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 content 1.Heamocytometer 2.Coulter counters 3.Flowcytometers 4.Auto Analyzers in clinical laboratories 5.Safety in the clinical laboratory

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.molecular biologypathologyimmunologyplant biology medicinesperm sortingsex 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