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Molecular Diagnostics and Genetics |
Departments of1
Pathology and
2 Pediatrics, New York University School of Medicine and Bellevue Hospital Center, New York, NY 10016.
aAuthor for correspondence. Fax 212-263-8099; e-mail michael.nardi{at}med.nyu.edu.
Background: Previous evaluations of HPLC as a tool for detection of hemoglobin variants have done so within newborn-screening programs and/or by use of stored samples. We describe a 32-month prospective study in a clinical diagnostic laboratory in which we evaluated the imprecision of HPLC retention times and determined the retention times for hemoglobin variants seen in a multiethnic setting.
Methods: We analyzed all samples on the Bio-Rad Variant II HPLC system. For normal hemoglobin fractions and hemoglobin variants, we recorded and analyzed their retention times, their proportion of the total hemoglobin (%), and the peak characteristics. We compared the imprecision of retention time with the imprecision of retention time normalized to the retention time of hemoglobin A0 (HbA0) and to the retention time of HbA2. Alkaline and acid hemoglobin electrophoresis, and in certain cases globin chain electrophoresis, isoelectric focusing, and DNA analysis, were performed to document the identities of the hemoglobin variants.
Results: The mean (SD) imprecision (CV) of the retention time was 1.0 (0.7)% with no statistical difference compared with the imprecision for normalized retention times. Among 60293 samples tested, we encountered 34 unique hemoglobin variants and 2 tetramers. Eighteen variants and 2 tetramers could be identified solely by retention time and 3 variants by retention time and proportion of total hemoglobin. Four variants could be identified by retention time and peak characteristics and eight variants by retention time and electrophoretic mobility. One variant (HbNew York) was missed on HPLC. Retention time on HPLC was superior to electrophoresis for the differentiation and identification of six members of the HbJ family, four members of the HbD family, and three variants with electrophoretic mobilities identical or similar to that of HbC. Six variants with electrophoretic mobilities identical or similar to that of HbS could be differentiated and identified by retention time and proportion of total hemoglobin. HPLC detected two variants (HbTy Gard and HbTwin Peaks) missed on electrophoresis.
Conclusions: The retention time on HPLC is reliable, reproducible, and in many cases superior to conventional hemoglobin electrophoresis for the detection and identification of hemoglobin variants. Confirmatory testing by electrophoresis can be eliminated in the majority of cases by use of retention time, proportion of total hemoglobin, and peak characteristics of HPLC.
The following articles in journals at HighWire Press have cited this article:
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  B. S. Shackley, T. A. Drake, and A. W. Butch Chronic Microcytic Anemia and Jaundice in a 36-Year-Old Male of Burmese Descent Lab Med, February 1, 2010; 41(2): 78 - 82. [Full Text] [PDF]
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 D. D. Mais, R. D. Gulbranson, and D. F. Keren The Range of Hemoglobin A2 in Hemoglobin E Heterozygotes as Determined by Capillary Electrophoresis Am J Clin Pathol, July 1, 2009; 132(1): 34 - 38. [Abstract] [Full Text] [PDF]
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 E. K. O'Keeffe, M. M. Rhodes, and A. Woodworth A Patient with a Previous Diagnosis of Hemoglobin S/C Disease with an Unusually Severe Disease Course Clin. Chem., June 1, 2009; 55(6): 1228 - 1231. [Full Text] [PDF]
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 H. D. Edwards, H. Sabio, and Z. K. Shihabi Rapid Quantitation of Hemoglobin S in Sickle Cell Patients Using the Variant II Turbo Analyzer Ann. Clin. Lab. Sci., January 1, 2009; 39(1): 32 - 37. [Abstract] [Full Text] [PDF]
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P. Kleinert, M. Schmid, K. Zurbriggen, O. Speer, M. Schmugge, B. Roschitzki, S. S. Durka, U. Leopold, T. Kuster, C. W. Heizmann, et al. Mass Spectrometry: A Tool for Enhanced Detection of Hemoglobin Variants Clin. Chem., January 1, 2008; 54(1): 69 - 76. [Abstract] [Full Text] [PDF]
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L. B. Thomas, S. J. Agosti, M. A. Man, and S. M. Mastorides Screening for Hemoglobinopathies During Routine Hemoglobin A1c Testing Using the Tosoh G7 Glycohemoglobin Analyzer Ann. Clin. Lab. Sci., January 1, 2007; 37(3): 251 - 255. [Abstract] [Full Text] [PDF]
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J. D. Hoyer and R. M. Scheidt Identification of Hemoglobin Variants by HPLC Clin. Chem., July 1, 2005; 51(7): 1303 - 1304. [Full Text] [PDF]
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A. Joutovsky, J. Hadzi-Nesic, and M. A. Nardi Identification of Hemoglobin Variants by HPLC - Reply Clin. Chem., July 1, 2005; 51(7): 1305 - 1305. [Full Text] [PDF]
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