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Viscoelastic testing (VET) by both TEG and ROTEM has demonstrated hypercoagulability early in Covid Associated Coagulopathy (CAC). Additional VET studies demonstrated fibrinolytic shutdown late in a majority of severely ill Covid-19 patients with associated elevation of D-dimer. Elevated d-dimer confirms that coagulation, followed by fibrinolysis, has occurred. These findings imply that, during CAC, three enzymes—thrombin, Factor XIIIa and plasmin—must have acted in sequence. However, limitations in standard VET analyses preclude exploration of the earliest phases of clot induction, as well as clot formation and clot dissolution in flowing blood. Herein we describe a novel method illuminating aspects of this unexplored area. In addition, we created an ex vivo blood flow model in which the interactions of thrombin, Factor XIII and plasmin with fibrinogen can be studied, allowing determination of Soluble Fibrin (SF), the highly unstable form of fibrin that precedes the appearance of a visible clot. This model allows determination of the SF level at which fibrin microclots begin to form and the length of time the resulting microclots persist under normal fibrinolytic attack. In addition, the flow model allows exploration of the late fibrinolytic shutdown that VET has previously demonstrated in CAC through standard methodology.

The primary focus of cardiopulmonary bypass management in Jehovah's Witness patients is the need to conserve blood. A consequence of these strategies inevitably results in hemodilution that is frequently extreme enough to dilute clotting factors and potentially impair coagulation. The purpose of this case report is to demonstrate that a hemodiluted patient requires less heparin to sustain anticoagulation and less protamine to reverse heparin at cardiopulmonary bypass termination. Patient harm may ensue unless the effects of extreme hemodilution are recognized.

Perioperative bleeding is a potentially devastating complication in neurosurgical patients, and plasma fibrinogen concentration has been identified as a potential modifiable risk factor for perioperative bleeding. The aim of this study was to evaluate preconditioning with Crotalus atrox venom (Cv-PC) as potential preventive therapy for reducing perioperative hemorrhage in the rodent model of surgical brain injury (SBI). C. atrox venom contains snake venom metalloproteinases that cleave fibrinogen into fibrin split products without inducing clotting. Separately, fibrinogen split products induce fibrinogen production, thereby elevating plasma fibrinogen levels. Thus, the hypothesis was that preconditioning with C. atrox venom will produce fibrinogen spilt products, thereby upregulating fibrinogen levels, ultimately improving perioperative hemostasis during SBI. We observed that Cv-PC SBI animals had significantly reduced intraoperative hemorrhage and postoperative hematoma volumes compared to those of vehicle preconditioned SBI animals. Cv-PC animals were also found to have higher levels of plasma fibrinogen at the time of surgery, with unchanged prothrombin time. Cv-PC studies with fractions of C. atrox venom suggest that snake venom metalloproteinases are largely responsible for the improved hemostasis by Cv-PC. Our findings indicate that Cv-PC increases plasma fibrinogen levels and may provide a promising therapy for reducing perioperative hemorrhage in elective surgeries.

Objectives Crystalloid administered during cardiopulmonary bypass may significantly dilute clotting factor concentrations, particularly in low blood volume patients. Should the administered fluid (pump prime plus IVs) drop the clotting factor concentrations below approximately 38%, almost all patients will bleed, heparin levels will be overestimated and excessive neutralizing protamine will be administered. This combination can render blood virtually unclottable. This paper describes algorithms that quantify dilution risk and the maximum fluid that can be safely administered. A confirmatory calculation to prevent excessive protamine administration is also described.

An exemplar sample is a small subset of a dataset that forms a characterization of the dataset and can also be used as a list of targets for further inspection. Traditionally exemplars are encountered in cluster analysis techniques such as k-medoids, where each exemplar represents a different cluster. This article extends the concept of exemplar sampling by constructing four criteria for choosing a sample: similarity, distinctness, exhaustiveness, and typicalness. Each criterion illustrates a different way of characterizing the dataset. We describe an adapted version of the CLARANS algorithm which can be used to efficiently draw exemplar samples, and show some examples on test datasets. Exemplar sampling is proposed for use in finite population inference, as an alternative to methods such as stratification or balancing.

Five multichannel hematology analyzers (four different models) were evaluated to determine the ability of analyzers to correctly measure packed cell volume (PCV) values across the clinically relevant range. A sixth analyzer (fifth model) was subsequently included using archival data. Twenty-two different blood samples with PCVs ranging from 0.2 to 0.6 L/L were run in duplicate on each analyzer. Duplicate reference PCV determinations were also done using the recently described ICSH-recommended reference method. Simultaneously, a simpler "surrogate reference" procedure was performed in duplicate. Analyzer values were compared to ICSH reference and to surrogate reference PCV values using Deming regression analysis. Compared to either reference, all analyzers showed a slope of less than 1.0, thereby overestimating the PCV at 0.2 L/L and underestimating the PCV at 0.6 L/L to some extent. This outcome was surprising. Deming regression slopes would be expected to average at 1.0. Instruments whose slopes are significantly less than 1.0 will show decreased sensitivity to both anemic and polycythemic patients. We know of no inherent reason why multichannel analyzers should behave in this fashion.

A majority of packed cell volume (PCV) assays performed in the United States are performed by multichannel hematology analyzers rather than by centrifugation of blood samples in glass capillary tubes. PCV results from both analytical approaches were compared to the recently described ICSH reference method to determine the effect of assay slope on clinical utility. Though not controllable by the end user, the slope of the PCV assay versus the reference was satisfactory when performed in centrifuged glass capillaries. In the small sample of multichannel analyzers surveyed, only two of six machines functioned as well in this respect as did the glass capillaries. The worst of the unsatisfactory multichannel analyzers would have identified correctly only about half of the anemic and the polycythemic patients in the 12,623 males surveyed in the Third National Health and Nutrition Examination Survey (NHANES III). The performance on the females in this U.S. Department of Health and Human Services study would have been similarly poor. It is important that manufacturers of multichannel analyzers attend to the slopes of their PCV assays if the results are to be diagnostically useful.

SummaryThrombosis accounts for a high proportion of disability and death in the West. Although soluble fibrin (SF) assays have been shown to be good predictors of thrombosis, current quantitative assays are too complex or lengthy to provide timely results, while simpler methods are qualitative and lack sensitivity. We here describe a rapid, new, protamine-based whole-blood screening method for SF which is quantitative and suitable for point-of-care use. Citrated whole blood is mixed with reagent under controlled conditions and the time until development of an SF precipitate is measured. Negative samples remain precipitate-free for 300 seconds. Strongly positive samples develop precipitate in as little as 10 seconds. SF times are mathematically converted to arbitrary SF units (SFU). This Rapid-SF test provides a simple and reliable means of detecting the presence of SF, and is well-suited for whole-blood rapid screening in the emergency department, operating room or clinic.

Objective To investigate the relationship of cardiopulmonary bypass-associated plasma dilution with blood product transfusion and postoperative morbidity. Design Retrospective chart review. Setting Single academic medical center. Participants Five hundred forty adults undergoing cardiac surgery between January 4, 2005 and September 19, 2007. Interventions Records were analyzed for demographics, blood volumes (BVs), and fluid balance. Plasma protein concentrations (% of baseline) at the end of bypass were calculated. The lowest and highest quartiles of plasma protein concentration were correlated with blood product administration and postoperative complications. Measurements and Main Results At the end of bypass, calculated plasma protein concentrations ranged from a low of 10% to a high of 111% of baseline. Concentrations below 45% of baseline were associated with increased blood product administration, longer ventilator support, and longer intensive care unit stay. Conclusions Patient morbidity and likelihood of transfusion were associated with calculated plasma protein concentrations below 45% of baseline. Bleeding and administered fluids decrease both hematocrit and plasma proteins. Infusion of washed, salvaged blood or red blood cells raises hematocrit, but further dilutes clotting factors. If this dilution is excessive, coagulopathy may ensue. Patients with the smallest BVs are at greatest risk, but dilution can negatively impact patients with large BVs as well if the fluid used for cardiopulmonary bypass prime and anesthesia management represents a significant fraction of total BV.

Platelets are important mediators of blood coagulation that lack nuclei, but contain mitochondria. Although the presence of mitochondria in platelets has long been recognized, platelet mitochondrial function remains largely unaddressed. On the basis of a small amount of literature that suggests platelet mitochondria are functional, we hypothesized that the inhibition of platelet mitochondria disrupts platelet function and platelet-activated blood coagulation. To test this hypothesis, members of the tetrazole, thiazole, and 1,2,3-triazole families of small molecule heterocycles were screened for the ability to inhibit isolated mitochondrial respiration and coagulation of whole blood. The families of heterocycles screened were chosen on the basis of the ability of the heterocycle family to inhibit a biomimetic model of cytochrome c oxidase (CcO). The strength of mitochondrial inhibition correlates with each compound's ability to deter platelet stimulation and platelet-activated blood clotting. These results suggest that for this class of molecules, inhibition of blood coagulation may be occurring through a mechanism involving mitochondrial inhibition.

We have identified a leukocyte activation syndrome that is occasionally associated with the transfusion of intraoperatively recovered erythrocytes. This syndrome appears to result from intravascular damage caused by leukocytes activated during the erythrocyte salvage process. We hypothesize that this syndrome is part of a larger disease grouping: disseminated intravascular inflammation (DII). DII is the analog of the coagulation disorder disseminated intravascular coagulation. In disseminated intravascular coagulation, the organ damage results from uncontrolled activation of the clotting pathway; in DII the damage is caused by leukocytes that have become activated by direct contact with bacteria or in rare instances--such as erythrocyte salvage--in the absence of bacteria and bacterial products. Recent studies of the hazards associated with intraoperative blood salvage indicate that activation of leukocytes can be achieved by exposure to activated platelets alone. If such activated leukocytes are reinfused along with the washed erythrocytes, widespread organ damage may result. The lung is the organ most severely affected by activated leukocytes. Adult respiratory distress syndrome is one outcome. It is likely that DII is a presently unrecognized pathophysiological process that complicates a variety of primary disease states and increases their lethality.

An independent assessment of the accuracy with which a large manufacturer assigned values to hematologic calibrators was performed. Data were collected from 1,767 hospitals and clinics distributed over North America. Statistical analysis of the mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration of these patient data confirmed the accuracy of the values for hemoglobin, erythrocyte count, hematocrit, and erythrocyte indices for the calibrators manufactured and released during a 9-month period. This approach proved both workable and effective in detecting inaccuracies of less than 1%. Few technologist-users have the time or the equipment to verify independently the accuracy of hematologic calibrators. The manufacturers should perform ongoing, independent assessments of the accuracy of their products. The statistical analysis of patient data provides manufacturers with a suitable method.

A static column of contrast agent or saline in an angiographic catheter will passively exchange with blood during angiography. The authors investigated the time course of this exchange in 5.5- and 7-F polyethylene catheters inclined at various angles. Passive blood exchange occurred 2 cm into the catheters within 7-15 seconds at most catheter tip angles, except for those catheters oriented so that their tips were nearly horizontal. In a separate series of experiments, the effect of contrast agent on platelet function and blood clotting was analyzed. The agent was sufficiently diluted in blood so as to simulate an angiographic procedure. The studies were performed in 60 cylindrical polyethylene containers with both unheparinized and heparinized blood. Use of an ionic contrast agent, more than a nonionic agent, lengthened the time for platelet aggregation (mean increases for ionic vs nonionic agents were 46.4 and 37.1 seconds, for unheparinized and heparinized blood, respectively), platelet adhesion to polyethylene surfaces (mean increases, 46.0 and 64.2 seconds), and platelet-stimulated coagulation (mean increases, 38.5 and 43.9 seconds). Conventional, intermittent flushing with saline or filling the catheter with contrast agent may be insufficient to prevent blood from rapidly back-filling the catheter tips. Contrast agents (ionic more than nonionic) distributed in the patient's blood volume inhibit platelet coating of catheter lumens and/or blood clotting under such circumstances.

A number of associations with post-bypass bleeding have been described in the accompanying paper. Herein we hypothesize that dilution is an underlying cause through a malign series of bypass-associated events. Heparinized blood behaves anomalously when diluted. Clotting times first shorten somewhat, then – as the dilution of whole blood approaches 50% – rapidly lengthen to unclottability. During cardiopulmonary bypass, low blood volume patients are at a significant risk of clotting factor dilution which will always be more severe than the level of whole blood dilution. If severe enough, this dilution may lower plasma clotting factors to a critical level and may result in excess protamine administration, secondary to overestimation of heparin. The presence of un-neutralized protamine combined with critically lowered clotting factors leads to marked coagulopathy.

With the advent of computerized databases, medical data has become easy to accumulate; however, effective use of this data continues to pose significant problems. In other circumstances, smoothing algorithms have been used to uncover non-obvious correlations, trends and relationships in noisy data. We have applied four such algorithms to a large dataset of postoperative blood replacement in cardiopulmonary bypass patients. When applied to this dataset, one of the algorithms proved surprisingly effective. It confirmed several previously observed correlations, and also provided an additional series of counterintuitive and apparently unrelated associations. These associations have been explored in an accompanying paper.

Extracorporeal devices have had limited effectiveness in liver failure due to consequences of inadequate anticoagulation. The purpose of this study was to evaluate an anticoagulation protocol developed for Liver Dialysis Unit (LDU) treatments. Twelve patients underwent 19 LDU treatments for acetaminophen overdose (n = 1), subacute liver failure (n = 1), and refractory encephalopathy in cirrhosis (n = 10). The initial 6 patients (group 1) were treated according to the manufacturer's recommendations. The subsequent 6 patients (group 2) were treated using a formal heparin anticoagulation protocol that included 2000 units in prime solution and 30 units/kg induction, activated clotting time (ACT) measurements, and heparin administered by dose-response curve to maintain 300-second ACT. Protamine reversal was used. Treatments were well tolerated and equally effective in both groups. Adequate (ACT > or = 250 seconds) and therapeutic (ACT 250-350 seconds) anticoagulation was maintained more consistently in group 2 (90.9% vs 50.0%, p < 0.0001; and 77.4% vs 45.8%, p < 0.001). There was less consumption of fibrinogen (12.1% vs 43.3%, p < 0.005) and platelets (13.8% vs 29.9%, p < 0.05) and a trend for less blood product used (8.3 vs 15.8 units/patient, p = 0.13) and fewer complications (16.7% vs 66.7%, p = 0.15) in group 2. Anticoagulation using ACT monitoring, heparin dose-response curves, and a target ACT of 300 seconds improves the safety of LDU treatments.

Usually, cotton laparotomy sponges are discarded when they become blood soaked. During bypass surgery, however, they are often wrung out into the pericardial sac and the contents of the sac are aspirated into the cardiopulmonary bypass (CPB) circuit. After cardiopulmonary bypass, many patients give evidence of mental confusion, excessive bleeding, and systemic inflammatory response syndrome (SIRS). We believe that a possible cause is reinfusion of blood that has been activated by contact with laparotomy sponges and contains fibrin microemboli and thrombin. Thrombin production, soluble fibrin (SF) development, and fibrinogen disappearance were measured, over time, in model systems composed of increasing amounts (0-10-20 microL) of tissue factor (TF) in 4 ml of anticoagulated blood distributed through the interstices of a 49-cm(2) laparotomy sponge. Clotting occurred in all of the sponges. Clotting was accelerated by the presence of TF. Without TF, clotting occurred in an average of 28 min (range 17-39). With 10 and 20 microl TF, clotting occurred in 20 (range 11-27) and 13 (range 10-16) min, respectively. Thrombin at a level of approximately 16 NIHU/ml whole blood was present in several donors for 10-20 min after clotting was complete. If 120 ml of blood was to be wrung from a full-sized laparotomy sponge at this point in time, it could contain as much as 2000 units of thrombin.

Blood accumulating in the pericardial sac is routinely reinfused during cardiopulmonary bypass (CPB) surgery. Such reinfusion has been associated with an increased incidence of serious complications such as coagulopathy, systemic inflammation, and neurologic sequelae. We hypothesize that some of these complications occur because the reinfused blood has been exposed to and activated by laparotomy sponges used to elevate the heart during vein graft emplacement. Such laparotomy sponges expose accumulating pericardial blood to a large, raw, cotton surface with an area approximately five times that of the CPB circuit (excluding the biocompatible oxygenator membrane). Because the reinfused blood has been exposed to this surface, the sponge becomes, in essence, a significant—though inapparent—part of the CPB circuit. Steps should be taken to either eliminate the sponge or to reduce the area of this foreign surface and make it more biocompatible.

Method comparison and calibration are important for clinical hematologists and for manufacturers of new methods and instruments. Automation of the white blood cell (WBC) differential count poses unique problems in this regard because the reference method is a microscopic manual cell count of 200 WBCs. A highly variable reference method can obscure the true relation between the reference and the test methods and reduce sensitivity to miscalibration. A computer model based on published cell distributions and on the statistical properties of proportions was created to enable testing of alternative approaches to calibration. Ordinary linear regression (OLR) and standard Deming regression (SDR) were applied to a comparison of the results obtained with a virtual cell counter (counting 2,000 WBCs) and that obtained with a virtual 200-cell reference method for monocytes (mean population percentage of 7.1%) and lymphocytes (mean population percentage of 29%). Both regression approaches can misrepresent the known mathematical relation between the methods. This problem almost always occurs when OLR is used and the proportion of the cell of interest is low, as in the case of monocytes (7.1% of the total count). OLR comparisons, however, introduce significant error even for cell counts of 29%. SDR performed much better for both cell types.

Background A substantial industry exists to provide formal review courses for Step 1 of the United States Medical Licensing Examination (USMLE). There are limited data on the usefulness of these courses. Aim To determine whether or not student participation in a commercial coaching course improves performance on Step 1 of the USMLE. Methods Scores achieved by 468 students on the National Board of Medical Examiners (NBME) Comprehensive Basic Science Examination (CBSE) were used to predict a score on Step 1 of the USMLE. The NBME is the organisation that prepares and administers the USMLE. Predicted USMLE scores were then regressed against the actual scores achieved by the students on Step 1. The students were divided into 2 groups: those who took a 3−4-week live commercial coaching course and those who studied on their own. Results The regression lines for the 369 students who studied on their own and the 99 students who took a commercial coaching course were statistically indistinguishable. The analysis was powerful enough to have picked up a difference of 1% on average (P = 0·05) or 2 questions out of the 350 constituting Step 1 of the USMLE. Neither the students who performed above average nor those who performed below average on the CBSE improved their performance on Step 1 as a result of the coaching courses. Conclusions Students who take a live, 3−4-week commercial coaching course to improve performance on Step 1 of the USMLE do not achieve higher scores than students who study on their own. Students should strongly consider whether or not a substantial investment in time and money for a commercial coaching course is justified in the light of such meagre returns.

Thrombosis accounts for a high proportion of disability and death in the West. Although soluble fibrin (SF) assays have been shown to be good predictors of thrombosis, current quantitative assays are too complex or lengthy to provide timely results, while simpler methods are qualitative and lack sensitivity. We here describe a rapid, new, protamine-based whole-blood screening method for SF which is quantitative and suitable for point-of-care use. Citrated whole blood is mixed with reagent under controlled conditions and the time until development of an SF precipitate is measured. Negative samples remain precipitate-free for 300 seconds. Strongly positive samples develop precipitate in as little as 10 seconds. SF times are mathematically converted to arbitrary SF units (SFU). This Rapid-SF test provides a simple and reliable means of detecting the presence of SF, and is well-suited for whole-blood rapid screening in the emergency department, operating room or clinic.

Purpose To determine why nonionic, low-osmolality contrast media (LOCM) at high concentrations (>30%) induce almost immediate platelet activation and to explore preventive measures. Materials and Methods Nonionic LOCM, when added to blood, activates platelets, raises the osmolality, and lowers the ionic strength. To clarify the mechanisms involved, platelet activation was studied in sodium chloride–sucrose solutions of varying osmolalities and varying ionic strengths. Several salts and Iloprost were evaluated as potential inhibitors of platelet activation. Results Platelets are activated by both osmolality and ionic strength changes. The salts most inhibitory to platelet activation were magnesium sulfate and sodium citrate. Iloprost lowered platelet activation to control levels at 4.3 ng/mL of nonionic LOCM and completely eliminated it at 8.6 ng/mL. Conclusion Three-quarters of nonionic LOCM–induced platelet activation appeared to be due to increased osmolality—the remainder to the decrease in ionic strength. Magnesium sulfate and Iloprost are potent inhibitors of this type of platelet activation.

Purpose To evaluate the platelet activation and aggregation produced by ionic high-osmolality contrast media (HOCM) and both ionic and nonionic low-osmolality contrast media (LOCM). Materials and Methods After each agent was mixed with heparinized blood, sequential platelet counts were used to monitor platelet aggregation, and flow cytometry was used to monitor both aggregation and activation. Aggregation was measured with CD41a-FITC (specific for glycoprotein IIb–IIIa) and activation was measured with CD62-PE (specific for P-selectin). Results High concentrations of the nonionic LOCM (>31% by volume in whole blood) induced more than 90% platelet activation within 2 minutes of exposure to freshly drawn heparinized whole blood. Aggregation followed immediately after activation and was somewhat reversible. High concentrations of the ionic HOCM (>31%) induced prominent activation, although it occurred at a much slower rate and to a lesser degree than with the nonionic media. There was approximately 70% activation after 45 minutes of exposure. Ionic HOCM inhibited platelet aggregation, however. The ionic LOCM ioxaglate produced minimal or no platelet aggregation or activation. Conclusion There is likely to be an agent-related difference in the risk of platelet activation and aggregation in the catheter lumen and in the immediate environment of the catheter tip, where high concentrations of contrast media are known to exist.

The question of whether to transfuse at all comes before the question of whether to use autologous versus homologous blood. Until recently, a hemoglobin level of 10 g/dL was considered the lowest acceptable level for a patient undergoing surgery. {1} There is now good evidence that this “action level” is too high. Indeed, oxygen delivery to the tissues is enhanced by a modest degree of anemia provided the patient can maintain an increased cardiac output. This is because the viscosity of whole blood is largely dependent upon the volume of red cells that it contains. High hematocrit blood can deliver increased oxygen to the tissues only if it can be moved swiftly through the microcirculation; and the high viscosity of such blood guarantees a prolonged circulation time. Why then does Mother Nature equip us with a hematocrit of 45 when there are so many physiological advantages that accrue when the hematocrit drops to 25? The answer is that most of us are upright and exercising large muscle groups most of the time. We are not resting quietly, lying in bed following major surgery. During vigorous physical activity a reserve capacity for oxygen transport is very helpful. Some athletes have received a one or two unit transfusion of their own blood just before competition for this very reason. Other athletes practice their sport at high altitude to achieve the same result.

Diatrizoate has been widely used clinically for many years and has been effective in inhibiting clot formation in catheters and syringes. The authors attempted to determine the amount of heparin required to raise the anticoagulant potency of ioversol, iohexol, or iopamidol to that of diatrizoate. The anticoagulant potency of ioxaglate was also examined. One hundred eighty glass syringes containing unmodified diatrizoate, unmodified ioxaglate, or a nonionic contrast agent mixed with incremental amounts of heparin were contaminated with venous blood from 10 healthy volunteers. The syringes were then repeatedly disturbed to simulate the frequent handling that occurs during an angiographic procedure. The weights of the clots in the syringes at 30 minutes were plotted against heparin levels and indicated that, for the average sample studied, the "diatrizoate-equivalent" heparin level for ioversol, iohexol, or iopamidol was less than 1.5 U (USP) of heparin per milliliter. An additional margin of anticoagulation is probably desirable. The 97.5 percentile of the distribution of the diatrizoate equivalents in the population was estimated to require a heparin dose of 3.5 U/mL.

This document is intended to assist towards the WHO objective that external quality assessment (EQA) schemes be established at national and/or regional levels world-wide. Quality assurance is defined as all steps taken by the director of a laboratory to ensure reliability of laboratory results and to increase accuracy, reproducibility and between-laboratory comparability. This includes the use of internal quality control procedures and participation in external quality assessment. Internal quality control provides the means for evaluation of analytic test results at the time of testing in order to decide whether they are reliable enough to be released to the requesting clinicians. EQA, on the other hand, refers to a system of retrospective and objective comparison of results from different laboratories by means of proficiency testing (PT) organised by an external agency. The main purpose is to establish between-laboratory and between-method (including between-instrument) comparability, and agreement with a reference standard where one exists. Internal quality control and EQA complement each other and must never be considered as alternatives.

To the Editor.— The advice that the National Blood Resource Education Program Expert Panel 1 has offered relative to the hazards of intraoperatively salvaged red blood cells is excellent. We wish to draw the attention of JAMA readers to an additional hazard. Recently, we have shown that when human blood diluted in a 1:2 ratio with saline is washed in a polycarbonate centrifuge bowl, a deposit of activated degranulated platelets forms against the inner plastic surface. 2 This layer has potent thromboplastic and leukoattractant properties. 3 In an animal model, 4 the reinfusion of washed red blood cells prepared in bowls coated with such platelet layers caused massive hemorrhage and increased vascular permeability. The Expert Panel suggests that "at the conclusion of cardiopulmonary bypass, all blood remaining in the oxygenator and bypass circuits should be salvaged and, if needed, infused." We agree, but wish to emphasize that this is one

Red cells arrested by fine fibers and subjected to drag forces in a moving stream of fluid can be stressed beyond the point of rupture. The technique, which is basically an in vitro analogue of the microangiopathic hemolytic state, thus permits a study of red cell behavior over the entire range of stress that the membrane can withstand. Measurements made by this approach show an exponential relationship between the velocity of fluid flow and the resultant membrane deformation. Such exponential membrane deformation is consistent with the morphology of red cell fragments and appears to explain their mechanism of formation.

Mathematical modeling was used to test two assumptions regarding red cell shape. The assumptions are that the elastic moduli of the red cell membrane are uniformly distributed throughout the membrane shell and that the biconcave shape results primarily from minimization of strain energy when this uniform shell is partially deflated. This strain energy is assumed to arise from bending (involving surface area strain) and shear (involving superficial tensile strain). The mathematical delineation demonstrated that it was impossible to produce a smooth symmetrical biconcave shape by minimizing shear energy in a partially deflated shell. It was possible to generate a symmetrical biconcave shape by minimizing bending energy; the shape was, however, thicker along the axis of symmetry than the measured red cell shape. A combination of bending and shear in the ratio of 6 to 1 produced a shape which matched the measured shape of a red cell to better than 1%, a deviation of the order of the thickness of the red cell membrane. The success of the mathematical model provides very strong evidence for the uniform shell-minimum bending energy hypothesis as the primary determinant of the discoid red cell shape.

Crenation can be thought of as a surface instability caused by intrinsic precurvature of the membrane. Mathematical modeling, on the presupposition that the red blood cell is a thin shell consisting of a connected (coupled) bilayer having uniformly distributed elastic properties shows that crenation can be initiated by negative precurvature, that is, intrinsic curvature having its concavity directed towards the outside of the cell. This is contrary to the currently accepted view which attributes the effect to positive precurvature of an unconnected bilayer. Crenation and the biconcave shape can coexist in the red cell. This suggests that the bilayer must be connected even when the cell is crenated because the biconcave shape could not otherwise be maintained. The progressive development of crenation to more advanced stages, such as the echinocyte type III and the spheroechinocyte can be accounted for if the outer layer of the membrane is stressed beyond the range where strain is proportional to stress. This is consistent with the extremely small radius of curvature at the tips of the crenations. Certain small variations in the uncrenated biconcave shape of the red cell can be interpreted mathematically as due either to negative intrinsic curvature or to shear resistance. Since, however, a small amount of negative precurvature has been shown to be capable of inducing crenation, it is unlikely to be the cause of the variations in the biconcave shape. These must therefore be due to shear resistance. In the light of this new approach, membrane molecular models based on the assumption that crenation is due to positive precurvature need reconsideration.

The extent to which semiturgid red cells could enter the tip of a glass micropipette of approximately 1-microgram internal diameter was used to measure the area expansion of freshly drawn erythrocytes. The pipette was mounted in a gas-tight glass observation chamber so that one single cell at a time could be observed while it was being perfused, either with buffer alone or with buffer containing an anesthetic at physiologic concentration. The effect of the drugs was studied at room temperature at doses of 1, 2, and 4 times ED50 for tadpoles. The drugs studied were: halothane, methoxyflurane, diethyl ether, fluroxene, isoflurane, hexanol, heptanol, octanol, decanol, dodecanol, tridecanol, tetradecanol, hexadecanol, and benzyl alcohol. The measured expansion closely approximated the expansion expected by incorporation of the molecules. Tetradecanol and hexadecanol are not anesthetic to tadpoles, but they did expand the membrane. Therefore, expansion may not be related to anesthesia.

A physical macromodel of the erythrocyte membrane was constructed to investigate the effect of intrinsic membrane precurvature on a unique non-isotropic, composite material that dissipated shear energy but stored bending energy. The intrinsic precurvature of the material could be modified from strong positive precurvature through neutral to strong negative precurvature. A spherical shell constructed of the negatively precurved variant of this non-isotropic material spontaneously assumed the characteristic shape of the echinocyte I.

Devices for intraoperative blood salvage remove plasma and, in theory, all of the cellular elements of blood except for rbcs. We have previously shown that complete white cell and platelet removal does not always occur and that the retained platelet-leukocyte deposit is potentially harmful (2). In this study we investigated the hydraulic conditions in the centrifuge bowl that allow activated platelets and leukocytes to adhere, the histology of the resulting cellular deposit, and the effects of reinfusing a saline extract of the deposit. Earlier work had suggested that the addition of calcium, of partially clotted blood, and of excessive saline should be avoided during intraoperative rbc salvage (2). The present observations explain, in part, why such measures would be expected to be beneficial.

Red cells moving in a stream of fluid can be arrested by means of a network of fibrin threads. Those cells which fold over a fibrin thread present to view a flat strap-like portion of membrane in which the strain can be easily observed and accurately measured both optically and by scanning electron microscopy. Preliminary results obtained by this method show elastic strains of nearly 300% and indicate that the stress required to produce these large strains is an order of magnitude greater than that reported by other methods.