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Detection of platelet-monocyte aggregates by the ADAM(®) image cytometer.

Jung BK, Cho CH, Moon KC, Sung Hur D, Yoon JA, Yoon SY - Int J Med Sci (2014)

Bottom Line: Moreover, the PMA measurements made by the ADAM(®) cytometer exhibited a high correlation with those made by a flow cytometric assay (R = 0.944).The ADAM(®) cytometer is a suitable alternative method to the flow cytometry-based assays.This, in turn, will provide valuable information regarding patient propensities to thrombotic diseases.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Seoul 152-703, South Korea.

ABSTRACT

Background: Inappropriate platelet activation is known to be associated with various thrombotic disorders. Platelet-monocyte aggregates (PMAs), whose formation is mediated by platelet surface P-selectin (CD62P), can be used as a reliable marker to detect platelet activation. Previous studies have generally detected PMAs through flow cytometry-based approaches. Recently, the ADAM(®) image cytometer (Nanoentek Inc., Seoul, Korea) was developed for image-based cellular analysis. In this study, we detected PMAs with the ADAM(®) cytometer, evaluated the reproducibility of the measurements made by the ADAM(®) cytometer, and compared the abilities of the ADAM(®) cytometer and a flow cytometric assay to detect PMAs.

Methods: Whole blood samples were collected from patients. Within 5 minutes of collection, anticoagulated whole blood samples were fixed in 10% paraformaldehyde and 5% glyoxal. Nineteen clinical specimens were collected; each was analyzed three times with the ADAM(®) cytometer in order to assess the reproducibility of its measurements. To compare the ability of the ADAM(®) cytometer with that of a flow cytometer to detect PMAs, each cytometer was used for 23 clinical samples and the correlation of the measurements was determined.

Results: The PMA measurements made by the ADAM(®) cytometer showed good reproducibility (CV < 10% for all specimens). Moreover, the PMA measurements made by the ADAM(®) cytometer exhibited a high correlation with those made by a flow cytometric assay (R = 0.944).

Conclusions: The ADAM(®) cytometer is a suitable alternative method to the flow cytometry-based assays. Since the ADAM cytometer does not need specialized instrument knowledge or software proficiency (unlike flow cytometry), the ADAM(®) cytometer can be used as a rapid and reliable POCT device to measure platelet activation in peripheral blood. This, in turn, will provide valuable information regarding patient propensities to thrombotic diseases.

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Flow cytometric features of leukocytes, monocytes, and platelet-monocyte aggregates (PMAs). Flow cytometric analysis of negative controls is shown in (A), (B), and (C), whereas flow cytometric analysis of a patient sample (the first to be analyzed) is shown in (D), (E), and (F). (A) All events were acquired by flow cytometry after lysing RBCs in whole blood specimens. In this plot, monocytes are characterized by high CD45 expression and intermediate side scatter. (B) All events shown in (A) were represented in a CD14 and CD45 plot. The monocytic region is characterized by high CD14 and CD45 expression (region within black box). (C) All events within black box in (B) were represented in a CD14 and CD41 plot. Events characterized by high expression of both CD14 and CD41 designate the PMAs.
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Figure 1: Flow cytometric features of leukocytes, monocytes, and platelet-monocyte aggregates (PMAs). Flow cytometric analysis of negative controls is shown in (A), (B), and (C), whereas flow cytometric analysis of a patient sample (the first to be analyzed) is shown in (D), (E), and (F). (A) All events were acquired by flow cytometry after lysing RBCs in whole blood specimens. In this plot, monocytes are characterized by high CD45 expression and intermediate side scatter. (B) All events shown in (A) were represented in a CD14 and CD45 plot. The monocytic region is characterized by high CD14 and CD45 expression (region within black box). (C) All events within black box in (B) were represented in a CD14 and CD41 plot. Events characterized by high expression of both CD14 and CD41 designate the PMAs.

Mentions: The gating strategy to identify platelet-monocyte aggregates (PMAs) is shown in Figure 1. Flow cytometric analysis of negative controls is shown in Figure 1(A), 1(B), and 1(C), whereas flow cytometric analysis of a patient sample (the first to be analyzed) is shown in Figure 1(D), 1(E), and 1(F). The numbers labeling the gates represent the percentages of events inside each gate. The target cell populations gated in the SSC and CD45-PC5 plot were transferred into the CD14-PE and CD45-PC5 plot. In this plot, monocytes, granulocytes, and lymphocytes were differentiated from one another according to the expression levels of CD14 (a monocyte marker) and CD45 (a ubiquitous marker of white blood cells). Among these cells, monocytes were identified as CD14high and CD45high cells. Then, CD14high and CD45high cells (gate B) were plotted on a CD14 vs. CD41 graph [Figure 1(C) and 1(F)], whereas the CD14high and the CD41high subpopulation was considered to represent PMAs, as shown in a previous study 11. The patient sample shown in Figure 1(F) exhibited 45.8% PMAs.


Detection of platelet-monocyte aggregates by the ADAM(®) image cytometer.

Jung BK, Cho CH, Moon KC, Sung Hur D, Yoon JA, Yoon SY - Int J Med Sci (2014)

Flow cytometric features of leukocytes, monocytes, and platelet-monocyte aggregates (PMAs). Flow cytometric analysis of negative controls is shown in (A), (B), and (C), whereas flow cytometric analysis of a patient sample (the first to be analyzed) is shown in (D), (E), and (F). (A) All events were acquired by flow cytometry after lysing RBCs in whole blood specimens. In this plot, monocytes are characterized by high CD45 expression and intermediate side scatter. (B) All events shown in (A) were represented in a CD14 and CD45 plot. The monocytic region is characterized by high CD14 and CD45 expression (region within black box). (C) All events within black box in (B) were represented in a CD14 and CD41 plot. Events characterized by high expression of both CD14 and CD41 designate the PMAs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4196123&req=5

Figure 1: Flow cytometric features of leukocytes, monocytes, and platelet-monocyte aggregates (PMAs). Flow cytometric analysis of negative controls is shown in (A), (B), and (C), whereas flow cytometric analysis of a patient sample (the first to be analyzed) is shown in (D), (E), and (F). (A) All events were acquired by flow cytometry after lysing RBCs in whole blood specimens. In this plot, monocytes are characterized by high CD45 expression and intermediate side scatter. (B) All events shown in (A) were represented in a CD14 and CD45 plot. The monocytic region is characterized by high CD14 and CD45 expression (region within black box). (C) All events within black box in (B) were represented in a CD14 and CD41 plot. Events characterized by high expression of both CD14 and CD41 designate the PMAs.
Mentions: The gating strategy to identify platelet-monocyte aggregates (PMAs) is shown in Figure 1. Flow cytometric analysis of negative controls is shown in Figure 1(A), 1(B), and 1(C), whereas flow cytometric analysis of a patient sample (the first to be analyzed) is shown in Figure 1(D), 1(E), and 1(F). The numbers labeling the gates represent the percentages of events inside each gate. The target cell populations gated in the SSC and CD45-PC5 plot were transferred into the CD14-PE and CD45-PC5 plot. In this plot, monocytes, granulocytes, and lymphocytes were differentiated from one another according to the expression levels of CD14 (a monocyte marker) and CD45 (a ubiquitous marker of white blood cells). Among these cells, monocytes were identified as CD14high and CD45high cells. Then, CD14high and CD45high cells (gate B) were plotted on a CD14 vs. CD41 graph [Figure 1(C) and 1(F)], whereas the CD14high and the CD41high subpopulation was considered to represent PMAs, as shown in a previous study 11. The patient sample shown in Figure 1(F) exhibited 45.8% PMAs.

Bottom Line: Moreover, the PMA measurements made by the ADAM(®) cytometer exhibited a high correlation with those made by a flow cytometric assay (R = 0.944).The ADAM(®) cytometer is a suitable alternative method to the flow cytometry-based assays.This, in turn, will provide valuable information regarding patient propensities to thrombotic diseases.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Seoul 152-703, South Korea.

ABSTRACT

Background: Inappropriate platelet activation is known to be associated with various thrombotic disorders. Platelet-monocyte aggregates (PMAs), whose formation is mediated by platelet surface P-selectin (CD62P), can be used as a reliable marker to detect platelet activation. Previous studies have generally detected PMAs through flow cytometry-based approaches. Recently, the ADAM(®) image cytometer (Nanoentek Inc., Seoul, Korea) was developed for image-based cellular analysis. In this study, we detected PMAs with the ADAM(®) cytometer, evaluated the reproducibility of the measurements made by the ADAM(®) cytometer, and compared the abilities of the ADAM(®) cytometer and a flow cytometric assay to detect PMAs.

Methods: Whole blood samples were collected from patients. Within 5 minutes of collection, anticoagulated whole blood samples were fixed in 10% paraformaldehyde and 5% glyoxal. Nineteen clinical specimens were collected; each was analyzed three times with the ADAM(®) cytometer in order to assess the reproducibility of its measurements. To compare the ability of the ADAM(®) cytometer with that of a flow cytometer to detect PMAs, each cytometer was used for 23 clinical samples and the correlation of the measurements was determined.

Results: The PMA measurements made by the ADAM(®) cytometer showed good reproducibility (CV < 10% for all specimens). Moreover, the PMA measurements made by the ADAM(®) cytometer exhibited a high correlation with those made by a flow cytometric assay (R = 0.944).

Conclusions: The ADAM(®) cytometer is a suitable alternative method to the flow cytometry-based assays. Since the ADAM cytometer does not need specialized instrument knowledge or software proficiency (unlike flow cytometry), the ADAM(®) cytometer can be used as a rapid and reliable POCT device to measure platelet activation in peripheral blood. This, in turn, will provide valuable information regarding patient propensities to thrombotic diseases.

Show MeSH
Related in: MedlinePlus