Structural Analysis Using Atomic Force Microscopy of the Surface of Red Blood Cells to Detect Anti-Red Blood Cell Autoantibody
Nobuhiko Kubo,1 Ryoji Hirota,2 Takao Okada,2 Tomoko Ohara,1 Mikihiko Kawano,1 Kouichi Itoh1
1Department of Clinical Laboratory Medicine, Jichi Medical School, Tochigi;
2Research Institute of Biomolecular Metrology, Ibaraki, Japan
INTRODUCTION:
Autoimmune hemolytic anemia (AIHA) consists of a group of disorders whose common characteristics are the presence of autoantibodies against red blood cells (RBCs) and, in turn, short RBC life caused by these antibodies. Direct visualization of antibody on the RBC surface poses an unprecedented challenge to the hematological laboratory. Atomic force microscopy (AFM) has recently been applied to detect nanometer-scale objects not only in the analysis of material science but also in the biomedical field [1]. AFM can reveal an image of the sample surface by monitoring the repulsive force generated by tip-surface interaction. In the system, the very fine sensitive tip at the end of the cantilever interacts with the sample (Figure 1). The position in X, Y, and Z directions is monitored by a piezoelectric scanner in the system (Figure 1). There are several scanning modes of AFM, such as contact mode and tapping mode. Tapping mode is generally used to visualize the surface structure of biological samples. Briefly, the tip of an oscillating cantilever briefly touches the sample surface line by line during each oscillation. Therefore, the tip is in intermittent contact with the sample. The response is used as an input to the feedback loop while the piezo height is adjusted so that the vibration amplitude remains constant. Thereby topographic information indicated by the piezo height change is recorded.
We used AFM to scan the surface of RBCs from an AIHA patient to detect anti-RBC autoantibody. RBCs from a control subject and from a patient with AIHA who showed positive results for direct Coombs test were washed several times in physiological saline and then fixed for more than 5 minutes in physiological saline containing 3% formaldehyde (PSCF). AFM examination was carried out using a Nanoscope IIIa Multimode system (Digital Instruments, Santa Barbara, CA, USA) in the tapping mode at room temperature in air.
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