| | Advantages | Disadvantages |
| Protein-based methods | - Extensive clinical experience
- Widely available
| - Not possible to make a definitive diagnosis of alpha or beta thalassemia
|
| High performance liquid chromatography (HPLC) | - Fully automated
- High precision
- Rapid, high throughput (short run times)
- Only a very small sample (eg, fraction of a blood spot for newborn screening) is required
- Can identify and quantify many variant hemoglobins
- Can identify and quantify normal hemoglobins (including Hb F, Hb A2, and Hb Barts)
- Information from the pattern of variant peaks guides further testing
| - Capital cost (equipment and reagents)
- Interpretation of results requires technical skill, experience
- Interference/interpretation of results affected by prematurity (in newborn), recent transfusion
- Decreased resolution with sample age due to Hb degradation
- Cannot distinguish Hb E, Hb Korle Bu, Hb Lepore from Hb A2 due to overlapping retention times
|
| Isoelectric focusing (IEF) | - Excellent resolution of common Hb variants and Hb Barts
- Distinguishes Hb E from Hb O and Hb S from Hb D and Hb G
- Hb A and Hb F are clearly resolved
- Requires only small sample
| - Cannot precisely quantify Hb variants
- Higher per test cost than electrophoresis
- Methemoglobin and glycosylated Hb appear as separate bands with sample age
- Bands on gel are less sharp with automated compared with manual IEF
|
| Capillary electrophoresis | - Fully automated
- High-throughput
- Identifies common Hb variants
- Separates Hb A2 from Hb E (unlike HPLC)
- Precise quantification of Hbs
- Information from zone pattern of variants guides further testing
- Complementary to HPLC
| - Capital cost (equipment, reagents)
- Requires technical skill, expertise
- Poor separation of Hb S from Hb D
- Electrophoresis zones are shifted in the absence of Hb A
- Decreased resolution with sample age
- Minimal sample required (1 mL), but if sample is <1 mL, dilution of other samples is required, adding labor
|
| Electrophoresis (cellulose acetate, citrate agar) | - Low cost
- Separation of major Hbs (Hb A, Hb F, Hb S/D, Hb C/E/O-Arab) and several less common Hb variants
- Acid pH (citrate agar) resolves Hb S and Hb C
| - Poor separation of Hbs other than Hbs A, F, S, C
- Cannot distinguish Hb E from Hb O, or Hb D from Hb G
- Labor intensive
|
| DNA-based methods | - Determines genotype
- Diagnostic for thalassemias
| - Cost is higher than most protein-based techniques
|
| Gap polymerase chain reaction (PCR) | - Identification of common alpha thalassemia deletions
- Identification of beta thalassemia and HPFH deletions
- Identification of alpha globin gene duplications
- Can be multiplexed to test several deletions in single assay
| - Testing is limited to selected/predefined deletions (cannot detect unknown deletions)
|
| Traditional DNA sequencing (Sanger) | - Identifies a spectrum of point mutations and small insertions and deletions (indels) that result in a hemoglobinopathy (thalassemia, Hb variants)
| - Cannot identify large deletions or duplications
- Only one DNA sequence (up to 1 kb) can be obtained at a time
|
| Multiplex ligation-dependent probe amplification (MLPA) | - Detects numerous deletions, duplications, and rearrangements of the alpha- and beta-globin gene loci in a single, multiplex assay
| - Capital cost, kits, reagents, software
- Requires technical skill, expertise
- If deletion breakpoints need to be further defined, additional DNA sequencing is needed
|
| Next-generation DNA sequencing (NGS) | - Simultaneous identification of complete spectrum of deletions, duplications, point mutations in alpha and beta globin genes (single parallel assay)
- Requires very small sample (less than that for Sanger sequencing)
- Rapid
- High accuracy, reliability
| - Capital cost, reagents (but less expensive than Sanger sequencing)
- Requires technical skill, expertise
|
| Allele-specific oligonucleotide testing (ASOT) | | - Testing is limited to selected/predefined mutations
|
| Restriction fragment length polymorphism (RFLP) testing | | - Testing is limited to selected/predefined mutations
|
