Løhne K, Urdal P, Leren T P, Tonstad S, Ose L
Department of Clinical Chemistry, Ullevål University Hospital, Oslo, Norway.
Cytometry. 1995 Aug 1;20(4):290-5. doi: 10.1002/cyto.990200404.
Flow cytometric methods for measurement of low-density lipoprotein (LDL) receptor activity on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia (FH). However, cellular LDL receptor activities measured in FH heterozygotes may overlap with those of healthy subjects. Analytical variation is probably responsible for some of this overlap. We have examined several technical details that may affect analytical variation. In each analysis, we included one standard and two control cell preparations. These were cells isolated from three donors and stored in aliquots at -135 degrees C. Use of standard cells reduced between-series analytical variation of the controls by approximately 50%. Preincubation-conditions used to induce the maximum number of receptors, the concentration of fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-perchlorate (DiI)-LDL, labelling time, and conditions during storage of labelled cells before flow cytometry were also examined in order to reduce analytical variation. Having standardized the assay, we found among 20 healthy subjects a median receptor activity of 100% vs. 51% among 26 patients who fulfilled clinical criteria for FH. However, four of the patients showed distinctly normal receptor activities, which may suggest either the presence of some other biochemical defect or that in vivo dysfunctional receptors may be measured as normal in some patients with our assay.
用于测量外周血单个核细胞（PBMC）上低密度脂蛋白（LDL）受体活性的流式细胞术方法可用于识别家族性高胆固醇血症（FH）患者。然而，FH杂合子中测得的细胞LDL受体活性可能与健康受试者的活性重叠。分析变异可能是造成这种重叠的部分原因。我们研究了几个可能影响分析变异的技术细节。在每次分析中，我们纳入了一份标准品和两份对照细胞制剂。这些细胞是从三名供体中分离出来的，并以等分试样的形式保存在-135℃。使用标准细胞使对照的系列间分析变异降低了约50%。为了减少分析变异，还研究了用于诱导最大受体数量的预孵育条件、荧光染料1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐（DiI）-LDL的浓度、标记时间以及流式细胞术检测前标记细胞的储存条件。在对检测方法进行标准化后，我们发现20名健康受试者的受体活性中位数为100%，而在26名符合FH临床标准的患者中为51%。然而，其中四名患者的受体活性明显正常，这可能表明存在其他一些生化缺陷，或者在我们的检测方法中，一些患者体内功能失调的受体可能被检测为正常。
