Volume 40, Issue 7

Hyperammonemia is mainly found in hepatic encephalopathy and in genetic defects of the urea cycle or other pathways of the intermediary metabolism. Clinically a difference has to be made between chronic moderate hyperammonemia and acutely increased concentrations. Pathogenetic mechanisms of ammonia toxicity to the brain are partly unraveled. In some animal models confounding variables, such as the reduced intake of food and amino acid imbalance due to liver insufficiency, do not allow to establish unequivocal causal relationships between the ammonia concentration and measured effects. In chronic moderate hyperammonemia an increased flux through the serotonin pathway is a key factor. It is caused by an increased transport of large neutral amino acids (including tryptophan) through the blood-brain barrier, accentuated by the imbalance of plasma amino acids in hepatic insufficiency. It is stimulated by D- or L-glutamine. Evidence is presented showing that a functioning γ-glutamyl cycle (glutathione formation) is a prerequisite. In acute hyperammonemia involvement of NMDA receptors, glutamate, NO and cGMP plays an additional role. In hyperammonemic crises the increased cerebral blood flow leads to brain edema; factors discussed here are increased osmolytes in astrocytes and serotoninergic activity. Recent data indicate that axonal development is affected by ammonia and can be normalized in vitro by creatine supplementation in developing mixed brain cell aggregate cultures, thus reviving the old hypothesis of the impact of hyperammonemia on energy metabolism in the developing brain that could cause mental retardation.

Neopterin is elevated in infections, autoimmune diseases and post-transplant. Recently neopterin elevation was linked to stress response and malignancy. To determine early changes of serum neopterin caused by surgical stress and to investigate their association with other inflammatory markers and with malignancy, we measured neopterin, C-reactive protein (CRP) and procalcitonin (PCT) levels at four predefined time-points within 24 hours in 27 patients admitted for liver resection. Our results show that neopterin increased during operation and the increase was not related to preoperative neopterin levels. On the first day after surgery neopterin level was not significantly different from postoperative levels. In patients with malignant disease neopterin concentration before operation was higher than in patients with non-malignant disease, however, the increase in neopterin concentration during operation was not different between both patient groups. During surgery CRP and PCT did not increase significantly. On the first postoperative day CRP and PCT were elevated and their levels correlated with neopterin (Pearson's correlation coefficient r=0.51 and r=0.76, respectively). We conclude that neopterin elevation during liver resection contributes major part to the increased levels observed on the first postoperative day. Perioperative neopterin release can/may be related to stress response and hypoxia produced during operation. Using this marker, hypoxic reperfusion damage could be detected earlier and more accurately.

Fetal nucleated red blood cells (NRBC) have been widely reported in maternal blood during pregnancy. However, there is no consensus with regard to their presence in all pregnancies. Therefore, the usefulness of developing a fetal NRBC-based noninvasive method suitable for clinical prenatal diagnosis remains uncertain. Fluorescence in situ hybridization (FISH) method was used to evaluate the ability of one of our own monoclonal antibodies (mAb), 2B7.4, to isolate fetal NRBC from maternal blood by magnetic activated cell sorting (MACS). Our mAb was able to isolate from 25 to 822 NRBC from all of the 45 maternal blood samples included in this study. A correct diagnosis was achieved in 21 out of 24 pregnancies carrying trisomic fetuses (87.5%), with a fetal/maternal NRBC frequency of 8.4%. In contrast, a significantly lower percentage of fetal NRBC (0.2%) was observed in 22% of pregnancies carrying a chromosomally normal male fetus, that were correctly predicted. In conclusion, using 2B7.4 mAb we succeeded in isolating NRBC from the maternal blood samples, but most of the isolated cells were maternal in origin. Nevertheless, a higher number of fetal NRBC was found in the peripheral blood of pregnant women carrying aneuploid fetuses, which could allow development of a screening method for prenatal diagnosis of fetal aneuploidies.

The in vivo assessment of free radicals concentration is hampered by their instability and extremely short half-life. The Diacron Reactive Oxygen Metabolites (D-ROM) test is a recently introduced method to evaluate the peroxidation of organic compounds. Since the manual performance of the test provides excessive analytical imprecision, the aim of this study was to evaluate the automation of this test. Within- and between-run imprecision and interference were assessed according to the guidelines proposed by the NCCLS. The reactive oxygen metabolites' (ROM) stability was evaluated in different physical conditions. For withinrun and between-run imprecision the coefficients of variation were consistently lower than 5%. The maximum allowable concentration was 28.2 mmol/l, 0.068 mmol/l and 171 mmol/l for triglycerides, haemoglobin and bilirubin, respectively. Serum storage at −20°C provided adequate ROM stability for up to 3 months, whereas storage at 4°C yielded non-reproducible results. In conclusion, our data provide evidence that the D-ROM assay has both an acceptable stability and an adequate imprecision. The automated assay may be regarded as a fast and reproducible method for the quantitative evaluation of oxidative stress. Since it is easily performed, the method is suitable for routine in clinical laboratories and may provide an accurate estimation of oxidative stress in vivo .

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-linked enzyme which appears to protect low-density lipoproteins (LDL) from oxidation. PON1 activity is associated with variation at the PON1 gene locus, specifically the common amino acid polymorphism at codon 192, for which the Q192 allele specifies low activity and the R192 allele specifies high activity. We investigated the association between the PON1 codon 192 polymorphism and fasting concentrations of glucose, lipids, lipoproteins and PON1 activity in 1380 subjects (724 men and 656 women). Several anthropometric and environmental factors were assessed in the present study. The PON1 Q192 allele frequency was 0.70 and 0.68 in men and women, respectively. In women, but not in men, significant associations were found between the PON1 codon 192 genotype and both total and LDL-cholesterol (p=0.004 and p=0.008, respectively), and subgroup analysis indicated that this relationship was predominant in postmenopausal women. Specifically, the Q192 allele was associated with increased total and LDL-cholesterol concentrations. Furthermore, these lipoprotein variables were higher among postmenopausal women with Q192/Q192 and Q192/R192 genotypes than in premenopausal women with the same genotypes (p<0.001). The findings suggest a gender-specific lipoprotein-genotype association with PON1 codon 192 genotypes in this study sample.

The levels of malondialdehyde and ceruloplasmin, and superoxide dismutase activity were higher, while transferrin concentration and the activities of glutathione peroxidase and catalase were lower in serum of patients with systemic lupus erythematosus (n=24) compared with healthy controls (n=20). Disease activity index correlated positively with serum malondialdehyde level (r=0.47, p<0.05), erythrocyte sedimentation rate (r=0.41, p<0.05) and C-reactive protein concentration (r=0.41, p<0.05), while it correlated negatively with serum superoxide dismutase (r=0.42, p<0.05) and glutathione peroxidase (r= −0.44, p<0.05) activities in patients. No such correlations were found in healthy control subjects. It remains to be seen whether correlations found between disease activity score and serum malondialdehyde level, and also activities of serum superoxide dismutase and glutathione peroxidase enzymes observed in the present study may be used to predict prognosis in patients with systemic lupus erythematosus.

Hereditary hemochromatosis (HHC) is an autosomal recessive disorder that damages various organs because of the deposition of excess iron. At the human hemochromatosis ( HFE ) gene, two mutations of C282Y and H63D have been reported. The frequencies of C282Y and H63D mutations vary among ethnic groups. At present, the most suitable screening test for HHC is the assessment of transferrin saturation (TS). We investigated the distribution of TS and the frequencies of C282Y and H63D mutations among Koreans. TS was measured in 2152 subjects who visited the health promotion center for a checkup. The mean (±SD) of TS was 41.7±15.4%. We randomly selected 240 subjects and tested them for C282Y and H63D mutations using PCR-restriction fragment length polymorphism (RFLP). All 240 randomly selected samples were found to be G/G homozygous non-mutated for C282Y. Of the 240 subjects, 18 (7.5%) were found to be C/G heterozygous and 222 subjects were C/C homozygous non-mutated for H63D. In this study, the C282Y mutation was not found in the Korean population, and the H63D mutation showed allele frequency of 3.8%. The mean TS in this study was higher than that of Caucasians.

The efficacy of the tandem mass spectrometry as a tool for the newborn screening of phenylketonuria, an inherited metabolic disorder, was investigated. Precision, reproducibility, selectivity and sensitivity were validated for phenylalanine and tyrosine measurements from dried blood spots. Bland-Altman plots were used to assess the agreement with conventional methods like fluorometry and ion exchange chromatography. The utility of the phenylalanine/tyrosine ratio for discrimination between mild hyperphenylalaninemia and classical types of phenylketonuria was investigated. Depending on concentration levels of phenylalanine and tyrosine the within-run and between-run assay variability ranged between 4.2% and 12.7%. Higher recoveries and a lower detection limit were found for the mass spectrometric method when compared to the fluorometric method. Pearson correlation coefficients of 0.91 for tandem mass spectrometry vs. fluorometric method, as well as 0.95 for tandem mass spectrometry vs. ion exchange chromatography were calculated. The closest agreement between methods was observed between tandem mass spectrometry and ion exchange chromatography. The results demonstrate a high efficacy of the tandem mass spectrometric method for quantitative determination of phenylalanine and tyrosine from dried blood spots. The phenylalanine/tyrosine ratio is crucial to improve the specificity and positive predictive value for the diagnosis of classical phenylketonuria.

Performance characteristics of the Abbott LCx ® HIV RNA Quantitative Assay (LCx HIV) were established in a multicenter study comparing it with the manual (Amplicor v1.5) and automated (Cobas) ultra-sensitive Roche Amplicor™ HIV-1 Monitor v1.5, the Bayer Quantiplex™ HIV RNA 3.0 (bDNA v3.0), and the Organon NucliSens ® HIV QT 2.0 (NucliSens). Within-run precision of LCx HIV assessed in clinical specimens was SD log 10 0.210 at ~50 copies/ml, and log 10 0.133 at ~400 copies/ml. Total precision in a reconstituted type B HIV-1 RNA panel was SD log 10 0.380 at 100 copies/ml, and SD log 10 0.180 at 1000 copies/ml. Type B HIV-1 RNA sensitivity (1 ml input) assessed at a 50%, 75% and 95% detection rate ranged from 29 to 41, 54 to 75 and 94 to 176 copies/ml, respectively. Overall specificity in HIV sero-negative individuals was 99.78%. Linear regression indicated close assay correlations and agreements for measurement of type B HIV-1 RNA. Pearson's correlations and (Log 10 LCx=aLog 10 x + b) linear regressions were 0.91 (y=0.892 Log 10 Amplicor + 0.595), 0.93 (y=0.827 Log 10 Cobas + 0.969), 0.93 (y=0.951 Log 10 bDNA + 0.550), and 0.79 (y=0.834 Log 10 nuclisens + 0.911). LCx HIV was least affected by the genetic variability of HIV-1. LCx HIV detected 99% of non-type B HIV-1 group M samples (subtypes A-G), Amplicor v1.5 detected 96%, and bDNA v3.0 detected 99%. The assays detected 10/11, 1/11 and 8/11, respectively of the HIV-1 group O samples. LCx HIV vs. Amplicor/bDNA Spearman's rank correlations for quantification of non-type B HIV-1 RNA were 0.76/0.84 (A), 0.93/0.93 (C), 0.73/0.99 (D), 0.86/0.98 (E), and 0.40/0.83 (group O). LCx HIV assays consistently detect and quantify type B, non-type B and group O HIV-1 RNA.

We investigated the interference of insulin antibodies in two insulin immunometric assays (Bio-Rad and Elecsys) by measuring direct and free insulin in plasma from 30 patients without insulin antibodies (group 1), as screened by a sensitive radio-binding assay, and in plasma from 80 patients with insulin antibodies (group 2). In group 1, the direct/free insulin ratio did not differ from 1, showing the equivalence of free and direct insulin results in theses samples. In group 2, this ratio was markedly increased (mean: Bio-Rad 2.63, Elecsys 5.02) and correlated positively with the insulin antibody radio-binding assay result (r=0.92 for the correlation between Bio-Rad and Elecsys assays after log-transformation of the ratios). In samples containing insulin antibodies, direct insulin concentration was frequently lower than total (bound and unbound) insulin measured with the Bio-Rad and Elecsys assays. This study underlines the interference of insulin antibodies in insulin immunometric assays and the importance of assessing an insulin immunometric assay for sensitivity towards the presence of these antibodies.

Important performance characteristics for any thyroid-stimulating hormone (TSH) assay include low-end sensitivity, precision across a wide dynamic range, and linear specimen dilution. Laboratories often characterize the performance of their TSH assay using many different sample matrices ( e.g. native human serum, synthetic buffer, processed human serum, etc .). However, this can lead to possible confusion as the relationships between sample matrix and assay performance are often poorly understood. The purpose of our study was to evaluate the performance of the ARCHITECT i 2000 TSH assay using a variety of different sample matrices. Functional sensitivity was found to be essentially equivalent in both hyperthyroid patient sera (0.0035 μIU/ml) and TSH affinity-stripped (0.0038 μIU/ml) sample matrices. Assay imprecision was also independent of sample matrix, with total imprecision ≤5.3% for both synthetic buffer and serum matrices. Similarly, specimens were found to dilute linearly over a wide range in both serum and synthetic buffer matrices. We conclude that the i 2000 TSH assay measures TSH similarly in a variety of sample matrices. This is an important design feature for this immunoassay which provides assurance that analytical performance assessed with matrices other than unprocessed human serum are representative of assay performance seen with patient specimens.

The erythrocyte sedimentation rate (ESR), now more appropriately referred to as the “length of sedimentation reaction in blood (LSRB)”, remains the most widely used laboratory test for monitoring the course of infections, inflammatory diseases and some types of cancer. Thanks to the several recently developed methods for the measurement of this reaction, the safety and reliability of LSRB testing procedures have improved. The method for LSRB measurement recommended by the International Council for Standardization in Hematology (ICSH) and the National Committee for Clinical Laboratory Standards (NCCLS) is based on the traditional Westergren method, using EDTA-anticoagulated samples. The present paper describes and evaluates a procedure for LSRB measurement with a new manual system, the Microtest 1, which requires only 30 μl of blood and is optimal for pediatric use. Microtest 1 results correlated satisfactorily with the ICSH recommended method (r=0.88, 95% CI 0.84–0.91; y=3.45+0.85x), had no significant bias (2.15, 95% CI −0.32–4.63) and had an imprecision of less than 7% for the reference range values. In addition, the results obtained with the Microtest 1 in “native” whole blood were comparable with those obtained with the reference method in K 3 EDTA and sodium citrate-anticoagulated specimens; (bias = 1.19, 95% CI −1.78 to 4.16 for K 3 EDTA-anticoagulated samples and bias = 2.43, 95% CI –0.58 to 5.44 for sodium citrate-anticoagulated samples).

This paper is the fourth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of γ-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of γ-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37°C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30°C IFCC reference method (1). Differences are tabulated and commented on in Appendix 2.

This paper is the fifth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of γ-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of γ-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37°C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30°C IFCC reference method (1). Differences are tabulated and commented on in Appendix 3.

This paper is the sixth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of γ-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37°C A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30°C IFCC reference method (1). Differences are tabulated and commented on in Appendix 1.

This paper is the seventh in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of γ-Glutamyltransferase. A document describing the determination of preliminary reference values is also in preparation. The certification of the catalytic activity concentrations as determined by the recently elaborated IFCC primary reference methods at 37°C of four enzyme preparations, namely IRMM/IFCC 452 γ-glutamyltransferase), IRMM/IFCC 453 (lactate dehydrogenase 1), IRMM/IFCC 454 (alanine aminotransferase) and IRMM/IFCC 455 (creatine kinase) is described. Homogeneity data were derived from previous results. Stability was assessed using recently obtained data as well as data from previous stability studies. The collaborative study for value assignment was performed under a strict quality control scheme to ensure traceability to the primary reference method. Uncertainty of the materials was assessed in compliance with the Guide to the Expression of Uncertainty in Measurement. The certified values obtained at 37°C are 1.90 μkat/l ± 0.04 μkat/l (114.1 U/l ± 2.4 U/l), for γ-glutamyltransferase, 8.37 μkat/l ± 0.12 μkat/l (502 U/l ± 7 U/l), for lactate dehydrogenase 1, 3.09 μkat/l ± 0.07 μkat/l (186 U/l ± 4 U/l), for alanine aminotransferase and 1.68 μkat/l ± 0.07 μkat/l (101 U/l ± 4 U/l), for creatine kinase. The materials are intended for internal quality control as well as for the evaluation of test systems as required by recent European Union legislation. Furthermore, the materials can be used to transfer accuracy from a reference method to a routine procedure provided the procedures exhibit the same analytical specificity and the certified materials are commutable.