Diapositiva 1

Definitions
n
Flow Cytometry
u
n
Measuring properties of cells in flow
Flow Sorting
u
u
Sorting (separating) cells based on properties
measured in flow
Also called Fluorescence-Activated Cell
Sorting (FACS)
Basics of Flow Cytometry
•Cells in suspension
Fluidics
•flow in single-file through
•an illuminated volume where they
Optics
•scatter light and emit fluorescence
•that is collected, filtered and
Electronics
•converted to digital values
•that are stored on a computer
Flow Cell
Injector
Tip
Sheath
fluid
Fluorescence
signals
Focused laser
beam
Purdue University Cytometry Laboratories
Forward Angle Light Scatter
Laser
FALS Sensor
Purdue University Cytometry Laboratories
90 Degree Light Scatter
Laser
FALS Sensor
90LS Sensor
Optics - Fluorescence Channels
n
n
The fluorescence emitted by each
fluorochrome is usually detected in a unique
fluorescence channel
The specificity of detection is controlled by
the wavelength selectivity of optical filters
and mirrors
Fluorescence Detectors
Laser
Freq
FALS Sensor
Fluorescence
Fluorescence detector
(PMT3, PMT4 etc.)
Common
Laser
Lines
350
300 nm
457 488 514
400 nm
500 nm
610 632
600 nm
700 nm
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
Example Channel Layout for
PMT
Laser-based Flow
Cytometry
4
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
Fluorescence Activated
Cell Sorting
488 nm laser
FALS Sensor
Fluorescence detector
Charged Plates
Single cells sorted
into test tubes
-
+
Emocromo, determinazione con
tecniche manuali e automatizzate
Blood Components: Cells
Blood Components:
"Blood Count" – % of Each Component
Figure 16-2: The blood count
Manual Hematocrit
• “PCV”, “HCT”, manual HCT, "spun crit“
• Centrifugation, chart reading as %
Manual hematocrit
• Advantages:
- “Gold standard” in animals?
- Examine plasma for color changes
- Plasma for protein/fibrinogen assays
• Errors:
- Falsely increased: trapped plasma between RBCs
- Falsely decreased: shrinkage induced by excess EDTA
- Transcription to report
Calculated Hematocrit
• Calculated hematocrit =
MCV x RBC count / 10
• No plasma trapping, no RBC shrinkage
• Accurate if cell counter validated for
veterinary species (modified human
instruments)
Hemoglobin
•
Colorimetric assay following lysis of
erythrocytes
•
Falsely increased by interference with Hb
assay: plasma change? cell debris?
- Lipemia
- Marked leukocytosis ( > 50,000/µL)
EMOCROMO
Erictrociti: (RBC)
-Diluizione 1/100 con fisiologica
- si contano RBC in 10 rettangoli
di 1/1000 mm3
- il risultato x 10.000 =
RBCx106/mm3
Leucociti (WBC)
- diluizione 1/10 con soluzione di
lisi (liquido di Türk: ac. acetico
4% + violetto di genziana 1%
- si contano WBC in 1 quadrato di
1/10 mm3
- il risultato x 100 =
WBCx103/mm3
Piastrine (PLT)
- Diluizione 1/20 con soluz. di lisi
- si contano PLT in 20 rettangoli
di 1/1000 mm3
- il risultato x 1000 =
PLTx103/mm3
VCS Technology