John R. Petersen

APRI : An easy and validated predictor of hepatic fibrosis in chronic hepatitis C

Goals To evaluate the aspartate aminotransferase/platelet ratio index (APRI) as a predictor of the presence or absence of significant fibrosis on liver biopsy of patients with chronic hepatitis C (HCV). Background The decision to treat HCV is often made on the basis of the presence or absence of significant fibrosis on the liver biopsy. Because liver biopsy is expensive and invasive a noninvasive marker to evaluate hepatic fibrosis would be useful. The APRI is an easy to calculate index that is one of several markers that have been proposed. Study We retrospectively reviewed the charts of 339 patients with chronic HCV who had liver biopsies from January 2000 to March 2003. We subsequently evaluated 151 patients receiving pretreatment evaluation liver biopsies who had serum aspartate aminotransferase, platelets, routine liver function tests, and demographic data obtained. All liver biopsies were staged by the Batts Ludwig criteria. Results The area under the curve of the receiver operator characteristics of the calculated APRI compared with the liver biopsy demonstrated that the fibrosis score was 0.889 in the prospective group and 0.790 in the retrospective group. To achieve predictive values of approximately 90%, useful cutoffs were found at 0.40 and 1.5 in the retrospective study, and 0.42 and 1.2 in the prospective study leaving intermediate zones of 58.9% and 41.1%, respectively. In the prospective group, 34 of 36 patients with a value of <0.42 were accurately predicted as having mild fibrosis, whereas 50 of 54 patients with a value >1.2 were accurately predicted to have significant fibrosis. Conclusions The APRI is a good estimator of hepatic fibrosis and was more accurate in a prospective group than a retrospective one. It potentially could be used to decrease the number of liver biopsies.

Capillary electrophoresis and its application in the clinical laboratory.

Over the past 10 years, capillary electrophoresis (CE) is an analytical tool that has shown great promise in replacing many conventional clinical laboratory methods, especially electrophoresis and high performance liquid chromatography (HPLC). The main attraction of CE was that it was fast, used small amounts of sample and reagents, and was extremely versatile, being able to separate large and small analytes, both neutral and charged. Because of this versatility, numerous methods for clinically relevant analytes have been developed. However, with the exception of the molecular diagnostic and forensic laboratories CE has not had a major impact. A possible reason is that CE is still perceived as requiring above-average technical expertise, precluding its use in a laboratory workforce that is less technically adept. With the introduction of multicapillary instruments that are more automated, less technique-dependent, in addition to the availability of commercial and cost effective test kit methods, CE may yet be accepted as a instrument routinely used in the clinical laboratories. Thus, this review will focus on the areas where CE shows the most potential to have the greatest impact on the clinical laboratory. These include analysis of proteins found in serum, urine, CSF and body fluids, immunosubstraction electrophoresis, hemoglobin variants, lipoproteins, carbohydrate-deficient transferrin (CDT), forensic and therapeutic drug screening, and molecular diagnostics.

Comparison of POCT and central laboratory blood glucose results using arterial, capillary, and venous samples from MICU patients on a tight glycemic protocol.

BACKGROUND Point of care (POC) glucose meters are routinely used to monitor glucose levels for patients on tight glycemic control therapy. We determined if glucose values were different for a POC glucose meter as compared to the main clinical laboratory for medical intensive care unit patients on a tight glycemic protocol and whether the site of blood sampling had a significant impact on glucose values. METHODS Eighty-four patients (114 paired samples) who were on a tight glycemic protocol in the period November 2005 through August 2006 were enrolled. After simultaneous blood draws, we compared the glucose levels for the glucose meter (arterial/venous/capillary), blood gas (arterial/venous), and central clinical laboratory (serum/plasma from arterial/venous samples). RESULTS The mean glucose levels of all arterial/venous/fingerstick samples using the glucose meter demonstrated a positive bias of 0.7-0.9 mmol/l (12.6-16.2 mg/dl) (p<0.001) relative to central laboratory venous plasma. There was also a smaller positive (0.1-0.3 mmol/l or 1.8-5.4 mg/dl, p<0.05) bias for arterial/venous blood gas samples and laboratory arterial serum/plasma glucose samples. Using Parkes error grid analysis we were able to show that the bias for arterial or venous POC glucose results would have not impacted clinical care. This was not the case, however, for fingerstick sampling where a high bias could have significantly impacted clinical care. Additionally, in 3 fingerstick samples a severe underestimation (<46% of the central laboratory plasma result) was found. CONCLUSION Glucose meters using arterial/venous whole blood may be utilized in the MICU; however, due to the increased variability of results we do not recommend the routine use of capillary blood sampling for monitoring glucose levels in the MICU setting.

Hyperbilirubinemia and Transcutaneous Bilirubinometry

BACKGROUND Neonatal jaundice or hyperbilirubinemia is a common occurrence in newborns. Although most cases of neonatal jaundice have a benign course, severe hyperbilirubinemia can lead to kernicterus, which is preventable if the hyperbilirubinemia is identified early and treated appropriately. CONTENT This review discusses neonatal jaundice and the use of transcutaneous bilirubin (TcB) measurements for identification of neonates at risk of severe hyperbilirubinemia. Such a practice requires appropriate serial testing and result interpretation according to risk level from a nomogram that provides bilirubin concentrations specific for the age of the neonate in hours. In this context, we have evaluated the potential impact on clinical outcome and limitations of TcB methods in current use. SUMMARY TcB measurement is a viable option in screening neonates to determine if they are at risk for clinically significant hyperbilirubinemia. Total serum bilirubin should be measured by a clinical laboratory if a newborn is shown to be at higher risk for clinically significant hyperbilirubinemia. In addition, external quality assessment to identify biases and operator training issues should be part of any TcB monitoring program.

Stability of prothrombin time and activated partial thromboplastin time tests under different storage conditions

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are common laboratory tests that are useful in the diagnosis of coagulation disorders and monitoring anticoagulant therapy. Recent expansions in the outreach laboratory services at our institution prompted us to investigate the shipping limitations for some tests, including PT and aPTT. Although we followed NCCLS guidelines for the collection of blood specimens, we observed falsely elevated PT and aPTT values due to the different storage conditions. The objective of this study is to determine the effect of conditions and duration of storage on PT and aPTT tests using plasma and whole blood samples, respectively. For this study, 36 plasma samples with normal and prolonged PT and aPTT were exposed to different storage conditions. Blood was centrifuged immediately and plasma was stored at room temperature (RT), refrigerated at 4 degrees C, or frozen at -20 degrees C. The samples were analyzed at 0 h and repeated at 6, 12 and 24 h under various conditions. Although statistically significant differences were observed for plasma samples for normal PT tests after 12 h at refrigerated and frozen storage conditions, the differences would not change the clinical interpretation of the results. On the other hand, samples stored refrigerated or at RT showed significant differences for aPTT at 24 h. These differences would change clinical interpretation, especially for samples with normal or near normal aPTT times. Interestingly, aPTT was significantly higher for samples stored frozen when compared to refrigerated and RT conditions at 6 h. Similar patterns were also observed on ten whole blood samples with normal PT and aPTT values. In conclusion, either plasma or whole blood samples can be accepted for PT testing up to 24 h and for aPTT testing up to 12 h only, when transported either at RT or at 4 degrees C.

Development of a urinary free cortisol assay using solid-phase extraction-capillary electrophoresis.

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE-CE method for the rapid analysis of UFC. UFC was concentrated using SPE C18 cartridges (3M Empore) under a vacuum and eluted with acetonitrile-SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile-SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10-15 min with a recovery of 89-94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 microg/l with SPE compared to 500 microg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.

Non-invasive markers of hepatic fibrosis in patients co-infected with HCV and HIV: Comparison of the APRI and FIB-4 index

INTRODUCTION The APRI and FIB-4 index are markers that have been proposed for the evaluation of hepatic fibrosis in patients co-infected with HIV and HCV. METHODS We retrospectively compared these 2 indices in 81 co-infected patients staged by liver biopsy. RESULTS The FIB-4 index was superior to the APRI for the differentiation of mild from significant fibrosis in both predictive values and area under the receiver operator curve (AUROC). The tests were comparable for the differentiation of mild/moderate from advanced fibrosis. CONCLUSION These tests could be used to evaluate co-infected patients for treatment, and exclude those that do not need periodic screening for hepatocellular carcinoma.

Biomarker Discovery for Early Detection of Hepatocellular Carcinoma in Hepatitis C–infected Patients

Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of 18O/16O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using 18O/16O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.

Micellar electrokinetic capillary chromatographic separation of steroids in urine by trioctylphosphine oxide and cationic surfactant.

Separation of the six structurally similar and hydrophobic neutral steroids, testosterone, dimethyltestosterone, testosterone propionate, cortisone, hydrocortisone and 17-deoxycorticosterone, was achieved by hydrophobic micellar electrokinetic chromatography. A triphasic separation involving micellar dodecyltrimethylammonium bromide (DTAB), a dynamic bilayer formed due to electrostatic interaction between the silica surface and DTAB, and aqueous phase is proposed to account for the observed separation of the steroids. The running buffer consisted of 0.05 M DTAB and 0.0052 M trioctylphosphine oxide in 0.01 M of phosphate buffer pH 7.4. A detection limit of 500 ng/ml was achieved for each steroid and the application of the method to urine samples is described.

Hemoglobin A1c: Assessment of three POC analyzers relative to a central laboratory method

BACKGROUND Glycosylated hemoglobin evaluation is very important for assessing the control of diabetes. Since the use of point-of-care (POC) devices for monitoring HbA1c is increasing, it is important to determine how these devices compare in relation to instrumentation used in the central laboratory (CL). METHODS Eighty-eight randomly selected samples previously analyzed using the Bio-Rad Variant™ II Hemoglobin Testing System were run on three POC Analyzers (Siemens DCA Vantage™ Analyzer, Axis-Shield Afinion™ AS100 Analyzer, and Bio-Rad In2it™ Analyzer). RESULTS All POC instruments showed good correlation to the CL method (R(2)>0.95 for all methods). HbA1c levels obtained using Variant II (mean=7.9; 95% CI=7.5-8.3%) and In2it (mean=7.9; 95% C.I.=7.5-8.2%) instruments were found to have no statistical mean difference (p=0.21), while the values obtained using DCA Vantage (mean=7.2% C.I.=6.9-7.5%) and Afinion (mean=7.3% C.I.=7.0-7.6%) instruments were different (p<0.001) from those of the CL method. The Afinion and DCA Vantage instruments increasingly underestimated the HbA1c compared to the CL as the HbA1c values increased. These differences were even more striking when the estimated average glucose is calculated. CONCLUSIONS Despite significant variation of results among the POC instruments evaluated relative to the CL method and pending resolution of HbA1c standardization issues, we conclude that all of the POC instruments can be used for HbA1c determination if clinicians are given instrument specific reference ranges.