|Year : 2014 | Volume
| Issue : 4 | Page : 195-201
Eight-hour versus 24-h whole-blood hold before preparation of platelet concentrates by the platelet-rich plasma method
Azza S El-Danasoury1, Mahira I El-Mogy1, Amal Farouk1, Ghada S Neseem1, Ghada M El-Gohary MD 2
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Adult Hematology/Internal Medicine Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||14-Nov-2014|
|Date of Acceptance||21-Nov-2014|
|Date of Web Publication||25-Mar-2015|
Ghada M El-Gohary
King Faisal Specialist Hospital & Research Center, PO Box 3354, Riyadh 11211, MBC 64
Source of Support: None, Conflict of Interest: None
Background Platelet concentrates (PCs) can be prepared by immediate processing of fresh whole blood (WB); alternatively, they can be prepared from WB stored overnight at room temperature. For blood centers, extension of WB storage time from 8 to 24 h would very useful because of significant operational and logistical benefits. This work was carried out to assess the quality of PCs prepared from WB that had been held overnight at ambient temperature compared with that of PCs prepared from fresh WB donations over a 5-day storage period.
Study design and methods Thirty units of WB were collected; 15 U were kept at room temperature for 6-8 h before platelet preparation (fresh group) and 15 U were kept in a controlled environment of 20-24°C for 22-24 h before platelet preparation (overnight group).
Results PCs prepared after an 8-h or a 24-h hold were comparable in platelet count per PC. CD62P expression and pH values were significantly lower in PCs prepared from 24-h hold donations.
Conclusion The quality of PC may in fact improve because of the ambient overnight hold period as evidenced by lower CD62P expression throughout the storage period, which indicates a lower level of platelet activation. The lower pH values detected in overnight PCs remained within the international acceptable range. Egyptian J Haematol 39:-0 © 2014 The Egyptian Society of Haematology.
Keywords: CD62P, platelet concentrates, transfusion
|How to cite this article:|
El-Danasoury AS, El-Mogy MI, Farouk A, Neseem GS, El-Gohary GM. Eight-hour versus 24-h whole-blood hold before preparation of platelet concentrates by the platelet-rich plasma method. Egypt J Haematol 2014;39:195-201
|How to cite this URL:|
El-Danasoury AS, El-Mogy MI, Farouk A, Neseem GS, El-Gohary GM. Eight-hour versus 24-h whole-blood hold before preparation of platelet concentrates by the platelet-rich plasma method. Egypt J Haematol [serial online] 2014 [cited 2022 Jan 22];39:195-201. Available from: http://www.ehj.eg.net/text.asp?2014/39/4/195/153944
| Introduction|| |
Platelets are an indispensable component for maintenance of vascular integrity, and thrombocytopenia can lead to bleeding symptoms ranging from petechiae and simple bruising to intracranial hemorrhage and death . The use of platelet transfusion has increased steadily to become an essential part of the treatment of cancer, hematological malignancies, marrow failure, and hematopoietic stem cell transplantation . For many years, the approach has been to produce platelet concentrate (PC) on the day of whole blood (WB) collection, with any unprocessed blood refrigerated overnight before processing into packed red blood cells (RBCs) alone . This processing method requires careful planning to ensure that adequate stocks of all component types are maintained .
Without doubt, both the deferral of female donors and the use of fresh PCs have marked effects on platelet availability .
Moreover, the demand for platelet transfusions continues to exceed supply in most countries, complicating the situation. Complementary approaches are needed to meet this demand .
Efforts are being made to lengthen the storage period of WB before component preparation to increase supplies. Longer storage of WB enables flexibility in practice, ensuring safer components for patients .
However, evaluation of the effects of the overnight hold of WB on the prepared PCs and how these effects might contribute toward the platelet storage lesion affecting the quality of the PCs at the end of the storage period is crucial.
The aim of this work is to evaluate the impact of extended hold of WB at room temperature - before processing - on PCs processed using the platelet-rich plasma (PRP) method. PCs were evaluated over a 5-day storage period.
| Materials and methods|| |
WB was collected from healthy donors subjected to a simple assessment of history and clinical examination according to Egyptian standards for blood banks and transfusion services . WB was collected using a triple blood bag system (JMS Singapore PTE LTD, Singapore Thermo Scientific Pierce, Rockford, IL, USA). A total of 450 ml of blood was drawn into the primary bag containing 63 ml of a citrate phosphate dextrose adenine-1 anticoagulant preservative. The collection process was not to exceed 10 min to avoid the possibility of platelet activation. A blood collection monitor was used, which impedes further blood flow once the desired amount (450 ml) has been drawn and agitates blood during collection to ensure adequate mixing with the anticoagulant-preservative mixture in the bag.
Thirty units of WB were collected; 15 U were kept at room temperature for 6-8 h before platelet preparation (fresh group) and 15 U were kept in a controlled environment of 20-24°C for 22-24 h before platelet preparation (overnight group). The controlled environment was an air-conditioned room, with the temperature maintained between 20 and 24°C.
After storage, WB bags were processed into packed RBC units, PCs, and fresh frozen plasma (FFP) according to the PRP method using a cooling centrifuge (Sorvall RC12BP; Thermo Scientific Inc.). The time and speed used for PC preparation were set after centrifuge calibration. The WB bags were turned upside down several times to mix the contents before centrifugation. PRP-PC was prepared using a standard two-centrifugation method: a soft spin, followed by a hard spin. In the soft spin, PRP was separated from WB by light spin centrifugation of WB units at 1800g for 2 min 30 s, with centrifuge brakes off and using the slow stop option with a deceleration curve of 5. PRP was transferred immediately into the empty PC bag using a manual extractor. The separation was not delayed to prevent variable further sedimentation of the cells. Then, in the hard spin, platelets were concentrated from PRP by heavy spin centrifugation at 5100g for 5 min and platelet-poor plasma was expressed into the FFP bag, except for 60 ml, which was retained with the platelet pellet. PC bags were left undisturbed with the label side down for a time period of 60-120 min, and then gently resuspended manually before being placed on a platelet agitator and kept under continuous horizontal agitation for storage. All centrifugation and storage steps were carried out at 20-24°C.
PCs were maintained at 20-24°C under continuous horizontal agitation for 5 days. Two-milliliter samples of well-mixed PC were drawn aseptically at days 1, 3, and 5.
Platelet count was performed using an automated cell counter SYSMEX KX-21N (Kobe, Japan Hanna Instruments, Woonsocket, USA). Platelet count per PC was calculated by multiplying the platelet count per ml by PC volume, which was determined by dividing the net PC weight by the specific density of PC (1.03 g/ml) [9,10]. Platelet count was determined on day 1. Measurement of pH in PC was performed using a pH meter (Hanna Bench pH 211 Meter) immediately after sampling. pH measurement was performed on days 1, 3, and 5.
Platelet activation was measured by determining CD62P expression on days 1, 3, and 5 using a flow cytometer (Epics XL; Beckman Coulter). Platelets were diluted to 50 × 10≤/ml using Isoton II, Beckman Coulter, as a diluent. Using plastic disposable tubes, two test tubes were used for each sample: a control tube and the test tube. Into each tube, 10 ml of the diluted platelets were added, to yield a final platelet count of 500 × 10≤ per tube. To the control tube, 5 ml of the PE-conjugated isotypic antibody control and 5 ml of the FITC-conjugated isotypic antibody control were added (clone 679.1Mc7; Beckman Coulter) (IgG1 isotype). To the test tube, 5 ml of CD62P - PE conjugated antibody (clone CLB-Thromb/6; Beckman Coulter) (IgG1 isotype) and 5 ml of CD61 - FITC of conjugated antibody (clone SZ21; Beckman Coulter) (IgG1 isotype) were added (labeling with anti-CD61 was used for precise identification of platelets). Mixing was performed by gentle drawing of the mixture up and down in the pipette tip. Tubes were incubated in the dark at room temperature for exactly 20 min. Then, 1 ml of PBS was added to each tube and mixed gently just before measurement. For each tube, a total of 10 000 platelet events were acquired on the flow cytometer. The isotypic control tube was run first to detect nonspecific labeling and to set the threshold for positivity. A gate was set on an intact platelet population defined by characteristic forward and side scatter. Out of the gated cells, the cells positive for CD61 and CD62P represent the activated platelets.
Statistical analysis of the data was carried out using the SPSS 16 software package under the Windows Vista operating system (SPSS Inc. Chicago, IL, USA). Continuous data parameters were analyzed for normality using the Shapiro - Wilk test; a parametric distribution of data was assumed and data were presented in the form of mean ± SD and range. A comparative study was carried out using the Student t-test for comparison of two independent samples and a paired t-test for comparison of paired-samples data sets. P value was used to determine 'statistical significance'. A P value less than 0.05 was considered significant and a P value less than 0.01 was considered as highly significant.
Graphical presentation of data was performed using EXCEL 2007 software under the Windows Vista operating system (Microsoft Corporation, Redmond, WA, USA).
| Results|| |
As shown in [Table 1] and [Figure 1], the mean platelet count measured at day 1 in fresh PCs was 6.09 ± 0.77 × 10 10 /PC, whereas in the overnight PCs, it was 5.73 ± 0.49 × 10 10 /PC. Comparison of these values indicated that they were not statistically significantly different (P > 0.05). However, the mean values of CD62P expression in overnight PCs measured at days 1, 3, and 5 were significantly lower than those of fresh PCs at all days (P < 0.05). The mean pH values of overnight PCs measured at days 1, 3, and 5 were significantly lower than those of fresh PCs on all days (P < 0.001). However, the pH in overnight PCs remained within the required range of 6.4-7.4.
|Figure 1: Comparison of the mean CD62P and pH values of fresh and overnight platelet concentrates (PCs). (a) Mean CD62P values in fresh and overnight PCs. (b) Mean pH values in fresh and overnight PCs. (-♦-) PC prepared from whole blood (WB) units held for 8 h; (-■-) PCs prepared from 24-h held WB. *Significant differences (P < 0.05) between the 8-h and 24-h conditions.|
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Comparison of CD62P expression during the storage period in fresh PCs showed a significant increase during storage (P < 0.001). Although in overnight PCs, the increase in CD62P expression from day 1 to day 3 was not statistically significant (P>0.05), it gained significance from day 3 to day 5 (P < 0.05) [Table 2] and [Table 3].
|Table 1: Comparison between fresh platelet concentrates and overnight platelet concentrates in day 1 mean platelet count and mean CD62P and pH values throughout the storage period|
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|Table 2: Paired comparison of CD62P and pH values throughout the storage period in fresh platelet concentrates|
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|Table 3 Paired comparison of CD62P and pH values throughout the storage period in overnight platelet concentrates|
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It is worth noting that, in the present study, all the values of CD62P expression remained below 50% throughout the 5-day storage period for both groups (except for the fifth-day values in 5 U of the fresh group and 2 U of the overnight group). This observation indicates that CD62P expression values below 50% correspond to good in-vivo viability of the transfused platelets .
Comparison of the pH values of each group on different days of storage versus each other showed a significant decrease in the pH during storage (P < 0.05) both in fresh and in overnight PCs [Table 2] and [Table 3].
All the overnight and fresh PCs fulfilled the American Association of Blood Banks (AABB) standard requiring end-of-storage pH to be 6.2 or greater  and the Canadian Standards Association requirement of pH greater than 6.2 .
Throughout storage, the pH of both fresh and overnight PCs was above the UK specifications of pH greater than 6.4  and within the range of 6.4-7.4 recommended by the Council of Europe guidelines  [except for one reading on day 1 in 1 U in the fresh PC group (7.43)].
Moreover, all the PCs had pH above 6.8, a value below which platelets begin to undergo some deleterious effects .
| Discussion|| |
PC preparation and storage are associated with several changes in platelets that result in a reduction in their recovery and survival after transfusion. These changes are known as platelet storage lesions. Concerns were raised that prolonged storage of WB at room temperature before PC preparation might add to the platelet storage lesion, affecting the quality of PCs.
In this study, we assessed the quality of PCs prepared from WB that had been held overnight at ambient temperature compared with that of PCs prepared from fresh WB donations over a 5-day storage period.
In the present study, we chose three in-vitro parameters to evaluate the impact that the extended WB hold had on the PCs prepared and to assess their quality and stability over the storage period, comparing the results against international specifications for the quality of PCs.
The first of these parameters was the platelet count per PC as the primary objective of platelet transfusion is to provide sufficient amount of platelets with adequate in-vivo viability .
In the present study, the mean platelet count measured at day 1 in fresh PCs was 6.09 × 10 10 /PC, ranging from a minimum of 5.2×10 10 /PC to a maximum of 7.8 × 10 10 /PC, whereas in overnight PCs, it was 5.73 × 10 10 /PC, ranging from a minimum of 5.2 × 10 10 /PC to a maximum of 7.3 × 10 10 /PC, the difference being statistically insignificant (P > 0.05), indicating that the overnight storage of WB at room temperature before processing by the PRP method did not affect the platelet count in the PCs prepared, which is consistent with the studies of Thibault et al. , in which the platelet count for fresh PCs was 6.4 ± 1.2 × 10 10 /PC versus 6.2 ± 1.0 × 10 10 /PC for overnight PCs, and Moroff et al. , in which the platelet count for fresh PCs was 6.8 ± 1.9 × 10 10 /PC versus 7.1 ± 2.5 × 10 10 /PC for overnight PCs.
In our study, 93.3% of fresh PCs had at least 5.5 × 10Ή°/PC and 86.6% of overnight PCs had at least 5.5 × 10Ή°/PC. From this perspective, both groups fulfilled the requirement of the Canadian Standards Association (≥75% of units should have ≥5.5×10Ή°/PC) .
Both groups did not fulfill the Council of Europe guidelines (≥75% of units should have ≥6.0 × 10Ή°/PC) ; only 40% of fresh PCs had at least 6.0 × 10Ή°/PC and 6.6% of overnight PCs had at least 6.0 × 10Ή°/PC.
The second parameter chosen was surface expression of CD62P as a marker of platelet activation during storage of PCs ,,. Platelets are activated during the collection procedure, processing, and storage. Their activation is associated with considerable morphological changes. Among these, the degranulation of the granular structures of platelets with subsequent translocation of the formerly intracellularly localized molecules to the surface of platelets is a hallmark of this process. The best example is the P-selectin molecule (CD62P), which is stored in a-granules of resting platelets. The translocation and membrane expression of this molecule occur very early during platelet activation .
The appearance of CD62P on the platelet membrane surface is one of the parameters that indicate a decrease in the quality of platelets during storage .
As for CD62P expression on day 1, the mean values in fresh PCs measured at days 1, 3, and 5 were 37.29 ± 4.74, 40.18 ± 5.58, and 45.87 ± 5.92%, respectively. The corresponding values for overnight PCs were 27.90 ± 11.84, 28.65 ± 10.15, and 35.53 ± 11.03%, respectively. The values of overnight PCs were significantly lower at all days (P < 0.05).
Comparison of CD62P expression over the storage period in fresh PCs showed a significant increase during storage (P < 0.001). Although in overnight PCs, the increase in CD62P expression from day 1 to day 3 was not statistically significant (P > 0.05), it gained significance from day 3 to day 5 and consequently from day 1 to day 5 (P < 0.05).
In agreement with our results, lower levels of platelet activation in overnight PCs throughout the storage period were reported in studies by Thomas et al.  and Dijkstra-Tiekstra et al. , who monitored the prepared PCs for 7 days and measured CD62P expression on days 1, 5, and 7.
It is worth noting that, in the present study, all the values of CD62P expression remained below 50% throughout the 5-day storage period for both groups, except for the fifth-day values in 5 U of the fresh group and 2 U of the overnight group. This observation indicates that CD62P expression values below 50% correspond to good in-vivo viability of the transfused platelets .
To identify the day on which changes become significant for PCs in each group, we compared values at consecutive time points. For fresh PCs, the difference was significant between day 1 and day 3 and between day 3 and day 5, whereas the values of overnight PCs were significantly increased only on comparing day 5 with day 3.
These results indicate that in fresh PCs, CD62P expression increases significantly throughout the entire storage period, whereas in overnight PCs, the increase in CD62P expression becomes significant only after day 3, meaning more stable and less activated platelets in the first 3 days of storage, during which they will mostly be used. The late increase in CD62P expression on day 5 is of minor significance.
The third parameter was pH. The choice of pH as a study measure was made on the basis of the fact that pH has been correlated with in-vivo platelet recovery ,,. Also, bacterial contamination can alter the pH of stored PCs .
The mean pH values in fresh PCs measured at days 1, 3, and 5 were 7.34 ± 0.05, 7.29 ± 0.05, and 7.24 ± 0.07, respectively. The corresponding values in overnight PCs were 7.27 ± 0.04, 7.20 ± 0.05, and 7.14 ± 0.06, respectively. Comparison of these values showed that they were significantly lower for overnight PCs on all days (P < 0.001). However, the pH in overnight PCs remained between the required range of 6.4 and 7.4.
Comparison of the pH values of each group on different days of storage versus each other showed a significant decrease in the pH during storage (P < 0.05) both in fresh and in overnight PCs.
Throughout storage, the pH of both fresh and overnight PCs was above the UK specifications of pH more than 6.4  and within the range of 6.4-7.4 recommended by the Council of Europe guidelines , except for one reading on day 1 in 1 U in the fresh PC group (7.43).
All the overnight and fresh PCs fulfilled the AABB standard requiring end-of-storage pH to be 6.2 or greater  and the Canadian Standards Association requirement of pH more than 6.2 .
Moreover, all the PCs had pH above 6.8, a value below which platelets begin to undergo some deleterious effects .
pH values in overnight PCs, although lower than the corresponding values in fresh PCs, were still acceptable. These results are in agreement with the results reported by other studies by Sandgren et al.  and Dijkstra-Tiekstra et al. .
The fact that overnight room temperature hold results in a lower pH can be attributed to the buildup of lactic acid produced by the metabolism of RBCs, platelets, and white blood cells (WBCs) in the stored WB .
Monitoring of pH values throughout the storage period in each group showed a significant decrease in pH. This finding is rather expected; because of storage at 24°C, the ongoing platelet metabolism can reduce the pH of PCs .
The finding that, in the present study, the three parameters measured were not compromised by overnight storage of WB before processing to PCs further supports the growing interest of blood banks to lengthen the current WB storage period from 8 to 24 h . This potentially has several operational advantages: first, the production of PC after overnight hold of WB markedly increases operational flexibility and logistics are easier when WB can be processed during the daytime, making night shifts unnecessary as (a) all WB blood units are available the following morning, enabling very efficient routine production by avoiding periods of waiting for WB to be supplied from different blood collection sites; (b) the staff for blood component preparation are basically needed only during routine working hours and the workload can be distributed evenly over time, which can indirectly lead to improvements in component quality and safety .
Second, a large percent of the blood supply is collected on mobile blood drives, often distant from blood component production laboratories. The standard processing of components required within 8 h often leaves only the RBCs and 24-h frozen plasma as products from such mobile collections. The 'overnight-hold method' solves the logistic problems associated with collection by distant mobile teams and donations collected in the evenings and enable the preparation of platelets from almost all blood donations .
In addition to the operational advantage, the hold time may allow reversal of some low-level activation of platelets caused by the donation procedure . Moreover, as WBCs in WB can ingest bacteria during the storage period , the risk of bacterial contamination is lower when WBCs are removed after 16-20 h ,. In fact, in a study that was designated to evaluate the effect of the overnight-hold method on the growth of bacteria in experimentally contaminated blood units, overnight PCs showed delayed bacterial growth on the second and fifth days of storage .
| Conclusion|| |
The platelet quality parameters measured in our study indicate that overnight storage of WB at room temperature before preparing PCs by the PRP method did not compromise the quality of the PCs produced.
The preserved quality of overnight PCs, besides the efficient production center operational issues such as working during business hours only and fewer wastage of potential PCs sources such as the WB units from mobile centers or late shifts, and aiding in choosing male only WB for PCs production, might all be important - even encouraging - reasons to shift to overnight hold of WB before the preparation of PCs.
| Acknowledgements|| |
The authors are appreciating the great effort made in reviewing the work which is an original manuscript.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]