Evaluation of an automated capillary electrophoresis system in the screening for hemoglobinopathies.

BACKGROUND: The biochemical diagnosis of hemoglobinopathies (thalassemias and hemoglobin (Hb) variants) is based on the separation and the quantification of Hb fractions. HPLC is the most commonly used method but capillary electrophoresis (CE) methods have also been developed successfully. The Capillarys II system is the first fully automated CE system that allows the quantification of Hb A2 and Hb F and the separation of Hb variants. We evaluated the ability of this system to separate and identify Hb variants and to quantify Hb A2 and Hb F. MATERIAL AND METHODS: The separation of 18 different Hb variants was studied and the imprecision on migration times was calculated for the three most frequent ones. The total imprecision on Hb A2 and Hb F quantification was determined. The results obtained for 44 patients were compared with those given by HPLC. The interference on Hb A2 measurement due to the presence of Hb S was studied. RESULTS: Fourteen out of the 18 variants tested, including all variants of clinical importance, were separated from Hb A. Imprecision on migration times was less than 1%. For Hb A2 quantification, imprecision was less than 3.5% and for Hb F, less than 7.0%. The comparison with HPLC showed an acceptable agreement between both methods but a systematic negative bias for Hb A2 and both proportional and systematic biases for Hb F. No interference from the presence of Hb S on the quantification of Hb A2 was observed. CONCLUSIONS: The fully automated Capillarys Hemoglobin method allows the detection and the separation of the most common Hb variants. It provides also a precise, quick, and very easy quantification of Hb F and Hb A2, even in the presence of Hb S. It is very suitable for routine investigation of hemoglopinopathies.

The determination of the levels of circulating galectin-1 and -3 in HNSCC patients could be used to monitor tumor progression and/or responses to therapy.

To evaluate galectin-1, -3 and -7 serum levels as diagnostic and/or prognostic markers for head and neck squamous cell carcinomas (HNSCCs). ELISA was employed to test sera from 102 patients with HNSCCs and from 38 healthy control volunteers for galectin-1, -3 and -7 serum levels. Serum galectin levels were assayed by ELISA and the levels of galectin expression in HNSCCs were determined by means of immunohistochemistry. HNSCCs display significant immunohistochemical amounts of galectin-7, but this galectin cannot be detected in the blood of HNSCC patients. Galectin-3 levels differ significantly (p=0.03) in healthy volunteers and HNSCC patients. Using a threshold value of 4.3 ng/ml, galectin-3 serum level enabled a significant level of discrimination (p=0.03) to be established between the cancer patients and the healthy volunteers, with 90% level of specificity and 36% level of sensitivity. The discrimination was even better when using a threshold value of 13.5 ng/ml for galectin-1 (p=0.001), with 100% level of specificity and 22% level of sensitivity. A subgroup of stage IV HNSCC patients displayed significantly reduced levels of circulating galectin-1 (p=0.003) and galectin-3 (p=0.001) after treatment as opposed to before. Galectin-3 concentrations in sera from the patients with a metastatic disease were significantly (p=0.01) higher than in sera from the patients with localized tumors. The determination of circulating levels of galectin-1 and -3 could be used to monitor the progression of their disease or their response to therapy.

Evaluation of oral corticosteroids and phosphodiesterase-4 inhibitor on the acute inflammation induced by inhaled lipopolysaccharide in human.

BACKGROUND: Endotoxins are pro-inflammatory substances present in the environment. In man, inhalation of its purified derivative lipopolysaccharide (LPS) induces inflammation related to macrophages and neutrophils. Corticosteroids and phosphodiesterase (PDE)-4 inhibitors have inhibiting effects on macrophages and neutrophils, respectively. This study investigated the effect of prednisolone and of the PDE-4 inhibitor cilomilast on the LPS-induced acute inflammation. METHODS: The study was a placebo-controlled, double-blind crossover design. On three occasions, at 2 weeks interval, 16 healthy subjects inhaled 50 microg LPS after a 6-day treatment with cilomilast (15 mg bd), prednisolone (10 mg bd) or placebo. For the assessment of the inflammatory response, induced sputum was obtained before inclusion and 6h post-LPS while blood samples were collected before, 6 and 24 h post-LPS. RESULTS: Inhaled LPS induced an increase in sputum neutrophils (p<0.0001), logMMP-9 (p<0.05), logMMP-9/TIMP-1 (p<0.01) and logTNF-alpha (p<0.02). At the blood level there were significant rise in neutrophilia (p<0.001), E-selectin (p<0.02), C-reactive protein (CRP) (p<0.001) and LPS-binding protein (p<0.001). There was both a slight, but not significant, increase in body temperature and decrease in forced expiratory volume in 1 s (FEV(1)). Neither prednisolone nor cilomilast had protective effect on the LPS-induced airways' inflammation. The LPS-induced CRP acute-phase protein of inflammation (0.58+/-0.13 and 3.52+/-0.41 mg/dL, before and after LPS, respectively) was significantly inhibited by a pre-treatment with prednisolone (1.39+/-0.32 mg/dL, p<0.01) and attenuated (2.65+/-0.30 mg/dL, p=0.09) with cilomilast. CONCLUSION: In healthy subjects, while the LPS-induced airways' inflammation was not modified either by oral prednisolone or by PDE-4 inhibitor cilomilast (at actual dosage), the LPS-induced acute phase of blood inflammation was reduced by prednisolone.

The place of capillary electrophoresis techniques in screening for haemoglobinopathies.

BACKGROUND: Capillary zone electrophoresis (CZE) methods are new laboratory diagnostic tools. The screening of haemoglobin (Hb) variants by a battery of two automated CZE methods at alkaline and acid pH, followed by micellar electrokinetic capillary chromatography (MECC), has been evaluated. METHODS: Three hundred and ninety-two patients' samples with an abnormal haemoglobin fraction, detected either by isoeletric focusing (IEF) or by automated cation-exchange exchange high-performance liquid chromatography (automated HPLC), were tested by both CZE methods. Their performances were compared with IEF and automated HPLC techniques. The place of MECC has been evaluated. RESULTS: Using both CZE methods, the clinically relevant variants (HbS, HbC, etc.) as well as 15 of 20 clinically silent variants tested were separated from HbA. Using the alkaline CZE method, a presumptive identification of Hb Bart's, HbH and Hb Constant Spring was obtained. Complementary testing by MECC has demonstrated it to be an aid in distinguishing the globin chain mutations before confirmation by DNA or protein structure analysis. CONCLUSION: Using only one device, alkaline CZE, acid CZE and MECC methods offer high resolution, automated sampling and rapid analysis. They are good tools for screening of haemoglobinopathies and can replace conventional techniques.