BIOCHEMISTRY
it introduced a potential variable that required assessment. A two-fold dilution with 25% PEG results in a final PEG concentration of 12.5% within the extraction mixture, whereas the three- fold dilution increases the final PEG concentration to 16.7%. The increased PEG concentration raised concerns that enhanced protein precipitation could remove a proportion of monomeric prolactin, resulting in an underestimation of %Recovery. To investigate this potential effect, patient sample comparison studies were performed. The results demonstrated that the 4.2% difference in final PEG concentration had minimal impact on calculated %Recovery, supporting the use of the three-fold dilution approach without significant alteration of assay interpretation (Fig 1). The optimised method demonstrated
close agreement with the original method for both monomeric prolactin concentration (R² = 0.9872) and calculated %Recovery (R² = 0.8544), indicating that the methodological changes had minimal impact on overall assay interpretation. The results of the patient comparison
study were encouraging; however, it was decided that further assessment of the optimised method would be beneficial through comparison with a method established in another laboratory. The biochemistry laboratory at The Christie NHS Foundation Trust kindly provided 10 samples previously identified as positive for macroprolactin for comparative analysis.
A particularly encouraging finding
was that the patient comparison results obtained using the new optimised method demonstrated closer agreement with the
%Recovery comparison: old method vs new method
100 90 80 70 60 50 40 30 20
10 00
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Old method %Recovery
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1000 500
0 0 500
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Monomeric prolactin = 0.9872
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Linear (old method %Recovery)
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00 20 Linear
(new method %Recovery)
%Recovery R2 = 0.8544 40 50 60
40 Fig 1. Patient comparison between the old and optimised macroprolactin methods.
results from The Christie NHS Foundation Trust than those obtained using the legacy method. This provided further confidence in the performance and reliability of the optimised assay (Fig 2).
Implementation challenges Finally, the macroprolactin assay had been optimised, the IQC challenge had been addressed, and the modified method had successfully undergone verification. The next step, and perhaps the most unexpectedly challenging one, was implementation; specifically staff training. Having experienced the difficulties
associated with the previous version of the %Recovery results comparison: Christie, legacy method vs optimised method
90 80 70 60 50 40 30 20 10 0 0
assay, staff were understandably cautious about learning another method. The new procedure initially appeared daunting, with multiple IQC levels, an additional dilution step, and several new processes to incorporate. As the saying goes, ‘beter the devil you know’. However, curiosity eventually prevailed.
Many members of staff had followed the assay development process over several months and were genuinely interested in seeing the final outcome. Gradually, training commenced, confidence increased, and the new procedure became part of routine practice. Sometimes the problem is not the
assay itself but the way it has evolved – or failed to evolve – within the laboratory. By questioning each stage of the process, and adapting the procedure to modern analytical systems, an assay that nobody particularly wanted to perform became one that could be controlled, reproduced and, most importantly, trusted.
2
Linear (Christie %Recovery) Christie %Recovery
4 6 Sample number Legacy method %Recovery Linear (legacy method %Recovery) Optimised method %Recovery Linear (optimised method %Recovery)
Fig 2. Method comparison across 10 macroprolactin-positive samples. The optimised method (green) shows closer agreement with the established method at The Christie NHS Foundation Trust (blue) than the legacy method (orange).
8 10 12
Elena Cohen MSc MIBMS is an advanced biomedical scientist working in biochemistry at Royal Oldham Hospital, Manchester, part of the Northern Care Alliance NHS Foundation Trust. With over 10 years’ experience in clinical biochemistry, Elena’s professional background also spans biochemistry, immunology, and haematology. Her specialist area is laboratory investigation of multiple myeloma, in particular advancing laboratory practice in the investigation of plasma cell disorders, optimising diagnostic pathways, and contributing to the continual improvement of patient care through evidence-based laboratory medicine.
September 2026
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