Quantifying morphological alteration of RBC population from light scattering data

Studies of RBC morphological alterations, despite their potential clinical and experimental application, are compromised due to lack of simple and rapid techniques. As a complementary approach toward quantitative microscopy, we have reconstituted morphological information from light scattering data obtained from flow cytometer. Normal and poikilocytic agent treated samples were analyzed by microscopy and respective morphological index (MI) was calculated from the morphology based scores assigned to RBC. The samples were simultaneously analyzed by flowcytometer and the scatter data were obtained. Accordingly, the best correlated parameters of both forward scatter and side scatter were chosen to formulate a suitable regression model with MI as response. Flow cytometry data was also verified with another instrument (BD FACS Verse) and the equation obtained was validated with separate set of samples. The multivariate regression analysis yields a quadratic model with MI as response (R² = 0.96, p < 0.001). The flow cytometric data from both instruments were in good agreement (Intra class correlation ~0.9, p < 0.001). The model was found to simulate the sample MI with high accuracy (R² = 0.97, p < 0.001). This proposed method was verified to be simple, rapid, quantitative and cost effective for the measurement of morphological alteration of RBC.