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.

EC4 European Syllabus for Post-Graduate Training in Clinical Chemistry and Laboratory Medicine: version 3 - 2005.

The EC4 Syllabus for Postgraduate Training is the basis for the European Register of Specialists in Clinical Chemistry and Laboratory Medicine. The syllabus: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. The syllabus is not primarily meant to be a training guide, but on the basis of the overview given (common minimal programme), national societies should formulate programmes that indicate where knowledge and experience is needed. The main points of this programme are: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. Knowledge in biochemistry, haematology, immunology, etc.; Pre-analytical conditions; Evaluation of results; Interpretations (post-analytical phase); Laboratory management; and Quality insurance management. The aim of this version of the syllabus is to be in accordance with the Directive of Professional Qualifications published on 30 September 2005. To prepare the common platforms planned in this directive, the disciplines are divided into four categories: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. Knowledge in biochemistry, haematology, immunology, etc.; Pre-analytical conditions; Evaluation of results; Interpretations (post-analytical phase); Laboratory management; and Quality insurance management. General chemistry, encompassing biochemistry, endocrinology, chemical (humoral), immunology, toxicology, and therapeutic drug monitoring; Haematology, covering cells, transfusion serology, coagulation, and cellular immunology; Microbiology, involving bacteriology, virology, parasitology, and mycology; Genetics and IVF.

Measurement of serum pralidoxime methylsulfate (Contrathion) by high-performance liquid chromatography with electrochemical detection.

Pralidoxime methylsulfate (Contrathion) is widely used to treat organophosphate poisoning. Despite animal and human studies, the usefulness of Contrathion therapy remains a matter of debate. Therapeutic dosage regimens need to be clarified and availability of a reliable method for plasma pralidoxime quantification would be helpful in this process. We here describe a high-performance liquid chromatography technique with electrochemical detection to measure pralidoxime concentrations in human serum using guanosine as an internal standard. The assay was linear between 0.25 and 50 microg mL(-1) with a quantification limit of 0.2 microg mL(-1). The analytical precision was satisfactory, with variation coefficients lower 10%. This assay was applied to the analysis of a serum from an organophosphorate poisoned patient and treated by Contrathion infusions (100 and 200 mg h(-1)) after a loading dose (400 mg).

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.