Thompson A B, Teschler H, Wang Y M, Konietzko N, Costabel U
Dept of Internal Medicine, University of Nebraska Medical Center, Omaha 68198-5300, USA.
Eur Respir J. 1996 Mar;9(3):603-8. doi: 10.1183/09031936.96.09030603.
The method of preparation of bronchoalveolar lavage fluid (BALF) for cytological examination can significantly affect the results of cellular quantitation. Investigations have shown that cytocentrifugation leads to an underestimation of the number of lymphocytes and membrane filter preparation to an underestimation of the number of neutrophils. As a simple alternative to these two techniques, BALF cells could be prepared by the microscope slide smear technique, which is familiar as the means for preparing peripheral blood for differential counts. In order to compare cell differentials determined by microscope slide technique with differentials resulting from cytocentrifugation, cells were isolated from 35 BALF samples using standard methods, and counted using a haematocytometer. Forty thousand cells in 200 microL were prepared by cytocentrifugation (3 min, 57 x g; Cytospin 2) and 5 x 10(5) cells in 5 microL by microscope slide smear. Both samples were air-dried, stained using May-Grünwald Giemsa stain, and 600 cells were counted to obtain differentials. To test the adequacy of sampling by the microscope slide smear technique, known quantities of lymphocytes or neutrophils were added to fixed numbers of BALF cells, microscope slide smears prepared, and differentials determined on 600 cells. The resulting differentials were compared to the calculated differentials. Preparation of BALF cells with the microscope slide smear technique yielded well-preserved cell morphology. Compared to cytocentrifugation, microscope slide smear preparations had significantly higher percentages of lymphocytes. The microscope slide smears for the samples with predetermined numbers of cells yielded lymphocyte and neutrophil percentages which did not differ from the calculated differentials (59.6 +/- 1.5 vs 59.6 +/- 5.2% and 54.6 +/- 6.0 vs 53.1 +/- 6.0%, respectively). Varying the number of cells counted from 100 to 800 confirmed the reproducibility of the counts for counting 600 cells. Using 5 x 10(5), 2.5 x 10(5), or 1 x 10(5) cells per preparation demonstrated that adequate specimens could be obtained from as few as 1 x 10(5) cells. Thus, microscope slide smear preparation is a simple and accurate method for the quantitation of bronchoalveolar lavage fluid cytology.
用于细胞学检查的支气管肺泡灌洗液（BALF）的制备方法会显著影响细胞定量结果。研究表明，细胞离心涂片法会导致淋巴细胞数量被低估，而膜滤器制备法会导致中性粒细胞数量被低估。作为这两种技术的一种简单替代方法，BALF细胞可以通过显微镜载玻片涂片技术制备，这是一种大家熟悉的用于制备外周血进行分类计数的方法。为了比较通过显微镜载玻片技术确定的细胞分类与细胞离心涂片法得到的分类结果，使用标准方法从35份BALF样本中分离细胞，并使用血细胞计数器进行计数。通过细胞离心涂片法（3分钟，57×g；Cytospin 2）制备200微升中含40000个细胞的样本，通过显微镜载玻片涂片法制备5微升中含5×10⁵个细胞的样本。两个样本均风干，用May-Grünwald Giemsa染色法染色，并计数600个细胞以获得分类结果。为了测试显微镜载玻片涂片技术采样的充分性，将已知数量的淋巴细胞或中性粒细胞添加到固定数量的BALF细胞中，制备显微镜载玻片涂片，并对600个细胞进行分类计数。将得到的分类结果与计算出的分类结果进行比较。用显微镜载玻片涂片技术制备BALF细胞可使细胞形态保存良好。与细胞离心涂片法相比，显微镜载玻片涂片制备的淋巴细胞百分比显著更高。对于含有预定数量细胞的样本，显微镜载玻片涂片得到的淋巴细胞和中性粒细胞百分比与计算出的分类结果无差异（分别为59.6±1.5%对59.6±5.2%和54.6±6.0%对53.1±6.0%）。将计数的细胞数量从100个变化到800个，证实了计数600个细胞时计数结果的可重复性。每份制备物使用5×10⁵、2.5×10⁵或1×10⁵个细胞表明，从低至1×10⁵个细胞就可以获得足够的样本。因此，显微镜载玻片涂片制备是一种简单而准确的支气管肺泡灌洗液体细胞学定量方法。
