Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review.

Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers of reagents. By use of a recombinantly produced factor C , based on the sequence of a coagulation enzyme present in horseshoe crab amebocyte lysates, the rFC tests are designed as substitutes for the traditional Limulus amebocyte lysate (LAL)/Tachypleus amebocyte lysate tests based on horseshoe crab blood. Comparative testing of samples with both the LAL and recombinant reagents has shown a high degree of correlation, suggesting that use of rFC is comparable to the more traditional LAL tests and may be technologically superior. Recombinant factor C does not recognize the factor G pathway, the alternate coagulation pathway that the lysate reagents detect. This feature allows rFC to detect endotoxin more selectively. As a recombinantly produced material, it avoids the use of the horseshoe crabs required for lysate production, thereby protecting this species, which is at risk in some parts of the world. Recombinant factor C is expected to further benefit from a more sustainable supply chain based upon a robust biotechnological production process. We summarize here the results of many studies that evaluated the use of recombinant technology for the detection of environmental endotoxin. Additionally, we include a review of the current compendia and regulatory status of the recombinant technologies for use in the quality control of pharmaceutical manufacturing. Our analysis confirms that the recombinant technologies are comparable in protecting patient safety.

A multinational study of the influence of health-related quality of life on in-hospital outcome after coronary artery bypass graft surgery.

BACKGROUND: The effect of health-related quality of life on in-hospital outcomes after coronary artery bypass grafting surgery has not been investigated in international multicenter studies. We hypothesized that poor preoperative health status is associated with mortality and length of hospital stay. METHODS: In the Multicenter Study of Perioperative Ischemia Epidemiology II, preoperative Short-Form 12, Mental Component Summary (MCS), and Physical Component Summary (PCS) scores were obtained prospectively from 4,811 patients (3,834 men, 977 women) undergoing coronary artery bypass grafting surgery at 72 centers in 17 countries. Primary outcome measures were in-hospital mortality and prolonged (>14 days) length of hospital stay. RESULTS: One hundred fifty-one patients (3.1%) died. After adjustment for regional differences, a 10-point reduction in MCS score was associated with higher mortality risk (odds ratio [OR] 1.17, 95% CI 1.004-1.37, P = .04) and prolonged hospital stay (OR 1.11, 95% CI 1.01-1.21, P = .03). The preoperative PCS score was not associated with mortality risk but significantly predicted prolonged length of hospital stay (OR 1.20, 95% CI 1.09-1.33, P < .001). There was no significant interaction between gender and either the MCS or the PCS score. DISCUSSION: The preoperative PCS predicted prolonged postoperative hospital stay, whereas the preoperative MCS score was an independent predictor of both prolonged length of hospital stay and mortality. Preoperative assessment of health-related quality of life factors with the Short-Form 12 might be a useful tool for risk stratification and planning for hospital discharge and rehabilitation.

Impact of hyperglycemia on perioperative mortality after coronary artery bypass graft surgery.

OBJECTIVE: The roles of perioperative hyperglycemia and diabetes in the risk stratification of patients undergoing coronary artery bypass graft surgery are unclear. The aim of this study is to explore the influence of perioperative hyperglycemia on postoperative mortality. METHOD: A prospective, observational study of 5050 patients undergoing cardiopulmonary bypass for coronary artery bypass graft surgery at 70 international centers was conducted, with 7500 measured variables per patient and outcomes adjudicated centrally. Postoperative blood glucose levels measured from the day of surgery to postoperative day 3 were available for 4799 patients. Multivariable logistic regression was used to determine the association of hyperglycemia with hospital mortality. RESULTS: A total of 164 patients died during hospitalization (3.2%). Mortality was significantly higher in the diabetic population compared with the nondiabetic population (4.2% vs 2.9%; P = .02). In nondiabetic patients, maximum postoperative blood glucose between 250 and 300 mg/dL (adjusted odds ratio, 2.56; 95% confidence interval, 1.18-5.57; P = .02) and maximum blood glucose of 300 mg/dL or greater (adjusted odds ratio, 2.74; 95% confidence interval, 1.22-6.16; P = .01), compared with maximum blood glucose less than 200 mg/dL, and postoperative insulin treatment (adjusted odds ratio, 2.04; 95% confidence interval, 1.12-3.70), were independent risk factors for an increased risk of in-hospital mortality. In diabetic patients, hyperglycemia was not associated with a higher mortality risk. CONCLUSIONS: Postoperative hyperglycemia is associated with increased in-hospital mortality in nondiabetic patients after coronary artery bypass graft surgery. In diabetic patients, hyperglycemia was not associated with mortality.

Postoperative red blood cell transfusion and morbid outcome in uncomplicated cardiac surgery patients.

OBJECTIVE: To evaluate postoperative red blood cell (RBC) transfusion and its association with postoperative cardiac events and multiorgan morbidity in uncomplicated cardiac surgery patients. METHODS: A cohort of 945 patients from the 5,436 coronary artery bypass grafting patients enrolled in the international Multicenter Study of Perioperative Ischemia (McSPI) Epidemiology II (EPI II) study was investigated. Inclusion criteria were low to moderate risk profile, postoperative hemoglobin level ≥ 10 g/dl, minimal postoperative blood loss, and no evidence of any morbid event on the day of surgery. RBC transfusion was assessed during the first 24 postoperative hours and cardiac as well as multiorgan outcomes from postoperative day 2 to hospital discharge. Multivariate analysis was applied to assess the effect of RBC transfusion on multiorgan outcomes. A secondary propensity score analysis was performed in 4,465 patients without early postoperative morbid outcomes. RESULTS: Transfused patients (193/945, 20.4%) were more likely to suffer cardiac events (P = 0.03), harvest-site infection (P = 0.002), and composite morbidity outcome (P = 0.04). RBC transfusion was associated with cardiac events on multivariate as well as on propensity score analysis (adjusted odds ratio, 1.39; 95% confidence interval, 1.01-1.92; P = 0.04), and with harvest-site infection on multivariate analysis. Additionally, propensity score analysis suggested possible associations of RBC transfusion with increased risks for composite morbidity outcome and in-hospital mortality, renal morbidity, pneumonia, and mediastinitis. CONCLUSIONS: The data suggest a potential association between postoperative RBC transfusion and increased morbidity for cardiac surgery patients with low to moderate mortality risk profiles, adequate hemoglobin levels, and low bleeding rates.

The ongoing variability in blood transfusion practices in cardiac surgery.

BACKGROUND: Although blood utilization has been under considerable scrutiny for the past two decades, particularly for surgery, studies comparing perioperative blood transfusion practices between countries are rare, and the evolution of international standards remains unknown. Therefore, the objective of this evaluation was to compare the perioperative transfusion of blood components in cardiac surgery in multiple centers in different countries. STUDY DESIGN AND METHODS: Transfusion practice was investigated prospectively in 70 centers among 16 countries. A total of 5065 randomly selected cardiac surgery patients of the Multicenter Study of Perioperative Ischemia Epidemiology II (EPI II) Study were evaluated. Utilization of red blood cells (RBCs), fresh-frozen plasma (FFP), and platelets (PLTs) was assessed daily, before, during, and after surgery until hospital discharge. RESULTS: Intraoperative RBC transfusion varied from 9 to 100 percent among the 16 countries, and 25 to 87 percent postoperatively (percentage of transfused patients). Similarly, frequency of transfusion of FFP varied from 0 to 98 percent intraoperatively and 3 to 95 percent postoperatively, and PLT transfusion from 0 to 51 and 0 to 39 percent, respectively. Moreover, there were not only marked differences in transfusion rates between centers in different countries but also in interinstitutional comparison of multiple centers within countries. CONCLUSION: In cardiac surgical patients, marked variability in transfusion practice exists between centers in various countries and suggests differences in perioperative practice patterns as well as possible inappropriate use. International standardization of perioperative practice patterns as well as transfusion regimes appears necessary.

Impact of preoperative anemia on outcome in patients undergoing coronary artery bypass graft surgery.

BACKGROUND: The risk of preoperative anemia in patients undergoing heart surgery has not been described precisely. Specifically, the impact of low hemoglobin per se or combined with other risk factors on postoperative outcome is unknown. Thus, we determined the effects of low preoperative hemoglobin and comorbidities on postoperative adverse outcomes in patients with coronary artery bypass graft in a large comprehensive multicenter study. METHODS AND RESULTS: The Multicenter Study of Perioperative Ischemia investigated 5065 patients with coronary artery bypass graft at 70 institutions worldwide, collecting approximately 7500 data points per patient. In 4804 patients who received no preoperative transfusions, we determined the association between lowest preoperative hemoglobin levels and in-hospital cardiac and noncardiac morbidity and mortality and the impact of concomitant risk factors, assessed by EuroSCORE, on this effect. In patients with EuroSCORE < 4 (n=2054), only noncardiac outcomes were increased, whereas patients with EuroSCORE > or = 4 (n=2750) showed an increased incidence of all postoperative events, starting at hemoglobin < 11 g/dL. Low preoperative hemoglobin was an independent predictor for noncardiac (renal > cerebral; P<0.001) outcomes, whereas the increase in cardiac events was due to other factors associated with preoperative anemia. CONCLUSIONS: Anemic patients undergoing cardiac surgery have an increased risk of postoperative adverse events. Importantly, the extent of preexisting comorbidities substantially affects perioperative anemia tolerance. Therefore, preoperative risk assessment and subsequent therapeutic strategies, such as blood transfusion, should take into account both the individual level of preoperative hemoglobin and the extent of concomitant risk factors.

Coronary artery bypass graft surgery--care globalization: the impact of national care on fatal and nonfatal outcome.

OBJECTIVE: In an international, prospective, observational study, we contrasted adverse vascular outcomes among four countries and then assessed practice pattern differences that may have contributed to these outcomes. METHODS: A total of 5065 patients undergoing coronary artery bypass graft surgery were analyzed at 70 international medical centers, and from this pool, 3180 patients from the 4 highest enrolling countries were selected. Fatal and nonfatal postoperative ischemic complications related to the heart, brain, kidney, and gastrointestinal tract were assessed by blinded investigators. RESULTS: In-hospital mortality was 1.5% (9/619) in the United Kingdom, 2.0% (9/444) in Canada, 2.7% (34/1283) in the United States, and 3.8% (32/834) in Germany (P = .03). The rates of the composite outcome (morbidity and mortality) were 12% in the United Kingdom, 16% in Canada, 18% in the United States, and 24% in Germany (P < .001). After adjustment for difference in case-mix (using the European System for Cardiac Operative Risk Evaluation) and practice, country was not an independent predictor for mortality. However, there was an independent effect of country on composite outcome. The practices that were associated with adverse outcomes were the intraoperative use of aprotinin, intraoperative transfusion of fresh-frozen plasma or platelets, lack of use of early postoperative aspirin, and use of postoperative heparin. CONCLUSIONS: Significant between-country differences in perioperative outcome exist and appear to be related to hematologic practices, including administration of antifibrinolytics, fresh-frozen plasma, platelets, heparin, and aspirin. Understanding the mechanisms for these observations and selection of practices associated with improved outcomes may result in significant patient benefit.

Mortality associated with aprotinin during 5 years following coronary artery bypass graft surgery.

CONTEXT: Acute safety concerns have been raised recently regarding certain hemorrhage-sparing medications commonly used in cardiac surgery. However, no comprehensive data exist regarding their associations with long-term mortality. OBJECTIVE: To contrast long-term all-cause mortality in patients undergoing coronary artery bypass graft (CABG) surgery according to use of 2 lysine analog antifibrinolytics (aminocaproic acid and tranexamic acid), the serine protease inhibitor aprotinin, or no antibleeding agent. DESIGN, SETTING, AND PARTICIPANTS: Observational study of mortality conducted between November 11, 1996, and December 7, 2006. Following index hospitalization (4374 patients; 69 medical centers), survival was prospectively assessed at 6 weeks, 6 months, and annually for 5 years after CABG surgery among 3876 patients enrolled in a 62-center international cohort study. The associations of survival with hemorrhage-sparing medications were compared using multivariable analyses including propensity adjustments. MAIN OUTCOME MEASURE: Death (all-cause) over 5 years. RESULTS: Aprotinin treatment (223 deaths among 1072 patients [20.8% 5-year mortality]) was associated with significantly increased mortality compared with control (128 deaths among 1009 patients [12.7%]; covariate adjusted hazard ratio for death, 1.48; 95% confidence interval, 1.19-1.85), whereas neither aminocaproic acid (132 deaths among 834 patients [15.8%]; adjusted hazard ratio for death, 1.03; 95% confidence interval, 0.80-1.33) nor tranexamic acid (65 deaths among 442 patients [14.7%]; adjusted hazard ratio for death, 1.07; 95% confidence interval, 0.80-1.45) was associated with increased mortality. In multivariable logistic regression, either with propensity adjustment or without, aprotinin was independently predictive of 5-year mortality (adjusted odds ratio with propensity adjustment, 1.48; 95% confidence interval, 1.13-1.93; P = .005) among patients with diverse risk profiles, as well as among those surviving their index hospitalization. Neither aminocaproic nor tranexamic acid was associated with increased risk of death. CONCLUSIONS: These findings indicate that in addition to the previously reported acute renal and vascular safety concerns, aprotinin use is associated with an increased risk of long-term mortality following CABG surgery. Use of aprotinin among patients undergoing CABG surgery does not appear prudent because safer and less expensive alternatives (ie, aminocaproic acid and tranexamic acid) are available.

Perturbations in laboratory values after coronary artery bypass graft surgery with cardiopulmonary bypass.

OBJECTIVE: The purpose of this study was to describe the sequential changes in commonly obtained laboratory values after coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB). METHODS: The authors examined laboratory data from 375 patients who underwent uncomplicated CABG with CPB in a multicenter clinical trial of a medication for postoperative pain. Data were obtained preoperatively, at the time of postoperative extubation, and at 4 subsequent intervals ending 14 days after extubation. Data obtained before study drug administration are reported for all patients; thereafter, only data from placebo patients without perioperative complications (n=123) are reported. RESULTS: Mean postoperative coagulation values remained within their reference ranges at the time of extubation. However, platelet counts increased to a peak value well above the reference range by the end of the study. Postoperative white blood cell counts rose above the reference range, mainly because of increased neutrophils. Serum chemistries were also altered; most patients showed a persistent postoperative hyperglycemia. Creatine kinase levels rose to nearly 4 times the upper limit of the reference range in the early postoperative period. Lactate dehydrogenase, serum aspartate aminotransferase, and alanine aminotransferase levels also increased above the reference range. Total protein and albumin values were below the reference range throughout the postoperative period. CONCLUSIONS: Laboratory values for hematology, blood coagulation, and serum chemistry change substantially after uncomplicated CABG with CPB. Recognition of these changes will facilitate the conduct of clinical research and may prevent inappropriate treatment based on abnormal laboratory findings that have no clinical significance.

Thrombopoietin responsiveness reflects the number of doublings undergone by megakaryocyte progenitors.

To assess the variation of thrombopoietin (TPO) responsiveness associated with megakaryocyte (MK) progenitor amplification, TPO dose-response curves were obtained for normal human, single-cell plated CD34(+)CD41(+) cells. The number of MKs per well was determined in situ and expressed as number of doublings (NbD). Dose-response curves of the mean frequency of clones of each size versus log TPO concentration showed highly significant differences in the TPO concentration needed for half-maximum generation of clones of different sizes (TPO(50)): 1.89 +/- 0.51 pg/mL for 1 MK clones; 7.75 +/- 0.81 pg/mL for 2 to 3 MK clones; 38.5 +/- 5.04 pg/mL for 4 to 7 MK clones, and 91.8 +/- 16.0 pg/mL for 8 to 15 MK clones. These results were consistent with a prediction of the generation-age model, because the number of previous doublings in vivo was inversely correlated with the number of residual doublings in vitro. TPO responsiveness decreased in vitro by a factor of 3.5 per doubling, reflecting the recruitment of progressively more ancestral progenitors. In support of this hypothesis, the more mature CD34(+)CD41(+)CD42(+) cell fraction had a lower TPO(50) (P < .001), underwent fewer NbD (P < .001), and expressed a 2.8-fold greater median Mpl receptor density (P < .001) than the CD34(+)CD41(+)CD42(-) fraction. Progenitors that have completed their proliferative program have maximum factor responsiveness and are preferentially induced to terminal differentiation.

Adverse gastrointestinal complications after cardiopulmonary bypass: can outcome be predicted from preoperative risk factors?

UNLABELLED: Adverse gastrointestinal (GI) outcome after cardiac surgery is an infrequent event but is a clinically important health care problem because of associated increased morbidity and mortality. The ability to identify patients at greatest risk before surgery may be helpful in planning appropriate perioperative management strategies. We examined the pre- and intraoperative characteristics of 2417 patients from 24 diverse United States medical centers enrolled in the Multicenter Study of Perioperative Ischemia Study who were undergoing cardiac surgery using cardiopulmonary bypass as predictors for adverse GI outcome. Resource utilization was evaluated for patients with and without adverse GI outcomes. Adverse GI outcomes occurred in 5.5% of patients (133 of 2417), increased in-hospital mortality 6.5-fold, prolonged the mean intensive care unit length of stay by 1 wk, and more than doubled the mean postoperative hospital stay (P < 0.0001). Predictors of adverse GI outcome included decreased left ventricular function, hyperbilirubinemia, thrombocytopenia, prolonged partial thromboplastin time, prior cardiovascular surgery, combined coronary artery bypass graft surgery and intracardiac or proximal aortic surgery, pharmacological cardiovascular support, and intraoperative transfusion. The literature suggests that adverse GI outcome after cardiac surgery is secondary to poor splanchnic perfusion, which many of these risk factors may predict. Therefore, patients deemed to be at risk before surgery may benefit from tightly controlled hemodynamic management and other strategies that optimize perioperative organ perfusion. IMPLICATIONS: We identified the preoperative and intraoperative predictors associated with an increased incidence of postoperative gastrointestinal complications after cardiac surgery using cardiopulmonary bypass. Because these complications are associated with frequent morbidity and mortality, these predictors may be helpful in identifying patients at increased risk so that risk stratification can be modified and perioperative management can be appropriately adjusted.

Estatística para ciências humanas / Statistics for social sciences

Em seus 13 capítulos, divididos em 5 partes, o livro pretende abordar, entre outros pontos, os métodos de descrição e comparação de dados, testes de significância, procedimentos para a obtenção de coeficientes de correlação e uma introdução à análise de regressão. Além disso, possui exemplos ilustrados e exercícios.

Cross-linked hemoglobin converts endotoxically inactive pentaacyl endotoxins into a physiologically active conformation.

The interaction of purified alpha alpha cross-linked hemoglobin (alpha alpha Hb) with a pentaacylated mutant lipopolysaccharide (pLPS) and the corresponding lipid A (pLA) was studied biophysically and the effects correlated with data from biological assays, i.e. cytokine induction (tumor necrosis factor-alpha) in human mononuclear cells and the Limulus amebocyte lysate assay. Fourier transform infrared spectroscopic and Zeta-Sizer experiments indicated an electrostatic as well as a non-electrostatic binding of alpha alpha Hb to the hydrophilic and to the hydrophobic moieties of the endotoxins with an increase of the inclination angle of the pLA backbone, with respect to the membrane surface, from 25 degrees to more than 50 degrees. Small angle synchrotron radiation x-ray diffraction measurements indicated a reorientation of the lipid A aggregates from a multilamellar into a cubic structure as a result of alpha alpha Hb interaction. Thus, in the absence of alpha alpha Hb, the molecular shape of the pentaacyl samples was cylindrical with a moderate inclination of the diglucosamine backbone, whereas, in the presence of the protein, the shape was conical, and the inclination angle was high. The cytokine-inducing capability in human mononuclear cells, negligible for the pure pentaacylated compounds, increased markedly in the presence of alpha alpha Hb in a concentration-dependent manner. In the Limulus assay, the pentaacylated samples were active a priori, and their activity was enhanced following binding to alpha alphaHb, at least at the highest protein concentrations. The data can be understood in the light of a reaggregation of the endotoxins because of alpha alpha Hb binding, with the endotoxin backbones then readily accessible for serum and membrane proteins. By using fluorescence resonance energy transfer spectroscopy, an uptake of the endotoxin-Hb complex into phospholipid liposomes was observed, which provides a basis for cell activation.

THE FINE STRUCTURE OF THE AMEBOCYTE IN THE BLOOD OF LIMULUS POLYPHEMUS. I. MORPHOLOGY OF THE NORMAL CELL.

The fine structure of the amebocyte (hemocyte) in the blood of Limulus polyphemus was reinvestigated regarding its normal state to resolve existing discrepancies in reporting before proceeding to studies of its participation in blood coagulation. The ovoid cell has all the organelles of a well equipped, metabolically active cell; having a nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, and ribosomes. However, no mitotic figures were seen. The most distinguishing characteristic of the cell is the presence of a population of large granules (predominant) and small, asymmetric dense granules (not always present). The large granules originate from the Golgi apparatus and the matrix of the developing granules undergoes a wide range of transitional patterns before assuming the mature dense condition. The transitional stages are interpreted as formative of new granules rather than stages of degranulation as previously reported. The small, asymmetric dense granules are considered to be a type of granule distinct from the larger more predominant type of granule.