Heinz-body anaemia

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Several Heinz bodies (arrows) project from the surface of red blood cells (Wright’s-Giemsa stain with 100x objective).
Heinz bodies are more prominent with the use of a vital stain (New methylene blue stain with 100x objective)
Feline blood. Heinz body hemolytic anemia. Morphologic evidence of regenerative anemia is present as anisocytosis and polychromasia. Smaller RBCs (arrows) have singular rounded membrane projections that are Heinz bodies. These inclusions are caused by oxidative injury due to drugs, toxic plants, certain chemicals, and metabolic diseases. Heinz bodies can be seen in cats that are clinically normal and not anemic (100x)
Feline blood. New methylene blue stain. Heinz bodies are noted on the edge of RBCs as turquoise inclusions (arrows). Aggregate and punctate reticulocytes can also be seen. Aggregate reticulocytes have definite clumps of RNA precipitate in the RBC cytoplasm. Punctate reticulocytes left of center have numerous small individual granules of RNA precipitate. When counting reticulocytes for the absolute reticulocyte count, only aggregate reticulocytes are enumerated to assess the regenerative response (100x)

Heinz bodies (HzB) are aggregates of denatured, precipitated haemoglobin within feline red blood cells (RBC). HzB can sometimes be seen on blood smears, but do not stain well and require new methylene blue stain to accurately diagnose in blood. They are easily identified as dark stainin structures near the periphery of erythrocytes.

Haemoglobin (Hb) protein globin chains are denatured through oxidative damage by reactive oxygen species. Oxidative damage to RBCs is ongoing due to the continuous generation of free oxygen radicals from cellular metabolic pathways. Reactive oxygen species include superoxide anion(O2·), hydrogen peroxide (H2O2), and hydroxyl radical (OH). Specifically, oxidation of reactive sulfhydryl (S-H) groups creates disulfide bonds that change the conformation of the globin protein chains, resulting in precipitation of the haemoglobin molecule. Oxidative damage to the globin chains also can create eccentrocytes. Eccentrocytes are formed with the shifting of Hb to one side of the cell after membrane cross-linking, creating a clear moon-shaped appearance on the other side. Oxidative damage can also affect the heme portion of haemoglobin, creating methemoglobin (MetHb) which is incapable of carrying oxygen. It is formed by oxidation of the iron in haemoglobin from a ferrous (Fe2+) to a ferric (Fe3+) state in methemoglobin. The overall effect of any oxidative damage to red blood cells is a decrease in their oxygen carrying capacity[1].

Red blood cells have metabolic antioxidant pathways that protect against ongoing oxidative processes and the production of HzB. The hexose monophosphate pathway produces reduced glutathione, a free radical scavenger that binds to reactive oxygen species before they can harm the cell and reduces disulfide bonds induced by oxidant stress. The methemoglobin reductase pathway reduces metHb to oxyHb, restoring the RBC's oxygen-carrying capacity. As red blood cells are anucleate and lack the ability to regenerate enzymes, these protective mechanisms are exhausted as the cell ages. Older RBCs are therefore most susceptible to oxidative damage.

HzB formation impairs RBC deformability by altering intracellular fluidity, and this decrease in malleability leads to increased entrapment in narrow splenic sinusoids as blood is filtered through the spleen. The HzB alone may be phagocytosed by the spleen in a process called "pitting", or the entire RBC may be culled. Sometimes, the production of large numbers of HzBs will result in intravascular lysis of HzB-containing RBCs and an intravascular haemolytic anaemia may occur. The degree of hemolysis and subsequent anaemia is dependent upon the rate of formation of HzB, the size and number of HzB, and any concurrent RBC membrane damage; these characteristics are highly variable depending upon the underlying cause. Other direct and indirect processes that may contribute to the removal of HzB-containing RBCs include membrane skeletal protein cross-linking, lipid peroxidation, glutathione depletion, antibody binding, and cation imbalance[2].

Cats have an increased incidence of HzB relative to other species due to both increased formation and decreased removal. Cats have eight reactive S-H Hb groups per Hb tetramer, which is two and four times greater than that of dogs and humans, respectively. An increased number of S-H groups increases the susceptibility to oxidative damage. A greater dissociability of Hb from tetramers to dimers in cats also increases vulnerability to HzB formation. The non-sinusoidal feline spleen does not entrap rigid HzB-containing RBCs efficiently, decreasing their removal from circulation. This results in approximately 1-2% HzB-containing RBCs circulating in healthy cats, with increased formation when exposed to oxidant drugs and in certain disease states. Haemolytic anaemia may or may not result, subject to the underlying mechanism of the HzB formation. However, the lifespan of HzB-containing RBCs may be shortened significantly from 60-70 days to 50-60 days or as low as 7-8 days depending upon the cause[3].

Causes

There are many substances that induce HzB formation in cats, with or without the development of anaemia including: propofol, acetaminophen, onions (thiosulfates), propylene glycol (carbohydrate food source, preservative), benzocaine products, phenols, methylene blue (urinary antiseptic, IV contrast dye), d-L methionine, vitamin K3 (>2.5 mg/lb/day), phenazopyridine (urinary analgesic), naphthalene, zinc, and copper. These compounds, or their metabolites, all directly or indirectly result in the formation of reactive oxygen species that cause oxidative damage. Increased HzB are also seen with systemic diseases in cats including hepatic lipidosis, diabetes mellitus with or without ketoacidosis, hyperthyroidism, and malignant lymphoma. Propofol, consumption of onions (thiosulfates), acetaminophen, and propylene glycol will be discussed further.

  • Propofol (2,6-Diisopropylphenol)

Propofol, a phenolic anaesthetic, is a non-cumulative drug used as an induction agent in cats. The non-cumulative property makes its selection for use in a consecutive day protocol a greater likelihood. Drugs containing -OH, -COOH, -NH2, -HN, and -SH (i.e. propofol and other phenolic compounds, acetaminophen, morphine, chloramphenicol, and salicylic acid) require glucuronide conjugation prior to renal excretion. Cats have low concentrations of the enzyme glucuronyl transferase required for the conjugation of these drugs. The lower concentrations of the enzyme means prolonged exposure of the RBCs to the oxidative parent compounds and/or metabolites[4].

A study conducted in which 6 cats were induced (6 mg/kg IV) and maintained (0.2-0.3 mg/kg/min for 30 min) on propofol for 7 consecutive days made the following findings: no hemolysis or anaemia in any cat, increased recovery time by approximately 25 minutes after day 2, clinical signs of malaise, anorexia, and/or diarrhoea on days 5, 6, and/or 7, increased HzB formation from 0.6% on day 1 to 22-31% on day 7, and facial oedema in 2 cats2. Although haemolytic anaemia was not detected, the RBC parameters were not measured after cessation of propofol (the author suggested this change in protocol for future studies). A decrease in RBC lifespan secondary to HzB formation with resultant regenerative anaemia may not have been detected for several days after propofol cessation. The results of this study suggest that propofol may be harmful in a consecutive day use protocol in cats and this use is not recommended.

  • Onions (Thiosulfates)

Heinz body formation in cats can result from ingestion of raw, cooked, or dehydrated onions. The addition of onion powder to baby food at as high as 1.8% content on a dry matter basis began in 1995 and one study found that as little as 0.3% onion powder in a feline diet resulted in HzB formation. Adverse effects are dose-dependent, with mild changes such as increased HzB formation, and mild decrease in packed cell volume with mild reticulocytosis with low dose exposure, to methemoglobinemia and haemolytic anaemia with high dose exposure.9 The decrease in PCV and increase in reticulocytes suggests an increase in RBC turnover. Several thiosulfate compounds have been implicated as the cause of oxidative injury to RBCs that occurs after onion consumption. Even in small quantities, onion products place an added oxidative stress that may be significant when being fed chronically or to an anorectic cat (with endogenous increased oxidative stress). Onions and onion products are not recommended for consumption by cats[5].

  • Acetaminophen (Tylenol)

Acetaminophen is metabolized by three major pathways including sulphate conjugation, glucuronide conjugation, and via the cytochrome P450 oxidase system. The lack of glucuronyl transferase in cats leads to a build up of toxic metabolites that cause severe oxidative injury, including HzB formation and methemoglobinemia. Acetaminophen is absorbed rapidly (within 60 minutes) and the toxic dose in cats is 50-60 mg/kg (i.e. a single tablet). A high dose of acetaminophen is considered to be 120 mg/kg and is usually associated with severe clinical signs. Administration of a single 90 mg/kg dosage of acetaminophen in one study resulted in methemoglobinemia, HzB formation, and decreased packed cell volume (within reference interval). There is no safe therapeutic dosage for acetaminophen in cats and its use should be avoided in this species[6].

  • Propylene Glycol

Propylene glycol (PG) is a common cause of HzB in cats. PG is a polyhydric alcohol used commonly as a solvent and preservative in pharmaceuticals, and as an inexpensive carbohydrate source in semi-moist foods for animals. PG does not directly cause oxidative damage. It is believed that aldehyde intermediates created during PG metabolism react with amino and sulfhydryl groups on the haemoglobin molecule. Cats fed PG at levels higher than what is typically found in commercial foods have a marked increase in HzB formation (up to 91% of RBCs affected), as well as other RBC abnormalities such as increased adhesion to surfaces, production of ghost cells, and increased membrane fragility.3 Studies have shown that cats on commercial PG-containing diets are more susceptible to the effects of oxidative stress induced by acetaminophen administration, displaying increased HzB and methemoglobin formation, relative to cats on PG-free diets.11 This increased susceptibility may have important clinical consequences in the presence of endogenous oxidative stress as occurs with diseases such as diabetes mellitus, hyperthyroidism, lymphoma, and hepatic lipidosis. The feeding of PG containing diets to cats should be avoided[7].

Diagnosis

HzB can be observed on a peripheral blood smear, frequently protruding from the cell membrane. Supravital stains (methylene blue, bromocresol green) produce a dark staining HzB. With Romanowsky-type stains, HzB may be a similar colour to RBCs if they are projecting outside the membrane or appear as a pale focal area within the RBC.

Treatment

Prevention of oxidative damage is paramount, as treatment can be challenging. Most important is to avoid administering known toxic compounds. Intravascular haemolytic anaemia secondary to HzB formation may require blood transfusion, fluid therapy (to counter acidosis and protect renal tubules from hemoglobinuric damage), and oxygen therapy[8].

Specific antioxidant therapy may include the use of N-acetylcysteine (NAC), vitamin E, or ascorbic acid. Oxygen therapy may also be required. Sometimes a single IV dose of methylene blue may be indicated.

NAC is a cytosolic sulfhydryl donor used to treat acute acetaminophen toxicity orally or intravenously. Recommended doses in cats is 140mg/kg IV ir orally once, followed by 70 mg/kg IV or orally every 4 hours for 7 doses.

Ascorbic acid is given at 30 mg/kg SQ or orally every 6 hours for 7 doses.

Vitamin E helps prevent lipid peroxidation within the RBC membrane and ascorbate scavenges free radicals and regenerates alpha-tocopherol. A study evaluating the short-term use of these three antioxidants showed no substantial prevention or diminishment of HzB formation in cats although protective increases in reduced glutathione were detected. Another study tested the daily use of a bioflavonoid antioxidant in cats given a single 90 mg/kg dose of acetaminophen and found a significant reduction in HzB formation with no affect on metHb formation. The affects of long-term antioxidant therapy may be beneficial especially in disease states producing higher levels of endogenous oxidative stress, as is seen with diabetes mellitus, hyperthyroidism, hepatic lipidosis, and lymphoma.

References

  1. Christopher MM et al (1990) Erythrocyte pathology and mechanisms of Heinz body-mediated hemolysis in cats. Vet Pathol 27:299-310
  2. Duncan, Prasse, and Mahaffey (1994) Erythrocytes. Veterinary Laboratory Medicine, 3rd ed. Ames, Iowa State University Press, pp:21-34
  3. Desnoyers M (2000) Anemias associated with heinz bodies. Schalm's Veterinary Hematology, 5th ed. Feldman BF, Zinkl JG, Jain NC (eds). Baltimore, Lippincott Williams & Wilkins, 2000, pp. 178-180.
  4. Andress JL et al (1995) The effects of consecutive day propofol anesthesia on feline red blood cells. Vet Surg 24:277-282
  5. Robertson JE et al (1998) Heinz body formation in cats fed baby food containing onion powder. J Am Vet Med Assoc 212:1260-1266
  6. Allison RW et al (2000) Effect of a bioflavonoid dietary supplement on acetaminophen-induced oxidative injury to feline erythrocytes. J Am Vet Med Assoc 217:1157-1161
  7. Weiss DJ et al (1990) Effects of propylene glycol-containing diets on acetaminophen-induced methemoglobinemia in cats. J Am Vet Med Assoc 196:1816-1819
  8. Hill AS et al (2001) Antioxidant prevention of Heinz body formation and oxidative injury in cats. Am J Vet Res 62:370-374