Volume 38, Issue 12

Background: The classical two-period crossover and two-parallel-groups designs for clinical drug trials are unable to answer many current scientific questions, and are sometimes ethically or financially difficult. For example, the classical designs do not allow the study of the effects of combined treatments and their interactions. Also, the generation of parallel data that are no more than a repetition of previous research is ethically debatable and in a sense a waste of money. Objectives are to identify what a classical clinical trial cannot manage, to summarize and discuss alternative trial designs that are helpful for such purposes. Results: A classical clinical trial cannot: (i) assess combined therapies, (ii) take historical data into account, (iii) safeguard ethics and efficacy during the course of long-term trials, (iv) study drugs, before well-established toxicity information is available, (v) account for the possibility of therapeutic equivalence between test and reference treatment, (vi) study multiple treatments in one trial, and (vii) adjust change scores for baseline levels. Alternative designs helpful for such purposes are respectively: (i) factorial designs, (ii) historical controls designs, (iii) group-sequential interim analysis designs, (iv) sequential designs for continuous monitoring, (v) therapeutic equivalence designs, (vi) multiple crossover-periods/multiple parallel-groups design, and (vii) increased precision designs through multivariate adjustment. Main problems include the increased risks of type I and type II errors and the loss of validity criteria. Conclusions: Non-classical trial designs are reviewed. They offer relevant scientific, ethical, and financial advantages. The increased risks of type I and type II errors should be accounted for in the design stage of the trial.

Hereditary haemochromatosis (HH) is one of the most common inherited diseases among Caucasians. Two mutations in the HFE gene have been implicated in HH: 80 to 90% of the patients with HH are homozygous for the point mutation CYS282Tyr, while the majority of the remaining patients displays either a compound heterozygosity for the mutation CYS282Tyr and the point mutation HIS63Asp, or are homozygous for HIS63Asp. Though the disease can be treated easily, symptoms are non-specific, and onset and severity are influenced by environmental factors, and therefore the disease can remain undetected until decades of iron overload lead to irreversible damage in a variety of organs, which may result in their failure. In order to detect patients with HH, simple and cost-effective tests are needed. We have developed a rapid, automated, PCR-based test which makes use of a diagnostic restriction site in each of two amplified fragments. The test employs off-the-shelf chemistry and uses the automated detection process of an immunoassay analyzer that is available in many clinical laboratories, thus avoiding an additional investment in a more specialized PCR analyzer. Because of its low costs and easy handling, the assay is particularly suited for the routine clinical laboratories.

Secretory group IIa phospholipase A 2 (PLA 2 -II) is an important regulator of proinflammatory lipid mediator production and may play a role in ileal inflammation in Crohn's disease. The enzyme has previously only been detected in epithelial Paneth cells. However, one characteristic feature of Crohn's disease is the transmural inflammation. Full thickness ileal sections from nine patients with Crohn's disease, and histologically normal sections from patients with colonic cancer (n=7) and chronic severe constipation (n=1) as controls, were used in this study. PLA 2 -II-positive cells were detected by immunofluorescence and in situ hybridization. Metachromatic staining and esterase staining were used to identify mast cells and macrophages, respectively. It was shown that mast cells and macrophages in the ileal submucosa in both patients and controls showed positive PLA 2 -II staining. The number of PLA 2 -II-labeled cells that did not react with metachromasia, e.g. macrophages, was significantly greater in inflamed Crohn's disease compared to controls. This is, to our knowledge, the first study that has described the presence in healthy, while presence and upregulation of PLA 2 -II-positive cells in inflamed human ileal submucosa. Our findings suggest a proinflammatory potential for secretory PLA 2 -II in submucosa, while proinflammatory stimulation of mast cells and macrophages in vitro has shown that the enzyme is responsible for delayed prostaglandin formation.

Ammonia is known to increase the secretion of Β-N-acetylhexosaminidase (Hex) to the extracellular medium in cultured human fibroblasts and Hep G2 cells. We examined 35 patients with liver cirrhosis and the results revealed a significant increase for the plasma activities of total Hex and its isoenzymes Hex A and Hex B (p < 0.001). The partial correlations, with other biochemical markers of liver injury constant, between plasma ammonia concentration and the activity (r = 0.658) and the proportion in percentage of the Hex B isoenzyme (r = 0.692) were statistically significant (p < 0.001). The increased concentration of ammonia could explain, at least partially, an increased secretion of Hex B isoforms to the plasma in patients with liver disease.

We have recently shown that low concentrations of copper ions increased the extracellular concentration of homocysteine in a HeLa cell culture system. This increase might be attributed to copper-induced oxidation of extracellular homocysteine, since earlier reports suggest that the oxidized fraction of homocysteine seems to be less available for cellular uptake than the reduced fraction. To investigate further this mechanism we have therefore, in the present study, determined intra- and extracellular concentrations of homocysteine and their relations to the extracellular thiol redox status. The results of this study show that the redox status of homocysteine is important for its extracellular concentration. Increased oxidation of thiols catalyzed by copper ions resulted in an increased extracellular concentration of homocysteine, whereas the addition of antioxidant agents (N-acetylcysteine or dithiothreitol) or a specific copper(I) ion chelator (bathocuproinedisulfonic acid) was accompanied by a decreased extracellular concentration of homocysteine. These effects are probably dependent on differences in cellular uptake between reduced and oxidized homocysteine.

Although serum total sialic acid has been shown to be a cardiovascular risk factor, with elevated levels associated with increased cardiovascular mortality and also with cerebrovascular disease, the reason for the elevation in serum sialic acid content remains obscure. It has been shown that an increased output of serum proteins by the liver due to some type of acute phase reaction may be one of the possible sources of an increased serum sialic acid concentration in patients with myocardial infarction. An increase in the activity of sialidase, which cleaves the terminal sialic acid residues from oligosaccharides, glycoproteins and gangliosides, may also play an important role in the elevation of serum total sialic acid in myocardial infarction. Elevated serum total sialic acid in the blood might result either from the shedding or secreting of sialic acid from the cell membrane surface, or releasing of cellular sialic acid from the cell into the bloodstream due to cell damage after myocardial infarction. The purpose of the present study is to investigate serum total and lipid-bound sialic acid and the enzymes serum lactate dehydrogenase, creatine kinase and aspartate aminotransferase in patients with acute myocardial infarction, at 24 h post-infarction (day 1), 48 h post-infarction (day 2) and 72 h post-infarction (day 3). A possible role of cell damage in the elevation of serum total and lipid-bound sialic acid levels in these patients was also evaluated. In this study, 40 patients with myocardial infarction ranging in age from 42 to 68 years, and 26 healthy volunteers ranging in age from 45 to 71 years were included. Serum total sialic acid determination was carried out by the thiobarbituric acid method of Warren and lipid-bound sialic acid by the method of Katopodis. Our data shows that a) there is a gradual increase in the levels of serum total sialic acid and lipid-bound sialic acid during the first three days after the acute myocardial infarction and b) the elevation in serum total sialic acid levels correlates with the elevation in lactate dehydrogenase activity only on day 1 following infarction. Therefore, either the shedding or secreting of sialic acid from the cell or cell membrane surface may be partly responsible for an increased serum sialic acid concentration especially on day 1 following myocardial infarction.

The concentration of leukocyte elastase/α 1 -proteinase inhibitor complexes in plasma and polymorphonuclear neutrophil extracts, and plasma trypsin inhibitory capacity were determined in 88 patients with type 2 diabetes and 47 control subjects. Higher values of these variables were found in patients as compared to controls (p < 0.001). The concentration of elastase was higher in obese patients than in lean ones (p < 0.05 for plasma, p < 0.01 for polymorphonuclear leukocytes). Only leukocyte elastase levels were significantly higher in the group with both micro- and macroangiopathy in comparison to the group with microangiopathy (p < 0.01) or macroangiopathy (p < 0.05) alone. Poor short-term glycaemic control was associated with higher elastase concentration in plasma and neutrophils (p < 0.05). The present study demonstrates that measurements of plasma polymorphonuclear neutrophil elastase level can be considered as a marker of development of diabetic angiopathy.

A high serum triglyceride (TG) concentration is associated with an increased serum concentration of small, dense low-density lipoprotein (LDL). To further characterize the hypertriglyceridemic condition, we examined sera from 240 subjects for small, dense LDL using non-denaturing polyacrylamide gradient gel electrophoresis. We focused on determining the frequency of the pattern B, which is characterized by a higher proportion of small, dense LDL, among hypertriglyceridemic individuals. The subjects were divided into four groups: a control group (TG ≤ 1.65 mmol/l, high-density lipoprotein cholesterol (HDL-C) ≥1.17 mmol/l; n = 71), a high TG group (TG >; 1.65 mmol/l, HDL-C ≥1.17 mmol/l; n = 36), a group with high TG and low HDL-C (TG > 1.65 mmol/l, HDL-C ≤ 0.91 mmol/l; n = 106), and a low HDL-C group (TG ≤ 1.65 mmol/l, HDL-C ≤ 0.91 mmol/l; n = 27). We found that pattern B occurs at a high frequency mainly in individuals with high TG and low HDL-C levels. We also observed an increased percentage of LDL within the 20.0 nm to 25.5 nm particle diameter range in this group. Analysis of the lipoprotein lipase gene in this group showed that some mutations seem to be associated with small, dense LDL, resulting in LDL pattern B.

The effect of sample collection, storage conditions (time and temperature), and freeze-thaw cycles on the stability of free prostate-specific antigen (fPSA), PSA complexed with α 1 -antichymotrypsin (ACT-PSA), and total PSA (tPSA) in serum was studied. The analytes were quantified using immunoassays for tPSA and fPSA on the Elecsys system 2010 and a research assay for ACT-PSA on the ES system (Roche Diagnostics). The stability of the analytes was calculated as percentages of the values measured in samples 1 h after blood collection. When the samples were stored at 37 °C, at room temperature or at 4 °C, the stability of ACT-PSA was less impaired than that of fPSA. To avoid erroneous results in the determination of PSA isoforms and their corresponding ratios, serum samples should be preserved at 4 °C when the analysis is performed within 8 h after blood collection, or they should be stored at −80 °C if the analysis is not feasible during that period.

There is abundant evidence that free radicals are involved in membrane pathology in the central nervous system and that they may play a role in neuropsychiatric disorders, including schizophrenia. In this study, we investigated erythrocyte superoxide dismutase and glutathione peroxidase activities as antioxidant enzymes, malondialdehydes as a sign of lipid peroxidation, and reduced glutathione levels in schizophrenic patients. Activities of superoxide dismutase and levels of malondialdehyde in erythrocytes were greater in all patients (n=48) and in patients with acute (n=16) and chronic schizophrenia (n=32) (p<0.001 for all patients and chronic patient group; p<0.05 for acute patient group). The activities of glutathione peroxidase were lower in patients (p<0.05 for all patients and acute patient group; p=0.051 for chronic patient group) compared with the control group. Mean erythrocyte reduced glutathione was lower in patients than in controls (p<0.05). In the patient group, erythrocyte superoxide dismutase activity was positively correlated with scales and duration of disease and erythrocyte malondialdehyde concentration. These data reveal that antioxidative defense mechanisms might be impaired in schizophrenic patients.

We estimated reference change value and steady state disease biological variation of glycohemoglobin (HbA 1c from serial measurements in 47 clinically stable type diabetes patients whose home-measured capillary glucose was stable throughout the study. Whole blood HbA 1c assays were performed by turbidimetric inhibition immunoassay. The analytical imprecision (coefficient of variation) was 7.1% and 5.0% for control materials with HbA 1c of 5.1% and 10.7%, respectively (n=152). The sampling interval was 6 months. Patients were classified into three groups (good, acceptable and poor control) according to the American Diabetes Association (ADA) clinical practice recommendations of 1999 based on HbA 1c reference interval. Steady state disease biological variation for each control group was 7.9%, 5.4% and 3.9%. HbA 1c absolute reference change value was 1.42, 1.50 and 1.37%, or as relative reference change value, 29.1%, 24.4% and 17.8%, respectively. The analytical goal, defined as one-half of the biological variation, was lower than 3.9% for well-controlled patients.

In a study of 50 full-term newborns, we found serum levels of ceruloplasmin, its oxidase activity and specific oxidase activity (activity per unit mass of enzyme protein) which were significantly lower than in adult subjects (p<0.001). A significant correlation was obtained between the specific oxidase activity of the neonatal ceruloplasmin and the transferrin/α-fetoprotein ratio (p<0.01), which reflects the maturity of the fetal hepatocyte through its ability to synthesise fetaland non-fetal-associated proteins. The lower specific oxidase activity of the ceruloplasmin in newborns would be due to a higher relative proportion of apoprotein, and/or other molecular forms with a poor copper content in the oxidase sites. This could be due to an inability to transfer copper into a common intracellular pool for holoceruloplasmin synthesis and biliary copper excretion. A high interindividual variability was found for the specific oxidase activity of the neonatal ceruloplasmin, and in the newborns with higher levels of ceruloplasmin this, from a catalytic point of view, is more similar to the adult form.

To evaluate the serum urate levels in AIDS patients with infections of the central nervous system (CNS), 46 patients who had at least two measurements of urate were included. A maximum of four measurements per patient were considered: prior to the CNS involvement (U-PRIOR), at the time of CNS involvement (U-CNS), after treatment for the CNS infection (U-AFTER), and the last measurement before death (U-LAST). Serum U-CNS levels were significantly lower than U-PRIOR values (p=0.038). U-AFTER levels were higher than U-CNS in the patients who improved (p=0.25), and lower in the patients who did not improve (p=0.026). There were no significant differences among the four diagnostic groups in U-CNS measurement (p=0.29) but they were found in U-AFTER determinations (p=0.018), probably as a result of the different response to treatment. Hypouricemia seemed to be associated with lower survival periods. We conclude that hypouricemia is common in AIDS patients with CNS infections, probably as a result of increased renal losses of urate, and that it may have prognostic significance. CNS infections are associated with significant decreases in serum urate levels in comparison with previous values. The urate concentrations seem to increase after successful treatment of the CNS infections, whereas they decrease further in patients who do not improve.

We have evaluated a newly introduced immunoturbidimetric transferrin receptor assay (IdeA TfR-IT, Orion Diagnostica, Finland) in healthy subjects and in a study population consisting of patients with rheumatoid arthritis and juvenile chronic arthritis. The IdeA TfR-IT assay was found to provide reproducible results which were in good agreement with the ELISA assays from Orion Diagnostica (IDeA-ELISA, correlation R 2 =0.8, n=102) and R&D systems (Quantikine TfR ELISA assay, correlation R 2 =0.95, n=39). The analysis of the patient samples suggested that, on the basis of serum transferrin receptor and ferritin concentrations, in approximately one third of patients with rheumatoid arthritis anemia is due to the depletion of iron stores. Apparently, in all patients with rheumatoid arthritis iron deficiency must be considered as a potential cause of the anemia. Now, that assays which are suitable for automated analyzers have become available for the measurement of serum transferrin receptor, this analyte has the potential to become a part of the routine evaluation of iron status.

A reference method is described for the determination of the substance concentration of ionized calcium in plasma by which ionized calcium (free or unbound) may be reliably determined on the basis of calibration with aqueous solutions with known concentration of ionized calcium. The composition of the calibration solutions is chosen such that the activity coefficient of the calcium ion is assumed to be identical both in the calibration solutions and in “normal” plasma, i.e. by convention, the ionic strength ( I m ) is 0.160 mol/kg. The convention is adopted of reporting ionized calcium measurements as concentration expressed as mmol/l. The proposed reference method for ionized calcium measurement in plasma is based on the use of a cell consisting of an external reference electrode with a saturated potassium chloride liquid/liquid junction in combination with a calcium ion-selective membrane electrode of defined construction and performance. Procedures for using the reference cell and a protocol for sample measurement are described. The preparation of the calibration solutions to be used are described in detail in Appendix A, secondary calibration solutions and check standards in Appendix B, and reference cell vessel design in Appendix C.