The patients were treated at the Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Israel. Partial results of this study were presented at the 17th Annual ECVIM-CA Congress, Septembr 13–15, 2007, Budapest, Hungary.
Peripheral Nucleated Red Blood Cells as a Prognostic Indicator in Heatstroke in Dogs
Version of Record online: 28 APR 2009
Copyright © 2009 by the American College of Veterinary Internal Medicine
Journal of Veterinary Internal Medicine
Volume 23, Issue 3, pages 544–551, May/June 2009
How to Cite
Aroch, I., Segev, G., Loeb, E. and Bruchim, Y. (2009), Peripheral Nucleated Red Blood Cells as a Prognostic Indicator in Heatstroke in Dogs. Journal of Veterinary Internal Medicine, 23: 544–551. doi: 10.1111/j.1939-1676.2009.0305.x
- Issue online: 19 MAY 2009
- Version of Record online: 28 APR 2009
- Submitted June 15, 2008; Revised January 20, 2009; Accepted January 21, 2009.
- Acute kidney injury;
- Disseminated intravascular coagulation;
Background: Heatstroke in dogs is often fatal and is associated with a high prevalence of secondary complications. Peripheral nucleated red blood cells (NRBC) occur in dogs with heatstroke, but their association with complications and the outcome is unclear.
Hypothesis: Peripheral NRBC are common in dogs with heatstroke and have prognostic significance.
Animals: Forty client-owned dogs with naturally occurring heatstroke.
Methods: Prospective, observational study. Dogs were followed from presentation to discharge or death. Serum biochemistry and coagulation tests were performed at presentation. CBC and evaluation of peripheral blood smears were performed at presentation and every 12 hours. The relative and the absolute NRBC numbers were calculated.
Results: Presence of NRBC was observed in 36/40 (90%) of the dogs at presentation. Median relative and absolute NRBC were 24 cells/100 leukocytes (range 0–124) and 1.48 × 103/μL (range 0.0–19.6 × 103/μL), respectively. Both were significantly higher in nonsurvivors (22) versus survivors (18) and in dogs with secondary renal failure and DIC versus those without these complications. Receiver operator curve analysis of relative NRBC at presentation as a predictor of death had an area under curve of 0.92. A cut-off point of 18 NRBC/100 leukocytes corresponded to a sensitivity and specificity of 91 and 88% for death.
Conclusions and Clinical Importance: Relative and absolute numbers of peripheral NRBC are clinically useful, correlate with the secondary complications, and are sensitive and specific markers of death in dogs with heatstroke, although they should never be used as a sole prognostic indicator nor should they replace clinical assessment.
absolute nucleated red blood cells
activated partial thromboplastin time
central nervous system
disseminated intravascular coagulation
Hebrew University Teaching Hospital
nucleated red blood cells
relative nucleated red blood cells
receiver operating characteristics
systemic inflammatory response syndrome
white blood cells
Heatstroke in dogs is a severe clinical syndrome characterized by core temperatures > 41°C (105.8°F) with central nervous system (CNS) dysfunction and tachypnea.1,2 It can result from exposure to a hot and humid environment (classical or environmental heatstroke) or from strenuous physical exercise (exertional heatstroke). Heatstroke is quite common in dogs and occurs particularly during the summer months, mainly in hot and humid environments.1,2 It is associated with a systemic inflammatory response syndrome (SIRS), leading to multiple organ dysfunction and death.3,4
Clinical heatstroke or heat-related illness in dogs has been described in 2 relatively large retrospective studies with an overall mortality of 50 and 64%.1,2 Risk factors for death include obesity, prolonged (> 90 minutes) time lag from the heat insult to presentation, hypoglycemia (< 47 mg/dL) on admission, azotemia (creatinine > 1.5 mg/dL at 24 hours after presentation), and development of disseminated intravascular coagulation (DIC) and acute kidney injury.2 Mental status on presentation (comatose versus noncomatose) is associated with death.1 Additionally, nonsurvivor dogs have lower glucose, total protein, albumin, and cholesterol concentrations and significantly higher concentrations of creatinine and total bilirubin than survivors. Nonsurvivors also have a higher occurrence of ventricular arrhythmias.1
Because of the high rate of systemic complications and fatality in heatstroke, treatment is challenging, complex, and costly, whereas the prognosis is uncertain. Although some of the above-mentioned risk factors can be used to assess the disease severity and the prognosis at presentation, others cannot be used at that time and, thus, cannot facilitate prognostic projections. Therefore, a readily available, cost-efficient tool that can accurately predict the severity of illness and the outcome in dogs with heatstroke and assist in therapeutic planning would be extremely useful.
Peripheral nucleated red blood cells (NRBC) are reported in blood smears of 15/26 dogs (58%) with heat-related illness.1 NRBC occur in mildly, moderately, and severely affected animals, with a median count of 2.5 and 1.0 cells per 100 white blood cells (WBC) in survivors and nonsurvivors, respectively. Counts in 16 survivors and 8 nonsurvivors were 0–3 and 0–95 cells per 100 WBC, respectively.1 Peripheral NRBC decreased by ≥ 66% in 3 dogs, which were sampled sequentially. However, there was no information when this was evident in relation to presentation or heat insult.1 NRBC are detected in 13/19 dogs with heatstroke (68%) (median relative NRBC 10, range 1–67 cells/100 leukocytes).2 The NRBC to polychromatophils ratio was > 1 in all cases, indicating a pathologic process unrelated to erythroid hyperplasia.2,3
The aims of this study were to prospectively investigate the prevalence, characteristics, and association of peripheral NRBC with clinical and clinicopathological data in naturally occurring heatstroke in dogs and to assess its usefulness as a marker of secondary complications, such as acute kidney injury and DIC, and as a prognostic indicator.
Materials and Methods
Selection of Cases and Collection of Data
This prospective study was approved by the Animal Care and Use Committee of the Hebrew University Veterinary Teaching Hospital (HUVTH). Dogs diagnosed with heatstroke based on the following criteria were enrolled prospectively and consecutively into the study. All dogs were presented to the Emergency Service, HUVTH, between May 2005 and September 2006, and diagnosed with heatstroke based on the history and presence of characteristics clinical signs, which had developed only after exposure to a warm environment, strenuous activity, or both. These signs included collapse, with or without CNS abnormalities. Collapse was defined by a history or physical examination findings of an acute loss of ambulation, recumbency, and inability to stand, which had occurred acutely. Dogs with coexisting medical conditions were excluded. Data included the signalment, history, clinical signs on admission and during hospitalization, laboratory test results, hospitalization time period (from presentation to discharge or death), outcome (survived to discharge, dead, or euthanized during hospitalization), and necropsy results. Environmental heatstroke was diagnosed if the clinical signs occurred after exposure to a hot and humid environment with no history of strenuous physical activity, whereas exertional heatstroke was diagnosed if the history indicated that the clinical signs appeared after strenuous physical exercise.2 The attending clinicians in charge of cases were blind to the NRBC count results evaluated in the current study. At presentation, dogs were assigned a neurological status score based on a 0–4 scale, where 0 = alert to mild depression, 2 = moderate to marked depression or delirium, 3 = stupor, and 4 = coma. Delirium was defined as a state of aimless walking, odd responses to stimuli, staggering, and partial response to the dog's name. Stupor was defined as a state of near-unconsciousness of severe diminished consciousness in which the dog responded only to strong stimuli, such as pinching with a hemostat. Coma was defined as a state of complete loss of consciousness with no response to strong stimuli.
Definition of Secondary Complications
Dogs diagnosed with DIC had thrombocytopenia (platelets < 150,000/μL, reference range 150,000–500,000/μL) and at least two of the following: prolongation (> 25%) of prothrombin time (PT) or activated partial thromboplastin time (aPTT) and clinical or postmortem signs compatible with DIC, including hematochezia, hematemesis, hematuria, and diffuse bleeding at any time point during the disease course.2 Dogs were diagnosed with acute kidney injury if the serum creatinine concentration was > 2 mg/dL after 24 hours of IV administration of fluids and the exclusion of pre- and postrenal components of their azotemia, or based on histological evidence of acute tubular necrosis at necropsy. Aggressive IV administration of fluids for 24 hours was assumed to have eliminated hypovolemia as a contributing prerenal mechanism for increased serum creatinine concentration. The urinary bladder of all dogs was routinely catheterized and the urine output was continuously monitored throughout the hospitalization thus excluding any postrenal mechanism for the increase in serum creatinine concentration.
Blood samples for CBCa and coagulation testsb were collected in potassium-EDTA and trisodium-citrate tubes, respectively, and analyzed within 30 minutes from collection. CBC, differential WBC, and NRBC counts and PT and aPTT were performed at presentation (and before treatment) and q8h–q12h until discharge from the hospital or until death or euthanasia. Differential WBC counts were performed manually by counting 100 leukocytes in modified Wright's-stained blood smears.c Total and differential NRBC counts and assessment of blood cell morphology were performed manually on examination of the same peripheral blood smears. NRBC were counted as NRBC per 100 WBC (rNRBC) when observed and their absolute number (aNRBC) was calculated based on the total WBC. The WBC count was corrected accordingly.5
Blood for glucose and creatinine concentration measurement was collected in sodium fluoride and serum tubes, respectively, and was analyzedd within 24 hours from collection.
For all continuous parameters, the normality of data distribution was evaluated by means of the Shapiro–Wilk test. Normally and nonnormally distributed continuous parameters are reported as mean ± standard deviation (SD) and as median and range, respectively, and were compared between the survivors and the nonsurvivors (euthanized cases excluded) by means of the Student t- or Mann–Whitney tests, respectively. Fisher's exact test was used to compare categorical variables between the outcome groups. Logistic regression analysis was performed to assess the relationship of different variables with the outcome. The association between aNRBC and rNRBC with the outcome was evaluated in all the dogs in the study as well as after exclusion of the euthanized animals by the receiver operating characteristics (ROC) procedure. The ROC analysis was used to select NRBC cut-off points and their corresponding sensitivities and specificities for prediction of the outcome. The area under the ROC curve was calculated by the trapezoidal rule. Fisher's exact test was used for the calculation of the 95% confidence interval (CI95%) for the specificity and sensitivity. The optimal cut-off point was selected as the point that was associated with the least number of misclassifications. Spearman's rank correlations were used to assess the correlation between continuous variables. For all tests applied, P < .05 was considered statistically significant. All calculations were performed using a statistical software.e
Signalment History and Clinical Signs
Forty dogs met the inclusion criteria, of which 23 and 17 had exertional and environmental heatstroke, respectively. All dogs were admitted from April to October, which corresponds to the warm season in Israel. The dogs had a median body weight of 34 kg (range 6–60) and included the following breeds: mixed-breed, Golden Retriever, Labrador Retriever, Belgian Malinois, Boxer, Chinese Shar-Pei, dog de-Bordeaux, Rottweiler (12, 5, 4, 4, 2, 2, 2, and 2 dogs, respectively), Great Dane, English Bulldog, Staffordshire Bull-Terrier, German Shepherd, English CockerSpaniel, Rhodesian Ridgeback, and Airedale Terrier (1 dog each). There was no significant difference between the proportion of males (25) and females (15) (P= .36). The median time lag from the insult of hyperthermia to presentation at the HUVTH was 3 hours (range 0.5–24). Twenty-five of 39 dogs (64%) in which this information was available were cooled by their owners before presentation at the HUVTH.
The most common clinical signs at presentation included collapse (39 dogs, 98%, CI95% 92–100%), shock (31 dogs, 79%, CI95% 66–93%), and seizures (14 dogs, 37%, CI95% 21–53%). The mental status was assessed in 36 dogs and abnormalities were observed in most of them, including coma (11 dogs, 31%, CI95% 15–46%), stupor (14 dogs, 39%, CI95% 22–56%), and delirium (10 dogs, 28%, CI95% 12–43%), whereas only 1 dog (3%, CI95% 0–8%) presented with mild depression with no additional neurological abnormalities. When comatose (mental status score 4) and no comatose (mental status 1, 2, and 3 combined) dogs were compared, the proportion of nonsurvivors was significantly (P= .01) higher in the comatose group. The median body temperature at presentation was 39.1 °C (SD 2.1, range 35.5–43.3, CI95% 38.5–39.8 °C). During hospitalization, petechiae or echymoses and bloody diarrhea were observed in 25 (63%) and 19 (48%) dogs, respectively.
The case fatality rate and the rapid disease progression along with technical limitations led to loss of data in some dogs. The most prevalent abnormalities at presentation included presence of peripheral NRBC (95%, CI95% 87–100%), increased hematocrit, hemoglobin concentration, and red blood cell (RBC) count (75, 78, and 68%, respectively, CI95% 61–89, 64–91, and 52–83%, respectively), thrombocytopenia (54%, CI95% 37–70%), absolute lymphopenia (38%, CI95% 22–54%), neutropenia (28%, CI95% 13–43%), leukocytosis (25%, CI95% 11–39%), monocytopenia (25%, CI95%11–39%), leukopenia (15%, CI95% 3–27%), and neutrophilia (15%, CI95% 3–27%). The median rNRBC and aNRBC were 24 per 100 WBC (range 0–124) and 1.48 × 103/μL (range 0.0–19.6 × 103/μL), respectively (Table 1). The majority of NRBC were metarubricytes (median 86%, range 50–100% of all NRBC) and the minority was rubricytes (median 14%, range 0–50% of all NRBC). Earlier erythroid precursors were not detected. The NRBC to polychromatophil ratio was > 1 in all smears. The relative and absolute NRBC as well as the number of dogs presenting NRBC of all dogs decreased whereas the percent metarubricytes of total NRBC increased from presentation to 36 hours after presentation; however, the number of dogs available for examination at subsequent time points after presentation was limited, mostly due to death (Table 2). When considering only data from dogs available at both presentation and 24 hours after presentation, the findings were similar to those of the entire population. Median rNRBC decreased from 18 to 2 per 100 WBC within the first 24 hours and median aNRBC decreased from 14.9 × 103/μL to 2.5 × 103/μL. Conversely, the metarubricyte percentage of total NRBC increased from 85 to 94%.
|Measure||n||Median||Range||% < RI (n)||% > RI (n)||Reference Interval|
|Red blood cells (106/mm3)||40||8.7||5.5–13.0||0 (0)||68 (27)||5.5–8.0|
|Hemoglobin (g/dL)||40||19.8||5.0–24.0||0 (0)||78 (31)||12.0–17.5|
|Hematocrit (%)||40||59.3||34.2–80.8||3 (1)||75 (30)||37–50|
|Mean corpuscular volume (fL)||40||66.0||61.0–72.0||0 (0)||0 (0)||60–77|
|Mean corpuscular hemoglobin (pg)||40||22.2||18.2–25.2||5 (2)||0 (0)||19.5–24.5|
|MCHC (g/dL)||40||33.2||27.0–36.6||13 (5)||5 (2)||32.0–36.0|
|RDW (%)||40||17.5||16.5–21.4||0 (0)||5 (2)||14.0–19.0|
|White blood cells (103/mm3)||40||10.80||1.31–45.40||15 (6)||25 (10)||6.00–17.00|
|Corrected white blood cellsa (103/mm3)||39||8.5||1.2–30.3||13 (5)||23 (9)||6.00–17.00|
|Platelets (103/mm3)||39||137||0–537||54 (21)||3 (1)||150–500|
|Segmented neutrophils (103/mm3)||39||6.10||0.83–18.13||28 (11)||15 (6)||3.00–11.50|
|Band neutrophils (103/mm3)||39||0.00||0.00–10.93||NA||8 (3)||0.00–0.30|
|Lymphocytes (103/mm3)||39||1.67||0.10–6.45||38 (15)||5 (2)||1.00–4.80|
|Eosinophils (103/mm3)||39||0.29||0.00–1.45||NA||8 (3)||0.00–1.00|
|Monocytes (103/mm3)||39||0.35||0.00–1.87||25 (10)||8 (3)||0.10–1.36|
|Basophils (103/mm3)||39||0.00||0.00–0.61||NA||1 (3)||Rare|
|rNRBC (cells/100 white blood cells)||39||23.5||0.0–124.0||NA||90 (36)||None|
|Absolute NRBC (103/mm3)||39||1.48||0.00–19.61||NA||90 (36)||None|
|Metarubricytes (% of total NRBC)||36||86||50–100||NA||NA||NA|
|Rubricytes (% of total NRBC)||36||14||0–50||NA||NA||NA|
|Prothrombin time (seconds)||37||10||5.1–60.0||3 (1)||76 (28)||6.0–8.5|
|aPTT (seconds)||37||19.3||9.9–70.0||5 (2)||49 (18)||11.5–19.5|
|Glucose (mg/dL)||36||45||12–266||53 (19)||8 (3)||70–110|
|Creatinine (mg/dL)||37||1.5||0.5–3.7||0 (0)||49 (18)||0.50–1.50|
|Time Postpresentation (hours)||Number (%) of Dogs with NRBC of All Dogs||Median (range) rNRBC (cells per 100 WBC)||Median (range) Absolute NRBC (× 103/mm3)||Median (range) % Metarubricytes of Total NRBC|
|0||36/38 (95)||24 (0–124)||1.48 (0.00–19.61)||86 (50–100)|
|12||12/15 (80)||6 (0–17)||0.00 (0.00–2.40)||96 (80–100)|
|24||7/13 (64)||1 (0–12)||0.00 (0.00–1.60)||100 (75–100)|
|36||2/4 (50)||0 (0–2)||0.06 (0.00–0.13)||100 (100–100)|
Prolongation of the PT and the aPTT at presentation were documented in 76% (CI95% 61–90%) and 49% (CI95% 32–66%) of the dogs, respectively. Hypoglycemia and increased serum creatinine were present in 53% (CI95% 37–70%) and 49% (CI95% 32–66%) of the dogs, respectively (Table 1).
Secondary Complications and Outcome
Information of the kidney status was available for 38/40 dogs. Sixteen (42%) and 23 (58%) dogs were diagnosed with acute kidney injury and DIC, respectively. Eighteen (45%), 19 (48%), and 3 (8%) dogs survived, died naturally, and were euthanized, respectively. The 3 dogs were euthanized at 2, 24, and 48 hours postpresentation due to financial considerations. Twenty of the 22 nonsurvivors died or were euthanized within 36 hours from presentation. The median hospitalization time periods of all dogs, nonsurvivors, and survivors were 24, 12, and 60 hours (all dogs range, 2 hours to 16 days), with a significant (P= .0002) difference between survivors and nonsurvivors. The death rates among patients with acute kidney injury and DIC were 81% (13/16) and 82% (19/23), respectively. The death rate among dogs with coexisting acute kidney injury and DIC was 87% (13/15).
Clinicopathologic Data in Survivors and Nonsurvivors
At presentation, nonsurvivors had significantly longer PT (P= .01), aPTT (P= .0001), higher counts of absolute lymphocytes (P= .018), rNRBC (P < .0001, Fig 1), aNRBC (P= .0001, Fig 1), percent rubricytes of all NRBC (P < .001), absolute metarubricytes and rubricytes (P= .0001 and .0001, respectively), lower platelets (P= .023), higher creatinine (P= .01), and lower glucose (P= .006) concentrations compared with survivors (Table 3). The median rNRBC of survivors and nonsurvivors on follow-up blood counts were available only for 15 dogs (9 survivors and 6 nonsurvivors) at 12 hours and for 13 dogs (8 survivors and 5 nonsurvivors) at 24 hours. The median rNRBC at 12 hours was 6 (range 0–17) and 10 (range 2–15.5) in survivors and nonsurvivors, respectively, and at 24 hours was 0.5 (range 0–12) and 2.5 (range 1–12), respectively. At these two time points, however, the rNRBC was not statistically different between the two outcome groups.
|Median (range)||% > RI||% < RI||Median (range)||% > RI||% < RI|
|Prothrombin time (seconds)||10||58.8||5.9||15.1||90||0||6–8.5||.01|
|aPTT (seconds)||17||11.8||11.8||29.9||80||0||11.5–19.5||< .001|
|Platelets (× 109/L)||217||6.3||75||108||0||100||150–500||.023|
|Lymphocytes (× 103/L)||0.9||0||75||2.2||9.5||23.8||1–4.8||.018|
|Relative NRBCa (%)||2||88.2||NA||50||100||NA||0||< .001|
|Absolute NRBC (× 103/L)||0.2||88.2||NA||4.6||100||NA||0||< .001|
|Metarubricyesb (%)||85||NA||NA||100||NA||NA||NA||< .004|
|Absolute metarubricytes (× 103/L)||0.2||88.2||NA||3||95.2||NA||0||< .001|
|Rubricytesc (%)||0||41.2||NA||7||NA||NA||NA||< .001|
|Absolute rubricytes (× 103/L)||0||41.2||NA||0.53||81||NA||0||< .001|
|Hospitalization period (days)||2.5||NA||NA||0.6||NA||NA||NA||< .001|
Correlations of NRBC with Clinicopathologic Measures
There were significant correlations between the relative rNRBC and PT (r= 0.45, P= .007), aPTT, (r= 0.75, P < .0001), WBC (r= 0.36, P= .026), absolute lymphocyte count (r= 0.46, P= .004), and glucose concentration (r=−0.59, P= .02). There were significant correlations between aNRBC and WBC (r= 0.62, P < .001) and lymphocyte count (r= 0.51, P= .012), but not with neutrophil and monocyte numbers.
NRBC at Presentation as a Predictor of Secondary Complications and Death
Dogs with DIC had significantly (P < .001) higher rNRBC (median 55, range 7–124 per 100 WBC) compared with those without DIC (median 2, range 0–41 cells per 100 WBC). Dogs with acute kidney injury had significantly (P= .006) higher rNRBC (median 48, range 0–76 cells per 100 WBC) compared with dogs without acute kidney injury (median 9, range 0–76 cells per 100 WBC). Receiver operator curve analysis of rNRBC (cells per 100 WBC) at presentation as a predictor of DIC had an area under the curve (AUC) of 0.94 (CI95% 0.86–1.00). A cut-off point of 13 rNRBC per 100 WBC corresponded to a sensitivity and specificity of 86% (CI95% 65–97%) and 87% (CI95% 60–98%), respectively. In a similar analysis, performed to assess rNRBC as a predictor of acute kidney injury, the area under the ROC curve was 0.76 (CI95% 59–93%) and a cut-off point of 17 rNRBC per 100 WBC yielded a sensitivity and specificity of 87% (CI95% 60–98%) and 67% (CI95% 43–85%), respectively.
Relative NRBC was found as a sensitive and specific marker of death. Receiver operator curve analysis of rNRBC (cells per 100 WBC) at presentation as a predictor of death yielded an AUC of 0.90 (CI95% 0.80–1.00). A cut-off point of 18 rNRBC corresponded to a sensitivity and specificity of 91% (CI95% 70–99%) and 88% (CI95% 64–99%), respectively. An rNRBC cut-off point of 5 cells/100 leukocytes corresponded to a sensitivity and specificity of 100% (CI95% 84–100%) and 59% (CI95% 33–82%), respectively. An rNRBC cut-off point of 38 cells/100 leukocytes corresponded to a sensitivity and specificity of 67% (CI95% 43–87%) and 94% (CI95% 71–100%), respectively. When the aNRBC were used instead of the rNRBC, the area under the ROC was 0.86 (CI95% 0.74–0.98) and the optimal cut-off point of 800 cells/μL corresponded to 91% (CI95% 70–99%) sensitivity and 77% (CI95% 50–93%) specificity. When the analyses were repeated with the 3 euthanized dogs excluded, the area under the ROC for relative and absolute NRBC increased further to 0.92 (CI95% 0.82–1.00) (Fig 2) and 0.89 (CI95% 0.77–1.00), respectively. Optimal cut-off points with corresponding sensitivities and specificities for these analyses were 18 cells per 100 leukocytes, 95% (CI95% 74–100%), and 88% (CI95% 64–99%) for rNRBC, respectively, and for 800 cells/μL, were 95% (CI95% 74–100%) and 77%, (CI95% 50–93%), respectively, for aNRBC.
The results of the present prospective study confirm that the presence of peripheral NRBC is common in dogs with heatstroke and are in agreement with previous observations.1,2 In fact, presence of NRBC was the most common hematological abnormality at presentation in dogs in this study. Most NRBCs were metarubricytes, the minority was rubricytes, and earlier erythroid precursors were absent. As the recent history of dogs with heatstroke is sometimes incomplete, and dogs are often presented with normal rectal temperatures and even hypothermia, presence of peripheral NRBC in a collapsed, tachypneic, and neurologically abnormal dog should thus raise suspicion of presence of heatstroke. The presence of peripheral NRBC was not associated with anemia and erythroid hyperplasia. At presentation, most of the dogs had increased hematocrit, hemoglobin concentration, and RBC count. Furthermore, the NRBC to polychromatophil ratio was > 1 in all dogs, suggesting a disease process unrelated to erythroid hyperplasia,3 as reported in a previous retrospective study.2 Finally, the acute onset and rapid progression of the disease in the dogs excludes a bone marrow reaction with increased erythropoiesis. Thus, the high prevalence of peripheral NRBC in dogs with heatstroke should be considered to be a direct or an indirect result of hyperthermia. Peripheral NRBC were also observed in human patients after thermal injuries and were most commonly present in patients with the largest burns.6
Appearance of peripheral NRBC in human heatstroke has never been described; however, in critically ill human patients, appearance of NRBCs in the peripheral blood has been associated with a variety of severe diseases and poor prognosis.7–12
The high prevalence of peripheral NRBC at presentation in the dogs in the present study made it possible to assess their usefulness as a prognostic marker for prediction of secondary complications and outcome. At presentation, significantly higher rNRBC and aNRBC were observed in nonsurvivors compared with survivors. It is clear from the results that NRBC numbers, whether relative or absolute, were predictors of death in dogs with heatstroke, as reflected by the high sensitivity and specificity. The presence of NRBC at presentation probably represents the severity of the thermal injury to the bone marrow and the latter probably correlates with the severity of the thermal injury of other organs and subsequent certain secondary complications, such as acute kidney injury and DIC as well. Thus, presence and magnitude of NRBC can be used as a simple, cost-effective, and readily available marker of overall organ injury in dogs with thermal injury. This is demonstrated by the finding that the higher the peripheral NRBC number at presentation, the higher was the probability that the disease has culminated in secondary complications such as DIC, acute kidney injury, and death. This assumption is supported by the significantly higher rNRBC at presentation in dogs diagnosed with DIC and acute kidney injury compared with those that had no such complications and by the fact that rNRBC was also a highly sensitive and specific predictor of DIC and death. In addition, there were highly significant positive correlations between rNRBC and PT as well as aPTT at presentation. Both DIC and acute kidney injury have been frequently previously reported in people and dogs with heatstroke and both have been found as risk factors for mortality in dogs.1,2,4,13,14
The presence and magnitude of NRBC should never be used as a sole prognostic indicator in dogs with heatstroke, nor should they replace proper clinical assessment, although both were found to be highly sensitive and specific markers of the outcome in this syndrome. The different cut-off points provided allow flexibility for clinicians, based on their preference, to either maximize sensitivity or specificity through individualized selection of cut-off points.
Although both rNRBC and aNRBC were highly sensitive and specific predictors of the outcome, there is no obvious explanation for the fact that rNRBC was a better one. Probably, the thermal insult and consequent bone marrow lesions were expressed more accurately in the rNRBC. Although rNRBC had a positive correlation with WBC, this correlation was weak, whereas aNRBC had a stronger correlation with the WBC and lymphocyte number. It seems that the aNRBC, which is calculated based on the WBC, is more influenced by changes in the WBC. Consequently, a similar thermal insult leading to a similar release of NRBC from the bone marrow will result in a lower aNRBC in leukopenic dogs compared with dogs with normal or increased WBC. Thus, rNRBC probably provides a better reflection of the lesion in dogs with leukopenia and serve as a better predictor of the outcome.
Another interesting observation of this study was that the proportion of metarubricytes of the total NRBC, the mature erythroid NRBC precursors, was significantly higher in survivors compared with nonsurvivors, whereas the proportion of rubricytes was higher in the nonsurvivor group. Most likely, the hyperthermia in nonsurvivors was more severe compared with survivors, resulting in more severe bone marrow lesions in the former group with subsequent release of earlier erythroid precursors. Thus, it is possible that the severity of the hyperthermia-induced bone marrow lesion that leads to release of NRBCs is positively associated not only with the numbers of peripheral NRBCs but also with their character: the more severe the injury, the higher is the proportion of rubricytes and lower is the proportion of metarubricytes of the total peripheral NRBCs population. This hypothesis gains further support from the gradual decrease in NRBC numbers and increase in the proportion of metarubricytes during hospitalization and healing. If this is true, prediction of the outcome and secondary complications in dogs with heatstroke can be improved by assessing the character of peripheral NRBCs in addition to their number. Further larger studies are needed to assess whether this observation is indeed clinically useful.
In a previous study of heat-related illness, the peripheral NRBC decreased by 66% within 12 hours from presentation; however, only 3 dogs had consecutive CBC evaluations.1 Similarly, in the present study, the median rNRBC markedly decreased from presentation to 12 and 24 hours after presentation (24, 6, and 1 cells/100 leukocytes, respectively). However; NRBC were still present in 2 of 4 dogs at 36 hours after presentation. Nonsurvivors consistently had a higher median rNRBC compared with survivors at all time points postpresentation; however, this difference did not reach statistical significance. Future studies with a higher number of animals are warranted to assess the clinical usefulness of NRBC during hospitalization for prediction of the outcome.
Hypoglycemia has been previously recorded in association with death in dogs with heatstroke, as observed in the present study as well, and, in the current study, was significantly, although moderately, correlated with rNRBC, which was a good predictor of death. It was also significantly correlated with prolongation of the PT and aPTT, which are potential markers of DIC.
Direct renal thermal injury, hypoxia, hypovolemia with consequent reduced glomerular filtration, coagulative necrosis due to DIC and microthrombosis, endotoxemia, and local and systemic release of cytokines and vasoactive mediators during SIRS probably contributed to the high prevalence of acute kidney injury in dogs with thermal injury, as has been previously suggested.15 In addition, as serious muscle damage is extremely common in dogs with heatstroke, it is possible that rhabdomyolysis with subsequent myoglobinemia and myoglobinuria could have also contributed to the development of acute kidney injury, as has been reported in people with heatstroke.2,15 This was manifested by an increased creatinine concentration at presentation in half of the dogs. Because peripheral NRBC were found to be significantly higher in dogs with acute kidney injury, presence of high numbers of peripheral NRBC at presentation in dogs with heatstroke patients should alarm clinicians to the presence of acute kidney injury, even in the absence of increased serum creatinine concentration. This early identification should prompt aggressive treatment to support kidney function and minimize further renal damage.
This study has several limitations; first, the definition of heatstroke in dogs is currently roughly based on human medicine criteria, with modification of the core temperature cut-off point. We have used this definition in our previous study of dogs with heatstroke2; however, in dogs, there is no clear cut line between heat-related illness and heatstroke, as the neurological abnormalities present in the latter condition could be mild in early disease stages and, thus, may be missed or hard to interpret. Thus, application of the present results in mild forms of heat-related illness in dogs should not be carried out without caution. Second, the number of available animals, especially during hospitalization, was limited due to the high mortality and discharge of animals from the hospital. This limited the statistical power of the comparisons of NRBC numbers and character with the outcome at different time points after presentation. Additionally, despite the efforts made to follow the study protocol, certain samples were missed and led to loss of data, which could potentially introduce bias to the statistical analyses. Third, there may be a bias in the diagnosis of secondary complications, such as DIC and acute kidney injury in the study, as these were diagnosed based on both antemortem clinicopathological measures as well as postmortem findings. Possibly, the incidence of both of these secondary complications may have been underestimated in the survivors due to the low sensitivity of the laboratory tests. On the other hand, inclusion of diagnoses based on postmortem results may have led to an erroneously high incidence of secondary complications in nonsurvivors compared with survivors. Fourth, the antemortem diagnosis of DIC could have been improved by more advanced tests that were not included in this study, such as antithrombin, d-dimer, fibrin degradation products, and protein C. Finally, as the number of animals that had no peripheral NRBC at presentation in the current study is small, any conclusions regarding their absence can only be a speculation.
In conclusion, this study shows that presence of peripheral NRBCs is a common phenomenon in dog with heatstroke and can be useful in the diagnosis of suspected cases of heatstroke in dogs. Both the rNRBC and the aNRBC had a high clinical correlation with secondary complications and were highly sensitive and specific predictors of the outcome. Examination of blood smears of dogs with heatstroke is simple, time and cost-effective, as well as readily available diagnostic tool and, thus, is very useful in clinical practice.
aAbacus or Arcus; Diatron, Wien, Austria
bKC 1A micro, Lemgo, Amelung, Germany, or ACL 200, Instrumentation Laboratory, Milan, Italy
cHema-Tek 2000 Slide Stainer, model 4488B; Bayer Corporation, Elkhart, IN, Stain: Hematek stainpack; Modified Wright's Stain
dCobas-Mira; Roche, Rottkreutz, Switzerland
eSPSS 14.0 for Windows; SPSS Inc, Chicago, IL
- 2Heat stroke in dogs: A retrospective study of 54 cases (1999–2004) and analysis of risk factors for death. J Vet Intern Med 2006;20:38–46., , , et al.
- 3Laboratory Profiles of Small Animal Diseases. A Guide to Laboratory Diagnosis, 3rd ed. St. Louis, MO: Mosby; 2001:104.
- 5Referral and in-office laboratories. In: WillardMD, TvedtenH, TurnwaldGH, eds. Small Animal Clinical Diagnosis by Laboratory Methods, 2nd ed. Philadelphia, PA: WB Saunders; 1994:17.