Die Analyse retikulierter Thrombozyten des Hundes mit dem Hämatologiegerät ProCyte Dx im Vergleich zur durchflusszytometrischen Bestimmung

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Objective The aim of this study was the method validation of whole blood analysis of reticulated platelets as marker of platelet regeneration with the ProCyte Dx (IDEXX Laboratories, Inc., USA) in comparison with flow cytometric analysis as reference method. In addition, repeatability was investigated and reference intervals were determined for this novel parameter on the ProCyte Dx. Material and methods 56 blood samples of dogs were prospectively analyzed between March 2017 and November 2017 for the method comparison study. Samples were obtained from healthy dogs as well as patients with thrombocytopenia, which were expected to have increased numbers of reticulated platelets. Exclusion criteria were the presence of moderate to high numbers of platelet aggregates grade 2-4 (of 0-4) by semiquantitative estimate on blood smear evaluation or invalid flow cytometric analysis. Flow cytometric analysis was considered invalid due to contamination of the washing buffer, unspecific binding of antibodies or when high numbers of events bound to antibodies were lacking the platelet specific light scatter properties. Exclusion of 14/56 samples resulted in 42 blood samples included for method comparison study. The population of reticulated platelets was detected by manual gating strategy in the optical channel of the ProCyte Dx using the manufacturer´s software. Flow cytometric measurement was performed with a dual labelling protocol. CD61 antibodies were used to detect platelets in whole blood and thiazole orange stained RNA-content in this population. Intra assay precision was evaluated in ten replicate measurements for both methods. Statistical analysis included determination of Spearman’s rank correlation coefficient, Bland-Altman analysis and a Passing-Bablok regression analysis. Microsoft excel 2016 (Microsoft corporation, Redmond, WA, USA) and the statistical software MedCalc® version 17.8.6 (MedCalc Software Ltd, Ostende, Belgium) were used for analysis of data. For establishment of reference intervals, measurements of 120 healthy adult dogs (age between 1 and 14 years) were included. The nonparametric method was performed with the software Reference Value Advisor version 2.1 (Biostatistiques, Ecole Nationale Vétérinaire de Toulouse, France). Results There was a fair correlation for reticulated platelet percentages between the two methods with a spearman rank’s correlation coefficient (rs) of 0.70. Bland-Altman analysis revealed a mean negative proportional bias of -8.2 % for the ProCyte Dx. Intra-assay CV in a sample with low percentages of reticulated platelets (mean 2.8 %) was 12.85 % for flow cytometry and 32.89 % for the ProCyte Dx. In a patient with thrombocytopenia and high values of reticulated platelets (mean 31.6 %), a lower intra-assay CV of 7.52 % was seen for the ProCyte Dx. The reference interval for the percentage of r-PLT ranged from 0 to 7.47 %. Conclusion The ProCyte Dx is capable to reliably detect reticulated platelets in blood samples of dogs. Compared to flow cytometry, there was an acceptable, but not a perfect correlation and a proportional bias. However, this is not surprising as the analyzers work with different methods. The low number of only 42 included samples is also a limitation as a broader range of measurements is preferable. The novel reference interval might be an aid to detect increased values of reticulated platelets in patients with thrombocytopenia that hints towards active megakaryopoiesis. The clinical value of this novel parameter with the ProCyte Dx could be investigated in future studies. The measurement of reticulated platelets with the ProCyte Dx as a point-of-care analyzer offers a new possibility for clinicians in small medical practices to use this parameter. In healthy dogs with low values of reticulated platelets, a certain degree of imprecision has to be taken into account. However, in more clinically relevant elevated values, intra-assay precision is satisfactory.

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Kristina Weiler

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