AUTOMATION IN HEMATOLOGY Flashcards
Automation started ..
Wallace H. Coulter (1913-1998)
Joseph R. Coulter, Jr (1924-1995)
• Used in order to count and size the different blood
One principle that hematology analyzers employ during the analysis of blood cells Automation in Hematology was developed
COULTER PRINCIPLE
Why do we employ analyzers in Hematology?
• Cell counting
• Diagnosis of hemoglobinopathies
• Immunophenotyping
• Diagnosis of Leukemias and Lymphomas
• Coagulation abnormalities
ADVANTAGES OF AUTOMATION
- Speed and efficient handling
- Greater Accuracy and Precision
- Multiple tests on single platform
- More efficient workload and management
- More timely diagnosis
DISADVANTAGES OF AUTOMATION
- Flagging
- RBC morphed limited
- Erroneous regulations
- Expensive
TYPES OF HEMATOLOGY ANALYZERS
- SEMI-AUTOMATED
- FULLY AUTOMATED
Measures only few parameters
Some steps like dilution is carried out manually
Semi-Automated
Measures multiple parameters
Requires only anticoagulated blood samples
Fully automated
BASIC COMPONENTS OF HEMATOLOGY ANALYZER
Hydraulics
Pnematics
Electrcals
Hydraulics
Mixing Chambers
Aperture baths/Flow cell
Hemoglobinometer
Aspirating unit
Dispensers
Diluters
→ where the sample is introduced
→ the sample will flow through and will be analyzed
Hydraulics
• Vacuums and pressure for operating valves and also to move the samples among the hydraulics
Pneumatics
• Analyzers and computing circuitry
Electricals
PRINCIPLES OF AUTOMATED BLOOD ANALYZERS
Electrical Impedance
Electrical impedance
Radio frequency
Optical light scatter
fluorescent flow cytometry
Detection and measurement of changes in electrical resistance produced by cells as they traverse a small aperture
Electrical Impedance
Makes use of the Coulter Principle
Where the sample IS suspended in_____
*solution is composed of electrically_____
• There is a flow of electric current within the solution
• 2 chambers filled with a conductive buffered electrolyte solutions separated by glass tube having a small aperture
• direct current is generated between the internal and external electrode
• Aperture for_____ is smaller than the____ aperture
Aperture bath
conductive diluent
RBC/platelet
WBC
- has the aperture where the cells pass through
→ detects/does the analysis of the blood cell that passes through here
Aperture tube
Sensing zone
• Our cells are____ conductors of electricity; they_____
• As the cell passes through the_____ in the aperture, it resists/impedes the electrical current and the resistance / electrical current creates a_____
poor; impede electrical current/ resist electrical current
sensing zone; voltage pulse
The voltage pulse is equivalent to the property of the cell
The height of the pulse is equivalent to the =
volume of the cell
The voltage pulses will be gathered by the
Oscilloscope
The pulses will be gathered and sorted out so they will be plotted into the…
histogram
X axis - volume of the cell
Y axis - number of the cell
Histogram
• The machine is set to identify a particular cell based on a_______
Example: The threshold discrimination for the platelets is between 2-30 fL; the machine is set to recognize particles as platelets when the particles under this range
treshold volume
Provides a sample stream surrounded by a sheath fluid as cells pass through the aperture
Allows the alignment of cells into a single-file passage through the sensing zone
Hydrodynamic Focusing
Hydrodynamic Focusing
Purpose (2)
• Prevents coincident passage of cells
• Prevents recirculation of cells back to the sensing zone
Different Variables Measured by Electrical Impedance
RBCs
WBCs (3 only)
platelets
is incorporated with another principle.
In this example, ______ is incorporated with electrical impedance/direct current
Radiofrequency
The radio frequency signal’s purpose is to
analyze the Internal complexity of the cell
Cell internal structure density:
• Nucleus: cytoplasm ratio
• Nuclear density
• Cytoplasmic granulation
• with radio frequency, the______ of the cell will be analyzed
internal structures
Involves light scattering
There is a source of light → in the form of______ LAMP
TUNGSTEN HALOGEN LAMP /
HALOGEN NEON LASER
OPTICAL LIGHT SCATTER
can identity
5 parts WBCs
LASER →
Light Amplification by Stimulated
Emission of Radiation
• Light source will emit a light and the light will heat the blood cell that is being analyzed
The blood cell will scatter the light in different directions
Light scattering
• Light source will emit a light and the light will heat the blood cell that is being analyzed
The blood cell will scatter the light in different directions
Light scattering
The light scattered by the cell will be captured
Absorption, diffraction, refraction, and reflection
The scattered light will convert the light signals into an electrical signal
The electrical signal will be analyzed by the electrical systems, and will be converted to results
There is a single file of cell; because this principle also employs_______
The cells will be analyzed one cell at a time
A light will be emitted by the source
As the light hits the cell, there will be a corresponding scattering of light
_______will be gathered by the detectors and converted to an electrical signal
HYDRODYNAMIC FOCUSING
Scattered light
Different angles of light scattered detected by detectors
- Forward angle scatter
- Side Scatter Light
- Forward Low Angel Scatter
a.0 degree scatter - straight line -
Volume of the call
Forward angle scatter
Side Scatter Light
_____fight scatering - intenal stracteres
a. A.k.a…
90°
Orthogonal Light Scatter
Sidescatter
b. Refraction and reflection of light
C. Internal structures of the cell
Forward Low Angle Scatter/Forward High Angle Scatter
a. → Differential scatter
b. Cell volume
Variables Measured by the Optical Light Scatter
RBC Count
Mean Cell Volume
5-part WBC differential
it is 5-part Neutrophils, eosinophils, basophils, lymphocytes, monocytes
FLUORESCENT FLOW CYTOMETRY
2 mechanisms employed
Light scattering
Fluorescence
- light emission when electrons are raised to an excited state; as they go back to their ground state; they emit a light of a particular wavelength
• There is a loss of energy, and that loss of energy is in the form of light
Fluorescence
Measures multiple cellular and fluorescent properties of cells when they flow as a single cell suspension through a laser beam
FLUORESCENT FLOW CYTOMETRY
FLUORESCENT FLOW CYTOMETRY
Side Scatter Light -
Side Fluorescence Light -
Forward Scatter Light -
internal cell structure
RNA/DNA information
cell volume
Components of the Fluorescent Flow Cytometry
1 - FLUIDICS (THE FLOW SYSTEM)
#2 - OPTICS
Fluorescent flow cytometry
The sample is injected into a stream of sheath fluid within the flow chamber
The cells are hydrodynamically focused so that they will line in a single file; so these cells will be analyzed one cell at a time
FLUIDICS (THE FLOW SYSTEM)
Fluorescent flow cytometry
Two types of flow rate
High flow rate =
Low flow rate =
immunophenotyping analysis
DNA analysis
Two types of flow rate
High flow rate =
Low flow rate =
immunophenotyping analysis
DNA analysis
LASER light is required to excite the cells
Fluorescent Flow Cytometry
A system of optical mirrors and filters then direct the specified wavelength of light to the designated photodetectors
Detectors for Fluorescence
converted to an electrical signal
ELECTRONICS
Converts optical signals (photons) to corresponding electronic signals (electronics)
Electronic signal produced is _____striking a cell
proportional to the amount of light
Fluorescent Flow Cytometry
___is converted to____ signal
_____receives the electric current and converts to____
_____assigns the voltage pulse is assigned a digital value representing a channel
The channel number is transferred to the computer
______the channel number to the appropriate position in the____
Light electronic
Amplifier; voltage pulse
Analog-to-Digital Converter (ADC)
Computer displays
data plot
Fluorescent Flow Cytometry
___is converted to____ signal
_____receives the electric current and converts to____
_____assigns the voltage pulse is assigned a digital value representing a channel
The channel number is transferred to the computer
______the channel number to the appropriate position in the____
Light electronic
Amplifier; voltage pulse
Analog-to-Digital Converter (ADC)
Computer displays
DATA ANALYSIS
Fluorescent Flow Cytometry
Data is collected and stored in the
computer
Forward Scatter, Side Scatter, Emitted Fluorescence
Data plots
Fluorescent flow cytometry
Single parameter =
Two parameters =
histogram
data
Fluorescent flow cytometry
Boundary that can be set to restrict the analysis to a specific population within the sample
Data selected by the ___ is then displayed in subsequent plots
GATING
Fluorescent flow cytometry
Consists of collecting cells of interests
• Defined through criteria of size and fluorescence
SORTING
ESTIMATION OF RETICULOCYTE COUNT
Reticulocyte counting makes use of______ stains to stain the residual
RNA of the reticulocyte
Supravital
+ Based upon the uptake of various dyes and fluorochromes by the RNA of reticulocytes
ESTIMATION OF RETICULOCYTE COUNT
ESTIMATION OF RETICULOCYTE COUNT
FLUORESCENT DYES USED FOR SUPRAVITAL STAINING
Auramine O
Thiazole Orange
CD4K 530
Oxazine 750
New Methylene Blue
Most widely used fluorochrome for [residual RNA]
Thiozole orange
Methylene Blue-
Fluorescent stain -
manual
Automated
Methylene Blue-
Fluorescent stain -
manual
Automated
Classification of reticulocytes into 3 maturation stages
a. Low Fluorescence Reticulocytes
b. Middle Fluorescence Reticulocytes
C. High Fluorescence Reticulocytes
Classification of reticulocytes into 3 maturation stages
a. Low Fluorescence Reticulocytes i.
__________
b. Middle Fluorescence Reticulocytes
C.______
High Fluorescence Reticulocytes i. Immature reticulocyte
More mature reticulocyte
Middle Fluorescence Reticulocytes
= the amount of RNA found in
the reticulocyte
Classification of reticulocytes into 3 maturation stages
The degree of fluorescence
OTHER METHODS
Identification and counting of granulocytes
Why granulocytes?
> Granulocytes contain the enzyme myeloperoxidase
• Lymphocytes are not stained
They will be ruled out
Peroxidase
Reticulocytes and platelets
Best for detecting immature platelets
Fluorescence
Accurate platelet counting using CD41/CD61 antibodies
Immunological
Graphical representation of numerical data of different cell populations in a cell counter
Histogram
Histogram
Gives information on:
• Average size of cell
• Distribution of size
identify the type of cell based on its volume
Discriminators separates the distribution curve for the volume
Threshold discriminator will be able to
Discriminators separates the distribution curve for the volume
Threshold discriminator will be
WBC Discriminator
RBC Discriminator
Platelet Discriminator
Fixed discriminator
LD = 30-60 fL
UD = fixed at 300 fL
LD = 25-75 fL
UD = 200-250 fL
LD = 2-6 fL
UD 12-30 fL
12 fL
Platelets have volume between____and counted between____
8-12 fL
2-25 fL
RBCs have volume____ and are counted between____
80-100 fL
25- 250 fL
The normal curve peaks between 80-100
This curve peaks at around peaks around 30-60
There is a shift going to the left, skewed more to the left
That means most of the cells in the sample are within this volume - between around 30-100
SHIFT TO THE LEFT
If most of the population of this cell falls around this volume [remember the normal curve range]
NORMAL
MICROCYTOSIS
More than 100
MACROCYTOSIS
There is a peak in the volume greater than 100 fL
CURVE SHIFTS TO THE RIGHT
TWO PEAKS IN THE CURVE
Seen in cases of_____ and seen in______ :
TAILS: Thalassemia, anemia of chronic disease, iron deficiency, lead poisoning, sideroblastic anemia
DIMORPHIC POPULATION
anisocytosis
microcytic hypochromic anemias
WBC HISTOGRAM
Cells > 35 fL = WBC in the WBC/Hb chamber
In the WBC chamber, it involves hemolysis of RBCs
RBCs will be hemolyzed so that only WBCs will remain in the solution
When RBCs are
hemolyzed, hemoglobin will be released
WBC and Hgb chamber
WBC HISTOGRAM
Lymphocytes =
Mononuclears =
Monocytes, blast cells, immature granulocytes, reache lymphaugtes
Neutrophils =
35-90 fL
90-160 fL
160-450 fL
WBC HISTOGRAM FOR IMPEDANCE
The plot has more number of cells that have a volume > 100
Around 160 - 200 fL
Normal: 160-450
More neutrophils vs. other cells
Lymphocytosis
Neutrophilia
WBC HISTOGRAM FOR IMPEDANCE
WBC-Histogram
Peak in the population of cells having a volume of 90-160 fL where the mononuclears are plotted
Monocytosis
WBC HISTOGRAM FOR IMPEDANCE
WBC-Histogram
Eosinophils
Eosinophilia
- To ensure readings from an instrument are consistent with other measurements
- To determine accuracy of the instrument readings
- To establish reliability of the instrument
Calibration
-Determines the accuracy and precision of the analyzers
→ “Tuning” of the instrument
Calibration
Calibration is done:
→ Upon installation of machine
* Every 6 moths
-Periodic after major repair
