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Commissie voor Klinische BiologieCommission de Biologie Clinique
Current practices in INR testing in Belgian laboratories
Marjan Van Blerk, WIV-ISP, Brussels
Rue Juliette Wytsmanstraat 14 | 1050 Brussels | BelgiumT +32 2 642 51 11 | F +32 2 642 50 01 | email: [email protected] | www.iph.fgov.be
• Belgian EQAS in Coagulation
• Survey 2008/3
Belgian EQAS in Coagulation
• 3 surveys per year• 3 lyophilized plasmas per survey screened
for hep B, hep C, syphilis and HIV 1 & 2• For PT testing:
- artificially depleted plasmas - pools of plasma samples from patients
treated with oral anticoagulants • Participation is mandatory
Belgian EQAS in Coagulation
• Statistical evaluation by a non-parametricapproach: method of Tukey
• Report of individual results• Unacceptable result:
outside ± 2 SD of method median value• Discussion of results within the advisory board• Global report
Evolution of overall CV, %
12.7 (n=223)
10.5 (n=329)3.5 – 4
9.7/10.4(n=223)
8.4 (n=329)2.5
20061997Median INR value
Survey 2006/3
• to assess the practices in INR testing in the Belgian and Luxembourg laboratories
• to determine the extent to whichrecommendations and guidelines are followed
Survey 2006/3
2 pools of plasma samples from patients on oral anticoagulants
1 artificially depleted plasma
Questionnaire
Survey 2006/3: Questionnaire
• Citrate concentration• Lot number thromboplastin• Calculation vs calibration of INR• If calculation:
ISI: Manufacturer/Local calibrationNormal PT: MNPT/Manufacturer/
Pool/Lyophilized plasma…• Validation of INR
Survey 2006/3: INR results
22412.73.8515.73.079.72.29
NCV,%INR 7400
CV,%INR7399
CV,%INR7395
Survey 2006/3: ConclusionsNeed for further standardization of PT testing in Belgium
Recommended to•use 0.105-0.109 mol/L trisodium citrate as anticoagulant•use homogeneous test systems (reagent/instrument)•determine the system-specific MNPT
Cave: local ISI calibration
Validation of INR: Need for guidelines and control materials
Evolution of overall CV, %
7.63.132008/26.63.152008/18.22.092007/39.43.792007/2
CV, %Median INR valueSurvey
Survey 2008/3
Repeat the study of 2006/3 to evaluate:
• whether the tendency to decrease of the overall CV could be confirmed
• and if so, if this could be related to changes in methodology
Survey 2008/3: Samples
3 pools of plasma samples frompatients on oral anticoagulants
CO/8610CO/8883CO/8884
Survey 2008/3: Questionnaire
• Citrate concentration• Lot number thromboplastin• Calculation vs calibration of INR• If calculation:
ISI: Manufacturer/Local calibrationNormal PT: MNPT/Manufacturer/
Pool/Lyophilized plasma…• Validation of INR
Survey 2008/3: Participation
Survey202 Belgian and 14 Luxembourg participants
Questionnaire (84.7%)
173 Belgian and 10 Luxembourg participants
Level of clotting factors, %
1136393XII989354XI395425IX1188597VIII12197X212915VII7210576V233115II
888488838610
KULeuven, UCL Mont-Godinne
Survey 2008/3: PT reagents
216Total
2.82.8
661
Rabbit brain extractRecombinant rabbit TFRabbit brain extract
IL PT-Fibrinogen HSIL PT-Fibrinogen RecombinantIL PT-Fibrinogen
3.27Recombinant human TFIL Recombiplastin4.29Rabbit brain extractKordia Simplastin Excel S6.915Recombinant human TFRoche Neoplastin R7.917Human placentaSiemens Thromborel S13.930Recombinant human TFIL Recombiplastin 2G23.651Recombinant human TFSiemens Innovin34.374Rabbit brain extractRoche Neoplastin CI Plus%NThromboplastinReagent
Survey 2008/3: INR Results
2158.92.868.02.058.83.74Overall173.12.653.81.965.33.48Thromborel S84.93.063.22.112.53.91Simplastin Excel S745.32.933.92.125.13.90Neoplastin CI Plus 303.62.684.61.953.83.50Recombiplastin 2G76.03.104.02.247.83.89Recombiplastin154.93.244.02.246.54.06Neoplastin R515.62.705.21.916.13.51InnovinNCV,%INRCV,%INRCV,%INRReagent
CO/8884CO/8883CO/8610
Survey 2008/3: INR Results
3.193.062.202.114.263.91Simplastin Excel S
2.782.652.041.963.673.48Thromborel S
2.922.681.951.953.903.50Recombiplastin 2G
2.792.701.951.913.663.51Innovin
RELAC8884
INR 8884
RELAC8883
INR8883
RELAC8610
INR8610
Survey 2008/3: Questionnaire
• Citrate concentration• Lot number thromboplastin• Calculation vs calibration of INR• If calculation:
ISI: Manufacturer/Local calibrationNormal PT: MNPT/Manufacturer/
Pool/Lyophilized plasma…• Validation of INR
Citrate concentration for specimen collection
1.120.109/0.1293.870.129
67.01220.1098.8160.10619.2350.105
%Number of participantsCitrate mol/L
0.129 mol/L citrate
March 2000: 84.8%March 2005: 32.5%November 2006: 12.3%November 2008: 4.9%
Survey 2008/3: Questionnaire
• Citrate concentration• Lot number thromboplastin• Calculation vs calibration of INR• If calculation:
ISI: Manufacturer/Local calibrationNormal PT: MNPT/Manufacturer/
Pool/Lyophilized plasma…• Validation of INR
Lot number
• Different lot numbers in use
• For Recombiplastin 2G and Neoplastin CI Plus: enough participants to compare resultsobtained by 2 different lot numbers
Lot number
222.82.94194.32.928884
222.72.12193.32.148883
223.63.91192.43.888610Lot number 100905Lot number 100719Neoplastin CI Plus
123.02.68133.42.648884
122.71.98132.31.938883
123.43.49134.33.478610Lot number 573983Lot number 1172514Recombiplastin 2G
NCV,%INRNCV,%INR
Survey 2008/3: Questionnaire
• Citrate concentration• Lotnumber thromboplastin• Calculation vs calibration of INR• If calculation:
ISI: Manufacturer/Local calibrationNormal PT: MNPT/Manufacturer/
Pool/Lyophilized plasma…• Validation of INR
Calculation vs calibration (n=183)
Calculation of INR: 73.8%Calibration of INR: 26.2%
PT-Multi Calibrator (Dade-Behring)16/17 users (94.1%) of Thromborel S reagent30/41 users (73.2%) of Innovin reagentAccuclot (Kordia)1 user of Simplastin Excel S (Thrombolyzer)AK Calibrant (Technoclone)1 user of Neoplastin CI Plus (AMAX 200)
InnovinCalculation (11) vscalibration (30)
1,5
1,75
2
2,25
2,5
2,75
3
3,25
3,5
3,75
4
INR
Inn
ovin
PT-
Mu
lti C
al 8
610
Inn
ovin
861
0
Inn
ovin
PT-
Mu
lti C
al 8
883
Inn
ovin
888
3
Inn
ovin
PT-
Mu
lti C
al 8
884
Inn
ovin
888
4
3.47
1.90
2.70
3.72
2.0
2.86
Median CV,%
5.3 3.7
3.9 2.6
4.6 3.6
PT-Multi CalibratorInnovin (30) vsThromborel S (16)
1,5
1,75
2
2,25
2,5
2,75
3
3,25
3,5
3,75
4
INR
Inn
ovin
PT-
Mu
lti C
al 8
610
Thro
mbo
rel P
T-M
ult
i Cal
861
0
Inn
ovin
PT-
Mu
lti C
al 8
883
Thro
mbo
rel P
T-M
ult
i Cal
888
3
Inn
ovin
PT-
Mu
lti C
al 8
884
Thro
mbo
rel P
T-M
ult
i Cal
888
4
Median CV, %
3.47 3.47
1.90 1.95
2.702.655.3 5.9
3.9 3.8
4.6 3.1
Survey 2008/3: Questionnaire
• Citrate concentration• Lotnumber thromboplastin• Calculation vs calibration of INR• If calculation:
ISI: Manufacturer/Local calibrationNormal PT: MNPT/Manufacturer/
Pool/Lyophilized plasma…• Validation of INR
ISI
0
5
10
15
20
25
30
35
40N
umbe
r of
par
tici
pant
s
,8 ,9 1 1,1 1,2 1,3 1,4ISI value
ISI (n=133)
• Recommended by manufacturer: 92.5%
• Local ISI calibration: 7.5%
Local ISI determination (n=10)
1Patient samplesSTAR Recombiplastin 2G1Patient samplesSTAR EvolutionRecombiplastin1Patient samplesACL TOPPT-Fibrinogen HS1EtaloquickSysmex CA-540Neoplastin CI Plus1Patient samples Innovin/BCSACL TOPInnovin
1Patient samples Recombiplastin 2G/ACL TOP
ACL TOPInnovin1Patient samples ThrombotestBCS XPInnovin1Patient samples ThrombotestSysmex CA-1500Innovin2RELAC Sysmex CA-1500InnovinNCalibration plasmasInstrumentReagent
Innovin: ISI manufacturer vs local ISI determination
63.72.8654.82.80888464.22.0052.22.00888361.63.7354.73.638610NCV, %INRNCV, %INR
Local ISI determinationISI manufacturer
Homogeneous↔non-homogeneous (12)
1.8 2.0 2.2 2.4 2.6 2.8
3.0
3.5
4.0
4.5
5.0
Recombiplastin 2006/3
7395
7400
Homogeneous↔non-homogeneous (7)
1.8 2.0 2.2 2.4 2.6 2.8
3.0
3.5
4.0
4.5
5.0
Recombiplastin 2008/3
8883
8610
Homogeneous↔non-homogeneous(15)
2.0 2.2 2.4 2.6 2.8
3.0
3.5
4.0
4.5
5.0
Neoplastin CI PLUS 2006/3
7395
7400
Homogeneous↔non-homogeneous (4)
2.0 2.2 2.4 2.6 2.8
3.0
3.5
4.0
4.5
5.0
Neoplastin CI PLUS 2008/3
8883
8610
Homogeneous↔non-homogeneous (9)
1.8 2.0 2.2 2.4 2.6
2.5
3.0
3.5
4.0
4.5
5.0
Innovin 2006/3
7395
7400
Homogeneous↔non-homogeneous (3)
1.8 2.0 2.2 2.4 2.6
2.5
3.0
3.5
4.0
4.5
5.0
Innovin 2008/3
8883
8610
Survey 2008/3: Questionnaire
• Citrate concentration• Lotnumber thromboplastin• Calculation vs calibration of INR• If calculation:
ISI: Manufacturer/Local calibrationNormal PT: MNPT/Manufacturer/
Pool/Lyophilized plasma…• Validation of INR
Normal PT (n=131)
46.6% MNPT23.7% 100% value of reference curve19.8% Manufacturer
4.6% Pool3.1% Lyophilized plasma2.3% Combination
MNPT: Number of healthy individuals
1Infinite1Hundreds2500135022002101-15010100270450-59840-49330-39
2420-29N laboratoriesN healthy individuals
Normal PT
1/1/1/1MNPT/Pool/Lyophilized plasma/Combination
450.0 100% reference curveSimplastin Excel S1/1/1Pool/Manufacturer/Combination666.7MNPTNeoplastin R
2/1Pool/Combination261.712.946.4MNPT270.612.848.2100% reference curveNeoplastin CI Plus119.211.337.9MNPT188.110.862.1ManufacturerRecombiplastin 2G2Manufacturer571.4MNPTRecombiplastin
2/1Lyophilized plasma/Pool872.7MNPT InnovinNCV,%Median%PT normal plasmaReagent
Normal PT
264.72.933.12.135.83.92MNPT
274.42.944.02.113.53.88100% reference curveNeoplastin CI Plus
116.22.674.81.937.33.45MNPT
183.32.684.11.953.03.52Manufacturer
Recombiplastin 2G
NCV%
INR 8884
CV%
INR8883
CV%
INR8610
Reagent
Survey 2008/3: Questionnaire
• Citrate concentration• Lotnumber thromboplastin• Calculation vs calibration of INR• If calculation:
ISI: Manufacturer/Local calibrationNormal PT: MNPT/Manufacturer/
Pool/Lyophilized plasma…• Validation of INR
CLSI (H54)
• Validation of current INR testing should beperformed in each laboratory
• Minimum of 3 certified plasmas with a range of INRs between 1.5 and 4.5
• Validation should take place with any change in reagent, reagent lot number, instrument or following a major instrument repair
• INR obtained using the local system shouldcompare to the assigned INR value of the certified plasmas by +/- 15%
Validation of INR (n=172)
47 laboratoires (27.3%) mentionned validatingthe accuracy of their INR results
However: the majority of those laboratories refer toa validation of the accuracy of theirINR results by means of IQC
4 of them refer to participation in EQAS
ConclusionsInsights into the current practices in PT testing in Belgium and Luxembourg
All laboratories use low-ISI thromboplastins
High diversity of techniquesDifferent techniques yield different resultsWithin a given technique: small CVNo technique is significantly better than another
Compared to 2006/3: improvement of overall CVindicating a closer agreement between the INR results of the different laboratories
Conclusions: INR results
2158.83.748.92.868.02.05
NCV,%INR 8610
CV,%INR8884
CV,%INR8883
22412.73.8515.73.079.72.29
NCV,%INR 7400
CV,%INR7399
CV,%INR7395
Conclusions: Methodologies
• 95.1 % of the laboratories use 0.105-0.109 mol/L trisodium citrate as anticoagulant
• 93.1% of the laboratories use homogeneous test systems (reagent/instrument) ↔ 83.6% in 2006
• 46.6% determine the system-specific MNPT ↔37.3% in 2006
Validation of INR: Need for guidelines and control materials
Thanks to
EQA advisory boardJ. ArnoutB. ChatelainK. DevreeseK. JochmansO. PradierW. Wijns
AMHP van den BesselaarParticipating laboratories