Flow Cytometry of Circulating Blood Microparticles Flow ... - MetroFlow
Flow Cytometry of Circulating Blood Microparticles Flow ... - MetroFlow
Flow Cytometry of Circulating Blood Microparticles Flow ... - MetroFlow
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<strong>Flow</strong> <strong>Cytometry</strong> <strong>of</strong> <strong>Circulating</strong> <strong>Blood</strong><br />
<strong>Microparticles</strong><br />
Arun S. Shet<br />
Laboratory <strong>of</strong> <strong>Blood</strong> and Vascular Biology<br />
Rockefeller University<br />
New York
Outline<br />
• What are microparticles and why are they<br />
important?<br />
• Pathophysiology <strong>of</strong> microparticles<br />
• <strong>Flow</strong> cytometric study <strong>of</strong> microparticles<br />
– Isolation from blood<br />
– <strong>Flow</strong> cytometry definition<br />
– Characterization <strong>of</strong> membrane antigens<br />
– Enumeration<br />
– Analysis strategies<br />
• Current flow cytometry approaches
<strong>Microparticles</strong> (MPs)<br />
<strong>Microparticles</strong> (MPs)<br />
• MPs are small, membrane derived vesicles released by<br />
activated or apoptotic cells.<br />
• They have excess phosphatidylserine (PS) on their<br />
outer leaflets exposed to plasma (label with the<br />
phopholipid probe annexin V)<br />
• MPs express surface antigens characteristic <strong>of</strong> their<br />
cell-<strong>of</strong>-origin allowing flow cytometric detection
<strong>Microparticles</strong>: pathophysiology<br />
Zwaal et al, <strong>Blood</strong> 1997
<strong>Microparticles</strong><br />
• Small (0.05 - 1.0 µm) and heterogeneous<br />
• Surface phosphatidylserine (+)<br />
• Membrane antigen (+)<br />
• Exosomes<br />
– Small (0.03-0.1 µm)<br />
– Exocytosis <strong>of</strong> multivesicular bodies<br />
– Enriched in tetraspanins (CD9, 63, 81 & 82) and MHC<br />
class II molecules<br />
– Play a role in antigen presentation
MPs - are they relevant?<br />
MPs - are they relevant?<br />
Although the mechanism <strong>of</strong> production, circulating<br />
time, fate and physiological function <strong>of</strong> MPs are areas<br />
<strong>of</strong> active investigation, their relevance is yet to be<br />
completely understood….
MPs contribute to physiological hemostasis….<br />
MPs contribute to physiological hemostasis….<br />
Humans with Scott syndrome have a defect in externalization <strong>of</strong> PS and<br />
membrane vesiculation that results in insufficient platelet MP formation<br />
and manifests clinically as a moderate to severe bleeding disorder.
MPs contribute to thrombosis….<br />
?CT = deletion <strong>of</strong> the cytoplasmic tail <strong>of</strong> p-selectin<br />
Andre et al PNAS 2000
MPs may be therapeutic….<br />
MPs may be therapeutic….<br />
Hrachavinova et al,<br />
Nature medicine 2003
Elevated blood microparticles in disease states<br />
Elevated blood microparticles in disease states<br />
• Heparin induced<br />
thrombosis<br />
• Thrombotic<br />
thrombocytopenic purpura<br />
• Hemolytic anemia<br />
• Paroxysmal nocturnal<br />
hemoglobinuria<br />
• Cancer - Leukemia<br />
• Lung & gastric cancer<br />
• Sepsis<br />
• Trauma<br />
• Diabetes mellitus<br />
• Renal failure<br />
• Coronary artery disease<br />
All <strong>of</strong> these conditions are associated with thrombosis
MPs - are they relevant?<br />
MPs - are they relevant?<br />
The mechanism <strong>of</strong> production, circulating time, fate and<br />
physiological function <strong>of</strong> MPs are areas worthy <strong>of</strong><br />
active investigation and require better understanding.
R1<br />
<strong>Flow</strong> cytometry: Methods - Gating<br />
<strong>Flow</strong> cytometry: Methods - Gating<br />
R1<br />
R2<br />
R2<br />
R1<br />
R1<br />
R2
<strong>Flow</strong> cytometry: Methods- annexin<br />
<strong>Flow</strong> cytometry: Methods- annexin<br />
Annexin V Cy 5<br />
Calcium buffer EDTA buffer<br />
Side scatter<br />
Side scatter<br />
Bead gate<br />
1.0μm beads<br />
MP gate<br />
Foward scatter
Side scatter<br />
Annexin V Cy 5<br />
Plasma contains annexin V labeling events<br />
R1<br />
R2<br />
R1<br />
Forward scatter<br />
R2<br />
Side scatter<br />
R1<br />
R2
Some annexin V labeling events in plasma are not removable<br />
by ultracentrifugation<br />
Side scatter<br />
Annexin V Cy 5<br />
R1 R1 R1<br />
Forward scatter<br />
Side scatter
• Removable by ultracentrifugation<br />
• Small in size (≤1.0μm)<br />
• Bind with annexin V<br />
(surface PS +)<br />
Microparticle definition<br />
Microparticle definition<br />
Annexin V Cy5<br />
Side scatter<br />
Side scatter<br />
Bead gate<br />
1.0μm beads<br />
MP gate<br />
Foward scatter<br />
PS (+) MPs<br />
PS (-) MPs<br />
plus noise
Sickle cell anemia<br />
Sickle cell anemia<br />
• Sickle cell anemia is the commonest inherited<br />
hematological disorder in the United States affecting<br />
~80,000 people<br />
• Point mutation in the ß-chain <strong>of</strong> the hemoglobin<br />
molecule<br />
• Results in hemoglobin that has decreased solubility and<br />
is prone to polymerization<br />
• Coagulation system is activated in patients with sickle<br />
cell disease
MPs in sickle cell anemia<br />
MPs in sickle cell anemia<br />
• Sickle blood contains MPs derived from<br />
– RBC : Allan et al, Nature 1982<br />
– Platelet : Wun et al, BJH 1998<br />
– ? Endothelial cells<br />
– ? Monocytes
Sickle crisis and steady state<br />
Sickle crisis and steady state<br />
• Crisis period<br />
– Pain having no cause other than SCD<br />
– Requiring morphine and/or NSAIDS and hospitalization<br />
– Samples obtained at time <strong>of</strong> onset <strong>of</strong> crisis or during hospital<br />
course<br />
• Steady state period<br />
– Any time exclusive <strong>of</strong> the 4 weeks prior to or after a crisis period<br />
– Samples obtained during routine clinic visit<br />
• Acute crisis (n=21) and steady state (n=16).<br />
• Normal subjects (n=13).
Sample collection and processing<br />
Sample collection and processing<br />
• Using a 21 G needle, after discarding 1 st 3 mL into a vacutainer<br />
tube containing buffered sodium citrate (1 part : 9 parts).<br />
• Processed immediately after collection.<br />
• Platelet free plasma (PFP) was prepared by a 2- step centrifugation<br />
procedure.<br />
• Stored at -80°C or analyzed immediately.<br />
• MPs extracted from PFP by ultracentrifugation<br />
• Labeled with annexin V Cy5 in Ca++ buffer in the dark for 30 min<br />
at 22 °C
• MP gate<br />
• Annexin V (+) quadrant<br />
Data analysis<br />
Bead gate<br />
MP gate<br />
• Quadrant settings determined by a sample <strong>of</strong> MPs labeled with<br />
annexin V in calcium buffer containing EDTA.<br />
• To enumerate MPs, we added a known # <strong>of</strong> 7 µm size beads<br />
which were adequately separated from the MP gate based on<br />
their size.<br />
• Annexin V (+) event count was multiplied by the following<br />
formula to obtain the total annexin (+) MP number in 1 mL <strong>of</strong><br />
platelet free plasma.<br />
MP = (+) event count X<br />
Beads added /Beads counted X plasma<br />
volume (mL)
Total Plasma <strong>Microparticles</strong> Are Elevated in Sickle<br />
Patients During Steady State and Crisis<br />
MP (x thousands / ml PFP)<br />
3500<br />
3000<br />
2500<br />
2000<br />
1500<br />
1000<br />
500<br />
0<br />
Normal St. state Crisis<br />
Shet et al, <strong>Blood</strong> 2003
Endothelial TF Expression in SCA<br />
Endothelial TF Expression in SCA<br />
Endothelial marker<br />
P1H12<br />
Tissue factor antigen<br />
<strong>Circulating</strong> endothelial<br />
cells in sickle cell<br />
anemia are activated<br />
and express tissue<br />
factor<br />
Solovey et al, NEJM 1997<br />
Solovey et al, JCI 1998
MP Origin and TF Expression<br />
MP origin and TF expression were identified by triplelabel<br />
flow cytometry using:<br />
– Annexin V<br />
– Cell specific monoclonals<br />
• Endothelial cells : VE cadherin<br />
• Monocytes: CD14<br />
• Platelets : aIIbß3<br />
• Red blood cells : Glycophorin A<br />
– TF monoclonal antibody
Cell specificity <strong>of</strong> anti-CD14 and<br />
anti-VE cadherin antibodies<br />
CD 14 + VE cad on monocytes<br />
Mononuclear cells Endothelial cells<br />
Fluorescence intensity (phycoerythrin)
Annexin Cy 5<br />
Endothelial cell (HUVEC) microparticles<br />
Phycoerythrin fluorescence
Fluorescein<br />
Phycoerythrin<br />
Endothelial cell (HUVEC) microparticles<br />
IgG-FITC<br />
CD146-FITC CD106-FITC<br />
IgG-PE VE cadherin-PE CD31-PE
R1<br />
Platelet-derived <strong>Microparticles</strong><br />
Washed unstimulated<br />
platelets obtained from<br />
a normal donor<br />
R2<br />
Annexin V CY 5<br />
R1 = microparticle gate<br />
R2 = platelet gate<br />
aIIbß3 FITC<br />
R1 microparticle gate<br />
R2 platelet gate
R1<br />
Platelet-derived <strong>Microparticles</strong><br />
Washed platelets treated with<br />
calcium ionophore (A23187)<br />
R2<br />
Annexin V CY 5<br />
aIIbß3 FITC<br />
R1 microparticle gate<br />
R2 platelet gate
Monocyte-Derived <strong>Microparticles</strong><br />
Mononuclear cells isolated from peripheral<br />
blood by a ficol gradient were enriched<br />
using CD 14 positive selection in a macs<br />
column. CD14 (+) cells were stimulated<br />
with calcium ionophore, centrifuged at<br />
2000 x g and the supernatant harvested for<br />
microparticle analysis.<br />
IgG 1 PE<br />
Annexin V CY 5<br />
CD 14 PE
Annexin V-Cy5<br />
FACS analysis <strong>of</strong> blood microparticles<br />
FACS analysis <strong>of</strong> blood microparticles<br />
1 2<br />
3 4<br />
Fluorescein-labeled monoclonal Antibody<br />
1-Isotype control<br />
2-glycophorin<br />
3-aIIbß3<br />
4-Tissue factor
MPs are derived from endothelial cells and monocytes<br />
in addition to RBCs and platelets<br />
MP (x thousands / ml PFP)<br />
1200<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
0<br />
RBC Platelet Monocyte<br />
Normal St. state Crisis<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
Endothelial<br />
Shet et al, <strong>Blood</strong> 2003
VE cadherin PE<br />
Example <strong>of</strong> Analysis <strong>of</strong> PS (+) MPs for Cell <strong>of</strong><br />
Origin and TF<br />
VE cadherin + TF<br />
Control IgG Sickle patient Normal control<br />
Tissue Factor FITC
Phycoerythrin labeled antibodies<br />
Medium control<br />
Fluorescein labeled antibodies
• MP gate<br />
• Annexin V (+) quadrant<br />
Data analysis<br />
• Quadrant settings determined by a sample <strong>of</strong> MPs<br />
labeled with equal concentration <strong>of</strong> isotype-matched<br />
IgG-PE and IgG-FITC<br />
• RUQ event number- RUQ background<br />
• Buffer with antibodies (‘medium’/electronic noise)<br />
• MP = (+) event count X Beads added/Beads counted X<br />
plasma volume (mL)
Monocytes and endothelial derived TF (+) MPs are<br />
significantly elevated during sickle crisis<br />
MP (x thousands) / ml PFP)<br />
140 Monocyte-derived TF<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Endothelial-derived TF<br />
Total TF<br />
Normal St. state Crisis<br />
Shet et al, <strong>Blood</strong> 2003
Procoagulant activity <strong>of</strong> microparticles<br />
Procoagulant activity <strong>of</strong> microparticles<br />
PT,seconds<br />
1200<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
0<br />
With TF Antibody<br />
With control Antibody<br />
Normal MPs Sickle MPs
Procoagulant activity <strong>of</strong> microparticles<br />
Aras et al, <strong>Blood</strong> 2004
Annexin V<br />
MP origin and TF expression in human endotoxemia<br />
Side scatter<br />
CD 14<br />
Tissue Factor<br />
Aras et al, <strong>Blood</strong> 2004
Electron micrographs <strong>of</strong> MP pellet<br />
Endothelial MPs in vitro <strong>Blood</strong> MPs (sickle)<br />
Bar = 100nm
Tissue factor<br />
VE cadherin<br />
EM <strong>of</strong> <strong>Blood</strong> MPs using immunogold labeling<br />
CD 14<br />
Bars = 100nm<br />
Control
Conclusions<br />
• Sickle blood contains microparticles from<br />
endothelial cells and monocytes.<br />
• A portion <strong>of</strong> endothelial and monocyte-derived<br />
microparticles express tissue factor.<br />
• Microparticle-associated tissue factor is<br />
functionally active.<br />
• Plasma coagulation markers correlate with<br />
total MP and total TF (+) MP number.<br />
Shet et al, <strong>Blood</strong> 2003
TF exposed<br />
PS exposed<br />
TF exposed<br />
No PS exposed<br />
Intravascular tissue factor<br />
Intravascular tissue factor<br />
Cell associated TF<br />
Microparticle associated TF<br />
TF exposed<br />
Soluble TF<br />
No transmembrane domain
Vessel lumen<br />
Intravascular tissue factor<br />
Vessel wall<br />
<strong>Blood</strong> Coagulation<br />
<strong>Blood</strong> Coagulation<br />
X Xa<br />
VIIa<br />
Activated<br />
endothelial cell<br />
Subendothelial tissue factor<br />
II<br />
IIa
<strong>Microparticles</strong> and blood coagulation<br />
<strong>Microparticles</strong> and blood coagulation<br />
VIIa<br />
Tissue factor<br />
X Xa<br />
VIIa<br />
Endothelial cell<br />
II<br />
IIa<br />
Fibrin clot
Standardization summary<br />
• Preanalytical step<br />
– Sample collection and preparation<br />
• Analytical step<br />
– Buffers<br />
– Reagents<br />
– Labeling procedure<br />
• Instrument<br />
– BD FACS calibur<br />
• Data analysis<br />
– Cellquestpro or Flojo<br />
• Confirmation <strong>of</strong> flow cytometry findings using independent<br />
methods
TF positive MPs in mice with high plasma sP-selectin<br />
Andre et al; PNAS 2000
Journal <strong>of</strong> vascular Surgery
<strong>Flow</strong> cytometric analysis <strong>of</strong> blood microparticles -<br />
published protocols<br />
http://www.sscvenice.it/ssc.pdf<br />
Shet et al & Jy et al, JTH 2004
Acknowledgements<br />
Univ <strong>of</strong> Minnesota<br />
Robert Hebbel<br />
Nigel Key<br />
James White & Marcie Krumwiede<br />
Hennepin County Medical Center<br />
Douglas Rausch & Louann Koopmeiners<br />
Rockefeller University<br />
Barry Coller<br />
SCDAA & LHI<br />
Clinical Scholars program (Rockefeller University)
Helix pomatia lectin and annexin V, two molecular probes for insect<br />
microparticles: possible involvement in hemolymph coagulation<br />
Ulrich Theopold* and Otto Schmidt<br />
Author Keywords: Coagulation; Lipophorin; Phosphatidylserine;<br />
<strong>Microparticles</strong>; Hemomucin
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