Szpechcinski Adam, Kopinski Piotr, Giedronowicz Dorota, Rozy Adriana, Jagus Paulina, Szolkowska Malgorzata, Chorostowska-Wynimko Joanna
Laboratory of Molecular Diagnostics and Immunology and Department of Pathomorphology, National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland.
Anal Quant Cytol Histol. 2011 Oct;33(5):289-96.
To validate the fast and accurate flow cytometric (FCM) protocol using blood-standardized antibodies for alveolar lymphocyte subtyping with respect to standard immunocytochemistry (IC).
FCM and IC were applied to immunophenotype T cell subsets in bronchoalveolar lavage (BAL) fluids from patients with interstitial lung diseases. Diagnostic BAL specimens from 50 patients with suspected sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis were evaluated by both IC and FCM. In FCM, CD4+ and CD8+ T cells were identified by light scatter gating with CD3 selection using basic tricolor cytometer.
Relative amounts of CD4+, CD8+ T cells, and CD4+/CD8+ ratios demonstrated by the FCM showed excellent, significant correlations with IC results. FCM values did not differ significantly from IC results. However, the sensitivity and specificity of conventional IC staining were not sufficient to assess CD4+/ CD8+ ratio in most idiopathic pulmonary fibrosis cases. Additionally, performing IC immunophenotyping in BAL samples with low lymphocyte content introduced a remarkable error into CD4+/CD8+ ratio assessment.
FCM allowed reliable, precise, and fast T-cell subset measurement in all BAL samples, overcoming the IC disadvantages. Our validated FCM protocol provides diagnostically relevant CD4+/CD8+ ratio determination by simple light scatter gating strategy with CD3 selection.
验证使用血液标准化抗体的快速准确流式细胞术（FCM）方案，用于肺泡淋巴细胞亚群分型，并与标准免疫细胞化学（IC）进行比较。
将FCM和IC应用于间质性肺疾病患者支气管肺泡灌洗（BAL）液中的T细胞亚群免疫表型分析。对50例疑似结节病、特发性肺纤维化和过敏性肺炎患者的诊断性BAL标本进行IC和FCM评估。在FCM中，使用基本三色细胞仪通过CD3选择的光散射门控来识别CD4 +和CD8 + T细胞。
FCM显示的CD4 +、CD8 + T细胞相对数量以及CD4 + / CD8 +比值与IC结果显示出极好的显著相关性。FCM值与IC结果无显著差异。然而，在大多数特发性肺纤维化病例中，传统IC染色的敏感性和特异性不足以评估CD4 + / CD8 +比值。此外，在淋巴细胞含量低的BAL样本中进行IC免疫表型分析会在CD4 + / CD8 +比值评估中引入显著误差。
FCM能够在所有BAL样本中进行可靠、精确和快速的T细胞亚群测量，克服了IC的缺点。我们验证的FCM方案通过简单的CD3选择光散射门控策略提供了与诊断相关的CD4 + / CD8 +比值测定。
