Cytometry Part B: Clinical Cytometry ( IF 3.4 ) Pub Date : 2020-10-14 , DOI: 10.1002/cyto.b.21959 Daniel Mazza Matos 1
Steric hindrance (SH) is a long-term known, albeit often forgotten, phenomenon in which the reduction, or less frequently the absence, of detectable fluorescence signals occurs because one monoclonal antibody hinders the binding of another one to its respective target antigen, when the reagents are used conjointly. The mechanism underlying SH is at least partially related to the presence of multiple monoclonal antibodies targeting the same macromolecular complex. Therefore, with the aim to avoid SH, each monoclonal antibody combination to be implemented in flow cytometry's routine should be beforehand tested against experiments of single-color staining. This procedure is part of the validation's studies of antibody and fluorochrome optimization recommended by the International Council for Standardization of Haematology (ICSH) and the International Clinical Cytometry Society (ICCS).
Notwithstanding the checks for SH, some unexpected behaviors of monoclonal antibodies can be seen in ordinary samples, even when the procedure of staining is part of an already validated protocol used on daily bases in a flow cytometry laboratory.
In our own experience, during a period of 9 years (2009–2017) performing systematic flow cytometry assays, we found only one patient with the a diagnosis of chronic lymphocytic leukemia (CLL), in which the peripheral blood sample showed the presence of the SH phenomenon with the variable combination of the monoclonal antibodies anti-kappa (FITC), anti-lambda (PE), anti-CD19 (PE-Cy5) and anti-CD20 (PE-Cy5).
The patient was a 53-year-old man who, in 2014, presented with a white blood cell count of 76.5 × 109/L and a lymphocytes count of 52.8 × 109/L. The review of peripheral blood smear slide showed the presence of small to medium-sized mature-looking lymphoid cells.
For flow cytometry procedure, the lyse-wash technique was employed in a sample of peripheral blood collected in EDTA. Briefly, 1.0 × 109 leucocytes/L were incubated with 5 μl of each monoclonal antibody. The following immunophenotypic panel, that has been employed in our facility for the analysis of all suspected cases of mature B-cell neoplasms, was used: tube 1: non-staining (control tube); tube 2: CD19 (FITC), CD5 (PE), CD45 (PE-Cy5); tube 3: CD2 (FITC), CD23 (PE); tube 4: kappa (FITC), lambda (PE), CD19 (PE-Cy5); tube 5: FMC7 (FITC), CD79 (PE); tube 6: CD10 (FITC), CD11c (PE), CD38 (PE-Cy5); tube 7: CD103 (FITC), CD20 (PE); tube 8: IgM (FITC), CD200 (PE), CD19 (PE-Cy5); tube 9: CD8 (FITC), CD4 (PE), CD3 (PE-Cy5).11 Immunophenotypic panel (target cells): tube (1): non-staining lymphocytes; tube (2): mature B-lymphocytes and CD5+ B-lymphocytes; tube (3): T-lymphocytes and CD23+ B-lymphocytes; tube (4): mature B-lymphocytes; tube (5): B-lymphocytes; tube (6): germinal center B- lymphocytes, terminally differentiated B- lymphocytes and other subsets of B-lymphocytes; tube (7): T-lymphocytes and abnormal B-cells (hairy cell leukemia); tube (8): mature B-lymphocytes and other subset of B-lymphocytes; tube (9): subsets of T-lymphocytes and rare CD8+ CLL. Of note: for a discussion about the uncommon inclusion of tube 9 (with only lineage T-cell markers) in an immunophenotypic panel specific for suspected cases of mature B-cell neoplasms, see: Matos DM. CD8 Antigen Expression in Chronic Lymphocytic Leukemia: Does it Have any Relevant Meaning? Cytometry Part B (Clinical Cytometry) 96B:96–98 (2019).
As traditionally recommended, the tubes containing antibodies against immunoglobulins were washed three times in PBS at room temperature. Of note, the clones of anti-kappa (FITC), anti-lambda (PE) and anti-CD19 (PE-Cy5) were G20-193 (isotype: mouse IgG1, k), JDC-12 (isotype: mouse IgG1, k) and HIB19 (isotype: mouse IgG1, k), respectively. All monoclonal antibodies were purchased from Becton Dickinson (San Jose, CA, USA), except anti-IgM (FITC), which were purchased from Dako (Carpinteria, CA, USA). A total of 10.000 cells per tube were acquired in a three-color COULTER EPICS XL-MCL flow cytometer (Beckman-Coulter).
Our first immunophenotypic analysis showed the presence of 75% of B-cells (CD19+) characterized by the co-expression of the CD5 antigen. The B-cells also exhibited positivity for the antigens CD11c, CD20low, CD23, CD45high, CD200, FMC7 and surface immunoglobulin IgMlow subtype. The B-cells were negative for the antigens CD2, CD3, CD4, CD8, CD10, CD25, CD38, CD79b and CD103. However, to our surprise, we were not able to detect either kappa or lambda expression on the surface of B-cells of tube 4 and, therefore, we were not initially able to confirm the light-chain restriction of them (Figure 1(a)).
We initially hypothesized to be dealing with: (a) a rare case of CLL characterized by the absence of surface light-chain immunoglobulin, that has previously been documented or, (b) the failure to detect the immunoglobulin light-chains as a consequence of anti-kappa and/or anti-lambda performance problems. However, the presence of surface IgM subtype heavy-chain on B-cells made us consider the alternative possibility that problems could be occurring with the set combination of reagents in the tube 4. Thus, we considered that the anti-CD19 (PE-Cy5) could be interfering with the binding of the anti-kappa (FITC) and/or the anti-lambda (PE) by means of SH. Alternatively, as a fourth hypothesis, we judged that fluorescence quenching could be the reason for the absence of kappa and/or lambda signals. So, we set about investigating the phenomenon: we initially performed another staining of tube 4, but now changing anti-CD19 (PE-Cy5) for anti-CD20 (PE-Cy5), clone 2H7, isotype mouse (C57BL/6 IgG2b), k. Yet, the phenomenon occurred anew and, thus, neither kappa nor lambda signals could be detected (data not shown).
We then tried an alternative approaching (the same blood specimen was used): we performed a new staining of tube 4, but this time adding sequentially the monoclonal antibodies: first, anti-kappa (FITC) and anti-lambda (PE) were added conjointly to the tube and, after 10 min of incubation, we finally added the anti-CD19 (PE-Cy5). The result is shown in Figure 1(b). The B-cells clearly exhibited lambdalow light-chain restriction. Therefore, our suspicion of SH was reinforced, to the detriment of the fluorescence quenching hypothesis, due to the fact that we would not expect that the sequential incubation procedure, which gave primacy of binding to anti-lambda (PE), would solve the problem of fluorescence quenching, because the possible energy transfer between PE and the tandem fluorochrome PE-Cy5 would still continue to occur. To confirm SH, we performed a new incubation, but this time without the anti-CD19 (PE-Cy5). The result is shown in Figure 1(C). Again, the B-cells exhibited lambdalow light-chain restriction.
Finally, to make the whole case more intriguing, we performed a minimal residual disease analysis of the bone marrow of the same patient, 17 months after the initial diagnosis. This time, tube 4 consisted of the set of monoclonal antibodies employed at diagnosis, namely, anti-kappa (FITC), anti-lambda (PE) and anti-CD19 (PE-Cy5), with the addition of anti-CD5 (APC). We found 0.4% of B-cells with the phenotype of CLL (CD5+, CD20low, CD43+, CD79b-). The CLL-cells exhibited lambdalow light-chain restriction. This time, curiously, no SH was seen.
Steric hindrance is a rare phenomenon mainly associated with the reduction of fluorescence intensity which, in the context where the level of expression of an antigen has clinical significance, could lead to an erroneous interpretation of flow cytometry results. In extreme cases, like the one that our report portrays, more than a “mere” decrease in fluorescence intensity, there is a total absence of the fluorescence signal.
In our case, we suggest that a possible explanation for the SH phenomenon was related to the spatial proximity of CD19 protein to the transmembrane immunoglobulin molecule of the B-cell receptor (BCR). However, it remains to be explained the reason why we found only a single case of SH in our series of immunophenotypic studies. In fact, between 2009 and 2017, we employed our standard immunophenotypic panel, specifically for the analysis of suspected cases of mature B-cell neoplasms, 62 times22 From 2015 onwards, we started working with a BD FACSCalibur 4-color flow cytometer and, thus, we modified tube 4, in such a way as to add the monoclonal antibody anti-CD5 (APC) on it. Thus, tube 4: kappa (FITC), lambda (PE), CD19 (PE-Cy5), CD5 (APC). Eventually, we customized the tube 4, changing CD19 (PE-Cy5) for CD19 (PerCP).
: in none of them the tube 4 showed the SH phenomenon. Additionally, in 101 times, the tube 4 was used as part of other immunophenotypic panels: again, we found no additional SH cases.
We do not have a complete explanation for the rarity of the phenomenon, even when the same combination of monoclonal antibodies is used so often, unless we assume that factors related to the distribution of the CD19 protein and the BCR in the membrane of B-cells of particular samples could contribute to SH. In any event, beyond the well-known influence of the molecular weights of the competing fluorochromes on the triggering of SH (with the combined use of heavy molecules, such as, for instance, PE and PE-Cy5, being more powerful to generate the phenomenon) (Table 1), the mechanisms involved in the emergence of SH are not plainly understood, being even possible that the degree of density of microvilli on cell surface could contribute to preventing the binding of the reagent involved (Wang et al., 2014). In this sense, other techniques could be of help to uncover the mechanism responsible to the appearance of SH. For example, Scanning Electron Microscopy – though be a technique not available for many centers around the world – could show the presence of artifacts on the surface of cells where the SH phenomenon has been documented. However, with the aim to clarify the molecular mechanisms that govern the interaction between antigen and antibody in SH, Atomic Force Microscopy could be more useful, whereas this technique allows the determination of the adhesion force, between antigen and antibody, as the parameter to be measured.33 The very preliminary idea would be to functionalize the tip of the Atomic Force Microscope with the antigen to be tested on the lymphocyte’s membrane and, then, measure the adhesion force between antigen and antibody. Of note, to use Atomic Force Microscopy, the cells need to be attached on a solid surface (a solid surface coated with fibrin, for example). I am very grateful to Professor Nicole Jaffrezic-Renault (Emeritus Research Director in CNRS, Institute of Analytical Sciences, University of Lyon, France) for our brief, but very stimulating and rich, discussion about the possibilities of using Atomic Force Microscopy for clarifying some underlying mechanisms responsible for the phenomenon of SH.
Fluorophorescc Sources: Thermo Fisher Scientific (personal communication), Biolegend (personal communication) and https://www.biolegend.com/en-us/fluorophore-families and Cell Signaling Technology (personal communication). |
Molecular weight (Daltons) | Excitation laser line (nm)aa Excitation Laser Line: (i) 405 nm = violet laser; (ii) 407 nm = krypton laser; (iii) 488 nm = blue laser (argon); (iv) 532 nm = green diode laser; (v) 561 nm = yellow-green laser; (vi) 594 nm = red-orange laser; (vii) 633 nm = red laser (HeNe); (viii) 640 nm = red laser. |
---|---|---|
Simple organic fluorophores | ||
FITC | 389 Da | 488 nm |
Pacific blue™ | 406 Da | 405 nm |
Cascade blue | 596 Da | 405 nm or 407 nm |
Violet Fluor 450 | 600 Da | 405 nm |
Texas red | 625 Da | 561 nm or 594 nm |
Alexa Fluor® 488 | 643 Da | 488 nm |
DyLight 405 | 793 Da | 405 nm |
Alexa Fluor® 594 | 820 Da | 561 nm or 594 nm |
Red Fluor 910 | 900 Da | 633 nm |
DyLight 488 | 1.0 kDa | 488 nm |
DyLight 650 | 1.1 kDa | 594 nm or 633 nm |
Alexa Fluor® 660 | 1.1 kDa | 633 nm |
Spark blue™ 550 | 1.2 kDa | 488 nm |
Alexa Fluor® 647 | 1.3 kDa | 633 nm or 640 nm |
Alexa Fluor® 700 | 1.4 kDa | 633 nm or 640 nm |
Spark NIR™ 685 | 3.1 kDa | 633 nm |
Protein-based fluorophores | ||
PerCP | 35 kDa | 488 nm |
PerCP-eFluor 710bb Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”. |
≅ 36–37 kDa | 488 nm |
PerCP-Cyanine5.5bb Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”. |
≅ 37 kDa | 488 nm |
APC | 105 kDa | 633 nm or 640 nm |
AmCyan | 108 kDa | 405 nm |
APC-Cyanine7bb Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”. |
109 kDa | 633 nm or 640 nm |
APC-fire™ 750bb Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”. |
110 kDa | 633 nm or 640 nm |
PE | 240 kDa | 488 nm or 532 nm or 561 nm |
PE-Cyanine5bb Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”. |
≅ 242 kDa | 488 nm or 532 nm or 561 nm |
PE-eFluor 610# | ≅ 242–245 kDa | 488 nm or 532 nm or 561 nm |
PE-Cyanine7bb Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”. |
245 kDa | 488 nm or 532 nm or 561 nm |
PE-dazzle™ 594bb Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”. |
245 kDa | 488 nm or 532 nm or 561 nm |
PE-fire™ 640bb Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”. |
260 kDa | 561 nm |
Organic polymers | ||
Brilliant violet 421™ | 60–80 kDa | 405 nm |
Brilliant violet 510™ | 60–80 kDa | 405 nm |
Brilliant violet 605™ | 60–80 kDa | 405 nm |
Brilliant violet 711™ | 60–80 kDa | 405 nm |
Kiravia Multimers | ||
KIRAVIA blue 520™ | 8 kDa | 488 nm |
- a Excitation Laser Line: (i) 405 nm = violet laser; (ii) 407 nm = krypton laser; (iii) 488 nm = blue laser (argon); (iv) 532 nm = green diode laser; (v) 561 nm = yellow-green laser; (vi) 594 nm = red-orange laser; (vii) 633 nm = red laser (HeNe); (viii) 640 nm = red laser.
- b Fluorophore:protein (F/P) ratio: The ratio of the PE, PerCP and APC molecules to small organic dyes (eFluor, Cyanine5, Cyanine5.5, Cyanine 7, Dazzle 594 and Fire 640) is often dependent on the conjugation process. Thus, it varies and can be “lot-specific” and “vendor-specific”.
- c Sources: Thermo Fisher Scientific (personal communication), Biolegend (personal communication) and https://www.biolegend.com/en-us/fluorophore-families and Cell Signaling Technology (personal communication).
Having said that, based on the results of our experiments performed to elucidate the staining inconsistences of tube 4, we can assert that SH is characteristically an asymmetrical phenomenon: when combined, a reagent “X” hampers a reagent “Y” to bind to its specific antigen “z”, but the reagent “Y” not-necessarily hampers the reagent “X” to bind to its specific antigen “w”. This contrasts with some staining problems related to the performance of monoclonal and polyclonal antibodies with specificity for the surface immunoglobulins light-chains, where the phenomenon has a symmetrical presentation: there is no fluorescence signal for both kappa and lambda light-chains.
In this train of thought, an equivalent experiment of sequential incubation was recently published and the presence of interference between the monoclonal antibodies, even when added at different times in the staining tube, suggested to the authors alternative mechanisms for the reduced fluorescence signal (De Vita et al., 2015). Of note, a related phenomenon of interference, fluorescence quenching, is also an asymmetrical phenomenon, but we believe that the experiment of sequential incubation that we performed could be used as simple method to help in the discrimination between SH and fluorescence quenching, without the use of complex experiments involving FRET (fluorescence-resonance energy transfer). FRET is possibly, although not exclusively, a mechanism of fluorescence quenching. FRET decreases the intensity of the donor fluorochrome and transfers the energy to an acceptor (De Vita et al., 2015).
In this sense, it is possible to speculate that, in our case, if fluorescence quenching was ex-hypothesi the very phenomenon underlying the absence of anti-lambda (PE) signal, the mechanism would be as follows: the fluorescence of the anti-lambda (PE) monoclonal antibody would be attenuated (or, as in our case, extinguished) by the anti-CD19 (PE-Cy5), which could absorb the anti-lambda (PE) derived energy (De Vita et al., 2015). But if this were true, the sequential incubation experiment performed by us would not restitute the anti-lambda (PE) signal. Thus, we sustain that the anti-lambda (PE) signal started to be seen because the mechanism underlying the absence of anti-lambda (PE) signal was SH, not fluorescence quenching. Accordingly, we suggest that the sequential incubation experiment we performed could be a suitable and easy test to discriminate between SH and fluorescence quenching in the practical of flow cytometry laboratories. Lastly, an alternative approach not tried by us, but that could eventually have solved the problem of SH as well, would be the exchange of PE-Cy5 for a fluorochrome of lower molecular weight (Table 1).
In the last decades, the number of fluorescent dyes available to be used in flow cytometry clinical and research studies has increased substantially. In fact, multiple lasers flow cytometers having two physical parameters (FSC and SSC) and 18 fluorescence detectors are relatively common nowadays. Instruments with more than 30 parameters, though of less common use, are also commercially available as, for instance, ID7000™ Spectral Cell Analyzer (more than 44 parameters; Sony Biotechnology), BD FACSymphony™ (50 parameters; Becton Dickinson) and Cytek® Aurora (67 parameters; Cytek Biosciences). Nevertheless, as it has been recently pointed out (De Vita et al., 2015), when creating reagent panels for immunophenotyping, the focus of attention is mainly on how to ensure consistency of reagents as well as color compensation issues, whereas the impact of a possible interference between monoclonal antibodies is generally not routinely tested. The problem is even more serious because, nowadays, most flow cytometry laboratories are relying on specialized softwares to manage color compensation, which can include over a dozen fluorochromes. Thus, in a nutshell, our report reinforces the necessity to look more carefully to the fluorochrome mutual interference issue, before abnormal levels of antigen expression can be asserted or even rare phenotypes of hematologic diseases can be considered and, consequently, incorrectly diagnosed in the landscape of polychromatic flow cytometry.
中文翻译:
立体障碍:流式细胞术中一个实际的(并且经常被遗忘的)问题
立体障碍(SH)是一种长期已知的现象,尽管经常被人们遗忘,但由于一种单克隆抗体阻碍了另一种单克隆抗体与其各自的靶抗原的结合,因此发生了可检测的荧光信号减少或减少的现象。试剂结合使用。SH的潜在机制至少部分与靶向相同大分子复合物的多种单克隆抗体的存在有关。因此,为了避免出现SH,应事先针对单色染色实验对流式细胞术常规中要使用的每种单克隆抗体组合进行测试。此过程是验证的一部分”
尽管对SH进行了检查,但即使在常规流式细胞仪实验室中已将验证步骤作为日常使用的已验证方法的一部分,在普通样品中仍会看到一些单克隆抗体的意外行为。
根据我们自己的经验,在9年(2009-2017年)期间,我们进行了系统的流式细胞仪检测,仅发现一名诊断为慢性淋巴细胞性白血病(CLL)的患者,其中外周血样本显示存在单克隆抗体抗kappa(FITC),抗lambda(PE),抗CD19(PE-Cy5)和抗CD20(PE-Cy5)可变组合的SH现象。
该患者是一名53岁的男子,2014年他的白细胞计数为76.5×10 9 / L,淋巴细胞计数为52.8×10 9 / L。对外周血涂片的回顾表明,存在着中小型成熟的淋巴样细胞。
对于流式细胞仪程序,在EDTA中收集的外周血样品中采用了裂解洗涤技术。简而言之,将1.0×10 9白细胞/ L与5μl每种单克隆抗体一起孵育。在我们的工厂中,使用以下免疫表型面板分析所有可疑的成熟B细胞肿瘤病例:试管1:不染色(对照管);管2:CD19(FITC),CD5(PE),CD45(PE‐Cy5); 管3:CD2(FITC),CD23(PE); 试管4:卡伯(FITC),λ(PE),CD19(PE‐Cy5); 管5:FMC7(FITC),CD79(PE); 软管6:CD10(FITC),CD11c(PE),CD38(PE-Cy5); 7号管:CD103(FITC),CD20(PE); 试管8:IgM(FITC),CD200(PE),CD19(PE‐Cy5); 管9:CD8(FITC),CD4(PE),CD3(PE-Cy5)。1个1免疫表型面板(靶细胞):试管(Wang等,2014):非染色淋巴细胞;管(De Vita et al。,2015):成熟的B淋巴细胞和CD5 + B淋巴细胞;试管(3):T淋巴细胞和CD23 + B淋巴细胞;试管(4):成熟的B淋巴细胞;试管(5):B淋巴细胞;管(6):生发中心B‐淋巴细胞,终末分化B‐淋巴细胞和B‐淋巴细胞的其他子集;试管(7):T淋巴细胞和异常B细胞(毛细胞白血病);管(8):成熟的B淋巴细胞和其他B淋巴细胞亚群;管(9):T淋巴细胞和稀有CD8 + CLL的子集。值得注意的是:关于在可疑的成熟B细胞肿瘤病例中特异的免疫表型中不常见地包含试管9(仅带有谱系T细胞标记)的讨论,请参见:Matos DM。CD8抗原在慢性淋巴细胞性白血病中的表达:有任何相关意义吗?细胞计数法B部分(临床细胞计数法)96B:96–98(2019)。
按照传统建议,将含有抗免疫球蛋白抗体的试管在室温下用PBS洗涤3次。值得注意的是,抗kappa(FITC),抗lambda(PE)和抗CD19(PE-Cy5)的克隆分别是G20-193(同种型:小鼠IgG1,k),JDC-12(同种型:小鼠IgG1, k)和HIB19(同种型:小鼠IgG1,k)。除anti-IgM(FITC)外,所有单克隆抗体均购自Becton Dickinson(美国加利福尼亚州圣何塞),而抗IgM(FITC)则购自Dako(美国加利福尼亚州卡平特里亚)。在三色COULTER EPICS XL-MCL流式细胞仪(Beckman-Coulter)中,每管总共采集了10.000个细胞。
我们的首次免疫表型分析显示,存在75%的B细胞(CD19 +),其特征是CD5抗原的共表达。B细胞还对抗原CD11c,CD20低,CD23,CD45高,CD200,FMC7和表面免疫球蛋白IgM低亚型表现出阳性。B细胞对抗原CD2,CD3,CD4,CD8,CD10,CD25,CD38,CD79b和CD103呈阴性。然而,令我们惊讶的是,我们无法在试管4的B细胞表面检测到kappa或lambda表达,因此,我们最初无法确认它们的轻链限制(图1(a ))。
我们最初假设是要处理:(a)罕见的特征在于以前没有文献报道的表面光链免疫球蛋白不存在,或者(b)由于以下原因未能检测到免疫球蛋白轻链:反kappa和/或反lambda性能问题。但是,B细胞上存在表面IgM亚型重链,这使我们考虑了在试管4中固定的试剂组合可能出现问题的另一种可能性。因此,我们认为抗CD19(PE-Cy5 )可能会通过SH干扰抗κ(FITC)和/或抗λ(PE)的结合。另外,作为第四个假设,我们认为荧光猝灭可能是缺乏κ和/或λ信号的原因。因此,我们着手研究这种现象:我们最初对试管4进行了另一次染色,但现在为抗CD20(PE-Cy5),克隆2H7,同型小鼠(C57BL / 6 IgG2b),k更换了抗CD19(PE-Cy5)。但是,该现象再次发生,因此无法检测到kappa和lambda信号(数据未显示)。
然后,我们尝试了另一种方法(使用了相同的血液样本):对试管4进行了新的染色,但是这次依次添加了单克隆抗体:首先添加了抗kappa(FITC)和抗lambda(PE)结合到试管中,孵育10分钟后,我们最终添加了抗CD19(PE-Cy5)。结果示于图1(b)。B细胞明显表现出低λ轻链限制。因此,由于我们不希望顺序培养程序能够很好地结合抗lambda(PE),因此我们对SH的怀疑得到了加强,损害了荧光猝灭假设。荧光猝灭的原因,因为PE和串联荧光染料PE-Cy5之间可能的能量转移仍将继续发生。为了确定SH,我们进行了新的孵育,但是这次没有抗CD19(PE-Cy5)。结果示于图1(C)。同样,B细胞表现出λ低轻链限制。
最后,为了使整个案例更引人入胜,我们在最初诊断后的17个月对同一患者的骨髓进行了最小残留病分析。这次,第4管由诊断时使用的单克隆抗体组成,即抗kappa(FITC),抗lambda(PE)和抗CD19(PE-Cy5),以及抗CD5(APC) )。我们发现0.4%的B细胞具有CLL表型(CD5 +,CD20低,CD43 +,CD79b-)。CLL细胞表现出λ低的轻链限制。奇怪的是,这次没有看到SH。
立体障碍是一种罕见的现象,主要与荧光强度的降低有关,在抗原表达水平具有临床意义的情况下,它可能导致流式细胞术结果的错误解释。在极端情况下,如我们的报告所描绘的那样,荧光强度的下降不仅仅是“仅仅”下降,而是完全没有荧光信号。
在我们的案例中,我们建议SH现象的可能解释与CD19蛋白与B细胞受体(BCR)的跨膜免疫球蛋白分子的空间接近性有关。但是,在我们的一系列免疫表型研究中仅发现一例SH的原因还有待解释。实际上,在2009年至2017年之间,我们采用了标准的免疫表型专家组,专门用于分析可疑的成熟B细胞肿瘤病例,为62次22从2015年起,我们开始使用BD FACSCalibur 4色流式细胞仪,因此,我们对4号管进行了改良,在其上添加了抗CD5单克隆抗体(APC)。因此,试管4:kappa(FITC),lambda(PE),CD19(PE-Cy5),CD5(APC)。最终,我们定制了电子管4,将CD19(PE-Cy5)更改为CD19(PerCP)。
:管4中没有一个显示SH现象。此外,在101次中,将试管4用作其他免疫表型面板的一部分:同样,我们没有发现其他SH病例。
即使经常使用相同的单克隆抗体组合,我们也没有对这种现象的稀有性的完整解释,除非我们假设与CD19蛋白和BCR在B细胞膜中的分布有关的因素的特殊样本可能有助于SH。无论如何,除了竞争性荧光染料的分子量对触发SH产生的众所周知的影响外(结合使用重分子,例如PE和PE-Cy5,更强大的能力来生成SH)现象(表1),尚不清楚SH出现的机制,甚至可能是细胞表面微绒毛的密度程度可能有助于阻止所涉及试剂的结合(Wang等,2014)。从这个意义上讲,其他技术可能有助于揭示导致SH出现的机制。例如,扫描电子显微镜虽然是世界上许多中心所不具备的技术,但可能显示出已记录SH现象的细胞表面存在伪影。但是,为了阐明控制SH中抗原和抗体之间相互作用的分子机制,原子力显微镜可能更有用,而该技术允许确定抗原和抗体之间的粘附力作为参数。测量。33最初步的想法是用待测抗原在淋巴细胞膜上功能化原子力显微镜的尖端,然后测量抗原与抗体之间的粘附力。值得注意的是,要使用原子力显微镜,必须将细胞附着在固体表面(例如,涂有纤维蛋白的固体表面)上。我非常感谢Nicole Jaffrezic-Renault教授(法国里昂大学分析科学研究所CNRS的Emeritus研究主任)对我们使用原子力显微镜阐明某些可能性的简短但非常刺激和丰富的讨论。造成SH现象的潜在机制。
荧光çc 资料来源:Thermo Fisher Scientific(个人通讯),Biolegend(个人通讯)和https://www.biolegend.com/en-us/fluorphore-families和Cell Signaling Technology(个人通讯)。 |
分子量(道尔顿) | 激发激光线(nm)的一个一个 激发激光线:(ⅰ)为405nm =紫色激光; (ii)407 nm = laser激光;(iii)488 nm =蓝色激光(氩);(iv)532 nm =绿光二极管激光器;(v)561 nm =黄绿色激光; (vi)594 nm =橘红色激光;(vii)633 nm =红色激光(HeNe);(viii)640纳米=红色激光。 |
---|---|---|
简单的有机荧光团 | ||
FITC | 389大 | 488纳米 |
太平洋蓝™ | 406达 | 405纳米 |
级联蓝色 | 596大 | 405纳米或407纳米 |
紫荧光450 | 600大 | 405纳米 |
德州红 | 625大 | 561 nm或594 nm |
AlexaFluor®488 | 643大 | 488纳米 |
dylight 405 | 793大 | 405纳米 |
AlexaFluor®594 | 820大 | 561 nm或594 nm |
红色荧光910 | 900大 | 633纳米 |
DyLight 488 | 1.0 kDa的 | 488纳米 |
dylight 650 | 1.1 kDa的 | 594 nm或633 nm |
AlexaFluor®660 | 1.1 kDa的 | 633纳米 |
Spark blue™550 | 1.2 kDa的 | 488纳米 |
AlexaFluor®647 | 1.3 kDa | 633 nm或640 nm |
AlexaFluor®700 | 1.4 kDa | 633 nm或640 nm |
Spark NIR™685 | 3.1 kDa | 633纳米 |
基于蛋白质的荧光团 | ||
PerCP | 35 kDa | 488纳米 |
PerCP‐eFluor 710 bb 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。 |
≅36–37 kDa | 488纳米 |
PerCP-花青5.5 bb 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。 |
≅37 kDa | 488纳米 |
装甲运兵车 | 105 kDa | 633 nm或640 nm |
AmCyan | 108 kDa | 405纳米 |
APC-花青7 bb 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。 |
109 kDa的 | 633 nm或640 nm |
APC-fire™750 bb 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。 |
110 kDa的 | 633 nm或640 nm |
聚乙烯 | 240 kDa的 | 488 nm或532 nm或561 nm |
PE-花青5 bb 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。 |
≅242 kDa | 488 nm或532 nm或561 nm |
PE-eFluor 610 # | ≅242–245 kDa | 488 nm或532 nm或561 nm |
PE-花青7 bb 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。 |
245 kDa的 | 488 nm或532 nm或561 nm |
PE‐dazzle™594 bb 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。 |
245 kDa的 | 488 nm或532 nm或561 nm |
PE-fire™640 bb 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。 |
260千达 | 561纳米 |
有机聚合物 | ||
艳紫421™ | 60–80 kDa | 405纳米 |
艳紫510™ | 60–80 kDa | 405纳米 |
绚丽的紫罗兰605™ | 60–80 kDa | 405纳米 |
绚丽的紫罗兰色711™ | 60–80 kDa | 405纳米 |
基拉维亚多聚体 | ||
KIRAVIA blue 520™ | 8 kDa | 488纳米 |
- 一个 激发激光线:(ⅰ)为405nm =紫色激光; (ii)407 nm = laser激光;(iii)488 nm =蓝色激光(氩);(iv)532 nm =绿光二极管激光器;(v)561 nm =黄绿色激光; (vi)594 nm =橘红色激光;(vii)633 nm =红色激光(HeNe);(viii)640纳米=红色激光。
- b 荧光团:蛋白质(F / P)比率:PE,PerCP和APC分子与小的有机染料(eFluor,Cyanine5,Cyanine5.5,Cyanine 7,Dazzle 594和Fire 640)的比率通常取决于共轭过程。因此,它有所不同,并且可以是“特定于批次”和“特定于供应商”的。
- c 资料来源:Thermo Fisher Scientific(个人通讯),Biolegend(个人通讯)和https://www.biolegend.com/en-us/fluorphore-families和Cell Signaling Technology(个人通讯)。
话虽如此,根据我们为阐明试管4的染色不一致性而进行的实验结果,我们可以断言SH是典型的不对称现象:结合使用时,试剂“ X”会阻碍试剂“ Y”与其结合特异性抗原“ z”,但试剂“ Y”不必要地阻碍试剂“ X”结合其特异性抗原“ w”。这与一些与表面免疫球蛋白轻链具有特异性的单克隆抗体和多克隆抗体的性能相关的染色问题形成了鲜明对比,该现象呈现对称现象:κ和λ轻链均无荧光信号。
在这种思路下,最近发表了一项等效的顺序孵育实验,即使在染色管中的不同时间添加单克隆抗体之间也存在干扰,这为作者们提出了减少荧光信号的另一种机制(De Vita等,2015)。值得注意的是,相关的干扰现象(荧光猝灭)也是一种不对称现象,但是我们认为,我们进行的顺序孵育实验可以用作帮助区分SH和荧光猝灭的简单方法,而无需使用涉及FRET(荧光共振能量转移)的复杂实验。尽管不是唯一,但FRET可能是荧光淬灭的机制。FRET降低了供体荧光染料的强度,并将能量转移到受体上(De Vita等,2015)。
从这个意义上讲,可以推测,在我们的情况下,如果荧光猝灭是对缺乏抗λ(PE)信号的潜在现象的假设,那么其机理如下: lambda(PE)单克隆抗体会被抗CD19(PE-Cy5)减毒(或在我们的案例中被熄灭),它可以吸收抗lambda(PE)衍生的能量(De Vita等,2015))。但是,如果这是真的,那么我们进行的顺序孵育实验将不会重新建立抗λ信号。因此,我们认为开始看到抗λ(PE)信号是因为缺乏抗λ(PE)信号的潜在机制是SH,而不是荧光猝灭。因此,我们建议在流式细胞仪实验室的实践中,我们进行的顺序孵育实验可能是区分SH和荧光猝灭的合适且简便的测试方法。最后,我们没有尝试过的另一种方法,但最终也可能解决了SH的问题,那就是用PE-Cy5交换低分子量的荧光染料(表1)。
在过去的几十年中,可用于流式细胞术临床和研究的荧光染料的数量已大大增加。实际上,如今具有两个物理参数(FSC和SSC)和18个荧光检测器的多台激光流式细胞仪相对普遍。具有30多个参数的仪器虽然不那么常用,但也可以通过商业途径获得,例如ID7000™光谱细胞分析仪(超过44个参数; Sony Biotechnology),BD FACSymphony™(50个参数; Becton Dickinson)和Cytek® Aurora(67个参数; Cytek Biosciences)。然而,正如最近指出的那样(De Vita et al。,2015),在创建用于免疫表型的试剂面板时,关注的焦点主要集中在如何确保试剂的一致性以及颜色补偿问题上,而单克隆抗体之间可能干扰的影响通常不会进行常规测试。这个问题更加严重,因为如今,大多数流式细胞实验室都依靠专门的软件来管理颜色补偿,其中可能包括十几种荧光染料。因此,简而言之,我们的报告强调在断定抗原表达异常水平或什至可以考虑罕见的血液病表型并因此在景观中被错误诊断之前,必须更仔细地研究荧光染料相互干扰的问题。流式细胞仪的研究。