Volume 31, Issue 6

During the 1960s to 1980s platelet function diagnostics was mainly performed using optical platelet aggregometry in platelet rich plasma. However, the last two decades witnessed an increasing application of methods which assess platelets directly in whole blood. Advantages include simpler test procedures, less sample manipulation and smaller sample volumes. Test methods are impedance aggregometry according to Cardinal and Flower, Impact analyzer (Cone and Plate analyzer), Verifynow (rapid platelet function test), “platelet mapping assay” (thrombelastography-based platelet function analysis), PFA-100 (in vitro bleeding time), and Multiplate analyzer (multiple electrode aggregometry). Differences between these methodologies include procedures for platelet activation and detection as well as the shear stress applied during the test. In the German-speaking countries the whole blood platelet function tests most frequently employed are the PFA-100 and Multiplate. The former is a global test which detects platelet activation and alterations of von Willebrand factor. The Multiplate has been designed mainly for monitoring anti-platelet agents (aspirin, clopidogrel, and GPIIb/IIIa antagonists). For the selection of the most appropriate platelet function test several factors have to be taken into account: basic goal of the examination (scientific evaluation vs. clinical application), underlying question (global assessment of primary hemostasis, monitoring of anti-platelet drugs, or vWF diagnostics), and logistic aspects (near-patient or laboratory analysis, complexity of the analysis, and costs).

Preoperative screening is the most frequent indication for coagulation testing in children. However, coagulation tests were developed and validated to clarify bleeding tendency. Most tests (PT, aPTT, thrombelastography, bleeding time or PFA) to detect so far unknown hemorrhagic diathesis, particularly von Willebrand disease, are inappropriate. Irrelevant coagulation abnormalities and false positive results further devaluate routine screening. It is necessary to reduce the number of tested children and investigate only when bleeding history or family history is indicative of possible bleeding tendency. The selected tests should initially rule out von Willebrand disease.

Thrombelastography/-metry provides information about clot firmness and stability. The use of standardized tests, such as FibTEM, allows estimation of platelet and plasmatic component of clot strength. This may lead to therapeutic consequences. However, estimation of clot strength by platelet count and fibrinogen determination according to Clauss is not feasible. This may be explained by the dynamic interaction of platelets and fibrinogen. Dysfibrinogenemia and factor XIII may also modify clot strength. Diagnosis of hyperfibrinolysis in the perioperative setting is impossible by the use of the D-dimer test. Thrombelastography/-metry is considered the “gold standard” of diagnosis of hyperfibrinolysis. Disorders in primary hemostasis, such as von Willebrand disease, and drug effects (ASS or clopidogrel) may not be detected by thrombelastography/-metry (with the exception of platelet mapping). Indications for thrombelastography/-metry are unexplained intra- and post-operative bleeding disorders. Thrombelastography/-metry may be useful in monitoring of fibrinogen substitution. Thrombelastometry (ROTEM ® ) allows generation of valid and reproducible thrombelastometric curves even under clinical conditions.

The Protein S Ac activity assay is based on the role of protein S as a cofactor to activated protein C (APC). Tests performed on three coagulation analyzers were studied to determine assay performance characteristics. The overall mean imprecision was 5.5%, 5.0% and 7.5% for within-run coefficient of variation (CV), run-to-run CV and total CV, respectively. A total of 239 sample results yielded a correlation to the STA Protein S Clotting assay of r=0.92 (slope=0.96). A group of patients with APC resistance and additional underlying conditions, such as oral contraception, oral anticoagulation, pregnancy and recurrent thrombosis showed pathologically low protein S values with activity assays as well as with free antigen assays. To differentiate between the effects of APC resistance and other influencing factors, samples from healthy blood donors with APC resistance were measured with both assay types. Based on comparison of antigen and activity results, it can be concluded that APC resistance lowers protein S activity values by approximately 15% (rel.). No interference was observed from physiologically relevant concentration of factor V, factor VIII, triglycerides, hemoglobin, bilirubin and heparin (low molecular weight heparin and unfractionated heparin). When samples are to be frozen, double centrifugation is the best procedure to reduce the platelet count. The stability of the sample was at least 4 h at room temperature, regardless of whether fresh or frozen samples were tested. Freezing had no observable effect on samples stored for up to 12 months at −70°C. Zusammenfassung Der Aktivitätstest Protein S Ac basiert auf der Rolle von Protein S als Kofaktor für aktiviertes Protein C (APC). Zur Bestimmung der Leistungsmerkmale wurden Untersuchungen auf drei verschiedenen Gerinnungsgeräten durchgeführt. Der durchschnittliche Variationskoeffizient betrug innerhalb einer Messreihe 5,5%, zwischen verschiedenen Messreihen 5,0% und insgesamt 7,5%. Die Messung von 239 Proben ergab eine Korrelation zu dem STA Protein S Clotting-Test von r=0,92 (Steigung=0,96). Eine Gruppe von Patienten, die APC-Resistenz kombiniert mit oraler Kontrazeption, oraler Antikoagulation, Schwangerschaft oder rezidivierender Thrombose aufwiesen, zeigte sowohl im Aktivitätstest wie auch im Freien-Antigen-Test pathologisch niedrige Werte. Um zwischen dem Einflussfaktor APC-Resistenz und anderen Einflussfaktoren zu unterscheiden, wurden gesunde Blutspender mit APC-Resistenz untersucht. Mit Hilfe des Vergleichs von Aktivitäts- und Antigen-Ergebnissen konnte geschlussfolgert werden, dass durch die APC-Resistenz die Aktivitäts-Ergebnisse um ca. 15% (relativ) erniedrigt wurden. Keine Interferenz wurde innerhalb physiologisch relevanter Konzentrationsbereiche bei folgenden Faktoren gefunden: Faktor V, Faktor VIII, Triglyzeride, Hämoglobin, Bilirubin, Heparin (LMWH und UFH). Die beste Prozedur, um zu hohe Restthrombozytenzahlen in eingefrorenen Proben zu vermeiden, ist eine doppelte Zentrifugation. Die Probenstabilität betrug sowohl bei frischen wie auch bei aufgetauten Proben mindestens vier Stunden bei Raumtemperatur. Durch Einfrieren und Lagerung bei −70°C bis zu 12 Monate konnte kein Einfluss auf die Messergebnisse beobachtet werden.

Hereditary spherocytosis is the most frequently encountered inherited hemolytic anemia among patients of Caucasian ethnic origin. Typical clinical laboratory features include anemia of varying degree, reticulocytosis, hyperbilirubinemia, reduced haptoglobin and increased lactate dehydrogenase activity. Red blood cells are typically described as microspherocytes. However, the presence of spherocytes alone does not confirm the diagnosis of hereditary spherocytosis. The classical diagnostic approach relies on testing red blood cell osmotic resistance or fragility. This test has not been standardized and remains difficult to interpret in neonatal blood samples. The addition of a flow cytometric assay based on reduced binding of eosin-5-maleimide (EMA) to spherocytes has largely improved the diagnostic algorithm. Several investigations have proved its diagnostic usefulness. Minimal sample volumes make the test particularly helpful in neonatal samples. The EMA test can be recommended as method of choice in diagnosing hereditary spherocytosis. The molecular genetic investigation of suspected hereditary spherocytosis remains a challenging method and should be performed in selected cases only.

Streptococcal tonsillopharyngitis still needs to be treated with antibiotics, especially to avoid non-infectious streptococcal disease sequelae. Diagnosis of streptococcal tonsillopharyngitis cannot be solely based on clinical symptoms. The classical microbiological diagnosis of streptococcal tonsillopharyngitis on solid media requires 1–3 days and thus may not meet the needs of daily practice. The development of easy-to-perform rapid streptococcal antigen detection tests has changed the routine management in the diagnosis of streptococcal pharyngitis within the last years. Nearly all modern rapid streptococcal antigen detection tests provide a high specificity (>95%). The sensitivity of this test system has also been improved within the last years (>85%). The selection of a rapid streptococcal antigen test for a specific medical unit must be performed with diligence. Before introducing a rapid streptococcal antigen test, the following aspects should be considered: type of unit (hospital vs. medical practice), costs and the availability of a laboratory performing classical microbiology. For paediatricians, it is advisable to perform both the rapid streptococcal antigen test and the classical microbiology culture in parallel, at least during the time of the introduction of the new test system until high specificity and sensitivity of the new method have been confirmed. Continuous education and basic training of medical personnel performing these rapid antigen tests is urgently needed and greatly influences test performance. In some selected antibiotic susceptibility testing of Streptococcus pyogenes isolates may be necessary, in particular, because macrolide resistance in Germany has been increasing within the last years.

Sepsis and septic shock are the major causes of morbidity and mortality in modern intensive care units. Despite recent advances in management of patients in intensive care units, incidence of sepsis is increasing worldwide and mortality still remains at 30–60% in developed countries. One explanation for the high mortality in these patients may be the lack of adequate diagnostic markers for early recognition, risk assessment and follow-up of sepsis. Therefore, numerous markers have been investigated during the last years, but only a few have proved clinically useful. However, particularly PCT, IL-6 and metabolic markers, such as cholesterol, are the most promising. The present review summarizes pathophysiology and clinical relevance in terms of early recognition and therapy monitoring of established and some new biomarkers.