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Read in one article – mass spectrometry flow cytometry (Part 1)

Introduction to mass spectrometry flow cytometry

Flow cytometry is the most classic single-cell analysis technology, which can detect multiple parameters in single cells, so as to analyze the subpopulation and function of samples. Fluorescence based flow cytometry has been developed for more than 50 years. It is a very mature cell detection technology. At present, it is widely used in various research fields of biology. It is the gold standard for single cell detection in major tertiary hospitals, especially the detection of leukemia. Traditional flow cytometry uses fluorescent groups to detect various proteins in cells, so it is also called fluorescent flow cytometry. However, due to the limitation of fluorescent groups, there are obvious technical bottlenecks in traditional flow cytometry. First, the number of fluorescent groups available is limited, generally no more than 20 at most, and commonly used in laboratories < 12; Second, the emission bands of each fluorescent group are wide, and the adjacent bands overlap seriously, resulting in the number of channels that can be detected simultaneously in one analysis is quite limited. When detecting the number of channels > After 8 hours, the panel design and compensation calculation of the experiment have begun to become extremely complex, making the multi parameter detection an extremely time-consuming and complex technical work, and the requirements for the operator are extremely high, which greatly limits its development.

The traditional streaming detection window is very crowded, and it is difficult to accommodate more detection channels

After entering the 21st century, new biological technologies emerge in endlessly. Researchers continue to deepen their research in the fields of hematopoiesis, immune cell differentiation and maturation, cancer cell transformation, stem cell self-renewal and differentiation, and the importance of single-cell heterogeneity has become a consensus. In order to understand the changes of various cytokines in cells more clearly, there are higher requirements for the number of detection channels and signal quality. Traditional flow cytometry has been out of reach in such scientific research fields, and there is an urgent need for a more efficient, easy-to-use flow cytometry with more parameters detected at the same time. Mass spectrometry flow cytometry combines the traditional flow cytometry technology and mass spectrometry technology, uses various isotopes of lanthanide elements as metal labels (or quality labels) to replace the previous fluorescent labels, and uses ICP-TOFMS to conduct high-speed, full spectrum and high-resolution quantitative analysis of metal isotopes, which completely solves the problem of fluorescent cross-linking in the traditional flow cytometry and realizes > Simultaneous detection of 50 parameters. By virtue of the ultra-high resolution of time-of-flight mass spectrometry, mass spectrometry flow cytometry can completely separate various metal isotopes, and the overlap between adjacent channels < 0.3%, so there is no need to calculate and compensate, which greatly simplifies the experimental process. Mass spectrometry flow cytometry, with its advantages of ultra-high flux, sensitivity and stability, is suitable for many research fields, such as immunity, tumor, blood, medicine, genetics and so on.

In summary, the main technical features of mass spectrometry flow cytometry are as follows:

There are many channels. Theoretically, there are more than 100, which are currently commercially available > 50. With technological progress, more elements can be used as labels, and the number of detection channels will further increase.

There is no interference between adjacent channels, and there is no need to calculate compensation. ICP mass spectrometry has super-high resolution, which can completely distinguish various elements used for labeling. The experimental data show that the interference between adjacent channels < 0.3%, basically negligible, no need to calculate compensation. This not only simplifies the experimental process, but also saves samples and reagents.

Using rare earth elements as labels has low background interference and high signal-to-noise ratio. Mass spectrometry flow cytometry uses rare earth elements that do not exist in life as detection tags, and uses the specific combination of antigen and antibody to achieve ultra-high detection signal-to-noise ratio.

Ultra high sensitivity ensures the detection of low abundance proteins. Mass spectrometry flow cytometry has both mass spectrometer and polymer metal chelation technology. As we all know, the unparalleled sensitivity of mass spectrometer is one of the main factors that make it the king of analytical instruments. Theoretically, one in ten thousand metal tag atoms can be detected by mass spectrometry flow cytometry. In addition, mass spectrometry flow cytometry chelates about 1000 metal elements on the polymer, and then combines with the sulfhydryl group on the antibody, which is equivalent to”amplifying” the protein to be tested by three orders of magnitude. At least hundreds of rare proteins can be detected in a single cell.

Diversified data processing methods to achieve in-depth analysis of samples. The rapid increase in the number of channels and the ultra fast detection speed of mass spectrometry make the amount of data increase sharply, which puts forward higher requirements for data processing methods. At present, various dimensionality reduction, clustering and visualization methods have been used to extract useful biological information from raw data for visual display. Common analysis methods include spade, PCA, visne and gemstone.

Mass spectrometryFlow workflow

History of mass spectrometry flow cytometry

In 1959, American chemists Rosalyn Yalow (female) and Solomon Berson developed radioimmunoassay (RIA) in the study of detecting plasma insulin content. Due to its high sensitivity, strong specificity, good accuracy and small sample consumption, RIA has developed rapidly and is widely used in the determination of proteins, enzymes, polypeptide hormones and various drugs. In 1977, Yalow shared the Nobel Prize in physiology or medicine with two other winners. The reason for winning the prize was”for the development of radioimunoassays of peptide hormones”.

Although radioimmunoassay has many advantages, the impact of radioisotopes on human, environment and biological sample activity is difficult to ignore. Since then, various immune detection technologies have been developed based on enzymes, chemiluminescence, fluorescence, metals (especially rare earth elements) and metal compounds, such as colloidal gold, enzyme-linked immunosorbent assay, time-resolved fluorescence (based on lanthanide chelates) and chemiluminescence immunity.

On July 17, 2001, Dmitry Bandura (physicist), Vladimir baranov (chemist), Scott Tanner (mass spectrometer) and Zoe Quinn, scientists of MDS SCIEX (later ab SCIEX and now SCIEX), submitted a patent, proposing a method of labeling antibodies with transition elements, and finally detecting them by ICP mass spectrometry or ICP emission spectrum by using the immune binding between antibodies and samples to be tested, This opened the era of mass spectrometry flow cytometry. Please remember the three gods above, which will be mentioned later. Coincidentally, the next day (July 18, 2001), Professor zhangxinrong of Tsinghua University submitted a chapter entitled”a novel combination of immunology and ICP-MS as a hyphenated technology for the determination of thioid stimulating hormone (TSH) in human serum”, proposing a novel technology of immune reaction and ICP-QMS combination using the rare earth element Europium (EU) as a label, It is used to detect thyroid stimulating hormone (TSH) in blood. In fact, on March 23rd, 2001, Professor zhangxinrong submitted another article,”Application of the biological adjust between antibody and colloid Au nanoparticles as analyze to inductively coupled plasma mass spectroscopy”, which was published on January 1st the following year. The article proposed to use colloidal gold nanoparticles as the label of immune detection, and ICPMS as the detector. Professor zhangxinrong is also a very respected scientist, who has done a lot of groundbreaking research work. He does not blindly follow the trend and likes to find another way. I guess Mr. Zhang doesn’t have a particularly strong engineering team, so although there are many achievements transformation projects, they are all XXX. It’s purely my guess. Don’t blame Mr. Zhang. Domestic mass spectrometry companies can talk to Mr. Zhang more and do some original transformation of scientific research achievements. Then, what’s more dramatic is that the above-mentioned patent was actually submitted by way of provisional application as early as December 28, 2000, and the formal application was submitted on July 17, 2001. According to the patent system of the United States, in order to seize the priority date, a simple patent text can be submitted through a temporary application, but at the same time, a formal application needs to be submitted within one year after the temporary application for replacement. At this time, the formally applied patent enjoys the priority date of the temporary application, and the text content of the temporary application will also be published as a priority document when the formal application is published. Therefore, in theory, Professor Zhang Xinrong made an important contribution to the embryonic stage of mass spectrometry flow cytometry, but research institutions are more interested in academic articles and pay far less attention to patents than commercial institutions.

Of course, at this time, there is still a wall between the patent and the article, and the mass spectrometry flow cytometry we see at present. It has not been mentioned for the detection of single cells, but it is a matter of getting close to becoming a mass spectrometry flow cytometry. There are certain historical reasons for this. On the one hand, single cell detection was not so popular at that time, and all kinds of immune detection technologies wanted to solve the disadvantages of radioimmunoassay; On the other hand, although people are aware of the advantages of this technology for multi parameter detection, however, few companies in the market were producing ICP-TOFMS 20 years ago, of course, 20 years later, today is not a lot. This is not because the technical threshold of ICP-TOFMS is too high, but because ICP-TOFMS has almost no corresponding application, so it lacks market drive. In addition, because ICP-QMS (quadrupole mass spectrometry) introduces the collision reaction cell technology (the inventor of this technology is also Scott Tanner), which solves the problem of multi atom interference and solves the problem of insufficient quadrupole resolution to a large extent, the application with high quantitative requirements and low resolution requirements can well meet the requirements with high cost performance. When the resolution is very high, there are also various inorganic magnetic mass spectra. If it is required to have a relatively high isotope abundance ratio, there are also multiple receiving magnetic mass spectra. Basically, ICP-TOFMS was useless, and it was a relatively small mass spectrometer at that time. At that time, only GBC company in Australia and LECO company in the United States were probably producing ICP-TOFMS, which were mostly used in the field of geological analysis.

In October, 2004, the three gods mentioned above and Olga ornatsky (biologist), a colleague at MDS SCIEX, jointly established a company called DVS sciences. The company’s name uses the initials of the three people’s names. It has to be said that foreigners really like to use their own names as company names, which are actually very similar to our time-honored brands. Scott Tanner, as the leader of the team, served as CEO/CTO. After obtaining the authorization of relevant technologies from his old owner SCIEX, they opened the company in Canada in 2005 and began to polish this new ICP-TOFMS instrument platform (the first generation of cytof in the future). Scott Tanner, a Canadian, joined MDS SCIEX after receiving his doctorate in physical chemistry from York University in Toronto and has been working there for 25 years. Scott Tanner believes that the University of Toronto is a perfect place to help them complete the development of cytof instruments and reagents. From 2005 to 2011, Scott Tanner served successively as a professor in the Department of bioengineering and the Department of chemistry at the University of Toronto. Since then, DVS from genome Canada and Ontario’s Ministry of research& Innovation and other Canadian government agencies received a total of US $17million. In 2011, another $15million investment was obtained from Mohr davidow ventures, 5am ventures, Roche, Pfizer and other commercial institutions. As of 2011, the financial support received by DVS from outside and the sales revenue of seven cytofs were almost $40million. value得一提的是,在第一代CyTOF定型之前,DVS团队先后经历了3代原型机和8代试剂的开发,可以说是非常的坎坷,技术创业之艰辛可见一斑。

2009年,加拿大DVS Sciences公司发布了第一个质谱流式平台CyTOF(Cytometry by Time-of-Flight)。很快,CyTOF的用户就开始遍布世界各处 ,除了加拿大的多伦多大学,还有美国NIH、斯坦福大学、日本、中国台湾等研究机构都有装机。

Scott Tanner和第一代质谱流式CyTOF


事实上,质谱流式早期的快速发展离不开一个人,那就是斯坦福大学医学院的Garry Nolan教授,这又是一位全面开花的骨灰级大佬,不仅学术做得好,科研转化还贼成功,是Rigel(NASDQ上市公司), Nodality(医疗诊断技术开发), BINA(基因组学计算技术,被Roche收购), Apprise(测序解决方案,被Roche收购)等公司的创始人,IONpath(另一家单细胞质谱成像技术公司,和质谱流式有比较大的渊源,后面会提到)和Akoya的联合创始人,以及多家公司(包括DVS)的董事和顾问。Garry Nolan教授在免疫学研究领域名声斐然,是传统流式技术的大牛。Scott Tanner带着自己的黑科技找上门后,Garry Nolan教授意识到这是一项至少能改变免疫学研究的革命性技术,随后便基于CyTOF对人类的骨髓细胞进行了研究,在单细胞水平同时进行了34个参数的检测,系统性揭示了造血系统免疫信号的传导机制,对人类造血系统中的细胞分化有了更详细的认识。相关研究成果刊登在Science杂志上,一经发表立刻引发了全球同行的巨大兴趣,至今全世界研究者使用CyTOF发表了不计其数的CNS(Cell、Nature、Science)文章。由此,质谱流式在生物医学领域名声大噪,持续保持一年几十台的装机量,仪器单价也从一开始的60万美元到了80万美元。毕竟,谁都明白掌握核心科技就能快人一步,贵一点怕什么。不得不说,Garry Nolan教授是一个极其合格的KOL,不仅让CyTOF声名远扬,还为CyTOF适配了很多开源的生信算法,制定了各种各样试验流程供后来者参考,同时还建立了CyTOF论文专门解答和讨论用户在使用CyTOF过程中遇到的各种疑难杂症,甚至还组织各细分领域的大佬们专门开设了CyTOF的小型课程(油管上有资源),可以说Garry Nolan教授为质谱流式的普及铺好了第三块基石。

2013年5月,DVS推出了第二代质谱流式CyTOF2以及新款MaxPar试剂盒。CyTOF 2采用了全新的底盘设计,能同时检测单细胞中最多达120种生物标记物,每秒最多可检测1000个细胞,改进了离子光学设计从而将灵敏度提高了1倍。5种新款MaxPar试剂盒含有开展高维度实验所需的所有必要试剂,提供最多达17种开箱即用的金属螯合抗体,并且能够与最多16种额外抗体同时结合,在单试管中实现多个细胞群体的高分辨率单细胞检测。

第二代质谱流式CyTOF 2

2014年1月29日,美国公司Fluidigm(富鲁达)宣布将以约2.075亿美元收购DVS Sciences。此时,质谱流式发展到了第二代——CyTOF 2。此后,Fluidigm又从Perkin Elmer公司手里买断了和质谱流式有关的所有专利,自此Fluidigm公司拥有了完整的专利、技术和团队,开始全力以赴推动质谱流式向前发展。不过,CyTOF在富鲁达手里并没有如一开始想象的那般美好,至少时至今日也还没到实现Scott Tanner设想的一年100台的装机量或者是每年1亿美元的销售额。富鲁达目前的市值还不到3亿美元,最近3年质谱流式年均销售收入不到7000万美元(包括仪器、试剂和服务),仪器占比~50%。按平均单价70万美元粗略估算,一年的新增装机大约是50台。这对于一个偏高端的单品质谱仪器其实也还算是一个不错的销量,但对于一个NASDQ的上市公司而言实在算不上是成功,而且科研领域如此火爆,几乎没有同类产品产品的竞争。不得不让人深思,到底是哪里不对劲儿呢?是富鲁达的管理层的问题吗?还是质谱流式的市场有限?亦或是传统流式的反围剿太猛?

近些年富鲁达的高层的确频繁换人,今年从Casdin Capital和 Viking Global Investors注入了2.5亿美元的战略融资之后,从丹纳赫等头部生命科学公司引入了大批高管。招商换帅的同时,富鲁达又把公司英文名改了,现在叫Standard BioTools Inc.,这不得不让人联想到我国五行文化中常见的“更名改运”的策略。换了班底,改了名字,又有大笔的现金流进账,且看3年内会不会有什么起色吧。目前质谱流式主要是在科研市场,虽然临床和制药领域已经开展了不少临床试验,但是还没有真正进入临床市场,比较还没有任何一家公司获得任何国家的医疗器械注册证。如果真能打开临床市场的大门,的确是会带来巨大的利润。不过奇怪的是,这么多年过去了一直没有听说富鲁达在进行医疗器械的注册,事出反常不应该啊。至于传统流式的反攻,我相信一定是有的。其实关于质谱流式和传统流式的讨论一直都存在,毋庸置疑质谱流式绝对是有优势的,只不过大部分情况下只是在多参数,高分辨方面有比较大的优势,其他不少指标实际上逊色不少的,比如传统流式的样品分析速度10000个细胞/秒,质谱流式通常低于1000个细胞/秒;传统流式的灵敏度和样品利用效率都高于质谱流式;另外传统流式可以进行细胞筛选和回收,质谱流式则不能。而且,基于荧光的流式技术也在发展,比如2004年提出的光谱流式也是主打多参数,创新点在于光路系统、光电检测和数据算法上,如今2014年成立的Cytek推出的Aurora光谱流式也可以实现5激光40色的多参数检测。而且,其24色的仪器和试剂分别已经拿到了NMPA医疗器械二类和试剂一类注册证,成功迈入了临床市场。2021年7月,Cytek公司成功敲钟NASDQ,市值一度高达25.4亿美元,目前回落到15.8亿美元。2021年,Cytek全年收入1.28亿美元,而今年第一季度净收入同比增长高达44%。


话说当年在Fluidigm之前,DVS其实是找了多个潜在买家的,包括Life Technologies,Agilent和Thermo Fisher等巨头。大概是当时的DVS过于弱小,而安捷伦和赛默飞这种质谱大厂一看,心想你这个质谱也没什么啊,比你这复杂的我都有,何必花钱买呢。巨头没有看在眼里。如今,Life Technologies已经被赛默飞收购,安捷伦以2.5亿美金买了中国的流式公司艾森生物,而赛默飞也推出了自己的Attune NxT流式细胞仪(基于声波聚焦技术,分析速度高达35000个/秒)。






质谱流式成像系统Hyperion Imaging System

关于质谱流式的“好戏”其实还没完,2019年9月,富鲁达一纸诉状起诉一家叫IONpath的创业公司,称其2018年发布了侵犯其专利技术的MIBITM(Multiplexed Ion Beam Imaging)高维空间蛋白质组学技术,同样可以在亚细胞水平进行40个生物标记物的多重质谱成像。富鲁达称自2019年起IONpath开始向市场积极推广,并顺服和默许富鲁达的用户搭配使用IONpath的MIBI仪器和富鲁达的试剂。IONpath何许人也?还记得我们前面列举过Garry Nolan教授一大堆的创始人头衔吗?没错,Garry Nolan教授是IONpath的联合创始人,而公司的主要创始人也是来自Garry Nolan教授实验室。Garry Nolan教授曾是DVS的科学顾问委员会的主席,DVS被收购之后,仍担任顾问职责直到2016年底。同时,同样来自Garry Nolan实验室的其他IONpath创始人也在富鲁达担任顾问,而且IONpath成立于2014年,是任职顾问不久之后的事情。看来IONpath的确是动了富鲁达的奶酪,据说IONpath的发展速度比DVS早期还要快,第一年的装机量就接近小10台。这恰好给了富鲁达管理层找到了一个业绩不佳的口实,好家伙,原来是你小子在偷偷吸血。作了他!富鲁达来势汹汹,且言之凿凿。其实,老实说从MIBI和Hyperion两者的硬件架构上来看,其实差别还挺大的。尤其是,MIBI的空间分辨率指标其实比Hyperion还高了好多,前者的空间分辨率可到280nm,是真正的亚细胞水平,而后者通常只有1μm。这主要取决于前端的采样技术。我们前面提到Hyperion用的激光烧蚀,那主要受限于激光聚焦的束斑尺寸,目前也基本上只能做到1μm这种水平,而MIBI采用的是二次离子质谱技术(SIMS),这也不是一个新鲜玩意儿,属于一个比较高端的配置,是使用聚焦的高能离子束(Primary Ions,叫它一次离子好了)对样品表面进行轰击,然后解吸和电离出二次待测样品离子(Secondary Ions)的技术。

富鲁达的Hyperion Imaging System(黄框和紫框)+Helios



不过,任何时候也不能坐以待毙,毕竟天无绝人之路,何况手里还有世界上最领先的质谱成像技术。2020年9月24日,IONpath宣布获得了1800万美元的B轮融资,众多的新老投资人中还包括老牌质谱公司Bruker。自从Bruke推出了timsTOF系列产品之后,在组学领域牢牢站稳了脚跟,现在又在逐步布局空间蛋白质组学了。2021年6月,Bruker还发布了针对单细胞蛋白组学的timsTOF SCP,可在数百个单细胞的分析中得到约 1,500 个蛋白质/细胞的定量分析结果,实现了真实无偏的单细胞蛋白组分析。不得不说,质谱这个已经发展了100多年的玩意儿目前看还是没有尽头的,而国内的质谱水平也还只是小学生,我们的视野应该放开一些,多一些合作开拓大市场,小一些争斗莫纠缠在小市场。话说回来,资本的嗅觉是最敏锐的,钱来了生路就来了。


2021年5月富鲁达发布了第四代质谱流式CyTOF XT,更加注重整机自动化运行,新增了样品自动上样、采集功能,配备了内部监控系统,自动感知管路堵塞,自动调谐,自动数据转换,一管样品可以实现50+标志物的多参数分析。

第四代质谱流式CyTOF XT

2022年5月11日,IONpath宣布完成了Corporate Round融资,巨头Thermo Fisher是唯一投资结构,同时这也是IONpath的第4次融资。被第二个质谱巨头加持,说明IONpath的路是走对了,这下妥了。而就上个月,2022年6月10号,Thermo刚宣布了已与TransMIT质谱开发中心合作,共同促进质谱成像技术在空间多组学领域的发展,具体是将前者的Orbitrap质谱和后者的Scanning Microscope Matrix-assisted Laser Desorption/Ionization(SMALDI) MSI和3D-surface MSI技术进行联用,可用于绘制包括代谢物、多肽、酶解的蛋白质等多种标记物分子的空间分布。由此可以看出,质谱成像技术是巨头们都在积极布局的一个市场。所以不管是Standard BioTools还是IONpath,日后无论能不能经营好,技术肯定是不愁找不到好买家的。


EXPEC 7910 ICP-QTOF(from谱育科技官网)

Starion星瀚 (from宸安生物官网)

       质谱流式的确是个不错的方向,没有那么多的巨头挡着,技术路径比较简单,市场前景又很明朗,还能彰显大国重器,为国分忧,是再好不过的潜力股了。在笔者看来,质谱流式技术的发展才刚刚开始,技术的迭代、细分和升级依然大有可为。未来的领先地位或许不会是Standard BioTools掌握,至少从目前的趋势看不会。国产质谱的崛起需要有一款旗帜产品在全球树立国产质谱的形象,质谱流式目前就具备这样的特质。


[1]Palladium-based Mass-Tag Cell Barcoding with a Doublet Filtering Scheme and Single Cell Deconvolution Algorithm


[2]Meeting the Challenges of High-Dimensional Single-Cell Data Analysis in Immunology


[4]Progress and Applications of Mass Cytometry in Sketching Immune Landscapes

DOI; 10.1002/ctm2.206

[5]The Nobel Prize in Physiology or Medicine 1977

[6]Standard Biotools财报

[7]多伦多大学杂志访谈:Seeing Into the “Soul” of Cells

[8]Standard BioTools官网

[9]中国第一台CyTOF 2质谱流式细胞仪震撼亮相厦门大学

[10]C&EN专访:Instrumental Efforts——Entrepreneurs take big risks to bring the latest scientific tools to market

[11]Garry Nolan’s Lab

[12]MIBI-TOF:A multiplexed imaging platform relates cellular phenotypes and tissue structure