Influence of feeding hematocrit and perfusion pressure on hematocrit reduction (Fåhraeus effect) in an artificial microvascular network.

OBJECTIVE: Hct in narrow vessels is reduced due to concentration of fast-flowing RBCs in the center, and of slower flowing plasma along the wall of the vessel, which in combination with plasma skimming at bifurcations leads to the striking heterogeneity of local Hct in branching capillary networks known as the network Fåhraeus effect. We analyzed the influence of feeding Hct and perfusion pressure on the Fåhraeus effect in an AMVN. METHODS: RBC suspensions in plasma with Hcts between 20% and 70% were perfused at pressures of 5-60 cm H2 O through the AMVN. A microscope and high-speed camera were used to measure RBC velocity and Hct in microchannels of height of 5 µm and widths of 5-19 µm. RESULTS: Channel Hcts were reduced compared with Hctfeeding in 5 and 7 µm microchannels, but not in larger microchannels. The magnitude of Hct reduction increased with decreasing Hctfeeding and decreasing ΔP (flow velocity), showing an about sevenfold higher effect for 40% Hctfeeding and low pressure/flow velocity than for 60% Hctfeeding and high pressure/flow velocity. CONCLUSIONS: The magnitude of the network Fåhraeus effect in an AMVN is inversely related to Hctfeeding and ΔP.

Influence of red blood cell aggregation on perfusion of an artificial microvascular network.

OBJECTIVE: RBCs suspended in plasma form multicellular aggregates under low-flow conditions, increasing apparent blood viscosity at low shear rates. It has previously been unclear, however, if RBC aggregation affects microvascular perfusion. Here, we analyzed the impact of RBC aggregation on perfusion and 'capillary' hematocrit in an AMVN at driving pressures ranging from 5 to 60 cm H2 O to determine if aggregation could improve tissue oxygenation. METHODS: RBCs were suspended at 30% hematocrit in either 46.5 g/L dextran 40 (D40, non-aggregating medium) or 35 g/L dextran 70 (D70, aggregating medium) solutions with equal viscosity. RESULTS: Aggregation was readily observed in the AMVN for RBCs suspended in D70 at driving pressures ≤40 cm H2 O. The AMVN perfusion rate was the same for RBCs suspended in aggregating and non-aggregating medium, at both 'venular' and 'capillary' level. Estimated 'capillary' hematocrit was higher for D70 suspensions than for D40 suspensions at intermediate driving pressures (5-40 cm H2 O). CONCLUSIONS: We conclude that although RBC aggregation did not affect the AMVN perfusion rate independently of the driving pressure, a higher hematocrit in the 'capillaries' of the network for D70 suspensions suggested a better oxygen transport capacity in the presence of RBC aggregation.

Optimal hematocrit in an artificial microvascular network.

BACKGROUND: Higher hematocrit increases the oxygen-carrying capacity of blood but also increases blood viscosity, thus decreasing blood flow through the microvasculature and reducing the oxygen delivery to tissues. Therefore, an optimal value of hematocrit that maximizes tissue oxygenation must exist. STUDY DESIGN AND METHODS: We used viscometry and an artificial microvascular network device to determine the optimal hematocrit in vitro. Suspensions of fresh red blood cells (RBCs) in plasma, normal saline, or a protein-containing buffer and suspensions of stored red blood cells (at Week 6 of standard hypothermic storage) in plasma with hematocrits ranging from 10 to 80% were evaluated. RESULTS: For viscometry, optimal hematocrits were 10, 25.2, 31.9, 37.1, and 37.5% for fresh RBCs in plasma at shear rates of 3.2 or less, 11.0, 27.7, 69.5, and 128.5 inverse seconds. For the artificial microvascular network, optimal hematocrits were 51.1, 55.6, 59.2, 60.9, 62.3, and 64.6% for fresh RBCs in plasma and 46.4, 48.1, 54.8, 61.4, 65.7, and 66.5% for stored RBCs in plasma at pressures of 2.5, 5, 10, 20, 40, and 60 cm H2 O. CONCLUSION: Although exact optimal hematocrit values may depend on specific microvascular architecture, our results suggest that the optimal hematocrit for oxygen delivery in the microvasculature depends on perfusion pressure. Therefore, anemia in chronic disorders may represent a beneficial physiological response to reduced perfusion pressure resulting from decreased heart function and/or vascular stenosis. Our results may help explain why a therapeutically increasing hematocrit in such conditions with RBC transfusion frequently leads to worse clinical outcomes.

Shape matters: the effect of red blood cell shape on perfusion of an artificial microvascular network.

BACKGROUND: The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphologic lesion. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of echinocytosis and normal discocytes. Here we studied how the change in shape of RBCs following along the path of the echinocytic transformation affects perfusion of an artificial microvascular network (AMVN). STUDY DESIGN AND METHODS: Blood samples were obtained from healthy consenting volunteers. RBCs were leukoreduced, resuspended in saline, and treated with various concentrations of sodium salicylate to induce shape changes approximating the stages of echinocytosis experienced by RBCs during hypothermic storage (e.g., discocyte, echinocyte I, echinocyte II, echinocyte III, spheroechinocyte, and spherocyte). The AMVN perfusion rate was measured for 40% hematocrit suspensions of RBCs with different shapes. RESULTS: The AMVN perfusion rates for RBCs with discocyte and echinocyte I shapes were similar, but there was a significant decline in the AMVN perfusion rate between RBCs with shapes approximating each subsequent stage of echinocytosis. The difference in AMVN perfusion between discocytes and spherocytes (the last stage of the echinocytic transformation) was 34%. CONCLUSION: The change in shape of RBCs from normal discocytes progressively through various stages of echinocytosis to spherocytes produced a substantial decline in the ability of these cells to perfuse an AMVN. Echinocytosis induced by hypothermic storage could therefore be responsible for a similarly substantial impairment of deformability previously observed for stored RBCs.

Effect of osmolality on erythrocyte rheology and perfusion of an artificial microvascular network.

Plasma sodium concentration is normally held within a narrow range. It may however vary greatly under pathophysiological conditions. Changes in osmolality lead to either swelling or shrinkage of red blood cells (RBCs). Here we investigated the influence of suspension osmolality on biophysical properties of RBCs and their ability to perfuse an artificial microvascular network (AMVN). Blood was drawn from healthy volunteers. RBC deformability was measured by osmotic gradient ektacytometry over a continuous range of osmolalities. Packed RBCs were suspended in NaCl solutions (0.45, 0.6, 0.9, 1.2, and 1.5 g/dL), resulting in supernatant osmolalities of 179 ± 4, 213 ± 1, 283 ± 2, 354 ± 3, and 423 ± 5 mOsm/kg H2O. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were determined using centrifuged microhematocrit. RBC suspensions at constant cell numbers were used to measure viscosity at shear rates ranging from 0.11 to 69.5s(-1) and the perfusion rate of the AMVN. MCV was inversely and MCHC directly proportional to osmolality. RBC deformability was maximized at isosmotic conditions (290 mOsm/kg H2O) and markedly decreased by either hypo- or hyperosmolality. The optimum osmolality for RBC suspension viscosity was shifted toward hyperosmolality, while lower osmolalities increased suspension viscosity exponentially. However, the AMVN perfusion rate was maximized at 290 mOsm/kg H2O and changed by less than 10% over a wide range of osmolalities. These findings contribute to the basic understanding of blood flow in health and disease and may have significant implications for the management of osmotic homeostasis in clinical practice.

Washing stored red blood cells in an albumin solution improves their morphologic and hemorheologic properties.

BACKGROUND: Prolonged storage of red blood cells (RBCs) leads to storage lesions, which may impair clinical outcomes after transfusion. A hallmark of storage lesions is progressive echinocytic shape transformation, which can be partially reversed by washing in albumin solutions. Here we have investigated the impact of this shape recovery on biorheologic variables. STUDY DESIGN AND METHODS: RBCs stored hypothermically for 6 to 7 weeks were washed in a 1% human serum albumin (HSA) solution. RBC deformability was measured with osmotic gradient ektacytometry. The viscosity of RBC suspensions was measured with a Couette-type viscometer. The flow behavior of RBCs suspended at 40% hematocrit was tested with an artificial microvascular network (AMVN). RESULTS: Washing in 1% albumin reduced higher degrees of echinocytes and increased the frequency of discocytes, thereby shifting the morphologic index toward discocytosis. Washing also reduced RBC swelling. This shape recovery was not seen after washing in saline, buffer, or plasma. RBC shape normalization did not improve cell deformability measured by ektacytometry, but it tended to decrease suspension viscosities at low shear rates and improved the perfusion of an AMVN. CONCLUSIONS: Washing of stored RBCs in a 1% HSA solution specifically reduces echinocytosis, and this shape recovery has a beneficial effect on microvascular perfusion in vitro. Washing in 1% albumin may represent a new approach to improving the quality of stored RBCs and thus potentially reducing the likelihood of adverse clinical outcomes associated with transfusion of blood stored for longer periods of time.

[What is the spleen needed for?]. / Wozu braucht es die Milz?

Although the spleen is not essential for survival, it has important functions such as immune defence, blood filtration, retention and on demand extramedullary hematopoiesis. The white pulp with its T-lymphocytes in perarteriolar lymphoid sheaths and B-lymphocytes in lymphoid follicules is responsible for the detection and removal of circulating pathogens. After splenectomy, this immune defence is missing, which may lead to overwhelming post- splenectomy sepsis by encapsulated bacteria, which has a high mortality rate. In the red pulp, the blood is filtered through narrow slits in the sinusoidal endothelium, which abnormal and senescent erythrocytes can not pass and are eliminated by macrophages. The spleen is, therefore, not a "spleen" of nature, but a sophisticated organ.

[Between thrombosis and bleeding - a case of paroxysmal nocturnal hemoglobinuria]. / Auf dem schmalen Grat zwischen Thrombosen und Verbluten.

A 67-year-old lady was admitted with a myocardial infarction. Coronarography revealed thrombotic material within coronary arteries. The underlying disease was paroxysmal nocturnal hemoglobinuria (PNH), a stem cell defect characterized by episodes of complement-induced hemolysis and thromboembolic events. Anticoagulation and antiaggregation were initiated. Subsequently, massive cerebral bleeding developed, to which the patient finally succumbed despite neurosurgical craniectomy. This tragic case illustrates that an antithrombotic treatment is always a balancing act with an unsecure outcome.

Stored erythrocytes have less capacity than normal erythrocytes to support primary haemostasis.

Primary haemostasis is mediated by platelet aggregation. Red blood cells (RBCs) are involved in this process. We hypothesised that stored RBCs could have less capacity to support primary haemostasis. This was tested with RBC units from 17 healthy volunteers stored for 45 days. Fresh citrated blood was taken again from the same donors and platelet-rich plasma was prepared, in which RBCs were resuspended with a constant haematocrit (40%), but changing fractions of stored versus fresh autologous RBCs (0, 25, 50, 75, and 100%, respectively). A platelet function analyser PFA-100((R)) was used. In this instrument blood is aspirated through a membrane pore coated with collagen and either epinephrine (EPI) or ADP, which causes platelets to adhere, aggregate, and form an occluding plug, which stops blood flow and is measured as closure time (CT). We found that the CT increased with increasing fractions of stored blood. CT-EPI was 121 +/- 17 seconds [s], 129 +/- 32 s, 164 +/- 45 s (p < 0.000, ANOVA), 214 +/- 54 s (p < 0.0001), and 273 +/- 36 s (p < 0.0001) for 0, 25, 50, 75, and 100% stored RBCs. For CT-ADP the values were 91 +/- 22 s, 95 +/- 12 s, 101 +/- 13 s, 124 +/- 44 s (p = 0.004), and 191 +/- 72 s (p < 0.0001), respectively. We conclude that stored RBCs have less capacity than normal RBCs to support primary haemostasis by platelet aggregation in vitro, suggesting a decreased capacity of stored RBCs to bring platelets into close contact with the wall, which may contribute to sustained bleeding seen after mass transfusion.

[Imaging of arteries and veins]. / Bildgebung von Arterien und Venen.

For all arteries, which are easily accessible by ultrasound, a colordoppler ultrasound investigation is the first choice. The method is cost effective, precise, without radiation hazard and generates not only morphological, but also useful hemodynamic information. For the investigation of veins (Varicosis and thrombosis) colordoppler ultrasound has almost uniformly replaced phlebography as the method of choice.

Influence of combinations of acetylsalicylic acid, acetaminophen, and diclofenac on platelet aggregation.

Acetylsalicylic acid (aspirin) is often given together with other nonsteroidal anti-inflammatory drugs and acetaminophen. The latter have been accused in epidemiologic studies to cause an increased cardiovascular risk. We have, therefore, analysed the influence of various such drug combinations on platelet aggregation in vitro. Citrated blood was incubated with either 25 microg/ml acetaminophen, 0.5 microg/ml aspirin, 0.04 microg/ml diclofenac, or buffer; followed by a second of the above-mentioned solutions. After a 20 min incubation, platelet aggregation was assessed with a platelet function analyser (PFA-100), which measures the pore closure time (CT) by aggregating platelets. The length of CT reflects the degree of platelet inhibition. Acetaminophen alone did not affect platelet aggregation. Aspirin and diclofenac both increased CT (184+/-69 s, P<0.01 and 196+/-54 s, P<0.001; control 120+/-13 s). Combinations of either aspirin and diclofenac, aspirin and acetaminophen, or diclofenac and acetaminophen increased CT further (290+/-22 s, 281+/-36 s, 288+/-25 s, respectively, P<0.001). The time sequence of drug application was important: when diclofenac or acetaminophen was added before aspirin, platelet aggregation was less inhibited than when given in opposite order, i.e. aspirin prior to diclofenac or acetaminophen. We conclude that acetaminophen by itself does not affect platelet aggregation, but potentiates the antiaggregatory effect of aspirin or diclofenac. Aspirin given before acetaminophen or diclofenac had a more potent antiaggregatory effect than vice versa. These observations may have clinical implications.

Platelet aggregation under high shear conditions during and after a 28-day administration of 100 mg acetylsalicylic acid in healthy volunteers.

Platelet primary hemostatic function occurs under high shear conditions. Cyclooxygenase inhibitors such as ibuprofen inhibit this platelet aggregation under high shear rates only to a limited extent. This prompted the present study on 10 healthy volunteers treated with 100 mg aspirin for 4 weeks. The platelet function analyser (PFA-100) was used to measure the closure time (CT) of a membrane pore coated with collagen and epinephrine by aggregating platelets under shear rates of 5000-6000 s(-1). A first dose of 100 mg aspirin prolonged the CT above the normal range in 4 of 10 individuals, but the CT for the whole group (153+/-42 s) was not different from baseline (112+/-18 s). After 7 and 28 days of treatment, CTs were >300 s in 8 individuals and the mean values for the group were significantly higher than baseline. However, one subject had an intermediate response and one had an aspirin non-responsiveness, which was not overcome by 300 mg aspirin daily. The CT was normalized in 4 individuals 48 h after the last aspirin dose and in 7 individuals after 72 h, when the mean value for the group became not different from baseline. We conclude that the platelet function measured with the PFA-100 is not inhibited significantly after a single dose of 100 mg aspirin, is thereafter inhibited consistently in the majority, but not all individuals during a 4 week treatment, and returns to normal in 48-72 h. Since large interindividual differences exist, monitoring of platelet inhibition at the beginning of an aspirin treatment should be considered and validated in a prospective study.

Influence of acute alcohol exposure on hemorheological parameters and platelet function in vivo and in vitro.

We have analysed the influence of acute alcohol exposure in vivo and in vitro on blood flow properties and platelet function. 12 healthy male volunteers drank either 4.36 ml red wine/kg body weight (=0.5 g ethanol/kg) or water at 06.00 p.m. under fasting conditions. Blood was drawn immediately before, and 1, 2, 4 and 13 h after alcohol ingestion. Alcohol had a detectable osmotic effect on erythrocytes; the mean cellular volume (MCV) was significantly smaller 1-4 h after ingestion. Whole blood viscosity remained unaffected, but blood viscosity at a standardized Hct of 45% measured at a high shear rate (94.5 s(-1)) was increased 2 h after wine ingestion. In the morning, 13 h after wine drinking, platelet aggregation measured with a platelet function analyser PFA-100 was increased to a greater extent than after water drinking. In vitro, no effect was seen when blood was incubated with 0, 12.5, 25, 50 and 100 mmol/l ethanol for 1 h at 37 degrees C. We conclude that an acute exposure to alcohol has only modest effects on hemorheological parameters and platelet aggregation in vivo and no effect in vitro, which suggests that other factors must be involved in both beneficial and harmful effects of wine drinking.

[An unconscious odyssey]. / Eine sinn- und bewusstlose Odyssee.

A 76-year-old ex-manager of a shipping company with preexisting pneumopathy suffered from massive pulmonary embolism after an operation in Germany and had to be mechanically ventilated. His wife requested a transportation by helicopter to the intensive care unit of a smaller hospital in the Engadin, Grisons, Switzerland, where he was later tracheotomized. Weeks later, the wife insisted on a transfer to another, even smaller hospital in the area. During that stay an emergency pericardiocentesis had to be performed. Thereafter, he was transferred to the intensive care unit of our hospital. The last journey was again by request of his wife against medical advices a flight back to his home in Germany, what he survived only for a few hours and was followed by the suicide of his wife. Why could nobody prevent or stop this insane odyssey?

The optimum hematocrit.

The hematocrit (Hct) determines the oxygen carrying capacity of blood, but also increases blood viscosity and thus flow resistance. From this dual role the concept of an optimum Hct for tissue oxygenation has been derived. Viscometric studies using the ratio Hct/blood viscosity at high shear rate showed an optimum Hct of 50-60% for red blood cell (RBC) suspensions in plasma. For the perfusion of an artificial microvascular network with 5-70µm channels the optimum Hct was 60-70% for high driving pressures. With lower shear rates or driving pressures the optimum Hct shifted towards lower values. In healthy, well trained athletes an increase of the Hct to supra-normal levels can increase exercise performance. These data with healthy individuals suggest that the optimum Hct for oxygen transport may be higher than the physiological range (35-40% in women, 39-50% in men). This is in contrast to clinical observations. Large clinical studies have repeatedly shown that a correction of anemia in a variety of disorders such as chronic kidney disease, heart failure, coronary syndrome, oncology, acute gastrointestinal bleeding, critical care, or surgery have better clinical outcomes when restrictive transfusion strategies are applied. Actual guidelines, therefore, recommend a transfusion threshold of 7-8 g/dL hemoglobin (Hct 20-24%) in stable, hospitalized patients. The discrepancy between the optimum Hct in health and disease may be due to factors such as decreased perfusion pressures (low cardiac output, vascular stenoses, change in vascular tone), endothelial cell dysfunction, leukocyte adhesion and others.

The influence of the haematocrit on primary haemostasis in vitro.

Primary haemostasis consists of platelet adhesion to subendothelial collagen, their activation and aggregation and finally the formation of a platelet plug. Erythrocytes are involved in this process because they flow in the center of the vessel and push platelets towards the site of action on the vessel wall and enhance shear forces, which activate platelets. In the platelet function analyzer PFA-100 (Dade Behring, Düdingen, Switzerland), the in vivo situation is simulated in vitro with blood being aspirated at high shear rates (5000s(-1)) through a capillary into a membrane pore with a diameter of 150 microm coated with type I collagen and either epinephrine or adenosine diphosphate. Aggregating platelets plug the pore and stop the flow, which is measured as the closure time. We analysed the influence of erythrocytes on platelet function analyzer measurements by systematic variation of the haematocrit (20, 30, 40, and 50%) at constant platelet counts of 289 +/- 61 x 10(3)/microl plasma, or 152 +/- 30 x 10(3)/microl blood, 96 +/- 9 x 10(3)/microl blood and 54 +/- 5 x 10(3)/microl blood, respectively. An inverse correlation was found between haematocrit and closure time under all circumstances. A decrease of the platelet count by 50 x 10(3)/microl could be compensated for by a 10% increase in haematocrit. The haematocrit must, therefore, be taken into consideration for the correct interpretation of PFA-100 measurements. Our data also provide a pathophysiological rationale to reduce the risk of bleeding in patients with thrombocytopenia and anaemia by normalizing the haematocrit with erythrocyte transfusions.

Epstein-Barr virus DNA in serum in a HIV-infected patient during development and treatment of non-Hodgkin's lymphoma.

Immunocompromised patients are at risk of developing Epstein-Barr virus (EBV)-related lymphoproliferative disorders. Quantitative determination of serum EBV DNA permits early diagnosis of an AIDS-related non-Hodgkin's lymphoma, and surveillance of treatment response and/or relapse, and thus may be a useful tool to monitor disease activity.