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Clinical Chemistry 48: 1625-1626, 2002;
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(Clinical Chemistry. 2002;48:1625-1626.)
© 2002 American Association for Clinical Chemistry, Inc.

Letters

Lot-to-Lot Inconsistency of Anticardiolipin Reagents

Daniel M. Hoefnera and Kiang-Teck J. Yeo

Dartmouth Medical School, Department of Pathology, Lebanon, NH 03756

aAddress correspondence to this author at: Dartmouth Medical School, Department of Pathology, HB7600, One Medical Center Dr., Lebanon, NH 03756.


To the Editor:

The diagnostic criteria for antiphospholipid syndrome include the presence of one or more typical clinical features plus one or more laboratory findings (1). The latter include positivity (on two or more occasions, >=6 weeks apart) of either lupus anticoagulant or anticardiolipin antibody (ACA).

We report inconsistencies among lots of anticardiolipin reagents from one supplier and suggest that the differences are related to changes in calibration materials that are also used by other suppliers of ACA reagents.

Several, perhaps most, ACA ELISAs are calibrated with Harris "standards" (Louisville APL Diagnostics, Inc.) or secondary calibrators that are traceable to them. Recently, there has been a change in the latest generation of calibration materials, the LAPL-GM-200 calibrators for IgG and IgM ACA. When the latest LAPL-GM-200 calibrators were produced, the manufacturer attempted to make these new calibrators agree with their three previous versions, LAPL-GM-100 (distributed 1997–2001), LAPL-GM-001 (1990–1997), and the originals (made before 1990).

We have been using ACA assays (QUANTA LiteTM Anticardiolipin IgG/IgM ELISA HRP Kit; INOVA Diagnostics) that use the Harris calibrators. In October 2001, we received a new shipment of both ACA IgM (lot no. 170264) and IgG (lot no. 170276) reagent sets, both based on the new LAPL-GM-200 calibrators. During routine checking of patient samples with the old and new reagent sets, we found a large negative proportional bias in the IgM results [y = 0.58x + 3 MPL (MPL is the conventional IgM ACA unit nomenclature; 1 MPL is the cardiolipin binding activity of 1 mg/L of an affinity-purified IgM); r = 0.992; Fig. 1A ] and a large positive proportional bias in the IgG results (y = 1.34x + 5 GPL; r = 0.997; data not shown). Concerns were relayed to INOVA. Subsequently (January 2002), we received reformulated reagent sets based on the GM-200 calibrators; these were prepared to better align the assays with results obtained with their previous reagents, which were calibrated with LAPL-GM-100 materials. The reformulated assay produced better agreement for the IgG when compared with assays calibrated with the LAPL-GM-100 materials (data not shown). However, the prior negative bias of the IgM was overcompensated for; when the revised reagent set (which contained INOVA’s in-house secondary calibrators traceable to the GM-200 material) was compared with the previous lot (no. 170264), we found the following bias: y = 2.45x - 16 MPL (r = 0.95; Fig. 1B ). Comparison of the revised lot and our last lot (170105) that was based on the prior GM-100 standards (170355) showed a slope >1.0 and a negative intercept (Fig. 1C ).



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  		Figure 1. Comparison of three lots of QUANTA Lite ACA IgM ELISA reagents.

Reagent lots 170105, 170264, and 170355 used INOVA calibrators 123105A, 122809A, and 132240A, respectively. All data were generated from actual patient specimens; dashed lines represent the line of identity. Regression statistics: (A), y = 0.58x + 3 MPL; r = 0.992; slope (95% confidence interval), 0.523–0.647; y-intercept (95% confidence interval), 0.62–5.3 MPL; (B), y = 2.45x - 16 MPL; r = 0.95; slope, 2.06–2.78; y-intercept, -24 to 6.0 MPL; (C), y = 1.78x - 20 MPL; r = 0.987; slope, 1.26–2.30; y-intercept, -47 to 6.8 MPL.

A semiquantitative assay with categorical limits (i.e., negative, low, medium, high positive) requires consistency across reagent lots. The INOVA product insert suggests that results <12.5 MPL be classified as negative, results >=12.5 to 20 MPL be classified as indeterminate, and results >20 MPL be reported as positive (with 20–80 MPL as low/medium and >80 MPL as high). For the last 397 patients that we tested with LAPL-GM-100 reagent sets, results for 31% of the patients were >20 MPL. Extrapolating from panels A and C in Fig. 1Up would suggest that this percentage would have been 16% with lot no. 170264 and 27% with lot no. 170355.

We appreciate that INOVA has listened to our concerns, but we feel it is important to alert the users of these products to the potential need to readjust their cutoff values when systematic changes occur with new lots of reagents.


References

  1. Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum 1999;42:1309-1311.[Web of Science][Medline] [Order article via Infotrieve]



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G. Endler, C. Marsik, B. Jilma, T. Schickbauer, R. Vormittag, O. Wagner, C. Mannhalter, H. Rumpold, and I. Pabinger
Anti-Cardiolipin Antibodies and Overall Survival in a Large Cohort: Preliminary Report
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[Abstract] [Full Text] [PDF]

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