Letter regarding “Plasma concentration of thrombopoietin in dogs with immune thrombocytopenia”

Abstract

I am writing in response to the article “Plasma concentration of thrombopoietin in dogs with immune thrombocytopenia” published in JVIM Early View on August 14, 2024. The authors conclude that “Plasma TPO concentration is paradoxically low at diagnosis for most dogs with pITP,” and that “This finding suggests that ineffective thrombopoiesis contributes to thrombocytopenia in pITP dogs and supports evaluating TPO receptor agonist treatment as used for pITP in humans.” I believe several key deficiencies in the TPO ELISA validation process described should be addressed before these statements can be accurately made. First, the sample matrix used in the spike-and-recovery experiment was comprised of pooled cryopoor plasma from healthy dogs and assay buffer. It is reasonable to assume that the pooled plasma contained endogenous TPO, however, the amount was not quantified. As such, it is not possible to determine whether the recovery of recombinant and endogenous TPO behaves in an additive (linear) or non-linear manner. If the relationship is non-linear, the spike-and-recovery experiment is not valid.¹ Second, plasma from the dogs with pITP may have contained interfering compounds (eg, inflammatory cytokines and growth factors²) that were not present or were present at a different concentration in the pooled healthy dog plasma. The authors acknowledge that they “do not know if the assay potentially cross-reacts with (these compounds).” Binding of these compounds with capture/detection reagents or TPO may have impaired the detection of TPO in dogs with pITP,³ potentially resulting in an erroneous conclusion.

Both of these problems could be overcome with a simple parallelism experiment, which would involve plotting measured TPO against various dilutions of pITP serum, and comparing these curves against the standard curve generated from serial dilutions of recombinant TPO in the sample matrix. Comparing the degree of “parallelism” between the curves would allow the relative accuracy of the ELISA in pITP serum to be assessed, as well as its selectivity and sensitivity.³ Other methods of validation could include the depletion of endogenous TPO from the pooled healthy plasma using an anti-recombinant TPO antibody, and the addition of various cytokines and growth factors to the assay buffer during the spike-and-recovery experiment. All three of these methods were used to validate the first human TPO ELISA.⁴