Flow Cytometry Laboratory Overview
The Laboratory of Flow Cytometry was formed in 1988 to provide a cutting edge core facility in flow cytometry under the direction of Dr. Carleton Stewart. His vision for the laboratory was to provide state of the art multicolor flow cytometry services to both the Clinical and Research staff at the Institute. In 2003, Dr. Paul Wallace was appointed Director and while continuing the original model in many ways expanded the capabilities to the laboratory, most notable the responsibility for microscopic imaging. In 2007, the Facility was renamed the Department of Flow and Image Cytometry and acquired an Amnis ImageStream cytometer capable of capturing multiparameter images of cells in flow. The facility also provides confocal and kinetic microscopy services but retains its roots in flow cytometry. Today most of the cytometers have been upgraded with digital electronics and 5 and 6 color clinical flow cytometry has become the norm. The more adventurous researchers at the Institute are performing 10 color experiments on the facility’s LSRII and FACSAria and Ed Podniesinski, the facilities engineer has been experimenting with a breadboard multi-spectral flow cytometer using a 26 channel APD array
The goals of the Department are to provide second to none multiparameter flow cytometry services for clinical diagnosis and follow-up and to basic and clinical research investigators. Both our clinical and basic research flow cytometry laboratories maintain GLP practices emphasizing a quality assurance program accredited by State and national accrediting agencies. The Imaging Facility provides researchers with qualitative and quantitative image analysis on the tissue, cellular and subcellular levels, time-kinetic multicolor image analysis and quantitative multispectral image analysis. To accomplish these goals, and, as a focal point for many interdisciplinary activities, the Department has evolved into a multipurpose laboratory that provides both core and reference laboratory services and maintains an active translational research program.
Our educational program provides didactic lectures and hands-on experience with (i) isolation, preparation, and staining of all types of human and animal cells, (ii) instrument setup and acquisition, and (iii) data analysis. The Resource provides mentoring and research studies to graduate and Post-Doctoral students through Roswell Park’s Educational Department and the University at Buffalo, SUNY. We also offer and an educational outreach program in clinical flow cytometry specializing in the diagnosis and follow-up of leukemias, myelodysplasias and lymphomas to laboratory professionals interested in advancing their knowledge of these techniques.
Finally, a major emphasis of the Department is to provide consultation and custom methods development services to investigators interested using flow and image cytometry. We are delighted to assist you or develop a technique internally for you. These labor intensive and highly specialized methodologies are often empirical in nature and cannot be easily obtained from outside sources.
Basic Research
In our experience, there are three types of research investigators who use the Flow and Image Cytometry Resource. The level 1 (advanced/proficient user), level 2 (clinical researcher) and level 3 (beginner)
Level1 users are those who are already proficient in the techniques and know the instruments’ capabilities. For these investigators the laboratory provides oversight and consultation as necessary but mainly provides the instrumentation, data analysis systems, and instruction on how to use them, while the investigator and his/her technical staff perform the work.Level 2 users depend exclusively on the expertise of the Department staff to provide sample preparation, data acquisition and analysis and interpretation. This includes all in-house and outside clinical research and clinical protocols as well as some basic research. Clinical research studies are defined by a protocol that has been approved by the Institutional Review Board (IRB).
Level 3 users include investigators and students who have just learned about flow or image cytometry or who are just beginning to consider application of this technology to address their research questions. For these individuals the Department staff may perform pilot experiments to determine what would be a useful approach. Staff then works one-on-one to train the investigators on proper sample preparation, instrument operation and data analysis for their protocol. If successfully developed and applied, our goal is to train these investigators to become proficient in the technique and attain level 1 status.
Most users of the flow cytometry components of the Resource and their technical staff are proficient in the instrumentation and associated data analysis software. These investigators perform their own flow cytometry data acquisition and analysis and have access to the facility 24 hours a day, seven days a week. Users schedule themselves for instrument use on a web-based calendar. The flow cytometry assisted sample preparation, acquisition, analysis and sorting service (for investigators who choose to have the facility personnel perform some or all of their flow cytometry) is available by arrangement from 8:00 A.M. to 6:00 P.M. Monday through Friday. Experiments at other times can be prescheduled with the Resource personnel.
Our staff, rather than the user, performs all cell-sorting experiments due to the complexity of the instrumentation. Users of the ImageStream, Confocal, and LiveCell Imaging components of the Resource bring stained samples to the facility for data acquisition and analysis by Resource staff. These users must schedule instrument time with staff, who are available from 8:00 A.M. to 6:00 P.M. Monday through Friday. In the long run, we believe many of these users will want to acquire their own data and will want 24/7 access to the ImageStream, Confocal and LiveCell Imaging instruments. If you are interested in learning how to operate this equipment please make our staff aware of your desire and we design together a training program for you.
IMAGE CYTOMETRY
The ImageStream platform acquires six multispectral images of each cell at rates that can exceed 10,000 cells per minute. In this process, a single cell image is optically decomposed into a set of spatially correlated sub-images, each corresponding to a different color component and this image processing occurs in real time during the image formation process, rather than via digital image processing of a conventional composite image. This spectral decomposition allows multimode imaging, the simultaneous detection of brightfield, darkfield, and multiple colors of fluorescence, by choosing distinct spectral bands for the different illumination modes. This technology is particularly useful in the quantitative analysis of intracellular distribution of molecular markers which has its applications in the study of cellular signal transduction pathways, and cellular morphological changes for example nuclear morphological changes associated with apoptosis. In summary, this technology combines the high throughput capability of conventional flow cytometry with the high image resolution capability of microscopy.
The Leica Live Cell Imaging Systems available at the satellite facility are advanced fluorescence imaging platforms which allow the study of fluorescent markers in live cells under temperature-, CO2-, and humidity-controlled conditions. Capabilities include overlaying of multi-channel images and acquisition of three dimensional and time lapse data and applications range from calcium ratio and FRET analysis to colocalization and motion analyses.
Confocal microscopy is performed on tissue sections and cells stained with various fluorescent markers. A Leica TCS SP2 Spectral Confocal Microscope, with a wide fluorochrome application range including capabilities for viewing Hoechst and DAPI nuclear stains (blue diode) forms the foundation of our confocal microscopy services. A confocal microscopist is available by appointment to assist users with the confocal microscope. The Leica TCS SP2 Spectral confocal fluorescence imaging system is equipped with 4 available laser excitation sources (405 nm diode laser, an argon laser with lines at 457, 477,488 and 514 nm, and two HeNe lasers, one at 543 and one at 633 nm). It has three continuously adjustable spectrophotometer detectors. Software allows 3-dimensional reconstruction of the obtained images
Electron microscopy services include transmission (Hitachi H-600 and scanning EM (Etec Autoscan)). In addition to routine tissue processing (including special processing of primary cultures, cell monolayers, and subcellular organelles), several specialized techniques are offered, including pre- and post-embedding immunocytochemistry, scanning immuno-EM, quantitative EM autoradiography, cryoultramicrotomy, negative staining, critical point drying, extreme angle rotary platinum shadowing, and Kleinschmidt technique. An electron microscopist, trained and experienced in all aspects of sample preparation, data acquisition and photographic documentation related to these techniques, is available to investigators by appointment. The Hitachi H-600 scanning transmission electron microscope is equipped with an electron energy loss filter which permits electron spectroscopy for elemental analysis and clarification of images blurred by inelastic electron scattering. The ETEC Autoscan scanning electron microscope is equipped with a backscattering electron detector.
LUMINEX SOLUBLE BEAD ARRAY (SBA)
There is a maturing technology that uses microspheres (beads) of different sizes and colors as a surface for capture reagents such as antibodies or oligonucleotide probes that are used for the measurement of analytes of all kinds. This technology has several advantages over the existing ELISA methodology. Since each bead is the substrate upon which the test occurs, multiple assays can be combined in a single tube. The SBA technology results in a dramatic reduction in the amount of sample required, reagents required and the throughput time for analysis. Colored beads are currently available for up to 100 different tests in a single tube, although 30-40 analytes per tube is more common. 100s of cytokines, chemokines and growth factors whose coordinate or discordant regulation is of clinical interest are available. In a nearly four year collaboration with Luminex Corporation (Austin, TX), we pioneered the SBA multiplex technology and currently offer 57 different human, 26 mouse, and 12 rat analytes consisting mostly of cytokines and chemokines. A list of human, mouse and rat cytokines currently offered is continuously updated at http://www.rpciflow.org/analytes_available.html. These multiplexed assays demonstrate a dynamic range of 2.5 to 3.5 logs with sensitivities typically less than 10 pg/ml.
The technology employs a flow cytometer with two lasers, a 635 nm diode laser to excite the red and infrared classifier fluorochromes and a 523 nm frequency doubling Nd-YAG laser to excite the phycoerythrin (PE) reporter fluorochrome. Multiple bead sets can be detected because each bead is impregnated with a unique ratio of red and infrared dyes. Thus each bead has a unique 2 parameter fluorescent address that can be resolved by the instrument. Monoclonal antibodies, to human, mouse, and rat cytokines or chemokines, are coupled to a specific bead set. Multiple bead sets, each with a unique address, are mixed and incubated with the sample. After washing, paired, analyte specific, biotinylated mAbs are incubated with the beads and strepavadin-PE is used as the reporter. The amount of analyte in a sample is quantified in pg/ml by interrogated a standard curve traceable to either a WHO, or International Biological Standard (NIBSC). We use a 5 parameter best fit model to mathematically fit the standard curve. While this model can extract data from the flat or near flat upper and lower portions of the curve, we only report values within its linear portion. Samples are initially diluted 1:2 in assay diluent to remove heterophilic antibodies; samples above the linear portion are further diluted and repeated until they fall within in the linear portion of the standard curev. Usually this is a 1:10 or 1:100 dilution, if you suspect your samples will be extremely high, please let us know ahead of time. Even with our strict data transforms, we typically have a dynamic range of from 2.5 to 3.5 logs, depending on the cytokine.
We offer several predefined panels, including TH1/TH2 and inflammatory panel. Since we do not rely on prepackaged reagents, a major benefit of using the Roswell Park Soluble Bead Array Laboratory is our capability to completely customize our service to your individual needs. This can be a tremendous cost saving when one or just a few analytes are requested, since we select and only charge for the reagents required. We have partnered with Invitrogen and offer all human, mouse and rat analytes they have made available. Multiplex beads can also be conjugated at Roswell Park for analytes not available from Invitrogen and are thoroughly tested in our system for sensitivity, dynamic range, reproducibility, and multiplexibility (i.e. lack of heterophilic cross-reactivity). In this manner, novel beads sets not available from Invitrogen can be configured provided there are suitable antibody pairs.Clinical Applications
There are five major applications offered by the clinical laboratory:
- Monitoring T cell subsets and HIV patients
- Immunophenotyping leukemia and lymphomas and monitoring residual disease
- Determining CD34 stem cell counts for hematopoietic reconstitution
- Diagnosis of paroxymal nocturnal hemoglobinuria(PNH)
- DNA analysis of S-phase fraction of solid and hematopoietic tumors
The laboratory accepts blood, bone marrow, CSF and other body fluids, node and tissue specimens.
The Director and Supervisor of the Flow & Image Cytometry Facility is available to discuss laboratory services regarding a particular patient in order to reduce costs and customize testing for the highest probability of obtaining clinically useful information. Interpretative and technical consultative services are also available at no additional costs to the physician or patient.
Please contact the laboratory whenever there is a question as to what test should be requested or the appropriateness of sending a specimen
Paul K. Wallace, PhD (716) 845-8471 Mary B. Dell (71) 845-3528Specimens Acceptable for Processing by the Flow Cytometry Laboratory:
- Peripheral Blood
- Acute leukemia
- Chronic (mature) B and Tlymphoid leukemia or lymphomas
- Chronic lymphocytic leukemia
- large granular lymphocyte proliferation
- Prolymphocytic leukemia
- Hairy cell leukemia
- Non-Hodgkin’s lymphoma
- Seazary’s T cell lymphoma
- Waldenstrom’s macroglobulinemia
- Plasma cell leukemia
- Lymphocytosis of undetermined etiology
- Suspected acquired immune deficiency syndrome (AIDS)
- Other immune deficiency
- Diagnosis of Paroxymal Nocturnal Hemoglobinuria(PNH)
- Paroxysmal Nocturnal Hemoglobinuria (PNH)
- Bone Marrow
- Acute leukemia at diagnosis or relapse
- Myelodysplastic syndrome (MDS)
- Chronic (mature) B and T lymphoid leukemia or lymphoma
- Multiple myeloma
- Nodal or Extranodal Tissues
- Malignant lymphoma
- Granulocytic Sarcoma (Chloroma)
- Carcinoma
- Sarcoma
- DNA index and cell cycle
- Blood or Bone Marrow
- All chronic myeloid leukemias or chronic myeloproliferative disorders
- All myelodysplastic syndromes
- Chronic (mature) B and T lymphoid leukemia or lymphoma
- Cerebrospinal and Synovial Fluids
- All acute leukemias, chronic lymphoid leukemias and non-Hodgkin’s lymphomas.
- All infectious leukocytoses.
- Pleural, Pericardial, Peritoneal and Synovial Fluids
- All acute leukemias, chronic lymphoid leukemia, and non-Hodgkin’s lymphomas
- Leukocytosis associated with infections
- Breast and Other Solid Tumors
- DNA Index and cell cycle
HOURS OF OPERATION
Flow Cytometry operates on evenings, weekends and holidays. A designated on-call Flow Cytometry Laboratory Technologists will process and tissue or STAT specimen for antibody labeling and fixation within four hours of collection. Have the Institute Operator contact the On-Call Flow Cytometry Technologist or page the Technologist directly at pager ext. 3062 . Unless an emergency exists which will impact on patient treatment or management, the flow cytometric assays themselves will be deferred until the first regular working day. Emergency Flow Cytometry results will be reported only to the requesting Staff Physician when available.
STAT results in four hours (from receipt in the laboratory), non-STAT report in 24 - 48 hours.Report includes interpretation and histograms of relevant cell populations.
Please contact the Laboratory for any special requirements.All tests use FDA approved Analyte Specific Reagents (ASRs) required for all clinical tests after November 23, 1998.
Laboratory certified by DOH and CLIA, approved by JCAHO and CAP.Education
The following lectures are taught throughout the year to sign up for notification of the next class email Marilyn Price or sign up outside the Science Flow Cytometry Lab.
- Introduction to Flow Cytometry
- Compensation and Fluorochrome Selection
- Immunophenotyping
- DNA staining and analysis
- Techniques to monitoring apoptosis
- Data analysis with WinList
- Data analysis with ModFit
- Data analysis with FCS Express
- EM techniques course
- Flow Cytometry
- Science Building: Earl Timm or Paul Wallace
- BLSC: Kieran O’Loughlin or Paul Wallace
- Image Cytometry
- BLSC ImageStream: Kieran O’Loughlin or Hans Minderman
- BLSC Confocal: Ed Hurley or Hans Minderman
- BLSC EM: Ed Hurley or Hans Minderman
LOCAL ACCOMMODATION
The Hilton Doubletree Hotel, formerly known as the Pillars Hotel, is immediately adjacent Roswell Park's Department of Flow and Image Cytometry. They have reasonable rates and each room features a king or two double beds, has high speed Internet access and is equipped with a microwave, refrigerator, coffee maker, iron and ironing board.. This hotel has shuttle service to both the Buffalo Niagara Airport and the Amtrak Station.
Doubletree Club Buffalo Downtown125 High Street, Buffalo
New York, USA 14203
Tel: (716) 845-0112
Fax: (716)-845-0125
Local restaurants abound within walking distance of Roswell Park. Other hotels and rental locations (for longer visits) are available.
DIRECTIONS AND TRANSPORTATION
Flow Cytometry is in the Science building easily accessed from High Street. Once in the RPCI complex, follow the directions below to the Hilton Doubletree (formerly known as the Pillars). The entrance to Science is just under the Pedestrian bridge connecting the Science building with Buffalo General Hospital. Take the elevators to the 6th floor and follow the signs to Flow Cytometry.
From points east, west, north, and south of Buffalo: Take the New York State Thruway (Interstate 90) to Exit 51W (Route 33 West). Exit from Route 33W at Locust Street. Turn right at the first traffic light (Michigan Avenue). Continue on Michigan Avenue for two blocks to Carlton Street. Turn left at the traffic signal. The parking ramp entrance is immediately on your left.
For the Hilton Doubletree continue up Michigan Avenue 1 more block to High Street. Turn left at the signal. The Pillars will be on the left. There is parking two blocks down on either your left or right.To return to Route 33, turn right on Carlton as you exit the parking ramp. Turn right at the signal onto Michigan Avenue. Travel two and one-half blocks to Cherry Street, immediately beyond the underpass. Turn left onto Cherry Street; keep left and enter 33 East. Follow 33 East to the New York State Thruway (I-90). The first ramp is for Erie-Cleveland and points south and west; the second ramp is for Albany and points north and east.
Flow Cytometry (6th floor Science Building)99 High Street (please do not use for shipping)
Buffalo, NY 14263
Voice: (716) 845-8471
Bus, Amtrak and Airline service to Buffalo is convenient and frequent. RPCI is 10 min. by taxi, limo, or subway from the bus (Oak St.) or train stations (Exchange St.) or 15 min. from the airport by taxis or limo.