quality control: in-house controls for elisa tests

1. Quality Control: In-house Controls for ELISA tests

3. Who should make and use In-House HIV Controls? Any lab which is conducting HIV EIA testing

4. Criteria for Developing Quality Controls for HIV Low Positive Between the cut off and positive control At a level where variability can be followed Generally ~2 times the cut off QC Sample Criteria The selection of the QC sample is critical to the QC programme. A QC should be selected at a level where the sample can identify variability. When plotting serial dilutions of a high positive sample this range is the linear portion of the sigmoidal curve, between the upper and lower plateau of the graph (Refer section 1.3.2 Selecting a suitable sample dilution). Generally, an appropriate level for a QC sample is a low positive which reacts between the cut off and the low positive control. For many assays a low positive which reacts at between 2 to 3 times the cut off is appropriate. A sample at this level will identify any minor changes in assay performance which may not be easily recognised by reviewing the negative and positive kit controls. For example, if a borderline QC sample proceeds out of range in the negative direction ie. below the lower control limit, this indicates an opportunity for the specimens with OD�s below the cut off to be falsely interpreted as negative. A decrease such as this may not be evident if monitoring the negative or high positive kit controls. The sample must be of good quality, of large volume and stable for long storage.QC Sample Criteria The selection of the QC sample is critical to the QC programme. A QC should be selected at a level where the sample can identify variability. When plotting serial dilutions of a high positive sample this range is the linear portion of the sigmoidal curve, between the upper and lower plateau of the graph (Refer section 1.3.2 Selecting a suitable sample dilution). Generally, an appropriate level for a QC sample is a low positive which reacts between the cut off and the low positive control. For many assays a low positive which reacts at between 2 to 3 times the cut off is appropriate. A sample at this level will identify any minor changes in assay performance which may not be easily recognised by reviewing the negative and positive kit controls. For example, if a borderline QC sample proceeds out of range in the negative direction ie. below the lower control limit, this indicates an opportunity for the specimens with OD�s below the cut off to be falsely interpreted as negative. A decrease such as this may not be evident if monitoring the negative or high positive kit controls. The sample must be of good quality, of large volume and stable for long storage.

5. Process for Preparing In-house Controls Serial Dilution of High Positive Stock Sample Select Suitable Dilution (pooled HIV- plasma) Produce Large Batch Test Stability Test Batch Variation Dispense, Label, Store Method of QC Production A protocol should be developed which details how to carry out each step. An original stock sample of high antibody titre is tested at different dilutions to identify a suitable antibody concentration to use as a QC sample Large volumes of the selected dilution are prepared and tested on different batch numbers to validate the samples stability and reproducibility. After validation the sample is aliquoted into volumes suitable for a weeks use and stored according to protocol which will be written to assure sample stability during storage. It may be appropriate to make a multi-marker QC sample which includes more than one analyte in the sample. For example, the NRL has produced a multi-marker QC sample which includes anti-HIV1/2, anti HTLV, anti-HCV and HBsAg markers. After production of a multimarker all analytes must be validated individually. Method of QC Production A protocol should be developed which details how to carry out each step. An original stock sample of high antibody titre is tested at different dilutions to identify a suitable antibody concentration to use as a QC sample Large volumes of the selected dilution are prepared and tested on different batch numbers to validate the samples stability and reproducibility. After validation the sample is aliquoted into volumes suitable for a weeks use and stored according to protocol which will be written to assure sample stability during storage. It may be appropriate to make a multi-marker QC sample which includes more than one analyte in the sample. For example, the NRL has produced a multi-marker QC sample which includes anti-HIV1/2, anti HTLV, anti-HCV and HBsAg markers. After production of a multimarker all analytes must be validated individually.

6. Making Suitable Dilutions 1.3 Method of QC Production 1.3.1 Making Suitable Dilutions A high antibody titre positive sample is diluted in normal human serum (NHS) or Basematrix (BioMedica BBI). The positive sample should be heat treated at 62�C for 20 minutes and filtered though a 0.2um Biological filter. A plasma diluent may need to be defibrinated, delipified and filtered (0.2um filter). An anti-bacterial agent such as Bronidox can be added to the dilutions. Check the package insert of the assay which the QC sample is to be produced for, to ensure that the preservative is appropriate and will not interfere with assay performance. A titration is conducted in tenfold or in doubling dilutions as follows. 100ul of NHS is added to each tube. 100ul of positive sample is added to the first tube. mix by moving the pipette plunger up and down at least 3 times. transfer 100ul from tube 1 to tube 2 and mix well. repeat this step for the remaining tubes. dilution in tube 10 is 1/1024 or in the case of tenfold dilution, 1010. Test each titration in the particular assay(s) for which the QC is (are) being synthesised. 1.3 Method of QC Production 1.3.1 Making Suitable Dilutions A high antibody titre positive sample is diluted in normal human serum (NHS) or Basematrix (BioMedica BBI). The positive sample should be heat treated at 62�C for 20 minutes and filtered though a 0.2um Biological filter. A plasma diluent may need to be defibrinated, delipified and filtered (0.2um filter). An anti-bacterial agent such as Bronidox can be added to the dilutions. Check the package insert of the assay which the QC sample is to be produced for, to ensure that the preservative is appropriate and will not interfere with assay performance. A titration is conducted in tenfold or in doubling dilutions as follows. 100ul of NHS is added to each tube. 100ul of positive sample is added to the first tube. mix by moving the pipette plunger up and down at least 3 times. transfer 100ul from tube 1 to tube 2 and mix well. repeat this step for the remaining tubes. dilution in tube 10 is 1/1024 or in the case of tenfold dilution, 1010. Test each titration in the particular assay(s) for which the QC is (are) being synthesised.

7. This titration will result in the following dilutions

8. Selecting a Suitable Sample Dilution 1.3 Method of QC Production 1.3.2 Selecting a Suitable Sample Dilution Plot the assay results against the sample dilutions to review the sigmoidal response curve. An appropriate QC dilution should be selected from the section of the graph were variability can be followed ie. the linear portion between 1/512 and 1/1638. Sample dilutions at the upper and lower plateau of the graph will not identify variation. The dilution should be between the positive kit control value and the calculated cut off value. If a QC sample is chosen with an OD which is too low, then the OD may fluctuate above and below the cut off due to normal variations. Although variation can still be monitored with this sample it is preferable to choose a QC which is consistently reactive. A sample chosen with an OD which is too high will be of limited use for border line monitoring and may not identify subtle changes. In the above example the 1/8192 dilution which produced a S/Co of 2.3 was chosen as the appropriate dilution. If the results achieved in the serial dilution are not suitable further dilutions between these titrations may be required. 1.3 Method of QC Production 1.3.2 Selecting a Suitable Sample Dilution Plot the assay results against the sample dilutions to review the sigmoidal response curve. An appropriate QC dilution should be selected from the section of the graph were variability can be followed ie. the linear portion between 1/512 and 1/1638. Sample dilutions at the upper and lower plateau of the graph will not identify variation. The dilution should be between the positive kit control value and the calculated cut off value. If a QC sample is chosen with an OD which is too low, then the OD may fluctuate above and below the cut off due to normal variations. Although variation can still be monitored with this sample it is preferable to choose a QC which is consistently reactive. A sample chosen with an OD which is too high will be of limited use for border line monitoring and may not identify subtle changes. In the above example the 1/8192 dilution which produced a S/Co of 2.3 was chosen as the appropriate dilution. If the results achieved in the serial dilution are not suitable further dilutions between these titrations may be required.

9. Calculation for control

10. Batch Production Prepare positive sample centrifuge heat inactivate (56�C for 30 min.) Mix positive sample in diluent magnetic stirrer Identify each batch with a unique ID number 1.3 Method of QC Preparation 1.3.3 Batch Production Centrifuge and filter (0.2um) positive sample. Positive sample may be heat inactivated at 62oC for 20 minutes. (Note: this may disturb the reactivity in some assays). Mix sample and the calculated volume of diluent for at least 1 hour on a magnetic stirrer in a biohazard cabinet, 3-4 hours may be required for volumes > 2 litres. Aliquot the batch into sterile large polypropylene storage containers such as Nalgene 250ml square bottles. Do not use polystyrene containers as these adsorb antibodies and will alter the antibody level of the QC sample. Number each bottle for future identification. Store at 4�C until ready to dispense. 1.3 Method of QC Preparation 1.3.3 Batch Production Centrifuge and filter (0.2um) positive sample. Positive sample may be heat inactivated at 62oC for 20 minutes. (Note: this may disturb the reactivity in some assays). Mix sample and the calculated volume of diluent for at least 1 hour on a magnetic stirrer in a biohazard cabinet, 3-4 hours may be required for volumes > 2 litres. Aliquot the batch into sterile large polypropylene storage containers such as Nalgene 250ml square bottles. Do not use polystyrene containers as these adsorb antibodies and will alter the antibody level of the QC sample. Number each bottle for future identification. Store at 4�C until ready to dispense.

11. Stability Testing Assess the rate of deterioration 1.3 Method of QC Production 1.3.4 Testing the Stability of the QC If the dilution selected is greater than 1/3000, it will be necessary to check the stability of the prepared sample to assess the rate of possible deterioration. Place aliquots of the diluted QC sample at -20?C, 4?C and room temperature (in areas where room temperature is 15-25?C) for one month. The samples in multiple aliquots are then tested throughout the month at 7, 14, 21 and 28 days. The results obtained at each temperature are reviewed and pass stability testing if the value is not statistically different from the original calculated results. The stability requirements are dependent on the future use of the sample. For example, if a sample is to be shipped to another region and may be at ambient temperature for a few weeks in transit, it is critical that the QC sample pass stability testing at this level. 1.3 Method of QC Production 1.3.4 Testing the Stability of the QC If the dilution selected is greater than 1/3000, it will be necessary to check the stability of the prepared sample to assess the rate of possible deterioration. Place aliquots of the diluted QC sample at -20?C, 4?C and room temperature (in areas where room temperature is 15-25?C) for one month. The samples in multiple aliquots are then tested throughout the month at 7, 14, 21 and 28 days. The results obtained at each temperature are reviewed and pass stability testing if the value is not statistically different from the original calculated results. The stability requirements are dependent on the future use of the sample. For example, if a sample is to be shipped to another region and may be at ambient temperature for a few weeks in transit, it is critical that the QC sample pass stability testing at this level.

12. Batch Validation Dispense aliquots Test aliquots Confirm desired titre level compare against target value Confirm minimal batch variation acceptable if CV <20% aim for <10% 1.3 Method of QC Production 1.3.5 Batch Validation The batch must be validated to ensure it has been sufficiently mixed and that the product is homogenous. Validation of the QC sample is extensive and should include the testing of an appropriate number of aliquots to represent the total volume. For example, if 1 litre of sample is produced and stored in 5 200 ml bottles, 20 aliquots from each of the bottles should be tested. The aliquots should be numbered so the results can be traced back to the original storage bottles. Test these aliquots in at least one assay system which the QC sample was designed to quality control. Compare the results with the target value to ensure correct result. Investigate the variability of the of the QC sample batch by calculating the coefficient of variation (CV) for selected samples. The batch is acceptable if the CV of the results is less than 20% (a discussion on statistical analyses and how to calculate a CV is described further in this chapter) . This validation process is a most important step in the methodology especially for large volume batches of QC with extensive dilution (e.g. 5 litres of 1/3000 dilution).1.3 Method of QC Production 1.3.5 Batch Validation The batch must be validated to ensure it has been sufficiently mixed and that the product is homogenous. Validation of the QC sample is extensive and should include the testing of an appropriate number of aliquots to represent the total volume. For example, if 1 litre of sample is produced and stored in 5 200 ml bottles, 20 aliquots from each of the bottles should be tested. The aliquots should be numbered so the results can be traced back to the original storage bottles. Test these aliquots in at least one assay system which the QC sample was designed to quality control. Compare the results with the target value to ensure correct result. Investigate the variability of the of the QC sample batch by calculating the coefficient of variation (CV) for selected samples. The batch is acceptable if the CV of the results is less than 20% (a discussion on statistical analyses and how to calculate a CV is described further in this chapter) . This validation process is a most important step in the methodology especially for large volume batches of QC with extensive dilution (e.g. 5 litres of 1/3000 dilution).

13. Storage of QC Samples Validated batch aliquoted into smaller �user friendly� volumes for storage Establish a storage protocol: store at -20oC �in use� vials stored at 4oC use 1ml vial maximum of one week freeze-dried requires accurate reconstitution) chemically preserved 1.3 Method of QC Production 1.3.6 Storing QC Samples The QC sample is dispensed into labelled aliquot's of a smaller �user friendly� volume. Aliquot's in use can be stored at 4?C. These should be discarded after 7 days and a new 1ml vial introduced. By aliqoting the sample into 1ml volumes all but the QC sample in use can be stored at -20?C. This prevents freeze thawing which can affect the QC sample result. 1ml aliquot's can be sub-aliquoted if the 1ml volume will not be exhausted in a week. The label on the QC Sample should include the name of the QC Sample, and may include the expiry date (usually 12 months from the date of production) and the production laboratory�s initials. 1.3 Method of QC Production 1.3.6 Storing QC Samples The QC sample is dispensed into labelled aliquot's of a smaller �user friendly� volume. Aliquot's in use can be stored at 4?C. These should be discarded after 7 days and a new 1ml vial introduced. By aliqoting the sample into 1ml volumes all but the QC sample in use can be stored at -20?C. This prevents freeze thawing which can affect the QC sample result. 1ml aliquot's can be sub-aliquoted if the 1ml volume will not be exhausted in a week. The label on the QC Sample should include the name of the QC Sample, and may include the expiry date (usually 12 months from the date of production) and the production laboratory�s initials.

15. When should you use your in-house control? Any time you run an EIA. On each plate in your EIA run.

16. Use the results of your in-house control as a part of testing validation.