Ultrastructural and Y-PCR analysis of trophoblast-like cells detected in peripheral maternal blood using antibodies and flow cytometry sorting

Biol. Cell (1991b 73 DNA STAINABILITY DEPENDS ON DNA QUANTITY AND DNA ACCESSIBILITY TO DYES

THYROID N E O P L A S M : ARE WE DOING ANY BETTER WXTH ~UANTXT&TXVEC~TOLOGY ?

v_Ac~~

LIAUTAUD FranQolse,

DUFER Jean, DELISLE MarieJoelle, CONINX Paul, Instltut Jean-Godlnot, BP ~71. 51056 Reims cedex. The d i f f e r e n ~ i a l

d i a g n o s i s o f t h y z o i d neoplasms

~hich can appear as quite difficult by routine =ytology prompted many authors to measure the nucleus DNA unsuccesfully (1,2). We therefore have though~ that image analysis could produce fureher information in orde~ to exclude the benign lesions. Seventy six patients whose thyroid cold nodule ~as surgically excised after fine needle asplraClon blopsy and examined by pathologlsC entered the s~udy (le : 56 ben~gn, -18 mallgnanC and 2 aeyplcal adenoma). A sac of 3662 cells from 33 benign cases was compared to another set of 1712 cells from 11 mallgnan~ nodules. The dlscrlmlnaEion begw~en the two populations was based on 4 nuclear features. From these da~8 the rate of nuclei which could be regarded as benign was computed for each case. The

average ra~e f o r t h e 33 benign c a s e s was 85%, In a p r o s p e c t i v e s~udy b e a r i n g on che 32 remaining patients, sensiClvlCy and specificity o f c o n v e n t i o n a l c y t o l o g y (CC) were r e s p e c ~ i v i l y 69% and 71% v e r s u s 92 and 88% f o r quancicaC~ve cytology (QC), However, logistic regression a n a l y s i s l n d i c a ~ e d tha~ CC (wi~h p • .0027) had a complementary r o l e to. ~ha~ of QC (p • .0002) In t h e d i a g n o s i s of ~hyrold neoplams. So f a r we have to consider both the me~hods as complementary t o each ocher. I ) 3OHANHESS£N J . , SOBRINHO-SIMOES H., T., TANGEN K. A.J.C.P., 1982, 77, 20-25.

2)

LXAUTAUD-ROGER F.. DUFER J . , DELISLE M.J., COHINX P. AnC~cancer 9, 231-234.

The detmninstion of cellularor nuclearD N A cement is one of the most common applications of flow cytometry (FCM) used by, researchers m monitor growth kineucs ~f cells and by cliniciansm

quannfy DNA content abnorn~dities and kinetics in tumor ceil pop~Intions. FCM of DNA conr~t can be done rapidly and conveniently using several fluorescent stains that bind smichinmetricellyand specifically to DNA. Propidium Iodide (Pl) is widely used in clinical zpplicl/ons, it forms complexes wire doublestranded D N A (and R N A ) by intercala,l,n 8 between base pairs. The

precision of measuremems may be ~ffe.~ by inslrumenml f a a ~ and by artifactsinlroduced M sample prepera(ionand sufiningas well as by eruebiologicvariance in cells populations. W e reporthere differentobservmons th.r emphasize the pwt played by chromatin slruct~al differences resulting in different degrees of accessibilityto PI. In physiologicalconditions,a decre~ed DNA staiubilKy is observed in human ejaculated spermatozoa which are c h ~ e d by the high degree of condensation of their cbrommn ; an incrwsed D N A stmn~bility,in relsion with metabolic

acuvity, is observed in routine bepaic ceils. In expenmauJ/ conditions, an increased DNA suunabil/ly of spermatozoa and lympbocytes is observed arts. thern~ deaamrl/on of DNA ( I$ nm al 97'C). In p.,hologiml conditions, an increased D N A swinabilitymay be observed in edenoma, m iaflammemry (issuesor in ~-~I bsfeaed cells, Several a ' i ~ a allow to distinguishthese ,bnormalities m D N A

sminsbUgy from q u a n ~ v e DNA content abnormalities (the DNA mde~ value, the missing of prolikming yetis, the non-Fopo~onal increase i. arm a.d pad( of fluorescence signal... ).

LIHDHO

PLUOT ~ . , Res., 1989,

ULTRASTRUCTURAL AND Y-PCR ANALYSIS OF TROPHOBLAST.LIKE CELLS DETECTED IN PERIPHERAL MATERNAL BLOOD USING ANTIBODIES AND FLOW CYTOMETRY SORTING, METEZEAU Philinne (I), BRUCH Jean.Fr(M~ric (2). GARCIAFONKNECHTEN-Nicole (3), KIEFER H~l~ne (1), JULIEN Christophe (4). RICHARD Yolnnde (5), KITZIS A lain (3), HS! BaeLi (6) and PAP[ERNIK Emile (7). !. lrutitut t'asteur, Paris, 2. Instimr P. & M. Curie, Paris, J. Instimt Cochin de &~n~tique moldculaire, Paris, 4. Couhronies SA Margency, J. INSERM U. IJl, Clamart, 6. IN$£RM U, 210, Nice, 7. IN$KRM U. 187, Paris. We report the detection of u'ophoblut-like cells (TLC) in the maternal circulation by using three monoclonal antibodies against uropboblast (GBI7, GB21, GB25) and flow cytomeury. Sone~i TLC from maternal blood of mothers carrying male (n=3) or female (n=2) fetuses at 28-38 weeks gestation were processed for electron microscopy and fetal DNA mnplificationof the Y-specific sequences. At the uimtserucmral level, most of the nucleated cells had the morphology of leucocytes suggesting maternal contaminants and we didn't find the characteristic features of the free intervillous tmphoblm cells. Nevetheless, PCR analysis showed an m~plification of Y-specific sequences in 2 out of 3 samples of enriched maternal DNA from mothers carrying male fetuses. However, Y specific sequences could not be detected in 2 samples of unenriched maternal DNA prepared from mothers carrying male fetuses. These results suggest that beside the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non invasive prcna~ diagnosis of fetal sex and sufficient Y (+) nuclei are found the diagnosis of selected num~cai chromosomes abnormalRies.

Ma

L~YB~

ILabor~oire d ~'.~olo~'e.Facolt~de M~dec/ne.34000/tlontpe~'e~. 2Sersicede C)~omt~'ieen Oar C7~7"S..2.YO00Be~ancon

In conclusion, these observ~ons conlirm the risk of detecuon of artef~ctualDNA quanritmve abnormalities through F C M analysis on the one hand and open new fieldsof F C M im,'estigatio~ through s qualitativeand a func(ionalstudy of D N A contenton the other hand.

PURIFICATION OF MOUSE CHROMOSOMES BY FLOW.CYTOMETRY: CONSTUCTION AND CHARACTERIZATION OF A GENOMIC LIBRARY FROM CHROMOSOME 19. BARON Bruno, ~'TF.~EAU Philine~ KEFER H~l~ne and GOLDBERG l~chel. ServiceINSERM.PASTKUR de Cyton~trieanalytiqueetprcfpararive, U n M de Bioc~nie Cellulaire,Ins:innPasteur 73013 Paris. As mouse chromosomes are quite similar in size, their flow ksuTotypes present only $ to 6 isolated peaks of fluorescence, precluding the separudon of individual chromosomes. In order to resolve this limitation, we established cell lines from cells including chromosomes issued from Robertsonian ~nslocations: their flowkaryotypes presents isolated peaks of fluonDscencospecific of normal mouse chromosomes. The sorting of mouse chromosomes X and Y (1) was done in that way, allowing the construction of their specific DNA libraries. We have also sorted mouse chromosomes 19 and constructed a DNA specific library containing about 3.104 cloned EcoRi restriction fragments. Flow-blot analysis of clones from the library showed that 8 clones on 9 must be issued from sorted chromosomes 19, which attests the purity of the library (2). Clones of the library were positioned on the chromosoa~ lg by analysis of the segregation of Restriction Fragment Lenght Polymorphism, allowing to improve the genetical map of this chromosome. (I) (2)

Baron,B., M~t4zeau.P., Hnmt, D., Robem, C., Goldbera, M. E.. and Bishop,C. (1986)Sorest. Celt Melee. Genet. 12, 289-295 Baron.B.. MP~au. P.. Kiefer~achelin,H. andGoldbetS.M. E. (1990). Biol. Cell. 69: I-8.