Journal of Clinical Laboratory Analysis

Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features. J. Clin. Lab. Anal. 16:47–51, 2002. © 2002 Wiley‐Liss, Inc.

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.

Neonatal sepsis, characterized by systemic signs of infection in the first month of life, remains an important clinical syndrome. Despite advances in neonatology, it has high rates of mortality and morbidity. The combine or alone usage of interleukin‐6 (IL‐6) and C‐reactive protein (CRP) has recently been proven to be useful in the early diagnosis of sepsis in newborns. The study included 282 patients; there were 232 in Group I (170 proven and 62 clinical sepsis) and 50 in Group II (control group). The optimum cut‐off value in the diagnosis of neonatal sepsis was found to be 24.65 pg/ml for IL‐6 and 4.82 mg/l for CRP. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of this IL‐6 cut‐off for neonatal sepsis were 72, 84, 95, and 42%, respectively. Sensitivity, specificity, PPV, and NPV of the CRP cut‐off for neonatal sepsis were 67, 97, 99, and 39%, respectively. The combination of IL‐6 (>24.65 pg/ml) and CRP (>4.82 mg/l) in the diagnosis of neonatal sepsis gave sensitivity, specificity, PPV, and NPV of 53, 100, 100, and 33%, respectively. To our knowledge, this is the largest reported study seeking to determine cut‐off levels for IL‐6 and CRP in the diagnosis of neonatal sepsis. In conclusion, we think that it is useful to evaluate IL‐6 and CRP, in combination, for the early diagnosis of neonatal sepsis. J. Clin. Lab. Anal. 24:407–412, 2010. © 2010 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.

This article reviews the clinical applications of C‐reactive protein (CRP). This acute‐phase protein is a distinct and sensitive marker for inflammation and tissue injury. It is a simple, fast, and relatively inexpensive latex agglutination test. The aspects of CRP reviewed include diagnostic support, serial measurements to evaluate disease course and therapeutic response, and screening studies.

Experimental evidence indicates a relationship between cholesterol α‐epoxide and skin cancer, and exposure of skin fibroblasts to ultraviolet radiation enduces formation of significant levels of this oxide. Colon cancer is also etiologically linked to cholesterol oxidation products. Higher than normal levels of cholestanetriol have been found in patients with colon cancer and also in those with precancerous disorders such as adenomatous polyps and ulcerative colitis. Higher than normal levels of cholesterol α‐epoxide have been found in breast fluid aspirates of women with benign breast disease, with or without atypical hyperplasia of the epithelium, and this may be a factor in the increased incidence of breast cancer associated with hyperplasia. Similarly, the observed increased levels of cholesterol α and β‐epoxides in prostatic fluid of men with benign prostatic hypertrophy may be associated with subsequent development of prostate cancer.

Cholesterol α‐epoxide has been found to be mutagenic to fibroblasts in culture and to induce morphological transformation in hamster embryo cells and in mouse C3H cells. 25‐Hydroxycholesterol and 20α‐hydroxycholesterol are potent suppressors of generation and proliferation of tumor‐specific cytotoxic T lymphocytes.

Although investigations into the role of cholesterol oxidation products in cancer are still in the early stages, evidence to date indicates a potentially significant role in the induction of some types of cancer.

The objective of the study was to determine the sensitivity and specificity of an indirect immunofluorescence (IIF) assay using transfected HEp‐2 cells to detect anti‐SS‐A/Ro autoantibodies in human sera. Seventy‐three sera having SS‐A/Ro autoantibodies as determined by double immunodiffusion (ID) and immunoblotting (IB) were tested by IIF on a HEp‐2 cell substrate that had been transfected with a full‐length cDNA encoding a human 60 kD SS‐A/Ro autoantigen. Controls included 30 normal human sera and 50 sera with a variety of other antinuclear antibodies. Prototype human and rabbit sera directed against the 60 kD SS‐A/Ro antigen produced intense speckled nuclear and nucleolar staining of transfected cells. Sixty‐nine of 73 (95%) SS‐A/Ro positive sera also produced this characteristic staining pattern. The endpoint autoantibody titers on transfected cells was fivefold greater than on untransfected cells. The 30 normal human sera and the 50 sera with other antinuclear antibodies did not produce this characteristic staining. Six of 32 (19%) unselected sera that were sent for autoantibody testing had reactivity with transfectants by IIF. Four of the six sera were confirmed to have anti‐SS‐A/Ro antibodies by ID and 5/6 by IB. By contrast, only three of these sera were scored as having a staining pattern compatible with SS‐A/Ro antibodies by IIF on standard HEp‐2 substrates. We conclude that SS‐A/Ro autoantibodies can be detected by an IIF assay using a HEp‐2 cell substrate transfected with a SS‐A/Ro cDNA. This new substrate detects SS‐A/Ro antibodies that were not identified on standard HEp‐2 substrates and by other immunoassays.©1995 wiley‐Liss, inc.

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.