Microchromatographic methods for hemoglobin A2 quantitation compared.

Abstract

On 20 consecutive work days during four weeks, one technologist performed 24 microchromatographic determinations of hemoglobin A2 (Hb A2) by each of four methods: the Efremov procedure requiring Tris/HCl buffer, the original Huisman technique with use of glycine developer, and two commercial test kits in which a modified glycine developer is used. The bloood samples tested were obtained from 12 adults with no hematological abnormality and from 12 beta-thalassemia carriers previously diagnosed by familial and hematologic studies. Results by the first method and the two commercial kits (one from Helena Laboratories and one from Isolab, Inc.) did not differ significantly in precision for either the normal or beta-thalassemia trait samples. For both sample types, the second method yielded larger coefficients of variation than those obtained with the other methods. Moreover, the second method was the only one with which values overlapped for normal samples and samples with above-normal Hb A2 concentrations.

Interlaboratory comparison of current high‐performance methods for HbA2

“…With regard to the imprecision, the data obtained in our study proven that all methods were performing better than 4.5% CV, as also recently reported (Anagnostopoulos et al. , 2009), thus confirming that the quality of the automated HPLC and CE methods is higher with respect to the imprecision obtainable years ago with the minicolumns methods (Brosius et al. , 1978), or by cellulose acetate electrophoresis followed by densitometry, or after elution of the HbA and HbA 2 bands and spectrophotometric quantitation (International Committee for Standardization in Haemathology 1978).…”

“…With regard to the imprecision, the data obtained in our study proven that all methods were performing better than 4.5% CV, as also recently reported (Anagnostopoulos et al, 2009), thus confirming that the quality of the automated HPLC and CE methods is higher with respect to the imprecision obtainable years ago with the minicolumns methods (Brosius et al, 1978), or by cellulose acetate electrophoresis followed by densitometry, or after elution of the HbA and HbA 2 bands and spectrophotometric quantitation (International Committee for Standardization in Haemathology 1978). However, from a recent study we have performed under rigorous conditions in agreement with the approach recommended (Fraser & Harris, 1989), HbA 2 seems to have a very narrow intra-individual biological variability, of the order of 0.4% (as CV), as for a typical genetic marker (Paleari et al, 2011).…”

Thalassemia Syndrome

“…The relative amounts of these Hbs can be estimated by calculating the peak areas of the absorbance, measured at 415 nm, of fractions eluted from the column. It has been shown that the results obtained from the batchwise micro-column are in agreement with those from HPLC, though the method lacks automation and yields lower precision (Dozy et al, 1968, Brosious et al, 1978, Srisawang et al, 2003. However, the result from ion exchange micro-column technique is acceptably accurate and precise and can be used to confirm some types of thalassemias such as βthalassemia trait.…”