Delayed-type hypersensitivity and chronic fatigue syndrome: the usefulness of assessing T-cell activation by flow cytometry--preliminary study.

Chronic fatigue syndrome or benign myalgic encephalomyelitis has been extensively described and investigated. Although numerous immunological abnormalities have been linked with the syndrome, none have been found to be specific. This article describes the detection of delayed-type hypersensitive responses to certain common environmental antigens in almost fifty per cent of patients with this syndrome. Such hypersensitivity can be detected by the intradermal administration of antigens derived from commensal organisms like the yeast Candida albicans albicans, and then monitoring for a systemic reaction over the following six to forty-eight hours. This approach can be consolidated by performing lymphocyte activation tests in parallel and measuring in vitro T-cell activation by Candida albicans albicans antigens by three-colour flow cytometry based on CD3, CD4 and either CD69 or CD25. Another useful parameter is the kinetics of neopterin excretion in the urine over the course of the skin test. The results showed that the intensity of the DTH response correlated with the number of T-cells activated in vitro. Various factors have been implicated in the fatigue of many patients, notably lack of sleep. However, it remains difficult to establish causality in either one direction or the other. This work is in the spirit of a multifactorial approach to the group of conditions referred to as "chronic fatigue syndrome".

Microdialysis study of amino acid neurotransmitters in the spinal dorsal horn of patients undergoing microsurgical dorsal root entry zone lesioning. Technical note.

The aim of this study was to develop, for the first time in the human spinal dorsal horn (DH), an in vivo method for the study of amino acids (AAs). A microdialysis technique was used to sample AAs in the extracellular fluid of the DH apex in eight patients in whom surgery in the dorsal root entry zone (DREZ) was performed. Before making microsurgical lesions, specific concentric-type microdialysis probes were implanted over a 60-minute period in the DREZ and directed to the DH apex (10 implantations). The AA concentrations in the dialysates were determined using high-performance liquid chromatography with fluorescence detection. The concentrations of excitatory AAs (glutamate and aspartate) and inhibitory AAs (gamma-aminobutyric acid and glycine) decreased and were stabilized by 45 minutes after probe implantation, whereas the levels of nonneurotransmitter AAs (alanine and threonine) were not stabilized at 60 minutes. The ability of the probe to track the changes of extracellular AAs was demonstrated. Neither intra- nor postoperative microdialysis-related complications were observed (with a follow up of 18 months). The present study demonstrates that microdialysis can be performed safely in the human DH during DREZ lesioning. Despite technical and analytical limitations related to the intraoperative conditions, this technique offers new possibilities for clinical research on neurotransmitters involved in some relevant pathological states, especially in chronic pain and spasticity.

[Role of isoelectric focusing of cerebrospinal fluid immunoglobulin G in the early biological assessment of multiple sclerosis]. / Apport de la focalisation isoélectrique des immunoglobulines G du liquide céphalorachidien dans le bilan biologique précoce de la sclérose en plaques.

A retrospective study was carried out at the Neurological and Neurosurgical Hospital of Lyon in order to evaluate the interest of detecting IgG oligoclonal bands by isoelectric focusing with IgG immunorevelation for the early diagnosis of multiple sclerosis (MS). Patients have been grouped according to their disorders: multiple sclerosis (281 cases), definite (182 cases) and possible (99 cases), others inflammatory neurological diseases (63 cases), various non-inflammatory neurological disorders (180 cases) and indefined neurological disorders (664 cases). The following examinations were performed: CSF cell count and cytology after concentration and cytocentrifugation, CSF and serum determination of albumin and IgG with CSF/serum ratios, agarose gel electrophoresis and isoelectric focusing of oligoclonal IgG. The technique used was isoelectric focusing using agarose gel, transfer onto PVDF membrane and then IgG immunorevelation with biotinylated anti-human IgG antibodies. Isoelectric focusing with IgG immunorevelation is the most sensitive (94%) and specific (96%) technique. Isoelectric focusing with immune detection can be recommended as the most efficient test (gold standard) for the detection of chronic CNS inflammation.

[Measurement of antiganglioside autoantibodies by immunodot-blot assay: clinical importance in peripheral neuropathies]. / Détection des autoanticorps antigangliosides par immunodot-blot: intérêt dans le diagnostic des neuropathies périphériques auto-immunitaires.

We retrospectively evaluated measurement data and clinical relevance of autoantibodies to gangliosides in peripheral neuropathies (PN). The IgG and IgM antiganglioside autoantibodies were determined by our own immunodot-blot assay on membrane and by enzyme-linked immunosorbent assay (Elisa) in sera of 1,342 patients with peripheral neuropathies. Anti-GM1 and anti-GD1b autoantibodies formed a part of the normal autoantibody repertoire and were common place in 12% of normal subjects and in 14% of disease control groups. Polyclonal IgM antiganglioside autoantibodies were detected in chronic PN, polyclonal IgG antiganglioside autoantibodies were detected in acute PN. Polyclonal IgM anti-GM1 and anti-GD1b autoantibodies were detected in 35 patients out of 48 with treatable multifocal motor neuropathy with persistent conduction blocks. These autoantibodies well discriminated between suspected motor peripheral neuropathies and motor neuron diseases (sensitivity 73%, specificity 83%, positive predictive value 60%, negative predictive value 91%). Monoclonal IgM autoantibodies reacted strongly with gangliosides in 15 patients out of 77 with M-IgM neuropathy (19%). M-IgM autoantibodies differed in their fine specificities with different principal target antigens as demonstrated with cross-reactivity. Such findings provide further evidence for a relationship between neurological syndromes and antiganglioside antibody profiles and also suggest that different gangliosides could be principal target antigens such as GM1, GD1b, GT1b, GD1a or GM2. Polyclonal IgG anti-GM1 and anti-GD1b autoantibodies were detected in 21 patients out of 22 with acute motor axonal Guillain-Barré syndrome with antecedent of infection by Campylobacter jejuni, polyclonal IgG anti-GQ1b autoantibodies in 9 patients out of 10 with Miller-Fisher syndrome. Detection of antiganglioside autoantibodies by immunodot-blot assay which is simple and quick in testing a large panel of gangliosides has become very important in the diagnosis and in the choose of expensive therapeutic strategies in chronic or acute autoimmune neuropathies.

[Detection of antibodies binding to myelin in 75 neuropathies associated with IgM gammopathy]. / Détection des anticorps anti-myéline: mise au point et évaluation dans 75 cas de neuropathies associées à une IgM monoclonale.

Polyneuropathies associated with IgM monoclonal gammopathy (IgM-MG) are the most frequent types of peripheral neuropathies associated with monoclonal gammopathy. The pathogenic relevance of IgM-MG in peripheral neuropathies is supported by several arguments. The more significant are pathological data on nerve biopsies and the demonstration of IgM antibody activity to peripheral nerve antigens, mainly myelin-associated glycoprotein (MAG) and, sulfated 3-glucuronyl paragloboside (SGPG) and sulfated 3-glucuronyl lactosaminyl paragloboside (SGLPG). The objective of this study was to determine the performance characteristics of 3 immunoassays for detection of anti-myelin antibodies in neuropathy associated with monoclonal IgM gammopathy. Monoclonal IgM may show antibody activity to carbohydrate epitopes shared by several nerve specific antigens. Results obtained with three reliable assay systems, indirect immunofluorescence for antibodies to myelin sheaths (33%), thin layer chromatography for anti-SGPG/SGLPG antibodies (54%) and Western-blot for anti-MAG antibodies (39%). Comparing the different assay systems for carbohydrate structure present on both MAG and SGPG/SGLPG on myelin sheath, the thin layer chromatography appeared to be more sensitive and reliable than Western blot.

[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.

[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.

[1995: a decisive progress for the interlaboratory transferability of results by using enzyme and protein reference materials]. / 1995: un progrès décisif dans la transférabilité interlaboratoire des résultats grâce aux matériaux de référence d'enzymes et de protéines.

Transferability of results in clinical enzymology and immunochemistry may be improved by using validated calibrators. For that purpose, certified reference materials (CRM) have been developed. Authors emphasize the need to make known and to use these tools properly, and on the potential efficiency of this approach in immunochemistry with CRM 470, certified for 14 plasma proteins and with enzyme CRMs produced at the international level. They recall the necessity to validate calibrators for specified techniques of measurement used in routine. They also indicate that this approach, if properly conducted, should allow to ensure the transferability of results in enzymology and immunochemistry and thus the clinical efficiency of laboratory results.

[Reference material (CRM 470) for serum proteins: preparation, characteristics and conditions of use]. / Le matériau de référence (CRM 470) pour les protéines sériques: préparation, caractéristiques et conditions d'utilisation.

Several national and international quality assurance schemes clearly demonstrated the discrepancies between the results given by different analytical systems designed for the immunochemical measurement of proteins. Thus, the need for the preparation of a new international secondary reference material appeared urgent to the International Federation for Clinical Chemistry (IFCC). In this paper, the authors describe the different steps of the preparation of the new standard CRM 470. This work financed by the Bureau Communautaire de Référence (BCR) has been done in collaboration with the College of American Pathologists (CAP). The physicochemical properties of the proteins present in the CRM 470 and the various controls undertaken on the preparation are described. The CRM 470 material (40,000 lyophylised 1 ml vials) is now certified for the 14 plasma proteins of major interest in clinical chemistry. The worldwide introduction of this material will considerably improve the accuracy of immunochemical proteins' determination and will allow the determination of the reference values as a function of age and sex.

A versatile injection system for flow-injection analysis.

Analyser injection systems based on the principle of flow-injection analysis depend on the technique used. They generally take the form of an injection loop valve; the injected sample volume is determined by the volume of the valve. Injection systems are seldom designed with a time factor to define this volume. The authors report on an original injection system, which enables the two techniques to be used. The paper describes the evaluation of this system using both injection techniques and the comparison between them. The results show good linearity (r = 0.999 to 1.000) and an average precision (CV = 1.04 to 1.51%) for the volume-based injection technique; (ii) good linearity (r = 1.000) and better precision (CV = 0.73 to 1.30%) for the time-based injection technique. The system can be used equally well by the loop and by the clock; however, the latter is preferable because of its practicability.

Assessment of the benzethonium chloride method for routine determination of protein in cerebrospinal fluid and urine.

We have tested the characteristics of the method of Iwata and Nishikaze (Clin Chem 25: 1317, 1979). The linearity, sensitivity, and precision are satisfactory and the reactivity of benzethonium chloride with various proteins (albumin, immunoglobulins) is the same. The method has been compared with Meulemans's technique (Clin Chim Acta 5: 757, 1960), routinely used in our laboratories, by analysis of 82 samples of cerebrospinal fluid (CSF) and 119 samples of urine. Our results for cerebrospinal fluid agree well with those of Iwata and Nishikaze (r = 0.976; y = 0.992x - 0.013), but we find their method unsuitable for urinary protein determination, probably because of interfering compounds in urine.

[Valve of the use of benzethonium chloride for turbidimetric analysis of cerebrospinal fluid proteins with an automatic analyzer]. / Intérêt de l'utilisation du chlorure de benzéthonium pour le dosage turbidimétrique des protéines du liquide céphalorachidien en analyse automatique.

We have adapted Iwata and Nishikaze's benzethonium chloride turbidimetric method for the assay of CSF proteins to the techniques of continuous flow and the centrifuge analyser. The proposed technique presents several advantages: benzethonium chloride forms a stable precipitate with the proteins with a slow kinetic of formation, enabling adaptation to the centrifuge analyser; the response obtained is identical for albumin and immunoglobulin G; this very sensitive technique allows the use of micro-samples. Studies of repeatability and the correlations made with other techniques give very satisfactory results.

Effect of middle molecules on immunological functions.

The cell-mediated immunodeficiency secondary to renal failure is well established and is largely dependent on toxic or inhibitory serum factors. Our approach was to investigate the effect of so-called middle molecules (MM) on in vitro and in vivo immunological functions. A crude fraction of MM isolated from the serum or urines of uremic patients by chromatography on Sephadex G-25 fine was shown to markedly inhibit the lymphocyte proliferation induced in vitro by various phytomitogens or by allogeneic cells. A marked depression of the graft-versus-host reaction was demonstrated in vivo. When rats were continuously infused with MM, a significant delay of skin allograft rejection was obtained. From these results it is clear that the MM fraction contains a potent inhibitor of several T lymphocyte functions.