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Anticoagulant-free preparation of platelets from human native blood - a methodical and morphological report
I'HROMBOSIS RESEARCH (c) Pergamon Press
18; Ltd.
149-158 1980.
Printed
in
the
United
States
oo49-3848/80/080149-lo~o2.00/0
ANTICOAG;yo;-FREE PREPARATIONOF PLATELETS FROM HUMAN NATIVE - A METHODICAL AND MORPHOLOGICALREPORT B. Hofmann, U. Till, J. Hofmann, W. Loesche, P. Spangenberg end G. Ostermann Medical Academy of Erfurt, Department of PathologicalBiochemistry, 506 Erfurt, Nordhauaer StraSe 74, GDR (Received 12.2.1979; in revised form Accepted by Editor F. Markwardt. Received by Executive Editorial Office
5.9.1979. 10.1.1980)
ABSTRACT Platelets are prepared from human native blood by a two-step procedure without use of anticoagulants.The first step involves Sephadex gel filtration of native blood to prevent clotting by the removal of plasma calcium. In a second step platelets are separated from other blood cells and plasma constituentsby centrifugation of the gel filtered blood on a Ficoll density gradient. During the whole procedure a pH of 7.4 is maintained. As a first measure for the cell integrity the morphological feature of the platelets was evaluated and compared to platelet-richplasma (PRP) which was prepared with different anticoagulantsat physiological pII.Considering the extent of pseudopod formation the anticoagulant-freeprepared platelets reveal a less activated state than the platelets in PRP.
INTRODUCTION In order to investigate biochemical and morphological parameters of blood platelets they have to be separated from other blood cells. Many separation techniqueshave been described all involving anticoagulationof native blood as the first step. This is achieved by mixing the blood with anticoagulating agents, followed by centrifugationto obtain PRP. Gel filtration or repeated washing procedures are used to transfer platelets into saline media (1,2).
Key words: blood platelets, anticoagulants,blood platelet cytology, gel chromatography,density gradient centrifugation. lb9
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However, anticoagulants not only prevent coagulation of blood by their effect on plasmatic clotting factors. They may also interfer with platelet metabolism and response to aggreation inducers as well as cause morphological alterations It is desirable, therefore, to prevent contamination 7 3-7). with anticoagulants during platelet separation, particularly when the initial phase of platelet activation is going to be studied. A method was elaborated that substitutes anticoagulants by passing native blood through a gel filtration column. Platelets are separated from other cells and plasma by a one-step density gradient centrifugation on Ficoll. The anticoagulantfree platelets (AFP) obtained by this method were studied in respect to their morphological feature by means of interference contrast microscopy. The data indicate that AFP are less altered during preparation in comparison to platelets of a homologous PBP. Functional characteristics of AFP will be communicated in a forthcoming paper. MATERIAL AND METHODS Blood was obtained from healthy donors by puncture of the cubital vein. The donors had not taken any antithrombotic drugs during the last two weeks. The different steps in platelet preparation procedur8 are shown (schematically) in Fig. 1. They are all run at 30 C. All chemicals used were reagent grade.
STEP
REMOVAL OF PLASMA CALCIUM
A;
GEL FILTRA-
I
TION OF NATIVE 01 OOD
STEP 8: DENSITY GRADIENT CENTRIFUCATION
4
CELLS PLASMA
:
;.“‘\.,:
SEPARATION PLATELETS FIUERW BLQOD
PLASMA
FICOLL DENSITY GRADIENT
PLATELETS
OF
RED and WHITE CELLS
FIG. 1 Schematical representation of the steps involved in platelet separation. The 4-way system is made from a piacryl block (2.5x2.5xl.Ocm) having two channels with a diameter of 2 mm.
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Preparation of Gel Filtration Columns and Density Gradients: Sephadex G-25, particle size 100-300 p (Pharmacia, Sweden) or Molselect G-25, particle size 120-320 p (Reanal, Hungary), respectively, were prepared according to the manufacturer's instructions and equilibrated with the elution medium which contained 14.5 mM triethanolamine-HCL (pH 7.41, 126 mM NaCl, and 5.5 mM glucose. The concentra5.4 mM KCl, 0.98 mM I@$1 tion of calcium in the rngiiurnamounted to 5-10 /uM resulting from impurities. The gels were filled into siliconized glass columns (2.5 cm in diameter) to a height of 22 cm. The gel bed was supported by adaptors covered with nylon nets having a pore size of 40 p. The upper adaptor was adjusted 0.5 cm above the gel surface. For density gradient centrifugation a linear increasin gradient was mixed from Ficoll solutions (Pharmacia, Sweden $ containing buffered saline and human serum albumin. Since both Ficoll and albumin are contaminated with considerable amounts of calcium, which cause platelet aggregation and fibrin formation during centrifugation, these substances have to be freed from calcium before use. This was done by ion-exchange chromatography of a 40% (w/v) aqueous Ficoll stock solution on Dowex 50W x 8 (Serva, GFR) and gel filtration of a 20% (w/v) albumin solution using Sephadex G-25. By these procedures calcium concentration of the Ficoll and albumin solutions could be diminished from 300 uM to 75 PM and from 45 PM to 10 m, respectively. The linear gradient was mixed from 12.5 ml of each a 2% and a 20% Ficoll solution which were obtained from the 40% stock solution by dilution with buffered saline. The ionic composition of the gradient solution was the same as of the elution medium of gel filtration. Additionally, albumin was added to a final concentration of 0.35%. The gradient was placed into siliconized glass centrifuge tubes (50 ml) which were prefilled with 2 ml of the 20% Ficoll sol'ution. The density of the gradient was between 1.015 and 1.073 g/ml. Procedure: According to the scheme, step A (Fig. 1) the cannula used for the withdrawal of blood from the cubital vein is directly connected to the inlet of the Sephadex column by polyethylene tubes and a 4-way system. After venipuncture the air present in the tube system and the first ml of blood are removed through the free opening of the 4-way system. To achieve a sufficient flow rate during the application of blood a vertical distance between cannula and column outlet of 120 cm was adjusted. 12.5 ml of blood were applied and eluted at a hydrostatic pressure of 180 cm. Collection of the eluate was started when red cells appeared. The first 5 ml of the eluate were discarded. The next 25 ml were carefully layered onto the top of the pre-mixed gradient. Centrifugation was carried out at 1,000 x g for 5 min. The platelets formed a turbid band which could be drawn by aspiration with a small polyethylene tube under eye control. Protein and Lipid Determinations: To prove the separation of plasma proteins and lipids from the platelets after the Ficoll gradient centrifugation two methods were employed: a> gel filtered blood was applied onto the Ficoll gradient as usually. The gradient was fractionated into 5 ml portions, the fraction@
diluted 1:3 with isotonic saline, the platelets pelleted and the supernatant used for analysis. b) from the gel filtered blood a tiplatelet-poor plasma" was made, centrifuged on the Picoll gradient and the gradient fractions were analysed. Proteins were measured according to LOWRY et al. (8) after precipitation with trichloroacetic acid and resolving with NaOH. Cholesterol was determined enzymatically using cholesterol oxidase (9). Triglycerides and phospholipids were extracted with chloroform/methanol (10) and assayed enzymatically with a test kit from Boehringer (Mannheim, GFR) and by total phosphorus analysis (111, respectively. Preparation of PRP: To compare AFP with PRP both were prepared from the blood of the same donor. The anticoagulants employed for PRP preparation were used in the following final concentrations: sodium citrate 11 mb!, hirudin 150 ATU/ml, heparin 10 U/ml, and EDTA 3 n&I. Calcium Measurements: Calcium concentrations were measured by atomic absorption spectroscopy with an AAS 1 (VEB Carl Zeiss, GDR). The number of platelets in AFP and PRP was counted =%== by p ase contrast microscopy after appropriate dilution of the platelet samples with a mixture of one volume of 2% lidocain and two volumes of 1% ammonium oxalate, The morphological feature of the platelets was evaluated by interference contrast microscopy (NOblARSKI optic) with the photomicroscope Dokuval (VEB Carl Zeiss, GDR). For this purpose the platelet samples were fixed in 1.2% glutardialdehyde.
r
-I
FIG, 2 Removal of calcium from native blood by gel filtration on Sephadex G-25. Hematocrit (x---x>, platelet count (o---o>, and extracellular calcium concentration (-1 were determined in 5 ml fractions of the eluate, The block on the abscissa indicates the pooled cell peak.
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RESULTS In Fig. 2 the elution profile for native blood on Sephadex G-25 is shown. About the same picture was obtained when filtration was carried out on Molselect G-25. Platelets and red cells are eluted with the same volume which corresponds to the void volume of the gel. The cell peak is not clearly separated from the plasma calcium which may be caused by overloading of the column. Furthermore, the blood applied is not eluted as a uniform band, but the upper front of the band extends small "fingers". This phenomenon cannot be explained satisfactorily. Clotting on the gel is not responsible for it, because the same picture is obtained when titrated blood is passed through the column. However, the extracellular calcium concentration of the pooled cell peak amounts to less than 50 @I which is too low to mediate rapid clotting. Within l-2 hours after filtration no formation of clots was visible. As a consequence of filtration, cells and plasma proteins are diluted about 3-4 times compared to the native blood. About 80% of the platelets are recovered in the pooled cell peak. The time elapsed between venipuncture and the elution of the cells amounts tq 4 min. In Fig. 3 separation of platelets from other cells and plasma proteins on Ficoll density gradient is shown (step B in the scheme given in Fig. 1). Red and white cells are sedimented. Platelets form a turbid white band between the sediment and the plasma proteins at the top of the tube. A small zone shortly above the sediment contains aggregated platelets. According to the separation profile fractions 6-8 (5 ml each) were pooled and used for further investigations. About 50% of the platelets applied onto the gradient are recovered in the pooled fractions. The platelet count in the suspension amounts to 40,000-90,000 platelets/ ~1. The concentration of extracellular calcium was in the range of 30-50 @I. A small contamination of platelets with plasma proteins is observed and ranges to 1.5 mg protein/ml suspension, i.e. 2% of the original protein content in the native plasma. This could be proved when platelets were separated on an albumin-free Ficoll gradient or when a platelet-poor plasma made from the gel filtered blood was applied onto the gradient. With respect to plasma lipids about the same relative amounts were found to be associ-
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FIG. 3 Separation of platelets on a Ficoll density gradient. Platelet count (x---xl and extracellular protein concentration (-1 were determined in 5 ml fraction numbered from the top to the bottom of the centrifuge tube. The block on the abscissa indicates the pooled fractions. Albumin was omitted from the gradient.
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TABLE 1 Amounts of Plasma Proteins and Lipids in the "Platelet Fractions" of the Ficoll Gradient Proteins PhosphoCholTriglyclipids ester01 erides Concentrations
61.0
1,300
(pghl)
48.7
5.1
7.0
4.7
76 of total amounts
applied onto the gradient
6.2
9.7
The values were obtained from a centrifugation of a plateletpoor plasma made from gel filtered blood. "Platelet fractions" refer to the fractions 6-8 of Fig. 30 ated with the platelets after the centrifugation. The separation of plasma proteins and lipids in a representative experiment is indicated in Table 1. The presence of fibrinogen in the final platelet suspension could be concluded from the ability of the platelets to undergo ADP-induced aggregation (not shown here). The presence of fibrinogen is a necessary requirement for the ADP-induced aggregation (12). The morphological feature of AFP obtained by interference contrast microscopy was compared to platelets in native blood and to PRP prepared with several anticoagulants. Typical microphotographs are presented in Fig. 4 a-f. Comparing the different forms of platelets and the extent of pseudopod formation it is obvious that AFP do more resemble platelets in native blood than do platelets in PRP (Table 2); The smallest differences between AFP and PRP were found when PBP was made from titrated blood. Hirudin- and EDTA-PRP exhibit altered platelet forms in a greater extent. TABLE 2 Platelet Shape in Different Preparations Platelet Discozytes Platelets Platelets with l-3 with more preparation without pseudosmall pseudo- than 3 small pods pods or 1 long pseudopods Native blood AFP Citrated PRP Hepsrin-PRP Hirudin-PEP EDTA-PRP
83.0 26.7 16,3 29:: 0.8
17.0 z;*z 60:5 26.8 14.7
Irregular forms
216 14.4
317
26.0 63.4 80.0
?Z 4.5
The differences in platelet shape were examined by interference contrast microscopy counting 250 platelets per sample. The values are given in percent.
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FIG. 4 Microphotographs obtained by interference contrast microscopy of different platelet preparations. Samples were fixed in glutardialdehyde. a - native blood, after fixation the platelets were separated by centrifugation; b - AFP; c - titrated PRP; d - heparin-PRP; e - hirudin-PRP; f - EDTA-PRP. Original magnification 1,000 x. DISCUSSION
The method presented allows the preparation of human platelets from whole blood avoiding contact of platelets with anticoagulants as commonly used for platelet preparation. Gel filtration of platelet suspensions including PRP is a well characterized technique to separate platelets from extracellular components (13,14). Therefore, we applied this method
in order to free native blood from plasma calcium and to prevent clotting. For this purpose both Sephadex G-25 and Molselect G-25 were suitable. Attempts to separate both calcium and plasma proteins from the blood cells in one step using Sepharose gels proved to be unsuccessful because erythrocytes hemolyzed resulting in ADP-liberation. After the gel filtration of native blood, platelets were separated from other cells and plasma constituents by centrifugation on a Ficoll density gradient containing 0.35% albumin. The presence of albumin was necessary to avoid spontaneous platelet aggregation during the centrifugation. Besides, it was reported that the addition of albumin to saline platelet suspensions is important for the preservation of platelet integrity (15,16). Density gradient centrifugation on Ficoll is an established method for cell separation (1'7,18). The use of Ficoll for separation of platelets from other blood cells leads to a less contamination of the platelets with red cells and leucocytes than simple differential centrifugation (19). For the evaluation of the platelet morphology interference contrast microscopy was used. This method enables a better evaluation of the platelet shape with respect to pseudopod formation and changes of the smoothness of the cell surface than phase contrast microscopy. However, by light microscopical methods it is difficult to distinguish between real discoid and spheroid platelet forms, particularly, when the platelets have formed pseudopods. Therefore, we classified the platelets in discocytes without pseudopods and platelets with pseudopods. AFP reveal a smaller extent of pseudopod formation than PRP prepared with sodium citrate or other anticoagulants. That means that AFP are in a less altered state than the different kinds of PRP, although AFP are made by a two-step procedure and finally suspended in a saline medium. The platelet surface appears to be relatively smooth with small and evenly distributed granular structures which seems to be a normal attribute of the platelet surface (20). Usual methods to transfer platelets from plasma into a saline medium and to remove the anticoagulant are gel filtration or repeated washing procedures. Both of these procedures cause an increase of the morphological alteration of the platelets compared to the original PRP (13,211. A comparison between AFP and other saline platelet suspensions prepared by usual methods is difficult as the latter often starts from PRP which was prepared at unphysiological low pH values, e.g. using ACD as anticoagulant. Lowering the pH results in an inhibition of the platelet activation during the preparation procedure (I). Preparing saline suspended platelets under such conditions may provide less or comparable pseudopod formation as observed in AFP (13). The advantage of AFP is, however, that the physiological pH of 7.4 is maintained during the whole procedure and that the platelets have never had any contact with anticoagulants. The influence of anticoagulants on the morphology of platelets can be clearly derived from the data of the different kinds of PRP. The results presented here indicate that AFP may be a good tool to investigate the influence of anticoagulants on platelets in a more direct way as it was previously possible.
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Further, it enables us to exclude any anticoagulant effects on platelet functions being under investigation. However, before to do so, AFP have to be characterized in functional respect. This will be done in a forthcoming paper. ACKNOWLEDGEMENTS The authors wish to had the possibility vestigations in its We Danz and Leman. Raithel for their C.
thank the VEB Carl Zeiss, Jena (GDR) for out a part of the microscopical to carry laboratories with the helpful support of thank further Mrs. E. Michel, C. Btlttner skillful technical assistance.
having inDrs. and
REFERENCES 1. DAY H.J., HOLMSEN, H. and ZUCKER, M.B.: Methods for separating platelets from blood and plasma. Thrombos. Diathes. Haemorrh. (StuttaO) 2, 648-654, 1975. 2. MJWON, R.G., READ, M.S. and SHERMER, R,W.: Comparison of certain functions of human platelets separated from blood by various means. Am. J. Pathol. 76, 323-332, 19740 3. MUSTARD, J.F., PERRY, D.W., KINLOUGH-RATHBONE, R.L. and PACKHAM, M.A.: Factors responsible for ADP-induced release reaction of human platelets. Am. J. Physiol. 228, 1757-1765, 1975. 4. O'BRIEN, J.R., SHOOBRIDGE, S.M. and FINCH, W.J.: Comparison
of the effect of heparin and citrate on platelet aggregation. J. Clin. Path. 22, 28-31, 1969. 5. GRANT, A.R, and ZUCKER, M.B.: EDTA-induced increase in
platelet surface charge associated with the loss of aggregability. Assessment by partition in aqueous two-phase polymer system and electrophoretic mobility. Blood 52, 515-523, 1978. 6. ZUCKER, M.B. and BORRELLI, B.A.: Reversible alterations in
platelet morphology produced by anticoagulants and by cold. Blood 9, 602-608, 1954. -7. WHITE
J.G.: Effects of ethylenediamine tetraacetic acid (EDTAI on platelet structure. Stand. J. Haematol. 2, 241254, 1968.
8. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J.:
Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-268, 1951. -9. RiJSCHLAU, Po, BERNT, E. and GRUBER, W.: Enzymatische Be-
stimmung des Gesamtcholesterins im Serum. Z. klin. them. klin. Biochem, 12, 403-407, 1974,
10, COHEN, P., BROEKMAN, M.Jo, VERKLEY, A., LISMAN, J.W.W. and DERKSEN, A.: Quantification of human platelet inositides and the influence of ionic environment on their incorporation of ortho-phosphate-32P. J. Clin. Invest. 50, 762-772, 1971. 11. SKIPSKI, V.P. and BARCLAY, M,: Thin-layer chromatography of lipids. In: Methods in Enzymology, Vol. XIV. J.M. Lowenstein (Ed.) New York: Academic Press, 1969, pp. 530-598. 12. MUSTARD, JOF., PACKHAM, M.A., KINLOUGH-RATHBONE, R.L., PERRY, D.W, and REGOECZI, E.: Fibrinogen and ADP-induced platelet aggregation. Blood 52, 453-466, 1978. 13. TANGEN, 0.. McKINNON, E,L. and BERMAN, H.J,: On the fine structure and aggregation requirements of el filtered platelets (GFP). Stand. J. Haematol. lC, 9% -105, 1973. 14. FINE, KoM., ASHBROOK, P.C., BRIGDEN, LoPo, MALDONADO, J.E. and DIDISHEIM, P.: Gel-filtered human platelets. Am. J. Pathol. 84, 11-22, 1976. 15. ROSSI, E,C,: The effect of albumin upon the loss of enzymes from washed platelets. J. Lab, Clin. Med. 2, 240 246, 1972, 16. TANGEN, O., ANDRAE, M,L. and NILSSON, B.E,: Nucleotide leakage from platelets in artificial media; Prevention by albumin and other macromolecules. Stand. J. Haematol. 11, 241-248, 1973. 17. TOMISAWA, S,: A new method for rapid separation of platelets from plasma by discontinuous Ficoll density gradient. Japan. J. EXP. Med, 45, 529-531, 19750 18. PEPPER, R,J., ZEE, T.W, and MICKELSON, M,M,: Purification of lymphocytes and platelets by gradient centrifugation. J, Lab, Clin, bled, 72, 842-848, 1968. 190 IMANDT, Lo, GENDERS, T., WESSELS, H. and HAANEN, C.: An improved method for preparing platelet rich plasma. Thrombos. Res, 11, 429-432, 1977. 20. GERRARD, J,M. and WHITE, J,G,: The structure and function of platelets with emphasis to their contractile nature. In: Pathobiology Annual 1976, H.L. Iochim (Ed.) New York: Appelton-Century-Crofts, 1976, pp. 31-58,, 21. HUTTON, RoA., HOWARD, M.A., DEYKIN, D, and HARDISTY, R.M.: Methods for the separation of platelets from plasma. Thrombos, Diathes. Haemorrh. (Stuttgo) 31, 119-132, 1974,
18; Ltd.
149-158 1980.
Printed
in
the
United
States
oo49-3848/80/080149-lo~o2.00/0
ANTICOAG;yo;-FREE PREPARATIONOF PLATELETS FROM HUMAN NATIVE - A METHODICAL AND MORPHOLOGICALREPORT B. Hofmann, U. Till, J. Hofmann, W. Loesche, P. Spangenberg end G. Ostermann Medical Academy of Erfurt, Department of PathologicalBiochemistry, 506 Erfurt, Nordhauaer StraSe 74, GDR (Received 12.2.1979; in revised form Accepted by Editor F. Markwardt. Received by Executive Editorial Office
5.9.1979. 10.1.1980)
ABSTRACT Platelets are prepared from human native blood by a two-step procedure without use of anticoagulants.The first step involves Sephadex gel filtration of native blood to prevent clotting by the removal of plasma calcium. In a second step platelets are separated from other blood cells and plasma constituentsby centrifugation of the gel filtered blood on a Ficoll density gradient. During the whole procedure a pH of 7.4 is maintained. As a first measure for the cell integrity the morphological feature of the platelets was evaluated and compared to platelet-richplasma (PRP) which was prepared with different anticoagulantsat physiological pII.Considering the extent of pseudopod formation the anticoagulant-freeprepared platelets reveal a less activated state than the platelets in PRP.
INTRODUCTION In order to investigate biochemical and morphological parameters of blood platelets they have to be separated from other blood cells. Many separation techniqueshave been described all involving anticoagulationof native blood as the first step. This is achieved by mixing the blood with anticoagulating agents, followed by centrifugationto obtain PRP. Gel filtration or repeated washing procedures are used to transfer platelets into saline media (1,2).
Key words: blood platelets, anticoagulants,blood platelet cytology, gel chromatography,density gradient centrifugation. lb9
ANTICOAGULANT-FREE
I 50
Vol.18,Nu.
PLATELETS
I/:_
However, anticoagulants not only prevent coagulation of blood by their effect on plasmatic clotting factors. They may also interfer with platelet metabolism and response to aggreation inducers as well as cause morphological alterations It is desirable, therefore, to prevent contamination 7 3-7). with anticoagulants during platelet separation, particularly when the initial phase of platelet activation is going to be studied. A method was elaborated that substitutes anticoagulants by passing native blood through a gel filtration column. Platelets are separated from other cells and plasma by a one-step density gradient centrifugation on Ficoll. The anticoagulantfree platelets (AFP) obtained by this method were studied in respect to their morphological feature by means of interference contrast microscopy. The data indicate that AFP are less altered during preparation in comparison to platelets of a homologous PBP. Functional characteristics of AFP will be communicated in a forthcoming paper. MATERIAL AND METHODS Blood was obtained from healthy donors by puncture of the cubital vein. The donors had not taken any antithrombotic drugs during the last two weeks. The different steps in platelet preparation procedur8 are shown (schematically) in Fig. 1. They are all run at 30 C. All chemicals used were reagent grade.
STEP
REMOVAL OF PLASMA CALCIUM
A;
GEL FILTRA-
I
TION OF NATIVE 01 OOD
STEP 8: DENSITY GRADIENT CENTRIFUCATION
4
CELLS PLASMA
:
;.“‘\.,:
SEPARATION PLATELETS FIUERW BLQOD
PLASMA
FICOLL DENSITY GRADIENT
PLATELETS
OF
RED and WHITE CELLS
FIG. 1 Schematical representation of the steps involved in platelet separation. The 4-way system is made from a piacryl block (2.5x2.5xl.Ocm) having two channels with a diameter of 2 mm.
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Preparation of Gel Filtration Columns and Density Gradients: Sephadex G-25, particle size 100-300 p (Pharmacia, Sweden) or Molselect G-25, particle size 120-320 p (Reanal, Hungary), respectively, were prepared according to the manufacturer's instructions and equilibrated with the elution medium which contained 14.5 mM triethanolamine-HCL (pH 7.41, 126 mM NaCl, and 5.5 mM glucose. The concentra5.4 mM KCl, 0.98 mM I@$1 tion of calcium in the rngiiurnamounted to 5-10 /uM resulting from impurities. The gels were filled into siliconized glass columns (2.5 cm in diameter) to a height of 22 cm. The gel bed was supported by adaptors covered with nylon nets having a pore size of 40 p. The upper adaptor was adjusted 0.5 cm above the gel surface. For density gradient centrifugation a linear increasin gradient was mixed from Ficoll solutions (Pharmacia, Sweden $ containing buffered saline and human serum albumin. Since both Ficoll and albumin are contaminated with considerable amounts of calcium, which cause platelet aggregation and fibrin formation during centrifugation, these substances have to be freed from calcium before use. This was done by ion-exchange chromatography of a 40% (w/v) aqueous Ficoll stock solution on Dowex 50W x 8 (Serva, GFR) and gel filtration of a 20% (w/v) albumin solution using Sephadex G-25. By these procedures calcium concentration of the Ficoll and albumin solutions could be diminished from 300 uM to 75 PM and from 45 PM to 10 m, respectively. The linear gradient was mixed from 12.5 ml of each a 2% and a 20% Ficoll solution which were obtained from the 40% stock solution by dilution with buffered saline. The ionic composition of the gradient solution was the same as of the elution medium of gel filtration. Additionally, albumin was added to a final concentration of 0.35%. The gradient was placed into siliconized glass centrifuge tubes (50 ml) which were prefilled with 2 ml of the 20% Ficoll sol'ution. The density of the gradient was between 1.015 and 1.073 g/ml. Procedure: According to the scheme, step A (Fig. 1) the cannula used for the withdrawal of blood from the cubital vein is directly connected to the inlet of the Sephadex column by polyethylene tubes and a 4-way system. After venipuncture the air present in the tube system and the first ml of blood are removed through the free opening of the 4-way system. To achieve a sufficient flow rate during the application of blood a vertical distance between cannula and column outlet of 120 cm was adjusted. 12.5 ml of blood were applied and eluted at a hydrostatic pressure of 180 cm. Collection of the eluate was started when red cells appeared. The first 5 ml of the eluate were discarded. The next 25 ml were carefully layered onto the top of the pre-mixed gradient. Centrifugation was carried out at 1,000 x g for 5 min. The platelets formed a turbid band which could be drawn by aspiration with a small polyethylene tube under eye control. Protein and Lipid Determinations: To prove the separation of plasma proteins and lipids from the platelets after the Ficoll gradient centrifugation two methods were employed: a> gel filtered blood was applied onto the Ficoll gradient as usually. The gradient was fractionated into 5 ml portions, the fraction@
diluted 1:3 with isotonic saline, the platelets pelleted and the supernatant used for analysis. b) from the gel filtered blood a tiplatelet-poor plasma" was made, centrifuged on the Picoll gradient and the gradient fractions were analysed. Proteins were measured according to LOWRY et al. (8) after precipitation with trichloroacetic acid and resolving with NaOH. Cholesterol was determined enzymatically using cholesterol oxidase (9). Triglycerides and phospholipids were extracted with chloroform/methanol (10) and assayed enzymatically with a test kit from Boehringer (Mannheim, GFR) and by total phosphorus analysis (111, respectively. Preparation of PRP: To compare AFP with PRP both were prepared from the blood of the same donor. The anticoagulants employed for PRP preparation were used in the following final concentrations: sodium citrate 11 mb!, hirudin 150 ATU/ml, heparin 10 U/ml, and EDTA 3 n&I. Calcium Measurements: Calcium concentrations were measured by atomic absorption spectroscopy with an AAS 1 (VEB Carl Zeiss, GDR). The number of platelets in AFP and PRP was counted =%== by p ase contrast microscopy after appropriate dilution of the platelet samples with a mixture of one volume of 2% lidocain and two volumes of 1% ammonium oxalate, The morphological feature of the platelets was evaluated by interference contrast microscopy (NOblARSKI optic) with the photomicroscope Dokuval (VEB Carl Zeiss, GDR). For this purpose the platelet samples were fixed in 1.2% glutardialdehyde.
r
-I
FIG, 2 Removal of calcium from native blood by gel filtration on Sephadex G-25. Hematocrit (x---x>, platelet count (o---o>, and extracellular calcium concentration (-1 were determined in 5 ml fractions of the eluate, The block on the abscissa indicates the pooled cell peak.
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RESULTS In Fig. 2 the elution profile for native blood on Sephadex G-25 is shown. About the same picture was obtained when filtration was carried out on Molselect G-25. Platelets and red cells are eluted with the same volume which corresponds to the void volume of the gel. The cell peak is not clearly separated from the plasma calcium which may be caused by overloading of the column. Furthermore, the blood applied is not eluted as a uniform band, but the upper front of the band extends small "fingers". This phenomenon cannot be explained satisfactorily. Clotting on the gel is not responsible for it, because the same picture is obtained when titrated blood is passed through the column. However, the extracellular calcium concentration of the pooled cell peak amounts to less than 50 @I which is too low to mediate rapid clotting. Within l-2 hours after filtration no formation of clots was visible. As a consequence of filtration, cells and plasma proteins are diluted about 3-4 times compared to the native blood. About 80% of the platelets are recovered in the pooled cell peak. The time elapsed between venipuncture and the elution of the cells amounts tq 4 min. In Fig. 3 separation of platelets from other cells and plasma proteins on Ficoll density gradient is shown (step B in the scheme given in Fig. 1). Red and white cells are sedimented. Platelets form a turbid white band between the sediment and the plasma proteins at the top of the tube. A small zone shortly above the sediment contains aggregated platelets. According to the separation profile fractions 6-8 (5 ml each) were pooled and used for further investigations. About 50% of the platelets applied onto the gradient are recovered in the pooled fractions. The platelet count in the suspension amounts to 40,000-90,000 platelets/ ~1. The concentration of extracellular calcium was in the range of 30-50 @I. A small contamination of platelets with plasma proteins is observed and ranges to 1.5 mg protein/ml suspension, i.e. 2% of the original protein content in the native plasma. This could be proved when platelets were separated on an albumin-free Ficoll gradient or when a platelet-poor plasma made from the gel filtered blood was applied onto the gradient. With respect to plasma lipids about the same relative amounts were found to be associ-
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No. ot tractions
9
FIG. 3 Separation of platelets on a Ficoll density gradient. Platelet count (x---xl and extracellular protein concentration (-1 were determined in 5 ml fraction numbered from the top to the bottom of the centrifuge tube. The block on the abscissa indicates the pooled fractions. Albumin was omitted from the gradient.
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I/'
TABLE 1 Amounts of Plasma Proteins and Lipids in the "Platelet Fractions" of the Ficoll Gradient Proteins PhosphoCholTriglyclipids ester01 erides Concentrations
61.0
1,300
(pghl)
48.7
5.1
7.0
4.7
76 of total amounts
applied onto the gradient
6.2
9.7
The values were obtained from a centrifugation of a plateletpoor plasma made from gel filtered blood. "Platelet fractions" refer to the fractions 6-8 of Fig. 30 ated with the platelets after the centrifugation. The separation of plasma proteins and lipids in a representative experiment is indicated in Table 1. The presence of fibrinogen in the final platelet suspension could be concluded from the ability of the platelets to undergo ADP-induced aggregation (not shown here). The presence of fibrinogen is a necessary requirement for the ADP-induced aggregation (12). The morphological feature of AFP obtained by interference contrast microscopy was compared to platelets in native blood and to PRP prepared with several anticoagulants. Typical microphotographs are presented in Fig. 4 a-f. Comparing the different forms of platelets and the extent of pseudopod formation it is obvious that AFP do more resemble platelets in native blood than do platelets in PRP (Table 2); The smallest differences between AFP and PRP were found when PBP was made from titrated blood. Hirudin- and EDTA-PRP exhibit altered platelet forms in a greater extent. TABLE 2 Platelet Shape in Different Preparations Platelet Discozytes Platelets Platelets with l-3 with more preparation without pseudosmall pseudo- than 3 small pods pods or 1 long pseudopods Native blood AFP Citrated PRP Hepsrin-PRP Hirudin-PEP EDTA-PRP
83.0 26.7 16,3 29:: 0.8
17.0 z;*z 60:5 26.8 14.7
Irregular forms
216 14.4
317
26.0 63.4 80.0
?Z 4.5
The differences in platelet shape were examined by interference contrast microscopy counting 250 platelets per sample. The values are given in percent.
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FIG. 4 Microphotographs obtained by interference contrast microscopy of different platelet preparations. Samples were fixed in glutardialdehyde. a - native blood, after fixation the platelets were separated by centrifugation; b - AFP; c - titrated PRP; d - heparin-PRP; e - hirudin-PRP; f - EDTA-PRP. Original magnification 1,000 x. DISCUSSION
The method presented allows the preparation of human platelets from whole blood avoiding contact of platelets with anticoagulants as commonly used for platelet preparation. Gel filtration of platelet suspensions including PRP is a well characterized technique to separate platelets from extracellular components (13,14). Therefore, we applied this method
in order to free native blood from plasma calcium and to prevent clotting. For this purpose both Sephadex G-25 and Molselect G-25 were suitable. Attempts to separate both calcium and plasma proteins from the blood cells in one step using Sepharose gels proved to be unsuccessful because erythrocytes hemolyzed resulting in ADP-liberation. After the gel filtration of native blood, platelets were separated from other cells and plasma constituents by centrifugation on a Ficoll density gradient containing 0.35% albumin. The presence of albumin was necessary to avoid spontaneous platelet aggregation during the centrifugation. Besides, it was reported that the addition of albumin to saline platelet suspensions is important for the preservation of platelet integrity (15,16). Density gradient centrifugation on Ficoll is an established method for cell separation (1'7,18). The use of Ficoll for separation of platelets from other blood cells leads to a less contamination of the platelets with red cells and leucocytes than simple differential centrifugation (19). For the evaluation of the platelet morphology interference contrast microscopy was used. This method enables a better evaluation of the platelet shape with respect to pseudopod formation and changes of the smoothness of the cell surface than phase contrast microscopy. However, by light microscopical methods it is difficult to distinguish between real discoid and spheroid platelet forms, particularly, when the platelets have formed pseudopods. Therefore, we classified the platelets in discocytes without pseudopods and platelets with pseudopods. AFP reveal a smaller extent of pseudopod formation than PRP prepared with sodium citrate or other anticoagulants. That means that AFP are in a less altered state than the different kinds of PRP, although AFP are made by a two-step procedure and finally suspended in a saline medium. The platelet surface appears to be relatively smooth with small and evenly distributed granular structures which seems to be a normal attribute of the platelet surface (20). Usual methods to transfer platelets from plasma into a saline medium and to remove the anticoagulant are gel filtration or repeated washing procedures. Both of these procedures cause an increase of the morphological alteration of the platelets compared to the original PRP (13,211. A comparison between AFP and other saline platelet suspensions prepared by usual methods is difficult as the latter often starts from PRP which was prepared at unphysiological low pH values, e.g. using ACD as anticoagulant. Lowering the pH results in an inhibition of the platelet activation during the preparation procedure (I). Preparing saline suspended platelets under such conditions may provide less or comparable pseudopod formation as observed in AFP (13). The advantage of AFP is, however, that the physiological pH of 7.4 is maintained during the whole procedure and that the platelets have never had any contact with anticoagulants. The influence of anticoagulants on the morphology of platelets can be clearly derived from the data of the different kinds of PRP. The results presented here indicate that AFP may be a good tool to investigate the influence of anticoagulants on platelets in a more direct way as it was previously possible.
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Further, it enables us to exclude any anticoagulant effects on platelet functions being under investigation. However, before to do so, AFP have to be characterized in functional respect. This will be done in a forthcoming paper. ACKNOWLEDGEMENTS The authors wish to had the possibility vestigations in its We Danz and Leman. Raithel for their C.
thank the VEB Carl Zeiss, Jena (GDR) for out a part of the microscopical to carry laboratories with the helpful support of thank further Mrs. E. Michel, C. Btlttner skillful technical assistance.
having inDrs. and
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