Platelet Aggregation Profiler Model PAP-8E...

Platelet Aggregation Profiler

Model PAP-8E

OPERATION REV: L

Operation Platelet Aggregation Profiler, PAP-8E

Bio/Data Corporation 2006, 2007, 2008, 2010, 2011 P/N 106221 Rev: L 2

TABLE OF CONTENTS Authorized Representative ..................................................................................................4 Declaration of Conformity ...................................................................................................4 Copyright Notice ..................................................................................................................4 Trademarks ...........................................................................................................................4 Safety Information ................................................................................................................5 Decontamination Procedure ...........................................................................................5 Symbols ............................................................................................................................6 Components and Accessories ............................................................................................8 Introduction ..........................................................................................................................8 Intended Use .....................................................................................................................9 Qualified Users .................................................................................................................9 Principle of Operation ..........................................................................................................9 Support Equipment ........................................................................................................ 10 Power Requirements ...................................................................................................... 10 Unpacking Components ................................................................................................ 10 Installation Instructions ..................................................................................................... 11 Software Installation ...................................................................................................... 13 System Initialization ....................................................................................................... 14 Language Support .......................................................................................................... 14 Software Operation ............................................................................................................ 17 Title Screen ..................................................................................................................... 17 Main Menu Screen .......................................................................................................... 17 Sign-In... .......................................................................................................................... 18 Back Up Data .................................................................................................................. 22 Restore Data ................................................................................................................... 23 Setup Screen .................................................................................................................. 27 Optical Calibration ......................................................................................................... 31 Test Selections ................................................................................................................... 32 Aggregation .................................................................................................................... 32 To Run a Test.................................................................................................................. 33 Test Templates ............................................................................................................... 34 Controls .......................................................................................................................... 35 To Run a Sample ............................................................................................................ 36 Inject/Event Button ......................................................................................................... 38 Filters.. ................................................................................................................................ 39 Create Test Type ................................................................................................................ 40 Result Reporting ................................................................................................................ 43 Clipboard Feature ........................................................................................................... 43 Aggregation Pattern Overlay ......................................................................................... 45 Collection and Preparation of Test Samples ................................................................... 45 The PDQ®, Platelet Function Centrifuge ...................................................................... 45 Platelet Aggregation ...................................................................................................... 48 Calculations ........................................................................................................................ 48 Area Under the Curve Calculation ................................................................................ 48 Quality Control ................................................................................................................... 48 Test Procedures ................................................................................................................. 49



Standard Aggregation Methods ........................................................................................ 49 Materials Required ...................................................................................................... 49

General Test Procedure ..................................................................................................... 49 Direct Mode ......................................................................................................................... 51 Special Aggregation Methods ........................................................................................... 53 Heparin Induced Thrombocytopenia (HIT) Method ..................................................... 53 Sticky Platelet Aggregation ........................................................................................... 55 Dose Response Testing ................................................................................................. 56 Leukocyte Aggregation ................................................................................................. 57 Ristocetin Cofactor Assay (RiCoF) Reagent Entry Screen ............................................. 59 Ristocetin Cofactor Activity Assay (RiCo) ....................................................................... 59 3 Point Standard Reference Curve Assay .................................................................... 60 4 Point Standard Reference Curve Assays .................................................................. 62 Standard Reference Curve Quality Control ................................................................. 63 Point Suppression .......................................................................................................... 63 Performing the Assay .................................................................................................... 65 Data Analysis ...................................................................................................................... 66 Command Functions ...................................................................................................... 66 Measurements ................................................................................................................ 66 Data Export ......................................................................................................................... 67 Prism Software ................................................................................................................... 68 Prism Software Installation Instructions ...................................................................... 68 Prism Technical Support ............................................................................................... 69 User Maintenance............................................................................................................... 70 Cleaning .......................................................................................................................... 70 Temperature Calibration ................................................................................................ 70 Fuse Replacement .......................................................................................................... 70 Cleaning the Test Wells ................................................................................................. 71 Certification ........................................................................................................................ 71 Trouble Shooting Guide .................................................................................................... 73 Ordering Information ......................................................................................................... 76 Disposables, Accessories and Reagents ..................................................................... 76 Warranties ........................................................................................................................... 77 Glossary .............................................................................................................................. 78 References .......................................................................................................................... 79 Platelet Aggregation References .................................................................................. 79 Platelet Aggregation Profiler and GraphPad Prism® .................................................. 80 Leukocyte Aggregation References ............................................................................. 80 Additional Information ....................................................................................................... 80 Standards ............................................................................................................................ 81 National Standards, USA ............................................................................................... 81 International Standards ................................................................................................. 81

155 Gibraltar Road Horsham, PA 19044 USA 215-441-4000 World Wide 800-257-3282 USA

FAX 215-443-8820 Email: [email protected] Internet: www.biodatacorp.com

mailto:[email protected]

http://www.biodatacorp.com/



Authorized Representative

EMERGO EUROPE, Molenstraat 15, 2513 BH The Hague, The Netherlands Declaration of Conformity

This Platelet Aggregation Profiler Model PAP-8E is designed to conform to the following standards: 1. European Union Directive 98/79/EC In Vitro Diagnostic Medical Devices 2. European Union Electromagnetic Compatibility Directive 89/336/EEC 3. European Union Low Voltage Directive 73/23/EEC. 4. European Standards: EN 61010-1:2001, Safety requirements for electrical equipment for

measurement, control, and laboratory use, EN 61010-2-101:2002 Particular requirements for in vitro diagnostic (IVD) medical equipment.

5. EMC standards - EN 61326, EN 61000-3-2, EN 61000-3-3 This analyzer is designed to be in compliance with the following standards: 1. UL 61010-1- Safety requirements for electrical equipment for measurement, control, and

laboratory use,- Part 1: General requirements 2. CAN/CSA C22.2 No. 61010-1- Safety requirements for electrical equipment for measurement,

control, and laboratory use,- Part 1: General requirements Copyright Notice This manual and associated software are copyrighted and protected by United States copyright laws and international treaties. The distribution and sales of this material are for the sole use of the original purchaser only. Disclosure, reproduction, copying, decompiling, disassembly or reverse engineering of this software is prohibited. This software and associated manuals are the property of Bio/Data Corporation and may be used only by licensed users. All copies of the software and associated manuals are to be returned to Bio/Data Corporation upon expiration of the license. ALL INFORMATION AND DATA CONTAINED IN THIS MANUAL IS CONSIDERED TO BE PROPRIETARY TO BIO/DATA CORPORATION. THEREFORE, NO PART OF THIS MANUAL MAY BE COPIED OR REPRODUCED WITHOUT THE EXPRESS AND ADVANCED WRITTEN AUTHORIZATION OF BIO/DATA CORPORATION. Trademarks Platelet Aggregation Profiler™ is a trademark of Bio/Data Corporation

vW Factor Assay is a registered trademark of Bio/Data Corporation

AggRecetin is a registered trademark of Bio/Data Corporation

BETA/Pak is a registered trademark of Bio/Data Corporation

PAR/Pak II is a registered trademark of Bio/Data Corporation

Windows is a registered trademark of Microsoft Corporation

Prism is a registered trademark of GraphPad Corporation

Biohit is a registered trademark of BIOHIT PLC.

PDQ is a registered trademark of Bio/Data Corporation

vW Select is a registered trademark of Bio/Data Corporation LTA Check™ is a trademark of Bio/Data Corporation Patents USA Patent# 7,453,555 – Aggregometer with near ultraviolet light source WW Patent Pending



Safety Information

Biohazardous Materials ALL PLASMAS AND BIOLOGICAL REAGENTS ARE POTENTIALLY BIOHAZARDOUS and should be handled with precaution. Follow the procedure for your laboratory or the Occupational Safety and Health Administration (OSHA, USA) Bloodborne Pathogen guidelines for handling of biohazardous materials and waste. Standard precautions must be observed when operating or servicing any laboratory instrument. Appropriate personal protective equipment shall be worn. Instruments shall be decontaminated in accordance with manufacturers and/or instrumentation protocol prior to service. Specimens from any patient could be infected with Human Immunodeficiency Virus (HIV) or Hepatitis B Virus (HBV) or other blood borne pathogens. Proper blood collection, transport and handling techniques must be followed to minimize risk to the laboratory staff and personal protective equipment must be worn according to your laboratory‟s procedures. See Clinical Laboratory Standards Institute formerly known as, “National Committee of Clinical Laboratory Standards (NCCLS)” (an organization based in the USA) document M29-T2 Protection of Laboratory Workers from Infectious Diseases Transmitted by Blood, Body Fluids and Tissue, Second Edition, Tentative Guidelines, Occupational Safety and Health Administration‟s (OSHA) (a government organization based in the USA) Bloodborne Pathogen Standards or the procedure established for your institution. Decontamination Procedure Test and Incubation Blocks, (interior and exterior)

Clean with a mild detergent solution (anti-microbial) and warm water. Use a cotton swab wetted with detergent. Do not use excessive amounts of liquid. Dry immediately.

Do not use solvents, sodium hypochlorite (bleach) solutions.

Decontaminate in accordance with the laboratory‟s standard practices on a routine basis.

Decontaminate prior to returning to Bio/Data Corporation or a distributor for service. A document stating that the decontamination was performed must be included with any returned PAP-8E.

The Biohit eLine pipettor shall be decontaminated as directed in the Biohit instruction manual.



Symbols

Caution Indicates either potential procedural problems, damage to hardware or loss of data

Electrical Hazard Warning - to reduce the risk of electrical shock, do not remove the cover. There are no user serviceable components.

Service should be performed by qualified personnel only. For continued protection against fire hazard, replace fuse only with the same type and size of fuse.

Do Not Reuse International symbol for Do Not Reuse, Single Use or Use Only Once

RS 232C Port IOIOI Connection port of PAP-8E data cable to the Personal Computer.

Fuse Symbol International symbol identifying the type of fuse required Replace only with the same size and type of fuse. Power Switch I/O International Symbol for On (I) and Off (O) positions of the power

switch.

No User International symbol indicating no user serviceable parts inside Serviceable Parts

WEEE Symbol Separate collection for electronic waste



Cautions

Should a liquid spill occur on the analyzer, immediately disconnect the main (power) cord. The affect of the spill must be determined by qualified service personnel prior to resuming operation of the analyzer. Any liquids that enter the analyzer must be completely removed before resuming operation of the analyzer.

Do not extend analyzer past the edge of the work bench as it may cause the analyzer to tip off the bench.

Do not use flammable liquids on or near the PAP-8E.

The use of test tubes and or stir bars other than those manufactured by Bio/Data Corporation for use with the PAP-8E will void all instrument warranties.

Research Use Only: Methods Identified as Research Use Only (RUO), are developing technologies or variations of current technologies that have not been reviewed by the US Food and Drug Administration. The user of RUO methods must ascertain by their own means that the RUO method is suitable to their intended purpose and validate the method for that intended use.

Test Tubes are to be used only once. Cross contamination will occur. Cleaning of test tubes removes the siliconized coating and thus exposes the sample to the glass surface, causing activation to occur.

The use of non-siliconized tubes will cause activation of the sample. Their use is not recommended.

Stir bars are to be used only once contamination will occur.

Stir bar coating will wear after use thereby exposing materials which will contaminate the test materials.

Reagent wells are for the insertion of reagent and sample vials for stirring and warming if required. Do not place reagents or samples directly into the wells.

All PAP-8E components and accessories are provided with operations and use documentation.

These documents must be read, understood and complied with for safe and proper operation of the PAP-8E.

Class 8 electronic waste, medical devices, recycle or disposal in accordance with Directive 2002/96/EC



Components and Accessories 1 Platelet Aggregation Profiler, Model PAP-8E Analyzer 2 Dell PC (Personal Computer) 3 PDQ®, Platelet Function Centrifuge (optional item) 4 Color Inkjet Printer (USA customers only) 5 Accessory kit, including:

a Serial Cable, DB9, Male/Female, 6 feet long b Software, PAP-8E c Stir Bars, Micro d Test Tubes, Micro, Siliconized e Inserts, Reagent Well, PAP-8E - 15mm, 17mm, 22mm, 24mm, and 29mm ID f Operations, Manual PAP-8E g Power Cords

6 Pipettor, Biohit®, eLine 10 - 300L a Stand and Charger, Pipettor, Biohit, eLine

b Tray, Pipettor Tips, Biohit, eLine 10 - 300L

7 Prism Software Package, GraphPad Inc 8 Barcode Scanner, Symbol Technologies (optional item) Introduction The Platelet Aggregation Profiler, Model PAP-8E brings platelet aggregation into the contemporary laboratory. The touch screen operation, onscreen procedure templates, and test menu make the PAP-8E easy to use. Eight test channels accommodate multipoint standard curves and patients in a single run, multiple patients with a single agonist, or a single patient with multiple agonists or dilutions. The time required to perform aggregation studies is shortened. Onscreen timers and audible alarms free the operator to perform other tasks during incubation and test sequences. Agonist induced platelet aggregation procedure templates are preprogrammed for ADP, Arachidonic Acid, Collagen, Epinephrine, Heparin (HIPA), Thrombin Induced Platelet Aggregation (TIPA_PRP) and (TIPA_WASHED), Ristocetin (RIPA) and LTA Check™, Performance Monitoring Check. Additional templates for the study of activated platelets, sticky platelets, Heparin Induced Thrombocytopenia (HIT) and dose response calculations are provided. Templates assure that tests are performed the same way, time after time.

User defined programmable templates, with separate “help” files, are also included. Each template may be developed, stored, and recalled as desired by the individual laboratory. The Direct Mode feature allows the user rapid access to the analyzer functions by passing the data entry screens and going directly to the Status Screen. When at the Status Screen, assays may be completed by simply setting up the (test/reagent) system, incubating the samples and running the assay.

The PAP-8E offers an enhanced Ristocetin Co-Factor Assay. Along with providing templates for the standard 3-point (100%, 50%, 25%) reference curve, the PAP-8E offers two user selectable 4-point reference curve templates. One 4-point (100%, 50%, 25%,12.5%) template may be used to extend the range of the assay below 25% activity. The other 4-point (100%, 80%, 40%, 20%) template provides optional methodology for research use only. All test related data, results and standard curves are permanently stored in a database which can be recalled, sorted or archived.



The PAP-8E accessories reduce many of the tedious tasks and eliminate much of the variability usually associated with platelet aggregometry.

GraphPad Prism for Windows is a powerful combination of basic laboratory and biostatistics, curve fitting (nonlinear regression) and scientific graphing in one comprehensive program. Prism is ideally suited to data plotting and analysis applications in the life sciences. The enhanced program gives you the ability to organize, analyze and graph repeated experiments, pick appropriate

statistical tests and interpret the results. It is compatible with Microsoft programs such as Word, Excel and PowerPoint. Additionally, Prism includes special features for different laboratory environments such as laboratory statistics and QC for the clinical laboratory, and dose response calculations and curves for research laboratories. Intended Use Platelet aggregation is a series of laboratory tests used to assess platelet function. Aggregation is a useful tool for laboratory evaluation of inherited or acquired abnormalities, clinical investigation of bleeding or thrombotic states, pharmacological study and research protocols. The Platelet Aggregation Profiler, Model PAP-8E measures platelet aggregation in platelet rich plasma (PRP). Platelet responses to a series of common agonists cause changes in optical density that are measured according to the method described by GVR Born.1 Results are compared to those from a known donor or established reference ranges. Responses to a series of common agonists are used to differentiate functional status and abnormalities. Ristocetin Cofactor Activity can be measured. Results are interpolated from a standard curve. The PAP-8E is suitable for use with human, animal or other blood specimens or samples used in clinical, pharmaceutical, CRO, biotechnology or other laboratories. Qualified Users All models of Platelet Aggregation Profilers are intended only for professional use by qualified laboratory personnel.

To obtain the maximum benefits from your PAP-8E, we strongly recommend that you read the manual completely and thoroughly familiarize yourself with the instrument's operation. Should questions remain after reading this manual, contact your local Bio/Data Corporation distributor or Bio/Data Corporation Customer Service Department at 155 Gibraltar Road, P.O. Box 347, Horsham, PA 19044 U. S. A., (215) 441-4000 (World-Wide) (800) 257-3282 (U.S.A.) Fax us at (215) 443-8820. Email: [email protected]

Principle of Operation The Platelet Aggregation Profiler, Model PAP-8E is designed to perform accurate and rapid platelet aggregation studies on platelet rich plasma utilizing eight independently operated channels. A platelet poor plasma specimen (PPP) is inserted into the selected channels; activation of the corresponding Blank button on the touch display sets the 100% baseline (100% optical transmission). The platelet rich plasma specimen (PRP) is then inserted into the same test well and the Test Start button is selected. The difference in optical density between the PPP and PRP specimens, expanded to a 100% scale with the PRP being 0% baseline (0% optical transmission), when the agonist reagent is added to the PRP and the change in optical transmission is recorded for several minutes.



As aggregation patterns are being generated, they are automatically stored in memory. After completion of the tests, results are automatically calculated to determine the appropriate data parameters. This information can be recalled either singularly or in any combination as often as required. This allows patterns to be separated or combined as required for reporting purposes. The aggregation patterns are compared to normal aggregation patterns to identify differences between the patterns caused by physiological state or condition of the sample.

The final test volumes are 250L as compared to the traditional standard volumes of 500L. Support Equipment The PAP-8E is designed to operate with a windows based personal computer (PC). Currently, the

Dell Optiplex PC with the ELO Touch Screen monitor has been validated for use with the PAP-8E. It was chosen for ease of use and its ability to maximize the function of the PAP-8E and software. It is recommended that this system not be altered. The minimum specifications for this PC are:

Processor: Pentium® 4; 3.2 GHz, Windows 7 Ultimate Operating System RAM: 512 MB, Hard Drive: 40 GB, DVD/CD Read Write 24X, Ports: (1) RS 232, (4) USB 2.0, Network: Ethernet Monitor: Touch Screen, 15 inch flat panel LCD Input Devices: Touch Screen, Keyboard, Mouse Output device – Printer, color Inkjet Sample Preparation – Platelet Function Centrifuge, Model PDQ™ Power Requirements The specific power requirement for your PAP-8E is selected with a switch located on the rear panel of the instrument. In all cases, the PAP-8E requires a grounded electrical receptacle. The use of "adapters" is not recommended. Should an adapter be used, it is essential that the adapter be securely grounded to the receptacle cover plate. It is important that the electrical line (receptacle) designated for your PAP-8E be substantially free of transient voltage spikes as may be caused by other types of equipment using the same circuit, such as centrifuges, refrigerators, freezers, water baths, compressors and x-ray equipment.

1. Equipment for 100-125 Volts Single Phase 3A F, 50/60 Hertz installations, verify that the voltage selector switch is set to the 115VAC position.

a. The voltage selection switch is located on the rear panel of the PAP-8E. 2. Equipment for 200-240 Volts Single Phase 3A F, 50/60 Hertz installations, verify that the

voltage selector switch is set to the 230VAC position a. The voltage selection switch is located on the rear panel of the PAP-8E.

Unpacking Components When the PAP-8E arrives, inspect the outer carton for any shipping damage. If damage is evident, inspect the contents for damage. Any damage of the contents should be reported to the carrier immediately.

During installation and routine operations of the PAP-8E: 1. Use only grounded electrical receptacles free of transient voltage spikes. 2. The use of a surge protector and an isolated grounded line are strongly recommended. 3. Do not remove any of the instrument's covers. 4. Do not use solvents of any nature on the PAP-8E. 5. When replacing a fuse, replace only with the same type and rating of the fuse to avoid fire

hazard. 6. The PAP-8E is intended for Installation Category II. 7. The enclosure rating code per EN 60529 is IP20



8. The PAP-8E is intended for indoor use at an altitude up to 6562 feet (2000m) within a

temperature range of 65F (18C) to 85F (29.5C) and a maximum relative humidity of 85%. Installation Instructions The PAP-8E should be installed in a controlled laboratory environment, which allows a total work space of 21 inches, (533mm) wide by 24 inches, (610mm) deep. The surface should be level and capable of supporting 75 pounds, (35kg); additional area is required for the samples, barcode scanner and pipettor. Proper configuration of the instrument places the monitor atop the PAP-8E and the PC beside them; all components may be stacked to conserve space if required.

The maximum load placed on top of the PAP-8E housing is limited to 50 pounds (23 kilograms); 4.76 pounds per square inch, (2.12 kilograms per square centimeter).

Do not extend PAP-8E past the edge of the work bench as it may cause the analyzer to tip off the bench.

Networked Computers Although not recommended, the PAP-8E software will operate on a PAP-8E PC that is connected to a network, however the software provides no specific network-related functions. The PAP-8E software must be installed on a Windows user account with administrative privileges. The PAP-8E software must be operated ONLY by way of this user account. Other user accounts on the PC will not be able to properly use the PAP-8E software. The PAP-8E program folder cannot be moved or altered in any way.

PAP-8E Integrated PC and Software: Acceptable Use and Software Restriction Notice

The factory installed software loaded on the integrated PC Includes: Windows® Operating System,

PAP-8E System Software© and Graphpad Prism® (“software”). Compliance with all proprietary and

restrictive notices that appear with the software or in its documentation is required by the software

licenses. You may not modify, adapt, copy, publish, or make derivative works of the software.

PAP-8E system users are not permitted to install additional off the shelf or user developed software

on the integrated PC except those approved by Bio/Data Corporation, or as listed below.

Approved Software:

Microsoft Office and its individual applications

IBM® Lotus® 123 or Lotus Smart Suite® and their individual applications

Google® docs and its individual applications

Corel® Home Office and its individual applications

Open Office and its individual applications

Intranet Interfaces

Unpacking and instrument assembly and wiring instructions

1. Remove the box labeled „Accessory Kit‟ box.

a. Open the kit and remove the following:

PAP-8E power cord

PAP-8E cable harness

Power strip (USA customers only)



2. Plug the power strip into the wall electrical outlet. Insure that the power switch is in the [OFF] position

Inspect exterior of shipping boxes for damage. If damage is noticed, make note of damage, photograph damage (if possible) notify shipping company and Bio/Data Corporation about the possibility of damage to instrument or accessories. 3. Remove the PAP-8E from Box

a. Remove the PAP-8E from the inner box b. Place onto the work surface with the rear of the instrument placed in a position that

allows access to rear panel. Check the voltage setting on the PAP-8E power module c. to ensure that it is in the correct position for use in the country it will be used in, this

will be either 110 or 220 Volts d. Plug the main power cable into the power inlet module; this is located next to the I/O

(on/off) switch. e. Plug the cable harness, (this cable has a DB-9 connector at both ends) into the

connector marked [IOIO] and only finger-tighten the screws. f. Plug the power cable into a slave outlet on the power strip g. Turn the PAP-8E so that it will be in the position it will be in the operational. h. Power on PAP-8E instrument.

4. Remove the monitor from the large box.

a. Remove the monitor from inner box b. Carefully place the monitor face down on the work surface with the base towards you. c. Remove the monitor connector cover, while pressing down on semi-circular recesses

at corners of monitor connector cover. Pull cover towards you to release it and place in safe location. This will be needed at a later time in the installation.

d. Position the monitor base in a vertical position (90° to the monitor). e. There are three cables from the cable harness and the power cable to connect. The

three cables that need to be connected are: USB (This is a black colored standard USB connector plug), monitor power (This is a black colored female plug) and graphic interface cable.

f. Each of the three cables must be inserted through the monitor base before connecting the cables to the monitor.

g. Replace monitor connector cover, make sure the 3 monitor cables are centered and grouped together. While holding the cables together, reinstall the monitor connector cover by placing it in towards the lower edge of the opening and sliding it away from you until it „clicks‟ into place. Rotate the monitor base approximately 90° so that it is almost parallel to the monitor.

h. Set monitor upright on the top of the PAP-8E i. Plug the monitor into a slave outlet on the power strip

5. Remove the PC (computer) from the outer box. a. Remove the PC, USB keyboard and USB mouse from the inner box. b. Place the PC on the work surface near the PAP-8E so that the cables from the PC

will be able to reach the PAP-8E. c. Plug the USB keyboard and USB mouse into the rear of the PC, these items are USB

devices and may be plugged into any available USB port. d. Plug the power cord into the power strip at the Master position. e. Cable Harness: Connect the monitor cable into the PC; these cords are color coded

as are the connections on the PC. f. Connect the USB cable from the monitor into any available USB port on the PC.



6. Remove the Printer from box. a. Remove the printer and cable from the inner box. b. Plug the printer cable flat USB connector into the CPU c. Place the printer into position. d. Plug the power cord into the printer and the plug into the slave outlet on the power

strip. e. Plug the USB cable into the printer.

7. Optional Item, remove the PDQ from box. a. Remove the PDQ™, power adapter and power cord from the box b. Remove shipping foam from inside the PDQ™ rotor and cover. c. Connect power cord from the power adapter to the PDQ™ d. Place PDQ™ in the position it will be used in on the work surface e. Connect power cord from the PDQ‟s power adapter to one of the slaved outlets on

the power strip 8. Remove the Pipettor box from box.

a. Remove the charging stand and power adapter from the Pipettor box b. Connect power adapter to the Pipettor charging stand

9. Remove Pipettor from its box and place onto the charging stand. The first time the Pipettor is charged, it should be left in the charging stand for 12 hours if possible. This will ensure maximum battery life of the Pipettor.

10. Turn the PC and the monitor on and switch the PAP-8E to the on position. The Pipettor charging stand will be powered as long as the charging stand is plugged into a non-switched electrical outlet.

11. For normal operations, the PAP-8E and related equipment may be left on.

Software Installation The PAP-8E software CD is included in the accessory kit. This CD should be retained and stored in accordance with laboratory policy.

The PAP-8E software is installed by Bio/Data Corporation prior to delivery. If required, the software installs automatically once the CD is loaded into the CD drive. The PAP-8E software is a standard Windows format auto-install. After the CD is placed in the loading tray and the tray is closed, the installation will begin.

To install the new PAP-8E Software Version:

1. Insert the CD into the loading tray. 2. The installation should start automatically. 3. If the setup program does not start then select Start from the taskbar and click Run. 4. Enter “d:\setup.exe" on the Open line. Click OK

An icon "Start PAP-8E" will be automatically placed on the desktop. When you run the PAP-8E Program your old data will be available as an archive database. The PAP-8E software may be installed on other PC‟s for review or analysis of data files. To install the Prism and barcode scanner programs, refer to the manufacturer‟s installation instructions. The documentation and CD are provided with each item. These also automatically initiate installation. If the installation program fails to initiate automatically, select the Start button located at the lower left of the screen. From the menu select Run, Browse, the CD drive letter and Install. This will initiate the automatic installation program.



The Prism software is a single user license. Do not install on more than one computer. Contact Bio/Data Corporation Technical Service or your local distributor if there are any problems associated with the installation of the software. System Initialization After the PAP-8E and the support equipment have been installed:

1. Turn on the PC. 2. Turn on the PAP-8E. The power switch is located on the left rear of the unit next to the power

cord. a. The indicator light located on the lower front right of the case will illuminate.

3. Initiate the PAP-8E program by double clicking the icon on the screen. a. The system status located at the lower left of the screen will indicate Warming. b. The warm up period will take approximately 20-30 minutes.

4. The printer will turn on automatically if plugged into the slave outlet of the power strip. 5. The Pipettor must be stored on the charging stand to be ready for use.

The time, date and language format in the PAP-8E software is controlled by the Windows 7 Ultimate software operating system. Language Support Set up the PAP-8E for non-English use. Windows 7 language packs are available for download and can be installed only from the Optional Updates section in Windows Update. However, these language packs are not available on the Microsoft Windows Server Update Services (WSUS) server or through the Microsoft Download Center. To download and install a Windows 7 language pack, follow these steps:

1. Start Microsoft Update. To do this, click Start, type Microsoft Update in the Start Search box, and then click Microsoft Update in the Programs list.

2. Click the optional update links for the language packs. 3. Under the Windows 7 Language Packs category, select the desired language pack.

Note: If you install many additional language packs, the disk space and system performance are affected. In particular, disk space and system performance are affected during servicing operations, such as Service Pack installations. Therefore, we recommend that you only add a language pack to your computer if you will use the language pack.

4. Click OK, and then click Install updates to start the download and installation process. To change the display language, follow these steps:

1. Click Start, and then type Change display language in the Start Search box. 2. Click Change display language. 3. In the drop-down list that appears, select the language that you want, and then click OK.

To uninstall a language pack, follow these steps:

1. Click Start, and then type Uninstall display language in the Start Search box. 2. Click Install or uninstall display languages. 3. Click Uninstall display languages.



4. Select the language or the languages that you want to uninstall, and then click Next. 5. Follow the rest of the steps to uninstall this language or languages.

After you install the desired language packs, you can hide unwanted language packs in Windows Update. To hide unwanted language packs in Windows Update, follow these steps:

1. Click the Windows 7 Language Packs category in the Select the updates you want to install page to highlight all the language packs.

2. Right-click the highlighted updates, and then select Hide update. In the Clock, Language, Region Options the data format must be changed from the standard European “decimal” format (3.12.07) to the front slash format (3/12/07). The database requires the front slash to define the number as a date.

1. Click Start 2. Click Control Panel 3. Click Clock, Language, Region 4. Select change the date, time, or number format 5. Under Format, Select Country / Language 6. Check short date, there should be a slash (/) between the m/d/yyyy. 7. If no slash, click on short date dropdown and select dash format 8. Click Apply, the screen will return to the previous screen, click Apply again and then OK

Operation This manual section is a description of the software operation. The Methods Section provides detailed information about each test type. The methods described in that section are the preferred test methods for use with the PAP-8E. Daily Procedure Visually inspect the outer surfaces of the analyzer as well as the incubation and test wells for spills or foreign materials. Instructions to remove liquid or debris from the test wells are detailed in the User Maintenance section of this manual. Clean with a mild detergent solution (anti-microbial) and warm water. Do not use excessive amounts of liquid. Dry immediately. Do not use solvents, hypochlorite (bleach) solutions or disinfectants.

The PC and PAP-8E program must be operating for the analyzer to function.

It is strongly recommended that no other software be running while the PAP-8E software is performing tests or other functions. Bio/Data Corporation cannot be responsible for any interactions of other software.

Check for software upgrades at the GraphPad Prism and optional Symbol barcode scanner manufacturer‟s web sites. The PC can be configured to automatically search for upgrades if connected to the internet.



Calibration and Certification With V2.0.0 PAP-8E analyzers, operator calibration may be performed with the Optics Calibration screen. The calibration is performed with distilled or reagent grade water. This calibration may be scheduled daily, weekly or as required by the laboratory standards. Calibration may be performed by all operator levels. See Main Menu Screen, Optical Calibration for detailed instructions. When updating PAP-8E‟s to V2.1.0, calibration is required after installation of software updates before any testing can be performed.

Calibration and Certification are required by the Clinical Laboratory Improvement Act (CLIA) and other international standards agencies. Refer to your laboratory‟s standard operating procedures for pertinent requirements and record keeping.

Annual calibration and certification as well as semi-annual calibration verification are recommended by Bio/Data Corporation and required by various regulatory agencies. The analyzer is provided with a Certificate of Analysis at time of purchase. Platelet Aggregation Profiler, Model PAP-8E: Contact Bio/Data Corporation Customer Service for Calibration and Certification services. All PAP-8E Calibration and Certification is performed in accordance with national and ISO 9001:2000 and 13485:2003 standards. BIOHIT eLine Electronic Pipettor: Contact your local Biohit distributor for Calibration and Certification services. Refer to the Biohit web site for additional information. Backing up Data To backup your data the PC has a CD read/write CD Drive. Backing up data is critical in all operations. The laboratory may have a procedure to specify the back up procedure and archiving. The Database Operation Screen provides an option to Backup Database and Retrieve Database. Follow the on screen directions for backing up and retrieving data.



Software Operation Title Screen

The first screen to appear at start up is the title screen. The Company information and copyright statement are displayed. This screen identifies the software revision level by the software version number. Select the Continue button to enter the program.

Main Menu Screen



This screen is the main menu screen. It is used to navigate to all other screens. The selections available on this screen are: System Status – Indicates when the PAP-8E is ready to operate. When the Incubation and Test Blocks are at temperature the color will change from yellow to green. Certain software functions may be used before the system is ready. However, tests cannot be run until the system is at the ready state and the Incubation and Test Blocks are at temperature. Active Data Base – This identifies which data base is active. The software defaults to the main or current database. Operator – Indicates user that is currently logged onto system. Optical Calibration – Optical calibration now accessible to all operators from the Main Menu Screen. LTA Check – This button will only appear when the LTA Check™ Software program is loaded on the PC. Selecting this button will open the LTA Check Software program. Refer to the LTA Check Software User Manual for detailed information on the use of this software.

Prism - GraphPad Prism is a powerful combination of laboratory and biostatistical, curve fitting (nonlinear regression) and scientific graphing features in one comprehensive program, developed specifically for bio-statistical applications. Selecting this button will open the program. Refer to the Prism User Manuals included in the accessory kit. Sign-In

To operate the PAP-8E, you must sign in. Select Sign-In and the Logon box will appear. Enter your operator identification and password and then select Enter. To add a new user you must have key operator status for your password. The status level of your password may restrict access to Database Operations and Setup/Maintenance functions. Refer to the user management screen description. The system is supplied with an operator ID of “bdc” and a password of “bdc” to allow initial access to all segments of the program. When “bdc” is entered the following security message will appear: “This operator name should be deleted and replaced with a permanent name.” This can be done in the Setup screen. As a security feature, a timeout of the operator logon will occur after 15-240 minutes of inactivity when the Main Menu is displayed. This timeout feature is defined by the supervisor in the setup screen.

http://www.graphpad.com/prism/Prism.htm



Note: After three consecutive unsuccessful attempts are made to logon to an operator‟s account, that account will be locked out until a supervisor logs on and resets the password for that account. The following warning message will be displayed before the third attempt.

The following warning message will be displayed after the third unsuccessful attempt.

If the operator attempts to logon again the following message will appear.

After the time of lockout, a brief message “Security Alert” will appear on the Main Menu Screen. This message will continue to appear until a supervisor logs onto the system and changes the password for the blocked user.



After the blocked password has been changed by a supervisor, the Security Alert message will disappear.

After an operator successfully logs on and chooses to Enable Password Change, the following screen will appear.

Select OK to assign new password or Cancel to exit. Note: Operators should not share passwords.



Database (Database Operation Screen) Selecting this tab allows you to archive data or change the working database to access archived data.

The options are: Select Database – Select a database from the dropdown list. To access archived data – Select the appropriate database, this will make the selected database active. View Memo – This screen allows you to review notes about the archived data. Once entered and saved, notes cannot be deleted. Create Archive – In this screen you can enter a name for the new archive and select the results to be archived.



The available choices are: Sort Data – In this screen you may select data by several parameters. These parameters may be date range, patient name, number and operator. This will prepare the data to transfer to the archive database. A sorting parameter must be selected prior to selecting the Sort option

Move Data to Archive – This will move the sorted or selected data from the current database to the archive database. The current database will be altered. Copy Data – This function will copy data from the current database into the new archive database. The current database will remain unchanged. Create Memo – This is used to add a description or other information to the database. Memos cannot be deleted once saved.

Back Up Data

This button allows the operator to backup database to CD ROM or USB flash drive.

1. Select Back Up Database and the following screen will appear.

2. Select “Yes” to back up data to the CD drive. 3. Select “No” to back up to a USB Flash drive. 4. CD or USB Flash Drive Instructions will appear.

or



5. After completing the CD or USB drive instructions, Select “OK”. 6. The following message will appear if there is a CD Create error.

7. The following message will appear if there is a CD Drive failure. Back Up to USB Flash Drive.

8. The database backup will start and the following message will appear.

9. After the back up is complete, exit then restart the program.

Restore Data

In case of a system failure, this button allows the operator to recover a database from CD ROM or USB flash drive.

1. Select Restore Data and the following screen will appear.



2. Select “Yes” to restore data from CD drive. 3. Select “No” to restore data from USB Flash drive. 4. The following message will appear if no backup data could be found on drive selected.

5. CD or USB Flash Drive Instructions will appear.

or

6. If the following operator error appears. Insert USB drive and try again.



7. After completing the CD or USB drive instructions, Select “OK”.

Select “Yes”.

8. The database restore will start and the following message will appear.



Select “OK”.

9. After the restore is complete, restart program.

Select “OK”.



Setup Screen

Setup - This screen allows users to select options for language, report headers, to enable or disable touch screen keyboard, and to change channel colors. The typical information for report headers to be put into the fields:

Field 1 - Institution‟s name Field 2 - Institution‟s address Field 3 - City, state, zip code or postal code and country Field 4 - Director's name Field 5 - Laboratory identification

Select Language The PAP-8E software provides a language option. This option provides translations of the PAP-8E labels and help files. During the initial setup of the PAP-8E software, a language selection may be made. The language selection is located at the lower left hand side of the screen. Selecting the down arrow of this box will display the available languages. Select the preferred language and follow the on screen instructions to install the language. If a desired language is not available, please contact Bio/Data Customer Service for assistance. Operator Login Timeout – Supervisor may enter 15 to 240 minutes. Operator Password Expiration – Supervisor may enter 1 to 365 days. If a user‟s password expires, the user will be prompted to enter a new password.



Sounds – Select to enable sounds.

Channel Colors – Selecting a channel with a double click opens this screen. Selecting a color from the pallet changes the color for that channel.

Enter the Block ID: This is the Serial Number (S/N) of the Unit. To Save, Select Exit. A message will appear to check the block temperature. Temperature Calibration is detailed in the User Maintenance section. Selecting Enable Keyboard will display the on-screen keyboard. Once enabled it will automatically appear when a data input block is selected.

Channel (trace) Filters – This function sets the standard trace data filters active when selected or deactivated if cleared. The selection affects all channels for all tests. The Test Result screen has a selection to change individual channels to the filtered/unfiltered setting.



Enable Confirmation - A user programmable 'Stop' and 'Abort' confirmation box in Status screen is a selection. If a test is stopped or aborted a confirmation window will appear. These confirmations can be selected to enable a confirmation response from the operator to allow the actions to avoid errors which can result in the loss of data.

Operator Management - This screen is used to assign password status levels and to track operator usage.

Add User: 1. Enter operator name and password in fields provided. 2. Enter operator level 1 or 2, 1= tech, 2=supervisor 3. Entered password is visible only as “*****”. 4. Click „Add Operator‟. 5. If the operator name already exists the following message will appear:

Delete User: 1. Select the operator from the data grid (click on the left edge of any row). 2. Press the Delete key. 3. All users can be deleted. If all operator names are deleted the supervisor may enter a

pass code provided by Bio/Data on the main sign in screen. This will log the operator on as a supervisor. You must then go to the Setup screen and enter a new name and password.



Change Password: Supervisors may delete, reset or lockout by selecting Change Password. 1. Select Change Password and the following screen will appear.

2. Select OK to assign new password or Cancel to exit. Temperature Calibration is detailed in the User Maintenance section. Maximum Test Time and Stir Speeds: This feature will allow the user to change the incubation time, maximum test time and stir speeds for each standard aggregation test type. This allows the modification of the standard test parameters without the operator having to create a new test type. Open the setup screen from the main menu and select Test Setup. Select a test type and enter the desired change in the appropriate box. Then select change and the information will become the parameter for that test type.

You must verify the test function after making changes to the incubation time or stir speed. The original parameters were optimized for Bio/Data‟s reagents and volumes.



The addition of a “Set to Default” button on the Setup Screen will allow the user to set the following defaults for all test types: Incubation Minutes – 2 minutes Maximum Test Minutes – 6 minutes Stir Speed RPM – 1200 RPM

Test Activation When creating tests the test type list can get very large. To keep only the current test in the list, the Selecting a test in the active column and clicking on the right arrow will remove the test from the active to the inactive list. To activate an inactive test select it on the inactive list and select the left arrow and it will be transferred to the active list.

Optical Calibration

With V2.1.0 PAP-8E analyzers, operator calibration may be performed with the Optics Calibration screen. The calibration is performed with distilled or reagent grade water. This calibration may be scheduled daily, weekly or as required by the laboratory standards. Calibration may be performed by all operator levels. When updating PAP-8E‟s to V2.1.0, calibration is required after installation of software before any testing can be performed. Calibration Procedure:

1. From the Main Menu Select Optics Calibration 2. Place 275 µL water into a siliconized test tube 3. Wipe the exterior of the tube with a lint free disposable wipe 4. Handle the tube by the top one inch (25mm) to avoid fingerprints in the optical path 5. Place the tube of water into channel 1 6. Select the Cal. Button 7. After about 5 seconds the Cal. Button will become enabled Note: In case of an instrument problem, calibration can take up to 4½ minutes for each channel. Stop can be clicked to stop the calibration. 8. Repeat steps 5 through 7 for the remaining channels 9. The values should be within a range of 6.2 to 7.8 volts 10. Select Print Certificate for a copy of the Calibration Report for your records



11. If the values are outside of the expected range contact your local representative or Bio/Data Service. In case of an error, a message will appear giving details of the problem.

Calibration and Certification are required by the Clinical Laboratory Improvement Act (CLIA) and other international standards agencies. Refer to your laboratory‟s standard operating procedures.

Annual calibration and certification as well as semi-annual calibration verification are recommended by Bio/Data Corporation and required by various regulatory agencies. The analyzer is provided with a Certificate of Analysis at purchase. Test Selections There are four categories of test selections listed on the Main Menu, these are:

1. Routine Platelet Aggregation – these are the standard platelet aggregation tests: Collagen, ADP,

Arachidonic Acid, Epinephrine, Spontaneous Platelet Aggregation, Heparin Induced Platelet Aggregation (HIPA), Ristocetin Induced Platelet Aggregation (RIPA), Thrombin Induced Platelet Aggregation (TIPA_PRP) and (TIPA_WASHED) and LTA Check™, Performance Monitoring Check.

2. Direct Mode – The Direct Mode allows the user rapid access to the analyzer functions by passing data entry screens and going directly to the Status Screen. When at the Status Screen, assays may be completed by simply setting up the system, incubating and running the assay.

3. Ristocetin Cofactor Assay (RiCoF)– there are three assays available, 25, 50, 100%; 12.5, 25, 50 100%; and 20, 40, 80,100%.

4. Special Aggregation – These include Heparin Induced Thrombocytopenia (HIT), Sticky Platelets and Dose Response Assays.

Aggregation This selection displays the available aggregation test selections. The PAP-8E software is installed with optimized test methods using Bio/Data Corporation‟s reagents and controls. These are



validated methods and cannot be changed, deleted or overwritten. See the Create/Edit Type for instructions on how to develop user defined test methods. Selecting the Routine Aggregation button from the Main Menu screen will display the test selection screen. By clicking on the Select a Test bar or arrow, the list of tests will appear. Once a test type is selected the options on the right will become active.

To Run a Test Select one test type or the multiple test type from the menu. Select Run Sample. The screen will switch to the Sample Data Entry screen.

Enter a Sample name. Enter or scan a Sample ID Number. Select a test Type, if more than one test type was originally selected.



Enter a concentration, if dilutions of a reagent are being used. Repeat the entries for the other seven channels as needed. Fill Down – Selecting this option allows the information in a column to be repeated several times. After selecting the Fill Down button, select the last row to include the information and it will be copied to that row automatically. Comment – this displays a screen that provides for the input of comments for an individual test or all channels. Protocol - In the test screens you may select the protocol button at any time. This will display the basic test protocol for the test type selected. If more than one test type is selected, a test type selection button will be displayed that allows the user to select which test type to view. Test Templates User defined Test Templates for profile test group entry. This feature allows the user to recall a complete test panel instead of re-entering the same tests each time. Select Templates from the Sample Data Entry Screen.

The Template Management screen will appear.



Select an Existing Template or Create a New Template. To recall an Existing Template, in the Select an Existing Template box, use the arrow to expose the drop down list of stored templates. Select a template and then select Load. The template will load into the Sample Data Entry Screen. Enter the patient identification and run the tests. To create a New Template, enter a name for the new template and enter a new template description, if required, then select Save. All of the new template information will be saved. Clicking “Cancel/Exit” will exit, take no action and close this screen. Clicking “Delete” will delete the selected template. Note: If you select a single test type on the “Test Type Selection” screen, the Template you load must contain only this one test type. To load a Template with multiple test types, select “Multiple Tests” on the “Test Type Selection” screen.

Controls When inputting test information, any test may be identified as a control test. This is done by clicking on the well number box. The well number will be highlighted in yellow once it is selected as a control. The test may also be deselected at this time by selecting the well number box again. A completed test identified as a control may be selected or deselected as a control test by selecting the Control button in the Stored Results Screen. First, click on the control button, a pop up will come up, select ok, then select the test row that you want as a control, a pop up box will let you know if it was selected or deselected as a control. Control test data may be compiled with the Data Sorting feature. Control results may be exported to the Prism software where they can be analyzed with statistical QC software. Special tests cannot be identified as controls, only aggregation and Ristocetin Cofactor Assay tests have this option.



Reagents The reagent information data should be verified to ensure the correct reagent information is recorded. A box will appear if Multiple Tests is selected where the test type is selected for the review of those reagents. Lot numbers should be verified and expiration dates should be reviewed to verify the correct reagent is being used and it is within its usable time period. Select Exit to return to the Sample Data Entry screen. If any information was changed, select “Save Changes.”

To Run a Sample Select the Next button and the Live Results screen will be displayed with the Test Control and Status Menu. A Blank prompt will be highlighted in the channel‟s status box for each of the tests entered. Test Control and Status Menu Status Screen Channel Testing Information Display As a help to the user, when the Test/Status Screen is displayed, the program will show the test type selected for each channel programmed for running. This is provided as an aid and does not affect the results on any channel or testing operation. This information will be displayed for all agonists and is to aid the user in the completion of the test run. The first 7 characters from the Sample Name on the Sample Data Entry Screen will appear over the test wells.



Place the test tube with a stir bar into the stirred incubation well, add the PRP sample and select the timer box for that channel. The timer will begin the count down. Repeat this for each test. The Timer box below the well number will begin displaying the count down time of the incubation period. The timer will count down to zero and sound an audible alarm. It will then count the elapsed time and show a negative sign to indicate that the time has exceeded the preset incubation time. While the sample is incubating, place the blank for the test in the channel and select the Blank button. The status will change to Start. If the blank is not set correctly, select the Back button to repeat the blanking process.

At the end of the incubation period, place the sample tube into the Test Well. Select Start, the status box will change from Start to Stop and the lower box will display Inject. The graph will start displaying the baseline. The percent aggregation will be displayed above the test well number. Place the pipette tip into the pipette guide, then inject the reagent and select Inject button on the screen for the channel. A point will appear on the graph to indicate the time the inject selection was made. The graph will adjust to 0% aggregation and the reaction will continue to be plotted. The time that the inject box is selected is the point from which calculations such as Lag Phase and AUC will be measured. The graph will also be corrected for any dilution effects and will automatically re-zero the plot for more accurate percent aggregation measurements. Once all of the tests have been initiated, the Test Control and Status Menu may be hidden by selecting the Hide button. To recall the menu select the Status button from the tool bar.



After the Inject Button is selected, it changes color (green to grey) to indicate that the reagent was added. It then becomes an Event Marker that marks the data stream and is displayed on the print out. Inject/Event Button Before setting the blank on a channel, the channel status display will change providing two buttons displaying:

If the “Abort” Button is pressed, this will abort the test for that specific channel and all setup data entered will be lost. An Aborted test cannot be re-setup and started until all channels in the current run are finished.

If the “Blank” Button is pressed, this will cause the instrument to set the blank for the channel

and the channel status display buttons will change to: Pressing the “<Back” will move program control back to the previous operation. Pressing the “Start” button will start the data generation for the channel and change the channel status display buttons to: The Sample should now be added and then: Pressing the “INJECT” Button causes the following events: After a delay for mixing, the baseline will be reset to 0% Aggregation Pressing the Inject button will start the timing for the “Lag Phase” and AUC “Area

Under the Curve” calculation. Note: If the Inject Button is not pressed the test will proceed normally showing

progression of curve generation until the test stops. The Baseline will not be adjusted and there will not be a Lag Phase or Area Under The Curve (AUC) calculated for the test. All other relevant parameters will be generated for the test.

Pressing the Inject Button will change the name to Event:

BLANK

ABORT

START

< BACK

STOP

INJECT

EVENT

STOP



From this point on the button will function as a “marker” to identify points on the data and will not affect the data stream. Up to 4 events may be marked through out the test run on each channel.

Pressing the “STOP” Button will stop the test run and recording of data. All data generated will be saved to memory

Filters During the tests or when results are recalled, the Filter selection may be made. When it is selected, a box appears where the channel to be filtered appears and the channel selection is made. The results are stored in a manner that allows them to be recalled either way at anytime. A Filtered aggregation results example is shown below.



Create Test Type This button displays the Create Test Type screen. You can create a user defined Routine Aggregation test type only.

The Create Test Type Screen will display which results will be provided with the created test type. Enter data for all fields. The program will prevent you from entering invalid values. The required information is:

Type Name Max Test Time, 30 Seconds to 480 minutes (8 Hours) Incubation Time, 1 to 15 minutes Stir Speed RPM, 50 to 1200, for most aggregation tests 1200 RPM is the most effective speed. Reagent Stir Speed, 50 to 300 RPM or 0 for off. Block Temperature, 32-45 Degree C. Select Sample Type – PRP or Washed Platelets. Select Create to save. Select Define Protocol to record test protocol. Select Finished. Select Define Reagents to identify reagents to be used. Select Save Exit



Routine Aggregation Results The results reported are identifies as:

1. PA – Primary Aggregation is the initial aggregation in a biphasic pattern, and this is the initial wave of aggregation. This is the final aggregation in normal aggregation patterns. This measurement is made on all patterns.

2. PS – Primary Slope is the initial or only slope. This measurement is made on all patterns. 3. SA – Secondary Aggregation is measured only when a biphasic pattern occurs. This is a

secondary aggregation wave. In a biphasic pattern this is the final aggregation result. The slope of the aggregation wave must change -2.5 (slow) for a period of 15 seconds before the 50% aggregation point and then begins again to produce a secondary aggregation wave with a slope increase of +2.0 for a period of 15 seconds. This is typically demonstrated in reagent dilution tests, epinephrine or certain pathological states.

4. SS – Secondary Slope is the secondary wave in the case of biphasic patterns. 5. AUC – Area Under the Curve is the calculation of the Percent Aggregation per Minute. The

result will include all aggregation activity from the time the inject button is pushed. 6. LP – Lag Phase is the time between the injection of the reagent and the initiation of the

aggregation. The Inject button must be used to identify the injection point; otherwise the measurement will not be made. Only reported with AA, Collagen, and HIT. The Lag must be greater than 15 seconds to be reported (this is the normal mixing time before aggregation begins).

7. DA – Disaggregation, This is the measurement after the maximum aggregation occurs where the aggregates break up and disaggregates. This is measured from the maximum aggregation to the point of maximum disaggregation.

8. MA – Maximum Aggregation is the highest aggregation seen during test. 9. FA – Final Aggregation is aggregation when test stops.

Tool Bar - The buttons along the bottom of the screen provide access to information and operations. The active buttons vary for each screen. Extend – This button will change the maximum test time from the pre-programmed maximum test time and allow the test to run an additional five minutes. Additional five minute increments may be added up to 8 times (40 minutes total). This is used if the operator observes the sample aggregation is occurring at an abnormal rate and will require additional time to achieve maximum aggregation or if disaggregation is occurring and the test requires additional time to complete. The test will continue until the pre-programmed maximum test time is reached or the Stop button is selected from the Status Menu. The buttons below the test well numbers will change from Accept or Reject. Once one of these buttons is selected, the boxes will change to Done and Cmnt (Comment). The Cmnt box selection allows you to type a comment about the test. This comment will become part of the database. Selecting Done saves the test as completed. A Rejected test will remain in the database. Under no circumstances can test data be deleted from the database. Help - The help screen may be selected at anytime. It contains the complete information from the Operation Manual for the open screen. Recall Test / Curve This button opens the active database. The tests in this database are displayed in descending order and the last test run is displayed first. Tests may be selected by the touch screen or with a mouse click. Up to eight tests may be selected at one time. The scroll bar at the side of the screen will allow you to move quickly through the test list.



Tests may also be selected by moving the pointer with the UP/DOWN buttons and placing it on the selected list by choosing the Select Current button. The last test selected may be removed from the list by selecting the Unselect Last button. The entire list may be cleared by selecting the Clear List button.

Once the tests are chosen, select the Show Results button. This displays the aggregation patterns and results for the selected tests. Sort Data - In this screen you may select data by any of several parameters. These parameters may be date range and/or patient name, number, date or date range and operator.

The Sort Screen entries are retained form previous sorting. Select the Clear button to remove the entries. Controls Selecting the Control button on the tool Bar allows the selected test to be identified as a control test. If a test was originally identified as a control, and the results are not acceptable, selecting that test and selecting the Control button will unselect the test.



Result Reporting Test results may be recalled for printing at a later time, when all testing is complete. This allows the tests to be run when the platelets are the freshest and most active. When the “Print” button is pressed, you will be asked – “Do you want to print reagent information on a second sheet Yes/No”? Reagent information may be printed each time a graph is printed.

To create a printout of stored result press print. The report includes:

1. Header information from the setup input. 2. The test graph as shown on the screen. 3. Test information, results and comments. 4. Acceptance sign off area.

The results may be selected individually in any order. The screen displays the results for review prior to printing. They may also be sorted by various parameters or combination of parameters, such as; date range, patient name, number, date and operator.

The Help button will display the pertinent section from the operation manual. Clipboard Feature Beginning with software version 1.0.8, PAP-8E users may copy test results into other Windows® documents such as Word®, Excel®, Powerpoint®, and Publisher®. The Clip button will appear on the upper left portion of the screen when this option is available.



The "Clipboard" feature creates an image very similar to the printed results and places it on the Windows® clipboard. From there the operator simply pastes the image into the selected document.

1. After running live tests or recalling previously run tests, click the "Clip" button at the upper-left of the "Live Results" or "Stored results" screen.

2. Wait briefly. The PAP-8E screens may change while this action is taking place. 3. A message box will appear telling you that you may now paste the clipboard into your

document. 4. Open the document you wish to paste the results into. 5. Right-click on the blank page and select "Paste".

6. The picture may be further formatted by right-clicking on the image and clicking "Show Picture Toolbar".

Note: The document must be set for landscape format for all test types except Sticky Platelets. The Clipboard feature has been tested successfully with Word®, Excel®, Powerpoint®, and Publisher®. It is not possible to paste the results directly into an Outlook or Outlook Express document. See the Export section of the manual. Windows

®, Word

®, Excel

®, Powerpoint

®, and Publisher

® are registered trademarks of Microsoft Corporation.



Aggregation Pattern Overlay The comparison of two recalled aggregation patterns can be performed by recalling the test results and selecting the Spacing button located at the top of the screen. This selection will either overlay the patterns or return them to their normal spacing.

Collection and Preparation of Test Samples Refer to the Specimen Preparation Section of the printed manual for detailed instruction about proper specimen collection and preparation.

The collection of acceptable specimens and preparation of quality samples for routine or special platelet aggregation and the Ristocetin Cofactor Assay is critical to obtaining accurate test results.

Collection and preparation errors affect the test results.

Platelets will activate if stored at a temperature below 20C. This activation changes the sample and alters the test results. Re-warming the platelets will not reverse this activation.

Testing must be completed within 4 hours of specimen collection. If not, platelet activity will decrease and test results will not be accurate after that point.

Sample Preparation

There are two methods for preparation of platelet rich and platelet poor plasmas and both are validated and accepted methods. The normal centrifugation method utilizes a standard laboratory centrifuge and requires about 40 minutes to prepare the samples. Bio/Data Corporation‟s PDQ™, Platelet Function Centrifuge method prepares the samples in approximately 5 minutes.

The PDQ®, Platelet Function Centrifuge The PDQ, Platelet Function Centrifuge, is intended to be used as a clinical centrifuge dedicated to the preparation of samples for platelet function testing. It has three pre-programmed modes that produce specific sample types: Platelet Rich Plasma, Platelet Poor Plasma and Platelet Free Plasma.



The three modes are: 1. Platelet Rich Plasma (PRP) 30 Seconds 2. Platelet Poor Plasma (PPP) 120 Seconds 3. Platelet Free Plasma (PFP) 180 Seconds

The overall time includes the following plus slowing and braking time.

SPIN TIME SECONDS

RAMP TIME SEC.

RAMP UP g-SECONDS

AT SPEED TIME SEC.

AT SPEED g-SECONDS

TOTAL g-SECONDS

PRP 30 20 44,000 10 44,000 88,000

PPP 60 20 44,000 40 176,000 220,000

PFP 120 20 44,000 100 440,000 484,000

As an example, the thirty second spin can be illustrated as a graph:

PRP Cycle The PDQ has reduced the time and effort of preparing samples for Platelet Function Testing. It has standardized the pre-analytic process by controlling the process from specimen to test. It prepares samples with reproducible sample quality, improved precision, and the samples are free from activation artifacts. It is highly recommended that the PDQ be used to prepare samples for use with the PAP-8E. The PDQ was validated under and complies with the requirements of Bio/Data Corporation‟s Quality System. Each sample type produced by the PDQ meets current published guidelines for platelet function test samples required for Light Transmission Aggregometry. PDQ generated samples can be used with all optical aggregometers. Studies performed by Bio/Data illustrate that the PDQ produces more platelets and a more complete range of platelet sizes when compared to the standard centrifugation method. The large platelets are not spun out as has been theorized. The greater number of platelets has no serious impact because the test methodology is based on a base line of the PPP / PRP difference, not absolute platelet count. Better test results will be obtained with more complete populations of platelets.



Platelet Size and Counts Note: Most cell counting instruments are not designed or validated to be used with Platelet Rich plasma Samples. Systems can not differentiate between debris and live cells of the same size. This is a common error when laboratories wish to automate platelet counting. Our studies were based on the CASY TT Cell Analyzer a particle counting device produced by Innovatis/Roach, Basel Switzerland. The detection method is a resistance measurement principle developed for cell analysis called Pulse Area Analysis. This analyzer was developed precisely for cell analysis and is used primarily in research settings. The principle is based on digital pulse processing a standard method of recording electrical signals. The concept is as cells pass through a precision orifice the resistance of a live cell is measured and a dead cell is sized by the size of the nucleus. The size range is 0.7 µM to 120µM. The measurement dynamics are a 1:40 ratio. This use for platelet counting was refined and validated by Bio/Data Corporation.

0.9 mL Volume Samples

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1.45 1.95 2.45 2.95 3.45 3.95 4.45 4.95 5.45 5.95

Size uM

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Platelet Aggregation Calculations The PAP-8E has a data reduction capability which calculates several parameters for each test performed. These are:

1. Primary slope 2. Primary aggregation 3. Secondary slope 4. Secondary aggregation 5. Area Under Curve 6. Lag phase 7. Disaggregation 8. Maximum aggregation 9. Final aggregation

Due to the natural variability of either normal or abnormal specimens, there may arise occasions in which the values reported in the Data Grid will not match values that may be visually interpreted from the generated pattern. If this situation occurs and causes an ambiguity in the diagnostic utility of the test, an “Analysis” of the pattern should be performed. Platelet aggregation analysis is usually a visual comparison of a Test Pattern against a Normal Pattern for differences of “Profile”. The utilization of reported numerical values, though important, should serve as an aid to the pattern comparison for the overall interpretation of a specific test pattern. The trained professional with the experience and skills required for correct analysis should only perform visual analysis of a generated pattern. Slope - When the test is started, the PAP-8E automatically starts monitoring the rate of aggregation and determines the maximal change over a standard increment of time. This provides uniform interpretation of slope values. Upon termination of the tests, the slope for each aggregation is displayed in the results section. The slope is displayed as a unit-less value. Studies have demonstrated that slope values determined by the PAP-8E correlate with manually determined slope values. All of these calculations as well as shape change, Area Under the Curve and Area Under a Slope can be made by the user in the Analysis section of the software. Area Under the Curve Calculation Area Under the Curve (AUC) is a calculation of the activity of aggregation from the base line (the injection zero point) to the final percent aggregation for the elapsed time for the test. The PAP-8E averages the data points at half second intervals, than divides that average by 120 seconds. The result is Percent Aggregation per minute. Expected results depend on the end of test time selected by the operator and the concentration of the agonist. Each laboratory must determine its own end time and references ranges. AUC is intended for Research Use Only. Quality Control To assure reagent performance, a control specimen should be evaluated each day that the tests are performed in the same manner as each test specimen.



For qualitative platelet aggregation studies, the control should consist of fresh platelet rich plasma collected from a known normal donor who has not ingested aspirin or aspirin-containing compounds within 10 days of testing.

Normal donors should be pre-screened for each test type and a history maintained by the laboratory. For Ristocetin cofactor normal donors include blood type and fibrinogen levels.

Test Procedures The PAP-8E is designed to use a total or final volume of 0.250 mL. All pre-programmed, standard test protocols are devised for “Micro-volume” methodology, using 0.225 mL of PRP and 0.025 mL of reagent volumes. This is the preferred method of operation for the PAP-8E. Test methods are dependent on the reagent manufacturer‟s technical product information. The following methods are based on Bio/Data Corporation‟s methods. Detailed test information is available in the Test Method section provided in this binder.

Testing must be completed within 3 hours of specimen collection. If not, platelet activity will decrease and test results will not be accurate after that point.

Standard Aggregation Methods The standard platelet aggregation tests are: Collagen, ADP, Arachidonic Acid, Epinephrine, Spontaneous Platelet Aggregation, Heparin Induced Platelet Aggregation (HIPA), Ristocetin Induced Platelet Aggregation (RIPA), Thrombin Induced Platelet Aggregation (TIPA_PRP) and (TIPA_WASHED) and LTA, Performance Monitor Check. Materials Required

1. Reagent, refer to manufacturer‟s package insert for reagent reconstitution and stability information

2. Test tubes, 7.5 X 55 mm, siliconized 3. Stir bars, micro, coated 4. Pipettes (0.225 mL and 0.025 mL volume capacities)

Reconstitution

Stored reagents must be returned to room temperature prior to use. Refer to manufacturer‟s package insert for reconstitution instructions.

General Test Procedure

1. Prepare a blank by pipetting 0.250 mL platelet poor plasma (PPP) into a tube. Do not add a stir bar.

2. Place empty test tube into the incubation wells a. add a stir bar

3. Pipette 0.225 mL of the sample platelet rich plasma (PRP) into one of the pre-warmed test tubes.

4. Place the tube of sample PRP plasma into the stirred sample incubation well. a. Select the timer and the count down will start. b. The sample will incubate at 37ºC for the preset time.

5. Set the 100% baseline by placing the blank into the Test Well. a. Select Set Blank.

6. Place the tube of sample PRP plasma into the test well



a. Select Start. 7. Add 0.025 mL reagent directly into the platelet rich plasma.

a. Select Inject. i. Do not allow reagent to run down the wall of the tube.

8. The test will run for pre-set time. a. Other manufacturer‟s test procedures may specify different times.

Standard aggregation test methods



Direct Mode A Direct Mode feature is included in this Version. The Direct Mode allows the user rapid access to the analyzer functions bypassing data entry screens and going direct to the “Status” screen. When at the “Status” Screen assays may be completed by simply setting up the test/reagent system, incubating and running the assay.

If required, the data entry that was bypassed in the Direct Mode may be included in the printouts of results by entering this information in the “Comments” before the test is saved. All comments entered will be saved along with the test results. When running test in the Direct Mode the patterns for each test will be displayed along with the DATA GRID, which will report the following parameters: PAP-8E Software Data Grid will show: PA PS SA SS AUC LP DA MA FA PA – Primary Aggregation PS – Primary Slope SA – Secondary Aggregation SS – Secondary Slope AUC – Area Under The Curve LP – Lag Phase DA – Disaggregation MA – Maximum Aggregation FA – Final Aggregation If the software detects any of the above conditions, a numerical value will be reported. These reported values may not always be pertinent or appropriate to the Assay Type performed. The user should review the reported values for their intended purpose. If the user disagrees with a reported parameter, that parameter may be ignored in reporting test results, or the parameter may be re-evaluated by recalling the test and performing an “Analysis” to generate data relating to the testing completed. DA, MA and FA may not be re-evaluated, as these parameters are “primary” tools for diagnosis as applicable. Primary Slope, Secondary Slope, Area Under The



Curve and Lag Phase may be re-evaluated in “Data Analysis” and Prism software. Primary Aggregation and Secondary Aggregation may be estimated directly from the patterns reported. The Direct Mode is provided as an “ease of use” feature to the users and should not be used if it does not meet the procedures and policies of the laboratory using the PAP-8E instrumentation for clinical diagnostic testing.



Special Aggregation Methods The special platelet aggregation tests are: Heparin Induced Thrombocytopenia (HIT), Sticky Platelets and Dose Response Assays. Heparin Induced Thrombocytopenia (HIT) Method Selecting the Heparin Induced Thrombocytopenia test method and Run Test will display this screen. Control Tests

Test Setup Control 1. Well 1: Donor serum / heparin 2. Well 2: HIT Positive sample / heparin 3. Well 3: Donor serum / saline 4. Well 4: PRP / Arachidonic Acid



Patient Tests The Setup Sample / Reagent

1. Well 1: Patient serum / heparin 2. Well 2: Patient serum / saline 3. Well 3: PRP /Arachidonic Acid

To print results for HIT:

1. Accept all results 2. Go to top right of screen “Go to Hit Screen” 3. Go back to Patient Screen, Exit 4. Go to “Select a Test” 5. From drop down – Select “HIT” 6. Select “Recall Data” 7. Select Donor to print or “Display Toggle” to display Patient results to print 8. Select Donor or Patient Results 9. Select “Show Results” 10. Select “Print”



Sticky Platelet Aggregation The Sticky Platelets sample input screen is shown below.

Test Setup The test setup for each sample consists of two sets of five tests.

1. Well 1 is for spontaneous aggregation. 2. Well 2 is for reagent stock dilution. 3. Wells 3, 4, 5 are for standard reagent dilutions. 4. 3 additional dilutions may be added in wells 6, 7, and 8. Enter the dilution concentration for

each test.

Enter the sample name and sample number. Brief instructions are shown at the lower left of the screen. The ADP tests must be completed before you can advance to the Epinephrine tests. If activity is observed in the 1:40 dilution sample then run three more reagent dilutions in wells 6, 7, and 8 to identify the dilution that produces no activity. Select the Go to Sticky Platelets Screen button to return to the Sticky Platelet Test screen. The need for additional dilutions is determined by the laboratory. Fill in additional dilutions and select Next.



If a test result is unacceptable the test should be rejected. Then select the Go To Sticky Platelets Screen button to return to the Sticky Platelet Test input screen. Rejected Test Well will say ready while all others will say completed. Select next. After running all ADP and selecting “accepted” then select Go to Sticky Platelets Screen. You will return to patient screen and then select “begin EPI series.” To print results for Sticky Platelets”:

1. Accept all results 2. Go to top right of screen “Go to Sticky Platelets” 3. Go back to Patient Screen, Exit 4. Pop up Screen will ask, “Are you sure you want to exit” 5. Select “yes” 6. Go to “Select a Test” 7. From the drop down – Select “Sticky Platelets” 8. Select “Recall Data” 9. Select Patient 10. Select “Show Results” 11. Select “Print”

The test results will be printed on two pages, one page for each of the test types. The Test Results screens can be viewed and toggled back and forth with the Go to Sticky Platelets Screen button at the top of the Results screen. When complete, Exit.

Dose Response Testing Dose Response testing, commonly referred to as IC50 or EC50 are performed in many different methods depending on the application. The following is a basic method.

1. Experimental design a. 6 to 8 concentrations based on geometric dilution series (x-fold) b. 1-2 replicates per concentration

i. Run 1-2 sets of tests in groups of dilutions. 2. Concentrations span the range of activity

a. at least one concentration is close to upper asymptote b. at least one concentration is close to the lower asymptote c. two concentrations span the 50%

The Dose Response test results utilize the Prism software to graph and analyze the results.

1. In the Special Aggregation test mode select the test type Dose Response 2. Run a set of six to eight dilutions 3. Run dilutions in highest to lowest order, highest in well 1 4. If in the proper range, repeat a second time 5. When the tests are completed,

a. Recall Data b. Show Results c. Select Export

6. A Data Export screen will appear



7. Select Export Type a. Text File – will create a raw data file containing calculated aggregation results and graph data that can be imported into Excel b. Prism – will export calculated aggregation results for PA, PS, SA, SS, LP, MA, FA c. Excel - will export calculated results for PA, PS, SA, SS, LP, MA, FA, graph data and will import directly into Excel. Must have Excel on your PC.

8. Begin Export 9. Close 10. Go to the PAP-8E Main Menu 11. Select Prism 12. Select the type of analysis method and perform the analysis. 13. Import the first set of test data

a. Select File; Import b. Go to C:\PAP-8_Export c. Select the file desired d. Select Import from the format Data Object menu e. The results will appear in the table

14. Refer to the online manuals for detailed information about the operation of the Prism software.

Leukocyte Aggregation User defined Test Type (Create Test Type) Leukocyte Adhesiveness/Aggregation, LAAT, WBC Aggregation, Neutrophil Aggregation, Cell Mediated Immunity Assay Purpose - Leukocyte aggregation [LA] is a biomarker for detecting and measuring the degree of inflammatory responses. 1 LA can be used to detect defects in leukocyte function. 2 LA is also used to study activation and platelet-leukocyte interactions, particularly in atherosclerotic disease and the effects of anti-inflammatory and anti-thrombotic drug therapy. Clinical Conditions

1. Differentiating bacterial from viral meningitis



2. Chronic Granulomatous Disease 3. Rheumatoid Arthritis and other autoimmune diseases such as Inflammatory Bowel Disease 4. Organ Transplantation 5. Sepsis

Procedure Description - The test principle is the same as that for optical platelet aggregation: 0% is set with cell free or buffer blank and 100% is set with cell free or buffer blank test sample, an inducer or agonist is added and total aggregation is measured. Cell preparation, cell count and inducer vary by protocol. Refer to the Methods Section for test methods. Limitations - Results are determined by comparing patient and normal donor curves. Expected Results - In certain conditions, spontaneous leukocyte aggregation [SLA] can occur. SLA requires as much as 60 minutes to occur and the sample must be stirred during that time.

Expected results depend on inducer and protocol used. Notes:

1. User Defined Protocol; Research Use Only. 2. Anticoagulant choice may affect test results. 3. Tests must be performed in siliconized test tubes. 4. Test parameters like stir speed and pre-incubation times vary by protocol and are not

necessarily the same as those for platelet aggregation. 5. Using of washed or unwashed cells has been reported, as have the testing of particular types

of leukocytes. 6. Each laboratory must prepare and validate its own procedure. 7. Each laboratory must establish its policy regarding singlet or duplicate testing.



Ristocetin Cofactor Assay (RiCoF) Reagent Entry Screen The PAP-8E Software allows the user to record all information relating to the Reagents used in the Ristocetin Cofactor test system. The information entered relates to the most current reagent used and will be associated with all testing completed. If the information is changed, that changed information will then be associated with all current testing completed. The screen below is an example of the reagent data entry screen.

Platelets – Enter current Platelet Lot data Ristocetin - Enter current Ristocetin Lot data Diluent – Enter data for the Platelet Diluent Reference Diluent – Enter data for diluent used for the reference plasma only if different than that used on the platelets Normal Reference Plasma – Enter data for the Normal Reference plasma used for the construction of the Standard Reference Curve Norm. Ref. Plasma % Factor Activity – Enter the assay value for the Normal Reference Plasma used to construct the Standard Reference Curve. The software allows values between 70% and 140% to be entered. Reference Curve linearity and acceptability will be based upon the value entered. Ristocetin Cofactor Activity Assay (RiCo) To perform the RiCo, select the Ristocetin Cofactor Assay button from the Menu screen. The test type screen will appear, select either the 3 point curve or one of the 4 point curves.



3 Point Standard Reference Curve Assay To review the current standard curve select Recall Curve, the curve results will be displayed. Select the Curve button and review the standard curve. If acceptable, normal and abnormal control plasmas should be run to verify that the curve is still viable.

Run a New Standard Reference Curve Select The Run Curve Button.



Prepare an aggregometer blank by pipetting 0.125mL reconstituted platelets and 0.125mL diluent into a tube. Mix thoroughly.

The standard reference curve dilutions must be placed in the test wells in descending order i.e. 100, 50, 25%



4 Point Standard Reference Curve Assays There are two types of four point assays available. After selecting Ristocetin Cofactor Assay, the

next selection is the curve type. The available curves are; 12.5 – 100%, and the 20 – 100%. Latter

type is used for Research Use Only (RUO). Each has specific standard curves, which are performed

in the same manner, except the dilutions.

To run a new 4 point standard curve:

Select the Run Curve button



The standard reference curve dilutions must be placed in the test wells in descending order i.e. 100, 50, 25, 12.5%. Do0 not use serial dilutions. See Methods Section.

Standard Reference Curve Quality Control The PAP-8E software includes an enhanced method to determine curve acceptability. When the tests are complete and the curve is displayed, the curve will be checked against four criteria to determine if it is an acceptable curve. These criteria are:

1. Minimum and maximum slope a. A curve that is too shallow or steep will not give accurate results.

2. R value of the curve data a. Defines the accuracy of the data points to the curve line.

3. Curve calculated value of the Reference Plasma. a. The slope of the reference plasma dilutions are calculated against the curve and the

activity compared to the assigned and calculated activities. 4. Linearity

a. “Check Linearity” For User review: the user will review the curve and decide to Accept/Reject. This will eliminate a curve to be labeled “Non Linear” that the user deems usable.

The Standard Reference Curve Results Screen If the criteria are out of specification, the term “examine” will be displayed. The operator will have the ability to accept or reject the curve. The curve print out will contain the graphics, test results. The sample assayed using this curve will have a curve status printed on the curve. Point Suppression When using the four point standard reference curves it is known that the curve linearity is affected by the 100% sample results. The effect is reduced accuracy at the 12.5% or the 0% end of the standard curve. The PAP-8E has a feature that allows the suppression of the 100% point.



After the standard curve data is reviewed, a selection can be made to suppress or eliminate the 100% point and recalculate the modified curve results. This can only be done by entering a level 2 password. In the case of the 100, 50, 25, 12.5% curve the curve will be identified as “Research Use Only”. The suppression is performed by following the steps below:

1. After the completion of the standard curve tests, open the Standard Curve Results screen 2. Select Suppress 3. If the operator is not using a level 2 password, a box will appear that requests a level 2

password. 4. Once the password is entered the curve point will be suppressed. The data is recalculated

for the remaining 3 points when the curve screen is closed. 5. Curve result screens and printouts will be labeled “RUO”.

After the curve point is suppressed, the values are recalculated once the screen is closed. The new values may be displayed by selecting the Curve button on the tool bar. For additional information, see Supplemental Bulletin ST-2009-04, Ristocetin Cofactor Assay – 100% Suppression located on our web site www.biodatacorp.com.




Performing the Assay

Prepare a sample blank by pipetting 0.125mL reconstituted platelets and 0.125mL diluent into a test tube. Mix thoroughly.

When all tests are complete the PAP-8E will calculate the percent factor activity for each test. Selecting the Curve button, the results will be displayed on the standard curve. The printout will include the aggregation patterns, standard curve and the calculated results. Note: “Out of Range” indicators: % Act = 999, result is > 120% % Act = - 999, result is < 15%



Data Analysis

Research Use Only (RUO) After the test is completed and stored, a test or tests must be selected and the Data Analysis option is enabled. It may be selected by clicking on the “Analysis” option in the Tool Bar. This function displays an additional grid with the analysis measurements and a brief description of the selected measurement.

Command Functions

1. Save - This button saves the points and data calculated for the test to the active database. 2. Clear selection: This button clears the measurement selected that has not been saved. 3. Exit - This ends the analysis session. 4. Color - This changes the color of the analysis points and lines to allow better visibility. 5. Channel - Selecting a number will display the results or graph from that channel from the

channels originally selected. 6. Dialog Window - Brief instructions are displayed when a parameter is selected. 7. Comment – This selection allows a comment to be added to the file. 8. Print – The print selection prints the Analysis graph, data and the original data.

Measurements All measurements are made in an identical manner. After selecting the type of measurement to be made, use the mouse to position the pointer and left click on the two points of the graph that mark the measurement to be made. The software will mark the points and connect them with a line, then perform the calculation. The result will appear in the Value box. Primary Slope, PS or Secondary Slope, SS Selecting either of these parameters allows user selection of the points on the curve from which the slopes will be measured. Lag Phase, LP In some assays there is a delay after the addition of the reagent and the initiation of the aggregation. This parameter may be used to measure this delay or any other time based function.



Disaggregation, DA In some conditions the aggregation is not stable and the platelets will disaggregate to a final level of aggregation that is less than the maximum aggregation. Area Under the Curve, AUC A standard method of comparing the magnitude of a reaction is calculating the area under the curve. Area Under a Slope, AS A standard method of comparing the magnitude of a slope is to use the area under the slope. Shape Change, SC Activated platelets undergo a shape change prior to the beginning of aggregation. Data Export

Research Use Only (RUO) Exporting Data to Prism, Excel® and other Software Programs To export data to Excel, Excel software must be loaded on the PAP-8E computer. Test results can be exported for further analysis. All test types except HIT and Sticky Platelets may be exported.

1. When the tests are completed on the PAP-8E: a. Recall data b. Show results c. Select export

2. A Data Export screen will appear

3. Select Export Type a. Text File – will create a raw data file containing calculated aggregation results and graph data that can be imported into Excel b. Prism – will export calculated aggregation results for PA, PS, SA, SS, LP, MA, FA



c. Excel - will export calculated results for PA, PS, SA, SS, LP, MA, FA, graph data and will import directly into Excel. Must have Excel on your PC.

4. Begin Export 5. Close

If you selected Text File or Prism from the Data Export Screen:

1. Go to the PAP-8EMain Menu 2. Select Prism 3. Select the type of analysis method and perform the analysis. 4. Import the first set of test data

a. Select File; Import b. Go to C:\PAP-8_Export c. Select the file desired d. Select Open e. Select Import from the format Data Object menu f. The results will appear in the table

5 To import a second data set a. In the Data Table screen, Select the first cell in the row below the last data row b. Select import again and repeat the action.

6 Refer to the Prism online manuals for detailed information about the operation of the Prism software. 7 For additional information, see Supplemental Bulletin ST-2007-02, Exporting Data to Prism Software located on our web site www.biodatacorp.com.

Prism Software

Research Use Only (RUO)

Prism Software Installation Instructions

GraphPad Prism is a powerful combination of basic laboratory and biostatistics, curve fitting and scientific graphing in one comprehensive program. It helps you organize, analyze and graph repeated experiments, pick appropriate statistical tests, and interpret the results; Prism is ideally suited to data plotting and analysis applications in the bio sciences. Easily set up project files, organize, analyze and graph repeated experiments; pick appropriate statistical tests and interpret the results. Prism is compatible with Microsoft programs such as Word, Excel and PowerPoint. Graphical presentation, organization and statistical analyses of test data are required tools for platelet aggregation assays used in clinical, research and pharmaceutical laboratories. Also data exporting for presentations, spreadsheets for web postings or other applications. The methods of data analysis and the various analysis parameters are beyond the content of this manual. The manuals provided with this software are clearly defined and comprehensive. References will be made below to the more common applications described in the support manuals provided with the Prism Software. The Prism online Manuals contain detailed step by step directions and are easy to follow. The manuals included are:

1. Prism Users Guide a. Contains the support information for all of the Prism features.

2. Regressions Book a. Defines the application of the Prism features in specific areas of analysis.

3. Prism Examples Manual a. Describes the step by step process for specific applications.




The standard Export function of the PAP-8E produces a semicolon delimited text file that is directly imported into Prism. When imported, the columns and rows can be selected to eliminate much of the unnecessary data. This data paring may be more efficiently performed with the use of scripts (macros). There are several methods for transferring your data to the Prism. These are direct importing, the writing of scripts and the standard cut and paste method. The first thing that must be done is to determine what type of analysis and graphing will be done with the data. Repetitive analytical and graphing tasks may be done with a number of Prism tools. The Prism Examples Manual describes three manual graphing methods;

1. Templates, duplicating a family of sheets 2. Applying a method by using a saved method or by following the proper example. 3. An automated method is scripting which automatically moves the selected data via a simple

programming language. This is described in Prism Examples Manual. The Use of Prism for dose response data calculations is described in the Prism Examples manual. The Regressions Book also has additional information about Dose Response Curves. This provides more detailed information on the analysis and data manipulations. This software is a fully supported copy of the GraphPad Prism licensed to your laboratory. For Technical Support you may go to the GraphPad web site or contact GraphPad directly. Prism Technical Support

1. Free Access to Online Calculators: www.graphpad.com 2. Quick Answer Database @ www.graphpad.com 3. Email: [email protected] 4. Phone support: email your phone number to [email protected]

Application Examples Clinical and Research Laboratories Clinical Lab Statistics Dose Response Curves EC 50/ IC 50 Calculations Output to reports, presentation software Pharmaceutical/Biotechnology/Device Companies Dose Response Cures EC 50/IC 50 Calculations Biostatistics Output to spreadsheet, presentation, publication software Clinical/Contract Research Organizations [CRO‟s] Data Management Sample Estimation Statistical Comparisons

http://www.graphpad.com/

http://www.graphpad.com/

mailto:[email protected]



User Maintenance Recommended intervals for routine maintenance: Daily – Inspect for spills or foreign materials on the surfaces or in the incubation or test wells. Check all connections. Daily, weekly or as required by laboratory standards - Calibrate Channels Monthly – Temperature Check 6 Months – Clean Test Wells. Yearly – Preventive Maintenance Procedure (PM). Contact Bio/Data Corporation Customer Service. Cleaning Inspect the PAP-8E for any spills or foreign materials on the surfaces or in the incubation or test wells. Clean with a mild detergent solution and warm water. Do not use excessive amounts of liquid. Dry immediately. Do not use solvents. For additional information refer to the Service section of this binder. Temperature Calibration Temperature calibration of the incubation and test block temperature may be performed by inserting a calibrated temperature probe or thermometer into a test tube filled with water placed in incubation well number 4 or 5 for 15 minutes. When using a wire probe or small diameter metal probe make sure the measuring tip is in contact with the block. The PC and PAP-8E software must be operating.

If the temperature is not 37C +/- 0.5, go to the Main Menu. Select Set Up and the Temperature tab. Enter the measured temperature in the box. Wait 15 minutes and read the temperature probe to verify the correction was successful. Note: If the temperature set point is not set to 37°C, the software will make the correction and the instrument will require fifteen minutes to calibrate and verify the new temperature. Fuse Replacement

This should only be performed by properly trained personnel.

The cause of the fuse failure should be determined prior to replacing the fuses.

1. Turn off the PAP-8E and remove the Power (main) cord. 2. Disconnect the power cord from the rear of the PAP-8E. 3. Using a small screw driver, open the fuse cover by inserting the screw driver into the end of

the cover. 4. Remove the fuse block by inserting the screw driver into the corner of the block and prying it

out. 5. Remove and replace the two fuses with the size and type indicated on the rear panel. 6. Reinsert the fuse block. 7. Close the cover and replace the power (mains) cord. 8. Turn on the power switch.



Cleaning the Test Wells Instructions to remove liquid or debris from the test wells.

Turn off the PAP-8E; the power switch is located at the left rear panel of the PAP-8E. This operation exposes sensitive electronic components and should be performed with care. Open the Pipette Guides Remove the two screws located at each end of the Test Wells. Lift the Pipette Guide and Test Well Insert assembly out of the Test Block.

The optical emitters and detectors are exposed at this point. Cleaning Vacuuming with a small adapter can remove debris. Do not use excessive amounts of liquid. Dry immediately.

Do not use solvents Test and Incubation Blocks, (interior and exterior)

1. Clean with a mild detergent solution (anti-microbial) and warm water. Use a cotton swab wet with detergent.

2. Do not use excessive amounts of liquid. 3. Dry immediately. 4. Use only foam, paper or cotton swabs to clean this area. 5. Use only moist swabs or wipes to clean this area. 6. Carefully remove any objects or liquids from the exposed cavity. 7. Clean the Test Well Insert. 8. Completely dry all surfaces and recesses. 9. Replace the insert and two screws.

Failure to remove all debris such as broken glass and all cleaning materials from the test well(s) will result in incorrect equipment operation and improper test results.

Certification Bio/Data Corporation provides calibration and certification services for the PAP-8E at its facility. Contact Customer Service for details. Certification documentation is in compliance with GMP, JCAHO, CLIA, and CE regulations.



PLATELET AGGREGATION PROFILER PAP-8E

ROUTINE MAINTENANCE RECORD YEAR: ___________________________ PAP-8E S/N: _____________

Visual Inspection and Check of Connections and Maintenance Cleaning ( ENTER INTITALS)

Optical Calibration (Enter OC)

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

1 6 MONTH INTERVAL – CLEANING OF TEST

WELLS 2

3 DATE: _____________

BY: _______________

DETAILS:

4

5

6

7

8

9

10

DATE: _____________

BY: _______________

DETAILS:

11

12

13

14

15

16

16

17

YEARLY PREVENTATIVE MAINTENANCE PROCEDURE

18

19

20

21

DATE: _____________

BY: _______________

DETAILS:

22

23

24

25

26

27

28

29

30

31

MONTHLY TEMPERATURE CHECKS (RECORD) JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC



Trouble Shooting Guide The PAP-8E software diagnostics operates in the background of the operating system. If a condition occurs that will affect the operation of the PAP-8E an error window will appear. If an operation cannot be executed or if an input error is made an error window will also appear. There are many operational errors that can appear and are self explanatory and self evident. Below is a list of errors that need additional description about the actions to be taken.

Error Message Action Description

Not Ready Error or Temperature Error Test block has not reached operating temperature. Testing cannot begin.

The test sample and blank are optically very similar. The test and results may be of poor quality.

Try to set the blank again. If the problem continues you must close this program and cycle power to the analyzer then restart this program. Notify Customer Service if the problem continues.

“There is a problem with Channel:# +10V. The Sample/blank is out of the optical range and cannot be measured by the instrument.” or “There is a problem with Channel:# -10V. The sample/blank is out of the optical range and cannot be measured.”

The sample or blank may be too opaque to be seen by the instrument or there may be a problem with the instrument electronics. Contact Customer Service.

No Connection Serial Communications Error Communication Error

Turn PAP-8E on before starting software. Check the RS232 cable connections. If the problem continues you must close this program and cycle power to the analyzer then restart this program. Notify Customer Service if the problem continues.

Cannot locate incubation value

Contact Customer Service.

An error has occurred in the program. 'No Type Selected'.

Contact Customer Service.

Electrical Offset

Close this program and cycle power to the PAP-8E then restart this program. Notify Customer Service if the problem continues.

Block Processor Error Block Temp/Proc Errors

You must close this program and cycle power to the PAP-8E then restart this program. Notify Customer Service if the problem continues.

Calibration Error

There is a problem with the calibration. Re-calibrate with water. If problem persists; Please contact Bio/Data Customer Service for assistance.

You must select a test type for each row containing a sample name and number, You must insert a sample name for each entry, You must insert a sample number for each entry

Incorrect data entry operation, correct the data or make the proper entry.



You must enter a valid number for the normal reference plasma percent activity

Invalid Selection, Analysis is allowed archived data only.

Cannot perform Analysis until test is accepted and saved (closed).

An error has occurred opening the export file. Either the filename is too short or incorrect characters have been used, or a file already exists under this filename. Enter another name.

Incorrectly entered file names.

All tests have not been completed. If you return to the menu screen all incomplete tests will be lost.

Complete the remaining tests, accept or reject then continue.

Point Selection Error You cannot plot analysis points beyond the total time of the test.

Reselect the type of measurement and select points on the displayed graph.

You must change the color assignment for each channel on the Setup screen.

The channel colors cannot be changed in the live Test or data recall screens.

This feature cannot be used while the active database is an archive.

Archiving Error, you cannot change or add to an archive.

See Help Section For Instructions For Fill Down.

Information must be entered in the field(s) above, the Fill Down button must be selected and the last row to be filled must be selected.

'Op_Level' must be set to 1 or 2 The operators access level must be defined, 1 = general operator, 2 = Full access.

'Last_On' and 'Last_Off' cannot be changed This record cannot be changed or deleted for regulatory control.

Entered Values Not Effective Until 'Initiate' Is Pressed

The Initiate button stores the value entered in operations like temperature calibration. If not pressed value will be lost.

You must enter some sort criteria before continuing

Select a sorting parameter.

You cannot calibrate the instrument until the test block reaches operating temperature

Wait until the test block reaches operating temperature

Calibration must be completed before a certificate can be printed Or Calibration must be performed before a certificate can be printed

Cannot print until a calibration is completed

Any channels that are out of range cannot be used for testing

Corrective action required

This channel is unstable and cannot be calibrated. Try again later or contact Bio/Data Corporation Customer Service


Calibration no voltage change (component failure or complete blockage of optical path)


There is a problem with this channel. The light level cannot be adjusted. There may be a failure in the instrument or the instrument is not compatible with this version of software. Please

Check instrument hardware version Corrective action required



contact Bio/Data Corporation customer service for assistance Or There is a problem with this channel. The light level cannot be adjusted. There may be a failure in the instrument or the instrument is not compatible with this version of software. Please contact Bio/Data Corporation customer service for assistance

This channel is out of calibration. Please clean the optical path as described in the operators manual or call Bio/Data Customer Service




Ordering Information

The use of Test Tubes and /or Stir bars other than those manufactured by Bio/Data Corporation for use with the PAP-8E will void all instrument warranties.

Test Tubes are to be used only once. Cross contamination will occur if tubes are reused. Cleaning of test tubes will remove the siliconized coating and expose the sample to the glass surface and activation will occur.

Stir bars are for single use only. Cross contamination will occur. Stir bars are coated with parylene, an excellent inert material, but will wear after one use exposing materials that will contaminate the test materials.

To order reagents, disposables or accessories contact Bio/Data Corporation or an authorized distributor:

Bio/Data Corporation Customer Service Department 155 Gibraltar Road P.O. Box 347 Horsham, PA 19044-0347 U. S. A. Phone (215) 441-4000 (World-Wide) or (800) 257-3282 (U.S.A.) FAX (215) 443-8820 Internet: www.biodatacorp.com or email: [email protected]

Disposables, Accessories and Reagents

Disposables Catalog Number

Quantity

Stir Bars, Plastic Coated, Micro 105990 50/pack

Test Tubes, Siliconized, Micro, 7.25 x 55 mm 101521 100/box

MagneTube, Test Tube with Stir Bar, Micro 107146 50sets/box

Pipette Tips

300L Refills

300L with plastic box

1000L Refills

1000L with plastic box

106554 106612 106610 106608

960/pack 960/pack 960/pack 960/pack

Accessories Catalog Number

Quantity

Reagent Well Inserts,15, 17, 19, 22 & 25 mm 106602 Two each of five sizes

Biohit Pipettors

eLine 10 - 300L, Electronic, Programmable

eLine 50 -1000L, Electronic, Programmable

Single Stand and Charger*

106232 106606 106560

1 1 1

GraphPad Prism Software 106548 1

*Chargers are country specific; please contact Customer Service for information.



Reagent Products Products Catalog

Number Quantity ADP (Adenosine-5'-Diphosphate) 101312 3 x 0.5 mL Arachidonic Acid (Sodium Archidonate) 101297 3 x 0.5 mL BETA/Pak® (ADP, Collagen, Ristocetin) 101580 1 x 0.5 mL each Collagen Type I, (Soluble Calf Skin) 101562 3 x 0.5 mL Epinephrine (Adrenalin) 101311 3 x 0.5 mL Lyophilized Platelets 101258 1 x 10.0 mL Lyophilized Platelets 101595 3 x 4.0 mL PAR/Pak® (ADP, Collagen, Epinephrine) 101310 2x 0.5 mL each Ristocetin AggRecetin® (1.5mg/mL) 100968 15mg AggRecetin® (1.0 - 1.5 mg/mL) 100970 15mg AggRecetin® (Bulk) 101241 100mg Sodium Citrate, 0.11 M 100994 237 mL Tris Buffered Saline, 0.06 M, pH 7.5 100991 473 mL vW Select® 15 (Ristocetin Cofactor) 107056 Kit vW Select® 50 (Ristocetin Cofactor) 106730 Kit vW Factor Assay® (Ristocetin Cofactor) 103025 Kit vW Normal Control Plasma 106426 3 x 0.5mL vW Abnormal Control Plasma 101270 3 x 0.5 mL vW Normal Reference Plasma 101269 3 x 0.5 Ml LTA Check™ √ Performance Monitoring Kit 107117 Kit LTA Check Software ™ 107149 Each Warranties

Instrument Warranty Limited Warranty: Bio/Data Corporation warrants, to the purchaser, this instrument to be free from defects in workmanship and material for a period of two years from date of installation. If an instrument defect should occur within this period, Bio/Data Corporation will (at its discretion) either repair, replace, or cause any necessary repairs to be made, free of charge, providing:

1. The purchaser completes and returns the Warranty Card to Bio/Data Corporation within ten (10) days from the original installation date.

2. The purchaser first contacts the dealer from whom he purchased the instrument to determine the extent of service required.

3. The purchaser pays the freight and insurance costs incurred if factory service is required. 4. The computer, monitor, printer, barcode scanner and programmable pipettor are covered

under their respective manufacturer‟s warranties. This warranty is in lieu of all other warranties, expressed or implied, and no responsibility is assumed for consequential damage or malfunction due to accident, abuse, or that caused by the use of unauthorized attachments or accessories.



Glossary AA - Arachidonic Acid reagent

ACP – Abnormal Control Plasma

ADP - Adenosine-5'-Diphosphate reagent

Archive Interval - The number of days that the software is programmed to remind the user to

archive their records in the current data file

AUC – Area under the curve

Box – A small window that contains a message

Chan or Ch - The channels or wells on the PAP-8E incubation and test blocks

Clicking - Using the mouse or the touch screen to locate your selection by pressing and releasing

the mouse left button when the pointer is in position

Cmnt – Comment

Coll –Collagen reagent

Conc – Concentration

DA – Disaggregation, measured in % aggregation change

Double Clicking - Using the mouse to locate your selection by pressing and releasing the mouse left

button twice when the pointer is in position

Epi - Epinephrine reagent

FA - Final Aggregation, aggregation when test stops

Icon - A graphical representation of an application

LP - Lag Phase, in seconds. The time before the reaction begins, from injection of the agonist

LTA Check – Performance Monitoring Check Kit

LTA Software – Companion Software for Performance Monitoring Check Kit

MA – Maximum Aggregation, highest aggregation during test

Menu Bar - Buttons from which choices initiate action within the program

NRP –Normal Reference Plasma

NCP – Normal Control Plasma

% Agg - Percent Aggregation

PA - Primary Aggregation, first or only aggregation, measured in percent

PDQ – Platelet Function Centrifuge

PS - Primary Slope, first or only slope

RUO – Research Use Only, not FDA approved must be validated by the laboratory for their use

SC - Shape Change

SS - Secondary Slope, biphasic aggregation

SA – Secondary Aggregation, biphasic aggregation

Text Box - A box in which alphanumeric information is entered using the keyboard

Tool Bar - Allows the user to access utilities for the displayed window.

U/mL - International Units per milliliter

Wells – The channels or wells on the PAP-8E incubation and test blocks

Window - A window is used to provide information, Help, error or menus



References Platelet Aggregation References

1. Born, GVR and Cross, MJ. The Aggregation of Blood Platelets. J. Physiol. 168: 178 – 195, 1963.

2. Eichelberger, JW. Kinetic (Slope) Measurement of Platelet Aggregation. Bio/Data Corporation. Horsham, PA. 1984.

3. Triplett, DA, ed. Platelet Function: Laboratory Evaluation and Clinical Application. American Society of Clinical Pathologist. Chicago. 1978.

4. Brinkous KM, Graham JE, Cooper HA, Allain JP, Wagner RH: Assay of von Willebrand factor in von Willebrand's disease and hemophilia. Use of microscopic platelet aggregation test. Thromb Res 6:267, 1975.

5. Olson JD, Brockway WJ, Fass DN, Magunson MA, Bowie EJW: Evaluation of ristocetin induced platelet aggregation. Am J Clin Path, 63:210, 1975.

6. Miller CH, Graham JB, Goldin LR, Elston RC: Genetics of classic von Willebrand's disease I. Phenotypic variation within families. Blood, 54:117, 1979.

7. Nilsson IM, Holmberg L: von Willebrand's disease today. Clinics in Hematology, Vol 8, No. 1, 1979.

8. Brinkhous KM, Read MS: Preservation of platelet receptors for platelet aggregating factor by air drying, freezing or lyophilization: new stable platelet preparations for von Willebrand factor assays. Thromb Res, 13:591, 1978.

9. Ramsey R, Evatt BL: Rapid assay for von Willebrand factor activity using formalin-fixed platelets and microfiltration technic. Am J Clin Path, 72:996, 1979.

10. Zimmerman TS, Abildgaard CF, Meyer D: The factor VIII abnormalities in severe von Willebrand's disease. N Eng J Med, 301:1307, 1979.

11. Allain JP, Cooper HA Wagner RH, et al: Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor. J Lab Clin Med, 85:318, 1975.

12. Boyum A: Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Inves. 21: Suppl 97:77 1968.

13. English D, Anderson BR: Single-step separation of red blood cell, granulocytes and mononuclear leukocytes on discontinuous density gradients of ficoll-hypaque. J.Immunol Methods 5:249, 1984.

14. Mackin WW, Rakich M, Marshall CL: Inhibition of rat neutrophil functional responses by azapropazone, an anti-gout drug. Biochem. Pharm. 35: 917, 1986.

15. Hammerschmidt DE, Et al: Lymphocyte Aggregation in Response to Adrenergic Stimulation. Blood 71: 1470, 1988.

16. Hammerschmidt DE, White JG, Craddock PR. Jacob HS: Cortocosteroids inhibit complement-induced granulocyte aggregation. A possible mechanism for their efficacy in shock states. J. Clin Invest 63:789, 1979.

17. Jacob HS, Hammerschmidt DE: Complement-induced granulocyte aggregation. Importance in myocardial infarction and shock lung. JAMA 245:2013, 1981.

18. O'Flaherty JT, Ward PA: Leukocyte Aggregation Induced by Chemotactic Factors. A Review .Inflammation: 3, 177, 1978.

19. Craddock PR, Hammerschmidt DE, Dalmasso AP, White JG, Jacob HS: Complement (C5a)-induced granulocyte aggregation in-vitro., J. Clin. Invest 60, 1977.

20. Craddock PR, Hammerschmidt DE, Moldow O, Jacob HS: Granulocyte aggregation as a manifestation of membrane interaction with complements possible role in leukocyte margination microvascular occlusion and endothelial damage. Semin. Hematol. 16, 140, 1979.Craddock PR, Fehr J, Dalmasso AP, Brigham KL, Jacob HS: Hemodialysis leukopenia. Pulmonary vascular leukostasis resulting from complement activation of dialyzer cellophane membranes. J. Clin. Invest. 59:879, 1977.



21. Hammerschmidt DE, Bowers TK. Lammi-Keefe CJ, Jacob HS, Craddock PR: Granulocyte Aggregometry: A sensitive technique for the detection of C5a and Complement Activation. Blood 55:898, 1980.

Platelet Aggregation Profiler and GraphPad Prism®

1. Fisher, J, Holland, TK, Pescador, R, Porta, R, and Ferro, L. Pharmacokinetics, Absoiption, Distribution and Distribution of [125I]-oligotide Following Intravenous or Oral Administration in the Rat. Thrombosis Res. Vol 85, Issue 6, Pgs 445 – 522. 15 Mar 97

2. Favaloro, EJ, Bonar, R, Sioufi, J, Hertzberg, M, Street, A, Lloyd, J, and Mardsen, K. Laboratory Diagnosis of von Willebrand Disorder; Current Practice in the Southern Hemisphere. Am J Clin Pathol 119(6): 882-893, 2003.

3. Joutsi-Korhonan, L, Smethurst, PA, Rankin, A, Gray, E, IJsseldijk, M, Onley, CM, Watkins, NA, Williamson, LM, Goodall, AH, deGroot, PG, Famdale, RW, and Ouwehand, WH. The low-frequency allele of the platelet collagen signaling receptor glycoprotein VI is associated with reduced functional responses and expression. Blood. Vol 1, No 11. pgs 4372-4379. 1 Jun 03.

4. Smith, SMG, Judge, HM, Peters, G, and Storey, RF. Multiple antiplatelet effects of Clopidogrel are not modulated by statin type in patients undergoing percutaneous coronarty intervention. Platelets. 15(8), pgs 465-474. 12/04.

5. Thompson, RD, Wakelin,MW, Larbi, KY, Dewar, A, Asimakopoulos, G, Horton, MA, Nakada, MT, and Nourshargh, S. Divergent Effects of Platelet-Endothelial Cell Adhesion Molecule-1 and β3 Integrin Blockade on Leukocyte Transmission in Vivo. J Immun. 165: 426-434. 2000.

6. Wang, X, Dorsam, RT, Lauver, A, Wang, H, Barbera, FA, Gibbs, S, Varon, D, Savion, N, Friedman, SM, and Feuerstein, GZ. Comparative Analysis of Various Platelet Glycoproteins IIb/IIIa Antagonists on Shear-Induced Platelet Activation and Adhesion. J Pharmacol & Exp Ther. Vol 303, No 3. pgs 1114-1120. 2002.

Leukocyte Aggregation References

1. Rotstein, R, Mardi, T, Justo, D, Berliner, S and Shapira, I. The Leukocyte Adhesiveness/Aggregation Test [LAAT] Converys Information of Biological Relevance and is not a Result of Chance Collision. Medical Hypotheses. 2002. Sep; 59 (3): 341.

2. Massey, DH. Acute and Chronic Inflammation. Powerpoint presentation. Department of Surgical Pathology, Medical College of Virginia, 1997.

3. McIntyre, TM, Prescott, SM, Weyrich, AS, and Zimmerman, GA. Cell-Cell Interactions: Leukocyte-Endothelial Interations. Current Opinion in Hematology. 2003.10:150-158.

4. Sadovsky, R. CSF Leukocyte Aggregation to Indentify Bacterial Meningitis. Am J Fam Med. 1 May 2000.

5. O‟Flaherty, JT and Ward, PA. Leukocyte Aggregation Induced by Chemotatic Factors: A Review. Inflammation. 1978. Vol 3, No 2. pg 177.

Additional Information

1. Sirridge MS: Laboratory Evaluation of Hemostasis, 2nd Edition, Lea and Ferbiger, Philadelphia, 1974.

2. Triplett DA, Harms CS, Newhouse P, Clark C: Platelet Function. Laboratory Evaluation and Clinical Application, ASCP, Chicago, 1978.

3. Williams WJ, Beutler E, Erslev AJ, Rundles RW: Hematology. McGraw-Hill, New York, 1977. 4. Caprino L, Rossi EC: Platelet Aggregation and Drugs. Proceedings of the Serono Symposia,

Vol. 3, 1974. 5. Day HJ, Holmsen H, Zucker MB: Platelet Function Testing. DHEW Publication 78-1087, 1976. 6. Coleman RW, Hirsh J, Marder Y, Salzman EW: Hemostasis and Thrombosis. Basic Principles

and Clinical Practice. J.B. Lippincott Co., Philadelphia, 1982.



Standards National Standards, USA

1. Centers for Disease Control and Prevention. Guidelines for Isolation Precautions in Hospitals. Centers for Disease Control and Prevention. 1996; Vol 17; 1:53 - 80.

2. National Committee for Clinical Laboratory Standards. NCCLS, Wayne, PA USA 3. Protection of Laboratory Workers from Occupationally Acquired Infections: Approved

Guideline. NCCLS document M29. 4. Transport and Processing of Blood Specimens Approved Guideline- Second Edition. NCCLS

Document H 18-A2. 5. Assays of von Willebrand Factor Antigen and Ristocetin Cofactor Activity: Approved

Guideline. HS1-A Vol 22 No 20. Sep'02. 6. The PAP-8E is designed to be in compliance with UL 61010-1- Safety requirements for

electrical equipment for measurement, control, and laboratory use,- Part 1: General requirements

International Standards

1. European Union Directive 98/79/EC In Vitro Diagnostic Medical Devices 2. European Union Electromagnetic Compatibility Directive 89/336/EEC 3. European Union Low Voltage Directive 73/23/EEC. 4. European Standards: EN 61010-1:2001, Safety requirements for electrical equipment for

measurement, control, and laboratory use, EN 61010-2-101:2002 Particular requirements for in vitro diagnostic (IVD) medical equipment.

5. The PAP-8E is designed to be in compliance with CAN/CSA C22.2 No. 61010-1- Safety requirements for electrical equipment for measurement, control, and laboratory use,- Part 1: General requirements

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