Volume 42, Issue 6

Background: Quality management systems should cover all the steps involved in the overall testing and non-testing processes. Aim: To identify quality indicators for extra-analytical processes in the clinical laboratory and to specify acceptability limits, in order to provide a useful tool for continuous improvement of laboratory service. Methods: A literature review by Medline search was performed using the keywords: Q-Tracks and Q-probes alone, and management , error , mistake , and indicator crossed with quality , laboratory and medicine . The indicators retrieved were organized according to the various laboratory processes. Their expression was standardized in relation to the total activity of each process reported in each paper reviewed. The magnitude of the errors reported was considered to be the current state of the art for the extra-analytical step and was proposed as the quality specification. Results: Examples of indicators and specifications for the pre-analytical process: • Analytical request: Error in patient identification (0.08%), request unintelligible (0.1%). • Sampling: Requested but not collected (7%), redraws (2%). • Transport and reception of samples: Inadequate transportation conditions (0.005%), hemolyzed sample (0.2%). Examples of indicators and specifications for the post-analytical process: • Analytical request: Error in patient identification (0.08%), request unintelligible (0.1%). • Sampling: Requested but not collected (7%), redraws (2%). • Transport and reception of samples: Inadequate transportation conditions (0.005%), hemolyzed sample (0.2%). • Report validation: Test not performed (1.4%), test performed but not requested (1.1%). • Intra-laboratory reports: Laboratory reporting errors (0.05%), delivery outside specified time (11%). • Consulting service: average time to communicate critical values for inpatients (6 min). Conclusions: These extra-analytical indicators and their specifications, expressed in a standardized manner, constitute a preliminary basis for comparison of individual laboratory performance with the purpose of improving laboratory quality.

Congenital disorders of glycosylation include a group of diseases, each of them caused by different protein (mostly enzyme) impairment due to a specific gene defect. The many subtypes are classified according to clinical features, enzymology and molecular genetic analyses. Problems in diagnostics arise from the great diversity in clinical presentation, usually age-related, and different severities of individual types of these, by far underdiagnosed, diseases. Also the biochemical findings tend to vary, even within a single type. No one screening test, common for all types, is available so far. Several methods of choice may be used in the first approach; other procedures must follow for detailed typing of the defect. Possible drawbacks and pitfalls in the diagnostics from the viewpoint of our 3-year studies and practical screening experience are presented.

Autoinflammatory phenomena, including autoantibody production and atopy, have been regarded as associated with endometriosis. The present study investigates the activity of eosinophils and the distribution of immunoglobulin E concentrations in the peritoneal fluid of women with early endometriosis. The study group consisted of 30 patients with laparoscopically diagnosed early endometriosis. The healthy control group consisted of 18 females with no evident changes in the abdominal cavity and no endometrial foci. Concentrations of immunoglobulin E in serum and peritoneal blood were determined by enzyme immunoassay. The activity of eosinophils was estimated according to the expression of the early activation molecule CD69 by the flow cytometry method. The concentrations of immunoglobulin E in the peripheral blood and peritoneal fluid were similar in both groups. However, the count of CD69+ eosinophils was higher in the peritoneal fluid of women with endometriosis. The results indicate that activated eosinophils accumulate in the peritoneal fluid in early endometriosis and can play a significant role in the pathogenesis of the disease.

Aberrations in the genomes of gliomas seem to correlate well with clinical parameters. Pertinent studies, however, rely on highly sophisticated methods, they require large amounts of high-quality sample material and/or they demand profound analytical expertise. Consequently, molecular tumour analysis has not yet been widely implemented in routine laboratory applications. We have developed an easy-to-perform approach for the rapid derivation of a detailed lossof-heterozygosity profile from individual gliomas. DNA of PCR quality is extracted in a one-step procedure from routinely obtained material. A microsatellite-based marker set is used to detect the deletion status of genomic regions (i) with established diagnostic relevance, (ii) recurrently found deleted in gliomas, or (iii) generally associated with tumour suppressor activity. The complete profile comprises 25 regions and is generated from 64 markers multiplexed into 18 reactions. Illustratively, we present findings from an anaplastic oligodendroglioma; the molecular data for this tumour allow refined diagnostic and prognostic statements that could not be derived from histology alone. Our approach should prove useful in the routine diagnosis of gliomas. Simultaneously, research data for many highly relevant regions are generated in a comparatively simple and inexpensive way.

Both secretory and cytosolic phospholipase A 2 enzymes have been implicated in the pathogenesis of arthritis in animal models, but the exact expression patterns of the enzymes in diseased human joint tissue are uncertain. We investigated the messenger RNA expression of group IIa, IVα and V phospholipase A 2 and localized the presence of group IIa and IVα phospholipase A 2 at protein levels in articular cartilage from patients with rheumatoid arthritis, osteoarthritis and patients with non-arthritic joints. Both group IIa phospholipase A 2 messenger RNA and protein were detected in all samples independent of diagnosis, but were far more prominent in cartilage from rheumatoid arthritis samples. In cartilage with rheumatoid arthritis, the enzyme was detected both within the chondrocytes and in the extracellular matrix, whereas only few osteoarthritic cartilage samples showed positive staining in the matrix. In the cartilage matrix of non-arthritic controls, group IIa phospholipase A 2 was totally absent. Messenger RNA for the group IVα and V phospholipase A 2 was, except for one osteoarthritic cartilage sample, exclusively detected in rheumatoid arthritic cartilage. For group IVα phospholipase A 2 this was also confirmed at the protein level. These results suggest that each phospholipase A 2 enzyme has distinct roles in both healthy and diseased joint tissue, and that the diversity and amount of enzyme correlate with the grade of inflammation and disease severity.

Neurokinin B levels were measured between the 10th–20th weeks of pregnancy, i.e., prior to the development of clinical symptoms, in women who developed preeclampsia or delivered a growth-restricted baby. No difference was found in plasma neurokinin B levels, although neurokinin B levels increased slightly towards term.

Cryoglobulins are pathological cold-precipitable immunoglobulins associated with a number of infectious, autoimmune and neoplastic disorders. Patients, when exposed to low temperatures, show symptoms related to intravascular precipitation of such immunoglobulins. The formation of cryoaggregates induced by exposure to cold temperature is the key pathogenetic mechanism. The subsequent intravascular precipitation can account for some clinical signs of peripheral vasculitis, but fails to explain the precipitation of cryoglobulins in regions where no significant temperature changes take place. We studied, in vitro, the activity of different ions on temperature-dependent aggregation of cryoglobulins and found that the concentration of Cl − present in solution is the most important variable that controls the size and the rate of formation of aggregates, both at low temperature and at 37°C. We suggest that chloride anion could be the most important factor involved in the pathogenesis of events in visceral regions, such as in the kidneys, where no temperature changes occur but where the local Cl − concentration changes to maintain blood electrolytic homeostasis and acid-basic equilibrium. Moreover, identification of a specific structural domain responsible for Cl − binding may provide new targets for drugs selectively designed to interfere with cryoglobulin aggregation.

The compliance of 581 drug addicts attending six methadone substitution outpatient clinics was determined over a period of 18 months. Urine from these patients was labeled following oral administration of low molecular weight polyethylene glycols as marker substances. These substances were measured in approx. 5800 urine samples. A protocol for applying marker substances and ways to prevent substitution of urine samples were evaluated. Normal values for marker substances in urine were determined. The results suggest that this labeling procedure is a new diagnostictool to prevent manipulation of urine samples by drug addicts receiving substitution therapy.

The derangement of neuro-endocrine control of circulation influences both disease evolution and response to treatment in patients with heart failure, but little data are available about the complex relationships between the degree of neuro-hormonal activation and clinical severity. We studied the relationships between cardiac natriuretic hormones (CNHs) and several neuro-hormones and immunological markers in a prospective cohort of 105 consecutive patients with cardiomyopathy (77 men and 28 women, mean age 66.7±12.4 years, range 33–89 years). We assayed the circulating levels of CNHs (atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)), plasma renin activity (PRA), aldosterone, cortisol, adrenaline, noradrenaline, thyroid hormones and thyroid stimulating hormone (TSH), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The concentrations of all CNHs and neuro-hormones were higher in patients with heart failure compared to normal subjects, except for free triiodothyronine (FT3), which was below normal values. ANP was positively related to NYHA class, IL-6, adrenaline, noradrenaline and cortisol, while negatively with ejection fraction and FT3. BNP was positively related to age, NYHA class, IL-6, TNF-α, adrenaline, noradrenaline and cortisol, while negatively with ejection fraction and FT3. A stepwise multiple linear regression indicated that plasma ANP depended only on ejection fraction, adrenaline and noradrenaline values, while for plasma BNP variation NYHA class contributed too. Our data confirm a progressive activation of hormonal and immunological systems in patients with heart failure. Furthermore, CNH circulating levels in heart failure are affected not only by cardiac function and disease severity, but also by activation of neuro-hormonal and stress-related cytokine systems, as well as by the thyroid hormones, even on usual medical treatment.

Early diagnosis of cobalamin deficiency is crucial, owing to the latent nature of this disorder and the resulting possible irreversible neurological damage. A normal serum cobalamin concentration does not reliably rule out a functional cobalamin deficiency and there does not at present seem to be any single diagnostic approach to achieve this diagnosis. A new marker for cobalamin status is the serum concentration of cobalamin bound to transcobalamin II (holoTC). Because methods suitable for routine use have been unavailable until recently, the clinical value of lowholoTC is still uncertain. Furthermore, there is at the moment no gold standard or true reference method to diagnose subtle cobalamin deficiency, which makes evaluation of the clinical usefulness of holoTC and the estimation of sensitivity and specificity problematic. In this study, we aimed to assess whether low holoTC concentrations are congruent with other biochemical signs of cobalamin deficiency in a group of psychogeriatric patients. The findings in the present study showthat holoTC is strongly related to serum cobalamin (0.68; p<0.001 in both patients and controls). Distribution of the different markers for cobalamin/folate status in the 33 patients with low levels of serum holoTC (below40 pmol/l) showed that 17 patients had normal levels of the other markers for cobalamin status. This may indicate poor specificity of lowholoTC for cobalamin deficiency. In 23 out of 176 patients with normal levels of holoTC we observed pathological levels of other markers for cobalamin deficiency. The use of holoTC in the present study group did not give significant additional information other than that given by serum cobalamin and therefore cannot be recommended in this clinical setting.

Owing to considerable physical, endocrinological and metabolic adaptations, the analysis of biochemical data in elite and top-class athletes requires caution. With the aim to identify metabolic and biochemical adaptations to particular lifestyle conditions, such as regular and strenuous physical exercise, we measured the concentration of serum albumin, creatinine, uric acid and glucose in 80 male professional cyclists, 37 male members of the Italian national cross-country ski team and 60 male healthy sedentary controls at rest. At variance with earlier investigations, endurance athletes showed significantly decreased concentrations of serum creatinine (controls: 83.1±11.0 μmol/l; skiers: 78.0±8.4 μmol/l; p<0.05; cyclists: 73.8±10.4 μmol/l; p<0.01), uric acid (controls: 362±69 μmol/l; skiers: 331±70 μmol/l; p<0.05; cyclists: 312±61 μmol/l; p<0.01) and glucose (controls: 5.35±0.54 mmol/l; skiers: 4.94±0.41 mmol/l; p<0.01; cyclists: 4.94±0.42 mmol/l; p<0.01). The concentration of serum albumin was also decreased in athletes, but the difference did not reach statistical significance (controls: 4.76±0.26 g/l; skiers: 4.71±0.22 g/l; p=0.384; cyclists: 4.68±0.22 g/l; p=0.393). Results of the present investigation demonstrate that values of laboratorytesting lying outside conventional reference limits calculated on sedentary populations might express physiological adaptations to regular and demanding physical aerobic activity, emphasizing the need for the estimation of reliable reference limits in elite and professional athletes, to avoid equivocal interpretation of results within clinical and anti-doping contests.

Background: S-adenosylmethionine is a methyl donor in many cellular reactions including detoxification of constantly produced hydrogen sulphide in the colon. A reduced capacity to detoxify hydrogen sulphide may be implicated in the pathogenesis of inflammatory bowel disease. S-adenosylmethionine could be low if this assumption is correct. We compared S-adenosylmethionine concentrations in whole blood in patients with severe and moderate inflammatory bowel disease with healthy reference persons. Methods: S-adenosylmethionine concentrations in whole blood were measured using high-pressure liquid chromatography. Patients with Crohn's disease (n=21), ulcerative colitis (n=7) and healthy age-matched reference persons (or controls) (n=17) were studied. Results: S-adenosylmethionine concentrations were significantly decreased in patients with severe inflammatory bowel disease (mean 1.10 mg/l) as compared to patients with moderate Crohn's disease and ulcerative colitis (mean 1.83 mg/l) and reference persons (mean 1.84 mg/l). Statistically significant inverse correlations were found between S-adenosylmethionine concentration and activity index (p<0.01 and R 2 =0.86) as well as Crohn's disease activity index (p<0.01 and R 2 =0.50) scores. Conclusions: Low concentrations of S-adenosylmethionine were found in patients with severe inflammatory bowel disease. Future studies will show whether S-adenosylmethionine is a marker for disease activity and a possible tool for investigation of sulphur toxicity as a causative mechanism in inflammatory bowel disease.

Assays for insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) have become essential tools in the diagnostic work-up of disorders of the somatotropic axis in children and adults. The aim of this study was to evaluate the automated IMMULITE ® IGF-I and IGFBP-3 assays and to establish reference limits–central 95% intervals, median, 0.1 and other centiles as clinically relevant–as a function of age from 797 females and 787 males, from the first week of life through the ninth decade. Pubertal children were classified by sex and by sexual maturation (Tanner stage). IGF-I and IGFBP-3 levels were also assayed in 20 pediatric patients each with growth hormone deficiency (GHD) and Turner syndrome (UTS), before and during 12 months of recombinant growth hormone (rhGH) therapy, as well as in 11 adult patients with GHD and seven with acromegaly before therapy. Both the IGF-I and IGFBP-3 assays were accurate, specific and sufficiently sensitive to measure IGF-I and IGFBP-3 in serum with good linearity and recovery. In the IGF-I assay, potential interference from IGFBPs was eliminated by blocking with excess IGF-II. Circulating IGF-I and IGFBP-3 concentrations, and their ratio IGF-I/IGFBP-3, were age-dependent, showing low levels immediately after birth, a typical pubertal peak for girls and boys, and a pronounced decline after puberty, reaching a plateau in early adulthood. In adults IGF-I and IGFBP-3 levels decreased smoothly but steadily with age. Children with GHD and UTS had low circulating IGF-I and IGFBP-3 levels which increased to normal reference limits under therapy with rhGH. Adult GHD patients showed IGF-I levels below the age-related median; untreated acromegalic patients mostly had IGF-I and IGFBP-3 levels above the age-related 97.5th centile. In conclusion, the automated IMMULITE ® IGF-I and IGFBP-3 assays are reliable tools in the diagnosis of pathologies of the GH/IGF axis and in the follow-up of their therapies.

This study was performed to quantify the effect of a 1-weekfreezer storage of urine on its calcium oxalate crystallization risk. Calcium oxalate is the most common urinary stone material observed in urolithiasis patients in western and affluent countries. The BONN-Risk-Index of calcium oxalate crystallization risk in human urine is determined from a crystallization experiment performed on untreated native urine samples. We tested the influence of a 1-weekfreezing on the BONN-Risk-Index value as well as the effect of the sample freezing on the urinary osmolality. In vitro crystallization experiments in 49 native urine samples from stone-forming and non-stone forming individuals were performed in order to determine their calcium oxalate crystallization risk according to the BONN-Risk-Index approach. Comparison of the results derived from original sample investigations with those obtained from the thawed aliquots by statistical evaluation shows that i) no significant deviation from linearity between both results exists and ii) both results are identical by statistical means. This is valid for both, the BONN-Risk-Index and the osmolality data. The differences in the BONN-Risk-Index results of both procedures of BONN-Risk-Index determination, however, exceed the clinically acceptable difference. Thus, determination of the urinary calcium oxalate crystallization risk from thawed urine samples cannot be recommended.

Lipoprotein(a) is an important predictor of cardiovascular disease risk. The lack of internationally accepted standardization has impeded the broad application of this lipoprotein in laboratory medicine. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), through its Working Group on Lipoprotein(a) and together with research institutions and several diagnostic companies, have succeeded in developing an international reference material that is intended for the transfer of a lipoprotein(a) concentration to manufacturers' master calibrators. IFCC SRM 2B has recently been accepted by the WHO Expert Committee on Biological Standardization as the ‘First WHO/IFCC International Reference Reagent for Lipoprotein(a) for Immunoassay’. The assigned unitage of 0.1071 nanomoles of lipoprotein(a) per vial is traceable to the consensus reference method for lipoprotein( a) and will enable conformity by diagnostic companies to the European Union's Directive on In vitro Diagnostic Medical Devices for the metrological traceability of calibrator materials.