Journal of Veterinary Internal Medicine最新文献

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

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.

I am writing in response to the article “Peripheral and intestinal T lymphocyte subsets in dogs with chronic inflammatory enteropathy” published in Issue 3, Volume 38 of JVIM. It is stated by the authors that the Wilcoxon's signed rank test was used to compare quantitative and ordinal qualitative variables between the healthy control group and the chronic inflammatory enteropathy group. As these groups are independent, the assumption of paired samples required for the use of Wilcoxon's signed rank test is not met and the results reported are therefore invalid. I am wondering if the data could be re-analyzed using the Mann-Whitney U-test, which is the appropriate statistical test for non-parametric ratio and ordinal data.¹

Understanding and correctly interpreting statistical results presented in scientific articles is a required skill for practicing evidence-based veterinary medicine. A prerequisite for doing so is the adequate reporting of the results in scientific journals. However, most authors of veterinary publications determine the importance of their findings based on statistical significance (ie, P < .05), indicating that neither the limitations of using P values for inference nor the existence of more appropriate alternatives are widely appreciated in veterinary medicine. This deficiency in knowledge indicates a need to increase awareness in veterinary medicine regarding reporting statistical measures that quantify the magnitude of an effect along with its level of uncertainty, and then interpreting these results for clinical decision making. We utilize a hypothetical randomized controlled trial of dietary management in cats with chronic kidney disease to discuss some common misconceptions about P values and provide practical suggestions for alternatives. Reporting appropriate effect estimates along with their confidence intervals will allow veterinarians to easily and correctly determine whether the magnitude of the effect of interest meets clinical needs while acknowledging uncertainty in the results. We also describe confidence interval functions and show their utility as visual tools in aiding interpretation of confidence intervals. By providing practical guidance, we show that a change in reporting and interpreting statistical results is feasible and necessary. We hope this crucial step will promote clinical decision making based on effect estimates and confidence intervals.