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Role of complement in patients with autoimmune hemolytic anemia and platelet transfusion refractoriness
Transfusion Clinique et Biologique 26 (2019) 152–154
Disponible en ligne sur
ScienceDirect www.sciencedirect.com
Update article
Role of complement in patients with autoimmune hemolytic anemia and platelet transfusion refractoriness Rôle du complément chez les patients présentant une anémie hémolytique auto-immune et une transfusion plaquettaire réfractaire Magali J Fontaine ∗ Department of Pathology, University of Maryland School of Medicine, Baltimore, MA, USA
a r t i c l e
i n f o
Article history: Available online 19 June 2019 Keywords: Complement Hemolysis Autoimmune Platelet transfusion refractoriness
a b s t r a c t The complement is a key player of the innate immune response. It provides defense mechanisms that are not specific, but very efficient at neutralizing any invader, accounting for 4% of the proteins in the peripheral blood. Nevertheless, there is a dark side to the complement system, as it may activate its machinery against healthy cells such as peripheral blood red blood cells and platelets resulting in undesired hemolysis and thrombocytopenia, respectively. Understanding and identifying the role of complement in these settings allow physicians to adjust their diagnostic and therapeutic modalities accordingly. The role of complement in the pathophysiology and management of autoimmune hemolytic anemia and of alloimmune-mediated thrombocytopenia is under investigation and discussed. ´ e´ franc¸aise de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights © 2019 Societ reserved.
r é s u m é Mots clés : Complément Hémolyse Auto-immune Thrombopénie réfractaire
Le système du complément est un élément important de l’immunité innée. Ce système comprend 4 % des protéines circulant dans le sang et procure une défense non spécifique mais immédiate en cas d’agression infectieuse. Ce système efficace est à double tranchant, car il est parfois actif par inadvertance contre des cellules vitales comme les globules rouges et les plaquettes, conduisant à des crises d’hémolyses et de thrombopénies réfractaires. Il est donc primordial d’analyser au mieux le rôle du complément dans ces pathologies sanguines aiguës et parfois réfractaires, pour améliorer leur diagnostic ainsi que la conduite thérapeutique, qui sont décrites ci-jointes. ´ e´ franc¸aise de transfusion sanguine (SFTS). Publie´ par Elsevier Masson SAS. Tous droits © 2019 Societ ´ ´ reserv es.
1. Introduction The complement was discovered over a hundred years ago as a set of heat-sensitive proteins in plasma that “complement” the antibacterial activity of antibodies and represents one of the most important human defense mechanisms against pathogens [1]. The complement belongs to the innate arm of the immune system rather than the adaptive arm, which involves primarily B and T cells
∗ Department of Pathology, University of Maryland School of Medicine, 22, S Greene Street N2W50a, 21201 Baltimore MD, USA. E-mail address: [email protected]
[2]. The defense mechanisms provided by the complement system are not specific, but very efficient at neutralizing any invader, accounting for 4% of the proteins in the peripheral blood. Indeed, the complement system is composed of a series of circulating enzymes that can initiate a cascade of enzymatic reactions similar to blood coagulation [2,3]. Crucial functional components therefore include microbe-recognition and attachment proteins, such as C3b and activating enzyme complexes (C3 convertase and C5 convertase) that allow the rapid accumulation of other components and allow generation of the membrane attack complex (MAC) on the surface of a pathogen and perforates its membrane [1,3]. Binding of complement fragments to immune cells such as macrophages can also stimulate the phagocytosis of the microbe bound to the
https://doi.org/10.1016/j.tracli.2019.06.232 ´ e´ franc¸aise de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved. 1246-7820/© 2019 Societ
M.J. Fontaine / Transfusion Clinique et Biologique 26 (2019) 152–154
complement fragment. Other small complement peptides (C5a) can stimulate the inflammatory response. Recent studies reveal that complement may also have a role in normal development, repair and regeneration of damaged tissues, by fixing dying cell debris and presenting them to macrophages for clearing [3]. Nevertheless, there is a dark side to the complement system, as it may activate its machinery against healthy cells such as peripheral blood red blood cells (RBC) and platelets resulting in hemolytic anemia and thrombocytopenia, respectively. The role of complement in the pathophysiology and management of autoimmune hemolytic anemia (AIHA) and of alloimmune-mediated thrombocytopenia is under investigation and discussed below [4].
2. The role of complement in AIHA Autoantibodies bound to self-erythrocyte antigens may induce hemolysis and cause AIHA. These autoantibodies are characterized by the presence of a positive Coombs test, also referred to as a direct antiglobulin test (DAT) [5]. The complement system is being recognized as enhancing the antibody mediated immune attack on cellular targets such as RBCs. The mechanisms of red cell destruction and clearance in AIHA depend on the ability of the autoantibody to activate complement and/or the interaction of the autoantibody with macrophage Fc-receptors [6]. The immune dysregulation associated with autoantibody formation, and the mechanism of the subsequent RBC destruction depend on the isotype of the autoantibody. Indeed IgM and IgG (IgG1, IgG2, IgG3 but not IgG4) are the only antibody isotypes that bind the C1 fragment to initiate the classic pathway activation [7]. The Fc regions of these antibody isotypes confer their ability to interact with C1. Variations in macrophage Fc-receptor polymorphisms, phagocytic activity, and expression of complement regulators, may dictate the clinical presentation as well as outcome in AIHA [6]. These specific complement-isotype complexes, IgM and certain IgG isotypes (mostly IgG1 and IgG3), can then cause intravascular hemolysis as described below [7]. The IgG or IgM molecules bound to RBC surface in the peripheral vasculature may fix complement protein 1q (C1q), initiating the classical pathway [4]. C1 esterase activates C2 and C4 to form C3 convertase, which then cleaves C3 into C3a and C3b. IgM/IgG then detach and separate from the RBC, leaving C3b-bound to the red cell surface. C3b-opsinized red cells are prone to extravascular hemolysis via phagocytic monocytes in the liver and spleen. Surviving C3b-bound erythrocytes can otherwise undergo an additional complement cleavage step to form C3d, a protein with protective properties against hemolysis that can be detected by a DAT. Alternatively, complement activation can also proceed past the C3 stage via the terminal complement pathway, in which C3b cleaves C5 into C5a and C5b, initiating the formation of the membrane attack complex (MAC) with other accessory complement proteins (C6, C7, C8, and C9) and trigger intravascular hemolysis [7]. Interestingly IgM coated RBCs can persist in the circulation without necessarily resulting in intravascular hemolysis, but it can be removed extravascularly by macrophages through C3 receptor (CR1 and CR3) binding. RBCs coated with IgM only (no complement), may also persist in the circulation normally because macrophages lack a receptor for IgM [4]. An interesting category of RBC autoantibodies is the one encountered in patients with paroxysmal cold hemoglobinuria (PCH), which is most often caused by transient viral or bacterial infections and presenting with hemoglobinemia, hemoglobinuria, or both [8]. These autoantibodies are usually IgG type and are characterized in the immunohematology laboratory by the Donath and Landsteiner (DL) test. The DL test is most commonly performed on RBC from a patient with a positive DAT result and evidence of C3 fragment on RBC surfaces with no evidence of autoantibody either
153
in the serum or in the eluate (bound to RBC surface). For the DL test, patient’s tested serum must be separated from a freshly collected blood sample maintained at 37 ◦ C until testing is performed in order to characterize the biphasic property of the IgG antibodies causing PCH. Indeed, these biphasic IgG antibodies bind to RBC and fix complement (C1) at 4 ◦ C in the peripheral blood but then cause intravascular hemolysis at 37 ◦ C, the temperature required for complement activation [5]. The therapeutic management of AIHA will depend on the characteristics of the autoantibody (i.e., cold reacting or warm reacting), and on its ability to bind complement. If the AIHA is associated with an underlying disease, such as malignancy, infection, or autoimmunity, these need to be treated first. Steroid therapy is first-line treatment and will inhibit autoantibody production as well as extravascular hemolysis by downregulating the expression of Fc␥ receptors on macrophages [9]. Monoclonal antibody against B cell CD20 antigen, causing B cell cytotoxicity, is often considered second-line treatment, more specifically in cold agglutinin disease (CAD), an IgM mediated AIHA [10]. Complement inhibitors are arising as novel therapies to improve morbidity and mortality in patients with frequent exacerbations and severe hemolysis. Eculizumab is a humanized monoclonal antibody that binds to C5 to inhibit the terminal complement pathway and has been considered for patients with CAD refractory to rituximab. But complement inhibitors that target the classical complement pathway (i.e. inhibitors against C1 esterase or C3 convertase), would be more effective in patients presenting with exacerbated hemolysis [7]. These severe exacerbations of hemolysis are most often extravascular and mediated by complement receptor- uptake of C3 opsonized RBCs by macrophages in the liver and spleen. Lastly, if patients present with symptomatic anemia, RBC transfusion may be required. But the immunohematologic reference workup is challenging due to the autoantibody masking any possible alloantibody. Shirey et al. showed that adsorption procedures can be significantly avoided by providing genotypically matched RBC units to patients with autoantibody, leading to a simplification in pretransfusion adsorption studies and overall increase in transfusion safety [11,12]. Thus advances in RBC genotyping technology may optimize the selection of RBC units for transfusion, even though the serologic crossmatch will be reactive due to the autoantibody.
3. The role of complement in platelet transfusion refractoriness The role of complement in managing patients who are highly immunized to platelet surface antigens and who have become refractory to platelet transfusion has been investigated by our group [13]. Platelet transfusion refractoriness is defined as repeated low post-transfusion platelet count increments (less than 5000 per l). Its evaluation consists of first ruling out non-immune causes such as sepsis, fever, disseminated intravascular coagulation (DIC), bleeding, splenomegaly, and drugs [13]. In the absence of these clinical conditions, alloimmunization to human leukocyte antigen (HLA) or to platelet-specific antigens should be considered [14,15]. HLA alloimmunization represents 75% of the causes associated with immune mediated platelet refractoriness [13]. A hospital based transfusion service has limited control over the nonimmune causes of refractoriness to platelet transfusion, but it can supply HLA compatible platelets [16,17]. These HLA compatible platelets, also referred to as antigen negative for the corresponding antibodies, result in platelet count increments, post-transfusion, equivalent to those obtained with HLA-matched platelets while increasing the donor pool of eligible donors [18]. Logistically, once the platelet antibodies are identified, the platelet units that are HLA
154
M.J. Fontaine / Transfusion Clinique et Biologique 26 (2019) 152–154
antigen negative for the corresponding antibodies can be searched for by the blood supplier and the HLA compatible units can then be allocated to the patient for transfusion; indeed, most blood suppliers keep track of the HLA type of their platelet donors. Thus the method of antibody detection is critical. Historic use of the complement dependent lymphocytotoxicity (CDC) assay for antibody detection has been replaced by flow cytometry based assays such as the IgG-single antigen bead (SAB) binding assay [19,20]. The flow cytometry screening and identification method for HLA antibody is also widely used for solid organ transplantation. But the classic flow cytometry screening method measures IgG binding only using mean fluorescence intensity (MFI) values as a readout and has high sensitivity and specificity [19]. However, it is widely considered to be “too sensitive” and the MFI value, used as a threshold for antibody identification, is debatable. Importantly, complement-binding antibodies have been known to be clinically relevant for 40 years in renal transplantation [21], and attempts have been made to develop complement dependent assays using the same flow-based SAB assay [22]. We independently developed a C1q assay that only detects those antibodies capable of binding the first component (C1q) of the classical complement pathway [22]. Our studies showed that HLA antibodies identified as binding C1q complement fragment are more immediately associated with platelet destruction than the HLA antibodies binding IgG only [13]. Thus in a case of highly alloimmunized patients, presenting with greater than 80% HLA antibody reactivity using the IgG-SAB method, HLA compatible donor platelets may be scarce. But these same patients, tested with the C1q based assay, may show a lower level of reactivity and allow for a greater number of compatible platelet donations to be allocated and transfused with adequate post-transfusion corrected count increments (greater than 5000 per l). Thus we concluded that the complement-binding property of a platelet HLA antibody may be most relevant in platelet recovery post-transfusion [13]. Interestingly, an analysis by Jackman et al. showed that the level of C1q-binding HLA class I antibodies did not differ significantly between patients presenting with refractoriness compared to patients with no refractoriness to platelet transfusion [23]. The finding from Jackman et al. confirms that antibody-independent mechanisms may also play a role in platelet transfusion refractoriness as previously reported [23,24]. Interestingly, in a study by Waterman et al., using a mouse model of platelet HLA alloimmunization, the role of the common gamma chain receptor was shown to play a more important role than complement C3 fragment in the extravascular clearance of platelets [25]. Indeed, mouse recipients that had a deletion of the common gamma chain of the Fc-receptor presented with decreased to no clearance of PLTs, caused by immune serum immunization, compared to mice with a deletion of the gene encoding complement factor C3 [25]. Thus far, the current complement inhibitors, such as eculizumab, are not likely indicated in patients with platelet transfusion refractoriness. On the other hand, using a complement C1q-dependent assay will more accurately identify the specificity of HLA antibodies to consider avoiding the corresponding antigens while selecting platelets for transfusion. 4. Conclusion We presented clinical scenarios, in which important peripheral blood cellular elements, red blood cells and platelets, may be affected by complement-mediated destruction. Understanding and identifying the role of complement in these settings allow
physicians to adjust their diagnostic and therapeutic modalities accordingly. Keeping in mind that the complement system is a key player in the innate arm of the immune system, one should also understand the infectious risk of modulating its effect. Disclosure of interest The authors declare that they have no competing interest. References [1] Kaufmann SHE. Immunology’s coming of age. Front Immunol 2019;10:684. [2] Hillion S, Arleevskaya MI, Blanco P, Bordron A, Brooks WH, Cesbron JY, et al. The innate part of the adaptive immune system. Clin Rev Allergy Immunol 2019, http://dx.doi.org/10.1007/s12016-019-08740-1 [Epub ahead of print]. [3] Hajishengallis G, Reis ES, Mastellos DC, Ricklin D, Lambris JD. Novel mechanisms and functions of complement. Nat Immunol 2017;18:1288. [4] Schreiber AD, Frank MM. Role of antibody and complement in the immune clearance and destruction of erythrocytes I. In vivo effects of IgG and IgM complement-fixing sites. J Clin Invest 1972;51:575–82. [5] Petz LD, Garratty G. Immune hemolytic anemias. 2nd ed. Philadelphia, Pa: Churchill Livingstone/Elsevier Science; 2004. [6] Meulenbroek EM, Wouters D, Zeerleder SS. Lyse or not to lyse: clinical significance of red blood cell autoantibodies. Blood Rev 2015;29:369–76. [7] Brodsky RA. Complement in hemolytic anemia. Blood 2015;126:2459–65. [8] Garratty G. Immune hemolytic anemia associated with negative routine serology. Semin Hematol 2005;42:156–64. [9] Barcellini W, Fattizzo B, Zaninoni A, Radice T, Nichele I, Di Bona E, et al. Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients. Blood 2014;124:2930–6. [10] Go RS, Winters JL, Kay NE. How I treat autoimmune hemolytic anemia. Blood 2017;129:2971–9. [11] Shirey RS, Boyd JS, Parwani AV, Tanz WS, Ness PM, King KE. Prophylactic antigen-matched donor blood for patients with warm autoantibodies: an algorithm for transfusion management. Transfusion 2002;42:1435–41. [12] Ziman A, Cohn C, Carey PM, Dunbar NM, Fung MK, Greinacher A, et al. Warm-reactive (immunoglobulin G) autoantibodies and laboratory testing best practices: review of the literature and survey of current practice. Transfusion 2017;57:463–77. [13] Fontaine MJ, Kuo J, Chen G, Galel SA, Miller E, Sequeira F, et al. Complement (C1q) fixing solid-phase screening for HLA antibodies increases the availability of compatible platelet components for refractory patients. Transfusion 2011;51:2611–8. [14] Doughty HA, Murphy MF, Metcalfe P, Rohatiner AZ, Lister TA, Waters AH. Relative importance of immune and non-immune causes of platelet refractoriness. Vox Sang 1994;66:200–5. [15] Fabris F, Soini B, Sartori R, Randi ML, Luzzatto G, Girolami A. Clinical and laboratory factors that affect the post-transfusion platelet increment. Transfus Sci 2000;23:63–8. [16] Schiffer CA. Diagnosis and management of refractoriness to platelet transfusion. Blood Rev 2001;15:175–80. [17] Schiffer CA. Management of patients refractory of platelet transfusion. Leukemia 2001;15:683–5. [18] Petz LD, Garratty G, Calhoun L, Clark BD, Terasaki PI, Gresens C, et al. Selecting donors of platelets for refractory patients on the basis of HLA antibody specificity. Transfusion 2000;40:1446–56. [19] Tait BD, Hudson F, Cantwell L, Brewin G, Holdsworth R, Bennett G, et al. Review article: luminex technology for HLA antibody detection in organ transplantation. Nephrology (Carlton) 2009;14:247–54. [20] Wahrmann M, Exner M, Regele H, Derfler K, Körmöczi GF, Lhotta K, et al. Flow cytometry based detection of HLA alloantibody mediated classical complement activation. J Immunol Methods 2003;275:149–60. [21] Smith JD, Hamour IM, Banner NR, Rose ML. C4d fixing, luminex binding antibodies – a new tool for prediction of graft failure after heart transplantation. Am J Transplant 2007;7:2809–15. [22] Chen G, Tyan TD. C1q assay for the detection of complement fixing antibody to HLA antigens. Methods Mol Biol 2013;1034:305–11. [23] Jackman RP, Lee JH, Pei R, Bolgiano D, Lebedeva M, Slichter SJ, et al. C1qbinding anti-HLA antibodies do not predict platelet transfusion failure in Trial to Reduce Alloimmunization to Platelets study participants. Transfusion 2016;56:1442–50. [24] Jackman RP, Muench MO, Heitman JW, Inglis HC, Law JP, Marschner S, et al. Immune modulation and lack of alloimmunization following transfusion with pathogen-reduced platelets in mice. Transfusion 2013;53:2697–709. [25] Waterman HR, Kapp LM, Munday A, Odem-Davis K, Zimring JC. Transfusioninduced alloimmunization and platelet refractoriness in a mouse model: mechanisms and interventions. Transfusion 2016;56:91–100.
Disponible en ligne sur
ScienceDirect www.sciencedirect.com
Update article
Role of complement in patients with autoimmune hemolytic anemia and platelet transfusion refractoriness Rôle du complément chez les patients présentant une anémie hémolytique auto-immune et une transfusion plaquettaire réfractaire Magali J Fontaine ∗ Department of Pathology, University of Maryland School of Medicine, Baltimore, MA, USA
a r t i c l e
i n f o
Article history: Available online 19 June 2019 Keywords: Complement Hemolysis Autoimmune Platelet transfusion refractoriness
a b s t r a c t The complement is a key player of the innate immune response. It provides defense mechanisms that are not specific, but very efficient at neutralizing any invader, accounting for 4% of the proteins in the peripheral blood. Nevertheless, there is a dark side to the complement system, as it may activate its machinery against healthy cells such as peripheral blood red blood cells and platelets resulting in undesired hemolysis and thrombocytopenia, respectively. Understanding and identifying the role of complement in these settings allow physicians to adjust their diagnostic and therapeutic modalities accordingly. The role of complement in the pathophysiology and management of autoimmune hemolytic anemia and of alloimmune-mediated thrombocytopenia is under investigation and discussed. ´ e´ franc¸aise de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights © 2019 Societ reserved.
r é s u m é Mots clés : Complément Hémolyse Auto-immune Thrombopénie réfractaire
Le système du complément est un élément important de l’immunité innée. Ce système comprend 4 % des protéines circulant dans le sang et procure une défense non spécifique mais immédiate en cas d’agression infectieuse. Ce système efficace est à double tranchant, car il est parfois actif par inadvertance contre des cellules vitales comme les globules rouges et les plaquettes, conduisant à des crises d’hémolyses et de thrombopénies réfractaires. Il est donc primordial d’analyser au mieux le rôle du complément dans ces pathologies sanguines aiguës et parfois réfractaires, pour améliorer leur diagnostic ainsi que la conduite thérapeutique, qui sont décrites ci-jointes. ´ e´ franc¸aise de transfusion sanguine (SFTS). Publie´ par Elsevier Masson SAS. Tous droits © 2019 Societ ´ ´ reserv es.
1. Introduction The complement was discovered over a hundred years ago as a set of heat-sensitive proteins in plasma that “complement” the antibacterial activity of antibodies and represents one of the most important human defense mechanisms against pathogens [1]. The complement belongs to the innate arm of the immune system rather than the adaptive arm, which involves primarily B and T cells
∗ Department of Pathology, University of Maryland School of Medicine, 22, S Greene Street N2W50a, 21201 Baltimore MD, USA. E-mail address: [email protected]
[2]. The defense mechanisms provided by the complement system are not specific, but very efficient at neutralizing any invader, accounting for 4% of the proteins in the peripheral blood. Indeed, the complement system is composed of a series of circulating enzymes that can initiate a cascade of enzymatic reactions similar to blood coagulation [2,3]. Crucial functional components therefore include microbe-recognition and attachment proteins, such as C3b and activating enzyme complexes (C3 convertase and C5 convertase) that allow the rapid accumulation of other components and allow generation of the membrane attack complex (MAC) on the surface of a pathogen and perforates its membrane [1,3]. Binding of complement fragments to immune cells such as macrophages can also stimulate the phagocytosis of the microbe bound to the
https://doi.org/10.1016/j.tracli.2019.06.232 ´ e´ franc¸aise de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved. 1246-7820/© 2019 Societ
M.J. Fontaine / Transfusion Clinique et Biologique 26 (2019) 152–154
complement fragment. Other small complement peptides (C5a) can stimulate the inflammatory response. Recent studies reveal that complement may also have a role in normal development, repair and regeneration of damaged tissues, by fixing dying cell debris and presenting them to macrophages for clearing [3]. Nevertheless, there is a dark side to the complement system, as it may activate its machinery against healthy cells such as peripheral blood red blood cells (RBC) and platelets resulting in hemolytic anemia and thrombocytopenia, respectively. The role of complement in the pathophysiology and management of autoimmune hemolytic anemia (AIHA) and of alloimmune-mediated thrombocytopenia is under investigation and discussed below [4].
2. The role of complement in AIHA Autoantibodies bound to self-erythrocyte antigens may induce hemolysis and cause AIHA. These autoantibodies are characterized by the presence of a positive Coombs test, also referred to as a direct antiglobulin test (DAT) [5]. The complement system is being recognized as enhancing the antibody mediated immune attack on cellular targets such as RBCs. The mechanisms of red cell destruction and clearance in AIHA depend on the ability of the autoantibody to activate complement and/or the interaction of the autoantibody with macrophage Fc-receptors [6]. The immune dysregulation associated with autoantibody formation, and the mechanism of the subsequent RBC destruction depend on the isotype of the autoantibody. Indeed IgM and IgG (IgG1, IgG2, IgG3 but not IgG4) are the only antibody isotypes that bind the C1 fragment to initiate the classic pathway activation [7]. The Fc regions of these antibody isotypes confer their ability to interact with C1. Variations in macrophage Fc-receptor polymorphisms, phagocytic activity, and expression of complement regulators, may dictate the clinical presentation as well as outcome in AIHA [6]. These specific complement-isotype complexes, IgM and certain IgG isotypes (mostly IgG1 and IgG3), can then cause intravascular hemolysis as described below [7]. The IgG or IgM molecules bound to RBC surface in the peripheral vasculature may fix complement protein 1q (C1q), initiating the classical pathway [4]. C1 esterase activates C2 and C4 to form C3 convertase, which then cleaves C3 into C3a and C3b. IgM/IgG then detach and separate from the RBC, leaving C3b-bound to the red cell surface. C3b-opsinized red cells are prone to extravascular hemolysis via phagocytic monocytes in the liver and spleen. Surviving C3b-bound erythrocytes can otherwise undergo an additional complement cleavage step to form C3d, a protein with protective properties against hemolysis that can be detected by a DAT. Alternatively, complement activation can also proceed past the C3 stage via the terminal complement pathway, in which C3b cleaves C5 into C5a and C5b, initiating the formation of the membrane attack complex (MAC) with other accessory complement proteins (C6, C7, C8, and C9) and trigger intravascular hemolysis [7]. Interestingly IgM coated RBCs can persist in the circulation without necessarily resulting in intravascular hemolysis, but it can be removed extravascularly by macrophages through C3 receptor (CR1 and CR3) binding. RBCs coated with IgM only (no complement), may also persist in the circulation normally because macrophages lack a receptor for IgM [4]. An interesting category of RBC autoantibodies is the one encountered in patients with paroxysmal cold hemoglobinuria (PCH), which is most often caused by transient viral or bacterial infections and presenting with hemoglobinemia, hemoglobinuria, or both [8]. These autoantibodies are usually IgG type and are characterized in the immunohematology laboratory by the Donath and Landsteiner (DL) test. The DL test is most commonly performed on RBC from a patient with a positive DAT result and evidence of C3 fragment on RBC surfaces with no evidence of autoantibody either
153
in the serum or in the eluate (bound to RBC surface). For the DL test, patient’s tested serum must be separated from a freshly collected blood sample maintained at 37 ◦ C until testing is performed in order to characterize the biphasic property of the IgG antibodies causing PCH. Indeed, these biphasic IgG antibodies bind to RBC and fix complement (C1) at 4 ◦ C in the peripheral blood but then cause intravascular hemolysis at 37 ◦ C, the temperature required for complement activation [5]. The therapeutic management of AIHA will depend on the characteristics of the autoantibody (i.e., cold reacting or warm reacting), and on its ability to bind complement. If the AIHA is associated with an underlying disease, such as malignancy, infection, or autoimmunity, these need to be treated first. Steroid therapy is first-line treatment and will inhibit autoantibody production as well as extravascular hemolysis by downregulating the expression of Fc␥ receptors on macrophages [9]. Monoclonal antibody against B cell CD20 antigen, causing B cell cytotoxicity, is often considered second-line treatment, more specifically in cold agglutinin disease (CAD), an IgM mediated AIHA [10]. Complement inhibitors are arising as novel therapies to improve morbidity and mortality in patients with frequent exacerbations and severe hemolysis. Eculizumab is a humanized monoclonal antibody that binds to C5 to inhibit the terminal complement pathway and has been considered for patients with CAD refractory to rituximab. But complement inhibitors that target the classical complement pathway (i.e. inhibitors against C1 esterase or C3 convertase), would be more effective in patients presenting with exacerbated hemolysis [7]. These severe exacerbations of hemolysis are most often extravascular and mediated by complement receptor- uptake of C3 opsonized RBCs by macrophages in the liver and spleen. Lastly, if patients present with symptomatic anemia, RBC transfusion may be required. But the immunohematologic reference workup is challenging due to the autoantibody masking any possible alloantibody. Shirey et al. showed that adsorption procedures can be significantly avoided by providing genotypically matched RBC units to patients with autoantibody, leading to a simplification in pretransfusion adsorption studies and overall increase in transfusion safety [11,12]. Thus advances in RBC genotyping technology may optimize the selection of RBC units for transfusion, even though the serologic crossmatch will be reactive due to the autoantibody.
3. The role of complement in platelet transfusion refractoriness The role of complement in managing patients who are highly immunized to platelet surface antigens and who have become refractory to platelet transfusion has been investigated by our group [13]. Platelet transfusion refractoriness is defined as repeated low post-transfusion platelet count increments (less than 5000 per l). Its evaluation consists of first ruling out non-immune causes such as sepsis, fever, disseminated intravascular coagulation (DIC), bleeding, splenomegaly, and drugs [13]. In the absence of these clinical conditions, alloimmunization to human leukocyte antigen (HLA) or to platelet-specific antigens should be considered [14,15]. HLA alloimmunization represents 75% of the causes associated with immune mediated platelet refractoriness [13]. A hospital based transfusion service has limited control over the nonimmune causes of refractoriness to platelet transfusion, but it can supply HLA compatible platelets [16,17]. These HLA compatible platelets, also referred to as antigen negative for the corresponding antibodies, result in platelet count increments, post-transfusion, equivalent to those obtained with HLA-matched platelets while increasing the donor pool of eligible donors [18]. Logistically, once the platelet antibodies are identified, the platelet units that are HLA
154
M.J. Fontaine / Transfusion Clinique et Biologique 26 (2019) 152–154
antigen negative for the corresponding antibodies can be searched for by the blood supplier and the HLA compatible units can then be allocated to the patient for transfusion; indeed, most blood suppliers keep track of the HLA type of their platelet donors. Thus the method of antibody detection is critical. Historic use of the complement dependent lymphocytotoxicity (CDC) assay for antibody detection has been replaced by flow cytometry based assays such as the IgG-single antigen bead (SAB) binding assay [19,20]. The flow cytometry screening and identification method for HLA antibody is also widely used for solid organ transplantation. But the classic flow cytometry screening method measures IgG binding only using mean fluorescence intensity (MFI) values as a readout and has high sensitivity and specificity [19]. However, it is widely considered to be “too sensitive” and the MFI value, used as a threshold for antibody identification, is debatable. Importantly, complement-binding antibodies have been known to be clinically relevant for 40 years in renal transplantation [21], and attempts have been made to develop complement dependent assays using the same flow-based SAB assay [22]. We independently developed a C1q assay that only detects those antibodies capable of binding the first component (C1q) of the classical complement pathway [22]. Our studies showed that HLA antibodies identified as binding C1q complement fragment are more immediately associated with platelet destruction than the HLA antibodies binding IgG only [13]. Thus in a case of highly alloimmunized patients, presenting with greater than 80% HLA antibody reactivity using the IgG-SAB method, HLA compatible donor platelets may be scarce. But these same patients, tested with the C1q based assay, may show a lower level of reactivity and allow for a greater number of compatible platelet donations to be allocated and transfused with adequate post-transfusion corrected count increments (greater than 5000 per l). Thus we concluded that the complement-binding property of a platelet HLA antibody may be most relevant in platelet recovery post-transfusion [13]. Interestingly, an analysis by Jackman et al. showed that the level of C1q-binding HLA class I antibodies did not differ significantly between patients presenting with refractoriness compared to patients with no refractoriness to platelet transfusion [23]. The finding from Jackman et al. confirms that antibody-independent mechanisms may also play a role in platelet transfusion refractoriness as previously reported [23,24]. Interestingly, in a study by Waterman et al., using a mouse model of platelet HLA alloimmunization, the role of the common gamma chain receptor was shown to play a more important role than complement C3 fragment in the extravascular clearance of platelets [25]. Indeed, mouse recipients that had a deletion of the common gamma chain of the Fc-receptor presented with decreased to no clearance of PLTs, caused by immune serum immunization, compared to mice with a deletion of the gene encoding complement factor C3 [25]. Thus far, the current complement inhibitors, such as eculizumab, are not likely indicated in patients with platelet transfusion refractoriness. On the other hand, using a complement C1q-dependent assay will more accurately identify the specificity of HLA antibodies to consider avoiding the corresponding antigens while selecting platelets for transfusion. 4. Conclusion We presented clinical scenarios, in which important peripheral blood cellular elements, red blood cells and platelets, may be affected by complement-mediated destruction. Understanding and identifying the role of complement in these settings allow
physicians to adjust their diagnostic and therapeutic modalities accordingly. Keeping in mind that the complement system is a key player in the innate arm of the immune system, one should also understand the infectious risk of modulating its effect. Disclosure of interest The authors declare that they have no competing interest. References [1] Kaufmann SHE. Immunology’s coming of age. Front Immunol 2019;10:684. [2] Hillion S, Arleevskaya MI, Blanco P, Bordron A, Brooks WH, Cesbron JY, et al. The innate part of the adaptive immune system. Clin Rev Allergy Immunol 2019, http://dx.doi.org/10.1007/s12016-019-08740-1 [Epub ahead of print]. [3] Hajishengallis G, Reis ES, Mastellos DC, Ricklin D, Lambris JD. Novel mechanisms and functions of complement. Nat Immunol 2017;18:1288. [4] Schreiber AD, Frank MM. Role of antibody and complement in the immune clearance and destruction of erythrocytes I. In vivo effects of IgG and IgM complement-fixing sites. J Clin Invest 1972;51:575–82. [5] Petz LD, Garratty G. Immune hemolytic anemias. 2nd ed. Philadelphia, Pa: Churchill Livingstone/Elsevier Science; 2004. [6] Meulenbroek EM, Wouters D, Zeerleder SS. Lyse or not to lyse: clinical significance of red blood cell autoantibodies. Blood Rev 2015;29:369–76. [7] Brodsky RA. Complement in hemolytic anemia. Blood 2015;126:2459–65. [8] Garratty G. Immune hemolytic anemia associated with negative routine serology. Semin Hematol 2005;42:156–64. [9] Barcellini W, Fattizzo B, Zaninoni A, Radice T, Nichele I, Di Bona E, et al. Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients. Blood 2014;124:2930–6. [10] Go RS, Winters JL, Kay NE. How I treat autoimmune hemolytic anemia. Blood 2017;129:2971–9. [11] Shirey RS, Boyd JS, Parwani AV, Tanz WS, Ness PM, King KE. Prophylactic antigen-matched donor blood for patients with warm autoantibodies: an algorithm for transfusion management. Transfusion 2002;42:1435–41. [12] Ziman A, Cohn C, Carey PM, Dunbar NM, Fung MK, Greinacher A, et al. Warm-reactive (immunoglobulin G) autoantibodies and laboratory testing best practices: review of the literature and survey of current practice. Transfusion 2017;57:463–77. [13] Fontaine MJ, Kuo J, Chen G, Galel SA, Miller E, Sequeira F, et al. Complement (C1q) fixing solid-phase screening for HLA antibodies increases the availability of compatible platelet components for refractory patients. Transfusion 2011;51:2611–8. [14] Doughty HA, Murphy MF, Metcalfe P, Rohatiner AZ, Lister TA, Waters AH. Relative importance of immune and non-immune causes of platelet refractoriness. Vox Sang 1994;66:200–5. [15] Fabris F, Soini B, Sartori R, Randi ML, Luzzatto G, Girolami A. Clinical and laboratory factors that affect the post-transfusion platelet increment. Transfus Sci 2000;23:63–8. [16] Schiffer CA. Diagnosis and management of refractoriness to platelet transfusion. Blood Rev 2001;15:175–80. [17] Schiffer CA. Management of patients refractory of platelet transfusion. Leukemia 2001;15:683–5. [18] Petz LD, Garratty G, Calhoun L, Clark BD, Terasaki PI, Gresens C, et al. Selecting donors of platelets for refractory patients on the basis of HLA antibody specificity. Transfusion 2000;40:1446–56. [19] Tait BD, Hudson F, Cantwell L, Brewin G, Holdsworth R, Bennett G, et al. Review article: luminex technology for HLA antibody detection in organ transplantation. Nephrology (Carlton) 2009;14:247–54. [20] Wahrmann M, Exner M, Regele H, Derfler K, Körmöczi GF, Lhotta K, et al. Flow cytometry based detection of HLA alloantibody mediated classical complement activation. J Immunol Methods 2003;275:149–60. [21] Smith JD, Hamour IM, Banner NR, Rose ML. C4d fixing, luminex binding antibodies – a new tool for prediction of graft failure after heart transplantation. Am J Transplant 2007;7:2809–15. [22] Chen G, Tyan TD. C1q assay for the detection of complement fixing antibody to HLA antigens. Methods Mol Biol 2013;1034:305–11. [23] Jackman RP, Lee JH, Pei R, Bolgiano D, Lebedeva M, Slichter SJ, et al. C1qbinding anti-HLA antibodies do not predict platelet transfusion failure in Trial to Reduce Alloimmunization to Platelets study participants. Transfusion 2016;56:1442–50. [24] Jackman RP, Muench MO, Heitman JW, Inglis HC, Law JP, Marschner S, et al. Immune modulation and lack of alloimmunization following transfusion with pathogen-reduced platelets in mice. Transfusion 2013;53:2697–709. [25] Waterman HR, Kapp LM, Munday A, Odem-Davis K, Zimring JC. Transfusioninduced alloimmunization and platelet refractoriness in a mouse model: mechanisms and interventions. Transfusion 2016;56:91–100.