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Abstract
Hemochromatosis was presumptively diagnosed using cytologic examination of liver tissue from an aged male Miniature Schnauzer. The dog was presented after receiving whole blood transfusion every 6–8 weeks for 3 years to treat pure red cell aplasia. The cytologic specimen contained clusters of hepatocytes with abundant intracytoplasmic gold-yellow pigment granules and clumps of extracellular, green-black, globular pigment, both interpreted to be hemosiderin. Histologic sections of liver revealed hepatocellular degeneration with bridging portal fibrosis, lobular atrophy, biliary hyperplasia, and diffuse, severe hemosiderin accumulation. Serum iron and ferritin levels, and dry-weight iron concentrations of liver, heart, and kidneys were markedly increased. Hemosiderin accumulation was confirmed in hepatocytes of cytologic and histologic specimens using Perl's Prussian blue staining. This report is the first description of transfusional hemochromatosis in a dog and is the first to describe its cytologic appearance in a veterinary patient.
Key words: Cytology; dogs; hemochromatosis; iron overload; liver.
Iron overload disorders are classified as hemosiderosis, which is an increased deposition of tissue iron without clinical signs, or hemochromatosis, which manifests clinically as organ dysfunction secondary to iron-induced injury.2 Hereditary hemochromatosis is a primary disorder of iron metabolism, and in human beings it involves mutations in the HFE gene.2 Secondary hemochromatosis occurs as a result of treatment of, or sequelae from, other disease processes. Examples include hemochromatosis due to multiple transfusions5 and pyruvate kinase deficiency in dogs.3 Parenchymal cells most frequently affected in all types of iron-overload disorders are those of the liver and the pancreas; however, iron deposition also can be seen in myocardial and skeletal muscle cells. In this article, we describe the clinical, cytologic, and histologic features of transfusional hemochromatosis in a dog with pure red cell aplasia (PRCA).
A 13-year-old, intact male Miniature Schnauzer was presented to the Colorado State University Veterinary Teaching Hospital, cachectic and dehydrated. The 10-kg dog had been receiving whole-blood transfusions (200–500 ml) every 6–8 weeks for 3 years as palliative therapy for PRCA. The PRCA, diagnosed 3 years before presentation, was characterized by a moderate to severe nonregenerative anemia accompanied by a marked bone marrow erythroid hypoplasia. A complete blood count revealed a nonregenerative anemia (packed cell volume 13%, reference range 43–58%; reticulocytes 7,840/µl, reference range 0–60,000/µl) and a normal leukocyte count. The serum biochemistry panel demonstrated evidence of hepatocellular injury with increased alanine aminotransferase (361 IU/liter, reference range 10–120 IU/liter) and aspartate aminotransferase (234 IU/liter, reference range 16–50 IU/liter), and cholestasis with increased alkaline phosphatase (214 IU/liter, reference range 18–160 IU/liter) and 
-glutamyl transferase (20 IU/liter, reference range 0–10 IU/liter). Preprandial (61.5 µmol/liter, reference range < 15 µmol/liter) and postprandial (242.1 µmol/liter, reference range < 25 µmol/liter) bile acids were increased, indicating decreased hepatic function. The presence of concurrrent hypocholesterolemia (98 mg/dl, reference range 130–300 mg/dl) and hypoalbuminemia (2.1 gm/dl, reference range 3.0–4.5 gm/dl) suggested that the dog had hepatic failure. Ultrasound of the liver revealed a hyperechoic liver parenchyma, with nodules and irregular borders consistent with chronic fibrosis and cirrhosis.
Liver tissue aspirates were prepared on glass slides and stained with Wright-Giemsa. The samples were moderately cellular, with sheets of hepatocytes that were slightly vacuolated. Abundant intra- and extracellular, fine, gold-yellow pigment granules and large, extracellular, green-black, irregularly shaped globules were interpreted to be hemosiderin (Figs. 1, 2). Several specimens contained sheets of cohesive polygonal cells with a high N:C ratio that were interpreted to be mesothelial cells (oval nuclei with finely stippled chromatin and a pink glycocalyx border) or biliary epithelial cells (darker blue and less abundant cytoplasm) (Fig. 2). Rare aggregates of relatively uniform spindle-shaped cells with ovoid nuclei interpreted to be fibrocytes also were noted (Fig. 3). Perl's Prussian blue staining of liver aspirates revealed a diffuse blue granular pigment within the hepatocyte cytoplasm and scattered in the background, confirming the presence of iron. The large globular pigment stained dark blue to black (Fig. 4). A few mesothelial cells contained small amounts of faint intracytoplasmic blue pigment. A cytologic interpretation of hemosiderin accumulation with possible biliary hyperplasia and fibroplasia, consistent with hemochromatosis, was made.
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At necropsy mild subcutaneous icterus was found. The left medial liver lobe was irregularly rounded. The mitral and tricuspid valves had moderate to severe endocardiosis with thickening of valve leaflets. Cross sections of ribs and long bones showed decreased marrow with replacement by adipose tissue. All other organ systems examined were free of gross lesions. Tissue samples were fixed in 10% neutral buffered formalin, routinely processed, and embedded in paraffin. Tissues from liver, heart, kidney, brain, and lung were sectioned to 5 µm and stained with hematoxylin and eosin (HE). Samples of liver, heart, and kidney were analyzed for dry-weight concentrations using the flame atomic absorption method (CSU Diagnostic Laboratory, Fort Collins, CO), and the results are listed in Table 1.
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Hemochromatosis has been reported in a variety of mammalian and avian species. Hepatic cirrhosis resembling hemochromatosis has been induced in dogs given chronic, large doses of parenteral iron sorbital or iron dextran for several years.6 Iron overload leading to liver failure also has been described in Basenjis, secondary to pyruvate kinase deficiency.3 Transfusional hemochromatosis has been described in people with refractory anemias associated with several disorders, including pure red cell aplasia, aplastic anemia, and leukemias.5,8 This report is the first description of transfusional hemochromatosis in a dog.
The pathogenesis of iron overload in this case likely resulted from infusion of red blood cells, with absence of blood loss and subsequent accumulation of stored iron (hemosiderin).1 Parenchymal cells become targets of iron deposition after transferrin has become saturated, which, in humans, can occur with only 10–15 units of transfused blood.7 The mechanism of iron toxicity in the liver is not completely understood; however, it has been shown that iron, which can exist in both oxidized and reduced forms, can act as a potent catalyst in the production of hydroxyl radicals, resulting in lipid peroxidation of organelle membranes.4 Fibrosis and collagen deposition is understood less well, and a causal relationship between iron deposition and fibrosis has not been firmly established. The diagnosis of hemochromatosis in this case, rather than hemosiderosis, was made because there was liver damage and decreased function secondary to iron deposition.
Cytologic impressions of hemochromatosis in liver have not been reported previously. In this case, hemochromatosis was presumptively diagnosed based on Wright-Giemsa–stained cytologic preparations of liver tissue aspirates, and there was a good correlation between cytologic and histologic interpretations. Cytologic preparations stained with Perl's stain can confirm the presence of intrahepatocyte hemosiderin and direct further diagnostic tests to verify the diagnosis of hemochromatosis.
Acknowledgments
We thank Dr. Martin David for critical review of the manuscript.
References
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