This work was supported in part by a Eurostars grant estar18105 and by an unrestricted grant provided by RR Mechatronics. The authors thank Sisto Hendriks and Jan de Zoeten for their technical support.

All procedures were approved by the ethical committee of the University Medical Center Utrecht (UMCU) and in accordance to the Declaration of Helsinki. Patients enrolled at the Texas Children&#8217;s Hematology Center (TCHC) were approved by the local IRB and in accordance with the Declaration of Helsinki.
1. General considerations
<ol>
	<li>Begin by performing a test measurement to warm up the bob and cup. Ensure that the temperature of the bob and cup is 37 &#730;C. This is important for good reproducibility.</li>
	<li>Ensure that the viscous polyvinylpyrrolidone (PVP) solution falls within the strict limits for osmolarity (282&#8211;286 mOsm/kg), pH (7.35&#8211;7.45) and viscosity (27.5&#8211;32.5 MPa) at room temperature (22 &#730;C). 
	NOTE: The PVP must be used at room temperature. If stored at a lower temperature, make sure it has warmed up to room temperature prior to taking any measurements.</li>
</ol>
2. Start-up of the ektacytometer
<ol>
	<li>Switch on the computer and the ektacytometer from the back. Start the software program (Table of Materials) on the computer.</li>
	<li>Make sure the nitrogen is available to deoxygenate the sample by opening the nitrogen cylinder.</li>
	<li>Lower the bob in the cup and make sure the cup can turn freely. Clean the cup on the inside and outside with a soft cloth and distilled water, because debris can hamper the EI measurements.</li>
	<li>When the software program is running, check for the following message on the screen: &#34;Make sure the gas valve is open&#34;&#160;and click OK.</li>
	<li>Ensure that the ektacytometer starts the pO2 self-check process that will appear on the screen. Select Start (enter). If it fails, rerun the self-check by clicking Hardware check | pO2 | Self check. 
	NOTE: If the self-check fails again, consider replacing the O2-spot. The O2-spot is replaced by gently pushing the spot out from the inside of the cup with a fingertip. A new spot is placed by gently pushing the spot from the outside into the cup.</li>
	<li>Choose pO2 scan from the different tests listed on the left. Choose Settings at the right of the screen and ensure they are set as per the parameters listed in Table 1. Keep the same settings for every measurement.</li>
	<li>In order to save these settings, press OK | OK. 
	NOTE: Preferred settings are listed in Table 1 but can be adjusted according to the user preferences and investigational purposes. For example, to study the sickling behavior more extensively, deoxygenation speed and duration can be altered.</li>
</ol>
3. Sample collection and preparation
NOTE: For the validation of the technique, ethylenediamine tetraacetic acid (EDTA)-treated blood from 38 SCD patients and 5 healthy controls included at the University Medical Center Utrecht or Texas Children&#39;s Hematology Center, in different clinical studies (Netherlands Trial Registry [NTR] identifier, NTR 6779 and NTR 6462), as well as anonymized leftover blood samples from patients who visited the outpatient clinic or were hospitalized were used.
<ol>
	<li>Collect blood samples by venipuncture (a minimum of 300 &#181;L/sample) in a tube containing EDTA. Make sure the blood has been stored for at least 30 min at 4 &#176;C, but no longer than 24 h. 
	NOTE: Citrate phosphate dextrose adenine (CPDA) or heparin can also be used, but the influence of these reagents on the sample preservation with respect to the oxygen gradient ektacytometry is not well-known.</li>
	<li>Mix the sample gently by inversion to homogenize. Do not shake the sample. Let the sample warm up to room temperature on a roller bench before the measurement. 
	NOTE: A sample tube (9&#8211;10 mL) that is stored for more than 1 h at 4 &#730;C must warm up for 15 min. When stored for less than 1 h at 4 &#730;C, it must warm up for 10 min. A sample tube (2&#8211;6 mL) that is stored for more than 1 h at 4 &#730;C must warm up for 10 min. When stored for less than 1 h at 4 &#730;C, it must warm up for 5 min.</li>
	<li>Measure the complete blood count on a hematology analyzer. To do so, take 20&#8211;200 &#181;L of whole blood in a tube containing EDTA. Place the aspiration needle in the tube and press on the button behind the needle of the hematology analyzer to start the measurement. 
	NOTE: In the complete blood count, the RBC number is measured, which is an important factor for standardizing the oxygen gradient ektacytometry measurements. RBC count is calculated from forward and sideward scatter by flow cytometry. Normal RBC count in healthy controls is 3.7&#8211;5.0 x 1012/L for females and 4.2&#8211;5.5 x 1012/L for males. RBC count in patients with SCD is generally decreased. Some hematology analyzers will also measure percent dense red blood cells (% DRBC) which can be of additional value in the interpretation of individual oxygen gradient ektacytometry curves.</li>
	<li>Standardize the whole blood sample to an RBC count of 200 x 106 RBCs in 5 mL PVP (200 x 106 RBCs/vial) by adjusting the volume of sample that will be added. If the total RBC count is less than 200 x 106, the diffraction pattern and EI will be affected.
	<ol>
		<li>Use the equation below to perform the counting. 
		4.0/xx (x 1012/L) x 50 = yy &#181;L whole blood/vial PVP 
		where xx is the calculated RBC count obtained from step 3.3 and yy is the amount of whole blood that is required for the actual measurement. Depending on the grade of anemia and other factors influencing RBC counts, the amount of whole blood required is 40&#8211;90 &#181;L.</li>
	</ol>
	</li>
</ol>
4. Oxygen gradient ektacytometry measurement
<ol>
	<li>Pipette the calculated sample volume (yy &#181;L of blood) into PVP to obtain a total volume of 5 mL. Prewet the tip by gently resuspending the blood 3x. Use a pipette tip with a wide opening to avoid additional stress on the RBCs. Gently mix the sample manually by inversion until it is homogeneous. 
	NOTE: Open the PVP vial for as short a time as possible to avoid air contact.</li>
	<li>Slowly draw 2.0 mL of the blood/PVP mixture into a 3 mL syringe without the needle. Push the plunger to remove any visible air bubbles and excessive sample solution until 1.5&#8211;1.8 mL is left in the syringe (depending on the cup volume).</li>
	<li>Inject the total sample volume slowly and evenly in the bob through the connector. Make sure the level of the sample is above the oxygen sensor (pink spot) and above the small suction hole. Do not leave any sample solution in the syringe.</li>
	<li>Click New and fill in the sample identifier, remarks, date of donation, and viscosity of PVP. Click OK |Aspirate. After 60 s, the cup will rotate and aspirate the sample for 15 s. Click OK when the rotation stops. Close the machine lid. Click Continue | Start now, as oxygen gradient ektacytometry is done with a fixed gain. The measurement will take about 28 min.</li>
	<li>After the measurement, print the report that shows the curve and parameters that are automatically calculated by the software. Ensure that the raw data is automatically stored in the designated folder in Settings. Maximum EI (EImax), minimum EI (EImin), pO2@95%EI (PoS), and area (area under the curve) are automatically calculated and added to the printed report and raw data.</li>
	<li>Manually obtain &#916;EI by calculating the difference between EImax and EImin. Calculate the percentage recovery by taking the difference in mean EI before deoxygenation (pO2 100&#8211;120 mmHg) and mean EI values during reoxygenation at 100&#8211;120 mmHg.</li>
</ol>
5. Cleaning of the ektacytometer
<ol>
	<li>Remove the sample syringe and replace it with a syringe filled with distilled water or deionized water.</li>
	<li>Press Clean, slowly flushing the connector during rinsing. Make sure to flush in both directions.</li>
	<li>Remove the syringe and lift the bob. Dry the bob, cup, and connector thoroughly with a soft cloth.</li>
	<li>Use a large syringe (10&#8211;50 mL) to flush the connector in order to remove any water remaining in the tubes and bob. Block the lower inlet/outlet of the bob to get back pressure in the tubes, thereby removing remaining water.</li>
	<li>Lower the bob in the cup. The machine is now ready for the next measurement.</li>
</ol>
6. Shutdown of the machine
<ol>
	<li>Ensure the machine is properly rinsed after the last measurement, as described above. Ensure the proper tubes relate to the cleaning solution.</li>
	<li>Close the software, press Close, and press Start to start end-of-day cleaning program.</li>
	<li>After completing the whole cleaning program, remove the syringe and lift the bob. Flush the connector with a big syringe.</li>
	<li>Empty the waste bottle and dry the bob and cup with a soft cloth. Flush the connector in order to remove the water remaining in the tubes and bob. Block the lower inlet/outlet of the bob to get back pressure in the tubes, thereby removing any remaining water.</li>
	<li>Close the lid of the machine. Close the nitrogen cylinder. Turn off the computer and the machine.</li>
</ol>

<ol><li>Clark, M. R., Mohandas, N., Shohet, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deformability+of+oxygenated+irreversibly+sickled+cells%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Deformability of oxygenated irreversibly sickled cells.</a> Journal of Clinical Investigation. 65 (1), 189-196 (1980).</li>
<li>Smith, C., Kuettner, J., Tukey, D., White, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variable+Deformability+of+Irreversibly+Sickled+Erythrocytes%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Variable Deformability of Irreversibly Sickled Erythrocytes.</a> Blood. 58 (1), 71-78 (1981).</li>
<li>Clark, M., Mohandas, N., Embury, S., Lubin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+laboratory+alternative+to+irreversibly+sickled+%28ISC%29+counts%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">A simple laboratory alternative to irreversibly sickled (ISC) counts.</a> Blood. 60 (3), 659-663 (1982).</li>
<li>DaCosta, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnostic+tool+for+red+blood+cell+membrane+disorders+Assessment+of+a+new+generation+ektacytometer%5BTitle%5D)+AND+%22Blood+Cells%2C+Molecules%2C+and+Diseases%22%5BJournal%5D)">Diagnostic tool for red blood cell membrane disorders Assessment of a new generation ektacytometer.</a> Blood Cells, Molecules, and Diseases. 56 (1), 9-22 (2016).</li>
<li>Rabai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deformability+analysis+of+sickle+blood+using+ektacytometry%5BTitle%5D)+AND+%22Biorheology%22%5BJournal%5D)">Deformability analysis of sickle blood using ektacytometry.</a> Biorheology. 51 (2-3), 159-170 (2014).</li>
<li>Ballas, S. K., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sickle+red+cell+microrheology+and+sickle+blood+rheology%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">Sickle red cell microrheology and sickle blood rheology.</a> Microcirculation. 11 (2), 209-225 (2004).</li>
<li>Connes, P., Alexy, T., Detterich, J., Romana, M., Hardy-Dessources, M. D., Ballas, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+blood+rheology+in+sickle+cell+disease%5BTitle%5D)+AND+%22Blood+Reviews%22%5BJournal%5D)">The role of blood rheology in sickle cell disease.</a> Blood Reviews. 30 (2), 111-118 (2015).</li>
<li>Hierso, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+oxidative+stress+on+red+blood+cell+rheology+in+sickle+cell+patients%5BTitle%5D)+AND+%22British+Journal+of+Haematology%22%5BJournal%5D)">Effects of oxidative stress on red blood cell rheology in sickle cell patients.</a> British Journal of Haematology. 166 (4), 601-606 (2014).</li>
<li>Mozar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Red+blood+cell+nitric+oxide+synthase+modulates+red+blood+cell+deformabilityin+sickle+cell+anemia%5BTitle%5D)+AND+%22Clinical+Hemorheology+and+Microcirculation%22%5BJournal%5D)">Red blood cell nitric oxide synthase modulates red blood cell deformabilityin sickle cell anemia.</a> Clinical Hemorheology and Microcirculation. 64 (1), 47-53 (2016).</li>
<li>Clark, M. R., Mohandas, N., Shohet, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osmotic+Gradient+Ektacytometry%3A+Comprehensive+Characterization+of+Red+Cell+Volume+and+Surface+Maintenance%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Osmotic Gradient Ektacytometry: Comprehensive Characterization of Red Cell Volume and Surface Maintenance.</a> Blood. 61 (5), 899-911 (1983).</li>
<li>Parrow, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurements+of+red+cell+deformability+and+hydration+reflect+HbF+and+HbA2in+blood+from+patients+with+sickle+cell+anemia%5BTitle%5D)+AND+%22Blood+Cells%2C+Molecules%2C+and+Diseases%22%5BJournal%5D)">Measurements of red cell deformability and hydration reflect HbF and HbA2in blood from patients with sickle cell anemia.</a> Blood Cells, Molecules, and Diseases. 65, 41-50 (2017).</li>
<li>Steinberg, M. H., Chui, D. H. K., Dover, G. J., Sebastiani, P., Alsultan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fetal+hemoglobin+in+sickle+cell+anemia%3A+A+glass+half+full%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Fetal hemoglobin in sickle cell anemia: A glass half full.</a> Blood. 123 (4), 481-485 (2014).</li>
<li>Ballas, S. K., Larner, J., Smith, E. D., Surrey, S., Schwartz, E., Rappaport, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheologic+predictors+of+the+severity+of+the+painful+sickle+cell+crisis%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Rheologic predictors of the severity of the painful sickle cell crisis.</a> Blood. 72 (4), 1216-1223 (1988).</li>
<li>Lande, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Incidence+of+Painful+Crisis+in+Homozygous+Sickle+Cell+Disease%3A+Correlation+with+Red+Cell+Deformability%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">The Incidence of Painful Crisis in Homozygous Sickle Cell Disease: Correlation with Red Cell Deformability.</a> Blood. 72 (6), 2056-2059 (1988).</li>
<li>Lemonne, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Does+increased+red+blood+cell+deformability+raise+the+risk+for+osteonecrosis+in+sickle+cell+anemia%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Does increased red blood cell deformability raise the risk for osteonecrosis in sickle cell anemia.</a> Blood. 121 (15), 3054-3057 (2013).</li>
<li>Ballas, S. K., Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Red+blood+cell+changes+during+the+evolution+of+the+sickle+cell+painful+crisis%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Red blood cell changes during the evolution of the sickle cell painful crisis.</a> Blood. 79 (8), 2154-2163 (1992).</li>
<li>Telen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+adhesion+and+the+endothelium%3A+E-selectin%2C+L-selectin%2C+and+pan-selectin+inhibitors%5BTitle%5D)+AND+%22Hematology%2FOncology+Clinics+of+North+America%22%5BJournal%5D)">Cellular adhesion and the endothelium: E-selectin, L-selectin, and pan-selectin inhibitors.</a> Hematology/Oncology Clinics of North America. 28 (2), 341-354 (2014).</li>
<li>Papageorgiou, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+polymerization+and+adhesion+under+hypoxia+in+sickle+cell+disease%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Simultaneous polymerization and adhesion under hypoxia in sickle cell disease.</a> Proceedings of the National Academy of Sciences. 115 (38), 201807405(2018).</li>
<li>Liu, J., Qiang, Y., Alvarez, O., Du, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+impedance+microflow+cytometry+with+oxygen+control+for+detection+of+sickle+cells%5BTitle%5D)+AND+%22Sensors+and+Actuators%2C+B%3A+Chemical%22%5BJournal%5D)">Electrical impedance microflow cytometry with oxygen control for detection of sickle cells.</a> Sensors and Actuators, B: Chemical. 255, 2392-2398 (2018).</li>
<li>Du, E., Diez-Silva, M., Kato, G. J., Dao, M., Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+sickle+cell+biorheology+and+implications+for+painful+vasoocclusive+crisis%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Kinetics of sickle cell biorheology and implications for painful vasoocclusive crisis.</a> Proceedings of the National Academy of Sciences. 112 (5), 1422-1427 (2015).</li>
<li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+assay+shows+that+increasing+red+cell+volume+could+be+a+treatment+for+sickle+cell+disease%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease.</a> Proceedings of the National Academy of Sciences. 114 (5), 689-696 (2017).</li>
<li>Rab, M. A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+reproducible+characterization+of+sickling+during+automated+deoxygenation+in+sickle+cell+disease+patients%5BTitle%5D)+AND+%22American+Journal+of+Hematology%22%5BJournal%5D)">Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients.</a> American Journal of Hematology. 94, February 575-584 (2019).</li>
<li>Renoux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Importance+of+methodological+standardization+of+ektacytometric+measures+of+red+blood+cell+deformability+in+sickle+cell+anemia%5BTitle%5D)+AND+%22Clinical+Hemorheology+and+Microcirculation%22%5BJournal%5D)">Importance of methodological standardization of ektacytometric measures of red blood cell deformability in sickle cell anemia.</a> Clinical Hemorheology and Microcirculation. 62 (2), 173-179 (2016).</li>
<li>Parrow, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+Deformability+and+Red+Cell+Heterogeneity+in+Blood+by+Ektacytometry%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry.</a> Journal of Visualized Experiments. (131), (2018).</li>
<li>Bessis, M., Feo, C., Jones, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitation+of+red+cell+deformability+during+progressive+deoxygenation+and+oxygenation+in+sickling+disorders+%28the+use+of+an+automated+Ektacytometer%29%5BTitle%5D)+AND+%22Blood+Cells%22%5BJournal%5D)">Quantitation of red cell deformability during progressive deoxygenation and oxygenation in sickling disorders (the use of an automated Ektacytometer).</a> Blood Cells. 8 (1), 17-28 (1982).</li>
<li>Sorette, M. P., Lavenant, M. G., Clark, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ektacytometric+measurement+of+sickle+cell+deformability+as+a+continuous+function+of+oxygen+tension%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Ektacytometric measurement of sickle cell deformability as a continuous function of oxygen tension.</a> Blood. 67 (6), 1600-1606 (1987).</li>
<li>Huang, Z., Hearne, L., Irby, C. E., King, S. B., Ballas, S. K., Kim-Shapiro, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+increased+deformability+of+deoxygenated+sickle+cells+upon+oxygenation%5BTitle%5D)+AND+%22Biophysical+journal%22%5BJournal%5D)">Kinetics of increased deformability of deoxygenated sickle cells upon oxygenation.</a> Biophysical journal. 85 (4), 2374-2383 (2003).</li>
<li>Antoniani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+fetal+hemoglobin+synthesis+by+CRISPR%2FCas9-mediated+editing+of+the+human+%CE%B2-globin+locus%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus.</a> Blood. 131 (17), 1960-1973 (2018).</li>
<li>Abdulmalik, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystallographic+analysis+of+human+hemoglobin+elucidates+the+structural+basis+of+the+potent+and+dual+antisickling+activity+of+pyridyl+derivatives+of+vanillin%5BTitle%5D)+AND+%22Acta+Crystallographica+Section+D%3A+Biological+Crystallography%22%5BJournal%5D)">Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin.</a> Acta Crystallographica Section D: Biological Crystallography. 67 (12), 1076(2011).</li>
<li>Oder, E., Safo, M. K., Abdulmalik, O., Kato, G. J., Discovery, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+Developments+in+Anti-Sickling+Agents%3A+Can+Drugs+Directly+Prevent+the+Polymerization+of+Sickle+Haemoglobin+In+Vivo%5BTitle%5D)+AND+%22British+Journal+of+Haematology%22%5BJournal%5D)">New Developments in Anti-Sickling Agents: Can Drugs Directly Prevent the Polymerization of Sickle Haemoglobin In Vivo.</a> British Journal of Haematology. 175 (1), 24-30 (2016).</li>
<li>Oksenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GBT440+increases+haemoglobin+oxygen+affinity%2C+reduces+sickling+and+prolongs+RBC+half-life+in+a+murine+model+of+sickle+cell+disease%5BTitle%5D)+AND+%22British+Journal+of+Haematology%22%5BJournal%5D)">GBT440 increases haemoglobin oxygen affinity, reduces sickling and prolongs RBC half-life in a murine model of sickle cell disease.</a> British Journal of Haematology. 175 (1), 141-153 (2016).</li>
<li>Xu, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis%2C+and+Biological+Evaluation+of+Ester+and+Ether+Derivatives+of+Antisickling+Agent+5-HMF+for+the+Treatment+of+Sickle+Cell+Disease%5BTitle%5D)+AND+%22Molecular+Pharmaceutics%22%5BJournal%5D)">Synthesis, and Biological Evaluation of Ester and Ether Derivatives of Antisickling Agent 5-HMF for the Treatment of Sickle Cell Disease.</a> Molecular Pharmaceutics. 14 (10), 3499-3511 (2017).</li>
</ol>

The authors declare no competing financial interests.

Here we describe oxygen gradient ektacytometry, a method that can be used to study the sickling behavior of red blood cells from SCD patients under a range of oxygen concentrations (Figure 4 and Figure 5). In order to obtain reproducible results, it is important to identify the factors that influence the results. For instance, temperature has a large impact on RBC deformability, mostly due to its effects on the thickness of the viscous solution (PVP). We recommend performing a test measurement at the start of the day to thoroughly heat the machine to 37 &#730;C. This will improve the reproducibility of the results. The osmolarity of the viscous solution should be within a narrow range (282&#8211;286 mOsm/kg for PVP), because osmolarity influences hydration status, which in turn affects RBC deformability. The pH and viscosity of PVP should also be tightly regulated. Differences in pH and temperature can influence curves dramatically22. Additionally, remaining water in the cup, bob, and tubes, may cause the lysis of RBCs, thereby resulting in incorrect data, because fewer intact RBCs present in the cup will be measured.
Settings to perform oxygen gradient ektacytometry can be adjusted to address specific investigational questions. Preferred settings are listed in Table 1. A deoxygenation time of 1,300 s was chosen based on observations showing that the extension of deoxygenation did not result in a lower EImin for most patients. In contrast, shortening of the deoxygenation time would hamper the discriminative power of the oxygen gradient ektacytometry. The reoxygenation time was set to 280 s due to the rapidly resolving HbS polymers during reoxygenation, and concomitant restoration of EI towards values measured prior to the deoxygenation. Shear stress was set to 30 Pa, which is analogous to the osmotic gradient ektacytometry. Lowering this parameter could hamper the discriminative power. Deoxygenation control can be used if a set of deoxygenation speed is applied to every patient sample. In our preferred settings, this option was switched off because the rate of deoxygenation is patient-specific due to the unique hemoglobin dissociation curve. Hence, switching on the deoxygenation control would eliminate this characteristic from the assay. However, this feature of oxygen gradient ektacytometry is still under investigation.
Several well-known factors influence oxygen gradient ektacytometry parameters, namely pH, temperature, and osmolarity. Ektacytometry, especially PoS, is influenced by 2,3-diphosphoglycerate (2,3-DPG)22. Also, there is a clear correlation between %HbF and the EImin, and to a lesser extent PoS (Figure 5A&#8211;D). EImax is associated with sickle cells at normoxia, which can explain the observation that shortly after a VOC, RBC deformability at normoxia (EImax), is higher. The latter is caused by the destruction of the most sickled cells, and hence less deformable RBCs during VOC16. As shown in Figure 5F, higher %dense RBCs (defined as RBCs with a hemoglobin concentration &#62;1.11 mg/mL) correlate strongly with a lower EImax. This indicates that dense cells are an important factor in RBC deformability at normoxia, similar to previously reported results1.
Standardization of samples is very important for obtaining reproducible results and for distinguishing between different genotypes and treatments. Correcting for RBC count is important, as the number of RBCs influence the intensity of the diffraction pattern. If lower RBC numbers are present in the gap between the bob and cup, the curve will shift upward and to the left. Additionally, the curve will fluctuate, hampering accurate calculation of the parameters, especially the PoS.
A limitation of this technique is that the EI value represents an average of all cells, including different subpopulations. Heterogeneity of RBC populations in SCD patients and its influence on ektacytometry measurement has been intensively studied. This resulted in standardization wherein the size of the diffraction pattern is adjusted to a fixed value instead of corrected for the RBC count23,24. Whether or not this way of standardization should also be applied to oxygen gradient ektacytometry measurements is currently under study.
Several techniques to measure RBC deformability under hypoxic conditions were developed based on a deoxygenation step that took place outside the ektacytometer25,26,27. Under these conditions, differences in cellular behavior were not observed between patients with HbS traits and healthy controls under physiological pH25. Oxygen gradient ektacytometry, however, clearly shows a low but evident PoS in individuals with HbS traits (Figure 4A). To date, in routine clinical practice, the only alternative methods to measure the tendency of an individual patient&#8217;s RBCs to sickle in vitro include a morphology-based sickling assay: RBCs are incubated under conditions that promote HbS polymerization, such as low oxygen tension or low pH. A fixative is added after incubation and the percentage of sickled cells is manually or digitally counted using light microscopy. Many preclinical and early phase pharmacologic trials use the sickling assay to generate a secondary outcome variable to be able to predict clinical efficacy in SCD28,29,30,31,32. However, it is time consuming, variability is high and sensitivity is low, the technique is not automated and, therefore, labor intensive. Moreover, morphological changes due to sickling might not correlate well with physiological parameters, such as RBC deformability, because it is a 2-dimensional static assay2.
Oxygen gradient ektacytometry provides a functional assay of sickling that is rapid and reproducible. This is an in vitro test that does not consider the endothelial surface. However, it does provide functional aspects of sickling behavior and RBC characteristics, making it a promising technique for sickle cell studies. Future applications of the technique include monitoring treatment efficacy in SCD patients, serving as a biomarker for new treatment strategies, studying sickling behavior, and monitoring chimerism after the stem cell transplantation in SCD.

In SCD, a single point mutation results in the production of HbS, which can polymerize upon deoxygenation. HbS polymerization causes sickling of RBCs and reduces RBC deformability. The combination of RBC sickling and RBC adherence to the endothelium leads to various SCD complications, including vaso-occlusive crises (VOC), stroke, organ damage, and chronic hemolytic anemia. Even at normoxic conditions, RBC deformability is compromised in patients with SCD. Deformability is further decreased at low oxygen concentrations. Key players that determine deformability at normoxia are dense cells, irreversibly sickled cells (ISC), and dehydrated cells, all of which have a decreased surface-to-volume ratio1,2,3.
Ektacytometry is an established method to measure RBC deformability, widely used for the diagnosis of hereditary hemolytic anemias, particularly membranopathies4. It can also be used to study hemorheology5,6,7,8,9. Osmotic gradient ektacytometry, in which RBC deformability is measured during a continuous change in osmolality, has been used to study SCD for over a decade10,11. The percentage of fetal hemoglobin (HbF) is one of the strongest inhibitors of HbS polymerization because neither HbF nor its mixed hybrid tetramer (&#8733;2&#946;S&#947;) can enter the deoxyHbS polymer phase12. Recent studies suggest that increasing HbF levels in SCD patients leads to a better surface-to-volume ratio, thereby improving the hydration state and thus the deformability in nontransfused patients11.
RBC deformability has been studied in the past as a biomarker for SCD complications, but with conflicting results. In studies performed cross-sectionally and at a steady state, individuals with higher levels of RBC deformability were found to have a higher incidence of osteonecrosis and more pain crises13,14,15. In contrast to these findings, when compared to the steady state values during an acute VOC, RBC deformability was decreased in longitudinal studies within the same individuals16. This discrepancy may be the result of studying RBC deformability under different conditions (i.e., during the steady state versus VOC). The percentage of sickled cells is high at the start of a VOC and the cells are rapidly destroyed as the crisis progresses, which may explain the difference between the steady state cross-sectional incidence data and longitudinal data obtained during the VOC. However, other factors, such as adherence of RBC subpopulations to the endothelial surface, may also be important in the occurrence of VOC. In SCD, it is more clinically relevant to measure the deformability during the deoxygenation, because vaso-occlusion typically occurs in the hypoxic postcapillary venules and not in the less hypoxic microcapillary network17. Additionally, the presence of ISCs may alter the ability of an ektacytometer to measure the deformability at normoxia. Distortion of the diffraction pattern is caused by ISCs, which results from the non-alignment during the flow1,2,3.
Alternative approaches to study the pathophysiology of VOC include measurements of RBC adherence to an artificial surface18, single cell electrical impedance microflow cytometry19, microfluidic-based models combining quantitative measurements of the cell sickling and unsickling with single cell rheology20, and laser-induced polymerization21. Although promising, these techniques are costly, labor intensive, and require extensive operator training. In addition, the assays that are morphology-based lack the ability to study cellular behavior, such as deformability, as a function of an oxygen gradient.
In this study, we describe a rapid and reproducible functional assay performed with an ektacytometer. This is a next generation ektacytometry measurement that measures the different qualitative aspects of RBC deformability expressed as the EI during deoxygenation (1,300 s) and swift reoxygenation (280 s). These time intervals allow for HbS polymer formation, and thereby the occurrence of morphological changes and then recovery. Deoxygenation occurs by introducing nitrogen gas, which slowly decreases the oxygen tension in the blood sample in the gap between the bob and cup of the ektacytometer. RBC deformability is continuously measured while oxygen tension is measured every 20 s by means of a small O2-spot present in the wall of the cup. During the test, approximately 80 pO2 measurements are coupled to the EI measured at that moment. The oxygen pressure drops below 20 mmHg during the deoxygenation, and reoxygenation is facilitated by the passive diffusion of ambient air. The experimental setup of the ektacytometer and oxygen gradient ektacytometry module is described in Figure 1 and Figure 2. The principle of ektacytometry is based on RBC-induced scattering of light from a laser beam. This results in an elliptical diffraction pattern when shear stress is applied at the same time (Figure 1).

<table><tbody><tr><td>ADVIA 120 Hematology Analyzer</td><td>Siemens</td><td>067-A004-14</td><td>Instrument</td></tr><tr><td>Cell-Dyn Sapphire Hematology Analyzer</td><td>Abbott</td><td>8H00-01</td><td>Instrument</td></tr><tr><td>Lorrca</td><td>RR Mechatronics</td><td>LORC109230 or LORC109110</td><td>Instrument</td></tr><tr><td>Lorrca Software version V5.08</td><td>RR Mechatronics</td><td>-</td><td>Software</td></tr><tr><td>Nitrogen gas 4.8 or 5.0</td><td>Local</td><td>-</td><td></td></tr><tr><td>O2-spot</td><td>RR Mechatronics</td><td>PO2S020153</td><td>O2 measurement</td></tr><tr><td>Oxygenscan module (pO2scan)</td><td>RR Mechatronics</td><td>PO2S109000</td><td>Add-on</td></tr><tr><td>Oxy-ISO</td><td>RR Mechatronics</td><td>QRR 030905</td><td>Viscous solution</td></tr><tr><td>X-Clean</td><td>RR Mechatronics</td><td>QRR 010946</td><td>Cleaning solution Lorrca</td></tr></tbody></table>

characterization of sickling during controlled automated deoxygenation with oxygen gradient ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Oxygen gradient ektacytometry can be used to characterize sickling behavior in patients with SCD. In this study, blood samples from a total of 38 SCD patients and five healthy controls were included. In healthy controls, the diffraction pattern is circular at rest and elliptical at higher shear stress4. From the elliptical diffraction pattern, the elongation index (EI) is calculated based on the height and width of the diffraction pattern. In oxygen gradient ektacytometry, slow and continuous deoxygenation of the sample by nitrogen gas is followed by swift reoxygenation by ambient air. Under these conditions, RBC sickling can be observed under deoxygenation. This will cause a distortion of the diffraction pattern because sickled red cells will not align properly under the applied shear stress. Hence, they appear to be less deformable as opposed to healthy RBCs (Figure 2).
Figure 3A shows how sickle RBCs change in shape upon deoxygenation, which mimicked conditions during oxygen gradient ektacytometry, whereas control sickle RBCs without deoxygenation show no change in shape. This process results in distortion of the diffraction pattern during oxygen gradient ektacytometry, and thus in a decrease in EI. Figure 3B shows the different diffraction patterns from which different parameters are generated.
A representative curve obtained by the ektacytometer is shown in Figure 3C. Six parameters reflect different characteristics of sickling behavior of RBCs: EImax is the maximum EI at the start of the measurement before deoxygenation. This parameter represents the baseline position and reflects the overall deformability of the total RBC population at ambient air. EImin is the minimum EI, which represents minimal deformability after deoxygenation. This parameter reflects changes in the shape and orientation of (sickle) RBCs upon deoxygenation. ΔEI is the difference between EImax and EImin, which indicates how many cells can sickle during one round of deoxygenation. 5% Point of Sickling (PoS5%) is the pO2 (mmHg) at which a 5% decrease of EImax during deoxygenation is measured. This represents the oxygen tension where the sickling process starts. Area reflects the area under the curve, which is determined by an integral calculation of EI and pO2 measurements between 100 mmHg and pO2min (mmHg). This is the result of previously described parameters EImax, EImin, and PoS. Recovery represents the difference of EI during the final part of reoxygenation compared to EI at baseline. Both EI values are measured at a pO2 of 100–120 mmHg. This parameter reflects the capacity of RBCs that sickle during deoxygenation to reverse sickling during reoxygenation22. Parameters from duplicate measurements generally had a coefficient of variation (CV) &lt;5% (median 1.83%). In case a CV &gt; 5% was obtained, a third measurement was performed. The parameters EImax and Recovery are the most reproducible with median CVs &lt;1%.
Representative curves of RBCs of healthy controls, patients with HbS traits (heterozygous HbS), and a homozygous SCD patient are shown in Figure 4A. The representative curve of the HbSC patient shows a lower recovery, which might indicate a different sickling process (Figure 4B). The representative curves of HbSS patients treated with hydroxyurea (HU) and transfusion are shown in Figure 4C and Figure 4D. Clearly, there is a big difference between the representative curves of HS traits (HbAS cells) and RBCs of HbSS patients treated with transfusion (consisting of a mixture of homozygous sickle (HbSS) and homozygous normal (HbAA) cells, Figure 4A,D). The clear differences in the curves of the untreated SCD patient and the HU and transfusion-treated patients highlights the usefulness of this assay (Figure 4C,D). Levels of HbF and HbS correlated significantly with EImin and, to a lesser extent, with PoS (Figure 5A–D). This indicates that those laboratory parameters that are important in the evaluation of the patient are also reflected in the oxygen gradient ektacytometry. The number of sickled cells at normoxia and percentage of dense RBCs (DRBCs) both influence EImax values, as they are significantly correlated (Figure 5E–F), which indicates that EImax reflects another important factor in the sickling process. These results show how different characteristics such has %HbS, %HbF, sickled cells at normoxia, and %DRBCs influence different parameters.
<img alt="Static equilibrium diagram; laser beam setup with camera, projection for material deformation analysis." class="xfigimg" src="/files/ftp_upload/60213/60213fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Schematic setup of the ektacytometer. The ektacytometer uses a Couette system to apply shear stress on the cells. A rotation outer cylinder (cup) and a static inner cylinder (bob) are used to induce shear stress by the creation of laminar flow at 37 ˚C. Between the bob and cup there is a small gap in which the blood suspension is injected. A laser beam shines from the bob through the blood suspension and is scattered by the presence of RBCs. The diffraction pattern is projected and analyzed by a camera. The elongation index (EI) is calculated with the height (a) and the width (b) of the diffraction pattern4. <a href="https://www.jove.com/files/ftp_upload/60213/60213fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Blood suspension rotation diffraction; experiment setup with diffraction pattern; optical analysis." class="xfigimg" src="/files/ftp_upload/60213/60213fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Schematic setup of the ektacytometer with oxygen gradient ektacytometry module. Schematic diagram of the module that shows deoxygenation of the blood suspension slowly with the infusion of nitrogen gas (N2). Oxygen tension is measured by the amount of quenching of the luminophore signal sent from the LED-fiber to the O2-spot. Upon deoxygenation, sickle RBCs will start to sickle, their deformability will decrease, and they will no longer align with elliptical RBCs. The sickled RBCs will distort the diffraction pattern, changing its shape from an ellipse to a rhomboid or diamond-like shape. This change in the shape of the diffraction pattern results in a decrease of EI. Measurements of pO2 and EI are not performed at the same height in the cup. This ensures better discrimination between the deoxygenation and reoxygenation curves and, hence, a better interpretation of the curve. This figure has been modified  from Rab et al.22 <a href="https://www.jove.com/files/ftp_upload/60213/60213fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Oxygenation-deoxygenation process shown through microscopy images, diagrams, and graph analysis." class="xfigimg" src="/files/ftp_upload/60213/60213fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Representative oxygen gradient ektacytometry curve and diffraction patterns. (A) Upon deoxygenation under conditions similar to the oxygen gradient ektacytometry, sickle RBCs were fixed. In control sickle RBCs, the same conditions were used, but without nitrogen gas. Deoxygenated sickle RBCs show a change in shape in contrast to control RBCs. (B) Upon deoxygenation and shear stress (30 Pa), the diffraction pattern changes from an ellipse to a rhomboid. (C) Representative curve of oxygen gradient ektacytometry. The maximum elongation index (EImax) represents the baseline position and shows an overall deformability of the total RBC population. Minimum EI (EImin) represents minimal deformability, which is caused by the change in shape and orientation of RBCs upon deoxygenation. ΔEI (dEI, the difference in EI between EImax and EImin) shows how many cells can sickle during one round of deoxygenation. Point of sickling (PoS, pO2 at 5% EI decrease) shows the oxygen tension when the first RBCs start to sickle. The area under the curve (from pO2min = 100 mmHg) is calculated in the parameter area. This summarizes EImax, EImin, and PoS. The capacity of sickled cells to unsickle during reoxygenation is represented in the parameter Recovery (percentage of EImax reached during reoxygenation). To aid in the interpretation, all data points were connected in every individual experiment by a line to graphically present the results. This figure has been modified  from Rab et al.22 <a href="https://www.jove.com/files/ftp_upload/60213/60213fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Blood oxygen dissociation curves for Hb variants; graphs show pO₂ vs EI, different Hb treatments." class="xfigimg" src="/files/ftp_upload/60213/60213fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Oxygen gradient ektacytometry parameters correlate with genotype and treatment regimens of SCD patients with SCD. (A) Representative graph of RBCs of HbS carriers (HbS trait) and healthy controls in relation to untreated HbSS patients. (B) Representative graph of RBCs of patients with Hemoglobin SC Disease (HbSC) in relation to untreated HbSS patients. (C) Representative graph of RBCs of hydroxyurea treated homozygous SCD patients (HbSS HU) in relation to untreated HbSS patients. (D) Representative graph of RBCs of HbSS patients treated with blood transfusion (HbSS transfusion) in relation to untreated HbSS patients. This figure has been modified  from Rab et al.22 <a href="https://www.jove.com/files/ftp_upload/60213/60213fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Correlation analysis graphs of hemoglobin saturation variables; scatter plots A-F with regression lines." class="xfigimg" src="/files/ftp_upload/60213/60213fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Oxygen gradient ektacytometry parameters are associated with %HbF, %HbS, %sickled cells at normoxia and %dense RBCs. (A) Linear correlation of minimum elongation index (EImin) and %HbF of 15 HbSS or HbS/β-thalassemia patients without transfusion. (B) Linear correlation of EImin and %HbS. (C) Linear correlation of PoS and %HbF. (D) Linear correlation of PoS and %HbS. (E) Linear correlation of maximum EI (EImax) and percent of sickled cells at normoxia measured with digital microscopy. (F) Linear correlation of EImax and percentage dense RBCs (%DRBCs) of 21 patients with HbSS. This figure has been modified  from Rab et al.22 <a href="https://www.jove.com/files/ftp_upload/60213/60213fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Settings</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Files</td>
			<td>Storage directory</td>
			<td></td>
		</tr>
		<tr>
			<td>General options</td>
			<td>Default medium viscosity</td>
			<td>Viscosity of PVP</td>
		</tr>
		<tr>
			<td>pO2 scan</td>
			<td>Minimum aspiration time (s)</td>
			<td>60</td>
		</tr>
		<tr>
			<td></td>
			<td>pO2 scan shear stress (Pa)</td>
			<td>30</td>
		</tr>
		<tr>
			<td></td>
			<td>Determine pO2 every (S)</td>
			<td>20</td>
		</tr>
		<tr>
			<td></td>
			<td>Moving average size</td>
			<td>2</td>
		</tr>
		<tr>
			<td></td>
			<td>pO2 scan step; Edit</td>
			<td>0 -OFF; 60 -ON; 1360 –OFF; 1640 -OFF</td>
		</tr>
		<tr>
			<td></td>
			<td>Cal. Area between (mmHg)</td>
			<td>10 and 100</td>
		</tr>
		<tr>
			<td></td>
			<td>pO2 control</td>
			<td>Off (unchecked)</td>
		</tr>
	</tbody>
</table>
Table 1. The preferred setting of the ektacytometer.

Watch this Scientific Journal Video about Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry at JoVE.com

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...

characterization-sickling-during-controlled-automated-deoxygenation

Research

JoVE Journal

Medicine

9.7K Views.  Utrecht University. Zuurstofgradiënt ektacytometrie is een functionele test die individuele sikkelneigingen beoordelen. Tot op heden zijn er geen andere functionele tests beschikbaar of gebruikt. Zuurstofgradiënt ektacytometrie is in staat om de zuurstofspanning te bepalen waarbij bloedcellen van de patiënt met sikkelcelziekte beginnen te sikkelen.Deze zuurstofspanning is patiëntspecifiek en kan dienen als biomarker voor de ernst van de ziekte. Bij sikkelcelziekte kan deze techniek worden gebruikt om ...

Video: Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Bioengineering

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Biochemistry

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca2+, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca2+ concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.

A protocol for the induction of eryptosis, programmed cell death in erythrocytes, using the calcium ionophore, ionomycin, is provided. Successful eryptosis is evaluated by monitoring the localization phosphatidylserine in the membrane outer leaflet. Factors affecting the success of the protocol have been examined and optimal conditions provided.

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Immunology and Infection

A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the most commonly used therapy to treat or prevent these devastating complications. Other therapies include hydroxyurea therapy as well as bone marrow transplantation. Chronic intermittent transfusion is especially indicated to prevent recurrent strokes. However, transfusion therapy is associated with significant adverse effects (e.g., alloimmunization and iron overload). The point-of-care (POC) lateral flow immunoassay used here quantifies the %HbS in 15 min using a small patient blood sample. Utilizing this information, the healthcare provider is able to adjust blood transfusion volume for SCD patients to achieve the desired target HbS (most often &#60;30%), while reducing the risk of transfusion-related complications. When compared to laboratory hemoglobin electrophoresis data for 38 whole blood samples, the POC test performed with very high correlation and linear fit (slope = 0.9949, R2 = 0.9751). The strong agreement between the two hemoglobin S percentage (%HbS) quantifying methods shows that 89.5% of samples fall within &#177;5.2% HbS bias. The calibration for quantifying %HbS is built into the device to allow for an automated quantification of %HbS. This time- and cost-effective POC test thus allows the healthcare provider to make timely informed decisions when treating SCD patients, using accurate and updated data.

We have developed a point-of-care immunoassay to rapidly quantify hemoglobin S (HbS) levels during transfusion therapy for sickle cell disease (SCD) patients. By applying a small amount of treated blood to the device, the healthcare provider can determine the %HbS in a SCD patient to immediately inform clinical decisions.

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Erythrocyte Sedimentation Rate: A Physics-Driven Characterization in a Medical Context

Erythrocyte (or red blood cell) sedimentation rate (ESR) is a physical derived parameter of blood which is often used in routine health checks and medical diagnosis. For instance, in the case of inflammation, a higher ESR is observed due to the associated increase in fibrinogen and other plasma proteins. It was believed that this increase was due to the formation of larger aggregates of red blood cells (RBCs) caused by the increase in fibrinogen. Indeed, fibrinogen is an agent-fostering aggregation of RBCs and in the Stokes regime-assumed to be observed in blood-larger aggregates sediment faster. However, all models of ESR measurements based on this hypothesis require further specific physical assumptions, not required in any other system. Besides, modern studies in the field of colloidal suspensions have established that attractive particles form percolating aggregates (i.e. aggregates as wide as the container). The sedimentation of these colloids then follows a so-called &#34;colloidal gel collapse&#34;. Recently, it has been shown that RBCs actually follow the same behavior. This hypothesis also allows to efficiently and analytically model the sedimentation curve of RBCs, from which robust and physically-meaningful descriptors can be extracted. This manuscript describes how to perform such an analysis, and discusses the benefits of this approach.

Erythrocyte sedimentation rate (ESR) is a physical parameter, often used in routine health checks and medical diagnosis. A theoretical model that allows to extract physically-meaningful parameters from the whole sedimentation curve, based on modern colloidal knowledge, has recently been developed. Here, we present a protocol to automatically collect the ESR over time, and extract the parameters of this recent model from this automated data collection. These refined parameters are also likely to improve the medical testimony.

Erythrocyte (or red blood cell) sedimentation rate (ESR) is a physical derived parameter of blood which is often used in routine health checks and medical ...

Non-Invasive Monitoring of Microvascular Oxygenation and Reactive Hyperemia using Hybrid, Near-Infrared Diffuse Optical Spectroscopy for Critical Care

The detection of levels of impairment in microvascular oxygen consumption and reactive hyperemia is vital in critical care. However, there are no practical means for a robust and quantitative evaluation. This paper describes a protocol to evaluate these impairments using a hybrid near-infrared diffuse optical device. The device contains modules for near-infrared time-resolved and diffuse correlation spectroscopies and pulse-oximetry. These modules allow the non-invasive, continuous, and real-time measurement of the absolute, microvascular blood/tissue oxygen saturation (StO2) and the blood flow index (BFI) along with the peripheral arterial oxygen saturation (SpO2). This device uses an integrated, computer-controlled tourniquet system to execute a standardized protocol with optical data acquisition from the brachioradialis muscle. The standardized vascular occlusion test (VOT) takes care of the variations in the occlusion duration and pressure reported in the literature, while the automation minimizes inter-operator differences. The protocol we describe focuses on a 3-min occlusion period but the details described in this paper can readily be adapted to other durations and cuff pressures, as well as other muscles. The inclusion of an extended baseline and post-occlusion recovery period measurement allows the quantification of the baseline values for all the parameters and the blood/tissue deoxygenation rate that corresponds to the metabolic rate of oxygen consumption. Once the cuff is released, we characterize the tissue reoxygenation rate, magnitude, and duration of the hyperemic response in BFI and StO2. These latter parameters correspond to the quantification of the reactive hyperemia, which provides information about the endothelial function. Furthermore, the above-mentioned measurements of the absolute concentration of oxygenated and deoxygenated hemoglobin, BFI, the derived metabolic rate of oxygen consumption, StO2, and SpO2 provide a yet-to-be-explored rich data set that can exhibit disease severity, personalized therapeutics, and management interventions.

We describe a protocol to non-invasively and continuously measure absolute microvascular blood flow index and blood oxygen saturation using a multi-modal device based on near-infrared diffuse optics. We then evaluate the metabolic rate of oxygen consumption and reactive hyperemia utilizing a vascular occlusion test.

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

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