Circulating microemboli in patients with myeloproliferative disorders.

Myeloproliferative disorders (MPD) are associated with an increased risk for thrombembolic events. In this study, we examined the prognostic value of transcranial Doppler (TCD) microemboli detection regarding clinical events and correlated TCD findings with results of blood cell counts and platelet flow cytometry to gain insight into the composition of circulating microemboli in these patients. In a cohort of 42 patients with MPD TCD microemboli detection was performed on a single occasion and correlated with thrombembolic events during a prospective follow up of 29.7 +/- 7.3 month. In all patients, a complete blood count and in 17 patients platelet flow cytometry were performed on the day of the TCD examination. Microembolic signals (MES) were recorded in 15 (35.7%) patients, however, without any correlation with the type of MPD, blood cell counts, or thrombembolic events [9 (21.4%)]. MES positive and negative patients did not differ regarding the levels of activated platelets, platelet microaggregates, or microparticles. We found a strong trend for higher rates of platelet-neutrophil conjugates in MES positive patients (P = 0.09). Detection of MES by TCD on a single occasion in MPD patients has only limited prognostic value. MES do not correlate with the type of MPD, nor blood cell counts. Flow cytometry suggests that MES in MPD may consist of platelet-neutrophil aggregates.

Comparison of GP IIB/IIIA inhibitors and their activity as measured by aggregometry, flow cytometry, single platelet counting, and the rapid platelet function analyzer.

BACKGROUND: GP IIb/IIIa inhibitors have primarily been used short-term e.g., during PTCA. They failed to show clinical benefit during long-term therapy. One reason might be the absence of a method to monitor inhibitor activity. This study compared platelet aggregometry, the rapid platelet function analyzer (RPFA) test, single platelet counting, and flow cytometric determination of receptor occupancy to measure GP IIb/IIIa-receptor inhibitor activity. METHODS: Increasing doses of abciximab, tirofiban, and eptifibatide were added to whole blood in vitro. Whole blood was used for the RPFA, for single platelet counting and flow cytometry. Platelet rich plasma was prepared for aggregometry. RESULTS: The correlation between aggregometry and RPFA results was linear for abciximab and eptifibatide. Tirofiban was a stronger inhibitor with the RPFA (IC(50) 7.7nM) than with aggregometry (IC(50) 19.6nM). The single platelet counting technique showed that even supratherapeutic concentrations of all three inhibitors could not completely suppress microaggregation. Abciximab concentrations that were equipotent to tirofiban with aggregometry were less potent with regards to the inhibition of microaggregation. This difference was more pronounced with TRAP induced microaggregation than with ADP. The flow cytometric receptor occupancy test showed that occupancy was 95% with 5 microg/ml abciximab and almost 97% with 10 microg/ml. Tirofiban reached a maximum receptor occupancy of 56%, eptifibatide 64%. CONCLUSIONS: While aggregometry is time consuming the RPFA provides results fast and with little variability. There is still a discrepancy between aggregometry and RPFA results for tirofiban. The single platelet counting technique detects the inhibition of microaggregation the relevance of which for the clinical outcome is not known. The flow cytometric receptor occupancy assay is best suited for abciximab.

Perioperative management of a patient with Fechtner syndrome.

Fechtner syndrome is a rare type of familial thrombocytopenia associated with large platelets, leukocyte inclusions, and features of Alport's syndrome. The bleeding tendency is usually mild, but severe hemorrhages have been reported. This is the case of a patient with Fechtner syndrome who was scheduled to undergo tonsillectomy. The patient had a history of easy bruising in childhood and a markedly prolonged bleeding time. Administration of DDAVP led to normalization of the bleeding time, and the patient underwent surgery without complications. With this approach the use of platelet concentrates could be avoided.

Effect of glycoprotein IIb/IIIa inhibitors on CD62p expression, platelet aggregates, and microparticles in vitro.

Flow cytometry can detect platelet activation (CD62p), aggregate formation, microparticle formation, and glycoprotein IIb/IIIa (GP IIb/IIIa) receptor occupancy in one sample at the level of single particles. We studied the effect of GP IIb/IIIa inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GP IIb/IIIa inhibitors (c7E3, DMP728, XJ757), then thrombin or adenosine diphosphate (ADP) was added, and after 1 minute the sample was fixed. Samples with thrombin but without c7E3 had a decrease in platelet count, from a mean of 260,000 platelets/microl to 56,000 platelets/microL, and aggregates increased. Samples with concentrations of c7E3 that resulted in 80% or more receptor blockade had no decrease in platelet count, and no aggregates were formed, but the number of CD62p-positive single platelets increased from 1200 to 7400 platelets/microL. The two other inhibitors (DMP 725, XJ757) or ADP instead of thrombin gave similar results. Microparticle formation did not change with platelet activation in the presence of a GP IIb/IIIa inhibitor. With small inhibitor doses resulting in <80% receptor blockade, the number of aggregates did not change or was even higher than that in samples without inhibitor. GP IIb/IIIa inhibitors do prevent aggregate formation but they do not prevent activation of platelets. With GP IIb/IIIa inhibition, more activated single platelets remain in the blood. One may expect an increasing number of circulating, activated platelets with the use of GP IIb/IIIa inhibitors.

Pharmacology of warfarin and clinical implications.

The history of oral anticoagulants started in the 1920s in North Dakota and Alberta when a new type of hemorrhagic disease struck cattle in these areas. The group of Karl Paul Link finally succeeded in isolating the causative agent, dicumarol. It was not before the 1940s, that dicumarol or its derivatives were introduced to medicine. Acenocoumarol, phenprocoumon, and warfarin are the most commonly used oral anticoagulants. There is no known difference in the pharmacodynamic activity of these agents on the vitamin K metabolism. They are completely absorbed from the gastrointestinal tract and are firmly bound to plasma albumin and metabolized in the liver. The different elimination half-lives of the coumarins have several implications for patient management. Absorption, protein binding, and anticoagulant activity of oral anticoagulants are affected in many different ways. Also the different pharmacological properties of coumarins require different strategies in the clinical management of patients.

Quantitative assessment of platelets, platelet microparticles, and platelet aggregates with flow cytometry.

With fluorescent beads it has become possible to determine absolute numbers of cells in a given sample instead of relative percentages on a standard flow cytometer. This study assesses the ability to count platelets, microparticles, and aggregates with a flow cytometer. Whole blood was stimulated with 0.1 U thrombin per milliliter. Platelet and microparticle counts decreased, while the number of aggregates increased. Unactivated whole blood was diluted with buffer and showed a corresponding decrease in the concentration of platelets, microparticles, and aggregates. The platelet count on the flow cytometer was always in good correlation with counts on an automated blood analyzer. Only the cytometer, and not the automated analyzer, was able to detect and count microparticles and aggregates. In highly diluted samples of unactivated whole blood there was a spurious relative increase in CD62p-positive platelets because of a surplus of anti-CD62p antibodies and a relative increase in microparticles. Flow cytometry is a valuable method for counting platelets, aggregates, and microparticles in unstimulated and activated blood samples. If the platelet count changes and drops to less than 50% of the count for which the amount of antibody and the cytometer settings have initially been adjusted, care has to be taken to avoid misinterpretation.

Relative and absolute changes of activated platelets, microparticles and platelet aggregates after activation in vitro.

Standard flow cytometers provide relative numbers of activated platelets, microparticles, and platelet aggregates. With fluorescent beads it is now possible to determine absolute numbers. Whole blood and platelet-rich plasma were incubated with agonists (ADP, collagen, thrombin). CD62p expression, microparticle and platelet aggregate formation were measured. Flow-Count Fluorospheres((R)) were added to calculate absolute concentrations. After activation there was an increase in the percentage of CD62p-positive platelets. However, the total number of platelets decreased and therefore the absolute number of CD62p-positive platelets did not increase but decreased. The number of CD62p-positive platelets decreased not as much as the number of CD62p-negative platelets, which explains why the relative percentage of CD62p-positive platelets increased. A similar increase in percent and decrease in absolute counts was found for microparticles. Platelet aggregates increased both in relative and absolute numbers. These results suggest that the detection of activated platelets by flow cytometry has to be complemented by the determination of the absolute concentrations to avoid misinterpretation.

Reactivity of platelets from healthy individuals and from patients with hematologic diseases following incubation with pegylated recombinant human megakaryocyte growth and development factor in vitro.

Platelets express the receptor for thrombopoietin. It is possible that thrombopoietin modulates platelet reactivity. We examined the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet activation in vitro using flow cytometry. We compared samples from healthy individuals and from patients with various hematologic diseases (AML, myeloma, postchemotherapy). Citrated whole blood was incubated with PEG-rHuMGDF (10 or 100 ng/ml), then a mild stimulus was added (0.1 U thrombin/ml). Blood from healthy individuals showed a significantly higher degree of platelet activation (CD62p expression), microparticle generation, and aggregate formation after incubation with PEG-rHuMGDF+thrombin versus thrombin alone (p < 0.05). However, this difference could not be shown for platelets from patients with thrombocytopenia or other hematologic diseases. The use of PEG-rHuMGDF should be safe and not cause an additional risk of thromboocclusive disease in these patients.

[Visceral leishmaniasis with an unusually long incubation time]. / Viszerale Leishmaniose mit ungewöhnlich langer Inkubationszeit.

HISTORY: A 25-year-old woman of Yugoslavian origin came to Germany two years before and did not leave Germany since this time. She developed a phlebothrombosis during pregnancy which was treated surgically and with subsequent heparinisation. The pregnancy had to be terminated by section because of abnormal liver functions and increased blood pressure. These values returned to normal within two months. Further tests again showed raised liver function tests (GOT 57 U/l, GPT 71 U/l) and antibodies against smooth muscle and actin. Autoimmune hepatitis was diagnosed and prednisolone given (100 mg daily). In the subsequent 4 months the patient progressively lost more weight and a pancytopenia developed. Suspected of having a systemic haematological syndrome she was admitted to hospital. FINDINGS: Physical examination was unremarkable except for hepato- and splenomegaly (spleen 15.6 cm in diameter by sonography). Laboratory tests showed hypergammaglobulinaemia (50 g/l, 53%), increased WBC count, as well as decreased haemoglobin concentration and platelet count (900 WBC/microliter, Hb 10.9 g/l, 146,000 platelets/microliter). Bone marrow puncture unexpectedly revealed a large number of Leishmania donovani. TREATMENT AND COURSE: Five-valent antimony was administered (sodium stibogluconate 20 mg/kg daily intravenously as bolus for 14 days). She has been free of symptoms since then (follow-up period of one year). CONCLUSION: Visceral leishmaniasis is a rare disease in Europe. Incubation periods of several years have been reported and the infection can be easily mistaken for other chronic liver disease, in this case for an autoimmune hepatitis. Leishmaniasis should be included in the differential diagnosis of unclear liver disease if there is a suggestive history (country of origin or journey into an endemic area).

Comparison of beta-thromboglobulin, flow cytometry, and platelet aggregometry to study platelet activation.

This study compares granule membrane protein (GMP)-140 expression measured by flow cytometry, release of beta-thromboglobulin (beta-TG), and platelet aggregometry as markers of platelet activation in vitro. Whole blood was activated with different concentrations of thrombin. There was a significant increase in beta-TG plasma levels after stimulation with 0.01 and 0.04 U thrombin/ml. There was also an increase in GMP-140 expression, but interindividual variability was high. Aggregometry of platelet-rich plasma did not detect platelet activation and formation of platelet aggregates with 0.05 and 0.1 U thrombin/ml, while flow cytometry showed an early and significant increase of GMP-140 expression with these doses. Beta-TG release is a more sensitive marker of platelet activation than GMP-140 while flow cytometry is easier to perform and less susceptible to artifacts.

Effect of recombinant aprotinin on platelet activation in patients undergoing open heart surgery.

The hemostatic effect of recombinant (r)-aprotinin was studied in 41 patients undergoing cardiopulmonary bypass surgery. Flow cytometry was used to measure the expression of glycoprotein 1b (GP1b) and alpha-granule membrane protein 140 (GMP-140) on platelets. Additional parameters studied were beta-thromboglobulin (beta-TG), fibrinogen, fibrinopeptide A, plasminogen, platelet count, and the amount of postoperative chest tube drainage. Chest tube drainage was significantly less in the r-aprotinin-treated patients (p < 0.001). The percentage of platelets expressing GMP-140 increased to 9.7% in r-aprotinin patients and to 12.1% in controls during the surgery. The difference between both groups was not significant. Both values returned to baseline the next day. GP1b expression decreased in both groups by 33-38% during the surgery and returned to baseline the next day. GP1b expression and the plasma concentrations of fibrinogen, fibrinopeptide A, beta-TG, and plasminogen were not different in r-aprotinin and control patients. We conclude that r-aprotinin reduces blood loss during cardiopulmonary bypass surgery, but the primary mechanism is likely not through a protective effect on platelets.

Deep vein thrombosis and pulmonary embolism: prevention, diagnosis, and treatment.

Pulmonary embolism (PE) is a significant cause of mortality in the elderly. More than 90% of pulmonary emboli originate from a thrombus in the deep veins of the legs. Proper diagnosis and treatment of deep vein thrombosis (DVT) are thus essential to prevent PE. Diagnosis of new or recurrent DVT is based on the results of one or more tests, including impedance plethysmography (IPG) or duplex venous scan; venography can often be avoided, based on results of initial testing. For suspected PE, perfusion lung scanning is the initial test of choice, followed by IPG/duplex or venography. Pulmonary angiography is indicated for patients with decreased cardiorespiratory reserve. Decisions governing prophylaxis of DVT are based on individual relative risk; prophylactic therapies include intermittent compression, low-dose heparin, and oral anticoagulants. Management of thromboembolism requires IV and oral anticoagulant therapy.