What light source is used in most ow cytometers?

Light scatter collected at 90 degrees to the laser beam is

A laser called:
What are the three main systems in a ow cytometer? Side-scattered light (SSC)
The uidics, the optics, and the electronics Side-scattered light is proportional to the Granularity or internal
What type of biological sample is best suited for ow cytometric complexity of the cell.
analysis? Side-scattered light is proportional to the Granularity or internal
A single cell suspension complexity of the cell.
What is the name given to the portion of the uid stream where Correlated measurements of both Forward-scattered light and
the cells are Side-scattered light can
located? allow differentiation of cells types in a heterogeneous cell
Sample core population.
When cells labeled with uorescent molecules pass through Correlated measurements of both Forward-scattered light and
the focused laser Side-scattered light can
beam, what two types of light signals are generated? allow differentiation of cells types in a heterogeneous cell
Scattered light and uorescence population.
Light emitted from a particle is collected by: When uorescent compounds absorb light energy and then
Lenses release excess energy,
The electronic signal produced by the detectors is proportional they emit Photons of light
to: When uorescent compounds absorb light energy and then
The amount of light striking them release excess energy,
All ow cytometric measurements can be made simultaneously they emit Photons of light
on a single cell. Characteristic wavelength ranges at which uorescent
True compounds can be excited
Particles must be in single-cell suspension before ow are called Absorption spectra.
cytometric analysis. Characteristic wavelength ranges at which uorescent
True compounds can be excited
The purpose of the uidics system in a ow cytometer is: are called Absorption spectra.
To position the sample core in the center of the laser beam The longer wavelengths of light emitted by a uorochrome
What two factors can affect illumination of the particles within make up its Emission spectrum
the laser beam? The longer wavelengths of light emitted by a uorochrome
The width and the positioning of the stream make up its Emission spectrum
How many cells or particles should pass through the laser Which laser is most commonly used in ow cytometry?
beam at a given time? The argon-ion laser
One The FITC and PE uorochromes are excited by this emission
The particle suspension is injected into Sheath uid; within the wavelength of an
ow chamber argon-ion laser:
The particle suspension is injected into Sheath uid; within the 488 nm
ow chamber Two uorescent dyes commonly used in ow cytometry are
The process of centering the sample core within the sheath FITC and PE
uid is known as: Two uorescent dyes commonly used in ow cytometry are
Hydrodynamic focusing FITC and PE
Which regulator controls the diameter of the sample core? Fluorochrome-labeled antibodies are used to detect Antigenic
The sample pressure regulator surface markers
What are the three possible pressure settings for a benchtop Fluorochrome-labeled antibodies are used to detect Antigenic
ow cytometer? surface markers
Low, Medium, and High The optical bench provides a stable surface for the interaction
Increasing sample pressure Increases,the sample ow rate of the laser light
and the width of the sample core. with The excitation and collection optics
Increasing sample pressure Increases,the sample ow rate The optical bench provides a stable surface for the interaction
and the width of the sample core. of the laser light
Good data resolution is required for DNA studies. What ow with The excitation and collection optics
rate is Which two components of the ow cytometer must be perfectly
recommended? aligned to
A lower ow rate illuminate cells uniformly?
A wider sample core decreases resolution. The laser beam and the sample stream
True The alignment of a stream-in-air cytometer requires daily
A high ow rate can be used when performing qualitative optimization.
measurements. True
True For benchtop ow cytometers, the xed Flow cell ensures that
When does light scattering occur? the
When a particle deects incident laser light laser intercepts the sample streamconsistently from day to day.
Which key cell components contribute to light scatter? For benchtop ow cytometers, the xed Flow cell ensures that
The cell membrane, nucleus, and internal granular components the
Light scattered in the same direction as the laser beam is laser intercepts the sample streamconsistently from day to day.
called: Optical lters are placed in front of detectors to:
Forward-scattered light (FSC) Limit the range of wavelengths reaching the detector
FSC is proportional to: _ A 530/30 bandpass lter transmits wavelengths of light
Cell surface area or size between 515 and 545 nm.
A 530/30 bandpass lter transmits wavelengths of light
between 515 and 545 nm.
A Beam splitter is used to split light signals by wavelength and
redirect
the light signals to the appropriate detector.
A Beam splitter is used to split light signals by wavelength and
redirect
the light signals to the appropriate detector.
Shortpass lters transmit wavelengths equal to or shorter than
a Sample Preparation Staining and washing
specied wavelength, while longpass lters transmit
wavelengths
equal to or longer than a specied wavelength.
Shortpass lters transmit wavelengths equal to or shorter than
a computer
specied wavelength, while longpass lters transmit
wavelengths
equal to or longer than a specied wavelength.
Forward-scattered light is detected by a Photodiode
Forward-scattered light is detected by a Photodiode
Side-scattered light and uorescence are detected by highly
sensitive
Photomultiplier tubes
The light detectors generate current, which travels to an
amplier. It converts the
current to voltages which are proportional to the intensity of
light striking them.
True
What does the ADC do?
Converts voltage pulses to channel numbers
History
Kohler and Milstein (Nobel prize 1984) for hybridomas studying
CD4:CD8 ratios in AIDS pts. HIV attack T helper cells which
have the CD4 marker so these will decrease.
Hybridomas
inject antigen into mouse and mouse make antibody in B
lymphocyte cells. Then remove spleen and harvest B cells.
Then harvest multiple myeloma plasma cells and fuse them
with the B cells.
Flow Cytometer
Fluorescent Activated Cell Sorter (measureing
Ag/Ab+flourescent dye). Flow cell counts how many cells are
marked with dye. They should go though one at time.
Advantage of Flow cytometer
Analysis of 800 cells/sec for a total of 100,000 in 2-3 mins.
Quicker and more accurate than other methods.
Ratio of CD4:CD8 in normal person versus a person with AIDS
2:1 is normal and 1:2 is abnormal due to a decrease in CD4.
Types of Specimens
Peripheral blood (whole blood), bone marrow, FNA (fine needle
aspiration) for lymph nodes or lymphoid tissue. BAL
(bronchioaveolar levage). Body fluids (CSF, pleural, synovial).
Volume: 50 uL for every test batch of CD you run
Sample Preparation of Blood
K2 or K3 EDTA, heparin (NO citrate = interference). RMT (NOT
refridgerated because interferes with attachment of antibody).
24 hours
Sample Preparation of Lymphoid tissue
Filtration (have a sieve to remove lumps) to allow for single cell
suspension
Sample Preparation of Lysis of RBC
Ammonium chloride 1:10 let sit for 30 min. RBC are unwanted.
Antibodies conjugated with fluorescent dye (15 min) added to
sample. Requires a wash step to get rid of unbound antibody.
Instrumentation
Fluidics, flow cell, flow chamber, laser light, photo detectors,
Instrument Components: Fluidics
Tubing, valves, syringes, vacuum, etc. Aspirates the sample
and transports it to the flow chamber. Shealth flow: Laminar
flow (one @ a time); Hydrodynamic focusing (in one direction)
[= fluid dynamic terms]
Instrument Components: FLow Chamber (Flow Cell)
Two types: 1)Quartz=high quality, very pure (disadvantage--
any imperfection gives false results and very expensive to
replace); 2)Stream-in-air= faucet/tap where little pieces drop
freely into the air (what is being measured is totally in the open)
(disadvantage--increase risk of biohazard when fixing
machiene due to out in open.)
Instrument Components: Laser light
light scattering: measures cell size (forward) and complexity
(side). Fluorescent labels: immunophenotyping (detect CD on
surface of cell)
Flourescent labels are aka
Flourescent dyes or markers or Flourochromes
Instrument Components: Photodetector
detects and amplifies signal
Instrument Components: Computer
results on a scattergram
Flow Cytometry Principle
Cell suspension surrounded by sheath fluid to flow chamber to
light scatters all direction (360 degrees) [photodetector in
certain places: forward and side] to light analysis: forward
angle light scatter (FS, FSC,FALS) and side scatter (SS, SSC)
and Fluorescence analysis (CD) to Scattergram
Light Scatter: Forward Scatter (FS, FSC, FALS)
(2 to 10 angle.) Correlates to SIZE.
Light Scatter: Side Scatter (SS, SSC)
(90 more or less angle) Correlates to COMPLEXITY (granules,
lobularity)
Which cells have Low FS and Low SS
FS: low are the smallest cells; SS: low are the lymphs which
are least complex

RBC Histogram: An Overview

The well-known Coulter principle of counting and sizing red cells provides the basis for generating the histogram. This method relies on

the change in conductance as each cell passes through an aperture. The change in conductance results in an electrical pulse, the

amplitude of which is proportional to the cell volume. The 256-channel pulse-height analyzer uses a number of thresholds to sort the

cells into several size (volume) channels from which the histogram is formed. Each channel on the X-axis represents a specific size

(volume) in femtoliter (24360 fL), increasing from left to right. The Y-axis represents the number of cells per channel, with each cell

being stored in the channel representing its size, so that after data accumulation is completed the relative number of cells (frequency) is
provided. This data is further processed by the computer, and the RBC curve is smoothed by a moving average technique and

displayed on a data management system.13Figure 1A shows a typical normal RBC histogram. For a detailed explanation on the

construction of histogram, please see reference 14.

Value of Histogram

A histogram, as shown in Figure 1 and Figure 2, is a graphic representation of a collection of data based on cell size and/or cell number

depicting variations in the process. It is sometimes referred to as a frequency of distribution curve. Because graphics can show data in

ways that are meaningful and quickly understood, the histogram is a very powerful tool in red cell morphological analysis. It enables

one to visualize, analyze, and interpret empirical data that displays morphological changes graphically as points, peaks or valleys, or as

a line of frequency curve. It allows the users to intuitively see the visual comparison of the center and spread of data and data

containing 2 or more variables: dimorphic red cells, subpopulations, and skewed data, leading to quick and cost-effective decision

making.

At times, a histogram can provide invaluable information that may not even be apparent in the automated numerical data.5,15 For

example, in megaloblastic anemia with developing iron deficiency, some of the hypochromic and microcytic red cells can be identified in

the histogram but may not be reflected in the numerical data, and this may well be the first clue to a not uncommon occurrence.

Likewise, iron deficiency may develop insidiously during the process of some other disease (eg, essential polycythemia), with normal

cell counts and indices, except the RDW, which is elevated. The RBC histogram is also abnormal, showing a shoulder of microcytic red

cells.16

Furthermore, a histogram can provide useful information for laboratorians in 1) monitoring the reliability of the results generated by the

analyzers; 2) investigating the potential cause(s) of the erroneous automated results; and 3) arriving at the presumptive diagnosis.7 For

example, certain conditions like the presence of fragmented red cells or red cell agglutination that could not have been identified earlier

without blood film examination can now be presumably detected on the red cell histogram.3,16 Likewise, in patients with iron deficiency

anemia (IDA) or megaloblastic anemia in treatment, a sequential histogram can clearly show the progressive appearance of a new

erythrocyte population well in advance of other numerical parameters.3

The presence of a right-sided shoulder usually corresponds to reticulocytosis (Figure 1J), and a trailer of erythrocyte populations on the

far right of the histogram correlates to red cell agglutination (Figure 1F). A leftward shift of the RBC histogram signifies microcytosis

(Figure 1B and Figure 1C), and a rightward shift suggests macrocytosis (Figure 1D and Figure 1E). Bimodal red cell histograms (Figure

1F, Figure 1G, Figure 1K, Figure 1L and Figure 2B to Figure 2E) are usually associated with therapeutic transfusion and/or hematinic

agent response to microcytic and macrocytic anemia, but they may also indicate other hematological disorders as shown in Table 1.

Although the size ranges for RBC histograms are between 24 fL and 360 fL, the instrument counts only those cells with volume sizes

between 36 fL and 360 fL as red cells. Those cells counted in the 24 fL to 36 fL range are rejected and not included in the RBC count.

They are enumerated and displayed in the histogram area between the 24 fL and 36 fL range, however, allowing the lower end of the
histogram to be monitored. Normally, the space below 36 fL remains clear, but in certain conditions the histogram may begin above the

baseline or show a high takeoff on the far left of the curve (Figure 1G to Figure 1I), which generally indicates the presence of small

particles. These particles include red cell fragments, microspherocytes, nucleated RBCs, nonlyzed RBCs, elliptocytosis,

macrothrombocytes, platelet clumps, bacteria, parasitic organisms, and other interfering substances such as cryoglobulin, cold

agglutinin, and macroglobinemia.7,1618

Figure 1

Red cell histograms in various hematological conditions. (Key hematological features of these conditions are summarized in Table 2.)

Figure (A) Normal histogram, (B) Microcytosis, iron deficiency anemia, (C) Microcytosis, beta thal trait, (D) Macrocytosis with normal

RDW, (E) Macrocytosis, megaloblastic anemia, (F) Cold agglutination, (G) Sideroblastic anemia, (H) Beta thalassemia major, (I)

Pyropoikilocytosis, (J) Reticulocytosis, (K) Post-iron therapy, (L) Post-iron therapy.