GCRC Core Laboratory
The GCRC Core Laboratory was established in 1972 to fulfill the following objectives:
1 - provide a consolidated, highly specialized, well-equipped, and professionally staffed unit capable of performing a variety of analytical procedures as required by the project investigators;
2 - develop new procedures to meet the requirements of approved protocols;
3 - establish by its expertise new or modified methodologies as required in a more efficient manner than if the investigators had to do so in their own individual laboratories with limited resources and equipment, and provide these services at a lower cost than that which can be arranged with an outside facility;
4 - maintain quality control and quality assurance;
5 - assist nursing staff with sample preparation and distribution, and assist investigators in the preparation and storage of genetic materials;
6 - provide consultation on laboratory issues which may arise among GCRC investigators;
7 - provide training in laboratory methodologies to young clinical investigators; and
8 - ensure subject privacy and confidentiality
The Core Laboratory is divided into two main functions: 1) Sample processing and distribution, and 2) Analytical procedures. The Core is responsible for performing non-routine analytical tasks and transforms to meet the changing needs of its investigators. The Core Laboratory provides a centralized resource for the preparation of DNA necessary for the growing number of genetic studies. In addition the Core performs several routine analyses for less expense than would be incurred were those samples sent to the hospital's Dynacare facility or other laboratories, and also provides a number of services not available in the routine laboratory or which are costly to perform elsewhere. Another important feature of this Core is that reproducible and reliable assay procedures are performed while economizing the use of personnel, supplies, and equipment. Whenever possible, samples for testing which cannot be performed here are sent to other GCRC Core Laboratories, while samples from other GCRCs are analyzed at this Center. The Core Laboratory thus represents an essential resource to assist GCRC-based investigators in conducting innovative, interdisciplinary research. The following sections detail the components of this Core.
Personnel
Glenn R. Krakower, Ph.D.
Director, GCRC Core Laboratory
Room 1161, Froedtert Hospital-West
Phone: 805-3802 E-mail: gkrakowe@mcw.edu
Dr. Krakower received his Ph.D. in biochemistry from Purdue University in 1980, with a strong interest in metabolic regulation. He came to Milwaukee and expanded his research interests into toxicology and pharmacotherapy with Drs. David Petering at the University of Wisconsin-Milwaukee and Barton Kamen at the MCW Department of Pharmacology & Toxicology. Dr. Krakower joined the research group of former GCRC Program Director Dr. Ahmed Kissebah in 1983, where he developed interests in the pathophysiology of obesity and the metabolic syndrome.
Dr. Krakower became the Associate GCRC Core Lab Director in 1989, then Laboratory Director in 1993, with the day-to-day responsibility for supervision of the Core Laboratory processing and distribution resource and for the analytical services, supervising staff, establishing new analytical methods, overseeing quality control procedures and SOPs, reviewing results before they are released to the investigators, maintaining laboratory certification, overseeing the maintenance of equipment, purchasing reagents, supplies, and equipment, and maintaining other laboratory records. In addition, he oversees the training of new technicians, graduate students, and young investigators with interests in patient-oriented research, and instructs participants of the Residents' Clinical Rotation in aspects of sample preparation and data interpretation, in addition to providing consultation with established investigators in laboratory ascertainments.
The preparatory procedures of the Core Laboratory is supported by Ms. Gwen Shadley (805-3849, gwenshad@mcw.edu), who has a Masters degree in Developmental Genetics and substantial laboratory experience in tissue culture, along with basic skills in chromatography and histology. Ms. Shadley has been with the Core Laboratory since August, 1999. Ms. Shadley is primarily responsible for DNA extraction and dilutions, in addition to maintaining quality control for these procedures and working to assure the smooth operation of the Autopure LS system. She prepares laboratory solutions, reagents, and media, provides instruction on DNA and white cell preparation to medical students and residents participating in clinical laboratory projects, and trains other laboratory personnel in the operation of the Autopure LS system.
Radioimmunoassays and other analytical procedures are primarily performed by Ms. Rae Ann Petersen, who oversees the distribution and processing of samples, provides investigators with periodic updates on their test results, and assists in instructing other laboratory personnel and residents participating in clinical laboratory projects. She also operates, calibrates, and performs routine maintenance on specialized equipment; performs basic quality control of laboratory refrigerators, freezers, and centrifuges; and assists GCRC Nursing staff in sample distribution and processing, in addition to assisting Ms. Shadley with preparatory procedures when needed.
Environment and Resources
The GCRC Core Laboratory (FH 1-West, Room 1160) is located opposite a walk-in cold room with walk-in freeze, and a storage room (total Core Laboratory space: 766 sq. ft.). This laboratory is used for DNA extraction and other preparatory work. The office of the Core Laboratory Director is adjacent to this laboratory. In addition to desk space for the 2 GCRC Core Laboratory technologists and small supporting equipment, Room 1160 houses:
• 1 Gentra Systems Autopure LS DNA processing system
• 1 Beckman DU800 double-beam spectrophotometer
• 1 Beckman Allegra 6R tabletop centrifuge
• 1 Zebra TLP 2844 bar-coding unit
• 2 4°C refrigerators
• 2 stand-up -20°C freezers
• 2 18 cu. ft. -80°C Revco Elite freezers
Shared Laboratory Space. The Core Laboratory has a longstanding close relationship with Endocrine Division personnel, who continue to share their expertise and resources. All Core Laboratory analytical testing is performed in space shared with the Dept. of Medicine's Division of Endocrinology, Metabolism and Clinical Nutrition (FH-W, Room 1134). This room houses the following GCRC equipment:
• 1 Packard 5650 gamma counter
• 2 Beckman II glucose analyzers
• 1 Mettler AE163 analytical balance
• 1 Mettler top-loading E200 preparatory balance
• 1 Bio-Tek Powerwavex 340 microplate reader
• 1 Bio-Tek ELx50 microplate strip washer
This room is also the location where radioactive substances are utilized. Dr. Krakower is the Authorized User for Radiation Safety for the GCRC. In addition, the adjacent FH-W area includes shared space that houses a culture area (with warm room), additional cold room/freezer space, and a room for shared equipment.
Sample Preparatory Laboratories. The sample preparation area of the Adult GCRC Unit is adjacent to the Nursing Station, in close proximity to the patient care area. This laboratory, maintained by the Core Laboratory, is supported by a Beckman Allegra refrigerated centrifuge, a small tabletop centrifuge, 1-1/2 refrigerators, and 1 -20° freezer for sample storage. The dedicated phlebotomy/sample preparation area in the Pediatric Unit includes one dedicated refrigerator/-20° freezer and one Revco freezer for -80°C sample storage.
Quality Control and Patient Privacy
Well-defined standard operating procedures (SOPs), which include quality control guidelines, are provided for all laboratory equipment and for each assay procedure. The SOP compilation folder (hard copy) is located in the office of the Core Laboratory Director, with a duplicate SOP compilation folder maintained in the office of the GCRC Center Administrator. In addition, laboratory personnel have electronic versions of individual SOPs, which are also available for sharing with other GCRC Core Laboratory Directors.
Calibration and standardization of laboratory instruments, including pipettors, are performed quarterly or when indicated, while temperatures of waterbaths and refrigerators are monitored on a daily basis, in accordance with the Clinical Laboratory Improvement Amendments (CLIA) requirements. Essential equipment (including the freezers, Autopure system, and gamma counter) are supported by emergency power lines. Freezer temperatures (-70° and -20°) are recorded daily and are connected to an alarm system which is also monitored by FH Security nights and weekends. Should the temperature drop beyond a certain point (defined by the freezer), security is instructed to phone a list of laboratory personnel.
Quality control is also assured in part through participation in the Proficiency Testing Program operated through the Wisconsin State Laboratory of Hygiene. Proficiency testing, performed triannually for routine clinical analytes (glucose and basic lipids), is an important requirement for CLIA compliance. Even though we are a research laboratory rather than a clinical one, the lab is evaluated by CLIA representatives at 2-year intervals, most recently in February, 2006; the Core Laboratory Certificate of Compliance is current through August 29, 2008.
Routine quality control and quality assurance is performed with every assay. Control samples of consistent matrix and range of values for the anticipated subject population are analyzed in a manner identical to those of the unknown samples. When controls for specific analytes are commercially unavailable, reference standards are purchased and prepared in the appropriate matrix. Controls/reference standards are run with each assay, often in multiple locations to ensure uniformity of results. Prior to releasing results, quality control values are checked against the means and standard deviations of the analysis to determine that the assay falls within control limits established for that particular analysis. Results of individual analyte determinations are reviewed for shifts and trends which might require attention. Controls which fall outside of two standard deviations are reported to Dr. Krakower; after consultation with Ms. Petersen and Ms. Marks, the assay will either be repeated using new controls or new test kits, or following re-calibration or maintenance of the instrument. Permanent laboratory records, including sample logbooks, assay worksheets, and instrument printouts, are stored for no fewer than 10 years.
Efficient communication with GCRC Nursing personnel is essential for the timely provision of supplies, maintenance of equipment in the sample processing laboratory, and seamless retrieval of samples for analysis. In addition to specific discussions between the Core Laboratory Director and the Nursing Supervisor or nursing staff regarding individual protocol requirements, frequent informal meetings or discussions between laboratory staff and the nurses assure smooth flow from sampling and processing to sample testing; summaries from these meetings are prepared at 2-month intervals and are on file in the office of Dr. Krakower and in the office of the Nursing Supervisor. The nursing staff have their own SOPs (FH formatting) for the operation of equipment and sample processing procedures; copies of these are included in the the Core Laboratory SOP book. Requests for the service of instruments located in the samples preparation areas are made through Dr. Krakower.
Patient Privacy and Confidentiality. Subject privacy and confidentiality is a priority for laboratory staff. Dr. Krakower works closely with the Research Subject Advocate to ensure subject data confidentiality and that DNA information designated in the consent form is strictly followed, in addition to discussing laboratory utilization issues on specific protocols. Results of these discussions, which are summarized monthly and stored in Dr. Krakower's office, are then communicated to Ms. Shadley and Ms. Petersen. All Core Laboratory personnel have been certified in Human Bioethics though the web-based CITI course in the Protection of Human Research Subjects (as overseen by the GCRC Research Subject Advocate), the NIH course in Human Participant Protections Education for Research Teams, as well as through the lecture series offered through the GCRC Training Program.
While each investigator has his/her own security measures written into the protocol, protection is also afforded by computerized bar-coding, where subjects receive a newly coded number that can be linked back to the patient. Bar code readers have been installed on laboratory equipment so that sample numbers can be read directly, obviating the need to manually transfer data, thus reducing the risk for errors. Bar codes are prepared by Core Laboratory staff on an as-needed basis and stored in spreadsheeted databases.
Core Laboratory staff work closely with the Informatics personnel to ensure patient privacy and data compilation into the appropriate databases, while Informatics personnel maintain laboratory computers and their software, provide instruction to laboratory staff on the de-identification or protection of subject data, and aid laboratory staff in sample tracking. Close collaboration with Informatics personnel also assures smooth flow from sample analysis to the reporting of data, in addition to ensuring HIPAA compliance and facilitating sample identification and data storage.
Core Laboratory Analyses
The following assay platforms are supported as specialized biochemical and analytical procedures: 1) radioimmunoassays (RIA), including peptide hormones related to obesity and diabetes, and steroid hormones; 2) colorimetric analyses, including basic lipids; 3) ELISA/EIAs, and 4) plasma glucose via dedicated glucose analyzers. DNA preparation from blood, saliva, and buccal samples is now completely automated, with the arrival of the Autopure LS system and the Beckman DU800 UV/VIS spectrophotometer, awarded through a Shared Instrumentation Grant submitted in March, 2003 (below), while reagents used in the Autopure system also facilitate white cell preparation in a recently updated procedure. Protocols to extract tissue DNA are in preparation.
The following tests are available through the GCRC Core Laboratory. Please contact Dr. Krakower for specific information about these tests and the amounts of plasma or serum required.
|
Analyte |
Method |
Matrix |
Vendor
|
|
Insulin |
RIA |
Plasma |
Linco Research |
|
C-Peptide |
RIA |
Plasma |
Linco Research |
|
Proinsulin |
RIA |
Plasma |
Linco Research |
|
Leptin |
RIA |
Plasma |
Linco Research |
|
Ghrelin |
RIA |
Plasma |
Linco Research |
|
Adiponectin |
RIA |
Plasma |
Linco Research |
|
Estradiol |
RIA |
Plasma |
DPC/Siemens |
|
Testosterone, free |
RIA |
Plasma |
DPC/Siemens |
|
Testosterone, total |
RIA |
Plasma |
DPC/Siemens |
|
Progesterone |
RIA |
Plasma |
DPC/Siemens |
|
DHEAS |
RIA |
Plasma |
DPC/Siemens |
|
FSH |
IRMA |
Serum |
DPC/Siemens |
|
LH |
IRMA |
Serum |
DPC/Siemens |
|
SHBG |
IRMA |
Serum |
DPC/Siemens |
|
Estrone |
RIA |
Serum |
DSL |
|
Cholecystokinin |
RIA: Double Ab |
Plasma |
Alpco |
|
IGF-1 |
RIA: Double Ab |
Serum |
DiaSorin |
|
Cholesterol |
Spectrophotometric |
Plasma |
Roche Diagnostics |
|
HDL-Cholesterol |
Spectrophotometric* |
Plasma |
Roche Diagnostics |
|
LDL-Cholesterol, direct |
Spectrophotometric |
Plasma |
Trinity Biotech |
|
Triglycerides |
Spectrophotometric |
Plasma |
StanBio |
|
Free Fatty Acids |
Spectrophotometric |
Plasma |
Wako Diagnostics |
|
C-Reactive Protein (hs) |
ELISA |
Serum |
MP Biomed |
|
TNF-alpha |
EIA |
Plasma |
R & D Systems |
|
Interleukin-6 |
EIA |
Plasma |
R & D Systems |
|
Glucose |
Beckman Analyzer II |
Plasma |
Beckman Coulter |
* Cholesterol assay follows phosphotungstate precipitation
The types of analyses done by the Core Laboratory fall into two categories: routine analyses and specialized tests. The routine chemistries, such as the glucose and simple lipids, which can be done by hospital labs, utilize long established methodologies that result in greater accuracy and/or sensitivity, for far less cost to the GCRC budget. These tests are performed only as study mid- or endpoints, and never for baseline values and/or testing to satisfy inclusion/exclusion criteria. The Core Laboratory participates in triannual proficiency testing for glucose, cholesterol, triglycerides, and HDL-cholesterol.
Statement of Information: Determinations for HDL- and direct LDL-cholesterol in the GCRC Core Laboratory are performed by manual procedures whose results do not necessarily conform to the values that would be obtained using the automated methods preferred by hospital laboratories. Our assay methods are gold standard procedures designed for accuracy, while automated procedures are designed for speed and precision. Because the recommended values for lipoprotein subfractions have been established using data determined by automated procedures, values provided by the Core Laboratory should never be used for clinical evaluations, and in no case should these results ever be given to the patients. Subjects whose Core Lab values fall outside guidelines for these tests should be advised to see their personal physicians for clinical testing.
Some specialized tests such as C-peptide and proinsulin, and other hormonal analyses not routinely available through the Dynacare Laboratory represent study endpoints of protocols approved by the GCRC Research Review Committee.
New tests can be established provided that they fall within the established assay platforms (i.e. colorimetric or radioimmunoassay) and are crucial to the accurate interpretation of the study. Alternatively, some tests not performed by the Core Laboratory may be available through other GCRC Core Laboratories. One service frequently provided is apoE genotyping, through an arrangement with the Core Lab at the University of Oregon Health Sciences Center. Dr. Krakower can assist you in searching for testing through other GCRCs.
Final approval for the types of analyses to be performed lies with the GCRC Advisory Committee (GAC). Analyses are reviewed to determine general applicability of tests to different researchers and to assess inclusion of routine analyses which may be performed by the Core Laboratory in a more timely and/or more cost effective manner. Priorities of testing are also overseen by the GAC. These are established in the following order:
1) NIH-funded research projects benefitting from GCRC assistance
2) Protocols funded by peer-reviewed grants from nonprofit organizations
3) Protocols funded through Children's Hospital, the VA, or other affiliated institutions
4) Pilot studies needing preliminary data for a subsequent grant application, deemed meritorious by the GCRC Research Review Committee
The GAC also provides oversight to assure that no particular investigator or investigator group monopolizes utilization of any single laboratory determination or group of determinations. Kits and supplies for these tests are paid from the grants of established investigators, including the Program Director. In some pilot or feasibility studies not funded by other means, the GCRC assumes these costs.
DNA Preparation: Shared Instrumentation Grant award. The GCRC Core Laboratory provides space for the Autopure ls System, along with the technical expertise of Dr. Glenn Krakower, who supervises administrative requirements for the instrumentation, and Ms. Gwen Shadley, who oversees its operation. A 7-member Internal Advisory Committee meets as necessary to develop policies and procedures for the use of the instrumentation, to review user fees, instrument performance and utilization, expansion of services, sample turnaround time, and other topics, within guidelines established through the GAC and the MCW guidelines for the use of shared instrumentation. Policies and procedures established by the Advisory Committee are reviewed by the MCW Core Facilities Committee, who assure that recommendations are consistent with institutional policy. Priorities assure that NIH-funded projects represent up to 75-80% of total utilization, the remainder including projects funded by other peer-reviewed organizations, and feasibility/pilot studies proposed by young investigators with high potential of obtaining NIH funding.
DNA is currently prepared for GCRC-sponsored investigations for no fee. However, DNA preparation is also available to all MCW investigators on a fee-for-use basis. Please contact Dr. Krakower for more information on DNA preparation.
Training
The Core Laboratory serves as a site for the training of both young clinical investigators and other Medical College staff. The institution of the Residents Clinical Rotation in 2004 saw an increase in training opportunity, where residents engage with Core Laboratory personnel to observe laboratory procedures, receive guidance in aspects of sample preparation, and participate in discussions on data interpretation. Infrequently local nursing students will also spend some time working with laboratory personnel. Dr. Krakower has provided guidance to several campus laboratory leaders in the preparation of materials for CLIA inspection and certification procedures, and he has worked with several dietetics students with interests in laboratory operations.
In addition, laboratory staff help train in appropriate procedures for sampling and storage, including DNA preparation, and provide consultation and assistance with analyses required by clinical investigators. With the arrival of the Autopure LS system for DNA extraction, MCW and FH staff members and residents have received training on the instrument from Ms. Shadley. A proposed 2-session course in laboratory sampling and data analysis, as part of the GCRC Clinical Research Curriculum, would cover principles of patient sampling, sample preparation, and interpretation of clinical results. Dr. Krakower also works individually with young investigators on the design and conduct of specific projects, and the implementation of laboratory analyses to meet their goals. He assists investigators in their preparation of GCRC protocols. In addition to discussing data collection and quality control issues, he is available to assist in their efforts to access pertinent literature, develop scientific presentations and manuscripts, and identify and obtain needed resources. As a useful resource, the CDC has provided us a Powerpoint presentation which discusses the many aspects of patient sampling.
Specimen Banking Powerpoint presentation