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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:


2

Where we are standing and where we are going.

p6 Isolated elevated levels of IgA-Anti- GPI


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 p14 INOVA Diagnostics APS ELISA test kits

right result? Comparison of five different commercial 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. 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.
GPL

Ta b l e 1

TEST VARIABILITY IN aCL-IgG AND IgM ELISA KITS


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)

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

MPL B

B1

M4

B3

B5

M1

M2

B2

B4

M3

M5

IgM aCL ELISA (results in MPL units)

1.

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

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.

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 More than one criterion present (any combination) Lupus Anticoagulant present alone Anti-cardiolipin antibody present alone 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

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 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 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.

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 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. 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.

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 Negative 24% (7/59) aPS/PT IgG or IgM Positive 76% (52/59)
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 |
5

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.
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 suspected.

are negative and APS is

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 |

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. Prothrombin (factor II) is another phospholipid binding protein with procoagulant activity. 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 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 Detects many ACA and 2GPI negative APS patients Close approximation of LAC High Specificity

| INOVA NEWS No. 6

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 ------------------ 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

PAT I E N T G R O U P 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) Syphilis Actin Antibody Positive H. Pylori Positive

No SAMPLES 247 24 71 6 2 2 5 8 14 12 1 2

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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PS/PT IgG (pos>30) 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 PS/PT IgM (pos>30) 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 LAC BORDERLINE SAMPLES 1 2 3 4 PS/PT IgG (pos>30) 22.1 75.2 217.5 278.5 PS/PT IgM (pos>30) 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 PS/PT + IgG GPI + 38 16* 10** 116

Relative Performance to GPI IgM IgM PS/PT + IgM GPI + 28 25 7 120

* 1 of the 16 was LAC positive and the other 15 were APS patients ** All 10 were from the APS group

1 of these was a normal and 6 were from the APS group 22 were from the APS group and 3 were LAC positives

10 | INOVA NEWS No. 6

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 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 lupus-like diseases.

erythematosus or other

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 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 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 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

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

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 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 10** 3 72 2 4 3 13 4 3 4 3 163 138 1256 856 Reaads 23** 11 51 8 10 15 33 18 4 9 45 166 78 815 551 Varelisa 15** 6 25 2 3 3 17 5 1 3 7 125 156 405 387 EliA 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)
Table 2

IN-HOUSE METHOD Positive (n) Negative (n) 8 6

QUANTA Lite 8 6

Orgentec 5 9

Reaads 6 8

Varelisa 4 10

EliA 6 8

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.
708620

DESCRIPTION
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 Q U A N TA L i t e A C A I g G I I I 2 Antigen:Purified cardiolipin Q U A N TA L i t e A C A I g M I I I 3 Antigen: Purified cardiolipin Q U A N TA L i t e A C A I g A I I I 4 Antigen: Purified cardiolipin

CALIBRATION
cutoff 5 point standard cur ve 5 point standard cur ve 5 point standard cur ve

INTERPRETATION
neg < decision point pos > decision point neg <15 GPL equiv 15-20 GPL pos >20 GPL neg <12.5 MPL equiv 12.5-20 MPL pos >20 MPL neg <12 APL equiv 12-20 APL pos >20 APL

708625

708630

708635

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.
708660

DESCRIPTION
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 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 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 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

CALIBRATION
cutoff 5 point standard cur ve 5 point standard cur ve 5 point standard cur ve

INTERPRETATION
neg < decision point pos > decision point neg < 20 pos > 20 neg < 20 pos > 20 neg < 20 pos > 20

708665

708670

708675

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.
708835

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

CALIBRATION
5 point standard cur ve 5 point standard cur ve

INTERPRETATION
neg < 30 Units pos > 30 Units neg < 30 Units pos > 30 Units

708845

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.
704625

DESCRIPTION
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 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 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

CALIBRATION
5 point standard cur ve 5 point standard cur ve 5 point standard cur ve

INTERPRETATION
neg <11 GPS U/mL pos > 11 GPS U/mL neg < 25 MPS U/mL pos > 25 MPS U/mL neg < 20 APS U/mL pos > 20 APS U/mL

704630

704635

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.
508668

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 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 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 info@inovadx.com www.inovadx.com

Authors Gary L. Norman, PhD Philipp von Landenberg, MD, PhD Mareike Lorenz, PhD Pier Luigi Meroni, MD, PhD Francesca Pregnolato, PhD Wally Binder, PhD Silvia S.Pierangeli, MD, PhD Kaija Javela, PhD Editor LeoPoldine Steindl Graphic Design Michael Kulwiec DesignLab

690120 February 10 Rev. 0

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