Hemorheological parameters in coronary artery disease detected by multi-slice CT

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1 Korea-Australia Rheology Journal, Vol.26, No.2, pp (May 2014) DOI: /s Andras Toth 1, Sandor Szukits 2, Edit Varady 2, Barbara Sandor 1, Miklos Rabai 1, Judit Papp 1, Istvan Juricskay 1, Gabor Kesmarky 1, Kalman Toth 1, *, Balazs Sumegi 3 and Istvan Battyani st Department of Medicine, University of Pecs School of Medicine, H-7624 Pecs, Ifjusag ut 13, Hungary 2 Department of Radiology, University of Pecs School of Medicine, H-7624 Pecs, Ifjusag ut 13, Hungary 3 Department of Biochemistry and Medical Chemistry, University of Pecs School of Medicine, H-7624 Pecs, Szigeti ut 12, Hungary (Received January 31, 2014; final revision April 8, 2014; accepted April 20, 2014) Epidemiological studies have confirmed that hemorheological parameters are primary risk factors in CAD; and their alterations in CAD have been described. We aimed to investigate both macro- and microrheological properties of blood in patients with CAD. The data of 121 patients (mean age: 58.8 ± 9.6 years) undergoing coronary CT were analyzed. Blood samples were obtained right before CT examinations. Hematocrit (Hct), plasma (PV) and apparent whole blood viscosity (WBV), red blood cell (RBC) aggregation and RBC deformability were measured. Patients were classified into four groups according to their coronary vessel state: Negative group (n = 32, mean age: 56.8 ± 11.1 years) without any coronary stenosis or atherosclerotic lesion and zero calcium-score, Non-significant group (n = 27, mean age: 59.2 ± 7.5 years) below 40% area stenosis, Single-vessel group (n = 32, mean age: 58.8 ± 8.5 years) over 40% area stenosis or history of PCI or CABG on one coronary vessel, Multi-vessel group (n = 30, mean age: 62.1 ± 8.4 years) with over 40% area stenosis or history of PCI or CABG on multiple coronary vessels. Hct was significantly (p < 0.05) higher in all CAD (Non-significant, Single-vessel, Multi-vessel) groups compared to the Negative group. WBV was significantly (p<0.05) higher in the Multi-vessel group compared to the Negative group. No significant (p 0.05) differences were observed in PV. RBC aggregation was significantly (p < 0.05) increased in the Multi-vessel group compared to the Negative group. RBC deformability showed a decreasing tendency with the increasing number of atherosclerotic vessels. Our results indicate that hemorheological variables are deteriorated in patients with CAD established by coronary CT, which is more pronounced in severe coronary disease. Keywords: coronary CT, red blood cell, aggregation, deformability, viscosity 1. Introduction Cardiovascular diseases (CVDs) are responsible for the vast majority of global deaths (World Health Organization, 2011). Over 80% of cardiovascular (CV) related deaths occur in the low- and middle-income countries, thus in spite of the prevention programs and improving healthcare in the high-income countries, the global mortality is still expected to rise in the future (Mathers and Loncar, 2006). The increasing burden of CVDs upholds the need of further research of their risk factors and pathogenesis. Coronary artery disease (CAD) is the leading cause of CV related deaths, mostly originating from the atherosclerosis of coronary vessels leading to progressive narrowing of the lumen and thus restricting blood flow toward the myocardium. The coronary vessel system is a unique part of the human vascular structure due to the periodic change in perfusion pressure and blood flow. At normal circumstances coronary flow is mainly determined # This article was presented at the 17th Conference of the European Society for Clinical Hemorheology & Microcirculation (ESCHM), held on 6-9 July, 2013, Hungary. *Corresponding author: toth.kalman@pte.hu by hemodynamic factors, but in certain conditions, such as pre-existing coronary stenosis, alteration of hemorheological parameters may impair myocardial microcirculation early (Baskurt et al., 1991; Toth and Kesmarky, 2007). The alterations of hemorheological parameters in CAD have been described by several prospective epidemiological studies; moreover the Framingham Study (Kannel et al., 1987), the Honolulu Heart Program (Carter et al., 1983), the Scottish Heart Health Study (Lowe et al., 1988), the Physicians' Health Study (Ma et al., 1999), the MONICA-Augsburg Cohort Study (Koenig et al., 1998), the Caerphilly and Speedwell Studies (Sweetnam et al., 1997; Yarnell et al., 1991) address altered hemorheological parameters as independent cardiovascular risk factors (Toth and Kesmarky, 2007). Previous clinical studies indicate significantly elevated hematocrit (Hct), whole blood viscosity (WBV), plasma viscosity (PV), fibrinogen level and red blood cell (RBC) aggregation in CAD (Lowe et al., 1980; Rainer et al., 1987; Lee et al., 2008), however only a few were able to detect statistically significant differences between the various stages of CAD (Neumann et al., 1989; Kesmarky et al., 1998) The Korean Society of Rheology and Springer 229

2 Andras Toth et al. Our previous study showed significantly increased Hct, WBV, PV and fibrinogen levels in CAD compared to healthy controls, moreover significant differences were found between CAD subgroups, suggesting a correlation with the severity of CAD (Kesmarky et al., 1998). 2. Aims Previous studies agreed in the alterations of hemorheological parameters in CAD, but not all were able to detect significant differences between different stages of the disease. We aimed to conduct a full scale hemorheological study on CAD patients, including measurement of RBC aggregation and deformability which have not been carried out previously. To the best of our knowledge, all previous studies used invasive coronary X-ray angiography to evaluate for CAD. In our current study, a new and well established method, coronary CT angiography was used for the evaluation of the coronary vessel system. This method is able to measure coronary stenosis precisely and can also determine the total extent of coronary calcification. 3. Methods 3.1. Patients The study was approved by the Regional Ethics Committee of the University of Pecs. 130 patients admitted to coronary CT angiography at the Regional Screening and Diagnostic Center of the University of Pecs participated in the study. Informed consent was signed by all subjects prior entering the study. Patients were questioned regarding previous medical history and existing medications. Patients with autoimmune disorders were not enrolled in the study Blood sampling Blood samples were obtained via antecubital veins just before coronary CT examinations. Samples were collected, using a 21-gauge butterfly needle set with minimal tourniquet, according to the latest hemorheological guideline (Baskurt et al., 2009), into sterile heparin (2x6 ml for hemorheological measurements), EDTA (3 ml for qualitative and quantitative blood cell analyses), citrate (2.7 ml for fibrinogen level) anticoagulated and native (5 ml for lipid profile) Vacutainer tubes. Blood samples were stored at ambient temperature until hemorheological measurements Coronary CT Coronary CT angiography examinations were performed at the Regional Screening and Diagnostic Centre of the University of Pecs with a first generation 64 slice, dual source Siemens Somatom Definition CT device. Prior to the examinations no food or caffeine containing fluid consumption was allowed for four hours. First a native scan was done to estimate total coronary calcification. After that a contrast enhanced scan was performed to determine coronary state. Coronary calcification was characterized by Agatson-score, lesions were characterized by area stenosis. Data processing and image evaluation was done with Siemens syngo software on Siemens Multimodality workstation Hemorheological and other laboratory measurements Hemorheological measurements were performed within 2 hours from blood sampling at the Hemorheological Laboratory of the 1 st Department of Medicine University of Pecs Medical School. Hematocrit was determined by microhematocrit centrifuge (Haemofuge Heraeus Instr., Germany). Plasma viscosity and whole blood viscosity were measured by Hevimet 40 capillary viscometer (Hemorex Ltd., Budapest, Hungary) (Toth et al., 2012) at 90 s -1 shear rate. Measurements were performed at 37 C temperature. Plasma was gained by centrifugation (Labofuge 400 R, Heraeus Instr., Germany) of blood samples for 10 minutes at 2,500 g. Red blood cell aggregation was determined by Myrenne (MA-1 Aggregometer, Myrenne GmbH, Roetgen, Germany) aggregometer (Vaya et al., 2003). Measurements were performed at room temperature (22 ± 1 o C); RBC aggregation was measured for 10 seconds. RBC aggregation was evaluated using both native samples and 40% Hct suspension of RBC in autologous plasma. Erythrocyte deformability was characterized by LORCA (Laser-assisted Optical Rotational Cell Analyzer; R&R Mechatronics Hoorn, Netherlands) using the laser diffraction ellipsometry technique (Hardeman et al., 1994). 25 µl of blood was suspended in 5 ml mpas viscosity polyvinylpyrrolidone solution dissolved in phosphate buffered saline (290 mosm/kg, ph = 7.4). RBCs were deformed at 9 different shear stresses from 30 Pa to 0.3 Pa. Deformability is characterized by the elongation index (EI). Central laboratory measurements were carried out at the Department of Laboratory Medicine of the University of Pecs Medical School. The following parameters were measured: Hct, Hgb concentration, RBC count, MCV, MCH, MCHC, lipid profile (LDL, HDL) and fibrinogen level Classification of patients Patients were classified into four groups based on their coronary vessel state (Table 1): 1) Negative group without any coronary stenosis or atherosclerotic lesion and zero 230 Korea-Australia Rheology J., Vol. 26, No. 2 (2014)

3 Table 1. Classification of patients. Negative (Neg) Non-significant (NS) Single-vessel (SV) Multi-vessel (MV) calcium-score, 2) Non-significant group below 40% area stenosis on one or more coronary vessels, 3) Single-vessel group over 40% area stenosis or history of percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) on one coronary vessel and 4) Multi-vessel group with over 40% area stenosis or history of PCI or CABG on multiple coronary vessels. Patients having stenosis over 40% on one coronary vessel plus PCI or CABG on another coronary artery were classified to the Multi-vessel group Statistical analysis Continuous variables are presented as mean ± S.E.M. Discrete variables are described through relative or absolute frequencies. Continuous variables were analyzed with one-way ANOVA; in case of dichotomous variables χ 2 test was used. A two-tailed p value less than 0.05 was considered statistically signicant. 4. Results no coronary stenosis or atherosclerotic lesion and zero calcium-score below 40% area stenosis on one or more coronary vessel over 40% area stenosis or history of PCI or CABG on one coronary vessel over 40% area stenosis or history of PCI or CABG on multiple coronary vessels over 40% area stenosis on one coronary vessel plus PCI or CABG on another coronary vessel Nine patients were excluded from the study due to classification-limiting CT image quality Subject characteristics There is an increasing trend in average age, body mass index (BMI) and proportion of males: Negative<Non-significant<Single-vessel<Multi-vessel. The average age in the Multi-vessel group is significantly higher compared to the Negative group. The proportion of males is greatly higher in the CAD (Non-significant, Single-vessel, Multivessel) groups compared to the Negative group, and continues to increase in a smaller extent in the CAD groups (Table 2). The prevalence of hypertension, diabetes mellitus (DM) and smoking has a similar increasing trend: Negative<Non-significant<Single-vessel<Multi-vessel; in the Single-vessel and Multi-vessel groups all of them are significantly higher compared to the Negative group. PCI and CABG are only present in the Single-vessel and Table 2. Demographic data, Ca-score, risk factors and revascularization procedures. Neg NS SV MV N Age (years) 56.8± ± ± ±1.5 a Males (%) a 75 a 90 a BMI (kg/m 2 ) 27.6± ± ± ±1.0 Ca-score a a a,b Hypertension 66% 70% 87% a 90% a DM 6% 22% 35% a 35% a Smoking 19% 33% 43% a 45% a PCI 0% 0% 19% a,b 33% a,b CABG 0% 0% 6% 30% a,b,c a significant (p < 0.05) difference compared to Negative group b significant (p < 0.05) difference compared to Non-significant group c significant (p < 0.05) difference compared to Single-vessel group Table 3. Medications. Neg NS SV MV Anti-platelet agents: Mono anti-platelet th. 41% 61% 55% 57% Dual anti-platelet th. 0% 0% 6% 21% a,b ASA 38% 46% 58% 68% a CLP 3% 15% 10% 32% a,c Statin 31% 62% a 58% a 64% a ACEi/ARB 59% 62% 68% 79% Beta-blocker 47% 63% 61% 75% a a significant (p < 0.05) difference compared to Negative group b significant (p < 0.05) difference compared to Non-significant group c significant (p < 0.05) difference compared to Single-vessel group Multi-vessel groups, being highest in the Multi-vessel group. Ca-score also has the previously described trend (Table 2). The use of anti-platelet, statin, ACEi/ARB and betablocker medication is already considerable in the Negative group and follows an increasing trend. The prevalence of ASA, CLP, statin and beta-blocker medication in the Multi-vessel group is significantly higher compared to the Negative group. Statin use is significantly increased in all CAD groups compared to the Negative group. Dual antiplatelet therapy is only used in the Single-vessel and Multi-vessel groups. The use of all the other drugs increases with the severity of CAD (Table 3) Hemorheological parameters Hematocrit increases in a Negative<Non-significant< Single-vessel<Multi-vessel manner; in the Non-significant, Korea-Australia Rheology J., Vol. 26, No. 2 (2014) 231

4 Andras Toth et al. Fig. 1. Hematocrit. Arrows represent significant (p < 0.05) differences. Fig. 4. Red blood cell aggregation in native and adjusted (Hct = 40%) samples measured by Myrenne. Arrows represent significant (p < 0.05) differences. Fig. 2. Apparent whole blood viscosity at 90 s -1 shear rate. Arrow represents significant (p < 0.05) difference. Fig. 5. Red blood cell aggregation in native and adjusted (Hct = 40%) samples measured by Myrenne. Arrow represents significant (p < 0.05) difference. Fig. 3. Plasma viscosity at 90 s -1 shear rate. Single-vessel and Multi-vessel groups it is significantly higher compared to the Negative group (Fig. 1). Apparent whole blood viscosity shows a similar increasing tendency. WBV in Multi-vessel group is significantly higher compared to the Negative group (Fig. 2). No significant difference was found in plasma viscosity (Fig. 3). RBC aggregation increases with the severity of coronary state. The M parameter was significantly higher in the Multi-vessel group compared to the Negative group (Fig. 4). The M1 parameter has the same increasing tendency (Fig. 5). In case of adjusted samples, the M and M1 parameters show similar elevating trend which are significantly higher Fig. 6. Red blood cell deformability measured by LORCA. Arrow represents significant (p < 0.05) difference. in the Multi-vessel group compared to the Negative group (Figs. 4&5), moreover M is significantly higher in the Multivessel group compared to the Non-significant group (Fig. 5). RBC deformability shows a decreasing trend at all shear stresses. EI at 30 and Pa was significantly lower in the Non-significant group compared to the Negative group (Figs 6&7) Central laboratory parameters RBC count, Hbg concentration, MCV, MCH, and 232 Korea-Australia Rheology J., Vol. 26, No. 2 (2014)

5 Fig. 7. Red blood cell deformability measured by LORCA. Arrow represents significant (p < 0.05) difference. Table 4. Relevant laboratory parameters. Neg NS SV MV RBC count (G/l) 4.5± ± ± ±0.1 a Hbg concentration 134.7± ±2.2 a 140.5±2.3 a 145.3±2.1 a (g/l) MCV (fl) 90.5± ± ± ±0.6 a MCH (pg) 30.0± ± ± ±0.3 a MCHC (g/l) MCHC have the same rank order: Negative<Non-significant<Single-vessel<Multi-vessel; in the Multi-vessel group all parameters are significantly higher compared to the Negative group (Table 4). LDL levels are similar in all groups (Table 4). HDL has a decreasing trend and it is significantly lower in the Multi-vessel group compared to all the other ones (Table 4). Fibrinogen level has an increasing tendency in the CAD groups, although no significant difference was found (Fig. 8). 5. Discussion 331.0± ± ± ±2.1 a LDL (mmol/l) 3.1± ± ± ±0.2 HDL (mmol/l) 1.5± ± ± ±0.1 a,b,c a significant (p < 0.05) difference compared to Negative group b significant (p < 0.05) difference compared to Non-significant group c significant (p < 0.05) difference compared to Single-vessel group Under normal conditions coronary blood flow is mainly regulated by hemodynamic factors, but under certain circumstances (eg. stenosis), the importance of fluid properties increases and their alterations can more easily affect flow rate through the coronary arteries. Furthermore, as the coronary system has the narrowest capillaries in the whole human circulatory system, alterations of hemorheological parameters may impair myocardial microcirculation early. Epidemiological studies have proven that altered hemorheological parameters are primary CV risk factors. Fig. 8. Plasma fibrinogen level. Clinical studies have reported the alteration of hemorheological parameters in CAD, but not all of them were able to identify significant differences between CAD subgroups, or measured both macro- and microrheological parameters. Age, male gender, obesity, hypertension, DM and smoking are major CV risk factors; thus their increasing trend was expected. The prevalence of hypertension is already high in patients with no CAD. The frequency of hypertension increases with age; therefore taking the relatively high average age of the Negative group into consideration, it is expected to have such prevalence. The same increasing trend was observed in the prevalence of PCI and CABG procedures. These procedures are indicated in hemodynamically significant coronary stenosis, therefore they are only present in the most severe CAD groups. Generally, the amount of applied medication increases with the deteriorating coronary status. The use of antiplatelet, statin, ACEi/ARB and beta-blocker is already notable in the Negative group and increases in CAD groups. According to the latest guideline for the management of CAD, prescription of these drugs is indicated in all cases if contraindications are not present (The Task Force on the management of stable coronary artery disease of the European Society of Cardiology, 2013). Unfortunately in the CAD groups not all patients receive proper medication. These drugs are also present in the Negative group. It is likely that these patients receive these drugs due to existing hypertension (ACEi/ARB, beta-blocker) or dyslipidemia (statin). Dual antiplatelet therapy is only present in the Single-vessel and Multi-vessel groups, where PCIs were performed and dual therapy is used routinely. Hct was significantly higher in CAD compared to patients with no vessel disease which is similar to the findings of our earlier study (Kesmarky et al., 1998). However we were not able to detect significant differences between CAD groups, although the increasing tendency is well visible. Elevated Hct has also been reported by Rainer et al. (1987) and Lowe et al. (1980). On the other hand Pfafferott et al. (1999) did not find significant difference between healthy controls, patients with stable/unstable Korea-Australia Rheology J., Vol. 26, No. 2 (2014) 233

6 Andras Toth et al. angina or acute myocardial infarct, but the epidemiological studies strongly support our findings. WBV was significantly elevated in the most severe vessel state, confirming our previous result (Kesmarky et al., 1998). This result is supported by Lowe et al. (1980), Rainer et al. (1987), Lee et al. (2008). Kesmarky et al. (1998) and Lee et al. (2008) reported statistically significant differences between CAD groups, while Vosseler et al. (2012) found no significant difference between CAD and healthy controls. Increased WBV has been observed in hypertension and diabetes mellitus, therefore the elevated WBV in the Multi-vessel group may be a consequence of the increased prevalence of these comorbidities. Elevated WBV was described as a CV risk factor in the MONICA study (Koenig et al., 1998), while on the other hand it has also been proposed to be a consequence of CAD. Our study found similar PV levels in all four groups, supporting the findings of Lowe et al. (1980). On the other hand, our earlier study, Rainer et al. (1987), Pfafferott et al. (1999) and Lee et al. (2008) found significantly elevated PV in CAD. No significant difference was found in fibrinogen level, although an increasing trend is evident in CAD subgroups. Lowe et al. (1980) reported similar plasma fibrinogen levels in CAD compared to healthy controls, while Kesmarky et al. (1998) and Rainer et al. (1987) observed a significant increase. The dissimilar findings in PV and fibrinogen levels may originate from the nowadays widely used statin medication. Ten years ago in the same region Marton et al. (2003) reported only 13% statin medication in patients admitted to hospital due to acute coronary syndrome. This is two times lower compared to our Negative group and four times lower compared to the CAD groups. RBC aggregation was significantly increased in CAD and significant difference was found between CAD subgroups. Rainer et al. (1987), Pfafferott et al. (1999) and Lee et al. (2008) confirm our results. RBC deformability has a decreasing trend being significantly lower in non-significant vessel disease compared to patients with no vessel disease. Pfafferott et al. (1999) reported no significant difference of RCB filterability. RBC count, Hgb concentration, MCV, MCH and MCHC were significantly increased in severe CAD. Increase of RBC count and MCV explains the same elevating trend of Hct which is calculated by multiplying these two values in automated cell analyzers. Increased Hgb concentration, MCH and MCHC may be a counter mechanism against stenosis caused low flow rate in order to maintain oxygen delivery. LDL levels were similar in each group. Though LDL is a CV risk factor and expected to be elevated in CAD patients, the much higher use of statins in CAD groups counteracts. The decreasing HDL levels were also expected in CAD. Our results suggest that hemorheological parameters gradually change with the severity of CAD. Increased WBV elevates flow resistance the significance of which is greater in case of an existing coronary stenosis. Increased WBV and RBC aggregation may elevate shear forces at the vascular endothelium (Cokelet, 1972) resulting in impaired endothelial function. Decreased RBC deformability negatively affects microcirculation, thus myocardial oxygen supply may be reduced by both macro- and microcirculatory causes. 6. Study limitations This is a cross-sectional study; therefore inference on the relationship between the measured variables may be speculative. According to the latest American (ACCF/ AHA/ACP/AATS/PCNA/SCAI/STS, 2012) and European guidelines (The Task Force on the management of stable coronary artery disease of the European Society of Cardiology, 2013), coronary CT is indicated in medium pretest probability of CAD, while coronary X-ray angiography is performed on high risk patients. In our study this resulted in a lower number of patients with severe CAD compared to our previous study where 65% of patient had multi-vessel disease. Due to these guidelines, much fewer multi-vessel CAD cases were expected therefore 40% area stenosis was chosen as a cut-off-point. Another important difference between our studies is the distance between control and severely ill groups. We did not have healthy controls, and the most severe cases are likely to be less severe compared to the earlier study. As a consequence, the differences in severity of CAD between the groups are lesser and significant differences are harder to be observed. Another possible reason for the lack of differences between different stages of CAD is the relatively poor discriminatory power of CT: this method is good at excluding CAD, but not as good in distinguishing highgrade from non-high grade stenoses. 7. 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