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Anti-cardiolipin antibodies (ACA), rst identied in syphilis patients as a result o the

presence o cardiolipin in the bovine heart extract used or the VDRL syphilis test, haveevolved into one o the primary components o antiphospholipid syndrome (APS)

diagnosis. The realization that the actual target o many antiphospholipid antibodies (aPL)

was the phospholipid binding protein 2GPI bound to cardiolipin antigen immobilized on

the ELISA well led to ELISA assays using puried 2GPI as the assay substrate. While some

labs continue to test or only IgG and IgM 2GPI isotypes, evidence suggests that

2GPI

IgA antibodies are associated with increased risk o adverse cardiovascular, thrombotic,

and pregnancy-associated events. In this issue o the INOVA newsletter, Aguilar-Valenzuela

et al. show some SLE patients are only positive or 2GPI IgA antibodies and recommend

2GPI IgA antibody testing in individuals suspected o 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 o the Sapporo monoclonal standards

to improve standardization and calibration o ACA kits. Javela and Mustonen describe

evaluation o ve commercial ACA IgG ELISA kits and document discouraging variation in

the interpretation o low and moderately positive specimens between kits.

The signicance o single and multiple ACA, 2GPI, and LAC positivities, as well as the

magnitude o the positivity, is discussed by Meroni and Pregnolato. Patients make

antibodies to a variety o phospholipid/protein targets, resulting in a heterogeneous

group o patient antibodies. Detection o 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 o phosphatidylserine /prothrombin, are described by Binder et al. Measurement

o both PS/PT IgG and IgM antibodies detected most LAC-positive

patients and close to 70% o the APS patients and identied some

APS patients missed by the conventional prole o ACA, 2GPI, and

LAC assays.

Antiphospholipid testing is evolving. New assays will allow ner

stratication o 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-phospholipdiagnostic s: Is there room or improvement o

standardization?

p3 High discrepancies in anti-phospholipid lare seen between laboratories

p4 Anti-phospholipid antibody detection:Where we are standing and where we are going.

p6 Isolated elevated levels o IgA-Anti-2GPI

are associated with clinical maniestations o the

antiphospholipid syndrome

p8 Clinical signiicance o IgG and IgMautoantibodies that target the complex o

phosphatidylserine and prothrombin (PS/PT)

p12 Anti-cardiolipin IgG ELISAs What is tright result? Comparison o ive dierent comme

test kits

p14 INOVA Diagnostics APS ELISA test kits

Gary L. Norman PhD

Senior Scientist

INOVA Diagnostics, Inc.


2/162 | INOVA NEWS No. 6

The anti-phospholipid syndrome (APS) is an

autoimmune disease which is characterized by

dierent clinical, haematological and serological

maniestations. These include venous and/or arteri-

al thrombosis, recurrent etal loss and low platelet

counts. Accordingly, the binding specicities o

anti-phospholipids (aPL) appear to be as hetero-

geneous as the clinical maniestations associated

with them.

Since the typical antigens are cardiolipin and

phosphatidylserine, it was thought that aPL can

be distinguished by their phospholipid specicity

alone.

Additionally, it could be shown that most o the aPLs

require a protein coactor to bind to their antigen.

One o these coactors is the apolipoprotein beta2

glycoprotein 1 (2GPI) with a postulated unction in

the lupus anticoagulant activity.

The clinical diagnosis o 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 dierent laboratories and assays even i

the laboratories are using the same assay.1

Discrepancies in results are even higher i labora-

tories use dierent brands o assays, as a result o

several variable actors (see table 1).

To overcome some o these problems, the use o

human and chimeric monoclonal antibodies or

standardization and calibration o the dierent

kits was introduced with the HCAL IgG Sapporo

Standard.2

However, using dierent monoclonal IgG and IgM

antibodies directed to 2GPI and/or cardiolipin

still does not lead to a reasonable agreement in

dierent test systems.

This might be due to the act that

monoclonal antibodies represent only one speci-

city to a certain epitope with a dened (high)

avidity, or does not contain the best match to theantibodies ound in the sera o antiphospholipid

patients.

Using monoclonal antibodies in aPL testing

especially in the case o cardiolipin and 2GP

diagnostics is not the be-all and end-all.

Remaining inconsistencies limit the clinical utility

and inter-laboratory transerability which in conclu-

sion indicates that the standardization regardingaCL testing still needs to be improved.

We are looking orward to the new upcoming

developments rom the companies in this highly

competitive eld o diagnostics.

Philipp von Landenberg

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

Monoclonal antibodies in anti-phospholipid diagnostics:

Is there room or improvement o 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 L T 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

Manuacturing and calibration o the assay

Source and purity o antigens

Specicity and isotype o detection antibodies

Heterogeneous avidity spectrum o antibodies

A variety o actors result in discrepancies between laboratories who usediferent brands o aCL assays.


3/16 TESTING FOR ANTIPHOSPHOLIPID SYNDROME | 3

Some years ago, a cross-laboratory evalu-

ation was perormed by the European

Antiphospholipid Forum. Basically, a set o

ten serum samples were tested at 24 dierent

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 ound or

aCL-IgG ELISA test kits as well as or the

aCL-IgM ELISA (Table 1). Some values in the

cut o range appeared to be positive in one

test kit but normal with another.

With the introduction o one IgG and one IgM

monoclonal antibody (HCAL and EY2C9) as

putative standards, a progressive decrease in

the variability o the values was obtained.

Even with monoclonal standardization the

dilemma o inconsistent comparability o

results still remains, since not all routine

laboratories are ollowing consensus

recommendations.

Although increasingly more laboratories and

manuacturers utilize standardized, uniorm

materials and procedures the relatively high

variability or a-PL antibodies assays are an

ongoing issue o discussion.

Mareike Lorenz

Institute o Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University,

Lagenbeckstrasse 1, 55131 Mainz, Germany

High discrepancies in anti-phospholipid levels are seen between laboratorie

Tincani A et al.,1. Thromb Haemost2001; 86:57583.

325

300

275

250

225

200

175

150

125

100

75

50

25

0

B1 M4 B3 B5 M1 M2 B2 B4 M3 M5A

GPL

IgG aCL ELISA (results in GPL units)

325

300

275

250

225

200

175

150

125

100

75

50

25

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


4/16

The diagnostic and prognostic

antiphospholipid prole

According to the revised classication crite-

ria, the positivity or lupus anticoagulant (LAC),

anti-cardiolipin (aCL) and anti-beta2 glycopro-

tein 1 (2GPI) antibodies are ormal classication

laboratory criteria. Hence, a single positivity stable

over time (at least 12 weeks) is sufcient to classi-

y a symptomatic patient as suering rom 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 specicity and the predictive value o each

single test and in particular o their combination

are not exactly the same. This is particularly true

taking into account that the most requent clini-

cal maniestations o the syndrome (i.e. deep vein

thrombosis or early miscarriages) are not speci-ic and rather common in the general population.

A sub-classication o patients according to their

positivities has been suggested in table 1.

The predictive value has been suggested to be the

highest or the category I.1

The identication o the risk prole oers the

rationale or both the secondary prophylactic

therapy (i.e. in order to prevent recurrences) and

the primary prophylaxis to avoid the occurrence o

the clinical events in the antiphospholipid antibody

(aPL) positive asymptomatic subjects.

While the clinical approach is not problematic or

patients displaying several positivities and/or high

aPL titres, the situation is dierent 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 specicity was suggested

to be related to the act that LAC is mediated by

antibodies against plasma proteins (anti-2GPI and

prothrombin) bound to anionic PL and ultimately

aecting their availability or the coagulation

cascade. To display this eect the antibodies need

to be at an elevated protein concentration and todisplay high avidity.

However, the clinical value o the presence o LAC

as an isolated assay or in asymptomatic subjects o

at low potency has been recently questioned.5

To improve specicity, the revised classica

tion criteria or the aCL assay require both a

stable positivity and a positivity threshold o 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-2GP

assays. A comparable high threshold has not been

suggested or the anti-2GPI assay since the norma

cut o should be calculated on the 99th percentile

o 50 normal subjects1. Hence, the sensitivity o the

anti-2GPI assay is high. Such a sensitivity combined

with its wider use in the laboratories makes the

possibility o isolated elevated anti-2GPI antibod-

ies more requent.

Pier Luigi Meroni, *Francesca Pregnolato

Division o Rheumatology Ist. Gaetano Pini, Dept. Internal Medicine University o 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 A T O R Y C R I T E R I A S A T 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

Classication o APS patients according to the positivity or the

antiphopsholipid assays

4 | INOVA NEWS No. 6


5/16

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

useul aPL assays?

There are preliminary results suggesting additional

assays could improve our diagnostic and prognostic

power as a second level o aPL testing.

This could be the case or anti-prothrombin

antibodies (anti-PT) as a second level assay to

conrm 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

unctional assay, it has also been suggested that

an anti-PT test may or may not conrm equivo-

cal unctional LAC. Although the heterogeneity o

the methods to detect anti-PT antibodies is still amatter o 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 maniestations.6-9

We recently analyzed a selected series o samples

rom APS patients and controls by using a new

ELISA assay or anti-PS/PT detection (kindly provid-

ed by Dr. W. Binder INOVA Diagnostics, USA).

Figure 1 reports our preliminary results showing agood specicity o the assay and correlation with

the clinical maniestations.

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

o Lupus Anti-coagulant (LAC) positive samples rom APS patients

aPS/PT IgG or

IgM Positive

76% (52/59)

LAC Positive Sera (n=59)

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

6/7 aPL positive

5/7 anti2GPI positive

4/7 aCL/anti2GPI positive

Tests detecting aCL IgA and anti-2GPI IgA

antibodies are available but are not ormally

included into the revised criteria because o the

lack o evidence that the assay may improve the

whole diagnostic power.

In act, IgA aPL appear to rather identiy subgroups

o patients, such as Aro-Americans or pure obstet-ric APS. However, the search or IgA aPL may be

useul in order to conrm the diagnosis o APS in

the case o an isolated positivity or a borderline

result in the other solid-phase assays.

Conclusions

The panel o aPL tests is still evolving and appar

ently, like other autoantibody amilies, more than

one assay and the use o second level tests appear

useul to improve our diagnostic and prognosticpower.

59 LAC positive sera have been tested. 52/59 (76%) resulted positive

or 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 or anti-PS/PT antibodie

Most o the positivities or anti-PS/PT antibodies were at high titres

and 44.1% o them were o the IgM isotype

Only 2 out o 40 pathological aPL negative control sera (30 with

autoimmune diseases, 10 with inectious diseases) displayed a low

positivity (1 IgG and 1 IgM)

The cut of was calculated on 91 NHS samples (43 AU or IgG and 44

AU or IgM)

TESTING FOR ANTIPHOSPHOLIPID SYNDROME | 5



Background and Purpose

Current diagnostic criteria recommend elevat-

ed titers o anti-Cardiolipin (aCL) and/or anti-

2Glycoprotein (

2GPI) antibody IgG or IgM by

ELISA and/or lupus anticoagulant (LAC) to conrmantiphospholipid syndrome (APS).

IgA aCL antibodies are ound more requently in

Aro-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 signicance remained elusive.

Several studies report a possible association

between elevated IgA anti-2GPI titers and APS-likemaniestations.

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 classied

as APS due the absence o aCL positive test.

Anti-2GPI IgA antibodies are more prevalent in

patients with SLE.

We recently reported ve isolated cases o exclusive

IgA anti-2GPI antibody sero-positivity with

concomitant APS clinical maniestations.

Objectives

Patients

Anti-phospholipid seropositivity was examined in

2799 SLE sera, whereo 599 samples came rom a

multi-ethnic, multi-center cohort (LUMINA) and

2200 samples were reerred to our laboratory

(APLS) or 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

rom INOVA Diagnostics, and an in-house protocol

IgA-2GPI titers were determined by two commer

cial ELISA tests, one rom INOVA Diagnostics..Results

Out o the 2799 samples, 50 samples were positive

exclusively or IgA-2GPI in at least one kit.

1Agui la r-Val en zu el a R, 1Seif AM, 2Al ar c n GS , 1Martnez-Martnez LA, 1Dang N, 1Papalardo E2Liu J, 4Vila LM, 1Najam S, 1McNearney T, 1Gonzalez EB, 6Binder W, 5Teodorescu M, 3Reveille JD1Pierangeli SS

1 University o Texas Medical Branch, Galveston, TX; 2University o Alabama at Birmingham, Birmingham, AL3University o Texas-Houston Heath Sciences Center, Houston, TX; 4University o Puerto Rico Medical Sciences

Campus, San Juan, PR; 5Theratest Laboratories, Lombard, IL; 6INOVA Diagnostics, San Diego, CA.

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

clinical maniestations o the antiphospholipid syndrome

O B J E C T I V E S

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

antibody positivity in a large cohort o patients with SLE

and in patients suspected o having APS

To correlate IgA-anti-2GPI antibody positivity with APS

associated clinical maniestations



A signicant number o subjects in the two groupshad at least one APS-related clinical maniestation,

that included:

Two o these samples were also LAC positive.

84% o samples were positive with the INOVA Kit,90% o 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 identiy additional patients

who have clinical eatures o APS but who do not

meet current diagnostic criteria.

It may be thereore recommended to test orIgA

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 totest forIgA

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 Immunology2003;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 A T E D C L I N I C A L M A N I F E S T A T I O N S

Deep vein thromboses

Pregnancy losses

Other APS-related pregnancy complications

Pulmonary inarctions, strokes, seizures, myocardial

inarctions

Thrombocytopenia

Non classical APS maniestations:

Skin ulcers, pulmonary hypertension, livedo reticularis,

cardiac valvular disease, seizures and migraines.



W. L. Binder, S. Lewis and Z. Shums

INOVA Diagnostics, San Diego, CA, USA

Clinical signicance o IgG and IgM autoantibodies that target

the complex o phosphatidylserine and prothrombin (PS/PT)

BackgroundAntiphospholipid antibodies represent a large

heterogeneous group o immunoglobulins o

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 or aiding in diagnosis o

antiphospholipid syndrome (APS).

More and more laboratories are now including

tests or detecting antibodies directed against

phospholipid binding proteins, the best studied o

which is 2GPI.

Prothrombin (actor II) is another phospholipid

binding protein with procoagulant activity.

A number o groups have denitively shown that

antibodies targeting the complex o phosphatidyl-

serine (PS) and prothrombin (PT) have signicant

clinical relevance due to their strong correlations

with clinical eatures o APS and with the presence

o 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 useul sensitivity o

APS and have high specicity. Their inclusion into

the laboratory criteria or classication o 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 o LAC

High Speciicity



Table 1

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

PAT I E N T G R O U P No SAMPLESPS/PT IgG

POSITIVE

PS/PT IgM

POSITIVE

PS/PT IgG

AND/OR IgM

POSITIVE

Normals 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%)

Inectious 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 rom an external and an internal study

Specic perormance characteristics o QUANTA Lite aPS/PT IgG and QUANTA Lite aPS/PT IgM kits

that detect the complex o phosphatidylserine and prothrombin (PS/PT) autoantibodies

Assay Characteristics

Antigen on solid phase is a layer o phosphatidylserine and human prothrombin, coated in the presence o

Ca++. Standard ELISA ormat 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 or IgG

and IgM antibodies to PS/PT. These results were used to calculate perormance characteristics and the new

assays were compared to traditional anti-GPI and LAC assays. Results are tabulated in Table 1.

Forty eight o the 71 APS patients (67.6%) were PS/PT positive and many o these individuals were ound to be negative using more

traditional assays such as anti-GPI and LAC.

Only 7 o 247 normals and 1 o the 52 disease controls were ound to be positive or either IgG or IgM PS/PT antibodies or a

combined specicity o 97.3% (8/299).

The assays were ound to have high inter and intra run precision.

Equivalent results were obtained with either serum or citrated plasma or both assays.

Amengual O. et al.,1. Arthritis Rheum 2003;48:886-895.DAgnillo P. et al.,2. The Journal of Immunology2003;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.


10/16



Most o the discrepant results are due to the higher

sensitivity o the IgG and IgM PS/PT kits or both

the LAC positives and especially the APS patients

although there were APS patients that were 2GPIpositive yet PS/PT negative.

The PS/PT IgG plus IgM kits detected all LAC

positive samples and most o the APS patients. It

was noticed that the vast majority o APS patients

were positive or PS/PT and/or 2GPI.

Conclusions

PS/PT IgG and IgM ELISA appears to be

very specic and detects a majority o LAC

positives

IgG and IgM autoantibodies that react with a

physiologic complex o phosphatidylserine

and prothrombin are sensitive markers or

anti-phospholipid syndrome

PS/PT IgG and IgM ELISA detects most APS

patients including many that are ACA, 2GPI,

LAC negativeThe tests exhibit high specicity 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 o ve diferent commercial test kits

Ja vela K. an d Mu st on en P.

Finnish Red Cross Blood Service, Helsinki, Finland

BackgroundAntiphospholipid syndrome (APS) is a disorder

characterized by recurrent thrombosis and/or

etal loss associated with characteristic laboratory

abnormalities.

Patients suspected to have APS should

be screened or anticardiolipin antibodies

(ACA), 2-glycoprotein 1 antibodies and lupus

anticoagulant.

More than 45 commercial kits or ACA detection

are available (n=45 in ECAT reerence list).

Objective

Evaluation o ve dierent commercially available

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

method.

Methods and Materials

Five dierent commercial ACA IgG assays were

chosen or 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 o all commercial ELISA assays are

calibrated against reerence sera rom E.N. Harris,Louisville.

Our in-house method was used as a reerence

method.

Ten positive, 4 strong positive and 14 negative

samples previously measured by our in-house

method were analyzed.

ResultsAll 14 negative samples were negative by all ACA

IgG assays.

The number o positive samples varied when the

cut-o o manuacturer (Table 1) and the laboratory

classication criteria or APS (ACA IgG results >40

GPL (Table 2) were used .

The Variation Coefcients o all assays were good

in the cut o range below 6.8% or all assays.

Conclusions

All tested commercial ELISAs had good

reproducibility and all strong positive samples

were positive by all assays

There was a signicant discrepancy between

assays when borderline, low positive or

intermediately positive ACA IgG samples were

analyzed

The correct recognition o high but also medium

titer ACA IgG is o high clinical signicance

The selection between reagents has to be

made or the method with the best correlation

to the existing one, since APL antibodies o APS

patients are repeatedly tested or monitoring

A potential problem will be i serum samples

are sent to a dierent laboratory which eitheruse a home made method as well, or a dierent

commercial test kit

The standardization o ACA IgG ELISAs remains

an unsolved problem

All trademarks are the properties of their respective companies



ACA IgG results14 positive samples measured by ve commercial ELISA assays. The 14

negative samples were negative by all ACA IgG assays.

Table 1

ACA IgG (GPL)

IN-HOUSE

METHOD

QUANTA

LiteOrgentec Reaads Varelisa EliA

29* 15** 10** 23** 15** 15**30 15 3 11 6 3

30 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-o value o the in-house method; **Cut-o value set by the manuacturer

The number o positive samples according to laboratory classication

criteria or 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 goo

reproducibility

and all strong

positive sampl

were positive b

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 i e d c a r d i o l i p i nc u t o

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 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



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 5

Q U A N T A L i t e A C A I g A I I I 4



c u r v e

n e g < 1 2 A P L

e q u i v 1 2 - 2 0 A P Lp 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

2G 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 i e d 2- g l y c o p r o te i n I

c u t o 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

2G P I I g G E L I S A 6



c u r v e

n e g < 2 0

p o s > 2 0

7 0 8 6 7 0Q U A N T A L i t e

2G P I I g M E L I S A 7



c u r v e

n e g < 2 0

p o s > 2 0

7 0 8 6 7 5

Q U A N T A L i t e 2

G P I I g A E L I S A 8



c u r v e

n e g < 2 0

p o s > 2 0

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

INOVA Diagnostics APS ELISA test kits

1. QUANTA Lite ACA Screen III is an enzyme-linked immunosorbe nt assay (ELISA) or the qualitative detection o cardiolipin antibod ies in human serum. The presence o cardiolipin antibodies can be used i

conjunction with clinical ndings and other laboratory tests to aid in assessing the risk o thrombosis in individuals with systemic lupus erythematosus (SLE) or lupus-like disorders.

2. QUANTA Lite ACA IgG III is an enzyme-l inked immunosorbent assay (ELISA) or the semi- quantitative detectio n o IgG cardiolipin antibodies in human serum. The presence o cardiolipin antibodies can be used i

conjunction with clinical ndings and other laboratory tests to aid in assessing the risk o 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) or the semi-quanti tative detection o IgM cardiolipin antibodies in human serum. The presence o cardiolipin antibodies can be used i


4. QUANTA Lite ACA IgA III is an enzyme-l inked immunosorbent assay (ELISA) or the semi- quantitative detectio n o IgA cardiolipin antibodies in human serum. The presence o cardiolipin antibodies can be used i


5. QUANTA Lite 2 GPI Screen is an enzyme-li nked immunosorbent assay (ELISA) or the qualitative detection o IgG, IgM and IgA antibodies to 2 glycoprotein I (2 GPI) in human serum. 2 GPI antibodies are use

as an aid in the diagnosis o 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 immunosor bent assay (ELISA) or the semi- quantitative detect ion o 2 GPI IgG antibodies in human serum. The presence o 2 GPI IgG antibodies can be used i

conjunction with clinical ndings and other laboratory tests to aid in the diagnosis o 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 immunosorbe nt assay (ELISA) or the semi-quantitative detect ion o 2 GPI IgM antibodies in human serum. The presence o 2 GPI IgM antibodies can be used i



8. QUANTA Lite 2

GPI IgA is an enzyme-linked immunosorbent assay (ELISA) or the semi-quantitative detection o 2

GPI IgA antibodies in human serum. The presence o 2GPI IgA antibodies can be used i




15/16

For more inormation on INOVA Diagnostics

complete product oferings visit www.inovadx.com

TESTING FOR ANTIPHOSPHOLIPID SYNDROME | 15

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 10

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 a c t o r


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 11



c u r v e

n 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 12



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 ls e r i n e a n d P r o t h r om b i n


c u r v e

n 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 ls e r i n e a n d P r o t h r om b i n


c u r v e

n 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

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

o r t h e q u a l i t y c o n t r o l o 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 ot e i n I (

2G 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) or the detection o IgG and IgM

class antibodies to phosphatidylserine/prothrombin complex (PS/PT) in serum or plasma. For use as an aid in the diagnosis o 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 ndings.

10. This assay is intended or the in-vitro measurement o IgG antiphosphatidylserine antibodies in human serum, as an aid in the diagnosis o anti-phospholipid syndrome (APS).

11. This assay is intended or the in-vitro measurement o IgM anti-phosphatidylserine antibodies in human serum, as an aid in the diagnosis o anti-phospholipid syndrome (APS).

12. This assay is intended or the in-vitro measurement o IgA anti-phosphatidylserine antibodies in human serum, as an aid in the diagnosis o anti-phospholipid syndrome (APS).


16/16

Published by

INOVA Diagnostics, Inc.

9900 Old Grove Road

San Diego, CA 92131

toll ree: (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

INOVA NEWSLETTERS ON OTHER AUTOIMMUNE TESTING TOPICS ARE AVAILABLE UPON REQUEST

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Third Generation CCP ELISA in Rheumatoid Arthritis Serology (No. 4)

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