DESCRIPTION Cluster type (n) poster 3JS/ABKARIAN_2013-2... · RESUME : The thesis project aims to...
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LABEX NUMEV Solutions Numériques Matérielles et Modélisation pour l’Environnement et le Vivant RESULTATS A microfluidic device with channels of ~ 12 mm x 12 mm x 30 mm in width, high and length was designed to investigate the cluster formation of RBCs on microcapillaries under flow conditions. At ~1 % of tube hematocrit and a pressure drop of 100 mbar, we observed cluster of two or more RBCs in pure buffer solution at four different channel position. An enhancement of cluster formation at the beginning of the channels is observed after add dextran of 70 kDa into the suspending media, mimicking physiological healthy conditions. Positionnement NUMEV : Projet intégré AAP2 2013 Mots clés : Red blood cells, sickle cell anemia, aggregation, microfluidics. RESUME : The thesis project aims to experimentally characterize the effects of red blood cell aggregation in microcirculatory flow. Starting from the non-pathological case of rouleaux formation it will be investigated how much RBC aggregation affects the flow dynamic of sickle cells disease. DESCRIPTION Viviana Clavería 1,2,* , Manouk Abkarian 1 and Christian Wagner 2 1 Laboratoire Charles Coulomb, Université Montpellier 2 - CNRS, UMR 5221, 34095 Cedex 5, Montpellier, France 2 Experimental Physics, Saarland University, 66123 Saarbrücken, Germany 1 http://www.coulomb.univ-montp2.fr/perso/manouk.abkarian 2 http://agwagner.physik.uni-saarland.de/ * [email protected] , 1 [email protected] , 2 [email protected] Red blood cell aggregation in Sickle cell anemia 13 14 16 35 29 40 0 2 4 6 8 10 12 14 16 0 20 40 60 80 Position 10 mm With 20 mg/ml Dex70 Without Dex70 Exp. fitting BSA sol. + 20 mg/ml Dex70 Exp. fitting BSA sol. Percentage (%) Cluster type (n) Model Exp3P2 Equation y =exp(a+b*x+c*x^2) Reduced Chi-Sqr 0.07731 0.03238 Adj. R-Square 0.99458 0.99305 Value Standard Erro Percentage a 5.33324 0.04935 b -1.25526 0.03396 c 0.05896 0.00334 Percentage without Dex70 a 5.31924 0.06332 b -1.23982 0.03973 c 0.05397 0.00394 1 2 3 4 5 6 7 0 20 40 60 80 100 Entrance With 20 mg/ml Dex70 Without Dex70 Exp. fitting BSA sol. + 20 mg/ml Dex70 Exp. fitting BSA sol. Percentage (%) Cluster type (n) Model Exp3P2 Equation y =exp(a+b*x+c*x^2) Reduced Chi-Sqr 0.05433 0.00791 Adj. R-Square 0.99598 0.9998 Value Standard Error Percentage a 4.71374 0.10902 b -0.59046 0.08791 c -0.06452 0.01613 a 6.69744 0.09019 b -2.29175 0.11932 c 0.06574 0.03175 1 2 3 4 5 6 7 0 20 40 60 80 100 With 20 mg/ml Dex70 Without Dex70 Exp. fitting BSA sol. + 20 mg/ml Dex70 Exp. fitting BSA sol. Position 1 mm Percentage (%) Cluster type (n) Model Exp3P2 Equation y =exp(a+b*x+c*x^2) Reduced Chi-Sqr 0.37224 0.00228 Adj. R-Square 0.97542 0.99988 Value Standard Error Percentage a 5.67875 0.21084 b -1.55433 0.1859 c 0.09171 0.02948 a 6.43801 0.05603 b -1.85858 0.07524 c -0.09576 0.02168 0 2 4 6 8 10 0 20 40 60 80 100 Position 2 mm With 20 mg/ml Dex70 Without Dex70 Exp. fitting BSA sol. + 20 mg/ml Dex70 Exp. fitting BSA sol. Percentage (%) Cluster type (n) Model Exp3P2 Equation y =exp(a+b*x+c*x^2) Reduced Chi-Sqr 0.26775 0.09138 Adj. R-Square 0.98374 0.99286 Value Standard Error Percentage a 5.58978 0.14258 b -1.41961 0.11317 c 0.06264 0.01687 Percentage without Dex70 a 6.54908 0.18957 b -2.29159 0.20597 c 0.15408 0.0445 Figure 4. Comparison between cluster ocurrence at 4 different channel positions in pure physiological buffer solution and a solution with 20 mg/ml of dextran 70. Although at the entrance and positions 1 and 2 mm deep into the microchannel, the cluster formation due to dextran 70 is predominant , surprisingly at position 10 mm the cluster ocurrence tend to be the same suggesting a strong cluster formation due to hydrodynamic interactions. Figure 5. a) Cluster length in pure physiological buffer solution and pure physiological buffer solution + 20 mg/ml of dextran 70. b) Distribution of the cluster size in the case of a cluster of 2 RBCs. In both cases 1 px ≈ 0,4 mm. a) b) After noticed the effect of dextran 70 in the cluster formation, we were interested in to compare the cluster length in order to quatify the effect of dextran in the suspending medium. We found that the difference in size is ~10% being the cluster length in buffer solution with dextran shorter. Figure 1. The rouleaux formation in PBS + dextran macromolecules (b) in the suspending media contrast the RBC behaviour in pure physiological buffer solution (a), where no aggregation is observed. a ) b) Figure 2. Ilustration of sickle cell formation. The homogeneous nucleation of a mutated hemoglobin produce self-assembly of the proteins and long rope-like fibers. On the other hand, the origin of Sickle cells lies in a recessive genetic disorder caused by a point mutation in the gene that encodes for the hemoglobin molecule of its carrier. This mutated hemoglobin, Is the responsible of the self-assembly of the proteins into long rope-like fibers (Fig. 2), causing, in certain conditions, the distortion of RBCs into the classical crescent or sickle shape and in all case a marked decrease of cells deformability. Therefore, unlike normal RBCs, sickled RBCs are believed to cause capillary blockages that deprive the organs and tissues of the oxygen -carrying blood hence painful crisis. Dr. Abkarian has established a microfluidic device that is suitable to study the aggregation and clogging process of sickle cell and it is planned to use this device for both aggregation/cluster formation and clogging of sickle cells. It should allow look at the dynamics of vaso-occlusion from a single channel to a suspension of RBCs. We will identify and characterize physical mechanisms, which contribute to the different adhesion processes of red blood cells and we will investigate how the aggregation of red blood cells affects the flow dynamics in healthy conditions and with a special focus on sickle cell disease. The linear aggregation of red blood cells (RBCs), know as rouleaux (Fig. 1), is typically related to regions of low shear rate. It is also known that thrombus is mostly formed at places of low shear rates as in aneurisms. However, increasing shear rates the rouleaux break and the viscosity of blood consequently decreases. Then, the rouleaux formation is assumed to be completely reversible. Finally, Dr. Wagner at the Saarland University suggested a signaling cascade that hypothesized even an active adhesion of red blood cells after stimulating with physiological substances released during blood coagulation. A test of this was recently possible and holographic optical tweezers were used to either validate or refute this hypothesis. The results of these measurements strongly support their proposed signaling cascade. To value the occurring adhesion to be of significance for the in vivo situation, it has to be strong enough. Therefore, it was quantified in further experiments by means of single cell force spectroscopy (Fig. 3). The results indicate that the inter cellular adhesion of red blood cells after stimulation is strong enough and therefore could be actively supportive for blood clot solidification. It will be an aim of the thesis project to clarify if a similar activation is responsible for the RBC aggregation in sickle cells. In the case of Sickle cell, to separate the effects of steric and hydrodynamic properties of adhesion to substrates, we will use artificial RBCs which consist of lipid vesicles enclosing hemoglobin S and whose production is controlled in laboratory (patent between the National Center of the Scientific Research, CNRS, and University Montpellier 2 in France). The behavior under flow to the one of sickle cells and the behavior of adhesive simple solid colloidal micron-size particles could be compared. Figure 3. Scheme of a single cell force spectroscopy testing.
Transcript of DESCRIPTION Cluster type (n) poster 3JS/ABKARIAN_2013-2... · RESUME : The thesis project aims to...
LABEX
NUMEV
Solutions
Numériques
Matérielles et
Modélisation pour
l’Environnement
et le Vivant
RESULTATS A microfluidic device with channels of ~ 12 mm x 12 mm x 30 mm in width, high and length was designed to investigate the cluster formation of RBCs on microcapillaries under flow conditions. At ~1 % of tube hematocrit and a pressure drop of 100 mbar, we observed cluster of two or more RBCs in pure buffer solution at four different channel position. An enhancement of cluster formation at the beginning of the channels is observed after add dextran of 70 kDa into the suspending media, mimicking physiological healthy conditions.
Positionnement NUMEV : Projet intégré AAP2 2013
Mots clés : Red blood cells, sickle cell anemia, aggregation, microfluidics.
RESUME : The thesis project aims to experimentally characterize the effects of red blood cell aggregation in microcirculatory flow. Starting from the non-pathological case of rouleaux formation it will be investigated how much RBC aggregation affects the flow dynamic of sickle cells disease.
DESCRIPTION
Viviana Clavería1,2,*, Manouk Abkarian1 and Christian Wagner2
1 Laboratoire Charles Coulomb, Université Montpellier 2 - CNRS, UMR 5221, 34095 Cedex 5, Montpellier, France 2 Experimental Physics, Saarland University, 66123 Saarbrücken, Germany
1 http://www.coulomb.univ-montp2.fr/perso/manouk.abkarian
2 http://agwagner.physik.uni-saarland.de/
* [email protected], 1 [email protected], 2 [email protected]
Red blood cell aggregation in Sickle cell anemia
13
14
16
35
29 40
0 2 4 6 8 10 12 14 16
0
20
40
60
80
Position 10 mm With 20 mg/ml Dex70
Without Dex70
Exp. fitting BSA sol. + 20 mg/ml Dex70
Exp. fitting BSA sol.
Pe
rcen
tag
e (
%)
Cluster type (n)
Model Exp3P2
Equation y =exp(a+b*x+c*x^2)
Reduced Chi-Sqr
0.07731 0.03238
Adj. R-Square 0.99458 0.99305
Value Standard Erro
Percentage
a 5.33324 0.04935
b -1.25526 0.03396
c 0.05896 0.00334
Percentage without Dex70
a 5.31924 0.06332
b -1.23982 0.03973
c 0.05397 0.00394
1 2 3 4 5 6 7
0
20
40
60
80
100
Entrance With 20 mg/ml Dex70
Without Dex70
Exp. fitting BSA sol. + 20 mg/ml Dex70
Exp. fitting BSA sol.
Pe
rcen
tag
e (
%)
Cluster type (n)
Model Exp3P2
Equation y =exp(a+b*x+c*x^2)
Reduced Chi-Sqr
0.05433 0.00791
Adj. R-Square 0.99598 0.9998
Value Standard Error
Percentage
a 4.71374 0.10902
b -0.59046 0.08791
c -0.06452 0.01613
a 6.69744 0.09019
b -2.29175 0.11932
c 0.06574 0.03175
1 2 3 4 5 6 7
0
20
40
60
80
100
With 20 mg/ml Dex70
Without Dex70
Exp. fitting BSA sol. + 20 mg/ml Dex70
Exp. fitting BSA sol.
Position 1 mm
Pe
rcen
tag
e (
%)
Cluster type (n)
Model Exp3P2
Equation y =exp(a+b*x+c*x^2)
Reduced Chi-Sqr
0.37224 0.00228
Adj. R-Square 0.97542 0.99988
Value Standard Error
Percentage
a 5.67875 0.21084
b -1.55433 0.1859
c 0.09171 0.02948
a 6.43801 0.05603
b -1.85858 0.07524
c -0.09576 0.02168
0 2 4 6 8 10
0
20
40
60
80
100
Position 2 mm With 20 mg/ml Dex70
Without Dex70
Exp. fitting BSA sol. + 20 mg/ml Dex70
Exp. fitting BSA sol.
Pe
rcen
tag
e (
%)
Cluster type (n)
Model Exp3P2
Equation y =exp(a+b*x+c*x^2)
Reduced Chi-Sqr
0.26775 0.09138
Adj. R-Square 0.98374 0.99286
Value Standard Error
Percentage
a 5.58978 0.14258
b -1.41961 0.11317
c 0.06264 0.01687
Percentage without Dex70
a 6.54908 0.18957
b -2.29159 0.20597
c 0.15408 0.0445
Figure 4. Comparison between cluster ocurrence at 4 different channel positions in pure physiological buffer solution and a solution with 20 mg/ml of dextran 70. Although at the entrance and positions 1 and 2 mm deep into the microchannel, the cluster formation due to dextran 70 is predominant , surprisingly at position 10 mm the cluster ocurrence tend to be the same suggesting a strong cluster formation due to hydrodynamic interactions.
Figure 5. a) Cluster length in pure physiological buffer solution and pure physiological buffer solution + 20 mg/ml of dextran 70. b) Distribution of the cluster size in the case of a cluster of 2 RBCs. In both cases 1 px ≈ 0,4 mm.
a)
b)
After noticed the effect of dextran 70 in the cluster formation, we were interested in to compare the cluster length in order to quatify the effect of dextran in the suspending medium. We found that the difference in size is ~10% being the cluster length in buffer solution with dextran shorter.
Figure 1. The rouleaux formation in PBS + dextran macromolecules (b) in the suspending media contrast the RBC behaviour in pure physiological buffer solution (a), where no aggregation is observed.
a)
b)
Figure 2. Ilustration of sickle cell formation. The homogeneous nucleation of a mutated hemoglobin produce self-assembly of the proteins and long rope-like fibers.
On the other hand, the origin of Sickle cells lies in a recessive genetic disorder caused by a point mutation in the gene that encodes for the hemoglobin molecule of its carrier. This mutated hemoglobin, Is the responsible of the self-assembly of the proteins into long rope-like fibers (Fig. 2), causing, in certain conditions, the distortion of RBCs into the classical crescent or sickle shape and in all case a marked decrease of cells deformability. Therefore, unlike normal RBCs, sickled RBCs are believed to cause capillary blockages that deprive the organs and tissues of the oxygen -carrying blood hence painful crisis. Dr. Abkarian has established a microfluidic device that is suitable to study the aggregation and clogging process of sickle cell and it is planned to use this device for both aggregation/cluster formation and clogging of sickle cells. It should allow look at the dynamics of vaso-occlusion from a single channel to a suspension of RBCs.
We will identify and characterize physical mechanisms, which contribute to the different adhesion processes of red blood cells and we will investigate how the aggregation of red blood cells affects the flow dynamics in healthy conditions and with a special focus on sickle cell disease. The linear aggregation of red blood cells (RBCs), know as rouleaux (Fig. 1), is typically related to regions of low shear rate. It is also known that thrombus is mostly formed at places of low shear rates as in aneurisms. However, increasing shear rates the rouleaux break and the viscosity of blood consequently decreases. Then, the rouleaux formation is assumed to be completely reversible.
Finally, Dr. Wagner at the Saarland University suggested a signaling cascade that hypothesized even an active adhesion of red blood cells after stimulating with physiological substances released during blood coagulation. A test of this was recently possible and holographic optical tweezers were used to either validate or refute this hypothesis. The results of these measurements strongly support their proposed signaling cascade. To value the occurring adhesion to be of significance for the in vivo situation, it has to be strong enough. Therefore, it was quantified in further experiments by means of single cell force spectroscopy (Fig. 3). The results indicate that the inter cellular adhesion of red blood cells after stimulation is strong enough and therefore could be actively supportive for blood clot solidification. It will be an aim of the thesis project to clarify if a similar activation is responsible for the RBC aggregation in sickle cells.
In the case of Sickle cell, to separate the effects of steric and hydrodynamic properties of adhesion to substrates, we will use artificial RBCs which consist of lipid vesicles enclosing hemoglobin S and whose production is controlled in laboratory (patent between the National Center of the Scientific Research, CNRS, and University Montpellier 2 in France). The behavior under flow to the one of sickle cells and the behavior of adhesive simple solid colloidal micron-size particles could be compared.
Figure 3. Scheme of a single cell force spectroscopy testing.
