Study Protocol and Study Population
The study included 120 IHD patients and 40 healthy subjects between 18-80 years of age. We selected a control group of 40 healthy subjects without overt heart disease and/or major cardiovascular risk factors (diabetes, smoking, hypertension, hypercholesterolemia, and familial history). A cardiovascular risk factors (CVRFs) score including age > 40 years, male sex, hypertension, diabetes, smoking, positive family history and hypercholesterolemia was calculated according to Vita et al.  Hypertension was defined as a history of hypertension for > 1 year that required the initiation of antihypertensive therapy by the primary physician. Smoking was defined as patients revealing a history of smoking for > two pack-years and current smoking. Positive family history was defined as documented evidence of coronary artery disease (CAD) in a parent or sibling before 60 years of age. Hypercholesterolemia was defined as fasting low-density-lipoprotein (LDL) cholesterol levels exceeding 130 mg/dl. Diabetes was defined as the need for oral antidiabetic drug therapy or insulin use.
Exclusion criteria were the presence of acutely decompensated heart failure with a New York Heart Association (NYHA) class of IV, infectious or inflammatory disease, active bleeding, surgery or trauma within two months, renal or liver dysfunction, thrombocytopenia, or anaemia, a severe comorbidity and alcohol or drug dependency, a history of other severe chronic diseases or cancer, or unwillingness to participate. The study conforms with the principles outlined in the Declaration of Helsinki and was approved by the local ethics committee. Written consent was obtained from each patient.
Coronary Angiography and Left Ventriculography
All IHD patients underwent left heart catheterization, left ventriculography and coronary angiography. Cardiac catheterization was performed according to the guidelines for coronary angiography of the American College of Cardiology and the American Heart Association . Cardiac function was determined by left ventriculography. Cardiac function was evaluated by global EF. Global Ejection Fraction (EF) was measured with Quantcor software (Siemens, Erlangen/Germany). The extent of coronary artery disease was scored by at least two independent interventional cardiologists as 0 (stenosis < 50 percent), 1 (stenosis of any main coronary artery > 50 percent), 2 (stenosis of two main coronary arteries > 50 percent), and 3 (stenosis of three main coronary arteries > 50 percent).
Frequency of CD34/45+ BM-CPCs
10 ml peripheral blood (PB) were taken from the study population to measure of BM-CPCs as well as conduct biochemical analyses. CD34/45+ BM-CPCs were quantified by flow cytometry (EPICS-XL, Beckmann Coulter) in 5 of 10 ml PB from each subject. Assessments in IHD patients (n = 120) were done during cardiac catheterization. For the control group (n = 40), measurements of CD34/45+ were performed on day 1 of admission. PB samples were analysed within two hours.
Samples were stained with fluorescein isothiacyanate (FITC) conjugate of a CD45+ antibody (clone J33, Coulter/Immunotech, Marseille/France) that detects all isoforms and glycoforms of the CD45 family, phycoerythrin (PE) conjugate of a CD34+ antibody (clone 581, Coulter/Immunotech, Marseille/France) that detects a class III epitope on all glycoforms of the CD34+ antigen. Control samples were stained with CD45+ FITC and an IgG1 PE (Coulter/Immunotech, Marseille/France) isotype.
For each patient, EDTA blood samples were labeled with CD34/45+ and IgG1/CD45. All tubes were incubated at room temperature in the dark. After incubation, mainly red blood cells were lysed with ammonium chloride and washed with phosphate-buffered saline (PBS). Samples were then stored on ice at 4°C in the dark for 20 min and analysed by flow cytometry [9, 10].
Samples were subjected to a 2D side scatter-fluorescence dot plot analysis. After appropriate gating, the concentration of BM-CPCs with low cytoplasmic granularity (low side ward scatter) was quantified and expressed as a concentration of cells per million white blood cells.
Serum creatine phosphokinase (CPK) values (normal range: 24 - 195 U/l), inflammatory markers such as C - reactive protein (CRP) (normal range < 0.5 mg/dl) leukocytes (normal range: 4 - 12 × 103/μl) and routine laboratory tests with HbA1c were measured in the remaining 5 of 10 ml PB from each study subject.
Continuous data are presented as mean ± SD. A comparison of the distributions of a continuous variable between two independent groups was performed using the two-sided nonparametric Mann-Whitney test. The type I error rate α was chosen as 5% and two-sided p-values equal or less than 0.05 were interpreted as statistically significant. Some qualitative baseline characteristics were compared using the Fisher's Exact-Test.
A bivariate regression analysis was presented in a graphical form and Pearson's correlation coefficient was obtained.
Statistical significance was accepted, if the corresponding two-sided p-value was smaller or equal to 0.05. Statistical analysis was performed with SPSS for Windows (Version 15.0).