Sickling kinetics and Image Flowcytometry.
This protocol exposes red cells to low oxygen in time. The spinning incubator of the sicklomat ensures rapid gas exchange with the cells, as experienced in the microvasculature. The spinning cycles ensure that all cells in the population are exposed to the gas mixture at the same time. At specified timepoints, samples are automatically collected, and fixed in morphology by mixing them with a fixation solution.
Image flowcytometry analysis is used to define the number of abnormal (sickled) cells at different timepoints. A total of 10,000 individual cell images are collected to provide proper statistical analysis as 100 images represent 1% of the population. Each picture (left) is represented in a dot plot (middle) that is analyzed by pixel analysis algorithms. Results can be plotted as the percentage of abnormal (sickled) cell in time. The curve adheres to a sigmoid curve fit defined by a starting value (Z) representing abnormal cells at time zero (ISC’s), a maximum (M) as the percentage of sickled cells after 10-20 minutes, the time (T) to reach ½ maximum change, and the sigmoidial parameter n. Together this data provides insight in the rate of sickling of sickle RBC under low oxygen.
Each event in image flowcytometry is a picture. Pixel analysis algorithms such as Circularity vs Shape ratio provide dot plots in which each dot is a cell picture. Specific subpopulations are defined as “sickled” cells, and the formation of such cells is plotted against time under low oxygen.
The figure represents three separate runs of the same sample exposed to 1% oxygen (PO2 = 8 mmHg).
