M. Heins

Storage of serum or whole blood samples ? Effects of time and temperature on 22 serum analytes

Information on the stability of serum analytes during storage of serum or whole blood samples is often incomplete and sometimes contradictory. Using a widely available analyser (Hitachi 737/Boehringer), we therefore determined the effects of storage time and temperature on the measured concentrations of the following serum analytes: sodium, potassium, calcium, chloride, inorganic phosphate, magnesium, creatinine, urea, uric acid, bilirubin, cholesterol, HDL- and LDL-cholesterol, triacylglycerols, creatine kinase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, alpha-amylase, lactate dehydrogenase and cholinesterase. When separated serum was stored at + 9 degrees C for seven days, the mean changes in inorganic phosphate and lactate dehydrogenase exceeded significantly (p < 0.05 or 0.001, respectively) the maximum allowable inaccuracy according to the Guidelines of the German Federal Medical Council; all other quantities were sufficiently stable. In serum at room temperature, inorganic phosphate, uric acid, HDL-cholesterol and triacylglycerols increased continuously, whereas bilirubin, LDL-cholesterol, creatine kinase and aspartate aminotransferase decreased more than the guidelines permit during the storage period (p < 0.05 for aspartate aminotransferase, p < 0.001 for the other analytes mentioned). In whole blood stored for 7 days at + 9 degrees C, only the following serum analytes satisfied the stability requirements of the guidelines: calcium, urea, cholesterol, HDL-cholesterol, LDL-cholesterol, triacylglycerols, creatine kinase, gamma-glutamyltransferase and cholinesterase. When stored at room temperature, only sodium, uric acid, bilirubin, cholesterol, triacylglycerols, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, alpha-amylase and cholinesterase were still stable after 3 days. The data collected show that all quantities examined are sufficiently stable for four days in separated serum stored at + 9 degrees C.

Influence of time and temperature on coagulation analytes in stored plasma.

Abstract There is no comprehensive study on the stability of coagulation analytes in plasma. We therefore determined the influence of storage time and temperature on prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, factors V and VIII, antithrombin III, protein C and S in plasma from 20 healthy subjects and 20 patients receiving heparin therapy. The stability in plasma, defined as the period during which there was a change of less than 10 % from the initial value, was 8 hours for activated partial thromboplastin time, 24 hours for prothrombin time, 48 hours for factor V and 7 days for thrombin time, fibrinogen, protein C and antithrombin III in healthy subjects at 6 °C. Factor VIII and protein S showed 19 and 12 % reduction in activity, respectively, after 8 hours. In volunteers not treated with heparin therapy, activated partial thromboplastin time was stable for 8 hours; prothrombin time for 48 hours; and thrombin time, fibrinogen and antithrombin III for 7 days with sample storage at room temperature. Factor VIII showed a decrease of 18 % after 8 hours. For patients receiving heparin therapy, the stability of the analytes in plasma stored at 6 °C was 8 hours for thrombin time, 24 hours for prothrombin time and activated partial thromboplastin time and 7 days for fibrinogen and antithrombin III. Factors V and VIII showed a decrease of 13 % and 20 % respectively after 8 hours. When the plasma of these patients was stored at room temperature, factor V was stable for 8 hours, and prothrombin time for 24 hours, whereas fibrinogen and antithrombin III remained unchanged for 7 days. Activated partial thromboplastin time showed an increase of 13 %, thrombin time a fall of 16 %, and factor VIII a decrease of 18 % after 8 hours.

Monitoring of osteoblast activity with an immunoradiometric assay for determination of bone alkaline phosphatase mass concentration in patients receiving renal transplants

We examined the diagnostic validity of an immunoradiometric assay for determination of mass concentration of bone alkaline phosphatase (EC 3.1.3.1) in 134 sera from 35 patients receiving renal transplants. Comparison between bone alkaline phosphatase concentration and total alkaline phosphatase activity yielded a strong correlation (r = +0.860; P < 0.001). Nine (17%) of 54 sera which were characterized by a total alkaline phosphatase activity between 100 units/l and the upper reference limit (178 units/l (males) and 160 units/l (females), respectively) showed an increased bone alkaline phosphatase concentration (> 21.3 micrograms/l (males) and > 15.0 micrograms/l (females), respectively). There was also a correlation between bone alkaline phosphatase values and parathyroid hormone levels both before (r = +0.640 (n = 23), P < 0.001) and after renal transplantation (r = +0.528 (n = 111), P < 0.0001). A follow-up of 15 patients after renal transplantation revealed that the median of bone alkaline phosphatase values increased from 5.5 micrograms/l before transplantation to 14.9 micrograms/l 3 months after transplantation (P < 0.0001). Nevertheless no correlation could be observed between parathyroid hormone concentrations and bone alkaline phosphatase values at any time following renal transplantation in these 15 patients (P > 0.1). Rise of bone alkaline phosphatase concentration following renal transplantation is most probably due to an activating effect of cyclosporin A upon osteoblasts.

Changes of hemostasis, endogenous fibrinolysis, platelet activation and endothelins after percutaneous transluminal coronary angioplasty in patients with stable angina.

OBJECTIVES This study investigated parameters of endogenous fibrinolysis, activation of coagulation and platelets, and endothelin levels before and after elective percutaneous transluminal coronary angioplasty (PTCA) in patients with stable coronary artery disease (CAD). BACKGROUND Abrupt vessel closure is a serious short-term complication after PTCA and is often unforeseeable. Detailed insight into the effect of PTCA on hemostasis, platelets and the release of vasoconstrictive substances, which are among the mainly discussed mechanisms of abrupt vessel closure, is needed to enhance the safety of coronary intervention. METHODS Plasma levels of markers of platelet activity, coagulation, endogenous fibrinolysis and endothelins were determined in 20 patients with stable CAD undergoing elective PTCA. The blood specimens were drawn before, immediately after, 1 h after intervention and on the next morning. RESULTS All patients showed an initially uncomplicated PTCA. Regarding the efficacy of anticoagulation after receiving 15.000 IU heparin during PTCA, two groups were compared. In eight patients with ineffective anticoagulation production of thrombin and platelet activation directly after and 1 h after PTCA was significantly higher compared with 12 patients with effective anticoagulation. Despite the strong activation of coagulation, only a low fibrinolytic response could be observed. Endothelins rose significantly after PTCA in both groups but stayed longer on higher levels in patients with distinct thrombin generation. Three of the eight patients without sufficient heparin treatment suffered abrupt vessel closure. CONCLUSIONS Initially uncomplicated dilation of coronary arteries leads to systemically measurable activation of coagulation and platelets in patients with ineffective doses of heparin and release of endothelins in all patients. Therefore, individual adjustment of anticoagulation and platelet inhibition in combination with effective antivasospastic substances are needed in every patient before, during and after initially uncomplicated PTCA to prevent this serious complication.

Monitoring of bone resorption after renal transplantation by measuring the urinary excretion of pyridinium cross-links

The urinary excretion of pyridinium cross-links was measured in 70 second morning urine samples from 49 patients following renal transplantation. One and three months after renal transplantation, the urinary excretion of pyridinium cross-links was higher (p < 0.05) than at one week after transplantation. At all times after transplantation, the values for the excretion of pyridinium cross-links were correlated with the bone alkaline phosphatase concentrations (p < 0.001). However, there was no correlation between parathyrin concentrations and the values for the excretion of pyridinium cross-links (p > 0.05). This rise in the excretion of pyridinium cross-links is probably due to an increase of bone resorption caused by cyclosporin A and/or glucocorticoids. In the case of 17 urines with excretion values of pyridinium cross-links above the upper reference limit (pyridinoline equivalents, 93 mumol/mol creatinine), only 2 (12%) of the corresponding sera showed increased bone alkaline phosphatase values. In patients following renal transplantation simultaneous assessment of bone formation and bone resorption (determined from bone alkaline phosphatase serum concentrations and the excretion of pyridinium cross-links) may therefore enhance the diagnostic sensitivity for detecting effects on bone metabolism.

Plasma levels of D‐dimer and circulating endothelial adhesion molecules in veno‐occlusive disease of the liver following allogeneic bone marrow transplantation

Abstract: Veno‐occlusive disease (VOD) of the liver is a frequent and life‐threatening complication of BMT. Recently, successful treatment by t‐PA has been reported but has been compromised by fatal bleeding events. Therefore, t‐PA application should be restricted to patients with severe VOD. However, moderate and severe forms of VOD are difficult to distinguish in early stages. We analyzed plasma levels of cross‐linked fibrin degradation products (D‐dimer) and soluble endothelial adhesion molecules such as sE‐selectin, sVCAM‐1 and sICAM‐1 in 10 consecutive patients undergoing allogeneic BMT to evaluate their use in identifying severe forms of VOD. During the observation period, 4 episodes of VOD occurred, 2 of which were fatal due to early onset of multiorgan failure. Concentrations of D‐dimer generally increased after transplantation. However, there was an additional significant increase in D‐dimer levels during severe VOD. Thus, D‐dimer levels above 1000 μg/1 were only found in 2 cases with severe VOD and fatal outcome. When compared with bilirubin concentrations substantial increases of D‐dimers appeared earlier during the course of severe VOD. In contrast, VOD episodes were not accompanied by significant increases in sE‐selectin, sVCAM‐1 and sICAM‐1 levels. It is concluded that measurement of D‐dimer concentrations may aid accuracy to the early diagnosis of severe VOD.

Chronic-intermittent Urokinase Therapy in Refractory Angina Pectoris

Summary Patients with coronary artery disease and severe angina pectoris refractory to conventional medical treatment (beta-blockers, nitrates, calcium antagonists) and without the option for invasive revascularization procedures, present an increasing clinical problem. For these patients, chronic-intermittent urokinase therapy has been developed as a combined rheological and thrombolytic approach to the improvement of myocardial perfusion. In chronic-intermittent urokinase therapy 500,000 IU of urokinase are applied 3 times a week over a period of 12 weeks as an intravenous bolus injection. The target fibrinogen level, which is the major determinant of rheological blood properties (plasma viscosity, red blood cell aggregation) in microcirculation should range between 200 and 250 mg/dl. Reductions of fibrinogen levels amount to an average of about 35%, with subsequent reductions of plasma viscosity (approx. −7%) and red blood cell aggregation (approx. −18%). These changes are accompanied by significant reductions of clinical symptoms (anginal events/week) by approximately 75% from initially 23 ± 12 events/week; the clinical therapeutic effect is achieved after 2–4 weeks of treatment. Apart from the clinical improvements, objective signs of myocardial ischemia during ergometric exercise testing — such as ‘average negative ST-segment deviation’, ‘endpoint at termination of exercise’ and ‘exercise time to angina pectoris and/or to negative ST-segment deviation > 0.1mV’ — change favorably after treatment. A dose-response study which compared the application of 3 × 50,000 IU/week (n = 49) with 3 × 500,000 IU/week (n = 49) in patients with refractory angina pectoris could demonstrate a beneficial dose-dependent clinical effect for the higher dosage, which seems to be closely related to the reductions of the fibrinogen levels. There was no cumulation of undesirable side effects like an increase of coronary ischemic events and bleeding complications in the high-dose group. After termination of therapy with 3 × 500,000 IU urokinase/week (n = 121), the therapeutic effect (reduction of anginal events > 66%/week) lasted for 13.9 ± 8.6 months during a follow up period of 19.3 ± 10 months. Conclusion: Chronic-intermittent urokinase therapy in a dosage of 3 × 500,000 IU/week over a period of 12 weeks represents an effective antiischemic and antianginal approach in patients with refractory angina pectoris and end-stage coronary artery disease.

Specific Pattern of Circulating Endothelial Adhesion Molecules in HIV-Associated Kaposi’s Sarcoma

BACKGROUND Circulating endothelial adhesion molecules have been found to be increased in states of immune activation, but little is known about their significance in the assessment of endothelial neoplasms. One of the most common tumors supposed to be derived from endothelial origin is HIV-associated Kaposis sarcoma (KS). METHODS Plasma concentrations of sCD54 (= intercellular adhesion molecule-1), sCD106 (= vascular cell adhesion molecule-1), and sCD62E (= E-selectin) were quantified by sandwich ELISA in 54 AIDS patients who were either free of active opportunistic disorders (n = 15, AIDS controls), or suffering from acute infections (n = 16), or exhibiting KS (n = 23), and in 18 age- and sex-matched healthy HIV-negative controls. RESULTS Both sCD54 and sCD106 plasma levels were consistently increased in all AIDS patients irrespective of concurrent opportunistic disorders (p < 0.005), while sCD62E levels were not altered in AIDS patients without KS (p > 0.05). In KS patients, sCD62E concentrations were decreased both compared to healthy (p = 0.0007) and to AIDS controls (p = 0.04), and stimulated sCD54 levels were less elevated than those of AIDS controls (p = 0.02). Plasma concentrations of all three adhesion molecules did not correlate to KS tumor stage. CONCLUSION There appears to be a specific pattern of circulating endothelial adhesion molecules in AIDS patients with associated KS. Although the present findings do not support a role for their determination as tumor markers, they might be involved in KS tumor pathogenesis.

Antithrombotic treatment for the preservation and enhancement of myocardial perfusion in chronic coronary artery disease

Summary Therapeutic principles in the conventional medical treatment of myocardial ischemia in clinically stable coronary artery disease e.g. nitrates, beta-blockers, and calcium antagonists are mainly guided by a reduction of cardiac work load, hence a reduction of cardiac pre- and afterload, as well as by a decrease of vasomotor tone in epicardial conductance vessels and changes of myocardial metabolism. Even though they enhance exercise-limited myocardial perfusion and improve the quality of life, they influence the actual pathogenesis of coronary atherosclerosis only marginally, probably a major reason for the lacking therapeutic effect of these agents on major cardiovascular events. In contrast, antithrombotic and lipid lowering therapy not only reduce acute events like cardiovascular death, myocardial infarction, and unstable angina but can also contribute to a preservation and an enhancement of coronary blood flow in the chronic state of the disease. It has been shown that anti-platelet agents may reduce new coronary lesion formation and probably have an impact on actual regression of atherosclerotic carotid plaques in a higher dosage, as presently used. Anticoagulants like low-molecular weight heparin have been shown to improve subjective and objective signs of exercise-induced myocardial ischemia. In addition to an enhancement of collateral function, antithrombotic effects on subclinical coronary thrombus formation seem likely. Long-term intermittent urokinase therapy, which has been developed for patients with refractory angina pectoris in end-stage coronary artery disease, reduces myocardial ischemia by an improvement of rheological blood properties mediated by a decrease of fibrinogen. In addition, thrombolytic properties of this regimen as well as actual plaque regression may contribute substantially to clinical effectiveness.

Erworbene Faktor Vlll·C-Hemmkörper bei Nichthämophilen

Antibodies against factor VIII:C occur in about 5-15 % of hemophilia A patients and induce refractoriness to factor VIII:C infusions. These antibodies are mostly of IgG class. In very rare cases facto