Brian S. Bull

Platelet Counting by the RBC/Platelet Ratio Method: A Reference Method

The International Council for Standardization in Haematology (ICSH) and the International Society of Laboratory Hematology (ISLH) recommend the counting of specifically labeled platelets relative to the RBCs with a fluorescence flow cytometer, together with an accurate RBC count determined with a semiautomated, single-channel aperture-impedance counter as a reference method for the enumeration of platelets. Fresh EDTA-anticoagulated venous blood specimens are measured within 4 hours of the draw. The specimen is prediluted (1:20) and the platelets labeled with two monoclonal antibodies specific to a cluster of differentiation common to all platelets. A final 1:1,000 dilution is made and at least 50,000 events with a minimum of 1,000 platelet events are counted with a flow cytometer to determine the RBC/platelet ratio. The platelet count is then calculated from this ratio and the RBC concentration of the original blood specimen.

Platelet Involvement in the Activated Coagulation Time of Heparinized Blood

The activated coagulation time (ACT), commonly used during cardiopulmonary bypass (CPB), is assumed to measure only differences in heparin levels. Studies were undertaken to determine whether platelet activation/inhibition might also influence the test. When either the platelet inhibitor prostacyclin or the platelet activator adenosine diphosphate (ADP) was added to healthy donor blood containing 2 1U of heparin per ml, there was significant prolongation of the ACT (prostacyclin: mean prolongation, 60.6%; ADP: mean prolongation, 52.3%). In blood taken from the extracorporeal circuit of ten CPB cases, the prolongation with the platelet inhibitor carbacyclin, a prostacyclin analogue, was infinite. It is concluded that the ACT as used during CPB must be interpreted as a measure of both platelet procoagulant activity and heparin activity. Furthermore, this dual sensitivity, while fortuitous, is probably advantageous; patients with hyporeactive platelets will automatically receive less heparin.

Evaluation of Tests Used to Monitor Heparin Therapy during Extracorporeal Circulation

Two tests, the activated coagulation time test (ACT), and the quantitative protamine titration test (QPT), were examined in detail as representative of a large number of tests potentially useful in determining dose of heparin needed during cardiopulmonary bypass and the dose of protamine needed for reversal of heparin. The variability introduced by the test methods (ACT 6 percent, QPT 8 per cent) was insignificant comparen (greater than 25 per cent) and the variation in plasma volume (14 per cent). Both of these variables affected not only QPT but also any modification of it that measures the level of heparin by titration with protamine solutions. Tests that measure the effect of heparin on the clotting time, of which the ACT is an example, were unaffected by either population variable when used in conjunction with a simply constructed dose-response curve.

The effect of three commercial coaching courses on Step One USMLE performance

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.

Estrogen effects on plasma volume, arterial blood pressure, interstitial space, plasma proteins, and blood viscosity in sheep

In adult castrated ewes the infusion of 17 beta-estradiol for 3 weeks was associated with a 12% increase in body weight, a 20% increase in whole blood volume (mainly due to a 27% increase of plasma volume), a 13% decrease in mean arterial blood pressure, and a 40% increase in heart rate. The change in plasma volume correlated with the change in estradiol concentration (r = 0.72). Most of the fluid was retained in the interstitial space, as represented by a 6 kg weight gain, 10% of which was in the intravascular compartment. Whole blood and plasma viscosity increased 16% and 21%, respectively, thus reversing some of the blood volume effects toward a hyperdynamic cardiovascular state. We conclude that many of the cardiovascular and hematologic changes with estrogen administration are similar to the changes observed during pregnancy, with the proposed requirement of decrease in mean arterial blood pressure as a condition for blood volume expansion.

Enhancing the safety of intraoperative RBC salvage.

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.

Inhibiting platelet-stimulated blood coagulation by inhibition of mitochondrial respiration

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 compounds 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.

Crenation and cupping of the red cell: A new theoretical approach. Part I. Crenation

Abstract 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 red cell shape from discocyte to hypotonic spherocyte—A mathematical delineation based on a uniform shell hypothesis☆

Abstract 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.

An Analysis of Platelet Activation and Aggregation Produced by Three Classes of Contrast Media

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.