Mechanisms of Clearing Incompatible RBC

Question: Discuss about the Mechanisms of Clearing Incompatible RBC. Answer: Introduction: Transfusion is done worldwide to treat anemia and hemorrhage. In case of incompatible transfusions, the antibodies bind to the red blood cells and induce fatal effects that includes hemolytic- transfusion reactions, hemolysis in fetus as well as newborn or autoimmune form of hemolytic- anemia. Though, compatibility tests have been performed to prevent an abnormal pathophysiology of incompatible transfusions, still incompatible blood- transfusions occurs. But, in case of few incompatible blood- transfusions, abnormal pathophysiology may not follow rather the incompatible RBCs might survive normally. This essay discusses about various mechanisms by which incompatible red blood cells are cleared off from the blood circulation. Cross matching- incompatible blood- transfusions involves infusing the red- blood cells from donor into the recipient with antibodies to fight against antigens that are present in the red blood cells of donor. Allo-antibodies were found to be generated against RBC antigen when there is a prior exposure to allogeneic RBCs, which could have been occurred during any previous blood- transfusions or pregnancy except for the blood- group antibodies (as the ABO system) that naturally occurs [1]. Generally, incompatible blood- transfusions were strictly prevented because of the fatal outcomes of hemolysis that is caused by the transfusion of incompatible RBCs leading to HRT (hemolytic- transfusion reactions) [2], [3]. They can occur due to the amnestic- antibody responses that are caused by the mis-transfusion. Additionally, in few circumstances, cross-match- incompatible RBCs were infused purposefully to person if he/she is at risk for developing hypoxia (because of anemia). It is done only if the risks of hypoxia outweigh the adverse effects of an induced HTR. Allo-antibodies developed against RBCs of fetus can lead to hemolytic disease in fetus that results in mortalities in which auto-antibodies bound to red blood cells causing (autoimmune) hemolytic- anemia [4]. Therefore, a clear understanding of the clearance mechanisms of RBC in which antibody- bounded with RBCs are cleared off from the blood circulation as well as the patho-physiology that ensues is much needed in many hospital settings. Hemolysis that is induced by antibody is basically thought to be occurred by any one of the two mechanisms. The first mechanism is that the RBCs could undergo an intra- vascular hemolysis at the time of activation of complement to develop the membrane- attack complex (i.e. MAC). Typically, this occurs due to the binding of immunoglobulin (Ig) M with the surfaces of RBC and sometimes it also occurs by binding with Immunoglobulin IgG [5]. Red- blood cells were also opsonized as well as ingested by the phagocytes which are called as extra- vascular hemolysis. When the complementary pathway is activated by the binding of antibody, C3 gets covalently attached to both cellular protein (surrounding it) and inciting antibodies through thioester bonding [6]. In case, if the complementary activation did not led to the formation of MAC to the level that a quick intra- vascular hemolysis occur, then C3 that is deposited on the surfaces of red- blood cells might be converted into C3b as well as i C3b that are ligands for the complement- receptors. In such cases, the complementary receptors that are located on the phagocytic cells (such as CR1, CR3 CR4) will consume the RBCs that are coated with C3b (phagocytosis). Additionally, C3 is usually needed to continue the complementary cascade so as to complete MAC assembly [7]. Secondly, an antibody- induced form of opsonization may develop because of the bounding of Fc- domains of the immunoglobulin IgG to red- blood cells that are identified by Fcg- receptors (FcgRs) on the surface of phagocytes extra- vascularly. The presence of these RBC clearance pathways after binding with antibodies were significantly proved by various studies in animal models [8]. In contrast to these two pathways, the alternative pathways of immunoglobulin IgG- mediated RBCs clearance were identified that does not involves both complement as well as FcgR pathways. These pathways involve direct effect of antibody- binding on RBCs. De-stabilization of the membrane of RBCs were found to induce programmed death of RBCs (eryptosis) [9] [10]. Antibodies were found to directly provoke the phosphatidylserine expression that are present on the surface of RBC that is able to cause phagocytosis because of the ligation of phosphatidylserine- scavenger receptors that are located on phagocytes [9]. In addition to that, the mechanical- induction of Ca2+ influx into red- blood cells results in eryptosis because of the direct effect of an antibody- surface binding [11] [12]. In 2010, Chadebech has stated that these pathways were not separated but they may overlap each other. Though these newly identified alternative mechanisms raise many mechanistic questions in regard to HTRs, they were analyzed in- vitro and hence it is not clear whether these findings are appropriate to the authentic HTRs- in- vivo. Ultimately, the clearance of red- blood cells from blood circulation in- vivo by the process of sequestration in spleen as well as liver was identified in regard to immunoglobulin IgG IgA autoimmune- hemolytic anemia whereas the role of sequestration was not observed in regard to incompatible transfusion [13]. A study was conducted by Liepkalns et al (2012) by using mono-clonal antibodies against the glycophorin- A (hGPA) of human blood- group as well as Duffy (as a part of fusion- protein known as HOD) antigens [14]. They found that anti- Duffy antibodies were found to remove RBCs that express HOD through Fc- receptors. In- contrast to that, anti- hGPA antibodies were assessed to remove off RBCs through a 3rd newer biphasic mechanism. During the 1st phase of mechanism, anti- hGPA antibodies agglutinate with RBCs, thus sequestering them out of circulation. In the 2nd phase of mechanism, phagocytes are needed for the removal of sequestered RBCs independently from both Fc- receptors and complement [14]. A collaborating cytokine burst was analyzed in regard to Fc- receptors that suggest that the de-coupling of phagocytosis and secretion of cytokine occurs at the time of clearance of incompatible hGPA- RBCs [15]. They have investigated the ability of RBC to survive in- vivo with the knowledge of clearance mechanism. They found that not all hGPA and HOD- RBCs where cleared when they face a bolus of anti- hGPA and anti- Duffy antibodies respectively. At the time of incompatible transfusions of hGPA and HOD, a group of RBCs were identified to be PREVIEW- resistant. The resistance of hGPA and HOD- RBCs was identified to not require C3 (Figure 1) [14]. The titration of anti- hGPA antibody- mediated clearance indicates that RBCs spectrum occurs among hGPA- RBCs. Many studies on incompatible- transfusion with HOD- RBCs indicate that the resistant RBCs do not acquire the resistance ability but instead the resistance power is an innate quality of this RBC population. They concluded that, the c learance pathways of incompatible- RBC appear to differ among blood- group antigens or binding- antibody and resistance do not require complement. Recent studies suggest that phagocytes are not needed for an early clearance of hGPA as well as HOD- RBCs but play a great role in preventing its return to blood- circulation. Phagocytes usually uses multi- scavenger receptors along with Fc- and complement- receptors) to digest the injured cells [14]. Generally, it is known that phagocytic cells are much needed for the clearance of RBC that is coated with IgG. In 2012, Liepkalns et al has evaluated the role of phagocytes in incompatible RBC clearance by injecting C57BL/6 samples with clodronate which is a toxic electron- transport chain (decoupling bi-phosphonate). They have injected clodronate (that is encapsulated in liposomes) directly into phagocytic cells that are digested by them selectively (Figure 2) [14]. They have injected control mice with liposomes that is similar to experimental group but without capsulated clodronate (empty liposome). After 24 hours, passive immunization was given along with transfusion of a combination of both hGPA and HOD. They observed that the samples, who received passive immunization of anti- hGPA, showed the clearance of RBCs to certain extent with both clodronate as well as empty liposome- treated mice at the 1st time i.e. 2 hrs after transfusion (Figure 2). Moreover, from 18 hours, many of the incompatible hGPA- RBCs were found to return back to blood- circulation that lead to the survival of more than half of incompatible- RBCs at two days after transfusion (Figure 2) [14]. On the other hand, the samples that received passive immunization with anti- Fy3 showed that liposomal- clodronate infusion has stopped the clearance of HOD- RBC initially, which confirms that clodronate is highly effective and lead to the depletion of sufficient phagocytic cells to prevent the clearance of RBC that is FcgR- dependent. They also examined the role of FcgRs in the incompatible RBC clearance by using the samples with a deleted common- g chain that is needed for expressing and functioning of the 3 murine FcgRs for phagocytosis (FcgR-I, FcgR-III, and FcgR-IV) [16]. A very little but apparent decrease in incompatible RBC clearance was found in sample which is passively immunized with 10F7 and/or 6A7 whereas the clearance of hGPA HOD- RBCs that is stimulated by anti- Fy3 was observed to be abrogated. These findings suggest that FcgRs are needed for the incompatible RBC clearance but has little effect on clearance by anti- hGPA. Liepkalns (2012) has tested whether RBC aggregation in- vivo affects the clearance that is mediated by anti- hGPA. He did incompatible- transfusions and analyzed the peripheral- blood at rapid- clearance phase and found that majority of aggregates were made up of hGPA HOD- RBCs and not control RBC. The findings suggests that the majority of complexes contains selective- agglutination in- vivo of the in-compatible RBCs. Further, the complexes were observed only during the early stage of reaction and had quickly decreased at the clearance phase and have stopped to be detectable as soon as the transfusion is completed (Figure 3D) [14]. Many studies state that spleens have no role in the clearance of incompatible RBC clearance and splenectomy does not has any effect on the clearance. The incompatible RBCs were cleared off from blood- circulation only in the extra- splenic areas. Hence, spleen is not needed but might be engaged in the clearance of in-compatible- RBC [14]. Then, though the sensitivity of different organs varies to clodronate, clodronate treatment leads to the return of RBCs that are incompatible to the blood- circulation after some time. These findings suggests that phagocytic cells are much needed in the prevention of initially cleared off RBCs from re- entering into the blood- circulation most probably by engulfing the RBCs that are bounded by antibody. But, as there was no return back of C3 KO and FcgR- KO to the blood- circulation, the signal for engulfing seems to be something that is different from that of opsonization with an Immunoglobulin IgG or a complement. Brain et al (2010) observed that the binding of poly-clonal human- IgG antibodies (that is present in the persons with hemolytic- transfusion reaction because of anti- Pr) could damage the RBCs directly by destroying the RBCs membrane, opening the channels of Ca2+ with exposure of phosphate-dylethanolamine [10]. Hence, it suggests that 10-F7and 6A-7 stimulate the expression of engulf me signal as like phosphate-idylserine and other that are recognized by a scavenger- receptor that are located on the phagocytic cells, which are sensitive to clodronate. It appears that both 10F-7 and 6A-7 causes eryptosis directly (Figure 4A) [14]. Thus to conclude, various mechanisms are involved in the clearance of incompatible RBCs from blood. 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