Whole blood aggregometry using the Multiplate analyser … NSM 2014/Morel-kopp.pdf · Whole blood...
Transcript of Whole blood aggregometry using the Multiplate analyser … NSM 2014/Morel-kopp.pdf · Whole blood...
18/08/2011
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Whole blood aggregometry using the Multiplate analyser for the
diagnosis of HIT
Marie-Christine Morel-Kopp, Chee Wee Tan and Christopher Ward
Northern Blood Research Centre, Haematology Department, Royal North Shore Hospital - Australia
Intermediate/high clinical probability
Positive Negative, high clinical probability
Positive
Suspected HIT
Cuker A, J Thromb Haemost 2011
Low clinical probability
HIT likely HIT indeterminate HIT unlikely – continue heparin, consider alternative
diagnosis
Negative
Obtain functional assay
Discontinue heparin, start alternative anticoagulant
Obtain immunologic assay
Negative, high clinical probability
HIT diagnostic and management algorithm
4T scoreLow (1-3)
4T scoreIntermediate
(4-5)
4T scoreHigh (6-8)
HDAA negative
HDAA positive
HDAA negative
HDAA positive
HDAA positive
HDAAnegative
OD <1.0 OD >1.0 OD <1.0 OD >1.0
AA not indicated
AA not indicated
Consider AA
AA indicated
AA indicated
AA not indicated
AA: Alternative AnticoagulantHDAA Heparin-Dependant Antibody Assay
Suspected HIT
Ruf et al, Thromb Haemost 2011
HIT diagnostic algorithm HIT diagnosis
Clinical assessment: 4T’s score
Laboratory investigationfirst step: rapid antibody detection (high sensitivity)
second step: platelet functional assay to identify the pathological antibodies (high specificity)
Not practical to test all samples by functional only
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Rapid antibody detection - 1
Particle gel immunoassaysPaGIA (Diamed) good alternative for rapid HIT diagnosis and patient management for numerous laboratories
PIFA Heparin/PF4 (Akers Biosciences) same principle as PaGIA, more expansive (A$ 100.00 per cartridge)
PaGIA not reliablefalse negative: serious problem
too many false positive
possible shortage of supply in Australia and New Zealand
Rapid antibody detection - 2
Elisa tests: Asserachrom IgG and IgGAM (Stago)
GTI-PF4 IgG and IgGAM (GTI Diagnostics)
Zymutest HIA IgG and IgGAM (Hyphen BioMed)
High sensitivity, better specificityhigh dose heparin confirmatory step possible but not 100% predictive
Not realistic to use these tests for urgent investigation. Patients’ samples are usually batched with one maximum 2 runs per week
HIT functional assays
Antibodies only bind to stoichiometric PF4-heparin complexes:→ Heparin must be stopped the day before blood collection for functional investigation
Functional assays must include low and high heparin concentrations
PRP 0.5 IU/ml 100 IU/ml
Washed platelets 0.1 - 0.2 IU/ml 100 IU/ml
HIT and donor selection
Fc γRII receptor polymorphisms:
• Trp27 Low responder - Gln27 High responder
• His131 Low responder - Arg131 High responder
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HIT functional assays - 1
Most commonly used methods:Light Transmission Aggregometry (LTA): PRP (less sensitive) or washed platelets
Heparin-Induced Platelet Activation assay (HIPA): washed platelets, micromethod
Serotonin Release Assay (SRA): washed platelets loaded with radioactive serotonine = Gold Standard
All techniques are time consuming and require skilledscientists⇒ not performed in a majority of haematology laboratories
Most of the laboratories performing functional assays use random donors (non-selected)
HIT functional assay: flow cytometry
9.35% 53.1%
Platelet microparticles
Platelet/Monocyte aggregates
Platelet CD62p exposure
HIT functional assay: flow cytometry-2
HIT Alert
(IQ Products)
A. unstimulated sample <5% pos in window 2B. B. Ca-ionophore stimulated sample >80% posC. patient sample without heparin <5% posD. patient sample with heparin >8% pos
HIT functional assay: CAT
Hep 0.2 HIT Pos
No heparin
Hep 0.2 Neg
Hep 0.2
No heparin
Hep 100
HIT positive sample
Tardy-Poncet B et al J Thromb Haemost. 2009, 7:1474-81
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Multiplate® Instrument
5 channels for parallel tests
easy to use Windows XP based software
automatic analysis and documentation
duplicate sensor forinternal quality control
electronic, interactive pipetting
On the market in Europe since 2005, first instrument in Australia end of 2008
single use test cell with twin impedancesensor
sample volume0.3 ml/test
firm adhesion and aggregation of platelets on the sensor surface enhances the electrical resistance between the 2 sensor wires
Multiplate® test cell
put the test cell into the measuring position
attach the sensorcable
pipette 150 µl heparin + 300 µl donor blood*
allow 3 minutes for warming
add 150 µl of patient‘s PPP and monitor for 15 or more minutes
* usually hirudin or citrate blood
Performing the test
test 1+2
velocity
aggr
egat
ion
[AU
]
time [min]
aggregation
Area under the curve = AUC
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5
test 1 test 2
• most important parameter• expressed in AU*min or U (10 AU*min = 1 U)
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Multiplate parameters
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Rapid diagnosis of HIT by WBIA
First study performed on consecutive blood samplesfrom patients with suspected HIT*
107 samples from 97 patients
WBIA superior to LTA and equivalent to SRA
*Morel-Kopp M-C et al Thromb Res. 125:e234-e239 2010
“Weakly” positive
Strongly positive
Negative
0.5 IU/ml heparin 100 IU/ml
No aggregation
8 min lagtime
LTA and WBIA tests recorded for 20 minutes
11
16 U
6 min lagtime
293 U
2 min lagtime
373 U
0 U
0 U
LTA WBIA WBIA
Elisa comparison
AsserachromIgGAM
Zymutest IgGAM
Zymutest IgG
GTI PF4 IgG WBIA N
neg neg neg neg neg 80
POS neg neg neg neg 6
neg POS POS neg neg 2
neg neg neg POS neg 1
POS POS neg neg neg 1
POS POS neg POS neg 1
POS POS POS POS POS 15
IgG > IgGAM without loss of sensitivity
Zymutest IgG = GTI PF4 IgG
*Morel-Kopp M-C et al IJLH 33(3):245-50 2011
N pos= 25 19 17 17
WBIA assay modificationHirudin better than citrate for donor blood collection
Shorter lagtimeIncreased velocity (13.7 → 16.1)Increased AUC (290 → 403)
Citrate
Hirudin
Neg C 0.5IU Pos 0.5 IU Pos 100 IU/ml
Monitoring for 15 minutes instead of 20
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Australian HIT multicentre study
Aim: to validate the WBIA as a suitable diagnostic tool in HIT and compare it to current gold standard SRA
8 centres from major states
181 samples positive for H-PF4 antibodies by PaGIA or ELISA (IgG or IgGAM) tested by:
Zymutest IgG
SRA (as described by Sheridan et al)
WBIA
Sheridan et al: A diagnostic test for heparin-induced thrombocytopenia. Blood 1986;67:27-30
Multiplate and HIT – protocol
Per disposable cell:
300 µl hirudin blood 150 µl Heparin in Saline (2 IU/ml or 400 IU/ml)
Incubation for 2 minutes
Addition of 150 µl citrated patient plasma or inactivated serum
Monitoring for 15 minutes
Donor selection for WBIA and SRA: FcγRII 131Arg homozygous and known to be a good responder
Results
WBIA
Negative Positive
SRANegative 98 12
Positive 7 65
181 samples positive for H-PF4 antibodies (IgG, IgA or IgM)
SRA positive result:• >20% serotonin released with 0.1IU/ml heparin • <20% with 100IU/ml heparin
WBIA positive result:• aggregation with 0.5IU/ml heparin • no aggregation or >70% AUC decrease with 100IU/ml heparin
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Discrepant samples - 1
SRA 0.1IU/ml
SRA 100IU/ml SRA WBIA
0.5IU/ml WBIA
100IU/ml WBIA SRA result
% release % release Result AUC AUC Result random donor
N=2 32.88 0.79 very weak Pos Neg 68 58 Neg
N=3 63.51 12.18 weak Pos Neg 0 0 Neg
N=2 74.63 0.95 weak Pos 0 Neg very weak Pos
7 samples SRA pos (0.1 IU/ml heparin average release 56%) and WBIA neg
Using random donor, only 2 remained positive
Using SRA new definition for positive result:>50% release with 0.1 IU/ml heparin, 2 samples would be classified as negative
Discrepant samples - 2
SRA 0.1IU/ml
SRA 100IU/ML SRA WBIA
0.5IU/ML WBIA
100IU/ML WBIA
% release % release Result AUC AUC ResultN=3 10.29 -3.07 Neg 167 0 Pos
SRA 0.5IU/ml
SRA 100IU/ML SRA
% release % release ResultP2 2.04 -.021 NegP3 53.89 8.19 Pos
No sample left for patient 1
3 samples WBIA pos and SRA neg
2 retested using 0.5 IU/ml heparin and 1 became positive
Variability of the high heparin step
Sample dilution
SRA 0.1IU/ml
SRA 100IU/ml SRA WBIA
0.5IU/ml WBIA
100IU/ml WBIA
% release % release result AUC AUC result
N=2undiluted 99.26 36.72 ? 232 64 ?diluted 1/2 95.19 -3.67 Pos 166 0 Pos
N=6undiluted 96.46 42.87 ? 168 0 Posdiluted 1/2 83.54 -2.99 Pos / /
N=1undiluted 97.91 51.19 ? 213 73 ?diluted 1/2 96.06 64.34 ? 105 0 Pos
9 samples exhibited >50% drop in serotonin release with 100IU/ml heparin but still >20%
All retested by SRA after a ½ dilution and 8 became clearly HIT positive (similar results using 250IU/ml heparin)
Sample which didn’t correct: LTA positive and 4T’s score of 6
Variability of the high heparin step
0.5IU/ml
100IU/ml
0102030405060708090
100
% s
erot
onin
rel
ease
d
undiluted diluted 1/2
0.1IU/ml 100IU/ml
0.5IU/ml
100IU/ml
Undiluted Diluted 1/2
0102030405060708090
100
% s
erot
onin
rel
ease
d
undiluted diluted 1/2
0.1IU/ml 100IU/ml
6 samples
2 samples
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Correlation WBIA and SRA
WBIA
Negative Positive
SRANegative 97 12Positive 7 65
WBIANegative Positive
SRANegative 99 3Positive 5 74
WBIA
Negative Positive
SRANegative 102 3Positive 2 74
Strict SRA definition and high responder donor:Sensitivity: 90.3%Specificity:89.0%PPV: 84.42%
Modified SRA definition and high responder donor (cut-off >50% release low dose, sample dilution, >50%drop for high dose):Sensitivity: 93.7%Specificity: 97.06%PPV: 96.1%
Modified SRA definition and use of random donor:Sensitivity: 97.3%Specificity: 97.14%PPV: 96.1%
Results on Ab detection WBIA
Negative Positive
SRANegative 53 0Positive 5 48
106 samples Diamed positive
All weak SRA pos
181 samples Diamed/IgGAM positive
40 Zymutest IgG neg
Zymutest IgG SRA WBIAOD Neg Pos Neg Pos<0.500 70 2 70 20.500-1.000 16 7 17 61.000-2.000 13 14 14 132.000-3.000 2 13 3 12>3.000 1 43 0 44
102 79 104 77
Weak SRA posRelease<50%
ISTH experts recommend no further testing if OD<1.00
HIT conclusion - 1
Rapid antibody detection assaysGel-based assays :
• expansive • low specificity, 100% more false positive than IgG ELISAs
especially with the “new” PaGIA (Diamed)
ELISAS IgGAM vs IgG :
• Same sensitivity• Higher specificity for IgG only assays
IgG ELISAS and OD cut-off:
• SSC recommendations: no functional testing for OD <1.00• 7 HIT positive cases in our cohort with 0.5 < OD <1.0
HIT conclusion - 2
Importance of functional assays:
• HIT diagnosis → heparin cessation, switch to alternative anticoagulants (danaparoid, lepirudin),
• Problems: more expensive and more likely to cause bleeding
• Failure to diagnose HIT or misdiagnosis of patient with thrombocytopenia unrelated to heparin may result in significant morbidity or mortality
SRA can also gives false positive result even with a positivity cut-off raised to >50% release AND false negative
Use laboratory result (including functional results) and clinical judgement for diagnosis and patient’s management
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Negative
70%
SRA-WBIALTA
HIT ?
PaGIA
IgG ElisaIgGAM Elisa
HIT-Thr
HIT: The Iceberg Model
By Warkentin TJ
HIT conclusion - 3WBIA is easy to performWBIA has a rapid turn-around time Only a small volume of unprocessed blood required per test (microcuvette now available 175 ul of blood only)Test sensitivity increased by using selected donors (high responders) and collecting blood in hirudin tube (not citrate)WBIA is a practical alternative for haematology laboratories that do not perform functional assays and rely only on PaGIA or ELISA for HIT diagnosis or have to wait an extended time to receive the SRA result and has been acknowledged as such by the “HIT International Experts”
HIT and WBIA: donor testing
Using a pool of HIT positive samples diluted ¼, we can compare bloods and select good responders
V: 17.0V: 12.7 V: 11.3
V: 10.2
V: 9.1
Plt: 312 Plt: 226 Plt: 212
V: 9.7
Plt: 175 Plt: 182Plt: 153Plt: 106
V: 12.2
Plt: 203
LT: 2.2 LT: 2.2 LT: 1.9 LT: 3.5
LT: 3.5 LT: 4.0 LT: 4.4
V: 10.6
LT: 4.0
200 AU
0 AU
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Thank You
All clinicians and scientists from:
QueenslandPeter MolleeConnie SolanoSarah Just
NSWChristopher WardChee Wee TanTimothy BrightonJoanne Joseph Dea Prawitha Anita GhevondianGeoffrey KershawJoyce Lo
VictoriaHuyen TranShuh Ying TanJennifer Butler
South AustraliaSimon McRaeElizabeth Duncan
Western AustraliaRoss BakerJim Thom