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

Summary

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.

Abstract

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.

Introduction

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 b....

Protocol

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: W....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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