Discriminating the hemolytic risk of blood type A plasmas using the complement hemolysis using human erythrocytes (CHUHE) assay.

BACKGROUND: The agglutination-based cross-matching method is sensitive for antibody binding to red blood cells but is only partially predictive of complement-mediated hemolysis, which is important in many acute hemolytic transfusion reactions. Here, we describe complement hemolysis using human erythrocytes (CHUHE) assays that directly evaluate complement-mediated hemolysis between individual serum-plasma and red blood cell combinations. The CHUHE assay is used to evaluate correlations between agglutination titers and complement-mediated hemolysis as well as the hemolytic potential of plasma from type A blood donors. STUDY DESIGN AND METHODS: Plasma or serum from each type A blood donor was incubated with AB or B red blood cells in the CHUHE assay and measured for free hemoglobin release. RESULTS: CHUHE assays for serum or plasma demonstrate a wide, dynamic range and high sensitivity for complement-mediated hemolysis for individual serum/plasma and red blood cell combinations. CHUHE results suggest that agglutination assays alone are only moderately predictive of complement-mediated hemolysis. CHUHE results also suggest that plasma from particular type A blood donors produce minimal complement-mediated hemolysis, whereas plasma from other type A blood donors produce moderate to high-level complement-mediated hemolysis, depending on the red blood cell donor. CONCLUSION: The current results indicate that the CHUHE assay can be used to assess complement-mediated hemolysis for plasma or serum from a type A blood donor, providing additional risk discrimination over agglutination titers alone.

High-throughput acoustic separation of platelets from whole blood.

Platelets contain growth factors which are important in biomedical and clinical applications. In this work, we present an acoustic separation device for high-throughput, non-invasive platelet isolation. In particular, we separated platelets from whole blood at a 10 mL min(-1) throughput, which is three orders of magnitude greater than that of existing acoustic-based platelet separation techniques. Without sample dilution, we observed more than 80% RBC/WBC removal and platelet recovery. High throughput, high separation efficiency, and biocompatibility make this device useful for many clinical applications.

Discriminating complement-mediated acute transfusion reaction for type O+ red blood cells transfused into a B+ recipient with the complement hemolysis using human erythrocytes (CHUHE) assay.

BACKGROUND: A patient with B+ sickle cell disease received 3 units of red blood cells (RBCs) from two O+ donors and developed fever and hypotension after the first unit, consistent with an acute transfusion reaction (ATR). Anti-B titers in plasma from each O+ donor were markedly elevated and nondiscriminatory. In order to evaluate the potential for the transfused units to produce complement-mediated hemolysis of B+ RBCs, hemolytic complement testing was performed. STUDY DESIGN AND METHODS: Plasma from each donor was diluted in veronal buffer and incubated with B+ RBCs, and free hemoglobin was measured by spectrophotometer in the complement hemolysis using human erythrocytes (CHUHE) assay. Peptide inhibitor of complement C1 (PIC1) was used to confirm antibody-initiated complement pathway activation. RESULTS: A 96-fold difference (p = 0.014) in hemolysis was measured between plasma samples from the two O+ donors using the CHUHE assay. The extremely high degree of hemolysis produced by the one plasma was inhibited by PIC1 in a dose-dependent manner. CONCLUSION: These results indicate that hemolytic complement testing with the CHUHE assay can be used to assess the risk of antibody-initiated, complement-mediated hemolysis from a transfusion beyond what can be achieved with antibody titers alone.

Additive solution-7 reduces the red blood cell cold storage lesion.

BACKGROUND: Transfusion of long-stored red blood cells (RBCs) is associated with decreased in vivo RBC recovery, delivery of RBC breakdown products, and increased morbidity and mortality. Reducing the burden of this RBC "storage lesion" is a major challenge in transfusion medicine. Additive solution-7 (AS-7) is a new RBC storage solution designed to improve RBC metabolism by providing phosphate and increasing buffering capacity. STUDY DESIGN AND METHODS: Storage quality in AS-7 was measured in a prospective, randomized, three-center trial using units of whole blood from healthy human subjects whose RBCs were stored for up to 56 days in AS-7 (n = 120) or for 42 days in the control solution AS-1 (n = 60). RESULTS: Hemolysis and shedding of protein-containing microvesicles were significantly reduced in RBCs stored in AS-7 for 42 and 56 days compared with RBCs stored in AS-1. Autologous in vivo recoveries of RBCs stored in AS-7 was 88 ± 5% at 42 days (n = 27) and 82 ± 3% at 56 days (n = 27), exceeding recoveries of RBCs stored in currently used solutions. CONCLUSION: Increasing the phosphate, pH range, and buffer capacity of a RBC storage system allowed RBCs to be stored better and longer than currently approved storage systems. AS-7 ameliorates the long-term storage lesion resulting in significantly increased viability in vitro and in vivo.

Peptide inhibitor of complement c1, a novel suppressor of classical pathway activation: mechanistic studies and clinical potential.

The classical pathway of complement plays multiple physiological roles including modulating immunological effectors initiated by adaptive immune responses and an essential homeostatic role in the clearance of damaged self-antigens. However, dysregulated classical pathway activation is associated with antibody-initiated, inflammatory diseases processes like cold agglutinin disease, acute intravascular hemolytic transfusion reaction (AIHTR), and acute/hyperacute transplantation rejection. To date, only one putative classical pathway inhibitor, C1 esterase inhibitor (C1-INH), is currently commercially available and its only approved indication is for replacement treatment in hereditary angioedema, which is predominantly a kinin pathway disease. Given the variety of disease conditions in which the classical pathway is implicated, development of therapeutics that specifically inhibits complement initiation represents a major unmet medical need. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement. In vitro studies have demonstrated that these peptide inhibitors of complement C1 (PIC1) bind to the collagen-like region of the initiator molecule of the classical pathway, C1q. PIC1 binding to C1q blocks activation of the associated serine proteases (C1s-C1r-C1r-C1s) and subsequent downstream complement activation. Rational design optimization of PIC1 has resulted in the generation of a highly potent derivative of 15 amino acids. PIC1 inhibits classical pathway mediated complement activation in ABO incompatibility in vitro and inhibiting classical pathway activation in vivo in rats. This review will focus on the pre-clinical development of PIC1 and discuss its potential as a therapeutic in antibody-mediated classical pathway disease, specifically AIHTR.

Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis.

BACKGROUND: Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement-mediated acute intravascular hemolysis. STUDY DESIGN AND METHODS: Human AB red blood cells (RBCs) were incubated with complement-sufficient or complement-deficient Wistar rat serum (WRS) in the presence and absence of human RBC antibody in vitro to elucidate the mechanism of hemolysis. To study the role of complement in acute intravascular hemolysis in vivo, Wistar rats were treated either with or without cobra venom factor (CVF) to deplete complement activity. Human AB RBCs were then injected into both groups of rats, followed by serial blood draws up to 2 hours. Venous blood clearance and lysis of transfused RBCs at each time point were measured by flow cytometry and spectrophotometry. RBC sequestration was determined in the liver, spleen, and kidney by immunohistochemistry. RESULTS: In vitro incubation of human RBCs with WRS demonstrated that RBC lysis was mediated via the classical complement pathway and that hemolysis was antibody dependent. Transfusion of human RBCs into rats showed significantly less hemolysis in the CVF group versus untreated group. RBC sequestration in the spleen and liver 2 hours posttransfusion were not quantitatively different between the two groups. CONCLUSIONS: Given the much higher degree of similarity for rat and human complement compared to mice, this simple rat model is ideal for testing novel inhibitors of classical pathway activation for the prevention and treatment of acute intravascular hemolysis.

In vitro and in vivo quality of leukoreduced apheresis platelets stored in a new platelet additive solution.

BACKGROUND: Platelets (PLTs) stored in additive solutions (PASs) may reduce the risk of several plasma-associated adverse transfusion reactions such as allergic reactions and potentially transfusion-associated lung injury. The objective of this study was to determine the in vitro characteristics and the in vivo radiolabeled recovery and survival of apheresis PLTs (APs) stored in a new PAS and compare the latter to Food and Drug Administration (FDA) criteria. STUDY DESIGN AND METHODS: Hyperconcentrated APs were collected from healthy subjects in a paired crossover study comparing PAS (35% plasma) and 100% plasma-stored APs (Part 1) up to 7 days and, in Part 2, to determine the in vivo recovery and survival of PAS stored AP at 5 days compared to fresh PLT controls. In vitro and in vivo assays were performed following standard methods. RESULTS: Sixty-six and 25 evaluable subjects successfully completed Parts 1 and 2, respectively. pH for PAS AP was maintained above 6.6 for 5 days of storage. P-selectin values were consistent with published values for commonly transfused PLT products. The PAS in vivo PLT recovery (54.3 ± 8.1%) was 86.7% of the fresh control, and survival (6.4 ± 1.3 days) was 78.0% of the fresh control, both meeting the FDA performance criteria. CONCLUSION: APs stored in PAS with 35% plasma carryover maintained pH over 5 days of storage and met current FDA criteria for radiolabeled recovery and survival. The use of PAS for storage of single-donor PLTs in clinical practice represents an acceptable transfusion product that reduces the volume of plasma associated with PLT transfusion.

Evaluation of the properties of components prepared and stored after holding of whole blood units for 8 and 24 hours at ambient temperature.

BACKGROUND: The capability of holding whole blood (WB) units at ambient temperature, overnight, should help in platelet (PLT) concentrate preparation logistics. We summarize the results of a study conducted in the early 1990s that compared, in particular, PLT and red blood cell (RBC) in vivo viability properties following storage after preparation after 8- and 24-hour WB hold periods. STUDY DESIGN AND METHODS: Individuals donated units of WB on two occasions. Centrifugation at 20 to 24°C to separate PLTs and additive system RBC placement at 1 to 6°C was completed 8 hours after phlebotomy or after 24 hours in randomized order. Components were not leukoreduced. Studies including in vitro biochemical and hematologic analyses and autologous in vivo RBC and PLT evaluations were conducted at two sites. RESULTS: RBC 24-hour in vivo (mean ± SD) recoveries (single-label approach), after 35 days of storage, were 79.2 ± 4.3 and 79.4 ± 3.9% (n = 9; p > 0.05), with WB holding periods of 8 and 24 hours, respectively. With 42 days of storage, recovery after a 24-hour hold was slightly less than with an 8-hour hold (72.9 ± 6.5% vs. 76.0 ± 5.4%; n = 17; p < 0.05). RBC 2,3-diphosphoglycerate acid levels were substantially less after the 24-hour hold compared to after the 8-hour hold (n = 18; p < 0.05). PLT recovery after 5 days of storage with 8- and 24-hour hold periods were similar, 51.1 ± 14.9 and 50.6 ± 17.7%, respectively (n = 18; p > 0.05). The PLT survival variable and in vitro properties reflecting storage quality also showed no significant difference. CONCLUSION: RBC and PLT in vivo variables, and most in vitro variables, were not significantly different after storage with WB holding times of 8 and 24 hours except for a slight diminution of RBC recovery with the 24-hour hold after 42 days of storage.

Automated collection of double red blood cell units with a variable-volume separation chamber.

BACKGROUND: Automated collection of blood components offers multiple advantages and has prompted development of portable devices. This study sought to document the biochemical and hematologic properties and in vivo recovery of red cells (RBCs) collected via a new device that employed a variable-volume centrifugal separation chamber. STUDY DESIGN AND METHODS: Normal subjects (n = 153) donated 2 units of RBCs via an automated blood collection system (Cymbal, Haemonetics). Procedures were conducted with wall outlet power (n = 49) or the device's battery source (n = 104). Units were collected with or without leukoreduction filtration and were stored in AS-3 for 42 days. The units were assessed via standard biochemical and hematologic tests before and after storage, and 24 leukoreduced (LR) and 24 non-LR RBCs were radiolabeled on Day 42 with Na(2)(51)CrO(4) for autologous return to determine recovery at 24 hours with concomitant determination of RBC volume via infusion of (99m)Tc-labeled fresh RBCs. RESULTS: Two standard RBC units (targeted to contain 180 mL of RBCs plus 100 mL of AS-3) could be collected in 35.7 +/- 2.0 minutes (n = 30) or 40.3 +/- 2.7 minutes for LR RBCs (n = 92). An additional 31 collections were conducted successfully with intentional filter bypassing. RBC units contained 104 +/- 4.1 percent of their targeted volumes (170-204 mL of RBCs), and LR RBCs contained 92 percent of non-LR RBCs' hemoglobin. All LR RBCs contained less than 1 x 10(6) white blood cells. Mean hemolysis was below 0.8 percent (Day 42) for all configurations. Adenosine triphosphate was well preserved. Mean recovery was 82 +/- 4.9 percent for RBCs and 84 +/- 7.0 percent for LR RBCs. CONCLUSIONS: The Cymbal device provided quick and efficient collection of 2 RBC units with properties meeting regulatory requirements and consistent with good clinical utility.