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Experientia 39 (1983), Birkhauser Verlag, CH-4010 Basel/Switzerland 121the vial, and put in to a fi re-proo f vessel inside the 1st tubein the presence of the 02 flow. The organic matrix of thesample was burned by placing the tube in a furnace whosetemperatur e reached 900 ~ After 10-15 min, the halogenswere carried by the 02 flow to the HMD resin, whichretains Br2 and C12, bu t not 12. 12 is f ixed by the Ag-wool,forming AgI. Finall y we placed the Ag-wool in a suitablevessel, in order to get the same counting geometry forstandards and samples treated with radiochemical separa-tion. The sensitivity of this method allows the mea sur ementof iodine conce ntrations as low as 0.02 ppm .R e s u l t s a n d d i s c u s s i o n . In the table we summarize theresults obtained; iopamidol is quickly eliminated from themost significant tissues. 15 min after its adminis trati on mosthas already passed through the kidneys and can be found i nthe bladder (see X-ray photograph). After 1 min we findthe greatest amount of iopamidol in the kidneys(15,000+ 1500 ppm); with blood, thyroid, liver an d br ainlevels following in decreasing order. With regard to thedecrease in the hematic level, diffusion of this comp ound toextravascular compartments seems to be slow; in fact wecan see that the amount of iopamidol in the blood remainsconstantly much higher than expected relatively to theamount in the liver and brain. Even total thyroid iodinelevels remai n significantly lower unti l thyroid basal valuesare reached. Brain iodine values remain much lower thanhematic values and 24 h after injection near basal levelshave been reached, suggesting that iodine measured in thebrain is due to iodine present in residual blood in thecerebral vessels and that iopamidol does not pass theblood-brain barrier. On the contrary, some extravasculardiffusion is seen at the hepatic level, where measuredvalues, even though remaining constantly lower thanhematic values, are significantly higher than those measur edin the brain and the elim inati on kinetics seem to be similarto those seen in the blood. This phenomenon could be dueto the structure of the liver itself, where plasma is known tofilter freely through the sinusoidal plexus into Disse space.The percent increase of iodine with regard to basal values isgreatly inferior for the thyroid when compared to othercompartments of the organism and after 24 h iodineconcentration has almost reached basal values. Subsequentexperimental data demonstrate a return to original values.From the results obtained in our study, the 60 day waitingperiod usually required before testing thyroid iodine incor-poration in patients who have undergone iodate RCMadminis tra tion seems to be excessive.Two months later, the sensitivity of the method employedallows us to ascertain that blood iodine levels are onlyslightly higher than basal values. At this time we can see

even less additio nal iodine present in the liver, but consid-ering the incidence of statistical error, this experimentalvalue can be considered as equal to physiological values.On the whole our results show that iopamidol is quicklyeliminate d and not likely to accumul ate in the most signifi-cant tissues; moreover it has no effect on thyroid iodineuptake.

1 Acknowledgments. The authors wish to acknowledge use ofthe nuclear reactor offered by Prof. Orvini, L.E.N.A., Paviaand the invaluable suggestions provided by Prof. G. Cittadiniand by Dr M. Gallorini. Moreover the authors wish to ac-knowledge the accurate revision of the English draft offered byDr S. Raffanti.2 Reprint requests to S.S., Istituto di Radiologia, Clinica Chirur-gica, Universith degli Studi V ia le Benedetto XV, 10,I- 16132 Genova (Italy).3 Cittadini, G., Ingrami, D., Piola, F., and Sartoris, F., Radiolo-go 16 (1977) 33.4 Levin, A.R., Grossman, H., Schubert, E.T., Winchester, P.,and Gilladoga, A., Am. J. Roentg. 10 5 (1969) 777.5 Mc Clennan, B.L., and Becker, J.A., Am. J. Roentg. 11 3(1971) 427.6 Meyer,M.W., and Read, R. C., Radiology 82 (1964) 630.7 Rapoport, S.I., and Levitan, H., Am. J. Roentg. 12 2 (1974)186.8 Alm6n, T., J. theor. Biol. 24(1969) 216.9 Rosa, M., Radiologo 19 (1980) 78.10 Pitr6,D., and Tirone, P., Farmaco 10 (1980) 826.11 Pitr~,D., and Felder, E., Invest. Radiol. $ 15 (1980) 301.12 Dean, J.A., Chemical separation methods. Van NostrandReinhold, Wokingham, Berks 1969.13 Morrison,G.H., Trace analysis. Physical Methods IntersciencePubl., New York 1965.14 Birks, J.B., The theory and practice of scintillation counting.Pergamon Press, Oxford 1964.15 Bertolini,G., and Coche, M., Semiconductor detectors. NorthHolland, Amsterdam 1968.16 Wat t, D.E., and Ramsden, D., High sensitivity counting tech-niques. Pergamon Press, Oxford 1964.17 Rapporto Finale di Sicurezza L.E.N.A., 1964.18 Price, W.J., Nuclear radiation detection. Mc Graw-Hill Book,New York 1958.19 Bonati , F., and Felder, E., 14th int. Congr. Radiol., Rio deJaneiro, Oct.23-29, 1977.20 Garlaschi, G., and Sartoris, F., Radiologo 19 (1980) 62.21 Lasser, E.C., Radiology 91 (1968) 63.22 Leroux,G.F. , J. Radiol. l~lectrol.56 (1975) 129.23 Mallarini, G., 7th int. Congr. of Lymphol. , Florence,Oct. 28-Nov. 2, 1979.

0014-4754/83/010119-0351.50 + 0.20/09 Verlag Basel, 1983

A holder for Ralph knives for cutting sec tions for high performance optical micr oscop y with a Mino t typem i c r o to m eD . Chappard and J.-L. Laurent lL a b o r a t o i r e d e B i o l o g i e d u T i s s u O s s e u x , U . E . R . d e M O d e c i n e , 3 0 , r u e F . G a m b o n , F - 4 2 0 2 3 S a i n t - E t i e n n e C e d e x ( F r a n c e ) ,Ju ly 1 4 , 1 9 8 1S u m m a r y . We present plans for an original holder for long-edged glass knives (Ralph knives). Knives are prepared byhand- break ing of commercial window glass (3 mm thick), for cutting large blocks of tissue emb edded in a water-mis cibleplastic resin, glycol methacrylate.Recent years have seen the development of high perfor-mance optical microscopy (HPOM) which allows a clearvisualization of the intimate structure of cells and tissue

organisation by standard light microscopes. HPOM makesuse of plastics as embedding media, instead of paraffinwax, which has been the traditi onal embeddin g med ium for

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122 Experientia 39 (1983), Birkhauser Verlag, CH-4010 Basel/Switzerlandgenerat ions of his tologis ts . S ince polymers produce lessd i s tor t ion and shr inkage than pa r a f f in , mor e de ta i l s a r enow obse r ved when l igh t mic r oscopy i s under taken . Suchhigh r eso lu t ion mic r oscopy wi l l f i l l the we l l - known gapbe tween t r ansmis s ion e lec t r on mic r oscopy and l igh t mic r os -copy . Var ious p las t ic embe dding med ia have been success -f u l ly in t r oduced f or HPOM , f or example methylm ethacr y-la te, N- buty l me thacr y la te , a r a ld i te and epoxies ( see r ev iewby Benne t t2) . However , g lyco l me thacr y la te ( GMA) , andvar ious mix tur es composed of GMA, a r e the mos t eas i lyhandled because of the i r wa te r - so lub i l i ty and the poss ib i l i tyof obt aining semi- th in sect ions ( i .e . : 1- to 3 ~tm in thickness)on a convent iona l Minot r o ta r y mic r o tome wi th la r ge a r eat issue blocks . We have recently descr ibed a method for thepur i f ica t ion and use of G M A 3 f or HPOM .Sectioning plas t ic blocks is eas ier with glass knives thanwi th s tee l kn ives . L indn er and Richar d 4 and o the r s 5 have

emphas ized the super ior i ty of long- edg ed g lass kn ives overthe shor t c lass ical t r iangu lar glass knife o f Latta andHa r tm ann 6 (6.5 mm in length) w hich is used for e lectronmicroscopy. Large glass knives were f ir s t introduced inh is to logy by Benne t t ( op . ci t .) a s 'Ra lp h Kniv es ' in honor ofDr P . Ra lph who invented the pr ocedur e f or the f r ac ture ofg las s. The i r o r ig ina l me thod makes use of hand br okenlarge s tr ips of f la t glass 6.5 m m thick.I n Sem ba ' s hands I, and in our own exper ience , i t was f oundto be dif f icult to obtain sui table knives by this technique.Al though devices have been desc r ibed f or mechanica l lyscor ing and breaking glass s tr ips4'5 we consider this proce-dur e r a the r expens ive . The method desc r ibed by Semba(op. c i t. ) with thin ner glass sl ides , 1.0-1.4 mm thick, gave usful l success, especial ly when a s tand ard win dow glass(3 mm thick) was used. We have been able to obtainexce l len t ly sha r pened Ra lph kn ives wi th an edge 20- 50 mm

36 5'

II

612 ,I . 3 6 - ~ IT Z 1 5 12 mic ro tome 1stee l knife holder

[ Ra lph k n i f e

in the main rod

S.crew rod ~ 5 fixed inhe main rod and in thbutterf ly nut ~ -- -A A SECTION

A

Hans for the Ra lph knife holder: A U pper view: all dimensions aregiven in mm. ~ = diameter. T he screw rods are fixed by Araldi te orLoctite. B Lateral view: section through AA. C Holder with aRalph knife in position for sectioning.

to~

but terf ly I LA A S E C T I O N ~ _ ~long; 25 mm was found to be the most sui table length forthe Leitz 1512 rotary microtome, be cause o f the des ign ofi ts s teel knife-holde r .Bennett (op. c i t . ) used Ralph-knives by gluing them on tos teel bars with a heat sof tening cement (dental cement forexample ) . Semba ( op . c i t . ) r ecommended a pa r a f f inmethod of adhes ion . However , these 2 techniques a r e t ime-consuming and need the pr epar a t ion of a l a r ge number ofknives before sect ioning.Several holders have been descr ibed for glass knives ;Behnke s and Shaw 9 have descr ibed holders for the tr iangu -la r La t ta and Har tm ann knives . A holde r i s ava i lab le fr omLei tz f or the 1515 MOT r o ta r y mic r o tome , which acceptsglass knives with an edge 12 mm long. G oryc ki 1~ hasadapted an u l t r a - mic r o tome holde r f or long kn ives andSzczesny 11 is the o nly one who has present ed plans for aholde r f or Ra lph kn ives pr epar ed accor d ing to Benne t t (op .cit.).W e have f oun d th i s la s t ho lde r exceedingly d if f icu l t tohandle . W hen i t s edge has de te r io r a ted , a g lass kn if e mus tbe changed ; with Szczesny' s holder , one m ust remove i tf rom the microtome, loosen 4 screws, change the knife and

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Experientia 39 (1983), Birkh~iuserVerlag, CH-4010 Basel/Switzerland 123make a new adjustment. To overcome these problems, wehave designed an original type of holder for Ralph knives25 mm long and 3 mm thick, to be used on a Leitz 1512microtome. We prefer standar d window glass, scored with adiamond and hand-broken. We present here detailed planswhich can be used by any workshop. The dimensions werecalculated for the Leitz 1512 microtome. The adapta tion ofthe holder for other microtomes like the Minot rotary typeis also possible by adjusting the dimensions.The Ralph knife is held by 2 lateral posterior clamps whichare firmly tightened by 2 anterior butterfly nuts. Thebacking bar is placed in the microtome steel-knife holder.Such a mounting does not require removal of the holderwhen the knife has to be changed. Alig nmen t and appr oachare not modified. These plans for an original holder forhand-broken Ralph glass knives may seem a little difficultor confused to a histologist, and we apologize in advance;the plans were designed by a professional industrialdraught sman for direct use by the workshop.

1 Acknowledgments. The authors are greatly indebted to MrG. Grange who designed and made the prototype. They wishto thank Miss C. Monzy for her help in preparing the manu-script.2 Bennett, H.S., Wyrick, A.D., Lee, S.W., and McNeil, J.H.,Stain Technol. 51 (1976) 71.3 Chappard, D., Laurent, J.L., Camps, M., and Montheard,J.P., Acta Histochem. 71(1982) 95.4 Lindner, M., and Richards, P., Sci. Tools 25 (1978) 61.5 Butler, .K., Stain Technol. 54 (1979) 53.6 Latt a, H., and Hartmann, J.F., Proc. Soc. exp. Biol. Med. 74(1950) 436.7 Semba,R., Stain Technol. 54 (1979) 251.8 Behnke,O., and Rostgaard, J., Stain Technol. 38 (1963) 299.9 Shaw,S.R., Stain Technol. 52 (1977) 291.10 Gorycki,M.A., and Sohm, E.K., Stain Technol. 54 (1979) 293.11 Szczesny,T.M., Stain Technol. 53 (1978) 50.

0014-4754/83/010121-0351.50+ 0.20/09 Verlag Basel, 1983

A microquantitative meth od for investigating the interaction betw een labeled lectins and the surface mem branesof human lymp hocytes using a semi-au tom atic harvesting m achineM.A. Jacquet and A. SharifCNRS LA 293, I N S E R M U 180 Biologie et Pathologie mol6culaires des glycoprot6ines, Facult6 de Mddecine, 45, rue desSain ts-Pbres, F- 752 70 Paris C~dex 06 (France), Jan uary 25, 1982Summary. We describe a simple, rapid and economical method for the study of the interaction of labeled lectins and thesurface membranes of human lymphocytes using a semi-automatic harvesting machine (Titertek Multiple Cell Harvester).The procedure requires both small numbers of cells and small amounts of lectin, moreover it reduces the number ofexperimen tal steps required.Lectins are bi-or mul tivalen t proteins which are foundpred omina ntly in the seeds of some also3 plants and occur ininvertebrates and m amma ls ' . These lectins bind to specif-ic cell surface saccharide determina nts and induce lymph o-cyte stimulation and cell agglutination2. It is generallyaccepted that the effects of lectins on the cell presuppose aprior binding to the glycoprotein receptor sites located onthe cell surface 4. Because of their part icula r prope rties,lectins have been found to be useful molecular probes forinvestigating the structure, topology and mobility of plasm amembr ane saccharide-bearing components5,6. Inhibit ion ofthe lectin-cell surface inter action with saccharides an d cellsurface components has been used to investigate the sac-charide specificity of lectins and also to evaluate the lectin

receptor activity of cell macromolecules. Such studies havebeen done using lectins labeled with radioactive com-pounds, and involve large amoun ts of both lectin and cells.Moreover, a long and repetitive centrifugation procedure isrequired 7's. In the present c ommu nicat ion we present asimple, rapid and economical procedure for studying theinteract ion of labeled lectins and the cell surfaces of huma nlymphocytes using a semiautomatic harvesting machine(Titertek Multip le Cell Harvester).Materials and methods. 1. Lectins. Concanavalin A(grade III) was obtained commercially from Sigma Chemi-cal Co.; Robinia lectin, Ricinus communis lectin (Var.sanguineus agglutinin, RCA 120) and anti-Robin ia lectinserum were prep ared by me thods described elswere 9' 11.

0~5Pg0.40.3

19 0.2g 0.1

J2 25 Total 3H ~ec hn adde d Z5 10~ug