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During microscopic quality control following an ICC run using several primary antibodies one positive control for a primary antibody gives weaker than expected results. What practical steps would you take to address this observation?
When weaker than expected staining of one positive control is observed, but other positive controls provide expected staining in an ICC run then it is unlikely that there is a general run issue.
Focusing therefore on the isolated weak staining the first consideration should be if any of the test slides stained with the primary antibody can be passed for reporting. If some show expected staining, for example of components where the antigen is normally present, and the preparations cannot be easily repeated, due to lack of spares slides or urgency of diagnosis, then these could go forward for reporting, but with a caveat that the positive control did not provide optimal staining. However, if on inspection of the test slides, no assurance that expected staining has been obtained then these slides should be pulled from reporting and an investigation started as to possible reasons for this failure before any further test slides are processed with the antibody. The following lines of investigation are suggested:
1. Check that the correct protocol was used for staining. If ‘yes’ then this can be eliminated as the cause, if ‘no’ then repeat ICC using the correct protocol on the positive control slide before using it on further test slides. If expected staining is achieved then the antibody and control are clear for use again.
2. After eliminating protocol deviation as the cause of the weaker than expected result then attention should be turned to the primary antibody. It should be checked as ‘in date’ for use and, if not in a ready to use format, that it was diluted and stored appropriately before use. If it has been used beyond its expiry date then it should either be replaced or re-validated as acceptable to use only if appropriate positive staining is achieved. If inappropriately diluted or stored then then these errors should be corrected and the antibody retested with the positive control.
3. If none of the above account for the weak staining of the positive control then the batch/lot number of the primary antibody should be checked to make sure that this was consistent with previous use when satisfactory staining was obtained. If a discrepancy is found this amounts to the use of an un-validated antibody that needs to be checked before further use on test slides. This may be done using the positive control supplemented, ideally, with a range of controls with differing quantities of the target antigen to assess sensitivity.
4. If there was no discrepancy in antibody batch/lot number then the sample from which the control slides have been cut should be checked. If this is near exhaustion then fixation conditions may be different than in the bulk of the sample and this may have been responsible for the weak staining. In this instance the safest thing to do is to test a replacement positive control with the antibody.
It should be noted that the above represent one tiered approach to investigation of the problem and they do not cover all eventualities. The issue must be addressed, but watch out for the unexpected!