dr. tarek el sewedy
DESCRIPTION
Pharos university Faculty of Allied Medical SCIENCE Clinical Laboratory Instrumentation (MELI-201 ). Dr. Tarek El Sewedy. Lecture 7. Cytometry & Flowcytometers AND Auto analyzers. Intended Learning Outcomes. Students will learn : Principles and instruments used in of Cytometry techniques - PowerPoint PPT PresentationTRANSCRIPT
Pharos university Faculty of Allied Medical SCIENCE
Clinical Laboratory Instrumentation
(MELI-201)
Dr. Tarek El Sewedy
Lecture 7
Cytometry & Flowcytometers
AND
Auto analyzers
Intended Learning Outcomes
Students will learn :
1. Principles and instruments used in of Cytometry techniques
2. Auto Analyzers in clinical laboratories
Lecture content1. Heamocytometer
2. Coulter counters
3. Flowcytometers
4. Auto Analyzers in clinical laboratories
5. Safety in the clinical laboratory
Cytometry
1. Heamocytometers Cytometry is a method for counting cells using an instrument called
a cytometer.
Cytometry was traditionally carried out using a microscope and a
special slide (Heamocytometer).
The slide has a grid engraved onto a surface where a specific
volume of liquid is held.
When viewed under a microscope, it is possible to count the cells
within the grid.
These slides allow the counting of cells in a small volume and
extrapolating the result to determine the total number.
A specific volume of cells is placed on the slide. The number of
cells present in each grid is counted and an average determined.
Conversion using a formula gives the number of cells per milliliter
in the culture.
This method is rapid and is easy to perform. However, cultures with
low number of cells are not appropriate for direct counts using
slide techniques since there will be too few cells/grid resulting in
high errors .
2. Electronic particle counters“Coulter Counters”
A Coulter counter is a type of electronic
particle counter in which there is a small
opening between two electrodes through
which electrolyte suspended particles pass.
In this sensing zone, each particle that
passes causes an electrolyte displacement
causing a current pulse. The pulse is noted
and recorded as one particle count.
By precisely controlling the rate at which
solution passes through the opening, it is
possible to get exact, reproducible counts at
high cell counting rate.
Advantages and Disadvantages of Coulter counters
• The advantage of this method is the simplicity of its operation
and it reproducibility. However, As in microscopic counts, the
machine cannot distinguish between living or dead cells or even
between dust and bacteria. Any reasonably sized particle in the
solution will be counted.
3. Flow cytometers
A flow cytometer is a sophisticated instrument for counting, sorting and
identifying cell populations by suspending them in a fluid and passing
them by an electronic detection apparatus. It also allows researchers to
determine various characteristics of cells.
Automated flow cytometers can sort, count, identify and Characterize
cells at a rate of 500 to 5,000 cells per second.
This far exceeds the rate of scientists using a Heamocytometer or a
coulter counter.
It is estimated that a skilled scientist can only hand-count cells at a rate
of 200 cells per minute using a hemocytometer.
Flow cytometers
How it Works• Cells of interest are labeled (e.g. with fluorescent markers) and suspended in solution.
• The cells are forced out in a liquid jet stream.
• A beam of laser light of a single frequency is directed onto the stream.
• Each suspended particle passing through the beam scatters the light in some way.
• Several detectors can pick up the scattered lights and florescence and is analyzed.
• The data from the light scattering can be plotted on a graph to visualize different cell
populations in the sample
DATA Analysis
Cell Sorting
Some flow cytometers have the capability of separating cells from a
mixture of cells based on characteristics determined by the scientist.
This is achieved adding a device called cell sorter to the
Flowcytometer. The cell sorter places cells into separate containers
based on size or other characteristics.
This ability provides scientists with a simple means of isolating
diseased cells or particular genetically modified cells from mixtures
of cells for further analysis.
Fluorescence-activated cell sorting (FACS) is a specialized type of
flow cytometry. It provides a method for sorting a heterogeneous
mixture of biological cells into two or more containers, one cell at a
time, based upon the specific light scattering and fluorescent
characteristics of each cell.
Applications of Flowcytometers
The technology has applications in a number of fields, including
molecular biology, pathology, immunology, plant biology. It has broad
application in medicine (especially in transplantation, hematology, tumor
immunology and chemotherapy, prenatal diagnosis, genetics and
sperm sorting for sex preselection.
AUTOMATED CHEMICAL ANALYZERS
(Autoanalyzers)
An autoanalyzer sequentially measures
blood chemistry through a series of steps of
mixing, reagent reaction and colorimetric
measurements .
The AutoAnalyzer profoundly changed the
character of the chemical testing laboratory
by allowing significant increases in the
numbers of samples that could be processed
Autoanalyzers main parts
Main Parts of autoanalyzers
Sampler: aspirates samples, standards, wash solutions into the system.
pump: It mixes samples with the reagents so that proper chemical color reactions can
take place, which are then read by the colorimeter.
Dialyzer: it controls selective passage of sample components through a semi
permeable membrane
Heating bath: The heating bath controls temperature (typically at 37 °C), as temp is
critical in color development
Main Parts of autoanalyzers
Colorimeter: It monitors the changes in optical density of the fluid stream
flowing through a tubular flow cell. Color intensities proportional to the
substance concentrations are converted to equivalent electrical voltages.
Recorder: The recorder displays the output information in a graphical form.
Safety in the clinical laboratory
Each laboratory should have a written manual of safe laboratory practices
which should be followed at all times.
Laboratory should have a first-aid box and at least one staff member trained
in first aid.
The laboratory should be a work area only; visitors should be restricted.
No food or drink should be consumed in the laboratory.
Wear protective clothing and remove it before leaving the laboratory.
Always consider any laboratory specimen as potentially infectious and
handle it carefully; wear protective gloves.
Place all specimens safely on a bench or in a rack to prevent spillage or
breakage.
Take great care when collecting and processing blood samples as they
may harbor infective agents (e.g. hepatitis B virus, parasites, etc.).
Do not contaminate yourself or the work areas with any specimen.
Do not pipette blood or other body fluids or any reagents by mouth.
Cover all cuts with an impervious dressing (plaster).
Dispose of used needles and lancets safely in a “sharps” container.
Once filled, containers should be autoclaved or soaked in disinfectant before
burning or burying in a deep pit.
Cover any spilled material or broken culture tubes with a cloth soaked in
disinfectant and leave for 30min. Then use a stiff brush or sheet of cardboard
to sweep it into a disposable specimen container.
At the end of the day swab the benches with a cloth soaked in disinfectant.
Wash your hands well after handling infective material and before leaving
the laboratory.
Assignment
ALI Mostafa Mahmoud: should prepare an assignment on the different types
and applications of flowcytometers
The Assignment should be delivered before next lecture
Study questions
Mentions 3 different applications of Flowcytometers in medicine.
Mention the main advantages and disadvantages of Heamocytometer
Mention the criteria for Sorting of cells using a flowcytometer.
Suggesting reading
Encyclopedia of Medical Devices and Instrumentation, 2nd ed. New York:
Wiley, 2006