Prevalence and Specificity of Red Blood Cell Alloimmunization: Insights from Transfusion-Dependent Populations in Serbia

Abstract

Background/Objectives: Red blood cell (RBC) alloimmunization is a significant challenge in transfusion medicine, particularly among transfusion-dependent patients, such as those with thalassemia. It arises from the production of antibodies against non-self RBC antigens and can lead to complications like hemolytic transfusion reactions. This study aimed to evaluate the prevalence, specificity, and clinical implications of RBC alloimmunization at the University Clinical Center of Serbia (UCCS), emphasizing transfusion-dependent populations. Methods: This retrospective study analyzed 27,530 transfusion records at UCCS between January 2023 and January 2024. Pre-transfusion testing included ABO and RhD typing, irregular antibody screening, and crossmatching. Data from 630 patients with positive antibody screening were reviewed. Alloantibody specificity was determined using indirect antiglobulin tests and advanced phenotyping methods. Results: Among 27,530 patients, 630 (2.29%) tested positive for irregular antibodies, predominantly males (57.14%) with a mean age of 49.6 years. Alloantibodies were detected in 70.47% of cases, most commonly targeting Rh (53.35%) and Kell (17.15%) systems. Anti-E (27.93%) and anti-D (18.02%) were the most frequent antibodies. Multiple alloantibodies were identified in 18.41% of patients, posing challenges for blood compatibility. In a total of 495 patients with thalassemia, antibodies were found in 9.69%. Alloimmunization was significantly associated with higher numbers of transfusions and pregnancies (p < 0.05). Conclusions: Our findings indicate that alloimmunization is predominantly associated with Rh and Kell antigens, suggesting that implementing targeted antigen matching may reduce the frequency of alloimmunization. While our study does not directly assess the impact of genotypic matching, the prior literature supports its role in enhancing transfusion safety, particularly for high-risk populations like thalassemia patients.

1. Introduction

Red blood cell (RBC) alloimmunization is a significant concern in transfusion medicine, particularly for transfusion-dependent patients, such as those with thalassemia. These patients require lifelong transfusions, which significantly increase their exposure to foreign RBC antigens and the risk of alloantibody production. Alloantibodies can lead to severe complications, including hemolytic transfusion reactions and difficulties in sourcing compatible blood, making it vital to optimize transfusion protocols for such high-risk populations [1]. Clinically significant RBC antibodies, typically produced in response to foreign antigens introduced during blood transfusions, organ transplants, or fetal–maternal incompatibility during pregnancy can mediate immune hemolysis [1]. This contributes to adverse outcomes, such as hemolytic transfusion reactions, hemolytic disease of the fetus and newborn (HDFN), and autoimmune hemolytic anemia (AIHA) [2]. Advances in immunohematology have enhanced the detection of these antibodies, enabling precise blood matching and reduced risk of hemolytic complications. The most encountered RBC antibodies are directed against the major blood group antigens Rh, Kell, Duffy, and Kidd systems [3,4,5]. However, alloimmunization to less common antigens can also occur, complicating transfusion and pregnancy management, especially among patients with multiple antibodies [1,2]. For patients requiring multiple transfusions, alloimmunization is a significant concern, leading to challenges in finding compatible blood units [3].

The University Clinical Center of Serbia (UCCS), the largest tertiary-level hospital in Serbia, provides transfusion care for patients with complex medical needs. Standard pre-transfusion testing at UCCS includes ABO and RhD antigen matching, along with crossmatches between donors and recipients, complemented by routine irregular RBC antibody screening. However, extended RBC antigen phenotyping is not performed, limiting the ability to preemptively address alloimmunization risks.

This study evaluates the prevalence and specificity of RBC alloimmunization at UCCS, with a focus on transfusion-dependent populations, such as thalassemia patients. By identifying the immunogenicity of specific antigens and assessing the need for targeted antigen-matching protocols, this study provides a database for evaluating transfusion safety and the prevalence of alloimmunization in a transfusion-dependent population.

2. Materials and Methods

2.1. Study Design

A retrospective study was conducted to estimate the prevalence of RBC alloimmunization in the transfused adult patients from January 2023 to January 2024 at the University Clinical Center of Serbia, which provides care for patients with surgical, obstetrics, onco-hematological conditions, etc. The study analyzed pre-transfusion test results from a total of 27,530 unique patients who underwent pre-transfusion testing during the study period. If an alloantibody was detected, a retrospective review of the patient’s transfusion history was conducted to determine prior exposure to RBC transfusions. The study assessed the prevalence rather than incidence of alloimmunization. In this manner, the study focused on all detected alloimmunization at the time of testing. The study did not monitor the development of RBC antibodies at the time after current transfusions. Pre-transfusion testing included ABO and RhD blood typing, irregular antibody screening, and crossmatch. Leukoreduction was not systematically applied to transfused RBC units during the study period, so this variable was not accounted for in alloimmunization analysis.

If a crossmatch test or irregular antibody screening test was positive, an antibody identification test was performed to identify the type of RBC antibody. Extended red blood cell phenotyping for antigens beyond ABO and RhD (such as Kell, Duffy, Kidd, and MNS systems) is not routinely performed at our institution. However, in cases where patients have a history of alloimmunization or require frequent transfusions (e.g., thalassemia or sickle cell disease patients), extended phenotyping may be considered on a case by case basis to improve transfusion compatibility. Data gathered from transfusion registers and information system from the Hospital Blood Bank Department for Pretransfusion Testing in the Emergency Center of UCCS included the age and gender of 630 patients who were positive for antibody screening or crossmatch. All patient data were anonymized before statistical analysis. Patient identifiers were removed at the stage of data extraction from hospital records, ensuring compliance with ethical guidelines.

Exclusion criteria were the following: (1) false positive results due to poor sampling, the presence of fibrin strands, sample contamination, or insufficient centrifugation of the sample; (2) patients who were not given antibody screening tests because they received only platelets (PLT) or fresh frozen plasma (FFP) transfusions. According to the current transfusion guidelines established by the National Expert Committee for Transfusion Medicine, routine antibody screening is not mandated before platelet or plasma transfusions unless clinically indicated.

2.2. Pre-Transfusion Laboratory Testing

Antibodies were detected through an indirect antiglobulin test (IAT) using commercial reagent RBCs (ID-DiacellPool, Biorad, Cressier, Switzerland) on commercial Liss/Coombs cards with a gel matrix containing anti-IgG and anti-C3d (Diamed GmbH, Cressier, Switzerland) on IH-500 (Biorad). The antiglobulin crossmatch was carried out to ensure that there were no antibodies presented in the patient’s serum that would react with donor cells (on commercial Liss/Coombs card at +37 °C). At the same time, crossmatch was performed in test tubes at room temperature (+22 °C).

If an antiglobulin crossmatch was positive, we performed antibody screening through IAT using commercial reagent RBCs (ID-DiaCell I+II, Bio-Rad, DiaMed GmbH, Cressier, Switzerland) on commercial Liss/Coombs cards (DiaMed GmbH, Switzerland). We also performed antibody identification using 11 cell panel (Bio-Rad Diamed GmbH, Cressier, Switzerland) and Liss/Coombs cards (DiaMed GmbH, Cressier, Switzerland). If the crossmatch in the test tube at room temperature (+22 °C) was positive, antibody identification was performed in the test tubes using ReaCell I, II, III test RBCs (Reagens Kft, Budapest, Hungary). After incubation at room temperature, centrifugation, and reading, in the case of a positive result, the test tubes were further incubated at +4 °C to enhance the reaction, followed by additional centrifugation and reading. RBCs treated with papain were used to increase the sensitivity of antibody detection in order to differentiate multiple antibodies, enhance the detection of weak antibodies, and use adsorption methods. Enzyme-treated RBCs were employed selectively when the presence of multiple antibodies was suspected or when weak antibodies required enhanced detection. Their application was particularly emphasized in cases of suspected Rh alloimmunization. Enzyme treatment was performed using a one-stage procedure.

2.3. Statistical Analysis

The Statistical Package for the Social Sciences (SPSS Statistics, Version 29.0) was utilized to create the database and process the statistical data. Standard descriptive values are summarized for every variable. Percentages were used for categorical variables. The chi-square (χ²) test of independence was used to determine whether there was a statistically significant association between the categorical variables. The Pearson correlation coefficient (PCC) was used to measure linear correlation between two sets of data. Correlations were considered statistically significant at p ≤ 0.05.

3. Results

A total of 27,530 patients’ individual samples were screened for the presence of irregular RBC antibodies. There were 13,297 (48.3%) male and 14,233 (51.7%) female patients. Antibody screening was positive in 630 (2.29%) patients, and 270/630 (42.86%) were female and 360/630 were (57.14%) male, with the difference between the two being statistically significant (χ2 = 19.83, p < 0.001). The age of the patients ranged from 18 to 86 years, with a mean age of 49.6 age for immunized patients and 50.4 for non-immunized. No correlation was observed between the age of the patient and rate of alloimmunization (r = 0.047, p > 0.05) (

Table 1. Characteristics of alloimmunization status.

Factors	Alloimmunization Rates
	Pearson’s Correlation (r)	p Value
Number of transfusions	0.9999	<0.001
Number of pregnancies	0.981	<0.05
Age	0.047	0.235

). Among the alloimmunized patients, 551 had received at least one blood transfusion in the past (range one to eight units). The median number of transfused RBC units was three (67 patients received one unit of blood, 114 patients received two units, 161 patients received three units, and 209 patients received four or more units). Among non-immunized patients, 2152 (8%) received a transfusion with a median of one unit administered. Out of 270 alloimmunized women, 101 had a history of previous pregnancies, distributed as follows: 21 women had one pregnancy, 33 had two pregnancies, and 47 had three or more pregnancies. A significant increase in the rate of alloimmunization was observed with an increase in the number of transfusions and pregnancies (p < 0.05) (Table 1).

Alloantibodies with determined specificity were found in 444 patients (70.47%), while antibodies of undetermined specificity were found in 143 patients (22.70%). In 11 patients (1.75%), only autoantibodies were identified, and 121 (19.20%) revealed autoantibodies with one or more underlying alloantibodies, while 498 patients (79.05%) had only alloantibodies. Among the 630 patients with detected antibodies, 10 had a negative antibody screening but were identified through a positive crossmatch. Of these, four had anti-Jka, three had anti-Fya, and three had anti-M.

A single alloantibody was identified in 328 (52.06%) sensitized patients, while 116 (18.41%) patients had developed more than one alloantibody. Alloantibodies against the Rh system were the most frequent, with 311 out of 583 alloantibodies (53.35%), while anti E was identified in 124 (21.27%) cases and anti D was identified in 80 cases (13.7%). Antibodies against the Kell system were present in 100 (17.15%) cases, and antibodies against the MNS system were present in 55 (9.43%) cases (

Table 2. Frequency and specificity of identified alloantibodies.

Blood Group System	Antibody	Antibody Detection Temperature (°C)	N (%)
Rh	D	37	80 (13.7)
	C	37	44 (7.55)
	E	37	124 (21.27)
	c	37	37 (6.35)
	e	37	9 (1.54)
	Cw	37	17 (2.92)
Kell	K	37	89 (15.27)
	Kpa	37	11 (1.89)
Duffy	Fya	37	16 (2.74)
	Fyb	37	5 (0.86)
Kidd	Jka	37	27 (4.63)
	Jkb	37	15 (2.57)
Lewis	Lea	37	5 (0.86)
	Leb	37	8 (1.37)
P1	P1 (IgM)	22	11 (1.89)
	P1 (IgG)	37	7 (1.20)
MNSs	M (IgM)	22	25 (4.29)
	M (IgG)	37	25 (4.29)
	N (IgM)	22	5 (0.86)
	N (IgG)	37	4 (0.69)
	S	37	6 (1.03)
	s	37	4 (0.69)
Lutheran	Lua	22	9 (1.54)
Total			583 (100)

).

Sensitized patients were predominantly those with pregnancy-related cases (16.03%) and malignancies, including hematological disorders (50.95%), accounting for a combined 66.98% (

Table 3. Clinical conditions associated with red blood cell allo- and autoantibodies.

Classification	Number of Patients (%)
Prenatal cases	101 (16.03)
Other gynecological conditions	43 (6.83)
Hematological disorders	108 (17.14)
Solid tumors	213 (33.81)
Hemodialysis	28 (4.44)
Other urinary system conditions	7 (1.11)
Autoimmune diseases	1 (0.16)
Trauma	19 (3.02)
Infections	3 (0.48)
Preoperative anemia correction	37 (5.87)
Gastroenterological diseases	27 (4.28)
Burns	2 (0.32)
Cardiovascular conditions	41 (6.51)
Total	630 (100)

). Of the 495/27,530 (1.8%) patients with thalassemia, 48/495 (9.69%) were found to have antibodies detected.

Out of the 132 identified autoantibodies, 46 were classified as warm-reactive (detected at 37 °C), while 86 were cold-reactive (detected at 4 °C). Cold-reactive autoantibodies were predominantly found in patients with hematological malignancies and other hemato-oncologic conditions. However, despite observing trends in antibody distribution among different patient groups, no statistically significant correlation was found between the specificity of the detected antibodies and the underlying diagnoses. No clinical complications related to alloimmunization were observed in the study population during the study period.

4. Discussion

Our goal was to detect potentially clinically significant antibodies, regardless of whether we identified previously undetected antibodies or reconfirmed antibodies that were identified in the past. The study confirmed that the antibody detection protocol we use, which initially includes crossmatch on +37 °C and +22 °C, is suitable to detect potentially clinically significant antibodies that are likely to cause accelerated destruction of a significant proportion of transfused cells. All alloantibodies detected in crossmatches were evaluated for specificity. We were not able to avoid detecting clinically “insignificant” antibodies related to blood group antigens. Clinically “insignificant” antibodies do not typically cause hemolytic transfusion reactions and do not necessitate antigen-negative donor units for transfusion, such as anti-Lea, anti-Leb, anti-P1, and certain anti-M or anti-N antibodies. While their detection may delay transfusion due to additional compatibility testing, they are generally not associated with significant clinical consequences. Our findings indicate that screening at multiple temperatures enhances antibody detection, particularly for cold-reactive antibodies. A 37 °C only approach could miss clinically relevant cold agglutinins, increasing transfusion risks.

Patients with repeated transfusions have greater exposure to foreign antigens, increasing the risk of antibody formation. Similarly, pregnancy-related alloimmunization, particularly against Rh and Kell antigens, is well-documented [6,7]. Our findings confirm a significant correlation between the number of transfusions and pregnancies and the rate of alloimmunization (p < 0.05). Patients with repeated transfusions have greater exposure to foreign antigens, increasing the risk of antibody formation. Similarly, pregnancy-related alloimmunization, particularly against Rh and Kell antigens, is well-documented [8]. The study also found that providing Rh and Kell antigen-matched blood products to patients could prevent the occurrence of 70.50% alloimmunization. While transfusion frequency is a key factor in alloimmunization, underlying clinical conditions also play a role. Patients with autoimmune diseases may exhibit heightened immune responses, predisposing them to alloimmunization. Conversely, patients on immunosuppressive therapy may have lower alloimmunization rates due to dampened immune responses [8,9]. Further research based on the current findings should explore the aforementioned impacts on the patient population described in this study. Additionally, some alloimmunized patients may have had undetectable antibody titers at the time of pre-transfusion testing. This could have led to an underestimation of the true alloimmunization rate, particularly for antibodies known to exhibit dosage effects (e.g., anti-Kidd).

The incidence of RBC antibodies varies significantly across different populations, influenced by transfusion practices, the frequency of pregnancies, and genetic diversity. In transfusion-dependent patients, such as those with sickle cell disease or thalassemia, alloimmunization rates tend to be higher due to the increased likelihood of exposure to foreign antigens [10,11]. The study confirms that thalassemia patients experience a significantly higher rate (9.69%) of alloimmunization compared to the general transfused population (2.29%). Despite the recognized benefits of antigen matching, extended red blood cell phenotyping for Kell, Duffy, Kidd, and MNS systems is not routinely performed at our institution. This may contribute to the higher risk of alloimmunization observed in transfusion-dependent patients, particularly those with thalassemia, who often develop multiple alloantibodies due to cumulative antigen exposure. Several studies have demonstrated that extended antigen matching reduces alloimmunization rates in these patients by minimizing exposure to immunogenic non-self-antigens [12]. On the other hand, such a practice might increase the burden on blood banks by limiting available donor units. Furthermore, the cost effectiveness of different levels of antigen matching should be considered. Future studies should explore the long-term outcomes of alloimmunization prevention strategies, particularly in resource-limited settings. Ultimately, the findings of this study emphasize the need for tailored testing protocols that strike a balance between reducing alloimmunization risk and maintaining transfusion efficiency.

Studies report an alloimmunization rate ranging from 5% to 50% depending on the population, antigenic exposure, and matching protocols in transfusion centers [6,7]. In contrast, in general hospital populations without repeated transfusion exposure, the prevalence of RBC antibodies is considerably lower, typically ranging between 1% and 2% [8]. In this study, the prevalence was 2.29%, considering that UCCS treats both patients who have not previously received transfusions and those who have undergone multiple transfusions. Patients with hematological disorders, along with those with other malignancies, represented the most likely group (50.95%) to develop antibodies to RBC transfusions, as transfusion therapy is a primary supportive treatment for these conditions. The association between disease type and specific antibody patterns observed in this study is consistent with previous research. Cold-reactive antibodies, predominantly seen in patients with hematological malignancies, have been linked to immune dysregulation in the literature, while warm-reactive alloantibodies were more frequently found in transfusion-dependent patients.

While our findings indicate that cold-reactive autoantibodies were predominantly observed in patients with hematological malignancies and other hemato-oncologic conditions, no direct correlation between antibody specificity and primary diagnosis was established. This suggests that alloimmunization risk in transfusion-dependent patients is multifactorial and likely influenced not only by the underlying disease. The absence of a disease-specific alloantibody pattern aligns with previous studies, which emphasize the complex interplay between antigenic exposure and immune response [13].

Rh and Kell antibodies were found combined with other alloantibodies in 10.13% of patients. Our findings also reveal that anti-E was the most prevalent antibody, which is understandable considering that “e” antigen is a high-incidence antigen. The high prevalence of anti-RhD antibodies is particularly surprising. The explanation can be sought in several possibilities. Even though anti-D prophylaxis is enforced routinely, some women still receive prenatal care without determining their RhD blood type or receiving prophylaxis. Immunization can also happen in patients with a weak D antigen or a partial D antigen who receive RhD-positive blood. Lastly, unfortunately, it may happen in RhD-negative patients who, due to a shortage of RhD-negative blood and vital medical indications, receive an RhD-positive blood unit.

In this study, Rh system antibodies (anti-D, anti-E, and anti-C) and Kell system antibodies (anti-K) were among the most frequently detected, corroborating their known immunogenicity. These findings align with previous reports that such antibodies are implicated in hemolytic transfusion reactions. In particular, anti-Kell has been associated with hemolytic disease of the fetus and newborn (HDFN), as it suppresses fetal erythropoiesis rather than causing direct hemolysis [1,9,10]. However, our study did not include an assessment of hemolytic severity in affected patients, and further investigation is needed to establish a direct link between these antibodies and clinical hemolysis in our cohort.

Immune hemolytic transfusion reactions occur when incompatible RBCs are transfused to a patient with RBC alloantibodies. These reactions can be acute or delayed, with delayed hemolytic transfusion reactions (DHTRs) being particularly deceptive, as they can occur days to weeks after transfusion, complicating diagnosis and management of anemia [11,12,13,14]. DHTRs happen frequently with antibodies like anti-Kidd when they are not detectable during pre-transfusion testing because of dosage phenomena or a decrease in titer in time after receiving transfusions [4,15,16,17,18,19]. In our study, 1.59% of patients showed a positive crossmatch and negative irregular antibody screening because of heterozygous reagent cells. The majority of these cases involved low-prevalence antigens, such as anti-Jka and anti-Lea. Anti-Kidd was detected in 7.20% of alloantibodies patients in our study.

In transfusion medicine, antibody identification precedes the selection of antigen-negative RBC units for transfusion, thus preventing HTRs. However, extended antigen matching is increasingly recognized as a strategy to reduce alloimmunization, particularly in patients with chronic transfusion needs, such as those with sickle cell disease. Genotypic matching, rather than traditional serologic matching, offers an opportunity to further reduce alloimmunization by accounting for genetic variation in blood group antigen expression [18,19,20].

Although this study provides valuable insights into RBC alloimmunization prevalence, certain limitations may have influenced the alloimmunization rates. One such factor is the lack of systematic leukoreduction, which would otherwise lead to minimizing residual white blood cells in order to reduce the risk of adverse reactions.

The findings of this study emphasize the importance of adapting transfusion practices to address alloimmunization risks not just within individual hospitals but globally. Given the high prevalence of Rh and Kell alloantibodies, the introduction of targeted antigen matching protocols could significantly improve patient outcomes.

Targeted antigen screening for high-risk populations is helpful to prevent complications related to alloimmunization. In the long run, extended antigen matching or genotypic matching in high-risk groups, such as thalassemia patients, should be a priority for optimizing transfusion safety. However, logistical and economic constraints often limit the routine implementation of such protocols. Under good blood supply conditions, future advancements in molecular genotyping may offer a more feasible approach to personalized antigen matching, reducing the risk of alloimmunization.

5. Conclusions

This study highlights the prevalence and specificity of clinically significant red blood cell alloantibodies, particularly within the Rh and Kell systems, and emphasizes the challenges of managing alloimmunization among transfusion-dependent patients. These findings underscore the need for targeted strategies, such as Rh and Kell phenotyping, to minimize alloimmunization risks. Globally, implementing these practices, even in resource-limited settings, could significantly enhance transfusion safety and reduce adverse outcomes. Furthermore, the adoption of genotypic matching techniques offers a promising approach to address genetic diversity in multiethnic and multicultural populations, ensuring better compatibility. By tailoring transfusion protocols to regional characteristics and high-risk populations, healthcare systems worldwide can improve resource allocation, optimize transfusion practices, and enhance patient outcomes.