1. Lymph node lymphoma A cheatsheet
Jean Oak

2. If you see a lot of granulocytes, may wonder about hemodilution
Larger/activated lymphs, PCs
Small lymphs
(should be the
predominant population)
Most cells should be in the lymph gate

3. Goal – Look for CD5+ B cells, plasma cells, GC B cells
Tube – CD5/CD38/CD45/CD19
Goal – Look for CD5+ B cells, plasma cells, GC B cells
Small number of CD5(dim) cells can be seen in reactive settings
Plasma cells
Likely
T cells
GC B cells
B cells are normally here
Plasma cells
GC B cells

4. Tube - Lambda/kappa/CD45/CD19
Goal – Look for surface light chain restriction
l-B cells
(these numbers should match)
k-B cells
(these numbers should match)
Monocytes with nonspecific binding via Fc receptors
Kappa:Lambda ratio is usually 2-3:1. Suspect a clonal population if ratio >4:1 (kappa-restricted) or <1:1 (lambda-restricted)

5. Goal – Look for B cells with aberrant CD10 expression or loss of CD20
Tube – CD20/CD10/CD45/CD19
Goal – Look for B cells with aberrant CD10 expression or loss of CD20
GC B cells
Reactive pattern looks like a hockey stick (continuous smear)
Look for B cells with CD20 loss
Probably plasma cells
(can verify on CD5/CD38/CD45/CD19 tube)
B cell
doublets

6. Goal – Look for aberrant T cells
Tube – CD8/CD3/CD45/CD4
Goal – Look for aberrant T cells
CD8 T cells
CD4 T cells
~5% of T cells are CD4/CD8 double negative = normal gd T cells
(Note % and are <5%)
T cells are ~70-80% of lymph nodes
CD4:CD8 ratio is around 2-3:1 but can be highly variable from >10:1 to <1:1

7. Usually naïve T cells predominate in the PB.
Tube – CD57/CD8/CD45/CD3
Goal – Look for aberrant CTL
Normal CD57
pattern
CD8+ T
CTL
(LGLs)
Mostly CD4+ T
NK, NKT
Naïve T cells
Tfh, NK
Usually naïve T cells predominate in the PB.

8. Goal – Look for aberrant T cell antigen loss
Tube #6 – CD7/CD3/CD45/CD2
Goal – Look for aberrant T cell antigen loss
Normal T cells should have CD3, CD2, CD7, and CD5 (can verify on CD5/CD38/CD45/CD19 tube)
Normal
gradient* NK NK
*Look for distinct populations – a gradual loss (“tail”) can be seen with aging or activation

9. Tube #7 – FMC7/CD23/CD45/CD22 (aka CLL tube)
Goal – Rule in/out CLL/MCL. This is the best tube to calculate %B cells (use CD22)
May want to consider adding HCL extension tube if there are monoclonal B cells
Mantle B cells
CLL
HCL
MCL
CLL
HCL*
MCL
GC B cells
CLL
MCL
FMC7 – Antibody against a different epitope of CD20

10. Goal – NK/NKT cell characterization
Tube #8 – CD56/CD16/CD45/CD3
Goal – NK/NKT cell characterization NK

11. Goal – Check specimen viability/cell membrane permeability*
Tube #8 – CD45/7AAD
Goal – Check specimen viability/cell membrane permeability*
Live Dead
*Dead cells have permeable membranes so 7AAD leaks into the cell
Why we care: dead cells can have nonspecific binding of antibodies

12. 2) Correlate with calculated k:l ratio here
Re-gating of Lambda/kappa/CD45/CD19 for all CD19+ cells
Goal – Look at B cells as one group for light chain restriction
Gate of all B cells
1) Visually
check the k:l ratio
2) Correlate with calculated k:l ratio here
Light chain expression of all cells