Effects on labile metabolites of temporal delay in freezing biopsy samples of dog myocardium in liquid nitrogen

Sudden hypothermia utilising liquid nitrogen has been used for immediate inhibition of metabolic reactions and preservation of labile compounds in heart muscle. It has been suggested that this rapid transfer of tissue into liquid nitrogen, within 1 to 2 s, is essential for accurate assessment of internal milieu conditions. We tested this hypothesis in normal dogs by measuring phosphocreatin, ATP, glycogen, and lactate concentrations in transmural layers of a core biopsy taken from the posterolateral wall of the left ventricle frozen immediately in liquid nitrogen or held at room temperature for varying times up to 300 s before freezing the tissue. The earliest significant change occurred in phosphocreatine levels after 60 s; only phosphocreatine demonstrated any changes within the first 120 s. These studies indicate that a delay of up to 30 s may be tolerated before freezing tissue without any change occurring in these labile metabolites.     

Studies on cold insoluble globulin. I Concentrations in citrated plasma in rheumatic disorders.

Cold insoluble globulin (CIG) is a normal glycoprotein of human serum and plasma. The physiological significance of this protein is unknown, but is shows a temperature-dependent relation to fibrinogen and fibrin. It is possible that it represents a substrate for activated fibrin-stabilising factor in the polymerisation of fibrin. CIG is found on the surface of fibroblasts. In the present study CIG was estimated in citrated plasma in 115 patients with rheumatic diseases. Increased amounts were found in patients with systemic lupus erythematosus, secondary amyloidosis in classical and definite rheumatoid arthritis, and in male patients with juvenile rheumatoid arthritis.     

Comparing techniques: the use of recalcified plasma in comparison with citrated plasma alone and in combination with thrombin in ultrastructural studies

Abstract

Fibrin plays a vital role in the coagulation process and fibrin fiber morphology can be studied using ultrastructural techniques. When studying the ultrastructure of fibrin networks, thrombin may be added to the plasma, ensuing fibrin network formation. The question that arises is whether there are differences in morphology when thrombin is added to plasma, versus morphology observed when plasma from citrated or recalcified citrated whole blood, is studied. The current study therefore aimed to compare ultrastructure of platelets and fibrin networks from these three techniques. Results indicated comparable platelet ultrastructure between smears formed from the plasma of citrated blood and that of the citrated recalcified blood. This method might give us further information regarding the ‘natural state’ fibrin assembly and association with platelets, when studying haemostasis. However, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibrin fibers.     

Comparing techniques: the use of recalcified plasma in comparison with citrated plasma alone and in combination with thrombin in ultrastructural studies

Fibrin plays a vital role in the coagulation process and fibrin fiber morphology can be studied using ultrastructural techniques. When studying the ultrastructure of fibrin networks, thrombin may be added to the plasma, ensuing fibrin network formation. The question that arises is whether there are differences in morphology when thrombin is added to plasma, versus morphology observed when plasma from citrated or recalcified citrated whole blood, is studied. The current study therefore aimed to compare ultrastructure of platelets and fibrin networks from these three techniques. Results indicated comparable platelet ultrastructure between smears formed from the plasma of citrated blood and that of the citrated recalcified blood. This method might give us further information regarding the 'natural state' fibrin assembly and association with platelets, when studying haemostasis. However, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibrin fibers.     

Determination of dabigatran in human plasma samples

The oral direct thrombin inhibitor dabigatran effectively prevents arterial and venous thromboembolism using fixed doses without the need for adjustment according to laboratory results. Dabigatran is eliminated from the circulation by ～80% through the kidneys. However, the in vitro anticoagulant effect of dabigatran may be necessary to determine in special patient populations such as in the elderly, for renal impairment, before operations, bleeding or thrombotic episodes, and to monitor self-compliance. Several clotting and thrombin-specific chromogenic substrate assays are available to analyze the biological activity of dabigatran. All of them are prolonged in the presence of dabigatran. This article reports the effects of dabigatran on clinical routine assays and the potential usefulness for determination in special risk groups of patients when overdose or lack of compliance are suspected.     

Determination of rivaroxaban in human plasma samples

Rivaroxaban is one of the novel oral direct factor Xa inhibitors, which is effective in preventing thromboembolic complications at fixed doses (i.e., once daily), without the need for dose adjustment according to laboratory monitoring. Nearly 60% of rivaroxaban is cleared from circulation by glomerular filtration, 30% of which is excreted as active drug. Therefore, as renal elimination plays a pivotal role in the metabolism of this drug, impairment of renal function may be important during anticoagulation with rivaroxaban over long periods of time. The assessment of the anticoagulant effect/concentration of rivaroxaban may thus be useful in special patient populations such as in the elderly and eldest, during acute diseases with concurrent dehydration, before surgery, during bleeding or thrombotic episodes, or to verify adherence to therapy. Rivaroxaban prolongs prothrombin time in a dose-dependent, linear fashion. Activated partial thromboplastin time (APTT) is also prolonged, but in an exponential manner. Substantial differences in test results might be generated by different thromboplastin and APTT reagents. One-step prothrombin-induced clotting time assay is sensitive to low concentrations of rivaroxaban. Chromogenic substrate assays specific for factor Xa are also sensitive to rivaroxaban. Several initiatives are currently ongoing to standardize the various methods to determine rivaroxaban in human plasma samples, some of which will be summarized in this article along with the dose-dependent effects of rivaroxaban on relevant coagulation parameters. Therefore, although rivaroxaban prolongs all coagulation assays used to assess the anticoagulant effects of most anticoagulants, the most specific assay cannot be identified at present. Moreover, clinical trials are needed to determine the relationship of assay results with bleeding or thrombotic complications.     

Clotting time analysis of citrated blood samples is strongly affected by the tube used for blood sampling.

Our aim was to establish whether differences in clotting times for recalcified blood and plasma samples might be explained by the use of different blood collection tubes. Samples obtained from different plastic vacuum tubes were recalcified and clotting times determined by free oscillation rheometry. The clotting times for blood collected in Vacutainer (Becton Dickinson, Rutherford, New Jersey, USA) or Vacuette (Greiner Bio-One, Kremsmünster, Austria) tubes decreased with time, with maximal effect after 30 min. Blood from Monovette (Sarstedt, Nümbrecht, Germany) tubes displayed longer clotting times, which did not decrease with time. Clotting times for plasma prepared after 1 h storage in Vacutainer or Vacuette tubes were unaffected by subsequent addition of corn trypsin inhibitor to inhibit factor XIIa, although an antibody against factor XI prolonged the clotting time markedly. In Monovette plasma, both corn trypsin inhibitor and anti-factor XI effectively prolonged the clotting time. When corn trypsin inhibitor or anti-factor XI was added to the tubes before blood collection, but both additions clearly prolonged the clotting times in all types of tubes, even though corn trypsin inhibitor was less effective in whole blood. Antibodies against human tissue factor did not affect the clotting times. The amounts of platelet or leukocyte microparticles in plasma were low and similar in all tubes. This indicates that blood collection in Vacutainer or Vacuette tubes induces a rapid activation of factor XII and factor XI.     

Antigen levels of plasminogen activator inhibitor type 1 in citrated plasma: determination of the contribution of platelets

To estimate the extent of the release of plasminogen activator inhibitor type 1 (PAI-1) from platelets following their accidental activation in vitro during blood collection, we studied the antigen levels of PAI-1 and beta-thromboglobulin (beta TG) in plasma and sera from 38 healthy volunteers. For each volunteer, we calculated the ratio of platelet-derived PAI-1/platelet-derived beta TG from the difference in their respective concentrations in serum and plasma. The mean +/- SD (0.012 +/- 0.003) and median (0.012) ratios were identical before and after 10 or 20 min of venous occlusion. The platelet contribution of PAI-1 in plasma was estimated by multiplying the plasma concentration of beta TG (as a marker of platelet activation) with the above-mentioned individual ratio. Under our conditions of blood collection (1/10 vol of 0.1 M citrate, pH 4.5, as anticoagulant, and handling of blood at 4 degrees C and within 30 min), the platelet contribution to the PAI-1 concentration in plasma was on average 12% and always negligible in plasma with high levels of PAI-1.     

Vitrification and levitation of a liquid droplet on liquid nitrogen

The vitrification of a liquid occurs when ice crystal formation is prevented in the cryogenic environment through ultrarapid cooling. In general, vitrification entails a large temperature difference between the liquid and its surrounding medium. In our droplet vitrification experiments, we observed that such vitrification events are accompanied by a Leidenfrost phenomenon, which impedes the heat transfer to cool the liquid, when the liquid droplet comes into direct contact with liquid nitrogen. This is distinct from the more generally observed Leidenfrost phenomenon that occurs when a liquid droplet is self-vaporized on a hot plate. In the case of rapid cooling, the phase transition from liquid to vitrified solid (i.e., vitrification) and the levitation of droplets on liquid nitrogen (i.e., Leidenfrost phenomenon) take place simultaneously. Here, we investigate these two simultaneous physical events by using a theoretical model containing three dimensionless parameters (i.e., Stefan, Biot, and Fourier numbers). We explain theoretically and observe experimentally a threshold droplet radius during the vitrification of a cryoprotectant droplet in the presence of the Leidenfrost effect.