The role of reduced red cell deformability in the pathogenesis of severe falciparum malaria and its restoration by blood transfusion.

As reduced red cell deformability (RCD) can contribute to derangement of the microcirculation, a central feature in the pathogenesis of severe malaria, RCD was measured with a laser diffraction technique in 232 consecutive patients with falciparum malaria on the Kenyan coast, of whom 99 had severe disease. RCD on admission (measured as elongation index [EI] at shear stress = 1.7 Pa) was reduced in proportion with severity of disease (fatal outcome: EI = 0.182 (SD = 0.048), survivors from severe disease: EI = 0.217 (SD = 0.043), uncomplicated malaria: EI = 0.249 (SD = 0.030), healthy controls: EI = 0.268 (SD = 0.022). All but 2 survivors with severe malaria and rigid erythrocytes received a blood transfusion restoring RCD. Reduced RCD may contribute to impaired microcirculatory flow and a fatal outcome in falciparum malaria. RCD can be improved by blood transfusion. Since severely reduced RCD has a strong predictive value for mortality, blood transfusion possibly improves disease outcome not only through its beneficial effect on anaemia but also on RCD.

The resistance to physiological shear stresses of the erythrocytic rosettes formed by cells infected with Plasmodium falciparum.

Rosetting forces are believed to be an important contributor to the microcirculatory obstruction that occurs in malaria caused by Plasmodium falciparum. In this study, rosettes of erythrocytes from cultures of this parasite were suspended in different media and exposed to shear stresses corresponding to those encountered on the arterial and venous sides of the human circulation. The rosettes formed by infected erythrocytes in malaria culture medium containing 10% AB serum were disrupted easily (approximately 50% being broken) when exposed to very low shear stresses of < 0.5 Pa. However, use of higher concentrations of serum strengthened the rosetting binding forces considerably. Suspension of rosettes in a viscous colloid (e.g. dextran) increased the adherence forces between infected and uninfected red cells. The results indicate that rosettes do resist the physiological shear forces that are encountered in the venular side of the circulation and could thus contribute to microvascular obstruction in falciparum malaria.

Abnormal blood flow and red blood cell deformability in severe malaria.

Obstruction of the microcirculation plays a central role in the pathophysiology of severe malaria. Here, Arjen Dondorp and colleagues describe the various contributors to impaired microcirculatory flow in falciparum malaria: sequestration, rosetting and recent findings regarding impaired red blood cell deformability. The correlation with clinical findings and possible therapeutic consequences are discussed.

Red cell deformability, splenic function and anaemia in thalassaemia.

Red cell deformability (RCD) was measured in 38 patients with alpha-thalassaemia and 48 patients with beta-thalassaemia, of whom 13 had undergone splenectomy. All splenectomized patients, but none of those with intact spleens, had very rigid erythrocytes with an elongation index <0.45 at a high shear stress of 30 Pa suggesting a splenic recognition threshold for removal of rigid red cells. At this shear stress RCD correlated strongly with the degree of anaemia in both the splenectomized (r = 0.81, P < 0.001) and non-splenectomized beta-thalassaemic patients (all patients r = 0.81, P < 0.001; homozygous beta-thalassaemic patients r = 0.51, P = 0. 01). These data suggest that reduced RCD is a major determinant of anaemia in thalassaemia.

Red blood cell deformability as a predictor of anemia in severe falciparum malaria.

Decreased erythropoiesis and increased clearance of both parasitized and noninfected erythrocytes both contribute to the pathogenesis of anemia in falciparum malaria. Erythrocytes with reduced deformability are more likely to be cleared from the circulation by the spleen, a process that is augmented in acute malaria. Using a laser diffraction technique, we measured red blood cell (RBC) deformability over a range of shear stresses and related this to the severity of anemia in 36 adults with severe falciparum malaria. The RBC deformability at a high shear stress of 30 Pa, similar to that encountered in the splenic sinusoids, showed a significant positive correlation with the nadir in hemoglobin concentration during hospitalization (r = 0.49, P < 0.002). Exclusion of five patients with microcytic anemia strengthened this relationship (r = 0.64, P < 0.001). Reduction in RBC deformability resulted mainly from changes in unparasitized erythrocytes. Reduced deformability of uninfected erythrocytes at high shear stresses and subsequent splenic removal of these cells may be an important contributor to the anemia of severe malaria.

Nitric oxides in plasma, urine, and cerebrospinal fluid in patients with severe falciparum malaria.

It has been suggested that nitric oxide (NO) plays an important role in the pathogenesis of severe falciparum malaria. Since NO has a very short half-life, nitrate and nitrite (NOx) levels, stable metabolites of NO, are used as measures of NO production. We measured plasma NOx levels in 24 adults with severe falciparum malaria on the Thai-Burmese border. After correction for renal function, there was no correlation between plasma NOx levels, or the total amount of NOx excreted in the urine, and disease severity. Plasma NOx levels decreased after the first 48 hr in all patients (P = 0.007), suggesting decreased NO production. The NOx levels in cerebrospinal fluid (CSF) correlated well with plasma NOx levels, but these did not show a correlation with coma depth, and were not significantly different from those in a healthy control group. These findings do not support the hypothesis that excessive NO production contributes to the pathogenesis of severe falciparum malaria. However, local changes in NO production, e.g., in the central nervous system, might not be reflected in the total NOx production or NOx levels in the CSF.

Red blood cell rigidification during cyclosporin therapy: a possible early warning signal for adverse reactions.

In a previous retrospective study with kidney-transplant patients, immunosuppressive treatment with Cyclosporin A (CsA) was found to be associated with impaired red blood cell (RBC) deformability. The aim of the present study was to evaluate and substantiate a possible causal relationship between the use of CsA and its effect on RBC deformability in a prospective study on non-transplant patients. Blood samples of 12 patients with psoriasis were taken before and after 2, 4, 8, 12 and 16 weeks of treatment with CsA (3-5 mg/day). Red cell deformability, expressed as Elongation Index (EI), was measured with the Laser-assisted Optical Rotational Cell Analyzer (LORCA), a new ektacytometric instrument. Mean values +/- SD for EI found after 16 weeks of treatment with cyclosporin (0.570 +/- 0.008) were significantly (p < 0.001) lower than the value before treatment (0.589 +/- 0.011). A dose-response relation could not be established within the small range of CsA doses used in this study. Irrespective of the dose, however, a significant correlation (r = -0.55; p = 0.0001) between duration of treatment and decrease in EI was demonstrated. In vitro incubation of blood with cyclosporin was not able to reproduce this effect, suggesting that a direct effect of the drug on RBCs is unlikely. Despite its use in relatively low doses, CsA causes a reduction in RBC deformability, an effect that increases during the course of treatment. It is suggested that this slow, but continuously increasing, RBC rigidification plays a role in the early pathogenesis of the adverse nephrotoxic complications frequently associated with this immunosuppressive regimen.

Prognostic significance of reduced red blood cell deformability in severe falciparum malaria.

Severe falciparum malaria is associated with microvascular obstruction resulting from sequestration of erythrocytes containing mature stages of the parasite. Since reduced red blood cell deformability (RBC-D) can contribute to impaired microcirculatory flow, RBC-D was measured in 23 patients with severe falciparum malaria (seven of whom subsequently died), 30 patients with uncomplicated malaria, and 17 healthy controls. The RBC-D, measured by ektacytometry, was significantly reduced in severe malaria and was particularly low in all fatal cases. At a low shear stress of 1.7 Pascal (Pa), a red blood cell elongation index less than 0.21 on admission to the hospital predicted fatal outcome with a sensitivity of 100% (confidence interval [CI] = 59-100%) and a specificity of 88% (CI = 61-98%). The reduction in the RBC-D appeared to result mainly from changes in unparasitized erythrocytes. Reduced deformability of unparasitized red blood cells in severe malaria may contribute to impaired microcirculatory flow and a fatal outcome in severe falciparum malaria.

The clinical value of lactate dehydrogenase in serum: a quantitative review.

The aim of this article is to describe guidelines for rational use of lactate dehydrogenase and its isoenzymes, in the diagnostic processes and during follow-up, based on a systematic review of relevant literature. Sources of data for this study were English-language scientific publications, obtained from the database of the National Library of Medicine (Medline), concerning the clinical application (diagnosis, monitoring or treatment of disease) of lactate dehydrogenase and lactate dehydrogenase isoenzyme measurements in serum in the following main clinical fields: cardiology, hepatology, haematology and oncology. For acceptance in the present review, studies had to include: a proper definition of the tested patient population, diagnostic criteria, sampling time, sampling frequency, and test characteristics. Estimation of the relation between lactate dehydrogenase or lactate dehydrogenase isoenzymes and specific diseases expressed as sensitivity, specificity, survival or remission rate were extracted. The application of serum lactate dehydrogenase is relevant in the diagnosis of myocardial infarction (late detection), haemolytic anaemia, ovarian dysgerminoma and testicular germ cell tumor. For monitoring the progress of a disease lactate dehydrogenase is relevant in establishing the survival duration and rate in Hodgkin's disease and non-Hodgkin's lymphoma, and in the follow-up of ovarian dysgerminoma. Rational use of lactate dehydrogenase can be achieved when requests for its determination are limited to the above mentioned conditions. No rationale could be found for measuring lactate dehydrogenase isoenzymes.

Recurrent thrombotic occlusions of arteries and veins caused by intravascular metastatic adenocarcinoma.

The occurrence of unexplained, rapidly recurring occlusions of arteries and veins in a previously healthy young woman is described. Post mortem examination showed no macroscopic abnormalities but revealed microscopic metastatic adenocarcinoma with remarkable intravascular localisation of the malignant cells. Whereas highly sensitive markers for the existence of systemic activation of blood coagulation remained within the normal range, it is suggested that specific characteristics of the tumour cells may have been responsible for this particular clinical presentation.

No decreased erythrocyte deformability in type 1 (insulin-dependent) diabetes, either by filtration or by ektacytometry.

A lower erythrocyte deformability, which causes impairment of the microcirculation, is postulated to contribute to diabetic organ complications. Erythrocyte deformability was measured in four groups of type 1 (insulin-dependent) diabetic subjects and 30 controls by filtration and ektacytometry. Twenty-five patients without organ complications, 21 with microalbuminuria, 13 with overt nephropathy and 12 with leg ulceration were studied. No decreased erythrocyte deformability was found in any of the diabetic groups with either technique, and neither did the total group of 71 diabetic subjects have a lower erythrocyte deformability when compared with the controls. In order to imitate local conditions in the kidney, erythrocyte deformability was also measured in hyperosmolar solutions. Again no differences were found between the diabetic groups separately or as a whole and the controls. Furthermore no correlation was found between erythrocyte deformability and the plasma glucose or glycosylated haemoglobin level.

Blood and plasma viscosity in diabetes: possible contribution to late organ complications?

It has been postulated that an increased whole blood and plasma viscosity contribute to diabetic organ complications. Blood viscosity was measured in 30 controls and four groups of insulin-dependent diabetic patients at three shear rates: 70 sec-1, 0.5 sec-1 and 0.05 sec-1. Results were compared before and after correction for a haematocrit of 0.45. Twenty-five patients without organ complications, 21 with microalbuminuria, 13 with overt nephropathy and 12 patients with leg ulcerations were studied. Blood and plasma viscosity were normal in the patients without organ complications and with microalbuminuria. Plasma viscosity was significantly elevated in the diabetic patient with nephropathy and leg ulceration. After correction for haematocrit blood viscosity was also higher in these two groups, although this was only significant in the group with leg ulceration. In conclusion blood and plasma viscosity were only elevated in the patients with major organ complications and not in the patients without or with early complications. Therefore it is unlikely that an elevation of blood or plasma viscosity contributes early in the pathogenesis of diabetic organ damage.

High mitomycin C concentration in tumour tissue can be achieved by isolated liver perfusion in rats.

To enable the treatment of hepatic metastasis with higher, theoretically more effective, doses of systemically toxic anticancer drugs, an isolated liver perfusion (ILP) technique was developed in WAG/Ola rats. First, in a toxicity study the maximally tolerated dose (MTD) of mitomycin C (MMC) was determined for a 25-min ILP and for hepatic artery infusion (HAI) after the administration of a bolus dose. The MTD in the ILP setting (4.8 mg/kg) was 4 times that using HAI (1.2 mg/kg). Subsequently, in a rat colorectal hepatic-metastasis model, concentrations of MMC in tumour, liver, plasma and perfusate were measured during a 25-min ILP to investigate the expected pharmacokinetic advantage of ILP. The mean plasma level determined after ILP (1.2 as well as 4.8 mg/kg MMC) was significantly lower (P less than 0.001) than that obtained following HAI. This may explain both the absence of severe systemic toxicity and the higher MTD in ILP-treated groups. No significant difference in mean tumour and liver tissue concentrations of MMC were found when the groups treated with 1.2 mg/kg drug via HAI vs ILP were compared. The mean MMC concentration in tumour tissue was significantly higher (almost 5 times; P less than 0.05) in rats treated by ILP with the MTD (4.8 mg/kg) than in those treated via HAI with the MTD (1.2 mg/kg). ILP of MMC can be safely performed using a dose 4 times higher than the MTD in the HAI setting, leading to an almost 5-fold concentration of MMC in hepatic metastasis. ILP of MMC may therefore represent a promising therapy for metastasis confined to the liver.

Platelet hyperreactivity and prognosis in survivors of myocardial infarction.

We tested the hypothesis that an increase in spontaneous aggregability of platelets in vitro predicts mortality and coronary events in patients who have survived a recent myocardial infarction. A cohort of 149 survivors of infarction entered our study three months after the index infarction and was followed for five years. At entry and at intervals of six months, spontaneous platelet aggregation (SPA) was tested and graded as positive (aggregation within 10 minutes), intermediate (aggregation after 10 to 20 minutes), or negative (no aggregation within 20 minutes). During follow-up, 6.4 percent (6 of 94) of the patients in the SPA-negative group died, as compared with 10.3 percent (3 of 29) in the SPA-intermediate group and 34.6 percent (9 of 26) in the SPA-positive group. As compared with the SPA-negative group, the SPA-intermediate group had a relative risk of death of 1.6 (95 percent confidence interval, 0.5 to 5.5) and the SPA-positive group had a risk of 5.4 (95 percent confidence interval, 2.2 to 13.4). At least one cardiac event (cardiac death or recurrent nonfatal myocardial infarction) occurred in 14.9 percent (14 of 94 patients) of the SPA-negative group, 24.1 percent (7 of 29) of the SPA-intermediate group, and 46.2 percent (12 of 26) of the SPA-positive group. A positive test result continued to have prognostic value throughout the five-year study. We conclude that spontaneous platelet aggregation in vitro is a useful biologic marker for the prediction of coronary events and mortality in this low-risk group of survivors of a myocardial infarction. A causal relation is suggested but not proved by our study.

Transient aggregation resistance of human blood platelets in fresh plasma. II. Physiological relevance: influence of fish oil.

In order to evaluate the physiological relevance of the blood platelet "transient aggregation resistance" (TAR) test we studied the effect on this test of two different amounts of fish oil, corresponding to .75 g g (2.5 mmol) and 1.5 g (5 mmol) of eicosapentaenoic acid respectively, added in a cross-over design to the normal diet of 16 healthy male volunteers. It appeared that the "baseline aggregation resistance" (BAR), equivalent to the classic platelet aggregation ADP-threshold, was not influenced by Maxepa while, in contrast, a significant prolongation of TAR occurred. Apparently platelet aggregation analysed early after blood withdrawal, measures aspects of physiological relevance which, due to their short half life, are missed in the original method.

Transient aggregation resistance of human blood platelets in fresh plasma. I. Methodology.

A novel analytical method, using turbidometry, for reporting the time-dependent decay in the threshold concentration of adenosine diphosphate (ADP), required to elicit a secondary aggregation response in fresh human citrated platelet-rich plasma (PRP), is described. The phenomenon, called "transient aggregation resistance" (TAR) ends, usually within one hour after venepuncture, in a steady state or "baseline aggregation resistance" (BAR). Back extrapolation of the mathematically transformed data to t = 0, yields a maximal threshold concentration of ADP, representing the initial aggregation resistance (TARmax) at the time of blood withdrawal, which threshold is usually many orders of magnitude higher than the BAR-value. The exponential decay of TAR can be characterized by its half-life (t1/2). Mixing fresh, rapidly prepared, plasma with PRP, kept long enough to show only the stable low BAR-value, could restore the initial high (transient) aggregation resistance found in fresh PRP, suggesting that it concerns a natural, labile plasmatic factor. One hour old PRP, deliberately made refractory to ADP, did not show the TAR phenomenon again, but had a higher BAR-value. It is suggested that the level of clinical relevance of these early in vitro aggregation measurements is higher than that of classical, delayed aggregometry (e.g. BAR-values).

Unexpected high prevalence of silent pulmonary embolism in patients with deep venous thrombosis.

In patients presenting with clinically suspected deep vein thrombosis, symptomatic pulmonary embolism is rarely apparent. To assess the prevalence of silent pulmonary embolism in outpatients with proven deep vein thrombosis but without symptoms of pulmonary embolism, perfusion ventilation lung scans were performed in 101 consecutive patients at presentation. Fifty-one percent of these patients had a high probability lung scan at the initiation of treatment. In comparison, in patients referred with suspected venous thrombosis, but who on subsequent objective testing did not have venous thrombosis (n = 44), the prevalence of a high probability-scan for pulmonary embolus was only 5 percent. At repeat lung scanning, performed after one week of anticoagulant treatment, complete to partial improvement was observed in 68 percent of the patients with initially abnormal scans. Lung-scan detected asymptomatic pulmonary embolism occurs frequently in patients presenting with symptomatic deep venous thrombosis, and the majority of these emboli showed significant to complete resolution within one week of anticoagulant treatment.

Serial impedance plethysmography for suspected deep venous thrombosis in outpatients. The Amsterdam General Practitioner Study.

Diagnosis of deep venous thrombosis by clinical signs and symptoms is unreliable, but contrast venography is relatively expensive and invasive. We therefore evaluated the use of impedance plethysmography as a noninvasive alternative in 426 consecutive outpatients with clinically suspected acute deep venous thrombosis. Four sequential impedance plethysmograms were obtained on days 1, 2, 5, and 10 of the study. In 289 patients (68 percent), the results of all four studies were normal, and these patients were not treated with anticoagulants. One of these patients may have had a minor pulmonary embolus during the 10-day study period. During a six-month follow-up of all patients, none of the 289 patients whose plethysmograms were normal died of venous thromboembolism or presented with suspected pulmonary embolism. In 137 patients (32 percent), the impedance plethysmograms were abnormal; 117 (85 percent) had the abnormal results on their first test, and 20 (15 percent) had them on subsequent tests. All patients with abnormal plethysmograms also underwent contrast venography, which confirmed the diagnosis of deep venous thrombosis in 92 percent. We conclude that the diagnostic accuracy of repeated impedance plethysmography compares favorably with that of venography and that the technique is a safe and effective noninvasive approach to the diagnosis and care of outpatients with clinically suspected acute deep venous thrombosis.