Thalassemia is one of the most heterogeneous diseases, with more than a thousand mutation types recorded worldwide. Molecular diagnosis of thalassemia by conventional PCR-based DNA analysis is time- and resource-consuming owing to the phenotype variability, disease complexity, and molecular diagnostic test limitations. Moreover, genetic counseling must be backed-up by an extensive diagnosis of the thalassemia-causing phenotype and the possible genetic modifiers. Data coming from advanced molecular techniques such as targeted sequencing by next-generation sequencing (NGS) and third-generation sequencing (TGS) are more appropriate and valuable for DNA analysis of thalassemia. While NGS is superior at variant calling to TGS thanks to its lower error rates, the longer reads nature of the TGS permits haplotype-phasing that is superior for variant discovery on the homologous genes and CNV calling. The emergence of many cutting-edge machine learning-based bioinformatics tools has improved the accuracy of variant and CNV calling. Constant improvement of these sequencing and bioinformatics will enable precise thalassemia detections, especially for the CNV and the homologous HBA and HBG genes. In conclusion, laboratory transiting from conventional DNA analysis to NGS or TGS and following the guidelines towards a single assay will contribute to a better diagnostics approach of thalassemia.

Alpha thalassemia (α-thalassemia) is an autosomal recessive disorder due to the reduction or absence of α globin chain production. Laboratory diagnosis of α-thalassemia requires molecular analysis for the confirmatory diagnosis. A screening test, comprising complete blood count, blood smear and hemoglobin quantification by high performance liquid chromatography and capillary electrophoresis, may not possibly detect all the thalassemia diseases. This review focused on the molecular techniques used to detect α-thalassemia, and the advantages and disadvantages of each technique were highlighted. Multiplex gap-polymerase chain reaction, single-tube multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, and loop-mediated isothermal amplification were used to detect common deletion of α-thalassemia. Furthermore, the reverse dot blot analysis and a single tube multiplex polymerase chain reaction could detect non-deletion mutation of the α-globin gene. Sanger sequencing is widely used to detect non-deletion mutations of α-thalassemia. Recently, next-generation sequencing was introduced in the diagnosis of both deletion and point mutations of α-thalassemia. Despite the advantages and disadvantages of different techniques, the routine method employed in the laboratory should be based on the facility, expertise, available equipment, and economic conditions.

Mutations of JAK2V617F, CALR, and MPL genes confirm the diagnosis of myeloproliferative neoplasm (MPN). This study aims to determine the genetic profile of JAK2V617F, CALR exon 9 Type 1 (52 bp deletion) and Type 2 (5 bp insertion), and MPL W515 L/K genes among Malaysian patients and correlate these mutations with clinical and hematologic parameters in MPN. Mutations of JAK2V617F, CALR, and MPL were analyzed in 159 Malaysian patients using allele-specific polymerase chain reaction, including 76 polycythemia vera (PV), 41 essential thrombocythemia (ET), and 42 primary myelofibrosis (PMF) mutations, and the demographics of the patients were retrieved. The result showed that 73.6% JAK2V617F, 5.66% CALR, and 27.7% were triple-negative mutations. No MPL W515L/K mutation was detected. In ET and PMF, the predominance type was the CALR Type 1 mutation. In JAK2V617F mutant patients, serum LDH was significantly higher in PMF compared to PV and ET. PV has a higher risk of evolving to post PV myelofibrosis compared to ET. A thrombotic event at initial diagnosis of 40.9% was high compared to global incidence. Only one PMF patient had a CALR mutation that transformed to acute myeloid leukemia. JAK2V617F and CALR mutations play an important role in diagnostics. Hence, every patient suspected of having a myeloproliferative neoplasm should be screened for these mutations.

Anemia is a condition in which red blood cells and/or hemoglobin (Hb) concentrations are decreased below the normal range, resulting in a lack of oxygen being transported to tissues and organs. Those afflicted with this condition may feel lethargic and weak, which reduces their quality of life. The condition may be manifested in inherited blood disorders, such as thalassemia and sickle cell disease, whereas acquired disorders include aplastic anemia, chronic disease, drug toxicity, pregnancy, and nutritional deficiency. The augmentation of fetal hemoglobin (HbF) results in the reduction in clinical symptoms in beta-hemoglobinopathies. Several transcription factors as well as medications such as hydroxyurea may help red blood cells produce more HbF. HbF expression increases with the downregulation of three main quantitative trait loci, namely, the XMN1-HBG2, HBS1L-MYB, and BCL11A genes. These genes contain single nucleotide polymorphisms (SNPs) that modulate the expression of HbF differently in various populations. Allele discrimination is important in SNP genotyping and is widely applied in many assays. In conclusion, the expression of HbF with a genetic modifier is crucial in determining the severity of anemic diseases, and genetic modification of HbF expression may offer clinical benefits in diagnosis and disease management.

The antiphospholipid syndrome (APS) manifests through venous or arterial thrombosis, with or without pregnancy complication alongside the continuous presence of antiphospholipid antibodies (aPL). APS classification relies on three aPL subtypes: anticardiolipin (aCL), anti-β2-glycoprotein I antibodies (anti-β2GPI), and lupus anticoagulants (LA) antibodies. Given that thrombosis and pregnancy issues are not unique to APS, the precise and reliable identification of aPL forms the basis for diagnosis. Semi-quantitative solid-phase assays identify two antibodies, aCL and anti-β2GPI, while LA detection occurs through various phospholipid-dependent coagulation assays that are based on antibody behaviour. LA, specifically, is conclusively associated with thrombosis, prompting discussions around the serological criteria for APS. Despite advancements in LA detection, the standardisation of all aPL detection assays remains imperative. The combined presence of aCL and anti-β2GPI with thrombosis inconsistently triggers concern. Initial presentations by APS patients commonly exhibit a heightened risk of stroke, miscarriages in the later stages of pregnancy, positive results of LA tests, and widespread thrombosis across multiple organs, often leading to adverse outcomes. Correctly diagnosing this condition is pivotal to avoid unnecessary long-term secondary thromboprophylaxis.