Ten-year-old, mare, quarterhorse, (Equus ferus caballus).This animal presented to the teaching hospital in November of 2014 for weight loss. At that time, blood work revealed hyperproteinemia and elevated blood ammonia (see laboratory results below). Ultrasound of the liver revealed rounded margins. A liver biopsy was performed and revealed nodular regeneration, bridging fibrosis, and arteriolar and bile duct proliferation. The clinical suspicion at that time was chronic pyrrolizidine alkaloid toxicity despite an absence of hepatocellular cytomegaly or karyomegaly. The patient re-presented in January of 2015 for increasing lethargy, dull mentation and waxing and waning appetite. The horse was treated with minocycline, metronidazole, pentoxifylline, lactulose, vitamin E, and prednisolone. The patient presented again in February of 2015 due to progression of clinical signs and failure to respond to treatment. Euthanasia was elected.

Gross Description:  

At necropsy, the liver was markedly reduced in size weighing 3.1 kg (0.7% of body weight) and had thick rounded margins. Some of the lobes appeared multinodular and there were multifocal areas of capsular fibrosis.

Histopathologic Description:

Liver: Within all sections, the hepatic capsule is variably thickened, and the lobular architecture is pronounced due to marked portal to portal bridging fibrosis. Dissecting bands of fibrosis variably infiltrate the surrounding parenchyma causing isolation of hepatic lobules and smaller clusters of hepatocytes. Rare isolated hepatocytes appear hyper-eosinophilic with pyknotic to karyolytic nuclei (individual cell apoptosis). Diffusely, hepatocytes contain variable amounts of coarse, dark brown, granular pigment (hemosiderin). Kupffer cells and macrophages, which course throughout areas of fibrosis, contain similar, but more variably sized, dark brown cytoplasmic granules (hemosiderophages). Areas of fibrosis also contain small to moderate numbers of lymphocytes and plasma cells, with rare neutrophils, and abundant variably sized bile ducts and small-caliber blood vessels. Occasional small aggregates of lymphocytes and plasma cells are observed throughout the parenchyma. In some areas, the hepatic parenchyma has been completely replaced by mature fibrous tissue and proliferating bile ducts. In addition, mature fibrous tissue occasionally appears to occlude central and portal veins with rare evidence of recanalization (venoocclusive disease). Within one section there are large, multifocal areas of acute hemorrhage and edema that cause separation and dis-organization of the hepatic cords. Occasional individual collagen bundles are segmentally darkly amphophilic (sidero-calcinosis). Within the most severely affected sections, there are frequent, irregular, refractile crystals associated with small numbers of multinucleated giant cells and epithelioid macrophages (foreign body response).

Morphologic Diagnosis:  

Liver: Severe, chronic, bridging portal and capsular fibrosis with lobular collapse, veno-occlusion, biliary hyperplasia and marked hepatocellular hemosiderosis.

Lab Results:  

February 2015: (reference ranges)
GGT 294 IU/L (8-22)
SDH 16 IU/L (0-8)
Resting bile acids 36 uMOL/L (4-11.5)
Plasma ammonia 242 UG/DL (5-59)
Postmortem liver heavy metal screen
Iron 5100 ppm (100-300)



Contributor Comment:  

The abundant hepatocellular and Kupffer cell pigment was confirmed as iron using Perls’ iron stain. Heavy metal analysis of the liver revealed an iron concentration of 5100 ppm, far exceeding the normal upper limit of 300 ppm. Perl’s staining also revealed iron deposits within smooth muscle trabeculae of the spleen, and within renal tubular epithelium at the corticomedullary junction. Iron within splenic trabeculae and within fibrous tissue in the liver is occasionally deposited in conjunction with calcium salts (siderocalcinosis). In addition, the most severely affected sections shows deposition of refractile clear crystals that are associated with a granulomatous foreign body response. These crystals are not birefringent under polarized light.

With the exception of certain avian species, including mynahs and toucans, hemo-chromatosis is a rare condition in domestic species. A hereditary form of hemochromatosis has been reported in Salers and Salers-cross cattle,8 and there have been individual case reports in horses.6 Cases of dietary iron overload have also been reported in sheep and cattle. Iron storage disease is classically divided into two entities: hemosiderosis (iron overload in the absence of clinical signs) and hemochromatosis (iron overload leading to hepatic damage including fibrosis, inflammation, and liver failure). In human medicine, iron storage disease is additionally subdivided into primary and secondary categories. Primary iron storage disease involves an inherent abnormality in iron metabolism. In humans, this is most commonly the result of one of two missense mutations in the HFE gene which encodes a protein involved in the interaction between transferrin and the transferrin receptor.9 Secondary hemochromatosis occurs from excessive intestinal absorption of iron either due to iron excess within the diet, or as a response to increased demand for erythro-poiesis. Secondary hemochromatosis can occur with any condition leading to chronic hemolysis. Although a very small amount of iron is eliminated through the bile, the body has no natural way of responding to excess iron and therefore iron accumulates over time, primarily in the liver. Iron accumulation results in hepatocellular toxicity through production of free radicals and organelle dysfunction, including lysosomal injury.8 This can lead to hepatocellular necrosis that progresses to bridging fibrosis, bile duct hyperplasia, and venoocclusive disease, as observed in this case.  In humans, hemochromatosis can also increase the risk of hepatocellular neoplasia9 and in birds, has been shown to predispose to certain bacterial infections such as Yersinia pseudotuberculosis.4
Distinguishing between primary and secondary iron storage disease can be especially difficult in chronic cases. In humans, the pattern of hemosiderin deposition can be helpful.6 In primary iron storage disease, hemosiderin accumulates first in hepatocytes while in secondary hemosiderosis, iron accumulates first within Kupffer cells and macrophages (reticulo-endothelial system). However, this requires that the liver is examined early in disease progression. In this case, a primary abnormality in iron metabolism is suspected based on the lack of clinical or histologic evidence of a second underlying disease process leading to chronic hemolysis, and feeding of a standard equine diet.

JPC Diagnosis:  

Liver: Fibrosis, portal and bridging, diffuse, marked with hepatocellular degeneration and loss and intra-hepatocellular hemo-siderosis, Quarterhorse, Equus ferus caballus.

Conference Comment:  

The contributor provides an outstanding example and thorough review of hemochromatosis in humans and veterinary species. Free iron is highly toxic to tissues due to its ability to participate in the generation of hydroxyl radical formation via the Fenton or Haber-Weiss reaction with hydrogen peroxide (H2O2) leading to lipid peroxidation and DNA damage.5 As a result, iron is typically bound to transferrin while in circulation and either ferritin or hemosiderin when stored and sequestered in tissue. When iron is bound to these proteins, it cannot participate in these injurious reactions. Ferritin concentration is highest in the liver, spleen, and bone marrow and is stored in hepatocytes and/or macrophages.1,3,5,7

In hepatocytes, iron is derived from plasma transferrin, while iron stored within macrophages is a result of erythrocyte breakdown. Normally, to offset the attritional loss of daily iron, duodenal enterocytes absorb approximately 1 to 2 mg of iron per day from the diet via divalent metal transporter-1 (DMT-1) and a heme carrier protein-1 (HCP-1) on the luminal surface of the enterocytes.5,9 Iron is transported from the cytoplasm of the enterocyte to the circulation by ferroportin.1,7 Absorbed iron circulates bound to transferrin and is used primarily by erythroid precursors in the synthesis of heme. Macrophages in the spleen clear dead and dying erythrocytes and release the iron from heme to export it to the circulation or store it in ferritin.

In iron-overload, transferrin is quickly saturated and iron is stored in the liver and various other tissues due to the lack of a regulated pathway for effective iron excretion.3

As mentioned above, hepatocytes are a major site of iron storage as ferritin and are also responsible for the production of type II acute phase protein, hepcidin, in response to inflammatory cytokine, interleukin-6.1,3,5,7 Hepcidin is transported in by blood by alpha-2-macroglubulin and blocks the release of iron from enterocytes and macrophages by degrading the iron exporter, ferroportin. In humans, decreased hepcidin synthesis caused by mutations in the hepcidin gene, HAMP, causes severe hemochromatosis in juveniles.1,5

Within the cytoplasm, iron is stored as ferritin, which is reconverted into iron as needed by the body. If tissue ferritin levels are high, ferritin aggregates into hemo-siderin globules, which is much more difficult to revert back to free iron. In hepatocytes overwhelmed with iron, most iron is stored as hemosiderin. Ferritin and hemosiderin readily stain with Pearls Prussian blue, demonstrated nicely in this case.1,5

Iron is a direct hepatotoxin and iron overload often results in the formation periportal bridging fibrosis with little inflammation.1,5 In addition to the markedly elevated postmortem liver heavy metal screen (Iron: 5100 ppm [100-300]), clinical pathology data from the provided serum biochemistry supports the histologic findings in this case.  Elevated sorbital dehydrogenase (SHD: 16 IU/L [0-8]) and elevated plasma ammonia (242 UG/dL [5-59]) indicate hepatocellular injury and decreased hepatic function respectively. In addition, elevated gamma-glutamyl trans-peptidase (GGT: 294 IU/L [8-22]) and elevated resting bile acids (36 uMOL/L [0-20]) indicate cholestasis and biliary hyperplasia with decreased hepatobiliary function.1,2,5,8


1. Bain PJ. Liver. In: Latimer KS ed. Duncan and Prasse’s Veterinary Laboratory Medicine Clinical Pathology. 5th ed. Ames, IA:Wiley-Blackwell; 2011:213-224.
2. Brockus CW. Erythrocytes. In: Latimer KS ed. Duncan and Prasse’s Veterinary Laboratory Medicine Clinical Pathology. 5th ed. Ames, IA:Wiley-Blackwell; 2011:4
3. Cullen JM, Stalker MJ. Liver and biliary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. Philadelphia, PA:Elsevier; 2016:272.
4. Galosi L, Farneti S, Rossi G, et al. Yersinia pseudotuberculosis, serogroup O:1A, infection in two amazon parrots (Amazona aestiva and Amazona oratrix) with hepatic hemosiderosis. J Zoo Wildl Med. 2015; 46(3):588-591.
5. Kumar V, Abbas AK, Fausto N. Red blood cell and bleeding disorders. In: Robbins and Cotran Pathologic Basis of Disease. 9th ed. Philadelphia, PA:Elsevier Saunders; 2015:650-651.
6. Lavoie JP, Tuescher JP. Massive iron overload and liver fibrosis resembling haemochromatosis in a racing pony. Equine vet J. 1993;25(6):552-554
7. Mazzaro LM, Johnson SP, Fair PA, Bossart G, Carlin KP, Jensen ED, Smith CR, Andrews GA, Chavey PS, Venn-Watson S. Iron indices in bottlenose dolphins (Tursiops truncatus). Comp Med. 2012; 62:508-515. 8. O’Toole D, Kelly EJ, McAllister MM, et al. Hepatic failure and hemochromatosis of Salers and Salers-cross cattle. Vet Pathol. 2001; 38(4):372-389.
9. Pietrangelo A. Hereditary hemochromatosis—A new look at an old disease. N Engl J Med. 2004; 350(23):2383-2397.
10. Weiss DJ. Iron and copper deficiencies and disorders of iron metabolism. In: Weiss DJ, Wardrop KJ, eds. Schalm’s Veterinary Hematology. 6th ed. Ames, IA:Wiley-Blackwell; 2010:170.

Click the slide to view.

2-1. Liver, horse.

2-2. Liver, horse.

2-3. Liver, horse.

2-4. Liver, horse.

2-5. Liver, horse.

Back | VP Home | Contact Us |