Incompatible erythrocyte transfusion with lipopolysaccharide induces acute lung injury in a novel rat model.

Acute transfusion reactions can manifest in many forms including acute hemolytic transfusion reaction, allergic reaction and transfusion-related acute lung injury. We previously developed an acute hemolytic transfusion reaction rat model mediated by transfusion of incompatible human erythrocytes against which rats have preexisting antibodies resulting in classical complement pathway mediated intravascular hemolysis. In this study, the acute hemolytic transfusion reaction model was adapted to yield an acute lung injury phenotype. Adolescent male Wistar rats were primed in the presence or absence of lipopolysaccharide followed by transfusion of incompatible erythrocytes. Blood was collected at various time points during the course of the experiment to determine complement C5a levels and free DNA in isolated plasma. At 4 hours, blood and lung tissue were recovered and assayed for complete blood count and histological acute lung injury, respectively. Compared to sham animals or animals receiving increasing amounts of incompatible erythrocytes (equivalent to a 15-45% transfusion) in the absence of lipopolysaccharide, lungs of animals receiving lipopolysaccharide and a 30% erythrocyte transfusion showed dramatic alveolar wall thickening due to neutrophil infiltration. C5a levels were significantly elevated in these animals indicating that complement activation contributes to lung damage. Additionally, these animals demonstrated a significant increase of free DNA in the blood over time suggestive of neutrophil extracellular trap formation previously associated with transfusion-related acute lung injury in humans and mice. This novel 'two-hit' model utilizing incompatible erythrocyte transfusion in the presence of lipopolysaccharide yields a robust acute lung injury phenotype.

ABO-incompatible platelets are associated with increased transfusion reaction rates.

BACKGROUND: ABO compatibility can affect platelet transfusion safety and efficacy, and ABO-incompatible (ABOi) platelets likely increases the risks of transfusion reactions though the magnitude of this risk is unclear. STUDY DESIGN AND METHODS: Data collected on all platelet transfusions administered over 36+ months were classified based on patient and product ABO blood group type and merged with a data set that included all transfusion reactions reported during that period. The transfusion reaction rates among various subsets was calculated. RESULTS: In patients greater than 1 year of age, the transfusion reaction rate in the ABO-compatible (ABO-identical) platelet group was 1.0%, while the ABOi platelet group had an elevated reaction rate of 1.7%. The increased reaction rate for ABOi platelets held true even if the analysis were limited to Centers for Disease Control and Prevention/National Healthcare Safety Network qualifying reactions or just allergic or febrile nonhemolytic reactions. The increased reaction rate with ABOi platelets was independent of unit age. Surprisingly, major-incompatible transfusions (A/B antigen incompatible) had the highest rate of reactions, at 2.0%. During the study period, three acute hemolytic reactions were reported out of 2522 plasma-incompatible platelet transfusions (0.12%). CONCLUSIONS: Our results find that compatible platelet transfusions have the lowest rate of transfusion reactions. While hemolytic reactions were observed with plasma-incompatible transfusions, the rate was low. Transfusion of ABO antigen-incompatible platelets had the highest rate of transfusion reactions and resulted in a transfusion reaction rate 1.5 to 2 times that of ABO compatible transfusions.

ABO-incompatible kidney transplantation can be successfully conducted by monitoring IgM isoagglutinin titers during desensitization.

BACKGROUND: Recent advances in desensitization techniques and immunosuppressive therapy have led to improved outcomes after ABO-incompatible (ABO-i) kidney transplantation (KT). However, questions remain unanswered, particularly regarding which type of ABO isoagglutinin-immunoglobulin M (IgM) or immunoglobulin M (IgG)-is significantly involved in antibody-mediated rejection (AMR). STUDY DESIGN AND METHODS: We retrospectively analyzed data from 120 patients who underwent ABO-i KT between 2012 and 2014. Preoperative plasma exchange was performed until the IgM isoagglutinin titer was 4 or less, regardless of the IgG titer. Clinical data were compared between patient groups with pre-KT IgG isoagglutinin titer 16 or greater (high IgG; titer range, 16-256; n = 39) and 8 or less (low IgG; titer range, -8; n = 81). RESULTS: The median follow-up periods were 59 (high IgG) and 55 (low IgG) months. Patient survival at 5 years (p = 0.314) was 100% (high IgG) and 97.4% (low IgG). Graft survival at 5 years (p = 0.480) was 100% (high IgG) and 98.7% (low IgG). AMR by anti-ABO antibody occurred in only one patient in the low-IgG group. CONCLUSION: Patients with high pre-KT IgG isoagglutinin titers had equally successful outcomes as those with low IgG titers. ABO-i KT can be successfully performed by reducing the pre-KT IgM isoagglutinin titer to 4 or less, as determined by the immediate spin tube method.

Identification of red blood cell antibodies in maternal breast milk implicated in prolonged hemolytic disease of the fetus and newborn.

BACKGROUND: Alloantibodies against more than 50 non-ABO blood group antigens have been implicated in hemolytic disease of the fetus and newborn (HDFN) and are expected to wane within weeks after delivery. Persistent anemia leads to the hypothesis of continued exposure to red blood cell (RBC) alloantibodies via breast milk, which has been shown in a murine model and suggested in rare case reports. CASE REPORT: We report three cases of prolonged HDFN in two neonates with anti-D HDFN and one with anti-Jka HDFN. Patient 1 demonstrated 4+ anti-D serologic testing beyond 2 months; therefore, antibody testing was performed on maternal breast milk. METHODS: Maternal serum samples were tested for the presence of unexpected antibodies using standard Ortho gel card and 37 °C 60 minutes with anti-human globulin (AHG) tube saline methods. Antibody titrations were performed using the standard 37 °C 60 minutes to AHG tube saline method. Fresh breast milk samples were tested using the standard 37 °C 60 minutes to AHG tube saline method for both unexpected antibodies and titration study. Fresh breast milk from an O-positive, antibody-negative donor was used as control for any reactivity that may have been due to milk solids or proteins alone. RESULTS: Using a known methodology applied in a novel way to test breast milk for RBC alloantibodies, antibodies against fetal RBCs were identified in the maternal breast milk of three patients. CONCLUSION: Maternal RBC alloantibodies are present in breast milk and may be clinically significant in patients with prolonged recovery from HDFN.

Multiplex blood group typing by cellular surface plasmon resonance imaging.

BACKGROUND: Blood-group typing of donors and patients is essential to avoid incompatible transfusions. Transfusion of incompatible RBCs may result in alloimmunization complicating future transfusions or in the presence of antibodies in adverse reactions. With more than 300 blood group antigens identified, it is difficult to provide fully compatible blood. Currently, standard practice is to match for the most immunogenic antigens. While the current agglutination-based RBC-typing methods are reliable for testing a selected number of antigens, they are not easily adaptable for high-throughput multiplex blood typing beyond the current standard. STUDY DESIGN AND METHODS: Surface plasmon resonance (SPR) is a label-free method to follow molecular-and, very recently, also cellular-interactions in real time. Demonstration of binding of RBCs to blood group antigen-specific antibodies by SPR has already been achieved. Here, we demonstrate the generation of an SPR array equipped with clinically relevant blood group antibodies (A, B, and Rh blood groups). To validate this method, we blindly compared typing of 946 blood donors with results of current diagnostic agglutination-based methods. RESULTS: RBC typing was achieved by monitoring RBC binding to blood group-specific antibodies on the sensor simultaneously within 5 minutes per sample. Regeneration of the chip was robust, allowing for typing of at least 100 samples. The typing results gave a 100% match with classical serology with all antibodies tested besides anti-E/e monoclonals, which gave inconsistent results due to low antibody specificity. CONCLUSION: This study demonstrates that SPR-based RBC typing for multiple antigens can be realized simultaneously with high-quality antibodies, enabling reduced hands-on time and possibly improving cost efficiency.

Underlying mechanism and specific prevention of hemolysis-induced platelet activation.

Thromboembolic complications significantly impair the outcome of hemolytic disorders. We hypothesized that red cell adenosine diphosphate (ADP) release results in significant platelet activation in hemolysis and that this prothrombotic state can be prevented by inhibition of the ADP P2Y12 receptor. In the current study, we therefore sought to investigate the mechanism and inhibition of hemolysis-induced platelet activation. The expression of activated integrin αIIbß3 was determined by flow cytometry, and platelet aggregation was assessed by multiple electrode platelet aggregometry. We demonstrate platelet activation and increased platelet aggregation by adding hemolytic blood (lysates) to whole blood, similarly to that achieved by the platelet agonist ADP. Enhanced platelet activation and reactivity in the presence of hemolytic blood were significantly abolished by apyrase, which catalyzes ADP degradation, and inhibited by blockade of the platelet ADP P2Y12 receptor with cangrelor. Platelets from patients treated with the ADP P2Y12 receptor antagonist clopidogrel showed a reduced response to lysates compared to platelets from healthy controls without antiplatelet treatment. Further, in vitro blood group ABO incompatibility induced hemolysis and led to increased platelet activation. Finally, "spontaneous" platelet aggregation seen in patients with cold agglutinin disease was completely abolished by cangrelor. In conclusion, hemolysis is associated with increased platelet activation and aggregation due to red cell derived ADP, which can be prevented by ADP receptor blockade.

Heliotherapy for Neonatal Hyperbilirubinemia in Southwest, Nigeria: A Baseline Pre-Intervention Study.

BACKGROUND: A novel filtered-sunlight phototherapy (FSPT) device has been demonstrated to be safe and efficacious for treating infants with neonatal jaundice in resource-constrained tropical settings. We set out to provide baseline data for evaluating the clinical impact of this device in a referral pediatric hospital. METHODS: We reviewed the medical records of infants admitted for neonatal hyperbilirubinemia in an inner-city Children's Hospital in Lagos, between January 2012 and December 2014 to determine the pattern, treatment and outcomes during the pre-intervention period. Factors associated with adverse outcomes were identified through multivariable logistic regression. RESULTS: Of the 5,229 neonatal admissions over the period, a total of 1,153 (22.1%) were admitted for neonatal hyperbilirubinemia. Complete records for 1,118 infants were available for analysis. The incidence of acute bilirubin encephalopathy (ABE) and exchange transfusion (ET) were 17.0% (95% CI: 14.9%-19.3%) and 31.5% (95% CI: 28.8%-34.3%) respectively. A total of 61 (5.5%, 95% CI: 4.3%-6.9%) of the jaundiced infants died. Weight on admission, peak total serum bilirubin (TSB), sepsis and exposure to hemolytic products were predictive of ABE, while age on admission, peak TSB, ABO incompatibility and ABE were predictive of ET. Rhesus incompatibility, asphyxia, exposure to hemolytic substances and ABE were associated with elevated mortality risk, while ET was a protective factor. Lack of routine irradiance monitoring and steady energy supply were frequent challenges for conventional blue-light phototherapy. CONCLUSIONS: Severe hyperbilirubinemia is associated with high rates of ABE and ET in this setting, and remains a significant contributor to neonatal admissions and mortality. To be impactful, FSPT, complemented with improved diagnostic facilities, should effectively curtail jaundice-related adverse outcomes in this and comparable settings.

C4d and C3d staining in biopsies of ABO- and HLA-incompatible renal allografts: correlation with histologic findings.

Biopsies of ABO-incompatible and positive crossmatch (HLA-incompatible) renal allografts were retrospectively examined to compare results of C4d and C3d staining, and the correlation between such staining and histologic findings suggestive of antibody-mediated rejection (AMR). A total of 75 biopsies (55 protocol, 17 for graft dysfunction, 3 for other indications) of 24 ABO-incompatible grafts and 244 biopsies (103 protocol, 129 for graft dysfunction, 12 for other indications) of 66 HLA-incompatible grafts were examined; all were stained for C4d and approximately 40% for C3d. In ABO-incompatible grafts, 80% of protocol biopsies and 59% performed for graft dysfunction showed C4d staining in peritubular capillaries (PTC); this staining was not correlated with neutrophil margination in PTC. In HLA-incompatible grafts, PTC C4d was present in 26% of protocol biopsies and 60% of biopsies for graft dysfunction; 92% of biopsies with >1+ (0-4+ scale), diffuse PTC C4d had > or =1+ margination and/or thrombotic microangiopathy (TMA), compared with 12% of C4d-negative biopsies. C3d was somewhat more predictive of margination than C4d in ABO-incompatible, but not HLA-incompatible, grafts. In summary, while PTC C4d deposition indicates probable AMR in biopsies of HLA-incompatible grafts, including protocol biopsies, there is no histologic evidence that C4d deposition is correlated with injury in most ABO-incompatible grafts.

Histologic findings one year after positive crossmatch or ABO blood group incompatible living donor kidney transplantation.

Recent protocols have allowed successful positive crossmatch (+XM) and ABO incompatible (ABOI) kidney transplantation, although their long-term outcome is not clear. To begin to assess this issue we compared protocol biopsies performed 12 months posttransplant in 37 +XM, 24 ABOI and 198 conventional allografts. Although the majority in all three groups had only minimal histologic changes, transplant glomerulopathy (TG) was significantly increased in +XM (22% vs. 13% ABOI vs. 8% conventional, p = 0.015), and correlated with prior humoral rejection (HR) by multivariate analysis (odds ratio 17.5, p < or = 0.0001). Patients with a prior history of HR also had a significant increase in interstitial fibrosis (No HR 54% vs. HR 86%, p = 0.045). In the absence of HR no difference in histologic changes was seen between groups, although all three groups had a demonstrable mild increase in interstitial fibrosis from biopsies performed at the time of transplant. Thus, although HR is associated with an increase in TG, in its absence allograft histology is similar in +XM, ABOI and conventional allografts 1 year posttransplant.

Monocyte chemoattractant protein production in red cell incompatibility.

BACKGROUND: Mononuclear phagocytes play a central role in hemolytic transfusion reactions by erythrophagocytosis and production of inflammatory mediators. Factors that affect the number or function of monocyters would be expected to alter the clinical course of hemolytic transfusion reactions, and thus the production of monocyte chemoattractant protein-1 (MCP-1), a recently described chemotactic and activating cytokine specific for monocytes, was investigated in two distinct settings of red cell (RBC) incompatibility. STUDY DESIGN AND METHODS: Fresh heparinized whole blood was incubated with ABO-compatible or -incompatible RBCs. Isolated peripheral blood mononuclear cells were incubated with anti-D-coated or uncoated RBCs. MCP-1 was measured in the plasma or culture medium by enzyme-linked immunosorbent assay. MCP-1 gene expression was detected by Northern blot analysis of buffy coat or mononuclear cell total RNA. RESULTS: Significant levels of MCP-1 protein in plasma or medium were detected 24 hours after the addition of incompatible RBCs, but not in the first 6 hours. Nonimmune hemolysis of added RBCs did not stimulate MCP-1 production. The inactivation of complement by heat treatment of plasma prior to the addition of RBCs to whole blood did not prevent MCP-1 production. Nor did neutralizing antibodies to tumor necrosis factor prevent MCP-1 production in ABO incompatibility. MCP-1 production was associated with increased steady-state levels of white cell MCP-1 mRNA, which occurred more rapidly in ABO than Rh incompatibility. CONCLUSION: The monocyte-specific chemotactic cytokine MCP-1 is produced by peripheral blood leukocytes in response to RBC incompatibility. MCP-1 may act in a positive feedback loop to recruit and activate monocytes during hemolytic transfusion reactions, thus contributing to the maintenance of these reactions.

Interleukin-8 production in red blood cell incompatibility.

Hemolytic transfusion reactions (HTR) are characterized by fever, shock, organ system failure, intravascular coagulation, and possibly death. The same findings may be associated with sepsis. Neutrophils have been implicated in the pathogenesis of HTR, although a mechanism for neutrophil activation has not been shown. In addition, the possible role that cytokines may play in HTR has not been investigated. We show that interleukin-8 (IL-8), a cytokine with chemotactic and neutrophil-activation properties, is produced in whole blood following addition of ABO-incompatible red blood cells, in a dose- and time-dependent manner related to the degree of hemolysis, and is inhibited by inactivation of complement. IL-8 production is accompanied by increased gene expression in the buffy coat. This observation has implications for the understanding of the pathogenesis of and for the treatment of HTR.

[Is amniotic fluid prolactin an osmoregulatory hormone of clinical importance?]. / Fruchtwasser-Prolaktin, ein osmoregulatorisches Hormon mit klinischer Bedeutung?

The present paper reports on the amniotic fluid prolactin concentrations in 61 pregnancies with undisturbed amniotic fluid volume from the 17th to the 41st week of pregnancy. The amniotic fluid prolactin concentrations reach a peak in the 19th week of pregnancy with a slight decrease to the 21st week. After a plateau from the 23rd to the 31st week of pregnancy, prolactin concentrations decrease to very low levels in late gestation. In pregnancies with disturbed amniotic fluid volume the prolactin concentrations are pathologically changed in comparison with "normal" pregnancies. The authors conclude that the amniotic fluid prolactin concentrations have a characteristic profile depending on the stage of pregnancy. From the pathologic prolactin concentrations in patients with hydramnios and otherwise pathologic pregnancies it may be concluded that prolactin is a clinically relevant osmoregulatory hormone for humans too.

Effect of dexamethasone on amniotic fluid absorbance in Rh-sensitized pregnancy.

Five Rh-sensitized pregnant women between 23 and 30 weeks gestation, with a poor obstetric history and initially high delta A450 values, were treated with weekly doses of 24 mg of dexamethasone over a period of 2-7 weeks to enhance fetal lung maturation. Four women showed a gradual decline in delta A450 during the treatment. All five deliveries were delayed until fetal lung maturity was confirmed by amniotic fluid lecithin/sphingomyelin (L/S) ratio and all five fetuses survived. It is possible that high doses of dexamethasone delayed the anticipated intrauterine deterioration of the fetuses and may have prevented the need for intrauterine transfusions.

Renin activity and concentrations of angiotensin I and II in amniotic fluid of normal and Rh-sensitized pregnancies.

Renin activity and the concentrations of angiotensin I and angiotensin II in amniotic fluid of second- and third-trimester pregnancies were determined by radioimmunoassay. Between the 28th and 38th wk of gestation, the mean renin activity in the amniotic fluid was higher than during early pregnancy (before the 18th wk of gestation). Both renin activity and the concentrations of angiotensin I and II were increased on some cases of Rh-incompatibility. One to two weeks after the administration of betamethasone to the mother with threatened premature delivery, the intra-amniotic renin--angiotensin system was slightly suppressed. In urine samples of newborns, angiotensin concentrations were in the same range as those found in the amniotic fluid; renin activity was very low or undetectable in the urine of male neonates (1--7 days of age). Thus, angiotensin II in the amniotic fluid may be derived both from fetal urine and/or as the product of enzymatic reactions in the amniotic sac; the latter is dependent not only on the presence of renin and converting enzyme but also on the local renin substrate (angiotensinogen) concentration.