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                    Echinocytosis Associated with Rattlesnake Envenomation in Dogs
                              D. E. Brown, D. J. Meyer, W. E. Wingfield and R. M. Walton
                                                Vet Pathol 1994 31: 654
                                         DOI: 10.1177/030098589403100604

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Vet Pathol 31:654-657 (1994)

                         Echinocytosis Associated with Rattlesnake
                                  Envenomation in Dogs
                    D. E. BROWN, J. MEYER, E. WINGFIELD, R. M. WALTON
                               D.        W.           AND

        Departments of Pathology (DEB, DJM, RMW) and Clinical Sciences (WEW), Colorado State University,
                      College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO

         Abstract. A retrospective study of 28 cases of rattlesnake bites in dogs indicated an association of enven-
      omation with echinocytosis; 25/28 dogs (89%) had echinocytosis within 24 hours of a rattlesnake bite being
      reported. Eighteen of the 28 dogs had marked type I11 echinocytosis (95-1 00% of mature erythrocytes affected),
      and seven dogs had moderate echinocytosis (15-30°/o of mature erythrocytes affected). The echinocytosis was
      transient, resolving within 48 hours of envenomation in those for which subsequent blood films were available
      (13/25). Hypokalemia was noted in 14 of 18 dogs for which biochemical data were available. The hypokalemia
      was not consistently associated with echinocytosis. The observation of echinocytosis in dogs supports a clinical
      diagnosis of rattlesnake envenomation.

        Key words: Dogs; echinocyte; erythrocyte; snakebite.

   Echinocytes are erythrocytes that have uniformly the nature of spiculation, as previously described.16 Eryth-
sized, regularly spaced projections from their mem- rocytes were counted in five oil-immersion fields at 1,000x
branes. The exact mechanism for echinocytic shape magnification to quantitatively assess percentage of echino-
change is unknown.12 Echinocyte formation can be an cytes present.
artifactual result of pH change from slow drying of                The packed cell volume (PCV), mean cell volume, and
                                                                 platelet count were reviewed from the 28 cases. Follow-up
blood films (~renation),'~ result from depletion of
                                                                 complete blood counts (CBCs) were evaluated when available
erythrocytic adenosine 5'-triphosphate (ATP) or in- (15/28 cases). Hemograms were performed with a multi-
creased cytosolic calcium and exposure to anionic am- channel blood cell counting system (Coulter Counter S-Plus
phipathic drugs,I2J6 and may be secondary to total body IV with histogram capability or Coulter T660, Coulter Co.,
cation depletion.'^^^ Echinocytosis has been associated Hialeah, FL).
with specific diseases, including glomerulonephritis and           Serum biochemical analyses were available for 18/28 dogs.
lymphosarcoma in the dog16 and pyruvate kinase de- Because many of these dogs were admitted on an emergency
ficiency in Basenjis.2 A burring phenomenon of eryth- basis to the critical care unit, complete serum biochemical
rocytes has been reported with snakebite in human profiles were not always done. The most common serum
 being^,^^^ a finding supportive of e n v e n ~ m a t i o n . ~ .analytes measured in this study were blood urea nitrogen
Erythrocyte burring has also been reported with coral (BUN), creatinine, sodium. and potassium. Serum analyses
                                                                 were performed on discrete automated analyzers (Hitachi
snake envenomation in dogs.8 This burring phenom-
                                                                 704, Boehringer-Mannheim, tndianapolis, IN, or Dupont
enon is morphologically compatible with echinocytes. ACAIII, Dupont, Wilmington, DE).
   A transient echinocytosis often develops rapidly in
dogs envenomated by rattlesnakes. The purpose of this In vitro studies
study was to retrospectively examine the association                Echinocyte formation can be an artifactual result of pH
of echinocytosis with rattlesnake envenomation in dogs. change from slow drying of blood films (~renation).'~            To
                                                                            evaluate this possibility, one drop of EDTA-preserved blood
                 Materials and Methods
                                                                            from each of five dogs with rattlesnake envenomation was
Retrospective studies                                                       mixed on a glass microscope slide with one drop of isotonic
  Wright-Giemsa-stained blood films were reviewed from                      saline. A coverslip was applied, and the slide was examined
28 dogs bitten by rattlesnakes from 1988 to 1993. The dogs                  by light microscopy with partial closure of the diaphragm.
were historically healthy; males and females of various breeds              As controls, saline wet preparations of blood were prepared
were represented. Initial blood films were collected within                 simultaneously from nonenvenomated healthy dogs.
24 hours of a reported bite, and subsequent blood films were                                               Results
available for 15/28 dogs. Blood films were made from eth-
ylenediaminetetraacetic acid (EDTA)-preserved blood.                          In eighteen of 28 dogs (64%), 95-100% of mature
Echinocytes were characterized as types 1-111, dependent on                 erythrocytes were type I11 echinocytes (Fig. l), which

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Vet Pathol 31:6, 1994                  Canine Echinocytosis and Rattlesnake Bite                                           655

  Fig. 1. Blood; Labrador Retriever. Dog was envenom-        Fig. 2. Saline preparation, EDTA-preserved blood; Lab-
ated by a rattlesnake approximately 4 hours prior to sam-  rador Retriever. Dog was envenomated by a rattlesnake ap-
pling. Echinocytic erythrocytes are numerous. Polychromat- proximately 4 hours prior to sampling. Echinocytic eryth-
ophilic erythrocytes retain their discoid shape (arrows). rocytes are present. No stain. Bar = 10 pm.
Wright-Giemsa stain. Bar = 10 pm.
                                                                        first 24 hours following envenomation. The echino-
are described as "ovoid or spherical cells with 10 to                   cytosis decreased from 100% to < 30% of cells affected
30 sharp, regularly spaced projections."I6 In seven dogs,               without normalization of serum potassium in five dogs.
within the first 24 hours 15-309'0 of mature erythro-                   Three dogs had echinocytosis with the serum potas-
cytes were echinocytes (type I1 and type III), and three                sium concentration within the reference range.
dogs had no echinocytes (one of these had spherocy-                        Complete blood counts showed a mild decrease
tosis). All polychromatophilic erythrocytes observed                    (mean decrease of 7.1%) in PCV in 10 of 28 dogs. In
had normal morphology (Fig. 1). Subsequent CBCs                         four of these dogs, PCV returned to reference range by
were available for 13/25 dogs with echinocytosis.                       day 2-6 following envenomation. In the remaining six
Echinocytes accounted for 5 30% of mature erythro-                      dogs, no follow-up CBC was performed. One anemic
cytes in 12 of these dogs by 48 hours; in one dog, 100%                 dog had spherocytosis and erythrocyte agglutination
of mature erythrocytes were echinocytes at 48 hours.                    with no echinocytes. Mean cell volumes were within
There did not appear to be a correlation in the reduc-                  reference range for all 28 dogs. There was thrombo-
tion of echinocytes and whether or not antivenin treat-                 cytopenia (platelet counts < 150,OOO/pl) in 18 of 28
ment was given.                                                         dogs; eight dogs normalized by day 2-6 following en-
   Hypokalemia was the only consistent biochemical                      venomation. The other 10 dogs had no follow-up. The
abnormality in 14 of 18 dogs (78%). Mean serum po-                      mean platelet count for envenomated dogs with throm-
tassium was 3.6 mEq/liter (range, 2.9-4.0 mEq/liter;                    bocytopenia was 70,000 plateletdpl (range, 11,000-
reference range, 4.1-5.5 mEq/liter). All 18 dogs had                    131,000 plateletslpl).
normal BUN and serum creatinine concentration. Only                        The majority of erythrocytes from envenomated dogs
one dog was hyponatremic. Nine of 14 dogs with hy-                      were echinocytic on saline wet preparations, supporting
pokalemia also had marked echinocytosis within the                      an in vivo phenomenon (Fig. 2). Saline wet prepara-

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656                                      Brown, Meyer, Wingfield, and Walton                                        Vet Pathol 3 1 5 , 1994

tions of blood prepared simultaneously from nonen-                     find hypokalemia in 14/18 dogs (78%). The cause of
venomated healthy dogs had normal discoid erythro-                     the hypokalemia was not apparent, and its association
cytes.                                                                 with echinocyte formation is unknown, although elec-
                                                                       trolyte alterations may be invo1ved.'J5 Hypokalemia
                      Discussion                                       in these cases was not consistently associated with
   Our study demonstrated a strong association be-                     echinoc ytosis.
tween echinocyte formation and envenomation.                              Sixty-four percent of the dogs had thrombocytope-
Echinocytes were present within 24 hours of a snake-                   nia, and 44% of those had returned to reference range
bite (89% of cases), and the shape change largely re-                  by day 2-6; no follow-up was available for the re-
solved by 48 hours. Although the majority (64%) of                     mainder of the dogs. The effects of rattlesnake enven-
dogs bitten had marked type I11 echinocytosis, there                   omation on platelets and coagulation have been pre-
was variation in percent echinocytosis among individ-                                                Platelet
                                                                       viously r e p ~ r t e d . ~ . ~ J ~ J ~ aggregation induced
uals. These differences may represent delayed time to                  directly by venom components and/or endothelial
presentation following bite, lower dose of venom re-                   damage with exposure of underlying collagen and
ceived, or no envenomation (no echinocytes). The ob-                   platelet aggregation at the site of vascular wall injury
servation that polychromatophilic erythrocytes ap-                     may contribute to the thrombocytopenia observed in
peared resistant to echinocyte transformation is not                   dogs in this study.'JOApproximately 20% of poisonous
explained. These cells are more resistant to crenation                 snakebites do not result in envenomation.ll Early di-
than are mature erythrocyte^.^ Polychromatophilic                      agnosis of envenomation is critical to appropriate ther-
erythrocytes, in contrast to mature erythrocytes, still                apeutic intervention. The observation of echinocyto-
have mitochondria and the ability to aerobically gen-                  sis, in dogs supports a clinical diagnosis of rattlesnake
erate ATP, therefore, they may be more resistant than                  envenomation. Use of a saline wet preparation is a
mature erythrocytes to potential ATPase effects of snake               quick, simple procedure that can be used to identify
venom.                                                                 echinoc ytosis.
   Mild anemia occurred in 10 of 28 dogs with return
of PCV to reference range by day 2-6 following en-                                                     References
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Vet Pathol 31:6, 1994                     Canine Echinocytosis and Rattlesnake Bite                                                   657

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