Detection and quantitation of normal and variant haemoglobins: an analytical review

Abstract

As well as six 'normal' haemoglobins that occur at various stages of development, more than 800 abnormal or variant haemoglobins have been described. Many of these variant haemoglobins have no significant clinical consequences apart from causing confusion to clinicians and in laboratories; however, some of the variant haemoglobins result in major morbidity or mortality. The laboratory challenge is to detect these clinically significant haemoglobins and to identify them with sufficient accuracy for clinical purposes, as well as to quantitate both these and the 'normal' haemoglobins. The techniques used to detect and quantitate these haemoglobins in routine service laboratories are discussed in detail. Methods used by referral laboratories, such as mass spectrometry and DNA analysis, are briefly discussed. Haemoglobin analysis is most often undertaken as part of neonatal, antenatal or pre-anaesthetic screening; these programmes are reviewed, together with possible changes to neonatal screening and antenatal screening that may occur as part of the NHS National Plan.

Assessment of malaria in pregnancy using rapid diagnostic tests and its association with HIV infection and hematologic parameters in South-Eastern Nigeria

“…The hemoglobin genotype was determined by the Hemoglobin electrophoresis technique at alkaline pH using cellulose acetate membrane (CAM). 4 The ABO blood grouping test was performed using the slide method 2 with commercially available reagents (Murex Diagnostics, Inc. Dartford, UK). The HIV Tri Line Test kits (Biosystem INC, Vienna, Austria) were first used to screen maternal serum samples and to detect antibodies to HIV-1 and HIV-2.…”

Quantification of Hemoglobin A2 by Tandem Mass Spectrometry

“…The prepared peptides are stable for at least 6 days at 10°C. A further advantage of directly targeting ␦ peptides by MSMS, in addition to speed and sensitivity, is the ability to detect and accurately quantify ␦ protein even in the presence of confounding factors which may prevent or interfere with HbA 2 quantification using traditional methods (2 ). These include variants that coelute with HbA 2 (e.g., HbE) or, in some formats, give rise to falsely increased HbA 2 values (e.g., HbS).…”

“…Initially, population screening programs were restricted to the use of erythrocyte indices (2 ), but screening for HbA 2 has been facilitated by use of automated HPLC of intact Hbs. Although MSMS appears an unlikely technique for accurately measuring HbA 2 , an ␣ 2 ␦ 2 tetramer, we postulated that the relative proportion of ␦-and ␤-globins should be equivalent to the proportion of HbA 2 .…”

Determination of unique amino acid substitutions in protein variants by peptide mass mapping with FT-ICR MS

“…A typical example is Hb, a number of mutations of which have been characterized by MS [2,8,14,15]. Analysis of the mass of intact proteins is useful for detecting mutations in small proteins such as globins, but the determination of the type and position of such a mutation awaits peptide mass mapping of an enzymatic digest.…”