Extended T-cell immunophenotyping is used in the evaluation for both primary and secondary immune disorders. The distinction between naïve and memory T-cells includes the expression patterns of CD45RO and CD45RA as well as other markers. CD45 isoform expression depends on the stage of T-cell maturation, activation, and differentiation. Naïve T-cells express the isoform CD45RA and the isoform CD45RO is primarily found on primed/memory T-cells. By flow cytometric assays using fluorochrome conjugated antibodies against CD45RA and CD45RO, there is a typical staining pattern seen within the CD4+ T lymphocyte and CD8+ T lymphocyte compartments (see reference cases in Figure 1). A well-studied polymorphism in the extracellular domain of CD45 is the C77G point mutation in a splice silencer region of exon 4, resulting in memory/effector lymphocytes of C77G carriers expressing both CD45RA and CD45RO. A characteristic CD45RA/CD45RO staining pattern of lymphocytes from C77G individuals has been reported (Tchilian et al., 2006).

In a retrospective study approved by our institutional review board (IRB #13310), we identified six patients with the characteristic CD45RA/CD45RO staining pattern out of 309 expanded lymphocyte immunophenotyping panels from 273 patients (M:F ratio of 0.9:1, average age 18 years, range 0–83) performed from January 2017 through August 2020. The patients were identified by natural language search and/or visual inspection of flow cytometry plots. Pertinent information was obtained from review of the electronic medical record. Five of the 6 patients had genotyping performed by Invitae (San Francisco, CA) as part of their clinical work-up. The presence or absence of the CD45 C77G polymorphism was included in the supplemental variant call file and denoted as NM_002838.4: PTPRC c.177C>G silent variant.

Flow cytometric analysis was performed using a 6-color FACSCanto II (Becton Dickinson Biosciences, BD, San Jose, CA) analyzer using the following antibodies (all from BD Biosciences): CD45RO (FITC), CD45RA (PE), CD8 (PerCP-Cy5.5), CD4 (PE-Cy7), CD3 (APC), and CD45 (APC-H7). The peripheral blood samples were processed by a stain-lyse method using a Beckman Coulter Whole Blood Lyse Kit (Immuno-Lyse followed by fixative). Data was analyzed in FACSDiva software (Becton Dickinson). Doublets were excluded by a forward scatter area by height (FSC-A by FSC-H) plot and debris were excluded by a forward by side-scatter plot. CD45 by side-scatter (SSC-A) was used to establish a lymphocyte gate. CD3 positive CD4 positive lymphocytes or CD3 positive CD8 positive lymphocytes were used as the input gate for CD45RA by CD45RO plots. Normalized mean fluorescence intensity (MFI) values were calculated for the CD45RO populations relative to non-T lymphocytes. Absolute values for lymphocyte subsets were calculated from the separately reported white blood cell differential count. A subset of the patients had the same samples re-run with additional wash steps with nearly identical results and all of the patients had repeat flow cytometry studies from different draw dates between 1 month and 15 months apart showing a similar CD45RA by CD45RO maturation pattern (data not shown).

Patient details for the six individuals with the characteristic CD45RA/CD45RO staining pattern are presented in Table 1. Five of the patients were under the age of 10 (average age for this subgroup 4 years old, range 6 months to 9 years), and one was an adult. Five of the patients presented with an infectious history, prompting evaluation in the Allergy/Immunology Clinic and laboratory evaluation with immunoglobulins, flow cytometry and genetic testing. Three had a history of allergic diseases, and one (the adult) had a history of autoimmune disease. Work-up led to formal diagnosis of primary immunodeficiency in only one patient (Case 4—specific antibody deficiency). All five patients tested were heterozygous for the CD45R C77G polymorphism.

Flow cytometry scattergrams showing the characteristic CD45RA by CD45RO maturation pattern are shown in Figure 1. There is acquisition of CD45RO expression with retained CD45RA expression of variable intensity, resulting in a “double-positive” population with a curved, or upside-down “U,” shape. In the six patient cohort, the CD45RO MFI relative to non-T lymphocytes ranged from 19 to 65 (average 51) in the CD4+ T cells and 11 to 47 (average 33) in the CD8+ T cells. The corresponding relative MFIs for the reference cases were 48 and 66 (for the CD4+ T cells) and 35 and 59 (for the CD8+ T cells). When quantitative assessment of the absolute value for the CD4 + CD45RO+ T-cell population was performed using the sum of the single positive CD45RA-CD45RO+ and the double positive CD45RA + CD45RO+ populations (using the right upper quadrant values in the plots shown), four patients fell within normal limits for age and two had slightly low levels. Regarding the patients with slightly low levels, the absolute value for the sum of the single positive CD45RA-CD45RO+ population and the double positive population in Case 4 was 172 cells per microliter (reference range for age 230–630) and in Case 5 was 179 (reference range for age 220–660).

The characteristic CD45RA/CD45RO curved pattern seen with the C77G polymorphism is different than either the CD45RA/CD45RO dual positive population expressing both isoforms in low density and falling along the linear negative slope line from CD45RA positive to CD45RO positive and the distinct CD45RA/CD45RO dual positive population expressing both isoforms with high density (“Ddull” and “Dbright” populations respectively) as described in the 1996 paper by Hamann, Baars, Hooibrink, and van Lier (1996).

Recognized since the mid-1990s, the exon 4 C77G polymorphism has a low mutation frequency, between 0% and 3.5% in studies of healthy control populations from Europe and the United States (Tchilian et al., 2006). All patients in our cohort identified as White/Caucasian; additional information on ancestry was not available to us. An increased frequency of the polymorphism has been reported in certain disease states including systemic sclerosis, hepatitis C, and autoimmune hepatitis. In our cohort, the characteristic CD45RA by CD45RO maturation pattern was seen in 2% of patients (6/273), which is concordant with the reported mutation frequency for the polymorphism in a healthy control population.

The aim of this letter to the editor is to bring the pattern of CD45RA/CD45RO staining associated with the C77G polymorphism to the awareness of the general flow cytometry community. Such an awareness could prevent unnecessary repeat flow cytometric studies which may be performed to confirm the immunophenotypic pattern. We recommend a description of the observed immunophenotypic pattern within the flow report with a reference to the association of the phenotypic pattern with the C77G polymorphism. In our opinion, for the semi-quantitative evaluation of memory/primed CD45RO T-lymphocytes in patients with this maturation pattern, the double positive CD45RA + CD45RO+ population should be used to compare to published reference ranges for the CD45RA-CD45RO+ population.

扩展 T 细胞免疫表型分析用于评估原发性和继发性免疫疾病。幼稚 T 细胞和记忆 T 细胞之间的区别包括 CD45RO 和 CD45RA 以及其他标志物的表达模式。CD45 异构体的表达取决于 T 细胞成熟、激活和分化的阶段。幼稚 T 细胞表达同种型 CD45RA，而同种型 CD45RO 主要存在于致敏/记忆 T 细胞上。通过使用针对 CD45RA 和 CD45RO 的荧光染料偶联抗体的流式细胞仪检测，在 CD4+ T 淋巴细胞和 CD8+ T 淋巴细胞隔室中可以看到典型的染色模式（参见图 1 中的参考案例）。CD45 细胞外结构域中一个经过充分研究的多态性是外显子 4 剪接沉默区的 C77G 点突变，导致 C77G 携带者的记忆/效应淋巴细胞表达 CD45RA 和 CD45RO。已经报道了来自 C77G 个体的淋巴细胞的特征性 CD45RA/CD45RO 染色模式（Tchilian 等人， 2006 年）。

在我们的机构审查委员会 (IRB #13310) 批准的一项回顾性研究中，我们从 273 名患者的 309 个扩大淋巴细胞免疫表型组中确定了 6 名具有特征性 CD45RA/CD45RO 染色模式的患者（M:F 比为 0.9:1，平均年龄18 岁，范围 0-83）从 2017 年 1 月到 2020 年 8 月进行。通过自然语言搜索和/或流式细胞仪图的目视检查来识别患者。相关信息来自对电子病历的审查。作为临床检查的一部分，6 名患者中有 5 名由 Invitae（加利福尼亚州旧金山）进行了基因分型。CD45 C77G 多态性的存在或不存在包含在补充变体调用文件中，并表示为 NM_002838.4：PTPRC c.177C>G 沉默变体。

使用 6 色 FACSCanto II (Becton Dickinson Biosciences, BD, San Jose, CA) 分析仪使用以下抗体（均来自 BD Biosciences）进行流式细胞术分析：CD45RO (FITC)、CD45RA (PE)、CD8 (PerCP- Cy5.5)、CD4 (PE-Cy7)、CD3 (APC) 和 CD45 (APC-H7)。使用 Beckman Coulter Whole Blood Lyse Kit（Immuno-Lyse 然后固定剂）通过染色裂解方法处理外周血样品。在 FACSDiva 软件 (Becton Dickinson) 中分析数据。双峰被高度前向散射面积（FSC-A by FSC-H）图排除，碎片被前向侧向散射图排除。侧向散射的 CD45 (SSC-A) 用于建立淋巴细胞门。CD3阳性CD4阳性淋巴细胞或CD3阳性CD8阳性淋巴细胞通过CD45RO图用作CD45RA的输入门。计算 CD45RO 群体相对于非 T 淋巴细胞的归一化平均荧光强度 (MFI) 值。淋巴细胞亚群的绝对值是从单独报告的白细胞分类计数中计算出来的。一部分患者使用额外的洗涤步骤重新运行相同的样本，结果几乎相同，并且所有患者在相隔 1 个月至 15 个月的不同抽取日期重复流式细胞术研究，显示 CD45RA 与 CD45RO 成熟模式相似（数据未显示）。

表 1 列出了 6 名具有特征性 CD45RA/CD45RO 染色模式的患者的详细信息。其中 5 名患者年龄在 10 岁以下（该亚组的平均年龄为 4 岁，范围为 6 个月至 9 岁），另外一名是一个成年人。其中五名患者有感染史，促使过敏/免疫学诊所进行评估，并使用免疫球蛋白、流式细胞术和基因检测进行实验室评估。三人有过敏性疾病史，一人（成人）有自身免疫病史。仅一名患者的检查导致正式诊断为原发性免疫缺陷（病例 4——特异性抗体缺陷）。测试的所有五名患者都是 CD45R C77G 多态性的杂合子。

通过 CD45RO 成熟模式显示特征性 CD45RA 的流式细胞术散点图如图 1 所示。获得了 CD45RO 表达，保留了不同强度的 CD45RA 表达，导致“双阳性”群体具有弯曲或倒置的“U” ，“ 形状。在 6 名患者队列中，CD4+ T 细胞相对于非 T 淋巴细胞的 CD45RO MFI 范围为 19 至 65（平均 51），CD8+ T 细胞为 11 至 47（平均 33）。参考病例的相应相对 MFI 分别为 48 和 66（对于 CD4+ T 细胞）和 35 和 59（对于 CD8+ T 细胞）。当使用单阳性 CD45RA-CD45RO+ 和双阳性 CD45RA + CD45RO+ 群体的总和（使用所示图中的右上象限值）对 CD4 + CD45RO+ T 细胞群体的绝对值进行定量评估时，四个患者的年龄在正常范围内，其中两名患者的水平略低。对于水平略低的患者，病例 4 中单阳性 CD45RA-CD45RO+ 群体和双阳性群体之和的绝对值为 172 个细胞/微升（参考范围为 230-630 岁），病例 5 为 179 （220-660 岁的参考范围）。

C77G 多态性所见的特征性 CD45RA/CD45RO 曲线模式不同于以低密度表达两种同种型的 CD45RA/CD45RO 双阳性群体，并沿着从 CD45RA 阳性到 CD45RO 阳性的线性负斜率线下降，以及不同的 CD45RA/CD45RO 双阳性群体如 Hamann、Baars、Hooibrink 和 van Lier（ 1996 年）在 1996 年的论文中所述，表达两种高密度亚型的阳性群体（分别为“Ddull”和“Dbright”群体）。

自 1990 年代中期以来，外显子 4 C77G 多态性的突变频率较低，在欧洲和美国的健康对照人群研究中为 0% 至 3.5%（Tchilian 等人，  2006 年）。我们队列中的所有患者都被确定为白人/高加索人；我们无法获得有关血统的其他信息。据报道，在某些疾病状态中多态性的频率增加，包括系统性硬化症、丙型肝炎和自身免疫性肝炎。在我们的队列中，2% 的患者 (6/273) 观察到 CD45RO 成熟模式的特征性 CD45RA，这与健康对照人群中报道的多态性突变频率一致。

这封致编辑的信旨在让一般流式细胞术界了解与 C77G 多态性相关的 CD45RA/CD45RO 染色模式。这种意识可以防止不必要的重复流式细胞术研究，这些研究可以用来确认免疫表型模式。我们建议在流量报告中描述观察到的免疫表型模式，并参考表型模式与 C77G 多态性的关联。我们认为，对于具有这种成熟模式的患者的记忆/致敏 CD45RO T 淋巴细胞的半定量评估，应使用双阳性 CD45RA + CD45RO+ 群体与公布的 CD45RA-CD45RO+ 群体参考范围进行比较。