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No significant improvement in diagnostic specificity of an anti‐PF4/polyanion immunoassay with use of high heparin confirmatory procedure

2005; Elsevier BV; Volume: 4; Issue: 1 Linguagem: Inglês

10.1111/j.1538-7836.2005.01698.x

1538-7933

Theodore E. Warkentin, Jo‐Ann I. Sheppard,

Intramuscular injections and effects

Journal of Thrombosis and HaemostasisVolume 4, Issue 1 p. 281-282 Free Access No significant improvement in diagnostic specificity of an anti-PF4/polyanion immunoassay with use of high heparin confirmatory procedure T. E. WARKENTIN, T. E. WARKENTIN Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, CanadaSearch for more papers by this authorJ. I. SHEPPARD, J. I. SHEPPARD Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, CanadaSearch for more papers by this author T. E. WARKENTIN, T. E. WARKENTIN Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, CanadaSearch for more papers by this authorJ. I. SHEPPARD, J. I. SHEPPARD Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, CanadaSearch for more papers by this author First published: 13 December 2005 https://doi.org/10.1111/j.1538-7836.2005.01698.xCitations: 38 Theodore E. Warkentin, Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences (General Site), 237 Barton St. E., Hamilton, Ontario L8L2X2, Canada. Tel.: +1 905 527 0271 ext. 46139; fax: +1 905 577 1421; e-mail: twarken@mcmaster.ca AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Enzyme-immunoassays (EIAs) for antiplatelet factor 4 (PF4)/polyanion antibodies are known to have low diagnostic specificity, and thus low positive predictive value, for heparin-induced thrombocytopenia (HIT) in certain clinical settings. For example, after cardiac surgery, as many as 30–60% of patients develop anti-PF4/heparin antibodies without evincing clinical evidence of HIT [1]. In March 2000, the manufacturer of an anti-PF4/polyanion EIA [2] recommended adding a test procedure whereby ‘inhibition of a positive reaction by 50% or more in the presence of excess heparin (100 IU mL−1) is considered confirmatory for heparin-dependent antibody characteristic of … HIT’ (GTI® Instruction For Use for ‘PF4 Enhanced®’ assay, 13 Jan 2005; Genetics Testing Institute, Waukesha, WI, USA). To our knowledge, no published data are available evaluating to what extent this additional test maneuver improves test specificity. We therefore determined the impact of this confirmatory procedure on test specificity in a patient group (postcardiac surgery) in which non-pathogenic antibodies are often observed. We used archived plasmas (stored at −70 °C) from our previous study [3] in which we found 50 of 100 (50.0%) patients had detectable anti-PF4/heparin antibodies of IgG class following the cardiac surgery; 20 of these 50 patients had platelet-activating antibodies detected by platelet serotonin release assay (SRA). Only one of these patients who tested positive in the SRA and EIA, however, developed clinical HIT [3]. We tested 98 available postoperative patient sera for anti-PF4/polyanion antibodies using the PF4 enhanced® assay (which collectively detects antibodies of the three immunoglobulin classes, IgG, IgA, and IgM). All positive samples were additionally tested in the presence and absence of high heparin (100 IU mL−1), with per cent inhibition calculated as per the manufacturer's instructions, in which the optical density (OD) of the negative control serum is subtracted from both patient measurements – with and without high heparin – before calculating per cent inhibition. We also tested for the individual immunoglobulin classes (IgG, IgA, IgM) of anti-PF4/heparin antibodies using our in-house assay [3]. We found that 78 of 98 (79.6%) patients tested positive in the commercial EIA using postoperative blood samples, of which 57 (73.1%) seroconverted from a negative (preoperative) baseline, nine (11.5%) seroconverted (more than a twofold increase) from a positive baseline, and 12 (15.4%) showed baseline reactivity without more than a twofold increase from baseline. Of these 78 positive plasma samples, 76 (97.4%) were inhibited by more than 50% by addition of 100 IU mL−1 heparin [median (IQR) inhibition, 98.2% (92.5%, 100.7%)], and thus were ‘confirmed’ as testing positive (Fig. 1). The two samples that were not inhibited had the following results (value in parentheses indicating OD in presence of 100 IU mL−1 heparin): 0.920 (0.829) and 2.977 (2.679) units. Interestingly, although neither sample could be inhibited by 50% or greater even when higher concentrations of heparin (300 and 1000 IU mL−1) were used, one sample nevertheless tested strongly positive in both the SRA [92% release at 0.1 U mL−1 heparin (normal <20% release); <20% release in the presence of anti-FcγIIa receptor-blocking monoclonal antibody and at 100 U mL−1 heparin] and in the in-house EIA (IgG, 1.750 units; IgA, 2.588 units; normal <0.450 units), raising the issue of whether failure to exhibit inhibition in the confirmatory procedure really excludes the presence of platelet-activating anti-PF4/heparin antibodies. As only one (1.0%) patient in the study developed clinical HIT [3], the positive predictive value of the commercial EIA was only marginally improved by using the high heparin confirmatory procedure (from 1.41% to 1.45%). Figure 1Open in figure viewerPowerPoint Inhibition of optical density (OD) in a commercial anti-PF4/polyanion EIA by high heparin confirmatory procedure. The box plots show the results of 76 patients whose plasma tested positive for anti-PF4/polyanion antibodies (left panel) and in which high heparin (100 IU mL−1) inhibited the reaction by 50 per cent or more (right panel). Results of two other patients are shown in which the confirmatory procedure did not show inhibition; one of these (*) tested strongly positive in the platelet SRA (see text for details). (Actual OD results, without subtracting negative control reactivity, are shown.) The boxes show the median (horizontal line within box), and the first (bottom of box) and third (top of box) quartiles. Mean values are shown as a plus sign (+). The upper and lower bars indicate the maximum and minimum observations within 1.5 × the interquartile range (distance between first and third quartiles). Circles indicate outliers beyond the upper and lower bars. We conclude that inclusion of the high heparin confirmatory procedure in the commercial EIA does little to improve diagnostic specificity, at least in a postcardiac surgery setting in which anti-PF4/polyanion antibodies are frequently generated. Rather, the routine inclusion of this test maneuver will either double test costs (by requiring that each assay be performed both in the absence and presence of high heparin) or will delay the reporting of a positive test result, in case an algorithm is used in which a tentative positive assay result is subsequently ‘confirmed’ by repeating the test in the absence and presence of high heparin. References 1 Lee DH, Warkentin TE. Frequency of heparin-induced thrombocytopenia. In: TE Warkentin, A Greinacher, eds. Heparin-Induced Thrombocytopenia, 3rd edn. New York: Marcel Dekker, Inc., 2004: 107– 48. Google Scholar 2 Visentin GP, Moghaddam M, Beery SE, McFarland JG, Aster RH. Heparin is not required for detection of antibodies associated with heparin-induced thrombocytopenia/thrombosis. J Lab Clin Med 2001; 138: 22– 31. CrossrefCASPubMedWeb of Science®Google Scholar 3 Warkentin TE, Sheppard JI, Horsewood P, Simpson PJ, Moore JC, Kelton JG. Impact of the patient population on the risk for heparin-induced thrombocytopenia. Blood 200; 96: 1703– 8. PubMedGoogle Scholar Citing Literature Volume4, Issue1January 2006Pages 281-282 FiguresReferencesRelatedInformation