Volume 43, Issue 4

The aim was to develop and validate an assay for measuring autoantibodies against human intrinsic factor (IF). For this purpose 1.25pmol of recombinant IF was coupled to each well. Samples (100μL of plasma diluted 1:1) were incubated for 1h, followed by 30min with the detection reagent (biotinylated IF, 3.3pmol/mL). Samples were obtained from healthy blood donors (n=141) and patients with suspected vitamin B 12 deficiency (n=355). The initial assay results (n=99 samples) were in reasonable agreement with those obtained using a commercial assay (Diagnostic Products Corporation). All donors but one showed a negative result. For the patient populations the fraction of positive results decreased with increasing levels of serum vitamin B 12 : 0.67 for <100pmol/L (n=13), 0.17 for 100–150pmol/L (n=23), 0.06 for 151–200pmol/L (n=65) and <0.01 for >200pmol/L (n=254). The imprecision was 11% as judged by repeat analyses. All samples remained positive when the catching reagent was exchanged with vitamin B 12 -saturated IF, suggesting that the samples contained both blocking (type I) and non-blocking (type II)antibodies. Results obtained with IF as the detection reagent correlated to those obtained when anti-immunoglobulin G was used instead. In conclusion, an ELISA using recombinant IF as both catching and detection reagent seems suitable for the detection of IF autoantibodies in plasma.

Antibody titer against malondialdehyde (MDA)-modified low-density lipoprotein (LDL) has been found to be associated with atherosclerosis, but it has not been established whether it would detect subjects with coronary artery disease (CAD). In the present study, receiver-operating characteristic (ROC) analysis was used to compare the diagnostic accuracy of the antibody titer against MDA-modified LDL and high-density lipoprotein (HDL) and LDL cholesterol levels in discrimination between subjects with (n=51) and without (n=35) angiographically verified 3-vessel CAD. As a result, the antibody titer against MDA-modified LDL was lower in subjects with CAD compared with subjects without CAD (p<0.0001). The area under the ROC plot was 0.822 (95% CI, 0.727 to 0.918) for the antibody titer and 0.769 (95% CI, 0.661 to 0.876) for the HDL cholesterol concentration. Both the antibody titer and the plasma HDL cholesterol level were more accurate markers of CAD than the LDL cholesterol level. As a conclusion, our results indicate that the antibody titer against MDA-modified LDL discriminates between subjects with widespread CAD and those without CAD similarly as the HDL cholesterol concentration. Moreover, the antibody titer against MDA-modified LDL is inversely correlated with the risk of severe CAD.

Increased expression of sialyl Lewis X or A antigens on metastatic cancer cells leads to their selectin-mediated extravasation. Profound fucosylation of the serum microenvironment may be a factor that interrupts adhesion and influences the formation of metastases. In this study we quantitatively analyzed fucosylation of serum glycoproteins in small-cell and non-small-cell lung cancer patients. Fucosylation of four chosen glycoprotein bands was measured as the reactivity with Aleuria aurantia lectin on nitrocellulose blots, preceded by polyacrylamide gel electrophoresis. Relative fucosylation and fucosylation coefficients were calculated by densitometric analysis. Fucosylated oligosaccharides were observed in higher amounts in cancer sera when compared to sera from healthy individuals in all bands analyzed. Glycoproteins of a molecular mass of 29kDa appear to carry more fucose residues than the 42-kDa band, comprising α 1 -acid glycoprotein and haptoglobin. Glycans of the 26-kDa band were fucosylated to a higher extent in non-small-cell vs. small-cell lung cancer. The results suggest that the extent of fucosylation could be a useful marker for estimation of the glycosylation status of serum proteins in cancer patients. Cluster analysis leads to the preliminary suggestion that the fucosylation status could serve as a predictive factor for patient survival.

Squamous cell carcinoma of the oesophagus is one of the most common cancers among black males in South Africa. Genetic polymorphism in the cytochrome P450 2E1 (CYP2E1) gene, coding for one of the main enzymes involved in the bioactivation of tobacco- and alcohol-related substances, was investigated for its role in the development of oesophageal cancer. Three single nucleotide polymorphisms −1053C→T, −1293G→A (both give rise to CYP2E1*5 ) and 7632T→A ( CYP2E1*6 ) in the 5′-untranslated region of CYP2E1 were investigated in 189 patients and 198 control individuals in South Africa. The mutant variants occurred between patients and controls at frequencies of 1% and 2% (−1053C→T), 1% and 3% (−1293G→A) and 18% and 7% (7632T→A), respectively. In comparing patients with controls, the heterozygous CYP2E1*6 genotype was associated with increased risk of the development of squamous cell carcinoma of the oesophagus (odds ratio, 5.90; p<0.001) after adjusting for age, sex, smoking and alcohol consumption. In contrast, we did not find a significant association between CYP2E1*5 and oesophageal cancer. Six novel mutations, −1371G→A, −1359C→G, −1342C→G, −1261T→del, −1189T→C and −1103C→G, were identified by DNA sequence analysis of the CYP2E1 promoter region. In summary, our case-control study of oesophageal cancer revealed an elevated risk associated with the CYP2E1*6 allele in South Africans.

The aim of our study was to evaluate the benefits of supplementation with 800mg/day of α-tocopherol with regard to cellular viability in HIV-1 seropositive patients undergoing anti-retroviral therapy. A total of 29 patients participated in the study, of whom 14 were given the supplement and 15 a placebo. The analyses were carried out before treatment commenced and after 60, 120 and 180days. The plasma levels ofHIV-1 RNA showed a significant decrease as a consequence of treatment time in the groups studied (p=0.0001), although the difference between the treatments over time was not verified (p=0.7343). The percentage of viable lymphocytes showed a significant increase as a consequence of treatment time in both groups studied (p=0.0002) and a significant difference between the treatments over time (p=0.0472). The percentage of lymphocytes in apoptosis showed a significant reduction over time (p=0.0003), as well as a significant difference between the treatments over time (p=0.0321). The significant increase in cellular viability indicates that supplementation with α-tocopherol offers an additional positive effect on cellular preservation in HIV-1 individuals undergoing anti-retroviral therapy; however, it represents an additional risk of anti-retroviral therapeutic failure, possibly due to drug-drug interaction involving up-regulation of metabolic clearance.

The effects of supplementation with a fixed combination of antioxidants (vitamins C and E, β-carotene and selenium) on superoxide dismutase activity, copper and zinc concentrations, and total antioxidant status were monitored in erythrocytes derived from a group of patients with Graves' disease treated with methimazole, with respect to the rate of achieving euthyroidism. Thyroid-stimulating hormone (TSH), thyroid hormones and the above-mentioned parameters were measured before therapy, and on days 30 and 60 after therapy initiation. The patients receiving antioxidant supplementation along with methimazole therapy (group A, n=27) achieved euthyroidism at a faster rate than those treated with methimazole alone (group B, n=28). The activity of superoxide dismutase decreased significantly in both patient groups during the treatment; however, there was no significant difference between the groups. There was no significant change in the erythrocyte concentration of copper, whereas the zinc concentration and total antioxidant status showed significant between-group differences. The study results clearly show that antioxidant supplementation in the treatment of Graves' disease is justified, while zinc and total antioxidant status in erythrocytes seem to be sensitive indicators of the efficacy of supplemental therapy.

We investigated concentrations of calcium, copper, iron, magnesium, potassium, sodium and zinc using atomic absorption spectroscopy in the hair of four groups of adult females (n=392), ranging in age from 20 to 50years, with different body mass index (BMI): BMI<18, slim group; BMI 18–25, normal group; BMI 26–35, overweight or obese group; and BMI>35, morbidly obese group. We found that the group with BMI<18 had the highest ratios for [Ca]/[Mg], [Fe]/[Cu] and [Zn]/[Cu], but the lowest ratio for [K]/[Na] in hair. On the contrary, the group with BMI>35 had the highest ratio for [K]/[Na], but the lowest for [Fe]/[Cu] and [Zn]/[Cu] in hair. Furthermore, when we compared concentrations of Ca, Cu, Fe, Mg, K, Na and Zn between the groups with BMI<18 and BMI>35, we found that there were significant differences (p<0.05) in zinc concentrations between these two groups. In addition, there were significant differences in Ca, Cu, Mg, K and Na concentrations, with p<0.01 at least. From this point of view, we suggest that hair concentrations of Ca, Cu, Fe, Mg, K, Na and Zn may be correlated with adult female BMI, but further studies are needed.

A recently proposed procedure for estimating the limit of detection (LoD) of an assay using a partly non-parametric approach is applied to five HPLC drug assays. The non-parametrically determined 95th percentile of the blank measurements (LoB) is obtained as the value of the N (95/100)+0.5 ordered observations. The LoD is the lowest level of analyte that is likely to yield a measured result exceeding the LoB. The LoD equals LoB+c β ×SD S , where SD S is the analytical SD at low concentrations, and c β = z 1–β /(1–1/(4× f )) ( f =degrees of freedom). c β ≈1.65 for a type II error of 5%. The blank distributions deviated significantly from normality for four of the five assays because of skewness to the right. The estimated LoB ranged from 3.3 to 10.2nmol/L. The LoDs were in the interval from 7.8 to 17.2nmol/L. A new procedure for estimation of the standard error (SE) of the estimated LoD that is partly based on the bootstrap principle showed good performance in simulation studies. In conclusion, the partly non-parametric procedure for estimation of the LoD of typical HPLC drug assays was found to be a suitable approach.

In a growing number of laboratories the technique of liquid chromatography-tandem mass spectrometry is used for the quantification of cyclosporin A in whole blood, employing cyclosporin D as the internal standard. Cyclosporin A is extensively metabolized in vivo; in liquid chromatography-tandem mass spectrometry respective metabolites can give rise to both parent and product ions that are isobaric with ions commonly used for the detection of cyclosporin A and cyclosporin D, respectively. In this article it is demonstrated that limited chromatography with co-elution of such metabolites together with cyclosporin A and cyclosporin D can lead to incorrect results.

Clinical activity indices are essential instruments in monitoring inflammatory bowel diseases such as Crohn's disease (CD) and ulcerative colitis (UC). To subclassify components of disease indices in CD and UC, investigate technical noise in estimation of the indices, establish a signal-to-noise ratio (SNR), evaluate correlation between indices and calculate the reference change value (RCV) for selected biochemical variables in individual cases, 50 patients with CD and 49 patients with UC were included in the study. Qualitative index variables were assessed for scoring errors. The standard deviation (SD) was estimated according to a rectangular model, while SD in biochemical variable scoring was estimated according to a Gaussian model; a combined SD was also calculated. These values were investigated for their individual contribution to variation. The 95% CI of an index value was based on ±1.96×SD combined and a change in separate biochemical variables was calculated as RCV 1.96×√2×SD combined . Correlation between different disease activity indices was assessed for unexplained variation. The Crohn's disease activity index (CDAI) had the highest variation compared to the van Hees (Hees) and the Harvey-Bradshaw index (HBI) in CD, but it also had the best SNR, whereas HBI had the lowest. In UC the clinical activity index (CAI) showed the highest variance, but the best SNR compared to Seo's activity index (AI). The 95% CI of the CDAI discriminatory activity sum of 150 in individual cases was 105–195, whereas the 95% interval for a change was ±62.4. Self-reported wellness contributed 40% to total variance in the CDAI. Factors of clinical importance increased errors in estimates and variance of the indices. Poor correlation was obtained between activity indices, with up to 70% unexplained variance. The SD combined for estimated errors was as high as 23 points, with the best SNR being approximately 20. Index factors increase the sensitivity of SNRs to errors and lower the disease specificity. Sensitivity optimisation may be achieved by standardisation of the variables and their use.

In proficiency testing (PT), the peer-group mean is conventionally computed after twice removing values exceeding the mean±3 SD. However, this adjustment fails if there are many outliers. In this study an iterative method was evaluated as a more robust way to estimate the means. The methodology repeatedly removes a proportion of the population (usually those exceeding the mean±1.6 SD), assuming the presence of a Gaussian distribution in the central portion, and reinflates the SD to compensate for the trimming. A computer simulation revealed that the estimated mean of a known Gaussian distribution was less affected by a subpopulation that overlaps the main population than was the conventional method. When the overlapping portions were removed, the iterative method predicted the true mean correctly. The method was applied to external PT results for 44 analytes. Although most peer-group distributions were clearly non-Gaussian, the segment included by the predicted mean±1.6 SD was regarded as Gaussian in 85.9% by the new method and 73.4% by the conventional method. The proposed methodology appears to be an improved way of estimating peer-group means.

In proficiency testing (PT), it is difficult to set rational criteria for determining target values and allowable limits. In Japan, the largest national PT program has for the past 8years used an evaluation scheme based on a “common coefficient of variation” (cCV) to overcome this difficulty. The cCV, the average within-method variation, is derived by one-way analysis of variance from data of peer groups after excluding those with large variations. A unit interval used for grading participants is obtained by multiplying the cCV by the peer-group mean which is computed using an iterative method featuring repeated removal of a large portion of the tails of the distribution and reinflation of the standard deviation (SD) to compensate for the trimming. The evaluation scheme gives a relatively better grade to those participants in peer groups with small CVs, compared to use of individual peer-group CVs. The scheme has expedited the transition to more reproducible methods, resulting in a notable overall improvement in laboratory performance. The cCV evaluation scheme is uniformly applicable to any analyte measured quantitatively. The iterative method, an integral part of the scheme, effectively precludes bias in the evaluation.

Small dense low-density lipoproteins (LDLs) have been associated with coronary heart disease (CHD) and type 2 diabetes in previous studies. However, the significance of small dense LDLs as a coronary risk factor for subjects with type 2 diabetes remains unclear. We measured mean LDL particle diameter by gradient gel electrophoresis (Quantimetrix Lipoprint™ LDL System) in 44 type 2 diabetes patients, 100 CHD patients, 35 CHD patients with type 2 diabetes and 88 age-matched control subjects. Mean LDL particle sizes were significantly smaller (p<0.05, Mann-Whitney test) in diabetes mellitus-only patients (25.7±0.8nm), CHD-only patients (25.2±1.4nm), and CHD patients with diabetes mellitus (24.9±1.6nm) than in controls (26.2±1.4nm). As for the mean LDL particle size, the prevalence of small dense LDLs (mean diameter ≤25.5nm) was higher (p<0.05, χ 2 test) in all disease groups than in controls. Furthermore, the LDL particle size in CHD patients with diabetes mellitus (24.9nm) was significantly smaller (p=0.049) than in diabetes mellitus-only patients (25.7nm). Multiple logistic regression analysis revealed that small dense LDL was independently associated with the incidence of CHD in all subjects [odds ratio (OR) 4.4, 95% confidence interval (CI) 1.2–16.1, and OR 4.5, 95% CI 1.2–17.3, p<0.05]. In this study the LDL particle size did not further decrease in CHD patients even when they had diabetes. However, the presence of CHD affects the LDL particle size in diabetes patients. These results suggest that identifying LDL size as a routine laboratory test could be used as a marker for CHD risk in both diabetic and non-diabetic Korean populations.

Regardless of its origin, any condition associated with inflammation is accompanied by an increase in serum C-reactive protein levels. This study compared the results of a rapid test for the bedside assay of C-reactive protein (QuikRead CRP, Orion Corporation, Orion Diagnostica, Espoo, Finland) with those of a standard laboratory assay in samples taken from 231 children aged less than 14years (126 males; median age 4.7years) attending the Emergency Department of Milan University's Institute of Pediatrics because of acute respiratory infection. The two methods showed similar median C-reactive protein levels (standard laboratory assay: 34.7mg/L, range 4–199mg/L; QuikRead CRP: 33.3mg/L, range <8–196mg/L; p=0.779) and a similar distribution of children with C-reactive protein levels of <20mg/L, 20–70mg/L and >70mg/L. This study shows for the first time that the rapid QuikRead CRP test can be performed at the bedside or in an outpatient clinic and, in less than 5min, gives the same quantitative results as those obtained using a more complex routine laboratory method.

The reference change value, defined as RCV=1.96×2 1/2 ×(s I 2 +s A 2 ) 1/2 , where s I is within-subject biological variation and s A is analytical variation, has been used for many years to take clinical decisions in patient monitoring. Furthermore, the index of individuality was defined as II=(s I 2 +s A 2 ) 1/2 /s G , where s G is the between-subject biological variation. This index has been simplified by later authors to s I /s G and has been used in monitoring situations to determine the utility of population-based reference intervals. Harris stated that when the index of individuality is lower than 0.6, the specific reference interval of the individual – when available – is better than the population-based reference interval. However, if a change within a patient is equivalent to the RCV applied for the significant difference between two measurements, the probability of detecting this change is only 50% (the same probability of missing it). Therefore, to obtain a higher probability of detecting a change by the RCV (e.g., 90%) the interpretation of the index of individuality has to be reconsidered. This contribution compares the power of the RCV to the use of cut-off points and population-based reference intervals. The benefits of the RCV compared to the distance to cut-off point or reference limit are also described in relation to the index of individuality.

Together with serum ferritin, erythrocyte ferritincan be a valuable diagnostic tool for evaluating the degree of impaired iron metabolism in different diseases. We collected peripheral blood samples from 64 subjects (22 healthy volunteers, 20 patients with hereditary hemochromatosis, and 22 patients on regular hemodialysis with secondary anemia) to evaluate whether an immunoenzymatic method generally used for serum ferritin can also be used to determine erythrocyte ferritin levels under various conditions of body iron status. Serum and erythrocyte ferritin levels were assayed in parallel using a microparticle enzyme immunoassay (MEIA) IMx-Ferritin kit and an IMx analyzer. The inter-assay imprecision of the serum and erythrocyte ferritin assays was 4.9% and 5.05%, the intra-assay imprecision was 2.2% and 2.3%, and the mean recovery was 102% (range 96–105%) and 101% (range 99–105%), respectively. Both serum and erythrocyte ferritin assays showed a detection limit of 1μg/L and good linearity (R 2 =0.99) in the intervals 13.9–443 and 3.9–135.6μg/L, respectively. Our findings demonstrate that the IMx-Ferritin assay currently used to measure serum ferritin levels can also be adopted to measure erythrocyte ferritin insofar as it clearly discriminates high and low erythrocyte ferritin levels in cases of both iron overload and deficiency.

Vitamin C interferes with assays involving the redox-reaction. However, the interference of Vitamin C with electrolytes has not been reported. In the present case, we describe a 61-year-old lady with severe electrolyte abnormalities after administration of high doses of vitamin C. This patient, who had terminal colon cancer, presented to hospital with anuria. Her electrolytes were extremely abnormal (determined on the Beckman Synchron LX20): serum sodium 200mmol/L, potassium 7.0mmol/L, and chloride 50mmol/L. Repeated measurements showed similar abnormalities. However, these critical abnormalities did not fit her clinical picture, as she was alert with normal vital signs. One of the specimens was also run on both the ABL700 and the Bayer644 analyzers, and the electrolytes appeared normal. Pooled serum from healthy individuals to which various amounts of vitamin C was added then was analyzed on Beckman Synchron LX20 for electrolytes, demonstrating the interference of vitamin C consistent with the initial finding. Thus, we eventually figured out that the aberrant results were due to the vitamin C caused analytical interference.