Andreas Salat

Influence of hematocrit and platelet count on impedance and reactivity of whole blood for electrical aggregometry

Previous studies have shown that differing qualities of blood specimen seem to influence whole blood electrical aggregometry (WBEA), making it difficult to standardize the method. The aim of this study was to investigate the impact of hematocrit (HCT) and platelet count (PLC) on in vitro platelet aggregation in citrated whole blood (CWB) in order to compensate for their possible effects on impedance aggregometry. Red blood cells and blood platelets were isolated from fresh citrated whole blood taken from 15 healthy donors (mean age = 26 years) and recombined to 20 physiologically relevant combinations of hematocrit and platelet count (HCT: 20-50, PLC: 100-500). Platelet aggregability was measured using WBEA with three different triggers. A special-purpose software package was used in this study, ensuring proper calibration, acquisition, and evaluation of analogue to digital converted data, allowing the calculation of a set of characteristic parameters of each impedance curve. Most of the linear regressions showed that all parameters significantly depend on HCT and PLC. Furthermore, we found interactions of both variables, making it impossible to focus on the effects of one of the investigated variables only. The outcome of this study is a set of dependences, allowing the calculation of regressions for in vitro aggregation in whole blood, enabling a comparison of blood of any quality with each other, regardless of the variables HCT and PLC. Together with the previously defined dependence of sample age on WBEA data, this step should help to make this technique a more reliable and practicable clinical tool, making it suitable for daily routine investigations.

Extensive coagulation monitoring in patients after implantation of the MicroMed Debakey continuous flow axial pump.

Ventricular assist device (VAD) implantation is associated with impaired primary hemostasis and thromboembolic complications. Recently, a new generation of implantable continuous flow axial pumps was introduced into clinical application. To study the potential thrombogenic properties of this type of pump, we applied extensive platelet monitoring was applied. In our institution, 13 patients received the MicroMed DeBakey VAD as a bridge to transplantation. Routine coagulation tests (platelet count, activated partial thromboplastin time, prothrombin time, antithrombin III activity) and platelet function tests (whole blood aggregometry, thrombelastography, flow cytometry) were performed. No clinically relevant thromboembolic events were detected. No correlation was found between global function tests, platelet aggregation, and thrombelastography. No correlation was detected between platelet activation and hemolysis parameters. Platelet aggregation and coagulation index were significantly suppressed early after operation. A subsequent phase of hyper-aggregability, starting around day 6, suggested the initiation of antiaggregation therapy. Platelet activation markers were upregulated in the postoperative period but were returned to preoperative levels after initiation of aspirin. In contrast to routine coagulation monitoring, platelet function tests reflect in detail the coagulation status of blood pump recipients and the efficiency of antiaggregation therapy. Aspirin and dipyridamole therapy in addition to oral anticoagulation using phenprocoumon may contribute to platelet function and clot mechanics restoration and is, therefore, recommended for patients after VAD implantation.

Early Detection of Preeclampsia by Determination of Platelet Aggregability

Preeclampsia is still a leading cause of maternal and fetal morbidity and mortality. There is evidence for the involvement of platelets. Therefore, we investigated the suitability of corrected whole blood impedance aggregometry as an early predictor of preeclampsia in 71 consecutive, high-risk pregnancies. According to the occurrence of preeclampsia, defined postpartum by an independent investigator, and the stage of pregnancy (early and late, cutoff: 25 weeks of gestation), four study groups were defined. Platelet aggregation data were corrected for the influence of hematocrit and platelet count by a special purpose software package. Women developing preeclampsia showed significantly higher platelet aggregation response compared to controls in early and late pregnancy. In early pregnancy, all women developing preeclampsia had aggregation responses to collagen higher than the highest responses among the controls. Hence, this test had a 100% positive predictive value of subsequent preeclampsia. Despite being significantly increased, platelet aggregability was of minor predictive value in late pregnancy. We conclude that preeclampsia is accompanied by exaggerated platelet aggregability, particularly perceptible early in the course of pregnancy. We propose collagen-induced whole blood platelet aggregation with correction for the influence of hematocrit and platelet count for early detection of preeclampsia.

Anal Human Papillomavirus Testing with Digene’s Hybrid Capture 2 Using Two Different Sampling Methods

PurposeThe incidence of human papillomavirus detection in the anal canal is rising. Efficient anal screening by cytology is hampered because of poor specificity. Human papillomavirus (HPV) testing is proposed in addition to Papanicolaou (Pap) testing for the detection of cervical neoplasia. The purpose of this study was to determine the usefulness of a human papillomavirus-DNA detection test to detect human papillomavirus-associated disease and to compare two different methods of sample collection.MethodsIn 555 patients, anal samples were obtained by using a cervical brush and a Dacron swab to test for high-risk and low-risk human papillomavirus-DNA. Patients positive for human papillomavirus-DNA underwent anoscopy. Biopsies were taken from visible lesions.ResultsLow-risk human papillomavirus-DNA was found in 325 of 555 patients (58.6 percent) and high-risk human papillomavirus-DNA in 285 of 555 patients (51.4 percent). Positive results confined to one single test method were higher for Dacron swab sampling (2.3 vs. 4.3 percent for low-risk human papillomavirus, P < 0.0001; 3.1 vs. 4.9 percent for high-risk human papillomavirus, P < 0.001). A positive correlation of relative light units was found for both sampling methods in the total cohort (P < 0.0001) as well as for patients who tested human papillomavirus-positive by both sampling techniques (P < 0.0001). Sampling with Dacron swabs yielded higher relative light units values compared with sampling with cervical brush for low-risk human papillomavirus-DNA and high-risk human papillomavirus-DNA.ConclusionsAnal screening for human papillomavirus-DNA by hybrid capture 2 is a useful method for detection of human papillomavirus-associated disease. Sample collection using Dacron swabs identifies more human papillomavirus-positive patients, and yields higher relative light unit values than using the cervical brush. Further studies are needed to determine the exact value of hybrid capture 2 in the screening for (pre)cancerous lesions of the anal canal.

Influence of Hypotensive and Normotensive Anesthesia on Platelet Aggregability and Hemostatic Markers in Orthognathic Surgery

This prospective randomized study investigated the influence of normotensive and hypotensive general anesthesia on platelet aggregability, intraoperative blood loss and parameters of plasmatic coagulation during extensive orthognathic surgery. A total of 30 patients were randomly allocated for either normotensive anesthesia maintained by continuous infusion of propofol and remifentanil (NORMO, n=10) or hypotensive anesthesia, whereby hypotension was induced by increasing the infusion rate of remifentanil (HYPO-R, n=10) or by administration of nitroglycerin (HYPO-N, n=10). Whole blood platelet aggregability was significantly reduced during hypotension compared to normotensive anesthesia (P<.01, HYPO-N and HYPO-R vs. NORMO). Mean arterial blood pressure during hypotension correlated well with adenosinediphosphate- (R=.712, P<.001) and collagen-induced platelet aggregability (R=.685, P<.001). Within hypotensive study groups, postoperative fibrinogen levels were significantly different, whereas intraoperative platelet aggregability, postoperative platelet count, prothrombin time, activated partial thromboplastin time and antithrombin levels were not different. Normotensive anesthesia, however, caused significant decreases in platelet count (-29%), prothrombin time (-24%), fibrinogen (-41%) and antithrombin (-28%) and a significant prolongation in activated partial thromboplastin time (+21%) and thrombin time (+18%). There was a trend to reduced intraoperative blood loss in hypotensive study groups; however, differences were not significant. In conclusion, induced hypotension--independent of substances used for induction of hypotension--reduces intraoperative platelet aggregability, subsequently protecting the coagulation system against subclinical consumption coagulopathy. Induced hypotension-caused platelet dysfunction does not lead to an increased intraoperative blood loss, but quite on the contrary shows a trend to reduced intraoperative blood loss, possibly by preventing platelet-induced subclinical consumption coagulopathy.

Changes of platelet surface antigens in patients suffering from abdominal septic shock.

Sepsis and related syndromes account for a high morbidity and mortality caused by the development of multiorgan failure. Pathogenesis of sepsis is complex, involving humoral as well as cellular factors. Since the role of platelets is still undefined in this concern, we investigated CD63, CD62P, CD36, and CD31 expression on platelets of patients in septic shock (n = 18) using a flow cytometric assay in whole blood. Samples were drawn within 24 hours of onset. We found thrombocytopenia accompanied by a significantly higher expression of CD63, CD62P, and CD31 and a significant downregulation of CD36 in comparison to healthy volunteers (n = 18). Changes in CD63 and CD62P expression indicates platelet activation. Because CD62P, CD36, and CD31 mediate interaction of platelets with leukocytes, subendothelial matrix and probably endothelial cells as well as platelet adhesion/aggregation, our findings suggest an involvement of platelets in leukocyte/endothelial cell interaction in septic shock. We suspect that thrombocytopenia is not due to bone marrow depression, but rather is due to consumption of highly activated platelets in the microcirculation. We feel that our observations may offer a rationale for potentially beneficial effects of antiplatelet therapy in sepsis; however, further studies have to evaluate its beneficial impact as well as its potential risk for bleeding complications.

The influence of sample age on collagen-induced platelet aggregation in whole blood

Whole blood electrical aggregometry (WBEA) has become an accepted method to gain quick information on platelet disorders. Compared to the optical method WBEA is closer to physiology and less complicated, but on the other hand more difficult to standardize. Different approaches have been attempted in the past to improve the reliability and practicability of this technique. The influence of sample age has not been defined so far. In a first step a mathematical modelling program was established, which is able to characterize the aggregation curves obtained after collagen stimulation. In a mathematical analysis various characteristics of the curve function were calculated and their sensitivity for aging investigated. Regression was performed for each characteristic, and correction factors defined. Our results indicate, that whole blood specimen for collagen induced aggregation can be used without correction factor up to 30 minutes. Data obtained with an age exceeding half an hour have to be corrected following a quadratic regression.

Comparison of in vitro closure time (PFA-100) with whole blood electrical aggregometry and platelet surface antigen expression in healthy volunteers

It was the aim of this study to compare in vitro closure time (PFA-100), reflecting platelet-related primary hemostasis, to more platelet-specific tests like whole blood electrical aggregometry and platelet surface antigen expression in healthy volunteers. In vitro closure time was measured using a PFA-100. Platelet surface antigen expression (CD63, CD62-P, CD42b, CD36, CD31) was determined in accordance with the consensus protocol for flow-cytometric characterisation of platelet function. Platelet aggregometry was performed using a whole blood electrical aggregometer (ADP and arachidonic acid as agonists). Analysis of the obtained data revealed only a few significant correlations between the different platelet function tests used. This finding can be explained by the various aspects of platelet function being focused by these tests in different extents. Whenever platelet function is analysed, the investigator should be aware of the specific and limited evidence of the method used. For screening purposes, it may be useful to introduce a platelet function index, referring to basal platelet activity, platelet adhesion and platelet aggregation at low and high shear stress forces.

Signifikante Verlängerung des Überlebens durch komplette Resektion isolierter Lungenmetastasen nach Mammacarcinom

Summary. At least 25 % of breast cancer patients develop distant metastases. In spite of increasingly sophisticated palliative therapies, the survival time of patients with metastasis did not appear to be significantly prolonged during the last 25 years (19–32 months following diagnosis) and 95 % of them die from metastatic disease. Therefore, it seems appropriate that the therapeutic risk/benefit ratio and impact on quality of life should be reassessed when asymptomatic patients are treated. Surgical treatment and pulmonary resection for metastatic disease has been proven a valuable therapeutic concept for a variety of malignancies. Three epidemiologically comparable collectives out of a total of 125 patients from our clinic were treated for isolated pulmonary metastasis following breast cancer (observation period: 1977–1997). Complete data sets could be established for 96 patients and were retrospectively analyzed following stratification into three groups according to their surgical therapy. Twenty-eight patients underwent complete resection (K), 34 had incomplete resections (I) and 34 had no surgical intervention for lung metastases (N). Comparison of the three therapy arms concerning stage, histology and receptor levels of the primary tumor, number of metastases, and the disease-free interval yielded no significant differences between groups K, I and N. Patients after complete resection of isolated lung metastases (group K) had a mean survival of 79 months (5-year survival 80 %, 10-year survival 60 %). This was significantly better than groups I and N (P < 0.00002). The mean survival of groups I and N was not significantly different (15.5 and 9 months respectively). The disease-free interval after operation of the primary tumor had no impact on the survival of group K, but showed a high correlation with the survival of group N (R2 = 0.81). Complete resection of isolated pulmonary metastases from carcinoma of the breast results in marked prolongation of survival with a low morbidity rate. Hence, routine chest X-ray should be considered an indispensable part of the oncological aftercare in breast cancer patients.Zusammenfassung. Zumindest ein Viertel aller Patienten mit Mammacarcinom kommen in ein metastasierendes Stadium. Ungeachtet der laufenden Adaptierung palliativer Therapiemodalitäten blieb das mittlere Überleben in diesem Stadium innerhalb von 25 Jahren unverändert (19–32 Monate nach Diagnose), und 95 % dieser Patientinnen starben an der Generalisierung des Malignoms. Das erste Therapieziel muß deshalb Schaffung und Erhaltung von Lebensqualität sein. Daneben hat sich die chirurgische Entfernung isolierter Lungenmetastasen nach anderen Malignomen als therapeutisches Konzept durchgesetzt. Berichtet wird von 3 epidemiologisch vergleichbaren Kollektiven aus insgesamt 125 Patientinnen unserer Abteilung mit isolierten Lungenmetastasen nach Mammacarcinom (Beobachtungszeitraum 1977–1997). Von 96 Patientinnen waren komplette Datensätze erhebbar, sodaß sie retrospektiv entsprechend der chirurgischen Therapie in 3 Gruppen stratifiziert werden konnten. 28 Patientinnen wurden komplett (K), 34 inkomplett (I) und 34 nicht (N) reseziert. Der Vergleich der 3 Therapiegruppen N, K und I untereinander hinsichtlich Tumorstadium, Histologie und Receptorstatus des Primärtumors, Anzahl der Metastasen und des tumorfreien Intervalls ergab keine statistisch signifikanten Unterschiede. Das mittlere Überleben der Gruppe K war mit 79 Monaten nach Resektion der Lungenmetastasen (5-Jahres-Überleben 80 %, 10-Jahres-Überleben 60 %) signifikant besser im Vergleich zu den Gruppen I und N (p <0,00002). Das mittlere Überleben der Gruppen I und N war mit 15,5 und respektive 9 Monaten nicht signifikant unterschiedlich. Das tumorfreie Intervall nach Operation des Primärtumors hatte auf die Prognose der Gruppe K keinen Einfluß, korrelierte aber mit dem Überleben in Gruppe N (R2 = 0,81). Die komplette Resektion isolierter Lungenmetastasen nach Mammacarcinom führt zu einer ausgeprägten Lebensverlängerung bei geringer Morbidität. Das Thoraxröntgen stellt daher einen unverzichtbaren Bestandteil des onkologischen Nachsorgeprogramms nach Mammacarcinom dar.

Anale HPV-Infektionen

Anogenitale Infektionen mit humanen Papillomaviren (HPV) verzeichnen in den letzten Jahren gerade in jüngeren Generationen durch frühe sexuelle Aktivität eine steigende Inzidenz. Neue Erkenntnisse der ätiologischen, epidemiologischen und pathophysiologischen Zusammenhänge erfordern eine Überarbeitung der proktologischen Position HPV-assoziierter Erkrankungen der Analregion. Dies ist auch aufgrund der ansteigenden Inzidenz präkanzeröser und invasiver Neubildungen nötig. In Ermangelung großer, kontrollierter Studien stellt dieser Konsensusbericht über weite Strecken die Expertenmeinung der auf dem Gebiet der HPVInfektion erfahrenen Autoren aus den Gebieten Proktologie, Dermatologie, Gynäkologie, Radiologie, Strahlentherapie und Pathologie dar. Anale HPV-Infektionen1