Insights into Therapeutic Mechanisms: Measuring Immature Platelet Fraction (IPF) Describes Response to Treatment in Immune Thrombocytopenic Purpura (ITP).

Abstract ITP is characterized by accelerated platelet destruction and also decreased platelet production. The inability to reliably quantify both platelet antibodies and platelet turnover has made it difficult to assess mechanisms of therapeutic effect with certainty. Two widely used therapies, IVIG and IV anti-D, are believed to primarily increase platelets by blocking Fcγ mediated platelet destruction; a similar effect in the marrow may also alter platelet production. Treatments for ITP are expanding as two thrombopoeitic agents (AMG 531 and Eltrombopag) complete phase 3 trials and a specific FcγRIII blocking agent (GMA161) is in phase I. One measure of thrombopoiesis is to use RNA-binding fluorochromes to identify RNA present in newly made, ‘reticulated’ platelets using flow cytometers. The Sysmex XE-2100 enables rapid and fully automated quantification of the absolute immature platelet fraction (IPF), equivalent to enumerating reticulated platelets. This study uses the IPF to assess acute response to treatment in 16 patients with ITP, providing insight into mechanisms of effect (table 1). Table 1: IPF & Platelet Acute Response to Treatment Patient Treatment Initial Plts Max Post-Treatment Plts Increase (Plts) Initial IPF Max Post-Treatment IPF Increase (IPF) E/P BSM = Eltrombopag vs placebo blind RCT study medication. Plts = platelet count 1 IVIG 1 121 120 0.7 33.5 32.8 2 IVIG 1 210 209 0.4 12 11.6 3 IVIG 10 157 147 1.7 8 6.3 4 IVIG 15 58 43 2.1 2.3 0.2 5 IVIG 7 171 164 1.5 6 4.5 6 IVIG 21 136 115 1.4 3.1 1.7 7 IVIG + Anti-D 3 160 157 1.8 17.3 15.5 8 Anti-D 7 335 328 2.8 8.4 5.6 9 Anti-D 9 79 70 2 5.5 3.5 10 Anti-D 1 11 10 0 3.9 3.9 11 Anti-D 13 167 154 2 5 3 12 GMA 14 108 94 3.4 13 9.6 13 GMA 7 45 38 1.2 5.1 3.9 14 Rituxan 24 187 163 6.6 14.4 7.8 15 E/P BSM 13 113 100 7.4 66.4 59 16 E/P BSM 16 558 542 5.9 29.4 23.5 In 4/6 patients treated with IVIG (example fig. 1), 4/4 patients treated with anti-D, and 2/2 GMA-treated patients, the IPF did not change appreciably while the platelet counts dramatically increased, substantiating the Fc inhibition mechanism of effect. In 3 patients with large platelet increases (120–209K) in response to IVIG or IVIG plus anti-D, the IPF also substantially increased, demonstrating that IVIG +/− anti-D may increase platelet production in certain cases. All had low pre-treatment IPF and platelet counts, suggesting antibody-mediated inhibition of platelet production. The two patients on the Eltrombopag/placebo blind RCT (example fig. 2) had 2 of the 3 largest increases in IPF, 23.5 and 59 × 10^3/uL. Using IPF to estimate platelet production provides novel insight into pathophysiology and mechanisms of effect of treatments in ITP. The dramatic increase in the absolute IPF seen with the thrombopoietic agent helps validate the clinical utility of this tool and allows discrimination of ITP patient heterogeneity. Fig. 1 (Patient 3) Response to treatment with IVIG Fig. 1. (Patient 3) Response to treatment with IVIG Fig. 2 (Patient 15): Response to Eltrombopag study medication Fig. 2. (Patient 15): Response to Eltrombopag study medication