Acute Hemolysis and Heme Suppress Anti-CD40 Antibody-Induced Necro-Inflammatory Liver Disease.

Clearance of red blood cells and hemoproteins is a key metabolic function of macrophages during hemolytic disorders and following tissue injury. Through this archetypical phagocytic function, heme is detoxified and iron is recycled to support erythropoiesis. Reciprocal interaction of heme metabolism and inflammatory macrophage functions may modify disease outcomes in a broad range of clinical conditions. We hypothesized that acute hemolysis and heme induce acute anti-inflammatory signals in liver macrophages. Using a macrophage-driven model of sterile liver inflammation, we showed that phenylhydrazine (PHZ)-mediated acute erythrophagocytosis blocked the anti-CD40 antibody-induced pathway of macrophage activation. This process attenuated the inflammatory cytokine release syndrome and necrotizing hepatitis induced by anti-CD40 antibody treatment of mice. We further established that administration of heme-albumin complexes specifically delivered heme to liver macrophages and replicated the anti-inflammatory effect of hemolysis. The anti-inflammatory heme-signal was induced in macrophages by an increased intracellular concentration of the porphyrin independently of iron. Overall, our work suggests that induction of heme-signaling strongly suppresses inflammatory macrophage function, providing protection against sterile liver inflammation.

In from the cold: M-protein light chain glycosylation is positively associated with cold agglutinin titer levels.

BACKGROUND: Primary cold agglutinin disease (CAD) is a monoclonal antibody (M-protein) and complement-mediated chronic hemolytic disease process. Antibody glycosylation can play a role in both antibody half-life and complement fixation. Recently, M-protein light chain (LC) glycosylation has been shown to be associated with AL amyloidosis. We hypothesized that M-protein LC glycosylation is also associated with cold agglutinin (CA) titers and CA-mediated hemolysis. STUDY DESIGN AND METHODS: A cross-sectional study of patients undergoing CA titer evaluation underwent mass spectrometric analysis for M-proteins and M-protein LC glycosylation. A subset of serum samples also underwent evaluation for the ability to trigger cold hemolysis in vitro. M-protein and M-protein LC glycosylation rates were compared across CA titer groups, clinical diagnosis, direct antiglobulin testing (DAT) results, and cold in vitro hemolysis rates. RESULTS: Both M-protein and M-protein LC glycosylation rates significantly differed across CA titer groups with the highest rates in those with elevated CA titers. M-protein LC glycosylation occurred almost exclusively on IgM kappa M-proteins and was significantly associated with positive DAT results and a clinical diagnosis of CAD. Cold in vitro hemolysis was demonstrated in two patients who both had a CA titer of more than 512 but there was no significant association with CA titer group or M-protein LC glycosylation status. CONCLUSION: M-protein LC glycosylation is significantly associated with higher CA titer levels. Given the role that antibody glycosylation can play in antibody half-life and complement fixation, further studies are needed to clarify the effects of LC glycosylation within the context of CAD.

Risk factors for hemolytic transfusion reactions resulting from ABO and minor red cell antigen incompatibility: From mislabeled samples to stem cell transplant and sickle cell disease.

Advances in laboratory testing, pathogen reduction and donor qualification have dramatically reduced the risk of acquiring an infection from a blood transfusion. Despite this progress, the most feared complication of transfusion - a hemolytic reaction due to incompatibility between donor and recipient - remains, with essentially no recent progress in the prevention or recognition of this rare but frequently lethal complication. Herein, the role that compatibility testing and transfusion practice play in the occurrence of acute hemolysis are described, with a special emphasis on clinical scenarios confer an increased risk of a severe hemolytic reaction in response to red blood cell or platelet transfusion. In addition, the signs and symptoms of a severe hemolytic reaction are summarized, along with the initial approach to clinical management.

Reduced red blood cell surface level of Factor H as a mechanism underlying paroxysmal nocturnal hemoglobinuria.

The absence of the cell-surface complement inhibitors CD55 and CD59 is considered the mechanism underlying the complement-mediated destruction of affected red blood cells (RBCs) in paroxysmal nocturnal hemoglobinuria (PNH) patients, but Factor H (FH), a fluid-phase complement inhibitor, has also been proposed to be involved. However, the status of FH on the PNH patient RBC surface is unclear and its precise role in PNH pathogenesis remains to be further defined. In this study, we identified significantly lower levels of surface-bound FH on the affected CD59- RBCs than on the unaffected CD59+ RBCs. Although this reduction in surface-bound FH on PNH RBCs was accompanied by decreased surface sialic acid levels, the enzymatic removal of sialic acids from these RBCs did not significantly affect the levels of surface-bound FH. We further observed higher surface levels of FH on the C3b/iC3b/C3dhigh RBCs than on C3b/iC3b/C3dlow RBCs within the affected PNH RBCs of patients treated with eculizumab. Finally, we determined that enhanced surface levels of FH on CD55/CD59-deficient RBCs from mice and PNH patients protected against complement-mediated hemolysis. Taken together, our results suggest that a reduced surface level of FH is another important mechanism underlying the pathogenesis of PNH.

Mitochondrial Reactive Oxygen Species Participate in Signaling Triggered by Heme in Macrophages and upon Hemolysis.

Hemolysis causes an increase of intravascular heme, oxidative damage, and inflammation in which macrophages play a critical role. In these cells, heme can act as a prototypical damage-associated molecular pattern, inducing TLR4-dependent cytokine production through the MyD88 pathway, independently of TRIF. Heme promotes reactive oxygen species (ROS) generation independently of TLR4. ROS and TNF production contribute to heme-induced necroptosis and inflammasome activation; however, the role of ROS in proinflammatory signaling and cytokine production remains unknown. In this study, we demonstrate that heme activates at least three signaling pathways that contribute to a robust MAPK phosphorylation and cytokine expression in mouse macrophages. Although heme did not induce a detectable Myddosome formation, the TLR4/MyD88 axis was important for phosphorylation of p38 and secretion of cytokines. ROS generation and spleen tyrosine kinase (Syk) activation induced by heme were critical for most proinflammatory signaling pathways, as the antioxidant N-acetyl-l-cysteine and a Syk inhibitor differentially blocked heme-induced ROS, MAPK phosphorylation, and cytokine production in macrophages. Early generated mitochondrial ROS induced by heme was Syk dependent, selectively promoted the phosphorylation of ERK1/2 without affecting JNK or p38, and contributed to CXCL1 and TNF production. Finally, lethality caused by sterile hemolysis in mice required TLR4, TNFR1, and mitochondrial ROS, supporting the rationale to target these pathways to mitigate tissue damage of hemolytic disorders.

New Insights in the Pathogenesis and Therapy of Cold Agglutinin-Mediated Autoimmune Hemolytic Anemia.

Autoimmune hemolytic anemias mediated by cold agglutinins can be divided into cold agglutinin disease (CAD), which is a well-defined clinicopathologic entity and a clonal lymphoproliferative disorder, and secondary cold agglutinin syndrome (CAS), in which a similar picture of cold-hemolytic anemia occurs secondary to another distinct clinical disease. Thus, the pathogenesis in CAD is quite different from that of polyclonal autoimmune diseases such as warm-antibody AIHA. In both CAD and CAS, hemolysis is mediated by the classical complement pathway and therefore can result in generation of anaphylotoxins, such as complement split product 3a (C3a) and, to some extent, C5a. On the other hand, infection and inflammation can act as triggers and drivers of hemolysis, exemplified by exacerbation of CAD in situations with acute phase reaction and the role of specific infections (particularly Mycoplasma pneumoniae and Epstein-Barr virus) as causes of CAS. In this review, the putative mechanisms behind these phenomena will be explained along with other recent achievements in the understanding of pathogenesis in these disorders. Therapeutic approaches have been directed against the clonal lymphoproliferation in CAD or the underlying disease in CAS. Currently, novel targeted treatments, in particular complement-directed therapies, are also being rapidly developed and will be reviewed.

Prothrombotic and Proinflammatory Activities of the ß-Hemolytic Group B Streptococcal Pigment.

A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1ß, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system.

Acute kidney injury as the presenting complaint of ceftazidime-induced immune-mediated haemolysis.

We present the case of ceftazidime-induced immune-mediated haemolysis with associated acute kidney injury in a 43-year-old woman. The patient initially presented to the regional cystic fibrosis centre for treatment of an infective exacerbation of cystic fibrosis. After initiation of ceftazidime (a third-generation cephalosporin), renal function rapidly deteriorated and a fall in haemoglobin was noted. On transfer to our care, a haemolysis screen identified immune-mediated haemolysis, and renal biopsy confirmed the finding of acute tubular necrosis secondary to haem pigment. The patient's renal function deteriorated such that she required haemodialysis, although she subsequently recovered and is now dialysis-independent. Although acute haemolytic reactions are recognised with third-generation cephalosporins, this is the first reported case of ceftazidime-induced immune-mediated haemolysis with acute kidney injury. Given the increased frequency of cephalosporin usage, it is important for both nephrologists and general physicians to be aware of this rare but very serious complication.

Distinguishing perforin-mediated lysis and granzyme-dependent apoptosis.

Perforin is an indispensable effector protein of primary cytotoxic lymphocytes (CTL or NK cells) that typically defend the host against virus infection, or gene-modified (chimeric antigen receptor-CAR) anticancer T cells. Perforin's pore-forming activity is necessary for the delivery of proapoptotic serine proteases, granzymes, into the cytosol of infected or cancerous target cells. The complete loss of perforin function is detrimental for the function of cytotoxic lymphocytes, and leads to fatal immune dysregulation in infants and predisposes the carriers of hypomorphic perforin mutations to various chronic inflammatory sequelae and blood cancers. Here, we describe several optimized and validated functional assays using purified effector proteins and cytotoxic lymphocytes that enable detailed analysis of perforin-mediated target cell death pathways.

Secretion of functionally active complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma Multiforme patients.

The complement system is an important humoral immune surveillance mechanism against tumours. However, many malignant tumours are resistant to complement mediated lysis. Here, we report secretion of complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma multiforme (GBM) patients. We investigated whether the secreted FHR5 exhibited functional activity similar to factor H, including inhibition of complement mediated lysis, acting as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase. Immunoblotting analysis of primary GBM cells (B30, B31 and B33) supernatant showed the active secretion of FHR5, but not of Factor H. ELISA revealed that the secretion of soluble GBM-FHR5 by cultured GBM cells increased in a time-dependent manner. Primary GBM-FHR5 inhibited complement mediated lysis, possessed co-factor activity for factor I mediated cleavage and displayed decay acceleration of C3 convertase. In summary, we detected the secretion of FHR5 by primary GBM cells B30, B31 and B33. The results demonstrated that GBM-FHR5 shares biological function with FH as a mechanism primary GBM cells potentially use to resist complement mediated lysis.

Molecular docking explores heightened immunogenicity and structural dynamics of acetaldehyde human immunoglobulin G adduct.

Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2-azobis 2-amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde-modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross-linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo-epitopes on IgG that enhances its immunogenicity.

S100A8 acts as an autocrine priming signal for heme-induced human Mϕ pro-inflammatory responses in hemolytic inflammation.

Intravascular hemolysis, in addition to reducing red cell counts, incurs extensive vascular inflammation and oxidative stress. One product of hemolysis, heme, is a potent danger associated molecular pattern (DAMP), activating leukocytes and inducing cytokine expression and processing, among other pro-inflammatory effects. We explored pathways by which heme-induced inflammation may be amplified under sterile conditions. Incubation of human Mϕs, differentiated from CD14+ cells, with heme induced time- and concentration-dependent gene and protein expression of S100A8, a myeloid cell-derived alarmin. Human Mϕ stimulation with recombinant S100A8, in turn, induced robust pro-IL-1ß expression that was dependent upon NF-κB activation, gene transcription, and partially dependent upon TLR4-mediated signaling. Moreover, heme itself stimulated significant Mϕ pro-IL-1ß gene and protein expression via an S100A8-mediated mechanism and greatly amplified S100A8-driven NLRP3 inflammasome-mediated IL-1ß secretion. In vivo, induction of acute intravascular hemolysis in mice induced a rapid elevation of plasma S100A8 that could be abolished by hemopexin, a heme scavenger. Finally, plasma S100A8 levels were found to be significantly elevated in patients with the inherited hemolytic anemia, sickle cell anemia, when compared with levels in healthy individuals. In conclusion, we demonstrate that hemolytic processes are associated with S100A8 generation and that some of the inflammatory effects of heme may be amplified by autocrine S100A8 production. Findings suggest a mechanism by which hemolytic inflammation could be propagated via leukocyte priming by endogenous proteins, even in sterile inflammatory environments such as those that occur in the hemolytic diseases. S100A8 may represent a therapeutic target for reducing inflammation in hemolytic disorders.

Fatal acute hemolytic transfusion reaction due to anti-B from a platelet apheresis unit stored in platelet additive solution.

BACKGROUND: Hemolytic transfusion reactions from out-of-group plasma in platelet (PLT) transfusions are uncommon, with most involving passive transfer of anti-A. Only rare reactions have ever been reported due to anti-B. STUDY DESIGN AND METHODS: An apheresis PLT product was donated by a blood group O male, processed using PLT additive solution, and pathogen reduced. Postreaction recipient testing included an antibody screen using gel technology, a direct antiglobulin test (DAT) using immunoglobulin G and C3, and an eluate against group O and B cells. Postreaction donor testing included measuring anti-B titers in saline, with and without anti-human globulin. RESULTS: A 60-year-old blood group B patient with relapsed acute myeloid leukemia developed confusion, fever, and hypotension within hours after a blood group O PLT transfusion. The posttransfusion reaction evaluation was remarkable for a positive DAT 3+ for C3; the eluate showed anti-B. Rapid extravascular hemolysis occurred, with a 50% decline in hemoglobin, a high lactate dehydrogenase, and a high bilirubin. She was resuscitated with fluids, blood products, pressors, and oxygen and died of asystole 60 hours later. The donor's anti-B titers were 128 by tube testing at immediate spin and 512 at the anti-human globulin phase. Notably, a group B patient at a different hospital received a split of the same apheresis unit, with no reaction. CONCLUSION: To our knowledge, this is the first fatality reported from passively transfused anti-B. The fact that one transfusion recipient died whereas another did not have any reported reaction highlights the potential importance of recipient variables in isohemagglutinin-mediated hemolysis.

Revisiting the putative role of heme as a trigger of inflammation.

Activation of the innate immune system by free heme has been proposed as one of the principal consequences of cell-free hemoglobin (Hb) exposure. Nonetheless, in the absence of infection, heme exposures within a hematoma, during hemolysis, or upon systemic administration of Hb (eg, as a Hb-based oxygen carrier) are typically not accompanied by uncontrolled inflammation, challenging the assumption that heme is a major proinflammatory mediator in vivo. Because of its hydrophobic nature, heme liberated from oxidized hemoglobin is rapidly transferred to alternative protein-binding sites (eg, albumin) or to hydrophobic lipid compartments minimizing protein-free heme under in vivo equilibrium conditions. We demonstrate that the capacity of heme to activate human neutrophil granulocytes strictly depends on the availability of non protein-associated heme. In human endothelial cells as well as in mouse macrophage cell cultures and in mouse models of local and systemic heme exposure, protein-associated heme or Hb do not induce inflammatory gene expression over a broad range of exposure conditions. Only experiments in protein-free culture medium demonstrated a weak capacity of heme-solutions to induce toll-like receptor-(TLR4) dependent TNF-alpha expression in macrophages. Our data suggests that the equilibrium-state of free and protein-associated heme critically determines the proinflammatory capacity of the metallo-porphyrin. Based on these data it appears unlikely that inflammation-promoting equilibrium conditions could ever occur in vivo.

Engulfment of Hb-activated platelets differentiates monocytes into pro-inflammatory macrophages in PNH patients.

The distinct response shown by different phenotypes of macrophages and monocytes under various clinical conditions has put the heterogeneity of these cells into focus of investigation for several diseases. Recently, we have described that after engulfing hemoglobin (Hb)-activated platelets, classical monocytes differentiated into pro-inflammatory phenotypes, which were abundant in the circulation of paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease patients. Our current study shows that upon engulfment of Hb-activated platelets, monocytes differentiate into M1-macrophages under M1-polarization stimulus (GM-CSF, IFN-γ + LPS). When grown under M2-polarization stimulus (M-CSF, IL-4 + IL13), the cells exhibited an M1-like phenotype, secreted elevated levels of pro-inflammatory cytokines including TNF-α and IL-1ß, and displayed loss of the secretion of cytokine such as IL-10 and also phagocytic ability unlike the conventional M2 macrophages. Interestingly, when differentiated under the above polarization stimulus, monocytes from PNH patients expressed high levels of CD80 and phospho-STAT1, like M1 macrophages. Hemolytic mice also exhibited a gradual increase in monocyte-platelet aggregates in circulation and accumulation of CD80high macrophages in thioglycollate-induced inflamed peritoneum. The spleen of the mice was also populated by CD80high macrophages with compromised phagocytic capacity. Our findings suggest that the hemolytic environment and specifically the Hb-activated platelets, which are abundant in circulation during intravascular hemolysis, closely regulate monocyte differentiation.

Heme Drives Susceptibility of Glomerular Endothelium to Complement Overactivation Due to Inefficient Upregulation of Heme Oxygenase-1.

Atypical hemolytic uremic syndrome (aHUS) is a severe disease characterized by microvascular endothelial cell (EC) lesions leading to thrombi formation, mechanical hemolysis and organ failure, predominantly renal. Complement system overactivation is a hallmark of aHUS. To investigate this selective susceptibility of the microvascular renal endothelium to complement attack and thrombotic microangiopathic lesions, we compared complement and cyto-protection markers on EC, from different vascular beds, in in vitro and in vivo models as well as in patients. No difference was observed for complement deposits or expression of complement and coagulation regulators between macrovascular and microvascular EC, either at resting state or after inflammatory challenge. After prolonged exposure to hemolysis-derived heme, higher C3 deposits were found on glomerular EC, in vitro and in vivo, compared with other EC in culture and in mice organs (liver, skin, brain, lungs and heart). This could be explained by a reduced complement regulation capacity due to weaker binding of Factor H and inefficient upregulation of thrombomodulin (TM). Microvascular EC also failed to upregulate the cytoprotective heme-degrading enzyme heme-oxygenase 1 (HO-1), normally induced by hemolysis products. Only HUVEC (Human Umbilical Vein EC) developed adaptation to heme, which was lost after inhibition of HO-1 activity. Interestingly, the expression of KLF2 and KLF4-known transcription factors of TM, also described as possible transcription modulators of HO-1- was weaker in micro than macrovascular EC under hemolytic conditions. Our results show that the microvascular EC, and especially glomerular EC, fail to adapt to the stress imposed by hemolysis and acquire a pro-coagulant and complement-activating phenotype. Together, these findings indicate that the vulnerability of glomerular EC to hemolysis is a key factor in aHUS, amplifying complement overactivation and thrombotic microangiopathic lesions.

Heme and hemolysis in innate immunity: adding insult to injury.

Heme is a vital, iron-containing prosthetic molecule present in a variety of proteins, of which hemoglobin is the most abundant. While the reactivity afforded by its central iron ion is essential for many cellular processes, it renders heme a potentially damaging molecule upon its release from hemeproteins, as it can catalyze the generation of reactive oxygen species. Severe intravascular hemolysis results in the leakage of vast amounts of hemoglobin, and subsequently, heme into the plasma. As such, heme is increasingly recognized as a major driving force for hemolysis-associated pathology including an increased risk for bacterial infections, due to its pro-oxidant, cytotoxic and immunomodulatory effects. Here, we provide a succinct review of recent, significant developments on how heme can influence innate immune functions, ranging from the maintenance of iron homeostasis by macrophages, the modulation of inflammatory responses, to its role in altering resistance mechanisms against bacterial infections.

Severe hemolysis after plasma transfusion in a neonate with necrotizing enterocolitis, Clostridium perfringens infection, and red blood cell T-polyagglutination.

BACKGROUND: Red blood cell (RBC) Thomsen-Friedenreich antigen exposure (T activation) in infants with necrotizing enterocolitis (NEC) has occasionally been associated with posttransfusional intravascular hemolysis thought to be due to anti-T antibodies in the donor plasma. STUDY DESIGN AND METHODS: We describe an infant with NEC and Clostridium perfringens infection complicated by severe hemolysis after plasma transfusion. After this case, infants with confirmed NEC were prospectively evaluated for T activation. We checked for hemolysis in patients with T activation receiving plasma-containing blood products. RESULTS: The infant had received 80 mL of fresh-frozen plasma (FFP). His RBCs displayed strong T activation, and agglutination was observed with four of six ABO-compatible FFP units. A direct antiglobulin test was negative. IgM-class anti-T antibodies were present in small amounts (titer of 8) in the transfused FFP. Anti-T antibodies from the blood donor were not hemolytic in vitro. In the prospective study, T activation was observed in three of 28 infants with NEC (11%). One infant presented moderate T activation and two infants presented very strong T activation but only moderate decreases in sialic acid expression on the RBC membrane. These three infants presented no signs of hemolysis after transfusion with unwashed blood products or FFP. CONCLUSION: Anti-T antibodies are unlikely to be the etiologic factor for the hemolytic reactions observed in infants with NEC and T activation. Massive RBC desialylation and the direct action of bacterial toxins are more probable causes. Strict avoidance of plasma-containing blood products does not seem justified in these infants.

Rare antibody-associated hemolytic transfusion reaction and transfusion-related acute lung injury: a case report.

BACKGROUND: Hemolytic transfusion reactions and transfusion-related acute lung injury (TRALI) are life-threatening complications associated with the transfusion of blood products. Hemorrhage is one of the most common surgical complications and the risk of bleeding is particularly acute in patients with hematologic deficiencies. Management of surgical bleeding can be divided into two phases. The first phase centers on immediate control of acute bleeding and the second phase focuses on keeping the patient stable and on reducing the sequelae associated with blood transfusions and blood loss. CASE PRESENTATION: We present the case of a 53-year-old woman with long-standing immune thrombocytopenia who underwent repair of a symptomatic ventral hernia. On post-operative day one the patient developed hemoperitoneum, requiring exploratory laparotomy and massive transfusion of blood products. The patient's recovery was complicated by consistently low hemoglobin, hematocrit and platelets, prompting frequent transfusion of additional blood products. Shortly after activation of the massive transfusion protocol, the patient developed TRALI. Compounding the situation, on post-operative day sixteen the patient's serum started to show hemolysis: lactate dehydrogenase (LDH) levels rose to 1,845 IU/L, with haptoglobin at less than 5.8 mg/dL and with a high reticulocyte count (4.38%). Previous testing had shown that the patient was positive for most major antigens implicated in antibody formation and was only producing anti-E and anti-K antibodies (considered for all transfusions). Initial pre- and post-transfusion direct antiglobulin tests (DAT) were indeed negative. However, repeat DATs in the days following the noted serum changes were consistent with new allo-antibody formation. These findings prompted immediate withholding of all blood products and a thorough blood bank work up. Despite strong evidence for new allo-antibody formation, no specific known antibody could be identified. The patient recover well when blood products were withheld. DISCUSSION: We present the case of a 53-year-old woman with long-standing immune thrombocytopenia who underwent repair of a symptomatic ventral hernia. On post-operative day one the patient developed hemoperitoneum, requiring exploratory laparotomy and massive transfusion of blood products. The patient's recovery was complicated by consistently low hemoglobin, hematocrit and platelets, prompting frequent transfusion of additional blood products. Shortly after activation of the massive transfusion protocol, the patient developed TRALI. Compounding the situation, on post-operative day sixteen the patient's serum started to show hemolysis: lactate dehydrogenase (LDH) levels rose to 1,845 IU/L, with haptoglobin at less than 5.8 mg/dL and with a high reticulocyte count (4.38%). Previous testing had shown that the patient was positive for most major antigens implicated in antibody formation and was only producing anti-E and anti-K antibodies (considered for all transfusions). Initial pre- and post-transfusion direct antiglobulin tests (DAT) were indeed negative. However, repeat DATs in the days following the noted serum changes were consistent with new allo-antibody formation. These findings prompted immediate withholding of all blood products and a thorough blood bank work up. Despite strong evidence for new allo-antibody formation, no specific known antibody could be identified. The patient recover well when blood products were withheld. Suspicion for hemolytic transfusion reactions should be high in patients with prior allo-antibody formation; these may present as acute hemolysis or as a delayed hemolytic transfusion reaction. Withholding blood products from these patients until compatible products have been identified is recommended. Moreover, TRALI is the leading cause of transfusion-related fatalities and should always be considered in transfusion settings. CONCLUSIONS: Suspicion for hemolytic transfusion reactions should be high in patients with prior allo-antibody formation; these may present as acute hemolysis or as a delayed hemolytic transfusion reaction. Withholding blood products from these patients until compatible products have been identified is recommended. Moreover, TRALI is the leading cause of transfusion-related fatalities and should always be considered in transfusion settings.