Volume 40, Issue 4

Pancreatic elastase-1 is a specific human protease synthetized by the acinar cells. Immunoreactive elastase-1 cannot be detected in either porcine or bovine pancreatic enzyme preparations. It is very stable and, in contrast to fecal chymotrypsin, elastase is unaffected by exogenous pancreatic enzyme treatment, and correlates well with exocrine pancreatic function tests. The measurement of this proteolytic enzyme in stool by means of an enzyme-linked immunosorbent assay (ELISA) is a sensitive, specific, and relatively inexpensive non-invasive test. It is an accurate function test for patients with chronic pancreatitis confirmed by endoscopic retrograde cholangiopancreatography and computerized axial tomography. It shows higher sensitivity and specificity for exocrine pancreatic insufficiency than fecal chymotrypsin determination and is comparable to oral pancreatic function tests such as the pancreolauryl test.

The acute-phase “panproteinase” inhibitor α 2 -macroglobulin α 2M), a protein involved in inflammatory reactions, has been identified in amyloid plaques in Alzheimer's disease (AD). In addition, α2M is involved in AD susceptibility at the genetic level, and a deletion polymorphism at the α2M gene has been found to be associated with sporadic AD. We analyzed the deletion polymorphism and α2M plasma levels in 93 ultraoctuagenarian patients with late-onset sporadic AD and in controls (n=157). α2M allele frequencies did not differ between AD patients α2M*2 =0.169) and controls ( α2M*2 =0.146). The mean plasma concentrations of α2M were similar in patients (271.8±79 mg/dl) and controls (269.5±81.2 mg/dl). No difference was found in the α2M mean plasma levels associated with the three α2M genotypes, indicating that the deletion has no effect on α2M protein level. However, in AD patients α2M mean plasma values differed significantly according to apolipoprotein E genotypes (p=0.03), with E3/E3 homozygotes showing the highest levels. Since in a previous work E3/E3 were found to be associated with the highest plasma levels of α 1 -antichymotrypsin, another acute-phase protein, the present findings seem to support the hypothesis that inflammation may be a relevant factor in AD pathogenesis peculiar to E3/E3 subjects.

Human paraoxonase (PON1) is a calcium-dependent esterase exclusively bound to apolipoprotein A-I and clusterin, containing high-density lipoprotein (HDL) particles that hydrolyzes organophosphates and aryl esters. Several studies have indicated that PON1 can prevent low-density lipoprotein (LDL) oxidation by hydrolyzing lipid peroxides in the lipoprotein, which is the crucial first step for atherogenesis. Therefore it may protect against the development of atherosclerosis. Serum PON1 activity has been shown to be decreased in familiar hypercholesterolemia and in diseases that are associated with accelerated atherogenesis. The PON1 gene has two common coding region polymorphisms, Leu 55 →Met and Gln 192 →Arg. Both polymorphisms have been identified as independent risk factors for cardiovascular disease in diabetic and non-diabetic patients. We have established high-speed and easy-to-perform genotyping for the two most significant PON1 gene polymorphisms, employing the LightCycler technology and melting curves. This technique eliminates PCR contamination related to sample handling and does not require digestion of PCR products with restriction enzymes and/or fragment separation on gels.

NOS3 (endothelial nitric oxide (NO) synthase) and p22phox (subunit of NAD(P)H oxidase) are two genes whose products are involved in formation and degradation of NO, a ubiquitous signaling molecule largely responsible for the maintenance of normal endothelial function. The G894T polymorphism of NOS3 and the C242T polymorphism of p22phox are reportedly associated with numerous cardiovascular diseases. For each polymorphism we developed a rapid and reliable method with the hybridization probes format on the Lightcycler TM and compared it with conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with regard to reliability, duration and cost. The new methods are more reliable, faster and less expensive than PCR-RFLP analysis and therefore represent a significant advantage in the detection of two candidate risk factors for cardiovascular diseases.

It is estimated that about one in 24 Belgian Caucasians is a cystic fibrosis (CF) heterozygote. Until now, CF heterozygotes can only be identified by genetic decoding ( e.g. Inno-Lipa CF2 test ® ), as they cannot be phenotypically distinguished from the general population. The aim of this study was to evaluate differences in salivary electrolyte concentrations (calcium, bicarbonate, chloride, potassium, sodium and phosphate) and salivary osmolarity between CF homozygotes (n=41), CF heterozygotes (n=56) and healthy controls (n=65). Differences between the three groups were investigated by means of non-parametric tests. Several significant differences between the three study groups and among, as well as between, the different cystic fibrosis transmembrane regulator (CFTR) genotypes were observed. Significant differences in salivary electrolyte concentrations between individuals with delta F508 mutation and those without, and between CF homozygotes and heterozygotes were observed. The concentrations of several salivary electrolytes, and salivary osmolarity were significantly higher in CFTR genotype individuals. Differences in salivary electrolyte concentrations may partly explain differences in caries between the three study groups.

We have investigated the serum level of granulocytecolony stimulating factor (G-CSF) and macrophagecolony stimulating factor (M-CSF) and the commonly accepted tumor markers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA 19–9) in colorectal cancer. Additionally, we have defined the diagnostic sensitivity, specificity, positive predictive value, negative predictive value and receiver-operating characteristics (ROC) curve for G-CSF and M-CSF. The serum levels of cytokines were measured in 49 patients with colorectal cancer and in 40 healthy subjects. G-CSF and M-CSF were determined using enzyme-linked immunosorbent assay (ELISA). CEA and CA 19–9 were measured by microparticle enzyme immunoassay. There were significant increases in the level of circulating G-CSF and M-CSF in the colorectal cancer patients compared to the control group. Moreover, the diagnostic sensitivity of M-CSF was higher (65%) than the sensitivity of CEA (31%) and CA 19–9 (20%). The diagnostic specificities of M-CSF and G-CSF were 95%, and the M-CSF predictive value was higher compared with the predictive value of G-CSF. These results suggest a potential role for M-CSF as a tumor

We measured serum levels of soluble intercellular adhesion molecule-1 and erythrocyte membrane sialic acid in patients with rheumatoid arthritis and studied their correlation with the disease activity. Serum soluble intercellular adhesion molecule-1 level was determined with sandwich ELISA and sialic acid level with the method of Shamberger in 42 patients with rheumatoid arthritis and in 30 healthy controls. Significantly lower erythrocyte membrane sialic acid and higher serum levels of soluble intercellular adhesion molecule-1 were found in patients with rheumatoid arthritis compared with healthy controls (p<0.001 for both). Significant negative correlation between soluble intercellular adhesion molecule-1 level and erythrocyte membrane sialic acid concentration (r=−0.49, p<0.001) and positive correlations between soluble intercellular adhesion molecule-1 level and Ritche Articular Index score and C-reactive protein (r=0.32, p<0.05; r=0.44, p<0.01, respectively) were observed. From these data we conclude that decreases in erythrocyte membrane sialic acid concentration and increases in soluble intercellular adhesion molecule-1, erythrocyte sedimentation rate and C-reactive protein levels are present in rheumatoid arthritis, and that the increased soluble intercellular adhesion molecule-1 in rheumatoid arthritis might be due to the decreased erythrocyte membrane sialic acid concentration. The levels of soluble intercellular adhesion molecule-1 may be a novel marker for the disease status and the activity of rheumatoid arthritis.

Pesticide use is one of several factors that have permitted maintenance of our supply of food in spite of continuing increase of the population. However, the use of biologically active compounds poses potential problems of toxicity. If the compound is used at any stage of food production, residues or derivatives may persist in food and the entire population may be exposed to the trace amounts of the material. The human body burden associated with long-term exposure may or may not be associated with illness. Persistent environmental contaminants such as pesticide residues have long been suspected to be implicated in cancer etiology. Organochlorine chemicals are persistent, lipophilic compounds commonly present in the environment. Some of them demonstrated carcinogenic activity in laboratory animals. Controversy still exists concerning their carcinogenic potential in humans. To answer this question, clinical toxicology laboratories should propose validated methodologies able to identify and quantify pesticide residues in biological samples. An example of chromatographic method dedicated to organochlorine residues is presented here and illustrated by results obtained in a healthy population (104 men, 147 women). Only 17.9% of the samples were free from detectable amounts of pesticides and p,p’-DDE 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene was the most frequently detected residue (66.5%). Hexachlorobenzene was found at detectable level in 13.5% of the samples.

Reference values for Ferritin Flex TM on the Dimension RxL analyzer calibrated against the 3rd International Standard for Ferritin (recombinant) and N-Latex Ferritin ® on the BNA II nephelometer calibrated against the 2nd International Standard for Ferritin (spleen) both from Dade Behring (Marburg, Germany) were established (77 men and 182 women). Exclusion criteria were iron deficiency or iron deficiency anemia, inflammation, liver disease, malignancy, and other hematological or chronic disorders. The reference values (5.0th–95th percentiles) were as follow: for N-Latex Ferritin ® –men, 12–399 μg/l; women <50 years, 11–102 μg/l and women ≥50 years, 17–219 μg/l; for Ferritin Flex TM –men, 14–415 μg/l; women <50 years, 11–111 μg/l and women ≥50 years, 22–224 μg/l. Both assays correlated very closely with each other (r=0.993). The linearity was acceptable down to 2 μg/l for the Ferritin Flex TM method, but only down to 15 μg/l for the N-Latex Ferritin ® assay. The mean recovery of the 3rd International Standard by N-Latex Ferritin ® and Ferritin Flex TM was comparable (~80%). We conclude that the new Ferritin Flex TM assay, which is based on the new 3rd International Standard, should be used for ferritin measurement in the routine medical laboratories in the future.

In order to study the relationships between sex hormones, aging, and circulating levels of cardiac natriuretic peptides and to define reference values for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) assays, we measured the plasma levels of cardiac natriuretic peptides in a large group of healthy adults divided according to age and sex. We studied 216 healthy subjects of both sexes (109 men and 107 women) with age ranging from 20 to 77 years (mean 43.2±14.8 years). All subjects were non-obese and had normal arterial blood pressure; they were free from acute diseases, including asymptomatic heart disease. Highly sensitive and specific IRMA methods were used to measure plasma ANP and BNP. The mean ANP value in healthy adult subjects of both sexes was 17.8±10.9 pg/ml with no significant difference between men (16.7±10.0 pg/ml) and women (18.8±11.7 pg/ml). The mean BNP value in healthy adult subjects of both sexes was 9.9±9.0 pg/ml with a significant difference (p<0.0001) between men (7.7±7.1 pg/ml) and women (12.2±10.2 pg/ml). There was a weak linear relationship between age and either ANP (r=0.350, p<0.0001) or BNP (r=0.254, p=0.0002) values. When the circulating levels of cardiac natriuretic hormones, and age and sex were analyzed by multiple stepwise regression analysis, both age and sex significantly and independently contributed to the regression. Our study indicates independent positive effects of aging and female sex hormones on ANP and BNP levels in healthy adult subjects. These effects should be taken into account in the calculation of appropriate reference values for cardiac natriuretic hormones.

Values of plasma amino acids (AA) in Taiwan Chinese in different age groups were determined. AA analysis was performed by ion-exchange chromatography. The results correlated well with the reference values published by Shapira in 1989, indicating that plasma AA concentrations are not evidently affected by race and dietary habits. We also confirmed the effect of age on plasma free AA levels. More than two thirds of 35 amino acids showed statistically significant differences between age groups (p<0.05, one-way ANOVA test). Levels of taurine, hydroxyproline, threonine, serine, glutamic acid, proline, citrulline and α-amino-η butyric acid varied with age (p<0.01). Only a few AA, including α-amino-adipic acid, glycine, alanine, cystine, homocystine, isoleucine, tyrosine, γ-aminobutyric acid and tryptophan, showed no statistically significant differences between the different age groups. When comparing the mean differences in individual AA among these three age groups using the post-hoc test, alanine, tyrosine, phenylalanine, glycine, glutamine, cystine, homocystine, isoleucine, γ-aminobutyric acid and tryptophan appeared to be constant across all age groups. Glutamic acid and serine correlated significantly with age (p<0.05), with a definite decline in concentration from infancy to adulthood (r=0.396 and 0.243; p<0.01, respectively). We conclude that AA values are not evidently affected by race, but they do vary with age.

Age-related variation of the urinary excretion of amino acids has been widely reported. Instead of the conventional reference intervals, continuous reference limits might be a more practical approach during periods of rapid changes such as infancy or childhood. Regression-based reference limits have been produced for the urinary excretion of 23 amino acids analyzed in 148 healthy individuals from 0 to 12 years of age. Urinary excretion was modeled as a function of age by two parametric procedures: the polynomial and the piecewise linear regression methods. Likewise, age-specific standard deviation was estimated by the regression of the absolute values of the residuals on age. Residual analysis was employed to select the best-fitting model and the 95% reference limits, and their 95% confidence intervals were calculated and plotted against the raw data. The urinary excretion of 19 amino acids decreased rapidly during the first year of life with a further slow decline thereafter. These amino acids fit better the piecewise model. The other four amino acids displayed a steady decrease in the urinary excretion from birth to puberty, and the excretion patterns fit better the quadratic or linear model. Fifteen amino acids showed a significant change in standard deviation with age. Regression-based reference limits differed consistently from conventional limits (<3 years) and narrower confidence intervals were obtained throughout the entire period studied. Avoiding partitioning gave rise to smoothly changing limits. Therefore, such alternative way of presenting amino acid reference limits may facilitate the follow-up of patients with inborn errors of amino acid metabolism.

We studied the uncertainty of measurement for the calcium and glucose (amount of) substance concentrations in serum. The evaluation follows a four-step procedure, which complies with the ISO document Guide to the Expression of Uncertainty in Measurement (GUM). The applications were chosen to represent commonly used measuring systems in medical laboratories. The uncertainty components are quantified using observations of the measuring system, and information from calibration certificates, instrument specifications and literature. The evaluation focuses on the measurement step but empirical terms are used to illustrate how the pre-analytical phase and patient-related issues can be accounted for. The software GUM Workbench ® was used to facilitate calculations and to visualize the importance of each uncertainty component. The combined standard uncertainties ( u c ) for the measurands were ≤2% including the pre-analytical uncertainty sources. The patient-related source is discussed in relation to clinician's diagnosis and decision-making. The evaluation, as carried out here for calcium and glucose substance concentration measurements, can easily be applied to many other measurands in clinical chemistry. This work emphasizes that the internal quality control can provide much of the information needed in the uncertainty evaluation, and that external quality assessment (EQA) schemes are important in the control of the uncertainty evaluated by the individual laboratories. Due to statistical and metrological limitations routine EQA schemes should themselves not be used as a means of uncertainty evaluation.

Prothrombin time (PT) is tested mostly to monitor patients on oral anticoagulant treatment. The International Normalised Ratio (INR) was introduced to improve and harmonise PT results and therapeutic range globally for patient care and the scientific literature. We studied the Quick PT in 179 patients and the Owren PT in 137 patients on oral anticoagulant therapy using two different reagents for the two methods of measuring PT. We assessed the clinical significance of the INR results obtained by each method using the two reagents and compared the Quick and Owren methods. We conclude that with the Quick method individual INR results differed from each other too much clinically, while using the Owren method individual INR results were clinically acceptable. Our opinion is that we should develop the INR system using the Owren PT method rather than the Quick to improve patient care.

Most equations used for calculation of the base excess ( BE , mmol/l) in human blood are based on the fundamental equation derived by Siggaard-Andersen and called the Van Slyke equation: BE = Z . [{ c HCO 3 -(P)– c 7.4 HCO 3 -(P)} + β. (pH–7.4)]. In simple approximation, where Z is a constant which depends only on total hemoglobin concentration ( c Hb, g/dl) in blood, three equations were tested: the ones proposed by Siggaard-Andersen (SA), the National Committee for Clinical Laboratory Standards (NCCLS) or Zander (ZA). They differ only slightly in the solubility factor for carbon dioxide αCO 2 , mmol/l.mmHg) and in the apparent p K (p K '), but more significantly in the plasma bicarbonate concentration at reference pH ( c> 7.4 HCO 3 -(P), mmol/l) and in Β, the slope of the CO 2 -buffer line (mmol/l) for whole blood. Furthermore, the approximation was improved either by variation in Z ( r c ), or in the apparent p K (p K ') with changing pH. Thus, from a total of seven equations and from a reference set for pH, p CO 2 and BE taken from the literature (n=148), the base excess was calculated. Over the whole range of base excess (−30 to +30 mmol/l) and p CO 2 (12 to 96 mmHg), mean accuracy Δ BE , mmol/l) was greatest in the simple equation according to Zander and decreased in the following order: ±0.86 (ZA); ±0.94 (ZA, r c ); ±0.96 (SA, r c ); ±1.03 (NCCLS, r c ); ±1.40 (NCCLS); ±1.48 (SA); and ±1.50 (p K' ). For all clinical purposes, the Van Slyke equation according to Zander is the best choice and can be recommended in the following form: BE = (1−0.0143 . cHb) . [{0.0304 . p CO 2 . 10 ph−6.1 −24.26} + (9.5+1.63 . c Hb) . (pH −7.4)] −0.2 . cHb . (1−sO 2 ), where the last term is a correction for oxygen saturation ( s O 2 ). Hence, base excess can be obtained with high accuracy (<1 mmol/l) from the measured quantities of pH, p CO 2 , c Hb, and s O 2 in used.

Clinical chemistry and laboratory medicine developed rapidly in the industrialised world after the Second World War, as evidenced by the recruitment of physicians and natural scientists, the intensive development of methods and technologies, the founding of societies such as the IFCC, and the organisation of scientific life. In East Germany a working group of clinical pathologists and chemists came together in 1960, out of which grew the Gesellschaft für Klinische Chemie und Laboratoriumsdiagnostik (Society for Clinical Chemistry and Laboratory Diagnostics) in 1968. Within the socialist health care system laboratory services were structured in a pyramid form based on the country's governmental districts. Each local diagnostic laboratory was part of a hierarchy; tests not possible in local laboratories were performed in larger ones. Groups of experts prescribed standardised methods and recommendations of quality control were administered at the district level. However, the funding of this quite acceptable structure was insufficient, resulting in difficulties. Although the development of equipment was carried out in close cooperation with users, it was hindered by shortages extant in the socialist economy. Postgraduate training of physicians, natural scientists, technicians and laboratory assistants was well organised, and clinical chemistry and pathobiochemistry were well established in the medical curriculum.