PBMC細胞的精確計數(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數(shù)板在顯微鏡下進行手工計數(shù)PBMCs；

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

3. 通過紅細胞的雙凹形形態(tài)鑒別紅細胞，需要經(jīng)驗豐富的操作者，且不停地調(diào)焦來鑒別；可想而知要把每個紅細胞鑒別出來，要耗費多長的時間，需要多強的工作量。

 

由于個體的差異（如正常人和病人）和人工分離提取的差異，紅細胞和血小板污染的程度差異性非常大，因此我們經(jīng)�？吹椒蛛x后的PBMC樣本千差萬別（見下圖），這也大大增加了在同一條件下，簡單快速準確計數(shù)PBMC的難度和檢測PBMC活性的難度。

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

 

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進一步擴增或進行不同的功能檢測。激活擴增后的細胞再回輸?shù)讲∪梭w內(nèi)進行細胞治療。在整個流程中，從分離到檢測，到培養(yǎng)，到回輸?shù)冗^程，細胞濃度和細胞活力都是必須監(jiān)測的參數(shù)。

 

密度梯度分離液是從外周血、骨髓，及臍血分離單個核細胞最常用的方法。但是不可避免的，分離后的細胞中，會有殘存的紅細胞及血小板混合在單個核細胞中。殘存的紅細胞和血小板的多少，與個體的差異，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)驗多豐富，都不可避免會存在紅細胞和血小板的污染。

 

1.       隨著時間的延長，細胞質(zhì)量下降；

2.       臨床試驗相關(guān)的樣品量很大；

3.       細胞樣品不純，分離之路依賴病人樣本和操作者

 

如何精確、快速、簡便計數(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.       使用血球計數(shù)板在顯微鏡下進行手工計數(shù)PBMC，即耗時耗人力，更不能達到精確計數(shù)的目的。

2.       通過明場的常規(guī)細胞計數(shù)儀同樣不能區(qū)分紅細胞，不能達到精確計數(shù)PBMC和精確活力分析的目的。

3.       迫切需要快速、簡便、精確計數(shù)PBMC的工具替代人工計數(shù)。Nexcelom公司走在了市場領(lǐng)先的位置，在常規(guī)明場的細胞計數(shù)儀基礎(chǔ)上，開發(fā)出了雙熒光細胞活力分析儀，可通過AOPI染料進行PBMC的快速精確計數(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AOPI染料進行雙熒光計數(shù)和活力分析，是可以排除紅細胞、血小板、細胞碎片等污染的精確計數(shù)方法。

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

活死單個核細胞可呈現(xiàn)熒光信號。而成熟的紅細胞及血小板，因為沒有細胞核，不能被AO/PI染色，因此可以完全被排除在外不被計數(shù)。

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

 

 

             

     

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

三款儀器型號可選：AUTO2000/K2/ VISION CBA，可精確、快速、簡便進行PBMC計數(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&細胞周期等

 

2.       明場的自動細胞計數(shù)儀進行PBMC計數(shù)，可通過臺盼藍排斥法進行PBMC的活力檢測。

雖然也能達到自動、快速的目的，但是仍無法有效的排除紅細胞的干擾，達到精確計數(shù)的目的。

臺盼藍排斥法檢測細胞死活，是通過臺盼藍這個細胞活性染料，其不能透過活細胞正常完整的細胞膜，故活細胞不著色，但死亡細胞的細胞膜通透性增加，可使染料通過細胞膜進入細胞內(nèi)，使死細胞著色呈藍色。是最常用的檢測細胞活率的方法。

但是臺盼藍排斥法并不能精確計數(shù)活死細胞，原因是細胞膜通透性不一樣，進入細胞內(nèi)的染料差異也很大，所以經(jīng)常會出現(xiàn)很難判定是死細胞還是活細胞。

如果對PBMC計數(shù)的準確性要求不是很高，但是需要快速、自動計數(shù)，而且對實驗的一致性和重復(fù)性的要求高，可選擇以下三款計數(shù)儀：AUTO1000/MINI/AUTO T4。

        

電腦操控，小巧美觀，性價比高           一體化設(shè)計，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