Automated analyzers Flashcards
Cell counts can be done by
Can be performed manually or by machines
Usually done on automated analyzers
Exotic and avian species counts are done manually
Cell counts are
Total red blood cell counts and platelet counts can be done using automated analyzers
When not available, estimates can be done
The white blood cell count is also known as the total leukocyte count
Types of hematology instruments
Blood cell analyzers fall into three categories
Impedance analyzers
Laser cytometry analyzers
Quantitative buffy coat analysis systems
Some analyzers will combine these methods
Each type of system has its advantages and disadvantages
You need to have a basic understanding of the different methods of analysis
This will allow you to understand their limitations to generate accurate, precise and therefore reliable results
Impedance analyzers work by
Classifies cells based on their size
An electric current passes across two electrodes that are separated by a glass tube that has a small opening/aperture
Electrolyte fluid is used as a conductor
A specific volume of cells in the electrolyte solution is passed through the opening of the glass tube either by vacuum or positive pressure
The cells do not conduct electrical current well, so they “impede” the flow while passing through the small opening
The small changes in current are counted to determine the blood cell concentration
The size of the cell is proportional to the change in current→ this system is able to differentiate between cell types (RBCs, WBCs and platelets)
When is a impedance analyzers inaccurate
Can be inaccurate in cats due to similarity in size of RBCs and platelets
Not reliable in exotic species as the size of RBCs, WBCs and thrombocytes are too similar
How is cell size displayed on an impedance analyzer
Cell size info may be displayed in a graphical format of the cell population
This is called a histogram
How to count WBC and RBC on a impedance analyzer
To count WBCs, a lytic or lysing agent is used
Lyses platelets and RBCs, therefore thy are not counted
Fragile WBCs can be lysed decreasing the total WBC count
RBCs are counted without the use of analytic agent and at a higher dilution
Erythrocyte size can also be determined
RBC analysis also provides information on cell volume and packed cell volume (PCV)
Can also use cell volume to determine mean corpuscular volume (MCV)
Disadvantages of impedance analyzers
Variations of cell size produces errors
Does not identify morphologic abnormalities
A thorough examination of the differential blood film must also be used to evaluate patients
Nucleated RBC, large platelets and platelet clumping create inaccurate results
These machines require daily cleaning or flushing as dust particles can affect results or cause blockages
Generally it is part of their start up procedure
Blood must be warmed to room temp
Quantitive buffy coat system works by
Uses centrifugation and staining to estimate cellular elements
The buffy coat layer is expanded within a specialized hematocrit tube and the use of a specialized bead
Provides hematocrit value, estimate of leukocyte concentration and platelet concentration
Gives a partial differential count
Granulocytes, monocytes and lymphocytes
Limitations because abnormalities may be undetected- blood smear examination is required
This type of system should only be used as a screening test
It only provides estimates of cell numbers, not actual cell counts
Laser flow cytometry analyzers work by
Uses focused laser beams to evaluate size and density of the cells
Cells scatter the light differently depending on their shape, volume, number and size of granules, and nuclei
Cells flow past the laser in a single file through a channel
How the light is refracted and picked up on the other side of the tube determines the cell type
Dyes can be added to the sample to help further classify cells
Can enumerate monocytes, lymphocytes, granulocytes, mature and immature RBCs, erythrocyte indices, red cell distribution width (RDW) and platelet parameters
Histograms look like
Provide a visual representation of the number and sizes of cell types present in a sample
Size on the “X” axis, numbers on the “Y” axis
Can also be represented by a scatter plot
Each dot represents a cell
Histograms are sued to determine
Average size of cells
Distribution of cell size
Detect subpopulations
Evidence of anomalies
What are histograms used for
There are RBC, WBC, and platelet histograms
WBC histograms evaluate lymphocytes, granulocytes and mononuclear cells
Histograms should be used as screening for pathology, but they are not diagnostic →they tell you that you need to look at a blood film
They are also used as a quality control measure for your machines
Help to verify differential blood film results
Total leukocyte count is
A specified volume of blood is used to count the total number of WBC
Your results will be reported as the # of WBCx109 /L (to 1 decimal place)
The total leukocyte count alone gives is limited info
It is essential to know what percentage of each WBC type the leukogram is composed of
This is known as the differential count
A total leukocyte count is performed with the Leuko-tic system OR by automated analyzers
The Leuko-tic system is a manual counting system that uses a blood diluting solution and a hemocytometer
Some automated analyzers use the same blood diluting solution as the Leuko-tic system
WBC estimation is
Used as a quality control measure for both manual and automated counts
Estimation of the total white blood cell count is done with the differential blood smear
Interpretation of the leukogram
Look at the total leukocyte and differential counts
Look for morphological abnormalities
Look or abnormal cell types
Immature cell stages
Why look at the total leukocyte and differential counts
This number is used to calculate the absolute differential counts
Total leukocyte count X cell %
Ex: neutrophils represent 60% of the WBCs in the differential count; total WBC count is 10.0x10^9/L
Absolute neutrophil count = 10.0x10^9/L x 60% = 6.0x10^9/L
Decreased total leukocyte count–look at the absolute count for each cell type & determine which is decreased
Increased total leukocyte count– look at the absolute count for each cell type & determine which is increased
Normal leukocyte count –look at the absolute count for each cell type & determine if it is within its normal reference range
What do abnormal leukocytes look like
These cells didn’t divide as often as they should have during development, or the cytoplasm and nucleus develop along different courses
Usually they are only classified as granulocyte or agranulocyte and are recorded as atypical cells on the differential blood film report sheet
1-2% is normal and are not recorded on the report sheet
Don’t be afraid to ask for a second opinion or send to a reference lab for evaluation
Total leukocyte counts are done by
Automated machines count all nucleated cells, this may include nucleated red blood cells
This will cause the total leukocyte count to be artificially high
Mathematical adjustments will be required when nucleated RBCs are seen on the differential smear
What are they types of chemistry analyzers
There are a variety of chemistry analyzers
Fall into one of 3 categories
Photometry- most common
Electromechanical
Light-scatter techniques
Photometry is
The machine used is a spectrophotometer: it produces light of various wavelengths cia filters, prisms or diffraction gratings
Spectrophotometers are designed to measure the amount of light transmitted through a solution
Basic components
Light source, prism, a wavelength selector, photodetector, and a read out device
The most common types of photometry and how they work
Absorbance- how much light did not reach the detector
-Used wet/rotor technology
Reflectance- how much light was reflected off a test substance
-Used by most in-house analyzers
-Uses a dry/slide chemistry system
Beers law is
A direct linear relationship exists between the concentration of an analyte and the light absorption when monochromatic light is passed through the sample
The transmission of the monochromatic light through a sample and the concentration of an analyte in the sample have an inverse exponential relationship
The degree of color change is proportional to the solution’s concentration
Beers law simplified
When we know how much light (of a single wavelength) was produced, and how much of that light passed through the solution to the detector, we can calculate the amount of light that was absorbed by the patient sample
Analyte–what we are measuring, ex. potassium
High concentrations of the analyte: more light is absorbed as it passes through the solution→and less light will reach the detector
Low concentrations of the analyte: less light is absorbed as it passes through the solution → more light will reach the detector
The higher the concentration of the analyte, the greater the color change at the detector
Two types of absorbance assay methods
End point
Kinetic
What is end point absorbance assay methods
Measures the concentration of preexisting substances in the serum or plasma
Involves a chemical reaction that reaches a stable end point and measures the color change
An internal standard curve is created when the instrument is calibrated
Must be recalibrated with each new reagent
A standard is used for calibration
Kinetic absorbance assay methods is
Generally used to measure enzyme assays or when enzyme based reagents are used
Reaction results are measured at a specific time after the initiation of the reaction
Does not reach a stable end point
Enzymes activity in inhibited by
Enzyme activity is greatly inhibited by low temp and accelerated high temp
Most assays are performed between 30-37 degree celsius
For every 10*C increase above the optimal temp, the enzyme activity doubles
Monitor incubators and water baths closely
Enzymes are
Enzymes are proteins
Denatured by temp, pH extremes, organic solvents, heavy metals or physical damage
Handle samples carefully
Electrochemical methods is used for
Also known as ion-selective electrode (ISE) technology
Some analyzers combine ISE and photometric methods
Most commonly used to measure electrolytes
The difference in electrical potential b/w the electrodes is proportional to the concentration of the ion in the sample
How does the electromechanical analyzers work
These analyzers are sometimes referred to as potentiometers
The potentiometer contains reference electrodes
Reference electrodes interact with only one type of ion and contain a known concentration of that ion
The patient sample interacts with a second ion specific electrode which is located within biosensor reagent strip or cartridge
There is a voltage difference b/w electrodes which is used to calculate the concentration of the ion in the samples
Light scatter analyzer is used by
Used to measure the concentration of larger molecules in fluids
Immunoglobulins
Antigen-antibody complexes
Drugs, etc
Detects the reduction of the intensity of a light as it passes front he light source, through a solution
Features and benefits of automated analyzers is
Automated analyzers use liquid reagents, dry reagents or slides that contain dry reagents
Almost all systems can now be integrated so that results are inserted directly into the patient’s electronic file
Eliminates missed charges
Accessible throughout the hospital
Dry systems work by
These include systems that use reagent impregnated slides, pads or cartridges
Catalyst Dx and VetTest
Most use reflectance photometry
There is little or no handling for reagents
Tend to be a little more expensive to run
Sample rejection is common particularly with lipemic, hemolyzed or large animal samples
Can preform individual or single assay tests
Slides do allow for loading of a large number of slides at once reducing the time needed to run a complete profile
Liquid systems work by
These include systems that use lyophilized reagent or already prepared liquid reagent
The most common is the lyophilized rotor technology
Vet scan
Rotor is made up of individual cuvettes that diluted sample is added to
Disadvantages of liquid systems
Cannot run individual tests
Tend to be quite accurate
Require more handling and storage space
Unitized systems require less handling but are more expensive
Liquid systems that use bulk reagents
There are also systems that use bulk reagents
These systems can run individual tests
Reagent solutions may or may not need to be diluted
Some have extensive maintenance requirements
Dedicated use analyzers are
Most utilize electrochemical methods
Test for a specific substance
Are used when only one substance is being tested
Blood glucose monitors
Instruments care and maintenance for analyzers
In order to generate reliable results, machines must be properly maintained
Follow manufacturer’s instructions carefully and completely
-Allow for warm-up periods, turn machines on first thing in the morning, and leave them on throughout the day
Make sure to document maintenance
Regular maintenance will prolong the life of the machine
