The Significance of Echinocytosis in Blood Smears

Sherri L. Stello, DVM; Kenneth S. Latimer, DVM, PhD, Perry J. Bain, DVM, PhD; Paula M. Krimer, DVM, PhD

Class of 2003 (Stello) and Department of Pathology (Latimer, Bain, Krimer), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Overview

Echinocytosis is a common observation in blood smears from a variety of animal species. It is often overlooked as an artifact of preparation. However, several disease processes and toxins have been found to alter the red blood cell membrane, leading to the formation of echinocytes. Therefore, the presence of echinoctyosis on blood smear examination or on hematology reports may have diagnostic significance.

Echinocyte Morphology

Echinocytes are morphologically altered red blood cells that appear to have numerous, fine, uniform spicules throughout the cell membrane.⁴ In mammals, echinocytes have been classified into five main types according to progressive stages in formation and maturation (Figs. 1 & 2). Type I echinocyte (echinodiscocytes) are described as irregularly shaped erythrocytes without defined spicules. Type II echinocytes have cellular projections that vary in length; however, these erythrocytes maintain a disc-shaped appearance. Type III echinocytes are more spherical erythrocytes with high spiculation. Spheroechinocytes I and II are slightly smaller erythrocytes that closely resemble spherocytes with blunted spicules.¹ These cells have been found to be rigid and nondeformable which may reduce blood flow in the microcirculation or cause microvascular injury.⁵ Some hematologists hypothesize that echinocyte formation is an anti-hemolytic tactic with an increase in plasma membrane surface area relative to cellular volume. Therefore, the cell volume may increase to a greater degree before lysis occurs.¹⁰

Figure 1. The sequential transformation of discocytes to echinocytes and spheroechinocytes (Bessis M: Blood Smears Reinterpreted, Springer-Verlag, 1977, p. 51).

Figure 2. Scanning electron micrographs of discocyte transformation into echinocytes. A. Discocyte, B. Early echinocyte, C. Well developed echinocyte (modified from Bessis M: Blood Smears Reinterpreted, Springer-Verlag, 1977, p. 53).

Erythrocyte Membrane Structure

The normal erythrocyte membrane is composed of a lipid bilayer and cytoskeleton. The lipid bilayer is mainly composed of cholesterol and phospholipids. The distribution of phospholipids vary in that some are located in the outer layer (glycolipids, phosphatidylcholine, sphingomyelin), while others occur in the interior layer toward the cytoplasm (phosphatidylinositols, phosphatidylethanolamine, phosphatidylserine). The distribution of phospholipids, transmembrane proteins, and cholestero,l in combination with the protein network of the cytoskeleton, are responsible for the integrity of the erythrocyte membrane.⁷

Differential Diagnosis for Echinocytosis

Differential diagnoses for the observation of echinocytes in blood smears or mention of echinocytes on the complete blood cell count is presented in Table 1.

Table 1. Differential diagnoses for the presence of echinocytosis.

Artifact: The Effects Of Glass and Blood Storage

Echinocytosis is often an artifact that results from basic substances diffusing out of the glass slide and cover slip resulting in an elevated pH (Fig. 3). This effect can be seen within minutes of blood smear preparation. If the erythrocytes are washed with physiologic saline before blood cell preparation, erythrocytes may transform into spheroechinocytes and hemolysis eventually may occur. This phenomenon can be avoided by using a plastic slides and coverslips to examine blood smears (but critical microscopic resolution is better achieved using glass slides and coverslips).

Figure 3. Echinocytes in the blood of a cat with peritonitis. Echinocytosis is probably an artifact of blood smear preparation (Wright-Leishman stain).

In order to diagnose echinocytosis in vivo, a fresh drop of blood that has had no contact with glass should be used. Oil can be substituted for physiologic saline to avoid the formation of spheroechinocytes.

Echinocytes also have been shown to increase in stored blood. The mechanisms responsible for echinocyte production include depletion of ATP (described below) and formation of lysolecithin in the plasma. Echinocytes can be observed within 24 hours of storage at 37^o C or after 3 weeks of storage at 4^o C. Storage-induced echinocytosis may be avoided by heating the sample to 56^oC for 30 minutes before storage. The heating is believed to destroy lecithin-cholesterol acyl transferase, an enzyme that produces lysolecithin in stored plasma. Echinocytosis may be reversed upon transfusion of the blood into a normal recipient or by restoring ATP with fresh plasma.¹

Erythrocyte Dehydration and ATP Depletion

Several mechanisms of echinocyte formation have been described in the medical literature. Most commonly mentioned is in vitro formation of echinocytes from red blood cell dehydration or expansion of the outer membrane leaflet by amphipathic drugs. Erythrocyte dehydration may be a response to cellular energy depletion (ATP) or increased intracellular calcium. These mechanisms are thought to cause RBC dehydration through loss of potassium. Decreased ATP inhibits the ATP-dependent sodium/potassium pump in the cellular membrane. In addition, an increase in intracellular calcium causes a rapid loss of potassium, water, and ATP.⁸ One study of echinocytosis in horses found elecrolyte imbalances, such as hyponatremia and hypochloremia, were commonly associated with an increased prevalence of echinocytes. Extracellular depletion of electrolytes leads to decreased extracellular fluid volume. In turn, a shift (osmotic effect) occurs as fluid moves from the intracellular compartment to the extracelluar compartment in a compensatory measure. Erythrocytes dehydrate as a result of this shift. Electrolyte depletion may be a complication in gastrointestinal or metabolic disease. It has also been found to be prominent in horses undergoing furosemide treatment.⁵

Amphiphilic Substances

Amphiphilic molecules, found in some detergents, intercalate into the outer lipid bilayer leading to subsequent morphologic changes in the erythrocyte's membrane. Band 3 is an anion exchange protein in the cell membrane that has been proposed to be a major factor in echinocyte formation. Mediation of influx and efflux of anions through the membrane cause inward- and outward-facing conformations of Band 3 protein, leading to contraction and relaxation of the membrane cytoskeleton by folding and unfolding spectrin (cytoskeletal component bound to Band 3). It is this change in the membrane skeleton that leads to morphologic transformation. Anionic and non-ionic amphiphilic drugs and detergents are inhibitors of Band 3 anion transport.⁹ Exogenous substances such as ozone, hexachlorocyclohaxane, acrolein and phenylhydrazine have been shown to induce echinocytosis at sublethal doses. However, the effect of chronic exposure to these substances at low concentrations remains to be determined .⁹

Snake Bite Envenomation

Several studies in dogs have found an increased incidence of echinocytosis after envenomation by the western diamond back rattlesnake (Crotalus atrox) and the coral snake (Micrurus fulvius). In one retrospective study of 28 cases of rattlesnake bites in dogs, 25 of 28 patients had echinocytosis within 24 hours of envenomation. Over 50% of patients had marked formation of type III echinocytes involving 95-100% of mature erythrocytes; the remaining patients had moderate echinocytosis that involved 15-30% of the erythrocyte population. The echinocytosis was transient and resolved within 48 hours. The pathogenesis leading to echinocytosis from envenomation is not well understood. Venom contains phospholipases, ATPases, and proteases. ATPases may lead to a depletion of ATP and alteration of membrane composition by phospholipases. In rattlesnake bites, phospholipase A2 (a calcium-dependent enzyme) may result in lysolecithin production in the plasma with subsequent echinocyte production. Electrolyte imbalances were a common finding that also could lead to dehydration of erythrocytes as described above.^2,6

Additional Considerations for Echinocytosis

Echinocytosis has been observed in lymphosarcoma, pyruvate kinase (PK) deficiency (Fig. 4), uremia, doxorubicin treatment, and glomerulonephritis.^3,8 The pathophysiology leading to echinocytosis in these diseases is speculative. PK deficiency is associated with energy depletion. Echinocytosis in lymphosarcoma may be partially the result of chemotherapy. Uremia and glomerulonephritis may be associated with acid-base and electrolyte disturbances. The mechanism for echinocytosis in doxorubicin treatment is unknown.

Figure 4. Echinocytes in the blood smear of a basenji with pyruvate kinase deficiency. Notice the polychromatophilic erythrocytes indicating erythroid regeneration (Wright-Leishman stain).

References

1. Bessis M: Blood Smears Reinterpreted. Springer-Verlag, Berlin, 1977, pp. 50-52, 64.

2. Brown DE, Meyer DJ, Wingfeld WE, Walton RM: Echinocytosis associated with rattlesnake envenomation in dogs. Vet Pathol 31:654-657, 1994.

3. Feldman B, Zinkl J, Jain N (eds): Schalm’s Veterinary Hematology, 5^th ed. Lippincott, Williams & Wilkins, Philadelphia, 2000, p. 147.

4. Glader BE, Lukens JL: Hereditary spherocytosis and other anemias due to abnormalities of the red cell membrane. In: Lee GR, Foerster J, Lukens J, Paraskevas F, Greer JP, Rodgers GM (eds): Wintrobe’s Clinical Hematology, 10^th ed, vol 1. Lippincott, Williams & Wilkins, Baltimore, 1998, pp. 1132-1159.

5. Geor R, Lund E, Weiss D: Echinocytosis in horses: 54 cases (1990). J Am Vet Med Assoc 202:976-979, 1993.

6. Marks S, Mannella C, Schaer M: Coral snake envenomation in the dog: Report of four cases and review of the literature. J Am Animal Hosp Assoc 26:629-633, 1990.

7. Smith JE: Erythrocyte membrane: Structure, function, and pathophysiology. Vet Pathol 24:471-476, 1987.

8. Weiss D, Kristensen A, Papenfuss N, McClay C: Quantitative evaluation of echinocytes in the dog. Vet Clin Pathol 19:114-117, 1990.

9. Wong P: A basis of echinocytosis and stomatocytosis in the disc-sphere transformations of the erythrocyte. J Theor Biol 196:343-361, 1999.

10. Zeni C, Bovolenta M, Stagni A: Occurrence of echinocytes in circulating RBC of black bullhead, Ictalurus melas (Rafinesque), following exposure to an anionic detergent at sublethal concentrations. Aquatic Toxicol 57:217-224, 2002.

Acknowledgement

Photograph of Solnhofen starfish (Barthel; © 1978 Ott Verlag Thun) is from the website of the UCLA IGPP Center for Astrobiology

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