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INOVA NEWS

THE EVOLVING STATE OF ANTIPHOSPHOLIPID ANTIBODY TESTING


Anti-cardiolipin antibodies (ACA), first identified in syphilis patients as a result of the
presence of cardiolipin in the bovine heart extract used for the VDRL syphilis test, have
evolved into one of the

primary components of antiphospholipid syndrome (APS)

diagnosis. The realization that the actual target of many antiphospholipid antibodies (aPL)
was the phospholipid binding protein 2GPI bound to cardiolipin antigen immobilized on
the ELISA well led to ELISA assays using purified 2GPI as the assay substrate. While some
labs continue to test for only IgG and IgM 2GPI isotypes, evidence suggests that 2GPI
IgA antibodies are associated with increased risk of adverse cardiovascular, thrombotic,
and pregnancy-associated events. In this issue of the INOVA newsletter, Aguilar-Valenzuela
et al. show some SLE patients are only positive for 2GPI IgA antibodies and recommend
2GPI IgA antibody testing in individuals suspected of 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 of the Sapporo monoclonal standards
to improve standardization and calibration of ACA kits. Javela and Mustonen describe
evaluation of five commercial ACA IgG ELISA kits and document discouraging variation in
the interpretation of low and moderately positive specimens between kits.
The significance of single and multiple ACA, 2GPI, and LAC positivities, as well as the
magnitude of the positivity, is discussed by Meroni and Pregnolato. Patients make
antibodies to a variety of phospholipid/protein targets, resulting in a heterogeneous
group of patient antibodies. Detection of 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 of phosphatidylserine /prothrombin, are described by Binder et al. Measurement
of both PS/PT IgG and IgM antibodies detected most LAC-positive
patients and close to 70% of the APS patients and identified some
APS patients missed by the conventional profile of ACA, 2GPI, and
LAC assays.
Antiphospholipid testing is evolving. New assays will allow finer
stratification of patients with APS, thrombotic, coagulation, and
pregnancy-related conditions into phenotypic groups with distinct
prognosis and management characteristics.

Gary L. Norman PhD


Senior Scientist
INOVA Diagnostics, Inc.

No. 6
IN THIS ISSUE

p2 Monoclonal antibodies in anti-phospholipid


diagnostics: Is there room for improvement of
standardization?

p3 High discrepancies in anti-phospholipid levels


are seen between laboratories

p4 Anti-phospholipid antibody detection:

Where we are standing and where we are going.

p6 Isolated elevated levels of IgA-Anti- GPI


2

are associated with clinical manifestations of the


antiphospholipid syndrome

p8 Clinical significance of IgG and IgM

autoantibodies that target the complex of


phosphatidylserine and prothrombin (PS/PT)

p12 Anti-cardiolipin IgG ELISAs What is the

right result? Comparison of five different commercial


test kits

p14 INOVA Diagnostics APS ELISA test kits

Monoclonal antibodies in anti-phospholipid diagnostics:


Is there room for improvement of standardization?
Philipp von Landenberg
Institut fuer Labormedizin, Solothurner Spitaeler AG, Baslerstrasse 15, 4600 Olten, Switzerland
The anti-phospholipid syndrome (APS) is an
autoimmune disease which is characterized by
different clinical, haematological and serological
manifestations. These include venous and/or arterial thrombosis, recurrent fetal loss and low platelet
counts. Accordingly, the binding specificities of
anti-phospholipids (aPL) appear to be as heterogeneous as the clinical manifestations associated
with them.
Since the typical antigens are cardiolipin and
phosphatidylserine, it was thought that aPL can
be distinguished by their phospholipid specificity
alone.
Additionally, it could be shown that most of the aPLs
require a protein cofactor to bind to their antigen.
One of these cofactors is the apolipoprotein beta2
glycoprotein 1 (2GPI) with a postulated function in
the lupus anticoagulant activity.
The clinical diagnosis of 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 different laboratories and assays even if
the laboratories are using the same assay.1
Discrepancies in results are even higher if laboratories use different brands of assays, as a result of
several variable factors (see table 1).
To overcome some of these problems, the use of
human and chimeric monoclonal antibodies for
standardization and calibration of the different
kits was introduced with the HCAL IgG Sapporo
Standard.2
2

| INOVA NEWS No. 6

Ta b l e 1

FA C T O R S R E S U LT 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 AY B R A N D S

Manufacturing and calibration of the assay


Source and purity of antigens
Specificity and isotype of detection antibodies
Heterogeneous avidity spectrum of antibodies
A variety of factors result in discrepancies between laboratories who use
different brands of aCL assays.

However, using different monoclonal IgG and IgM


antibodies directed to 2GPI and/or cardiolipin
still does not lead to a reasonable agreement in
different test systems.
This might be due to the fact that
monoclonal antibodies represent only one specificity to a certain epitope with a defined (high)
avidity, or does not contain the best match to the
antibodies found in the sera of antiphospholipid
patients.
Using monoclonal antibodies in aPL testing,
especially in the case of cardiolipin and 2GPI
diagnostics is not the be-all and end-all.
Remaining inconsistencies limit the clinical utility
and inter-laboratory transferability which in conclusion indicates that the standardization regarding
aCL testing still needs to be improved.
We are looking forward to the new upcoming
developments from the companies in this highly
competitive field of diagnostics.

1.
2.

Wong RCW et al., Thrombosis Research 2004;114:559-571.


Koike T et al., Arthritis & Rheumatism 1999; 42:309-1311.

High discrepancies in anti-phospholipid levels are seen between laboratories


Mareike Lorenz
Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University,
Lagenbeckstrasse 1, 55131 Mainz, Germany
Some years ago, a cross-laboratory evaluation was performed by the European
Antiphospholipid Forum. Basically, a set of
ten serum samples were tested at 24 different
European centers, using either home made
internally standardized or commercially available assays.

Ta b l e 1

TEST VARIABILITY IN aCL-IgG AND IgM ELISA KITS

With the introduction of one IgG and one IgM


monoclonal antibody (HCAL and EY2C9) as
putative standards, a progressive decrease in
the variability of the values was obtained.
Even with monoclonal standardization the
dilemma of inconsistent comparability of
results still remains, since not all routine
laboratories are following consensus
recommendations.
Although increasingly more laboratories and
manufacturers utilize standardized, uniform
materials and procedures the relatively high
variability for a-PL antibodies assays are an
ongoing issue of discussion.

1.

325
300
275
250
225
200
175
150
125
100
75
50
25
0

B1

M4

B3

B5

M1

M2

B2

B4

M3

M5

IgG aCL ELISA (results in GPL units)

MPL

A wide variability in results was found for


aCL-IgG ELISA test kits as well as for the
aCL-IgM ELISA (Table 1). Some values in the
cut off range appeared to be positive in one
test kit but normal with another.

GPL

Results were reported to the organizers and


compared to each other.1

325
300
275
250
225
200
175
150
125
100
75
50
25
0

B1

M4

B3

B5

M1

M2

B2

B4

M3

M5

IgM aCL ELISA (results in MPL units)

Tincani A et al., Thromb Haemost 2001; 86:57583.

TESTING FOR ANTIPHOSPHOLIPID SYNDROME |

Antiphospholipid antibody detection:


Where we are standing and where we are going
Pier Luigi Meroni, *Francesca Pregnolato
Division of Rheumatology Ist. Gaetano Pini, Dept. Internal Medicine University of Milan
*IRCCS Istituto Auxologico Italiano Milan (Italy)
The diagnostic and prognostic
antiphospholipid profile
According to the revised classification criteria, the positivity for lupus anticoagulant (LAC),
anti-cardiolipin (aCL) and anti-beta2 glycoprotein 1 (2GPI) antibodies are formal classification
laboratory criteria. Hence, a single positivity stable
over time (at least 12 weeks) is sufficient to classify a symptomatic patient as suffering from 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 specificity and the predictive value of each
single test and in particular of their combination
are not exactly the same. This is particularly true
taking into account that the most frequent clinical manifestations of the syndrome (i.e. deep vein
thrombosis or early miscarriages) are not specific and rather common in the general population.
A sub-classification of patients according to their
positivities has been suggested in table 1.
Ta b l e 1

L A B O R AT O R Y C R I T E R I A S AT 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

Classification of APS patients according to the positivity for the


antiphopsholipid assays

The predictive value has been suggested to be the


highest for the category I.1

| INOVA NEWS No. 6

The identification of the risk profile offers the


rationale for both the secondary prophylactic
therapy (i.e. in order to prevent recurrences) and
the primary prophylaxis to avoid the occurrence of
the clinical events in the antiphospholipid antibody
(aPL) positive asymptomatic subjects.
While the clinical approach is not problematic for
patients displaying several positivities and/or high
aPL titres, the situation is different 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 specificity was suggested
to be related to the fact that LAC is mediated by
antibodies against plasma proteins (anti-2GPI and
prothrombin) bound to anionic PL and ultimately
affecting their availability for the coagulation
cascade. To display this effect the antibodies need
to be at an elevated protein concentration and to
display high avidity.
However, the clinical value of the presence of LAC
as an isolated assay or in asymptomatic subjects or
at low potency has been recently questioned.5
To improve specificity, the revised classification criteria for the aCL assay require both a
stable positivity and a positivity threshold of 40
International Units1. As a consequence, a single
aPL positivity is quite rare with titres > 40 IU usually associated with positive LAC and/or anti-2GPI
assays. A comparable high threshold has not been
suggested for the anti-2GPI assay since the normal
cut off should be calculated on the 99th percentile
of 50 normal subjects1. Hence, the sensitivity of the
anti-2GPI assay is high. Such a sensitivity combined
with its wider use in the laboratories makes the
possibility of isolated elevated anti-2GPI antibodies more frequent.

Do we have (or are we going to have) new


useful aPL assays?

Tests detecting aCL IgA and anti-2GPI IgA


antibodies are available but are not formally
included into the revised criteria because of the
lack of evidence that the assay may improve the
whole diagnostic power.

There are preliminary results suggesting additional


assays could improve our diagnostic and prognostic
power as a second level of aPL testing.

In fact, IgA aPL appear to rather identify subgroups


of patients, such as Afro-Americans or pure obstetric APS. However, the search for IgA aPL may be
useful in order to confirm the diagnosis of APS in
the case of an isolated positivity or a borderline
result in the other solid-phase assays.

This could be the case for anti-prothrombin


antibodies (anti-PT) as a second level assay to
confirm 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
functional assay, it has also been suggested that
an anti-PT test may or may not confirm equivocal functional LAC. Although the heterogeneity of
the methods to detect anti-PT antibodies is still a
matter of debate, recent studies have once again
raised the possibility that anti-PT and in particular anti-PS/PT antibodies may display a diagnostic/
prognostic value on vascular manifestations.6-9

Conclusions
The panel of aPL tests is still evolving and apparently, like other autoantibody families, more than
one assay and the use of second level tests appear
useful to improve our diagnostic and prognostic
power.

We recently analyzed a selected series of samples


from APS patients and controls by using a new
ELISA assay for anti-PS/PT detection (kindly provided by Dr. W. Binder INOVA Diagnostics, USA).
Figure 1 reports our preliminary results showing a
good specificity of the assay and correlation with
the clinical manifestations.

1.
2.
3.
4.
5.
6.
7.
8.
9.

Miyakis S. et al., J Thromb Haemost 2006; 4:295-306.


Lee RM. et al., Obstetrics Gynecol 2003; 102:294300.
Pengo V. et al., J Thromb Haemost 2009.
Galli M. et al., . Blood 2003; 101: 18271832.
Pengo V. et al., J Thromb Haemost 2009; 7: 1737-1740.
Galli M. et al., Blood 2003; 102: 2717-2723.
Tincani A. et al., Clin Exp Rheumatol 2007; 25: 268-274.
Galli M. et al.,. Blood 2007; 110:1178-1183.
Sakai Y.et al., Arthritis Rheum 2009; 60: 2457-2467.

Figure 1

Detection of anti-PS/PT antibodies by ELISA in a selected series


of Lupus Anti-coagulant (LAC) positive samples from APS patients

aPS/PT IgG or
IgM Positive
76% (52/59)

aPS/PT IgG or IgM Negative


24% (7/59)

6/7 aPL positive


5/7 anti2GPI positive
4/7 aCL/anti2GPI positive

LAC Positive Sera (n=59)


59 LAC positive sera have been tested. 52/59 (76%) resulted positive
for 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 for anti-PS/PT antibodies
Most of the positivities for anti-PS/PT antibodies were at high titres
and 44.1% of them were of the IgM isotype
Only 2 out of 40 pathological aPL negative control sera (30 with
autoimmune diseases, 10 with infectious diseases) displayed a low
positivity (1 IgG and 1 IgM)
The cut off was calculated on 91 NHS samples (43 AU for IgG and 44
AU for IgM)
TESTING FOR ANTIPHOSPHOLIPID SYNDROME |

Isolated elevated levels of IgA-anti-2GPI are associated with


clinical manifestations of the antiphospholipid syndrome
Aguilar-Valenzuela R, 1Seif AM, 2Alarcn GS, 1Martnez-Martnez LA, 1Dang N, 1Papalardo E,
2
Liu J, 4Vila LM, 1Najam S, 1McNearney T, 1Gonzalez EB, 6Binder W, 5Teodorescu M, 3Reveille JD,
1
Pierangeli SS
1
University of Texas Medical Branch, Galveston, TX; 2University of Alabama at Birmingham, Birmingham, AL;
3
University of Texas-Houston Heath Sciences Center, Houston, TX; 4University of Puerto Rico Medical Sciences
Campus, San Juan, PR; 5Theratest Laboratories, Lombard, IL; 6INOVA Diagnostics, San Diego, CA.
1

Background and Purpose


Current diagnostic criteria recommend elevated titers of anti-Cardiolipin (aCL) and/or anti2Glycoprotein (2GPI) antibody IgG or IgM by
ELISA and/or lupus anticoagulant (LAC) to confirm
antiphospholipid syndrome (APS).
IgA aCL antibodies are found more frequently in
Afro-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 significance remained elusive.
Several studies report a possible association
between elevated IgA anti-2GPI titers and APS-like
manifestations.
Anti-2GPI IgA antibodies were strongly associated (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 classified
as APS due the absence of aCL positive test.
Anti-2GPI IgA antibodies are more prevalent in
patients with SLE.
We recently reported five isolated cases of exclusive
IgA anti-2GPI antibody sero-positivity with
concomitant APS clinical manifestations.
6

| INOVA NEWS No. 6

Objectives
OBJECTIVES

To examine the prevalence of exclusive IgA-anti-2GPI


antibody positivity in a large cohort of patients with SLE
and in patients suspected of having APS
To correlate IgA-anti-2GPI antibody positivity with APS
associated clinical manifestations
Patients
Anti-phospholipid seropositivity was examined in
2799 SLE sera, whereof 599 samples came from a
multi-ethnic, multi-center cohort (LUMINA) and
2200 samples were referred to our laboratory
(APLS) for APS work-up.
Laboratory methods
aCL (IgG, IgM, IgA) Screen, aPL (IgG and IgM), anti2GPI (IgG and IgM) antibodies were determined
by using two commercially available test kits, one
from INOVA Diagnostics, and an in-house protocol.
IgA-2GPI titers were determined by two commercial ELISA tests, one from INOVA Diagnostics..
Results
Out of the 2799 samples, 50 samples were positive
exclusively for IgA-2GPI in at least one kit.

A significant number of subjects in the two groups


had at least one APS-related clinical manifestation,
that included:
A P S - R E L AT E D C L I N I C A L M A N I F E S TAT I O N S

Deep vein thromboses


Pregnancy losses
Other APS-related pregnancy complications
Pulmonary infarctions, strokes, seizures, myocardial
infarctions
Thrombocytopenia
Non classical APS manifestations:
Skin ulcers, pulmonary hypertension, livedo reticularis,
cardiac valvular disease, seizures and migraines.

...elevated IgA

anti-2GPI antibody
titers may identify
additional patients who
have clinical features
of APS but who do not
meet current diagnostic
criteria.

Two of these samples were also LAC positive.

It may be therefore

84% of samples were positive with the INOVA Kit,


90% of samples were positive with the competitive
kit (correlation between the two kits was 0.93%).

recommended to test for

Conclusions

when other aPL tests

It may be therefore recommended to test for


IgA 2GPI antibodies when other aPL tests are
negative and APS is suspected.

1.
2.
3.
4.

This study supports that elevated IgA anti-2GPI


antibody titers may identify additional patients
who have clinical features of APS but who do not
meet current diagnostic criteria.

IgA 2GPI antibodies

are negative and APS is


suspected.

Amengual O. et al., Arthritis Rheum 2003;48:886-895.


DAgnillo P. et al., The Journal of Immunology 2003;170:3408-3422.
Atsumi T. et al., Arthritis Rheum 2000; 43:1982-1993.
Atsumi T. et al., Thrombosis Research 2004;114:553-538.

TESTING FOR ANTIPHOSPHOLIPID SYNDROME |

Clinical significance of IgG and IgM autoantibodies that target


the complex of phosphatidylserine and prothrombin (PS/PT)
W. L. Binder, S. Lewis and Z. Shums
INOVA Diagnostics, San Diego, CA, USA
Background
Antiphospholipid antibodies represent a large
heterogeneous group of immunoglobulins of
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 for aiding in diagnosis of
antiphospholipid syndrome (APS).
More and more laboratories are now including
tests for detecting antibodies directed against
phospholipid binding proteins, the best studied of
which is 2GPI.

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 useful sensitivity for
APS and have high specificity. Their inclusion into
the laboratory criteria for classification of 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

Prothrombin (factor II) is another phospholipid


binding protein with procoagulant activity.

Detects many ACA and 2GPI negative APS patients

A number of groups have definitively shown that


antibodies targeting the complex of phosphatidylserine (PS) and prothrombin (PT) have significant
clinical relevance due to their strong correlations
with clinical features of APS and with the presence
of LAC.1

High Specificity

| INOVA NEWS No. 6

Close approximation of LAC

Specific performance characteristics of QUANTA Lite aPS/PT IgG and QUANTA Lite aPS/PT IgM kits
that detect the complex of phosphatidylserine and prothrombin (PS/PT) autoantibodies
Assay Characteristics
Antigen on solid phase is a layer of phosphatidylserine and human prothrombin, coated in the presence of
Ca++. Standard ELISA format 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 for IgG
and IgM antibodies to PS/PT. These results were used to calculate performance characteristics and the new
assays were compared to traditional anti-GPI and LAC assays. Results are tabulated in Table 1.
Combined results from an external and an internal study
Table 1

PAT I E N T G R O U P

No SAMPLES

Normals
Lupus Anticoagulant Positive (LAC)
Antiphospholipid Syndrome (APS)
Rheumatoid Arthritis
Crohn's
Ulcerative Colitis
Celiac
LAC negative
Infectious disease (CMV, Toxo, Rubella, HSV HBV HCV)

247
24
71
6
2
2
5
8
14

Syphilis
Actin Antibody Positive
H. Pylori Positive

12
1
2

------------------ NUMBER POSITIVE (%) -------------------PS/PT IgG


PS/PT IgG
PS/PT IgM
AND/OR IgM
POSITIVE
POSITIVE
POSITIVE
3 (1.2%)
4 (1.6%)
7 (2.8%)
21 (87.5%)
19 (79.2%)
24 (100%)
33 (46.5%)
34 (47.9%)
48 (67.6%)
0
0
0
0
0
0
0
0
0
0
0
0
0
1 (12.5%)
1 (12.5%)
0
0
0
0
0
0

0
0
0

0
0
0

Forty eight of the 71 APS patients (67.6%) were PS/PT positive and many of these individuals were found to be negative using more
traditional assays such as anti-GPI and LAC.
Only 7 of 247 normals and 1 of the 52 disease controls were found to be positive for either IgG or IgM PS/PT antibodies for a
combined specificity of 97.3% (8/299).
The assays were found to have high inter and intra run precision.
Equivalent results were obtained with either serum or citrated plasma for both assays.
1.
2.
3.
4.

Amengual O. et al., Arthritis Rheum 2003;48:886-895.


DAgnillo P. et al., The Journal of Immunology 2003;170:3408-3422.
Atsumi T. et al., Arthritis Rheum 2000; 43:1982-1993.
Atsumi T. et al., Thrombosis Research 2004;114:553-538.

TESTING FOR ANTIPHOSPHOLIPID SYNDROME |

Performance of PS/PT IgG and PS/PT IgM ELISA with 20 LAC positive samples
and 4 borderline positive samples
LAC POSITIVE
SAMPLES

PS/PT IgG
(pos>30)

PS/PT IgM
(pos>30)

LAC BORDERLINE
SAMPLES

PS/PT IgG
(pos>30)

PS/PT IgM
(pos>30)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

141
146
139
151
144
112
213
217
21.2
125.6
51.3
224
97
157
152
173
147
136
15.5
88.3

81.2
61.6
60.2
67.6
54.4
39.7
11.5
10.4
98.7
51.6
406
16.6
138
132
65.3
305
253
63.7
114
109

1
2
3
4

22.1
75.2
217.5
278.5

132.6
7.8
21.6
35.6

All LAC positive patients were found to be strongly


positive for either IgG PS/PT, IgM PS/PT antibodies
or both. The combined use of aPS/PT IgG and aPS/PT
IgM detected all 24 known lupus anticoagulant
positives and 67.6% of the APS patients.

Agreement for both the IgG and IgM PS/PT kits with respect to the 2GPI kits
The relative agreement for both the IgG and IgM PS/PT kits with respect to the 2GPI assay is 85.6% and 82.2%.
Relative Performance to GPI IgG

IgG GPI

IgG PS/PT

IgM PS/PT

38

10**

28

16*

116

25

120

* 1 of the 16 was LAC positive and the other


15 were APS patients
** All 10 were from the APS group

10 | INOVA NEWS No. 6

Relative Performance to GPI IgM

IgM GPI

1 of these was a normal and 6 were from


the APS group
22 were from the APS group and 3 were
LAC positives

The PS/PT IgG plus IgM kits detected all LAC


positive samples and most of the APS patients. It
was noticed that the vast majority of APS patients
were positive for PS/PT and/or 2GPI.
Conclusions
PS/PT IgG and IgM ELISA appears to be
very specific and detects a majority of LAC
positives
IgG and IgM autoantibodies that react with a
physiologic complex of phosphatidylserine
and prothrombin are sensitive markers for
anti-phospholipid syndrome
PS/PT IgG and IgM ELISA detects most APS
patients including many that are ACA, 2GPI,
LAC negative
The tests exhibit high specificity 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

Most of the discrepant results are due to the higher


sensitivity of the IgG and IgM PS/PT kits for both
the LAC positives and especially the APS patients
although there were APS patients that were 2GPI
positive yet PS/PT negative.

erythematosus or other
lupus-like diseases.

TESTING FOR ANTIPHOSPHOLIPID SYNDROME | 11

Anti-cardiolipin IgG ELISAs What is the right result?


Comparison of five different commercial test kits
Javela K. and Mustonen P.
Finnish Red Cross Blood Service, Helsinki, Finland
Background

Results

Antiphospholipid syndrome (APS) is a disorder


characterized by recurrent thrombosis and/or
fetal loss associated with characteristic laboratory
abnormalities.

All 14 negative samples were negative by all ACA


IgG assays.

Patients suspected to have APS should


be screened for anticardiolipin antibodies
(ACA), 2-glycoprotein 1 antibodies and lupus
anticoagulant.
More than 45 commercial kits for ACA detection
are available (n=45 in ECAT reference list).

The number of positive samples varied when the


cut-off of manufacturer (Table 1) and the laboratory
classification criteria for APS (ACA IgG results >40
GPL (Table 2) were used .
The Variation Coefficients of all assays were good,
in the cut off range below 6.8% for all assays.
Conclusions

Objective

Evaluation of five different commercially available


ACA IgG/IgM ELISA test kits to replace our in-house
method.

Methods and Materials


Five different commercial ACA IgG assays were
chosen for 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 of all commercial ELISA assays are


calibrated against reference sera from E.N. Harris,
Louisville.
Our in-house method was used as a reference
method.
Ten positive, 4 strong positive and 14 negative
samples previously measured by our in-house
method were analyzed.

All trademarks are the properties of their respective companies

12 | INOVA NEWS No. 6

All tested commercial ELISAs had good


reproducibility and all strong positive samples
were positive by all assays
There was a significant discrepancy between
assays when borderline, low positive or
intermediately positive ACA IgG samples were
analyzed
The correct recognition of high but also medium
titer ACA IgG is of high clinical significance
The selection between reagents has to be
made for the method with the best correlation
to the existing one, since APL antibodies of APS
patients are repeatedly tested for monitoring
A potential problem will be if serum samples
are sent to a different laboratory which either
use a home made method as well, or a different
commercial test kit
The standardization of ACA IgG ELISAs remains
an unsolved problem

ACA IgG results


14 positive samples measured by five commercial ELISA assays. The 14
negative samples were negative by all ACA IgG assays.
Table 1

IN-HOUSE
METHOD
29*
30
30
33
35
36
38
41
42
43
43
59
87
173
646

QUANTA
Lite
15**
15
47
23
26
12
43
48
42
26
13
185
112
391
447

Orgentec

Reaads

Varelisa

EliA

10**
3
72
2
4
3
13
4
3
4
3
163
138
1256
856

23**
11
51
8
10
15
33
18
4
9
45
166
78
815
551

15**
6
25
2
3
3
17
5
1
3
7
125
156
405
387

15**
3
114
2
7
1
243
4
1
6
1
87
442
85
327

All tested

commercial
ELISAs had good
reproducibility
and all strong

positive samples
were positive by
all assays.

*Cut-off value of the in-house method; **Cut-off value set by the manufacturer

The number of positive samples according to laboratory classification


criteria for APS (>40 GPL)

ACA IgG (GPL)

Table 2

IN-HOUSE
METHOD

QUANTA
Lite

Orgentec

Reaads

Varelisa

EliA

Positive (n)

Negative (n)

10

TESTING FOR ANTIPHOSPHOLIPID SYNDROME | 13

INOVA Diagnostics APS ELISA test kits


INOVA Diagnostics, Inc. offers a wide range of Antiphospholipid Syndrome (APS) ELISA test kits.
QUANTA Lite ACA
PRODUCT No.

DESCRIPTION

CALIBRATION

INTERPRETATION

708620

Q U A N TA L i t e A C A S c r e e n I I I 1
Antigen: Purified cardiolipin

cutoff

neg < decision point


pos > decision point

708625

Q U A N TA L i t e A C A I g G I I I 2
Antigen:Purified cardiolipin

5 point standard
cur ve

neg <15 GPL


equiv 15-20 GPL
pos >20 GPL

708630

Q U A N TA L i t e A C A I g M I I I 3
Antigen: Purified cardiolipin

5 point standard
cur ve

neg <12.5 MPL


equiv 12.5-20 MPL
pos >20 MPL

708635

Q U A N TA L i t e A C A I g A I I I 4
Antigen: Purified cardiolipin

5 point standard
cur ve

neg <12 APL


equiv 12-20 APL
pos >20 APL

1. QUANTA Lite ACA Screen III is an enzyme-linked immunosorbent assay (ELISA) for the qualitative detection of cardiolipin antibodies in human serum. The presence of cardiolipin antibodies can be used in
conjunction with clinical findings and other laboratory tests to aid in assessing the risk of thrombosis in individuals with systemic lupus erythematosus (SLE) or lupus-like disorders.
2. QUANTA Lite ACA IgG III is an enzyme-linked immunosorbent assay (ELISA) for the semi-quantitative detection of IgG cardiolipin antibodies in human serum. The presence of cardiolipin antibodies can be used in
conjunction with clinical findings and other laboratory tests to aid in assessing the risk of 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) for the semi-quantitative detection of IgM cardiolipin antibodies in human serum. The presence of cardiolipin antibodies can be used in
conjunction with clinical findings and other laboratory tests to aid in assessing the risk of thrombosis in individuals with Systemic Lupus Erythematosus (SLE) or lupus-like disorders.
4. QUANTA Lite ACA IgA III is an enzyme-linked immunosorbent assay (ELISA) for the semi-quantitative detection of IgA cardiolipin antibodies in human serum. The presence of cardiolipin antibodies can be used in
conjunction with clinical findings and other laboratory tests to aid in assessing the risk of thrombosis in individuals with Systemic Lupus Erythematosus (SLE) or lupus-like disorders.

QUANTA Lite 2 GPI


PRODUCT No.

DESCRIPTION

CALIBRATION

INTERPRETATION

708660

Q U A N TA L i t e 2 G 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 f i e d 2- g l y c o p r o t e i n I

cutoff

neg < decision point


pos > decision point

708665

Q U A N TA L i t e 2 G P I I g G E L I S A 6
A n t i g e n : P u r i f i e d 2- g l y c o p r o t e i n I

5 point standard
cur ve

neg < 20
pos > 20

708670

Q U A N TA L i t e 2 G P I I g M E L I S A 7
A n t i g e n : P u r i f i e d 2- g l y c o p r o t e i n I

5 point standard
cur ve

neg < 20
pos > 20

708675

Q U A N TA 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 f i e d 2- g l y c o p r o t e i n I

5 point standard
cur ve

neg < 20
pos > 20

5. QUANTA Lite 2 GPI Screen is an enzyme-linked immunosorbent assay (ELISA) for the qualitative detection of IgG, IgM and IgA antibodies to 2 glycoprotein I (2 GPI) in human serum. 2 GPI antibodies are used
as an aid in the diagnosis of 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 immunosorbent assay (ELISA) for the semi-quantitative detection of 2 GPI IgG antibodies in human serum. The presence of 2 GPI IgG antibodies can be used in
conjunction with clinical findings and other laboratory tests to aid in the diagnosis of 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 immunosorbent assay (ELISA) for the semi-quantitative detection of 2 GPI IgM antibodies in human serum. The presence of 2 GPI IgM antibodies can be used in
conjunction with clinical findings and other laboratory tests to aid in the diagnosis of 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) for the semi-quantitative detection of 2 GPI IgA antibodies in human serum. The presence of 2GPI IgA antibodies can be used in
conjunction with clinical findings and other laboratory tests to aid in the diagnosis of certain autoimmune disease thrombotic disorders, such as those secondary to systemic lupus erythematosus (SLE) or other
lupus-like thrombotic diseases.

14 | INOVA NEWS No. 6

INOVA Diagnostics APS ELISA test kits


QUANTA Lite aPS/PT Phosphatidylserine/Prothrombin
PRODUCT No.

DESCRIPTION

CALIBRATION

INTERPRETATION

708835

Q U A N TA L i t e a P S / P T I g G 9
Antigen:Phosphatidylserine and Prothrombin

5 point standard
cur ve

neg < 30 Units


pos > 30 Units

708845

Q U A N TA L i t e a P S / P T I g M 9
Antigen:Phosphatidylserine and Prothrombin

5 point standard
cur ve

neg < 30 Units


pos > 30 Units

9. QUANTA Lite aPS/PT IgG and/or QUANTA Lite aPS/PT IgM kits are semi-quantitative and qualitative enzyme-linked immunosorbent assays (ELISA) for the detection of IgG and IgM
class antibodies to phosphatidylserine/prothrombin complex (PS/PT) in serum or plasma. For use as an aid in the diagnosis of 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 findings.

HUMAN ANTI-PHOSPHATIDYLSERINE
PRODUCT No.

DESCRIPTION

CALIBRATION

INTERPRETATION

704625

H 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
Antigen: Phosphatidylserine and cofactor

5 point standard
cur ve

neg <11 GPS U/mL


pos > 11 GPS U/mL

704630

H 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
Antigen: Phosphatidylserine and cofactor

5 point standard
cur ve

neg < 25 MPS U/mL


pos > 25 MPS U/mL

704635

H 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
Antigen: Phosphatidylserine and cofactor

5 point standard
cur ve

neg < 20 APS U/mL


pos > 20 APS U/mL

10. This assay is intended for the in-vitro measurement of IgG antiphosphatidylserine antibodies in human serum, as an aid in the diagnosis of anti-phospholipid syndrome (APS).
11. This assay is intended for the in-vitro measurement of IgM anti-phosphatidylserine antibodies in human serum, as an aid in the diagnosis of anti-phospholipid syndrome (APS).
12. This assay is intended for the in-vitro measurement of IgA anti-phosphatidylserine antibodies in human serum, as an aid in the diagnosis of anti-phospholipid syndrome (APS).

ANTIPHOSPHOLIPID SYNDROME (APS) - COMPONENTS


PRODUCT No.

DESCRIPTION
IgG Sapporo Standard (HCAL)
The IgG Sapporo Standard (HCAL) is used as an international standard
for the quality control of anti- cardiolipin IgG (aCL)
a n d a n t i - 2- g l y c o p r o t e 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

508668

IgM Sapporo Standard (EY2C9)


The IgM Sapporo Standard (EY2C9) is used as an international standard
for the quality control of anti- cardiolipin IgM (aCL) and
a n t i - 2- g l y c o p r o t e i n I ( 2 G 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

508673

For more information on INOVA Diagnostics


complete product offerings visit www.inovadx.com

TESTING FOR ANTIPHOSPHOLIPID SYNDROME | 15

INOVA NEWS

No. 6

INOVA NEWSLETTERS ON OTHER AUTOIMMUNE TESTING TOPICS ARE AVAILABLE UPON REQUEST
Celiac Disease Serology with Deamidated Gliadin Peptide (DGP) Assays (No. 3)
Third Generation CCP ELISA in Rheumatoid Arthritis Serology (No. 4)
Diagnosis of Primary Biliary Cirrhosis - Utilizing MIT3 Antigen Assays (No. 5)

Published by

Authors

INOVA Diagnostics, Inc.

Gary L. Norman, PhD

9900 Old Grove Road

Philipp von Landenberg, MD, PhD

San Diego, CA 92131

Mareike Lorenz, PhD

toll free: (800) 545-9495 (US only)

Pier Luigi Meroni, MD, PhD

phone: (858) 586-9900 (outside the US)

Francesca Pregnolato, PhD

Fax (858) 586-9911

Wally Binder, PhD

info@inovadx.com

Silvia S.Pierangeli, MD, PhD

Graphic Design

www.inovadx.com

Kaija Javela, PhD

Michael Kulwiec DesignLab

Editor
LeoPoldine Steindl

690120 February 10 Rev. 0