WHAT MAKES A GOOD D-DIMER ASSAY

FOR VTE EXCLUSION?

Guido Reber

ARL, Fribourg, octobre 2009

FIXa

Blood reactivity

Wall damage

Thrombogenicclot

Wall directedfibrinolysis

Flow

Vessel size

Clot size

Clot shape

Clot density

VTEDVTED

A A dynamicdynamic systemsystem

RISQUE

A A dynamicdynamic systemsystem

BENEFICE

RISQUE

A A dynamicdynamic systemsystem

RISQUE

Fibrinogen structure

Covalent binding between adjacent D-domains

- reactivity of antibodies

- format of the assays

- calibrators

- coefficient of variation at the cut-off level

- cut-off assessment and validation

- sensitivity, specificity, negative predictive value and negative

likelihood ratio

- lessons from meta-analyses

FACTORS INFLUENCING D-DIMER ASSAYS

GENERAL REQUIREMENTS FOR A D-DIMER ASSAY

- single assay

- random access mode

- available 24h/day

- short turn-around time

- quantitative

- observer-independent

- POCT: easy to use

ANTIBODY SPECIFICITY

Binding of crosslinked fragments, fibrinogen and fibrinogen lysate to mAb 3B6/22

Whitaker et al.,J. Clin Pathol 1984

Specificity of antibodies combinations

mAb 10B5E12C9 + anti-D-AP conjugated mAb 2C5A10 + anti-D-AP conjugated

mAb 10B5E12C9 + 2C5A10-AP conjugated

Pittet et al., Clin Chem 1996;42;410

DDFragment X

Fragment Y

Fragment D

ASSAY FORMATS

1. QUANTITATIVE SANDWICH ELISA

- microplate

- tips

- membrane

- beads

- immunochromatography

2. QUALITATIVE SANDWICH ELISA

3. SANDWICH TYPE IMMUNOASSAYS

4. QUANTITATIVE LATEX-ENHANCED IMMUNOASSAYS

5. QUALITATIVE LATEX IMMUNOASSAYS

Observer-dependantObserver-independant

Dempfle et al., T&H 2001;85:671.

FACT Study

FACT study

Thromb Haemost 2001;85:671.

Slopes of the calibration linesobtained by dilutions in normal plasma pool of a pool of fibrin degradation productsisolated from patients withDIC (A).

The fibin degradationproducts were treated withplasmin

Ratio of the slopes after/beforeplasmin treatment (B)

Coefficient of variation at the cut-off level

Collaborative D-dimer comparison Trial: ECAT Foundation (P. Meijer) and INSTAND (M. Spannagl)

0

5

10

15

20

25

166 306 593 1011 1236 3009 5145

Asserachrom D-dimères (ng/ml)

coef

ficie

nt d

e va

riatio

n (%

)

n=52

n=50

n=65

n=60

Adapted from Freyburger et al., Semin Vasc Med 2005; 5:328

coef

ficie

nt o

f var

iatio

n (%

)

Asserachrom D-dimer (ng/ml)

CUT-OFF VALUE AND MEASURING RANGE

Δ OD

Freyburger et al., Semin Vasc Med 2005; 5:328

Relationship between CV at the cut-off point and misclassification

Validation of a diagnostic test or strategyBüller et al, Thromb Haemost 1991;66:133-7

Phase I Technical study Elaboration of the technical aspects,e.g. type of antibody, or image-acquisitionprotocol

Phase II Performance study Comparison of the test’s characteristicswith an accepted diagnostic standard

Phase III Outcome study Study with follow-up of patients in whomthe clinical decision was made based onthe test’s results and monitoring of unfavorableoutcomes

Phase IV Cost-effectivenessstudy

Comparison of the cost-effectiveness ofstrategies incorporating the test to existingstrategies

Diagnostic PerformancesDiagnostic Performances

D + D -

Test + TP FP

Test - FN TN

D-dimer Test

Gold standard

Intrinsic performances-sensitivity-specificity- ROC curve

Practical values- negative predictive- positive predictive

- Likelihoodratios

- Likelihood ratios

•• Positive Positive likelihoodlikelihood ratio (PLR)ratio (PLR)–– ProbabilityProbability to have a positive test in to have a positive test in individualsindividuals

withwith diseasedisease / / ProbabilityProbability to have a positive test to have a positive test in in individualsindividuals withoutwithout diseasedisease

–– ComputeCompute : (TP+/ D+ )/(FP+/M: (TP+/ D+ )/(FP+/M--) = ) = Se / (1Se / (1-- SpSp))

•• NegativeNegative likelihoodlikelihood ratio (NLR)ratio (NLR)–– ProbabilityProbability to have a to have a negativenegative test in test in

individualsindividuals withwith diseasedisease / / ProbabilityProbability to have a to have a negativenegative test in test in individualsindividuals withoutwithout diseasedisease

–– ComputeCompute : (FN/M+)/(TN/M: (FN/M+)/(TN/M--) = ) = (1(1--Se) / Se) / SpSp

LikelihoodLikelihood Ratios Ratios

Fagan TJ. N Engl J Med 1975; 293: 275

nn ExclusionExclusionnn LowLow ≤≤ 0.20.2nn FairFair ≤≤ 0.10.1nn VeryVery highhigh : : ≤≤ 0.020.02

nn ConfirmationConfirmationnn LowLow ≥≥ 55nn FairFair ≥≥ 1010nn VeryVery highhigh ≥≥ 5050

Sensitivity, specificity, negative predictive value

The required sensitivity will depend on the place of the D-dimer

testing in the diagnostic work-up:

- if used as stand-alone test a high sensitivity is required

- if used in low clinical probability patients only the sensitivity

could be lower

Assays displaying high sensitivity usually have lower specificity

Negative predictive value will also depend on disease

prevalence

Clinical probability

< 500 µg/LNo Tt

Proximal CUSFemoral - popliteal

Computed Tomographyscan

+

-Positive

Tt+

NegativeNo Tt

-

-

V/Q scan +/- Angiography

PositiveTt

NegativeNo Tt-

+Unconclusive or High CP

Low Intermed. High

lack of compressibility

Tt+

D-dimères

Perrier A. Am J Med 2004

D-dimer (vidas)

Marik et al. N Engl J Med 2008

0102030405060708090

100

1980 1988 1996 1998 2001

DVTPE

Prevalence of DVT and PE in populations of clinically suspected individuals

Data from Geneva University Hospital

%

Can meta-analyses’ conclusions help in the choice of a

D-dimer assay?

Stein et al. Ann Intern Med 2004

ELISA quantitative(< 500 µg/L)

Latex quantitative(< 500 µg/L)

ELISA semi-quant.(< 500 µg/L)

Latex semi-quant.(< 500 µg/L)

Hemagglutinationqualitative (negative)

AssayType

Sensitivity SpecificityNegativelikelihood

ratio

Positive likelihood

ratio

Diagnostic performances of D-dimer assays for DVT exclusion (mean and 95% confidence interval)

NLR

0.13

0.220.24

0.150.13

0.09

0.25

De Nisio. JTH 2007

Can meta-analyses’ conclusions help in the choice of a D-dimer assay?

Performance of DPerformance of D--dimer dimer assaysassays SeSe SpSp NLRNLR PLRPLR

for PE exclusionfor PE exclusion (IC (IC àà 95%)95%)

ELISA quantitative (< 500ELISA quantitative (< 500µµg/L)g/L) 98%98% 44%44% 0.08 (0.040.08 (0.04--0.18)0.18) 1.41.4

Latex quantitative (<500 Latex quantitative (<500 µµg/L)g/L) 90%90% 45%45% 0.20 (0.100.20 (0.10--0.39)0.39) 1.71.7

Latex semiLatex semi--quantitative (<500 quantitative (<500 µµg/L)g/L) 85%85% 54%54% 0.29 (0.030.29 (0.03--2.46)2.46) 3.03.0

HemagglutinationHemagglutination qualitative qualitative ((negativenegative) )

78%78% 74%74% 0.31 (0.180.31 (0.18--0.56)0.56) 2.22.2

Roy et al. BMJ 2005 ; 331 : 259Stein et al. Ann Intern Med 2004

Can meta-analyses’ conclusions help in the choice of a D-dimer assay?

De Nisio. JTH 2007

NLR

0.09

0.180.19

0.120.09

0.07

0.21

Can meta-analyses’ conclusions help in the choice of a D-dimer assay?

CAN WE RELY ON META-ANLYSES CONCLUSIONS?

Stein et al. Ann Intern Med 2004

The ELISA in general dominate the comparative ranking among D-dimer assays for sensitivity and NLR. For excluding PE or DVT a negative result on quantitative rapid ELISA is as diagnostic useful as a normal lung scan or negative ultrasonography finding.

Assay sensitivity and negativepredictive value were frequently < 90% uncharateristc of a good-rule-out test. General use of D-dimer assay as a stand-alone test for the diagnosis of DVT is not supported by the literature.

Heim et al. Clin Chem 2004

D-dimer tests using latexturbidimetric methods appear to have tests characteristicscomparable to those for ELISA methods.

Brown et al. Clin Chem 2003

Compared to other D-dimer assays, the ELFA, microplate ELISA and latex quantitative assays have highersensitivity but lower specificity, resulting in a more confident exclusion of the disease at the expense of more additional imaging

Di Nisio et al. JTH 2007

CONCLUSIONS

- D-dimer assays should be quantitative (observer-independent)

- they should be single tests or available in random access mode

- the coefficient of variation at the cut level should not exceed5%

- turn-around time should not exceed 60 min

- whichever their place in the diagnostic work-up, their sensitivity should be as high as possible and their negative likelihood ratio around 0.1