Vladimir Palicka

Preanalytical quality improvement: from dream to reality

Abstract Laboratory diagnostics (i.e., the total testing process) develops conventionally through a virtual loop, originally referred to as “the brain to brain cycle” by George Lundberg. Throughout this complex cycle, there is an inherent possibility that a mistake might occur. According to reliable data, preanalytical errors still account for nearly 60%–70% of all problems occurring in laboratory diagnostics, most of them attributable to mishandling procedures during collection, handling, preparing or storing the specimens. Although most of these would be “intercepted” before inappropriate reactions are taken, in nearly one fifth of the cases they can produce inappropriate investigations and unjustifiable increase in costs, while generating inappropriate clinical decisions and causing some unfortunate circumstances. Several steps have already been undertaken to increase awareness and establish a governance of this frequently overlooked aspect of the total testing process. Standardization and monitoring preanalytical variables is of foremost importance and is associated with the most efficient and well-organized laboratories, resulting in reduced operational costs and increased revenues. As such, this article is aimed at providing readers with significant updates on the total quality management of the preanalytical phase to endeavour further improvement for patient safety throughout this phase of the total testing process.

Preanalytical quality improvement: in quality we trust

Abstract Total quality in laboratory medicine should be defined as the guarantee that each activity throughout the total testing process is correctly performed, providing valuable medical decision-making and effective patient care. In the past decades, a 10-fold reduction in the analytical error rate has been achieved thanks to improvements in both reliability and standardization of analytical techniques, reagents, and instrumentation. Notable advances in information technology, quality control and quality assurance methods have also assured a valuable contribution for reducing diagnostic errors. Nevertheless, several lines of evidence still suggest that most errors in laboratory diagnostics fall outside the analytical phase, and the pre- and postanalytical steps have been found to be much more vulnerable. This collective paper, which is the logical continuum of the former already published in this journal 2 years ago, provides additional contribution to risk management in the preanalytical phase and is a synopsis of the lectures of the 2nd European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)-Becton Dickinson (BD) European Conference on Preanalytical Phase meeting entitled “Preanalytical quality improvement: in quality we trust” (Zagreb, Croatia, 1–2 March 2013). The leading topics that will be discussed include quality indicators for preanalytical phase, phlebotomy practices for collection of blood gas analysis and pediatric samples, lipemia and blood collection tube interferences, preanalytical requirements of urinalysis, molecular biology hemostasis and platelet testing, as well as indications on best practices for safe blood collection. Auditing of the preanalytical phase by ISO assessors and external quality assessment for preanalytical phase are also discussed.

Evaluation of oxidative stress and inflammation in obese adults with metabolic syndrome

Abstract Background: Obesity and metabolic syndrome increase the risk of cardiovascular morbidity and mortality. Oxidative stress seems to be involved in the pathophysiology of diabetes and cardiovascular complications of metabolic syndrome. The aim of our study was to evaluate the level of oxidative stress and inflammation in obese adults with and without metabolic syndrome. Methods: Oxidative stress and inflammation markers (total amount of free radicals, malondialdehyde, allantoin, α1-antiproteinase, oxidized/reduced glutathione ratio, high-sensitive C-reactive protein, fibrinogen), total antioxidant capacity and lipid standardized α-tocopherol were determined in obese subjects fulfilling at least three criteria of metabolic syndrome according to the National Cholesterol Education Program-Adult Treatment Panel III guidelines (n=20 patients), in obese subjects without metabolic syndrome (n=20 patients) and in 48 healthy controls. Results: Oxidative stress and inflammation markers were significantly elevated in the obese subjects, especially in those exhibiting metabolic syndrome. According to multidimensional statistical analysis, oxidative stress was independently related to triacylglyceride concentration, abdominal fat, low high-density lipoprotein cholesterol and low lipid standardized α-tocopherol in the patients with metabolic syndrome. Conclusions: High levels of free radicals together with low antioxidant capacity detected in obese adults indicate elevated oxidative stress, which is – together with systemic inflammation – further potentiated in the case of obese patients with metabolic syndrome. This imbalance in oxidative/antioxidative status and subclinical inflammatory state leads to higher risk of atherosclerotic and diabetic complications. Clin Chem Lab Med 2008;46:499–505.

Multicenter evaluation of the hemolysis index in automated clinical chemistry systems

Abstract Background: In vitro hemolysis, the prevailing cause of preanalytical error in routine laboratory diagnostics, might influence the reliability of several tests, affect the quality of the total testing process and jeopardize patient safety. Although laboratory instrumentation is now routinely equipped with systems capable of automatically testing and eventually correcting for hemolysis interference, to our knowledge there are no reports that have compared the efficiency of different analytical platforms for identifying and classifying specimens with hemolysis. Methods: Serum from a healthy volunteer was spiked with varying amounts of hemolyzed blood from the same volunteer, providing a serum free hemoglobin concentration ranging from 0.0 g/L to 2.0 g/L as measured by the reference cyanmethemoglobin assay. The spiked serum samples were shipped to seven separate laboratories and the hemolysis index (HI) was tested in triplicate on the following analytical platforms: Roche Modular System P (n=4) and Integra 400 Plus (n=1), Siemens Dimension RxL (n=3), ADVIA 2400 (n=1) and ADVIA 1800 (n=1), Olympus AU 680 (n=1) and Coulter DXC 800 (n=1). Results: Satisfactory agreement of HI results was observed among the various analytical platforms, despite a trend toward overestimation by the ADVIA 2400 and 1800. After normalizing results according to the instrument-specific alert value, discrepancies were considerably reduced. All instruments except for the Dimension RxL gave values normalized to the instrument-specific alert value, <1.0 for the sample with 0.048 g/L free hemoglobin, and >1.0 for the sample with 0.075 g/L free hemoglobin. The results of the four Modular System P tests were also highly reproducible among the different facilities. When evaluating instruments that provided quantitative HI results, the mean intra-assay coefficient of variation (CV) calculated for the triplicate determinations was always between 0.1% and 2.7%. Conclusions: The results of this multicenter evaluation confirm that efficiency of different analytical platforms to correctly identify and classify unsuitable samples is satisfactory. However, more effort should be placed on the standardization of reporting HI. All the instruments that we tested provide either quantitative or qualitative results that are essentially comparable, but which should always be compared with the instrument-specific alert values to harmonize their efficiency. Clin Chem Lab Med 2009;47:934–9.

Epidemiology of Helicobacter pylori Infection in the Czech Republic

Background:  Prevalence of Helicobacter pylori infection has been estimated to range from 60 to 95% in the former communist countries of Central and Eastern Europe. The aim of this study was to evaluate H. pylori infection prevalence in a representative sample of the Czech population. The second objective was to describe difference of H. pylori prevalence between different social groups of children and adults.

Biochemical profile and survival in nonagenarians

OBJECTIVES Old age is associated with an increase in frequency of disorders involving virtually all organ systems, resulting in a rise of mortality. The aim of the project was to study the relationship between biochemical markers and all-cause mortality in a defined age group. DESIGN AND METHODS Thirty-eight nonagenarians, aged 92 +/- 2 (range 90-100) years entered the study. At the start of the study, a sample of peripheral blood and urine were obtained for analysis of 50 basic biochemical, hematologic and biologic parameters. The assessment was then repeated in 6 to 12 months intervals. The significance of difference between surviving subjects and those who died was examined by Mann-Whitney U test and the correlation between the variables was studied by Spearman rank correlation coefficient. RESULTS During the observation period, 21 of the studied subjects died leaving 17 persons still alive at the end of the study. The mean time from the first measurement to the death was 12 +/- 10 (range 0-33) months. The mean follow-up time in surviving subjects was 31 +/- 12 (range 4-45) months. Serum vitamin E and calcium were significantly higher, and serum alanine aminotransferase (ALT) and urinary neopterin were significantly lower in survivors compared to the subjects who died. No other parameters were significantly different in survivors and in persons who died. Urinary neopterin exhibited a significant negative correlation with serum sodium concentration (RS = -0.50, p < 0.01), but the other parameters did not correlate significantly. CONCLUSION In conclusion, among the parameters studied, differences between survivors and nonsurvivors were observed only for serum vitamin E, calcium, ALT and urinary neopterin. These findings may form a basis for prospective interventional trials in this group of patients.

Cardiac troponin T as a marker of myocardial damage caused by antineoplastic drugs in rabbits

Abstract Anthracycline derivatives are among the most effective antineoplastic drugs but their therapeutic use is limited by their adverse effects. The cardiac side-effects of antineoplastic drugs were investigated in rabbits in vivo from the viewpoint of release of cardiac troponin T (cTnT) measured by Elecsys Troponin T STAT immunoassay (Boehringer Mannheim, Germany). No increase in cTnT was found following administration of a single dose of daunorubicin (3 mg/kg i.v., n = 4). During development of daunorubicin-induced cardiomyopathy (daunorubicin 3 mg/kg i.v., once a week; maximum nine administrations, n = 7), the levels of cTnT were within the physiological range (i.e. cTnT < 0.1 μg/1) at the beginning of the experiment and before and after the 5th administration, but the pathological values of cTnT after the 8th administration in 43% animals (0.22 ± 0.08 μg/l) correlated with their premature death. In the control group, the levels of cTnT were always lower than 0.1 μg/l during the experiment. Following administration of a new antineoplastic drug – Oracin {6-[2-(2-hydroxyethyl) aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno [1,2-c]-isoquinoline hydrochloride, 10 mg/kg i.v., once weekly, ten administrations, n = 7}, there was no increase in cTnT levels. These findings correlated with the PEP: LVET index, histological examination and no animal succumbing to premature death. It is possible to conclude that cTnT is a useful marker for the prediction of experimentally induced anthracycline cardiomyopathy and for the evaluation of cardiotoxic (and, possibly, cardioprotective) effects of new drugs in rabbits.

Inhibitory effect of glycation on catalytic activity of alanine aminotransferase

Non-enzymatic glycation is a common post-translational modification of tissue and plasma proteins which can impair their functions in living organisms. In this study, the authors have demonstrated for the first time an inhibitory effect of in vitro glycation on the catalytic activity of alanine aminotransferase (ALT, EC 2.6.1.2), a pyridoxal phosphate enzyme with several lysine residues in the molecule. The porcine heart enzyme was incubated with 50 mmol/l D-fructose, D-glucose, D,L-glyceraldehyde, or D-ribose in 0.1 mol/l phosphate buffer (pH 7.4) at 25°C for up to 20 days. The strongest glycation effect was shown by D,L-glyceraldehyde, which caused complete enzyme inhibition within 6 days. After 20 days of incubation, the ALT activity in samples with D-fructose and D-ribose was less than 7% of the initial enzyme activity. A statistically significant effect of D-glucose on the enzymatic activity of ALT was not found. Incubation of ALT with D-fructose, D,L-glyceraldehyde and D-ribose minimized its catalytic activity both in the glycated and non-glycated fractions of the samples. Markedly higher activity was found in the glycated fraction with glucose. The inhibitory effect of glycation of ALT with D-fructose and D-ribose was found to be more intensive in the presence of L-alanine and weaker in the presence of 2-oxoglutarate. The findings suggest that glycation of the e-amino group of Lys313 as a crucial part of the catalytic site of ALT may contribute to ALT inactivation in the presence of glycating sugars. Nevertheless, glycation of lysine residues outside the active center of ALT seems to be primary.

Impact of cardiopulmonary bypass on peripheral tissue metabolism and microvascular blood flow.

The aim of this study was to monitor and compare the changes in metabolism and blood flow in the skeletal muscles during cardiac operations performed with cardiopulmonary bypass (CPB) and operations without CPB (off-pump) by means of interstitial microdialysis (Figure 1). Surgical revascularization, coronary artery bypass grafting (CABG), was performed in 40 patients randomized to two groups. Twenty patients (On-Pump Group) were operated on using CPB, 20 patients (Off-Pump Group) were operated on without CPB. Interstitial microdialysis was performed by 2 probes of a CMA 60 (CMA Microdialysis AB, Solna, Sweden) inserted into the patient’s deltoid muscle. Microdialysis measurements were performed at 30-minute intervals. Glucose, lactate, pyruvate and glycerol as markers of basic metabolism and tissue perfusion were measured in samples from the first probe, using a CMA 600 Analyzer (CMA Microdialysis AB). Blood flow through the interstitium was monitored by means of dynamic microdialysis of ethanol as a flow-marker in the dialysates taken from the second probe (ethanol dilution technique). Results in both the groups were statistically processed and compared. Both the groups were similar in respect of preoperative characteristics. Dynamic changes of interstitial concentrations of the measured analytes were found in both the patient groups (on-pump vs. off-pump) during the operation. There was no significant difference in dialysate concentrations of glucose and lactate between the groups. Significant differences were detected in pyruvate and glycerol interstitial concentrations, lactate/pyruvate ratio and lactate/glucose ratio between the on-pump vs. off-pump patients. In the Off-Pump Group, pyruvate concentrations were higher and the values of concentrations of glycerol lower. The lactate/pyruvate ratio and the lactate/glucose ratio, indicating the aerobic and anaerobic tissue metabolism status, were lower in the Off-Pump Group. There was no significant difference in dialysate concentrations of ethanol as a flow-marker during the surgery in either of the groups. There was no statistically significant difference between the groups (On-Pump Group vs. Off-Pump Group) comparing the postoperative clinical outcome (ICU stay, ventilation duration, length of hospital stay). The dynamic changes in the interstitial concentrations of the glucose, glycerol, pyruvate and lactate were found in both the groups of patients (On-Pump Group and Off-Pump Group), but there was no difference in local blood flow when the ethanol dilution technique was used. These results showed significantly higher aerobic metabolic activity of the peripheral tissue of patients in the Off-Pump Group vs. the On-Pump Group during the course of cardiac revascularization surgery. Results suggest that extracorporeal circulation, cardiopulmonary bypass, compromises peripheral tissue (skeletal muscles) energy metabolism. These changes have no impact on the postoperative clinical outcome; no significant difference between the groups was found.

Cardiac troponin T in pregnant women having intravenous tocolytic therapy.

Abstract. We studied drug-induced cardiotoxic effects in 22 pregnant women having tocolysis with intravenous fenoterol and verapamil. Because CK-MB is released from the uterus and placenta, we used the determination of cardiac troponin T (cTnT) as it is one of the most sensitive and specific indicators of myocardial necrosis. Cardiac troponin T levels were within physiological range (0.08± 0.01 μg/l) in all healthy pregnant women tested between 32 and 36 weeks of gestation (control group). In the pregnant women having tocolysis cTnT levels started to increase slightly during the first day of treatment (0.10±0.03 μg/l) and were significantly higher (p<0.05) during the third day (0.35±0.14 μg/l) of tocolytic therapy. The cTnT levels in cord blood (0.13±0.03 μg/l) did not correspond with maternal cTnT concentrations.