Hemostasis Research Core Laboratory

Purpose and Background

The purpose of the Hemostasis Research Core Laboratory at the Cleveland Clinic Foundation is to (1) provide routine and specialized hemostasis testing for the detection of coagulation and platelet activation during pharmacologic or mechanical intervention in coronary artery disease, (2) to provide functional in vivo monitoring of the effect of drugs that block coagulation or platelet function, (3) to provide centralized specimen collection, processing and assay to reduce inter-laboratory variability, (4) to provide standardized specimen collection kits, instructions and shipping to ensure the suitability of specimens for sensitive hemostasis testing.

The Hemostasis research Core Laboratory is dedicated to the study of coagulation and platelet activation with the implantation of cardiovascular devices and in cardiovascular diseases. We also emphasize the study of anticoagulant and anti-platelet drugs on the hemostatic system. Facilities are available for the hemostatic study of drugs and devices in vitro, in animal models and in small and large-scale clinical trials. For large-scale, multi-institutional studies, the Hemostasis Research Core Laboratory works closely with the nurse coordinators at the trial sites and the central coordinating center to facilitate all steps of a study, from protocol planning, to patient instruction sheets, to preparation of sample collection kits, to coordination of specimen collection and shipping, to frozen specimen achiving, to performance of the assays, to results reporting, database preparation and data analysis. The technologists in the laboratory are available on an on-call basis for performance of assays on a 24hr basis, if needed.

Facilities and Assays Available

All of our instruments have both written quality control and routine procedures available upon request. We are also able to customize procedures, i.e., rewrite them according to the specific instructions of the study concerned. When a customized procedure is requrested, an in-service is then held to convey study requirements and answer staff questions.

Instrumentation available in the Hemostasis Research Core Laboratory includes the following:

1. A recirculating laminar flow system based on the flow system of J. Sixma with a Masterflex pump. This system is capable of producing laminar flow from 40sec-1 to 1000sec-1. It has been used for study of platelet interaction with biomaterials and for study of the hemostatic function of endothelial cells on biomaterial substrates. Two rotating disc systems (Pine Instrument Corporation) for the study of coagulation and platelet interaction with biomaterial substrates or the in vitro study of anticoagulants and anti-platelet drugs.
2. A PAP-4 Platelet Aggregometer (BioData Corporation) for the study of platelet aggregation and platelet functional inhibition by anti-platlelet drugs.
3. Simplate (Organon Technica) bleeding time devices for the performance of bleeding times
4. A System 7000 (Dynatech). This is an automated instrument for the performance of enzyme linked immunosorbent assays (ELISA)
5. A FACScan three channel flow cytometer (Becton Dickinson). This instrument is useful for the study of platelet surface proteins and activation markers. It can also be used to study platelet aggregation, dense granule release and leukocyte/platelet interactions.
6. A complete cell culture facility with liquid nitrogen storage, 6ft and 4ft biological hoods, CO2 incubators, centrifuges, inverted phase microscopes, sterilization, and photomicroscopy.
7. Computer facilities include
   1. an IBM 486DX computer with 80MD hard drive and color ink jet printer
   2. a Macintosh Quadra 900 AV with 16 MB RAM and 2 GM hard drive, 230 MB optical drive, 100 MB Zip Drive, color flat bed scanner and color ink jet printers.
   3. Standard software: Microsoft Word 6.0, Microsoft Excel, FoxBase Data Base, Mac Draw, Netscape is available.

1. Coagulation Activation:

Platelet Activation: w PT, APTT e Platelet Factor 4 w Fibrinogen e b Thromboglobulin w Fibrin Monomer e Thromboxane B2 w Fibrinopeptide A e Flow Cytometry w Prothrombin F1.2 CD41a (GPIIb) w Thrombin/Antithrombin Complex (TAT) CD62 (P-selectin) w Factor Assays CD36 (TS Receptor) (II, V, VII, VIII, IX, X, XI, XII, XIIIa) LIBS-1 CD63 (gp53) Dense Granule Release (Mepacrine) Endothelial Cell Coagulant Function Platelet Aggregation w in vitro under laminar shear Platelet Adhesion w tPA/PAI-1 release e in vitro under laminar shear w platelet adhesion e epifluorescent video microscopy w thrombomodulin, tissue factor e atomic force microscopy Fibrinolytic Activation: Others: w Euglobulin Lysis Time e Heparin Activity (Plasma and w D dimer Surface) w Fibrin(ogen) Degradation Products e von Willebrand's Antigen w tPA e Ristocetin Cofactor w PAI e Antithrombin III w tPA/PAI-1 complexes e Heparin Cofactor II e von Willebrand Multimers e Protein C, Protein S e Activated Protein C Resistance

Other auxilliary services that we provide include:

1. Phlebotomy
2. Specimen collection tubes prepared with the anticoagulant of choice
3. Specimen processing and storage (-70oC)
4. Test Report Form Preparation (Excel)
5. Database Capabilities (Excel and FoxBase)
6. Statistical Analysis of Results
7. Graphical Data Representation (Kaleidagraph, Sigma Plot)

Experience of the Hemostasis Research Core Laboratory

We have recently taken on the role of Core Coordinating Laboratory for several Cardiology studies that involve the collection of samples throughout the United States and Canada. Working in conjunction with coordinators at various sites located throughout North America, eligible patients are identified. Then a customized study kit is prepared that may include a letter of introduction to the patient explaining the purpose of the study, an instruction letter to the patient detailing where he needs to go and what he needs to do when having his blood drawn, testing laboratory site instructions for sample collection and primary processing, as well as specimen collection tubes, lables, tourniquets, needles, centrifuge/freezing tubes, mailing labels, and mailing supplies. Examples of kits and instruction sheets are available upon requrest. Close telephone contact is maintained between the Core Laboratory and the testing sites to answer questions and facilitate specimen collection. For some studies (i.e., GUSTO IIb) a slide presentation detailing all aspects of specimen collection, processing and shipment was prepared and distributed to all sites.

Some of our recent clinical trial experience is listed below:

GUSTO IIb
Desirudin vs. heparin in patients with acute coronary syndromes
Role: We were the Noth American Core Laboratory for the Hemostasis substudy.
230 patients

IMPACT-II
Integrelin in coronary intervention
Role: platelet aggregation studies
Harrington RA, Kleiman NS, Kottke-Marchant K, Lincoff AM, Tcheng JE, Sigmon KN, Joseph D, Rios G, Trainor K, Rose D, Greenberg CS, Kitt MM, Topol EJ, Califf RM: Immediate and reversible platelet inhibition after intravenous administration of a peptide glycoprotein IIb/IIIa inhibitor during percutaneous coronary intervention.Amer J Cardiol 76:1222-1227 (1995). ORBIT
Study of oral glycoprotein IIb/IIIa receptor antagonist in patients undergoing percutaneous coronary revascularization.
Role: platelet aggregation studies and bleeding times.

ARG 911
An Open-Label Study of NOVASTAN in patients with heparin-induced thrombocytopenia
Role: Heparin-induced platelet aggregation tests

SC-54684A
Safety study of oral glycoprotein IIb/IIa receptor antagonist
Role: platelet aggregation studies and bleeding times.

PARADIGM
Dose finding study of lamifiban in acute MI
Role: platelet aggregation studies and bleeding times

GENEQUEST
Study of genetic markers in patients with atherosclerosis and their sibilings
2200 patients
Role: Core laboratory for collection, processing, assay and storage of specimens for lipid profiels, glucose, hemoglobin A1C, homocysteine, lipoprotein (a), factor VII, fibrinogen

PURSUIT
Study of Integrelin and deaminated Integrelin in patients with acute coronary syndromes
Role: platelet aggregation

Organization

The organization of the Hemostasis Research Core Laboratory

Kandice Kottke-Marchant, MD, PhD
Medical Director
Dept. of Clinical Pathology

Linda Brooks, MT (ASCP)
Technical Director
of Hemostasis Research Core Laboratory

Carlumandarlo Zaramo, BS
Medical Technologist

Laura Pergi, BS
Medical Technologist

The Medical Director provides direction to the Technical Director in development of methodologies, selection of assays appropriate for particular clinical studies and directs data analysis and preparation of protocols. The Medical Director also meets with current and potential project sponsors, presents Core Laboratory data at national and international meetings and prepares manuscripts from the study data. Dr. Kottke-Marchant has a PhD in Pathology and Macromolecular Science from Case Western Reserve University, an MD from Case Western Reserve University and has AP/CP pathology boards with subspecialization in Hematopathology, Cardiovascular Pathology and Coagulation.

The Technical Director is responsible for the day-to-day operation of the laboratory. She establishes and reviews all technical and quality control procedures on a yearly basis. She is responsible for customizing procedures to suit a partcular clinical study. She is responsible for interacting with sites for the collection and processing of specimens. She is also responsible for the design of specimen collection kits and instruction sheets. Additionally, the Technical Director is responsible for helping with the analysis of the various assays offered. Linda Brooks, MT (ASCP) is a medical technologist with subspecialty training in Laboratory Hematology, Chemistry, and Hemostasis.

The Medical Technologist is responsible for the performance of the hemostasis assays, processing and archiving of specimens, data base entry, and results reporting. The Medical Technologist is also available for phlebotomy and performance of bedside bleeding times. Carlumandralo Zaramo has a BS in Biology and has 5years of research laboratory experience in hemostasis and gastroenterology. Laura Pergi is a recent addition to the lab and is Coordinator of the Gusto III project.

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