**3.1 Serology typing**

Knowledge of the role of blood groups with their antigens and variants in alloimmunization was pivotal for the development of transfusion practices and medical interventions that require blood transfusion such as trauma, hematological diseases (e.g. SCD, MDS, thalassaemia, and aplastic anemia) and later for transplantation and cancer treatment.

Serology has been considered the gold standard technique for blood group typing for a long time. Serological methods detect the antigen expressed on the red cell using specific antibodies and can be carried out manually or by automated platforms. Typing of blood group antigens using this method is easy, fast, reliable, and accurate for most of the antigens. However, serology has limitations, some of which cannot be overcome when it is used as a standalone testing platform (**Table 1**). Scarcity of serological reagents for some blood group systems for which there is no monoclonal antibody available is a major limitation. In addition, human serum samples from different donors vary in reactivity, which is an issue when a nearly exhausted batch of reagent needs to be replaced. This is especially problematic when an alloantibody for that antigen is suspected to be causing adverse events after transfusion. In those circumstances, molecular methods can be used as an alternative or as a complementary test for identification of genes associated with the blood group antigens expression and prediction of antigenic profile (see **Table 1**).

### **3.2 Molecular typing**

The identification of genes that encode proteins carrying blood group antigens and the molecular polymorphisms that result in distinct antigenicity of these proteins is possible using molecular typing methods, which facilitate blood typing


**205**

*Accuracy of Blood Group Typing in the Management and Prevention of Alloimmunization*

resolution in complex cases and overcome limitations of serological techniques when dealing with alloimmunized and multitransfused patients. In addition, molecular techniques have allowed identification of genes encoding clinically relevant antigens for which serological reagents are not available (see **Table 1**). In

Blood group genotyping is performed to predict blood group antigens by identifying specific polymorphisms associated with the expression of an antigen. Most variations in the blood group antigens are linked to point mutations, but for some, other molecular mechanisms are responsible, such as deletion or insertion of a gene, an exon or a nucleotide sequence (i.e. ABO, RH, and DO blood group systems), sequence duplication, (i.e. RHD gene and GE blood group system), nonsense mutation (i.e. RHD gene), and hybrid genes (i.e. RH, MNS, ABO, and CH/RG blood group systems) [30]. In contrast to serology, molecular tests are performed on DNA obtained from nucleated cells and are not affected by the presence of donor's red cells in patient's sample, which is a common occurrence in samples of patients with recent/multiple blood transfusions. Thus, RBC genotyping can resolve blood group typing discrepancies in multitransfused patients presenting with mixed field reactions, alloantibodies, or autoantibodies (**Table 1**). Also, blood group genotyping can substantially help patients who were not previously phenotyped and need regular transfusions by facilitating management of these patients and preventing alloimmunization [31]. Studies comparing serology and genotyping in multitransfused population such as patients with thalassaemia and SCD have shown that genotyping is superior to serology for resolving discrepancies [31–35]. Use of genotyped matched units has been shown to decrease alloimmunization rates [36], increase hemoglobin levels and in vivo RBC survival, and diminish frequency of transfusions [37–39].

SCD is the most common congenital red blood cell disorder affecting millions of people worldwide with high mortality and morbidity rates [40]. It is considered a major public health issue by the WHO. Characterized by an abnormal synthesis of hemoglobin, this genetic trait is most common among people of African ancestry. Abnormal hemoglobin carried in red cells causes these cells to sickle (thus the name SCD), which as early symptoms produces swelling of the hands and feet, anemia, fatigue, and jaundice. Long-term effects of the disease include serious damage in spleen, brain, eyes, lungs, liver, heart, kidneys, bones, and/or skin that can accumulate over a person's lifetime. Patients can survive beyond their 50s, and most fatalities are not associated with chronic organ failure but occur due to an acute episode of one of the SCD complications. SCD can be cured by bone marrow transplanta-

Blood transfusion therapy is part of treatment for SCD patients and it is mainly indicated for prevention of stroke and vaso-occlusive crisis. However, transfusion benefits are limited upon development of alloantibodies, a common adverse event of transfusion. The high incidence of RBC alloimmunization in SCD is multifactorial, but lack of blood group compatibility between donor and recipient is a key factor. This is more evident in countries where donors' and patients' ethnicities significantly diverge, that is, in North America, where blood donors are mostly Caucasians while SCD patients are predominantly of African descent; thus, SCD patients are frequently exposed to foreign antigens and, consequently, have higher

Transfusion protocols for management of SCD and prevention of alloimmunization vary among the hospitals and transfusion services. In many centers that provide care to SCD patients, transfusions are phenotypically matched for RH

those instances, genotyping is critical to resolve clinical challenges.

*DOI: http://dx.doi.org/10.5772/intechopen.90095*

**3.3 Sickle cell disease**

tion, but only a few patients get transplant.

risk of developing multiple alloantibodies.

#### **Table 1.**

*Benefits of genotyping over serology.*

#### *Accuracy of Blood Group Typing in the Management and Prevention of Alloimmunization DOI: http://dx.doi.org/10.5772/intechopen.90095*

resolution in complex cases and overcome limitations of serological techniques when dealing with alloimmunized and multitransfused patients. In addition, molecular techniques have allowed identification of genes encoding clinically relevant antigens for which serological reagents are not available (see **Table 1**). In those instances, genotyping is critical to resolve clinical challenges.

Blood group genotyping is performed to predict blood group antigens by identifying specific polymorphisms associated with the expression of an antigen. Most variations in the blood group antigens are linked to point mutations, but for some, other molecular mechanisms are responsible, such as deletion or insertion of a gene, an exon or a nucleotide sequence (i.e. ABO, RH, and DO blood group systems), sequence duplication, (i.e. RHD gene and GE blood group system), nonsense mutation (i.e. RHD gene), and hybrid genes (i.e. RH, MNS, ABO, and CH/RG blood group systems) [30].

In contrast to serology, molecular tests are performed on DNA obtained from nucleated cells and are not affected by the presence of donor's red cells in patient's sample, which is a common occurrence in samples of patients with recent/multiple blood transfusions. Thus, RBC genotyping can resolve blood group typing discrepancies in multitransfused patients presenting with mixed field reactions, alloantibodies, or autoantibodies (**Table 1**). Also, blood group genotyping can substantially help patients who were not previously phenotyped and need regular transfusions by facilitating management of these patients and preventing alloimmunization [31].

Studies comparing serology and genotyping in multitransfused population such as patients with thalassaemia and SCD have shown that genotyping is superior to serology for resolving discrepancies [31–35]. Use of genotyped matched units has been shown to decrease alloimmunization rates [36], increase hemoglobin levels and in vivo RBC survival, and diminish frequency of transfusions [37–39].

#### **3.3 Sickle cell disease**

*Human Blood Group Systems and Haemoglobinopathies*

Fyb

), JK (Jk<sup>a</sup>

**3.1 Serology typing**

and cancer treatment.

**3.2 Molecular typing**

antisera (i.e. Doa

and VS)

hybrids)

rare RBC phenotypes

*Benefits of genotyping over serology.*

No available antisera, weak or limited

, Dob , Jsa , Jsb , Kpa , Kpb , V

Mixed field caused by the presence of donor's RBCs in patient's sample (i.e. patients with recent blood transfusions) Presence of interfering antibodies (i.e. autoantibodies, multiple antibodies, antibodies against high prevalence antigens)

Presence of variant antigens (i.e. hybrid RH types, FY silencing mutations, MNS

Detection of blood type of fetus at risk of HDFN without invasive procedure

Mass screening for antigen-negative and

, Jkb

Verification of compatibility for Rh (D, E, C, c, e) and K, which are the most frequent antigens involved in alloimmunization, is considered partial matching. Extended matching should include at least RH (D, C, E, c, e), KEL (K), FY (Fya

), MNS (S, s) and, if available, additional antigens.

Knowledge of the role of blood groups with their antigens and variants in alloimmunization was pivotal for the development of transfusion practices and medical interventions that require blood transfusion such as trauma, hematological diseases (e.g. SCD, MDS, thalassaemia, and aplastic anemia) and later for transplantation

Serology has been considered the gold standard technique for blood group typing for a long time. Serological methods detect the antigen expressed on the red cell using specific antibodies and can be carried out manually or by automated platforms. Typing of blood group antigens using this method is easy, fast, reliable, and accurate for most of the antigens. However, serology has limitations, some of which cannot be overcome when it is used as a standalone testing platform (**Table 1**). Scarcity of serological reagents for some blood group systems for which there is no monoclonal antibody available is a major limitation. In addition, human serum samples from different donors vary in reactivity, which is an issue when a nearly exhausted batch of reagent needs to be replaced. This is especially problematic when an alloantibody for that antigen is suspected to be causing adverse events after transfusion. In those circumstances, molecular methods can be used as an alternative or as a complementary test for identification of genes associated with the blood

group antigens expression and prediction of antigenic profile (see **Table 1**).

and the molecular polymorphisms that result in distinct antigenicity of these proteins is possible using molecular typing methods, which facilitate blood typing

**Serology limitation Genotyping application**

The identification of genes that encode proteins carrying blood group antigens

Blood group typing can be easily performed by single PCR

Detection of genes and molecular mechanisms associated

Detection of blood type and zygosity on DNA extracted from

Use of high throughput platforms for donor screening allows

mass scale typing and creation of databases

Genotyping is performed with extracted DNA from nucleated cells (e.g. leukocytes, epithelial). The presence of donor's RBCs or interfering antibodies in patient's sample

and/or high throughput platform

does not interfere with the results

with variant antigen expression

maternal plasma

,

**204**

**Table 1.**

SCD is the most common congenital red blood cell disorder affecting millions of people worldwide with high mortality and morbidity rates [40]. It is considered a major public health issue by the WHO. Characterized by an abnormal synthesis of hemoglobin, this genetic trait is most common among people of African ancestry. Abnormal hemoglobin carried in red cells causes these cells to sickle (thus the name SCD), which as early symptoms produces swelling of the hands and feet, anemia, fatigue, and jaundice. Long-term effects of the disease include serious damage in spleen, brain, eyes, lungs, liver, heart, kidneys, bones, and/or skin that can accumulate over a person's lifetime. Patients can survive beyond their 50s, and most fatalities are not associated with chronic organ failure but occur due to an acute episode of one of the SCD complications. SCD can be cured by bone marrow transplantation, but only a few patients get transplant.

Blood transfusion therapy is part of treatment for SCD patients and it is mainly indicated for prevention of stroke and vaso-occlusive crisis. However, transfusion benefits are limited upon development of alloantibodies, a common adverse event of transfusion. The high incidence of RBC alloimmunization in SCD is multifactorial, but lack of blood group compatibility between donor and recipient is a key factor. This is more evident in countries where donors' and patients' ethnicities significantly diverge, that is, in North America, where blood donors are mostly Caucasians while SCD patients are predominantly of African descent; thus, SCD patients are frequently exposed to foreign antigens and, consequently, have higher risk of developing multiple alloantibodies.

Transfusion protocols for management of SCD and prevention of alloimmunization vary among the hospitals and transfusion services. In many centers that provide care to SCD patients, transfusions are phenotypically matched for RH

(D, C/c, E/e) and K [41], while others provide extended matching including RH (D, C, c, E, e), KEL (K, k), FY (Fya , Fyb ), JK (Jk<sup>a</sup> , Jkb ), and MNS (S, s) in addition to the standard ABO and Rh(D). Less frequently, extended matching is performed by genotyping [36]. However, a wide range of institutions do not request phenotypically matched RBC units until the patient has produced an alloantibody [42].

It has been reported that antibodies against Rh antigens are the most frequently identified antibodies in multitransfused SCD patients despite transfusion from Rh phenotype matched donors [15]. The main reason for that is the high frequency of Rh variants in people of African descent. It has been reported that 90% of SCD patients and donors of African ancestry have at least one variant *RHD* or *RHCE* allele [15, 43]. The term "variant" is used when *RHD* and/or *RHCE* genes are carrying genetic alterations that may affect the RhD and RhCe protein expression. Variant alleles can encode weak and/or altered antigens and serological methods are limited in which variant Rh antigens can be identified and might not be reliable [44, 45].

The prophylactic RBC matching performed by serology typing, before exposure to RBC transfusions, can decrease transfusion complications in SCD patients substantially, but does not eliminate the occurrence of alloimmunization against Rh variants and other non-matched antigens that can cause DHTR [41]. Currently available molecular typing methods can predict several blood group antigens allowing a more precise RBC matching and can support transfusion decision-making. *RHD* and *RHCE* genotype matching particularly benefits SCD patients carrying Rh variants. For instance, SCD patients presenting D+ or e+ phenotype can make alloantibodies to these antigens despite receiving Rh phenotyped matching RBCs. The molecular analysis in such individuals may identify polymorphisms in *RH* genes responsible for the phenotypic alteration, confirming the alloimmune status of the antibody. In such cases, provision of *RH* genotype matched units or units negative for the specific antigen would be recommended, because the antibodies produced may be clinically relevant [13, 15].

An additional benefit of blood group genotyping on transfusion management of SCD patients is the capability of identifying silence mutations like −67T>C in the ACKR1 gene (Duffy gene). Patients carrying the mutation can receive Fy(b+) units, because the mutation only abolishes expression of Fy(b) on red cells but not in other tissues. The detection of this mutation avoids unnecessary use of Fy(b−) and increases the chances to find compatible units available even for highly restrictive matching.

Extended genotyping including Dombrock: Do<sup>a</sup> , Dob , Joa , Hy; Kell: Kpa , Kpb , Js<sup>a</sup> , Jsb ; Rh: V, VS; Colton: Co<sup>a</sup> , Cob ; Cartwright: Yta , Ytb ; Lutheran: Lua , Lub ; Diego: Di<sup>a</sup> , Dib ; and Scianna: Sc1, Sc2 may help prevent development of clinically significant antibodies that can be potentially life-threatening.

#### **3.4 Thalassaemia**

Thalassaemia is an inherited blood disorder associated with a mutation in one of the genes involved in hemoglobin production resulting in abnormal form or an inadequate amount of hemoglobin. RBCs carrying abnormal hemoglobin do not function properly and are destroyed in large numbers leading to anemia. People with thalassaemia may have mild or severe anemia depending on the type of thalassaemia. Severe anemia requires regular blood transfusions to maintain the hemoglobin and RBCs levels, and to suppress the ineffective erythropoiesis but can lead to alloimmunization.

The prevalence of alloimmunization in patients with thalassaemia varies among geographical locations and may be related to the heterogeneity of population, transfusion exposure frequency, patients age, antigen matching policy, recipient related

**207**

*Accuracy of Blood Group Typing in the Management and Prevention of Alloimmunization*

decreasing rates of alloantibody and autoantibody formation [46–48].

procedures for phenotyping and transfusion matching [42].

**3.5 Rare type blood donor selection**

for clinically relevant antigens.

genotyping matching strategy.

matching policies, therefore the phenotyping might not be reliable.

factors, and other factors [46]. The most common alloantibodies reported in these patients are against RH (primarily anti-E and anti-C) and K, followed by antigens of the FY, JK, MNS, and other blood group systems. Development of autoantibodies is also commonly observed in these patients. A policy for RH and KEL matching been introduced worldwide and its effectiveness has been demonstrated by the

The *Thalassaemia International Federation* guidelines for transfusion-dependent thalassaemia published in 2014 recommends that all patients should receive prophylactic ABO, RH (D, C, c, E, e), and KEL (K) matched transfusions identified either with serology or genotyping. In addition, it is indicated that antigen typing should be performed using molecular rather than serologic testing if the patient had received transfusions previously [49]. However, surveys have reported that there is still a lack of adherence to recommendations and a large variation in transfusion practices for thalassaemia and SCD patients among the health care systems [42]. Some of the challenges for transfusion centers include difficulty in obtaining a reliable antibody and transfusion history and the lack of standards regarding

Molecular typing has been introduced in several centers to confirm extended blood group profiles obtained through serological methods, however, it is not routine yet [31, 32, 42]. This approach is particularly important for thalassaemia patients because these patients are transfusion-dependent and, in many circumstances, might have received transfusions at hospitals with different transfusion

Alloimmunized patients require transfusion of RBCs that are negative for a particular antigen. Historically, serology methods, which are labor-intensive and time-consuming, have been used to screen for antigen-negative units. However, the standard practice is likely to change with the high-throughput platforms for blood group genotyping being approved by regulatory bodies and becoming more widely used. High-throughput platforms allow identification of a higher number of antigens compared with serology, increasing the availability of blood characterized

The implementation of RBC mass scale genotyping for donor screening has started in blood centers, especially in large collecting facilities [50–53]. The successful establishment of a blood group genotype database has already been accomplished aiming to fulfill antigen-negative requests, especially for SCD patients receiving regular transfusions, and to create an inventory of frozen red cell units with rare blood types [51]. The refereed database comprises 43,066 non-Caucasian blood donors genotyped for 32 single nucleotide polymorphisms, related to the expression of 42 blood group antigens. The report showed that within 4 years of starting RBC genotyping, the blood group antigen database generated on blood donors was fivefold larger than that obtained by serology methods over 30 years. In addition, most antigen-negative units

requests to that center were met using exclusively the genotyping database.

Strategies for finding units to fulfill transfusion requests for SCD patients have included RBC genotyping of non-Caucasian blood donors and donors with altered Rh antigen expression [54, 55]. The genotyping selection of donors with a genetic background similar to that of SCD patients' increases the chances of finding compatible blood for these patients, including RH-genotype matching. However, the low percentage of blood donors with African ethnic background combined with the cost of genotyping are limiting factors for widespread use of extended RBC

*DOI: http://dx.doi.org/10.5772/intechopen.90095*

#### *Accuracy of Blood Group Typing in the Management and Prevention of Alloimmunization DOI: http://dx.doi.org/10.5772/intechopen.90095*

factors, and other factors [46]. The most common alloantibodies reported in these patients are against RH (primarily anti-E and anti-C) and K, followed by antigens of the FY, JK, MNS, and other blood group systems. Development of autoantibodies is also commonly observed in these patients. A policy for RH and KEL matching been introduced worldwide and its effectiveness has been demonstrated by the decreasing rates of alloantibody and autoantibody formation [46–48].

The *Thalassaemia International Federation* guidelines for transfusion-dependent thalassaemia published in 2014 recommends that all patients should receive prophylactic ABO, RH (D, C, c, E, e), and KEL (K) matched transfusions identified either with serology or genotyping. In addition, it is indicated that antigen typing should be performed using molecular rather than serologic testing if the patient had received transfusions previously [49]. However, surveys have reported that there is still a lack of adherence to recommendations and a large variation in transfusion practices for thalassaemia and SCD patients among the health care systems [42]. Some of the challenges for transfusion centers include difficulty in obtaining a reliable antibody and transfusion history and the lack of standards regarding procedures for phenotyping and transfusion matching [42].

Molecular typing has been introduced in several centers to confirm extended blood group profiles obtained through serological methods, however, it is not routine yet [31, 32, 42]. This approach is particularly important for thalassaemia patients because these patients are transfusion-dependent and, in many circumstances, might have received transfusions at hospitals with different transfusion matching policies, therefore the phenotyping might not be reliable.
