Publications by authors named "Rosemary L Sparrow"

41 Publications

Higher donor body mass index is associated with increased hemolysis of red blood cells at 42-days of storage: A retrospective analysis of routine quality control data.

Transfusion 2021 Feb 24;61(2):449-463. Epub 2020 Nov 24.

Melbourne Dental School, The University of Melbourne, Melbourne, Victoria, Australia.

Background: For reasons unclear, some stored red blood cells (RBCs) have low hemolysis, while others have high hemolysis, which impacts quality consistency. To identify variables that influence hemolysis, routine quality control (QC) data for 42-days-stored RBCs with corresponding donor information were analyzed.

Study Design And Methods: RBC QC and donor data were obtained from a national blood supplier. Regression models and analyses were performed on total cohort stratified by donor sex and by high hemolysis (≥90th percentile) vs control (<90th percentile) samples, including matching.

Results: Data included 1734 leukoreduced RBCs (822 female, 912 male), processed by buffy coat-poor or whole blood filtration methods. Male RBCs had larger volume, hemoglobin content, and higher hemolysis than female RBCs (median hemolysis, 0.24% vs 0.21%; all P < .0001). Multivariable regression identified increased body mass index (BMI) and RBC variables were associated with higher hemolysis (P < .0001), along with older female age and buffy coat-poor processing method (P < .002). Logistic regression models comparing the high and control hemolysis subsets, matched for RBC component variables and processing method, identified overweight-obese BMI (>27 kg/m ) in males remained the single donor-related variable associated with higher hemolysis (P < .0001); odds ratio, 3 (95% confidence interval [CI], 1.3-6.7), increasing to 4 (95% CI, 1.8-8.6) for obese males (BMI > 30 kg/m ). Female donor obesity and older age trended toward higher hemolysis.

Conclusion: Donor BMI, sex, and female age influence the level of hemolysis of 42-days-stored RBCs. Other factors, not identified in this study, also influence the level of hemolysis.
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http://dx.doi.org/10.1111/trf.16203DOI Listing
February 2021

Re-introducing whole blood for transfusion: considerations for blood providers.

Vox Sang 2021 Feb 30;116(2):167-174. Epub 2020 Sep 30.

Epidemiology and Preventive Medicine, Monash University, Melbourne, Vic, Australia.

Whole blood is the original blood preparation but disappeared from the blood bank inventories in the 1980s following the advent of component therapy. In the early 2000s, both military and civilian practice called for changes in the transfusion support for massive haemorrhage. The 'clear fluid' policy was abandoned and replaced by early balanced transfusion of platelets, plasma and red cells. Whole blood is an attractive alternative to multi-component therapy, which offers reduced hemodilution, lower donor exposure and simplified logistics. However, the potential for wider re-introduction of whole blood requires re-evaluation of haemolysins, storage conditions and shelf-life, the need for leucocyte depletion/ pathogen reduction and inventory management for blood providers. This review addresses these questions and calls for research to define the optimal whole blood product and the indications for its use.
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http://dx.doi.org/10.1111/vox.12998DOI Listing
February 2021

Critical peptic ulcer bleeding requiring massive blood transfusion: outcomes of 270 cases.

Intern Med J 2020 Aug 12. Epub 2020 Aug 12.

Transfusion Research Unit, Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Victoria, Australia.

Background: Critical peptic ulcer bleeding requiring massive transfusion is a gastroenterological emergency. Few data exist on management and outcomes. The Australian and New Zealand Massive Transfusion Registry collects comprehensive data on adult patients receiving massive transfusion across all bleeding contexts.

Aim: To evaluate clinical factors, management (procedural interventions, transfusions) and outcomes after massive transfusion for critical peptic ulcer bleeding.

Method: Demographics, diagnosis, procedures, and mortality data were available for 5,482 massive transfusion cases from 23 hospitals. International Classification of Diseases-Australian modification, 10 Edition codes were used to determine peptic ulcer bleeding and the Australian Classification of Health Intervention for interventions (endoscopic, radiological, surgical).

Results: Peptic ulcer bleeding accounted for 270 (4.9%) of all in-hospital massive transfusion cases. 70% were male. Median number of red blood cell (RBC) units transfused was 7 [interquartile-range, 6 to 10]. 30-day mortality was 19.6%. Age (75 vs 67 years; p=0.009) and Charlson Comorbidity Index (3 vs 1; p<0.001) were higher in those who died. Highest 24-hour INR (1.5 vs 1.4; p<0.001) and creatinine (118 μmol/L vs. 96 μmol/L; p=0.03), and nadir platelet count (86 x10 /L vs 118 x10 /L; p=0.01) were also associated with 30-day mortality. There were no differences in mortality according to number of RBC, platelets or plasma units transfused, gastroscopy (with or without intervention), interventional radiology or surgery.

Conclusion: One in five patients with critical peptic ulcer bleeding requiring massive transfusion died by 30 days. Mortality was associated with patient characteristics rather than clinical interventions (procedures, blood product transfusion). This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1111/imj.15009DOI Listing
August 2020

Haematological features, transfusion management and outcomes of massive obstetric haemorrhage: findings from the Australian and New Zealand Massive Transfusion Registry.

Br J Haematol 2020 08 16;190(4):618-628. Epub 2020 Feb 16.

Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Vic, Australia.

Massive obstetric haemorrhage (MOH) is a leading cause of maternal morbidity and mortality world-wide. Using the Australian and New Zealand Massive Transfusion Registry, we performed a bi-national cohort study of MOH defined as bleeding at ≥20 weeks' gestation or postpartum requiring ≥5 red blood cells (RBC) units within 4 h. Between 2008 and 2015, we identified 249 cases of MOH cases from 19 sites. Predominant causes of MOH were uterine atony (22%), placenta praevia (20%) and obstetric trauma (19%). Intensive care unit admission and/or hysterectomy occurred in 44% and 29% of cases, respectively. There were three deaths. Hypofibrinogenaemia (<2 g/l) occurred in 52% of cases in the first 24 h after massive transfusion commenced; of these cases, 74% received cryoprecipitate. Median values of other haemostatic tests were within accepted limits. Plasma, platelets or cryoprecipitate were transfused in 88%, 66% and 57% of cases, respectively. By multivariate regression, transfusion of ≥6 RBC units before the first cryoprecipitate (odds ratio [OR] 3·5, 95% CI: 1·7-7·2), placenta praevia (OR 7·2, 95% CI: 2·0-26·4) and emergency caesarean section (OR 4·9, 95% CI: 2·0-11·7) were independently associated with increased risk of hysterectomy. These findings confirm MOH as a major cause of maternal morbidity and mortality and indicate areas for practice improvement.
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http://dx.doi.org/10.1111/bjh.16524DOI Listing
August 2020

Clinical coding data algorithm to categorize type of gastrointestinal bleeding as a primary reason for massive transfusion: results from the Australian and New Zealand Massive Transfusion Registry.

Vox Sang 2019 Nov 6;114(8):853-860. Epub 2019 Sep 6.

Transfusion Research Unit, Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Vic, Australia.

Background: Management of major gastrointestinal bleeding (GIB) may require massive transfusion (MT), but limited data are available. Upper and lower GIB have different aetiologies, prognosis, bleeding patterns and outcomes. Better understanding of current transfusion management and outcomes in these patients is important. We sought to define and validate an algorithm based on clinical coding data to distinguish critical upper and lower GIB using data from the Australian and New Zealand Massive Transfusion Registry (ANZ-MTR).

Study Design And Methods: Australian and New Zealand Massive Transfusion Registry hospital-source data on adult patients receiving a MT (defined as ≥5 red cell units within 4 h) for any bleeding context were used. An algorithm allocating ICD-10-AM codes into 'probable' or 'possible' causes of GIB was developed and applied to the ANZ-MTR. Source medical records of 69 randomly selected cases were independently reviewed to validate the algorithm.

Results: Of 5482 MT cases available from 25 hospitals, 716 (13%) were identified as GIB with 538/716 (75%) categorized 'probable' and 178/716 'possible' GIB. Upper and lower GIB causes of MT were identified for 455/538 (85%) and 76/538 (14%) 'probable' cases, respectively; 7/538 (1·3%) cases had both upper and lower GIB. Allocation by the algorithm into a 'probable' GIB category had a 95·7% (CI: 90-100%) positive predictive value when validated against source medical records.

Conclusion: An algorithm based on ICD-10-AM codes can be used to accurately categorize patients with luminal GIB as the primary reason for MT, enabling further study of this critically unwell and resource-intensive cohort of patients.
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http://dx.doi.org/10.1111/vox.12840DOI Listing
November 2019

Intravenous immunoglobulin for adjunctive treatment of severe infections in ICUs.

Curr Opin Crit Care 2019 10;25(5):417-422

Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia.

Purpose Of Review: This review focuses on the emerging literature regarding the use of intravenous immunoglobulins (IVIg) in critically ill patients with severe infections. The aim is to provide an accessible summary of the most recent evidence of IVIg use in sepsis and septic shock and to help clinicians to understand why there is still equipoise regarding the potential benefit of this adjunctive therapy in this setting.

Recent Findings: Observational studies with propensity score matching analyses and investigating the effect of IVIg in severe infections including necrotizing soft tissue infection have been recently published. These studies suffer important flaws precluding robust conclusion to be drawn. Some recent randomized controlled trials raised interesting findings supportive of personalized medicine but are likely to be underpowered or confounded.

Summary: Insufficient evidence is available to support IVIg use in sepsis and septic shock, apart from the specific case of streptococcal toxic shock syndrome. Current literature suggests that IVIg efficacy in sepsis or septic shock could depend on the IVIg preparation (IgM-enriched or minimal IgM), time of administration (<24 h), dose, and the inflammatory/immunomodulation profile of the patients. Investigator-initiated research, incorporating these parameters, is warranted to determine whether IVIg benefits critically ill patients with severe infection.
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http://dx.doi.org/10.1097/MCC.0000000000000639DOI Listing
October 2019

Preparation of Platelet Concentrates for Research and Transfusion Purposes.

Methods Mol Biol 2017 ;1619:31-42

Transfusion Science, Melbourne, VIC, Australia.

Platelets are specialized cellular elements of the blood that play central roles in physiologic and pathologic processes of hemostasis, wound healing, host defense, thrombosis, inflammation, and tumor metastasis. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion, signaling molecules, and release of bioactive factors. Transfusion of platelet concentrates is an important treatment component for thrombocytopenia and bleeding. Recent progress in high-throughput mRNA and protein profiling techniques has advanced the understanding of platelet biological functions toward identifying novel platelet-expressed and secreted proteins, analyzing functional changes between normal and pathologic states, and determining the effects of processing and storage on platelet concentrates for transfusion. It is important to understand the different standard methods of platelet preparation and how they differ from the perspective for use as research samples in clinical chemistry. Two simple methods are described here for the preparation of research-scale platelet samples from whole blood, and detailed notes are provided about the methods used for the preparation of platelet concentrates for transfusion.
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http://dx.doi.org/10.1007/978-1-4939-7057-5_3DOI Listing
March 2018

A Protocol for the Preparation of Cryoprecipitate and Cryo-depleted Plasma for Proteomic Studies.

Methods Mol Biol 2017 ;1619:23-30

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia.

Cryoprecipitate is a concentrate of high-molecular-weight plasma proteins that precipitate when frozen plasma is slowly thawed at 1-6 °C. The concentrate contains factor VIII (antihemophilic factor), von Willebrand factor (vWF), fibrinogen, factor XIII, fibronectin, and small amounts of other plasma proteins. Clinical grade preparations of cryoprecipitate are mainly used to treat fibrinogen deficiency caused by acute bleeding or functional abnormalities of the fibrinogen protein. In the past, cryoprecipitate was used to treat von Willebrand disease and hemophilia A (factor VIII deficiency), but the availability of more highly purified coagulation factor concentrates or recombinant protein preparations has superseded the use of cryoprecipitate for these coagulopathies. Cryo-depleted plasma ("cryosupernatant") is the plasma supernatant remaining following removal of the cryoprecipitate from frozen-thawed plasma. It contains all the other plasma proteins and clotting factors present in plasma that remain soluble during cold-temperature thawing of the plasma. This protocol describes the clinical-scale preparation of cryoprecipitate and cryo-depleted plasma for proteomic studies.
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http://dx.doi.org/10.1007/978-1-4939-7057-5_2DOI Listing
March 2018

Red blood cell components: time to revisit the sources of variability.

Blood Transfus 2017 Mar;15(2):116-125

Department of Immunology and Pathology, Monash University, Melbourne, VIC, Australia.

Quality and safety of red blood cell (RBC) components is managed by screening of donors and strict regulatory controls of blood collection, processing and storage procedures. Despite these efforts, variations in RBC component quality exist as exemplified by the wide range in storage-induced haemolysis. This article provides a brief overview of the variables that contribute or potentially contribute to the quality of stored RBC components, including blood collection, processing, and donor-related variables. Particular focus is made on donor health and lifestyle factors that are not specifically screened and may impact on the physicobiochemical properties of RBCs and their storability. Inflammatory and oxidative stress states may be especially relevant as RBCs are susceptible to oxidative injury. Few studies have investigated the effect of specific donor-related variables on the quality of stored RBC components. Donor-related variables may be unaccounted confounders in the "age of blood" clinical studies that compared outcomes following transfusion of fresher or longer-stored RBC components. The conclusion is drawn that the blood donor is the greatest source of RBC component variability and the least "regulated" aspect of blood component production. It is proposed that more research is needed to better understand the connection between donor-related variables and quality consistency of stored RBC components. This could be very important given the impact of modern lifestyles that sees escalating rates of non-communicable health conditions that are associated with increased oxidative stress, such as hypertension, obesity and diabetes in children and adults, as well as an ageing population in many countries. The effect of these changes to global health and population demographics will impact on blood donor panels, and without significant new research, the consequences on the quality of stored blood components and transfusion outcomes are unknown.
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http://dx.doi.org/10.2450/2017.0326-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336332PMC
March 2017

Stored red blood cell susceptibility to in vitro transfusion-associated stress conditions is higher after longer storage and increased by storage in saline-adenine-glucose-mannitol compared to AS-1.

Transfusion 2015 Sep 13;55(9):2197-206. Epub 2015 May 13.

formerly Research and Development, Australian Red Cross Blood Service, West Melbourne, Victoria, Australia.

Background: Biochemical changes induced in red blood cells (RBCs) during storage may impair their function upon transfusion. Transfusion-associated stresses may further amplify storage lesion effects including increased phosphatidylserine (PS) exposure at the RBC membrane, microparticle (MP) release, and adhesion to endothelial cells (ECs). RBC stress susceptibility in vitro was investigated in relation to storage time and additive solution.

Study Design And Methods: Leukoreduced whole blood donations (n = 18) were paired, mixed, and resplit before separating the RBCs for storage in saline-adenine-glucose-mannitol (SAGM) or AS-1. Samples were taken after 3, 21, or 35 days. For oxidative stress treatment, RBCs were exposed to 0.5 mmol/L tert-butylhydroperoxide. Transfusion-associated stress was simulated by overnight culture at 37 °C with plasma containing inflammatory mediators. PS exposure and MPs were measured by flow cytometry and adhesion to ECs was tested under flow conditions. PS specificity of adhesion was tested by blocking with PS-containing lipid vesicles.

Results: Oxidative stress induced significantly higher PS exposure and adhesion to ECs in RBCs stored for 35 days compared to 3 days (p < 0.04). PS-containing vesicles blocked RBC-EC adhesion. After overnight culture with or without plasma, PS exposure and EC adhesion were significantly increased (p < 0.05). MP numbers increased with longer RBC storage and after RBC culture with plasma. Culture conditions influenced MP numbers from Day 35 RBCs. RBCs stored in SAGM had significantly higher PS exposure after stress treatment than AS-1 RBCs (p < 0.02).

Conclusion: Storage for 35 days significantly increased RBC susceptibility to oxidative and in vitro transfusion-associated stresses and was higher for RBCs stored in SAGM compared to AS-1.
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http://dx.doi.org/10.1111/trf.13138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573361PMC
September 2015

Red blood cell storage duration and trauma.

Transfus Med Rev 2015 Apr 18;29(2):120-6. Epub 2014 Dec 18.

Department of Immunology, Monash University, Melbourne, Victoria, Australia. Electronic address:

Numerous retrospective clinical studies suggest that transfusion of longer stored red blood cells (RBCs) is associated with an independent risk of poorer outcomes for certain groups of patients, including trauma, intensive care, and cardiac surgery patients. Large multicenter randomized controlled trials are currently underway to address the concern about RBC storage duration. However, none of these randomized controlled trials focus specifically on trauma patients with hemorrhage. Major trauma, particularly due to road accidents, is the leading cause of critical injury in the younger-than-40-year-old age group. Severe bleeding associated with major trauma induces hemodynamic dysregulation that increases the risk of hypoxia, coagulopathy, and potentially multiorgan failure, which can be fatal. In major trauma, a multitude of stress-associated changes occur to the patient's RBCs, including morphological changes that increase cell rigidity and thereby alter blood flow hemodynamics, particularly in the microvascular vessels, and reduce RBC survival. Initial inflammatory responses induce deleterious cellular interactions, including endothelial activation, RBC adhesion, and erythrophagocytosis that are quickly followed by profound immunosuppressive responses. Stored RBCs exhibit similar biophysical characteristics to those of trauma-stressed RBCs. Whether transfusion of RBCs that exhibit storage lesion changes exacerbates the hemodynamic perturbations already active in the trauma patient is not known. This article reviews findings from several recent nonrandomized studies examining RBC storage duration and clinical outcomes in trauma patients. The rationale for further research on RBC storage duration in the trauma setting is provided.
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http://dx.doi.org/10.1016/j.tmrv.2014.09.007DOI Listing
April 2015

AS-7 improved in vitro quality of red blood cells prepared from whole blood held overnight at room temperature.

Transfusion 2015 Jan 15;55(1):108-14. Epub 2014 Jul 15.

Research & Development, Australian Red Cross Blood Service, Melbourne, Victoria, Australia.

Background: Extended room temperature (RT) hold of whole blood (WB) may affect the quality of red blood cell (RBC) components produced from these donations. The availability of better RBC additive solutions (ASs) may help reduce the effects. A new AS, AS-7 (SOLX, Haemonetics Corporation), was investigated for improved in vitro quality of RBCs prepared from WB held overnight at RT.

Study Design And Methods: Sixteen WB units were held for 21.4 hours ± 40 minutes at 22°C on cooling plates before processing. Each pair of ABO-matched WB units were pooled, divided into a WB filter pack containing saline-adenine-glucose-mannitol (control) and a LEUKOSEP WB-filter pack containing SOLX, and processed according to manufacturer's instructions. RBCs were stored at 2 to 6°C and sampled weekly until expiry. Glycophorin A (GPA+) and annexin V-binding microparticles (MPs) were quantitated using flow cytometry. Osmotic fragility, intracellular pH (pHi), adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), and routine quality variables were measured. Adhesion of RBCs to human endothelial cells (ECs) was evaluated by flow perfusion under low shear stress (0.5 dyne/cm(2) ), similar to low blood flow in microvessels.

Results: ATP and 2,3-DPG levels were improved for SOLX-RBCs. SOLX-RBCs maintained higher pHi, increased resistance to hypotonic stress, and reduced numbers of GPA+ MPs. No significant difference was observed between annexin V binding to MPs or adhesion of RBCs to ECs under shear stress.

Conclusion: SOLX-stored RBCs showed increased osmotic resistance, pHi, and reduced GPA+ MPs and together with higher ATP and 2,3-DPG levels demonstrated improved in vitro RBC quality measures during 42 days of storage.
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http://dx.doi.org/10.1111/trf.12779DOI Listing
January 2015

Microparticle profile and procoagulant activity of fresh-frozen plasma is affected by whole blood leukoreduction rather than 24-hour room temperature hold.

Transfusion 2014 Aug 18;54(8):1935-44. Epub 2014 Mar 18.

Research and Development, Australian Red Cross Blood Service, Melbourne, Victoria, Australia.

Background: Microparticles (MPs) are small phospholipid-containing vesicles that have procoagulant properties. MPs are thought to contribute to the hemostatic potential of plasma. This study investigated the effects of whole blood (WB) hold time and leukoreduction (LR) on the MP profile and hemostatic potential of fresh-frozen plasma (FFP).

Study Design And Methods: WB units (n=12) from healthy donors were divided into two pairs and each pair was held at 20 to 24°C for 6 or 24 hours. At the designated hold time, 1 unit from the pair was LR while the other unit was not LR. FFP was prepared by standard procedures, aliquoted, and frozen. The MP content was determined by flow cytometry using an absolute count assay and specific labels for red blood cells (CD235a), platelets (CD41), and phosphatidylserine (PS). The hemostatic potential was determined by thrombelastography (TEG) and coagulation factor assays.

Results: Compared to non-LR-FFP, LR-FFP had significantly lower numbers of MPs, particularly CD41+ MPs and PS-positive MPs (p<0.03). LR-FFP, compared to non-LR-FFP, had a slower clot formation time (p=0.002); lower clot strength (p<0.001); and lower Factor (F)VIII, FXII, and fibrinogen levels (p<0.01). With longer WB hold time, the TEG profile was unchanged, although FVIII levels were decreased as expected (p<0.01). On average FFP units met quality requirements.

Conclusion: LR of WB resulted in lower hemostatic potential of FFP in conjunction with depletion of MPs and coagulation factors. Longer WB hold time did not significantly affect the hemostatic potential of FFP as measured by TEG. Acceptable hemostatic quality was maintained for all FFP processing conditions studied.
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http://dx.doi.org/10.1111/trf.12602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164532PMC
August 2014

In vitro measures of membrane changes reveal differences between red blood cells stored in saline-adenine-glucose-mannitol and AS-1 additive solutions: a paired study.

Transfusion 2014 Mar 22;54(3):560-8. Epub 2013 Jul 22.

Research and Development, Australian Red Cross Blood Service, Melbourne, Victoria, Australia; Blood Systems Research Institute, San Francisco, California.

Background: Saline-adenine-glucose-mannitol (SAGM) and a variant solution, AS-1, have been used for more than 30 years to preserve red blood cells (RBCs). Reputedly these RBC components have similar quality, although no paired study has been reported. To determine whether differences exist, a paired study of SAGM RBCs and AS-1 RBCs was conducted to identify membrane changes, including microparticle (MP) quantitation and in vitro RBC-endothelial cell (EC) interaction.

Study Design And Methods: Two whole blood packs were pooled and split and RBCs were prepared (n=6 pairs). One pack was suspended in SAGM and one in AS-1. Samples were collected during 42 days of refrigerated storage. RBC shape and size and glycophorin A (GPA)(+) and phosphatidylserine (PS)(+) MPs were measured by flow cytometry. RBC adhesion to ECs was determined by an in vitro flow perfusion assay. Routine variables (pH, hemolysis) were also measured.

Results: Compared to SAGM RBCs, AS-1 RBCs had lower hemolysis (p<0.04), lower GPA(+) MPs (p<0.03), and lower PS(+) MPs (p<0.03) from Day 14 onward. AS-1 RBCs had higher (p<0.02) side scatter from Day 28 onward compared to SAGM RBCs. SAGM RBCs were more adherent to ECs on Day 28 of storage compared to AS-1 RBCs (p=0.04), but reversed on Day 42 (p=0.02).

Conclusion: SAGM RBCs lose more membrane during storage. SAGM RBCs had increased adherence to ECs on Day 28 of storage, while AS-1 RBCs were more adherent on Day 42. The effect of these differences on the function and survival of SAGM RBCs and AS-1 RBCs after transfusion remains to be determined.
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http://dx.doi.org/10.1111/trf.12344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4173075PMC
March 2014

Time to revisit red blood cell additive solutions and storage conditions: a role for "omics" analyses.

Blood Transfus 2012 May;10 Suppl 2:s7-11

Research and Development, Australian Red Cross Blood Service, Melbourne, VIC 3205, Australia. rsparrow@ redcrossblood.org.au

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http://dx.doi.org/10.2450/2012.003SDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418618PMC
May 2012

Induction of multi-functional T cells in a phase I clinical trial of dendritic cell immunotherapy in hepatitis C virus infected individuals.

PLoS One 2012 14;7(8):e39368. Epub 2012 Aug 14.

Burnet Institute, Melbourne, Victoria, Australia.

We have previously reported a world-first phase I clinical trial to treat HCV patients using monocyte-derived dendritic cells (Mo-DC) loaded with HCV-specific lipopeptides. While the brief treatment proved to be safe, it failed to reduce the viral load and induced only transient cell-mediated immune responses, measured by IFNγ ELIspot. Here we reanalysed the PBMC samples from this trial to further elucidate the immunological events associated with the Mo-DC therapy. We found that HCV-specific single- and multi-cytokine secreting T cells were induced by the Mo-DC immunotherapy in some patients, although at irregular intervals and not consistently directed to the same HCV antigen. Despite the vaccination, the responses were generally poor in quality and comprised of primarily single-cytokine secreting cells. The frequency of FOXP3(+) regulatory T cells (Treg) fluctuated following DC infusion and eventually dropped to below baseline by week 12, an interesting trend suggesting that the vaccination may have resulted in a more subtle outcome than was initially apparent. Our data suggested that Mo-DC therapy induced complex immune responses in vivo that may or may not lead to clinical benefit.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039368PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419178PMC
February 2013

Microparticle content of plasma for transfusion is influenced by the whole blood hold conditions: pre-analytical considerations for proteomic investigations.

J Proteomics 2012 Dec 16;76 Spec No.:211-9. Epub 2012 Jul 16.

Research and Development, Australian Red Cross Blood Service, Melbourne, Victoria, Australia.

Microparticles (MPs) are shed from normal blood cells and may contribute to the coagulation potential of plasma. Transfusion of fresh frozen plasma (FFP) is used to correct coagulopathies and blood loss in trauma or major surgery. The role of MPs in FFP clinical efficacy is unknown. Regulations that govern the preparation of FFP vary in different countries. The aim of this study was to determine the effect of whole blood (WB)-hold conditions before FFP preparation on the MP profile. WB units were held at room temperature (RT) or combination of RT and refrigeration for up to 24h before FFP preparation. The MP content in thawed FFP was measured to reflect transfusion practice. The absolute number of MPs in FFP increased with longer WB hold time. Refrigeration of WB may also promote increased generation of MPs. In particular the number of platelet-derived and phosphatidylserine-containing MPs, which are known to have procoagulant properties, increased. Lipid peroxidation increased with longer WB-hold time. Donor-related factors appear to govern lipid peroxidation levels. Holistic proteomic and coagulant analyses of FFP MPs are warranted. Such information could guide the choice of the optimal handling conditions of WB and the most relevant quality control procedures for FFP. This article is part of a Special Issue entitled: Integrated omics.
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http://dx.doi.org/10.1016/j.jprot.2012.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177036PMC
December 2012

Properties of stored red blood cells: understanding immune and vascular reactivity.

Transfusion 2011 Apr;51(4):894-900

Blood Systems Research Institute and University of California, San Francisco, California, USA.

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http://dx.doi.org/10.1111/j.1537-2995.2011.03103.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3081134PMC
April 2011

Preparation of platelet concentrates.

Methods Mol Biol 2011 ;728:267-78

Research Unit, Australian Red Cross Blood Service, South Melbourne, VIC, Australia.

Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, wound healing, host defense, thrombosis, inflammation, and tumor metastasis. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion, signaling molecules, and release of bioactive factors. Transfusion of platelet concentrates is an important treatment component for thrombocytopenia and bleeding. Recent progress in high-throughput mRNA and protein profiling techniques has advanced the understanding of platelet biological functions toward identifying novel platelet-expressed and secreted proteins, analyzing functional changes between normal and pathologic states, and determining the effects of processing and storage on platelet concentrates for transfusion. It is important to understand the different standard methods of platelet preparation and how they differ from the perspective for use as research samples in clinical chemistry. Two simple methods are described here for the preparation of research-scale platelet samples from whole blood, and detailed notes are provided about the methods used for the preparation of platelet concentrates for transfusion.
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http://dx.doi.org/10.1007/978-1-61779-068-3_18DOI Listing
July 2011

A protocol for the preparation of cryoprecipitate and cryodepleted plasma.

Methods Mol Biol 2011 ;728:259-65

Research Unit, Australian Red Cross Blood Service, South Melbourne, VIC, Australia.

Cryoprecipitate is a concentrate of high-molecular-weight plasma proteins that precipitate when frozen plasma is slowly thawed at 1-6°C. The concentrate contains factor VIII (antihemophilic factor), von Willebrand factor (vWF), fibrinogen, factor XIII, fibronectin, and small amounts of other plasma proteins. Clinical-grade preparations of cryoprecipitate are mainly used to treat fibrinogen deficiency caused by acute bleeding or functional abnormalities of the fibrinogen protein. In the past, cryoprecipitate was used to treat von Willebrand disease and hemophilia A (factor VIII deficiency), but the availability of more highly purified coagulation factor concentrates or recombinant protein preparations has superseded the use of cryoprecipitate for these coagulopathies. Cryodepleted plasma ("cryosupernatant") is the plasma supernatant that remains following removal of the cryoprecipitate from frozen-thawed plasma. It contains all the other plasma proteins and clotting factors present in plasma that remain soluble during cold-temperature thawing of the plasma.
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http://dx.doi.org/10.1007/978-1-61779-068-3_17DOI Listing
July 2011

Effect of additive solutions on red blood cell (RBC) membrane properties of stored RBCs prepared from whole blood held for 24 hours at room temperature.

Transfusion 2011 Jan;51 Suppl 1:25S-33S

Research and Business Development Division, Australian Red Cross Blood Service, Melbourne, Victoria, Australia.

Background: The quality of RBC components is influenced by collection, processing and storage conditions. Regulations require that whole blood (WB) units be refrigerated within 8 hours and processed into RBCs within 24 hours of collection. Overnight room temperature hold of WB has logistical advantages, but the effect on RBC quality has not been fully investigated. RBC additive solutions were compared for their ability to provide improved quality of RBCs prepared from WB held at room temperature for 24 hours.

Study Design And Methods: Leukocyte-reduced RBCs were prepared from WB held at 20°C on cooling plates for 24 hours prior to processing. RBCs were stored in additive solutions, SAG-M (control), Erythrosol-4, and PAGGSM, under standard blood banking conditions and sampled during 49 days of storage. Stored RBCs were evaluated for RBC shape and microparticle (MP) accumulation using flow cytometry. Osmotic fragility, adhesion of RBCs to endothelium under shear stress conditions (0.5 dyne/cm(2) ), and routine RBC quality parameters were assessed.

Results: RBCs stored in Erythrosol-4 and PAGGSM had decreased cell size, reduced osmotic fragility, and decreased accumulation of glycophorin A-positive MPs and annexin V-binding MPs compared with RBCs stored in SAG-M. RBCs stored in erythrosol-4 had increased adherence to endothelium at days 42 and 49 compared with RBCs stored in SAG-M or PAGGSM.

Conclusion: RBCs stored in PAGGSM or Erythrosol-4 had improved retention of RBC membrane and osmotic resilience. The development of new additive solutions may offer improved quality of RBC components prepared from WB held overnight at room temperature.
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http://dx.doi.org/10.1111/j.1537-2995.2010.02960.xDOI Listing
January 2011

Evaluation of overnight hold of whole blood at room temperature before component processing: effect of red blood cell (RBC) additive solutions on in vitro RBC measures.

Transfusion 2011 Jan;51 Suppl 1:15S-24S

Department of Product and Process Development, Sanquin Blood Bank North West Region, Amsterdam, the Netherlands.

Background: Whole blood (WB) can be held at room temperature (18-25°C) up to 8 hours after collection; thereafter the unit must be refrigerated, rendering it unsuitable for platelet (PLT) production. Overnight hold at room temperature before processing has logistic advantages, and we evaluated this process in an international multicenter study for both buffy coat (BC)- and PLT-rich plasma (PRP)-based blood components and compared three red blood cell (RBC) additive solutions (ASs) for their ability to offset effects of overnight hold.

Study Design And Methods: Nine centers participated; seven used the BC method, and two used the PRP method. Four WB units were pooled and split; 1 unit was processed less than 8 hours from collection (Group A), and the other three (Groups B, C, and D) were held at room temperature and processed after 24 to 26 hours. RBCs in Groups A and B were resuspended in saline-adenine-glucose-mannitol, Group C in phosphate-adenine-guanosine-glucose-saline-mannitol, and Group D in ErythroSol-4 RBCs were stored at 2 to 6°C for 49 days.

Results: RBCs from overnight-held WB had lower 2,3-diphosphoglycerate (2,3-DPG) and higher adenosine triphosphate (ATP). At the end of storage there were no differences between groups, apart from a slightly higher hemolysis in Group B. ErythroSol-4 showed a slightly higher initial ATP and 2,3-DPG content, but at the end of storage no differences were found.

Conclusion: Overnight hold of WB before processing has no lasting deleterious effects on in vitro quality of subsequently prepared components. The use of different RBC ASs did not appear to offer significant advantages in terms of RBC quality at the end, regardless of the processing method.
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http://dx.doi.org/10.1111/j.1537-2995.2010.02959.xDOI Listing
January 2011

The effects of long-term storage of human red blood cells on the glutathione synthesis rate and steady-state concentration.

Transfusion 2011 Jul 20;51(7):1450-9. Epub 2011 Jan 20.

Faculty of Science, Macquarie University, Sydney, New South Wales, Australia.

Background: Banked red blood cells (RBCs) undergo changes that reduce their viability after transfusion. Dysfunction of the glutathione (GSH) antioxidant system may be implicated. We measured the rate of GSH synthesis in stored RBCs and applied a model of GSH metabolism to identify storage-dependent changes that may affect GSH production.

Study Design And Methods: RBC units (n = 6) in saline-adenine-glucose-mannitol (SAGM) solution were each divided into four transfusion bags and separate treatments were applied: 1) SAGM (control), 2) GSH precursor amino acids, 3) aminoguanidine, and 4) glyoxal. RBCs were sampled during 6 weeks of storage. Rejuvenated RBCs were also analyzed.

Results: After 6 weeks, the ATP concentration declined to 50 ± 5.5% (p < 0.05) of that in the fresh RBCs. For control RBCs, the GSH concentration decreased by 27 ± 6.5% (p < 0.05) and the rate of GSH synthesis by 45 ± 8% (p < 0.05). The rate of GSH synthesis in rejuvenated and amino acid-treated RBCs was unchanged after 6 weeks. Modeling identified that the decline in GSH synthesis was due to decreased intracellular substrate concentrations and reduced amino acid transport, secondary to decreased ATP concentration.

Conclusion: This study has uniquely shown that the glutathione synthesis rate decreased significantly after 6 weeks in stored RBCs. Our results have identified potential opportunities for improvement of banked blood storage.
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http://dx.doi.org/10.1111/j.1537-2995.2010.03026.xDOI Listing
July 2011

A phase I clinical trial of dendritic cell immunotherapy in HCV-infected individuals.

J Hepatol 2010 Oct 20;53(4):599-607. Epub 2010 Jun 20.

Virology Program, Burnet Institute, G.P.O. Box 2284, Melbourne, Vic. 3001, Australia.

Background & Aims: HCV patients who fail conventional interferon-based therapy have limited treatment options. Dendritic cells are central to the priming and development of antigen-specific CD4(+) and CD8(+) T cell immunity, necessary to elicit effective viral clearance. The aim of the study was to investigate the safety and efficacy of vaccination with autologous dendritic cells loaded with HCV-specific cytotoxic T cell epitopes.

Methods: We examined the potential of autologous monocyte-derived dendritic cells (MoDC), presenting HCV-specific HLA A2.1-restricted cytotoxic T cell epitopes, to influence the course of infection in six patients who failed conventional therapy. Dendritic cells were loaded and activated ex vivo with lipopeptides. In this phase 1 dose escalation study, all patients received a standard dose of cells by the intradermal route while sequential patients received an increased dose by the intravenous route.

Results: No patient showed a severe adverse reaction although all experienced transient minor side effects. HCV-specific CD8(+) T cell responses were enumerated in PBMC by ELIspot for interferon-gamma. Patients generated de novo responses, not only to peptides presented by the cellular vaccine but also to additional viral epitopes not represented in the lipopeptides, suggestive of epitope spreading. Despite this, no increases in ALT levels were observed. However, the responses were not sustained and failed to influence the viral load, the anti-HCV core antibody response and the level of circulating cytokines.

Conclusions: Immunotherapy using autologous MoDC pulsed with lipopeptides was safe, but was unable to generate sustained responses or alter the outcome of the infection. Alternative dosing regimens or vaccination routes may need to be considered to achieve therapeutic benefit.
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http://dx.doi.org/10.1016/j.jhep.2010.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930140PMC
October 2010

Red blood cell storage and transfusion-related immunomodulation.

Blood Transfus 2010 Jun;8 Suppl 3:s26-30

Research Unit, Australian Red Cross Blood Service, Melbourne, Victoria, Australia.

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http://dx.doi.org/10.2450/2010.005SDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897195PMC
June 2010

Red blood cell hemolysis during blood bank storage: using national quality management data to answer basic scientific questions.

Transfusion 2009 Dec;49(12):2599-603

Department of Pathology, University of Maryland, Baltimore, Maryland 21201, USA.

Background: Hemolysis of red blood cells (RBCs) during blood bank storage is the most obvious manifestation of RBC storage system failure. However, its analysis is made difficult because the largest source of interunit difference is donor specific. Availability of data from national blood systems on large numbers of RBC units used for internal quality control (QC) purposes and stored and processed in uniform ways permits statistical analysis.

Study Design And Methods: Measures of hemolysis during and at the end of storage on randomly selected donor units observed for QC purposes were obtained from four national blood systems. Groups of these measures from units that had undergone similar processing and storage were sorted to create histograms and the histograms were compared statistically.

Results: A total of 14,087 measures were obtained under seven storage conditions, including more than 12,000 measures made in a single country under four closely related conditions. Distributions of percent hemolysis are skewed normal and outliers are random. Additive solutions appear to be equivalent, except that the 42 mmol/L mannitol in AS-1 reduces hemolysis compared to conventional 30 mmol/L mannitol in saline, adenine, glucose, and mannitol. Increasing storage from 35 to 42 days increased measured hemolysis by 30% and leukoreduction decreased it by 53%.

Conclusions: Large national data sets provide useful information about the distribution of hemolysis at the end of RBC storage. This information can aid blood storage system development and regulatory science.
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http://dx.doi.org/10.1111/j.1537-2995.2009.02275.xDOI Listing
December 2009

Level of platelet-derived cytokines in leukoreduced red blood cells is influenced by the processing method and type of leukoreduction filter.

Transfusion 2010 Jan 18;50(1):185-9. Epub 2009 Aug 18.

Research Unit, Australian Red Cross Blood Service, Melbourne, Victoria, Australia.

Background: In contrast to the well-documented effect of white blood cells on the quality of red blood cells (RBCs), the effect of platelets (PLTs) has received little consideration. In this study, the PLT content and level of PLT-derived cytokines in RBCs prepared using different types of leukoreduction methods were investigated.

Study Design And Methods: Buffy coat-poor RBCs and five types of leukofiltered (LF) RBCs, including RBCs prepared with a whole blood (WB) PLT-saving filter, were prepared and stored according to standard blood bank conditions. PLT content was measured on Day 1, and levels of PLT-derived cytokines were measured by enzyme-linked immunosorbent assay at nominated timepoints during 42 days of storage.

Results: The PLT content of leukoreduced RBCs varied widely depending on the processing method and/or leukoreduction filter used, with some types of RBCs containing very low PLT counts while other units contained PLT counts comparable to those of unprocessed WB. The PLT content of RBCs directly influenced the concentration and accumulation of PLT-derived cytokines. Several PLT-derived factors exhibited significant accumulation throughout 42 days of storage. RBCs with high PLT content exhibited concentrations of RANTES (CCL5) and soluble CD40 ligand equivalent to those previously reported to show significant biologic and clinical effects.

Conclusion: The PLT content and levels of PLT-derived cytokines in leukoreduced RBCs are influenced by the processing method and types of leukoreduction filters used. It may be inappropriate to consider LF-RBCs prepared with different types of leukoreduction filters as equivalent products based on their differing levels of PLT factors.
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http://dx.doi.org/10.1111/j.1537-2995.2009.02353.xDOI Listing
January 2010

International blood collection and storage: clinical use of blood products.

J Proteomics 2010 Jan 4;73(3):386-95. Epub 2009 Aug 4.

Joint Proteomics Laboratory, Ludwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia.

Human blood transfusion is the process of transferring blood or blood-based products from an individual into the circulatory system of another. From the theory of circulation of blood to the early practice of blood transfusion, transfusion medicine has been an important concept for many centuries. The practicality of transfusion, however, only became a possibility during and shortly after the Second World War. Today, blood and its derivatives play a critical role in worldwide health care systems, with blood components having direct clinical indications. Over the past several years worldwide organizations including the World Health Organization (WHO) have made a number of substantial improvements to the regulation of the worlds blood supply. This continuous supply plays a critical role throughout health care systems worldwide, with procedures for blood collection, processing, and storage now complex, standardised processes. As the areas of clinical validation of different disease states from blood-derived sources (i.e., disease biomarkers) move towards validation stages, the importance of controlled- and standardised-protocols is imperative.
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http://dx.doi.org/10.1016/j.jprot.2009.07.011DOI Listing
January 2010

Enrichment of human platelet membranes for proteomic analysis.

Methods Mol Biol 2009 ;528:245-58

Ludwig Institute for Cancer Research, Melbourne, Australia.

Platelets (thrombocytes) are the smallest human blood cells and are pivotal in processes of hemostasis and thrombosis. Central to their function, the activation of platelets includes a complex interplay of adhesion and signalling molecules mediated via the plasma and inner membrane. Because platelets are enucleated, the analysis of the proteome is the best way to approach their biology. Here, we employ mass spectrometry (MS)-based proteomics to characterise membrane proteins derived from non-stimulated human platelets. This protocol details the extraction and purification of platelet membrane proteins from whole blood using SDS-PAGE in conjunction with LC-MS/MS. This method allowed the identification, and characterization of 207 platelet membrane proteins (PMP) from approximately 9.95 x 10(9) platelets (16).
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http://dx.doi.org/10.1007/978-1-60327-310-7_17DOI Listing
March 2009

Comparison of human platelet membrane-cytoskeletal proteins with the plasma proteome: Towards understanding the platelet-plasma nexus.

Proteomics Clin Appl 2008 Jan 17;2(1):63-77. Epub 2007 Dec 17.

Joint Proteomics Laboratory, Ludwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria, Australia.

Platelets are essential for maintaining vascular integrity. Given the anucleate nature of platelets, definition of their proteome is essential for understanding platelet pathophysiology. We describe here a detailed MS-based proteomic analysis of the platelet membrane/cytoskeletal sub-proteome from purified, normal, non-activated human platelets. In contrast to previous platelet proteomic purification strategies, the buffy-coat method was utilized in this study to isolate and purify minimally activated platelets, yielding significantly reduced contaminants for leukocytes (0.02 ± 0.007×10(6) /L) and erythrocytes (0.21 ± 0.02%). Using a false discovery rate of 1%, 203 proteins were identified and characterized with respect to their subcellular localization, biological function, and cellular processes. Of these, 16 have not been identified in previous human platelet proteome studies. As a first approach towards understanding the dynamic platelet-plasma protein composition nexus, we re-analysed the entire HUPO plasma proteome project dataset (647 plasma proteins identified) and compared these data with our platelet proteome dataset. Co-identified proteins (41) were further analysed with respect to their relative abundances (exponentially modified protein abundance index) and functional enrichment in these two proteomes, as well as their correlation with the platelet transcriptome. Both platelet membrane/cytoskeletal and plasma proteome reference datasets, comprising both processed and unprocessed MS/MS spectra, are publicly accessible (http://www.ludwig.edu.au/archive/).
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http://dx.doi.org/10.1002/prca.200780067DOI Listing
January 2008