Pathology in Practice

Marigold E. Ernst Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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Nicole Weinstein Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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Katie M. Boes Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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Katherine E. Wilson Department of Large Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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William F. Gilsenan Department of Large Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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History

A 14-year-old 423-kg (931-lb) Tennessee Walking Horse mare was evaluated at the Virginia-Maryland College of Veterinary Medicine Veterinary Teaching Hospital because of a sudden onset of vulvar bleeding, signs of depression, and lethargy.

Clinical and Clinicopathologic Findings

Abnormal physical examination findings included mental dullness, tachycardia (90 beats/min [reference interval, 28 to 44 beats/min]), muddy brown oral mucous membranes, icteric sclerae, and dried blood on the medial aspect of the tibiotarsal regions. Results of a transrectal ultrasonographic examination indicated that the mare was pregnant (week 2 to week 3 of gestation) but were otherwise unremarkable. Urethral catheterization yielded 4 L of opaque brown-red urine (Figure 1), and it was concluded that the medial aspects of the tibiotarsal regions were stained with dried bloody urine, rather than with blood from the vagina. Centrifugation of a microhematocrit tube containing EDTA-anticoagulated whole blood revealed red plasma and a PCV of 14% (reference interval, 32.0% to 53.0%); plasma protein concentration was 8.2 g/dL (reference interval, 5.8 to 8.7 g/dL) as determined via refractometry.

Figure 1—
Figure 1—

Photographs of a tube of cloudy brown-red urine obtained by catheterization (A) and a microhematocrit tube of blood (after centrifugation [B]) from a mare that was evaluated because of mental dullness, tachycardia, muddy brown oral mucous membranes, icteric sclerae, and presumed vulvar bleeding. In panel B, the PCV is 14% and the plasma is bright red.

Citation: Journal of the American Veterinary Medical Association 249, 1; 10.2460/javma.249.1.59

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page →

Additional Clinicopathologic and Hematologic Findings

Further client communication revealed that the horse was exposed to dried red maple tree cuttings 2 weeks previously. Results of a CBC performed with a hematology analyzera confirmed severe anemia (RBC count, 2.70 × 106 RBCs/μL [reference interval, 6.8 × 106 RBCs/μL to 12.9 × 106 RBCs/μL]; Hct, 14.1% [reference interval, 32.0% to 53.0%]) and provided evidence of hemoglobinemia attributable to hemolysis (hemoglobin concentration, 7.0 g/dL [reference interval, 11.0 to 19.0 g/dL]; mean corpuscular hemoglobin concentration, 49.6 g/dL [reference interval, 33.6 to 36.5 g/dL]; and cell hemoglobin concentration mean, 39.1 g/dL [reference interval, 33.6 to 36.5 g/dL]). Blood smear evaluation revealed frequent eccentrocytes, pyknocytes, and ghost cells and rare Heinz bodies, indicative of oxidant-induced hemolytic anemia (Figure 2). Additional hematologic findings included evidence of inflammation (neutrophil count, 5.870 × 103 neutrophils/μL [reference interval, 2.450 × 103 neutrophils/μL to 6.500 × 103 neutrophils/μL]; band count, 0.452 × 103 bands/μL [reference interval, 0.000 × 103 bands/μL to 0.000 × 103 bands/μL]; and toxic change) and a normal platelet concentration (179 × 103 platelets/μL [reference interval, 100 × 103 platelets/μL to 350 × 103 platelets/μL]). Clinically important plasma biochemical and urinalysis results supported dehydration (urine specific gravity, > 1.060; plasma total protein concentration, 8.1 mg/dL [reference interval, 6.0 to 7.6 mg/dL]), hemolysis and anorexia (direct bilirubin concentration, 0.3 mg/dL [reference interval, 0.0 to 0.4 mg/dL]; indirect bilirubin concentration, 9.6 g/dL [reference interval, 0.0 to 2.2 mg/dL]; and 3+ bilirubinuria), and hemoglobinuria (urine findings, 3+ blood, 0 to 2 RBCs/hpf, 3+ protein, and 4+ sulfosalicylic acid test result). Plasma creatine kinase activity was 1,125 U/L (reference interval, 183 to 542 U/L); blood methemoglobin concentration was 27% (reference interval, < 3%), as measured by differential spectrophotometry with the cyanmethemoglobin method.

Figure 2—
Figure 2—

Photomicrograph of a blood smear preparation from the horse in Figure 1. Eccentrocytes (thick black arrows), pyknocytes (arrowheads), ghost cells (white arrows), and rare Heinz bodies (thin black arrow) are present, indicating oxidative erythrocyte damage. Modified Wright stain; bar = 6 μm.

Citation: Journal of the American Veterinary Medical Association 249, 1; 10.2460/javma.249.1.59

Clinicopathologic Diagnosis and Case Summary

Clinicopathologic diagnosis: oxidant-induced hemolytic anemia and methemoglobinemia.

Case summary: red maple toxicosis in a horse.

Comments

Initial differential diagnoses for a horse with anemia, red plasma, and dark red urine include several causes of hemolytic anemia, such as immune-mediated hemolytic anemia (eg, penicillin- or heparin-induced anemia or neonatal isoerythrolysis), infectious hemolytic anemia (eg, babesiosis, Clostridium perfringens infection, or equine infectious anemia), and oxidant-induced hemolytic anemia (eg, red maple toxicosis or glucose-6-phosphate dehydrogenase deficiency). In the horse of the present report, the blood smear findings of eccentrocytes, pyknocytes, and Heinz bodies, along with methemoglobinemia and a history of being exposed to dried red maple clippings identified the disease as red maple toxicosis.

Red maple (Acer rubrum) toxicosis predominantly develops in horses1–3 and other equids such as Grevy's zebra,4 although 2 suspected alpaca cases have been documented.5 Although ingestion of fresh red maple leaves does not typically cause toxicosis, ingestion of wilted or dried red maple leaves at a dose as low as 1.5 g/kg (0.68 g/lb) induces clinical signs attributable to oxidative damage to erythrocytes with subsequent intravascular and extravascular hemolysis and methemoglobinemia.1 A specific toxic agent has not been identified, although gallic acid, tannic acid, and pyrogallol have all been implicated.6 Results of a recent study7 suggest that pyrogallol has the largest role in oxidative injury, and it has been proposed that pyrogallol is metabolized from gallic acid and tannic acid by equine gastrointestinal microbes, such as Klebsiella pneumoniae and Entero-bacter cloacae.

Red blood cells contain several enzymes that limit oxidative damage, including reduced glutathione and superoxide dismutase. When these enzymes are depleted as a result of an overwhelming dose of an oxidizing agent, irreversible oxidative damage occurs. Damage to an RBC's membrane causes opposing sides of the membrane to adhere, leading to eccentrocyte formation. Pyknocytes subsequently form when the fused membranes fragment off the cell. Eccentrocytes and pyknocytes are more fragile than normal erythrocytes and lyse easily under normal hemodynamic forces (intravascular hemolysis). These abnormal cells are also targeted for removal by the reticuloendothelial system (extravascular hemolysis).8 Oxidation of sulfhydryl groups on globin chains causes denaturing and precipitation of clumps of hemoglobin, which are visualized as Heinz bodies. Methemoglobin forms when iron molecules within hemoglobin are oxidized from the ferrous (Fe2+) to the ferric (Fe3+) form, which is unable to bind oxygen.9 Horses appear to be relatively more susceptible to oxidative red cell damage than several other species, including humans. In 1 study,10 equine erythrocytes were slow to reduce methemoglobin back to oxyhemoglobin in the presence of nitrates. Also, reduced glutathione is depleted more quickly in equine erythrocytes than in human erythrocytes.11

Two clinical syndromes of red maple toxicosis in horses have been described: death within a postingestion period of 18 hours as a result of severe methemoglobinemia, and hemolytic anemia with or without methemoglobinemia.1 Horses and ponies with the latter clinical syndrome often have lethargy, signs of depression, cyanosis, brown mucous membranes, and tachycardia on physical examination.1,2 Fever may be present and is positively associated with survival rate.12 Methemoglobinemia may result in brown or dark red blood, and a spot test to compare color of blood from an ill animal with that from a healthy animal can provide strong evidence of methemoglobinemia in a field setting (Figure 3). Frequently observed clinicopathologic abnormalities in affected equids include anemia, hemoglobinemia, hemoglobinuria, methemoglobinemia, and hyperbilirubinemia as well as the presence of eccentrocytes, pyknocytes, schistocytes, and Heinz bodies in blood smears.1,2,12 Additionally, hemoglobinuric nephrosis may cause acute renal failure.3,13

Figure 3—
Figure 3—

Photograph of a methemoglobin spot test (on absorbent white paper) to compare blood from the horse in Figure 1 (left) with blood from a healthy horse (right). The affected horse's blood is darker than that of the healthy horse, indicating a higher concentration of methemoglobin.

Citation: Journal of the American Veterinary Medical Association 249, 1; 10.2460/javma.249.1.59

In equids with red maple toxicosis, clinical signs are a result of hemoglobic hypoxia, specifically anemia and methemoglobinemia. Results of a CBC with blood smear examination best identify hemolytic anemia, whereas co-oximetry is the preferred method for diagnosis of methemoglobinemia in a clinic setting. Blood gas analysis and pulse oximetry do not aid in the diagnosis of methemoglobinemia.14 In affected animals, Pao2 is usually normal because oxygen delivery to blood is not impaired, and oxygen saturation (measured by blood gas analysis or pulse oximetry) is erroneously high when methemoglobin is present.

In cases of hemolytic anemia, endogenous substances may affect interpretation of data for other analytes. Free hemoglobin, Heinz bodies, and hyperbilirubinemia may increase the hemoglobin concentration relative to the Hct, resulting in a falsely high mean corpuscular hemoglobin concentration with an unaffected cell hemoglobin concentration mean.8 These substances likely explain the high mean corpuscular hemoglobin concentration in the horse of the present report. However, it is interesting to note that eccentrocytosis and pyknocytosis may contribute to a true hyperchromic state because fusion and fragmentation of cell membranes cause loss of cell volume without proportionate loss of hemoglobin.15 Free hemoglobin also may interfere with many biochemical assays. Hyperbilirubinemia may falsely decrease serum creatinine concentration measured by the Jaffe reaction, a method commonly employed in many clinical chemistry laboratories. Pigmenturia may cause overestimation of urine protein and ketone concentrations when assessed by use of a dipstick. A sulfosalicylic acid test, which is not affected by pigmenturia, is useful to verify proteinuria due to overflow (hemoglobin) or renal tubular injury (albumin or other low-to-medium-molecular-weight proteins). In this test, proteins are denatured by acids and form a precipitate; the resulting increased turbidity is subjectively scored or is quantified by spectrophotometry. Proteinuria may falsely increase urine specific gravity measured by refractometry.16

Even among treated horses, the mortality rate associated with red maple toxicosis is high (60% to 65%).1,12 Possible sequelae include laminitis, colic, and abortion12,17,18; hence, hospitalized horses should be frequently monitored for signs of lameness or abdominal pain. Treatment for red maple toxicosis is supportive, with the aim of promoting tissue perfusion and oxygenation. Intravenous administration of crystalloid or colloid fluids is a major component of treatment in most cases, and severely anemic animals may require transfusion with blood products such as fresh whole blood. Affected horses may benefit from treatment with NSAIDs to reduce pain and decrease the inflammation associated with hemolysis.12 Ascorbic acid (vitamin C) has been suggested as an adjunct treatment because of its antioxidant properties. In 1 case report,17 2 horses with red maple toxicosis were administered ascorbic acid and survived the initial crisis. Methylene blue is used to treat nitrite-induced methemoglobinemia in ruminants, but its use in horses should be avoided because of a lack of efficacy in promoting methemoglobin reduction in equine erythrocytes.3,10 Glucocorticoids are frequently used to treat red maple toxicosis in horses, but their efficacy is controversial because a negative correlation between glucocorticoid administration and survival rate has been documented.12

During a 9-day period of hospitalization, the horse of the present report received lactated Ringer solution (1 L/h [2.4 mL/kg/h {1.1 mL/lb/h}], IV, on days 1 to 8), 3 fresh whole blood transfusions (8 L/transfusion/d, IV, on days 1, 2, and 4), ascorbic acid (50 mg diluted in 1 L of saline [0.9% NaCl] solution, IV, q 24 h on days 1 to 4), and flunixin meglumine (0.25 mg/kg [0.11 mg/lb]), q 8 h, on days 5 to 9). Altrenogest (0.05 mg/kg [0.02 mg/lb], PO, q 24 h) was also administered during hospitalization to maintain pregnancy. After the first 48 hours, the horse's condition improved steadily. Prior to hospital discharge on day 9, transrectal ultrasonographic examination revealed that the fetus was likely not viable, but it was unclear whether this was due to fetal hypoxia secondary to the dam's anemia or other factors.

Footnotes

a.

Advia 2120, Siemens Medical Solutions USA Inc, Malvern, Pa.

References

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