Postoperative transfusion hemoglobin threshold and functional recovery after high-risk oncologic surgery: A randomized controlled pilot study.

BACKGROUND: Robust evidence to inform best transfusion management after major oncologic surgery, where postoperative recovery might impact treatment regimens for cancer, is lacking. We conducted a study to validate the feasibility of a larger trial comparing liberal versus restrictive red blood cells (RBC) transfusion strategies after major oncologic surgery. STUDY DESIGN AND METHODS: This was a two-center, randomized, controlled, study of patients admitted to the intensive care unit after major oncologic surgery. Patients whose hemoglobin level dropped below 9.5 g/dL, were randomly assigned to immediately receive a 1-unit RBC transfusion (liberal) or delayed until the hemoglobin level dropped below 7.5 g/dL (restrictive). The primary outcome was the median hemoglobin level between randomization to day 30 post-surgery. Disability-free survival was evaluated by the WHODAS 2.0 questionnaire. RESULTS: 30 patients were randomized (15 patients/group) in 15 months with a mean recruitment rate of 1.8 patients per month. The median hemoglobin level was significantly higher in the liberal group than in the restrictive group: 10.1 g/dL (IQR 9.6-10.5) versus 8.8 g/dL (IQR 8.3-9.4), p < .001, and RBC transfusion rates were 100% versus 66.7%, p = .04. The disability-free survival was similar between groups: 26.7% versus 20%, p = 1. DISCUSSION: Our results support the feasibility of a phase 3 randomized controlled trial comparing the impact of liberal versus restrictive transfusion strategies on the functional recovery of critically ill patients following major oncologic surgery.

Characterization of freeze-dried oxidized human red blood cells for pre-transfusion testing by synchrotron FTIR microspectroscopy live-cell analysis.

Oxidative treatment of human red blood cells (RBCs) prior to freeze-drying appears to stabilize the RBCs to withstand dried storage at room temperature. To better understand the effects of oxidation and freeze-drying/rehydration on RBC lipids and proteins, single-cell measurements were performed by synchrotron-based Fourier transform infrared (FTIR) microspectroscopy 'live-cell' (unfixed) analysis. Lipid and protein spectral data of tert-butyl hydroperoxide (TBHP)-oxidized RBCs (oxRBCs), FDoxRBCs and control (untreated) RBCs were compared using principal component analysis (PCA) and band integration ratios. The oxRBCs and FDoxRBCs samples had similar spectral profiles that were clearly different to control RBCs. Spectral changes in the CH stretching region of oxRBCs and FDoxRBCs indicated the presence of increased saturated and shorter-chain lipids, consistent with lipid peroxidation and stiffening of the RBC membrane compared to control RBCs. The PCA loadings plot for the fingerprint region of control RBCs corresponding to the α-helical structure of hemoglobin, shows that oxRBCs and FDoxRBCs have conformational changes in the protein secondary structure to ß-pleated sheets and ß-turns. Finally, the freeze-drying process did not appear to compound or induce additional changes. In this context, FDoxRBCs could become a stable source of reagent RBCs for pre-transfusion blood serology testing. The synchrotron FTIR microspectroscopic live-cell protocol provides a powerful analytical tool to characterize and contrast the effects of different treatments on RBC chemical composition at the single cell level.

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.

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/m2 ) 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/m2 ). 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.

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

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.

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

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 5482 massive transfusion cases from 23 hospitals. International Classification of Diseases 10th Edition, Australian Modification codes were used to determine peptic ulcer bleeding and the Australian Classification of Health Intervention for interventions (i.e. 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, 6-10). Thirty-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-h international normalised ratio (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 × 109 /L vs 118 × 109 /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 (e.g. procedures, blood product transfusion).

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

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.

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.

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.

Intravenous immunoglobulin for adjunctive treatment of severe infections in ICUs.

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.

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

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.

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.

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.

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

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.

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.

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.

PROACTIVE SCREENING ALGORITHM FOR EARLY-ONSET PNEUMONIA IN PATIENTS WITH OUT-OF-HOSPITAL CARDIAC ARREST: A BEFORE-AFTER IMPLEMENTATION STUDY.

ABSTRACT: Introduction : Early-onset pneumonia (EOP) occurs in around 50% of critically ill patients with out-of-hospital cardiac arrest (OHCA) and is associated with increased morbidity. Prompt diagnosis of EOP in these patients is difficult because of targeted temperature management and the postcardiac arrest syndrome. We hypothesized that an algorithm for proactive screening of EOP would improve patient outcomes. Methods : We conducted a single-center observational study comparing the outcomes of mechanically ventilated adult patients with OHCA, before (study period 1) and after (study period 2) implementation of an algorithm for proactive diagnosis of EOP, including an early distal pulmonary specimen. An inverse probability treatment weighted multivariable regression was performed to identify independent parameters associated with duration of mechanical ventilation. A subgroup analysis was conducted in patients alive on day 5 after intensive care unit admission. Results : Over the 4-year study period, 190 patients (99 and 91 for study periods 1 and 2, respectively) were enrolled. The overall incidence of EOP was 57.4% and was similar between both study periods. Although there was no difference in the time interval to antibiotic initiation, study period 2 was independently associated with higher SpO 2 /FiO 2 ratios on days 3 and 4. We also observed a decrease in mechanical ventilation time in study period 2 (4.5 [1-11.3] vs. 3 [2-5.8] days; P = 0.07), and this reached statistical significance in the subgroup analysis of patients alive at day 5 (10 [5-17] vs. 5 [3-9] days, P = 0.01). Conclusion: In critically ill patients with OHCA, proactive diagnosis of EOP was not associated with a significant change in the time to antibiotic initiation. Further research is warranted to better define optimal diagnosis and management of EOP in this setting.

Ultra-Massive Transfusion: Predictors of Occurrence and In-Hospital mortality From the Australian and New Zealand Massive Transfusion Registry (ANZ-MTR).

Few data exist on patient clinical characteristics, predictors of occurrence and short- and long-term outcomes of ultra-massive transfusion (UMT), defined as receiving 20 units or more of red blood cells (RBCs) within 48h. This study analyses UMT events from the Australian and New Zealand Massive Transfusion Registry (ANZ-MTR). The ANZ-MTR captured all patients at 29 participating sites receiving a massive transfusion (MT), defined as ≥5 units of RBCs within 4h. Of 9028 patients, 803 (8.9%) received an UMT. UMT patients were younger than other MT patients (median age 57y vs 62y; P < .001). In UMT and MT, males predominated (66.3% and 62.9%, respectively); and context was predominantly trauma (28.8% and 23%) and cardiothoracic surgery (CTS) (21.7% and 20.3%). Median RBC units received within 4h were 16 (UMT) and 6 (MT). In UMT, 4h FFP:RBC ratio (0.6 vs 0.4, P < .001), and 4h cryoprecipitate use (72.9% vs 39.9%, P < .001) were higher. Independent predictors of UMT (Odds Ratio; 95% CI) were age <60y (1.52; 1.28-1.79), baseline Hb >100g/L (1.31; 1.08-1.59), INR >1.5 (1.56; 1.24-1.96), and APTT >60s (4.49; 3.40-5.61). Predictors of in-hospital mortality in UMT included Charlson Comorbidity Index score ≥3 (11.20, 0.60 - 25.00) and bleeding context, with mortality less likely in liver transplant (0.07, 0.01-0.41) and more likely in vascular surgery (8.27, 1.54-72.85), compared with CTS. In-hospital mortality was higher in the UMT group compared with MT group (20.5% vs 44.2%, P < .001), however 5y survival following discharge was not significantly different between the groups (HR=0.87 [95%CI 0.64-1.18], P = .38). UMT patients are more commonly younger, with baseline coagulopathy, and have higher in-hospital mortality compared with MT. However, UMT is not futile: 55.8% survived to discharge, without significant difference in survival postdischarge between the groups.

Protocols for the Isolation of Platelets for Research and Contrast to Production of Platelet Concentrates for Transfusion.

Platelets are specialized cellular elements of blood and play a central role in maintaining normal hemostasis, wound healing, and host defense but also are implicated in pathologic processes of thrombosis, inflammation, and tumor progression and dissemination. Transfusion of platelet concentrates is an important treatment for thrombocytopenia (low platelet count) due to disease or significant blood loss, with the goal being to prevent bleeding or to arrest active bleeding. In blood circulation, platelets are in a resting state; however, when triggered by a stimulus, such as blood vessel injury, become activated (also termed procoagulant). Platelet activation is the basis of their biological function to arrest active bleeding, comprising a complex interplay of morphological phenotype/shape change, adhesion, expression of signaling molecules, and release of bioactive factors, including extracellular vesicles/microparticles. Advances in high-throughput mRNA and protein profiling techniques have brought new understanding of platelet biological functions, including identification of novel platelet proteins and secreted molecules, analysis of functional changes between normal and pathologic states, and determining the effects of processing and storage on platelet concentrates for transfusion. However, because platelets are very easily activated, it is important to understand the different in vitro methods for platelet isolation commonly used 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 human whole blood, and detailed notes are provided about the methods used for the preparation of platelet concentrates for transfusion.

Preparation of Cryoprecipitate and Cryo-depleted Plasma for Proteomic Research Analysis.

When frozen plasma is slowly thawed in cold conditions (1-6 °C), high-molecular-weight plasma proteins precipitate forming a concentrate known as cryoprecipitate. The concentrate is enriched with several important coagulation proteins, including fibrinogen, antihemophilic factor (factor VIII), von Willebrand factor, fibrin stabilizing factor (factor XIII), fibronectin, and small amounts of other plasma proteins. In current medical practice, clinical-grade preparations of cryoprecipitate are used mostly to correct fibrinogen deficiency caused by acute blood loss or due to 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 (also called cryosupernatant) is the plasma supernatant remaining following removal of the cryoprecipitate from frozen-thawed plasma and contains all the remaining soluble plasma proteins. This protocol describes the research-scale preparation of cryoprecipitate and cryo-depleted plasma suitable for proteomic studies and is based on the procedures used to prepare clinical-grade cryoprecipitate.

Red blood cell alloantibodies in the context of critical bleeding and massive transfusion.

BACKGROUND: In the context of critical bleeding and massive transfusion (CB/MT), little is known about the development of new red blood cell (RBC) alloantibodies. We performed a retrospective, observational study to examine the frequency of RBC alloantibodies (pre-existent, anamnestic, or new) in patients with CB/MT, defined as transfusion of five or more RBC units in any 4-hour period, for any cause of CB. MATERIALS AND METHODS: Data on 2,585 New Zealand patients (date/time of MT initiation, demographic data, blood group, clinical context, and transfused RBCs) were obtained from the Australian and New Zealand Massive Transfusion Registry. RBC alloantibody screening/identification data were extracted from the New Zealand Blood Service database. We calculated summary statistics, compared proportions between different independent groups using the Chi-squared test, and performed logistic regression analysis to examine the effects of variables on alloantibody presence or formation. We also determined the immunogenicities of selected RBC antigens in the context of CB/MT. RESULTS: Of 1,234 assessable patients, 1,166 (94.5%) showed no evidence of any alloantibody. Pre-existent, anamnestic, and new alloantibodies were found, respectively, in 4.3%, 0.4%, and 7.2% of assessable patients. By multivariable regression analysis, transfusion of D-positive RBC to D-negative patients was independently associated with new alloantibody formation. Neither the quantum of RBC transfused nor trauma as clinical context were so associated although the latter trended towards a predisposition. "Antibodies of undetermined specificity" were the commonest pre-existent and new alloantibodies. The immunogenicity of Jka was the highest in this setting. DISCUSSION: RBC alloantibodies of any type were rare in this CB/MT population. Patients undergoing CB/MT appear to have low risks of re-stimulating anamnestic alloantibodies, or of developing new RBC alloantibodies.

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.

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.

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

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.

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.

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.