The erythrocyte adhesiveness/aggregation test (EAAT): A new biomarker to reveal the presence of low grade subclinical smoldering inflammation in individuals with atherosclerotic risk factors - ScienceDirect

Atherosclerosis

Volume 165, Issue 2, 1 December 2002, Pages 343-351

Atherosclerosis

https://doi.org/10.1016/S0021-9150(02)00250-2 Get rights and content

Abstract

Background: Multiple acute phase proteins and atherosclerotic risk factors increase the aggregability of erythrocytes. Methods and results: We used a simple slide test and image analysis to determine the degree of erythrocyte adhesiveness/aggregation in the peripheral blood of 222 women and 221 men with no, one, two or more atherosclerotic risk factors. The degree of erythrocyte adhesiveness/aggregation correlated significantly with the concentration of commonly used variables of the acute phase response. We also showed that individuals with low erythrocyte adhesiveness/aggregation tend to be younger and to have fewer risk factors for atherosclerosis, including diabetes mellitus, hypertension, hyperlipidemia and smoking. Conclusions: The association between increased erythrocyte adhesiveness/aggregation, higher concentrations of acute phase proteins, and increased atherosclerotic risk factors points to a possible clinical applicability of the erythrocyte adhesiveness/aggregation test (EAAT) to reveal the presence of both low-grade subclinical smoldering inflammation and morbid biology in individuals with risk factors for atherosclerosis.

Introduction

Atherothrombosis is associated with the presence of a low-grade, subclinical smoldering inflammatory response [1]. Several inflammatory markers, among them high-sensitive C-reactive protein (hs-CRP) [2], the erythrocyte sedimentation rate (ESR) [3], fibrinogen concentrations [4], interleukin-6 (IL6) [5], serum amyloid A [6] and other markers of the acute phase response [7] have been shown to be related to the degree of the atherosclerotic process. These markers are relevant not only for the determination of the intensity of the inflammatory response, but have prognostic implications as well [8]. The identification and quantitation of this response might be relevant once therapeutic interventions are considered [9].

We recently introduced a new diagnostic concept for the evaluation of the degree of the inflammatory response [10], [11], [12], [13]. This concept is practically a global assessment of the acute phase response, based on the observation that various acute phase proteins participate in the induction and maintenance of increased erythrocyte aggregability [14], [15], [16]. Thus, by using simple electro-optical devices, we could easily quantitate the degree of the adhesiveness/aggregation of the cells [17]. We found that the degree of this adhesiveness/aggregation does, indeed, correlate with the intensity of the inflammatory response as determined by the classical markers, i.e. hs-CRP, ESR, quantitative fibrinogen and the white blood cell count (WBCC). In addition, we could show that the direct observation of the erythrocyte adhesiveness/aggregation [18] might be superior to either erythrocyte sedimentation or quantitative fibrinogen in the differentiation between individuals with ischemic vascular diseases and controls [11].

The aim in the present study was to characterize the clinical and laboratory profiles of patients with various degrees of erythrocyte adhesiveness/aggregation and to find the relation between multiplicity of atherosclerosis risk factors and the degree of erythrocyte adhesiveness/aggregation.

Section snippets

Participants

This prospective study was preformed between May 2000 and April 2001 at the Tel Aviv Medical Center. The local ethics committee approved the study and all the subjects gave their written informed consent to participate in it. Enrolled were apparently healthy members of the medical staff and individuals with established risk factors for atherosclerosis, including diabetes mellitus, hypertension and hyperlipidemia, who were followed in the respective outpatient clinics. Exclusion criteria

Results

The present investigation included a total of 443 individuals, 222 women with a mean±SD age of 49±16 years (range 19–86) and 221 men aged 46±13 years (range 21–82). Analysis was performed separately for women and men due to the sex-related differences in the baseline level of the inflammatory markers (i.e. hs-CRP concentrations, ESR or quantitative fibrinogen).

The Pearson correlation coefficients between the various clinical and laboratory variables and the VR (the degree of erythrocyte

Discussion

Our findings clearly demonstrate that the use of sensitive markers of the acute phase response reveals the presence of low-grade inflammation in apparently healthy individuals and in those with a spectrum of risk factors for atherothrombosis. Recent studies have presented several lines of evidence that the down-regulation of the inflammatory response might prove beneficial [20]. Hence, the use of these markers in clinical practice might have a role in the risk stratification of the patients and

Acknowledgements

The authors thank Esther Eshkol for editorial assistance, Einav Ivanov for secretarial assistance and Ron Berliner, technical engineer, for his excellent technical support.

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Cited by (35)

Quantitative Measurement of Erythrocyte Aggregation as a Systemic Inflammatory Marker by Ultrasound Imaging: A Systematic Review
2018, Ultrasound in Medicine and Biology
Citation Excerpt :
Some studies noted significant reductions in erythrocyte aggregation and improved hemorheological profiles (Allegra et al. 1995; Nicolaides 2003) after lowering the inflammatory state through lifestyle modifications (Raz et al. 2007; Sandor et al. 2014) or by the use of anti-inflammatory therapeutic agents (Ge et al. 2016; Jiang and Lian 2015; Kelly and Dominguez 2010; Li et al. 2015; Szentkereszty et al. 2014). Lastly, some studies (n = 15) underscored erythrocyte aggregation as a superior or independent marker of inflammation (Anuk et al. 2002; Assayag et al. 2005, 2008; Berliner et al. 2002, 2005; Gyawali et al. 2016; Levin et al. 2006; Maharshak et al. 2002; Rotstein et al. 2002a; Sharshun et al. 2003; Urbach et al. 2002, 2003, 2005; Vayá et al. 2013b) or sepsis (Yeom et al. 2017). Only 1 study reported increased erythrocyte aggregation with aging and associated it with increase in inflammation (Vayá et al. 2013a).
This systematic review is aimed at answering two questions: (i) Is erythrocyte aggregation a useful biomarker in assessing systemic inflammation? (ii) Does quantitative ultrasound imaging provide the non-invasive option to measure erythrocyte aggregation in real time? The search was executed through bibliographic electronic databases CINAHL, EMB Review, EMBASE, MEDLINE, PubMed and the grey literature. The majority of studies correlated elevated erythrocyte aggregation with inflammatory blood markers for several pathologic states. Some studies used “erythrocyte aggregation” as an established marker of systemic inflammation. There were limited but promising articles regarding the use of quantitative ultrasound spectroscopy to monitor erythrocyte aggregation. Similarly, there were limited studies that used other ultrasound techniques to measure systemic inflammation. The quantitative measurement of erythrocyte aggregation has the potential to be a routine clinical marker of inflammation as it can reflect the cumulative inflammatory dynamics in vivo, is relatively simple to measure, is cost-effective and has a rapid turnaround time. Technologies like quantitative ultrasound spectroscopy that can measure erythrocyte aggregation non-invasively and in real time may offer the advantage of continuous monitoring of the inflammation state and, thus, may help in rapid decision making in a critical care setup.
In vivo biodistribution and clearance of peptide amphiphile micelles
2015, Nanomedicine: Nanotechnology, Biology, and Medicine
Citation Excerpt :
DPH was excited at 350 nm and fluorescence emission was measured at 428 nm. The erythrocyte aggregation test was performed using a simple slide test as previously reported.35 Whole blood was drawn from female, 6 months old, transgenic mice homozygous for the Apoetm1Unc mutation (The Jackson Laboratory, Bar Harbor, ME, USA) via an open chest cardiac puncture and collected in tubes containing sodium citrate.
Peptide amphiphiles (PAs) are promising biomaterials for medical applications. To translate the use of PAs successfully from laboratories to clinics, in vivo studies regarding the safety of these nanomaterials are required. To examine the toxicity and clearance of PA biomaterials, we intravenously administered cy7-labeled, spherical PA micelles, control micelles without a peptide sequence, or PBS in a murine model and investigated biocompatibility, biodistribution, and clearance. Both peptide and non-peptide labeled micelles were approximately 8 nm in diameter, but of opposite surface charge. Neither micelle type caused aggregation or hemolysis of red blood cells. All micelles primarily accumulated in the bladder and were present in urine samples confirming elimination through renal clearance. Ex vivo imaging showed that micelles were also found in the liver suggesting some involvement of the reticuloendothelial system. However, no evidence of toxicity was found within the liver, spleen, kidney, bladder, intestines, lung, and heart.
Safety studies related to peptide amphiphile biomaterials are discussed in this paper, demonstrating that organotoxicity is unlikely with these materials, however, RES activation in the liver may be of consideration in further studies and needed for potential applications.
Increased erythrocyte adhesiveness and aggregation in obstructive sleep apnea syndrome
2008, Thrombosis Research
Citation Excerpt :
The lower the erythrocyte percentage and the higher the vacuum range, the greater the EAA activity (Fig. 1). This test is well documented and has been validated in previous studies [15,18–20,27–32]. Univariate analyses (ANOVA, chi-squared test, and partial and Pearson correlation) were used to investigate the association among the sleep parameters, epidemiologic data, and laboratory values.
Obstructive sleep apnea (OSA) is associated with an increased incidence of stroke and myocardial infarction as well as increased prothrombotic and inflammatory processes. Although erythrocyte adhesiveness/aggregation is known to be elevated in cardiovascular diseases, it has never been evaluated in OSA. The aim of this study was to examine the possible association of OSA and erythrocyte adhesiveness/aggregation.
The study was conducted in the Sleep Laboratory of a tertiary university-affiliated medical center in 79 patients (age 57.1 ± 12.9 years) with a diagnosis of OSA (apnea hypopnea index 41.2 ± 25.9). Findings were compared with data on 1079 controls without clinical symptoms of OSA, matched for sex, age, and body mass index. Overnight polysomnography and blood sampling for erythrocyte sedimentation rate, levels of fibrinogen, high-sensitivity C-reactive protein, and erythrocyte adhesion/aggregation test consisting of measures of erythrocyte percentage and vacuum range on image analysis.
The study group had significantly higher values than controls of all three markers of inflammation (p < 0.001 for erythrocyte sedimentation rate and fibrinogen; p = 0.037 for C-reactive protein). Erythrocyte percentage was significantly lower in the sleep apnea group (84.05 ± 15.97 vs. 90.79 ± 11.23%, p < 0.001), and vacuum range was significantly higher (8.22 ± 7.98 vs. 4.63 ± 4.05 μm, p < 0.001), indicating stronger erythrocyte adhesion/aggregation. Both these factors were significantly correlated with erythrocyte sedimentation rate and to hs-CRP (percentage: r = − 0.630; 0.258, p = 0.005; 0.031; vacuum range: r = 0.494; − 0.328, p = 0.001; 0.005 respectively).
OSA is associated with increased erythrocyte aggregation/adhesion, which is correlated with an increase in inflammation markers. These findings might help explain the increased cardiovascular morbidity in OSA.
Inflammation-sensitive proteins and erythrocyte aggregation in atherothrombosis
2005, International Journal of Cardiology
Objective: To find the relative contribution of various inflammation-sensitive proteins including fibrinogen, immunoglobulins (IgG, IgM and IgA), ceruloplasmin and high sensitivity C-reactive protein (hs-CRP) to the induction and/or maintenance of enhanced erythrocyte adhesiveness/aggregation in the peripheral blood of individuals with atherothrombotic risk factors. Methods: The degree of erythrocyte adhesiveness/aggregation was determined by a simple slide test and image analysis. In addition, we measured various inflammation-sensitive protein levels including fibrinogen, ceruloplasmin, immunoglobulins and hs-CRP in a group of 234 individuals with atherothrombotic risk factors and healthy ones. Pearson partial correlations and multiple linear regression analysis were performed. Results: Fibrinogen was found to be the major protein contributing to the enhanced erythrocyte adhesiveness/aggregation, explaining 30% of the model. Fibrinogen and IgG together explained 32.4% of the model. Other inflammation-sensitive proteins did not reach statistical significance and were excluded from the model. Conclusions: Among inflammation-sensitive proteins measured in our cohort, fibrinogen is the dominant contributor to erythrocyte adhesiveness/aggregation in the peripheral blood of individuals with atherothrombotic risk factors and healthy ones. These findings may pave the way for the development of therapeutic strategies directed at the attenuation of erythrocyte aggregability in individuals with atherothrombosis.
Erythrocyte adhesiveness/aggregation: A novel biomarker for the detection of low-grade internal inflammation in individuals with atherothrombotic risk factors and proven vascular disease
2005, American Heart Journal
Citation Excerpt :
We therefore turned to the erythrocyte as a possible sensor for the detection of adhesive proteins in the peripheral blood. In fact, we could show that increased erythrocyte adhesiveness/aggregation follows the infusion of immunoglobulins,34 that it can be abolished after plasmapheresis,35 and that the direct quantitation might be superior to the indirect method of Zeltser et al.36 Moreover, we could earlier show a consistent and significant link between the presence of atherosclerotic risk factors, the intensity of the inflammatory response, and the degree of cell adhesiveness/aggregation in the peripheral blood.18,19 We presented our results separately for men and women as a result of baseline differences that exist between the sexes regarding hemoglobin concentrations, ESR, fibrinogen, and even hs-CRP.
We have introduced a concept of using the erythrocyte as a sensor for the detection of enhanced inflammation-sensitive protein concentrations. We presently evaluated the capability of this new biomarker to detect the presence of inflammation in individuals with a history of a vascular disease as opposed to individuals with atherothrombotic risk factors but no clinically evident vascular disease.
The degree of erythrocyte adhesiveness/aggregation was determined in the peripheral venous blood by using a simple blood test. Blood was drawn into a syringe containing sodium citrate and trickled onto a slide at an angle of 30°. The slides were than scanned by a blinded technician by using an image analyzer to determine the area that is covered by the erythrocytes.
One hundred fifty-six subjects (61 women and 95 men) of 2586 (1238 women and 1348 men) met the criteria of a definite vascular disease (history of stroke, myocardial infarction, coronary artery bypass grafting, or peripheral vascular disease). The degree of erythrocyte aggregation was significantly (P = .008) higher in men, but not in women, with vascular disease as opposed to these without a vascular disease. The results of receiver operating characteristic curve analysis confirmed the diagnostic superiority of the erythrocyte aggregation biomarker over other commonly used markers of the acute phase in men. Similar results were obtained by using discriminant analysis. Finally, a significant correlation was found between the degree of erythrocyte aggregation and other markers of the acute phase suggesting its relevance for the detection and quantitation of low-grade inflammation in individuals with atherothrombosis.
Erythrocyte adhesiveness/aggregation may be a useful biomarker to detect internal inflammation in individuals with atherothrombosis.
The degree of red blood cell aggregation on peripheral blood glass slides corresponds to inter-erythrocyte cohesive forces in laminar flow
2004, Thrombosis Research
Objective: To determine the degree of correlation between red blood cell (RBC) aggregation on peripheral blood glass slides (PBGS) as determined by image analysis and the inter-erythrocytic cohesive forces as determined in a computerized cell flow properties analyzer (CFA). Study design: RBC aggregation was assessed using both systems simultaneously in healthy volunteers, obese patients and hypercholesterolemic individuals before and following LDL apheresis. Results: A significant (r=0.5, p<0.001) correlation was noted between the flow-dependent average aggregate size (AAS) obtained in the CFA and the degree of RBC aggregation on peripheral blood glass slides. Moreover, the enhanced RBC aggregation on the slides was positively associated with the appearance of larger aggregates in the CFA (r=0.5, p<0.001) and inversely with the formation of smaller aggregates (r=−0.27, p<0.04). A similar reduction in RBC aggregation following LDL apheresis was noted in both systems. Conclusions: The phenomenon of RBC aggregation on peripheral blood glass slides is governed by significant inter-erythrocytic cohesive forces and is not a result of a mere coincidental superimposition of cells. The slide test offers a rapid and simple method of evaluating rheologically significant RBC aggregation, and may allow stratification of patients at risk for atherothrombosis.

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¹: Professor Shlomo Berliner is a shareholder in the Inflamet Ltd. Company, Tel Aviv, Israel.

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