APTT-based assays

Commonalities and contrasts in ISTH, BCSH &
CLSI guidelines for lupus anticoagulant detection
Dr Gary Moore
Sub-atomic particles
Lupus anticoagulants
Extra-solar planets
Detected by inference
Based on exclusion of other possible causes of our findings
The problem with detection by inference is specificity…..
All LA assays are ‘global’ tests designed to detect the antibodies based on the
assumption (hope?) that everything else about the patient’s coagulation is normal
Intrinsic pathway-based assays
APTTs
Non-LA causes of screening test elevation
LA-responsive APTT
Deficiencies of factors I, II, V, VIII, IX, X, XI, XII, PK, HMWK
dAPTT
Anticoagulation with VKA, UFH, (LMWH), Direct-FXa, DTI
KCT
Non-phospholipid-dependent inhibitor
SCT
Aprotinin
Shortening of screening test
Elevated FVIII, FIX
Elevated fibrinogen
Extrinsic pathway-based assays
Non-LA causes of screening test elevation
dPT
Deficiencies of factors I, II, V, X
(dPT only FVII, VIII, IX)
ASLA
Anticoagulation with VKA, Direct-FXa, DTI, (UFH)
Non-phospholipid-dependent inhibitor
Common pathway-based assays
FX activation
Non-LA causes of screening test elevation
dRVVT
Deficiencies of factors I, II, V, X
VLVT
Anticoagulation with VKA, Direct-FXa, DTI, (UFH)
Non-phospholipid-dependent inhibitor
Common pathway-based assays
FII activation
Non-LA causes of screening test elevation
Textarin time
Deficiencies of factors I, II
TSVT
Anticoagulation with UFH, DTI
Non-phospholipid-dependent inhibitor
‘Traditional’ diagnostic criteria
Prolongation of at least one phospholipid-dependent coagulation test
Evidence of inhibitory activity demonstrated by the effect of test plasma on NPP
Confirmation of the phospholipid-dependent nature of the inhibitor
Screen
Mix
Confirm
Exclusion of other causes of elevated clotting times that can mask, mimic or co-exist with LA
Routine PT & APTT
Thrombin & Reptilase time
Factor assays
anti-Xa or DTI assay
Telephone call
Why do we need guidelines?
Antibody heterogeneity
epitope specificity
concentration / avidity / affinity
Reagent variation
activators
phospholipid
Analyser end-point detection
tilt-tube
mechanical
photo-optical
No gold standard assay
no such thing as a LA assay
No reference preparation
what do you compare with?
Different interpretation strategies
clotting times
normalised ratios
calculations for PL-dependence
mixing test interpretation
2009
2012
H60-A
2014
Pre-analytical
Preparation of plasma samples:
Collect blood into 0.109 mol/L tri-sodium citrate
(Double) centrifugation
Platelet count <10 x 109/L
Filtration through 0.2 μm filters or ultracentrifugation not recommended
Samples should not be repeatedly thawed and frozen
Pre-analytical
Preliminary coagulation screen:
Coagulation screen helpful to exclude undiagnosed coagulopathies and anticoagulant treatment
Prothrombin time
APTT
Thrombin time
Further suggests employing LA-unresponsive ‘routine’ APTT
reduce serendipitous findings of LA in asymptomatic patients
if normal, can interpret results from ‘LA-responsive’ APTT at face value
LA screening tests
dRVVT
APTT
dRVVT & LA-responsive APTT only
other assays not recommended
dRVVT specifically recommended
2nd assay would normally be a suitable APTT
other assays not excluded
2 tests of different principles/pathways
dRVVT & LA-responsive APTT preferred 1st line assays
other assays not excluded as 1st or 2nd line assays
dRVVT variation
dRVVT widely used
Specific for LA in patients at high risk of thrombosis
International EQA show dRVVT most robust test in detecting LA
Pengo V, Biasiolo A, Gresele P, Maronqui, F, Erba N, Veschi F, Ghirarduzzi A, de Candia E, Montaruli B, Testa S, Barcellona D, Tripodi A; participating centres of
Italian Federation of Thrombosis Centres (FCSA). Survey of lupus anticoagulant diagnosis by central evaluation of positive plasma samples. J Thromb Haemost
2007; 5: 925-930
Jennings I, Kitchen S, Woods TA, Preston FE, Greaves M. Potentially clinically important inaccuracies in testing for the lupus anticoagulant: an analysis of results
from three surveys of the UK national external quality control scheme (NEQAS) for blood coagulation. Thromb Haemost 1997; 77: 934-937
Gardiner C, Mackie IJ, Malia RG, Jones DW, Winter M, Leeming D, Taberner SA, Machin SJ, Greaves M. The importance of locally derived reference ranges and
standardized calculation of dilute Russell’s viper venom time results in screening for lupus anticoagulant. Br J Haematol 2000; 111: 1230-1235
Lawrie AS, Mackie IJ, Purdy G, Machin SJ. The sensitivity and specificity of commercial reagents for the detection of lupus anticoagulant show marked differences in
performance between photo-optical and mechanical coagulometers. Thromb Haemost 1999; 81: 758-762
Moore GW, Savidge GF, Smith MP. Improved detection of lupus anticoagulants by the dilute Russell’s Viper venom time. Blood Coagul Fibrinolysis 2000; 11: 767-774
Moore GW & Savidge GF. Heterogeneity of Russell’s viper venom affects the sensitivity of the dilute Russell’s viper venom time to lupus anticoagulants. Blood
Coagul Fibrinloysis 2004; 15: 279-282
Arnout J, Meijer P, Vermylen J. Lupus anticoagulant testing in Europe: An analysis of results from the first European Concerted Action on Thrombophilia (ECAT)
survey using plasmas spiked with monoclonal antibodies against human β2-glycoprotein I. Thromb Haemost 1999; 81: 929-934
Jennings I, Greaves M, Mackie IJ, Kitchen S, Woods TA, Preston FE. UKNEQAS for Blood Coagulation. Lupus anticoagulant testing: improvements in performance in
a UK NEQAS proficiency testing exercise after dissemination of national guidelines on laboratory methods. Br J Haematol 2002;119: 364-369
Moffat KA, Ledford-Kraemer MR, Plumhoff EA, McKay H, Nichols WL, Meijer P, Hayward CP. Are laboratories following published recommendations for lupus
anticoagulant testing? An international evaluation of practices. Thromb Haemost 2009; 101: 178-184
Triplett DA. Use of the dilute Russell’s viper venom time (DRVVT): its importance and pitfalls. J Autoimm 2000; 15: 173-178
Moore GW, Tugnait S, Savidge GF. Evaluation of a new generation dilute Russell’s viper venom time assay system for lupus anticoagulant detection utilising frozen
reagents and controls. Br J Biomed Sci 2005; 62: 127-131
Tripodi A, Biasiolo A, Chantarangkul V, Pengo V. Lupus anticoagulant (LA) testing: performance of clinical laboratories assessed by a national survey using
lyophilised affinity-purified Immunoglobulin with LA activity. Clin Chem 2003; 49: 1608 -1614
McGlasson DL & Fritsma GA. Comparison of six dilute Russell Viper venom time lupus anticoagulant screen/confirm assay kits. Semin Thromb Hemost 2013; 39:
315-319
dRVVT variation
The Sensitivity and Specificity of Commercial Reagents
for the Detection of Lupus Anticoagulant show
Marked Differences in Performance between Photo-optical
And Mechanical Coagulometers
A.S. Lawrie, I.J. Mackie, G Purdy, S.J. Machin
5 dRVVT kits
2 analysers
Common LA+ve & LA-ve samples
Local reference intervals (n=?20)
Thromb Haemost 1999; 81: 758-62
5 RVV preparations
Common phospholipid
Common LA+ve & LA-ve samples
Local reference intervals (n=75)
6 dRVVT kits
Clotting times vs ratios
Common LA+ve & LA-ve samples
Local reference intervals (n=42)
dRVVT variation
Mechanical
Photo-optical
The Sensitivity and Specificity of Commercial Reagents
for the Detection of Lupus Anticoagulant show
Marked Differences in Performance between Photo-optical
And Mechanical Coagulometers
dRVVT kit
Sens %
Spec %
Sens %
Spec %
Manchester
76
96
76
62
Unicorn
83
81
90
69
A.S. Lawrie, I.J. Mackie, G Purdy, S.J. Machin
IL
90
42
90
46
Thromb Haemost 1999; 81: 758-62
Am Diagnostica
72
100
90
100
Gradipore
90
54
97
23
20 normals, 10 LA+ve, 10 LA+ve on VKA, 10 VKA, 10 UFH
300 samples from thrombotic population; 48 (16%) LA+ve
dRVVT reagent combination
No. samples +ve in this combination
Sigma only
20
All five RVV reagents
15
American Diagnostica only
12
Diagen, Manchester, D Stago, American Diagnostica
8
Sigma, American Diagnostica
5
Manchester only
4
Sigma, Diagen, American Diagnostica
3
Diagen, Manchester, D Stago
3
Diagen, D Stago
2
Diagen only
2
Diagen, Manchester, American Diagnostica
2
Other combinations where only one sample was positive
10
Correctly
classified
% correctly
classified
Diagnostica Stago
43/43
100
Precision Biologic
40/42
95
Siemens
41/43
95
IL
41/41
100
TCoag
40/42
95
Sekisui
25/35
71
Distributor
43 LA+ve
APTT-based assays
LA-responsive (‘routine’) APTT
Dilute APTT
Kaolin Clotting Time
Silica Clotting Time
Silica activator
Low phospholipid content
Proven LA sensitivity
LA-responsive
Low phospholipid content
Diluting a phospholipid preparation of LA-unresponsive
composition may not perform as well as an undiluted LAresponsive preparation
LA-sensitive/responsive
risks missing weaker LA if the phospholipid is too concentrated
APTT-based assays – only employ silica activator?
4.0
20 LA+ve samples
Silica
APTT (s)
RI mean (s)
Kershaw et al, Semin Thromb Hemost 2012;38:375-384
3.0
Median
normalised
screen ratio
2.0
Silica
Silica
Ellagic
acid
Polyphenols
Ellagic
acid
Ellagic
acid
1.0
KCT
not recommended
poorer reproducibility compared with other available assays
sample handling issues
long clotting times
variation in residual lipid
problematic behaviour (of kaolin) in automated coagulometers
recommendation to use APTT of proven sensitivity includes KCT
low turbidity, slow settling reagents available
sensitive assay in experienced hands
KCT
Perhaps more important:
1:4 dilution in normal plasma may dilute less potent antibodies:
loss of sensitivity
no commercially available PL-dependence confirmatory test:
loss of specificity
dPT not recommended because of thromboplastin variability
Just as much variation as APTT reagents (maybe less with recombinant reagents)
UK NEQAS reports reveal that no two laboratories use the same dilutions/procedure
High sensitivity with recombinant thromboplastin
Clinical experience suggests that dPT detects clinically significant antibodies
Standardised kit with screen/confirm recently available & has been subjected to scrutiny
Suggestion that LA detection improved when dRVVT & APTT accompanied by dPT
Evidence that some LA preferentially manifest in extrinsic pathway-based assays
Liestøl S, Jacobsen EM, Wisløff F. Dilute prothrombin-time based lupus ratio test. Integrated LA testing with recombinant tissue thromboplastin. Thromb
Res 2002;105:177-182
Mackie IJ, Lawrie AS, Greenfield RS, Guinto ER, Machin SJ. A new lupus anticoagulant test based on dilute prothrombin time. Thromb Res 2004;114:673674
Devreese KMJ. Evaluation of a new commercial dilute prothrombin time in the diagnosis of lupus anticoagulants. Thromb Res 2008;123:404-411
Lawrie AS, Mackie IJ, Purdy G, Greenfield RS, Guinto ER, Machin SJ. Lupus anticoagulant testing using a dilute prothrombin time with confirm procedure.
J Thromb Haemost 2005;3 (Suppl 1) Abstract P1817
Galli M, Borrelli G, Jacobsen EM, Marfisi RM, Finazzi G, Marchioli R, Wisloff F, Marziali S, Morboeuf O, Barbui T. Clinical significance of different
antiphospholipid antibodies in the WAPS (warfarin in the antiphospholipid syndrome) study. Blood. 2007;110:1178-1183
Moore GW, Patel Y, Savidge GF, Smith MP. The activated seven lupus anticoagulant (ASLA) assay: A new sensitive and specific assay for lupus
anticoagulant detection. Blood 2000;96:648-649
Moore GW, Smith MP, Patel Y, Savidge GF. The activated seven lupus anticoagulant (ASLA) assay: a new test for lupus anticoagulants (LAs). Evidence
that some LAs are detectable only in extrinsic pathway based assays. Blood Coagul Fibrinolysis 2002;13:261-269
Martinuzzo M, Adamczuk M, Varela ML, Pombo G, Forastiero R. The activated seven lupus anticoagulant (ASLA) test has comparable sensitivity to
classical assays for screening of lupus anticoagulant. Thromb Haemost 2005;93:1007-1009
Moore GW, Rangarajan S, Savidge GF. The activated seven lupus anticoagulant assay detects clinically significant antibodies. Clin Appl Thromb/Haemost
2008;14:332-337
110 samples from patients with VTE previously classified as LA negative
Assays based on snake venom fractions Taipan, Textarin & Ecarin not recommended:
no standardised commercial assays
require further critical evaluation
Prothrombin activator groups
A
no co-factors
B
Ca++ dependent
C
Ca++ & PL dependent
Taipan
D
Ca++, PL & FVa dependent
Textarin
Insensitive to VKA effect
Ecarin
T/E ratio = Textarin time (s)
Ecarin time (s)
TSVT screen ratio via NPP
TSVT PNP ratio
% correction of ratio
(1) % correction of ratio
(2)
TSVT (s)
ET (s)
(3)
TSVT (ratio)
ET (ratio)
Taipan, Textarin & Ecarin venoms
Triplett DA, Stocker KF, Unger GA, Barna LK. The Textarin/Ecarin ratio: a confirmatory test for lupus anticoagulants. Thromb Haemost. 1993; 70:
925-931
Rooney AM, McNally T, Mackie IJ, Machin SJ. The Taipan snake venom time: a new test for lupus anticoagulant. J Clin Pathol 1994;47:497-501
Moore GW, Smith MP, Savidge GF. The Ecarin time is an improved confirmatory test for the Taipan snake venom time in warfarinised patients with
lupus anticoagulants. Blood Coagul Fibrinolysis 2003;14:307-312
Forastiero RR, Cerrato GS, Carreras LO. Evaluation of recently described tests for detection of the lupus anticoagulant. Thromb Haemost
1994;72:728-783
Luddington R, Scales C, Baglin T. Lupus anticoagulant testing with optical end point automation. Thromb Res 1999; 96:197-203
Lawrie AS, Mackie IJ, Purdy G, Machin SJ. The sensitivity and specificity of commercial reagents for the detection of lupus anticoagulant show
marked differences in performance between photo-optical and mechanical coagulometers. Thromb Haemost. 1999; 81:758-62.
Parmar K, Connor P, Hughes GRV, Hunt B J. Validation of the Taipan snake venom assay in routine practice to assess lupus anticoagulant status
in patients being assessed for lupus anticoagulant and not receiving oral anticoagulant. J Thromb Haemost 2003;1 Suppl 1 July: abstract number
PI553
Moore GW, Kamat AV, Gurney DA, O'Connor O, Rangarajan S, Carr R, Savidge GF. Alteration in the laboratory profile of a lupus anticoagulant in a
patient with non-Hodgkin’s lymphoma. Clin Lab Haematol. 2004; 26:429-34.
Parmar K, Lefkou E, Doughty H, Connor P, Hunt BJ. The utility of the Taipan snake venom assay in assessing lupus anticoagulant status in
individuals receiving or not receiving an oral vitamin K antagonist. Blood Coagul Fibrinolysis 2009;20:271-275
Moore GW. Combining Taipan snake venom time/Ecarin time screening with the mixing studies of conventional assays increases detection rates of
lupus anticoagulants in orally anticoagulated patients. Thromb J 2007;5:12
van Os GM, de Laat B, Kamphuisen PW, Meijers JC, de Groot PG. Detection of lupus anticoagulant in the presence of rivaroxaban using Taipan
snake venom time. J Thromb Haemost. 2011; 9:1657-1659
Moore GW, Bromidge ES, Polgrean RF, Archer RA, Squires I. Taipan snake venom time coupled with ecarin time testing enhances lupus
anticoagulant detection in non-anticoagulated patients. J Thromb Haemost 2013;11(Suppl 2) Abstract 3.62-6
LA screening tests
dRVVT
APTT
Numbers of screening tests
No single test is sensitive to all LA – use 2 tests of different principles
2 tests of different principles/pathways
dRVVT & LALA-responsive APTT preferred 1st line assays
other assays not excluded as 1st or 2nd line assays
dRVVT specifically recommended
2nd assay would normally be a suitable APTT
other assays not excluded
dRVVT & LALA-responsive APTT only
other assays not recommended
Risk of false-positive results increased to unacceptable level if >2 tests performed
Potential inconsistency between techniques used for additional test methods
Some patients will generate an elevated screening test with at least one test/reagent type
Chances of this occurring increase as more tests performed
genuine LA unreactive in other reagents
‘weak’ LA
discrete analytical error
merely because the patient is a natural statistical outlier for that reagent/analyser pairing
ethnic differences
LA screening tests
dRVVT
APTT
Numbers of screening tests
2 tests of different principles/pathways
dRVVT & LALA-responsive APTT preferred 1st line assays
other assays not excluded as 1st or 2nd line assays
dRVVT specifically recommended
2nd assay would normally be a suitable APTT
other assays not excluded
dRVVT & LALA-responsive APTT only
other assays not recommended
>2 screening tests may well result in more positive individual screening test results
Application of the confirmatory test(s) will not lead to more positive overall interpretations
Instead, genuine LA that were unreactive in first-line assays may be identified
Some perform 3 assays, covering intrinsic, extrinsic & common pathways to minimise this problem
CLSI supports limiting to 2 although LA heterogeneity may necessitate additional screening tests
Clotting times vs ratios
Table 1: section (E) Expression of results
Results should be expressed as ratio of patient-to-PNP for all procedures (screening, mixing and confirm)
Normalised ratios for all results
What do you gain from normalising the data?
Improves intra- and inter- assay variation by mitigating for:
operator variability
analyser performance variation
reagent quality/stability
NPP variation between reagents
NPP variation between paired reagents
Ratio calculations
Screen & confirm ratios calculated using normal pool plasma clotting time as the denominator
Screen & confirm ratios calculated using RI mean clotting time as the denominator
Not all NPP generate the same clotting
times with different reagents for the same
test type
Gardiner C, Mackie IJ, Malia RG, Jones DW, Winter M, Leeming D, Taberner SA, Machin SJ,
Greaves M. The importance of locally derived reference ranges and standardized calculation
of dilute Russell’s viper venom time results in screening for lupus anticoagulant. Br J
Haematol. 2000;111:1230-1235
Jennings I, Greaves M, Mackie IJ, Kitchen S, Woods TA, Preston FE. UKNEQAS for Blood
Coagulation. Lupus anticoagulant testing: improvements in performance in a UK NEQAS
proficiency testing exercise after dissemination of national guidelines on laboratory methods.
Br J Haematol. 2002;119:364-369
Results from NPPs taken into different
sample tubes &/or lyophilised may not
correlate with local patient samples
Hirst CF, Poller L. The cause of turbidity in lyophilised plasmas and its effects on coagulation
tests. J Clin Pathol 1992; 45: 701-703
Local RI mean negates variability between
different NPP preparations or different
batches of a given NPP
Moore GW, Brown KL, Bromidge ES, Drew AJ, Ledford-Kraemer MR. Lupus anticoagulant
detection: out of control? Int J Lab Haematol 2013;35:128-136
De Laat B, Derksen RH, Reber G, Musial J, Swadzba J, Bozic B, Cucnik S, Regnault V,
Forastiero R, Woodhams BJ, de Groot PG. An international multicentre-laboratory evaluation
of a new assay to detect specifically lupus anticoagulants dependent on the presence of antibeta2-glycoprotein autoantibodies. Thromb Haemost 2011;9:149–53
Comparison of NPP mean clotting times
dRVVT
screen
dRVVT
confirm
dAPTT
screen
dAPTT
confirm
CRYOcheck™ frozen normal pool mean (s)
44.0
37.8
36.0
42.8
Locally prepared normal pool mean (s)
44.8
34.8
38.1
40.3
Technoclone lyophilised platelet poor plasma
mean (s)
47.4
35.9
42.8
46.8
Reference interval mean (s)
43.8
37.6
41.4
45.9
NPP
CRYOcheck™ frozen normal pool virtually identical to RI means for dRVVTs
Technoclone lyophilised platelet poor plasma closest to RI means for dAPTTs
Moore GW et al. Lupus anticoagulant detection: out of control? Int J Lab Haematol 2013;35:128-136
False positive or negative results with unsuitable NPP
Ref. interval
dRVVT screen
dRVVT confirm
dAPTT screen
dAPTT confirm
Clotting times (s)
37.1 – 51.1
33.8 – 41.4
33.1 – 49.7
37.6 – 54.2
Ratios
0.85 – 1.17
0.90 – 1.10
0.80 – 1.20
0.82 – 1.18
False negative dRVVT screen:
False positive dAPTT screen:
54.7 s
% correction (<10)
1.15
54.7 s
47.4 s
43.8s
Technoclone NPP
RI mean
47.0 s
47.0 s
=
1.31
36.0 s
41.4s
CRYOcheck NPP
RI mean
False negative dAPTT interpretation: 51.6 s
Confirmatory tests
=
=
1.35
51.6 s
38.1 s
41.4s
Local NPP
RI mean
50.5 s
=
1.25
50.5 s
40.3 s
45.9s
Local NPP
RI mean
7.4
12.0
=
1.25
=
1.14
=
1.25
=
1.10
Mixing test
Perform on 1:1 mixture with NPP
Evaluate with Index of Circulating Anticoagulant (ICA) or mixing test-specific cut-off
Detection and Quantitative Evaluation of Lupus Circulating
Anticoagulant Activity
Rosner E, Pauzner R, Lusky A, Modan M, Many A.
Thromb Haemost 1987; 57: 144-7
ICA (Rosner Index):
Cut-off ~ 15%
(Mixture clotting time – NPP clotting time)
test clotting time
x 100
Mixing test
Mixing test RIs narrower than undiluted plasma
Clotting times of normal samples at the extremes are compensated for upon mixing with NPP
Thus, lower cut-off & increased sensitivity
Mixing test
Mandated
Screen - Mix - Confirm
Mixing test improves specificity but introduces dilution factor that can mask weak LA
If screen & confirm on undiluted plasma appear positive and no evidence of other
causes of elevated clotting times, consider LA positive even if mixing test negative
Mixing test unnecessary only if:
(i) LA screen elevated
(ii) Associated confirm test corrects mathematically AND into reference range
(iii) No evidence of other causes of elevated clotting times
Dilution effect in LA mixing tests
Reference range
Coagulation screen
(DFXa-sensitive)
1.0
0.8 – 1.2
(LA-unresponsive)
1.1
0.8 – 1.2
Fibrinogen (g/L)
3.5
1.67 – 5.43
Thrombin time ratio
1.10
0.80 – 1.23
dAPTT ratio
1.33
0.81 – 1.23
dAPTT confirm ratio
1.00
0.81 – 1.13
dAPTT 50:50 mix ratio
1.09
0.86 – 1.15
dAPTT 50:50 mix confirm ratio
0.97
0.85 – 1.09
dRVVT ratio
1.42
0.86 – 1.19
dRVVT confirm ratio
1.09
0.83 – 1.13
dRVVT 50:50 mix ratio
0.95
0.90 – 1.10
dRVVT 50:50 mix confirm ratio
0.94
0.94 – 1.13
INR
APTT ratio
LA assays
Authorisation comment:
Consistent with the presence of a lupus anticoagulant by dAPTT & dRVVT analysis.
Suggest repeat in no less than 12 weeks to confirm persistence of the LA.
Paradigm shift
Even 1:1 mixing studies can dilute LA to give clotting time/ratio below cut-off
Reber G, Meijer P. In ECAT veritas? Lupus 2012; 21: 722-724
Hong SK, Hwang SM, Kim JE, Kim HK. Clinical significance of the mixing test in laboratory diagnoses of lupus anticoagulant: the fate of the mixing test in integrated lupus
anticoagulant test systems. Blood Coagul Fibrinolysis 2012; [Epub ahead of print]
Devreese KM. No more mixing tests required for integrated assay systems in the laboratory diagnosis of lupus anticoagulants? J Thromb Haemost 2010; 8: 1120-1122
Moore GW, Savidge GF. The dilution effect of equal volume mixing studies compromises confirmation of inhibition by lupus anticoagulants even when mixture specific
reference ranges are applied. Thromb Res 2006;118:523-528
Thom J, Ivey L, Eikelboom J. Normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulant. J Thromb Haemost 2003; 1: 2689-2691
Clyne LP, Yen Y, Kriz NS, Breitenstein MG. The lupus anticoagulant. High incidence of ‘negative’ mixing studies in a human immunodeficiency virus-positive population.
Arch Pathol Lab Med 1993; 117: 595-601
Moore GW, Savidge GF, Smith MP. Improved detection of lupus anticoagulants by the dilute Russell’s Viper venom time. Blood Coagul Fibrinolysis 2000; 11: 767-74
Kaczor DA, Bickford NN, Triplett DA. Evaluation of different mixing study reagents and dilution effect in lupus anticoagulant testing. Am J Clin Pathol 1991; 95: 408-411
Moore GW, Henley A, Greenwood CK, Rangarajan S. Further evidence of false negative screening for lupus anticoagulants. Thromb Res 2008;121:477-484
Devreese KMJ. Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants. Thromb Res 2007;119:369-376
Moore GW. Combining Taipan snake venom time/Ecarin time screening with the mixing studies of conventional assays increases detection rates of lupus anticoagulants in
orally anticoagulated patients. Thromb J 2007; 5: 12
Male C, Lechner K, Speiser W, Pabinger I. Transient lupus anticoagulants in children: stepwise disappearance of diagnostic features. Thromb Haemost 2000; 83: 174-175
Brandt JT, Triplett DA, Musgrave K, Orr C. The sensitivity of different coagulation reagents to the presence of lupus anticoagulants. Arch Pathol Lab Med 1987;111:120-124
Screen – Confirm – (Mix)
Confirmatory test for phospholipid dependence
Screen and confirm must be based on the same test principle
% correction of ratio
Normalised test/confirm ratio
(screen ratio – confirm ratio) x 100%
screen ratio
screen normalised ratio
confirm normalised ratio
BCSH guidelines 2000 suggested flow chart
APTT
abnormal
normal
Mixing test
correction
no correction
dRVVT
normal
Factor assays
Stop or do other tests
abnormal
Correction/confirm test
no correction
equivocal
correction
LA
Other tests
Interpretations based on BCSH 2000
1.
2.
3.
4.
5.
APTT
Mixing test
dRVVT
Elevated
Elevated
Positive for LA
APTT
Mixing test
dRVVT
Elevated
Normal
Positive for LA
APTT
dRVVT
Normal
Positive for LA
APTT
Mixing test
dRVVT
Elevated
Elevated
Normal
APTT
Mixing test
dRVVT
Elevated
Normal
Normal
Antibody detected
Antibody detected
Antibody detected
Antibody presumed to
be LA
APTT-reactive LA
missed
False negative screening tests
The ability of a given concentration of antibody to prolong a clotting time above the reference range may
depend on the clotting time an individual plasma would have had without the influence of the LA
For example:
Baseline ratios:
Appearance of LA:
dRVVT screen
0.90
(RI 0.84 – 1.18)
dRVVT confirm
0.91
(RI 0.88 – 1.12)
dRVVT screen
1.15
dRVVT confirm
0.91
% ratio correction
20.9
(<10)
Normalised ratio
1.26
(<1.15)
Integrated testing
Perform paired screen and confirm in parallel on every patient
Assess for concordance/discordance irrespective of whether screen elevated or not
CLSI reserves its definition of integrated testing to screen & confirm on 1:1 mixing tests
“ In principle, these tests do not require performance of the mixing test….”
Circumvents traditional testing order of Screen – Mix - Confirm
Integrated testing - limitations
Confirm result in LA +ve patients does not always shorten to within the RI
dRVVT screen ratio
1.98
(RI 0.84 – 1.18)
dRVVT confirm ratio
1.35
(RI 0.88 – 1.12)
% correction
31.8
(<10)
Screen/confirm ratio
1.47
(<1.15)
Elevated confirm:
potent/avid LA
co-existing abnormality
dRVVT screen ratio
1.98
(RI 0.84 – 1.18)
Mixing test screen ratio
1.59
(RI 0.90 – 1.10)
dRVVT confirm ratio
1.85
(RI 0.88 – 1.12)
Mixing test confirm ratio
1.54
(RI 0.89 – 1.10)
(<10)
Non-phospholipid dependent inhibitor
% correction
Screen/confirm ratio
6.6
1.07
(<1.15)
Mixing test screen ratio
1.01
(RI 0.90 – 1.10)
Mixing test confirm ratio
1.02
(RI 0.89 – 1.10)
Mixing test screen ratio
1.42
(RI 0.90 – 1.10)
Mixing test confirm ratio
1.08
(RI 0.89 – 1.10)
dRVVT screen ratio
1.29
(RI 0.84 – 1.18)
dRVVT confirm ratio
1.12
(RI 0.88 – 1.12)
Mixing test screen ratio
1.98
(RI 0.90 – 1.10)
Mixing test confirm ratio
1.08
(RI 0.89 – 1.10)
Factor deficiency
Lupus anticoagulant
Lupus anticoagulant co-factor effect
Cut-off values
Cut-off values should be specific for reagent/analyser pairing
Do not use cut-offs from elsewhere
99th percentile from at least 40 donors
Cut-off values
Cut-off values should be specific for reagent/analyser pairing
Cut-offs may be available from manufacturer but local validation advised
Historically: mean + 2SD (97.5th percentile)
99th percentile (mean + 2.3SD if Gaussian) would improve specificity but reduce sensitivity
Large numbers of suitably prepared normal donors needed to estimate 97.5th or 99th with accuracy
Previously established cut-offs (manufacturer or different analyser); validate with fewer donors (20 – 60)
Cut-off values
Cut-off values should be specific for reagent/analyser pairing
Aligns with CLSI C28-A3 How to define and determine reference intervals in the clinical laboratory
Clotting assays, including APTT, dRVVT & dPT have Gaussian distributions (parametric appropriate)
≥40 donors & calculate mean ±2SD
Will generate 2.5% tails but composite LA testing not just screen result reveals whether LA present or not
Reference intervals can be established by transference
LA testing during therapeutic anticoagulation
Most patients can wait for LA testing until the period of anticoagulation is complete
VKAs
Result interpretation is difficult because of prolonged basal clotting times
Recommend testing 1 - 2 weeks after discontinuation of treatment or when INR <1.5
If INR 1.5 - 3.0, consider 1:1 mixing studies; interpretation may be difficult & titre diluted 2-fold
Textarin / Taipan / Ecarin testing not recommended as they require further critical evaluation
LA testing during therapeutic anticoagulation
VKAs
Utility of testing undiluted plasma is disputed
Perform screen & confirm on 1:1 mixtures with NPP
Positive result is diagnostic but negative result does not exclude a weak LA
TSVT + Ecarin time or platelet neutralisation procedure useful secondary testing
No limits placed on INR values
Detecting LA in VKA anticoagulated patients
Mixing studies often inconclusive when using assays sensitive to the warfarin effect
INR
dRVVT
Confirm
1:1 screen
1:1 confirm
2.0 – 4.5
0.86 – 1.19
0.83 – 1.13
0.90 – 1.10
0.94 – 1.13
3.51
2.87
2.39
1.46
1.07
Neat plasma unreliable (?)
>10% correction
1:1 confirm returns into reference range
1:1 screen
1:1 confirm
1.07
1.00
Mixing studies negative
BUT a weak LA cannot be excluded
TSVT/ET will detect many LA
dRVVT &/or dAPTT mixing tests may detect TSVT-unreactive LA (if sufficiently potent to overcome dilution)
Negative TSVT and dRVVT/dAPTT mixing tests - does not exclude LA
Triple test strategy increases detection rates
LA testing during therapeutic anticoagulation
Heparins
LA screening not possible if plasma unclottable or heparin level exceeds neutraliser capacity
don’t perform LA screening if patient receiving therapeutic doses of UFH
heparin neutralisers should quench low-dose subcutaneous UFH & LMWH
avoid platelet neutralisation procedure
gives examples of when LA can and can’t be detected
avoid platelet neutralisation procedure
LA testing during therapeutic anticoagulation
NOACs
Effect of DTIs or direct FXa inhibitors unknown (written in 2009)
Not covered
Provides table of interferences
Most LA assays affected by DTIs and direct FXa inhibitors
Summary
ISTH 2009
BCSH 2012
CLSI 2013
Area of
recommendation
Sample preparation
Assays to use
Double centrifugation
dRVVT & APTT
Testing order
dRVVT
plus APTT or others
Screen – Mix - Confirm
Screen – Confirm - Mix
NPP denominator
RI mean denominator
Ratio derivation
Reference
interval/cut-offs
99th percentile
Phospholipiddependence
calculations
Mixing test
97.5th percentile
(if Gaussian)
Testing patients on
UFH
Interpretive
reporting
97.5th percentile
(if Gaussian)
% correction of screen by confirm
LA ratio (screen/confirm)
Perform on 1:1 mixture with NPP
Perform on 1:1 mixture with NPP
Interpret with ICA
or
mixing test-specific cut-off
Testing patients on
VKAs
dRVVT & APTT
&/or others
Perform on 1:1 mixture with NPP
Interpret with ICA
or
mixing test-specific cut-off
Undiluted plasma if INR <1.5
Screen & confirm on 1:1 mix with NPP
Screen & confirm on 1:1 mix with NPP
Mix with NPP if INR >1.5 <3.0
TSVT + ET or PNP
TSVT + ET or PNP
Interpret with caution
Can detect LA in some cases where heparin neutraliser is effective
Recommended
ELISA for the pathological
domain I β2GPI antibodies
Dr Gary Moore
Haemostasis & Thrombosis
St. Thomas’ Hospital
Westminster Bridge Road
London SE1 7EH
UK
[email protected]
www.viapth.co.uk

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