Various PCR-based methods have increased the accuracy of blood group typing and effectively resolved certain complications of conventional serological methods (13). These include conventional PCR, real-time PCR (qPCR), multiplex PCR, and digital droplet PCR (ddPCR). These methods utilize designed primers specific to known alleles to amplify DNA fragments, Then the results are analyzed through gel electrophoresis or restriction enzyme digestion (13). Among these, real-time PCR enables the rapid and accurate identification of blood group antigens by detecting polymorphism responsible for antigen expression (13). This technique uses fluorescently labeled probes designed to bind specific DNA sequences. During amplification, Taq polymerase cleaves the probe, releasing a fluorescent signal that is proportional to the amount of target DNA, thereby enabling both qualitative detection and quantitative analysis (14). The high sensitivity and specificity of this method make it well-suited for genotyping applications (14, 15).

Multiplex PCR has been used to simultaneously amplify several target DNA sequences in a single reaction by employing multiple primer sets. This approach facilitates the detection and analysis of multiple genes or genetic variations, saving time, reagents, and effort compared to performing separate individual reactions (16). In this method, two reaction mixtures, containing allele-specific primers and the DNA fragments for different blood group antigens (Duffy, Kidd, Rh, Diego, and MNS) are amplified (17). The technique has been validated against standard serology and polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) and proved to be valid and suitable for supplying antigen-matched units to transfusion-dependent patients (17).

Digital droplet PCR (ddPCR) is another sensitive and specific technique used in blood group genotyping. Unlike traditional PCR, wherein the amplification is done on bulk DNA, ddPCR allows for partitioning a sample into thousands or millions of nanodroplets, by which DNA can be individually amplified and quantified on a single-molecule basis (18). This method enables the detection of low-frequency variants and minor alleles, making it especially useful for transfusion-dependent populations (19). ddPCR has the advantage of accuracy in mixed populations, effectively solving challenges associated with multiply transfused patients (19).

MALDI-TOF MS is a highly accurate analytical technique that is used to measure the mass of DNA fragments. In this method specific primers will bind to the polymorphic site and will be extended by DNA polymerase by adding one or a few nucleotides complementary to the target sequence. Then the amplified extension products are ionized by laser, electrostatically accelerated, and separated based on their mass-to-charge ratio (m/z), facilitating accurate differentiation between variants (20). To improve reliability, it is often combined with PCR-SSP, a targeted approach in which primers amplify DNA only when a specific allele is present. The integration of MALDI-TOF with PCR-SSP, provides a powerful strategy for identifying both common and rare blood group variants (21).

In transfusion medicine, Microarray provides the molecular resolution needed to define blood group antigens, thus assessing transfusion compatibility, minimizing adverse reactions, and enhancing transfusion safety (22). Single Nucleotide Polymorphisms (SNPs), defined as single-base variations in the DNA sequence, and can be detected using SNP array-based genotyping. In this method, DNA is hybridized to immobilized oligonucleotide probes on chips allowing detection of multiple SNPs simultaneously. This technique is valuable in cases where serological methods are inconclusive or unavailable. However, most commercially available SNP arrays are limited by the number of loci and often fail to capture many transfusion-relevant variants (22). To overcome these challenges, a specialized genome-wide SNP array, called the transfusion medicine array (TM-Array), was developed using the Affymetrix Axiom platform to facilitate the comprehensive study of both common and rare transfusion-associated variants across genetically diverse donor and recipient populations. The TM-Array includes probes with targets involved in RBC and platelet structure and function, the human leukocyte antigen (HLA) system, and blood groups, including human platelet antigen (HPA) (22). The Affymetrix Axiom platform is a hybridization-based genotyping array that offers a flexible choice of predesigned modules and customizable content. The TM-Array features approximately 1.38 million hybridization probes, representing about 879,000 SNPs and copy number polymorphism markers (22). The array demonstrated exceptional technical performance, with over 99% of SNPs reliably genotyped, as well as strong reproducibility, showing an error rate below 0.03%. It also exhibited excellent parent–child trio accuracy of 99.97%. Blood group genotyping results were in concordance with serological findings, and the array could detect rare alleles with a minor allele frequency as low as 0.5% (22).

NGS has been utilized for analyzing genome variations, including SNPs, structural variations, and copy number variations (23). NGS has the advantage of enabling the complete genotyping of many blood group systems in a single test, allowing for the detection of both common and rare variants (23). NGS has successfully characterized complex genomic configurations in systems like Rh and MNS, which are often difficult to interpret using conventional methods (24). By utilizing high-throughput sequencing technologies, NGS facilitates complete analysis of the entire blood group gene loci, improving accuracy and resolution in transfusion-related genotyping (25). In the context of NGS-based genotyping, massively parallel sequencing, also known as high-throughput sequencing, has been used for profiling full blood group genotypes, allowing for the identification of known and novel genetic variants affecting blood group antigens (26). This sequencing method is based on fragmenting genomic DNA, then sequencing it in parallel and aligning the resulting reads to a reference genome for variant detection (26). This approach enables the discovery of novel polymorphisms that cannot be detected by microarray or traditional genotyping methods (26). Short-read NGS has been applied to whole-genome blood group genotyping (27). For instance, the Ion Torrent system, employing NGS technology, detects nucleotide incorporation by measuring pH changes during DNA synthesis (28). This allows for real-time and label-free sequencing. It provides reads with an average insert size of 200 base pairs, suitable for targeted sequence amplification and the sequencing of specific genome regions, enabling an efficient and cost-effective focus on clinically relevant variants (28). Whole-genome sequencing, used for the genetic analysis of variants associated with the Kidd, Duffy, and Kell blood group systems, was performed by generating 150 bp paired-end reads with 30X coverage depth employing Illumina technology, thus revealing all known and novel variants across the genome (29). These findings highlight the potential of NGS technologies to improve transfusion safety through more accurate and individualized blood group genotyping (29).

A CRISPR/Cas13a-based SNP was developed to detect specific ABO types, allowing for the resolution of weak and subgroup alleles. Unlike the widely known CRISPR/Cas9 gene-editing system which uses Cas9 endonuclease to target DNA, the CRISPR/Cas13a system targets RNA molecules. In this method, DNA from the ABO gene was amplified using PCR. Then the amplified DNA was transcribed into RNA, making it a target for Cas13a. A specifically designed guide RNA (gRNA) was used to recognize the RNA sequence with the SNP site, the Cas13a activates and cleaves the RNA target. This cleavage will produce fluorescent signal that confirms the presence of the SNP. This approach demonstrated accurate ABO genotyping with a sensitivity of approximately 50 pg. per reaction and a detection time of 60 min (30).

Blood group antigen expression holds significance in transfusion medicine, organ transplantation, and the understanding of disease susceptibility (43). The regulation of blood group antigen expression is determined by specific genes and their encoded glycosyltransferases (44).

Blood group antigens are found on the surface of RBCs and other cell types and are essential for immunological recognition (44). Obtaining knowledge on their expression and silencing and using techniques such as gene knockdown or knockout can significantly affect transfusion medicine (12). Blood group systems such as ABO, Rh, Kell, and Duffy are encoded by specific loci that impact glycoprotein and glycolipid synthesis on the cell surface (45). The ABO gene encodes glycosyltransferases, forming an ABO system (8). Genetic variations in this region, especially single nucleotide polymorphisms (SNPs), can affect enzyme activity and lead to variations in antigen expression levels (44, 46). For instance, alterations in the RHD gene, such as SNPs and hybrid alleles can alter antigen expression and give rise to weak D, partial D, or Del phenotypes (47).

Techniques like RNAi can assist in the selective suppression of the specific blood group antigens, for example, targeting the ABO gene to reduce its expression and lowering the risks linked with ABO incompatibility during transplantation (48, 49).

Moreover, overexpressing antigens such as ABO RhD in cell models helps in understanding the immunogenic and biochemical pathways involved in their synthesis (49). In addition, studies on ABH antigen regulation help explain the disappearance of ABH antigens during carcinogenesis and highlighted potential therapeutic targets in ABO-mismatched organ transplantation (45).

A pressing challenge in transfusion medicine is the availability of RBCs with extremely rare phenotypes (50), such as Rh-null (in which Rh antigens are absent from RBC membranes) (50). Rh-null blood cells are important for transfusion in patients with the same phenotype and for the diagnosis of Rh alloimmunization, especially in complicated cases like pregnancy (50).

Recent advances have focused on the production of RBCs from different cell sources. Human-induced pluripotent stem cells (hiPSCs) can be used as they offer an unlimited number of hemopoietic progenitor cells that can differentiate into erythroid cells (51), which can be produced from peripheral blood mononuclear cells and modified through gene editing, thus offering a novel system to produce customized red cells (51). Cells derived from hiPSCs could remedy the shortage in situations where rare blood types like Rh-null and Kell-negative phenotypes would provide greater compatibility for patients with alloimmunization (52, 53).

Induced pluripotent stem cells (iPSCs) have been generated from the dermal fibroblasts of an individual with a Bombay blood phenotype using Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc). These iPSCs were established to be pluripotent and capable of significant cellular differentiation into various types. Furthermore, the hiPSCs maintained the genetic mutations associated with the Bombay phenotype, notably, the FUT1 and FUT2 gene mutations. Cells differentiated from hiPSCs can efficiently produce hematopoietic lineage cells positive for markers such as CD34 and Runx1, along with the erythroid markers α-globin and γ-globin. The abovementioned study represents a step toward the production of universal donor RBCs (53).

Park et al. (54) established the generation of autologous iPSC-derived RBCs from peripheral blood mononuclear cells without chromosomal mutations, as shown in Figure 1. Another group of researchers also established hiPSCs from the primary bone marrow CD34 + cells of an O-negative blood donor (53). Table 2 summarizes these technologies and their usage.

miRNAs have been recognized as vital modulators of blood group antigens (62). miRNAs are small, non-coding RNAs used for post-transcriptional gene regulation that regulate the expression of blood group antigens on RBCs and other cell types (63). In transfusion medicine, miRNA-based therapeutic approaches have been studied to reduce the risks of alloimmunization and enhance compatibility (64, 65). Kronstein-Wiedemann et al. (12) determined the role of miRNAs in the regulation of the expression of ABO blood group antigens during erythropoiesis. They found that miR-331-3p and miR-1908-5p directly regulate glycosyltransferase A and B mRNA, as increasing expression of these miRNAs in hematopoietic stem cells significantly reduced A and B antigen expression on RBCs (12, 45). The Rhesus protein RhAG is regulated by miR-9. RhAG was one among 170 putative target genes with an inverse correlation with miR-9 expression of miR-9 (66). The expression of the KLF1 protein was also downregulated upon the overexpression of miR-326 and increased expression of γ-globin. This decreased the amounts of Lutheran and other blood group antigens (the Indian, P1PK, Landsteiner-Wiener, Knops, OK, RAPH, and I blood group systems) (67). In autoimmune hemolytic anemia, silencing miRNAs involved in autoantibody production showed potential in the reduction of hemolysis (68). Moreover, downregulation of the miRNAs that target RhD expression may reduce the immune response in sensitized Rh-negative individuals, resulting in better transfusion outcomes (12).

In transplantation, miRNA-based interventions have been used to control the expression of ABO and HLA antigens on donor tissues to reduce organ rejection risk (68). For example, silencing miR-150, which directly regulates erythropoiesis, was found to reduce immunogenic antigen levels, thus reducing the immune reaction (69, 70). Another example is miR-223, whose effects on HLA antigen expression on megakaryocytes could reduce HLA-mediated alloimmune responses and thereby improve the compatibility of platelet transfusions for patients suffering refractory thrombocytopenia (58, 71). Silencing miR-181a in transplantation models reduces the rates of graft rejection by decreasing the immunogenicity of transplanted tissues. This miRNA has been shown to regulate HLA-DR antigen expression on donor tissues (72). miR-451 is involved in erythrocyte maturation and the expression of blood group antigens. Its downregulation may improve the survival and compatibility of transfused RBCs by minimizing the effect of immune-mediated clearance (73).