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CS-5100 SOP – Prothrombin Time / INR

Document ID: SUKBMS-13-384

Date: 12/10/2015

Version: 3.0

Contents1.0 Purpose and Scope of SOP......................................................................................22.0 Responsibility............................................................................................................23.0 References................................................................................................................. 34.0 Definitions..................................................................................................................35.0 Documentation...........................................................................................................46.0 Actions and Methods.................................................................................................5

6.1 Health and Safety.................................................................................................................56.2 Principle of Test...................................................................................................................6

6.2.1 CS-5100 Analyser...........................................................................................................66.2.2 Prothrombin Time / INR...................................................................................................7

6.3 Personnel Level of Training Required...............................................................................86.4 Specimen Requirements.....................................................................................................96.5 Equipment..........................................................................................................................106.6 Reagents.............................................................................................................................10

6.6.1 Reagents Required, Reconstitution and Storage..........................................................106.7 Calibration..........................................................................................................................12

6.7.1 Using PT Multi-Calibrator..............................................................................................126.7.2 ISI Input Calibration Curves - Manual entry of ISI value and MNPT.............................19

6.7.3 Validating Reagent for Use...........................................................................................216.8 Quality Control...................................................................................................................226.9 Error Log.............................................................................................................................22

7.0 Additional Notes/Comments...................................................................................24

Document Ref: SUKBMS-13-384

Version: 3.0Page | 1

Date: 12/10/2015 Classification: Unrestricted[Security Classification]

1.0 Purpose and Scope of SOP

The following SOP describes the procedures for the Prothrombin Time assay, also used for INR calculation on the Sysmex CS-5100 Blood Coagulation Analyser.

2.0 Responsibility

It is the responsibility of the Senior/Chief BMS/Laboratory Manager, along with the Quality Manager, to ensure implementation and update of this procedure.

It is the responsibility of the Senior/Chief BMS/Laboratory Manager and Training Officer to ensure competency and training for this procedure.

Staff able to undertake these duties are trained, qualified Biomedical Scientists, and Trainee Biomedical Scientists under supervision and where stated, suitable trained MLA staff.

Before carrying out this procedure you must have;

Read and understood the laboratory safety guidelines. Read and understood the Standard Operating Procedure along with the associated health

and safety documentation. Undertaken any internal associated Competency Assessment necessary.

If there is any part of the SOP which you do not understand, you must discuss this with a senior member of staff.




3.0 References

IFU – Sysmex CS-5100

IFU – Siemens Dade Innovin Rev. 06

IFU – Siemens Thromborel S Rev. 06

IFU – Siemens PT-Multi Calibrator Rev 05

4.0 Definitions

BMS Biomedical Scientist

MLA Medical Laboratory Assistant

IFU Instructions for use

PT Prothrombin Time

INR International Normalised Ratio

MNPT Mean Normal Prothrombin Time

ISI International Sensitivity Index

MSDS Material Safety Data Sheets

IPU Information Processing Unit

TAV Table of Assigned Values

SOP Standard Operating Procedure




5.0 Documentation

Electronic copies of the following documentation can all be found on the Sysmex Customer Portal at www.sysmex.co.uk

Sysmex CS-5100 IFU

Sysmex CS-5100 Software Guide

Sysmex and Siemens material safety data sheets

Sysmex and Siemens Declarations of Conformity

Siemens Reagent IFUs

Siemens Reagent Application Sheets For CS-5100

Siemens Reagents - Certificates of Analysis

Siemens Reagents - Certificate of Traceability

References for Calculating Uncertainty

Specific ISI values for CS-Series




http://www.sysmex.co.uk/

6.0 Actions and Methods

6.1 Health and Safety

Good laboratory practice dictates that gloves should be worn when handling biological specimens. If there is a chance of spray or aerosol material then safety goggles should also be worn (for example; when removing a sample cap, or cleaning up a breakage in the centrifuge). If you have any doubts about the procedure you are carrying out, you must seek the advice of a senior member of staff.

The analyser must ALWAYS be run with the light shield down.

The cap-piercing mode uses a sharp probe and care must be taken when opening the cover. Gloves must be worn and care needs to be taken in order to avoid a needle stick injury when cleaning the probe.

In the event of exposure to or spillage of chemicals/reagents please refer to laboratory COSHH assessments and MSDS.

The CS-5100 is connected to an uninterrupted power supply (UPS) and should there be a loss of mains power the UPS will provide a temporary source of power. When this situation arises there will be a bleeping sound from the UPS. The UPS will provide power for approximately 15 minutes. If this occurs then remove all the racks from the analyser that are still waiting to be processed. If there is a rack in the middle of being processed then this can be left to exit the sampling area as normal. Wait for the analyser to complete all the tests that it is processing and once the instrument has returned to the “Ready” state power down the analyser and IPU. Wait for the power to be restored before powering up the IPU and analyser.

Do not store heavy reagents or consumables on shelves above chest height and be aware of your posture at all times when carrying out any procedure described in this SOP. Avoid any stooping posture and ensure a straight back at all times.

When replacing the rinse water it is important that the keg is not completely filled to the top as it will make it difficult to lift. Use a trolley to transport the rinse kegs, do not carry by hand.

If the liquid waste is connected to a waste container then take carry when handling and emptying the waste. Use protective clothing (e.g. gloves and safety goggles) when discarding the waste into a sink/sluice. Use a trolley to transport the waste, do not carry by hand.

If you need to open the light shield the analyser should be in ready state with the green LED lit and the green “Ready” symbol visible in the bottom left hand corner of the IPU screen. In the event of an emergency, the red ‘Stop’ button on the front of the analyser can be pressed in order to bring the instrument to stand-by; this will cause all sample processing to be aborted and allow the main covers to be opened.




6.2 Principle of Test

6.2.1 CS-5100 Analyser

The Sysmex CS-5100 coagulation analyser measures the change in transmitted light through the reaction cuvette during the coagulation process. Light from the halogen lamp is dispersed in 340, 405, 575, 660 and 800 nm wavelengths by five different filters.

Fiber

The spectroscopic light is carried from the lamp unit to the cuvette by an optical fibre where it shines through the mixture of sample and reagent in a reaction cuvette. The light transmitted through the mixture at all wavelengths is detected every 0.1 second by the photo diode and is converted into an electrical signal. The clotting time and the dOD/min (change in optical density) are derived from this signal by a microprocessor. All detectors can be used for the Clotting, Chromogenic and Immunoturbidometric assays. Various error flags provide warnings if the assay kinetics do not meet the set criteria.

A HIL (haemolysis, icterus and lipaemia) check is automatically carried out on each patient plasma sample (excludes micro mode and QC). Absorbance measurements at 405nm (icterus), 575nm (haemolysis) and 660nm (lipaemia) are made and this is used to classify the appropriate interfering substance into 6 judgement levels (0-5) before sample measurement begins. Each judgement level is distinguished by the range of absorbance. The judgement level can be seen by accessing the browser screen and selecting the “measurement information” tab. If the sample absorbance is greater than judgement level 5, a (*) symbol will be shown. The default setting is to output values with a low reliability flag. If an interfering substance is detected the analyser will continue to process the sample, however an “H, I or L” will be displayed on the ‘joblist’ in the ‘Sample Info’ column and a (*) symbol will be attached to the left side of the numeric value.




6.2.2 Prothrombin Time / INR

Innovin® or Thromborel S® Reagent is used on the CS-5100 coagulation analysers to determine PT and INR.

Innovin® reagent is prepared from purified recombinant human tissue factor produced in E. coli, combined with synthetic phospholipids (thromboplastin), calcium, buffers and stabilizers.

Innovin® reagent initiates clotting via the extrinsic and common pathways in a global screening test, the Prothrombin time (PT) and has three major applications based upon the PT:

Rapid screening test to detect single or combined deficiencies of the extrinsic coagulation system, indicative of hereditary and acquired coagulation disorders, liver disease or vitamin K deficiency;

Sensitive monitoring test for oral anticoagulant therapy with vitamin K antagonists;

Assay for specific coagulation factors.

Innovin® Reagent is manufactured using recombinant human tissue factor and synthetic phospholipids which do not contain any other clotting factors such as prothrombin, FVII and FX. Therefore, it is highly sensitive to factor deficiencies and oral anticoagulant-treated patient plasma samples. The sensitivity of Dade® Innovin® Reagent is very similar to the WHO human brain reference thromboplastin and has an INR close to 1.0.

Innovin® Reagent is insensitive to therapeutic levels of heparin. The high sensitivity of Innovin® Reagent to coagulation factors and its insensitivity to therapeutic heparin make it beneficial in monitoring oral anticoagulant therapy with vitamin K antagonists. In addition, its high sensitivity (i.e. the responsiveness of the reagent to moderately depleted factor activity) allows differentiation of abnormal plasmas, even in the mildly pathological range.

Thromborel® S Reagent contains lyophilized human placental thromboplastin, calcium chloride, stabilizers and preservatives.

Thromborel® S Reagent is used for the determination of the PT according to Quick and, in conjunction with the relevant deficient plasmas, for the determination of the activity of coagulation factors II, V, VII and X.

The PT measured with Thromborel® S Reagent is a rapid, sensitive screening test for coagulation disorders in the domain of the extrinsic system (Factors II, V, VII and X). Due to its high sensitivity for these coagulation factors Thromborel® S Reagent is especially well suited for;

The induction and monitoring of oral anticoagulant therapy.

Diagnosing genetically caused deficiencies in coagulation factors of the extrinsic system.

Diagnosing acquired deficiencies in coagulation factors.

Checking the synthesis performance of the liver in hepatic diseases.




With Thromborel® S Reagent and the relevant deficient plasma, it is possible to determine the activity of coagulation factors II, V, VII and X.

According to the joint recommendations of the World Health Organization (WHO) and the International Committee on Thrombosis and Haemostasis, the PT results for patients on vitamin K antagonist oral anticoagulants (warfarin) should be reported as INR values. Reported INR results are independent of the reagents and methods used, and are specifically intended for assessing patients stabilized on long-term oral anticoagulant therapy.

[ ]

The ISI for the reagent/instrument combination can be found on the IFU included in the Innovin or Thromborel S packs, or on the Sysmex Customer Portal under Resources/Haemostasis/CS-5100/ISI values.

The MNPT can be calculated locally using the geometric mean >20 normal samples.

Alternatively both can be calculated locally using Siemen’s PT multi-calibrator.

Additionally, the CS-5100 coagulation analyser is able to derive a fibrinogen value from the determination of the PT.

The fibrinogen concentration is derived by analysing the change in optical density during the PT determination, using a derived fibrinogen calibration curve. This calibration curve (master curve) is provided by Siemens in the lot dependant table of assigned values included in each reagent pack or on the Sysmex Customer Portal under Resources/Haemostasis/CS-5100/ISI values.

However, although useful, this derived fibrinogen is not reliable outside the normal reference range; it often gives slightly elevated levels compared to the Clauss fibrinogen assay and can be inaccurate in some disease states such as liver disease and also in patients that are anti-coagulated.

If the derived fibrinogen value is outside the normal reference range, a Fibrinogen level should be determined using the Clauss fibrinogen assay.

6.3 Personnel Level of Training Required

All BMS personnel who have had appropriate training may carry out this procedure.




ISIPatient PT

MNPT

INR =

6.4 Specimen Requirements

The sample required for PT analysis on the CS-5100 analyser is citrated whole blood (1:9 ratio of 3.2% or 3.8% sodium citrate solution to whole blood).

Centrifuge at 1500 x g for no less than 15 minutes at room temperature.

If immediate testing is to be done, the plasma may remain on the packed cells. Otherwise plasma should be separated from the cells. To separate the plasma, use a plastic transfer pipette, remove plasma to a plastic tube.

Do not store on ice or at +2 to +8 °C as cold activation of F VII may alter the results.

Plasma containing unfractionated heparin should be centrifuged within one hour of blood collection, stored at room temperature and tested within four (4) hours.

Non-heparinized plasma should be tested within four hours of blood collection.

Platelet-poor plasma may be frozen at ≤ -20 °C for up to two (2) weeks in a non-frost-free freezer. Frozen plasma should be rapidly thawed at +37 °C, gently mixed and tested immediately. Samples should not stand at +37 °C for more than five minutes.

Please refer to CLSI document H21–A5 for detailed information on sample preparation and storage.

The analysers are run in cap piercing mode which does not require the removal of caps, but they can also be used to analyse plasma samples pipetted into cups. In the latter situation, samples may be run in “micro mode” which does not require the aspiration of a daughter aliquot, thus saving sample volume. Paediatric sample tubes should also be run in ‘micro mode’ and require a carrier tube to allow a snug fit into the sample racks.

Samples run in STAT mode must have caps removed.

All sample tubes should be clearly labelled with the locally agreed minimum labelling requirements.




6.5 Equipment

A Sysmex CS-5100 Coagulation analyser.

The supplier details are as follows:

6.6 Reagents

6.6.1 Reagents Required, Reconstitution and Storage

Innovin® B4212-50 (10x10ml) or B4212-100 (12x20ml)

Or Thromborel S OUHP 29 (10x10ml) or OUHP 49 (10x10ml)

Ci-Trol® Level 1 291070 (10x1ml)

CiTrol® Level 2 291071 (10x1ml)

Ci-Trol® Level 3 (optional) 291072 (10x1ml)

Control Plasma N ORKE 41 (10X1ml) alternative to Ci-Trol® Level 1

Control Plasma P OUPZ 17 (10X1ml) alternative to Ci-Trol® Level 2

PT Multi-calibrator OPAT 03 (6x1ml)

CA Clean I 96406313 (1x50ml)

CA Clean II 97405810 (1x5L)

Reaction Cuvettes 06414810 (3000)

4ml cups EC65.1 (250)

In addition there is a requirement for approximately 20 litres of de-ionised water per day.




Switchboard: 0870 902 9210

Reagent Hotline: 0870 902 9213

Main Fax: 0870 902 9211

Customer Support Centre

Product hotline: 0870 902 9216

Service hotline: 0870 902 9217

Sysmex UK LTDSysmex HouseGaramonde DriveWymbushMilton KeynesMK8 8DF

Innovin® Reagent is reconstituted with distilled or deionised water using the volume stated on the vial label.

Allow time for the vial to reach room temperature (30 minutes) before reconstitution with the volume of distilled water indicated on the vial label. Mix carefully and leave to stand for 30 minutes at room temperature. Remix gently before loading onto analyser and allow 15 minutes for the reagent to equilibrate before use.

Note: Do not use water that contains preservatives.

Store lyophilised reagent at 2-8 °C. At this temperature, the unopened reagent can be used until its expiry date (see vial label).

Stability after opening: See Reagent insert.On-board Stability: See Application sheet for CS-5100 analyser.Do not freeze.

Thromborel® S Reagent is reconstituted with the amount of distilled or deionised water stated on the vial label and warmed to 37 °C before use. Please note: after reaching 37 °C the reagent must be incubated at this temperature for 45 minutes, even if it is warmed at another temperature on certain analysers. Before use the reagent should be mixed carefully. The reagent must be mixed every 24 hours whilst on board to achieve the stated stability.

Store Thromborel® S Reagent unopened at 2-8 °C and use by the expiry date given on the label.

Stability after opening: See Reagent insert.On-board Stability: See Application sheet for CS-5100 analyser.Do not freeze.

Ci-Trol® Level 1, Level 2, Level 3, Control Plasma N and Control Plasma P. Allow time for the vials to reach room temperature (30 minutes) before reconstitution by adding exactly 1.0 mL of distilled water to each. Gently tilt the vial to mix and leave to stand for 15 minutes with the vial closed. Before use, gently mix once more.

Stored at 2-8 °C, lyophilized Ci-Trol® can be used until the labelled expiry date. Do not freeze.

Stability after opening: See Reagent insert.On-board Stability: See Application sheet for CS-5100 analyser.

Whenever a new batch of Innovin is started, the ISI and MNPT must be changed. If calibrating locally you will need the PT-Multi Calibrator set which contains 6 plasmas.

Reconstitute the calibrators 1-6 with the amount of distilled water indicated on the label. Allow to stand for at least 30 minutes at 15-25 °C. Agitate carefully before using (prevent foam formation).

The lyophilised calibrators must be stored at 2-8 °C and may be used up to the expiry date given on the label.

Stability after opening: See Reagent insert.On-board Stability: See Application sheet for CS-5100 analyser.The calibrators may be frozen and thawed again once after reconstitution. They must be frozen as quickly as possible in the original vials, well-sealed. Thaw in a water bath at 37 °C for a




maximum of 10 minutes. The calibrators must then be used for calibration within 2 hours at 15-25 °C.

6.7 Calibration

A new calibration curve is required on each change of PT reagent batch.

6.7.1 Using PT Multi-Calibrator

1) Prepare reagents and PT Multi-calibrators and load onto the analyser.

2) Select [Order] followed by [Switch Order].

3) The “Switch Order” dialogue box appears, select [Holder Calib. Curve Order].




4) Select the [PT Multi] assay group from the bottom of the screen.

5) The “Calibration Curve Order Entry” dialogue box appears. Select the [Change] box and choose the “Reagent Lot” batch combination required for calibration from the drop down list. Press [OK].




6) Click on the [Lot] box, and select the relevant calibrator lot from the scroll box. Press [OK].

7) If the data has been input using the 2D barcode; the “Assay Sheet Value” will automatically be populated in the box.If not, use the keypad to type in the value from the TAV sheet. Press [OK].




8) Select the [Regist.] icon.

9) Select [Joblist] to conform the correct assay has been ordered, followed by the [Start] icon and the calibration will be performed. The joblist will show the progress of the calibration curve.




10) Once the calibration curve has completed, Check to see if there are any error or review messages on the Joblist. If errors or review flags present, troubleshoot accordingly.

11) Select the [Calibration Curve] icon. Press [Change], select Test e.g. [PT Multi], then choose current lot combination from dropdown list. The following screens will be displayed:




12) Check that the calibration curve is acceptable by checking the r value, this should ideally be 1.00 but a value between 0.996 - 1.00 is acceptable.

13) By selecting the [Detailed Display] icon then select the [Compared Calib. Curve] option, it is possible to view previous calibration curves for the same lot number or another lot number against the current calibration curve. The previous curve is displayed in green and is often hidden directly behind the new curve which is blue.




14) Once you are ready to accept the new curve, the curve can be validated, by selecting [Validate]. The dialog box below will be shown.

If you wish the new reagent lot (group) to be available for sample analysis using this new Calibration curve, tick the checkbox and press [OK].




If you do not wish to introduce this reagent into current use for sample analysis you can still validate the new Calibration curve but un-tick the checkbox and press [OK]. NB: If you un-tick this box a “reagent not set” flag will occur if staff try to run with this reagent.

6.7.2 ISI Input Calibration Curves - Manual entry of ISI value and MNPT

1) The ISI value can be found on the TAV sheet for the CS-series.

2) Make sure that the new Innovin lot number has been loaded onto the analyser or entered

into Reagent Master using the 2D barcode scanner.

3) From the menu select the [Calib. Curve] icon, then [Change], followed by [PT], then select the correct reagent combination form the dropdown list. Select the [Edit] button.




4) In the [Edit Calibration Curve] entry box, enter the ISI value as shown on the TAV sheet or select [Lot Master] for automatic entry of ISI Value if you have input lot information via a 2D scanner. Enter the locally derived “Normal Value”.

5) Press [OK] when finished.




6) Press [Validate]. At the [Validation of Calibration Curve] dialogue box make sure that the “This reagent is used for sample analysis” check box is ticked, then press “OK”.

If you wish the new reagent lot (group) to be available for sample analysis using this new Calibration curve, tick the checkbox and press [OK].

If you do not wish to introduce this reagent into current use for sample analysis you can still validate the new Calibration curve but un-tick the checkbox and press [OK]. NB: If you un-tick this box a “reagent not set” flag will occur if staff try to run with this reagent.




6.7.3 Validating Reagent for Use

If you chose not to set the reagent for sample analysis at the time of validation you will need to modify the reagent settings when you decide to make this reagent available for use.

1) Display the [Reagent] screen

2) Select the reagent holder which contains the relevant reagent i.e. Thrombin

3) Press [Lot Group Settings]

4) Select the relevant parameter tab and tick the checkbox next to the relevant Reagent Lot (Group), press [OK].




6.8 Quality Control

See CS-5100 SOP - QC.

It is essential that all lyophilised controls are reconstituted as described in the respective IFU and left for the stated time and temperature prior to running on the CS-5100. Failure to follow these instructions may lead to incorrect results.

Ci-Trol® 1 / Control Plasma N and Ci-Trol® 2 / Control Plasma P should be run for the PT assay at least twice a day and with every reagent vial change. If the lamp is changed, or any repairs are carried out on the CS-5100 analysers, such as a change of probe, tubing or pumps then the controls should be run again.

QC can be run from either the sample rack or from within the instrument.

6.9 Error Log

Depending on the error, if there are any problems with the analyser then it will generate an audible alarm and the “Error Help” screen will appear (the message column can be widened to display the whole message if it is not showing).




Message Board

Action Message

The message board will contain a list of all of the errors that have occurred today along with the time. The most recent error message will be at the top of the list. When you highlight an error message the “Action Message” box on the lower half of the screen will provide suggestions in order to resolve the problem.

It is worth looking at the times of the error messages underneath the most recent/last error message. On occasions an error message may be caused by a sequence of actions leading to a range of error messages. In this instance the problem is more likely to have been caused by the first error in the sequence of error messages.

1) A comment regarding the action taken to rectify the problem can be added by selecting [Log Corrective Action].

2) Select the appropriate corrective action or select [Direct Entry] to add a free text comment, click [OK] once done.

3) Error messages can be viewed by clicking the [Warning symbol] at the bottom of the IPU screen.

If all the error messages have been acknowledged then this symbol will appear white.




The [Log Corrective Action] icon can be used to record corrective actions for previous error message in order to ensure the warning triangle returns to the white icon.

4) Click the [Error Log] button to show a list of all the errors that have occurred on the analysers.

There is a detailed table of Corrective action for errors in the CS-5100 Instructions for Use.

7.0 Additional Notes/Comments

It is essential that the sample is filled to the correct fill level, any sample which is over filled or under filled should not be processed. The CS-5100 has the ability to detect under and over-filled samples. If the sample volume check is enabled then the sample will be flagged when these conditions have been triggered.

Grossly haemolysed, grossly lipaemic samples and samples with clots should be rejected. The CS-5100 has the ability to detect haemolysis, icterus and lipaemia. If this functionality is activated then samples will be flagged when these conditions are triggered.

Results of tests should always be interpreted in conjunction with the patient’s medical history, clinical presentation and other findings.

Many commonly administered drugs may affect the results of the prothrombin time. This should be kept in mind especially when unusual or unexpected abnormal results are obtained.

Innovin reagent is insensitive to concentrations of unfractionated heparin up to approximately 2.0 units per ml.

Hirudin or other direct thrombin inhibitors in therapeutic doses can result in prolonged PT.

Inhibitors such as lupus anticoagulants may interfere with the PT and result in INR values that do not reflect the exact dose of anticoagulation.

The derived fibrinogen obtained from the PT, often gives slightly elevated levels compared to the Clauss assay and can be inaccurate in some disease states such as liver disease and also in patients that are anti-coagulated.

Unexplained abnormal APTT/PT results should always be followed by additional coagulation studies to determine the source of abnormal results.

Should there be any problem with the analyser it will alarm and the error Help screen will appear. The message column can be widened if the whole message is not showing.




Sysmex UK LtdSysmex House, Garamonde Drive.WymbushMilton Keynes,MK8 8DF

Tel: 0870 902 9216Fax: 0870 902 9211

WWW.SYSMEX.CO.UK

http://WWW.SYSMEX.CO.UK/

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