This work was supported by the Intramural Research Program of the National Institutes of Diabetes, Digestive and Kidney Diseases and the National Heart, Lung and Blood Institute of the National Institutes of Health. The opinions expressed herein are the sole responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

All subjects in this study gave written informed consent in accordance with the Declaration of Helsinki and the National Institutes of Health Institutional Review Board approved protocols.
1. Turning on the ektacytometer
<ol>
	<li>Connect the tubing from the cleaning solution to the low and high osmolar polyvinylpyrrolidone (PVP) solutions. Be careful to connect the 0 osmolar tube to the low osmolar solution and the 500 osmolar tube to the high osmolar solution. 
	Note: The low osmolar PVP solution should have an osmolality between 35 and 55 milliosmoles per kilogram (mOsm/kg), a pH of 7.25-7.45 at 25 &#176;C and a viscosity between 27.0 and 33.0 centipoise (cP) at 37.0 &#177; 0.5 &#176;C. The high osmolar PVP solution should have an osmolality between 764 and 804 mOsm/kg, a pH of 7.25-7.45 at 25 &#176;C and a viscosity measure of 27.0-33.0 cP at 37.0 &#176; 0.5 &#176;C.</li>
	<li>Ensure that the bob is lowered completely into the cup. Launch the software and prime the machine (Hardware Check | Instrument IO). Allow the instrument to complete the priming cycle. Once the cycle is complete, lift the bob out of the cup and completely dry the bob and the cup with a low lint cleaning tissue. 
	Note: Residual water will lyse red cells, producing interference.</li>
</ol>
2. Measuring deformability as a function of increasing shear stress
<ol>
	<li>Obtain blood (less than 1 mL is adequate to perform these techniques with replicates) in vial containing an appropriate anticoagulant. 
	Note: EDTA is preferred over heparin because it has less influence over hemorheological parameters.33 Keep blood at room temperature if measurements are performed within 6 hours of blood draw.</li>
	<li>Gently mix whole blood sample before testing by inverting the vial several times. Add 25 &#181;L of whole blood to 5 mL of iso-osmolar PVP solution by pipetting, cap the vial and mix gently by inverting several times. The iso-osmolar PVP solution should have an osmolality between 284 and 304 mOsm/kg, a pH of 7.3-7.4 at 25 &#176;C and a viscosity of 27.0-33.0 cP at 37.0 &#177; 0.5 &#176;C.</li>
	<li>On the software choose deformability from the main menu. Create a new analysis and add experimental details (Deformability | Add desired details | Okay).
	<ol>
		<li>Lift lid to ektacytometer, verify that the bob is fully lowered into cup and the cup is turning.</li>
		<li>Add 1 mL of PVP blood solution into the space between the cup and bob by pipetting.</li>
		<li>Lift the bob slightly to bring samples down. Wait until all bubbles have moved out of the solution, then close the lid to the ektacytometer. If needed, press the aspiration button to help remove bubbles.</li>
	</ol>
	</li>
	<li>Adjust the gain to 200 by moving the arrow along the scroll bar on the software (see note). When the temperature is stable at 37 &#176;C and the diffraction image is stable, press start (Start). 
	Note: For many studies, a good diffraction image can be obtained from healthy blood (hemoglobin concentration &#62; 12.0 g/dL, mean corpuscular volume of 80-96 fL and mean corpuscular hemoglobin concentrations of 33-36 g/dL) with the camera gain set to 200. For studies of blood from sickle cell anemia, adjusting the camera gain to generate a 4.5 cm diffraction image has been suggested as the default setting to allow comparison of results across studies and laboratories.9</li>
	<li>Observe diffraction patterns as data acquisition progresses to ensure that they remain circular, elliptical or diamond shaped. When data acquisition is complete, save or print the report (File | Save or File | Print). EI Max and SS &#189; values will be reported automatically, along with elongation indices corresponding to user-specified or default shear stresses. Data will also be saved automatically by the software.</li>
	<li>At the end, press the clean option on the dialog box on the computer monitor (Clean). After the sample is aspirated, rinse the space between the cup and bob by squirting deionized water into the space while the instrument remains in the clean cycle. Once the clean cycle is complete, lift the bob out of the cup and completely dry the bob and the cup with a low lint cleaning tissue (critical: Residual water will lyse red cells, producing interference).</li>
	<li>Click on the Main Menu button on the software to return to the main page (Main Menu).</li>
</ol>
3. Measuring cellular heterogeneity
<ol>
	<li>Gently mix whole blood sample before testing by inverting the vial several times. Pipet 25 &#181;L of whole blood to a new 5 mL vial of iso-osmolar PVP solution, cap the vial and mix gently by inverting until the mixture is homogenous.</li>
	<li>Choose deformability from the main menu. Create a new analysis and add experimental details (Deformability | Add desired details | Okay).
	<ol>
		<li>Lift lid to ektacytometer, verify that the bob is fully lowered into cup and the cup is turning.</li>
		<li>Pipet 1 mL of the PVP blood solution into the space between the cup and bob.</li>
		<li>Wait until all bubbles have moved out of the solution, then close the lid to the ektacytometer.</li>
		<li>Ensure that a stable diffraction image is present on the screen. Adjust the camera gain by moving the arrow along the scrollbar on the software until it produces a 3.8 cm diffraction height. Use a ruler to verify the height of the image on the computer screen.</li>
	</ol>
	</li>
	<li>When the temperature is stable at 37 &#176;C and the diffraction image is stable, press start (Start).</li>
	<li>Observe diffraction patterns as data acquisition progresses to ensure that they remain circular, elliptical or diamond-shaped. When data acquisition is complete, save or print the report (File | Save or File | Print).</li>
	<li>At the end, press the clean option on the dialog box on the computer monitor (Clean). After the sample is aspirated, rinse the space between the cup and bob by squirting deionized water from a squirt bottle into it while the instrument remains in the clean cycle. Once the clean cycle is complete, lift the bob out of the cup and completely dry the bob and the cup with a low lint cleaning tissue (critical: Residual water will lyse red cells, producing interference).</li>
	<li>Repeat steps 2.1-2.5 and adjust camera gain to obtain a 4.5 cm diffraction pattern height (step 2.2).</li>
	<li>Repeat steps 2.1-2.5 and adjust camera gain to obtain a 5.4 cm diffraction pattern height (step 2.2).</li>
	<li>At the end, click on the main menu button to return to the main page of the software (Main Menu).</li>
	<li>To determine the degree of diffraction pattern distortion based on EI Max as a percentage, use the following equation (the same equation can be performed with the data from the 4.5 cm diffraction pattern height if desired): 
	<img alt="Equation 1" src="/files/ftp_upload/56910/56910eq1v2.jpg" /></li>
	<li>Similarly, to determine the degree of diffraction pattern distortion based on SS1/2 use the same equation with the reported SS1/2 value: 
	<img alt="Equation 2" src="/files/ftp_upload/56910/56910eq2v2.jpg" /></li>
</ol>
4. Osmotic gradient ektacytometry
<ol>
	<li>Obtain sample as described in 1.1. Gently mix whole blood sample before testing by inverting several times. Add 250 &#181;L of whole blood to 5 mL iso-osmolar PVP vial by pipetting, cap the vial and mix gently by inverting until the mixture is homogenous.</li>
	<li>Choose osmoscan from the main menu (Osmoscan). Place vial containing the blood PVP solution underneath the needle on the left-hand side of the machine. Lower the needle until it touches the bottom of the vial. Make sure tubing is properly connected to the low and high osmolar solutions for gradient production. Close the lid to the ektacytometer and open the door on the lower half so that you can watch the blood enter the tubing.</li>
	<li>Press new analysis and type in experimental details (Osmoscan | New Analysis | Enter desired details | Okay). Adjust the camera gain to 200 by moving the arrow controlling it on the software and allow the machine run until blood is seen entering the cup from the tubing beneath the instrument.
	<ol>
		<li>Once the blood has entered the cup and a stable diffraction pattern image is on the computer screen, begin data acquisition by pressing the start now button on the dialog box (Start Now).</li>
	</ol>
	</li>
	<li>Allow the ektacytometer to acquire data up to approximately 500 mOsm/kg, then stop the instrument. Save or print report (File | Save or File | Print). Data will also save automatically.</li>
	<li>Remove the old PVP-blood vial. Replace it with a clean vial containing deionized water. Place it beneath the needle, bring the needle down so that it touches the bottom of the vial and press the rinse button in the dialog box to rinse the gradient system (Rinse).</li>
	<li>Once the rinse is complete, press the clean option on the dialog box on the computer monitor (Clean). Once the clean cycle is complete, lift the bob out of the cup and completely dry the bob and the cup with a low lint cleaning tissue (critical: Residual water will lyse red cells, producing interference). 
	Note: The osmoscan report provides elongation indices across the osmotic gradient. EI Min, O (EI Min), EI Max, O (EI Max), EI hyper and O hyper are generated automatically and included in the report. The range of osmotic gradient ektacytometry parameters obtained from blood from 9 healthy volunteers is: EI Min 0.12-0.196 arbitrary units (a.u.); O Min 117-144 mOsm/kg; EI Max 0.551-0.573 a.u.; O (EI Max) 272-312 mOsm/kg; EI hyper 0.278-0.286; O Hyper 454-505 mOsm/kg.</li>
</ol>
5. Turning off the ektacytometer
<ol>
	<li>Clean the instrument properly before it is shut down.
	<ol>
		<li>To do this, connect the tubing from the low and high osmolar solutions to the y-adapter leading to the cleaning solution. Place a vial containing cleaning solution below the needle on the left-hand side of the machine and lower the needle until it touches the bottom of the vial. Ensure that the bob is lowered completely into the cup.</li>
		<li>Close the software, and press start on the end of day clean dialog box on the computer monitor (Close | Start). Allow the instrument to cycle through cleaning completely.</li>
	</ol>
	</li>
	<li>Disconnect the tubing to the waste bottle and remove it from the instrument to discard waste. Dry the bob completely. Turn the machine off.</li>
</ol>

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<li>Uyuklu, M., Meiselman, H. J., Baskurt, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+hemoglobin+oxygenation+level+on+red+blood+cell+deformability+and+aggregation+parameters%5BTitle%5D)+AND+%22Clin+Hemorheol+Microcirc%22%5BJournal%5D)">Effect of hemoglobin oxygenation level on red blood cell deformability and aggregation parameters.</a> Clin Hemorheol Microcirc. 41 (3), 179-188 (2009).</li>
<li>Embury, S. H., Clark, M. R., Monroy, G., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concurrent+sickle+cell+anemia+and+alpha-thalassemia.+Effect+on+pathological+properties+of+sickle+erythrocytes%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Concurrent sickle cell anemia and alpha-thalassemia. Effect on pathological properties of sickle erythrocytes.</a> J Clin Invest. 73 (1), 116-123 (1984).</li>
<li>von Tempelhoff, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlation+between+blood+rheological+properties+and+red+blood+cell+indices%28MCH%2C+MCV%2C+MCHC%29+in+healthy+women%5BTitle%5D)+AND+%22Clin+Hemorheol+Microcirc%22%5BJournal%5D)">Correlation between blood rheological properties and red blood cell indices(MCH, MCV, MCHC) in healthy women.</a> Clin Hemorheol Microcirc. 62 (1), 45-54 (2016).</li>
<li>Da Costa, L., Galimand, J., Fenneteau, O., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hereditary+spherocytosis%2C+elliptocytosis%2C+and+other+red+cell+membrane+disorders%5BTitle%5D)+AND+%22Blood+Rev%22%5BJournal%5D)">Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders.</a> Blood Rev. 27 (4), 167-178 (2013).</li>
</ol>

The authors have nothing to disclose.

The ektacytometry techniques described are straightforward and well automated, ensuring valid and reproducible results. Nonetheless, some critical steps exist. Proper temperature control of the blood is important. Storage at room temperature for more than eight hours may affect SS &#189; values.34 Ensuring that the temperature of the machine is stable at 37 &#176;C is also important, as viscosity of the suspending medium is temperature dependent. Blood should be fully oxygenated to avoid decreased...

Ektacytometry provides a convenient measure of red cell deformability in response to alterations in shear stress (measured in pascals (Pa)) or suspending medium osmolality. Pertinent parameters of red cell deformability include the maximum elongation index (EI Max), a measure of the maximum deformability of a red cell in response to increasing shear stress, and shear stress &#189; (SS &#189;), the shear stress required to achieve half maximal deformability.1 Osmotic gradient ektacytometry has several informative parameters. These include the elongation index minimum (EI Min), a measure of surface-to-volume ratio and the osmolality at which it occurs (O Min), which is a measure of osmotic fragility. EI Max and the osmolality at which it occurs (O (EI Max)) provide information on membrane flexibility and cell surface area. Half maximal elongation in the hypertonic arm of the osmotic gradient is represented by EI hyper. EI hyper and the osmolality at which it occurs, O hyper, provide information about the intracellular viscosity of the red cell which is determined by hemoglobin concentration.2,3 Measuring deformability in heterogenous blood is complicated by the fact that rigid cells, such as sickled red blood cells, do not properly align with the direction of flow such as deformable cells in response to increasing shear stress. Rather than producing a characteristic elliptical diffraction image, rigid cells produce a spherical pattern which results in a diamond-shaped diffraction pattern when overlaid on the ellipse produced by deformable cells.4,5,6 The spherical pattern has been shown to correspond to irreversibly sickled cells by performing ektacytometry on isolated fractions of cells following density centrifugation.6 The elongation index calculation includes measures of both the long and short axis of the ellipse; a diamond shape therefore produces an apparent decrease in elongation by increasing the width of the short axis.7 It has been previously shown that the degree of diffraction pattern distortion is correlated with both the percentage of sickle hemoglobin (HbS) and the percentage of sickled cells in the blood from patients with sickle cell anemia.5 The degree of diffraction pattern distortion can be obtained by complex mathematical analyses.8 It can also be obtained by adjusting the opening of the camera aperture on the ektacytometer or the grey level of the fitting software to alter the diffraction pattern height.5 However, details regarding how to adjust the grey level are not well defined and the camera aperture is not readily accessible on the latest generation of the commercially available ektacytometer. To circumvent these issues, the easily accessible camera gain can be used to adjust diffraction pattern heights.9 Using this method to estimate cellular heterogeneity, the degree of diffraction pattern distortion can be correlated with the percentage of fetal hemoglobin in the blood of patients with sickle cell anemia.10 Several osmotic gradient ektacytometry parameters are likewise correlated with the percentage of fetal or sickle hemoglobin in blood from patients with sickle cell anemia. Diffraction pattern distortion correlations likely reflect the contribution of hemoglobin composition to the percentage of rigid, non-deformable cells. Of additional interest, the entire osmotic gradient ektacytometry profile undergoes biphasic changes that correspond to the percentage of dense cells in circulation during sickle cell crisis.11
Ektacytometry is likewise useful in the study of several other disorders. Osmotic gradient ektacytometry is diagnostic for the inherited red cell membrane disorders, such as hereditary spherocytosis, hereditary elliptocytosis and hereditary pyropoikilocytosis.3,12,13,14 Decreased deformability occurs in iron deficiency.15 Characterization of the &#34;storage lesion&#34; of blood has employed ektacytometry and future studies investigating both the nature of the lesion and interventions to prevent its formation during the storage of banked blood are likely to benefit from the techniques presented here.16 Decreased red cell deformability has also been correlated with microvascular disease in diabetes.17 Recent studies linking hyperglycemia, red cell ascorbate concentrations and osmotic fragility suggest these factors may be important in the development of microvascular disease.18 Ektacytometry studies are currently underway to investigate this hypothesis (Parrow and Levine, unpublished data). Blood stage malarial infection is another interesting avenue of red cell deformability investigations. Cellular deformability of Plasmodium falciparum infected red blood cells decreases dramatically during the 48 hours of intracellular maturation of the parasite from ring stage to schizont stage. Evidence indicates that this decreased deformability is reversed upon maturation of the parasite. The reversal coincides with release of infected red cells into the circulation. Decreased deformability is thought to be mediated by Plasmodium proteins that promote sequestration of the red cell.19 These studies represent a small sampling of clinically important conditions where measuring erythrocyte deformability and osmotic gradient parameters are relevant. Several additional areas of study exist.
Alternative techniques for measuring red cell deformability include optical tweezers (also known as laser traps) which use the physical properties of photons to stretch single red cells in one or more directions.20 This technique has the advantage of measuring the deformability of single erythrocytes, but some uncertainty in force calibration has produced considerable variability across studies 21 and data analysis can be labor-intensive unless automated.22 Micropipette aspiration, which uses negative pressure to aspirate an erythrocyte into a micropipette, has also been used to measure deformability of red cells.7,23 Multiple measurements, such as the pressure required to aspirate the red cell, are possible with each measure defining different characteristics of the red cell.23 Atomic force microscopy is a high resolution technique that measures membrane stiffness by quantifying laser beam deflection as an indicator of cantilever deflection along the surface of a red cell.24 These techniques provide information about individual erythrocytes, are not easily adapted to measure changes in populations of red blood cells, and, in general, require considerable technical expertise.
The desire to sample both individual and populations of cells simultaneously has led to advances in automation and the development of microfluidics and array-based methods. Like ektacytometry, rheoscopy measures deformability as a function of shear stress but images are acquired directly via microscope.25 For higher through-put analyses, automated cell imaging has been employed to produce deformability distributions using the rheoscope.26 Cellular heterogeneity can be quantified by this method if data from a healthy control subject are available.27 Microfluidics techniques also allow for high through-put analyses of single cells; multiple designs using adaptations of filtration,28 cell transit analyzers,29 which measures the time required for an erythrocyte flow through a micropore, and alternatives that measure the pressure required for erythrocyte transit rather than time 30 have been developed. Another platform for high through-put analysis of individual cells is the single cell microchamber array chip, which has the additional advantage of allowing for downstream fluorescence-based characterization of the cells.31 Although each of these techniques is potentially useful and may be superior for particular applications, the comparative advantages of ektacytometry includes sensitivity, ease of use, and precision.32 The latest generation of commercially available ektacytometers also possess considerable versatility in the number of assays that can be performed.

<table><tbody><tr><td>LoRRca MaxSis standard version</td><td>Mechatronics</td><td>LORC109000</td><td></td></tr><tr><td>LoRRca MaxSis Osmoscan</td><td>Mechatronics</td><td>LORC109001</td><td></td></tr><tr><td>Polyvinylpyrrolidone solution (PVP) 0mOsm</td><td>Mechatronics</td><td>QRR030910</td><td></td></tr><tr><td>Polyvinylpyrrolidone solution (PVP) 500mOsm</td><td>Mechatronics</td><td>QRR030930</td><td></td></tr><tr><td>Polyvinylpyrrolidone solution (PVP) 5mL vials</td><td>Mechatronics</td><td>QRR030901</td><td></td></tr><tr><td>X clean</td><td>Mechatronics</td><td>QRR010946</td><td></td></tr><tr><td>P1000&amp;nbsp;</td><td>MilliporeSigma</td><td>Z646555</td><td></td></tr><tr><td>P200</td><td>MilliporeSigma</td><td>Z646547</td><td></td></tr><tr><td>P200 filter tips</td><td>MidSci</td><td>AV200-H</td><td></td></tr><tr><td>P1250 filter tips</td><td>MidSci</td><td>AV1250-H</td><td></td></tr><tr><td>Kimwipes</td><td>MidSci</td><td>8091</td><td></td></tr><tr><td>1.5 mL eppendorf tubes</td><td>MidSci</td><td>AVSS1700</td><td></td></tr><tr><td>15 mL conical vial</td><td>MidSci</td><td>C15R</td><td></td></tr></tbody></table>

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.

The ektacytometry results described in this manuscript can be used to measure red cell deformability in any condition. A schematic of the general set up of an ektacytometer is shown in Figure 1. Homogeneous populations of erythrocytes will produce an elliptical diffraction pattern in response to increasing shear stress that can be used to calculate the elongation index as shown in Figure 2. Diffraction pattern distortion occurs i...

Watch this Scientific Journal Video about Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry at JoVE.com

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

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 ...

measuring-deformability-red-cell-heterogeneity-blood

Research

JoVE Journal

Immunology and Infection

14.5K Views.  Saint Louis University School of Medicine. Hier presenteren we technieken voor het meten van de vervormbaarheid van de rode cel en cellulaire heterogeniteit door ektacytometry. Deze technieken zijn van toepassing op de algemene onderzoeken van rode cel vervormbaarheid en specifieke onderzoeken van bloed ziekten gekenmerkt door de aanwezigheid van zowel stijve als vervormbare erytrocyten in omloop, zoals sikkelcelanemie.

Video: Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Developmental Biology

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Bioengineering

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Mechanical Fatigue Testing of RBCs by Amplitude-Modulated Electrodeformation

Amplitude-Modulated Electrodeformation to Evaluate Mechanical Fatigue of Biological Cells

Red blood cells (RBCs) are known for their remarkable deformability. They repeatedly undergo considerable deformation when passing through the microcirculation. Reduced deformability is seen in physiologically aged RBCs. Existing techniques to measure cell deformability cannot easily be used for measuring fatigue, the gradual degradation in cell membranes caused by cyclic loads. We present a protocol to evaluate mechanical degradation in RBCs from cyclic shear stresses using amplitude shift keying (ASK) modulation-based electrodeformation in a microfluidic channel. Briefly, the interdigitated electrodes in the microfluidic channel are excited with a low voltage alternating current at radio frequencies using a signal generator. RBCs in suspension respond to the electric field and exhibit positive dielectrophoresis (DEP), which moves cells to the electrode edges. Cells are then stretched due to the electrical forces exerted on the two cell halves, resulting in uniaxial stretching, known as electrodeformation. The level of shear stress and the resultant deformation can be easily adjusted by changing the amplitude of the excitation wave. This enables quantifications of nonlinear deformability of RBCs in response to small and large deformations at high throughput. Modifying the excitation wave with the ASK strategy induces cyclic electrodeformation with programmable loading rates and frequencies. This provides a convenient way for the characterization of RBC fatigue. Our ASK-modulated electrodeformation approach enables, for the first time, a direct measurement of RBC fatigue from cyclic loads. It can be used as a tool for general biomechanical testing, for analyses of cell deformability and fatigue in other cell types and diseased conditions, and can also be combined with strategies to control the microenvironment of cells, such as oxygen tension and biological and chemical cues.

Author Spotlight: Studying Biomechanics of Circulating Cells by Modulating Their Electrodeformation Behavior 

Presented here is a protocol for mechanical fatigue testing in the case of human red blood cells using an amplitude-modulated electrodeformation approach. This general approach can be used to measure the systematic changes in morphological and biomechanical characteristics of biological cells in a suspension from cyclic deformation.

Red blood cells (RBCs) are known for their remarkable deformability. They repeatedly undergo considerable deformation when passing through the ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Biology

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.

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 ...

Medicine

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

Quantitative Analysis of Viscoelastic Properties of Red Blood Cells Using Optical Tweezers and Defocusing Microscopy

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Here, an integrated protocol based on optical tweezers and defocusing microscopy is described to measure the rheological properties of cells. This protocol has wide applicability in studying the viscoelastic properties of erythrocytes under variable physio-pathological conditions.

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

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