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Sep 2019
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One-step White Blood Cell Extracellular Staining Method for Flow Cytometry
流式细胞术白细胞细胞外一步染色法   

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Abstract

Flow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one-step method in which white blood cell staining and red blood cell lysis are carried out in a single step, using a gentle lysis solution mixed with fluorescent antibody conjugates or probes in a dry or liquid format. The blood sample may be obtained from a routine venipuncture or directly from a fingerprick, allowing for near-patient analysis. This procedure enables the analysis of common white blood cell markers as well as markers related to infections or sepsis. This simpler and faster protocol may help to democratize the use of flow cytometry in the research and medical fields.


Graphic abstract:



One-step White Blood Cell Extracellular Staining Method for Flow Cytometry.


Keywords: Blood (), Leukocytes (白细胞), Flow cytometry (流式细胞术), One-step method (一步法), Point-of-care (医疗点)

Background

Flow cytometry is increasingly used in scientific investigations and healthcare; however, it requires a time-consuming, multi-step sample preparation procedure, which limits its use to specialized laboratories. Most whole blood sample preparation protocols for extracellular staining consist of at least three steps: using a venous blood sample collected in an anticoagulated sampling tube, 50-100 µl is pipetted into a reaction tube; as a second step, fluorescent antibodies are mixed and incubated with the blood to allow white blood cell staining; as a third step, the lysis solution is added to the reaction tube to allow for red blood cell removal; finally, an optional washing step is performed to reduce non-specific fluorescence.


Due to these multiple timed steps that need to be carried out in specialized laboratories, the use of flow cytometry is limited. It is, for example, not yet routinely used in the clinical field, even though the assessment of patient immune cells has proven helpful in the indication of potential disease activity (Brown and Wittwer, 2000). Some authors have tried to establish protocols with only one step, proposing to remove the lysis step; however, this method necessitates a dedicated flow cytometer, restricting its use(Petriz et al., 2018).


In this study, we propose a new universal method in which white blood cell staining and red blood cell lysis are carried out in a single step. The lysis solution is stable at room temperature (RT) and does not interfere with the staining owing to its composition, which comprises a substrate that is specifically cleaved by an enzyme uniquely present in RBC membranes (van Agthoven, 2007). White cells are maintained at neutral pH under isotonic conditions and are therefore not affected. The lysis solution can include low-dose formaldehyde (0.05%) to stabilize markers of interest (Bourgoin et al., 2020c).


The fine titration of the conjugates and their direct dilution in the lysis solution enable the final washing step to be eliminated while maintaining low background levels. Using the new Dried Unitized Reagent Assays (DURA) Innovations technology, the conjugates can be dried, which enables RT storage and removes the need for pipetting, thereby supporting better standardization. The blood sample may be of venous origin or from a fingerprick, allowing for less invasive sampling suitable for a point-of-care setting. This approach is possible thanks to an excess volume of lysis solution, which permits efficient RBC lysis in up to 50 µl of blood. The conjugate quantities have also been adjusted to stain white blood cells contained in 2-50 µl blood, with a small quantity of blood no longer being a limitation because 2 µl blood contains an average of 10,000 leukocytes. As a 100-cell subpopulation is usually considered statistically representative, using 2 µl of blood allows for analysis of subpopulations as low as 1%, which is sufficient for most routine applications.


Since management of patients with infections in the Emergency Department (ED) is challenging for practitioners, we developed a panel that enables rapid patient triage in the ED using flow cytometry. It has been shown that monitoring the expression of CD169 on monocytes (mCD169), CD64 on neutrophils (nCD64), and HLA-DR on monocytes (mHLA-DR) by flow cytometry can be indicative of viral or bacterial infection, or sepsis, respectively. We therefore established a panel consisting of antibodies targeting these three markers and evaluated the one-step method in subjects with infection and septic conditions by measuring the expression of the three infection-related markers (Bourgoin et al., 2019a, 2019b, 2020a, 2020b, 2020c, and 2021; Bedin et al., 2020; Michel et al., 2020).


Many other applications can be envisioned in fields where flow cytometry is routinely performed, such as measuring T, B, and NK cell proportions, detecting leukemias and lymphomas, and enumerating CD34+ stem cells for transplantation.

Materials and Reagents

  1. Regular flow cytometry 5-ml test tubes, polypropylene or polystyrene (12 × 75 mm), or 1.4-ml microtubes (e.g., from Micronic), or deep-well plates (any vendor).

  2. Lysis solution (Beckman Coulter, VersaLyseTM, catalog number: IM3648, store at 18-24°C, 2-year shelf-life)

  3. Fixative solution (IOTest®3 10×; Beckman Coulter, catalog number: A07800, store at 2-8°C, 1-year shelf-life)

  4. Antibody cocktail (IOTest Myeloid Activation CD169-PE (Phycoerythrin)/HLA-DR-APC (Allophycocyanin)/CD64-PB (Pacific-Blue) Antibody Cocktail from Beckman Coulter, catalog number: C63854, store at 2-8°C, 1-year shelf-life)

  5. Easy batch preparation of the lysis buffer (see Recipes)

  6. Batch preparation of the staining and lysis buffer for n tests (see Recipes)

Equipment

  1. Pipettes (Gilson, PIPETMAN®, catalog numbers: FA10003M [2-20 µl] and FA10006M [100-1000 µl])

  2. 3-laser, 10-color cytometer (Beckman Coulter, Navios, catalog number: B47904) or 3-laser 13-color cytometer (Beckman Coulter, CytoFLEX, catalog number: B53000). Any other 3-laser cytometer can be used (e.g., Becton Dickinson FACSCanto, Cytek Aurora)

Software

  1. Kaluza Software version 2.1 (Beckman Coulter, https://www.beckman.fr/flow-cytometry/software/kaluza)

    Note: Any other flow cytometry software can be used (e.g., Becton Dickinson Flowjo and Cytek SpectroFlo).

Procedure

  1. Prepare the lysis buffer (wear common Personal Protective Equipment).

    Dilute 1:200 the fixative solution in the lysis solution under chemical hood protection and mix.

  2. Perform the lysis–staining step.

  3. In a tube containing 500 µl lysis buffer and 10 µl conjugate panel, add 2-50 µl blood sample (venous or capillary), mix, and incubate for 15 min in the dark at RT (18-25°C).

  4. Acquire the data on a 3-laser cytometer (then dispose of the waste in containers dedicated to biohazard waste).

Data analysis

  1. Acquiring data on the flow cytometer

    Unstained cells are used to set the parameters of the flow cytometer.

    1. Turn on the flow cytometer.

    2. Open the acquisition software and create four dot plots:

      1. Side Scatter (SSC) on the x-axis and Forward Scatter (FSC) on the y-axis.

      2. Channel dedicated to PE (FL2 on Navios) detection on the x-axis and SSC on the y-axis.

      3. Chanel dedicated to APC (FL6 on Navios) detection on the x-axis and SSC on the y-axis.

      4. Chanel dedicated to PB (FL9 on Navios) detection on the x-axis and SSC on the y-axis.

    3. Set the voltages/gains for the SSC-FSC plot such that most of the cell population is in the middle of the graph (Figure 1a). The discriminant/threshold should be set up in such a way that minimum debris is acquired.

    4. Set all the compensations to 0.

    5. Set the voltages for FL9, FL2, and FL6 such that the lymphocyte mean or median fluorescence intensity is around 0.3 (Figure 1b).



      Figure 1. Flow cytometry parameters on (a) SSC-FSC plot and (b) FL2-, FL6-, and FL9-SSC plots


    6. Save the protocol.

    7. Acquire the first stained sample using a high flow rate (~1 µl/s) without any compensation. Make sure to acquire at least 500 monocytes for a robust analysis (usually 30 to 90 s).

    8. On the SSC-FSC plot, draw a leukocyte gate, avoiding debris (Figure 2a).

    9. On the SSC-FL9 (CD64-PB) plot, draw the lymphocyte, monocyte, and neutrophil gates (Figure 2b).

    10. CD169 and HLA-DR expression levels should be assessed on monocytes. CD64 expression levels should be assessed on neutrophils (Figure 2b and 2c).



      Figure 2. Data analysis procedure. (a and b) Gating strategy of lymphocytes, monocytes, and neutrophils. (c) CD169, CD64, and HLA-DR expression level assessment strategy.


    11. Marker levels should be expressed as the Mean of Fluorescence Intensity, Median of Fluorescence Intensity, or Signal-to-noise. Signal-to-noise is calculated as follows: CD169 expression levels on monocytes should be divided by CD169 expression levels on lymphocytes; CD64 expression levels on neutrophils should be divided by CD64 expression levels on lymphocytes; and HLA-DR expression levels on monocytes should be divided by HLA-DR expression levels on neutrophils (Figure 3).

    12. Results are considered as follows: High mCD169 expression is indicative of viral stimuli, high nCD64 expression is indicative of bacterial stimuli, high mHLA-DR expression is indicative of infectious stimuli, and low mHLA-DR expression is indicative of immune exhaustion (Figure 3).



      Figure 3. mCD169, nCD64, and mHLA-DR expression assessment in whole blood using the one-step method. Examples are given for one healthy volunteer, one virus‐infected patient, one bacteria‐infected patient, and one patient with sepsis.


    13. More research will be necessary to precisely set the thresholds in each laboratory setting, but as an indication, previous evaluations have shown that thresholds should be close to 3 for the CD169 and CD64 indexes.

    14. Further analysis can be performed using software such as Kaluza (re-analysis, other gating and auto-gating, other histograms or dot plots, coloring, compensation adjustments, refined percentages and fluorescent values, overlay and merge data sets, batch analysis for higher throughput, etc.).

Notes

Depending on the target marker, application, and required stability, the method should be adapted in each laboratory setting. For example, and only for indication and not as an established performance, it has been observed in some applications that:

  1. Blood sample can be venous or capillary.

  2. Blood can be anticoagulated with EDTA or heparin.

  3. Thanks to the gentle lysis reagent and added fixative, blood can be processed up to 3-4 days after sampling when stored at RT or 2-8°C. Moreover, samples can be analyzed on the cytometer up to 3 days after processing when stored at 2-8°C.

  4. The lysis solution can include 0.05% fixative solution.

  5. The lysis buffer (lysis solution containing fixative solution) can be stored for up to one month at RT.

  6. The targeted marker can be any membrane marker.

  7. Antibodies can be liquid or dried depending on product availability.

    WARNING:

    1. If a liquid cocktail is used, an antibody volume higher than 100 µl could impact lysis efficiency.

    2. If a commercial DURAClone format is used, the lysis volume should be increased to 1 ml (for 2-50 µl blood) or 2 ml (for 50-100 µl blood as per IFU).

  8. Antibodies can be premixed with the lysis buffer up to a few days prior.

Recipes

Note: To be adapted depending on each application; performances are not established.

  1. Easy batch preparation of lysis buffer

    One bottle Versalyse solution (100 ml)

    500 µl fixative solution (10×)

    Mix and store at RT

  2. Batch preparation of the staining and lysis buffer for n tests

    Pipet (n+1) × 500 µl lysis buffer

    Add (n+1) × 10 µl myeloid activation cocktail (or any other panel at its optimal dose)

    Mix well and distribute 510 µl per reaction tube, store in the dark

    Tube is now ready to receive the blood sample

Acknowledgments

IAB is the recipient of a CIFRE Ph.D. grant (N°2018/1212) from the ANRT (National Agency for Research and Technology). This work was supported by Beckman Coulter through donation of the research reagents used in the flow cytometry experiments and participation of the four employees: IAB, PB, JMB, and FM. The method described was modified from ‘VersaLyse and the mechanism of ammonium chloride lysis’ (van Agthoven, 2007) and resulted in recent publications (Bourgoin et al., 2019a, 2019b, 2020a, 2020b, 2020c, and 2021; Bedin et al., 2020; Michel et al., 2020).

Competing interests

There are no conflicts of interest or competing interests.

Ethics

All enrolled patients provided informed consent, and the procedures followed were in accordance with the Helsinki Declaration. Routine care of the subjects was not modified; analyses were performed on anonymized left-over blood, and all data collected in the study were part of routine clinical practice and retrieved from subject records. Results of this study had no influence on subject management.

References

  1. Bedin, A.S., Makinson, A., Picot, M.C., Mennechet, F., Malergue, F., Pisoni, A., Nyiramigisha, E., Montagnier, L., Bollore, K. and Debiesse, S. J. (2020). Monocyte CD169 expression as a biomarker in the early diagnosis of COVID-19. J Infect Dis 223(4): 562-567.
  2. Bourgoin, P., Biechele, G., Ait Belkacem, I., Morange, P. E. and Malergue, F. (2020a). Role of the interferons in CD64 and CD169 expressions in whole blood: Relevance in the balance between viral- or bacterial-oriented immune responses. Immun Inflamm Dis 8(1): 106-123.
  3. Bourgoin, P., Hayman, J., Rimmele, T., Venet, F., Malergue, F. and Monneret, G. (2019a). A novel one-step extracellular staining for flow cytometry: Proof-of-concept on sepsis-related biomarkers. J Immunol Methods 470: 59-63.
  4. Bourgoin, P., Lediagon, G., Arnoux, I., Bernot, D., Morange, P. E., Michelet, P., Malergue, F. and Markarian, T. (2020b). Flow cytometry evaluation of infection-related biomarkers in febrile subjects in the emergency department. Future Microbiol 15: 189-201.
  5. Bourgoin, P., Soliveres, T., Ahriz, D., Arnoux, I., Meisel, C., Unterwalder, N., Morange, P. E., Michelet, P., Malergue, F. and Markarian, T. (2019b). Clinical research assessment by flow cytometry of biomarkers for infectious stratification in an Emergency Department. Biomark Med 13(16): 1373-1386.
  6. Bourgoin, P., Soliveres, T., Barbaresi, A., Loundou, A., Belkacem, I. A., Arnoux, I., Bernot, D., Loosveld, M., Morange, P. E., Michelet, P., Malergue, F. and Markarian, T. (2021). CD169 and CD64 could help differentiate bacterial from CoVID-19 or other viral infections in the Emergency Department. Cytometry A 99(5): 435-445.
  7. Bourgoin, P., Taspinar, R., Gossez, M., Venet, F., Delwarde, B., Rimmele, T., Morange, P. E., Malergue, F. and Monneret, G. (2020c). Toward Monocyte HLA-DR Bedside Monitoring: A Proof of Concept Study. Shock 55(6): 782-789.
  8. Brown, M. and Wittwer, C. (2000). Flow cytometry: principles and clinical applications in hematology. Clin Chem 46(8 Pt 2): 1221-1229.
  9. Michel, M., Malergue, F., Belkacem, I. A., Bourgoin, P., Morange, P.-E., Arnoux, I., Miloud, T., Million, M., Tissot-Dupont, H., Mege, J. L., Busnel, J. M. and Vitte, J. (2020). An ultra-sensitive, ultra-fast whole blood monocyte CD169 assay for COVID-19 screening. medRxiv.
  10. van Agthoven, A. (2007). VersaLyse and the mechanism of ammonium chloride lysis. Int J Lab Hematol 29: 65-66.

简介

[摘要]流式细胞术是一种强大的分析技术,越来越多地用于科学研究和医疗保健;ħ H但是,它需要耗时的多步骤程序的样品,这限制了它的使用,以专业实验室。在这项研究中,我们提出在其中白血细胞染色和红血细胞裂解在单个步骤中进行一个新的通用的一步方法,使用温和的LYSI小号溶液在干燥的或液体的荧光标记抗体或探针混合格式。血液样本可以从常规静脉穿刺或直接从指尖获取,以便在患者附近进行分析。此过程使在普通的白血细胞标记物以及相关的感染或败血症标志物分析。这种更简单、更快速的协议可能有助于使流式细胞术在研究和医学领域的使用民主化。

图文摘要:

流式细胞术的一步法白细胞细胞外染色法。

[背景]流式细胞术越来越多地用于科学研究和医疗保健;然而,它需要一个耗时的、多步骤的样品制备程序,这限制了它在专业实验室的使用。为胞外染色最全血样品制备协议至少包含三个步骤:ü唱在抗凝采样管中收集静脉血样,50 - 100微升被移液到反应管; 一个是个第二步骤中,荧光抗体混合,并用血一起温育,以允许白细胞染色; 一个是个第三步骤中,LYSI小号溶液加入到反应管中,以允许对红血细胞去除; ˚F inally,任选的洗涤步骤被执行以重新达斯非特异性荧光。

由于这些需要在专业实验室中进行的多个定时步骤,流式细胞术的使用受到限制。例如,尽管已经证明对患者免疫细胞的评估有助于指示潜在的疾病活动,但它尚未常规用于临床领域(Brown 和 Wittwer,2000)。一些作者试图建立只有一个步骤的协议,建议取消裂解步骤;然而,这种方法需要专用的流式细胞仪,限制了这种方法的使用(Petriz等,2018)。

在这项研究中,我们提出了一种新的通用方法,其中白细胞染色和红细胞裂解在一个步骤中进行。该LYSI小号溶液是在室温下(RT)稳定和不Ñ ö t用染色干扰由于其组合物,其包括具体地由唯一地存在于红细胞细胞膜的酶切割的底物的(van Agthoven,2007) 。白细胞在等渗条件下保持在中性 pH 值,因此不受影响。该LYSI小号溶液C的包括低剂量甲醛(0.05%)吨Ö稳定感兴趣的标记(布尔昆等人,2020C) 。

偶联物的精细滴定和它们在裂解液中的直接稀释能够消除最后的洗涤步骤,同时保持低背景水平。使用新的干成套试剂测定(DURA)的创新技术,该缀合物Ç的被干燥,这使得RT贮存和消除了对吸移的需要,从而更好地支持标准化。血液样本可能来自静脉或来自指尖,允许进行适合床旁护理环境的侵入性较小的采样。由于裂解液体积过大,这种方法成为可能,它允许在多达 50 µl 的血液中进行有效的 RBC 裂解。所述缀合物的量也被调节至污点瓦特海特血细胞包含在2 - 50微升的血液,用少量的血液不再是一个限制,因为2微升血液中含有平均10 ,000亮氨酸ķ ocytes。由于 100 个细胞亚群通常被认为具有统计代表性,因此使用 2 µl 血液可以分析低至 1% 的亚群,这对于大多数常规应用来说已经足够了。

由于急诊科 (ED) 感染患者的管理对从业者来说具有挑战性,因此我们开发了一个面板,可以使用流式细胞术在 ED 中快速对患者进行分类。已经表明,通过流式细胞术监测单核细胞 (mCD169) 上的 CD169、中性粒细胞 (nCD64) 上的 CD64 和单核细胞 (mHLA-DR) 上的 HLA-DR 的表达可分别指示病毒或细菌感染或败血症. 因此,我们建立了由抗体靶向这些三个标记的面板和评估的一步法我n,其中由感染和感染性的条件的受试者测量的三个感染相关的标志物的表达(布尔昆等人。,2019a,2019b,2020A, 2020b、2020c 和 2021;贝丁等人,2020 年;米歇尔等人,2020 年)。

在常规进行流式细胞术的领域中,可以设想许多其他应用,例如测量 T、B 和 NK 细胞比例,检测白血病和淋巴瘤,以及计数用于移植的CD34 +干细胞。

关键字:血, 白细胞, 流式细胞术, 一步法, 医疗点

材料和试剂
 
定期˚F低血细胞计数5 -米升试管,聚丙烯或聚苯乙烯(12 × 75 mm)时,或1.4 -米升微管(例如。,,或从深Micronic公司)-孔板(任何供应商)。
雷瑟小号溶液(Beckman Coulter公司,VersaLyse TM ,目录号:IM3648,保存于18-24℃,2 -年的保质期) 
固定液(IOTest ® 3 10 × ;Beckman Coulter,目录号:A07800,在 2-8°C 下储存,1 年保质期)
Antibod ÿ鸡尾酒(IOTest髓激活CD169-PE(藻红蛋白)/ HLA-DR-APC(别藻蓝蛋白)/ CD64-PB(太平洋蓝)从Beckman Coulter公司,目录号抗体混合物:C63854,储存在2-8℃, 1 年保质期)
在LYSI易批量制备小号缓冲液(见食谱)
染色的批量制备和LYSI小号n个测试缓冲(见食谱)
 
设备
 
移液器(吉尔森,移液器® ,目录号小号:FA10003M [ 2-20微升]和FA10006M [ 100-1000微升] )
3 激光、10 色细胞计数器(Beckman Coulter ,Navios,目录号:B47904)或 3 激光 13 色细胞计数器(Beckman Coulter ,CytoFLEX,目录号:B53000)。任何ø疗法3-激光流式细胞仪都可以使用(例如。,Becton Dickinson公司的FACSCanto,Cytek极光)
 
软件
 
Kaluza 软件 2.1 版(Beckman Coulter,https://www.beckman.fr/flow-cytometry/software/kaluza)
注意:可以使用任何其他流式细胞术软件(例如,Becton Dickinson Flowjo 、Cytek SpectroFlo)。
 
程序
 
准备LYSI小号BUF FER(w ^耳普通个人防护装备)。
在化学罩保护下将裂解液中的固定液稀释 1:200 并混合。
执行lys是–染色步骤。
在含有500个微升赖氨酸的管是缓冲液和10微升共轭面板中,添加2-50微升的血液样品(VE理性或毛细管),混合,并孵育对于在黑暗中在15分钟RT (18-25℃)。
获取关于一个3激光cytomete数据R(吨母鸡处置的在专用于生物危害废物容器废物)。
 
数据分析
 
A.在流式细胞仪上获取数据      
未染色的细胞用于设置流式细胞仪的参数。
打开流式细胞仪。
打开采集软件并创建四个点图:
x 轴上的侧向散射 (SSC) 和 y 轴上的前向散射 (FSC)。
信道专用于PE(FL2上NAVIOS)检测上吨他X轴和SSC在y -轴。
Chanel的专用于APC(FL6上NAVIOS)检测上吨他X轴和SSC在y轴。
Chanel 专用于x 轴上的 PB(Navios 上的 FL9)检测和 y 轴上的 SSC 。
设置了电压/增益的SSC-FSC情节,使得大部分的细胞群是在曲线图(图1a)的中间。应以获取最少碎片的方式设置判别式/阈值。
将所有补偿设置为 0。
设置 FL9、FL2 和 FL6 的电压,使淋巴细胞平均或中值荧光强度约为 0.3 (图 1b)。
 
 
图1.流cytomet RY上的(a)SSC-FSC情节和(b)FL2-,FL6-,参数和FL9-SSC图
 
保存协议。
使用高流速 ( ~ 1 µl/s)获取第一个染色样品,无需任何补偿。确保获得至少 500 个单核细胞以进行可靠的分析(通常为 30 到 90 秒)。
在SSC-FSC情节,画出一个亮氨酸ķ ocyte栅极,避免碎片(图2a) 。
在SSC-FL9(CD64-PB)图,画出淋巴细胞,单核细胞,和嗜中性粒细胞闸门(图2b) 。
CD169 和 HLA-DR 表达水平应在单核细胞上进行评估。CD64表达水平应该在嗜中性粒细胞进行评估(图2b和2 C) 。
 
 
图 2. 数据分析程序。(a和b)的淋巴细胞,单核细胞的门控策略,和嗜中性粒细胞。(c) CD169、CD64 和 HLA-DR 表达水平评估策略。
 
标记水平应该表示为在荧光的平均强度,荧光强度的中位数,或信号-到-噪声。信号-到-噪声被计算如下:在单核细胞CD169表达水平应当由淋巴细胞CD169表达水平被划分; 中性粒细胞上的 CD64 表达水平应除以淋巴细胞上的 CD64 表达水平;单核细胞上的 HLA-DR 表达水平应除以中性粒细胞上的 HLA-DR 表达水平(图 3)。
结果被认为如下:高 mCD169 表达指示病毒刺激,高 nCD64 表达指示细菌刺激,高 mHLA-DR 表达指示感染性刺激,低 mHLA-DR 表达指示免疫衰竭(图 3).
 
 
图3.米CD169,nCD64 ,在全血中使用一步法和mHLA-DR表达的评估。例如一个用于一个健康志愿者,一个VIR再给予我们-感染的病人,一个细菌-感染的病人,1例败血症。
 
需要更多的研究来精确设置每个实验室环境中的阈值,但作为一个指标,之前的评估表明,CD169 和 CD64 指数es 的阈值应该接近 3 。
可以使用 Kaluza 等软件进行进一步分析(重新分析、其他门控和自动门控、其他直方图或点图、着色、补偿调整、精炼百分比和荧光值、叠加和合并数据集、批量分析以提高吞吐量,等。)。
 
笔记
 
根据目标标记、应用和所需的稳定性,该方法应适用于每个实验室设置。例如,仅用于指示而不是作为既定性能,在某些应用中观察到:
血液试料c的是静脉或毛细血管。
血液Ç一个用EDTA或肝素抗凝来。
多亏了温和裂解试剂和固定液加入,血液Ç一个采样后进行处理达3-4天,当储存在室温或2-8℃。此外,样品C的被分析上的流式细胞仪的处理后3天,当储存在2-8℃。
裂解液可以包括 0.05% 的固定液。
裂解缓冲液(LYS是溶液含固定溶液)C的在RT贮存长达一个月。
靶向标记可以是任何膜标记。
抗体Ç一个是液体或取决于产品可用性干燥。
警告:
如果使用液体混合物,超过100 µl 的抗体可能会影响裂解效率。
如果使用的是商业DURAClone格式,所述裂解体积应当增加至1ml(为2-50微升血液)或2毫升(50-100微升血液按照IFU)。
抗体Ç一个与裂解预混缓冲高达几天之前。
 
食谱
 
注意:根据每个应用进行调整;表演不成立。
LYSI易一批准备小号缓冲
一瓶 Versalyse 溶液(100 毫升)
500 μ升固定溶液(10 × )
混合并储存在室温下
染色的批量制备和LYSI小号n个测试缓冲
移液管 (n+1) × 500 µl 裂解缓冲液
添加 (n+1) × 10 µl 髓样激活混合物(或任何其他面板的最佳剂量)
混匀,每管分装 510 µl,避光保存
试管现在准备好接收血样
 
致谢
 
IAB是在收件人一CIFRE博士 ANRT(国家研究与技术局)的资助(N°2018/1212)。这项工作得到了贝克曼库尔特 (Beckman Coulter) 的支持,捐赠了流式细胞术实验中使用的研究试剂,以及四位员工的参与:IAB、PB、JMB 和 FM。所描述的方法是从“VersaLyse 和氯化铵裂解机制” (van Agthoven,2007 年)修改而来的,并产生了最近的出版物(Bourgoin等人,2019a、2019b、2020a、2020b、2020c 和 2021;Bedin等人。 2020;米歇尔等人,2020) 。
 
利益争夺
 
有没有利益或竞争利益冲突小号。
 
伦理
 
所有入选患者签署知情同意书,并在随后的程序均符合赫尔辛基宣言。受试者的日常护理没有改变;分析在匿名留下执行-过血,并在研究中收集的所有数据都是常规的临床实践的一部分,从主题的记录取得。次的结果是研究对受管理没有影响。
 
参考
 
Bedin, A. S., Makinson, A., Picot, MC, Mennechet, F., Malergue, F., Pisoni, A., Nyiramigisha, E., Montagnier, L., Bollore, K. 和 Debiesse, SJ ( 2020)。单核细胞 CD169 表达作为 COVID-19 早期诊断的生物标志物。J Infect Dis 223(4): 562-567。             
Bourgoin, P.、Biechele, G.、Ait Belkacem, I.、Morange, PE 和 Malergue, F. (2020a)。干扰素在全血 CD64 和 CD169 表达中的作用:病毒或细菌免疫反应之间平衡的相关性。免疫炎症 8(1): 106-123。
Bourgoin, P.、Hayman, J.、Rimmele, T.、Venet, F.、Malergue, F. 和 Monneret, G. (2019a)。一种用于流式细胞术的新型一步细胞外染色:脓毒症相关生物标志物的概念验证。J 免疫学方法470:59-63。
Bourgoin, P.、Lediagon, G.、Arnoux, I.、Bernot, D.、Morange, PE、Michelet, P.、Malergue, F. 和 Markarian, T. (2020b)。急诊科发热患者感染相关生物标志物的流式细胞术评估。未来微生物15:189-201。              
Bourgoin, P., Soliveres, T., Ahriz, D., Arnoux, I., Meisel, C., Unterwalder, N., Morange, PE, Michelet, P., Malergue, F. 和 Markarian, T. (2019b )。通过流式细胞术对急诊科感染分层生物标志物的临床研究评估。Biomark Med 13(16): 1373-1386。
Bourgoin, P., Soliveres, T., Barbaresi, A., Loundou, A., Belkacem, IA, Arnoux, I., Bernot, D., Loosveld, M., Morange, PE, Michelet, P., Malergue, F. 和 Markarian, T. (2021)。CD169 和 CD64 可以帮助在急诊科区分细菌与 CoVID-19 或其他病毒感染。 细胞计数法 A 99(5):435-445 。
Bourgoin, P., Taspinar, R., Gossez, M., Venet, F., Delwarde, B., Rimmele, T., Morange, PE, Malergue, F. 和 Monneret, G. (2020c)。走向单核细胞 HLA-DR 床边监测:概念研究的证明。休克55(6):782-789。
Brown, M. 和 Wittwer, C. (2000)。流式细胞术:血液学的原理和临床应用。临床化学46(8 Pt 2):1221-1229。
Michel, M., Malergue, F., Belkacem, IA, Bourgoin, P., Morange, P.-E., Arnoux, I., Miloud, T., Million, M., Tissot-Dupont, H., Mege , J.-L., Busnel, J.-M. 和 Vitte, J. (2020)。用于 COVID-19 筛查的超灵敏、超快速全血单核细胞 CD169 检测。医学Rxiv 。
van Agthoven, A. (2007)。VersaLyse和氯化铵裂解的机制。 诠释J实验室血液学29:65 - 66。
 
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引用:Belkacem, I. A., Bourgoin, P., Busnel, J. M., Galland, F. and Malergue, F. (2021). One-step White Blood Cell Extracellular Staining Method for Flow Cytometry. Bio-protocol 11(16): e4135. DOI: 10.21769/BioProtoc.4135.
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