Core Laboratories

The Drug Assay Core is utilized for preclinical and clinical research studies that require the quantification of parent drug and metabolite concentrations in human plasma and urine samples. The analytical facility is well-equipped with a variety of instruments that include:

HPLC systems with flourescence, UV-visible, radiochemical, electrochemical and photodiode array detection capabilities
UV-visible spectrophotometer
GC mass spectrometer
GLC with chiral/nonchiral columns and nitrogen- phosphorous detection
Beta and gamma scintillation counters
Refrigerated high-speed and ultra-centrifuges

Drug Assay Core personnel have expertise in the design of preclinical and clinical pharmacology studies, and in the modeling of pharmacodynamic and pharmacokinetic systems.

Contact Information

Lefteris Zacharia
412-648-5979
zacharial@dom.pitt.edu
100 Technology Drive, Suite 450, Pittsburgh, PA 15219

Clinical Investigation Core (CIC)

Mission Statement...

We offer our investigators a quality translational research management service with expertise tailored to their needs.

Scope

A HIPAA compliant integrated pathway for the access of patient data
Professional clinical research management
A centralized sample and tissue repository equipped with tracking capability

Organization

See chart in slide show in 'documents' area.

Goals

Provide clinical study coordination. Assist in the preparation of protocols and consent forms for IRB and GCRC management requirements.
Support patient recruitment endeavors.
Assure quality control by internally auditing protocols.
Maintain tissue and sample banks.
Track samples via front-end interface with Oracle-based information system, maintained by our Information Technology and Biostatistics Center (ITBC).

Summary and Conclusion

The Clinical Investigation Core provides professional assistance from protocol development, coordination, and management to the electronic storage of samples and databases.

Contact Information

Amy Steele
Research Coordinator
RN, BSN
Center for Clinical Pharmacology, University of Pittsburgh
Kaufmann Building
3471 Fifth Avenue, Suite 900
Pittsburgh, PA 15213
Phone: 412-802-3160
Email: steelea@dom.pitt.edu
Fax: 412-647-9846

Pharmacogenetics Core Laboratory (PCL)

Quick Links:

Assay Development

Quality Control Program

Confidentiality

The University of Pittsburgh GCRC Pharmacogenetics Core Laboratory (PCL) was established in 1999. The PCL is a sophisticated, centralized, efficient, low-cost resource for providing pharmacogenomic technology and expertise on drug metabolism to clinical researchers investigating the implications of interindividual variation in expression of drug metabolizing enzymes.

The PCL has several important components. Examples of key laboratory procedures include the following:

Genotype variant alleles of human drug metabolizing enzymes and other endpoints

The PCL genotyping service component utilizes one of four approaches to identify alleles of the known genetic polymorphisms of phase I and II human drug metabolizing enzymes and other genes. These methods include the Applied Biosystems GeneAmp 7700® Sequence Detection system for TaqMan based allele discrimination assay, the Luminex multiplexed microsphere approach, the Nanogen NanochipT technology and restriction fragment length polymorphism PCR (RFLP-PCR). The first three approaches allow for a rapid and high throughput analysis of samples with minimized risk of contamination. As new allelic variants become identified, this battery of tests is constantly expanded and undergoes extensive quality control procedures.

Quantitate gene expression levels

These analyses are completed using real-time quantitative RT-PCR (TaqMan technology). TaqMan technology, more accurately called 5' nuclease QPCR, is a new combination of instrumentation and chemistry which allows easy and fast QPCR. Previously, QPCR was a very difficult and labor intensive technique but with the advent of newer technology and instrumentation, this is no longer the case. TaqMan technology currently represents the state of the art approach for mRNA quantitation. In the PCL, the QPCR analyses are performed using the Perkin Elmer Applied Biosystems GeneAmp 7700® Sequence Detection system instrument, which is capable of quantitating PCR product accumulation in real time, in 96 tubes simultaneously. TaqMan based gene expression analysis has several advantages over conventional analysis methods. First, it is very fast as there is no need for gel or hybridization steps. When the PCR has finished, the data are exported for immediate analysis. Second, it is extremely sensitive and can detect expression of low copy mRNAs even when starting from very small amounts of starting material. Finally, the RNA to be analyzed can be significantly degraded without affecting the quantitation, a factor that can be very valuable when working with clinical specimens.

A PCL Steering Committee has been created to oversee this laboratory and is responsible for making recommendations in prioritization of projects.

Assay Development

With recent advances in the human genome sequencing project, one of the initial applications is the emerging field of pharmacogenomics. Pharmacogenomics encompasses several major areas:

The study of polymorphic variations to drug response and disease susceptibility
Identification of the effects of drugs/xenobiotics at the genomic level
Genotype/phenotype associations

The promise of pharmacogenomics is that studies using genome-based technology will lead to the identification of novel SNPs, and the characterization of their impact on human health. Therefore, the development and application of screening technologies in clinical and translational research is a high priority. It is likely that the need for a state-of-the-art pharmacogenetics core lab is only going to increase. To date the focus in the PCL has been on phase I and II enzyme endpoints, but broadly defined, pharmacogenetic based analyses includes a wide range of target genes and we anticipate expanding the panel of assays available.

Quality Control

The core laboratory is committed to providing accurate and reliable laboratory results using the most current technology and maintaining the privacy of genetic information. We are also committed to providing an intellectual resource for the development and application of novel methodological approaches and biomarker endpoints. The Clinical Laboratory Improvement Act (CLIA) does not yet have standards specific to genetic testing except for cytogenetics. We have instituted standard operating procedures based on the College of American Pathologists/American College of Medical Genetics' (CAP/ACMG) Molecular Pathology accreditation program. Our laboratory has a Standard Operating Procedures (SOP) manual which includes sections on quality assurance, quality control, personnel, method descriptions, safety procedures and equipment operating methods. For example, in addition to positive and negative PCR controls included with each amplification reaction, the following quality control measures are routinely performed. Blinded duplicate sample analyses are performed for all genotyping and mRNA expression endpoints. An additional 10% of samples are repeated to further avoid misclassification and verify reproducibility. To maintain optimum performance of the thermal cycler, the sample block will be cleaned, reproducibility of the cycle time will be confirmed, and instrument diagnostic tests for the heater and chiller will be performed. Pre-PCR and post-PCR areas are physically separated to prevent carry-over of amplified DNA sequences. A solution of 10% bleach will be used to clean the biological hazard hood used for pre-PCR procedures before and after every use to reduce DNA amplicon contamination. A separate set of supplies and pipettors will be dedicated for each phase of the PCR reaction preparation and analysis. Filtered pipette tips are utilized to eliminate carryover contamination. Two or more qualified personnel will interpret all gel electrophoresis data and perform mRNA data calculations.

In addition to following the federal guidelines to protect patient privacy, the PCL is aware that there is increasing general public concern over personal privacy issues of individuals who participate in genetic and pharmacogenomic research, and how this genetic information would be best protected to avoid any potential abuse or misuse of genetic data, without compromising its scientific content and value.

Confidentiality

Protecting patient privacy is of utmost concern and confidentiality is maintained by computer security access codes and locked file cabinets. Currently, every biological sample is labeled with a bar code sample identifier number. The sample identification number is distinct from a patient name, medical record number or social security number and reflects both the study type and sample date. All personnel, including the phlebotomist, research nurse and laboratory personnel, coming into contact with biological samples are required to initial the sample and an accompanying sample flow sheet, verifying that receipt of signed informed consents has been obtained. Recently the PCL purchased the FreezerWorks software package, a software system designed for barcode sample labeling and sample tracking.

Both the PCL barcode system and laboratory databases are protected with computer security access codes. The laboratory generated data information to be included in the laboratory database consists of sample number, date sample collected, study specific pharmacogenetic endpoint results matched to lab personnel responsible for data generation and date of analysis.

Contact Information

Marjorie Romkes, PhD
Director
(412) 383-8885
romkes@dom.pitt.edu
Center for Clinical Pharmacology, University of Pittsburgh, 100 Technology Drive, Pittsburgh, PA 15219

Information Technology & Biostatistics Center (ITBC)

The ITBC is a University-wide resource for clinical scientists and organizations. Research investigators are assisted through the use of custom designed, internet-based applications for protocol management, biological sample tracking and inventory, input forms, database design, statistical analysis and decision support.

Informatics Expertise

Various ITBC developed tools (Protocol Document Management System, MyWebDesk) provide the investigator and administrators access to important information about study protocols. Decision support software (DORAS) provides administrator and investigator access to advanced data mining techniques. The ITBC will design custom Internet-based applications for specific purposes; these applications will be hosted and maintained by the centralized ITBC Staff.

A centralized freezer inventory system, which has the ability to track samples in real-time, is available. This real-time biological sample tracking and inventory system is designed to interface with other custom developed Internet based decision support systems.

Technical Information:

The Information Technology section of the Information Technology & Biostatistics Center (ITBC) currently has seven rack mounted servers with internal DAT tape drive and two 300 VA rack mounted UPS systems. Each system is configured as a Raid 5 array with a fifth global spare and each contains a Hot Plug redundant power supply to improve system uptime reliability. A DVD reader/writer is used for storage of the Oracle cold backup data files as well as all development and internet based files.

The ITBC has access to site licenses for the full line of standard Oracle and Microsoft products and well as a number of other desktop publishing, analysis, data recovery and utility programs. The ITBC is specializing in the development of secure web based data collection applications (3-tier design) that connect to a central Oracle or MS SQL database as well as other remote data servers. The architecture is designed to be global, secure, and scalable and allow authorized access by investigators, statisticians and other authorized individuals.

The development and production servers are covered under the University of Pittsburgh firewall (Lucent Brick 80). Both within and outside the firewall, password protection and IP tracking are the basic security methods used for Intranet/Internet access. Security outside of the firewall is also maintained by use of a secure socket layer (SSL) for sensitive information and/or a virtual private network (VPN) for absolute security through encryption. All data is screened for privacy and identified and de-identified views are created with access to these views controlled by password and IP address authentication.

In addition to the above, the center for Clinical Pharmacology is part of a Novell-based local area network that serves approximately 400 IBM-compatible and Macintosh personal computers within the UPMC. A series of servers support file storage and backup, e-mail communications, remote access and security. Ethernet links through T1 lines support internal electronic communication as well as access to the Internet and the University of Pittsburgh's VAX and UNIX system. The network also permits searches of computerized databases, such as protein/DNA sequences, Current Contents, Medline reference services, UPMC Medical Archive Retrieval System, etc. from individual desktop PCs, which are available in each laboratory and each faculty member office.

Documents

ITBC Investigator Package