The number of positive axillary lymph nodes (LNs) is the only node-related factor for prognostic evaluation of breast cancer recognized by AJCC (TNM staging). However, N staging may not completely reflect LN tumor involvement due to the erroneous count of LNs in the presence of matted LNs and different tumor volume in LNs. Additionally, the positive/total LN ratio (LNR) has been shown to outperform N staging in survival prediction. In our study, to better quantify the tumor involvement of axillary LNs, we measured the cross-sectional cancer area (CSCA) of the positive LNs in 292 breast cancer patients diagnosed between 1998 and 2000 in our institution and compared its prognostic value to that of number of positive LNs (metLN)/N stage and LNR. Statistical analyses of these three LN-related factors were performed by Kaplan-Meier method and multivariate Cox's regression model. Patients were divided into three groups based on the different LN CSCA (<50, 50-500, and >500 mm(2)), or LNR (<0.1, 0.1-0.65, and >0.65), or N stage (N1-N3). Multivariate analysis demonstrated LNR was the most significant LN-related survival predictor with hazard ratio (HR) 25.0 (P = 0.001), compared to the metLN (HR 0.09, P = 0.052) and CSCA (HR 2.24, P = 0.323).

Lung cancer is the second most common cancer in the United States. Since therapeutic regimens target specific mutations, molecular testing has become routine practice. Molecular analysis is expensive and poses a burden on rising health care costs. The present study was undertaken to see if pathologic features are predictive of molecular alterations in lung cancers. A retrospective search of …

Mass spectrometric analysis of serum is a promising approach to biomarker discovery and PCa diagnosis. We determined serum protein expression patterns in 148 patients that were subsequently evaluated by biopsy of the prostate. Serum biomarkers were selected by metal affinity chromatography on copper-activated IMAC 30 chips and displayed by SELDI -TOF-MS using the PBS II analyzer (Ciphergen). Detected peaks with m/z ratios in the range of 1000 to 40,000, S/N ratios > 1.2, and intensities > 0.1 were grouped by m/z ratio so as to define 573 parameters of the serum biomarker profile. Parameters that discriminated the Ca from the non-Ca group were identified by binary logistic regression. Logistic regression models were evaluated in a randomly chosen set of 98 training cases (28 Ca, 70 Non-Ca), and tested in an independent set of 50 test cases (21 Ca, 29 Non-Ca). We derived a 10-parameter model that had a sensitivity (Se) of 0.79 and a specificity (Sp) of 0.89, for predicting the presence of PCa in a subsequent biopsy for the cases in the training set. However, Se and Sp for this model decreased to 0.50 and 0.76 respectively, in the test set. As the number of parameters in the models was reduced from 10 to 2, there was a progressive decrease in Se, but Sp remained at 0.70 or above in models that included the parameter corresponding to a protein peak with an m/z ratio of 14,900. This biomarker might prove to be useful for increasing the specificity of serum screening for PCa. Our results indicate that protein profiling by this method does not have the level of diagnostic accuracy required for routine clinical applications, possibly owing to excessive run-to-run analytical variability and/or to the limited number of cases that were analyzed.

Serum free light chain measurements have been shown to be useful in the diagnosis and monitoring of patients with monoclonal gammopathies. The present study was undertaken to evaluate the effect of adding the measurement of serum free light chain kappa to lambda ratios to the serum protein electrophoresis evaluation that we typically use as an initial screen for the detection of monoclonal proteins. We retrospectively tested 347 consecutive samples from individuals who had no previous history of plasma cell dyscrasia and had not previously had a serum or urine electrophoresis or immunofixation electrophoresis test at our institution. The quantitative serum protein electrophoresis test that was ordered was performed using Hydragel Beta 1- Beta 2 gels and Hydrasis instrument (Sebia, Inc., Norcross, GA). The protein content of the electrophoresis zones were quantitated by scanning densitometry and the electrophoresis pattern of each sample was qualitatively examined for abnormal bands and suspicious findings by a single, experienced observer. Serum free light chain concentrations and the serum free light chain kappa to lambda ratios were determined using the Freelite Human Kappa and Lambda Kits (The Binding Site Ltd, Birmingham, UK) and the Immage analyzer (Beckman Coulter Inc., Brea, CA). The serum free light chain kappa to lambda ratios were outside the reference interval (0.25 to1.65) in 23 of the samples. Ten abnormal ratios were observed among a group of 57 samples that had either positive or suspicious qualitative evaluations for the presence of a restriction or that demonstrated hypo-gammaglobulinemia. Both abnormalities led to recommendations for follow-up testing, which confirmed the presence of a monoclonal protein in 21 of the samples. Six abnormal ratios were observed among a group of 159 specimens that had quantitative abnormalities in albumin or one or more of globulin fractions (hypo-gammaglobulinemia excepted) and normal qualitative evaluations. Seven abnormal ratios were observed among a group of 131 samples that had normal quantitative results and normal qualitative evaluations. Follow-up testing is not usually recommended for serum protein electrophoresis results like those in the latter two groups. We found that the addition of the serum free light chain kappa to lambda ratio to the serum protein electrophoresis test increased the number of abnormal screens that would have required further clinical and/or laboratory evaluation by 23%(i.e. from 57 to 70). Given the high specificity of the serum free light chain kappa to lambda ratio for monoclonal light chains, the additional 13 abnormal samples identified by this test are expected to have a high likelihood of harboring a monoclonal protein that would have otherwise eluded detection. Pending a definitive prospective study, we estimate that the addition of a serum free light chain kappa to lambda ratio to the serum protein electrophoresis screen would increase the rate of detection of serum monoclonal proteins by as much as 1.6-fold.

Purpose: The most widely accepted method for the evaluation of intestinal barrier integrity is the measurement of the permeation of sugar probes following an oral test dose of sugars. The most-widely used sugar probes are sucrose, lactulose, mannitol and sucralose. Measuring these sugars using a sensitive gas chromatographic (GC) method, we noticed interference on the area of the lactulose and mannitol peaks. Methods: We tested different sugars to detect the possible makeup of these interferences and finally detected that the lactose interferes with lactulose peak and fructose interferes with mannitol peak. On further developing of our method, we were able to reasonably separate these peaks using different columns and condition for our assay. Sample preparation was rapid and simple and included adding internal standard sugars, derivitization and sily-lation. We used two chromatographic methods. In the first method we used Megabore column and had a run time of 34 minutes. This resulted in partial separation of the peaks. In the second method we used thinner and longer capillary column and was able to reasonably separate the lactose and lactulose peaks and the mannitol and fructose peaks with run time of 22 minutes. Results: The sugar probes including mannitol, sucrose, lactulose and sucralose and fructose and lactose were detected precisely, without interference. The assay was linear between lactulose concentrations of 0.5 and 40 g/L(r2 = 1.000, p <0.0001) and mannitol concentrations of 0.01 and40g/L (r2 = 1.000). The sensitivity of this method remained high using new column and assay condition. The minimum detectable concentration calculated for both methods was 0.5 mg/L for lactulose and 1 mg/L for mannitol. Conclusions: This is the first report of interference of commonly used sugars with test of intestinal permeability. These sugars are found in most of fruits and dairy products and could easily interfere with the result of permeability tests. Our new GC assay of urine sugar probes permits the simultaneous quantitation of sucralose, sucrose, mannitol and lactulose, without interference with lactose and fructose. This assay is a rapid, simple, sensitive and reproducible method to accurately measure intestinal permeability.

We developed a capillary column gas chromatography (CCGC) method for the measurement of urinary sucralose (S) and three other sugar probes including, sucrose, lactulose (L) and mannitol (M) for use in in vivo studies of intestinal permeability. We compared the capillary method with a packed column gas chromatography (PCGC) method. We also investigated a possible role for sucralose as a probe for the measurement of whole gut permeability. Sample preparation was rapid and simple. The above four sugars were detected precisely, without interference. We measured intestinal permeability using 5- and 24-h urine collections in 14 healthy volunteers. The metabolism of sugars was evaluated by incubating the intestinal bacteria with an iso-osmolar mixture of mannitol, lactulose and sucralose at 37°C for 19 h. Sugar concentrations and the pH of the mixture were monitored. The use of the CCGC method improved the detection of sucralose as compared to PCGC. The average coefficient of variation decreased from 15% to 4%. It also increased the sensitivity of detection by 200–2000-fold. The GC assay was linear between sucralose concentrations of 0.2 and 40 g/l (r=1.000). Intestinal bacteria metabolized lactulose and acidified the media but did not metabolize sucralose or mannitol. The new method for the measurement of urinary sucralose permits the simultaneous quantitation of sucrose, mannitol and lactulose, and is rapid, simple, sensitive, accurate and reproducible. Because neither S nor M is metabolized by intestinal bacteria, and because only a tiny fraction of either sugar is absorbed, this pair of sugar probes appears to be available for absorption throughout the GI tract. Thus, the 24-h urinary concentrations of S and M, or the urinary S/M ratio following an oral dose of a sugar mixture, might be good markers for whole gut permeability.

BACKGROUND:A number of variables influence the effect(s) of alcohol on distinct segments of the intestine. In these studies, we examined the effect of T-cell activation on gastric and small bowel permeability in alcohol-fed mice.METHODS:Gastric permeability was assessed using sucrose absorption, whereas small bowel permeability was followed using the ratio of lactulose to mannitol absorption and inulin absorption. T cells were activated by injecting antigen OVA(323-339) into DO11.10 T-cell receptor transgenic mice.RESULTS:T-cell activation increased gastric and small bowel permeability through a pathway mediated by interferon-gamma and tumor necrosis factor. In mice that were fed a liquid diet that contained 30% ethanol-derived calories for 2 weeks, T-cell activation increased gastric permeability to levels greater than that observed in solid diet or pair-fed, liquid control diet. By comparison, changes in small bowel permeability induced by T-cell activation were abrogated in alcohol-fed mice. Analysis of intestinal cytokine mRNA levels (interferon-gamma and tumor necrosis factor) indicated that relevant mucosal T-cell function was preserved in alcohol-fed mice.CONCLUSIONS:Overall, these data suggest that alcohol potentiates the effects of T-cell activation on gastric permeability, at the same time blunting effects on small bowel permeability

The hepatic levels of three protein markers of oxidative stress, polymerase beta, Ref-1, and PCNA, and of the pro-apoptotic protein, Bax, were quantitated after exposure to WY 14,643 (500 ppm in the feed) for 6 or 34 days in a rodent that is susceptible peroxisome proliferator (PP)-induced liver tumors (the Sprague Dawley rat) and in a rodent that is relatively resistant PP-induced liver tumors (the Syrian hamster). The analysis of detergent-extracted whole liver homogenates by immunoblotting showed a marked increase in the abundance of a 45-kDa variant of polymerase beta immunoreactivity and significant increases in the expression of Ref-1 and PCNA in WY 14,643-exposed rats. In contrast. WY 14,643-exposed hamsters expressed only trace levels of the polymerase beta variant and showed significant decreases in the expression of Ref-1 and PCNA. Long-term WY 14,643 exposure was associated with marked decreases in Bax expression in both species. Dose-response studies in the rat showed that the hepatic expression of the polymerase beta and Ref-1 were significantly increased after 6 days of exposure to WY 14,643 at levels of 5 and 50 ppm, respectively. The analysis of subcellular fractions of rat liver showed that the pathological increases in the levels of polymerase beta, Ref-1, and PCNA were especially prominent in mitochondria-enriched particulate liver subfractions. These results indicate that WY 14,643 exposure is associated with an increase in oxidative stress to the liver and that liver mitochondria are a major target of WY 14,643-associated liver damage. Our data are consistent with the hypothesis that the chronic overexpression of mutagenic or oncogenic effectors like polymerase beta and Ref-1 in a setting of increased hepatocyte proliferation and decreased apoptosis may facilitate peroxisome proliferator-induced hepatocellular carcinoma in the rat.

Edited by Danielle B. Freedman, James Hooper, Philip J. Wood, David J. Worthington, and Christopher P. Price, 252 pp, with illus, Washington, DC, AACC Press, 2001.This collection of 48 clinical case presentations, which were contributed by a group of clinical biochemists and chemical pathologists from institutions located throughout the United Kingdom, is intended to provide the holistic view of clinical problems, diagnostic processes, and intervention strategies that may be lacking in laboratory professionals whose primary training is in the sciences rather than in medicine. The cases are presented in a standard format in which the initial presentation, the history and physical examination, provisional diagnoses, initial laboratory test results, working diagnoses, further investigations, final diagnoses, and management are presented in a progressive fashion. Each case concludes with a review of the key features of the case, a list of special points to remember, and several relevant references.The cases are presented in a concise outline format that is ideal for readers who already have some background in the field and intend to use the text as a means of reviewing important concepts and learning new approaches to test interpretation. On the other hand, this format will not be as appealing to less experienced practitioners who are looking for more detailed discussions of fundamental concepts. An important negative feature of the text is that the reference ranges that are necessary for the interpretation of the test results associated with each case are presented in tables that are located in the back of the book. The need to flip back and forth between a case and the table of reference values disrupts a reader's train of thought and makes it more difficult to evaluate and interpret the laboratory data and to recognize patterns of abnormal test results. I also found some inconsistencies and errors in the units of measure given for aldosterone, a test result that appears in 7 of the cases. This raises the concern that the text could have been edited more carefully.These negative features are offset by the fact that the text presents an interesting assortment of unusual cases that effectively focus the reader's attention on important problems and issues pertaining to test use and interpretation. The book provides a review of some of the more advanced concepts in the interpretation of clinical chemistry test results and, as such, will be of value to any reader who is seeking to gain more expertise in this subspecialty of pathology.

3rd ed, edited by Steven J. Soldin, Carlo Brugnara, and Jocelyn M. Hicks, 232 pp, Washington, DC, American Association for Clinical Chemistry, 1999.The third edition of this text provides reference values for approximately 150 clinical chemistry parameters and 25 hematology parameters in children. The values that are presented were obtained either from the recent literature or from original investigations in the authors' own laboratories. The ranges presented for each analyte are typically partitioned by age and sex (where appropriate) and have been derived from reference samples of between 50 and 100 subjects. When available, literature references to articles in which the original studies were published are presented. In addition, the entry for each analyte includes the specimen type used for analysis and some information about the reference subjects and the methods of analysis. A laboratory director might consult a reference like this to compare a reference value that was estimated in his or her laboratory with an established value from the literature or to evaluate the validity of transferring a published reference value to her or his laboratory. Therefore, a text that facilitates these goals would be of great value.Although this book presents a large collection of pediatric reference values, references of this type are often of limited value to laboratorians. One flaw that decreases my enthusiasm for this type of book in general (and a shortcoming of this particular book) is that the protocols used in the original reference studies are not described in enough detail to allow the reader to evaluate the quality or the applicability of the reference values that are presented. In the absence of a complete set of specifications for the original reference study (for the details that should be provided, see Sunderman FW Jr. Clin Chem. 1973;21:1873.), the reader must go back to the primary literature to obtain the necessary information. Thus, a reader might just as well start with primary references (which can be readily found using simple search strategies in public domain databases such as PubMed) that are available on the Internet. A second limitation of this particular text is that many of the reference values presented are estimated by an indirect method using results determined in hospitalized patients. The validity of indirect methods for the establishment of reference values (especially ones that are suitable for transfer to other populations) is still not widely accepted in the field of laboratory medicine.In the book's preface, one of the authors states that “…reference ranges are guidelines for the clinician and cannot be used as definitive indicators of health or pathological states.” Yet, one of the claims printed on the book's back cover states that “…this book will allow you to interpret patient results….” Such a claim misrepresents the utility of the information that the book contains, especially in view of the deficiencies I have mentioned.

Edited by Ishwarlal Jialal, William E. Winter, and Daniel W. Chan, 250 pp, with illus, Washington, DC, American Association for Clinical Chemistry, 1999.This multiauthored text focuses on laboratory approaches to the diagnosis and management of endocrine disorders involving the pituitary gland, the thyroid gland, the adrenal cortex and medulla, the gonads, and the gastrointestinal tract as well as hyperglycemia and hypoglycemia, metabolic bone disease, and endocrine malignancies. The book is intended to serve as a guide to the selection and interpretation of endocrine-related testing and is aimed at clinical pathologists, clinical chemists, endocrinologists, and medical technologists. Although numerous resources are already available on this subject, a concise, practically oriented text covering the most commonly ordered tests and pertaining to the most prevalent endocrine disorders is always of value to the intended audience. The contributors to this handbook are a distinguished group of clinical endocrinologists, pathologists, and clinical chemists, most of whom practice in an academic teaching hospital environment. All of the authors have considerable experience in endocrine laboratory testing.Each chapter presents a brief review of relevant physiological and clinical aspects of a topic; however, the major focus is fixed on the biochemical tests that have become a mainstay in the evaluation of endocrine function. The authors present test strategies (which are often depicted as algorithms), details of patient preparation, potential sources of analytical or physiological interferences, the more commonly used protocols for provocative testing, reference values, and guidelines for interpretation and follow-up testing. I especially liked the chapter on the thyroid gland for its concise presentation of the essentials of this high-volume branch of endocrine testing, the chapter on metabolic bone disease for its timeliness and its clear treatment of the relatively complex assortment of biochemical markers available in this area, and the chapter on endocrine malignancies for its up-to-date review of the literature on the subject. One general criticism I have concerns the currentness of the references. Excluding the chapter on endocrine malignancies, more than 95% of the literature cited in the other chapters was published before 1997. A second criticism is that although the text contains an appropriate number of tables and figures that are generally useful and well conceived, many of them are set in small typefaces that are difficult to read.Despite these limitations, this handbook is a good and practical review of the topics covered. The authors present distillates of useful laboratory-centered information in a readily accessible format, which will be appreciated by laboratorians who are looking for a concise summary of selected areas of endocrine testing.

Only 30% of alcoholics develop liver disease (ALD) suggesting that additional factors are needed. Endotoxin is one such factor, but its etiology is unclear. Since the gut is the main source of endotoxin, we sought to determine whether an increase in intestinal permeability (leaky gut) is required for alcohol-induced endotoxemia and liver injury and whether the gut leakiness is preventable. For 10 weeks, rats received by gavage increasing alcohol doses (to 8 g/kg/day) and either oats (10 g/kg) or chow b.i.d. Intestinal permeability was then assessed by urinary excretion of lactulose and mannitol. Liver injury was evaluated histologically, biochemically (liver fat content), and by serum aminotransferase. Alcohol caused gut leakiness that was associated with both endotoxemia and liver injury. Oats prevented these changes. We conclude that chronic gavage of alcohol in rats is a simple experimental model that mimics key aspects of ALD, including endotoxemia and liver injury, and can be useful to study possible mechanisms of endotoxemia in ALD. Since preventing the gut leakiness by oats also prevented the endotoxemia and ameliorated liver damage in rat, our results suggest that alcohol-induced gut leakiness 1) may cause alcohol-induced endotoxemia and liver injury and 2) may be the critical cofactor in the 30% of alcoholics who develop ALD. Further studies are needed to determine whether ALD in humans can be prevented by preventing alcohol-induced gut leakiness, studies that should lead to the development of useful therapeutic agents for the prevention of ALD.