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sample analysed in duplicate. By the time samples requiring macroprolactin testing reach the bench, there is often very litle serum remaining, making analysis challenging. Given these limitations, it was


decided that the existing method should be optimised and the modified procedure subsequently verified before implementation.


The IQC problem… One of the most significant shortcomings of the original macroprolactin assay is the complete absence of internal quality control (IQC). In an era where virtually every laboratory assay is accompanied by multiple levels of quality control, relying on an assay with none felt rather unsetling. It was therefore considered essential that an IQC strategy be developed alongside the optimisation of the method. Many laboratories use both positive


and negative IQC materials for macroprolactin analysis. Positive controls are commonly prepared by reducing pooled patient serum with anti-human prolactin antibody to generate artificial macroprolactin complexes. While this approach is scientifically sound, it also comes with a high price tag that, unfortunately, our laboratory budget was not prepared to embrace. As no additional funding was available (and no hidden pot of money miraculously appeared), an alternative strategy had to be developed using materials already available within the laboratory. Rather than viewing this as a


limitation, it became an opportunity to design a practical and sustainable IQC system that could be reproduced without the need for expensive specialised reagents. Four IQC levels were established


to monitor different aspects of assay performance: IQC Level 1. Testing for interference This IQC level contains the analyser diluent which is routinely used to dilute prolactin samples on board and a pooled patient serum of prolactin concentration >1000 mU/L. The diluent is mixed with a serum pool at a three- fold dilution and tested for prolactin. Unlike PEG, the analyser diluent has no protein-precipitating properties. Consequently, the prolactin concentration obtained following dilution should reflect the expected dilution factor, demonstrating that the diluent itself does not facilitate protein precipitation or extraction. This control confirms that any reduction in prolactin concentration observed following PEG treatment is atributable


The optimisation process aimed


to identify the lowest possible patient serum volume that could be used during PEG treatment while still generating sufficient volume for reliable analysis


to PEG-mediated protein precipitation rather than an effect of the analyser diluent. IQC Level 2. Negative control A pooled patient serum with a prolactin concentration >1000 mU/L was used as the negative control. Following PEG precipitation, this material was expected to demonstrate a prolactin recovery greater than 60%, indicating the absence of significant macroprolactin and confirming acceptable assay performance. To ensure that the selected pool was truly negative, patient samples with prolactin concentrations >1000 mU/L were anonymised and individually assessed following PEG precipitation. Following confirmation of negative status, samples with %Recovery values of >70% were pooled to create the negative IQC material. IQC Level 3. PEG functionality control A pooled serum sample with a high IgG concentration was subjected to PEG precipitation, after which the IgG concentration was measured. Since PEG effectively precipitates immunoglobulins, the post-extraction IgG concentration should be below the assay’s limit of detection. This control provides assurance that the PEG is doing exactly what it is supposed to do and has not quietly lost its enthusiasm for protein precipitation. IQC Level 4. Equivocal control A level 3 commercial immunoassay quality control material routinely used on the immunoassay platform was selected to provide an equivocal control. Following PEG extraction, this material consistently produced a prolactin recovery between 40% and 60%, representing the equivocal reporting range. This control provides ongoing assurance that the assay can reliably identify samples that fall into the equivocal category.


Although unconventional, this four- level IQC strategy proved to be robust, inexpensive and readily reproducible. More importantly, it transformed an assay


36 WWW.PATHOLOGYINPRACTICE.COM September 2026


with no quality control into one with a comprehensive monitoring system, all without purchasing costly materials.


Assay optimisation The next stage of the project focused on assay optimisation. Several key questions needed to be addressed: What is the minimum acceptable sample volume required to perform the assay reliably? How could the viscosity of PEG-treated samples be reduced to improve analyser compatibility?


The automated immunoassay system used within our laboratory requires a minimum sample volume of 100 µL for prolactin measurement. Therefore, the optimisation process aimed to identify the lowest possible patient serum volume that could be used during PEG treatment while still generating sufficient volume for reliable analysis. Several approaches were evaluated, and the most suitable was identified as a three-fold dilution using 50 µL of patient serum. Although this approach provided sufficient sample volume for analysis, it did not resolve the viscosity issue. Following the centrifugation step, the sample remained incompatible with the automated immunoassay system and continued to generate analyser errors. To overcome this limitation, an additional dilution step was introduced before the sample was presented to the analyser. The centrifuged sample was subjected to a further two-fold dilution, resulting in a final dilution factor of six. This additional dilution successfully reduced sample viscosity while maintaining a sufficient prolactin concentration for reliable measurement. The prolactin recovery rate formula


was adjusted to accommodate final six- fold dilution:


The three-fold dilution with 25% PEG successfully addressed the issue of excessive assay sample volume; however,


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