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ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 3  |  Page : 880-886

Reticulated platelets in acute coronary syndrome patients


1 Clinical Pathology Department, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Shebin El-Kom Teaching Hospital, Menoufia, Egypt

Date of Submission02-Mar-2016
Date of Acceptance22-May-2016
Date of Web Publication15-Nov-2017

Correspondence Address:
Asmaa M Mohammed Mousa
Shebin El-Kom, Menoufia, 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.218251

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  Abstract 

Objective
The aim of this study was to evaluate reticulated platelets (RP) as a predicting factor of the outcome in acute coronary syndrome (ACS) patients.
Background
ACS is due to rupture of an arterial plaque. RPs are newly formed platelets with a greater mass, residual amount of RNA, and an increased prothrombotic potential compared with mature platelets. In this study, the RPs were investigated as participating factors in the pathogenesis of ACS.
Patients and methods
A total of 50 patients with ACS [33 acute myocardial infarction (AMI) and 17 unstable angina] and 15 clinically healthy individuals as controls were investigated for RPs by means of flowcytometry analysis.
Results
RPs were highly significantly increased in AMI than in unstable angina and controls.
Conclusion
High levels of RPs are found in ACS patients and it can be used as a predictor for adverse outcome. Moreover, it can participate in the development of AMI due to its high thrombogenic potential.

Keywords: acute coronary syndrome, flowcytometry, reticulated platelets


How to cite this article:
Khalifa KA, Helwa MA, Mohammed Mousa AM. Reticulated platelets in acute coronary syndrome patients. Menoufia Med J 2017;30:880-6

How to cite this URL:
Khalifa KA, Helwa MA, Mohammed Mousa AM. Reticulated platelets in acute coronary syndrome patients. Menoufia Med J [serial online] 2017 [cited 2019 Nov 17];30:880-6. Available from: http://www.mmj.eg.net/text.asp?2017/30/3/880/218251


  Introduction Top


Acute coronary syndrome (ACS) describes the clinical manifestations that follow the disruption of coronary arterial plaque complicated by thrombosis, embolization, and varying degrees of obstruction to myocardial perfusion. The clinical features depend upon the extent and the severity of myocardial ischemia [1].

Platelets play a pivotal role in the occurrence of atherothrombotic events [2]. Reticulated platelets (RPs), as they are analogous to erythroid reticulocytes, comprise the youngest component of the circulating platelet pool [3]. They contain measurable amounts of cytosolic mRNA [4]. RPs also tend to be larger in size and contain more dense granules, metabolically and enzymatically more active, thus having a greater prothrombotic potential [5].

In ACS patients, the acute event is able to elicit an inflammatory response with the release of a variety of proinflammatory cytokines, which may influence platelet turnover with the subsequent mobilization and release of newly formed large, reticulated, reactive platelets from the bone marrow [6]. Immature platelets with an increased hemostatic potential may contribute to coronary thrombus progression and acute myocardial infarction (AMI) development [7]. RPs can be identified by their RNA content using flowcytometry [8].

The aim of the present study was to evaluate RPs as a participating factor in the pathogenesis of ACS and its ability to predict the outcome of ACS.


  Patients and Methods Top


This study was conducted on 65 individuals selected from Menoufia University Hospital. All participants provided written informed consent, and approval from the ethics committee of Faculty of Medicine was obtained. They were divided into two groups:

Group I (patients with ACS) included 50 patients with ACS [33 AMI patients and 17 unstable angina (UA) patients], 36 male and 14 female; their ages ranged from 47 to 75 years, with a mean ± SD age of 61.32 ± 7.20 years.

Group II (the control group) included 15 apparently healthy individuals, 12 male and three female, who served as the control group; their ages ranged from 45 to 74 years, with a mean ± SD age of 57.40 ± 7.89 years.

All participants in this study were subjected to the following:

  1. Full medical history, emphasizing on family history and past history, including history of diabetes mellitus, hypertension, smoking, recurrent chest pain, and history of medication.
  2. Clinical examination.
  3. Laboratory investigations, including complete blood count (CBC) and flowcytometry analysis of RPs and calculation of RP fraction (RP %).


Sample collection

A volume of 2 ml of venous blood sample was drawn from every patient at the time of hospitalization in the ACS group and from every control. The blood sample was obtained through antecubital venous access under complete aseptic technique and emptied into a BD Vacutainer plastic blood collection tube containing K2 EDTA (di-potassium ethylendiamine tetra-acetic acid) as anticoagulant (Fisher Scientific Safety Services, MA, USA). The tubes were immediately inverted 8–10 times and analyzed in an automated ADVIA-2120 hematological analyzer (Bayer Diagnostics, Newbury, UK) with examination of Leishman-stained smears for red blood cell morphology, differential white blood cell count and platelet count, and morphology.

The remaining blood from CBC examination was taken for measurement of RP % with immunophenotyping of peripheral blood samples.

Assessment of reticulated platelets by immunophenotyping

The principle depends on enumeration of CD61 and thiazole orange-positive cells. Detection of CD61 was performed using monoclonal antibodies (IMMUNOSTEP is located in Salamanca [Spain]). This test depends on the ability of a monoclonal antibody to bind to the surface of the cells expressing CD61. Staining of RNA remnants of immature platelet (RP) was performed with thiazole orange reagent [9].

RPs were measured with flowcytometry using the Becton-Dickinson fluorescence-activated flowcytometer (FACS Calibur; BD Immunecytometry Systems, San Jose, California, USA).

Procedure

  1. Platelet-rich plasma (PRP) was prepared by centrifugation of EDTA blood at 200g for 10 min.
  2. The supernatant PRP was separated into another clean tube.
  3. Thereafter, we added 20 μl of Monoclonal Anti-Human CD61 PE to 10 μl of PRP and mixed gently with a vortex mixer.
  4. The mixture was incubated in a dark at room temperature (20–25°C) for 15 min.
  5. Thereafter, we added 1 ml of thiazole orange (BD RETIC-COUNT; Becton, Dickinson, Hilden, Germany) to the previous tube, and then the tube was capped and vortexed gently and incubated in the dark at room temperature (20–25°C) for 15 min.
  6. The previous mixture was washed three times using 2 ml PBS and finally suspended in 200 μl PBS.
  7. Negative auto control tube was prepared containing 200 μl PBS + 10 μl PRP.


Flowcytometric analysis

Data were acquired using Becton-Dickinson fluorescence-activated flowcytometer (FACS Calibur; BD Immunecytometry Systems) in the Clinical Pathology Department, Faculty of Medicine, Menoufia University.

The instrument set up was checked weekly using QC windows beads (flowcytometry standard; San Juan, Puerto Rico, West Midlands, UK). Forward scatter and side scatter measurements were made using linear amplifiers, and fluorescence measurements were made with logarithmic amplifiers.

  1. An electronic gate was made around platelets according to light scatter characteristics after adjustment of the software to a linear histogram.
  2. To exclude cell autofluorescence and instrument noise of fluorescence, a negative autocontrol was examined and the histogram was adjusted in a way that more than 99% of the unstained platelet population was in the double negative left lower quadrant. In a second histogram, the percentage of (CD61 PE, TO) positive platelets was calculated automatically using the software of the flowcytometer apparatus [10].


Calculation

  1. Absolute reticulated platelet count (also called immature platelet count) was calculated as follows: IPC = RP %×platelet count).


Statistical analysis

The data were collected, tabulated, and analyzed using SPSS version 22.0. Data were expressed as percentage (%), and mean and SD. The c2-test was used to study association between two qualitative variables. Student's t-test was used as a test of significance for comparison between two groups having normally distributed quantitative variables. The Mann–Whitney test for unpaired data was used for comparison between two groups. Spearman's correlation coefficient (r) (nonparametric test) is a test used to measure the association between two quantitative variables. To examine the association between RP and cardiovascular risk factors, we performed binary logistic regression analysis. To test the independent risk factor for AMI, the level of significance was set as P value less than 0.05.


  Results Top


There was no statistically significant difference between the studied groups as regards age and sex (P > 0.05 = nonsignificant) [Table 1].
Table 1: Comparison between studied groups as regards age and sex

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There was a highly significant difference between cases and controls as regards RP % (P < 0.001) and RP count (P < 0.001) [Table 2], and there was a significant difference between AMI and UA as regards RP % (P < 0.003) and RP count (P < 0.043) [Table 2].
Table 2: Comparison between studied groups as regards RP % and RP count

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Comparison between cases and controls as regards RP % and RP count is shown in [Figure 1].
Figure 1: Comparison between cases and controls as regards reticulated platelet percentage (RP %) and absolute reticulated platelet (ARP).

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Comparison between AMI and UA as regards RP % and RP count is shown in [Figure 2].
Figure 2: Comparison between acute myocardial infarction (AMI) and unstable angina (UA) as regards reticulated platelet percentage (RP %) and absolute reticulated platelet (ARP).

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Comparison between subtypes of acute cardiac conditions as regards RP % and RP count showed the highest level of RP % in the ST-elevation myocardial infarction (STEMI) group [Table 2] and [Figure 3].
Figure 3: Comparison between subtypes of acute cardiac conditions as regards reticulated platelet percentage (RP %) and absolute reticulated platelet (ARP).

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The comparison between AMI and UA cases showed a significant difference as regards previous cardiovascular intervention (P < 0.05) [Table 3].
Table 3: Comparison between AMI and UA as regards clinical data

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RP % and RP count showed a statistically significant relationship with cardiac risk factors such as smoking, hypertension, diabetes mellitus, and previous cardiovascular intervention (P < 0.05) [Table 4].
Table 4: Relationship between RP % and ARP and other risk factors

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The correlation study between MPV and RP % and RP count among cases revealed a highly significant positive correlation (P < 0.001) [Figure 4] and [Figure 5]).
Figure 4: Spearman correlation between reticulated platelet percentage (RP %) and MPV among cases.

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Figure 5: Spearman correlation between reticulated platelet (RP) count and MPV among cases.

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Binary logistic regression analysis for independent risk factors of acute cardiovascular diseases showed that RP % is an independent risk factor for acute cardiovascular diseases (odds ratio = 1.9 and 95% confidence interval = 1.01–19.87) [Table 5].
Table 5: Binary logistic regression analysis for independent risk factors of acute cardiovascular diseases

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  Discussion Top


ACS are a set of signs and symptoms due to rupture of atherosclerotic plaque and are a consequence of platelet-rich coronary thrombus formation. The thrombus leads to partial or complete coronary artery occlusion, which, in turn, leads to myocardial ischemia and various clinical manifestations ranging from UA to AMI [11]. Patients with ACS include UA, non-ST-elevation myocardial infarction (NSTEMI), and STEMI [12].

RPs are the youngest forms of circulating platelets that contain residual mRNA. These platelets are larger and possibly more active compared with non-RPs [13].

Platelets and their activity have an important role in the initiation of atherosclerotic lesions and coronary thrombus formation. Larger platelets are enzymatically and metabolically more active and have a higher potential thrombotic ability as compared with smaller platelets [14].

In the light of the previous postulations, the aim of this study was to evaluate RPs as a participating factor in the pathogenesis of ACS and its ability to predict the outcome of ACS.

In this study, there was a nonsignificant decrease in platelet count in group I (ACS) compared with group II (controls) (P > 0.05 = nonsignificant).

This finding is in agreement with that of Fatih et al. [15], who concluded that there was no difference in platelet counts between control, STEMI, NSTEMI, and UA patients, and it is also similar to the finding of Lippi et al. [16], who found that the platelet count did not differ significantly between ACS cases and controls.

This study found that MPV level was significantly increased in group I (ACS) compared with group II (controls) (P ≤ 0.001) and significantly increased in AMI than in UA (P = 0.008).

These findings are in agreement with the studies by Arevalo-Lorido et al.[17] and Murat et al. [18], who reported that MPV is an independent risk factor for myocardial infarction in coronary artery disease and that it might be a determinant factor for future ischemic episodes and death.

As regards RPs, we find that RP % and RP count level were significantly higher in group I (ACS) compared with group II (control) (P ≤ 0.001). This is in agreement with the finding of Lakkis et al. [9], who reported that RP % is higher in cases than in controls using flowcytometric analysis and platelet staining with thiazole orange stain.

In this study, the comparison between different types of ACS as regards RP % and RP count revealed a statistically significant difference between STEMI, NSTEMI, and UA patients.

These findings are in agreement with those of Lakkis et al. [9] and Grove et al. [7], who reported that immature platelet fraction (IPF) % was higher in STEMI patients than in NSTEMI and UA patients (P < 0.001).

In this study, there was an association between RP % and risk factors for coronary disease, such as smoking. There was a highly statistically significant relationship between IPF% and smoking (P < 0.001). This is in agreement with the findings of Grove et al. [7], who reported that, in active smokers, RP was 18% higher than that in nonsmoking individuals (P = 0.007).

In addition, the present study showed a highly significant association between RP % and diabetes (P < 0.001). This is in agreement with the finding of Cesari et al. [5], who showed that platelet turnover was accelerated in smokers and diabetic patients.

The binary logistic regression analysis showed that RP % is an independent risk factor for acute cardiovascular diseases (odds ratio = 1.9 and 95% confidence interval = 1.01–19.87). This is in agreement with the findings of Ibrahim et al. [19], who reported that there is an association of immature platelets with adverse cardiovascular outcomes, and hence immature platelet is a novel biomarker for major adverse cardiovascular events and can help in risk stratification in patients with coronary artery disease. Moreover, Cesari et al.[5] found that RPs are significantly associated with an increased risk for cardiovascular death in ACS patients.


  Conclusion Top


The present study demonstrates that RP % is higher in ACS patients than in controls, and higher in STEMI than in NTEMI and UA patients.

RP % is positively correlated with MPV.

Logistic regression for factors affecting AMI indicates that RP % is an independent risk factor for AMI.

High levels of RPs are found in ACS patients and it can be used as a predictor for adverse outcome. Moreover, it can participate in the development of AMI due to its high thrombogenic potential.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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