Capillary Electrophoresis

Asbsestos Related Diseases Non-Gynaecological Cancers and Some Benign Diseases

Tumor Biology - Vol. 17, No. 1, 1996  by Assoc. Prof. Halis Simsek, MD, Section of Gastroenterology, Hacettepe University Medical School, Resat Nuri sokak No: 21/13, Yukariayranci, Ankara, 06100 (Turkey).  Here is an excerpt: "Abstract - Elevated levels of CA 125 have been shown to be present in the serum of patients with ovarian carcinoma, non-gynaecological cancers and some benign diseases. However, the value of CA 125 in malignant peritoneal mesothelioma has not been studied in detail. Therefore, 7 patients with diffuse malignant mesothelioma were included in this study. Median age was 52.4 (range 16-73), and 6 of them were women. The mean serum CA 125 level was 308kU/l (range 8-1,300 kU/1). Serum CA 125 concentrations were found to be elevated in all 6 female patients. In 3 patients, serum CA 125 levels were followed prospectively and showed a very close correlation with the response to chemotherapy. In 2 responder patients, initially elevated CA 125 levels returned to normal during remission after chemotherapy. In 1 non-responder patient, the serum CA 125 level continued to rise. In conclusion, the serum CA 125 level might be helpful in the diagnosis and follow-up of malignant peritoneal mesothelioma."

Another interesting study is called, "High-performance capillary electrophoretic analysis of hyaluronan in effusions from human malignant Mesothelioma" - Journal of Chromatography B: Biomedical Sciences and Applications - Volume 697, Issues 1-2, 12 September 1997, Pages 277-281 by Nikos K. Karamanosa and Anders Hjerpeb – Here is an excerpt: "Abstract - A procedure to quantify hyaluronan in effusions from human malignant mesothelioma using a highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method is presented. Following ethanol precipitation, hyaluronan and galactosaminoglycans were degraded to Δ4.5-dissacharides with a mixture of chondroitinases ABC and AC. Heparan sulphate and proteins/glycoproteins were separated by ultrafiltration on a Centricon 3 membrane, and hyaluronan-derived disaccharides were analysed by direct injection of the filtrate into a HPCE system. Determination of hyaluronan in effusions from five healthy individuals and three patients with mesothelioma gave values comparable to those found using the HPLC method. One of the advantages of the HPCE method as compared to HPLC is the low solvent consumption. The much lower detection limit (attomole level) of the HPCE method may also allow the analysis of hyaluronan content in serum. The contribution of HPCE in diagnosis of a neoplasm, such as human malignant mesothelioma, illustrates the great potential of this technique in the field of life sciences."

Another interesting study is called, "Protein expression of the RB-related gene family and SV40 large T antigen in mesothelioma and lung cancer" - Nature Publishing Group, Basingstoke, ROYAUME-UNI  (1987) (Revue).  Here is an excerpt: "Abstract - Mutational inactivation of the RB-related gene RBL2/ p130 has been reported as a common and important prognostic factor in human lung cancer. To examine the role of the RB-related gene family in lung cancer we analysed the protein expression of the RB gene in cell lines obtained from 83 patients with small cell lung cancer (SCLC) and 114 patients with non-SCLC that included 21 novel lung tumor samples. While we detected five new SCLC with mutant RB [removed]RB inactivation in 75/83; 90.4%), we did not detect any RB mutations in the new non-SCLC cell lines (RB inactivation in 13/114 non-SCLC and mesothelioma; 11.4%). In addition, we detected expression of a full-length RBL1/p107 and RBL2/p130 species in every sample tested (RBL1 or RBL2 inactivation in 0/69) and confirmed that both RB-related gene products retain functional binding activity to the E1A viral oncoprotein. Since expression of SV40 Large T antigen (Tag) has been reported in a subset of human lung tumors where it may inactivate RBL1 and RBL2, we also examined mesothelioma and non-mesothelioma lung tumors for Tag expression. Although we detected a faint 85 kDa protein species using specific anti-Tag antibodies, this signal migrated slightly faster than Tag extracted from Cos7 cells and did not exhibit binding activity to the RB or RBL1 proteins. Finally, we subjected 11 lung cancer cell lines to nucleotide sequencing and did not detect mutations within the C-terminal RBL2 exons 19-22 as recently reported. While the RB/p16 tumor suppressor pathway is targeted for mutations in 100% of lung cancers, mutational inactivation of the related RBL1 and RBL2 genes is a rare event."

Another interesting study is called, "Benign cystic mesothelioma involving the female genital tract: report of four cases" by Schneider V, Partridge JR, Gutierrez F, Hurt WG, Maizels MS, Demay RM. - Am J Obstet Gynecol. 1983 Feb 1;145(3):355-9.  Here is an excerpt: "Abstract - Four cases of benign cystic mesothelioma are described. The disease affects young white women (mean age, 30 years), and they present with chronic pelvic pain. At laparoscopy or laparotomy, multiple cysts ranging in size from 0.5 to 4 cm in diameter and containing clear fluid are seen. The disease commonly affects the pelvic organs and/or omentum. With the electron microscope, the cell of origin of this proliferative process is shown to be the mesothelial cell. The disease has been previously described under a variety of terms. There seems to be a tendency for recurrence, but no malignant potential is apparent. Treatment may be conservative with preservation of pelvic organs. Benign cystic mesothelioma should be considered in the differential diagnosis of cystic lesions of the female genital tract."

Another interesting study is called, "Monoclonal antibody Ber-EP4: Its use in the differential diagnosis of malignant mesothelioma and carcinoma in cell blocks of malignant effusions and FNA specimens" by Dr. Brigid Maguire M.B.B.S., Darrel Whitaker Ph.D., C.F.I.A.C., F.I.M.L.S., A.A.I.M.S., Salvatore Carrello B.App.Sc., Dominic Spagnolo M.B.B.S., F.R.C.P.A. - Diagnostic Cytopathology - Volume 10, Issue 2, pages 130–134, March 1994.  Here is an excerpt: "Abstract - Formal sublimate-fixed cell blocks derived from 129 malignant pleural (and some peritoneal) effusions, 8 benign effusions with reactive mesothelial cells, and 23 FNA specimens, were immunostained with monoclonal antibody Ber-EP4 to assess its ability to distinguish malignant mesothelioma (MM) from carcinoma.

Only 2 of 44 (4%) well-characterized MM were Ber-EP4+, while none of 8 benign mesothelial proliferations reacted with the antibody. Fifty-seven percent of 23 pulmonary adenocarcinomas (AC) and 60% of 43 pulmonary carcinomas of all other histological types were Ber-EP4+. Of 40 metastatic AC originating from breast, gastrointestinal tract, ovary, endometrium, and kidney, 80% were Ber-EP4+. The predictive value of positive Ber-EP4 staining in distinguishing AC from MM was 96%. The predictive value of a negative Ber-EP4 in excluding MM was 70%, when the differential diagnosis was adenocarcinoma. These results suggest that Ber-EP4 is helpful in differentiating MM and AC if used together with other discriminating antibodies."

We all owe a debt of gratitude to these fine researchers.  If you found any of these excerpts interesting, please read the studies in their entirety.

About the Author

Monty Wrobleski is the author of this article.  For more information please click on the following links

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Testing Diagnostics: Getting Personal

November 2011 - Personalized medicine, one goal of molecular diagnostics that is already being clinically realized, offers the opportunity to treat individuals on a case-by-case basis with greater efficacy and efficiency than ever before.

Molecular diagnostics is heading the way of personalized medicine, aiming to help physicians devise particular treatment plans for individuals based on their genetic makeup.

"This is a very exciting time. We're seeing this incredible focus on molecular diagnostics from the medical community and also from the investment community," says Paul Beresford, vice president of Business Development and Strategic Marketing for Biodesix, Broomfield, Colo, a fully integrated molecular diagnostics company developing products that support excellence in clinical decision-making and improve patient care.

Much of the molecular diagnostics applications have found clinical relevance in a variety of cancers. Genetic tests can evaluate tumors to reveal individual biomarkers, which are becoming increasingly relevant in predicting prognosis and choosing treatment plans.

COLORECTAL CANCER

KRAS and BRAF mutations have been in the spotlight in cancer diagnostics and treatment, as mutations of these types may indicate a lower response likelihood for common cancer therapies.

"Should patients be subjected to difficult-to-tolerate therapy, as well as the economic burden of that therapy, when there's a chance that it may be ineffective in treating the cancer?" asks Julian Walker, senior manager, Product Management, Genetic Systems Division of Life Technologies, a global biotechnology tools company based in Arcadia, Calif, that provides premier systems, consumables, and services for scientific researchers around the world. "The FDA has limited the recommended use of certain chemotherapy drugs for patients with KRAS mutations for this very reason."

Anti-EGFR therapies are commonly used in a number of cancer types. The epidermal growth factor receptor (EGFR) pathway is associated with the development and progression of many cancer conditions. However, approximately 35% to 45% of metastatic colorectal cancer (mCRC) tumors may have a KRAS or BRAF mutation, which makes them less likely to respond to anti-EGFR therapies. Identifying these mutations is therefore of great importance in clinical and pharmaceutical research. Life Technologies currently has KRAS and BRAF Mutation Analysis Reagent sets under the Applied Biosystems brand available for research purposes only, and the companies are in the process of determining the regulatory path for these reagents.

"It is often cited that we have not seen the same stepwise advance in the treatment of cancer that we have seen in other major diseases. Genetic and genomic analysis promise to give us the insights to help make those potential advances. We have seen the number of molecular diagnostic tests grow from hundreds to thousands, and we should continue to see this trend with cancer being a primary driver—especially as research strives toward more sensitive, specific, and targeted therapeutics," Walker says.

While most attention to date has been paid to KRAS and BRAF mutations in mCRC tumors, these mutations have also commonly been found in several other cancers, including pancreatic cancer, non-Hodgkin's lymphoma, malignant melanoma, papillary thyroid carcinoma, non-small-cell lung carcinoma, and adenocarcinoma of the lung.

LUNG CANCER

According to the American Cancer Society, 222,000 Americans were newly diagnosed with lung cancer last year. Advanced non-small-cell lung cancer is the most common type of lung cancer.

Limited treatment options currently exist for second-line lung cancer, generally a choice between monotherapy, chemotherapy, or an EGFR-TKI. The decision is often based on prognostic factors—such as sex, race, and whether the patient smokes or not—that may not actually be indicative of treatment response.

Biodesix offers a serum-based proteomic test called VeriStrat, which is intended to help physicians make more informed treatment decisions for patients with advanced non-small-cell lung cancer based on their likelihood to respond to EGFR-TKI therapy. The test was released in May 2009 and is currently available as a CLIA-certified lab test.

The VeriStrat test provides a binary result, either VeriStrat Good or VeriStrat Poor, based on the protein patterns in a patient's serum. A result of VeriStrat Poor indicates that the patient will receive little to no benefit from erlotinib (Tarceva) therapy, while VeriStrat Good indicates that the patient's outcome compares favorably with other treatment options available.

One advantage of VeriStrat is that, as a serum test, it is not dependent on a biopsy, where the tumor may evolve in a patient's body during the time of analysis. VeriStrat offers a 72-hour turnaround guarantee, and it has been turning around samples in about 24 hours, according to Beresford.

"Because we're analyzing a patient's serum, it allows our test to bridge across multiple tumor types. VeriStrat appears to work in colorectal, head, and neck cancer, based on clinical trials," Beresford says.

Biodesix, founded in 2005, is devoted to advancing molecular diagnostics via its unique ProTS biomarker discovery and diagnostic platform. ProTS is a fast, nonhypothesis-based platform used for discovery, validation, and commercialization, and it is not dependent on the existence of immunoassays.

BREAST CANCER

According to the American Cancer Society, approximately 210,000 new cases of breast cancer were diagnosed in 2010. Breast cancer is the second most commonly diagnosed cancer and the second leading cause of cancer deaths among women in the United States.

Molecular diagnostics of breast cancer have focused largely on the expression of the HER2 gene in tumors, particularly with the success of the drug Herceptin in treating these tumors. Generally, about 25% of breast cancers consist of HER2 positive tumors, and these cases are likely to respond to Herceptin. Patients with more than two copies of the gene are considered "amplified" or "positive," and are likely to respond to Herceptin treatment.

An emerging leader in molecular diagnostics and the analysis of DNA, RNA, and proteins at the molecular level, Abbott Molecular, Abbott Park, Ill, offers the PathVysion test, which is based on fluorescence in situ hybridization (FISH) technology and allows a physician to determine how many copies of the HER2 gene are present in a patient. FISH uses labeled single-stranded DNA probes to identify the location of genes on an individual's chromosomes. The test can be performed in less than 24 to 36 hours and can be run manually, in a semiautomated or fully automated manner. PathVysion is also indicated as an aid in predicting survival and remission in patients with stage II, node-positive breast cancer treated with adjuvant cyclophosphamide, docorbuicin, and 5-fluorouracil (CAF) chemotherapy.

The PathVysion assay demonstrates a hybridization efficiency of 98% in formalin-fixed, paraffin-embedded biopsy specimens, and published studies using the PathVysion DNA Probe Kit have demonstrated a concordance of 97% to 99% with known HER2 positive specimens. The test's use in gastric cancer is currently being developed, and its use may extend to other types of cancer in the future.

Ventana Medical Systems, Oro Valley, Ariz, offers a panel test for biomarkers of breast cancer, including HER2.

The panel employs a combination of antibodies (immunohistochemistry) and molecular probes (in situ hybridization) to reveal individual biomarkers of breast cancer tumors. HER2 IHC tests provide a protein-based diagnostic to identify overexpression of the HER2 protein as a result of too many copies of the HER2 gene. HER2 ISH tests provide a gene-based diagnostic to identify amplification of HER2 genes and the resulting production of too much HER2 protein. The Ventana IHC breast panel and ISH assay are available in a ready-to-use form and fully automated on the Ventana BenchMark series of instruments.

More specifically, the ant-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody is FDA-approved for the semiquantitative determination of HER2 protein expression status as an aid in the assessment of breast cancer patients for whom Herceptin therapy is being considered. Currently under review with the FDA is the INFORM HER2 Dual ISH DNA Probe Cocktail assay, which is presently CE Marked (Europe) for both breast and gastric samples and is available for commercial use outside the United States and Canada.

GASTRIC CANCERS

The American Cancer Society estimates that there were 37,000 new cases of stomach and esophageal cancer in 2010, and that they caused more than 25,000 deaths last year.

The HER2 gene also shows significant relevance in gastric cancers, although a different evaluation lens may be required in the determination of HER2 positivity in gastric tumors compared to breast tumors. In gastric cancer, staining of a HER2 positive tumor results in a U-shaped stain, which may be considered HER2 negative in the lens of breast cancer. Because HER2 clusters differently in the cells of the stomach or esophagus compared to breast cancers, it is becoming clear that different modes of evaluation are necessary. For example, more tumor tissue samples are generally required for determining HER2 status in gastric cancers, the staining pattern of the cell membrane is different, and the scoring and interpretation of the test results are different.

Krysta Pellegrino, a company spokesperson for Genentech, headquartered in San Francisco, sees that as another step in the direction toward personalized medicine.

"This is what companies like Roche and Ventana are trying to do, is develop treatment plans that work for individuals," she says.

MACHINERY

Life Technologies offers a new series of genetic analyzer, the 3500 Series Genetic Analyzer, which includes 8- and 24-capillary Genetic Analyzers and are designed for research use only. Compared to the 3130 Series, the new series offers higher throughput, advanced software allowing real-time data quality assessment, multiplexing capabilities for DNA fragment analysis with up to six different dyes, a standard power supply, and less bench space required.

The 3500 Dx Series Systems are Life Technologies' first CE-IVD marked capillary electrophoresis instruments, designed to comply with the Directive 98/79/EC on In Vitro Diagnostic Medical Devices, currently registered in most European countries and intending to file the application for 510(k) registration with the FDA before the end of the calendar year 2011.

With rapid improvements in equipment, assays, and the elucidation of relevant biomarkers, the field of molecular diagnostics is burgeoning. Personalized medicine, one goal of molecular diagnostics that is already being clinically realized, offers the opportunity to treat individuals on a case-by-case basis with greater efficacy and efficiency than ever before.

"The future of personalized health care may include the pathologist delivering a comprehensive molecular ‘profile' that identifies initial targets of therapy as well as molecular pathways or specific mutations that the cancer may use to circumvent first-line therapy," says Eric Walk, Ventana's chief medical officer.

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why we use TWO buffer solutions in capillary electrophoresis?

Electroosmotic flow is observed when an electric field is applied to a solution in a capillary that has fixed charges on its interior wall. Charge is accumulated on the inner surface of a capillary when a buffer solution is placed inside the capillary. In a fused-silica capillary, silanol (Si-OH) groups attached to the interior wall of the capillary are ionized to negatively charged silanoate (Si-O-) groups at pH values greater than three. The ionization of the capillary wall can be enhanced by first running a basic solution, such as NaOH or KOH through the capillary prior to introducing the buffer solution. Attracted to the negatively charged silanoate groups, the positively charged cations of the buffer solution will form two inner layers of cations (called the diffuse double layer or the electrical double layer) on the capillary wall . The first layer is referred to as the fixed layer because it is held tightly to the silanoate groups. The outer layer, called the mobile layer, is farther from the silanoate groups. The mobile cation layer is pulled in the direction of the negatively charged cathode when an electric field is applied. Since these cations are solvated, the bulk buffer solution migrates with the mobile layer, causing the electroosmotic flow of the buffer solution. Other capillaries including Teflon capillaries also exhibit electroosmotic flow. The EOF of these capillaries is probably the result of adsorption of the electrically charged ions of the buffer onto the capillary walls The rate of EOF is dependent on the field strength and the charge density of the capillary wall. The wall's charge density is proportional to the pH of the buffer solution. The electroosmotic flow will increase with pH until all of the available silanols lining the wall of the capillary are fully ionized

Capillary Electrophoresis of Free Amino Acids in Physiological Fluids Without Derivatization Employing Direct or Indirect Absorbance Detection (Med Worm)

Whole blood and/or plasma amino acids are useful for monitoring whole body
protein and amino acid metabolism in an organism under various physiological
and pathophysiological conditions. Various methodological procedures are in
use for their measurement in biological fluids. From the time when capillary
electrophoresis was introduced as a technology offering rapid separation of
various ionic and/or ionizable compounds with low sample and solvent
consumption, there were many attempts to use it for the measurement of amino
acids present in physiological fluids. As a rule, these methods require
derivatization procedures for detection purposes. (Source: Springer protocols
feed by Protein Science)

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Med Worm

Fluorescent DNA sequencing with Capillary Electrophoresis