**3.1 Intravascular haemolysis**

*Human Blood Group Systems and Haemoglobinopathies*

transfused red blood cell concentrate [5].

1.69/100,000 per year [7].

**3. Mechanisms of haemolytic transfusion reactions**

between 1:10,000 and 1:50,000 transfused blood components [3, 4]. In contrast, the incidence for patients receiving a transfusion is estimated to be higher (about 1:500–1:800 patients) because most recipients receive more than one blood unit. It is worth noting that the estimation of the frequency of haemolytic reactions depends on the number of transfusions in a given centre. Thus, in large clinical centres, where severely ill patients are treated, more of these events are recorded [4]. A report issued by the Quebec Haemovigilance System covering 5 years of observation described 47 ABO incompatibility reactions, 55 cases of acute haemolytic transfusion reaction and 91 cases of delayed transfusion reaction in reference to 7059 all reported transfusion reactions. It was estimated that the frequency of reactions resulting from the ABO incompatibility was 1:27,318, acute haemolytic transfusion reactions 1:14,901 and delayed haemolytic transfusion reactions 1:9313 per unit of

The most common reaction among the acute (approximately 30%) was haemolysis resulting from ABO incompatibility [5]. In the annual report Serious Hazards of Transfusion (SHOT), published in England, in 2017, 42 haemolytic transfusion reactions were reported in reference to 3230 of all reactions observed following transfusion of blood components, of which 13 cases of acute haemolytic transfusion reaction and 29 cases of delayed haemolytic reaction (including 6 cases of hyperhemolysis) were reported. The number of reported cases of delayed haemolytic transfusion reaction was higher than in 2016, but comparable with previous years [6]. Factors that can affect the increase in the number of delayed haemolytic reactions include correctness in carrying out serological tests, longer survival of patients after transfusions and an increase in the number of transfused blood components. Since most patients receive more than one unit of red blood cell concentrate, the estimated incidence of delayed haemolytic transfusion reactions is from 1:854 to 1:524 per patient who has been transfused and is higher than per transfused unit [7]. In the population, delayed haemolytic transfusion reactions occur with a frequency of

Red blood cells undergo haemolysis in the intravascular mechanism, in blood or extravascular vessels, that is, organs involving cells of the reticuloendothelial system, primarily spleen and/or liver. Clinically significant differences between the above mechanisms of red blood cells destruction are based on the time of onset of haemolysis and the destruction rate of red blood cells. Intravascular haemolysis is characterised by the destruction of red blood cells at a rate of about 200 ml of transfused cells within 1 h of transfusion. It is manifested by a rapid decrease in haemoglobin, haemoglobinemia and haemoglobinuria and can potentially be life threatening [2]. In contrast, extravascular haemolysis is less dramatic, with a rate of destruction of red blood cells of approximately 0.25 ml/h/1 kg of recipient's body weight. For example, for 70 kg recipient, about 18 ml of transfused red blood cells are destroyed per hour. However, it is worth noting that despite the low intensity of haemolysis, the survival time of red blood cells after transfusion is significantly reduced [2]. In general, intravascular haemolysis is called as an early acute haemolytic transfusion reaction. It can occur during transfusion or up to 24 h after transfusion of red blood cells. In comparison extravascular haemolysis is called delayed haemolytic transfusion reaction and usually occurs 24 h or days after the end of the transfusion. The quoted breakdown of reactions is somewhat artificial, because the symptoms associated with haemolytic reactions sometimes overlap [1].

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Most often intravascular haemolysis is the result of the destruction of red blood cells by the complement system, stimulated by the presence of alloantibodies or autoantibodies. Among alloantibodies, such haemolysis is induced by anti-A and anti-B, rarely anti-Jka, anti-Jkb, anti-Vel, anti-P, anti-Lea and very unique antibodies with other specificities [10, 11]. In all these cases, haemolysis takes place via the classical pathway of complement activation. Its occurrence and severity, in addition to the class of antibodies, is also affected by the number of antigenic determinants with which the antibodies react. The reaction is most severe in the case of antigens A and B, because their number is estimated at about 5 × 105 per cell [12, 13]. In contrast, the presence of antigens from the Rh, Kell, Kidd and Duffy systems on the surface of red blood cells is determined in the range of 103 –104 per cell [12]. **Table 1** shows the number of antigenic determinants on the cell surface for selected red blood cell antigens.

Antibodies combined with antigens by triggering the complement cascade lead to cell lysis. This mechanism is called the classic pathway for complement activation and is shown in **Figure 1**.

The starting point is the antigen-antibody complex present on the surface of the cell membrane [14, 15]. Antibodies of the IgM and IgG class (outside the IgG4 subclass) bind the C1q protein in the initial stage of activation. The condition for complement activation is the binding of the C1q molecule by two Fc fragments of adjacent IgG antibodies or by one IgM molecule. It should be noted here that the IgM class is more efficient in starting the process of complement activation than the IgG class [2, 15]. The C1qrs complex is created and activates the C2 and C4 components and their distribution into C2a and C2b as well as C4a and C4b. The C4b2a


#### **Table 1.**

*Number of antigenic determinants on the cell surface of the red blood cell (according to [12, 13]).*

**Figure 1.** *The classic pathway for complement.*

complex has proteolytic properties and is called C3 convertase. Convertase breaks down molecules of C3 into C3a, C3b, C3c and C3d. The C3b and C3d components bind with the red blood cell membrane and in many cases the complement cascade process ends. In other cases, the C3b component activates C5 and C5a and C5b are formed. C5b binds to C6, then to C7. This creates a complex of three C5b-6-7 particles, which is partially incorporated into the cell membrane and further binds C8. The C5b-8 complexes create holes in the cell membrane that increase when exposed to the C9 component. The C5B-C9 complex called membrane attack complex (MAC) creates pores in the cell membrane of a red blood cell that are 1/700 of its size. Haemoglobin escapes from the cells into the plasma, and the effects of haemolysis are visible macroscopically in the plasma of the blood sample [15].

The alternative path of complement activation and the lectin path of complement activation do not play a role in the destruction of red blood cells. Although the mechanism of the lectin route may be the reason for the in vivo ineffectiveness of the use of monoclonal and recombinant antibodies, which are thus eliminated from the body before they fulfil their function, for example, anti-D Ig for prevention purposes in RhD maternal-foetal conflict [16].

However, the complement system does not work specifically. The safety of body cells is enabled by factors that regulate complement activity present in plasma and on cells of various tissues, including red blood cells. Membrane inhibitor of reactive lysis (MIRL) (CD59) and decay accelerating factor (DAF) (CD55) are essential to protect red blood cells from haemolysis. The expression of these membrane inhibitors is associated with Cromer group system and CD59. On blood cells with the Cromer mull phenotype, known as Inab, DAF inhibitor expression is absent [17, 18]. DAF regulates C3a-converting activity. MIRL inhibits membrane attack complex [15, 17]. Lack of these particles may increase the susceptibility of red blood cells to intravascular haemolysis due to complement activation [19].

#### **3.2 Extravascular haemolysis**

In a situation in which, despite activation of the complement system, through antigen-antibody reaction, there is no intravascular haemolysis, red blood cells with detectable C3b component remain in the circulation. This kind of mechanism of red blood cell destruction occurs for IgG antibodies with complement system [13]. They may interact with CR1 and CR3 receptors on macrophages and consequently undergo phagocytosis. Most of the cells coated by the complement C3b component are destroyed by liver macrophages, that is, by Kupffer cells, while the cells coated with antibody molecules are mainly destroyed by spleen macrophages. They have surface receptors that recognise antibody classes and subclasses, and complement components,

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*Post-Transfusion Haemolytic Reactions DOI: http://dx.doi.org/10.5772/intechopen.91019*

**reactions**

of which the Fc R1 receptor is specific for red cells coated with antibodies [1]. Blood cells connected to this receptor are destroyed in the process of antibody-dependent cytotoxicity. Red blood cells can be absorbed and completely "digested" inside the macrophage. They can also be partially absorbed and then the integrity of the cell membrane is disturbed by the loss of proteins and lipids, which changes its osmotic properties. Such a blood cell, after being released from the macrophage, circulates in the blood as a spherocyte, whose survival is short. The macrophage cytotoxins are another mechanism that plays a role in the destruction of red blood cells. As a consequence of antibody-dependent cell-mediated cytotoxicity (ADCC) haemoglobinemia and haemoglobinuria may occur similarly to intravascular haemolysis, although the

**4. Mediators of inflammatory reactions in haemolytic transfusion** 

development of intravascular haemolytic transfusion reaction [15].

CCL2 is mainly a chemotactic and activating factor for monocytes [1, 12].

In incompatibility, in which non-complement IgG antibodies cause extravascular haemolysis, cytokines belonging to two categories differing in response rates are produced: (1) synthesised at a concentration higher than 1 μg/ml within 24 h and (2) synthesised at a concentration of about 100 pg/ml. Low concentration cytokines include IL-1β, IL-6 and TNF-α. CXCL8 concentration is similar to that in intravascular haemolysis, whereas TNF-α is synthesised at low concentration, estimated at <100 pg/ml [1, 2]. IL-1ra (receptor antagonist) is produced in extravascular haemolysis, which is an IL-1 receptor antagonist. Its presence to some extent affects some clinical differences between extravascular and intravascular haemolysis [23]. IL-1β concentration and IL-6 produced by monocytes in response to red blood cells coated with IgG antibodies increase progressively within 24 h to a concentration of 100 pg/ml. Since IL-1β and IL-6 affect proliferation and differentiation

Receptors for complement activation products C3a and C5a are found on many cells: monocytes, macrophages, neutrophils, platelets, endothelium and smooth muscle. Their release causes an increase in the concentration of oxygen radicals, leukotrienes, nitric oxide and cytokines. The increase in cytokine release may also be due to the interaction of Fcγ R1 receptors with IgG molecules associated with red blood cells. Udani et al. [20] showed in vitro that in the case of ABO incompatibility, monocytes are directly involved in the formation of acute haemolytic transfusion reaction [15]. Incompatible red blood cells reduce CD14 expression and increase CD44 expression on monocytes in whole blood. After 24 incubations with incompatible red blood cells, monocytes show a significant increase in CD44 levels. The results of these studies indicate a critical role of monocyte activation in the

In ABO incompatibility, in which anti-A, anti-B and anti-AB antibodies activate complement leading to intravascular haemolysis, a large amount of tumour necrosis factor-α (TNF) and interleukins CXCL8 (IL-8) and CCL2 are released into the plasma (MCP-1) [19–21]. TNF-α is released first, its elevated concentration is already detected within first 2 h. It carries a pro-inflammatory potential that is responsible for fever, leukocyte activation, stimulation of procoagulant activity, increased antibody production and vascular wall permeability [22]. TNF-α also stimulates endothelial cells to synthesise adhesion molecules and chemotactic cytokines [22]. CXCL8 and CCL2 produced in the blood during ABO incompatibility will appear later than TNF-α in very high concentrations. CXCL8 primarily activates neutrophils, which leads to the accumulation of leukocytes in the lung vessels of small diameter and damage to the endothelium of blood vessels and their higher permeability [1, 12].

antibodies that caused it do not bind complement components.

#### *Post-Transfusion Haemolytic Reactions DOI: http://dx.doi.org/10.5772/intechopen.91019*

*Human Blood Group Systems and Haemoglobinopathies*

purposes in RhD maternal-foetal conflict [16].

**3.2 Extravascular haemolysis**

intravascular haemolysis due to complement activation [19].

complex has proteolytic properties and is called C3 convertase. Convertase breaks down molecules of C3 into C3a, C3b, C3c and C3d. The C3b and C3d components bind with the red blood cell membrane and in many cases the complement cascade process ends. In other cases, the C3b component activates C5 and C5a and C5b are formed. C5b binds to C6, then to C7. This creates a complex of three C5b-6-7 particles, which is partially incorporated into the cell membrane and further binds C8. The C5b-8 complexes create holes in the cell membrane that increase when exposed to the C9 component. The C5B-C9 complex called membrane attack complex (MAC) creates pores in the cell membrane of a red blood cell that are 1/700 of its size. Haemoglobin escapes from the cells into the plasma, and the effects of haemolysis are visible macroscopically in the plasma of the blood sample [15]. The alternative path of complement activation and the lectin path of complement activation do not play a role in the destruction of red blood cells. Although the mechanism of the lectin route may be the reason for the in vivo ineffectiveness of the use of monoclonal and recombinant antibodies, which are thus eliminated from the body before they fulfil their function, for example, anti-D Ig for prevention

However, the complement system does not work specifically. The safety of body cells is enabled by factors that regulate complement activity present in plasma and on cells of various tissues, including red blood cells. Membrane inhibitor of reactive lysis (MIRL) (CD59) and decay accelerating factor (DAF) (CD55) are essential to protect red blood cells from haemolysis. The expression of these membrane inhibitors is associated with Cromer group system and CD59. On blood cells with the Cromer mull phenotype, known as Inab, DAF inhibitor expression is absent [17, 18]. DAF regulates C3a-converting activity. MIRL inhibits membrane attack complex [15, 17]. Lack of these particles may increase the susceptibility of red blood cells to

In a situation in which, despite activation of the complement system, through antigen-antibody reaction, there is no intravascular haemolysis, red blood cells with detectable C3b component remain in the circulation. This kind of mechanism of red blood cell destruction occurs for IgG antibodies with complement system [13]. They may interact with CR1 and CR3 receptors on macrophages and consequently undergo phagocytosis. Most of the cells coated by the complement C3b component are destroyed by liver macrophages, that is, by Kupffer cells, while the cells coated with antibody molecules are mainly destroyed by spleen macrophages. They have surface receptors that recognise antibody classes and subclasses, and complement components,

**94**

**Figure 1.**

*The classic pathway for complement.*

of which the Fc R1 receptor is specific for red cells coated with antibodies [1]. Blood cells connected to this receptor are destroyed in the process of antibody-dependent cytotoxicity. Red blood cells can be absorbed and completely "digested" inside the macrophage. They can also be partially absorbed and then the integrity of the cell membrane is disturbed by the loss of proteins and lipids, which changes its osmotic properties. Such a blood cell, after being released from the macrophage, circulates in the blood as a spherocyte, whose survival is short. The macrophage cytotoxins are another mechanism that plays a role in the destruction of red blood cells. As a consequence of antibody-dependent cell-mediated cytotoxicity (ADCC) haemoglobinemia and haemoglobinuria may occur similarly to intravascular haemolysis, although the antibodies that caused it do not bind complement components.
