Retrospective evaluation of anemia and erythrocyte morphological anomalies in dogs with lymphoma or inflammatory bowel disease

Cyril Parachini-Winter Department of Veterinary Clinical Science, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.

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Lisa M. Carioto Department of Veterinary Clinical Science, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.

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Carolyn Gara-Boivin Department of Pathology and Microbiology, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.

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Abstract

OBJECTIVE To assess the prevalences of anemia and various RBC anomalies in dogs with lymphoma versus inflammatory bowel disease (IBD) and to evaluate potential relationships between these variables and the severity of lymphoma.

DESIGN Retrospective cross-sectional study.

ANIMALS 82 client-owned dogs.

PROCEDURES Medical records and blood smears were reviewed for dogs in which IBD or lymphoma had been diagnosed between January 1, 2006, and December 31, 2014, and for healthy dogs evaluated during that time frame. Hematologic data were analyzed, and results were compared among groups of healthy dogs, dogs with IBD, and dogs with lymphoma. Results were also compared within the lymphoma group between dogs further grouped on the basis of lymphoma clinical stage, substage, and cell size.

RESULTS Prevalence of anemia was significantly higher in dogs with lymphoma (17/32 [53%]) than in dogs with IBD (5/23 [22%]). The total number of different RBC anomalies was significantly higher in dogs with lymphoma than in dogs that were healthy or had IBD. A cutoff of 3 different RBC anomalies/dog enabled differentiation between lymphoma and IBD, with a sensitivity and specificity of 71% and 70%, respectively (area under the fitted curve, 0.7239 ± 0.0727). The presence of eccentrocytes was the only individual RBC anomaly significantly more common in dogs with lymphoma (8/28 [29%]) versus dogs with IBD (1/23 [4%]).

CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that detection of anemia combined with ≥ 3 RBC morphological anomalies, particularly eccentrocytes, on blood smears should increase the clinical suspicion of lymphoma, compared with IBD, in dogs.

Abstract

OBJECTIVE To assess the prevalences of anemia and various RBC anomalies in dogs with lymphoma versus inflammatory bowel disease (IBD) and to evaluate potential relationships between these variables and the severity of lymphoma.

DESIGN Retrospective cross-sectional study.

ANIMALS 82 client-owned dogs.

PROCEDURES Medical records and blood smears were reviewed for dogs in which IBD or lymphoma had been diagnosed between January 1, 2006, and December 31, 2014, and for healthy dogs evaluated during that time frame. Hematologic data were analyzed, and results were compared among groups of healthy dogs, dogs with IBD, and dogs with lymphoma. Results were also compared within the lymphoma group between dogs further grouped on the basis of lymphoma clinical stage, substage, and cell size.

RESULTS Prevalence of anemia was significantly higher in dogs with lymphoma (17/32 [53%]) than in dogs with IBD (5/23 [22%]). The total number of different RBC anomalies was significantly higher in dogs with lymphoma than in dogs that were healthy or had IBD. A cutoff of 3 different RBC anomalies/dog enabled differentiation between lymphoma and IBD, with a sensitivity and specificity of 71% and 70%, respectively (area under the fitted curve, 0.7239 ± 0.0727). The presence of eccentrocytes was the only individual RBC anomaly significantly more common in dogs with lymphoma (8/28 [29%]) versus dogs with IBD (1/23 [4%]).

CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that detection of anemia combined with ≥ 3 RBC morphological anomalies, particularly eccentrocytes, on blood smears should increase the clinical suspicion of lymphoma, compared with IBD, in dogs.

Lymphoma is one of the most commonly diagnosed malignancies in dogs, accounting for 7% to 24% of all neoplasias1 and 83% of hematopoietic malignancies.2 Although the gastrointestinal form of lymphoma is less common than the multicentric form, it still accounts for 5% to 7% of all lymphomas diagnosed in dogs. Difficulties can be encountered in differentiating gastrointestinal lymphoma from IBD, especially lymphocytic-plasmacytic enteritis, on the basis of histopathologic lesions alone, especially in dogs.3,4 Further, the possibility that an immunoproliferative enteritis could precede subsequent development of gastrointestinal lymphoma in dogs has been raised,5 and a recent study6 of cats shows that misdiagnosis between gastrointestinal lymphoma and IBD could have occurred in 39% (7/18) of cats if only a single segment of the gastrointestinal tract had been biopsied.

Several new diagnostic tools have emerged in recent years to help differentiate gastrointestinal lymphoma from IBD with more certainty. A recent recommendation4 to improve the accuracy of distinguishing between IBD and gastrointestinal lymphoma is to use a stepwise approach, starting with histologic assessment of H&E-stained biopsy samples; followed by immunohistochemical staining to detect CD20, CD3, and Ki67 antigens; and finally, a PCR assay for antigen receptor gene rearrangements to assess clonality. The importance of performing this combination of procedures is underscored by results of a study7 in which the PCR assay identified a monoclonal population of neoplastic cells in only 22 of 29 (76%) dogs with gastrointestinal lymphoma. Also, 10 of 19 (53%) cats in which IBD had been diagnosed on the basis of histologic examination results alone were reclassified as having T-cell lymphoma when a combination of H&E staining, immunohistochemical staining, and the PCR assay were used.8 Hence, none of these individual techniques alone can guarantee a definitive diagnosis of lymphoma or IBD, but collectively, they often require general anesthesia and gastrointestinal biopsy, which may be considered invasive and cost-prohibitive.

Hematologic evaluation, including a CBC and cytologic examination of a blood smear, however, is inexpensive and commonly performed during diagnostic testing and treatment of dogs with lymphoma or IBD. Hematologic abnormalities have been noted in 50% to 85% of dogs with lymphoma, and anemia is one of the most common paraneoplastic syndromes reported in dogs with lymphoma, with reported prevalence ranging from 30% to 77%.3,9–13 Further, lymphoma has been associated with RBC abnormalities, including acanthocytes, codocytes, eccentrocytes, poikilocytosis,14 polychromasia, schistocytes, sideroblasts and siderocytes,15 and spherocytes.3,16–20 Although investigation of the prevalence of each specific RBC abnormality in blood smears of dogs with lymphoma is lacking, in a previous study,21 lymphoma was the most common (7/20 [35%]) disease encountered in a population of dogs with irregularly spiculated RBCs (eg, acanthocytes, keratocytes, and schistocytes). In dogs with IBD, however, RBC morphological characteristics are rarely reported, and results of CBCs, with the exception of occasionally noted anemia, are commonly within reference limits.22,23 In addition, oxidative stress, which may contribute to eccentrocytosis, has been associated with lymphoid neoplasia in humans and animals.24–28 A retrospective study20 of 4,251 dogs with various conditions found that 60 had eccentrocytosis and that all 5 dogs with T-cell lymphoma had eccentrocytosis. However, the actual prevalence of eccentrocytosis in a group of dogs with lymphoma has yet to be evaluated.

The objectives of the study reported here were to assess the prevalences of anemia and various RBC morphological anomalies in dogs with lymphoma versus IBD and to evaluate potential relationships between these variables and the severity of lymphoma. We hypothesized that anemia and RBC anomalies would occur more commonly in dogs with lymphoma than in dogs with IBD. We also believed that there would be more pronounced RBC anomalies in dogs with versus dogs without advanced-stage, T-cell, or high-grade lymphoma.

Materials and Methods

Animals

Client-owned dogs in which lymphoma or IBD had been diagnosed between January 1, 2006, and December 31, 2014, were identified from a search of the medical records of the Centre Hospitalier Universitaire Vétérinaire, Faculté de Médecine Vétérinaire of the Université de Montréal by use of the following search terms: alimentary, gastrointestinal, intestinal, colorectal, and multicentric lymphoma; lymphadenomegaly; IBD; lymphangiectasia; lymphocytic-plasmacytic, eosinophilic, and granulomatous gastritis, enteritis, or colitis; gastrointestinal biopsies; vomiting; diarrhea; and weight loss. Inclusion criteria for dogs were an unequivocal diagnosis of lymphoma or IBD on the basis of cytologic (for lymphoma only) or histologic (for lymphoma or IBD) examination, a CBC that had been performed ≤ 2 weeks prior to diagnosis, and availability of blood smears saved from analyses performed ≤ 2 weeks prior to diagnosis. Dogs with less specific diagnoses (eg, round-cell neoplasia or possible emerging lymphoma) and dogs in which the diagnosis was only suspected (eg, likely compatible with a given disease) were excluded. Additional exclusion criteria were lack of a CBC; a CBC performed ≥ 2 weeks before the diagnosis, any time after the diagnosis, or after any intervention (eg, anesthesia and biopsies); a lack of blood smear saved from analyses performed ≤ 2 weeks prior to diagnosis; a diagnosis made previously at another hospital; and treatment with any type of cytotoxic chemotherapy prior to referral. Dogs were not excluded if they had received corticosteroids before referral.

Client-owned healthy dogs included in the study were randomly selected from the population of dogs that were examined during the same time period at the Université de Montréal for annual health assessment or vaccination and that did not have any abnormalities on clinical examination or any history of medical problems. To avoid a biased selection of healthy dogs with normal CBCs and a type 1 error, a clinically normal CBC was not part of the inclusion criteria for the healthy dog population.

Medical records review

Medical records for dogs fitting the inclusion criteria were reviewed for signalment, medical history, abnormal findings on physical examination, results of hematologic analyses and diagnostic imaging (eg, thoracic radiography, abdominal ultrasonography, CT, and MRI), and diagnosis. In addition, sampling sites and cytologic or histologic results (eg, IBD type, lymphoma cell size, and histologic grade) for fine-needle aspirates and biopsy samples were also recorded as well as the form and immunotype of lymphoma, when determined. Dogs were grouped according to whether they were healthy, had IBD, or had lymphoma, and those in the lymphoma group were further subdivided by clinical stage (I to V) and substage (a or b) of lymphoma according to the WHO classification.2 Dogs with lymphoma affecting any location other than the lymph nodes, liver, or spleen were classified as having stage V lymphoma.

Hematologic assessment

Hematologic variables recorded for each dog included anemia (yes or no), Hct (reference range, 40% to 56%), Hgb concentration (reference range, 139 to 198 g/L), RBC count (reference range, 5.4 to 8.6 × 106/μL), MCV (reference range, 62 to 73 fL), MCHC (reference range, 325 to 373 g/L), total number of different RBC anomalies, and individual RBC anomaly semiquantitative value (on a scale of 0 to 4+). For each dog, anemia was considered to have been present if ≥ 2 of the following criteria were met: Hct < 40%, Hgb concentration < 139 g/L, or RBC count < 5.4 × 106 RBCs/μL. All CBC data were generated at the Service de Diagnostic, Faculté de Médecine Vétérinaire, Université of Montréal with a hematology analyzer.a

All blood smears saved from analyses performed ≤ 2 weeks prior to diagnosis were reassessed by a board-certified clinical veterinary pathologist (CGB) blinded to each final diagnosis. For each blood smear, 10 hpf (ie, 1,000X) were randomly selected from the monolayer region, counts of 10 different RBC morphological anomalies (acanthocytes, codocytes, eccentrocytes, HBs, keratocytes, nucleated RBCs, poikilocytes, polychromasia, schistocytes, and spherocytes) were individually recorded, the mean count for each of the 10 different RBC anomalies was calculated, and the mean count was then converted to a value on a semiquantitative scale from 0 to 4+ as previously described29 (Appendix). Of the 10 different RBC anomalies considered, the total number of different anomalies with a semiquantitative value ≥ 1+ was also calculated for each dog.

Statistical analysis

Results were compared among the healthy, IBD, and lymphoma groups as well as within the lymphoma group (ie, stages I to III, compared with stages IV and V; substage a, compared with substage b; and small-cell lymphoma, compared with intermediate- and large-cell lymphoma). A general linear model that included mean Hct, MCV, and MCHC was used to evaluate condition (ie, lymphoma, IBD, or healthy; stages I to III or stages IV and V; substage a or b; and small-cell lymphoma or intermediate- and large-cell lymphoma) as factors. The model also took into account unequal variances among conditions. Tukey post hoc tests were used to compare the means of pairs. In addition, anemia and distribution of morphological anomalies were compared among groups with a Cochran-Mantel-Haenszel test, which accounted for the ordinal scale used for the RBC anomalies. Values of P ≤ 0.05 were considered significant, and statistical analyses were performed with standard software.b

The ideal cutoff for the total number of RBC anomalies per dog that could differentiate dogs in the lymphoma group from dogs in the IBD group was identified with an ROC curve calculation.c Once the best threshold was established, the sensitivity, specificity, positive predictive value, and negative predictive value for the total number of RBC anomalies per dog were calculated.

Results

Animals

Eighty-two dogs (lymphoma group, n = 32; IBD group, 23; and healthy group, 27) met the inclusion criteria for the study. Mean ages at diagnosis for dogs in the lymphoma, IBD, and healthy groups were 7.9 years (range, 2 to 14 years), 6.3 years (range, 1 to 13 years), and 6.4 years (range, 2 to 14 years), respectively. The lymphoma group had 14 males and 18 females, the IBD group had 18 males and 5 females, and the healthy group had 15 males and 12 females. The lymphoma group included 5 Golden Retrievers, 4 Bernese Mountain Dogs, 4 Labrador Retrievers, 4 mixed-breed dogs, 2 Boxers, 2 Poodles, 2 pit bull-type dogs, and 9 other purebred dogs. The IBD group consisted of 4 Yorkshire Terriers, 3 Bernese Mountain Dogs, 3 mixed-breed dogs, 2 Golden Retrievers, 2 German Shepherd Dogs, and 9 other purebred dogs. The healthy dogs consisted of 17 mixed-breed dogs, 5 Golden Retrievers, and 5 Labrador Retrievers.

Previous treatments and comorbidities

No dog had received cytotoxic chemotherapy at the time of diagnosis. For the lymphoma group, 15 dogs had not received any other form of treatment, whereas the remaining dogs had received antimicrobials (n = 10), prednisone (8), or NSAIDs (5). Six dogs in the IBD group had not received any medication before referral; however, the remaining dogs had received antimicrobials (n = 11), prednisone (5), antacids (5), sucralfate (4), or fenbendazole (4), alone or in combination. Thirty-three of the 55 (60%) dogs in the IBD (14/23 [61%]) and lymphoma (19/32 [59%]) groups combined had concurrent diseases at the time of referral. These diseases included allergic dermatitis or otitis (n = 13), hypothyroidism (3), urinary incontinence and anxiety disorders (2 each), and chronic kidney disease, hypertriglyceridemia, hypoadrenocorticism, Cushing disease, subaortic stenosis, and borreliosis (1 each).

For dogs in the IBD group, IBD had been diagnosed on the basis of results of histologic evaluation of gastrointestinal biopsy specimens. Clinical features of these dogs were recorded (Table 1). The clinical form and affected segment of the gastrointestinal tract were not mentioned in the medical records of 5 dogs; however, 2 to 3 segments of the gastrointestinal tract were noted to have been affected in 4 dogs. Although no dogs had simultaneous diagnoses of IBD and lymphoma, a 4-year-old spayed female Bernese Mountain Dog in which lymphocytic-plasmacytic enteritis had initially been diagnosed developed large-cell hepatosplenic lymphoma 3 years later. This dog was included in the IBD group because there was no evidence of lymphoma at the time of IBD diagnosis and initial hematologic assessment.

Table 1—

Clinical characteristics of 23 client-owned dogs in which IBD was diagnosed at a veterinary teaching hospital between January 1, 2006, and December 31, 2014, and for which the presence of anemia and RBC morphological anomalies were assessed.

CharacteristicNo. of dogs
Clinical form of IBD
 Lymphocytic-plasmacytic7
 Eosinophilic4
 Lymphangiectasia4
 Mixed3
 Unknown5
Gastrointestinal segment affected*
 Stomach8
 Duodenum and jejunum7
 Colon and rectum7
 Unknown5

Some dogs had affected segments in more than one category.

For the lymphoma group, we initially intended to include only cases with primary gastrointestinal lymphoma; however, only 7 dogs satisfied the inclusion criteria. A descriptive subanalysis of the gastrointestinal lymphoma group, compared with the IBD group, was performed, but a decision was made to include all cases of lymphoma to achieve a sample size that could be statistically compared with the IBD group. Clinical features of dogs in the lymphoma group were recorded (Table 2). The diagnosis of lymphoma in all 7 dogs with gastrointestinal lymphoma and in some dogs with cutaneous or multicentric lymphoma was made on the basis of histopathologic lesions identified in biopsy samples (gastrointestinal tract [n = 6], lymph node [3], bone marrow [2], and skin [1]). The diagnosis of lymphoma in the remaining dogs was made on the basis of results of cytologic examination of fine-needle aspirates. In dogs with gastrointestinal lymphoma, the jejunum (n = 3) was most commonly affected, followed by the entire gastrointestinal tract (ie, multifocal lesions in the stomach and small intestines; 2), duodenum (1), and ileum and cecum (1). Eleven dogs had stage V lymphoma, which included all 7 dogs with gastrointestinal lymphoma (these dogs also had involvement of abdominal lymph nodes [n = 7] and pancreas [1]); 2 dogs with leukemic lymphoma; 1 dog with nodal, ocular, and CNS lymphoma; and 1 dog with nodal and pulmonary lymphoma. The lymphoma cell size was not specified in the medical records of 4 dogs, and these 4 dogs were excluded from comparisons of results for dogs with small-cell lymphoma versus intermediate- and large-cell lymphoma. Comparisons of results between dogs with B-cell versus T-cell lymphoma could not be performed because of the small number of dogs in which immunophenotype had been determined (n = 8).

Table 2—

Clinical characteristics of 32 client-owned dogs ir which lymphoma was diagnosed at a veterinary teaching hospital between January 1, 2006, and December 31, 2014, and for which the presence of anemia and RBC morphological anomalies were assessed.

CharacteristicNo. of dogs
Clinical form
 Multicentric24
 Gastrointestinal7
 Cutaneous1
Immunophenotype
 T-cell (CD3+)5
 B-cell (CD79+)2
 T-cell-rich B-cell1
 Undetermined24
Cell size
 Large15
 Intermediate6
 Small7
 Undetermined4
Clinical stage*
 I0 (0; 0)
 II1 (0; 1)
 III7 (4; 3)
 IV13 (4; 9)
 V11 (2; 9)

Numbers in parentheses represent number of dogs with substage a lymphoma; number of dogs with substage b lymphoma.

Hematologic findings

All hematologic data (eg, Hct, Hgb concentration, RBC count, MCHC, and MCV) were tested for normality with the Shapiro-Wilk test and followed a normal distribution. The prevalence of anemia in the lymphoma group (17/32 [53%]) was significantly (P = 0.02 and P < 0.001, respectively) higher, compared with the IBD group (5/23 [22%]) and healthy group (1/27 [4%]). The only dog considered anemic in the healthy group had no abnormal findings on physical examination, no known abnormalities in its medical history, and no other abnormalities noted in the results of CBC or serum chemistry analyses. Hence, this dog was not excluded from the healthy group. Within the lymphoma group, a particular emphasis was placed on anomalies encountered in dogs with primary gastrointestinal lymphoma; however, the low number of cases precluded a statistical comparison of results with those of the IBD group. Nonetheless, it was interesting that 5 of the 7 dogs with primary gastrointestinal lymphoma were anemic.

When stage and substage of lymphoma were considered, the prevalence of anemia was significantly (P = 0.045) higher in dogs with stages IV and V lymphoma (15/24 [62.5%]), compared with stages II and III (1/8 [12.5%]). The prevalence of anemia did not differ significantly (P = 0.52 and P = 0.29, respectively) between dogs with small-cell lymphoma (n = 7) versus intermediate- or large-cell lymphoma (21) or between dogs with substage a (10) versus substage b (22) disease.

The linear model indicated that, when considered individually, mean Hct and mean Hgb concentration differed significantly (P = < 0.001) among the 3 groups (lymphoma group [mean Hct, 40.3%; mean Hgb concentration, 134.8 g/L], IBD group [mean Hct, 43.8%; mean Hgb concentration, 148.2 g/L], and healthy group [mean Hct, 48%; mean Hgb concentration, 170.1 g/L]). However, mean Hct and Hgb concentrations did not differ significantly (P = 0.27 and P = 0.21, respectively) between the lymphoma group and IBD group.

Mean ± SEM of the MCHC was within reference limits for all groups (lymphoma group, 333.2 ± 2.9 g/L; IBD group, 338.4 ± 3.5 g/L; and healthy group, 355.3 ± 1.6 g/L). Similarly, mean ± SEM of the MCV was within reference limits for all groups (lymphoma group, 66.7 ± 0.94 fL; IBD group, 67.4 ± 0.96 fL; and healthy group, 67.2 ± 0.53 fL]). Further, 12 of the 17 anemic dogs in the lymphoma group and 3 of the 5 anemic dogs in the IBD group had normocytic and normochromic anemia.

Blood smear assessment

The blood smears of 4 dogs in the lymphoma group were not available for reassessment. Although data from these cases were still included in the rest of the analyses, they were excluded from RBC anomaly analyses.

Total number of different RBC morphological anomalies per dog for the lymphoma group (mean, 3.7; median, 4; range, 1 to 8) was significantly (P = 0.01 and P < 0.001, respectively) higher than that for the IBD group (mean, 2.5; median, 2; range, 1 to 6) and the healthy group (mean, 0.4; median, 0; range, 0 to 3). In addition, the total number of different RBC anomalies per dog was significantly (P < 0.001) higher for the IBD group than for the healthy group (Figure 1). Within the lymphoma group, the total number of RBC anomalies per dog was significantly (P = 0.02) higher for dogs with substage b (mean, 4.1; median, 4; range, 2 to 8; n = 20) than for dogs with substage a (mean, 2.5; median, 2.5; range, 1 to 4; 8; Figure 2) lymphoma. No significant (P = 0.91 and P = 0.60, respectively) differences were noted between small-cell lymphoma (n = 7), compared with intermediate- and large-cell lymphoma (21), or between stages II and III (8), compared with stages IV and V (20), for all RBC anomalies evaluated individually.

Figure 1—
Figure 1—

Histogram depicting the distribution of total number of different RBC morphological anomalies with a semiquantitative value ≥ 1 + (on a scale from 0 to 4+) identified per dog on blood smears from 78 client-owned dogs that were healthy (light gray bars; n = 27) or that had IBD (dark gray bars; 23) or lymphoma (black bars; 28). Blood smears for 4 dogs with lymphoma were not available for reassessment.

Citation: Journal of the American Veterinary Medical Association 254, 4; 10.2460/javma.254.4.487

Figure 2—
Figure 2—

Histogram depicting the distribution of total number of different RBC morphological anomalies with a semiquantitative value of ≥ 1+ identified per dog on the basis of results from blood smears reviewed for dogs in the lymphoma group in Figure 1 that were further categorized as having lymphoma substage a (gray bars; n = 8) or substage b (black bars; 20).

Citation: Journal of the American Veterinary Medical Association 254, 4; 10.2460/javma.254.4.487

Results of the ROC curve analysis indicated that the ideal balance between sensitivity and specificity to differentiate lymphoma from IBD was obtained with a threshold of 3 different RBC morphological anomalies/dog (area under the fitted curve, 0.7239 ± 0.0727; P = 0.001) out of the maximum number of 10 possible different RBC morphological anomalies considered in the study. At this threshold, the sensitivity, specificity, and positive and negative predictive values were 71%, 70%, 74%, and 67%, respectively. In other words, 74% of dogs that had ≥ 3 different RBC morphological anomalies with a semiquantitative score of ≥ 1+ had lymphoma and not IBD. Conversely, 67% of dogs that had ≤ 2 different RBC anomalies had IBD and not lymphoma. Interestingly, although no statistical conclusions could be made regarding the 7 dogs with gastrointestinal lymphoma, all 7 had ≥ 3 RBC morphological anomalies, and the mean number of different RBC morphological anomalies for these dogs was 3.7 (median, 4; range, 3 to 7).

When the different kinds of RBC morphological anomalies were considered individually, 5 anomalies (eccentrocytes, keratocytes, polychromasia, schistocytes, and spherocytes) were significantly (P < 0.05) more common in the lymphoma group than in the healthy group (Figure 3). Polychromasia and keratocytes were significantly (P < 0.05) more common in the IBD group, compared with the healthy group. Presence of eccentrocytes, on the other hand, was the only anomaly significantly (P = 0.03) more common in the lymphoma group, compared with the IBD group, with 0, 1+, and 2+ values for this anomaly found in 71% (20/28), 25% (7/28), and 4% (1/28) of dogs in the lymphoma group, compared with 96% (22/23), 4% (1/23), and 0% of dogs in the IBD group.

Figure 3—
Figure 3—

Histogram depicting percentages of dogs in Figure 1 with a semiquantitative value of ≥ 1+ for 5 specific RBC morphological anomalies. *Prevalence was significantly (P < 0.05) higher in the lymphoma group, compared with the healthy group. †Prevalence was significantly (P < 0.05) higher in the IBD group, compared with the healthy group. ‡Prevalence was significantly (P = 0.03) higher in the lymphoma group, compared with the IBD group. See Figure 1 for remainder of key.

Citation: Journal of the American Veterinary Medical Association 254, 4; 10.2460/javma.254.4.487

Although the small number of dogs with primary gastrointestinal lymphoma hindered the completion of statistical tests, the prevalence of all 5 anomalies (eccentrocytes, keratocytes, polychromasia, schistocytes, and spherocytes) identified as being more common for dogs with lymphoma than for healthy dogs was even higher in the 7 dogs with primary gastrointestinal lymphoma. In particular, all 7 dogs with primary gastrointestinal lymphoma had polychromasia (1+ to 2+), 4 had spherocytes (1+ to 2+), 3 had keratocytes (1+ to 2+), 3 had schistocytes (1+ to 2+), and 3 had eccentrocytes (1+ to 2+).

Discussion

Findings indicated that the prevalence of anemia for dogs with lymphoma in the present study was 53%, which was higher than previously reported in certain studies3,10–12 but similar to the prevalence in others.9,13 The mean ± SEMs of the MCV and MCHC were within reference limits for the lymphoma group and IBD group, and most of the anemias were normochromic and normocytic. A likely explanation for the prevalence of anemia in the present study being higher in the lymphoma group than in the IBD group could have been that dogs with lymphoma had more severe anemia of chronic disease secondary to production of interleukin-1 and interleukin-6 by neoplastic lymphocytes, stimulation of hepcidin production by hepatocytes, and inhibition of intestinal absorption of iron. Results of the present study also indicated that the lymphoma group had a high prevalence of RBC anomalies associated with pathological conditions that cause fragmentation of RBCs (eg, schistocytes and keratocytes). This finding suggested that RBC fragmentation (eg, secondary to disseminated intravascular coagulation and microangiopathy) could have been another reason for the higher prevalence of anemia among dogs with lymphoma. Although gastrointestinal bleeding can be encountered with both IBD and gastrointestinal lymphoma, the latter is more invasive and could trigger more severe bleeding, which also could have contributed to the higher prevalence of anemia in the lymphoma group. Autoimmune hemolytic anemia, pure red cell aplasia, hypersplenism, and bone marrow infiltration are also potential causes of anemia in dogs with lymphoma.12,30–36

Overall, the aforementioned mechanisms could explain the higher prevalence of 5 RBC anomalies (eccentrocytes, keratocytes, polychromasia, schistocytes, and spherocytes) found in the lymphoma group in the present study. Schistocytes and keratocytes can result from microangiopathy, but can also result from increased RBC membrane fragility (eg, secondary to anemia of chronic disease and iron deficiency), liver disease (eg, secondary to stage IV lymphoma),18,19 or myelofibrosis (eg, secondary to bone marrow invasion).16,32,37,38 Spherocytes and polychromasia are frequently associated with regenerative anemia, which can be caused by autoimmune-mediated hemolytic anemia or gastrointestinal bleeding.30,35,39 Eccentrocytes may ensue with oxidative stress and are commonly observed in various cancers including lymphoma and chronic leukemia.24–28

In the present study, the area under the fitted curve obtained on ROC analysis for the number of different RBC anomalies for the lymphoma group, compared with the IBD group, was 0.7239 ± 0.0727 (P = 0.001) and indicated that the cutoff of 3 RBC anomalies was a fair predictor to help differentiate dogs with lymphoma from dogs with IBD. Dogs in the present study were more likely to have had lymphoma if they had ≥ 3 different RBC anomalies found on the blood smear assessment.

Many hypotheses have been proposed to explain the rise in oxidative stress associated with various types of cancer, including lymphoma. These include increased production of reactive oxygen species by neoplastic lymphocytes, decreased superoxide dismutase activities, reduced glutathione concentration, mutations in mitochondrial DNA or reduction of mitochondrial burdens (leading to anomalies in respiratory chain function and increased superoxide production), alteration in nicotinamide adenine dinucleotide phosphate-oxidase synthesis within glucose-lacking tissues (eg, necrotic areas of rapidly growing tumors), and production of endothelin (chemoattractant for macrophages and neutrophils that produce reactive oxygen species) by tumorous cells.24–28,40–42

The 29% prevalence of eccentrocytes found in the lymphoma group of the present study was much lower than the 100% prevalence previously reported in 5 cases of T-cell lymphoma.20 But when only dogs with gastrointestinal lymphoma in the present study were considered, the prevalence of eccentrocytes was 43%. The discrepancy between studies could be related to differences in sample size (ie, 28 dogs with lymphoma in the present study, compared with 5 dogs in the study by Caldin et al20). In addition, dogs with B-cell or T-cell lymphoma were evaluated in the present study, whereas only dogs with T-cell lymphoma were evaluated in the other. Taken together, our results suggested that more RBC morphological anomalies indicative of oxidative stress were observed on blood smears from dogs with lymphoma, and these anomalies appeared to have been even more common among dogs with gastrointestinal lymphoma. A prospective study performed on a larger population of dogs with gastrointestinal lymphoma alone is warranted to confirm this finding.

Another potential RBC anomaly arising from oxidative stress is HB formation, but HBs were not observed in the present study. The lack of HBs was consistent with results of previous studies that indicate oxidative stress commonly results in eccentrocyte formation in dogs43,44 and HB formation in cats.37,45,46 A proposed mechanism for HB formation is oxidation of thiol groups on the Hgb chain by reactive oxygen species, leading to clumping of Hgb, which then appears as projections extending from the cell. Feline Hgb has 8 thiol groups, whereas canine Hgb has 2 thiol groups. In addition, cats have a nonsinusoidal spleen architecture (ie, has large pores in splenic pulp venules) that is more permissive than that of a dog's spleen for RBCs with HBs.37,45,46 A study evaluating cats with IBD and gastrointestinal lymphoma would be warranted to determine whether cats with lymphoma have a higher prevalence of HBs than do cats with IBD.

Dogs with substage b lymphoma generally have a worse prognosis than do dogs with substage a.2 Results of the present study supported this in that the total number of RBC anomalies was higher in dogs with substage b lymphoma than in dogs with substage a lymphoma. Further, although no statistical conclusion could be drawn because of the small number of affected dogs, high prevalences of keratocytes, acanthocytes, schistocytes, and codocytes were detected in dogs with substage b lymphoma in the present study. Many of these RBC anomalies can be associated with severe and potentially life-threatening disorders, such as disseminated intravascular coagulation and microvascular injury (associated with keratocytes, acanthocytes, and schistocytes), severe hepatic injury (associated with acanthocytes and codocytes), or myelofibrosis and myelophthisis (associated with schistocytes). Prospective studies with predetermined end points, such as progression-free and survival time analyses, would be helpful in investigating the potential prognostic value of these RBC anomalies in dogs with lymphoma. Moreover, it could be beneficial to include a broader panel of RBC anomalies to help determine with more certainty the causes of these anomalies. For example, the presence of ovalocytes and dacryocytes could strengthen the suspicion of myelofibrosis.

A major limitation of the present study was its retrospective nature. This limitation was partially offset by the fact that all blood smears were reassessed by a single board-certified clinical veterinary pathologist. In addition, the RBC morphological anomaly values were calculated on the basis of RBC anomalies per hpf instead of the number of anomalies per RBC. Although anemia could have affected the number of RBC anomalies per field, the method used most resembled that used when the blood smears were originally evaluated by the institution's clinical pathology laboratory.

Another major limitation was the small number (n = 7) of dogs with primary gastrointestinal lymphomas that met the inclusion criteria. The initial goal of the present study was to evaluate anemia and various RBC anomalies associated with the gastrointestinal form of lymphoma versus IBD. Our preliminary results were promising; however, statistical conclusions could not be made. Hence, to increase the number of cases and permit statistical comparisons, a decision was made to include dogs with all forms of lymphoma in this study. The authors are fully aware that cutaneous or multicentric lymphoma and IBD have very different pathological processes and can usually be differentiated easily by their respective clinical manifestations. Therefore, our results should only be used to lay the groundwork for a larger, prospective study investigating primary gastrointestinal lymphoma, compared with IBD, in dogs and eventually in cats.

Comorbidities were another limitation in that 33 of the 55 (60%) dogs in the IBD and lymphoma groups combined had concurrent diseases at the time of referral, and these comorbidities could have been confounding factors in the present study. Some of these diseases could have contributed to anemia or affected RBC morphological characteristics, including hypothyroidism (n = 3) as well as hypoadrenocorticism, chronic kidney disease, hypertriglyceridemia, and borreliosis (1 each). However, many of the comorbidities noted in the present study are likely inherent to any study involving a population of older dogs. Although we acknowledge that it might have introduced a bias to our results, we chose not to exclude dogs with comorbidities because we believe they reflected the reality of many other dogs affected by diseases such as gastrointestinal lymphoma or IBD.

The most important result of the present study was that the total number of different RBC morphological anomalies per dog, as calculated from blood smear assessments, was significantly higher in dogs with lymphoma, compared with dogs with IBD. Further, the RBC anomalies were even more common in dogs with gastrointestinal lymphoma, although the number of such affected dogs was too small to allow for statistical analyses. Moreover, the prevalence of 5 anomalies (eccentrocytes, keratocytes, polychromasia, schistocytes, spherocytes), when evaluated individually, was higher in both the lymphoma group and the gastrointestinal lymphoma subgroup, compared with the healthy group. Presence of eccentrocytes, however, was the only RBC anomaly significantly more common in the lymphoma group (8/28 [29%]), compared with the IBD group (1/23 [4%]). Further studies evaluating larger populations of dogs with gastrointestinal lymphoma or IBD may confirm the preliminary findings of the present study and potentially result in the use of blood smears to help differentiate lymphoma from IBD in dogs.

Acknowledgments

Presented in abstract form at the 2016 American College of Veterinary Internal Medicine Forum Research Abstract Program, Denver, June 2016.

No third-party funding or support was received in association with the present study or the writing or publication of the manuscript. The authors declare that there were no conflicts of interest.

The authors thank M. Guy Beauchamp for his assistance in statistical analysis.

ABBREVIATIONS

HB

Heinz body

Hgb

Hemoglobin

IBD

Inflammatory bowel disease

MCHC

Mean corpuscular hemoglobin concentration

MCV

Mean corpuscular volume

Footnotes

a.

Advia 120 hematology system, Siemens Healthcare GmbH, Erlangen, Germany.

b.

SAS, version 9.4, SAS Institute Inc, Cary, NC.

c.

ROC analysis, Johns Hopkins University School of Medicine, Baltimore, Md. Available at: www.jrocfit.org. Accessed May 11, 2016.

References

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    • Search Google Scholar
    • Export Citation
  • 2. Vail DM, Pinkerton ME, Young KM. Canine lymphoma and lymphoid leukemias. In: Withrow SJ, Vail DM, Page RL, eds. Withrow & MacEwen's small animal clinical oncology. St Louis: Elsevier Saunders, 2013;608638.

    • Search Google Scholar
    • Export Citation
  • 3. Couto CG, Rutgers HC, Sherding RG, et al. Gastrointestinal lymphoma in 20 dogs. A retrospective study. J Vet Intern Med 1989;3:7378.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Carrasco V, Rodriguez-Bertos A, Rodriguez-Franco F, et al. Distinguishing intestinal lymphoma from inflammatory bowel disease in canine duodenal endoscopic biopsy samples. Vet Pathol 2015;52:668675.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Breitschwerdt EB, Waltman C, Hagstad HV, et al. Clinical and epidemiologic characterization of a diarrheal syndrome in Basenji dogs. J Am Vet Med Assoc 1982;180:914920.

    • Search Google Scholar
    • Export Citation
  • 6. Scott KD, Zoran DL, Mansell J, et al. Utility of endoscopic biopsies of the duodenum and ileum for diagnosis of inflammatory bowel disease and small cell lymphoma in cats. J Vet Intern Med 2011;25:12531257.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Ohmura S, Leipig M, Schöpper I, et al. Detection of monoclonality in intestinal lymphoma with polymerase chain reaction for antigen receptor gene rearrangement analysis to differentiate from enteritis in dogs. Vet Comp Oncol 2017;15:194207.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Kiupel M, Smedley RC, Pfent C, et al. Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples. Vet Pathol 2011;48:212222.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Martini V, Melzi E, Comazzi S, et al. Peripheral blood abnormalities and bone marrow infiltration in canine large B-cell lymphoma: is there a link? Vet Comp Oncol 2015;13:117123.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Miller AG, Morley PS, Rao S, et al. Anemia is associated with decreased survival time in dogs with lymphoma. J Vet Intern Med 2009;23:116122.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Abbo AH, Lucroy MD. Assessment of anemia as an independent predictor of response to chemotherapy and survival in dogs with lymphoma: 96 cases (1993–2006). J Am Vet Med Assoc 2007;231:18361842.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Lucroy MD, Christopher MM, Kraegel SA, et al. Anaemia associated with canine lymphoma. Comp Haematol Int 1998;8:16.

  • 13. Tasca S, Carli E, Caldin M, et al. Hematologic abnormalities and flow cytometric immunophenotyping results in dogs with hematopoietic neoplasia: 210 cases (2002–2006). Vet Clin Pathol 2009;38:212.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Badylak SF, Van Vleet JF, Herman EH, et al. Poikilocytosis in dogs with chronic doxorubicin toxicosis. Am J Vet Res 1985;46:505508.

  • 15. Canfield PJ, Watsond ADJ, Ratcliffed RCC. Dyserythropoiesis, sideroblasts/siderocytes and hemoglobin crystallization in a dog. Vet Clin Pathol 1987;16:2128.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Hoff B, Lumsden JH, Valli VE. An appraisal of bone marrow biopsy in assessment of sick dogs. Can J Comp Med 1985;49:3442.

  • 17. Weiss DJ. Sideroblastic anemia in 7 dogs (1996–2002). J Vet Intern Med 2005;19:325328.

  • 18. Keller SM, Vernau W, Hodges J, et al. Hepatosplenic and hepatocytotropic T-cell lymphoma: two distinct types of T-cell lymphoma in dogs. Vet Pathol 2013;50:281290.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Fry MM, Vernau W, Pesavento PA, et al. Hepatosplenic lymphoma in a dog. Vet Pathol 2003;40:556562.

  • 20. Caldin M, Carli E, Furlanello T, et al. A retrospective study of 60 cases of eccentrocytosis in the dog. Vet Clin Pathol 2005;34:224231.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Weiss DJ, Kristensen A, Papenfuss N. Qualitative evaluation of irregularly spiculated red blood cells in the dog. Vet Clin Pathol 1993;22:117121.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Boyle TE, Bissett SA. Idiopathic inflammatory bowel disease. Vetlearn 2007;9:712.

  • 23. Fogle JE, Bissett SA. Mucosal immunity and chronic idiopathic enteropathies in dogs. Compend Contin Educ Vet 2007;29:290302.

  • 24. Winter JL, Barber LG, Freeman L, et al. Antioxidant status and biomarkers of oxidative stress in dogs with lymphoma. J Vet Intern Med 2009;23:311316.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Abou-Seif MA, Rabia A, Nasr M. Antioxidant status, erythrocyte membrane lipid peroxidation and osmotic fragility in malignant lymphoma patients. Clin Chem Lab Med 2000;38:737742.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Honda M, Yamada Y, Tomonaga M, et al. Correlation of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, and clinical features of hematological disorders: a pilot study. Leuk Res 2000;24:461468.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Halliwell B. Oxidative stress and cancer: have we moved forward? Biochem J 2007;401:111.

  • 28. Vajdovich P, Kriska T, Mézes M, et al. Redox status of dogs with non-Hodgkin lymphomas. An ESR study. Cancer Lett 2005;224:339346.

  • 29. Weiss DJ. Uniform evaluation and semiquantitative reporting of hematologic data in veterinary laboratories. Vet Clin Pathol 1984;13:2731.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Morley P, Mathes M, Guth A, et al. Anti-erythrocyte antibodies and disease associations in anemic and nonanemic dogs. J Vet Intern Med 2008;22:886892.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Madewell BR, Feldman BF. Characterization of anemias associated with neoplasia in small animals. J Am Vet Med Assoc 1980;176:419425.

    • Search Google Scholar
    • Export Citation
  • 32. Weiss DJ, Smith SA. A retrospective study of 19 cases of canine myelofibrosis. J Vet Intern Med 2002;16:174178.

  • 33. Weiss DJ. Primary pure red cell aplasia in dogs: 13 cases (1996–2000). J Am Vet Med Assoc 2002;221:9395.

  • 34. Moullet I, Salles G, Ketterer N, et al. Frequency and significance of anemia in non-Hodgkin's lymphoma patients. Ann Oncol 1998;9:11091115.

  • 35. McGovern KF, Lascola KM, Davis E, et al. T-cell lymphoma with immune-mediated anemia and thrombocytopenia in a horse. J Vet Intern Med 2011;25:11811185.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Graff EC, Spangler EA, Smith A, et al. Hematologic findings predictive of bone marrow disease in dogs with multicentric large-cell lymphoma. Vet Clin Pathol 2014;43:505512.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Christopher MM, Lee SE. Red cell morphologic alterations in cats with hepatic disease. Vet Clin Pathol 1994;23:712.

  • 38. Albitar M, Manshouri T, Shen Y, et al. Myelodysplastic syndrome is not merely “preleukemia.”. Blood 2002;100:791798.

  • 39. Ehrenfeld M, Abu-Shakra M, Buskila D, et al. The dual association between lymphoma and autoimmunity. Blood Cells Mol Dis 2001;27:750756.

  • 40. Benz CC, Yau C. Ageing, oxidative stress and cancer: paradigms in parallax. Nat Rev Cancer 2008;8:875879.

  • 41. Zhou Y, Hileman EO, Plunkett W, et al. Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents. Blood 2003;101:40984104.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Blackburn RV, Spitz DR, Liu X, et al. Metabolic oxidative stress activates signal transduction and gene expression during glucose deprivation in human tumor cells. Free Radic Biol Med 1999;26:419430.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Yamato O, Kasai E, Katsura T, et al. Heinz body hemolytic anemia with eccentrocytosis from ingestion of Chinese chive (Allium tuberosum) and garlic (Allium sativum) in a dog. J Am Anim Hosp Assoc 2005;41:6873.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Yamoto O, Maede Y. Susceptibility to onion-induced hemolysis in dogs with hereditary high erythrocyte reduced glutathione and potassium concentrations. Am J Vet Res 1992;53:134137.

    • Search Google Scholar
    • Export Citation
  • 45. Christopher MM, White JG, Eaton JW. Erythrocyte pathology and mechanisms of Heinz body-mediated hemolysis in cats. Vet Pathol 1990;27:299310.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. Christopher MM. Relation of endogenous Heinz bodies to disease and anemia in cats: 120 cases (1978–1987). J Am Vet Med Assoc 1989;194:10891095.

    • Search Google Scholar
    • Export Citation

Appendix

Semiquantitative scoring system for the frequency of RBC morphological anomalies observed during microscopic examination of blood smears from dogs (From Weiss DJ. Uniform evaluation and semiquantitative reporting of hematologic data in veterinary laboratories. Vet Clin Pathol 1984;13:27–31. Reprinted with permission from John Wiley and Sons Publishing.).

 Score
 01+2+3+4+
Poikilocytes< 33–1011–5051–200> 200
Polychromasia< 22–78–1415–29> 30
Codocytes< 33–56–1516–30> 30
Spherocytes< 55–1011–5051–150> 150
Nucleated RBC01–23–89–20> 20
Acanthocytes01–23–89–20> 20
Keratocytes01–23–89–20> 20
Schistocytes01–23–89–20> 20
Eccentrocytes01–23–89–20> 20
Heinz bodies01–23–89–20> 20

Scores were assigned on the basis of mean number of abnormal cells per 1,000X microscopic monolayer field.

  • Figure 1—

    Histogram depicting the distribution of total number of different RBC morphological anomalies with a semiquantitative value ≥ 1 + (on a scale from 0 to 4+) identified per dog on blood smears from 78 client-owned dogs that were healthy (light gray bars; n = 27) or that had IBD (dark gray bars; 23) or lymphoma (black bars; 28). Blood smears for 4 dogs with lymphoma were not available for reassessment.

  • Figure 2—

    Histogram depicting the distribution of total number of different RBC morphological anomalies with a semiquantitative value of ≥ 1+ identified per dog on the basis of results from blood smears reviewed for dogs in the lymphoma group in Figure 1 that were further categorized as having lymphoma substage a (gray bars; n = 8) or substage b (black bars; 20).

  • Figure 3—

    Histogram depicting percentages of dogs in Figure 1 with a semiquantitative value of ≥ 1+ for 5 specific RBC morphological anomalies. *Prevalence was significantly (P < 0.05) higher in the lymphoma group, compared with the healthy group. †Prevalence was significantly (P < 0.05) higher in the IBD group, compared with the healthy group. ‡Prevalence was significantly (P = 0.03) higher in the lymphoma group, compared with the IBD group. See Figure 1 for remainder of key.

  • 1. Kaiser HE. Animal neoplasms: a systemic review. In: Kaiser HE, ed. Neoplasms: comparative pathology of growth in animals, plants, and man. Baltimore: Williams & Wilkins Co, 1981;747812.

    • Search Google Scholar
    • Export Citation
  • 2. Vail DM, Pinkerton ME, Young KM. Canine lymphoma and lymphoid leukemias. In: Withrow SJ, Vail DM, Page RL, eds. Withrow & MacEwen's small animal clinical oncology. St Louis: Elsevier Saunders, 2013;608638.

    • Search Google Scholar
    • Export Citation
  • 3. Couto CG, Rutgers HC, Sherding RG, et al. Gastrointestinal lymphoma in 20 dogs. A retrospective study. J Vet Intern Med 1989;3:7378.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Carrasco V, Rodriguez-Bertos A, Rodriguez-Franco F, et al. Distinguishing intestinal lymphoma from inflammatory bowel disease in canine duodenal endoscopic biopsy samples. Vet Pathol 2015;52:668675.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Breitschwerdt EB, Waltman C, Hagstad HV, et al. Clinical and epidemiologic characterization of a diarrheal syndrome in Basenji dogs. J Am Vet Med Assoc 1982;180:914920.

    • Search Google Scholar
    • Export Citation
  • 6. Scott KD, Zoran DL, Mansell J, et al. Utility of endoscopic biopsies of the duodenum and ileum for diagnosis of inflammatory bowel disease and small cell lymphoma in cats. J Vet Intern Med 2011;25:12531257.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Ohmura S, Leipig M, Schöpper I, et al. Detection of monoclonality in intestinal lymphoma with polymerase chain reaction for antigen receptor gene rearrangement analysis to differentiate from enteritis in dogs. Vet Comp Oncol 2017;15:194207.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Kiupel M, Smedley RC, Pfent C, et al. Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples. Vet Pathol 2011;48:212222.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Martini V, Melzi E, Comazzi S, et al. Peripheral blood abnormalities and bone marrow infiltration in canine large B-cell lymphoma: is there a link? Vet Comp Oncol 2015;13:117123.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Miller AG, Morley PS, Rao S, et al. Anemia is associated with decreased survival time in dogs with lymphoma. J Vet Intern Med 2009;23:116122.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Abbo AH, Lucroy MD. Assessment of anemia as an independent predictor of response to chemotherapy and survival in dogs with lymphoma: 96 cases (1993–2006). J Am Vet Med Assoc 2007;231:18361842.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Lucroy MD, Christopher MM, Kraegel SA, et al. Anaemia associated with canine lymphoma. Comp Haematol Int 1998;8:16.

  • 13. Tasca S, Carli E, Caldin M, et al. Hematologic abnormalities and flow cytometric immunophenotyping results in dogs with hematopoietic neoplasia: 210 cases (2002–2006). Vet Clin Pathol 2009;38:212.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Badylak SF, Van Vleet JF, Herman EH, et al. Poikilocytosis in dogs with chronic doxorubicin toxicosis. Am J Vet Res 1985;46:505508.

  • 15. Canfield PJ, Watsond ADJ, Ratcliffed RCC. Dyserythropoiesis, sideroblasts/siderocytes and hemoglobin crystallization in a dog. Vet Clin Pathol 1987;16:2128.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Hoff B, Lumsden JH, Valli VE. An appraisal of bone marrow biopsy in assessment of sick dogs. Can J Comp Med 1985;49:3442.

  • 17. Weiss DJ. Sideroblastic anemia in 7 dogs (1996–2002). J Vet Intern Med 2005;19:325328.

  • 18. Keller SM, Vernau W, Hodges J, et al. Hepatosplenic and hepatocytotropic T-cell lymphoma: two distinct types of T-cell lymphoma in dogs. Vet Pathol 2013;50:281290.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Fry MM, Vernau W, Pesavento PA, et al. Hepatosplenic lymphoma in a dog. Vet Pathol 2003;40:556562.

  • 20. Caldin M, Carli E, Furlanello T, et al. A retrospective study of 60 cases of eccentrocytosis in the dog. Vet Clin Pathol 2005;34:224231.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Weiss DJ, Kristensen A, Papenfuss N. Qualitative evaluation of irregularly spiculated red blood cells in the dog. Vet Clin Pathol 1993;22:117121.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Boyle TE, Bissett SA. Idiopathic inflammatory bowel disease. Vetlearn 2007;9:712.

  • 23. Fogle JE, Bissett SA. Mucosal immunity and chronic idiopathic enteropathies in dogs. Compend Contin Educ Vet 2007;29:290302.

  • 24. Winter JL, Barber LG, Freeman L, et al. Antioxidant status and biomarkers of oxidative stress in dogs with lymphoma. J Vet Intern Med 2009;23:311316.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Abou-Seif MA, Rabia A, Nasr M. Antioxidant status, erythrocyte membrane lipid peroxidation and osmotic fragility in malignant lymphoma patients. Clin Chem Lab Med 2000;38:737742.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Honda M, Yamada Y, Tomonaga M, et al. Correlation of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, and clinical features of hematological disorders: a pilot study. Leuk Res 2000;24:461468.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Halliwell B. Oxidative stress and cancer: have we moved forward? Biochem J 2007;401:111.

  • 28. Vajdovich P, Kriska T, Mézes M, et al. Redox status of dogs with non-Hodgkin lymphomas. An ESR study. Cancer Lett 2005;224:339346.

  • 29. Weiss DJ. Uniform evaluation and semiquantitative reporting of hematologic data in veterinary laboratories. Vet Clin Pathol 1984;13:2731.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Morley P, Mathes M, Guth A, et al. Anti-erythrocyte antibodies and disease associations in anemic and nonanemic dogs. J Vet Intern Med 2008;22:886892.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Madewell BR, Feldman BF. Characterization of anemias associated with neoplasia in small animals. J Am Vet Med Assoc 1980;176:419425.

    • Search Google Scholar
    • Export Citation
  • 32. Weiss DJ, Smith SA. A retrospective study of 19 cases of canine myelofibrosis. J Vet Intern Med 2002;16:174178.

  • 33. Weiss DJ. Primary pure red cell aplasia in dogs: 13 cases (1996–2000). J Am Vet Med Assoc 2002;221:9395.

  • 34. Moullet I, Salles G, Ketterer N, et al. Frequency and significance of anemia in non-Hodgkin's lymphoma patients. Ann Oncol 1998;9:11091115.

  • 35. McGovern KF, Lascola KM, Davis E, et al. T-cell lymphoma with immune-mediated anemia and thrombocytopenia in a horse. J Vet Intern Med 2011;25:11811185.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Graff EC, Spangler EA, Smith A, et al. Hematologic findings predictive of bone marrow disease in dogs with multicentric large-cell lymphoma. Vet Clin Pathol 2014;43:505512.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Christopher MM, Lee SE. Red cell morphologic alterations in cats with hepatic disease. Vet Clin Pathol 1994;23:712.

  • 38. Albitar M, Manshouri T, Shen Y, et al. Myelodysplastic syndrome is not merely “preleukemia.”. Blood 2002;100:791798.

  • 39. Ehrenfeld M, Abu-Shakra M, Buskila D, et al. The dual association between lymphoma and autoimmunity. Blood Cells Mol Dis 2001;27:750756.

  • 40. Benz CC, Yau C. Ageing, oxidative stress and cancer: paradigms in parallax. Nat Rev Cancer 2008;8:875879.

  • 41. Zhou Y, Hileman EO, Plunkett W, et al. Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents. Blood 2003;101:40984104.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Blackburn RV, Spitz DR, Liu X, et al. Metabolic oxidative stress activates signal transduction and gene expression during glucose deprivation in human tumor cells. Free Radic Biol Med 1999;26:419430.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Yamato O, Kasai E, Katsura T, et al. Heinz body hemolytic anemia with eccentrocytosis from ingestion of Chinese chive (Allium tuberosum) and garlic (Allium sativum) in a dog. J Am Anim Hosp Assoc 2005;41:6873.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Yamoto O, Maede Y. Susceptibility to onion-induced hemolysis in dogs with hereditary high erythrocyte reduced glutathione and potassium concentrations. Am J Vet Res 1992;53:134137.

    • Search Google Scholar
    • Export Citation
  • 45. Christopher MM, White JG, Eaton JW. Erythrocyte pathology and mechanisms of Heinz body-mediated hemolysis in cats. Vet Pathol 1990;27:299310.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. Christopher MM. Relation of endogenous Heinz bodies to disease and anemia in cats: 120 cases (1978–1987). J Am Vet Med Assoc 1989;194:10891095.

    • Search Google Scholar
    • Export Citation

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