Name: microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro
Uploaded: 2016-03-19T14:00:00
Description: Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

The authors have no acknowledgements.

This protocol follows the institutional ethical guidelines for research on human samples and informed consent was obtained from all donors involved. Approval for the experiments described here was obtained from the institutional review board of the Antwerp University Hospital.
Note: Temperature indications are always room temperature, unless specified.
1. Preparation Flow Chamber Setup
<ol>
	<li>Preparing Lanes, Tubing and Pins
	<ol>
		<li>Vortex the collagen susp...

<ol>
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F., Yam, K., Gerace, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hemostasis+in+flowing+blood.">Evaluation of hemostasis in flowing blood.</a> Am J Hematol. 87 (Suppl 1), S51-S55 (2012).</li><li>Roest, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+chamber-based+assays+to+measure+thrombus+formation+in+vitro:+requirements+for+standardization.">Flow chamber-based assays to measure thrombus formation in vitro: requirements for standardization.</a> J Thromb Haemost. 9, 2322-2324 (2011).</li><li>Heemskerk, J. W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+surfaces+to+measure+thrombus+formation+under+flow:+possibilities+for+standardization.">Collagen surfaces to measure thrombus formation under flow: possibilities for standardization.</a> J Thromb Haemost. 9, 856-858 (2011).</li><li>Shrivastava, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+platelet+storage+lesion.">The platelet storage lesion.</a> Transfus Apher Sci. 41, 105-113 (2009).</li><li>Miyaji, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+platelet+aggregation+of+platelet+concentrate+during+storage+recovers+in+the+body+after+transfusion.">Decreased platelet aggregation of platelet concentrate during storage recovers in the body after transfusion.</a> Transfusion. 44, 891-899 (2004).</li><li>Goodrich, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+in+vitro+platelet+quality+measurements+with+in+vivo+platelet+viability+in+human+subjects.">Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects.</a> Vox Sang. 90, 279-285 (2006).</li><li>Van Aelst, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riboflavin+and+amotosalen+photochemical+treatments+of+platelet+concentrates+reduce+thrombus+formation+kinetics+in+vitro.">Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro.</a> Vox Sang. 108, 328-339 (2015).</li><li>Van Aelst, B., Devloo, R., Vandekerckhove, P., Compernolle, V., Feys, H. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+of+blood+platelets+by+adenosine+diphosphate+and+its+reversal.">Aggregation of blood platelets by adenosine diphosphate and its reversal.</a> Nature. 194, 927-929 (1962).</li><li>Cattaneo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+Standardization+of+Light+Transmission+Aggregometry:+A+Consensus+of+the+Working+Party+from+the+Platelet+Physiology+Subcommittee+of+SSC/ISTH.">Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.</a> J Thromb Haemost. 11, 1183-1189 (2013).</li><li>Deckmyn, H., Feys, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+quality+control+of+platelets+for+transfusion.">Assays for quality control of platelets for transfusion.</a> ISBT Sci Series. 8, 221-224 (2013).</li><li>Andre, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticoagulants+(thrombin+inhibitors)+and+aspirin+synergize+with+P2Y12+receptor+antagonism+in+thrombosis.">Anticoagulants (thrombin inhibitors) and aspirin synergize with P2Y12 receptor antagonism in thrombosis.</a> Circulation. 108, 2697-2703 (2003).</li><li>Casari, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Willebrand+factor+mutation+promotes+thrombocytopathy+by+inhibiting+integrin+alphaIIbbeta3.">von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin alphaIIbbeta3.</a> J Clin Invest. 123, 5071-5081 (2013).</li><li>Gitz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+platelet+survival+after+cold+storage+by+prevention+of+Glycoprotein+Ib&#945;+clustering+in+lipid+rafts.">Improved platelet survival after cold storage by prevention of Glycoprotein Ib&#945; clustering in lipid rafts.</a> Haematologica. , (2012).</li><li>Maurer-Spurej, E., Labrie, A., Brown, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+Quality+Testing+of+Blood+Platelet+Transfusions+with+Dynamic+Light+Scattering.">Routine Quality Testing of Blood Platelet Transfusions with Dynamic Light Scattering.</a> Part Part Syst Char. 25, 99-104 (2008).</li><li>Van Kruchten, R., Cosemans, J. 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Microfluidic flow chamber experiments are an excellent tool to investigate platelet function in flowing blood and are used to evaluate hemostasis in vitro in varying experimental contexts. Despite poor interlaboratory standardization9, we demonstrate that within our laboratory the experimental variation is acceptable. This allows to reliably compare (paired) samples within a given study. This was validated using the well documented phenomenon of platelet storage lesion, which is a detrimental conseque...

Hemostasis requires the combined and regulated activity of cells, proteins, ions and tissues in a restricted spatiotemporal context1. Uncontrolled activity may lead to hemorrhage or thrombosis and morbidity or mortality in a spectrum of disorders related to blood coagulation. A microfluidic flow chamber experiment is a challenging technique that mimics hemostasis in vitro. This approach allows investigation of the complex interplay of processes that take part in hemostasis with a leading role for blood platelets.
Following vascular injury, platelets adhere to the exposed subendothelial matrix (glyco)proteins to prevent blood loss. Following adhesion, platelets activate and aggregate in response to auto- and paracrine signaling which finally leads to the formation of a platelet network, stabilized by fibrin and resulting in a firm, wound sealing thrombus2. Unlike most other platelet function tests, experiments with flow chambers take into account the physical parameter of blood flow and therefore the influence of rheology on the participating cells and biomolecules3,4.
Flow chamber experiments have generated landmark insights in hemostasis and thrombosis by varying key parameters that influence hemostatic (sub)processes including the adhesive matrix, rheology and flow profiles, cellular composition, presence of toxins or drugs, ionic strength and many more. In the past two decades, low throughput flow chamber experiments requiring large sample volumes (10-100 ml) have evolved to microfluidic chambers often consisting of small parallel-plate chambers and including modern technology for perfusing whole blood at controlled wall shear conditions5. Microscaling has significantly increased assay throughput mostly because the hardware setup has simplified and less (blood) volume is required, rendering the experiment more accessible and versatile. For instance, blood from small laboratory animals can now be used without the need to sacrifice animals. Blood samples of genetically modified mice have thus aided in the identification of key molecules promoting or inhibiting hemostasis and in novel basic insights6.
Specialized research laboratories often still use custom made flow chambers for instance from polydimethylsiloxane (PDMS)7 that polymerizes on lithographed molds which can be blueprinted by software. The resulting chamber is inexpensive, disposable and can be easily disassembled for post hoc analysis. Furthermore, basically any design of vessels, including bifurcations or sharp turns can be built on command. This advantage is also its downside since standardization was already the primary problem with flow chamber experiments, and PDMS custom made chambers have not aided this. On top of this particular issue, coating (conditions), fluorescent probes, anticoagulant, temperature and time between sampling and analysis are all poorly standardized8. Standardization of these variables is challenging, but nonetheless required to permit comparison of results between laboratories. This topic is the major subject of the International Society on Thrombosis and Haemostasis in Scientific and Standardization subcommittee on Biorheology9,10.
Platelet concentrates (PC) are transfused in patients suffering from various diseases that cause thrombocytopenia and/or bleeding. But platelets in PC are known to desensitize, especially in function of storage time11, a deterioration process linked to ageing and commonly referred to as platelet storage lesion. It is sometimes claimed that such platelets restore in circulation once transfused12, but evidence for this is scarce. Furthermore, the functionality of platelets making up a PC is not routinely tested because the relationship between such assays and therapeutic or prophylactic efficacy is unclear13. Microfluidic flow chambers offer a means to investigate platelet function in PC to optimize the chain of manipulations between collection and issuing. It is a powerful research tool for direct (paired) comparisons of PC as we have previously published14,15 and is described here.

<table border="0" cellpadding="0" cellspacing="0" width="1113">
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer tube with EDTA&#160;</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368856</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Heparin</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368480</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Sodium Citrate</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">366575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hirudin Blood tube</td>
			<td style="width:220px;">Roche</td>
			<td style="width:268px;">6675751 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer Eclipse</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368650</td>
			<td>Blood collection needle with preattached holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 100-1000</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">740290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 2-200</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">739280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 1-10</td>
			<td style="width:220px;">Eppendorf</td>
			<td style="width:268px;">A08928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tube 5mL</td>
			<td style="width:220px;">Simport</td>
			<td style="width:268px;">11691380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 15mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">1888271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 50mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:268px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision wipes</td>
			<td style="width:220px;">Kimtech</td>
			<td style="width:268px;">5511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Fluoro+ Biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">188V8CF-400-100-02P10</td>
			<td>Named in figure S1 A as &#39;Biochip&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Tubing</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">TUBING-TYGON-B1IC-B1OC-ROLL 100F</td>
			<td>Named in figure S1 B as &#39;Disposable tubing&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Needles</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">SS-P-B1IC-B1OC-PACK200</td>
			<td>Named in figure S1 B as &#39;Pin&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Connectors for single inlet cables of biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">CONNECTORS-B1IC-PACK100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multiflow8 connect</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">MF8-CONNECT-BIC3-N-THROMBOSIS</td>
			<td>Named in figure S1 B as &#39;Reusable tubing&#39; and &#39;Splitter&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Humidified box</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">HUMID-BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microfluidic pump</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">N/A</td>
			<td>Venaflux Assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Horm Collagen</td>
			<td style="width:220px;">Takeda/Nycomed</td>
			<td style="width:268px;">1130630</td>
			<td style="width:409px;">Native equine tendon collagen (type I) 
			Isotonic glucose solution to dilute collagen is supplemented</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES buffered saline (HBS)</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blocking buffer</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcein AM</td>
			<td style="width:220px;">Molecular probes</td>
			<td style="width:268px;">C1430</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach 10%</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1M NaOH</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Denaturated alcohol</td>
			<td style="width:220px;">Fiers</td>
			<td style="width:268px;">T0011.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mirus Evo Nanopump</td>
			<td>Cellix</td>
			<td>188-MIRUS-PUMP-EVO</td>
			<td>with Multiflow8. Named in figure S1 A as &#39;Pump&#39; and &#39;Manifold&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer Z1</td>
			<td>equipped with a colibri-LED and high resolution CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:268px;">N/A</td>
			<td>ZEN 2012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematology analyzer</td>
			<td>Sysmex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge</td>
			<td>Eppendorf</td>
			<td>521-0095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Platelet incubater</td>
			<td style="width:220px;">Helmer</td>
			<td style="width:268px;">PF-48i</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubation water bath</td>
			<td>GFL</td>
			<td>1013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette</td>
			<td>Brand</td>
			<td>A03429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Roller</td>
			<td>Ratek</td>
			<td>BTR5-12V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile docking device</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">TSCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing Sealer</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">AC-155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:220px;">VWR</td>
			<td style="width:268px;">58816-121</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

To demonstrate intra-assay variation, three identical reconstituted whole blood samples were perfused simultaneously over collagen coated surfaces (Figure 1). This resulted in a coefficient of variation of 8.7%. This statistic suggests acceptable intra-assay and intralaboratory variation permitting reliable comparison between related samples.
The inlet of the commercial flow chamber we describe here is perpendic...

Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

The overall goal of this experiment is to mimic platelet transfusion in vitro and test it using hemostasis on collagen, with hydrodynamic flow and real time microscopy. Patients with thrombocytopenia are treated with stored platelet concentrates. Our method is intended to help answer key questions in the field of thrombosis and hemostasis, with specific emphasis on the quality and function of such stored platelet concentrates.The added value of this technique is that it mimics blood flow, and thus takes into account rheology influences on participating cells and biomolecules. The current method assesses platelet binding to collagen. It can, however, also be applied to other adhesive tissue, including atherosclerotic plaque, endothelial cells, or specific matrix proteins like Von Willebrand factor and fibrinogen.Begin with preparing the biochip. Vortex and dilute a collagen stock one to 20 in the manufacturer's isotonic glucose solution to a final concentration of 50 micrograms per milliliter. Then, pipette 0.8 microliters into the lanes of a new disposable biochip.Fill all the lanes on one end of the chip, and mark this as the outlet end. Then inspect the chip to make sure that each lane is filled 5/6 of the way with the solution, and no air bubbles. Then, incubate the chip at four degrees celsius for four hours or overnight in a humidified and closed container.Later, block the coded channels by pipetting blocking buffer into the other end of the lanes. Then for each lane, cut 12-centimeter lengths of tubing, and attach a pin to each length of tubing to make them attachable to the biochip. Rinse the tube lengths with distilled water from a syringe and a connector.Then, saturate them with blocking buffer and seal them into a humidified container for at least one hour. Next, prepare the pump and the manifold. First, rinse the pump and manifold with distilled water to remove any air bubbles.Next, attach the biochip to the automated microscope stage. Put one end of the lengths into a 1.5 milliliter tube filled with HBS, then attach them with the other end to the biochip inlets. Put the manifold pins in the outlets of the biochip.Then, rinse the system with HBS using the pump. Any remaining blocking buffer or poorly adhered collagen will be thus removed. This completes the flow chamber preparations.Begin with pooling recent blood collections from healthy volunteers into a conical centrifuge tube. Spin the sample for about 15 minutes at 250 Gs.Do not use the brake, because it will disturb the loosely-packed lower phase. Remove and discard the platelet-rich plasma and the buffy coat.Save the packed red blood cells containing as few platelets as possible, to make the reconstituted blood. Now, start thawing four milliliters of type AB plasma at 37 degrees celsius for five minutes and 20 seconds. Meanwhile, determine the hematocrit of the prepared packed red blood cells using an automated hematology analyzer.Then, determine the platelet concentration of a blood bank prepared platelet concentrate to use for reconstitution of the whole blood. Calculate the required volumes for one milliliter of reconstituted blood with 40%hematocrit, and 250, 000 platelets per microliter. Now, using a clipped pipette tip, transfer the required volume of red blood cells, plasma, and platelet concentrate to the desired concentration, and a final volume of one milliliter.Mix the reconstituted blood with gentle inversions, and perform a complete blood count. Then, get a prepared microcentrifuge tube containing Calcein AM, and add one milliliter of reconstituted blood to it. Mix the blood with dye using gentle inversions.Then, incubate the reconstituted blood for five minutes at 37 degrees celsius. In the software, select the region of interest in the perfusion lane. Focus on the collagen fibers adhered at the bottom of the lanes at 100x magnification.Ideally, use phase contrast or differential interference contrast. Choose the set current Z for selected tile regions option to digitally fix the selected Z positions. The optical focus prior to perfusion should be on ameloblast collagen, but this can be tricky.An alternative is to set the microscope focus on the initial adhering platelets. Now, gently mix the samples and position them next to the biochip. Then, put the tubing connected to the observed lane into the reconstituted blood.Next, using the software controlling the pump, apply a 50 dyne per square centimeter pressure to move the blood sample into the lane and start the experiment. During the experiment, record images every 15 seconds for five minutes. Determine the thrombus growth kinetics using image analysis software.In this case, Zen 2012 is utilized. Begin with opening the plugin image analysis to determine the surface coverage of the platelets. Set the fluorescence threshold in the analyze interactive tab to define the pixel intensity that correlates with a positive signal, which would come from adhering platelets.Then, use create tables to automatically generate a spreadsheet with a list of the surface areas of the positive signals for each time point. Save these spreadsheets in the XML format, and open them in a spreadsheet program for further calculations. At every time point, calculate the total surface areas of the selected objects, and express this value as the percentage of surface that is covered.Then, plot the data as a function of the perfusion time, and calculate the slope by linear regression. This models the thrombus growth kinetics of that particular experimental condition. Three identical reconstituted whole blood samples were perfused simultaneously over collagen-coated surfaces.This resulted in a coefficient of variation of 8.7%suggesting acceptable intra-assay and intra-laboratory variation for test comparison. Transfusion was simulated by reconstituting the blood with the deficient component. Several reconstitutions were performed with decreasing platelet concentrations.As expected, lower platelet counts resulted in less adhesion as a function of perfusion time. Next the effect of decreased temperatures after blood collection and during perfusion were investigated. Thrombi were found to build more slowly when the blood was cooled to room temperature.As compared to a sample kept at 37 degrees Celsius throughout the study. While circulating, platelets bind to vessel injury sites in conditions of elevated wall sheer stress. As expected, varying the sheer rate in the microfluidic flow chambers changed the total platelet adhesion.These conditions also change the thrombus growth kinetics. After watching this video, one should have a good understanding of how to perform microfluidic flow chamber experiments using reconstituted blood to test the quality and function of stored platelet concentrates. This technique can be done in roughly seven hours.Microfluidic flow chamber experiments paved the way for researchers in the field of platelet transfusion to explore the impact of donation, preparation and storage variables. One relevant example is pathogen inactivation. We have given example of just one experimental subject.However, variations in calcium concentration, sheer stress and surface coatings can be used to address different research questions. Similar to this procedure other measurements in microfluidic flow chambers can be performed to answer additional questions. For instance the performance of stored platelets to coagulation on the flow.

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