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.

Oxygen gradient ektacytometry is a functional assay that assess individual sickling tendency. To date, no other functional assays are available or used. Oxygen gradient ektacytometry is able to determine the oxygen tension at which blood cells of patient with sickle cell disease start to sickle.

This oxygen tension is patient-specific and it can serve as a biomarker for disease severity. In sickle cell disease, this technique can be used to monitor treatment efficacy and it can be used for the development of new treatments. Visual demonstration of this technique is important because it is very sensitive.

There are numerous factors that will influence the measurement which you do not know when you start working with this technique. Somebody who tries this for the first time may experience that the technique is very sensitive for sample preparation and sample handling. My advice is to work as standardized as possible and to develop a routine.

To begin this procedure, switch on the computer and the ektacytometer and open the software on the computer. Open the nitrogen cylinder to make sure the nitrogen is available to deoxygenate the sample. Lower the bob in the cup and make sure the cup can turn freely.

When the software is running, check for the message make sure the gas valve is open and click OK.Ensure that the ektacytometer starts the self-check process and select start. If the self-check fails, re-run it by clicking hardware check, pO2, self-check. 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 one of the text protocol making sure to keep the same settings for every measurement. Save these settings by clicking OK, then clicking OK again. First, collect blood samples by venepuncture in a tube containing EDTA and store the blood at four degrees Celsius for a minimum of 30 minutes but no longer than 24 hours.

After this, mix the samples gently by inversion to homogenize. Let the samples warm up to room temperature on a roller bench before the measurement. Use a hematology analyzer to measure the complete blood count by placing the aspiration needling into the tube and pressing the button behind the needle of the hematology analyzer to begin the measurement and the analyzer will use between 20 and 200 microliters of whole blood.

Then standardize the whole blood sample to an RBC count of 200 million red blood cells in five milliliters of PVP by calculating the volume of the sample that will be added. To begin, cut a pipette tip and pre-wet it by resuspending the blood three times. Then pipette the calculated sample volume into a PVP vial.

Gently mix the sample manually by inversion until it is homogenous. Slowly draw two milliliters of the blood and PVP mixture into a three milliliter syringe without the needle. Push the plunger to remove any visible air bubbles and excess sample solution until between 1.5 and 1.8 milliliters are left in the syringe.

Inject the total sample volume slowly and evenly into the bob through the connector. Make sure that the level of the sample is above the oxygen sensor and the small suction hole. In the software, click new and fill in the sample identifier, remarks, date of donation, and the viscosity of PVP.

Click OK and aspirate. After 60 seconds of aspirating, the cup turns and aspirate again for 15 seconds. When the rotation stops, click OK.Close the machine lid.

Click continue and start now as oxygen gradient ektacytometry is done with a fixed gain. 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 the settings.

First, remove the sample syringe and replace it with a syringe filled with distilled water or deionized water. Press clean and slowly flush the connector during rinsing making sure to flush in both directions. Next, remove the syringe and lift the bob.

Use a soft cloth to thoroughly dry the bob and connector. Then use a large syringe to flush the connector in order to remove any water remaining in the tube and bob. Keep the syringe at the inlet and block the outlet of the bob to get back pressure in the tubes thereby removing the remaining water.

Then dry the cup while avoiding touching the oxygen spot. Lower the bob and the cup readying the machine for the next measurement. After ensuring that the machine is properly rinsed, check to make sure that the proper tubes relate to the cleaning solution.

Close the software, press close, and press start to begin the end of day cleaning program. Then empty the waste bottle and use a soft cloth to dry the bob and cup. Close the lid of the machine.

Close the nitrogen cylinder and turn off the computer and machine. Oxygen gradient ektacytometry can be used to study the sickling behavior of red blood cells under a range of oxygen concentrations. In oxygen gradient ektacytometry, continuous deoxygenation of the sample by nitrogen gas is followed by swift reoxygenation by ambient air.

Under these conditions, red blood cell 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. Sickle red blood cells show a change in shape during deoxygenation that mimics conditions during oxygen gradient ektacytometry but show no change in shape when oxygenated.

This process results in distortion of the diffraction pattern during oxygen gradient ektacytometry and thus in a decrease in the elongation index. A representative curve obtained by the ektacytometer shows the six parameters that reflect different characteristics of the sickling behavior:the maximum elongation index, the minimum elongation index, the delta EI, the point of sickling, the area under the curve, and the recovery. Representative curves of red blood cells from healthy controls, patients with HbS traits, and a homozygous sickle cell disease patient are shown here.

The clear differences in the representative curves of homozygous sickle cell patients treated with hydroxyl urea and transfusion highlights the usefulness of this assay. There are numerous factors that influence oxygen gradient ektacytometry such as temperature, pH, and osmolarity of the PVP, but also remnants of water and the degree of mixing of the blood sample solution can affect the measurements. Therefore, standardized sample handling is essential.

The outcome of oxygen gradient ektacytometry may be directly implemented in clinical practice, thus contributing to personalized medicine. This technique makes it possible to study sickling behavior to investigate the potential of new treatments for sickle cell disease and can serve as a clinically relevant biomarker for disease severity.