The effect of variation in donor platelet function on transfusion outcome: a semirandomized controlled trial.

The effect of variation in platelet function in platelet donors on patient outcome following platelet transfusion is unknown. This trial assessed the hypothesis that platelets collected from donors with highly responsive platelets to agonists in vitro assessed by flow cytometry (high-responder donors) are cleared more quickly from the circulation than those from low-responder donors, resulting in lower platelet count increments following transfusion. This parallel group, semirandomized double-blinded trial was conducted in a single center in the United Kingdom. Eligible patients were those 16 or older with thrombocytopenia secondary to bone marrow failure, requiring prophylactic platelet transfusion. Patients were randomly assigned to receive a platelet donation from a high- or low-responder donor when both were available, or when only 1 type of platelet was available, patients received that. Participants, investigators, and those assessing outcomes were masked to group assignment. The primary end point was the platelet count increment 10 to 90 minutes following transfusion. Analysis was by intention to treat. Fifty-one patients were assigned to receive platelets from low-responder donors, and 49 from high-responder donors (47 of which were randomized and 53 nonrandomized). There was no significant difference in platelet count increment 10 to 90 minutes following transfusion in patients receiving platelets from high-responder (mean, 21.0 × 109/L; 95% confidence interval [CI], 4.9-37.2) or low-responder (mean, 23.3 × 109/L; 95% CI, 7.8-38.9) donors (mean difference, 2.3; 95% CI, -1.1 to 5.7; P = .18). These results support the current policy of not selecting platelet donors on the basis of platelet function for prophylactic platelet transfusion.

Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets.

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions.

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.

A randomized noninferiority crossover trial of corrected count increments and bleeding in thrombocytopenic hematology patients receiving 2- to 5- versus 6- or 7-day-stored platelets.

BACKGROUND: Bacterial screening offers the possibility of extending platelet (PLT) storage to Day 7. We conducted a noninferiority, crossover trial comparing PLTs stored for 6 or 7 days versus 2 to 5 days. STUDY DESIGN AND METHODS: Stable hematology patients were allocated to receive blocks of 2- to 5- and 6- or 7-day PLTs in random order. The primary outcome was the proportion of successful transfusions during the first block, defined as a corrected count increment (CCI) of more than 4.5 at 8 to 24 hours posttransfusion. RESULTS: Of 122 patients with an evaluable first block, 87 (71%) and 84 (69%) had successful transfusions after 2- to 5- and 6- or 7-day PLTs of mean (SD) ages of 3.8 (1.0) and 6.4 (0.5) days, respectively. Six- or 7-day PLTs were declared noninferior to 2- to 5-day PLTs since the upper confidence interval (CI) limit was less than the predefined noninferiority margin of 10% (95% CI, -14.0% to 9.1%; p = 0.766). Logistic regression analysis gave an adjusted odds ratio of 0.86 (95% CI, 0.47-1.58; p = 0.625). Mean (SD) 8- to 24-hour CCIs were 9.4 (7.9) and 7.7 (7.1) after transfusion with 2- to 5- or 6- or 7-day PLTs (95% CI, -3.31 to 0.03; p = 0.054). The proportions of days with bleeding scores of WHO Grade 2 or higher were 13% (38/297 days) and 11% (32/296 days; 95% CI, -3.2 to 7.2; p = 0.454). Median interval to next PLT transfusion (2 days) was unaffected (95% CI, -10.5 to 5.4; p = 0.531). CONCLUSION: In hematology patients, there was no evidence that 6- or 7-day PLTs were inferior to 2- to 5-day PLTs, as measured by proportion of patients with successful transfusions, bleeding events, or interval to next transfusion.

Challenges in the management of the blood supply.

Although blood suppliers are seeing short-term reductions in blood demand as a result of initiatives in patient blood management, modelling suggests that during the next 5-10 years, blood availability in developed countries will need to increase again to meet the demands of ageing populations. Increasing of the blood supply raises many challenges; new approaches to recruitment and retainment of future generations of blood donors will be needed, and care will be necessary to avoid taking too much blood from these donors. Integrated approaches in blood stock management between transfusion services and hospitals will be important to minimise wastage--eg, by use of supply chain solutions from industry. Cross-disciplinary systems for patient blood management need to be developed to lessen the need for transfusion--eg, by early identification and reversal of anaemia with haematinics or by reversal of the underlying cause. Personalised medicine could be applied to match donors to patients, not only with extended blood typing, but also by using genetically determined storage characteristics of blood components. Growing of red cells or platelets in large quantities from stem cells is a possibility in the future, but challenges of cost, scaling up, and reproducibility remain to be solved.

The Transfusion Alternatives Preoperatively in Sickle Cell Disease (TAPS) study: a randomised, controlled, multicentre clinical trial.

BACKGROUND: No consensus exists on whether preoperative blood transfusions are beneficial in patients with sickle-cell disease. We assessed whether perioperative complication rates would be altered by preoperative transfusion. METHODS: We did a multicentre, randomised trial. Eligible patients were aged at least 1 year, had haemoglobin SS or Sß(0)thalassaemia sickle-cell-disease subtypes, and were scheduled for low-risk or medium-risk operations. Patients were randomly assigned no transfusion or transfusion no more than 10 days before surgery. The primary outcome was the proportion of clinically important complications between randomisation and 30 days after surgery. Analysis was by intention to treat. FINDINGS: 67 (96%) of 70 enrolled patients-33 no preoperative transfusion and 34 preoperative transfusion-were assessed. 65 (97%) of 67 patients had the haemoglobin SS subtype and 54 (81%) were scheduled to undergo medium-risk surgery. 13 (39%) of 33 patients in the no-preoperative-transfusion group had clinically important complications, compared with five (15%) in the preoperative-transfusion group (p=0.023). Of these, 10 (30%) and one (3%), respectively, had serious adverse events. The unadjusted odds ratio of clinically important complications was 3.8 (95% CI 1.2-12.2, p=0.027). 10 (91%) of 11 serious adverse events were acute chest syndrome (nine in the no-preoperative-transfusion group and one in the preoperative-transfusion group). Duration of hospital stay and readmission rates did not differ between study groups. INTERPRETATION: Preoperative transfusion was associated with decreased perioperative complications in patients with sickle-cell disease in this trial. This approach could, therefore, be beneficial for patients with the haemoglobin SS subtype who are scheduled to undergo low-risk and medium-risk surgeries. FUNDING: NHS Blood and Transplant.

Transfusion of prion-filtered red cells does not increase the rate of alloimmunization or transfusion reactions in patients: results of the UK trial of prion-filtered versus standard red cells in surgical patients (PRISM A).

This study, conducted for the UK Blood Transfusion Services (UKBTS), evaluated the clinical safety of red cells filtered through a CE-marked prion removal filter (P-Capt™). Patients requiring blood transfusion for elective procedures in nine UK hospitals were entered into a non-randomized open trial to assess development of red cell antibodies to standard red cell (RCC) or prion-filtered red cell concentrates (PF-RCC) at eight weeks and six months post-transfusion. Patients who received at least 1 unit of PF-RCC were compared with a control cohort given RCC only. About 917 PF-RCC and 1336 RCC units were transfused into 299 and 291 patients respectively. Twenty-six new red cell antibodies were detected post-transfusion in 10 patients in each arm, an overall alloimmunization rate of 4.4%. Neither the treatment arm [odds ratio (OR) 0.93, 95% confidence interval (CI) 0.3, 2.5] nor number of units transfused (OR 0.95, 95% CI 0.8, 1.1) had a significant effect on the proportion of patients who developed new alloantibodies. No pan-reactive antibodies or antibodies specifically against PF-RCC were detected. There was no difference in transfusion reactions between arms, and no novel transfusion-related adverse events clearly attributable to PF-RCC were seen. These data suggest that prion filtration of red cells does not reduce overall transfusion safety. This finding requires confirmation in large populations of transfused patients.

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.

Developing recombinant HPA-1a-specific antibodies with abrogated Fcgamma receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia.

Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A SNP in the gene encoding integrin beta3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a-specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a-specific scFv (B2) with an IgG1 constant region modified to minimize Fcgamma receptor-dependent platelet destruction (G1Deltanab). B2G1Deltanab saturated HPA-1a+ platelets and substantially inhibited binding of clinical HPA-1a-specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Deltanab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Deltanab inhibited chemiluminescence induced by B2G1 and HPA-1a-specific sera. In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a-specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia. As the Deltanab constant region is uninformative in mice, F(ab')2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a-specific antibodies. These results provide rationale for human clinical studies.

Practices associated with ABO-incompatible platelet transfusions: a BEST Collaborative international survey.

BACKGROUND: There is a lack of evidence for guiding the best strategy for ABO selection of platelet (PLT) transfusions. As a baseline for future studies, the BEST Collaborative performed an international survey of current practices in this area. STUDY DESIGN AND METHODS: An international survey was sent via BEST members to transfusion services and hospitals requesting the demographics of the transfused patient population, ABO matching policies, anti-A and anti-B measurements in PLT concentrates (PCs), and practices regarding ABO-incompatible PC transfusions to adult and pediatric patients. RESULTS: We received 126 responses from 14 countries, 59% from Europe. Most of them were from local/community (42%) and university hospitals (39%) serving between 500 and 1500 beds; 50.4% transfused fewer than 1000 PCs per year. One-fifth of respondents (19.4%, mainly local/community hospitals) did not have a written policy for selecting ABO-incompatible PCs. Significant practice variation was reported when ABO-mismatched PLTs were given to adults: for PCs suspended in 100% plasma, 29% to 43% of respondents selected any ABO group available; 52% to 61% selected units with compatible supernatant; and, in the case of minor ABO incompatibility, 43% to 54% did not take any specific action. In contrast if ABO-identical PCs were not available for a pediatric recipient, for PCs resuspended in 100% plasma, 71% to 82% selected PCs so the supernatant plasma would be compatible with patient's red blood cells. CONCLUSION: Considerable practice variation exists when transfusing ABO-incompatible PCs, suggesting an opportunity for research to inform evidence-based practices.

Ten years of hemovigilance reports of transfusion-related acute lung injury in the United Kingdom and the impact of preferential use of male donor plasma.

BACKGROUND AND METHODS: From 1996 through 2006, 195 cases were reported as transfusion-related acute lung injury (TRALI) to the Serious Hazards of Transfusion scheme and from 1999 onward classified by probability, using clinical features and HLA and/or HNA typing. From late 2003, the National Blood Service provided 80 to 90 percent of fresh-frozen plasma (FFP) and plasma for platelet (PLT) pools from male donors. RESULTS: Forty-nine percent of reports were highly likely/probable TRALI, and 51 percent possible/unlikely. Of 96 investigations, donor antibodies recognizing recipient antigens were found in 73 cases (65%), with HLA Class I in 25 of those (40%), HLA Class II antibodies in 38 (62%), and granulocyte antibodies in 12 (17%). A review in 2003 revealed that the TRALI risk/component was 6.9 times higher for FFP and 8.2 times higher for PLTs than for red blood cells, and that in donors of implicated FFP/PLTs, white blood cell antibodies were found 3.6 times more often than by chance (p

Reduced FcRn-mediated transcytosis of IgG2 due to a missing Glycine in its lower hinge.

Neonatal Fc-receptor (FcRn), the major histocompatibility complex (MHC) class I-like Fc-receptor, transports immunoglobuline G (IgG) across cell layers, extending IgG half-life in circulation and providing newborns with humoral immunity. IgG1 and IgG2 have similar half-lives, yet IgG2 displays lower foetal than maternal concentration at term, despite all known FcRn binding residues being preserved between IgG1 and IgG2. We investigated FcRn mediated transcytosis of VH-matched IgG1 and IgG2 and mutated variants thereof lacking Fc-gamma receptor (FcγR) binding in human cells expressing FcRn. We observed that FcγR binding was not required for transport and that FcRn transported less IgG2 than IgG1. Transport of IgG1 with a shortened lower hinge (ΔGly236, absent in germline IgG2), was reduced to levels equivalent to IgG2. Conversely, transport of IgG2 + Gly236 was increased to IgG1 levels. Gly236 is not a contact residue between IgG and FcRn, suggesting that its absence leads to an altered conformation of IgG, possibly due to a less flexible Fab, positioned closer to the Fc portion. This may sterically hinder FcRn binding and transport. We conclude that the lack of Gly236 is sufficient to explain the reduced FcRn-mediated IgG2 transcytosis and accounts for the low maternal/fetal IgG2 ratio at term.

National Blood Service TRALI Reduction Policies: Implementation and Effect.

SUMMARY: This article describes the TRALI reduction policies which were introduced by the National Blood Service in late 2003. Consideration was given to the reasons for their introduction and how the changes were implemented. The observed effects which followed the introduction of these policies were examined by analysis of reports to the Serious Hazards of Transfusion (SHOT) Scheme.

Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport.

We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions.

Serious hazards of transfusion: a decade of hemovigilance in the UK.

The Serious Hazards of Transfusion (SHOT) scheme is a UK-wide, independent, professionally led hemovigilance system focused on learning from adverse events. SHOT was established in 1996 as a confidential reporting system for significant transfusion-related events, building an evidence base to support blood safety policy decisions, clinical guidelines, clinician education, and improvements in transfusion practice. Recommendations are formulated by an independent steering group drawn from medical royal colleges and professional bodies. Ten years after its inception, SHOT has analyzed 2630 transfusion safety events, published 8 annual reports with recommendations, and presented data nationally and internationally. These recommendations have underpinned key initiatives, in particular the UK Department of Health "Better Blood Transfusion" strategy. SHOT has encouraged open reporting of adverse events and "near-misses" in a supportive, learning culture, vigilance in hospital transfusion practice, and evaluation of information technology to support this process. The importance of education and training has been emphasized. Detailed analysis of events has identified weaknesses in the transfusion chain. A collaborative initiative between SHOT, the Chief Medical Officer for England's National Blood Transfusion Committee, and the National Patient Safety Agency aims to reduce ABO-incompatible transfusions by improving bedside practice. Cumulative SHOT data have documented the decline in transfusion-related graft vs host disease after implementation of leucodepletion and have highlighted transfusion-related acute lung injury and bacterial contamination of platelets as important causes of death and morbidity. The UK blood services have developed strategies to reduce these risks. Future SHOT data will evaluate the success of these and other blood safety improvements.

Differential binding to human FcgammaRIIa and FcgammaRIIb receptors by human IgG wildtype and mutant antibodies.

We are investigating the interactions of recombinant human IgG antibodies with Fc receptors to enable selection of a constant region giving minimal depletion of antigen-bearing cells. Eight variant constant regions were made by substituting motifs between human IgG subclasses in the lower hinge region and/or a specially close loop of the CH2 domain. Mutations in the lower hinge region were shown to eliminate FcgammaRI binding and monocyte activation [Eur. J. Immunol. 29 (1999) 2613]. Here, we detail interactions with FcgammaRIIa of the 131R and 131H allotypes and FcgammaRIIb. Lower hinge mutations caused large reductions in binding whereas modification of residues 327, 330 and 331 had less dramatic effects. However, like the wildtype IgG subclass binding hierarchies, the effect of the mutations varied between different receptors. We identified IgG1 variants which react with the activating receptor, FcgammaRIIa, at least 10-fold less efficiently than wildtype IgG1 but whose binding to the inhibitory receptor, FcgammaRIIb, is only four-fold reduced. Manipulation of interactions with FcgammaRIIb separately from those with activating receptors provides potential for designing antibodies with novel and effective combinations of attributes. In addition, insight is gained into the evolution of functional differences in human IgG subclasses.

Neonatal thrombocytopenia and platelet transfusion - a UK perspective.

Five percent of newborn infants admitted to UK neonatal units during a recent study developed a platelet count <60 × 10(9)/l, and 60% of these were transfused platelets. This review summarises the common causes and mechanisms of thrombocytopenia in the newborn. Relevant evidence relating the platelet count to the risk of haemorrhage is reviewed, and current UK guidance on transfusion thresholds outlined. The UK policy for the provision of platelets for transfusion to neonates is described, including the particular requirements for neonatal allo-immune thrombocytopenia. Finally, we look towards the future and prospects for reducing the need to expose newborns to donor-derived platelets.

The effect of recombinant IgG antibodies against the leucine-33 form of the platelet beta3 integrin (HPA-1a) on platelet function.

Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-1a1b platelets to fibrinogen was reduced by 20%, and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human alphaIIbbeta3 or alphaVbeta3 transfectants of HPA-1a (Leu(33)) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on beta3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy.

Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

Neonatal transfusion.

Neonates and particularly preterm neonates are frequent recipients of large volumes of blood products relative to their size. Good quality evidence for transfusion practice in this patient group has been lacking but is now increasing. Triggers for red cell transfusion are now better defined, with on-going trials of platelet transfusions likely to yield similar evidence. Transfusion is now extremely safe, but complications such as transfusion associated acute lung injury (TRALI) and transfusion associated circulatory overload (TACO) are likely to be under recognised, particularly in the sick extremely preterm neonate with respiratory symptoms. This review summarises the rationale and current practice with regard to blood component therapy. Background data on component specifications and hazards of transfusion are provided. Indications for transfusion of specific products including red cells, platelets, and plasma are discussed, and their use is illustrated by case examples.