Transfusion Medicine has evolved becoming increasingly safer for patients requiring therapy with blood components and hemoderivatives.1,2 The first successful blood transfusion in animals was performed by physician Richard Lower in 1666. However, it was only after the discovery of blood types by Karl Landsteiner at the beginning of the 20th century that the first blood transfusion preceded by tests of compatibility using the ABO system could be performed. This was carried out by Reuben Ottenberg in 1907. Despite this historical breakthrough, blood transfusion continued to be considered a risky procedure.2 The need to treat wounded soldiers who died of acute bleeding in the two world wars intensified research on the subject, and blood transfusion began to be used on a large scale after that period. This culminated in the creation of transfusion commissions aimed at ensuring the correct use of hemotherapy.2 With the development of transfusion medicine as a medical specialty, blood processing has undergone technological innovations. Today, good practices and surveillance ensure safety throughout the blood cycle, providing safe hemotherapy for patients.2

To enable individual screening of blood components, it is necessary not only to consider the interaction between donor antigens and recipient antibodies, but also to select the most appropriate blood component based on the patient's clinical condition and individual characteristics.3 Both assessments aim to detect compatibility through cross-reactivity and minimize the risk of adverse reactions.4 According to Brazilian regulations, the pre-transfusion tests that must be performed include ABO and Rh typing, irregular antibody screening (IAS), and crossmatching.3 These tests are performed to determine the patient's erythrocyte profile and select the most suitable blood component.3

Exposure to antigens, not only of the ABO and Rh systems but also of several other systems present in the erythrocyte membrane, all of which are catalogued in 45 blood systems, may result in alloimmunization.5 When the body is exposed to non-self-antigens, the immune system responds by activating B lymphocytes, leading to the production of antibodies that neutralize these antigens.6 The risk of developing alloantibodies depends on factors such as the number and frequency of transfusions, pregnancy, antigen immunogenicity, recipient immune response, patient's ethnicity, and differences in the pattern of antigens, both of the donor and of the recipient.6,7 Alloimmunization predisposes patients to acute or delayed hemolytic transfusion reactions, in addition to making the selection of compatible blood components difficult.7,8

The need to minimize transfusion reactions has led to the adoption of increasingly precise immunohematological techniques in transfusion practices, as well as the optimization of strategies to prevent alloimmunization. The latter requires an understanding of the characteristics of the alloimmunized population. Therefore, the objectives of this study were to determine the frequency of alloimmunization among patients evaluated during the study period, investigate the demographic, clinical, and epidemiological characteristics of patients with a positive result in the IAS test, and to assess the frequency of erythrocyte antigens and anti-erythrocyte alloantibodies in the analyzed population.

Between January 1 2019 and December 31 2020, 15,307 patient samples underwent IAS at AGETRA (HC-UFU/EBSERH). Among these, 599 samples tested positive for IAS, constituting 3.9% of the total samples. After excluding duplicate samples (n = 398), the study focused on 201 patients with confirmed positive IAS results out of 14,909 patients attended by AGETRA during the specified period. The prevalence of positive IAS samples in the unit averaged 1.3% (201/14,909 - Figure 1).

Figure 1.

Flow diagram of the patients analyzed in the study.

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The median age of the 201 patients was 53 years (interquartile range [IQR]: 32-65 years), with alloimmunization occurring more frequently in women (64.2%) than in men (35.8%). Blood groups A (39.8%) and O (38.8%), and RhD positive samples (69.1%) were the most common. Approximately half (48.2%) of the patients were transfused due to preoperative procedures. During the hospitalization period, 32.8% of the patients received 1-3 transfusions, and 71.6% had a history of transfusions (Table 1).

Out of the 201 alloimmunized patients identified in AGETRA, 121 had their alloantibodies characterized by the Hemominas Foundation. The remaining 80 patients did not undergo alloantibody characterization due to non-reactivity against the red blood cell panel employed by the reference laboratory, a situation that arises when the reactivity of the test conducted by AGETRA is low, prompting the need for sample confirmation. Another contributing factor was the impossibility to recollect samples by the healthcare team for submission to the reference laboratory. Among the clinically significant alloantibodies characterized, the most frequently found were those belonging to the Rh and Kell blood systems, with prevailing anti-D (27.2%), anti-E (15.0%), and anti-Kell (11.5%) antibodies (Figure 2A and 2B).

Figure 2.

Frequency of alloantibodies detected in 121 of 201 irregular antibody-positive patients evaluated by the transfusion service (AGETRA) of Hospital de Clínicas of the Federal University of Uberlândia (HC-UFU/EBSERH), from January 2019 to December 2020 according to the type of alloantibody (A) and the blood system (B). aAntibodies not identified by the RBC panel used.

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Of 201 patients, 121 were confirmed IAS-positive by the Hemominas Foundation, 30.6% (37/121) had profiles with multiple associations of alloantibodies, as shown in Table 2. The association of anti-D and anti-C was the most frequent (Profile 1), followed by anti-c and anti-E (Profile 2) and Anti-C, Anti-Kell and unidentified (Profile 3). Several other associations were identified, but with single occurrences in the studied population.

According to the distribution of alloantibodies by gender, as shown in Table 3, alloantibodies belonging to different blood systems were identified in the female population. The most frequent were anti-D (28.7%), anti-E (14.7%), and anti-Kell (10.8%), while other clinically significant alloantibodies such as anti-Jka, anti-Jkb, anti-Fya, anti-Lea, anti-Leb, anti-M, anti-S, and anti-Dia were also detected. In men, a smaller number of alloantibodies were characterized, with anti-D (13.9%), anti-E (9.7%), and anti-Kell (8.3%) being the most prevalent.

Seventy-six patients (37.8%; 76/201) underwent erythrocyte immunophenotyping as shown in Table 4. The female gender (55.3%) was more prevalent than the male gender (44.7%); 31.6% of patients were transfused due to preoperative procedures and 26.3% due to malignancies. During the hospitalization period, 46.1% of patients received 1-3 transfusions, and 31.6% had a previous history of transfusion. Additionally, 39.5% of patients had undergone previous surgical procedures. The antigens identified in immunophenotyped patients were e (100%), c (86.8%), C (40.8%) and E (17.1%).

In this study, complete data was retrieved for 14 out of 18 IAS-positive pregnant women, whose alloantibodies were characterized and are presented in Table 5. The average age of the patients was 27 years, and most were multiparous. Among the 14 patients, 12 (85.7%) had anti-D as the most prevalent antibody, 7 (7/14; 50%) had previous use of anti-Rh immunoglobulin (anti-RhIg), and blood type A negative was the most frequent (4/12; 33.3%). A history of previous transfusion was identified in only one pregnant woman. Notably, Patient 9, who was A positive, presented with anti-Lea alloantibody, an unusual profile for alloimmunization induced by isoimmunized pregnancy. Erythrocyte phenotyping was not performed for most of the pregnant women.

In this study the prevalence of positive IAS samples was 1.3%, a similar rate to that of Pereira Bueno et al., who reported a rate of 1.1%.9 Despite the advances in the safety of transfusion medicine, adverse effects such as alloimmunization, acute and late transfusion hemolytic reactions, and iron overload are still observed, conditions that are more prevalent among polytransfused patients specifically those with hematological/oncohematological diseases.10,11 However, early recognition and proper management can prevent more severe outcomes for patients.

Due to miscegenation, a characteristic of the Brazilian population, there is a heterogeneity of phenotypic frequencies of blood systems, which explains the wide range of polymorphisms.12 Corroborating the data available in the literature, which report that blood groups A and O, both RhD positive, are the most frequent in Brazil, these systems were predominant in this research.12-14 Furthermore, alloimmunization was common in older patients. This fact was also reported by another study, in which over 30-year-old patients had a greater chance of developing alloimmunization with the risk increasing with age.15-17 Therefore, in addition to the transfusion history, age can also influence the positivity of IAS.

Regarding ethnicity, the distribution between whites and non-whites within this study was similar, which suggests that this is not a factor related to the development of alloimmunization. Gender, in another way, seems to be related to this adverse effect, since more women had positive IAS. Other studies have also shown a higher frequency of alloimmunization in female patients, which is explained by the gestational history and exposure to different antigens.18-20

The erythrocyte antigens detected in this study, D, E, e, C and c, are highly immunogenic. For this reason, their respective antibodies (anti-D, anti-E, anti-e, anti-C and anti-c) can induce acute or delayed post-transfusion hemolytic reactions and hemolytic disease of the newborn (HDN).21,22 The results presented confirm the high detection and significance of the Rh system, which is consistent with previous studies.8,21

The Rh system is one of the most polymorphic and immunogenic known in humans and represents the most significant cause of hemolytic transfusion reactions.10 The system has about 55 erythrocyte antigens, five of which are of greater clinical importance: D, E, e, C, and c.3 These antigens are glycoproteins important to the integrity of the erythrocyte membrane, with structural function and responsible for gas transport.2 Various polymorphisms in the proteins of this system make it more likely to induce intense immune responses.23 The Kell system, also considered one of the most immunogenic, has 36 antigens, with K and k being the most clinically important.3 The antibodies Anti-K and Anti-k, belong to the IgG class and are fully formed at birth, a relevant characteristic, particularly in pregnancy, since they can attack erythrocyte precursors of the fetus, resulting in severe anemic conditions.14,24 Together with the effects of the antibodies against the Rh system, they are closely related to severe acute or delayed hemolytic reactions, in addition to HDN.2,14,25

The distribution of alloantibodies varies according to the study population, however, the prevalence of Anti-D, Anti-E, and Anti-Kell demonstrated in this work, corroborate the data available in the literature, which show a higher prevalence of these antibodies in several countries, including Brazil.10,19,26 The literature also demonstrates the frequent identification of other systems in different populations, as is the case of Anti-E, Anti-D, and Anti-M in Chinese,26 and Anti-E, Anti-Lea, Anti-K and anti-D in North Americans.27 The high immunogenicity, individual immune response, dose, and frequency of transfusion are characteristics that increase the probability of irregular antibodies against blood systems such as Rh and Kell.16,17,28 In addition, immune response, dose, and frequency of transfusion are also characteristics that increase the probability of irregular antibodies developing.28

Several studies highlight the concern with patients at high risk of alloimmunization due to the need for multiple transfusions, for example, patients with hematological diseases such as sickle cell anemia and thalassemia, and oncohematological diseases, such as myelodysplastic syndromes, leukemias and lymphomas.17,21,22,29 These individuals are exposed to different erythrocyte antigens more and are therefore more likely to produce alloantibodies.7 Otherwise, in this study, transfusion was indicated mainly as part of preoperative procedures, which is justified by the profile of the population treated at the hospital. Although a limited number of studies have explored the characteristics of alloimmunized patients due to preoperative procedures, it is well known that phenotyping is not typically conducted during emergency transfusions. Furthermore, even in the absence of repeated transfusions, immunological effects can be induced. These effects can persist beyond their detectability and result in delayed transfusion reactions.19,28,30

Proper management of blood components, particularly with regards to the Rh and Kell blood systems, aims to prevent the co-occurrence of alloantibodies, as this association is often more frequent in patients who have received multiple transfusions and are therefore more likely to experience post-transfusion hemolytic reactions.26 Due to the immunogenicity of these two systems, the results of this study are consistent with the literature, as the most prevalent associations were found between Rh and Kell.26,31 However, the Rh system was the most frequently involved, likely due to the high genetic variability of RhD23 as demonstrated by Politou et al.19 The high frequencies of Anti-E and Anti-Kell, in addition to the data on associations, warrant special attention to these systems due to the high prevalence of Anti-D, Anti-E, and Anti-Kell antibodies in the population, particularly in women.

Regarding the formation of antibodies, the ‘unidentified’ antibodies that were detected and found relevant in this study may have been due to pan reactivity with autoantibodies. This class of antibodies can lead to a masking effect on the presence of alloantibodies, making patient management challenging.10,17 However, it is known that additional dilution and adsorption techniques can aid in the separation and identification of alloantibodies.17,32

The care aimed at preventing alloimmunization in women should primarily focus on pregnancies due to the higher frequency of alloimmunization in this population caused by exposure to different antigens.10,14,20 In the current study, a high frequency of antibodies against Rh system antigens was observed in correlation with gender and blood systems. This is due to the higher frequency of the Rh system in the Brazilian population.16,33 The MNS system was the third most common blood system with antibody development, and interestingly, alloantibodies from this system only appeared in women, which differs from other studies.19,26 Although not clinically significant, Anti-M is part of a complex blood system and was frequently detected in our study data.2,14 Finally, the proportion of alloimmunized men and women followed the pattern of 1.8% to 2.7% found in other studies.19,34,35

The epidemiological and clinical characteristics of immunophenotyped patients were evaluated thus identifying the most frequent erythrocyte antigens. The highest frequencies were the e, c, and C antigens. This phenotypic screening is essential for enhancing transfusion safety and preventing future alloimmunization. Therefore, measures such as performing extended phenotyping can help prevent and reduce this condition.10,21,22,36,37

Concerning alloimmunized pregnant women, it was observed that, despite the various clinical indications for transfusion, most were multiparous, supporting the possibility of maternal-fetal sensitization, as demonstrated in other studies.21,28 The most frequently found antibodies were of the Rh blood system, indicating possible alloimmunization induced by isoimmunized pregnancies.20

The use of human anti-D immunoglobulin in pregnant women with RhD antigen incompatibility with their partner is one of the methods employed to prevent HDN and maternal alloimmunization during prenatal care.18 In this study, it was not possible to distinguish whether the production of alloantibodies in pregnant women occurred due to sensitization in previous pregnancies, the use of the medication (Rho(D) immune globulin), or a history of transfusions outside HC-UFU/EBSERH, due to the absence of prior negative results and previous medical records. Nevertheless, it is undeniable that the rates of alloimmunization in pregnant women who use immunoglobulin during prenatal care are reduced.38

The main feature of the present study was the utilization of a retrospective design, limiting patient analysis by not providing longitudinal follow-up information about their outcomes. Furthermore, being a single-center study, it was not possible to verify previous transfusions in other health facilities, which could directly impact the laboratory results analyzed. Additionally, only the initial positive sample from each patient was evaluated in this study, potentially limiting the identification of new alloantibodies produced after the patient's exposure to transfusion and other antigenic stimuli. Nevertheless, the impossibility of following patients over time, as in a prospective study, justifies this methodological decision. At the national level, it is important to acknowledge that Brazil is a developing country, and certain regions, such as the one in this study, may lack the necessary resources to perform phenotyping for all patients at the local hospital.

This study found that the majority of patients who received transfusions were female and underwent preoperative procedures. Alloantibodies from the Rh and Kell blood systems, which are highly immunogenic, were frequently detected. Therefore, the screening and characterization of alloantibodies, along with erythrocyte immunophenotyping, are necessary to better understand the alloimmunized population. This approach can improve the safety and efficacy of transfusion therapy, reducing the risk of serious reactions. This is particularly important for oncohematological, chronic renal, transplanted, and polytransfused patients.