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)

6

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