Quality assessment of the preparation and storage of leukocyte-depleted pooled platelet concentrates

ABSTRACT

Objective:

To explore the feasibility of using a disposable platelet storage bag containing a leukocyte filter to prepare leukocyte-depleted pooled platelet concentrates with the buffy coat method.

Methods:

150 bags of whole blood samples (400 mL/bag) were stored overnight at 22 ± 2°C, and buffy coats were separated on Day 2, then 5 units of ABO homotypic buffy coat and 1 unit of plasma were pooled into a disposable platelet storage bag containing a leukocyte filter to prepare leukocyte-depleted pooled platelet concentrates and stored in a Platelet Agitator. On Day 2, 4, 5 and 7 after the collection of whole blood, platelet content, pH value, pO₂, pCO₂, glucose (GLU), ATP, and other quality indicators were measured.

Results:

The quality indicators of leukocyte-depleted pooled platelet concentrates met the requirements for leukocyte-depleted aphaeresis platelets in the Chinese national standard Quality Requirements for Whole Blood and Blood Components (GB18469-2012). With the prolongation of storage time, MPV and PDW of platelets gradually increased, pH value, bicarbonate, and GLU gradually decreased, LA, LDH, and ATP gradually increased, pO₂ slightly increased, pCO₂ decreased, and HSR had no significant change. ESC decreased significantly on Day 7, CD62p decreased first and then increased, sP-selectin and GP V increased first and then decreased, but the results on Day 7 were higher than those on Day 2.

Conclusion:

The quality of leukocyte-depleted pooled platelet concentrates prepared by the buffy coat method using disposable platelet storage bags containing a leukocyte filter was comparable to that of leukocyte-depleted apheresis platelets, and could be used clinically.

KEYWORDS:

• Buffy coat method
• leukocyte-depleted pooled platelet concentrates
• platelet quality
• leukocyte filtration
• apheresis platelets
• quality indicators
• in vitro storage quality
• platelet storage bag

Introduction

Platelets play an important role in hemostasis and are the main blood preparations used in the treatment of thrombocytopenia caused by various reasons [1]. The platelet preparations currently used around the world are mainly platelet concentrates prepared from whole blood or apheresis platelets. In accordance with the Research Report on the Current Situation of Blood Component Preparation in China released in 2019, only approximately 25.9% of blood collection and supply institutions in China provide concentrated platelets separated from whole blood [2]. This may be related to the advantages of apheresis platelets such as low risk of infectious diseases, low incidence of transfusion reactions, and low rate of bacterial contamination [3]. In addition, it is also related to the lack of optimization of functional study and preparation methods of platelet concentrates, high scrap rate of expired products, and insufficient amount of homotypic blood to prepare adult therapeutic platelets [4,5].

With the improvement of preparation technologies such as blood separators, platelet storage bags, and platelet storage solutions, platelet concentrates derived from whole blood account for the majority of clinical usage in European countries such as Denmark, Spain, and the Netherlands [6,7]. Studies have shown similar in vitro quality during storage of platelet concentrates and apheresis platelets [8], with no difference in corrected count increment (CCI) between 1 and 24 h after transfusion [3]. Recent studies have shown that for patients who fail to respond to transfusions, platelet concentrates pooled from random donors have a slight advantage over single-donor platelets [9]. Moreover, platelet concentrates separated and prepared from whole blood have the advantages of low source cost and saving blood resources [7]. Through leukocyte filtration, multi-sample pooling, and storage in platelet storage bags, platelet concentrates could achieve a quality similar to that of apheresis platelets, which would be an important supplement in case of shortage of apheresis platelets.

There are two preparation methods for platelet concentrates, the platelet rich plasma method (PRP method) and the buffy coat method (BC method). The BC method is widely used around the world due to its high platelet recovery rate, low residual leukocytes, and good platelet activation function [10–14]. The BC method is relatively popular in China, but there is a lack of systematic data on the quality of platelet concentrates. Therefore, this study would use a disposable platelet storage bag containing a leukocyte filter to pool and store platelet concentrates, and monitor the quality changes to explore the possibility of clinical use.

Materials and methods

Preparation of leukocyte-depleted platelet concentrates

150 bags of whole blood (400 ml/bag) from random donors were collected between February to June 2019. All blood donors met the national Whole Blood and Component Donor Selection Requirements. The blood was obtained with their informed consent and approval from the hospital ethics committee.

400 ml of whole blood was collected, and placed at 22 ± 2°C overnight, however separated within 24 h. On Day 2, the whole blood was centrifuged at 4000 g for 12 min at 22 ± 2°C, and approximately 45–50 mL of buffy coat was separated and allowed to stand at 22 ± 2°C for 2 h. 5 units of buffy coat and 1 unit of plasma were aseptically connected to the pooling bags on the same set of disposable platelet storage bags with leucodepletion filters (Jiaxing Tianhe Pharmaceutical Co. Ltd.) respectively to obtain buffy coat mixture, and centrifuged at 500 g for 9 min at 22 ± 2°C. On an automatic blood separator (Fresenius Kabi, Germany), the upper platelet mixture entered the platelet storage bag through a leucodepletion filter to prepare leukocyte-depleted pooled platelet concentrates, and stored in a Platelet Agitator (HELMER, USA)

Samples

For quality control indicators such as mixed amount of red blood cells and residual leukocytes, samples were retained after the preparation of leukocyte-depleted platelet concentrates (i.e. Day 2 after the collection of whole blood); for the sterility test, samples were retained on Day 5 after the collection of whole blood; for quality control indicators such as platelet content and pH value, as well as quality monitoring indicators for in vitro storage of platelets such as metabolic indicators, activation indicators and in vitro function evaluation indicators, samples were retained on Day 2, 4, 5 and 7 after the collection of whole blood.

Measurement of platelet content, mixed amount of red blood cells, and residual leukocytes

After preparation, leukocyte-depleted platelet concentrates were weighed with a blood collection scale (CZK-IICT, China), and the capacity was calculated based on the specific gravity of 1.026. On this basis, the platelet concentration was measured with a blood cell analyzer (Sysmex, Japan), and the platelet content in each bag was calculated. The red blood cell concentration was measured with a NAGEOTTE counting plate, and the mixed amount of red blood cells was calculated. The leukocyte concentration was measured with a residual leukocyte counter (NanoEnTek-inc, South Korea), and residual leukocytes were calculated.

Sterility test

After the preparation of leukocyte-depleted pooled platelet concentrates, 20 mL of samples were taken. 10 mL was aseptically inoculated into aerobic and anaerobic culture bottles respectively and incubated in the culture wells of the bacterial culture instrument (Merieux, France) at 37°C for 7 days to obtain the results of sterility test. All instrument operations were performed in accordance with the manufacturer’s instructions.

Measurement of platelet volume

Mean platelet volume (MPV) and platelet distribution width (PDW) were measured with a hematology analyzer (BC3000 plus, Mindray, China).

Metabolic indicators

Platelet lactate (LA), Bicarbonate, pH, partial pressure of oxygen (pO₂), and partial pressure of carbon dioxide (pCO₂) were measured with a blood-gas analyzer (Radiometer, Denmark); platelet glucose (GLU) and lactate dehydrogenase (LDH) were measured with a fully automatic biochemical analyzer (Beckmancoulter, USA); the supernatant of the platelet lysate was taken and the ATP content was measured with ATP Assay Kit (beyotime, China). All operations were performed according to the instructions.

Activation indicators

The positive rate of CD62p was measured by flow cytometry. The platelet concentration was adjusted to 200 × 10⁹/L, and 5 μL was taken and added into 2 special tubes for flow cytometry. 5 μL of mouse IgG1-PE (BD, USA) and 5 μL of Mouse Anti-Human CD61-FITC (BD, USA) were added to the first tube as the control group, and 5 μL of Mouse Anti-Human CD61-FITC (BD, USA) and 5 μL of Mouse Anti-Human CD62P-PE (BD, USA) were added to the second tube as the test group. 50 μL of phosphate-buffered saline (PBS, BIOISCO, China) was added to each tube, mixed gently and incubated for 30 min at room temperature in the dark, fixed with 400 μL of PBS solution (pH 7.38) containing 1% paraformaldehyde, and then tested by flow cytometry (FACS-Verse, BD, USA). Compensatory regulation was performed by simple staining with anti-CD61-FITC antibody and anti-CD62P-PE antibody; FSC/SSC was set as the logarithmic coordinate axis, the cell aggregation point was selected as the P₁ gate, and CD61 positive within the P₁ gate was selected as the P₂ gate, and then the expression levels of CD62P in the P₂ gate were compared between the control group and the test group, respectively.

Soluble P-selectin (sP-selectin) and glycoprotein V (GPV) were measured using double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). 1 mL of platelets (10⁶) was centrifuged at 800–1500 g for 20 min, and the supernatant was taken. In the microwells pre-coated with capture antibodies, the supernatant or standard of platelet concentrates was added, and then the HRP-conjugated detection antibody was added to form an antibody-antigen-enzyme-antibody complex. After incubation and washing, the substrate TMB was added for color development, the absorbance value was measured with a microplate reader (iMark^TM, BIO-RAD, USA) at a wavelength of 450 nm, and the concentration of sP-selectin or GPⅤ was calculated through the standard curve. It was performed according to the operating instructions of ELISA kit for human soluble P-selectin and ELISA kit for human platelet GP Ⅴ (Shanghai Jianglai, China).

In vitro functional evaluation indicators

The extent of shape change (ESC) of platelets was measured with a spectrophotometer (SHIMADZU, Japan). The mixed platelet concentrates were centrifuged at 3000 g for 10 min, the supernatant was taken to obtain platelet poor plasma (PPP), and the pooled platelet concentrates were diluted to 300 × 10⁹/L using PPP to obtain platelet rich plasma (PRP). With 1 mL of PPP as blank, 1 mL of PRP was taken and measured at 610 nm. Absorbance was measured every 10 s for a total duration of 5 min. 10 μL of 0.1 mol/L EDTA (Sinopharm, China) was added and mixed 1 min after the start of the measurement, and 10 μL of 1.0 mmol/L ADP (Solarbio® life science, China) was added and mixed at an interval of 5–10 s. After the measurement, the OD_{0 min}, OD_min and OD_{5 min} values on the curve were read. $\mathrm{E}\mathrm{S}\mathrm{C}\left(\text{\%}\right)={\displaystyle \frac{\mathrm{O}{\mathrm{D}}_{5\mathrm{m}\mathrm{i}\mathrm{n}}-\mathrm{O}{\mathrm{D}}_{\mathrm{m}\mathrm{i}\mathrm{n}}}{\mathrm{O}{\mathrm{D}}_{0\mathrm{m}\mathrm{i}\mathrm{n}}}\times 100\text{\%}}$The hypotonic shock response (HSR) of platelets was measured with a spectrophotometer. Under the condition of a wavelength of 610 nm, PPP and PBS were mixed in a 2:1 ratio to test the zero point; PRP and PBS were mixed in a ratio of 2:1 to measure the change curve of OD value within 15 min as the baseline; PRP and purified water were mixed in a ratio of 2:1 to measure the change curve of OD value within 15 min as the test sample curve. The OD value at 15 min (OD₁), the minimum OD value (OD₂) of the sample, and the baseline OD value (OD₀) at 15 min in the sample curve were read. The results were calculated according to the formula: $\mathrm{H}\mathrm{S}\mathrm{R}={\displaystyle \frac{\mathrm{O}{\mathrm{D}}_1-\mathrm{O}{\mathrm{D}}_2}{\mathrm{O}{\mathrm{D}}_0-\mathrm{O}{\mathrm{D}}_2}\times 100\text{\%}}$

Statistical analysis

Data were analyzed using IBM-SPSS version 25.0 (IBM, Chicago, IL, USA) statistical software. The Schapiro-Wilk (S-W) test method was used to perform normal distribution test and Levene’s test of homogeneity of variance on the quality index data of leukocyte-depleted pooled platelet concentrates stored for different times. For normal distribution data, the multiple comparison analysis of variance was used, and the Bonferroni method was used for pairwise comparison; for skewed distribution data, the multiple independent rank sum test (Kruskal Wallis H test) was used. P < 0.05 indicated that the difference was statistically significant.

Results

Results of quality control indicators for leukocyte-depleted pooled platelet concentrates

As seen in Table 1, after the preparation of leukocyte-depleted pooled platelet concentrates (2 days after collection), the platelet content was 2.69–4.1 × 10¹¹, the mixed amount of red blood cells was 1.3–7.3 × 10⁹, the residual leukocytes were 0.5–3.9 × 10⁶, the pH value at the end of storage period (Day 5) was 6.9–7.2, and there was no bacterial growth, which met the requirements for leukocyte-depleted aphaeresis platelets in the Chinese national standard: Quality Requirements for Whole Blood and Blood Components (GB18469-2012), as shown in Table 1.

Table 1. Results of quality control indicators for leukocyte-depleted pooled platelet concentrates.

Test No.	Platelet content in whole blood (×1011)	Platelet content (×1011)	Platelet yield (%)	Mixed amount of red blood cells (×109)	Residual leukocytes (×106)	pH value (storage for 5 days)	Sterility test
1	3.83	2.71	71	6.2	3.8	7	No bacterial growth
2	4.30	2.91	68	5.1	3.1	7	No bacterial growth
3	4.12	2.83	69	6.6	2.6	6.9	No bacterial growth
4	3.90	2.89	74	7.3	3.9	6.9	No bacterial growth
5	4.26	3.36	79	2.6	1.2	7.1	No bacterial growth
6	3.65	2.72	75	3.7	1.6	7.1	No bacterial growth
7	4.42	3.49	79	4.1	3.2	7.1	No bacterial growth
8	4.48	3.13	70	1.3	0.8	7	No bacterial growth
9	3.70	2.69	73	1.9	1.4	7.1	No bacterial growth
10	5.23	3.81	73	3.2	1.2	7.2	No bacterial growth
11	5.57	4.10	74	3.5	1.8	7.1	No bacterial growth
12	4.66	3.21	69	1.8	1.3	7.1	No bacterial growth
13	4.55	3.59	79	3.8	0.6	7.2	No bacterial growth
14	4.63	3.38	73	4.2	0.8	7	No bacterial growth
15	4.26	2.90	68	3.9	1.6	7.1	No bacterial growth
16	4.69	3.03	65	4.0	2.2	7.2	No bacterial growth
17	4.12	2.99	72	3.9	1.2	7	No bacterial growth
18	3.75	2.76	74	4.3	2.4	7	No bacterial growth
19	4.63	3.24	70	1.8	0.8	7	No bacterial growth
20	4.35	3.28	75	7.1	1.2	7.1	No bacterial growth
21	4.96	3.60	73	1.8	0.7	7	No bacterial growth
22	4.18	3.00	72	2.3	1.3	7.1	No bacterial growth
23	4.56	3.57	78	1.6	2.1	7	No bacterial growth
24	3.76	2.81	75	1.9	1.2	6.9	No bacterial growth
25	4.10	3.37	82	2.2	2.4	7	No bacterial growth
26	4.45	3.29	74	2.6	0.5	6.9	No bacterial growth
27	4.97	3.52	71	2.3	1.3	7	No bacterial growth
28	4.46	3.09	69	2.2	1.4	7	No bacterial growth
29	3.82	2.92	77	3.3	0.6	7.1	No bacterial growth
30	4.13	2.97	72	1.9	0.9	7	No bacterial growth
S	/	≥2.5	/	≤8	≤5	6.4–7.4	No bacterial growth

S: National standard;/: No data.

The results of platelet volume

With the prolongation of storage period, the platelet content gradually decreased, and became statistically significant on Day 7. The MPV gradually increased and and became statistically significant on Day 7. PDW began to show statistically significant changes on Day 5, as shown in Figure 1.

Figure 1. The volume of leukocyte-depleted pooled platelet concentrates. A: The platelet content at each time point showed a skewed distribution, and the multiple independent rank sum test (Kruskal Wallis H test) was used. The results showed that the difference in platelet content at different storage times was statistically significant (H = 9.768, P = 0.021). B: The MPV at each time point showed a normal distribution and homogeneity of variance, the multiple comparison analysis of variance was used, and the Bonferroni method was used for pairwise comparison. The results showed that the difference in MPV at different storage times was statistically significant (F = 18.157, P < 0.001). C: The PDW at each time point showed a skewed distribution, and the multiple independent rank sum test (Kruskal Wallis H test) was used. The results showed that the difference in PDW at different storage times was statistically significant (H = 17.248, P = 0.001).

The results of metabolic indicators

In addition to platelet pO₂, significant changes were observed in platelet pH, pCO₂, LA, Bicarbonate, GLU, LDH, and ATP with the prolongation of storage time, as shown in Figure 2. Among them, the pH, pCO₂, Bicarbonate, and GLU of platelets gradually decreased (Figure 2A, C, E, F). pCO₂ and Bicarbonate became statistically significant on Day 2, GLU on Day 4, and pH value on Day 5. However, LA, LDH and ATP of platelets increased with the prolongation of storage time (Figure 2D, G, H), and LA of platelets became statistically significant on Day 4, LDH on Day 5, and ATP on Day 7.

Figure 2. The metabolic indicators of leukocyte-depleted pooled platelet concentrates. The test results of pH, pO2, pCO2, LA, Bicarbonate, GLU, LDH, and ATP of platelets at different storage time points showed a skewed distribution, and the multiple independent rank sum test (Kruskal Wallis H test) was used. A: The difference in pH value at different storage times was statistically significant (H = 17.248, P = 0.001). B: There was no statistically significant difference in pO2 of platelets at different storage times (H = 4.976, P = 0.174). C: The difference in pCO2 of platelets at different storage times was statistically significant (H = 105.885, P < 0.001). D: The difference in LA of platelets at different storage times was statistically significant (H = 75.418, P < 0.001). E: The difference in Bicarbonate of platelets at different storage times was statistically significant (H = 98.862, P < 0.001). F: The difference in GLU of platelets at different storage times was statistically significant (H = 78.895, P < 0.001). G: The difference in LDH of platelets at different storage times was statistically significant (H = 58.070, P < 0.001). H: The difference in ATP of platelets at different storage times was statistically significant (H = 29.568, P < 0.001).

The results of activation indicators

With the prolongation of storage time, CD62p, sP-sel, and GPV of platelets showed different changes, as shown in Figure 3. The change of CD62p showed a trend of first decreasing and then increasing (Figure 3A), with a statistically significant increase on Day 7 compared to Day 2. The sP-sel and GPV showed a synchronous change of first increasing and then decreasing (Figure 3B, C), both reaching their peak on Day 5, with a statistically significant change compared Day 7 to Day 2.

Figure 3. The activation indicators of leukocyte-depleted pooled platelet concentrates. The test results of CD62p, sP-sel, and GPV of platelets at different storage times showed a skewed distribution, so the multiple independent rank sum test (Kruskal Wallis H test) was used. A: The difference in CD62p of platelets at different storage times was statistically significant (H = 44.354, P < 0.001). B: The difference in sP-sel of platelets at different storage times was statistically significant (H = 18.474, P < 0.001). C: The difference in GPV of platelets at different storage times was statistically significant (H = 20.964, P < 0.001).

The results of in vitro functional evaluation indicators

With the prolongation of storage period, there was no statistically significant change in HSR value of platelets, but the extent of shape of platelets change showed a statistically significant change on Day 7 of storage, as shown in Figure 4B.

Figure 4. The in vitro functional evaluation indicators of leukocyte-depleted pooled platelet concentrates. A: The extent of shape change (ESC) of platelets at different storage times showed a skewed distribution, and the rank sum test was used. The difference between the groups was statistically significant (H = 12.729, P = 0.005). B: The hypotonic shock response (HSR) of platelets at different storage times showed a skewed distribution, and the rank sum test was used. There was no statistically significant difference between the groups (H = 6.931, P = 0.074).

Discussion

In this study, 150 bags of whole blood were stored at overnight 22 ± 2°C, and the buffy coat was separated by centrifugation within 24 h and pooled according to ABO homotype, and then 30 bags of leukocyte-reduced platelet concentrates were formed through a leukocyte filter. Due to the current lack of relevant standards for leukocyte-depleted pooled platelet concentrates in China, we compared them with the quality control requirements for leukocyte-depleted apheresis platelets in the Chinese national standard Quality Requirements for Whole Blood and Blood Components (GB18469-2012). The results showed that the quality of all products met the requirements (Table 1), indicating that the quality was equivalent to that of leukocyte-depleted aphaeresis platelets, and was suitable for clinical use.

The changes in platelet lifespan shortening and hypofunction can be roughly divided into three categories: platelet activation, metabolic changes, and platelet aging [15,16]. The monitoring results of platelet morphology, metabolism, activation, function and other indicators can objectively reflect the storage damage of platelets and comprehensively evaluate the quality of in vitro storage of platelets. In this study, with the prolongation of storage time, the volume of leukocyte-depleted pooled platelet concentrates increased (Figure 2A, D, F), pH value and GLU gradually decreased, and LA production significantly increased, which was basically consistent with the results reported by Zeng et al. [10]. pCO₂ decreased, which was basically consistent with the results reported by Levin et al. [14]. ATP did not decrease as reported by other researchers [10], but increased. We examined four days of data from all 30 samples and found that not all platelet ATP increased, but some samples significantly increased the overall trend. However, the reason is not clear at present, and the detection method and sample processing need to be further verified. The HSR did not change significantly during the storage period, which was consistent with the results reported by Zeng, and Levin et al., but the HSR results in this study were slightly lower than the normal range of 50–90%, and were generally lower than the results reported by Zeng et al. [10,14]. In terms of ESC results, Zeng et al. reported that the leukocyte-reduced platelet concentrates prepared by the PRP method decreased significantly on Day 5, while in this study, the significant decrease did not occur until Day 7 (Figure 4A), which was basically consistent with Levin et al.’s report that there was no significant change on Day 1–6 [10,14], indicating that the platelet concentrates prepared by the BC method had certain advantages in retaining the extent of shape change. CD62p, also known as P-selectin, granular membrane protein 140 (GMP-140), and platelet activation-dependent granule-external membrane protein (PADGEM), is a specific marker for activated platelets. After platelet activation, the particles in platelets fuse with platelet membrane proteins, causing the granule membrane proteins to flip to the surface of the platelet membrane, significantly increasing the expression level of CD62p on the platelet plasma membrane, and releasing soluble proteins into the plasma [17]. GP V is one of the most abundant glycoproteins on the surface of platelet membranes. It is released into the plasma as platelets are activated and hydrolyzed, and can also be used as an indicator of platelet activation [18]. In this study, CD62p of platelets first decreased and then increased, while sP-selectin and GP V first increased and then decreased, but the results on Day 7 were higher than those on Day 2 (Figure 3), indicating a certain degree of platelet activation; the values on Day 2 and Day 7 were significantly higher than the results reported by Zeng et al., which might be due to the differences caused by centrifugation, leucocyte filtration and other treatments [10].

There are many factors that affect the quality of leukocyte-depleted pooled platelet concentrates, and the mechanism of platelet storage lesion is also very complex, which may be related to blood collection methods and storage media, preparation methods, container and filter selection, etc. However, the platelet yield prepared in this study was lower than the results obtained by Zeng et al. by the PRP method [10], which might be due to the fact that they only compared the results before and after filtration, while the platelet yield in this study was the result of the comparison of the platelet content in the final product and the initial whole blood, including the overall loss of centrifugation and filtration by leukocyte filter. Studies has shown that compared to top-and-top bags, top-and-bottom bags can significantly increase platelet yield [19,20], so improving blood collection bags can improve preparation results. Due to differences in the initial blood and platelet storage systems used in the study by Zeng et al., this study could not find significant advantages of the BC method (Table 2).

Table 2. Quality changes of pooled platelet concentrate stored at different times (mean ± SD; n = 30).

	D2	D4	D5	D7
Plt (10^11/Bag)	3.17 ± 0.35	3.00 ± 0.34	2.94 ± 0.34*	2.92 ± 0.34*
pH value	7.12 ± 0.09	7.07 ± 0.09	7.04 ± 0.09*	6.95 ± 0.10*#Δ
MPV (fL)	7.35 ± 0.28	7.31 ± 0.30	7.48 ± 0.30#	7.84 ± 0.34*#Δ
PDW	15.12 ± 0.20	15.31 ± 0.21*	15.22 ± 0.16*	15.33 ± 0.20*Δ
pO2 (mmHg)	90.23 ± 21.65	82.46 ± 24.13	78.54 ± 28.84	89.87 ± 30.59
pCO2 (mmHg)	71.7 ± 11.69	26.93 ± 2.75*	21.48 ± 2.91*#	15.32 ± 2.62*#Δ
LA (mmol/L)	5.19 ± 1.29	6.91 ± 1.05*	7.74 ± 1.81*#	11.64 ± 2.58*#Δ
Bicarbonate (mmol/L)	11.42 ± 1.30	8.10 ± 0.76*	6.84 ± 0.79*#	4.83 ± 1.40*#Δ
GLU (mmol/L)	21.46 ± 0.80	20.17 ± 1.05*	19.45 ± 0.71*#	17.86 ± 1.36*#Δ
LDH (U/L)	140.57 ± 10.60	160.33 ± 48.55*	207.23 ± 134.73*	278.03 ± 128.22*#Δ
CD62p positive rate (%)	52.33 ± 13.43	39.56 ± 17.88*	38.43 ± 16.16*	66.36 ± 8.47*#Δ
ATP (μmol/platelets)	9.52 ± 3.87	10.21 ± 6.56	12.91 ± 7.08*	18.28 ± 7.06*#Δ
sP-sel (pg/ml)	306.97 ± 56.57	344.57 ± 57.62*	372.92 ± 72.00*	316.83 ± 68.19*Δ
GPV (ng/ml)	57.77 ± 14.64	65.21 ± 8.38*	73.46 ± 9.98*	70.62 ± 21.90*
HSR (%)	47.63 ± 7.44	50.69 ± 10.58	44.34 ± 6.57#	47.85 ± 9.07
PMC (%)	1.81 ± 1.27	1.19 ± 0.89*	1.11 ± 0.77*#	1.05 ± 1.10*

Plt: Platelet count; MPV: mean platelet volume; PDW: Platelet distribution width; LA: platelet lactate; GLU: Platelet glucose; LDH: Lactate dehydrogenase; sP-sel: soluble p-selectin; GPV: Glycoprotein V; HSR: hypotonic shock response; PMC: Platelet morphology changes; D2: Day2; D4: Day4; D5: Day5; D7: Day7; Note: *: Compared with D2, p < 0.05; #: compared with D4, p < 0.05; Δ: compared with D5, p < 0.05.

In summary, this study used a domestic disposable leukocyte-depleted platelet storage bag and the buffy coat method to prepare leukocyte-depleted pooled platelet concentrates. The quality indicators of in vitro storage of platelets showed a downward trend, but still met the requirements for clinical use. At the same time, it is recommended to improve the collection bag to further improve the quality of platelets and ensure the clinical therapeutic effect of platelets.

Author contributions

Feng Chen and Xiaoqing Dai: design & statistical analysis; Feng Chen and Azhong Li: wrote the first draft of the manuscript. Wei Hu: provision of study material or patients; Feng Chen and Yinhong Zheng: writing-review & editing. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

The authors are grateful to all of the participants and contributors.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.