Showing papers in "Clinical Chemistry in 1999"
TL;DR: Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats, and may play a key role either in conjunction with, or in place of, antibodies in the form of aptamer-based diagnostic products in the market.
Abstract: Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.
2,178 citations
TL;DR: Development of modulatory strategies targeting this transcription factor may provide a novel therapeutic tool for the treatment or prevention of various diseases.
Abstract: Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor that governs the expression of genes encoding cytokines, chemokines, growth factors, cell adhesion molecules, and some acute phase proteins in health and in various disease states. NF-κB is activated by several agents, including cytokines, oxidant free radicals, inhaled particles, ultraviolet irradiation, and bacterial or viral products. Inappropriate activation of NF-κB has been linked to inflammatory events associated with autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS. In contrast, complete and persistent inhibition of NF-κB has been linked directly to apoptosis, inappropriate immune cell development, and delayed cell growth. Therefore, development of modulatory strategies targeting this transcription factor may provide a novel therapeutic tool for the treatment or prevention of various diseases.
741 citations
TL;DR: The Sixth Conference on the "Standards of Laboratory Practice Series", sponsored by the National Academy of Clinical Biochemistry (NACB), was held on August 4-5, 1998, at the Annual Meeting of the American Association for Clinical Chemistry, in Chicago, IL, and an expert committee was assembled to write recommendations on the use of cardiac markers in coronary artery diseases.
Abstract: The Sixth Conference on the "Standards of Laboratory Practice Series", sponsored by the National Academy of Clinical Biochemistry (NACB), was held on August 4-5, 1998, at the Annual Meeting of the American Association for Clinical Chemistry, in Chicago, IL. An expert committee was assembled to write recommendations on the use of cardiac markers in coronary artery diseases. The NACB Committee prepared a preliminary draft of the guidelines, made them available on the World Wide Web (www.nacb.org), and distributed them before the presentations. The recommendations were divided into four areas: the use of markers in the triage of patients with chest pain, acute coronary syndromes, clinical applications other than acute myocardial infarction and research, and assay platforms and markers of acute myocardial infarction. The recommendations were revised and subsequently re-presented in part at the "Biomarkers in Acute Cardiac Syndromes Conference", sponsored by the Jewish Hospital Heart and Lung Institute, Louisville KY, on October 16-17, 1998. This report lists each recommendation, its scientific justification, and a summary of discussions from conference participants and reviewers. Approximately 100 individuals responded to various versions of these recommendations via direct correspondences, telephone calls to Committee members, electronic mail correspondence to the Committee Chairman, or oral questions and comments raised during one of the two conference presentations. Some of the recommendations were changed to reflect the consensus opinion. In cases in which there was no consensus, the Committee included pertinent discussion without necessarily changing the original recommendations. At times, the Committee members felt that although a particular recommendation might not be the current standard of care today, they anticipate that it likely will be adopted in the near future.
675 citations
TL;DR: The findings indicate that the Latex method is equally as efficacious as the validated ELISA in classifying patients into cutoff points established by prospective studies for risk stratification for coronary and cerebrovascular disease.
Abstract: Background: Prospective studies have shown that C-reactive protein (CRP) can be used to predict risk of future cardiovascular events. High-sensitivity methods for CRP (hs-CRP) measurement are needed for this purpose.
Methods: We compared the clinical efficacy of an automated and commercially available latex-enhanced assay (Latex) for hs-CRP (Dade Behring) to a validated in-house ELISA, previously shown to predict future peripheral arterial disease (PAD) in asymptomatic populations. Using a prospective, nested, case-control design, we measured baseline hs-CRP concentrations in 144 apparently healthy men who subsequently developed symptomatic PAD and 144 age- and smoking habit-matched controls who remained free of vascular disease over the follow-up period of 60 months.
Results: The two hs-CRP assays correlated highly ( r = 0.95; P <0.001), and all but two participants were classified into concordant quartiles or varied by only one quartile. The median hs-CRP of the case group was significantly higher than that of controls when measured by either the ELISA (1.34 vs 0.99 mg/L; P = 0.034) or the Latex method (1.80 vs 1.20 mg/L; P = 0.042). Furthermore, for both ELISA and the Latex method, the calculated relative risks of developing PAD increased significantly with each increasing quartile of hs-CRP. The calculated interquartile increase in relative risk of PAD was 31% (95% confidence interval, 5.2–62.2%; P = 0.01) for ELISA and 34% (95% confidence interval, 8.2–66.1%; P = 0.007) for the Latex method.
Conclusions: Our findings indicate that the Latex method is equally as efficacious as the validated ELISA in classifying patients into cutoff points established by prospective studies for risk stratification for coronary and cerebrovascular disease.
641 citations
TL;DR: A heightened awareness on the part of laboratory staff and clinicians of the problems caused by this type of interference in routine immunoassay tests is desirable, and efforts should be directed at improving methods for identifying and eliminating thistype of analytical interference.
Abstract: Purpose: The scope and significance of human anti-animal antibody interference in immunological assays is reviewed with an emphasis on human anti-animal immunoglobulins, particularly human anti-mouse antibodies (HAMAs).
Issues: Anti-animal antibodies (IgG, IgA, IgM, IgE class, anti-isotype, and anti-idiotype specificity) arise as a result of iatrogenic and noniatrogenic causes and include human anti-mouse, -rabbit, -goat, -sheep, -cow, -pig, -rat, and -horse antibodies and antibodies with mixed specificity. Circulating antibodies can reach gram per liter concentrations and may persist for years. Prevalence estimates for anti-animal antibodies in the general population vary widely and range from <1% to 80%. Human anti-animal antibodies cause interferences in immunological assays. The most common human anti-animal antibody interferent is HAMA, which causes both positive and negative interferences in two-site mouse monoclonal antibody-based assays. Strategies to prevent the development of human anti-animal antibody responses include immunosuppressant therapy and the use of humanized, polyethylene glycolylated, or Fab fragments of antibody agents. Sample pretreatment or assay redesign can eliminate immunoassay interferences caused by anti-animal antibodies. Enzyme immunoassays, immunoradiometric assays, immunofluorescence, and HPLC assays have been designed to detect HAMA and other anti-animal antibodies, but intermethod comparability is complicated by differences in assay specificity and lack of standardization.
Conclusions: Human anti-animal antibodies often go unnoticed, to the detriment of patient care. A heightened awareness on the part of laboratory staff and clinicians of the problems caused by this type of interference in routine immunoassay tests is desirable. Efforts should be directed at improving methods for identifying and eliminating this type of analytical interference.
606 citations
TL;DR: Interactions between systemic and local factors are important in the pathogenesis of osteoporosis as well as the skeletal changes in hyperparathyroidism and hyperthyroidism.
Abstract: The skeleton is a metabolically active organ that undergoes continuous remodeling throughout life. This remodeling is necessary both to maintain the structural integrity of the skeleton and to subserve its metabolic functions as a storehouse of calcium and phosphorus. These dual functions often come into conflict under conditions of changing mechanical forces or metabolic and nutritional stress. The bone remodeling cycle involves a complex series of sequential steps that are highly regulated. The "activation" phase of remodeling is dependent on the effects of local and systemic factors on mesenchymal cells of the osteoblast lineage. These cells interact with hematopoietic precursors to form osteoclasts in the "resorption" phase. Subsequently, there is a "reversal" phase during which mononuclear cells are present on the bone surface. They may complete the resorption process and produce the signals that initiate formation. Finally, successive waves of mesenchymal cells differentiate into functional osteoblasts, which lay down matrix in the "formation" phase. The effects of calcium-regulating hormones on this remodeling cycle subserve the metabolic functions of the skeleton. Other systemic hormones control overall skeletal growth. The responses to changes in mechanical force and repair of microfractures, as well as the maintenance of the remodeling cycle, are determined locally by cytokines, prostaglandins, and growth factors. Interactions between systemic and local factors are important in the pathogenesis of osteoporosis as well as the skeletal changes in hyperparathyroidism and hyperthyroidism. Local factors are implicated in the pathogenesis of the skeletal changes associated with immobilization, inflammation, and Paget disease of bone.
535 citations
TL;DR: The results suggest that preeclampsia is associated with disturbances in the liberation and/or clearance mechanisms of circulating DNA and raise the possibility that measurement of circulatingDNA may prove useful as a marker for the diagnosis and/ or monitoring of preeClampsia.
Abstract: Background: There is much recent interest in the biologic and diagnostic implication of cell-free non-host DNA in the plasma and serum of human subjects. To determine if quantitative abnormalities of circulating non-host DNA may be associated with certain pathologic processes, we used circulating fetal DNA in preeclampsia as a model system. Methods: We studied 20 preeclamptic women and 20 control subjects of comparable gestational age (means, 32 and 33 weeks, respectively). Male fetal DNA in maternal serum was measured using real-time quantitative PCR for the SRY gene on the Y chromosome. Results: The imprecision (CV) of the assay was 2.7%. The median circulating fetal DNA was increased fivefold in 20 preeclamptic women compared with 20 control pregnant women (381 vs 76 genome-equivalents/mL, P <0.001). Conclusions: These observations suggest that preeclampsia is associated with disturbances in the liberation and/or clearance mechanisms of circulating DNA. These results also raise the possibility that measurement of circulating DNA may prove useful as a marker for the diagnosis and/or monitoring of preeclampsia.
482 citations
TL;DR: Preliminary evidence is provided and cautious optimism is allowed that NIR diffuse reflectance spectroscopy using the 1050-2450 nm wavelength range can be used to predict blood glucose concentrations noninvasively.
Abstract: Background: Self-monitoring of blood glucose by diabetics is crucial in the reduction of complications related to diabetes. Current monitoring techniques are invasive and painful, and discourage regular use. The aim of this study was to demonstrate the use of near-infrared (NIR) diffuse reflectance over the 1050–2450 nm wavelength range for noninvasive monitoring of blood glucose.
Methods: Two approaches were used to develop calibration models for predicting the concentration of blood glucose. In the first approach, seven diabetic subjects were studied over a 35-day period with random collection of NIR spectra. Corresponding blood samples were collected for analyte analysis during the collection of each NIR spectrum. The second approach involved three nondiabetic subjects and the use of oral glucose tolerance tests (OGTTs) over multiple days to cause fluctuations in blood glucose concentrations. Twenty NIR spectra were collected over the 3.5-h test, with 16 corresponding blood specimens taken for analyte analysis.
Results: Statistically valid calibration models were developed on three of the seven diabetic subjects. The mean standard error of prediction through cross-validation was 1.41 mmol/L (25 mg/dL). The results from the OGTT testing of three nondiabetic subjects yielded a mean standard error of calibration of 1.1 mmol/L (20 mg/dL). Validation of the calibration model with an independent test set produced a mean standard error of prediction equivalent to 1.03 mmol/L (19 mg/dL).
Conclusions: These data provide preliminary evidence and allow cautious optimism that NIR diffuse reflectance spectroscopy using the 1050–2450 nm wavelength range can be used to predict blood glucose concentrations noninvasively. Substantial research is still required to validate whether this technology is a viable tool for long-term home diagnostic use by diabetics.
425 citations
TL;DR: The gap between the established need and current technology limitations for in vivo glucose measurements is discussed, and several technologies have potential for leading to viable measuring devices, but most of the data are based on in vitro experimentation.
Abstract: Frequent determination of glucose concentrations in diabetic patients is an important tool for diabetes management. This requires repetitive lancing and finger bleeding. Use of noninvasive (NI) detection techniques offers several advantages, such as the absence of pain and exposure to sharp objects and biohazard materials, the potential for increased frequency of testing, and hence, tighter control of the glucose concentrations, and the potential for a closed-loop system including a monitor and an insulin pump. These potential advantages have led to considerable interest in the commercialization of NI glucose monitoring devices. Review of the scientific, patent, and commercial literature indicates that the spectroscopic basis for NI determination of glucose is not yet well established, and attempts at commercialization may be several steps ahead of our understanding the origin and characteristics of an in vivo glucose-specific or glucose-related signal. Several technologies have potential for leading to viable measuring devices, but most of the data are based on in vitro experimentation. Because of the technical complexity of in vivo glucose measurements, this review aims at discussing the gap between the established need and current technology limitations.
393 citations
TL;DR: The method of Vester and Rasmussen is modified by using tris(2-carboxyethyl)phosphine (TCEP), a newer stable, water-soluble phosphine derivative introduced by Gilfix et al. (6), as the reducing agent, cystamine as the internal standard, and isocratic separation of the thiols extracted from only 50 μL of pla standard, asma within 6 min.
Abstract: Although several approaches for measuring plasma total homocysteine by HPLC have been described during the last few years (1)(2)(3)(4), none combines all the desired features for a rapid, user-friendly, and robust assay: (a) a stable, efficient, and nonhazardous reducing agent; (b) incorporation of a suitable internal standard; and (c) rapid, isocratic separation of the thiols of interest, using a mobile phase of mild pH. We have therefore modified the method of Vester and Rasmussen (5) by using tris(2-carboxyethyl)phosphine (TCEP), a newer stable, water-soluble phosphine derivative introduced by Gilfix et al. (6), as the reducing agent, cystamine as the internalnd isocratic separation of the thiols extracted from only 50 μL of pla standard, asma within 6 min.
A mixture of 50 μL of plasma, 25 μL of internal standard, and 25 μL of phosphate-buffered saline (PBS, pH 7.4) was incubated with 10 μL of 100 g/L TCEP (Pierce Chemical Co.) for 30 min at room temperature to reduce and release protein-bound thiols, after which 90 μL of 100 g/L trichloroacetic acid containing 1 mmol/L EDTA was added for deproteinization. After the sample was centrifuged for 10 min at 13000 g , 50 μL of the supernatant was added to an autosampler vial containing 10 μL of 1.55 mol/L NaOH; 125 μL of 0.125 mol/L borate buffer containing 4 mmol/L EDTA, pH 9.5; and 50 μL of 1 g/L SBD-F (Wako Chemicals) in the borate buffer. The sample was then incubated for 60 min at 60 °C. HPLC was carried out on a 2690 Alliance solvent delivery system …
379 citations
TL;DR: Abnormally high concentrations of circulating fetal DNA are found in a proportion of women carrying fetuses with trisomy 21, and the robustness and reproducibility of real-time PCR analysis of maternal plasma makes it a valuable tool for cross-institutional collaboration involving centers located in different parts of the world.
Abstract: Background: The recent discovery of the presence of circulating cell-free fetal DNA in maternal plasma opens up new prenatal diagnostic applications and provides new avenues for clinical investigation. It is of research and potential diagnostic interest to determine whether fetal trisomy 21 may be associated with quantitative abnormalities of circulating fetal DNA in maternal plasma.
Methods: Maternal plasma samples were prospectively collected from two centers situated in Hong Kong and Boston. Samples collected from Boston consisted of 7 women carrying male trisomy 21 fetuses, 19 carrying euploid male fetuses, and 13 carrying female fetuses. Samples collected from Hong Kong consisted of 6 women carrying male trisomy 21 fetuses, 18 carrying euploid male fetuses, and 10 carrying female fetuses. Male fetal DNA in maternal plasma was measured using real-time quantitative Y-chromosomal PCR.
Results: For patients recruited from Boston, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 46.0 genome-equivalents/mL and 23.3 genome-equivalents/mL, respectively ( P = 0.028). For patients recruited from Hong Kong, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 48.2 genome-equivalents/mL and 16.3 genome-equivalents/mL, respectively ( P = 0.026). None of the samples from women carrying female fetuses had detectable Y-chromosomal signals.
Conclusions: Abnormally high concentrations of circulating fetal DNA are found in a proportion of women carrying fetuses with trisomy 21. The robustness and reproducibility of real-time PCR analysis of maternal plasma makes it a valuable tool for cross-institutional collaboration involving centers located in different parts of the world.
TL;DR: Bone markers have been useful in clinical practice and have been helpful in understanding the pathogenesis of osteoporosis and the mechanism of action of therapies, and in clinical trials, markers aid in selecting optimal dose and inUnderstanding the time course of onset and resolution of treatment effect.
Abstract: Remodeling is essential for bone health. It begins with resorption of old bone by osteoclasts, followed by the formation of new bone by osteoblasts. Remodeling is coupled (formation is linked to resorption). After middle age or perhaps beginning earlier, bone loss occurs because resorption exceeds formation. This imbalance is accentuated by estrogen deficiency as well as by many diseases and conditions. Biochemical markers that reflect remodeling and can be measured in blood or urine include resorption markers (e.g., collagen cross-links) and formation markers (e.g., alkaline phosphatase). Bone markers exhibit substantial short-term and long-term fluctuations related to time of day, phase of the menstrual cycle, and season of the year, as well as diet, exercise, and anything else that alters bone remodeling. These biological factors, in addition to assay imprecision, produce significant intra- and interindividual variability in markers. Bone marker measurements are noninvasive, inexpensive, and can be repeated often. Unfortunately, most of the studies that provided insight on clinical situations did not focus on markers as a primary endpoint. Bone markers have been useful in clinical practice and have been helpful in understanding the pathogenesis of osteoporosis and the mechanism of action of therapies. In clinical trials, markers aid in selecting optimal dose and in understanding the time course of onset and resolution of treatment effect. Clinical questions that might be answered by bone markers include diagnosing osteoporosis, identifying "fast bone losers" and patients at high risk of fracture, selecting the best treatment for osteoporosis, and providing an early indication of the response to treatment. Additional information is needed to define specific situations and cut points to allow marker results to be used with confidence in making decisions about individual patients.
TL;DR: Software-optimized DHPLC is a highly sensitive method for mutation detection, however, where sensitivity >96% is required, the data suggest that in addition to the recommended temperature, fragments should also be run at the recommendedTemperature plus 2 degrees C.
Abstract: Background: Denaturing HPLC (DHPLC) is a semi-automated method for detecting unknown DNA sequence variants. The sensitivity of the method is dependent on the temperature at which the analysis is undertaken, the selection of which is dependent on operator experience. To circumvent this, software has been developed for predicting the optimal temperature for DHPLC analysis. We examined the utility of this software.
Methods: To maximize the relevance of our data for other investigators, we have screened 42 different amplimers from CFTR, TSC1, and TSC2. The samples consisted of 103 unique sequence heterozygotes and 126 wild-type homozygous controls.
Results: At the temperature recommended by the software, 96% (99 of 103) of heterozygotes and all of the wild-type controls were correctly classified. This compares favorably with sensitivities of 85% for single-stranded conformation polymorphism and 82% for gel-based heteroduplex analyses of the same fragments.
Conclusions: Software-optimized DHPLC is a highly sensitive method for mutation detection. However, where sensitivity >96% is required, our data suggest that in addition to the recommended temperature, fragments should also be run at the recommended temperature plus 2 °C.
TL;DR: A new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.
Abstract: Background: Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations. Methods: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA. Results: The dynamic range was >1000-fold. The relationship between Ct and log starting concentration was linear (r2 ≥0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (>5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio ( Conclusions: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.
TL;DR: This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLC-based methods.
Abstract: Affinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLC-based methods. Some traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on- or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with other analytical methods, such as reversed-phase liquid chromatography, gas chromatography, and capillary electrophoresis. Indirect analyte detection methods are also described in which immunoaffinity chromatography is used to perform flow-based immunoassays. Other applications that are reviewed include affinity-based chiral separations and the use of affinity chromatography for the study of drug or hormone interactions with binding proteins. Some areas of possible future developments are then considered, such as tandem affinity methods and the use of synthetic dyes, immobilized metal ions, molecular imprints, or aptamers as affinity ligands for clinical analytes.
TL;DR: Clinically, ZnPP quantification is valuable as a sensitive and specific tool for evaluating iron nutrition and metabolism, and has a potential therapeutic application in controlling bilirubin formation in neonates as a preventive measure for hyperbilirubinemia.
Abstract: Zinc protoporphyrin (ZnPP) is a normal metabolite that is formed in trace amounts during heme biosynthesis. The final reaction in the biosynthetic pathway of heme is the chelation of iron with protoporphyrin. During periods of iron insufficiency or impaired iron utilization, zinc becomes an alternative metal substrate for ferrochelatase, leading to increased ZnPP formation. Evidence suggests that this metal substitution is one of the first biochemical responses to iron depletion, causing increased ZnPP to appear in circulating erythrocytes. Because this zinc-for-iron substitution occurs predominantly within the bone marrow, the ZnPP/heme ratio in erythrocytes reflects iron status in the bone marrow. In addition, ZnPP may regulate heme catabolism through competitive inhibition of heme oxygenase, the rate-limiting enzyme in the heme degradation pathway that produces bilirubin and carbon monoxide. Physiological roles, especially relating to carbon monoxide and possibly nitric oxide production, have been suggested for ZnPP. Clinically, ZnPP quantification is valuable as a sensitive and specific tool for evaluating iron nutrition and metabolism. Diagnostic determinations are applicable in a variety of clinical settings, including pediatrics, obstetrics, and blood banking. ZnPP analytical methodologies for clinical studies are discussed. In addition to diagnostic tests and metabolic studies, ZnPP has a potential therapeutic application in controlling bilirubin formation in neonates as a preventive measure for hyperbilirubinemia. Biochemical research techniques, both in vivo and in vitro, are described for further studies into the role of ZnPP in metabolism and physiology.
TL;DR: In vitro and in vivo measurements showed that the sensitivity of the glucose measurement is unaffected by the presence of common blood analytes but that there can be substantial shifts in baseline values, indicating the need for spectroscopic data to develop algorithms for the detection of glucose in the absence of other analytes.
Abstract: We report here on in vitro and in vivo experiments that are intended to explore the feasibility of photoacoustic spectroscopy as a tool for the noninvasive measurement of blood glucose. The in vivo results from oral glucose tests on eight subjects showed good correlation with clinical measurements but indicated that physiological factors and person-to-person variability are important. In vitro measurements showed that the sensitivity of the glucose measurement is unaffected by the presence of common blood analytes but that there can be substantial shifts in baseline values. The results indicate the need for spectroscopic data to develop algorithms for the detection of glucose in the presence of other analytes.
TL;DR: The major hormones that are responsible for normal calcium homeostasis are parathyroid hormone and 1,25-dihydroxyvitamin D; these hormones control extracellular fluid calcium on a chronic basis and are the major causes of aberrant extrace cellular fluid calcium concentrations.
Abstract: Calcium homeostasis in the extracellular fluid is tightly controlled and defended physiologically. Hypercalcemia always represents considerable underlying pathology and occurs when the hormonal control of calcium homeostasis is overwhelmed. The major hormones that are responsible for normal calcium homeostasis are parathyroid hormone and 1,25-dihydroxyvitamin D; these hormones control extracellular fluid calcium on a chronic basis. Over- or underproduction of these hormones or the tumor peptide, parathyroid hormone-related peptide, are the major causes of aberrant extracellular fluid calcium concentrations. These hormonal defense mechanisms are reviewed here.
TL;DR: The ability of flow cytometry to simultaneously acquire data for numerous particles and subsequently analyze multiple characteristics for each particle makes it a powerful technique for cell sorting and cell analyses.
Abstract: The study of cytokines has been of great interest to researchers in many scientific disciplines, including cell biology and immunology. Cytokines form a sophisticated network that serves to modulate a myriad of cellular events. Within such a network, and through complex feedback mechanisms, cytokine functions are mostly interdependent. Because of the extreme complexity of the cytokine network, a simultaneous measurement of multiple cytokines in a single sample represents a desirable and effective approach.
Several methods are available to measure cytokines and their messenger RNAs. To measure cytokines secreted from cells, researchers commonly use conventional ELISA techniques. ELISA methods are generally cost-effective but restricted to measuring one cytokine at a time. Consequently, the ELISA approach requires not only multiple sample aliquots but also repetitive execution of the procedures for each cytokine of interest. The ability of flow cytometry to simultaneously acquire data for numerous particles and subsequently analyze multiple characteristics for each particle makes it a powerful technique for cell sorting and cell analyses. The application is fundamentally built on the high sensitivity of a flow cytometer to discern characteristics either within or on the surface of a cell. Several investigators have constructed microparticle-based immunoassays that use a flow cytometer …
TL;DR: This work has developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry, and was able to quantitatively detect 10 β-Actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA.
Abstract: The use of common reverse transcription (RT)-PCR reference target sequences can produce false-positive results by amplification of either contaminating DNA or processed pseudogenes. Furthermore, qualitative RT-PCR alone cannot distinguish between high- and poor-quality cDNA preparations, which again may be crucial for the interpretation of low-abundance transcripts. We have developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry. Through this technique, we were able to quantitatively detect 10 β-actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA. Thus, the presented method may be advantageous for the interpretation of quantitative RT-PCR results.
PCR analysis may be difficult to interpret unless a reference target sequence is amplified in parallel. This control reaction is necessary to evaluate whether a sufficient amount of amplifiable material is present in the sample investigated. Thus, negative PCR can be defined as such only when amplification of a reference gene reveals a positive result. Amplifying a reference is especially important in RT-PCR because RNA can be degraded rapidly before or during cDNA synthesis.
As reference sequences for RT-PCR, so called “housekeeping” genes are preferred because they are constitutionally expressed by all cell types. β-Actin is an attractive candidate for reference coamplification because it exhibits only minor intraindividual kinetic changes and is not primarily affected by any human disease (1). However, a major concern with β-actin and other commonly used references such as glyceraldehyde-3-phosphate dehydrogenase is that processed pseudogenes can be coamplified by primers that are originally restricted to cDNA. This problem is difficult to circumvent because sequence homologies of >90% exist between mRNA and the respective pseudogenes (2); thus, the pseudogenes usually cannot be distinguished in gel electrophoresis. Furthermore, as long as genomic DNA contamination cannot safely be excluded, DNA may be coamplified together with cDNA. Thus, positive analysis of …
TL;DR: Clinical intervention studies have demonstrated that reduction in the chronic microvascular and macrovascular complications of type 2 diabetes requires treatment of hyperglycemia, and oral antihyperglycemic agents increase endogenous insulin secretion, decrease insulin resistance, or lower postprandial plasma glucose rise by delaying absorption of complex carbohydrates.
Abstract: Type 2 diabetes is a heterogeneous disorder. Clinical expression of the disorder requires both genetic and environmental factors. One theory concerning its etiology is that it is the result of the evolution of a thrifty genotype that had survival benefits in the past but is detrimental in the current environment. An opposing theory is that it represents an adult metabolic response to fetal malnutrition. Hyperglycemia in type 2 diabetes results from absolute or relative insulin deficiency. Most often relative insulin deficiency is attributable to an inability to adequately compensate for insulin resistance. Insulin resistance may be caused by a variety of genetic or metabolic factors. The most common etiological factor in insulin resistance is central obesity. Insulin resistance is associated with a cluster of metabolic abnormalities that include glucose intolerance, hypertension, a unique dyslipidemia, a procoagulant state, and an increase in macrovascular disease. Clinical intervention studies have demonstrated that reduction in the chronic microvascular and macrovascular complications of type 2 diabetes requires treatment of hyperglycemia to achieve hemoglobin A1c <7.0%, blood pressure ≤130/80 mmHg, and plasma LDL-cholesterol ≤2.6 mmol/L (≤100 mg/dL). Oral antihyperglycemic agents increase endogenous insulin secretion, decrease insulin resistance, or lower postprandial plasma glucose rise by delaying absorption of complex carbohydrates. Long-term glycemic control in type 2 diabetes requires progressive, stepwise, combination treatment with oral agents and eventually combination treatment with oral agents and insulin.
TL;DR: The Triage panel offers clinicians a whole blood, point-of-care analysis of multiple cardiac markers that provides excellent clinical sensitivity and specificity for the detection of acute MI.
Abstract: This multicenter study evaluated the Biosite Triage® Cardiac Panel as a quantitative, multimarker, whole blood system for the detection of acute myocardial infarction (MI). Optimum cutoffs for the discrimination of acute MI (n = 192 patients, 59 with MI) as determined by ROC curve analyses were as follows: 0.4 μg/L for cardiac troponin I (cTnI); 4.3 μg/L for the creatine kinase MB isoenzyme (CK-MB); and 107 μg/L for myoglobin. The Triage Panel showed the following concordances for detection or rule-out of MI compared with established devices: cTnI >89%; CK-MB >81%; myoglobin >69%. No significant differences were present between methods for the same marker. Diagnostic efficiencies demonstrated comparable sensitivities and specificities for the diagnosis of MI in patients presenting with symptoms compared with the Dade, Beckman, and Behring CK-MB, cTnI, and myoglobin assays; the ratio of sensitivity to specificity for each marker was as follows: cTnI, 98%:100%; CK-MB, 95%:91%; myoglobin, 81%:92%. The areas under the ROC curves for the Biosite myoglobin, CK-MB, and cTnI were 0.818, 0.905, and 0.970, respectively; the areas were significantly different, P <0.05. In patients with skeletal muscle injury and renal disease, the Triage cTnI showed 94% and 100% specificity, respectively. The Triage panel offers clinicians a whole blood, point-of-care analysis of multiple cardiac markers that provides excellent clinical sensitivity and specificity for the detection of acute MI.
TL;DR: Quality specifications can be derived from analysis of performance on clinical decision-making, and the idea of utilizing quality specifications in assessing the acceptability of method performance was firmly stated in 1974.
Abstract: The discipline of clinical chemistry is dynamic. Even a superficial glance at contents pages of the Journal over recent years demonstrates a marked evolution of the field. Appropriately, the Information for authors has also changed over the years to reflect new concepts of what constitutes an acceptable publication.
Readers of the Journal may have noticed an addition to the 1999 Information for authors (1), under the heading Description of Analytical Methods and Results , namely:
“ Analytical quality. Results obtained for the performance characteristics should be compared objectively to well-documented quality specifications, e.g., published data on the state of the art, performance required by regulatory bodies such as CLIA ‘88, or recommendations documented by expert professional groups”. In addition, quality specifications can be derived from analysis of performance on clinical decision-making.
Many manuscripts deal with the development of new analytical methods or evaluation of commercially available analytical systems. Experimental designs and statistical techniques used to derive data on imprecision and bias are generally more than satisfactory. This is hardly surprising in view of the very many published protocols for the evaluation of methods (2). In contrast, objective analysis of whether the imprecision and bias are satisfactory is often less well done.
This is not a new phenomenon. Although the idea of utilizing quality specifications in assessing the acceptability of method performance was firmly stated in 1974 (3), it was pointed out more than a decade ago that few evaluators actually did compare the performance achieved with preset quality specifications (4)(5). This seems rather difficult to understand because quality specifications based on the state of the art (6), the views of an expert individual (7), and biological variation (8) had been available for many years. Indeed, even a superficial reading of the more recent literature would demonstrate that many …
TL;DR: The results of measuring the concentrations of both GFAP and S-100 protein in the blood of patients with acute severe head trauma and healthy controls are described, using the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) system for detection of GFAP.
Abstract: Glial fibrillary acidic protein (GFAP), found only in glial cells of the central nervous system (CNS) (1)(2), was first isolated by Eng et al. (1) in 1971. GFAP is a monomeric molecule with a molecular mass between 40 and 53 kDa (3)(4) and an isoelectric point between 5.7 and 5.8 (2). GFAP represents the major part of the cytoskeleton of astrocytes. DeArmond et al. (5) reported on the spontaneous degradation of GFAP in vitro and in vivo; under physiological conditions, GFAP polymerizes spontaneously to astrofibrils with a length of 0.8–1.06 μm.
Numerous reports in the literature document the usefulness of measuring GFAP in cerebrospinal fluid (CSF) as a specific indicator of CNS pathology (6)(7)(8)(9)(10)(11)(12)(13). Measuring GFAP in blood would have clinical advantages over CSF measurements; however, to our knowledge, the only method attempted to date to measure GFAP in blood (14) was not successful.
We describe the results of measuring the concentrations of both GFAP and S-100 protein in the blood of patients with acute severe head trauma and healthy controls, using the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) system for detection of GFAP (DELFIA can also be used to measure the concentration of GFAP in CSF).
Blood samples were obtained from 25 patients (19 males and 6 females; ages, 16–72 years; mean age, 38.6 ± 19.0 years) who were admitted <24 h after severe head trauma (a Glasgow Coma Scale score of ≤6 on admission). Three samples were obtained from each patient: the first sample was obtained on admission to the hospital, the second was obtained 24 h after the head injury, and the third was obtained 48 h after the injury.
Blood samples were also obtained from 70 healthy blood donors …
TL;DR: The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C. trachomatis and N. gonorrhoeae and other organisms.
Abstract: Background: Amplified DNA probes provide powerful tools for the detection of infectious diseases, cancer, and genetic diseases. Commercially available amplification systems suffer from low throughput and require decontamination schemes, significant hands-on time, and specially trained laboratory staff. Our objective was to develop a DNA probe system to overcome these limitations.
Methods: We developed a DNA probe system, the BDProbeTecTMET, based on simultaneous strand displacement amplification and real-time fluorescence detection. The system uses sealed microwells to minimize the release of amplicons to the environment. To avoid the need for specially trained labor, the system uses a simple workflow with predispensed reagent devices; a programmable, expandable-spacing pipettor; and the 96-microwell format. Amplification and detection time was 1 h, with potential throughput up to 564 patient results per shift. We tested 122 total patient specimens obtained from a family practice clinic with the BD ProbeTecET and the Abbott LCx® amplified system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae .
Results: Based on reportable results, the BDProbeTecET results for both organisms were 100% sensitive and 100% specific relative to the LCx.
Conclusions: The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C. trachomatis and N. gonorrhoeae and other organisms.
TL;DR: This work used nasopharyngeal carcinoma (NPC) as a model system and attempted to detect Epstein-Barr virus-latent gene transcripts in cell-free plasma samples from NPC patients, suggesting that the EBV genome and latency products may serve as potential markers for the screening and diagnosis of this cancer.
Abstract: Chen et al. (1) and Nawroz et al. (2) have reported that tumor-derived DNA is detectable in the plasma and serum of cancer patients and have opened up a new molecular approach for the early detection of malignancy. It is not known, however, whether circulating tumor-derived RNA is also present in plasma, because of the lability of RNA. To address this possibility, we used nasopharyngeal carcinoma (NPC) as a model system and attempted to detect Epstein-Barr virus (EBV)-latent gene transcripts in cell-free plasma samples from NPC patients.
NPC constitutes one of the commonest cancers in Hong Kong and Southern China (3). Previous studies have indicated that EBV is consistently detected in all undifferentiated NPC cases and is present in all cancer cells (3). Latent EBV infection is an early event in the development of this cancer (4). These findings suggested that the EBV genome and latency products may serve as potential markers for the screening and diagnosis of this cancer. Among the EBV-latent genes, the small EBV-encoded RNAs (EBERs) are expressed in all NPC cases and are the most abundant latency-associated transcripts in NPC cells (∼105 to 106 …
TL;DR: This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion and can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.
Abstract: Background: A genetic syndrome of cutaneous malignant melanoma and nervous system tumors recently has been characterized and shown to be linked to the INK4 locus in the 9p21 region. Hemizygosity at adjacent physically mapped microsatellite markers indicated deletion of p16 , p19 , and p15 clustered tumor suppressors. Because individuals from this family could benefit from predictive testing in terms of cancer prevention, we developed a direct test without need to analyze parental DNAs to comply with the rules of individual consent and secrecy.
Methods: We developed an assay using TaqManTM real-time quantitative PCR, with p15 as the test sequence and albumin ( ALB ) as the reference gene. The normalized ratio of p15 / ALB is expected to yield a value of ∼1 in individuals without the deletion, whereas a ratio of ∼0.5, indicating p15 haploinsufficiency, is expected in predisposed individuals.
Results: All patients harboring the previously defined at-risk haplotype were correctly identified using this approach. In six individuals with deletions, the p15 / ALB ratios were 0.472–0.556 (SD, 0.013–0.078). In the five individuals without deletions, the ratios were 0.919–1.019 (SD, 0.006–0.075).
Conclusions: This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion. This approach, not limited by marker informativeness or the need for harvesting live cells, can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.
TL;DR: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.
Abstract: Background: Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround.
Methods: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 μL of either plasma or urine with homocystine- d 8 (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy- d 4 were detected through the transition from the precursor to the product ion ( m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy- d 4 was 1.5 min in a 2.5-min analysis.
Results: Daily calibrations between 2.5 and 60 μmol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 μmol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method ( y ) and the LC-MS/MS method ( x ) was y = 1.097 x − 1.377 ( r = 0.975; S y|x =1.595 μmol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y ) and LC-MS/MS ( x ) was y = 1.039 x + 0.025 ( r = 0.969; S y|x =1.146 μmol/L; n = 367). Inter- and intraassay CVs were 2.9–5.9% and 3.6–5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 μmol/L. Mean recovery of tHcy was 94.2% (20 μmol/L) and 97.8% (50 μmol/L).
Conclusions: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.
TL;DR: Genotype-based reference intervals may be a way to increase the clinical utility of CA 19-9 and investigate the biological variation of CA19-9, and evaluate the utility of Lewis and secretor genotyping on a group of individuals with serologically defined Lewis phenotypes.
Abstract: The concentration of the tumor marker CA 19-9 is influenced by the patient’s secretor status and Lewis genotype. The aim of this study was to establish novel reference intervals for CA 19-9 in serum based on secretor and Lewis genotypes, to investigate the biological variation of CA 19-9, and to evaluate the utility of Lewis and secretor genotyping on a group of individuals with serologically defined Lewis phenotypes. CA 19-9 was measured in serum of 500 healthy individuals. Secretor and Lewis genotypes were determined by sequencing and PCR-cleavage methods. Significant differences were found between subgroups with different Lewis and secretor genotypes. Genotype-based reference intervals for CA 19-9 are presented. The upper reference limit for all individuals was 28.7 kilounits/L; for secretors and nonsecretors, the upper reference limits were 12.4 and 61.2 kilounits/L, respectively. The analytical imprecision (CVA) was 9.8%, the within-subject variability (CVI) was 15.8%, and the between-subject variability (CVG) was 102.2%. Good agreement was found between Lewis and secretor genotyping and conventional blood grouping. Genotype-based reference intervals may be a way to increase the clinical utility of CA 19-9. On the basis of the calculation of a critical difference for sequential values (significant at P ≤0.05) of 51.5%, a 40–50% change in marker concentration is suggested as the limit for significant change when the marker is used for follow up. PCR-based genotyping is a reliable method for secretor and Lewis histo-blood grouping.
TL;DR: Direct evidence of the absorption of anthocyanins in humans is reported by combining an octadecylsilane (ODS) solid-phase extraction procedure for plasma sample preparation and an HPLC system with diode array for Anthocyanin separation and detection.
Abstract: Anthocyanins are a group of natural antioxidants (1)(2)(3)(4) widely distributed in fruits and vegetables. Anthocyanins have two absorbance peaks, at 270–280 nm and 510–540 nm, respectively. The intake of anthocyanins in humans has been estimated to be 180–215 mg/day in the US (5), which is much higher than the intake (23 mg/day) of other flavonoids, including quercetin, kaempferol, myricetin, apigenin, and luteolin (6). Various biological and pharmacological activities of anthocyanins have been reported using crude fruit extracts, which are rich in anthocyanins (7). However, the absorption of dietary anthocyanins has never been shown clearly in humans, although one substance with an absorbance spectrum similar to those of the anthocyanins was reported in the plasma of nonsupplemented human subjects (8), and anthocyanin-like compounds have been found in human urine (9). We report here direct evidence of the absorption of anthocyanins in humans, which was obtained by combining an octadecylsilane (ODS) solid-phase extraction procedure for plasma sample preparation and an HPLC system with diode array for anthocyanin separation and detection.
One male subject (one of the authors), 35 years of age, consumed 25 g of elderberry extract containing 1.5 g of total anthocyanins after fasting overnight. The elderberry extract is commercially available as a nutritional supplement for humans. Its main constituents are anthocyanins, mainly cyanidin 3-glucoside and cyanidin 3-sambubioside; other polyphenols were very limited (10). Blood samples were obtained before and 30 and 60 min after anthocyanin consumption. The EDTA blood samples were centrifuged at 500 g for 10 min at 4 °C, and the plasma was quickly removed. A …