Clinical Biochemistry 40 (2007) 744 – 746
Effect of HbS in the determination of HbA2 with the Biorad Variant II analyzer Christoforos Kalleas c , Ioannis Tentes a , Dimitrios Margaritis b , Kostas Anagnostopoulos a,⁎, Athina Toli c , Dimitrios Pendilas c , Georgios Bourikas b , Constantinos Tsatalas b , Alexandros H. Kortsaris a a
Laboratory of Biochemistry, Department of Medicine, Democritus University of Thrace, University Campus, 68100 Alexandroupolis, Greece b Hematology Clinic, Department of Medicine, Democritus University of Thrace, Alexandroupolis, Greece c Hematology Laboratory, General Hospital of Poligiros, Poligiros, Greece Received 19 January 2007; received in revised form 5 March 2007; accepted 7 March 2007 Available online 23 March 2007
Abstract Objectives: The effect of the presence of HbS in the determination of HbA2 using the Biorad Variant II analyzer. Design and methods: The effect of HbS presence in the samples was quantified using the HELENA SAS-MX alkaline gel electrophoresis kit as the reference method. Results: The %HbA2 values from the Variant II analyzer and the HELENA SAS-MX alkaline gel electrophoresis kit show a good linear correlation in the absence of HbS. A strong positive bias in the %HbA2 values from the Variant II is apparent in the presence of HbS in the samples, when compared to the alkaline electrophoresis gel. Conclusion: The Variant II analyzer gives reliable results for %HbA2 determination when no HbS is detectable in the samples. When HbS is present, the gel electrophoresis method gives more accurate results. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Haemoglobin A2; Haemoglobin S; HPLC; Method comparison; Gel electrophoresis
Introduction Measurement of the percentage of haemoglobin A2 (HbA2) is very important in the diagnosis and characterization of haemoglobinopathies, especially in identifying β-thalassemia traits in the β-thalassemia screening [1]. A lot of effort is being directed towards the development of analytical methods for the percentage assessment of the various types of haemoglobins in clinical samples, including haemoglobin A2. The latter is particularly important for the diagnosis of the β-thalassemia trait, in genetic counselling and in the prevention of β-thalassemia. In most cases where the percentage value of HbA2 is considerably lower or higher than reference levels, it is easy to come to a clinical decision. However, in cases where the values of HbA2 are in the
“gray” or borderline area, as a result of a potential iron deficiency [2], identifying the β-thalassemia trait can be problematic. To this end, an accurate determination would be of valuable clinical use. There are many methods available for the determination of HbA2 levels in routine use in the clinical laboratory [3–7], including HPLC. An advantage of the HPLC methods is that they can be fully automated, especially when performed on dedicated instruments, as usually is the case. In this investigation we study the effect of the presence of haemoglobin S (HbS) in the assessment of HbA2 using the Biorad Variant II HPLC haemoglobin analyzer. Materials and methods HPLC system
⁎ Corresponding author. Fax: +30 25510 30502. E-mail addresses:
[email protected],
[email protected] (K. Anagnostopoulos).
The Biorad Variant II (Biorad Corporation, USA) uses a cation-exchange HPLC column for the separation of haemo-
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.03.008
C. Kalleas et al. / Clinical Biochemistry 40 (2007) 744–746
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Results Comparison of the HELENA SAS-MX alkaline Hb gel to the Biorad Variant II in the presence and absence of HbS
Fig. 1. Comparison of the HELENA SAS-MX alkaline Hb gel kit to the BIORAD Variant II in the absence of HbS. The thick solid line is the calculated linear regression line.
globins F, A2, A, S, D and C using a gradient of sodium phosphate buffers in a 6.5 min program. The absorbance of the eluted haemoglobins is measured at 415 nm with correction at 690 nm. The calibrator used is the Biorad Variant haemoglobin A2/F calibrator, provided by the manufacturer. Alkaline agarose gel chromatography The HELENA SAS-MX alkaline gel Hb kit is based on an electrophoretic method for the separation and identification of haemoglobins. The electrophoresis is performed in alkaline buffer. After the separation of the haemoglobins, the gel is stained by acid blue stain and the results are obtained after scanning of the gel and integration using the DE400+ densitometer and the accompanying analysis software (Biomidi Electrophoresis, France).
Upon comparing the two methods we found that when no HbS is present in the samples the two methods correlate well. Presented in Fig. 1 are the results of the comparison between the Biorad Variant II and the HELENA alkaline Hb gel, in samples that do not contain HbS. A good linear correlation is shown (r = 0.974, p < 0.0001, 95% CI: 0.953 to 0.986), taking into consideration the different nature of the two methods. The mean bias is −0.21%. It should be noted that, although for lower % HbA2 values the linear correlation is very strong, for high % HbA2 values there seems to be a small negative bias. In Fig. 2 the same comparison of the two methods has been performed in samples containing HbS. These samples were all from subjects with the sickle trait. It is evident that the linear correlation between the two methods is much weaker, regarding the %HbA2 determination (r = 0.730, p < 0.0001, 95% CI: 0.528 to 0.853). The mean bias is 0.50%. From the bias plot versus the HbS levels (as determined by the gel method) it is clear that a correction formula cannot be established (data not shown). Discussion There are many techniques for the separation and quantification of haemoglobin fractions. Electrophoretic methods are the first ones used and they are cheap and easy to perform but suffer from some limitations. The most serious drawback of gel-based methods is in the quantification of the separated fractions, which is performed by scanning and densitometry, procedures that are inherently not so accurate. HPLC techniques are the most reliable methods which can be fully automated, with respect to primary tube sampling, barcode reading and integrated quality-control software. Such is the case with the Biorad Variant II instrument.
Treatment of samples Blood samples containing EDTA as anticoagulant were used. Samples were processed from the primary tubes. Calibrators and controls were provided by the manufacturer and handled as directed. Method comparison All blood samples were measured in duplicate with each of the two methods. The threshold for within-method duplicate outlier detection was set as four times the mean absolute differences between duplicates for each method and four times the relative absolute differences. Data points that fell outside of both limits were eliminated. The threshold for between-method outliers was set as four times the mean absolute differences between methods and their average and four times the relative absolute differences between methods and their average. Data points that fell outside of both limits were eliminated [8]. In the remaining data points, linear regression analysis was performed.
Fig. 2. Comparison of the HELENA SAS-MX alkaline Hb gel kit to the BIORAD Variant II in the presence of HbS. The thick solid line is the calculated linear regression line.
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It is well known in the clinical haematology laboratories that the presence of HbS can interfere with the determination of HbA2 when using chromatographic techniques [5,9–11]. One reason might be the reduced affinity of the βS chain for the α chain, which leaves more free α chains to combine with the δ chains, resulting in increased HbA2 (α2δ2) percentage [5]. However, this cannot be the only reason since, if it were so, the percentage of HbA2 would also be significantly increased in the case of gel electrophoresis. Another reason could be the coelution of HbS adducts (including, but not limited to, glycated HbS) [9]. In the case of gel electrophoresis these adducts do not co-migrate with HbA2. However, in the case of chromatography, these adducts could co-elute with HbA2 and falsely elevate its percentage. In a previous report we have examined the effect of the HbS presence in the determination of HbA2 using the TOSOH HLC273G7 instrument and found out that the presence of HbS can lead to falsely elevated values of %HbA2 [11]. The same seems to be the case when comparing the HELENA SAS-MX alkaline Hb gel kit to the Biorad Variant II instrument. In the absence of HbS, a good linear correlation is evident, presented in Fig. 1 (r = 0.974, p < 0.0001, 95% CI: 0.954 to 0.986). The correlation coefficient is lower compared to the one reported for the TOSOH HLC723G7 instrument (r = 0.985, p < 0.0001, 95% CI: 0.974 to 0.991) [11], but since there is a significant overlap in the two confidence intervals the lower value may very well be the result of the experimental statistical variance present in all measurements. In the presence of HbS, as shown in Fig. 2, the linear correlation is much weaker (r = 0.730, p < 0.0001, 95% CI: 0.528 to 0.853). It should be noted that the two confidence intervals (absence and presence of HbS) do not overlap. The mean bias is 0.50%. From the findings above alone it cannot be certain that it is the HPLC method that is affected since it could be that the presence of HbS affects the gel electrophoresis method. However, in the past we examined the effect of HbS presence when comparing the HELENA SASMX alkaline Hb gel kit to the HELENA Beta-Thal Quik column kit and found out that the ion exchange column is affected by the presence of HbS too, and to a greater extent [11]. No correction formula can be calculated from the bias plot versus %HbS since there is no correlation between the bias and the HbS levels (data not shown). When comparing the performance of the two HPLC systems in the presence of HbS we can see that the %HbA2 values seem
to be equally affected in both cases. For the TOSOH instrument the reported correlation coefficient is 0.757 (p < 0.0001, 95% CI: 0.559 to 0.873) [11] and for the Biorad instrument it is 0.730, p < 0.0001, 95% CI: 0.528 to 0.853. We can see that there is a very significant overlap in the two confidence intervals. We can therefore conclude that both instruments are affected by the presence of HbS to the same degree. The determination of the percentage of HbA2 is of particular use in distinguishing between homozygous sickle cell disease (HbSS) and heterozygous HbS/β0 thalassemia. These disorders show similar clinical and haematological symptoms with the main difference being that in HbS/β0 thalassemia the MCV and MCH are lower and the HbA2 percentage is increased. Since the presence of HbS results in a high positive bias in the determination of HbA2 levels, this could make the diagnosis of these conditions more complicated. References [1] Gu X, Zeng Y. A review of the molecular diagnosis of thalassemia. Hematology 2002;7(4):203–9. [2] Giordano PC. The effect of iron deficiency anemia on the levels of haemoglobin subtypes: possible consequences for clinical diagnosis. Clin Lab Haematol 2003;25(3):203. [3] Deyde VM, Fattoum S, Lo BB, Plaseska D, Efremov Gd. Haemoglobin A2′ (HbA2δ′) in the Mauritian population: first results of a preliminary study. Ann Hematol 2002;81:386–8. [4] Center for Disease Control, U.S. Department of Health and Human Services/Public Health Service, Laboratory methods for detecting haemoglobinopathies, Sept. 1984. [5] Head CE, Conroy M, Jarvis M, Phelan L, Bain BJ. Some observations on the measurement of haemoglobin A2 and S percentages by high performance liquid chromatography in the presence and absence of alpha thalassaemia. J Clin Pathol 2004;57(3):276–80. [6] Ravindran MS, Patel ZM, Khatkhatay MI, Dandekar SP. Beta-thalassaemia carrier detection by ELISA: a simple screening strategy for developing countries. J Clin Lab Anal 2005;19(1):22–5. [7] Gerritsma J, Sinnige D, Drieze C, et al. Quantitative and qualitative analysis of haemoglobin variants using capillary zone electrophoresis. Ann Clin Biochem 2000;37(Pt 3):380–9. [8] NCCLS Approved Guideline, “Method comparison and bias estimation using patient samples”, NCCLS Document EP9-A (ISBN 1-56238-283-7), 1995. [9] Shu DD, Kraus JS, Bures K. Influence of haemoglobin S adducts on haemoglobin A2 quantification by HPLC. Clin Chem 1996;42(7):1113–4. [10] Shokrani M, Terrell F, Turner EA, Aguinaga MD. Chromatographic measurements of haemoglobin A2 in blood samples that contain sickle haemoglobin. Ann Clin Lab Sci 2000;30(2):191–4. [11] Kalleas C, Tentes I, Margaritis D, et al. Effect of HbS in the determination of HbA2 with the TOSOH HLC-723G7 analyzer and the HELENA BetaThal Quik column. Clin Biochem 2007;40:242–7.