|Year : 2020 | Volume
| Issue : 1 | Page : 17-21
Precious platelets: The utility of cold-stored and cryopreserved platelets
Rizwan Javed1, Frozan Ahmadi2, Asheer Jawed3
1 Department of Clinical Haematology and BMT, Tata Medical Center, Kolkata, West Bengal, India
2 Molecular Genetics, Guys Hospital/Kings College London, London, United Kingdom
3 Department of Respiratory Medicine at William Harvey Hospital, Ashford, United Kingdom
|Date of Submission||28-Feb-2020|
|Date of Decision||30-Mar-2020|
|Date of Acceptance||31-Mar-2020|
|Date of Web Publication||17-Apr-2020|
Department of Clinical Haematology and BMT, Tata Medical Center, Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
The current storage temperature of platelets prepared by both pooled buffy coats and by apheresis is 22°C + 2°C (room temperature [RT]). The adverse microbiological and metabolic implications associated with RT storage have restricted the shelf life of platelets from 5 to 7 days. The proposed alternative is cold-stored (4°C) platelets that could be stored for 21 days and cryopreserved platelets with dimethyl sulfoxide for up to 2 years at −80°C. An increase in shelf life could be of great utility in rural and military settings. Based on the proven quality and properties of cold-stored platelets, the US Food and Drug Authority has approved its use for up to 3 days in patients having active bleeding.
Keywords: Bleeding, cold-stored platelets, cryopreserved platelets
|How to cite this article:|
Javed R, Ahmadi F, Jawed A. Precious platelets: The utility of cold-stored and cryopreserved platelets. Glob J Transfus Med 2020;5:17-21
| Introduction|| |
The storage temperature of platelets has been the subject of much controversy and scrutiny within transfusion science. The current storage temperature of platelets prepared by both pooled buffy coats and by apheresis is 22°C + 2°C. However, room temperature (RT) storage of platelets proves to be optimum conditions for the growth of bacteria. Consequently, upon transfusion, contaminated platelet products can induce life-threatening infections in patients. Moreover, during RT storage of platelets, metabolic processes continue without any hindrance. Therefore, in order to maintain gaseous exchange, platelets are stored in gas-permeable bags which have an increased surface area and undergo constant gentle agitation. Despite these current practices, during storage, there is a gradual deterioration in platelet quality with regard to various metabolic, functional, and morphologic derangements. This is globally defined as storage lesions, which are thought to increase when platelets are stored at RT. Consequently, the microbiological and metabolic implications associated with RT storage have restricted the shelf life of platelets from 5 to 7 days. Logistically, the unique storage conditions are difficult to maintain and the short shelf life means a greater demand for platelet donors while equally driving waste. The proposed alternative to RT storage is cold (4°C) storage of platelets.
The aim of this study was to review the current literature on the utility of cold-stored and cryopreserved platelets
| Methodology|| |
PubMed and Google databases were searched for advantages of cold-stored and cryopreserved platelets. Reference lists were cross-checked for relevant citations, and more searches were undertaken till the desired information was obtained.
| Advantages of Cold-Stored Platelets|| |
Cold-stored platelets would resolve the problem of microbial contamination as most bacteria do not propagate in cold storage conditions. In addition, the metabolic rate would slow down subsequently preserving hemostatic function. Consequently, cold storage of platelets provokes discussion about a longer shelf life. As cold-stored platelets could be stored for 21 days and cryopreserved platelets with dimethyl sulfoxide (DMSO) for up to 2 years at −80°C. An increase in shelf life could be of great utility in rural and military settings. Moreover, logistically, cold storage of platelets could easily be initiated as the infrastructure for other refrigerated blood components such as red cells is already well established. However, despite these advantages, cold storage of platelets was abandoned and replaced with RT storage in the 1970s. This is because cold storage of platelets is associated with rapid platelet clearance posttransfusion.
| Physiology of Cold-Stored Platelets|| |
It has been shown through studies in murine models that cooling of platelets to 4°C results in irreversible clustering of glycoprotein Ib (GPIb) (von Willebrand factor receptor) on platelet surface. In the clustered configuration, the platelet surface GPIb molecules are sequestered by liver macrophages through their αMβ2 integrin receptors. This leads to the subsequent demise of cold-stored platelet as they are rapidly phagocytosed by the liver macrophages. This made RT-stored platelets indispensable for patients requiring prophylactic platelet transfusions – accounting for 70% of platelet transfusion recipients – who require long-acting platelets.
However, does high platelet recovery and survival then translate into high hemostatic function? Since there is no way of accurately determining platelet functionalityin vivo in fact, the posttransfusion platelet count simply measures circulating platelets and disregards platelet viability. Therefore, this begs the question, by storing platelets at RT is platelet quantity more important than platelet quality? Cold-stored platelets recently got the Food and Drug Authority approval for use up to 3 days in patients having active bleeding., This development resulted in renewed interest in cold storage among transfusion medicine experts.
| Comparative Studies|| |
Two independent studies conducted by Johnson et al. and Nair et al. have contributed to the accumulation of evidence in support of cold storage of platelets. Moreover, investigators have compared RT storage with cold storage with regard to platelet function. Although, Johnson et al. have included cryopreserved platelets and have a biochemical perspective on platelet function. Whereas, Nair et al. have interpreted platelet function with regard to clot properties. Therefore, naturally, they have taken very different approaches to demonstrate the effect of storage temperature on functionality. Since there are no direct measures of platelet function,in vitro studies involve assessing several indicators of function which include morphology and metabolism. Moreover, functional tests such as aggregometry and clot formation may also be employed to define functional capacity.
| Metabolic Changes|| |
Subsequently, Johnson et al. measured several metabolic variables such as pH, lactate, glucose over a 21-day storage period. Interestingly, the results demonstrated that at the end of the 21-day storage period, the pH was actually lower in cold-stored platelets (6.99) compared with RT storage (7.43). Moreover, it is seen that by day 14, glucose exhausted faster (0.1 mmol/L) in RT-stored platelets compared with cold-stored platelet which demonstrated a slower consumption (0.3 mmol/L) of glucose. These results are of high importance, as they suggest that despite the increased rate of metabolism in RT-stored platelets, pH remains stable and contests claim that RT storage leads to a drop in pH and subsequently diminishedin vivo survival. In addition, during the 21-day storage period, there was a gradual decline in acetate levels from 22.7 mmol/L to 7.4 mmol/L; in contrast, cold-stored platelet had 21.1 mmol/L of acetate at the end of storage period. Moreover, RT-stored platelets maintained ATP production until day 14, whereby glucose had been exhausted. In contrast, ATP levels showed a gradual decline in cold-stored platelets. The acetate and ATP results demonstrate that oxidative phosphorylation was negatively impacted by cold storage. The ATP depletion observed with cold-storage platelets may be attributed to shape change, which is an ATP-expensive process.
| Morphological Changes and Clot Properties|| |
Interestingly, the subsequent morphological studies carried demonstrated that during the 21-day storage period, cold-stored platelets lost their swirl after day 2, whereas RT-stored platelets maintained a 3+ swirl score throughout the storage period. In addition, cryopreserved platelet displayed a 2+ swirl score 6 h postthaw. A high swirl reflects the retention of a discoid morphology, whereas the absence of swirl is indicative of spherical morphology. The spherical shape is typical of activated platelets, whereas discoid platelets are not activated and correlate with bestin vivo viability. Therefore, results suggest that RT-stored platelets have highin vivo viability in comparison to cold-stored platelets. In contrast, the morphological results of Nair et al. are in support of cold-stored platelets. Scanning electron microscopy has, revealed that the clot ultrastructure of cold-stored platelets is comparable to that of fresh platelets. More specifically, the results demonstrated that the fiber density of clots produced by fresh and cold-stored platelets was similar. However, the fiber density of the RT-stored platelets was 25%–30% lower. Similarly, fresh platelets and cold-stored platelet had a similar length distribution with a mean length of 5 μm, whereas RT-stored platelets were characterized with a mean length of 10 μm. In addition, the fresh and cold-stored platelets had a straighter curvature in comparison to RT-stored platelets. Consequently, the ultrastructure of fresh and cold-stored platelets had denser clots with thinner fibers and more branching points, whereas RT-stored platelet clots were characterized as less dense and thicker fiber with fewer branching points.
The functional consequences of the morphological and metabolic differences were then determined by both using thromboelastography (TEG) and rheology, respectively., The TEG analyzers are composed of a torsion wire, a pendulum, and a cuvette. The cuvette typically contains a small sample of citrated blood in addition to calcium chloride, kaolin, and phospholipid. The purpose of the latter three additions is activation of coagulation cascade. The cuvette is heated to 37°C and rotated at an angle of 4° 45′, six times per minute, the purpose of which is to imitate the blood flow in the venous circulation. The torsion wire with a stationary pin attachment is inserted into the cuvette. As the blood clots, fibrin production increases which confers increased resistance against the pin. The mechanical energy of the clot is transduced into an electrical current which is then received and analyzed by a computer. The variables measured include reaction time (R-time), maximum amplitude (MA), kinetic value (K-value), and alpha-angle. The R-time is time from the start of the test to the beginning of clot formation; as such, the normal R-time values range between 7.515 min. Studies demonstrated that on day 5 of storage, the R-time of cold-stored platelets (~9 min) was lower compared with RT storage (~12 min). Interestingly, cryopreserved platelets had the shortest R-time on day 5 of storage (~7 min). However, all storage temperatures are within the normal ranges of R-time. Moreover, the MA is a measure of maximum attainable clot strength. Interestingly, Johnson et al. reported no difference in MA between cold-stored and RT-stored platelets, although cryopreserved platelets demonstrated a low MA in comparison to the liquid stored platelets. Similarly, there was no difference in K-value (time taken to reach a certain level of clot strength) and alpha-angle (speed of fibrin buildup and crosslinking) between cold-stored and RT-stored platelets.
In contrast to Johnson et al., Nair et al. used cone and plate rheometry combined with dynamic mechanical analysis to define clot properties – clot stiffness and strength. The former is defined as the measure of resistance by the clot to reversible deformation, whereas the latter is defined as the ability of a clot to carry large load before irreversible deformation. The measurement of stiffness was determined by a small-amplitude oscillatory test using the Rheoplus instrument. Small-amplitude oscillatory testing was carried out, whereby the sample is loaded between the plates at a known gap, after which the upper plate is oscillated back and forth at a given stress/strain. To determine stiffness, the Rheoplus was adjusted to 0.5% strain and 1 Hz to track the evolution of the elastic modulus (resistance to deformation) of clots. The results demonstrated that cold-stored platelet showed a ~4 fold increase in clot stiffness compared to RT-stored platelets. To determine clot strength, the rheometer applied progressively increasing strains (1%–250%) to the point where the clot failed (irreversible deformation), so that critical stress can be estimated. The results demonstrated that the critical stress for clots formed with cold-stored and fresh platelets was comparable at ~200 Pa and ~300 Pa, respectively. In contrast, the critical stress for clots formed with RT-stored platelets was ~100 Pa. Therefore, it can be concluded that the rheology study showed that clot strength of cold-stored platelets was significantly stronger (P < 0.05).
Therefore, it can be gathered that the clot strength results in both studies were in contrast. The clot strength ability of cold-stored platelets is attributed to exogenous plasma FXIII. In fact, cold-stored platelets have significantly higher levels (2-fold increase) of plasma FXIII than in RT-stored platelets. Cold-induced plasma FXIII contributes to crosslinking and lateral aggregation of fiber which subsequently decreases fibrin thickness and increases clot stiffness by ~4-fold compared with uncrosslinked fibers. Therefore, the lack of difference in clot strength between cold and RT-stored platelets in the study by Johnson et al. may be attributed to the fact they stored their platelet concentrate in 30% plasma/70% SSP (additive solution) instead of FFP.
The “activated” phenotype associated with cold stored platelets and more specifically demonstrated by activation of GPIIb/IIIa facilitates platelet aggregation and adherence. This corroborated with the results of Johnson et al. which revealed through platelet aggregometry that cold-stored platelets reached a higher maximal aggregation in response to ADP compared with RT-stored platelets. Therefore, this “activated” phenotype may explain superior clot properties and hemostatic function of cold-stored platelets. However, equally, this raises the concern of an increased risk of prothrombotic action by cold-stored platelets.
| Manufacturing and Thawing of Cryopreserved Platelets|| |
“O” group fresh apheresis platelets having platelet content of >2.8 × 109 is mixed with 75 ml of 27% DMSO and 0.9% saline to get a final concentration of 5%–6% DMSO in the cryobag and centrifuged for 12 min at 1250 g (Heraeus 6000i centrifuge, GmbH)., As per Valeri method, the supernatant was removed using a manual extractor and the final platelet product is dump frozen at −80°C in a mechanical deep freezer. Cryopreserved platelets are thawed at 30°C (plasma thawer) and reconstituted with type AB plasma. The reconstituted platelets have a shelf life of 6 h.
| Advantage of Cryopreserved Platelets|| |
Inventory of human leukocyte antigen/human platelet antigen-cryopreserved platelets would be beneficial in platelet refractory., Since they are easily thawed and reconstituted, they could be effectively used in trauma management. At Prague, the military university hospital uses cryopreserved platelets as a standard blood product. Manufacturing, thawing, and reconstitution of cryopreserved platelets are financially and technologically feasible. Hence, the cost of transfusion therapy is not significantly altered by the use of cryopreserved platelets.
| Relevance to Developing Asian Countries|| |
According to the World Health Organization Factsheet 2013,, there are around 100,000 deaths annually in the developing countries due to the unmet transfusion needs of pregnant women alone. Majority of them succumb to postpartum hemorrhage. Along with rising trauma-related hemorrhage in the developing world, the readily availability of cold-stored platelets could be of great hemostatic utility to these patients in austere settings.
| Conclusion|| |
There is a general consensus that cold storage of platelets is long overdue for the treatment of active bleeding rather than in those requiring prophylaxis. However, this suggests that perhaps, two inventories need to be maintained for platelets which raise logistical and financial concerns. Moreover, thein vitro results demonstrate some optimistic results with regard to increased clot strength and increased hemostatic ability of cold-stored platelets. However,in vitro tests alone are not sufficient to reliably predictin vivo functionality of cold-stored/RT-stored platelets. This is because there is evidence to suggest that many of the storage abnormalities are actually reversible posttransfusion. In the context of developing Asian countries, cold-stored platelets could be of great utility in patients with acute hemorrhage, even in remote places, where peripheral blood centers cannot procure specialized platelet agitators. However, it cannot be overemphasized that large and well-designed clinical trials are required to define the therapeutic efficacy, dosage, and transfusion intervals of differently stored platelets. In conclusion, it is worth considering the utility of cold-stored platelets in cases of acute hemorrhage, especially in resource deficient settings.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Murphy S, Gardner FH. Effect of storage temperature on maintenance of platelet viability-deleterious effect of refrigerated storage. N
Engl J Med 1969;280:1094-8.
Ketter PM, Kamucheka R, Arulanandam B, Akers K, Cap AP. Platelet enhancement of bacterial growth during room temperature storage: Mitigation through refrigeration. Transfusion 2019;59:1479-89.
Braathen H, Sivertsen J, Lunde THF, Kristoffersen EK, Assmus J, Hervig TA, et al
quality and platelet function of cold and delayed cold storage of apheresis platelet concentrates in platelet additive solution for 21 days. Transfusion 2019;59:2652-61.
Reddoch KM, Pidcoke HF, Montgomery RK, Fedyk CG, Aden JK, Ramasubramanian AK, et al
. Hemostatic function of apheresis platelets stored at 4°C and 22°C. Shock 2014;41 Suppl 1:54-61.
Kaufman RM. Platelets: Testing, dosing and the storage lesion-recent advances. Hematology Am Soc Hematol Educ Program 2006(1):492-6.
Berzuini A, Spreafico M, Prati D. One size doesn't fit all: Should we reconsider the introduction of cold-stored platelets in blood bank inventories? F1000Res 2017;6:95.
Miles J, Bailey SL, Fang L, Osborne B, Barbara, Corson J, et al
. Evaluation of efficacy and safety of cold-stored platelets in healthy human subjects treated with dual antiplatelet therapy. Blood 2019;134:718.
Stubbs JR, Tran SA, Emery RL, Hammel SA, Haugen AL, Zielinski MD, et al
. Cold platelets for trauma-associated bleeding: Regulatory approval, accreditation approval, and practice implementation-just the “tip of the iceberg”. Transfusion 2017;57:2836-44.
Johnson L, Tan S, Wood B, Davis A, Marks DC. Refrigeration and cryopreservation of platelets differentially affect platelet metabolism and function: A comparison with conventional platelet storage conditions. Transfusion 2016;56:1807-18.
Nair PM, Pandya SG, Dallo SF, Reddoch KM, Montgomery RK, Pidcoke HF, et al
. Platelets stored at 4°C contribute to superior clot properties compared to current standard-of-care through fibrin-crosslinking. Br J Haematol 2017;178:119-29.
Badlou BA, Ijseldijk MJ, Smid WM, Akkerman JW. Prolonged platelet preservation by transient metabolic suppression. Transfusion 2005;45:214-22.
Getz TM. Physiology of cold-stored platelets. Transfus Apher Sci 2019;58:12-5.
Bose E, Hravnak M. Thromboelastography: A Practice Summary for Nurse Practitioners Treating Hemorrhage. J Nurse Pract 2015;11:702-9.
Bohonek M, Kutac D, Landova L, Koranova M, Sladkova E, Staskova E, et al
. The use of cryopreserved platelets in the treatment of polytraumatic patients and patients with massive bleeding. Transfusion 2019;59:1474-8.
Valeri CR, Ragno G, Khuri S. Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at − 80 degrees C without post-thaw processing. Transfusion 2005;45:1890-8.
Cohn CS, Dumont LJ, Lozano M, Marks DC, Johnson L, Ismay S, et al
. Vox Sanguinis International Forum on platelet cryopreservation. Vox Sang 2017;112:e69-e85.
Johnson L, Coorey CP, Marks DC. The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserine-expressing platelets and platelet microparticles. Transfusion 2014;54:1917-26.
Cohn CS, Williams S. Cryopreserved platelets: The thaw begins … (Article, p. 2794). Transfusion 2019;59:2759-62.
Reddoch-Cardenas KM, Bynum JA, Meledeo MA, Nair PM, Wu X, Darlington DN, et al
. Cold-stored platelets: A product with function optimized for hemorrhage control. Transfus Apher Sci 2019;58:16-22.