An Evaluation of Random Donor Platelets Prepared from Overnight Stored Buffy Coat Method: A Better Alternative to the 6-h Restriction

Abstract

Preparing maximum platelets from all daily blood donations is essential to maintain adequate stock of platelets that may be feasible by storing buffy coat overnight. The overnight stored buffy coat method is prevalent in Europe and was not studied much in India. Therefore, a comparative study was planned to compare RDPs prepared from fresh and overnight stored buffy coat methods. In this study total of eighty RDPs were prepared. Forty RDPs were prepared by each method by matching similar platelet counts in whole blood in pairs and avoiding other bias. Quality analysis was done in various hematological, biochemical, metabolic, and activation parameters and with National guideline. RDPs prepared by the overnight stored buffy coat method had statistically higher mean platelet counts, lesser WBC contamination, lesser glucose concentration, higher lactate concentration, lesser pH, and higher MPV & PDW than RDPs prepared by the fresh buffy coat method. P selectin levels were lesser in the overnight stored category. The platelet yield in RDPs (extraction from the whole blood) was more in the overnight stored category. Compared with the national guideline, 87.5% of units passed the quality criteria in the fresh category compared to 100% of units in the overnight category. RDPs prepared from overnight stored buffy coats not only had better quality parameters but also were in a less activated state. Because of better quality and feasibility in blood centers, the overnight stored buffy coat method may also be used along with the traditional preparation method.

Introduction

In the present era of transfusion medicine, the requirement for blood components, including platelets, is increasing day by day. It is of utmost importance to maintain adequate stock of platelets because of their shortest expiry compared to other blood products. It is prudent to maximize the preparation of platelets from donated blood to maintain adequate Random Donor Platelets (RDPs) stock. Unlike Packed Red Blood Cells (PRBC) and plasma prepared from donated whole blood after initial centrifugation, RDPs are prepared by the buffy coat method after the second centrifugation. This second centrifugation may delay RDP preparation in centers where daily donation is high or in big camps. Other factors include having limited equipment for blood component separation, limited workforce and avoiding separation work in the evening & night hours, and lack of supervision of trained staff. These may lead to a lesser interest in the preparation of RDPs from daily high donations 1.

According to the drug and cosmetic act 1940 of India, blood components are to be processed within 6 h of collection for the preservation of the functionality of coagulation factors. Beyond this period, there are chances of deterioration of labile coagulation factors in the collected blood, and hemolysis may be expected in whole blood units. Platelets show maximum vitality at room temperature, that is, when stored around 22 °C 2. Having a similar temperature requirement for the storage of the platelet component before and after the separation of platelets will raise a general query about the preparation of RDPs from overnight storage at room temperature. The preparation of buffy coat RDPs after overnight hanging (16–20 h) is prevalent in European countries. The overnight hung method of RDP preparation was not much studied in India. Introducing such a technique in India can be an answer to many issues faced by the blood centres like increasing the quality of platelet concentrates prepared, conducting big & far distance camps and proper usage of the available workforce along with overcoming the restriction of preparation of platelets within 6 h of collection. Hence, this study was planned to compare the quality of RDP units prepared by both overnight and fresh buffy coat methods with the hypothesis that the platelet preparation prepared from the overnight stored buffy coat would be of similar or better quality as compared to platelet concentrates prepared from fresh buffy coat.

Materials and Methods

This study was done in a tertiary care institute of North India and the ethical approval of the study was taken. In this study total of eighty RDPs (40 each from fresh as well as overnight stored buffy coat method) were prepared and analyzed. Whole blood was collected in 450 ml quadruple Top and Bottom blood bags. Many factors might influence the quality parameters in both types of RDPs like inherent differences in whole blood volume and platelets count in the same, equipment, manpower and procedure of preparation. The usage of identical, properly calibrated and tested machinery was ensured. All units were prepared by the same manpower. Proper following of Standard Operating Procedures (SOP) and process flow was ensured. For avoiding inherent difference in whole blood volume and platelet count in whole blood unit, one pair was selected that had less than 5% difference in whole blood volume and donors’ platelet count/mm³ by the following formulas.

$$\frac{\text{Volume of whole blood collected in method}1-\text{Volume of whole blood collected in method}2}{\text{Volume of whole blood collected in method}1}\times 100$$

$$\frac{\text{Platelet count}/{\text{mm}}^3\text{of the donors in method}1-\text{Platelet count}/{\text{mm}}^3\text{of the donors in method}2}{\text{Platelet count}/{\text{mm}}^3\text{of the donors in method}1}\times 100$$

One each in this whole blood pair was randomly processed for preparation of RDPs by fresh as well as overnight stored buffy coat method and paired analysis was done.

In the fresh buffy coat method, RDPs were prepared within 6 h of the collection while in the overnight stored method, the first separation was done within 6 h of the collection while the second separation was done after storing the buffy coat units in room temperature overnight and separating platelets from buffy coat next morning. Quality analysis of the units were done within 12–24 h of preparation of RDPs i.e. on day 1 of storage. Testing was done only once during storage. Samples from the RDPs were taken as per the standard procedure of quality control like proper mixing of the unit, proper stripping of segments and analysis of the samples using the same analyzers for various hematological, biochemical, metabolic, activation parameters like; Volume, Platelets Count/Unit, White Blood Cells (WBC) Contamination, RBC Contamination, Platelet Indices, Swirling, pH, PO₂, PCO₂, Lactate Concentration, Glucose Concentration, Bicarbonate Concentration and p Selectin. Swirling was assessed for the functional status of the platelets whereas the soluble p Selectin testing was done to study the platelet activation. Quality Analysis was done in all 80 units while soluble p selectin was done in 28 randomly selected units in both hands assuming 95% power and 95% confidence interval. The comparison was also done with the quality criteria given by Indian regulatory authorities 3. The sterility testing was not done because RDPs of fresh buffy coat methods were issued to the patients. On the other hand, RDPs prepared from overnight stored buffy coat method were not issued to the patients as, without proper approval from DCGI, the same cannot be used for transfusion to the patients. The distribution of data in normal and non-normal was calculated using paired t-test and Wilcoxon Mann Whitney test respectively. P value less than 0.05 was considered statistically significant. The mean percentage yield in the RDPs in the form of extraction from whole blood unit was also calculated by the following formula 4.

$$\frac{\text{Percentage of platelet yield in RDP}=\text{Number of platelets in RDP unit}}{\text{Number of platelets in whole blood}}\times 100$$

Results

The mean percentage difference with the range of the units of overnight from fresh buffy coat for volume of whole blood and donors’ platelet count/µl was + 0.09% (− 4.31% to + 4.34%) and + 0.24% (− 3.63–3.22%) respectively. The statistical difference in both the groups in both the parameters was non-significant. The comparison of various quality parameters of both groups is shown in Table 1. There was a significant difference between the two groups in terms of soluble P selectin concentration (ng/ml) (W = 658.500, p ≤ 0.001), with the mean soluble P selectin concentration (ng/ml) being more in the fresh RDP units (median value with IQR 52.3 (43.14–56.45) as compared to overnight stored RDP units [median value with IQR 39.13 (31.78–46.51)]. The strength of association (Point-Biserial Correlation) was 0.47 (Large effect size). All units in both methods showed grade 3 swirling. The platelet yield obtained in the overnight method (mean 72.95% range 52.02–97.88%) was statistically higher as compared to the fresh buffy coat method (mean 57.49% range 42.59–85.30%). On comparing quality criteria with the National guideline of year 2022, 87.5% of RDPs of fresh buffy coat method as compared to 100% of the units of RDPs of overnight stored buffy coat method passed the quality criteria of > 6 × 10¹⁰ platelets count/unit. All units passed the criteria of pH > 6.

Table 1.

Comparison of quality parameters of RDPs prepared by two methods

Parameters	Fresh (n = 40)	Overnight (n = 40)	p value
Volume of platelet units (ml)	81.67 ± 4.46	82.75 ± 4.45	0.284
	70–89	71–90
Unit platelet concentration (× 106/ml)	807.18 ± 56.99	1010.12 ± 66.58	< 0.001
	728–898	913–1121
Platelet count/unit  (× 1010)	6.59 ± 0.55	8.35 ± 0.66	< 0.001
	5.64–7.86	7.17–9.88
WBC count/unit  (× 107)	3.86 ± 0.63	3.44 ± 0.96	0.024
	2.5–5.1	1.3–5.9
pH	7.13 ± 0.11	7.05 ± 0.12	0.002
	6.86–7.27	6.8–7.19
RBC contamination (ml)	0.188 ± 0.08	0.190 ± 0.08	0.945
	0.05–0.3	0.1–0.3
pO2 (mmHg)	118.50 ± 12.40	118.80 ± 11.87	0.912
	100–149	101–144
pCO2 (mmHg)	52.12 ± 12.35	52.08 ± 12.16	0.977
	35–77	36–79
Glucose (mmol/L)	458.70 ± 37.03	437.02 ± 38.47	0.004
	355–498	355–488
Lactate (mmol/L)	5.34 ± 0.38	5.57 ± 0.41	0.002
	4.85–6.25	5.01–6.48
Mean platelet volume (MPV) (fL)	9.29 ± 0.47	9.69 ± 0.47	< 0.001
	8.5–10.3	8.9–10.7
Platelets distribution width (PDW) (fL)	12.90 ± 1.21	13.44 ± 1.18	0.049
	35–77	36–79

The table shows the mean ± standard deviation and the range

Discussion

Different preparations of platelet concentrates have been used in different parts of the world. Initially, it was the PRP method of platelet extraction from whole blood collection which was widely used. Later Buffy coat method was introduced in many places of Europe. In the current study, the overnight stored RDPs had higher platelet counts than freshly prepared ones. Similar results were obtained in other studies 1, 5, 6. The platelets prepared from the over-night stored buffy coat method have enough time to settle down and thus have a better platelet count. So, there are fewer possibilities that the platelets would get trapped among WBCs and fail to come into RDP during soft centrifugation in the fresh buffy coat method. On the other hand, more platelets would be released into the plasma during the second centrifugation in the overnight stored method of preparation. Also, a neat interface would have been formed between the platelet layer and the white blood cells in the overnight stored buffy coat method, hence it would have been easier for the optical sensor in the automated component separator to separate the platelets 7. In the study done by Boeri et al. also the platelet yield was increased on holding the buffy coat for 12 h in comparison to the 3-hour initial hold period 8.

Mean WBCs contamination in RDPs prepared by fresh buffy coat method was statistically higher than RDPs prepared by overnight stored buffy coat method (p = 0.024). There may be some factors that can contribute to this fact like more settling down of the WBCs from the buffy coat or plasma into the RBC layer in overnight stored method or lysis of WBCs in overnight stored buffy coat as these buffy coats are stored at room temperature (20–24 °C). 9 This result was similar to the studies conducted by Tiwari et al. 1 and Philip et al. 5 Ojha et al. showed opposite findings in which units prepared by overnight stored method had a higher count as compared to the fresh buffy coat method 6. More resting time would have caused the WBCs also to get released to the plasma layer according to Ojha et al. The Mean Platelet Volume (MPV) of RDPs prepared from overnight stored buffy coat was higher (p = < 0.001) when compared to the freshly prepared ones. The overnight stored buffy coat was having a higher hold period. With storage, the changes in the shape of the platelets from discoid to spherical cells occur and this would have led to a higher MPV 10. The mean Platelet Distribution Width (PDW) of the RDPs prepared from overnight stored buffy coat was higher as compared to the fresh buffy coat method (p = 0.049). The results were similar to the study done by Singh et al. 10.

The glucose concentration of freshly prepared units was statistically higher (p = 0.004) as compared to the units prepared from overnight stored buffy coat. This result was similar to the studies conducted by Tiwari et al. 1, Philip et al. 5 and Ojha et al. 6. In the current study, the lactate concentration was statistically higher (p = 0.002) in the RDPs prepared from the overnight stored buffy coat. This finding was in accordance with the findings in the study by Tiwari et al. 1 and Ojha et al.  6. The fall in glucose and rise in lactate concentration may be attributed to the fact that during anaerobic glycolysis glucose is consumed for a generation of ATP and lactate is produced. Glucose metabolism generates 15% of ATP. In glycolysis, lactate and free hydrogen ion are formed. This can in turn make the platelet concentrate more acidic too 11. A statistically significant difference (p = 0.002) in the pH of RDPs prepared by both methods was seen. The RDPs prepared from overnight stored buffy coat (7.05 ± 0.12) were more acidic pH compared to the freshly prepared RDP units (7.13 ± 0.11). These differences in the pH were comparable to the studies done by Tiwari et al. 1, Philip et al. 5, and Ojha et al. 6. In all these studies the RDPs from overnight stored buffy coat were having more acidic pH. The reason for a higher acidic pH can be mainly due to the larger holding time of the platelet concentrates in the case of overnight stored ones which would have resulted in more lactate production due to anaerobic glycolysis 11. In each case, the units were able to maintain pH. The lower glucose, higher lactate concentration and lower pH may be attributed to the higher platelet count in the overnight stored buffy coat method in addition to the overnight holding.

In the current study, no significant differences were noticed in pO₂ concentrations between the RDPs prepared from overnight stored buffy coat and the fresh ones (p = 0.912). In the study by Philip et al. similarly, no significant differences were seen between both the groups 5. In the study by Tiwary et al. a significant difference was noted in the pO₂ values between the two groups on day 1 but on day 5 no significant differences were noticed 1. No significant differences were noticed in pCO₂ concentrations between RDPs prepared from overnight stored buffy coats and fresh ones in the current study (p = 0.977). In the study by Philip et al. similarly, no significant differences were seen between both the groups 5.

In the current study, it was found that the platelet concentrates prepared from overnight stored buffy coat method were in less activated than freshly prepared ones. Soluble P selectin concentration present in the storage plasma was assessed for the same and it was statistically more in RDPs prepared from the fresh buffy coat method (p < 0.001). The longer holding period for overnight ones would have given enough time for the platelets to become less activated. Whereas in fresh ones since the resting period between two centrifugations is less the final product would be more activated. In a study conducted by Philip et al. similar results were found upon studying the CD62P expression percentage by flow cytometry method 5. A study done by Tiwary et al. also showed similar results 1. Pujol et al. in their similar study found that the buffy coat when stored for 4 h showed more activation in comparison to the ones stored for 18 h with no detrimental effects on the quality parameters till the shelf life of 5 days 12.

Passing the criteria for a new product is essential for licensure and regulatory point of view. Comparing the quality criteria by latest national guidelines of the year 2022, RDPs prepared by overnight stored buffy coat method passed all the quality criteria and better maintained these quality criteria as compared to fresh buffy coat method. RDPs prepared from the overnight stored buffy coat method had a platelet yield (in terms of extraction from whole blood) of 72.95% whereas the fresh preparation method had only 57.49%. In the preparation of RDPs, platelet loss may occur. This loss may be minimized by storing buffy coat units overnight as shown by the results of the platelet yield of the RDPs. Getting good yield in RDPs from donated whole blood is one of the important major quality assurance steps for making a high-quality product.

This study had some limitations like, in vivo effects of the transfusions were not analysed. RDPs prepared from the overnight stored buffy coat method were not transfused to the patients due to regulatory issues, however, RDPs of the fresh buffy coat method were issued to the patients for avoiding any wastage. All tests were performed on Day 1, and therefore these results are preliminary, based on the present pilot testing and further studies are needed to observe the parameters over the standard shelf life as per the regulations of the land. Another limitation was that no bacterial culture testing was done because RDPs of fresh buffy coat methods were issued to the patients. Whereas the strength of the study was that it was able to analyze both types of platelet concentrates in pairs and rigorous efforts were done to avoid all confounding factors that can affect the quality of RDPs. As a result, the duration of storage of the buffy coat was the only factor responsible for quality difference. Another strength of the study was it was able to bring out statistically significant benefits of the overnight stored buffy coat method. The invitro analysis findings and methodology of analysis in pairs can be utilized for further study for invivo analysis of overnight buffy coat stored RDPs as a clinical trial after DCGI approvals. After proving invivo effectiveness of RDPs of the overnight stored method as a superiority trial or non-inferiority trial over the fresh method, the RDP preparation method may be changed. Before such trials invitro comparison is essential and this study provided significant insight regarding the same.

In the current study, it was found that the preparation of RDPs from both the methods was feasible. Considering the preparation of overnight stored RDPs regularly in blood centers, especially on camp days provide more ease of preparation and better utilization of manpower. By implementing this method of component separation, RDPs preparations may be avoided in non-routine hours and hence stricter following of SOP can be ensured. The authors recommend to implement both methods of preparation in the blood center. In the current study, all parameters were better in overnight method except glucose and lactate concentration. In this condition, overnight RDPs can be used for transfusion early as compared to fresh RDPs in the total RDPs stock. The benefits and drawbacks of RDPs prepared by overnight stored buffy coat method is described in Fig. 1.

Fig. 1.

The benefits and drawbacks of RDPs prepared by overnight stored buffy coat method

Conclusion

Within the context of the present pilot testing, the observation of the current study implies that the platelet concentrates prepared from the overnight stored buffy coats not only had better hematological quality parameters but also were in a less activated state. All the quality control parameters of the platelet concentrates were met by the overnight buffy coat stored RDPs. The platelet count per unit was comparatively higher and the WBC count per unit was comparatively lesser in overnight buffy coat stored RDPs. The pH of overnight buffy coat stored RDPs was comparatively more acidic with significantly higher lactate concentration and lower glucose concentration but was found fulfilling the quality criteria.