Volume 134, Issue 1, Pages 174-181 (July 2014) New insights in heparin-induced thrombocytopenia by the use of fluid-phase assays to detect specifically platelet factor 4/heparin complex antibodies and antibody-secreting cells  Annika Schulze, Inga Jensch, Krystin Krauel, Adnan Alahmad, Hans-Peter Müller, Andreas Greinacher, Matthias Hundt  Thrombosis Research  Volume 134, Issue 1, Pages 174-181 (July 2014) DOI: 10.1016/j.thromres.2014.04.014 Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 1 Schematic of the solid- and fluid-phase immunoassays. (A) Solid-phase ELISA. The binding of the serum Ab to immobilized antigen (e.g. PF4 or PF4/heparin) leads to the formation of antigen-Ab complexes. This is detected by an anti-immunoglobulin Ab-enzyme conjugate and the corresponding specific substrate. (B) Fluid-phase ELISA. The serum Ab are captured by immobilized anti-immunoglobulin Ab. Subsequently, specific serum Ab bind the biotinylated antigen (e.g. PF4 or PF4/heparin) in solution. The formed biotinylated antigen-Ab complex is then detected by a streptavidin-enzyme conjugate and its substrate. In the fluid-phase ELISPOT cells are added to the wells and the secreted Ab are captured by immobilized anti-immunoglobulin Ab. The remaining steps follow the fluid-phase ELISA principle. Thrombosis Research 2014 134, 174-181DOI: (10.1016/j.thromres.2014.04.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 2 The fluid-phase ELISA detects IgG and IgM Ab against the murine PF4/heparin complexes with high specificity. Mice were immunized with murine PF4/heparin, serum was obtained at different time points (day 1=first day of injection) and binding of IgG (A, C) and IgM (B, D) Ab from respective sera to murine PF4 alone (circle) and murine PF4/heparin (square) was evaluated by solid-phase ELISA (A, B) and fluid-phase ELISA (C, D). Inhibition of Ab binding to murine PF4/heparin was tested by excess of heparin (100IU/ml; triangle). Data are mean OD±SD of five individual mice. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. The dotted lines represent the upper limit of the normal range. Thrombosis Research 2014 134, 174-181DOI: (10.1016/j.thromres.2014.04.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 3 The immunization with ovalbumin and PBS did not lead to the production of anti-murine PF4/heparin Ab. Mice were immunized either with ovalbumin (circle), murine PF4/heparin (square) or received only the vehicle PBS (triangle). IgG (A) and IgM (B) anti-murine PF4/heparin Ab were determined by PF4/heparin fluid-phase ELISA. IgG (C) and IgM (D) anti-ovalbumin Ab were measured by the ovalbumin ELISA. Data are mean OD±SD of each three individual mice per group each. (**P<0.01, ***P<0.001, ns: not significant). The dotted lines represent the upper limit of the normal range. Thrombosis Research 2014 134, 174-181DOI: (10.1016/j.thromres.2014.04.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 4 Murine PF4/heparin ASC can be specifically detected and are localized in the spleen. (A) Determination of the optimal ratio between biotinylated and unbiotinylated murine PF4 in the murine PF4/heparin complexes for the ELISPOT. Different ratios of biotinylated and unbiotinylated murine PF4 within the murine PF4/heparin complex were tested for the detection of murine PF4/heparin-specific IgG (upper row) and IgM (lower row) ASC (2.5x105 cells/well) in the spleen of an immunized mouse (day 4). Spots represent the individual ASC. (B) Validation of murine PF4/heparin-specific ELISPOT. Splenocytes of control mice (day 0) and murine PF4/heparin immunized mice (day 4 and 8) were analyzed for total and murine PF4/heparin-specific IgG (left) and IgM (right) ASC. For the detection of total ASC 1.25x105 cells/well and for murine PF4/heparin-specific ASC 1.25 x105, 2.5 x105, 5 x105 and 10 x105 cells/well were used. (C, D) Percentage of murine PF4/heparin-specific ASC. Splenocytes of PF4/heparin immunized mice (day 4) were analyzed for the binding of secreted IgG (left) and IgM (right) Ab to murine PF4 (circle), murine PF4/heparin (square) and murine PF4/heparin in the presence of an excess of heparin (100IU/ml) (triangle). Three individual measurements as well as means are shown. The number of murine PF4/heparin-specific ASC was expressed as a percentage of the total number of IgG or IgM ASC. (**P<0.01, ***P<0.001). (E, F) Localization of murine PF4/heparin-specific ASC. Investigation of the spleen, bone marrow and peritoneal cavity with respect to murine PF4/heparin IgG (E) and IgM (F) specific ASC. (**P<0.01, ***P<0.001). Thrombosis Research 2014 134, 174-181DOI: (10.1016/j.thromres.2014.04.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 5 The cross-reactivity of anti-PF4/heparin Ab is species-dependent. (A) Analysis of the cross-reactivity of murine anti-PF4/heparin Ab with the human PF4/heparin complex. The binding of IgG Ab from sera of mice (n=5; day 11) immunized with murine PF4/heparin to murine PF4/heparin (circle) and human PF4/heparin (square) was tested by fluid-phase ELISA. (B) Analysis of the cross-reactivity of human anti-PF4/heparin Ab with the murine PF4/heparin complex. The binding of IgG Ab from human serum of patients (n=5) tested positive for anti-human PF4/heparin Ab by an in-house PF4/heparin EIA to human PF4 alone (closed circle), human PF4/heparin (square), murine PF4 alone (inverted triangle) and murine PF4/heparin (diamond) was evaluated by fluid-phase ELISA. All sera were also tested for inhibition of Ab binding to the human PF4/heparin (triangle) and murine PF4/heparin (open circle) complexes, dissolved by excess of heparin (100IU/ml). The dotted lines represent the upper limit of the normal range. Individual measurements as well as the median are shown. Thrombosis Research 2014 134, 174-181DOI: (10.1016/j.thromres.2014.04.014) Copyright © 2014 Elsevier Ltd Terms and Conditions