Sysmex® CS-2500 System Reference Documents

Sysmex® CS-2500 SystemReference Documents

T 05013.008 l Effective Date: 04/12/202104-2021 l © Siemens Healthcare Diagnostics Inc., 2021

Source Documents

1. SYSMEX Automated Blood Coagulation Analyzer CS-2500 Reference Guide, Version 2.10,2020-09

2. Using Quality Control Features of Sysmex CS Systems to Prepare for Lot Rollover,11316580, Rev. A

3. Establishing APTT Values Corresponding to the Unfractionated Heparin TherapeuticRange (U.S.), 11533694, Rev. A

______________________________________________________________________________

All rights, including rights created by patent grant or registrationof a utility model or design, are reserved. All other trademarksand brands are the property of their respective owners.

Product availability may vary from country to country and issubject to varying regulatory requirements. Please contactyour local representative for availability.

04-2021 | All rights reserved© 2021 Siemens Healthcare Diagnostics Inc.

Note: This document is for supplemental use only, and notmeant to be used in place of primary technical materials.

This resource guide, and the software described within, arecopyrighted. No part of this may be copied, reproduced, translated, or reduced to any electronic medium or machine-readable form without the prior written consent of Siemens Healthcare Diagnostics, Inc.

Global Siemens HeadquartersSiemens AGWittelsbacherplatz 280333 MuenchenGermany

Global Siemens HealthcareHeadquartersSiemens AGHealthcare SectorHenkestrasse 12791052 ErlangenGermany

Global DivisionSiemens Healthcare Diagnostics Inc.511 Benedict AvenueTarrytown, NY 10591-5005USAwww.siemens.com/diagnostics

Application SheetPT INR /Dade® Innovin®

For additional information, limitations and interferences please refer to the Instructions for Use of the analyzer andcheck current Instructions for Use for reagents, controls and calibrators and tables of assigned/analytical values.The parameters defined in this application sheet have been developed by Siemens Healthcare Diagnostics to provideoptimal product performance with the assay and instrument combination. Any modification to these parameters mayaffect performance of this and other assays in use on your system and the resulting assay values. It is the responsibilityof the user to validate any modifications and their impact on all assay results. Results of this test should always beinterpreted in conjunction with the patient's medical history, clinical presentation and other findings.The application sheet lists all combinations of controls and calibrators for use with the reagent and instrument system;other combinations are not validated or supported by Siemens.

Version 05Release Date 2019-05

Software Version ≥ 01–68USA only

Materials RequiredProduct Order No. Order No. Order No. Order No. Name in Test Pro‐

tocol• Component(s) Package Size 1 Package Size 2 Package Size 3 Package Size 4Dade® Innovin® REF B4212-51 REF B4212-101

10 × 10 mL 12 × 20 mL PT InnCi-Trol CONTROL 1 REF B4244-10

20 × 1 mL Citrol 1Ci-Trol CONTROL 2 REF B4244-20


20 × 1 mL Citrol 3CONTROL N REF ORKE45

10 × 1 mL CtlNCA CLEAN II REF 10708787 REF 964-0611-9 REF 974-0581-0

1 × 45 mL 1 × 500 mL 1 × 5 L Clean IISLD mini Cup REF 10709524

500 pcs. n/aSample Cup Conical 4 mL REF 424-1160-8

100 pcs. n/aReagent Cap S GW 5 REF AS143226

200 pcs. n/aCompatible cover ring GW 5 REF CC907148

10 pcs. n/aReagent Cap L GW 15 REF AF504574

100 pcs. n/aCompatible cover ring GW 15 REF BB564291

10 pcs. n/a

Additional NotesTo perform QC it is recommended to transfer controls into SLD mini Cup.Performance characteristics have been established using the CS-2100i and were confirmed for the CS-2500.

On-board StabilityComponent Position Condition Time (h)Dade® Innovin® reagent table – 72Ci-Trol CONTROL 1 reagent table SLD mini Cup 24Ci-Trol CONTROL 2 reagent table SLD mini Cup 21Ci-Trol CONTROL 3 reagent table SLD mini Cup 24CONTROL N reagent table SLD mini Cup 24CA CLEAN II reagent table GW 50a 120

a If required CA CLEAN II has to be transferred into an appropriate vial.

CS-2500 PT INR / Dade® Innovin® (V. 05)Reference Guide Page 1 of 4

RG_36_EN-U Rev. 2.10 USA only 21/154

The stability data presented here were established under controlled laboratory conditions. Due to differences inlaboratory environmental conditions and reagent vial filling volumes, the on-board stability may deviate from theabove-mentioned values.

Interference Studies

No interferences up to ...

path. lowsample

normal sam‐ple

path. highsample

– (mg/dL) (mg/dL)HIL

IndexHemoglobin n/a 1 000 1 000 5b

Bilirubin (not conjugated) n/a 60 60 n/aBilirubin (conjugated) n/a 40 40 n/aTriglyceridesc n/a 218.3 288.8 4b

b Further details see Addendum "HIL Index".c evaluated with INTRALIPID equivalent (LIPOVENOES) spiked samples

Lipoglycopeptide antibacterial drugs (such as oritavancin or telavancin) may interfere with PT based assays. ConsultInstructions for Use of respective drugs.

Performance CharacteristicsThe following studies were conducted using specimens collected in 3.2 % sodium citrate solution.The performance of this device has not been established in neonate and pediatric patient populations.Test results obtained from pediatric patient populations may be age dependent. Therefore, each laboratory must followtheir regulatory agency guidelines to qualify additional age groups.

Method ComparisonPredicate Device n Regression Equation rd

PT INR / Dade® Innovin® on SYSMEX CA-1500 454 y = 1.047 × − 0.052e 0.999d r = Correlation Coefficiente no unit (INR)

PrecisionThe coefficient of variation of the analytical system (Total Precision CV) on the same lot of control plasma should be asfollows:

< 7.5 %In a multicenter study the following results were obtained:

MeanINR

Repeatability CV(%)

Between-Site CV(%)

Total Precision CV(%)

Ci-Trol CONTROL 1 1.042 0.49 0.00 0.76Ci-Trol CONTROL 2 2.753 1.17 0.00 2.12Ci-Trol CONTROL 3 4.493 1.06 0.00 2.31

Measuring IntervalComments INR– 0.93 – 8.00

Factor SensitivityThe factor sensitivity of Dade® Innovin® was investigated for FACTOR V DEFICIENT (FV) and FACTOR VII DEFICIENT (FVII)on the CS-2100i analyzer according to the recommendations of CLSI guideline H47- A2. In this study using fivedifferent reagent lots of Dade® Innovin® factor sensitivity levels ranged from 37 to 56 % of Norm for FV and from 46 to56 % of Norm for FVII.

Expected ValuesIn a study with ostensibly healthy subjects the following data were obtained:

Comments nMedian

INR2.5th to 97.5th percentile

INR– 204 1.01 0.93 – 1.15

PT INR / Dade® Innovin® (V. 05) CS-2500Page 2 of 4 Reference Guide

22/154 USA only RG_36_EN-U Rev. 2.10

Reference intervals vary from laboratory to laboratory depending on the population, the technique and reagent lot.Therefore, each laboratory must establish its own reference intervals or verify them whenever one or more of theaforementioned variables are changed.For more information on establishing reference intervals see CLSI document C28-A3, "How to Define and DetermineReference Intervals in the Clinical Laboratory; Approved Guideline."

BibliographyRefer to the Instructions for Use of the reagent.

CS-2500 PT INR / Dade® Innovin® (V. 05)Reference Guide Page 3 of 4


Test ProtocolSettings - Assay Group Settings - Assay Parameter

Management ID Assay Parameter Comments1023 PT INN~INR –

Standard Curve CalibrationThe assay is not calibrated.Enter the MNPT and the ISI.

RemarksSystem specific MNPT and ISI have to be used.The mean normal PT (MNPT) is defined as the mean value of the normal range. Follow the appropriate CLSI guidelinefor establishing an MNPT.ISI values for prothrombin time must be entered directly as they appear on the lot-specific table of ISI values. Anychanges of the reagent lot, software (upgrades), major service, etc., require verification of the ISI value. Failure to enterthe correct ISI value will cause incorrect International Normalization Ratio (INR) results.For further information, please refer to the Instruction for Use of the reagent.In rare cases the Error Code "0032.0002.000 (Flat Curve)" may appear for QC measurements. When the error "Flatcurve" occurs, the QC result is released if "QC sample" was selected in the settings "Automatic Validation" and"Automatic Output". A QC result with a "Flat curve" error is considered to be valid. Therefore it is recommended to usethe QC function of the software to measure controls.

Siemens Healthcare Diagnostics Products GmbHEmil-von-Behring-Str. 7635041 Marburg/GermanyUSA DistributorSiemens Healthcare Diagnostics Inc.Newark, DE 19714 USA

Siemens Healthineers HeadquartersSiemens Healthcare GmbHHenkestraße 12791052 Erlangen/GermanyPhone: +49 9131 84-0siemens-healthineers.com

RxOnly

PT INR / Dade® Innovin® (V. 05) CS-2500Page 4 of 4 Reference Guide


http://www.siemens-healthineers.com

Application SheetAPTT /Dade® Actin® FSL Activated PTT Reagent





tocol• Component(s) Package Size 1 Package Size 2 Package Size 3 Package Size 4ACTIN FSL REF B4219-1 REF B4219-2

10 × 2 mL 10 × 10 mL APTT FSLCi-Trol CONTROL 1 REF B4244-10




10 × 1 mL CtlNCaCl2 SOLUTION REF ORHO37

10 × 15 mL CaCl2SLD mini Cup REF 10709524




10 pcs. n/aReagent Cap L GW 15 REF AF504574

100 pcs. n/aCompatible cover ring GW 15 REF BB564291

10 pcs. n/a

Additional NotesTo perform QC it is recommended to transfer controls into SLD mini Cup.Performance characteristics have been established using the CS-2100i and were confirmed for the CS-2500.

On-board StabilityComponent Position Condition Time (h)ACTIN FSL reagent table – 72Ci-Trol CONTROL 1 reagent table SLD mini Cup 24Ci-Trol CONTROL 2 reagent table SLD mini Cup 24Ci-Trol CONTROL 3 reagent table SLD mini Cup 24CONTROL N reagent table SLD mini Cup 24CaCl2 SOLUTION reagent table – 72

CS-2500 APTT / Dade® Actin® FSL Activated PTT Reagent (V. 07)Reference Guide Page 1 of 4





path. lowsample

normal sam‐ple

path. highsample

– (mg/dL) (mg/dL)HIL

IndexHemoglobin n/a 1 000 1 000 5a

Bilirubin (not conjugated) n/a 60 60 n/aBilirubin (conjugated) n/a 40 40 n/aTriglyceridesb n/a 544.8 263.6 4a

a Further details see Addendum "HIL Index".b evaluated with INTRALIPID equivalent (LIPOVENOES) spiked samples

Lipoglycopeptide antibacterial drugs (such as oritavancin or telavancin) may interfere with APTT based assays. ConsultInstructions for Use of respective drugs.


Method ComparisonPredicate Device n Regression Equation rc

APTT / Dade® Actin® FSL Activated PTT Reagent on SYSMEX CA-1500 432 y = 1.077 × − 2.305 s 0.996c r = Correlation Coefficient



Mean(s)

Repeatability CV(%)

Between-Site CV(%)


Ci-Trol CONTROL 1 28.52 0.87 0.35 1.20Ci-Trol CONTROL 3 58.73 0.88 0.18 0.96

Measuring IntervalComments (s)– 20.0 – 139.0

Factor SensitivityThe factor sensitivity of ACTIN FSL was investigated for factor VIII deficiency (FVIII) and factor IX deficiency (FIX) on theCS-2100i analyzer according to the recommendations of CLSI guideline H47- A2. In this study using five differentreagent lots of ACTIN FSL factor sensitivity levels ranged from 45 to 51 % of Norm for FVIII and from 37 to 47 % ofNorm for FIX.

Heparin SensitivityIn a correlation study comparing the CS-2100i to the SYSMEX CA-1500 system, the results of two lots of ACTIN FSL onsamples from patients under UFH therapy revealed the following comparability using Passing-Bablok regressionanalysis:n = 118, y = 1.067 × − 2.003, r = 0.993; and n = 107, y = 1.066 × − 1.629, r = 0.996.In a study comparing APTT using ACTIN FSL on CS-2100i with Anti Xa testing using Berichrom HEPARIN on SYSMEXCA-1500 system a correlation coefficient of 0.718 was obtained. Classifying the samples using the Anti Xa therapeuticrange (0.3 to 0.7 IU/mL) and the calculated APTT therapeutic range demonstrated discordant results in 17 out of 54samples.

APTT / Dade® Actin® FSL Activated PTT Reagent (V. 07) CS-2500Page 2 of 4 Reference Guide



Comments nMean

(s)Median

(s)2.5th to 97.5th percentile

(s)– 204 27.3 27.3 23.9 – 30.7



CS-2500 APTT / Dade® Actin® FSL Activated PTT Reagent (V. 07)Reference Guide Page 3 of 4



Management ID Assay Parameter Comments1252 APTT FSL~sec The default setting of the sub measurement time is 320 seconds.1291 APTT FSL e~sec The default setting of the sub measurement time is 600 seconds.

Data CheckSettings – Assay Group Settings – Assay Parameter – Data CheckReport Limit Check Lower Limit 20.0

Upper Limit 139.0

Standard Curve CalibrationThe assay is not calibrated.



RxOnly

APTT / Dade® Actin® FSL Activated PTT Reagent (V. 07) CS-2500Page 4 of 4 Reference Guide



Application SheetFibrinogen /Dade® Thrombin Reagent





tocol• Component(s) Package Size 1 Package Size 2 Package Size 3 Package Size 4THROMBIN REAGENT REF B4233-25 REF B4233-27

10 × 1 mL 10 × 5 mL FbgSTANDARD PLASMA REF ORKL19

10 × 1 mL n/aCi-Trol CONTROL 1 REF B4244-10


10 × 1 mL CtlNCONTROL P REF OUPZ19

10 × 1 mL CtlPData-Fi FIBRINOGEN CONTROL REF B4233-22

10 × 1 mL AbnFbgCA SYSTEM BUFFER REF B4265-37

8 × 250 mL OVBOV BUFFER REF B4234-25

10 × 15 mL OVBCA CLEAN I REF 964-0631-3

1 × 50 mL Clean ISLD mini Cup REF 10709524


100 pcs. n/aReagent Cap S GW 5 (optional) REF AS143226

200 pcs. n/aCompatible cover ring GW 5 (optional) REF CC907148

10 pcs. n/aReagent Cap L GW 15 (optional) REF AF504574

100 pcs. n/aCompatible cover ring GW 15 (optional) REF BB564291

10 pcs. n/a

Additional NotesTo perform QC it is recommended to transfer controls into SLD mini Cup.To perform calibration it is recommended to transfer STANDARD PLASMA into Sample Cup Conical 4 mL or SLD mini Cup.As calibrators are intended for immediate use, no on-board stability data has been established.Performance characteristics have been established using the CS-2100i and were confirmed for the CS-2500.

CS-2500 Fibrinogen / Dade® Thrombin Reagent (V. 04)Reference Guide Page 1 of 4


On-board StabilityComponent Position Condition Time (h)THROMBIN REAGENT reagent table – 72STANDARD PLASMA reagent table – n/aCi-Trol CONTROL 1 reagent table SLD mini Cup 24CONTROL N reagent table SLD mini Cup 24CONTROL P reagent table SLD mini Cup 24Data-Fi FIBRINOGEN CONTROL reagent table SLD mini Cup 24CA SYSTEM BUFFER buffer table GW 15a 24OV BUFFER buffer table GW 15 24CA CLEAN I reagent table A, pos. 5 GW 50 120

a For positioning on the analyzer, the buffer has to be transferred into an appropriate vial.The stability data presented here were established under controlled laboratory conditions. Due to differences inlaboratory environmental conditions and reagent vial filling volumes, the on-board stability may deviate from theabove-mentioned values.



path. lowsample

normal sam‐ple

path. highsample

(mg/dL) (mg/dL) (mg/dL)HIL

IndexHemoglobin 150 600 600 5b,c

Bilirubin (not conjugated) 12 24.8 60 n/aBilirubin (conjugated) 20 40 40 n/aTriglyceridesd 171.0 335.2 537.5 5b,c

HESe 21.2 g/L 18.8 g/L 28.6 g/L n/ab Further details see Addendum "HIL Index".c The "HIL Index" assignment follows the lowest interference concentration, found for normal and/or pathological high samples or pathological

low samples x 4 (4 times higher sample concentration because of 4/1 re-analysis condition).d evaluated with INTRALIPID equivalent (LIPOVENOES) spiked samplese hydroxyethyl starch


Method ComparisonPredicate Device n Regression Equation rf

Fibrinogen / Dade® Thrombin Reagent on SYSMEX CA-1500 356 y = 1.048 × − 4.417 mg/dL 0.994f r = Correlation Coefficient



Mean(mg/dL)

Repeatability CV(%)

Between-Site CV(%)


CONTROL N 245.6 3.02 1.23 3.41CONTROL P 83.9 4.60 0.00 4.62

Measuring IntervalComments (mg/dL)– 50. – 860.

Fibrinogen / Dade® Thrombin Reagent (V. 04) CS-2500Page 2 of 4 Reference Guide



Comments nMean

(mg/dL)Median(mg/dL)

2.5th to 97.5th percentile(mg/dL)

– 204 292. 278. 187. – 446.



CS-2500 Fibrinogen / Dade® Thrombin Reagent (V. 04)Reference Guide Page 3 of 4



Management ID Assay Parameter Comments1321 Fbg DT –

Data CheckSettings – Assay Group Settings – Assay Parameter – Data CheckReport Limit Check Lower Limit 50. mg/dL

Upper Limit 860. mg/dL

Standard Curve (example only)A new standard curve must be established when changing a reagent lot, after major maintenance or service, ifindicated by quality control results and when required by laboratory control procedures and/or governmentregulations.Fbg DT

(mg/dL) (s)711. 3.2474. 3.8237. 7.3119. 14.2

59. 24.7

RemarksThe automatic redilution of samples is not available in the "Micro Mode". Sample results outside the concentrationrange of the standard curve are extrapolated, and therefore, have a higher potential to be inaccurate. Therefore,results of samples measured in the "Micro Mode" may only be released if the results are within the concentration rangeof the standard curve.The setting comprises an automated redilution rule of defined order as follows:

1. Assay Parameter Fbg DT | Upper: 450 mg/dL | Lower: 0 mg/dL | Dil. Ratio: 1/22. Assay Parameter Fbg DT | Upper: 1 200 mg/dL | Lower: 80 mg/dL | Dil. Ratio: 4/1

The values 0 mg/dL and 1 200 mg/dL do not describe the lower and upper limit of the measuring range, but constitutean essential element of the setting.The automated redilution requires an extrapolation rule setting as follows:Extrapolation ON | Min. X 0.10 | Max. X 2.00If redilution is chosen a dilution ratio of 1:2 or 4:1 should be selected.



RxOnly

Fibrinogen / Dade® Thrombin Reagent (V. 04) CS-2500Page 4 of 4 Reference Guide



Application SheetD-dimer /INNOVANCE® D‑Dimer



Software Version ≥ 01-68USA only


tocol• Component(s) Package Size 1 Package Size 2 Package Size 3 Package Size 4INNOVANCE® D‑Dimer REF OPBP09 REF OPBP11• INNOVANCE D‑Dimer REAGENT 3 × 4 mL 6 × 4 mL DDiReag• INNOVANCE D‑Dimer BUFFER 3 × 5 mL 6 × 5 mL DDiBuf• INNOVANCE D‑Dimer SUPPLEMENT 3 × 2.6 mL 6 × 2.6 mL DDiSup• INNOVANCE D‑Dimer DILUENT 3 × 5 mL 6 × 5 mL DDiDil• INNOVANCE D‑Dimer CALIBRATOR 2 × 1 mL 2 × 1 mL DDiCal• EMPTY VIAL 1 × 12 pcs. n/aINNOVANCE® D-Dimer Controls REF OPDY09• INNOVANCE D-Dimer CONTROL 1 5 × 1 mL DDiCtl1• INNOVANCE D-Dimer CONTROL 2 5 × 1 mL DDiCtl2INNOVANCE D‑Dimer DILUENT REF OPBR03

10 × 5 mL DDiDilSLD mini Cup REF 10709524


100 pcs. n/aReagent Cap S GW 5 (optional) REF AS143226

200 pcs. n/aCompatible cover ring GW 5 (optional) REF CC907148

10 pcs. n/a

Additional NotesTo perform QC it is recommended to transfer controls into SLD mini Cup.To perform calibration it is recommended to transfer INNOVANCE D‑Dimer CALIBRATOR into Sample Cup Conical 4 mLor SLD mini Cup.As calibrators are intended for immediate use, no on-board stability data has been established.Performance characteristics have been established using the CS-2100i and were confirmed for the CS-2500.

On-board StabilityComponent Position Condition Time (h)INNOVANCE D‑Dimer REAGENT reagent table – 48INNOVANCE D‑Dimer BUFFER reagent table – 48INNOVANCE D‑Dimer SUPPLEMENT reagent table – 48INNOVANCE D‑Dimer DILUENT buffer table – 48INNOVANCE D‑Dimer CALIBRATOR reagent table – n/aINNOVANCE D-Dimer CONTROL 1 reagent table SLD mini Cup 24INNOVANCE D-Dimer CONTROL 2 reagent table SLD mini Cup 24

CS-2500 D-dimer / INNOVANCE® D‑Dimer (V. 06)Reference Guide Page 1 of 6





path. lowsample

normal sam‐ple

path. highsample

(mg/dL) (mg/dL) (mg/dL)HIL

IndexHemoglobin 1 000 1 000 1 000 5a

Bilirubin (not conjugated) 60 60 60 n/aBilirubin (conjugated) 40 40 40 n/aTriglyceridesb 190.8 300.3 294.3 3a

a Further details see Addendum "HIL Index".b evaluated with INTRALIPID equivalent (LIPOVENOES) spiked samples

LimitationsHigher levels of lipids or turbid samples can lead to falsely elevated or decreased values. It is therefore recommendedto perform an additional centrifugation step of the plasma (10 minutes at approx. 15.000 x g) before analyzing lipemicpatient specimens.


NoteFor the exclusion of deep vein thrombosis (DVT) the diagnostic performance was assessed in a population of patientswith the suspicion of a first event of DVT. For other patient populations (e. g. with recurrent or chronic DVT) theeffectiveness of the device to exclude DVT has not been verified. References to scientific literature1,2,3 about thediagnosis of recurrent or chronic DVT can be found in the bibliography section of this application sheet.

Method ComparisonPredicate Device n Regression Equation rc

D-dimer / INNOVANCE® D‑Dimer on SYSMEX CA-1500 349 y = 0.982 × + 0.015 mg/L FEU 0.997c r = Correlation Coefficient



Mean(mg/L FEU)

Repeatability CV(%)

Between-Site CV(%)


INNOVANCE D-DimerCONTROL 1 0.310 3.36 2.13 4.33INNOVANCE D-DimerCONTROL 2 2.335 2.92 2.95 5.05

Measuring IntervalComments (mg/L FEU)– 0.19 – 35.20

Clinical PerformanceThe INNOVANCE® D‑Dimer assay was evaluated on the CS-2100i in a multi-center study to validate the exclusion ofPulmonary Embolism (PE) and a first event of Deep Vein Thrombosis (DVT). The specimens were tested with theINNOVANCE® D‑Dimer assay and results were compared to a cut-off value of 0.50 mg/L FEU. A D-dimer result< 0.50 mg/L FEU was considered negative and a D-dimer result ≥ 0.50 mg/L FEU was considered positive.

D-dimer / INNOVANCE® D‑Dimer (V. 06) CS-2500Page 2 of 6 Reference Guide


The instrument-specific sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) withtheir respective lower bound (LCL) of the two-sided 95 % confidence interval were calculated. Results obtained for eachstudy population are detailed below.

Exclusion of PEThe INNOVANCE® D‑Dimer assay was evaluated on the CS-2100i in a multi-center study to validate the exclusion of PEusing frozen specimens collected prospectively from 1930 consecutive outpatients presenting to the emergency orambulatory department with suspected PE. Of these 1930 patients, 96 were excluded for a total of 1834 patients.All potentially eligible patients were evaluated using the Wells’ rules to estimate their pre-test probability (PTP) withregard to PE, and then categorized into high, intermediate or low PTP. Patients with a high PTP score were excludedfrom enrollment.Patients with no or a positive D-dimer result with the D-dimer assay used at the respective study center were evaluatedby imaging methods, e.g. spiral CT and/or VQ scan. Patients with a negative D-dimer result with the D-dimer assay usedat the respective study center underwent imaging at the physician’s discretion.All patients with a negative diagnosis of PE at presentation were followed up after three months to evaluate potentialdevelopment of PE. Patients with unobtainable follow-up data were excluded from analysis resulting in n=1467patients available for final analysis. The overall prevalence of PE in the 1467 patients was 6.9 % (101 of 1467) with6.0 % in the US population and 37.2 % in the European population.

US sites (PE) Reference (Imaging and 3-month follow-up)Positive Negative Total

INNOVANCE® D‑Dimeron CS-2100i

Positive 82 616 698Negative 3 723 726

Total 85 1 339 1 424 Sensitivity % = 96.5 95 % LCL= 90.0 Specificity % = 54.0 95 % LCL= 51.3 NPV % = 99.6 95 % LCL= 98.8 NPVd % = 98.9 95 % LCL= 96.7 PPV % = 11.7 95 % LCL= 9.6 PPVd % = 27.0 95 % LCL= 22.7

d standardized to a prevalence of 15 %

OUS sites (PE) Reference (Imaging and 3-month follow-up)Positive Negative Total



Total 16 27 43 Sensitivity % = 100.0 95 % LCL= 79.4 Specificity % = 81.5 95 % LCL= 61.9 NPV % = 100.0 95 % LCL= 85.1 NPVe % = 100.0 95 % LCL= 95.1 PPV % = 76.2 95 % LCL= 54.9 PPVe % = 48.8 95 % LCL= 26.6

e standardized to a prevalence of 15 %

US and OUS sites (PE) Reference (Imaging and 3-month follow-up)Positive Negative Total



Total 101 1 366 1 467 Sensitivity % = 97.0 95 % LCL= 91.6 Specificity % = 54.5 95 % LCL= 51.9 NPV % = 99.6 95 % LCL= 98.8 NPVf % = 99.0 95 % LCL= 97.2



US and OUS sites (PE) Reference (Imaging and 3-month follow-up)Positive Negative Total

PPV % = 13.6 95 % LCL= 11.3 PPVf % = 27.4 95 % LCL= 23.3

f standardized to a prevalence of 15 %Exclusion of DVT

The INNOVANCE® D‑Dimer assay was evaluated on the CS-2100i in a multi-center study to validate the exclusion of afirst event of DVT using frozen specimens collected prospectively from 1907 consecutive outpatients presenting to theemergency or ambulatory department with suspected DVT. Of these 1907 patients, 368 were excluded from analysis(including 213 patients reported to have a previous documented or chronic DVT) resulting in a total of 1539 patients.All potentially eligible patients were evaluated using the Wells’ rules to estimate their pre-test probability (PTP) withregard to DVT, and then categorized into likely or unlikely, or alternatively as high, intermediate or low PTP. Patientswith a high or likely PTP score were excluded from enrollment.Patients with no or a positive D-dimer result with the D-dimer assay used at the respective study center were evaluatedby imaging methods, e.g. ultrasound. Patients with a negative D-dimer result with the D-dimer assay used at therespective study center underwent imaging at the physician’s discretion.All patients with a negative clinical diagnosis of DVT at presentation were followed up after three months to evaluatepotential development of DVT. Patients with unobtainable follow-up data were excluded from analysis resulting inn=1317 patients available for final analysis. The overall prevalence of DVT in the 1317 patients was 6.1 % (80 of 1317)with 7.0 % in the US population and 4.7 % in the European population.

US sites (DVT) Reference (Imaging and 3-month follow-up)Positive Negative Total



Total 56 747 803 Sensitivity % = 98.2 95 % LCL= 90.4 Specificity % = 39.8 95 % LCL= 36.2 NPV % = 99.7 95 % LCL= 98.1 NPVg % = 99.2 95 % LCL= 95.7 PPV % = 10.9 95 % LCL= 8.5 PPVg % = 22.3 95 % LCL= 17.9

g standardized to a prevalence of 15 %

OUS sites (DVT) Reference (Imaging and 3-month follow-up)Positive Negative Total



Total 24 490 514 Sensitivity % = 95.8 95 % LCL= 78.9 Specificity % = 55.7 95 % LCL= 51.2 NPV % = 99.6 95 % LCL= 98.0 NPVh % = 98.7 95 % LCL= 93.0 PPV % = 9.6 95 % LCL= 6.5 PPVh % = 27.6 95 % LCL= 20.0

h standardized to a prevalence of 15 %

US and OUS sites (DVT) Reference (Imaging and 3-month follow-up)Positive Negative Total



Total 80 1 237 1 317 Sensitivity % = 97.5 95 % LCL= 91.3



US and OUS sites (DVT) Reference (Imaging and 3-month follow-up)Positive Negative Total

Specificity % = 46.1 95 % LCL= 43.3 NPV % = 99.7 95 % LCL= 98.7 NPVi % = 99.1 95 % LCL= 96.6 PPV % = 10.5 95 % LCL= 8.5 PPVi % = 24.2 95 % LCL= 20.2

i standardized to a prevalence of 15 %


Comments nMedian

(mg/L FEU)2.5th to 97.5th percentile

(mg/L FEU)– 203 0.26 < 0.19 – 1.12



1. Van der Hulle, T.; et al. (2016) Current standings in diagnostic management of acute venous thromboembolism:Still rough around the edges, Blood Reviews Vol.30 Issue 1, p.21-26

2. Bates, S.M.; et al. (2012) Diagnosis of DVT - Antithrombotic Therapy and Prevention of Thrombosis, 9th ed:American College of Chest Physicians Evidence-Based Clinical Practice Guidelines, Chest Vol.141 Suppl.2, p.351-418

3. Tripodi, A. (2011) D-Dimer Testing in Laboratory Practice, Clinical Chemistry Vol.57 Issue 9, p.1256-1262




Management ID Assay Parameter Comments2321 INN DDi –

Data CheckSettings – Assay Group Settings – Assay Parameter – Data CheckReport Limit Check Lower Limit 0.19 mg/L

Upper Limit 35.20 mg/L

Standard Curve (example only)A new standard curve must be established when changing a reagent lot, after major maintenance or service, ifindicated by quality control results and when required by laboratory control procedures and/or governmentregulations.INN DDi

(mg/L) (dOD)4.54 0.20182.27 0.13681.13 0.06840.57 0.02890.28 0.01130.14 0.0051

RemarksThe automatic redilution of samples is not available in the "Micro Mode". Sample results outside the concentrationrange of the standard curve are extrapolated, and therefore, have a higher potential to be inaccurate. Therefore,results of samples measured in the "Micro Mode" may only be released if the results are within the concentration rangeof the standard curve.The setting comprises an automated redilution rule of defined order as follows:

1. Assay Parameter INN DDi | Upper: 4.4 mg/L | Lower: 0 mg/L | Dil. Ratio: 1/102. Assay Parameter INN DDi~dOD | Error Type Antigen Excess | Dil. Ratio: 1/103. Assay Parameter INN DDi~dOD | Error Type Range in non-linear | Dil. Ratio: 1/10

The automated redilution requires an extrapolation rule setting as follows:Extrapolation ON | Min. X 0.01 | Max. X 20.00If redilution is chosen a dilution ratio of 1:10 should be selected.Only D-Dimer concentrations below 4.4 mg/L FEU obtained by redilution measurement have to be confirmed withoutredilution.



RxOnly




Application SheetHeparin /INNOVANCE® Heparin



Software Version ≥ 01-68USA only


tocol• Component(s) Package Size 1 Package Size 2 Package Size 3 Package Size 4INNOVANCE HEPARIN REF OPOA05• INNOVANCE HEPARIN REAGENT 5 × 3.2 mL IHepRea• INNOVANCE HEPARIN SUBSTRATE 5 × 4.0 mL IHepSubINNOVANCE HEPARIN CALIBRATOR REF OPOB05• INNOVANCE HEPARIN CALIBRATOR 1 1 × 1.0 mL IHepCal1• INNOVANCE HEPARIN CALIBRATOR 2 1 × 1.0 mL IHepCal2• INNOVANCE HEPARIN CALIBRATOR 3 1 × 1.0 mL IHepCal3• INNOVANCE HEPARIN CALIBRATOR 4 1 × 1.0 mL IHepCal4• INNOVANCE HEPARIN CALIBRATOR 5 1 × 1.0 mL IHepCal5INNOVANCE HEPARIN LMW CONTROL 1 REF OPOE05

5 × 1 mL IHepLMW1INNOVANCE HEPARIN LMW CONTROL 2 REF OPOF05

5 × 1 mL IHepLMW2INNOVANCE HEPARIN UF CONTROL 1 REF OPOC05

5 × 1 mL IHepUF1INNOVANCE HEPARIN UF CONTROL 2 REF OPOD05

5 × 1 mL IHepUF2STANDARD PLASMA a REF ORKL19

10 × 1 mL n/aCA SYSTEM BUFFER REF B4265-37

8 × 250 mL OVBOV BUFFER REF B4234-25

10 × 15 mL OVBCA CLEAN I REF 964-0631-3

1 × 50 mL Clean ISample Cup Conical 4 mL REF 424-1160-8

100 pcs. n/aSLD mini Cup REF 10709524



10 pcs. n/aa STANDARD PLASMA is only required to measure low molecular weight heparin samples above the analytical measuring interval

Additional NotesTo perform calibration of the assay and/or quality control testing on the CS-2500 System it is recommended to transferthe INNOVANCE HEPARIN CALIBRATOR , INNOVANCE HEPARIN LMW CONTROL 1 , INNOVANCE HEPARIN LMW CONTROL 2 ,

CS-2500 Heparin / INNOVANCE® Heparin (V. 05)Reference Guide Page 1 of 4


INNOVANCE HEPARIN UF CONTROL 1 and INNOVANCE HEPARIN UF CONTROL 2 into Sample Cup Conical 4 mL orSLD mini Cup.On-board Stability data have been established using the CS-2100i and are valid for the CS-2500.

On-board StabilityComponent Position Condition Time (h)INNOVANCE HEPARIN REAGENT reagent table Reagent Cap S GW 5; Compatible cover ring GW 5 72INNOVANCE HEPARIN SUBSTRATE reagent table Reagent Cap S GW 5; Compatible cover ring GW 5 72INNOVANCE HEPARIN CALIBRATOR 1 reagent table SLD mini Cup 8

reagent table Sample Cup Conical 4 mL 8INNOVANCE HEPARIN CALIBRATOR 2 reagent table SLD mini Cup 8




reagent table Sample Cup Conical 4 mL 8INNOVANCE HEPARIN LMW CONTROL 1 reagent table SLD mini Cup 8

reagent table Sample Cup Conical 4 mL 8INNOVANCE HEPARIN LMW CONTROL 2 reagent table SLD mini Cup 8

reagent table Sample Cup Conical 4 mL 8INNOVANCE HEPARIN UF CONTROL 1 reagent table SLD mini Cup 8

reagent table Sample Cup Conical 4 mL 8INNOVANCE HEPARIN UF CONTROL 2 reagent table SLD mini Cup 8

reagent table Sample Cup Conical 4 mL 8STANDARD PLASMA n/a – n/aCA SYSTEM BUFFER buffer table GW 15b 72OV BUFFER buffer table GW 15 72CA CLEAN I reagent table A, pos. 5 GW 50 120

b For positioning on the analyzer, the buffer has to be transferred into an appropriate vial.The stability data presented here were established under controlled laboratory conditions. Due to differences inlaboratory environmental conditions and reagent vial filling volumes, the on-board stability may deviate from theabove-mentioned values.

Interference StudiesNo interferences up to ... (mg/dL)

HILIndex

Hemoglobin 692 5c

Bilirubin (not conjugated) 37 n/aBilirubin (conjugated) 39 n/aTriglyceridesd 414 5c

c Further details see Addendum "HIL Index".d evaluated with INTRALIPID equivalent (LIPOVENOES) spiked samples


Method ComparisonA study was performed with frozen samples to compare Heparin / INNOVANCE® Heparin on the the CS-2500 toHeparin / INNOVANCE® Heparin on the BCS® XP System for the measurement of heparin.The results from the Passing-Bablok regression analysis are summarized in the following table:

Heparin / INNOVANCE® Heparin (V. 05) CS-2500Page 2 of 4 Reference Guide


Predicate Device Regression Equation re

Heparin / INNOVANCE® Heparin on BCS® XP (UFH (n=147) and LMWH (n=130) containingsamples)

y = 0.97 × − 0.01 IU/mL 1.00

e r = Correlation Coefficient

PrecisionThe standard deviation (SD) of the analytical system should be as follows:Samples with heparin activities of≤ 0.50 IU/mL [SD] ≤ 0.03 IU/mLSamples with heparin activities of> 0.50 IU/mL [CV] ≤ 7.5 %

Precision studies were conducted on the CS-2500 according to CLSI guideline EP05-A2, usingINNOVANCE HEPARIN UF CONTROL 1 , INNOVANCE HEPARIN UF CONTROL 2 , INNOVANCE HEPARIN LMW CONTROL 1 ,INNOVANCE HEPARIN LMW CONTROL 2 and 6 plasma pools covering the measuring range.The results from the precision studies are summarized in the following table (Calculation of CV (%): CV (%) = SD(IU/mL) × 100 / Mean (IU/mL)):

Mean(IU/mL)

Repeatability SD(IU/mL)

Within-Device/Lab SD

(IU/mL)INNOVANCE HEPARINLMW CONTROL 1 0.41 0.006 0.011INNOVANCE HEPARINLMW CONTROL 2 0.98 0.013 0.017INNOVANCE HEPARINUF CONTROL 1 0.28 0.006 0.010INNOVANCE HEPARINUF CONTROL 2 0.65 0.006 0.012Plasma pool 1 containingLMWH 0.15 0.007 0.009Plasma pool 1 containing UFH 0.15 0.007 0.009Plasma pool 2 containingLMWH 0.68 0.015 0.017Plasma pool 2 containing UFH 1.09 0.015 0.016Plasma pool 3 containingLMWH 1.36 0.023 0.026Plasma pool 3 containing UFH 1.38 0.016 0.020

Measuring IntervalComments (IU/mL)– 0.10 – 1.50

Samples with low-molecular weight heparin (LMWH) concentrations above 1.50 IU/mL can be diluted manually bymixing two parts of the sample with one part of STANDARD PLASMA . The result of the manually diluted samples must bemultiplied by the dilution factor 1.5 prior to reporting. This procedure results in an extended measuring range of0.10 to 2.25 IU/mL for LMWH.When applying the above described procedure to dilute samples into the analytical measuring interval for low-molecular weight heparin, the antithrombin activity in samples with pathological low Antithrombin activities will becompensated. This may lead to an overestimation of the effect of anticoagulation in these patients.

Expected Valuesn/a


CS-2500 Heparin / INNOVANCE® Heparin (V. 05)Reference Guide Page 3 of 4


Test ProtocolSettings – Assay Group Settings – Assay Parameter

Management ID Assay Parameter Comments2050 INN Hep~IU_mL –

Standard Curve (example only)A new standard curve must be established when changing a reagent lot, after major maintenance or service, ifindicated by quality control results and when required by laboratory control procedures and/or governmentregulations.INN Hep~IU_mL

(dOD) (IU/mL)0.6724 0.000.4934 0.410.3740 0.790.2822 1.170.2238 1.55

Remarks–

Siemens Healthcare Diagnostics Products GmbHEmil-von-Behring-Str. 7635041 Marburg/Germany


RxOnly

Heparin / INNOVANCE® Heparin (V. 05) CS-2500Page 4 of 4 Reference Guide



1

Sysmex® CS-2500 System Sysmex® CS-5100 System

Siemens Healthcare Diagnostics Inc.

Customer Bulletin October 2018

Using Quality Control Features of Sysmex CS Systems to Prepare for Lot Rollover 11316580, Rev. A

Important Information

Siemens Healthineers is providing information regarding the quality control features of the Sysmex® CS Systems software to prepare for implementation of new control and reagent lots, or “lot rollover.”

The information in Appendix A will enable the operator to run and establish ranges on new Quality Control (QC) lots before the current QC lot is depleted. The following recommendations should be used as a guideline when converting to new lots of control and reagents for Sysmex CS Systems. All recommendations are based on Clinical & Laboratory Standards Institute (CLSI) documents to meet CLIA/CAP requirements for hemostasis.

Part I provides guidance on implementation of new reagent and unassayed control material for the PT and aPTT assays using the QC Software.

Part II provides guidance on implementation of new control lots for other hemostasis assays with the QC Software.

Your laboratory is responsible for determining internal policies and procedures for new lot implementation requirements in compliance with your respective accrediting agencies. The laboratory is responsible for determining what procedures are appropriate, and which performance limits or specifications are applicable.

Lot Rollover Considerations

Typically reagent and control lots for Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT) assays are changed once per year when unassayed control products are used. This is commonly referred to as “lot rollover.” Lot rollover activities should be started approximately one (1) month prior to the desired date of use of the new products. Assayed control products may be changed more or less frequently, and change of the reagent lot simultaneously is not required.

Note: If the laboratory participates in the Quality Assurance Program (QAP) program, the QAP Coordinator will contact the laboratory to set up delivery of the new reagent and QC lots. The QAP program is designed for participants to use a specific combination of control and reagent lots for a 12-month period. Using the sequestered lot of reagent and controls provided by the QAP coordinator provides consistency in recovery within the control program.3

Using Quality Control Features of Sysmex CS Systems to Prepare for Lot Rollover

11316580, Rev. A, 2018-10 Page 2 of 43

Before using the new reagent lot with new QC lots it is common practice to perform the following procedures:

Establish or verify reference interval (reference range).

Establish mean normal PT (MNPT) value for new PT reagent.

Establish QC ranges using new reagent with new control material.

Perform a lot-to-lot method correlation with patient samples on both your current reagent lots and the new reagent lots.

Verify heparin therapeutic range for aPTT if monitoring unfractionated heparin therapy.

Verification of Reference Interval

If a lab has previously established a reference interval (using 120 samples) for its population, it may verify that reference interval. A lab may verify a reference interval by collecting a minimum of 20 samples representing the adult reference population (10 males and 10 females).

Note: A separate range should be established for pediatric populations.

Fresh samples are preferred but frozen platelet poor plasma may be used if prepared and thawed per CLSI guidelines. Run samples with the current reagent lot assays, PT and aPTT, and with new reagent lot assays, PTN and aPTTN. This should be conducted simultaneously or within one (1) hour of each other. (Note: This data can be used for lot-to-lot correlations).

The original study's reference interval may be considered valid if no more than two of the 20 donors' values (or 10% of the results) fall outside of the original study's reference interval. If more than two (2) values exceed the original study reference interval, an additional 20 samples should be assessed. If more than two (2) values of this new set of samples exceed the original study limits, refer to CLSI EP28-A3c section 11.2 for more information on additional testing required.

Laboratories are responsible for determining appropriate studies (i.e. number of samples) for reference interval verification and method verification. For more information on establishing reference intervals see CLSI document EP28-A3c, How to Define and Determine Reference Intervals in the Clinical Laboratory; Approved Guideline.

Notes:

After review of data, medication and patient history may be used for excluding aberrant results.

The MNPT for INR calculation must be the geometric mean.

For data pool with a Gaussian distribution the mean and geometric mean will be essentially the same. With equal distribution mean/median values will be similar. A skewed data pool is most likely the result of improper specimen type and additional samples may be required.

Lot-to-Lot Method Correlation

Your laboratory may choose to compare recovery between the new and old reagent lots with a method correlation study. The normal samples used for verification of the reference range can be applied to this lot-to-lot method correlation.

A lot-to-lot method correlation is recommended but not required by regulatory agencies.

Minimum of 40 samples: 20 normal, 20 abnormal.

Abnormal samples should span the reportable range of the assay.


11316580, Rev. A, 2018-10 Page 3 of 43

Test samples on current and new reagent lot numbers simultaneously or within one (1) hour of each other.

Calculate linear regression statistics: – Use statistical software to complete this. If not available consider a website calculator such

as one found at www.westgard.com.

Retain documentation of study data to ensure quality laboratory records.

aPTT Therapeutic Range for Heparin Dosing

With each new aPTT reagent lot number, the unfractionated heparin therapeutic range should be verified as required by CAP/CLIA. Therapeutic range establishment is not required if the laboratory is not monitoring unfractionated heparin therapy with the aPTT assay.

The ex vivo heparin Xa assay vs. aPTT study is recommended by CAP and CLSI.

Method correlation of heparinized samples (minimum 20 covering the therapeutic range) may be used in subsequent years when initial therapeutic range is established with an ex vivo heparin Xa study. Agreement is based on average bias between the reagent lots. This is also referred to as the cumulative summation.

The in vitro heparin response curve is an alternative if the laboratory is unable to obtain proper samples for the ex vivo study. This method is not preferred.

Further detail on the above options is available in Siemens Healthineers Technical Bulletin – Establishing aPTT Values Corresponding to the Unfractionated Heparin Therapeutic Range.

Calibrated Assays

A calibration is required with reagent lot number change.

Calibration/calibration verification is required every six (6) months per CLIA.

Calibration may be required after major preventive maintenance or replacement of critical parts.

Calibration may be required with control shift, trend or outside acceptable limits. Ensure analyzer is performing properly before recalibration.

Regulatory Information

Product and system availability are subject to local regulatory requirements and, therefore, vary by country. If you have any questions or need additional information, please contact your local technical support provider or distributor.

Additional Assistance

Hemostasis QAP Coordinator:

Responsible to set up customers on lot specific programs for the QAP.

Coordinators are assigned to specific areas of the country.

Phone: 1 800 242 3233, option 5

Email: [email protected]

Hemostasis QAP LabLink Online technical support:


11316580, Rev. A, 2018-10 Page 4 of 43

Provide guidance and troubleshooting assistance for the QAP LabLink Online program.

Phone: 1 800 242 3233, option 2, option 4

Email: [email protected]

Hemostasis Customer Care Center - Technical Solutions

Provide general assistance and troubleshooting guidance with Hemostasis products.

Phone: 1 800 242 3233, option 1

Technical information is available at https://www.healthcare.siemens.com/doclib/. If you need additional assistance, please contact your Siemens Healthineers Customer Care Center.

Please retain this bulletin with your records for future reference.

References 1. CLSI Document, EP9-A3: Measurement Procedure and Bias Estimation Using Patient Samples;

Approved Guideline-Third Addition, August 2013.

2. CLSI Document EP28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory: Approved Guideline, Third Addition, October 2010.

3. CLSI Document H47-A2: One-Stage Prothrombin Time (PT) Test and Activated Partial Thromboplastin Time (APTT) Test; Approved Guideline-Second Addition, May 2008

4. Siemens Healthineers Technical Bulletin – Establishing aPTT Values Corresponding to the Unfractionated Heparin Therapeutic Range

5. Siemens Healthineers Technical Bulletin – Understanding the Hemostasis LabAssurance Quality Assurance Program.

6. Sysmex CS-2500 Instructions for Use, USA

7. Sysmex CS-5100 Instructions for Use, USA

Trademark Information

Actin, Ci-Trol, Innovin are trademarks of Siemens Healthcare Diagnostics Inc.

Sysmex is a trademark of the Sysmex Corporation.

All other trademarks are the property of their respective owners.


11316580, Rev. A, 2018-10 Page 5 of 43

Appendix A

Part I:

Implementation of PT and PTT new reagent and unassayed control lots

Note: Auto QC for PT and PTT should be deactivated prior to start of the lot-rollover process. Path: Assay Group Settings > Select Test (PT, PTT) > QC Tab > Select control > Deselect Auto QC. Repeat for each level of control. Reboot of the system is required for this to take effect.

Joblist Verify assays PTN and PTTN are listed

1. Navigate to Joblist. Scroll to the right to view all assays listed.

2. Identify if PTN and PTTN are available.

a. If available go to page 6: Reagent Lot Master.

b. If not available continue with step 3.

3. Navigate to Assay Group Settings.

NOTE: Sysmex CS-5100 users will need to first select Settings to access Assay Group Settings.

4. Highlight PTN in list. Check box for Valid. (If needed) Select Save.

5. Highlight PTTN in list. Check box for Valid. (If needed) Select Save.

6. Select Close. Close software using Red X. Turn off instrument.

7. Start software with CS Startup icon. Turn on instrument.

8. Navigate to Joblist. Scroll to the right to view all assays listed.

9. To add assays to Joblist, Select More to navigate to Customize.

a. Highlight PTN in left list.

b. Highlight last Parameter in right list. Select Add.

c. Highlight PTTN in left list.

d. Highlight last item in right list. Select Add.

NOTE: Use Delete to move item back to left listing.

e. Select OK.


11316580, Rev. A, 2018-10 Page 6 of 43

10. Scroll in the Joblist to the right to confirm PTN and PTTN are present.


11316580, Rev. A, 2018-10 Page 7 of 43

Reagent Lot Master

Enter new QC lots

NOTE: New PT and PTT lot numbers are not entered until go live. During Lot Rollover, use New Inn and New AFSL reagent designations.

1. Select Menu > Reagent Lot Master.

NOTE: Sysmex CS-5100 users will need to first select Settings to access Reagent Lot Master.

2. Verify that New Inn and New AFSL are listed. Scroll down the list to view all reagents.

3. To add new QC lot, click in Lot No. field.

4. Scan the QC vial barcode with 2D barcode reader or manually enter the 6 digit Siemens Healthineers lot number.

5. Click in the Exp. Date field.

6. Enter expiration date on vial using the calendar.

NOTE: An incorrect default date is loaded if not entered manually.

7. Select Add to register the new QC lot number to the list.

8. Repeat for each new level of QC.

9. Select Save.

10. Select Close.


11316580, Rev. A, 2018-10 Page 8 of 43

QC Chart

Register new QC lots to chart

1. Select QC Chart. Select Lot Roll tab.

2. Select on chart at the top of screen- PTN Ci-Trol 1.

3. Select More until Add Lot is displayed.

4. Select Add Lot (the software will move through each step).

5. Select down arrow for Lot No. and select lot (see lot just added).

6. Verify Use QC barcode checkbox is enabled, disable Use Preset checkbox.

7. Select Next.

8. Assay Parameter Selection- all assays that use Ci-Trol 1 will be selected. Do not make any changes to this screen.


11316580, Rev. A, 2018-10 Page 9 of 43

9. Select Next.

10. QC Chart Settings- no changes on this screen. The new Ci-Trol 1 is setup for both PTN and PTTN. No target limits are added at this time.


11316580, Rev. A, 2018-10 Page 10 of 43

11. Select Next.

12. QC Barcode Settings- Highlight QC11 (Ci-Trol 1).


11316580, Rev. A, 2018-10 Page 11 of 43

13. Select Next.

14. Confirmation of the Settings Completion.

Select OK for CS-2500 to save.

Select Complete for CS-5100 to save.


11316580, Rev. A, 2018-10 Page 12 of 43

15. Repeat Add Lot for Ci-Trol 3:

Select Lot Roll tab, select on chart - PTN Ci-Trol 3.

Repeat steps 4-14 for Ci-Trol 3.

Select barcode ID QC13 for Ci-Trol 3.

NOTE: Repeat for all levels of QC.


11316580, Rev. A, 2018-10 Page 13 of 43

View charts: Current vs. New

1. Select More until Switch Display is listed.

2. Select New. All new lots are displayed to the “New” side of the QC chart. Go to Lot Roll tab.

3. To view your current PT and PTT charts, Select Switch Display and select Current. Go to Routine tab. All current lots will be seen in this view.

Establish new QC ranges

QC Ranges should be established using reagents and QC exactly the same way they typically are used. Consider age of QC/reagent, claimed onboard stability, claimed reconstituted stability, etc.

Reconstitute new QC and reagents lots according to package insert directions.

Have different technologists reconstitute reagents/QC.

At least 30 points for each level of QC should be collected.

1. Load new reagent.

a. Select an empty green rack from the Reagent Table inner wheel.

b. Place new PT reagent in reagent rack position 2 with barcode hidden.

c. Place new PTT reagent in reagent rack position 1 with barcode hidden.

d. Load to Reagent Table and let barcode reading occur.


11316580, Rev. A, 2018-10 Page 14 of 43

e. Highlight position 2 showing red question mark.

f. Select Edit Reagent Info.

g. Select New Inn from list (list is in alphabetical order).

h. Click in lot number field and select lot number listed (636901).

i. Click in vial field and select correct vial size. (GW15 for example)

j. Select OK to save information.


11316580, Rev. A, 2018-10 Page 15 of 43

k. Highlight position 1 showing red question mark.

l. Select Edit Reagent Info.

m. Select New AFSL from the list.

n. Click in lot number field and select lot number listed (627301).

o. Click on vial field and select correct vial size (GW15 for example).

p. Select OK to save information.

2. Highlight/Select New Inn position on Reagent screen.

3. Select Lot Group Settings.

4. Add check mark to “Used in Sample Analysis” checkbox.

5. Click OK. (This gives instrument permission to run this reagent for the assay).

6. Highlight/Select New AFSL position.

7. Select Lot Group Settings.

8. Add check mark to “Used in Sample Analysis” checkbox.

9. Select OK.


11316580, Rev. A, 2018-10 Page 16 of 43

10. Load new QC vials.

a. Select a yellow C rack.

b. Place QC vials containing SLD minicup with barcodes facing out to be read.

c. Load onto the Reagent table. Select OK to barcode read.

11. Order new QC for PTN and PTTN.

a. Select Order. Select Switch Order.

b. Select Holder QC Order.

c. Select Order Entry.

d. Select the radio button QC01-QC20 on the left.

e. Select QC11 from list. Select PTN and PTTN from test list.


11316580, Rev. A, 2018-10 Page 17 of 43

f. Arrow down.

g. Repeat to order QC13 PTN/ PTTN.

h. Select OK.

12. Press Start to run your new QC.

Are you ready to go Live?

Cumulated 30 points of valid QC for each level.

Go to PTN and PTTN QC charts and verify all the data.

Exclude erroneous data using Confirm Data. See Quick Reference Guide Quality Control tab.

Verified reference range.

Calculated geometric mean for new PT lot.

Completed lot-to-lot method correlation.

Verified aPTT therapeutic range- if run patients on unfractionated heparin.

Performed a backup to save data before rolling over to new lots.

See Quick Reference Guide Backup tab.


11316580, Rev. A, 2018-10 Page 18 of 43

Steps to go live

Enter new PT and PTT lot information in Reagent Lot Master

1. PT Innovin® - Use package insert barcode to enter ISI, lot number and expiration date.

a. Select Import.

b. Select Barcode.

c. Scan barcode on sheet with 2D barcode reader.

d. Select Save.

e. Select Exit.

2. Actin® FSL- Manually enter lot number and exp. Date.

a. Click in Lot No. field.

b. Enter 6 digit lot number (or scan vial barcode with 2D barcode reader).

c. Place cursor in Exp. Date field.

d. Enter expiration date using calendar.

e. Select Add.

f. Select Save.

3. Delete old PT and PTT lot numbers.

a. Highlight old lot, Select Delete.

4. Remove old lots of reagent and QC from the analyzer reagent table.

5. Load new QC.

6. Load PT Innovin and AFSL with barcodes visible.

a. The system must read the barcode. Do NOT call them New Inn or New AFSL.

7. Enter ISI and Normal Value (MNPT) for new PT lot.

a. Use Quick Reference Guide- Enter ISI and Normal Tab to enter the information.


11316580, Rev. A, 2018-10 Page 19 of 43

8. Print current QC if a hard copy is desired. See Quick Reference Guide/ Quality Control tab.

a. Select QC Chart icon.

b. Select Routine tab PT and PTT QC.

c. Select Switch Display and choose Current.

d. Select Print Report/Select Active Tab/Select Date Range/enter date range/Select List/Select Print.

e. Note your Historical SD for your QC. This SD can be used with your new lots.

9. Make new lots current.

a. Go to Lot Roll tab.

b. Select Switch Display.

c. Select New.

d. Highlight PTN Ci-Trol 1 Levey Jennings Chart.

e. The statistics are shown to the right of the chart.

f. Print PTN and PTTN data if hard copy is desired.

g. Are the SD’s similar to your current lots?


11316580, Rev. A, 2018-10 Page 20 of 43

NOTE: Historical SD’s may also be used instead of the auto calculated SD to provide a more robust QC range.

Change Lot

1. With PTN Ci-Trol 1 chart highlighted, Select Change Lot.

2. New QC lot number is displayed. This is the lot that will be switched to Current.

3. Select Next.

4. Confirm that “Monitoring” is checked.

5. All assays that use the control listed are checked. Do not change this setting!


11316580, Rev. A, 2018-10 Page 21 of 43

6. Select Next.

QC Chart Setting is displayed.

7. To calculate PTN and PTTN QC target limits Select Auto Calc.


11316580, Rev. A, 2018-10 Page 22 of 43

8. Select the 3rd and 4th options in the Auto Calc. screen to calculate target, 2SD/3SD limits.

9. Select Ok.

10. Warning box appears - Select OK

11. Enter PTN & PTTN target limit values into the PT & PTT settings manually.

12. Place cursor/highlight PT.

13. Select Detailed Settings.

14. Enter PTN values into the PT target/limits using the keypad.

15. Select OK on Detailed Settings screen.


11316580, Rev. A, 2018-10 Page 23 of 43

16. Repeat steps for PTT. Highlight PTT and select Detailed Settings.

17. Enter Target/Limits of PTTN for PTT.

18. Select OK on Detailed Settings Screen.

19. Select Next. A screen asking for a Fbg target will display.

20. Select OK. 21. Add a mean for Fbg if you do not have points to auto calculate.

a. Highlight Fbg C. Select Detailed Settings.

b. Enter a Target (mean).


11316580, Rev. A, 2018-10 Page 24 of 43

c. NOTE: This would only be for Fbg/ Ci-Trol 1.

22. Select Next.

23. QC Barcode Settings: Change QC01 lot (Ci-Trol 1) by Selecting on the QC01 key. This action

changes QC01 to your new Ci-Trol 1 lot number.

24. Click Next. 25. Confirmation of the Settings Completion: All settings are completed.

NOTE: Old QC Ci-Trol 1 lot data for PT, PTT, Fbg is stored automatically in a folder on C Drive. (See screenshot on page 43).


11316580, Rev. A, 2018-10 Page 25 of 43

26. Select OK. (For CS-5100: Select Complete).

27. Repeat the above steps for Change Lot for new Ci-Trol 3 (Ci-Trol 2 also as required).

28. Navigate to Lot Roll tab, PTN Ci-Trol 3 QC Chart.

29. Select Change Lot. Continue steps from above.

30. Update QC Barcode settings to deselect QC11 & QC13 lot numbers.

31. Select More (Operation panel) until QCBarcode Setting is available.

32. Select QCBarcode Setting.

33. Clear out QC11 & QC13 as desired. Use down arrow and click on blank space.


11316580, Rev. A, 2018-10 Page 26 of 43

34. Navigate to Routine tab and find the charts you just changed.

The chart will be devoid of points.

35. Order QC for PT and PTT. Use QC01 and QC03.

Note: If your laboratory uses the Auto QC function for PT and PTT this should be activated. Path: Assay Group Settings > Select Test (PT, PTT) > QC Tab > Select control > Deselect Auto QC. Repeat for each level of control.


11316580, Rev. A, 2018-10 Page 27 of 43

QC Chart Additional information:

QC data points for PTN and PTTN after Change Lot, will transfer from New to Current.

The current QC data points will be green.

Current data points are judged by the Target/Limit settings which indicate your QC is in range or not.

PTN and PTTN data can remain in the current QC chart until next lot rollover.

PTN and PTTN “New” charts are ready for the next lot rollover.

The new QC data points will be blue. There are no Target/Limit settings when establishing a “new” control lot.

Different shapes for the data points indicate different reagent lots.


11316580, Rev. A, 2018-10 Page 28 of 43

Lot Rollover Check List -

Reference Interval verification

Establish MNPT value

Establish QC ranges –Print PTN and PTTN QC points (minimum 30 points each)

Perform lot-to-lot method correlation

Print QC data for current PT QC

Print QC data for current aPTT QC

Enter new QC lot numbers into Reagent Lot Master

Input new PT reagent lot and Exp. Date in Reagent Lot Master

Input new aPTT reagent lot and Exp. Date in Reagent Lot Master

Change Lot: for new Ci-Trol 1 (PTN, PTTN, Fbg, PT, PTT)

This step enters new QC range for PT, PTT using PTN and PTTN statistics

Change Lot: for new Ci-Trol 3 (PTN, PTTN, PT, PTT)

This step enters new QC range for PT, PTT using PTN and PTTN statistics

Input new PT Normal value (MNPT) and ISI and validate

LIS: Make changes as needed

Run QC for PT and PTT

Heparin Therapeutic Range study for new lot PTT reagent


11316580, Rev. A, 2018-10 Page 29 of 43

Part II:

Uploading new control lots for other hemostasis assays with the QC Software

This process will enable the operator to upload assayed control ranges or run and establish ranges on unassayed QC lots before the current QC lot is depleted. This process is explained with Innovance® D-Dimer (assayed controls) and Fibrinogen (unassayed controls) as exemplary assays, however you can apply this process to other assays and control material distributed by Siemens Healthineers. This process is based on the assumption the reagent lot in use is not being changed.

Refer to the CS Quick Reference Guide for detailed instructions to enter reagent information in the Reagent Lot Master.

Reagent Lot Master

Enter new QC lots

1. Select Menu > Reagent Lot Master.

NOTE: Sysmex CS-5100 users will need to first select Settings to access Reagent Lot Master.

2. Select Import.

3. Select Barcode.

a. Use the 2D barcode reader to scan the 2D barcode on the DDi QC package insert sheet.

b. For fibrinogen QC or other QC materials that do not have a 2D barcode package insert sheet, enter the lot number and expiration date manually.

4. Select Save.

5. Select Close.


11316580, Rev. A, 2018-10 Page 30 of 43

QC Chart

Register new QC lots to chart

1. Select QC Chart.

2. Select appropriate control group tab (example: Routine, DDi).

3. Highlight the chart for the assay/QC material by Selecting on the center of the chart. The current lot is displayed.

4. Select More until Add Lot is displayed.

5. Select Add Lot (the software will move through each step).


11316580, Rev. A, 2018-10 Page 31 of 43

6. Select down arrow for Lot No. and select lot. The lot number, control name and expiration

date are displayed.

Note: This displays QC lots listed in the Reagent Lot Master.


11316580, Rev. A, 2018-10 Page 32 of 43

7. Verify Use QC barcode checkbox is enabled. Disable Use Preset checkbox.

8. Select Next.

9. Select Next. No action is needed for the Assay Parameter Selection.

10. QC Chart Settings is used to enter QC ranges.

a. To input TAV ranges (i.e. DDi QC):

i. Select the Lot Master Key.

ii. Select Yes. The QC ranges are now entered.


11316580, Rev. A, 2018-10 Page 33 of 43

iii. Select Next.

b. To input unassayed QC for a calibrated assay (i.e. Fibrinogen) select Next.

Note: The range will not be entered.


11316580, Rev. A, 2018-10 Page 34 of 43

11. QC Barcode Settings.

a. Select an unused QC file box. For ease of use, select a QC file box next to the current lot QC file. (if QC06=current, select QC16 for new lot).

b. Select Next.

12. Confirmation of the Settings Completion.

a. Select OK for CS-2500.

b. Select Complete for CS-5100.


11316580, Rev. A, 2018-10 Page 35 of 43

Note: To add a lot to another QC level, select the appropriate chart and repeat.

View charts: Current vs. New

1. Select More until Switch Display is listed.

2. Select New. All new lots are displayed to the “New” side of the QC chart. Go to Lot Roll tab.


11316580, Rev. A, 2018-10 Page 36 of 43

3. To view your current QC charts, Select Switch Display and select Current. Go to Routine tab. All current lots will be seen in this view.

DDi Current lot is displayed in screen below.


11316580, Rev. A, 2018-10 Page 37 of 43

Running the New QC 1. Verify correct assay reagent lots are currently on the analyzer. Only the reagent lots to be

used with the new lot of QC should be onboard!

Note: This is important when a new reagent lot is being started at the same time as new QC lot.

2. Load new lot of QC in C Rack.

3. Order assays using the file number identified in QC Charts >QC barcode settings.

4. The new QC points will display on the New QC Chart.

5. Use QC Chart>Switch Display>New to view New chart.

Make new QC lots current

Note: For assayed QC (DDi QC Level 1 &2) with package insert ranges, it is possible to change new QC to current at any time. Calibrated assays such as Fibrinogen which may use unassayed QC (Ci-Trol, Abnormal Fib Control) require an adequate number of points to establish a range in order to change the lot from new to current.

1. Select QC Chart.

2. Select appropriate control group tab (example: Routine, DDi).

3. Highlight the chart for the assay/QC material by selecting on the center of the chart. The current lot is displayed.

4. Select Change Lot.

5. Control Information Registration is displayed with new lot information.

6. Select Next.


11316580, Rev. A, 2018-10 Page 38 of 43

7. Assay Parameter Selection is displayed.

8. Confirm that “Monitoring” is checked.

9. Select Next.

10. QC Chart Setting is displayed. There are 3 choices to enter ranges.

The range is entered on this screen. For assayed controls (INN DDi) the manufacture range was entered with the new control process and will be displayed here.


11316580, Rev. A, 2018-10 Page 39 of 43

Note: For assayed controls such as Innovance D-Dimer for which it is desired to use the manufacturers provided range, select Detailed Settings and enter an appropriate 1SD value.

Detailed Settings is used to manually enter range.

I. Select Detailed Settings.

II. Use keypad to enter range next to each target limit settings.

a. Upper stop = 3 SD.

b. Upper limit = 2 SD.

c. Target = Mean.

d. Lower limit = 2 SD.

e. Lower stop = 3 S.

III. Select OK.

Use the current settings: DO NOT USE. This will enter the settings from the current lot in use.

Auto Calc.: Calculates the range from the points in chart.

I. Select Auto Calc.

II. Select 1SD Target Upper/Lower Limit Upper/Lower Stop limit.

III. Select OK.


11316580, Rev. A, 2018-10 Page 40 of 43

11. QC range will display when complete.

12. Select Next. QC Barcode Settings Screen will display.

13. Select the desired QC file, it will display in blue. Select Next.


11316580, Rev. A, 2018-10 Page 41 of 43

14. Confirmation of the Settings Completion: All settings are completed.

Select OK for the CS-2500.

Select Complete for the CS-5100.

Note: Data file path for storage of old lot QC data is indicated. (See screen shot page 43)

15. The new chart is now empty. The points have moved to the current chart.

Select Switch Display.

Select Current to view your chart.


11316580, Rev. A, 2018-10 Page 42 of 43

16. The new lot has been converted to the current lot.


11316580, Rev. A, 2018-10 Page 43 of 43

Screen showing the data file path to find file with old QC chart information

Files are identified by the QC material name and lot number.

This is a CSV file that can be viewed in Excel.

This file must be viewed on another computer that has the Excel program.

11533694, Rev. A, 2020-06 © Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany Page 1 of 8

BCS® XP Systems Sysmex® CA-Series Systems Sysmex® CS-Systems

Siemens Healthcare Diagnostics Products GmbH

Customer Bulletin June 2020

Establishing APTT Values Corresponding to the Unfractionated Heparin Therapeutic Range (U.S.) 11533694, Rev. A

Introduction

Siemens Healthineers is providing information regarding acceptable methods for establishing APTT values corresponding to the Unfractionated Heparin Therapeutic range when using APTT Reagents for Unfractionated Heparin Monitoring. This range needs to be established with each change of lot number, type of APTT reagent, or instrument platform.

Product Ordering Information

Product Name Product Description Siemens Material Number (SMN)

INNOVANCE® Heparin Kit 180 tests/kit 10873535

INNOVANCE Heparin Calibrator 1 set, 5 levels 10873530

INNOVANCE Heparin UF Control 1 5 x 1 mL 10873531

INNOVANCE Heparin UF Control 2 5 x 1 mL 10873532

Actin® 10 x 2 mL 10445709

10 x 10 mL 10445711

Actin FS 10 x 2 mL 10445712

10 x 10 mL 10445710

Actin FSL 10 x 2 mL 10445713

10 x 10 mL 10445714

Pathromtin® SL 10 x 5 mL 10446066

Background

Heparin is a widely used anticoagulant in the treatment and prevention of thrombosis. The anticoagulant effect is achieved via heparin’s interaction with antithrombin, a naturally occurring inhibitor of thrombin and other activated clotting factors. The APTT is the most popular laboratory test for monitoring the effectiveness of unfractionated heparin therapy. There are many in-vivo variables that can affect the test results, such as the patient’s antithrombin level, clotting factor levels, type of heparin used in therapy, method of administration, and drugs administered in combination with the heparin therapy. The most

Establishing APTT Values Corresponding to the Unfractionated Heparin Therapeutic Range (U.S.)

11533694, Rev. A, 2020-06 Page 2 of 8

useful information on how an APTT reagent responds to heparinized plasmas is obtained by testing patient samples.

This customer bulletin will describe the three methods used to determine the APTT values corresponding to the unfractionated heparin therapeutic range:

• Comparison of APTT values to Unfractionated Heparin concentrations (ex-vivo method) • Validation of the existing APTT reagent established Unfractionated Heparin therapeutic range, for

use with the new lot of APTT reagent (Cumulative Summation method) • Heparin Response Curve (in-vitro method)

Variables Affecting the APTT Results

Establishing APTT values corresponding to the heparin therapeutic range is required when monitoring unfractionated heparin therapy with APTT reagents. Although the APTT has been widely accepted for the last 50 years to monitor unfractionated heparin therapy, the determination of a heparin therapeutic range continues to be difficult.

Sample collection and handling are critical to any testing performed in the coagulation laboratory. Proper collection and handling techniques must be strictly followed, in order to provide valid test results. Variables that can affect the results obtained include: phlebotomy technique, the concentration of the anticoagulant used to collect the sample, the blood-to-anticoagulant ratio, storage temperature of sample prior to testing, centrifugation speed, length of time before testing, and platelet concentration after centrifugation.1,2 For current recommendations regarding standard laboratory practice, please refer to the Clinical and Laboratory Standard Institute (CLSI) Documents H47-A2 and H21-A5.

The reagent chosen to perform the APTT has considerable influence on the therapeutic range. The phospholipid, as well as activator type and content of the reagent have a large effect in determining the sensitivity of the reagent to heparin. The optical, mechanical, and turbidimetric instrument methodology can also affect the results generated.

The physiological status of the patient during heparin administration has an impact on the effectiveness of the therapy. Increased levels of Factor VIII and to a lesser degree fibrinogen are relatively common and can shorten the APTT. Concurrent therapy with Coumadin, factor deficiencies, and lupus-like anticoagulants may cause increased APTT results which may not indicate the true anticoagulation status of the patient.

Determination of APTT Values Corresponding to the Unfractionated Heparin Therapeutic Range

The recommendations presented by the College of American Pathologist (CAP) during Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy state that laboratories must determine the appropriate therapeutic range for their own APTT system used to monitor unfractionated heparin therapy.3 Laboratories have three procedures that can be followed to accomplish the validation of heparin sensitivity of the APTT: comparison of APTT values to Unfractionated Heparin concentrations (ex-vivo method), validation of the existing APTT reagent established Unfractionated Heparin therapeutic range for use with the new lot of APTT reagent (Cumulative Summation), or an in-vitro Heparin Response Curve.

This validation should be achieved by determining both the APTT and unfractionated heparin concentration for samples from patients receiving unfractionated heparin for the treatment of thromboembolism. A dose response curve can be calculated from the data using regression analysis, and


11533694, Rev. A, 2020-06 Page 3 of 8

the APTT values corresponding to an unfractionated heparin concentration of 0.3 – 0.7 IU/ml (by factor Xa inhibition assay) can then be derived. Validation of the existing APTT reagent established unfractionated heparin therapeutic range is appropriate for use in the years following an initial ex-vivo study. The cumulative summation process can be used to complete this process. Mixing pooled normal plasma with heparin in-vitro to determine the heparin therapeutic range (In-Vitro Heparin Response Curve) is no longer a preferred method.4 However, it may be the only viable option for laboratories unable to collect enough samples from patients receiving unfractionated heparin therapy to complete an ex-vivo study.

Comparison of APTT Values to Unfractionated Heparin Concentrations (Ex-Vivo Method)

The ex-vivo method determines both the APTT and unfractionated heparin concentration for samples from patients receiving unfractionated heparin for the treatment of thromboembolism. A dose response curve can be calculated from the data using regression analysis, and the APTT values corresponding to a unfractionated heparin concentration of 0.3 – 0.7 IU/ml (by Factor Xa inhibition assay) can then be derived. The anti-Xa based assays have become the preferred method to determine unfractionated heparin concentration. These assays have excellent sensitivity and specificity and are least affected by the variables discussed above.

When run in parallel with protamine titration, which has been the reference method used for many studies throughout the last 30 years, the unfractionated heparin therapeutic range using the anti-Xa assay is 0.3 – 0.7 IU/ml. The protamine titration range has been established as 0.2 – 0.4 IU/ml. Protamine titration is based on inhibition of clotting at the Factor IIa level whereas the anti-Xa assay measures the inhibition reaction at the Factor Xa level. The difference in test principle can be used to explain the difference in ranges for the two assays.

Determining the Heparin Therapeutic Range by Ex-Vivo Method 1. Set up an anti-Xa heparin assay on the system using INNOVANCE Heparin. 2. Calibrate the assay with INNOVANCE Heparin calibrator. 3. Collect acceptable patient samples, ideally collecting citrated blood from 30 – 40 patients receiving

unfractionated heparin therapy. The samples selected must have heparin concentrations that span the entire therapeutic range.

4. Measure the PT/INR and APTT; if the INR is less than 1.3, this sample can be included in the study. 5. Measure the unfractionated heparin concentration with the anti-Xa assay. 6. Perform a linear regression analysis with the trend line of the data with the unfractionated heparin

concentration on the X axis and the APTT result on the Y axis. 7. Using the corresponding regression equation, determine the APTT values corresponding to the ex-

vivo heparin results at 0.3 IU/ml and 0.7 U/ml: • Regression equation: Y = 50x + 60

Example: APTT corresponding to 0.3 IU/ml is 75 seconds. APTT corresponding to 0.7 IU/ml is 95 seconds. APTT values corresponding to the unfractionated heparin therapeutic range are 75 – 95 seconds.


11533694, Rev. A, 2020-06 Page 4 of 8

Note: All APTT testing should be performed on fresh samples. The unfractionated heparin anti-Xa testing may be performed on frozen samples if the sample was double spun as described in the preparation of the fresh normal plasma pool. Frozen samples should be rapidly thawed at 37°C, then gently mixed before testing.2 (Refer to CLSI Guideline H21-A5).

Note: For laboratories that struggle to collect an adequate number of patients undergoing Unfractionated Heparin Therapy, the following guidelines can be considered: the “absolute minimum number of samples for an accurate [Heparin Therapeutic Range] is 20, with fewer than 10% of the samples from the same patient or 50% of the samples with international normalized ratio of 1.3 to 1.5.” 5

Validation of Existing APTT Reagent Established Unfractionated Heparin Therapeutic Range, For Use with the New Lot (Cumulative Summation)

Comparison of the unfractionated heparin sensitivity of a new lot of reagent with an existing validated lot of reagent can be achieved by using the Cumulative Summation method. This is achieved ideally by selecting a new reagent lot that has the similar heparin responsiveness as the lot currently in use. It is important to control for (and prevent) drift with multiple changes over time.6 This method should only be used in years directly following an establishment of the Heparin Therapeutic Range with an ex-vivo study.

Validating Subsequent Lots of Reagent 1. Accumulate plasma samples from patients receiving unfractionated heparin therapy by performing

the following procedure: Note: The specimens can be collected before the time that the new reagent will be evaluated. a. Centrifuge specimens. b. Using a plastic pipette, remove the plasma and place in a polypropylene tube. c. Re-centrifuge for 10 – 15 minutes to remove platelets. d. Transfer into aliquots for future APTT reagent comparisons. e. Freeze until ready to use (-20°C to -70°C).

2. Select the new APTT reagent to validate. 3. Using the previously aliquoted samples, perform side-by-side testing between the previously

validated reagent and the new reagent. 4. Plot the comparison data with the old reagent on the X axis and the new reagent on the Y axis.

Note: Visual or regression analysis can be used to both determine the acceptability of the comparison data and identify discrepant and outlier results. The sum, mean, and standard deviation should be determined for each APTT reagent and the differences between the two reagents should be documented.

Document the cumulative summation of differences. In addition to recording the difference in the mean, the laboratory should prepare and maintain a cumulative summation of the differences that have occurred over time (See Table 2 below). By documenting these changes, the cumulative shift in the reagent performance in the presence of heparin can be determined and monitored. CAP suggests a “cumulative change of more than seven seconds is reason for concern.”5 CLSI guideline H47-A2 suggests a cumulative change greater than five seconds is reason for concern. It is the laboratory’s responsibility to determine what is considered acceptable variation and the actions that will be taken if acceptable variation is exceeded. Acceptable actions for reagents that exceed the acceptable limit are testing an alternate lot of reagent or completing a new ex-vivo study to confirm the Unfractionated Heparin Sensitivity of the new APTT reagent lot.5


11533694, Rev. A, 2020-06 Page 5 of 8

Cumulative Summation Difference Record5

Reagent/ Year

Mean Current Lot (sec)

Mean New Lot (sec)

Difference [New-Old] (sec)

Cumulative Summation (sec)

Accept / Reject

1 (2010) 65.4 62.9 -2.5 -2.5 Accept

2 (2011) 67.0 74.1 7.1 4.6 Accept

3 a (2012) 74.2 54.8 -19.4 -14.8 Reject

3 b (2012) 72.8 67.3 -5.5 -0.9 Accept

In-Vitro Heparin Response Curve

Mixing pooled normal plasma with unfractionated heparin in-vitro to determine the unfractionated heparin therapeutic range is no longer the preferred method. The individual in-vivo variability in heparin response is unaccounted for in the pooled plasma. Considering the nature of in-vitro mixed pooled plasma, laboratories with limited numbers of samples from patients receiving unfractionated heparin therapy can use an in-vitro heparin response curve as an alternative method to establish the APTT values corresponding to the unfractionated heparin therapeutic range. The comparison study with the heparin anti-Xa study (described on page 3) used on the same reagent should be performed to determine which dilutions of mixed pooled normal plasma corresponds with the anti-Xa concentrations of 0.3 to 0.7 IU/mL for that particular reagent.

Determining the Heparin Therapeutic Range with the In-Vitro Heparin Response Curve: 1. In order to prepare the dilutions, fresh pooled normal plasma or commercial pooled normal plasma

should be used. The following is the suggested process for preparation of Fresh Pooled Normal Plasma: a. Collect 6 – 10 fresh citrated samples from normal individuals using the collection technique

routinely used by the laboratory.

Note: Perform a routine APTT on each specimen to ensure each specimen is within normal range before pooling plasmas together.

b. Centrifuge. Using a plastic pipette, remove the plasma and place in a polypropylene tube. c. Re-centrifuge for 10 – 15 minutes to remove platelets; keep plasma covered and store at 2 – 8° C.

Note: Fresh plasma must be Platelet Poor (<10,000/mm3).

Note: Calibrator Material should not be used. d. Pool the plasmas together just prior to preparing the dilutions.

2. Make a stock solution of the unfractionated heparin solution used for patient therapy in the facility at 10 USP IU/ml heparin in 0.85 % saline. Refer to Table 3 for dilution recommendations.

Note: Request the lowest concentration of stock solution the pharmacy can provide. This will reduce errors that can occur during the manual dilution process.

Note: Heparin bagged with dextrose should not be used.


11533694, Rev. A, 2020-06 Page 6 of 8

Preparation of Heparin Stock Solution

Pharmaceutical Heparin Concentration: 10,000 IU/mL

Tube # UFH Saline Concentration

1 (1:10) 1.0 ml 9.0ml 1000 IU/mL

2 (1:10) 1.0ml from Tube #1 9.0ml 100 IU/mL


Pharmaceutical Heparin Concentration: 5,000 IU/mL


1 (1:5) 1.0ml 4.0ml 1000 IU/mL



Pharmaceutical Heparin Concentration: 1000 IU/mL


1 (1:10) 1.0ml 9.0ml 100 IU/mL


Pharmaceutical Heparin Concentration: 100 IU/mL


1 (1:10) 1.0ml 9.0ml 10 IU/mL

3. Prepare the 1.0 U/ml Heparin calibrator. Make a 1:10 dilution of the stock heparin solution formulated above with either Fresh Pooled Normal Plasma or commercial pooled normal plasma. Let the 1.0 U/ml calibrator equilibrate for 10 – 20 minutes. This allows for the heparin protein binding to take place, which is essential for curve reliability. See Table 4 for dilution recommendations.

Preparation of the 1.0 IU/ml Heparin Calibrator

Stock Heparin Solution Concentration: 10 IU/mL

Tube # “Stock” 10 IU/mL Heparin PNP Concentration

1 (1:10) 0.5 4.5ml 1 IU/mL

4. To prepare dilution points for the in-vitro Heparin Response curve, use the dilution scheme in Table 5.

Note: A specific analyzer configuration may allow this step to be automatically performed by the analyzer.

Preparation of Calibrator Dilutions Heparin Std.

Concentration PNP

1 IU/mL of “Working Stock”

0.0 IU 1000 µl 0

0.1 IU 900 µl 100 µl

0.2 IU 800 µl 200 µl

0.3 IU 700 µl 300 µl

0.4 IU 600 µl 400 µl

0.5 IU 500 µl 500 µl

0.6 IU 400 µl 600 µl

5. Perform APTT measurements on each dilution.


11533694, Rev. A, 2020-06 Page 7 of 8

6. Select the APTT results in seconds for the dilutions that correspond to the Heparin Therapeutic Range as determined in Table 6.

Siemens Specific APTT Reagent

Reagent Type Heparin Std. Concentration

correlating to the Low end of HTR (IU/ml)

Heparin Std. Concentration correlating to the High end

of HTR (IU/ml)

Actin® 0.2 0.4

Actin FS 0.2 0.4

Actin FSL 0.3 0.5

Pathromtin® SL 0.2 0.4

Note: Siemens Healthineers studies show that for Actin FSL the dilutions of 0.3 and 0.5 IU/mL correspond to an ex-vivo heparin range by heparin anti-Xa of 0.3 IU/ml to 0.7 IU/ml.

Conclusion

The recommended method for establishing APTT values corresponding to the unfractionated heparin therapeutic range for APTT monitoring of unfractionated heparin therapy is by the ex-vivo method. This method is the most specific and is least affected by the variables inherent to unfractionated heparin therapeutic range establishment. The cumulative summation method can be used to validate subsequent lots of reagent once the anti-Xa method has been completed once. Although the in-vitro heparin response curve method is not preferred, this method can be an alternative under special circumstances. The heparin response curve should only be used if the availability of samples from patients receiving heparin is limited.

References 1. CLSI. One-Stage Prothrombin Time (PT) Test and Activated Partial Thromboplastin Time (APTT) Test;

Approved Guideline-Second Edition. CLSI document H47.A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.

2. CLSI. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline-Fifth Edition. CLSI document H21-A5. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.

3. Olson JD, Arkin CF, Brandt JT, and others. College of American Pathologist Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy. Laboratory monitoring of unfractionated heparin therapy. Arch Pathol Lab Med 1998; 122:765-816.

4. Hirsh Jack, Raschke Robert, Warkentin Theodore and others. Heparin: Mechanism of Action, Pharmacokinetics, Dosing Considerations, Monitoring, Efficacy, and Safety. CHEST Supplement October 1995; 260S.

5. Marlar RA, Gausman J. The Optimum Number and Types of Plasma Samples Necessary for an Accurate Activated Partial Thromboplastin Time-Based Heparin Therapeutic Range. Arch Pathol Lab Med 2013; 137:77 – 82.

6. Olson JD. How to validate heparin sensitivity of the APTT. CAP Today 2004.


11533694, Rev. A, 2020-06 Page 8 of 8

Regulatory Information

Product and system availability are subject to local regulatory requirements and, therefore, vary by country. If you have any questions or need additional information, please contact your local technical support provider or distributor.

Additional Assistance

Technical information is available at https://www.healthcare.siemens.com/doclib/. If you need additional assistance, please contact your Siemens Healthineers Remote Services Center.

Please retain this bulletin with your records for future reference.

Trademark Information

Actin, BCS, INNOVANCE and Pathromtin are trademarks of Siemens Healthcare Diagnostics Products GmbH.

Sysmex is a trademark of the Sysmex Corporation.

All other trademarks are the property of their respective owners.