A 11-COLOR FLOW CYTOMETRY PANEL TO ASSESS THE ABSOLUTE NUMBER OF LYMPHOCYTE, MONOCYTE and DENDRITIC CELL IN HUMAN WHOLE BLOOD — Australasian Cytometry Society
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  3. A 11-COLOR FLOW CYTOMETRY PANEL TO ASSESS THE ABSOLUTE NUMBER OF LYMPHOCYTE, MONOCYTE and DENDRITIC CELL IN HUMAN WHOLE BLOOD

A 11-COLOR FLOW CYTOMETRY PANEL TO ASSESS THE ABSOLUTE NUMBER OF LYMPHOCYTE, MONOCYTE and DENDRITIC CELL IN HUMAN WHOLE BLOOD (24110)

Ya Wang 1 2 , Maryam Shojaei 1 2 , Anthony Mclean 2 , David Booth 1 , Maggie Wang 1 , Suat Dervish 1 , Benjamin Tang 2
  1. Westmead Millennium Institute, Westmead, NSW, Australia
  2. Intensive Care Medicine, Nepean Hospital, Penrith, NSW, Australia

Aim: To develop a 11-color flow cytometry panel for assessing the absolute number of different immune cell populations in human whole blood

Methods: Peripheral venous blood was collected into tubes containing anticoagulant heparin. Total white blood cells were counted using Sysmex XT-1800i automated hematology analyzer (Sysmex Corporation, Hyogo, Japan) for calculation of absolute number.  200µl of human blood were incubated with a premixed antibody cocktail for 30min at 4°C in the dark, which was followed by erythrocyte lysis for 10min at room temperature in the dark. After lysis, samples were washed once with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and resuspended in PBS before analyzing on BD LSRII Flow Cytometer (BD Biosciences, San Jose, CA). Flow cytometry standard (FCS) files were analyzed using FlowJo software V.10.0.8 to determine the percentage of each cell population. Absolute numbers of different cell populations (peripheral blood lymphocytes, monocytes, and dendritic cells (DCs)) were calculated by multiplying the total white blood cell count with the total percentage of each cell population.

Results: Using this panel, we are able to detect T lymphocyte (CD3+CD4+/-CD8+/-), NK cell (CD3-CD56bright/dim), B cell (CD19+), monocyte (HLADR+CD14+/-CD16+/-) and DC subsets: myeloid DC1 (mDC1) (CD3-CD19-CD14-CD56-HLADR+CD1c+CD303-), mDC2 (CD3-CD19-CD14-CD56-HLADR+CD141+CD303-) and plasmacytoid DC (pDC) (CD3-CD19-CD14-CD56-HLADR+ CD303+ CD1c-) in human whole blood.

Discussion and conclusion: This 11-color flow cytometry panel allows us to analyze all the immune cell populations simultaneously, which is critical in understanding the roles and interactions of different immune cells in different disease settings. And we are particularly interested in investigating the importance of different immune cells in influenza virus infection by comparing the number of cells in normal versus influenza infected individuals.

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