A 13-year-old 480-kg mixed breed gelding was examined because of a 2-day history of anorexia, fever, mild colic, and icterus. Three days before the onset of clinical signs, the horse received vaccinations for rabies, tetanus, West Nile virus, Western equine encephalomyelitis, and Eastern equine encephalomyelitis. On examination the horse was quiet, alert, and responsive. Heart rate was 54 beats/min, respiration rate was 18 breaths/min, and temperature was 37.9°C. Mucous membranes and sclera of the eyes were markedly icteric and capillary refill time was 2 seconds. Heart, lung, and abdominal auscultation were within normal limits. Peripheral lymph nodes were not enlarged and no abnormalities were detected on the remainder of the physical examination. Naso-gastric intubation revealed no net gastric reflux. Abdominal ultrasonography revealed a small area of moderately thickened (0.8 cm) ventral colon. Thoracic ultrasound did not reveal abnormalities. Rectal examination revealed splenomegaly but was otherwise within normal limits. Abdominocentesis was attempted but peritoneal fluid was not obtained. Complete blood count (CBC) revealed anemia (PCV 16%; reference range 33–42%) and thrombocytopenia (26,000/μL [reference range 100,000–600,000/μL]). Total white blood cell count and differential were within normal limits. Cytological evaluation of peripheral blood revealed rare nucleated red blood cells but otherwise abnormalities were not detected. Serum biochemistry abnormalities were limited to hyperglycemia (135 mg/dL; reference range 60–107 mg/dL) and hyperbilirubinemia (10.9 mg/dL; reference range 0.5–2.5 mg/dL). Direct and indirect bilirubin values were not measured. Total protein (TP) was 6.1 g/dL (reference range 5.5–7.5 g/dL). Hyperfibrinogenemia was detected (275 g/dL; reference range 125–262 g/dL). Venous blood gas analysis1 identified hyperlactatemia (7.1 mmol/L; reference range <1 mmol/L) but was otherwise normal. Prothrombin time and partial thromboplastin time were normal. A direct Coombs test was negative. ELISA and agar gel immunodiffusion (Coggins test) for equine infectious anemia and ELISA for Anaplasma phagocytophilum were negative. Volume replacement included lactated Ringer's2 (20 mL/kg bolus followed by 2 mL/kg/h IV maintenance). Urine specific gravity (USG) after the initial fluid bolus was 1.025 and dipstick analysis did not reveal any abnormalities. Following the fluid bolus, PCV was 13% and TP 5.6 g/dL. The horse was lethargic and tachycardic (66 beats/min). Commercial fresh frozen plasma3 was administered (4 mL/kg IV). Cross matching was performed and 8 L whole blood was administered from a donor horse. Treatment included dexamethasone4 (0.2 mg/kg, IV, q24h), trimethoprim sulfamethoxazole5 (TMS) (30 mg/kg, PO, q12h), and sucralfate6 (20 mg/kg, PO, q6h). Because of the history of colic, feed was withheld for the first 18 hours. No signs of colic or fever were noted. The day after initial examination the horse was brighter and vital parameters were within normal limits. PCV was 17% and blood lactate concentration was 2.2 mmol/L. Marked thrombocytopenia persisted (22,000/μL). Gastroscopy was performed and did not reveal any abnormalities. Feeding with small amounts of grass hay was initiated. Intravenous administration of fluids was discontinued 36 hours after initial examination. On day 3, PCV was 20%. USG was 1.020 and further urine dipstick analysis did not reveal any abnormalities. Mild hyperlactatemia persisted (2.8 mmol/L). Whole blood was submitted7 for flow cytometry to assess platelet and red blood cell surface associated antibodies. For this test 10,000 red blood cells or platelets are evaluated and the values given represent the percentage of the total number of cells affected. Platelet and red blood cell surface associated IgA antibody was normal (2% and <1%, reference range <4% and <3%, respectively). Surface IgG antibody values were 9% for platelets and 10% for red blood cells (reference range <4% and <3%, respectively). Surface IgM antibody values were 8% and 4% (reference range <4% and <3%). These findings are consistent with immune-mediated thrombocytopenia (IMT) and immune-mediated hemolytic anemia (IMHA) but do not differentiate between primary and secondary causes. On day 5, PCV was 24% and thrombocytopenia was improving (68,000/μL). Hyperbilirubinemia persisted (6.2 mg/dL) and rare nucleated red blood cells were still noted in peripheral blood. Dexamethasone was reduced to 0.1 mg/kg, IV q24h and antimicrobial therapy was discontinued. An increase in respiratory rate and effort was noted. Rectal examination, thoracic, and abdominal ultrasound remained unchanged. Venous blood gas revealed worsening hyperlactatemia (4.8 mmol/L). Intravenous administration of fluids (lactated Ringer's 2 mL/kg/h) was reinitiated and PCV and TP closely monitored. On day 6, fever of 103.3°F was noted, responsive to phenylbutazone (2.2 mg/kg, IV). Over the next several days, fevers of up to 104.7°F persisted but would resolve for 12 hours after phenylbutazone administration. Hyperlactatemia persisted (3–6 mmol/L). Platelet count and PCV remained stable at 70,000/μL and 22–25% until day 8 when PCV declined (16%). Rare nucleated red blood cells were still noted and the CBC otherwise revealed no abnormalities. The horse was intermittently dull but maintained a moderate appetite. Treatment with dexamethasone, TMS, and sucralfate was continued. Given the close association between vaccination administration and the onset of IMHA and IMT, an immune-mediated response to vaccination was considered likely. However, the poor response to corticosteroids along with recurrent fever and hyperlactatemia led to a suspicion that IMHA and IMT were secondary to neoplasia. Because of the thickened ventral colon, a rectal biopsy was performed and results did not reveal any abnormalities. Abdominocentesis was attempted again but remained unrewarding. The horse's condition deteriorated on day 11. Marked tachycardia (70 beats/min), further decline in PCV (13%), and hyperlactatemia (11.1 mmol/L) were noted. A second whole blood transfusion (8 L) was administered, dexamethasone was increased (0.2 mg/kg, IV q24h), and treatment with oxytetracycline8 (6.6 mg/kg, IV q12h) was initiated. Sucralfate continued to be given and administration of TMS was discontinued. Lactate was 8–15 mmol/L and PCV was 9–14% for the next 48 hours. A further blood transfusion was declined by the owner. Oxygen (10 L/min intranasal) was administered. A bone marrow biopsy was obtained from the tuber coxae. Bone marrow cytology was nondiagnostic. Histopathology from the same site revealed sparse amounts of actual marrow consisting of collapsed hemopoietic stroma with no active hemopoietic cells present. Although regions were suspicious of myeofibrosis, the sample was considered insufficient for a conclusive interpretation. Results of immunophenotyping of peripheral blood7 were not consistent with neoplasia. Mild decreases in CD4+ T lymphocytes and mild increases in CD8+ T lymphocytes were noted and considered nonspecific. Cell-cycle analysis7 detected populations of aneuploid cells in peripheral blood which although inconclusive could indicate the presence of circulating neoplastic cells. Serum IgM levels9 were decreased (<25 mg/dL [reference range 67–543 mg/dL]) which suggested lymphoma. On day 16 cytological analysis of peripheral blood revealed the presence of rare lymphoblasts. The horse was euthanized because of the high index of suspicion of lymphoma and lack of response to treatment. Gross necropsy findings revealed locally extensive, red spongy areas in the diaphysis of both femurs. The gastrosplenic, tracheobronchial, and cranial mediastinal lymph nodes were mottled red and markedly enlarged. Other lymph nodes were grossly unremarkable. Several 2-cm arboriform depressions were noted in the spleen. The remainder of the necropsy including the colon (as an abnormality was suspected on the ultrasound) was unremarkable. Histopathology of left femur bone marrow revealed approximately 60% of hemopoietic cells to consist of a monomorphic population of neoplastic cells. The neoplastic cells were round to ovoid with occasionally cleaved central euchromatic nucleus, finely stippled chromatin, and 1–3 prominent magenta nucleoli. There was moderate anisocytosis, moderate anisokaryosis, and karyomegaly. Approximately 45 mitotic figures were present per 10 high power fields (400X). Approximately 40% of the hemopoietic cells in bone marrow from one rib showed similar changes. Similar neoplastic cells were seen in cranial mediastinal, gastrosplenic, and tracheobronchial lymph nodes (Fig 1). In the spleen the periarteriolar lymphoid nodules were expanded by similar neoplastic cells, occasionally coalescing with adjacent nodules. Immunohistochemical stains were performed to determine the cell phenotype which included CD3 which labels T cells and CD20, CD79a, and BLA36 which label B cells. The neoplastic cells labeled positive with CD3 and CD20 (Figs 2, 3) and negative for CD79a and BLA36. A final diagnosis of T-cell lymphoma was made. Lymphoma is one of the most common neoplasms in horses with prevalence estimated at 2.5%.1 Clinical signs are often varied and may include anemia and fever. Although well reported in the human literature, only a single publication exists documenting IMHA and IMT associated with neoplasia in horses.2 In that report, the diagnosis of IMHA in 3 horses was based on clinical evidence of hemolysis and a positive Coombs test. Although platelet factor 3 levels were abnormal in 1 horse, the diagnosis of IMT was presumptive in the other 2 horses. Since that publication, more sensitive diagnostic modalities have become available to diagnose IMHA and IMT. Immune-mediated hemolytic anemia and IMT are caused by antibody-mediated destruction of red blood cells and can occur independently or concurrently in horses. IMHA occurs rarely as a primary disease. IMHA can occur secondary to bacterial infection, neoplasia, inflammatory bowel disease, and purpura hemorrhagica in horses.3 IMT in horses can be primary (idiopathic) or secondary to drug administration, neoplasia, or infection.4 Flow cytometry to assess platelet and red blood cell surface-associated antibodies in this horse revealed increases in IgG and IgM suggesting that hemolytic anemia and thrombocytopenia were caused by an immune-mediated process. Flow cytometry techniques have been adapted for equine and canine erythrocytes and platelets to detect membrane-bound antibodies in cases of immune-mediated anemia and thrombocytopenia.5 Assays with isotype-specific antibodies to equine immunoglobulins can elucidate the class of cell-bound antibodies.6 The horse described in this report was negative for a direct Coombs test. A negative Coombs test does not rule out IMHA and it is estimated that approximately 30% of humans and canines with IMHA are negative on a Coombs test.7 Proposed explanations include limitations in detection of globulins,2 insufficient quantities of bound antibody,8 or corticosteroid therapy.9 The horse in this case report had not received corticosteroids before blood collection for the Coombs test. Autoagglutination in the absence of a positive direct Coombs test may occur in the presence of low affinity auto antibodies that eluted from erythrocytes when washed.10 Anemia is observed in approximately 1/3 of horses with lymphoma and proposed causes include anemia of chronic inflammatory disease (most common), blood loss, IMHA hematopoietic dysplasia and leukoerythroblastic anemia.2 In humans, lymphoma appears to be the most common cancer associated with immune-mediated destruction of red blood cells and platelets (6–10% prevalence).11-15 Several theories have been proposed to explain the association between neoplasia and immune-mediated diseases. Neoplasia and immune-mediated disease can be caused by a common immune system defect (inappropriate activation or inactivation of T cells).16 Auto-antibodies may be produced by the neoplastic process itself.16 In some T-cell lymphomas production of anti-red cell antibodies can be part of a general stimulation of the lymphoma.12 Finally, cell molecular changes might be responsible for the association between immune-mediated disease and neoplasia as both conditions have been associated with oncogene over-expression in lymphocytes.16 Fever is reported in approximately 50% of horses with lymphoma and is thought to be caused by tumor necrosis, pyrogens associated with neoplastic growth, and secondary infections.17 Lymphoma was a possible cause of fever in this present horse; however, the contribution of hemolysis is possible. Peripheral blood cytological abnormalities were initially limited to the presence of rare nucleated red blood cells. This is a very unusual finding in horses as the equine bone marrow only rarely releases nucleated red blood cells into the peripheral circulation in marked anemia.18, 19 In other species, nucleated red blood cells can be seen in peripheral blood with regenerative anemia, often accompanied by findings such as reticulocytosis, polychromasia, or macrocytosis. Other potential differentials for the presence of nucleated red blood cells could include myeloproliferative disorders, impaired splenic function, or infiltrative marrow diseases such as neoplasia.19 Given the clinical and histopathological findings in this horse and the lack of additional signs of regenerative anemia in the peripheral blood or bone marrow, the presence of nucleated red blood cells in the peripheral circulation was considered most likely secondary to neoplasia in the bone marrow. Rare lymphoblasts were found in peripheral blood on the day the horse was euthanized. The presence of these cells suggests a secondary leukemic process which has been reported in horses with lymphoma.20 Possible causes for the persistent hyperlactatemia include tissue hypoxia secondary to inadequate tissue perfusion, increased lactate release from the gastrointestinal tract, or anemia. The hyperlactatemia worsened in this horse despite normovolemia and no clinical evidence of cardiac disease, making impaired tissue perfusion unlikely. Hyperlactatemia can occur with severe anemia. Although the anemia may have contributed to the hyperlactatemia, a direct association between reductions in PCV and increases in lactate concentration was not found making it unlikely that this was the sole explanation for the increased lactate. Although a small area of the ventral colon appeared thickened on abdominal ultrasound, clinical evidence of gastrointestinal disease was not found at necropsy. Studies have demonstrated that malignant transformation is associated with increases in anaerobic and aerobic cellular lactate excretion. In a human study, in all tumor types investigated, high concentrations of lactate were correlated with a high incidence of distant metastasis already in early stages of disease. Lactate dehydrogenase (LDH) was found to be upregulated in most tumors compared to surrounding normal tissue.21 In human studies of lymphoma, increased LDH activity in serum are reported.15, 22 Similarly, lymphoma in dogs has been associated with increased serum lactate concentration and LDH activity.23 Although speculative, it is possible that the hyperlactatemia noted in this horse was associated with lymphoma. Immunophenotyping and cell cycle analysis were performed. Immunophenotyping (normal in this horse) is performed with flow cytometry and may be normal with equine lymphoma if the horse is not leukemic and lymphocytes do not have abnormal protein expression. Cell-cycle analysis is a research tool and is not routinely available. In this horse, the presence of aneuploid cells in peripheral blood was further evidence of neoplasia. Aneuploid cells are seen in lymphoma cases as cells are in an abnormal state of cellular division. IgM deficiencies can occur in horses with lymphoma. Deficiency can be secondary to a lack of production of IgM if B-cell lines are obliterated (T-cell lymphoma), or can occur because of inability of B cells to synthesize IgM (B-cell lymphoma). The value of decreased serum IgM values in equine lymphoma has been evaluated.24 At a lower limit of <23 mg/dL sensitivity was poor (23%). However, the test had good specificity (88%) for supporting a diagnosis of lymphoma. This T-cell lymphoma was concurrently positive with CD20, a membranous antigen commonly associated with B-cell phenotype as in humans.25 Due to negative CD79a/BLA36 stains, it is highly unlikely that this is B-cell neoplasia and the unique aspect of CD20 labeling suggests an atypical subset of equine lymphoma. The authors express their gratitude to Dr. Ted Valli and Dr. Luke Borst for their expertise and assistance with this case report.

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