Validación de una metodología analítica para determinar bifenilos policlorados en muestras de plasma sanguíneo / Validation of an analytical methodology to determine polychlorinated biphenyls in samples from blood plasma

Resumen Introducción. Los bifenilos policlorados se encuentran entre los cinco contaminantes orgánicos persistentes más tóxicos para los organismos vivos, según la Agency for Toxic Substances and Disease Registry (ATSDR) de los Estados Unidos. Objetivo. Estandarizar y validar un método analítico para la determinación y cuantificación de los bifenilos policlorados indicadores en muestras de plasma sanguíneo, mediante cromatografía de gases acoplada a espectrometría de masas. Materiales y métodos. Se fortificó un pool de plasma para hacer los ensayos en la matriz. Además, se utilizó el material de referencia NIST SRM ® 1958 (Organic Contaminants in Fortified Human Serum, Freeze-Dried) para los ensayos de veracidad y precisión intermedia. Resultados. Los porcentajes de recuperación obtenidos con la metodología estuvieron entre 88,4 y 97,5 %, y el sesgo fue menor del 20 %. Los límites de detección y cuantificación de los bifenilos policlorados indicadores policlorados fueron de 0,04 µg/L y 0,10 µg/L, respectivamente. La linealidad representada por el coeficiente de determinación (R2) varió entre 0,9866 y 0,9886. La precisión expresada como desviación estándar relativa fue menor del 20 % en todo el rango lineal de trabajo (0,5-500 µg/L). Por último, se analizaron 115 muestras de población colombiana de diferentes zonas del país y se encontraron 65 muestras positivas, de las cuales dos estuvieron por encima de los valores de control biológico en humanos (Human Biomonitoring Values, HBM- II): 7,0 µg/L, 2XΣPCB 138, 153, 180 , y otras dos, por encima del HBM-I: 3,5 µg/L, 2XΣPCB 138, 153, 180. Conclusión. El método desarrollado resultó ser preciso para el análisis de los bifenilos policlorados en muestras de plasma sanguíneo y se puede utilizar para el control biológico de estos contaminantes en población colombiana.

Abstract Introduction: Polychlorinated biphenyls are among the five most toxic persistent contaminants for living organisms according to the Agency for Toxic Substances and Disease Registry (ATSDR). Objective: To standardize and validate an analytical method to determine and quantify polychlorinated biphenyl indicators in samples from blood plasma by means of gas chromatography-mass spectrometry. Materials and methods: We fortified a plasma pool to do the matrix assays. Additionally, we used the NIST SRM® 1958 reference material for the veracity and intermediate accuracy assays. Results: Methodology recovery percentages ranged between 88.4 and 97.5%, and the bias was less than 20%. Detection and quantification limits were 0.04 µg/L and 0.10 µg/L, respectively, for all polychlorinated biphenyl indicators. The linearity represented by the determination coefficient (R2 ) varied between 0.9866 and 0.9886. Accuracy, expressed as relative standard deviation was less than 20% in all the linear work range (0.5-500 µg/L). Finally, we analyzed 115 samples from Colombian population in various zones of the country and we found 65 positive samples, from which two samples were above HBM-II (7.0 µg/L, 2XΣPCB 138, 153, 180), and two, above HBM-I (3.5 µg/L, 2XΣPCB 138, 153, 180 ). Conclusion: The method we developed is accurate for PCB analysis in blood plasma samples and could be used for biological surveillance of these contaminants in the Colombian population.

Urinary Stone Composition Analysis Using Dual Source CT Dual Energy Imaging / 中国医学影像学杂志

PurposeTo analyze the urinary stone chemical composition using dual source CT (DSCT) dual energy imaging.Materials and MethodsNinety-seven stone samples with known chemical composition were placed in fresh swine kidneys and examined with DSCT dual energy scan, including 55 calcium oxalate stones, 13 hydroxyapatite stones, 9 uric acid stones, 4 cystine stones, 2 struvite stones and 14 mixed stones. The mean attenuation values and dual energy index (DEI) were measured at 80 kV and 140 kV. The stone composition was analyzed to determine the sensitivity, specificity and accuracy.ResultsDual energy analysis showed 59 calcium oxalate stones, 11 hydroxyapatite stones, 9 uric acid stones, 4 cystine stones and 14 mixed stones. 2 hydroxyapatite stones were erroneously marked as calcium oxalate stones and 2 struvite stones as calcium oxalate stones. The accuracy was 95.88% (93/97). The difference CT value at 80 kV and 140 kV and DEI value were statistically significant (P<0.05). The sensitivity for detecting calcium oxalate stones, hydroxyapatite stones, uric acid stones and cysteine stones were 100.00%, 84.60%, 100.00% and 100.00%, respectively; with specificity of 85.70%, 100.00%, 100.00% and 100.00%, respectively; and the accuracy were 95.18%, 97.59%, 100.00% and 100.00%, respectively.ConclusionDSCT dual energy imaging accurately analyzes the chemical composition of urinary stone in vitro, which provides important clinical value in analyzing the urinary stonesin vivo.

Development and clinical application of microscopy review criteria of automated urine chemistry and sedimental analysis / 中华检验医学杂志

Objective To integrate urine strip chemistry analysis with urine sedimental analysis and set up the criteria for urine microscopy review following automated urine analysis.Methods A total of 1 714 urine samples were collected from Peking Union Medical College Hospital from November 2008 to October 2010.Out of 1 714 samples, 1 300 samples were used for the establishment of review criteria, and 214 samples were used for criteria verification.The other 200 samples from healthy donors were used to set up the normal reference range of fully automated urine sedimental analyzer UF-1000i.RBC,WBC,PRO and CAST in all the samples were measured by Siemens Bayer Clinitek 500 urine strip chemistry analyzer, Sysmex UF-1000i urine sedimental analyzer and microscopic examination.Based on the different laboratory automation in urine analysis, four microscopic review protocols were defined: (1) Protocol 1: based on chemistry results only, microscopy review was performed when any of WBC, RBC and PRO was positive; (2) Protocol 2: based on fully automated sedimental analyzer only,microscopy review was performed when any of WBC, RBC and CAST was over the upper limit of the reference range; (3) Protocol 3: All the results of urine chemistry analyzer and sedimental analyzer were integrated.If two WBC results were different between two systems (in one system WBC was positive or over the upper limit of the reference range but in another system WBC was negative or within the reference range), and any of RBC, PRO/CAST was positive or over the upper limit, microscopic review was performed; (4) Protocol 4: if any of WBC, RBC, PRO/CAST was different between two systems, microscopic review was performed.Review criteria were performed with Sysmex Laboman UriAccess 3.0 software.Results The reference ranges of UF-1000i parameters were RBC 0-7.5/μl (male), 0-15.9/μl (female); WBC 0-11.6/μl (male), 0-12.7/μl (female); Epithelial cell were 0-6.5/μl (male), 0-21.4/μl (female); CAST 0-1.3/μl.The results of microscopic examination revealed that positive samples were 47.46% (617/1 300) and negative samples were 52.54% (683/1 300). Among positive samples, majority showed the presence of RBC (60.13%,371/617), followed by CAST (8.43%,52/617).The false negative rates of four protocols were 8.38% (109/1 300), 4.69% (61/1 300), 0.62% (8/1 300) and 0.54% (7/1 300), respectively.The review rates were 47.85% (622/1 300), 59.38% (772/1 300), 72.85% (947/1 300) and 52.23% (679/1 300), respectively.Although there were false negative cases in protocol 4, all the patients had normal serum creatine level.In those 214 patients for verification, the false negative rate using protocol 4 was zero, the review rates were 53.74% (115/214).Conclusions Protocol 4 shows lest false negative rate and lower review rate.Importantly, there was no patients with serious renal function abnormality missed using protocol 4.Therefore, protocol 4 is an ideal criteria for microscopy review following automated urine analysis.

Study and application of the combination of urine dry chemistry examination and urine sediment flow cytometry examination on the microscopic screening of the urine formed component / 中华检验医学杂志

Objective To establish the proper review rules for the microscopic screening of urine analyzed by UF-1000i automatic urinalysis work station (composed of UF-1000i urine flow cytometer and AX-4030 urine dry chemical analyzer).Methods A total of 2 839 random urine samples were collected at Chinese People′s Liberation Army General Hospital from September 2009 to February 2010, and were analyzed using UF-1000i urinalysis work station.The parameters obtained from UF-1000i and AX-4030 included RBC, WBC, CAST and ERY, LEU, PRO.After analysis by urinalysis work station, each urine sample was examined microscopically by two technologists using double-blind method.The average results got from the two technologists were regarded as the judging criterion.Based on the criterion, the review rules for the 2 839 urine samples tested by urinalysis work station were created and adjusted, and the true positive rate, false positive rate, true negative rate, false negative rate and review rate of these review rules were calculated.After that, 299 randomly selected urine samples were tested to validate these review rules.Omission diagnostic rate and review rate were used to assess the clinical practicability of the review rules.Results Thirty seven rules for microscopic review and twenty seven rules without further microscopic examination were set up based on six parameters using UriAccess 3.0 Software.The microscopic examination result was taken as the judging criterion, the consistency rate of these rules was 81.11%(2 311/2 839), the true positive rate was 40.51%(1 150/2 839), the false positive rate was 16.17%(459/2 839), the true negative rate was 41.00%(1 164/2 839), the false negative rate(omission diagnostic rate) was 2.43%(69/2 839) and the review rate was 18.28% (519/2 839).Additional 299 urine samples were assayed using UriAccess3.0 software to further verify these review rules.The consistency rate was 82.27%(246/299), the true positive rate was 36.12%(108/299), the false positive rate was 16.39%(49/299), the true negative rate was 46.15%(138/299), the false negative rate(omission diagnostic rate) was 1.34%(4/299), the review rate was 19.06%(57/299). The 4 false negative samples selected by these review rules did not come from the nephropathy department or the urology department.Microscopic results of RBC and WBC form these 4 samples ranged 3-8 cells/HP. Thus, these review rules could avoid the missed diagnosis of those patients with severe renal dysfunction.Conclusion The review rules established from this study for the UF-1000i urinalysis work station can effectively detect abnormal urine samples and improve the efficiency and the quality of urinalysis in routine clinical practice.

Examination of the urine sediment and establishment of the criterion for microscopy review / 中华检验医学杂志

Urine sediment analysis is of importance for the diagnosis, differential diagnosis and prognosis in urinary system diseases.To understand the standardization of sediment analysis and its development, This article analyzes the advantage and disadvantage of automated instrument used in urine sediment analysis, and then developes the criteria for microscopy review following automated urinalysis.

The progression of laboratory diagnosis for urinary tract infection / 中华检验医学杂志

Urinary tract infection (UTI)is a commonly encountered disease during routine clinical care. In the mean time, it is also a commonly encountered hospital acquired infection. UTI possesses various clinical symptoms, and usually the symptoms are atypical. The detection of leukocyturia and bacteriuria is an essential index for screening and confirmatory diagnosis of UTI. This article summarized the pathogenesis of UTI, diagnostic criteria, clinical significance and application evaluation of detection of the WBC and bacteria in urine.

Evaluation of urine analysis by flow cytometry and strip test in diagnosing urinary tract infection / 中华检验医学杂志

Objective To evaluate the clinical application of automated urine formed elements analyzer and/or urine dipstick analyzer for examination of urinary formed elements in screening urinary tract infection (UTI). Methods 148 fresh midstream clear-catch urine samples from the UTI patients and 284 fresh midstream clear-catch urine samples from non-UTI subjects were selected. Bacteria culture was performed for bacterial colony counting and identification. Bacteria counts ( BACT), yeast-like fungus and WBC were performed by UF-looOi automated urine formed elements analyzer. Leukocyte esterase test (LEU) and nitrite test (NIT) were performed by URISYS 2400 urine dipstick analyzer. We evaluated data obtained from urine dipstick analyzer, UF-1000i and combination of UF-1000i with urine dipstick analyzer and the results was compared with those obtained from quantitative bacterial culture. Then we evaluated the sensibility, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. Results Among the 148 patients with UTI, the positive rate of the quantitative bacterial culture was 73.6% (109/148), the positive rate of LEU and NIT detected by dipstick test 26. 4% (39/148).There was significantly statistical difference between bacterial culture and strip test(χ2 = 55.68 ,P < 0. 05 ). The positive rate of urine flow cytometry by UF-1000i with either positive of BACT and WBC was 91.2%(135/148), which was higher than the positive rate of the quantitative bacterial culture. There was significant difference between two methods (χ2 = 14. 70, P < 0. 05 ). The positive rate of anyone positive among BACT, WBC, LEU and NIT was 94. 6% (140/148) when detected with combination of dipstick test and UF-1000i, which was higher than the positive rate of the quantitative bacterial culture. And there was significant difference between two methods (χ2 = 20. 45, P < 0. 05 ). The sensitivity of dipstick test was low (26. 4% ,39/148 ), and specificity was high ( 99. 3%, 282/284 ) . The sensitivity, specificity, positive predictive value, negative predictive value of BACT detected by UF-1000i in diagnosing urinary tract infection were 92. 6% ( 137/148 ), 39. 8% ( 113/284 ). 44. 5% ( 137/308 ) and 91.1% ( 113/124 ), respectively. If the dipstick test was combined with UF-1000i, the sensitivity, negative predictive value, specificity, positive predictive value and accuracy were 98.0% ( 145/148 ), 97.1% ( 100/103 ). 35.2% (100/284) ,44. 1% (145/329) and 56. 7% (245/432), respectively. Conclusions The combination of urine dipstick test and automated urine formed elements analyzer UF-1000i plays an important role in early diagnosis of UTI. And it has significant value in diagnosis of UTI, especially for the patients with negative bacterial cultures of urine sample.

Applied fundamental research of echinacea species / 北京大学学报(医学版)

Echinacea is a most famous "immune herb" in western countries, and continues to be the best selling herb for many years. For the last five years, our research group has cooperated with Institute of Medicinal Plants in Huairou District of Beijing, carrying out studies on Echinacea purpurea, which involved botany, cultivation, pharmacognosy, phytochemistry, quality control, pharmacology and toxicology of the species. Two other species introduced from Canada, Echinacea angustifolia and Echinacea pallida, were also included in the taxonomic, cultivated and pharmacognostic studies. The results acquired have opened up the path to introduce Echinacea species into Traditional Chinese Medicine, thus established the possibility of developing more promising drugs from them.

Protocolo de validación de métodos analíticos para la cuantificación de fármacos

Se presenta un resumen de las consideraciones generales para la confección del protocolo de validación de métodos analíticos utilizados en la determinación cuantitativa de fármacos en forma de materia prima o en formulaciones y en estudios de estabilidad. Se describe detalladamente el proceso de validación que incluye los requisitos exigidos para la utilización de las materias primas, materiales de referencia, equipamiento, personal y determinación de los parámetros de linealidad, precisión, exactitud y selectividad (con el procesamiento estadístico de los resultados experimentales y criterios de aceptación), así como la presentación de los resultados en el informe final de la validación.

A summary of the general considerations fot the formulation of a validation protocol of the analytical methods used in drug quantitative determination in a raw material form, or in formulations and firmness studies. The validation process is fully described; it includes the necessary requirements for the usage of raw materials, reference materials, supplying, personnel, and determination of linearity, preciseness, exactness, and selectiveness (with the statistical processing of experimental results, and acceptance criteria), as well as the presentation of outcomes in the validation final report.