Brigitte Dircks
Platelet-bound antibodies and neutrophil-platelet aggregates in dogs with different diseases
Besides their functions in hemostasis and thrombus formation, platelets also take part in a multitude of physiologic as well as pathophysiologic processes including inflammation, immunology, tumor spreading, and tissue repair. In the first, retrospective part of this study underlying diseases and clinical and clinicopathologic variables of thrombocytopenic dogs with and without platelet-bound antibodies (PBAs) as well as clinicopathologic variables of dogs with suspected primary immune-mediated thrombocytopenia (pIMT) (i.e. without detected associated disease) were characterized. For this purpose, medical records of thrombocytopenic dogs, in which a PBA test was carried out by use of flow cytometry between January 2004 and December 2006, were reviewed.
In 37 of the 83 dogs (45%) included into the study, PBAs were detected. Underlying diseases of dogs with positive and negative PBA test results, however, were of great diversity.
Thirteen dogs in which no underlying disease was found were suspicious of having pIMT.
Platelet counts were significantly lower in dogs with positive PBA test results (16 × 103 cells/μl; 2–119 × 103 cells/μl, [median; minimum–maximum]) than in dogs with negative PBA test results (44 × 103 cells/μl; 3–115 × 103 cells/μl) (P = 0.005). Dogs with pIMT had significantly lower platelet counts (14 × 103 cells/μl; 2–44 × 103 cells/μl), compared with platelet counts of dogs having secondary IMT (sIMT) (31 × 103 cells/μl; 3–119 × 103 cells/μl). Hemorrhage was detected significantly more often in dogs with positive PBA test results (24/37 [65%]) than in dogs with negative PBA test results (18/46 [39%]). The mean platelet volume (MPV) was significantly lower in PBA-positive dogs (12.5 fl; 7.4–20 fl versus 14.8 fl; 7.8–24 fl) (P = 0.01). An increase in MPV (defined as MPV > 14.3 fl) was found significantly more often in dogs negative for PBAs (19/33 [58%]) than in dogs positive for PBAs (7/26 [27%]). None of the dogs suspected of having pIMT had an increase in MPV.
Analysis of bone marrow aspirates revealed an increase in megakaryopoietic activity in 14 of 21 (67%) PBA-positive dogs and in 7 of 18 (39%) PBA-negative dogs; these proportions did not differ significantly. An increase in megakaryopoiesis was detected in all 9 dogs with suspected pIMT in which bone marrow analysis was performed. This was significantly higher than the proportion of dogs with sIMT (6/13).
The results of the first part of the present study indicate that immune-mediated destruction is a potent pathogenic mechanism for thrombocytopenia in a variety of underlying conditions.
Dogs with a positive PBA test result, and additionally severe thrombocytopenia, an MPV value within or below the reference range, and an increased megakaryopoiesis are suspicious for having pIMT. The MPV appears to be a helpful diagnostic tool in the evaluation of dogs with thrombocytopenia, however, its importance with regard to megakaryopoiesis remains unclear.
The second part of this study is addressing PNA in dogs. Increased PNA formation has been detected previously in different diseases in human medicine; however, no data exist regarding the presence of PNA in vivo in dogs. Therefore, the objective of the current study was the measurement of PNA in dogs with systemic inflammatory diseases. In order to characterize relevant factors for the aggregate formation, the influence of various platelet- and neutrophil agonists on PNA formation was analysed in vitro. Furthermore, neutrophil shape characteristics (neutrophil size and granularity), as indicator for the activation of these cells, were studied in dogs with systemic inflammatory diseases and after stimulation with different agonists.
Twenty dogs with systemic inflammatory response syndrome (SIRS) of which 6 had sepsis and 12 had disseminated intravascular coagulation (DIC) were included into the study. Blood of 10 clinically owned healthy beagle dogs was used as control blood as well as for incubation with different agonists in the following final concentrations and combinations: phorbol myristate acetate (PMA) (5 µmol/L), collagen (COL) (20 µg/mL), adenosine diphosphate (ADP) (20 µmol/L), epinephrine (EPI) (20 µmol/L), COL (20 µg/mL) + ADP (20 µmol/L), EPI (20 µmol/L) + ADP (20 µmol/L), lipopolysaccharide (LPS) (1, 2, 5, and 10 µg/mL), und arachidonic acid (AA) (0.5, 1, and 2 mmol/L). Platelet-neutrophil aggregates were measured flow cytometrically after early separation of platelets from neutrophils by use of a Ficoll density gradient immediately after blood sampling or after incubation with agonists,
respectively, in order to minimize artificial PNA formation. PNA were measured based on the detection of CD61 associated with neutrophils. Quantification of PNA was done by the mean fluorescence intensity (MFI) of all neutrophil cells, and the percentage of CD61-positive neutrophils. Mean forward angle (correlating with cell size) and side angle light scatter values (correlating with granularity and complexity) were recorded as parameters for the neutrophil shape.
The MFI (5.1 1.6 median standard deviation versus 3.3 0.1) (P = 0.003) and the percentage of CD61-positive neutrophils (10.5% ± 7.9versus 2.3% 0.2) (P = 0.003) were significantly higher in dogs with SIRS compared to healthy control dogs. Furthermore, increased cell size (P < 0.001), and decreased granularity (P = 0.013) of neutrophils were observed in these dogs. When dogs with and without sepsis or DIC were compared, however, no difference was seen with regard to the previously mentioned parameters.
A significant increase in PNA formation of variable degree was seen after incubation of blood with all agonists. Most marked increases in MFI (62.4 29.2 versus 4.2 0.5) (P < 0.001), and percentage of CD61-positive neutrophils (89.4% 9.8 versus 4.7% 0.6) (P < 0.001) were observed after stimulation with PMA in comparison with unstimulated samples.
Incubation with a combination of COL and ADP showed no further increase in PNA formation when compared to incubation with COL alone, whereas a combination of ADP and EPI led to a nearly cumulative response. Incubation of blood with LPS and AA at different dosages resulted in a dose-dependent increase in the MFI as well as the percentage of CD61-positive neutrophils.
Incubation with all tested agonists except EPI resulted in an increased cell size and decreased granularity of neutrophils with PMA, again, having the most prominent effect. Combinations of ADP and COL or EPI and ADP showed no additive effect on these parameters. Similar to the PNA formation, incubation with both, LPS and AA, resulted in a dose-dependent increase of cell size and decrease of granularity of neutrophils.
In conclusion, increased PNA formation and neutrophil shape changes were found in dogs with systemic inflammatory diseases. The fact, that these changes were also detected after incubation with pure platelet agonists, is indicating the role of platelet activation in aggregate formation. Further studies are necessary to predict the significance of PNA and neutrophil shape changes in the pathogenesis of SIRS and sepsis and whether the measurement of these
parameters provides a useful diagnostic tool for the detection of pro-inflammatory states in dogs.