Read E J, Cardine L L, Yu M Y
Department of Transfusion Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland.
Transfusion. 1991 Jul-Aug;31(6):502-8. doi: 10.1046/j.1537-2995.1991.31691306246.x.
In vivo survival studies of human red cells (RBCs) are commonly carried out by using chromium-51 (51Cr), a gamma-emitting radionuclide, as the cell label. The effects of labeling human RBCs with PKH-2, a nonradioactive lipophilic fluorescent dye that binds to the cell membrane, and the feasibility of detecting the labeled cells by flow cytometric analysis were investigated. Optimal labeling, defined as maximum mean fluorescence intensity with minimal cell-to-cell variability in fluorescence intensity and minimal cell loss, was achieved with the use of 15.0 x 10(-6) M (15.0 x 10(-6) mol/L) PKH-2 and a cell concentration of 4.0 x 10(9) RBCs per mL. Both freshly drawn and stored RBCs could be labeled, but RBCs stored for more than 20 days did not take up the label as uniformly as fresher cells. Although labeling with PKH-2 did not interfere with the detection of ABO, Rh(D), or common minor RBC antigens by routine serologic methods, it resulted in a morphologic appearance resembling echinocytosis and an increased resistance to osmotic lysis by hypotonic saline. RBCs labeled by this method could be quantitated accurately in blood samples in which their proportion was 0.01 percent, or 1 labeled cell in 10,000 cells. This method holds promise as a simple, reliable, and sensitive method for the detection of labeled human RBCs, but the in vivo significance of the label's effects on cell morphology and osmotic fragility is not known. Further studies directly comparing PKH-2-labeled and 51Cr-labeled RBCs will be necessary to establish the accuracy of the former method in determining the in vivo survival of human RBCs.
人类红细胞（RBC）的体内存活研究通常通过使用铬-51（51Cr）（一种γ发射放射性核素）作为细胞标记物来进行。研究了用PKH-2（一种与细胞膜结合的非放射性亲脂性荧光染料）标记人类红细胞的效果以及通过流式细胞术分析检测标记细胞的可行性。使用15.0×10（-6）M（15.0×10（-6）mol/L）PKH-2和每毫升4.0×10（9）个红细胞的细胞浓度可实现最佳标记，即最大平均荧光强度，同时荧光强度的细胞间变异性最小且细胞损失最小。新鲜抽取的红细胞和储存的红细胞均可被标记，但储存超过20天的红细胞摄取标记不如新鲜细胞均匀。尽管用PKH-2标记不干扰通过常规血清学方法检测ABO、Rh（D）或常见的次要红细胞抗原，但它导致红细胞出现类似棘形红细胞增多症的形态外观，并增加了对低渗盐水渗透裂解的抵抗力。通过这种方法标记的红细胞在其比例为0.01%（即10000个细胞中有1个标记细胞）的血液样本中可被准确定量。该方法有望成为一种简单、可靠且灵敏的检测标记人类红细胞的方法，但标记对细胞形态和渗透脆性影响的体内意义尚不清楚。有必要进一步开展直接比较PKH-2标记和51Cr标记红细胞的研究，以确定前一种方法在测定人类红细胞体内存活情况方面的准确性。
