Clinical laboratory

instrumentation
Chapter one
Introduction

• Clinical laboratory is responsible for

– Analyzing patient specimens
– Diagnosis of disease
– Evaluate the effectiveness of therapy.
• The major sections of the clinical laboratory
– Clinical Chemistry
• Performs analyses on blood, urine, cerebrospinal fluid (CSF), and other fluids to
determine how much of various clinically important substances they contain
• Most applications of electronic instrumentation in the clinical laboratory take place in the
chemistry section.
– Hematology
• Performs determinations of the numbers and characteristics of the formed elements in
the blood (red blood cells, white blood cells, and platelets) as well as tests of the function
of physiological systems in the blood (clotting studies are an example).
• Use automated cell Counter
– Microbiology
• Performs studies on various body tissues and fluids to determine whether pathological
microorganisms are present.
• Devices that automatically monitor the status of blood cultures
Introduction..
• Definition
– Any device used to analyze and measure the physical and chemical
prosperity of analytes in patient sample for the purpose of
diagnosis and monitoring
• Eg of analytes
– Cells
– Proteins
– Carbohydrates
– Lipids
– Gases
– End product if metabolism
 Introduction
• Responsibility of laboratory
 Designing of workflow

 Selection of appropriate materials (reagents, equipment and instruments).

 Consulting clinicians on

 Test selection

 Reference values of analytes

 Guiding patients on sample collection

 Calibration

 Analyzing control and patient specimens

 Recording of patient results

Selecting Analytical Instruments

 What accuracy is required?

 How reproducible?

 How much sample is available?

 What is the concentration range of the analyte?

 What components of the sample will cause interference?

 What are the physical and chemical properties of the sample

matrix?

 How many samples are to be analyzed?

 Precision and accuracy

• Precision
 The closeness of agreement between independent test
results obtained under stipulated conditions
• Accuracy
 The closeness of the agreement between the result of a
measurement and a true value of the measurement
 Precision Vs accuracy

Not precise Not precise Precise Precise

Not accurate But accurate And accurate But not accurate
Instrument Management

 Selection
 Acquisition
 Installation
 Calibration / Validation
 Maintenance
 Troubleshooting
 Service and repair
 Retiring equipment / disposition
Selection Criteria

 Use

Matching equipment with service provided

 Performance characteristics

 Facility requirements

 Cost

 Supply of reagents

 Ease of operation

 Warranty

 Availability of manufacturer technical support

 Service Contracts
 Acquiring Equipment
 Purchase, Lease, or Rent
 Donor provided
 Contract considerations:
 Parts Manual
 Installation
 Operators’ Manual
 Trial period
 Contents of service contracts
 Installation Checklist

• Prior to installation:
– verify physical requirements have been met
 Safety checks, electrical, space, ventilation, water supply,
ambient temperature, etc.
– confirm responsibility for installation
• Upon receipt:
– verify package contents
– do not attempt to use prior to proper installation
• If required, ensure the equipment is installed by the
manufacturer
Installation

• After installation
– Establish inventory record
– Define conditions
– Develop and implement protocols for calibration,
performance verification, and operating procedures
– Establish maintenance program
– Provide training for all operators
Calibration of instruments

• Calibration a set of operation that establish under specific

condition on the relationship between reagent system
/instrument response and corresponding concentration
/activity values of an analyte.
• It is the Measurement of Accuracy.
• The purpose of calibration is to ensure that the measuring
accuracy is known over the whole measurement range under
specified environmental conditions
When do we need calibration?

 During installation
 After maintenance
 After displacement of instrument
 After systemic error
 Periodic calibration

• Periodic calibration has to be repeated at prescribed intervals

because the characteristics of any instrument change over a
period of time
Factors deciding the frequency of calibration

Conditions of use
Usage rate
Skill level of personnel
Degree of accuracy expected
Costs of calibration
 Instrument maintenance
1. Preventive maintenance
 Schedule Maintenance before the breakdown of the
instrument, the schedule can be
 Daily
 Weekly
 Monthly
 This type of maintenance is performed by laboratory
professionals
2. Curative maintenance
 Unscheduled Maintenance after the breakdown of the
instrument and this type of maintenance is
 performed by trained biomedical engineer
 Maintenance Program

Systematic and routine cleaning, adjustment, or replacement

of instrument and equipment parts

– Performed periodically, daily, weekly, monthly

• Monitoring of instrument parameters to verify that that your

equipment is working according to the manufacturer’s
specification

• Performed after major instrument repair

Implementing a Maintenance Program

 Assign responsibility
 Oversight of all laboratory equipment
 Individual responsibilities
 Develop written policies and procedures
 Train staff
 Keep records
 For each piece of equipment:
 Establish routine maintenance plan
 Establish required function checks
 Develop a list of spare parts
Troubleshooting

• Check manufacturer’s instructions

• Determine source of problem
– Sample problem
– Reagent problem
– Equipment problem
– Check electrical supply
– Check water supply
• Make one change at a time
Service and Repair

• Schedule service that must be periodically performed by the

manufacturer
• Options
– Centrally service small equipment, e.g., microscopes,
washers, pipettes
– Team of biomedical service technicians
Benefits of a Maintenance Program

• Safety
• Fewer interruptions of work
• Lower repair costs
• Elimination of premature replacement
• Less standby equipment
• Identification of high maintenance cost
• Reduction of variation in test results
• Greater confidence in the reliability of results
Retiring Equipment / Disposition

• When?
– When experts indicate not repairable

– Outmoded, will replace with new equipment

• Why?

– Prevent inaccurate test results

– Free up valuable space

– Hazardous

• How?

– Salvage any useable parts

– Consider biohazard, follow safety disposal procedures

Equipment Documentation

• Develop a problem log record for each piece of equipment

• Date problem occurred, removed from service
• Reason for breakdown or failure
• Corrective action taken
• Date returned to use
• Change in maintenance or function checks
Water Purity and Distillation System Use
 Purpose of Water Purification

To remove dirt, chemicals such as ions and substances so that only pure H2O
remains for laboratory uses.

There are different levels of purity:

– Type I

• Quantitative reagents, dilutions of samples

– Sterilized type I: microbiologic testing
– UV Oxidized sterilized type I: cell culture and nucleic acid testing

– Type II
• Qualitative urinalysis, rinsing laboratory ware, qualitative reagents or
stains
– Type III
• Tap water for washing laboratory ware
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Criteria established to determine levels of purity

Characteristic | Type I | Type II | Type III

• Colony forming unit | 10 | 1000 | NA
(CFU/mL)
• Resistance (megaohm-cm) |<10 | 2 -9 |1.9-0.1
• Higher resistance to current conductance means that fewer
ions are present.

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 Four Main Methods of Water Purification

• Distillation
• Ion Exchange
• Reverse Osmosis
• UV Oxidation

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 Distillation

• Oldest and time-tested method

• Principle:
– Heat the liquid to boiling point (vaporisation) and collect steam
into condensation coils (condensation).
• Large contaminating particles in water are first removed by:
– sedimentation
– decanting of supernatant
– filtering through a sieve
• Vaporisation and condensation removes volatile contaminants.

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 Distillation

• Type II water is produced by this method

• Advantages: inexpensive and simple to operate equipment
• Problems with carry-over

volatiles and electrolytes

Safe Operation of Distiller

• Check that power cord is grounded
• Do not allow to boil dry
• Periodically clean out built-up residue

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Ion Exchange Filtration

• Principle:
– some or all ions are removed with a membrane ion exchange filter
• Good secondary water purification step
• Produces Type I water if start with distilled water and then filtered
with anion and then cation filters

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Ion Exchange Filtration

• Water is called deionised by this process.

• Conductance meter will check level of ions removed
• Higher ohm/ cm indicates fewer ions remain

Disadvantages:
• Relatively expensive process especially if two-column system
• Ion exchange columns need replacement periodically

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Reverse Osmosis Filtration
• Principle:
– pressure forces water through semi-permeable membrane to
remove many organic and inorganic chemicals.
• Dissolved gases remain.
• Type I water is produced from reverse osmosis filtration if first start
with distilled water

Disadvantages:
• Very expensive process
• Reverse osmosis filter columns need replacement periodically

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UV Oxidation
• Principle:
– Water exposed to UV wavelengths and ozone will sterilize bacteria
but not remove them.
• Type I sterilized water is produced after membrane purification
followed by UV oxidation process.
Disadvantages:
• Very expensive process
• Not commonly available in developing countries
• Filter columns and UV lamp need replacement periodically

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Sterilized type I water

• Sterilized type I water is used in cell culture.

• Glass distillation or other water purification techniques (e.g.
deionisation) may be used but the water for cell culture should be
autoclaved before use.

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Laboratory Sterilization
Autoclave
 Basic Definitions

• Sterilization is the use of a physical or chemical procedure to destroy

all microbial life, including highly resistant bacterial endospores.
• Disinfect ion eliminates virtually all pathogenic non-spore-forming
microorganisms but not necessarily all microbial forms on inanimate
objects.
– Effectiveness is influenced by the kinds and numbers of organisms,
the amount of organic matter, the object to be disinfected and
chemical exposure time, temperature and concentration.

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Purpose

• To kill microorganisms and endospores

• To decontaminate reusable equipment, specimens or other infectious
wastes prior to routine disposal
• To prepare sterile media for cell cultures.

Methods
– Moist heat (autoclave)
– Dry heat
– Chemical sterilisation
– Filtration

The most commonly employed method in medical laboratories is with

moist heat, an autoclave. 37
Autoclave Conditions
These main factors must be considered for sterilization with an
autoclave:
– Temperature
– Pressure
– Timing

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 Moist Heat Steriliser/ Autoclave

Most Heat Sterilizer/ Autoclave can:

– Agar and liquid culture media
– Swabs for use in microbiology work.
– Specimen containers
– Pipettes
– Petri dishes
– Laboratory glassware or heat-stable equipment using a higher
temperature.

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Temperature

• Steam is produced when pure water boils at 1000 C.

• Autoclaving at 1210C for 15-20 min. is required for the fastest route.

• In an autoclave, all air is removed and pressure is used to produce high

temperature steam.
• At a pressure of 15 pounds per square inch (psi), the temperature of
saturated steam rises to 1210C.
• If air remains behind in the steam it will not properly sterilize some
autoclaves have continuous pressure purge which removes cold air
pockets for uniform sterilization.
• Pressure may be also noted in bars. 1.1 bar is equivalent to 104 k Pa or
15psi.
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Pressure
• An autoclave cycle that ensures a sterilizing temperature of 121°C for at
least 15 minutes at a pressure of 15 psi (100 kPa) is adequate for liquid
reagents; for equipment sealed in autoclave bags a temperature of
126°C is desirable, or 121°C for 20 minutes.

Time
• 1210C for 15-20 min. is required for the fastest route of sterilization but a lower
temperature and longer sterilization time can be used.
• It takes certain period of time for the saturated steam to penetrate the entire
load and for heat transfer to occur. Time for heat up needs to be added to
total time

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 Autoclave Safety

• Follow operating and maintenance instructions supplied by

manufacturer
• To prevent accidents and injury it is important to allow sufficient time
after sterilization for the pressure to return to zero and for the load to
cool.
• Many newer autoclaves have locks that won’t release until temperature
and pressure have lowered to safe levels.

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 Methods of monitoring the performance of Autoclave

Daily
– Follow manufacturer’s instructions and monitor time,
temperature and pressure gauges
– Simple screen with Sterilization strip quarterly or annually
– Spore strips or Indicator chemicals

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 Other Less Used Sterilization Methods

• Dry Heat (oven) requires 1600C for 120 minutes

• recommended for sterilisation of some materials prior to use such as
– serum or egg based media
– glassware, petri dishes, inoculating materials that may not withstand
high pressure steam
• Chemical Sterilization with disinfectants
– More expensive and some concerns for persistence of chemical
pollution
• Membrane Filtration
– Most frequently used for sterilization of water prior to purification
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Biological Safety Cabinets
Biological safety cabinets are:
– Containment device with HEPA (high efficiency particulate air flow)
filter designed to provide personal environmental or product
protection from biohazard materials
– Used to prevent the escape of aerosols into the laboratory
environment
– Protect the experiment from airborne contamination
• Selection of a biological safety cabinet is based on:
– Hazard of the agent used in the experiment
– Potential of the laboratory technique to produce aerosols
– Need to protect the experiment from airborne contamination
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Types of Biological Safety Cabinet

• There are three types of biological safety cabinets used in the

microbiological laboratory:
– Class I cabinet
– Class II cabinet
• A1
• A2
• B1
• B2
– Class III cabinet

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Class I Safety cabinets
• It is an open-fronted work chamber which is exhaust-ventilated to
provide personnel and environmental protection only by means of an
inward air flow away from the operator
• 100% air exhaust
• The exhaust air being filtered through a HEPA filter before being
discharged from the cabinet.
• Class I cabinets should be used with agents of low to moderate risk.

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Class I Safety cabinets
Class II Safety Cabinets
• A Class II biological safety cabinet is a partially open-fronted work
chamber which provides protection for personnel, the surrounding
and/or experiment against contamination by means of HEPA filtered air
flowing in a downward uniform and unidirectional (laminar) manner.
• The means of creating a “barrier air flow” at the work opening is not
only by means of an inward air flow from the operator but also from
the inside to create air curtain.
• The downward air velocity should be sufficient to afford protection
against cross-contamination and prevent outflow.
• There are three types of class II cabinet

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Type A1 cabinet
• The Type A1 cabinet has a minimum inflow velocity of 75 ft./min.
• The filtered makeup air is divided equally over the work surface at
about two to six inches above the work surface.
• Exhaust is drawn at the bottom of the cabinet where it rises to the
top.
• At the top of the cabinet, 70% of the air recirculates through the
supply HEPA filter, the other 30% of air exhausted through the
exhaust HEPA filter.
Type A1 cabinet
 Type A 2
• Has a minimum inflow velocity of 100 ft/min.
• A negative air pressure plenum surrounds all contaminated plenums
that are under positive pressure
Type A 2
 Type B1
• The Type B1 cabinets have a minimum inflow velocity of 100 ft/min.
• These cabinets must be hard‐ducted to an exhaust system( In contrast
to the type A1 and A2 cabinets, 70% of air from the rear grille is
exhausted and only 30% is recirculated).
 Type B2
• Type B2 cabinets have a minimum inflow velocity of 100 ft/min
• Type B2 cabinet is expensive to operate because no air is recirculated
within.
• this type is mainly found in such applications as toxicology laboratories.
• cabinets of these types generally monitor the exhaust flow, shutting off
the supply blower and sounding an alarm if the exhaust flow is
insufficient.
• Used in contaminated environment
Class III cabinets
• It is a totally enclosed and gas-tight structure.
• Work procedures in the cabinet are carried out through replaceable
arm-length gloved sleeves.
• The cabinet is supplied with air through a HEPA filter and exhausted
through two HEPA filters mounted in series.
• The cabinet is operated at negative pressure and provides total
protection for use with all categories of biological agent.
• The negative pressures within a Class III cabinet should be a minimum
of 200 pa(pascal).
• The air flow should also be sufficient to allow for two of the glove ports
to be open and achieve a minimum velocity of 0.75m/s through the 59
Class III cabinets
Safety cabinet effectiveness

• The place of safety cabinets are important. The main problems are
caused by current of air from doors and windows and the movement
of people and therefore, safety cabinets should not be sited near
doors.
• HEPA filters are highly efficient in removing viable microorganisms
with a quoted efficiency rate is 99.997%.
• The achievement of barrier air flow should be confirmed by
contamination challenge tests.
• When successful containment is reached, exhaust velocity should
then be measured and recorded

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 Decontamination of safety cabinets

• The working surfaces and walls of safety cabinets should be

decontaminated on a day-to-day basis by swabbing with disinfectant.
• Glutaraldehyde is probably the best disinfectant for this purpose as
phenolics may leave sticky residues and hypochlorite may, in time,
corrode the metal.
– For thorough decontamination e.g. after spillages, before
maintenance, filter changing and testing, fumigation is necessary
• Before fumigation, the installation should be checked to ensure that
none of the gas can escape to the room or other rooms.
• Some safety cabinets have built in UV light for disinfection.
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End of chapter one