Dieter Ratge

Two-round rapid-cycle RT-PCR in single closed capillaries increases the sensitivity of HCV RNA detection and avoids amplicon carry-over

BACKGROUNDnFor the detection of hepatitis C virus (HCV) specific nucleic acids the polymerase chain reaction (PCR) is widely used. Rapid-cycle PCR is performed in glass capillaries with the LightCycler instrument and allows PCR including product analysis to be performed within a closed system in about 1 h. Thus, rapid-cycle PCR appears especially suitable for routine diagnostic applications. However, the volume of the PCR vessel is restricted to about 20 microl, which may limit the sensitivity of the PCR. To increase its sensitivity two-round or nested primer PCR protocols have been developed. In rapid-cycle PCR first-round PCR products are usually collected from the capillaries by centrifugation, a procedure prone to cross-contamination.nnnOBJECTIVESnDevelopment of a two-round rapid-cycle reverse transcription-polymerase chain reaction (RT-PCR) in single closed LightCycler capillaries for the sensitive detection of HCV RNA in serum or plasma.nnnSTUDY DESIGNnA set of two pairs of nested primers was selected. The first-round RT-PCR reaction mixture was separated from the second-round PCR mixture by silicone oil. Reverse transcription followed by the first-round PCR was performed. Then, the second-round mixture was combined with first-round products by a centrifugation step followed by second round PCR during which fluorescence intensities were recorded and used for quantification.nnnRESULTSnTo establish the sensitivity of this novel assay a serial dilution of HCV reference standard was used. In plasma samples about 100 IU/ml HCV were consistently detected using the high pure viral RNA kit for nucleic acid purification. This detection limit was found to be about 20 fold increased compared with single-round RT-PCR and corresponded to 3.4 IU of HCV per capillary. Using a panel of HCV genotype standards the novel assay exhibited similar sensitivity for all HCV genotypes. The applicability for clinical routine testing was demonstrated by examining 156 clinical samples.nnnCONCLUSIONnTwo-round RT-PCR with the LightCycler instrument using a single closed capillary throughout the procedure was found ideally suited for rapid (100 min), accurate and sensitive molecular diagnosis of active HCV infections. Since the capillaries remained closed during the procedure carry-over contamination was precluded.

Agonist-induced down-regulation of the β2-adrenoceptor and its mRNA in human mononuclear leukocytes

Agonist-mediated regulation of beta2-adrenoceptors in mononuclear leukocytes has been examined at the protein but not at the mRNA level. In the present study, incubation of mononuclear leukocytes with the beta-agonist (-)-isoproterenol (10(-6) M) for up to 42 hr led to a maximum decrease in both beta2-adrenoceptor mRNA concentration and total receptor number of ca. 56 and 70%, respectively. The decrease in the mRNA level, however, was slower than for the protein level. After 4 hr of incubation with the beta-agonist, the protein level decreased to a minimum of 65% of the initial amount, while an incubation of 8 hr was necessary to reach a similar decrease in the level of mRNA (69% of the initial level). Measurements of mRNA stability revealed a reduction in the half-life of beta2-adrenoceptor mRNA from 2.7 to 1.1 hr following 4 hr of incubation with (-)-isoproterenol. Our data clearly demonstrate that treatment of human mononuclear leukocytes with (-)-isoproterenol induces a beta2-adrenoceptor down-regulation together with a slower time course of mRNA down-regulation which is partly due to a reduction of mRNA stability.

Minimally Invasive Extracorporeal Bypass in Minimally Invasive Heart Valve Operations: A Prospective Randomized Trial

BACKGROUNDnMinimally invasive extracorporeal circulation (MECC) is predominantly used in coronary operations. Data supporting the benefits of MECC in minimally invasive valve operations are still absent.nnnMETHODSnPatients undergoing either isolated minimally invasive mitral or aortic valve procedures were prospectively randomized to a minimally invasive group (MECC; nxa0= 101) or a conventional extracorporeal circulation group (CECC; nxa0= 99). The procedural and postoperative outcomes were compared, including the levels of inflammation factors (procalcitonin, interleukin [IL]-6, IL-8, and IL-10), tumor necrosis factor-α [TNF-α], and interferon-gamma [IFN-γ]).nnnRESULTSnThe demographics were comparable between the groups regarding age (MECC versus CECC, 70.5 ± 10.2 years versus 73.1 ± 8.9 years; Pxa0= 0.086), left ventricular function (59.2% ± 13.4% versus 62.1% ± 14.0%; pxa0= 0.302), EuroSCORE (7.4% ± 7.9% versus 6.8% ± 4.0%; pxa0= 0.256), and other comorbidities. Hospital mortality (nxa0= 1 versus nxa0= 3; pxa0= 0.339) and other complications were similar. However, hemoglobin level (111.9 ± 19.0 g/L versus 103.8 ± 14.6 g/L; pxa0= 0.001), the number of packed red blood cells (PRBCs) (1.1 ± 1.9 versus 1.7 ± 1.8; pxa0= 0.003), the levels of ILs (IL-6, 194.0 ± 131.8 pg/mL versus 289.2 ± 62.5 pg/mL; pxa0= 0.020; IL-8, 38.1 ± 27.3 pg/mL versus 45.8 ± 43.4 pg/mL; pxa0= 0.012; IL-10, 29.0 ± 123.9 pg/mL versus 49.9 ± 85.6 pg/mL; pxa0= 0.012), TNF-α (3.8 ± 6.7 ng/mL versus 10.8 ± 47.7 ng/mL; pxa0= 0.049), and IFN-γ (1.9 ± 1.9 pg/mL versus 4.5 ± 2.7 pg/mL; pxa0= 0.027) were in favor of patients in the MECC group. Additionally, those patients had shorter postoperative ventilation time (7.7 ± 8.4 hours versus 9.3 ± 12.9 hours; pxa0= 0.010) and intensive care unit (ICU) stay (1.2 ± 1.2 days versus 2.2 ± 3.8 days; pxa0= 0.047).nnnCONCLUSIONSnThe intraprocedural data were excellent and comparable in the groups, but postoperative outcomes were better in the MECC group. Thus MECC is preferable to CECC even for minimally invasive valve procedures. These findings strongly support a combined strategy of minimally invasive valve operations and minimally invasive extracorporeal circulation.

Acetylcholine-induced coronary spasm in patients with unobstructed coronary arteries is associated with elevated concentrations of soluble CD40 ligand and high-sensitivity C-reactive protein.

Objectives To assess whether epicardial and microvascular coronary artery spasm in response to acetylcholine (ACH) is associated with markers of inflammation, platelet stimulation, and endothelial activation in patients with angina and unobstructed coronary arteries. Background Patients with angina pectoris despite angiographically normal coronary arteries represent a diagnostic and therapeutic challenge. Both impaired coronary microvascular dilatory responses as well as diffuse distal epicardial and microvascular coronary artery spasm have been described as possible pathogenic mechanisms. Although inflammation has been proposed to play a pathogenic role in angina, an association between ACH-induced coronary vasospasm and inflammation in Caucasians has not been reported previously in this context. Patients and methods We assessed 62 consecutive patients (26 men, age 60±10 years) with chest pain despite angiographically unobstructed coronary arteries (<50% stenosis) who underwent intracoronary ACH testing for the diagnosis of coronary artery spasm. High-sensitivity C-reactive protein (hs-CRP), e-selectin, neopterin, and sCD40L concentrations were measured in all patients before ACH testing. The ACH test was considered to be ‘positive’ in the presence of (a) angina and at least 75% coronary diameter reduction (epicardial coronary artery spasm) or (b) ischemic ST-shifts and angina in the absence of epicardial spasm (microvascular spasm). Eight patients without angina pectoris served as a control group. Results The ACH test was positive in 48 patients (77%). Twenty-seven patients had epicardial spasm (56%) and 21 patients had microvascular spasm (44%). Epicardial spasm was diffuse in 26 patients (96%) and focal in one patient (4%). Elevated hs-CRP, e-selectin, and sCD40 ligand concentrations were significantly (P⩽0.05) associated with a positive ACH-test response. Hs-CRP (odds ratio 1.54, confidence interval 1.02–2.33, P=0.04) and sCD40 ligand (odds ratio 1.001, confidence interval 1.00–1.001, P=0.003) were predictors for a positive ACH test on multivariate analysis. None of the patients in the control group developed epicardial or microvascular spasm during ACH testing. Conclusion Epicardial and microvascular coronary spasm in response to ACH correlate significantly with hs-CRP and sCD40 ligand concentrations in patients with angina pectoris and angiographically unobstructed coronary arteries. These results suggest that an association exists between inflammation and coronary artery spasm in patients with angina pectoris despite unobstructed coronary arteries and studies are needed to explore the mechanisms underlying this association.

Severe hyperphosphatemia in a patient with chronic kidney disease and multiple myeloma–to strengthen the case toward renal replacement therapy?

We report a patient with multiple myeloma and chronic kidney disease who presented with severe hyperphosphatemia in the outpatient clinic without any related symptoms. Initial differential diagnosis: Tumor lysis syndrome or chronic kidney disease. Further work‐up revealed pseudohyperphosphatemia. In general, treatment is not necessary if the true phosphate level is within the reference range and the patient is asymptomatic.

Evaluation of sample integrity measurement with VITROS® 5,1 FS chemistry system / Evaluation der Messung der Probenintegrität am VITROS® 5,1 FS

Abstract The measurement of sample integrity on a clinical laboratory system may help to avoid misleading results. The VITROS® 5,1 FS Chemistry System is a new analyzer which offers this feature. Approximately 50 diagnostic specimens were evaluated for the presence of hemolysis, icterus, and turbidity using MicroSensor™ technology onboard the VITROS® 5,1 FS. The samples were also assessed for hemoglobin concentration on a Lange photometer using the Harboe method and values were compared with the MicroSensor™ technology hemolysis index (H-index). For the icterus index (I-index), comparison was based on total bilirubin measured on the VITROS® system. To correlate the turbidity index (T-index), a comparison with triglycerides measured on the VITROS® 5,1 FS was undertaken. In addition, samples run on the VITROS® 5,1 FS were measured on a manual photometer at 700 nm. Good correlation was established between the manual Harboe method for measurement of hemoglobin and the VITROS® 5,1 FS H-index. For the I-index, a good correlation was observed compared to the quantitative bilirubin (TBIL slide) measurement. A poor correlation was expected for the comparison of the T-index with the triglycerides as measured on the VITROS® system. An additional study comparing the VITROS® MicroSensor T-index vs. manual photometer measurement at 700 nm yielded a very good correlation. The VITROS® 5,1 FS chemistry system MicroSensor™ technology measures the index for hemolyzed, icteric, and turbid samples. The indices correlate well compared to other methods for measuring these indices. Therefore, the use of MicroSensor™ technology offers a useful feature to identify samples containing optical interferents during routine work and to avoid misleading results by providing flags. Zusammenfassung Die Messung der Probenintegrität auf einem klinisch-chemischen Laborsystem kann helfen, irreführende Ergebnisse zu vermeiden. Das VITROS 5,1 FS-Analysensystem ist ein neuer Analysator, der diese Messungen automatisch durchführt. Etwa 50 Patientenproben wurden auf hämolytische und ikterische Färbung sowie auf Trübung mit Hilfe der sogenannten MicroSensor-Technologie am VITROS 5,1 FS geprüft. Die Hämoglobin-Konzentration wurde photometrisch, unter Einsatz der Harboe-Methode ermittelt, während die MicroSensor-Technologie den Hämolyse-Index (H-Index) erfasste. Für die ikterische Färbung wurde der I-Index mittels der MicroSensor-Messung bestimmt und mit der Gesamt-bilirubin-Konzentration (Diazo-Methode) verglichen. Der Grad der Trübung (T-Index) wurde sowohl mit der Triglyzerid-Konzentration verglichen, als auch mit der Messung der verdünnten Probe bei 700 nm mit Hilfe eines manuellen Photometers. Eine gute Korrelation wurde zwischen der manuellen Harboe-Methode zur Messung des Hämoglobins und dem VITROS 5,1 FS H-Index gefunden. Analoge Daten konnten für den Vergleich der I-Indizes und der quantitativ gemessenen Bilirubin-Konzentrationen beobachtet werden. Zu erwarten war die schlechte Korrelation für den Vergleich des T-Indexes mit der quantitativ bestimmten Triglyzerid-Konzentration. Eine zusätzliche Studie zur Messung der Absorption mit dem MicroSensor T-Index, im Vergleich zur manuellen Photometermessung bei 700 nm, lieferte eine sehr gute Korrelation. Die Messung der Indizes am VITROS 5,1 FS während der Pipettierung, ohne zusätzliches Reagenz und Probenmaterial sowie ohne Reduktion der Durchsatzrate ist ein sehr wertvolles Instrument zur zuverlässigen und fehlerfreien Befundermittlung.

ABNORMAL CORONARY VASOMOTION IN PATIENTS WITH UNOBSTRUCTED CORONARY ARTERIES IS ASSOCIATED WITH BIOMARKERS OF INFLAMMATION, ENDOTHELIAL FUNCTION AND PLATELET ACTIVATION

Background: Patients with typical angina despite unobstructed coronary arteries represent a diagnostic and therapeutic challenge. Endothelial dysfunction leading to abnormal vasomotion has been suggested to be a pathogenic mechanism. However, this has not been systematically investigated. We assessed the relationship between abnormal coronary vasomotion during intracoronary acetylcholine testing (ACH-test) and biomarkers of inflammation, endothelial dysfunction and platelet activation (hs-CRP, e-selectin, neopterin, soluble CD40ligand).

Caucasian patients suffering from coronary vasospastic angina have an intact peripheral endothelium-dependent vasoreactivity

We sought to evaluate whether Caucasian patients suffering from vasospastic angina have a decreased brachial artery flow-mediated dilation (FMD) like their Japanese counterparts and whether certain serum factors known to be associated with impaired vasomotility or endothelial dysfunction are abnormal. In this prospectively conducted study, 33 subjects presenting with resting angina were identified to suffer from coronary vasospastic angina (coronary spasm group). A control group of 19 subjects with matched cardiovascular risk profiles was defined out of patients admitted to our hospital for evaluation of atypical chest pain. Intracoronary acetylcholine(ACh)-testing for vasospasm was performed in all patients after coronary artery disease (CAD) had been ruled out. Brachial artery FMD was measured using high-resolution ultrasound. There was no significant difference in brachial artery FMD between the coronary spasm and the control group (7.05+/-2.24% vs. 7.12+/-2.50%; p=0.93). The endothelium-independent vasodilator response of the brachial artery to sublingual nitroglycerin did not differ either between the two groups (21.88+/-6.13% vs. 21.48+/-7.38%; p=0.84). Simple and multiple linear regression analysis revealed that only baseline brachial artery diameter was a significant determinant of FMD (p<0.0001). No relationship could be detected between impaired coronary vasomotility and peripheral endothelium-dependent or independent vasodilation in Caucasian patients suffering from coronary vasospastic angina illustrating a further clue for racial differences in the pathophysiology of vasospastic angina.