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Discuss the quality control of serological assays

 

Serological testing remains the bulk of the work carried out by a routine virus diagnostic laboratory. It is essential to have good quality control protocols in all sections of serology in order to ensure the validity of the test results. Strictly speaking, quality control refers to the measures that must be included during each assay to verify that the test is working properly. However, the term is often more loosely used to cover aspects of quality assurance and also quality assessment. Quality assurance is defined as the overall program that ensures that the final results reported by the laboratory are correct, and quality assessment (also known as proficiency testing) is a means to determine the quality of the results generated by the laboratory. It is usually an external evaluation of the laboratory's performance.

Quality Control: Monitoring the Testing Process

As mentioned previously, quality control refers to those measures that must be included during each assay in order to verify that the test is working properly. The following items are essential elements of quality control that must be performed during every assay:

1. Each run must include one full set of controls

2. The controls for each test run must yield results within the limits of the manufacturer's criteria for acceptability and validity of the run.

3. All test kits must be used before the expiration date to ensure valid results

4. Physical parameters of the test such as incubation time and temperature must be followed to ensure proper performance.

Ordinarily, each test kit has a set of positive and negative control that are to be included in each test run. These controls are considered to be internal controls, while any other controls included in the run are referred to as external controls. Internal controls are essential for QC measures for each run and are intended for use only with the lot number of the corresponding test kit. External controls can be included on a run to monitor consistent performance, lot to lot variation between kits, and to serve as an indicator of assay performance on samples that are borderline reactors.
 
 

Quality Assurance

QA is an ongoing process that requires daily attention by all laboratory staff. Many variables can affect the quality of results so that the monitoring of which is essential in a quality assurance program.

1. Documentation - this is probably the most important part of quality assurance. There must be an up to date standard operating procedure of the test. The original source of the procedure, and all subsequent changes  must be documented and traceable. All worksheets and testing records must be kept up to date and traceable, as are all computer records.

1. The condition of the specimens - the specimen should be clearly labeled with patient details using at least two different identifiers. the specimen to be tested should not be haemolysed or lipaemic. Utmost care must be taken during the splitting of the specimen so that the correct specimen is placed into the correctly labeled tube.

2. The educational background and training of the laboratory personnel. A continuing education program for laboratory workers should be included and individual laboratory personnel should be evaluated to identify areas for improvement

3. The controls used in the test runs - appropriate internal and possibly external controls should be used in each test run.

4. Up to date performance records of each test run and quality control procedures should be kept.

5. The interpretation of results - results should be interpreted according to the manufacturer's criteria. Where the interpretation of the test is potentially subjective, as in the case of haemagglutination-inhibition, complement-fixation, or single-radial haemolysis, it may be advisable for the results to be read and counterchecked by a second person.

6. The transcription of results - Transcriptional or clerical errors include mistakes made during the transfer of information from the test readout to the worksheet, and from the worksheet to the computer or report form. These type of errors probably account for the majority of errors in the laboratory. Possible mechanisms include having a second technologist to check the final result and the supervisor to check the results before releasing.

7. The reporting of results - the results should be reported in a timely manner to the appropriate individual. The report should be authorized by a suitably qualified individual.

8. Internal audit - regular audits should be carried out to look at the quality assurance program.

Quality Assessment

Quality assessment is a means to determine the quality of results. It is usually an external evaluation of a laboratory's performance that relies on incorporating proficiency panels of well-characterized sera into the testing routine. External quality assessment (EQA) is now recognized as an essential component of quality assurance and is the only means to give the laboratory manager an independent means of ensuring that his routine quality control is adequate and effective. The National External Quality Assessment Scheme (NEQAS) is administered by the PHLS. It is important for the participating laboratory to treat NEQAS specimens in the same manner as normal routine specimens. All NEQAS assessment results should be widely circulated and discussed in the laboratory.

Increasingly, internal quality assessment schemes are being put in place in individual laboratories. These usually involve the use of internal control specimens. These o.d. values of the internal quality control specimens are plotted in a Shewhart-type chart (usually, it is the o.d of IQC/o.d. negative control which is used), and evaluated against Westgard rules. Westgard rules are based on statistical considerations and thus probability. An assay run where the IPC is >2 or 3 s.d., or there is a continual trend on one side of the chart, would raise suspicions of either or both random and systematic errors. It may be prudent to reject the run altogether.

Accreditation

Accreditation is an external audit of an applicant department’s organization and quality assurance program is an external audit of an applicant department’s organization and quality assurance program. There is now a trend towards accepting ISO 15189 as the standard for all accreditation bodies in order to provide uniformity of standards and cross-recognition.  Examples of accreditation authorities include the CPA (College of American Pathologists) in the U.S., CPA (Clinical Pathology Accreditation) in the UK, and NATA in Australia. In addition the the whole department, individual assays may be accredited at the same time on an individual basis as well.  Accurate and up to date documentation, with complete traceability forms a very important part of the accreditation process.

 

Quality control considerations for different serological tests.

The following considerations apply to the following serological assays;-

Commercial EIAs and RIAs - on the whole, currently available commercial assays are now of a high standard. It is important to ensure that the manufacturer's controls are included in each run and that the tests are interpreted according to the manufacturer's instructions. If wells or beads from more than one kit is used for the assay run, it is important to ensure that the kits come from the same batch, otherwise, a different set of controls should be put up for each different batch.

In house RIAs and EIAs - on the whole, in house tests are considerably more difficult to perform and more likely to go wrong than commercial assays. The conditions specified for the test should be followed exactly each time to ensure that the test is reproducible. If possible, freshly made up reagents should be used each time to minimize the chance of deterioration of the reagent eg. fetal calf serum, affecting the result of the test.

Immunofluorescence (IFA) - the interpretation of IFA result is subjective. Difficulty may arise in the interpretation of borderline positive results which may require an experienced reader to interpret.

Complement-fixation tests - problems often arise with CFTs because of problems with one of the reagents such as the antigen, complement, the read blood cells or the VBS diluent. Vigilance must be exercised at all times. It is important to put up controls for each antigen and carry out a complement back titration. Each new batch of antigen must be titrated using chessboard titration method.

Haemagglutination-inhibition tests - again problems are commonly seen with HAI. Any of the reagents used may be implicated. It is important to carry out an antigen titration prior to each test.

Single radial haemolysis - as in the case of CFTs and HAIs, problems are relatively common. It is essential to use freshly made up reagents.

Agglutination tests - Latex agglutination and particle agglutination tests are very simple to perform. However, there may be problems in the interpretation of results which is subjective. Care must be taken to avoid the contamination of one specimen with another specimen in an adjacent well when setting up the test.

Others - the same principles apply to other serological tests such as western blots, radioimmunoprecipitation assays, virus neutralization assays and counter-immune electrophoresis.

To conclude, quality control in serological assays is an on-going process and requires vigilance by the laboratory personnel at all times. Quality control of serological assay is a continuously evolving subject, but there is a general consensus that a laboratory should aim to be accredited. Where possible, the laboratory should aim for accreditation under ISO 15189 with all individual tests accredited.

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