Der Inhalt dieses Dokuments dient lediglich als Beispiel, die aktuellste Version senden wir gerne zu: [email protected]. The content of this document is an example only. You may request the latest version at: [email protected] stored closed, of: stored is they beuntil expiry can used dateconditions, the printed Unopened STORAGE for stabilize to use. homogenize before allow method, manual For guide application instruction. for stabilize to use. allow homogenize before method, manual f For of formation avoiding while the on analyzer following directly application guide dissolution instruction. complete until vigorously Shake removeGently freeze the R • • • • • • AND PRECAUT WARNINGS achieve to order in batch each for adjusted is concentration and optimum assay. for the linearity reactivity Xa Factor the necessary, If 2 x 4 g/L) substance. (0.9 azide sodium of amounts small x 3 11(65)). x 3 stabilisers. REAGENTS: + Substrate (residual)] [FXa +[FXa (excess)] [DiXaI] of concentration 405 at absorbance by (measured released pNa of amount being DiXal FXa the the by inhibition, hydrolyses and the constant on a based of method assayed, chromogenic a is DiXal BIOPHEN™ PRINCIPLE: or thrombotic overdose) suspected associate presenting factor patients risk for a accident, presenting patients for surgery, emergency help situations, clinical some in may, it or required be may concentrations inhibitor Xa Factor direct of Measurement Clinical: to (UFH heparins and and insensitive LMWH). to method direct FXa inhibitors specific two chromogenic a stages BIOPHEN™is kit The DiXaI Technical: AND EXPLANA SUMMARY method suitable suchmethod.for not is Fondaparinuxindirect as inhibitors heparins.This or Xa Edoxaban Factor direct anti of an determination is kit DiXaI BIOPHEN™ The USE INTENDED stored closed, of: stored is

R2 R1 R3 R1 R3 R2 R1

EAGENT PREPARATION EAGENT gn suis hw ht h raet cn e hpe a ro tmeaue without temperature room at shipped be of device This can reagents the degradation. that show studies Aging safety the only fromUse reagents samekits. the of batch with compliance full should be accordance disposed of Waste applicable with in regulations. local in care of extreme with users or contact pipes,lead copper In can sodium generate compounds. explosive azide Therefore, exercise absent. must are types manipulation the in precautions agents these infectious of that reagents assurance complete offer can approved reagents, for and theseHCV, andand antigen, 2 surface B HIV of hepatitis 1, HIV to preparation antibodies the for the plasma the test for to used are requiredmethods is plasma human thes Whenever in provided reagents Some

2.5 mL mL 2.5 mL 2.5

0

155 rue d’Eragny, Neuville rue d’Eragny, 155 95000 HYPHENBioMed Reagent stability after reconstitution, free from any contamination or evaporation, and and evaporation, or contamination any from free reconstitution, after stability Reagent and evaporation, or contamination any from free reconstitution, after stability Reagent egn 3: Reagent Reagent is ready to use; homogenize and load it directly on the analyzer following following analyzer the on directly it load and homogenize use; to ready is Reagent egn 2: Reagent R2

egn 1: Reagent mL Chromogenic method for the assay of directChromogenic of methodassay the for

i ctae hmn lsa o prfe mdu) uig n uoae o manual or automated an using medium), purified (or plasma human citrated in , Reconstitute theeach contentsof with vial exactly ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪

vial

AND STABILITY AND

vial vial egns hud e trd t 2 at stored be should reagents

s. s.

s. s. Stability on of board theanalyzer:Stability se months2 days7 months2 onof board theanalyzer:Stability the ap see specific months2 days7 days 15 :

DiXal the sample. in

in vitro

ufr Tris Buffer: X () Prfe, freeze Purified, (h): FXa usrt: Freeze Substrate:

- 1 pcfc hooei sbtae rlaig aairaiie pa. The (pNa). paranitroaniline releasing substrate, chromogenic specific - 4

. in the management of management the in at room at (18 temperature room at (18 temperature

diagnostic use professional is diagnostic intended for use the in laboratory. ➔ at 2 at -

dryingto stopper, when avoid product any opening theloss vial. frozen at at frozen 2 at at frozen : R1

TION: [FXa : IONS: ➔

-

8°C.

- - xes uniy f atr a Fa. h rsda Fco Xa Factor residual The (FXa). Xa Factor of quantity excess

NaCl 8°C. Peptide +pNA

-

DiXaI] +[FXa residual]

BIOPHEN™DiXaI R2

of these materials of biological they as wereif infectious.

results are found to be negative. However, no test method test no However, negative. be to found are results

- - -

EDTA reaction buffer, pH 7.85. Contains 1% PEG and and PEG 1% Contains 7.85. pH buffer, reaction EDTA niios ( inhibitors 20°C or20°C less* or20°C less* kt cnan aeil o hmn n aia origin. animal and human of materials contain kits e

- re Fco Xa Factor dried - - sur 3 x 2.5 mL; 3 x2.5 mL; Xa chromogenic method for the the for method chromogenic Xa

- REF Oise, Oise, France - as re hmn atr a Cnan BA Ti and Tris BSA, Contains Xa. Factor human dried

- patients receiving DiXal treatment (e.g.: prior to to prior (e.g.: treatment DiXal receiving patients ° i ter rgnl akgn. ne these Under packaging. original their in 8°C 30 30 preservative. DiXaIs - - 25°C). 25°C).

hemorrhagic 221030 iue a ro tmeaue (18 temperature room at minutes (18 temperature room at minutes , uh s Rivaroxaban as such ),

e the e application. specific -

pcfc hooei sbtae (CS substrate chromogenic specific

on theon R3

nm) is inversely proportional to the the to proportional inversely is nm) 2.5 mL 2.5 of distilled water

Factor (DiXaIs) Xa inhibitors otis a Contains

4 x kit psds o i te vn of event the in or episodes, .

20

d with an an with d

mL plication.

eai neutralizing heparin n vitro in a ad od it load and oam

,

Apixaban hemorrhagic

quantitative quantitative . - - 25°C), 25°C),

or or -

Also refer to the specific to refer specific the Also application guide • • • Reagents: AND MATERIA REAGENTS contaminatedDiscardAa vial color substrate. yellow the use indicates newand a one. once, rapidly possible37°C as *Thaw as use at and immediately. only of: closed, is 2. Dilute the samples the Dilute 2. in curve,dilute the calibration the Resuspend 1. Apixaban assay: absorbance. Mea test: the of that of citricAcetic Xa(h), or Factor sample. diluted substrate, (20%) test order acid (2%), acid reverse the in reagents the mixing by obtained is blank sample The (20%). acid The resulting*Or yellow2 acetic stable for hours. colour is acid(2%)* Citric following a Add the to 3. tube plastic at incubated concentrations are the control on flyer the with provided indicated (18 at stored If controls. quality the with test and curve calibration the Perform samples the Dilute 2. curve,dilute the calibration 1. Rivaroxaban assay: specific See request. on available are specific and application guide guides application method, automated an For 37 at test the Perform methods. (endpoint) manual or automated kinetic, for used be can kit The PROCEDURE: plasma For please references storage, to refer storage).handling and collection, see CLSIH21 States, the United the ( guidelines local applicable with accordance in stored and prepared be should Specimens venipuncture. clean M,volume) (0.109 by 3.2%) thetube. Discard first (1 anticoagulant citrate trisodium the onto collected carefully be should volumes) (9 blood The COLLECTION SPECIMEN • • • Materials:

Calibrators R3 R2 R1 References References Calibrators eupn the Resuspend Rivaroxaban Rivaroxaban °C Controls Calibrators 20% water. Distilled Stopwatch, calibrated pipettes. Stopwatch,calibrated Water Spectrophotometer - low range l Apixaban Apixaban Apixaban

Samples Samples ow range 25 Dosage Dosage ue h otcl est a 405 at density optical the sure

Substrate (h) FXa Reagent stability after opening, free from any contamination or evaporation, and stored and evaporation, or contamination any from free opening, after stability Reagent and read colorand at intensity C, h dltd samples diluted the °C), Mix and measure optical the Mix density at and acetic acid or (endpoint acid acid 2%acetic citric method) - bath.

▪ ▪ ▪

, test or plasmas, controls dilutedor Mix and incubate Mix incubate and

pre

and specific controls with specific suchand known as: titration,

Stability onof board theanalyzer:Stability the see specific days7 months2

pre - incubated incubated at calibrators calibrators calibrator - incubated

Apixaban Calibrator Apixaban Calibrator in

Mix and incubate at at Mix incubate and and

at room at (18 temperature 225301 / / 225201225301 / 226201 Calibrator Calibrator

Reference R3 R3 reference Control / Low/ Control

BIOPHEN™ BIOPHEN™ Reagents

226101 226201 226001 222701 AND PREPARATION at 2 at chromogenic chromogenic assay Apixaban calibrator calibrators LS REQUIRED BUT NOT NOT BUT REQUIRED LS

NA NA

ad otos s ecie i te pcfc ntutos Fr the For instructions. specific the in described as controls and s buffer, as as buffer, table the below:described in as buffer, table the below:described in at for 37°C / Low /

n cnrl a dsrbd n h seii isrcin. o the For instructions. specific the in described as controls and 37°C - at 37°C 8°C.

405 hud e etd quickly tested be should precautions eachanalyzer. for

Stop the reaction by reactionby the Stop adding: 226101

s s

- nm s in

A5 nm. Subtract the measured blank value from the test test the from value blank measured the Subtract nm.

in in

. 6

exactly for further specimenguideline concerning information R3 R3 ofthe analyzer used. 37°C, 37°C,

buffer, the described as buffer, table below.in the described as buffer, table below.in 405 7 analyzer . : reference reference in

Rivaroxaban Control - Control Control

for 25°C). 225201 225301 225101 224501 1 minute 1 37°C

: y n a m r e G r o f t c a t n o c 33-3 66 Fax:0800-24 e e | 33-0 66 0800-24 Tel: r f - l l o T Tel: +43-1-236 222 1 | Fax: 1 222 Tel: +43-1-236+43-1-236 222 111 GmbH [email protected] | www.coachrom.com Diagnostica CoaChrom Support: and Sales

Calibrator / / Calibrator Low

224501 / / 225101224501 / 226001222701 nm NA R3 NA

English, revision: last English, 45 seconds 45 Rivaroxaban BIOPHEN™ BIOPHEN™ PROVIDED:

against the corresponding the against blank. :

.

s s / Low /

Fr ah ac, the batch, each For .

, then add the add the then ,

exactly

kit.

D750

Dilution in reagent

Dilution in reagent

1/15 (standardrange) 1/15 1/40 (standard range) 1/40 application.

1/3 (low range) (low 1/3 1/6 (low range) (low 1/6 following: 0 Edoxaban Calibrator Edoxaban Calibrator - 3 Edoxaban Control 0 Volume - 400 µL 400 µL 200 µL 200 µL 200 room temperature room 201 2255 226 2 1/15 1/40 1/3 1/6 /BI/1030/v BIOPHEN™ BIOPHEN™ calibrator 9 501 / / 501 2264

/ 01

/

Low

Low

2254

R3 R3

and

01 01

for for

7

/

Perform the calibration curve and test with the quality controls. If stored at room temperature QUALITY CONTROL: (18-25°C), the diluted samples should be tested quickly. For each batch, the calibrators and The use of quality controls serves to validate method compliance, along with between-test control concentrations are indicated on the flyer provided with the kit. assay homogeneity for a given batch of reagents. 3. Add the following to a plastic tube incubated at 37°C: Include the quality controls with each series, as per good laboratory practice, in order to Reagents Volume validate the test. A new calibration curve should be defined, preferably for each test series, Calibrators, or test plasmas, or controls diluted in R3 200 µL and at least for each new reagent batch, or after analyser maintenance, or when the measured R1 FXa (h) pre-incubated at 37°C 200 µL quality control values fall outside the acceptable range for the method. Mix and incubate at 37°C for exactly 1 minute, then add the following: Each laboratory must define its acceptable ranges and verify the expected performance in its R2 Substrate pre-incubated at 37°C 200 µL analytical system.

Mix and incubate at 37°C, for 45 seconds exactly RESULTS: Stop the reaction by adding: Citric acid (2%)* 400 µL • For the manual endpoint method, plot the calibration curve, with the OD 405 nm along the Mix and measure the optical density at 405 nm against the corresponding blank. Y-axis and the analyte concentration along the X-axis: *Or acetic acid (20%). The resulting yellow colour is stable for 2 hours. - Rivaroxaban low range, use a Lin-Log scale (ng/mL – OD). The sample blank is obtained by mixing the reagents in the reverse order of that of the test: - Rivaroxaban standard range, use a Lin-Lin scale (ng/mL – OD). Acetic acid (20%) or citric acid (2%), substrate, Factor Xa(h), diluted test sample. - Apixaban, use a Lin-Lin scale (ng/mL – OD) for both ranges. Measure the optical density at 405 nm. Subtract the measured blank value from the test - Edoxaban low range, use a Lin-Lin scale (ng/mL – OD). absorbance. - Edoxaban standard range, use a Lin-Log scale (ng/mL – OD). • The concentration of DiXal (in ng/mL) in the test sample is inferred directly from the Edoxaban assay: calibration curve, when the standard dilution is used. 1. Resuspend the calibrators and controls as described in the specific instructions. For the • The results should be interpreted according to the patient's clinical and biological status. calibration curve, dilute the calibrators in R3 buffer, as described in the table below. 2. Dilute the samples in R3 buffer, as described in the table below: LIMITATIONS: Calibrators Controls Dosage Dilution in reagent R3 • To ensure optimum test performance and to meet the specifications, the technical reference reference instructions validated by HYPHEN BioMed should be followed carefully. Edoxaban 226501 225501 1/15 • Any reagent presenting an unusual appearance or showing signs of contamination must be Edoxaban rejected. 226401 225401 1/4 low range • Any suspicious samples or those showing signs of activation must be rejected. 1/15 (standard range) • Highly concentrated samples can be pre-diluted in a pool of normal plasmas. The measured Samples NA NA 1/4 (low range) concentrations should then be multiplied by the supplementary dilution factor. Perform the calibration curve and test with the quality controls. If stored at room temperature (18-25°C), the diluted samples should be tested quickly. For each batch, the calibrators and EXPECTED VALUES: control concentrations are indicated on the flyer provided with the kit. Apixaban, Rivaroxaban and Edoxaban are not found in normal plasma. 3. Add the following to a plastic tube incubated at 37°C: The normal interval, therapeutic range and hemorrhagic risk range should be defined Reagents Volume according to applicable local guidelines. Calibrators, or test plasmas, or controls diluted in R3 200 µL R1 FXa (h) pre-incubated at 37°C 200 µL PERFORMANCE: Mix and incubate at 37°C for exactly 1 minute, then add the following: • The lower limit and the measurement range are defined by the analytical system R2 Substrate pre-incubated at 37°C 200 µL used. Mix and incubate at 37°C, for 45 seconds exactly • For the standard range, the calibration range is about 0 to 500 ng/mL Stop the reaction by adding: Rivaroxaban/Edoxaban and about 0 to 600 ng/mL Apixaban. Citric acid (2%)* 400 µL • For the low range, the calibration range is about 0 to 100 ng/mL Rivaroxaban and about 0 Mix and measure the optical density at 405 nm against the corresponding blank. to 120 ng/mL Apixaban/Edoxaban. *Or acetic acid (20%). The resulting yellow colour is stable for 2 hours. • Performance studies were conducted internally on 1 batch of reagent on Sysmex CS-series. The sample blank is obtained by mixing the reagents in the reverse order of that of the test: Performance was assessed using the laboratory's controls. The following results were Acetic acid (20%) or citric acid (2%), substrate, Factor Xa(h), diluted test sample. obtained: Measure the optical density at 405 nm. Subtract the measured blank value from the test Rivaroxaban assay Apixaban assay absorbance. Standard range Low range Standard range Low range N CV% N CV% N CV% N CV% Create a plasma blank if sample is icteric, lipaemic, haemolysed, or if its color differs from the Intra-test 30 1.5 30 1.7 30 1.8 30 1.8 standard plasmas. Inter-test 20 2.3 20 2.2 20 3.2 20 2.9 When employing the kinetic method, use ΔOD 405 instead of OD 405. Edoxaban assay Standard range Low range If a reaction volume other than that indicated above is required for the method used, the N CV% N CV% volume ratio must be strictly observed in order to guarantee assay performance. The user is Intra-test 40 2.1 40 2.0 responsible for validating any changes and their impact on all results. Inter-test 120 2.7 120 5.1

• By the assay principle, no coagulation factor interference, such as Factor II and X, is CALIBRATION: expected. The assay is completely insensitive to heparins (UFH and LMWH) at usual The BIOPHEN™ DiXal test can be calibrated for the analysis of various anti-Xa analytes: concentrations. Apixaban, Rivaroxaban, Edoxaban. Kits containing calibrators specific to these analytes and • Specific, sensitive assay, offering a high degree of flexibility over the measurement range covering the dynamic test range are available from HYPHEN BioMed (see the 'REAGENTS according to the working dilution used. AND MATERIALS REQUIRED BUT NOT PROVIDED' paragraph) and can be used to • The test is optimized and calibrated relative to the Rivaroxaban/Apixaban/Edoxaban generate the calibration curve specific to the assayed analyte. concentration. The calibration curves are established with a concentration expressed in The following calibration curves are given by way of example only. The calibration curve ng/mL. If another direct Factor Xa inhibitor is used, the user must take into consideration the established for the assay series must be used. specific anti-Xa activity of the substance used. • Correlation with reference method (LCMS :MS vs BIOPHEN™ DiXaI Edoxaban) : Low range Standard range Sysmex CS-5100 : n = 142 y = 1.00x + 13.10 r = 0.997 Refer to the specific application guide for each analyzer. • Interferences: see specific application guide for each analyzer.

REFERENCES: 1. Weitz JI, et al. American College of Chest Physicians. New antithrombotic drugs: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence- Based Clinical Practice Guidelines. Chest. 2012. 2. Pernod G, et al.. Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors. Proposals of the Working Group on Perioperative Haemostasis (GIHP). Ann Fr Anesth Reanim. 2013. 3. Douxfils J, et al. Comparison of calibrated chromogenic anti-Xa assay and PT tests with LC-MS/MS for the therapeutic monitoring of patients treated with rivaroxaban. Thromb Haemost. 2013. 4. Douxfils J, et al. Impact of Apixaban on routine and specific coagulation assays: a practical laboratory guide. Thromb Haemost. 2013 June. 5. Ruff CT et al. Association between edoxaban dose, concentration, anti-Factor Xa activity, and outcomes: an analysis of data from the randomised, double-blind ENGAGE AF-TIMI 48 trial. Lancet. ;385(9984):2288-95. 2015. 6. CLSI Document H21-A5 : “Collection, transport, and processing of blood specimens for testing plasma -based coagulation assays and molecular hemostasis assays; approved guideline”. Fifth Edition, 28, 5, 2008. 7. Douxfils J, et al. Non-VKA Oral Anticoagulants: Accurate Measurement of Plasma Drug. BioMed Research International. 2015. 8. Mauge L. and Alhenc-Gelas M. Stabilité pré-analytique des paramètres de la coagulation: revue des données disponibles. Ann Biol Clin. 2014.

SYMBOLS: Symbols used and signs listed in the ISO 15223-1 standard, see Symbol definitions document.

R1 H315: Causes skin irritation H319: Causes serious eye irritation

HYPHEN BioMed 155 rue d’Eragny, 95000 Neuville-sur-Oise, France D750-02/BI/1030/v7