Calibration Issues – Phospholipid Antibodies and ANCA

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Calibration Issues –Phospholipid Antibodies and ANCA

Workshops in Portgual 2006 Peter WhiteUK NEQAS for Immunology, Allergy and ImmunochemistryNorthern General Hospital, Sheffield

Neutrophil Cytoplasmic Antibodies - 2006

UK Participants 108Non-UK Participants 308

Web entry system: 35%Response rate: Approx 80%

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Pos Neg Equivocal

C. ANCA 4 5 1P. ANCA 1 9

PR3 5 5MPO 1 9


C. ANCA IIF TitreMinimum 10 users

1264716918010921All Manufacturers

065206510632063106120611Manufacturer

1324416620712723Inova

15410515018511925In House

240120200160800Immunoconcepts

1865617630414340Immco

96361681187515Euroimmun

60401081206826Bio-Rad/Kallestad/S

1515202020BioDiagnostics

106371441686418Binding Site

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Inova

In House

Immunoconcepts

Immco

Euroimmun

Bio-Rad/Kallestad/S

Binding Site

All Manufacturers

Manufacturer

80282920695223

132020206425258

000013300

404040201104040

14121415851416

202626261312026

22242416892018

40212719882821

0652063206310622062106120611

P. ANCA IIF TitreMinimum 10 users


11128105117102Wieslab

1011154011Phadia Varelisa

1421239717Phadia Elia

2418305321ORGen Tec

4234568150Inova

35454813633 In House

29264310030Hycor Biomedical

472612524145Grifols AESKU

1819255015Fenning

884610814290Euroimmun

34475814444Diamedix

172123242362161BMD

68578310659BioDiagnostics

2616335625Binding Site

244152Axis Shield/Diastat


06520651063206310612Manufacturer

PR3 Units/ml

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238810Wieslab

13211Phadia Varelisa

13211Phadia Elia

21322ORGen Tec

42334Inova

32955In House

22811Hycor Biomedical

11900Grifols AESKU

32512Fenning

56956Euroimmun

11611Diamedix

06700BMD

22533BioDiagnostics

21344Binding Site

2721Axis Shield/Diastat


0652062106120611Manufacturer

MPO Units/ml

-20

0

20

40

60

80

100

120

140

160

0 10 20 30 40 50 60 70 80 90 100

M LT M

Axis Shield

Binding Si te

BioDiagnosti cs

Eur oimmun

Inova

ORGenTec

Phadia El ia

Phadia Var el isa

Wieslab

Neutrophil Cytoplasmic Antibodies –PR3 ‘Linearity’/Calibration

5

0

20

40

60

80

100

120

140

160

180

200

220

0 20 40 60 80 100 120

M LT M

Axis Shield

Binding Sit e

BioDiagnost ics

Inova

ORGenTec

Phadia Elia

Phadia Varelisa

Wieslab

Euroimmun

Neutrophil Cytoplasmic Antibodies –MPO ‘Linearity’/Calibration

Phospholipid Antibodies - 2006

UK Participants 135Non-UK Participants 255

Response rate: approx 83%

Incorporates IgG and IgM assays for antibodies to

Cardiolipinβ2 Glycoprotein 1Phosphatidylserine

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Some laboratories using ‘in house’ELISAs

but

most use one the commercially available products


Commercial assays usually calibrated with ‘Harris Standards’Louisville, KentuckyProvided as a series of ‘calibrators’ to construct a calibration curvebutthere have been successive versions of the Harris Standard which are not cross referencedDifferent versions have different values of GPLU/ml and MPLU/ml

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Scheme standards (IgG) produced in an attempt to allow analysis of EQA data in common unitage

Calibrated against Harris version of the time (? Version 1) in GPLU/ml

Current standard 97/514 produced from the same single donor source as original material and calibrated carefully from it –provides continuity of calibration and antibody specificity

Used by some manufacturers for calibrating their commercial assays


Generally acknowledged that the Scheme standards 97/656 and 97/514 have not achieved the degree of uniformity which had been expected

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IUIS/WHO/AF/CDC Committee for the Standardisation of Autoantibodies

Initiative to produce monoclonal IgG and IgM anticardiolipinstandards

Proposal to proceed with the release of the standards approved

But

Some confusion as to whether they will only be suitable for quantitation of anti-β2-GP1


Current position appears to be that lypholised standards have been produced and are scheduled to be released by CDC Atlanta but may already be purchased from Inova

Plans to quantitate and assign values as µg/ml rather than GPL or MPL units

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Intended Use

97/514 is a serum standard for cardiolipinantibody (IgG) for use in performance evaluation only in the UK NEQAS.

97/514 is prepared from human serum obtained from volunteer donors.


Quality Control

Appropriate internal controls should be included in any assays performed where possible. If controls are out of range then potential problems with the assay should be investigated before releasing any results. The scheme standard should not be used as a primary internal control material

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Instructions for reconstitution and storage

Upon arrival the material shall be stored at below -18°C. To make it ready for use, the freeze dried standard has to be reconstituted according to the following procedure by an appropriately experienced member of staff –this is a critical step and an error will impact on your EQA returns


Product : 97/514 NEQAS scheme standard (lyophilised)

: Reconstitute in 1mL of distilled water

Lot : 001

Expiry Date : 2007-03

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Assigned value of the standard

Positive test result for cardiolipin antibody (IgG) – assigned value 61 GPLU/ml

Autoantibodies of other specificities may be present


97/514 should be assayed in parallel with the two UK NEQAS samples sent on each distribution. Your assay values can be converted to 97/514 related values by following the worked example

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If the value obtained for 97/514 in the assay run was 65 kit units/ml and the value for the QA sample was 78 kit units/ml then:

61 x 78 = 73 (97/514) GPLU/ml65


Interpretation

The following reference intervals should be used for the semi quantitative interpretation of results:

<20 GPLU/ml Negative20-40 GPLU/ml Weak positive41-60 GPLU/ml Moderate positive>60 GPLU/ml Strong positive

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Anticardiolipin Antibody - 2006

43.712.027.431Phadia

24.316.869.289Orgentec

21.611.452.918Inova

45.820.043.614In House

40.225.262.620Grifols

38.629.576.59Diamedix

22.68.738.521CLS Autozyme

13.46.447.98Binding Site

CV%SDmeann

ACA ‘G’0642


1451033612Overall Total

41862Phadia

791011Orgentec

51020Inova

1940In House

13502Grifols

7101Diamedix

01370CLS Autozyme

0801Binding Site

Strong PosMod PosWk PosNeg

ACA ‘G’0642

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7.929Phadia

19.891Orgentec

13.818Inova

7.811In House

26.520Grifols

6.121CLS Autozyme

7.66Binding Site

meann

ACA’G’0641


69101180Overall Total10227Phadia155035Orgentec00116Inova00212In House02153Grifols0081Diamedix00020CLS Autozyme0009Binding Site

Strong PosMod PosWk PosNegACA ‘G’0641

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83.419Phadia

114.058Orgentec

143.912Inova

76.312In House

75.417Grifols

108.010CLS Autozyme

meann

ACA’M’0641


2132466Overall Total18200Phadia74211Orgentec15000Inova9301In House

10601Grifols8001Diamedix

13000CLS Autozyme7010Binding Site

Strong PosMod PosWk PosNegACA ‘M’0641

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International consensus statement on an update of the classification criteria for definite antiphospholipidsyndrome (APS)

Journal of Thrombosis and Haemostasis, 4: 295-306

‘threshold used to distinguish moderate –high levels from low levels has no standard’

‘definition of the level that best corresponds to the risk of clinical manifestations is difficult’

‘the committee introduces a clear statement on threshold for positive: >40 GPL or MPL units



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Anticardiolipin assay‘Interlaboratory agreement on aCL measurement remains marginal with both home-based and commercial assays’

‘Discrepancies are mainly because of cut-off, calibration and other methodological issues’

‘Expression of aCL assays in ranges of positivity achieves better interlaboratory and inter-run agreement than do quantitative read outs’



‘Methodology and standardisation limitations expressed for aCL also apply for anti-β2 GP1’


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‘To optimise standardisation, new reference samples (monoclonal antibodies, named HCAL and EY2C9) will be distributed from the Centre for Disease Control ……. free of charge’

‘They will have to be validated against existing calibrators, and their specificity, avidity and stability over time should be monitored’



Cardiolipin Study

ACAG ACAGMenarini Orgentec Ratio

97/656 84.4 53.5 1.6

97/514 84.6 54.7 1.6

HCAL 102.5 61.9 1.7

0531 51.1 29.9 1.7

0541 92.1 44.4 2.1

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Information about the programmescan be found on website www.immqas.org.uk

©Copyright: The data from UK NEQAS is confidential to the NHS and may not be produced without the permission of the Organiser

Documents

Calibration Issues – Phospholipid Antibodies and ANCA