Betaglobin StripAssay IME from Vienna Lab

Thalassemia is an inherited blood disorder in which the body makes an abnormal form of hemoglobin, the protein in red blood cells that carries oxygen. The disorder results in excessive destruction of red blood cells, which leads to anemia. Hemoglobin is made of two proteins: Alpha globin and beta globin. Beta-thalassemia occurs when a gene or genes related to the beta-globin protein are missing or changed (mutated). The best and most sensitive way to diagnose beta-thalassemia is by DNA analysis. DNA analysis can performed using a variety of methods; some labs use qPCR and high resolution melt (HRM), others use Amplification Refractory Mutation System (ARMS) PCR, but in my opinion, the best method of diagnosis of beta-thalassemia is the Betaglobin StripAssay® from Vienna Lab. This is an in vitro diagnostic (IVD) test, which doesn’t need any pre-validation. Based on reverse-hybridization of biotinylated PCR products, it covers 22 mutations, greater than 96% of relevant mutations (they have an exclusive strip for India). Also, no carcinogenic substances, like ethidium bromide, are required.
The kit contains the following components which are characterized into 3 divisions: Extraction (Lysis Solution, GenXTRACT Resin), Amplification (Dilution buffer, Amplification Mix; Taq Polymerase is not supplied in the kit), Hybridization & Washing (Denaturing solution (“DNAT”), Typing Trays, TestStrips, Hybridization Buffer, Wash Solution A, Conjugate Solution, Wash Solution B, Color Developer).

The Betaglobin StripAssay is based on the reverse-hybridization principle, and includes three successive steps. First, the DNA is isolated from anticoagulated blood. Extraction requires just 100 uL of whole blood (fresh/frozen). Next, incubate the blood with the Lysis Solution for 15 minutes and then centrifuge (for 5 minutes at 3000 rpm). Remove and discard the supernatant, add another 1 ml lysis solution and centrifuge again (5 min at 12,000 rpm). Remove and discard the supernatant, add the GenXTRACT Resin to the pellet (GenXTRACT Resin will act as a binding medium), incubate at 56°C for 20 minutes and then at 98°C for 10 minutes. Finally, centrifuge (5 minutes at 12,000 rpm) and take the supernatant for PCR amplification. The entire extraction process does not take more than 80 minutes.

Following DNA extraction, a multiplex PCR reaction is performed which amplifies the relevant betaglobin genes. An important recommendation is the DNA concentration should not exceed 200 ng/reaction. The amplification products are hybridized to biotinylated, oligonucleotide probes on the test strip. This hybridization step requires a couple of dedicated instruments: a water bath with a shaking platform and adjustable temperature (45°C ± 0.5°C), and an orbital shaker. The hybridization has a couple of incubations and takes 120 minutes. Samples can be processed using the typing trays; each typing tray has 6 reservoirs allowing you to process 6 samples simultaneously. The bound biotinylated sequences are then detected using streptavidin-alkaline phosphatase and color-generating substrates.

Results can be interpreted easily with the naked eye; no documentation system is needed. A positive reaction of the uppermost Control line indicates the correct function of Conjugate Solution and Color Developer. This line should always stain positive. The entire protocol takes 6 hours. Possible results: normal genotype, heterozygous genotype or homozygous mutant genotype.