Evaluation of mean corpuscular volume difference as a marker for serum hypertonicity during water deprivation in dogs

Jennifer M. Reinhart Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Misty R. Yancey Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Lisa M. Pohlman Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Thomas Schermerhorn Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Abstract

OBJECTIVE To evaluate mean corpuscular volume difference (dMCV) as a marker for hypertonicity induced by water deprivation in dogs.

ANIMALS 5 healthy Greyhounds maintained in a research colony.

PROCEDURES Water was withheld for 24 hours. Blood and urine samples were collected before (time 0) and every 6 hours during water deprivation. Serum and urine osmolality were measured on the basis of freezing point depression, and dMCV was calculated from routine hematologic variables.

RESULTS Serum and urine osmolality significantly increased and body weight decreased over time in healthy Greyhounds during water deprivation, although most dogs developed only a slight increase in serum osmolality. The dMCV also increased over time, but the value at 24 hours did not differ significantly from the value at time 0. However, a significant correlation was found between serum osmolality and dMCV. A dMCV ≥ 5 fL yielded 100% specificity for predicting hypertonicity when hypertonicity was defined as serum osmolality ≥ 310 mOsM.

CONCLUSIONS AND CLINICAL RELEVANCE dMCV may be a useful marker for detection of mild hypertonicity in dogs and may have clinical and research applications for use in screening canine populations for hypertonicity.

Abstract

OBJECTIVE To evaluate mean corpuscular volume difference (dMCV) as a marker for hypertonicity induced by water deprivation in dogs.

ANIMALS 5 healthy Greyhounds maintained in a research colony.

PROCEDURES Water was withheld for 24 hours. Blood and urine samples were collected before (time 0) and every 6 hours during water deprivation. Serum and urine osmolality were measured on the basis of freezing point depression, and dMCV was calculated from routine hematologic variables.

RESULTS Serum and urine osmolality significantly increased and body weight decreased over time in healthy Greyhounds during water deprivation, although most dogs developed only a slight increase in serum osmolality. The dMCV also increased over time, but the value at 24 hours did not differ significantly from the value at time 0. However, a significant correlation was found between serum osmolality and dMCV. A dMCV ≥ 5 fL yielded 100% specificity for predicting hypertonicity when hypertonicity was defined as serum osmolality ≥ 310 mOsM.

CONCLUSIONS AND CLINICAL RELEVANCE dMCV may be a useful marker for detection of mild hypertonicity in dogs and may have clinical and research applications for use in screening canine populations for hypertonicity.

Plasma tonicity is a reflection of the osmotic pressure gradient across a cell membrane. In healthy animals, tonicity is maintained by multiple mechanisms that regulate solute concentrations and extracellular water concentration.1 Maintaining physiologically normal extracellular fluid tonicity is an important factor in volume homeostasis of cells and tissues. Hypertonicity is an abnormal state characterized by an increased extracellular solute load that can cause detrimental effects, such as osmotic stress, that alter cell volume and function.1–3 Chronic hypertonicity is known or suspected to have a role in numerous diseases in human and veterinary medicine.4–6 Tonicity is difficult to quantify in a clinical setting because it is the numeric sum of all effective osmole concentrations, not all of which are readily identified or routinely measured.2,3

Rather than attempting to quantify individual solute concentrations, it is possible to infer total tonicity from a physiologic effect on cell size.7 Spurious increases in RBC MCV occur when blood from hypertonic patients is assessed with an automated cell analyzer. The RBCs are acclimated to hypertonic patient plasma and swell as a result of uptake of extracellular fluid when placed in isotonic media used with automated hematology analyzers because the analyzer solutions are hypotonic relative to the RBC intracellular environment.8 Investigators in 1 study9 exploited the physiologic basis for autoanalyzer error and developed the dMCV as an index for plasma hypertonicity. The dMCV is the difference between the MCV measured with an automated cell analyzer and the MCV calculated by use of the value for the centrifugation Hct, which is performed with RBCs in the patient plasma.9

In a recent retrospective study,7 our laboratory group found that dMCV is a marker for hypertonicity in hospitalized dogs. We found that dMCV ≥ 3 fL predicted hypertonicity with moderate sensitivity and specificity (76% and 71%, respectively) when total serum osmolality was ≥ 320 mOsM.7

Sensitivity and specificity of dMCV indexed to total serum osmolality may be decreased in hospitalized dogs for several reasons. First, although serum osmolality approximates tonicity, it likely overestimates tonicity because total serum osmolality includes ineffective osmoles such as urea.1 Increased concentrations of urea and other diffusible osmoles in the serum of hospitalized dogs contribute a proportionally higher amount of ineffective osmoles to the serum total osmolality, compared with the contribution in healthy dogs. Second, the relationship between osmolality and tonicity is further obscured because the tonic nature of many solutes has not been defined and the pattern of osmoles with undefined tonic effects that accumulate in hospitalized patients may differ with the disease state.

To further clarify the relationship between hypertonicity and dMCV and to validate dMCV as a hypertonicity marker in dogs, an in vivo model of induced hypertonicity is needed. In healthy dogs, hypertonicity can be induced by restriction of oral fluids.10 Acute changes in tonicity in dogs with physiologically normal renal function should not be accompanied by a significant increase in ineffective osmole concentrations; thus, any increase in serum osmolality in these dogs should reflect an increase in tonicity. A water-deprivation method would also allow dMCV to be evaluated in a group of dogs that develop hypertonicity through the same physiologic mechanism; such a method would eliminate problems encountered in hospitalized dogs in which hypertonicity can develop through a variety of physiologic or pathological mechanisms. Because hypertonic plasma stimulates vasopressin release, detection of an increased plasma vasopressin concentration is an ideal way to verify the presence of hypertonicity in water-deprived dogs. Currently, direct measurement of vasopressin concentrations is not possible because an assay for use on canine samples is not commercially available. However, release of vasopressin can be indirectly assessed by identifying an increase in the concentration of urine after a hypertonic stimulus. Therefore, production of concentrated urine can be used to verify a hypertonic state during water deprivation and allow for controlled assessment of dMCV as a marker for hypertonicity.

The purpose of the study reported here was to validate dMCV as a marker for hypertonicity in dogs and to determine a cutoff value for dMCV that indicates hypertonicity. We hypothesized that dMCV would increase with induction of hypertonicity during water restriction and that a dMCV of approximately 3 fL would be a useful cutoff for identification of serum hypertonicity.

Materials and Methods

ANIMALS

Five healthy Greyhounds (3 neutered males and 2 spayed females) that were part of a university research colony were used in the study. Each dog was 5 years old. Body weight of the dogs ranged between 33.4 and 40.7 kg. The study protocol was approved by the Kansas State University Institutional Animal Care and Use Committee.

PROCEDURES

Water was withheld from each dog for 24 hours. Body weight was recorded, and blood and urine samples were collected before (time 0) and every 6 hours during water deprivation. For each dog, maximum dehydration was limited to 5% (as determined on the basis of 5% loss of body weight); dogs were removed from the study if weight loss exceeded 5% of initial body weight.

Routine hematologic variables were assessed with an automated cell analyzera and manually with the Hct determined by use of a microcentrifuge and card reader. The dMCV was calculated as described elsewhere7,9 by use of the following equation: dMCV = MCVM – ([Hct × 10]/RBC concentration), where MCVM is the measured MCV. Sodium and BUN concentrations were determined by use of a clinical biochemistry analyzer.b Azotemia and hypernatremia were defined when concentrations exceeded the upper end of the reference intervals (BUN, > 33 mg/dL; sodium, > 154 mmol/L). Serum and urine osmolality were measured by use of a freezing-point depression osmometer.c Osmolality samples were analyzed in duplicate; intra-assay and interassay coefficients of variation for the osmometer were 0.48% and 1.37%, respectively.

STATISTICAL ANALYSIS

The effect of water deprivation on body weight, BUN concentration, sodium concentration, serum osmolality, urine osmolality, and dMCV was assessed with the Friedman test for repeated measures and Tukey test for post hoc multiple comparisons. Effectiveness of water deprivation to induce hypertonicity was determined by use of changes in body weight, serum osmolality, and urine osmolality. To evaluate the validity of serum osmolality as a surrogate standard for tonicity, the BUN concentration was monitored to evaluate the contribution of ineffective osmoles, and the sodium concentration was monitored to evaluate the contribution of effective osmoles to serum osmolality. The Pearson correlation coefficient was calculated to determine the association between serum osmolality and dMCV. An ROC curve was generated to assess the ability of dMCV to predict mild hypertonicity, and sensitivity, specificity, and the area under the ROC curve were calculated. For the purposes of the ROC curve analysis, mild hypertonicity was defined as serum osmolality ≥ 310 mOsM. All statistical analyses were performed with commercial software.d,e Values of P ≤ 0.05 were considered significant.

Results

Body weight decreased significantly (P < 0.001) over time but did not exceed 5% in any dog at any time. Osmolality of serum (P = 0.02) and urine (P = 0.01) increased significantly over time (Figure 1). Three of 5 dogs had serum osmolality > 310 mOsM at 1 or more time points during the study. The dMCV increased in 4 of 5 dogs after water deprivation for 24 hours. Mean dMCV also increased over time (nearly double in 24 hours), but this increase was not significant (P = 0.53; Figure 2). However, dMCV was positively correlated with serum osmolality (r = 0.71; P < 0.001; Figure 3). The area under the ROC curve for dMCV for predicting mild hypertonicity (serum osmolality ≥ 310 mOsM) was 0.84. A cutoff for dMCV of 3 fL yielded sensitivity of 100% and specificity of 31%, whereas a cutoff of 5 fL yielded sensitivity of 64% and specificity of 100%. There was no significant (P = 0.36) change in BUN concentration during the study, but sodium concentration increased significantly (P = 0.001) from 0 to 24 hours. None of the dogs became azotemic during the study, but 3 of 5 dogs became hypernatremic; these were the same 3 dogs that became hyperosmolar (serum osmolality ≥ 310 mOsM).

Figure 1—
Figure 1—

Changes in mean ± SD osmolality of serum (A) and urine (B) over time in 5 dogs from which water was withheld for 24 hours. Start of water deprivation was time 0. *Value differs significantly (P ≤ 0.05) from the value at time 0.

Citation: American Journal of Veterinary Research 76, 2; 10.2460/ajvr.76.2.170

Figure 2—
Figure 2—

Change in dMCV over time in dogs in response to water deprivation for 24 hours. Each pattern with squares represents results for a particular dog; the mean value for the 5 dogs is indicated (circles and thick solid line).

Citation: American Journal of Veterinary Research 76, 2; 10.2460/ajvr.76.2.170

Figure 3—
Figure 3—

Scatterplot of serum osmolality compared with dMCV for 5 dogs from which water was withheld for 24 hours. Samples were obtained before and at 6-hour intervals during water deprivation. There is a strong correlation (r = 0.71; P < 0.001).

Citation: American Journal of Veterinary Research 76, 2; 10.2460/ajvr.76.2.170

Discussion

Analysis of the results of the study reported here suggested that dMCV can be a useful marker for serum hypertonicity in dogs. Body weight decreased while osmolality of serum and urine increased significantly during water deprivation. The increase in urine osmolality was likely a result of hypertonic stimulation of vasopressin release and could be considered an indirect indicator of plasma hypertonicity. Thus, on the basis of physiologic changes identified in the present study, water deprivation was an effective method for induction of hypertonicity in dogs. Mean dMCV increased over time, although not significantly, which is the expected response of RBCs obtained from hypertonic dogs.7 Moreover, dMCV increased in 4 of 5 dogs in the present study, and the mean dMCV increased (nearly doubled) within 24 hours. The reason that this was not a significant increase is unclear. It is possible that the tonic stimulus induced by water deprivation for 24 hours may not have been sufficient or have persisted long enough to elicit a significant change in dMCV in all 5 dogs. Response of an individual to a mild tonic stimulus can be variable and is dependent on multiple physiologic factors, including renal concentrating ability and hypothalamic osmotic set point.1 It is also possible that typical intraindividual variation in dMCV combined with a small population size prevented us from detecting a significant effect. A reference interval and day-to-day variability for dMCV in healthy, euhydrated dogs has not yet been established and should be addressed in future studies.

A significant correlation was detected between dMCV and serum osmolality. As previously discussed, serum osmolality is less than ideal as a criterion-referenced standard for tonicity measurement, but it may be an appropriate surrogate index in healthy dogs. Quantitatively, the difference between osmolality and tonicity is the total concentration of ineffective osmoles. In healthy animals, the major ineffective serum osmole is urea and the major effective osmole is sodium. The urea concentration (ie, BUN concentration) did not change significantly during water deprivation, whereas the sodium concentration significantly increased over time; furthermore, the 3 dogs that became hyperosmolar were the same 3 dogs that became hypernatremic. These findings suggested that the observed increase in serum osmolality was caused by increased concentrations of effective osmoles. Tonicity is determined by summation of all effective osmole concentrations; therefore, in healthy dogs, it is appropriate to use serum osmolality in lieu of tonicity for validation of dMCV. Analysis of the ROC curve yielded 2 useful cutoff values for dMCV. All dogs with a dMCV ≤ 3 fL were normotonic at the time of measurement. Conversely, a dMCV ≥ 5 fL yielded specificity of 100% for hypertonic serum. Values for dMCV in the range of 3 to 5 fL may be difficult to interpret without clinical context. Analysis of the ROC curve was designed to detect mild hypertonicity, defined as serum osmolality ≥ 310 mOsM, which represents an increase in serum osmolality of < 5% (reference value for canine serum osmolality = 300 mOsM).1 Thus, a dMCV ≥ 5 fL predicts hypertonicity, but extremely mild tonic increases may not always be detected.

Results of the present study are similar to those of a recently published study7 conducted by our laboratory group in which dMCV ≥ 3 fL predicted hypertonicity with moderate sensitivity (76%) and specificity (71%) in a group of dogs with various clinical illnesses. In that study7 and the present study, different severities of hypertonicity (≥ 310 mOsM vs ≥ 320 mOsM) were evaluated, so the performance characteristics cannot be directly compared; furthermore, differences in serum solute compositions between healthy and ill study populations also likely influenced the results. That previous study7 involved evaluation of hospitalized canine patients in the intensive care unit, some of which had increased ineffective osmole concentrations (eg, BUN concentration). The accumulation of ineffective osmoles in serum of hospitalized canine patients means that serum osmolality is not as useful as a surrogate for tonicity in ill dogs as is serum osmolality in the healthy dogs of the present study.

The dMCV may provide more information than other methods used to estimate tonicity because it responds to increased effective osmolar concentrations and also reflects functional effects of hypertonicity on cells. Thus, the dMCV represents physiologic implications of tonic derangements and provides additional information over purely quantitative estimates, such as serum osmolality. Although hypertonicity develops during many diseases in dogs, the clinical importance of hypertonicity remains undefined in most situations. The dMCV is a novel marker for serum hypertonicity that, in conjunction with assessment of osmolality and solute concentrations, may better elucidate the role of hypertonicity in canine medicine.

Acknowledgments

This manuscript represents a portion of a thesis submitted by Dr. Reinhart to the Kansas State University Department of Clinical Sciences as partial fulfillment of the requirements for a Master of Science degree.

Supported by an intramural grant through the Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University.

The authors thank Dr. James Roush for assistance with the statistical analysis and Dr. Butch KuKanich for providing the animals.

ABBREVIATIONS

dMCV

Mean corpuscular volume difference

MCV

Mean corpuscular volume

ROC

Receiver operating characteristic

Footnotes

a.

Advia 2120, Siemens Medical Solutions Inc, Malvern, Pa.

b.

COBAS C501, Roche Diagnostics, Indianapolis, Ind.

c.

Micro-OSMETTE, Precision Systems Inc, Natick, Mass.

d.

Winks Kwikstat, Texasoft, Cedar Hill, Tex.

e.

Statplus:mac, version 2009, AnalystSoft Inc, Alexandria, Va. Available at: www.analystsoft.com/en/. Accessed Oct 1, 2014.

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