INCREASED PLATELET LEUKOCYTE INTERACTIONS IN TYPE 2 DIABETIC INDIVIDUALS COMPARED TO HEALTHY CONTROLS
(Abstract release date: 05/19/16)
EHA Library. Mkandla Z. 06/09/16; 133101; E1552
Mr. Zibusiso Mkandla
Contributions
Contributions
Abstract
Abstract: E1552
Type: Eposter Presentation
Background
Type 2 diabetes mellitus (T2DM) is considered as a form of low grade inflammatory response. Activated platelets are involved in inflammatory processes and bind to inflammatory cells monocytes and neutrophils to form platelet leukocyte aggregates (PLAs). Platelet bound neutrophils and monocytes become activated (expressing CD69) and undergo a phenotypic change into pro-coagulant phenotypes expressing tissue factor (CD142).
Aims
1) To investigate the baseline levels of platelet activation in pre-diabetes and diabetes and compare this to normal controls.2) To investigate the percentage of monocytes and neutrophils forming aggregates with platelets (PMA’s and PNA’s) in pre-diabetes and diabetes and comparing this to normal individuals.3) To investigate the up regulation of pro-thrombotic and activation antigens on the surface of monocytes and neutrophils (Tissue Factor and CD69) in pre-diabetic and diabetic individuals.
Methods
Peripheral blood was collected into 4.5ml sodium citrate tubes containing 3.2% sodium citrate, samples were analysed within 1 hour of collection on the Navios 8 colour flow cytometer (Beckman Coulter, Miami, Florida). To ensure reporting on standardized results, flow check pro fluorescent labelled beads (Beckman Coulter, Miami, Florida) were used to standardise the optical path and laminar flow of the instrument. Blood was also collected into serum separator tubes (SST), heparin and EDTA for both biochemical and haematology testing.50µl of citrated blood was incubated with 5µl of an antibody cocktail containing the antibodies CD42b-FITC, CD142-PE, CD69-PC5, CD16-PC7 and CD14-APC.After 10 minute incubation in the dark at room temperature each sample was lysed with 500µl Versalyse lysing solution for 15 minutes. 500µl of PBS was added to the sample and it was immediately analysed on the flow cytometer.
Results
Our results showed increased levels of platelet leukocyte aggregates %PLA in pre-diabetes and diabetes therefore indicating increased interactions between platelets and leukocytes in the peripheral blood of pre-DM and DM individuals. The diabetic group showed increased baseline levels of circulating platelets monocyte aggregates (%PMAs) median 49.04[36.78-62] compared to the control group 7.2[5.4-9], p<0.0001. Baseline platelet neutrophil aggregates (%PNAs) were significantly increased in the DM group [median 13.56(10.17-16.95)] compared to the control group [median 6.01(4.51-7.51)] p<0.0001. We also showed increased levels of tissue factor expression, a main part of the coagulation cascade, on the monocytes and neutrophils possibly is causing the hyper-coaguable state seen in diabetic individuals causing CVDs. Tissue factor (TF) expression on both monocytes for the diabetic group were significantly higher than the control group and correlated with increased levels of %PMAs (r=0.6744, p<0.0001).
Conclusion
In this study we showed increased PLAs in diabetic individuals than normal controls which may be responsible for the cardiovascular complications in diabetic patients. The increased tissue factor expression on monocytes and neutrophils in diabetic individuals may also contribute to the pro-coagulant state.
Session topic: E-poster
Keyword(s): Flow cytometry, Inflammation, Platelet activation
Type: Eposter Presentation
Background
Type 2 diabetes mellitus (T2DM) is considered as a form of low grade inflammatory response. Activated platelets are involved in inflammatory processes and bind to inflammatory cells monocytes and neutrophils to form platelet leukocyte aggregates (PLAs). Platelet bound neutrophils and monocytes become activated (expressing CD69) and undergo a phenotypic change into pro-coagulant phenotypes expressing tissue factor (CD142).
Aims
1) To investigate the baseline levels of platelet activation in pre-diabetes and diabetes and compare this to normal controls.2) To investigate the percentage of monocytes and neutrophils forming aggregates with platelets (PMA’s and PNA’s) in pre-diabetes and diabetes and comparing this to normal individuals.3) To investigate the up regulation of pro-thrombotic and activation antigens on the surface of monocytes and neutrophils (Tissue Factor and CD69) in pre-diabetic and diabetic individuals.
Methods
Peripheral blood was collected into 4.5ml sodium citrate tubes containing 3.2% sodium citrate, samples were analysed within 1 hour of collection on the Navios 8 colour flow cytometer (Beckman Coulter, Miami, Florida). To ensure reporting on standardized results, flow check pro fluorescent labelled beads (Beckman Coulter, Miami, Florida) were used to standardise the optical path and laminar flow of the instrument. Blood was also collected into serum separator tubes (SST), heparin and EDTA for both biochemical and haematology testing.50µl of citrated blood was incubated with 5µl of an antibody cocktail containing the antibodies CD42b-FITC, CD142-PE, CD69-PC5, CD16-PC7 and CD14-APC.After 10 minute incubation in the dark at room temperature each sample was lysed with 500µl Versalyse lysing solution for 15 minutes. 500µl of PBS was added to the sample and it was immediately analysed on the flow cytometer.
Results
Our results showed increased levels of platelet leukocyte aggregates %PLA in pre-diabetes and diabetes therefore indicating increased interactions between platelets and leukocytes in the peripheral blood of pre-DM and DM individuals. The diabetic group showed increased baseline levels of circulating platelets monocyte aggregates (%PMAs) median 49.04[36.78-62] compared to the control group 7.2[5.4-9], p<0.0001. Baseline platelet neutrophil aggregates (%PNAs) were significantly increased in the DM group [median 13.56(10.17-16.95)] compared to the control group [median 6.01(4.51-7.51)] p<0.0001. We also showed increased levels of tissue factor expression, a main part of the coagulation cascade, on the monocytes and neutrophils possibly is causing the hyper-coaguable state seen in diabetic individuals causing CVDs. Tissue factor (TF) expression on both monocytes for the diabetic group were significantly higher than the control group and correlated with increased levels of %PMAs (r=0.6744, p<0.0001).
Conclusion
In this study we showed increased PLAs in diabetic individuals than normal controls which may be responsible for the cardiovascular complications in diabetic patients. The increased tissue factor expression on monocytes and neutrophils in diabetic individuals may also contribute to the pro-coagulant state.
Session topic: E-poster
Keyword(s): Flow cytometry, Inflammation, Platelet activation
Abstract: E1552
Type: Eposter Presentation
Background
Type 2 diabetes mellitus (T2DM) is considered as a form of low grade inflammatory response. Activated platelets are involved in inflammatory processes and bind to inflammatory cells monocytes and neutrophils to form platelet leukocyte aggregates (PLAs). Platelet bound neutrophils and monocytes become activated (expressing CD69) and undergo a phenotypic change into pro-coagulant phenotypes expressing tissue factor (CD142).
Aims
1) To investigate the baseline levels of platelet activation in pre-diabetes and diabetes and compare this to normal controls.2) To investigate the percentage of monocytes and neutrophils forming aggregates with platelets (PMA’s and PNA’s) in pre-diabetes and diabetes and comparing this to normal individuals.3) To investigate the up regulation of pro-thrombotic and activation antigens on the surface of monocytes and neutrophils (Tissue Factor and CD69) in pre-diabetic and diabetic individuals.
Methods
Peripheral blood was collected into 4.5ml sodium citrate tubes containing 3.2% sodium citrate, samples were analysed within 1 hour of collection on the Navios 8 colour flow cytometer (Beckman Coulter, Miami, Florida). To ensure reporting on standardized results, flow check pro fluorescent labelled beads (Beckman Coulter, Miami, Florida) were used to standardise the optical path and laminar flow of the instrument. Blood was also collected into serum separator tubes (SST), heparin and EDTA for both biochemical and haematology testing.50µl of citrated blood was incubated with 5µl of an antibody cocktail containing the antibodies CD42b-FITC, CD142-PE, CD69-PC5, CD16-PC7 and CD14-APC.After 10 minute incubation in the dark at room temperature each sample was lysed with 500µl Versalyse lysing solution for 15 minutes. 500µl of PBS was added to the sample and it was immediately analysed on the flow cytometer.
Results
Our results showed increased levels of platelet leukocyte aggregates %PLA in pre-diabetes and diabetes therefore indicating increased interactions between platelets and leukocytes in the peripheral blood of pre-DM and DM individuals. The diabetic group showed increased baseline levels of circulating platelets monocyte aggregates (%PMAs) median 49.04[36.78-62] compared to the control group 7.2[5.4-9], p<0.0001. Baseline platelet neutrophil aggregates (%PNAs) were significantly increased in the DM group [median 13.56(10.17-16.95)] compared to the control group [median 6.01(4.51-7.51)] p<0.0001. We also showed increased levels of tissue factor expression, a main part of the coagulation cascade, on the monocytes and neutrophils possibly is causing the hyper-coaguable state seen in diabetic individuals causing CVDs. Tissue factor (TF) expression on both monocytes for the diabetic group were significantly higher than the control group and correlated with increased levels of %PMAs (r=0.6744, p<0.0001).
Conclusion
In this study we showed increased PLAs in diabetic individuals than normal controls which may be responsible for the cardiovascular complications in diabetic patients. The increased tissue factor expression on monocytes and neutrophils in diabetic individuals may also contribute to the pro-coagulant state.
Session topic: E-poster
Keyword(s): Flow cytometry, Inflammation, Platelet activation
Type: Eposter Presentation
Background
Type 2 diabetes mellitus (T2DM) is considered as a form of low grade inflammatory response. Activated platelets are involved in inflammatory processes and bind to inflammatory cells monocytes and neutrophils to form platelet leukocyte aggregates (PLAs). Platelet bound neutrophils and monocytes become activated (expressing CD69) and undergo a phenotypic change into pro-coagulant phenotypes expressing tissue factor (CD142).
Aims
1) To investigate the baseline levels of platelet activation in pre-diabetes and diabetes and compare this to normal controls.2) To investigate the percentage of monocytes and neutrophils forming aggregates with platelets (PMA’s and PNA’s) in pre-diabetes and diabetes and comparing this to normal individuals.3) To investigate the up regulation of pro-thrombotic and activation antigens on the surface of monocytes and neutrophils (Tissue Factor and CD69) in pre-diabetic and diabetic individuals.
Methods
Peripheral blood was collected into 4.5ml sodium citrate tubes containing 3.2% sodium citrate, samples were analysed within 1 hour of collection on the Navios 8 colour flow cytometer (Beckman Coulter, Miami, Florida). To ensure reporting on standardized results, flow check pro fluorescent labelled beads (Beckman Coulter, Miami, Florida) were used to standardise the optical path and laminar flow of the instrument. Blood was also collected into serum separator tubes (SST), heparin and EDTA for both biochemical and haematology testing.50µl of citrated blood was incubated with 5µl of an antibody cocktail containing the antibodies CD42b-FITC, CD142-PE, CD69-PC5, CD16-PC7 and CD14-APC.After 10 minute incubation in the dark at room temperature each sample was lysed with 500µl Versalyse lysing solution for 15 minutes. 500µl of PBS was added to the sample and it was immediately analysed on the flow cytometer.
Results
Our results showed increased levels of platelet leukocyte aggregates %PLA in pre-diabetes and diabetes therefore indicating increased interactions between platelets and leukocytes in the peripheral blood of pre-DM and DM individuals. The diabetic group showed increased baseline levels of circulating platelets monocyte aggregates (%PMAs) median 49.04[36.78-62] compared to the control group 7.2[5.4-9], p<0.0001. Baseline platelet neutrophil aggregates (%PNAs) were significantly increased in the DM group [median 13.56(10.17-16.95)] compared to the control group [median 6.01(4.51-7.51)] p<0.0001. We also showed increased levels of tissue factor expression, a main part of the coagulation cascade, on the monocytes and neutrophils possibly is causing the hyper-coaguable state seen in diabetic individuals causing CVDs. Tissue factor (TF) expression on both monocytes for the diabetic group were significantly higher than the control group and correlated with increased levels of %PMAs (r=0.6744, p<0.0001).
Conclusion
In this study we showed increased PLAs in diabetic individuals than normal controls which may be responsible for the cardiovascular complications in diabetic patients. The increased tissue factor expression on monocytes and neutrophils in diabetic individuals may also contribute to the pro-coagulant state.
Session topic: E-poster
Keyword(s): Flow cytometry, Inflammation, Platelet activation
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