Human Blood Groups: Clinical Importance in Transfusion Medicine

2.1 Foundations and clinical applications of human blood groups in “non-malignant”, “benign” hematology

In patients with hemoglobinopathies, sickle cell anemia, and aplastic anemia, it is of paramount importance to avoid any alloimmunization because, in addition to requiring long-term supportive transfusion therapy, some of them also undergo allo-HSCT as a treatment method.

Sickle cell anemia (SCD) is an inherited disorder with a genetic basis, characterized by the presence of mutant beta globin. The mutation occurs in the HBB gene, which in turn leads to the production of abnormal hemoglobin S. Since they have an abnormal shape, sickle-shaped red cells block blood flow, resulting in organ damage. According to data from the World Health Organization (WHO) [2] for 2021, approximately 7.74 million people worldwide have SCD (August 6, 2025). Although the disease is not typical for Bulgaria and other regions at this stage, it is unclear what challenges the future will bring due to increasing population migration. The clinically significant blood group systems in this disease are determined by the immunogenic activity of the antigens. Conrath S. et al. have described an alloimmunization incidence ranging from 20% to 50% [3] to antigens outside the ABO system. This is due to genetic polymorphisms in erythrocyte antigens between donors and recipients. Different variants of expression exist for both the D and C antigens. This requires that, in cases of serologically established weak antigen expression and until the genetic variant is clarified, blood that is negative for the respective antigen be transfused. In emergency situations, the time available to secure safe blood is extremely important. While serological methods for determining ABO/D and Rh phenotypes take 15–20 minutes, genetic (molecular) testing may take several days. In patients with SCD, the first priority is to determine the phenotype with regard to strong immunogens outside the ABO system and to transfuse a phenotypically compatible blood product. In the absence of time for pre-transfusion screening for antierythrocyte antibodies, transfusion of phenotypically matched red cell concentrates (RCCs) is part of the safety measures for transfusion therapy.

The next places in the alphabet of clinically significant blood group antigens are occupied by the antigens of the Kell, Kidd, and MNSs blood group systems. Kell antibodies, which are immune antibodies, can cause both extremely severe post-transfusion reactions and severe, even fatal, hemolytic disease of the fetus. Despite all the modern possibilities of fetal medicine and intrauterine exchange transfusion, prevention remains paramount in relation to Kell HDF.

The Jk^a (Kidd) antigen, our erythrocyte urea transporter, is responsible for delayed hemolytic transfusion reactions if the patient is immunized. Barouqa M, et al. [4] described a clinical case of a patient with SCD and serologically proven Jk^a alloantibodies, with immunization established in a variant of the Jk^a antigen with missing epitopes. This is evidence that serological methods for antigen typing will not always answer the question of immunization – whether it is the result of autoimmunity or an alloimmune process. We must not forget that once anti-Jk^a alloantibodies have appeared, even if they are not subsequently detected, they can be reactivated and rapidly depleted in the antigen-antibody reaction during transfusion of Jk^a (+) positive blood. In this case, despite the negative result of pre-transfusion antibody screening, it is very likely that they are present. One of the reasons they are not detected is that the antibodies are below the sensitivity threshold of serological techniques. They have the ability to activate the complement system, so it is unclear how severe a post-transfusion reaction may be. Liang, S. et al. have proven that the reason for the weakened expression of the antigen in the Jk^a variant is a result of promoter polymorphisms of the variant allele [5]. Given the high prevalence of the Jk^a antigen – 77% in the Caucasian race, over 90% in Black people, and over 70% in the Asian race – it is not an easy task to quickly find antigen-negative blood.

Among the hemoglobinopathies, thalassemia, which affects about 5% of the world’s population, has a reported average frequency of 15–22 per 100,000 people [6]. Migration has led to changes in the epidemiology of this hereditary disease. The severe form of β-thalassemia – the major form, transfusion-dependent β-thalassemia (TDT), requires lifelong supportive transfusion therapy. Allo-HSCT and gene therapy, which have the potential to cure the disease and change this transfusion dependence, have their indications and are not applicable to absolutely all patients. More important than treatment are screening programs for the prevention of the severe form of the disease. In our country, the carrier frequency of the β-thalassemia gene is 2.4–2.5% [7]. Approximately 170,000 Bulgarians are carriers of the β-thalassemia gene. In our medical facility, the Department of Disorders of Hemostasis and the Department of Thalassemia are registered with Orhanet Bulgaria and are part of EuroBloodNet. Although the disease is classified as rare, there are cases in which emergency situations arise, requiring a good understanding of transfusion policy in the diagnosis of thalassemia. Improved blood quality makes it possible to extend survival with a good quality of life. Gianesin, B. et al., in their first nationwide study, found an overall median survival in TDT of 71.2 years, and in SCD an overall median survival of 73.4 years [8]. Since TDT requires monthly maintenance transfusion therapy, a key factor is to minimize the risk of alloimmunization to erythrocyte antigens. In the 5th Edition Guidelines for the management of TDT by the Thalassemia International Federation [9], the recommendations are that blood transfusions should be consistent with the ABO, Rh, and Kell blood group systems. Serological phenotyping is performed before the first blood transfusion, and if one has already been performed, blood group antigen typing is done by molecular (genetic) testing. New technologies, such as pathogen inactivation of blood components and the production of universal blood, require time for additional research, refinement of methods, and assessment of cost and benefits, which necessitates knowledge of the extent of all risks associated with blood transfusion in these patients. Given the frequency of alloimmunization in thalassemia, which Franceschi L. et al. report to be 11.4%, but with varying ranges from 2.9% to 37% in different studies [10], this means that blood transfusions must first and foremost be compatible with clinically significant immunogens such as Rh and Kell antigens. Furthermore, when blood transfusions begin in early childhood, the risk of alloimmunization decreases, which can be explained by the fact that a not fully developed childhood immune system is capable of developing tolerance to foreign antigens.

The main challenge in transfusion therapy is rare Rh phenotypes, such as ccDEE, which present us with the dilemma of choosing ABO-nonidentical but phenotypically compatible blood. Given that leukocyte-depleted RCCs are also produced in additive solutions, which remove as much donor plasma as possible, this means that this ABO-nonidentical but compatible blood carries virtually no risk of a hemolytic transfusion reaction. The reason is that when donor plasma is removed, donor agglutinins are also removed. This represents the optimal alternative to low-titer RCCs (anti-A/B <1:16) for ABO-compatible, nonidentical transfusion therapy. In addition, additive solutions prolong the survival of allogeneic red cells, which leads to longer intervals between transfusions. Any infection, pregnancy, or childbirth is a factor that increases transfusion needs in patients with thalassemia. In this case, however, it is more important to meet these needs in a timely manner than to wait for ABO-identical blood with the corresponding recipient phenotype, which is rare. The leading blood group systems in this case are Rh (with all antigens except D, such as CcEe) and Kell.

The C^w antigen, which is rare in populations but immunogenic, is a factor in the formation of clinically significant antibodies that are active at 37 °C, including in the indirect antiglobulin test. In addition to their immune origin, anti-C^w alloantibodies also occur naturally, but with the accompanying risk of hemolytic complications (post-transfusion reaction, hemolytic disease of the fetus). A meta-analysis by Indriani V. et al. found that the frequency of anti-C^w alloimmunization is below 0.5% (0.26%) in thalassemia [11], but it is possible that it may be higher in different populations. Regardless of the fact that pre-transfusion screening for antibodies becomes negative after a certain time interval because antigen-negative blood has been transfused, the rules require the transfusion of RCC that do not contain the antigen to be continued. The low prevalence logically allows us to find C^w – negative blood easily and quickly. Regarding genetic (molecular) characteristics, it is important to note that it is most commonly found in haplotypes that contain C and D antigens and can very rarely be detected if these antigens are absent. It is important to prevent any risk associated with previous alloimmunization.

In the Kell blood group system, with more than 30 known antigens, the KEL 1 antigen is of greatest clinical significance due to its strong immunogenicity. Alloimmunization to KEL 1 (K) is a barrier to emergency blood transfusion if there is no time to perform all pre-transfusion immunohematological tests (screening for erythrocyte antibodies in auto – and allo-systems, crossmatch test between recipient and donor), and at the same time the patient is alloimmunized and has antibodies. The K antigen is present on fetal erythrocytes from as early as 10 weeks of gestation, which makes it a risk factor for severe and sometimes even fatal HDF. In addition to fetal anemia, it can also provoke thrombocytopenia, as it is proven on precursors and the megakaryocyte line. For this reason, we must not forget that Kell (-) negative blood must be transfused to all women of childbearing age, as well as to all transfusion-dependent patients. Given the prevalence (no higher than 20% for different races), Kell (-) negative blood components are easily available. Franceschi L. et al. [10] report a 25.6% prevalence of Kell alloimmunization in TDT. Unlike KEL 1, the k antigen (Cellano – KEL2) has an extremely high population prevalence (over 99%), which is why only isolated cases of alloimmunization have been described. However, it is extremely difficult to find homozygous K (+) positive blood. Serological testing with only a K test reagent is not sufficient to prove the absence of Cellano. To establish homozygosity for the K antigen, testing with an anti-Cellano test reagent is mandatory. Considering that globally, Cellano (-) negative blood donors account for less than 1% of the population, finding suitable blood in such situations is a real challenge.

The Kp^a (KEL3) antigen has a low prevalence (2% in Caucasians), but once alloantibodies appear, they are always clinically significant because, in addition to acute and delayed hemolytic reactions, they also cause HDFN. In addition to being immune, anti-Kp^a alloantibodies can also be natural. Since they are of the IgG immunoglobulin class (active in the indirect Coombs test), the crossmatch test is sufficient to ensure compatible blood if it is impossible to test the donor’s antigen. For each specific situation and for each specific patient, the safest transfusion approach is selected, which, however, requires knowledge of the clinically significant immunogens from the blood group systems in transfusion medicine.

From the MNS blood group system, anti-M alloantibodies are clinically significant for transfusion practice, especially in cases where they are active according to the IAT. In addition to being immune, they can also be natural antibodies, which, in rare cases, cause HDFN. In a study by Gholamrezazade A. et al. [12], in which 104 patients with thalassemia were genotyped, the following frequencies of MNS alleles were found: MNSs alleles incidence – 25%, MNss-23%, MMSs-21%, while MMSS and MMss were 9% each. They also found that the lowest allele frequencies were for the following combinations of antigens: NNss-8%, MNSS-only 2%, and NNSS, NNSs was 1% each. Alloimmunization with respect to Ss antigens also has clinical significance. Based on the frequency of these antigens, an alloimmunized patient requires sufficient time to find a suitable blood unit. Once again, we may encounter the dilemma of transfusing ABO-nonidentical (compatible) but Rh, Kell, and MNSs antigen-compatible blood.

Aplastic anemia is a rare disease which, according to data from the International Foundation for Aplastic Anemia and MDS [13], is diagnosed in 600–900 people in the U.S. each year. The presence of severe pancytopenia, the occurrence of hemorrhagic complications, and immunosuppressive treatment with antithymocyte globulin (ATG) pose a serious challenge for supportive transfusion therapy if it is needed in large volumes, and the patient has a rare blood type and phenotype – for example, AB/Rh D (+) positive with Rh phenotype CCDee. The leading factors for choosing a transfusion policy for this diagnosis are age, gender, and whether Allo-HSCT is being considered as part of the individual treatment plan. Despite the use of leukocyte-depleted blood components, the risk of HLA alloimmunization has not yet been completely eliminated, as not every country has introduced 100% transfusion therapy with leukocyte-depleted blood. It is not routine practice to perform 100% leukocyte reduction of blood during the manufacturing process. The formation of HLA antibodies is a key risk factor for the development of donor-specific antibodies, as well as in some cases of refractoriness to transfusion with platelet concentrates. Once again, we come back to risk prevention, which takes precedence over the treatment of any complications (Figure 1).

Autoimmune hemolytic anemias (the puzzle of immunohematology), whether in the context of idiopathic forms or in the course of underlying hematological (or other) diseases, pose a difficult challenge for immunohematologists and clinicians when free panreactive warm autoantibodies, with or without specificity, are found in the serum. When antierythrocyte autoantibodies are active only by enzyme methods, the risk of post-transfusion hemolytic reaction is significantly lower because we do not transfuse enzymatically treated blood. The clinical situation is different when these free autoantibodies are active in an indirect antiglobulin test. The degree of activity is one of the leading factors in the choice of transfusion strategy – antigen-negative blood, which carries a risk of alloimmunization, or blood with the patient’s phenotype, which, however, may increase the degree of hemolysis. The choice of approach is mainly determined by the specificity of the autoantibodies, which, in most cases, are directed against antigens of the Rh blood group system. In cold agglutinin disease, the clinical significance of cold autoantibodies depends on their temperature range of action – active only up to 22 °C or from 4 °C to 37 °C. Let us not forget that supportive transfusion therapy can be life-saving in cases of critically low hemoglobin levels and severe tissue hypoxia with organ complications. Immunohematological diagnosis sometimes requires hours for blood group typing, determination of antibody specificity, and searching for a suitable blood unit. However, the patient may not have all this time, and instead of wasting it on finding the least incompatible blood, it is more important to transfuse O/Rh D (-) negative RCC (with additive solution), as in this case, the risk of alloimmunization is not a major concern. In such critical situations, the type of transfusion O/Rh D (-) negative RCC—is paramount. It should be leukocyte-depleted (to prevent febrile post-transfusion reactions) and include an additive solution (to prevent hemolytic post-transfusion reactions from donor natural agglutinins), as low-titer RCCs are not always available. Given the specificity of autoantibodies and maximum indirect Coombs activity (4 +), it is natural that we will not transfuse maximally incompatible blood. In this case, alloimmunization poses a lower risk than worsening the degree of hemolysis, although it is important to consider whether a female child or a woman of childbearing age is being transfused. With the current development of medicine and the possibilities of intrauterine blood transfusion, alloimmunization is now a solvable problem. A multidisciplinary approach to severe forms of AIHA is crucial because such patients do not always end up in hematology clinics during their first hospitalization. Their first encounter may even be in the emergency room or abdominal surgery due to clinical manifestations of jaundice caused by hyperbilirubinemia. Clinical, laboratory, and serological markers are key to determining the severity of hemolytic anemia. First in clinical significance here is, again, the ABO system, followed by Rh and Kell, especially in conditions of urgent need for transfusion therapy for vital indications. Pouchelon C. et al. emphasize that insufficient compensatory function of the bone marrow in severe hemolysis is the main indication for transfusion therapy in patients requiring resuscitation care [14]. As described by Versino F., Revelli N., Villa S., Pettine L., Zaninoni A., Prati D., Passamonti F., Barcellini W., and Fattizzo B. [15], postponing blood transfusion (abstaining from blood transfusion) is often the result of fear of a reaction. From their study of 305 patients with AIHA, they found that 33% required transfusion support, with only 7% experiencing post-transfusion reactions, none of which were hemolytic (reactions were minor febrile). For transfusion therapy to be safe, it is important to match the transfused blood in terms of clinically significant, highly immunogenic antigens, as well as the serological (warm, cold, with or without specificity, degree of activity by different methods), immunological (immunoglobulin class), and temperature characteristics of autoantibodies. Let us not be guided by fear, but rather be courageous and assertive in critical situations, while possessing the necessary knowledge to protect human life.

Another rare disease is thrombotic thrombocytopenic purpura (TTP), which may be acquired (autoimmune) or congenital, with manifestations of microangiopathic hemolytic anemia and thrombocytopenia [16]. The acquired form requires therapeutic plasmapheresis (exchange) with replacement of the recipient’s plasma with donor plasma. In about 40% of patients, the classic pentad of TTP symptoms is present (thrombocytopenia, hemolytic anemia, fever, neurological, and renal disorders). Reynolds’ pentad includes the characteristic combination of Charcot’s triad, such as right upper quadrant pain, jaundice, and fever, with shock. Here, the “golden blood group” for us transfusion specialists is the patient’s O blood group. The reason is that therapeutic plasma exchange over a 10-day period for a patient weighing 80–100 kg requires over 200 units of fresh frozen plasma (FFP), which means that such a number of blood donors is needed when the plasma is from a whole blood unit. In this case, the O blood type of the patient is “gold” because FFP from all ABO blood groups can be transfused, as the patient does not express A or B antigens, and FFP contains only agglutinins. This allows for quick and easy provision of the necessary volume of plasma for therapeutic plasma exchange, especially considering how many blood donors are needed for a single case. Given the varying number of blood donors worldwide, which, according to the World Health Organization, ranges from 5 to 31.5 per 1,000 people in different countries, ensuring a sufficient volume of plasma for blood groups other than O is not always an easy task [17]. While a patient with blood group A or B can be transfused with both ABO-identical and AB plasma, the only option for blood group AB is FFP from an AB donor. However, the low population frequency of the AB group is associated with limited options. Although rare, this disease is a challenge for a transfusion system with limited resources, as resource provision depends on the patient’s ABO blood type.

2.2 Malignant hematology

The pace of development in oncohematology and cell therapy poses challenges for transfusion medicine in terms of the type and volume of supportive transfusion therapy. The ABO blood group system is once again of primary clinical importance, both in terms of erythrocyte and platelet immunology. ABO antigens have different levels of expression on glycoproteins on the surface of platelets. This determines the alternatives to supportive platelet transfusion therapy in the absence of ABO-identical platelet concentrates. The role of natural anti-A and anti-B agglutinins in donor plasma is key, as they are a possible cause of refractoriness to transfusion therapy with platelet concentrates in cases of ABO – nonidentical transfusions. As early as 2020, Nancy M. Dunbar [18] published that in patients with high titers of anti-A/B agglutinins (from group O, A, or B), better efficacy may be observed in platelet transfusion therapy with ABO-nonidentical platelet concentrates, type “major” ABO incompatibility (without “bidirectional”, such as group “A” platelets in a group “B” patient or “B” platelets in a group “A” patient). We have a “major” ABO-incompatible platelet transfusion when a patient from group O receives PCAP from groups A, B, or AB, or when a patient from groups A or B receives PCAP from the AB blood group [18]. In acute leukemia, especially during induction chemotherapy and severe thrombocytopenia, the prevention of hemorrhagic complications is paramount and includes the management of supportive platelet transfusion therapy with its possible alternatives. The transfusion policy for oncohematology patients that we apply in our hospital with regard to platelet concentrate by apheresis (PCAP) is as follows (Table 1).

In cases where the patient has blood type AB and is subject to Allo-HSCT, and there is no possibility of ABO-identical (Rh phenotypically compatible) blood, we transfuse A or B RCCs. In the absence of AB PCAP, we transfuse platelets with the same A or B group as the transfused blood. We are guided by the fact that donor plasma in PCAP (with natural anti-A/B agglutinins) is a risk factor for the ineffectiveness of platelet transfusion therapy when one of the two corresponding A or B antigens becomes dominant. We use this approach in both oncohematology and aplastic anemia.

The primary clinical importance of the ABO blood group system is paramount in patients with blood group A2 and the presence of natural anti-A1 antibodies. In these cases, during chemotherapy and when supportive transfusion therapy is needed, our policy is to transfuse RCCs from group O, but only those produced with additive solution, while FFP and platelet concentrates are from group A. Furthermore, we should remember that weakened antigen expression of A or B antigen may be the first marker pointing us toward a diagnosis of leukemia, even before the results of molecular diagnostics and immunophenotyping are ready. Weakened expression requires an individual transfusion strategy for these patients. Given the high immunogenicity of Rh blood group system antigens, the prevention of Rh alloimmunization is key to reducing the risk of post-transplant hemolytic anemia in subsequent Allo-HSCT. This is related to the fact that Rh incompatibility, as well as ABO incompatibility, is not a barrier to donor selection. The conditioning regimen plays a role, as in cases of reduced intensity, it is assumed that in the post-transplantation period there are two immune systems – that of the donor and that of the recipient. In this case, prior erythrocyte alloimmunization is a prerequisite for an increased risk of hemolytic complications after grafting if the relevant antigen is present in the donor.

In addition, the use of monoclonal antibodies to treat diseases such as multiple myeloma or AIHA may cause positive results in indirect Coombs immunohematological tests, as is the case with the use of Darzalex (Daratumumab). A positive IAT requires several months after the last application of the medication to become negative. This requires knowledge of immunohematological methods, such as enzyme and agglutination, which are also used to detect clinically significant alloantibodies for transfusion practice. This necessitates our transfusion policy to take into account both the biological characteristics of the patient, such as gender and age, and the immunogenicity of the different blood group antigens. A clear, evidence-based transfusion strategy saves us valuable time, especially in critical situations, which is necessary for performing additional tests, such as dithiothreitol (DTT) for inactivating the CD 38 antigen and eliminating interference in IAT. In their retrospective study, Eritzpokhoff L. et al. [19] found that none of the transfused patients treated with Daratumumab developed new alloantibodies or had a hemolytic post-transfusion reaction. This demonstrated both the low immunization risk and the immunosuppressive effect of the drug. In this case, the Rh and Kell phenotyping of these patients and screening for alloantibodies prior to treatment initiation are key. In emergency situations, transfusion of Rh and Kell-phenotypically matched blood is the safest transfusion policy for the patient if there is no time for serological pre-transfusion testing. It should be remembered that clinically significant antibodies to antigens from the Rh, Kell, MNSs, and Jk systems are detected by enzyme and/or agglutination methods, in addition to IAT. Just as the transfusion of Kell (-) negative blood during treatment with Daratumumab is a leading strategy for preventing anti-Kell alloimmunization, the immunosuppressive effect of the drug is crucial to the absence of new antibody formation.

Allogeneic hematopoietic stem cell transplantation, as a potential cure for high-risk acute leukemia, requires a thorough understanding of transfusion policy in cases of both ABO and Rh incompatibility between recipient and donor. Alloimmunization to erythrocyte antigens outside the ABO system can occur in the direction of donor/recipient or vice versa, posing a risk for post-transplant hemolytic anemia. Furthermore, no one can predict:

• when a possible relapse of the underlying disease occurs,

• whether there will be an indication for a second transplant from another donor,

• whether there will be a match (correspondence) between the erythrocyte and serum blood group, or a lasting (persistent) chimerism will be observed, such as donor-type red blood cells but natural agglutinins of the recipient type.

According to a retrospective analysis by the European Society for Blood and Marrow Transplantation (EBMT) of 30,487 transplant patients between 2010 and 2021, 43.7% of transplants involved ABO incompatibilities [20]. The ABO system is not relevant for donor selection, but it is relevant for transfusion policy both during and after the transplant itself. Transfusion therapy, regardless of when it is required after the procedure and in accordance with the protocol [21] for ABO-incompatible transplantation, is a guarantee of safety and poses no risk of adverse reactions. This is especially important in cases of mismatch between the recipient’s erythrocyte and serum group at the time of testing. The ABO blood group system, with its natural IgM/IgG agglutinins, may not be the main factor in donor selection, but it is the main factor in the selection of the ABO type of transfused blood components and applied alternatives. The Rh blood group system is key in the transfusion therapy of a severe form of AIHA that has developed after transplantation. Rh recipient/donor relationships are crucial to the management of alloimmunization. Based on our analysis of 232 Allo-HSCT cases at our transplant center between 2017 and 2024 [22], we identified a 2.16% incidence of post-transplant AIHA. This established incidence is associated with different risk and concomitant factors, such as leukemia relapse, graft-versus-host disease, graft failure, Evans syndrome, and reactivation of the Epstein-Barr Virus. In the transfusion therapy of AIHA, although it was performed only with partially compatible blood, no reactions were observed. Blood transfusion was consistent with both the ABO and Rh donor/recipient relationships and the results from immunohematology diagnostics on the ABO/Rh phenotype of the recipient at the time before transfusion. The serological characteristics and properties of the established antierythrocyte autoantibodies are decisive for the approach in these cases. The timely provision of leukocyte-depleted RCCs, consistent with the Rh blood group system (Rh phenotype), although not fully compatible, is vital for a favorable outcome in this complication.

Data from the National Center for Transfusion Hematology in our country for 2024 [23] indicates that the number of blood donors in Bulgaria is 27.7 ‰ (Figure 2), which proves the need to use clear alternatives in transfusion therapy when ABO/Rh phenotypically identical blood components are not available.