J Vet Intern Med 2003;17:360–363
Intravascular Hemolysis Associated with Liver Disease in a Horse with Marked Neutrophil Hypersegmentation Shashi K. Ramaiah, John W. Harvey, Steeve Gigue`re, Robert P. Franklin, and P. Cynda Crawford
H
emolytic anemia in horses results from the increased destruction of circulating erythrocytes, either by phagocytosis and degradation within macrophages of the mononuclear phagocyte system or, less commonly, by lysis within the circulation (intravascular hemolysis).1 A rare fulminant intravascular hemolytic syndrome with prominent hemoglobinuria has been reported near the time of death in horses with liver failure.2 However, to the authors’ knowledge, the clinicopathologic findings during recovery from this syndrome have not been described. In addition, hemolysis associated with liver disease concurrent with marked hypersegmentation of neutrophils has not been reported in the horse. A 10-year-old Quarter Horse mare was presented to the University of Florida Veterinary Medical Teaching Hospital with a 4-day history of anorexia and an 8-hour history of passing dark red urine. Three weeks before referral, she had an episode of colic that was treated medically by the referring veterinarian. At presentation, the mare was severely depressed, was approximately 5% dehydrated, and had icteric mucus membranes. She was febrile (102.08F [38.98C]) with heart and respiratory rates of 48 beats/min and 32 breaths/min. Mild ventral edema and poor body condition were noted, and the mare passed a small amount of dark red urine. Other findings on physical examination and rectal palpation were within normal limits. An initial CBC showed a high total white blood cell count with mature neutrophilia (Table 1) along with marked hemolysis that was apparent in plasma. Microscopic evaluation of the blood smear revealed marked neutrophil hypersegmentation and echinocytosis (Fig 1). The marked hemolysis in the initial plasma sample made several of the biochemical test results of questionable reliability. Prominent increases were noted in plasma alkaline phosphatase, g-glutamyltransferase, and aspartate aminotransferase activities along with marked increases in the total bilirubin, total protein, and globulin concentrations (Table 2). Plasma albumin and blood urea nitrogen concentrations were decreased, and the plasma ammonia concentration was markedly increased. Other chemistry values were within normal limits. UrinalFrom the Departments of Physiological Sciences (Ramaiah, Harvey), Large Animal Clinical Sciences (Gigue`re, Franklin), and Pathobiology (Crawford), College of Veterinary Medicine, University of Florida, Gainesville, FL. Previously presented as a case summary in the glass slide review section of the 36th ASVCP Annual Meeting, December 4, 2001, Salt Lake City, UT. Reprint requests: John W. Harvey, DVM, PhD, Professor and Chair, Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0144; e-mail:
[email protected]. Submitted May 2, 2002; Revised October 6, 2002; Accepted November 12, 2002. Copyright q 2003 by the American College of Veterinary Internal Medicine 0891-6640/03/1703-0017/$3.00/0
ysis showed marked hemoglobinuria, proteinuria, and bilirubinuria. Erythrocytes were not seen in the urine sediment. Clinicopathologic findings were consistent with intravascular hemolysis and liver disease.3 An enzyme-linked immunosorbent assay (ELISA) for equine infectious anemia was negative. Thorough examination of a blood smear did not identify babesial organisms. Complement fixation tests for Babesia equi and Babesia caballi and an ELISA test for Ehrlichia equi were negative. No eccentrocytes (WrightGiemsa stain) or Heinz bodies (new methylene blue stain) were present in stained blood films, and the methemoglobin concentration (0.5%) was within the reference interval for horses (0–1.04%). The direct antiglobulin (Coombs) test was negative on 2 occasions. There was no history of transfusion of blood products or administration of hypo-osmolar fluids. The osmolality (270 mmol/kg) was slightly below the reference interval (288–302 mmol/kg) but was not believed to be low enough to account for the hemolysis present. It is suspected that Clostridium septicemia causes intravascular hemolysis in horses,4 but it was considered highly unlikely in this horse because of the lack of clinical signs of gangrenous inflammation. Abdominal ultrasound showed that the volume of peritoneal fluid was increased. The liver parenchyma was within normal limits. Abdominocentesis showed dark reddish brown fluid with a total protein concentration of 4.2 g/dL and a nucleated cell count of 17.7 3 103/mL (74% markedly hypersegmented neutrophils, 2% lymphocytes, and 24% mononuclear phagocytes). No erythrocytes were seen in the smear of abdominal fluid. Aerobic and anaerobic bacterial cultures of the fluid were negative. Activated partial thromboplastin time was within normal limits, whereas prothrombin time was slightly prolonged (14.1 seconds) compared to that of a healthy control (10.8 seconds). Ultrasound-guided liver biopsies were obtained for histological examination and bacterial culture. Initial therapy included administration of polyionic isotonic fluid, broad-spectrum antimicrobials (potassium penicillin G 25,000 IU/kg IV q6h and enrofloxacin 5.5 mg/kg IV q24h) for possible bacterial cholangiohepatitis and metronidazole (25 mg/kg PO q12h) in an attempt to decrease ammonia production by intestinal bacteria. The mare was also fed a low-protein diet. The PCV decreased to 22% the day after admission (day 2), and marked hemolysis persisted. Therapy with dexamethasone (0.1 mg/kg IV q24h) was initiated because a negative Coombs’ test was not considered sufficient to definitively rule out immune-mediated hemolytic anemia. In an attempt to better characterize neutrophil hypersegmentation and rule out a myeloproliferative disease, a bone marrow aspirate and core biopsies were performed 2 days after admission. Cytological examination of the aspirate suggested low-normal overall cellularity with low-normal numbers of megakaryocytes; however, the core biopsy was 80% cellular with normal numbers of megakaryocytes. Or-
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Table 1. Sequential CBC findings in a mare with intravascular hemolysis associated with liver failure. Parameter
Day 1
Day 5
Day 12
Day 27
Day 81
Day 240
Reference Interval
Plasma WBC (3103/mL) PCV (%) MCV (fL) MCHC (g/dL) RDW (%) Platelets (3103/mL) Fibrinogen (mg/dL)
Hemolysis 25.5a 34 49 33 21 232 NA
Clear 17.7a 16 53 32 22 160 200
Clear 21.2a 13 58 38 31 201 200
Clear 8.9a 27 56 35 26 286 400
Clear 9.6a 38 48 36 26 281 300
Clear 9.6a 41 44 37 22 150 300
Clear 5.2–13.9 32–47 43–54 34–37 20–24 100–270 100–400
WBC, white blood cells; MCV, mean cell volume; MCHC, mean cell hemoglobin concentration; RDW, red cell distribution width; NA, not available. a Marked neutrophil hypersegmentation was present in all smears.
derly maturation was present in the erythroid series, and polychromatophilic erythrocytes were present. The myeloid series was complete with orderly maturation; however, most mature neutrophils were markedly hypersegmented. The myeloid : erythroid ratio was 0.46 : 1, suggesting that mild erythrocyte hyperplasia was present in light of the overall marrow cellularity and the increased blood neutrophil count. Cobalamin (patient, .1.2 mg/L; control, .1.2 mg/ L) and folate (patient, 8.6 mg/L; control, 12.0 mg/L) concentrations were within the reference ranges of horses.5 On day 5, the mare was more alert, was afebrile, and had a better appetite. However, there was a marked increase in the amount of peripheral edema, and the plasma albumin concentration was 1.6 g/dL. Plasma and urine were of normal appearance for the 1st time since admission. The PCV had decreased to 16%, and echinocytosis was still present. Liver enzyme activities and bile acids were high (Table 2). Urinalysis was normal except for persistent bilirubinuria. The mare was administered hydroxyethyl starch (10 mL/kg IV) in an attempt to limit interstitial edema formation by restoring capillary colloid oncotic pressure. Protein electrophoresis confirmed hypoalbuminemia and showed a polyclonal hyperglobulinemia with increased b-globulins (2.96 g/dL). Serum immunoglobulin quantification by radial immunodiffusion showed increased immunoglobulin G (IgG)
Fig 1. Hypersegmented neutrophils and echinocytes in blood from a Quarter Horse. Bar 5 10 mm.
(3,200 mg/dL; reference interval, 670–1,650 mg/dL) and normal IgA and IgM concentrations. Histopathologic examination of liver biopsies taken on the day of admission revealed portal to random neutrophilic necrotizing hepatitis, biliary hyperplasia, hepatovacuolar degeneration, moderate hepatocellular anisokaryosis, and occasional megaloblastic hepatocytes. These findings were suggestive of a pyrrolizidine alkaloid toxicosis. Aerobic and anaerobic bacterial cultures of liver biopsies were negative. A farm visit showed a good-quality pasture and failed to identify pyrrolizidine-containing plants. Analysis of pond water for blue-green algae was negative. Plasma biochemical profiles from 5 other horses in the pasture were within reference intervals. On day 8, fluid therapy was discontinued, and penicillin and enrofloxacin were replaced with trimethoprim-sulfamethoxazole (30 mg/kg PO q12h). Dexamethasone therapy was tapered progressively. Erythrocyte reduced glutathione (GSH) content was approximately half that of the healthy control (patient, 1.09 mmol/L; control, 1.98 mmol/L) and was below the reference interval for horses (1.76–3.32 mmol/L) when the PCV was 13% on day 9. The erythrocyte GSH content (patient, 2.13 mmol/L; control, 2.43 mmol/L) was subsequently within normal limits when measured after the PCV had returned to normal. Echinocytosis was no longer present in blood films on day 12, but 31 anisocytosis, 31 spherocytosis, and 11 schistocytosis were noted. Erythrocyte morphology was normal in subsequent blood films except for 11 anisocytosis on day 27. The mare’s condition remained unchanged until day 13, when she developed bronchopneumonia. Culture of a transtracheal aspirate yielded a predominant growth of Escherichia coli that was resistant to trimethoprim-sulfamethoxazole. Therapy with ceftiofur (2.2 mg/kg IM q12h) and phenylbutazone (1 g PO q12h) was initiated. The mare made an uneventful recovery, and all laboratory abnormalities progressively resolved (Table 1), except for the marked neutrophil hypersegmentation. Despite their abnormal morphology, the neutrophils’ oxidative burst and ability to phagocytize Staphylococcus aureus were within normal limits and similar to the values from a healthy control. Although the hepatocyte megalocytosis observed histologically was suggestive of pyrrolizidine alkaloid toxicity, the etiology of the presumptive toxic insult to the liver
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Ramaiah et al
Table 2. Selected sequential plasma chemistry findings in a mare with intravascular hemolysis associated with liver failure. Parameter
Day 1
Day 5
Day 12
Day 27
Day 81
Day 240
Reference Interval
Plasma Bilirubin (mg/dL) Alk phos (IU/L) AST (IU/L) SDH (IU/L) GGT (IU/L) Total protein (g/dL) Albumin (g/dL) Globulin (g/dL) BUN (mg/dL) Glucose (mg/dL) Bile acids (mmol/L) Ammonia (mmol/L)
Hemolysis 17.1 451 1,148 NA 56 9.2 2.3 6.9 3 84 NA 423
Clear 10.6 392 1,098 44.4 128 7.3 1.6 5.7 7 114 116 31
Clear 7.0 470 608 NA 173 6.7 1.8 4.9 15 211 NA NA
Clear 1.5 579 484 61.8 139 7.3 1.9 5.4 3 85 27 NA
Clear 0.4 178 421 NA 47 7.1 2.6 4.5 13 71 6 NA
Clear 0.7 157 481 13.2 16 8.0 3.1 4.9 14 90 3 11
Clear 0.6–3 68–200 180–365 2–6 10–40 5.5–8.0 3.0–4.5 2.5–5.0 12–25 65–139 ,15 ,40
Alk phos, alkaline phosphatase; AST, aspartate aminotransferase; SDH, sorbitol dehydrogenase; GGT, g-glutamyltransferase; BUN, blood urea nitrogen; NA, not available.
could not be determined. The intravascular hemolysis in the horse of this study was likely the result of severe liver disease, because other known causes of intravascular hemolysis were ruled out, and the intravascular hemolysis resolved as the liver disease subsided (Tables 1, 2). In a previous report, intravascular hemolysis developed near the time of death in 9 horses with liver failure, with pyrrolizidine toxicity being the most common cause.2 However, other types of hepatic disease were also apparently present in these horses, suggesting that intravascular hemolysis may be the result of hepatic disease or hepatic insufficiency per se, rather than a direct effect of a toxic agent on erythrocytes. The mechanism for intravascular hemolysis in cases of liver failure in horses is unknown, but bile acids or their salts have been considered possible hemolytic factors in horses, and the total bile acid concentration was markedly increased in the patient when 1st measured on day 5.6 Bile salts can cause hemolysis in vitro, with some bile salts being more potent than others.7 Overall erythrocyte injury by bile salts also depends on bile salt concentration, pH, and bile salt contact time with erythrocyte membranes. In addition to the above factors, the susceptibility of erythrocytes for lysis by bile salts varies among animal species.8 Bile salts initially appear to bind to the outer layer of the membrane lipid bilayer, causing the preferential release of phospholipids from this half of the membrane.9 Membrane lysis probably occurs after bile salts penetrate the membrane and alter the structure of both leaflets of the lipid bilayer.10 Total cholates and total chenodeoxycholates account for most of the bile acids in normal horses and in horses with liver disease.11 Studies that use human erythrocytes indicate that chenodeoxycholates are much more prone to cause hemolysis than cholates, but it is not known whether the same holds true for horse erythrocytes.7 Erythrocytes appearing as ‘‘burr cells’’ have previously been reported in the blood of horses with intravascular hemolysis and liver failure.6 However, the nature of this abnormal erythrocyte morphology is unclear because both acanthocytes and echinocytes have been referred to as burr cells in the past. The most common cause of echinocyte
formation in horses is systemic electrolyte depletion.12 Electrolyte depletion may have contributed to the echinocyte formation in this horse, although hyponatremia and hypochloremia were not present. Increased bile salt concentrations might also account for the prominent echinocytosis initially noted in the horse of the present study. Echinocyte formation has been shown to occur in the presence of various amphipathic drugs that distribute preferentially in the exoplasmic leaflet of the lipid bilayer.13 Echinocytosis was absent when the bile acids had decreased to 27 mmol/L on day 27. The presence of spherocytes on day 12 does not necessarily indicate that the presenting intravascular hemolysis was primarily an immune-mediated process. The transition from echinocytosis to spherocytosis might reflect a continuum of membrane injury, as has been recognized in dogs after coral snake and rattlesnake bites.14 The low erythrocyte GSH concentration that was present when the horse was anemic suggests that an oxidant insult may have contributed to the erythrocyte destruction.15 Pyrrolizidine alkaloids are metabolized to highly reactive electrophilic compounds that can produce lipid peroxidation and other forms of cell injury. These reactive metabolites are conjugated with GSH within the liver, resulting in reduced liver GSH concentrations.16 There is evidence that metabolites of pyrrolizidine alkaloids enter erythrocytes.17 Although erythrocyte GSH concentration has not been reported in animals with pyrrolizidine toxicity, erythrocyte GSH concentration would likely be decreased if reactive metabolites were present in high amounts.15 Low erythrocyte GSH concentrations might also occur secondary to unknown oxidants generated in association with liver disease. Evidence of lipid peroxidation has been measured in the blood of rats after experimental extrahepatic cholestasis along with reduced concentrations of liver and erythrocyte GSH.18 Neutrophil hypersegmentation was another prominent finding in the horse of the present study. Neutrophil hypersegmentation has been reported as a result of glucocorticoid administration, chronic stress, or myeloproliferative disorders.14 It has also been reported in dogs with an inherited defect in cobalamin absorption and in a cat with folate de-
Hemolysis and Liver Disease in a Horse
ficiency.14 There was no history of glucocorticoid administration or evidence of myeloproliferative disorder (on the basis of bone marrow findings) in the horse of the present study. Chronic stress is unlikely, as the hypersegmentation persisted after recovery, and a stress leukogram was not present. Deficiencies of cobalamin and folate were ruled out in the horse of our study because serum concentrations were similar to values from a healthy control and within published reference intervals. Idiopathic hypersegmentation of neutrophils has been reported as an incidental finding in a 6-year-old Quarter Horse mare.19 Because the current patient is also a Quarter Horse, we suggest that the idiopathic neutrophil hypersegmentation is an inherited defect. In the earlier report, neutrophil hypersegmentation did not seem to be associated with increased susceptibility to infection, but neutrophil function assays were not performed.19 Neutrophil function was normal as measured in the horse of our study.
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