MLSP 5213: Flow Cytometry

CD markers: B cells

CD10, CD19, CD20, CD22, CD23, CD25

-immunoglobulin heavy chains, keppa and lambda chains

CD markers: T cells

CD2, CD3, CD5, CD7, CD4 and CD8, CD10, CD25

CD markers: natural killer cells

CD16, CD56

CD markers: myeloid cells

CD13, CD33, (CD14, CD15), CD117

CD markers: stem cells and leukemic blasts

CD34, (CD117)

CD markers: all nucleated hematopoietic cells

CD45

Forward scatter

cell size

Side scatter

internal complexity

Where are lymphocytes in flow cytometry

lower left

-small and agranular

Where are monocytes in flow cytometry

up from lymphocytes and to the right

-little bigger in size and few more granules

Where are granulocytes in flow cytometry

upper right

-largest in size and most granules

In a CD45 flow cytometry, what cells express a lot of CD45

lymphocytes and monocytes

In a CD45 flow cytometry, what cells express little of CD45

Blasts and granulocytes

Fluorescent labels

-absorbs excitation wavelengths of light

-emit light of longer wavelength

-lower energy as electrons return to ground state

Fluorescent labels should have

-high intensity

-stable

-have high molar extinction coefficient (absorb excitation wavelength well)

FITC

-emits light at 530 nm

-green light

PE

-emits light at 580 nm

-orange light

Different emission wavelengths allow

-to be distinguished from one another

-collected by different light detectors

fluorescence intensity

The amount of antibody attached is proportional to the intesity of light emitted from that cell

Quadrant marker

drawn around the negative population of cells to separate the negative population from the positive population

CD10 is present in

early in lymphoid development

-disappears as the cell matures

What is an important tool to diagnosis and treat leukemias

Assessing expression of various CD markers

What CD marker is typically expressed on T cells but can be misexpressed on B cell neoplasms

CD5

In a normal person, the ratio of kappa to light chains should be

less than 10:1

What happens to kappa and lambda chains in malignancy

cell has undergone somatic recombination and light chains are restricted

Kappa lambda ratio in malignancies

kappa lambda ratio becomes skewed

-greater than 10 or less than 1

In normal peripheral blood, the majority of lymphocytes are

T cells

-more CD4 cells

Fluorescence activated cell sorting

Cells with a specific combination of surface marker expression are diverted by the analyzer out of the main flow stream into a collection tube and other cells are discarded

What is FACS used for

purify stem cells for hematopoietic stem cell transplant using peripheral blood

What is used to increase the number of circulating hematopoietic stem cells

the donor is treated with G-CSF

-drives stem cell expansion in the bone marrow and increases the numer of circulating stem cells

How is the stem cell product collected

leukapheresis

Aspheresis

peripheral blood is removed from the donor, mononuclear cells are collected and returned to the patient

CD22

expression is present in the majority of the leukemic blasts

-target for immune based cytotoxic therapies

Which CD markers can rule out a myeloid leukemia

CD13 and CD33

Majority of blasts are negative for

T cell markers

Which CD marker is used to collect cells in FACS

CD34

Tdt is a marker of

leukemic lymphoblasts

-can ID very immature lymphoid blasts

Blasts express CD34, Tdt, CD19, CD20, and CD22 is consistent with

B cell ALL

An auer rod is indicative for

AML

CD14 and CD15 tend to be expressed more in

monocytic and monoblastic cells

-unreliable

What can be used to help distinguish between monoblastic and monocytic leukemia

CD117

-also expressed in a high percentage of myeloid leukemias

Acute monocytic leukemias lack expression of

CD117

Monoblastic leukemias express what CD marker in high percentage

CD117

-low CD14 and CD15 as well

HLA-DR expresed on

myeloid, monocytic and B cells

Expression of these three antigens suggest AML

HLA-DR

CD13

CD33

CD34, CD13, CD33, and CD117 expression and low expression of CD14 and CD15 suggest

acute myeloid leukemia

What CD marker is misexpressed in CLL

CD5

CD79b

expressed exclusively in B cells

-tends to be lower in CLL

HLA-DR state in CLL

absent

Increased number of kappa chains reflects

proliferation of the malignant B cell clone and ratio exceeds 10:1

Patients with a benign expansion of B cells rarely exceed

a 10:1 kappa to lambda ratio

Mixexpression of CD5, along with CD19, CD20, and CD23 indicates

mature B cell neoplasm that kappa light chain restricted

Lack of other T cell markers excludes

malignant T cell process

Lack of CD10 indicates

it is not an acute lymphocytic leukemia