Follicular lymphoma (FL) is one of the most common types of lymphoma in the United States and Western Europe, accounting for ~20% of all cases. It is an indolent neoplasm of germinal center centrocytes and centroblasts with at least a partially follicular pattern in most cases, although rare cases have an entirely diffuse pattern. FL is a neoplasm of B-cell lineage and expresses germinal center-associated antigens. Immunophenotyping by flow cytometry (FC) is a fast and cost-effective technique that is widely used for diagnosis and follow-up of hematological disorders. CD45, also known as leukocyte common antigen, is a ubiquitous membrane glycoprotein that expressed with variable intensity on all nucleated cells of hematopoietic origin, except for erythroid cells, and a very small subset of normal plasma cells. In the process of B-cell maturation, B-cell precursors (hematogones) show relatively low levels of CD45. In comparison, mature B-cells and most B-cell lymphomas show a high surface CD45 intensity, although some large B-cell lymphomas and high-grade B-cell lymphomas, such as double/triple-hit lymphoma, can show a dim CD45 expression (Starr, Kwon, & Kallakury, 2019). Complete loss of CD45 expression in low-grade B-cell lymphomas including FL is rare (Wang, Xu, & Cui, 2015). We present a case of CD45-negative low-grade FL. This case posed a diagnostic challenge and highlighted the need for an alternative gating strategy for FC immunophenotypic analysis and integration of other diagnostic modalities.

A 62-year-old woman with a history of low-grade FL, initially diagnosed in 1997, status post chemotherapy using fludarabine, novantrone, and dexamethasone (FND) therapy, followed by relapsed disease in 1999, treated with six-cycles of FND followed by an allogeneic stem cell transplant in 2001, and chimerism studies showed 100% recipient cells at 1-year. The patient remained in remission for 18 years until recently when she presented with night sweats, bilateral axillary lymphadenopathy, and a cecal mass without hepatosplenomegaly, and she was diagnosed with left breast ductal carcinoma. The immunophenotype of the original FL was not available, except that the lymphoma cells were CD10-positive and expressed monotypic lambda-light chain. A complete blood count showed hemoglobin 9.8 g/dl (normal: 12.0–16.0 g/dl), platelets 482 K/μl (normal: 140–440 K/μl), and white blood cells 7.3 K/μl (normal: 4.0–11.0 K/μl) with a differential count of neutrophils 80%, lymphocytes 10%, monocytes 6%, eosinophils 2%, and basophils 1%. The serum lactate dehydrogenase level was 220 U/L (normal: 135–214 U/L). A staging bone marrow aspiration and biopsy showed no involvement by lymphoma. A right axillary lymph node needle core biopsy was performed. The concurrent lymph node fine needle aspirate specimen was collected in RPMI for flow cytometry immunophenotypic study. The sample was incubated with monoclonal antibodies for 10 min at 4°C, followed by erythrocyte lysis with ammonium chloride (PharmLyse, BD Biosciences, San Diego, CA) at room temperature for 10 min using a standard lyse/wash technique. Samples were acquired on FACSCanto II instruments (BD Biosciences). The following fluorochrome-tagged antibodies were used in a four-tube panel: CD3 (APC), CD4 (PerCP-Cy5.5), CD5 (PerCP-Cy5.5, V450), CD8 (FITC), CD10 (APC), CD 11c (FITC), CD14 (APC-H7), CD19 (PE-Cy7, PE), CD20 (APC-H7), CD22 (PerCP-Cy5.5), CD23 (PE), CD34 (PE-Cy7), CD38 (V450), CD43 (FITC), CD45 (V500), CD56 (V450), CD79b (APC), CD200 (PE), surface kappa (FITC), and surface lambda (PE). A total of 100,000 events were acquired for each tube. Data were analyzed using FCS Express software (De Novo Software, Los Angeles, CA). Lymphocytes were defined by CD45/SSC (side scatter).

In the current case, FC immunophenotypic analysis based on CD45 (clone HI30)/side scatter showed a lymphocyte gate composed of unremarkable CD3+ T-cells (64%), and CD19+ polytypic B-cells (36%). However, another CD19+ population was identified in the CD45-negative region. These cells, 21% as highlighted in black, were CD10 (weak+), CD19+, CD20+, CD22+, CD34−, CD45−, and surface lambda immunoglobulin light chain restricted (Figure 1(a)). Histologic examination of this specimen showed effacement of the lymph node architecture by a vaguely nodular neoplasm composed predominantly of small centrocytes and a few centroblasts (<15/HPF) consistent with grade 2 FL (Figure 1(b–d)). Immunohistochemical stains (Figure 1(e–j)) showed that the lymphoma cells were positive for CD10 (weak), CD20, CD79a, Bcl-6, Bcl-2, and Pax-5 (weak) and were negative for CD34 and TdT. The antibody specific for CD21 highlighted follicular dendritic cell meshworks associated with neoplastic nodules supporting follicular architecture. The proliferation index as assessed by Ki-67 was variable, but overall was 20–30%. Although it was challenging to assess due to the presence of many CD45+ background T-cells and normal B-cells, CD45 (clone 2B11 + PD7/26) confirmed that the lymphoma cells were negative. In contrast to previously reported cases of CD45- FL, FISH in the current case was negative for IGH-BCL2 fusion, which has been reported in ~15% of low-grade follicular lymphomas (Wang et al., 2015).

The absence of CD45 expression in this case of FL is unequivocal. CD45 was negative by both FC and immunohistochemistry analysis, using two different CD45 monoclonal antibodies, HI30 and 2B11 + PD7/26, respectively. These results also confirm that the lack of CD45 expression by the neoplastic cells is not epitope dependent. Interestingly, PAX-5 was extremely weak by immunohistochemistry, but other B-cell markers such as CD19, CD20, CD22, CD79a, and Bcl-6 were brightly expressed by the FL cells. Concurrent loss of CD45 and specific lineage associated B-cell antigens (in the absence of immunotherapy) has been reported in large B-cell lymphoma (Starr et al., 2019).

CD45 negative FL is rare and only two cases have been reported to date (Wang et al., 2015). FC analysis for potential lymphoma cases often uses a lymphocyte gate, based on CD45/SSC, but such a gate would have missed these cells. The CD45 negative population might have been considered as erythrocytes, platelets or debris, and therefore overlooked completely. Furthermore, this case had many polytypic B-cells in the lymphocyte gate, further distracting attention from the CD45-negative cell population. Therefore, it is advisable to assess the B-cell markers in unselected cells before moving to a specific gate. This analysis strategy is pivotal in the era of targeted therapy when neoplastic cells may lose certain markers following treatment.

In summary, the absence of CD45 in the case we report was a diagnostic challenge and raised a differential diagnosis that included B-lymphoblastic lymphoma or B-cell lymphoma undergoing dedifferentiation. These possibilities were excluded based on morphologic features, absence of CD34 and TdT, and the presence of many mature B-cell markers including CD10 (weak), CD20, CD79a, Bcl-6, and lambda light chain restriction. Awareness that FL can be CD45 negative is needed to avoid potential misdiagnosis.

滤泡性淋巴瘤 (FL) 是美国和西欧最常见的淋巴瘤类型之一，约占所有病例的 20%。它是一种由生发中心中心细胞和中心母细胞组成的惰性肿瘤，在大多数情况下至少具有部分滤泡型，但极少数情况下具有完全弥散型。FL 是 B 细胞谱系的肿瘤，表达生发中心相关抗原。通过流式细胞术 (FC) 进行免疫表型分析是一种快速且具有成本效益的技术，广泛用于血液病的诊断和随访。CD45，也称为白细胞共同抗原，是一种普遍存在的膜糖蛋白，在除红细胞和一小部分正常浆细胞外，在所有造血来源的有核细胞上以不同强度表达。在 B 细胞成熟的过程中，B 细胞前体（血细胞）显示出相对较低水平的 CD45。相比之下，成熟 B 细胞和大多数 B 细胞淋巴瘤表现出较高的表面 CD45 强度，尽管一些大 B 细胞淋巴瘤和高级别 B 细胞淋巴瘤，例如双打击/三打击淋巴瘤，可以显示较暗的 CD45表达式（Starr、Kwon 和 Kalakury， 2019 年）。在包括 FL 在内的低级别 B 细胞淋巴瘤中，CD45 表达完全丧失是罕见的（Wang、Xu 和 Cui，  2015 年）。我们提出一例 CD45 阴性低级别 FL 病例。该病例提出了诊断挑战，并强调需要为 FC 免疫表型分析和其他诊断方式整合采用替代门控策略。

一名 62 岁女性，有低级别 FL 病史，最初于 1997 年诊断，化疗后状态使用氟达拉滨、诺凡酮和地塞米松 (FND) 治疗，随后于 1999 年复发，接受 6 个周期的 FND 治疗随后在 2001 年进行了同种异体干细胞移植，嵌合体研究显示 1 年时受体细胞达到 100%。患者在缓解期长达 18 年，直到最近出现盗汗、双侧腋窝淋巴结肿大和无肝脾肿大的盲肠肿块，并被诊断为左乳导管癌。原始 FL 的免疫表型不可用，除了淋巴瘤细胞为 CD10 阳性并表达单型 λ-轻链。全血细胞计数显示血红蛋白 9.8 g/dl（正常：12.0–16.0 g/dl），血小板 482 K/μl（正常：140–440 K/μl)，白细胞 7.3 K/μl（正常：4.0–11.0 K/μl），中性粒细胞 80%、淋巴细胞 10%、单核细胞 6%、嗜酸性粒细胞 2% 和嗜碱性粒细胞 1 的分类计数%。血清乳酸脱氢酶水平为 220 U/L（正常：135-214 U/L）。分期骨髓抽吸和活检显示没有淋巴瘤受累。进行了右腋窝淋巴结针芯活检。在RPMI中收集并发淋巴结细针抽吸标本用于流式细胞术免疫表型研究。将样品与单克隆抗体在 4°C 下孵育 10 分钟，然后使用标准裂解/洗涤技术在室温下用氯化铵 (PharmLyse, BD Biosciences, San Diego, CA) 裂解红细胞 10 分钟。样品是在 FACSCanto II 仪器（BD Biosciences）上采集的。在四管面板中使用以下荧光标记抗体：CD3 (APC)、CD4 (PerCP-Cy5.5)、CD5 (PerCP-Cy5.5、V450)、CD8 (FITC)、CD10 (APC)、 CD 11c (FITC)、CD14 (APC-H7)、CD19 (PE-Cy7、PE)、CD20 (APC-H7)、CD22 (PerCP-Cy5.5)、CD23 (PE)、CD34 (PE-Cy7)、 CD38 (V450)、CD43 (FITC)、CD45 (V500)、CD56 (V450)、CD79b (APC)、CD200 (PE)、表面 kappa (FITC) 和表面 lambda (PE)。每个管总共获得了 100,000 个事件。使用 FCS Express 软件 (De Novo Software, Los Angeles, CA) 分析数据。淋巴细胞由 CD45/SSC（侧向散射）定义。CD79b (APC)、CD200 (PE)、表面 kappa (FITC) 和表面 lambda (PE)。每个管总共获得了 100,000 个事件。使用 FCS Express 软件 (De Novo Software, Los Angeles, CA) 分析数据。淋巴细胞由 CD45/SSC（侧向散射）定义。CD79b (APC)、CD200 (PE)、表面 kappa (FITC) 和表面 lambda (PE)。每个管总共获得了 100,000 个事件。使用 FCS Express 软件 (De Novo Software, Los Angeles, CA) 分析数据。淋巴细胞由 CD45/SSC（侧向散射）定义。

在当前病例中，基于 CD45（克隆 HI30）/侧向散射的 FC 免疫表型分析显示淋巴细胞门由不显着的 CD3+ T 细胞（64%）和 CD19+ 多型 B 细胞（36%）组成。然而，在 CD45 阴性区域发现了另一个 CD19+ 群体。这些细胞（21% 以黑色突出显示）是 CD10（弱+）、CD19+、CD20+、CD22+、CD34-、CD45- 和表面 λ 免疫球蛋白轻链受限（图 1(a)）。该标本的组织学检查显示淋巴结结构被主要由小中心细胞和少数中心母细胞（<15/HPF）组成的模糊结节性肿瘤消除，符合 2 级 FL（图 1（b-d））。免疫组化染色（图 1（e-j））显示淋巴瘤细胞对 CD10（弱）、CD20、CD79a、Bcl-6、Bcl-2、和 Pax-5（弱），CD34 和 TdT 呈阴性。CD21 特异性抗体突出了与支持滤泡结构的肿瘤结节相关的滤泡树突细胞网。Ki-67 评估的增殖指数是可变的，但总体为 20-30%。尽管由于存在许多 CD45+ 背景 T 细胞和正常 B 细胞而难以评估，但 CD45（克隆 2B11 + PD7/26）证实淋巴瘤细胞是阴性的。与先前报道的 CD45-FL 病例相比，当前病例中的 FISH 为阴性 尽管由于存在许多 CD45+ 背景 T 细胞和正常 B 细胞而难以评估，但 CD45（克隆 2B11 + PD7/26）证实淋巴瘤细胞是阴性的。与先前报道的 CD45-FL 病例相比，当前病例中的 FISH 为阴性 尽管由于存在许多 CD45+ 背景 T 细胞和正常 B 细胞而难以评估，但 CD45（克隆 2B11 + PD7/26）证实淋巴瘤细胞是阴性的。与先前报道的 CD45-FL 病例相比，当前病例中的 FISH 为阴性IGH-BCL2融合，已在约 15% 的低级别滤泡性淋巴瘤中报道（Wang 等人，  2015 年）。

在这种 FL 病例中 CD45 表达的缺失是明确的。分别使用两种不同的 CD45 单克隆抗体 HI30 和 2B11 + PD7/26 进行 FC 和免疫组织化学分析，CD45 均为阴性。这些结果还证实肿瘤细胞缺乏CD45表达不是表位依赖性的。有趣的是，PAX-5 在免疫组织化学中非常弱，但其他 B 细胞标志物，如 CD19、CD20、CD22、CD79a 和 Bcl-6 在 FL 细胞中明显表达。在大 B 细胞淋巴瘤中报告了 CD45 和特定谱系相关 B 细胞抗原（在没有免疫治疗的情况下）的同时丢失（Starr 等人，  2019 年）。

CD45 阴性 FL 很少见，迄今为止仅报告了两例（Wang 等人，  2015 年）。潜在淋巴瘤病例的 FC 分析通常使用基于 CD45/SSC 的淋巴细胞门，但这样的门会漏掉这些细胞。CD45 阴性人群可能被认为是红细胞、血小板或碎片，因此完全被忽视了。此外，该病例的淋巴细胞门中有许多多型 B 细胞，进一步分散了对 CD45 阴性细胞群的注意力。因此，建议在移动到特定门之前评估未选择细胞中的 B 细胞标记。这种分析策略在靶向治疗时代至关重要，因为肿瘤细胞在治疗后可能会失去某些标志物。

总之，我们报告的病例中缺乏 CD45 是一个诊断挑战，并提出了包括 B 淋巴细胞淋巴瘤或正在去分化的 B 细胞淋巴瘤的鉴别诊断。基于形态学特征、缺乏 CD34 和 TdT 以及存在许多成熟 B 细胞标志物，包括 CD10（弱）、CD20、CD79a、Bcl-6 和 lambda 轻链限制，这些可能性被排除在外。需要意识到 FL 可能是 CD45 阴性以避免潜在的误诊。