Analysis of the qPCR Assay Efficacy as Molecular Diagnostic in Patients with Chemotherapy and/or Antibiotic Therapy

Abstract High sensitivity of qPCR assay can be compromised by the presence of PCR inhibitors in samples analyzed. The aim of this study was to analyze the RT-qPCR assay efficiency considering the RNA quality/quantity and the presence of PCR inhibitors in patients with chemotherapy and/or antibiotic therapy. We analyzed 60 samples using RT-qPCR from individuals suspected of leukemia and 44 samples were quantified by fluorimetry and spectrophotometry. The efficiency of the RT-qPCR assay was evaluated comparing the threshold cycle (Ct) from tested samples and the standard curve. The 260/280 and 260/230 ratios, the presence of PCR inhibitors and the amount of sample (ng) used in the RT-qPCR reaction can be associated with 56.8% (R²=0.56, p<0.05) in the Ct obtained. The decrease of the RT-qPCR efficiency can be explained in 42,8% due to the variation of the 260/280 ratio (R²=0.42,p<0.05). The presence of antibiotics in the blood sample can be associated in 11.3% with the variability of 260/280 ratio (R²=0.11,p<0.05). Presence of chemotherapeutic drugs in the blood sample was not correlated with Ct variation (p=0.17). The spectrophotometer determines a RNA quantification with 2.2 times higher than the fluorimeter (t=2.2, p=0,03) and this difference is correlated with the 260/280 ratio (R²=0.36, p<0.05). Samples with low purity had a reduction in the qPCR efficiency, although we did not observe false results.

Utility of plasma fluorometric emission scanning for diagnosis of the first 2 cases reports of variegate porphyria: a very rare type of porphyrias in Thai.

Two Thai women who are siblings presented with a history of recurrent pruritic vesicles on dorsum of both hands and extensor surface of forearms where the sun-exposed areas are. The excoriated vesicles were healed with depressed scars. They had no previous history of intense abdominal pain, seizure, or psychiatric disorder Urinary porphyrins were analyzed by High Performance Liquid Chromatography (HPLC). The level of coproporphyrin III was detected to be higher than the uroporphyrin level. Fluorescence emission scanning of both patients' plasma was performed and demonstrated typical emission peak at 626 nm, that confirmed the diagnosis of variegate porphyria.

Automated determination of glucose-6-phosphate dehydrogenase (G6PD) on a SPOTCHECK Microflow analyzer.

Glucose-6-phosphate dehydrogenase (G6PD) is the initial enzyme in the hexose monophosphate pathway of glucose metabolism. Deficiency of G6PD has been linked to increased sensitivity of red cells to hemolytic anemia due to certain oxidant drugs, infectious agents or fava beans. It is an inherited error in metabolism and has a high incidence in certain ethnic groups. Astoria-Pacific has developed an automated assay for use on the SPOTCHECK Microflow Analyzer for the semi-quantitative determination of G6PD activity in erythrocytes. After sample extraction, all assay steps are automated including reagent addition, incubation and data collection. Use of on-line dialysis removes interferences. The assay is intended primarily as a screening tool in the diagnosis and treatment of disease states associated with G6PD deficiency in newborns. G6PD in the dried blood spot is extracted and placed on the instrument. Samples are then aspirated into the system at a rate of 90 samples/hour. All other reagents are added by the SPOTCHECK Analyzer on-line during sample processing. Incubation of each sample occurs on-line at 37 degrees C, and after dialysis the NADPH reaction product is excited at 365 nm. Fluorescence is measured at 500 nm. A lack of fluorescence indicates a probable G6PD deficiency. Data reduction occurs real time through a FASPac software thus individual results are available during a run as soon as each sample analysis is complete. The Astoria-Pacific International G6PD reagent kit paired with the SPOTCHECK Microflow Analyzer provides an effective and easy to use screening tool for determining G6PD deficiency in newborns.

Optimización del análisis de ácidos biliares séricos por método enzimático fluorométrico / Improvement of an enzymatic fluorometric assay for serum bile acids

Con el objeto de optimizar la determinación fluorométrica de los ácidos biliares séricos (ABS) en cuanto a selectividad y sensibilidad, se desarrolló una metodología de preparación de muestra y preconcentración utilizando columnas de extracción en fase sólida (SPE-C18). Previa desproteinización del suero con acetonitrilo frío, la fase orgánica evaporada y reconstituida con acetonitrilo: agua (3:70), se aplicó a una columna SPE-C18 y los ABS fueron eluidos con metanol. En el extractivo metanólico, evaporado a sequedad y reconstituido con metanol se dosaron los ABS por método enzimático fluorométrico empleando 3Ó-hidroxiesteroide deshidrogenasa, ß-NAD, diaforasa y resazurina. En la validación de la preparación de muestra se utilizó [24-14C] ácido glicocólico. La recuperación fue del 89,0 ñ 1,3 por ciento (SD), con DSR de 1,4 para n=9 (3 días). Se determinaron los ABS en sujetos controles, resultando un valor medio de 2,61 ñ 0,39 µM (SEM) (n=27). La metodología propuesta combina las siguientes ventajas: aumento de la selectividad del método enzimático, eliminación de interferencias y preconcentración de los ABS liberados, previamente, de la unión a proteínas plasmáticas