Scand J Haematol (1976) 17,71-77

Preparation of Concentrated Platelet Suspensions by Dehydration against Polyethyleneglycol 20,000 5. W. N. AKKERMAN, G. GORTER,J. WESTER& J. J. SIXMA Depariment of Haematology, University Hospital, Utrecht, The Netherlands

Gel filtered platelet suspensions were concentrated by repeated dehydration against polyethyleneglycol 20,000 followed by dialysis against slightly hypotonic buffer. This method increased the platelet concentration 2-3 times with a recovery of 80-100 %. The final cell suspension closely resembled the original platelet rich plasma as tested by a number of platelet tests. K e y words: dehydration - gel filtration


integrity - platelets

Accepted for publication March 20, 1976 Correspondence to: Dr. J. W. N. Akkerrnan, Department of Haematology, University Hospital, Utrecht, The Netherlands

Platelet separation from plasma is generally taken as disadvantageous for the preservation of cell integrity (Day et a1 1975). Platelet studies are therefore best made directly in platelet rich plasma (PRP). Unfortunately a number of biochemical techniques are disturbed by plasma constituents or require higher cell concentrations than those normally found in PRP. Concentrated cell suspensions in artificial media are usually prepared by repeated centrifugation with or without albumin (Mustard et a1 1972, Walsh 1972). Centrifugation of platelets changes cell morphology and induces leakage of ADP and other metabolic alterations (Mustard et a1 1972, Hutton et a1 1974,

Zucker et a1 1974, Day et a1 1975). Platelet separation from plasma is therefore better achieved by gel filtration which shows the mentioned disadvantages to a much lesser extent (Tangen et a1 1973, Zucker et a1 1974). A disadvantage of gel filtration is the dilution of the sample, which is the more pronounced as more of the effluent is collected. The present paper describes a useful extension of the gel filtration technique for the preparation of concentrated platelet suspensions in artificial media: the gel filtered platelets (GFP) are collected in a dialysis bag and ‘dialyzed’ against polyethylene glycol 20,000. Suspensions pre-



pared by this technique closely resemble the original PRP as tested by a number of platelet tests. MATERIALS AND METHODS Preparation of concentrated platelet suspensions

160 ml of freshly drawn blood was obtained from 4 normal volunteers, 40 ml each, collected in citrate (0.1 vol 129 mM trisodium citrate dihydrate) and centrifuged (room temp., 200 x g, 10 min) to obtain platelet rich plasma (PRP). The PRP was immediately placed on a siliconized column (column size 5 x 20 cm) containing Sepharose 2B (Pharmacia, Uppsala), which was equilibrated with Caz+-freeTyrode solution (Walsh 1972) (pH 7.2, osmolality 300 mOs kg-l) containing 137 mM NaCI; 2.68 mM KCl; 0.42 mM NaHzP04; 12 mM NaHC03; 1.7 mM MgC12; 5.5 mM glucose and 2 g 1-l albumin (Sigma, St. Louis). Chromatography was performed at room temperature according to Tangen et a1 (1971). The platelet yield normally was between 90-100 % but only the most concentrated part of the effluent was collected (50-70 % of total platelets). Subsequently the platelet suspension was collected in a dialysis bag and placed in polyethyleneglycol 20,000 (Fluka, Buchs) for 15 min at room temperature. This was followed by dialysis for 20 min at room temperature against the Ca2+-free Tyrcrde solution which was diluted with distilled water t o an osmolality of 270 mOs kg-l. The whole concentration procedure was repeated once. Integrity studies

The integrity studies consisted of tests on the following parameters: 1. Ultrastructural appearance. Platelet suspensions were fixed in 6 volumes of 1 % glutaraldehyde in 0.07 M sodium phosphate buffer (pH 7.4), postfixed in osO4 and embedded in epon (Sixma et a1 1972).

2. Aggregability. Platelet suspensions were incubated in a Payton Dual Channel Suspension

Aggregation Module (Payton Associates, Scarborough, Canada; settings were: range 5; level 3; temp. 37O C; stirring speed 900 rpm; cuvettes 0.312). To 0.40 ml of platelet suspension 0.05 ml of a 10 g I-' fibrinogen solution (Kabi, Stockholm) in saline was added. N o fibrinogen was added to PRP. After 15 sec aggregation was initiated by addition of 0.05 ml ADP solution (Sigma, St. Louis) in saline. Variations in optical density were recorded on an Omniscribe TM 5000 recorder (Houston Instruments, Bellaire, USA; recorder speed 1 inch min-'). The signal from the platelet poor plasma or the buffer was adjusted to 1 mV and that from the PRP or cell suspensions to 9.6 mV. 3. Platelet factor 3 availability. The availability of platelet factor 3 in non-stimulated cell suspensions was measured according to Sixma & Nijessen (1970). 4. Metabolism of 3H-adenine and adenylate energy charge. Platelet suspensions were incubated with 1 p M 3H-adenine for 30 min at 37O C. Adenine nucleotides were extracted with cold EDTAethanol (1 part of platelet suspension for 2 parts of 86 % ethanol containing 10 mM EDTA, disodium salt) and subsequently separated by electrophoresis at 60 V cm-l (Holmsen & Weiss 1970). Radioactivity of separated nucleotides was measured in a mixture of 2 g PPO and 0.1 g POPOP per 1 of toluene in a liquid scintillation counter (Packard Tricarb, model 3380).

5. Total A T P and A D P and endogenous serotonin. Total ATP and ADP in platelets were measured according to Holmsen et a1 (1972) using the luciferin-luciferase mixture of Sigma. Light deflection was detected with an Amino Chem Glow photometer, coupled on a Perkin Elmer 56 recorder. The variation coefficient for this test for both ATP and ADP was 4 %. For the measurement of endogenous serotonin according to Rao et a1 (1976) 1 ml of platelet suspension was spun off in an Eppendorf microfuge (12,000 x g, 4 min, room temp.) and 1 ml cold 1 N perchloric acid was added t o the pellet. This pellet was resuspended by sonication (30 sec 24 K.c. sec-l) and again centrifuged (12,000 x g, 4 min, room temp.). 0.6 ml 5 N HCI was added to the supernatant. This mixture was used for assay of serotonin (including its derivates) in a

CONCENTRATION OF PLATELET SUSPENSIONS Perkin Elmer MPF-3 fluorescence spectrophotometer (excitation wavelength 310 nm, emission wavelength 540 nm, slit widths 10 nm). The variation coefficient for this test was 4 %. 6. Lactate dehydrogenase ( L D H ) activity, lactate accumulation and oxygen tension. LDH activity was measured according to Bergmeyer (1974). Lactate accumulation and oxygen tension were measured as described elsewhere (Akkerman et a1 1976).


Preparation of concentrated platelet suspensions

The present available isolation procedures all have serious disadvantages. Centrifugation disrupts the cells and liberates ADP which must be counteracted by high amounts of EDTA or ADP removing enzymes (Zirkovic & Detwiler 1970, Mustard et a1 1972). Furthermore centrifugation induces shape change and increases stickiness when performed at low temperature (Mannucci 1972, Day et a1 1975). Albumin gradient centrifugation (Walsh 1972) drastically disturbs platelet morphology (Hutton et a1 1974, Zucker et a1 1974) and in our hands often shifted the label in platelets prelabelled with 3H adenine from the ATP into the hypoxanthine-inosine fraction, which is typical for irreversible platelet injury (Miirer et a1 1974). We separated the cells from plasma by gel filtration on Sepharose 2B which led to only slight loss of cell integrity (Tangen et a1 1973, Mason et a1 1974). The dilution inherent tu this step was first counteracted by albumin gradient centrifugation, but this proved to be unsuccessful for reasons already mentioned. We therefore concentrated the suspension by collecting the effluent in a dialysis bag placed in poly-


ethyleneglycol 20,000 (PEG). PEG withdraws water and ions from the suspension but water is drawn at a faster rate. This resulted in a slight alteration in buffer composition and a concomitant increase in osmolality. Therefore PEG dialysis was followed by dialysis for 20 min at room temperature against slightly hypotonic buffer (270 mOs k g l ) . During this step the volume of the cell suspension was kept constant. Both types of dialysis were repeated once. This repeated dialysis against PEG and hypotonic buffer increased the platelet concentration between 2 and 3 times. The method is highly reproducible in terms of concentration and yield (between 80 and 100 %, Table 1). The variation in osmolality and buffer composition are disadvantageous, but if performed under standardized conditions these changes are highly reproducible and probably lead to a constant effect on the platelets (Table 2). During the dialysis scheme described here osmolality never exceded 360 mOs kg-l. The dialysis bag was impermeable for PEG: distilled water ‘dialyzed’ against PEG for 5 h at room temperature showed no change in osmolality. Furthermore the typical emission spectrum of PEG was absent which indicates that contamination was less than 20 mg 1-‘. Integrity studies

1. Morphology. Platelets in the concentrated suspensions had a similar morphology as in GFP or PRP kept at room temperature during the gel filtration and concentration steps (Figure 1). However, platelet morphdugy deteriolrated in time whether the cells were separated from plasma (GFP and concentrated suspension, Figure 1, B-C) or not (aged PRP, Figure 1 D).



TABLE 1 Gel filtration of human platelets and subsequent concentration of ihe suspension by dehydration against PEG 20,000



280 103

16,520 x lo6

Gel filtered platelets


260 103

8,320 x 1P



Concentrated platelet suspension


624 103

6,864 x lo6





TABLE 2 Variations in buffer composition before and after concentration of gel filtered platelets

After chromatography

304k 9

2.61 0.10


0.48 f0.02

12.25 f 1.57

1.76 k 0.26

After concentration against PEG 20,000

330+ 14

2.68 k 0.13


13.00 k 1.36

2.22 & 0.16

Variations in osmolality (expressed as mOs kg-l) and K’, Pi, HC03- and Mgz’ content (expressed as mrnol I-’ in Ca*+-freetyrode after gel filtration on Sepharose-2B and concentration against PEG 20,000 (mean k SD of 6-8 determinations). Osmolality was measured in a Fiske Osrnometer (Fiske Associates, Uxbridge, USA). K+ was measured in the flamephotometer and HC03 in the colorimeter of a SMA-6 (Technicon). Mgz’ was measured by atomic absorption in an Unicarn SP-1900 and Pi in an Autoanalyzer (Technicon). Differences in osmolality and Pi content were significant (student t test: p < 0.05).

2. Aggregability. Concentrated suspensions hardlji showed an aItered susceptibility towards ADP compared to GFP, but both were slightly less responsive than fresh PRP (Figure 2).

3. Platelet factor 3 availability. There was a small but reproducible reduction in platelet factor 3 availability after gel filtration which was restored during the concentration steps. The reason for this change is not understood (Table 3). 4. Metabolism of 3H adenine and adenylate energy charge. Irreversible platelet injury is often accompanied by an increase of 3Hlabel in the hypoxanthine-inosine fraction at the cost of the ATP fraction (Murer et a1

1974). Such a shift was absent in GFP and the concentrated suspensions. The adenylate energy charge varied between 0.88 and 0.91 in all suspensions (Table 3).

5 . Endogenous ATP, ADP and serotonin. The data show no significant decrease of ATP, ADP or serotonin. Release of storage granules during gel filtration or concentration against PEG is therefore not likely to occur. 6 . Cytoplasmatic markers. Since the lactate dehydrogenase (EC 1. l .1.27) activity was similar in PRP, GFP and concentrated suspensions cytoplasmic leakage was probably negligible.


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ficiency in the storage pool of platelet adenine nucleotides. Br J Haematol 19, 643-49. Hutton R A, Howard M A, Deykin D & Hardisty R M (1974) Methods for the separation of platelets from plasma. Thromb Diath Haemorrh 31, 119-32. Mannucci P M (1972) In P M Mannucci & S Gorini (eds) Platelet function and thrombosis. A d v Exp Med Biol 34, 57-78. Mason R G, Read M S & Shermer R W (1974) Comparison of certain functions of human platelets separated from blood by various means. A m J Pathol 76, 323-32. Miirer E H, Day H J & Lieberman J E (1974) Metabolic aspects of the secretion of stored compounds from blood platelets. Biochim Biophys Acta 362, 266-75, Mustard J F, Perry D W, Ardlie N G & Packham M A (1972) Preparation of suspensions of washed platelets from humans. Br J Haematol 22, 193-204. Rao G H R, White J G, Jachimowicz A A & ACKNOWLEDGEMENTS Witkop C J (1976) An improved method for The authors are grateful to Dr S. Holme for perthe extraction of endogenous platelet serotonin. forming the aggregation studies, Dr J. Lips and J Lab Clin M e d 87, 129-37. Dr B. Beukers for measuring the adenylate energy Sixma J J, Linssen W H & Geuze H J (1972) charge and to Miss Anneke Starkenburg for techGlutaraldehyde fixation of human blood platenical assistance. The authors also thank Dr K. H. lets. Thromb Diath Haemorrh 27, 272-17. Peuker, Department of Clinical Biochemistry, Sixma J J & Nijessen J G (1970) Characteristics University Hospital, Utrecht for measuring buffer of platelet factor 3 release during ADP-induced compositions. aggregation. Thromb Diath Haemorrh 24, 20613. Tangen 0, Berman H J & Marfey P (1971) Gel REFERENCES filtration: a new technique for separation of blood platelets from plasma. Thromb Diath Akkerman J W N, Gorter G, Sixma J J & Staal Haemorrh 25, 268-78. G E J (1976) Variations in levels of glycolytic Tangen 0,McKinnon E L & Berman H J (1973) intermediates in human platelets during plateletOn the fine structure and aggregation requirecollagen interaction. Biochim Biophys Acta 421, ments of gel filtered platelets. Scand J Haematol 296-307. 10, 96-105. Bergmeyer H U (1974) Methods of enzymatic analysis, p 481. Verlag Chemie, Academic Press, Walsh P N (1972) Albumin density gradient separation and washing of platelets and the study of New York. platelet coagulant activities. Br J Haematol 22, Day H J, Holmsen H & Zucker M B (1975) 205-17. Methods for separating platelets from blood Zirkovic R V & Detwiler T C (1970) Thrombin and plasma. Thromb Diath Haemorrh 33, 648induced release of glycolytic intermediates from 54. rat platelets. Biochim Biophys Acta 201, 502-05. Holmsen H, Storm E & Day H J (1972) DeterZucker W H, Shermer R W & Mason R G (1974) mination of ATP and ADP in blood platelets. Ultrastructural comparison of human platelets Anal Biochem 46, 489-501. separated from blood by various means. A m J Holmsen H & Weiss H J (1970) Hereditary defect Pathol 77, 255-67. in the platelet release reaction caused by a de-

2.45 k 1.29 pmol min-' lo-" platelets (mean k SD, n x 11). This level was increased to 3.68 k 0.78 (n = 8; t test: 0.05) after gel filtration but stayed p constant during the concentration procedure (3.60 1.26, n = 12; t test: p > 0.50). The alterations in buffer composition that occur during dehydration of platelet suspensions against PEG are therefore of minor importance to glycolytic flux. We conclude that dehydration of gel filtered platelets against polyethyleneglycol 20,000 hardly effects platelet integrity and is therefore useful in preparing concentrated platelet suspensions in artificial media.

Preparation of concentrated platelet suspensions by dehydration against polyethyleneglycol 20,000.

Scand J Haematol (1976) 17,71-77 Preparation of Concentrated Platelet Suspensions by Dehydration against Polyethyleneglycol 20,000 5. W. N. AKKERMAN,...
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