ANNALS OF CL IN ICAL AND LABORATORY SCIENCE, Vol. 6, No. 2Copyright © 1976, Institute for Clinical Science

Cytology of Cerebrospinal Fluid: Technical and D iagnosis Considerations

B ER N A R D G O N D O S , M .D .

Department o f Pathology, University o f California,

San Francisco, CA 94143

ABSTRACTT he usefu ln ess o f c e reb ro sp in a l flu id cytology in th e d iagnosis o f m alig ­

nancy has b e e n e n h a n c e d in rec e n t years by th e d e v e lo p m en t o f n ew tech n iq u es for in c reas in g ce llu la r y ield . M icropore filters an d a specia lly ad ap ted cy tocen trifuge h av e b e e n u se d for th is p u rp o se w ith a re su ltin g in crease in ce ll co n cen tra tio n an d in d iagn ostic accuracy. C ereb ro sp in a l flu id exam ination has b e e n found to b e o f particu la r va lue in th e d iagnosis o f m etasta tic carcinom a, lym phom atous an d leu kem ic in v o lv em en t o f th e m en inges an d ce rta in p rim ary cen tra l nervous system tum ors. C ytologic c rite ria for th e d iffe ren tia l d iagnosis o f th e p rin c ip a l types o f tu m ors e n ­co u n te red in ce reb ro sp in a l flu id are d iscussed .

In tro d u c tio nT he ap p lica tio n o f cy to logic d e tec tio n

o f m alignancy to th e exam ination o f c e re ­b ro sp in a l flu id has b e e n w e ll e s tab lish ed over th e p ast tw o d ecad es an d th e re have b e e n s e v e ra l r e v ie w s on th is s u b ­je c t .2,5*6,7,9,11,13,18 N ev erth e le ss , th e va lue o f th is p ro ced u re has b e e n som ew hat lim ­ited b y th e g en era lly sparse ce llu la rity o f s p e c im e n s p r e p a r e d b y s ta n d a r d m eth ods an d b y th e re la tive lack o f ex­p e r ie n c e o f m o s t la b o r a to r ie s in th e c y to lo g ic e v a lu a t io n o f c e re b ro s p in a l flu id. In th e p ast few years, re fin em en ts a n d m o d if ic a tio n s o f te c h n iq u e h a v e b e e n in t r o d u c e d w h ic h p r o v id e in ­c reased ce llu la r con cen tra tio n an d e n a ­b le a m ore p rec ise an d easily u n d e rs to o d c lassifica tion o f th e ty p es o f m alig n an t cells found. T h e p u rp o se o f th e p re se n t rep o rt is to d escrib e an d com pare th e var­

io u s te c h n iq u e s a v a i la b le fo r th e cy to logic ex am in a tio n o f c e re b ro sp in a l flu id an d to d e fin e th e cy tologic c rite ria for th e d iagnosis o f d iffe ren t tu m o r types.T ech n iq u es

T h e s ta n d a rd m e th o d s o f cy to lo g ic ev a lua tion o f ce reb ro sp in a l flu id involve m icroscop ic exam ination o f th e sed im en t ob ta in ed fo llow ing cen trifu g a tio n o f th e flu id . T h e sp ec im en s m ay b e w e t film s s ta in ed w ith a dye such as to lu id in e b lu e or th e m ore u su a l sm ears fixed in 95 p e r­c en t alcoho l an d s ta in ed b y th e rou tine P ap an ico lao u te c h n iq u e . T h e w et-film p rep a ra tio n s can b e ra p id ly an d eas ily p re p a re d .7,9 M in im al c e llu la r d is to rtion is p re sen t. H o w ev er, if c e llu la rity is sparse as is th e case in m any C S F spec im ens, th e n u m b er o f cells p re se n t m ay b e in su f­fic ien t to e s tab lish a d iagnosis ev en in th e

152

CYTOLOGY O F CEREBROSPINAL FLU ID 153

case o f c le a r-o u t m alig n an cy . F u r th e r ­m ore, w et-film p repara tion s are no t p e r ­m an en t w h ich is a m ajor d isadvantage. M ore gen e ra lly u sed are th e fixed Pap- s ta in ed sm ears w h ich can b e re ta in ed in ­d efin ite ly . T h e y do suffer from lack o f su ffic ien t ce llu la r con cen tra tio n and th is has b e e n a s ig n ifican t d e te rre n t to th e w id e r ap p lica tio n o f ce reb ro sp in a l flu id (CSF) cytology.

B e c a u s e o f th is d i f f ic u l ty , n e w e r m e th o d s h a v e b e e n d e v e lo p e d to in ­crease th e ce ll recovery rate. T h ese have b e e n o f tw o m ain types: (1) m icropore fil­te rs for co n cen tra tin g cells an d (2 ) a sp e ­c ia l c y to c e n tr ifu g e in w h ic h a ir -d r ie d film s can b e p rep a red on a glass slide. T h ese te c h n iq u e s have b e e n d e sc rib ed in d e ta il e ls e w h e re .3,8*11-12,14 T h e d iscu s­sion h e re w ill b e lim ited to a com parison o f th e ir u se fu ln ess in eva lua tion o f C SF .

T w o ty p e s o f m ic ro p o re filte rs have b e e n u se d in th e cytology laboratory, th e M illipo re filter* an d th e N u c lep o re fil­t e r ! T h e M il l ip o re t e c h n iq u e , as em p lo y ed in o u r laboratory , u tilizes a 19 by 42 m m rec tan g u la r filte r w ith pore size o f 5 m/i,. U sing a 1000 m l filte rin g flask an d a suc tion dev ice p ro d u c in g m inim al n e g a t iv e p r e s s u r e , th e s p e c im e n is p o u red in to th e fu n n e l so as to ju s t cover th e filter. A fter s ta in in g w ith a m od ified P a p a n ic o la o u m e th o d , th e f i l t e r is m o u n ted on a glass slide an d a coverslip is ad d ed . S ince th e filte r i tse lf is sta ined , an o p aq u e b lu e b ack g rou nd is p resen t. T h e cells are w ell co n cen tra ted on th e filte r a n d c lea rly d efin ed . S light d isto r­tion is p re se n t ow in g to shrinkage.

T he N u clep o re is a la te r d ev e lo p m en t w h ic h e l im in a te s th e o p a q u e b a c k ­g ro u n d . T h e f i l te r is a d is c w h ic h is one in ch in d iam e te r w ith 7 to 8 m/i, pore size. P ores are v is ib le u n d e r th e m icro­scope w ith th is ty pe o f filter, occasionally

in te rfe rin g w ith screen ing . T his p rob lem can b e overcom e b y disso lv ing th e filte r w ith ch loroform ,10 b u t th e use o f th is toxic chem ical is som ew hat hazardous in th e cyto logy laborato ry . F u rth e rm o re , com ­p ariso n o f th e M illipo re an d N u clep o re filters has show n a sign ifican tly g rea te r c e ll c o n c e n tra t io n w ith th e M illip o re te c h n iq u e .1

T h e cy tocen trifuge | is o f spec ia l value for th e ro u tin e ce ll coun t and d iffe ren tia l o n C S F s p e c im e n s . S p e c im e n s a re p laced in th e cham bers o f th e cy tocen ­trifuge w ith glass slides in app ositio n to th e ex it ports o f th e cham bers an d c e n ­tr ifu g e d a t 1200 rpm for 10 m in . T h e slides are th e n a ir-d ried an d s ta in ed by th e W right-G iem sa m ethod. T h e p ro ce ­d u re is ra p id a n d s im p le , r e q u ir in g a m in im um o f in s tru c tio n to u se .4,14 C e llu ­la r c o n c e n t r a t io n is g r e a te r th a n b y ro u tin e cen trifugation , b u t less th an w ith filte r te c h n iq u e s .1 A m ajor d raw b ack is th e variab le an d som etim es co n sid e rab le ce llu la r d isto rtion th a t is p rod uced , p a r­tic u la r ly in n u c le a r ap p ea ran ce . W hile th e filte r te ch n iq u e s m ay p ro d u ce sligh t ce llu la r con traction , n u c lea r m orphology is w e ll p reserv ed . T h e la tte r is, o f course , a c r i t i c a l fa c to r in th e e v a lu a t io n o f m alignancy.

In o u r exp erience , th e M illipo re filte r p ro v id es co n sis ten tly h igh ce llu la r con­cen tra tio n an d satisfactory ce ll p re se rv a ­tion . A lth oug h so m ew h at tim e-consum - in g an d re q u ir in g carefu l a tten tio n d u r­in g p ro c e s s in g , th e s e a re c o n s id e re d m in o r d ra w b a c k s in r e la t io n to th e su p e rio r q u a lity o f p repara tion s ob ta ined . In co m p ariso n to cy to cen trifu g e sp e c i­m ens, th e filters are m uch eas ie r to in te r­p re t ow in g to b e tte r an d m ore co n sis ten t ce ll p rese rv a tio n and are m ore like ly to p ro v id e an accu rate d iagnosis ow ing to g rea te r ce ll concen tra tion .

* M illipore F ilte r Corporation, Bedford, MA. f G en era l E lec tric Com pany, P leasanton, CA.

t S h an d o n S c ien tific C om pany L td ., L o ndon , E ngland.

154 GONDOS

O u r g e n e ra l p ro c e d u re in h a n d l in g C S F s p e c im e n s is to p r e p a r e b o th a ro u tin e P apan ico laou -sta ined fixed sm ear and a M illip o re filte r. A th ird W right- sta in ed sm ear is p rep a red i f th e re is suffi­c ien t m ateria l o r if th e re is a c lin ica l in d i­cation o f lym phom a or leu kem ia . T h e ce l­lu la r c h a ra c te r is t ic s o f th e la t te r le n d th em se lv es to exam ination w ith W righ t’s stain. T h e M illipo re filte r is also qu ite sa tisfacto ry for th e e v a lu a tio n o f th e se cell types.

T h e advan tages an d d isadv an tag es of the d iffe ren t te ch n iq u e s are sum m arized in tab le I. A d e ta ile d com p ariso n o f th e ce ll re co v e ry ra te s w ith th e f ilte r an d cy tocen trifuge te ch n iq u es can b e found in a separa te rep o rt .1D iagnosis

T he types o f m alig nan t tum ors th a t can b e d iag n o sed b y C S F cytology fall in to th r e e m a jo r c a te g o r ie s : ( 1 ) p r im a ry tum ors o f th e cen tra l nervous system ; (2 ) leukem ias an d lym phom as in vo lv ing th e cen tra l nervo us system ; an d (3) m etas ta ­tic carcinom a. T h ese occu r in a ratio of app roxim ately 2 :1:2, as sho w n in tab le II. T h e figures in d ica ted are d raw n from th e re v ie w o f S p rig g s a n d B o d d in g to n ,13 w h ich in c lu d e d 72 cases w h e re m alig ­n an t ce lls w e re found . C o m p arab le r e ­sults have b e e n o b se rv ed in o u r m ateia l. O f 275 C S F sp ec im en s re c e iv e d in th e

U n iv ersity o f C alifo rn ia at San F rancisco C y to lo g y L a b o ra to ry o v e r a 12 m o n th p e rio d from Ju ly 1973 to Ju n e 1974, th e re w ere 26 in w h ich m alig n an t cells w ere p resen t, in c lu d in g 11 w ith p rim ary CNS tum ors, 5 w ith leu k em ia or lym phom a, an d 10 w ith m etasta tic carcinom a.

T h e p re sen ce o f m alig nan t cells in C S F is d e p e n d e n t on access o f th e tum or cells to th e m en inges an d v en tricu la r spaces. T h u s, th e re are ce rta in ty p es o f tum ors th a t are like ly to p ro d u ce positive C S F find ings. T his is gen e ra lly tru e o f m etas­ta tic tu m ors an d th e lym phom a-leukem ia group, w h ich m ay b eco m e d issem in a ted a n d in v o lv e th e m e n in g es . A m ong th e p rim ary CN S tum ors, m edu llob lastom a, b e c a u s e o f its in v a s iv e c h a ra c te r , fre ­q u e n t ly p r o d u c e s p o s i t iv e f in d in g s , w hile gliomas and m eningiom as are less lik e ly to do so. C e lls from tum ors o f glial o rig in can b e foun d w ith som e freq uency in flu id rem o v ed a t cranio tom y, b u t th is is less often th e case in spec im ens ob ta ined b y lu m b a r p u n c tu re . M en in g io m as, in sp ite o f th e ir lo ca tio n , ra re ly exfo lia te cells in to th e ce reb ro sp in a l flu id ow ing to th e ir re stric ted an d d e lim ite d grow th p a t­te rn .

In ou r m ateria l, th e p rin c ipa l d iagnos­tic p rob lem s en c o u n te re d in th e exam ina­tion o f C S F re la ted to four tum or types: ly m p h o m a a n d leu k em ia , m e d u llo b la s ­to m a, a s tro cy to m a a n d m e ta s ta tic car-

TABLE I

Techniques for Cytologic Examination of Cerebrospinal Fluid

Method Advantages Disadvantages

Wet-film (Toluidine blue) Fixed smear (Pap stain)Fixed smear (Wright's stain)

Millipore filter

Nuclepore filter

Cytocentrifuge

Simple, rapidSimple, good cell preservation Useful in case of leukemia,

lymphoma Good cellular concentration

Good cellular concentration

Rapid, simple

Scant cellularity, non-permanent Scant cellularityInadequate nuclear detail for other cell types

Cell distortion, complex, time- consuming

Cell distortion, complex, time- consuming, cumbersome

Poor cell preservation

CYTOLOGY O F CEREBROSPINAL FLU ID 155

Relative Frequency of Different Types of Tumors Producing Positive Cerebrospinal Fluid Cytology

TABLE II

Type Percent

Primary central nervous system neoplasm 38medulloblastoma, astrocytoma,ependymoma, meningioma, pineal teratoma

Leukemia and lymphoma 18Metastatic carcinoma 44

lung, breast, kidney, stomach, colon

cinom a. T h e d is tin g u ish in g cytologic fea­tu res o f th e se tum ors are sum m arized in tab le I II . S pecific p rob lem s in d iffe ren ­tial d iagnosis are d e sc r ib e d in detail.

C ells from lym phocy tic lym phom a an d le u k e m ia m u s t f i r s t o f a ll b e d i s t i n ­g u ish e d from c irc u la tin g ly m p h o cy tes . T h e la tte r re p re se n t a norm al com p onen t o f c e reb ro sp in a l flu id and , in m ost M illi- pore p repara tion s, sca tte red m atu re lym ­phocy tes are p resen t. In th e p re sen ce o f a m alig nan t ly m pho pro lifera tive d isorder, large n u m b ers o f lym pho id e lem en ts o f vary ing d eg ree s o f m atu rity are found. T h e m alignan t cells have large n u c le i, ir­regu lar n u c lea r o u tlines an d p ro m in en t n u c leo li (figure 1). D iffe ren tia tio n from m edu llo b lastom a is an im p ortan t con sid ­e ra tio n , s in ce th e tw o ty p e s o f m a lig ­nancy m ay occur in th e sam e young age g ro u p a n d b o th a re c h a r a c te r iz e d b y

TABLE III

Distinguishing Features of Malignant Tumors Most Often Found in Cerebrospinal Fluid

Leukemia, lymphoma

Medulloblastoma

Astrocytoma

Metastatic carcinoma

Large number single cells with hyperchromatic nuclei, little or no visible cytoplasm, individually distributed.

Groups of cells and single cells with hyperchromatic nuclei, little or no visible cytoplasm, molding of adjacent cell borders.

Generally few cells, single with vesicular hyperchromatic nuclei, granular cytoplasm, irregular cell borders.

Variable cell numbers, single and grouped, hyperchromatic nuclei, granular cytoplasm, cellular pleomorphism.

sm all ro u n d ce lls w ith h y p erch ro m atic n u c le i and scant cytop lasm . In th e case o f m e d u llo b la s to m a , c e ll a g g re g a te s a re p re se n t in w h ich th e re is m old in g an d su p erim p o sitio n o f ad jacen t cells (figure 2 ), w h ile in m a lig n an c ies o f ly m p h o id orig in th e cells are in d iv id u a lly d is tr ib ­u ted w ith o u t d irec t contact. C lin ica l h is ­tory is also usefu l in d is tin g u ish in g th e se tw o groups.

A stro cy to m a ce lls m u s t b e d if fe re n ­tia te d from norm al h istiocy tes an d m on o­cytes th a t are occasionally en c o u n te re d in C S F , since th e se d iffe ren t ce ll types are sim ilarly s ing le an d ovoid w ith e c c e n ­tric nucle i. T h e cells from an astrocy tom a differ in th a t th e ir shape is m ore ir reg u ­lar, th e n u c leo cy to p lasm ic ratio h ig h e r an d o th e r c rite r ia of m alignancy such as coarse ch rom atin c lu m pin g an d n u c lea r h y p e rch ro m asia are e v id e n t (figure 3). D is tin c tio n from m e ta s ta tic ca rc in o m a can b e a p rob lem , since th e la tte r m ay also occu r as s ing le cells. H o w ev er, ca re ­ful search w ill g en era lly revea l a t le a s t a few groupings in m etasta tic carcinom a, an d th ese are ch arac te rized b y re la tiv e ly little cy top lasm an d p leom orph ic a p p ea r­ance (figure 4). T h e m ost com m on p ri­m ary sources o f m etasta tic carc inom a are b reas t an d lung. In th e form er case, th e cells are sm all an d round , w ith arran g e­m en t in lin e a r or ball-like group ings, a l­th o u g h occasionally large s ing le ce lls can b e s e e n . M e ta s ta tic lu n g tu m o rs m ay sh o w sq u am o u s d iffe re n tia tio n , g la n d form ation or u n d iffe ren tia ted ap p ea ran ce d e p e n d in g on th e h isto log ic type . H isto ry is h e lp fu l in such cases, b u t on occasion in it ia l d iag n o s is m ay b e m ad e on th e basis o f C S F find ings. T h e p re se n c e o f ce ll g roups w ith h ig h nu cleo cy to p lasm ic ratios, n u c lea r h y perch rom asia a n d p rom ­in e n t n u c le o li p o in ts to a d iag n o sis o f m e ta s ta tic tu m or. C e lls from an e p e n ­dy m om a m ay p re s e n t sim ila r fe a tu re s , b u t th e d is tin c tio n can g en era lly be m ade in term s o f c lin ica l find ings.

1 5 6 GONDOS

+

Figure 1. A cute lym phoblastic leukem ia, cerebrosp inal flu id (CSF). P leom orphic single cells w ith large, irregular nucle i and scant cytoplasm are dense ly arranged, b u t not in d irect contact. N ote prom inent nucleoli. M illipore filter, Papanicolaou stain, (x 1000). F igure 2. C ells from m edulloblastom a, CSF, show ing sim i­larity to im m ature lym phocytes, b u t w ith clustering an d superim position of adjacent cells. C oarse chrom atin p a tte rn a n d n u c le a r p le o m o rp h ism a re e v id e n t. M ill ip o re f i l te r , P a p a n ic o la o u s ta in , (x 1000). F igure 3. Single cell from astrocytom a, CSF. N ucleus is slightly eccentric and show s prom inent chrom atin clum ping. C ell has irregular ovoid shape and granular cytoplasm. M illipore filter, Papanicolaou stain, (x 1000). F igure 4. M etastatic carcinom a of the breast, CSF. Note cell grouping, high nucleocyto- plasm ic ratio and nuclear hyperchrom asia. M illipore filter, Papanicolaou stain, (x 1000).

O th e r ty p es o f p rim ary C N S tu m ors w hich have b e e n en co u n te red in ce re ­b ro s p in a l f lu id in c lu d e m e n in g io m a , p in e a l te r a to m a a n d r e t in o b la s to m a . T h e s e h a v e d i s t in c t iv e c l in ic a l a n d cytologic features and, in the p resen ce of an ad eq uate ce llu la r sam ple and clin ical

history, do not p re sen t a p rob lem in diffe­ren tia l diagnosis.

A cknow ledgm en tPhotographic assistance was p rov ided by Richard

H. Renston, C. T. (ASCP).

CYTOLOGY O F CEREBROSPINAL FLU ID 157

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11. Ric h , J. R.: A m em brane filter te ch n iq u e of c e re b ro sp in a l f lu id cy tology. J. N eu ro su rg . 36:661-666, 1972.

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