Cs-5100 Sop - Aptt

Contents

1.0 Purpose and Scope of SOP

The following SOP describes the procedures for the Activated Partial Thromboplastin Time assay on the Sysmex CS-5100 Blood Coagulation Analyser. Document Ref: SUKBMS-13-385 Version: 2.0

Page | 2 Date: 12/10/2015 Classification: Unrestricted 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.

Document Ref: SUKBMS-13-385 Version: 2.0

Page | 3 Date: 12/10/2015 Classification: Unrestricted 3.0 References

IFU – Sysmex CS-5100 IFU – Siemens Dade Actin FS Activated PTT Reagent

IFU – Siemens Dade Actin FSL Activated PTT Reagent

IFU – Siemens Pathromtin SL

4.0 Definitions

BMS Biomedical Scientist

MLA Medical Laboratory Assistant

IFU Instructions for use

APTT Activated Partial Thromboplastin Time

MNAPTT Mean Normal Activated Partial Thromboplastin Time

MSDS Material Safety Data Sheets

TAV Table of Assigned Values

SOP Standard Operating Procedure

Page | 4 Date: 12/10/2015 Classification: Unrestricted 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 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

Page | 5 Date: 12/10/2015 Classification: Unrestricted 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.

Page | 6 Date: 12/10/2015 Classification: Unrestricted 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.

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. Document Ref: SUKBMS-13-385 Version: 2.0

Page | 7 Date: 12/10/2015 Classification: Unrestricted 6.2.2 Activated Partial Thromboplastin Time

The activated partial thromboplastin time, a global screening procedure is used primarily to evaluate coagulation abnormalities in the intrinsic pathway, but will also detect severe functional deficiencies in factors II, V, X, or fibrinogen. The APTT has also been widely advocated as a means to monitor the effectiveness of unfractionated heparin therapy where the clotting time is prolonged in proportion to the level of heparin. The presence of non-specific inhibitors, such as lupus anticoagulants may prolong the APTT, but this effect is variable and generally recognized as being related more to the nature of the APTT reagent employed. In summary, the APTT is a clinically important screening test with wide applicability for the diagnosis of coagulant disorders and therapeutic monitoring of both haemorrhagic and thrombotic disease. Factors of the intrinsic coagulation system are activated by incubating the plasma with the optimal amount of phospholipids and a surface activator. The addition of calcium ions triggers the coagulation process, and the clotting time is then measured. The reagents provided by Sysmex UK to measure APTT are:  Actin FS® Reagent contains liquid purified soy phosphatides with plasma activator for use in the determination of the activated partial thromboplastin time (APTT) and other coagulation procedures requiring an activated partial thromboplastin reagent. In patients receiving oral anticoagulants, the circulating levels of factors II, VII, IX, and X are depressed, therefore the APTT can be expected to be prolonged. Actin FS® Reagent is relatively insensitive to circulating lupus anticoagulants.

 Dade Actin® FSL Reagent contains purified soy phosphatides and rabbit brain phosphatides in 1.0 x 10-4 M ellagic acid with added buffer, stabilizers and preservative. No standard potency has been established and accepted for purified phosphatides. Actin FSL is relatively sensitive to circulating lupus anticoagulants. The APTT has been reported to show variable sensitivity to the presence of lupus-like inhibitors. Using Dade® Actin® FSL Reagent, the APTT exceeded the normal range in 74 % of 65 patient plasma samples with positive tissue thromboplastin inhibition (TTI) tests.

 Pathromtin ® SL Reagent contains silicon dioxide particles (1.2 g/L), plant phospholipids (0.25 g/L), sodium chloride, HEPES, pH 7.6, preservative: Sodium azide (< 1 g/L). Pathromtin ® SL Reagent enables rapid screening for disorders of the intrinsic coagulation system and sensitively detects Factors VIII and IX as well as the contact factors. In conjunction with deficient plasmas it enables the individual factors of the intrinsic system to be quantified and permits diagnosis of Document Ref: SUKBMS-13-385 Version: 2.0

Page | 8 Date: 12/10/2015 Classification: Unrestricted haemophilia. In addition it can be used for monitoring therapy with unfractionated heparin. For specimens suspected of contamination with heparin from lines, Hepzyme can be used to neutralise it and confirm contamination.  Dade® Hepzyme® is a freeze-dried preparation of purified bacterial heparinase I with added stabilizers. The presence of heparin in a specimen may interfere with the interpretation of various coagulation tests. Heparinase I, the active component of Dade® Hepzyme®, is specific for heparin. Heparin contained in plasma samples can be digested by the addition of heparinase prior to prothrombin time (PT) and activated partial thromboplastin time (APTT) assays. Dade® Hepzyme® can neutralize up to 2 units of unfractionated heparin in 1 ml of citrated plasma.

6.3 Personnel Level of Training Required

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

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.

Page | 9 Date: 12/10/2015 Classification: Unrestricted 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

Page | 10 Date: 12/10/2015 Classification: Unrestricted 6.6.1 Reagents Required, Reconstitution and Storage

Please see section 6.2.2 for information on the sensitivities of the different APTT reagents, Actin FS®, Actin FSL® and Pathromtin SL® and their intended use. Actin FS® or B4218-100 (10x10ml), B4218-20 (10x2ml) Actin FSL® or B4219-1 (10x2ml), B4219-2 (10x10ml) Pathromtin SL® OQGS 35 (20x5ml)

Ci-Trol® Level 1 291070 (10x1ml) Ci-Trol® Level 2 291071 (10x1ml) Ci-Trol® Level 3 (optional) 291072 (10x1ml)

Calcium Chloride ORHO 37 (10x15ml) Hepzyme B4240-10 (10x1ml) CA Clean I 96406313 (1x50ml) CA Clean II 97405810 (1x5L)

Trash bags CM957174 (50) 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.

Dade Actin® FS Reagent must be mixed gently by inversion (5 to 8 times) before use. Stored unopened at 2-8 °C the reagent can be used until the expiry date on the vial. If the reagent is left to stand, a green deposit may form consisting of ellagic acid and lipids. Before use, mix by inverting. Avoid contamination with plasma. Stability after opening: See Reagent insert. On-board Stability: See Application sheet for CS-5100 analyser. Do not freeze.

Dade Actin® FSL Reagent must be mixed gently by inversion (5 to 8 times) before use. Stored unopened at 2-8 °C the reagent can be used until the expiry date on the vial. If the reagent is left to stand, a green deposit may form consisting of ellagic acid and lipids. Before use, mix by inverting. Avoid contamination with plasma. Stability after opening: See Reagent insert. On-board Stability: See Application sheet for CS-5100 analyser. Do not freeze. Pathromtin SL ® Reagent must be mixed gently by inversion (5 to 8 times) before use. Every 24 hours of use, the reagent must be inverted to re-suspend any sediment. Stored unopened at 2-8 °C the reagent can be used until the expiry date on the vial. Avoid contamination with plasma. Stability after opening: See Reagent insert. On-board Stability: See Application sheet for CS-5100 analyser. Do not freeze.

Page | 11 Date: 12/10/2015 Classification: Unrestricted Calcium Chloride (CaCl2) Solution 0.025 mol/L is ready to use. The unopened vials can be stored at 2-25 °C until the expiry date indicated on the label. Stability after opening: See Reagent insert. On-board Stability: See Application sheet for CS-5100 analyser.

Ci-Trol® Level 1, Level 2 and Level 3 (optional) are reconstituted by adding exactly 1.0 mL of distilled water to each. Gently tilt the vial 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. Stability after reconstitution: See Reagent insert. On-board Stability: See Application sheet for CS-5100 analyser.

Dade Hepzyme ® Reconstitute each vial with 1 ml patient citrated plasma. Allow to stand at room temperature (15-25 °C) for 15 minutes before testing. Mix carefully once more before using. If stored unopened at 2-8 °C, the reagent can be stored and used up to the expiry date indicated on the label. Stability after reconstitution: See Reagent insert. On-board Stability: See Application sheet for CS-5100 analyser.

Ci-Trol® Level 1 may be used as a QC for Hepzyme neutralisation. Reconstitute two vials of the same lot and combine. Perform APTT determination. The result must fall within the determined confidence range. Prepare a heparin stock solution with approximately 19.6 U/ml heparin (use the same heparin as administered for heparin therapy) in physiological saline as follows: For heparin with 1000 U/ml, add 0.1 mL heparin to 5.0 mL saline and mix well. To 1.5 ml Dade® Ci-Trol® Level 1 add 0.02 mL (19.6 U/ml) heparin stock solution. This control will contain approximately 0.26 U/ml heparin. Gently invert to mix. Perform APTT determinations of heparin-spiked Dade® Ci-Trol® Level 1. The results must lie within the allowed confidence range established within each laboratory for this control when spiked with heparin from a specific source/manufacturer. Neutralize 1 ml of the heparin-spiked Dade® Ci-Trol® Level 1 with Dade® Hepzyme®, handling it in the same manner as a patient plasma. Perform APTT determination of the neutralized Dade® Ci-Trol® Level 1. The results should lie within the confidence range established in the laboratory for an APTT or within an allowable confidence range for this control (also calculated in each individual laboratory) when neutralized with Dade® Hepzyme®.

Page | 12 Date: 12/10/2015 Classification: Unrestricted 6.7 Calibration

Calibration is not required for the APTT. However, whenever the batch changes you will need to enter the new mean normal APTT on the calibration screen if you require APTT ratios for patients on heparin therapy. The MNAPTT is the geometric mean of the APTT results in seconds of at least 20 normal samples (usually obtained in-house).

6.7.1 Input of Normal Value for APTR-R Calculation

1) Load the required reagents on-board the analyser.

2) Select [Calib. Curve] followed by [Change], and select [APTT].

3) Select [Edit].

Page | 13 Date: 12/10/2015 Classification: Unrestricted 4) Enter the MNAPTT into the “Normal Value” box and the expiry date will automatically default to the expiry date of the reagent. Press [OK].

5) Press [Validate].

Page | 14 Date: 12/10/2015 Classification: Unrestricted If you wish the new reagent lot (group) to be availble 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 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]

Page | 15 Date: 12/10/2015 Classification: Unrestricted 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).

Page | 16 Date: 12/10/2015 Classification: Unrestricted 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 acion taken to rectify the problem can be added by selecting [Log Corrective Action].

Page | 17 Date: 12/10/2015 Classification: Unrestricted 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.

Page | 18 Date: 12/10/2015 Classification: Unrestricted 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. APTT testing encompasses the entire clotting process from contact activation to fibrin formation and is therefore more susceptible to variations than specific individual tests. The control and use of APTT is therefore subject to inherent limitations.

APTT testing may be affected by a number of commonly administered drugs. A decrease in APTT times has been noted in males on oestrogen therapy and females on oral contraceptives. Increase in APTT times has been seen in diphenylhydantoin, heparin, warfarin, naloxone and radiographic agent administration. Therapeutic doses of hirudin or other direct thrombin inhibitors may prolong clotting times.

When the APTT is used as a control of heparin treatment the results should be expressed as a ratio (of the time obtained for a normal pool containing no heparin) as APTT reagents have different sensitivities to heparin. Another problem with using APTT as a heparin control is that it is affected by a number of variables not related to heparin such as fibrinogen and factor VIII concentration and the presence of fibrinogen degradation products. When these factors are abnormal there may be a dissociation of the APTT and heparin level causing an apparent heparin resistance and in such circumstances a heparin assay should be performed.

When using the APTT for monitoring heparin therapy the following should be taken into consideration:

o The half-life of unfractionated heparin in vivo is approximately 45-60 minutes in vivo, when administered it has an immediate anticoagulant effect, but the degree of the effect decreases rapidly with time. Thus the time of collection of sample is important, when the patient is having intermittent single intravenous injections. o Platelet factor 4, a heparin neutralising factor in platelet alpha granules can be released by platelet damage or aggregation. Thus samples should be collected with the minimum amount of trauma. o Platelet aggregation can occur at cold temperatures thus centrifugation should occur at room temperature for heparin studies.

Page | 19 Date: 12/10/2015 Classification: Unrestricted Samples with unexplained high APTT results can be treated with Hepzyme which neutralizes the action of Heparin. This can therefore be used as additional information to identify or exclude Heparin as a cause of the raised APTT.

If neutralization does not return the APTT to within the normal range and high levels of heparin are suspected, sequential neutralization may be tried. A plasma specimen should not be subjected to more than two sequential neutralizations, and heparin levels should not exceed 4 USP units. Shortened APTT results may indicate some abnormal pro-coagulant condition in the patient’s coagulation system or the pre-activation of the sample during phlebotomy.

Inhibitors such as lupus anticoagulants may interfere with the APTT.

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

Page | 20 Date: 12/10/2015 Classification: Unrestricted Document Ref: SUKBMS-13-385 Version: 2.0

Page | 21 Date: 12/10/2015 Classification: Unrestricted Sysmex UK Ltd Sysmex House, Garamonde Drive. Wymbush Milton Keynes, MK8 8DF

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