G. Pindur

Prediction of fetal Rh D and Rh CcEe phenotype from maternal plasma with real-time polymerase chain reaction

Real-time PCR methods for the detection of RHD and the C, c, and E allele of RHCE were applied for the prediction of fetal Rh phenotype using maternal plasma. In one of 36 samples investigated the DNA extraction failed. When we tested the remaining 35 samples for Rh antigens which were absent on the mothers red cells, the fetal D-status was correctly determined in 26 of 27 cases (1 false negative). Fetal C was tested correctly in 23 samples, c was true positive in the only c-negative woman and the fetal E-status was correctly determined in 35 cases. In conclusion real-time PCR of maternal plasma is a non-invasive method to determine fetal RH genotype. However, more studies are required for routine applications because the method is not 100% sensitive.

Highly Substituted Hydroxyethyl Starch (HES 200/0.62) Leads to Type-l von Willebrand Syndrome after Repeated Administration

Hydroxyethyl starch (HES) is a frequently used plasma substitute that is popular due to a high degree of therapeutic safety. However, the administration of large volumes of highly substituted, high-molecular-weight starch often leads to iatrogenic von Willebrand syndrome (vWS) with hemorrhagic complications. In patients with cerebral circulatory disturbances we carried out hemodilution therapy during 9-10 days, infusing HES 200/0.62. A von Willebrand factor (vWF) multimeric analysis was carried out in 6 patients using a modified western blot according to the sodium dodecyl sulfate agarose gel electrophoresis method. The vWF multimeric analysis showed that all multimers decreased to the same degree, corresponding to type-I vWS.

Influence of rheological parameters on the velocity of erythrocytes passing nailfold capillaries in humans

One thousand two hundred and fifty-six subjects (apparently healthy subjects and patients with cardiovascular diseases) were registered in a prospective study including demographical and clinical data, rheological parameters (hematocrit, plasma viscosity, erythrocyte aggregation, erythrocyte deformability) as well as the erythrocyte velocity in human nailfold capillaries under resting and postischemic conditions. A multivariate regression analysis showed that under resting conditions there was no correlation between rheological parameters and erythrocyte velocity in capillaries. The blood flow regulation seemed to be so effective, that pathological changes of the blood fluidity showed no effect on the velocity of an erythrocyte passing the capillaries. During vessel paralysis in the early phase of the postischemic hyperemia following a stasis of three minutes in the vasculature distal to a pressure cuff at the upper arm a very clear correlation between the plasma viscosity and the maximum postischemic erythrocyte velocity in ipsilateral cutaneous capillaries could be observed (p < 0.0001) while none of the other rheological parameters seemed to play a role. In a subgroup of diabetic patients the erythrocyte aggregation (measured during stasis) also correlated with the erythrocyte velocity (p = 0.0175) besides the plasma viscosity. This shows that a correlation of rheological parameters with the capillary perfusion could only be found during vessel paralysis. In of diabetic patients besides the plasma viscosity also the erythrocyte aggregation correlated with the mean capillary erythrocyte velocity. Theses results are in agreement with the hypothesis from Barras that plasma viscosity determines the perfusion of microvessels. Under certain conditions e.g. diabetic disorder, also the erythrocyte aggregation plays a role.

Influence of Low Molecular Weight Hydroxyethyl Starch (HES 40/0.5-0.55) on Hemostasis and Hemorheology

Hydroxyethyl starch (HES) with a high or medium molecular weight (MW) and a high degree of substitution is difficult to degrade and leads to an accumulation of large molecules. These molecules have a negative effect on hemostasiological parameters. In 10 patients with cerebrovascular diseases, a hemodilution therapy was carried out with low MW HES for 10 days. Due to the low MW of the HES used (56-61 kD), the rheological parameters erythrocyte aggregation and plasma viscosity were significantly lowered (p < 0.01). No coagulation parameters studied were affected beyond the dilution effect, which was measured using the decline in hematocrit. Low MW starch is a volume substitute that is well-suited for repeated infusion or hemodilution therapy, particularly for patients with increased hemorrhagic diathesis, because it does not affect hemostasis. The disadvantage of a relatively short volume effect can be compensated through a continuous infusion of a larger volume.

Decrease of Fibronectin Following Repeated Infusion of Highly Substituted Hydroxyethyl Starch

OBJECTIVE Fibronectin (Fn) plays an important part in unspecific defense mechanisms because of its ability to mediate the binding of foreign-body particles, bacteria, collagen, and other macromolecules to phagocytising cells of the reticulo-endothelial system (RES). The aim of the present study was to examine the effect of a 10-day hemodilution therapy on Fn concentration in humans. DESIGN The patients were randomized and treated with either 10% hydroxyethyl starch (HES) 200/0.62 or 6% HES 200/0.62. SETTING Neurology department of an university clinic. PATIENTS We examined 12 patients with cerebrovascular perfusion disturbances. INTERVENTIONS The Fn concentration was determined using simple radial immunodiffusion. RESULTS The Fn concentration dropped significantly in all 12 patients (p < 0.01) in a dose-dependent manner beyond the dilution effect. 10% HES 200/0.62 caused a Fn decrease from 26.6 +/- 9.2 to 10.0 +/- 2.2 mg/dl (-62.2%), 6% HES reduced Fn from 25.5 +/- 9.9 to 15.0 +/- 3.2 mg/dl (-41.1%). In one patient there was a continuous decrease of Fn from 41.0 down to only 6.4 mg/dl. CONCLUSION According to the results of animal experiments, the decrease of Fn seems to indicate depression of the RES. Besides its defense function, Fn probably plays a role in embryogenesis, wound healing, and blood clotting. Therefore, we assume that the drug-induced reduction of Fn possibly has clinical relevance.

Factor VII Half‐life after Transfusion of a Steam‐treated Prothrombin Complex Concentrate in a Patient with Homozygous Factor VII Deficiency

Factor VII (FVII) deficiency is a rare bleeding disorder which can be treated effectively with prothrombin complex concentrate (PCC) [ 1, 21. However, these preparations may transmit viral diseases or induce accelerated intravascular coagulation. PCCs are frequently used for the treatment of acquired defects in hemostasis, such as result from excessive loss of blood, liver disease or coumarin treatment. Transfusions of PCCs in cases of inherited deficiencies of coagulation factors are well suited to assess the efficacy of these preparations. We report on a male patient with moderate, crossreacting material (CRM)-positive FVII deficiency who underwent extraction of 12 teeth with prophylactic transfusion of a steam-treated PCC. He was 32 years of age with no history of bleeding and had a FVI1:C (as determined with a one-stage assay using reagents and standards from Immuno, Vienna) of 0.03 U/ml and a FVI1:Ag of 26% (using antibodies from Behring, Marburg). Reduced FVII levels could also be detected in his mother (0.56 U/ml) and his son (0.34 U/ml). One day prior to surgery the patient received 2,000 U of a steam-treated PCC (Prothromblex TIM4, Immuno, Vienna, containing 2,220 U FVII, as determined using the above-mentioned one-stage assay) to assess the in vivo recovery and half-life as described recently [3]. After transfusion of 2,OOOU PCC (28.6U FVII:C/kg body weight), FVII reached a peak level of 0.88 U/ml. The decay of FVII activity is shown in figure 1. Using a monoexponential model, a half-disappearance time of 6.6 h was estimated. Using a biexponential model, the distribution and elimination half-lives were 3.8 and 15.4 h, respectively (fig. 1). The calculated distribution volume was 2,571 ml, the in vivo recovery was 133%. After infusion of PCC, no signs of Fig. 1. Factor VII activity (triangles) after transfusion of 2,000 U PCC. The broken line represents the calculated curve of decay of FVII activity (y=0.708* exp (-0.003*t) + 0.155* exp(-O.O0075*t) + 0.03).

The use of antithrombotic agents in microvascular surgery

Despite increasing advances in microvascular free tissue transfer, flap failures, most commonly resulting from thrombosis at the anastomotic vascular site, remain a significant concern. Although several experimental and clinical studies have been carried out, no consensus has been reached so far on the efficacy, dosage and timing of anticoagulant agents available for the prevention and treatment of thrombosis in microvascular surgery. Inhibition of fibrin formation and platelet function or the use of thrombolytic agents is a common approach in the antithrombotic management. However, some agents exhibit serious side effects and all of them carry the risk of bleedings. The current literature on the use of antithrombotic agents, targeting at clinical trials in microvascular surgery, is therefore reviewed, to provide an informative basis for recommendations for an appropriate pharmacological approach.

Elimination kinetics of different hydroxyethyl starches and effects on blood fluidity

In a single blind randomized cross-over study 6 volunteers each received infusions of 500 m1 of 6 different types of hydroxyethyl starch (HES) solutions. Between the test periods there was a regular wash-out phase of 3 months. The concentration and molecular weight distribution of the intravascular HES-molecules as well as the blood fluidity were determined before and up to 24 hours after the infusions. The C2/C6 substitution ratio and the molar substitution exert an essential influence on the elimination of the HES-molecules. An increase in the C2/C6 substitution ratio from 4.6 to 10.8 with unchanged molecular weight (MW) and molar substitution brings about an increase in the half-life of the elimination in the 13 -phase t1I2-13 from 10.4 to 19.5 hours. In case a high C2/C6 substitution ratio is combined with a high molar substitution (0.62 instead of 0.5), half-life rises up to 36.1 hours. Whereas the molecular weights of all medium-molecular HEStypes (MW 200,000) decrease, the medium molecular weight of the

On the prophylactic and therapeutic use of danaparoid sodium (Orgaran®) in patients with heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a rare but dangerous complication of heparin prophylaxis or treatment. The present laboratory tests to measure heparin-associated antibodies are not specific. The diagnosis of HIT mainly depends on the decrease in platelet count and on clinical symptoms. To evaluate clinical outcome, bleeding complications and platelet counts were evaluated in 45 patients with HIT type II (HIT II) treated prophylactically (subcutaneous injections) or therapeutically (intravenous infusion) with danaparoid. Group I included 24 patients with HIT II without thromboembolic complications who received danaparoid twice daily subcutaneously (10 IU/kg) for a mean of 16 days. Group II included 21 patients with thromboembolic complications. They were treated with intravenous danaparoid (2.6 IU/kg/h ± 1.1) for a mean of 17 days. During subcutaneous prophylaxis, mean anti-Xa levels of 0.2 U/mL and during intravenous treatment, mean anti-Xa levels of 0.4 U/mL were reached. No deaths, amputations, or serious bleeding complications occurred, and no new thromboses were observed in both patient groups.Treatment with danaparoid led to a fast normalization of the platelet counts. This normalization occurred earlier and the concentration of platelets was higher in patients treated with intravenous doses. Danaparoid with subsequent vitamin K-antagonist treatment effectively prevents thromboembolic complications in patients with HIT.

Fibrinolytic and procoagulant activity in septic and haemorrhagic shock.

Septic and haemorhagic shock carry the risk of high mortality. Failure of microcirculation secondary to alterations of haemostasis and fibrinolysis play a major role in the pathogenesis of shock. The aim of this study was to evaluate the clinical relevance of procoagulatory and fibrinolytic activities referring to survival. Therefore, 39 patients (23 to 80 yrs, 16 females, 23 males) suffering from haemorrhagic (n = 21) and septic shock (n = 18) were screened prospectively for plasmatic coagulation and fibrinolysis parameters. Thirteen patients (33.3%) developed lethal outcome. Concerning fibrinolysis, plasminogen was significantly lower in non-survivors by day 1 and plasmin-antiplasmin complex significantly higher by day 4 compared to survivors. Consecutive increase of plasminogen over day 4 and 7 was significantly stronger in survivors. Concerning haemostasis activation, thrombin-antithrombin complex was higher and D-dimers or fibrinogen levels were lower, but not significantly different, in non-survivors compared to survivors. We conclude from these data, that procoagulant activities are increased, but not significantly predictive for the clinical outcome in septic and haemorrhagic shock. By contrast, fibrinolysis, as measured by enhanced capacity and responsiveness, is clearly predictive and plays a significant role for survival, possibly due to its clearing function in microcirculation.