Epigenetics and assisted reproductive technology.

During gametogenesis, the female and male germ cells undergo a process whereby imprinting marks are erased from the genome. During the later stages of germ-cell development, the methylation marks of the female and male germ lines are re-established. A second phase of demethylation of the genome occurs at the time of fertilization, and during development of the early embryo. Assisted reproductive technology involves several steps that subject the gametes and early developing embryos to environmental stress, and this is the primary reason for an increased interest in the putative link between these techniques and imprinting disorders. Although animal studies support a link between assisted reproductive techniques (ARTs) and imprinting disorders, via altered methylation patterns, data in humans are inconsistent. Here we provide an overview of the field of epigenetics in relation to ARTs.

Evaluation of the DPC IMMULITE random access immunoassay analyser.

The automated immunoassay analyser, IMMULITE, developed by DPC, was evaluated. IMMULITE is an automated system that allows random access in combination with immediate and continuous access. In this study we evaluated the IMMULITE on four panels of analytes: thyroid, fertility, tumour and "non-routine" markers. We observed good within-rum reproducibility (ranging from 2.3-15.9% CV, for low controls, to 2.7-8.7% CV for high controls) as well as between-day imprecision (ranging from 3.7-24.6% CV for low controls, to 2.5-11.8% cv for high controls). The analytical sensitivity of the assays ranged generally from very sensitive to acceptable. The dilution curves for all assays were nearly linear i.e. the maximum deviation of the observed from the expected recovery was 8%. Correlation between IMMULITE and other assays (AxSYM, IMx, TDx, AIA-1200, DPC-c. a. c., Medgenix) varied from r = 0.931 to r = 0.994, except for lutropin and parathyrin with coefficients of correlation of r = 0.594 and r = 0.591. The slopes of the regression lines ranged from 0.745 to 1.327, except for parathyrin where a slope of 2.389 was found. Inter-laboratory correlation was very good between the two locations (Sittard and Apeldoorn) and varied from r = 0.984 to r = 0.999; slopes of the regression lines varied from 0.924 to 1.086. We conclude that the DPC-IMMULITE system is suitable for testing routine analytes in random access mode as well as for testing "non-routine" analytes on an automated immunoassay analyser.

Evaluation of performance characteristics of automated measurement systems for allergy testing.

Reliability of test results, convenient handling and flexibility are major requirements on automated immunoassays systems. To investigate to what extent these requirements were met by the Pharmacia CAP and DPC IMMULITE and DPC Microplate Systems, we evaluated several performance characteristics of assays of specific IgE against some common inhalant allergens as well as the atopy tests Phadiatop (Pharmacia CAP System) and AlaTOP (DPC IMMULITE and Microplate System). Comparing Phadiatop and AlaTOP results (n = 95) to clinical data, the sensitivity was found to be 97% in the Pharmacia CAP System and 82% in the AlaTOP-DPC Microplate System and 88% with AlaTOP-IMMULITE. Specificity was in all cases higher than 90%. The pooled total variation was more than twice as high with the DPC Microplate System as compared to the Pharmacia CAP System in our first investigation. A second investigation showed similar values. The investigation of systematic differences showed that the error contribution of sample related differences between the systems was even larger and far exceeded the analytical variation. Thus the two methods do not seem to be measuring the same specific IgE antibodies. In 8 out of 8 cases with the Pharmacia CAP System positive and DPC negative results and in 2 out of 2 cases with DPC positive and Pharmacia CAP System negative results, the presence of IgE antibodies could be confirmed by IgE immunoblotting. Serum dilutions showed very irregular O/E patterns for the DPC Microplate System. There was no effect when adding non-specific IgE to serum samples. Addition of competing IgG antibodies showed a moderate decrease in binding of specific IgE in the Pharmacia CAP System when increasing amounts of IgG were added. The effect in the DPC Microplate System was more pronounced with large decreases, or increases of measured values even at lower concentration of the competing antibody. The results may indicate insufficient allergen concentration in the DPC assay and draw attention to the risk for undesirable complex formation between allergen and antibody in solution. The combination of the two DPC systems did not offer any advantages over Pharmacia CAP System from the handling or work flow point of view.

Evaluation of the Abbott automated random, immediate and continuous access immunoassay analyser, the AxSYM.

The new Abbott automated immunoassay analyser called AxSYM was evaluated in our laboratories. The AxSYM is a completely automated system that allows random access in combination with immediate and continuous access. Here we describe the evaluation of the AxSYM on three panels of analytes, thyroid, fertility and tumour markers. We observed good within-run reproducibility (ranging from %CV = 3.15 to 11.37 for low assay control to %CV = 1.18 to 6.38 for high controls) as well as between-day and between-laboratory precision (low controls 3.44 to 14.6% CV and high controls 2.56 to 8.9% CV). The analytical sensitivity of the assays ranged from very sensitive to acceptable, but always better than claimed by the manufacturer. The throughput of the AxSYM in our hands was 68 tests per hour. Correlation between the AxSYM assays and other Abbott assays (IMx) was excellent (slopes ranging from 0.965 to 1.071, correlation, r = 0.964 to 0.999). Compared to the Tosoh AIA 1200, good correlation was observed (slopes ranging from 0.964 to 1.111, correlation, r = 0.740 to 1.000), except for thyrotropin (slope = 0.689, r = 0.998) and carcinoembryonic antigen (slope = 0.737, r = 0.949). We concluded that the AxSYM represents a new generation of random access automated immuno analysers with very good performance in a daily routine use. We also concluded that the AxSYM was very technician friendly compared to the Tosoh AIA 1200.

A European Quality Control Programme as a cooperative tool between users and a diagnostic company.

Achievement of appropriate precision is a major challenge for the clinical laboratory, particularly in specialized fields such as enzymology and immunochemistry. Especially in immunochemistry, where accuracy can only poorly be checked because of a lack of reference methods and a multitude of detectable epitopes, precision remains an important index of quality. In immunochemical methods there are three main sources of variation: differences in methodology, lot-to-lot variation and performance of the assay, the latter including the skill of the technicians and instrument performance. The results of a European Quality Control Programme for the users of one type of automated immunoassay analyser were compared with a similar Italian Quality Control Programme for users of different immunometric techniques. The precisions (%CV) of the two programmes were compared, as well as the number of values lying within one half of the biological variation of the analysed serum components. Feedback between users and manufacturers leads to a clear improvement in analysis performance, so that most of the considered values come to lie within the acceptable limits of 1/2 of the biological variation. It is concluded that both national and international quality control programmes are very useful for indicating the intrinsic quality of the quantities used in the clinical laboratory.

Comparison of the automated random access immunoassay analysers, ACS-180 (Ciba Corning) and AIA-1200 (Tosoh).

Two random access immunoassay analysers, the ACS-180 (Ciba Corning) and the AIA-1200 (Tosoh Corp.) were compared with respect to performance and user-friendliness. Precision studies revealed an almost equal intra-assay variation coefficient, but day-to-day reproducibility was better on the AIA-1200. Recovery of dilution series of tumour markers was too high with the ACS-180. Both systems were free from carry-over effects, on account of the extra wash step in the ACS-180 (for thyrotropin, carcinoembryonic antigen and human chorionic gonadotropin) and the separate pipetting tip in the AIA-1200. Reagent stability is better in the AIA-1200 than in the ACS-180. The AIA-1200 is more user-friendly and produces a higher throughput than the ACS-180 in daily routine operation (92 results/hour vs. 78 results/hour).

Clinical relevance of lysozyme in the faeces.

To elucidate the value of faecal lysozyme determination in the differential diagnosis of patients with atypical abdominal complaints, stool samples of healthy controls, patients with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) were analysed. Faecal lysozyme concentration in healthy controls ranged from 0 to 6 mg/l with a mean of 3 mg/l. Patients with IBS had similar faecal lysozyme levels. In contrast, faecal lysozyme concentrations in patients with IBD were increased (range 6 to 104 mg/l). The difference between patients with IBS and IBD was highly significant (P less than 0.001). The determination of faecal lysozyme concentration may provide a useful test in the work-up of patients with abdominal complaints. In addition, the faeces lysozyme concentration appeared to be an objective parameter of the inflammatory activity of IBD in 11 patients investigated.

Greater than expected alanine aminotransferase activities in plasma and in hearts of patients with acute myocardial infarction.

Early increases in the activity of alanine aminotransferase (ALT, EC 2.6.1.2) in plasma are observed in about 7% of patients with acute myocardial infarction (AMI), of whom about half die. Some type of liver injury, secondary to AMI, could be responsible for this phenomenon. However, quantitative analysis shows that the release of ALT in most of these patients conforms to the myocardial release pattern. Moreover, extra release of hepatic aspartate aminotransferase (EC 2.6.1.1) is not observed. These findings suggest that the heart may occasionally contain a high ALT activity. This hypothesis was verified by determination of enzyme activities in 10 hearts obtained from patients who died after AMI. The mean ALT activity in these hearts, 21 (SD 12) U per gram wet weight, significantly (P less than 0.01) exceeds the value of 7.7 (SD 4.9) U/g found for seven control hearts and may reflect increased amino acid metabolism in the energy-depleted heart muscle, as described earlier for skeletal muscle.

Analytical performance of three commercially available nephelometers compared for quantifying proteins in serum and cerebrospinal fluid.

We investigated the analytical performance and imprecision of three commercially available nephelometers for the quantification of various proteins in pooled serum and cerebrospinal fluid. Albumin, transferrin, IgG, IgA, and IgM in serum were determined nephelometrically (BNA, Behring; Array, Beckman) and turbidimetrically (Cobas Bio, Hoffmann-La Roche). All these proteins in cerebrospinal fluid except transferrin were determined nephelometrically with all three systems. CVs and lower limits of detection were compared for all instruments, and stability of calibration curves was evaluated.

Faecal lysozyme: determination, reference intervals and some data in gastro-intestinal disease.

After diluting faecal samples with a solution of Brij and saline and subsequently ultrafiltrating the faecal mixtures, lysozyme concentration can be reproducibly measured in the obtained faecal fluids, using a turbidimetric method. Measuring faecal lysozyme concentration enables discrimination normal individuals and patients with irritable bowel syndrome between patients with inflammatory bowel disease and colonic cancer. Lysozyme distribution in stools appears to be homogeneous. Faecal lysozyme concentration is stable when samples are stored during at least 1 wk at 6 degrees C. It appears that the lysozyme activity is directly correlated with the clinical status and severity of the disease. Faecal lysozyme may thus serve as an important tool both in diagnosis and in follow-up in the out-patients clinic for gastroenterology.

Rapid determination of five coagulation parameters in one sample with a centrifugal analyzer.

Spectrophotometric clotting assays using chromogenic substrates were adapted for use in a routine laboratory batch analyzer. Contrary to another described method the same plasma dilution can be used for each of the different coagulation parameters. Optimal assay conditions were determined. The within- and between-assay variation, the intraindividual variation during 1 day and from month-to-month were determined; the chromogenic assay was compared to a clotting assay. No significant difference (p less than 0.01) between manual and automated methods was found.

A new method for chemical analysis of faeces.

By using a centriflo membrane cone filter it has become possible to obtain an ultrafiltrate from a 24-h stool specimen. In this faecal fluid several clinical chemical parameters were analysed, such as pH, osmolality, creatinine, sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate and lactate. Reference intervals for these substances were obtained in healthy individuals. The data of this control group were compared to those of patients with diarrhoea due to active inflammatory bowel disease, irritable bowel syndrome, lactose intolerance and persons with an ileostomy.

Short-term and long-term intra-individual variations and critical differences of coagulation parameters.

The intra-individual variations of 9 coagulation parameters were studied during short-term (within-one-day) and long-term (six months) periods. Two groups of healthy individuals (viz. 60 and 274 persons) were involved. Moreover, critical differences have been calculated from the total variations, comprising both biological and analytical variations. The influences of external factors such as sex, smoking and the use of oral contraceptives have also been studied. The variability and critical differences found in coagulation parameters in this study appeared to be of the same order as those observed in parameters usually determined in clinical chemistry and haematology. The application of critical differences in the evaluation and use of laboratory data, also in coagulation studies, enhances the objectivity of the judgement. Moreover, in the follow-up of patients the sensitivity of the parameters is increased.

PIP: The purpose of this study was to assess intra-individual variation in 9 coagulation parameters and calculate critical differences in a population of healthy volunteers in both the shortterm (1 day) and longterm (6 months). Intra-individual variations ranged from 0-6.6% and from 3.9-16.4% for the 3 screening tests and 6 specific coagulation tests investigated; critical differences varied from 9.9-19.5% and 14.8-46.7%, respectively. Significant differences for the intra-individual variations of plasminogen were found between male and female volunteers. Smoking and oral contraceptive (OC) use, however, did not cause significant differences in the variation in the shortterm. In the long-term investigation, only the plasminogen levels between female users versus nonusers of OCs were significantly different. The variability of the measured parameters was strikingly low for thrombin time and factor V. Overall, the dk90 values for the coagulation parameters analyzed fell in the same range as other clinical and hematologic laboratory parameters in terms of their clinical interpretation, suggesting that determination of these parameters is reliable for clinical use.

Short-term and long-term intra-individual variations and critical differences of haematological laboratory parameters.

The complete blood cell count and the differential leukocyte count have been studied for their variations within-one-day and during one week (short-term variations) and during a period of six months (long-term variations) with an automatic haematology analyser (H-6000, Technicon), in which some new parameters (large unstained cells, high peroxidase cells, red cell distribution width, platelet distribution width, mean platelet volume, plateletcrit) are included. The influence of external factors such as sex-differences, smoking/non-smoking, use of oral contraceptives etc. have also been studied. It appears that the use of critical differences together with haematological data improves the value of these parameters in diagnosis and treatment of patients.

Short-term and long-term intra-individual variations and critical differences of clinical chemical laboratory parameters.

Twenty eight clinical chemical blood parameters were studied with respect to intra-individual variations. Both short term variations, within one day and during one week, and long term variations, during a six months period, were investigated in groups of 62, 16 and 274 individuals. Moreover, critical differences were calculated from the total variations, i.e. physiological (personal) and analytical variations. The influences of external factors (sex, smoking, and oral contraceptives) were being studied. It was concluded that the use of laboratory data in clinical diagnosis is considerably improved when intra-individual variations and critical differences are used.

Effects of biological and analytical variations on the appropriate use of "reference intervals" in clinical chemistry. Proposal of a scheme for longitudinal assessment of laboratory values.

For twenty one different laboratory parameters the separate contributions of both biological (age, sex, hospitalisation) and analytical variations to the total variation have been examined. For each of the different laboratory parameters the reference interval and critical difference are given for the longitudinal evaluation of analysis results. These critical differences are not only calculated on the basis of the analytical imprecision from day to day, as has been usual until now, but the mean individual deviations of the reference samples have also been included. The critical differences given in this paper are therefore essentially higher for some parameters compared with the critical differences on the basis of the analytical deviation, and are therefore much more realistic quantities to be used for longitudinal evaluation. Finally for practical purposes a scheme is presented for use with longitudinal assessment of laboratory values, in order to enable the practising physician to consider these data in daily decision making.