Does hemodilution enhance coagulability?

UNLABELLED: Recent publications reported enhanced coagulability in hemodilution determined by TEG. In contrast, earlier reports have shown prolongation of in-vivo bleeding time in anemia. In order to take a closer look at this discrepancy undiluted and diluted anticoagulated blood samples (20 % with saline solution, hydroxyl-ethyl starch 6 % (HES), autologous platelet poor plasma (PPP)) were investigated by TEG (n = 10), ball (n = 10), and hook coagulometer (n = 15) as well as tests simulating primary hemostasis ex vivo (Platelet Function Analyzer PFA-100, n = 10). RESULTS: Dilution with plasma changed TEG parameters in a way, when started by recalcification of the blood sample, which is characteristic of enhanced coagulability (r decreased in all and k in 8 of 10 samples, maximal amplitude increased in 9 out of 10). With HES, changes in TEG parameters mainly indicated reduced coagulability (k increased in 7 out of 10, MA decreased in 10 out of 10). When the coagulation was additionally activated by PTT reagent (InTEG) the TEG parameters also mainly showed hypocoagulation with the three dilution solutions. Coagulation times with ball and hook coagulometers were significantly prolonged by dilution especially with saline (+ 25 % and + 17 %, p < 0.001). Dilution always significantly (often abnormally) prolonged closure time in PFA-100 (saline + 41 +/- 18 %, PPP + 37 +/- 20 %, HES + 69 +/- 24 %) demonstrating disturbance of primary hemostasis, particularly with HES. CONCLUSIONS: From the results obtained it can be concluded that the changes in the classical TEG (without addition of PTT-reagent), suggesting an enhanced coagulability, may be caused methodically as they are also found with autologous PPP. On the other hand, a disturbance of the primary hemostasis in hemodilution has to be taken into account from the results seen with the PFA-100 and a number of published data.

Measurement of primary haemostasis using a pressure clamp technique.

UNLABELLED: With the PFA-100 a sensitive and specific screening test for primary haemostasis has recently become available. An important part of the device is a capillary, providing a defined haemodynamic resistance for the perfusion of the aperture. A modified method to measure platelet function (VCP2) is presented in which the capillary essentially is replaced with an 'electronic capillary' by clamping the pressure/flow relationship. RESULTS AND CONCLUSION: Closure time (CT) and blood volume (BV) as determined by PFA-100 and VCP2 correlated well within (r = 0.922 - 0.952) and between the two methods (r = 0.86). The test variability (CV) of CT could be significantly reduced in the VCP2 method (collagen/epi 3.9 vs. 5.9%, p<0.05; collagen/ADP 3.3 vs. 6.9%, p<0.001), thus considerably increasing test reliability and reducing test variance. In preliminary clinical studies the VCP2 system showed comparable sensitivity for vWD and slightly less sensitivity regarding ASA ingestion. The test spectrum of VCP2 could be extended to more thrombocytopenic samples (< or =20 000/microl) even in combination with low haematocrit levels (20%), thus perhaps permitting the determination of the bleeding risk in bone marrow hypoplasia. Additionally, the sensitivity and applicability can easily be adapted to the desired need only by software modifications.

[The effects of aprotinin. Blood loss and coagulation parameters in orthotopic liver transplantation: A clinical-experimental, prospective and randomized double-blind study]. / Die Wirkung von Aprotinin. Blutverlust und Gerinnungsparameter bei orthotoper Lebertransplantation: Eine klinisch-experimentelle, prospektive und randomisierte Doppelblindstudie.

The effect of aprotinin (2,000,000 IU as a bolus +500,000 JU/h until the end of the operation) on transfusion requirements and coagulation parameters in orthotopic liver transplantation (study group: n = 9; placebo group: n = 9) was investigated in a randomised, double-blind study. Coagulation parameters were monitored intraoperatively using a mobile laboratory. In contrast to the published results, no effect on transfusion requirements could be demonstrated. However, aprotinin showed a positive effect on some coagulation parameters in the reperfusion phase. The mechanism appeared to be inhibition of the contact activation of the intrinsic system with less thrombin generation in the study group.

Prediction of the transfusion effect of platelet concentrates as measured by a model of primary hemostasis ex vivo.

A method to determine primary hemostasis ex vivo (Thrombostat) was modified to monitor the transfusion effect of platelet concentrates (PC) in 12 patients with thrombocytopenia following bone marrow transplantation. It was possible to measure platelet function in patients with a platelet count lower than 2 x 10(10)/l. In addition, the platelet aggregometer (Born) was adapted to determine cell function in PC anticoagulated with acid citrate dextrose of citrate phosphate dextrose. It was possible to make a prediction (r = 0.89) of the effect of a given PC on a patient's ex vivo primary hemostasis parameters. Platelet aggregation following addition of 20 muM ADP to PC, obtained from 12 single donors, resulted in an average maximal light transmission (light transmission/age of concentrate in days) of 61%/1 day and 37%/5 days, respectively. The same experiment gave only 39%/1 day and 13%/4 days for pooled platelets. To avoid possible immunization and bleeding complications, a reliable monitoring of platelet transfusion seems highly desirable.

Interaction of antiplatelet drugs in vitro: aspirin, iloprost, and the nitric oxide donors SIN-1 and sodium nitroprusside.

The interaction of three antiplatelet drugs was studied in vitro: aspirin, an inhibitor of the cyclooxygenase pathway of platelet activation; iloprost, a stable analog of prostacyclin that increases platelet cAMP; and the nitrix oxide donors SIN-1 and sodium nitroprusside (SNP), which both raise platelet cGMP. Platelet adhesion and aggregation evoked by collagen/ADP were measured in anticoagulated blood under physiological flow conditions using the new Thrombostat. Aggregation was also measured in platelet-rich plasma (PRP) upon stimulation by a low (2.5 micrograms/ml) and high (20 micrograms/ml) dose of collagen, ADP, or thrombin-receptor activating peptide (TRAP). We found a synergism between iloprost and aspirin in inhibiting platelet adhesion/aggregation in flowing blood and aggregation of PRP stimulated by collagen. The mean inhibitory concentrations (IC50) of iloprost in the presence of aspirin were much lower (0.7 nM and 0.5 nM in flowing blood and low-dose collagen-stimulated PRP, respectively) than in the absence of aspirin (3 and 3.6 nM, respectively). Synergism between SIN-1 and aspirin was observed in inhibiting platelet activation in flowing blood but was much less pronounced in inhibiting collagen-induced aggregation of PRP. SIN-1/SNP and iloprost synergistically inhibited the aggregation of PRP induced by collagen as well as platelet adhesion/aggregation in blood. We found that two protein substrates of cAMP- and cGMP-dependent protein kinases, rap1B and a 50 kD protein, were associated with the functional synergism between SIN-1 and iloprost and were synergistically phosphorylated by platelet treatment with both iloprost and SIN-1. Platelet inhibition by SIN-1, iloprost, and aspirin was synergistic when measured in blood. In contrast, only additive effects of SIN-1 and iloprost were observed when platelet aggregation was measured in aspirin-treated PRP stimulated by ADP, TRAP, or collagen. Our study defines the basis for a more effective antiplatelet therapy using a combination of cGMP- and cAMP-elevating and cyclooxygenase-inhibiting drugs. The results also emphasize the importance of using various methods for the evaluation of antiplatelet drugs.

The Thrombostat system. A useful method to test antiplatelet drugs and diets.

UNLABELLED: The use of platelet inhibitory drugs, like aspirin, has resulted in a significant reduction of thrombotic complications in primary and secondary prevention of heart attacks. To find more effective substances or better drug combinations, inhibition of primary hemostasis in vitro (Thrombostat system) was investigated, with different drugs and fish diet, using small samples (1 ml) of anticoagulated (Na- citrate 3.8%, 1/9) human blood. RESULTS: 1. In the presence of 1 mM aspirin, which had no effect on bleeding volume, only 0.6 nM iloprost were necessary to show a 50% inhibition, in contrast to 2.5 nM without aspirin. 2. At aspirin concentrations of 1 mM, 50% inhibition of primary hemostasis could be achieved with 20 microM SIN-1, or with 7 microM SIN-1 together with iloprost (500 pM). The same effect was seen only with very high doses of SIN-1 (1000 microM) alone. 3. For 50% inhibition of primary hemostasis in vitro, RGDS concentrations were reduced from 250 microM to 160 microM when blood was pretreated with 1 mM aspirin and to 75 microM when 500 pM iloprost were added additionally. 4. Japanese fishermen (eating 270 g fish/day) demonstrated significantly longer in-vivo bleeding times and in-vitro bleeding volumes (6.49 min/224 microliters), respectively, as compared to Japanese farmers (90 g fish/day, 4.85 min/137 microliters). 5. In Japanese subjects in-vivo bleeding times correlated with in-vitro bleeding volumes (0.69). The Thrombostat system proved to be a sensitive method to detect synergistic effects of various antiplatelet drugs in vitro and of a platelet inhibitory diet ex vivo.

Quality control of platelet concentrates by the Thrombostat 4000.

Quality control of platelet concentrates (PC) is an important prerequisite for good transfusion praxis. However, direct measurement of platelet function is complex, since available methods (e.g. aggregometry, serotonin release) are time consuming and require special equipment. Therefore a test system is needed, which is easy to handle, fast, and achieves reliable results. The present paper compares the results of conventional platelet function tests with those of a modified in-vitro bleeding test (IVBT) (Thrombostat 4000) in liquid-stored and cryopreserved PCs. A high correlation between aggregometry, serotonin release, GMP 140 expression upon stimulation, and IVBT was demonstrated. Therefore IVBT seems to be a good alternative to the conventional platelet function tests for quality control of PCs. In addition, a good correlation between the results of IVBT of patients' blood after PC transfusion and IVBT of patients blood before transfusion supplemented with platelets of the respective PC could be found. Therefore IVBT seems to be able to predict PC transfusion success. However, since these data were obtained in a small sample undergoing bone marrow transplantation, further studies are needed to verify this hypothesis.

[Quality control of platelet concentrates. Functional assessment of stored platelets in vitro]. / Qualitätskontrolle von Thrombozytenkonzentraten. Funktionsmessung konservierter Thrombozyten in vitro.

Platelet concentrates transfused for correction of thrombocytopenia or reduced platelet function do not consistently improve primary haemostasis in the recipient. Insufficient therapeutic effects may be caused by impaired donor platelet function and by unfavourable donation and storage conditions, as well as by immunological interactions with the recipient blood. The present study was designed to investigate whether the effect of platelet transfusion on recipient platelet function can be predicted by in vitro methods. METHODS. Blood samples were taken from 12 thrombocytopenic patients before (20 ml, P0) and after (10 ml, P(vivo)) transfusion of one unit of platelets previously stored for 24-120 h in acid citrate dextrose. An additional sample was taken from the platelet concentrate (TK) immediately before transfusion. P0 was divided into two specimens and TK platelets were added to one of them (P(vitro) in order to obtain a platelet count similar to that in P(vivo). Bleeding time (BT) and bleeding volume (BV) of the samples P0, P(vivo) and P(vitro) were measured using the method of Kratzer and Born (Fig. 2); mean values were calculated for each sample from six measurements. Aggregability of TK platelets was determined in addition by aggregometry. In contrast to previous studies, physiological Ca2+ concentrations were restored and secondary haemostasis was inhibited by low-molecular-weight heparin (Fragmin P, Pfrimmer Kabi GmbH und Co. KG, Erlangen) in the platelet-rich plasma used for aggregometry. RESULTS. Platelet counts increased in all patients after transfusion (P(vivo) vs P0, Table 1) and were nearly identical in P(vitro) and P(vivo) (r = 0.94, P < 0.001; Fig. 3). Parameters of primary haemostasis were significantly improved by addition of platelets to P0 in vitro (BT P < 0.05, BV P < 0.01) as well as by platelet transfusion (BT P < 0.05, BV P < 0.01). Direct comparison of P(vitro) and P(vivo) yielded a very close correlation of BT (r = 0.88, P < 0.001) and BV (r = 0.89, P < 0.01) in both samples. Although aggregometry revealed decreasing platelet function with increased storage time, aggregability was considerably higher compared to previous studies of platelet concentrates stored for 2-5 days. CONCLUSION. A new technique has been developed which allows reliable prediction of the effect of platelet concentrates on primary haemostasis of the recipient by in vitro measurement of bleeding time and bleeding volume prior to transfusion using the method of Kratzer and Born.

[Monitoring blood coagulation in larger liver operations]. / Monitoring der Blutgerinnung bei grossen Leberoperationen.

Bleeding is causally related to about 50% of postoperative deaths following liver resection. Main factors contributing to increased perioperative bleeding in liver surgery include surgical trauma, reduced activity of clotting factors and inhibitors due to impaired hepatic synthesis, low platelet count and poor platelet function as well as impaired clearance of activated clotting factors by the reticuloendothelial system of the liver (Kupffer cells). Hemostasis may be further impaired by transfusion of blood components, since citrate added for conservation is not adequately metabolized by the failing liver. Surgical bleeding leads to a loss of pro- and anticoagulatory factors as well as to activation of coagulation. Finally, hyperfibrinolysis induced by release of tissue plasminogen activator (t-PA, primary hyperfibrinolysis) and disseminated coagulation (secondary hyperfibrinolysis) contribute to increased bleeding. Therefore early diagnosis and treatment of coagulation disorders is of paramount importance during liver surgery. Screening parameters of hemostasis and fibrinolysis should be available on a 24-hour basis in centers performing liver surgery. Screening for disorders of secondary hemostasis includes evaluation of prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration and the activity of the most important inhibitor, antithrombin III (AT III). Thrombelastography is the leading method for diagnosis of hyperfibrinolysis, which can also be assessed by determination of D-dimer, fibrinogen and fibrin degradation products. Evaluation of primary hemostasis is frequently restricted to platelet count, which is only a rough parameter. In contrast, measurement of in vitro bleeding time and volume enables repeated quantification of platelet function in patients with impaired hemostasis.

Neuronal network analysis of serum electrophoresis.

AIMS: To advise a system of neuronal networks which can classify the densitometric patterns of serum electrophoresis. METHODS: Digitised data containing 83 normal and 132 pathological serum protein electrophoresis patterns were presented to four neuronal networks containing 1900 neurons. Network 1 evaluates the integrated values of the albumin, alpha 1, alpha 2, beta and gamma fractions together with total protein (Biuret method). Networks 2, 3, and 4 analyse the shape of the albumin, beta and gamma fractions. To increase the sensitivity for the detection of monoclonal gammopathies a Fourier transformation was applied to the beta and gamma fractions. RESULTS: After a learning period of 20 minutes (back-propagation learning algorithm) the system was tested with a set of electrophoresis patterns comprising 446 routinely collected samples. It differentiated between physiological and pathological curves with a sensitivity of 97.5% and a specificity of 98.8%, with 86% correct diagnoses. All monoclonal gammopathies were recognised by the Fourier detector. CONCLUSIONS: Neuronal networks could be useful for certain medical uses. Unlike rule based systems, neuronal networks do not have to be programmed but have the capacity to "learn" quickly.

[Combination of a simple hollow fiber system with leukocyte filter for production of leukocyte depleted erythrocyte concentrates and plasma]. / Kombination eines einfachen Hohlfaserfiltersystems mit einem Leukozytenfilter zur Herstellung leukozytendepletierter Erythrozytenkonzentrate und von Plasma.

Leukocyte-depleted red cell concentrate (RCC) and plasma were separated by a hollow fiber filter system combined with a leukocyte filter without any additional devices. The RCC with 100 ml additive solution had a weight of 329 g; hematocrit was 0.55, free hemoglobin 16 mg/dl; leukocytes were (0.6 +/- 0.6) x 10(9)/l. The plasma (268 g) contained 5.4 g/dl of total protein, and only a few blood cells; clotting factor VIII activity 75%, all satisfying the guidelines.

Increasing platelet aggregability after venepuncture is platelet, not plasma derived.

The time course of ADP induced aggregation of human platelets was determined in aliquots of stored platelet rich plasma 3.5, 10, 30 and 100 minutes after venepuncture. The maximal rate of aggregation was found to increase throughout this entire period, even though pH (7.4), CO2 (7 volume per cent) and temperature (35 degrees C) of the samples were kept constant. The mean acceleration (+/- SEM) between 3.5 and 100 minutes was 41.7 +/- 6.9 per cent (n = 67) at an ADP-concentration of 1 mumol/l and 18.3 +/- 6.2 per cent (n = 23) at 2 mumol/l ADP. The effect did not result from changes of any platelet regulatory factors putatively present alone in the plasma. Acceleration of aggregability was only found when the platelets themselves underwent storage, but not when freshly prepared plasma was given to prestored platelets. The change in aggregability was not diminished after inhibition of platelet cyclooxygenase by oral administration of acetylsalicylic acid.

Streamline pattern and velocity components of flow in a model of a branching coronary vessel--possible functional implication for the development of localized platelet deposition in vitro.

A simplified model of the left descending coronary artery was perfused with a suspension of tungstic acid particles in dimethyl sulfoxide/water solution. To visualize the streamline pattern within the main tube, flow was illuminated with laser light using the dark field technique. Moving particles appeared as points, or, when illuminated with pulsating light, as a series of points. They were photographed and evaluated with respect to the velocity vector normal (vl) and parallel (v =) to the vessel wall. Wall shear stress (tw) was calculated from (v =) and fluid viscosity. At Reynolds (Re) numbers comparable to the in vivo situation, defined flow disturbances were observed in the main tube: (i) Separation of the flow on the wall facing the opening of the side branch and (ii) development of a stagnation point flow at defined locations. Comparison of our results with experiments using platelet suspensions, allowed those hydrodynamic conditions to be determined which are responsible for the deposition of platelet microthrombi in vitro. These conditions were identified as being positive velocity components toward the wall (vl greater than 0).

Simulation of primary haemostasis in vitro.

Primary haemostasis was simulated in vitro under standardized geometrical, rheological and biochemical conditions. The system was based on the hypothesis that adenine nucleotides, especially adenosine 5'-diphosphate, released from injured vessel wall cells play an important role for the initial platelet aggregation following lesion of small vessels. The device allows the reproducible measurement of in vitro bleeding time and volume in small samples of whole blood.