A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes by Yenan T. Bryceson, Daniela Pende, Andrea.

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A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes by Yenan T. Bryceson, Daniela Pende, Andrea Maul-Pavicic, Kimberly C. Gilmour, Heike Ufheil, Thomas Vraetz, Samuel C. Chiang, Stefania Marcenaro, Raffaella Meazza, Ilka Bondzio, Denise Walshe, Gritta Janka, Kai Lehmberg, Karin Beutel, Udo zur Stadt, Nadine Binder, Maurizio Arico, Lorenzo Moretta, Jan-Inge Henter, and Stephan Ehl Blood Volume 119(12):2754-2763 March 22, 2012 ©2012 by American Society of Hematology

Evaluation of a consensus protocol for the analysis of NK-cell and CTL degranulation. Evaluation of a consensus protocol for the analysis of NK-cell and CTL degranulation. (A-B) Degranulation of resting NK cells analyzed with protocol 1. (A) FACS plots illustrating the induction of CD107a expression in CD3−CD56+ NK cells using PBMCs from a healthy donor and a patient with FHL5 after incubation with medium or with NK-sensitive K562 target cells. (B) Results from the same sample analyzed in 3 different laboratories (▴, Genoa; □, Freiburg; and ●, Stockholm). (C-D) Degranulation of IL-2–stimulated NK cells. (C) NK-cell degranulation assay using PBMCs that had been stimulated for 48 hours with PHA and IL-2. (D) Results from the same sample analyzed in 3 different laboratories. (E-F) Degranulation of T-cell blasts. (E) FACS plots illustrating the induction of CD107a expression in 48h PHA/IL-2–stimulated CD8+ T cells from a healthy donor and from a patient with FHL5 after incubation with medium alone or medium plus anti-CD3/anti-CD28 beads. (F) Overlay of stimulated (white) and unstimulated (shaded gray) samples. Numbers inside quadrants represent ΔCD107a (A,C) and ΔMFI of CD107a (E-F). ΔCD107a indicates the difference in the percentage of cells expressing CD107a before stimulation subtracted from the percentage of cells expressing CD107a after stimulation. ΔMFI indicates the respective difference in MFI. Yenan T. Bryceson et al. Blood 2012;119:2754-2763 ©2012 by American Society of Hematology

NK-cell and CTL CD107a assays identify most patients with genetic disorders of degranulation. NK-cell and CTL CD107a assays identify most patients with genetic disorders of degranulation. Results of CD107a degranulation assays using resting NK cells (A-C), IL-2–activated NK cells (D-F), or short-term CTL blasts (G-H) from patients with FHL3 (A,D,G), FHL4 (B,E), or FHL5 (C,F,H). ΔCD107a (%) indicates the difference in the percentage of cells expressing CD107a before stimulation subtracted from the percentage of cells expressing CD107a after stimulation; ΔCD107a (MFI), respective difference in MFI; Pr. 1, protocol 1; and Pr. 2, protocol 2. The gray shaded areas represent the range from the 10th to the 90th percentile of values obtained in healthy donors. Closed symbols represent patients manifesting with HLH before age 2; open symbols indicate manifestation of HLH after age 2. Yenan T. Bryceson et al. Blood 2012;119:2754-2763 ©2012 by American Society of Hematology

Impaired NK-cell and CTL degranulation in patients with immunodeficiency and albinism. Impaired NK-cell and CTL degranulation in patients with immunodeficiency and albinism. Results of CD107a degranulation assays using resting NK cells (A), IL-2–activated NK cells (B), or short-term CTL blasts (C) from patients with CHS (triangles) and GS2 (circles). Closed symbols represent patients manifesting with HLH before age 2; open symbols indicate manifestation of HLH after age 2. For additional explanations, see legend to Figure 2. Yenan T. Bryceson et al. Blood 2012;119:2754-2763 ©2012 by American Society of Hematology

NK-cell degranulation assays are normal in most patients with FHL2, XLP, and secondary HLH. Results of CD107a degranulation assays using resting NK cells (A-C) and IL-2–activated NK cells (D-F) from patients with FHL2 (A,D), XLP1 and XLP2 (B,E), or secondar... NK-cell degranulation assays are normal in most patients with FHL2, XLP, and secondary HLH. Results of CD107a degranulation assays using resting NK cells (A-C) and IL-2–activated NK cells (D-F) from patients with FHL2 (A,D), XLP1 and XLP2 (B,E), or secondary HLH (2° HLH; C,F). In panels A and C, closed symbols represent patients manifesting with HLH before age 2 and open symbols indicate manifestation of HLH after age 2. In panels B and E, triangles represent patients with XLP1 and circles represent patients with XLP2. For additional explanations, see legend to Figure 2. Yenan T. Bryceson et al. Blood 2012;119:2754-2763 ©2012 by American Society of Hematology

Immunosuppressive therapy does not significantly impair the performance of the degranulation assays. Immunosuppressive therapy does not significantly impair the performance of the degranulation assays. Results were pooled from those patients with FHL2, XLP, and secondary HLH for whom information on HLH-2004 or other immunosuppressive treatment (IS) at the time of analysis was available. (A) Analysis of resting NK cells. (B) Analysis of IL-2–activated NK cells. Yenan T. Bryceson et al. Blood 2012;119:2754-2763 ©2012 by American Society of Hematology

Summary of results obtained in this study and statistical evaluation. Summary of results obtained in this study and statistical evaluation. (A) Distribution of results in the different categories. “Other” indicates patients who either did not fulfill the criteria for HLH or for whom clinical and genetic information was insufficient for final classification. (B-C) Empirical ROC curves for resting (B) and activated (C) NK-cell degranulation assays based on a combined dataset generated with the 2 protocols. Yenan T. Bryceson et al. Blood 2012;119:2754-2763 ©2012 by American Society of Hematology

Proposed laboratory diagnostic algorithm based on degranulation assays for patients presenting with HLH. Normal values have to be determined in the diagnostic laboratory for the evaluation of resting NK-cell degranulation. Proposed laboratory diagnostic algorithm based on degranulation assays for patients presenting with HLH. Normal values have to be determined in the diagnostic laboratory for the evaluation of resting NK-cell degranulation. For the centers participating in this study, resting NK-cell degranulation was considered defective if < 5%, abnormal if 5%-10%, and normal if > 10%, cutoffs that have proven to be useful. Analyses of activated NK-cell degranulation is recommended for all patients. AICD indicates activation-induced cell death. Yenan T. Bryceson et al. Blood 2012;119:2754-2763 ©2012 by American Society of Hematology