Reproducibility and Harmonization in Research Using Biological Standards: The Example of Platelet Agonist Collagen-Related Peptide

Researchers at the MHRA South Mimms Laboratories have been developing manufacturing biological standards for over 60 years, and these reference reagents are used to ensure the quality, consistency, and safety of biological medicines and diagnostics. NIBSC standards from the MHRA support translation of cutting-edge therapeutic products, as well as the control of established medicines. At the MHRA South Mimms Laboratories, we use state-of-the-art technologies to produce materials that support the development and regulation of novel therapeutic innovations.

Biological standards remain underutilized by academics in the biological sciences due to a lack of knowledge exchange. This negatively impacts the quality of research and contributes to the reproducibility crisis. Biological standardization ensures experiments are reproducible, regardless of where or when they are performed.

A lack of proper knowledge in basic metrological principles and training compromises scientific rigor and negatively impacts reproducibility. Although our protocol focuses on standardizing platelet function measurement, the practice can be applied to any biological system. Applying biological standardization to research practices will not only standardize biological experiments, but also help to address inconsistencies and the lack of harmonization that undermines research.

To begin the assay, using the platelet receptor agonist, collagen-related peptide cross-linked, or CR-PXL, dilute an appropriate amount of the CR-PXL stock in the assay buffer to a concentration suitable to induce maximum platelet aggregation. The result in solution will be henceforth referred to as the standard. Next, starting from the same highest prepared concentrations, sequentially perform twofold serial dilutions for both the standard and the test reagent.

Prepare a six to eight-point dilution series for both the test and the standard in assay buffer that generates aggregation responses from 100%down to 0%When the platelet-rich plasma or PRP is ready to use, add an aliquot of PRP into the light transmission aggregometry cuvette. Allow the samples to warm to 37 degrees Celsius in the holding wells for a couple of minutes. Once appropriately warmed, place the cuvettes in the aggregometer to start recording the traces.

With constant stirring, add the agonist, and record the data. Perform data collection for each prepared dilution of the standard and the test reagent. Use GraphPad Prism or any suitable software package to check if the concentration curves for the standard and test are parallel.

Enter the data such that the X axis contains the concentrations, and the Y-axis contains the responses indicated, by either the percentage of maximum aggregation or the area under the curve. Next, transform the concentration values to the corresponding logarithms or log concentration. Select Nonlinear regression from the Analysis functions and use the equation for dose response stimulation.

Select log agonist vs. response Variable slope, and use a three or four-way fit, depending on which is best for the data. Determine if the curves generated are parallel by clicking the Compare tab and comparing the data sets.

Ensure that the slope and asymptotes of the test and the standard curves are equivalent. If these conditions are met, proceed to calculate the potency or EC50 using nonlinear regression curve fitting. Calculate the relative potency by dividing the EC50 value of the standard by that of the test.

Adjust the mass per volume concentration of the test reagent to account for the difference in potency with respect to the standard. The platelet aggregation traces for the standard and the test reagent revealed the difference between the two aggregation responses. At 0.075 micrograms per milliliter, there was a clear response to the standard, while for the test, the corresponding concentration showed no response.

The EC50 values of the standard and test reagent were calculated by plotting the concentration response curve.