High performance liquid chromatography is the diagnostic method of choice for haemoglobin variants in a large number of laboratories worldwide. It is able to detect and resolve a large number of haemoglobin variants, especially when used in conjunction with another method of screening / identification (e.g. haemoglobin electrophoresis). In addition, it is able to quantify the amount of variant haemoglobin present, which itself is a useful parameter in identification.
Method:
- Exchange column containing small sphreres of silica which are modified to be weakly cationic
- Anticoagulated specimen is lysed and diluted in buffer, then injected into column
- Hb is absorbed into column and eluted by means of a gradient of increasing ionic strength
- Eluate passes through a photometer which measures changes in absorbance
Reasons to use:
- Major discriminatory value:
- Separates haemoglobin S from Hb Lepore, Hb D-Punjab and Hb G-Philadelphia
- Separates haemoglobin E from Hb C, Hb O-Arab
- Pitfalls: cannot discriminate between HbA2 and HbE
Typical Retention Times:
Window / Peak | Retention Time (min) | Causes / Variant Haemoglobin |
---|---|---|
P1 | 0.63 – 0.85 | Bilirubin, haemoglobin H, haemoglobin Bart’s |
F | 0.98 – 1.2 | Haemoglobin F |
P2 | 1.24 – 1.4 | Glycated haemoglobin, haemoglobin Hope |
P3 | 1.4 – 1.9 | Post-translationally modified haemoglobin A, haemoglobin J-Oxford |
A | 1.3 – 3.1 | Haemoglobin A, glycated S, Köln |
A2 | 3.3 – 3.9 | Haemoglobin A2, E, Lepore |
D | 3.9 – 4.3 | Haemoglobin D-Punjab, G-Philadelphia |
S | 4.3 – 4.7 | Haemoglobin S, Q-Thailand |
C | 4.9 – 5.3 | Haemoglobin C, O-Arab |
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