Quality Control in Serology

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Quality control in serology

• Dr.T.V.Rao MD

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What is Quality Control?

• Process or system for monitoring the quality of
  laboratory testing, and the accuracy and
  precision of results
• Routinely collect and analyze data from every
  test run or procedure
• Allows for immediate corrective action

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Definitions

• Quality Control - QC refers to the measures that
  must be included during each assay run to verify
  that the test is working properly.
• Quality Assurance - QA is defined as the overall
  program that ensures that the final results
  reported by the laboratory are correct.
• The aim of quality control is simply to ensure
  that the results generated by the test are
  correct. However, quality assurance is concerned
  with much more that the right test is carried
  out on the right specimen, and that the right
  result and right interpretation is delivered to
  the right person at the right time

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Definitions

• Quality Assessment - quality assessment (also
  known as proficiency testing) is a means to
  determine the quality of the results generated by
  the laboratory. Quality assessment is a challenge
  to the effectiveness of the QA and QC programs.
• Quality Assessment may be external or internal,
  examples of external programs

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Designing a QC Program

• Establish written policies and procedures
• Corrective action procedures
• Train all staff
• Design forms
• Assure complete documentation and review

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The Quality System
Information Management
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The Quality Assurance Cycle
Pre-Analytic
Patient/Client Prep Sample Collection
Personnel Competency Test Evaluations
Reporting

• Data and Lab Management
• Safety
• Customer Service

Post-Analytic
Sample Receipt and Accessioning
Record Keeping
Sample Transport
Quality Control
Testing
Analytic
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Need for Serological Tests

• SEROLOGICAL TESTS are performed to demonstrate
  antigens in the serum, or the response of the
  human body to these infectious agent ( Antibodies
  ) to establish its contact with the immune
  system. Their diagnostic importance stems from
  demonstration of a rising titre of antibodies to
  the agent which inter alia indicates a
  progressive infection. In rare instances is the
  presence of antibody in a single sample
  indicative of infection and disease. Serological
  tests are of importance in epidemiological
  studies and to ascertain the response of the
  population to vaccines and other
  immunopotentiators.

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Importance of Serological Tests

• Serological tests are of importance in
  epidemiological studies and to ascertain the
  response of the population to vaccines and other
  immunopotentiators.

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Serology performed mainly as vitro tests

• Serological tests are also useful for the in
  vitro detection of microbial infections, and for
  the classification and sub classification of
  infectious agents (e.g. Salmonella, Shigella,
  Streptococcus, etc.).

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COLLECTION OF SPECIMEN

• There must be a system for the orderly and
  efficient requesting of tests collection and
  identification of specimens and transporting,
  preparation, and storage of specimens. Nothing is
  more important than having an adequate amount of
  an appropriate specimen in good condition for
  examination. If each specimen is not properly
  collected, labeled, and handled, or is not
  representative, the laboratory may do more harm
  than good by testing it.

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Haemolysed specimens are not suitable for testing

• Haemolysed blood specimens are not suitable for
  serological studies. It is always advisable to
  avoid factors which cause hemolysis (Table 14.1).
  Specimens containing precipitates should be
  centrifuged prior to testing.

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Avoidable causes of hemolysis

• Blood sampling through too small bore of a needle
•       Forced suction of blood in the syringe
  during blood collection
•       Vigorous shaking of blood from the syringe,
  especially through a needle
•       Centrifuging blood sample at a high speed
  before clotting
•       Freezing and thawing of blood
•       Unclean tubes with residual detergents
•      

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Serology can Detect either antigen or antibody

• Serological reactions detect either a specific
  antigen produced by the microorganism or a
  specific immune response of the human body.
  Serological tests may detect
• 1 an immunological principle (antigen-antibody
  reaction ELISA, Widal)
•  2 a non-specific reaction (VDRL test)
•   3 a reaction mediated by complement (complement
  fixation test)

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advantages with serological methods.

•  Rapid identification of agent
•      High specificity of detection of antigen
•      Simplicity of performance
•      Safe procedures
•      Diagnostic aids
•      Epidemiological tools
•      Retrospective confirmation of diagnosis

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List of tests added every day

• A wide variety of serological tests are now
  available and every day new ones are added to an
  already impressive list. Every laboratory must
  define a policy for conducting these tests
  because some may be expensive, all require
  certain reagents (sera or antigens etc.) which
  have limited shelf life, and all require
  standardised techniques which must be documented
  in SOPM.

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Standard operating procedures are foundations in
all protocols

• An important element in maintaining day-to-day
  uniformity in laboratory results is an
  established procedure manual (SOPM) which details
  all phases of the laboratorys operation
  (including safety precautions) and is used by all
  laboratory personnel. It should include
  instructions for collecting, transporting, and
  storing specimens, for preparing and storing
  reagents, and for performing tests. In addition,
  the controls and calibrators to be used should be
  listed along with directions for their use,
  expected results, and instructions for corrective
  measures if the expected results are not obtained.

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Control Sera

• Source
• Some control sera are available commercially.
  Small volumes are generally available as
  components in kits but are intended to be used
  only with a single kit. A few may be available in
  larger quantities.
• Preparation
• Sera to be used as controls should be kept
  sterile to avoid deterioration. In general each
  procedure should have a normal control serum
  (negative), a strong positive control serum, and
  another positive control serum which is reactive
  at the critical concentration (borderline
  positive). With some tests, controls with a low
  concentration of analyze should be included.
  Controls recommended by the manufacturer of a
  particular test should always be used and
  additional control sera can be included if a test
  involves special problems.

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Storage

• Sera to be used as controls should be
  standardized against international reference
  materials when they are available. "Standards"
  included in commercial kits are not calibrated
  with each other and often are not
  interchangeable. These should be stored in
  aliquotes in frozen forms. Repeated freezing and
  thawing should be avoided.

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Quality reagents give optimal results

• Quality reagents are necessary for quality
  performance. A record should be kept of any
  changes in reagents in case the performance of a
  test changes. Before new reagents are introduced
  into a system they should be tested in parallel
  with the old reagents against a panel of
  appropriate reference sera to be sure that
  consistent reactions are obtained. The results
  obtained with the panel should reflect the
  sensitivity and specificity of the reagents being
  compared.

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Label all the reagents

• Reagents should be clearly labeled to indicate
  their identity, hazards involved in their use,
  recommended storage conditions, and preparation
  and expiration dates.

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EQUIPMENT AND INSTRUMENTS

• All glassware used in immunologic tests must be
  clean and free of detergent. Chipped or etched
  glassware should be discarded. Calibrated
  glassware should be checked for accuracy.
• The users accuracy and precision requirements
  should be met or exceeded when equipment is
  tested under working conditions. The
  manufacturers specifications for performance
  should be checked and met. Instruments and
  equipment should be monitored routinely. The
  temperature of water baths, incubators,
  refrigerators, and freezers should be checked
  periodically and records maintained. Maintenance
  should be performed and records kept on a regular
  basis by individuals who are trained and are
  familiar with the equipment.

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Quality control of instruments

• Instruments used for measurements including
  spectro-photometers, spectrometers, dilutors, and
  automatic pipettes should be calibrated on a
  regular basis.

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Selecting a procedure or a protocol
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Choose the appropriate test to your laboratory

• As new tests and methods are developed for
  various analytes (antibodies or antigens), the
  most appropriate must be chosen for each
  laboratorys needs. A number of factors must be
  considered, including bias, specificity,
  sensitivity, precision, cost and ease of
  performance. Bias, specificity and sensitivity
  may be related. Frequently the more sensitive a
  test, the less specific it is. Bias may result
  from low specificity or sensitivity.

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How to reduce the presence of Bias

• To determine the presence of bias, the proposed
  method should be compared with other reliable
  methods, preferably with a standard method or
  clinical data. The same specimens should be run
  with both methods in the same laboratory and the
  results compared, although interlaboratory
  comparisons are also useful. If the results from
  the different methods do not agree, one must
  determine the reason for the difference and then
  decide which result is more useful.

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What is clinical specificity

• The clinical specificity of a method is evaluated
  by testing negative samples and samples
  containing substances which might cause
  interference. Closely related or cross-reacting
  substances frequently found in clinical specimens
  should be included.

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Making Suitable Dilutions
100 ul serum in tube 1
Mix and Transfer
Discard
100ul diluent in each tube
Each tube is a 12 dilution of the previous tube
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Selecting a Suitable Sample Dilution
Serial Dilutions on Abbott AxSYM HIV-1/HIV-2 MEIA
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18
16
14
12
S/Co Ratio
10
8
6
Pos Cont 3.3
4
Cut Off 1.0
2
Neg Cont 0.38
0
Doubling Dilutions
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What is clinical Sensitivity

• The clinical sensitivity of a method being
  evaluated should be compared to that of other
  methods, but the purpose of the test must also be
  considered. In general, a definitive test need
  not be as sensitive as a screening test. The test
  should distinguish between normal and abnormal
  levels of analyze.

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Evaluation on precision

• The precision of a quantitative or
  Semiquantitative test must be evaluated in light
  of the precision required for the clinical
  application of the test results. Many factors
  affect precision, but one that is frequently
  overlooked in serologic tests is the size of the
  dilution increments. If all other variables are
  held constant, serologic tests tend to become
  less precise as the size of the dilution
  increment increases. For example, it should be
  expected that a test based on a four fold
  dilution would be less precise than the same test
  with a two-fold dilution.

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Errors in measurement

• True value - this is an ideal concept which
  cannot be achieved.
• Accepted true value - the value approximating the
  true value, the difference between the two values
  is negligible.
• Error - the discrepancy between the result of a
  measurement and the true (or accepted true
  value).

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When you need a highly sensitive test

• A test with maximum possible sensitivity is
  desirable when a disease is serious and its
  diagnosis should not be missed when the disease
  is treatable, and when false-positive results do
  not lead to serious problems. Similarly a test
  with maximum specificity is desirable when a
  disease is serious but is not treatable, the
  knowledge that the disease is absent has
  psychological or public health value, and
  false-positive results can lead to serious
  problems. A high predictive value of a positive
  test result is desirable when treatment of a
  false positive might have serious consequences.

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Quality control of tests detecting antibodies

• The performance of tests is monitored with
  controls. Antigenic serum panels as well as sera
  with known quantities of antibodies are available
  and should be routinely used. Correct performance
  of reagents is reflected by the expected reaction
  in tubes which lack one or more of the components
  necessary for the reaction. For example, the
  presence of anti-streptolysin O reagent is
  demonstrated by haemolysis in the tube containing
  the reagent buffer and cells but no antibody to
  inhibit haemolysis.

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Antibody test

• Flocculation test(RPR)
• control procedures required
• Nonreactive serum controlWeakly reactive serum
  controlReactive serum control
• Expected results
• No clumpingClumping of graded activityClumping
  of graded activity

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Antibody test

• Antibody test
• Latex agglutination test(ASO)
• Control procedures required
• Negative control serumPositive control serum
• Expected results
• No clumpingClumping

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Antibody test

• Antibody test
• Direct agglutination(Widal test, STA for
  Brucellosis)
• Control procedures required
• Antigen controlNegative control serumPositive
  control serum
• Expected results
• No clumpingNo clumpingClumping

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Antibody test

• Antibody test
• Passive haemagglutination (ASO)
• Control procedures required
• Streptolysin controlRed cell control
• Expected results
• HemolysisNo hemolysis

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Antigen test

• Antigen test
• Coagglutination test(Haemolytic streptococci
  meningitis antigens)
• Control material
• Group A,B,C streptococciN.meningitidis
• Expected result
• Agglutination with corresponding serum,

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Quality control procedures for tests detecting
antigens

• Antigen test
• capsular Quelling reaction(Omni serum,
  H.influenzae type b)
• Control material
• PneumococciHaemolytic streptococciH.influenzae
  type bAcinetobacter anitratum
• Expected result
• Capsular swellingNo reactionCapsular
  swellingNo reaction

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REPORTING AND RECORD KEEPING

• Complete and accurate records must be maintained
  in a good quality assurance programme. These
  records should include personnel information
  details of equipment, preventive maintenance,
  service, and repair copies of reports to
  physicians or other clients accession records
  records of reagents and materials used records
  of observations made concurrently with the
  performance of each step in the examination of
  specimens proficiency testing results and
  internal quality control results.
•  

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Accuracy and Precision

• The degree of fluctuation in the measurements is
  indicative of the precision of the assay.
• The closeness of measurements to the true value
  is indicative of the accuracy of the assay.
• Quality Control is used to monitor both the
  precision and the accuracy of the assay in order
  to provide reliable results.

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Precision and Accuracy

• Precise and inaccurate

• Precise and accurate

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Designing a QC Program

• Establish written policies and procedures
• Corrective action procedures
• Train all staff
• Design forms
• Assure complete documentation and review

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Qualitative QC

• Quality control is performed for both, system is
  somewhat different
• Controls available
• Blood Bank/Serology/Micro
• RPR/TPHA
• Dipstick technology
• Pregnancy

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Establishing Control Ranges

• Select appropriate controls
• Assay them repeatedly over time
• at least 20 data points
• Make sure any procedural variation is
  represented
• different operators
• different times of day
• Determine the degree of variability in the data
  to establish acceptable range

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Measurement of Variability

• A certain amount of variability will naturally
  occur when a control is tested repeatedly.
• Variability is affected by operator technique,
  environmental conditions, and the performance
  characteristics of the assay method.
• The goal is to differentiate between variability
  due to chance from that due to error.

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Sources of error

• Input data required - such as standards used,
  calibration values, and values of physical
  constants.
• Inherent characteristics of the quantity being
  measured - e.g. CFT and HAI titer.
• Instruments used - accuracy, repeatability.
• Observer fallibility - reading errors, blunders,
  equipment selection, analysis and computation
  errors.
• Environment - any external influences affecting
  the measurement.
• Theory assumed - validity of mathematical methods
  and approximations.

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Avoiding the errors

• The accessioning and reporting system should
  minimize the possibility of clerical errors.
  Precautions should be taken to prevent reporting
  results on the wrong specimen and transposing
  digits in reporting quantitative data. The system
  should be so designed that the history associated
  with a sample can be reconstructed in detail if
  necessary. Who performed which tests, what
  reagents and lot numbers they used, what the
  control results were for that run, and how and
  when the results were reported should also be
  documented

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Random Error

• An error which varies in an unpredictable manner,
  in magnitude and sign, when a large number of
  measurements of the same quantity are made under
  effectively identical conditions.
• Random errors create a characteristic spread of
  results for any test method and cannot be
  accounted for by applying corrections. Random
  errors are difficult to eliminate but repetition
  reduces the influences of random errors.
• Examples of random errors include errors in
  pipetting and changes in incubation period.
  Random errors can be minimized by training,
  supervision and adherence to standard operating
  procedures.

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Sources of error

• Input data required - such as standards used,
  calibration values, and values of physical
  constants.
• Inherent characteristics of the quantity being
  measured - e.g. CFT and HAI titer.
• Instruments used - accuracy, repeatability.
• Observer fallibility - reading errors, blunders,
  equipment selection, analysis and computation
  errors.
• Environment - any external influences affecting
  the measurement.
• Theory assumed - validity of mathematical methods
  and approximations.

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How to implement a QC program?

• Establish written policies and procedures
• Assign responsibility for monitoring and
  reviewing
• Train staff
• Obtain control materials
• Collect data
• Set target values (mean, SD)
• Establish Levey-Jennings charts
• Routinely plot control data
• Establish and implement troubleshooting and
  corrective action protocols
• Establish and maintain system for documentation

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Monitoring QC Data

• Use Levey-Jennings chart
• Plot control values each run, make decision
  regarding acceptability of run
• Monitor over time to evaluate the precision and
  accuracy of repeated measurements
• Review charts at defined intervals, take
  necessary action, and document

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Internal Quality Control Program for Serological
Testing

• An internal quality control program depend on the
  use of internal quality control (IQC) specimens,
  Shewhart Control Charts, and the use of
  statistical methods for interpretation.
• Internal Quality Control Specimens
• IQC specimens comprises either (1) in-house
  patient sera (single or pooled clinical samples),
  or (2) international serum standards with values
  within each clinically significant ranges.

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