Tissue factor bearing microparticles: optimization of flow cytometric detection Jérémie Bez, Tarik Bouriche, Philippe Poncelet
Research and Technology/BioCytex, Marseille, France
Results: AnnV‐FITC staining of MPs in presence of Ca²+ and FCM‐based detection of micro‐clots MPs in buffer
2000 MP/uL
1000 MP/uL
500 MP/uL
250 MP/uL
125 MP/uL
62 MP/uL
31 MP/uL
16 MP/uL
8 MP/uL
4 MP/uL
FS Log
Tissue factor (TF) is a transmembrane glycoprotein that is present on the plasma membrane of many cells and microparticles. TF is a receptor for factor VII/VIIa that is required for the initiation of blood coagulation. Positive microparticles (TF‐MPs) are highly procoagulant and have been tightly linked to thrombosis in a variety of diseases such as sepsis or cancer…. Flow cytometry (FCM) is the most often used method for detection of TF‐MPs in Platelet‐Free Plasma samples (PFPs) . Such detection is challenged by the weak surface expression of TF antigen and its high intrinsic procoagulant potential.
SBTF1-PE
Introduction:
P8C‐304
MPs in plasma
SS Log
AnnV-FITC
Effect of direct thrombin inhibitors (DTI)
MP minimum size
T=0 h a‐DNP‐PE
Small size of MP requires specific adaptations of FCM protocols
AnnV‐FITC
AnnexinV‐FITC: TAU Technologies BV Binding buffer:10xC with AnnV‐F, TAU Technologies BV rHirudin STAGO, ref.00830: Each 1 mL vial (2000 ATU/mL) is distributed into 100*10µL aliquots, kept frozen at ‐80°C and used extemporaneously at 2 ATU/mL final concentration in Ca²+ containing binding buffer. MPFP: Normal human plasma pool (STAGO, F) was submitted to 2 round of ultra‐centrifugation (100,000g 90 mn) and frozen at ‐80°C into aliquots for future use. TF‐MPs: Purified from culture supernatants of a pancreatic cell line bxpc‐3 (kind gift from Dr. A Pélegrin, IRCM, Montpellier, F). Anti‐TF MAbs: ‐ HTF1‐PE (BDIS/Pharmigen) ‐ SBTF1‐PE: Prototype reagent (BioCytex, Marseille, F), characterised as shown below against well‐known, previously mapped anti‐TF MAbs.
MAb SBTF1 TF9‐1D10
HTF1
TF9‐5B7
SBTF1 (B4C9)
TF
T=2 h
New standardized limit
VD8
TF
SBTF3 (B5C3)
TF9‐10H10 TF8‐5G9
SBTF1‐PE
0.3 µm 0.1 µm
Old standardized limit
DABIGATRAN
0.5 µm
AnnV‐FITC
TF‐MPs spiked plasma after dual staining with AnnV‐FITC in DTI‐containing binding buffer. DTI provided either as r‐Hirudin (2 ATU/mL, Stago), GPRP (2 mM), Dabigatran (2 µM). Note: staining without DTI induced short time clotting, impeding FCM analysis (not shown).
Clot formation in Ca²+
buffer
AnnV‐FITC
SSC
FSC
1 µm (MP maximum size)
T=1 h
HIRUDINE
MP‐size window standardized using "Megamix Plus" Megamix Plus beads
0 ATU/mL
2 ATU/mL GPRP
FCM analysis was performed on a Gallios cytometer, (Beckman‐ Coulter). MP‐size window was standardized using Megamix‐Plus FSC beads with cut‐off at 0.3 µm‐eq
DTI protecting effect
SBTF1‐PE
Materials and Methods:
FSC
Our aim was to optimize FCM‐based detection protocol for TF‐MPs. We here show that, despite using adequate fluorescent anti‐TF antibody conjugates, a yet un‐recognized plasma micro‐clotting artefact impedes the detection of such a highly pro‐coagulant material.
When purified TF‐MPs derived from highly TF pancreatic tumor cells (bxpc‐3) where spiked into MPFP and analyzed after AnnV‐FITC staining in presence of Ca²+, MPs were not detected as expected and replaced in the MP gate by new non fluorescent events. Note the presence in highest concentrations of spiking of events with elevated levels of Side and Forward scatter corresponding to µ‐aggregates of MP +/‐ fibrin strands or µ‐clots. Prevalence and size of such µ‐aggregates seems to be linked to the level of TF‐MPs spiking. Clot is visible in tube with highest TF‐MPs level after only a few minutes following addition of Ca²+ containing binding buffer, highly suggesting TF‐MPs induced coagulation despite the 1:10 final dilution of plasma.
SBTF1‐PE
Aim:
AnnV‐FITC
SSC
TF‐MPs rich plasma was stained with/without Hirudin in Binding Buffer (2 ATU/mL) and FCM analysis operated immediately , 1h or 2h after addition of bb+. In the absence of DTI, detection of TF‐MPs is: Almost feasible just after dilution (T= 0h), although µ‐aggregation is already visible (arrows). Difficult at T= 1h with major interference of dual‐stained µ‐aggregates which entraps fluorescent reagents and appears as a "comet" of dual positive events (red arrows) and large MP‐like events with high scatter levels (black arrows). Impossible at T= 2h with major clot having entrapped all MP (no more events seen, same as with buffer alone).
clot buffer with Ca²+
buffer wo Ca²+
Comparing AnnV+ staining with Ca2+ buffer (left) with its negative control tube diluted with Ca2+‐free buffer (right) shows the presence of visible clot (image courtesy of Dr Sukesh C. Nair & coll., Hematology unit, CMC Vellore, India).
In the presence of DTI, detection of TF‐MPs remains feasible at any time; no µ‐aggregate detected by FCM and no clot formed in the tubes.
Lactadherin‐FITC staining of MPs DNP PE
a‐TF SBTF1 (B4C9)
a‐TF HTF1
Premature clot formation, initially observed in TF‐MPs rich plasma has also been occasionnaly observed in pathological plasma samples. Here, it happened in normal fresh PFP stored for one night at 2‐8°C. The likely explanation lies in the previously reported cold‐induced activation of plasma factors (mainly FVII and FIX), which may create artefactual, TF‐MPs independent, coagulation via the intrinsic pathway.
Staining of TF+MP spiked in MPFP using anti‐TF MAb‐PE conjugates SBTF1 (clone B4C9) or HTF1 and Lactadherin‐FITC. This shows higher intensity TF staining with SBTF1 and illustrates presence of TF‐MPs. a‐DNP‐PE is used as matched irrelevant IgG1 control.
Conclusion: Bez et al, @ Symposium focused on TF & FVII, UNC, Chapel-Hill (May 2012)
3D map of TF molecules using well defined anti‐TF MAbs from Scripps Institute (kind gift from Pr W. Ruf, La Jolla) SBTF‐1 is a high affinity MAb detecting TF on a very accessible epitope, located on the head of TF, far from the cell membrane in contrast to TF8‐5G9, and not in a partially cryptic domain, as is the case with HTF‐1
[email protected] [email protected]
FCM analysis of TF‐MPs needs to be optimized using adequate anti‐TF MAb conjugates. Since premature clot formation can also be encountered in normal plasma samples, especially when these have been kept some time in the cold, adding anti‐coagulant in Annexin binding buffer appears mandatory in all situations. Although Heparin has been initially proposed (ref. 1), we advocate for the use of hirudin, a direct thrombin inhibitor that shows simpler and more direct mode of action (e.g. no influence on AT, nor on liberation of membrane TFPI, …). ref. 1) Iversen et al, J Immunol Methods, 2013
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