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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 biomolecules^3,4.

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 Biorheology^9,10.

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 published^14,15 and is described here.

Protokół

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

1. Preparing Lanes, Tubing and Pins
   1. Vortex the collagen suspension vigorously and dilute 1/20 in the isotonic glucose solution supplied by the provider to a final concentration of 50 µg/ml.
      Note: We use equine tendon collagen, mainly made up of type I fibrils. The equine collagen type I is often referred to as "Horm" collagen and is the golden standard for this type of assay⁹ for both historical as well as biological reasons. Human type III collagen can also be used, but the fibrils coat less well and the platelet response is not as strong. Other coating surfaces can also be used, for example von Willebrand Factor (VWF), fibrinogen, fibronectin, laminin, vitronectin, thrombospondin-1 or combinations of these¹⁶.
   2. Take a new disposable biochip from the provider's container. The dimensions of the biochips used here are 0.4W x 0.1H x 20L in mm³.
   3. Pipet 0.8 µl into the lane(s) of the microfluidic biochip on one end of the chip and mark as outlet. Make sure that the lane is filled 5/6th with the collagen containing coating solution prepared in 1.1.1. Ensure that there are no air bubbles.
      Note: Channels are partially coated to avoid accumulation of collagen fibers at the entrance of the channel (see discussion).
   4. Incubate at 4 °C for 4 hr or overnight in a humidified and closed container.
   5. Block the coated channels by pipetting blocking buffer (1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (HBS; 0.9% (w/v) NaCl, pH 7.4) at the other end and mark as inlet. Make sure that the lane is completely filled with the blocking buffer avoiding air bubbles.
   6. Cut tubing at equal length (12 cm). Use one per lane and connect each tubing with a pin. For example, an experiment comparing two conditions, run in duplicate will require four tubing stretches and four pins to be prepared.
   7. Rinse the tubing with distilled water using a syringe and a 26 G needle or the accompanying connectors.
   8. Saturate the tubing with blocking buffer. Store in a closed and humidified container for a minimum of 1 hr.
2. Preparing Pump and Manifold
   1. Rinse the pump and manifold with distilled water, remove air bubbles.
   2. Aspirate the blocking buffer out of the biochip lane(s) using the fixed 10 µl tip at the outlet. Clean the surface of the biochip with a precision dust free wipe and denaturated alcohol to remove prints and dust.
   3. Fix the biochip on an automated microscope stage. If more than one lane is used simultaneously in one run, connect the eight-lane manifold splitter to the biochip outlet.
      Note: The eight-lane manifold splitter is a piece of hardware (Figure S1) connected to the pump and the biochip. It allows operation of all available (eight) lanes on a biochip or a combination of lanes which can be operator-defined in the accompanying software.
   4. Use the pins in the tubing to fix them in the biochip inlet. Place the other end of the tubing (without pin) in a 1.5 ml conical test tube filled with HBS. Rinse all tubing and their connected lanes with 1 ml HBS using the pump, so as to remove remainder blocking buffer and poorly adhering collagen.

2. Preparation of Blood Samples

1. Collection and separation of fresh whole blood from a healthy volunteer.¹⁷
   1. Collect the first milliliters of blood in an evacuated tube containing Ethylenediaminetetraacetic acid (EDTA) as an anticoagulant and exclusively use this sample for complete blood count (CBC) with an automated hematology analyzer.
   2. Collect a volume of blood in a suitable anticoagulant using evacuated tubes. Standard anticoagulants for flow chamber experiments are heparin or hirudin when fibrin formation is not part of the study protocol and sodium citrate when it is.
      Note: Heparin was used as an anticoagulant for all the experiments described in the results.
      Note: The amount of blood depends on the number of experiments to be performed. Approximately 1 tube (7 ml) for 3 lanes.
   3. Place the tubes on a rotator pending blood reconstitution.
      Note: The assay should be completed within 3 hr of phlebotomy.
   4. Centrifuge for 15 min at 250 g to prepare platelet rich plasma (PRP). Do not use the centrifuge break to prevent disturbance of the loosely packed pellet.
      1. When more than one tube was collected, pool the blood in a single conical centrifugation tube.
         Note: Centrifugation can be done more slowly or less long, depending on the PRP yield and differential cell "contamination" preferred.
   5. Remove and discard the PRP and buffy coat yielding packed red blood cells with few platelets.
      Note: The platelet count in the packed red cell fraction is 13 ±5 x 10³ per µl (mean ±SD, n = 12) on average in our hands.
2. Blood Reconstitution
   1. Thaw blood group AB (Rhesus D negative) plasma at 37 °C for 5 min and 20 sec per 4 ml.
   2. Determine the hematocrit of the packed red blood cells prepared in 2.1.5 using an automated hematology analyzer.
   3. Determine the platelet concentration in the blood bank prepared platelet concentrate that will be used to reconstitute the red cell fraction above.
   4. Calculate the volume of packed red blood cells and platelet concentrate that will yield 40% hematocrit and 250 x 10³ platelets/µl in a 1 ml sample.
      Note: Other target titers of cells can be set arbitrarily, depending on the study protocol.
   5. Transfer packed red blood cells and plasma into a fresh tube using a clipped pipet tip and add platelet concentrate until a sample volume of 1 ml is reached.
      Note: Depending on the variables studied, the plasma fraction should be equal in all reconstituted samples because plasma has a significant influence on thrombus formation rate. For instance, repeated freeze-thawing or plasma taken from different donors or on different anticoagulants may influence the result.
   6. Mix the reconstituted blood gently by inverting and perform a CBC.
   7. Prepare a "blank" control sample in which the volume of platelet fraction is replaced by the same volume of 0.9% (m/v) sodium chloride in water to determine the concentration of endogenous platelets (i.e. non-blood bank platelets) in the reconstituted blood using a CBC.
3. Labeling
   1. Pipet 1 ml reconstituted blood in a test tube containing 1 µl 5 mM Calcein AM (5 µM final concentration).
      Note: Other cell dyes can be used¹⁴.
   2. Mix gently by inverting.
   3. Incubate for 5 min at 37 °C prior to use.

3. Perfusion Assay

1. Focus the objective on the collagen fibers adhered at the bottom of the lanes. Ideally, use phase-contrast or differential interference contrast (DIC) settings for this focusing strategy. Select ‘Set current Z for selected tile regions’ in the experiment software to digitally fix the selected Z-positions.
2. Select a region of interest (ROI) in the lane (xy) in the experiment software of the microscope that will be recorded during the experiment.
   Note: The ROI can be any surface area arbitrarily chosen within a perfusion lane. It is advisable not to analyze thrombus formation close to the in- and outlet of a lane so to avoid side effects of the variable flow profile in that region, even though this is relatively small. The ROI surface area should contain a significant number of platelets or thrombi to allow leveling of the signal. In this protocol the ROI is a digitally stitched aggregate of three equally sized side-by-side images resulting in 0.62 mm² in the middle of the 2 cm long lane.
3. Mix the samples gently by inverting and position these next to the biochip on the automated stage.
4. Place the tubing that is connected with the inlet of the biochip in the test tubes containing the reconstituted blood samples.
5. Launch the pump at 50 dyne/cm² (or other shear stresses as desired) for those channels linked to test tubes containing the reconstituted blood samples using the software of the pump.
   Note: Other shear stresses can be used.
6. Record images every 15 sec for 5 min in real-time using the acquisition and experiment software of the microscope.
   Note: Other time series can be used depending on the experimental set-up.
   Note: We generally use a 100X magnification (10X objective and 10X lenses), but higher (or lower) magnification can easily be used as an alternative.

4. Wash Out

1. Wash out all tubing attached to the outlet and connected to the multichannel manifold or pump using distilled water, followed by sodium hypochlorite (bleach) 0.5% (v/v) and finally 0.1 M NaOH in water. Discard the tubing pinned to the biochip inlet as hazardous waste.

5. Data Analysis

1. Determine thrombus growth kinetics with the image analysis software. The following commands are specific for ZEN2012.
   1. Open the plugin Image Analysis to determine the surface coverage of the platelets.
   2. Set the fluorescence threshold in the Analyze Interactive tab to define the pixel intensity that correlates with a positive signal, i.e. an adhered platelet or adhering platelets.
   3. Use Create Tables to automatically generate a spreadsheet that will contain the separate surface areas (in µm²) of those "objects" containing pixels with a signal between the selected thresholds. This is performed for each time point.
      Note: Once fluorescence thresholds have been chose, the analysis software automatically detects 'objects' in the view field that fulfill the criteria. These objects are thrombi, small platelet aggregates or single platelets and cover a number of pixels. Every object is listed in the spreadsheet separately.
   4. Save these spreadsheets in xml format and open them in a spreadsheet program for further calculations.
   5. Total the surface areas of the selected objects by summation and divide the result by the total area of the measurement field (µm²). This will yield the relative surface coverage (%). Do so for every time point.
   6. Plot these surface coverages in function of the perfusion time and calculate the slope by linear regression, yielding the thrombus growth kinetic of that particular condition.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
BD vacutainer tube with EDTA	Becton, Dickinson and Company	368856
BD vacutainer tube with Heparin	Becton, Dickinson and Company	368480
BD vacutainer tube with Sodium Citrate	Becton, Dickinson and Company	366575
Hirudin Blood tube	Roche	6675751 001
BD vacutainer Eclipse	Becton, Dickinson and Company	368650	Blood collection needle with preattached holder
Pipette tips 100-1,000	Greiner bio-one	740290
Pipette tips 2-200	Greiner bio-one	739280
Pipette tips 1-10	Eppendorf	A08928
Tube 5 ml	Simport	11691380
Conical tube 15 ml	Greiner bio-one	1888271
Conical tube 50 ml	Greiner bio-one	227261
10 ml Syringe	BD	309604
Precision wipes	Kimtech	5511
Vena8 Fluoro+ Biochips	Cellix	188V8CF-400-100-02P10	Named in Figure S1 A as 'Biochip'
Vena8 Tubing	Cellix	TUBING-TYGON-B1IC-B1OC-ROLL 100F	Named in Figure S1 B as 'Disposable tubing'
Vena8 Needles	Cellix	SS-P-B1IC-B1OC-PACK200	Named in Figure S1 B as 'Pin'
Connectors for single inlet cables of biochips	Cellix	CONNECTORS-B1IC-PACK100
Multiflow8 connect	Cellix	MF8-CONNECT-BIC3-N-THROMBOSIS	Named in Figure S1 B as 'Reusable tubing' and 'Splitter'
Humidified box	Cellix	HUMID-BOX
Software microfluidic pump	Cellix	N/A	Venaflux Assay
Horm Collagen	Takeda/Nycomed	1130630	Native equine tendon collagen (type I) Isotonic glucose solution to dilute collagen is supplemented
HEPES buffered saline (HBS)	in house preparation	in house preparation	10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4)
Blocking buffer	in house preparation	in house preparation	1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS
Calcein AM	Molecular probes	C1430
Bleach 10%	in house preparation	in house preparation
0.1 M NaOH	in house preparation	in house preparation
Denaturated alcohol	Fiers	T0011.5
Mirus Evo Nanopump	Cellix	188-MIRUS-PUMP-EVO	with Multiflow8. Named in Figure S1 A as 'Pump' and 'Manifold'
Microscope	Zeiss	Axio Observer Z1	equipped with a colibri-LED and high resolution CCD camera
Software microscope	Zeiss	N/A	ZEN 2012
Hematology analyzer	Sysmex	N/A
Table Top Centrifuge	Eppendorf	521-0095
Platelet incubater	Helmer	PF-48i
Incubation water bath	GFL	1013
Pipette	Brand	A03429
Tube Roller	Ratek	BTR5-12V
Sterile docking device	Terumo BCT	TSCD
Tubing Sealer	Terumo BCT	AC-155
Vortex	VWR	58816-121