Investigation of the effect of pre‐analytical factors on particle concentration and size in cryoprecipitate using nanoparticle tracking analysis

Cryoprecipitate is used primarily to replenish fibrinogen levels in patients. Little is known about the presence of micro‐ or nano‐sized particles in cryoprecipitate. Therefore, we aimed to quantify these particles and investigate some pre‐analytical considerations.


| INTRODUCTION
Cryoprecipitate is a clinically important blood component used primarily to replenish fibrinogen levels in patients, in cases such as during traumatic haemorrhage. 1 It is manufactured by slowly thawing fresh frozen plasma (FFP) overnight at 1-6 C. 2 The resulting precipitate is recovered by centrifugation and solubilised in a minimal volume of the remaining plasma.Cryoprecipitate is enriched in high molecular weight plasma proteins, such as factor VIII, von Willebrand factor (vWF), fibrinogen, fibronectin, and factor XIII. 3 Human plasma contains a diverse range of micro-and nanoparticles, such as lipoproteins, protein complexes, viruses, and extracellular vesicles (EVs).Despite the critical role of cryoprecipitate in transfusion medicine, there is a lack of up-to-date literature regarding these particles in cryoprecipitate, with the last report being more than 30 years old. 4 Recently, improvements in analytical techniques such as nanoflow cytometry, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA) have improved our ability to study the physical and phenotype properties of these particles. 5A is a modern technique designed for visualising and analysing nanoparticles in liquid suspension. 6Currently, there is no literature about the use of NTA to measure particles contained in cryoprecipitate.
We therefore investigated the size distribution and concentration of particles in individual cryoprecipitate units using NTA.In addition, we assessed the impact of preanalytical sample preparation methods (freeze/thaw cycles and filtration) on data acquired using NTA.

| Preanalytical preparation of cryoprecipitate
Each frozen cryoprecipitate unit was rapidly thawed at 37 C in a water bath and smaller aliquots were made with volumes appropriate for single-use assays.One aliquot from each unit was stored at room temperature, designated as having undergone 1st freeze/thaw, and was analysed with NTA.The remaining aliquots were immediately snap-frozen with dry ice and stored at À80 C.An aliquot was thawed after 48 h and analysed with NTA (2nd freeze/thaw).
Particle measurements were performed using NTA with each sample diluted 1:500 with 0.2 μm filtered PBS (Sartorius, Göttingen, Germany) and clarified using 0.45 μm polytetrafluoroethylene (PTFE) filters.Due to the potential risk of microfluidics blockage, the routine F I G U R E 1 No significant changes in the particle concentration, mean and mode of cryoprecipitate from two freeze-thaw cycles.Ten cryoprecipitate units were subjected to two consecutive freeze-thaw cycles.Samples collected after each freeze-thaw were diluted and filtered using a 0.45 μm PTFE membrane before nanoparticle tracking analysis.Data shown are the particle (A) concentration, (B) mean and (C) mode for each cryoprecipitate unit after 1st or 2nd freeze-thaw.Statistics were performed using a paired t-test.ns, not significant (p > 0.05).

| Statistical analysis
All statistical analyses were performed using Graphpad Prism (GraphPad Prism 9.1.0Software, San Diego, CA) with data presented as mean ± standard deviation.A paired t-test for was used for the comparison between the first and second freeze-thaw cycles.A oneway analysis of variance (ANOVA) was used with Tukey-Kramer's multiple comparison test for comparison involving multiple groups.
Significance was defined as p < 0.05.

| Freeze-thaw cycles did not affect particle concentration or size in cryoprecipitate
Freezing and thawing can damage nanoparticles resulting in an increase in particle size and a decrease in concentration. 7Ten individual cryoprecipitate units were used to investigate the effect of two freeze/thaw cycles on the concentration and size of nanoparticles (Figure 1).No significant difference in mean particle concentration was observed between the first and second freeze/thaw cycles (2.87 Â 10 11 ± 1.56 Â 10 11 particles/mL vs. 2.39 Â 10 11 ± 7.57 Â 10 10 particles/mL; p = 0.180).Similarly, there was no significant difference in mean particle size for these freeze/thaw cycles (112.0 ± 6.20 nm vs. 114.3± 5.48 nm; p = 0.211).The particle mode also showed no significant difference between these freeze/thaw cycles (89.57± 10.12 nm vs. 91.98 ± 7.52 nm; p = 0.250).The results revealed that the particles in cryoprecipitate were not physically altered from an additional freeze/thaw cycle.

| Filter material impacted particle size but not concentration in cryoprecipitate
Samples were filtered prior to NTA to reduce the risk of a blockage occurring within the microfluidics.However, it was unclear whether this would impact quantification.To assess the possible effects of PTFE or RC filtration on NTA analysis, 10 units of diluted filtered cryoprecipitate were compared with three units of unfiltered samples (Figure 2).No significant difference in particle concentration was observed between the filtered and unfiltered samples (p > 0.05).PTFE filtration, however, significantly reduced particle mean size compared to both RC filtered (112.7 ± 6.03 nm vs. 133.8± 7.50 nm; p < 0.0001) and unfiltered (112.7 ± 6.03 nm vs. 133.7 ± 13.63 nm; p = 0.0015) cryoprecipitate.No significant differences were detected in particle mode size between filtered and unfiltered cryoprecipitate, but differences were shown between samples filtered with PTFE and RC (89.79 ± 9.09 nm vs. 107.9± 11.76 nm, p = 0.0039).As RC filters demonstrated no difference compared to the unfiltered cryoprecipitate, these filters were used for subsequent analyses.

| Cryoprecipitate contained nanoparticles of various sizes and concentrations
The previous experiments had demonstrated that the second freeze/thaw cycle aliquots were suitable and that RC filters were the best choice for NTA.Therefore, we then used these optimised protocols and NTA to characterise the nanoparticles present in 10 units of cryoprecipitate (Figure 3).The average particle concentration was 2.50 Â 10 11 ± 1.10 Â 10 11 particles/mL and the mean particle size was 133.8 ± 7.5 nm (mode of 107.9 ± 11.1 nm).The size profiles were similar for all samples with most of the particles ranged in size from 50 to 300 nm.

| DISCUSSION
Little is known regarding the concentration and size distribution of micro-and nanoparticles present within cryoprecipitate.Particles may exhibit procoagulant activities, potentially mediated by tissue factor, phosphatidylserine, or other moieties contained on or with the particles, that may have important clinical implications when transfused into recipients. 8We therefore determined optimal preanalytical and storage considerations for NTA analysis of particles in cryoprecipitate.
We then used these protocols to profile, in 10 cryoprecipitate units, the concentration and the size of these particles.
In the clinical setting, frozen cryoprecipitate is thawed and transfused into recipients.Ideally, to model the clinical setting, we would perform all downstream analyses on freshly thawed cryoprecipitate.However, practically this is hard to perform multiple downstream analyses for all 10 units at once.Given that cryopreservation and freeze/thawing may have an impact on nanoparticles contained in plasma, 7 we therefore investigated whether it was possible to thaw cryoprecipitate units and then aliquot and refreeze samples for later analyses.We found that this additional freeze/thaw cycle did not affect either the mean or mode particle size or the particle concentration as measured using NTA.This suggested that the particles maintained their membrane integrity and may be suitable for downstream analyses such as next-generation sequencing and mass spectrometry.
We observed that the preanalytical filtration material could influence the NTA results, and more specifically that measured particle size was significantly altered when the PTFE filter was used, suggesting that RC filters may be better for the preanalytical filtration of samples for NTA.The increase in pressure for PTFE compared to RC is noticeable during filtration and may have contributed to the alteration of the particle size observed, although their biomechanical properties are not well defined. 9However, given the high viscosity and high protein content of cryoprecipitate, clarification using filtration is necessary to prevent the microfluidic degradation within the Nanosight, especially when analysing many samples.Therefore, RC filters are the better choice for clarifying cryoprecipitate samples for NTA.
We observed a concentration of 0.25 Â 10 12 particles/mL in cryoprecipitate.Other studies using NTA on human platelet free plasma report concentrations of 1-5 Â 10 12 particles/mL. 10When comparing the values obtained for cryoprecipitate units in our study, the similarity of particle concentration suggests no enrichment of the number particles in cryoprecipitate compared to plasma.The particles found in cryoprecipitate are likely from the residual cryoprecipitate poor plasma used to solubilise the precipitate during manufacturing.
However, there may be a compositional difference in the population of particles in cryoprecipitate as previously reported. 4Further studies could address these questions by sampling the source plasma unit prior to cryoprecipitate manufacture and then comparing this with both the final cryoprecipitate unit and its paired cryodepleted plasma unit.Additionally, this study investigated whole blood derived cryoprecipitate, so further studies could compare this with aphaeresis derived cryoprecipitiate.
The size distribution of the particles in cryoprecipitate (mean: 133.8 ± 7.5 nm, mode: 107.9 ± 11.06 nm) that we measured using Nanosight overlaps with a diverse range of nanoparticles.These include EVs such as exosomes ($50-150 nm) and microvesicles/microparticles ($100-1000 nm), as well as lipid-based but non-vesicular structures such as the very low-density lipoprotein and chylomicrons (20-1200 nm). 11 did not include any enrichment protocols or identification of specific subtypes of particles, such as EVs.Future studies could employ various available techniques to isolate these particles. 12Perhaps more importantly, to investigate the potential therapeutic role these particles play when transfused into patients, future studies could characterise the protein, lipid and nucleic acid content of these particles using techniques such as flow cytometry, western blotting, and electron microscopy. 5Other studies investigating the role of EVs in plasma and red cell units have found they contained molecules such as tissue factor 13 and phosphatidylserine, 14 which can mediate coagulation pathways.
Overall, this study presents data profiling the particles contained in cryoprecipitate and suggests that NTA is simple and valuable tool to quantify particles in cryoprecipitate when preanalytical filtration using RC is used.Furthermore, no differences were observed between two freeze/thaw cycles of cryoprecipitate particle concentration and size distribution, which is ideal for laboratory investigations where sample storage is warranted prior to analysis.
This study was approved by the Australian Red Cross Lifeblood Ethics Committee (2020#08) and the University Human Research Ethics Committee (UHREC) at Queensland University of Technology (2000000548).Ten units of cryoprecipitate derived from Group O positive whole blood, from different single donors, were supplied by Australian Red Cross Lifeblood.

F I G U R E 2
PTFE filtration impacted the mean and mode size of particles in cryoprecipitate.Stored cryoprecipitate aliquots from different units were thawed and either left unfiltered (n = 3) or were filtered with PTFE (n = 10) or RC (n = 10) prior to Nanoparticle tracking analysis (NTA).The (A) concentration, (B) mean, and (C) mode of particles were measured by NTA.Data shown are mean ± SD and statistics performed with one-way ANOVA.***p < 0.001; ****p < 0.0001.PTFE, polytetrafluoroethylene; RC, regenerated cellulose.protocol of our laboratory was to filter diluted samples before analysis using 0.45 μm PTFE filters.To compare the possible impact of the filter material on particle quantification, cryoprecipitate samples which had undergone 1st freeze/thaw were diluted and filtered with either hydrophobic PTFE or hydrophilic regenerated cellulose (RC) before NTA analysis.

F
I G U R E 3 Single-particle analysis of particles contained in different cryoprecipitate units.Ten cryoprecipitate units were interrogated using nanoparticle tracking analysis.Data shown are the particle size and concentration from two independent experiments (rep #1, #2), with each consisting of 10 video recordings.The solid or dotted pattern line represents the mean, while the shaded area is the standard deviation.

2. 2 |
NTA measurement of size and concentration of particles NTA using the Nanosight NS300 instrument (Malvern technologies, Malvern, UK) was used to measure particle concentration and size.Diluted samples were injected into Nanosight using a 1 mL syringe (Becton, Dickinson and Company, New Jersey, USA) and analysed under constant flow at 50 units/min with the instrument temperature set above 23 C.Ten videos of 60 s were recorded for each sample with a camera level set at 13.All samples were analysed in duplicate.