Flow Cytometry

Front 1

flow fsc alone

Back 1

for review

Front 2

flow ssc alone

Back 2

for review

Front 3

cd45 vs. ssc

Back 3

for review

Front 4

cd45 alone

Back 4

for review

Front 5

fwd vs ssc

Back 5

for review

Front 6

what is the most commonly used laser in flow and at what wavelength does it excite at

Back 6

argon - excites at 488 nm

Front 7

secondary options for laser

Back 7

hene

Front 8

wavelength of excitation and emission: FITC

Back 8

use argon laser with 488nm excitation
emits around 520 nm

Front 9

wavelengths of excitation and emission: PE

Back 9

excitation, can use argon at 488nm (though rather broad excitation options), emits around 580nm

Front 10

wavelengths of excitation and emission: PI

Back 10

can have excite with argon (488 nm), emits at 620 ish

Front 11

wavelengths of excitation and emission: PE-TR conjugate

Back 11

conjugated molecule so takes advantage of PE's excitation with Argon (488nm) but can emit higher more like texas red or more 630-660

Front 12

reverse forward and side scatter

Back 12

for review

Front 13

what is often in FL1

Back 13

fitc

Front 14

what is often in fl2

Back 14

Pe

Front 15

what is often in fl3

Back 15

percp or ecd (could use PI)

Front 16

what is often in fl4

Back 16

far red fluorescence - could use a couple/conjugate dye or a series of dyes called allophycocyanins (using a wavelength higher than 488 nm)

Front 17

in HIV infected patients how use flow

Back 17

monitor number of CD4+ tcells by looking for CD3+CD4+ population

Front 18

in hiv testing by flow how are the antibodies examined and which ones

Back 18

anti-cd4 - FL1
anti-cd8 - FL2
anti-cd45 - FL3 (gating parameter)
anti-cd3 - FL4

Front 19

what are the options to get an "absolute" CD4 + T cell population

Back 19

- combine % CD4 plus total count from hematology (variable results)
- bead technology where flow counts directly

Front 20

for initial immunodeficiency w/u, what ag are reviewed

Back 20

- CD45 for gating
- CD3 (if absent, DiGeorge's)
- CD4
- CD8
- CD19 (total B cells)
- CD56/CD16 (NK or LGL)
note if CD3+CD56+ NK T cells
if CD3-CD56+ NK cells (often CD8+ too)

Front 21

important information re; blasts for gating

Back 21

- Blasts have low CD45
– Myeloblasts have increased side scatter
– Lymphoblasts may have increased side scatter

Front 22

flow panels for: B-ALL

Back 22

CD19, 34, 10, TdT, 45 (less bright as the
level of differentiation drops).
(note: some T cell lymphomas can have CD20)

Front 23

flow panel to include for preB ALL

Back 23

Cytoplasmic mu and CD20

Front 24

flow panels for T ALL

Back 24

CD2, 5, 7
 CD4 and 8 are negative in Pre T cell,
double positive in the cortical thymocyte
stage and singularly positive in mature T
cells.
 TdT may be found in early T cell leukemias

Front 25

hairy cell leukemia flow panel

Back 25

CD19, 20, 25, 11c, CD103

Front 26

flow panel for CLL

Back 26

CD19, 20, Likely to have CD5 and 23

Front 27

myeloid markers in flow

Back 27

CD13, 33
Possibly CD34 in M0/M1
More differentiated will have CD14 and 15.
Low level expression of CD4 is possible

Front 28

what is the quantitative difference in DNA total amount between Go/G1 vs. G2

Back 28

in G2, there is double the amount of DNA total (post DNA synthesis but pre cell division)

Front 29

what is true of aneuploidy (IN GENERAL) in tumors and major exception

Back 29

generally more aggressive tumors;
major exception: neuroblastoma where diploidy is worse prognosis

Front 30

what does PI stain

Back 30

double stranded nucleic acids (if remove double stranded RNA then PI is linearly related to ds DNA)

Front 31

define hyperdiploid

Back 31

greater than the normal 2n
number of chromosomes

Front 32

define hypodiploid

Back 32

Less than the normal 2n
number of chromosomes

Front 33

define tetraploidy

Back 33

Containing double the
number of chromosomes

Front 34

define DNA index

Back 34

The ratio between the amount
of DNA in the G0/G1 phase of the tumor
cell divided by the amount of DNA in a
normal diploid cell.

Front 35

dna histogram

Back 35

for review - normal histogram

Front 36

what is the DNA index

Back 36

2.8/2 = 1.4

Front 37

what is a flow cytometry crossmatch

Back 37

mix donor cells with patient serum looking for antibodies in patient serum that might react to patient's cells - add in a fluorescently labeled anti-IgG -can additional determine if the cells of the donors are CD3 or CD19 to see if B or T cells are reacting against the patient's serum

Front 38

what is special about phenotyping in prolymphocytic leukemia

Back 38

bright kappa

Front 39

poor prognostic markers for CLL

Back 39

recall CLL CD5+ CD23+
add in CD138 and Zap 70 as poor prognostic markers

Front 40

what are the two principle myeloid markers for flow

Back 40

CD13, CD33

Front 41

what is useful to know about CD34 in myeloid neoplasms in flow

Back 41

CD34 is better expressed in the less differentiated myeloid neoplasms (see more in MO/M1); low levels in M6 and M7

Front 42

what "special" phenotyping can you see in M4e

Back 42

CD2

Front 43

what can be found on M4 and M5 immunophenotypically

Back 43

low levels of CD4

Front 44

what is the most persistent immunophenotypic marker in B cell neoplasms

Back 44

CD19

Front 45

what are three very early B cell markers (but not necessarily specific at all)

Back 45

CD34, CD10 and tdt

Front 46

what might appear is a slightly more differentiated b cell neoplasm

Back 46

Cmu, CD22 and CD20

Front 47

what phenotypic marker increases with myeloid differentiation AND with b cell maturation

Back 47

CD45 expression

Front 48

what is one of the earliest markers for T cell differentiation

Back 48

CD2

Front 49

how can CD34 be used in stem cell transplants

Back 49

since CD34 is a stem cell marker, donors (?) can be tracked with CD34 with colony stimulating factors for stem cell harvesting

Front 50

three markers that lymphs can express upon activation

Back 50

CD25, CD69 and CD154

Front 51

what is PNH a defect in

Back 51

lack of expression of GPI. so can't link certain proteins to cell surface (e.g. CD55 and CD59)

Front 52

how can flow be used in PNH

Back 52

looking for CD55 or CD59 expression (if absent, then PNH b/c GPI couldn't bind them to cell surface)
- look in PMNs or rbcs

Front 53

how is flow more useful than the Ham's test in detection of PNH

Back 53

if examine PMNs in flow, don't have to worry about recent transfusions altering conclusions (as in Hams' test)

Front 54

does PNH have hemolysis

Back 54

yes, but not caused by ab

Front 55

in very brief, what is Ham's test

Back 55

putting patients blood in very mild acid, if lysed then might be PNH - indicative of membrane fragility

Front 56

in brief, what is the nitro blue terazolium chloride test

Back 56

incubate cells with dye. if blue, cells able to produce ROS. If not blue (in case of CGD) can't produce ROS

Front 57

how do pmn functional assays work by flow

Back 57

-activated/functional PMNs can reduce rhodamine (thus get bright signal)
- those from CGD patients can't reduce rhodamine (don't get bright signal)

Front 58

sample of flow

Back 58

CGD - note: gates on pmns - only a few studies will gate on pmns - cgd (rhodamine), pnh (cd55, 56) and maybe LAD (with CD18)

Front 59

what is PNH

Back 59

defect in which GPI linked
antigens (CD55, CD59) are not present on
the cell surface

Front 60

with flow for pnh, what could be gated

Back 60

pmns or rbcs

Front 61

luminex multiplexed beads

Back 61

review