Article

Atherosclerosis Risk in Communities (ARIC) Carotid MRI Flow Cytometry Study of Monocyte and Platelet Markers: Intraindividual Variability and Reliability

August 2008
Clinical Chemistry 54(8):1363-71

August 2008
54(8):1363-71

DOI:10.1373/clinchem.2007.102202

Source
PubMed

Authors:

Cellular markers help identify different components of a pathological process and may contribute to the diagnosis, prognostic assessment, and management of patients with suspected syndromes. Flow cytometry can be used to accurately assess markers of platelet and leukocyte activation and cellular aggregation in whole blood. To use cell markers as predictors of disease requires that they be measured reliably and show modest within-individual, day-to-day variation. We used whole blood flow cytometry to analyze monocyte and platelet markers in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI study. We estimated laboratory variability using 20 split samples, process variation using replicate blood tubes taken from 112 subjects, and biologic plus process variation using replicate blood samples taken 4-8 weeks apart from 55 people. For most analytes, the laboratory CV was <10% (mean 3.6%, range 0%-14.5%) and reliability was excellent (75% of analytes had R > 0.90). Reliability coefficients based on repeat-visit data indicated substantial to high repeatability (R > 0.60) for CD14, Toll-like receptor (TLR)-2, CD162, CD61, CD41, CD62P, CD154, and platelet-leukocyte aggregates. In contrast, TLR-4, CD45, myeloperoxidase (MPO), and cyclooxygenase (COX)-2 had slight to moderate repeat visit reliability. The high repeatability results for selected platelet and monocyte markers indicate that they can be reliably measured in multicenter studies with delayed sample processing, provided that rigorous standardization of sample collection, shipping, and flow cytometry procedures is applied.

Interactions among variants in TXA2R, P2Y12 and GPIIIa are associated with carotid plaque vulnerability in Chinese population

Article

Full-text available

Apr 2018

Purpose The associations between variants in platelet activation-relevant genes and carotid plaque vulnerability are not fully understood. The aim of the present study was to investigate the associations of the variants in platelet activation-relevant genes and interactions among these variants with carotid plaque vulnerability. Results There were no significant differences in the frequencies of genotypes of the 11 variants between patients and controls. Among 396 patients, 102 patients had not carotid plaque, 106 had VP, and 188 had SP. The 11 variants were not independently associated with risk of carotid plaque vulnerability after adjusting for potential confounding variables. However, the GMDR analysis showed that there were synergistic effects of gene-gene interactions among TXA2Rr s1131882, GPIIIa rs2317676 and P2Y12 rs16863323 on carotid plaque vulnerability. The high-risk interactions among the three variants were associated with high platelet activation, and independently associated with the risk of carotid plaque vulnerability. Methods Eleven variants in platelet activation-relevant genes were examined using mass spectrometry methods in 396 ischemic stroke patients and 291controls. Platelet-leukocyte aggregates and platelet aggregation were also measured. Carotid plaques were assessed by B-mode ultrasound. According to the results of ultrasound, the patients were stratified into three groups: non-plaque group, vulnerable plaque (VP) group and stable plaque (SP) group. Furthermore, gene-gene interactions were analyzed using generalized multifactor dimensionality reduction (GMDR) methods. Conclusions The rs1131882, rs2317676, and rs16863323 three-loci interactions may confer a higher risk of carotid plaque vulnerability, and might be potential markers for plaque instability.

Precision and Reliability of 5 Platelet Function Tests in Healthy Volunteers and Donors on Daily Antiplatelet Agent Therapy

Article

Full-text available

Oct 2014

Background: Anticoagulation protocols used during mechanical circulatory support call for titration of antiplatelet agents. We compared the precision and reliability of 5 platelet function tests in healthy volunteers and donors on daily antiplatelet therapy to distinguish their efficacy for titrating antiplatelet therapy. Methods: We assessed arachidonic acid-induced platelet function by light transmission aggregometry (LTA), Multiplate impedance aggregometry, VerifyNow, and platelet mapping by thromboelastography (TEG PM). We assessed ADP-induced platelet function by the same methods and flow cytometry. Forty healthy volunteers and 10-13 volunteers on daily aspirin and/or clopidogrel therapy were evaluated. We compared tests for intraassay precision, interassay precision (samples from 2 separate blood draws), and reliability coefficient. Results: For arachidonic acid-induced platelet aggregation in healthy volunteers, intra- and interassay CVs were ≤ 10% for all methods. Intra- and interassay precision among donors on daily aspirin was ≤ 30% for all methods except LTA (38% interassay CV) and TEG PM (95% intraassay and 104% interassay CV). For ADP-induced platelet function, intra- and interassay precision was ≤ 10% and ≤ 30% for all methods. Only Multiplate demonstrated moderate or greater (R > 0.40) reliability coefficients for arachidonic acid-induced platelet function among all subjects. All methods of ADP-induced platelet function, except TEG PM, demonstrated substantial or greater (R > 0.60) reliability among all subjects. Conclusions: TEG PM is least suited to monitor effects of antiplatelet agents. Multiplate impedance aggregometry was the only method to demonstrate an acceptable reliability coefficient among healthy volunteers and donors on both aspirin and clopidogrel therapy.

Risk factor correlates of platelet and leukocyte markers assessed by flow cytometry in a population-based sample

Article

Nov 2008

Platelet and leukocyte products are involved in atherothrombosis. However, the determinants of platelet and leukocyte markers assessed by flow cytometry have not been documented in a population-based sample. We performed flow cytometry on blood from participants (n=1894) in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI Study. Cellular aggregates and multiple platelet and leukocyte markers, such as myeloperoxidase in granulocytes and toll-like receptor-4, CD14, and CD45 in monocytes, were quantified. Their cross-sectional associations with demographic and risk factors were assessed using multiple linear regression. Mean values of most cellular markers and aggregates were considerably higher in blacks than whites (p<0.01). There were some differences in cellular markers between men and women, but little association with age. LDL-cholesterol was associated positively with several markers (toll-like receptor-4 and myeloperoxidase in granulocytes and CD162 in lymphocytes). Cholesterol-lowering therapy tended to show opposite associations. Smokers had much higher granulocyte myeloperoxidase than nonsmokers. However, most other correlations between risk factors and cellular markers were nonsignificant. Race/ethnicity, sex, and to a lesser degree LDL-cholesterol and cholesterol-lowering therapy, but few other risk factors, were correlated with markers of cellular activation in this population-based study.

Association of monocyte myeloperoxidase with incident cardiovascular disease: The Atherosclerosis Risk in Communities Study

Article

Full-text available

Oct 2018
PLOS ONE

Myeloperoxidase (MPO) is a heme-containing peroxidase found in azurophilic granules of neutrophils and monocytes. Epidemiological studies have reported greater plasma MPO concentration to be associated with increased incidence of several cardiovascular diseases (CVD), but the association of intracellular monocyte MPO (mMPO) with CVD is unclear. The prospective population-based Atherosclerosis Risk in Communities (ARIC) cohort study measured mMPO using flow cytometry in 1,465 participants. The association of mMPO with incident cardiovascular disease (CVD, comprising incident coronary heart disease (CHD), heart failure, stroke, peripheral artery disease, and cardiovascular mortality) was examined over a median 9.6 years of follow-up (n = 290 CVD events). There was no statistically significant association between mMPO and all incident CVD events in either age, sex, and race-adjusted proportional hazards models (HR (95% CI) across tertiles of mMPO: 1, 1.09 (0.76, 1.57), and 0.78 (0.52, 1.15), P-trend = 0.21) or adjusted for other major CVD risk factors (HR (95% CI): 1, 1.17 (0.81, 1.69), and 0.87 (0.58, 1.29), P-trend = 0.50). There also was no association between mMPO tertiles and incident CHD, heart failure, or all-cause mortality, examined separately. In conclusion, intracellular monocyte myeloperoxidase was not associated with incident cardiovascular disease in this prospective population-based study.

Genetic variants in TLR2 and TLR4 are associated with markers of monocyte activation: the Atherosclerosis Risk in Communities MRI Study

Article

Full-text available

Feb 2011
HUM GENET

Markers of monocyte activation play a critical role in atherosclerosis, but little is known about the genetic influences on cellular levels. Therefore, we investigated the influence of genetic variants in monocyte differentiation antigen (CD14), toll-like receptor-4 (TLR4), toll-like receptor-2 (TLR2), and myeloperoxidase (MPO) on monocyte surface receptor levels. The study sample consisted of 1,817 members of a biracial cohort of adults from the Atherosclerosis Risk in Communities Carotid MRI Study. Monocyte receptors were measured using flow cytometry on fasting whole blood samples. TLR2 rs1816702 genotype was significantly associated with CD14+/TLR2+ percent of positive cells (%) and median fluorescence intensity (MFI) in whites but not in blacks (p < 0.001). Specifically, the presence of the minor T-allele was associated with increased receptor levels. In blacks, TLR4 rs5030719 was significantly associated with CD14+/TLR4+ monocytes (MFI) with mean ± SE intensities of 16.7 ± 0.05 and 16.0 ± 0.14 for GG and GT/TT genotypes, respectively (p < 0.001). Variants in TLR2 and TLR4 were associated with monocyte receptor levels of TLR2 and TLR4, respectively, in a biracial cohort of adults. To our knowledge, this is the first study to look at associations between variants in the toll-like receptor family and toll-like receptor levels on monocytes.

Novel Scoring System for Identifying Impending Complete Fractures in Incomplete Atypical Femoral Fractures

Article

Full-text available

Nov 2016
J CLIN ENDOCR METAB

Context: Although impending incomplete atypical femoral fractures (AFFs) require prophylactic fixation, there is still a lack of study on predicting complete fracture among the incomplete AFFs. Objective: Our purposes are (1) to develop a scoring system to predict progression into complete fracture and (2) to evaluate its reliability and validity. Design, setting, and patients: We reviewed 46 incomplete AFFs in 44 patients, who did not undergo prophylactic fixation. A weighted scoring system, including four identified risk factors; the site, severity of pain, status of the contralateral femur, and the extent of radiolucent line, was developed. We evaluated its inter-observer reliability by using Intraclass Correlation Coefficiency (ICC), and its accuracy using Receiver Operator Characteristic (ROC) curve. The validity of the scoring system was tested in a different cohort. Intervention: Observational study Main Outcome Measure: Progression to complete fracture within 6 months Results: Among 46 incomplete fractures, 13 developed a complete fracture within 6 months. The probability of complete fracture increased abruptly when the score was 8 points or more. The proposed scoring system showed an almost perfect reliability (ICC, 0.997; 95% CI, 0.995 to 0.998), and higher accuracy than any single risk factor in ROC curve. In the different series, the positive predictive value was 100% and the sensitivity was 75%, when cut-off value was 8 points. Conclusion: The progression to complete fracture could be predicted by using our scoring system. Incomplete AFF with scores < 8 points can be treated conservatively, while lesions with scores ≥ 8 require prophylactic fixation.

Comprehensive Proteome Profiling of Platelet Identified a Protein Profile Predictive of Responses to An Antiplatelet Agent Sarpogrelate

Article

Full-text available

Sep 2016

Sarpogrelate is an antiplatelet agent widely used to treat arterial occlusive diseases. Evaluation of platelet aggregation is essential to monitor therapeutic effects of sarpogrelate. Currently, no molecular signatures are available to evaluate platelet aggregation. Here, we performed comprehensive proteome profiling of platelets collected from 14 subjects before and after sarpogrelate administration using LC-MS/MS analysis coupled with extensive fractionation. Of 5,423 proteins detected, we identified 499 proteins affected by sarpogrelate and found that they strongly represented cellular processes related to platelet activation and aggregation, including cell activation, coagulation, and vesicle-mediated transports. Based on the network model of the proteins involved in these processes, we selected three proteins (cut-like homeobox 1; coagulation factor XIII, B polypeptide; and peptidylprolyl isomerase D) that reflect the platelet aggregation-related processes after confirming their alterations by sarpogrelate in independent samples using Western blotting. Our proteomic approach provided a protein profile predictive of therapeutic effects of sarpogrelate.

Medium-Term Variability of the Human Serum Metabolome in the Atherosclerosis Risk in Communities (ARIC) Study

Article

Full-text available

Jun 2014

Abstract Metabolomics is a systems biology tool providing small molecule signatures of disease etiology. In order to estimate the biologic variability of the human serum metabolome, this study calculated intraclass correlation coefficients (ICCs) for 178 stably-detected metabolites measured by untargeted chromatography/mass spectrometry. We studied a subsample of 60 participants (57% males, 70% Caucasians, aged 73.77±5.3 years) in the Atherosclerosis Risk in Communities (ARIC) Study who provided two fasting serum samples 4-6 weeks apart. The median ICC across all metabolites was 0.60, and 82% of metabolites had at least fair variability (i.e., ICC>= 0.40). There was variation in the medium-term variability among metabolites, with those in the pathways of amino acid and lipid metabolism showing relatively high ICCs, and those in the carbohydrate pathway showing relatively low ICCs. The results of this study provide a valuable resource for future study design and outcome interpretation of mass spectrometry-based metabolomic studies in epidemiology.

About CD45(-)/CD31(+)/CD105(+) Circulating Cells in Patients with Gynecologic Malignancies-Response

Article

Mar 2014

Monocyte and T-Cell Responses to Exercise Training in Elderly Subjects

Article

Jun 2011
J STRENGTH COND RES

The purpose of this study was to examine the effects of exercise training on age-related impairment of immune parameters related to T-cell activation in elderly individuals. Twenty-four elderly subjects were assigned to an exercise training group (EXC: 3 men, 9 women; age 61-76 years) or a nonexercise control group (CON: 4 men, 8 women; age 62-79 years). Subjects in EXC participated in exercise sessions 2 d·wk(-1) for 12 weeks. The training session included stretching and endurance exercise (10 minutes), resistance training comprised leg extension, leg press, hip abduction, and hip adduction using exercise machine and each subject's body weight. Subjects in CON maintained their normal physical activity levels during the study period. Blood samples were collected before and after the training period. Samples were measured for the numbers of leukocytes, lymphocytes, and monocytes, and for CD3(+), CD4(+), CD8(+), CD28(+)CD4(+), CD28(+)CD8(+), TRL-4(+)CD14(+), and CD80(+)CD14(+) cells. The number of leukocytes, lymphocytes, monocytes, CD3(+), CD4(+), and CD8(+) cells did not change after 12 weeks in either EXC or CON. The number of CD28(+)CD8(+) cells increased significantly after training in EXC (p ≤ 0.05), although CON showed no significant change. In the EXC group, CD80(+)CD14(+) cell counts were significantly higher after training (p ≤ 0.05), but the TLR-4(+)CD14(+) cell counts were unchanged. In the CON group, no significant alteration existed in TLR-4(+)CD14(+) and CD80(+)CD14(+) cell numbers. In conclusion, exercise training in elderly people is associated with increased CD28-expressing Tc cells and CD80-expressing monocytes. Therefore, exercise training might upregulate monocyte and T-cell-mediated immunity in elderly people.

Sources of Variability in Measurements of Cardiac Troponin T in a Community-Based Sample: The Atherosclerosis Risk in Communities Study

Article

Jun 2011
CLIN CHEM

Application of cardiac troponin T (cTnT) as a marker of myocyte damage requires knowledge of its measurement variability. Using a highly sensitive assay for measurement, we evaluated the long-term storage stability of plasma cTnT at -70 °C and the sources of cTnT variability. Samples from the Atherosclerosis Risk in Communities study collected in 1996-1998 and 2005-2006 were assayed centrally to quantify variability in cTnT attributable to processing (replicates from same blood draw, n = 87), laboratory (replicates after freeze thaw, n = 29), short-term (n = 40) and long-term biological variation (repeat visit, n = 38), and degradation in frozen storage (n = 7677). Approximately 30% of this population-based cohort had cTnT concentrations below the detection limit (3 ng/L). Reliability coefficients for all paired comparisons exceeded 0.93 except for samples drawn 8 years apart (r = 0.36). Sources of cTnT variation (as CVs) were: laboratory, 2.1% and 11.2% in those with and without heart failure, respectively; processing, 18.3%; biological, 16.6% at 6 weeks and 48.4% at 8 years. The reference change value at 6 weeks (68.5%) indicated that 4 samples are needed to determine a homeostatic set point within ±25%. The estimated cTnT degradation rate over the first year in long-term frozen storage was 0.36 ng/L per year. cTnT was detectable in approximately 70% of community-dwelling middle-aged study participants and stable in -70 °C storage. The variability in cTnT attributable to 1 freeze-thaw cycle is of small magnitude. The observed high laboratory and intraindividual (biological) reliability of cTnT support its use for population-based research, and in clinical settings that rely on classification and serial measurements.

Influences of Weight Loss on Monocytes and T-Cell Subpopulations in Male Judo Athletes

Article

Apr 2011
J STRENGTH COND RES

The purpose of this study was to examine weight loss effects on immune function in judo athletes. Six elite male Japanese judo athletes (20.3 ± 0.4 years) were enrolled in this study. They completed usual weight loss programs during 2 weeks preceding an actual competition. Subjects noted the appearance of upper-respiratory tract infection (URTI) symptoms during the study period. Blood samples were obtained at 40 (baseline period: BL) and 3 (weight loss period: WL) days before and 1 day after the competition (AC). The CD3, CD4, CD8, CD56CD3, CD28CD4, CD28CD8, and Toll-like-receptor-4 (TLR-4) CD14 cells were counted by using flow cytometer analysis. The 6 subjects reported 1 headache, 3 runny nose conditions, and 1 coughing instance during the WL. The CD3, CD4, CD8, and CD28CD4 cell counts were significantly lower at WL than at BL (p ≤ 0.05); they reverted to the baseline value at AC. The TLR-4CD14 cells were significantly fewer at WL (p ≤ 0.05); they remained fewer than they had been at BL, even at AC. These results suggest that 2 weeks of weight loss before a competition can impair cell-mediated immune function and induce high susceptibility to URTI in judo athletes. Coaches, support staff, and athletes should monitor athletes' weight loss, hydration status, appearance of URTI symptoms, and immunocompetence such as lymphocytes and monocytes to prevent the physical condition from becoming worse.

Decline in platelet microparticles contributes to reduced hemostatic potential of stored plasma

Article

Mar 2011

In an effort to administer life-saving transfusions quickly, some trauma centers maintain thawed plasma (TP). According to AABB, TP is approved for transfusion for up to five days when stored at 1-6° C. However, the alterations in microparticles (MP) contained in the plasma, which are an integral component of plasma's hemostatic capacity, are not well characterized. We report on MP changes in TP between its initial thaw (FFP-0) and five days (FFP-5) of storage. FFP units (n=30) were thawed at 37° C and kept refrigerated for five days. Phenotypes of residual cells, which include platelets, erythrocytes, leukocytes, monocytes, endothelial cells, and MP counterparts of each cell type, were analyzed by flow cytometry. Functional assays were used for MP procoagulant activity, plasma thrombin generation, and clotting properties (thromboelastography). In FFP-0 the majority (94%) of residual cells were platelets, along with significant levels of platelet MPs (4408 × 10(3)/L). FFP-5 showed a decline in MP count by 50% (p<0.0001), and procoagulant activity by 29% (p<0.0001). FFP-5 exhibited only 54% (p<0.0001) of the potential for thrombin generation as FFP-0, while thromboelastography indicated a slower clotting response (p<0.0001) and a longer delay in reaching maximum clot (p<0.01). Removal of MP by filtration resulted in reduced thrombin generation, while the MP replacement restored it. Decline in MP with storage contributes to FFP-5's reduced ability to provide the hemostatic potential exhibited by FFP-0, suggesting the presence of platelet MPs in freshly TP may be beneficial and protective in the initial treatment of hemorrhage.

The ARIC Carotid MRI Study of Blood Cellular Markers: An Inverse Association of Monocyte Myeloperoxidase Content With Peripheral Arterial Disease

Article

Apr 2011
Angiology

We evaluated the association of blood monocyte and platelet activation markers with the risk of peripheral artery disease (PAD) in a multicenter study of atherosclerosis among African American and Caucasian patients. Flow cytometric analysis of blood cells was performed in 1791 participants (209 cases with PAD and 1582 noncases) from the cross-sectional Atherosclerosis Risk in Communities Carotid Magnetic Resonance Imaging ([MRI] ARIC Carotid MRI) Study to assess platelet glycoproteins IIb and IIIa, P-selectin, CD40 ligand, platelet-leukocyte aggregates, monocyte lipopolysaccharide receptor, toll-like receptors (TLRs) 2 and 4, P-selectin glycoprotein ligand 1, cyclooxygenase 2, and myeloperoxidase. Multivariate regression analyses evaluated the association of cellular markers with the risk of PAD. After adjusting for age, race, and gender, platelet CD40L, and monocyte myeloperoxidase (mMPO) levels were significantly lower (P < .001), and monocyte TLR-4 levels were higher (P = .03) in patients with PAD. With additional adjustments for conventional risk factors, mMPO remained inversely and independently associated with the risk of PAD (odds ratio [OR]: 0.35, P = .01).

Association of the platelet GPIIb/IIIa polymorphism with atherosclerotic plaque morphology: The Atherosclerosis Risk in Communities (ARIC) Study

Article

Feb 2011

Platelet activation and aggregation play an important role in the pathogenesis of cardiovascular disease. We examined the association of a single nucleotide polymorphism (SNP) in the GPIIIa platelet glycoprotein (Leu33Pro) with carotid artery plaque morphology and with expression of platelet markers using data from the Atherosclerosis Risk in Communities (ARIC) Carotid MRI study. The study sample consisted of 1202 Caucasian members of the ARIC study cohort recruited in 2004-2005 to participate in the Carotid MRI Substudy under stratified sampling based on maximum carotid artery wall thickness. The Leu33Pro polymorphism was identified as SNP rs5918 in the ITGB3 gene. Plaque visualization was accomplished with contrast enhanced MRI examination of the thickest segment of the carotid artery. Expression of platelet markers was measured using fasting whole blood flow cytometry. This cross-sectional analysis based on age and gender adjusted weighted linear regression models suggests that those homozygous for the Leu33Pro risk allele (C) have decreased mean and minimum fibrous cap thickness. We did not observe differences in plaque lipid volume or maximum carotid artery wall thickness across SNP rs5918 genotypes. Carriers of the Leu33Pro polymorphism, as compared to major allele homozygotes, had greater percent of platelets expressing P-selectin, a platelet glycoprotein indicating activation status. Prevalent coronary heart disease did not affect estimates of fibrous cap thickness or of platelet activation. Our results suggest that individuals with Leu33Pro polymorphism of the GPIIIa glycoprotein may be predisposed to increased risk of atherosclerotic plaque rupture.

Correlation between leukocyte‐platelet aggregates and thrombosis in myeloproliferative neoplasms

Article

Nov 2021

Introduction: The impact of activated blood and endothelial cells on the thrombosis in myeloproliferative neoplasms (MPN) has not yet been clarified. We prospectively analyzed correlation between circulating leukocyte-platelet aggregates and soluble selectins to thrombosis occurrence in MPN, in the context of standard and cardiovascular risk factors, and different clinical and biological characteristics. Methods: Flow cytometric analysis of neutrophil-platelet (Neu-Plt) and monocyte-platelet (Mo-Plt) aggregates in peripheral blood, as well as quantification of soluble E-/L-/P-selectins by enzyme immunoassay, was performed on 95 newly diagnosed MPN patients. Results: During the follow-up, thrombosis occurred in 12.6% MPN patients (arterial 9.4%, venous 3.2%), with a mean time of 39 months. The overall incidence rate of main thrombotic events was 4.36 per 100 patient-years. The incidence of arterial hypertension (HTA) was significantly higher in patients with thrombosis, compared to those without thrombosis (P < .05). The level of soluble P-selectin was significantly higher in patients with thrombosis compared to those without thrombosis (346.89 ng/mL vs 286.39 ng/mL, P = .034). The mean level of Neu-Plt (26.7% vs 22.4%) and Mo-Plt (17.8% vs 12.3%) aggregates did not differ significantly between the groups with and without thrombosis. A multivariate COX proportional hazard regression model confirmed an independent predictive significance of Mo-Plt aggregates (HR = 1.561, 95% CI: 1.007-2.420, P = .046), as well as the cumulative effect of Mo-Plt aggregates and HTA (HR = 1.975, 95%CI: 1.215-3.212, P = .006) for thrombosis occurrence. Conclusion: Monocyte-platelet aggregates represent an independent risk factor for thrombosis occurrence, further on supported by HTA.

Estimating Short- and Long-term Reference Change Values and Index of Individuality for Tests of Platelet Function

Article

Oct 2019

Background: In order to manage risks of bleeding and thrombosis after some surgical procedures, platelet function is often measured repeatedly over days or weeks using laboratory tests of platelet function. To interpret test results in the perioperative period, it is necessary to understand analytical, biological and between-person variation. Methods: We collected three separate blood specimens from 16 healthy volunteers on the first study day, and one additional specimen from each volunteer 1, 2, and 3 months later. Arachidonic acid-induced and adenosine diphosphate (ADP)-induced platelet function were measured in duplicate by whole blood impedance aggregometry using Multiplate (ASPI/ADP tests) and VerifyNow (Aspirin Reaction Units [ARU] and P2Y12 Reaction Units [PRU]). The analytical variation (CVA), within-subject variation (CVI), between-subject variation (CVG), index of individuality (II), and reference change values (RCV) were calculated. Results: VerifyNow ARU demonstrated the smallest short-term and long-term variability (CVA, CVI, and CVG ∼1%), resulting in short- and long-term RCV values <5%. II was also higher (1.92) for VerifyNow ARU than other platelet function tests. Multiplate ASPI and ADP tests had the highest RCV both short-(19.0% and 25.2%, respectively) and long-term (32.1% and 39.6%, respectively) due to increased CVA (>5%) and CVI (3.9-13.1%). VerifyNow PRU had a lower RCV than Multiplate ADP; but was the only test with II <0.6. Conclusions: VerifyNow ARU results can be interpreted relative to a fixed cut-off or population-based reference interval; or relative to small changes in an individual's previous values. VerifyNow PRU and Multiplate ASPI and ADP tests should only be interpreted based upon relative change; and can only distinguish relatively large (>23%) changes over several weeks.

Effects of n-3 Fatty Acid Treatment on Monocyte Phenotypes in Humans with Hypertriglyceridemia

Article

Aug 2017

Background: Hypertriglyceridemia increases risk for atherosclerotic cardiovascular disease and may contribute to atherosclerosis by changing circulating monocyte phenotypes. High-dose n-3 polyunsaturated fatty acids reduce blood triglyceride levels. Effects of triglyceride-lowering therapy on monocyte phenotypes are not well known. Objective: We examined effects of n-3 polyunsaturated fatty acid treatments (eicosapentaenoic acid [EPA] plus docosapentaenoic acid [MAT9001] vs EPA ethyl esters [EPA-EE]) on monocyte phenotypes in individuals with hypertriglyceridemia. Methods: Individuals with triglycerides 200 to 400 mg/dL were recruited. Subjects received 2 treatments in randomized order for 14 days each: MAT9001 and EPA-EE, at 4 g/d. At 2 days before the start of, and on the last day of, each treatment, nile red staining for lipids and phenotypes of each monocyte subset were examined by flow cytometry after an overnight fast and postprandially after a high-fat meal. Results: Treatment with MAT9001 or EPA-EE reduced fasting triglyceride levels and decreased proportions of intermediate monocytes. Only MAT9001 decreased postprandial blood triglyceride levels, lowered fasting nile red levels, indicating less lipid in classical and intermediate monocytes, and reduced postprandial CD11c levels on nonclassical monocytes. MAT9001 and EPA-EE each reduced fasting and postprandial CD11c and CD36 levels on classical and intermediate monocytes and postprandial CCR5 levels on intermediate and nonclassical monocytes, with no significant differences between the 2 treatments. Conclusions: Treatment with MAT9001 in individuals with hypertriglyceridemia reduced fasting nile red staining for lipids in classical and intermediate monocytes. MAT9001 and EPA-EE each improved fasting and postprandial monocyte phenotypes, which could potentially help to protect against atherosclerosis.

Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis

Article

Full-text available

Aug 2016

Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis.

A single controlled exposure to secondhand smoke may not alter thrombogenesis or trigger platelet activation

Article

May 2016

Introduction: Chronic secondhand smoke (SHS) exposure increases cardiovascular events, particularly acute thrombotic events. There are little human data on acute SHS exposure. The aim of this study was to determine whether a single controlled exposure of humans to SHS increased thrombogenesis. Methods: After 6-8 hours fast, subjects (n = 50) were exposed to constant dose SHS (particulate level of 500 μg/m3) for 120 minutes in a temperature-regulated and ventilated, simulated bar environment. Blood was drawn before and immediately after SHS exposure for thromboelastography (TEG) and flow cytometry. Maximum clot strength (MA) was measured using TEG and platelet leukocyte aggregates (LPA) were measured as an index of platelet activation. Anti-CD 14 antibodies were used as leukocyte markers and anti-CD 41 antibodies as platelet markers for cytometry. Data were analyzed using students' t test for paired samples. Results: There was no effect of acute exposure to SHS on platelet activation or thrombogenesis. Also, intra group (smokers [n = 19] and nonsmokers [n = 31]) comparisons of LPA and TEG parameters did not show changes with SHS exposure. Conclusions: While there are abundant data showing enhanced thrombogenesis and platelet activation following repeated exposure to SHS, our study suggests that a single exposure does not appear to significantly alter thrombin kinetics nor result in platelet activation. The effects of SHS on thrombogenesis might be nonlinear.

Monocytes in metabolic disorders - Opportunities for flow cytometry contributions

Article

Full-text available

Mar 2010

Chronic inflammation has arised as a major underlying cause of atherosclerosis, obesity and diabetes. It is mediated by cells of innate immune system like macrophages but also by their antecedents, circulating monocytes. Roles of monocyte subsets and different markers of monocyte activation in the context of metabolic disorders have been reviewed. Applying cell based approach through flow cytometry in this field has resulted with new understanding of pathophysiologic mechanisms. Possible implications of these insights in diagnosis, prognosis and revealing of therapeutic targets in metabolic disorders remain a challenge for future.

Preliminary Findings of Morphometric Analysis of Ankle Joint in Korean Population

Article

Feb 2014

The clinical success of ankle joint arthroplasty depends on the availability of information on the morphology of the relevant bones. Thus, the implant design and surgical technique should be adjusted to the ankle morphology. However, few reports have described the characteristics of ankle morphometry in Korean populations. The present study evaluated the characteristics of ankle morphometry in a Korean population sample. Weightbearing ankle radiographs of 100 Korean patients were retrieved, and 13 representative indexes were measured after establishing the reliability of the measurements. Ankle morphometry was analyzed in terms of (1) size diversity, (2) aspect (anteroposterior/mediolateral) ratio, (3) distal anteroposterior inclination angle, and (4) complication-related anatomy. The measurements were compared with those of previous studies of white populations. In terms of size diversity, the ankle morphometry in Koreans was smaller in all parameters, except for the talar width. Koreans had a different aspect ratio than whites. The increase in the distal anteroposterior inclination angle was statistically significant (p < .001), and complication-related indexes were also significantly increased. In addition to the smaller dimensions in Korean populations, surgeons should be aware of the characteristics of Korean patients, such as the steep slope and vulnerability to iatrogenic malleolar fractures.

Mean platelet volume predicting carotid atherosclerosis in atherothrombotic ischemic stroke

Article

Sep 2011
IRISH J MED SCI

Platelets are involved in atherosclerosis. Mean platelet volume (MPV) could be a marker of platelet activation. We aim to determine whether MPV levels were correlated with the presence of atherosclerotic disease in carotid arteries of patients with stroke. We recruited 215 patients with atherothrombotic stroke. All the participants underwent ultrasonographic evaluation of their extracranial carotid arteries. MPV was measured in automated hematology analysis system. The subjects were divided according to plaques and severity of carotid stenosis. Univariate and multivariate statistical analyses and a ROC curve to predict carotid stenosis were performed. Univariate analysis showed a positive relationship between MPV and the degree of carotid atherosclerosis (p < 0.00007), and with carotid intima to media thickness (p < 0.00002). In ROC curve, a MPV cut-off of 11.25 fl was obtained for a sensibility of 70% and a specificity of 71% (p < 0.02). Multivariate analysis showed significant correlation with severity of carotid stenosis, when MPV was higher than 11.25 fl (OR: 2.9, p < 0.00007). Our results indicate that an elevated MPV could be an easily measurable marker of severity of carotid stenosis in patients with atherothrombotic stroke.

Dose-dependent decrease of platelet activation and tissue factor by omega-3 polyunsaturated fatty acids in patients with advanced chronic heart failure

Article

Full-text available

Jul 2011

Chronic heart failure (CHF) is characterised by activation of neuroendocrine and inflammatory pathways, and both are linked to a prothrombotic state. Treatment with omega-3 polyunsaturated fatty acids (n3-PUFA) showed significant benefits including mortality reduction in CHF, but exact mechanisms of action are still unclear. We investigated the effects of n3-PUFA on markers of platelet activation and thrombogenesis in patients with severe CHF. Thirty-six patients with non-ischaemic CHF (LVEF<35%, NYHA class>2) under optimised therapy were randomised to supplementation with 1g/day or 4g/day n3-PUFA, or placebo for 12 weeks. Using whole-blood flow cytometry, monocyteplatelet aggregates characterised by CD14+/CD42b+ co-expression and monocytic tissue factor (TF) were determined. Plasma levels of P-selectin, sCD40L, fibrinogen, prothrombin fragment F1.2, TF and proinflammatory markers (high sensitive[hs] interleukin-6, hsCRP, hsTNFalpha, monocyte chemotactic protein-1) were measured by immunoassay. Supplementation with 1g/day and 4g/day n3-PUFA but not placebo significantly reduced monocyte-platelet aggregates in a dose-dependent manner (p for trend=0.02 across the groups). A dose of 4g/day but not 1g/day n3-PUFA significantly decreased P-selectin (p=0.03). Plasma TF decreased dose-dependently upon n3-PUFA supplementation (p for trend=0.02), paralleled by a significant decrease of TF+-monocytes (p for trend=0.01). The amount of 4g/day n3-PUFA exhibited modest anti-inflammatory effects with a significant reduction of hs interleukin-6 (p<0.01) and a trend-wise reduction of hsTNF-alpha (p=0.09). No changes were seen for sCD40L, fibrinogen, hsCRP and monocyte chemotactic protein-1, while F1.2 was decreased by 4g/day n3-PUFA (P=0.03). In patients with severe non-ischaemic CHF, treatment with n3-PUFA leads to a dose-dependent decrease of platelet activation and TF. Higher dosage exhibits also anti-inflammatory effects. * ClinicalTrials.gov registration number: NCT00149409

Association of Blood Monocyte and Platelet Markers with Carotid Artery Characteristics: The Atherosclerosis Risk in Communities Carotid MRI Study

Article

May 2011
CEREBROVASC DIS

Atherosclerosis is characterized by infiltration of inflammatory cells from circulating blood. Blood cell activation could play an important role in plaque formation. We analyzed the relationship between blood cellular markers and quantitative measures of carotid wall components in 1,546 participants from the ARIC (Atherosclerosis Risk in Communities) Carotid MRI Study. Carotid imaging was performed using a gadolinium contrast-enhanced MRI and cellular phenotyping by flow cytometry. Monocyte Toll-like receptor (TLR)-2 is associated with larger plaques, while CD14, myeloperoxidase, and TLR-4 associate with smaller. Platelet CD40L is associated with smaller plaques and thinner caps, while P-selectin is associated with smaller core size. Blood cell activation is significantly associated with atherosclerotic changes of the carotid wall.

Measuring acetabular dysplasia in plain radiographs

Article

Mar 2011
ARCH ORTHOP TRAUM SU

Several radiologic parameters have been used to qualify an acetabular coverage in studies determining whether an association exists between acetabular dysplasia and osteoarthritis of hip. However, it is not known which parameter is optimum for these epidemiologic studies. We evaluate the reliability, validity, and robustness of the radiologic parameters of acetabular coverage used in these studies. Center-edge angle (CEA), acetabular depth (AD), acetabular angle (AA), acetabular roof obliquity (ARO), and roof angle (RA) were evaluated. The components of intra- and interobserver reliability were tested. The correlations between each parameter were used to depict convergent validity. The robustness of the parameters to different projection (urogram), different definitions of the lateral acetabular margin, and a differing pelvic tilt were evaluated. The intra- and interobserver reliabilities of CEA, AD and AA ranged from 0.777 to 0.925. The CEA, AD and AA showed acceptable validity in the correlation. The AD on the urograms was 22.0% higher than those on the standing hip radiographs (P < 0.001). When the osteophyte was included in the definition of lateral acetabular margin, the CEA and AD increased significantly (P < 0.001). In simulating pelvic tilting, the AD increased significantly with the anterior pelvic tilt (P < 0.001). The ARO and RA showed poor clinical relevance. When measuring acetabular dysplasia, the AD is unsuitable for use, because it is not robust to different projection of beam and different pelvic tilts. Furthermore, one should consider that the CEA and AA are significantly influenced by different definitions of lateral acetabular margin.

Associations between dietary patterns and flow cytometry-measured biomarkers of inflammation and cellular activation in the Atherosclerosis Risk in Communities (ARIC) Carotid Artery MRI Study

Article

Sep 2010

Specific foods and overall dietary patterns are associated with soluble biomarkers of systemic inflammation and endothelial activation. However, no large epidemiological studies have evaluated relationships between such dietary factors and cell-specific markers of activation and inflammation as measured by flow cytometry. Cell aggregates and multiple platelet and leukocyte markers were quantified by flow cytometry in fresh whole blood from 1101 white adults participating in the Carotid Artery MRI Study, a subset of the larger Atherosclerosis Risk in Communities (ARIC) Study. Two dietary patterns ("Healthy" and "Western") were empirically derived via principal components analysis using data collected by food frequency questionnaire. Cross-sectional associations between dietary patterns and flow cytometry-measured biomarkers were evaluated, adjusting for demographics and lifestyle factors, including medications use. After multivariable adjustment, monocyte lipopolysaccharide receptor (CD14), monocyte toll-like receptor-2, and platelet glycoprotein IIb (CD41) showed inverse associations with the Healthy dietary pattern (p=0.01, 0.04, and 0.01, respectively). In contrast, the Western dietary pattern was positively associated with CD41 and platelet-granulocyte aggregates (p=0.01 and 0.04, respectively). Independent of other dietary factors, alcohol consumption was inversely associated with levels of pan-leukocyte marker (CD45), P-selectin (CD62P) on PLA1 and on PLA2 platelets, and platelet-monocyte, platelet-granulocyte, and platelet-lymphocyte aggregates. Dietary patterns and alcohol intake were each cross-sectionally associated with select markers of cellular activation and inflammation measured by flow cytometry. These data are consistent with the hypothesis that holistic measures of dietary intake are associated with inflammation.

Large-scale genomic studies reveal central role of ABO in sP-selectin and sICAM-1 levels

Article

Full-text available

Feb 2010
HUM MOL GENET

P-selectin and intercellular adhesion molecule-1 (ICAM-1) participate in inflammatory processes by promoting adhesion of leukocytes to vascular wall endothelium. Their soluble levels have been associated with adverse cardiovascular events. To identify loci affecting soluble levels of P-selectin (sP-selectin) and ICAM-1 (sICAM-1), we performed a genome-wide association study in a sample of 4115 (sP-selectin) and 9813 (sICAM-1) individuals of European ancestry as a part of The Cohorts for Heart and Aging Research in Genome Epidemiology consortium. The most significant SNP association for sP-selectin was within the SELP gene (rs6136, P = 4.05 × 10-61) and for sICAM-1 levels within the ICAM-1 gene (rs3093030, P = 3.53 × 10-23). Both sP-selectin and sICAM-1 were associated with ABO gene variants (rs579459, P = 1.86 × 10-41 and rs649129, P = 1.22 × 10-15, respectively) and in both cases the observed associations could be accounted for by the A1 allele of the ABO blood group. The absence of an association between ABO blood group and platelet-bound P-selectin levels in an independent subsample (N = 1088) from the ARIC study, suggests that the ABO blood group may influence cleavage of the P-selectin protein from the cell surface or clearance from the circulation, rather than its production and cellular presentation. These results provide new insights into adhesion molecule biology. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] /* */

SELP and SELPLG genetic variation is associated with cell surface measures of SELP and SELPLG: The atherosclerosis risk in communities carotid MRI study

Article

May 2009
CLIN CHEM

P-selectin (SELP) and its ligand, P-selectin glycoprotein ligand 1 (SELPLG), play key roles in both the inflammatory response and the atherosclerotic process. Previous studies have shown genetic variation in the SELP gene [selectin P (granule membrane protein 140 kDa, antigen CD62)] to be associated with plasma SELP concentrations; however, the major biological function of SELP (and SELPLG) is at the cell surface. We therefore investigated the association of SELP polymorphisms with platelet SELP measures and polymorphisms in the SELPLG gene (selectin P ligand) with lymphocyte, granulocyte, and monocyte SELPLG measures among 1870 participants in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI Study. Whole-blood flow cytometry was used to analyze leukocyte and platelet markers in the ARIC Carotid MRI Study. The allele frequencies for the SELP and SELPLG polymorphisms of whites and African Americans were markedly different; therefore, all analyses were race specific. SELP T715P was significantly associated with lower values for platelet SELP measures in whites (P = 0.0001), whereas SELP N562D was significantly associated with higher values for SELP measures in African Americans (P = 0.02). SELPLG M62I was significantly associated with lower granulocyte and monocyte SELPLG measures in African Americans (P = 0.003 and P = 0.0002, respectively) and with lower lymphocyte SELPLG measures in whites (P = 0.01). Specific SELP and SELPLG polymorphisms were associated with cell surface measures of SELP and SELPLG in both whites and African Americans in the ARIC Carotid MRI Study. To our knowledge, this study is the first to examine the association of SELP and SELPLG genetic variation with measures of cell surface SELP and SELPLG.

Increased Platelet Reactivity and Circulating Monocyte-Platelet Aggregates in Patients With Stable Coronary Artery Disease

Article

Full-text available

Feb 1998
J AM COLL CARDIOL

We sought to examine whether patients with stable coronary artery disease (CAD) have increased platelet reactivity and an enhanced propensity to form monocyte-platelet aggregates. Platelet-dependent thrombosis and leukocyte infiltration into the vessel wall are characteristic cellular events seen in atherosclerosis. Anticoagulated peripheral venous blood from 19 patients with stable CAD and 19 normal control subjects was incubated with or without various platelet agonists and analyzed by whole blood flow cytometry. Circulating degranulated platelets were increased in patients with CAD compared with control subjects (mean [+/- SEM] percent P-selectin-positive platelets: 2.1 +/- 0.2 vs. 1.5 +/- 0.2, p < 0.01) and were more reactive to stimulation with 1 micromol/liter of adenosine diphosphate (ADP) (28.7 +/- 3.9 vs. 16.1 +/- 2.2, p < 0.01), 1 micromol/liter of ADP/epinephrine (51.4 +/- 4.6 vs. 37.5 +/- 3.8, p < 0.05) or 5 micromol/liter of thrombin receptor agonist peptide (TRAP) (65.7 +/- 6.8 vs. 20.2 +/- 5.1, p < 0.01). Patients with stable CAD also had increased circulating monocyte-platelet aggregates compared with control subjects (percent platelet-positive monocytes: 15.3 +/- 3.0 vs. 6.3 +/- 0.9, p < 0.01). Furthermore, patients with stable CAD formed more monocyte-platelet aggregates than did control subjects when their whole blood was stimulated with 1 micromol/liter of ADP (50.4 +/- 4.5 vs. 28.1 +/- 5.3, p < 0.01), 1 micromol/liter of ADP/epinephrine (60.7 +/- 4.3 vs. 48.0 +/- 4.8, p < 0.05) or 5 micromol/liter of TRAP (67.6 +/- 5.7 vs. 34.3 +/- 7.0, p < 0.01). Patients with stable CAD have circulating activated platelets, circulating monocyte-platelet aggregates, increased platelet reactivity and an increased propensity to form monocyte-platelet aggregates.

A practical approach to multicolor flow cytometry for immunophenotyping

Article

Full-text available

Oct 2000
J IMMUNOL METHODS

Through a series of novel developments in flow cytometry hardware, software, and dye-chemistry it is now possible to simultaneously measure up to 11 distinct fluorescences and two scattered light parameters on each cell. Such advanced multicolor systems have a number of advantages over current two- and three-color flow cytometric measurements. They provide a large amount of novel information for each sample studied, an exquisitely accurate quantitation of even rare cell populations, and allow identification and characterization of novel cell subsets. In particular, this technology is proving crucial to identifying functionally homogeneous subsets of cells within the enormously complex immune system; such identification and enumeration is important for understanding disease pathogenesis. However, multicolor flow cytometry comes with a new and sometimes difficult set of technical problems that must be overcome by users to derive meaningful results. In this manuscript, we describe the basic aspects of multicolor flow cytometry, including the technical hurdles and artefacts that may occur, and provide some suggestions for how to best overcome these hurdles. While inspired by the 11-color technology that we currently use, these principles apply to all flow cytometric experiments in which more than one fluorescent dye is used.

Validation and quality control in clinical flow cytometry

Article

Full-text available

Oct 2000
J IMMUNOL METHODS

Clinical flow cytometry has evolved from two-parameter quantitative assessment of peripheral blood lymphocytes to six-parameter qualitative evaluation of bone marrow for hematopathology. Leukemia and lymphoma immunophenotyping represent an extremely important complement to morphology in the diagnosis and monitoring of hematopoietic malignancies. The complexity of five- and six-parameter analyses and the interpretation of the data rely on standardization and validation of the instrument, the reagents and the procedure. In addition, flow cytometry laboratories in the U.S. are required to document proficiency testing, sample preparation, method accuracy, specificity, sensitivity and precision. NCCLS and the U.S.-Canadian Consensus Conference have provided recommendations, but each laboratory is ultimately responsible for validating its own qualitative and quantitative procedures. This paper reviews procedures for validation and quality control of all aspects of the operation of a clinical flow cytometry service.

Association Between Myeloperoxidase Levels and Risk of Coronary Artery Disease

Article

Full-text available

Nov 2001

Myeloperoxidase (MPO), a leukocyte enzyme that promotes oxidation of lipoproteins in atheroma, has been proposed as a possible mediator of atherosclerosis. To determine the association between MPO levels and prevalence of coronary artery disease (CAD). Case-control study conducted from July to September 2000 in a US tertiary care referral center, including 158 patients with established CAD (cases) and 175 patients without angiographically significant CAD (controls). Association of MPO levels per milligram of neutrophil protein (leukocyte-MPO) and MPO levels per milliliter of blood (blood-MPO) with CAD risk. Leukocyte- and blood-MPO levels were both significantly greater in patients with CAD than in controls (P<.001). In multivariable models adjusting for traditional cardiovascular risk factors, Framingham risk score, and white blood cell counts, MPO levels were significantly associated with presence of CAD, with an OR of 11.9 (95% CI, 5.5-25.5) for the highest vs lowest quartiles of leukocyte-MPO and an OR of 20.4 (95% CI, 8.9-47.2) for the highest vs lowest quartiles of blood-MPO. Elevated levels of leukocyte- and blood-MPO are associated with the presence of CAD. These findings support a potential role for MPO as an inflammatory marker in CAD and may have implications for atherosclerosis diagnosis and risk assessment.

Role of Monocytes in Atherogenesis

Article

Full-text available

Nov 2003

This review focuses on the role of monocytes in the early phase of atherogenesis, before foam cell formation. An emerging consensus underscores the importance of the cellular inflammatory system in atherogenesis. Initiation of the process apparently hinges on accumulating low-density lipoproteins (LDL) undergoing oxidation and glycation, providing stimuli for the release of monocyte attracting chemokines and for the upregulation of endothelial adhesive molecules. These conditions favor monocyte transmigration to the intima, where chemically modified, aggregated, or proteoglycan- or antibody-complexed LDL may be endocytotically internalized via scavenger receptors present on the emergent macrophage surface. The differentiating monocytes in concert with T lymphocytes exert a modulating effect on lipoproteins. These events propagate a series of reactions entailing generation of lipid peroxides and expression of chemokines, adhesion molecules, cytokines, and growth factors, thereby sustaining an ongoing inflammatory process leading ultimately to lesion formation. New data emerging from studies using transgenic animals, notably mice, have provided novel insights into many of the cellular interactions and signaling mechanisms involving monocytes/macrophages in the atherogenic processes. A number of these studies, focusing on mechanisms for monocyte activation and the roles of adhesive molecules, chemokines, cytokines and growth factors, are addressed in this review.

TLR Signaling: An Emerging Bridge from Innate Immunity to Atherogenesis

Article

Full-text available

Dec 2004

Chronic inflammation and disordered lipid metabolism represent hallmarks of atherosclerosis. Considerable evidence suggests that innate immune defense mechanisms might interact with proinflammatory pathways and contribute to development of arterial plaques. The preponderance of such evidence has been indirect clinical and epidemiologic studies, with some support from experimental animal models of atherosclerosis. However, recent data now directly implicate signaling by TLR4 in the pathogenesis of atherosclerosis, establishing a key link between atherosclerosis and defense against both foreign pathogens and endogenously generated inflammatory ligands. In this study, we briefly review these and closely related studies, highlighting areas that should provide fertile ground for future studies aimed at a more comprehensive understanding of the interplay between innate immune defense mechanisms, atherosclerosis, and related vascular disorders.

Thematic review series: The Immune System and Atherogenesis. Paying the price for pathogen protection: toll receptors in atherogenesis

Article

Full-text available

Apr 2005

Atherosclerosis is a chronic inflammatory response characterized by the accumulation of cells of innate and acquired immune systems within the intima of the arterial wall. Macrophages are the predominant participant in innate immune responses in atherosclerosis. Protein receptors expressed by macrophages and endothelial cells recognize components and products of microorganisms and play a vital role in innate immunity. In particular, the members of the toll-like receptor (TLR) family play a critical role in the inflammatory components of atherosclerosis. Both exogenous ligands involved in microbial recognition as well as endogenous ligands involved in sterile inflammation pathways are implicated in the pathology of atherosclerosis. In this review, we discuss our current understanding of the role of TLRs and their coactivators in atherosclerosis, with particular emphasis on studies in atherosclerosis-prone hypercholesterolemic mice.

Modulation of atherosclerosis in mice by Toll-like receptor 2

Article

Full-text available

Dec 2005

Epidemiologic evidence has established a relationship between microbial infection and atherosclerosis. Mammalian TLRs provide clues on the mechanism of this inflammatory cascade. TLR2 has a large ligand repertoire that includes bacterial-derived exogenous and possibly host-derived endogenous ligands. In atherosclerosis-susceptible low-density lipoprotein receptor-deficient (Ldlr-/-) mice, complete deficiency of TLR2 led to a reduction in atherosclerosis. However, with BM transplantation, loss of TLR2 expression from BM-derived cells had no effect on disease progression. This suggested that an unknown endogenous TLR2 agonist influenced lesion progression by activating TLR2 in cells that were not of BM cell origin. Moreover, with intraperitoneal administration of a synthetic TLR2/TLR1 agonist, Pam3CSK4, disease burden was dramatically increased in Ldlr-/- mice. A complete deficiency of TLR2 in Ldlr-/- mice, as well as a deficiency of TLR2 only in BM-derived cells in Ldlr-/- mice, led to striking protection against Pam3CSK4-mediated atherosclerosis, suggesting a role for BM-derived cell expression of TLR2 in transducing the effects of an exogenous TLR2 agonist. These studies support the concept that chronic or recurrent microbial infections may contribute to atherosclerotic disease. Additionally, these data suggest the presence of host-derived endogenous TLR2 agonists.

Stabilization of white blood cells and immunologic markers for extended analysis using flow cytometry

Article

Full-text available

Nov 2005
J IMMUNOL METHODS

We evaluated whole blood samples drawn from 25 healthy donors and 20 HIV-infected donors into K3EDTA and Cyto-Chex BCT blood collection tubes for CD4, CD8, and CD3 cell counts (HIV Panel). Samples collected in Cyto-Chex BCT were stored at room temperature and tested by 4-color flow cytometry at 6 h, 3 days, and 7 days after isolation for CD4, CD8, and CD3 absolute cell counts/microl and compared with samples collected in K3EDTA tubes and tested at 6 h. Regardless of donor type, the samples collected in Cyto-Chex BCT and tested on day 7 yielded results that were statistically indistinguishable (with correlation coefficients of 0.96 or greater) from samples collected in K3EDTA tubes and tested at 6 h. We conclude that whole blood samples collected in Cyto-Chex BCT are stabilized for their marker phenotype for at least 7 days after phlebotomy.

Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry

Article

Jul 1987

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light- scatter profile. Alternatively, platelets could be identified with FITC- AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin- PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose- dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.

THE ATHEROSCLEROSIS RISK IN COMMUNIT (ARIC) STUI)Y: DESIGN AND OBJECTWES

Article

Apr 1989
AM J EPIDEMIOL

THE ARIC INVESTIGATORS

Atherosclerosis Risk in Communities (ARIC) is a new prospective study to investigate the etiology of atherosclerosis and its clinical sequelae and variation in cardiovascular risk factors, medical care, and disease by race, sex, place, and time. in each of four US communities—Forsyth County, North Carolina, Jackson, Mississippi, suburbs of Minneapolis, Minnesota, and Washington County, Maryland—4, 000 adults aged 45–64 years will be examined twice, three years apart. ARIC has coordinating, ultrasound, pulmonary, and electrocardiographic centers and three central laboratories. Three cohorts represent the ethnic mix of their communities; the Jackson cohort, its black population. Examinations include ultrasound scanning of carotid and popliteal arteries; lipids, lipoprotelns, and apolipoproteins assayed in the Lipid Laboratory; and coagulation, inhibition, and platelet and fibrinolytic actmty assayed in the Hemostasis Laboratory. Surveil lance for coronary heart disease will involve review of hospitalizations and deaths among community residents aged 35–74 years. ARIC aims to study atheroscle rosis by direct observation of the disease and by use of modem biochemistry.

Statistical Methods for Rates and Proportions. New York:

Article

Jan 1981

J.L. Fleiss

Platelet activation and arterial thrombosis

Article

Oct 1994

Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a

Article

Aug 2000
J IMMUNOL METHODS

Detection of intracellular myeloperoxidase (MPO), CD79a and CD3 has become the most specific tool for the assignment of myeloid, B- and T-lymphoid lineages in acute leukemias. In order to establish the best combination of monoclonal antibody reagent and sample preparation technique for the intracellular detection of these three markers, we compared six different cell fixation–permeabilization kits (Cytofix/Cytoperm™, Fix and Perm™, Intraprep™, Intrastain™, Permeacyte™ and Permeafix™) using 12 fluorochrome conjugates derived from seven monoclonal antibody (mAb) clones. A total of 21 samples corresponding to normal peripheral blood (n=4), normal bone marrow (n=3), acute myeloblastic leukemia (AML, n=6), precursor B-acute lymphoblastic leukemia (ALL, n=6) and T-ALL (n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side scatter and mostly increased forward scatter as compared to erythrocyte-lyse-washed and 1% paraformaldehyde fixed samples. The autofluorescence levels of the leukocyte populations was only significantly increased with use of the Cytofix/Cytoperm™ kit and mildly with the other techniques. In addition, non-specific staining increased significantly for combinations of any anti-MPO mAb with the Cytofix/Cytoperm™ kit and for the CD3 clone S4.1 combined with any intracellular method. Anti-MPO antibodies gave a stronger fluorescence signal when conjugated to PE than when coupled to FITC. In conclusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in combination with Fix and Perm™, Intraprep™, Intrastain™ or Permeafix™, provided specific staining of the respective markers in sufficient intensities. Thus, combined selection of fixation/permeabilization kits and monoclonal antibody reagents against CD3, CD79a and MPO is required for obtaining optimal cytoplasmic detection of these antigens.

Cytometric Analysis of Cell Phenotype and Function

Article

Jun 2002
TRANSFUSION MED

1. Principles of flow cytometry Marion G. Macey 2. Introduction to the general principles of sample preparation Desmond A. McCarthy 3. Fluorescence and fluorochromes Desmond A. McCarthy 4. Quality control in flow cytometry David Barnett and John T. Reilly 5. Data analysis in flow cytometry Mark W. Lowdell 6. Laser scanning cytometry: application to the immunophenotyping of hematological malignancies Richard J. Clatch 7. Leucocyte immunobiology Marion G. Macey and Timothy M. Milne 8. Immunophenotypic analysis of leucocytes in disease Jamie D. Cavenagh and Marion G. Macey 9. Analysis and isolation of minor cell populations Terry Hoy 10. Cell cycle, DNA and DNA ploidy analysis Paul D. Allen and Adrian C. Newland 11. Cell viability, necrosis and apoptosis Paul D. Allen and Adrian C. Newland 12. Phagocyte biology and function Marion G. Macey 13. Intracellular measures of signalling pathways James W. Jacobberger and David W. Hedley 14. Cell-cell interactions Marion G. Macey and Mary R. Cahill 15. Nucleic acids Charles L. Goolsby 16. Microbial infections David Lloyd, David J. Mason and Marc T. E. Suller 17. Leucocyte cell surface antigens Marion G. Macey 18. Recent and future developments: conclusions Desmond A. McCarthy Appendix 1. Abbreviations Appendix 2. Useful Internet sites.

Detection of activated platelets in whole blood using activation–dependent monoclonal antibodies

Article

Aug 1987

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light-scatter profile. Alternatively, platelets could be identified with FITC-AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.

Guideline for flow cytometric immunophenotyping: A report from the National Institute of Allergy and Infectious Diseases, Division of AIDS

Article

Oct 1993

Nationwide quality control trial on lymphocyte phenotyping and flow cytometry performance in Italy

Article

Jan 1993

The Italian Society for Cytometry (Gruppo Italiano di Citometria, GIC) promoted this nationwide large-scale quality control trial on cellular immunophenotyping and flow cytometer performance in 1991. The aim of this independent study was to evaluate instrument performance with calibrated fluorescence microbeads (minimum threshold for FITC, linearity, coefficients of variation and resolution indexes for fluorescence), and to correlate it to the measurement of lyophilized lymphocyte surface immunofluorescence staining with a wide spectrum of antibodies (percentage of positive cells and fluorescence mean intensity).

Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study

Article

Sep 1997

Fifty-five laboratories participated in a send-out study of four peripheral blood samples comparing a standard protocol vs. local protocols for flow cytometric lymphocyte immunophenotyping. The standard protocol included centrally provided reagents, instrument setup using triple-fluorescent microbeads and a three-color, whole-blood immunostaining technique based on fluorescein isothiocyanate and phycoerythrin-labeled monoclonal antibodies, erythrocyte lysis, washing, fixation, and identification of nucleated cells by the DNA/RNA stain LDS-751. Data analysis guidelines included lymphocyte selection using CD45,CD14-assisted "backgating" on forward (FSC) and sideward (SSC) light scatter and placement of fluorescence (FL) markers on the basis of the isotype control staining. Most (i.e., 77%) of the variation in results of percentage lymphocyte subset assessments using the standard protocol was explained by laboratory, sample, background FL, and the interaction between laboratory and sample. Purity and completeness of the FSC,SSC lymphogate, background FL, flow cytometer type, and flow cytometer setup (which were either partly or entirely determined by laboratory) contributed significantly to the variation. The effect of the leukocyte differential count on the variation in absolute numbers of lymphocyte subsets was particularly large in lymphopenic samples. The use of this standard protocol vs. local protocols did not reduce the interlaboratory variation. Instrument incompatibility with the standard protocol (e.g., incompatible filter combinations for LDS-751 detection) and lack of experience of many participants with three-color flow cytometry (in particular with the use of LDS-751) may have contributed to that result. We suggest that training and experience in a universally applicable standard protocol are critical for minimization of interlaboratory variation in flow cytometric immunophenotyping.

European Working Group on Clinical Cell Analysis: Consensus Protocol for the Flow Cytometric Characterisation of Platelet Function

Article

Jun 1998

Introduction An increased or disturbed activation and aggregation of platelets plays a major role in the pathophysiology of thrombosis and haemostasis and is related to cardiovascular disease processes. In addition to qualitative disturbances of platelet function, changes in thrombopoiesis or an increased elimination of platelets, (e. g., in autoimmune thrombocytopenia), are also of major clinical relevance. Flow cytometry is increasingly used for the specific characterisation of phenotypic alterations of platelets which are related to cellular activation, haemostatic function and to maturation of precursor cells. These new techniques also allow the study of the in vitro response of platelets to stimuli and the modification thereof under platelet-targeted therapy as well as the characterisation of platelet-specific antibodies. In this protocol, specific flow cytometric techniques for platelet analysis are recommended based on a description of the current state of flow cytometric methodology. These recommendations are an attempt to promote the use of these new techniques which are at present broadly evaluated for diagnostic purposes. Furthermore, the definition of the still open questions primarily related to the technical details of the method should help to promote the multi-center evaluation of procedures with the goal to finally develop standardized operation procedures as the basis of interlaboratory reproducibility when applied to diagnostic testing.

Performance of calibration standards for antigen quantitation with flow cytometry

Article

Nov 1998

In the frame of the activities initiated by the Task Force for Antigen Quantitation of the European Working Group on Clinical Cell Analysis (EWGCCA), an experiment was conducted to evaluate microbead standards used for quantitative flow cytometry (QFCM). An unified window of analysis (UWA) was established on three different instruments (EPICS XL [Coulter Corporation, Miami, FL], FACScan and FACS Calibur [Becton Dickinson, San Jose, CA]) with QC3 microbeads (FCSC, PR). By using this defined fluorescence intensity scale, the performance of several monoclonal antibodies directed to CD3, CD4, and CD8 (conjugated and unconjugated), from three manufacturers (BDIS, Coulter [Immunotech], and DAKO) was tested. In addition, the QIFI system (DAKO) and QuantiBRITE (BDIS), and a method of relative fluorescence intensity (RFI, method of Giorgi), were compared. mAbs reacting with three more antigens, CD16, CD19, and CD38 were tested on the FACScan instrument. Quantitation was carried out using a single batch of cryopreserved peripheral blood leukocytes, and all tests were performed as single color analyses. Significant correlations were observed between the antibody-binding capacity (ABC) values of the same CD antigen measured with various calibrators and with antibodies differing in respect to vendor, labeling and possible epitope recognition. Despite the significant correlations, the ABC values of most monoclonal antibodies differed by 20-40% when determined by the different fluorochrome conjugates and different calibrators. The results of this study indicate that, at the present stage of QFCM consistent ABC values may be attained between laboratories provided that a specific calibration system is used including specific calibrators, reagents, and protocols.

Standardization of lymphocyte antibody binding capacity - A multi-centre study

Article

May 2000

As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.

Fluorescence Intensity Calibration for Immunophenotyping by Flow Cytometry

Article

Aug 2000

Fluorescence intensity (FI) is the basis for classifying phenotypes by fluorescence-label flow cytometry. FI is customarily recorded as an arbitrary relative value, but with proper calibration it can be expressed in stoichiometric units called molecules of equivalent soluble fluorochrome (MESF) that reflect the concentrations of the fluorescent conjugates and the receptors they stain. Forthcoming availability of authoritative standards and consensus methods will alleviate many of the difficulties encountered in making valid MESF measurements. FI calibration establishes the true values for the critical parameters of the fluorescence measurement, a useful feature for quality control. It further allows the establishment of a comparable window of analysis across different times and laboratories, and it permits numeric assessment of antibody-binding capacity (ABC) values in selected cell populations. The relation between ABC values and receptor expression is complicated by several factors, but careful assessment of the binding chemistry can establish the actual number of receptors on cells stained by fluorescent conjugates.

Evaluation of Platelet Function by Flow Cytometry

Article

Aug 2000

Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.

Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols

Article

Sep 2000
J IMMUNOL METHODS

Detection of intracellular myeloperoxidase (MPO), CD79a and CD3 has become the most specific tool for the assignment of myeloid, B- and T-lymphoid lineages in acute leukemias. In order to establish the best combination of monoclonal antibody reagent and sample preparation technique for the intracellular detection of these three markers, we compared six different cell fixation-permeabilization kits (Cytofix/Cytoperm, Fix and Perm, Intraprep, Intrastain, Permeacyte and Permeafix) using 12 fluorochrome conjugates derived from seven monoclonal antibody (mAb) clones. A total of 21 samples corresponding to normal peripheral blood (n=4), normal bone marrow (n=3), acute myeloblastic leukemia (AML, n=6), precursor B-acute lymphoblastic leukemia (ALL, n=6) and T-ALL (n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side scatter and mostly increased forward scatter as compared to erythrocyte-lyse-washed and 1% paraformaldehyde fixed samples. The autofluorescence levels of the leukocyte populations was only significantly increased with use of the Cytofix/Cytoperm kit and mildly with the other techniques. In addition, non-specific staining increased significantly for combinations of any anti-MPO mAb with the Cytofix/Cytoperm kit and for the CD3 clone S4.1 combined with any intracellular method. Anti-MPO antibodies gave a stronger fluorescence signal when conjugated to PE than when coupled to FITC. In conclusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in combination with Fix and Perm, Intraprep, Intrastain or Permeafix, provided specific staining of the respective markers in sufficient intensities. Thus, combined selection of fixation/permeabilization kits and monoclonal antibody reagents against CD3, CD79a and MPO is required for obtaining optimal cytoplasmic detection of these antigens.

Stability of fresh and preserved fetal and adult lymphocyte cell surface markers

Article

Feb 2000

Delay between blood collection and immunophenotyping of peripheral blood (PB) and umbilical cord blood (UCB) lymphocytes occurs frequently. Holding media address this problem, but there are few reports of their limitations. We tested the ability of Cyto-Chex (CC) to preserve the ability of lymphocyte subpopulations to be immunophenotyped after time delays. Ten UCB and 10 PB specimens were kept up to 48 hr and then placed in CC for 1 week, with removal of aliquots for staining with CD3, CD4, CD8, CD19, and CD45 at intervals. There were no significant changes in the number of nonpreserved PB or UCB cells stained with any of the reagents after 48 hr. Aside from a small decrease in UCB B-cell numbers, there were no changes in UCB or PB lymphocyte numbers after placing them in CC for 1 week. However, PB and UCB CD19+ cells and CD8+ T cells lost capacity for bright staining after 1 week in CC. The results suggest that UCB and PB can be held for up to 48 hr before being placed in CC and then kept for up to 1 week in CC with no decrease in T-cell numbers and only a minor reduction of B-cell numbers, albeit with a marked reduction in staining intensity.

Circulating Monocyte-Platelet Aggregates Are a More Sensitive Marker of In Vivo Platelet Activation Than Platelet Surface P-Selectin Studies in Baboons, Human Coronary Intervention, and Human Acute Myocardial Infarction

Article

Sep 2001
CIRCULATION

Platelet surface P-selectin is considered the "gold standard" marker of platelet activation. Degranulated, P-selectin-positive platelets, however, aggregate with leukocytes in vitro and rapidly lose surface P-selectin in vivo. Flow cytometric tracking of autologous, biotinylated platelets in baboons enabled us to directly demonstrate for the first time in vivo that (1) infused degranulated platelets very rapidly form circulating aggregates with monocytes and neutrophils, and (2) 30 minutes after infusion of the degranulated platelets, the percentage of circulating monocytes aggregated with infused platelets persist at high levels, whereas the percentage of circulating neutrophils aggregated with infused platelets and the platelet surface P-selectin of nonaggregated infused platelets return to baseline. We therefore performed 2 clinical studies in patients with acute coronary syndromes. First, after percutaneous coronary intervention (n=10), there was an increased number of circulating monocyte-platelet (and to a lesser extent, neutrophil-platelet) aggregates but not P-selectin-positive platelets. Second, of 93 patients presenting to an Emergency Department with chest pain, patients with acute myocardial infarction (AMI) (n=9) had more circulating monocyte-platelet aggregates (34.2+/-10.3% [mean+/-SEM]) than patients with no AMI (n=84, 19.3+/-1.4%, P<0.05) and normal control subjects (n=10, 11.5+/-0.8%, P<0.001). Circulating P-selectin-positive platelets, however, were not increased in chest pain patients with or without AMI. As demonstrated by 3 independent means (in vivo tracking of activated platelets in baboons, human coronary intervention, and human AMI), circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin.

Circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction

Article

Oct 2001
J AM COLL CARDIOL

We investigated whether elevated levels of circulating monocyte-platelet aggregates (MPA) can be used to identify patients with acute myocardial infarction (AMI). Commonly used blood markers of AMI reflect myocardial cell death, but do not reflect the earlier pathophysiologic processes of plaque rupture, platelet activation and resultant thrombus formation. Circulating MPA form after platelet activation. In a single center between October 1998 and November 1999, we measured circulating MPA in a blinded fashion by whole blood flow cytometry in 211 consecutive patients who presented to the emergency department (ED) with chest pain and were admitted to rule out AMI. Acute myocardial infarction was diagnosed by a CK-MB fraction greater than three times control. Patients with AMI (n = 61), as compared with those without AMI (n = 150), had significantly higher numbers of circulating MPA (11.6 +/- 11.4 vs. 6.4 +/- 3.6, mean +/- SD, p < 0.0001). After controlling for age, the adjusted odds of developing AMI for patients in the 2nd, 3rd and 4th quartiles of MPA, in comparison with patients in the lowest quartile (odds ratio = 1.0), were 2.1 (95% confidence interval [CI]: 0.7, 6.8), 4.4 (95% CI: 1.5, 13.1) and 10.8 (95% CI: 3.6, 32.0), respectively. The number of circulating MPA in patients with AMI presenting within 4 h of symptom onset (14.4) was significantly greater than those presenting after 4 h (9.4) and after 8 h (7.0), (p < 0.001). Of the 61 patients with AMI, 35 (57%) had a normal creatine kinase isoenzyme ratio at the time of presentation to the ED, but had high levels of circulating MPA (13.3). Circulating MPA are an early marker of AMI.

Platelet-Monocyte Aggregates: Bridging Thrombosis and Inflammation

Article

Jun 2002
CIRCULATION

Thrombus formation within a coronary vessel is the precipitating event in unstable coronary syndromes. The angiographic severity of coronary stenoses may not predict sites of subsequent acute coronary events, inasmuch as rupture of atheromatous plaque with thrombosis in relatively mildly stenosed vessels may underlie many acute coronary syndromes. The intact endothelium normally prevents platelet activation, but the intimal injury associated with endothelial denudation and plaque rupture exposes subendothelial collagen and von Willebrand factor, which support prompt platelet adhesion and activation. Circulating platelets can adhere either directly to collagen or indirectly, via the binding of von Willebrand factor, to the glycoprotein Ib/IX complex. Local platelet activation promotes thrombus formation and additional platelet recruitment by supporting cell surface thrombin formation and releasing potent platelet agonists, such as adenosine 5′-diphosphate, serotonin, and thromboxane A2. The central role of platelet activation in acute coronary syndromes is supported by increased platelet-derived thromboxane and prostaglandin metabolites detected in patients with acute coronary syndromes1 and the clear clinical benefit of treatment with aspirin for prevention of acute coronary events.2 See p 2166 The importance of platelet-dependent thrombosis has made the platelet aggregate a common therapeutic target in acute coronary syndromes. Recently, such therapies have included the use of aspirin, thienopyridines, and direct glycoprotein IIb/IIIa inhibitors. Although the mechanism of action of these various agents differs, they all inhibit fibrinogen-dependent platelet–platelet associations (ie, platelet aggregation). However, as highlighted by the recent disappointing clinical results of trials using oral glycoprotein IIb/IIIa inhibitors,3 merely preventing the process of platelet aggregation may not always translate into clinical efficacy. Recent data suggest that patients with acute coronary syndromes have not only increased interactions between platelets (homotypic aggregates) but also increased interactions between platelets and leukocytes (heterotypic aggregates) detectable in circulating blood. These aggregates form when platelets are …

Atherosclerosis in Inflammation

Article

Dec 2002

Peter Libby

Abundant data link hypercholesterolaemia to atherogenesis. However, only recently have we appreciated that inflammatory mechanisms couple dyslipidaemia to atheroma formation. Leukocyte recruitment and expression of pro-inflammatory cytokines characterize early atherogenesis, and malfunction of inflammatory mediators mutes atheroma formation in mice. Moreover, inflammatory pathways promote thrombosis, a late and dreaded complication of atherosclerosis responsible for myocardial infarctions and most strokes. The new appreciation of the role of inflammation in atherosclerosis provides a mechanistic framework for understanding the clinical benefits of lipid-lowering therapies. Identifying the triggers for inflammation and unravelling the details of inflammatory pathways may eventually furnish new therapeutic targets.

Emerging role of myeloperoxidase and oxidant stress markers in cardiovascular risk assessment

Article

Sep 2003

Myeloperoxidase, an abundant leukocyte protein that generates reactive oxidant species, is present and catalytically active within atherosclerotic lesions. Numerous lines of evidence suggest mechanistic links between myeloperoxidase, inflammation and both acute and chronic manifestations of cardiovascular disease. Myeloperoxidase generates reactive oxidant species as part of its function in innate host defense mechanisms. The reactive species formed, however, may also damage normal tissues, contributing to inflammatory injury. Recent studies suggest that MPO-generated oxidants participate in multiple processes relevant to cardiovascular disease development and outcomes, including induction of foam cell formation, endothelial dysfunction, development of vulnerable plaque, and ventricular remodeling following acute myocardial infarction. Of note, measurements of myeloperoxidase mass and activity may be useful in cardiac risk stratification, both for chronic disease assessment, as well as in identification of patients at risk in the acute setting. The inflammatory protein myeloperoxidase is present, active and mechanistically poised to participate in the initiation and progression of cardiovascular disease. The many links between myeloperoxidase, oxidation and cardiovascular disease suggest this leukocyte protein may have clinical utility in risk stratification for cardiovascular disease status and outcomes.

Formalization of the MESF Unit of Fluorescence Intensity

Article

Jan 2004

This report summarizes the work performed during the past two years at the National Institute of Standards and Technology (NIST) in the refinement and formal definition of the MESF unit of fluorescence intensity. In addition to the theory underlying the MESF unit, considerations of error analysis are also presented. The details of this work may be found in the three publications of the NIST Journal of Research (www.nist.gov) listed as the references 2-4. The use of the fluorescence intensity unit provides a tool to compare quantitative fluorescence intensity measurements over time and across platforms.

Myeloperoxidase and Plaque Vulnerability

Article

Aug 2004
ARTERIOSCL THROM VAS

Stanley Hazen

Sudden cardiac death remains the leading cause of mortality in industrialized societies, outpacing all cancer related deaths combined. The majority of sudden cardiac deaths arise from acute myocardial infarction secondary to intracoronary artery thromboses. Remarkably, the culprit lesions involved are typically not flow-limiting stenoses,1,2 but rather inflamed lipid-laden lesions.3,4 Whereas plaque fissuring or rupture, which exposes the intensely prothrombogenic lipid core, occurs in a majority of cases, fully 40% of intracoronary artery thromboses arise at sites of superficial erosions, where endothelial cell (EC) loss and denudation occurs.3 The mechanisms responsible for plaque vulnerability leading to acute coronary artery thrombosis remain poorly understood. Mounting evidence, however, points toward a critical role for inflammatory processes.5–8 Macrophages serve as the dominant cell type in the immediate site of both plaque ruptures and superficial erosions in subjects who experience acute coronary thrombosis,8 and recent clinical investigations reveal important associations between leukocytes,9,10 their enzymes,11–13 and their activation,14,15 in subjects with unstable angina and acute coronary syndromes. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Sugiyama and colleagues significantly extend our knowledge of potential pathophysiologic inflammatory processes within vulnerable atheroma.16 Using a combination of biochemical, cellular, and immunohistologic studies, they describe unifying mechanistic links between the activity of the leukocyte enzyme myeloperoxidase (MPO) and two cardinal features of vulnerable plaque: EC loss/denudation and development of a prothrombotic phenotype. See page 1309 First identified within human atherosclerotic plaque nearly a decade ago,17 MPO has emerged as an important potential participant in the atherosclerotic process. MPO, a member of the heme peroxidase superfamily, generates reactive oxidants and diffusible radical species as part of its normal function in innate host defenses.18 A unique activity of MPO is its ability to use the halide chloride as cosubstrate …

Toll-like receptors in the pathogenesis of human disease

Article

Nov 2004

Members of the Toll-like receptor (TLR) family are key regulators of both innate and adaptive immune responses. The function of TLRs in various human diseases has been investigated by comparison of the incidence of disease among people having different polymorphisms in genes that participate in TLR signaling. These studies have shown that TLR function affects several diseases, including sepsis, immunodeficiencies, atherosclerosis and asthma. As this body of data grows, it will provide new insights into disease pathogenesis as well as valuable information on the merits of various therapeutic options.

A general method for bead-enhanced quantitation by flow cytometry

Article

Jan 2007
J IMMUNOL METHODS

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry.

Platelets as Mediators of Inflammation

Article

Mar 2007
HEMATOL ONCOL CLIN N

Steven R Steinhubl

An expanding body of evidence continues to build on the central role of inflammation in the progression and clinical manifestations of atherosclerosis. Platelets, long thought to play only a reactionary role at the time of endothelial disruption, are now recognized as important mediators of the inflammatory process. Platelet activation, which is modulated by both inflammatory and hemostatic factors, can lead to the release of hundreds of proteins--many with known proinflammatory functions. Although compelling evidence is lacking that antiplatelet therapies directly lower markers of inflammation, there are intriguing, although preliminary, data suggesting that markers of inflammation predict the clinical benefit of antiplatelet therapies.

Platelets in Inflammation and Atherothrombosis

Article

Apr 2007

Jan 1987

Wa Fuller

Fuller, WA. Measurement Error Models. Wiley; New York: 1987.

Fifth Edition. Clinical and Laboratory Standards Institute Procedures for the collection of diagnostic blood specimens by venipuncture

Jan 2003

Clinical and Laboratory Standards Institute. Approved Standard. Vol. Fifth Edition. Clinical and Laboratory Standards Institute; Wayne, PA: 2003. Procedures for the collection of diagnostic blood specimens by venipuncture. CLSI document H3-A5

Paying the price for pathogen protection: Toll receptors in atherogenesis

Jan 2005
404-415

P Tobias
Lk Curtiss

Tobias P, Curtiss LK. Paying the price for pathogen protection: Toll receptors in atherogenesis. J Lipid Res 2005;46:404–11. [PubMed: 15654120]

Atherosclerosis Risk in Communities (ARIC) Carotid MRI Flow Cytometry Study of Monocyte and Platelet Markers: Intraindividual Variability and Reliability

Abstract

No full-text available

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