RESUMEN
Quantification of hepatitis B virus (HBV) DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log10 HBV DNA dynamic range, were thawed and stored at -70, 4, 23, 37, and 45 degrees C (+/-1.5 degrees C) for 0, 24, 72, and 120 h (+/-2 h) and were refrozen at -70 degrees C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4 degrees C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at < or =4 degrees C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45 degrees C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at < or =4 degrees C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of > or =20% at 23 or 37 degrees C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at -70 or 4 degrees C.
Asunto(s)
ADN Viral/sangre , ADN Viral/metabolismo , Virus de la Hepatitis B/aislamiento & purificación , Virus de la Hepatitis B/metabolismo , Hepatitis B/sangre , Hepatitis B/metabolismo , ADN Viral/aislamiento & purificación , Monitoreo de Drogas , Hepatitis B/tratamiento farmacológico , Humanos , Modelos Lineales , Modelos Logísticos , Juego de Reactivos para Diagnóstico , Manejo de EspecímenesRESUMEN
We have developed a single-stage assay for heparin, using reagents modified from the two-stage Dade Protopath heparin synthetic substrate assay. The single-stage assay involves simultaneous mixing of a plasma sample, an antithrombin III source, alpha-thrombin, and the alpha-thrombin fluorogenic substrate. The synthetic substrate, antithrombin III, and heparin-antithrombin III complex compete for the alpha-thrombin active site. The alpha-thrombin is inactivated by the heparin-antithrombin complex while substrate is being hydrolyzed, so that total product formation decreases with heparin concentration. Day-to-day CV was 9.3% at a heparin concentration of 246 USP units/L. Comparison of results of the single-stage heparin assay with those of a two-stage esterolytic assay yielded the linear regression equation: esterolytic = 0.834 (single-stage)--7 USP units/L (r = 0.94, n = 47). Bilirubin interfered with the single-stage assay, resulting in an apparent increase in sample heparin concentration. The single-stage heparin assay can be automated for centrifugal analyzers capable of double-reagent addition and fluorometric detection, substantially decreasing reagent requirements and therefore costs.
Asunto(s)
Heparina/sangre , Centrifugación , Fluorometría , Humanos , Métodos , Valores de ReferenciaRESUMEN
This assay for heparin is based on the heparin-accelerated rate of alpha-thrombin III. The rate or product formation from the residual active thrombin is inversely proportional to plasma heparin content. The assay can be performed manually, but our results were obtained with a discrete analyzer, the ABA-100. The assay is insensitive to concentrations of antithrombin III in plasma. Precision studies gave CVs of less than 10%. This assay was compared to the Dade Protopath heparin assay and a correlation coefficient of 0.90 was obtained (n = 62). The correlation between activated partial thromboplastin times and heparin concentrations (r = 0.67) was calculated frm results on 78 plasma specimens from 10 patients.
Asunto(s)
Heparina/sangre , Antitrombina III/metabolismo , Humanos , Métodos , Tiempo de Tromboplastina Parcial , Trombina/metabolismoRESUMEN
OBJECTIVE: Irritable bowel syndrome (IBS) is diagnosed by the presence of a constellation of symptoms fulfilling the Manning or Rome Criteria, after exclusion of organic disease. To exclude other diagnoses that might contribute to the abdominal pain or bowel symptoms experienced by subjects with IBS, numerous screening algorithms have been advocated, incorporating lactose hydrogen breath tests, thyroid function tests, fecal ova and parasite determination, and colonic endoscopy/radiography. The utility of these tests in uncovering alternative diagnoses, other than IBS, was examined in 1452 patients. METHODS: Data were combined from two large multinational studies of IBS patients. All patients exhibited symptoms meeting the Rome criteria for IBS for at least 6 months before study entry. If prior evaluation had been > 2 yr previously, patients underwent colonic endoscopy/radiography at study entry. In addition, thyroid function tests, fecal ova and parasite determination, and a lactose hydrogen breath test were performed. RESULTS: Lactose malabsorption was diagnosed in 23% (256/1122) of patients. Colonic abnormalities were detected in 2% (7/306) of patients; in four patients, colonic inflammation (n = 3) or obstruction (n = 1) may have contributed to symptoms of abdominal pain or altered bowel habits. Abnormal thyroid-stimulating hormone levels were detected in 6% (67/1209) of patients, of whom half were hypothyroid and half were hyperthyroid. Positive fecal ova and parasite tests were noted in 2% (19/1154) of patients. CONCLUSIONS: Examination of screening tests in 1452 patients with an established history of IBS revealed an incidence of lactose malabsorption comparable to that in the general U.S. population and a low incidence of thyroid dysfunction, ova and parasite infestation, or colonic pathology. The limited detection rates, added costs, and inconvenience of these tests suggest that their routine use in the diagnostic evaluation of established IBS patients should be scrutinized.