Preparation of discoid washed platelets by differential centrifugation

Clinica Chimica Acta 275 (1998) 99–105

Short communication

Preparation of discoid washed platelets by differential centrifugation a, b b Yukiko Hayashi *, Susumu Takenaka , Chikashi Kohmura , a Hisami Ikeda a

Department of Laboratory Medicine, Asahikawa Medical College, Nishikagura 4 -5 -3 -11, Asahikawa 078 8510, Japan b Department of Clinical Laboratory, Asahikawa Medical College, Nishikagura 4 -5 -3 -11, Asahikawa 078 8510, Japan Received 17 October 1997; received in revised form 6 April 1998; accepted 9 April 1998

Keywords: Additive free platelet; Differential centrifugation; High BSA buffer

1. Introduction Platelets prepared by differential centrifugation and gel filtration techniques neither retain good morphology nor function as they are in plasma without inhibitors of arachidonic acid metabolism and / or scavengers of its metabolites [1]. However, additives such as aspirin, prostaglandinE 1 (PGE 1 ) and apyrase are inappropriate in experiments involving arachidonic acid (AA) metabolism. Albumin is known to act as stabilizer and inhibitor of arachidonic acid metabolites and nucleotides [2–4], and can be used in situations regardless of AA metabolism. We report that low g centrifugation in combination with high concentration of bovine serum albumin (BSA) brings about the results substantially equivalent to those with unwashed platelets in platelet rich plasma (PRP). 2. Materials and methods Wash buffer and reaction buffer were prepared according to the method of Tandon et al. [5]. Constituents of wash buffer were 5.5 mmol / l dextrose, 128 mmol / l NaCl, 4.26 mmol / l Na 2 HPO 4 , 7.46 mmol / l NaH 2 PO 4 , 4.77 mmol / l *Corresponding author. 0009-8981 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00072-2

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trisodium citrate, and 3.5 g / l BSA (pH 6.5). In modified wash buffer, BSA concentration was increased to 20 g / l (pH 5.8). Reaction buffer was the modified Ca 21 free HEPES-Tyrode solution containing 136 mmol / l NaCl, 5.5 mmol / l dextrose, 1 mmol / l MgCl 2 , 0.47 mmol / l NaH 2 PO 4 11.62 mmol / l NaHCO 3 , 2.7 mmol / l KCl, 10 mmol / l HEPES and 3.5 g / l BSA (pH 7.3). Five ml of blood from apparently healthy volunteers was drawn in 9 ml polyethylene tubes containing 0.55 ml of 0.12 mol / l sodium citrate and centrifuged at 50–200 g for 15 min to obtain PRP. After centrifugation of PRP at 220–380 g for 15 min, the pellets were carefully suspended in 1.5–2 ml of the modified wash buffer, and centrifuged at 250 g for 10 min. After the pellets were resuspended in the reaction buffer, tube was left at room temperature for 30 min, so that erythrocytes could settle down to the bottom. The upper layer was carefully removed and used for experiments. All the operations were carried out at room temperature. About 70% of platelets in PRP were recovered in this procedure. Prostaglandins in the platelet-free supernatants were assayed by gas chromatography–mass spectrometry (GC / MS) according to the methods of Knott and co-workers [6] with slight modification. Briefly, the particle free-supernatant in the presence of 20 mmol / l indomethacin was acidified to pH 3 by adding 1 mol / l HCl and passed through Sep-Pak C18 cartridge (Waters Corporation, USA) prewashed with methanol–water (22:78). The prostaglandins in the samples were eluted with ethyl acetate and derivatized to pentafluorobenzyl, methoxime, trimethylsilyl forms, followed by vaporization in the injector preheated to 2808C. The samples were carried to capillary column of fused silica (30 m 3 0.3 mm 3 0.1 mm, DB-1, J&W Scientific) by helium gas. The initial temperature 508C of the column was maintained for 1 min, increased to 2008C at 508C / min and finally up to 2808C at a rate of 38C / min. The samples were analyzed by JMS-SX 102 GC / MS spectrometer equipped with a data processing system (JEOL Ltd, Japan) in the negative ion ionization mode.

3. Results and discussion In order to reduce close cell to cell contact during centrifugation that might induce platelet activation and release of AA and its metabolites, we employed lower g value centrifugation to prepare PRP. In centrifugation at 75 g for 15 min, leukocytes remaining in the PRP were much fewer (0.002%) than those at 50 g (0.17%). With increasing g values, elimination of leukocytes was attained at the expense of size and yields of platelets. Although no leukocytes remained on centrifugation at 200 g for 15 min, harmful effects to platelet counts and mean platelet volume (MPV) were inevitable. The percentage of erythrocytes to platelets at 200 g was not different from that at 75 g (Table 1).

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Table 1 Optimal g value to prepare PRP G max

n

WBC / PLT(%)

RBC / PLT(%)

Platelet count ( 3 10 3 / ml)

MPV(fL)

50 75 200

6 15 15

0.1760.22 0.00260.002 0.00

not tested 0.1960.11 0.1560.23

298659 271644 23617

8.160.9 7.360.5 4.360.3

Freshly collected citrated blood from healthy volunteers was centrifuged at the indicated g value (maximum) for 15 min. Approximately 70–80% of PRP was sampled. Percent of the cells to platelets are expressed by Mean6SD. n: Total number of subjects analyzed at each g value.

In order to harvest large platelets without irreversible aggregate formation, optimal g values for pelleting platelets were next studied. By centrifugation for 15 min at 250 g. MPV of platelets in the supernatant became smaller than that at 220 g, suggesting that most of the large platelets were moved to the pellet without irreversible aggregation (Table 2). Aggregates were observed at centrifugation higher than 250 g. It has been reported that serum albumin is inhibitory to platelet aggregation induced by AA, ADP and collagen through its converting action of prostaglandin H 2 (PGH 2 ) to prostaglandin D 2 (PGD 2 ) [2–4,7]. In order to inhibit platelet aggregation during centrifugation, platelets were resuspended in the buffer containing low albumin (3.5 g / l BSA, i.e. wash buffer) or high albumin (20 g / l, i.e. modified wash buffer). The 20 g / l BSA should be equivalent to 100 g / l human serum albumin (HSA) in terms of PGH 2 converting activity because BSA is reported to have five times high PGH 2 converting activity as HSA [3]. The 20 g / l BSA will have sufficient PGH 2 converting activity to inhibit platelet aggregation as in PRP because concentration of HSA in PRP is about 40 g / l. Table 2 Optimal g value for pelleting platelets G max

n

MPV a

Aggregates

220 250 300 350

6 6 5 6

8.360.92 6.960.53 6.260.83 6.160.61

0/6 0/6 3/5 4/6

PRP from healthy volunteers was centrifuged at the indicated g value. The MPV of platelets in the supernatant was analyzed by Technicon H-1. The pellets were suspended in buffer and aggregates in the suspensions were examined microscopically. a MPV in the supernatant. n: Total number of subjects tested at the each g value.

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The platelets in the modified wash buffer solution centrifuged at 250 g for 15 min were completely dispersed in reaction buffer and showed consistent swirling. Microscopically, more than 95% of platelets remained single and discoid after washing with high BSA buffer, while the preparation with low BSA (3.5 g / l) buffer contained aggregates and fewer discoid platelets. In analysis of prostaglandins (PGD 2 , PGE 2 and TxB 2 ) in the supernatant, only PGD 2 was detected in the samples showing no aggregates. On the contrary, PGE 2 was detected invariably in the samples with aggregates (Table 3, Fig. 1). In spite of high albumin concentration, both PGE 2 and aggregate formation were observed in samples prepared at 800 g centrifugation ( a No 5 and a No 6 of (Table 3)). The results are consistent with the assumption that albumin inhibits platelet aggregation by its converting activity of PGH 2 synthesized by activated platelets during centrifugation. Overproduction of PGH 2 exceeding converting capacity of albumin results in PGE 2 production and aggregate formation. It was reported that discoid washed platelets showed good responses comparable to PRP platelets [4,8,9]. The platelets washed by the modified buffer were resuspended in the reaction buffer which contained 0.5 mg / mL fibrinogen and 0.2 mmol / l Ca 21 and their functions were compared with those of unwashed platelets in PRP. In the platelets washed with high BSA, responses to collagen, epinephrine and ADP were almost the same as those of PRP (Fig. 2). The responses to weak agonists were gradually decreased and lost in 90–120 min after separation from plasma, while morphological properties were retained

Table 3 Appearance of PGE 2 and PGD 2 in the supernatant of platelet samples during centrifugation No

BSA in buffer (g / l)

PGE 2 / PGD 2

Aggregates

1 2 3 4 5 6 7 8 9

20 20 20 20 20 20 3.5 3.5 3.5

2/1 2/1 2/1 2/1 1/2 1/2 1/1 1/1 1/1

1a 1a 1 1 1

Nine samples from 9 individuals were assayed for prostaglandins by GC / MS. Samples were centrifuged at 250 g except No 5 and No 6. The samples No 5 and No 6 were centrifuged at 800g a . Results were expressed as presence ( 1 ) or absence (2) of peaks corresponding to PGE 2 or PGD 2 , since the GC / MS assay, as a qualitative rather than quantitative assay, gave different size of peaks in different occasion even with the same sample.

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Fig. 1. GC / MS profiles of cell-free supernatants. Prostaglandins extracted from the cell-free supernatants after platelet centrifugation were derivatized and analyzed as described in the section entitled ‘‘Materials and methods’’. Ion masses were given after analysis for authentic PGD 2 , PGE 2 and TxB 2 (Sigma Aldrich, Japan) derivatized by the same methods as the samples. Representative selected ion monitoring (SIM) chromatogram of authentic PGD 2 , PGE 2 (m / z 524) and TxB 2 (m / z 614) (A), prostaglandins from the cell free supernatant of platelets which were centrifuged at 250 g in buffer containing 20 g / l BSA (B) and 3.5 g / l BSA (C).

even after 4 h. The preparation with low BSA buffer contained fewer discoid platelets, and showed consistently weak responses particularly to weak agonists such as ADP (Data not shown). In conclusion, our modification of differential centrifugation with high BSA concentration to prepare platelet suspension has advantages mainly in two

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Fig. 2. Typical aggregation responses of PRP and washed platelets to various inducers. PRPs from freshly collected citrated blood were divided into two parts. One was washed as described in the text and served as washed platelets and the other was used as unwashed PRP. Aggregation of 0.2 ml samples (2.5–3.5 3 10 8 / ml) were monitored using Aggretec TE-500 (Erma Optical Works ltd, Japan), at 378C with stirring at 900 rpm.

respects; (1) it is a simple process with conventional apparatus, (2) more than 70% of the platelets preserving native properties were recovered from PRP. Probably, BSA stabilizes platelets through its PGH 2 converting activity.

Acknowledgements We are much indebted to Mr Hiroaki Akutsu, a technical staff of Central Laboratory for Research and Education, for GC / MS analysis.

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