Name: measuring deformability and red cell heterogeneity in blood by ektacytometry
Uploaded: 2018-01-12T14:00:00
Description: Watch this Scientific Journal Video about Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry at JoVE.com

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
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	<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:...

<ol>
	<li>Bessis, M., Mohandas, N., Feo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+ektacytometry:+a+new+method+of+measuring+red+cell+deformability+and+red+cell+indices.">Automated ektacytometry: a new method of measuring red cell deformability and red cell indices.</a> Blood Cells. 6 (3), 315-327 (1980).</li><li>Clark, M. R., Mohandas, N., Shohet, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotic+gradient+ektacytometry:+comprehensive+characterization+of+red+cell+volume+and+surface+maintenance.">Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance.</a> Blood. 61 (5), 899-910 (1983).</li><li>Da Costa, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tool+for+red+blood+cell+membrane+disorders:+Assessment+of+a+new+generation+ektacytometer.">Diagnostic tool for red blood cell membrane disorders: Assessment of a new generation ektacytometer.</a> Blood Cells Mol Dis. 56 (1), 9-22 (2016).</li><li>Clark, M. R., Mohandas, N., Shohet, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformability+of+oxygenated+irreversibly+sickled+cells.">Deformability of oxygenated irreversibly sickled cells.</a> J Clin Invest. 65 (1), 189-196 (1980).</li><li>Rabai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformability+analysis+of+sickle+blood+using+ektacytometry.">Deformability analysis of sickle blood using ektacytometry.</a> Biorheology. 51 (2-3), 159-170 (2014).</li><li>Bessis, M., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Diffraction+Patterns+of+Sickle+Cells+in+Fluid+Shear+Fields.">Laser Diffraction Patterns of Sickle Cells in Fluid Shear Fields.</a> Blood Cells. 3, 229-239 (1977).</li><li>Kim, Y., Kim, K., Park, Y., Moschandreou, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Blood Cell - An Overview of Studies in Hematology. , (2012).</li><li>Streekstra, G. J., Dobbe, J. G., Hoekstra, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+fraction+poorly+deformable+red+blood+cells+using+ektacytometry.">Quantification of the fraction poorly deformable red blood cells using ektacytometry.</a> Opt Express. 18 (13), 14173-14182 (2010).</li><li>Renoux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+methodological+standardization+for+the+ektacytometric+measures+of+red+blood+cell+deformability+in+sickle+cell+anemia.">Importance of methodological standardization for the ektacytometric measures of red blood cell deformability in sickle cell anemia.</a> Clin Hemorheol Microcirc. 62 (2), 173-179 (2016).</li><li>Parrow, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+red+cell+deformability+and+hydration+reflect+HbF+and+HbA2+in+blood+from+patients+with+sickle+cell+anemia.">Measurements of red cell deformability and hydration reflect HbF and HbA2 in blood from patients with sickle cell anemia.</a> Blood Cells Mol Dis. 65, 41-50 (2017).</li><li>Ballas, S. K., Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+changes+during+the+evolution+of+the+sickle+cell+painful+crisis.">Red blood cell changes during the evolution of the sickle cell painful crisis.</a> Blood. 79 (8), 2154-2163 (1992).</li><li>Johnson, R. M., Ravindranath, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotic+scan+ektacytometry+in+clinical+diagnosis.">Osmotic scan ektacytometry in clinical diagnosis.</a> J Pediatr Hematol Oncol. 18 (2), 122-129 (1996).</li><li>Mohandas, N., Clark, M. R., Jacobs, M. S., Shohet, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+factors+regulating+erythrocyte+deformability.">Analysis of factors regulating erythrocyte deformability.</a> J Clin Invest. 66 (3), 563-573 (1980).</li><li>Lazarova, E., Gulbis, B., Oirschot, B. V., van Wijk, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+osmotic+gradient+ektacytometry+for+the+diagnosis+of+hereditary+spherocytosis:+interlaboratory+method+validation+and+experience.">Next-generation osmotic gradient ektacytometry for the diagnosis of hereditary spherocytosis: interlaboratory method validation and experience.</a> Clin Chem Lab Med. 55 (3), 394-402 (2017).</li><li>Anderson, C., Aronson, I., Jacobs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+Deformability+is+Reduced+and+Fragility+increased+by+Iron+Deficiency.">Erythrocyte Deformability is Reduced and Fragility increased by Iron Deficiency.</a> Hematology. 4 (5), 457-460 (1999).</li><li>Reinhart, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Washing+stored+red+blood+cells+in+an+albumin+solution+improves+their+morphologic+and+hemorheologic+properties.">Washing stored red blood cells in an albumin solution improves their morphologic and hemorheologic properties.</a> Transfusion. 55 (8), 1872-1881 (2015).</li><li>Shin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+impairment+of+erythrocyte+deformability+as+indicator+of+microangiopathy+in+type+2+diabetes+mellitus.">Progressive impairment of erythrocyte deformability as indicator of microangiopathy in type 2 diabetes mellitus.</a> Clin Hemorheol Microcirc. 36 (3), 253-261 (2007).</li><li>Tu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+Red+Blood+Cell+Vitamin+C+Concentrations+Induce+Red+Blood+Cell+Fragility:+A+Link+to+Diabetes+Via+Glucose,+Glucose+Transporters,+and+Dehydroascorbic+Acid.">Low Red Blood Cell Vitamin C Concentrations Induce Red Blood Cell Fragility: A Link to Diabetes Via Glucose, Glucose Transporters, and Dehydroascorbic Acid.</a> EBioMedicine. 2 (11), 1735-1750 (2015).</li><li>Tiburcio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+switch+in+infected+erythrocyte+deformability+at+the+maturation+and+blood+circulation+of+Plasmodium+falciparum+transmission+stages.">A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages.</a> Blood. 119 (24), e172-e180 (2012).</li><li>Henon, S., Lenormand, G., Richert, A., Gallet, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+determination+of+the+shear+modulus+of+the+human+erythrocyte+membrane+using+optical+tweezers.">A new determination of the shear modulus of the human erythrocyte membrane using optical tweezers.</a> Biophys J. 76 (2), 1145-1151 (1999).</li><li>Mills, J. P., Qie, L., Dao, M., Lim, C. T., Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+elastic+and+viscoelastic+deformation+of+the+human+red+blood+cell+with+optical+tweezers.">Nonlinear elastic and viscoelastic deformation of the human red blood cell with optical tweezers.</a> Mech Chem Biosyst. 1 (3), 169-180 (2004).</li><li>Moura, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+real+time+evaluation+of+red+blood+cell+elasticity+by+optical+tweezers.">Automatic real time evaluation of red blood cell elasticity by optical tweezers.</a> Rev Sci Instrum. 86 (5), 053702 (2015).</li><li>Evans, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+membrane+concept+applied+to+the+analysis+of+fluid+shear-+and+micropipette-deformed+red+blood+cells.">New membrane concept applied to the analysis of fluid shear- and micropipette-deformed red blood cells.</a> Biophys J. 13 (9), 941-954 (1973).</li><li>Chen, X., Feng, L., Jin, H., Feng, S., Yu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+erythrocyte+deformability+using+atomic+force+microscopy:+correlation+study+of+the+erythrocyte+deformability+with+atomic+force+microscopy+and+hemorheology.">Quantification of the erythrocyte deformability using atomic force microscopy: correlation study of the erythrocyte deformability with atomic force microscopy and hemorheology.</a> Clin Hemorheol Microcirc. 43 (3), 243-251 (2009).</li><li>Musielak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell-deformability+measurement:+review+of+techniques.">Red blood cell-deformability measurement: review of techniques.</a> Clin Hemorheol Microcirc. 42 (1), 47-64 (2009).</li><li>Dobbe, J. G., Streekstra, G. J., Hardeman, M. R., Ince, C., Grimbergen, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+distribution+of+red+blood+cell+deformability+using+an+automated+rheoscope.">Measurement of the distribution of red blood cell deformability using an automated rheoscope.</a> Cytometry. 50 (6), 313-325 (2002).</li><li>Dobbe, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+red+blood+cell-deformability+distributions.">Analyzing red blood cell-deformability distributions.</a> Blood Cells Mol Dis. 28 (3), 373-384 (2002).</li><li>Kikuchi, Y., Arai, T., Koyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+filtration+method+for+red+cell+deformability+measurement.">Improved filtration method for red cell deformability measurement.</a> Med Biol Eng Comput. 21 (3), 270-276 (1983).</li><li>Moessmer, G., Meiselman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+micropore+filtration+approach+to+the+analysis+of+white+cell+rheology.">A new micropore filtration approach to the analysis of white cell rheology.</a> Biorheology. 27 (6), 829-848 (1990).</li><li>Guo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+analysis+of+red+blood+cell+deformability.">Microfluidic analysis of red blood cell deformability.</a> J Biomech. 47 (8), 1767-1776 (2014).</li><li>Doh, I., Lee, W. C., Cho, Y. H., Pisano, A. P., Kuypers, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformation+measurement+of+individual+cells+in+large+populations+using+a+single-cell+microchamber+array+chip.">Deformation measurement of individual cells in large populations using a single-cell microchamber array chip.</a> Appl Phys Lett. 100 (17), 173702-173703 (2012).</li><li>Baskurt, O. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+commercially+available+ektacytometers+with+different+shearing+geometries.">Comparison of three commercially available ektacytometers with different shearing geometries.</a> Biorheology. 46 (3), 251-264 (2009).</li><li>Baskurt, O. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+guidelines+for+hemorheological+laboratory+techniques.">New guidelines for hemorheological laboratory techniques.</a> Clin Hemorheol Microcirc. 42 (2), 75-97 (2009).</li><li>Uyuklu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+storage+duration+and+temperature+of+human+blood+on+red+cell+deformability+and+aggregation.">Effects of storage duration and temperature of human blood on red cell deformability and aggregation.</a> Clin Hemorheol Microcirc. 41 (4), 269-278 (2009).</li><li>Uyuklu, M., Meiselman, H. J., Baskurt, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hemoglobin+oxygenation+level+on+red+blood+cell+deformability+and+aggregation+parameters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+sickle+cell+anemia+and+alpha-thalassemia.+Effect+on+pathological+properties+of+sickle+erythrocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+blood+rheological+properties+and+red+blood+cell+indices(MCH,+MCV,+MCHC)+in+healthy+women.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+spherocytosis,+elliptocytosis,+and+other+red+cell+membrane+disorders.">Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders.</a> Blood Rev. 27 (4), 167-178 (2013).</li></ol>

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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">LoRRca MaxSis standard version</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">LORC109000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">LoRRca MaxSis Osmoscan</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">LORC109001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Polyvinylpyrrolidone solution (PVP) 0mOsm</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">QRR030910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Polyvinylpyrrolidone solution (PVP) 500mOsm</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">QRR030930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Polyvinylpyrrolidone solution (PVP) 5mL vials</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">QRR030901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">X clean</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">QRR010946</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P1000&#160;</td>
			<td style="width:115px;">MilliporeSigma</td>
			<td>Z646555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">P200</td>
			<td style="width:115px;">MilliporeSigma</td>
			<td style="width:119px;">Z646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">P200 filter tips</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">AV200-H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">P1250 filter tips</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">AV1250-H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Kimwipes</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">8091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">1.5 mL eppendorf tubes</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">AVSS1700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">15 mL conical vial</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">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 ...

The overall goal of this technique is to measure red cell deformability and cellular heterogeneity by ektacytometry. This method can help answer key questions in ektacytometry such as how deformable erythrocytes are, how heterogeneous a blood sample is, and how the experiment does change as a function of disease. The main advantages of this techniques are accessibility, convenience, and reliability in measuring deformability and heterogeneity of red blood cells populations.The techniques may be useful for monitoring the response to therapy in sickle cell anemia patients. Because red cell deformability and heterogeneity are correlated with hemoglobin composition and concentration. These methods can be applied to other diseases characterized by decreased red cell deformability, such as diabetes, where decreased red cell deformability might contribute to microvascular disease.Begin by connecting the zero osmolar tube to the low osmolar PVP solution, and the 500 osmolar tube to the high osmolar PVP solution. Ensure that the bob is lowered completely into the cup. Launch the software and prime the machine by selecting Hardware Check, and then 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. Gently mix a freshly drawn whole blood sample by inverting the vial several times. Then, pipette 25 microliters of whole blood into five milliliters of iso-osmolar PVP solution.After capping the vial, mix gently by again inverting several times. Choose deformability from the software main menu, then create a new analysis and add the experimental details. Next, lift the lid of the ektacytometer, and verify that the bob is fully lowered into the cup and that the cup is turning.Pipette one milliliter of PVP blood solution into the space between the cup and the bob. Then lift the bob slightly to bring the sample down. Wait until all bubbles have moved out of the solution, then close the lid to the ektacytometer.Adjust the gain to 200 by moving the arrow along the scroll bar in the software. When the temperature is stable at 37 degrees Celsius and the diffraction image is stable, press start. Observe the diffraction patterns as data acquisition progresses to ensure that they remain circular, elliptical, or diamond shaped.When data acquisition is complete, print the report and clean. A diffraction pattern obtained from healthy blood in response to shear stress shows the expected elliptical pattern. Elongation index is calculated using the equation shown here.When 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. Proper drying is critical, as residual water will lyse red cells, producing interference.Select export to save the report. Last, click on the main menu button on the software to return to the main page. After loading another sample into the ektacytometer as before, ensure that a stable diffraction image is present on the screen.Adjust the camera gain by moving a ruler along the screen until it produces a 3.8 cm diffraction height. Use a ruler to verify the height of the image on the computer screen. Then, press start and observe the data acquisition as before.This image shows a 3.8 cm diffraction pattern obtained from blood from a sickle cell anemia patient. After cleaning the ektacytometer as previously shown, repeat the procedure to obtain diffraction patterns at 4.5 cm and 5.4 cm heights. Diffraction pattern distortion occurs in a heterogeneous blood samples because rigid red cells do not align properly with deformable cells, resulting in a diamond shaped diffraction pattern.This distortion pattern is increasingly detectable as the camera gain is adjusted to generate larger diffraction patterns. For osmotic gradient ektacytometry, begin by adding 250 microliters of whole blood to a vial containing five milliliters of iso-osmolar PVP and gently mix. Choose Osmoscan from the main menu in the software.Then place the 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 the 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 to enable a view of the blood entering the tubing. Press New Analysis and type in the experimental details. After adjusting the camera gain to 200, allow the machine to run until blood enters the cup from the tubing beneath the instrument.When the computer screen shows a stable diffraction pattern, begin data acquisition by pressing the Start Now button on the dialogue box. Allow the ektacytometer to acquire data up to approximately 500 milliosmoles per kilogram, and then stop the instrument. Print the report.Next, remove the old PVP blood vial and replace it with a clean vial containing deionized water. Bring the needle down so that it touches the bottom of the vial, then press the rinse button in the dialogue box to rinse the gradient system. Once the rinse is complete, press the clean option on the dialogue box on the computer monitor, and select export to save the report.Dry the bob and cup as before. Homogeneous blood populations, as found in healthy volunteers, do not produce different deformability curves when diffraction sizes are measured. In contrast, heterogeneous blood populations such as blood from patients with sickle cell anemia, show significant decreases in deformability measures as a function of diffraction pattern size.In sickle blood, when the degree of diffraction pattern distortion is measured as a function of EI max, it is correlated with hemoglobin S, and the adult hemoglobin variant, hemoglobin A2.Transfusion corrects the distortion, as indicated by its inverse relationship with the percentage of normal adult hemoglobin in the blood. Whether the degree of diffraction pattern distortion is measured as a function of the shear stress required to achieve half maximal deformability, or as a function of EI max, it is correlated with fetal hemoglobin. Key osmotic gradient ektacytometry parameters, such as EI max, EI min, and O min, provide additional information about the cellular hydration, surface to volume ratios, and osmotic fragility, respectively.Once mastered, these techniques can be done in approximately one hour, if performed properly. Following this procedure, other methods like microscopy can be performed, to answer additional questions pertaining to the deformability of individual erythrocytes. After watching this video, you should have a good understanding of how to measure deformability and serum heterogeneity in blood samples.

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Research

JoVE Journal

Immunology and Infection

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

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 ...

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. 
In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...