Journal of Clinical Laboratory Analysis

Real‐time polymerase chain reaction (PCR) is a frequently used technique in molecular diagnostics. To date, practical guidelines for the complete process of optimization and validation of commercial and in‐house developed molecular diagnostic methods are scare. Therefore, we propose a practical guiding principle for the optimization and validation of real‐time PCR assays. Based on literature, existing guidelines, and personal experience, we created a checklist that can be used in different steps of the development and validation process of commercial and in‐house developed real‐time PCR assays. Furthermore, determination of target values and reproducibility of internal quality controls are included, which allows a statistical follow‐up of the performance of the assay. Recently, we used this checklist for the development of various qualitative and quantitative assays for microbiological and hematological applications, for which accreditation according to ISO 15189:2007 was obtained. In our experience, the use of the proposed guidelines leads to a more efficient and standardized optimization and validation. Ultimately, this results in reliable and robust molecular diagnostics. The proposed checklist is independent of environment, equipment, and specific applications and can be used in other laboratories. A worldwide consensus on this kind of checklist should be aimed at. J. Clin. Lab. Anal. 23:145–151, 2009. © 2009 Wiley‐Liss, Inc.

Background: Microalbuminuria is an indicator of kidney damage and a risk factor for the progression kidney disease, cardiovascular disease, and so on. Therefore, accurate and precise measurement of urinary albumin is critical. However, there are no reference measurement procedures and reference materials for urinary albumin. Methods: Nephelometry, turbidimetry, colloidal gold method, radioimmunoassay, and chemiluminescence immunoassay were performed for methodological evaluation, based on imprecision test, recovery rate, linearity, haemoglobin interference rate, and verified reference interval. Then we tested 40 urine samples from diabetic patients by each method, and compared the result between assays. Results: The results indicate that nephelometry is the method with best analytical performance among the five methods, with an average intraassay coefficient of variation (CV) of 2.6%, an average interassay CV of 1.7%, a mean recovery of 99.6%, a linearity of R=1.00 from 2 to 250 mg/l, and an interference rate of <10% at haemoglobin concentrations of <1.82 g/l. The correlation (r) between assays was from 0.701 to 0.982, and the Bland–Altman plots indicated each assay provided significantly different results from each other. Conclusion: Nephelometry is the clinical urinary albumin method with best analytical performance in our study. J. Clin. Lab. Anal. 25:324–329, 2011. © 2011 Wiley‐Liss, Inc.

In this study, we have the objective of evaluating the lymphoproliferative response and determining interferon (IFN)‐γ and interleukin (IL)‐10 cytokine production in the peripheral blood mononuclear cells (PBMC) of patients with American tegumentary leishmaniasis prior and post 12 months of chemotherapy treatment with meglumine antimoniate compared with the PBMC of noninfected donors. Lymphoproliferation, such as cytokine production, was evaluated through in vitro stimulus with the soluble antigenic fraction from Leishmania (Viannia) braziliensis promastigotes (1.25 µg/ml) and Concanavalin A (2.5 µg/ml). Patients showed a significant lymphoproliferative response prior and post treatment compared with the control group. Similar result, prior to chemotherapy treatment, was observed in IFN‐γ and IL‐10 production when patients were compared with the control group. After chemotherapy treatment, PBMC lymphoproliferative response of the patients revealed an increase, whereas patients have shown a decrease in IFN‐γ levels and an increase in IL‐10, although without statistical difference. These results may indicate that the patients produced a specific cellular response to the soluble antigenic fraction suggesting that besides Th1 and Th2 dichotomy, immunological regulation mechanisms with the participation of memory T cells and regulatory T cells could be present in the clinical evolution of these patients. This understanding will allow the study and identification of new L. (V.) braziliensis molecules potentially candidates to vaccines. J. Clin. Lab. Anal. 23:63–69, 2009. © 2009 Wiley‐Liss, Inc.

The debate continues regarding the possible interference of phenytoin metabolites in phenytoin immunoassays, and its clinical importance for patients with renal failure. The aim of this study was to compare the results obtained using the Abbott fluorescence polarization immunoassay (FPIA), Dade enzyme‐multiplied immunoassay technique (EMIT), and high‐performance liquid chromatography (HPLC) to establish the significance of the differences in conditions of renal failure. Thirty‐six adult patients who had been treated with phenytoin and whose renal function ranged from normal to severely impaired were chosen for this study. In accordance with previously established validation criteria for analytical methods for the determination of drugs, a 15% bias from the HPLC phenytoin values was considered an acceptable limit. The mean (±SEM) glomerular filtration rate (GFR) of the patients was 37.5±4.6 mL/min (range = 10–102 mL/min).The mean values found using FPIA (10.8±1.2 µg/mL) and EMIT (10.8±1.3 µg/mL) presented acceptable deviations with respect to HPLC (10.5±1.2 µg/mL), and a high correlation was found among the results (N = 36) of the different methods (r≥0.987, P<0.001). An FPIA deviation above the 15% bias limit with respect to HPLC was found only in two cases with very low serum phenytoin concentrations and low GFR values (<20 mL/min), although it does not appear to be important in terms of adjusting drug dosage. According to our data, FPIA and EMIT gave accurate results for total phenytoin in serum samples from patients with renal failure. J. Clin. Lab. Anal. 21:119–123, 2007. © 2007 Wiley‐Liss, Inc.

The aim of the present study was to investigate the serum total protein (TP), total sialic acid (TSA), lipid‐associated sialic acid (LSA), LSA/TP, and LSA/TP values in type 2 diabetes mellitus (DM) patients. Two study groups (healthy controls and type 2 DM subjects) were examined. For the type 2 DM group, 120 patients (60 females and 60 males) who had been diagnosed and treated for type 2 DM in the Yuzuncu Yil University Hospital, Van, Turkey, were selected consecutively. Forty healthy individuals (20 females and 20 males) were selected from hospital staff and other outpatient clinics to serve as the control group. They were matched for age, sex, body mass index, and smoking status. None of the participants had taken vitamin or mineral supplements for at least 2 weeks before sampling. To determine serum glucose, TP, TSA, and LSA levels, blood samples were drawn after all of the subjects fasted overnight. It was found that diabetics had higher TSA, LSA, TSA/TP, and LSA/TP levels than controls. However, the TP levels were not significantly different between the groups. Our results showed that TSA, LSA, TSA/TP, and LSA/TP have interactive connections with DM. These parameters can be used as a diagnostic index for patients with DM. J. Clin. Lab. Anal. 17:124–126, 2003. © 2003 Wiley‐Liss, Inc.

We studied 103 patients seen in our Prostate Cancer Detection Clinic to determine whether a correlation exists between serum prostate‐specific antigen (PSA) values and ultrasound‐calculated prostate gland volume. Seventy men (68%) had a PSA value ≤ ng/ml (our upper limit of normal). The men were subclassified by prostate gland volume at arbitrary break points. Twenty‐five men (24%) had a prostate gland volume ≤ 25 cm³; in 96%, the PSA value was ≤ 4 mg/ml. Further analysis revealed that the percentage of men with a normal serum PSA value decreased as the prostate gland volume increased; 65.6% of the group with a gland volume between 25 and 50 cm³ (40 of 61) and 35.5% of the group whose prostate volume exceeded 50 cm³ (6 of 17) had PSA values ≤4 ng/ml. Four men had PSA values ≥20 ng/ml; all had prostate cancer. Cancer was diagnosed in four additional patients, three with PSA values between 5 and 10 ng/ml and one with a PSA value <4 ng/ml. There appears to be a direct relationship between prostate gland volume and PSA value, as well as a cancer value threshold. The clinical implications of these findings are discussed.

Because of differences in the types and quantities of glycosaminoglycans (GAGs) in various mucopolysaccharidoses (MPSs), MPS screening tests, including the Berry spot and acid turbidity tests, are not specific or sensitive enough for the preliminary diagnosis of MPS. A false‐negative result is common. We analyzed urine samples collected from 492 patients who were examined for inborn errors of metabolism using the Berry spot and acid turbidity (qualitative and quantitative) tests. Of those, 48 MPS patients (seven with MPS I, 17 with MPS II, nine with MPS III, 11 with MPS IV, and four with MPS VI) underwent preliminary differentiation between MPS types by two‐dimensional electrophoresis (2D‐EP), and were confirmed by enzymatic assay. Approximately 21.0% and 7.1% of the 492 samples showed positive reactions in the Berry spot and acid turbidity tests, respectively. Of these, a total of 35 samples with MPS types I, II, and VI showed strong positive reactions in both tests. Five patients with Sanfilippo (MPS III) and six patients with Morquio (IV) syndromes showed false‐negative results in both tests. In our study, approximately 13.8% (68 in 492 samples) samples showed a positive reaction in the Berry spot test but a negative one in the acid turbidity test, for unknown reasons. The Berry spot and acid turbidity tests are used extensively for the preliminary diagnosis of MPS in Asia; however, the possibility of a misdiagnosis of MPS type III and IV with both tests should be kept in mind. For accurate diagnosis and confirmation of MPS, the 2D‐EP method and enzymatic assay are recommended. They provide high sensitivity, specificity, and efficiency in diagnosing MPS. J. Clin. Lab. Anal. 16:253–258, 2002. © 2002 Wiley‐Liss, Inc.