Sickle cell anaemia (SCA) is the consequence of abnormal haemoglobin production due to an inherited point mutation in the β‐globin gene. The resulting haemoglobin tetramer is poorly soluble when deoxygenated, and when this is prolonged, intracellular gelation of sickle haemoglobin occurs, followed by haemoglobin polymerisation. If many cycles of sickling and unsickling occur, the red cell membrane will be disrupted leading to haemolysis and vaso‐occlusive events. Recent studies have also shown that leucocyte adhesion molecules and nitric oxide (NO) depletion are involved in endothelial damage. New insights in SCA pathophysiology and vascular biology have shown that cell‐derived microparticle (MP) generation is also involved in the vaso‐occlusion. Endothelial damage is perpetuated by impaired production or increased consumption of protective modulators such as protein C, protein S and NO. New therapeutic interventions should address these aspects of SCA pathogenesis. To date, the only US‐FDA‐approved therapy to prevent painful vaso‐occulsive episodes is hydroxyurea that reduces haemoglobin polymerisation in sickle cells by increasing the production of foetal haemoglobin and L‐glutamine. However, several new drugs have been tested in the last years in randomised clinical trials. We here report an update on the current status of knowledge on SCA.

IntroductionCirculating microparticles (MP) have been described in sickle cell anaemia (SCA); however, their interaction with endothelial markers remains unclear. We investigated the relationship between MP, protein C (PC), free protein S (PS), nitric oxide (NO), endothelin-1 (ET-1) and adrenomedullin (ADM) in a large cohort of paediatric patients.MethodA total of 111 children of African ethnicity with SCA: 51 in steady state; 15 in crises; 30 on hydroxyurea (HU) therapy; 15 on transfusion; 17 controls (HbAA) of similar age/ethnicity. MP were analysed by flow cytometry using: Annexin V (AV), CD61, CD42a, CD62P, CD235a, CD14, CD142 (tissue factor), CD201 (endothelial PC receptor), CD62E, CD36 (TSP-1), CD47 (TSP-1 receptor), CD31 (PECAM), CD144 (VE-cadherin). Protein C, free PS, NO, pro-ADM and C-terminal ET-1 were also measured.ResultsTotal MP AV was lower in crisis (1.26×106 ml−1; 0.56–2.44×106) and steady state (1.35×106 ml−1; 0.71–3.0×106) compared to transfusion (4.33×106 ml−1; 1.6–9.2×106, p<0.01). Protein C levels were significantly lower in crisis (median 0.52 IU ml−1; interquartile range 0.43–0.62) compared with all other groups: HbAA (0.72 IU ml−1; 0.66–0.82, p<0.001); HU (0.67 IU ml−1; 0.58–0.77, p<0.001); steady state (0.63 IU ml−1; 0.54–0.70, p<0.05) and transfusion (0.60 IU ml−1; 0.54–0.70, p<0.05). In addition, levels were significantly reduced in steady state (0.63 IU ml−1; 0.54–0.70) compared with HbAA (0.72 IU ml−1; 0.66–0.80, p<0.01). PS levels were significantly higher in HbAA (0.85 IU ml−1; 0.72–0.97) compared with crisis (0.49 IU ml−1; 0.42–0.64, p<0.001), HU (0.65 IU ml−1; 0.56–0.74, p<0.01) and transfusion (0.59 IU ml−1; 0.47–0.71, p<0.01). There was also a significant difference in crisis patients compared with steady state (0.49 IU ml−1; 0.42–0.64 vs. 0.68 IU ml−1; 0.58–0.79, p<0.05). There was high correlation (R>0.9, p<0.05) between total numbers of AV-positive MP (MP AV) and platelet MP expressing non-activation platelet markers. There was a lower correlation between MP AV and MP CD62P (R=0.73, p<0.05) (platelet activation marker), and also a lower correlation between percentage of MP expressing CD201 (%MP CD201) and %MP CD14 (R=0.627, p<0.001). %MP CD201 was higher in crisis (11.6%) compared with HbAA (3.2%, p<0.05); %MP CD144 was higher in crisis (7.6%) compared with transfusion (2.1%, p<0.05); %CD14 (0.77%) was higher in crisis compared with transfusion (0.0%, p<0.05) and steady state (0.0%, p<0.01); MP CD14 was detectable in a higher number of samples (92%) in crisis compared with the rest (40%); %MP CD235a was higher in crisis (17.9%) compared with transfusion (8.9%), HU (8.7%) and steady state (9.9%, p<0.05); %CD62E did not differ significantly across the groups and CD142 was undetectable. Pro-ADM levels were raised in chest crisis: 0.38 nmol L−1 (0.31–0.49) versus steady state: 0.27 nmol L−1 (0.25–0.32; p<0.01) and control: 0.28 nmol L−1 (0.27–0.31; p<0.01). CT-proET-1 levels were reduced in patients on HU therapy: 43.6 pmol L−1 (12.6–49.6) versus control: 55.1 pmol L−1 (45.2–63....

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This study investigated whether filtration of leucodepleted red cells in SAG-M through the P-CAPT filter in order to prevent the potential risk of vCJD infection associated with prion transmission through transfusion has any deleterious effect on red-cell quality. Bottom-and-top SAG-M leucodepleted red-cell concentrates (24 units) were prion-reduction filtered on the day following collection, with half of the units undergoing irradiation on day 14. A control group (12 units) was not prion filtered. Units were sampled at 7-day intervals up to day 35 and tested using standard measures of red-cell quality as well as prothrombin content (to examine prion filter efficacy). Haemoglobin loss per unit was approximately 9 g and in some cases levels were below standard specification (40 g). Haemolysis increased significantly after filtration [0.01 (0.00-0.05) vs. 0.23 (0.07-0.52, p<0.001)]. Prothrombin levels were reduced 41.6-fold compared to leucodepleted red-cell units. Product specifications were within or close to routine acceptable levels. Owing to the reduction in haemoglobin levels below those specified, it may be preferable to reduce haemoglobin specification levels and transfuse more prion-filtered units rather than transfuse potentially unsafe blood product. The risk of transfusing more units with less haemoglobin should be offset against the risk of transfusing unfiltered blood.

3157 Poster Board III-94 Introduction Transfusion circulatory overload (TACO) is a complication of transfusion, often confused with transfusion acute lung injury (TRALI), and is probably under-reported. The pathophysiology is believed to be caused by increased hydrostatic blood pressure leading to fluid leakage in the alveolar space, emulating congestive cardiac failure. We report on a large cohort of TACO cases in Ireland. Methods All consecutive TACO cases reported to the National Haemovigilance Office of Ireland between 2000-2008 were retrospectively reviewed. The incidence of TACO was calculated on units issued from the Irish Blood Transfusion Service (IBTS). Comparative analysis of patient demographic and clinical data was made using Fisher's exact test. Results 179 TACO cases were reviewed. 149 (83%) involved red cell concentrate (RCC), platelets (PLT) n=4 (2%); fresh frozen plasma (FFP) /solvent detergent plasma (SDP) n=16 (8%); multiple components n=10 (6%). During the study period, a total 1,614,973 blood components (RCC, FFP, SDP and PLT) were issued corresponding to a TACO incidence of 1/10.000 of all components issued. TACO incidence per RCC, platelets and plasma issued was 1/8000, 1/48000 and 1/15000 respectively, excluding the cases where multiple components were transfused. While the majority of patients developing TACO were elderly (> 70years)(n= 120, 67%), up to eight cases (4%) were reported in young patients (<30 years). There was no difference in gender. An underlying cardiovascular condition was reported in 73% (131) cases. Patient outcome was specified in 178 cases. One hundred and fifty three (85%) patients recovered post reaction. However, 15 (8%) patients experienced morbidity ranging from prolonged resolution of symptoms, to myocardial infarction (n=2), cancellation of surgery (n=1), and admission to intensive care (n=4). 25 (14%) deaths were reported, and TACO was identified as possible cause of death in n=5 (2%) cases. Four of these patients were male and one patient was female. A total of 158 (88%) patients received diuretics before (n=36), during (n=26) or post (n=124). Nineteen patients (11%) did not receive diuretics; four of these patients died, one of which was possibly attributed to transfusion. Information was unavailable in two cases. Patients treated with diuretics post transfusion were more likely to survive (80% vs. 40%); however statistical significance was not reached (p=0.07). The median transfused volume was 275 mls (range 80-9500 mls). There was no statistical significance between actual time of transfusion and time over which transfusion were prescribed. Approximately 10 % (18) cases involved large volume transfusion most likely in an emergency situation. While only three of these transfusion events met the clinical definition of a massive transfusion (150mls per minute), patients in this cohort received between 300 and 8200 mls per hour. The median volume transfused was 1500mls (range 1000mls – 9500mls). Eleven (61%) patients had underlying cardiovascular conditions. Seven patients were > 70 years, all of whom had underlying cardio–vascular conditions. Four (22%) patients died, one death which was possibly attributed to transfusion, and a further three patients (17%) required admission to intensive care. Four (22%) cases involved young female patients (age < 30 years) who had no underlying cardiovascular condition, but one patient had asthma. These patients were being treated for obstetric bleeding, and all recovered. Conclusion This study reports a TACO incidence of 1 /10 000 units issued from the IBTS. Although this incidence is based on units issued rather than transfused, it probably reflects significant underreporting, and perhaps even under-recognition. This study identified TACO has significant impact on patient outcome with approximately 8% (15) of patients suffering significant morbidity and a further 2% (5) of patients dying following onset of TACO. Finally, patients receiving large volume transfusion in emergency situations even young patients are at risk of developing TACO. Disclosures No relevant conflicts of interest to declare.

The cover image is based on the Review Article INSIGHT INTO THE COMPLEX PATHOPHYSIOLOGY OF SICKLE CELL ANAEMIA AND POSSIBLE TREATMENT by Andrea Piccin et al., https://doi.org/10.1111/ejh.13212.

Microparticles (MP) originate from blebbing and shedding from cell membrane surfaces in physiological and pathological conditions. Increased levels are generated by a number of mechanisms including platelet activation, vascular endothelial damage, thrombin activity, C5b-9 activation, and PF4-heparin-antibody interaction. Increased circulating MP have been described in patients with sickle cell anemia (SCA). Elevated monocyte-derived MP expressing tissue factor have been reported in patients in crisis. The lack of a standardised method for MP quantification remains problematic. We measured MP (numbers and functional markers), protein C and free protein S, in a large cohort of pediatric patients to investigate the role of MP in SCA and their relation to protein C and free protein S plasma levels. One hundred and eleven children of sub-Saharan African ethnicity with SCA (hemoglobin (Hb) SS) were studied: 51 without previous history of crisis in steady state (mean age 5.3 years); 15 in crisis (9 chest crises, 6 other, mean age 5.4 years); 30 on hydroxyurea (mean age 7.2 years); 15 on transfusion therapy (mean age 6.5 years); 17 children of sub-Saharan African ethnicity of similar age (mean age 4.6 years) were used as control group (Hb AA). MP were analyzed by flow cytometry, according to Biró et al (J Thromb Haemost.2004; 2(10):1842–51), using Annexin V and antibodies against, CD61, CD42a, CD62P (P-selectin), CD235a, CD14, CD142 (tissue factor), CD201 (endothelial protein C receptor or EPCR), CD62E (E-selectin), CD36 (thrombospondin or TSP-1), CD47 (TSP-1 receptor), CD31 PECAM (platelet-endothelial cellular adhesion marker), CD144 (VE-cadherin). Protein C (chromogenic) and free protein S (latex based assay) were measured in all subjects. Correlation was measured by Pearson Rank test, and comparisons between groups were analyzed by Mann-Whitney test. Total MP AV were lower in crisis (1.26 × 106/ml; 0.56–2.44 × 106) and steady state (1.35 × 106/ml; 0.71– 3.0 × 106) compared to transfusion (4.33 × 106/ml; 1.6–9.2 × 106p&lt;0.01). Protein C levels were significantly lower in crisis (median 0.52 IU/ml; interquartile range 0.43–0.62) compared to all other groups: HbAA (0.72 IU/ml; 0.66–0.82, p&lt;0.001); hydroxyurea (0.67 IU/ml; 0.58–0.77, p&lt;0.001); steady state (0.63 IU/ml; 0.54–0.70, p&lt;0.05) and transfusion (0.60 IU/ml; 0.54–0.70, p&lt;0.05). In addition levels were significantly reduced in steady state (0.63 IU/ml; 0.54–0.70) compared to HbAA (0.72 IU/ml; 0.66–0.80, p&lt;0.01). Protein S levels were significantly higher in HbAA (0.85 IU/ml; 0.72–0.97) compared with crisis (0.49 IU/ml; 0.42–0.64, p&lt;0.001), hydroxyurea (0.65 IU/ml; 0.56–0.74, p&lt;0.01), and transfusion (0.59 IU/ml; 0.47–0.71, p&lt;0.01). There was also a significant difference in crisis patients compared to steady state (0.49 IU/ml; 0.42–0.64 v 0.68 IU/ml; 0.58–0.79, p&lt;0.05). There was high correlation (R&gt;0.9, p&lt;0.05) between total numbers of Annexin V positive MP (MP AV) and platelet MP expressing non-activation platelet markers. There was a lower correlation between MP AV and MP CD62P (R=0.73, p&lt;0.05) (platelet activation marker), and also a lower correlation between percentage of MP expressing CD201 (%MP CD201) and %MP CD14 (R=0.627, p&lt;0.001). %MP CD201 was higher in crisis (11.6%) compared to HbAA (3.2%, p&lt;0.05); %MP CD144 was higher in crisis (7.6%) compared to transfusion (2.1%, p&lt;0.05); %CD14 (0.77%) was higher in crisis compared to transfusion (0.0%, p&lt;0.05) and steady state (0.0%, p&lt;0.01); MP CD14 was detectable in a higher number of samples (92%) in crisis compared to the rest (40%); %MP CD235a was higher in crisis (17.9%) compared to transfusion (8.9%), hydroxurea (8.7%) and steady state (9.9%, p&lt;0.05); %CD62E did not differ significantly across the groups and CD142 was undetectable. These studies indicate that there are significantly lower levels of protein C and free protein S in children with sickle cell crisis. In addition there are significantly lower numbers of circulating platelet MP in steady state and crisis patients; however in crisis a significantly higher percentage of MP express markers of endothelial and vascular damage, and of red cell origin. Among these are composite hybrid microparticles expressing markers of more than one cell type, probably brought about by severe vascular stress and close contact of various circulating cell types with vascular endothelium.