MIDTERMS: L3 - LAB EVAL OF CELLS (AUTOMATED) Flashcards
The _____ Principle is the most widely used method for counting and sizing microscopic particles suspended in a fluid
Coulter Principle
Founder of Coulter Corp founded in 1958:
Wallace H. Coulter (1913-1998) and Joseph R. Coulter, Jr. (1924-1995)
This principle has been called “the first viable basis for flow cytometry”
Coulter Principle
The Coulter brothers were the early pioneers in the
different automated hematological tests such as:
○ Cell counting ○ Diagnosis of Hemoglobinopathies ○ Immunophenotyping ○ Diagnosis of Leukemias and Lymphomas ○ Coagulation abnormalities ○ Monoclonal antibodies
ALL TRUE ABOUT ADVANTAGES OF AUTOMATION EXCEPT:
- Speed and efficient handling
- Multiple tests on a single platform
- Demands intensive labor manual examination of blood smear
- More timely diagnosis
3
T OR F
Automation
- Comments on RBC morphology can be generated
- Can recognize fragmented cells
False
Types of hematology analyzers:
○ Measures only a few parameters
○ Some steps like dilution is carried out manually
Semi-Automated
Types of hematology analyzers:
○ Measures multiple parameters
○ Requires only anticoagulated blood samples
Fully Automated
3 basic components of hematology analyzer:
Hydraulics, Pneumatics, Electricals
Generates vacuum and pressure that is necessary for operating the valves as well as moving the sample to your hydraulics system
Pneumatics
Controls the operational sequences of total
system and includes the electronic analyzers and computing circuitry for processing the data generated
Electricals
- Analyzers and computing circuitry
MOTF
- Aperture current composed of external and internal electrode
- Blood cell suspension is a solution composed of electrically conductive diluent (ex saline)
BOTH TRUE
Detection and measurement of changes in electrical resistance produced by cells as they traverse a small aperture - uses the principle:
Coulter Principle
T OR F
In the counting chamber, your low frequency current is applied between external (aperture tube) and internal (cell dilution) electrode
False
- external (cell dilution) and internal(aperture tube) electrode
Electrical resistance occurs as cells pass through the sensing aperture causing ______ that are measurable
Voltage pulses
MOTF
- Aperture for RBC or platelet are smaller than WBC
- 1 pulse counts for 1 cell
TRUE
- Data are plotted in a frequency distribution graph
- Relative number of y axis (number of your cells) vs x axis (channel number equivalent to the specific volume or volume of the x axis which is the femtoliters)
Histogram
- Display the pulses that are generated by the cells as they interrupt the current
(old display) - In which, The number of pulses are proportional to the number of cells counted and the height of the pulse is directly proportional to the volume of the cell
Oscilloscope
Pulses - collected and sorted according to its amplitude by _______
Pulse height analyzers
Provides a sample stream surrounded by sheath fluid as cells pass through the aperture
Hydrodynamic Focusing
MC Protein build up causes A. Increase in cell count B. Increase in volume of the cell C. Decrease in cell count D. BOTH A AND B E. BOTH B AND C
E
Allows the central sample
stream to narrow, to separate, and to align the cell in a single line for passage in the sensing zone.
A. Sheath fluid
B. Burn circuit
C. Laminar flow
D. Hydrodynamic focusing
C
Show simultaneous use of direct current and
radiofrequency in one measurement system.
RFDC detection method (2 methods: impedance and
radio frequency)
- Attenuated by nucleus to cytoplasm ratio, nuclear density, cytoplasmic granulation
- Measured by radiofrequency
Conductivity
Displays your cell population as clusters with the number of that representing now the concentration of the cell type.
Two dimensional distribution cytogram (scatterplot)
Optical light scatter
- To prevent the non scattered light from entering the detectors
Lens blocker
MOTF
- Diffraction - bending around corners the corners of the cell
- Refraction - bending due to the change in speed
- Reflection - backwards scatter of rays caused by an obstruction.
ALL TRUE
MOTF
- Laser - Emit a monochromatic light
- Source of light - Tungsten halogen lamp and halogen neon laser
- Photodetector - detect scattered light and convert it into electrical signal
ALL TRUE
MOTF
- Forward Angle Scatter - Correlates to the Internal structures of the cell
- Forward Angle Scatter - 0 degrees
- Forward angle scatter - diffraction of light
1 - false
2,3 - true
1 - correlates with cell volume
MOTF
- Side Scatter Light - also known . Orthogonal Light Scatter
- Side Scatter Light - refraction of light only
- Side Scatter Light - internal structure of cell
- Side Scatter Light - 90 degrees
1,3,4 - true
2 - false
2 - refraction and reflection
MOTF
- Forward low angle scatter - 2-3 degrees
- Forward high angle scatter - 2-3 degrees
- Differential scatter - used for cellular analysis
- Differential scatter - Combination of Forward Low Angle Scatter/Forward High Angle Scatter
1,3,4 - true
2 - false
FHA - 5-15 degrees
MC
- Measures multiple and fluorescent properties of cells when they flow as a single cell suspension through a laser beam
- Light scattering and fluorescence
A. Radiofrequency
B. FLUORESCENT FLOW CYTOMETRY
C. Optical light scatter
B. FLUORESCENT FLOW CYTOMETRY
T OR F
- Side Fluorescence Light – RNA/DNA information
- Forward Scatter Light – cell volume
TRUE
T OR F
○ High flow rate = immunophenotyping analysis
○ Low flow rate = DNA analysis
TRUE
Arrange the ff:
Fluorescent flow cytometry
A. Light is converted to electronic signal
B. Computer displays the channel number to the appropriate position in the data plot
C.Amplifier receives the electric current and converts to voltage pulse
D. Analog-to-Digital Converter (ADC) assigns the voltage pulse is assigned a digital value representing a channel
E.The channel number is transferred to the computer
ACDEB
- Consists of collecting cells of interests
- Defined through criteria of size
and fluorescence
Sorting
- Boundary that can be set to restrict the analysis to a specific population within the sample
- Data selected by the gate is then displayed in subsequent plots
Gating
- Data is collected and stored in the computer
- Forward Scatter, Side Scatter, Emitted Fluorescence
Data analysis
T OR F
○ Single parameter = data plot
○ Two parameters = histogram
FALSE
T OR F
One important property or usefulness of fluorescence is:
Estimation of Reticulocyte count
TRUE
T OR F
Fluorescence - Purpose: Provide us a quantitative measure of erythropoiesis
TRUE
MOTF
- The degree of your fluorescence emission caused by the excitation of the bound dye is proportional to the amount of RNA in your erythrocyte
- More mature RBC, Higher fluorescence
1 true, 2 false
2 - more immature = higher fluoresence
MOTF
- Fluorescence - best detecting immature platelets
- Peroxidase -Identification and counting of granulocytes
- Immunological - Accurate platelet counting using CD41/CD61 antibodies
ALL TRUE
Graphical representation of numerical data of different cell populations in a cell counter
Histogram
ALL TRUE ABOUT Histogram
- Gives information - Average size of cell
- Distribution of size
- X-axis - number of cells
- Y-axis - number of cells
- X-axis - volume of cells
- Contains discriminators that separates the distribution curve for the volume
1, 2, 4, 5, 6
Enumerate discriminators in histogram:
● WBC Discriminator ○ LD = 30-60 fL ○ UD = fixed at 300 fL ● RBC Discriminator ○ LD = 25-75 fL ○ UD = 200-250 fL ● Platelet Discriminator ○ LD=2-6fL ○ UD 12-30 fL ○ Fixed discriminator 12 fL
MOTF
RBC and PLATELET histogram
1. Shift to the left - macrocytosis
2. Shift to the right - microcytocis
BOTH FALSE
- Shift to the left - microcytocis
- Shift to the right - macrocytocis
WBC discriminators
● Lymphocytes = 35-90 fL
● MID = 90-160 fL
○ Refer to your monocytes, basophils and eosinophils
● Neutrophils = 160-450 fL
T OR F
MID cells are hardest to distinguish
True - Could be among monocyte, basophil, or
eosinophil
WBC SCATTER PLOT
X-axis refers to
Y-axis refers to
X-axis refers to the slide scatter = granularity of the cell
Y-axis refers to forward scatter = size of the cell
T OR F
Calibration - To determine accuracy the instrument readings
TRUE
MOTF
- Lipemia - higher turbidity = High hgb, high mch
- Cold agglutinins - Low RBC and High MCV
BOTH TRUE
MOTF
- Hemolysis - low rbc, high hct
- Mirocytes - low rbc, high platelet
1 - false, low rbc and hct
2 - true
T OR F
Nucleated rbc results to high wbc
TRUE
Lysis-resistant RBCs with abnormal hemoglobins causes: 1 - HIGH WBC 2 - LOW WBC 3 - HIGH HGB 4 - LOW HGB
1,3
Platelet clumps results to: 1 - Low platelet 2 - High platelet 3 - High wbc 4 - low wbc
1,3
Leukemic cells results to 1 - low wbc 2 - high wbc 3 - high platelet 4 - low platelet
1,3
