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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.
No. 6
IN THIS ISSUE
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
1.
2.
Ta b l e 1
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
MPL
GPL
325
300
275
250
225
200
175
150
125
100
75
50
25
0
B1
M4
B3
B5
M1
M2
B2
B4
M3
M5
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
IIa
IIb
IIc
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.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Figure 1
aPS/PT IgG or
IgM Positive
76% (52/59)
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 |
Objectives
OBJECTIVES
...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
Conclusions
1.
2.
3.
4.
High Specificity
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
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.
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
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
IgM GPI
erythematosus or other
lupus-like diseases.
Results
Objective
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
Table 2
IN-HOUSE
METHOD
QUANTA
Lite
Orgentec
Reaads
Varelisa
EliA
Positive (n)
Negative (n)
10
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
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
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
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
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.
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
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.
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
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
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
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
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
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).
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
508673
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
info@inovadx.com
Graphic Design
www.inovadx.com
Editor
LeoPoldine Steindl