Establishment of an in-house ELISA and the reference range for serum amyloid A (SAA): complementarity between SAA and C-reactive protein as markers of inflammation.

INTRODUCTION: Serum amyloid A (SAA) and C-reactive protein (CRP) are both acute-phase reactants synthesized by the liver upon stimulation by proinflammatory cytokines reflecting both the acute and chronic inflammatory states. METHODS: We have established a one-step, sandwich ELISA on microplate for SAA using commercial antibodies for coating and detection. RESULTS: This in-house ELISA has a sensitivity of 0.12 mg/l. Both within-day and between-day CVs were <10%. The in-house assay correlated well with the commercial ELISA kit from Anogen (r=0.95). We also established the reference range for apparently healthy Chinese. Statistically higher SAA values were found in those >50 years old. No difference was found between genders. We found only slightly increased levels of SAA in early stage of type 2 diabetics, but highly increased levels of SAA were detected in patients with acute myocardial infarction, generally associated with intense inflammation. At the early stage of type 2 diabetes associated with low inflammation, SAA was found to be complementary to CRP in test sensitivity. CONCLUSIONS: Based on our data and reports from the literature we believe that SAA responds differently than CRP in inflammatory diseases such as in type 2 diabetes and acute myocardial infarction, and is complementary to CRP in test sensitivity.

Establishment of a sandwich ELISA using commercial antibody for plasma or serum 3-nitrotyrosine (3NT). Elevation in inflammatory diseases and complementary between 3NT and myeloperoxidase.

BACKGROUND: Amino acid tyrosine residue of a protein can be nitrated to form 3-nitrotyrosine (3NT), which is now being considered as a marker of inflammation, oxidative and nitrosative stress. METHOD: An in-house ELISA has been established using the same commercial antibody for both binding and detection of 3NT containing proteins. RESULTS: The sensitivity of the in-house ELISA was 1.8 nmol/l. The imprecision was <10% at all concentrations. The in-house assay correlates well with a commercial kit (r=0.89). In addition to EDTA plasma, we found that both heparinized plasma and serum can also be used to quantify 3NT concentration. Using the in-house ELISA we have detected increased concentrations of 3NT in diseases known to be associated with inflammation and also in subjects with polyps. As marker of oxidative stress and inflammation, both 3NT and myeloperoxidase are complementary to each other in test sensitivity. CONCLUSION: This ELISA can be used in the clinical laboratories to monitor the inflammatory disease activity and assess early risks that are associated with inflammation, oxidative and nitrosative stress.

Esophageal perforations: new perspectives and treatment paradigms.

Despite significant advances in modern surgery and intensive care medicine, esophageal perforation continues to present a diagnostic and therapeutic challenge. Controversies over the diagnosis and management of esophageal perforation remain, and debate still exists over the optimal therapeutic approach. Surgical therapy has been the traditional and preferred treatment; however, less invasive approaches to esophageal perforation continue to evolve. As the incidence of esophageal perforation increases with the advancement of invasive endoscopic procedures, early recognition of clinical features and implementation of effective treatment are essential for a favorable clinical outcome with minimal morbidity and mortality. This review will attempt to summarize the pathogenesis and diagnostic evaluation of esophageal injuries, and highlight the evolving therapeutic options for the management of esophageal perforation.

Linking inflammation and atherogenesis: Soluble markers identified for the detection of risk factors and for early risk assessment.

Increasing evidence has shown that atherogenesis is not only caused by hypercholesterolemia. Several risk factors including abdominal obesity, dyslipidemia, hyperglycemia, bacterial and viral infection, hyperhomocysteinemia have been identified recently, all mediated through inflammation, which can lead to atherosclerosis. Several events have also been identified to be involved in the overall inflammation reaction in the blood vessel which include endothelium dysfunction, expression of adhesion molecules, recruitment of leukocytes to the injured endothelium, migration of monocytes to the arterial intima, and transformation of monocytes to macrophages. In order to facilitate the assessment of early risk for atherogenesis we have made an effort in this review to identify soluble markers that will allow the detection of these risk factors and the identification of associated inflammation events. Since early risks for atherogenesis are largely preventable with dietary modification and lifestyle changes, capable of detecting early risks by monitoring soluble risk markers is conceivably important for asymptomatic individuals to avoid serious or fatal consequences of atherosclerosis. These soluble markers should also be useful for monitoring the effectiveness of intervention and for the identification of therapeutic targets.

Development of an ELISA for myeloperoxidase on microplate: normal reference values and effect of temperature on specimen preparation.

BACKGROUND: Myeloperoxidase (MPO), a leukocyte enzyme, is implicated in both the pathogenesis and the progression of atherosclerosis. METHODS: We developed a sandwich ELISA on microplate using commercial antibodies for the measurement of plasma MPO. RESULTS: The in-house ELISA has a sensitivity of 15 ng/ml. Both within-day and between-day imprecision were <10%. The in-house assay was well correlated with the commercial kit from Oxis (gamma=0.96). We have established normal reference range for MPO for apparently healthy Chinese. No statistical difference was found between males and females and the various age groups. Because the coating antibodies used by two different kits are different in their affinities for MPO, the analysis by the in-house ELISA that was approximately three times that of the Oxis kit when testing the same specimens. We found that it is necessary to keep the heparinized whole blood on ice before centrifugation in order to prevent further release of MPO from the leukocytes at room temperature. For the same reason, serum is not recommended for MPO measurement. We also found that either pooled human plasma or serum containing MPO can be used as calibrators. CONCLUSIONS: We believe that this ELISA for MPO is useful to assess risk for inflammation, oxidative stress, nitrosative stress, and for predicting cardiac events.

Prognostic significance of DCC and p27Kip1 in colorectal cancer.

The progression of colorectal cancer is a multistage process associated with specific molecular alterations. The stepwise accumulation of these multiple genetic mutations progressively results in the acquisition of neoplastic cell behavior. The genetic abnormalities associated with the expression of metastatic phenotype, therefore, may be of prognostic significance in the clinical treatment of colorectal cancer patients. In this study, the immunohistochemical expression of the deleted in colorectal cancer gene (DCC) and p27Kip1 was assessed in 168 paraffin-embedded, formalin-fixed tumors of patients with stage II and III colorectal cancer. Kaplan-Meier survival curves and log-rank statistics were used to analyze survival times after curative primary tumor resection, and Cox proportional hazards models were used to adjust the assessment of demographic and clinical covariates. Loss of DCC or p27Kip1 expression had no influence on survival in patients with stage II or III colorectal cancer. The 5-year survival rates of DCC-positive and DCC-negative tumors were 51.8% and 35.7% (P=0.40), respectively. The 5-year survival rate of patients with p27Kip1-positive tumors was 47.9%, whereas the rate for patients with p27Kip1-negative tumors was 38.8% (P=0.68). After adjustment for all evaluated variables, neither DCC or p27Kip1 was found to be a predictor of survival (risk ratio for DCC, 0.98; 95% confidence interval, 0.66-1.56; P=0.92; risk ratio for p27Kip1, 0.87; 95% confidence interval, 0.58-1.29; P=0.49). The present study demonstrated that the expression of neither DCC nor p27Kip1 was predictive in poor survival outcome in patients with stage II or III colorectal cancer.

Association of soluble markers with various stages and major events of atherosclerosis.

The purpose of this review is to identify soluble markers from the recent literature that facilitate the early prevention and early detection of atherosclerosis, and that may serve as therapeutic targets. Soluble markers associated with various stages and major events of atherosclerosis were identified. We divided the process of atherosclerosis into several stages, including stages for the early risk, plaque expansion, and stable and unstable angina-though excluding the end stage of myocardial infarction. For major events taking place prior to and during the progression of atherosclerosis we included events such as endothelial dysfunction in the artery, expression of adhesion molecules at the injured endothelium, continued inflammatory responses, oxidative stress, and ischemia. We found that reactions such as cell injury, adhesion, inflammation, and oxidative stress occur not only at the early stage of risk but persist throughout the process of atherosclerosis. Most markers associated with these major events are clustered together at any time of the disease. Few markers are characteristic of individual stages. We noted that reactions such as inflammation are continuously intensified with the progression of the disease. Finally, we underscore the importance of measuring a panel consisting of minimal numbers of multiple markers with the maximal sensitivity for early risk assessment, diagnosis, and prognosis. We envision that patterns characteristic of various stages of atherosclerosis may be identifiable with the use of the multiple markers described in this review.

Enzymatic assay of homocysteine on microtiter plates or a TECAN analyzer using crude lysate containing recombinant methionine gamma-lyase.

An enzymatic assay for plasma homocysteine was developed that uses a crude lysate of E. coli containing the recombinant enzyme, methionine gamma-lyase. The assay uses a commercially available fluorophore and 96-well microtiter plates; it can be performed manually or with the TECAN automated analyzer. The CVs for within-run and between-run precision are < 10%. Close correlation (r > 0.9) was obtained between results by this enzymatic method vs a reference HPLC procedure. In a Chinese population, the concentration of plasma total homocysteine was found to be gender- and age-dependent. Mean concentrations of plasma total homocysteine increased with age and were higher in men than women. Serum homocysteine concentrations did not differ significantly from those in plasma, provided the whole blood specimens were kept at 4 degrees C for 2 hr, or at room temperature for < 45 min, between venepuncture and centrifugation.

Microplate ELISA for urine microalbumin: reference values and results in patients with type 2 diabetes and cardiovascular disease.

An ELISA for urine microalbumin using microtiter plates has been developed. The assay uses polyclonal anti-human albumin antibody for coating the microtiter plates and the same antibody conjugated with horseradish peroxidase for detection. The assay sensitivity is 1.6 microg/ml. Results by this in-house ELISA show good correlation (r = 0.99) with those obtained by a commercial assay using the Behring BNII autoanalyzer. Within-day and between-day CVs are 10%. Reference values for microalbumin in 769 urine specimens from healthy Chinese subjects were higher in women than men and higher in subjects 50 yr than in those <50 yr of age. Elevated mean concentrations of urine microalbumin were observed in patients with type 2 diabetes and CVD. This in-house ELISA is simple, sensitive, precise, and especially suited for laboratories without expensive autoanalyzers.

Microplate ELISAs for soluble VCAM-1 and ICAM-1.

Soluble vascular cell adhesion molecule (sVCAM-1) and soluble intercellular adhesion molecule (sICAM-1) are adhesion molecules that are detectable in the serum of patients with cancer, cardiovascular diseases (CVD), and type 2 diabetes. This report describes enzyme-linked immunosorbent assays (ELISAs) on microplates for sVCAM-1 and sICAM-1. The ELISAs have the sandwich test format; polyclonal antibodies are coated on microwells and a one-step procedure is used in which the serum specimen and detecting antibody are added simultaneously to an antibody-coated well. These assays both use HRP-conjugated sheep anti-mouse-IgG to generate the color for quantification. Sensitivities for detecting sVCAM-1 and sICAM-1 are 49 and 40 ng/ml, respectively. Coefficients of variation for within-day and day-to-day replicate analyses are <10%. Results by these in-house ELISAs for serum sVCAM-1 and sICAM-1 compared well with those obtained with commercial kits from R&D Systems, Inc. (correlation coefficients = 0.98 and 0.99 for sVCAM-1 and sICAM-1, respectively). Reference values for serum sVCAM-1 and sICAM-1 levels were measured in 369 apparently healthy Chinese adults, age 30 to 79 yr. There was no significant effect of gender on the reference values for sVCAM-1 or sICAM-1. Serum sVCAM-1 levels (mean +/- SD) were higher in subjects 60 yr old (625 +/- 126 ng/ml), compared to those <60 yr old (525 +/- 110 ng/ml) (p <0.001). Age did not significantly affect the reference values for serum sICAM-1 levels (mean +/- SD, 249 +/- 86 ng/ml). The authors believe that these simple, inexpensive ELISAs will be useful for assessing the risks for development of cancer, CVD, and type 2 diabetes.

C-erbB2 oncoprotein and its soluble ectodomain: a new potential tumor marker for prognosis early detection and monitoring patients undergoing Herceptin treatment.

c-erbB-2 oncoprotein (or p185) coded by c-erbB-2 oncogene can be extracted with detergent from cell membrane of breast tissue and breast tumor cell line. The ectodomain of p185 can be cleaved proteolytically from the transmembrane receptor and released into solution. In blood circulation, only the ectodomain can be found, whereas only p185 exists in the extracts of tissue and cell line. p185 can also be quantified in the fine-needle aspirate biopsies and the concentration of p185 from the biopsies of malignant breast tissue is much higher than that of the normal and benign tissue. Measuring the ectodomain of the c-erbB-2 oncoprotein not only is useful to identify breast cancer patient who will benefit from Herceptin treatment but also can be used to monitor patients during the treatment. Overexpression of p185 and the elevation of circulating ectodomain is usually associated with poor prognosis. Overexpression of p185 in breast cancer patients with positive estrogen receptor identifies a subgroup of patients who will not respond to the endocrine therapy. Activation of p185 appears to be an early event in tumorigenesis for some cancers. It is possible that the ectodomain could be used as an early tumor marker detecting benign disease.

Hyperhomocysteinemia is a risk factor for cancer and a new potential tumor marker.

Plasma homocysteine (Hcy), a well-known independent risk factor for coronary heart disease, is also a risk factor for cancer. Results from our studies indicate that Hcy could be used as a tumor marker. We found elevated circulating total homocysteine (tHcy) in cancer patients even though they were not treated with anti-folate drugs. In serial specimens from cancer patients undergoing treatment, the change of tHcy coincided with the concentration of tumor markers. The rapid proliferation of tumor cells contributed to the much higher concentrations of circulating tHcy. Both concentrations of tHcy and tumor marker would increase in parallel during the growth of tumor cell, but only the Hcy concentration would decline in response to tumor cell death. Several biochemical changes, including folate deficiency, oxidative stress, aberrant DNA methylation, and production of homocysteine thiolactone have been identified in association with hyperhomocysteinemia, which explained why elevated homocysteine eventually led to carcinogenesis. Conceivably, tHcy may be used as a more accurate tumor marker for monitoring cancer patients during treatment, and hyperhomocysteinemia as a risk factor for carcinogenesis.

Serum total homocysteine increases with the rapid proliferation rate of tumor cells and decline upon cell death: a potential new tumor marker.

BACKGROUND: We were interested to know why cancer patients are frequently associated with elevated circulating total homocysteine (tHcy) even though they are not treated with anti-folate drugs. METHODS: We employed tissue cultures to compare both the homocysteine (Hcy)-released and production of tumor markers between tumor and normal cell lines. RESULTS: We detected much higher concentrations of homocysteine (Hcy) released by the tumor cells. However, much less difference was found between normal and tumor cell lines when Hcy concentration was expressed per the same number of cells. During the cell culture, the increase of Hcy and the increase of tumor marker concentration paralleled each other for the first 7 days. After the seventh day of the culture when cells started dying, tumor markers continued to rise, whereas levels of Hcy and cell numbers leveled off. We found that the serum concentration of Hcy fluctuated in circulation coinciding with that of tumor marker in individual cancer patients unless taking anti-neoplastic drug. CONCLUSIONS: The elevation of tHcy concentration may be caused by the rapid tumor cell proliferation and reflect only the number of live cells. Serum Hcy may be a potentially useful tumor marker to monitor tumor activity.

Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics.

Reactive oxygen species (ROS) produced either endogenously or exogenously can attack lipid, protein and nucleic acid simultaneously in the living cells. In nuclear and mitochondrial DNA, 8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Numerous evidences have indicated that urinary 8-OHdG not only is a biomarker of generalized, cellular oxidative stress but might also be a risk factor for cancer, atherosclerosis and diabetes. For example, elevated level of urinary 8-OHdG has been detected in patients with various cancers. In human atherosclerotic plaques, there were increased amounts of oxidatively modified DNA and 8-OHdG. Elevated urinary 8-OHdG and leukocyte DNA were also detected in diabetic patients with hyperglycemia, and the level of urinary 8-OHdG in diabetes correlated with the severity of diabetic nephropathy and retinopathy. We have discussed various methods for determining 8-OHdG in the tissue and urine, including HPLC with and without extraction, and ELISA. Using the ELISA we developed, we found that the normal range of urinary 8-OHdG for females was 43.9 +/- 42.1 ng/mg creatinine and 29.6 +/- 24.5 ng/mg creatinine for males, respectively. We found that the normal value between females and males is significantly different (p < 0.001).

Development of ELISA on microplate for serum C-reactive protein and establishment of age-dependent normal reference range.

BACKGROUND: C-reactive protein (CRP), a marker of systemic inflammation, has been proposed to predict outcome in patients with unstable angina; and elevated levels of CRP were found to be associated with an increased risk of coronary events. METHODS: Two enzyme-linked immunosorbent assays (ELISA) of different sensitivities were developed on microplate for CRP. Both ELISA established used Dako polyclonal anti-CRP antibody for coating and Dako horse radish peroxidase (HRP)-conjugated polyclonal anti-CRP antibody for detection. RESULTS: The sensitivity of the high and regular sensitivity ELISA was 0.16 and 0.6 mg/l, respectively. Our assays demonstrated an excellent correlation with commercial CRP assays performed on a Behring Nephelometer Analyzer II (BNII) at both regular and ultrasensitive levels, with both correlation coefficients above 0.98 and slopes of approximately 1. Using our microplate assays, we established normal reference value for serum CRP. Based on ANOVA statistical test, we found that the mean +/- S.D. was 1.3 +/- 1.27 mg/l (n=202) for normal individuals of 50-80 years and 0.43 +/- 0.42 mg/l (n=148) for the group of 20-50 years. CONCLUSIONS: The normal serum CRP mean concentrations for two age groups were distinctively different (p value<0.001). Our study suggests two different normal cutoffs of serum CRP to be employed for individuals in different age groups.

Cell-free DNA: measurement in various carcinomas and establishment of normal reference range.

BACKGROUND: Cell-free DNA is detectable in circulating blood. Numerous reports in the literature have pointed out that cell-free DNA in plasma or serum has the clinical potential to be a more specific tumor marker for the diagnosis and prognosis, as well as the early detection, of cancer. METHODS: In order to adapt cell-free DNA to a routine clinical laboratory test, we used commercial kits such as the QIAamp blood kit for DNA extraction and the PicoGreen DNA kit for DNA quantification. This was done so our results and the normal reference value established would allow to be compared by other laboratories. We have established the normal reference level of cell-free DNA for females and males from age 20-70 years. We also detected elevated cell-free DNA in all cancers that were tested in this study, including carcinomas, leukemia and lymphoma. RESULTS: Our study indicates that the elevation of serum cell-free DNA was usually detected in specimens containing elevated tumor markers and is most likely associated with tumor metastases. The electrophoretic pattern of cell-free DNA showed that cell-free DNA from cancer patient is fragmented, containing smaller DNA (100 bp) not found in normal cell-free DNA. CONCLUSIONS: Measuring cell-free DNA may complement currently used tumor markers for the management of cancer patients.

Elevated cell-free serum DNA detected in patients with myocardial infarction.

BACKGROUND: Cell-free DNA is detectable in the circulation. Increased cell-free DNA has been detected in cancer patients and individuals with trauma. We want to know whether patients with myocardial infarction (MI) also have increased cell-free DNA in their blood. METHODS: We used a QIAamp blood kit for DNA extraction from serum and the PicoGreen DNA kit for quantification. DNA patterns of serum DNA were established by gel electrophoresis on 2.5% metarphor gel. RESULTS: The average serum DNA in MI patients (N=55) was 511+/-398 ng/ml, more than 10-fold higher than normal (36.3+/-23.8 ng/ml, n=274). Patients with increased CK-MB (>4%) were associated with highly increased concentrations of cell-free DNA (93.4%). There was no correlation between the concentration of cell-free DNA and the concentrations of CK-MB, troponin I and C-reactive protein. In serial specimens, we found that the cell-free DNA rose early, but peaked behind CK-MB. A slightly diffused DNA ladder could be found with pooled cell-free DNA from MI patients by electrophoresis with the smallest DNA band at only a few hundred base pairs. CONCLUSIONS: Cell-free DNA in MI patients is increased in patients diagnosed with MI, and may complement troponin and CK-MB in a multiple marker test format.

Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers.

BACKGROUND: 8-hydroxydeoxyguanosine (8-OHdG) is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Urinary 8-OHdG is now considered as a biomarker of generalized, cellular oxidative stress and is linked to degenerative diseases including cancer. METHODS: We developed a competitive enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG by coating BSA conjugated 8-hydroxyguanine (8-OHG) on a microplate. Urine specimens containing 8-OHdG and monoclonal anti-8-OHdG antibody were incubated together in the microwell. Final quantification of bound anti-8-OHdG antibody was estimated by the addition of HRP-conjugated sheep-anti-mouse antibody. RESULTS: The concentration range of the calibration curve was 0-60 ng/ml. The sensitivity of the assay was 0.5 ng/ml. The within-day precision and day-to-day precision were <10%. The ELISA correlated well with a commercial kit (r=0.9). Our assay measured not only 8-OHdG but also 8-OHG and 8-hyroxyguanine in urine. Increased urinary concentration of 8-OHdG and its analogs were detected in both patients with bladder cancer (70.5+/-38.2 ng/mg creatinine) and prostate cancer (58.8+/-43.4 ng/mg creatinine) as compared to the healthy control (36.1+/-24.5 ng/mg creatinine). CONCLUSION: Our preliminary data suggest that the competitive ELISA for 8-OHdG and its analogs appears to be a simple method for quantifying the extent of oxidative stress and may have potential for identifying cancer risk.

Objective review of mediastinal lymph node examination in a lung cancer resection cohort.

BACKGROUND: Accurate staging of resected lung cancer requires mediastinal lymph node (MLN) examination. MLN dissection (MLND) and systematic sampling (SS) are acceptable procedures; random sampling (RS) and no sampling (NS) are not. Forty percent of US lung cancer resections have NS. We closely examined the pattern of MLN examination in a lung resection cohort. METHODS: This is a retrospective review of all lung cancer resections in Memphis, TN, from 2004 to 2007. We compared operating surgeons' claims to the pathology report and an audit of the operation narrative by an independent surgeon. RESULTS: Forty-five percent of resections were reported by surgeons as MLND, 8% RS, and 48% NS. None met pathology criteria for MLND, 9% were SS, 50% were RS, and 42% were NS. The concordance rate between the operating surgeon and pathology report was 39%. The surgeon audit suggested 29% of resections had MLND, 26% RS, and 45% NS. Concordance between operating and auditing surgeons was 71%. Sublobar resection, T1 stage, and age were associated with NS. CONCLUSIONS: Most resections had suboptimal MLN examination. Concordance was poor between surgeon claims, objective review of pathology reports, and an independent surgeon audit. The higher concordance between operating and auditing surgeons may suggest incomplete pathology examination of MLN material. The terms used by operating surgeons to describe MLN retrieval were often inaccurate.

A novel surgical approach to cardiac autotransplantation in complex cardiac sarcoma resection.

A 28-year-old woman was admitted to our institution, reporting progressive dyspnea, cough, and weight loss of 14 kg. Two-dimensional echocardiography revealed a left atrial mass, and cardiac magnetic resonance imaging showed localized involvement of the mass with adjacent structures. These clinical signs and radiographic images were highly suggestive of cardiac sarcoma. The patient underwent emergent mediastinal exploration, and an incisional biopsy of the mass showed high-grade sarcoma. Removing the tumor required radical en bloc resection of the left atrium, including the mitral valve, the left pulmonary vein, and the left lower lobe of the lung. Autotransplantation was necessary for the resection and reconstruction. We report a unique method of handling the right atrium to avoid the potential complications associated with bicaval anastomoses after autotransplantation.