Clinical use of blood,blood components and blood products.

The goal of modern transfusion therapy is to provide appropriate replacement therapy with blood components as opposed to whole blood for patients with specific hematologic deficiencies. A prerequisite of component therapy is, therefore, correct identification of the deficiency. Appropriate use of components avoids many of the hazards associated with the use of whole blood, and at the same time makes maximal use of this valuable resource. Blood components separated from whole blood soon after collection and appropriately stored can, in combination, provide all the factors present in fresh whole blood. Red cell concentrates prepared from multiple packs have a hematocrit of approximately 70%. They may be stored for up to 3 weeks at 4 degrees C and are recommended for most situations requiring red cell transfusions. Platelet concentrates, which can be stored for up to 72 hours at 22 degrees C, may be used for thrombocytopenic patients. Fresh frozen plasma, stored plasma, cryoprecipitated factor VIII, factor VIII concentrate and factor IX complex concentrate are available for the proper treatment of patients with hemorrhagic disorders due to coagulation factor deficiencies. Similarly, albumin and immune serum globulin are available for their oncotic and antibody properties respectively. Thus, the availability and appropriate use of the various blood products allows not only optimal transfusion therapy for each patient, but also fuller utilization of national blood resources.     

Proteomics and transfusion medicine: future perspectives

Limited number of important discoveries have greatly contributed to the progresses achieved in the blood transfusion; ABO histo-blood groups, citrate as anticoagulant, fractionation of plasma proteins, plastic bags and apheresis machines. Three major types of blood products are transfused to patients: red cell concentrates, platelet concentrates and fresh frozen plasma. Several parameters of these products change during storage process and they have been well studied over the years. However, several aspects have completely been ignored; in particular those related to peptide and protein changes. This review presents what has been done using proteomic tools and the potentials of proteomics for transfusion medicine.     

Effects of simulated microgravity (HDT) on blood fluidity.

Exposures to microgravity and head-down tilt (HDT) produce similar changes in body fluid. This causes an increase in hematocrit that significantly affects hemorheological values. Lack of physical stimulation under bed rest conditions and the relative immobility of the crew during spaceflight also affects the blood fluidity. A group of six healthy male subjects participated as volunteers, and blood samples were collected 10 days before, on day 2 and day 9, and 2 days after the HDT phase. Blood rheology was quantified by plasma viscometry, red cell aggregability, and red cell deformability. A reduced red cell deformability, an indication of the diminished quality of the red blood cells, was measured under HDT conditions that finally led to the so-called "space flight anemia." Enhanced red cell membrane fragility induced by diminished physical activity and an increase in hemoglobin concentration are responsible for this effect. Plasma viscosity is reduced as a result of diminished plasma proteins. However, despite the reduction in plasma proteins, including fibrinogen, alpha 2-macroglobulin, and immunoglobulin M, red cell aggregation was enhanced, principally because of the increase in hematocrit. Our results of hemorheological alterations under HDT conditions may help to elucidate the formerly documented hematologic changes during spaceflight.     

Magnetic resonance microscopy determined velocity and hematocrit distributions in a Couette viscometer

Magnetic resonance microscopy is used to non-invasively measure the radial velocity distribution in Couette flow of erythrocyte suspensions of varying aggregation behavior at a nominal shear rate of 2.20 s(-1) in a 1 mm gap. Suspensions of red blood cells in albumin-saline, plasma and 1.48% Dextran added plasma at average hematocrits near 0.40 are studied, providing a range of aggregation ability. The spatial distribution of the red blood cell volume fraction, hematocrit, is calculated from the velocity distribution. The hematocrit profiles provide direct measure of the thickness of the aggregation and shear rate dependent red blood cell depletion at the Couette surfaces. At the nominal shear rate studied hematocrit distributions for the red blood cells in plasma show a depletion zone near the inner Couette wall but not the outer wall. The red blood cells in plasma with Dextran show cell depletion regions of approximately 100 mum at both the inner and outer Couette surfaces, with greater depletion at the inner wall, but approach the normal blood hematocrit distribution with a doubling of shear rate due to decreased aggregation. The material response of the blood is spatially dependent with the shear rate and the hematocrit distribution non-uniform across the gap.     

Detection of red cell aggregation by low shear rate viscometry in whole blood with elevated plasma viscosity

The viscosity of whole blood measured at low shear rates is determined partly by shear resistance of the red cell aggregates present, stronger aggregation increasing the viscosity in the absence of other changes. Effects of cell deformability can confound interpretation and comparison in terms of aggregation, however, particularly when the plasma viscosity is high. We illustrate the problem with a comparison of hematocrit-adjusted blood from type 1 diabetes patients and controls in which it is found the apparent and relative viscosities at a true shear rate of 0.20 s-1 are lower in the patient samples than age matched controls, in spite of reports that aggregation is increased in such populations. Because the plasma viscosities of the patients were higher on average than controls, we performed a series of experiments to examine the effect of plasma protein concentration and viscosity on normal blood viscosity. Dilution or concentration by ultrafiltration of autologous plasma and viscosity measurements at low shear on constant hematocrit red cell suspensions showed (a) suspension viscosity at 0.25 and 3 s-1 increased monotonically with plasma protein concentration and viscosity but (b) the relative viscosity increased, in concert with the microscopic aggregation grade, up to a viscosity of approximately 1.25 mPa-s but above this the value the relative viscosity no longer increased as the degree of aggregation increased in concentrated plasmas. It is suggested that in order to reduce cell deformation effects in hyperviscous pathological plasmas, patient and control plasmas should be systematically diluted before hematocrit is adjusted and rheological measurements are made. True shear rates should be calculated. Comparison of relative viscosities at low true shear rates appears to allow the effects of red cell aggregation to be distinguished by variable shear rate viscometry in clinical blood samples.     

Preparation of leukocyte-poor platelet concentrates from buffy coats by a new type of quadruple bag

A new quadruple blood bag system for collection of platelet concentrates is described. The principle modification consists of a smaller (150 ml) buffy coat bag in conjunction with SAG-mannitol for red cell preservation. Platelet yield and function were comparable to conventional techniques, leukocyte contamination was lower (1.7 +/- 1.3 x 10(7) per unit). The system is recommended for specialized institutions with a high demand of blood components.     

Analysis of the shelf life and transfusion life of whole blood and erythrocyte concentrates

The shelf life of 60,000 units of whole blood and red cell concentrates in the blood center were analysed before delivering to hospitals as well as the transfusion age of 18,000 units in relation to the stock-size and the outdated units. The mean transfusion age of whole blood units and red blood cell concentrates amounted to 11 to 12 days and 16 to 17 days, respectively. The shelf life of blood transfused within 24 hours after collection was 1.6% and 3.8% transfused within 48 hours according to the total number of whole blood and red blood cell concentrates. Washed red cell concentrates were prepared from buffy coat-free resuspended red cell concentrates on an average at the 7th day of storage. A high stock-size of about 1,000 units rather than of 600 units in the blood center increased the shelf life and also the number of outdated units from less than 0.5% up to more than 3%.     

Effect of purified recombinant human erythropoietin on anemia in rats with experimental renal failure induced by five-sixth nephrectomy

Recombinant human erythropoietin (rHuEPO) was purified from the conditioned media of Chinese hamster ovary cells with a transfected human erythropoietin gene. We investigated the effects of the rHuEPO in rats with renal anemia induced by partial nephrectomy. Five-sixth nephrectomy resulted in renal failure with anemia. Twenty-five days after the operation plasma urea nitrogen was increased about 2.5 times, and the red blood cell count, hematocrit, and hemoglobin concentration fell to 85% of normal. The reticulocyte count and plasma erythropoietin level did not change such as they do in patients with anemia due to chronic renal failure. Both total red blood cell volume and the plasma iron turnover rate were depressed in five-sixth nephrectomized rats compared with normal rats.The five-sixth nephrectomized rats were injected with rHuEPO (60 IU/kg) intravenously every second day for a total of six injections. After three injections of rHuEPO, circulation volume of total red blood cells was increased from 9.9 ml to 14.6 ml, and the plasma iron turnover rate was increased from 1.03 mg/kg/day to 2.12 mg/kg/day, and the reticulocyte count was also increased. After six injections, a marked increase of the red blood cell count, hematocrit, and hemoglobin concentration were observed. Plasma urea nitrogen and the creatinine levels as indications for renal function did not change after rHuEPO administration in both normal and five-sixth nephrectomized rats.In conclusion rHuEPO has a potent erythropoietic action and it is possible to cure the anemia caused by renal failure.     

Effect of nonaxisymmetric hematocrit distribution on non-Newtonian blood flow in small tubes

Hematocrit distribution and red blood cell aggregation are the major determinants of blood flow in narrow tubes at low flow rates. It has been observed experimentally that in microcirculation the hematocrit distribution is not uniform. This nonuniformity may result from plasma skimming and cell screening effects and also from red cell sedimentation. The goal of the present study is to understand the effect of nonaxisymmetric hematocrit distribution on the flow of human and cat blood in small blood vessels of the microcirculation. Blood vessels are modeled as circular cylindrical tubes. Human blood is described by Quemada's rheological model, in which local viscosity is a function of both the local hematocrit and a structural parameter that is related to the size of red blood cell aggregates. Cat blood is described by Casson's model. Eccentric hematocrit distribution is considered such that the axis of the cylindrical core region of red cell suspension is parallel to the axis of the blood vessel but not coincident. The problem is solved numerically by using finite element method. The calculations predict nonaxisymmetric distribution of velocity and shear stress in the blood vessel and the increase of apparent viscosity with increasing eccentricity of the core.     

Effect of dilution on sedimentational separation of bacteria from blood

Bacteria must be separated from septic whole blood in preparation for rapid antibiotic susceptibility tests. This work improves upon past work isolating bacteria from whole blood by exploring an important experimental factor: Whole blood dilution. Herein, we use the continuity equation to model red blood cell sedimentation and show that overall spinning time decreases as the blood is diluted. We found that the bacteria can also be captured more efficiently from diluted blood, up to approximately 68 ± 8% recovery (95% confidence interval). However, diluting blood both requires and creates extra fluid that end users must handle; an optimal dilution, which maximizes bacteria recovery and minimizes waste, was found to scale with the square root of the whole blood hematocrit. This work also explores a hypothesis that plasma backflow, which occurs as red cells move radially outward, causes bacterial enrichment in the supernatant plasma with an impact proportional to the plasma backflow velocity. Bacteria experiments carried out with diluted blood demonstrate such bacterial enrichment, but not in the hypothesized manner as enrichment occurred only in undiluted blood samples at physiological hematocrit.     

Splenic storage volume in the unanesthetized resting beagle

Determination of the dog's splenic storage volume without surgical procedures requires measurement of both circulating and total red cell volumes. The estimation of circulating red cell volume by radioisotope techniques is impeded by the rapid uptake of tagged cells in the spleen. The circulating cell volume might be calculated from plasma volume and large vessel hematocrit, provided that the latter is corrected for the unequal distribution of red cells in the circulatory system. However, the correction factor can only be estimated in the splenectomized dog. We describe here a method to determine the factor in the intact dog, "physiologically splenectomized" by the severe exercise. The values obtained by this method slightly exceed those in the resting dog, as shown by studies in splenectomized exercising beagles in which splenic function was simulated by infusion of packed cells. The method was tested in beagles exercised by swimming and treadmill running and it was concluded that in the unanesthetized resting beagle about one-third of all erythrocytes is stored in the spleen. Labeled cells are equilibrated with about one-half of the splenic storage volume within 10 min after their injection. During maximal exertion the mean increase in large vessel hematocrit was 38.6 +/- 3.3%, the mean decrease in plasma volume 13.6 +/- 1.7% and the mean increase in plasma osmolarity 2.8 +/- 0.9% (percentages of control values).     

A new method for measuring the yield stress in thin layers of sedimenting blood.

A new method is presented to describe the low shear rate behavior of blood. We observed the response of a thin layer of sedimenting blood to a graded shear stress in a wedge-shaped chamber. The method allows quantitation of the degree of phase separation between red cells and plasma, and extracts the yield stress of the cell phase as a function of hematocrit. Our studies showed that the behavior of normal human blood underwent a transition from a solid-like gel to a Casson fluid. This transition began at the Casson predicted yield stress. The viscoelastic properties of blood were examined at shear stresses below the yield stress. The measured Young's elastic moduli were in good agreement with published data. The yield stress of blood showed a linear dependence on hematocrit up to 60%, and increased more rapidly at higher hematocrit.     

Preservation of erythrocytes by freezing in liquid nitrogen. Use of an I.B.M. blood regenerator]

The freezing of blood permits preservation of red cells over long periods of time, several months or years. Leucocyte and platelet contamination of red cell concentrates to be frozen is negligible. The amount of the various red cell metabolites (2.3 D.P.G., A.T.P., etc.) is maintained. Washing of thawed red cells removes the remaining plasma proteins and cell residues. The freezing method employed is that of Row et al. The protector used is 28% glycerol added in equal amounts to red cell concentrate to be frozen. The blood bag is kept in liquid nitrogen at -- 196 degrees C. Thawing takes place in a water bath at 45 degrees C. Wash solution is the IBM Blood regenerator. The solution used for removing glycerol is hypertonic natrium chloride. The following parameters have been investigated: --hemoglobin level; --osmotic fragility; --the amount of 2.3 D.P.G.; --residual glycerol after thawing; and clearance of leucocytes and platelets following each step of the protocol. Preliminary data regarding these features and therapeutic efficiency of processed blood are satisfactory.     

Regional lung hematocrit in humans using positron emission tomography

Regional lung hematocrit ratio (R) was measured in five normal subjects and five patients (2 with pneumonia, 2 with nephrotic syndrome with anemia, and 1 with pancreatitis) using positron emission tomography, a red cell marker 11CO, and a plasma marker [methyl-11C]albumin). The measurements were made in a transaxial thoracic section at midheart level with the subject in supine posture and with a spatial resolution of 1.7 cm. The normal regional hematocrit ratio (means +/- SE) calculated for the lung was 0.90 +/- 0.014, 0.94 +/- 0.023 for the thoracic wall, and 1.00 +/- 0.003 for the heart chambers. The regional lung hematocrit ratio in the patients ranged between 0.81 and 0.86. No correlation was found among the regional lung hematocrit ratio and regional blood volume, lung extravascular density, and the peripheral hematocrit (obtained from venous blood samples). To the extent that 70% of the pulmonary blood in the field of view is in larger vessels with normal hematocrit, the hematocrit in the capillary bed is approximately two-thirds that of the peripheral venous value. Blood volume measurements on the basis of single vascular tracers need to take account of these results.     

Hemorheology and oxygen transport in vertebrates. A role in thermoregulation?

We studied the effect of temperature on blood rheology in three vertebrate species with different thermoregulation and erythrocyte characteristics. Higher fibrinogen proportion to total plasma protein was found in turtles (20%) than in pigeons (5.6%) and rats (4.2%). Higher plasma viscosity at room temperature than at homeotherm body temperature was observed in rats (1.69 mPa x s at 20 degrees C vs. 1.33 mPa x s at 37 degrees C), pigeons (3.40 mPa x s at 20 degrees C vs. 1.75 mPa x s at 40 degrees C), and turtles (1.74 mPa x s at 20 degrees C vs. 1.32 mPa x s at 37 degrees C). This fact allow us to hypothesize that thermal changes in protein structure may account for an adjustment of the plasma viscosity. Blood viscosity was dependent on shear rate, temperature and hematocrit in the three species. A different behaviour in apparent and relative viscosities between rat and pigeon at environmental temperature was found. Moreover, the blood oxygen transport capacity seems more affected by a reduction of temperature in rats than in pigeons. Both findings indicate a greater influence of temperature on mammalian erythrocyte than on nucleated red cells, possibly as a consequence of differences in thermal sensitivity and mechanical stability between them. A comparison between the three species revealed that apparent blood viscosity measured at homeotherm physiological temperature was linearly related to the hematocrit level of each species. However, when measured at environmental temperature, rat blood showed a higher apparent viscosity than those found in species with non-nucleated red cells, thus indicating a higher impact of temperature decrease on blood viscosity in mammals. This suggest that regional hypothermia caused by cold exposure may affect mammalian blood rheological behaviour in a higher extent than in other vertebrate species having nucleated red cells and, consequently, influencing circulatory function and oxygen transport.     

In?Vitro Measurement of Particle Margination in the Microchannel Flow: Effect of Varying Hematocrit

It has long been known that platelets undergo margination when flowing in blood vessels, such that there is an excess concentration near the vessel wall. We conduct experiments and three-dimensional boundary integral simulations of platelet-sized spherical particles in a microchannel 30 μm in height to measure the particle-concentration distribution profile and observe its margination at 10%, 20%, and 30% red blood cell hematocrit. The experiments involved adding 2.15-μm-diameter spheres into a solution of red blood cells, plasma, and water and flowing this mixture down a microfluidic channel at a wall shear rate of 1000 s⁻¹. Fluorescence imaging was used to determine the height and velocity of particles in the channel. Experimental results indicate that margination has largely occurred before particles travel 1 cm downstream and that hematocrit plays a role in the degree of margination. With simulations, we can track the trajectories of the particles with higher resolution. These simulations also confirm that margination from an initially uniform distribution of spheres and red blood cells occurs over the length scale of O(1 cm), with higher hematocrit showing faster margination. The results presented here, from both experiments and 3D simulations, may help explain the relationship between bleeding time in vessel trauma and red blood cell hematocrit as platelets move to a vessel wall.     

The mean red cell volume in long distance runners

Red cell indices were determined in 6 well trained runners before and after a 100 km race, and Coulter Counter (CC) determinations compared with calculated values derived from centrifuged hematocrit (ctrf), red cell count (CC) and hemoglobin measurements. The following changes were observed immediately after the race, as compared to values 3 days before: MCV(ctrf) decreased by 4.9% (p less than 0.001), MCV(CC) increased by 1.9% (p less than 0.05), MCHC(ctrf) increased by 4% and MCHC(CC) decreased by 3%. The increase in MCV(CC) suggests that intraerythrocyte osmolality was increased, this probably leading to swelling of the cells induced by a shift of water from the diluting Coulter Counter solution into the red cells prior to the MCV measurement. The decrease in MCV(ctrf) immediately after the race was not correlated with the increase in plasma osmolality. This suggests that plasma osmolality alone was not the key factor for regulation of red cell volume. The changes in MCV(ctrf), which contributed to a surprising stability of the hematocrit value and plasma volume, might represent a physiological principle for the maintenance of a favourable blood viscosity.     

Blood flow in the capillary bed

From arteries to veins, the blood has to go through the ‘capillary’ blood vessels. These blood vessels are so small that often their diameter is smaller than that of the red blood cells. Intimate interactions occur, therefore, between the blood cells and the blood vessels.

A general survey of recent works on capillary blood flow is given in this article. Some details are presented for two problems: the problem of deformation of the flexible red blood cells, their motion in the capillary blood vessels, and the pressure drop due to the red cell blood vessel interaction; and the problem of the flow of plasma ‘bolus’ between neighboring red cells.

The solution supplies many details about the microcirculation phenomenon. Taken together, a method is offered for the calculation of pressure drop in the capillary as a function of various physical parameters: the red cell volume per unit blood volume, (hematocrit), the ratio of the cell diameter to the blood vessel diameter, the ratio of the length of the blood vessel to its length, the volume of individual red cells, and a parameter relating the cell membrane elasticity, plasma viscosity and the cell velocity.     

Hematocrit reduction in bifurcations due to plasma skimming

Expressions and numerical values for hematocrit reduction are calculated as blood flows from a cylindrical feeding tube into a cylindrical capillary at a right-angle branch. Blood is considered to consist of two Newtonian fluids, plasma and red cell suspension, which have equal densities but different viscosities. The concentration profile of the red cells is concluded to depend on the size of the feeding tube. An estimate for the thickness of the plasma layer adjacent to the wall is obtained.     

Deduction of pulmonary microvascular hematocrit from indicator dilution curves

We have developed a new model describing the relationship between plasma and red cell tracers flowing through the lung. The model is the result of an analysis of the transport of radiolabeled plasma albumin between two flowing phases and shows that differences between red cell and plasma tracer curves are related to microvascular hematocrit. The model was tested in an isolated, blood-perfused dog lung preparation in which we injected⁵¹Cr-labeled red cells and¹²⁵I-labeled plasma albumin into the pulmonary artery. From the tracer concentration-time curves at the venous outflow, we calculatedh _r, the ratio of microvascular hematocrit to large-vessel hematocrit. In 18 baseline experiments,h _r=0.92±0.01 (mn±sem) at a blood flow rate of 10.7±0.3 ml s⁻¹. We determined the effects of (a) glass bead embolization, (b) alloxan, and (c) lobe ligation onh _r. Embolization attenuated the separation between plasma and red cells (increasedh _r), probably as a consequence of passive vasodilation. Alloxan enhanced separation of plasma and red cells (decreasedh _r), possibly as a result of arteriolar vasoconstriction. Ligation of a fraction of the perfused tissue at constant flow did not cause significant change inh _r in the remaining perfused tissue. The model assumes that large-vessel transit times are uniform and that all dispersion occurs in the microvasculature. A theoretical analysis apportioning dispersion between large and small vessels disclosed that the error associated with these assumptions is likely to be less than 15% of the measuredh _r. We conclude from this study that the microvascular hematocrit model describes experimental plasma and red cell curves. The results imply thath _r can be readily deduced from tagged red cells and plasma and can be accounted for in calculating permeability-surface area in diffusing tracer experiments.