Chrono-log 490 4+4 User manual

INSTRUCTION MANUAL

FOR THE

CHRONO-LOG

PLATELET AGGREGOMETER

MODEL 490 4+4

490 4+ Four Channel Optical Aggregometer

490 4+4 Eight Channel Optical Aggregometer

●2 West Park Road ●Havertown PA 19083

●TEL 800-247-6665 ●610-853-1130

FOR IN

VITRO DIAGNOSTIC USE

For Measuring platelet aggregation in platelet rich plasma

Document # 49044IM1

Revision 7.5 1

Dated February 16, 017

TABLE OF CONTENTS

Manufacturer/EC Rep Details Instrument Function Verification 7

Power Requirements / Fuses Operating Instructions 7

Warnings Specimen Collection 7

Ventilation Equipment & Materials 8

Hazards Preparation 8

Misuse Calibration 11

Summary and Explanation Quality Control 11

Overview of Hemostasis 3 Procedure Stepwise 11

Abnormalities of the hemostatic

mechanism

3 Calculations 13

Platelet function 3 Reporting Results 13

Formation of the primary hemostatic

plug

3 Interpretation 16

Qualitative platelet function disorders 3 Procedure Notes 16

Principles of Platelet Aggregation 4 Limitations of the Procedure 16

Instrument Specifications 5 Service/Preventive Maintenance 16

Intended Use 5 Cleaning System Components 16

Operating Specifications 5 Self-Calibration of Optical Circuits 17

Environmental Specifications 5 Troubleshooting Guide 19

General 5 Assistance and Distributor Information 19

Instrument Controls 6 Bibliography 0

Instrument Calibration Appendix A 1

Installation 6 Appendix B

Document # 49044IM1

Revision 7.5

Dated February 16, 017

Manufacturer: Chrono-log Corp.

West Park Road

Havertown, PA 19083

USA

Phone: 1-610-853-1130

Email: chronolog@chronolog.com

EC Rep: BioTop Medical

Poortgebouw Noord,

Rijnsburgerweg 10

333 AA Leiden

Netherlands

Phone 31 71 5 8 01 1

Fax 31 71 5 8 01 15

Power requirements: 115 or 30VAC; 50 or 60 Hz 60 watts max.

The main supply voltage fluctuations are

not to exceed 10% of the nominal supply

voltage.

Fuse: For 115 VAC: 630 mA, 50V; Time lag,

5x 0mm or equivalent;

For 30 VAC: 315 mA, 50V; Time Lag,

5x 0mm or equivalent.

Warnings:

WARNING- FIRE HAZARD For continued protection replace only

with the same type and rating of fuse.

Caution – This Instrument is intended for indoor use only. Failure

to operate in a protected environment in accordance with

instructions may present a shock hazard.

Warning: Patient specimens should be handled as if they contain

infectious materials, in accordance with national guidelines for

Biosafety/Hazard Group . In the United States, the universal

precautions recommended by the Department of Labor,

Occupational Safety and Health Administration apply.

(Occupational Exposure to Blood Borne Pathogens; final rule ( 9

CFR 1910, 1030) FEDERAL REGISTER.

This device complies with part 15 of the FCC Rules. Operation is

subject to the following two conditions: (1) This device may not

cause harmful interference, and ( ) this device must accept any

interference received, including interference that may cause

undesired operation.

NOTE: This equipment has been tested and found to comply with

the limits for a Class B digital device, pursuant to part 15 of the

FCC Rules. These limits are designed to provide reasonable

protection against harmful interference in a residential

installation. This equipment generates, uses and can radiate radio

frequency energy and, if not installed and used in accordance with

the instructions, may cause harmful interference to radio

communications. However, there is no guarantee that

interference will not occur in a particular installation. If this

equipment does cause harmful interference to radio or television

reception, which can be determined by turning the equipment off

and on, the user is encouraged to try to correct the interference

by one or more of the following measures:

•Reorient or relocate the receiving antenna.

•Increase the separation between the equipment and

receiver.

•Connect the equipment into an outlet on a circuit different

from that to which the receiver is connected.

•Consult the dealer or an experienced radio/TV technician for

help.

Caution: Federal law restricts this device to sale by or on the order

of a physician for measuring platelet aggregation in platelet rich

plasma.

VENTILATI N

To ensure that the system has adequate ventilation, please

adhere to the following instructions carefully:

1. Do not place Aggregometer within a closed in wall or on top

of cloth material which can act as insulation.

. Do not place Aggregometer where it will receive direct

sunlight.

3. Do not place Aggregometer next to a heat source of any kind

including heating vents.

4. Make sure that all openings on the Aggregometer remain

unobstructed, especially the fan guard on the back of the

unit.

HAZARDS

There are no hazards associated specifically with use of the

Aggregometer.

However, normal precautions, which apply to the handling of

blood, should be observed in handling the samples.

Non-factory authorized service personnel should not remove the

instrument cover.

MISUSE

To prevent electrical shock, the Aggregometer and peripheral

power cables must be plugged into a properly grounded power

source. Do not use adaptor plugs or remove grounding prong

from cable. If an extension cable must be used, use a three wire

cable with properly grounded plugs.

Do not spill liquids or food on your Aggregometer.

Do not put objects or materials other than cuvettes in the heater

block wells.

Do not push any object through the fan guard.

SUMMARY AND EXPLANATI N

When a blood vessel is damaged, platelets adhere to the wound

edges, aggregate, synthesize prostaglandins and release

serotonin, ADP and ATP.8 Prostaglandin synthesis and release

products cause further aggregation. The coagulation cascade is

initiated, thrombin generated, fibrin formed and the platelet plug

anchored to the damaged vessel.15

Defects in platelet function due to lack of a cell membrane

glycoprotein, cytoplasmic storage granules, platelet enzymes or a

plasma factor often result in excessive bleeding after trauma,

frequent bruising or nosebleeds, or excessive menstrual blood

loss. Yet patients who present with one or more of these clinical

signs are much more likely to be tested initially for coagulation

abnormalities than for platelet dysfunction. Further, surgical

Document # 49044IM1

Revision 7.5 3

Dated February 16, 017

patients are not routinely screened for platelet defects despite

the fact that many cases of platelet dysfunction are discovered

only after excessive or recurrent post-surgical bleeding.

In 196 , Born described the aggregation of platelets by ADP and

modified a colorimeter to monitor continuously this aggregation

in platelet rich plasma. These modifications included incubation

at 37°C, stirring and recording the change in light transmission

over time on a pen recorder.1

VERVIEW F HEM STASIS

Hemostasis - from the Greek word for blood; and, the Greek word

meaning standing - is a complex, delicately balanced system of

interactions that keeps blood circulating as a fluid through the

blood vessels. A simplified representation of the process is shown

below.

There are three elements of the hemostatic mechanism:

• Blood vessels

• Plasma proteins known as coagulation factors

• Platelets

A defect or abnormality in the interactive process of hemostasis

may lead to abnormal bleeding or to inappropriate clotting.

Abnormalities of the hemostatic mechanism

Blood vessels - abnormalities of the endothelial cell lining of the

blood vessels can arise from injury, inflammation, infection or

atherosclerosis. The endothelium may lose its normal anti-

thrombotic properties and begin to synthesize and release

compounds that promote thrombosis.9 Vascular disorders that

result in abnormal blood vessel support structures may contribute

to disordered hemostasis.

Coagulation proteins - abnormal levels of coagulation factor

levels or defective function of the coagulation factors can disturb

hemostatic balance. These defects can be hereditary or they can

be acquired, through a pathological process, for example.

Platelets - disorders of the platelet component of hemostasis can

arise from abnormal numbers of platelets (quantitative defects)

or functional impairment (qualitative defects). Defects in the

platelet component of the hemostatic system can also be

acquired or inherited.

Platelet function

The platelets (or thrombocytes) are small, discoid cells that

circulate in the blood along with red cells and leukocytes. The

cell's nucleus is lost during the maturation process. Platelets have

cytoplasmic granules known as dense granules and alpha

granules. These contain compounds that amplify the platelet

response if they are exocytosed (released, secreted).

The main function of platelets is the maintenance of blood vessel

integrity by prevention of red cell migration through the vessel

wall. Platelets also prevent vascular leakage by plugging any sites

of damage or injury. In the case of damage or injury that exposes

the subendothelium and/or basement membrane, circulating

platelets are recruited to the site to form a platelet aggregate.

This physiologic reaction is known as formation of the primary

hemostatic plug.15

Formation of the primary hemostatic plug

Platelets adhere and aggregate at any site of sub endothelial

exposure. 15 Exposure of the subendothelium results in the

unmasking of the structural protein collagen. Collagen is a

platelet stimulus.

Platelets adhere to the now exposed collagen fibrils. The platelets

change shape and pseudopods are formed. The shape change

and pseduopods result in closer contact with other individual

platelets. Granule contents are exocytosed. More platelets are

recruited and stimulated to undergo shape change, pseudopod

formation and granule release. This aggregated mass physically

prevents leakage at the site. 16

Von Willebrand factor (found in the alpha granules of the platelet

and circulating in the blood in association with Factor VIII) is

classified as an adhesive protein. It interacts with a binding site

on the platelet membrane and acts to strengthen the platelets'

adherence to the endothelium. 17

Qualitative platelet function disorders

•Defective adhesion

oVon Willebrand Disease -quantitative or qualitative

defect in plasma von Willebrand Factor (vWF)

oBernard-Soulier Syndrome (BSS) -lack of platelet

membrane glycoprotein Ib (GPIb)

•Defective aggregation

oAfibrinogenemia - deficiency of plasma fibrinogen

oGlanzmann's Thrombasthenia -defective or deficient

platelet membrane glycoprotein IIb-IIIa (GP IIb-IIIa)

•Defective platelet granule secretion

oStorage pool deficiency (SPD) -deficiency in dense

granule contents (ADP, ATP and/or serotonin)

oGray platelet syndrome -deficiency in alpha granule

contents [platelet factor 4 (PF4), platelet vWF,

thrombospondin, and platelet derived growth factor

(PDGF)]

oArachidonic acid metabolic pathway abnormalities

Document # 49044IM1

Revision 7.5 4

Dated February 16, 017

Defective liberation of arachidonic acid from the

platelet membrane

Deficiency of the enzyme cyclo-oxygenase

Deficiency of the enzyme thromboxane

synthetase

oSecretion defects with normal granule contents and

normal Arachidonic Acid metabolic pathway

oDefective cytosolic calcium mobilization

oDefective early responses -myosin light chain

phosphorylation; phosphatidylinositol metabolism

oDefective or Blocked Receptors to Specific Agonists (in

addition to BSS and thrombasthenia)

Defective response to epinephrine -

myeloproliferative disorders (MPD)

Defective response to collagen

Defective response to U46619 (the stable analog

of thromboxane A )

oDefective Platelet Coagulant Activities - the platelet

contribution to and interaction with the coagulation

scheme.

•Miscellaneous defects

oCongenital

May-Heggelin Anomaly

Down's Syndrome

Thrombocytopenia with absent radii (TAR

syndrome)

oAcquired

Uremia

Extracorporeal circulation

PRINCIPLES F PLATELET AGGREGATI N TESTS

Platelets are known to aggregate under a variety of conditions

and in the presence of a number of different reagents. "Platelet

aggregation" is a term used to denote the adherence of one

platelet to another. The phenomenon can be induced by adding

aggregating agents to platelet-rich plasma. Platelet aggregation

depends on the presence of calcium, fibrinogen and one or more

plasmatic factors, and an aggregating agent. Platelet aggregation

will vary with different aggregating agents and concentrations.

For optical aggregometry, ADP, epinephrine, collagen and

ristocetin are used extensively for screening purposes and provide

the most immediate information for basic diagnostic

considerations.18

The selection of these reagents has some basis in theory. Both

ADP and epinephrine (adrenaline) are contained within the

platelet in storage organelles and are released from the platelet

during formation of the primary hemostatic plug and may thereby

induce further platelet aggregation. 3 Consequently, in-vitro

platelet response to these reagents has proven to be of help in

determining the nature of a patient's bleeding disorder.

Collagen, on the other hand, is not contained in the platelet but is

found in the supporting connective tissue of the blood vasculature

and is considered to be the first aggregating or pro-coagulant

factor that the platelet encounters following vascular trauma.

Hence, in-vitro study of the platelet response to collagen has

assumed considerable importance.4,1

Other reagents such as thrombin3, the calcium ionophore A 3187,

arachidonic acid, ristocetin, Bovine Factor VIII, and serotonin have

also been used to study platelet response for more specific

purposes.

Platelet aggregation is the most useful in-vitro test of platelet

function presently available. It is a diagnostic tool, which can

provide insight that is difficult or impossible to obtain by other

techniques, thus aiding in patient diagnosis and proper selection

of treatment or therapy. Experience with this technique has

delineated a spectrum of inherited and acquired platelet

dysfunctional states.

Platelet aggregation is clinically significant in the detection and

diagnosis of acquired or congenital qualitative platelet defects.

The platelet's ability or inability to respond to particular

aggregating reagents is the basis for differentiating platelet

dysfunctions6 as shown in the table below:

AGGREGATI N STUDIES N SELECTED PLATELET FUNCTI N

DEFECTS

DEFECT

Platelet

Aggregation

By ADP

Platelet

Aggregation

by Collagen

Platelet

Aggregation

Ristocetin

Thrombasthenia Decreased Decreased Normal

Thrombopathia or

Thrombocytopathy

Normal

(1st phase) Decreased Normal

von Willebrand's

Disease Normal Normal Abnormal

Non

steroidal,

Anti-inflammatory

drugs

Normal

(1st phase) Decreased Abnormal

ptical Aggregation Tests

In-vitro platelet aggregation is an effort to characterize the in-vivo

ability of the platelets to form the primary hemostatic plug.

Platelets in a suspension of plasma are isolated from an anti-

coagulated blood sample by a relatively low centrifugal force

centrifugation. This material is known as platelet rich plasma

(PRP). Platelet poor plasma (PPP) is prepared by centrifuging the

blood sample at a relatively high force.

The Born type aggregometer or optical aggregometer is a fixed

wavelength spectrophotometer with a sample chamber (or

chambers) heated to 37°C. Provision is made for stirring of the

sample because platelet to platelet contact is necessary to the

determination of in vitro platelet aggregation.

The CHRONO-LOG® sample chambers are designed so that a

beam of infra-red light shines through two cuvettes, one

containing PRP (the sample) and one containing PPP (the

reference). Silicon photodiodes detect the light able to pass

through the samples: PRP is arbitrarily considered to be 0% light

transmission or 0% aggregation; PPP is considered to be 100%

light transmission or 100% aggregation. The difference in light

transmission outputs from the photodiodes is transferred to

recording devices.

The twin well, dual infra-red beam design ensures reproducibility.

Each channel has a separate well for the test (PRP or washed

platelets; O% light transmission) and corresponding reference

(PPP or buffer; 100% light transmission) samples. A SELECT/SET

switch allows the use of either a separate or a common reference

sample. The optical aggregation output is proportional to the

continuously measured difference in light transmission between

the test and reference samples. Pressing a single pushbutton sets

the 0% and 100% light transmission baselines. With the sensitivity

of the twin-well dual-beam detection system, a count difference

of only 75 x 109/L between the test and reference sample is

Document # 49044IM1

Revision 7.5 5

Dated February 16, 017

needed for testing. If the light transmission difference between

the test and reference samples is insufficient for accurate testing,

the aggregation output cycles continuously between the baselines

to warn the operator and a Range Error will be indicated on the

front panel.

When a stimulus is added to the cuvette containing PRP and the

platelets respond, changes in light transmission occur and are

recorded over time by the recording device.

PRP, which is turbid, is stirred in a test cuvette maintained at

37° C. The light transmittance through this turbid sample is

measured relative to the PPP blank. When the agonist is added,

the platelets will form increasingly larger aggregates and the PRP

will begin to clear, allowing more light to pass through. This

increase in light transmittance is directly proportional to the

amount of aggregation and is amplified and recorded as a signal

on chart paper or digitized into a computer using AGGRO/LINK®

Opti8TM Software.

When the platelets undergo shape change in response to a

stimulus (agonist; aggregating agent), their larger size allows less

light to pass through the PRP: this is recorded as less light

transmission through the sample relative to the PPP. If the dose

of aggregating agent is strong enough to cause the platelets to

adhere to each other and form aggregates, more light is able to

pass through the PRP sample. The change in light transmission

recorded, over time, shows a trend towards the platelet poor

plasma, or 100% light transmission. In-vitro aggregation

recordings are characterized by their appearances:

•shape change

•a first wave of aggregation (primary aggregation) that may

reverse and return towards the PRP baseline

•Irreversible second wave aggregation that occurs when the

platelets’ secreted granule contents become the stimulus

and cause additional aggregation.

Aggregation curves are also characterized by:

•the maximum amount of change in light transmission caused

by the agonist (percent aggregation)

•the slope, or rate of aggregation, reported in % of

aggregation per minute.

Multiple aggregating agents and concentrations are usually used

to stimulate the platelets. Different aggregating agents stimulate

different pathways of activation in the platelets: either binding

sites or metabolic pathways. Different concentrations of agonists

are used to elicit a family of curves (dose response curves).

The pattern of responses to these test panels is compared to

established normal response patterns and established abnormal

response patterns. This information is considered to relate to the

platelet function component of hemostasis.

CHR N -L G® M DEL 490 4+, 490 4+4

AGGREGATI N SYSTEM INSTRUMENT

SPECIFICATI NS

Intended Use

The CHRONO-LOG® Model 490 4+4 Aggregometer is intended for

use for in-vitro diagnostic use for measuring Platelet Aggregation

in Platelet Rich Plasma. This device is intended to be used in a

clinical laboratory environment by laboratory technicians. For use

only with light transmission aggregometry assays cleared for use

with the CHRONO-LOG® Platelet Aggregometry systems.

perating Specifications

•Display - Liquid Crystal Display (LCD) unit with a 4 character

by -line capability. The LCD displays the actual

temperature in degrees Celsius, stirring in RPM’s,

PPP/reference Select, and calibration mode and warning

messages.

•Heater Block - Electronically controlled heater block can be

set between 35.0°C and 39.0°C in 0.1°C steps. Both the

temperature setting and actual temperature of heater block

are displayed on front panel. Operation is prevented while

temperature is outside ±0. °C of the temperature setting.

•Stirrer - Nine stirring speeds from 400 RPM to 1 00 RPM in

100 RPM steps and a stirrer stopped position. The selected

stirring speed is displayed on the front panel. Stirrer speed

accuracy is better than 0.01%. There is a feature that

prevents operation if stirrer is not within ± 10 RPM of the

selected setting.

•Optical/Aggregation/Turbidometric - Automatic baseline

setting from a front panel push button with an over-range

detection to prevent operation if baseline does not set.

•Output resistance for analog output - Less than 10,000

ohms.

•Computer Interface – USB

•Power requirements - 115 or 30VAC; 50 or 60 Hz 60 watts

max. The main supply voltage fluctuations are not to exceed

10% of the nominal supply voltage.

•Fuse – For 115 VAC: 630 mA, 50V; Time lag, 5x 0mm or

equivalent; For 30 VAC: 315 mA 50V, Time Lag, 5x 0mm

or equivalent.

•For continued protection only the correct stated fuse values

may be used. Failure to use the correct value may result in a

hazard.

•DIN input connector for an eight (8) channel configuration.

Environmental Specifications

Caution – This Instrument is intended for indoor use only.

Failure to operate in a protected environment in accordance

with instructions may present a shock hazard. Use of the

instrument in a manner contrary to manufacturer guidelines

may result in protection impairment.

•Operating Temperature: 15° to 30° C (60° to 86° F)

•Storage Temperature: - 0° to 60° C (-4° to 140° F)

•Relative Humidity: 5% to 85% (Noncondensing)

•Operating Altitude: -16 to 000 M (-50 to 6,560 ft.)

•Storage Altitude: -16 to 10,600 M (-50 to 35,000 ft.)

General

•Four (4) Channels [One, 4-Channel Module] or Eight (8)

Channels [Two, 4-Channel Modules] with connecting cable

•Sample Volume - 500 µL or 50 µL [spacer not required for

micro-volume testing]

•Cuvettes - P/N 31

•Stir Bars - reusable teflon coated P/N 313, disposable P/N

311

•Incubation Wells – two ( ) wells for each channel for P/N

31 cuvettes at 36.5° ± 1.0°C.

Document # 49044IM1

Revision 7.5 6

Dated February 16, 017

•Dimensions - 14" (35.5cm) wide, 8.5" ( 1.6cm) high, 15"

(38cm) deep [per Module].

•Weight – 19.3 lbs. (8.75kg) [per Module]

•Output

1. Analog: Connectors on rear chassis marked 1, , 3 and

4 [Module 1] and 5, 6, 7 and 8 [Module ], for interface

to a strip chart recorder. Normal operating voltages

are in the Millivolt range.

. USB: Internal AGGRO/LINK interface for use with a

computer. This option includes AGGRO/LINK® Opti8TM

and vW CoFactor Opti8TM software.

3. Interconnecting: To connect two, 4-channel modules

for eight (8) channel configuration.

•Pollution Degree

•Installation Category II

•Protection Class 1

•Equipment Mobility – Desktop

•Continuous Operation - Continuous

Recommended Optional Recorder(s) - CHRONO-LOG® Model 709

Dual Pen (Quantity 4 Recorders required). Other recorders must

have 1 megohm minimum input impedance and 100 mV range.

The minimum computer requirements for the AGGRO/LINK® are

as follows:

•Windows-based PC with a Pentium/compatible processor

running at 133 MHz or higher.

•3 Meg of system memory.

•Windows 7 or later operating system.

•Super VGA or Higher Monitor (17 inch or larger).

•Screen resolution of 800x600 or better.

•Minimum Hard Disk Space required for installation: 3MB.

•Additional Hard Disk Space for storage of tests.

•Mouse or compatible pointing device.

Instrument Controls

•Power ON/OFF switch - powers instrument on and off.

•BASELINE Pushbutton(s), one per channel - zeros instrument

output when held down, allowing the recorder baseline to

be set using the Pen Recorder Zero Control. The Pen

Recorder is set on the right of the chart. Pressing and

releasing, sets optical aggregation gain.

•SELECT Pushbutton(s) – allows for toggling between the

Stirring Speed, Temperature and PPP/Reference Setting,

using the SET pushbutton switch. One SELECT and SET

pushbutton controls two channels. For example, Channel 1

and , Channels 3 and 4, Channels 5 and 6 and Channels 7

and 8 are set in pairs.

•Stirring Range: 400 to 1 00 RPM in 100 RPM increments.

•Temperature Range: 35.0°C to 39.0°C in 0.1°C increments.

•PPP/Reference Setting … each pair of channels can be set to

reference a PPP sample in the well of that channel or can be

set to reference the PPP sample in the well of Channel 1.

•SET Pushbutton(s) - sets the Stirring Speed, Temperature

and PPP/Reference depending on which feature is selected.

•Each time the SET button is pushed, the selected function is

incremented until it reaches the maximum setting.

•Calibration Switch – located on the front of the instrument

and is key-activated.

Instrument Calibration

The optical technique is calibrated automatically during the

testing procedure.

INSTALLATI N

NOTE: For a four channel instru ent, approxi ately 1.5 sq. ft.

(0.139 sq. .) 14 x 15” of bench space is required. For an eight

channel syste , approxi ately 3 sq. ft. (0.278 sq. .) of bench

space is required.

NOTE: Software packages cannot be run si ultaneously on one

Aggregation syste . Be sure to close the AGGR /LINK® pti8TM

Software prior to opening the vW CoFactor Opti8 TM Software

and vice versa.

1. Unpack the CHRONO-LOG® Aggregometer and supplies from

their shipping containers.

. Check that the items received match the Packing List.

3. Check that the Instruments were not damaged in shipping.

4. Store the reagents as specified on each label.

5. Place the Aggregometer and Computer in a clean dry area

on a bench top at a height of 8-35’’.

6. Check that the voltage select switch on the Aggregometer is

set to the proper voltage. If the voltage select switch is

changed, then the fuse also needs to be changed to

maintain protection. Plug the power cords, as appropriate,

into a 115 or 0 Volts AC filtered power strip and then plug

the power strip into a grounded power outlet.

7. Turn power ON for the Aggregometer(s).

8. Computer Connections: (See Connection Charts in Appendix

B ) If the computer was purchased through Chrono-log the

software was pre-installed, proceed to step 9.

a. Turn power ON for the computer.

b. Place the Chrono-log supplied AGGRO/LINK® Opti8TM

Software Installation CD in the drive and run the setup

file “CDM v*.*WHQL Certified” located in the folder

named USB Drivers.

c. Connect the USB cable to the Aggregometer. Windows

should complete setting up the USB drivers on the

computer.

NOTE: If the steps above are not followed in sequence, the USB

driver ay not install co pletely. This will be indicated by FTDI

or USB Serial port devices showing up in Device Manager under

the Other Devices Category. Right Clicking these and selecting

Update driver ay co plete the installation. Please consult

your IT depart ent or Chrono-log service depart ent for

assistance.

d. The AGGRO/LINK® Opti8 TM and vW CoFactor pti8 TM

Software packages are in separate folders on the CD.

Double click each Setup.exe file to start installation of

each software and follow on-screen instructions to

complete the installation.

e. It is recommended that the software be run with

desktop composition disabled (a setting in the

Windows Operating System) and as Administrator for

all Windows user accounts. Insufficient privileges may

result in run time errors. Please consult your IT staff if

technical support is required.

9. Start the AGGRO/LINK® Opti8 TM Software on the Computer.

During the software start-up, the computer will test the

communication. The Program will respond with

AGGREGOMETER READY on the lower right side of the status

bar when communication is established.

a. If communication is not established, the Program will

continue searching and respond with AGGREGOMETER

NOT CONNECTED on the lower right side of the status

bar if connection cannot be established.

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Dated February 16, 017

b. Check the cable and make sure the Aggregometer has

power.

c. To retry, select “Aggregometer” from the ribbon, then

“Connect” from the dropdown menu.

d. Once AGGREGOMETER READY appears on the lower

right side of the status bar, close the AGGRO/LINK®

Opti8 TM Software.

10. Start the vW CoFactor pti8 TM Software on the Computer.

During the software start-up, the computer will test the

communication. The Program will respond with

AGGREGOMETER READY on the lower right side of the status

bar when communication is established.

a. If communication is not established, then the Program

will continue searching and respond with A/L NOT

CONNECTED on the lower right side of the status bar if

connection cannot be established.

b. Check the cable and make sure the Aggregometer has

power.

c. To retry, select “Aggregometer” from the ribbon, then

“Connect” from the dropdown menu.

d. Once AGGREGOMETER READY appears on the lower

right side of the status bar, close the vW CoFactor

Opti8TM Software.

11. The LCD display on each channel will show a temperature

reading. This reading will settle at the desired temperature

in approximately 15 minutes.

1 . The System is now installed and ready for use.

INSTRUMENT FUNCTI N VERIFICATI N

1. Temperature Check – set to 37° in all channels.

. Stirring - take a cuvette with stir bar and place in each

reaction well to observe it spinning.

3. Optical Circuit Function - insert a BLACK cuvette from P/N

3 Calibration Kit into the reaction well for PRP. Insert a

water cuvette from P/N 3 calibration kit into the reaction

well for PPP. Press and HOLD the SET BASELINE button and

observe the tracing at the 100% light transmission baseline.

Release the SET BASELINE button and observe a return to

the PRP 0% baseline.

4. Replace the BLACK cuvette with the other water cuvette

from P/N 3 calibration kit and observe the tracing near the

100% light transmission baseline.

PERATING INSTRUCTI NS

For Model 490 4+/490 4+4, install the AGGRO/LINK® Opti8TM

Software as described previously under INSTALLATION section of

this manual.

For Model 490 4+DR/490 4+4DR the software for running the

AGGRO/LINK® Opti8TM programs were installed at the factory.

Before attempting to run aggregation testing, read the Software

section on page 10 and PROCEDURE – STEPWISE on page 11 of

this manual to learn the operation of AGGRO/LINK® Opti8TM

software.

SPECIMEN C LLECTI N:

Preparation and Handling of Blood Specimens:

It is extremely important that care be taken in the collection,

handling, and preparation of the patient's blood specimen.

Warning: Patient specimens should be handled as if they contain

infectious materials, in accordance with national guidelines for

Biosafety/Hazard Group . In the United States, the universal

precautions recommended by the Department of Labor,

Occupational Safety and Health Administration apply.

(Occupational Exposure to Blood Borne Pathogens; final rule ( 9

CFR 1910, 1030) FEDERAL REGISTER.

Essential precautions can be summarized as follows:

•Do not pipette by mouth.

•Wear disposable gloves during all specimen and assay

manipulations.

•Avoid use of sharp-pointed or glass liquid handling devices,

which may puncture skin.

•Do not smoke, eat, or drink in the laboratory area.

•Avoid splashing any liquid specimens and reagents and the

formation of aerosols.

•Wash hands thoroughly on completion of a manipulation.

Patient Preparation:

Subjects for Optical platelet aggregation tests should be resting,

fasting and non-smoking. Subjects should avoid taking any

prescription or over-the-counter medications known to affect

platelet function for ten (10) days to two ( ) weeks prior to the

test.

A partial list of medications with known antiplatelet effects

follow: 19

•C X-1 Inhibitors (Acetylsalicylic acid)

Aspirin and all proprietary or over-the-counter (OTC)

preparations containing acetylsalicylic acid

•C X-1 and C X-2 Inhibitors (Nonsteroidal anti-

inflammatory drugs [NSAIDs])

Ibuprofen, Indomethacin, naproxen, Mefenamic acid

•C X-2 Inhibitors (Coxibs)

Celecoxib

•Inhibitors of Platelet Receptors

Abciximab (αIIbβ3), Clopidogrel (P Y1 ), Prasugral (P Y1 )

•RGD Peptomimetics

Eptifibatide, Tirofiban

•Phosphodiesterase Inhibitors

Dipyridamole, Cilostazole

•Anticoagulants

Heparin, Warfarin, Direct Thrombin Inhibitors (lepirudin,

argatroban, bivalirudin)

•Cardiovascular Agents

β-adrenergic blockers (propranolol), Vasodilators

(nitroprusside, nitroclygerin), Diuretics (furosemide),

Calcium channel blockers

•Antimicrobials

β-lactams (penicillin, cephalosporins), Amphotericin

(antifungal), Hydroxychloroquines (antimalarial),

Nitrofurantoin

•Chemotherapeutics Agents

Asparaginase, Plicamycin, Vincristine

•Psychotropics and Anesthetics

Thricyclic antidepressents (imipramine), Phenothiazines

(chlorpromazine), Local and general anesthesia (fluothane)

•Miscellaneous Agents

Clofibrate, Dextrans, Guaifenesin (expectorant),

Radiographic contrast

•Foods/Herbals

Alcohol, Caffeine (methylxanthine), Garlic, Onion, Ginger,

Fish Oil, Vitamins C and E.

Document # 49044IM1

Revision 7.5 8

Dated February 16, 017

Type:

For testing with 500 µL PRP samples: Five (5) 4.5 mL blue top

evacuated tubes per patient. For testing with 50 µL PRP Samples:

Three (3) 4.5 mL blue top evacuated tubes per patient.

Specimen should be drawn with a minimum of trauma or stasis at

the venipuncture site and anti-coagulated with 3. % or 3.8%

sodium citrate, in the ratio of one (1) part anticoagulant to nine

(9) parts of blood.

An EDTA blood specimen must be collected from the patient for

hematocrit and platelet count. The blood specimen must be

collected using plastic equipment throughout.

Plastic or non-contact surfaced (siliconized) materials should be

used throughout in order to minimize activation of the platelets

during sample preparation.

Handling Conditions:

Testing can start 30 minutes after venipuncture and continue for

about .5 hours after. Specimen should be kept at room

temperature ( 4° to 7°C).

EQUIPMENT AND MATERIALS:

Equipment:

1. CHRONO-LOG® 490 4+ or 490 4+4 Aggregation system with

internal AGGGRO/LINK® Interface.

. Windows®-Compatible Computer and AGGRO/LINK® Opti8TM

software installed.

Materials:

1. P/N 31 Cuvettes

. P/N 311 Disposable Siliconized Stir bars

3. P/N 3 Calibration Kit containing 1 Black Cuvette and

Sealed Water Cuvettes.

4. P/N 331 .5-10 µL Adjustable Pipette

5. P/N 335 Tips for P/N 331

6. P/N 33 10-100 µL Adjustable Pipette

7. P/N 337 Tips for P/N 33

8. P/N 333 100-1000 µL Adjustable Pipette

9. P/N 339 Tips for P/N 333

10. P/N 384 CHRONO-PAR® ADP

11. P/N 385 CHRONO-PAR® Collagen

1 . P/N 390 CHRONO-PAR® Arachidonic Acid

13. P/N 393 CHRONO-PAR® Epinephrine

14. P/N 396 CHRONO-PAR® Ristocetin

15. P/N 397 Saline (0.9%) 15mL

Avoid blood bank saline because it ay be an incorrect

os olality. Cell counter diluents are not suitable because they

contain EDTA, which inhibits platelet aggregation. So e

infusion salines are inappropriate because they contain benzyl

alcohol (or other preservatives). Such preservatives/additives

inhibit platelet function.

16. P/N 398 Purified Water 15mL.

Should be pyrogen free (ATP free) for reconstituting reagents.

Avoid any sterile water for injection that contains benzyl alcohol

or other preservatives/additives because they inhibit platelet

function.

17. 15 mL conical test tube and cap, or similar (per test subject):

for storing the PRP and PPP specimens.

18. Ice bucket: for maintenance of the reagents during the

course of the working day

19. Vortex-type mixer: for mixing the Arachidonic Acid

0. Long-stemmed plastic transfer pipettes to take off PRP and

PPP for placing into 15 mL test tubes.

1. Lintless wipes, such as KimWipes .

Gauze squares are NOT suitable.

PREPARATI N:

1. Aggregometer

a. Turn on the unit and let it heat up for 10-15 minutes or

until the heater block stabilizes at 37°C.

b. Place P/N 311 Stir bars in P/N 31 Cuvettes.

c. Put cuvettes containing stir bar in the incubation wells

to warm up.

d. When the Model 490 4+4 test channels are set to

Reference Channel 1, a single sample of PPP can be

used as the reference sample for all tests run with the

same patient's blood, so the amount of PPP required is

only enough for one sample, about 500 µL.

2. Preparation of Sample:

a. Mix sample by gentle inversion; DO NOT SHAKE.

b. To prepare the platelet rich plasma (PRP):

1) Centrifuge sample at approximately 100-170g for

15 minutes.

) Take off the PRP with a polypropylene transfer

pipette and place into a polypropylene plastic

tube and add cap.

3) Recap the blue top tubes.

NOTE: Per CLSI Docu ent H58-A, the pH of the PRP sa ple can

be preserved as follows:19

•Place PRP in a plastic tube with li ited surface area-to-

volu e ratio (place large volu e of PRP in a s all tube)

•Cap the PRP tube as PRP in uncapped tube undergoes a rise

in pH due to diffusion of CO2 fro plas a

•Avoid frequent ixing/agitation of PRP

•Introduce PRP directly into the tube and don’t allow it to

flow down the sides of the tube.

4) Properly label the tube, include the patient's

name and sample type. Parafilm or cap the top.

Keep at room temperature ( 4°C to 7°C).

c. To prepare the platelet poor plasma (PPP):

1) Place blue top tubes into centrifuge.

) Centrifuge sample at approximately 1500- 400 g

for 0 minutes.

3) Take off the PRP with a polypropylene transfer

pipette and put it into a polypropylene plastic

tube.

4) Properly label the tube, include the patient's

name and sample type. Parafilm or cap the top.

Keep at room temperature ( 4°C to 7°C).

When ready to begin testing, dispense one aliquot per channel of

adjusted or non-adjusted PRP of 500 µL volume into P/N 31

cuvettes with stir bars. Warm for a minimum of 3 minutes. It is

not recommended to incubate a sample beyond 30 minutes.

3. Reagent Preparation

The following reagents are sourced from:

Chrono-log Corp.

W. Park Road

Havertown, PA 19083 USA

Tel: 610-853-1130

Document # 49044IM1

Revision 7.5 9

Dated February 16, 017

a. Water

Catalog No.: 398

Supplied As: Purified Water 15mL

Sterile distilled, bottled water suitable for CHRONO-

PAR® Reagent preparation.

Pyrogen free (ATP free) for reconstituting reagents

and not containing preservatives/additives such as

benzyl alcohol which inhibits platelet function. Do not

use water fro the lab purification syste .

b. Saline

Catalog No.: 397

Supplied As: Saline (0.9%) 15mL

Sterile, physiological, saline for CHRONO-PAR® Reagent

preparation.

Avoid blood bank saline because it ay be an

incorrect os olarity. Cell counter diluents are not

suitable because they contain EDTA, which inhibits

platelet aggregation. Infusion salines are

inappropriate because they contain benzyl alcohol (or

other preservatives). Such preservatives/additives

inhibit platelet function.

c. ADP

Catalog No.: 384

Supplied As: .5 mg of lyophilized preparation of

adenosine diphosphate.

Stock Conc.: 1 mM

Stock Storage: Store frozen at below 0°C

Stock Shelf life: Until expiration date.

Working Conc.: 1 mM

Working Storage: -8°C

Working Shelf life: 8 hours

Reconstitute with 5.0 mL of irrigation grade

physiological saline.

Preparation: Tap vial gently to get contents to the

bottom. Reconstitute with 5 mL of irrigation grade

physiological saline. Allow to sit for 10 minutes with

occasional inversion. Add 5 µL of reagent to 500 µL

sample or .5 µL of reagent to 50 µL sample for a final

concentration of 10 µM. Normal aggregation is seen in

PRP with final concentrations of 5-10 µM.

Stability: The reconstituted ADP reagent can be stored

frozen at -70°C volumes suitable for a days testing for

one year or until expiration date, whichever comes

first.

d. Arachidonic Acid

Catalog No.: 390

Supplied As: Minimum of 10 mg of Arachidonic Acid

with a purity of better than 99%. Albumin contains

100mg of bovine albumin, fraction V powder, 96 to 99%

pure.

Stock Conc.: 50 mM

Stock Storage: Frozen below - 0°C for Arachidonic Acid,

Refrigerate at -8°C for albumin.

Stock Shelf Life: Until expiration date.

Working Conc.: 50 mM

Working Storage: -8°C [in the dark]

Working Shelf Life: 8 hours

Reconstitute with 0.7 mL of the saline-albumin

solution.

Preparation: First tap contents gently to the bottom of

the vial of albumin. Reconstitute the albumin with 1

mL of irrigation grade physiological saline. Allow to sit,

then mix with occasional swirling. Allow 15 to 30

minutes for the albumin to fully absorb the saline

(check visually). The Arachidonic Acid in the vial is an

oily drop which must be shaken or tapped to the

bottom of the vial. Break vial tip with Cap CrackerTM

supplied. Pipette reconstituted albumin into both the

tip and body of the vial in 100 µL aliquots to a total

volume of 700 µL. Make sure any Arachidonic Acid

remaining on the tip or body of the vial is mixed by

rotating the vial as the albumin is added. Repeat a few

times in each section of the vial then vigorously mix

the Arachidonic Acid into the albumin using a transfer

pipette. Combine the suspension from the tip with

that in the body of the vial and continue mixing until

the Arachidonic Acid suspension reaches maximum

turbidity. Transfer reagent to micro centrifuge tube

and vortex for 5 minutes. The reconstituted

Arachidonic Acid suspension should appear very milky

with numerous small bubbles.

Add 5 µL of reagent to 500 µL PRP sample or .5 µL of

reagent to 50 µL sample for a concentration of 0.5

mM. Normal aggregation is seen with final

concentrations of 0.5 -1.0 mM.

Stability: The reconstituted Arachidonic Acid can be

stored frozen at -70°C in the dark in 100 µL volumes

for 3 months or until expiration date, whichever comes

first. When stored frozen at - 0°C in the dark

Arachidonic Acid is stable for 1 month or until

expiration date, whichever comes first. Aliquots can be

hand thawed and vigorously re-suspended with a

vortex mixer just before use.

e. Collagen

Catalog no.: 385

Supplied as: 1 mg of native Collagen fibrils (type I) from

equine tendons suspended in isotonic glucose solution

of ph .7/vial.

Stock Conc.: 1 mg/mL

Stock Storage: Refrigerate at -8°C

Stock Shelf Life: Until expiration date.

Working Conc.: 1 mg/mL

Working Storage: -8°C

Working Shelf life: Until expiration date.

Preparation: Collagen can be used directly as supplied.

Invert or swirl vial before use, as Collagen fibrils are in

suspension. Do not freeze. If required, Collagen can

Document # 49044IM1

Revision 7.5 10

Dated February 16, 017

be further diluted in isotonic glucose pH .7. Do not

dilute entire bottle, only enough for a day’s testing.

Add 1 µL of reagent to 500 µL sample or 0.5 µL of

reagent to 50 µL sample for a final concentration of

µg/mL. Normal aggregation is seen with final

concentrations of 1-5 µg/mL.

Stability: Collagen does not contain any preservative,

but because of its very low pH, organisms do not grow

as readily. If asceptic techniques are used (sterile

syringe and needle to remove one day’s use),

remaining reagent, if stored at - 8°C, is stable until

expiration date. Reagent removed from the vial is

stable for one week at -8°C.

f. Epinephrine

Catalog No.: 393

Supplied As: Lyophilized preparation of l-Epinephrine

bitartarate with stabilizers

Stock Conc.: 10 mM for Whole Blood Testing; 1mM for

PRP testing

Stock Storage: Refrigerate at -8°C

Stock Shelf life: Until expiration date

Working Conc.: 1 mM for PRP testing

Working Storage: -8°C in dark container

Working Shelf life: 8 hours (in the dark)

Reconstitute with 5.0 mL sterile distilled water (dilute

1:10 with physiological saline for PRP testing).

Preparation: Tap vial gently to get contents to the

bottom. Remove stopper and reconstitute with 5.0 mL

sterile, distilled water. Dilute the stock 1:10 with

physiological saline for PRP testing. Allow to sit for ten

minutes with occasional inversion. Adding 5µL of 1:10

Diluted Solution to 500 µL sample of platelet rich

plasma or .5 µL of reagent to 50 µL sample gives a

final concentration of 10 µM. Normal aggregation is

seen with final concentrations of 5-10 µM in platelet

rich plasma.

Stability: Epinephrine is a comparatively unstable

reagent. The unused reconstituted Epinephrine can be

stored frozen at -70°C in the dark and in 100 µL

aliquots for 3 months or until the expiration date,

whichever comes first.

NOTE: Nor al subjects exhibit considerable variability that is

not correlated with age, sex, stress, diet, platelet count or

he atocrit.

g. Ristocetin

Catalog No.: 396

Supplied As: 6 .5 mg of stabilized freeze dried

Ristocetin.

Stock Conc.: 1 5 mg/mL

Stock Storage: Refrigerate at -8°C (in the dark)

Stock Shelf Life: Until expiration date

Working Conc.: 1 5 mg/mL

Working Storage: -8°C

Working Shelf Life: 8 hours (in the dark)

Reconstitute with 0.5 mL of sterile distilled water.

Preparation: Tap vial gently to get contents to the

bottom. Remove stopper and reconstitute with 0.5 mL

of sterile distilled water. Do not shake or invert vial.

Re-stopper and allow to sit for 10-15 minutes. Visually

inspect bottom of vial to confirm reagent is fully in

suspension. Do not shake the reagent, invert gently, to

take up any reagent remaining in stopper and allow

vial to sit for another 10-15 minutes until all particulate

matter is well dissolved. Reagent may have a clear-to-

brownish color suspension after reconstitution. Never

shake reagent. Swirl gently just before use.

Add 5 µL of reagent to 500 µL PRP sample or .5 µL of

reagent to 50 µL sample for a concentration of 1. 5

mg/mL. Normal aggregation is seen with final

concentration of 0.63 - 1.5 mg/mL.

Stability: The unused reconstituted Ristocetin reagent

can be stored frozen at - 0°C in volumes suitable for a

days testing for 3 months or until the expiration date,

whichever comes first. DO NOT STORE AT -70oC.

4. AGGR /LINK® pti8TM Software

a. Turn on Computer and Start AGGRO/LINK® Opti8TM for

Windows® program. Be sure “Aggregometer Ready”

appears in the bottom right-hand corner of the screen.

If not, check USB Port, cables and connectors. If

USB/Com Port is changed, go to AGGREGOMETER then

CONNECT.

b. Click on EDIT and CONFIGURATION. Type in your

institutions information under Report Header. This will

be printed out on top of Report Format when Report

Batch Print is selected under File. Click on OK when

completed.

c. To have Area Under Curve and Lag Time Calculated

and printed on test data, click on the box to select. To

disable, leave this box unchecked.

d. To display and print Start Indicators, click on the box to

select. To disable, leave this box unchecked.

e. Under the AGGREGOMETER window, select or set-up a

test Procedure page for Optical mode. To save the

procedure for future use, change the name in the

Procedure Name field. Click on OK and select Run New

Patient under AGGREGOMETER.

N TE – AGGR /LINK® pti8TM Software features:

•CREATE MERGE – Creates a blank document for pasting

curves

•EXP RT - Export test data for use with another program

•ADJUST SL PE LINE – Change slope calculation time

•FFSET CURVES – Change the physical start time of a test

Channel

•C PY SCREEN – Copies the test grid and curves to the

system Clipboard

•MERGE C PY ALL or SELECTED - To copy all the curves from

a file or to copy only selected curves from a file to a merge

document.

•START & ST P TIMES – Automatic marking at addition of

reagent.

•CUST M C L R SETUP – Use Default tracing colors or

customize

Document # 49044IM1

Revision 7.5 11

Dated February 16, 017

•ACTIVATE Bar – provides the ability to control each test

individually or in sets of 2 or 4 channels, eliminating the

risk of some samples sitting in test well for extended

period of time before tests are started.

1. To run up to Eight (8) tests at the same time:

a. After a 3-minute incubation, place test cuvettes

in PRP test wells – from 1 to 8 channels

b. Click on “Activate” Bar and press the Set Baseline

button for each channel.

c. Monitor all tracings for stability.

d. Once tracings are stable and, if a clean test

screen is needed, Click on “Aggregometer” and

“Reset” for all channels.

e. If required that the Baseline setting appear on

test printout, press Set Baseline for all channels.

f. Click on “Start” Bar for Channel 1 and add

reagent.

g. If Baseline setting not required on test printout,

Click on the “Start” Bar for Channel 1 and add

reagent.

h. Repeat “e and f” or “g” for remaining channels.

. To run tests in 4 channel sets:

a. After a 3-minute incubation, place 4 test

cuvette(s) in PRP test well(s) in Channels 1 thru 4.

b. Click on “Activate” Bar and press the Set Baseline

for Channels 1 thru 4.

c. Monitor tracings for stability. [N TE: At this

point, place 4 additional test cuvettes in

Incubation wells for Channels 5 thru 8 for a 3-

minute incubation.]

d. Once tracings are stable and, if a clean test

screen is needed, Click on “Aggregometer” and

“Reset” for Channels 1 thru 4.

e. If required that the Baseline setting appear on

test printout, press Set Baseline for Channels 1

thru 4.

f. Click on “Start” Bar for Channel 1 and add

reagent.

g. If Baseline setting not required on test printout,

Click on the “Start” bar for Channel 1 and add

reagent.

h. Repeat “e and f” or “g” for remaining channels.

i. After 3-incubation, place 4 test cuvette(s) in PRP

test well(s) in Channels 5 thru 8.

j. Repeat “b” thru “h” for Channels 5 thru 8.

5. Micro-Pipettes with 2-Stop Control Button

a. Description of 2 Stops

1) First Stop – The measuring stroke for aspirating

and dispensing the selected volume

) Second Stop – To blow out any liquid remaining

in the pipette tip after dispensing.

b. Volume Setting

When adjusting the volume setting from a higher value

to a lower value, turn the knob past the desired

volume and then back to the required setting.

c. Filling the Pipette Tip

1) Press the control button down to the first stop

) Immerse the pipette tip vertically into the liquid

3) Aspirate and dispense the liquid three times by

using the first stop only.

4) Remove the tip slowly from the liquid. For large

volumes, wait approximately 3 seconds before

removing the tip from the liquid.

5) Wipe off the outside of the tip with a lint-free

tissue to remove any excess liquid, taking

precaution that the tissue does not touch the tip

opening.

NOTE: When pipetting Whole Blood, PRP or Platelets do NOT

aspirate/dispense 3 ti es. Only take up the sa ple one ti e.

d. Dispensing

Place the pipette tip into the cuvette, so that the end

of the tip is immersed in the sample.

1) Slightly angle the pipette so that the tip is angled

to touch the wall of the cuvette.

) Press the control button down to the first stop,

then press the control button down to the

second stop (blow-out) to empty the pipette tip.

3) Hold the control button down at the second

stop and, while keeping the pipette tip at a slight

angle, pull the pipette tip out of the sample.

4) Once the pipette tip is completely outside of

the cuvette, release the control button.

CALIBRATI N:

For testing platelets, the Optical mode is calibrated automatically

during the testing procedure by the setting of 0% (PRP) and 100%

(PPP) baselines.

QUALITY C NTR L:

It is good laboratory practice to run a drug free normal control

whenever reagents are reconstituted or thawed. Test results

should fall within Normal Ranges established in each laboratory.

If desired, positive controls can be provided by collecting samples

from aspirin volunteers or subjects previously diagnosed with a

platelet disorder.

Positive controls can also be made in-vitro by the addition of

aspirin or the depletion of plasma. A final concentration of 1 mM

aspirin in citrated blood will inhibit the response to arachidonic

acid. Centrifugation, removal of platelet poor plasma and

replacement with an equal volume of saline, while leaving the

buffy coat in place will inhibit response to Ristocetin.

PR CEDURE - STEPWISE:

1. Check Aggregometer to be sure heater block has stabilized

to 37°C.

. Place P/N 311 Stir bars in P/N 31 Cuvettes. Put cuvettes

containing stir bar in the incubation wells to warm up.

3. For testing the same donor in all channels, set all channels to

reference channel #1 PPP. [If each channel is set to its own

PPP, a cuvette with 500 µL PPP is required for each channel.]

SELECT Pushbutton(s) – allows for toggling between the Stirring

Speed, Temperature and PPP/Reference Settings.

SET Pushbutton(s) - sets the Stirring Speed, Temperature and

PPP/Reference, depending on which feature is selected. Each

time the SET button is pushed, the selected function is

incremented until it reaches the maximum setting.

Document # 49044IM1

Revision 7.5 1

Dated February 16, 017

N TE: ne SELECT and SET pushbutton controls two channels in

tandem. For example, Channels 1 & 2, Channels 3 & 4, Channels

5 & 6 and Channels 7 & 8 are set in pairs.

4. Place the cuvettes containing 500 µL PPP in the reference

well(s) …stir bar is not required. Check for bubbles and be

sure to wipe cuvette with a clean KimWipe.

5. Place 500 µL or 50 µL Platelet-RICH-Plasma (PRP) into pre-

warmed cuvettes with stir bars. [Prepare (1) for each test

channel.] Incubate for three (3) minutes in incubation wells

6. Start AGGRO/LINK® Opti8TM for Windows® program and

select TEST PROCEDURE under the AGGREGOMETER

window. Under PROCEDURE NAME, set-up or load the

procedure that corresponds to the reagent and method

being used. Optical tests with PRP should run for a

ini u of 5 inutes. Slope length can be set from 16 to

99 seconds. Chrono-log standard setting is 16 seconds. Click

OK.

7. Select “RUN NEW TEST” under AGGREGOMETER window.

Patient information page will appear. Patient information

can be completed at this time or can be entered during or

after test is completed. [Click on EDIT then TEST

INFORMATION] When “Run Sa e Test” is selected, patient

data fro previous test will be used.

NOTE – Patient data fro Trace 1 can be copied to other

tracings. Click on Select All if testing sa e donor in all channels

or Click on appropriate Trace Nos. and then Click on COPY.

NOTE – The Test Identification field at the top of the page is used

to identify and select test(s) in the Test Directory. For Clinical

testing, place patient identifier in this field.

8. Place the cuvettes containing PRP in the PRP wells (One for

each Optical Channel). Check for bubbles and be sure to

wipe cuvette with a clean.

9. Click OK to begin test. Click on color-coded “Activate” bar for

each channel.

10. Push the SET BASELINE buttons for each channel. The

tracing should move to 100% when the button is depressed

and to 0% when the button is released, using the numbers

on the left side of the graph. Be sure to press and hold the

Baseline button until the tracing reaches 100% of the graph

and then release.

NOTE: The Graph Range ti e can be adjusted under VIEW and

then Set Graph Range. When the test tracings have reached the

end of the Graph Range, additional ti e is added auto atically

in one inute incre ents, up to a total of 60 inutes.

11. Monitor tracings for stability.

1 . When tracing(s) have reached stability, take up the

appropriate reagent and click on the color-coded “Start” bar

for Channel 1. [ r … See N TE 2 below].

13. Add reagent

a. Use 1 µ

µµ

µL of Collagen with 500 µL sample volume or

0.5 µ

µµ

µL with 50 µL sample for 2 µ

µµ

µg/mL final

concentration…or for a 5 µ

µµ

µg/mL final concentration

use 2.5 µ

µµ

µL with 500 µL sample volume or 1.25 µ

µµ

µL with

50 µL sample.

NOTE: As shown above, reagent volu es are cut in half when

testing with 250

µµ

L PRP sa ples.

b. Use 5 µ

µµ

µL of ADP with 500 µL sample volume for a final

concentration of 10 µ

µµ

µM…or 2.5 µ

µµ

µL for 5 µ

µµ

µM.

c. Use 5 µ

µµ

µL of Arachidonic Acid with 500 µL sample

volume for a final concentration of 0.5 mM…or 2.5 µ

µµ

µL

for 0.25mM.

d. Use 5 µ

µµ

µL of Ristocetin with 500 µL sample volume for a

final concentration of 1.25 mg/mL.

e. Use 2 µ

µµ

µL of Ristocetin with 500 µL sample volume for a

final concentration of 0.5 mg/mL (Type B vW).

f. Use 2.5 µ

µµ

µL of Epinephrine with 500 µL sample volume

for a final concentration of 5 µ

µµ

µM.

NOTE – CHRONO-PAR® Reagents do not require preparation of

ultiple stock solutions. To change final concentration, adjust

pipette volu es as shown above.

14. Repeat steps 1 and 13 for each test channel.

15. Allow all tests to run for a minimum of five (5) minutes.

NOTE: The “Activate” bar in Step 9 provides the ability to control

each channel individually and to stagger the start of each test.

NOTE: To have the baseline setting visible on the final printout,

when a tracing has reached stability, Click on Aggrego eter …

Reset Channel … and select appropriate Channel #. Click on the

“Activate” bar and reset the baseline. Then, click on the “Start”

bar and add the appropriate reagent for that Channel.

16. While tests are running, prepare one test cuvette for each

channel (Cuvette, stir bar and 500 µL or 50 µL PRP) & begin

three (3) minute incubation.

17. There are a number of Options to Stop Tests as each test can

be stopped individually or all tests stopped at the same

time, as follows:

a. To Stop Tests individually, click on Aggregometer, click

on Stop Test and select the appropriate Channel.

b. STOP Icon at top of screen stops all tests at the same

time.

18. Clicking on the “Start” bar prior to adding the reagent sets

the Start and Stop time for each channel. If there is a

baseline shift or some other artifact that should not be

included in the final calculation, Click on EDIT and SET START

& STOP TIMES (or use ICONS at top of screen).

a. If only calculating Slope and Amplitude:

1) A small box will appear with Trace 1 selected.

) If the Automatic Start Time Feature was used

before the addition of the reagent, a dotted line

will appear on the screen at that point (Start

Line) with another dotted line (Stop Line) placed

“X” number of minutes after (Test Time set up on

procedure page).

3) If required, Start & Stop times can be moved:

a) Individually – Left Click, Hold and Drag to

new position. (Be sure Start Line is placed

on stable baseline just before or just after

adding the reagent. Stop Line should be

placed five (5) minutes after the Start

Time.)

b) Simultaneously – Place cursor near Start

Line. Right click, Hold & Drag Start Line to

new position. Stop Line will move in

tandem.

4) Click on other Tracings and repeat, if needed.

Document # 49044IM1

Revision 7.5 13

Dated February 16, 017

b. If calculating Slope, Amplitude, Lag Time and Area

Under the Curve at the same settings:

1) Click on Trace 1

) Click on “Set All” to calculate all parameters with

one setting.

3) Follow Steps ) through 4) above.

NOTE – When calculating Lag Ti e individually, only a start ti e

is displayed.

19. After setting the Start and Stop Times, Click on DONE, then

select Calculate Results under the EDIT window (or use

ICON). This command will calculate aggregation percentage,

slope and if selected, lag time and area under the curve.

Check Duration times to be sure Start & Stop Lines were set

correctly. Click on OK and calculations will appear in Data

Box.

0. After the calculations have been completed, SAVE the test,

using the SAVE command under FILE. Tests can be printed

using the PRINT command in the FILE WINDOW. PRINT is for

printing one test per page.

1. To print multiple test graphs separately at one time or in a

Report Format, click on File and Report Batch Print, followed

by:

a. Select the tests to be printed by placing (√) in boxes to

left of tests listed.

b. Click on the “Insert Selected” button to move tests to

lower panel.

c. The order the tests are printed out can be arranged by

using UP or DOWN buttons.

d. Click on “Report Print” to include all selected tests in a

single report … or,

e. Click on “Batch Print” to print each test separately.

. Remove the samples from the reaction well and discard.

CALCULATI NS:

1. Amplitude – Optical aggregation results are expressed as a

percentage of aggregation at a given time interval from

reagent addition; 100% aggregation is defined as the

difference between the 0% (PRP) baseline and the 100%

(PPP) baseline.

. Slope – Slope is determined by drawing a tangent through

the steepest part of the curve. A right triangle is then

constructed over an interval of one minute. The height of

the triangle is the rate of change of aggregation in one

minute, which is defined as the slope. AGGRO/LINK®

Opti8TM

Software can be set from 16 to 99 seconds (3 point to 198

point) sliding curve. To change the length of a slope line,

click on EDIT, then ADJUST SL PE LINE. Click on Trace

number and use arrows to adjust slope length up to 99

seconds. Chrono-log standard setting is 16 seconds (3

points).

REP RTING RESULTS:

1. Report Hematocrit.

. Report Platelet Count.

3. Report Platelet Aggregation percent and slope.

Reference Ranges:

NOTE: The following Nor al Ranges were obtained fro

various laboratories and publications. They should be used as a

guideline only. Nor al ranges should be established for

aggregation in each and every laboratory.

Normal Ranges in

Platelet Rich Plasma

(Mean ±

±±

±1 SD)

Reagent

Conc.

Agg. (%)

Collagen

2 µg/mL

–

Arachidonic Acid

0.5 mM

–

99*

ADP

µM

–

Epinephrine

µM

–

Ristocetin

1.25mg/mL

–

102

(*+ 2 SD)

Procedures for Abnormal Results:

1. When Normal Control Test(s) Is Abnormal

a. No patient result shall be reported if any reagent does

not recover findings within reference range limits

when tested with the normal control.

b. Repeat both Normal Control and Patient test with new

aliquot of frozen reagent or reconstitute new vial of

reagent. Be sure each test cuvette contains a stir bar.

. When Normal Control Test(s) Is Normal

a. Repeat abnormal patient test(s) to be sure result is not

due to a technical variable. Be sure each test cuvette

contains a stir bar.

b. If repeated test(s) continue to be abnormal, report the

abnormal result(s) and request a retest on another day

to confirm findings.

Platelet Abnormalities

Platelet aggregation is clinically significant in the detection and

diagnosis of acquired14 or congenital qualitative platelet defects.

The platelet’s ability or inability to respond to particular

aggregating reagents is the basis for differentiating platelet

dysfunctions as shown in the table on the following page.

Document # 49044IM1

Revision 7.5 14

Dated February 16, 017

* Second-wave Inhibited

** Type B and Platelet-type von Willebrand increased at low concentration 0. -0.6 mg/mL. In addition, when cryoprecipitate is

added to test sample from patient with Platelet-Type [pseudo] VWD, enhanced response to low concentration Ristocetin will

continue, a Type B patient will show no response.7

*** To distinguish between von Willebrand & Bernard Soulier, add normal plasma or cryoprecipitate to patient sample, vW patient

will respond, Bernard Soulier will not. 19

Key: A – Absent H – Hyper N – Normal R - Reduced

(Compared to Normal Ranges)

AGGREGATION RESPONSE WITH SELECTED ABNORMALITIES

Reagent Final

Concentration Aspirin Effect Von Willebrand &

Bernard Soulier

Storage Pool/

Secretion Defect Glanzmann's Thrombasthenia

ADP 5 – 10 µ

µµ

µM N, R * N N, R * A

Arachidonic Acid 0.5 mM A N N A

Collagen 1 – 5 µ

µµ

µg/mL

1 or 2

µµ

µg/mL 5 µ

µµ

µg/mL N

Epinephrine 10 - 50 µ

µµ

µM R* N R * A

Ristocetin 0.5 – 1.5

mg/mL

Qualitative

Defect

** A,R,H ***

N N

Document # 49044IM1

Revision 7.5 15

Dated February 16, 017

Reporting Format:

PLATELET AGGREGATI N STUDIES:

DATE __________________________________

PHYSICIAN _________________________________ TIME BL D DRAWN: ___________

INSTITUTI N: ______________________________ DEPARTMENT: ____________________

PATIENT ___________________ I.D. _______________ DATE F BIRTH: ______________

AGE: ________ SEX: (M) (F) HCT ___________ PLT CT _________ BT _________

CLINICAL HIST RY: ______________________________________________________________________________________

________________________________________________________________________________________________________

PTICAL AGGREGATI N TEST RESULTS

AG NIST N RMAL RANGES

(Mean ±

±±

±1 SD)

C NTR L

VALUE

PATIENT

VALUE

TEST

RESULTS

(Normal, Reduced,

Increased)

ADP, 5 µ

µµ

µM

69 - 88%

ADP, 10 µ

µµ

µM

71 - 88%

C LLAGEN, 2 µ

µµ

µg/mL

70 - 94%

C LLAGEN, 5µ

µµ

µg/mL

ARACHID NIC ACID,

0.5 mM

74 - 99%*

EPINEPHRINE, 5 µ

µµ

µM

78 - 88%

RIST CETIN, 1.25 mg/mL

87 - 102%

RIST CETIN, 0.5 mg/mL

* ±

±±

±2SD

INTERPRETATI N:

_____________________________________________________________________________________________________________________

TEST PERF RMED BY: ___________________________________ INTERPRETED BY: _______________________________________________

Document # 49044IM1

Revision 7.5 16

Dated February 16, 017

INTERPRETATI N:

Aggregation curves in PRP can be interpreted as follows:

•By direct comparison to a normal drug free control which

also provides real time quality control.

•Comparison to published normal values that can be verified

and reproduced by any laboratory.

With Collagen: Collagen is useful for checking the platelet’s

general ability to aggregate. A lag phase of up to a minute is

typically seen with this agonist.

With Arachidonic Acid: Arachidonic Acid is converted to

thromboxane A in the presence of cyclooxygenase. Aspirin

inhibits the cyclooxygenase pathway, causing a significant

reduction in aggregation with this agonist. Normal aggregation is

seen with concentrations of 0.5 mM to 1.0 mM.

With ADP: ADP exposes the fibrinogen binding site on the

membrane glycoprotein GPIIb/IIIa complex. Aggregation testing is

typically performed in PRP with concentrations ranging from 1 µM

to 10 µM. At the lower concentrations up to 3 µM, a first wave of

aggregation will be followed by disaggregation. At the higher

concentrations, the first wave of aggregation will blend into the

second wave, masking the biphasic wave. It is often necessary to

perform dose-response testing with multiple concentrations to

obtain a biphasic response. Aspirin effect may be seen with mid-

range concentrations such as 5 µM.

With Epinephrine: Shape change is not seen with this agonist.

Higher concentrations (≥ 5 µM) produce a biphasic curve with

second-wave aggregation dependent on thromboxane A

synthesis. “Epinephrine is the least consistent agonist used in the

assessment of platelet aggregation and, if the Epinephrine

response is the only abnormality seen in testing, one should be

very hesitant to make the diagnosis of a ‘disorder’ based on this

result.” 18

With Ristocetin: This antibiotic is used for the detection of von

Willebrand Disease (a quantitative or qualitative defect in plasma

vW Factor) and Bernard Soulier (a lack of platelet membrane

glycoprotein (GPIb). Normal results are seen with concentrations

ranging from 0.75 to 1.5 mg/mL. To detect Type B or Platelet-

Type vW, test for a Hyper-response at low concentrations (0. –

0.6 mg/mL). 7 To distinguish between vW and Bernard Soulier,

add normal plasma or cryoprecipitate to patient sample. vW

patient will respond, Bernard Soulier will not. A Qualitative defect

such as a saw-tooth pattern may be seen with subjects taking

aspirin.

PR CEDURE N TES:

1. With the Model 490 4+4, spacers are not required when

testing micro-volume samples ( 50 µL of PRP) and all

CHRONO-PAR® reagent volumes are reduced by half. For

example: 5 µ

µµ

µL of ADP = a final concentration of 10 µ

µµ

µM with

a 500 µ

µµ

µL PRP sample… use only 2.5 µ

µµ

µL with a 250 µ

µµ

µL PRP

sample.

. It is important that the pipette tip be pushed to the bottom

of the cuvette & the reagent forcefully injected into the

sample. D N T introduce the reagent above the sample in

the cuvette since the reagent will cling to the side of the

cuvette and will not mix with the sample. (DO NOT forcefully

inject reagent if testing with smaller volume of 50 µL PRP.)

LIMITATI NS F THE PR CEDURE:

•In a study of one hundred and six patients with storage pool

deficiency (SPD), 3% had normal optical (PRP) aggregation

responses to ADP, Epinephrine and Collagen; and 44% had

miscellaneous aggregation abnormalities. The authors

concluded that SPD is common, heterogeneous and not

necessarily associated with optical (PRP) aggregation

abnormalities. 10

•Tests should be performed within 3 hours of venipuncture.

•Many drugs inhibit platelet function. 5,11,19 Unless the aim of

testing is to demonstrate drug-induced inhibition, patients

should be drug free for ten (10) days to two ( ) weeks prior

to testing.

•Further Clinical and Laboratory evaluation may be required

to confirm diagnosis.

•Red Blood Cells in PRP can inhibit the ability of the

Aggregometer to detect changes in light intensity. This may

cause the appearance of a decrease in platelet

aggregation.19

•Hemolysis results in release of nucleotides from the red cells

which may cause activation or desensitization of platelets,

especially to ADP.19

•Lipids in PRP can interfere with light transmission readings &

prevent recording of aggregation.

•Platelet counts below 100,000/µL may cause problems with

the setting of optical baseline, preventing the recording of

aggregation

•This device has not been evaluated for pediatric use.

SERVICE/PREVENTATIVE MAINTENANCE

This Unit does not require Preventative Maintenance; however,

yearly calibration is recommended. Calibration and service, if

required, should be obtained from the factory or a factory

authorized representative.

In the 48 continental United States a Factory Service and Loaner

Contract program is available. On-site field service is also

available in some areas. Contact the factory for details.

Outside of the United States contact your distributor for service.

In countries where there is no distributor, contact Chrono-log

directly.

Replacement of Parts: Critical components should only be

replaced with manufacturer approved parts. Maintenance should

be performed according to service manuals and PM checklists.

Protective Earth Ground: The instrument is connected to ground

at the instrument chassis. This sole purpose connection point is

made between the power entry module and the chassis with

green/yellow wire and is marked with the IEC 60417-5019

symbol. This connection must be reestablished if it is

removed for any purpose during servicing.

CLEANING SYSTEM C MP NENTS

Recommended Tools and Accessories

A Liquid Dishwashing Detergent -- Use a mixture of one part liquid

dishwashing detergent and three parts water to clean the exterior

of the Aggregometer.

Document # 49044IM1

Revision 7.5 17

Dated February 16, 017

A Soft, Lint-Free Cleaning Cloth -- Moisten the cleaning cloth with

the dishwashing detergent solution to clean the exterior of the

Aggregometer.

A Small Vacuum Cleaner with a Brush attachment -- Use the

vacuum cleaner to remove dust and dirt from the exterior of the

Aggregometer.

Cleaning the Aggregometer

1. Turn off the Aggregometer and any attached peripherals.

Disconnect them from the Aggregometer. Unplug the

instrument before cleaning to prevent a shock hazard should

any fluid enter the power switch.

. Use a vacuum cleaner to remove any dust from the slots and

holes in the Aggregometer.

3. Moisten a soft cleaning cloth with a solution of three parts

water and one part liquid detergent.

N TE -- D N T S AK THE CL TH IN THE S LUTI N; D N T

LET S LUTI N DRIP INSIDE THE AGGREG METER.

4. Use the moistened cloth to wipe the Aggregometer exterior.

SELF-CALIBRATI N F PTICAL CIRCUITS

This procedure enables the Optical wells (PRP & PPP) to be

calibrated automatically with the push of a button and verifies

that the instrument's optical circuits are operating properly.

During the performance of this procedure, the light intensity of

the LEDs are adjusted and set to the proper calibrated voltage.

Use a stop watch or timer since some of the steps must be timed.

The calibration switch located on the front of the instrument is

key-activated. This is to prevent unauthorized use or accidental

initialization of the procedure. DO NOT LEAVE THE KEY IN THE

SWITCH WHEN NOT PERFORMING THE CALIBRATION

PROCEDURE. The key should be stored in a safe location

accessible only to personnel authorized by laboratory

management. If the key is lost, a replacement key may be

purchased from the factory. Please note your Key # here

____________________.

Failure to follow this procedure exactly may result in an

inaccurate calibration. The Post Calibration Test checks the

accuracy of the calibration and enables the operator to have a

record of the results. This calibration procedure is not a

substitute for the recommended yearly instrument calibration

and PM.

Preparation:

1. Materials Required

a. P/N 3 . Calibration Kit with (1) black cuvette and ( )

sealed water cuvettes.

b. Lint-Free Wipe

c. Calibration Key

. Turn on the unit and let it heat up until the heater blocks

stabilize at 37°C.

3. Since each Channel is calibrated independently, confirm

that that PPP/Reference Select Switches are set to

reference each Channel's PPP sample, and are not

referencing Channel #1 PPP.

NOTE: Additional steps required for CHRONO-LOG® instru ents

that have a PPP Reference switch allowing ultiple channels to

reference the PPP in Channel. [See “Additional Post Calibration

test for Instru ents with PPP Select Switches” on Page 19 ]

4. Place the ( ) water cuvettes from P/N 3 in the incubation

wells and pre-warm for a minimum of five (5) minutes. Be

sure to always wipe cuvettes with Kim Wipe® before placing

in test or reference wells.

5. Turn on computer and Start AGGRO/LINK® Opti8TM for

Windows® program.

6. Under the AGGREGOMETER window, select or set-up a Test

Procedure page for Optical mode. Click on OK.

7. Select RUN NEW PATIENT under AGGREGOMETER and Click

on OK.

8. Type, “Optical Calibration Pre”, in Test Identification box at

top of screen. Click on OK and test will begin running.

9. After the five (5) minute incubation, check cuvettes for air

bubbles. Eliminate bubbles by tapping the cuvette.

Pre-Calibration Test

NOTE: Throughout the entire procedure, the Water Cuvette used

in the PRP well should only be used in PRP test wells and the PPP

Water Cuvette should only be used in PPP reference wells. Be

sure to use the Align ent Mark on side of each cuvette to ensure

consistent place ent of cuvettes into test and reference wells.

1. Click on Aggregometer, select Reset Channel and select ALL

Channels. Click on OK.

. After wiping the outside with a Kim Wipe®, place BLACK

cuvette in Channel 1 PRP well and a water cuvette in the PPP

well of Channel 1, with the alignment mark placed left

center.

3. Click on the Channel 1 “Activate” bar. [NOTE: If this pre-test

is to be SAVED, Click on the “Start” Bar for Channel 1. If not

saving, the following steps can be performed after clicking

on the “Activated” button. ]

4. Push and Hold the Set Baseline button until the tracing

reaches a full 100% of graph before releasing. Tracing will

then return to 0% of graph.

5. Replace the BLACK cuvette in the PRP well with the other

water cuvette, after wiping the outside with a Kim Wipe®.

Be sure alignment mark is placed left center. The tracing

should move near 100% on the graph. Allow to run for a

few seconds and note the position where the tracing

stabilizes. (Ex: 97%)

6. Repeat Steps through 5 above for Channel , and then

continue to repeat for Channels 3, 4, 5, 6, 7 and 8. Be sure

to use the water cuvette in the PPP well in all PPP wells and

the water cuvette in the PRP well in all PRP wells. Also be

sure to wipe the outside with a Kim Wipe® and to place the

Alignment Mark to the left center of the test well.

7. If saving the test … Once all channels are completed, Click on

the STOP icon followed by the SAVE icon and the PRINT icon

to save and print the test.

8. If any tracing is > 4% from 0% or 100% on the graph,

proceed with calibration steps below, calibrating all eight (8)

channels.

Document # 49044IM1

Revision 7.5 18

Dated February 16, 017

Calibration

NOTE: Calibrate ALL test channels, even if only one channel is

out of tolerance. This will ensure that all LEDs are set to the

sa e intensity.

1. Click on Aggregometer, select Reset Channel and then select

ALL Channels. Click on OK.

. After wiping the outside with a Kim Wipe®, place BLACK

cuvette in PRP well of Channel 1 and place PPP water

cuvette in PPP well.

3. Click on the “Activate” bar for Channels 1 through 8.

4. Press the Baseline button for Channel 1 and hold until the

tracing reaches a full 100% of graph before releasing.

Tracing will then return to 0% of graph.

5. Transfer the BLACK cuvette in PRP well and water cuvette in

PPP well to Channel and repeat Step 4.

6. Repeat Step 5 for Channels 3 through 8.

7. Remove Black cuvette from test well.

8. Click on the “Start” Bar for Channels 1 through 4.

9. After wiping the outside with a Kim Wipe® and keeping the

Alignment Marks left center, place the PRP water cuvette in

Channel 1 PRP well and the “P” water cuvette in Channel 1

PPP well.

10. Place the key in the calibrate switch on the module

containing Channels 1 through 4 and turn the key clockwise

90°and keep it in this position. Since calibration does not

start until the baseline is set, all four (4) channels can be

calibrated.

11. Set the timer for one minute but do not start. Press the Set

Baseline button for Channel 1, hold for a second, then

release. Start the timer as it may take up to a minute for the

channel to calibrate. During this minute DO NOT touch or

move the samples. Moving the samples may cause

inaccuracies in the calibration. Wait for the one minute [or

when tracing runs stable for around 20 seconds] before

proceeding to the next step.

NOTE: Disregard traces during calibration.

1 . After waiting one minute, or when tracing stabilizes in

Channel 1 … Repeat steps 9 through 11 for Channel , then

3, then 4. Calibration key remains ON throughout this

process.

13. Once step 1 is completed for all four (4) channels, turn OFF

the calibration mode by turning calibrate key counter-

clockwise 90° and remove the key from the lock.

Calibration of Module 1 is now completed.

14. To calibrate Channels 5, 6, 7 and 8 in Module 2, beginning

with Channel 5, repeat steps 8 through 13above, using the

calibrate key in that odule.

15. If required, SAVE and PRINT the test.

Post Calibration test

[Confirm the Calibration Key has been removed from

lock]

1. Under the AGGREGOMETER window, select or set-up a Test

Procedure page for Optical mode. Click on OK.

. Select RUN NEW PATIENT under AGGREGOMETER and Click

on OK.

3. After wiping the outside with a Kim Wipe®, place BLACK

cuvette in Channel 1 PRP well and the PPP water cuvette in

the PPP well of Channel 1, with the alignment mark placed

left center.

4. Click on the Channel 1 “Activate” bar. [NOTE: If this test is to

be SAVED, Click on the “Start” Bar for Channel 1. If not

saving, the following steps can be performed after clicking

on the “Activate” button.]

5. Push and Hold the Set Baseline button until the tracing

reaches a full 100% of graph before releasing. Tracing will

then return to 0% of graph.

6. After wiping the outside with a Kim Wipe®, replace the

BLACK cuvette in the PRP well with the PRP water cuvette

with the alignment mark placed left center. The tracing

should move near 100% on the graph. Allow to run for a

few seconds and note the position where the tracing

stabilizes. (Ex: 97%)

7. Repeat Steps 3 through 6 for Channel , then repeat for

Channels 3, 4, 5, 6, 7 and 8. Be sure to use the PPP water

cuvette in the PPP wells and the PRP water cuvette in the

PRP wells. Also be sure to place the Alignment Mark to the

left center of each test well and to wipe the outside of the

cuvettes with a Kim Wipe® when placing into the test well.

8. If any tracing is > 4% from 0% or 100% on the graph,

calibration of that Channel will need to be repeated,

following the previous calibration steps.

9. If saving the test … Once all channels are completed, Click on

the STOP icon followed by the SAVE icon and the PRINT icon

to save and print this test.

NOTE: If the tracings are > 4% fro 0% or 100% on the graph,

this ay indicate that Auto-Calibration was not set properly.

Recheck the two water cuvettes to be sure they atch. See

Appendix A for further instructions on atching water cuvettes,

then repeat the entire calibration procedure. Be sure to wipe the

cuvettes with a Ki Wipe® each ti e they are placed into a test

or reference well. If, after the second atte pt, the results are

> 4%, the syste ay need Service. Contact Chrono-log Service

Depart ent at 1-800-247-6665 for further assistance.

Additional Post Calibration test for Instruments

with PPP Select Switches - This section provides for the

ability to check each channel’s calibration against the Channel 1

PPP Reference.

[Confirm the Calibration Key has been removed from

lock]

SELECT Pushbutton(s) – allows for toggling between the Stirring

Speed, Te perature and PPP/Reference Settings.

SET Pushbutton(s) - sets the Stirring Speed, Te perature and

PPP/Reference, depending on which feature is selected. Each

ti e the SET button is pushed, the selected function is

incre ented until it reaches the axi u setting.

NOTE: One SELECT and SET pushbutton controls two channels in

tande . For exa ple, Channels 1 & 2, Channels 3 &4, Channels 5

& 6 and Channels 7 & 8 are set in pairs.

1. Set all Channels to Reference Channel 1 PPP

a. Push SELECT pushbutton for Channels 1 and until

PPP/Reference setting is displayed on the LCD screen.

b. Push SET pushbutton for Channels 1 and until the

reference to Channel 1 PPP is displayed.

Document # 49044IM1

Revision 7.5 19

Dated February 16, 017

c. Push SELECT pushbutton for Channels 1 and to return

to Default screen … which will inactivate the SET

pushbutton for those two channels.

d. Repeat steps a through c for remaining channels.

. Under the AGGREGOMETER window, select or set-up a Test

Procedure page for Optical mode. Click on OK.

3. Select RUN NEW PATIENT under AGGREGOMETER and Click

on OK.

4. After wiping the outside with a Kim Wipe® , place BLACK

cuvette in Channel 1 PRP well and the PPP water cuvette in

the PPP well of Channel 1, with the alignment mark placed

left center.

5. Click on the Channel 1 “Activate” bar. [NOTE: If this test is to

be SAVED, Click on the “Start” Bar for Channel 1. If not

saving, the following steps can be performed after clicking

on the “Activate” button.]

6. Push and Hold the Set Baseline button until the tracing

reaches a full 100% of graph before releasing. Tracing will

then return to 0% of graph.

7. After wiping the outside with a Kim Wipe®, replace the

BLACK cuvette in the PRP well with the PRP water cuvette.

The tracing should move near 100% on the graph. Allow to

run for a few seconds and note the position where the

tracing stabilizes. (Ex: 97%).

8. Repeat Steps 4 through 7 for remaining channels, except …

leave the PPP cuvette in the Channel 1 PPP well. Also be

sure to place the Alignment Mark to the left center in each

test well and to wipe the outside of the cuvettes with a Kim

Wipe® when placing into the test well.

9. If any tracing is > 4% from 0% or 100% on the graph,

calibration of that Channel will need to be repeated,

following the previous calibration steps.

10. If saving the test … Once all channels are completed, Click on

the STOP icon followed by the SAVE icon and the PRINT icon

to save and print this test.

NOTE: If the tracings are > 4% fro 0% or 100% on the graph,

this ay indicate that Auto-Calibration was not set properly.

Recheck the two water cuvettes to be sure they atch. See

Appendix A for further instructions on atching water cuvettes,

then repeat the entire calibration procedure. Be sure to wipe the

cuvettes with a Ki Wipe® each ti e they are placed into a test

or reference well. If, after the second atte pt, the results are

still > 4%, the syste ay need service. Contact Chrono-log

Service Depart ent at 1-800-247-6665 for further assistance.

Discussion

The Black cuvette blocks all light from passing through. The clear

water samples allow the maximum amount of light through. Each

instrument is adjusted at the factory for a set voltage with water

samples. The automatic calibration procedure resets the voltage

while the water samples are in the wells.

The water samples have equal optical density and should allow

the same amount of light to pass through the samples. This will

result in a tracing at 100% of the test graph. This tracing is used

to check that the calibration is set properly.

Sometimes the difference is caused by variations in the cuvettes.

If the baseline is not within 4% of the 100% baseline, the P/N 3

Kit may need to be replaced.

The PRE-calibration, Calibration and POST- Calibration steps

described above will catch calibration errors, including conditions

where one channel has matching wells that do not match the

remaining channels. Calibration with empty wells is one instance

that would create this problem. Faults may occur at any time due

to spills, broken glass, etc. Daily Quality Control checks can be

performed to prevent false readings.

If the tracing is still > 4% after following this procedure, contact

Chrono-log Service Department at 1-800- 47-6665 to inquire

about instrument service.

Troubleshooting Guide

Many factors will affect the performance and reproducibility of

test runs on the Aggregometer. These can be broken down into

several categories; Instrument failure or Calibration problems,

Sample and Materials problems, and Operator technique related

problems. Below is a brief list of common problems.

Missing stir bar

Bubbles in sample blocking optical path

Sample volume too low

Platelet count- PRP below 75K, PPP above 3K

Platelet count- mismatch between PPP samples for each

Cuvette dirty, scratched

Stirring RPM setting mismatch

Stirring motor failure

Inconsistent pipetting; force, position, tip too short.

ROUTINE QC CHECKS should be performed on a quarterly basis, or

if any of the following conditions should exist during testing.

Optical calibration should be checked with Water Samples as

described under the Pre-Calibration procedure. If required,

Calibration of the Optical Circuits can be performed using the

same Water Samples as described under Calibration.

Test Results > 100%

Test Results on all channels do not fall within +/- 10% of the

average

If these suggestions do not solve the problem, then contact

Chrono-log or your local distributor to inquire about service.

Assistance and Distributor Information

For Assistance on training, service, software updates and other

information, contact your Chrono-log representative or your local

distributor. Information on Chrono-log Distributors can be found

on the Chrono-log website at www.chronolog.com.

Table of contents

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