Improved platelet recovery in cryopreserved platelets reconstituted in freeze‐dried plasma

Abstract

Background

Cryopreserved platelets can support availability in settings where fresh platelets are inaccessible. Freeze‐dried plasma (FDP) may serve as an alternative reconstitution medium to fresh frozen plasma (FFP) when plasma thawing is impractical. The effects of FDP on cryopreserved platelet quality remain underexplored. Thus, we aimed to evaluate its suitability.

Study Design and Methods

Cryopreserved platelets were prepared from double‐dose buffy‐coat concentrates, split, prewashed, and frozen using dimethyl sulfoxide (DMSO) (5–6%). After thawing, paired units were resuspended in FFP (AB = 8) or FDP (OctaplasLG Powder, AB = 8). Platelet count and mean platelet volume were analyzed alongside blood gas parameters. Additionally, extracellular lactate dehydrogenase (LDH), sP‐selectin, and VEGF were measured in the supernatant. Platelet markers (aggregation, adhesion, and activation) and microparticles were analyzed by flow cytometry. Clotting ability was evaluated using ROTEM.

Results

After thawing, FDP units contained significantly more platelets than FFP units 206 ± 27 versus 176 ± 18 ×10⁹/units (p = .0006). LDH activity was lower (p = .040), whereas VEGF levels were higher (p = .0003) in the FDP group. Oxygen and carbon dioxide pressure differed significantly, yet pH was normal. Phenotypic expression and microparticle content demonstrated no significant differences. FDP units showed a shorter clotting time in ROTEM EXTEM (50 ± 5 vs. 58 ± 9 s, p = .035), although clot strength was similar.

Discussion

FDP‐reconstituted platelets were functionally comparable to those reconstituted with FFP, while demonstrating improved recovery and enhanced clot initiation, potentially due to differences in plasma composition. Given its logistical advantages, particularly in resource‐limited settings, FDP represents a promising reconstitution medium for cryopreserved platelets.

Abbreviations

CFT

clot formation time

CT

clotting time

DMSO

dimethyl sulfoxide

FDP

freeze‐dried plasma

FFP

fresh frozen plasma

FLyP

French lyophilized plasma

LDH

lactate dehydrogenase

MCF

maximum clot firmness

MFI

median fluorescence intensity

MPV

mean platelet volume

PMPs

platelet microparticles

ROTEM

rotational thromboelastometry

sP‐selectin

soluble P‐selectin

VEGF

vascular endothelial growth factor

ΔΨm

mitochondrial membrane potential

1. INTRODUCTION

Cryopreserved platelets are an alternative to conventionally stored platelets that permit long‐term storage at −80°C. ¹ , ² The extended shelf life of up to several years enables blood banks to stockpile platelets, thereby mitigating the challenges of unpredictable platelet supply. The limited storage time of conventional platelets (5–7 days) is particularly problematic in remote or austere environments such as military operations, where cryopreserved platelets provide a valuable alternative. ³ , ⁴ , ⁵ Cryopreserved platelets exhibit both phenotypic and functional differences from conventional platelets, including procoagulant properties that render them suitable for treating actively bleeding patients. ⁵ , ⁶ , ⁷

The most widely used cryopreservation method, developed by Valeri, includes 5–6% dimethyl sulfoxide (DMSO) as a cryoprotectant. In this method, platelets are prewashed, and only a small volume (10–30 mL) is frozen. ⁸ Upon request, cryopreserved platelets are thawed and resuspended, typically in thawed fresh frozen plasma (FFP), although other liquids such as saline and additive solutions have also been tested. ⁸ , ⁹ , ¹⁰ Plasma is generally preferred due to its glucose content, which may support platelet recovery, and its supply of coagulation factors essential for managing bleeding. ¹¹ , ¹² However, thawing FFP takes at least 20 min, which can delay transfusion preparation.

Freeze‐dried plasma (FDP) offers logistical advantages over FFP and has historically been used mainly in a military context, with limited access in civilian healthcare. FDP is commonly produced through lyophilization, although spray drying is an alternative technology. Depending on the manufacturer, FDP may be derived from pooled donor plasma or single‐donor units, and some products undergo pathogen reduction while others do not. ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ Before use, FDP is reconstituted in sterile water and can be prepared within approximately 10 min. Unlike FFP, FDP can be stored at ambient temperatures, making it highly suitable for austere or resource‐limited environments. In vitro studies have demonstrated that FDP maintains comparable quality to FFP, with only a moderate (~10%) reduction in labile clotting factors and no increased risk of adverse reactions. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ Given the advantages, FDP represents an alternative resuspension medium for cryopreserved platelets, particularly in military or remote settings. A previous study reported promising results with French Lyophilized Plasma (FLyP), ²² but the impact of other FDP formulations on platelet phenotype and function is not investigated. To address this, our study aims to compare the effects of OctaplasLG Powder and FFP as reconstitution media on the quality and function of cryopreserved platelets in vitro.

2. MATERIALS AND METHODS

2.1. Cryopreservation

Cryopreserved platelets were prepared from double‐dose platelet concentrates (n = 8), produced from pools of eight ABO‐identical buffy coats, the day after collection, as previously described. ²³ These double‐dose platelets are suspended in about 65% additive solution (SSP+) and 35% plasma. Each double‐dose concentrate was evenly divided into two separate freezing bags (Macopharma, Tourcoing, France) using a sterile welding device (Terumo BCT, Lakewood, CO, USA). Thereafter, freezing medium containing 25% DMSO in NaCl (9 mg/mL; 50 mL) was sterilely added to each platelet concentrate (n = 16). The platelet concentrates were then centrifuged at 1200 × g for 10 min, after which nearly all the supernatant was removed. Resulting in an approximately 10‐mL frozen platelet suspension in 5% DMSO. The prepared units were immediately put in metal boxes (Ninolab, Stockholm, Sweden) and placed in a −80°C freezer, subsequently stored for 29–210 days.

2.2. Plasma preparation

FFP (AB = 8) was prepared from whole blood donations at Karolinska University Hospital, processed, and frozen at ≤ − 25 °C within 16 h of collection. For reconstitution, FFP was thawed at 37°C for ~20 min using a Barkey plasma thawer (Barkey GmbH & Co. KG, Griesheim, Germany).

Lyophilized FDP, OctaplasLG Powder (AB = 8) (Octapharma, Lachen, Switzerland), is a mega‐pooled and solvent/detergent‐treated product. ¹⁴ FDP was kept at +4°C until use and reconstituted in 200‐mL sterile water with a spike‐and‐needle set, as previously described. ¹³

2.3. Thawing of platelets

Paired platelet units were thawed simultaneously using a Barkey plasma thawer (Barkey GmbH & Co. KG, Griesheim, Germany) at 37°C for <5 min. Each platelet unit was sterile welded (Terumo BCT, Lakewood, CO, USA) and resuspended in either 200 mL of FFP (Karolinska University Hospital) or 200 mL of FDP (Octaplas LG Powder), resulting in a final DMSO concentration of less than 1%. All units were gently massaged to dissolve aggregates and then maintained at room temperature without agitation for 30 min before further testing.

2.4. Intra‐ and extracellular metabolic parameters

Platelet counts and mean platelet volumes were measured using the hematology analyzer Swelab Alfa (Boule Medical, Stockholm, Sweden). Blood gas parameters, including pH, glucose, lactate, bicarbonate, CO₂, and O₂, along with electrolytes (cNa⁺, cK⁺, and cCl⁻), were analyzed using the ABL 800 (Radiometer Medical ApS, Copenhagen, Denmark). To assess platelet lysis, extracellular lactate dehydrogenase (LDH) activity was measured via spectrophotometry using an assay kit (Sigma‐Aldrich N6660, St. Louis, USA). Samples from the supernatant (centrifuged for 15 min at 3000 rpm) and platelet units (pretreated 1:10 with 0.5% Triton) were prepared and frozen at −80°C in phosphate buffer. Upon analysis, absorbance readings were recorded at 340 nm at 1‐minute intervals over 3 min using the UV5Bio spectrophotometer (Mettler Toledo, Greifensee, Switzerland). LDH activity was calculated and expressed as a percentage of total available activity.

2.5. Flow cytometry

The expression of various platelet markers was analyzed by flow cytometry. Samples were single stained with CD41‐FITC (Beckman Coulter Immunotech, Marseille, France); CD42b‐FITC (Beckman Coulter Immunotech, Marseille, France); CD61‐PE (Beckman Coulter Immunotech, Marseille, France); CD62P‐PE (Beckman Coulter Immunotech, Marseille, France); CD63‐FITC (Beckman Coulter Immunotech, Marseille, France); CD31‐FITC (Sigma‐Aldrich, St. Louis, MI, USA); GPVI‐PE (Pharmingen BD Biosciences, San Jose, CA, USA); PAC1‐FITC (BD Biosciences, California, USA); and Annexin V‐ FITC (BD Biosciences, Erembodegem, Belgium). The proportion (%) of positive cells was calculated from 5000 gated events, analyzed on the CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences). Median fluorescence intensity (MFI) was calculated for the positive population of each marker, representing the MFI of positive events within the platelet gate.

Changes in the platelet mitochondria transmembrane potential (Δψ) were assessed using the mitochondrial permeability transition detection kit MitoPT JC‐1 (Immuno‐Chemistry Technologies, LCC, Bloomington, MN, USA), analyzed on CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences). Dual‐color detection distinguished polarized (red fluorescence) from depolarized mitochondria (green fluorescence). The percentage of positive cells corresponds to the fraction with intact Δψ.

Platelet microparticles (PMPs) were analyzed directly from platelet units without centrifugation to minimize sample manipulation. Microparticles (<1 μm) were identified using size‐calibrated beads and dual staining with CD61‐PE (Beckman Coulter Immunotech, Marseille, France) and Annexin V‐FITC (BD Biosciences, Erembodegerm, Belgium), as previously described. ²⁴ For each sample, 10,000 gated events (<1 μm) were acquired. PMP proportion was based on CD61+ single‐positive or CD61+/Annexin V double‐positive events, and Annexin V+ fractions within CD61+ were calculated to assess phosphatidylserine exposure. Analyses were performed on a CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences).

2.6. Soluble factors

Levels of VEGF‐A and sP‐selectin were quantified using the Ella™ microfluidic automated immunoassay system (Bio‐Techne, Minneapolis, USA), according to the manufacturer's instructions. In short, samples were stored at −80°C until batch analysis. Each batch also included a sample from a randomly selected FFP and FDP unit, which served as an indicative baseline control. Before analysis, samples were thawed and centrifuged at 10,000 × g for 5 min at 4°C. VEGF‐A levels were measured using 16‐sample cartridges, with samples diluted 1:2 in sample diluent. Levels of sP‐selectin were measured using 32‐sample cartridges, with samples diluted 1:100 in dilution buffer. For each assay, 50 μL of diluted sample was loaded into each well. Data acquisition was performed using the Simple Plex Runner Software v.4.1.0.22 (ProteinSimple).

2.7. Thromboelastometry

Thromboelastometry was performed using a ROTEM delta 3000 (TEM International, GmbH, Munich, Germany). A set volume of the platelet sample was mixed 1:1 with red cell concentrate and directly analyzed on the EXTEM and FIBTEM channels according to the manufacturer's instructions.

2.8. Statistics

The mean values and standard deviations are given if not further specified. Normality was assessed using the Shapiro–Wilk test. For normally distributed data, paired t‐tests were performed to determine statistical significance (p < .05). For non‐normally distributed data, differences were assessed using the Wilcoxon matched pairs signed rank test. For comparisons involving more than three parameters, multiple comparison correction was applied using the Holm‐Šidák method. All the statistical analyses were carried out using GraphPad Prism version 10.0.3.

3. RESULTS

3.1. Operational assessment of preparing the platelets

The thawing time for FFP was about 20 min, while the reconstitution of FDP was completed in less than 10 min. All FDP units dissolved easily, produced an acceptable amount of foam, and showed no visible aggregates. The preparation time for cryopreserved platelets was consistent regardless of whether FFP or FDP was used, with all units processed within 10 min, typically around 5 min. No significant differences in aggregate formation were observed. Only one paired unit per arm showed increased presence of small aggregates, which resolved with gentle manual agitation.

3.2. Improved platelet recovery in FDP‐reconstituted units

The platelet count per unit was similar in both groups before freezing, with a mean of 262 ± 26 × 10⁹ (FFP) and 260 ± 24 × 10⁹ (FDP). After freezing, the units reconstituted with FDP contained significantly more platelets, 206 ± 27 × 10⁹/unit, compared to those reconstituted with FFP, 176 ± 18 × 10⁹/unit (p = .0006) (Figure 1A). Accordingly, the average platelet recovery was 79% for FDP and 67% for FPP. Extracellular LDH was lower in the FDP group, with a mean of 31% ± 6%, compared to 36% ± 4% in the FFP group (p = .040) (Figure 1B). The mean platelet volume (MPV) also differed significantly, with the FDP platelets being larger than those in the FFP group, 9.8 ± 0.3 vs. 9.5 ± 0.3 (p = .003).

FIGURE 1.

Improved platelet recovery in FDP units. Platelet integrity following reconstitution with FFP or FDP was assessed using platelet count (A) and extracellular LDH (B). Both assays demonstrated significant differences, with FDP showing a higher platelet concentration and lower LDH extracellularly compared to FFP. Data are presented as bars (mean ± SD) with dots representing each unit (n = 8). *p < .05, **p < .001, ***p < .0001.

3.3. Differences in oxygen and carbon dioxide pressure

In general, most blood gas parameters, including pH, remained comparable between FFP and FDP (Table 1). However, a notable difference was observed in oxygen and carbon dioxide pressure. The FFP platelets contained significantly more carbon dioxide (p < .0001) and less oxygen (p < .001) than FDP platelets. Additionally, reconstitution with FFP was associated with higher glucose and bicarbonate levels compared to FDP (p < .0001).

TABLE 1.

Blood gas and electrolytes after reconstitution. Data presented as mean ± SD.

	FFP n = 8	FDP n = 8	p‐value (adjusted)
pH (37°C)	6.989 ± 0.029	7.003 ± 0.011	.544
pCO2 (kPa)	10.28 ± 0.49	1.72 ± 0.27	<.0001*
pO2 (kPa)	11.9 ± 2.0	17.5 ± 2.8	.005*
K+ (mmol/L)	4.0 ± 0.2	4.1 ± 0.1	.179
Na+ (mmol/L)	151 ± 4.8	146 ± 1.8	.097
Cl− (mmol/L)	86 ± 6.0	83 ± 0.9	.574
Glucose (mmol/L)	16.4 ± 2.2	4.2 ± 0.3	<.0001*
Lactate (mmol/L)	2.0 ± 1.1	1.9 ± 0.1	.814
Bicarbonate (mmol/L)	17.7 ± 1.2	3.1 ± 0.4	<.0001*

Note: Bold formatting is applied to all significant p‑values from *p < 0.05.

3.4. Similar phenotypic expression

The expression of various platelet‐specific receptors involved in aggregation, adhesion, and activation did not differ significantly between FDP and FFP units when assessed by both the proportion of positive events (%) (Figure 2) and median fluorescence intensity (MFI) (Table 2). Platelets reconstituted in both FFP and FDP exhibited high levels of spontaneous activation, with over 60% positivity as determined by CD62P and PAC‐1. Likewise, phosphatidylserine expression was elevated in both groups, exceeding 90%. Staining of the mitochondrial membrane potential (ΔΨ) by JC‐1 showed no significant difference, with a mean of 74% ± 15% for FFP and 80% ±12% for FDP (Figure 2).

FIGURE 2.

FDP use did not affect platelet phenotype compared to FFP. The phenotypic expression of extracellular and intracellular structures was assessed by flow cytometry in platelets reconstituted in either FFP or FDP. No significant differences were observed between the groups (p > .05). Data are presented as mean ± SD (error bars) (n = 8).

TABLE 2.

Flow cytometric assessment of platelet phenotype in FDP and FFP units.

	FDP n = 8		FFP n = 8		p‐value (adjusted)
	% positive gated events	Median MFI	% positive gated events	Median MFI	% positive gated events	Median MFI
CD41	96.5 ± 1.1	427 k ± 21 k	94.6 ± 2.3	429 k ± 41 k	.68	>.99
CD61	96.4 ± 2.2	548 k ± 70 k	94.3 ± 2.1	599 k ± 60 k	.58	.39
CD42b	95.4 ± 2.1	79 k ± 15 k	94.3 ± 2.7	71 k ± 16 k	.74	.068
PECAM	95.5 ± 1.6	34 k ± 4.5 k	93.7 ± 3.2	39 k ± 2.7 k	.68	.15
GPVI	85.6 ± 4.7	11 k ± 4.2 k	79.4 ± 8.9	10 k ± 3.5 k	.08	.44
CD63	91.5 ± 2.0	16 k ± 2.1 k	88.4 ± 4.0	15 k ± 2.4 k	.19	.79
CD62p	66.2 ± 7.5	9.3 k ± 1.5 k	63.9 ± 7.7	9.9 k ± 1.4 k	.68	.76
PAC‐1	74.7 ± 9.9	5.1 k ± 0.4 k	68.6 ± 11.1	4.5 k ± 0.9 k	.68	.47
Annexin V	95.4 ± 3.2	324 k ± 127 k	92.7 ± 3.7	250 k ± 91 k	.48	.12
JC‐1 (ΔΨm)	74.6 ± 14.9	n/a	80.0 ± 11.9	n/a	.68	n/a

Note: Median MFI calculated for positive gated events; values represent mean ± SD for % positive events.

3.5. Release of soluble factors and microparticles

Flow cytometric analysis of platelet‐derived microparticles revealed no significant differences between FFP and FDP reconstitution of cryopreserved platelets. The proportion of CD61+ microparticles was similar between groups, averaging 32% in the FFP group and 33% in the FDP group (Figure 3A). The proportion of phosphatidylserine‐positive (annexin V+) particles among CD61+ microparticles was also consistent, with a mean of 79% in the FFP group and 75% in the FDP group (Figure 3B). Additionally, one control plasma sample was analyzed, indicating a higher presence of CD61⁺ PMPs in FDP (23%) compared to FFP (5%). Phosphatidylserine expression also appeared higher in FDP control plasma (89%) than in FFP (56%).

FIGURE 3.

Microparticles and α‐granule secretion in the reconstituted cryopreserved platelets. (A) PMPs were analyzed by flow cytometry and categorized as CD61⁺/Annexin V⁺ (double‐positive) or CD61⁺ only (single‐positive). (B) The proportion of Annexin V⁺ events within CD61⁺ microparticles was compared between FFP and FDP groups. (C) VEGF and (D) soluble P‐selectin concentrations were measured in plasma supernatants. Each group included one control plasma unit (Ctrl FFP and Ctrl FDP) as indicative examples of baseline values. Data are presented as mean ± SD (error bars) (n = 8 platelet units per group; control plasma shown as single bars). ***p < .0001.

In addition, the release of VEGF and sP‐selectin was measured in the two groups, along with one randomly selected control unit per group, to indicate baseline values. The concentration of VEGF was significantly higher in the FDP group compared to the FFP, at 409 ± 113 pg/mL versus 331 ± 101 pg/mL, respectively (p = .0003) (Figure 3C). In contrast, sP‐selectin concentrations were statistically comparable, with mean values of 360 ± 57 ng/mL for FDP and 347 ± 56 ng/mL for FFP (Figure 3D). Notably, FDP control plasma contained higher concentrations of these factors than FFP control plasma, suggesting that a smaller proportion of the measured concentrations in the FDP group originated from platelets rather than from the plasma itself.

3.6. Faster clotting time (CT) in FDP units

ROTEM was used to assess the in vitro coagulation capacity of the thawed platelets. The use of FDP significantly shortened the EXTEM clotting time (CT) compared to FFP. The mean CT for FDP was 50 ± 5 s, while for FFP it was 58 ± 9 s (p = .035) (ref. 38–79 s) (Figure 4A). However, no significant differences were observed in EXTEM clot formation time (CFT) or maximum clot firmness (MCF) (Figure 4B,C). The average CFT exceeded the reference range < 159 s, with mean values around 210 s in both groups. Similarly, MCF was slightly below the reference threshold of >50 mm, with a mean value of 48 mm in both groups. In addition, MCF values assessed using the FIBTEM channel showed no significant differences between groups, 12 ± 3 mm for FFP and 12 ± 2 mm for FDP (ref. 9–25 mm) (Figure 4D). The platelet‐dependent contribution to clot strength, calculated as the difference between EXTEM MCF and FIBTEM MCF, did not differ significantly between groups (FFP: 34.8 ± 3.5; FDP: 36.5 ± 5.0; p = .1602).

FIGURE 4.

Reduced clotting time in FDP units. Thromboelastography using the EXTEM channel assessed (A) clotting time, (B) clot formation time, and (C) maximum clot firmness for cryopreserved platelets reconstituted with either FFP or FDP. (D) Maximum clot firmness using the FIBTEM channel. (E) Representative ROTEM EXTEM graphs for FFP and FDP groups: upper panels show plasma alone before reconstitution; lower panels show reconstituted platelet units. Data are presented as box‐and‐whisker plots (5th–95th percentile) (n = 8). *p < .05.

4. DISCUSSION

Utilizing FDP as a reconstitution medium may facilitate the availability of cryopreserved platelets and potentially offer an advantage in emergency transfusion scenarios. In this study, we compared the functional characteristics of cryopreserved platelets reconstituted in FDP (Octaplas LG Powder) and in‐house FFP. Interestingly, reconstitution with FDP resulted in higher platelet recovery and lower extracellular LDH activity, indicating improved structural integrity. These findings are consistent with previous reports suggesting FDP as a feasible alternative, though not necessarily superior to FFP. ²²

The observed improvement in platelet recovery is noteworthy, though the underlying mechanism remains unclear. After reconstitution, FDP units exhibited lower CO₂ levels compared to FFP units. While elevated CO₂ is associated with pH reduction and impaired platelet viability during storage, ²⁵ , ²⁶ this typically affects long‐term storage rather than short‐term reconstitution. Both groups had pH values above 6.4, as recommended by the Council of Europe, ²⁷ making CO₂‐driven pH changes unlikely to account for the difference. All analyses were performed shortly after reconstitution, so potential effects of prolonged storage remain unassessed. Furthermore, mitochondrial membrane potential was comparable between groups, indicating that no major metabolic disruption occurred under the studied conditions. Instead, the observed reduction in platelet counts points toward non‐mitochondrial mechanisms.

Bearing that in mind, it is plausible that only the short‐term exposure during reconstitution could transiently impact platelet integrity. Hypothetically, the higher recovery observed in the FDP units may be attributed to the composition of FDP, creating a more protective or stabilizing environment for platelets following reconstitution. Differences in protein content, osmolality, or ionic strength could influence platelet stability, aggregation tendency, or susceptibility to non‐apoptotic lysis. ²⁸ The FDP environment may therefore help reduce platelet loss by limiting aggregation or lysis, potentially offering a more favorable condition post‐reconstitution.

While FDP appears to support platelet integrity, its impact on platelet phenotype was further investigated. It is widely recognized that cryopreservation significantly alters platelet morphology, leading to the emergence of distinct subpopulations, including activated, procoagulant, and novel phenotypes. ²⁹ , ³⁰ These alterations are partly characterized by increased expression of activation markers such as P‐selectin, along with slight reductions in key surface receptors like CD42b (GPIbα) and GPVI, while the overall expression of CD61 (GPIIb/IIIa) remains largely unchanged. ²⁴ , ³¹ , ³² Our findings align with previous reports, although the observed effects appear comparatively mild. The phenotypic patterns were highly consistent across both plasma types, reinforcing the feasibility of using FDP as a viable alternative for platelet reconstitution.

Cryopreservation not only alters platelet morphology but also triggers granule secretion, a process that shapes platelet function and their interactions with other cells. Among these, sP‐selectin and VEGF are modulators released from α‐granules associated with platelet activation, a process known to be elevated in cryopreserved platelets. ²⁹ , ³³ , ³⁴ , ³⁵ When comparing the two groups, VEGF levels were significantly higher in the FDP units, presumably due to the naturally higher VEGF content in FDP. Similarly, sP‐selectin levels were also initially higher in FDP; however, no difference was observed after platelet reconstitution. Suggesting that the release of sP‐selectin post‐reconstitution may be lower when using FDP. Consequently, this indicates that FDP may contribute to a lower release of intracellular substances, supporting earlier conclusions that reconstitution in FDP reduces cell disintegration in cryopreserved platelets.

A major limitation of this study is that only one randomly selected plasma from each study group was measured as a control. This provides illustrative rather than representative data and does not account for donor‐to‐donor variability in FFP. Ideally, all plasma units should have been tested prior to reconstitution to accurately assess baseline levels of biological modulators and their potential impact on platelet integrity. An increase in biological modulators could potentially contribute to adverse immunomodulatory effects in the recipient, ³⁵ , ³⁶ , ³⁷ underscoring the need for systematic pre‐reconstitution testing in future studies. Furthermore, this limitation extends to include other study parameters such as LDH, microparticles, blood gases, and ROTEM. Without testing plasma prior to reconstitution, it is not possible to provide a comprehensive understanding of plasma composition and its influence on platelet recovery, phenotype, and function.

To assess whether platelet clotting function remained effective after reconstitution, ROTEM analysis was performed. Despite the higher platelet count in FDP units, no improvement in clot strength was observed in the EXTEM channel. Both EXTEM CFT and MCF were similar between the groups, indicating comparable overall clotting capacity. However, CFT and MCF values in both groups were outside the normal reference range, consistent with previous studies showing reduced clot strength compared to liquid‐stored platelets. ³¹ , ³⁸ , ³⁹ , ⁴⁰ Prolonged CFT and reduced MCF in EXTEM may also be attributed to decreased levels of fibrinogen or factor XIII. ⁴¹ , ⁴² Notably, FIBTEM MCF, which assesses clot firmness independent of platelet contribution, remained within the normal range. This suggests that EXTEM impairment is more likely due to platelet dysfunction rather than deficiencies in plasma coagulation factors. This is further supported by earlier studies, demonstrating similar levels of FXIII and fibrinogen in FDP and FFP. ¹³ , ¹⁴

Interestingly, FDP was associated with a significantly shorter EXTEM CT, indicating accelerated clot initiation. This effect may be attributed to differences in the composition of plasma coagulation factors, which can influence the initiation phase of clot formation. ⁴³ Octaplas LG Powder, a pooled plasma product, offers more consistent quality, whereas FFP, derived from single donors, exhibits greater variability, as reflected in our results. However, in a previous study, ¹³ we found no difference in clot initiation between Octaplas LG Powder and FFP. Notably, that study employed TEG rather than ROTEM, which may account for the discrepancy. Similarly, another study ⁴⁴ also failed to demonstrate a shortened CT when comparing Octaplas LG Powder to FFP. Yet, the use of only three FFP units in that study may have limited its ability to detect donor variability.

An alternative explanation for the shortened CT could be the presence of procoagulant platelet‐derived particles, which are believed to enhance coagulation initiation. ⁴⁵ , ⁴⁶ , ⁴⁷ However, in the present study, we found no significant differences in the proportion of microparticles between the groups, nor did we observe any differences in phosphatidylserine expression, both of which are considered contributors to procoagulant activity. However, the single control plasma analyzed from the FDP group appeared to contain more PMPs than its FFP counterpart. Although this observation is not representative of the entire group, it suggests that plasma composition may influence PMP levels and warrants further investigation, including assessment of the total number of PMPs rather than just their proportion.

So far, only a limited number of studies have explored reconstitution media other than plasma. Two studies have examined the use of platelet additive solutions, SSP+ and PAS‐G, with supportive results. ¹² , ⁴⁸ In the study by Johnson et al., ¹² platelets resuspended in glucose‐containing media, such as PAS‐G or plasma, demonstrated better recovery and in vitro quality than those resuspended in glucose‐free SSP+. This supports previous assumptions that glucose is essential for optimal platelet storage. ⁴⁹ Another study reconstituted cryopreserved platelets in saline, which resulted in lower in vitro recovery but acceptable in vivo survival after transfusion. ¹⁰ The lack of studies highlights the need for further research to identify the most effective resuspension methods. To date, FFP has remained the preferred medium, primarily due to its hemostatic properties, which are critical in managing bleeding trauma. ⁵⁰ , ⁵¹ , ⁵²

In summary, this study demonstrates that FDP is a viable alternative to FFP for the reconstitution of cryopreserved platelets. FDP was associated with faster clot initiation and improved platelet recovery, as indicated by higher platelet counts and lower extracellular LDH activity. While the underlying mechanisms remain to be fully elucidated, differences in plasma composition, such as blood gas parameters or coagulation factor consistency, may contribute to the observed effects. Importantly, FDP offers significant logistical advantages, particularly in remote resource‐limited settings, by reducing preparation time and eliminating the need for thawed plasma. The study findings support the potential implementation of FDP as a reconstitution medium, particularly in settings where rapid availability and logistical efficiency are critical.

FUNDING INFORMATION

Research grant (ALF) from the Stockholm County, no 530783.

CONFLICT OF INTEREST STATEMENT

The authors have disclosed no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.