Search

Oxidative treatment of human red blood cells (RBCs) prior to freeze-drying appears to stabilize the RBCs to withstand dried storage at room temperature.

Transfusion-related respiratory complications can be challenging to diagnose especially in mechanically-ventilated patients in the intensive care unit (ICU) due to the concurrent respiratory symptoms associated with the patients’ primary diagnoses. In this narrative review, transfusion-related respiratory complications, including transfusion-associated dyspnea (TAD), transfusion-related acute lung injury (TRALI), transfusion-associated circulatory overload (TACO), and transfusion-related allergic reaction (TRAR), are briefly presented in light of the recent consensus or experts’ definitions; and the diagnosis issues for ICU patients are discussed. Acute respiratory failure occurring during, or within 6 to 24 hours, of transfusion might be a transfusion-related respiratory complication. The recent updated definitions for TRALI and TACO should assist clinicians to differentiate between possible diagnoses. The issues for ICU clinicians are first to recognize the acute respiratory deterioration and the possible causality between the deterioration and blood transfusion and secondly to make the proper diagnosis. This remains challenging for mechanically-ventilated patients. Clinical assessment to identify ICU patients at particular risk of transfusion-related respiratory complications and non-invasive investigation tools could be beneficial and may help to remind clinicians to be alert to the link between transfusion and worsening of respiratory symptoms in these vulnerable critically ill patients.

BACKGROUND: Studies comparing mortality following massive transfusion (MT) with fresher versus longer-stored red blood cells (RBCs) have focused on trauma patients. The Australian and New Zealand Massive Transfusion Registry collects data on all adult MT cases (≥5 RBCs within 4 hours, any bleeding context, ≥18 years) at participating hospitals. METHODS: Years 2007 to 2018 data from 29 hospitals were analyzed to quantify the association between mortality and RBC storage time in adult MT cases. We ran three logistic regression models separately on each of seven bleeding contexts, with in-hospital mortality as the outcome and, in turn, (1) mean storage time (STmean) quartiles, (2) proportion of RBCs ≥30 days old (propOLD), and (3) scalar age of blood index as predictors. RESULTS: A total of 8,685 adult MT cases involving transfusion of 126,622 RBCs were analyzed with Australian and New Zealand data analyzed separately. Mean storage times for these cases were (by quartile in ascending order) as follows: Australia, 12.5 days (range, 3.1-15.5 days), 17.7 (15.5-19.9), 22.3 (19.9-24.9), and 29.8 (24.9-41.7); New Zealand, 11.3 days (3.6-13.7), 15.3 (13.7-16.8), 18.7 (16.8-20.7), and 24.5 (20.7-35.6). The odds ratios comparing in-hospital mortality for each quartile with that of the control first quartile (freshest blood), proportion of longer-stored (≥30 days) RBCs, and scalar age of blood index were not statistically significant across all bleeding contexts. CONCLUSION: We find no correlation between in-hospital mortality and storage time of transfused RBCs in a large cohort of adult MT patients representing all bleeding contexts. These results are consistent with those of recent large multicenter trials. LEVEL OF EVIDENCE: Epidemiologic, level III; Therapeutic, level IV.

Prior to transfusion of red blood cells (RBCs), recipients must be tested for the presence of alloantibodies to avoid immune complications. Liquid-preserved reagent RBCs with known blood group antigen phenotypes are used for testing. However, these reagents have practical constraints, including limited shelf-life and require constant refrigeration. To address these issues, we explore the effects of rapid freeze-drying conditions with trehalose cryoprotectant (0.1-1 M concentrations) on human RBCs and storage of freeze-dried RBCs (FDRBCs) at room temperature (RT) for up to 12 months. We report that rapid freeze-drying of RBCs for 2.5 hr with 0.5 M trehalose achieves recoverable cells with near-normal morphological shape, although size-reduced. The FDRBCs are metabolically active and functional in antibody-agglutination tests by the column agglutination test (CAT) for ABO and Rhesus-D blood group antigens. Expression of the Duffy blood group protein (CD234) decreases by 50% after freeze-drying RBCs. The initial recovery rate is ≤25%; however, 43% of these FDRBCs are still recoverable after RT storage for 12 months. In this proof-of-principle study, we show that rapid freeze-drying can stabilize RBCs. Further refinements to improve the recovery rate and preservation of antigenic epitopes will make FDRBCs a practical alternative source of reagent RBCs for pre-transfusion alloantibody identification.

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.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.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 JkRBC 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.

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 (

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 (

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, 10th 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

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.

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.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.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.

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.

The European trauma guidelines were developed to assist clinicians in the early phase of trauma management to diagnose and treat coagulopathy and bleeding. This study aimed to determine compliance with these European trauma guidelines in a French referral trauma centre.Medical charts of trauma patients with an injury severity score≥16 admitted between January 2013 and December 2014 were reviewed. Compliance with 21 recommendations in the first 24-hours of patient management was assessed.There were 145 patients with median ISS of 34 [IQR 25-41]. A good level of compliance (i.e. applied in≥80% of patients) was identified for nine recommendations, inconsistent compliance (i.e. applied in 50 to 79% of patients) for six recommendations, including fibrinogen levels at hospital admission and achievement of a target mean arterial blood pressure (MAP)80mmHg in patients with major bleeding and TBI (55.5%), and poor compliance (i.e. applied in50% of patients) for another six recommendations. Poorly applied recommendations included early measurement of lactate or base deficit (32%), early administration of tranexamic acid (18%), and achievement of normocapnia in patients with TBI undergoing invasive ventilation (3%).In a referral trauma centre, nine of the 21 evaluable recommendations in the European trauma guidelines were applied in≥80% of patients. Early diagnosis and treatment of trauma-related coagulopathy was identified as an area for significant practice improvement. In patients with TBI, efforts should be made to achieve the targeted MAP and to maintain normocapnia.

Objectives Transfusion-related adverse events (TRAE) can contribute to patient morbidity and mortality. In this brief narrative review, the strategies that clinicians can apply at the bedside to avoid TRAE are discussed. Methods Strategies to avoid the following five types of TRAE were reviewed: transfusion-associated circulatory overload (TACO), transfusion-related acute lung injury (TRALI), transfusion-associated hypothermia (TAH), transfusion-related allergic reactions (TRAR) and acute haemolytic transfusion reactions (AHTR). Results Minimizing exposure to blood components is fundamental to TRAE avoidance. Pre-transfusion assessment can identify patients at risk of TACO, TRAR and TAH, and avoidance steps implemented. Preventive strategies for TACO include lower transfusion rate, ‘one unit at a time’ transfusion policy and possibly diuretic medication. Patients with past history of TRAR should preferably be given plasma-free blood components; anti-histamine medication prior to transfusion could be considered. TAH is common in the massive transfusion setting, particularly trauma patients. Warming of patients are key strategies to avoid TAH. Identification of patients at risk of TRALI is more opaque; however, any measures that limit pulmonary inflammation prior to transfusion may decrease the risk of TRALI. Causes of AHTR are commonly due to human error and failure to apply rigorous cross-checks of patient and issued RBC component blood groups. Conclusions Beneficial strategies to avoid TRAE include judicious use of blood components, identification of high-risk patients, adherence to recommended clinical processes and awareness of TRAE pathophysiology. More evidence is warranted to better guide clinicians in the prevention of TRAE.

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

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.

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.

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.

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/cm2), 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.

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

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

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.

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.

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.

Blood transfusion is an integral component of life-saving fluid resuscitation for anemic, surgical, oncological, and severely injured patients. However, there have been well-publicized studies suggesting that blood may have harmful side-effects. One possible factor contributing to adverse effects of transfusion is the duration of red blood cell storage prior to transfusion. Our group is exploring possible mechanisms of a putative “storage lesion”, including measuring modulation of the immune system or changes in adhesion properties between endothelial cells and transfused red blood cells. The following review will explore changes that occur during red blood cell storage and how these might impact pathways that are important in transfusion recipients’ clinical outcome, as well as provide a broad overview of the research our group is performing on the topic.

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

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/cm2), 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.

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.

Hemolysis of red blood cells (RBCs)during blood bank storage is the most obvious manifes-tation of RBC storage system failure. However, itsanalysis is made difficult because the largest source ofinterunit difference is donor specific. Availability of datafrom national blood systems on large numbers of RBCunits used for internal quality control (QC) purposesand stored and processed in uniform ways permits sta-tistical analysis.

Tissue deficiency of vitamin B12 and folate results in an increase in serum homocysteine (sHcy). We have measured sHcy in patients with reduced serum vitamin B12 and/or red cell folate (RCF) to determine its usefulness as a discriminant for the diagnostic interpretation of reduced vitamin levels. Of 3846 patients who had serum vitamin B12 and RCF assayed, 335 (9%) had reduced vitamin levels. Multivariate analysis showed a significant association between sHcy and serum creatinine (p = 0.0001), positive intrinsic factor (IF) antibody or neutrophil hypersegmentation (NHS) (p = 0.001), increased MCV (p = 0.014) and low RCF (p = 0.025) but no relationship with the level of serum vitamin B12 or haemoglobin. After censoring the patients with renal impairment (n = 54), the distribution of the remaining 72 patients with elevated sHcy was 37/151 (25%) with low serum vitamin B12 with or without low RCF and 35/130 (27%) with low RCF alone. sHcy correctly identified response to vitamin therapy in 33/35 (94%) patients who had adequate parameters to assess response. The positive predictive values of IF antibody/NHS, macrocytosis and/or low RCF for elevated sHcy were 100% and 34% respectively. Twenty-four percent of patients with a low serum vitamin B12 and elevated sHcy had no abnormal haematologic parameters as determined by the routine laboratory staff. These data suggest that the usefulness of measuring sHcy in a routine diagnostic setting is limited and a careful review of the peripheral blood for macrocytosis and NHS plus determination of RCF may be a more cost-effective process than sHcy assay in most instances to determine the presence of tissue deficiency.

Adherence of red blood cells (RBCs) to vascular endothelium impairs blood flow, decreases oxygen delivery and leads to vaso-occulusion. RBCs are relatively non-adherent however little is known of how changes to RBCs for transfusion during storage may affect their adherence properties. The aim of this study was to monitor adherence of stored RBCs to vascular endothelium under conditions of continuous flow in vitro. Specifically the influence of RBC storage time and leucocyte burden of stored red cell preparations was investigated. Human umbilical vein endothelial cells (ECs) were grown to confluence on fibronectin-coated coverslips. Nonleucocyte- reduced, buffy-coat-reduced and leucocyte-filtered RBC products were prepared according to standard blood bank procedures. RBC samples were collected at multiple time points until product expiry and perfused across an EC monolayer using a parallel flow chamber mounted to an inverted microscope. Perfusion of RBCs was controlled for shear stress and temperature. RBC-EC interactions were recorded using a digital camera attached to the microscope. The number of RBCs adhering to the EC layer progressively increased with product storage time. RBCs from products stored for 28 and 42 days were significantly more adherent than fresher cells. RBCs from products containing leucocytes were also significantly more adherent to the EC layer on days 28 and 42 of storage than RBCs from leucocyte-reduced products. Our findings indicate that product storage time and leucocyte burden increase the adhesion of RBCs to an EC layer. These results may lead to greater understanding of the interaction of transfused RBCs with recipient endothelium and the biological consequences of this adherence.

The aim of this project was to identify potential protein mediators of adverse transfusion reactions in stored red cell concentrates. Previously, research into protein mediators of adverse transfusion reactions has focussed on specific proteins, such as cytokines. In this study, a global approach was taken which involved two-dimensional electrophoresis based Proteomics, which has the potential to uncover previously unrecognised mediators or novel proteins. Red cell products with and without pre-storage leukoreduction (each n = 6) were prepared and stored according to standard blood bank procedures. Supernatant samples were taken at several time points until product expiry. Proteins were separated by two-dimensional electrophoresis and those that accumulated in the supernatant were selected for identification by mass spectrometry. The protein profile of supernatant from leukoreduced red cell products was less complex compared to non-leukoreduced products (from 4.4 fold fewer proteins at day 1 to 1.6 fold fewer at day 43). Several proteins were observed to be predominantly present in leukoreduced products which may potentially be beneficial to red cell survival. These proteins were identified as being involved in the maintenance of a stable extracellular environment. Conversely, a number of proteins, which may have detrimental effects, were predominantly expressed in non-leukoreduced products. These proteins included a neutrophil chemoattractant (activator) and a potential acute-phase reactant. A number of proteins identified by mass spectrometry were matched to theoretical proteins of unknown function. The findings confirm the usefulness of Proteomics to investigate storage effects on blood products. The clinical relevance of these proteins is the focus of our future investigations.

Red blood cell (RBC) transfusion has been implicated in certain adverse patient outcomes. Immunomodulation may play a central role. The aim of this project was to determine the ability of supernatants and cellular fractions of RBC concentrates to modulate the immune response of allogeneic mononuclear cells (MNCs), particularly monocytes. Non-leucocytereduced, buffy-coat-depleted and leucocyte-filtered RBC concentrates were prepared and stored according to standard blood bank procedures. RBC samples were collected on day 1 and fortnightly until product expiry, and centrifuged to obtain supernatant and cellular fractions. On the day of RBC sample collection, whole blood (WB) and MNCs were prepared from ABO-compatible allogeneic donors. Induction of monocyte CD11b and CD54 was determined by flow cytometry by incubating allogeneic WB with RBC supernatants or cellular fractions, followed by staining with fl uorescently-labelled anti-CD14, anti-CD11b and anti-CD54. Cytokine release was determined by incubating MNCs with RBC supernatants and culture supernatants were assessed by ELISA for IL-8, TNFalpha; and IL-10. Supernatant and cellular fraction from non-leucocyte-reduced RBC concentrates induced expression of CD11b and CD54 on allogeneic monocytes. Buffy-coatdepleted and leucocyte-filtered RBC concentrates had minimal effect on monocyte CD11b or CD54 expression. Cytokine release from MNCs incubated with RBC supernatant suggested a tenuous balance between proinflammatory (IL-8 and TNF alpha;) and immunosuppressive (IL-10) responses. All RBC product types induced cytokine release from MNCs. The results from this study indicate that stored RBC concentrates can modulate allogeneic MNCs. Both proinflammatory and immunosuppressive responses were evident. Leucocytes in RBC concentrates appear to favour a proinflammatory response by MNCs.

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 × 106/ 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/). © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discrimination between live and apoptotic cells is important for accurate determination of viable CD34(+) cells in hematopoietic stem cell transplant products. SYTO16 is a sensitive fluorescent dye for discriminating live from apoptotic leukocytes. The incidence of apoptotic leukocytes in paired samples of fresh and cryopreserved-thawed cord blood (CB) was determined by the SYTO16/7-AAD flow cytometric assay. Cell migration and expression of the cell homing molecule L-selectin (CD62L) was determined in relation to SYTO16 staining. SYTO16 detected significant proportions of apoptotic lymphocytes and CD34(+) cells in fresh and thawed CB buffy-coat samples that were not detected by 7-AAD. Compared to fresh CB, the proportion of apoptotic lymphocytes and CD34(+) cells significantly increased following thawing. Significantly higher proportions of live SYTO16(bright) lymphocytes and CD34(+) cells were found in the migrated cell population compared to the non-migrated population. Significantly fewer lymphocytes and CD34(+) cells expressed CD62L following thawing. Absence of CD62L expression was strongly correlated with apoptotic/SYTO16(dim) lymphocytes and CD34(+) cells. Cryopreserved-thawed CB contains significant proportions of apoptotic lymphocytes and CD34(+) cells that are not detected by 7-AAD. SYTO16 offers a sensitive method for discrimination of live from apoptotic leukocytes and assists in accurate assessment of CB quality and suitability for use in clinical transplantation.

BACKGROUND: The contribution of RBC transfusion to adverse patient outcomes is controversial. There is conflicting clinical data and limited biologic data that provide an underpinning biologic rationale for any adverse impacts from RBC transfusion. This study used in-vitro measures of PMN stimulation to determine the ability of supernatant from RBCs to stimulate allogeneic WBCs and to determine the influence of residual donor WBCs and storage time on the proinflammatory potential of RBCs. STUDY DESIGN AND METHODS: Three types of RBCs were assessed: standard non-WBC-reduced RBCs (S-RBCs), buffy coat-poor RBCs (BCP-RBCs), and prestorage WBC-filtered RBC (LF-RBCs). Supernatant was collected weekly up to Day 42 of storage. PMN priming by supernatant from RBCs was determined by three methods: induction of CD11b expression on PMNs, induction of IL-8 release from PMNs, and the chemotactic effect of supernatant on PMNs. RESULTS: Supernatant from S-RBCs induced the expression of CD11b on PMNs, primed PMNs to release IL-8, and was chemotactic for PMNs. The magnitude of this PMN-priming progressively amplified with storage time. In contrast, supernatant from BCP-RBCs or LF-RBCs did not significantly prime PMNs. The PMN-priming effect of supernatant from RBCs correlated more closely with the level of MNCs in the RBCs than PMN content. CONCLUSION: Supernatant from stored S-RBCs prime unstimulated allogeneic PMNs in vitro. Prestorage buffy-coat WBC reduction was as effective as WBC depletion in abrogating this proinflammatory response elicited by supernatants from RBCs. The clinical consequences, if any, of these findings for transfusion recipients are unknown.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

Single-platform flow cytometric absolute cell counting protocols provide increased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris, such as cord blood samples, provide challenges for these assays. We describe a simple, robust absolute CD34+ cell counting protocol, suitable for cord blood, using TRUCOUNT absolute count tubes (BD Biosciences, San Jose, CA) and a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that each tube is supplied with a known number of lyophilized fluorescent beads. The method includes no-wash fixative-free ammonium chloride red blood cell lysis and the viability dye, 7-amino actinomycin D, to exclude dead cells. The threshold was set on CD45 expression in the FL1 channel and an exclusion gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal condition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual-platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) showed high correlation (R(2) = 0.949 and 0.989, respectively) and no significant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect of a fixative-containing lysis reagent when used in a lyse-and-wash procedure. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes and the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration.

A chimeric individual possesses two or more genetically distinct ceil populations. Although the chimerism may not be evident in all gene systems, various loci display greater numbers of alleles than genetically “normal” individ uals. The proposit’a was referred for further laboratory investigation due to a mixed-field ABO blood group reaction following routine antenatal testing. Various molecular (HLA class II, ABO genotyping, and 10 short tandem repeat [STR] microsatellites) and serologic (HLA class I and red cell blood groups) typing techniques were employed to investigate a number of polymorphic loci located on different chromosomes. Chimerism was identified in 8 out of the 1.4 chromosomes tested: chromosome 1 (Duffy), 6 (HLA class I and Iï), 9 (ABO), 11 (HUMTH01), 12 (HUMPLA2A1), 15 (HUMFES/FPS). 18 (Kidd) and 21 (D21S11). The proposita was determined to be a probable dispermic chimera, based on the results of the serology and molecular studies.

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.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.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.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.

The ability of bone marrow (BM) stroma derived from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML) to support normal hematopoiesis was investigated using a two-stage long-term bone marrow culture (LTBMC) procedure. Of particular interest was whether leukemia-derived stroma were capable of supporting the very immature, uncommitted hematopoietic progenitor cells (HPC) which are considered to have a higher dependence and association with the BM stroma than the more mature committed HPC. Confluent stromal layers were recharged with selected populations of normal HPC enriched for the CD34+CD38− cells (immature, uncommitted HPC) or the CD34+CD38+ cells (mature, committed HPC). The weekly output of clonable granulocyte-macrophage progenitor cells (CFU-GM) was used as an indicator of the hematopoietic-supporting ability of the cultures. Stromal layers derived from 5 7 patients newly diagnosed with AML, showed significantly depressed ability to support the CD34+CD38− cells compared to stroma derived from normal donors. The supporting function of the AML-derived stroma for the more mature CD34+CD38+ cells was similar to that of the normal stroma ( 3 3 cases). Stromal layers derived from patients with chronic-phase CML showed normal or enhanced supporting function for the CD34+CD38− cells ( 5 6 cases), and likewise for the CD34+CD38+ cells ( 3 3 cases). This study revealed a selective defect in the ability of BM stroma from patients with AML to support the maturation of normal early uncommitted HPC, represented by the CD34+CD38− cells, whilst the ability to support the committed CD34+CD38+ cells was not affected. This suggests that the BM microenvironment may be implicated in the disease mechanism of AML. It does not, however, appear to be as clearly implicated in chronic-phase CML.

Background and Objectives Refrigerated storage of red blood cells (RBCs) induces numerous changes that may target the cells for erythrophagocytosis following transfusion. The influence of storage upon the phagocytosis of unseparated and fractionated young and old stored RBCs was investigated using two in vitro quantitative phagocytosis assays. Materials and Methods Leucocyte-depleted RBC units were sampled at day 1 or 42 of storage. Young and old RBCs were fractionated at day 1 by density centrifugation and stored in paediatric packs for up to 42 days. RBCs were labelled with the fluorescent dye PKH26 and incubated with the human monocytic cell line THP-1. Erythrophagocytosis was quantified by flow cytometry and plate fluorometric assays. Results A higher proportion of THP-1 cells phagocytosed RBCs stored for 42 days compared with 1 day (41% and 24% respectively; P

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.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.Compared to SAGM RBCs, AS-1 RBCs had lower hemolysis (p0.04), lower GPA(+) MPs (p0.03), and lower PS(+) MPs (p0.03) from Day 14 onward. AS-1 RBCs had higher (p0.02) 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).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.

ABO polymorphism at the gene level has been investigated by molecular methods, predominantly sequencing and restriction fragment length polymorphism (RFLP). We describe the application of the polymerase chain reaction–sequence specific oligonucleotide (PCR-SSO) method, which is considered to be more versatile for large sample numbers, compared with conventional ABO genotyping by PCR-RFLP. PCR-SSO, while maintaining accurate and reliable results, reduces costs and labor. A population of 155 random individuals was investigated for the three O alleles, O1, O1*, and O2 . The allelic frequencies were 35 percent, 26 percent, and 5 percent, respectively. PCR-SSO results correlated completely with both serologic and PCR-RFLP results.

Genotyping enables the identification of both maternally and paternally derived alleles. A number of protocols have been described for the genotyping of the ABO blood group system. Generally, these methods have a number of disadvantages including the use of hazardous reagents, being technically demanding, and the excessive use of materials. In this study, a relatively simple polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method is described. Four different amplifications were used that were specific for nucleotides sites 261, 526, 703, and 796 to distinguish the A, B, O1 and O2 alleles. The ABO genotypes of 294 random individuals were determined and were found to completely correlate with the serologic phenotypes. The protocol is applicable for investigations of weak or nonexpression of ABO alleles, paternity determinations, and population analysis.