Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme.

Strain-specific spleen remodelling in Plasmodium yoelii infections in Balb/c mice facilitates adherence and spleen macrophage-clearance escape

Knowledge of the dynamic features of the processes driven by malaria parasites in the spleen is lacking. To gain insight into the function and structure of the spleen in malaria, we have implemented intravital microscopy and magnetic resonance imaging of the mouse spleen in experimental infections with non-lethal (17X) and lethal (17XL) Plasmodium yoelii strains. Noticeably, there was higher parasite accumulation, reduced motility, loss of directionality, increased residence time and altered magnetic resonance only in the spleens of mice infected with 17X. Moreover, these differences were associated with the formation of a strain-specific induced spleen tissue barrier of fibroblastic origin, with red pulp macrophage-clearance evasion and with adherence of infected red blood cells to this barrier. Our data suggest that in this reticulocyte-prone non-lethal rodent malaria model, passage through the spleen is different from what is known in other Plasmodium species and open new avenues for functional/structural studies of this lymphoid organ in malaria.

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.

Treatment of thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP), characterized by thrombocytopenia and microangiopathic haemolytic anaemia, was almost universally fatal until the introduction of plasma exchange (PE) therapy in the 1970s. Based on clinical studies, daily PE has become the first-choice therapy since 1991. Recent findings may explain its effectiveness, which may include, in particular, the removal of anti-ADAMTS13 autoantibodies and unusually large von Willebrand factor multimers and/or supply of ADAMTS13 in acquired idiopathic or congenital TTP. Based on currently available data, the favoured PE regimen is daily PE [involving replacement of 1-1.5 times the patient's plasma volume with fresh-frozen plasma (FFP)] until remission. Adverse events of treatment are mainly related to central venous catheters. The potential reduction of plasma related side-effects, such as transfusion-related acute lung injury (TRALI) or febrile transfusion reactions by use of solvent-detergent treated (S/D) plasma instead of FFP is not established by controlled clinical studies. Uncontrolled clinical observations and the hypothesis of an autoimmune process in a significant part of the patients with acquired idiopathic TTP suggest a beneficial effect of adjunctive therapy with corticosteroids. Other immunosuppressive treatments are not tested in controlled trials and should be reserved for refractory or relapsing disease. There is no convincing evidence for the use of antiplatelet agents. Supportive treatment with transfusion of red blood cells or platelets has to be evaluated on a clinical basis, but the transfusion trigger for platelets should be very restrictive. Further controlled, prospective studies should consider the different pathophysiological features of thrombotic microangiopathies, address the prognostic significance of ADAMTS13 and explore alternative exchange fluids to FFP, the role of immunosuppressive therapies and of new plasma saving approaches as recombinant ADAMTS13 and protein A immunoadsorption.

[Examination of urine in the child]

The examination of urine in children can be very complex, due to the difficulty to obtain clean urine specimens in infants and toddlers. Clean catch is an easy system to obtain urine but patience is needed. Transurethral catheterization or suprapubic aspiration is useful in infants and toddlers with sign of pyelonephritis. Urine bag specimens are not useful in the diagnosis of urinary tract infection because of the high rate of false positive cultures. The 24 hours urine collection is frequently replaced by a spot urine and the ratio of the measured substances with the urine creatinine are calculated. Urine microscopy is needed for the evaluation of pathological results in the dipstick testing: confirm that red urine is due to haematuria by demonstration of red blood cells on urine microscopy, dysmorphic cells and red-cell casts are pathognomonic of glomerular bleeding, white-cell casts signify glomerular inflammation and bacteria are easily seen in unstained urine. A urine culture is pathologic if the colony count exceeds 10(4) in the transurethral catheterization or clean void. In the suprapubic aspiration is any number of colony pathologic. Urate crystals in the urine of infants may cause a pink discoloration to nappies. Urine screenings are not very useful and should be performed only at the age of 5 years or by sexual-active adolescents.

A KEL gene encoding serine at position 193 of the Kell glycoprotein results in expression of KEL1 antigen

BACKGROUND: The KEL2/KEL1 (k/K) blood group polymorphism represents 578C>T in the KEL gene and Thr193Met in the Kell glycoprotein. Anti-KEL1 can cause severe hemolytic disease of the fetus and newborn. Molecular genotyping for KEL*1 is routinely used for assessing whether a fetus is at risk. Red blood cells (RBCs) from a KEL:1 blood donor (D1) were found to have abnormal KEL1 expression during evaluation of anti-KEL1 reagents. STUDY DESIGN AND METHODS: Kell genotyping methods, including KEL exon 6 direct sequencing, were applied. KEL cDNA from D1 was sequenced. Flow cytometry was used to assess KEL1 and KEL2 RBC expression. RESULTS: RBCs from the donor, her mother, and an unrelated donor gave weak or negative reactions with some anti-KEL1 reagents. Other Kell-system antigens appeared normal. The three individuals were homozygous for KEL C578 (KEL*2) but heterozygous for a 577A>T transversion, encoding Ser193. They appeared to be KEL*2 homozygotes by routine genotyping methods. Flow cytometry revealed weak KEL1 expression and normal KEL2, similar to that of KEL*2 homozygotes. CONCLUSION: Ser193 in the Kell glycoprotein appears to result in expression of abnormal KEL1, in addition to KEL2. The mutation is not detected by routine Kell genotyping methods and, because of unpredicted KEL1 expression, could lead to a misdiagnosis.

Early resuscitation with polymerized bovine hemoglobin reverses acidosis, but not peripheral tissue oxygenation, in a severe hamster shock model

Awake hamsters equipped with the dorsal window chamber preparation were subjected to hemorrhage of 50% of the estimated blood volume. Initial resuscitation (25% of estimated blood volume) with polymerized bovine hemoglobin (PBH) or 10% hydroxyethyl starch (HES) occurred in concert with an equivolumetric bleeding to simulate the early, prehospital setting (exchange transfusion). Resuscitation (25% of estimated blood volume) without bleeding was performed with PBH, HES, or autologous red blood cells (HES-RBCs). Peripheral microcirculation, tissue oxygenation, and systemic hemodynamic and blood gas parameters were assessed. After exchange transfusion, base deficit was -8.6 +/- 3.7 mmol/L (PBH) and -5.1 +/- 5.3 mmol/L (HES) (not significant). Functional capillary density was 17% +/- 6% of baseline (PBH) and 31% +/- 11% (HES) (P < 0.05) and arteriolar diameter 73% +/- 3% of baseline (PBH) and 90% + 5% (HES) (P < 0.01). At the end, hemoglobin levels were 3.7 +/- 0.3 g/dL with HES, 8.2 +/- 0.6 g/dL with PBH, and 10.4 +/- 0.8 g/dL with HES-RBCs (P < 0.01 HES vs. PBH and HES-RBCs, P < 0.05 PBH vs. HES-RBCs). Base excess was restored to baseline with PBH and HES-RBCs, but not with HES (P < 0.05). Functional capillary density was 46% +/- 5% of baseline (PBH), 62% + 20% (HES-RBCs), and 36% +/- 19% (HES) (P < 0.01 HES-RBCs vs. HES). Peripheral oxygen delivery and consumption was highest with HES-RBCs, followed by PBH (P < 0.05 HES-RBCs vs. PBH, P < 0.01 HES-RBCs and PBH vs. HES). In conclusion, the PBH led to a correction of base deficit comparable to blood transfusion. However, oxygenation of the peripheral tissue was inferior with PBH. This was attributed to its negative impact on the peripheral microcirculation caused by arteriolar vasoconstriction.

Transfusion efficacy of ABO major-mismatched platelets (PLTs) in children is inferior to that of ABO-identical PLTs

BACKGROUND: ABO major compatibility is essential in transfusions of red blood cells but is not requisite in PLT transfusions. In adults there is some evidence that transfusion efficacy of ABO blood group-identical platelets (PLTs) is superior to major-mismatched PLTs. However, in children this question has not been investigated for more than 30 years. STUDY DESIGN AND METHODS: In a prospective study, the efficacy (based on the 1-hour percentage of PLT recovery [PPR(1hr)]) of 400 eligible ABO blood group-identical or out-of-group apheresis PLT concentrates (APCs), transfused mainly prophylactically to 50 children with hematologic malignancies, solid tumors, or aplastic anemia was investigated. The primary objective was to compare PPR(1hr) between ABO-identical and major-mismatched transfusions. RESULTS: After ABO major-mismatched transfusions, PPR(1hr) was significantly lower than after ABO blood group-identical transfusions (median 21% vs. 32%; p = 0.034). Multivariate analysis showed major-mismatched transfusions to be significantly more often unsuccessful than identical transfusions (odds ratio [OR], 3.97; 95% confidence interval [CI], 1.52-10.39; p = 0.005). Using flow cytometry and fluorescent microscopy, it could be demonstrated that PLTs of subgroup A(1), significantly expressing A antigen on their surface, were rapidly cleared from the circulation of group O or B recipients. In contrast, major-mismatched transfusions of A(2) PLTs, expressing no detectable A antigen, were as successful as identical transfusions (OR, 1.13; 95% CI, 0.16-7.88; p = 0.90). CONCLUSION: These data clearly indicate that in children ABO major-mismatched PLT transfusions result in inferior transfusion efficacy, with the only exception of group A(2) PLTs. ABO minor-mismatched PLTs showed comparable efficacy to identical transfusions.

Comparison of low potassium Euro-Collins solution and standard Euro-Collins solution in an extracorporeal rat heart-lung model

OBJECTIVE: Euro-Collins solution (EC) is routinely used in lung transplantation. The high potassium of EC, however, may damage the vascular endothelium, thereby contributing to postischemic reperfusion injury. To assess the influence of the potassium concentration on lung preservation, we evaluated the effect of a "low potassium Euro-Collins solution" (LPEC), in which the sodium and potassium concentrations were reversed. METHODS: In an extracorporeal rat heart-lung model lungs were preserved with EC and LPEC. The heart-lung blocks (HLB) were perfused with Krebs-Henseleit solution containing washed bovine red blood cells and ventilated with room air. The lungs were perfused via the working right ventricle with deoxygenated perfusate. Oxygenation and pulmonary vascular resistance (PVR) were monitored. After baseline measurements, hearts were arrested with St. Thomas' solution and the lungs were perfused with EC or LPEC, or were not perfused (controls). The HLBs were stored for 5 min or 2 h ischemic time at 4 degrees C. Reperfusion and ventilation was performed for 40 min. At the end of the trial the wet/dry ratio of the lungs was calculated and light microscopic assessment of the degree of edema was performed. RESULTS: After 5 min of ischemia oxygenation was significantly better in both preserved groups compared to the controls. Pulmonary vascular resistance was elevated in all three groups after 30 min reperfusion at both ischemic times. After 2 h of ischemia PVR of the group preserved with LPEC was significantly lower than those of the EC and controls (LPEC-5 min: 184 +/- 65 dynes * sec * cm-5, EC-5 min: 275 +/- 119 dynes * sec * cm * cm-5, LPEC-2 h: 324 +/- 47 dynes * sec * m-5, EC-2 h: 507 +/- 83 dynes * sec * cm-5). Oxygenation after 2 h of ischemia and 30 min reperfusion was significantly better in the LPEC group compared to EC and controls (LPEC: 70 +/- 17 mmHg, EC: 44 +/- 3 mmHg). The wet/dry ratio was significantly lower in the two preserved groups compared to controls (LPEC-5 min: 5.7 +/- 0.7, EC-5 min: 5.8 +/- 1.2, controls-5 min: 7.5 +/- 1.8, LPEC-2 h: 6.7 +/- 0.4, EC: 6.9 +/- 0.4, controls-2 h: 7.3 +/- 0.4). CONCLUSIONS: We thus conclude that LPEC results in better oxygenation and lower PVR in this lung preservation model. A low potassium concentration in lung preservation solutions may help in reducing the incidence of early graft dysfunction following lung transplantation.

Does point of care prothrombin time measurement reduce the transfusion of fresh frozen plasma in patients undergoing major surgery? The POC-OP randomized-controlled trial

BACKGROUND: Bleeding is a frequent complication during surgery. The intraoperative administration of blood products, including packed red blood cells, platelets and fresh frozen plasma (FFP), is often live saving. Complications of blood transfusions contribute considerably to perioperative costs and blood product resources are limited. Consequently, strategies to optimize the decision to transfuse are needed. Bleeding during surgery is a dynamic process and may result in major blood loss and coagulopathy due to dilution and consumption. The indication for transfusion should be based on reliable coagulation studies. While hemoglobin levels and platelet counts are available within 15 minutes, standard coagulation studies require one hour. Therefore, the decision to administer FFP has to be made in the absence of any data. Point of care testing of prothrombin time ensures that one major parameter of coagulation is available in the operation theatre within minutes. It is fast, easy to perform, inexpensive and may enable physicians to rationally determine the need for FFP. METHODS/DESIGN: The objective of the POC-OP trial is to determine the effectiveness of point of care prothrombin time testing to reduce the administration of FFP. It is a patient and assessor blind, single center randomized controlled parallel group trial in 220 patients aged between 18 and 90 years undergoing major surgery (any type, except cardiac surgery and liver transplantation) with an estimated blood loss during surgery exceeding 20% of the calculated total blood volume or a requirement of FFP according to the judgment of the physicians in charge. Patients are randomized to usual care plus point of care prothrombin time testing or usual care alone without point of care testing. The primary outcome is the relative risk to receive any FFP perioperatively. The inclusion of 110 patients per group will yield more than 80% power to detect a clinically relevant relative risk of 0.60 to receive FFP of the experimental as compared with the control group. DISCUSSION: Point of care prothrombin time testing in the operation theatre may reduce the administration of FFP considerably, which in turn may decrease costs and complications usually associated with the administration of blood products. TRIAL REGISTRATION: NCT00656396.

The association of preoperative anemia and perioperative allogeneic blood transfusion with the risk of surgical site infection

BACKGROUND: The purpose of the study was to investigate allogeneic blood transfusion (ABT) and preoperative anemia as risk factors for surgical site infection (SSI). STUDY DESIGN AND METHODS: A prospective, observational cohort of 5873 consecutive general surgical procedures at Basel University Hospital was analyzed to determine the relationship between perioperative ABT and preoperative anemia and the incidence of SSI. ABT was defined as transfusion of leukoreduced red blood cells during surgery and anemia as hemoglobin concentration of less than 120 g/L before surgery. Surgical wounds and resulting infections were assessed to Centers for Disease Control standards. RESULTS: The overall SSI rate was 4.8% (284 of 5873). In univariable logistic regression analyses, perioperative ABT (crude odds ratio [OR], 2.93; 95% confidence interval [CI], 2.1 to 4.0; p < 0.001) and preoperative anemia (crude OR, 1.32; 95% CI, 1.0 to 1.7; p = 0.037) were significantly associated with an increased odds of SSI. After adjusting for 13 characteristics of the patient and the procedure in multivariable analyses, associations were substantially reduced for ABT (OR, 1.25; 95% CI, 0.8 to 1.9; p = 0.310; OR, 1.07; 95% CI, 0.6 to 2.0; p = 0.817 for 1-2 blood units and >or=3 blood units, respectively) and anemia (OR, 0.91; 95% CI, 0.7 to 1.2; p = 0.530). Duration of surgery was the main confounding variable. CONCLUSION: Our findings point to important confounding factors and strengthen existing doubts on leukoreduced ABT during general surgery and preoperative anemia as risk factors for SSIs.

Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0.870 and 0.783, respectively (n = 240; P < 0.001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.

Histo-blood group antigens as allo- and autoantigens

The science of blood groups has made giant steps forward during the last decade. Blood-group typing of red blood cells (RBCs) is performed on more than 15 million samples per year in Europe, today much less often for forensic reasons than for clinical purposes such as transfusion and organ transplantation. Specific monoclonal antibodies are used with interpretation on the basis of RBC agglutination patterns, and mass genotyping may well be on its way to becoming a routine procedure. The discovery that most blood group systems, whose antigens are by definition found on RBCs, are also expressed in multiple other tissues has sparked the interest of transplantation medicine in immunohematology beyond the HLA system. The one and only "histo-blood group" (HBG) system that is routinely considered in transplantation medicine is ABO, because ABO antigen-incompatible donor/recipient constellations are preferably avoided. However, other HBG systems may also play a role, thus far underestimated. This paper is an up-to-date analysis of the importance of HBG systems in the alloimmunity of transplantation and autoimmune events, such as hemolytic anemia.

Clinical experience with the use of bivalirudin in a large population undergoing endovascular abdominal aortic aneurysm repair

PURPOSE: To retrospectively evaluate the safety and effectiveness of the use of bivalirudin, a direct thrombin antagonist, compared with unfractionated heparin in endovascular aneurysm repair (EVAR). MATERIALS AND METHODS: Between March 1994 and September 2007, 740 consecutive patients (mean age, 75.7 y +/- 7.7; 69 women) underwent elective EVAR for infrarenal abdominal aortic aneurysm. Bivalirudin was used in 98 of these 740 (13.2%) and unfractioned heparin was used in the other 642 (86.8%). Complications were classified according to the Society of Vascular Surgery/International Society for Cardiovascular Surgery criteria. Major bleeding was defined as clinically overt blood loss resulting in a decrease of hemoglobin of more than 3 g/dL, any decrease in hemoglobin of more than 4 g/dL, transfusion of 2 U or more of red blood cells, or intracranial or retroperitoneal hemorrhage. RESULTS: Grade 1 major complications were observed in 161 of 642 patients (25.2%) in the heparin group and 12 of 98 patients (12.2%) in the bivalirudin group (P = .0046), whereas the incidences of grade 3 major complications were not significantly different between groups (P = .57). The rate of total complications was higher in the heparin group than in the bivalirudin group (247 of 642 [38.5%] vs 21 of 98 [21.4%]; P = .001). Major bleeding occurred in 10 of 98 patients (10.2%) receiving bivalirudin and in 91 of 642 patients (14.2%) receiving heparin (P = .34). One of 21 major complications (4.76%) in the bivalirudin group and 12 of 247 major complications (4.86%) in the heparin group were attributable to thrombosis (P = 1.0). CONCLUSIONS: Bivalirudin is a safe and feasible alternative to unfractionated heparin in patients undergoing EVAR.

Diseases in neonatal foals. Part 2: potential risk factors for a higher incidence of infectious diseases during the first 30 days post partum

REASON FOR PERFORMING STUDY: The development of clinical illness in foals is usually predetermined by perinatal history, management or stressful environmental conditions. OBJECTIVES: To determine potential risk factors for an increased incidence of infectious diseases during the first 30 days post partum. METHODS: The population consisted of Thoroughbred foals born on stud farms in the Newmarket (UK) area in 2005 (n = 1031). They were followed for their first 30 days. Factors suspected to influence the incidence of infectious neonatal diseases were examined in a logistic regression approach for each of the 3 outcomes (total infectious diseases, systemic disease with diarrhoea and total infectious diseases excluding diarrhoea). All 28 factors were either foal or mare or stud farm related. RESULTS: Several significant risk factors for a higher disease incidence, such as birth complications, colostrum intake by stomach tube and leucocytosis 12-48 h post partum were identified. The factor 'boarding stud' seemed to be protective against disease. CONCLUSION: Some factors, such as the mare's time at stud before foaling, the mare's rotavirus vaccination schedule and fibrinogen-values that empirically had been linked to the outcome previously were not confirmed as relevant. This included the reported useful prophylactic treatment with antimicrobial drugs. POTENTIAL RELEVANCE: Factors to be considered when evaluating newborn foals include: stud management, the birth process, route of colostrum intake, white and red blood cells, and the date of birth. These may help to detect foals at risk to develop an infection so that targeted prophylactic measures can be initiated.