1. health and fitness
2. disease

Variability in laboratory
interpretation of data: The
example of phospholipid
antibody tests.
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 the 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)
 aCL (anticardiolipin [antibodies])
 a 2GPI (anti-beta 2 glycoprotein I [antibodies])
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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.
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 & ab2GPI 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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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, >99th percentile?
 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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Cross laboratory testing of LA (RCPA QAP)
Favaloro et al. STH 2012;38:404–11
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Cross laboratory testing of LA (RCPA QAP)
Favaloro et al. STH 2012;38:404–11
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INR CAP data:
Olson et al. Arch Pathol Lab Med. 2007;131:1641–7
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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 & ab2GPI 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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Solid phase aPL assays.
 aCL and ab2GPI are the most commonly employed and are identified in the
classification guidelines*
 IgG subclass most commonly investigated (and most significantly associated
with clinical features of APS)
 IgM less commonly investigated; IgA rarely investigated (less associated to APS).
 Typically performed by immunology labs as part of auto-immune profile.
 Most commonly performed by ELISA (new platforms emerging)
 Serum (not double spun plasma)
 Not affected by anticoagulant therapy (heparin, warfarin, new DOACs)
 Reported in either GPL / MPL or arbitrary units (Manufacturer/product
dependent)
 GPL / MPL = 1 g/mL IgG / IgM aCL
* Miyakis et al. JTH 2006;4:295-306
 Polyclonal / monoclonal calibrators
** Pierangeli et al. Lupus. 2011;20(2):182-90
Lakos et al. Arthritis Rheum. 2012;64:1-10.
 Suggested reading**
Pierangeli et al. Clin Chim Acta. 2012;413:358-60
H61 CLSI aPL guidelines in progress
(http://www.clsi.org/standards-development/publicdocuments/)
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Cross laboratory testing of LA (RCPA QAP)
Favaloro et al. STH 2012;38:404–11
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Cross laboratory testing of aPL (RCPA QAP)
Favaloro et al. STH 2012;38:404–11
Favaloro et al. STH 2012;38:390–403
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Cross laboratory testing of aPL
(European Forum on Antiphospholipid Antibodies)
Reber et al. STH
2008;34:340–346
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Cross laboratory testing of LA (RCPA QAP)
Favaloro et al. STH 2012;38:404–11
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Cross laboratory testing of aCL (RCPA QAP)
Favaloro et al. STH 2012;38:390–403
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Explaining the variability LA vs solid phase aPL assays.
 Laboratory LA diagnostic procedures are heterogeous
 different instruments, APTT reagents (variously LA ‘sensitive’), dRVVT reagents, use or not of other
test procedures, use or not of mixing studies, variable mixing ratios, source/type of mix plasma,
derivation of normal ranges/cut-off values [number of individuals, +/- 2SD, +/- 3 SD, >99th percentile],
method to assess correction [ratio, normalized ratio, source of denominator in ratio, % correction,
Rosner index/ICA, etc, etc.
 Within dRVVT
method – reasonable standardization/harmonization across different
manufacturers
 Similar normalized ratio cut-off value for 5/6 reagent manufacturers (~‘1.2’) *
 Possible that dRVVT formulation similar in different manufacturer reagents
 Normalized ratio akin to INR system
 harmonizes data to a normal value
 minimizes cross lab / cross instrument / cross reagent variability
 Solid phase aPL assays **
 Absolutely no standardization / harmonization across instrument platforms / reagent kits
 Differ in terms of calibrators (source & type)
 Differ in terms of assignment of values (GPL / MPL vs arbitrary units)
* McGlasson & Fritsma. STH 2013; 39:315-319
** Pierangeli et al. Lupus. 2011;20(2):182-90
Lakos et al. Arthritis Rheum. 2012;64:1-10.
Pierangeli et al. Clin Chim Acta. 2012;413:358-60
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Explaining the variability LA vs solid phase aPL assays.
 Normalized
LA ratio akin to INR system
 harmonizes data to a normal value
 minimizes cross lab / cross instrument / cross reagent variability
 Initial LA results (dRVVT, APTT, SCT) are in seconds
 These will typically yield higher inter-laboratory CVs than ratios
 Reagents / instruments
 Cross lab study by Pradella et al*
 200 normal subjects (40 for each of 5 centres)
 6 LA functional assays (same procedure, reagent lot and analyser type at each site).
 IL reagents/ ACL top
 Statistically significant differences in results obtained between study centres
when times reported in seconds.
 Results not statistically different when reported as ratios.
*Pradella et al. Clin Chem Lab Med 2013; 51(2): 379–85
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Significance?
LA correlates with thrombosis risk more than solid phase assays*
 Hypothesis 1:**
 LA (clot based assays) relatively insensitive to aPL (thus only high titre /
‘clinically more significant’ aPL detected).
 Solid phase assays over-sensitive to aPL (thus detects both ‘clinically
significant’ and ‘‘clinically insignificant’ aPL).
 Hypothesis 2:***
 LA detection by dRVVT relatively harmonized/standardized (thus
consistency observed in different research studies).
 Solid phase assays totally disharmonized / non-standardized (thus no
consistency observed in different research studies).
* Galli et al. Blood; 2003; 101:1827-32 & 102:2717-23
** Roubey RA. Lupus 2010;19:440–5.
*** Favaloro EJ. Int J Lab Hem. 2013, 35, 269–74
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Barriers to standardization / harmonization
 aPL reagent / instrument manufacturers
 Expert groups write guidelines and provide evidence for clinical utility
benefits from standardization / harmonization*
 No commercial (financial) benefit
 Indeed, probable financial disincentives.
 IVD regulators
 No regulatory requirement
 Why would a manufacturer change their product to match other products (ie
standardization) without financial benefit or regulatory requirement?
 Regulatory approved products – very expensive to change product and obtain
new approval.
* Pierangeli et al. Lupus. 2011;20(2):182-90
Lakos et al. Arthritis Rheum. 2012;64:1-10.
Pierangeli et al. Clin Chim Acta. 2012;413:358-60
H61 CLSI aPL guidelines in progress
(http://www.clsi.org/standards-development/publicdocuments/)
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Barriers to standardization / harmonization
FDA ‘cardiolipin’ related approvals / clearances by year
Favaloro EJ et al. STH. 2013;39:235-49.
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Barriers to standardization / harmonization
FDA ‘cardiolipin’ related approvals / clearances in 2010
HemosIL (IL AcuStar aCL IgG and IgM immunoassays, and controls).
 Phadia (EliA aCL IgG and IgM plus aB2GPI IgG and IgM immunoassays,
and controls).
 Both cleared under a 510(k) Substantial Equivalence Determination ruling.

Favaloro EJ et al. STH. 2013;39:235-49.
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Barriers to standardization / harmonization
FDA Decision Summary
“Traceability, Stability, Expected values (controls, calibrators, or methods)”
HemosIL (IL AcuStar aCL )
Phadia (EliA aCL )
An international reference material for anti-cardiolipin
antibodies is not available. The assay is calibrated in
relative arbitrary units (U/mL). The two calibrator
values are assigned using in-house standards and a
four-parameter Master Curve. The assignment values
of the two calibrators are used to create a lot-specific
four-parameter logistic curve, using two stored
parameters from the Master Curve and two lot-specific
parameters based on the calibrator values. Control
values assignments are performed using two QC
approved batches of reagents (with corresponding
calibrators) and two different ACL AcuStar instruments.
The controls are analyzed in replicates of 10 on both
instruments, then the batches of reagent are switched
between instruments and the controls are re-analyzed.
The IgG and IgM calibrators are traceable (via an
unbroken chain of calibrations) to the International
Reference Preparation (IRP) 67/86 of Human Serum
Immunoglobulins A, G and M from WHO. New batches
of IgG and IgM Calibrators are compared to a secondary
standard (standardized with the IRP) or the IRP directly
and adjusted accordingly to meet the correct
concentration. The instrument measures specific
IgG/IgM concentrations in μg/L. By using a conversion
factor given by the lot-specific code of the EliA
Cardiolipin IgG and IgM Well, the results are
automatically converted to GPL-U/mL or MPL-U/mL,
respectively.
Favaloro EJ et al. STH. 2013;39:235-49.
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Barriers to standardization / harmonization
“Traceability, Stability, Expected values (controls, calibrators, or methods)”
HemosIL vs Phadia
1. HemosIL aCL summary says “An international reference material for anticardiolipin antibodies is not available. The assay is calibrated in relative arbitrary
units (U/mL).”
2. Phadia aCL summary says “The IgG and IgM calibrators are traceable (via an
unbroken chain of calibrations) to the International Reference Preparation (IRP)
67/86 of Human Serum Immunoglobulins A, G and M from WHO.”
3. Statements appear contradictory, but are not.
4. Solid phase aPL assays (aCL) initially reported in 1983.
5. Still in 2010 - Distinct methods used for calibration & reporting
6. We may be waiting for many more years before standardization takes hold for
solid phase aPL assays.
7. Variability in LA assays? Comparatively ‘no problem’.
Favaloro EJ et al. STH. 2013;39:235-49.
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Testing for aCL in 2013
Acknowledgments
 Meeting organisers
 RCPAQAP
 Diagnostic Haemostasis laboratory staff
 Soma Mohammed
 Jane McDonald
 Ella Grezchnik
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