RELATIONSHIPS OF MEAN CORPUSCULAR VOLUME WITH DIAMETER AND SURFACE AREA OF CANINE ERYTHROCYTES

INTRODUCTION value. The RDW is more sensitive than MCV to detect erythrocyte sub populations during response to ironExamination of blood is virtually the universal first deficiency anemia (Jain, 1993). However, a significant step in the evaluation of health and disease in human and proportion of human patients with RBC macrocytosis animal patients as physiological and pathological due to pernicious anemia had normal RDW rather than changes in most tissues are reflected directly or an elevated RDW (Perry et al, 1985). Furthermore, red indirectly in blood. Erythrocyte morphology is cell agglutination can falsely elevate MCV values, and routinely assessed microscopically and is an important even falsely lower HCT measurement as the upper limit aid in diagnostic haematology (Thrall, 2006; Jain, 1993; in automated analyzers is usually 360fl, thereby Proctor et al, 1976). The morphology of red cells is excluding counting large RBC clumps (Greer, 2009). categorized according to colour, size, shape, intra or Immature RBCs and oxidative damage due to metabolic epicellular structures, and their arrangement on blood diseases and conditions can be detected only by films, which are unique for each animal species (Ford, examining erythrocyte morphology (Udroiu, 2014). 2013;Thrall, 2006). Erythrocyte morphometry provide valuable data on The typical 3D biconcave disk shape of RBCs in changes in erythrocyte morphology and could animal species show very little variability in size and com ple men t the res ult s obt ain ed by sta nda rd shape between cells (Adili et al., 2017). Anisocytosisis hematology methods. For example, morphometric defined as size variations resulting from microcytes, studies have demonstrated that the erythrocytes in male macrocytes or both (Proctor et al, 1976). Parameters of rabbits are smaller than in females, though the average red cell volumes, such as, Mean Corpuscular Volume cell volume was not significantly different (Poljièak(MCV), Red Cell Distribution Width (RDW) and/or Milaset al, 2009). RDW-SD are tools genera lly used to quanti fy Changes in diameter may not be reflected in the MCV anisocytosis. Changes in RBC size correspond to obtained as a direct measurement from automated changes in cell diameter and not necessarily to changes hematology analyzers, which is the average volume of in RBC volume, and changes in cell volumes are not RBCs in a mixture of cells (Weiss and Wardrop, 2010), always reflected in the MCV. Furthermore, the surface and does not indicate individual changes in RBC sizes. area and volume changes in disease do not occur equally Therefore, it is important and timely to study in all RBCs in a sample (Jay and Rowlands, 1975). morphometry of RBCs, such as, the relationship Hypochromic microcytic ery throcytes in ironbetween the diameter, the surface area (SA) and the deficiency show decreased MCV when determined MCV of individual RBCs. Several techniques have been electronically, but show normal diameter (Thrall, 2006). proposed for morphological studies of biological The small diameter of spherocytesis not reflected in the specimens, such as optical coherent tomography and MCV value as the volume of spherocytes is always confocal microscopy. The objective of this study was to within the reference range (Thrall, 2006). Presence of analyze the relationship of MCV with SA and diameters schistocytes in blood film is not reflected in the MCV in 2D views of RBCs of clinically healthy dogs using SUMMARY: Assessment of erythrocyte morphology is an important aid in diagnostic haematology. Anisocytosis which is changes in RBC size, correspond to changes in diameter and surface area (SA) of the cell, and are not always reflected in the Mean Corpuscular volume (MCV). This paper discusses the relationship of MCV with surface area and diameters (vertical and horizontal) in 2D views of erythrocytes of clinically healthy dogs using confocal microscopy. This information would be valuable for early detection of cellular changes in dogs in subclinical or clinical diseases. The average diameters, average SA, and MCV values of RBCs studied were in normal distribution and the mean values of 7.169 ± 0.648 ìm for horizontal diameter (D1), 7.1245 ± 0.6646 ìm for vertical diameter (D2), 2 and 41.061 ìm ± 6.866 for SA did not reveal a significant correlation or a strength of association with the MCV values, indicating that the MCV value has limitations as an objective measurement of detecting anisocytosis. DOI: http://doi.org/10.4038/slvj.v64i2.23

confocal microscopy. The information presented would predict the value of the dependent variable (MCV) based be valuable for early detection of cellular changes in dogs on the value of the independent variable (SA, D1 and D2). with subclinical or clinical diseases.
Box plots were created separately to visualize the pattern of association between the MCV, D1, D2 and SA using the

MATERIALS AND METHODS
Box plot R software.
Dogs presented to Veterinary Teaching Hospital (VTH), RESULTS AND DISCUSSION Peradeniya with normal Full Blood Counts (FBC) analyzed using a fully automated veterinary hematology Mo rp ho met ry pr ov ide s hig her rel iab ili ty and analyzer (MS9-5V, MELET Schloesing laboratories, reproducibility of cytological and histopathological France) were selected for this study. A volume of 1ml diagnoses in human medicine (Albertini et al., 2003). This blood was collected to K EDTA coated tubes with the paper compares the relationship of MCV with surface area 2 (SA) of erythrocytes and diameters in 2D views of RBCs. consent of the owners. The anticoagulant K EDTA is 2 The Full Blood Count (FBC) on blood collected from 14 recommended by ICSH (International Council for apparently healthy dogs (9 females, 5 males) of different Standardization in Hematology) and CLSI (Clinical and breeds (German shepherd, Lion shepherd, and Rottweiler, Laboratory Standards Institute) as the choice of Doberman and Cross breds), with varying ages (4 months anticoagulant for blood cell counting. to 13 years) were measured within 4 hours of collection. Blood smears were prepared from each sample, stained The perpendicular diameters (horizontal, D1 and with Leishman stain and transported to the Parasitology vertical, D2) and surface area (SA) of each RBC in laboratory, Faculty of Medicine, University of Jaffna to micrometer up to 3 decimals using the ZEN lite (blue study morphometry of RBC in 2D view using a Laser edition) software from Carl Zeiss are shown in Figure  scanning confocal microscope (Carl Zeiss LSM 700, 1.The above software allowed accurate morphometric Germany).Up to 500 RBCs in the middle one third of the measurements, similar to other software, such as monolayers of each slide were randomly examined by OPTIKATM Vision Pro software (Adili et al, 2017), than cross sectional method under oil immersion (×100) using the conventional method of measuring the diameters of the above microscope. A laser beam (405 nm -639 nm erythrocytes in a blood film using a scale on an oil wavelength) was passed through the confocal optical path, immersion lens in a light microscope. The use of software and images were obtained with Zeiss Axio Imager Z2 reduces errors due to the human factor that is involved in (high resolving and high sensitive cameras) on the studies with ocular micrometer. monitor at 2560 ×1920 resolution to generate the 2- The reference values of MCV for dogs in Sri Lanka dimensional object information of the RBCs ( The Pearson Correlation coefficient did not reveal (horizontal, D1 and vertical, D2) and surface area (SA) of significant relationships between MCV and SA (r=each RBC was measured in micrometers up to 3 decimals 0.023, p=0.140), MCV and D1 (r=0.011, p=0.140), or using the ZEN lite (blue edition) software from Carl Zeiss.
between MCV and D2 (r=-0.05, p=0.001). Linear These values were analyzed with the MCV values in the regression analysis between MCV (dependent variable) FBC report. and SA did not reveal a significant correlation (r-0.011, p=0.140), or a strength of association (P-0.474, R-sq. -Data analysis 0.1%). A significant linear relationship or significant Anderson Darling normality test (ADNT) was strength of association was not evident between MCV and performed using 'MINITAB' software to analyze whether D1(p-0.474, R-sq. -0.0%) or between MCV and D2 the data obtained for MCV, diameters and SA of RBCs (p=0.001, R-sq.0.1%).The box plots also did not reveal a follows a normal distribution (Ghasemi and Zahediasl, pattern of association between the MCV and SA (Figure 2) 2012). Mean diameter, standard deviation and variance, or between MCV and D1 ( Figure 3) or D2 (Figure 4).Adili minimum, maximum data for D1, D2 and SA of 500 RBC et al, 2017 also have not seen significant differences per sample were calculated using the 'MINITAB' between the diameter and the SA of RBCs of dogs.

software. The Pearson Correlation Coefficient test was
Anisocytosis in a blood sample, depicted by parameters performed using the same software to assess the strength of red cell volumes, such as, MCV and RDW do not and direction of association of MCV values with complement the changes in RBC size in relation to diameters (D1, D2, average diameters) and SA diameter or surface area (Proctor et al, 1976). Our results individually. The Linear regression test was performed to show that diameters and surface areas of RBCs do not results as significant, when in reality, they are close to correspond with the MCV values calculated by the reference range, and they also should be able to automated analyzer, indicating that the MCV value has recognize changes in a patient's test results that are due limitations as an objective measurement of detecting to random error before the test results are reported anisocytosis. Practitioners may interpret changes in test (Houwen, 1989). Electronic hematology analyzers are hemoanalyzers can occur at a rate of approximately 1 per Houwen, B. (1989). Delta checks for random error detection in 500 samples or more, while the frequency of random hematology tests. Https://academic.oup.com/ errors in most laboratories exceeds 1 per 200 to 500 labmed/article-pdf/20/6/410/8267391/labmed20-0410.pdf samples (Houwen, 1989). Therefore, a blood smear must Jain, N.C. (1993). Chapters 1 and 7: Essentials of Veterinary be examined if and when an automated instrument Hematology. Lea &Feiger, Philadelphia.