Clinical Chemistry
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Clinical Chemistry 43: 34-39, 1997;
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(Clinical Chemistry. 1997;43:34-39.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants

Jean Riou, Christian Godart, Didier Hurtrel, Mireille Mathis, Catherine Bimet, Josiane Bardakdjian-Michau, Claude Préhu, Henri Wajcmana and Frédéric Galactéros

Department of Biochemistry and INSERM U91, Hôpital Henri Mondor, 94010 Créteil, France.
a Author for correspondence. Fax (33-1) 49 81 28 95; e-mail wajcman{at}im3.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A battery of relatively simple tests allows the presumptive identification of hemoglobin (Hb) variants, making unnecessary structural analysis by protein chemistry methods or DNA sequencing. The primary step in this strategy involves the use of a matrix of electrophoretic mobilities obtained under various experimental conditions. This leads to an unambiguous result in ~90% of the cases. Additional tests are required to characterize with more confidence the remaining 10%. We describe here the use of cation-exchange HPLC on the Bio-Rad Variant automated analyzer with the "ß Thalassemia Short" program. By comparing the elution time of 125 human Hb mutants, we found that some variants with almost identical pI values or produced by the same type of amino acid substitution displayed different elution times. We present several examples in which use of the HPLC profile helped establish the diagnosis.


Key Words: indexing terms: sickle cell disease • thalassemia • electrophoresis • isoelectric focusing • globin chains


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The use of cation-exchange high-performance liquid chromatography (CE-HPLC) to separate and quantify various normal and abnormal hemoglobin (Hb) fractions has been increasing (1)(2)(3)(4).1 This method has also been proposed for screening Hbs of clinical significance (3)(5)(6). The Bio-Rad Variant Hemoglobin Testing System (Bio-Rad Labs., Hercules, CA), a totally automated CE-HPLC instrument, has been used in our laboratory for routine quantification of HbA2 and HbF for >2 years. In a recent evaluation of the Bio-Rad Variant "ß Thalassemia Short" program for quantification of Hbs A, S, C, and F, Papadea and Cate (7) obtained results that were comparable with or exceeded those of traditional methods. We think CE-HPLC is also a valuable additional tool for presumptive identification of Hb variants.

The strategy used in our laboratory to characterize rare mutant Hbs primarily involves multiple electrophoreses in several experimental conditions (8)(9). This leads to an unambiguous result in >90% of the cases. Nevertheless, a few variants may behave similarly during electrophoresis and share several common biochemical or functional properties; for those cases, any additional test that would allow discrimination will be of interest. Here we describe the use of the Bio-Rad Variant analyzer with the ß Thalassemia Short program to discriminate Hb variants. Working under the experimental conditions specified by the manufacturer, with a few technical modifications, we obtained highly reproducible retention times for repeated assays with the same column and reagent batch. Therefore, we evaluated the possibility of using a normalized elution time as an additional variable in our approach to the presumptive identification of Hb variants.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
blood samples
Samples (Hb concentrations ~2 g/L) were obtained by hemolyzing 20 µL of blood in 1 mL of a buffer of potassium hydrogen phthalate (5 g/L) and potassium cyanide (5 g/L). We applied 20 µL of hemolysate onto the column for analysis.

The blood samples were obtained either from patients who required an investigation of their Hb because of the presence of an abnormal Hb component or from our reference collection of rare Hb variants, stored in liquid nitrogen. In all cases, the structural abnormality was checked by protein structure analysis (10). A flow diagram showing the techniques used in our laboratory to identify Hb variants is shown in Fig. 1 .



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  		Figure 1. Flow diagram showing the techniques used to evaluate a Hb variant.

When the electrophoretic data match for several Hbs, additional tests (e.g., reversed-phase HPLC, measurement of functional properties) may be performed before a structural determination is made. IEF agarose, CE-HPLC, citrate agar electrophoresis (CAE), and solubility tests are systematically performed on each sample in our laboratory—a strategy that aids detection of unusual variants, such as a common Hb with more than one substitution within a given chain.

hplc analysis
The Bio-Rad Variant, a fully automated HPLC system, uses double-wavelength detection (415 and 690 nm). Several elution methods, including specific columns, buffers, and softwares, are available from the manufacturer. The ß Thalassemia Short program, the most widely used Variant program, has been designed to separate and determine in 5–6 min the area percentages for HbA2 and HbF and to provide qualitative determinations of abnormal Hbs. Windows for retention times have been established for presumptive identification of the most commonly occurring Hb variants. In the ß Thalassemia Short program, a 3 x 0.46 cm nonporous cation-exchange column is eluted at a flow rate of 2 mL/min by a gradient of two phosphate buffers that differ in pH and ionic strength. The various Hb components give slight differences in elution time from one column to another and from one reagent batch to another. The elution time for a Hb component also varies slightly according to its concentration in the sample. We found that, for a given column, a more accurate calibration than that proposed by the manufacturer could be obtained by using HbA2 as a reference. This Hb is present only between narrow concentration limits, which prevents any significant modification of its elution time.

electrophoretic studies
The methods for electrophoretic analysis of Hbs included electrophoresis on cellulose acetate at alkaline pH, citrate agar electrophoresis, isoelectric focusing (IEF) of Hb, and electrophoresis of globin chains in 6 mol/L urea in Tris-EDTA buffer at pH 6.0 and 9.0 or in the presence of Triton X-100 (7)(8). In our laboratory, the electrophoretic mobilities of >350 Hb variants are available in a data bank under a format convenient for comparison. In this approach, IEF is expressed in mm from HbA, and the mobilities of the other electrophoretic systems are scaled by using several slow and fast-moving Hbs as references. Migration of the globin chains in urea is estimated from a scale in which the values for normal {alpha}- and ß-chain are +10 and +20, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
When the blood samples were prepared according to the manufacturer's instructions, we found that some Hb components that were present in low amounts (~1% of total Hb) were eluted together with HbF in the HbF retention time frame. These additional fractions, most probably reversibly glycated Hbs (11), disappeared when we hemolyzed the samples with a potassium hydrogen phthalate buffer instead of the Bio-Rad Hemolysis reagent (Fig. 2 ). This procedure for sample preparation is the same as that used for HPLC determination of HbA1c. Under these experimental conditions, the elution profile of the HbF window in the ß Thalassemia Short program was improved. It allowed an excellent agreement between chromatographic measurement of HbF, starting at <1%, and resistance of the variant to alkaline denaturation. Linear regression of a study involving 140 samples gave a slope of 1.07 (r = 0.963). Aged Hb specimens display some degraded products, which are eluted in the P2 and P3 windows.



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  		Figure 2. Elution profile of a hemolysate prepared (A) according to the manufacturer's instructions and (B) with potassium hydrogen phthalate–potassium cyanide buffer to improve resolution of the HbF window.

The small artifactual peak eluted at the position of HbF in A has disappeared in B. This method for sample preparation allows analysis of samples that contain a low amount of HbF or a weakly expressed mutant Hb eluting in the HbF window.

The most commonly occurring variants are HbS (ß6Glu->Val), HbC (ß6Glu->Lys), HbE (ß26Glu->Lys), and HbD-Punjab (ß121Glu->Gln). Presumptive identification of these abnormal Hbs is made by using the retention time windows "S-Window," "D-Window," "A2-Window,"and "C-Window" specified by the manufacturer. A study of the retention times of these common variants, determined in >200 samples, is shown in Table 1 . The actual elution time of these variants, if present in the hemolysate at a similar range of concentration, was highly reproducible in relation to that of HbA2 as a calibrator. Moreover, these Hbs were found to be eluted within a window of smaller size than that given as an example by the manufacturer. Nevertheless, the elution time of a given Hb component differs slightly according to its concentration in the hemolysate. We have observed this for adult Hbs in neonatal screening, or when the variant is unstable or associated with a thalassemic trait.


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  		Table 1. Elution times and "windows" for normal Hb and some of the more frequently occurring variants.1

As shown in Table 2 , some of the frequently encountered {alpha}- or ß-chain variants, for which the pI is identical or very close to that of HbS, may be easily recognized by CE-HPLC. This is important for differentiating the diagnosis, already suggested by electrophoresis, between HbD-Punjab and other HbD-like variants, which are frequently found in association with HbS but have opposite clinical consequences. Hb Korle Bu, the most common of these, elutes at the same position as HbA2, whereas HbD-Punjab elutes just after (Fig. 3 ). Hb variants with an identical amino acid substitution displayed elution times that varied from one case to another, as exemplified in Table 3 for those carrying a Glu->Lys substitution. The most extreme situation is that of HbE and HbC, which elute near HbA2 and at 5.0 min, respectively. As Fig. 4 shows, combining the IEF mobilities and the HPLC retention times unambiguously identifies all the abnormal Hbs belonging to this group.


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  		Table 2. Difference in the retention time, relative to HbA2, of variants with pI near that of Hb S.



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  		Figure 3. Elution pattern of hemolysates from patients carrying HbD-Punjab or Hb Korle Bu: (A) adult with HbA and HbD-Punjab; (B) adult with HbA and Hb Korle Bu; (C) newborn baby, composite heterozygote for HbS and Hb Korle Bu; and (D) patient, composite heterozygote for HbS and HbD-Punjab.


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  		Table 3. Difference in retention time, relative to HbA2, of variants carrying a Glu->Lys substitution.*



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  		Figure 4. CE-HPLC and IEF combined analysis of the Hb variants carrying the Glu->Lys amino acid substitution.

The elution times of several {alpha}- and ß-chain Hb variants, in comparison with those of HbA, HbF, and HbA2, and the position of the different windows are shown in Fig. 5 . To show in the same diagram the elution times observed with different columns or reagent batches, one must normalize the experimental data. A convenient way to do this is to convert the observed rough data into a scale wherein the reference value is the mean elution time of HbA2 given by the manufacturer.



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  		Figure 5. Order of elution of several normal and abnormal Hbs.

To eliminate between-column and between-(reagent)batch variabilities, we normalize the elution times in terms of the mean elution value for HbA2 given by the manufacturer (3.8 min).

Some Hbs that are difficult to distinguish from HbA electrophoretically, such as Hb Rainier, Alzette, or Puttelange, are clearly separated by HPLC. HbH, which it is important to recognize in {alpha}-thalassemic patients, elutes before the start of the standard integration program. Hb Constant Spring, an elongated thalassemic {alpha}-chain variant seen frequently in Southeast Asian populations, elutes in the C-window. The presence of an abnormal HbA2 eluting after the main abnormal peak is a convenient way to distinguish between {alpha}- or ß-chain variants that have identical elution times. The retention times of >125 different Hb variants are now available in our data bank.

The sensitivity of the method is as efficient as that of polyacrylamide gel IEF and allows recognition of an abnormal adult Hb in a blood sample obtained in a neonatal screening program for the main hemoglobinopathies. However, the concentration effect, which might slightly modify the retention time, has to be taken into account. Calibration of the elution time with a lesser amount of abnormal Hb (<10%) is required.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As of 1995, ~700 Hb mutations have been structurally characterized (12). Each year, our laboratory, which works as a reference center for abnormal Hbs, characterizes several hundreds of samples thought to contain a rare Hb variant. To avoid performing a complete structural determination by protein chemistry methods or by DNA sequencing for each unusual sample—which usually confirms a rare but already described variant—we have developed a multicriteria approach to reach a presumptive diagnosis. This strategy is based primarily on electrophoretic characterization of the variant in various experimental conditions by comparison with a data bank that contains references for the >350 variants we have observed. Including additional fast, reliable, and relatively simple biochemical methods may improve the electrophoresis approach. Recently, we proposed the use of reversed-phase perfusion chromatography for chain analysis as a complementary tool for the characterization of Hb variants (13). Additional considerations to take into account include the patient's ethnic origin (14) and clinical presentation and the functional properties of the erythrocytes or blood lysate. Measuring the molecular mass of the variant by electrospray mass spectrometry may indicate which kind of amino acid exchange has occurred (15).

Taken together, all of these informative results lead to a presumptive diagnosis that, even in the more exotic cases, is almost always found to be accurate. This was indeed the case for >100 cases, where we also checked the defect at the molecular level. Including another simple method such as the well-standardized CE-HPLC described here will certainly decrease the number of incomplete matches between the variant under investigation and a reference from the data bank and hence will minimize the need to work up an identification at the molecular level.


   Acknowledgments
 
We acknowledge the support of the Bio-Rad Co. in this work.


   Footnotes
 
1 Nonstandard abbreviations: Hb, hemoglobin; CE-HPLC, cation-exchange HPLC; and IEF, isoelectric focusing.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wilson JB, Headlee ME, Huisman THJ. A new high-performance liquid chromatographic procedure for the separation and quantitation of various hemoglobin variants in adults and newborn babies. J Lab Clin Med 1983;102:174-185. [Web of Science][Medline] [Order article via Infotrieve]
  2. Kutlar A, Kutlar F, Wilson JB, Headlee ME, Huisman THJ. Quantitation of hemoglobin components by high-performance cation-exchange liquid chromatography: its use in diagnosis and in the assessment of cellular distribution of hemoglobin variants. Am J Hematol 1984;17:39-53. [Web of Science][Medline] [Order article via Infotrieve]
  3. Rogers BB, Wessels RA, Ou CN, Buffone GJ. High-performance liquid chromatography in the diagnosis of hemoglobinopathies and thalassemias. Am J Clin Pathol 1985;84:671-674. [Web of Science][Medline] [Order article via Infotrieve]
  4. Samperi P, Mancuso GR, Dibenedetto SP, Di Cataldo A, Ragusa R, Schiliro G. High performance liquid chromatography (HPLC): a simple method to quantify HbC, O-Arab, Agenogi, and F. Clin Lab Haematol 1990;13:169-175.
  5. Shapira E, Miller VL, Miller JB, Qu Y. Sickle cell screening using a rapid automated HPLC system. Clin Chim Acta 1989;182:301-308. [Web of Science][Medline] [Order article via Infotrieve]
  6. Ou CN, Rognerud CL. Rapid analysis of hemoglobin variants by cation-exchange HPLC. Clin Chem 1993;39:820-824. [Abstract/Free Full Text]
  7. Papadea C, Cate JC. Identification and quantification of hemoglobins A, F, S, and C by automated chromatography. Clin Chem 1996;42:57-63. [Abstract/Free Full Text]
  8. Schneider RG, Barwick RC. Electrophoretic mobilities of mutant hemoglobins and mutant globin chains. In: Schmidt RM, Fairbanks VF, eds. CRC handbook series in clinical laboratory science. Section I: Hematology, Vol. 4. Boca Raton, FL: CRC Press, 1986:125–39..
  9. Lacombe C, Riou J, Godard C, Rosa J, Galacteros F. Characterization approach of "silent" beta-chain hemoglobin variants. Acta Haematol 1986;78:119-122.
  10. Wajcman H, Bardakdjian J, Ducrocq R. Structural characterization of abnormal hemoglobins from dried blood specimens in a neonatal screening program. Ann Biol Clin 1993;50:867-870.
  11. Tan GB, Aw TC, Dunstan RA, Lee SH. Evaluation of high-performance liquid chromatography for routine estimation of haemoglobins A2 and F. J Clin Pathol 1993;46:852-856. [Abstract/Free Full Text]
  12. International Hemoglobin Information Center. Variant list. Hemoglobin 1995;19:39–124..
  13. Wajcman H, Ducrocq R, Riou J, Mathis M, Godart C, Préhu C, Galacteros F. Perfusion chromatography on reversed-phase column allows fast analysis of human globin chains. Anal Biochem 1996;237:80-87. [Web of Science][Medline] [Order article via Infotrieve]
  14. Chami B, Braconnier F, Riou J, Bardakdjian-Michau J, Préhu C, Blouquit Y, et al. Geographic distribution of 119 alleles of the {alpha} and ß globin genes detected in 432 French Caucasian carriers of haemoglobin variant. Ann Génét 1995;38:206-216. [Web of Science][Medline] [Order article via Infotrieve]
  15. Lacombe C, Promé D, Blouquit Y, Bardakdjian J, Arous N, Bost M, et al. New results of hemoglobin variant structure determinations by fast atom bombardment mass spectrometry. Hemoglobin 1990;14:529-548. [Web of Science][Medline] [Order article via Infotrieve]



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