High pressure liquid chromatography and electrospray ionization mass spectrometry are advantageously integrated into a two-levels approach to detection and identification of haemoglobin variants

Abstract

Detecting and correctly identifying haemoglobin (Hb) variants is typically achieved by a two-levels laboratory approach. We report our experience in dealing with 91 Hb variants, including a number of frequent and a few rare variants. Screening included akaline agarose gel electrophoresis (AGE), ion-exchange automated high-performance liquid chromatography (HPLC) and a test for deoxyhaemoglobin solubility. Identification was based on electrospray ionization-mass spectrometry (ESI-MS). Our results confirmed the advantages of HPLC over AGE for screening, because of the occurrence of some electrophoretically 'silent' variants. ESI-MS permitted the definitive identification of 90 of the 91 variants included in the study, in some cases (e.g. HbS) through the application of a simple protocol (direct injection of the sample), in other cases requiring the application of more demanding procedures (purification of the variant chain and peptide analysis after enzymatic or chemical cleavage). In an additional case (Hb J-Oxford), ESI-MS assay did not lead to definitive identification, but gave indications for designing the appropriate primers to focus DNA sequence analysis on the specific region of the gene. Deoxyhaemoglobin solubility test was positive only in the presence of HbS. We conclude that HPLC and ESI-MS are advantageously integrated into a two-level analytical system for the detection and confirmation of variant Hbs.

Separation of haemoglobin HbE and HbA2 by the fully automated, high‐pressure liquid chromatography Tosoh HLC‐723 G7 analyzer

“…In the recent years low‐pressure liquid chromatography and HPLC instruments specifically devised for separating Hb fractions have been increasingly employed by the hospital laboratories, particularly for the monitoring of diabetes (Chambers, Phillips & Chapman, 1993; Caruso et al. , 2005).…”

“…Moreover, because of the β‐thal legacy affecting some European areas, several clinical laboratories of the most relevant European hospitals are equipped with additional suitable instrumentation and methods to diagnose such syndromes. Generally, the presumptive identification of the principal Hb alterations is made at the peripheral hospitals equipped with only HPLC, and the samples are then sent to the major centres for the definitive diagnosis (Caruso et al. , 2005).…”

Triagem neonatal para hemoglobinopatias: experiência de um ano na rede de saúde pública do Rio Grande do Sul, Brasil

“…Em nosso estudo, encontramos três pais com o perfil Hb SC e uma mãe com perfil Hb SS, que puderam realizar tratamento para aliviar sintomas e melhorar sua qualidade de vida. Além disso, a presença de 14 amostras classificadas como variantes demonstra a existência de outras hemoglobinas em nosso meio, ainda não identificadas corretamente pela falta de estudos mais avançados, tais como reação da cadeia polimerase, seqüenciamento e espectrometria de massa 19,20,21 .…”

The First Case of Hb G-Honolulu [α30(B11)Glu→Gln (GAG>CAG); HBA2:c.91G>A] Observed in Association with Hb S [β6(A3)Glu→Val, GAG>GTG] in a Healthy Italian Child

“…The solubility test for Hb S detection was performed according to standard laboratory procedures (8). Identification of the Hb variants was performed by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) (9). A red cell lysate was prepared, treating washed erythrocytes with 1 volume of water and 0.4 volumes of carbon tetrachloride.…”