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