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. 1993 Sep;46(9):852–856. doi: 10.1136/jcp.46.9.852

Evaluation of high performance liquid chromatography for routine estimation of haemoglobins A2 and F.

G B Tan 1, T C Aw 1, R A Dunstan 1, S H Lee 1
PMCID: PMC501524  PMID: 7693766

Abstract

AIMS--To compare high performance liquid chromatography (HPLC) with conventional methods for the estimation of blood haemoglobin A2 (HbA2) and haemoglobin F (HbF) concentrations in routine thalassemia screening. METHODS--An HPLC system (the VARIANT Hemoglobin Testing System) was tested for precision and reproducibility in the measurement of HbA2 and HbF, and reference ranges were obtained for a local healthy adult population. HPLC was compared with column anion exchange chromatography for HbA2 measurement, and radial immunodiffusion, or alkaline denaturation for HbF measurement. The reliability of HbA2 measurement by HPLC for the detection of beta thalassaemia and HbE was assessed in 200 consecutive samples for routine thalassaemia screening. RESULTS--HPLC was rapid, technically easy, and gave good precision and reproducibility. In all comparisons linear regression analysis showed good correlation between HbA2 or HbF concentrations determined by HPLC and by the respective conventional methods. All beta thalassaemia or haemoglobin E carriers presumptively identified by conventional methods in 200 consecutive samples were detected by HbA2 measurement using HPLC, without any false positive or false negative results. CONCLUSIONS--The measurement of HbA2 and HbF by HPLC is rapid, reproducible, and precise. It is as reliable as column chromatography for the measurement of HbA2 and radial immunodiffusion or alkaline denaturation for the measurement of HbF. HPLC may be an appropriate method for rapid screening in population surveys for beta thalassaemia and HbE carriers.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson H. M., Ranney H. M. Southeast Asian immigrants: the new thalassemias in Americans. Semin Hematol. 1990 Jul;27(3):239–246. [PubMed] [Google Scholar]
  2. BETKE K., MARTI H. R., SCHLICHT I. Estimation of small percentages of foetal haemoglobin. Nature. 1959 Dec 12;184(Suppl 24):1877–1878. doi: 10.1038/1841877a0. [DOI] [PubMed] [Google Scholar]
  3. Deacon-Smith R., Lord I. Haemoglobin A2 measurement using high performance liquid chromatography. Med Lab Sci. 1992 Jun;49(2):138–140. [PubMed] [Google Scholar]
  4. Huisman T. H., Gardiner M. B., Wilson J. B. Experiences with the quantitation of human hemoglobin types by high pressure liquid chromatography. Prog Clin Biol Res. 1981;60:69–82. [PubMed] [Google Scholar]
  5. Kazazian H. H., Jr The thalassemia syndromes: molecular basis and prenatal diagnosis in 1990. Semin Hematol. 1990 Jul;27(3):209–228. [PubMed] [Google Scholar]
  6. Kutlar A., Kutlar F., Gu L. G., Mayson S. M., Huisman T. H. Fetal hemoglobin in normal adults and beta-thalassemia heterozygotes. Hum Genet. 1990 Jun;85(1):106–110. doi: 10.1007/BF00276333. [DOI] [PubMed] [Google Scholar]
  7. Kutlar A., Kutlar F., Wilson J. B., Headlee M. G., Huisman T. H. 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 Jul;17(1):39–53. doi: 10.1002/ajh.2830170106. [DOI] [PubMed] [Google Scholar]
  8. Rogers B. B., Wessels R. A., Ou C. N., Buffone G. J. High-performance liquid chromatography in the diagnosis of hemoglobinopathies and thalassemias. Report of three cases. Am J Clin Pathol. 1985 Nov;84(5):671–674. doi: 10.1093/ajcp/84.5.671. [DOI] [PubMed] [Google Scholar]
  9. Samperi P., Mancuso G. R., Dibenedetto S. P., Di Cataldo A., Ragusa R., Schilirò G. High performance liquid chromatography (HPLC): a simple method to quantify Hb C, O-Arab, Agenogi and F. Clin Lab Haematol. 1991;13(2):169–175. doi: 10.1111/j.1365-2257.1991.tb00266.x. [DOI] [PubMed] [Google Scholar]
  10. Steinberg M. H., Adams J. G., 3rd Hemoglobin A2: origin, evolution, and aftermath. Blood. 1991 Nov 1;78(9):2165–2177. [PubMed] [Google Scholar]
  11. Wilson J. B., Headlee M. E., Huisman T. H. 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 Aug;102(2):174–186. [PubMed] [Google Scholar]

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