Engström K G, Möller B, Meiselman H J
Department of Physiology and Biophysics, University of Southern California, School of Medicine, Los Angeles.
Blood Cells. 1992;18(2):241-57; discussion 258-65.
Although red blood cell (RBC) geometry has been extensively studied by micropipette aspiration, the small size of RBC and pipettes vs. the optical resolution of light microscopy suggests the need to consider potential errors. The present study addressed such difficulties and investigated four specific problems: (1) use of exact equations to calculate RBC membrane area and volume; (2) calibration of the pipette internal diameter (PID); (3) correction for a noncylindrical pipette barrel; (4) diffraction distortion of the RBC image. The observed PID represents a cylinder lens enlargement that can be theoretically derived from the glass/buffer refractive index ratio (1.49/1.33 = 1.12). This enlargement was experimentally confirmed by: (1) studying pipettes bent to allow measurement through the barrel (horizontal) and at the orifice (vertical), with a resulting diameter ratio of 1.12 +/- 0.01; (2) and by replacing the surrounding buffer with immersion oil and hence abolishing the lens phenomenon (ratio = 1.12 +/- 0.02). In addition, use of aspirated oil droplets demonstrated a 3.2 +/- 0.2% error when the PID is focused at a sharp, maximum diameter. The average pipette cone angle was 1.49 +/- 0.09 degrees and varied considerably with pipette pulling procedures; calculated tongue geometry inside the pipette was affected by the noncylindrical pipette barrel. The RBC diffraction error, demonstrated by touching two aspirated cells held by opposing pipettes, was 0.091 +/- 0.002 microns. The PID, cone angle, and diffraction artifacts significantly (p < 0.001) affected calculated RBC geometry (average errors up to 5.4% for area and 9.6% for volume). Two new methods to calculate, rather than directly measure, the PID from images of a single RBC, during either osmotic or pressure manipulation, were evaluated; the osmotic method closely predicted the PID, whereas the pressure method markedly underestimated the PID. Our results thus confirm the need to consider the above-mentioned phenomena when determining RBC geometric parameters via micropipette aspiration.
尽管通过微量移液管抽吸对红细胞（RBC）的几何形状进行了广泛研究，但红细胞和移液管的尺寸较小，与光学显微镜的光学分辨率相比，这表明有必要考虑潜在误差。本研究解决了这些困难，并研究了四个具体问题：（1）使用精确方程计算红细胞膜面积和体积；（2）校准移液管内径（PID）；（3）校正非圆柱形移液管腔；（4）红细胞图像的衍射畸变。观察到的PID代表圆柱透镜放大，理论上可从玻璃/缓冲液折射率比（1.49/1.33 = 1.12）推导得出。这种放大通过以下方式得到实验证实：（1）研究弯曲的移液管，以便通过管腔（水平）和管口（垂直）进行测量，得出的直径比为1.12±0.01；（2）用浸油替换周围的缓冲液，从而消除透镜现象（比率 = 1.12±0.02）。此外，当PID聚焦在尖锐的最大直径时，使用抽吸的油滴显示出3.2±0.2%的误差。移液管的平均锥角为1.49±0.09度，并且随移液管拉制程序有很大变化；移液管腔内计算出的舌状几何形状受非圆柱形移液管腔的影响。通过接触由相对的移液管固定的两个抽吸细胞所证明的红细胞衍射误差为0.091±0.002微米。PID、锥角和衍射伪像显著（p < 0.001）影响计算出的红细胞几何形状（面积平均误差高达5.4%，体积平均误差高达9.6%）。评估了两种在渗透或压力操作期间从单个红细胞图像计算而非直接测量PID的新方法；渗透法能很好地预测PID，而压力法明显低估了PID。因此，我们的结果证实了在通过微量移液管抽吸确定红细胞几何参数时需要考虑上述现象。
