Blood Grouping and Typing Methods: Current Insights and Recent Advancements

Abstract

Blood grouping is a vital process in transfusion medicine. Usually, blood grouping is performed using serological techniques based on antigen–antibody interactions such as test tube method that is the gold standard method for grouping of blood. Blood grouping reagents are crucial instruments in immunohematology, for identifying blood group antigens on red blood cells, such as Rh and ABO. In this review, we have outlined various blood grouping systems and several techniques for blood grouping including test tubes, gel cards, rapid cards, and more recent technologies like molecular imprinting, ultrasound backscattering along with blood group genotyping, molecular typing of blood cells based on SNV and real-time PCR are discussed. The role of current computational techniques in blood group prediction methods for blood group determination is also included in the review. With the advances in bioinformatics, many new processes have been developed that predicts blood group antigens directly from genetic sequences through (computational) methods that are based on genome sequencing, and structural bioinformatics, thereby providing high-throughput, accurate, and cost-effective alternatives to traditional tube testing methods

Keywords

Blood Grouping, Blood Grouping Systems, Transfusion, Genotyping

Introduction

 

 

 

 

 

 

 

Gel Card Method

The gel technique for determination of blood group is based on the principle described by Yves Lapierre on the red blood cell agglutination reactions. A typical gel card is composed of 6-8 microtubes/wells, each having gel suspended in isotonic buffer containing specific antisera, Eg. Anti-A, Anti-B, Anti AB, Anti-D or any other antisera. When red blood cell suspension is added through the opening at the top of each well, the agglutination occurs if the red blood cell antigens react with the corresponding antisera, present in the gel suspension or in the serum or plasma sample (in the case of reverse grouping test). The gel column acts as a strainer that blocks the agglutinated red blood cells on the top of the well of the gel cards upon centrifugation of the entire gel card. However, all the negative cells that have not agglutinated with the antisera pass through the gel column upon centrifugation of the card. The agglutinated red blood cells get captured at the top of the well whereas the non-agglutinated red blood cells settle down at the bottom of the well forming a pellet. ²³

 

Microtitre Plate Method

In this method, Grouping is carried out in micro wells as it is quick, very simple, cost-effective and easy to read, infer and verify the hemagglutination reactions. These are composed of rigid polystyrene microtiter plates containing 96 U or v shaped bottom wells and the plates are covered to prevent evaporation and are incubated 10 - 30 min at room temperature. After centrifugation 30 - 60 sec at 100xg, using a centrifuge adapter for plates, the plates are agitated by a shaker for 10 - 30 sec, to resuspend the cells completely. The agglutination reactions are read from the bottom of the plate by a reading device with an illuminated and magnifying mirror. The advantages of the microtiter hemagglutination method is clearly evident by rapid detection. This method is sensitive and ideal for large number of samples.²⁴

Rapid Blood Grouping Cards

These are type of rapid detection device in which the absorbant paper strip is impregnated with a matrix of antibodies and dye. When a drop of blood is applied, lines of colour develop as the blood spreads across the strip and reacts with the antibodies. Such type of test is based on the principle of lateral flow guided by capillary action. Reagents are pre-dried with the appropriate antisera and packed as an easy-to-use device that can be self-performed easily. However, it is only useful for the preliminary testing and the confirmation may require advanced laboratory tests. The autocontrol usually present serves as a negative control that does not contain any antibodies in Control well (Ctrl) that validates the test results. ²⁵

Solid Phase Red Cell Adherence (SPRCA)

Also known as the Pad system, it is the paper-based blood grouping systems. In such paper-based analytical device the antisera are immobilized antigens on the absorbant material for simultaneous determination of ABO and Rh blood groups. This kit is made of two different sheets of paper having two different zones. The reverse side has a blood separation zone combined with Whatman paper to separate plasma and serum for reverse grouping. Only monoclonal antisera can be used as DAT samples with such kind of pad systems. ¹⁰

Automated Blood Bank Test System

The Blood Center can cater the needs of the patients by focusing on specific blood components during donation with the help of automated blood bank system. Component separation is made easier with the adoption of numerous automated blood bank testing systems. High-quality blood collection that is appropriate for blood component preparation is facilitated by automatic electronic blood collection monitors (BCM) or blood mixers. In order to address patient needs as soon as possible, automated blood donation enables the Blood Center to obtain specific blood components during donation. Red blood cells (RBC), platelets, and plasma are separated using semiautomatic machinery, which has the advantage of removing leucocytes from blood components.²⁶

In an Apheresis, the healthy donor's blood is drawn by a process cell separator machine, which separates the required component and returns the remaining blood to the donor. It is possible to obtain collect separate blood components from the donor, including peripheral blood stem cells, plasma, RBC, platelets, leukocytes and neocytes. Amongst numerous benefits, these devices can offer larger product quantities, due to which a component's entire effective dose is administered all at once. Moreover, there is less exposure to many donors, which lowers the risk of transfusion-transmitted illnesses and alloimmunization. In addition, the product's quality and purity are superior to those of the manually gathered component. ²⁶ . In one of the studies conducted by Shin Et al, it was found that ABO/Rh(D) typing a single sample with an automated analyzer had a greater overall cost than testing multiple samples at once, but it was less expensive than testing a single sample manually. The entire price of an unexpected antibody. Compared to the manual method, screening with an automated analyzer was lower.²⁷ A number of factors, including rising transfusion demand, donor safety and selection, donor base changes, product purity, numerous components, product quality, and economics, influences the need for or desirability of RBCs through apheresis or other automated methods.²⁸. An automated blood processor can help further standardize the processing of blood components, which will improve the quality of blood products used in patient treatment.

RECENT METHODS FOR BLOOD GROUPING

Since last few decades, the test tube method blood typing relies on serological assays that detect antigen–antibody reactions. While being effective, these methods have limitations such as reagent dependency, inability to detect weak or rare antigens, and difficulties in patients who have recently received transfusions. Few of the newer techniques are being discussed here

Blood Typing by Plastic Microinjection Moulding

Reaction microchambers, flow splitting microchannels, chaotic micromixers, and detection microfilters are all fully integrated into the biochip. The flow splitting microchannel can split the loaded sample blood into two or four equal amounts so that two or four blood agglutination tests can be performed simultaneously. To effectively react agglutinogens on red blood cells (RBCs) with agglutinins in serum, a serpentine laminating micromixer was incorporated into the biochip. Chaotic advection and splitting/recombination are two chaotic mixing techniques that are combined in this micromixer. Additionally, relatively large area reaction microchambers were used to maintain the mixing of the sample blood and serum during the reaction phase before filtering.²⁹.

Ultrasound Backscattering—Quantitative Measurements of Agglutinates

This novel method is based on ultrasonic backscattering blood group typing. In an acoustic heterogeneous medium, specific alterations in the direction, phase, amplitude, and velocity of sound waves are observed on the red cells without the conversion of mechanical energy into heat. The volume and acoustic properties (elasticity, viscosity, and density) of the red cell surface affect the degree and angle of the dispersed energy. The Doppler effect can be used to record the scattering characteristics of agglutinated red blood cells in comparison to non-agglutinated cells.³⁰

Human Blood Group Typing Based on Digital Photographs of RBC Agglutination Process

A technique based on the In vitro agglutination of human red blood cells has been developed for identifying human blood types (groups). The statistical analysis of digital images captured during the agglutination process forms the basis of this technique. Experimental evidence suggests that these images can be used to assess the probability that the erythrocytes in the blood sample would agglutinate in response to the related iso-hemagglutinating sera. The biological specimen is exposed to ultrasonic waves in order to speed up the erythrocyte agglutination process and increase the method's sensitivity, as previously suggested by the authors. After that, the agglutination reaction's outcomes are assessed visually.³¹

Molecular imprinting: Synthetic materials as substitutes for biological antibodies and receptors.

The versatile technique of molecular imprinting creates useful materials that can identify and, in some cases, react to specific chemical and biological substances of interest. The synthesis of molecularly imprinted polymers (MIPs) is guided by templates. As a result, it would be more accurate to characterize molecular imprinting as a method founded on "rational design," which permits the resolution of problems pertaining to research and applications. Material scientists may now produce unique synthetic molecules with greatly increased stability using basic chemical building blocks, which can either complement or replace natural receptors. ³²

It has been demonstrated by Piletsky, S. S., et al. that natural antibodies in blood group analysis can be replaced with molecularly imprinted nanoparticles specific to blood antigens. Instead of using the current agglutination-based theories, this method is based on magnetically-induced decolorization.³³.

Molecular imprinting is one of the most significant contemporary techniques for blood group typing because the synthetic antibodies it produces have similar sensitivity and selectivity to natural antibodies, are easy to make, and can be reused for several tests with sufficient stability. These features make synthetic antibodies superior choices for blood typing and other modern biosensor technologies. ³

 

 

Blood Group Genotyping

The process of identifying blood group characteristics from specific DNA sequences is known as blood group genotyping. Genotyping is sometimes known as blood group genomic testing or molecular blood grouping. These days, phenotyping methods are less common than genotyping. For instance, information from genomic testing is more precise and dependable if blood group phenotypic information is required but a particular red cell sample is not available; genomic testing is also favored since it might be less costly than serological approaches.³⁴.

The genes for the majority of the pertinent blood group systems have been defined as a result of human genome sequencing, and the polymorphisms causing the majority of the clinically significant blood group antigens have been identified. When erythrocytes are unavailable or when serological testing is either impossible or inconclusive due to a lack of antisera, molecular blood group typing is utilized. Additionally, in some circumstances, molecular testing could be more economical. The majority of molecular typing techniques rely on DNA hybridization, DNA sequencing, or PCR using certain primers. In particular, the shift from Sanger-based sequencing to next-generation sequencing (NGS) technologies has created intriguing new opportunities for blood group genotyping.³⁵. Next-generation sequencing, sometimes referred to as massively parallel sequencing, has a high throughput capacity and maps every point of variation from a reference sequence, making it possible to find new SNVs. In general, serological phenotyping and the identification of a complete profile of blood group SNVs serve as a foundation for the supply of compatible blood, improving transfusion safety. SNVs are the most common genetic alteration linked to blood type antigens. SNV mapping, which entails highly multiplexed genotyping, can be carried out on commercial microarray technologies to predict blood group antigen phenotypes. ³⁶

The International Society of Blood Transfusion (ISBT) has identified 48 blood group systems, and single nucleotide variations (SNVs) provide the molecular basis for the expression of all blood type antigens. Numerous DNA assays for blood group antigen prediction were developed using this as their basis. Many clinical troubleshooting problems that hemagglutination was unable to resolve have been resolved by molecular typing of blood type genes in diagnostics. They are essential for determining antigen types for which typing reagents are unavailable.

Patients with warm autoantibodies or those who have recently received a transfusion should be specifically typed; blood group variations should be identified; prenatal testing should be conducted; rare blood types should be sought after; and repositories of antigen-negative red blood cells (RBCs) for transfusion should be made more dependable. Molecular typing of blood group genes is recommended as part of the antibody identification procedure for patients who are transfusion dependent. This is due to the fact that by determining the patient's anticipated phenotype, the lab can ascertain which antigens the patient can and cannot react to in order to generate alloantibodies. Furthermore, molecular typing increases precision and provides more details about the patient's antigenic profile, especially when serological reagents such as unusual blood types and Dombrock (DO) antigens are not available. ^{37 38}
The power of next-generation sequencing (NGS) of complete genomes or exomes, or by concentrating on specific blood type loci in conjunction with pretransfusion serologic testing, will improve immunohematology in routine transfusion practice.
Real-time quantitative PCR is one medium-throughput PCR technique for SNP analysis and allele discrimination. Perhaps the most important use of real-time quantitative PCR in immunohaematology is the identification and amplification of the RHD gene from free fetal DNA taken from maternal plasma. The presence of freely circulating fetal DNA in mothers For more than a decade, high-throughput single nucleotide polymorphism-based blood type microarray technology has been available. It was initially developed for the analysis of gene expression in both sick and healthy tissues.

This method uses SNP microarrays with synthetic oligonucleotide probes that contain the desired mutation. A solid phase, such as a glass slide, is where the probes are affixed.
The blood group gene around the target mutation is amplified by PCR prior to the products being hybridized with the probe. After rigorous cleaning procedures to ensure specificity, a scanning device analyzes the signals and software evaluates the reactions.
The cost and reimbursement of molecular diagnostics have hindered their widespread usage. However, the cost of molecular detection can be significantly reduced by processing a large number of samples per response using a centralized testing methodology and evaluating many polymorphisms concurrently. Since the introduction of short-read NGS techniques, sequencing costs have also dropped. It is important to keep in mind that molecular testing would only need to be performed once in a lifetime and may be included in the patient transfusion record. ³⁹

COMPUTATIONAL APPROACHES FOR BLOOD GROUPING

With the advancement of Bioinformatics, genomics and computational approaches have emerged as powerful tools for predicting blood group phenotypes from genetic information. In-silico analysis enables researchers and clinicians to interpret DNA sequence data to identify blood group alleles and predict antigen expression and reduced reagent dependency.

A novel, safe, all-inclusive web server algorithm called RBCeq has been developed to effectively evaluate NGS data in the context of transfusion medicine applications. As web server-based blood group genotyping software, it can characterize both known blood group alleles and potential novel alleles that can reduce and silence antigen expression. This solves the computational and storage issues related to processing large raw NGS datasets. It is difficult and time-consuming to analyze NGS data to forecast blood types; RBCeq fills the gaps in this field and will make it easier to apply and translate this technology to enhance patient and blood donor safety. ⁴⁰

BloodAGENT is an open-source, flexible tool for determining blood type alleles and evaluating genetic diversity. Under the normal circumstances, bloodAGENT obtains good concordance in blood group allele determination. The findings of this tool demonstrate the resilience of bloodAGENT and its capacity for managing intricate genomic analysis.⁴¹

For thorough and high-throughput RH genotyping from WGS data, Chang et al. designed a computational approach called RHtyper. This method can be applied to patients who already have WGS data and can detect a variety of RH genetic variants found in SCD patients. Currently, the identification of antibodies and the selection of donor units for Rh alloimmunized patients are aided by knowledge of each patient's unique RH genotype. Further investigation is required to ascertain the immunogenicity of particular RH variant alleles in order to improve red cell matching by genotype for SCD patients.⁴²

With its capabilities for data analysis, pattern recognition, and workflow optimization, artificial intelligence (AI) has grown in importance as a tool in the healthcare industry. AI in transfusion medicine (TM) tackles issues such patient safety, inventory management, complex immunohematology, and donor recruitment and retention. Clients can improve decision-making, efficiency, and risk reduction by integrating technologies such as machine learning (ML), deep learning, and natural language processing (NLP). The advent of High-throughput computational tools has enabled detection of rare alleles with high accuracy. With the advancements in bioinformatics in blood grouping techniques, which support blood group genotyping, the costs of data processing, storage, and analysis will lower the costs of the sequencing, reducing the overall expenses of the process. However, the bioinformatics algorithms that enable sequencing and blood group characterization techniques need to be optimized, as personal genome sequencing is expected to become more common in the near future.Without improvements to the bioinformatics underlying blood group genotyping, the costs associated with data processing, storage, and analysis will exceed the costs of the sequencing itself, thereby reducing the economic viability of such a strategy. As personal genome sequencing is forecast to become increasingly prevalent in the near future, there is a clear need for the optimization of the bioinformatics algorithms underlying sequencing and blood group characterization approaches.

Blood typing by modern computational techniques helps identify compatible donors and recipients, especially for patients requiring repeated transfusions. Moreover, such analysis can detect rare or novel variants that may not be identifiable through serology. It can remarkable investigation in blood group distribution and genetic diversity among diverse populations. Genomic blood typing supports precision medicine by predicting individual blood group profiles.

CONCLUSION

Blood grouping methods have progressed from simple agglutination-based tests to advanced automated and molecular systems. While conventional serological methods remain essential, future developments—especially genotyping, microfluidics, and AI integration—are transforming transfusion medicine toward greater precision, speed, and safety. There are many alternative technologies documented in the literature to identify the agglutination reactions because visual inspection is no longer suitable for collecting exact quantitative information. Modern biosensors and molecular blood type techniques have also been taken into consideration for simple, accurate, and exact analysis, despite the traditional approaches.

It is widely claimed that novel multiplexing methods combined with current blood testing assays will be the future testing algorithms used in blood centres. In transfusion medicine, DNA microarray technology aids in the discovery of novel genes and advances our understanding of infectious diseases. It provides a potentially useful tool for high throughput platelet and red cell antigen genotyping. Future immunohaematology labs may be dominated by this automated method for extended blood group genotyping with multiplex analysis capability.

Though the computational techniques for blood group determination have a few some limitations of specialized infrastructure requirements, Dependency on high-quality sequencing data and Inadequate knowledge of rare variants, Computational bioinformatics approaches have revolutionized blood group determination by enabling genotype-based prediction of blood group antigens. These methods provide a powerful complement to traditional serological testing, particularly for identifying rare variants and understanding antigen structure and function. Continued advancements in sequencing technologies, databases, and computational algorithms will further improve the reliability and clinical applicability of genomic blood typing in transfusion medicine. Large-scale genomic databases and improved algorithms will enable real-time blood typing and facilitate global rare donor registries.

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