Veterinary Clinical Pathology ISSN 0275-6382
CASE REPORT
Botryoid nuclei in the peripheral blood of a dog with heatstroke
Cinzia Mastrorilli1, Elizabeth G. Welles1, Bryan Hux2, Peter W. Christopherson1
Departments of 1Pathobiology and 2Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL, USA
Key Words
Apoptosis, blood smear, hyperthermia,
inappropriate rubricytosis, karyolysis
Correspondence
Cinzia Mastrorilli, DVM, PhD, Diplomate ACVP,
Department of Pathobiology, College of
Veterinary Medicine, 166 Greene Hall, Auburn
University, Auburn, AL 36849, USA
E-mail: czm0010@auburn.edu
DOI:10.1111/vcp.12041
Case Presentation
An EDTA-anticoagulated blood sample collected from
a 1.5-year-old, intact male, English Bulldog was sub-
mitted to the Clinical Pathology Laboratory of the Col-
lege of Veterinary Medicine, Auburn University, for a
CBC. The sample was analyzed by an ADVIA 120
hematology instrument (Siemens Healthcare Diagnos-
tics, Deerfield, IL, USA; software version 3.1.8.0-MS).
A peripheral blood smear, stained in an automated
stainer (Aerospray 7150 Hematology Slide Stainer –
cytocentrifuge, Wescor Inc., Logan, UT, USA) with an
alcohol-based modified Wright’s stain, was reviewed
for the presence of atypical cells.
Pertinent CBC data are given in Table 1. On blood
film examination, a short monolayer area suggested
increased erythrocyte density. There were minimal
anisocytosis and polychromasia and numerous meta-
rubricytes (Figure 1A, Table 1). There were mild
mature neutropenia, and lymphopenia. Numerous
Vet Clin Pathol 42/2 (2013) 145–149 ©2013 American Society for Veterinary Clinical Pathology
Abstract: An EDTA-anticoagulated blood sample collected from a 1.5-
year-old, intact male, English Bulldog was submitted for a CBC. The CBC
data and blood smear evaluation revealed borderline high hematocrit
(54%, reference interval 37–55%), inappropriate rubricytosis, moderate
leukopenia due to both mature neutropenia and lymphopenia, and mild
thrombocytopenia. Numerous leukocytes showed evidence of karyolysis,
pyknosis, and karyorhexis, and apoptotic bodies were frequent in the back-
ground. Many neutrophils had botryoid nuclei characterized by increased
numbers of nuclear segments radially arranged with spoke-like, delicate
chromatin filaments connecting the segments centrally. The finding of bot-
ryoid nuclei and inappropriate rubricytosis was indicative of severe hyper-
thermia, such as heatstroke. The dog had been exercised a long time during
conditions of high temperature and humidity until he collapsed. The dog
was diagnosed with severe heatstroke, hypovolemic shock, disseminated
intravascular coagulation, and multiorgan dysfunction syndrome. Despite
aggressive treatment, the patient died of cardiopulmonary arrest. Botryoid
nuclei are frequent in people with heatstroke. In the authors’ experience,
botryoid nuclei are seen commonly in dogs with heatstroke, but they have
never been reported in veterinary medicine. The presence of petechiation
with only mild thrombocytopenia and inappropriate rubricytosis also is
suggestive of heatstroke and manifests ongoing life-threatening vascular
derangement.
leukocytes showed evidence of karyolysis, pyknosis,
and karyorhexis, and occasional apoptotic bodies
were present in the background (Figure 1B–F). Many
neutrophils had finely stippled chromatin and
increased numbers of nuclear segments radially
arranged with spoke-like, delicate chromatin fila-
ments connecting the segments to the cell center (Fig-
ure 1G–I). Platelets were unevenly distributed, but
appeared to be mildly decreased (estimated platelet
density 100,000–140,000/lL). Final interpretation
was borderline erythrocytosis, likely due to hemocon-
centration or splenic contraction, and mild to moder-
ate leukopenia, likely due to both oncotic and
apoptotic cell death. The peculiar nuclear segmenta-
tion of many of the neutrophils was typical of botry-
oid nuclei. The finding of botryoid nuclei and
inappropriate rubricytosis was indicative of severe
hyperthermia, such as a heatstroke.
The dog had been exercised for a long time during
extreme temperature and humidity conditions (early
145
Botryoid nuclei in heatstroke Mastrorilli et al

Table 1. Hematologic profile of a dog with heatstroke at presentation veterinarian, the dog was laterally recumbent and
to the Auburn University Small Animal Teaching Hospital. dyspneic with a body temperature of 43.8°C (110.9°F).
Analyte Patient’s Values Reference Interval He was treated with oxygen by face mask, water baths
Hematocrit (%) 54 37–55
and fans, IV fluids, and an antibiotic (ampicillin). As
RBC (106/lL) 7.21 5.5–8.5 the dog developed neurologic signs, it was treated with
Hemoglobin (g/dL) 18.2 12–18 5 mg diazepam IV and 11 g mannitol IV over 20
MCV (fL) 74.9 60–77 minutes, and was then referred to the Critical Care Ser-
MCHC (g/dL) 33.7 32–36 vice of the Auburn University Small Animal Teaching
Nucleated RBCs (/100 WBC) 95 – Hospital (CCS-AUSATH).
Noncorrected WBC (/lL) 6570 6000–17,000
Upon presentation to CCS-AUSATH, the dog was
Corrected WBC (/lL) 3370 6000–17,000
Neutrophils (/lL) 2359 3000–11,400
stuporous, with cold, pale to slightly cyanotic mucous
Bands (/lL) 0 0–300 membranes, prolonged capillary refill time, and weak
Lymphocytes (/lL) 842 1000–4000 pulses (heart rate 120 bpm). There was diffuse epider-
Platelets (/lL) 127,000 164,000–510,000 mal and mucosal petechiation. Foul smelling mucoid
MPV (fL) 11.3 8.4–13 hematochezia with flecks of sloughed tissue was ooz-
ing from the rectum. During examination, the dog
afternoon, recorded minimum and maximum temper- vomited a large amount of hemorrhagic mucus, while
atures of that day, 23.8–35°C [75–95°F])1 until he sud- his body temperature continued to decrease (lowest
denly collapsed. Upon presentation to the referring temperature 35.2°C [95.4°F]).

A B C D

E F G

H I J

Figure 1. Peripheral blood smear of a dog with heatstroke. (A) Metarubricyte; (B) Karyolytic cell with typical pale-stained nucleus due to dissolution
and leakage of chromatin from the nuclear membrane; (C) Pyknotic cell (upper) and karyorhectic cell (lower); (D-F) Apoptotic bodies with organelles and/
or condensed nuclear fragments; (G-I) Botryoid nuclei characterized by increased numbers of nuclear segments radially arranged with spoke-like, deli-
cate chromatin filaments that connect them to the center of the cell. Chromatin condensation is lacking; (J) Blood from a healthy dog with normal body
temperature with an aged neutrophil characterized by condensed chromatin and hypersegmentation with linear arrangement of the nuclear segments.
Modified Wright’s stain, 9100 objective.

146 Vet Clin Pathol 42/2 (2013) 145–149 ©2013 American Society for Veterinary Clinical Pathology
Mastrorilli et al
The dog was diagnosed with severe heatstroke and
subsequent hypovolemic shock, disseminated intra-
vascular coagulation (DIC), and multiorgan dysfunc-
tion syndrome. The following day, the dog developed
pulmonary edema, went into cardiopulmonary arrest,
and could not be re-animated.
Discussion
The major findings in this case are the presence of bot-
ryoid nuclei, karyolytic, pyknotic, and karyorhectic
leukocytes with apoptotic bodies, petechiation despite
only mild thrombocytopenia, and inappropriate rubri-
cytosis.
Botryoid nuclei (or botryoid neutrophils) are
described in people with burns and hyperthermia and
are considered a characteristic feature supporting the
diagnosis of heatstroke.2–4 The term botryoid derives
from the Greek “botrys,” meaning bunch of grapes,
and was proposed because the nuclear segments in
botryoid cells are clustered in a fashion that is reminis-
cent of grapes on a stem.2
To the authors’ knowledge, botryoid nuclei have
never been reported in veterinary medicine, although,
in our experience (authors CM, EGW, PWC), they are
seen commonly in dogs with heatstroke. Pictures of
botryoid neutrophils in dogs have been published in a
case of Adderall toxicity with related hyperthermia5
and in dogs treated for cancer with whole body hyper-
thermia,6 but the neutrophil nuclei were described
as being simply “hypersegmented”5 or “atypically
hypersegmented with pinwheel appearance.”6 The
hypersegmentation was hypothesized to be the result
of accelerated cell aging or heat-induced damage.5
The hypersegmentation typically observed in aged
neutrophils is characterized by chromatin condensa-
tion and linear arrangement of nuclear segments
(Figure 1J), whereas in botryoid nuclei chromatin
condensation is lacking and nuclear segments have a
characteristic radial distribution (Figure 1G–I).
Although botryoid nuclei appear to be radially
segmented, from a strict point of view “radial segmenta-
tion” is a phenomenon observed in human mononu-
clear cells cultured or stored with sodium oxalate and in
certain human leukemias of mononuclear cells, which
were first described by Rieder in 1893 and named “Rie-
der cells.”7,8 Radial segmentation develops during an
aberrant mitotic division because of perinuclear con-
traction of the mitotic spindle, which on light micros-
copy is seen as bright cytoplasmic areas of “less stainable
matter” between the nuclear segments.8 Although it has
been reported that botryoid nuclei result from radial seg-
Vet Clin Pathol 42/2 (2013) 145–149 ©2013 American Society for Veterinary Clinical Pathology
Botryoid nuclei in heatstroke
mentation,9 cytoplasmic brightening is not present in
botryoid cells and, in the authors’ opinion, botryoid cells
are not undergoing mitosis, but are more likely to have
been damaged by high temperature.
The pathogenesis of cell damage in heatstroke is
not well understood. The effect of heat on the body is
determined by the degree to which the temperature
rises and its duration.10,11 At temperatures of 39.5°C
(103.1°F)12, 41.5°C (106.7°F)10, and 42.5–43°C
(108.5–109.4°F)13, cell death has been reported to
result from apoptosis via an unknown initiator of the
caspase cascade.10,12,13 During febrile illnesses, neutro-
phil apoptosis may be triggered as a defensive mecha-
nism to limit collateral tissue injury.12 Extreme
temperatures (49–50°C [120.2–122°F]) induce dena-
turation and unfolding of cytoskeletal proteins and cel-
lular enzymes, liquefaction of membrane lipids, and
damage to mitochondria with resulting cell death by
oncosis in less than 5 minutes.14
In the authors’ opinion, the morphologic changes
seen in this patient’s peripheral blood smear may be
attributed to both oncotic and apoptotic cell death. It is
not always possible to make the distinction between
oncosis and apoptosis based on cell morphology, but,
as a general rule, karyolysis is typical of oncotic cell
death, whereas, karyorhexis and pyknosis suggest
apoptosis, but are not pathognomonic.15 In the present
case, it is possible that the apoptotic pathway was trig-
gered during the phase of heat exhaustion. With pro-
gression to heatstroke, the increasing temperature
may have resulted in abortion of apoptosis and signifi-
cant cell damage to cause cell death by oncosis. The
exaggerated nuclear segmentation of botryoid nuclei
may have resulted from a sudden alteration of the
cytoskeleton of cells that were in an early phase of
apoptosis, before the DNA had been broken down in
the organized fashion typical of apoptosis.
It is worth pointing out that karyorhexis, as first
described by Klebs, is spelled with a single “r.”16 The
current incorrect spelling with a double “r” derives
from a typographical error during the printing of the
title of a subsequent manuscript, and the misspelled
“karyorrhexis” then became fixed in the English litera-
ture.15,17
Apoptotic cells and bodies were frequent in the
peripheral blood of this dog. Apoptotic cells are fre-
quently found in the peripheral blood of people with
cobalamin deficiency due to unrepaired excision of
uracil, but they have never been described in dogs with
similar or other disorders.18 Hypersegmented neu-
trophils in cobalamin deficiency resemble aged neu-
trophils and appear to form because of a defect in
nuclear maturation.18,19
147
Botryoid nuclei in heatstroke
The occurrence of petechiation with only mild
thrombocytopenia has been reported in dogs with
heatstroke and has been attributed to heat-induced
platelet dysfunction and vascular abnormalities.20 In
heatstroke, thrombocytopenia is likely due to periph-
eral consumption because of DIC and heat-induced
vasculitis, gastrointestinal bleeding, and hyperthermia-
induced platelet aggregation and destruction.20
Inappropriate rubricytosis is a frequent finding in
dogs with heat-related illness such as heatstroke.20–22
It is believed that hyperthermia may cause direct dam-
age to the bone marrow sinusoidal epithelium with
subsequent inappropriate release of nRBCs, but so far
histopathologic evidence of disruption of the blood-
bone marrow barrier has not been confirmed.20–23 It
has been postulated that light microscopy may not be
sensitive enough to detect such changes, or the release
of nRBCs may be mediated by cytokines produced
during the systemic inflammatory response.23
Another known cause of inappropriate rubricyto-
sis is splenic contraction.5 However, on histopathologic
examination, the spleen of many dogs with fatal heat-
stroke is characterized by red pulp congestion.23 It is
possible that the nRBCs are released from the spleen in
the early phase of heat exhaustion when splenic con-
traction develops in response to poor visceral perfu-
sion. In advanced heatstroke, vasoconstriction is
prevented by the hypothalamic sympathetic center
with resultant splanchnic vasodilation and conges-
tion.23 The fact that the number of circulating nRBCs
decreases rapidly after hospitalization (usually within
12–24 hours)20,22 makes the hypothesis of splenic
contraction more likely than the disruption of the
blood-bone marrow barrier. Tissue perfusion is likely
re-established within 24 hours of intensive care,
whereas evidence of endothelial integrity derange-
ment, such as DIC, persists in many patients.
Circulating nRBCs likely occur in human heat-
stroke, but their presence has not been emphasized in
the recent literature. The presence of circulating
nRBCs in critically ill people has been seen with a vari-
ety of severe diseases and is associated with increased
mortality risk.24 Similarly, the presence and magni-
tude of rubricytosis at the time of presentation were
found to be predictors of death in dogs with heat-
stroke.22 In our opinion, the fact that the relative num-
ber of nRBCs was found to be a better prognostic
indicator than the absolute number22 is because higher
relative numbers of nRBCs occur in the presence of
leukopenia, and it is likely that the leukopenia itself is
indicative of a more severe disease status.
Inappropriate rubricytosis is seen also in lead toxico-
sis, where the number of nRBC can be from 1/100 WBC
148
Mastrorilli et al
up to > 200/100 WBC, while anemia is only present in
the late phase of the chronic form of toxicosis.25 Baso-
philic stippling is not a consistent finding in dogs, but its
presence makes the diagnosis of lead toxicosis likely.25,26
In conclusion, botryoid nuclei have never been
described in veterinary literature, but are a common
finding in dogs with heatstroke. The identification of
botryoid nuclei on peripheral blood smears may aid in
the diagnosis of heatstroke especially when history is
lacking, the index of suspicion is low (eg, moderate
environmental temperature), or the body temperature
has already returned within physiologic limits. The pre-
sence of petechiation with only mild thrombocytopenia
and inappropriate rubricytosis also is suggestive of heat-
stroke and should alert the clinician that widespread
vascular activation has been triggered and multiorgan
failure must therefore be anticipated, regardless of how
rapidly or effectively the patient has been cooled.
Disclosure: The authors have indicated that they have
no affiliations or financial involvement with any
organization or entity with a financial interest in, or in
financial competition with, the subject matter or
materials discussed in this article.
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