Production of single unit platelet concentrates in Latin American countries has been a common practice for several years. Particularly in Mexico, the subtraction of platelets from whole blood (WB) is carried out in stages at 22°C temperature: hard spin centrifugation, removing the buffy-coat (BC) layer from WB with automatic separator, second soft spin centrifugation to BC layer, and the supernatant platelets removing to a storage bag [1]. To provide an adequate platelet dose to an adult patient, it is necessary to transfuse 4 to 8 platelet concentrates obtained from WB [2].

Further, to optimize the production and increase the availability of platelet concentrates (PC), the production of leukodepleted platelet concentrates (LDPC) from BC pooling is validated with two different storage methods: pooled plasma (PP) during five (5) days storage, and platelet additive solution (PAS) for seven (7) days storage. Both methodologies have been widely evaluated in past years, and it is critical to understand the structural and biochemical changes of LDPC through the storage period [3],[4].

Several factors, such as the preparation method, type of storage bag, type of additive solutions, and storage temperature, can influence platelets’ quality during this period. These parameters can lead the storage units through structural and biochemical changes collectively referred to as platelet storage lesion [5],[6]. The benefits of platelet production, suspended in additive solutions, have been described in other publications.

Halumain and Sweeny (Journal of Clinical Apheresis, 2012) summarized different PAS types and their composition, concluding that PAS presented some critical advantages to the hospital transfusion services and the manufacturing blood centers [7]. Gulliksson et al. (Vox Sanguinis, 2003) studied the benefits of PAS III modified (PAS-IIIM) with Magnesium and Calcium in the composition. They found that storage of platelets in PAS-IIIM reduced the rate of glycolysis and improved the pH and hypotonic shock response reactivity [8]. Leitner et al. (Vox Sanguinis 2015) examined the quality of platelets resuspended in three different PAS, stored for up to 7 days, which indicated that the optimization of these additive solutions became a focus of interest not only for the extended period of storage but for the possibility to introduce pathogen inactivation methods [9].

In Mexico, the Health Authorities endorsed a five-day storage period after donation, extendable to 7 days, if bacterial reduction systems or bacterial contamination detection methods are used. Still, the PAS method has not been standardized yet [10]. There are not many studies about platelet preparation in pooled plasma (PP). The reason is why a comparative analysis between the 5-days and the 7-days storage would be of significant support for Latin American countries’ different Health Authorities.

During the past few years, there have been many developments supporting the benefits of using an additive solution (PAS) for PC storage. Using the current generation of PAS, the storage quality of platelets, evaluated in vitro and in vivo, is at least, as good, if not better, in PAS compared to PP, in addition to other benefits, such as reduced of transfusion allergies reactions (TARs), being, in turn, essential, the reduction of exposure to donor plasma greater than 65% in PAS PCs products, which may have a beneficial impact on the decrease of TRALI (transfusion-related acute lung injury), since this transfusion reaction is commonly due to bioactive substances and/or antibodies against HLA (human leukocyte antigens) or neutrophil antigens in donor plasma.

Furthermore, transfusion of ABO-incompatible plasma PCs is associated with hemolytic transfusion reactions. Therefore, the reduced volume of plasma in PAS PCs will likely decrease the risk of ABO-incompatible hemolysis. The reduction in PP used in PCs will also release additional plasma for fractionation in the industry [11],[12],[13]. This comparison aims to analyze the in vitro quality results of the platelet components. It is to evaluate the benefits of extending to 7 days of storage of platelets. For that reason, a comparison between the 5-day PP and the 7-day LDPC in SSP+ is hypothesized to be compliant with the available literature findings.

The pool of BC from WB

Whole blood was collected from 120 (process 1—2018) and 136 (process 2—2019) volunteer donors, meeting Mexican guidelines criteria for blood donation [10]. 450 mL of WB was drawn into a quadruple Top and Bottom (T&B) system (Macopharma, Tourcoing—France) containing citrate base anticoagulant (CPD). Collected packs were centrifuged for 12 minutes at 3600 rpm (PRESVAC DP-2065 R Plus, Buenos Aires—Argentina). Using an automatic separator (Macopress Smart, Macopharma, Tourcoing—France), the plasma and the red blood cells were placed into separate bags, with the remaining BCs in the collection bag. Approximately 25 mL (process 1) and 15 mL (process 2) of plasma were returned to the BC bags before they were sealed and separated. Buffy-coats, the first intermediate component in the procedure, were held without agitation at room temperature approximately for 18 hours (overnight).

After the screening tests, between the day of collection and the next morning, four BCs were obtained with identical ABO and pooled with a sterile connecting device (TCD Sterile Tube Welder B40, Genesis, Ramsey, NJ-US) in an octopus-designed bag packet: connected to the pooling bag (PVC-DEHP), the system has one platelet storage, designed with a specific platelets storage plasticizer container (PVC-BTHC Oxy-Pl) and an in-line filter, to reduce the leukocytes from the platelets concentrate (PC) during the transfer. One unit of PP (process 1) and one bag of 300 mL of PAS (SSP+, Macopharma) (process 2) was mixed with the BC pooling system (TRV8006XU, Macopharma).

Platelet Concentrate from BC Pooling

Centrifuge setting for BC pools storage in pooled plasma (PP) was 1900 rpm × 7 minutes (process 1), and for the BC pool storage in SSP+, it was at 1350 rpm × 7 min (PRESVAC DP-2065 R Plus). Both processes used the Macopress Smart (Macopharma) to obtain LDPC, which were tested for CD45, CD42b, CD62P, and Annexin V (CyAn ADP-Beckman Coulter); pCO₂, pO₂, and pH (OPTI CCA-TS2); glucose, DHL (lactic dehydrogenase), and Total Proteins (Unicel DXC800-Beckman Coulter); and platelet count and MPV (Swilab Alfa—Boule).

Plasma Carry Over—PCO

The residual plasma or plasma carry over (PCO) was calculated to determine each BC’s hematocrit to measure the plasma volume BCs conforming the four BC pooling units for the 34 units of LDPC process and storage in 300 mL of SSP+ additive solution [14].

The following formula was applied to obtain the PCO value:

It was possible to calculate BC’s average volume of 68.6 ± 1.33 mL with 68.81 ± 2.89% mean hematocrit with a standardized separation process resulting in an average total plasma volume for each LDPC 85.96 ± 9.04 mL. After processing and separating LDPC according to the established methodology (300 mL SSP+), the calculated PCO resulted in an average value of 22.24 ± 1.75%.

Sampling and testing

For LDPC storage in PP (process 1), the test samples were taken on day 1 (2 hours rest after collection) and day 5 of storage. For LDPC in SSP+ (process 2), the samples were taken on days 1, 5, and 7 of storage. Both groups of LDPC were stored in the Presvac AP 48 L agitator (Buenos Aires, Argentina) at room temperature under constant agitation.

The evaluation of in vitro PC quality was made as follow:

Platelet concentration and media platelet volume (MPV)

Platelets were sampled after 2 hours of resting, and the sample was immediately tested on a hematology analyzer (Swilab Alfa—Boule, Spånga, Sweden) located in the blood bank.

CD62P, Annexin V, CD45, and CD42b by flow cytometry

Platelet concentrates were transported under temperature control (room temperature) from Puebla to Mexico City and processed in the flow cytometer following every marker’s laboratory protocols (CyAn ADP-Beckman Coulter, Indianapolis, USA) on the same day of the sample collection.

Evaluation in vitro PC metabolism

On days 1 and 5 for both groups, and day 7 for SSP+, the pools were sample to measure pH, pO₂, pCO₂ (OPTI CCA-TS2, Opti Medical Systems, Atlanta, USA); and glucose, DHL, and total proteins were analyzed during the same days with Unicel DXC800 (Beckman Coulter, Indianapolis, USA).

Statistical Analysis

A software performed the statistical analysis (SPSS for Windows package, release 27, SPSS Inc., Chicago, IL). The number of cases, the arithmetic mean, the standard deviation, and the 95% confidence interval were obtained for all the quantitative parameters. The comparative analysis of the evaluated parameters’ mean values, both their absolute values and their percentage changes between the two groups, was carried out using the T-Student test for independent samples. All the tests were performed with bilateral contrast, considering the differences statistically significant when the type error (p) represented <0.05.

Data from Unidad Médica de Alta Especialidad, Hospital de Especialidades (UMAE HE) Blood Bank, in Puebla (Mexico – 2019), on the evaluation of leukodepleted platelet, concentrates from buffy-coat pooling (LDPC) suspended in SSP+ (Macopharma), were analyzed, measured on days of storage 1, 5, and 7, and then it was compared with the results obtained by the same blood center in 2018 when pooling LDPC suspended in plasma (PP) and evaluated results on days 1 and 5.

Volume values of LDPC suspended in SSP + were higher than the LDPC suspended in plasma (p < 0.05): The media volumes of units were 239 mL (233–345) for PP and 265 mL (256–274) for SSP+, with platelet recovery on day 1 of 2.97 (2.54–3.4) × 10¹¹/unit and 3.08 (2.7–3.51) × 10¹¹/unit, respectively. Platelet recoveries from BCs did not present significant differences between PP and SSP+ methods (p >0.05) on day 1 of storage. Media platelet volume (MPV) changed during storage was found not significant (p > 0.05) (Table 1).

The results for CD42b antigen in PP and SSP+ presented no significant differences, with media values: 3.32 (2.86–3.78) × 10¹¹/mL for PP and 3.24 (2.75–3.73) × 10¹¹/mL for SSP+ during day 1 of storage (Table 1). The measure of platelet content using CD42b antigen by flow cytometry is a powerful technique deemed as an essential tool for investigating cellular activity since 1989, including platelet function and activation. Among other markers, CD42b are frequently used as platelet identifiers, as they are only present on platelets and not any other circulating blood cell [15].

Although no differences were obtained from platelet recovery between the two storage media on day 1, the decrease in platelets counts during days of storage was different: The mean percentage decrease of platelet count, from day 1 to day 7 suspended in SSP+ was 14.3% (6.6–22.0%), higher than the LDPC group suspended in PP from day 1 to day 5. This is a 4.8 ± 5.6% mean percentage decrease. Besides, the mean percentage decrease of CD42b antigen of LDPC suspended in SSP+ from day 1 to day 7 was significantly higher than the LDPC suspended in PP from day 1 to 5 (p < 0.05) (Figure 1A and Figure 1B).

Residual leukocyte count (WBC) was measured with flow cytometry (CD45 antigen, a pan-leukocyte marker) to qualify the leukodepletion in all the LDPC. The media results in both groups presented at least two logarithms below than EU guidelines target (< 1 × 10⁶ cell/mL): 48.2 × 10⁴ cell/mL for PP and 12 × 10⁴ cell/mL for SSP+, which means that the mean of WBC with the expression of CD45 on day 1 of PLTs suspended in PP was significantly higher than that WBC suspended in SSP+ (p < 0.05) (Table 2) [16].

Platelet activation was detected by CD62P expression. The mean of the adhesion protein CD62P or P-Selectin on day 1 for LDPC suspended in SSP+ and suspended in PP were similar (p > 0.05). However, during days of storage, the mean increase of CD62P adhesion from day 1 to day 5 of LDPCs suspended PP was significantly higher than in the group of LDPC suspended in SSP+ (p < 0.05); and the mean increase of CD62P protein adhesion from day 1 to day 5 of LDPC suspended in PP was also significantly higher than LDPC suspended in SSP+ from day 1 to 7 of storage (p < 0.05) (Figure 2).

Annexin V, procoagulant activity, and apoptosis marker were detected by flow cytometry in both groups. The mean of the Annexin V on day 1, of the LDPCs, suspended in SSP +, was higher than the LDPC suspended in PP (p < 0.05), but for day 5 storage, the mean of the Annexin V of the PLTs suspended in SSP+ and suspended in PP was not different (p > 0.05). For the final day of storage (day 7), there was a similar mean percentage increase of Annexin V from day 1 to 5 of PLTs in PP and suspended in SSP+ (p > 0.05) (Table 2).

Platelet metabolism

The partial pressures of oxygen (pO₂) presented comparable average value for both groups of LDPCs (49 mmHg for PP and 55 mmHg for SSP+) (p > 0.05); and the mean percentage decrease of pO₂, from day 1 to day 5 of PLTs suspended in PP and SSP+, was not different either (p > 0.05). The situation was similar for the difference values between days 1 and 5 for PP, days 1 and 7 for SSP+ (Table 3).

For pCO₂ measurements, the values also decreased. The mean pCO₂ on day 1 of the LDPC suspended in PP was significantly higher (67 ± 7 mmHg) than the LDPC suspended in SSP+ (33 ± 6 mmHg). The mean pCO₂ on day 5 of the LDPC in PP was also significantly higher than the PLTs in SSP+ (p < 0.05). Finally, the percentage decrease of pCO₂ of PLTs in PP, from day 1 to day 5, was more significant than the PC suspended in SSP+, from day 1 to 7 (Table 3).

Glucose was consumed continuously during storage period in all LDPC, and the mean of LDH (increase) in LDPCs suspended in SSP+, from day 1 to 7, was higher than that of LDPC suspended in PP, from day 1 to day 5 (p < 0.05). It is important to note that the initial glucose concentration in the LDPC store in PP was three times the LDPC stored in SSP+. There was a higher glucose consumption for LDPC in SSP+ than in PP (p < 0.05) (Figure 3A and Figure 3B).

It was also expected that the mean concentration of Total Proteins on day 1 of PLTs suspended in PP was significantly higher than the PLTs suspended in SSP+ (5.9 g/dL for PP vs. 1.4 g/dL for SSP+); however, during the storage period, the mean percentage increase of PC in SSP+, from day 1 to 7, was significantly higher than the PC suspended in PP, from day 1 to day 5 (p < 0.05) (Table 3).

The pH on day 1 presented values of 7.0 ± 0.1 in both groups, with a 2.5% and 2.7% increase for PP and SSP+ respectively on day 5, and with a non-significant difference in the mean percentage increase of the PC, suspended in SSP+, from day 1 to 7 and the PC suspended in PP, from day 1 to day 5 (p > 0.05) (Table 3).

UMAE HE Blood Bank of Puebla, Mexico is one of the leading hospitals in Puebla state (Puebla de Zaragoza is a city in Mexico’s eastern central area); collecting and processing approximately 24,000 blood donors per year. Platelets are obtained by the BC removal method to separate single platelet concentrates on fulfilling the transfusion needs. In 2018, the center accepted to evaluate platelet quality in 5 days of storage after preparing LDPC with the pooling BC method. With positive results from the first test, a second evaluation was made in 2019 using the same platelet methodology. This time with platelet additive solution SSP+ (20% PCO) and extending the storage time to 7 days.

To our knowledge, this is the first project carried out in a Latin American country where the quality of LDPC prepared from BC pooling stored in plasma and platelet additive solution is compared. The production of platelets by pooling BC is not yet a standardized procedure established in UMAE HE Blood Bank or any other center in Mexico. However, the validations intend to support future implementation, with the evidence-based proof that the SSP+ would be a solution for the shortage of platelets availability and as a complement of the single donor platelets produces in the center, taking into account that this is a type of platelet products with demonstrated satisfactory storage quality. There have been no reports of increased adverse transfusion reactions, reduced hemostatic effectiveness, or increased incidence of transfusion-related sepsis with such pre-storage pooled platelet products [17],[18].

Platelet recovery could depend on centrifugation settings and the storage medium’s content to pool the buffy coats. SSP+ has a similar viscosity to water, while the soluble proteins in plasma enhance its viscosity; thus, pooling with plasma should results in higher recoveries than pooling with SSP+. However, in our evaluation, the platelet recovery shows no significant difference (p > 0.05), validating both storage methods as quality transfusion products [19].

Although the reduction percentage, during the storage period, was significantly higher with SSP+ (14.3 ± 7.7%) than PP (4.8 ± 5.6%), all the LDPC by the end of the storage with SSP+ comply with the minimum quantity required to be considered useful according to with European Directorate for the Quality of Medicines & Health Care of the Council of Europe (EDQM) [16], with an average of 2.62 × 10¹¹ platelets/mL (Table 4). These phenomena have been attributed to activation during storage time, resulting in microaggregation, fragmentation, or platelet integrity losses. Previous studies have ruled out apoptosis as a significant contributor to platelet loss in vitro [20],[21].

Additive solutions in platelet production have provided the possibility of including elements with specific effects on platelets during their storage, elements that would not be present in pooled plasma, or the anticoagulant used for their production. Among the effects observed using PAS include reducing platelet activation and improving platelets’ metabolism and function, showing an improved storage environment, representing useful tools to optimize platelet storage conditions [22]. CD62P variability has been the most widely used measure for platelet activation over the years [4], and it has been used in this study to perform the comparison.

With the results with our own storages LDPC, this concept of PAS stability for platelet preparation could be considered substantiated, since the activation tests showed a significant difference with PP in comparison with SSP+ storage (p < 0.05), being lower activation activity with SSP+ (Figure 2). Platelet viability remained high in both groups throughout storage. Annexin V protein increased gradually during the platelet units’ storage, but the percentage increase was not significantly different between PP or SSP+ (p > 0.05) (Table 4).

Platelet metabolism

The pH of PP and SSP+ units was maintained within a narrower range below 7.4 during 5 and 7 days of storage, respectively, showing increasing values with no significant differences (p > 0.05). Meaning, the pH was maintained well above the EDQM recommendations in all units (Table 4) [16].

Glucose was present at the end of storage in both LDPC storage media. However, the LDPC storage in PP had a higher starting glucose concentration than PC in SSP+ (p < 0.05). The mean decrease in glucose was not statistically different in both groups (Figure 3). Consequently, lactate production (LDH) was significantly higher in PP than SSP+ during the storage period. The percentage increase was also significantly different between PP and SSP+, being higher in SSP+ (p < 0.05) (Figure 3).

High rates of glucose consumption can deplete this fuel source and result in higher pH (not our case) and may have some implications in the platelets’ total metabolic balance. High lactate production rates also produce more acid that requires buffering to prevent precipitous declines in pH over storage. In general, the lactate generation rates were lower in SSP+ units, thus reducing the buffering burden throughout storage [22].

All units exhibited a time-dependent storage decrease in pO₂: 4% by day 5 in PP and 11% by day 7 in SSP+, showing no significant differences for both storage media; and the decrease presented with pCO₂ show values significant higher on PP than SSP+ (Table 3 and Table 4). The significant interactions observed for pCO₂ confirm that the absolute changes of CO₂ levels were different, with PCs stored in plasma showing the highest mean decrement (66.0 ± 4.6%), indicating the fastest rate of CO₂ decrease than SSP+ (48.6 ± 9.7%).

Platelet additive solutions have shown advantages over the use of pooled plasma for LPDC preparation. These benefits include reducing the number of transfusion reactions, particularly allergic reactions and transfusion-related acute lung injury (TRALI), where plasma proteins have been implicated. In contrast, more plasma becomes available for fractionation. Due to the absence of regular antibodies, such as anti-A and anti-B, ABO-incompatible transfusions are better tolerated [23].

The standard storage time of PC is 5 days in many countries because of bacterial contamination and platelet injury concerns. When implementing new processing methods for PCs, the platelets’ stability in vitro should prove safe and effective because in vivo patient studies of platelet quality are not available everywhere.

The preparation of PC has been improved and standardized during the years by process optimization and wise production method selections. The final products have been continuously improving, searching for more efficient ways to have better therapeutic effects. Storage of 34 LDPCs showed that PCs, made from 4 BCs, suspended in SSP+, stored for seven days, in a BTHC plasticized PVC container (TRV8006XU, Macopharma) and 30 LDPC, also made from 4 BCs, suspended in PP, storage for five days, 100 percent comply to the requirements of EDQM: platelet recovery > 2.0 × 10¹¹ cell/mL, pH greater than 6.8 at the end of the storage period and WBC < 1.0 × 10⁶ cell/mL. Based on the in vitro variable studies in this investigation, a PC storage period of 7 days in SSP+ appears to be feasible since the results did not show statistically significant differences with PP, which could let us conclude that the seven-day storage produces platelet products with the same quality that the standardized five-days storage, opening a place for potential advantage to happen when adding the extended two days storage become possible for blood banks already struggling with blood availability and donors shortage.