Volume 38, Issue 7

Background: Systematic reviews have gradually replaced single studies as the highest level of documented effectiveness of health care interventions. Systematic reviewing is a new scientific method, concerned with the development and application of methods for identifying relevant literature, analysing the material while increasing validity and precision, and presenting and discussing the results in a way that does justice to the research question and to the available evidence. The objective of this study was to review the systematic reviews in laboratory medicine, to evaluate the methods applied in these reviews and the applicability of guidelines of the Cochrane Methods Working Group on Screening and Diagnostic Tests, and identify areas for future research. Methods: All the systematic reviews in the field of clinical chemistry and laboratory haematology that could be identified in Medline, EMBASE and other literature databases up to December 1998, were evaluated. Results: We studied 23 reviews of diagnostic trials. Although all reviews share the same basic methodology, there was a wide variation in the methods applied. There was no consensus on the quality criteria for inclusion of primary studies. The results of the primary studies were heterogeneous in most cases. This was partly due to design flaws in the primary studies, but was also inherent in the diverse study designs in diagnostic trials. We observed differences in the analysis of the factors that cause heterogeneity of the results, and in the summary statistics used to pool the data from the primary studies. The additional diagnostic value of a test, after other test results are taken into consideration, was only addressed in one study. Conclusion: This overview of 23 reviews of diagnostic trials identifies areas in the methods of systematic reviewing where consensus is lacking, such as quality rating of primary studies, analysis of heterogeneity between primary studies and pooling of data. Guidelines need to be improved on these points.

The reference values concept has been adopted by health care professionals, including clinical chemists, laboratory scientists, and clinicians and simultaneously by all the official organizations in charge of the establishment of legislation. But the estimation of reference limits, and the evaluation of biological variability need to be improved at the level of the procedures, which are currently too long and too expensive and not feasible easily for all laboratories. The procedures for obtaining reference values, if we follow the original documents, are complex, and that is the main reason that clinical chemists or diagnostic kit manufacturers have not used them systematically. There is clearly a need that scientific societies and international organizations propose practical recommendations: 1) Recommendations to describe methods linked to systematic error. · How to transfer reference limits from one laboratory to another laboratory using different methods? · Should we determine reference limits for each method? · How can we differentiate bias due to the populations from these due to the method? Clear collaborations with manufacturers involved in kits and diagnostic systems are needed. 2) Practical recommendations linked to the reference population. · How to transfer reference limits from one laboratory to another laboratory using different methods? · Should we determine reference limits for each method? · How can we differentiate bias due to the populations from these due to the method? Clear collaborations with manufacturers involved in kits and diagnostic systems are needed. · How to select a homogenous population? (Careful recommendations on the choice between healthy individuals, blood donors and individuals hospitalised for other diseases should be given.) · How to estimate ethnic differences? · How to define the exclusion and inclusion criteria according to quantity? · How to deal with the question of reference limits for unstable periods, aging or old people particularly, when the difference between aging and disease is very difficult to define? 3) Practical recommendations on the statistical methods to be used. · How to transfer reference limits from one laboratory to another laboratory using different methods? · Should we determine reference limits for each method? · How can we differentiate bias due to the populations from these due to the method? Clear collaborations with manufacturers involved in kits and diagnostic systems are needed. · How to select a homogenous population? (Careful recommendations on the choice between healthy individuals, blood donors and individuals hospitalised for other diseases should be given.) · How to estimate ethnic differences? · How to define the exclusion and inclusion criteria according to quantity? · How to deal with the question of reference limits for unstable periods, aging or old people particularly, when the difference between aging and disease is very difficult to define? · How to make a good choice of the interquantile interval? Should we use and present only the centiles 2.5 or 97.5, or on the contrary should we give other centiles in addition, for example 5, 10, 75, 80, 85, 90? 4) Practical recommendations linked to the use of the concept of the reference values. · How to transfer reference limits from one laboratory to another laboratory using different methods? · Should we determine reference limits for each method? · How can we differentiate bias due to the populations from these due to the method? Clear collaborations with manufacturers involved in kits and diagnostic systems are needed. · How to select a homogenous population? (Careful recommendations on the choice between healthy individuals, blood donors and individuals hospitalised for other diseases should be given.) · How to estimate ethnic differences? · How to define the exclusion and inclusion criteria according to quantity? · How to deal with the question of reference limits for unstable periods, aging or old people particularly, when the difference between aging and disease is very difficult to define? · How to make a good choice of the interquantile interval? Should we use and present only the centiles 2.5 or 97.5, or on the contrary should we give other centiles in addition, for example 5, 10, 75, 80, 85, 90? · How to make this concept more concrete and to have official definitions which are better understandable and not only abstract? · How to demonstrate the value of using simultaneously reference limits and decision limits, and what does each of these limits bring to results interpretation? · How to improve the presentation of the results? How to give more information on biological variability in the laboratory data, taking into account the scientific validity of their determination? Should we use new information techniques and new communication systems for reaching these objectives? The responses to all these questions could only be provided if there is a concerted effort at the international level. Practical recommendations should be given, which would be very useful for a better understanding and use of reference values by laboratory scientists and clinicians.

The high prevalence of subjects in the general population and among patients with thyroid diseases who are positive for serum anti-thyroglobulin antibodies, together with the inconsistency of hybridoma techniques to obtain large amounts of anti-thyroglobulin antibodies, prompted us to prepare anti-thyroglobulin antibodies from human serum. An anti-thyroglobulin antibody positive serum was absorbed with CNBr-activated sepharose 4B conjugated to thyroglobulin and anti-thyroglobulin antibodies eluted by acid pH (3.0), basic pH (10.7) and detergent (SDS 3 g/l or 7 g/l). Preliminary experiments carried out with rabbit anti-human thyroglobulin allowed us to purify an Ig-fraction that retained its anti-thyroglobulin activity by immunoprecipitation and tanned red cell haemagglutination. When subjected to agarose gel electrophoresis and Silver stain, this fraction has the mobility of rabbit Ig. Further experiments were carried out using Hashimoto's serum (tanned red cell haemagglutination 1:40620). pH 3.0 was found to give the best yield of stable antibodies. An IgG fraction was eluted at a concentration of 10±1.2 mg/l of serum. This fraction has the same electrophoretic mobility as human IgG and is close to pure human anti-thyroglobulin antibody. A dose-response curve was built up at a concentration range between 0.016 mg–2 mg of IgG anti-thyroglobulin antibody per litre. The slope of the curve parallels that of a dilution curve of the whole serum, suggesting that the purified antibodies are representative of the whole antibody population. In conclusion, we provide a method for preparing considerable amounts of highly purified anti-thyroglobulin antibodies from human serum for application in clinical medicine and basic research, and in particular which provides a standard for measuring anti-human thyroglobulin serum antibodies by weight rather than the presently used standard expressed in U/ml. Furt h e rmore, this reagent could act as an ideal immuno-vector for the diagnosis and therapy of differentiated thyroid carcinoma.

Wharton's jelly is abundant in extracellular matrix, which is known as a storage site to concentrate and stabilise growth factors in the vicinity of cells. It was previously found that Wharton's jelly contains significant amounts of insulin-like growth factor (IGF)-1 and IGF-binding proteins (BPs). IGF-1 is a stimulator of biosynthetics of collagen and sulphated glycosaminoglycans. Preeclampsia (edema, proteinuria, hypertension (EPH)-gestosis) is accompanied by an accumulation of sulphated glycosaminoglycans in Whart on's jelly. IGF-1 and BPs may play an important role in such a remodelling of this tissue. It was decided to evaluate the alterations in amounts of IGF-1 and BPs in Wharton's jelly of newborns delivered by mothers with preeclampsia. Studies were performed on Wharton's jelly of 10 controls and 10 newborns delivered by mothers with preeclampsia (edema, proteinuria > 500 mg/l, arterial pressure: systolic > 140 mm Hg, diastolic >90 mmHg). Radioimmunological techniques were employed to determine IGF-1 and IGF-BPs (BP-1 and BP-3). It was found that preeclampsia is associated with a decrease in IGF-1-and IGF-BP-1 in Wharton's jelly. A slight increase in IGF-BP-3 was found. Ligand blotting demonstrated that BP-3 (not BP-1) is a main component of Wharton's jelly, which binds IGF-1. Heparin drastically inhibited the binding of IGF-1 by BP-3. It is known from our previous studies that preeclampsia is associated with an increase in the amount of sulphated glycosaminoglycans (heparin, heparan sulphate, dermatan sulphate) in Wharton's jelly. This may be a factor, which prevents the binding of IGF-1 by BPs and facilitates the binding of IGF-1 to cells, stimulating them to produce sulphated glycosaminoglycans in Wharton's jelly.

Similarly to many immune molecules of human milk, C3 and C4 levels decrease during lactation. We investigated the influence, over the first three weeks of lactation, of both prematurity and parity on the sequential evolution of these levels. Milk C3 and C4 concentrations were measured by immunonephelometry in 494 individual samples collected from 76 lactating mothers. C3 and C4 concentrations were higher in milk from preterm or primiparous mothers. The major differences were observed in milk from days 5–8 and 9–20, likely due to pronounced interindividual variations in levels of days 1–4 milk. Milk from mothers of precocious (33 weeks' gestation or less) preterm newborns presented higher concentrations and a slower decrease of C3 and C4 levels than that from mothers of late (33–37 weeks' gestation) preterm newborns, when compared to term mothers. Finally, the inversion of the C3/C4 ratio occuring over time, previously reported, appeared later in milk from mothers of preterm newborns. The influence of prematurity was even greater in primiparous than in multiparous mothers. Both C3 and C4 levels therefore appear to be influenced in human milk by the parity and prematurity of the delivery. Mothers from preterm newborns seem to provide higher levels of C3 for a longer period post delivery.

We evaluated the performance of the Emit ® tox benzodiazepine and barbiturate assays (Dade Behring) and fluorescence polarisation immunoassay (FPIA) (Abbott) for use with serum determinations in preliminary therapeutic drug monitoring or acute drug intoxication detection. Performance, as indicated by CVs, of the Emit ® tox benzodiazepine and barbiturate assays and FPIA showed that both immunochemical techniques are precise and have good reproducibility. For within-run studies, results from benzodiazepine determinations showed maximum CV values of 1.91% for the Emit ® method and 2.65% for FPIA; results from barbiturate determinations showed maximum CV values of 2.01% for the Emit ® method and 1.89% for FPIA. For between-run studies, results from benzodiazepine determinations showed maximum CV values of 1.79% for the Emit ® method and 1.12% for FPIA; results from barbiturate determinations showed maximum CV values of 2.09% for the Emit ® method and 2.02% for FPIA.

Inflammatory bowel disorders are characterized by an accumulation of eosinophilic granulocytes, mast cells, lymphocytes and neutrophilic granulocytes in the intestinal mucosa. The aim of this study was to examine the concentration of eosinophilic granulocytes in the blood of patients during active ulcerative colitis in comparison with patients during remission and apparently healthy control subjects. Besides counting, the activity grade of eosinophilic granulocytes has been studied by estimation of their degranulation product eosinophil cationic protein. Subjects with active ulcerative colitis could be distinguished from patients with quiescent ulcerative colitis by establishment of the eosinophil cationic protein concentration, neutrophilic granulocyte count, erythrocyte sedimentation rate, C-reactive protein and albumin concentration. After two weeks of corticosteroid treatment, serum eosinophil cationic protein concentrations and eosinophil counts in blood were significantly decreased. A decrease in blood eosinophil count was accompanied by a decrease in eosinophil cationic protein concentrations in serum in most subjects with ulcerative colitis. After twelve weeks of corticosteroid administration, serum albumin concentrations were significantly increased, whereas serum concentrations of C-reactive protein were significantly decreased. During treatment with corticosteroids, serum eosinophil cationic protein concentrations and blood eosinophil counts are appropriate laboratory markers to detect the effect of medication in the course of ulcerative colitis.

We describe a simple, rapid and reproducible method for measuring hypoxanthine, xanthine and uric acid in urine samples of infants by capillary electrophoresis with an uncoated fused silica capillary and ultraviolet detection. Conditions were 40mmol/l borate buffer pH 9.4 with sodium dodecyl sulphate (0.1mol) 3s hydrodynamic load and the voltage 20kV. The calibration curves for hypoxanthine and xanthine were linear to 150μmol/l and for uric acid to 300μmol/l. The limits of detection for hypoxanthine, xanthine and uric acid were 0.5, 2.0 and 20μmol/l, respectively. Analytical recovery of the three purines ranged from 93 to 105%. Overall CVs were <5.2%. Using this method, we measured purine concentrations in the urine samples of 103 infants, divided into three groups, according to different degrees of hypoxia. There was a significant difference among the three groups only in hypoxanthine concentrations. In 35 urine samples from the control group, 43 urine samples from infants with a history of an apparent life threatening episode and in 25 urine samples from infants with infantile apnoea, the mean concentrations of hypoxanthine (mean ± SD) were 13.1 ± 4.0, 25.1 ± 8.1 and 58.2 ± 17.4 μmol/mmol creatinine, respectively. The diagnostic accuracy of the purine measurement was evaluated by receiver-operating characteristic curve analysis.

During the process of aging red blood cells become denser and smaller. Counterflow centrifugation separates particles of lower density and smaller diameter from those of higher density and bigger diameter. Thus, the question arises: which property of the red cells, density or size, governs the age-related separation by counterflow centrifugation? It is shown that it is the size which dominates the balance between sedimentation and streaming. Age-related separation of human red blood cells by counterflow centrifugation (elutriation) was analysed by the standard hematological parameters (hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration), hemoglobin A 1c and the membrane protein ratio 4.1a/(4.1a+4.1b). Red blood cells with a high hemoglobin A 1c content and a high 4.1a/(4.1a+4.1b) ratio were found in the early fractions of the elutriation. This proves that old cells make up early fractions, while the “youngsters” constitute later ones. The elutriation technique used (yielding human red blood cells in a “healthy state”) and the age parameters studied show that the membrane protein ratio 4.1a/(4.1a+4.1b) is another reliable age parameter for the assessment of red blood cell age.

Reference intervals in clinical chemistry are commonly based on results of measurements in reference populations or are taken from the literature. A reference population should represent a defined group of individuals and be as similar as possible to the patients under investigation. Frequently, reference populations have been recruited from institutionalised healthy young people who do not necessarily fulfill these criteria. In the present study we describe the temporal changes in 37 commonly measured quantities in men and women from childhood to late in life. The samples were collected in the primary health care and sorted according to an assumed decision by the physician. The emerging group of individuals forms a reference population that was regarded as “non-diseased” and the results of measurements in this population are reference values. A remaining group of “non-healthy” were likewise identified for comparison. The central 95 percentile was wider than those usually assigned to the quantities whereas the medians almost coincided. In the “non-healthy” group the medians were shifted in a direction that would be expected from pathophysiology aspects.

The association of apolipoprotein E concentration and common polymorphism (codons 112/158) with lipid and apolipoprotein concentrations was studied in two Chinese healthy population samples from Beijing (n=99) and Shanghai (n=67). Body mass index and apolipoproteins E and AI and triglyceride concentrations were significantly different between the two populations (0.001<p<0.05), but apolipoprotein E common allele frequencies were not significantly different. Apolipoprotein E concentration was independently associated with apolipoproteins B and AI, total cholesterol and triglyceride concentrations in the Beijing group, but only with total cholesterol and apolipoprotein AI concentrations in the Shanghai group. Apolipoprotein E polymorphism was significantly associated with apolipoproteins E and AI, and triglyceride concentrations in the Beijing subjects (0.001<p<0.01), whereas apolipoprotein E polymorphism was significantly related only to apolipoprotein E concentration in the Shanghai group (p<0.001). This preliminary study with a limited number of subjects shows that the relations between both apolipoprotein E genotypes and concentration with lipid traits are not the same in the two studied Chinese populations.

Uric acid is known to be an end product of purine metabolism. Increases in uric acid may be found in clinical conditions associated with tissue hypoxia. We have investigated the value of uric acid to differentiate between a transudate and exudate. In this study, we measured uric acid in the pleural fluid and the serum of 110 patients, 30 women and 80 men with a mean age of 49.5±19 years. Light's criteria were used to differentiate between a transudate and exudate. Mean serum uric acid was 496.7±153.4 μmol/l in patients with transudates and 291.3±143.1 μmol/l in patients with exudates. Mean pleural fluid uric acid was 487.7±165 μmol/l in patients with transudates and 279.9±142.1 μmol/l in patients with exudates. These data showed that the levels of serum and pleural uric acid were higher in transudates than exudates (p<0.01). However, there was no significant difference between pleural fluid/serum uric acid ratio of the two patient groups (p>0.05). The specificity and sensitivity of pleural uric acid for diagnosis of transudate effusions were 73% and 80.6%, respectively. The specificity and sensitivity of pleural uric acid for diagnosis of transudate effusions from exudates without malignancy were 71.8% and 91.7%, respectively. The sensitivity and specificity of pleural lactate dehydrogenase for diagnosis of exudates were 82% and 89%; the sensitivity and specificity of pleural fluid/serum lactate dehydrogenase were 85% and 89%; the sensitivity and specificity of pleural fluid/serum protein were 91% and 89%, respectively. Using all three of Light's criteria together, the sensitivity was 91% and its specificity was 94%. Our findings indicate that determination of uric acid in pleural fluid may be of diagnostic value in differential diagnosis of transudates and exudates. The sensitivity of pleural uric acid measurement was higher for exudates without malignancy. However, Light's criteria remain the best means of separating transudates from exudates.

Familial defective apolipoprotein (apo) B-100 is an autosomal codominant disorder associated with hypercholesterolemia and an increased risk of coronary artery disease. Two independent mutations affecting the codon 3500 (Arg 3500 →Gln and Arg 3500 →Trp) have been shown to cause ligand-defective apo B-100. Identification of carriers of these mutations is an important step in the risk stratification of individuals and families with hypercholesterolemia. We evaluated a homogeneous assay for detection of mutations at codon 3500 that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. This single-tube analysis is performed on the LightCycler™, a microvolume fluorimeter integrated with a thermal cycler. Continuous acquisition of fluorescence data during a melting curve analysis at completion of PCR allows the detection of mutations, as loss of fluorescence occurs in an allele-specific manner. By plotting melting peaks, the three apo B-100 alleles were readily distinguishable. Using this method, genotyping of 32 samples is completed within 40 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The specific detection of mutations at codon 3500 of the apo B gene on the LightCycler™ is a rapid and reliable method that is ideally suitable for typing both small and large numbers of samples.