Strategies to improve platelet cryopreservation: A narrative review

Abbreviations

2FA

N‐(2‐fluorophenyl)‐d‐gluconamide

DES

deep eutectic solvent

DMSO

dimethyl sulfoxide

EVs

extracellular vesicles

GMP

good manufacturing practice

PVA

polyvinyl alcohol

1. INTRODUCTION

Platelets for transfusion are conventionally stored at room temperature (20–24°C), in gas‐permeable bags, with constant agitation. These storage conditions are required to preserve platelet quality and support survival following transfusion. ¹ However, platelet quality and function progressively deteriorate over the storage period, known as the platelet storage lesion. ² Additionally, the risk of bacterial growth limits the platelet shelf life to 7 days, after which the components are discarded if they have not been transfused. The combined impact of a short shelf life and unpredictable supply can, at times, strain transfusion services, ³ and this pressure could be alleviated if platelets could be stockpiled in times of excess supply. Accordingly, platelet cryopreservation is a promising solution to extend the platelet shelf life, reduce component wastage, and ensure adequate inventory.

Cryopreservation of platelets offers several unique advantages over conventional platelet storage. Platelet cryopreservation extends the shelf life from 7 days to at least 2 years, although recent data suggest that platelet quality is maintained for up to 10 years. ⁴ , ⁵ , ⁶ The extended shelf life afforded by cryopreservation has the potential to reduce component wastage and enable stockpiling to ensure a stable supply is available. Additionally, the long shelf life supports the supply of platelets to remote and austere environments, which are ordinarily difficult to supply with room temperature platelets, as unpredictable use leads to high wastage. ⁷ , ⁸ Cryopreservation of platelets would also allow blood centers to freeze platelets in times of excess supply and stockpile product for future use. This approach is also advantageous among growing fears of global conflicts and the ability to maintain blood supplies. ³ Further, the ability to freeze specific HLA‐ or HPA‐phenotyped platelets to support refractory patients would be advantageous. ⁹ , ¹⁰ , ¹¹ , ¹² Ultimately, cryopreserved platelets offer flexibility to healthcare providers, enabling better inventory management as platelets are thawed on demand.

The most widely accepted method for platelet cryopreservation uses 5%–6% dimethyl sulfoxide (DMSO) as a cryoprotectant, followed by centrifugation to remove the majority of the DMSO‐containing supernatant, and storage below −65°C. ¹³ , ¹⁴ , ¹⁵ When required, platelets are thawed, resuspended in a suitable media, such as freshly thawed plasma or saline, ⁴ , ¹⁶ , ¹⁷ , ¹⁸ and are ready for transfusion in 30 min or less. If cryopreserved platelets are not transfused immediately, they can be stored at room temperature for up to 6 h. ¹⁹ , ²⁰ The post‐thaw shelf life is limited due to the addition of either DMSO or the resuspension media via an “open” system, and alterations to platelets that are observed when stored beyond this. ¹⁷ , ¹⁸

Platelets frozen using this method are phenotypically and functionally different from platelets stored at room temperature. The freeze–thaw process results in a loss of up to 30% of platelets due to damage. ¹⁶ , ²¹ , ²² , ²³ Typical characteristics of cryopreserved platelets include a lower abundance of functional surface receptors such as GPIbα and GPVI, externalization of phosphatidylserine, and extensive shedding of extracellular vesicles (EVs). ¹⁶ , ¹⁷ , ²² , ²³ , ²⁴ Functionally, cryopreserved platelets initiate rapid clot formation but form weaker clots than conventionally stored platelets in vitro, when assessed by viscoelastic testing. ¹⁷ , ²⁵ , ²⁶ Cryopreserved platelets also demonstrate enhanced thrombin generation with elevated peak thrombin and faster clotting times compared with conventionally stored platelets. ²¹ , ²⁷ , ²⁸ These functional changes may be due to the exposure of phosphatidylserine on the platelet membrane and EVs. ²¹ , ²⁴ The ability to initiate clot formation quickly is thought to make cryopreserved platelets most suitable for the treatment of active bleeding, where prompt cessation of bleeding is the priority. ²⁹

Cryopreserved platelets have been assessed in in vivo studies, both in healthy volunteers and selected patient populations. The recovery of autologous cryopreserved platelets in healthy volunteers is reported to be ~33%–42%, compared with ~63%–68% for fresh platelets. ¹⁶ , ³⁰ In terms of survival, cryopreserved platelets showed a reduction in platelet survival (~8.4–8.6 days), compared with ~7.0–7.5 days for fresh platelets. ¹⁶ , ³⁰ However, cryopreserved platelets exceeded the FDA survival criterion. ¹⁶

There is clear international interest in cryopreserved platelets as a clinical product for specific indications. While cryopreserved platelets have been used successfully in military settings for over two decades, ⁵ their use in civilian settings is increasing, ⁸ but would likely be broader and more accessible if the preparation process were simplified. Currently, cryopreserved platelets are used by The Netherlands Armed Forces and The Australian Defence Force for military deployments, the Military University Hospital in Prague, and in Poland for clinical indications such as immune refractory patients or stem cell transplant patients when conventionally stored platelets are not available. ⁵ , ¹⁴ , ²⁰ , ³¹ Cryopreserved platelets have also been used extensively to support thrombocytopenic, refractory hematology/oncology patients. ¹² , ³² In terms of clinical studies, there have been a limited number of small randomized controlled trials investigating cryopreserved platelets. ³³ , ³⁴ , ³⁵ Importantly, the clinical use and trial data suggest that cryopreserved platelets are safe and the occurrence of adverse events is low. ³¹ , ³² , ³³ , ³⁴ , ³⁵ However, large, appropriately powered trials are currently underway to confirm safety and address efficacy outcomes in terms of bleeding in specific patient populations. CLIP‐II (NCT03991481), ¹⁹ which recently completed recruitment, CLIPNZ‐II (ACTRN12621000271808), ¹⁹ and the CRYPTICS (NCT04709705) ³⁶ trials are all multi‐center, randomized, blinded, controlled clinical non‐inferiority trials comparing postoperative blood loss in bleeding cardiac surgery patients receiving either cryopreserved platelets or standard room temperature stored platelets. The outcomes of these trials, combined with the advantages of longer shelf‐life, will inform the best clinical practice regarding cryopreserved platelets.

This review discusses the development of the most commonly used platelet cryopreservation technique (the “Valeri method”), highlights advances that have occurred in the field, and considers new directions to be explored for the continued enhancement of cryopreserved platelets.

2. DEVELOPMENT OF THE MOST COMMONLY ADOPTED METHOD OF PLATELET CRYOPRESERVATION

Valeri et al. pioneered platelet cryopreservation in the 1970s, utilizing DMSO as a cryoprotective agent for platelets. ³⁷ However, due to toxicity, minimizing the concentration of DMSO in the transfused platelet component was a priority during method development. DMSO toxicity is characterized by headaches, nausea, gastrointestinal problems, vasoconstriction, and skin reactions, which have been reported when the dose of DMSO exceeds 1 g/kg of the recipient body weight. ⁷ , ³⁸ , ³⁹ Initially, excess DMSO was removed in a post‐thaw washing step. ³⁷ However, this process requires specialized equipment (a blood bag centrifuge) at the time of thawing, which is a major logistical challenge when considering the preparation of the platelets for transfusion in both hospital and military settings. As cryopreserved platelets have a short post‐thaw shelf life, the post‐thaw washing step had to be performed at the site of use. Subsequent modifications simplified the process by removing the excess DMSO‐containing supernatant before freezing. ¹⁵

The “Valeri method” for platelet cryopreservation has been frequently adopted since it was established around 2 decades ago. This method involves the addition of 5%–6% DMSO, followed by centrifugation and removal of the majority of the DMSO‐containing supernatant, and resuspension of the pellet in the residual supernatant. The hyperconcentrated product is then frozen in a −80°C freezer and stored below −65°C (Figure 1A). When required, platelets are rapidly thawed in a water bath and resuspended in a larger volume of solution to facilitate transfusion. Of note, there are country‐/institute‐specific variations in the cryopreservation and thawing methods (Table 1).

FIGURE 1.

Most commonly adopted method and idealized method for platelet cryopreservation. (A) Dimethyl sulfoxide (DMSO) is added to an apheresis platelet component to achieve a final concentration of 5%–6%. Platelets are then pelleted via centrifugation to allow for the removal of the supernatant before freezing, minimizing the residual DMSO. Platelets are then resuspended in the residual supernatant by gentle rubbing in a circular motion. The concentrated platelet component is then frozen in a −80°C mechanical freezer. In preparation for transfusion, the platelet hyperconcentrate is thawed in a water bath at 37°C for ~5 min. The platelet component is then resuspended in the desired media (plasma or saline). (B) To simplify the platelet cryopreservation process, several modifications should be investigated. The use of a non‐toxic cryoprotectant or an acceptable amount of DMSO to eliminate the removal step would negate the need for the centrifugation step, saving both time and reducing damage to the platelets. Furthermore, this modification would support a simplified thawing process, where the platelet component could simply be removed from the freezer and thawed ready for transfusion without the need to use a resuspension media. [Color figure can be viewed at wileyonlinelibrary.com]

TABLE 1.

Examples of country‐specific variations for platelet cryopreservation.

Starting component			Freezing process		Thawing and resuspension		References
Country	Type	Suspension	DMSO (%)	Freezing volume (mL)	Solution	Solution volume (mL)
Australia	Apheresis	40% plasma/60% SSP+	5.5	20–30	Plasma	250–310	[7, 19]
Czech Republic	Apheresis	Plasma	6	12–14	Plasma	200–280	[71]
The Netherlands	Apheresis	35% plasma/65% Intersol	6	10–20	Plasma	225–315	[4, 5]
New Zealand	Apheresis	30% plasma/70% SSP+	6	20–35	Plasma	100	[50]
Sweden	Pooled buffy coat	35% plasma/65% SSP+	5	10	Plasma	200	[8]
USA	Apheresis	Plasma	6	20–35	0.9% NaCl	25	[16, 36]

3. KEY ASPECTS OF THE PLATELET CRYOPRESERVATION PROCESS

While the “Valeri method” is the most commonly used, there have been numerous advances in the field of cryobiology in the decades that have followed. Therefore, it may be time to revisit this technique to see whether improvements could be made to the platelet cryopreservation process.

3.1. Cryoprotectants

Cryoprotectants are used to minimize detrimental changes to cell membranes and internal structures during the cryopreservation process. While DMSO is the most widely used cryoprotectant for platelets, the process to remove excess DMSO via centrifugation and concentration of platelets is time‐consuming and introduces its own set of drawbacks, as discussed below. Alternative cryoprotective agents including trehalose, propylene glycol, ethylene glycol, glycerol–glucose, nitric oxide, and dextran have previously been investigated. ⁴⁰ , ⁴¹ , ⁴² , ⁴³ , ⁴⁴ However, either the platelet quality was deemed inferior, or the method was more cumbersome with these agents compared with cryopreservation using the standard DMSO method. Of note, the freezing and thawing conditions used in previous studies were not always aligned with the current method. As such, it may be worth revisiting these cryoprotectants, either alone or in combination, to improve the cryopreservation process.

3.2. Centrifugation and concentration

Platelets must be centrifuged before freezing to enable the removal of excess DMSO, to reduce the risk of DMSO toxicity following transfusion. Platelets containing DMSO are centrifuged at 1250g for 10 min at room temperature in a blood bag centrifuge, which subsequently allows for the removal of the majority of the DMSO‐containing supernatant. ¹⁵ While essential to the current method, the centrifugation step takes time and limits the number of components that can be prepared at any one time. Centrifugation is also damaging to platelets, contributing to the reduced recovery and loss of surface receptors following thawing. ⁴⁵ Interestingly, Valeri and colleagues found that the removal of the DMSO‐supernatant before freezing resulted in an increased proportion of thawed platelets with reduced GPIbα and increased phosphatidylserine externalization when compared with washing platelets following thawing. ¹⁵ However, this trade‐off was accepted as the benefits provided by removing the post‐thaw washing step outweighed the reduction in platelet quality.

Currently, there are differences in how the concentration step is performed in centers internationally (Table 1). The volume of the hyperconcentrate varies, ranging from 10 to 35 mL. This variation may contribute to minor alterations in platelet quality that arise due to different shear forces experienced during resuspension, the concentration of platelets within the bag, and freezing rates. If an alternative cryopreservation method were developed where the centrifugation step were not required, this would greatly simplify the process and potentially reduce variations in international manufacturing practices.

3.3. Freezing rate/approach

An optimal rate of cooling is required to prevent damage from osmotic rupture or by uncontrolled ice recrystallization, which is essential to achieve high recovery and viability rates following cryopreservation. ⁴⁶ , ⁴⁷ The standard method of platelet cryopreservation uses uncontrolled “dump freezing,” which results in a freezing rate of ~3–5°C/min. ⁴ While this may not sound ideal, it is effective, as demonstrated in a number of studies. ⁵ , ¹³ , ²² Specifically, Tynngård et al. compared uncontrolled and controlled‐rate freezing protocols on various platelet quality parameters and concluded that the controlled‐rate method was not superior to uncontrolled freezing in the context of standard DMSO cryopreserved platelets. ⁴⁸ A similar conclusion was reached when a downscaled method of platelet cryopreservation was optimized, as freezing rates between 2 and 6°C/min did not alter the platelet phenotype or function. ⁴⁹ These studies suggest that there is a range of acceptable freezing rates within which platelets can be frozen. Given that the volume of platelets frozen for transfusion is small (10–35 mL), the freezing rate likely remains similar between manufacturing processes and may not make a significant difference to the overall quality of the product.

3.4. Thawing and resuspension

As platelets are frozen as a low volume hyperconcentrate, they are reconstituted in a volume of plasma or saline before transfusion. The approach and rationale for reconstitution vary, depending on the cryopreservation protocol (Table 1). Many protocols use a large volume of plasma (100–315 mL) to dilute the platelets to a concentration that aligns with standard platelet components. ⁸ , ¹⁷ , ¹⁸ , ²¹ , ²⁵ , ⁵⁰ Plasma is commonly used as the resuspension media, as patients who are bleeding will likely benefit from plasma. ⁴ An alternate approach for reconstitution uses a low volume of saline (25 mL) and is aimed at diluting the residual DMSO. ³⁶ , ⁵¹ The reconstitution step may also include a rest period on a platelet agitator after thawing, before resuspension. The reconstitution solution is most commonly added after thawing, with the exception of New Zealand, where platelets are frozen in tandem with the resuspending plasma (100 mL) separated by a clip, allowing the plasma to mix with the platelets during thawing. ⁵⁰ If cryopreserved platelets did not require resuspension following thawing, this would save both time and resources, streamlining the process.

3.5. Post‐thaw shelf life

Following thawing and resuspension, the post‐thaw shelf life is limited to 6 h. While cryopreserved platelets are intended to be thawed on demand, transfusion within this time frame is not always achievable, particularly when a patient requires fewer platelet components than anticipated, resulting in the component being discarded. In vitro studies assessing the quality of cryopreserved platelets when stored beyond this time demonstrate dramatic changes to platelets, ¹⁷ , ¹⁸ suggesting that an extension of the shelf life at room temperature is not viable. However, in vitro data demonstrate that refrigerating platelets after thawing maintains platelet quality parameters such as glycoprotein expression, activation markers, and platelet function (as measured by thromboelastography) for at least 10 days and may represent a feasible and simple approach to extending the post‐thaw shelf life. ⁵²

3.6. Unknown factors

Despite decades of research into cryopreserved platelets, the specific mechanism(s) that lead to cryopreservation‐induced changes remain unclear. Alterations to the platelet membrane and functionality following thawing suggest signaling pathways may be altered during cryopreservation, but this has not been extensively investigated. Therefore, further efforts should focus on exploring potential mechanisms of cryopreservation‐induced alterations so that they can be specifically addressed. This may also help delineate which changes are inherent to the cryopreservation process and exposure to low temperatures, and which changes are able to be mitigated. A greater understanding of these processes could also support the development of novel cryoprotectants to best support platelets during cryopreservation.

Donor attributes, including sex and health, have been associated with platelet storage performance, ⁵³ , ⁵⁴ , ⁵⁵ although this has not been considered in the context of cryopreservation. Areas of specific interest include assessment as to whether certain platelet phenotypes may be more resistant/susceptible to cryopreservation‐induced damage, as has been shown for red cells. ⁵⁶ Further, platelets from females have been shown to possess greater hemostatic potential than males, ⁵⁷ which may make them more attractive for cryopreservation, given the targeted recipients. As such, research efforts directed towards identifying specific attributes that are associated with good/poor quality profiles following cryopreservation could be harnessed to generate a superior quality product.

4. NOVEL APPROACHES TO PLATELET CRYOPRESERVATION

To continue advancing platelet cryopreservation, it is important to consider what should be achieved by the ideal cryopreservation process. Given the challenges highlighted in relation to current methods of platelet cryopreservation, several areas could be improved. A generalized process for the ideal platelet cryopreservation method is outlined in Figure 1B. Platelet cryopreservation should involve minimal or no component manipulation before freezing and following thawing. This would not only reduce labor and training requirements for both those manufacturing the components but also for laboratory staff who are responsible for the preparation of blood components before transfusion. Reducing component manipulation would also reduce the use of other resources, such as resuspension media, which saves money, and in the case of plasma, would make this resource available for other clinical needs. The ideal cryopreserved platelet component would also need to be of high quality, with a long shelf life and have the capacity for extended storage following thawing.

Recently, alternative methods for platelet cryopreservation have been explored to improve the cryopreservation process and/or to better preserve platelet quality parameters. These approaches include automation, a novel approach to remove residual DMSO, the use of small molecule cryoprotective agents, and controlled rate freezing. ²⁶ , ⁴⁸ , ⁵¹ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ , ⁶² A summary of these strategies is shown in Table 2.

TABLE 2.

Summary of recent novel approaches to improve platelet cryopreservation.

	References
Automation
Spinning membrane device	[51]
Novel DMSO reduction strategies
Dialysis Reduced DMSO concentration	[45, 63]
Novel cryoprotectants
Deep eutectic solvents (DES) Ice recrystallization inhibitors (IRI) Calcium chelation Saline Matrix metalloproteinase inhibitors	[26, 58, 59, 60, 61, 62]
Freezer conditions
Controlled rate	[48, 59]

4.1. Automation of cryopreserved platelet manufacture

Automating the manufacturing process for cryopreserved platelets would be advantageous to reduce manual handling and staff labor. Dumont et al. recently reported using the CUE Cell Processing System (Fresenius Kabi) to achieve this. The system uses spinning membrane technology to concentrate platelets, add the desired concentration of DMSO (currently 6%) and aliquot the selected volume into a freezing bag. ⁵¹ Platelets frozen using this system demonstrated a decrease in activation markers accompanied by a reduction in thrombin generation potency, ⁵¹ which may reflect a decrease in the hemostatic potential of these platelets compared with standard methods. While the thawed platelets contained a final concentration of 3% DMSO (following reconstitution), this can be adjusted by the instrument. Thus, if a lower percentage of DMSO was deemed suitable for cryoprotection of platelets, this would be readily achievable with this system. While the processing times using this system were not reported, the automated nature provides potential to tailor the product once more is known about the ideal conditions for platelet cryopreservation. This automated system could likely be assimilated into a good manufacturing practice (GMP) environment to create a product that is suitable for clinical use.

4.2. Novel DMSO reduction strategies

The ability to remove excess DMSO without centrifugation could potentially reduce platelet damage and improve platelet quality following thawing. Removal of DMSO by dialysis has recently been assessed. ⁶³ Platelets frozen with 5%–6% DMSO without pre‐freeze concentration were washed for up to 30 min following thawing using a dialysis machine. ⁶³ Several reported platelet quality parameters, including recovery, phosphatidylserine externalization, and function (by thromboelastography) appear to be largely comparable to existing literature on standard cryopreserved platelets. ⁶³ This unique approach for DMSO removal presents a potential alternative to the current method while also maintaining platelets of suitable quality. While the freezing process would essentially be comparable to current methods in a GMP environment, post‐thaw processing is not ideal for use in military or hospital settings due to the requirement for specialized dialysis equipment.

4.3. Novel cryoprotectants

Efforts to improve the quality of cryopreserved platelets have also led to the assessment of alternative cryoprotectants and various small molecules in combination with the standard amount of DMSO. These studies aimed to alleviate cryopreservation‐induced alterations in the existing process. Investigations into two different ice recrystallization inhibitors (IRIs), polyvinyl alcohol (PVA) and N‐(2‐fluorophenyl)‐d‐gluconamide (2FA), when used in addition to DMSO, had minimal protective effects compared with standard cryopreservation methods, with only a small capacity to rescue certain platelet quality parameters observed. ⁶⁰ , ⁶² PVA was found to reduce the percentage of platelets with surface‐expressed CD62P, but the effect was deemed too small to be of biological relevance. ⁶² Similarly, supplementation with 2FA improved platelet recovery and retention of GPVI following cryopreservation when compared with standard methods; however, the majority of parameters remained unchanged. ⁶⁰ Thus, ice recrystallization does not appear to be a major source of damage in the context of platelet cryopreservation. The release of calcium from internal stores was hypothesized to contribute to cryopreservation‐induced alterations, including receptor shedding. To investigate this, a calcium chelator, BAPTA‐AM, was added to DMSO‐containing platelets before cryopreservation. Significant improvements to the retention of the highly labile GPIbα and GPVI receptors were observed, alongside a reduction in platelet activation markers such as phosphatidylserine and P‐selectin externalization. ²⁶ Six et al. also demonstrated that the addition of a matrix metalloproteinase inhibitor, marimastat, to DMSO‐containing platelets before cryopreservation significantly reduced the loss of GPIbα. ⁵⁸ Despite the increased abundance of GPIbα when platelets were frozen with either BAPTA‐AM or marimastat, no improvements to platelet function were observed. ²⁶ , ⁵⁸ While these studies have expanded our understanding of the mechanisms leading to cryopreservation‐induced changes to platelets, these methods are unlikely to be implemented as they involve significant component manipulation and do not eliminate the need for DMSO.

While the concentration of DMSO used for platelet cryopreservation (5%–6%) is already lower than what is standard for many cell types (generally 10%), recent investigations have focused on reducing the DMSO content or eliminating DMSO altogether. One potential strategy that has recently been reported is to reduce the concentration of DMSO required to freeze platelets to a level that would be safe to transfuse, thereby eliminating the need for centrifugation. Cryopreservation with 3% DMSO without pre‐freeze or post‐thaw DMSO removal did not adversely affect platelet quality or stability over a 12‐month period. ⁴⁵ Importantly, this approach also eliminated the need for post‐thaw resuspension, thus improving the logistics of preparing platelets for transfusion. Ehn et al. demonstrated that saline could be used to cryopreserve platelets in the absence of DMSO. ⁵⁹ Broadly, although the preservative effects observed were marginally less than what can be achieved with the conventional DMSO method, the DMSO‐free cryopreserved platelets remained viable and hemostatically active in vitro, as assessed by thromboelastometry. ⁵⁹ Interestingly, the use of controlled rate freezing for platelet cryopreservation with saline was shown to be required for optimal platelet quality in the absence of DMSO. ⁵⁹ This suggests that the freezing rate of platelets could be further optimized for specific cryoprotective agents. While this approach eliminates DMSO, the optimized saline cryopreservation method still involved centrifugation and post‐thaw resuspension in plasma. Thus, while this approach does not alleviate all challenges associated with the conventional cryopreservation method, it warrants further investigation. More recently, a deep eutectic solvent (DES, 10% Proline‐Glycerol 1:3) was shown to provide comparable platelet recovery, phenotype, and function to standard DMSO methods. ⁶¹ Interestingly, this study also reported that platelets frozen in the absence of any cryoprotective agent were of reasonable quality, ⁶¹ suggesting that platelets may be more resilient to cryopreservation‐induced alterations than other cell types. Freezing platelets with DES or in the absence of any cryoprotective agent enabled the elimination of the centrifugation step and the need for platelets to be resuspended following thawing, greatly simplifying the cryopreservation and thawing process from end to end. Further, notable advantages with both saline and DES are cost‐effectiveness and low toxicity, ⁵⁹ , ⁶⁴ making them attractive substitutes for the standard DMSO method and feasible to introduce as a clinical product. Together, these studies provide strong support that platelet cryopreservation in the absence of DMSO is achievable. This research also supports exploration of other novel types of cryoprotectants, such as anti‐freeze proteins and osmolyte solutions, ⁶⁵ in the context of platelet cryopreservation, as well as the re‐investigation of previously explored non‐toxic cryoprotectants, including trehalose, using revised techniques.

5. FUTURE DIRECTIONS TO IMPROVE PLATELET CRYOPRESERVATION

While headway is being made, some important factors should be considered when designing future studies. The most appropriate baseline for comparisons of quality changes needs to be considered. Within the literature, novel strategies are most commonly compared with the “Valeri method” to assess improvement or detriment. However, this may no longer present the best option. Given that there are several processing steps that could be improved in the “Valeri method,” perhaps a more appropriate approach would be to compare new techniques to platelets frozen in both the presence (e.g. “Valeri method” as this is the current product transfused) and absence of DMSO. Freezing and thawing a conventional room temperature‐stored platelet component without a cryoprotectant is as simple as the process can be. Thus, any comparisons made to this approach would allow for a comparison considering all logistical pros and cons of each method. It would also be clinically relevant to compare thawed platelets to the current transfusible products, which include platelets stored at room temperature (for up to 7 days), as well as cold‐stored platelets (up to 14 days). ⁶⁶

The techniques used to assess the quality of cryopreserved platelets must also be considered carefully. The phenotype and function of cryopreserved platelets prepared using the “Valeri method” are well documented within the literature, and the differences to conventionally stored platelets are well established. Assays typically used to assess the quality and function of conventionally room temperature stored platelets are not necessarily appropriate for the assessment of cryopreserved platelets. ²⁸ If the suite of assays is not carefully selected, then good quality platelets may be considered poor quality, and the converse, as has historically happened with these components. The functionality of thawed platelets has been demonstrated using assays such as viscoelastic testing, thrombin generation, and microfluidic models, rather than light transmission aggregomtery. ⁶ , ²² , ²⁸ Microfluidic flow perfusion models are attractive as they mimic the biophysical environment of blood vessels to assess platelet function under conditions of hydrodynamic shear. ⁶⁷ , ⁶⁸ The models can be tailored to assess platelet adhesion to different physiologically relevant substrates and the deposition of fibrin following adhesion, under different shear rates (venous vs. arterial). ⁶⁹ Although platelet swirl and concentration (post‐thaw recovery) are often used to define the quality of platelets, they seem to provide a poor reflection of platelet quality in the context of cryopreservation. Inherent limitations with hematology analyzers can lead to inaccurate measurement of platelet recovery, due to counting debris, platelet fragments, or microparticles as platelets, resulting in false elevation of platelet recovery. ⁷⁰ The pre‐determined size range and light scatter properties of hematology analyzers mean that potentially only a subset of platelets is accurately measured, while small, large, aggregated, or degranulated platelets may not be accounted for. Newer fluorescence‐based methods may also be inappropriate for enumeration of cryopreserved platelets as the ability of platelets to retain the dyes following cryopreservation is impaired. Further, it is unknown what these in vitro quality markers predict in terms of in vivo quality following transfusion, highlighting the need for further clinical trials. With a number of clinical trials ongoing, findings from these studies provide an opportunity to better understand how in vitro quality markers relate to in vivo function. This information could be harnessed to direct future investigations into other patient cohorts, which will be crucial to assess how cryopreserved platelets can be best used in a clinical setting.

Substantial progress has been made in improving access to platelets through cryopreservation; however, more work remains to be done. By expanding our understanding of the mechanics of the freezing process in the context of platelets, further enhancements to platelet cryopreservation will be possible. Overall, simplifying the process of preparing cryopreserved platelets will support broader implementation of this product, which would be beneficial to the transfusion community globally.

FUNDING INFORMATION

Australian Governments Fund Australian Red Cross Lifeblood to provide blood, blood products, and services to the Australian community.

CONFLICT OF INTEREST STATEMENT

The authors have no conflicts of interest to disclose.

Waters L, Marks DC, Johnson L. Strategies to improve platelet cryopreservation: A narrative review. Transfusion. 2025;65(4):740–749. 10.1111/trf.18204