Clinical Hemorheology and Microcirculation 62 (2016) 45–54 DOI 10.3233/CH-151944 IOS Press
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Correlation between blood rheological properties and red blood cell indices (MCH, MCV, MCHC) in healthy women Georg-Friedrich von Tempelhoffa,c,∗ , Olga Schelkunova,c , Attila Demirhana,c , Panagiotis Tsikourasc,e , Werner Rathf , Eva Veltenb,c and Roland Csorbab,c,d a
Department of Obstetrics and Gynaecology, St. Vinzenz Hospital, Hanau, Germany Department of Obstetrics and Gynaecology, City Hospital of Aschaffenburg, Aschaffenburg, Germany c Institut of Coagulation Disorders in Obstetrics and Gynaecology, Hausen, Germany d Department of Obstetrics and Gynaecology, University of Debrecen, Hungary e Democritus University of Thrace, Department of Obstetrics and Gynaecology, Alexandroupolis, Greece f Department of Obstetrics and Gynaecology, RWTH University of Aachen, Germany b
Abstract. OBJECTIVE: Structure and mechanical properties of red blood cells are markedly influenced by pathophysiology of many diseases which in turn potentially impair microcirculatory blood flow. The physiological association between blood rheological parameters and red blood cell indices was investigated in otherwise healthy unselected mid-age women prior to elective gynaecological surgery. METHODS: Red Blood Cell-deformability (RBC 1.2, 3.0; 6.0, 12.0; 30.0, 60.0) during exposure to low (RBC 1.2, 3.0), moderate (RBC 16.0, 12.0) and high shear forces (RBC 30.0, 60.0; Rheodyn; Myrenne), -aggregation (MA1; Myrenne) during low shear (E1; 4−1 S) and in stasis (E0) and plasma viscosity (Pv; KSV 1; Fresenius) were correlated with red blood cell indices (RBC-I: MCV, MCH and MCHC) and subjects’ characteristics in 286 healthy women the day before undergoing gynaecologic standard surgery. Women with known pregnancy, malign-, infective-, chronic-disease or extreme BMI (40 Kg/m2 ) were excluded from this trial. RESULTS: From June 2014 to December 2014 a total of 286 healthy women (age: 46.5 ± 17.6 y; BMI: 25.5 ± 5.2 kg/m2 ) were eligible for inclusion into this prospective evaluation. Pv (mean ± SD: 1.17 ± 0.12 mPa s) and RBC aggregation (E0:12.6 ± 6.3; E1:17.9 ± 7.3) were not significantly correlated with RBC-I but with age and BMI. In contrast, RBCdeformability correlated significantly with MCV and MCH but significantly inversely correlated with MCHC. Deformability significantly increased with age but was unaffected by BMI of women. The correlation between RBC-I and RBC deformability was most remarkable during moderate shear force exposure. Neither haemoglobin nor haematocrit were correlated with RBC deformability or RBC-I. CONCLUSIONS: Cell volume and haemoglobin content had a strong impact on deformability in apparently healthy mid age women, whereas low MCHC and large MCV were associated with an increase in deformability while high MCHC and small MCV correlated with increased rigidity of RBC. BMI had no impact on deformability while age was associated with an increase in all determinants of blood viscosity. RBC aggregability was not affected by MCV, MCHC or MCH in mid-age women. Keywords: Plasma viscosity, red blood cell aggregation, red blood cell deformability MCH, MCV, MCHC
∗ Corresponding author: Georg-Friedrich von Tempelhoff, Institute of Coagulation Disorders in Obstetrics and Gynaecology, Freiherr vom Stein Str.29, 63179 Hausen, Germany. Tel.: +49 15221 554988; Fax: +49 6106 610682; E-mail: [email protected].
1386-0291/16/$35.00 © 2016 – IOS Press and the authors. All rights reserved
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G.-F. von Tempelhoff et al. / Correlation between blood rheological properties
1. Introduction From hemodynamic point of view blood is a two-phase suspension of formed elements where cells are suspended in plasma which is an aqueous solution of organic molecules, proteins and salts. The viscosity of blood depends on the existing shear forces and is determined by hematocrit, plasma viscosity (PV), Red Blood Cell (RBC) aggregation and the bio-mechanical properties of RBCs [4]. Deformability describes the ability of RBCs to change shape in response to deforming forces which not only improves their flow properties but also protects against cell disruption under bulk flow conditions and in the microcirculation when passing through capillary vessels as small as 3 m in diameter in particular [2]. Early during erythropoiesis, the number of cell organelles is reduced to a minimum through extrusion of the nucleus, endoplasmic reticulum and mitochondria ensuring optimal plasticity of the cellular membrane. Variability of cell morphology may involve a change in cell curvature, uniaxial deformation or area expansion [4]. Under physiological conditions the deforming force is predominantly exert from outside the cell by fluid shear stress. However, it may be generated in the cell itself, caused by a change of the osmotic milieu [8], by formation of rigid fibers of cytoplasmic hemoglobin S, such as in sickle cell anemia [6], or by increased density of the intracellular fluid mainly as a result of hemoglobin-concentration and -properties [10]. The extent, vector and mode of deformation depend on the magnitude, rate and direction of the exposed stress. While RBCs behave as elastic bodies, and thus the shape change is reversible when the deforming forces are removed [7] they also exhibit viscous behavior so they respond as a viscoelastic body as well. Internal viscosity, viscoelastic properties and surface area to volume ratio are the main determinants of RBC deformability [14]. Plastic changes of the RBC membrane may be irreversibly upon exposure to excessive shear forces or in pathologic conditions associated with numerous diseases such as metabolic Syndrome, diabetes mellitus [12], iron deficient anemia [23], hemoglobinopathy [5, 13], pre-eclampsia/HELLPSyndrome [18], and autoimmune disease. Changes in RBC indices, e.g. mean corpuscular volume (MCV), corpuscular hemoglobin content (MCH) of erythrocytes, as well as mean hemoglobin concentration of the erythrocyte mass were found to correlate with impaired RBC deformability in effected patients. In a first step it was the aim of this community based study to investigate the association between blood rheological parameters and RBC deformability in particular and RBC indices in otherwise healthy mid-age women prior to elective gynaecological surgery.
2. Patients and methods 2.1. Recruitment of healthy individuals and blood sampling Within a 6-months period, from June 2014 to December 2014 all women scheduled for elective gynaecologic surgery, were ask to participate in the trial. Women with known pregnancy, malign-, infective-, chronic-disease, extreme BMI (40 Kg/m2 ) or receiving permanent medication were excluded from this part of the trial. Elective surgery included hysterectomy, minor vulvo-vaginaluterine or laparoscopic surgery for benign, non-infective diseases. Blood sampling for rheological estimations and pre-operative blood routine was performed at the same time by venepuncture from the antecubital vein between 8:00 h and 10:00 h a.m. the day before scheduled elective surgery and after 12 hours overnight fasting. The blood was collected into standard vacutainer tubes containing EDTA K3 for measuring blood rheological variables and RBC indices. All laboratory estimations were instantly performed within 2 hours after blood collection in the Hemorheological-Hemostaseological Laboratory Unit of the Gynaecologic Department.
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2.2. Laboratory methods The red blood cell count, haemoglobin concentration (Hb), haematocrit (Hct), white blood cell count, platelet count, C reactive protein, and erythrocyte indices i.e. mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined by an Advia 2120i Haematology System with Autoslide (Siemens, Germany). Erythrocyte deformability was determined by means of ektacytometric techniques in a Rheodyn shear stress diffractometer (Myrenne Gmbh, Germany) [17]. A sample size of 30 L of whole blood was solved in 2 ml Dextran 24 mPa s and exposed to increasing shear forces (low shear: 1.2, 3.0; moderate shear: 6.0, 12.0; high shear: 30.0 and 60.0 Pascal). Results are expressed as percental elongation (EI) whereas a high EI corresponds with an increased RBC deformability [1]. RBC aggregation was estimated using a photometric rheoscope developed by Schmid-Sch¨onbein et al. [19]. Average RBC aggregation is determined by the quantity of light transmission which is measured by photo sensors in two modes, during stasis and while samples are subjected to low shear rate of 3 s−1 . Light transmission increases proportionally with extend of RBC aggregation and after processing in an integrated computer results are expressed in arbitrary units (s−1 ). The EDTA-containing blood sample is centrifuged for 10 min at 300 U/min, so that a suspension of erythrocyte in its own autologous plasma is produced corresponding to a hematocrit level of 45%. 20 L of this sample is then placed into the Myrenne Aggregometer 1 (Myrenne, Roetgen, Germany). For determination of plasma viscosity vacuum tubes were centrifuged for 20 min (2.000 g at 4◦ C) whereas probes from the middle-layer of the plasma were obtained and inserted into and measured with the system of a Capillary tube viscometer (KSPV 1 Fresenius, Bad Homburg, Germany) at 37◦ C according to Jung et al. [11]. 2.3. Statistical analysis Descriptive analysis included calculation of mean values ± standard deviations, inter quartile ranges and 95 percentiles. Two-sided Pearson’s correlation coefficient was used to correlate different parameters. P values of less than 0.05 were considered statistically significant. All tests were performed by assuming a confidence interval of 95%. Statistical analyses were conducted using PSPP-project version 0.7.9, released February 2012. 3. Results This is a cross-sectional observational study. During the study period from June 2014 to December 2014 a total of 286 healthy women (age: 46.5 ± 17.6 y; BMI: 25.5 ± 5.2 kg/m2 ) were eligible for inclusion into this prospective evaluation. All the operations were indicated due to benign and non-infectional elective gynaecological illnesses, mainly one-day surgery: 58% hysteroscopy and laparoscopy; 26% dilatation and curettage of the uterus; 16% hysterectomy. The patients‘ characteristics are presented in Table 1. 3.1. Rheological parameters Preoperative results of rheological parameters (Table 2) and Blood count in addition to RBC indices (Table 3) are separately shown. Plasma viscosity and RBC aggregation for both stasis and low shear was statically significantly correlated with the age and BMI of patients. Conversely, RBC deformability was only statistically significantly correlated with the age of patients but not with their BMI. There was no statistically significant correlation between Pv and RBC aggregation (stasis and low shear)
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G.-F. von Tempelhoff et al. / Correlation between blood rheological properties Table 1 Descriptive statistics of Characteristics of 286 women undergoing elective gynaecologic surgery: Means, Range and Standard deviation
Age (y) Weight (kg) Height (cm) BMI (kg/m2 )
N
Mean
Range
SD
286 282 283 282
48.54 70.6 166.46 25.46
46.5–50.59 68.82–72.37 165.76–167.16 24.85–26.07
17.578 15.124 6.018 5.211
BMI = Body Mass Index.
Table 2 Descriptive statistics of rheological parameters: Means, Range and Standard deviation
Pv (mPa s) E0 E1 RBC 1.2 (%) RBC 3.0 (%) RBC 6.0 (%) RBC 12 (%) RBC 30 (%) RBC 60 (%)
N
Mean
Range
SD
283 282 282 285 285 285 285 285 285
1.1749 12.664 17.930 11.291 22.878 30.919 37.196 43.586 47.131
1.1604–1.1895 11.922–13.406 17.072–18.788 10.958–11.625 22.483–23.273 30.560–31.278 36.845–37.547 43.214–43.957 46.736–47.525
0.12422 6.3290 7.3206 2.8576 3.3874 3.0789 3.0127 3.1854 3.3832
Pv = plasma viscosity, E0 = erythrocyte aggregation in stasis, E1 = erythrocyte aggregation under low shear forces, RBC = Erythrocyte deformability when exposed to shear force of 1.2, 3.0, 6.0, 12.0, 30.0 and 60.0 Pascal.
Table 3 Descriptive statistics of Hematologic parameters: Means, Range and Standard deviation
Hb (g/dL) Hct (%) Leu (/L) Thr (/L) MCV (fL) MCHC (g/dL) MCH (pg)
N
Mean
Range
SD
277 277 277 276 277 277 277
13.204 39.262 7,819.89 281,079.71 89.77 33.68 30.32
13.041–13.368 38.795–39.729 7,492.28–8,147.50 271,834.2–290,325.1 89.05–90.49 33.55–33.81 30.08–30.55
1.3810 3.9484 2769.727 78022.194 6.098 1.108 1.973
Hb = haemoglobin, Hct = hematocrit, MCV = mean cell volume, MCHC = mean corpuscular haemoglobin concentration, MCH = mean corpuscular volume, Leu = Leucocyte, Thr = Thrombocyte.
and MCV, MCHC nor MCH (Table 4). While MCV (r = 0.223; p < 0.001) and MCHC (r = 0.156; p = 0.009) was statistically significantly correlated with the age of women the correlation was inverse for MCHC (r = −0.114; p = 0.059). None of the RBC indices was statistical significant correlated with BMI. RBC-deformability was statistically significantly positive correlated with MCV and MCH as well, while statistically significantly inversely correlated with MCHC during all shear exposures. Correlation coefficient for MCV and RBC deformability was most remarkable during moderate shear
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Table 4 Bivariate Pearson Correlation of haematological- and rheological parameters, age and BMI
age N R P BMI N R P MCV N R P MCHC N R P MCH N R P ∗
Pv
E0
E1
RBC 1.2
RBC 3.0
RBC 6.0
RBC 12.0
RBC 30
RBC 60
283 0.165 0.005
282 0.131 0.028
282 0.185 0.002
285 0.046 0.443
285 0.132 0.026
285 0.145 0.014
285 0.158 0.007
285 0.147 0.013
285 0.131 0.027
279 0.144 0.016
278 0.182 0.002
281 0.224 0.001
281 0.012 0.846
281 0.047 0.435
281 0.096 0.110
281 0.113 0.058
281 0.094 0.115
281 0.075 0.213
274 −0.013 0.832
273 −0.082 0.179
273 −0.054 0.371
276 0.128 0.034∗
276 0.198 0.001∗
276 0.209 0.0001∗
276 0.214 0.0001∗
276 0.197 0.001∗
276 0.160 0.008∗
274 −0.086 0.157
273 −0.038 0.533
273 0.038 0.527
276 −0.145 0.016∗
276 −0.160 0.008∗
276 −0.155 0.010∗
276 −0.176 0.003∗
276 −0.200 0.001∗
276 −0.202 0.001∗
274 −0.033 0.583
273 −0.078 0.201
273 −0.022 0.712
276 0.183 0.002∗
276 0.201 0.001∗
276 0.198 0.001∗
276 0.161 0.008∗
276 0.122 0.043∗
276 0.108 0.073
Statistically significant p value (p < 0.05).
force exposure at 6.0 Pascal (r = 0.206) and 12.0 Pascal (r = 0.214; p < 0.001) as shown in Fig. 1A–F. The same association was found between MCH and RBC deformability whereas during shear force exposure at 6.0 Pascal (r = 0.201; p < 0.001) and 12.0 Pascal (r = 0.198; p < 0.001) correlation coefficients were highest (Fig. 2A–F). In contrast, RBC deformability continuously decreased with increasing MCHC (Fig. 3A–F), whereas negative correlation coefficients were strongest at high shear force exposure at 40 Pascal (r = −0.20; p = 0.01) and 60 Pascal (r = −0.202; p = 0.003). Neither haemoglobin concentration (Range of r = −0.046–0.04), haematocrit (Range of r = −0.001–0.06), platelet- (Range of r = −0.072 – −0.018) nor leukocyte-counts (Range of r = −0.106 – −0.048) were correlated with RBC deformability (p > 0.05). A summary of all correlation coefficients, p values and number of estimated individuals is presented in Table 4. There was a statistical significant correlation between RBC deformability and RBC indices for neither haemoglobin values nor haematocrit. 4. Discussion The combination of a high degree flexibility of the erythrocyte membrane (viscoelasticity), in the presence of a low intracellular fluid viscosity and a high surface area/cell volume ratio provides optimal constellation for maximal cell deformability in the microcirculation or passing through the spaces between the endothelial cells of the splenic sinusoids smaller than 3 m in diameter [4]. Biconcave discoid geometry of RBCs with a major diameter of 8 m and MCV of about 90 fL (89,77 fL in our study) under normal conditions results in a 40% greater mean surface area (SF) of about 140 m2 compared to that of a spherical cell shape of the same MCV which allows uniaxial deformation
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G.-F. von Tempelhoff et al. / Correlation between blood rheological properties
Fig. 1. A–F, Correlation between MCV and RBC deformability during low, moderate and high shear force exposure. ∗ Statistically significant p value (p < 0.05).
G.-F. von Tempelhoff et al. / Correlation between blood rheological properties
51
Fig. 2. A–F, Correlation between MCH and RBC deformability during low, moderate and high shear force exposure. ∗ Statistically significant p value (p < 0.05).
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G.-F. von Tempelhoff et al. / Correlation between blood rheological properties
Fig. 3. A–F, Correlation between MCHC and RBC deformability during low, moderate and high shear force exposure. ∗ Statistically significant p value (p < 0.05).
G.-F. von Tempelhoff et al. / Correlation between blood rheological properties
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[4]. Hemoglobin-concentration and its biochemical properties primarily determine intracellular fluid viscosity which is about 7 mPa s and which as such has no elastic behavior [4]. Our results confirm a close relation between RBC deformability and MCV, and although correlation coefficients between 0.128 and 0.214 were within in a narrow range, significantly highest correlation was found when RBCs were exposed to moderate to high shear forces, which approximately corresponds to the shear stress found in the splenic sinusoids. Moreover, the reliance of RBC deformability on MCHC could be clearly demonstrated, whereas increasing MCHC went along with a continuous reduction in RBC deformability, which was most pronounced during high shear stress exposure. The role of MCV on the impact of RBC deformability has been impressively shown in patients with macrocytosis greater than 97 fL (mean: 115.9 ± 5.2 fL) where elongation indices for RBCs were significantly higher compared to normocytic controls at low, moderate and high shear stress test conditions (r = 0.548; p < 0.01) [22]. The trend towards improved RBC deformability in extreme sized RBCs in vitro may be limited when passing through very narrow vessel in vivo. MCV reduces with the aging of RBC while rigidity increases which likely is a main cause of their clearance in the spleen. Bosch et al. [3] could demonstrate reduced deformability in the fraction of older RBCs characterized by increased MCHC and lower MCV which is in line with our observation. Numerous study have shown reduced RBC deformability in patients with hemoglobinopathia such as sickle cell anemia [5, 6, 13] or thalassemia [20]. However, reduced deformability associated with these diseases unlikely is the result of anemia itself but rather is caused by impaired viscoelasticity of the membrane through generation of rigid fibers of HbS that distort and damage the cytoskeleton and membrane in sickle cell anemia, alternatively by attachment of unstable globin precipitates to the membrane that induce oxidation of the skeletal proteins in thalassemia. Vaya et al. [23] found significantly lower deformability of RBC - using same ektacytometric technics as in our trial - in 50 patients with iron deficiency anemia compared to age- and sex- matched healthy controls. Correlation analyzes between RBC-indices and -deformability was not performed, but compared to healthy subjects anemia was characterized by significantly lower MCV and MCHC as well. It is unclear to what extend each indices represents a dominating effector of reduced RBC deformability in iron deficient anemic patients. Blood rheological results in our trial were closely related with the age of women. While RBC aggregation and PV were also correlated with BMI, the latter had no effect on RBC deformability in women of this trial whose mean BMI was 25.5 kg/m2 which is in the range of moderate overweight according to WHO (25–30 Kg/m2 ) [15]. The influence of obesity on hemorheology has been addressed in a number of trials and while a trend towards increased viscosity was found in most of them, improvement in hemorheological parameters after weight loss and treatment with low calorie diet was reported as well [9, 16]. A recent trial reconfirmed significant correlation between BMI and whole blood viscosity, hematocrit, Pv and RBC aggregation. As in our trial BMI in these patients was 27.54 ± 0.34 kg/m2 indicating moderate overweight whereas RBC rigidity was indifferent for high and low BMIs as well [21]. The results of the current trial based on notable number of apparently healthy women show close relation between RBC deformability, MCV and MCHC which is age dependent but independent of the BMI in mid-age women with normal to moderate increased weight. The results of all blood rheological parameters indicate an age dependent increase in blood viscosity.
Acknowledgments The authors declare no conflict of interest regarding this published material. “Detailed data of this manuscript are part of a thesis by Mr. Attila Demirhan”.
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Correlation between blood rheological properties and red blood cell indices (MCH, MCV, MCHC) in healthy women Georg-Friedrich von Tempelhoffa,c,∗ , Olga Schelkunova,c , Attila Demirhana,c , Panagiotis Tsikourasc,e , Werner Rathf , Eva Veltenb,c and Roland Csorbab,c,d a
Department of Obstetrics and Gynaecology, St. Vinzenz Hospital, Hanau, Germany Department of Obstetrics and Gynaecology, City Hospital of Aschaffenburg, Aschaffenburg, Germany c Institut of Coagulation Disorders in Obstetrics and Gynaecology, Hausen, Germany d Department of Obstetrics and Gynaecology, University of Debrecen, Hungary e Democritus University of Thrace, Department of Obstetrics and Gynaecology, Alexandroupolis, Greece f Department of Obstetrics and Gynaecology, RWTH University of Aachen, Germany b
Abstract. OBJECTIVE: Structure and mechanical properties of red blood cells are markedly influenced by pathophysiology of many diseases which in turn potentially impair microcirculatory blood flow. The physiological association between blood rheological parameters and red blood cell indices was investigated in otherwise healthy unselected mid-age women prior to elective gynaecological surgery. METHODS: Red Blood Cell-deformability (RBC 1.2, 3.0; 6.0, 12.0; 30.0, 60.0) during exposure to low (RBC 1.2, 3.0), moderate (RBC 16.0, 12.0) and high shear forces (RBC 30.0, 60.0; Rheodyn; Myrenne), -aggregation (MA1; Myrenne) during low shear (E1; 4−1 S) and in stasis (E0) and plasma viscosity (Pv; KSV 1; Fresenius) were correlated with red blood cell indices (RBC-I: MCV, MCH and MCHC) and subjects’ characteristics in 286 healthy women the day before undergoing gynaecologic standard surgery. Women with known pregnancy, malign-, infective-, chronic-disease or extreme BMI (40 Kg/m2 ) were excluded from this trial. RESULTS: From June 2014 to December 2014 a total of 286 healthy women (age: 46.5 ± 17.6 y; BMI: 25.5 ± 5.2 kg/m2 ) were eligible for inclusion into this prospective evaluation. Pv (mean ± SD: 1.17 ± 0.12 mPa s) and RBC aggregation (E0:12.6 ± 6.3; E1:17.9 ± 7.3) were not significantly correlated with RBC-I but with age and BMI. In contrast, RBCdeformability correlated significantly with MCV and MCH but significantly inversely correlated with MCHC. Deformability significantly increased with age but was unaffected by BMI of women. The correlation between RBC-I and RBC deformability was most remarkable during moderate shear force exposure. Neither haemoglobin nor haematocrit were correlated with RBC deformability or RBC-I. CONCLUSIONS: Cell volume and haemoglobin content had a strong impact on deformability in apparently healthy mid age women, whereas low MCHC and large MCV were associated with an increase in deformability while high MCHC and small MCV correlated with increased rigidity of RBC. BMI had no impact on deformability while age was associated with an increase in all determinants of blood viscosity. RBC aggregability was not affected by MCV, MCHC or MCH in mid-age women. Keywords: Plasma viscosity, red blood cell aggregation, red blood cell deformability MCH, MCV, MCHC
∗ Corresponding author: Georg-Friedrich von Tempelhoff, Institute of Coagulation Disorders in Obstetrics and Gynaecology, Freiherr vom Stein Str.29, 63179 Hausen, Germany. Tel.: +49 15221 554988; Fax: +49 6106 610682; E-mail: [email protected].
1386-0291/16/$35.00 © 2016 – IOS Press and the authors. All rights reserved
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G.-F. von Tempelhoff et al. / Correlation between blood rheological properties
1. Introduction From hemodynamic point of view blood is a two-phase suspension of formed elements where cells are suspended in plasma which is an aqueous solution of organic molecules, proteins and salts. The viscosity of blood depends on the existing shear forces and is determined by hematocrit, plasma viscosity (PV), Red Blood Cell (RBC) aggregation and the bio-mechanical properties of RBCs [4]. Deformability describes the ability of RBCs to change shape in response to deforming forces which not only improves their flow properties but also protects against cell disruption under bulk flow conditions and in the microcirculation when passing through capillary vessels as small as 3 m in diameter in particular [2]. Early during erythropoiesis, the number of cell organelles is reduced to a minimum through extrusion of the nucleus, endoplasmic reticulum and mitochondria ensuring optimal plasticity of the cellular membrane. Variability of cell morphology may involve a change in cell curvature, uniaxial deformation or area expansion [4]. Under physiological conditions the deforming force is predominantly exert from outside the cell by fluid shear stress. However, it may be generated in the cell itself, caused by a change of the osmotic milieu [8], by formation of rigid fibers of cytoplasmic hemoglobin S, such as in sickle cell anemia [6], or by increased density of the intracellular fluid mainly as a result of hemoglobin-concentration and -properties [10]. The extent, vector and mode of deformation depend on the magnitude, rate and direction of the exposed stress. While RBCs behave as elastic bodies, and thus the shape change is reversible when the deforming forces are removed [7] they also exhibit viscous behavior so they respond as a viscoelastic body as well. Internal viscosity, viscoelastic properties and surface area to volume ratio are the main determinants of RBC deformability [14]. Plastic changes of the RBC membrane may be irreversibly upon exposure to excessive shear forces or in pathologic conditions associated with numerous diseases such as metabolic Syndrome, diabetes mellitus [12], iron deficient anemia [23], hemoglobinopathy [5, 13], pre-eclampsia/HELLPSyndrome [18], and autoimmune disease. Changes in RBC indices, e.g. mean corpuscular volume (MCV), corpuscular hemoglobin content (MCH) of erythrocytes, as well as mean hemoglobin concentration of the erythrocyte mass were found to correlate with impaired RBC deformability in effected patients. In a first step it was the aim of this community based study to investigate the association between blood rheological parameters and RBC deformability in particular and RBC indices in otherwise healthy mid-age women prior to elective gynaecological surgery.
2. Patients and methods 2.1. Recruitment of healthy individuals and blood sampling Within a 6-months period, from June 2014 to December 2014 all women scheduled for elective gynaecologic surgery, were ask to participate in the trial. Women with known pregnancy, malign-, infective-, chronic-disease, extreme BMI (40 Kg/m2 ) or receiving permanent medication were excluded from this part of the trial. Elective surgery included hysterectomy, minor vulvo-vaginaluterine or laparoscopic surgery for benign, non-infective diseases. Blood sampling for rheological estimations and pre-operative blood routine was performed at the same time by venepuncture from the antecubital vein between 8:00 h and 10:00 h a.m. the day before scheduled elective surgery and after 12 hours overnight fasting. The blood was collected into standard vacutainer tubes containing EDTA K3 for measuring blood rheological variables and RBC indices. All laboratory estimations were instantly performed within 2 hours after blood collection in the Hemorheological-Hemostaseological Laboratory Unit of the Gynaecologic Department.
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2.2. Laboratory methods The red blood cell count, haemoglobin concentration (Hb), haematocrit (Hct), white blood cell count, platelet count, C reactive protein, and erythrocyte indices i.e. mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined by an Advia 2120i Haematology System with Autoslide (Siemens, Germany). Erythrocyte deformability was determined by means of ektacytometric techniques in a Rheodyn shear stress diffractometer (Myrenne Gmbh, Germany) [17]. A sample size of 30 L of whole blood was solved in 2 ml Dextran 24 mPa s and exposed to increasing shear forces (low shear: 1.2, 3.0; moderate shear: 6.0, 12.0; high shear: 30.0 and 60.0 Pascal). Results are expressed as percental elongation (EI) whereas a high EI corresponds with an increased RBC deformability [1]. RBC aggregation was estimated using a photometric rheoscope developed by Schmid-Sch¨onbein et al. [19]. Average RBC aggregation is determined by the quantity of light transmission which is measured by photo sensors in two modes, during stasis and while samples are subjected to low shear rate of 3 s−1 . Light transmission increases proportionally with extend of RBC aggregation and after processing in an integrated computer results are expressed in arbitrary units (s−1 ). The EDTA-containing blood sample is centrifuged for 10 min at 300 U/min, so that a suspension of erythrocyte in its own autologous plasma is produced corresponding to a hematocrit level of 45%. 20 L of this sample is then placed into the Myrenne Aggregometer 1 (Myrenne, Roetgen, Germany). For determination of plasma viscosity vacuum tubes were centrifuged for 20 min (2.000 g at 4◦ C) whereas probes from the middle-layer of the plasma were obtained and inserted into and measured with the system of a Capillary tube viscometer (KSPV 1 Fresenius, Bad Homburg, Germany) at 37◦ C according to Jung et al. [11]. 2.3. Statistical analysis Descriptive analysis included calculation of mean values ± standard deviations, inter quartile ranges and 95 percentiles. Two-sided Pearson’s correlation coefficient was used to correlate different parameters. P values of less than 0.05 were considered statistically significant. All tests were performed by assuming a confidence interval of 95%. Statistical analyses were conducted using PSPP-project version 0.7.9, released February 2012. 3. Results This is a cross-sectional observational study. During the study period from June 2014 to December 2014 a total of 286 healthy women (age: 46.5 ± 17.6 y; BMI: 25.5 ± 5.2 kg/m2 ) were eligible for inclusion into this prospective evaluation. All the operations were indicated due to benign and non-infectional elective gynaecological illnesses, mainly one-day surgery: 58% hysteroscopy and laparoscopy; 26% dilatation and curettage of the uterus; 16% hysterectomy. The patients‘ characteristics are presented in Table 1. 3.1. Rheological parameters Preoperative results of rheological parameters (Table 2) and Blood count in addition to RBC indices (Table 3) are separately shown. Plasma viscosity and RBC aggregation for both stasis and low shear was statically significantly correlated with the age and BMI of patients. Conversely, RBC deformability was only statistically significantly correlated with the age of patients but not with their BMI. There was no statistically significant correlation between Pv and RBC aggregation (stasis and low shear)
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G.-F. von Tempelhoff et al. / Correlation between blood rheological properties Table 1 Descriptive statistics of Characteristics of 286 women undergoing elective gynaecologic surgery: Means, Range and Standard deviation
Age (y) Weight (kg) Height (cm) BMI (kg/m2 )
N
Mean
Range
SD
286 282 283 282
48.54 70.6 166.46 25.46
46.5–50.59 68.82–72.37 165.76–167.16 24.85–26.07
17.578 15.124 6.018 5.211
BMI = Body Mass Index.
Table 2 Descriptive statistics of rheological parameters: Means, Range and Standard deviation
Pv (mPa s) E0 E1 RBC 1.2 (%) RBC 3.0 (%) RBC 6.0 (%) RBC 12 (%) RBC 30 (%) RBC 60 (%)
N
Mean
Range
SD
283 282 282 285 285 285 285 285 285
1.1749 12.664 17.930 11.291 22.878 30.919 37.196 43.586 47.131
1.1604–1.1895 11.922–13.406 17.072–18.788 10.958–11.625 22.483–23.273 30.560–31.278 36.845–37.547 43.214–43.957 46.736–47.525
0.12422 6.3290 7.3206 2.8576 3.3874 3.0789 3.0127 3.1854 3.3832
Pv = plasma viscosity, E0 = erythrocyte aggregation in stasis, E1 = erythrocyte aggregation under low shear forces, RBC = Erythrocyte deformability when exposed to shear force of 1.2, 3.0, 6.0, 12.0, 30.0 and 60.0 Pascal.
Table 3 Descriptive statistics of Hematologic parameters: Means, Range and Standard deviation
Hb (g/dL) Hct (%) Leu (/L) Thr (/L) MCV (fL) MCHC (g/dL) MCH (pg)
N
Mean
Range
SD
277 277 277 276 277 277 277
13.204 39.262 7,819.89 281,079.71 89.77 33.68 30.32
13.041–13.368 38.795–39.729 7,492.28–8,147.50 271,834.2–290,325.1 89.05–90.49 33.55–33.81 30.08–30.55
1.3810 3.9484 2769.727 78022.194 6.098 1.108 1.973
Hb = haemoglobin, Hct = hematocrit, MCV = mean cell volume, MCHC = mean corpuscular haemoglobin concentration, MCH = mean corpuscular volume, Leu = Leucocyte, Thr = Thrombocyte.
and MCV, MCHC nor MCH (Table 4). While MCV (r = 0.223; p < 0.001) and MCHC (r = 0.156; p = 0.009) was statistically significantly correlated with the age of women the correlation was inverse for MCHC (r = −0.114; p = 0.059). None of the RBC indices was statistical significant correlated with BMI. RBC-deformability was statistically significantly positive correlated with MCV and MCH as well, while statistically significantly inversely correlated with MCHC during all shear exposures. Correlation coefficient for MCV and RBC deformability was most remarkable during moderate shear
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Table 4 Bivariate Pearson Correlation of haematological- and rheological parameters, age and BMI
age N R P BMI N R P MCV N R P MCHC N R P MCH N R P ∗
Pv
E0
E1
RBC 1.2
RBC 3.0
RBC 6.0
RBC 12.0
RBC 30
RBC 60
283 0.165 0.005
282 0.131 0.028
282 0.185 0.002
285 0.046 0.443
285 0.132 0.026
285 0.145 0.014
285 0.158 0.007
285 0.147 0.013
285 0.131 0.027
279 0.144 0.016
278 0.182 0.002
281 0.224 0.001
281 0.012 0.846
281 0.047 0.435
281 0.096 0.110
281 0.113 0.058
281 0.094 0.115
281 0.075 0.213
274 −0.013 0.832
273 −0.082 0.179
273 −0.054 0.371
276 0.128 0.034∗
276 0.198 0.001∗
276 0.209 0.0001∗
276 0.214 0.0001∗
276 0.197 0.001∗
276 0.160 0.008∗
274 −0.086 0.157
273 −0.038 0.533
273 0.038 0.527
276 −0.145 0.016∗
276 −0.160 0.008∗
276 −0.155 0.010∗
276 −0.176 0.003∗
276 −0.200 0.001∗
276 −0.202 0.001∗
274 −0.033 0.583
273 −0.078 0.201
273 −0.022 0.712
276 0.183 0.002∗
276 0.201 0.001∗
276 0.198 0.001∗
276 0.161 0.008∗
276 0.122 0.043∗
276 0.108 0.073
Statistically significant p value (p < 0.05).
force exposure at 6.0 Pascal (r = 0.206) and 12.0 Pascal (r = 0.214; p < 0.001) as shown in Fig. 1A–F. The same association was found between MCH and RBC deformability whereas during shear force exposure at 6.0 Pascal (r = 0.201; p < 0.001) and 12.0 Pascal (r = 0.198; p < 0.001) correlation coefficients were highest (Fig. 2A–F). In contrast, RBC deformability continuously decreased with increasing MCHC (Fig. 3A–F), whereas negative correlation coefficients were strongest at high shear force exposure at 40 Pascal (r = −0.20; p = 0.01) and 60 Pascal (r = −0.202; p = 0.003). Neither haemoglobin concentration (Range of r = −0.046–0.04), haematocrit (Range of r = −0.001–0.06), platelet- (Range of r = −0.072 – −0.018) nor leukocyte-counts (Range of r = −0.106 – −0.048) were correlated with RBC deformability (p > 0.05). A summary of all correlation coefficients, p values and number of estimated individuals is presented in Table 4. There was a statistical significant correlation between RBC deformability and RBC indices for neither haemoglobin values nor haematocrit. 4. Discussion The combination of a high degree flexibility of the erythrocyte membrane (viscoelasticity), in the presence of a low intracellular fluid viscosity and a high surface area/cell volume ratio provides optimal constellation for maximal cell deformability in the microcirculation or passing through the spaces between the endothelial cells of the splenic sinusoids smaller than 3 m in diameter [4]. Biconcave discoid geometry of RBCs with a major diameter of 8 m and MCV of about 90 fL (89,77 fL in our study) under normal conditions results in a 40% greater mean surface area (SF) of about 140 m2 compared to that of a spherical cell shape of the same MCV which allows uniaxial deformation
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G.-F. von Tempelhoff et al. / Correlation between blood rheological properties
Fig. 1. A–F, Correlation between MCV and RBC deformability during low, moderate and high shear force exposure. ∗ Statistically significant p value (p < 0.05).
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Fig. 2. A–F, Correlation between MCH and RBC deformability during low, moderate and high shear force exposure. ∗ Statistically significant p value (p < 0.05).
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G.-F. von Tempelhoff et al. / Correlation between blood rheological properties
Fig. 3. A–F, Correlation between MCHC and RBC deformability during low, moderate and high shear force exposure. ∗ Statistically significant p value (p < 0.05).
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[4]. Hemoglobin-concentration and its biochemical properties primarily determine intracellular fluid viscosity which is about 7 mPa s and which as such has no elastic behavior [4]. Our results confirm a close relation between RBC deformability and MCV, and although correlation coefficients between 0.128 and 0.214 were within in a narrow range, significantly highest correlation was found when RBCs were exposed to moderate to high shear forces, which approximately corresponds to the shear stress found in the splenic sinusoids. Moreover, the reliance of RBC deformability on MCHC could be clearly demonstrated, whereas increasing MCHC went along with a continuous reduction in RBC deformability, which was most pronounced during high shear stress exposure. The role of MCV on the impact of RBC deformability has been impressively shown in patients with macrocytosis greater than 97 fL (mean: 115.9 ± 5.2 fL) where elongation indices for RBCs were significantly higher compared to normocytic controls at low, moderate and high shear stress test conditions (r = 0.548; p < 0.01) [22]. The trend towards improved RBC deformability in extreme sized RBCs in vitro may be limited when passing through very narrow vessel in vivo. MCV reduces with the aging of RBC while rigidity increases which likely is a main cause of their clearance in the spleen. Bosch et al. [3] could demonstrate reduced deformability in the fraction of older RBCs characterized by increased MCHC and lower MCV which is in line with our observation. Numerous study have shown reduced RBC deformability in patients with hemoglobinopathia such as sickle cell anemia [5, 6, 13] or thalassemia [20]. However, reduced deformability associated with these diseases unlikely is the result of anemia itself but rather is caused by impaired viscoelasticity of the membrane through generation of rigid fibers of HbS that distort and damage the cytoskeleton and membrane in sickle cell anemia, alternatively by attachment of unstable globin precipitates to the membrane that induce oxidation of the skeletal proteins in thalassemia. Vaya et al. [23] found significantly lower deformability of RBC - using same ektacytometric technics as in our trial - in 50 patients with iron deficiency anemia compared to age- and sex- matched healthy controls. Correlation analyzes between RBC-indices and -deformability was not performed, but compared to healthy subjects anemia was characterized by significantly lower MCV and MCHC as well. It is unclear to what extend each indices represents a dominating effector of reduced RBC deformability in iron deficient anemic patients. Blood rheological results in our trial were closely related with the age of women. While RBC aggregation and PV were also correlated with BMI, the latter had no effect on RBC deformability in women of this trial whose mean BMI was 25.5 kg/m2 which is in the range of moderate overweight according to WHO (25–30 Kg/m2 ) [15]. The influence of obesity on hemorheology has been addressed in a number of trials and while a trend towards increased viscosity was found in most of them, improvement in hemorheological parameters after weight loss and treatment with low calorie diet was reported as well [9, 16]. A recent trial reconfirmed significant correlation between BMI and whole blood viscosity, hematocrit, Pv and RBC aggregation. As in our trial BMI in these patients was 27.54 ± 0.34 kg/m2 indicating moderate overweight whereas RBC rigidity was indifferent for high and low BMIs as well [21]. The results of the current trial based on notable number of apparently healthy women show close relation between RBC deformability, MCV and MCHC which is age dependent but independent of the BMI in mid-age women with normal to moderate increased weight. The results of all blood rheological parameters indicate an age dependent increase in blood viscosity.
Acknowledgments The authors declare no conflict of interest regarding this published material. “Detailed data of this manuscript are part of a thesis by Mr. Attila Demirhan”.
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