Recombinant HPA-1a antibody therapy for treatment of fetomaternal alloimmune thrombocytopenia: proof of principle in human volunteers.

Fetomaternal alloimmune thrombocytopenia, caused by the maternal generation of antibodies against fetal human platelet antigen-1a (HPA-1a), can result in intracranial hemorrhage and intrauterine death. We have developed a therapeutic human recombinant high-affinity HPA-1a antibody (B2G1Δnab) that competes for binding to the HPA-1a epitope but carries a modified constant region that does not bind to Fcγ receptors. In vitro studies with a range of clinical anti-HPA-1a sera have shown that B2G1Δnab blocks monocyte chemiluminescence by >75%. In this first-in-man study, we demonstrate that HPA-1a1b autologous platelets (matching fetal phenotype) sensitized with B2G1Δnab have the same intravascular survival as unsensitized platelets (190 hours), while platelets sensitized with a destructive immunoglobulin G1 version of the antibody (B2G1) are cleared from the circulation in 2 hours. Mimicking the situation in fetuses receiving B2G1Δnab as therapy, we show that platelets sensitized with a combination of B2G1 (representing destructive HPA-1a antibody) and B2G1Δnab survive 3 times as long in circulation compared with platelets sensitized with B2G1 alone. This confirms the therapeutic potential of B2G1Δnab. The efficient clearance of platelets sensitized with B2G1 also opens up the opportunity to carry out studies of prophylaxis to prevent alloimmunization in HPA-1a-negative mothers.

Pathogen inactivation of platelets using ultraviolet C light: effect on in vitro function and recovery and survival of platelets.

BACKGROUND: We evaluated the effect of treating platelets (PLTs) using ultraviolet (UV)C light without the addition of any photosensitizing chemicals on PLT function in vitro and PLT recovery and survival in an autologous radiolabeled volunteer study. STUDY DESIGN AND METHODS: For in vitro studies, pooled or single buffy coat-derived PLT concentrates (PCs) were pooled and split to obtain identical PCs that were either treated with UVC or untreated (n = 6 each) and stored for 7 days. PLT recovery and survival were determined in a two-arm parallel autologous study in healthy volunteers performed according to BEST guidelines. UVC-treated or untreated PCs (n = 6 each) were stored for 5 days and were compared to fresh PLTs from the same donor. RESULTS: There were no significant differences on Day 7 of storage between paired UVC-treated and control PC units for pH, adenosine triphosphate, lactate dehydrogenase, CD62P, CD63, PLT microparticles, and JC-1 binding, but annexin V binding, lactate accumulation, and expression of CD41/61 were significantly higher in treated units (p < 0.05). Compared with control units, the recovery and survival of UVC-treated PC were reduced after 5 days of storage (p < 0.05) and when expressed as a percentage of fresh values, survival was reduced by 20% (p = 0.005) and recovery by 17% (p = 0.088). CONCLUSION: UVC-treated PLTs stored for 5 days showed marginal changes in PLT metabolism and activation in vitro and were associated with a degree of reduction in recovery and survival similar to other pathogen inactivation systems that are licensed and in use.

In vitro quality of single-donor platelets treated with riboflavin and ultraviolet light and stored in platelet storage medium for up to 8 days.

BACKGROUND: The Mirasol pathogen reduction technology system is known to increase the activation and metabolic rate of platelets (PLTs). Storage of Mirasol PLTs in PLT storage medium (PSM) has the potential to slow this accelerated PLT storage lesion. We investigated the quality of Mirasol-treated PLTs stored in either 50% SSP+ or 50% Composol for 8 days. STUDY DESIGN AND METHODS: Single-donor double hyperconcentrates were divided between control and Mirasol-treated arms and after treatment were suspended in approximately 50% (vol/vol) SSP+ (n = 8) or Composol (n = 7). In vitro markers of PLT activation and/or apoptosis were measured over an 8-day storage period. RESULTS: Mirasol treatment resulted in increased spontaneous PLT activation and glycolysis and these effects were worsened when PLTs were treated below a certain volume (150 mL). At higher treatment volumes there were no significant differences between treated units stored in either Composol or SSP+. When low-volume units were stored in Composol the median pH fell below 6.4 on Day 5 and bicarbonate was undetectable, whereas in SSP+ the median pH value was greater than 6.9 and bicarbonate remained at detectable levels, despite other markers of in vitro function being similar to those of Composol. CONCLUSION: Mirasol treatment of PLTs followed by storage in PSM results in increased PLT activation and metabolism to a level similar to that reported for PLTs treated and stored in plasma. Units treated at a low volume (<150 mL) showed poor in vitro quality.

Thrombin generation and clot formation in methylene blue-treated plasma and cryoprecipitate.

BACKGROUND: Methylene blue (MB) treatment of plasma is known to reduce the activity of clotting factors, but its effect on thrombin generation and clot formation is not well documented. STUDY DESIGN AND METHODS: Individual clotting factors and inhibitors and global tests of thrombin generation and clot formation using rotational thrombelastometry (ROTEM) were assessed in a paired study of standard or MB plasma and cryoprecipitate (n = 20 each). RESULTS: MB treatment resulted in a 10 percent reduction in endogenous thrombin potential and 30 percent decrease in peak thrombin as well as the expected 20 to 35 percent loss of Factor (F)VIII, fibrinogen, and FXI activity. MB treatment had no effect on the rate of clot formation and increased the clot firmness by 20 percent as assessed by ROTEM. There were minimal further changes in either coagulation factor levels or thrombin generation when thawed plasma was stored for an additional 24 hours. FVIII and fibrinogen content of MB cryoprecipitate was reduced by 30 and 40 percent, respectively, but this was not associated with altered clot time or rate of clot formation by ROTEM and only an 8 percent decrease in clot firmness. CONCLUSIONS: It is concluded that MB treatment is associated with a reduction in the thrombin-generating capacity of plasma, but has very little effect on the strength of clot formation as assessed by thrombelastometry. The thrombin-generating capacity of standard and MB plasma is relatively unaltered by subsequent storage of thawed plasma at 4 degrees C for 24 hours.