A 73 year old woman presented with shortness of breath and was found to have bilateral pleural effusions. She had a history of marginal zone B-Cell lymphoma involving the bone marrow, which was diagnosed 3 months before this presentation and was treated with Rituximab.
一名 73 岁女性因呼吸急促就诊，发现双侧胸腔积液。她有累及骨髓的边缘区 B 细胞淋巴瘤病史，在就诊前 3 个月被诊断为该病，并接受了利妥昔单抗治疗。
Thoracentesis revealed an atypical lymphoid population comprised of intermediate and large sized cells with eccentrically placed nuclei, multiple prominent nucleoli and scant to moderate amounts of basophilic cytoplasm (Image 1). Initial evaluation of the cytology material was concerning for large-cell transformation of the patient’s previously diagnosed marginal zone B cell lymphoma. A representative portion of the fine needle aspirate sample was sent for flow cytometric immunophenotyping.
胸腔穿刺术显示非典型淋巴细胞群，由中等和大型细胞组成，具有偏心放置的细胞核、多个突出的核仁和少量至中等量的嗜碱性细胞质（图 1）。对细胞学材料的初步评估涉及患者先前诊断的边缘区 B 细胞淋巴瘤的大细胞转化。将细针抽吸物样品的代表性部分送去进行流式细胞术免疫表型分析。
Image 1. Cytology (Diff Quik, 400X). The atypical lymphoid population is comprised of intermediate and large sized cells with eccentrically placed nuclei, multiple prominent nucleoli and scant to moderate amounts of basophilic cytoplasm.
图 1.细胞学（Diff Quik，400X）。非典型淋巴细胞群由中等和大型细胞组成，具有偏心放置的细胞核、多个突出的核仁和少量至中等量的嗜碱性细胞质。
Flow cytometric immunophenotyping showed a distinct population of atypical cells with moderate CD45 expression and increased side scatter in keeping with cytoplasmic complexity (Figure 1, black arrows). On an initial screening B cell lymphoma panel these cells were negative for CD19 and positive for CD30 (partial), and CD44 (Figure 2).
流式细胞术免疫表型分析显示，具有中等CD45表达的非典型细胞群，侧向散射增加，与细胞质复杂性一致（图1，黑色箭头）。在初步筛查 B 细胞淋巴瘤面板上，这些细胞的 CD19 呈阴性，CD30（部分）和 CD44 呈阳性（图 2）。
Figure 1. The neoplastic population shows expression of CD30 and CD44.
图 1.肿瘤群体显示 CD30 和 CD44 的表达。
The population of interest lacked expression of CD10, CD20, CD22 and surface immunoglobulin light chains and CD138 (Figure 2 and 3).
目标群体缺乏 CD10、CD20、CD22 和表面免疫球蛋白轻链以及 CD138 的表达（图 2 和 3）。
Figure 2. The neoplastic population lacks expression of CD10, CD19, CD20, and CD22.
图2.肿瘤群体缺乏 CD10、CD19、CD20 和 CD22 的表达。
Figure 3. The neoplastic population of cells are negative for surface immunoglobulin light chains and CD138.
图3.肿瘤细胞群对表面免疫球蛋白轻链和 CD138 呈阴性。
CD30 expression prompted the investigation of additional T-cell markers to rule out a T cell lymphoma (Figure 4). This population showed dim expression of CD7 but was otherwise negative for pan T cell markers (CD2, CD3, CD5) as well as CD4 and CD8 (Figure 4).
CD30 表达促使研究其他 T 细胞标志物以排除 T 细胞淋巴瘤（图 4）。该人群的 CD7 表达微弱，但其他方面对 pan T 细胞标志物（CD2、CD3、CD5）以及 CD4 和 CD8 呈阴性（图 4）。
Figure 4. The neoplastic population of cells are positive for CD7 (dim) and CD30 and negative for CD3, CD4, CD8, and CD26.
图4.肿瘤细胞群的 CD7 （dim） 和 CD30 呈阳性，CD3、CD4、CD8 和 CD26 呈阴性。
Given the unusual immunophenotype of the neoplasms, a diagnosis of diffuse large B cell lymphoma (transformation of the known marginal zone lymphoma) seemed less likely and other possibilities were considered.
鉴于肿瘤的异常免疫表型，弥漫性大B细胞淋巴瘤（已知边缘区淋巴瘤的转化）的诊断似乎不太可能，并考虑了其他可能性。
The presence of CD30 expression and the plasmablastic morphologic features together with the clinical presentation with effusions raised the possibility of primary effusion lymphoma. IHC for anti-HHV8 was performed on the cell block sample (Image 2).
CD30 表达的存在和浆母细胞形态学特征以及积液的临床表现增加了原发性积液淋巴瘤的可能性。对细胞块样品进行抗HHV8的IHC（图2）。
Image 2. Cytology (cell block 400X) A. Hematoxylin and eosin stain of the cell block reveals large, atypical lymphoid cells; Small and large atypical lymphoid cells are highlighted by CD30 (B), HHV8 (LANA-1) (C), and CD138 (focally) (D).
图 2.细胞学检查（细胞块400X） A.苏木精和细胞块的伊红染色显示大的，非典型淋巴细胞;CD30 （B）、HHV8 （LANA-1） （C） 和 CD138 （局灶性） （D） 突出显示小的和大的非典型淋巴样细胞。
Primary effusion lymphoma (PEL) is a large B-cell neoplasm usually presenting as serous effusions without a detectable tumor mass [1]. It is universally associated with the human herpesvirus 8 (HHV8). It usually occurs in the setting of immunodeficiency [2]. Some patients with PEL secondarily develop solid tumors in adjacent structures such as the pleura [3-5].
原发性积液淋巴瘤（primary epfusion lymphoma， PEL）是一种大型B细胞肿瘤，通常表现为浆液性积液，但无可检测到的肿瘤肿块[1]。它普遍与人类疱疹病毒 8 （HHV8） 相关。它通常发生在免疫缺陷的情况下[2]。一些PEL患者继发性在邻近结构（如胸膜）发生实体瘤[3-5]。
NEGATIVE:
pan- B-cell markers (CD19, CD20, and CD79a), surface and cytoplasmic Ig, and BCL6.
阴性：泛 B 细胞标志物（CD19、CD20 和 CD79a）、表面和细胞质 Ig 以及 BCL6。
PEL is usually negative for T/NK-cell antigens, although aberrant expression of T-cell markers may occur. PEL is usually positive for EBV-encoded small RNA (EBER) by
in situ
hybridization but negative for EBV latent membrane protein 1 (LMP1) by IHC. This could be explained by EBV virus latency. It is ability of a pathogenic virus to lie dormant (latent) within a cell, denoted as the lysogenic part of the viral life cycle. EBV expresses its genes in one of three patterns, known as latency programs. EBV can exhibit one of three latency programs: Latency I, Latency II, or Latency III. Each latency program leads to the production of a limited, distinct set of viral proteins and viral RNAs. The Epstein-Barr virus encoded RNAs (EBERs): EBER1 and EBER2 are expressed during all latency forms [6], whereas LMP1 is expressed only in latency 2 and 3 rendering it a less sensitive marker for detection of EBV infection. EBV-negative PEL is common in elderly, HIV-negative patients from HHV8-endemic regions (Mediterranean) [7].
PEL 通常对 T/NK 细胞抗原呈阴性，但可能会出现 T 细胞标志物的异常表达。通过原位杂交，PEL 通常对 EBV 编码的小 RNA （EBER） 呈阳性，但通过 IHC 对 EBV 潜伏膜蛋白 1 （LMP1） 呈阴性。这可以用EBV病毒潜伏期来解释。它是致病病毒在细胞内休眠（潜伏）的能力，表示为病毒生命周期的溶原部分。EBV以三种模式之一表达其基因，称为潜伏期程序。EBV 可以表现出三种延迟程序之一：延迟 I、延迟 II 或延迟 III。每个潜伏期程序都会导致产生一组有限的、不同的病毒蛋白和病毒RNA。EB病毒编码的RNA（EBERs）：EBER1和EBER2在所有潜伏期均表达[6]，而LMP1仅在潜伏期2和3中表达，因此其检测EBV感染的敏感性较低。EBV阴性PEL常见于HHV8流行地区（地中海）的老年HIV阴性患者[7]。
Differential Diagnosis
鉴别诊断
Most common cavities involved by PEL: pleural, pericardial, and peritoneal [8-10].
PEL最常见的蛀牙：胸膜腔、心包腔和腹膜腔[8-10]。
It was thought that PEL can involve an artificial cavity related to the capsule of a breast implant [11] although it was described only in one case report without appropriate HHV8 staining and before recognition of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), which this case probably was presenting [12].
据认为，PEL可能涉及与乳房植入物包膜相关的人工空洞[11]，但仅在1例病例报告中进行了描述，但未进行适当的HHV8染色，并且在识别为乳房植入物相关间变性大细胞淋巴瘤（burimplant-associated interagile large cell lymphoma， BIA-ALCL）之前，该病例可能就诊于此[12]。
The prognosis is very unfavorable. Median survival is < 6 months. Rare cases have been reported that responded to chemotherapy and/or immune modulation [13].
预后非常不利。中位生存期< 6 个月。据报道，对化疗和/或免疫调节有反应的罕见病例[13]。
Flow Cytometry Utility
流式细胞术实用程序
The importance of utility of flow cytometry in establishing a diagnosis of PEL has been previously shown by others [14]. In the series by Galan et al. the authors described a case of PEL in an 88-year-old HIV-negative female with right-sided pleural effusion without significant lymphadenopathies or other effusions. The cytological study of the pleural fluid revealed a dense proliferation of large plasmablastic cells. A six-color multiparametric flow cytometry immunophenotyping study revealed 45% of large in size and high cellular complexity cells positive for CD45 (dim), CD38, CD138, CD30 and HLA-DR; and negative for CD19, CD20, cytoplasmatic CD79a, surface and cytoplasmic light chains Kappa and Lambda, CD3, CD4, CD5, CD7, CD8, CD28, CD56, CD81, and CD117.
In situ
hybridization for EBV-encoded small RNA was negative and immunohistochemistry for Kaposi sarcoma herpesvirus (HHV8) confirmed the diagnosis of PEL. These results in addition to the current case highlight the utility of flow cytometry in the diagnosis of lymphomas involving body cavities.
流式细胞术在确定PEL诊断中的重要性已有其他研究[14]。在 Galan 等人的系列研究中，作者描述了一例 88 岁的 HIV 阴性女性的 PEL 病例，该女性右侧胸腔积液没有明显的淋巴结肿大或其他胸腔积液。胸腔积液的细胞学研究显示大浆母细胞密集增殖。一项六色多参数流式细胞术免疫表型研究显示，45% 的大尺寸和高细胞复杂性细胞对 CD45 （dim）、CD38、CD138、CD30 和 HLA-DR 呈阳性;CD19、CD20、细胞质 CD79a、表面和细胞质轻链 Kappa 和 Lambda、CD3、CD4、CD5、CD7、CD8、CD28、CD56、CD81 和 CD117 阴性。EBV 编码的小 RNA 的原位杂交结果为阴性，卡波西肉瘤疱疹病毒 （HHV8） 的免疫组化证实了 PEL 的诊断。除了目前的病例外，这些结果还突出了流式细胞术在诊断涉及体腔的淋巴瘤中的效用。
In Summary
总结
PEL is associated with a proliferation of large B-cells which are positive for HHV8, CD45 (dim), CD30, CD38, and CD138 and negative for lineage defining B cell markers (CD19, CD20, and CD79a). Although PEL is a very rare lymphoma, it is important to consider it in patients with pleural, pericardial, and peritoneal effusions by sending a sample for cytological examination and flow cytometric immunophenotyping. Due to the absence of a mass lesion, cytology and flow cytometry are essential for establishing the diagnosis of PEL.
PEL 与大 B 细胞的增殖有关，这些 B 细胞对 HHV8、CD45 （dim）、CD30、CD38 和 CD138 呈阳性，对谱系定义 B 细胞标志物（CD19、CD20 和 CD79a）呈阴性。虽然 PEL 是一种非常罕见的淋巴瘤，但对于胸腔、心包和腹腔积液患者，通过送去样本进行细胞学检查和流式细胞术免疫表型分析来考虑 PEL 是很重要的。由于没有占位性病变，细胞学检查和流式细胞术对于确定 PEL 的诊断至关重要。
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引用
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This case was previously presented by authors as eCSI Case on International Clinical Cytometry Society website. For more information please follow:
https://www.cytometry.org/public/newsletters/eICCS-10-1/article7.php
