Laboratory testing for LA

Diagnosing lupus
anticoagulants
Emmanuel J Favaloro, Haematology, ICPMR, Westmead Hospital
ISLH, Toronto, May, 2013

Disclosures
No disclosures or conflicts of interest
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Dedication
Dedicated to memory of Prof Jerry Koutts.
November 27, 1944 - April 2, 2013
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Talk overview – ‘attack of the acronyms’
‣ APS (antiphospholipid [antibody]
syndrome)
‣ aPL (antiphospholipid [antibodies])
‣ LA (Lupus anticoagulants
[antibodies]/inhibitor)
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Talk overview – ‘attack of the acronyms’
‣ APTT (Activated Partial Thromboplastin Time)
‣ dRVVT (dilute Russell Viper Venom Time)
‣ SCT (Silica Clotting Time)
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Talk overview – ‘attack of the acronyms’
‣ aCL (anticardiolipin [antibodies])
‣ a 2GPI (anti-beta 2 glycoprotein I [antibodies])
‣ aPT (anti-prothrombin [antibodies])
‣ aPS (anti-phosphatidylserine [antibodies])
‣ aPS/PT (anti-phosphatidylserine-prothrombin
[antibodies] complex)
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APS – more acronyms
‣ PAPS - Primary Antiphospholipid Syndrome (APS not
associated with another disease process)
‣ SAPS - Secondary Antiphospholipid Syndrome (APS
associated with another disease process; e.g., SLE).
‣ CAPS - Catastrophic Antiphospholipid Syndrome (an
aggressive form of APS; widespread thrombosis, multiple
organ sites; significant morbidity and/or mortality).
‣ SNAPS - Seronegative Antiphospholipid Syndrome
(patients with typical clinical manifestations of APS but
negative for a range of aPL tests/assays).
Favaloro & Wong. Autoimmunity Highlights. 2010; 1:5-14
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APS (antiphospholipid syndrome)
‣ An auto-immune disease associated with the
presence of antiphospholipid antibodies (aPL)
‣ May present with a wide variety of clinical
manifestations:
‣ Haematologists – may see this as a prothrombotic
disorder (& perhaps as thrombocytopaenia)
‣ Obs & Gyn – pregnancy morbidity
‣ Dermatologists – dermatological presentations
‣ Neurologist - migraine, memory loss, chorea
‣ other clinical specialists = other clinical presentations
Favaloro & Wong. Autoimmunity Highlights. 2010; 1:5-14
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Revised classification criteria for APS.*
APS is present if at least one of the following clinical
criteria (and one of the laboratory criteria) are met:
1. Clinical criteria
a. Vascular thrombosis:
One or more clinical episodes of arterial, venous, or small vessel thrombosis, in
any tissue or organ. Thrombosis must be confirmed by objective validated criteria.
b. Pregnancy morbidity:
(i) One or more unexplained deaths of a morphologically normal fetus at or
beyond the 10th week of gestation, with normal fetal morphology, or
(ii) One or more premature births of a morphologically normal neonate before
the 34th week of gestation because of eclampsia, severe preeclampsia, or
placental insufficiency, or
(iii) Three or more unexplained consecutive spontaneous abortions before the
10th week of gestation, with exclusion of anatomic, hormonal and chromosomal
causes.
* Summarised from Miyakis et al. JTH 2006;4:295-306.
‘APS classification criteria’ to establish ‘definite APS’ for inclusion in
research studies; however, popularly used as ‘APS diagnostic criteria’
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Revised classification criteria for APS.*
APS is present if at least one of the following (clinical
criteria and one of the) laboratory criteria are met:
2. Laboratory criteria**
a. LA present detected according to ISTH SSC guidelines***
b. aCL antibody of IgG and/or IgM isotype, present in medium or high titer.
c. a 2GPI antibody of IgG and/or IgM isotype.
**aPL must be detected on two or more occasions at least 12
weeks apart. aCL & a 2GPI should be “measured by a
standardized ELISA according to recommended procedures”.
* Miyakis et al. JTH 2006;4:295-306
*** Pengo et al. JTH 2009;7:1737-40
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Revised classification criteria for APS.*
APS is present if at least one of the following (clinical
criteria and one of the) laboratory criteria are met:
2. Laboratory criteria
a. LA present detected according to ISTH SSC guidelines**
• May soon be challenged by ‘CLSI guidelines’
• What about the British guidelines?***
b. aCL antibody of IgG and/or IgM isotype, present in medium or high titer.
• IgM?
• No standard accepted definition of medium or high titer
c. a 2GPI antibody of IgG and/or IgM isotype.
• IgM?
• No standard accepted definition of medium or high titer
d. aCL & a 2GPI should be “measured by a standardized ELISA according to
recommended procedures”.
• standardized ELISA? No such beast.
* Miyakis et al. JTH 2006;4:295-306
** Pengo et al. JTH 2009;7:1737-40
*** Keeling et al. BJH 2012;157:47-58
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Laboratory testing to identify LA requires*:
(1) Prolongation of at least one of two phospholipid dependent
clotting assays based on different principles and coagulation
pathways (‘screening’);
(2) Evidence that prolongation of the screening test(s)
demonstrates phospholipid dependence by using a similar second
test(s) using altered concentrations and/or composition of
phospholipids (‘confirmation’);
(3) Mixing assays are recommended; if performed, these must
show evidence of inhibitory activity by the effect of patient plasma
on an equal volume of normal pooled plasma (‘mixing’);
(4) LA should be distinguished from other causes of prolonged
clotting times that may mask, mimic, or coexist with LA, such as
anticoagulant therapies or other coagulopathies (‘exclusion’).
* annotated from ISTH SSC** & CLSI guidelines
(**Pengo et al. JTH 2009;7:1737-40 & prior)
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Laboratory testing for LA:
‣ Two screening tests ‘negative’ in order to exclude LA;
recommended*:
‣ APTT (Activated Partial Thromboplastin Time) &
‣ dRVVT (dilute Russell Viper Venom Time)
‣ Either one ‘positive’ in order to identify LA
‣ Possible that no two labs use exactly the same LA procedure
‣ APTT: reagents are LA ‘sensitive’ (variably) vs LA insensitive (variably)
‣ dRVVT – some reagents are more sensitive to LA than others
‣ APTT (mix vs no mix)
‣ dRVVT (mix or no mix)
‣ Mix (1:1 or other?)
‣ Mix plasma (normal) used (commercial, in house, lyophilised, frozen)
‣ Normal ranges – 20, 40 or more individuals? +/- 2SD, +/- 3 SD, >99 th percentile, etc?
‣ Ratio, normalised ratio, % correction, Rosner index/ICA
‣ Ratio – denominator = normal plasma value or median of reference interval (etc)
‣ etc, etc
* Pengo et al. JTH 2009;7:1737-40
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Laboratory testing for LA - Cut-off values :
‣ Individual screen & confirm assays:
‣ ISTH SSC* & CLSI guidelines – require ‘local validation’ of
cut-off value
‣ ISTH SSC: based on testing of ‘at least 40 adult healthy
donors 99 th percentile
‣ CLSI recommendation – will be different
‣ source of normal donors?
‣ are hospital ‘normals’ true normals?
‣ exclude outliers/LA positive samples?
‣ statistical anomaly of 40 donors vs 99 th percentile for nonnormal
distributions.
* Pengo et al. JTH 2009;7:1737-40
** McGlasson & Fritsma. STH 2013; 39:315-319
*** Kershaw & Orellana. STH 2013;39:283-290
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Laboratory testing for LA - Cut-off values :
‣ Screen & confirm assay cut-offs:
‣ Support for ‘generic’ ratio of (around) 1.2*
‣ 200 normal subjects (40 for each of 5 centres)
‣ 6 LA functional assays (same procedure, reagent
lot and analyser type at each site).
‣ dRVVT LA screen and confirm assays, SCT (silica
clotting time) screen and confirm assays, based on
APTT, with low and high phospholipid content
‣ 2 mixing assays (dRVVT & SCT)
‣ IL reagents/ ACL top
* Pradella et al. Clin Chem Lab Med 2013; 51(2): 379–385
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Laboratory testing for LA - Cut-off values for screening:
* Pradella et al. Clin Chem Lab Med 2013; 51(2): 379–385
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Laboratory testing for LA - Cut-off values :
Pradella et al. Clin Chem Lab Med 2013; 51(2): 379–85
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Laboratory testing for LA - Cut-off values for LA pos/neg:
‣ Ratio of screen/confirm:
‣ Most labs probably use 1.2 used as ‘generic’ normalised ratio
‣ Many reagent manufacturers mention a ‘generic’ ratio of 1.2
‣ ‘Generic’ ratio of 1.2 recently ‘validated’*
‣ Values as low as 1.06 are used by some labs
‣ Too low a value = increase false positive; too high a value =
increase false negative.
‣ Alternatives using mixing such as % correction/Rosner
index (ICA – index of circulating anticoagulant)
‣ ICA = [(b-c)/a] x 100 (where a, b, c = clotting times of patient,
mixture, normal, respectively)
‣ For Rosner index/ICA - local determination of value (generally
between 10-20%)**
* McGlasson & Fritsma. STH 2013; 39:315-319
** Kershaw & Orellana. STH 2013;39:283-290
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Cross laboratory testing of LA (RCPA QAP)
Favaloro et al. STH 2012;38:404–11
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Laboratory testing for LA - Cut-off values :
‣ Ratio of dRVVT screen/confirm:
‣ ‘Generic’ ratio of 1.2 recently ‘validated’*
‣ Normal subjects (n = 42) vs LA-positive plasmas (n
= 43) vs 6 DRVVT LA screen and confirm systems on
STAR instrument.
‣ Diagnostica Stago, Precision BioLogic, Siemens
Healthcare, Tcoag, Instrumentation Laboratories
(Werfen), Sekisui Diagnostics (previously American
Diagnostics).
‣ Uncorrected screen/confirm ratios and screen/
confirm ratios that were normalized using MRI and
mean PNP results
* McGlasson & Fritsma. STH 2013; 39:315-319
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Laboratory testing for LA - Cut-off values :
‣ Ratio of dRVVT screen/confirm:
‣ Similar mean screen/confirm ratios for normal specimens for
all systems (all close to 1.0).
* McGlasson & Fritsma. STH 2013; 39:315-319
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Laboratory testing for LA - Cut-off values :
‣ Ratio of dRVVT screen/confirm:
‣ ‘Grand mean action limit, MRI + 3 SD, derived from the local normal
plasmas (1.2) confirmed manufacturers’ recommended ‘cut-off’.
McGlasson & Fritsma. STH
2013; 39:315-319
x6 1.16 1.14 1.20
x5 1.16 1.16 1.21 (Exclude SK)
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Laboratory testing for LA - Cut-off values :
‣ Ratio of dRVVT screen/confirm:
‣ Similar mean dRVVT screen/confirm ratios for LA-positive specimens
(1.91 to 2.04) for 5/6 systems (Sekisui outlier @ 1.2 – ie relatively insensitive
to LA).
McGlasson & Fritsma. STH 2013; 39:315-319
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Laboratory testing for LA –
To mix or not to mix – that is the question!:
‣ Latest ISTH SSC guidelines vague on requirement for mixing studies
(‘recommended’ but not ‘mandated’)*
‣ Later clarification from SSC guideline authors ‘strong recommendation’ for
mixing studies**
‣ New CLSI guidelines appear to reduce ‘relative importance’ of mixing
studies.
‣ Integrated test systems (in theory) ‘not requiring mixing’ becoming very
popular.
‣ Mixing reported to ‘dilute’ ‘weak’ LA and potentially lead to false negative
LA.
‣ Mixing adds complexity and cost to test process (more testing &
requirement for normal plasma pool).
‣ Not mixing can lead to false negative LA in some cases of strong LA.
* Pengo et al. JTH 2009;7:1737-40
** Tripodi & Pengo. JTH 2011;9:2126-7
** Tripodi. STH 2012;38:385–9.
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Laboratory testing for LA –
To mix or not to mix – that is the question!:
‣ Not mixing can lead to false negative LA in some cases of strong LA.
‣ Case 1: ‘diagnostic challenge’
‣ Very strong LA, dispatched to 93 RCPA QAP participants
‣ Participants were blind to presence of LA and asked to perform ‘usual testing as
if this sample was received for a specific patient investigation’.
‣ Clinical information provided:
‣ Sample referred from a satellite facility
‣ 76 year old female patient, B-cell lymphoma diagnosed 4years ago
‣ Put on 3-month follow-up with no specific therapy.
‣ During recent follow-up visit identified to have increasing abdominal distension, night
sweats, weight loss and a significant drop in haemoglobin.
‣ Further clinical investigation might require exploratory surgery.
‣ Routine coagulation tests performed at satellite facility showed prolongation of both
prothrombin time (PT) and activated partial thromboplastin time (APTT). Mixing tests did
not show any substantial correction.
‣ Satellite facility is otherwise unable to provide any additional information
Favaloro et al. JTH 2010; 8: 2828–31.
Bonar et al. Pathology 2012;44:240–7
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Laboratory testing for LA –
To mix or not to mix – that is the question!:
Favaloro et al. JTH 2010; 8: 2828–31.
Bonar et al. Pathology 2012;44:240–7
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Laboratory testing for LA –
To mix or not to mix – that is the question!:
Favaloro et al. JTH 2010; 8: 2828–31.
Bonar et al. Pathology 2012;44:240–7
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Laboratory testing for LA –
To mix or not to mix – that is the question!:
Favaloro et al. STH 2012;38:404–11
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Laboratory testing for LA –
Mix then what? That is another question!
APTT as example
‣ 1:1 mixing tests
‣ 55 samples with factor deficiencies
(solid triangles) vs 44 samples with
inhibitors, lupus anticoagulants,
unfractionated heparin, or dabigatran
(open circles).
‣ STA-R & TriniCLOT S
‣ Subtracting the NPP CT from the
1:1mix CT
‣ Corrections within 4 seconds of the NPP
consistent with factor deficiency
‣ More than 8 seconds difference
consistent with an ‘inhibitor’.
‣ Between 4-8 s, inhibitors and factor
deficiencies could not be distinguished.
Kershaw & Orellana. STH 2013;39:283–90.
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Laboratory testing for LA –
Mix then what? That is another question!
APTT as example
‣ Same test, same samples, same
method, different approach.
‣ Ratio of the 1:1 mix APTT to normal
pool APTT
‣ Ratio below 1.1 excludes inhibitors
‣ Ratio above 1.2 suggests the presence
of an ‘inhibitor’.
‣ Between 1.1-1.2, inhibitors and factor
deficiencies could not be distinguished.
Kershaw & Orellana. STH 2013;39:283–90.
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Laboratory testing for LA –
Mix then what? That is another question!
APTT as example
‣ Same test, same samples, same
method, different approach.
‣ Index of circulating anticoagulant
(ICA, or Rosner index).
‣ Level of < 5% consistent with factor
deficiency.
‣ Level of > 11% consistent with the
presence of an inhibitor.
‣ Between 5-11%, inhibitors and factor
deficiencies could not be distinguished.
‣ Cut-off of 15% is shown for comparison.
Kershaw & Orellana. STH 2013;39:283–90.
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Laboratory testing for LA –
Mix then what? That is another question!
APTT as example
‣ Same test, same samples, same
method, different approach.
‣ Percent correction formula of
Chang
‣ All factor deficiencies showed a level of
>72% correction.
‣ Level of 72% correction.
Kershaw & Orellana. STH 2013;39:283–90.
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Laboratory diagnosis of LA – Summary 1
‣ Follow the guidelines*
‣ But which ones? (ISTH SSC, UK BCSH, CLSI).
‣ Screening by APTT, SCT, dRVVT - nor/abn cut-off :
‣ Ratio will be somewhere between 1.10 and 1.25 (I like 1.15 or 1.20)
‣ ICA will be somewhere between 5-15% (11-12%?)
‣ Difference cut-off will be somewhere between 4-10 sec
‣ No single cut-off value will be 100% sensitive & 100% specific
‣ Ratio of dRVVT screen/confirm – LA neg /LA pos cut-off:
‣ 1.2 is a good starting point for a normalised ratio (Stago, Precision
BioLogic, Siemens, Tcoag, IL)
‣ Value in ‘inter-laboratory’ harmonisation
‣ Sekisui Diagnostics assay not recommended
* Pengo et al. JTH 2009;7:1737-40
Keeling et al. BJH 2012;157:47-58
http://www.clsi.org/standards-development/public-documents/
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Laboratory diagnosis of LA – Summary 2
‣ Follow manufacturer instructions
‣ Validate manufacturer claims:
‣ To keep them ‘honest’ (they sometimes make mistakes)
‣ Be warned that validation of cut-off targets with 1.2)
‣ ‘Weak’ LA can be missed if mixing performed
‣ ‘Strong’ LA can be missed if mixing not performed
‣ Recommendation for labs not mixing
‣ Perform mixing study if non-mixed screen and confirm assays
(eg dRVVT) equally prolonged (ie normal ratio) – you may have a
strong LA.
* Pengo et al. JTH 2009;7:1737-40
Keeling et al. BJH 2012;157:47-58
http://www.clsi.org/standards-development/public-documents/
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Clinical utility of aPL testing:
‣ LA correlates with thrombosis risk more than solid phase assays
‣ Multiple positivity correlates with adverse events risk more than
single positivity
Annual incidence of
thrombosis according to
aPL profile
Galli et al. Blood; 2003; 101:1827-32 & 102:2717-23
Galli. STH;2012;38:348-52
Pengo et al. STH;2012;38:322-7
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An algorithm for identification of APS
Favaloro. IJLH 2013. 35; 269-74.
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Acknowledgments
‣ Meeting organisers
‣ RCPAQAP Haematology,
‣ Diagnostic Haemostasis laboratory staff
‣ Soma Mohammed
‣ Jane McDonald
‣ Ella Grezchnik
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