Response to letter regarding “Plasma concentration of thrombopoietin in dogs with immune thrombocytopenia”

We appreciate Dr. Wun's interest in our article and the opportunity to clarify the thrombopoietin assay's configuration. Dr. Wun raises a concern regarding the addition of cryopoor plasma to the canine plasma samples. There was no addition of cryopoor plasma to any of the canine plasma samples, including plasma from healthy dogs, ITP dogs, or spike-and-recovery plasmas. All canine plasma samples were diluted in assay buffer alone. Rather, the assay standards and blanks were diluted in assay buffer containing cryopoor plasma. The composition of the diluents is stated in the manuscript section 3.3 “ELISA development and optimization: To minimize effects of high background absorbance values associated with plasma proteins, standards and blanks were diluted in 1 part canine pooled cryopoor plasma added to 4 parts assay buffer. Test plasma samples were diluted in assay buffer without addition of cryopoor plasma.”

The addition of cryopoor plasma to the standard curve diluent corrected for high absorbance values consistently observed in canine plasma, considered a “matrix” effect.¹ Because the content of TPO in the cryopoor plasma was unknown, the assay was configured with “blank” wells containing only cryopoor plasma in buffer to account for any canine TPO present in this matrix. All OD readings were then adjusted based on the blank OD (stated in section 3.3 “The mean absorbance value of the blank wells was subtracted from all standard and test sample wells”). We recognize that this correction may preclude quantitation of low TPO concentrations in test samples and acknowledged this limitation in the manuscript's discussion. “Our assay lacks precision at low TPO concentrations. The assay cannot accurately quantitate plasma TPO concentrations below approximately 60 pg/mL.”

Dr. Wun also mentions non-additive TPO recovery and signal quenching by plasma interferents. Quantitative rcTPO recovery (buffer with cryopoor plasma) is depicted in Figure 2, with sample raw OD values and calculated TPO concentrations included as Supplemental Figure 4. The mean spike-in recoveries of rcTPO (buffer with canine plasma) ranged from 87.5% (200 pg/mL) to 84% (800 pg/mL) with reproducibility for replicate determinations summarized in section 3.5 “TPO performance characteristics.”

We believe, within the assay's stated limitations, that the described TPO ELISA will be useful for studying thrombopoietic response in dogs with ITP and bone marrow disorders.