10-color Phenotyping of Human TBNK and Mature Erythrocytes using 3 Lasers

In this panel, all lymphocytes were identified by staining with CD45. From the CD45+ population, B cells (CD19), NK cells (CD56), and T cells (CD3) were identified. In addition, mature erythrocytes were identified using CD235. The T-cell population was further analyzed to find effector T cells (CD8) and helper T cells (CD4). These T-cell populations were analyzed to find subset populations, Naïve CD4 and CD8 T cells, and Activated CD8 T cells using HLA-DR and CD45RA.

Fixed human whole blood was stained with 10 colors and analyzed on an SA3800 with 405-nm, 488-nm, and 638-nm lasers. Populations were gated on forward (FCS) and side scatter (SSC), then CD45+. From that population, several subsets were identified.

Figure A shows single positive control overlays of the spectral curves for CD45, CD3, CD4, and CD8 from a human TBNK sample. When compared to the TBNK sample (Figure B), the single positive control overlays match. CD4 Alexa Fluor® 647 and CD8 Alexa Fluor® 532 are used to distinguish the presence of T-helper cells and T-cytotoxic cells in the sample.

Comparative Data on Human Lymphocytes

A four-color comparative analysis of fixed human lymphocytes was done to evaluate resolution and sensitivity. The same sample was used on the same day on four different instruments. Each instrument had the recommended cleaning and instrument QC performed before running the sample. When compared with competitors, the Sony SA3800 and SP6800 spectral analyzer results showed better separation between the negative and positive populations.

In addition, a comparison was performed of the removal of autofluorescence. Each instrument showed autofluroescent populations stemming from the negative population. The SA3800 was able to identify the population, create it as its own color using the Autofluorescence Finder, and subtract the populations to allow the user to see the true populations.

4-Color Comparative Data on Human Lymphocytes

Comparative Data with Autofluorescence Removal

Using 10 Colors with 405-nm and 488-nm Lasers to find Multiple Populations

In an experiment using fixed human blood, B cells (CD19), macrophages (CD33), basophils (CD123), NK cells (NKp46), and helper T cells (CD45/CD3/CD4) were identified. Also, a smaller subset population of NKT cells was analyzed using CD57. CD57 shows expression mostly on negative CD4 cells, since it is associated with CD8 killer T cells.

Spectral Fingerprinting using a TBNK panel

Cytometrists use many forms of controls to determine cell populations from a heterogeneous mixture of cells. In this example, a 6-color TBNK panel was analyzed completely by spectral fingerprints. By comparing the single positive spectral fingerprints, the need for an FMO control to identify a population is decreased. In addition, since the autofluorescence from the unstained cells is treated as a separate color, it is subtracted to be able to identify the cellular lineage easily.

Single Positive Control Spectral Graphs