Post on 11-Feb-2018
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Anti-cardiolipin antibodies (ACA), rst identied in syphilis patients as a result o the
presence o cardiolipin in the bovine heart extract used or the VDRL syphilis test, haveevolved into one o the primary components o antiphospholipid syndrome (APS)
diagnosis. The realization that the actual target o many antiphospholipid antibodies (aPL)
was the phospholipid binding protein 2GPI bound to cardiolipin antigen immobilized on
the ELISA well led to ELISA assays using puried 2GPI as the assay substrate. While some
labs continue to test or only IgG and IgM 2GPI isotypes, evidence suggests that
2GPI
IgA antibodies are associated with increased risk o adverse cardiovascular, thrombotic,
and pregnancy-associated events. In this issue o the INOVA newsletter, Aguilar-Valenzuela
et al. show some SLE patients are only positive or 2GPI IgA antibodies and recommend
2GPI IgA antibody testing in individuals suspected o APS in whom other aPL antibodies
are negative.
A long-standing problem with aPL testing has been inter-assay and inter-lab variability.
von Landenberg and Lorenz each discuss the use o the Sapporo monoclonal standards
to improve standardization and calibration o ACA kits. Javela and Mustonen describe
evaluation o ve commercial ACA IgG ELISA kits and document discouraging variation in
the interpretation o low and moderately positive specimens between kits.
The signicance o single and multiple ACA, 2GPI, and LAC positivities, as well as the
magnitude o the positivity, is discussed by Meroni and Pregnolato. Patients make
antibodies to a variety o phospholipid/protein targets, resulting in a heterogeneous
group o patient antibodies. Detection o all patients requires more than one assay and
the authors suggest that new assays such as PS/PT will provide improved diagnostic and
prognostic power.
INOVAs new aPS/PT IgG and IgM assays, which recognize antibodies to a physiological
complex o phosphatidylserine /prothrombin, are described by Binder et al. Measurement
o both PS/PT IgG and IgM antibodies detected most LAC-positive
patients and close to 70% o the APS patients and identied some
APS patients missed by the conventional prole o ACA, 2GPI, and
LAC assays.
Antiphospholipid testing is evolving. New assays will allow ner
stratication o patients with APS, thrombotic, coagulation, and
pregnancy-related conditions into phenotypic groups with distinct
prognosis and management characteristics.
THE EVOLVING STATE OF ANTI-PHOSPHOLIPID ANTIBODY TESTING
IN THIS ISSUE
INOVA NEWS
No. 6p2 Monoclonal antibodies in anti-phospholipdiagnostic s: Is there room or improvement o
standardization?
p3 High discrepancies in anti-phospholipid lare seen between laboratories
p4 Anti-phospholipid antibody detection:Where we are standing and where we are going.
p6 Isolated elevated levels o IgA-Anti-2GPI
are associated with clinical maniestations o the
antiphospholipid syndrome
p8 Clinical signiicance o IgG and IgMautoantibodies that target the complex o
phosphatidylserine and prothrombin (PS/PT)
p12 Anti-cardiolipin IgG ELISAs What is tright result? Comparison o ive dierent comme
test kits
p14 INOVA Diagnostics APS ELISA test kits
Gary L. Norman PhD
Senior Scientist
INOVA Diagnostics, Inc.
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The anti-phospholipid syndrome (APS) is an
autoimmune disease which is characterized by
dierent clinical, haematological and serological
maniestations. These include venous and/or arteri-
al thrombosis, recurrent etal loss and low platelet
counts. Accordingly, the binding specicities o
anti-phospholipids (aPL) appear to be as hetero-
geneous as the clinical maniestations associated
with them.
Since the typical antigens are cardiolipin and
phosphatidylserine, it was thought that aPL can
be distinguished by their phospholipid specicity
alone.
Additionally, it could be shown that most o the aPLs
require a protein coactor to bind to their antigen.
One o these coactors is the apolipoprotein beta2
glycoprotein 1 (2GPI) with a postulated unction in
the lupus anticoagulant activity.
The clinical diagnosis o APS depends in most cases
on positive anti-cardiolipin antibodies (aCL) and/or
positive lupus anticoagulant (LA) test results.
Ongoing reports are showing that there is
considerable variation in aCL results obtained
between dierent laboratories and assays even i
the laboratories are using the same assay.1
Discrepancies in results are even higher i labora-
tories use dierent brands o assays, as a result o
several variable actors (see table 1).
To overcome some o these problems, the use o
human and chimeric monoclonal antibodies or
standardization and calibration o the dierent
kits was introduced with the HCAL IgG Sapporo
Standard.2
However, using dierent monoclonal IgG and IgM
antibodies directed to 2GPI and/or cardiolipin
still does not lead to a reasonable agreement in
dierent test systems.
This might be due to the act that
monoclonal antibodies represent only one speci-
city to a certain epitope with a dened (high)
avidity, or does not contain the best match to theantibodies ound in the sera o antiphospholipid
patients.
Using monoclonal antibodies in aPL testing
especially in the case o cardiolipin and 2GP
diagnostics is not the be-all and end-all.
Remaining inconsistencies limit the clinical utility
and inter-laboratory transerability which in conclu-
sion indicates that the standardization regardingaCL testing still needs to be improved.
We are looking orward to the new upcoming
developments rom the companies in this highly
competitive eld o diagnostics.
Philipp von Landenberg
Institut uer Labormedizin, Solothurner Spitaeler AG, Baslerstrasse 15, 4600 Olten, Switzerland
Monoclonal antibodies in anti-phospholipid diagnostics:
Is there room or improvement o standardization?
Wong RCW et al., Thrombosis Research 2004;114:559-571.1.Koike T et al., Arthritis & Rheumatism 1999; 42:309-1311.2.
T a b l e 1
F A C T O R S R E S U L T I N G I N V A R I A B I L I T Y
B E T W E E N a C L A S S A Y B R A N D S
Manuacturing and calibration o the assay
Source and purity o antigens
Specicity and isotype o detection antibodies
Heterogeneous avidity spectrum o antibodies
A variety o actors result in discrepancies between laboratories who usediferent brands o aCL assays.
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Some years ago, a cross-laboratory evalu-
ation was perormed by the European
Antiphospholipid Forum. Basically, a set o
ten serum samples were tested at 24 dierent
European centers, using either home made
internally standardized or commercially avail-
able assays.
Results were reported to the organizers and
compared to each other.1
A wide variability in results was ound or
aCL-IgG ELISA test kits as well as or the
aCL-IgM ELISA (Table 1). Some values in the
cut o range appeared to be positive in one
test kit but normal with another.
With the introduction o one IgG and one IgM
monoclonal antibody (HCAL and EY2C9) as
putative standards, a progressive decrease in
the variability o the values was obtained.
Even with monoclonal standardization the
dilemma o inconsistent comparability o
results still remains, since not all routine
laboratories are ollowing consensus
recommendations.
Although increasingly more laboratories and
manuacturers utilize standardized, uniorm
materials and procedures the relatively high
variability or a-PL antibodies assays are an
ongoing issue o discussion.
Mareike Lorenz
Institute o Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University,
Lagenbeckstrasse 1, 55131 Mainz, Germany
High discrepancies in anti-phospholipid levels are seen between laboratorie
Tincani A et al.,1. Thromb Haemost2001; 86:57583.
325
300
275
250
225
200
175
150
125
100
75
50
25
0
B1 M4 B3 B5 M1 M2 B2 B4 M3 M5A
GPL
IgG aCL ELISA (results in GPL units)
325
300
275
250
225
200
175
150
125
100
75
50
25
0
B1 M4 B3 B5 M1 M2 B2 B4 M3 M5B
MPL
IgM aCL ELISA (results in MPL units)
T a b l e 1
TEST VARIABILITY IN aCL-IgG AND IgM ELISA KITS
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The diagnostic and prognostic
antiphospholipid prole
According to the revised classication crite-
ria, the positivity or lupus anticoagulant (LAC),
anti-cardiolipin (aCL) and anti-beta2 glycopro-
tein 1 (2GPI) antibodies are ormal classication
laboratory criteria. Hence, a single positivity stable
over time (at least 12 weeks) is sufcient to classi-
y a symptomatic patient as suering rom the
antiphospholipid syndrome (APS).1
Patients displaying multiple positivities and/or
high antibody titres have more severe disease and
higher recurrence rate despite treatment. However,
the specicity and the predictive value o each
single test and in particular o their combination
are not exactly the same. This is particularly true
taking into account that the most requent clini-
cal maniestations o the syndrome (i.e. deep vein
thrombosis or early miscarriages) are not speci-ic and rather common in the general population.
A sub-classication o patients according to their
positivities has been suggested in table 1.
The predictive value has been suggested to be the
highest or the category I.1
The identication o the risk prole oers the
rationale or both the secondary prophylactic
therapy (i.e. in order to prevent recurrences) and
the primary prophylaxis to avoid the occurrence o
the clinical events in the antiphospholipid antibody
(aPL) positive asymptomatic subjects.
While the clinical approach is not problematic or
patients displaying several positivities and/or high
aPL titres, the situation is dierent when we are
dealing with patients with one positivity only.2,3
It is largely accepted that LAC better correlates with
thrombosis and pregnancy morbidity than aCL or
anti-2GPI.4 Such a high specicity was suggested
to be related to the act that LAC is mediated by
antibodies against plasma proteins (anti-2GPI and
prothrombin) bound to anionic PL and ultimately
aecting their availability or the coagulation
cascade. To display this eect the antibodies need
to be at an elevated protein concentration and todisplay high avidity.
However, the clinical value o the presence o LAC
as an isolated assay or in asymptomatic subjects o
at low potency has been recently questioned.5
To improve specicity, the revised classica
tion criteria or the aCL assay require both a
stable positivity and a positivity threshold o 40
International Units1. As a consequence, a single
aPL positivity is quite rare with titres > 40 IU usual-
ly associated with positive LAC and/or anti-2GP
assays. A comparable high threshold has not been
suggested or the anti-2GPI assay since the norma
cut o should be calculated on the 99th percentile
o 50 normal subjects1. Hence, the sensitivity o the
anti-2GPI assay is high. Such a sensitivity combined
with its wider use in the laboratories makes the
possibility o isolated elevated anti-2GPI antibod-
ies more requent.
Pier Luigi Meroni, *Francesca Pregnolato
Division o Rheumatology Ist. Gaetano Pini, Dept. Internal Medicine University o Milan
*IRCCS Istituto Auxologico Italiano Milan (Italy)
Antiphospholipid antibody detection:
Where we are standing and where we are going
T a b l e 1
L A B O R A T O R Y C R I T E R I A S A T I S F I E D
I More than one criterion present (any combination)
IIa Lupus Anticoagulant present alone
IIb Anti-cardiolipin antibody present alone
IIc Anti-Beta-2 glycoprotein I antibody present alone
Classication o APS patients according to the positivity or the
antiphopsholipid assays
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Do we have (or are we going to have) new
useul aPL assays?
There are preliminary results suggesting additional
assays could improve our diagnostic and prognostic
power as a second level o aPL testing.
This could be the case or anti-prothrombin
antibodies (anti-PT) as a second level assay to
conrm an isolated LAC positivity or to overcome
the technical problems related to LAC testing in
patients under anticoagulation. Moreover, since
the antibodies are detected by a solid-phase
assay displaying a higher sensitivity than the LAC
unctional assay, it has also been suggested that
an anti-PT test may or may not conrm equivo-
cal unctional LAC. Although the heterogeneity o
the methods to detect anti-PT antibodies is still amatter o debate, recent studies have once again
raised the possibility that anti-PT and in particu-
lar anti-PS/PT antibodies may display a diagnostic/
prognostic value on vascular maniestations.6-9
We recently analyzed a selected series o samples
rom APS patients and controls by using a new
ELISA assay or anti-PS/PT detection (kindly provid-
ed by Dr. W. Binder INOVA Diagnostics, USA).
Figure 1 reports our preliminary results showing agood specicity o the assay and correlation with
the clinical maniestations.
Miyakis S. et al., J Thromb Haemost 2006; 4:295-306.1.Lee RM. et al., Obstetrics Gynecol 2003; 102:294300.2.Pengo V. et al., J Thromb Haemost 2009.3.Galli M. et al., . Blood 2003; 101: 18271832.4.Pengo V. et al., J Thromb Haemost 2009; 7: 1737-1740.5.
Galli M. et al., Blood 2003; 102: 2717-2723.6.Tincani A. et al., Clin Exp Rheumatol 2007; 25: 268-274.7.Galli M. et al.,. Blood 2007; 110:1178-1183.8.Sakai Y.et al., Arthritis Rheum 2009; 60: 2457-2467.9.
F i g u r e 1
Detection o anti-PS/PT antibodies by ELISA in a selected series
o Lupus Anti-coagulant (LAC) positive samples rom APS patients
aPS/PT IgG or
IgM Positive
76% (52/59)
LAC Positive Sera (n=59)
aPS/PT IgG or IgM Negative24% (7/59)
6/7 aPL positive
5/7 anti2GPI positive
4/7 aCL/anti2GPI positive
Tests detecting aCL IgA and anti-2GPI IgA
antibodies are available but are not ormally
included into the revised criteria because o the
lack o evidence that the assay may improve the
whole diagnostic power.
In act, IgA aPL appear to rather identiy subgroups
o patients, such as Aro-Americans or pure obstet-ric APS. However, the search or IgA aPL may be
useul in order to conrm the diagnosis o APS in
the case o an isolated positivity or a borderline
result in the other solid-phase assays.
Conclusions
The panel o aPL tests is still evolving and appar
ently, like other autoantibody amilies, more than
one assay and the use o second level tests appear
useul to improve our diagnostic and prognosticpower.
59 LAC positive sera have been tested. 52/59 (76%) resulted positive
or IgG or IgM anti-PS/PT antibodies
Only 7 samples were LAC positive and anti-PS/PT antibody negative
but displayed a reactivity against CL or 2GPI coated plates.
3 samples with equivocal LAC were negative or anti-PS/PT antibodie
Most o the positivities or anti-PS/PT antibodies were at high titres
and 44.1% o them were o the IgM isotype
Only 2 out o 40 pathological aPL negative control sera (30 with
autoimmune diseases, 10 with inectious diseases) displayed a low
positivity (1 IgG and 1 IgM)
The cut of was calculated on 91 NHS samples (43 AU or IgG and 44
AU or IgM)
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Background and Purpose
Current diagnostic criteria recommend elevat-
ed titers o anti-Cardiolipin (aCL) and/or anti-
2Glycoprotein (
2GPI) antibody IgG or IgM by
ELISA and/or lupus anticoagulant (LAC) to conrmantiphospholipid syndrome (APS).
IgA aCL antibodies are ound more requently in
Aro-Caribbean populations usually in association
with other IgG and/or IgM aCL antibodies and have
been shown to be pathogenic in animal models,
their clinical signicance remained elusive.
Several studies report a possible association
between elevated IgA anti-2GPI titers and APS-likemaniestations.
Anti-2GPI IgA antibodies were strongly associ-
ated (Odds Ratio 1.77) with thrombosis episodes
in a retrospective study that involved 472 APS
patients.
IgA anti-2GPI has been associated with stroke in
normal patients. Interestingly the subjects had
recurrent miscarriages but they were not classied
as APS due the absence o aCL positive test.
Anti-2GPI IgA antibodies are more prevalent in
patients with SLE.
We recently reported ve isolated cases o exclusive
IgA anti-2GPI antibody sero-positivity with
concomitant APS clinical maniestations.
Objectives
Patients
Anti-phospholipid seropositivity was examined in
2799 SLE sera, whereo 599 samples came rom a
multi-ethnic, multi-center cohort (LUMINA) and
2200 samples were reerred to our laboratory
(APLS) or APS work-up.
Laboratory methods
aCL (IgG, IgM, IgA) Screen, aPL (IgG and IgM), anti-
2GPI (IgG and IgM) antibodies were determined
by using two commercially available test kits, one
rom INOVA Diagnostics, and an in-house protocol
IgA-2GPI titers were determined by two commer
cial ELISA tests, one rom INOVA Diagnostics..Results
Out o the 2799 samples, 50 samples were positive
exclusively or IgA-2GPI in at least one kit.
1Agui la r-Val en zu el a R, 1Seif AM, 2Al ar c n GS , 1Martnez-Martnez LA, 1Dang N, 1Papalardo E2Liu J, 4Vila LM, 1Najam S, 1McNearney T, 1Gonzalez EB, 6Binder W, 5Teodorescu M, 3Reveille JD1Pierangeli SS
1 University o Texas Medical Branch, Galveston, TX; 2University o Alabama at Birmingham, Birmingham, AL3University o Texas-Houston Heath Sciences Center, Houston, TX; 4University o Puerto Rico Medical Sciences
Campus, San Juan, PR; 5Theratest Laboratories, Lombard, IL; 6INOVA Diagnostics, San Diego, CA.
Isolated elevated levels o IgA-anti-2GPI are associated with
clinical maniestations o the antiphospholipid syndrome
O B J E C T I V E S
To examine the prevalence o exclusive IgA-anti-2GPI
antibody positivity in a large cohort o patients with SLE
and in patients suspected o having APS
To correlate IgA-anti-2GPI antibody positivity with APS
associated clinical maniestations
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A signicant number o subjects in the two groupshad at least one APS-related clinical maniestation,
that included:
Two o these samples were also LAC positive.
84% o samples were positive with the INOVA Kit,90% o samples were positive with the competitive
kit (correlation between the two kits was 0.93%).
Conclusions
This study supports that elevated IgA anti-2GPI
antibody titers may identiy additional patients
who have clinical eatures o APS but who do not
meet current diagnostic criteria.
It may be thereore recommended to test orIgA
2GPI antibodies when other aPL tests are
negative and APS is suspected.
...elevated IgA
anti-2GPI antibody
titers may identify
additional patients who
have clinical features
of APS but who do not
meet current diagnostic
criteria.
It may be therefore
recommended totest forIgA
2GPI antibodies
when other aPL tests
are negative and APS is
suspected.
Amengual O. et al.,1. Arthritis Rheum 2003;48:886-895.DAgnillo P. et al.,2. The Journal of Immunology2003;170:3408-3422.Atsumi T. et al.,3. Arthritis Rheum 2000; 43:1982-1993.Atsumi T. et al.,4. Thrombosis Research 2004;114:553-538.
A P S - R E L A T E D C L I N I C A L M A N I F E S T A T I O N S
Deep vein thromboses
Pregnancy losses
Other APS-related pregnancy complications
Pulmonary inarctions, strokes, seizures, myocardial
inarctions
Thrombocytopenia
Non classical APS maniestations:
Skin ulcers, pulmonary hypertension, livedo reticularis,
cardiac valvular disease, seizures and migraines.
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W. L. Binder, S. Lewis and Z. Shums
INOVA Diagnostics, San Diego, CA, USA
Clinical signicance o IgG and IgM autoantibodies that target
the complex o phosphatidylserine and prothrombin (PS/PT)
BackgroundAntiphospholipid antibodies represent a large
heterogeneous group o immunoglobulins o
considerable clinical importance due to their
association with arterial and/or venous thrombosis,
recurrent pregnancy loss, neurological disorders,
pulmonary hypertension and thrombocytopenia.
Clinical laboratories routinely use the anticardiolipin
antibody ELISA and the lupus anticoagulant(LAC) clotting assay or aiding in diagnosis o
antiphospholipid syndrome (APS).
More and more laboratories are now including
tests or detecting antibodies directed against
phospholipid binding proteins, the best studied o
which is 2GPI.
Prothrombin (actor II) is another phospholipid
binding protein with procoagulant activity.
A number o groups have denitively shown that
antibodies targeting the complex o phosphatidyl-
serine (PS) and prothrombin (PT) have signicant
clinical relevance due to their strong correlations
with clinical eatures o APS and with the presence
o LAC.1
It was also shown that it is the antibody to the
PS/PT complex rather than antibodies that
target prothrombin alone that correlate with
LAC and APS.2,3
The PS/PT antibodies provide useul sensitivity o
APS and have high specicity. Their inclusion into
the laboratory criteria or classication o APS has
been proposed.4
GOALS AND CHARACTERISTICS OF
PS/PT IgG AND IgM ELISA ASSAY
Does not detect 2GPI reactive antibodies
Sapporo monoclonals do not react
Strong positive 2GPI do not react
Detects many ACA and 2GPI negative APS patients
Close approximation o LAC
High Speciicity
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Table 1
------------------ NUMBER POSITIVE (%) --------------------
PAT I E N T G R O U P No SAMPLESPS/PT IgG
POSITIVE
PS/PT IgM
POSITIVE
PS/PT IgG
AND/OR IgM
POSITIVE
Normals 247 3 (1.2%) 4 (1.6%) 7 (2.8%)
Lupus Anticoagulant Positive (LAC) 24 21 (87.5%) 19 (79.2%) 24 (100%)
Antiphospholipid Syndrome (APS) 71 33 (46.5%) 34 (47.9%) 48 (67.6%)
Rheumatoid Arthritis 6 0 0 0
Crohn's 2 0 0 0
Ulcerative Colitis 2 0 0 0
Celiac 5 0 0 0
LAC negative 8 0 1 (12.5%) 1 (12.5%)
Inectious disease (CMV, Toxo, Rubella, HSV HBV HCV) 14 0 0 0
Syphilis 12 0 0 0
Actin Antibody Positive 1 0 0 0
H. Pylori Positive 2 0 0 0
Combined results rom an external and an internal study
Specic perormance characteristics o QUANTA Lite aPS/PT IgG and QUANTA Lite aPS/PT IgM kits
that detect the complex o phosphatidylserine and prothrombin (PS/PT) autoantibodies
Assay Characteristics
Antigen on solid phase is a layer o phosphatidylserine and human prothrombin, coated in the presence o
Ca++. Standard ELISA ormat with 3 thirty minute incubations and a 5 point standard curve.
Method
We tested 71 patients with APS, 24 known LAC positives, 247 random normals and 52 disease controls or IgG
and IgM antibodies to PS/PT. These results were used to calculate perormance characteristics and the new
assays were compared to traditional anti-GPI and LAC assays. Results are tabulated in Table 1.
Forty eight o the 71 APS patients (67.6%) were PS/PT positive and many o these individuals were ound to be negative using more
traditional assays such as anti-GPI and LAC.
Only 7 o 247 normals and 1 o the 52 disease controls were ound to be positive or either IgG or IgM PS/PT antibodies or a
combined specicity o 97.3% (8/299).
The assays were ound to have high inter and intra run precision.
Equivalent results were obtained with either serum or citrated plasma or both assays.
Amengual O. et al.,1. Arthritis Rheum 2003;48:886-895.DAgnillo P. et al.,2. The Journal of Immunology2003;170:3408-3422.Atsumi T. et al.,3. Arthritis Rheum 2000; 43:1982-1993.Atsumi T. et al.,4. Thrombosis Research 2004;114:553-538.
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Most o the discrepant results are due to the higher
sensitivity o the IgG and IgM PS/PT kits or both
the LAC positives and especially the APS patients
although there were APS patients that were 2GPIpositive yet PS/PT negative.
The PS/PT IgG plus IgM kits detected all LAC
positive samples and most o the APS patients. It
was noticed that the vast majority o APS patients
were positive or PS/PT and/or 2GPI.
Conclusions
PS/PT IgG and IgM ELISA appears to be
very specic and detects a majority o LAC
positives
IgG and IgM autoantibodies that react with a
physiologic complex o phosphatidylserine
and prothrombin are sensitive markers or
anti-phospholipid syndrome
PS/PT IgG and IgM ELISA detects most APS
patients including many that are ACA, 2GPI,
LAC negativeThe tests exhibit high specicity and
reproducibility and can be run with serum
or plasma specimens
The detection of IgG
and IgM class antibodies
to phosphatidylserine/
prothrombin complex
(PS/PT) is an aid
in the diagnosis of
autoimmune thrombotic
disorders, such as
anti-phospholipid
syndrome (APS) and
those secondary
to systemic lupus
erythematosus or other
lupus-like diseases.
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Anti-cardiolipin IgG ELISAs What is the right result?
Comparison o ve diferent commercial test kits
Ja vela K. an d Mu st on en P.
Finnish Red Cross Blood Service, Helsinki, Finland
BackgroundAntiphospholipid syndrome (APS) is a disorder
characterized by recurrent thrombosis and/or
etal loss associated with characteristic laboratory
abnormalities.
Patients suspected to have APS should
be screened or anticardiolipin antibodies
(ACA), 2-glycoprotein 1 antibodies and lupus
anticoagulant.
More than 45 commercial kits or ACA detection
are available (n=45 in ECAT reerence list).
Objective
Evaluation o ve dierent commercially available
ACA IgG/IgM ELISA test kits to replace our in-house
method.
Methods and Materials
Five dierent commercial ACA IgG assays were
chosen or comparison:
QUANTA Lite ACA IgG III, INOVA
Anti-Cardiolipin IgG/IgM, Orgentec
Reaads Anti-Cardiolipin IgG/IgM, Corgenix
Varelisa Cardiolipin IgG Antibodies; Phadia
EliA Cardiolipin IgG, Phadia
The standards o all commercial ELISA assays are
calibrated against reerence sera rom E.N. Harris,Louisville.
Our in-house method was used as a reerence
method.
Ten positive, 4 strong positive and 14 negative
samples previously measured by our in-house
method were analyzed.
ResultsAll 14 negative samples were negative by all ACA
IgG assays.
The number o positive samples varied when the
cut-o o manuacturer (Table 1) and the laboratory
classication criteria or APS (ACA IgG results >40
GPL (Table 2) were used .
The Variation Coefcients o all assays were good
in the cut o range below 6.8% or all assays.
Conclusions
All tested commercial ELISAs had good
reproducibility and all strong positive samples
were positive by all assays
There was a signicant discrepancy between
assays when borderline, low positive or
intermediately positive ACA IgG samples were
analyzed
The correct recognition o high but also medium
titer ACA IgG is o high clinical signicance
The selection between reagents has to be
made or the method with the best correlation
to the existing one, since APL antibodies o APS
patients are repeatedly tested or monitoring
A potential problem will be i serum samples
are sent to a dierent laboratory which eitheruse a home made method as well, or a dierent
commercial test kit
The standardization o ACA IgG ELISAs remains
an unsolved problem
All trademarks are the properties of their respective companies
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ACA IgG results14 positive samples measured by ve commercial ELISA assays. The 14
negative samples were negative by all ACA IgG assays.
Table 1
ACA IgG (GPL)
IN-HOUSE
METHOD
QUANTA
LiteOrgentec Reaads Varelisa EliA
29* 15** 10** 23** 15** 15**30 15 3 11 6 3
30 47 72 51 25 11433 23 2 8 2 235 26 4 10 3 736 12 3 15 3 138 43 13 33 17 24341 48 4 18 5 442 42 3 4 1 143 26 4 9 3 643 13 3 45 7 159 185 163 166 125 8787 112 138 78 156 442
173 391 1256 815 405 85646 447 856 551 387 327
*Cut-o value o the in-house method; **Cut-o value set by the manuacturer
The number o positive samples according to laboratory classication
criteria or APS (>40 GPL)
Table 2
IN-HOUSE
METHOD
QUANTA
Lite Orgentec Reaads Varelisa EliA
Positive (n) 8 8 5 6 4 6
Negative (n) 6 6 9 8 10 8
All tested
commercial
ELISAs had goo
reproducibility
and all strong
positive sampl
were positive b
all assays.
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QUANTA Lite ACA
PRODUCT No. DESCRIPTION CALIBRATION INTERPRETATION
7 0 8 6 2 0Q U A N T A L i t e A C A S c r e e n I I I 1
A n t i g e n : P u r i i e d c a r d i o l i p i nc u t o
n e g < d e c i s i o n p o i n t
p o s > d e c i s i o n p o i n t
7 0 8 6 2 5Q U A N T A L i t e A C A I g G I I I 2
A n t i g e n : P u r i i e d c a r d i o l i p i n
5 p o i n t s t a n d a r d
c u r v e
n e g < 1 5 G P L
e q u i v 1 5 - 2 0 G P L
p o s > 2 0 G P L
7 0 8 6 3 0Q U A N T A L i t e A C A I g M I I I 3
A n t i g e n : P u r i i e d c a r d i o l i p i n
5 p o i n t s t a n d a r d
c u r v e
n e g < 1 2 . 5 M P L
e q u i v 1 2 . 5 - 2 0 M P L
p o s > 2 0 M P L
7 0 8 6 3 5
Q U A N T A L i t e A C A I g A I I I 4
A n t i g e n : P u r i i e d c a r d i o l i p i n
5 p o i n t s t a n d a r d
c u r v e
n e g < 1 2 A P L
e q u i v 1 2 - 2 0 A P Lp o s > 2 0 A P L
QUANTA Lite 2 GPI
PRODUCT No. DESCRIPTION CALIBRATION INTERPRETATION
7 0 8 6 6 0Q U A N T A L i t e
2G P I S c r e e n i n g E L I S A 5
A n t i g e n : P u r i i e d 2- g l y c o p r o te i n I
c u t o n e g < d e c i s i o n p o i n t
p o s > d e c i s i o n p o i n t
7 0 8 6 6 5Q U A N T A L i t e
2G P I I g G E L I S A 6
A n t i g e n : P u r i i e d 2- g l y c o p r o te i n I
5 p o i n t s t a n d a r d
c u r v e
n e g < 2 0
p o s > 2 0
7 0 8 6 7 0Q U A N T A L i t e
2G P I I g M E L I S A 7
A n t i g e n : P u r i i e d 2- g l y c o p r o te i n I
5 p o i n t s t a n d a r d
c u r v e
n e g < 2 0
p o s > 2 0
7 0 8 6 7 5
Q U A N T A L i t e 2
G P I I g A E L I S A 8
A n t i g e n : P u r i i e d 2- g l y c o p r o te i n I
5 p o i n t s t a n d a r d
c u r v e
n e g < 2 0
p o s > 2 0
INOVA Diagnostics, Inc. ofers a wide range o Antiphospholipid Syndrome (APS) ELISA test kits.
INOVA Diagnostics APS ELISA test kits
1. QUANTA Lite ACA Screen III is an enzyme-linked immunosorbe nt assay (ELISA) or the qualitative detection o cardiolipin antibod ies in human serum. The presence o cardiolipin antibodies can be used i
conjunction with clinical ndings and other laboratory tests to aid in assessing the risk o thrombosis in individuals with systemic lupus erythematosus (SLE) or lupus-like disorders.
2. QUANTA Lite ACA IgG III is an enzyme-l inked immunosorbent assay (ELISA) or the semi- quantitative detectio n o IgG cardiolipin antibodies in human serum. The presence o cardiolipin antibodies can be used i
conjunction with clinical ndings and other laboratory tests to aid in assessing the risk o thrombosis in individuals with Systemic Lupus Erythematosus (SLE) or lupus-like disorders.
3. QUANTA Lite ACA IgM III is an enzyme -linked immunosorbent assay (ELISA) or the semi-quanti tative detection o IgM cardiolipin antibodies in human serum. The presence o cardiolipin antibodies can be used i
conjunction with clinical ndings and other laboratory tests to aid in assessing the risk o thrombosis in individuals with Systemic Lupus Erythematosus (SLE) or lupus-like disorders.
4. QUANTA Lite ACA IgA III is an enzyme-l inked immunosorbent assay (ELISA) or the semi- quantitative detectio n o IgA cardiolipin antibodies in human serum. The presence o cardiolipin antibodies can be used i
conjunction with clinical ndings and other laboratory tests to aid in assessing the risk o thrombosis in individuals with Systemic Lupus Erythematosus (SLE) or lupus-like disorders.
5. QUANTA Lite 2 GPI Screen is an enzyme-li nked immunosorbent assay (ELISA) or the qualitative detection o IgG, IgM and IgA antibodies to 2 glycoprotein I (2 GPI) in human serum. 2 GPI antibodies are use
as an aid in the diagnosis o certain autoimmune thrombotic disorders, such as those secondary to systemic lupus erythematosus (SLE) or other lupus-like disorders.
6. QUANTA Lite 2 GPI IgG is an enzyme-linked immunosor bent assay (ELISA) or the semi- quantitative detect ion o 2 GPI IgG antibodies in human serum. The presence o 2 GPI IgG antibodies can be used i
conjunction with clinical ndings and other laboratory tests to aid in the diagnosis o certain autoimmune disease thrombotic disorders, such as those secondary to systemic lupus erythematosus (SLE) or other
lupus-like thrombotic diseases.
7. QUANTA Lite 2 GPI IgM is an enzyme-linked immunosorbe nt assay (ELISA) or the semi-quantitative detect ion o 2 GPI IgM antibodies in human serum. The presence o 2 GPI IgM antibodies can be used i
conjunction with clinical ndings and other laboratory tests to aid in the diagnosis o certain autoimmune disease thrombotic disorders, such as those secondary to systemic lupus erythematosus (SLE) or other
lupus-like thrombotic diseases.
8. QUANTA Lite 2
GPI IgA is an enzyme-linked immunosorbent assay (ELISA) or the semi-quantitative detection o 2
GPI IgA antibodies in human serum. The presence o 2GPI IgA antibodies can be used i
conjunction with clinical ndings and other laboratory tests to aid in the diagnosis o certain autoimmune disease thrombotic disorders, such as those secondary to systemic lupus erythematosus (SLE) or other
lupus-like thrombotic diseases.
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For more inormation on INOVA Diagnostics
complete product oferings visit www.inovadx.com
TESTING FOR ANTIPHOSPHOLIPID SYNDROME | 15
HUMAN ANTI-PHOSPHATIDYLSERINE
PRODUCT No. DESCRIPTION CALIBRATION INTERPRETATION
7 0 4 6 2 5H u m a n A n t i - P h o s p h a t i d y l s e r i n e I g G 10
A n t i g e n : P h o s p h a t i d y l s e r i n e a n d c o a c t o r
5 p o i n t s t a n d a r d
c u r v e
n e g < 1 1 G P S U / m L
p o s > 1 1 G P S U / m L
7 0 4 6 3 0H u m a n A n t i - P h o s p h a t i d y l s e r i n e I g M 11
A n t i g e n : P h o s p h a t i d y l s e r i n e a n d c o a c t o r
5 p o i n t s t a n d a r d
c u r v e
n e g < 2 5 M P S U / m L
p o s > 2 5 M P S U / m L
7 0 4 6 3 5H u m a n A n t i - P h o s p h a t i d y l s e r i n e I g A 12
A n t i g e n : P h o s p h a t i d y l s e r i n e a n d c o a c t o r
5 p o i n t s t a n d a r d
c u r v e
n e g < 2 0 A P S U / m L
p o s > 2 0 A P S U / m L
QUANTA Lite aPS/PT Phosphatidylserine/Prothrombin
PRODUCT No. DESCRIPTION CALIBRATION INTERPRETATION
7 0 8 8 3 5Q U A N T A L i t e a P S / P T I g G 9
A n t i g e n : P h o s p h a t i d y ls e r i n e a n d P r o t h r om b i n
5 p o i n t s t a n d a r d
c u r v e
n e g < 3 0 U n i t s
p o s > 3 0 U n i t s
7 0 8 8 4 5Q U A N T A L i t e a P S / P T I g M 9
A n t i g e n : P h o s p h a t i d y ls e r i n e a n d P r o t h r om b i n
5 p o i n t s t a n d a r d
c u r v e
n e g < 3 0 U n i t s
p o s > 3 0 U n i t s
ANTIPHOSPHOLIPID SYNDROME (APS) - COMPONENTS
PRODUCT No. DESCRIPTION
5 0 8 6 6 8
I g G S a p p o r o S t a n d a r d ( H C A L )
T h e I g G S a p p o r o S t a n d a r d ( H C A L ) i s u s e d a s a n i n t e r n a t i o n a l s t a n d a r d
o r t h e q u a l i t y c o n t r o l o a n t i - c a r d i o l i p i n I g G ( a C L )
a n d a n t i - 2- g l y c o p r o te i n I ( 2 G P I ) I g G a n t i b o d y E L I S A p r o d u c t s
5 0 8 6 7 3
I g M S a p p o r o S t a n d a r d ( E Y 2 C 9 )
T h e I g M S a p p o r o S t a n d a r d ( E Y 2 C 9 ) i s u s e d a s a n i n t e r n a t i o n a l s t a n d a r d
o r t h e q u a l i t y c o n t r o l o a n t i - c a r d i o l i p i n I g M ( a C L ) a n d
a n t i - 2- g l y c o p r ot e i n I (
2G P I ) I g M a n t i b o d y E L I S A p r o d u c t s
INOVA Diagnostics APS ELISA test kits
9. QUANTA Lite aPS/PT IgG and/or QUANTA Lite aPS/PT IgM kits are semi-quantitative and qualitative enzyme-linked immunosorbent assays (ELISA) or the detection o IgG and IgM
class antibodies to phosphatidylserine/prothrombin complex (PS/PT) in serum or plasma. For use as an aid in the diagnosis o certain autoimmune thrombotic disorders, such as
anti-phospholipid syndrome (APS) and those secondary to systemic lupus erythematosus or other lupus-like diseases, in conjunction with other laboratory and clinical ndings.
10. This assay is intended or the in-vitro measurement o IgG antiphosphatidylserine antibodies in human serum, as an aid in the diagnosis o anti-phospholipid syndrome (APS).
11. This assay is intended or the in-vitro measurement o IgM anti-phosphatidylserine antibodies in human serum, as an aid in the diagnosis o anti-phospholipid syndrome (APS).
12. This assay is intended or the in-vitro measurement o IgA anti-phosphatidylserine antibodies in human serum, as an aid in the diagnosis o anti-phospholipid syndrome (APS).
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Published by
INOVA Diagnostics, Inc.
9900 Old Grove Road
San Diego, CA 92131
toll ree: (800) 545-9495 (US only)
phone: (858) 586-9900 (outside the US)
Fax (858) 586-9911
ino@inovadx.com
www.inovadx.com
Authors
Gary L. Norman, PhD
Philipp von Landenberg, MD, PhD
Mareike Lorenz, PhD
Pier Luigi Meroni, MD, PhD
Francesca Pregnolato, PhD
Wally Binder, PhD
Silvia S.Pierangeli, MD, PhD
Kaija Javela, PhD
Editor
LeoPoldine Steindl
Graphic Design
Michael Kulwiec DesignLab
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