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An automated chemiluminescence immunoassay may detect mostly relevant

IgG anticardiolipin antibodies according to revised Sydney criteria

Article in Acta clinica Belgica · August 2012

DOI: 10.1179/ACB.67.3.2062653 · Source: PubMed

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Chemiluminescence immunoassay and IgG anticardiolipin 1

Original Article

AN AUTOMATED CHEMILUMINESCENCE
IMMUNOASSAY MAY DETECT MOSTLY
RELEVANT IgG ANTICARDIOLIPIN ANTIBODIES
ACCORDING TO REVISED SYDNEY CRITERIA
Noubouossie D1, Valsamis J2, Corazza F3, Rozen L1, Debaugnies F1, Demulder A1
1
Laboratory of Hematology and Haemostasis, 2Laboratory of Hormonology and 3Laboratory of
Immunology, CHU Brugmann, Brussels, Belgium

Correspondence and offprint requests to: Denis Noubouossie, E-mail: noubouossie75@yahoo.fr

ABSTRACT observed between CLIA results and APS (χ² = 12.25;

Background: Detection of anticardiolipin antibodies
(ACA) is an independent laboratory criterion for diagnosis
of antiphospholipid syndrome (APS). Alternative methods
to ELISA were recently developed such as automated
chemiluminescence immunoassay (CLIA).
Patients and methods: We compared a CLIA to an
ELISA kit for the detection of IgG isotype of ACA. 87 rou-
tine samples from 75 patients suspected of having APS
were tested using each method. Cut-off values were cal-
culated in our laboratory for each test using 99th percen-
tile of 50 normal controls.
Results: Cut-off values were > 20 GPL for ELISA and
> 2 GPL for CLIA. Overall agreement (OA), agreement for
positive (AP) and agreement for negative (AN) cases
were 56.3%, 49.2% and 77.2% respectively. Most dis-
crepant results were positive with ELISA and negative
with CLIA. However, OA, AP and AN increased to 82.1%,
84.6% and 80% respectively when CLIA was compared
to the repeated ELISA performed at least 12 weeks later.
When correlated with APS-related clinical background,
CLIA showed lower sensitivity, higher specificity and
higher likelihood ratio (LR) as compared to first ELISA
whereas these parameters were similar to those of the
repeated ELISA. No association was found between any
test results and APS-related clinical background of the
patients. Using our own cut-off value (> 2GPL), sensitiv-
ity, specificity and LR of CLIA to identify patients with
APS were respectively 100%, 72.3% and 3.6. A ROC
curve showed that at 7.5 GPL cut-off value, specificity
and LR improved to 91.1% and 11.25 respectively, with-
out affecting sensitivity. A strong correlation was
p < 0.001).
Conclusion: The performance of CLIA is as good as a
repeated ELISA test to detect IgG ACA in suspected APS
patients. It is fully automated, which represents several
advantages over semi-manual ELISA techniques for its
implementation in a routine laboratory.
Key words: anticardiolipin antibodies, ELISA, chemiluminescence
immunoassay, automated assay, agreement
INTRODUCTION
Antiphospholipid antibodies (aPLs) represent a heteroge-
neous group of antibodies that recognize phospholipids (PL),
PL-binding proteins or PL-protein complexes. There is strong
evidence that aPLs are pathogenic in vivo, leading to a large
variety of clinical manifestations among which vascular
thrombosis and recurrent foetal loss/morbidity are the most
prevalent. The presence of these clinical events associated to
the detection of aPLs in the blood characterizes the antiphos-
pholipid syndrome (APS). Criteria for classification as APS
were first defined in Sapporo in 1999 (1) and were later
revised in Sydney in 2006 (2). According to this last consensus
statement, laboratory tests that are useful to define the labo-
ratory criteria of APS are the lupus anticoagulant, anticardi-
olipin antibodies (ACA) and anti-β2-glycoprotein I antibodies
(anti-β2GPI). All three tests are considered as independent
risk factors; therefore the positivity in any of them is sufficient
to consider it as a laboratory criterion of APS. ACA were first
detected using a radioimmunoassay (RIA) in 1983 (3). From
1985, the detection of ACA was replaced by enzyme-linked

doi: 10.2143/ACB.67.0.2062000 Acta Clinica Belgica, 2012; 67-?

2 Chemiluminescence immunoassay and IgG anticardiolipin
immuno-sorbent assays (ELISA). Until the early 1990s, ACA
testing was mainly performed using home-made ELISAs. As
commercial kits became available, routine laboratories
started to use them with a high inter-laboratory variability of
the results. Despite considerable efforts, standardization of
ACA testing is far from being achieved, as shown by the
results of external quality surveys (4,5). Nowadays, emerging
technologies lead to the development of fully automated
methods for the detection of anticardiolipin antibodies. This
study aimed to compare a fully automated chemilumines-
cence immunoassay (CLIA) to an ELISA assay for the detection
of IgG isotype of ACA.
PATIENTS AND METHODS
Samples analyzed in this study were selected from
patients referred to our laboratory for the detection of
antiphospholipid antibodies. Positive and negative samples
were selected according to the results obtained from the
ELISA method routinely used in our laboratory for the
detection of IgG ACA (REAADS® Anticardiolipin IgG Semi
Quantitative test kit, CORGENIX, USA) with a cut-off value
of > 23 GPL. The selection was made to obtain a distribution
of samples results with IgG ACA levels ranging from 4 GPL
to the maximum detected (82 GPL). Samples were analyzed
in batch using the fully automated method (Liaison® Cardi-
olipin IgG, DIASORIN, Italy) which is based on the CLIA prin-
ciple as described by Capuano et al (6). Briefly, highly puri-
fied cardiolipin is coated on the magnetic particles in the
presence of β2GP1 (solid phase). During the first incuba-
tion, IgG ACA present in calibrators, samples and controls
bind to the solid phase. During the second incubation, a
mouse monoclonal isoluminol-antibody conjugate reacts
with IgG ACA already bound to the solid phase. A wash
cycle removes unbound material after each incubation.
Subsequently, the starter reagent is added and a flash
chemiluminescence reaction is produced. The light signal is
measured by a photomultiplier as relative light units (RLU)
and is proportional to the amount of IgG ACA present in
calibrators, samples and controls. Results are expressed in
GPL units (adopted unit of IgG antiphospholipids antibody
Table 1: Assay-specific characteristics of chemiluminescence (CLIA) and enzyme-linked immunosorbentassays (ELISA) for
the detection of IgG anticardiolipin antibodies
Characteristics ELISA
Solid phase Stabilized beef heart cardiolipin coated microwells
Immunoconjugate Goat anti-human IgG antibody
Signal detected Optical density
Calibrators Traceable to the Harris standard (University of Louisville)
Manufacturer’s cut-off < 23 (calculated as the mean + 2SD)
Units GPL (1 µg/ml)
Intra-assay CV (manufacturer) Low (20.1 GPL): 7.7% (n = 28)
High (48.8 GPL): 9.4% (n = 28)
Inter-assay CV (manufacturer) Low (20.1 GPL): 11.9% (n = 3)
High (48.8 GPL): 13.2% (n = 3)
ELISA: enzyme linked immunosorbentassay, CLIA: chemiluminescence immunoassay, CV: coefficient of variation, GPL: unit of IgG antiphospholipids antibody
titers
Acta Clinica Belgica, 2012; 67-?
titer), the cut-off value as indicated by the manufacturer is
> 20 GPL. Table 1 compares assay-specific characteristics of
the two methods.
We also selected a set of samples that were repeatedly
positive for the ELISA test after at least 12 weeks. Clinical
records of all selected samples were retrospectively examined
and patients were classified into 2 groups. A first group con-
sisted of patients presenting APS-related clinical features,
including venous or arterial thrombosis and obstetric morbid-
ity. The second group consisted of patients without any APS-
related clinical feature including auto-immune disease (AID)
without thrombosis or obstetric morbidity, and other dis-
eases.
Normal controls (NC) used for cut-off values determina-
tion were selected among samples tested for routine coagu-
lation before a minor surgery. The controls had no history of
coagulation disorder, no treatment interfering with haemo-
static parameters, had a normal CRP and normal routine
coagulation tests.
Patients and NC samples were collected into Vacutainer®
tubes (BD, Plymouth, UK) containing buffered sodium citrate
(0.109M). Platelets-poor plasma were obtained from each
sample after double centrifugation at 3200 g, stored at − 80°C
and analyzed in batches.
Statistical analyses
All results were expressed as positive or negative accord-
ing to our own cut-off values. The overall agreement between
both tests (OA), the agreement for positive (AP) and for nega-
tive (AN) cases were expressed as percentage. OA is the sum
of positive and negative results concordant with both tests
divided by the total number of results. AP is the number of
positive results with both methods divided by all positive
ELISA results. AN is the number of negative results with both
methods divided by all negative ELISA results. Sensitivity,
specificity and likelihood ratio (LR) for each test were calcu-
lated against the clinical background and APS classification of
the patients. Chi-square with Yates’correction (χ²) was used to
assess the correlations between test results and clinical fea-
tures. Statistical calculations were computed using the soft-
ware Graphpad Prism® version 5 (GraphPad Software Inc,
USA). A p < 0.05 was considered significant.
CLIA
Purified Cardiolipin and human β2GP1 Coated magnetic
beads
Mouse anti-human IgG antibody
Relative light units
Traceable to the Sapporo standard (HCAL)
< 20 (calculated as the 99 percentile)
GPL/ml
Negative control: 5.4% (n = 20)
Positive control: 7.6% (n = 20)
Negative control: 20.2%
Positive control: 7.3% (n = not available)
 Chemiluminescence immunoassay and IgG anticardiolipin 3

RESULTS Agreement between results of CLIA and repeated ELISA test

Eighty seven samples were consecutively selected, includ-
ing 65 positive and 22 negative ELISA results. These samples
corresponded to 75 patients. Demographics of NC and
patients, as well as the distribution of patients according to
the clinical features are shown in Table 2.
Cut-off values determination for each method
A total of 50 NC samples were tested using each method.
Each sample was tested in duplicate. The mean of ACA levels
was calculated in both methods. For ELISA, the range of all
mean values was 2-19.5 GPL. For CLIA, all the NC samples
tested had ACA level < 2 GPL (detection threshold of the test).
The 99th percentile values was 19.5 GPL for ELISA and < 2 GPL
for CLIA. Consequently, we set our own cut-off values at
20 GPL for ELISA and 2 GPL for CLIA.
Agreement between results of CLIA and first ELISA test
All results were interpreted using our own cut-off values.
Accordingly, OA, AP and AN were 56.3%, 49.2% and 77.2%
respectively. As shown in Table 3, the OA ranged between 42.8%
and 58.8% for the different subgroups of patients according to
their clinical features. In all clinical groups, most of the discord-
ant cases were positive with ELISA and negative with CLIA.
Twenty eight ELISA results were retested after at least
12 weeks interval. For those samples, OA with CLIA was 50%
when the ELISA test was performed only one time, with dis-
cordant cases observed in all groups according to clinical fea-
tures. OA considerably improved to 82.1% while AP and AN
were 84.6% and 80% respectively when the repeated ELISA
test results were considered. As shown in Table 4, all the dis-
cordant results were found in patients who did not fulfil any
clinical criteria for APS. Those patients included 4 having AID
and 1 breast cancer patient classified as “other”.
Correlations between tests results and clinical features of APS
We classified patients according to their clinical background,
as APS-related (thrombosis and obstetric morbidity) and non
APS-related (AID without APS-related clinical features or other
diseases). The sensitivity, specificity and LR were computed for
each test against this clinical classification and summarized in
Table 5. Sensitivity was lower for CLIA whereas specificity and
LR was higher when compared with the first ELISA (see
Table 5(a)). These parameters were almost similar between CLIA
and the repeated ELISA, although a slightly higher specificity
and LR was observed for ELISA (see Table 5(b)). As shown in
Table 5, no significant association was found between any test
results and APS-related clinical features, as assessed with χ².

Table 2: Demographic and clinical features of patients

Normal Total APS-related Clinical features No APS-related clinical feature
controls patients
All Thromb PM All AID Others
Number of individuals
All 40 75 28 17 11 47 19 28
Female 17 49 20 9 11 29 15 14
Male 23 26 8 8 0 18 4 14
Median age (Percentile 25 – Percentile 75)
All 48 52 40 63 34 57 47 61
(44-66) (36-71) (33-71) (37-83) (30-40) (40-71) (36-63) (46-73)
Female 52 46 39 62 34 52 46 60
(47-71) (33-62) (30-61) (34-76) (30-40) (34-63) (33-60) (38-69)
Male 48 71 73 73 – 67 62 67
(43-54) (48-80) (37-84) (37-84) (51-77) (40-76) (53-80)
APS: antiphospholipid syndrome, Thromb: thrombosis (arterial and/or venous), PM: pregnancy morbidity, AID: autoimmune disease.

Table 3: Agreement between results of CLIA and the first ELISA: distribution according to clinical features
All With APS-related clinical features Without APS-related clinical feature
patients
All Thromb PM All AID Others
Total number 75 28 17 11 47 19 28
OA (%)* 50.6 53.5 58.8 45.4 48.9 57.8 42.8
(39-62) (35-72) (35-82) (16-75) (35-63) (36-80) (25-61)
Discordant (%)* 49.3 46.4 41.1 54.4 51.0 42.1 57.1
(38-61) (28-65) (18-65) (25-84) (37-65) (20-64) (39-75)
Elisa+/Clia- (%)* 42.6 35.7 29.4 45.4 46.8 36.8 53.5
(31-54) (28-65) (8-51) (16-75) (33-61) (15-59) (35-72)
Elisa-/Clia+ (%)* 6.6 10.7 11.7 9.0 4.2 5.2 3.5
(1-12) (0-22) (0-27) (0-26) (0-10) (0-15) (0-10)
* In percent of the corresponding column, in brackets are 95% confidence intervals, OA: overall agreement, APS: antiphospholipid syndrome, Thromb:
thrombosis (arterial and/or venous), PM: pregnancy morbidity, AID: autoimmune disease

Acta Clinica Belgica, 2012; 67-?

4 Chemiluminescence immunoassay and IgG anticardiolipin

Table 4: Agreement between results of CLIA and the repeated ELISA: distribution according to clinical features
All With APS-related clinical features Without APS-related clinical feature
patients
All Thromb PM All AID Others
Total number 28 12 6 6 16 9 7
OA (%)* 82.1 100 100 100 68.7 55.5 85.7
(68-96) (46-91) (23-88) (60-100)
Discordant (%)* 17.8 0 0 0 31.2 44.4 14.2
(4-32) (9-54) (12-77) (0-40)
Elisa+/Clia- (%)* 7.1 0 0 0 12.5 22.2 0
(0-17) (0-29) (0-49)
Elisa-/Clia+ (%)* 10.7 0 0 0 18.7 22.2 14.2
(0-22) (0-38) (0-49) (0-40)
* In percent of the corresponding column, in brackets are 95% confidence intervals, OA: overall agreement, APS: antiphospholipid syndrome, Thromb:
thrombosis (arterial and/or venous), PM: pregnancy morbidity, AID: autoimmune disease.

Table 5: Comparison of the relationship between the test results and clinical features of antiphospholipid syndrome
Sensitivity (%) Specificity (%) LR Chi-square
Comparison of CLIA with results of the first ELISA (n = 75)
ELISA 71.4 (51-86) 29.7 (17-44) 1.01 0.02NS
CLIA 46.4 (27-66) 72.3 (57-84) 1.67 1.96NS
Comparison of CLIA with results of the repeated ELISA (n = 28)
ELISA 66.6 (34-90) 68.7 (41-88) 2.13 2.18NS
CLIA 66.6 (34-90) 62.5 (35-84) 1.77 1.31NS
CLIA: chemiluminescence immunoassay, ELISA: enzyme linked immunosorbentassay, LR: likelihood ratio, NS chi-square with Yate’s correction not significant
(p > 0.05).

Performances of CLIA for diagnosis of APS patients in our

cohort
According to Sydney recommendations, 8 patients
(7 fe­male and 1 male) were classified as having APS on the
basis of the presence of APS-related clinical features and a con-
firmed positive ELISA result. The median (P25-P75) age of these
APS positive patients was 34 (31-63). We classified a total of
47 patients without any clinical feature of the syndrome as not
having APS, including 19 AID patients. The remaining
20 patients who had APS-related clinical features but who were
not retested, or who had a negative result with the second
ELISA were excluded. We then analyzed the performance of
CLIA for the diagnosis of APS patients in this population. At the
cut-off value determined in our laboratory (2 GPL), sensitivity,
specificity and LR were 100%, 72.3%, and 3.6 respectively.
There was a strong correlation between CLIA results and APS
(χ² = 12.25; p < 0.001). The Receiver Operator Characteristic
(ROC) curve shown in the figure represents the trade-off of Figure 1: ROC curve for diagnosis of patients having antiphospholipid
sensitivity versus specificity at different cut-off values. As syndrome (APS) in our cohort. Eight patients were classified
as having APS as they manifested APS-related clinical fea-
shown by this curve, the best performance of CLIA was
tures and had a positive result with the repeated ELISA per-
observed at the cut-off of 7.5 GPL with 100% sensitivity, 91.1% formed at least 12 weeks interval. Forty-seven patients not
specificity and a LR of 11.25. having any APS-related clinical feature were classified as
controls. Cut-off values: 2 GPL (own laboratory), 7.5 GPL (best
performances), 20 GPL (manufacturer).
DISCUSSION
This study aimed to compare a fully automated CLIA to was included because, as shown in previous studies, both
an ELISA kit used in our laboratory for the detection of IgG methods were strongly concordant for the negative cases
ACA. Samples selected were among those sent to the labo- (9). We considered it interesting to evaluate the perfor-
ratory for routine aPL detection in a context of a suspicion mance of CLIA in a group of patients including majority of
of primary or secondary APS. A minority of negative ­samples positive cases with ELISA. We focused only on IgG ACA

Acta Clinica Belgica, 2012; 67-?

 Chemiluminescence immunoassay and IgG anticardiolipin
i­sotype which is the most prevalent, and usually present
when other isotypes are detected. IgG isotype is also
reported to be more strongly associated with clinical mani-
festations of APS than IgA or IgM aPLs, the later often being
reported as clinically irrelevant and related to non-APS
related false positive results (7,8).
We calculated our own cut-off values for each test as the
99th percentile of 50 individuals attending our hospital for a
minor surgery. These individuals were matched to demo-
graphic characteristics of the patients included in the study.
We found an important difference between the cut-off value
recommended by the manufacturer (20 GPL) and that calcu-
lated in our laboratory (2 GPL) for the CLIA test. This high-
lights the importance of determining own cut-off values as
advised by the European Forum for aPLs (10).
Because a golden standard for detection of ACA does not
exist to date, true sensitivity and specificity cannot be esti-
mated. Therefore, we used the percentage of agreement and
tests relevance against clinical features of APS to compare the
results of both tests. The overall agreement observed in this
study is close to that previously reported between several
ELISA kits (11). A recent study comparing another CLIA kit to
the same ELISA kit we used in this study showed a higher
overall agreement between the two tests (12) . Assessing the
relationship between ELISA test results and APS-related clini-
cal features, Decavele et al (13) observed lower sensitivity and
higher specificity than we observed in our study. Although an
interesting approach, these are not true sensitivity and spec-
ificity as the test results are not evaluated against well defined
APS patients. The differences observed between our results
and those cited above are probably due to the fact that we
compared smaller number of cases, and we included a major-
ity of patients with positive ELISA results. Discrepant results
in our study were more often those positive with ELISA and
negative with CLIA.
The percentage of agreement greatly improved when
the results of the CLIA test were compared to repeatedly
positive ELISA results. Interestingly, remaining discrepant
results were observed only in patients that did not present
any APS-related clinical feature. Similar observations were
made when comparing the relevance of tests against APS-
related clinical features. Indeed, CLIA showed a lower sensi-
tivity but a higher specificity and higher LR when compared
to the first ELISA, whereas the two tests showed comparable
sensitivity, specificity and LR when CLIA was compared to
repeated ELISA results. Despite the ELISA test was repeated
for rather a small number of samples, the similar perfor-
mance we observed between both tests was mainly due to
the disappearance of transient IgG ACA detected by the first
ELISA. These findings suggest that the CLIA test may detect
mostly persistent IgG ACA. This could be explained by dif-
ferences in the assay-specific characteristics of the two tests,
particularly the source of antigens (cardiolipin and β2GPI)
and the β2GPI-dependency of tests. The cardiolipin-coated
wells of the ELISA kit used in this study were (otherwise
specified by the manufacturer), saturated using bovine
serum whereas purified human β2GP1 is used in the CLIA
test (6). It is well known that one of the drawbacks of ACA
ELISAs is their low specificity (7,14,15). However, Levy et al
(16) demonstrated that performing ACA detection in a
β2GPI-dependent manner (which is the case with the CLIA
5
test) allowed discriminating between pathogenic and non
pathogenic ACA, therefore increasing the specificity of the
test.
We also assessed the performance of the CLIA test to dis-
criminate patients having APS. Those patients presented both
APS-related clinical features and a positive ELISA result that
was confirmed at least 12 weeks later according to the Syd-
ney recommendations (2). A limitation was that our APS
patients were tested neither for IgM ACA, nor for anti- β2GPI
antibodies. Also, results of lupus anticoagulant detection
were not considered. This way, we could have missed some
APS positive patients. Nevertheless, using our own cut-off
value (2 GPL), the CLIA test was able to detect all so classified
APS patients with a specificity of 72.3%. Using ROC curve
analysis, we found that by increasing the cut-off value to
7.5 GPL, the specificity was improved to 91.1% with 100%
sensitivity and LR of 11.25. These findings are close to those
of Capuano et al (6) which showed that CLIA were positive in
94% of 70 clinically defined patients having primary or sec-
ondary APS.
As previously reported (13), we did not find any correla-
tion between CLIA results and APS-related clinical features
alone. However, a strong correlation was observed between
CLIA results and APS. This highlights the importance of clas-
sifying APS patients based on both clinical and laboratory
criteria, as recommended in the Sydney consensus (2).
In conclusion, our observations showed that the perfor-
mance of CLIA is as good as repeated ELISA test to detect IgG
ACA in suspected APS patients. Furthermore, the test also
fulfils the minimal requirements as advised by the European
Forum on aPLs. It is fully automated; therefore minimizing
errors associated with human handling, with improved repro-
ducibility. It is more convenient to use in routine clinical labo-
ratories, and could be performed on a single automate that
is also used for other immunoassays.
Acknowledgements
The authors would like to thank Roger Janssens in the
laboratory of Hormonology and Stephanie D’Hondt of the
Laboratory of Haematology and Haemostasis for technical
assistance.
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