Chapter 1 · QC for qualitative and semi-quantitative immunoassays

Immunology involves both quantitative (e.g. IgE, complement levels) and qualitative or semi-quantitative methods (e.g. ANA, ANCA, anti-dsDNA titres). For quantitative assays, QC runs as in other sections. For qualitative assays, a positive control and a negative control must be run with each batch. For semi-quantitative methods, a weak positive control near the cut-off is essential to detect drift toward false-negative results.

Chapter 1 · Practical example

QC for qualitative immunoassays — what a negative control tells you

Qualitative and semi-quantitative immunoassays require controls that validate the assay at multiple points — not just a positive and a negative at the extremes.

▲ The situation

A laboratory runs ANA screening by IIF. The daily QC consists of one known strongly positive serum (to confirm the assay is working) and one blank (to confirm no non-specific staining). The quality manager is reviewing whether this is sufficient.

Reference: ISO 15189:2022 Clause 7.3.7

✓
Good practice

The quality manager reviews the clinical QC requirements: the assay is used to distinguish strongly positive, weakly positive, and negative results. A weakly positive control at 1:40 or 1:80 is needed to confirm the assay can detect weak positivity — the blank and strong positive alone do not validate the performance at the clinical cut-off. A weak positive control is added. On the third week of use, the weak positive control fails while the strong positive passes — revealing a calibration shift that would have caused weakly positive patient samples to read as negative.

✗
Poor practice

The strong positive and blank QC continue as the sole controls. An ANA IIF assay performed using a batch with a reagent preparation error produces negative results for all samples — including four known positive patient samples being retested. The strong positive control also reads negative, triggering investigation. However, without the weak positive control, the team does not know how long the assay has been producing false negatives at the clinically relevant titre range.

● What this means for your practice

Qualitative assay QC must validate performance across the clinical range, not just confirm the assay is producing any result. A weak positive control at the clinical cut-off is essential for ANA IIF, HIV testing, and any other qualitative assay where the distinction between weakly positive and negative is clinically significant.