Flow Cytometry

Normal B CD38/10

Loses 10 and 38

Normal B 38 and 20

Loses 38, gains 20 but becomes a bit dimmer at full maturation

Blast gating options

Low SSC, Dim CD45, CD34 Low SSC CD45/SSC usually favored as it will miss less abnormal populations

HLA-DR in monocyte maturation

Maintained. Different than myeloids that lost it early on

CD48,CD117, CD33, CD9, CD68

Mast Cells

CD26

Mature T

When is CD14 acquired by mono line.

Mature monocytes

CD45 + ,CD13 + , CD33 + , CD11b + , CD15 + , CD16 +

Mature neutrophils

How to narrow the coaxial stream? Why would you do so?

Reduce sample stream pressure. Useful when too many cells are passing at once.

Type 2 beads.

Reference beads: Have varying bright fluorescent intensity, can also have antibody binding capacity.

Best B cell lineage markers

CD19,CD79a,CD22,CD10(s)

Easy way to isolated B lymphs from lymph gating.

CD19P CD2N population

Most frequent abberancies in AML

CD2,CD7,CD19,CD56

In what condition do neuts often express CD64

sepsis. be careful when interpreting

ETP Phenotype

+cCD3 + , CD1a - , CD8 - , CD5 dim phenotype, with common co-expression of myeloid antigens (including CD117,CD13 and CD33) and markers of immaturity (i.e. CD34,HLA-DR, TdT and CD133)

Normal K:L Ratio

1:2

Normal RI for hematogones in pediatric marrow

7%~ vs 1-2% in adults

CD9 in ALL

Aberrant when uniform. Usually variable

B cell clonality suspicion when?

Abnormal K:L, weak SIg, aberrant CD5, CD10 e xpression, CD25, CD23, Weak mature B markers, Abberant T markers

Type 1 beads.

Alignment beads: Small beads used for optical alignment.

Other cells found in conventional blast gate

Basos, Monos, early myeloid, hypogranular neuts, dendritic cells

T cells and CD5

Becomes strong while acquiring CD3.

Type 0 beads. Explanation and use.

Blanks: Similar size to lymphocytes but with no added fluorescence. Used for setting thresholds.

Follicular Lymphoma

CD10 positive small cells, pan B with dim CD19. usually CD5 negative.

Alternative for CD34 when looking at myeloid blasts

CD117 or even TdT

Most frequent abberancies in ALL

CD13,CD33,CD15

best monocytic lineage markers

CD14,CD64,CD11c

Markers used to confirm immaturity of T-ALL

CD34,TdT, CD1a, sCD3 lack, HLA-DR lack

Blastic plasmacytoid dendritic cell neoplasm

CD4/CD56/CD103 POS CD34/CD117/MPO/CD64/CD14 NEG

Common megakaryocytic markers?

CD41b,CD42b,CD61

Suspicions for clonal T disorders

CD4:8 abnormal, loss of CD4/CD8 or coexpression, loss of CD7/5, low intensity for T markers, excess of CD2+/CD3- cells. Abberant antigens.

T LGL

CD8+. seen with neutropenia, CD5/CD7 weaker, CD16 pos(strange)

Type 3 beads.

Calibration beads: these beads are equivalent insize to lymphocytes but have varying fluorescence inten-sity, ranging from dim to bright. They share the sameproperties as type IIa, IIb and IIc beads. These beads areused in quantitative flow cytometry where a calibrationcurve is required to determine the antibody bindingcapacity of the cell.

Most common ALL in children

Common > Pre-B > Pro-B

CD34 + , TdT + ,CD19 + , CD10 +

Common ALL immunophenotype

What determines side scatter?

Complexity, granularity of the cell

Doublet exclusion

Doublet exclusion is performed by plotting the height or width against the area for forward scatter or side scatter (Figure 24). Doublets will have double the area and width values of single cells whilst the height is roughly the same. Therefore disproportions between height, width, and area can be used to identify doublets.

CD34 + TdT + CD10 bright CD19 dim CD22 dim CD79b + CD20

Early B precursors

CD34, TdT, DR,CD19 and CD10

Early Hematogones

Sezary syndrome

Expanded CD4 with loss of CD7/CD26 and dim other antigens

Isotype control

Fluorochrome of same class as diagnostic antibody but without any specific affinity

T cell development of CD3

Gained as CD45 increases

HLA-DR in mature T lymphs

Indicates activation. Consider reactive causes

Common cause of CD8 proliferation

Infectious Mono. Other viral lymphocytosis.

Burkitt's Lymphoma

Medium cells with Pan-B and weak SIg, CD10*, TdT Neg. Bright CD45. CD5 neg.

MPO Negative AML. What is the differential

Monoblastic, Erythroid, Mega, Mast Cell, Basophilic, Early AML

When is CD117 usually lost in granulocytes? What about CD34.

Myelocyte stage. CD34 often lost first in pros along with HLA-DR

Use of KIR antibodies

NK neoplasms

CD16

Nk,Neuts

A CD34 + CD117 - CD45 dim CD13 -

Normal B cell precursors

Why are samples warmed to RT?

Optimizes appropriate antibody binding.

Mantle Cell

Pos for most mature b markers including CD5, FMC7, whilst CD10 negative and CD23 negative

T cell development of CD4/8

Precursors negative for both then gain both as thymocytes then develop into one or the other

CD117 in blast populations

Pretty lineage specific to AML, but less so than MPO

CD19 in AML

Pretty rare outside of t(8:21) so quite predictive of such. Often faint positive. Very rarely positive in normal myeloblasts.

TdT in Acute leukemia

Pretty ubiquitous in ALL, sometimes expressed in AML, particularly in early variants

When do monos acquire CD4?

Promonocyte stage where it is brightest

DLBCL

Rare ever present in PB. Often evolves from another disorder. Large cells with strong CD20, FMC7, CD79a, CD22 and CD45 bright. CD10 depends on subtype

CD25 expression in B-ALL

Should prompt investigation of Ph-like BCRABL disease

What determines forward scatter?

Size of the cell

Coexpression of CD34 and bright CD20

Suspicious. Consider Blasts.

CD4

T Helper

CD2

T/NK

CD71

Transferrin marker. Indicates active cells. Not entirely specific to RBC

What beads are usually used for daily QC?

Type 2b.

Which AML most frequently expresses CD2 abberantly

Variant M3

CD38D/CD138B

abnormal plasma cells

CD3 in T-Lymphs

cCD3 gained by precusors, sCD3 only on mature lymphs

When does CD15 appear in grans

later in mature cells

What cell population tend to autofluoresce the most

myeloid due to granules. dead cells.

A CD34 + CD117 + CD45 dim CD13 +

normal mono/myeloid maturing progenitors

CD45d, CD19, CD38b, CD138, restricted cyto Lc, no SIG

plasma cells

best lineage markers for TCell

sCD3, cCD3

Steric hindrance

this is the phenomenon where two antibodies directed at adjacent epitopes can mechanically interfere with each other's binding. This reduced binding results in apparent reduced antigen expression.

CD4:CD8 in HIV

very low