[Prescription of bone remodeling markers in hospitals]. / Enquête sur la prescription des marqueurs du remodelage osseux en milieu hospitalier.

Biochemical markers of bone turnover have for several years been considered as valuable parameters in research clinical studies, but their use in individual patients is still debated. Recently several position papers have proposed guidelines for their use in clinical practice in patients with post menopausal osteoporosis. In the present article, we report the results of a survey which aims at comparing the actual modalities of prescription of French physicians with the above-mentioned recommendations. We contacted by phone clinical chemists from 158 different hospitals and asked them to transmit to the concerned physicians of their hospital a detailed questionnaire for assessing which bone marker(s) is (are) prescribed and for which purpose (s), and if not prescribed, the reason of non prescription. We were able to analyze 309 questionnaires from 89 hospitals including 5 specialties, rheumatology (35.9%), endocrinology (18.1%), gynecology (11.0%), internal medicine (22.0%) and geriatry (12.9%). The results showed large discrepancies between the mode of prescription of a subset of physicians and the guidelines. The most often evoked reason for non prescription was a lack of information about bone markers suggesting a need for teaching courses. This survey has also shown that many physicians do not know exactly which parameters are effectively measured in their hospital and which are addressed to specialized laboratories underlining the importance of the dialogue between clinicians and clinical chemists. We propose that in a given hospital, the present article may serve as a basis for a discussion between clinicians and biologists about the development and/or the optimization of the measurements of these markers of bone turnover.

[Biochemical markers of bone remodeling: pre-analytical variations and guidelines for their use. SFBC (Société Française de Biologie Clinique) Work Group. Biochemical markers of bone remodeling]. / Les marqueurs biologiques du remodelage osseux: variations pré-analytiques et recommandations pour leur utilisation. Groupe de travail SFBC Marqueurs biochimiques du remodelage osseux.

Biochemical markers of bone turnover have been developed over the past 20 years that are more specific for bone tissue than conventional ones such as total alkaline phosphatase and urinary hydroxyproline. They have been widely used in clinical research and in clinical trials of new therapies as secondary end points of treatment efficacy. Most of the interest has been devoted to their use in postmenopausal osteoporosis, a condition characterized by subtle modifications of bone metabolism that cannot be detected readily by conventional markers of bone turnover. Although several recent studies have suggested that biochemical markers may be used for the management of the individual patient in routine clinical practice, this has not been clearly defined and is a matter of debate. Because of the crucial importance to clarify this issue, the Société Francaise de Biologie Clinique prompted an expert committee to summarize the available data and to make recommendations. The following paper includes a review on the biochemical and analytical aspects of the markers of bone formation and resorption and on the sources of variability such as sex, age, menstrual cycle, pregnancy and lactation, physical activity, seasonal variation and effects of diseases and treatments. We will also describe the effects of pre-analytical factors on the measurements of the different markers. Finally based on that review, we will make practical recommendations for the use of these markers in order to minimize the variability of the measurements and improve the clinical interpretation of the data.

[Tamm-Horsfall protein]. / La protéine de Tamm-Horsfall.

Human Tamm-Horsfall glycoprotein, the major urinary protein, is a glycosyl-phosphatidyl-inositol (GPI) - anchored membrane protein which mainly resides at the luminal face of cells of the thick ascending limb of Henle's loop (TAL) and early distal convoluted tubules of nephron. Tamm-Horsfall protein contains exclusively N-linked glycans, mainly of polyantennary type largely sialylated and fucosylated, but also high-mannose glycans. Only a portion of the Tamm-Horsfall protein is released as soluble protein by the action of proteases and in a minor amount by a cell-associated GPI-specific phospholipase. The physiological function of Tamm-Horsfall glycoprotein has not been clarified to date. Since the urinary Tamm-Horsfall protein has a high gel-forming tendency, it has been postulated that it takes part in the water impermeability of TAL. It is also proposed that the Tamm-Horsfall protein plays a protective role towards pyelonephritogenic pathogens such as Escherichia coli. The Tamm-Horsfall protein may inhibit the colonization of these pathogens in the renal mucosa in that the soluble form competes with that exposed at the plasma membrane. Recently, urinary Tamm-Horsfall protein has been implicated in tubulointerstitial nephritis.

Quantitative analysis of C60 fullerene in blood and tissues by high-performance liquid chromatography with photodiode-array and mass spectrometric detection.

A high-performance liquid chromatographic (HPLC) assay method for C60 fullerene, in blood, liver and spleen using photodiode-array detection or mass spectrometric detection (LC-MS) and C70 fullerene, as the internal standard, is described. The recovery from mouse blood and tissues spiked with micronized C60 exceeds 90%. The method is linear from 0.05 to 200 mg of C60 per liter of blood and from 0.05 to 5.00% of C60 per tissue weight. The limit of detection of the method is 0.1 ng of C60 per injection. This method was applied to mouse blood and tissue samples after intraperitoneal administration of a micronized C60 suspension.

[Improvements from the reference material CRM 470 in the standardization of the determination of serum proteins]. / Améliorations apportées par le matériau de référence CRM 470 dans la standardisation du dosage des protéines sériques.

The introduction of the CRM 470 in 1993 (certified reference material for 14 serum proteins) and its utilization by industrial companies for cross-calibrating their commercial standards has been an important break-through in protein standardization. This improvement has been clearly illustrated by the last national quality control survey performed in France in may 1995. At this time, about 60% of the 1,870 participants have already adopted the new standardization. The between-run precision (interlaboratory and intertechnique) has been considerably improved by the use of the new international standard (5.8 to 12.2% versus 10 to 24.1% before standardization); the same is true for accuracy. These results should convince the last reluctant laboratories to adopt the new standardization. Thus, it seems now possible to define reference ranges for serum proteins: this is the new task assigned to the Committee for Plasma Protein Standardization of the IFCC.

Evaluation of human IgG subclass assays on Beckman array.

Automated immunonephelometric assays were developed to measure human IgG subclasses in serum, on the Beckman Array Protein System (APSR), with sheep antihuman IgG subclass antisera. The interassay imprecision was judged to be satisfactory in each case (CV: IgG1: 2.3%; IgG2: 2.9%; IgG3: 4.0% and IgG4 5.4%). The standard curves were found to be linear in the ranges 1.5-30.0 g/L for IgG1, 1.5-15.0 g/L for IgG2, 0.1-1.0 g/L for IgG3 and 0.1-1.0 g/L for IgG4. The lower limit for quantification of the immunephelometric assays was 0.5 g/L for IgG1, 0.04 g/L for IgG2, 0.06 g/L for IgG3 and 0.03 g/L for IgG4. We found no antigen excess to at least 60.0 g/L for IgG1 and IgG2, 3.5 g/L for IgG3 and 2.0 g/L for IgG4. Nephelometric results correlated with those of radial immunodiffusion (r = 0.96 for IgG1, r = 0.93 for IgG2, r = 0.90 for IgG3 and r = 0.96 for IgG4). The reaction monitored by the Beckman Array Protein System is easy to perform, rapid and precise. The performance makes the immunonephelometric method suitable for the determination of IgG subclass concentrations in routine analysis.

Comparison of six serum ferritin immunoassays and isoferritin spectrotypes in malignancies.

Serum ferritin was measured by six enzyme immunoassays in specimens from patients with digestive cancers (n = 30) and hematologic malignancies (n = 33). Most mean comparisons show significant differences in both groups of patients. In digestive cancers correlations between any two methods are very satisfactory (r > 0.99) but a proportional bias is often observed. In hematologic malignancies, correlations are bad (r < 0.80 in 8 out of 15 correlations) because of many discrepant values. Isoelectric focusing separation of isoferritins was performed in most specimens and the pattern of each serum was compared to the between kit CV. We conclude that an 'acid' spectrotype increases between-kit analytical variability. We try to explain the results taking into account the nature of the immunological systems and the cross-reactions with tissular isoferritins. In conclusion, our results indicate that large differences may be observed in sera from hematologic malignancies (leukemias, lymphomas ... ) We recommend that monitoring be achieved by the same method of measurement.

[Serum determination of IgG, IgA, IgM, transferrin and haptoglobin. I. Attempt at normalization of results]. / Dosages sériques d'IgG, IgA, IgM, transferrine et haptoglobine. I. Essai d'harmonisation des résultats.

This study demonstrates the advantages and limitations of normalizing results for five serum proteins (IgG, IgA, IgM, transferrin and haptoglobin), analysed in liquid phase on ten different systems (open clinical chemistry and dedicated protein analysers). Seven sets of results from normal and pathological sera (without monoclonal proteins) were compared using: - calibrators supplied by each manufacturer; - - serial dilutions of a single stabilized pool of liquid serum. In addition to validating the quality of the stabilised serum, we have been able to identify: - significant variations in results using different analytical systems for the same sample; - a major reduction in these variations, often greater than 50%, through normalization using a common serum pool. In some two thirds of the cases, this reduction brought the degree of variation into an acceptable range.

[Serum levels of IgG, IgA, IgM, transferrin and haptoglobin. III. Results obtained with monoclonal immunoglobulins]. / Dosages sériques d'IgG, IgA, IgM, transferrine et haptoglobine. III. Résultats obtenus en présence d'immunoglobulines monoclonales.

This study is the third part of a multicenter evaluation carried out with ten analysers. Five proteins (IgG, IgA, IgM, transferrin, haptoglobin) were assayed in three sera, each containing one monoclonal Ig (IgG, IgA, or IgM). The expected agreement was not obtained with these particular sera, except in the case of haptoglobin. The marked imprecision of the monoclonal Ig quantification is highlighted, including the fact that many erroneous results are due to the antigen excess phenomenon. It is also shown that in the serum containing monoclonal IgM, quantification of the other proteins, remaining polyclonal Ig, as well as transferrin and haptoglobin, is difficult. The authors recall that electrophoresis and immunoelectrophoresis, which are indispensable for the characterization of the monoclonal components, are of great help in solving such difficulties.

[Serum levels of IgG, IgA, IgM, transferrin and haptoglobin. II. Analytical precision and comparison of results from different analysers]. / Dosages sériques d'IgG, IgA, IgM, transferrine et haptoglobine. II. Précision analytique et comparaison des résultats fournis par différents analyseurs.

We compare the precision of immunochemical techniques for determining immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), transferrin and haptoglobin. Precision was found satisfactory. Results of samples containing monoclonal Ig, showed substantial discrepancies, especially the serum containing monoclonal IgM. In this case, the nephelometric analysers gave the best results. In conclusion, when different techniques are used for monitoring protein immunoassays, a good knowledge of the performance of the analyser is necessary for correct interpretation and to avoid potentially misleading results.

[Multicenter study of commercial kits for the determination of human IgG subclasses, using the ELISA technique]. / Etude multicentrique des trousses commercialisées pour le dosage des sous-classes d'IgG humaines par technique ELISA.

We evaluated two commercially available sandwich type Elisa procedures for the measurement of IgG subclasses in human serum. Assay kits from The Binding Site and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service were tested in six laboratories. The performance of spectrophotometers, pipettes and dilutors were assessed at each center. Within-run precision was estimated according to the Valtec method (Société Française de Biologie Clinique). The overall coefficient of variation ranged from 4 to 50% depending on subclass and kit. We also evaluated the IgG2 and IgG4 specificity using four sera containing a monoclonal IgG2 or IgG4 (kappa or lambda type). Using total IgG and immunoelectrophoresis as a comparative technique, IgG2 kappa and IgG4 kappa were both underestimated, IgG2 lambda was overestimated while IgG4 lambda compared favorably. Polyclonal IgG subclasses were frequently overestimated in these sera suggesting cross-reactions with either monoclonal IgG or other polyclonal IgG. Antigen excess was investigated and not encountered with either kit. Our results demonstrate that these procedures are insufficiently accurate or precise for routine clinical use.

[Erythrocyte ferritin]. / La ferritine érythrocytaire.

Red cell ferritin is a residue of erythroblast ferritin. It reflects the balance between the iron supply to the erythroid marrow and the need for haemoglobin synthesis. Erythrocyte ferritin can be measured in haemolysates after discarding plasma and leucocytes by different methods. The decrease in erythrocyte ferritin content indicates manifest iron deficiency anaemia. Ferritin levels do not appear to be influenced by inflammation, infection, tissue necrosis or tumors and may be a reliable indicator of iron status in inflammatory diseases. Erythrocyte ferritin is markedly increased in patients with iron overload allowing early diagnosis of hereditary haemochromatosis and the monitoring of phlebotomy therapy. Finally, in some pathologies erythrocyte assay is a more reliable indicator of the iron status than serum ferritin.

Study of six antisera used for the immunonephelometric assay of human IgG.

The composition and performance of six commercial antisera for the immunonephelometric assay of IgG were studied. The analysis of the protein content of these reagents revealed significant differences in composition: the total protein contents ranged from 41 to over 130 g/l and gamma-globulins were twice to thirteen times higher than in a normal serum of the species. The antibodies found in these reagents were mostly IgG, with traces of IgM, and no IgA. Precipitation curves yielded by these antisera with a standard nephelometer showed a great diversity of performance. The very different shapes, the span of antigen concentrations and antibody dilutions necessary to obtain comparable quantities of precipitates further demonstrated the wide differences between these reagents. A method previously proposed by other authors was found to be inadequate. The failure of this method is attributed to the different conditions for the antigen-antibody reaction, as determined by the instrumentation used. It is concluded that the fundamental and classical criteria for testing antisera remain valid: specificity and the study of the precipitation curve to define the quantitative limits of the reaction. A simple universal method is inapplicable.

[Hemodialysis and ferritinemia]. / Hémodialyse et ferritinémie.

Iron-deficiency is a common phenomenon in chronic renal diseases and haemodialysis patients, a treatment with iron or transfusions is always provided in an early preventive way; yet, an overload may appear. Serum ferritin, in spite of analytic variability, remains at the present time a good witness to appreciate patients' iron stores. Authors report the results obtained with four commercial reagents in healthy population and in haemodialysed children or adults. The comparison of results for this parameter shows that haemodialysed sera, with or without treatment, have the same behaviour with reagents as those of healthy subjects.

Enzyme immunoassay of serum erythropoietin in healthy children: reference values.

A solid phase enzyme immunoassay for erythropoietin which required only a small specimen of blood was evaluated. It permitted measurement up to 150 IU/L. Recovery was between 86.2% and 110.0% and between-batch precision between 4.7% and 5.7%. We determined the levels of erythropoietin in infants aged 10 months (geometric mean 11.2 IU/L; 95% range 3.6-34.9) and in children aged 2 years (geometric mean 10.5 IU/L; 95% range 4.3-25.8). Both were significantly higher than the values found in children aged 4 years (geometric mean 7.2 IU/L; 95% range 2.4-21.8). There were no differences between the values in children at 4 years, those aged 4-15 years and adults.

Usefulness of a prognostic inflammatory and nutritional index in pediatric clinical practice.

The clinical usefulness of a prognostic inflammatory and nutritional index (PINI) was evaluated in 47 hospitalized infants and 72 age-matched controls from 1 mth to 14 yr of age. The PINI formula is a combination of two markers each of infection (C-reactive protein and orosomucoid) and of malnutrition (albumin and transthyretin). Healthy children are identified by a PINI value less than 1 whereas sick patients are characterized by a progressive rise above 1 as the conditions worsens. The PINI scoring system provides the clinician with a sensitive and universal tool, allowing the correct follow-up of both inflammatory and nutritional poles of the disease spectrum. The formula is of particular help in pediatric practice in recognising early subclinical complications and efficacy of specific therapeutic strategies. The micromethod is simple, rapid and may be regularly repeated since it requires only 46 microliters of serum.

Automated determination of urinary Na+, K+, chloride, inorganic phosphate, urea, and creatinine without sample dilution, with the "RA-XT".

We describe how concentrations of chloride, urea, inorganic phosphate, and creatinine in urine can be measured directly, without manual sample dilution, in a discrete analyzer (the Technicon "RA-XT"). These methods were accurate for concentrations of chloride up to 280 mmol/L, urea up to 500 mmol/L, inorganic phosphate up to 50 mmol/L, and creatinine up to 30 mmol/L. CVs are less than 3% nd results correlate well with those obtained by continuous-flow analysis (SMA-II). All these reagents are stable at room temperature for three weeks. Analyses are easy to perform and infrequent calibration is required.

[Reference values of erythrocyte ferritin in children and adults]. / Valeurs de référence de la ferritine érythrocytaire chez l'enfant et l'adulte.

The erythrocyte ferritin content was measured in 183 healthy subjects in age from 4 to 68 years; 80 were male and 103 were female. In children between the ages of 4 and 12 years there is no significant difference in the mean value between boys and girls. In females the erythrocyte ferritin concentration is independent of age. After 12 years of age the erythrocyte ferritin content is higher in men. Reference intervals were determined by the two quantiles 0.05 and 0.95. The reference interval is 3-24 age by cell in boys between 4 and 12 and females between 4 and 63; the reference interval is 5-38 age by cell in males between 13 and 68 years of age.