Counting Nucleated Cells from Whole Blood

Nucleated blood cell, or white blood cell (WBC), concentration is a critical parameter in immunology research. Blood samples are often used as starting material in life science and contain a mixture of different cells e.g. lymphocytes, monocytes, granulocytes, hematopoietic stem cells as well as erythrocytes and platelets. The nucleated cell fraction is very interesting for scientists working in vaccine and assay development, transplant immunology and cancer research^1,2.

As the isolation procedure of different subpopulations from blood is usually time-consuming and expensive, the NucleoCounter^® instruments can determine the number of nucleated cells prior to, and throughout, the isolation process for routine monitoring. Evaluate the quality of blood samples after shipping or storage and use the NucleoCounter^® instruments for assay readout in pre-clinical animal models and for determining cell count from bone marrow aspirates.

The NucleoCounter^® instruments are a rapid, cost-effective alternative to conventional hematology analyzers, using red blood cell lysis and dual-fluorescence nuclear staining to measure the nucleated cell count and viability in whole blood.

The analysis itself only takes 50 seconds and does not require instrument warm-up or calibration. Thus, the NucleoCounter® is the ideal instrument for research and bioprocessing laboratories with moderate sample processing needs. In contrast to hematology analyzers, the NucleoCounter^® can be used more broadly for counting cultured cells, with unparalleled precision.

Hematology analyzers are often used to count and characterize blood cells; however, they can be complex and expensive to operate. The NucleoCounter^® instruments offer an easy and fast protocol to precisely determine viability and cell count directly from whole blood. Adding Solution 17 to a blood sample followed by a short incubation time lyses the erythrocytes, allowing the NucleoCounter^® instrument to analyze the blood sample using the Via2-Cassette™, NC-Slide A2™ or NC-Slide A8™. The NucleoCounter^® can be used for a broad range of counting applications e.g. in adoptive immunotherapies and for counting bone marrow-derived mesenchymal stem cells.

Counting Nucleated Cells in Whole Blood Samples

The following method is used to obtain precise results when counting count nucleated cells in a whole blood sample with a NucleoCounter^®. First, you add Solution 17, a blood lysis buffer, to the whole blood sample. The ammonium chloride within the solution leads to swelling and osmotic rupture of erythrocytes³ and the small amount of acridine orange (AO) compensates for the quenching effect of the hemoglobin.

After a short incubation time, the sample is stained with the two fluorescent dyes, AO and DAPI. These dyes are pre-loaded and contained within the Via2-Cassette™ and used with the NucleoCounter^® NC-202™ for analysis. AO will stain all cells green and DAPI will only stain the dead cells blue. To determine viability and cell count, the NucleoCounter^® instruments will automatically perform fluorescent image acquisition and calculate the results. The NC-View™ software allows for easy coupling between the obtained image and scatter plots showing quantitative fluorescence for precise control of the analysis.

1. M Zhang and B Huang: The multi-differentiation potential of peripheral blood mononuclear cells. Stem Cell Research & Therapy.  2012; 3(6): p. 48-48
2. B Park, KH Yoo and C Kim: Hematopoietic stem cell expansion and generation: the ways to make a breakthrough. Blood research. 2015; 50(4): p. 194-203
3. TL Crippen, LM Bootland, JAC Leong et al.: Analysis of salmonid leukocytes purified by hypotonic lysis of erythrocytes. Journal of Aquatic Animal Health. 2001; 13: p. 234-245