Red Blood Cell Alloimmunization: Life-Threatening Response

Abstract

Alloimmunization is the formation of antibodies against non-self-antigens from a different member of the same species due to exposure to them via transfusion, pregnancy, or transplantation. Further to ABO(H) alloantigens, more alloantibody reactivity toward RBCs appeared as a result of transfusion evolution. Considering that nowadays RBC polymorphisms include more than 300 distinct alloantigens, alloantibodies produced against these antigens can cause various complications such as hemolytic disease of the fetus and newborn (HDFN) or hemolytic transfusion reactions (HTRs) which are related to significant morbidity and mortality. It seems that different factors can influence alloimmunization such as genetic factors, underlying diseases, infection, and inflammation. It is said that expanded antigen matching of RBCs is the only way to reduce transfusion-associated alloimmunization in the future but there is no way to fully eliminate the development and consequences of alloimmunization. So, it seems additional investigations are needed in this field.

1. Introduction

Alloimmunization is the formation of antibodies against non-self-antigens from a different member of the same species due to exposure to them via transfusion, pregnancy, or transplantation [1, 2]. The likelihood of alloimmunization can differ from population to population according to blood group antigens expression frequencies [3]. Despite many researches in this field, alloimmunization still remains a common and serious issue in blood transfusion and medical sciences [4]. Various complications such as hemolytic disease of the fetus and newborn (HDFN), erythroblastosis fetalis, or hemolytic transfusion reaction (HTR)s can happen through alloimmunization which can lead to significant morbidity and mortality [1, 4]. Because the increasing complexity of alloimmunization and the importance of improving blood transfusion safety, pregnancy-related care, and fetal/neonatal outcomes in patients, it seems additional investigations are needed to improve knowledge of the development and consequences of red blood cell (RBC) alloimmunization for earlier prevention, diagnosis, and effective treatment of it [5].

2. RBC alloantibody formation, detection, and evanescence

Alloimmunization can trigger an immune response, leading to the production of antibodies against the foreign RBC antigens [6]. A small number of blood recipients produce detectable alloantibodies because of having three conditions: (1) exposure to non-self RBC antigens, (2) sufficient dosage of antigen to provoke the immune system, and (3) having the human leukocyte antigen (HLA) to present those antigens [7, 8]. But unfortunately it has to be said that only 30% of induced RBC alloantibodies are detected which can be due to alloantibody evanescence (reduced over time) prior to the next alloantibody screen and/or insufficient sensitivity of commonly employed assays [5, 7]. Studies have indicated that around 70% of alloantibodies become undetectable just a few years after their initial formation [9]. To identify important alloantibodies, “screen” test is used, which is actually the indirect antiglobulin test (IAT) [7]. Traditional simple tube testing or newer methods such as solid-phase, gel technology, flow cytometry, or the enzyme-linked immunosorbent assay may help in completing antibody screening tests [1, 5].

3. RBC alloimmunization in the pregnant patient

Pregnancy is another common cause of alloimmunization, as maternal antibodies (IgG) which may be as a result of fetal/maternal hemorrhage during pregnancy/delivery, or via intrauterine transfusion (IUT), can cross the placenta and target fetal RBC surface antigens [1, 10]. Some factors such as prior major surgery, RBC or platelet transfusion, multiparity, prior male child, or operative removal of a prior placenta may be responsible for increased risk of alloimmunization [10]. It is said that HLA-DRB1*15-positive women are also more susceptible to antibody production [11]. In addition, a pregnant woman who has a history of drug use is at a higher risk of alloimmunization and it is probably because of needle-sharing [12]. Without intervention, maternal alloantibodies can cause hemolysis and suppress erythropoiesis, resulting in marked anemia and possibly immunosuppression in the fetus [1]. According to research, it is said that the major cause of fetal anemia is maternal RBC alloimmunization [13]. Up to 1 in 600 pregnancies are affected by maternal RBC alloimmunization and despite primary prevention strategies against RhD antigen, HDFN is mostly caused by anti-D alloantibodies [7]. Interestingly, some antibodies may not be clinically important because they are against antigens with low expression on RBC (anti-Lewis) or they are IgM antibodies that are incapable of crossing the placental barriers(anti-N) [10]. To check the presence or absence of maternal antibodies, IAT is recommended during pregnancy [13].

3.1 Hemolytic disease of the fetus and newborn

HDFN is a life-threatening disease that occurs due to the destruction of fetal erythrocytes by maternal IgG alloantibodies that persist for up to 6 months after birth and cause HDFN consequences till neonatal time [2, 14]. The risk of this disease increases in the second and third trimesters of pregnancy because of the increase in transplacental transfer [15]. It can be caused by more than 50 RBC alloantibodies [16]. It seems antigens that antibodies against them cause HDFN in order of importance are: D, c, K, E, Fya/Fyb, Jka/Jkb, and MNS [14]. Approximately 1.25% of pregnant women have clinically important RBC alloantibodies which affect approximately 1/300 to 1/600 of live births by causing HDFN [2]. It is said about 83% of HDFNs that occur are due to previous pregnancies, 3% are due to previous transfusions, and 14% are undetermined [17]. The history of HDFN in the mother’s previous pregnancy is very important because if there is HDFN in the previous pregnancy, the condition will be worse in the following one so previous obstetric history should be checked [2]. If the mother has clinically significant antibodies, the fetus should be examined for the expression of the relevant antigens [18]. Today, non-invasive techniques such as testing cell-free DNA from maternal plasma are used for this purpose [18]. Ultrasound-based techniques are also used for high-risk pregnancies to determine if the fetus is at risk of HDFN if required [19]. If after these investigations, the mother’s antibodies are considered dangerous for the fetus, then intermittent monitoring like examining the severity of the disease using the antibody titer (which is considered 1:8 for anti-kell and 1:16 or 1:32 for others as a critical titer) is needed [1]. If the critical titer is reached because one of the most clinically important manifestations of HDFN is fetal and neonatal anemia, Noninvasive detection of moderate and severe anemia can be achieved through the use of Doppler ultrasonography, which relies on the observation of an elevation in the peak velocity of systolic blood flow in the middle cerebral artery [15, 20]. Finally, if needed, IUT, intravenous immune globulin (IVIg), or plasma exchange can be used as therapeutic measures [1].

4. Factors suggested to modify alloimmunization risk

The process of alloimmunization is influenced by various factors [21]. According to research, some factors such as the female gender due to increased vulnerability during pregnancy, miscarriages, abortions, and childbirth or maybe it is based on the hypothesis that women have a stronger immune system than men, pro-inflammatory cytokines (due to promote antigen presentation), longstanding infection, diabetes, allogeneic hematopoietic stem cell transplantation, acute chest syndrome, Vaso-occlusive crisis, and solid tumors increase the risk of antibody production and alloimmunization while some others like T regulatory cells (through suppressing immune responses), lymphoproliferative disease, leukemia (because of lymphocyte dysfunction and immunosuppression), and symptomatic atherosclerosis have the opposite effect on it [22, 23, 24, 25, 26, 27, 28]. In addition, it is said that an elevated count of reticulocytes in RBC units increases the possibility of alloimmunization in patients receiving them [29]. The genetics of the patient also play a role in the alloimmunization process, particularly with HLA because of its role in the regulation of the immune system [3, 22]. Certain HLA alleles are associated with an increased risk of alloimmunization [3]. It is also interesting that some HLA-IIs are associated with some specific antibody formation, for example, HLA-DRB1*04 with Anti-Fya, HLA-DRB1*11 & -DRB1*13 with Anti-K, and HLA*DRB1*15 with Rh system [3]. In addition to genetic factors, other environmental factors such as antigen disparity between patients and donors, age at first transfusion, and severity of underlying diseases may also contribute to the development of red blood cell alloimmunization [9, 22]. It is believed that factors such as the age and number of red blood cell units transfused and the type and amount of immunogenic antigens encountered during transfusion also may be responsible for modifying alloimmunization [8, 22]. Generally, the risk of alloimmunization depends on both the donor and recipient, which will be discussed below separately.

4.1 Donor factors

Genetic factors, length of RBC storage, contamination, and damage to RBCs are responsible for the increased risk of RBC alloimmunization [5, 9]. According to research, it seems that the age of RBC unit has a significant relationship with alloantibody formation, so older RBC units, due to having a higher amount of intracellular heme with a negative effect on the heme oxygenase system, lead to an increase in the level of oxidative stress, inflammation and as a result, alloimmunization [30]. Also, it is said, RBC units that obtained from male donors exhibit a greater susceptibility to storage-related degeneration and hemolysis probably because of testosterone [31].

4.2 Recipient factors

Recipients are divided into three groups based on the production of antibodies: (1) the individual who does not produce alloantibodies despite repeated exposure to foreign blood group antigens called “non-responder,” (2) the one who produces just one antibody regardless of the number of exposures called “responder,” and (3) an individual who produces more than one antibody independent of the number of exposures called “hyper responder” [8]. Various factors cause these differences, for example, some factors such as having certain genetic factors like TNF, MALT1, TLR1, STAT1, TANK, IKK1, IL-2, ADRA1b, IL-6, IL-1B, CTLA4, and some HLA variants including HLA-DRB1*04, -DRB1*15, and -DQB1*03, female sex, prior exposure, method of exposure, antigen dose, viral infection, autoimmunity, myelodysplastic syndrome (MDS), sickle cell disease (SCD), thalassemia, experiencing febrile transfusion reactions, and inflammatory bowel disease (IBD) are responsible for increased risk of RBC alloimmunization [3, 5, 8, 9, 32, 33, 34]. In opposite, Some others like older age (it has been reported that individuals aged over 77 years are at a lower risk of blood group antigen alloimmunization probably because of immunosuppression), gram-negative infection, bone marrow failure, acute myeloid or lymphoid leukemia, immunosuppressive drugs, chronic liver or renal failure (CRF), and various genetic factors (like IL-10, TLR7, STAM, OX40L, IFNAR1, STAT4, IRF7, and FCGR2) can reduce the risk of alloimmunization [5, 9]. Some diseases that affect the rate of alloimmunization are summarized in Table 1.

Disease	Alloimmunization rate (%)	Effect on alloimmunization	Reason of effect	References
Sickle cell disease	19–43	Increase	Frequent blood transfusion reduces Treg suppressive function	[3, 6, 35]
Thalassemia major	5–45	Increase	Repeated RBC transfusions	[6, 36]
Myelodysplastic syndrome	15–59	Increase	Higher utilization of blood transfusions, changes in the immune system	[6, 8]
Chronic liver or renal failure	1.3	Decrease	Hampered (humoral) immune response, renal replacement therapy (RRT) mechanistically modulates RBC alloimmunization	[37, 38]
Inflammatory bowel disease	8–9	Increase	Inflammation	[6, 33]
Aplastic anemia	11	Increase	Repeated RBC transfusions	[39, 40]

Table 1.

A review of some diseases affecting the rate of alloimmunization.

5. Clinical significance of RBC alloimmunization

Various complications happen through alloimmunization which can lead to significant morbidity and mortality [4, 5]. Among these complications, alloimmunization has the greatest contributions to HDFN and delayed hemolytic transfusion reactions (DHTRs) [9, 41]. In addition, there are some diseases in which the high level of alloimmunization has brought consequences [42]. For example, in acute myeloid leukemia (AML), aplastic anemia (AA), hematopoietic progenitor cell transplant, non-Hodgkin lymphoma (NHL), solid tumors, and especially MDS and SCD patients due to the high-risk of alloimmunization, it is very difficult to find compatible blood and blood transfusions may be delayed in them, which can be dangerous [42].

Among other important changes that occur during alloimmunization, we can mention decreased CD4/CD8 ratio, increased B lymphocytes as well as CD8+ lymphocytes, and Treg lymphocyte deficiency which upset the balance of the immune system and bring consequences [43].

6. Clinical management

The easiest and most effective strategy to prevent alloimmunization is to limit blood transfusion and use it only in necessary cases [7]. Using AABB clinical guidelines can help us to make a more accurate diagnosis [44]. The next step to reduce the possibility of alloimmunization is to find the most antigenically similar RBC unit to the recipient by genotyping the recipient for various minor RBC antigens to pick up the most antigen-matched RBC units [8, 45]. Because this practice may not be economical in many cases, it is suggested that the prophylactic matching for antigens other than ABO and Rh be performed for people at risk such as thalassemia major, SCDs, AA, MDS, chronic myeloproliferative disease and other malignancy, CRF who require repeated RBC transfusions, and also pregnant women [8, 40, 46]. It should be noted that it is better to record and store the information obtained in the first hospital or center that the patient visited and make it available to other medical centers using electronic databases [7, 47]. This strategy in addition to limiting the transfusion record fragmentation and duplicated tests and procedures, helps to save time and money and increases the safety of blood transfusion; because if the antibodies are not detectable to a hospital for any reason, then the information collected by previous hospitals can be helpful [7, 48]. However, despite the advantages of this method, its use is still controversial and needs improvement due to the errors that have sometimes occurred [1, 48]. According to recent researches, leukoreduced RBC units may decrease the incidence of RBC alloimmunization [49]. Using immunosuppressants like corticosteroids also has been shown a significant protective effect against alloimmunization [50]. The effect of splenectomy on alloimmunization is controversial, however, in a study by Evers et al., splenectomy was found to be significantly associated with protection against primary alloimmunization [51]. These strategies are used to prevent the formation of alloantibodies but if alloantibodies are formed, an action should be taken to limit their further development and destructive effects [8]. One approach to prevent the further spread of alloantibodies and reduce the rate of RBCs destruction is immunosuppression using IVIg and corticosteroids [9]. Also, it is said that the use of anti-CD20 antibodies, B cell depletion, or plasma cell targeting in humans has been associated with preventing the formation of new antibodies; however, their definitive effect on alloimmunization needs further investigation [5, 52]. In addition, C5 inhibitor and eculizumab may be advantageous in restricting alloantibody-mediated hemolysis [9].

7. Conclusions

Numerous influential factors play a role in the decrease or increase of alloimmunization, making it a multifactorial phenomenon [24]. Despite many researches that have been done in this field, the effect of some factors on alloimmunization is controversial and the researchers have not yet reached an agreement regarding the definitive effect of these factors on alloimmunization. For example, the effect of splenectomy on alloimmunization has been different in several studies, so that in some studies it has been introduced as a risk factor for alloimmunization and in others as a factor to reduce it [51, 53, 54]. The complexity of alloimmunization, along with the variable titer of antibodies during different times and the difficulty of identifying alloantibodies, has made it still have many hidden aspects [5]. Additionally, inadequate recognition of pregnancy alloimmunization causes HDFN to still remain as a serious complication [2, 55]. So, new mitigation and detection strategies and novel therapies of RBC alloimmunization are needed to improve transfusion and pregnancy safety and limit its associated morbidity and mortality, and also it is critical to conduct more investigations regarding better understanding of risk factors for alloantibodies development.

Conflict of interest

The authors declare no conflict of interest.