Anti-Granulocyte Antibodies in Transfusion Medicine

Granulocyte antibodies, in contrast to antibodies recognizing red blood cells and platelets, have been widely neglected in transfusion medicine for several decades, although their clinical relevance is well-described. Autoimmune neutropenia of infancy and neonatal alloimmune neutropenia are probably the most well-known granulocyte antibody-induced disorders. The facts that antibody-induced neutropenias are rare disorders with a favorable prognosis and that sophisticated, labor-intensive in-house techniques are required for granulocyte antibody detection may both have contributed to this negligence. When transfusion-related acute lung injury (TRALI) was delineated as the leading cause of transfusion-associated mortality and, subsequently, granulocytes were identified as key players in TRALI, granulocyte antibodies received progressively higher attention. It took only a short period of time to unravel that transfusion of granulocyte antibodies can lead to activation of the transfusion recipient’s own granulocytes with subsequent breakdown of the pulmonary endothelial barrier and lung edema. Clinical and regulatory pressure has prompted the identification of new target antigens on granulocytes, the development of better diagnostic approaches for antibody identification, and drastic changes in blood component production in order to reduce the risk of TRALI. There were two major fields of controversy in this rather straight-forward process. First, there are two groups of antibodies associated with TRALI: 1) granulocyte antibodies in sensu strictu, i.e., those which recognize human neutrophil antigens (HNA); and 2) human leukocyte antigen (HLA) antibodies which can bind to granulocytes, but also to other cells. These observations led to a debate on whether granulocytes are primary target cells or, at least in some cases, “auxiliary” cells in TRALI. Evidence from animal studies supports the idea that HLA class I antibodies may bind to endothelial cells and recruit granulocytes to the vessel wall, where they get activated; and that HLA class II antibodies, since their target antigen is not expressed on quiescent granulocytes, may bind to monocytes which then activate granulocytes. Recent data on HNA antibodies which cause TRALI by direct binding to pulmonary endothelium in the absence of granulocytes have added to the complexity, as has the observation from prospective clinical trials that HLA class I antibodies are of less relevance than previously anticipated. Today, there is consensus that different antibodies can cause TRALI by different pathways which do partially overlap. Second, the presence of granulocyte antibodies in blood components as the only causative factor for TRALI appeared partially doubtful: a minority of components produced from the same donor and transfused to different recipients led to TRALI. These doubts were further substantiated by the fact that in some TRALI cases, no antibodies whatsoever could be identified. Other substances often referred to as biological response modifiers (BMR) were demonstrated causative for TRALI, but only in animals pre-exposed to priming agents. This observation was crucial in understanding that not only factors present in a blood component (antibody or BMR) need to be considered, but also factors present in the recipient; this led to the “two-hit model” or “threshold model” of TRALI. Nowadays, there is consensus that antibody-mediated TRALI and non-antibody-mediated TRALI exist, with the latter one posing new challenges for transfusion medicine. This overview will start from pre-TRALI knowledge of granulocyte antibodies, consider antibody-mediated TRALI in detail, and will finally return to non-TRALI disorders in the light of relevant new findings in the field. Disclosures No relevant conflicts of interest to declare.

Ivig, but Not a Monoclonal Anti-RBC Antibody, Requires the Presence of Gr-1+ Cells to Ameliorate Immune Thrombocytopenia in a Murine Model

Abstract 268 Although there are many theories as to the mechanism of action of IVIg in the treatment of autoimmune disease, the exact pathway by which IVIg functions remains unclear. Many cell populations have been implicated in the IVIg pathway, including dendritic cells, which are considered to be one of the central initiators of IVIg effects, and macrophages, which are involved in platelet destruction. In addition, there is evidence from several groups that additional intermediary cell types may be involved. IVIg administration can induce a suppressive effect on peripheral blood neutrophil counts in ITP patients. In fact, alloimmunized thrombocytopenic patients, who display low neutrophil counts, do not respond to IVIg therapy. Here, we questioned whether Gr-1+cells (consisting primarily of neutrophils) are a critical cell type required for IVIg function, in a murine model of ITP. Another IVIg product which has ameliorative effects similar to IVIg but appears to function via a different mechanism is anti-D. We have previously shown that IVIg and a monoclonal antibody with “anti-D like” activity, TER-119, can successfully ameliorate thrombocytopenia in a murine model of ITP. In human patients as well as murine models of ITP, these 2 therapeutics appear to function via different mechanisms. Some work has shown that IVIg and anti-D work by the same, or overlapping mechanism, while other work shows a notable difference in that IVIg can cause neutropenia under conditions where it works to ameliorate autoimmune inflammation. Mice pretreated with 50 mg IVIg (∼2g/kg), or 50 ug TER-119 thirty min prior to administration of anti-platelet antibody MWReg30, show protection from thrombocytopenia compared with untreated mice. To assess the potential role for Gr-1+ cells in IVIg vs TER-119 mediated amelioration of murine ITP, we used RB6-8C5, a well described rat antibody for Gr-1+ cell depletion. Mice were injected with RB6-8C5 or control rat IgG 24 hr prior to thrombocytopenia induction. Mice pretreated with RB6-8C5 failed to respond to IVIg therapy compared with control mice. In contrast, Gr-1+ cell depletion had no effect on the ability of TER-119 to ameliorate the thrombocytopenia. This suggests that Gr-1+ cells likely play an essential role in IVIg function. In contrast, TER-119, does not depend on the presence of Gr-1+cells, suggesting that the mechanisms of action for IVIg and RBC specific antibodies are different for this requirement. In line with these observations, it has been observed that IVIg can modulate neutrophil activity, suggesting that in the murine ITP model, IVIg may function through a neutrophil dependent pathway. Experiments using more specific granulocyte antibodies will help ascertain whether neutrophils or some other Gr-1+ cell population is involved in IVIg function. Disclosures: No relevant conflicts of interest to declare.

A Novel Tool for High-Throughput Screening of Granulocyte-Specific Antibodies Using the Automated Flow Cytometric Granulocyte Immunofluorescence Test (Flow-GIFT)

Transfusion-related acute lung injury (TRALI) is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT) has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti—HNA 3a, n = 3; anti—HNA-1b, n = 1) and GAT (anti—HNA-2a, n = 1). The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of blood products.