Determination of vitamin D in tears of healthy individuals by the electrochemiluminescence method.

BACKGROUND: Vitamin D is a fat-soluble steroid hormone which can be converted into various forms and is of extreme physiological importance to our body. However, its functions and local metabolic pathways in some organs, such as the eye, have not yet been well studied. We aimed to verify the correlation between vitamin D levels in blood and tear fluid and the possibility of using tear fluid as a biological material for monitoring eye disorders in the future. METHODS: The electrochemiluminescence method was used to examine blood and tear samples collected with Schirmer test strips from 21 individuals without ocular disease. RESULTS: At the 95% confidence interval, mean tear fluid vitamin D = 37.8 ± 3.6 ng/mL, which is higher than the serum level, with a mean of 30.3 ± 7.7 ng/mL; Lin's concordance correlation coefficient = -0.018 (-0.174; 0.139), Pearson's coefficient = -0.070, and the Bland-Altman coefficient = -11.12 (-30.40; 8.16). Results were obtained using the program Stata version 11.0. CONCLUSION: It is possible to determine vitamin D levels in tear fluid using the electrochemiluminescence method, and as the results do not correlate with blood, there is possibility of using tear fluid as a biological matrix for detection of vitamin D, which may increase the possibilities of new studies in eye disorders.

Fasting conditions: Influence of water intake on clinical chemistry analytes.

INTRODUCTION: Currently available recommendations regarding fasting requirements before phlebotomy do not specify any maximum water intake volume permitted during the fasting period. The aim was to study the effects of 300 mL water intake 1 h before phlebotomy on specific analytes. MATERIALS AND METHODS: Blood was collected from 20 women (median age (min-max): 24 (22 - 50) years) in basal state (T0) and 1 h after 300 mL water intake (T1). Glucose, total proteins (TP), urea, creatinine, cystatin C, total bilirubin (BT), total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides (Tg), uric acid (UA), high-sensitivity C-reactive protein, gamma-glutamyl transferase (GGT), aspartate-aminotransferase (AST), alanine-aminotransferase and lactate-dehydrogenase (LD) were studied. Results were analyzed using Wilcoxon test. Mean difference (%) was calculated for each analyte and was further compared with reference change value (RCV). Only mean differences (%) higher than RCV were considered clinically significant. RESULTS: Significant differences (median T0vs median T1, P) were observed for TP (73 vs 74 g/L, 0.001); urea (4.08 vs 4.16 mmol/L, 0.010); BT (12 vs 13 µmol/L, 0.021); total cholesterol (4.9 vs 4.9 mmol/L, 0.042); Tg (1.05 vs 1.06 mmol/L, 0.002); UA (260 vs 270 µmol/L, 0.006); GGT (12 vs 12 U/L, 0.046); AST (22 vs 24 U/L, 0.001); and LD (364 vs 386 U/L, 0.001). Although the differences observed were statistically significant, they were not indicative of clinically significant changes. CONCLUSIONS: A water intake of 300 mL 1 h prior to phlebotomy does not interfere with the analytes studied in the present work.

The coming of age of liquid chromatography coupled to tandem mass spectrometry in the endocrinology laboratory.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been rapidly incorporated in the routine of the endocrinology laboratory. Most endocrinologists are aware of the benefits afforded by this technique and tandem mass spectrometers are clearly no longer a mere research method but an important tool widely used for diagnosis. In the last 15 years, LC-MS/MS has replaced techniques such as immunoassay and HPLC for the analysis of hormones because it provides higher specificity and good sensitivity. Also, it permits simultaneous measurement of several analytes and sample preparation and acquisition are fast and simple. Although several strategies based on LC-MS/MS have been described in the last 15 years, there is still room for improvement. The impact of matrix effects and isobaric interferences have been addressed by only a few studies, and standardization with reference materials is available for a limited number of analytes. This review summarizes the application of LC-MS/MS in analyzing three classes of hormones: steroids, derivatives of the aromatic amino acids, and peptides and proteins. The benefits and current limitations of LC-MS/MS will be discussed for these hormone categories.

Analytical interference of quinolone antibiotics and quinine derived drugs on urinary protein determined by reagent strips and the pyrogallol red-molybdate protein assay.

OBJECTIVES: To investigate the analytical interference of drugs in urinary protein and to estimate the lowest interfering concentrations. DESIGN AND METHODS: Drug supplemented urine samples were compared to the control with two reagent strips and the total protein was determined using Pyrogallol Red-Molybdate (PRM). RESULTS: False-positive interferences occurred with Multistix 10 SG for hydroxychloroquine, levofloxacin and ofloxacin. No interference was observed with Combur 10 Test M. Statistically significant false-positive interferences were observed in the PRM assay with all tested drugs, and lowest interfering concentrations were mostly above estimated therapeutic concentrations. The PRM assay "confirmed" the results of the Multistix dipstick, so a real proteinuria could be presumed from the double analytical interference. CONCLUSIONS: This is the first report of analytical interference by quinolone and quinine derivatives in the PRM assay. Special attention to patients using these drugs is needed to minimize errors in the interpretation of urinary protein results.

A simple and inexpensive automated technique for measurement of serum nitrite/nitrate.

OBJECTIVE: We described an automated technique for measurement of serum nitrite/nitrate (NO(x)) using the Cobas Mira clinical chemistry analyzer. DESIGN AND METHODS: NO(x) was measured by the modified Griess method. Precision, accuracy, linearity, instrument carry-over and lower limit of quantitation (LLOQ) were assessed. RESULTS: The automated technique for measurement of serum NO(x) was linear, precise, and accurate. It has a LLOQ of 2.0 µmol/L. CONCLUSION: Serum NO(x) measured by the modified Griess method can be applied easily to the Cobas Mira clinical chemistry analyzer.

17alpha-hydroxyprogesterone and cortisol serum levels in neonates and young children: influence of age, gestational age, gender and methodological procedures.

To determine the influence of age, gestational age, gender and methodological protocol on serum 17OHP and cortisol concentrations. 17OHP in non-extracted (NE) and extracted (E) sera was measured by RIA in 319 full-term (FT) (1 d-5 yr) infants, 38 pre-term (PT) and in 19 neonates with classical CAH at diagnosis. 17OHP (NE- and E-) decreased with age in normal children. The extraction procedure significantly reduced 17OHP by eliminating interfering steroids in children < 1 year. Sexual dimorphism was only observed in NE-17OHP. 17OHP in PT was always higher than in FT up to 2 months of age (p < 0.001). Neither NE- nor E-17OHP in CAH overlapped with those of FT or PT (p < 0.001) allowing to omit the extraction procedure to confirm CAH diagnosis. Cortisol levels were within normal range in neonates with CAH, thus not adding useful information about adrenal function. Chronological and gestational age, gender, and extraction for 17OHP measurement are important factors to know when assessing adrenal function during the first year of life.

Serum IGF-I and IGFBP-3 reference values from a chemiluminescent assay in normal children and adolescents of hispanic and italian origin: presence of sexual dimorphism in IGF-I values.

Serum IGF-I and IGFBP-3 assays are used to monitor rhGH treatment. Some discrepancies in results obtained by means of different assays have been reported. The aim of this study was to establish normal ranges for circulating IGF-I and IGFBP-3 in children and adolescents of Hispanic and Italian origin. Circulating levels of IGF-I and IGFBP-3 were measured in 169 Hispanic and Italian prepubertal children and 66 adolescents of both sexes, using a chemiluminescent assay. Serum levels of IGF-I and IGFBP-3 increased from early childhood into adolescence. After pubertal peaks of IGF-I and IGFBP-3, slight decreases were observed with increasing age. Furthermore, serum IGF-I levels were significantly higher in girls than in boys, suggesting a sexual dimorphism in serum IGF-I values in late prepuberty and early puberty. Differences in IGF-I and IGFBP-3 absolute values between our study and previous studies suggest the need to establish reference ranges for each ethnic group.

Determination of alpha- and beta-amanitin in clinical urine samples by Capillary Zone Electrophoresis.

Amanitins are toxins found in species of the mushroom genera Amanita, Lepiota and Galerina. Intoxication after ingestion of these mushrooms can be fatal with an estimated 20% of mortality rate. An early diagnosis is necessary in order to avoid invasive and expensive therapy and to improve patient's prognosis. In this paper, a Capillary Zone Electrophoresis method was developed and validated to determine alpha- and beta-amanitin in urine in less than 7 min using 5 mM, pH 10 borate buffer as background electrolyte. The separation conditions were: capillary: 75 microm I.D., 41 cm effective length, 48 cm total length, 25 degrees C, 20 KV and PDA detection at 214 nm. Sample treatment for analysis only required urine dilution in background electrolyte. The method was validated following established criteria and was found to be selective, linear in the range 5-100 ng/ml. Intra- and inter-day precision and accuracy were within required limits. Limit of detection (LOD) and limit of quantification (LOQ) were 1.5 and 5 ng/ml, respectively. Eight urine samples from suspected cases of intoxication with amanitins were analyzed after 2 years of storage at -20 degrees C, and beta-amanitin was determined in two samples with concentrations of 53 and 65 ng/ml, respectively. The method here described includes the use of non-aggressive reagents to the capillary or the system and is the first Capillary Electrophoresis method used to determine amanitins in clinical samples.

Computer-assisted bar-coding system significantly reduces clinical laboratory specimen identification errors in a pediatric oncology hospital.

OBJECTIVE: To assess the ability of a bar code-based electronic positive patient and specimen identification (EPPID) system to reduce identification errors in a pediatric hospital's clinical laboratory. STUDY DESIGN: An EPPID system was implemented at a pediatric oncology hospital to reduce errors in patient and laboratory specimen identification. The EPPID system included bar-code identifiers and handheld personal digital assistants supporting real-time order verification. System efficacy was measured in 3 consecutive 12-month time frames, corresponding to periods before, during, and immediately after full EPPID implementation. RESULTS: A significant reduction in the median percentage of mislabeled specimens was observed in the 3-year study period. A decline from 0.03% to 0.005% (P < .001) was observed in the 12 months after full system implementation. On the basis of the pre-intervention detected error rate, it was estimated that EPPID prevented at least 62 mislabeling events during its first year of operation. CONCLUSIONS: EPPID decreased the rate of misidentification of clinical laboratory samples. The diminution of errors observed in this study provides support for the development of national guidelines for the use of bar coding for laboratory specimens, paralleling recent recommendations for medication administration.

Molecular identification of coccidioides isolates from Mexican patients.

Molecular studies of the genome of the fungus Coccidioides have demonstrated two nearly identical, but well-identified species, Coccidioides immitis and C. posadasii, known as "California" and "non-California" species, respectively. The objective of this study was to determine, through molecular methods, whether both species of Coccidioides are present in Mexican patients with coccidioidomycosis and to estimate, their geographical distribution in Mexico. We analyzed 56 clinical isolates of Coccidioides spp. from Mexican patients. Molecular identification of each strain was done by means of real time PCR using TaqMan(R) probes to amplify single nucleotide polymorphisms (SNPs) in four target sequences, loci, named proline 157, proline 174, hexokinase 149 and glucose-synthase 192. SNP analysis identified two of the 56 isolates as Coccidioides immitis and the remaining 54 as C. posadasii. The dual probe assay that included proline 157, proline 174 and glucose-synthase 192 gave consistent results on SNP differentiation between the two species. In contrast, the template matching hexokinase 149 gave negative results for any species in 34 samples. Our results did not show geographical overlap of the species, and they also confirmed that C. posadasii is the most frequent species in Mexico. A vast majority of C. posadasii strains were localized in the north-central region of the country.

Preservation of urine samples for metabolic evaluation of stone-forming patients.

Metabolic evaluation of stone-forming (SF) patients is based on the determination of calcium, oxalate, citrate, uric acid and other parameters in 24-h urine samples under a random diet. A reliable measurement of urinary oxalate requires the collection of urine in a receptacle containing acid preservative. However, urinary uric acid cannot be determined in the same sample under this condition. Therefore, we tested the hypothesis that the addition of preservatives (acid or alkali) after urine collection would not modify the results of those lithogenic parameters. Thirty-four healthy subjects (HS) were submitted to two non-consecutive collections of 24-h urine. The first sample was collected in a receptacle containing hydrochloric acid (HCl 6 N) and the second in a dry plastic container, with HCl being added as soon as the urine sample was received at the laboratory. Additionally, 34 HS and 34 SF patients collected a spot urine sample that was divided into four aliquots, one containing HCl, another containing sodium bicarbonate (NaHCO(3 )5 g/l), and two others in which these two preservative agents were added 24 h later. Urinary oxalate, calcium, magnesium, citrate, creatinine and uric acid were determined. Urinary parameters were also evaluated in the presence of calcium oxalate or uric acid crystals. Mean values of all urinary parameters obtained from previously acidified 24-h urine samples did not differ from those where acid was added after urine collection. The same was true for spot urine samples, with the exception of urinary citrate that presented a slight albeit significant change of 5.9% between samples in HS and 3.1% in SF. Uric acid was also not different between pre- and post-alkalinized spot urine samples. The presence of crystals did not alter these results. We concluded that post-delivery acidification or alkalinization of urine samples does not modify the measured levels of urinary oxalate, calcium, magnesium, creatinine and uric acid, and that the change on citrate was not relevant, hence allowing all parameters to be determined in a single urine sample, thus avoiding the inconvenience and cost of multiple 24-h urine sample collections.

Evaluation of tumour necrosis factor alpha, interleukin-2 soluble receptor, nitric oxide metabolites, and lipids as inflammatory markers in type 2 diabetes mellitus.

This study compared the results of tumour necrosis factor alpha (TNF-alpha), interleukin-2 soluble receptor (sIL-2R), nitric oxide metabolites (NO(x)), C-reactive protein (CRP), and lipids (total cholesterol, high-density lipoprotein (HDL-cholesterol), low-density lipoprotein (LDL-cholesterol), and triglycerides) between control group (nondiabetic subjects) and overweight type 2 DM subjects. To restrict the influence of variables that could interfere in the interpretation of data, subjects with obesity and/or acute or chronic inflammatory disease, haemoglobinopathies, recent use of antibiotics, antiinflammatory drugs, and trauma were excluded. Type 2 DM patients (n = 39; age 53.3 +/- 9.0 years; median glycated haemoglobin A(1c)< 8%) presented higher levels of TNF-alpha, triglycerides (P < .01), NO(x) and sIL-2R (P < .05) than control group (n = 28; age 39.7 +/- 14.1 years). CRP, LDL-cholesterol, total cholesterol, and HDL-cholesterol did not differ among groups. Diabetic women (n = 21) had higher levels of TNF-alpha, total cholesterol, LDL-cholesterol, and HDL-cholesterol than diabetic men (n = 18) (P < .05), but there were no differences among sexes in the control group. This study indicates that increased level of proinflammatory markers occurs in type 2 DM even in the absence of obesity and marked hyperglycaemia, confirming that the inflammation course of the atherosclerotic process is more severe in diabetic patients than in nondiabetic subjects.

Development of restricted-access media supports and their application to the direct analysis of biological fluid samples via high-performance liquid chromatography.

A quick overview of published methods for analyzing compounds in complex biological samples reveals that the most difficult step is the clean-up or extraction of a required compound from the matrix. The strategy required to analyze exogenous compounds in biological fluids depends greatly upon the nature of the compound and upon the biomatrix. Coupled-column separation using restricted-access media as the first dimension in order to exclude macromolecules and retain micromolecules has been successfully used for a number of biological fluids. This paper presents the history of the development of restricted-access media supports and of their application to the direct injection of biological fluid samples in high-performance liquid chromatography.

El rol del bioquímico clínico en los comentarios interpretativos: Criterios para su desarrollo / The role of the clinical biochemist in the interpretative commentaries: Criteria for its development

El establecimiento de la Mejora Contínua de la Calidad en los servicios de los laboratorios, ha llevado a la fijación de estándares de trabajo, monitoreo de indicadores de gestión y a la creación de ambientes donde se trata de optimizar permanentemente la atención a los pacientes. La clarificación de la posible significación de los resultados y reportes y su adecuada comunicación es una parte de la etapa post-analítica cada vez más importante dentro de la tarea de los bioquímicos. Consideramos que los informes interpretativos deben ser emitidos en todos los casos por el profesional que valida el informe, convenientemente entrenado para este tipo de comunicaciones y con el conocimiento y supervisión de sus superiores. Para ser útiles, los comentarios interpretativos deben ser certeros, sucintos y estar adaptados a los conocimientos y experiencia del receptor a fin de proporcionar la mayor información clínicamente útil. Excepto en los casos en los que es posible un contacto directo con el médico para discutir los resultados o cuando se conoce la evolución de un caso, no existe oportunidad para el bioquímico de aprender por medio del feedback y así aumentar sus habilidades interpretativas. Si somos capaces de producir resultados útiles, de alta calidad analítica y de poder interpretarlos, debemos procurar que la comunicación con los demás integrantes del Equipo de Salud sea fluída para la mejor utilización de todo lo que el laboratorio puede ofrecer. Es parte de nuestra tarea la interpretación de los datos desde la visión bioquímica como aporte valioso para el desempeño de la labor médica y la armoniosa integración del Equipo de Salud.

El rol del bioquímico clínico en los comentarios interpretativos: Criterios para su desarrollo / The role of the clinical biochemist in the interpretative commentaries: Criteria for its development

El establecimiento de la Mejora Contínua de la Calidad en los servicios de los laboratorios, ha llevado a la fijación de estándares de trabajo, monitoreo de indicadores de gestión y a la creación de ambientes donde se trata de optimizar permanentemente la atención a los pacientes. La clarificación de la posible significación de los resultados y reportes y su adecuada comunicación es una parte de la etapa post-analítica cada vez más importante dentro de la tarea de los bioquímicos. Consideramos que los informes interpretativos deben ser emitidos en todos los casos por el profesional que valida el informe, convenientemente entrenado para este tipo de comunicaciones y con el conocimiento y supervisión de sus superiores. Para ser útiles, los comentarios interpretativos deben ser certeros, sucintos y estar adaptados a los conocimientos y experiencia del receptor a fin de proporcionar la mayor información clínicamente útil. Excepto en los casos en los que es posible un contacto directo con el médico para discutir los resultados o cuando se conoce la evolución de un caso, no existe oportunidad para el bioquímico de aprender por medio del feedback y así aumentar sus habilidades interpretativas. Si somos capaces de producir resultados útiles, de alta calidad analítica y de poder interpretarlos, debemos procurar que la comunicación con los demás integrantes del Equipo de Salud sea fluída para la mejor utilización de todo lo que el laboratorio puede ofrecer. Es parte de nuestra tarea la interpretación de los datos desde la visión bioquímica como aporte valioso para el desempeño de la labor médica y la armoniosa integración del Equipo de Salud. (AU)

Purification and characterization of human hemoglobin: effect of the hemolysis conditions.

Human hemoglobin was isolated and purified by anion exchange chromatography. To isolate hemoglobin, outdated red blood cells (RBC) were transformed into carbonylhemoglobin, by reaction with carbon monoxide, and submitted to washing/centrifugation procedures, to eliminate other plasma proteins. Albumin was quantified in each supernatant, by the bromcresol green method. Hemolysis was performed in three different hypotonic media (water, 0.01 M NaCl and 5 mM Tris/HCl buffer at pH 7.4), at 8 degrees C for 24 h. Sonication for 5 min was also used to lyse RBC. After isolation of hemoglobin, additional purification was carried out by anion exchange chromatography on AG MP-1, Q-SFF and both exchangers. Hemoglobin concentration of hemolysates and of purified solutions were determined by the hemiglobincyanide method. Residual phospholipids were extracted from the four different hemolysates, as well as from the purified hemoglobin solutions, and were analyzed by high performance liquid chromatography. Native and SDS-polyacrylamide gel electrophoresis experiments were performed on purified hemoglobin samples to verify the presence of proteins other than hemoglobin. According to the results, the hemolysis conditions have influence on the purification of hemoglobin.