[Inappropriately undetectable thyroglobulin after exclusion of interference from thyroglobulin antibodies]. / Discordances entre différents immunodosages de la thyroglobuline en l'absence d'anticorps anti-thyroglobuline.

Thyroglobuline (Tg) is a large molecule of high molecular weight mainly indicated in monitoring differentiated thyroid cancer (DTC), its measurement remains difficult. We report the case of a patient who underwent total thyroidectomy for a poorly differentiated thyroid insular carcinoma. Despite several (131)I treatments, a progressive elevation of serum Tg is observed. A control performed in another laboratory using an immunoradiometric assay (IRMA Cisbio) returns an undetectable value (< 0,2 mg/L). A new sample was simultaneously sent to different laboratories. Three nonisotopic immunometric assays showed high values of Tg while the IRMA assay, considered the gold standard, gave a result below the detection threshold. The absence of Tg antibodies and of anti-mouse antibodies was confirmed. The IRMA Kit manufacturer agreed to carry out an expertise. After changing their detection antibody, the presence of a high Tg was demonstrated, in agreement with non-isotopic techniques. The expertise conclusion was a lack of detection by the IRMA Tg assay. This incident was notified to AFSSAPS by the manufacturer.

Lectin affinity electrophoresis of serum alkaline phosphatase in metastasized breast cancer.

The use of serum alkaline phosphatase (ALP) isoenzymes as markers of breast cancer metastases and treatment efficacy has received little attention. Twenty-six breast cancer women (56+/-13 years, all post-menopausal) were prospectively evaluated during their first and third course of chemotherapy (4-week interval). Serum samples were analyzed for ALP isoenzymes (bone, liver, and intestine) using a lectin affinity electrophoresis kit (Hydragel 15 ISO-PAL, Sebia) adapted on a semi-automated Hydrasys system (Sebia). Results were compared with imaging techniques for the presence of metastases; bone ALP isoenzyme (B-ALP) results were compared with C-Terminal degradation products of type I collagen (S-CTX) (CrossLaps, IDS Nordic). Serum B-ALP, but not S-CTX, confirmed the presence of bone metastases (BM) (n=15) with 67/100% sensitivity/specificity (using a 69 UI/L ROC cut-off); ROC AUC was 0.806 (P=0.0004) (NS for S-CTX). Chemotherapy reduced serum B-ALP by 24% over 4 weeks (P=0.0012); there was no change for S-CTX. There was no specific clinical pattern for other ALP isoenzymes (liver and intestine). In conclusion, serum B-ALP, but not S-CTX, could help confirm the presence of BM in breast cancer patients.

Clinical value of combined determination of plasma L-DOPA/tyrosine ratio, S100B, MIA and LDH in melanoma.

AIM OF THE STUDY: L-DOPA/tyrosine ratio (an index of tyrosinase activity), melanoma antigens S100B and MIA, lactate deshydrogenase (LDH) and their combinations were evaluated for clinical value as tumour markers in melanoma. METHODS: Blood samples were obtained in 170 melanoma patients (stage I-II: n=57, III: n=54, IV: n=59) at inclusion and in a sub-group of 82 subjects during follow-up for up to 4 years. Laboratory analyses were performed by HPLC (L-DOPA, L-tyrosine), immunoassays (S100B, MIA) and colourimetry (LDH). RESULTS: All markers, except LDH, were elevated in stage IV versus other stages. S100B and MIA highly correlated, especially in stage IV (r(s): 0.849, p<0.001). The combination of L-DOPA/tyrosine ratio with S100B displayed the highest sensitivity/specificity (73/70%) to confirm stage III-IV or stage IV alone (69/75%) (ROC optimised cut-off). Only the L-DOPA/tyrosine ratio significantly increased (+36% over 5 months, p=0.001) during progression from stage I-III to higher stages. S100B, MIA and LDH, but not the L-DOPA/tyrosine ratio, responded to progression towards death in stage IV. All markers exhibited a prognostic value in deceased patients (n=44); S100B and MIA were the best predictors of survival time by Cox proportional-hazards regression. CONCLUSION: The combination of plasma L-DOPA/tyrosine ratio and S100B appears an attractive approach for the biological follow-up of melanoma patients.

Simultaneous determination of monodesethylchloroquine, chloroquine, cycloguanil and proguanil on dried blood spots by reverse-phase liquid chromatography.

A method for simultaneous analysis of chloroquine, proguanil and their metabolites from a whole blood sample (80 microL) dried on a filter paper was developed. Sample preparation included a liquid extraction from the filter paper, followed by a solid-phase extraction (C18 Bond Elut cartridge). Separation was obtained by reverse-phase liquid chromatography (HPLC) using a gradient elution on an X-Terra column; UV detection was made at 254 nm. This assay was linear between 150 and 2500 ng mL(-1) for chloroquine (and metabolite) and 300 and 2500 ng mL(-1) for proguanil and cycloguanil. The lower limit of quantification was close to 50 ng mL(-1) for chloroquine (and its metabolite) and 100 ng mL(-1) for proguanil (and its metabolite). No chromatographic interference from endogenous compounds or other tested anti-malarial drugs was evidenced. Chromatographic separation takes about 40 min with a coefficient of variation below 10.3% for within- and between-batch precision. The paper sampling method was validated in 10 healthy subjects treated by Savarine. The stability of compounds and metabolites on the filter paper was evaluated at four temperatures (-20, +4, 20 and 50 degrees C) and for 1, 5 and 20 days. Cycloguanil concentrations were not influenced by storage conditions, whereas, high temperatures and prolonged storage decreased chloroquine and proguanil levels. The proposed HPLC assay is accurate, precise and cost-effective; it can be used for pharmacokinetic and epidemiological studies on anti-malarial treatments.

Quantification of pralidoxime methylsulfate (Contrathion) in human urine by capillary zone electrophoresis.

Pralidoxime methylsulfate (Contrathion) is widely used to treat organophosphate poisoning. For the first time, we developed a specific assay for urinary pralidoxime using capillary zone electrophoresis (CZE) in the following conditions: fused-silica capillary (length: 47 cm, internal diameter: 75 microm), electrolyte solution: 25 mM sodium borate (pH 9.1), voltage: 15 kV, temperature: 25 degrees C, injection time: 1 or 2s, on-line UV detection: 280 nm. Sample preparation did not require a deproteinization step (1:5 dilution in water). The method was linear between 0.125 and 2 mg mL-1 of pralidoxime (quantification limit: 0.10 mg mL-1). Coefficients of variation for intra- and inter-assay precision were below 10% for all three control levels (0.15-1.15 mg mL-1). This assay was successfully applied to urine specimens from organophosphate poisoned patients treated by Contrathion (n=10). This CZE method allows the measure of pralidoxime in urine within 15 min with excellent precision, selectivity, and sensitivity. It is simple (no pretreatment) and convenient, thus suitable for the monitoring of Contrathion therapy in organophosphate poisoned patients.

Evaluation of renal function in intensive care: plasma cystatin C vs. creatinine and derived glomerular filtration rate estimates.

Plasma cystatin C, a new marker of glomerular filtration rate (GFR), was prospectively evaluated in surgical intensive care. Cystatin C was measured (immunonephelometry, Dade-Behring) in 10 patients selected to cover a full range of GFR (phase I) and in 28 unselected consecutive patients followed for 5 days post-admission (phase II). Results were compared with (51)Cr-EDTA clearance (phase I only), plasma creatinine (kinetic Jaffe, Roche), 24-h or estimated by Cockcroft and Gault (CG) creatinine clearance (CrCl), and modified diet in renal disease (MDRD)-estimated GFR. In phase I, the highest correlation with(51)Cr-EDTA clearance (22-198 mL/min) was noted for CG CrCl (r(2): 0.883, p<0.001). During phase II follow-up, 24-h CrCl could not be calculated in 25% of daily evaluations. Cystatin C correlated with creatinine (0.856, p<0.0001) and CG CrCl with MDRD GFR (0.926, p<0.0001) in renal failure (10-78 mL/min, n=60). There was a +40% (p<0.001) median difference between cystatin C and creatinine (as a % of upper normal cut-off). Sensitivity/specificity to detect a <80 mL/min CG CrCl was 88/97% for cystatin C vs. 48/100% for creatinine (laboratory cut-off). In patients with normal and stable renal function (n=14), day-to-day intra-individual variation was 7.4% for cystatin C (vs. 10.6% for creatinine). In intensive care unit surgical adult patients, CG CrCl provides an easy and cost-effective estimate of GFR. Superior to creatinine, plasma cystatin C can be measured in selected patients where CG CrCl is known to be inaccurate.

Cystatin C as a marker of GFR--history, indications, and future research.

OBJECTIVE: To summarize recent knowledge on the small molecular weight protein cystatin C (cys-C) and its use as a marker of the glomerular filtration rate (GFR). METHODS: A multinational expert meeting was held in April 2002 in Marburg, Germany. Contributors summarized their main findings. CONCLUSIONS: Cys-C is at least equal if not superior to serum creatinine as a marker of GFR. The independence from height, gender, age, and muscle mass is advantageous. Select patient groups such as children, the elderly, and patients with reduced muscle mass benefit in particular.

Evaluation of the serum L-dopa/L-tyrosine ratio as a melanoma marker.

A wide range of molecules have been investigated as tumour markers in melanoma, most of which are not suitable for use by clinical oncologists for the detection of fast and unpredictable metastatic dissemination. We have already shown that the serum L-dopa/L-tyrosine ratio (an index of tyrosinase functional activity) correlates with the tumour burden and in some cases predicted disease progression in metastatic melanoma patients. We examined the potential value of this ratio for the follow-up, therapy monitoring and prognosis in melanoma compared with a reference marker (S100B, a melanoma-associated antigen). Sixty melanoma patients (24 stage I-II, 18 stage III, 18 stage IV, American Joint Committee on Cancer staging) were entered into the study, sampled two to eight times (before and after therapy) and were followed for up to 30 months. Serum L-dopa and L-tyrosine were determined by high performance liquid chromatography and S100B by an immunoluminometric assay. In stage III patients with elevated marker concentration, lymph node dissection decreased the S100B level (from 0.27 to < 0.13 microg/l, P=0.008), but not the L-dopa/L-tyrosine ratio. Chemotherapy decreased the L-dopa/L-tyrosine ratio by 38% (P =0.04) and the S100B level by 45% (P = 0.02) in stage IV responders. During follow-up, patients with marker levels within normal limits (n=19) had stable disease, except for two stage II patients. In patients with progressive disease (n=20), an increase in one or both markers was observed. Stage IV patients with high L-Dopa/L-Tyrosine ratio (above 20 x 10-5) at inclusion had shorter survival (3 months), while patients with low levels had longer survival (15 months). Levels of S100B had no impact on survival, as all stage IV patients (with levels below or above 0.38 microg/l) had the same survival (5 months). The serum L-dopa/L-tyrosine ratio may be influenced by successful therapy and levels at inclusion may correlate with prognosis in stage IV patients. Levels of these two markers in other biological fluids such as cerebrospinal fluid and tumour exudates may be useful diagnostically and prognostically in difficult cases.

Automated multicapillary electrophoresis for analysis of human serum proteins.

BACKGROUND: We evaluated a new, automated multicapillary zone electrophoresis (CE) instrument (Capillarys), 4.51 software version; Sebia) for human serum protein analysis. METHODS: With the Capillarys beta1-beta2+ reagent set, proteins were separated at 7 kV for 4 min in 15.5 cm x 25 micro m fused-silica capillaries (n = 8) at 35.5 degrees C in a pH 10 buffer with online detection at 200 nm. Serum samples with different electrophoretic patterns (n = 265) or potential interference (n = 69) were analyzed and compared with agarose gel electrophoresis (AGE; Hydrasys)-Hyrys, Hydragel protein(e) 15/30 reagent set; Sebia). RESULTS: CVs were <3.5% for albumin, <11% for alpha(1)-globulin, <4.1% for alpha(2)-globulin, <7.4% for beta-globulin, and <5.8% for gamma-globulin (3 control levels); measured throughput was 60 samples/h. In patients without paraprotein (n = 116), the median differences between CE and AGE were -5.4 g/L for albumin, 4.0 g/L for alpha(1)-globulin, 0.7 g/L for alpha(2)-globulin, 0.6 g/L for beta-globulin (P <0.001 for all fractions), and -0.1 g/L for gamma-globulin (not significant). More samples had at least one gamma-migrating peak detected by CE (n = 135 vs 130; paraprotein detection limit, approximately 0.5-0.7 g/L), but fewer were quantified (n = 84 vs 91) because of gamma- to beta-migration shifts. There was a 1.2 g/L median difference between CE and AGE for gamma-migrating paraprotein quantification (n = 69; P <0.001). Several ultraviolet-absorbing substances (lipid emulsion, hemoglobin) or molecules (contrast agent, gelatin-based plasma substitute) induced CE artifacts. CONCLUSIONS: The Capillarys instrument is a reliable CE system for serum protein analysis, combining advantages of full automation (ease of use, bar-code identification, computer-assisted correction of alpha(1)-globulins) with high analytical performances and throughput.

Urinary free light chain analysis by the Freelite immunoassay: a preliminary study in multiple myeloma.

Evaluate the Freelite free light chain immunoassay for urine analysis in myeloma. Urine specimens from 20 patients were analyzed by Freelite (The Binding Site) and SDS-agarose gel electrophoresis (Hydragel protéinurie, Sebia). Using the kappa/lambda ratio, Freelite was more sensitive than electrophoresis to detect free light chains, but concentration was overestimated in 75% of cases. Despite high sensitivity and full automation, Freelite inaccurately measures monoclonal free light chains in urine.

Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis.

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.

Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.