This protocol aligns the erythrocyte sedimentation rate measurements with the latest physical model and provides a more robust analysis of the test. This protocol allows a more accurate and systematic use of the erythrocyte sedimentation rate, but also provides the conventional values. We previously demonstrated that this technique is particular relevant for disorders presenting a low erythrocyte sedimentation rate value.
It can be used for diagnostic screenings for neuroacanthocytosis syndromes. To begin, collect the blood in standard 9-milliliter EDTA tubes, and the serum in standard 9-milliliter serum tubes. For optimal compaction of the packed erythrocytes, centrifuge the blood samples at 3, 000 g for at least seven minutes, and aspirate the supernatant.
Now prepare mixtures of autologous plasma and serum with determined proportions. For example, when preparing 2.5 milliliters of the 25%to 75%plasma serum mixture, add 0.625 milliliters of plasma to 1.875 milliliters of serum. Resuspend the pellet in PBS or the desired suspending liquid, and mix gently after including the supernatant for washing.
Repeat thrice, and perform the last wash with the desired suspending liquid. Extract the required volume of packed cells. Process the packed cells as a highly-viscous liquid for a 4-milliliter sample at a hematocrit of 45%Suspend 1.8 milliliters of packed cells within 2.2 milliliters of the plasma serum mixture or any other desired liquid.
To extract the required amount of sample for hematocrit determination, build the microhematocrit capillaries by immersing its lower tip in the liquid. When the required amount of sample rises in the tube with capillary suction, cover the opening to stop the flow. Next, seal the capillaries with sealing wax, place them in the microhematocrit centrifuge, and run them at 15, 000 g for five minutes, as per the manufacturer's instructions.
Read the hematocrit level on the capillary and write it down for reference. Now, to record the sample's sedimentation, set up a steady camera in front of the holder where the ESR tubes will be left at rest, and use a white, illuminated background for better contrast. Using empty Westergren tubes without markings, set the focus and the field of view of the camera to obtain the highest resolution where the samples will be placed.
Ensure that the borders of the pictures are aligned in vertical and horizontal directions. Then take a picture of a scaled tube to extract the pixel resolution. Set the camera's light and exposure time to have a white background but no saturation.
Fill the bottom container of the Westergren tube with the volume of the samples corresponding to the manufacturer's instructions, and insert them in the bottom container, as indicated by the manufacturer. Once the first tube is ready, place it in the holder, and start the camera recording. Recording one picture every minute usually gives a good resolution of the ESR kinetic curve.
Prepare and place the next samples. Ensure not to stand before any sample when a picture is taken. After recording, to extract the ESR curve using the MATLAB code, select a region of interest where only one tube is visible, the part located under the lowest position of the erythrocyte cell-free plasma interface but within the sample.
Note down the values of X, Y, W, and H, and use these values in the MATLAB script. Convert the region of interest of the RGB picture into a grayscale picture or matrix. Usually, combining the three color channels is very efficient with a clear background.
Then binarize the picture. For a healthy sample, using the ARC2 threshold usually gives a consistent result. For samples with a high hematocrit or with some hemolysis, it might be better to adjust it manually or use another automatic method, depending on the exact contrast obtained between the various phases inside the tube.
Obtain the average of the pixel values of GR along the horizontal direction. This step minimizes the noise and efficiently averages the possible interface irregularities. Before computing the variations, smooth the curve with a moving average, especially if the tubes contain some horizontal markings.
Then identify the interface position as the point with the highest intensity variation, and repeat for each picture and each sample. For each sample, use any suitable software to save the positions of the interface over time in an appropriate format to fit the physical model. Using any suitably fit software, knowing the hematocrit and the initial height of the blood column, find the values of the delay time, the dimensionless time, and the final packed volume fraction of the erythrocytes, which minimize the sum of squared residual deviations for the physical model.
Once the quantitative curve is extracted from the picture, save the physical parameters of the sample. Traditional ESR values at 30 minutes, one hour, or two hours can still be extracted from the curve for reference. The sedimentation velocity decreases with increasing hematocrit or initial volume fractions.
Sedimentation of erythrocytes become slower when the concentration of fibrinogen decreases. Variation of the extracted maximal sedimentation velocity as a function of the sample hematocrit and values obtained for the dimensionless time, the final packed volume fraction of the erythrocyte and the delay time, is shown. It could be seen that the correction removes the overall dependency on the initial hematocrit as well as most of the data dispersion.
Characteristic values of the extracted parameters for various fibrinogen concentrations are shown. The initial sedimentation speed at the beginning of the sedimentation process increases with an increase in the fibrinogen concentration. The dimensionless characteristic time of sedimentation decreases with increasing fibrinogen concentration.
The final volume fraction of sedimented erythrocytes is nearly independent of fibrinogen concentration. The delay time decreases with increasing fibrinogen concentration, indicating that stronger attractive interactions accelerate the rearrangement of initial erythrocyte structures that leads to the establishment of fluid channels. One of the critical points regarding the image acquisition is to have a uniform bright background, right?
Avoid pronounced shadows around the tubes. The technique could be used for the characterization of other diseases associated with lower erythrocyte sedimentation rate, such as sickle cell anemia or chronic mountain sickness.
