Quality control in serology

DR.T.V.RAO MD 1

Dr.T.V.Rao MD

QUALITY CONTROL IN SEROLOGY

DR.T.V.RAO MD 2

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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• Establish written policies and procedures• Corrective action

procedures• Train all staff• Design forms • Assure complete

documentation and review

DESIGNING A QC PROGRAM –

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Purchasing & Inventory

AssessmentOccurrence

Management

Information Management

Process Improvement

Customer Service

Facilities & Safety

THE QUALITY SYSTEM

Organization Personnel Equipment

Documents & Records

Process Control (QC & EQA) &

Specimen Management

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The Quality Assurance Cycle

•Data and Lab Management•Safety•Customer Service

Patient/Client PrepSample Collection

Sample Receipt and Accessioning

Sample TransportQuality Control

Record Keeping

ReportingPersonnel CompetencyTest Evaluations

Testing

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

IMPORTANCE OF SEROLOGICAL TESTS

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• 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.).

SEROLOGY PERFORMED MAINLY AS VITRO TESTS

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• 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.

COLLECTION OF SPECIMEN

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• 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.

HAEMOLYSED SPECIMENS ARE NOT SUITABLE FOR TESTING

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• 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

•

AVOIDABLE CAUSES OF HEMOLYSIS

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• 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)

SEROLOGY CAN DETECT EITHER ANTIGEN OR ANTIBODY

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• Rapid identification of agent

• High specificity of detection of antigen

• Simplicity of performance

• Safe procedures

• Diagnostic aids

• Epidemiological tools

• Retrospective confirmation of diagnosis

ADVANTAGES WITH SEROLOGICAL METHODS.

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• 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.

LIST OF TESTS ADDED EVERY DAY

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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 laboratory’s 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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• 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.

STORAGE

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• 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.

QUALITY REAGENTS GIVE OPTIMAL RESULTS

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

LABEL ALL THE REAGENTS

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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 user’s accuracy and precision requirements should be met or exceeded when equipment is tested under working conditions. The manufacturer’s 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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• Instruments used for measurements including spectro-photometers, spectrometers, dilutors, and automatic pipettes should be calibrated on a regular basis.

QUALITY CONTROL OF INSTRUMENTS

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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 laboratory’s 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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• 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.

HOW TO REDUCE THE PRESENCE OF BIAS

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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 DILUTIONS100 ul serum

in tube 1

100ul diluent in each tube

Mix and Transfer

Each tube is a 1:2 dilution

of the previous tube

Discard

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SELECTING A SUITABLE SAMPLE DILUTION

Serial Dilutions on Abbott AxSYM HIV-1/HIV-2 MEIA

Doubling Dilutions2 4 8 16 32 64 128

256

512

1024

2048

4096

8192

16384

32768

65536

131072

262144

524288

S/C

o Ratio

0

2

4

6

8

10

12

14

16

18

20

Pos Cont 3.3

Neg Cont 0.38Cut Off 1.0

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• 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.

WHAT IS CLINICAL SENSITIVITY

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• 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.

EVALUATION ON PRECISION

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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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• Flocculation test(RPR)

• control procedures required

• Nonreactive serum controlWeakly reactive serum controlReactive serum control

• Expected results

• No clumpingClumping of graded activityClumping of graded activity

ANTIBODY TEST

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• Antibody test

• Latex agglutination test(ASO)

• Control procedures required

• Negative control serumPositive control serum


• No clumpingClumping

ANTIBODY TEST

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• Antibody test• Direct agglutination

(Widal test, STA for Brucellosis)


• Antigen controlNegative control serumPositive control serum


• No clumpingNo clumpingClumping

ANTIBODY TEST

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• Antibody test

• Passive haemagglutination (ASO)


• Streptolysin controlRed cell control


• HemolysisNo hemolysis

ANTIBODY TEST

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• Antigen test

• Coagglutination test(Haemolytic streptococci meningitis antigens)

• Control material

• Group A,B,C streptococci

N.meningitidis

• Expected result

• Agglutination with corresponding serum,

ANTIGEN TEST

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• 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

QUALITY CONTROL PROCEDURES FOR TESTS DETECTING ANTIGENS

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• 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.

•

REPORTING AND RECORD KEEPING

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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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• Precise and inaccurate • Precise and accurate

PRECISION AND ACCURACY

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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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• 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.

MEASUREMENT OF VARIABILITY

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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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• 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

AVOIDING THE ERRORS

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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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• 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

MONITORING QC DATA

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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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Quality control in serology

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