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

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

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The authors declare no competing financial interests.

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

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

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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">ADVIA 120 Hematology Analyzer</td>
			<td style="width:159px;">Siemens</td>
			<td style="width:119px;">067-A004-14</td>
			<td style="width:167px;">Instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Cell-Dyn Sapphire Hematology Analyzer</td>
			<td style="width:159px;">Abbott</td>
			<td style="width:119px;">8H00-01</td>
			<td>Instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Lorrca</td>
			<td style="width:159px;">RR Mechatronics</td>
			<td style="width:119px;">LORC109230 or LORC109110</td>
			<td>Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Lorrca Software version V5.08</td>
			<td style="width:159px;">RR Mechatronics</td>
			<td style="width:119px;">-</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Nitrogen gas 4.8 or 5.0</td>
			<td style="width:159px;">Local</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">O2-spot</td>
			<td style="width:159px;">RR Mechatronics</td>
			<td>PO2S020153</td>
			<td>O2 measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Oxygenscan module (pO2scan)</td>
			<td style="width:159px;">RR Mechatronics</td>
			<td>PO2S109000</td>
			<td>Add-on</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Oxy-ISO</td>
			<td style="width:159px;">RR Mechatronics</td>
			<td>QRR 030905</td>
			<td>Viscous solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">X-Clean</td>
			<td style="width:159px;">RR Mechatronics</td>
			<td>QRR 010946</td>
			<td>Cleaning solution Lorrca</td>
		</tr>
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characterization of sickling during controlled automated deoxygenation with oxygen gradient ektacytometry

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

Oxygen gradient ektacytometry can be used to characterize sickling behavior in patients with SCD. In this study, blood samples from a total of 38 SCD patients and five healthy controls were included. In healthy controls, the diffraction pattern is circular at rest and elliptical at higher shear stress4. From the elliptical diffraction pattern, the elongation index (EI) is calculated based on the height and width of the diffraction pattern. In oxygen gradient ektacytometry, slow and continuous deoxygenation of the sample by nitrogen gas is followed by swift reoxygenation by ambient air. Under these conditions, RBC sickling can be observed under deoxygenation. This will cause a distortion of the diffraction pattern because sickled red cells will not align properly under the applied shear stress. Hence, they appear to be less deformable as opposed to healthy RBCs (Figure 2).
Figure 3A shows how sickle RBCs change in shape upon deoxygenation, which mimicked conditions during oxygen gradient ektacytometry, whereas control sickle RBCs without deoxygenation show no change in shape. This process results in distortion of the diffraction pattern during oxygen gradient ektacytometry, and thus in a decrease in EI. Figure 3B shows the different diffraction patterns from which different parameters are generated.
A representative curve obtained by the ektacytometer is shown in Figure 3C. Six parameters reflect different characteristics of sickling behavior of RBCs: EImax is the maximum EI at the start of the measurement before deoxygenation. This parameter represents the baseline position and reflects the overall deformability of the total RBC population at ambient air. EImin is the minimum EI, which represents minimal deformability after deoxygenation. This parameter reflects changes in the shape and orientation of (sickle) RBCs upon deoxygenation. &#916;EI is the difference between EImax and EImin, which indicates how many cells can sickle during one round of deoxygenation. 5% Point of Sickling (PoS5%) is the pO2 (mmHg) at which a 5% decrease of EImax during deoxygenation is measured. This represents the oxygen tension where the sickling process starts. Area reflects the area under the curve, which is determined by an integral calculation of EI and pO2 measurements between 100 mmHg and pO2min (mmHg). This is the result of previously described parameters EImax, EImin, and PoS. Recovery represents the difference of EI during the final part of reoxygenation compared to EI at baseline. Both EI values are measured at a pO2 of 100&#8211;120 mmHg. This parameter reflects the capacity of RBCs that sickle during deoxygenation to reverse sickling during reoxygenation22. Parameters from duplicate measurements generally had a coefficient of variation (CV) &#60;5% (median 1.83%). In case a CV &#62; 5% was obtained, a third measurement was performed. The parameters EImax and Recovery are the most reproducible with median CVs &#60;1%.
Representative curves of RBCs of healthy controls, patients with HbS traits (heterozygous HbS), and a homozygous SCD patient are shown in Figure 4A. The representative curve of the HbSC patient shows a lower recovery, which might indicate a different sickling process (Figure 4B). The representative curves of HbSS patients treated with hydroxyurea (HU) and transfusion are shown in Figure 4C and Figure 4D. Clearly, there is a big difference between the representative curves of HS traits (HbAS cells) and RBCs of HbSS patients treated with transfusion (consisting of a mixture of homozygous sickle (HbSS) and homozygous normal (HbAA) cells, Figure 4A,D). The clear differences in the curves of the untreated SCD patient and the HU and transfusion-treated patients highlights the usefulness of this assay (Figure 4C,D). Levels of HbF and HbS correlated significantly with EImin and, to a lesser extent, with PoS (Figure 5A&#8211;D). This indicates that those laboratory parameters that are important in the evaluation of the patient are also reflected in the oxygen gradient ektacytometry. The number of sickled cells at normoxia and percentage of dense RBCs (DRBCs) both influence EImax values, as they are significantly correlated (Figure 5E&#8211;F), which indicates that EImax reflects another important factor in the sickling process. These results show how different characteristics such has %HbS, %HbF, sickled cells at normoxia, and %DRBCs influence different parameters.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60213/60213fig1.jpg" /> 
Figure 1. Schematic setup of the ektacytometer. The ektacytometer uses a Couette system to apply shear stress on the cells. A rotation outer cylinder (cup) and a static inner cylinder (bob) are used to induce shear stress by the creation of laminar flow at 37 &#730;C. Between the bob and cup there is a small gap in which the blood suspension is injected. A laser beam shines from the bob through the blood suspension and is scattered by the presence of RBCs. The diffraction pattern is projected and analyzed by a camera. The elongation index (EI) is calculated with the height (a) and the width (b) of the diffraction pattern4. <a href="https://www.jove.com/files/ftp_upload/60213/60213fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60213/60213fig2.jpg" /> 
Figure 2. Schematic setup of the ektacytometer with oxygen gradient ektacytometry module. Schematic diagram of the module that shows deoxygenation of the blood suspension slowly with the infusion of nitrogen gas (N2). Oxygen tension is measured by the amount of quenching of the luminophore signal sent from the LED-fiber to the O2-spot. Upon deoxygenation, sickle RBCs will start to sickle, their deformability will decrease, and they will no longer align with elliptical RBCs. The sickled RBCs will distort the diffraction pattern, changing its shape from an ellipse to a rhomboid or diamond-like shape. This change in the shape of the diffraction pattern results in a decrease of EI. Measurements of pO2 and EI are not performed at the same height in the cup. This ensures better discrimination between the deoxygenation and reoxygenation curves and, hence, a better interpretation of the curve. This figure has been modified&#160; from Rab et al.22 <a href="https://www.jove.com/files/ftp_upload/60213/60213fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60213/60213fig3.jpg" /> 
Figure 3. Representative oxygen gradient ektacytometry curve and diffraction patterns. (A) Upon deoxygenation under conditions similar to the oxygen gradient ektacytometry, sickle RBCs were fixed. In control sickle RBCs, the same conditions were used, but without nitrogen gas. Deoxygenated sickle RBCs show a change in shape in contrast to control RBCs. (B) Upon deoxygenation and shear stress (30 Pa), the diffraction pattern changes from an ellipse to a rhomboid. (C) Representative curve of oxygen gradient ektacytometry. The maximum elongation index (EImax) represents the baseline position and shows an overall deformability of the total RBC population. Minimum EI (EImin) represents minimal deformability, which is caused by the change in shape and orientation of RBCs upon deoxygenation. &#916;EI (dEI, the difference in EI between EImax and EImin) shows how many cells can sickle during one round of deoxygenation. Point of sickling (PoS, pO2 at 5% EI decrease) shows the oxygen tension when the first RBCs start to sickle. The area under the curve (from pO2min = 100 mmHg) is calculated in the parameter area. This summarizes EImax, EImin, and PoS. The capacity of sickled cells to unsickle during reoxygenation is represented in the parameter Recovery (percentage of EImax reached during reoxygenation). To aid in the interpretation, all data points were connected in every individual experiment by a line to graphically present the results. This figure has been modified&#160; from Rab et al.22 <a href="https://www.jove.com/files/ftp_upload/60213/60213fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60213/60213fig4.jpg" /> 
Figure 4. Oxygen gradient ektacytometry parameters correlate with genotype and treatment regimens of SCD patients with SCD. (A) Representative graph of RBCs of HbS carriers (HbS trait) and healthy controls in relation to untreated HbSS patients. (B) Representative graph of RBCs of patients with Hemoglobin SC Disease (HbSC) in relation to untreated HbSS patients. (C) Representative graph of RBCs of hydroxyurea treated homozygous SCD patients (HbSS HU) in relation to untreated HbSS patients. (D) Representative graph of RBCs of HbSS patients treated with blood transfusion (HbSS transfusion) in relation to untreated HbSS patients. This figure has been modified&#160; from Rab et al.22 <a href="https://www.jove.com/files/ftp_upload/60213/60213fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60213/60213fig5.jpg" /> 
Figure 5. Oxygen gradient ektacytometry parameters are associated with %HbF, %HbS, %sickled cells at normoxia and %dense RBCs. (A) Linear correlation of minimum elongation index (EImin) and %HbF of 15 HbSS or HbS/&#946;-thalassemia patients without transfusion. (B) Linear correlation of EImin and %HbS. (C) Linear correlation of PoS and %HbF. (D) Linear correlation of PoS and %HbS. (E) Linear correlation of maximum EI (EImax) and percent of sickled cells at normoxia measured with digital microscopy. (F) Linear correlation of EImax and percentage dense RBCs (%DRBCs) of 21 patients with HbSS. This figure has been modified&#160; from Rab et al.22 <a href="https://www.jove.com/files/ftp_upload/60213/60213fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Settings</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Files</td>
			<td>Storage directory</td>
			<td></td>
		</tr>
		<tr>
			<td>General options</td>
			<td>Default medium viscosity</td>
			<td>Viscosity of PVP</td>
		</tr>
		<tr>
			<td>pO2 scan</td>
			<td>Minimum aspiration time (s)</td>
			<td>60</td>
		</tr>
		<tr>
			<td></td>
			<td>pO2 scan shear stress (Pa)</td>
			<td>30</td>
		</tr>
		<tr>
			<td></td>
			<td>Determine pO2 every (S)</td>
			<td>20</td>
		</tr>
		<tr>
			<td></td>
			<td>Moving average size</td>
			<td>2</td>
		</tr>
		<tr>
			<td></td>
			<td>pO2 scan step; Edit</td>
			<td>0 -OFF; 60 -ON; 1360 &#8211;OFF; 1640 -OFF</td>
		</tr>
		<tr>
			<td></td>
			<td>Cal. Area between (mmHg)</td>
			<td>10 and 100</td>
		</tr>
		<tr>
			<td></td>
			<td>pO2 control</td>
			<td>Off (unchecked)</td>
		</tr>
	</tbody>
</table>
Table 1. The preferred setting of the ektacytometer.

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

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

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

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

characterization-sickling-during-controlled-automated-deoxygenation

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9.6K Views.  Utrecht University. 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.

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

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

Drug-Induced Sleep Endoscopy (DISE) with Target Controlled Infusion (TCI) and Bispectral Analysis in Obstructive Sleep Apnea

The aim of this study was to establish a standardized protocol for drug-induced sleep endoscopy (DISE) to differentiate obstruction patterns in obstructive sleep apnea (OSA). Target-controlled infusion (TCI) of the sedative propofol was combined with real-time monitoring of the depth of sedation using bispectral analysis.
In an observational study 57 patients (mean age 44.8 years, &#177; SD 10.5; mean apnea hypopnea Index (AHI) 30.8/hr, &#177; SD 21.6, mean BMI 28.2 kg/m2, &#177; SD 5.3) underwent cardiorespiratory polysomnography followed by DISE with TCI and bispectral analysis. Sleep was induced solely by the intravenous infusion of propofol with a TCI-pump, with an initial target plasma level of 2.0 &#181;g/ml. Under continuous monitoring of the patient&#39;s respiration, state of consciousness and value of the bispectral analysis, the target plasma propofol level was raised in steps of 0.2 &#181;g/ml/2 min until the desired depth of sedation was reached. The mean value of the bispectral analysis at the target depth of sedation was determined and the obstruction patterns during DISE-TCI-bispectral analysis then classified according to the VOTE-system. Subsequently the results were analyzed according to polysomnographic and anthropometric data. The occurrence of multilevel obstruction sites across all degrees of severity of OSA clarifies the need for sleep endoscopy prior to upper airway surgery.
The advantage of this technique is the reproducibility of the protocol even for heterogeneous groups of patients. In addition, the gradual controlled and standardized increase of the plasma level of propofol with real-time control of the bispectral index leads to a precisely controllable depth of sedation. The DISE-TCI-bispectral analysis procedure is a step towards a required reproducible protocol of sleep endoscopy &#8212; capable of standardization. However it is not yet known whether these observed obstruction patterns also correspond to findings in natural sleep.

Drug-Induced Sleep Endoscopy DISE with Target Controlled Infusion TCI and Bispectral Analysis in Obstructive Sleep Apnea

The aim of this study was to establish a standardized protocol for sleep endoscopy to differentiate obstruction patterns in obstructive sleep apnea (OSA). Target-controlled infusion (TCI) of the sedative was combined with real-time monitoring of the depth of sedation using bispectral analysis.

The aim of this study was to establish a standardized protocol for drug-induced sleep endoscopy (DISE) to differentiate obstruction patterns in ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...