Burger S R, Kadidlo D, McCullough J
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, USA.
Transfusion. 1999 May;39(5):451-6. doi: 10.1046/j.1537-2995.1999.39050451.x.
Progenitor assays are the principal method for evaluating hematopoietic cell function. The magnitude of assay variability and the assay steps contributing to variability were determined, and modifications intended to increase assay consistency were evaluated.
Assays were performed using a serum-free progenitor assay medium with cells plated at 5.0 x 10(4) and 1.0 x 10(5) cells per plate. A peripheral blood progenitor cell component collected from a normal donor after administration of granulocyte-colony-stimulating factor was divided into identical aliquots. Each experiment involved at least 5 technologists, each performing assays in duplicate on five aliquots, with each person scoring all assay plates. Three sample preparation methods were tested: 1) ficoll mononuclear cell enrichment and sample dilution, 2) sample dilution without ficoll separation, and 3) sample dilution without ficoll separation, with cell counts performed before and after each dilution step, dilution volumes calculated on the basis of each cell count, automated electronic pipettors used in dilution steps, and colony frequency calculated on the basis of cell counts from the final specimen.
Global variability for colony-forming units-granulocyte-macrophage, represented by the percentage of CV for all specimens and all technologists, was 89.6 percent at 5.0 x 10(4) cells per plate and 81.3 percent at 1.0 x 10(5), when ficoll separation was used. Subjective differences in scoring plates did not account for most of the variability observed, as results for any individual plate read by multiple technologists had a mean CV of 15.6 percent and 19.7 percent at the two plating concentrations. Method 3 resulted in the greatest improvement, reducing CV to 24.4 percent at 5.0 x 10(4) cells per plate and to 24.2 percent at 1.0 x 10(5) cells per plate. Similar results were obtained for erythroid-burst-forming units.
Baseline assay results were extremely inconsistent. Interindividual differences in colony interpretation did not contribute significantly to assay variability, although sample preparation and plating did. Improved control over cell concentration decreased assay variability by 70 to 73 percent.
祖细胞检测是评估造血细胞功能的主要方法。确定了检测变异性的大小以及导致变异性的检测步骤，并评估了旨在提高检测一致性的改进措施。
使用无血清祖细胞检测培养基进行检测，每孔接种5.0×10⁴和1.0×10⁵个细胞。从正常供体在给予粒细胞集落刺激因子后采集的外周血祖细胞成分被分成相同的等分试样。每个实验至少有5名技术人员参与，每人对五个等分试样进行一式两份的检测，并对所有检测孔进行评分。测试了三种样品制备方法：1）菲可分离单个核细胞并稀释样品，2）不进行菲可分离直接稀释样品，3）不进行菲可分离直接稀释样品，在每个稀释步骤前后进行细胞计数，根据每次细胞计数计算稀释体积，在稀释步骤中使用自动电子移液器，并根据最终标本的细胞计数计算集落频率。
当使用菲可分离时，以所有标本和所有技术人员的CV百分比表示的粒细胞-巨噬细胞集落形成单位的总体变异性，在每孔接种5.0×10⁴个细胞时为89.6%，在每孔接种1.0×10⁵个细胞时为81.3%。多个技术人员读取的任何单个孔的结果在两种接种浓度下的平均CV分别为15.6%和19.7%，因此评分孔的主观差异并未占观察到的大部分变异性。方法3带来了最大的改进，在每孔接种5.0×10⁴个细胞时将CV降至24.4%，在每孔接种1.0×10⁵个细胞时降至24.2%。红系爆式集落形成单位也获得了类似结果。
基线检测结果极不一致。尽管样品制备和接种会导致检测变异性，但个体间在集落解读上的差异对检测变异性的贡献并不显著。对细胞浓度的更好控制使检测变异性降低了70%至73%。
