Leukocyte aggregation-related pseudoleukopenia in pediatrics: a sporadic event or a systematic error?

OBJECTIVE: To determine whether electronic counter-related pseudoleukopenia is a rare phenomenon or a systematic underestimation in children with acute infection/inflammation. METHODS: We have used a simple slide test and image analysis to reveal the number of white blood cells and their degree of aggregation. The number of leukocytes counted by an electronic cell analyzer was divided by the number of cells counted on the slides creating an electronic cell-to-slide leukocyte count ratio. RESULTS: A significant (P < 0.0005) negative (r = -0.314) correlation between the above mentioned ratios and the percent of aggregated leukocytes in the peripheral blood was found in a group of 239 children with various acute infections. Thus elevated leukocyte aggregation is associated with a relatively lower electronic analyzer cell count. CONCLUSIONS: The appearance of aggregated leukocytes in the peripheral blood during acute infections might be associated with pseudoleukopenia. This phenomenon has been extensively described in the adult population and seems to exist in children as well.

Automatic 3-dimensional visualization of peripheral blood slides: a new approach for the detection of infection/inflammation at the point of care.

CONTEXT: The identification and quantitation of the intensity of the acute-phase response at the point of care might be of clinical relevance. OBJECTIVE: To report the possibility of automatic screening of unstained peripheral blood slides by using a 3-dimensional image analysis system. DESIGN: Peripheral venous blood was obtained from children with acute inflammation/infection and examined by an automatic 3-dimensional image analyzer to detect the number of white blood cells as well as to reveal the degree of erythrocyte aggregation, a marker of the humoral phase response. RESULTS: We included 66 children with acute bacterial infections and 59 with nonbacterial inflammation/infection; mean ages of the 2 groups were 4.3 +/- 3.9 years and 4.2 +/- 3.7 years, respectively (P = .91). The percentages of correct classifications based on discriminant analysis in predicting between bacterial and nonbacterial inflammation/infection were 61.3% by using the white blood cell count, 64.5% by using the percentage of granulocytes, 61.6% by using the degree of erythrocyte aggregation, and 59.2% by using the number of leukocytes counted on the unstained slides. The results of the receiver-operated characteristic curve analysis yielded an area under the curve of 0.714 (P < .001) for the number of granulocytes, 0.699 (P < .001) for the white blood cell count, 0.685 (P < .001) for the number of leukocytes on the slides, and 0.685 (P = .001) for the degree of erythrocyte aggregation. The correlation between the number of leukocytes by the electronic cell analyzer and the number of cells counted on the slides was highly significant (r = 0.85, P < .001). CONCLUSIONS: It is feasible to use an automatic 3-dimensional image analyzer to reveal the different intensities of the acute-phase response between a group of children with an acute bacterial infection and another with nonbacterial inflammation/infection. These findings might be relevant for potential application at the point of care.

Erythrocyte adhesiveness/aggregation: a novel biomarker for the detection of low-grade internal inflammation in individuals with atherothrombotic risk factors and proven vascular disease.

BACKGROUND: 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. METHODS: 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 degrees. The slides were than scanned by a blinded technician by using an image analyzer to determine the area that is covered by the erythrocytes. RESULTS: 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. CONCLUSION: Erythrocyte adhesiveness/aggregation may be a useful biomarker to detect internal inflammation in individuals with atherothrombosis.

Inflammation-sensitive proteins and erythrocyte aggregation in atherothrombosis.

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.

Roxithromycin-induced torsades de pointes.

Roxithromycin is an oral macrolide antibiotic agent frequently used in the clinical setting. The risk of pro-arrhythmia during roxithromycin use has always been considered extremely remote. We describe a patient who developed prolongation of the QT interval and torsades de pointes under roxithromycin treatment.

Clinical and laboratory characteristics of patients with atherothrombotic risk factors presenting with low concentrations of highly sensitive C-reactive protein.

AIMS: To clarify whether individuals with low concentrations of highly sensitive C-reactive protein (hs-CRP) are free of atherothrombotic risk factors or low-grade inflammation. METHODS AND RESULTS: This cross-sectional study included 1266 individuals with risk factors (diabetes, hypertension, hyperlipidemia, smoking, obesity). Eighty percent of women and the same percentage of men with a low concentration (0.8 +/- 0.4 mg/l) of hs-CRP had at least one atherothrombotic risk factor. About one-fourth of the females and a one-third of the males with a low concentration of hs-CRP had at least one laboratory marker of chronic inflammation (erythrocyte sedimentation rate (ESR), white blood cell count (WBCC) or fibrinogen concentration) in the upper 75th percentile. CONCLUSION: Individuals presenting with low concentrations of hs-CRP are not necessarily free of atherothrombotic risk factors. One-fourth of the females and a third of the males in this group presented laboratory evidence of a low-grade inflammation as detected by increased erythrocyte sedimentation rate, white blood cell count or fibrinogen concentration.

Gender difference in C-reactive protein concentrations in individuals with atherothrombotic risk factors and apparently healthy ones.

Recent studies have shown that C-reactive proteins have a pathogenetic role in atherothrombosis and concentrations of these substances could be used as a marker for future vascular events. The objective of this study was to determine gender differences in highly sensitive C-reactive protein (hs-CRP) in individuals with atherothrombotic risk factors and apparently healthy ones. We have presently matched 469 females and 469 males having the same age and body mass index (BMI). Of these, 210 men and 210 women had no atherothrombotic risk factors. In this group the hs-CRP concentrations were 1.6+/-3.4 mg l(-1) in women and 1.0+/-2.7 mg l(-1) in men (p<0.0005). These values were 2.1+/-3.4 mg l(-1) and 1.5+/-2.8 mg l(-1), respectively, in the entire cohort (p<0.0005), which included also individuals with atherothrombotic risk factors. We conclude that significant gender differences exist in hs-CRP concentrations despite perfect matching for age and BMI. These differences should be reflected in guidelines that suggest hs-CRP cut-off points for the stratification of vascular risk.

Rheological determinants in patients with Gaucher disease and internal inflammation.

The purpose of this study was to determine whether the inflammatory response in patients with Gaucher disease (GD) is accompanied by enhanced adhesiveness/aggregation of both red and white blood cells. Sixty patients with GD and matched controls were included. The degree of erythrocyte and leukocyte adhesiveness/aggregation was determined by using a simple slide test and image analysis. Patients with GD had significantly elevated concentrations of fibrinogen (328 vs. 262 mg/dl, P < 0.0001) and accelerated erythrocyte sedimentation rates (27 vs. 13 mm/H, P < 0.005). This was accompanied by a significantly enhanced degree of erythrocyte (75 vs. 85, P < 0.0001) and leukocyte (3.5 vs. 1.3, P < 0.002) adhesiveness/aggregation. The low-grade, smoldering, and subclinical internal inflammation in individuals with GD is accompanied by an increased degree of erythrocyte and leukocyte adhesiveness/aggregation. These findings might have rheological consequences in terms of microcirculatory slow flow and tissue hypoxemia.

Telemedicine-based application for the detection of inflammation in pediatrics.

To assess the feasibility of using telemedicine for the diagnosis of inflammation in a pediatric population. The degree of erythrocyte adhesiveness/aggregation and the number of white blood cells on peripheral blood slides were evaluated using an image analysis system and transferred to an operator in a remote location. A highly significant correlation was found between the degrees of erythrocyte adhesiveness/aggregation as well as estimated white blood cell count determined by a remote operator and the objective measurements of both erythrocyte sedimentation rate and white blood cell count. A reasonable estimate of both erythrocyte sedimentation rate and white blood cell count can be obtained by using telemedicine in a group of children with acute infection/inflammation.

The appearance of aggregated erythrocytes in the peripheral blood of individuals with insulin resistance.

BACKGROUND: Insulin resistance is associated with low-grade inflammatory response. The probability that the acute-phase response is associated with enhanced erythrocyte adhesiveness/aggregation was not explored. METHODS: The degree of erythrocyte adhesiveness/aggregation was evaluated by using a simple slide test. The insulin resistance was evaluated by insulin and glucose concentrations after a night of fasting. The inflammatory response was evaluated by variables of acute-phase response. RESULTS: A significant correlation (r = -0.2, p = 0.02) was noted between insulin resistance expressed as the HOMA index and the degree of erythrocyte adhesiveness/aggregation. This was probably due to the concomitant acute-phase response and the presence of increased amounts of inflammation-sensitive proteins that were found to correlate significantly with the degree of erythrocyte adhesiveness/aggregation. In the multiple linear regression analysis, erythrocyte sedimentation rate and fibrinogen concentration but not HOMA index were found to correlate significantly (p < 0.0001 and p = 0.0007 respectively) with the degree of red blood cell adhesiveness/aggregation. CONCLUSIONS: Insulin resistance is associated with an enhanced degree of erythrocyte adhesiveness/aggregation and this is related to the presence of enhanced inflammation-sensitive plasma proteins that are part of the acute-phase response. These findings might have hemorheological consequences and might contribute to the pathophysiology of the insulin-resistance syndrome.

A synergistic effect of albumin and fibrinogen on immunoglobulin-induced red blood cell aggregation.

Therapeutic administration of immunoglobulins (Ig) has the potential to precipitate thrombotic events. This phenomenon may be explained by red blood cell (RBC) aggregation, which can be potentiated by Ig. The contribution of plasma albumin and fibrinogen to Ig-induced RBC aggregation is unclear. We examined RBC aggregation in three settings: 1) patients receiving therapeutic infusions of Ig; 2) patients receiving plasma supplemented in vitro with Ig; and 3) patients receiving RBC suspensions in standard buffer with varying concentrations of albumin, Ig, and fibrinogen. Ig infusion augmented aggregation of RBCs from patients with normal or high plasma levels of albumin but decreased aggregation in those with lower plasma albumin concentrations. In vitro, RBC aggregation was significantly increased only when all three components, fibrinogen, albumin, and Ig, were present at or above normal concentrations in the suspension but was unaffected when any one of the components was absent from the suspension. Our results suggest a three-way interaction among fibrinogen, Ig, and albumin that synergistically induces RBC aggregation in plasma. Understanding these interactions may help predict clinically important phenomena related to RBC aggregation, such as thrombotic complications of Ig infusion.

Inflammation at a glance: erythrocyte adhesiveness/aggregation test to reveal the presence of inflammation in people with atherothrombosis.

The erythrocyte adhesiveness/aggregation test is a new biomarker to detect low-grade inflammation in patients with atherothrombosis. In a group of 30 individuals with an acute ischemic event, the variability of EAAT during a follow-up period was similar to those obtained for other commonly used markers of the acute phase response, suggesting the potential clinical use of this novel marker.

Enhanced erythrocyte adhesiveness/aggregation in obesity corresponds to low-grade inflammation.

OBJECTIVE: Previous studies have suggested that obesity enhances the inflammatory response, producing macromolecules involved in the induction and/or maintenance of increased erythrocyte aggregation. The objectives of this study were to evaluate the correlation between inflammation markers, erythrocyte adhesiveness/aggregation, and the degree of obesity and to assess phosphatidylserine expression on erythrocyte surface membrane of obese vs. nonobese individuals. RESEARCH METHODS AND PROCEDURES: Erythrocyte adhesiveness/aggregation in the peripheral venous blood was evaluated by using a new biomarker, phosphatidylserine expression was assessed by means of flow cytometry, and markers of inflammation were measured in 65 subjects: 30 obese [body mass index (BMI) = 41 +/- 7.7 kg/m(2)] and 35 nonobese (BMI = 24 +/- 2.7 kg/m(2)) individuals. Pearson correlations and Student's t test were performed. RESULTS: A highly significant difference was noted in the degree of erythrocyte adhesiveness/aggregation and markers of inflammation between the study groups. BMI correlated with erythrocyte adhesiveness/aggregation (r = 0.42, p = 0.001), erythrocyte sedimentation rate (r = 0.42, p = 0.001), high-sensitive C-reactive protein (r = 0.55, p < 10(-4)), fibrinogen (r = 0.37, p = 0.004), and white blood cell count (r = 0.45, p < 10(-4)). The degree of erythrocyte adhesiveness/aggregation correlated with erythrocyte sedimentation rate (r = 0.5, p < 10(-4)), high-sensitive C-reactive protein (r = 0.56, p < 10(-4)), fibrinogen (r = 0.54, p < 10(-4)), and white blood cell count (r = 0.32, p = 0.01). DISCUSSION: Our results suggest that obesity-related erythrocyte adhesiveness/aggregation is probably mediated through increased concentrations of adhesive macromolecules in the circulation and not necessarily through hyperlipidemia or phosphatidylserine exposure on erythrocyte's membrane.

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.

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.

A simple biomarker to exclude the presence of low grade inflammation in apparently healthy individuals.

BACKGROUND: Atherosclerosis is accompanied by a low grade inflammatory response. HYPOTHESIS: To use erythrocyte aggregability as a biomarker to exclude the presence of low grade inflammatory response in apparently healthy individuals. METHODS: The adhesiveness/aggregation of red blood cells was quantitated by using a simple slide test and image analysis. RESULTS: We included 121 apparently healthy individuals and found a significant correlation between the degree of erythrocyte adhesiveness/aggregation and either the concentration of high sensitive CRP (r = 0.6, P < 0.001), erythrocyte sedimentation rate (r = 0.5, P < 0.0001) or fibrinogen (r = 0.5, P < 0.0001). By using certain cutoff points for the erythrocyte adhesiveness/aggregation test we could define individuals with a very low grade inflammatory response. CONCLUSIONS: By using this inexpensive and rapid assessment, we could clearly discriminate between individuals with a very low inflammatory response and those with a more intense one. This biomarker should be further evaluated as a possible screening test for use in large populations of apparently healthy individuals in whom the detection of low grade inflammation might contribute to guiding appropriate lifestyle modifications and therapeutic interventions.

Iodide-induced sialadenitis complicating coronary angiography.

Sialadenitis is an uncommon complication of intravenous administration of iodinated contrast medium. We present two cases of iodide-induced sialadenitis following coronary angiography. Clinical features and clues to differentiation from suppurative sialadenitis are discussed. The mechanism for aseptic iodide-induced sialadenitis remains unclear, but prognosis with conservative treatment is apparently good. Sialadenitis may recur with subsequent administration of contrast medium.

Detection of a thrombolysis-related reduction in red blood cell adhesiveness/aggregation using a simple slide test.

Increased red cell aggregability might have unfavorable rheological effects in the microcirculation. It has been suggested that thrombolysis-related hypofibrinogenemia might be associated with a reduced erythrocyte adhesiveness/aggregation. We followed the reduction in erythrocyte adhesiveness/aggregation using a simple slide test and image analysis that measures the spaces that are formed between the aggregated erythrocytes. A significant (p = 0.01) reduction in the degree of erythrocyte adhesiveness/aggregation was noted in patients with acute myocardial infarction who received thrombolysis as opposed to individuals with Braunwald Class IIIB unstable angina who had normal fibrinogen concentrations. No change was found in the Westergren erythrocyte sedimentation rate, which is an indirect method to detect changes in aggregability of red blood cells. The present study shows the superiority of using a direct measurement of red blood cell adhesiveness/aggregation. This extremely rapid, cheap and almost bedside methodology to detect changes in erythrocyte adhesiveness/aggregation might be useful to detect changes of hemorheological relevance following thrombolysis.

Superiority of a functional leukocyte adhesiveness/aggregation test over the white blood cell count to discriminate between mild and significant inflammatory response in patients with acute bacterial infections.

Electronic cell counters may underestimate the white blood cell count (WBCC) in the presence of aggregated leukocytes. In the present study we focused on the possibility of using a functional, as opposed to an anatomic, count to circumvent this eventual underestimation. A model of bacterial infection was used because of the importance of leukocytosis in the physician's clinical decision-making process. There were 35 patients with low C-reactive protein (CRP) concentrations (0.5-4.9 mg/dL), 45 with intermediate (5-9.9 mg/dL), and 120 with relatively high (>10 mg/dL) CRP concentrations. A significant (P=0.008) difference was noted between the state of leukocyte adhesiveness/aggregation in the peripheral blood of individuals with low CRP concentrations (3.5%+/-4.3%) and those with high CRP concentrations (7.4%+/-8%), while there was no significant difference in the respective number of WBCs per cubic millimeter (cmm) (11,600 +/- 5,500 and 14,000 +/- 7,200, respectively). We raise the possibility that a functional test might be superior over an anatomic count in patients with acute bacterial infection and a significant acute phase response.