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Clinica Chimica Acta, 192 (1990) 115-120 Elsevier

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CCA 04844

A new, rapid, one-step method for the isolation of platelets from human blood
Terence
Department

C. Ford

2, John

Graham

and David

Rickwood

of Cellular and Molecular Sciences, St. Georges Hospital Medical School, London
and Department of Biology, University of Essex, Colchester (UK)

(Received 28 September 1989; revision received and accepted 13 August 1990) Key words: Blood platelet; Nycodenz gradient; Centrifugation

Summary We describe a new, rapid method for the isolation of platelets from human blood using a single centrifugation step through Nycodenz, an inert, nontoxic, nonionic medium. As judged by aggregation and nucleotide release studies, the platelets are recovered in high yield and in excellent functional condition.

Introduction For the investigation of the biochemical and physiological properties of blood platelets it is obviously desirable to be able to isolate them by a swift and simple method which does not damage their functional properties. Platelets are relatively fragile structures and require gentle treatment to retain their in vivo properties. The most common methods of isolation involve centrifugation which, in itself, can be a harsh treatment, especially when the cells are pelleted a number of times during the washing procedures which cause severe alterations to the platelets. A number of methods have been devised to optimise the recovery and minimise the damage to platelets. Ganguly and Sonmchsen [l] and Imandt et al. [2] developed methods using sodium metrizoate and Ficoll as the separation medium, while Levy-Toledano et al. [3] reported success with Metrizamide gradients. Wilson and Graham [4] isolated platelets from human blood using free-flow electrophoresis. Although these methods were reported to result in acceptable preparations of platelets, they are time-consuming and involve a number of steps. Here we report a new, single step method giving a very high recovery of platelets in excellent functional condition

Correspondence to: Dr. D. Rickwood, Department of Biology, University of Essex, Wivenhoe Park, Colchester COH 354 UK. 0009-8981/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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with minimal contamination by other cells, which is eminently routine isolation of pure platelets.
Materials and methods

suitable for the

Nycodenz in powder form was obtained from Nycomed Pharma, Oslo, Norway. Fresh blood samples were collected from volunteer donors using citrate (0.32% final concentration) as the anti-coagulation agent. For the best results, the blood sample should be freshly drawn and processed within 2 h. If blood older than 2 h is used, while the purity of the isolated platelets is m~nt~ned, a deterioration of their integrity is observed. Blood from each donor was divided to allow a direct comparison of two methods to be made. Platelet separating solution: 12.0% (w/v) Nycodenz was made up in 0.1 mol/l NaCl buffered with 5 mmol/l Tricine-NaOH (pH 7.4). The density of the solution was 1.063 g/ml and the osmolality 315 mOsm. Platelet preparation using the Nycodenz solution: one undiluted 5 ml aliquot from each blood sample was carefully overlayered onto 5 ml of the Nycodenz solution, in a lo-ml centrifuge tube, 1 cm diameter. Centrifugation was at 350 x g for 15 min at 20C. Platelet preparation by differential ~n~fugation: an aliquot of each blood sample was cent~fuged at 800 X g for 10 min to obtain a platelet-rich plasma (PRP). Platelets were counted using an improved Neubauer haemocytometer and the concentration adjusted to 2-4 X lO/ml by dilution with autologous plasma. Aggregation studies: aggregating agents used were collagen 4 p&/ml (Sigma Chem.), ristocetin 1 mg/ml and arachidonic acid 250 pg/ml (Paramount Diagnostics) and the tests were carried out in a Coulter Dual Channel Aggrometer. Nucleotide (ATP, ADP) levels were determined both before and after aggregation (Table II), using a bioluminescence assay [5,6].
ReSldtS

After centrifugation (Fig. 1) a pellet of erythrocytes with a buffy coat of leucocytes was formed. At the sample/medium (loading) interface a whitish band was seen gradually becoming more diffuse as it extended towards the pelleted material. This band was found to consist, almost exclusively, of platelets in excellent morphological condition. The top 2 ml of the original sample layer provided the platelet-poor plasma (PPP) for subsequent aggregation studies. The interface band was harvested, using a Pasteur pipette, to a depth of about 1 cm below the interface (fraction A) and examined by phase contrast microscopy. The cells were counted using an improved Neubauer haemocytometer. Fraction A was found to contain 70% of the total platelets of the sample, with less than 0.1% ~ont~nation by other biood cells (Table I>_Harvesting the rest of the band (Fraction B) to within about
OS cm of the pellet, made the final recovery of platelets up to at least 95% of the

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NYCOUEtQ SEPARATION 12%(w/vJ O.~%(W/V)

PLATELET SOLUTION NYCOD~N~ N&l (pH 7.0)

BLOOD SWLE SHOULD BE FRESHLY TAKEN WITH ACD AS THE ANTICOAGULANT

5nM TRICINE

SAMPLE CENTRIFUGE 350x9 1SMin 18-23'C NYCODENZ SOLUTION RBCs AND LEUCOCYTES REMAINDER OF PLATELETS 7W OF PLATELETS

Fig. 1. A diagramatic representation of the appearance of the gradient before and after centrifugation.

TABLE I Recovery of platelets and other blood cells from Nycodenz barrier fractions Fraction Whole blood Fraction A Fraction B Pellet Platelets 3.6 x 2.4x 1.1 x 6.5 x 109 10 109 10 Erythrocytes n.d. 2.5 x 10s 1.0 x 106 n.d. Leucocytes n.d. 3.0 x 103 7.5 x lo4 n.d.

n.d., not determined. Figures are the total numbers of cells from 10 ml of blood. They are from a single preparation. Three other preparations showed an identicat pattern of cell numbers, the percentage recoveries for each cell type in each fraction from the other preparations varied by less than 6%.

TABLE II Levels of nucleotides in platelets, before and after aggregation Aggregating agent None Collagen Ristocetin Aracbidonic acid nmol Nucleotide/lOg ADP 57 14.2 12.4 14.4 platelets ATP 76 6.0 6.6 7.8

These data are averages from three experiments.

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Time

(Minutes)

Fig. 2. The time-course for platelet aggregation: after addition of 25 pl of arachidonic acid (- - - - - -); after addition of 2 pl of collagen (- .- .-); after standing 2 h at room temperature, collagen added (---).

total population. However, the closer to the pellet, the greater was the contamination by other blood cells. Harvesting the Fraction A (70% plus of the platelets) provided a sufficient concentration of platelets to allow the tests for platelet integrity to be carried out without the necessity of pelleting the platelets, which tends to decrease the yield and increase the damage to them. The presence of Nycodenz did not interfere with the subsequent tests. When the operations described were carried out at normal room temperature, (18-22OC) the results were as above, but when carried out at 27OC, it was found that the sample started sedimenting immediately into the medium. Centrifugation under these conditions resulted in a very low recovery of platelets. Therefore, if the room temperature is above 22 o C, the medium should be equilibrated at 20 C in the centrifuge tube before the sample is added. Excellent aggregation was achieved in all cases (Fig. 2) and, moreover, exposure of the platelets to the Nycodenz medium for up to 2 h at room temperature had no detectable effect on platelet aggregation. For comparison, an aggregation plot for platelets prepared by routine differential centrifugation was carried out. The timecourses for aggregation were very similar, but the extent of agggregation as determined by the incremental change in light-scattering, was greater in the case of platelets isolated by the single-step Nycodenz method. Levels of nucleotides (ATP and ADP), determined both before and after aggregation, were comparable with those present in platelets isolated by routine differential centrifugation, as is their release upon aggregation. Discussion
Human blood platelets are not only counted routinely in haematology service departments, but are isolated for the study of blood-clotting, as a model for a number of important ailments, for example, essential hypertension [7] and increas-

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ingly as a source of growth factors [8]. A rapid method producing a high yield of uncontaminated platelets in a reproducible fashion is therefore central to all such work. Methods which rely upon differential centrifugation suffer the dual problems of low yield and relatively high levels of contamination by other blood cells unless the platelet and other cell pellets are resuspended and washed several times. This is not only time-consuming but potentially damaging to the platelets. Some workers even discard part of the platelet-containing pellet to reduce ~nta~nation by erythrocytes and leucocytes [9]. Other methods, using iodinated density-gradient media or free-flow electrophoresis [l-4] also require a number of steps, including the preliminary production of a platelet-rich fraction by differential centrifugation. The method reported here requires no preliminary preparation of the blood sample, only a simple one-step procedure using whofe, undiluted blood. Moreover, exposure of the platelets to the Nycodenz medium for up to 2 h has no detectable adverse effect upon the platelet functions. Aggregation studies can be carried out directly on the harvested material without recourse to washing. Fraction A contains 70% of the total platelets with only 0.01% contamination by other cells. Fractions A and B together contain over 95% of the total platelet population with only 0.04% conta~nation by other blood cells. Acknowledgements We wish to thank the Cell Surface Research Fund (Department of Molecular Sciences, St. Geoges Hospital Medical School) for financial Sheila Talbot and Andrew Chitohe (Department of Haematology, Hospital Medical School) for carrying out the aggregation studies and tide estimations. References
1 Ganguly P, Sonnichsen WJ. A simple method for the isolation of blood platelets. J Clin Path01 1973;26:635. 2 Imandt L, Genders T, Wessels H, Haanen C. An improved method for preparing platelet-rich plasma. Thrombosis Res 1977; 11:429. 3 Levy-Toledano S, Rendu F, Bredoux R, de La Baume H, Dmozynska A, Savariau E, Caen JP. Human platelet isolation from whole blood on metrizamide gradients. In: Protides of the biological fluids XXVII Colloquium, Brussels, Peeters H, ed. UK: Permagon Press, 1979. 4 Witson RBJ. Graham JM. Isolation of platelets from human blood by free-fIow electrophoresis. Clin Chim Acta 1986;159:211. 5 Summerfield GP, Keenen JP, Brodie HJ, Bellingham AJ. Bioluminescent assay of adenine nucleotides: rapid analysis of ATP and ADP in red cells and platelets Using the LKB luminometer. Clin Lab Haemat 1981;3:259. 6 Shattil SJ, Bennett JS. Platelets and their membranes in hemostasis: physiology and pathophysiology. Ann Intern Med 1980;94:108. 7 Resink TJ. Platelet calcium-linked abnormalities in essential hypertension. Ann NY Acad Sci 1986;488:252. 8 Rossi R, Raines EW, Bowen-Pope DF. Biology of platelet derived growth-factors. Cell 1986;46:155. 9 Rittenhouse-Simmons S. Production of diglyceride from phosphotidylinositol in activated human platelets. J Clin Invest 1979;63:580.

Cellular and support and St. Georges the nucleo-