ATS-13 Activity Assay
Contents
1. 044104 0 a r 4 Test i eee 4 CALCULATIONS AND 6 75 0 20 2200 4 naana iawa 5 T 5 ATS 13 Activity Assay 1 303280 IFUEN REV F INTENDED USE ATS 13 Activity Assay is for the quantitative measurement of ADAMTS 13 protease activity SUMMARY AND EXPLANATION It was recently discovered that ADAMTS 13 is the protease responsible for cleaving von Willebrand Factor deficiency of ADAMTS 13 activity has been demonstrated in the plasma of thrombotic thrombocytopenic purpura TTP patients The lack of ADAMTS 13 activity results in the accumulation of multimers of von Willebrand Factor in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP Mild or moderately decreased levels of ADAMTS 13 activity have also been associated with other disease states and conditions PRINCIPLE OF THE PROCEDURE The ATS 13 Activity Assay is based on fluorescence resonance energy transfer FRET technology A synthetic fragment of the von Willebrand Factor protein is used as the Substrate Cleavage of this peptide between two modified residues releases the fluorescence quenching capabilities This assay is2. Control Recording Sheet Discard after single use ATS 403577 Calibrator A Store at 15 to 30 C Contains human source material Thaw and mix thoroughly before use CALA Ready for use Values can be found on ATS 13 Activity Assay Calibrator and Control Recording Sheet Discard after single use ATS 403578 Calibrator B Store at 15 to 30 C Contains human source material Thaw and mix thoroughly before use CALB Ready for use Values can be found on ATS 13 Activity Assay Calibrator and Control Recording Sheet Discard after single use ATS 403579 Calibrator C Store at 15 to 30 C Contains human source material Thaw and mix thoroughly before use CALC Ready for use Values can be found on ATS 13 Activity Assay Calibrator and Control Recording Sheet Discard after single use ATS 13 Activity Assay 2 303280 IFUEN REV F ATS 403580 Calibrator D Store at 15 to 30 C Contains human source material Thaw and mix thoroughly before use CALD Ready for use Values can be found on ATS 13 Calibrator and Control Recording Sheet Discard after single use ATS 403581 Calibrator Store at 15 to 30 C Contains human source material Thaw and mix thoroughly before use CALE Ready for use Values can be found on ATS 13 Activity Assay Calibrator and Control Recording Sheet Discard after single use PRECAUTIONS Do not use reagents that are turbid or contaminated Care MUST be taken to avoid contamination of Calibrators and Substrate
3. based on quantifying the cleavage of a small fragment of von Willebrand Factor by the ADAMTS 13 protease The cleavage of this synthetic substrate is detected by reading the fluorescence that results when the substrate is cleaved REAGENTS Maximum number of tests per kit ATS 13 40 tests per kit All reagents should be stored as directed by the label REF ATS 403575 Black Microwell Strips Once removed from the foil pouch take care not to expose the strips to dust or MS particulates Take care to protect from moisture Strips should be stored at room temperature Ready to use ATS 403614 Substrate lyophilized Keep substrate protected from light Store lyophilized material at 15 to 30 C SUB Hydrated substrate should be stored upright in the original stoppered vial sealed with Parafilm wrap at 15 to 30 C non cycling freezer in the dark ATS 403600 Specimen Diluent Ready for use Store at 2 to 8 C SD 403612 Substrate Buffer Ready for use Store at 2 to 8 C SB 403597 Positive Control High Store at 15 to 30 C Contains human source material Thaw and mix thoroughly PCH before use Ready for use Values can be found on ATS 13 Activity Assay Calibrator and Control Recording Sheet Discard after single use PCL 403598 Positive Control Low Store at 15 to 30 C Contains human source material Thaw and mix thoroughly before use Ready for use Values can be found on ATS 13 Activity Assay Calibrator and
4. 000 RCF may cause tube breakage and exposure to blood and possible injury Following centrifugation transfer the top 2 3 of the plasma layer into a new plastic tube Re centrifuge the collected plasma at 1500 to 1800 RCF with the brake off for an additional 15 20 minutes to remove any red cells or platelets 7 Transfer the top 2 3 of the plasma into new plastic tube taking care not to disturb any cells at the bottom of the tube Sample Storage 1 Plasma should be stored at 2 to 8 C and assayed within 4 hours OR aliquoted and frozen at 70 C or colder for up to 6 months 2 Frozen plasma should be thawed rapidly at 37 C Thawed plasma should be stored at 2 to 8 C and assayed within 4 hours ATS 13 Activity Assay 3 303280 IFUEN REV F PROCEDURE Materials Provided Box A 303281 1 6x 130 uL Positive Control High 2 6 130 Positive Control Low 3 6 sets of Calibrators 5 levels 130 uL each Calibrator A Calibrator B Calibrator C Calibrator D Calibrator E 4 1 0 10 mg Substrate Box B 303282 1 2 Microwell frames each containing 6 2 x 8 Black Microwell Strips 2 1 14 Specimen Diluent 3 1x14mL Substrate Buffer Additional Materials Required Polypropylene plastic test tubes for patient sample dilutions and substrate dilution Transfer pipets Adjustable micropipets to deliver 10 100 uL and 100 1000 uL Disposable tips DMSO Reagent Grade Fluorescent plate reader capable of measuring fluoresce
5. INSTRUCTIONS FOR USE _ IMMUCOR REF ATS 13 For Research Use Only Not for use in diagnostic procedures 30 C 8 C ri 5 303281 303282 TABLE OF CONTENTS INTENDED USE canes senet ances svenvecaeeiazaseosuzede 2 SUMMARY 2 2222 2 2 PRINCIPLE OF THE PROCEDURE oie 550 2 5 45 2 PREGA UT 3 os ooo ace 65 A 3 SPECIMEN COLLECTION AND STORAGE ccccseesseesesseeeeeeeenseneeeeeeeeeeeesaeeasaneesaeeesaeessaeeeseeesseeeeseeesseeeeaeeesseeeenseaseeeeeas 3 Sample Collection and Preparation 3 Sample Storage a aeren ea ree seule Sole at a ed ae 3 4 Materials Provided alee 4 Additional Materials
6. Inadvertent contamination of these reagents with human plasma will invalidate the assigned values of the calibrators Unopened and lyophilized reagents are stable until the expiration date printed on the box when stored as directed Do not use reagents beyond their expiration date Microwells and reagents contained in the kit are not to be used in conjunction with any other test system Discard any unused portions of Calibrators Controls and used Black Microwell Strips after each run Substitution of components other than those provided in this kit may lead to inconsistent or erroneous results When making dilutions follow pipette manufacturer s instructions for appropriate dispensing and rinsing techniques The enzyme substrate reaction is temperature sensitive and should be performed in a controlled area at 22 to 25 C Only plasma should be used in the assay Serum will give inaccurate results Care MUST be taken to avoid the introduction of particulate material cardboard paper towel fiber foam debris dust etc into the wells of the assay CAUTION human plasma used in the Calibrators and Positive Controls for this product has been tested and found negative for antibody to HIV HCV and HBsAg by FDA approved methods No test method however can offer complete assurance that HIV Hepatitis C virus Hepatitis B virus or other infectious agents are absent Therefore these materials should be handled as potentially infectious Di
7. NOTE Remaining Stock Substrate Solution should be stoppered and stored upright the original vial with original stopper sealed with Parafilm wrap at 20 C non cycling freezer in the dark Re hydrated stock can be used for up to 6 months following re hydration 12 Place plate in fluorimeter with Excitation 340 350 nm and Emission 440 450 nm at room temperature Read and start timer for 30 minutes Record results as time zero NOTE Reading must be taken within 5 minutes of addition of substrate 13 Remove plate from fluorimeter store plate at room temperature not in plate reader and protect from light NOTE cover plate with paper or cardboard Fibers in the plate can cause random fluorescence Cover with aluminum foil 14 At 30 minutes place plate fluorimeter with Excitation 340 350 nm and Emission 440 450 nm at room temperature Read and record results CALCULATIONS AND RESULTS Use the ATS 13 Analysis Workbook provided on the CD 503001 to obtain the results Normal ADAMTS 13 Activity for controls and samples Instructions for the workbook are provided in the User Manual included on the CD compact disc The values calculated for the Positive Control High and Positive Control Low should fall within the allowable range identified on the lot specific ATS 13 Reporting Card Assays where the controls do not meet these criteria should be considered invalid and should be repeated Plasma sample
8. gnated on the Recording Sheet Do not dilute 8 Add 50 uL of the prediluted sample plasma solution prepared in step 4 in duplicate to the appropriate microwells of the black microwell strips as designated on the Recording Sheet NOTE If multiple patient samples are tested at the same time only one set of calibrators and controls are required 9 Prepare Stock Substrate Solution Remove stopper carefully as some Substrate may cling to the plastic Reconstitute the lyophilized Substrate by adding 37 uL of reagent grade DMSO to the Substrate vial Mix solution and add 113 uL reagent grade Replace the stopper and close the cap tightly Mix well by gently swirling until all contents are dissolved ATS 13 Activity Assay 4 303280 IFUEN REV F 10 Prepare the assay Substrate Solution 8 in a plastic tube according to the table below Patient Samples Volume Stock Substrate Volume Substrate to Test Solution pL Buffer L 1 25 795 5 37 1193 10 53 1714 40 150 4850 This be prepared by adding substrate buffer directly to stock substrate vial if being used for testing within one assay NOTE A repeating pipette should not be used 11 Mix the solution thoroughly Protect from light Immediately following preparation add 50 uL of Substrate Solution into each microwell containing a patient sample calibrator or control Gently tap the sides of the microwell frame to ensure even distribution
9. nce at Excitation 340 350 nm and Emission 440 450 nm Timer Centrifuge Aluminum Foil 0 CD Compact Disc 503001 available from Immucor Customer Service e 5 13 Analysis Workbook e User Manual 11 Reporting Card available at www immucor com 12 Plate Layout Sheet optional available at www immucor com ROT Go Ne Test Procedure 1 Allow all reagents to warm to room temperature NOTE Only remove one set of Calibrators and Controls per assay 2 Determine the number of patient samples to be tested Using the Recording Sheet assign each sample to a location consisting of two duplicate wells Record the identity of each sample on the Recording Sheet Place the sample replicates horizontally e g CALA in wells A1 and 2 3 Remove microwell frame from pouch Promptly remove unneeded strips from frame and reseal the protective pouch 4 Ina plastic test tube dilute each patient plasma sample to be tested by adding 18 uL plasma into 132 uL Specimen Diluent 5 Add 50 uL of each Calibrator in duplicate to the appropriate microwells of the black microwell strips as designated on the Recording Sheet Do not dilute 6 Add 50 uL of Positive Control Low in duplicate to the appropriate microwells of the black microwell strips as designated on the Recording Sheet Do not dilute Add 50 uL of Positive Control High in duplicate to the appropriate microwells of the black microwell strips as desi
10. onged or repeated exposure if swallowed P260 Do not breathe dust fume gas mist vapours spray P261 Avoid breathing dust fume gas mist vapours spray P264 Wash skin thoroughly after handling P270 Do not eat drink or smoke when using this product P272 Contaminated work clothing should not be allowed out of the workplace P280 Wear protective gloves protective clothing eye protection face protection P301 P312 P330 IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell Rinse mouth P302 P352 IF ON SKIN Wash with plenty of soap and water P314 Get medical advice attention if you feel unwell P333 P313 If skin irritation or rash occurs Get medical advice attention 501 Dispose of contents container to approved waste disposal plant ATS 13 Activity Assay 6 303280 IFUEN REV F
11. s with calculated ADAMTS 13 activities greater than the value assigned for Cal E will be reported as greater than the assigned value for Cal E by the Analysis Workbook REFERENCES Collection Transport and Processing of Blood Specimens for Coagulation Testing and General Performance of Coagulation Assays Approved Guideline H21 A4 NCCLS 2003 23 35 Kokame K Matsumoto M Fujimura Y Miyata T Blood 2004 103 607 Kokame K Nobe Y Kokubo Y Okayama A Miyata T Br J Haematol 2005 129 93 L mmle B George JN Seminars in Hematology 2004 41 1 1 L mmle B Kremer Hovinga JA Alberio L J Thromb Haemost 2005 3 1663 ATS 13 Activity Assay 5 303280 IFUEN REV F EC REP Immucor GTI Diagnostics Inc Immucor Medizinische Diagnostik GmbH 20925 Crossroads Circle Adam Opel Strasse 26A Waukesha WI 53186 USA Rodermark 63322 Germany US and International Contact Information Technical Support waukeshatechsupport immucor com www immucor com MICROSOFT and EXCEL are trademarks of Microsoft Corporation PARAFILM is a trademark of Bemis Company Inc Any other brand name that may appear in this package insert belongs to its respective owner 2005 2015 Immucor GTI Diagnostics Inc 303280 IFUEN Rev F 2015 05 15 Warning Warning H302 Harmful if swallowed H317 May cause an allergic skin reaction H373 May cause damage to organs Kidney through prol
12. scard all components when completed according to local regulations SPECIMEN COLLECTION AND STORAGE Sample Collection and Preparation NOTE Only platelet poor plasma collected in 3 2 sodium citrate may be used for this assay Do not use plasma that has been collected in or treated with EDTA See Collection Transport and Processing of Blood Specimens for Coagulation Testing and General Performance of Coagulation Assays Approved Guideline H21 A4 NCCLS Volume 23 Number 35 December 2003 for details Plasma collection should be performed as follows 1 Collect blood in buffered sodium citrate light blue top 3 2 plastic tubes available in 4 5 mL 2 7 mL or 1 8 mL full draw tubes NOTE Partial draw tubes should NOT be processed Since the tubes are pre calibrated to draw the specified amount of blood the resulting sample will not have the proper 9 1 ratio of blood to anticoagulant if a full sample is not collected 2 After collection store tube upright at room temperature until centrifugation NOTE Blood samples should be centrifuged between fifteen minutes and two hours after blood collection for best results 3 Remix the blood sample immediately prior to centrifugation by gently inverting the tube 8 to 10 times 4 Centrifuge blood sample at room temperature in a horizontal rotor Swing out rotor for 15 20 minutes at 1500 to 1800 RCF Relative Centrifugal Force with the brake off WARNING Excessive centrifuge speed over 2
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