Lecture 10

fluidics, optics, electronics

FSC (forward scatter)
SSC (side scatter)
Fluorescence

sample prep/ processing
FC clinical application
Data analysis, QC, troubleshooting

natural fluorescence found within cells
(needs to be minimized)

any particle which generates signal after passing through the laser beam
(units are events per second!)

light that is scattered in the forward direction (up to 20deg) after interacting with a particle

cell size

light that is scattered at 90deg after interacting with particle

internal complexity like granularity, presence of vacuoles, or inclusions

plots which display a single measurement parameter

plots which display 2 measurement parameters (bi-variate plots)

identification of cells in population through the staining and identification of specific markers using monoclonal abs conjugated with fluorochromes
(detects signal surface markers)

dye which allows ID of dead cells through a reduction in cell membrane integrity
(cells are still alive?)

lymphocytes
bc they don’t have granules
(if not then debris)

measuring the physical and chemical properties of cells which are flowing in a stream of fluid

delivers the cells singly past a point of measurement (laser beam) at a speed of several thousands per second

based on lights scattered and the fluorescence emitted by cells/ particles

cells AND particles (from bacterium (0.2 um) to human blastocyst (100 um)
and chromosomes/ proteins

information Is combination of processed lights scattered and forward/side angles, and fluorescence

1. able to analyze multiple parameters in large # of cells
2. can ID rare-event cells in heterogenous cell pop (MRD in leukemia/ lymphoma)
3. central role in diagnostic algorithm for leukemias

fluidics, optics, electronics, detectors

sheath fluid tank
flow cell

lasers
filters

FSC, SSC
FL detectors

ADC system
data processing system

sheath fluid tank pressurized by air which injects sheath fluid by laminar flow to a flow cell

a faster outer sheath fluid around the sample stream allows narrowing of the stream creating a stream of single cells

allows cells to be interrogated one by one by the laser beam

fluidics
What is definition of hydrodynamic focusing?

central alignment of cells within the flow cell essential for consistent illumination of cells as they pass the laser beam

cell enters beam > generates scattered lights/ fluorescence > makes current in PMT > when center of beam, PMT reaches max, cell fully illuminated > cell flows out and PMT drops to baseline

1. sample prep
2. instrument sample
3. run the data through FC
4. analyze data

BM aspirate in heparin
BM FNB
Peripheral blood in EDTA
lymphoid tissues
body cav fluids and solid tissues

samples must be single cell suspensions
cell viability is crucial (85% needs to be alive)

have to become homogenized and prepared as single cell suspensions
(using mech disruption)

transported to lab as anticoagulated;
tissue samples and BM transported in culture media to maintain viability (RPMI 1640)

aggregates, micro clots, or cell clumps
(use cell strainer)

LIVE and intact cells (live cell analysis)

1. to obtain stats (to report pop %, abs cell cts, fluorescence intensity)
2. to ID abnormal pop based on light scatter, staining patterns, or both

IDing normal pop using the same parameters

clusters of differentiation

cell surface markers useful for the ID and characterization of leukocytes

through human leukocyte differentiation antigens (HLDA) workshop started in 1982

provide standardization of monoclonal abs to human antigens across labs

mAbs conjugated with FL

CD3+ CD4+

CD3+ CD8+

CD19+ CD20+ CD3-

CD16+ CD56+ CD3-

CD20- CD25- CD34+

CD11c CD123

CD34

CD13 CD33

CD66b

CD41 CD61 CD62

using 2 cell properties picked up by FC detectors

bi-variate plots

SS vs FS
SS vs 1 CD ab (single color)
1 CD ab vs another CD ab (2 colors)

right: positive (more intense signal)
therefore higher expression of ag

one parameter vs count or time

when cell pop have similar physical properties:
size, cytoplasmic complexity, and expression of spec ag form clusters on the plots generated by FC

an electronic boundary an operator uses to delineate cell clusters;
gating selects population of interest and excludes the other

continue with SSC vs CD45

blasts

allows initial ID of the target population

eval of antigenic profile of target population by gating

immune deficiency
stem cell enumeration
PNH
Reticulocytes
FMH
Cell cycle analysis
Transplantation

SCID, HIV, etc

CD34

CD55, CD59

RNA binding dyes
e.g. TO

Ab against HbF, RhD, or both
or CA

DNA content

HLA, flow cross match
donor lymph + pt serum, incubate with antihuman IgG F(ab)/ FITC

1. WB or BM samples stored for long time with anticoag (>4hrs)
2. tissue samples not transferred in approp media (culture media or saline-moistened gauze)
3. harsh vortex (only spacial vortex for cell pellets)
4. agg mech disruption of tissues
5. cell pellets are kept dry for long time (resuspend in relevant buffer no bubbles)
6. high conc suspension used (adjust and dilute)

keeping cells on ice can improve viability bc it slows down necrosis and cell metabolism

delivered fresh to lab
Even stored at 4C, samples deteriorate (but more slowly than at RT)

within 48 hrs, influenced by type of anticoag

at 6 hrs when incubated in EDTA
Heparin delays process significantly (preferred anticoag)

promptly, as many cells deteriorate/ die

heparin