NeoGenomics Medical Flow Cytometry Flashcards
The first two gates
FSC/SSC non-debris gate, then D45 by SSC
CD45 dim represents the Blast gate. They include normal myeloid and lymphoid precursors as well as blasts.
CD45 negative represent the non-WBCs. These are erythroid precursors, debris, a few nonhematopoietic cells, as well as some abnormal cells that do not express or have lost expression of CD45 (tumor cells, abnormal plasma cells, etc).
CD45 intermediate and high SSC represents the granulocytes.
CD45 bright and SSC low/intermediate defines the mononuclear gate, including lymphocytes (tend to be SSC low) and monocytes (tend to be SSC intermediate).
Which hematopoietic cells cannot be easily localized by SSC vs CD45 gating?
Plasma cells are the most important type
They can be quite variable in both CD45 expression and SSC.
Instead, a CD45 by CD38 gating strategy is used to find these guys. Plasma cells are almost always bright for CD38 and CD138.
Abnormal plasma cells are usually dinstinguished by loss of CD45 and CD19 and gain of CD56.
What type of cell is identified by these flow plots?
Abnormal plasma cells
They are definitely plasma cells, as defined by their CD138 and CD38 brightness.
However, the abnormally express CD56, have lost expression of CD45, and are uniformly kappa chain expression (basically no lambdas). FSC also shows that they are unusually large.
This probably represents a case of myeloma.
If you treat a patient’s myeloma with anti-CD38. . .
. . . they may lose CD38 expression.
Initial B cell gate
CD20 by CD19
Double positives are mature B cells, which can then be tracked throughout other B cell gates.
Gates to investigate for CLL/SLL
CD5 by CD19 (assess for CD5 positivity)
CD23 by CD19 (assess for CD23 positivity)
A CLL/SLL cell is classically CD19/CD5/CD23 triple positive.
MCL’s leukemic component may appear as a CD19/CD5 double positive cell, but will be CD23 negative.
Sorting by B cell size
Usually done as a FSC by CD19 gate on the lymphocyte population (CD45 bright CD SS low)
Initial T cell gating
- FSC by CD3, to categorize CD3+ cells by size
- Small T cell CD4/CD8
- Large T cell CD4/CD8
NK/NKT cell gating
Clinically, NKs are defined as CD56+CD3- cells while NKTs are defined as CD56+CD3+ cells
Both populations are seprately plotted on CD4 by CD8, CD7 by CD38, and CD2 by CD5
There should almost always be more __ cells than __ cells.
There should almost always be more T cells than B cells.
Otherwise, a B cell leukemia or lymphoma should be suspected.
5 features suggestive of a B cell lymphoma/leukemia
- K/L ratio > 3 or < 0.3
- Abnormal pan-B marker expression (loss of either CD19 or CD20)
- Aberrant expression of CD5, CD10, or CD11cbright.
- Mature B cells (CD45+, CD20+) without any surface light chain expression
- Large B cells
3 Features suggestive of T cell leukemia/lymphoma
- Markedly abnormal CD4:CD8 ratio (>10 or <0.1)
- Non-thymocyte CD4+CD8+ cells or CD4-CD8- T cells
- Variable loss of pan-T markers (CD2, CD3, CD5, CD7)
Features suggestive of an NK cell leukemia/lymphoma
Relative increase in NK cells to > 20% of all lymphocytes
NKs are defined as CD3-CD7+CD56+ lymphocytes
Add on for B cells with no surface light chain
Cytoplasmic light chain panel
Add on for suspected Hairy cell leukemia
Hairy cell markers: CD22, CD25, CD103
Along with: CD19, CD20, CD11c, kappa, lambda
The above is available as a package. CD123 is also often seen on HCL and is available separately.
Add on for lymphoma companion tube
Useful in discriminating CLL/SLL from MCL
CD3, CD22, CD36, CD43, CD45, CD200, FMC-7
“ZAP-70” add on tube
ZAP-70 is prognostic for CLL/SLL, so this should be used when the diagnosis is known CLL/SLL.
CD3, CD5, CD19, CD45, cZAP-70
TCR Receptor add on tube
To work up a suspected T cell lymphoma
CD3, CD4, CD8, CD25, CD38, CD45, CD56, CD57
TCR-gamma/delta, TCR-alpha/beta
CD30 is available and may be requested. It may be seen in peripheral T cell lymphomas and anaplastic large cell lymphoma.
“T Cell Therapy” panel add on
Module that assesses neoplastic T cells for the presence of targetable antigens. Useful for therapeutic decisions in a known T cell lymphoma.
CD3, CD4, CD7, CD8
CD25, CD26, CD30, CD52, CD279 (aka PD-1), CD45
V-beta T cell add on
Assesses the V-beta component of the TCR for clonality.
This test detects 24 human V-beta families (V of VDJ, the variable region of the beta TCR chain), or about ~70% of possible V-beta components. So, it will not give a concrete answer to clonality 30% of the time.
The two forms of CD16
CD16a: The full CD16 you know and love. FcgRIIIa.
CD16b: Truncated and GPI-anchored for of FcgRIII which is incapable of cytoplasmic signal transduction. FcgRIIIb.
The granulocyte “U”
Think of this as similar to the MΦ waterfall.
CD13 by CD11b.
A good “U” indicates good granulocyte maturation. The “U” is distorted in MDS and MPN (dyssynchronous maturation states). CD13 by CD15 produces a similar plot.
The MΦ Waterfall
Ly6C by MHCII gated on CD45+CD11b+CD64+ cells.
Population 1: Top left, Ly6ChighMHCIIlow. Newly arrived tissue M0.
Population 2: Top right, Ly6ChighMHCIIhigh. Intermediate/early activated M0.
Population 3: Bottom right, Ly6ClowMHCIIhigh. Mature MΦ.
The granulocyte ✓
Same idea as the granulocyte “U”. Shows good granulocyte maturation and distortions are present in MDS and MPN.
CD13 by CD16
This will be the first graph you see/look at in the granulocyte section of the standard flow panel.
Defining a hematogone by flow cytometry
Hematogones, or precursor B cells, are defined as CD19+, CD10+, CD45dim cells. Note that they therefore arise from the blast gate.
Differentiating them from lymphoblasts can be challenging. The CD10/CD20 plot can be useful as hematogones typically gain CD20 and lose CD10 as they mature, forming a streak. Meanwhile, lymphoblasts form a tight cluster on this plot.
The attached plot shows hematogones (H) of three maturation stages (H1, H2, H3). LyB are mature B lymphocytes demonstrating CD20+ and CD10dim/-. The H2 population demonstrates the “streak.”
If all else fails, the hematogone add on tube is always at your disposal.
Normal B cell maturation by flow cytometry
