Anti Phospholipid Inova Orgentec

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.

THE EVOLVING STATE OF ANTI-PHOSPHOLIPID ANTIBODY TESTING

IN THIS ISSUE

INOVA NEWS

No. 6p2 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-2GPI 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

Gary L. Norman PhDSenior Scientist

INOVA Diagnostics, Inc.

2 | INOVA NEWS No. 6

The anti-phospholipid syndrome (APS) is an autoimmune disease which is characterized by different clinical, haematological and serological manifestations. These include venous and/or arteri-al thrombosis, recurrent fetal loss and low platelet counts. Accordingly, the binding specificities of anti-phospholipids (aPL) appear to be as hetero-geneous 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 labora-tories 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

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 speci-ficity 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 conclu-sion 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.

Philipp von Landenberg

Institut fuer Labormedizin, Solothurner Spitaeler AG, Baslerstrasse 15, 4600 Olten, Switzerland

Monoclonal antibodies in anti-phospholipid diagnostics: Is there room for improvement of 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 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 A Y 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.

TESTING FOR ANTIPHOSPHOLIPID SYNDROME | 3

Some years ago, a cross-laboratory evalu-ation 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 avail-able assays.

Results were reported to the organizers and compared to each other.1

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.

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.

Mareike LorenzInstitute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University, Lagenbeckstrasse 1, 55131 Mainz, Germany

High discrepancies in anti-phospholipid levels are seen between laboratories

Tincani A et al., 1. Thromb Haemost 2001; 86:57583.

325300275250225200175150125100

755025

0

B1 M4 B3 B5 M1 M2 B2 B4 M3 M5A

GPL

IgG aCL ELISA (results in GPL units)

325300275250225200175150125100

755025

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

The diagnostic and prognostic antiphospholipid profile

According to the revised classification crite-ria, the positivity for lupus anticoagulant (LAC), anti-cardiolipin (aCL) and anti-beta2 glycopro-tein 1 (2GPI) antibodies are formal classification laboratory criteria. Hence, a single positivity stable over time (at least 12 weeks) is sufficient to classi-fy 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 clini-cal manifestations of the syndrome (i.e. deep vein thrombosis or early miscarriages) are not specif-ic and rather common in the general population. A sub-classification of patients according to their positivities has been suggested in table 1.

The predictive value has been suggested to be the highest for the category I.1

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 classifica-tion 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 usual-ly 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 antibod-ies more frequent.

Pier Luigi Meroni, *Francesca PregnolatoDivision of Rheumatology Ist. Gaetano Pini, Dept. Internal Medicine University of 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 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


Do we have (or are we going to have) new useful aPL assays?

There are preliminary results suggesting additional assays could improve our diagnostic and prognostic power as a second level of aPL testing.

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 equivo-cal 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 particu-lar anti-PS/PT antibodies may display a diagnostic/prognostic value on vascular manifestations.6-9

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 provid-ed 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.

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 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 Positive76% (52/59)

LAC Positive Sera (n=59)

aPS/PT IgG or IgM Negative24% (7/59)

6/7 aPL positive 5/7 anti2GPI positive4/7 aCL/anti2GPI positive

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.

In fact, IgA aPL appear to rather identify subgroups of patients, such as Afro-Americans or pure obstet-ric 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.

Conclusions

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

59 LAC positive sera have been tested. 52/59 (76%) resulted positive for IgG or IgM anti-PS/PT antibodiesOnly 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 antibodiesMost of the positivities for anti-PS/PT antibodies were at high titres and 44.1% of them were of the IgM isotypeOnly 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)



Background and Purpose

Current diagnostic criteria recommend elevat-ed titers of anti-Cardiolipin (aCL) and/or anti-2Glycoprotein (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 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 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.

Objectives

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), anti-2GPI (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 commer-cial 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.

1Aguilar-Valenzuela R, 1Seif AM, 2Alarcn GS, 1Martnez-Martnez LA, 1Dang N, 1Papalardo E, 2Liu J, 4Vila LM, 1Najam S, 1McNearney T, 1Gonzalez EB, 6Binder W, 5Teodorescu M, 3Reveille JD, 1Pierangeli SS1 University of Texas Medical Branch, Galveston, TX; 2University of Alabama at Birmingham, Birmingham, AL; 3University 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.

Isolated elevated levels of IgA-anti-2GPI are associated with clinical manifestations of the antiphospholipid syndrome

O B J E C T I V E S

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


A significant number of subjects in the two groups had at least one APS-related clinical manifestation, that included:

Two of these samples were also LAC positive.

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%).

Conclusions

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.

It may be therefore recommended to test for IgA 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 to test for IgA 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 Immunology 2003;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 AT E D C L I N I C A L M A N I F E S T AT I O N S

Deep vein thromboses

Pregnancy lossesOther 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.


W. L. Binder, S. Lewis and Z. ShumsINOVA Diagnostics, San Diego, CA, USA

Clinical significance of IgG and IgM autoantibodies that target the complex of phosphatidylserine and prothrombin (PS/PT)

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.

Prothrombin (factor II) is another phospholipid binding protein with procoagulant activity.

A number of groups have definitively shown that antibodies targeting the complex of phosphatidyl-serine (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

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 OFPS/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 of LAC

High Specificity


Table 1

------------------ NUMBER POSITIVE (%) --------------------

PAT I E N T G R O U P No SAMPLES PS/PT IgGPOSITIVE

PS/PT IgMPOSITIVE

PS/PT IgG AND/OR IgM

POSITIVENormals 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%)

Infectious 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 from an external and an internal study

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.

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.

Amengual O. et al., 1. Arthritis Rheum 2003;48:886-895.DAgnillo P. et al., 2. The Journal of Immunology 2003;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.


LAC POSITIVESAMPLES

PS/PT IgG(pos>30)

PS/PT IgM(pos>30)

1 141 81.22 146 61.63 139 60.24 151 67.65 144 54.46 112 39.77 213 11.58 217 10.49 21.2 98.7

10 125.6 51.611 51.3 40612 224 16.613 97 13814 157 13215 152 65.316 173 30517 147 25318 136 63.719 15.5 11420 88.3 109

Performance of PS/PT IgG and PS/PT IgM ELISA with 20 LAC positive samples

and 4 borderline positive samples

Relative Performance to GPI IgG

IgG PS/PT

+ -

IgG GPI + 38 10**

- 16* 116

* 1 of the 16 was LAC positive and the other 15 were APS patients

** All 10 were from the APS group

Relative Performance to GPI IgM

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.

LAC BORDERLINESAMPLES

PS/PT IgG(pos>30)

PS/PT IgM(pos>30)

1 22.1 132.62 75.2 7.83 217.5 21.64 278.5 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%.

IgM PS/PT

+ -

IgM GPI + 28 7

- 25 120

1 of these was a normal and 6 were from the APS group

22 were from the APS group and 3 were LAC positives


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.

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 positivesIgG and IgM autoantibodies that react with a physiologic complex of phosphatidylserine and prothrombin are sensitive markers for anti-phospholipid syndromePS/PT IgG and IgM ELISA detects most APS patients including many that are ACA, 2GPI, LAC negativeThe 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 erythematosus or other lupus-like diseases.


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

Antiphospholipid syndrome (APS) is a disorder characterized by recurrent thrombosis and/or fetal loss associated with characteristic laboratory abnormalities.

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).

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.

Results

All 14 negative samples were negative by all ACA IgG assays.

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

All tested commercial ELISAs had good reproducibility and all strong positive samples were positive by all assaysThere 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 significanceThe 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 monitoringA 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 kitThe standardization of ACA IgG ELISAs remains an unsolved problem

All trademarks are the properties of their respective companies


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 ACA IgG (GPL)

IN-HOUSE METHOD

QUANTA Lite

Orgentec Reaads Varelisa EliA

29* 15** 10** 23** 15** 15**30 15 3 11 6 330 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-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)

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 good reproducibility and all strong positive samples were positive by all assays.


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 f i e d c a r d i o l i p i n c u t o f f

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 f 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 f i e d c a r d i o l i p i n5 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 5Q 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 f 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 L

p o s > 2 0 A P L

QUANTA Lite 2 GPI


7 0 8 6 6 0Q U A N T A 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 Ic u t o f f

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 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 I5 p o i n t s t a n d a r d

c u r v en e g < 2 0 p o s > 2 0

7 0 8 6 7 0Q U A N T A 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 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 5Q 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 f i e d 2 - g l y c o p r o t e i n I 5 p o i n t s t a n d a r d

c u r v en e g < 2 0 p o s > 2 0

INOVA Diagnostics, Inc. offers a wide range of Antiphospholipid Syndrome (APS) ELISA test kits.

INOVA Diagnostics APS ELISA test kits

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.

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.

For more information on INOVA Diagnostics complete product offerings visit www.inovadx.com


HUMAN ANTI-PHOSPHATIDYLSERINE


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 1 0 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 f 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 1 1

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 f a c t o r5 p o i n t s t a n d a r d

c u r v en 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 1 2

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 f a c t o r5 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


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 l s e r i n e a n d P r o t h r o m b i n5 p o i n t s t a n d a r d

c u r v en 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 l s e r i n e a n d P r o t h r o m b i n5 p o i n t s t a n d a r d

c u r v en 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 f o r t h e q u a l i t y c o n t r o l o f 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 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

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 f o r t h e q u a l i t y c o n t r o l o f 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 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

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) 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.

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).

690120 February 10 Rev. 0

Published by

INOVA Diagnostics, Inc.

9900 Old Grove Road

San Diego, CA 92131

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

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

Fax (858) 586-9911

[email protected]

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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