Validation of serum non-invasive tests of liver fibrosis as prognostic markers of clinical outcomes in people with fatty liver disease in Australia.

BACKGROUND AND AIM: The validity of non-invasive tests (NITs) of liver fibrosis for the prediction of liver and mortality outcomes in an Australian cohort is unknown. We aimed to verify the utility of available NITs to predict overall and cause-specific mortality and major adverse liver outcome (MALO). METHODS: This was an analysis from the Crossroads 1 clinic sub-study of a randomly sampled adult cohort from regional Australia between 2001 and 2003. Baseline variables included demographic details, anthropometry, health and lifestyle data, and laboratory tests. Non-alcoholic fatty liver disease (NAFLD) and metabolic-(dysfunction) associated fatty liver disease (MAFLD) were defined by fatty liver index ≥ 60 and other accepted criteria. Outcomes were defined by the International Statistical Classification of Diseases and Related Health Problems 10th Revision codes for linked hospitalization and death registry data. Available serum-based NITs were analyzed as predictors of overall, cardiovascular disease-related, and cancer-related mortality and MALO in those with fatty liver disease (FLD). RESULTS: In total, 1324 and 1444 participants were included for NAFLD and MAFLD analysis (prevalence 35.4% and 40.7%, respectively). There were 298 deaths (89 cardiovascular disease-related and 98 cancer-related) and 24 MALO over a median 19.7 years of follow-up time. In both forms of FLD, fibrosis-4 index, Steatosis-Associated Fibrosis Estimator score, and Forns fibrosis score consistently had the highest area under the receiver operating characteristic curve (AUROC) for overall and cause-specific mortality, with AUROC > 0.70 for each outcome. However, all had poor discriminatory ability for determining MALO in each FLD. CONCLUSIONS: Several liver fibrosis NITs perform similarly reasonably well in predicting the risk of mortality outcomes in those with FLD but are poorly discriminatory for MALO prediction.

NAFLD and MAFLD independently increase the risk of major adverse cardiovascular events (MACE): a 20-year longitudinal follow-up study from regional Australia.

BACKGROUND AND AIMS: The association between fatty liver disease (FLD) and cardiovascular disease (CVD) in an Australian context has yet to be defined. The primary aim of this study was to investigate the association between FLD and 3-point major adverse cardiovascular events (MACE). METHODS: This was a longitudinal follow-up study of a randomly sampled adult cohort from regional Australia between 2001 and 2003. Baseline covariates included demographic details, anthropometry, health and lifestyle data, and laboratory tests. Non-alcoholic fatty liver disease (NAFLD) and metabolic-(dysfunction) associated fatty liver disease (MAFLD) were diagnosed in participants with fatty liver index (FLI) ≥ 60 and meeting other standard criteria. ICD-10 codes were used to define clinical outcomes linked to hospitalisations. Three-point MACE defined as non-fatal myocardial infarction (MI) and cerebrovascular accident (CVA) and CVD death. RESULTS: In total, 1324 and 1444 participants met inclusion criteria for NAFLD and MAFLD analysis, respectively. Over 23,577 and 25,469 person-years follow-up, NAFLD and MAFLD were independent predictors for 3-point MACE, adjusting for demographic covariates and known cardiometabolic risk factors, whilst considering non-CVD death as a competing event (NAFLD: sub-hazard ratio [sHR] 1.56, 95% confidence interval [CI 1.12-2.19]; MAFLD: sHR 1.51, 95% CI 1.11-2.06). The results held true on several sensitivity analyses. CONCLUSIONS: Both forms of FLD increase the risk for CVD independent of traditional cardiometabolic risk factors.

Chronic pain and the use of complementary and alternative medicine in rural Victoria, Australia.

OBJECTIVE: The relationship between chronic pain and complementary and alternative medicine (CAM) use is poorly understood, and the situation in rural Australia is particularly unclear. The objective here was to determine the socio-demographic factors associated with the use of CAM for the treatment of chronic pain in a region of rural Australia. METHODS: This secondary analysis used data from a population health survey, Crossroads-II, to assess the relationships of various socio-demographic factors with the use of CAM by those suffering from chronic pain. DESIGN: Face-to-face surveys at households randomly selected from residential address lists. SETTING: A large regional centre and three nearby rural towns in northern Victoria, Australia. PARTICIPANTS: Sixteen years of age and older. MAIN OUTCOME MEASURES: Use of a CAM service to treat chronic pain. RESULTS: Being female (2.40 [1.47, 3.93], p < 0.001) and having a bachelor's degree (OR 2.24 [1.20, 4.20], p < 0.001) had a significant positive relationship with the use of CAM overall to redress chronic pain and those 50 years and older had greater odds of using manipulation therapies relative to those below 50 years (50-64: OR 0.52 [0.32, 0.86], p = 0.010; 65+: 0.37 [0.18, 0.75], p = 0.005). CONCLUSION: In the studied region, females and those with university education have the greatest odds of using CAM to treat chronic pain. This study needs to be complemented with more mechanistic investigations into the reasons people make the decisions they make about using CAM for the management of chronic pain.

Positive change in intent to practice rurally is strongly associated with nursing and allied health students of metropolitan origin after rural clinical placement.

OBJECTIVE: To: (1) assess whether rural clinical placements influence change in intention to practice rurally for nursing and allied health students, (2) to assess whether residential origin (metropolitan or rural) or university location (metropolitan or non-metropolitan) influence this change, and (3) assess residential origin of health professionals practicing rurally after graduation. DESIGN: Cross-sectional survey (2014-2022) conducted in Northern/Northeastern Victoria and Grampians region by nursing and allied health students completing rural clinical placements. Survey explored student placement satisfaction, intention to practice rurally and demographic information, with a follow-up two years' post-placement. FINDINGS: Experience of a rural placement increased students' intention of practicing rurally. Importantly, 57% of metropolitan origin students studying at metropolitan campuses exhibited positive change. Logistic regression analysis suggested that after controlling for age, gender and satisfaction with placement and supervision, students of metropolitan origin at metropolitan campuses were 6.4 times more likely to report positive change in intent to practice rurally after placement than students of rural origin studying at non-metropolitan campuses.Follow-up data suggested that a substantial proportion of health professionals providing services in rural areas were of metropolitan origin. RESULTS: These findings concur that rural origin and rural training are important predictors of working rurally. Additionally, metropolitan students can change their intention to practice rurally after a rural placement. CONCLUSIONS: This study supports strategies to enhance intention to practice rurally that are not restricted to rural origin students to build rural workforce.

Non-alcoholic fatty liver disease prevalence in Australia has risen over 15 years in conjunction with increased prevalence of obesity and reduction in healthy lifestyle.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver condition globally. The aim of this study was to evaluate the change in age- and sex-standardized prevalence of NAFLD in regional Victoria over a 15-year period and explore the underlying factors associated with differences over time. METHODS: Repeated comparative cross-sectional studies in four towns in regional Victoria, Australia. Individuals randomly selected from households from residential address lists from local government organizations in 2001-2003 (CrossRoads I [CR1]) and 2016-2018 (CrossRoads II [CR2]) with 1040 (99%) and 704 (94%) participants from CR1 and CR2 having complete data for analysis. Primary outcome was change in prevalence estimates of NAFLD (defined by a fatty liver index ≥ 60 in the absence of excess alcohol and viral hepatitis) between 2003 and 2018. RESULTS: Crude prevalence of NAFLD increased from 32.7% to 38.8% (P < 0.01), while age-standardized/sex-standardized prevalence increased from 32.4% to 35.4% (P < 0.01). Concurrently, prevalence of obesity defined by BMI and elevated waist circumference increased 28% and 25%, respectively. Women had a greater increase in the prevalence of NAFLD than men, in parallel with increasing prevalence of obesity. Proportion of participants consuming takeaway food greater than once weekly increased significantly over time. Up to 60% of NAFLD patients require additional tests for assessment of significant fibrosis. CONCLUSIONS: Crude and age-standardized/sex-standardized prevalence of NAFLD have both increased significantly over the last 15 years, particularly among women, in association with a parallel rise in the prevalence of obesity.

Impact of renaming NAFLD to MAFLD in an Australian regional cohort: Results from a prospective population-based study.

BACKGROUND AND AIMS: Clinical and public health implications of the recent redefining of non-alcoholic fatty liver disease (NAFLD) to metabolic-associated fatty liver disease (MAFLD) remain unclear. We sought to determine the prevalence and compare MAFLD with NAFLD in a well-defined cohort. METHODS: A cross-sectional study was conducted in regional Victoria with participants from randomly selected households. Demographic and health-related clinical and laboratory data were obtained. Fatty liver was defined as a fatty liver index ≥ 60 with MAFLD defined according to recent international expert consensus. RESULTS: A total of 722 participants were included. Mean age was 59.3 ± 16 years, and 55.3% were women with a median body mass index of 27.8 kg/m2 . Most (75.2%) participants were overweight or obese. MAFLD was present in 341 participants giving an unadjusted prevalence of 47.2% compared with a NAFLD prevalence of 38.7%. Fifty-nine (17.5%) participants met the criteria of MAFLD but not NAFLD. The increased prevalence of MAFLD in this cohort was primarily driven by dual etiology of fatty liver. All participants classified as NAFLD met the new definition of MAFLD. Compared with NAFLD subjects, participants with MAFLD had higher ALT (26.0 [14.0] U/L vs 30.0 [23] U/L, P = 0.024), but there were no differences in non-invasive markers for steatosis or fibrosis. CONCLUSION: Metabolic-associated fatty liver disease is a highly prevalent condition within this large community cohort. Application of the MAFLD definition increased prevalence of fatty liver disease by including people with dual etiologies of liver disease.

The Pandemic Is Not Occurring in a Vacuum: The Impact of COVID-19 and Other Disasters on Workforce Mental Health in Australia.

OBJECTIVE: Prior to coronavirus disease (COVID-19), many Australians experienced extreme bushfires, droughts, and floods. A history of experiencing these events might be a risk factor for increased psychological distress during COVID-19. This study aimed to provide insight into the mental health of Australian workers during the initial COVID-19 outbreak, with an additional focus on whether previous disaster exposure and impact from that disaster is a risk factor for increased psychological distress. METHODS: A snowball recruitment strategy was used. Participants (n = 596) completed an online survey, which included the Depression Anxiety Stress Scales-21, and questions related to mental health and disaster exposure. RESULTS: Overall, 19.2%, 13.4%, and 16.8% of participants were experiencing moderate to extremely severe depression, anxiety, and stress symptoms, respectively. Multiple regression found that higher depression, anxiety, and stress symptoms were associated with a pre-existing mental health diagnosis; only higher stress symptoms were associated with having experienced a disaster, with impact, in addition to COVID-19. CONCLUSIONS: People who have experienced impact from an additional disaster might need additional support to protect their mental health during COVID-19. A focus on the cumulative mental health impacts of multiple disasters and the implications for organizational communities where recovery work is undertaken, such as schools and workplaces, is needed.

Detrimental Health Behaviour Changes among Females Living in Rural Areas during the COVID-19 Pandemic.

Women are predicted to be disproportionately impacted by the COVID-19 pandemic due to increased carer responsibilities, loss of income, worry about the virus and a predominantly female healthcare workforce. Whilst there is emerging evidence that negative mental health impacts associated with the COVID-19 pandemic may be more pronounced for women than men, less attention has focussed on changes to health behaviours and health seeking experienced by women. Similarly, the impact of the pandemic in rural areas has not been investigated in detail. Our research questions were 'have females residing in rural areas experienced changes in alcohol consumption, unhealthy food consumption, smoking, exercise or health seeking during the COVID-19 pandemic?' and 'are there differences in health behaviour changes between rural females living with or without children?'. Net increases (scale of 0-1) in consumption of unhealthy food (95% CI 0.05, 0.22) and alcohol (95% CI 0.12, 0.29) were observed. Net decreases (scale of -1 to 0) in visits to the doctor (95% CI -0.23, -0.35) and other health professionals (95% CI -0.40, -0.54) were observed. Compared with females living without children, females who lived with children were significantly associated with increased alcohol consumption (OR 2.4 (95% CI 1.4, 4.1), decreased visits to the doctor (OR 1.9 (95% CI 1.1, 3.2) and decreased visits to other health professionals (OR 1.9 (95% CI 1.1, 3.3). Results suggest that public health approaches may be required to support females residing in rural areas to optimise their health behaviours during the pandemic, particularly for those living with children. Policies must be gender responsive to counteract worsening health and social inequities both during and after the pandemic.

Characteristics of patients who access zero, one or multiple general practices and reasons for their choices: a study in regional Australia.

BACKGROUND: Most people in Australia visit a General Practitioner each year and are free to choose their General Practitioner and/or practice on each occasion. A proportion of people visit multiple general practices, which can reduce continuity of care, a core value of general practice. Utilisation of multiple general practices is associated with metropolitan residence and younger age. However, it is unclear which factors are associated with utilisation of multiple general practices in rural areas, where there are often General Practitioner workforce shortages and higher proportions of patients who may benefit from continuity of care, including older people and people living with chronic disease. The aim of this study was to compare the characteristics of people in a rural Australian area who accessed multiple general practices in the previous year with people who had accessed one practice, or none. METHODS: A cross-sectional survey assessed self-reported utilisation and perspective of general practice services, uses of multiple practices, associated reasons, lifestyle advice and screening services received in four regional Victorian towns. Households were randomly selected and residents aged 16+ were eligible to participate in the adult survey. RESULTS: Most people had attended a single general practice (78.9%), while 14.4% attended more than one practice and 6.7% attended no practices in the previous 12 months. Compared with utilisation of a single general practice, multiple general practice attendance in the previous year was associated with younger age (adjusted odds ratio (aOR 95% confidence interval) 0.98 per year (0.97-0.99), residence in the regional centre aOR 2.90(2.22-3.78), emergency department (ED) attendance in the last 12 months aOR 1.65(1.22-2.21) and no out of pocket costs aOR 1.36(1.04-1.79)). Reasons for multiple general practice attendance included availability of appointments, cost and access to specific services. Compared with multiple general practice attendance, those attending single practices reported more screening tests but similar frequency of lifestyle advice. People who accessed multiple practices were less likely to report very high satisfaction (51.7% vs 62.9% p < 0.001) or excellent degree of confidence in their doctor (42.0% vs 49.8% p = 0.006) than single practice attendees. CONCLUSIONS: Those attending single practices report higher satisfaction and confidence in their GP and were less likely to attend ED. Further studies are required to test whether increasing availability of appointments and reducing out-of-pocket expenses would increase single practice attendance and/or decrease healthcare costs overall.

Hearing loss and access to audiology services in rural victoria: Findings from the crossroads study.

CONTEXT: Rural residents can be exposed to high levels of agricultural machinery noise and are at risk of hearing loss. AIMS: This study aimed to determine audiology service use and rates of hearing loss in a regional area of Australia, using both self-report and audiology testing. SETTING AND DESIGN: A survey of randomly selected households was undertaken and 6432 participants were interviewed face to face about their health, hearing, and use of audiology services. A total of 1454 participants were randomly selected to undertake standard audiology testing. MATERIAL AND METHODS: Material Hearing was evaluated using conventional audiometry. STATISTICAL ANALYSIS USED: Independent t-tests, cChi-squared tests, and logistic regression were used to examine the association amongbetween hearing loss, use of audiology services, and demographic factors. RESULTS: Hearing issues were present in 12.5% of the survey participants. The rate of hearing loss increased significantly with age. Males were significantly more likely to have hearing loss than females (9.5% vs. 5.2%, pP << 0.01). The majority of people who reported accessing audiology services in the past 12 months were satisfied with the care they received (85.2%), and experienced short waiting times for these services (68.2% waited ≤≤ 7 days). CONCLUSIONS: Males had higher rates of hearing issues than females in this rural area. Audiology services in the region were accessible within short waiting times, and clients were satisfied with the service.

Longitudinal study of health, disease and access to care in rural Victoria: the Crossroads-II study: methods.

BACKGROUND: High quality, contemporary data regarding patterns of chronic disease is essential for planning by health services, policy makers and local governments, but surprisingly scarce, including in rural Australia. This dearth of data occurs despite the recognition that rural Australians live with high rates of ill health, poor health behaviours and restricted access to health services. Crossroads-II is set in the Goulburn Valley, a rural region of Victoria, Australia 100-300 km north of metropolitan Melbourne. It is primarily an irrigated agricultural area. The aim of the study is to identify changes in the prevalence of key chronic health conditions including the extent of undiagnosed and undermanaged disease, and association with access to care, over a 15 year period. METHODS/DESIGN: This study is a 15 year follow up from the 2000-2003 Crossroads-I study (2376 households participated). Crossroads-II includes a similar face to face household survey of 3600 randomly selected households across four towns of sizes 6300 to 49,800 (50% sampled in the larger town with the remainder sampled equally from the three smaller towns). Self-reported health, health behaviour and health service usage information is verified and supplemented in a nested sub-study of 900 randomly selected adult participants in 'clinics' involving a range of additional questionnaires and biophysical measurements. The study is expected to run from October 2016 to December 2018. DISCUSSION: Besides providing epidemiological and health service utilisation information relating to different diseases and their risk factors in towns of different sizes, the results will be used to develop a composite measure of health service access. The importance of access to health services will be investigated by assessing the correlation of this measure with rates of undiagnosed and undermanaged disease at the mesh block level. Results will be shared with partner organisations to inform service planning and interventions to improve health outcomes for local people.

The importance of adequate referrals for chronic kidney disease

DISCUSSION: Regional Australia remains a district of workforce shortage for nephrology. Thus, it is imperative that patients who have the greatest need for nephrologist services are effectively identified upon referral. The aim of this study was to assess referrals to a regional nephrology service against Australian guidelines by assessing the patient's renal function and the information contained in the referral document at the time of first consultation. We conducted a retrospective study of all referrals to a regional Australian nephrology service between 2013 and 2015. The 582 referrals that met the inclusion criteria were compared with Australian nephrology referral guidelines. Less than half of the referral documents (n = 253; 43.5%) described a clinical situation that met referral guidelines, typically due to insufficient pathology investigation. However, after consideration of renal functional test results performed at the initial consultation, an additional 82 cases met referral guidelines (n = 335; 57.6%). More than 40% of nephrology referrals to a regional Australian service did not meet Australian nephrology referral guidelines. This has implications for a regional nephrology service that is experiencing workforce pressures, in addition to the health system more broadly, and for patients. Many referrals contained insufficient information to allow differentiation of patients who would benefit most from nephrology care from patients who could be appropriately managed within primary care.

Barriers to effective conversations regarding overweight and obesity in regional Victoria.

BACKGROUND: General practitioners (GPs) have a crucial role to play in engaging patients in discussions about overweight and obesity. However, such discussions are currently uncommon. The aim of this study was to examine how GPs in rural areas talk about overweight and obesity with their patients, specifically to identify key barriers to effective conversations. METHODS: This study used a qualitative methodology. Semi-structured interviews were conducted with GPs (n = 7) and patients (n = 7) across two rural areas. RESULTS: Key barriers to effective conversations between GPs and patients about overweight and/or obesity include: uncertainty about appropriate language; lack of time; concerns about compromising mutual trust and rapport; concerns about patient readiness; concerns about patients' mental health and how this may be impacted by discussing a potentially upsetting and stigmatising topic; and lack of effective and individualised treatment and/or referral options. DISCUSSION: The findings suggest that responses to overweight and obesity need to be localised and tailored. Structural-level change is required to enable better responses to overweight and obesity, including multidisciplinary team approaches.

Age of blood and recipient factors determine the severity of transfusion-related acute lung injury (TRALI).

INTRODUCTION: Critical care patients frequently receive blood transfusions. Some reports show an association between aged or stored blood and increased morbidity and mortality, including the development of transfusion-related acute lung injury (TRALI). However, the existence of conflicting data endorses the need for research to either reject this association, or to confirm it and elucidate the underlying mechanisms. METHODS: Twenty-eight sheep were randomised into two groups, receiving saline or lipopolysaccharide (LPS). Sheep were further randomised to also receive transfusion of pooled and heat-inactivated supernatant from fresh (Day 1) or stored (Day 42) non-leucoreduced human packed red blood cells (PRBC) or an infusion of saline. TRALI was defined by hypoxaemia during or within two hours of transfusion and histological evidence of pulmonary oedema. Regression modelling compared physiology between groups, and to a previous study, using stored platelet concentrates (PLT). Samples of the transfused blood products also underwent cytokine array and biochemical analyses, and their neutrophil priming ability was measured in vitro. RESULTS: TRALI did not develop in sheep that first received saline-infusion. In contrast, 80% of sheep that first received LPS-infusion developed TRALI following transfusion with "stored PRBC." The decreased mean arterial pressure and cardiac output as well as increased central venous pressure and body temperature were more severe for TRALI induced by "stored PRBC" than by "stored PLT." Storage-related accumulation of several factors was demonstrated in both "stored PRBC" and "stored PLT", and was associated with increased in vitro neutrophil priming. Concentrations of several factors were higher in the "stored PRBC" than in the "stored PLT," however, there was no difference to neutrophil priming in vitro. CONCLUSIONS: In this in vivo ovine model, both recipient and blood product factors contributed to the development of TRALI. Sick (LPS infused) sheep rather than healthy (saline infused) sheep predominantly developed TRALI when transfused with supernatant from stored but not fresh PRBC. "Stored PRBC" induced a more severe injury than "stored PLT" and had a different storage lesion profile, suggesting that these outcomes may be associated with storage lesion factors unique to each blood product type. Therefore, the transfusion of fresh rather than stored PRBC may minimise the risk of TRALI.

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

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.

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

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.

Enrichment of human platelet membranes for proteomic analysis.

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).

Proteomic analysis of supernatant from pooled buffy-coat platelet concentrates throughout 7-day storage.

BACKGROUND: The platelet (PLT) storage lesion remains incompletely understood. To gain a greater insight into the PLT storage lesion, a proteomic analysis of supernatant from leukofiltered pooled buffy-coat PLT concentrates (PCs) was undertaken. STUDY DESIGN AND METHODS: PCs were prepared in PLT additive solution and stored according to standard blood bank procedures. Supernatant samples were collected throughout 7 days of storage. Maps of supernatant proteins were generated by two-dimensional (2D) gel electrophoresis and mass spectrometry. Cytokine antibody microarrays and enzyme-linked immunosorbent assay were used to investigate bioactive molecules. RESULTS: The 2D gel maps of PC supernatant proteins displayed many features of plasma protein maps. Several storage-induced protein changes were identified including modifications to major plasma proteins. PLT-derived proteins were also identified, including tremlike transcript 1 and integrin-linked kinase, which may influence PLT-endothelium interactions. Cytokine antibody microarrays revealed a number of bioactive proteins that have not been previously associated with PCs produced for transfusion, such as brain-derived neurotrophic factor (BDNF). The concentration of PLT-derived cytokines including BDNF, CXCL7, epidermal growth factor, PLT-derived growth factor (PDGF), and CCL5 significantly increased during storage of PCs. Extended storage from Day 5 to Day 7 caused significantly increased levels of BDNF, PDGF, and CCL5 in PC supernatant. CONCLUSION: Proteomic techniques provide valuable new insight into the effects of storage on PCs and the contribution of soluble proteins to the development of the PLT storage lesion and recipient responses to PLT transfusion.

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

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 ±â€…0.007×10(6) /L) and erythrocytes (0.21 ±â€…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/).

Proteomic analysis of supernatants of stored red blood cell products.

BACKGROUND: The development of the red blood cell (RBC) storage lesion remains incompletely understood. To gain a greater insight into the mechanisms involved, a proteomics analysis was used to identify proteins that accumulate in supernatants of standard nonleukoreduced RBC products (S-RBCs) and prestorage leukofiltered RBC products (LF-RBCs) during storage. STUDY DESIGN AND METHODS: S-RBCs and LF-RBCs were collected and stored in accordance with standard blood bank procedures. Supernatant samples were collected at fortnightly intervals until product expiry (at Day 42). Maps of supernatant proteins were generated by two-dimensional (2D)-gel electrophoresis and selected proteins were identified by mass spectrometry. RESULTS: 2D-gel mapping revealed that greater numbers of proteins accumulated in supernatants of S-RBCs compared to LF-RBCs. Abundant plasma proteins were strongly represented in both products. Several potentially bioactive proteins were found to predominantly accumulate in supernatant of S-RBCs. Among these, a promoter of neutrophil adhesion and an acute-phase scavenger protein were identified. In contrast, proteins found to accumulate predominantly in supernatant of LF-RBCs were RBC-regulatory proteins. CONCLUSION: Proteomics provides a valuable approach to examine storage-related effects on RBCs. Such analytical approaches may help to elucidate the mechanisms involved in the RBC storage lesion and provide insights into the biologic consequences of transfusion of stored RBC products.