Plasma pooling in combination with amotosalen/UVA pathogen inactivation to increase standardisation and safety of therapeutic plasma units

Abstract Objectives Assessment of the impact of pooling five single‐donor plasma (SDP) units to obtain six pathogen‐reduced therapeutic plasma (PTP) units on standardisation and the retention of labile coagulation factors. Background SDP shows a high inter‐donor variability with potential implications for the clinical treatment outcome. Additionally, there is still an existing risk for window‐period transmissions of blood borne pathogens including newly emerging pathogens. Methods/Materials Five ABO‐identical SDP units were pooled, treated with the INTERTCEPT™ Blood System (Cerus Corporation, U.S.A.) and split into six PTP units which were frozen and thawed after 30 days. The variability in volume, labile coagulation factor retention and activity was assessed. Results The variability of volumes between the PTP units was reduced by 46% compared to SDP units. The variability in coagulation factor content between the PTP units was reduced by 63% compared to SDP units. Moderate, but significant losses of coagulation factors (except for vWF) were observed in PTPs compared to SDPs. Conclusion The pooling of five SDP units to obtain six PTP units significantly increases product standardisation with potential implications for safety, economics as well as transfusion‐transmitted pathogen safety, making it an interesting alternative to quarantine SDP (qSDP) and pathogen‐reduced SDP.


| INTRODUCTION
The total protein profile (including contents of coagulation factors) in individual fresh frozen plasma (FFP) units shows a high variability due to the impact of genetic factors on the human proteome. 1  The majority of infected individuals shows no symptoms, despite positive NAT results. 5-8 SARS-CoV-2 (the virus causing COVID-19) genomic RNA has been detected in blood products from asymptomatic donors. 9 Evidence for transmission by blood transfusion has not been shown yet but cannot be excluded. 10 The current situation shows that the qSDP concept only works for specific pathogens if testing is applied, which is not the case for SARS-CoV-2 as well as potentially newly emerging pathogens in the future.
Amotosalen/UVA (AS) pathogen inactivation technology (INTERCEPT™ Blood System, Cerus Corporation), a targeted photochemical reaction irreversibly crosslinking nucleic acids, 11 allows the treatment of pools of five plasma units and splitting into six standardised therapeutic units. 12 The key objectives for this production method are the prevention of transmission of infectious agents and the preservation of the haemostatic capacity as well as the clinical effectiveness. AS-treatment of plasma effectively inactivates a broad spectrum of pathogens, including newly emerging pathogens. 13,14 In vitro studies revealed a moderate loss of coagulation factors post PI-treatment, with an overall retention meeting the regulatory criteria for therapeutic use. 15,16 Also, plasma treated with the INTERCEPT Blood System has been analysed in a series of clinical studies including patients with acquired and inherited coagulopathies as well as patients requiring therapeutic plasma exchange. In all these studies, the plasma showed a high level of tolerability and a safety profile comparable to conventional plasma. 17,18 Our Institute, the Lodz Regional Blood Transfusion Center, is a Polish mid-size blood centre with almost 61.000 whole blood donations in 2018. We produced almost 61.000 plasma units (30% of them for clinical use, 6% of those pathogen-reduced) in 2018. In the current study, we intended to evaluate the impact of pooling five SDP units to obtain six PTP units and AS pathogen inactivation treatment on plasma standardisation and the content of labile coagulation factors as a potential alternative to qSDP and PI-SDP to potentially improve the clinical safety of our plasma products.

| Sampling and analytical testing
Samples were collected from the SDPs before pooling, from the pools directly after pooling and mixing (pre-inactivation) and from the PTP units immediately after pathogen inactivation, into Eppendorf ® Safe-Lock microcentrifuge tubes (volume 1.5 ml). Samples were frozen at

| Data analysis
Comparison of plasma pre and post pathogen-inactivation treatment was performed using the two-sample paired t test. Two-tailed p values of <0.01 are considered statistically significant.

| The impact of plasma pooling on plasma volume standardisation
Twenty-five SDP units have been collected to generate five pools of five ABO-identical SDP units respectively. Three pools were blood group B, one blood group A and one blood group O. The total volume loss during processing (pooling, pathogen inactivation, splitting) was 5.5 ± 1.9%. However, since six PTP units were produced from originally five SDP units, the total volume reduction per plasma unit was 21.1 ± 3.5%, an additional volume reduction of 15.6% (Table 1). The difference between the highest and lowest volume was reduced from 40 ml between SDPs to 22 ml between PTPs, a reduction of 46.5%. in PTPs, a reduction of the spectrum of 54.8% (Table 2). In total, the distribution of values found for the contents of fibrinogen and labile coagulation factors was reduced 63.4% in PTPs compared to SDPs.

| The impact of plasma pooling and pathogeninactivation treatment on the content of fibrinogen, labile coagulation factors and coagulation time
The average fibrinogen retention was 78.7% with respect to the concentration (Table 3). However, the average total fibrinogen content per PTP unit was reduced 37.2 ± 2.5% compared to the SDP units, 24.6% due to processing and 12.6% due to volume reduction by splitting five units into six. The average FVIII retention was 67.8% with respect to the concentration. However, the average total FVIII content per PTP unit was reduced 42.1 ± 11.3% compared to the SDP units, 30.5% due to processing and 11.6% due to volume reduction by splitting five units into six. The average FIX retention was 74.8% with respect to the concentration. The average total FIX content per PTP unit was reduced 40.9 ± 2.6% compared to the SDP units, 29.1% due to processing and 11.8% due to volume reduction by splitting five units into six. Since the average vWF activity was not significantly different pre-and post-treatment (p value 0.014) we also analysed the vWF antigen content, which was also not significantly different (p value 0.377). The average PT was prolonged for 5.2%, the average APTT for 14.5% (Table 3)  T A B L E 1 Volumes (ml) of the initially collected single donor plasma units (SDP), the total volume of the SDPs before pooling, the final pathogen-reduced therapeutic plasma units (PTP), the total volume of the originally pooled units post processing (PP) and the total volume loss during processing (pooling, PI-treatment, splitting) as well as the blood groups (BG) of each ABO-identical pool implications for patient clinical outcome. 22 But also, beyond the "standard" factors a standardised product has advantages. The authors of the above-mentioned study furthermore concluded that also the con-

| Conclusion
The pooling of five SDP units to obtain six PTP units significantly increases plasma standardisation with potential implications for safety of the transfusion recipient, making it an interesting alternative to qSDP and pathogen-reduced SDP, especially in the light of pandemic preparedness.