Abstract
BALF cellular analysis results are greatly impacted by processing technique #AdvancesinILD @ERSTalkhttp://///wly/u9js30i1dup.
To the Editor:
We read with great interest the study by Bollmannet al。[1] wherein the authors describe the impact of collection protocols and bronchoscope lumen size on bronchoalveolar lavage fluid (BALF) cellular analysis. Their findings highlight the importance of standardising BALF collection protocols in order to meaningfully interpret and compare BALF analysis results, for both clinical and research purposes. As a complement to their study, we would like to emphasise the importance of laboratory methods used to process the collected sample. We present our experience with highly discordant BALF cell counts and differentials, using two processing protocols in our local laboratory.
Multiple elements in the laboratory method used for BALF processing, including cytocentrifugation technique and staining, can impact BALF cell differential [2–4]。Published guidelines for BALF processing and interpretation recommend that cytocentrifugation with Giemsa type staining (CY) be used for differential cell count analysis [5]。ThinPrep (TP) technology with Papanicolaou staining is a liquid-based preparation method for cytological specimens which has been adopted by many pathology laboratories as it is automated and results in a clean background with an even distribution of cells on the slide. The Papanicolaou stain used for TP is optimal for detecting atypical and malignant cells and but not for differentiating inflammatory cells [6]。缺乏数据比较BALF细胞分析技术,并且临床实践中的任何一种技术的普遍存在未知[5]。Until recently, BALF cellular analysis was performed exclusively using TP at our institution, due to the relatively higher cost and resource intensity of CY. As a quality improvement initiative, we compared the BALF cell count differentials obtained using both techniques concurrently during this time of transition from TP to CY in accordance with current guidelines.
We retrospectively identified BALF samples collected for clinical purposesviaflexible bronchoscopy over a 13 month period from four hospitals in Calgary, AB, Canada. Each sample was analysed using both TP and CY methods concurrently, with all samples analysed at a single laboratory.
TP (Cytyc, Marlborough, MA, USA) samples were prepared according to the manufacturer's instructions. The sample was placed into an alcohol-based preservative solution, Cytolyte (Cytyc), centrifuged to obtain cell pellet that was resuspended in Preservcyt (Cytyc), then an even monolayer of cells was collected using a filter. The cells were subsequently transferred to a glass slide, and stained with the Papanicolaou technique. The bronchoalveolar lavage (BAL) differential was performed by a cytopathology technologist and a cytopathologist performed the final review.
For the CY method, initial total white blood cell (WBC) and red blood cell (RBC) counts were analysed using a Sysmex Cell count analyser (Sysmex Canada, Mississauga, ON, Canada). If WBC count was >10 000×106L−1or RBC >5 000 000×106L−1a dilution was performed to 1000×106L−1。如果BALF是粘稠的,则加入几粒透明质酸酶,混合良好10分钟;如果仍然粘性,则将样品在37℃温育5分钟,彻底混合,然后通过Sysmex分析仪进行。然后使用Shandon Cytospin 3细胞缺口(Thermo Fisher Scientific,Waltham,Ma,Ma,Ma,Ma,USA)制备两种细胞螺旋液载玻片3分钟。将载玻片用赖特-Giemsa染色染色,并且通过血质病学技术专家进行血质病学家进行最终审查的血管病理学家。
对于每个样品,根据美国胸部社会(ATS)标准BAL诊断模式(淋巴细胞> 15%;中性粒细胞> 3%)分类细胞组合物。嗜嗜酸性钙> 1%),并根据建议特异性间质性肺病(ILD;淋巴细胞差分计数> 50%;中性粒细胞计数> 50%;嗜酸性粒细胞计数> 25%)[5]。描述性比较用于评估两种方法之间的模式的差异。使用CY值作为每个特定模式的参考标准计算TP的敏感度和特异性。
从这两种方法都是avai BALF细胞分析lable from 28 samples (26 patients; two patients with BAL from two lobes). BALF sampling was undertaken to assess for infection in 9/28 (32%), airspace disease in 5/28 (18%), suspected eosinophilic lung disease in 3/28 (11%), malignancy in 1/28 (3%), and “other” in 10/28 (36%).
Classification of BALF according to the analysis method used for standard and ILD specific cellular patterns is shown in thetable 1。More samples were classified as an ILD specific pattern (for all cell types) when analysed by CYversusTP, whereas there was no trend in classification by the standard cellular pattern. Cellular pattern was discordant between analysis techniques for a lymphocytic pattern in 5/28 (18%), neutrophilic pattern in 7/28 (25%), and eosinophilic pattern in 12/28 (43%). There were fewer discordant samples when classified by ILD specific pattern, however, 10/28 samples (36%) were still discordant by at least one ILD specific pattern. The absolute difference between techniques ranged from −47% to 70%, with no consistent directionality. TP had poor sensitivity for ILD specific lymphocytic and eosinophilic patterns (0% for both).
BALF cellular analyses were discordant when TP was compared to CY in a majority of samples for both BAL and ILD specific patterns. TP had very poor sensitivity for ILD specific lymphocytic and eosinophilic patterns. Our findings highlight the impact of BALF processing and analysis techniques, and emphasise that standardised techniques must be broadly implemented. Our findings support previous literature demonstrating BALF processing techniques can impact the BALF cell differential. Saltiniet al。[2] compared BALF cell differential counts from a cytocentrifugeversusnon-cytocentrifuge technique and found cytocentrifugation was consistently associated with a loss in lymphocyte percentage. A more recent study demonstrated variations within the cytocentrifugation conditions can also impact the BALF cell differential [3]。
BALF细胞分析是在诊断某些肺病的诊断中,最常见的肺炎,非特异性肺炎,非特异性间质性肺炎和嗜酸性肺炎(嗜酸性肺炎)是一种信息性临床工具5]。However, for BALF to contribute meaningful pathobiological information, results must be accurate, with accuracy dependent upon processing technique. An informal survey (personal communications, K.A. Johannson) of ILD practitioners from different centres suggests that many clinicians are unfamiliar with the BALF processing techniques in use at their institution. Clinicians should enquire as to their local BALF processing and analytic techniques if BALF cellular analyses are being used for clinical or research purposes, and aim to implement the currently recommended technique.
我们的数据具有重要的局限性,包括小样本大小和回顾性研究设计。我们只比较TP结果与ATS标准,Cy,因此不和谐的结果不能推广到其他制剂和染色方法。数据收集主要用于当地质量评估,因此我们不能考虑可能影响BALF的患者特定因素,例如吸烟状态或BALF收集技巧。然而,我们将两种分析方法进行了比较了同一样本中的两种分析方法,因此这些因素不太可能影响我们的内部内容。
We hope that our data, combined with that of Bollmannet al。[1],将在BALF分析的作用方面通知中心和中心之间的潜在来源,鉴于对超敏感性肺炎等疾病的广泛兴趣,及时的话题。准确的结果对于BALF分析的有意义应用至关重要,我们鼓励正在进行的合作努力来标准化BALF收集,加工和分析,以指导研究和优化患者护理。
Footnotes
利益冲突:K.A.Johannson已从Hoffman-La Roche Ltd和Boehringer-Ingelheim获得个人费用和非财政支持,并从UCB Biopharma Sprl和Chest Foundation授予提交的工作。
- 收到August 29, 2017.
- Accepted2018年1月10日。
- 复制right ©ERS 2018