BASICSCIENCES

Indium-I11 oxine,a chelateof In-i 11 with 8-hydroxyquinoline, was introduced in 1976 by Thakur andassociates as a label for granulocytes (1 ) and platelets(2). Since then we and others have optimized the variousplatelet-labeling parameters, performed comparativesurvival studies with Cr-SI-labeled platelets, and usedlabeled platelets in thrombosis research (3—8).However,platelet labeling in plasma with In-i I 1 oxine, as recommended by a panel on diagnostic application of radioisotopes in hematology (9), resulted in inconsistentand low labeling efficiency. Also, because of the poorsolubility of oxine in aqueous solution, a small amountof ethyl alcohol is needed as a solvent for In-i 11 oxine,but the probabilityof injuriouseffectsof alcoholonplatelet functions was demonstrated by Haut and Cowan(JO).

We havedevelopeda newwater-soluble,high-affinityplatelet label that has demonstrated a higher stability

ReceivedApr. 29, 1981; revisionacceptedJuly 20, 1981.For reprints contact: M. K. Dewanjee,PhD, Mayo Clinic, Roch

ester,MN 55905.

constant and a higher (olive oil/citrate buffer) partitioncoefficient than In-i i I oxine. The chelating ability oftropolone was used by Dyrssen (1 1) in the separation ofmetal ionsby solventextraction and by Pitt and Gupta(12) for iron removal in iron-storage disease. IndiumI 1i-labeled tropolone permits efficient, consistent, andconvenient platelet labeling in either an acid-citratedextrose (ACD)-saline or an ACD-plasma medium.We havealsodevelopeda kit for platelet labeling,whichmay lead to more widespread use of In-i I i-labeledplatelets in investigations of arterial thrombosis andthrombocytopenia.

MATERIAL AND METHODS

Preparation of sterile ACD anticoagulant kit. Fourgrams of anhydrous citric acid, 11.2 g of anhydrous tnsodium citrate (or i 2.78 g of tnisodium citrate dihydrate), and 6.0 g of dextrose were dissolved in 500 ml ofdistilled water. The solution was membrane filtered (0.22@.tm),and 8-ml and 25-ml aliquots were stored in sterile10-mI or 50-ml vials. The former are used for bloodcollection, the latter for preparation of the ACD-saline

Volume22,Number ii 981

RADIOCHEMISTRY AND RADIOPHARMACEUTICALS

Indium-i 11 Tropolone, A New High-Affinity PlateletLabel: Preparationand

Evaluationof LabelingParameters

Mrinal K. Dewanjee, Shyam A. Rao, and Paul Didisheim

Mayo Cilnic and Mayo Foundation,Rochester, Minnesota

Platelets were labeled with a new neutral, lipid-solublemetal complex of indium-ill and tropoione.Unlike oxlne, which mustbe dissolvedIn ethyl alcohol,tropoloneis solublein isotonicsaline.Platelet labelingwith in-I I I tropoionecanbe performedin bothacid-citrate-dextrose(ACD)-piasma and ACD•salinemediawfthintwo hours'time. Labelingefficiencyhas been 80-90 % in ACD-saIineand60-70 % in the ACD-plasmamedium.

Optimumconcentrationsfor the labelingof plateletswfthin-Ill tropoionewere5 @ig/mlIn ACD-saline and 10 @g/mlin ACD-plasma, using a 15-mm incubation atroomtemperature.A kit formulationfor convenientroutinepreparationof In-Illlabeledplateletshasbeendeveloped.Sevenparametersof plateletlabelingwerestudied:concentrationof tropolone,citrate, plasmaproteins,and calcium ions;also platelet density,temperature,and pH of Incubationmedium.Their effects onthe mechanismof platelet labelingwith lipid-solubletracersare discussed.

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DEWANJEE,RAO, AND DIDISHEIM

kit. The pH of the ACD solution was “—5.0and osmolarity was ‘@300mOsm.

Sterile ACD-Salinekit for platelet labeling with In-I 11tropolone. Thirty-six milliliters of ACD solution weremixed with 250 ml of sterile isotonic saline, and pH wasadjusted to 6.5, with i -ml aliquots transferred to a disposable polystyrene tube; 4.0 ml of sterile I N NaOHwas used to titrate. The solution was membrane filtered(0.22 zm) and aliquots of 10-30 ml were transferred tosterile vials. This stock solution contained 3.02 mg ofcitrate ion per milliliter. We find stainless steel needlesmore suitable than aluminum needles in the transfer ofi N NaOH solution. The batches were tested for osmolarity (“—‘300mOsm), sterility (culture for 7 days withbrain-heart infusion broth with 5% beef extract, and 5days with blood-agar medium), and apyrogenicity (Limulus amebocyte lysate test*).

Preparation of In-I I I tropolone. Eight to ten mg oftropolone t (weighed by microbalance) was dissolved byvortexing in sterile isotonic saline in a sterile polystyrenetube. The volume was adjusted to a concentration of 1@.ig/@tlof saline. The pH was adjusted to 7.4 with 0.1 NNaOH and the solutionwassterilizedby membranefiltration(0.22 @tm).Whenit wascappedat roomtemperature, we found that this stock solution yielded constant labeling efficiency for almost 3 mo.

Twenty microliters of tropolone solution was transferred to a 12-ml polypropylene tube with a sterile micropipette, and 300- 1,200 zCi of In- I 11 chloride wasadded dropwise. The solution was mixed for 2 mm, 3.5ml of ACD-saline was added, and the pH of the solutionwas adjusted to 6.5 with sterile 0. 1 N NaOH, using aprecalibrated pH meter. We found this solution of In-i 11tropolone in ACD-saline suitable up to I wk afterpreparation. We also found that the I-ml syringe witha polypropylene holder (No. 7022D) for the needle (27gauge, 1/2in. long) introduced minimal aluminum contamination in the preparation of ACD-saline. Ten micrograms of tropolone were used in the In-i 11 tropolonepreparation for platelet labeling in plasma, and the pHof the final In- 111 tropolone solution in a small volumewas adjusted to 7.0 with 0.1 N sterile NaOH. Sterilestock solutions of 0. 1 N NaOH and 0. 1 N HC1 for pHadjustment were kept in sterile polystyrene tubes in therefrigerator.

For the determination of optimum conditions ofplatelet labeling with In-I 11 tropolone, we studied sevenparameters: plasma proteins, citrate, tropolone, calciumion concentration, platelet density, temperature, and pHof incubation medium. Platelets were obtained from fivehealthy mongrel dogs and counted with a commercialcounter.@The details of each of these experiments aredescribed below.

Tropolone concentration and platelet-labeling efficiency. Fifty microcuries of ‘IIInCl3 was added to aliquots of stock solution of tropolone in saline; the pH was

adjusted to 6.5. Four-milliliter aliquots of ACD-salinestock (pH = 6.5) were added and 2.2 X iO@plasma-freeplatelets were added and incubated for 30 mm at roomtemperature. They were then washed with ACD-plasma.Labeling efficiency was determined after removal of thesmall fraction of platelet aggregates (less than 5% of totalradioactivity). For plasma labeling, the platelet pelletwas resuspended in 0.5 ml of plasma and transferred toIn-i I I tropolone in a small volume at pH 7. Labelingefficiency in percent was then determined by dividing theradioactivity of In- 111 in the platelet pellet by the totalradioactivity in the platelet pellet and washings, andmultiplying by 100. The radioactivity was measured inan ionization chamber.

In mostof theselabelingexperiments,dogbloodwascollected in ACD-anticoagulant (86 ml of blood/ 16 mlof ACD) with a 19-gauge needle. Platelet-rich plasmawas obtained by spinning the whole blood in a 40-mIsterile centrifuge tube (round-bottom polycarbonate,screw cap) at 200 g for 10 mm. Four to eight millilitersof platelet-rich plasma was transferred to a 12-mIpolypropylene centrifuge tube with screw cap. (Thesecaps maintain sterility during labeling and also preventloss of carbon dioxide and the consequent increase inplasma pH, which can adversely affect platelet function.)The platelet-rich plasma was then spun at I600 g toobtain the platelet pellet. For plasma labeling, theseplatelets were suspended in 0.5 ml ACD-plasma with asterile polyethylene pipette. After incubation for 15 mmwith In-I 11 tropolone at room temperature, 2.5 ml ofACD-plasma was added and the platelets were washedby spinningat I 600 g for 10 mm, then resuspendedandwashed again with 3 ml of ACD-plasma. Finally, anyresidual platelet aggregates were removed by spinningat 100 g for S mm. Residual red blood cells were lysedby incubation with 5 ml of 0.2% saline for 30 mm. Hemoglobin-associated radioactivity was removed bycentrifugation at 1600 g for 10 mm. The platelet-labelingefficiency was determined by measuring radioactivitywith an ionization chamber in the platelet-pellet washings and platelet aggregates. The only difference in labeling procedure between In- 111 oxine and In- 111 tropolone was that with the latter no washing of the platelet

•0

Platelet •°Iab&ing

efficiency(%) @o

20

0 10203040100 200 300 400 S00

Tropoloneconcentration( ,u.g/mI)

FIG. 1. Effect of tropolone concentration on platelet labeling inplasma and ACD-saline. Dashed line is platelet in-i 11 after erythrocytelysis.

.oo

982 THE JOURNAL OF NUCLEAR MEDICINE

--.@ OAcO.S.@

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100

80

60

40

20

of In-i i 1 tropoione solution (50 @tgof tropolone) wasthen added and final pH adjustments were made to theabove-mentioned values. Platelets, 2.2 X i0@, from 4 ml

of platelet-rich plasma were incubated in triplicate for30 mm at room temperature, and platelet-labeling efficiency wasdetermined.

Plasma incubation on release of In-i 11 label. Indium-i 1i-labeled platelets, tagged in ACD-saline orplasma medium, were resuspended in ACD-plasma andchecked for In-i 1i release during 6 hr at room temperature. Aliquots of platelets were centrifuged at 1, 2, 4,and 6 hr, and radioactivity in plasma and platelets wasdetermined with the ionization chamber. More than 95%of the In-i i I was bound to platelets at 6 hr after labelingin eitherACD-salineor ACD-plasma.

Aggregation of control and In-I I 1-labeled platelets.Blood of conscious, healthy mongrel dogs that had notreceived any drugs or participated in any experimentsin thepreviousmonthwascollectedfromajugularveininto ACD solution. Platelet countst of all tested suspensions were adjusted to 300,000/mm3 with autologousplatelet-poor plasma. Platelet aggregation (13) was then

examined at 37°C with a single-channel plateletaggregometer.11The ADP used was from equine muscle.To control any effect of aging on aggregation, plateletrich plasma and suspensions prepared from it were testedalternately and within 4 hr of the time of venepuncture.

RESULTS

Increasing tropolone concentration decreased platelet-labeling efficiency, with a peak value of about 5—6@zg/mlin ACD-saline and iO jzg/ml in ACD-plasma

(Fig. I). It is also evident from Fig. i that labeling inplasma rather than ACD-saline led to lower plateletlabelingefficiency.Increasingthe plasmaproteinsdecreased platelet-labeling efficiency (Fig. 2). Slight lossof labeling efficiency resulted after red-cell lysis forplatelets labeled in either ACD-saline or ACD-plasma,but the general shape and peak of tropolone concentration did not change.

The platelet-labeling efficiency increased with bothincreasingincubationtimeandincreasingtemperature(Fig. 3). The rate ofincrease in labeling efficiency with

0

FIG.3. Effectof temperatureandtimeof incubationonplateletlabeling efficiency.

PlateletLabeling

Efficiency(%)

0 50 100 160 200 260 300Platelet-poorplasma(pt/mI)

FIG.2.Eftectofplasmaproteinsonplatelet-labelingefficiency.

pellet with ACD-saline was necessary, thus eliminatingone step.

Plasma protein concentration. The effect of plasmaon platelet-labeling efficiency was determined by addingincreasing amounts of plasma to 2.2 X i0@platelets obtamed from 4 ml of platelet-rich plasma. Variableamounts of platelet-poor plasma were added directly to50 @Ciof In-i 11 tropolone containing 50 @tgof tropolonein 4 ml of ACD-saline. The platelets were labeled andwashed as before, and platelet-labeling efficiency determined.

Incubationtemperatureand time. This was studied byadding 2.2 X i0@platelets to 50 @Ciof In-i 11 tropolone(20 @gtropolone) in 4 ml ACD-saline. The platelet Iabelingwasdeterminedafter incubationat 4°C,roomtemperature (@-“25°C),and 37°Cfor periods of 5—120mm.

Numberof platelets. Platelets obtained from i , 2, 3,5, 8, and 12 ml of platelet-rich plasma were incubated

with In-i i 1 tropolone in 0.5 ml of plasma or 4 ml ofACD-saline at room temperature for 30 mm, and labeling efficiency was determined.

Concentration of citrate ion in ACD-saline. Citratechelates calcium ion and a host of trace-metal impuritiesin the incubation medium, and chelation of free Ca2+prevents activation of the coagulation cascade. Sincecitrate ion is an essential anticoagulant for plateletharvesting and labeling, we studied the effect of in

creasing amounts of citrate ion on platelet-labeling efficiency. Platelets, 2.2 X iO@,from 4 ml of platelet-richplasma were incubated for 30 mm at pH 6.5 with 50 zCiof In-i i 1 tropolone containing 50 j.ig of tropolone. Thecitrate ion concentration varied from 1.5 to 177 mg/ml,and platelet-labeling efficiency was determined.

Concentration of calcium ion in ACD-saline. Platelets,1.2 X iOu,from4 ml of platelet-richplasmawerecentrifuged to form a platelet pellet. Variable amounts ofCa2@ion were mixed with 50 @Ciof In-i 11 tropolone in4 ml of ACD-saline. These mixtures were added to theplatelet pellet and incubated for 30 mm at room temperature, and platelet-labeling efficiency was deter

mined.Hydrogen ion concentration in ACD-saline. The pH

ofACD-saline was adjusted to 5, 6, 6.5, 7, 8, 9, 10, andi I with 0.5 N HCI or 0.5 N NaOH. An aliquot of 50 @tCi

100

80

60

40

20. 25C

. 37C

PlateletLabeling

Efficiency(%)

20 40 60 80 100 120Timeof Incubation(mm)

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DEWANJEE,RAO, AND DIDISHEIM

100

80

60

40

20

100

80

PlatsIst80LabelIng

EffIciency(%) 4o

P@t.I.ILabeling

Efficiency(S)

20

0 04 08 12 16 20 24 2.8

Platelet denalty ( 109/ml)I 2 3 4 5 6

Ca@IonConcentration(mg/mI)

FiG. 4. Effect of platelet density on platelet-labeling efficiency.

incubation time was higher at 4°Cand lower at 37°C.No further gain in labeling efficiency was observed after60 mm of incubation.

With either ACD-saline or ACD-plasma as incubation medium, the labeling efficiency increased andreached almost a plateau value at 2.8 X i0@platelets.ACD-saline labeling led consistently to higher labelingefficiency (Fig. 4).

Increasing concentration of citrate ion decreasedplatelet-labeling efficiency (Fig. 5). If this curve is cxtrapolated to zero citrate concentration, the platelet labeling increases to its maximum value of about 87%.When unlabeled In- 111 tropolone was kept for a weekat roomtemperature,dissolvedin ACD-salinewith 3mg/mi added citrate, there was no loss of platelet-labeling potential.

Calcium ions at various concentrations did not affectplatelet-labeling efficiency when platelets were labeledwith In-I I I tropolone in ACD-saline (Fig. 6). There isalways excess citrate ion available to chelate excesscalcium ions. The platelet pellets were also easily dispersed in the ACD-plasma solution, although excessCa2+ was available for promoting aggregation.

The highest platelet-labeling efficiency was obtainedat pH 9 (Fig. 7) when platelets were labeled with in- I 11tropolone in ACD-saline. At this pH the platelet membrane may be most permeable because of reversible lossof phospholipid and cholesterol. At higher or lower pHvalues, the platelet-labeling efficiency decreased.

Similar effects of these parameters on platelet-labelingefficiency have been observed by several investigators(2—6)when platelets were labeled with In-I 11 oxine inTyrode buffer, ACD-saline, or ACD-plasma.

Aggregation of platelets to which tropolone was

FIG. 6. Effect of Ca2@ion concentration on platelet-labeling eff I-ciency.

added, in the presence of ACD-plasma or ACD-saline,was diminished in comparison with the parent plateletrich plasma (Fig. 8). The reduction was comparablewhether the In-i I 1 tropolone was added in the presenceof ACD-plasmaorof ACD-saline.Plateletaggregationwas also comparable when tropolone was used in placeof In-i 11 tropolone.

DISCUSSION

The structures of the three chelating agents, oxine,acetylacetone, and tropolone, are shown in Fig. 9. Bothoxine and tropolone molecules form five-membered octahedral and neutral complexes with In-i 11 and a varietyof other divalent and trivalent cations (1 J ) and are usedfor the analytic separation of metal ions by solvent cxtraction. Acetylacetone forms a six-membered ring withmetal ions. Most of these complexes are efficiently cxtracted at low pH in chloroform and other lipid-solubilizing organic solvents. The structure of the I :3 complex of In- I I I (tropolone)3 is shown in Fig. 10; indiumion loses its ionic characteristics as it is buried in theorganic envelope of tropolone. It may then diffusethrough a lipid membrane like a neutral ionophore. Ourpreliminary studies indicate that In-i I I tropolone hasa higher lipid solubility than In-i I I oxine and in-I I Iacetylacetone. The partition coefficient for olive oil/ACD-saline was found to be 3.54 ±0.28 for In-I I Ioxine, 7.93 ±I .04 for In- 111 acetylacetone,and I 8. I 8±I .79fortheIn-I I 1tropolonecomplexes.Thisfive-foldincrease in lipid solubility permits more efficient cellularextraction of In-i I 1 tropolone from both ACD-salineand ACD-plasma. Tropolone has been evaluated for thesequestration of ferric ion in iron-storage disease (12).

100

80

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40

20

100

80

LabelingEfficiency 60

(3@)

40

20

PlatelstLabelingEfficiency

(3@)

0 40 80 120 160

Citrate IonConcentration(mg/mI)

FIG. 5. Effect of citrate ion concentration on platelet-labelingeffI-ciency.

0 2 4 6 8 10 12

pH of C@nTr@p..j@5in

FIG.7. Effectof pHonplateletlabelingwithIn-i11 tropoloneinACD-salinesolution.

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/“TT\

BASICSCIENCESRADIOCHEMISTRYAND RADIOPHARMACEUTICALS

H2H3C C CH3 (V@

@ c\ii0 0 HO 0

Oxine Acetylacetone Tropolon

FIG.9. Structuresofthreechelatingagentsthatformneutrallipidsoluble complexes with In-i 11 cation.

The results of platelet labeling with excess citrate ion(Fig. 5) indicate that when platelets are labeled inACD-saline, the labeling efficiency will not be affectedby the metal-ion contaminants—for example, traces ofCd2+ ion from@ IIInC13; A13+ion from the aluminumneedle; or Ca2+, Mg2+, or Fe3+ from the saline solutionor the glass vial or residual plasma. The kinetics of reverse exchange leading to the formation of In-I I I citratemay be slow, but excess citrate might increase intracellular citrate ion. This might lead to the formation ofIn-i I i citrate, which could diffuse out again duringwashing (11).

Although several investigators (1—3)have suggestedchloroform extraction of In- 111 oxine, with removal ofchloroform by evaporation and redissolution in ethylalcohol before platelet labeling, we found this step unnecessary. Because In-i 11 ion is carrier-free, the formation of In-i i 1 oxine in the presence of excess oxineis assured. Although 75—95%of the In- 111 complex isextracted with chloroform, it is not known how much ofthe oxine is extracted along with the corresponding Ini 1i complexes, for we also found that most of theselipid-soluble tracers were absorbed to glass and polymersurfaces. Solvent extraction of In-i I I oxine or In- 1i 1tropolone and other manipulations thus introduce anunknown parameter about the level of oxine or tropolone,and these levels are of critical importance in plateletlabeling (Fig. 1).

Aggregation of platelets by ADP was reduced by thehandling procedures required to label the platelets withIn-i I 1 tropolone. The presence or absence of plasmaduringthelabelingproceduredidnotaffectthedegreeof reduction of platelet aggregation. Neither the In-i 11radioactivity nor the tropolone appeared to affect plateletfunction, inasmuch as the reductions of platelet aggre

UCC

EI,CC

I-.

Dl

-.1

DlC

I,CC

U‘a

FIG. 8. Typical aggregationtracing of control and labeled canineplatelets with ADP.As platelets aggregate, light transmittance increases. Left: normalplatelet-richplasma(PRP).Center(controls):platelet suspensions containing tropolone. Right (labeled): plateletsuspensionscontaining In-i 11tropolone. Top:tropolone addedinpresence of plasma. Bottom: tropolone added in presence ofAGO-saline. Time and concentration of ADP additions are mdicated.

Its toxicity is minimal in the microgram amounts usedfor platelet labeling. The LD50 of tropolone in mice byintraperitoneal administration was in the range 15—200mg/kg, and that of oxine by subcutaneous and intraperitoneal administration 30 mg/kg and 88.8 mg/kg,respectively (14). This toxicity is of no significance,because most of the tropolone carrier, like oxine, isprobably released as a result of ligand exchange insidethe platelets and is thus removed from them duringwashing with ACD-plasma solution.

The major advantage of In-i 1i tropolone is its solubility in isotonic saline; hence, no solvent that could adversely affect platelet function—such as ethyl alcoholin the case of oxine and HEPES or Tris buffer in the caseof acetylacetone—is necessary for cell labeling.

The decrease in labeling efficiency at low temperatures (Fig. 3) may be due to freezing of cholesterol andreduction of permeability at lower temperature. Whenplatelets are suspendedin ACD-saline, there is a rapidloss of cholesterol and phospholipid from the plateletmembrane (15—17).The enhancement of membranepermeability increases incorporation of In-i I 1 tropolone,and therefore labeling efficiency, when platelets are incubated in ACD-saline rather than plasma. Similarobservations were made by Scheffel and associates (3)when human platelets were incubated with In-i 1i oxinein plasma. For an investigator with minimal experiencein cell labeling, the ACD-saline medium might be moresuitable than ACD-plasma, because the former couldprovide higher labeling efficiency and viability and couldtolerate trace metals better than the plasma medium.

t1lndium@ (Tropolone)3

FIG.10.StructureofIn-ii i tropolone.Threemoleculesofmonovalent tropolone anion combine with one central trivalent In-i I 1ion to form an octahedral complex of In-i 1i(tropolone)3.

Volume 22, Number 11 985

DEWANJEE,RAO, AND DIDISHEIM

gation of control and labeled platelets were comparable.Thus the reduction in platelet aggregation appears to betotally explicable by physical handling (for example,centrifugation, resuspension) and not by the labelingitself. During labeling manipulations—especiallycentrifugation, pelleting, and dispersion—platelets mightlose some ADP and require more exogenous ADP fortheir aggregation. This loss of ADP from platelet organelles is a reversible process. Reimers and associates(15) pointed out that a partial degranulation processdoes not affect platelet survival. These observations arein agreement with our preliminary platelet-survivalstudies.

The intracellular ligand exchange between In-l 1ioxine and cytoplasmic protein was studied by Hwang(16) and by Pandian and associates (17). The timeintegrated perturbation factors can be related to therotational correlation time of the environment of theIn-i I I nucleus. Indium-I I 1 platelets showed rotationalcorrelation times of half to a third of that for In-i I ioxine. This suggests that, inside the platelet, the environment of the In-I 11 nucleus changes from that ofoxine to that of a large molecule. Our work on In-i 11oxine and In-I i I tropolone with red blood cells suggestedthat red-cell membrane retained 50-60% of the totalIn-l I I inside the red cell (18). This result suggests thatan equilibrium is established inside the cell whereby thephospholipid and proteins of membranes of cell and organelles share In-i I I along with cytoplasmic proteins.Perturbed angular correlation studies (16,17) of different In-i I I oxineand In-i I i-labeled plateletsalsosupport this hypothesis. It is only after lysis of the cellthat these In-I I 1-bound protein and in-i I 1-labeledorganelle fragments are released into the circulation andsequestered mainly by the reticuloendothelial system.

Our preliminary work suggests that both lipoproteinsand transferrin sequester the neutral In-I 11 complexes.As we increase the amount of plasma proteins, moreIn-I i I complex is incorporated into lipoproteins andtransferrin, and less is available for cellular uptake. Also,the variability ofcell labeling in plasma within the samespecies of animals could easily be accounted for byvariationsin theamountof lipoproteins,transferrin,andmetal ions in plasma.

Our preliminary studies with Sephadex gel chromatography indicate that the cytoplasmic protein carrierof In-I I I has a molecular weight of 50,000—55,000daltons. A similar molecular weight for In-I I 1-boundplatelet protein was reported by Hudson and associates(19) for platelets labeled with In-l I I oxine. Variationsof intraplatelet distribution of In- 111 were observed byEakins and associates (20) when platelets were labeledin ACD-saline and ACD-plasma. As long as In-I I I isbound to platelet organelles or cytoplasmic plateletproteins,and the membrane integrity is intact, plateletrecovery, survival, and imaging may not be affected

whether the platelets are labeled in an ACD-saline or anACD-plasma medium. The present knowledge of simplified platelet labeling with In-i 1 1, and the acceptable

radiation dose (21 ) for platelet-survival and imagingstudies, should lead to more widespread use of In-i 1ilabeled platelets in the future.

FOOTNOTES

S Microbiological Associates.

t Aldrich Chemical Co.

2Coulter ZBI or S-Plus counter.I Model 330,Chrono-LogCorp.

ACKNOWLEDGMENTS

The authorsgreatly appreciatethe encouragementof Heinz W.Wahner, M.D., and the expert technical assistanceof Mr. SushitalChowdhury,Mr. JamesA. Rosemark,and Mr. JohnQ. Stropp.

This investigationwassupportedin part by GrantsHL-24602 andHV-929I5 from the National Institutes of Health, Public HealthService.

REFERENCES

I. MCAn@E JG, THAKUR ML: Survey of radioactive agents forin vitro labeling of phagocytic leukocytes. I. Soluble agents.iNuciMed 17:480-487,1976

2. THAKUR ML, WELCH MJ, JOIST JH, et al: Indium-hIlabeled platelets: studies on preparation and evaluation of invitro and in vivo functions. Thromb Res 9:345—357,1976

3. SCHEFFEL U, TSAN M-F, MCINTYRE PA: Labeling ofhumanplateletswith [I I‘In]8-hydroxyquinoline.J Nuci Med20:524—531,1979

4. HEATON WA, DAvis HH, WELCH Mi, et al: Indium-ill:a newradionuclide label for studying human platelet kinetics.Br J Haema:ol 42:61 3-622, 1979

5. DEWANJEE MK, FUSTERV. KAYE MP, et al: Imagingplatelet deposition with ‘‘‘In-labeledplatelets in coronaryartery bypassgrafts in dogs. Mayo Clin Proc 53:327—331,1978

6. DIDISHEIM P. DEWANJEE MK, FASS DN, et al: Bloodcompatibility ofcirculatory assistdevices.First Annual ReportPrepared for Devices and Technology Branch, Division ofHeart and Vascular Diseases, National Heart, Lung, andBlood Institute, I 980. Report No. PB8O2026 09, availablefrom National Technical Information Service, Springfield,Virginia 22151

7. HEYNS AP, BADENHORSTPN, PIETERSH, Ctal: Preparation of a viable population of indium-I I I-labelled humanblood platelets. Thromb Haemost 42:1473—1482, I 979

8. SINN H, SILVESTER DJ: Simplified cell labelling with indium-i I I acetylacetone.Br J Radio! 52:758-759, 1979

9. By the Panel on Diagnostic Application of Radioisotopes inHematology, International Committee for Standardizationin Hematology: Recommended methods for radioisotopeplatelet survival studies.Blood 50:1137—1144, 1977

10. HAUT Mi, COWAN DH: The effect ofethanol on hemostaticproperties of human blood platelets. Am J Med 56:22—33,1974

I 1. DYRSSEN D: Studies on the extraction of metal complexes.XXI.Thecomplexformationofthoriumwithtropolone.AdaChem Scand9:1567-1574,I955

12. PITT CG, GUPTA G: The design and synthesis of chelating

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BASIC SCIENCESRADIOCHEMISTRY AND RADIOPHARMACEUTICALS

agentsfor the treatment of iron overloadin Cooley'sanemia.In Development of Iron Chelators for Clinidal Use: Prodeedingsofa Symposium. (DHEW Publication No. [NIH]76-994.)WF Anderson,MC Hiller, Eds.Bethesda,Maryland,U.S. Department of Health, Education, and Welfare, 1975,pp 137—168

13. BORN GVR: Aggregation of blood platelets by adenosinediphosphateand its reversal.Nature 194:927—929,1962

14. ALBERT A, HAMPTON A, SELBIE FR, et al: The influenceof chemical constitution on antibacterial activity. Part VII.The site of action of 8-hydroxy-quinohine (oxine). Br J ExpPathol35:75-84,1954

15. REIMERS H-J, KINLOUGH-RATHBONE RL, CAZENAVEJ-P, et al: In vitro and in vivo functions of thrombin-treatedplatelets. Thromb Haemost 35:151—166,1976

16. HWANG KJ: Modes of interaction of (In3@)-8-hydroxyquinoline with membrane bilayer. J Nuci Med 19:1162—1170,I978

/7. PANDIAN S, MATHIASCJ, WELCH MH: Useof perturbedangular correlation studies in the elucidation of the mechanism of indium-I I I labeling of human platelets.J Lab CompRadiopharmChem 18:67-68, 1981(abst)

18. DEWANJEE MK, RAO SA: Red cell membrane permeabilityand metal oxine-hemoglobin transchelation: Implications incell labeling. J Lab Comp Radiopharm Chem I 8:278, 1981(abst)

19. HUDSON EM, RAMSEY RB, EVATT BL: Subcellular localization of indium-I 11 labeled platelets. J Lab C/in Med 97:577—582,1981

20. EAKINS MN, BAKER JRJ, BUTLER KD, et al: Intracellulardistribution of 111-In in rabbit platelets labelled with I I I-In-oxine usingelectronmicroscopicautoradiography.ThrombHaemost 42:469, 1979 (abst)

21. ROBERTSON JS, DEWANJEE MK, BROWN ML, Ctal: Distribution and dosimetry of indium-I 1I-labeled platelets.RadiologyI40:169-176,I981

Volume 22, Number 1i 987

RADIOPHARMACEUTICAL SCIENCE COUNCILWORKSHOP ON

The Choice of the Appropriate AnImal Model in Radlotracer Design

January 28, 1982 PhoenIx Hilton Phoenix, Arizona

The Radiopharmaceutical Science Council will hold a one-dayworkshop held in conjunction with the midwinter meetin9 of the Society of Nuclear Medicine. The program for the workshop will include the following invited papers aswell as contributed papers.

Edward A. Carr, M.D. “AnimalModels for Study of Radiopharmaceuticals that are Substrates for(Catecholamine) Uptakei and Uptake2―

Brian M. Gallagher, Ph.D. “MonoclonalAntibodies: The Design of the Appropriate Carrier andEvaluation System―

Adrian Nunn, Ph.D. “SpeciesDifferences and the Need for Multiple Animal Models for Hepatobiliary Agents―Michael J. Welch, Ph.D. “LabeledCells―Leonard I. Wiebe, Ph.D. “OncologicalModels for Screening Potential Diagnostic Radiopharmaceuticals―

Participants are encouraged to make hotel reservations at the meeting site by contacting the Registrar, SNM, 475 ParkAve. So., New York, NY iOOi6. A registration fee of $35.00will be collected at the entrance to the meeting room.

For further information contact the Co-Organizers: William Eckelman, President, RPSC, or Richard M. Lambrecht,President-Elect, RPSC, do the National Office.

Abstracts should be sent to Richard M. Lambrecht, Ph.D., Dept. of Chemistry, Brookhaven National Laboratory,Upton, NY 11973.

DeadlineforthereceiptofabstractsIsJanuary2,1982.

SNM REFERRALSERVICEThe SNM Referral Service is accepting applications from employers and job applicants. The Service lists positionswanted and positions available in the following nuclear medicine fields: Physician, Technologist, Scientist, Commercial, and Other.

The fee forjob applicants is $5.00 for SNM members and $50.00 for nonmembers. For employers, thefee is $50.00foreach position listed.

To obtain more information and an application form, please write to:Referral Service

SocietyofNuclearMedicine475ParkAvenueSouthNewYork,NY 10016