PBMC細(xì)胞的精確計(jì)數(shù)和活性分析

 

1. 目前，絕大多數(shù)的科研工作者仍在使用血球計(jì)數(shù)板在顯微鏡下進(jìn)行手工計(jì)數(shù)PBMCs；

2. 但是顯微鏡下由于白細(xì)胞和紅細(xì)胞大小非常接近，不能完全區(qū)分白細(xì)胞和紅細(xì)胞；

3. 通過(guò)紅細(xì)胞的雙凹形形態(tài)鑒別紅細(xì)胞，需要經(jīng)驗(yàn)豐富的操作者，且不停地調(diào)焦來(lái)鑒別；可想而知要把每個(gè)紅細(xì)胞鑒別出來(lái)，要耗費(fèi)多長(zhǎng)的時(shí)間，需要多強(qiáng)的工作量。

 

由于個(gè)體的差異（如正常人和病人）和人工分離提取的差異，紅細(xì)胞和血小板污染的程度差異性非常大，因此我們經(jīng)常看到分離后的PBMC樣本千差萬(wàn)別（見(jiàn)下圖），這也大大增加了在同一條件下，簡(jiǎn)單快速準(zhǔn)確計(jì)數(shù)PBMC的難度和檢測(cè)PBMC活性的難度。

PBMC（外周血單個(gè)核細(xì)胞）是免疫學(xué)功能研究中最常用的細(xì)胞模型，如細(xì)胞增殖、細(xì)胞毒性、細(xì)胞因子分泌等。

 

如在癌癥免疫治療領(lǐng)域中，需從病人全血中分離出PBMC，分離的PBMC進(jìn)一步擴(kuò)增或進(jìn)行不同的功能檢測(cè)。激活擴(kuò)增后的細(xì)胞再回輸?shù)讲∪梭w內(nèi)進(jìn)行細(xì)胞治療。在整個(gè)流程中，從分離到檢測(cè)，到培養(yǎng)，到回輸?shù)冗^(guò)程，細(xì)胞濃度和細(xì)胞活力都是必須監(jiān)測(cè)的參數(shù)。

 

密度梯度分離液是從外周血、骨髓，及臍血分離單個(gè)核細(xì)胞最常用的方法。但是不可避免的，分離后的細(xì)胞中，會(huì)有殘存的紅細(xì)胞及血小板混合在單個(gè)核細(xì)胞中。殘存的紅細(xì)胞和血小板的多少，與個(gè)體的差異，以及分離的效果相關(guān)；但是不管分離的技術(shù)多高超，經(jīng)驗(yàn)多豐富，都不可避免會(huì)存在紅細(xì)胞和血小板的污染。

 

1.       隨著時(shí)間的延長(zhǎng)，細(xì)胞質(zhì)量下降；

2.       臨床試驗(yàn)相關(guān)的樣品量很大；

3.       細(xì)胞樣品不純，分離之路依賴病人樣本和操作者

 

如何精確、快速、簡(jiǎn)便計(jì)數(shù)PBMC以及精確分析PBMC活力，是免疫學(xué)研究以及免疫治療領(lǐng)域至關(guān)重要的步驟。

1.       使用血球計(jì)數(shù)板在顯微鏡下進(jìn)行手工計(jì)數(shù)PBMC，即耗時(shí)耗人力，更不能達(dá)到精確計(jì)數(shù)的目的。

2.       通過(guò)明場(chǎng)的常規(guī)細(xì)胞計(jì)數(shù)儀同樣不能區(qū)分紅細(xì)胞，不能達(dá)到精確計(jì)數(shù)PBMC和精確活力分析的目的。

3.       迫切需要快速、簡(jiǎn)便、精確計(jì)數(shù)PBMC的工具替代人工計(jì)數(shù)。Nexcelom公司走在了市場(chǎng)領(lǐng)先的位置，在常規(guī)明場(chǎng)的細(xì)胞計(jì)數(shù)儀基礎(chǔ)上，開(kāi)發(fā)出了雙熒光細(xì)胞活力分析儀，可通過(guò)AOPI染料進(jìn)行PBMC的快速精確計(jì)數(shù)和活力分析。

 

1.     Preliminary Report: Evaluation of Storage Conditions and Cryococktails during Peripheral Blood Mononuclear Cell Cryopreservation", L.M. Cosentino, W. Corwin, J.M Baust, N.Diaz-Mayoral, H.Cooley, W. Shao, R. Van Buskirk, and J.G Baust, Cell Preservation Technology, Volume 5 Number 4, 2007

2.     "Viability and Functional Activity of Cryopreserved Mononuclear Cells", A. Weinberg, L. Zhang, D. Brown, A. Brice, B.Polsky, M. S. Hirsch, S. Owens, and K. Lamb Clincal and Diagnostic Laboratory Immunology, July, 2000, P714-716

3.     "Cell loss and recovery in umbilical cord blood processing: a comparison of postthaw and postwash samples", V. Laroche, D. H. McKenna, G. Moroff, T. Schierman, D. Kadidlo, and J. McCullough, Transfusion, Vol., 45, Dec. 2005.

4.     "Viability and Recovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study", C. A. Kleeberger, R. H. Lyles, J. B. Margolick, C. R. Rinaldo, J. P. Phair, and J. V. Giorgi, Clinccal and Diagnostic laboratory Immunology, Vol. 6, No. 1, Jan. 1999.

 

通過(guò)AOPI染料進(jìn)行雙熒光計(jì)數(shù)和活力分析，是可以排除紅細(xì)胞、血小板、細(xì)胞碎片等污染的精確計(jì)數(shù)方法。

AO（吖啶橙）和PI（碘化丙啶）是可對(duì)DNA染色的細(xì)胞核染色試劑。其中AO可以通過(guò)完整的細(xì)胞膜，嵌入所有細(xì)胞（活細(xì)胞和死細(xì)胞）的細(xì)胞核，呈現(xiàn)綠色熒光；PI只能通過(guò)不完整的細(xì)胞膜，即死細(xì)胞的細(xì)胞膜，嵌入所有死細(xì)胞的細(xì)胞核，呈現(xiàn)紅色熒光。

活死單個(gè)核細(xì)胞可呈現(xiàn)熒光信號(hào)。而成熟的紅細(xì)胞及血小板，因?yàn)闆](méi)有細(xì)胞核，不能被AO/PI染色，因此可以完全被排除在外不被計(jì)數(shù)。

通過(guò)AOPI染料進(jìn)行雙熒光計(jì)數(shù)和活力分析，可以精確計(jì)數(shù)分離后的PBMC以及活力分析，亦可進(jìn)行全血中的PBMC。

 

 

             

     

1.       雙熒光計(jì)數(shù)和活力分析是PBMC計(jì)數(shù)的最佳選擇方法。

三款儀器型號(hào)可選：AUTO2000/K2/ VISION CBA，可精確、快速、簡(jiǎn)便進(jìn)行PBMC計(jì)數(shù)和活力分析。

      

觸屏操控                                       電腦操控          電腦操控，高配置，可分析凋亡&細(xì)胞周期等

 

2.       明場(chǎng)的自動(dòng)細(xì)胞計(jì)數(shù)儀進(jìn)行PBMC計(jì)數(shù)，可通過(guò)臺(tái)盼藍(lán)排斥法進(jìn)行PBMC的活力檢測(cè)。

雖然也能達(dá)到自動(dòng)、快速的目的，但是仍無(wú)法有效的排除紅細(xì)胞的干擾，達(dá)到精確計(jì)數(shù)的目的。

臺(tái)盼藍(lán)排斥法檢測(cè)細(xì)胞死活，是通過(guò)臺(tái)盼藍(lán)這個(gè)細(xì)胞活性染料，其不能透過(guò)活細(xì)胞正常完整的細(xì)胞膜，故活細(xì)胞不著色，但死亡細(xì)胞的細(xì)胞膜通透性增加，可使染料通過(guò)細(xì)胞膜進(jìn)入細(xì)胞內(nèi)，使死細(xì)胞著色呈藍(lán)色。是最常用的檢測(cè)細(xì)胞活率的方法。

但是臺(tái)盼藍(lán)排斥法并不能精確計(jì)數(shù)活死細(xì)胞，原因是細(xì)胞膜通透性不一樣，進(jìn)入細(xì)胞內(nèi)的染料差異也很大，所以經(jīng)常會(huì)出現(xiàn)很難判定是死細(xì)胞還是活細(xì)胞。

如果對(duì)PBMC計(jì)數(shù)的準(zhǔn)確性要求不是很高，但是需要快速、自動(dòng)計(jì)數(shù)，而且對(duì)實(shí)驗(yàn)的一致性和重復(fù)性的要求高，可選擇以下三款計(jì)數(shù)儀：AUTO1000/MINI/AUTO T4。

        

電腦操控，小巧美觀，性價(jià)比高           一體化設(shè)計(jì)，觸屏操控          經(jīng)典款，符合GLP/GMP

 

1.     Chan, L.L., Wilkinson, A.R., Paradis, B.D. and Lai, N. (2012b) Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods. Journal of Fluorescence 22, 1301-1311.

2.     Almeida, C.-A.M., Roberts, S.G., Laird, R., McKinnon, E., Ahmed, I., Pfafferott, K., Turley, J., Keane, N.M., Lucas, A., Rushton, B., Chopra, A., Mallal, S. and John, M. (2009) Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses. Journal of Immunological Methods 344, 1-5.

3.     Constantino, B.T. and Cogionis, B. (2000) Nucleated RBCs - Significance in the Peripheral Blood Film. Laboratory Medicine 31, 223-229.

4.     Laroche, V., McKenna, D.H., Moroff, G., Schierman, T., Kadidlo, D. and McCullough, J. (2005) Cell loss and recovery in umbilical cord blood processing: a comparison of postthaw and postwash samples. Transfusion 45, 1909-1916.

5.     Sigfusson, A. and Souhami, R. (1984) The Effects of Erythrocyte Contamination on Pokeweed Mitogen Induced Immunoglobulin-Synthesis in Man. Journal of Immunological Methods 72, 167-170.

6.     Szabo, S.E., Monroe, S.L., Fiorino, S., Bitzan, J. and Loper, K. (2004) Evaluation of an Automated Instrument for Viability and Concentration Measurements of Cryopreserved Hematopoietic Cells. Laboratory Hematology 10, 109-111.

 

Author	Date	Title	Journal	Cell Type	Cellometer / Applications
Mahato, Ram I	November 2013	Synthesis and Characterization of an Anti-Apoptotic Immunosuppressive Compound for Improving the Outcome of Islet Transplantation	Bioconjugate Chemistry	PBMC	Cellometer Auto T4... Cell Counting
Banerjee, Subhadra	October 2013	Expression of the B-Cell Receptor Component CD79a on Immature Myeloid Cells Contributes to Their Tumor Promoting Effects	PLOS ONE	PBMC	Cellometer (Not Specified)... Cell Concentration
Bogoslovsky, Tanya	September 2013	Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials	Blood Disorders and Transfusion	PBMC	Cellometer Auto T4... Trypan Blue Exclusion
Singh, Harjeet	May 2013	Manufacture of Clincal-Grade CD-19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artifical Antigen Presenting Cells	PLoS one	PBMC, Daudi, NALM-6, EL-4	Cellometer Unspecfied... Trypan blue viability
Shankar Pandey, Gouri	January 2013	Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry	BioMed Research International	PBMCs	Cellometer Unspecfied... Trypan blue viability
Filbert, Helene	October 2012	Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols	Cancer Immunology and Immunotherapy	PBMC	Nexcelom Cellometer... Trypan blue viability
Duran, M.C.	July 2012	Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti-human CD14 mAb and automated magnetic activated cell sorting	Equine Veterinary Journal	PBMCs	Cellometer Auto T4... Trypan blue viability
O'Connor, Colleen M.	February 2012	Adoptive T-cell therapy improves treatment of canine non-Hodgkin lymphoma post chemotherapy	Scientific Reports	T-cells and PBMC	Cellometer Auto T4... Trypan blue viability
Clark, Eva H.	January 2012	Plasmodium falciparum Malaria in the Peruvian Amazon, a Region of low Transmission, Is Associated with Immunologic Memory	Infection and Immunity	PBMC	Cellometer Auto T4... Trypan blue viability
Sethu, Palaniappan	December 2011	Clinical application of microfluidic leukocyte enrichment protocol in mild phenotype sickle cell disease (SCD)	Biomedical Microdevices	Leukocyte-enriched sample, PBMC	Cellometer (Not Specified)... Cell Number
Uhl, Elizabeth	August 2011	Identification of Altered MicroRNA Expression in Canine Lymphoid Cell Lines and Cases of B- and T-Cell Lymphomas	Genes, Chromosomes, & Cancer	GL-1; CL-1, LN, PBMC	Cellometer Auto T4... Other
Lee, Moo-Seung	June 2011	Shiga toxins induce autophagy leading to differential signalling pathways in toxin-sensitive and toxin resistant human cells	Cellular Microbiology	Primary PBMCs, THP-1	Cellometer Automated Cell counter... Other
Zierold, Claudia	May 2011	Developing mechanistic insights into cardiovascular cell therapy: Cardiovascular Cell Therapy Research Network Biorepository Core Laboratory rationale	American Heart Journal	Bone Marrow, PBMC	Cellometer Auto T4... Trypan blue viability
Pathak, Shresh	February 2011	IL-1B; Is Overexpressed and Aberrantly Regulated in Corticosteroid Nonresponders with Autoimmune Inner Ear Disease	The Journal of Immunology	PBMC	Cellometer Auto T4... Trypan blue viability
Wullner, Danika	June 2010	Considerations for optimization and validation of an in vitro PBMC derived T cell assay for immunogenicity prediction of biotherapeutics	Clinical Immunology	PBMC, Dendritic, CD4	Cellometer Auto T4... Trypan blue viability
Bruchova, Hana	March 2009	Erythropoiesis in polycythemia vera is hyper-proliferative and has accelerated maturation	Blood Cells, Molecules, and Diseases	PBMC	Cellometer Auto T4... Trypan blue viability
Meyers, John A.	February 2009	Chronic Lymphocytic Leukemia and B and T Cells Differ in their Response to Cyclic Nucleotide Phosphodiesterase Inhibitors	The Journal of Immunology	CLL cells, PBMC	Cellometer Auto T4/M10... Normalizati
White, William N.	December 2008	Clinical application of microfluidic leukocyte enrichment protocol in mild phenotype sick cell disease (SCD)	Biomedical Microdevices	PBMC, PNM, Erythrocytes	Cellometer Auto T4... Other
Bruchova, Hana	August 2007	Regulated expression of microRNAs in normal and polycythemia vera erythropoiesis	Experimental Hematology	PBMC, JAK2 V617F	Cellometer Auto T4... Trypan blue viability