Prognosis in canine idiopathic immune-mediated haemolytic anaemia

Introduction

Idiopathic immune-mediated hemolytic anaemia (IMHA), characterized by antibody-mediated red cell destruction, is recognized as one of the most frequently occurring immune-mediated diseases in the dog [62]. Idiopathic IMHA is diagnosed based on a Coombs’ positive hemolytic anaemia, the presence of spherocytes (Figure 1), and exclusion of infectious or neoplastic disorders, vaccination, and medication that can cause a secondary IMHA [40]. The presentation of a dog diagnosed with idiopathic IMHA varies from a mild anaemia to a severe hemolytic crisis with a mortality of 20 % - 70 % that mainly occurs in the first 2 weeks post diagnosis [1] [37] [44]. The central theme of this study was to analyze clinical, therapeutic, and pathophysiological factors that contribute to disease outcome in dogs with idiopathic IMHA [16].

Figure 1. Blood smear in canine IMHA. It demonstrates the typical features canine IMH: a marked anisocytosis of the erythrocytes caused by the presence of polychromatic macrocytes and hyperchromic microcytes. The latter cells lack the central lucent area due to a change from biconcave to spherical shape. Hence they are called microspherocyts. They are the result of partial phagocytosis of the erythrocytic membrane due to the presence of anti-erytrocyte antibodies and are one of the diagnostic criteria of IMHA.

Retrospective studies report a poor outcome in dogs with autoagglutination [37], non-regenerative or severe anaemia [49], thrombocytopenia [2], severe leucocytosis [1], high plasma bilirubin concentration [2] [37] [49], or increased prothrombin time [5]. Not every dog diagnosed with idiopathic IMHA displays all these characteristics, however, and it is unclear if they are part of the process of antibody-mediated red cell destruction or result from independent pathophysiological processes secondary to the hemolysis. The relationship between various characteristics and an event may be explored by statistical multivariate models that analyze the probability that death occurs in a specified time period. Lifelong immunosuppression has been recommended, but evidence to support this recommendation is lacking [34] [40]. Therefore, a study [20] was devised to estimate survival time in dogs with idiopathic IMHA and to explore which clinical and laboratory characteristics determine the probability of survival; in addition, the question was asked if immunosuppression for three months is sufficient to maintain remission of idiopathic IMHA.

Immunomodulation is the mainstay of treatment in IMHA and may be combined with whole blood or packed red cell transfusions and anticoagulation medication [34] [35] [40]. It has been suggested to combine glucocorticoids with other immunosuppressive agents or cytotoxic drugs if the clinical condition worsens, if side effects of glucocorticoids are unacceptable, or as part of standard treatment protocols [25] [40] [58]. Azathioprine, a thiopurine analog, is a cytotoxic drug that interferes with DNA synthesis by competition with adenosine [67]. It has a synergistic effect with prednisolone and if used in combination the prednisolone dose may be reduced [73]. A beneficial effect of treatment with azathioprine in idiopathic IMHA has been reported in two retrospective studies [1] [22]. However, the duration of azathioprine therapy in one study must be judged suboptimal since the clinical effect of azathioprine may be expected only after at least 11 days of treatment [1] [73]. In the other study the efficacy of azathioprine alone could not be determined since dogs were treated with cyclophosphamide on top of azathioprine and prednisolone [1]. Therefore, a study was devised to assess the additional beneficial therapeutic effect of azathioprine compared to prednisolone therapy in dogs with idiopathic IMHA [21].

The laboratory diagnosis of IMHA rests upon the demonstration of an immune-mediated mechanism for hemolysis [40]. The direct agglutination test (DAT) demonstrates the presence of anti-erythrocyte antibodies by incubating a suspension of washed patient erythrocytes with polyvalent or monovalent antisera specific for dog immunoglobulin or complement [26]. There is a lack of uniformity between laboratory procedures for the DAT [26]. Instead of testing only a few dilutions, testing of more dilutions in a microtitre tray has been advocated to result in less false-negative results due to a prozone effect [26] [53]. Incubation is generally performed at 37°C, but some laboratories also incubate at 4°C, a procedure debated by others [26] [40] [65] [71]. The monovalent DAT is suggested to have a higher sensitivity than the polyvalent DAT, and the sensitivity and specificity of reagents from different manufacturers differ considerably [26] [61]. A gel-based polyvalent DAT has been developed that offers the possibility to standardize anti-erythrocyte antibody testing between laboratories [9]. To assess the performance of this gel-based DAT in comparison to the traditional DAT based on erythrocyte agglutination and to assess the usefulness of this gel-test as a diagnostic tool in the diagnosis of IMHA a study was devised [16].

Abundant evidence has been presented for a state of hypercoagulability during the hospitalization period in dogs with idiopathic IMHA [2] [24] [27] [46] [50]. Post-mortem examinations have demonstrated thromboembolisms in many organs [2] [12] [24]. Leucocytosis and a left shift are present in most dogs, and increased leucocyte counts have been associated with moderate to marked tissue damage [1] [2] [12] [22] [24] [55]. Changes in acute phase protein concentrations have been measured fitting the presence of an acute phase response [4] [8] [14] [66] [75]. Interleukin-8 (Il-8) is one of the cytokines that is increased in the acute phase response. It is a major chemotaxin for leucocytes and orchestrates the margination and extravasation process of leucocytes through increases in selectin expression on endothelial cells [10] [64]. Leucocytes, in particular monocytes, play an important role in thrombogenesis by producing tissue factor (TF) which initiates the extrinsic pathway of coagulation [63] [70]. It was hypothesized that dogs with idiopathic IMHA have increased blood levels of IL-8 and TF. To validate reference genes for future quantitative RT-PCR in idiopathic IMHA, canine whole blood was studied [18]. To assess the contribution of whole blood gene expressions of TF and IL-8 to the inflammatory response and the coagulation abnormalities, in another study was devised [19].

Our study presents another literature review and critical evaluation the diagnostic tests and the results of therapy [17]. This part was motivated by a remarkable observation: the mortality rates in canine idiopathic IMHA have not decreased since the earliest publications [1] [6] [37] [44] [52]. Azathioprine, cyclophosphamide, other immunomodulators, and heparin are often used for treatment of canine idiopathic IMHA, despite the fact that evidence of their efficacy is lacking [1] [5] [11] [13] [22] [39] [48] [68] [72].

Discussion

Mortality percentages in canine idiopathic IMHA range between 20 % - 70 % with most deaths occurring in the first two weeks after diagnosis [1] [22] [37] [41]. Calculations of survival times were performed in three different cohorts [19] [20] [21]. In the cohort of 149 dogs treated with prednisolone and azathioprine (AP protocol), the half-year survival was 72.6% (95% CI: 64.9 – 81.3%) [20], in another cohort of 73 dogs treated with prednisolone (P protocol), the 1-year survival was 64% (95% CI: 54 – 77%) [21], and in a cohort of 24 dogs, treated with prednisolone, the half-year survival was 75% (95% CI: 59.5 – 94.5%) [19] [20] [21].

Eighty percent of dogs in the cohort treated with azathioprine and prednisolone (n=149) showed a leucocytosis and a left shift at presentation [20] similar to the findings in the cohort treated with prednisolone alone (n=73) [21]. The cohort of 24 dogs [19] had significantly increased leucocytes, band neutrophils, and monocytes. The summary of CBC data from other studies [17] supports the conclusion that an inflammatory response consisting of a pronounced leucocytosis [2] [11] [20] [21] [22] [24] [55] and a left shift [2] [11] [20] [21] [24] are common laboratory features at presentation.

Our literature survey [17] suggests that as many as half of the deaths are related to thromboembolisms [2] [4] [12] [46] [50] [51]; from the summary of clinical observational studies we conclude that these dogs present with abnormalities in coagulation parameters, suggesting the presence of disseminated intravascular coagulation (DIC). The results for coagulation times, fibrinogen concentration, and thrombocyte counts support this assumption [20] [21]. The prothrombin and activated partial thromboplastin times were increased in 46% and 67% (n=98), respectively, and a decreased fibrinogen concentration and thrombocyte counts below 50 x 109/l were found in 18% (n=96) and 25% (n=148), respectively, of the cohort treated with azathioprine and prednisolone [20]. These coagulation en thrombocyte count results were not significantly different from those found in the cohort treated with prednisolone [21]. Decreases in individual coagulation factor activities fitting DIC were also found in the cohort of 24 dogs [19]. A low mean platelet content (MPC), indicative of platelet activation, was found in the group of dogs diagnosed with DIC and in the group with idiopathic IMHA [15] [19] [45] [47]. In addition, large platelets, characterized by an increased mean platelet volume (MPV) and mean platelet mass (MPM) were found, most likely as a result of increased platelet production rates [45] [47] [54]. It has been reported that large platelets are associated with an increased hemostatic capacity [47]. In conclusion, the dogs with idiopathic IMHA [19] have decreased MPC and increased MPV and MPM, reflecting a high platelet turnover due to the continuous platelet activation occurring in DIC.

Uni- and multivariate analysis with the aim to identify prognostic variables and their relationship have been performed in 3 cohorts of dogs with idiopathic IMHA [19] [20] [21]. The findings of increased plasma urea or creatinine concentrations, icterus, increased band neutrophil, and monocyte counts, thrombocytopenia, and prolonged APTT as prognostic variables in the respective multivariate models indicate that parameters signalling renal and/or liver failure, inflammation, and DIC - alone or in combination - are robust independent predictors of mortality. Other authors have found the same or related prognostic factors [17, table 8].

Hypoxia is a risk factor found by some workers [7] [49] could not be confirmed in the univariate analyses [19] [20] [21]. Nevertheless, a pathology study that established a positive relation between the presence of hypoxic necrosis, especially in the liver, and increased leucocyte counts [12] and a study that related increased duration of hyperlactemia to mortality [6] both support that anaemia may be central in the development of a high mortality risk. Oxygen delivery was impaired in a canine isovolemic anaemia model below a hematocrit of 10% [32] [33]. The fact that icterus and failing liver functions are important risk factors is in agreement with hypoxia, since the failing liver functions can be attributed to centrolobular hypoxic hepatocyte necrosis resulting from severe anaemia.

Both the inflammatory response and the thrombotic tendency as major and independent prognostic factors led us to investigate the underlying pathophysiological mechanisms. Tissue factor (TF) expression by inflammatory cells, especially monocytes has been reported as a link between inflammation and coagulation [3] [38]. This occurs mainly through the NF-kB signaling pathway and leads to increases in IL-8, a major chemotaxin for leucocytes [10] [64]. It was hypothesized that blood levels of TF and IL-8 are increased in dogs with idiopathic IMHA, due to their increased expression by inflammatory cells [19]. Total leucocyte counts and band neutrophils are increased in 80% of dogs with idiopathic IMHA at presentation [20] [21], and increase further during hospitalization [6]. The high leucocyte turnover suggests a continuing production of IL-8. Similarly, to explain a thrombotic tendency, a continuous intravascular source for TF must be present. We therefore chose to measure TF and IL-8 gene expression during the hospitalization using quantitative RT-PCR. Gene expressions was measured in whole blood, since isolation of leucocytes may cause up-regulation of cytokine expressions [42].

One of the solutions to control for the internal variation affecting quantitative RT-PCR results is the use of reference genes as an internal standard [69] [74]. These are selected based on the assumption that their expression is stable in all cells, regardless of the tissue or individual [31] [43]. The suitability of nine frequently used canine genes was investigated [18]. The analysis revealed that white blood cell count and disease category had a statistically significant effect on the expression of the potential reference genes in canine whole blood. Two software applications were used to select genes with the most stable expression and the required number to provide an optimal normaliser within the experimental setting [19]. The Normfinder package selects reference genes with the most stable expression between groups and GeNorm selects the genes with the least variation in individual samples [28] [69]. It was concluded that multiple reference genes are necessary to provide the stable normalizer; since expression may be influenced by the experimental conditions, it is necessary to assess the stability of expression for each experimental situation anew [18].

The hypothesis was rejected that hypercoagulability and an inflammatory response are due to increased expression of IL-8 and TF by monocytes [6]. Our study demonstrated that whole blood TF expression was increased, but IL-8 expression was not significantly different from that in healthy dogs and significantly lower than in the groups with systemic disease, neoplasia and DIC, and sepsis. The decreases in coagulation factors FII, FV, FVII, FIX, FXI, FXII confirmed the presence of DIC in many patients. The high FVIII and fibrinogen activity suggested an acute phase response. Much evidence has been accumulated in the literature for an acute-phase response in dogs with idiopathic IMHA [4] [8] [14] [66] [75]. Increased leucocyte counts and turnover such as documented in [17] [20] [21] have been related to the severity of centrolobular hepatocyte necrosis [12]. In the first part of the acute-phase response, macrophages activated by liver hypoxia release interleukin-1 and tumor necrosis factor, which is followed by the release of IL-8 and monocyte chemoattractant protein by local fibroblasts and endothelial cells [30]. We identified an increased monocyte count as independent negative prognostic parameter in the multivariate model, which predict death in dogs with idiopathic IMHA [19]. In a recent study, monocyte counts were not identified as a prognostic factor. But serum cytokine concentrations related to monocyte recruitment (monocyte chemoattractant protein-1, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 15 and interleukin-18) were increased, and both, monocyte chemoattractant protein -1 and interleukin-18, were independently associated with higher mortality [8]. Similar to our findings, the results for the serum IL-8 concentrations (median 2.6 µg/l, range (1.2–32.0), n=20) in this study were not significantly different from those in the healthy controls (median 1.6 µg/l, range (0.6–5.4), n=6) [8].

Whole blood TF expression was increased in dogs with idiopathic IMHA [19] and thus contributes to the consumptive coagulopathy [19] [20] [21]. TF expression in monocytes is regulated via the NF-kB signaling pathway and its activation is expected to result in increased expression of IL-8 as well [3]. Since IL-8 expression was not increased in patients, our results suggest that another source than blood monocytes for the increased whole blood TF expression. Platelets have been reported to express TF but not IL-8 and may be the alternative source of TF expression in our study [57]. This assumption is supported by the fact that p-selectin, a platelet activation marker, was elevated in dogs with idiopathic IMHA [15] [23] [27].

The summarized evidence [17] demonstrates that results supporting the use of immunomodulators in addition to glucocorticoids are lacking, despite all the efforts invested in observational studies and randomized clinical trials [1] [5] [11] [21] [22] [39] [48] [72]. Analysis of the trials with regard to inclusion criteria [17] suggests be advantageous to categorize dogs with idiopathic IMHA based on their probability of survival in addition to the randomization. Power analyses to estimate sample sizes necessary for clinical trials have been too optimistic and led to false negative outcomes [13]. An example of a power analysis shows that larger sample sizes are needed than hitherto used. In fact, our retrospective cohort study [21] had 149 dogs in the AP protocol group and 73 dogs in the P protocol group, respectively, and is one of the largest studies comparing treatments in canine idiopathic IMHA. However, this study is observational, using a historical control group, and not a randomized controlled trial.

Observational studies [17] may be more suited than controlled randomized trials to establish the required duration of immunosuppression, the natural history of the disease, and occasional side effects. We were able to demonstrate in the retrospective cohorts [20] [21] that - in contrast with general recommendations, which include lifelong immunosuppression - an immunosuppressive regime with prednisolone alone or in combination with azathioprine for 3 months is sufficient to obtain remission. Side-effects due to azathioprine were observed in 8% (n=222) [21]. Recurrences of hemolytic crisis may occur in at least 10% (n=222) [20] [21]. A lack of additional therapeutic effect of azathioprine in the cohort treated with azathioprine and prednisolone in comparison to the cohort treated with prednisolone alone was reported [21]. We discussed the use of historical controls, which may have resulted in confounding due to improvement of supportive care within the time span of both cohorts [17] [21].

It was concluded [17] that a mortality risk based classification of dogs with idiopathic IMHA is needed to ensure that dogs that enter a trial have similar mortality risks. Indeed, the survival probabilities between the treatment arms may have differed in study [21] since the dogs in the trial arm treated with azathioprine and prednisolone had lower thrombocyte counts and longer duration of clinical signs which may have obscured an additional treatment effect of the azathioprine. Therefore a blinded randomized clinical trial is still needed to establish the true effect of azathioprine. In view of the expected slow onset of azathioprine it is unlikely that a benefit of azathioprine can be discerned in the first 1-2 weeks after the start of treatment. Therefore such a trial should be conducted in the subset of dogs with idiopathic IMHA that are likely to survive the initial hazardous hospitalization period.

We concluded [17] that progress in treatment is slow, and multi-center trials may be the only solution to obtain study groups with enough power to find differences in outcome due to treatment. The datasets of the cohorts [19] [20] [21], and previous data sets may be utilized to provide the basics for a mortality risk-based scoring system. Such a system, however, should be validated in a prospective study that preferably incorporates data from dogs from different research groups working on canine idiopathic IMHA to ensure that the resulting scoring system is properly validated for general application.

Agreement upon the diagnostic criteria for canine idiopathic IMHA will be a necessary prerequisite for the postulated multi-center trials. The use of similar inclusion and exclusion criteria as summarized in [17], predicts that generally accepted criteria will include moderate to marked anaemia, and diagnostic procedures to exclude pathophysiological routes of development of anaemia other than haemolysis [1] [5] [11] [21] [22] [39] [49] [72].

However, the diagnosis of immune-mediated hemolysis ultimately depends upon the demonstration of anti-erythrocyte antibodies commonly achieved by conventional DAT [26], the execution of which is poorly standardized and source for much debate [26]. We have shown [16] that the results of a fast polyvalent gel-based DAT agree very well with those of the conventional format, as performed in two veterinary university clinic laboratories specialized in hematology. Since a gel-based DAT may be commercially produced, these results are encouraging with regard to future standardisation. A potential additional advantage may be that the gel-based DAT was less often positive in secondary IMHA [16].

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Appendix

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