillary Electrophoresis,CE; Amplification Refracted Mutation System, ARMS; Single Base Extension,SBE.⁎ Corresponding author. Department of Laboratory Medicine, Kaohsiung

Medical University Hospital, 100 Shih-Chuan 1st Rd., Kaohsiung, Taiwan.E-mail address: jgchang@mail.kmuh.org.tw (J.-G. Chang).

0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemistsdoi:10.1016/j.clinbiochem.2009.07.017

Some single substitutions can lead to amino acid replacementsthat cause hemolytic anemias, such as sickle cell disease, orhemoglobins that are unstable or have altered oxygen affinity.Other sequence changes have little or no effect on hemoglobinfunction, but are useful polymorphisms for genetic studies.

β-thalassemia is an endemic disease in many regions of theworld. The most common genetic lesion of β-thalassemia ispoint mutations. Patients of each ethnic population carry theirown specific types of mutations, consisting of a few verycommon ones and a variable number of rare ones [2,3]. Inthe Southeast-Asian population, the common β-thalassemiamutations include c.-78 ANG, c.2 TNA, c.52 ANT, c.84_85insC, c.125_128 delTCTT, c.130 GNT, c.216_217 insA, andc.316–197 CNT [4]. In Chinese, more than 20 types ofmutations have been found, and half of these types are alsofound in Taiwanese. In Orientals, it is one of the most commonhereditary diseases with carrying rates of 1–3% [5,6]. The mostcommon mutations are point mutations of β-globin gene; four

. Published by Elsevier Inc. All rights reserved.

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types of the point mutation account for over 85% of cases of β-thalassemia in Taiwan. These are promoter −28 A→G, codon17 AAG→TAG, codon 41–42 deleted 4 bases, and IVS-2 nt654 C→T mutations [7]. The most common β-thalassemiamutation was the IVS-2 nt 654 C→T mutation; and the mostcommon Hemoglobin (Hb) variant was the HbE [8].

High-resolution melting (HRM) analysis is a new gene scantool that quickly performs the Polymerase Chain Reaction(PCR) and identifies sequences alterations without requiringpost-PCR treatment [9–12]. Recently, it has also been used inthe detection of α- and β-thalassemia of deletion form [13,14].The authors utilized real-time gap-PCR with SYBR Green1 andHRM analysis for diagnosis of β-thalassemia 3.5 kb deletion.So far, there is no thorough study on the capability of HRM toidentify HBB (hemoglobin, beta) gene point mutations. In thisstudy, we report a method for rapid detection of HBB genemutations.

Materials and methods

DNA samples

DNA samples were obtained from 10 prenatal casesincluding parents, and 62 subjects including 30 β-thalassemiaminor, 3 β-thalassemia major, Hb variants including 2 cases ofHbE, 6 cases of Hb J-Kaohsiung and 1 cases of Hb J-Meinungand 20 normal individuals at Kaohsiung Medical UniversityHospital. Genomic DNA was collected from peripheral wholeblood using the NucleoSpin® Blood Kit (Macherey-Nagel)according to the manufacturer's instructions. The genotypingfrom each samples were determined by the PCR-RestrictionFragment Length Polymorphism (PCR-RFLP) previously.

Primers

We designed the primer sets on the HBB DNA sequences(NCBI Reference Sequence: NG_000007.3). Table 1 shows theprimer sets for the detection of HBB gene mutations inpromoter, exon 1, exon 2, intron 3 and exon 3. For the prenatal

Table 1Primers use for HRM analysis of HBB gene mutations.

Detection for Sequence (5′ to 3′)

Promoter and Exon 1 P1 5′-CCAATCTACTCCCAGGAGCA-3′ (forP2 5′-GGCAGAGAGAGTCAGTGCCTA-3′ (r

Promoter and Initiation codon ⁎ P3 5′-ACTTCTCCTCAGGAGTCAGGT-3′ (reExon 1 ⁎ P4 5′-AGACACCATGGTGCACCTGAC-3′ (fExon 2 P5 5′-GAAGACTCTTGGGTTTCTGA-3′(forw

P6 5′-TCATTCGTCTGTTTCCCATTCTAAACExon 2 ⁎ P7 5′-GAGCCTTCACCTTAGGGTTT-3′ (reveExon 2 ⁎ P8 5′-CTCCTGATGCTGTTATGGGC-3′ (forw

P9 5′-AGAAAACATCAAGGGTCCCA-3′ (reIntron 2 P10 5′-GTGTACACATATTGACCAAATCAG

P11 5′-GGTAGCTGGATTGTAGCTGC-3′ (revIntron 2 ⁎ P12 5′-ATTTATATGCAGAAATATTG-3′ (reveExon 3 P13 5′-CTGGATTATTCTGAGTCCAAGC-3′(

P14 5′-ATTAGGCAGAATCCAGATGCTC-3′⁎ PCR primers were redesigned to block SNP interference.

diagnosis of β-thalassemia, we used primer P1+P3 fordetecting the promoter and initiation codon mutations, primerP2+P4 for detecting the mutations in exon 1 region except theinitiation codon, primers P5+P9 for detecting the mutation inthe exon 2 and primers P10+P11 for detecting the c.316–197CNT. The primers synthesized were all of standard molecularbiology quality (Protech Technology Enterprise Co., Ltd,Taiwan).

High-resolution melting

PCR reactions were performed in duplicate in 20 μL finalvolume using LightCycler ® 480 High-Resolution MeltingMaster (Roche Diagnostics) ×1—contains Taq, nucleotides andthe dye ResoLight— and 50 ng DNA. The primers and MgCl2were used at 2.5 mM, for HBB gene mutations. In each assay,we included DNAs with known HBB gene mutation and wild-type control DNAs.

The HRM assays were performed using the LightCycler®480 Instrument (Roche Diagnostics) provided with the softwareLightCycler® 480 Gene Scanning Software Version 1.0 (RocheDiagnostics).

The PCR program requires SYBR Green I filter (533 nm)and it consists of an initial denaturation–activation step at 95 °Cfor 10 min, followed by a 45-cycle program (denaturation at95 °C for 15 s, annealing at 56 °C or 52 °C (according toTable 1) 15 s and elongation at 72 °C for 15 s with reading of thefluorescence; acquisition mode: single). The melting programincludes three steps: denaturalization at 95 °C for 1 min,renaturation at 40 °C for 1 min and then melting that consists ofa continuous fluorescent reading from 60 to 90 °C at 25acquisitions per °C.

The shapes of difference plot curves of the duplicate of eachDNA sample must be reproducible both in shape and peak height.

Gene scanning

The melting curve analysis performed by the Gene ScanningSoftware comprises three steps: normalization of melting

Length of PCRamplicon (bp)

Nucleotides (nt) Annealingtemp. ( °C)

ward) 323 nt70469–nt70488 52everse) nt70771–nt70791verse) 154 nt70602–nt70622 56orward) 204 nt70588–nt70608 56ard) 404 nt70751–nt70770 52-3′ (reverse) nt71129–nt71154rse) 164 nt70895–nt70914 56ard) 193 nt70876–nt70895 56verse) nt71049–nt71068GGTA-3′ (forward) 293 nt71496–nt71523 56erse) nt71769–nt71788rse) 223 nt71699–nt71718 52forward) 309 nt71820–nt71841 52(reverse) nt72107–nt72128

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curves, equaling to 100% the initial fluorescence and to 0% thefluorescence remnant after DNA dissociation, shifting of thetemperature axis of the normalized melting curves to the pointwhere the entire double-stranded DNA is completely denatured,and finally, the difference plot analyzes the differences inmelting curve shape by subtracting the curves from wild-typeand HBB mutations DNA, therefore the difference plot helpsclustering the samples into groups.

Sequencing

To confirm HRM analysis results, sequencing analysis wasalso performed in all samples. After HRM analysis, sampleswere purified with PCR-M™ clean up system (VIOGEN). ThePCR products generated after HRM can be sequenced directly.The sequence reaction was performed in 10 μL final volumeusing 5 μL of the purified PCR product, 2.5 μM of one of PCRprimers and 1 μL of ABI PRISM terminator cycle sequencingkit v3.1 (Applied Biosystems). The sequencing program is a 25-cycle PCR program (denaturation 96 °C 10 s; annealing 50 °C5 s and elongation 60 °C 4 min). The sequence detection wasperformed in the ABI Prism 310 Genetic Analyzer (AppliedBiosystems).

Fig. 1. Screening of theHBB genes mutations in promoter and initiation codon. (A) isnew primer set (P1+P3). The c.−78ANG, c.−79ANG and c.2TNG are easily to be

Results

Screening of the HBB genes mutations: promoter and exon1 regions

In this section, we used first-designed primer (P1+P2) toidentify HBB genes mutations in promoter and exon 1 includingc.−78ANG, c.−79ANG, c.2TNG, c.52ANT, c.79GNA, andc.84_85insC. Finally, we found 1 single nucleotide polymorph-ism [SNP] (c.9CNT) in the region and this SNP interfere theidentification of HBB genes mutations in promoter and exon 1using HRM analysis (Supplementary data 1). We were unable todifferentiate the c.−78ANG, c.−79ANG, c.2TNG, c.52ANT,c.79GNA, and c.84_85insC from the complicated meltingcurve. Therefore, we redesigned two new primer sets (P1+P3and P4+P2) which overlay the SNP in order to block the SNPinterference. We successfully used the new primers set toidentify c.− 78A NG, c.− 79A NG, c.2T NG, c.52A NT,c.79GNA, and c.84_85insC. Fig. 1 shows that we were ableto differentiate the c.−78ANG, c.−79ANG, c.2TNG from themelting curve by using primers P1 and P3. Fig. 2 shows that wewere able to differentiate the c.52ANT, c.79GNA, andc.84_85insC from the melting curve by using primers P4 and

the high-resolution melting curves and (B) is the difference plots with redesigneddistinguished in the normalized and temp-shifted difference plot.

Fig. 2. Screening of the HBB genes mutations in exon 1. (A) is the high-resolution melting curves and (B) is the difference plots with redesigned new primer set(P4+P2). The c.79GNA, c.52ANT and c.84_85insC are easily to be distinguished in the normalized and temp-shifted difference plot.

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P2. Those results were confirmed by direct sequencing of PCRproducts (Supplementary data 1).

Screening of the HBB genes mutations: exon 2 region

In this section, we use first-designed primers (P5+P6) toidentify HBB genes mutations in exon 2 includingc.123_124insT, c.125_128delTCTT, c.130 GNT, c.216_217insA and Hb J-Meinung. Finally, we found 3 SNPs (c.171CNG,c.315+16GNC and c.315+74TNG) in this region and the 3SNPs interfere the identification of HBB genes mutations inexon 2 using HRM analysis (Supplementary data 2). Also,we were unable to differentiate the c.123_124insT,c.125_128delTCTT, c.130 GNT, c.216_217 ins A and Hb J-Meinung from the complicated melting curve. Therefore, weredesigned a new primer set (P5+P9) which overlaid the 2 SNPsin order to block the SNPs interference. We successfully usedth is new pr imer se t to ident i fy c .123_124insT,c.125_128delTCTT, c.130 GNT, c.216_217 ins A and Hb J-Meinung (Fig. 3). On the other hand, we tried to redesign twonew primers sets (P5+P7 and P8+P9) to block 3 SNPs,however, the curve of c.170GNA (Hb J-Meinung or Hb J-Bangkok) shift to the group of wild-type DNA by using theprimer sets P5 and P7 (Supplementary data 3). The c.216_217ins

A can be easily distinguished from wild-type DNA using newprimer set (P8+P9) (Supplementary data 4). These results wereconfirmed by direct sequencing of the PCR products.

Screening of the HBB genes mutations: intron 2

In this section, we used first-designed primer (P10+P11) toidentify HBB genes mutations in intron 2, c.316–197 C. Wediscovered 1 SNP (c.316–185CNT) in intron 2, however, wewere still able to identify c.316–197 C from wild-type DNA.Fig. 4 shows that the SNP did not interfere with the meltingcurve. The c.316–197 C could be easily seen in the normalized,temperature-shifted plot. Even though, we also redesigned anew primers set (P10+P12) in order to block the SNP. The SNPwas successfully blocked by the redesigned primer and we wereable to see the grouping in the normalized, temperature-shiftedplot (Supplementary data 5). These results were confirmed bydirect sequencing of the PCR products.

Screening of the c.341TNA (Hb Kaohsiung or New York):exon 3

The HRM analysis successfully differentiated the six clinicalsamples (c.341TNA) from the wild-type control DNA on the

Fig. 3. Screening of the HBB gene mutations in exon 2. (A) is the high-resolution melting curves and (B) is the difference plots with redesigned new primer set(P5+P9). The c.171CNT (heterozygous), c.170GNA, c.216_217ins A, c.130GNT, c.123_124insT, c.125_128delTCTT and c.125_128delTCTT (homozygous) areeasily to be distinguished in the normalized and temp-shifted difference plot.

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basis of the shape of normalized, temperature-shifted plot anddifference plot curves (Fig. 5). These c.341TNA were clearlydistinguishable from the wild-type control DNA. HRM analysisprofiles of c.341TNA were confirmed by DNA sequencing.These results were confirmed by direct sequencing of the PCRproducts.

Application of HRM analysis in prenatal diagnosis ofβ-thalassemia

In this section, we would like to evaluate the practicality ofthis methodology for prenatal diagnosis. Prenatal samples withknown HBB gene mutation were made by a blindedinvestigator. These clinical samples were obtained from 10families including parents and probands. Four primer sets(P1+P3, P2+P4, P5+P9 and P10+P11) were used to analyzethe samples using HRM analysis in the same PCR condition.

All of the positive controls and negative controls were alsoadded in the same run. We are able to distinguish the unknownsample whether it is mutant or normal according to the meltingcurves of positive and negative controls in the normalized and

temp-shifted difference plot. For a rare case, we use the directsequencing for rechecking the result.

The HRM were able to detect the mutations precisely. Fig. 6showed that the representative cases were successfullyidentified by only a single run.

Discussion

There are many techniques that have been used to screen anddiagnose Hb variants and thalassemia. For example, highperformance liquid chromatography (HPLC) [15], capillaryelectrophoresis (CE) [16], restriction fragment length poly-morphism (RFLP) [17], amplification refracted mutationsystem (ARMS-PCR) [18] and single base extension (SBE)[19]. The request for rapid and reliable detection andidentification of Hb variants and thalassemia is rapidlyincreasing for the definition of clinical samples and orientatingtargeted therapies or counseling. Some methods for large-scaledetection of HBB gene mutations are expensive and technicallytime-consuming. HRM analysis represents the next generationof mutation scanning technology and offers considerable time

Fig. 4. Screening of the HBB genes mutations in intron 2. (A) is the high-resolution melting curves and (B) is the difference plots with redesigned new primer set 0+P11). The c.316–197 CNT (heterozygous), wild-type (heterozygous), wild-type (homozygous) and c.316–197 CNT (homozygous) are easily to be distinguished in the normalized and temp-shifted difference ot. wt: c.316–185CNT.

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(P1pl

Fig. 5. Screening of the Hb variant, c.341TNA. (A) is the high-resolution melting curves and (B) is the difference plots with primer set (P13+P14). The c.341TNA iseasily to be distinguished in the normalized and temp-shifted difference plot.

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and cost savings over those methods previously described [20].In the presence of a saturating double-stranded DNA-bindingdye, amplicons are slowly heated until fully denatured while thefluorescence is monitored [9]. Amplicons heterozygous for asequence variant yield altered melting curves compared withnormal control samples.

HRM is rapidly becoming the most important mutationscanning methodology. It is a closed-tube method, meaning thatPCR amplification and subsequent analysis are sequentiallyperformed in the well. This makes it more convenient than otherscanning methodology. Good amplicon design is essential toobtain robust and reproducible HRM analysis. SNPs near theprimers usually altered the low temperature region of the curve,making overlay across this region most effective in presentingshape difference. Besides, the difference between wild-type andheterozygote curves became smaller and difficult to differenti-ate if the product length increases [21]. Although HRM analysisis a powerful screening tool, the presence of unexpectedpolymorphisms close to the mutations of interest may interferewith genotyping. Extra care needs to be taken in designing PCRreactions to avoid primer dimers and non-specific amplificationin HRM analysis.

Apparently, HRM is not able to detect mutations encom-passing the whole gene or entire exon due to sensitivity of

detection. In addition, the design primers should flank the exonor intron as closely as possible. If a SNP is close to the exon orintron boundary, the primer can be placed over the SNP and amismatched base with no allelic preference can be introduced atthe SNP position [22,23]. At the beginning, we anticipatedusing the 4 primer sets (P1+P2, P5+P6, P10+P11 and P13+P14) for the detection of HBB gene mutations and Hb variantsas we previously described by HRM analysis. The presence ofinheritable polymorphisms will affect HRM analysis. There-fore, PCR primers were redesigned to generate an ampliconsuitable for HRM analysis of HBB gene.

In this study, we found 5 SNPs of HBB gene in Taiwanesewhere 1 SNP (c.9CNT) in exon 1 [25], 3 SNPs (c.171CNG,c.315+16GNC and c.315+74TNG) in exon 2 and 1 SNP inintron 2 (c.316–185CNT), respectively. The c.9C→T (silentvariant) is a common variant with an allele frequency of 38%, asdetermine in review of clinical samples submitted for β-globinsequencing (courtesy of Dr. Elaine Lyon, ARUP Laboratories)[24]. Those SNPs were confirmed by direct sequencing of PCRproducts. Indeed, they interfered with the identification of HBBgenes mutations using HRM analysis. We cannot obviouslydifferentiate the HBB genes mutations from the complicatedmelting curve. Therefore, we redesigned a new primer setswhich overlaid the SNPs in order to block the SNPs interference.

Fig. 6. Application of HRM analysis in prenatal diagnosis of β-thalassemia. Sample a (mother) is c.316–197 CNT (D), sample b (father) is c.125_128delTCTT (C), and sample c (child) is c.316–197 CNTcombines withc.125_128delTCTT (C, D). wt: c.316–185CNT.

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Particularly, it is suitable to use the primer set whichoverlays the SNP for the detection of HBB gene mutations byHRM analysis. In our study, we successfully blocked the 2SNPs in exon 2 and the mutant DNAs were obviouslydifferent from control DNAs. Nevertheless, the curve ofc.170GNA (Hb J-Meinung or Hb J-Bangkok) shifts to thegroup of wild-type DNA (Supplementary data 3). The reasonis the location of SNP (c.171CNG) is just only located by theside of c.170GNA. If we used the primer set (P5+P7), we areunable to detect c.170GNA in exon 2 by HRM analysis. Onthe other hand, 1 SNP (c.316–185CNT) was found in intron 2and we also redesigned the new primer set (P10+P12) whichoverlaid the SNP. Unluckily, the primer set cannot beamplified at the annealing temperature 56 °C but at 52 °C.Consequently, we need to use the primer set (P10+P11)which includes the SNP for identify c.316–197 CNT in theprenatal diagnosis. Finally, four primer sets (P1+P3, P2+P4,P5+P9 and P10+P11) were used as prenatal diagnosis byHRM analysis in the same PCR condition. Prenatal diagnosisof thalassemia is very important because approximately 300babies may be born with a severe form of thalassemia per yearin Taiwan. As aforementioned, β-thalassemia is one of themost common hereditary diseases with carrying rates of 1–3%. We believe that HRM is a fast, reliable and accuratescreening method for β-thalassemia and important for theprevention of this disease.

Hb E, Hb J-Meinung and Hb Kaohsiung are three commonHb variants in Taiwan. Hb Kaohsiung or New York wasdiscovered in 1967 by Ranney et al. [25]. This hemoglobinvariant is due to a glutamic acid amino acid residue substitutionat position 113 of the β-globin chain for the usual neutral valine.We have previously demonstrated a successful method toidentify Hb Kaohsiung or New York, a T→A substitution atcodon 113 of the β-globin chain in a Taiwanese family [26].However, this method requires post-PCR treatment prior to gelelectrophoresis, being time-consuming and expensive. In thisstudy, we easily distinguished Hb Kaohsiung from wild-typeDNA using HRM analysis (Fig. 5).

Traditionally, PCR-RFLP is the methodology for thediagnosis of β-thalassemia in our laboratory. It costsapproximately US ⁎$100 per run and needs more than aday for screening and identification. Including all consum-ables for HRM analysis but excluding manpower andequipment amortization, HRM analysis costs approximatelyUS ⁎$20 per run (including all positive controls and negativecontrols). In addition, a single medical technologist can handleall of the procedures easily and provide the result within aday. With this highly sensitive and specific diagnostic tool, wewill be able to manage a large quantity of clinical samples inour laboratory.

HRM analysis offers several benefits including loweringmanpower, time-saving, and decreasing the risk of PCRcarryover contamination. Additionally, the HRM analysis isthe most cost-effective in diagnostic laboratories withmoderate to high patient sample volumes. This is because upto 96 or 384 DNA samples can be analyzed within 2 h by asingle medical technologist (including data interpretation). In

summary, we have set up a very fast, simple and non-expensive HRM for the detection of HBB gene mutationshitherto known. Our results suggest that HRM is a feasible andhighly accurate method for the screening and identification ofβ-thalassemia, therefore, it could replace the currently methodsapplied in the screening of β-thalassemia and also prenataldiagnosis.

Declaration of interestThe authors report no conflicts of interst. There is no patent application regardingthe method.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.clinbiochem.2009.07.017.

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