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.
