Vorkommenshäufigkeit und pathophysiologische Faktoren der Thrombozytopenie bei Hunden mit portosystemischen Shunts

Beschreibung

Background The versatile use of reticulated platelets has already been demonstrated in various studies in human medicine and is increasingly gaining importance in veterinary medicine. The ADVIA 120/2120 is capable of determining canine r-PLTs using a fixed gating strategy and flow cytometric data analysis software. Recently, a new method has been introduced for software version v6.11.7 using a moving threshold that has not been evaluated before for dogs. Objective The aim of this study was to evaluate the novel method of r-PLT detection using a moving threshold (r-PLTmt) in comparison with flow cytometry as refence method and to provide reference intervals. Moreover, the impact of validation of results by visual inspection of scattergrams was assessed as well as intra-assay coefficient of variation (CV) in samples of low, medium, and high concentrations of r-PLTs and samples with potential interferences with the measurement such as microcytic erythrocytes. Materials and Methods Forty-two blood samples were compared with the reference method of flow cytometry using the automated r-PLT method of the ADVIA 120/2120 (r-PLTmt) and a visually validated r-PLT method (r-PLTmtv). The novelty of the automated measurement consists of a moving threshold (mt), which creates an individual adjustment of the gate for the detection of reticulated platelets to the individual platelet counts. The method of visual validation (r-PLTmtv) takes into account the characteristics of canine reticulated platelets, which appear further right in the scattergram and outside the preset gate. The calculation of the coefficient of variation was assessed in samples with low, medium, and high concentrations of r-PLTs and samples with potential interference with the measurement such as microcytic erythrocytes evaluated. Spearman's rank correlation coefficient, Bland Altman analysis, and Passing-Bablok regression were used for statistical evaluation of the method comparison. Reference intervals for percentage and absolute r-PLTs were established using 120 blood samples from healthy dogs for both the r-PLTmt and the r-PLTmtv method. Results The coefficient of variation (CV) for percentage r-PLTs assessed with flow cytometry was 12.9 %. The CVs for percentage and absolute r-PLTs assessed with the automated method were 29.1 % and 29.6 % and for the validated method 22.6 % and 24.3 % for low r-PLT counts. The CVs for percentage and absolute r-PLTs assessed with the automated method were 15.7 % and 14.4 % and assessed with the validated method 12.9 % and 10.9 % for medium r-PLT counts. The CVs for percentage and absolute r-PLTs with the automated method were 20.2 % and 14.8 % and for the validated method 18.3 % and 14.5 % for high r-PLT counts. The coefficients of variation for percentage and absolute r-PLTs by automated method showed values of 65.7 % for %r-PLTmt and 35.8 % for %r-PLTmt and values of 65.0 % for r-PLTmt and 36.7 % r-PLTmt for measurement of a-r-PLT, respectively, for the samples with high interference by microcytic hypochromic erythrocytes. The coefficients of variation for the validated method showed values of 19.8 % for %r-PLTmtv and 13.1 % for %r-PLTmtv and 17.5 % and 14.6 % for a-r-PLTmtv. Method comparison showed a correlation coefficient of 0.75 for the automated method and 0.76 for the modified method compared to flow cytometry. The mean deviation for %r-PLTmt was -10.76 (range: 42.63) and for %r-PLTmtv -9.68 (range: 39.22). Reference intervals for percentage and absolute r-PLT were 0.2 to 3.8 % and 0.6 to 10.2 x 109/l (r-PLTmt) and 0.3 to 4.5 % and 1.1 to 10.3 x 109/l (r-PLTmtv). Conclusion The new r-PLT parameter of the ADVIA 120/2120 showed moderate correlation with flow cytometry for both measurement methods with visual validation showing slightly higher correlation than the standard automated method. Visual validation of scattergrams is particularly recommended for samples with high interference from microcytic, hypochromic erythrocytes. Similarly, inclusion of signals located further right in scattergrams of dogs leads to more precise results. In summary, both measurement methods are suitable for the detection of reticulated platelets. The optimization of the gate towards adaption to characteristics of canine reticulated platelets could offer an entry into routine diagnostics and is therefore desirable.

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Anja Kuhn

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