The retrospective study investigated hematological and biochemical differences in dogs with neoplastic splenic disease (NSD), comparing malignant versus non-malignant, as well as malignant lymphoid versus malignant non-lymphoid tumors. The research included 20 dogs with splenic disease admitted at the University Veterinary Hospital in Skopje within three years. Each dog underwent clinical examination, abdominal ultrasound, blood, serum, and cytology analysis. Based on cytological findings, dogs were divided into two groups: malignant (M, n=14) and benign (B, n=6) spleen disease. The M group was further divided into malignant lymphoid (ML, n=6) and malignant non-lymphoid (MNL, n=8) subgroups. Data (mean±SEM) was analyzed with Kruskal-Wallis and Mann-Whitney U Test with significance levels set at pp<. The M group had significantly higher values than B group for white blood cell (32.08±9.29 and 7.37±0.96x109/L, respectively), monocyte (2.89±1.09 and 0.64±0.12x109/L, respectively), neutrophil counts (17.46±3.80 and 5.39±0.88x109/L, respectively), alanine aminotransferase (82.52±12.49 and 36.89±4.14 IU/L, respectively), and alkaline phosphatase levels (279.16±62.06 and 111.80±15.40 IU/L, respectively). Within the M group, ML subgroup had significantly higher values than MNL for white blood cell (62.80±19.65 and 15.02±3.03x109/L, respectively) and neutrophil counts (29.02±7.86 and 11.04±2.22x109/L, respectively), but lower for alkaline phosphatase (252.80±56.02 and 293.80±93.76 U/L, respectively). Ultrasonographic findings of heterogenous hypoechoic nodules (‘leopard’ pattern), pleomorphic lymphoblasts infiltration, and elevated leucocytes, neutrophils, monocytes, alanine transaminase, and alkaline phosphatase, may aid in differentiating malignant from benign NSD in dogs. Additional to the ultrasonographic and cytology findings, elevated leucocytes, neutrophils, and alkaline phosphatase effectively differentiate lymphoid from non-lymphoid NSD in dogs.
The spleen is a significant part of the lymphatic system in dogs related with lymphopoiesis, erythrocyte and platelet storage, phagocytosis of old and damaged erythrocytes, blood filtration, extramedullary hematopoiesis, and iron metabolism (
Ultrasonographic finding of splenomegaly is commonly observed during NSD and is highly correlated with neoplastic changes (
The high variability of clinical, hematological, and biochemical findings in dog NSDs renders these parameters to be non-specific and of low diagnostic importance. However, the current study hypothesized that there may be higher specificity and difference between the benign, malignant lymphoid, and malignant non-lymphoid NSDs for the hematological and biochemical parameters. The aim of the current study was to compare the hematological and biochemical parameters between malignant and benign NSDs confirmed by ultrasonographic and cytology diagnosis, and more specifically, to elucidate the differences among the malignant lymphoid, non-lymphoid, and benign NSDs in dogs.
The study included 20 dogs with splenic-related disorders of various age, sex, and breed admitted at the University Veterinary Hospital at the Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje over a period of 3 years. A diverse range of dog breeds was included: 4 Labrador Retrievers, 3 Golden Retrievers, 3 German Shepherds, 2 Shar-Peis, 1 Corgi, 1 Maltese, 1 French Bulldog, 2 Boxers, 1 Cocker Spaniel, 1 Pug, and 1 Staffordshire Terrier. The patients were between 5 and 11 years of age. Twelve of the patients were male and eight were female. All animals included in this study underwent standard clinical and diagnostic procedures with respect to animal welfare. Clinical records were obtained and used for research purposes with the owner’s consent. Under these circumstances, no Ethical Approval was deemed necessary since the procedures were routine and the data was used for scientific purposes with consent of the owners. Upon admission, the dogs were subjected to clinical examination, abdominal ultrasound, complete blood count, serum, and cytology analysis, routinely performed as part of the diagnostic procedures.
The ultrasound examination was performed with an Esaote MyLab30Gold ultrasound machine (Esaote, Italy) or Vetus 8 (Myndray, China) with a microconvex probe. Abdominal retrocostal or intercostal left approach was used on the left caudal thoracic wall. Size, shape, echogenicity, and echotexture of the spleen were determined for each dog. Splenic formations were noted and characterized.
Blood samples were taken from the cephalic vein in EDTA vacutainers and were analyzed on a veterinary hematology analyzer Exigo (Boule, Sweden). The following hematological parameters with their respective reference ranges were obtained: erythrocyte blood count (RBC-5.50-8.50x1012/L), packed cell volume (PCV-47-55%), hemoglobin (HGB-12-18 g/dL), platelets (PLT-200-500x109/L), white blood cells count (WBC-6-17x109/L), lymphocytes (LYM-1.2-5.0x109/L), monocytes (MON-0.3-1.5x109/L), and neutrophils (NEU-3.5-12.0x109/L).
Blood samples for biochemical analysis were collected in a vacutainer without anticoagulant. The sera of these blood samples were analyzed on an automatic biochemical spectrophotometer ChemWell 2910 (AwarenessTechnology INC, USA), according to the manufacturer instructions (Human, Germany). The following biochemical parameters with their respective reference ranges were obtained: alanine transaminase (ALT-8.2-57.3 IU/L), aspartate aminotransferase (AST-8.9-48.5 IU/L), alkaline phosphatase (AP-10.6-100.7 IU/L), total proteins (TP-54.1-75.2 g/L), albumins (ALB-25.8-44.7 g/L), creatinine (CRT-44.3-138.4 umol/L), and urea (UREA-3.14-9.25 mmol/L). Reference values for hematology and biochemistry were obtained from Merck Veterinary manual.
The cytology samples were taken by using ultrasound guidance with a 21G needle with the ‘fine needle aspiration’ method described elsewhere(
Following the clinical, ultrasonographic, and cytology findings, the dogs were divided in two major groups, malignant - M (n=14) and benign - B (n=6) splenic disease. The M group was additionally divided into malignant lymphoid - ML (n=6) and malignant non-lymphoid - MNL (n=8) splenic disease subgroups.
The data (mean±standard deviation) were compared with Kruskal-Wallis and Mann-Whitney U Test with effect size of 0.25, error probability of 0.05, total sample size of 20, number of groups 2 (Statistica v8, StatSoft Inc.). The power of the tests was estimated with G*Power (v. 3.1.9.2, University test was employed with power of 0.16 (df=17), whereas for Kruskal-Wallis, it was 0.14 (df=17). Significant differences were considered at p<0.05.
Lymphoid malignancies (lymphoma) (n=6) were mostly with heterogeneous echotexture visualized as multiple hypo-echoic nodules with a leopard spots appearance of the spleen (n=5) (Fig. 1), and as single heterogeneous focal mass (n=1). Non-lymphoid malignancies (sarcomas) (n=8) appeared mostly as single or multiple hypo-echoic heterogenous masses with variable size (0.3-5.0 cm). The larger tumor lesions had anechogenic cavity formations with irregular shapes localized inside the neoplastic mass. Benign spleen changes (n=6) were mostly manifested with splenomegaly and inhomogeneous splenic echotexture.
Microscopy evaluation of spleen cell cytology revealed various cellular and extra cellular matrix characteristics. Benign spleen lesions had typical lymphoid cell population composed of small mature lymphocytes, reactive lymphocytes, intact neutrophils, monocytes, fibrocytes, and erythrocytes characterized by normochromic erythroisocytosis. Malignant spleen lesions had numerous pleomorphic atypical cells with immature characteristics. Lymphoblasts were predominant cell population in the malignant lymphoid neoplasia, with round shape and immature cellular and nuclear morphology characteristics (anisocytosis and anisonucleosis). Hemangiosarcoma/hemangiopericytoma, or soft tissue sarcoma, had pleomorphic cell cytology of different cell lines. Individual mesenchymal cells were with indistinct cytoplasmatic borders, pale bluish cytoplasm with discrete vacuolization, oval or elongated anisokariotic nucleus with coarse chromatin and prominent metachromatic nucleolus. Histiocytic sarcoma was characterized with discrete round cells with characteristic cellular, cytoplasmatic, and nuclear changes with mitotic formations (Fig. 2).
RBC and PCV were decreased bellow the lower reference value in all groups, but they did not differ significantly. HGB and PLT were in the reference ranges for all groups and did not differ significantly. WBC were in the reference range for B (7.37±0.96x109/L) and MNL (15.02±3.03x109/L, but were significantly higher for M (32.08±9.29x109/L) and ML (62.80±19.65x109/L). ML had significantly higher WBC compared to B and MNL (15.02±3.03x109/L). MNL had significantly lower WBC compared to ML. The LYM were non significantly different among the groups due to the high variances, despite the high numerical differences. M (7.39±3.41x109/L) and ML (17.68±8.00x109/L) had higher LYM than the reference range, whereas B had small negative deviation from the lower reference value. M was had non-significantly higher LYM compared to B (1.12±0.06x109/L). MON were above the reference range in the M group (2.89±1.09x109/L) mostly in B (0.64±0.12x109/L) and MNL (1.06±0.20x109/L) were in the reference range. The M group had significantly higher MON compared to B, whereas no significant differences were observed between MNL and ML. The NEU were above the reference range in the M group (17.46±3.80x109/L) mostly contributed by the ML group (29.02±7.86x109/L). NEU were significantly higher in M compared to B (5.39±0.88x109/L). Significant difference in NEU was ML and MNL (11.04±2.22x109/L). No significant difference was observed in NEU between MNL and B. More detailed data for the hematological parameters can be found in Table 1.
The urea, creatinine, and total proteins were in the reference range for all groups and they were non-significantly different. The albumins were non-significantly different among the groups and were in the reference range for all groups with exception of MNL (24.33±2.44 g/L) which was below the lower reference value. AST value in M (73.81±23.75 U/L) was above the reference range mostly due to MNL group (85.15±36.01 IU/L).
Comparison of means ±SE of hematological parameters between benign (B), malign (M), malign non-lymphoid (MNL), and malign lymphoid tumors (ML)
Comparison of means ±SE of biochemical parameters between benign (B), malign (M), malign non-lymphoid (MNL), and malign lymphoid tumors (ML)
The AST in ML (53.40±17.43 IU/L) was above the reference range but had little contribution to M. However, there was no significant difference in the AST level between the groups. ALT had higher than the reference range values in M (82.52±12.49 IU/L), and more specifically in MNL (87.30±14.24 IU/L) and ML (73.90±25.69 IU/L). ALT was within the reference range in B (36.89±4.14 IU/L). Significant differences were observed between B and M, however, no significant differences were found between MNL and ML, neither between these two groups and B. AP level was above the reference range in all groups. M group (279.16±62.06 IU/L) had significantly higher AP value compared to B (111.80±15.40 IU/L). MNL (293.80±93.76 IU/L) had significantly higher AP value compared to B, but non-significantly higher compared to ML (252.80±56.02 IU/L). ML was non-significantly different compared to B. More detailed data for the biochemical parameters can be found in Table 2. (
Lymphoma is a common spleen malignancy in dogs which can be observed on ultrasound as a solitary formation or as multiple hypo-echoic nodules with a “leopard-spot” pattern. This ultrasonographic finding was the most frequently observed in this study. Unlike lymphoma, nonlymphoid spleen tumors (e.g., sarcomas) appeared with or without splenomegaly with a poorly circumscribed, inhomogeneous mass of variable echogenicity (
Fine needle aspiration and Tru-cut needle biopsy are routinely performed to help distinguish benign from malignant lesions (
The current study hypothesized that NSD cases confirmed by cytology and ultrasonography can be correlated with biochemical and hematological indices. Additionally, the study hypothesized that these indices could potentially differentiate between malignant lymphoid from malignant nonlymphoid NSD cases.
The observed hematological findings indicated on mild anemia with no significant differences in RBC between the benign and malignant groups. Usually, spleen lesions are concomitant with microangiopathies. Erythrocytes with poikilocytosis that pass through the altered vascular endothelium can be damaged or aggregated in the intercellular spaces of the red pulp. The occurrence of hypochromic anemia, erythrocyte sequestration, schistocytes, other types of altered erythrocyte morphologies, and hemophagocytic syndrome, could be considered as a predictive pattern of malignant metaplasia from nonlymphoid neoplasia (
Similarly, packed cell volume and hemoglobin concentration were non-significantly different between the benign and malignant subgroups (MNL, ML), and could not be directly associated with NSD. However, it has been reported that anemia and nucleated red blood cells are associated with malignant tumors in dogs with splenomegaly (
The significantly higher leucocyte count in the malignant groups could be considered as an expected finding due to the immunological or systemic inflammatory response. Malignant diseases in spleen causes increased WBC as a result of chronic inflammation which is accompanied by release of interleukins (IL-1, IL-6, TNF-α), ischemic and necrotic changes of the malignant secondary bacterial growth. The role of circulating lymphocytes remains complex, as both tumorsuppressing and tumor-promoting mechanisms are involved (
Total protein concentration, urea, and creatinine were not changed beyond the upper and lower reference ranges nor they were significantly different between the groups. This was expected as the spleen is not directly associated with the protein metabolism and renal perfusion. Similar finding was reported even in the terminal stage of the disease (
The malignant group had twofold significantly higher value of ALT compared to the benign group, but no significant differences were observed between MNL and ML. Oliveira et al. (
AST had 2.45-fold higher value in the malignant compared to the benign group, however, even though it was markedly higher in the MNL, no significant differences were observed between either group. necrotic alterations, these findings were expected. Additionally, the higher values may be related with muscle tissue alterations induced by neoplasia in the abdominal muscles, elevated catabolism, as well as metastasis. The non-significant differences were also expected due to the low sample size and increasing levels of ALT and AST were associated with tumor progression (
The malignant group had 2.5-fold significantly higher value of AP compared to the benign group. MNL had significantly higher value than ML. The higher concentration in the malignant groups was expected due to the enzyme’s non-specific nature regarding pathological conditions and its high prevalence in different tissues, including the spleen. It could be considered as an indirect biomarker of intensive mitotic division in the spleen or with increased metastatic potential of hepatic or hepatobiliary malignant cells. Metabolic stress syndrome with endogenous cortisol level can induce elevated AP levels (
The limitations of the study are the low sample size in the groups which yielded low statistical power of 0.20 (20%). Therefore, the findings could be considered as merely indicative and could be used for further research by increasing the sample size.
The ultrasonographic finding of heterogenous hypoechoic nodules with ‘leopard’ pattern morphology of the spleen, and pleomorphic lymphoblasts infiltration with increased values of leucocytes, neutrophils, monocytes, alanine transaminase, and alkaline phosphatase, are clear indicators for distinguishing malignant from benign NSD in dogs. The ultrasonographic and cytology findings, combined with higher values of leucocytes, neutrophils, and alkaline phosphatase, can be used for distinguishing lymphoid from nonlymphoid NSD in dogs. None of these parameters alone could be used as a direct diagnostic indicator for NSD.
The authors declare that they have no financial or non-financial conflict of interest regarding authorship and publication of this article.
The study was conducted on admitted patients at the University Veterinary Hospital at the Faculty of Veterinary Medicine-Skopje, together with colleagues from private practice.
IC performed the hematology, biochemistry, and cytology analyses and wrote the manuscript. AA collected and analyzed the data. IU revised the written text. EAP and TN performed the ultrasonographic diagnostics. MN conducted a detailed statistical analysis and made corrections to the final version of the manuscript.