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986 Alan R. Liss. Inc. Cytometry 7101-103 (1986)

TECHNICALNOTE

A Protocol for Papanicolaou Staining of CytologicSpecimens Following Flow Analysis

Todd K. Berkan, Jay E . Reeder, Peter A. Lopez, Jr., Kevin M. Gorman, and Leon L. Wheeless, Jr.Analytical Cytology Unit, Department of Pathology and Laboratory Medicine (T.H.B., J.E.R., L.L.W.), Department of

Biology K.M.G.),University of Rochester Medical Center, Rochester, New York 14642, nd Fox Chase Cancer Center,Cell Sorter Facility (P.A.L.), Philadelphia, Pennsylvania 19111

Received for publication April 1, 1985; ccepted August 7, 1985

A protocol has been developed for restainingcytologic specimens th at have been analyzed

on a multidimensional slit-scan l o w system,The technique involves Papanicolaou stain-ing of cells on membrane filter th at hasbeen previously stained with acridine orangeand fixed with glutaraldehyde buffer. Thespecimen and staining solutions were sequen-tially added to a 5-micrometer pore size, 47-mm diameter Gelman Metricel filte r whileit remained in a glass filtration apparatus.

The practice of retaining the filter in thefiltration apparatus throughout the stainingprocedure minimizes cell loss and eliminates

specimen cross contamination when com-pared with conventional filter dip staining.

The availability of thi s postflow specimen Pa-panicolaou staining protocol permits accu-rate determination of th e performance char-acteristics of a multidimensional slit-scanflow system and should be useful wheneverstaining o a limited number o cells withminimal cell loss is desired.

Key terms: Multidimensional slit-scan, Pa-panicolaou staining, postflow specimens, ac-ridine orange, cell loss

procedure has been developed for eval uati ng post-flow analysis aliquots of cytologic specimens analyzedon a multidimensional slit-scan MDSS) flow system (1,8).This procedure is used in assessing the performance ofthe flow system in screening cytologic specimens fromthe female genital tract and urinary bladder for carci-noma or its precursors,

The postflow aliquot is collected through the exit portof the MDSS flow chamber. Because of the number ofcells contained in this aliquot (approximately 50,000cells) and t he large collection volume (30-60 ml distilledwater), th e utilization of a technique t ha t resu lts in highcell recovery is essential. In addition, the cellular mate-rial m ust be presented so th at accurate cytological eval-uation can be accomplished using l ight microscopy, Thisrequires th at the cells, previously stained with acridineorange AO) and fixed in glutaraldehyde, be restainedwith conventional Papanicolaou st ains . To satisfy thesecriteria, a protocol has been developed to Papanicolaoustain cells on a cellulosic filter without removing thefilter from the filtration apparatus. In this protocol, thestaining solutions and washes are sequentially addedthrough the filter to restain the cells while the filterremains in the apparatus.

The use of membrane filters for preparing cytologic

samples with limited cellularity is common in cytopa-thology laboratories. Typically, a suspension of cells is

filtered onto a &micrometer pore size cellulosic filterwith subsequent fixation and staining of the cells bydipping the filter through a series of solutions (5). Al-though useful, this procedure can result in a portion ofthe sample being lost t o the stainin g and fixation solu-tions as well as possible cross contamination of the sam-ple. Improved cell recovery was reported by mainta iningth e filter i n the filtration a ppar atus during fixation andPapanicolaou staining. However, unsolved technicalstai ning problems were reported (4).

This report presents a protocol for Papanicolaou sta in-in g cytologic specimens previously sta ined with A 0 andfixed in glutaraldehyde buffer. Cell loss and overallstaining characteristics are compared with results ob-tai ned by alcohol fixation with subsequent Papanicolaoustain ing of cells on a membrane filter.

MATERIALS ANT) METHODSA total of 20 gynecological samples containing cells

derived from a broad spectrum of abnormality presentin the female genital tract was collected. Both normaland abnormal samples were obtained by scraping theute rine ectocervix using a plastic Ayer-type spatu la. The

This work was supported by the National Cancer Institute underGrant R 1 CA 33148.

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102 BERKAN ET AL.

FIG. 1. Normal cells (left) an d abno rmal cells (righ t) previouslystained with AO, fixed with glutarald ehyde and subsequently Papani-colaou stained .

cells were suspended in a mucus-dissolving solution(Mucosol R), Lerner Labs). Each sample was syringedfor cell dispersal and divided into two equal aliquots.Syringing was accomplished by a pneumatically drivenautomatic syringer (6,7).

A 5-micrometer pore size, 47-mm diameter GelmanMetricel filter (Gelman Sciences f60003) was pre-pared for each aliquot by expanding the filter in 95%ethanol for 3 s in a glass petri dish. Each filter wasthen clamped into an analytical filter holder (MilliporeCorporation XX10047-30), an d secured on a filter man-ifold. Low vacuum (2-5 cm Hg) was appl ied to th e filter

s it was hydrated.

Papanicolaou Staining and EvaluationThe first specimen aliquot of ten samples was pro-

cessed according to a poststain fixation protocol devel-oped for preparing gynecological specimens for analysi son the X-Y-Z mutidimensional slit-scan flow system (2,3).Following low-speed centrifugation (2,000 r p d 2 min),

th e Mucosol was removed and th e cells stained in sus-pension using 10 cc of 0.01% A 0 solution, washed, a ndresuspended in 10 cc of Millonigs glu taraldehyde buffer(3.8 aq.). The sample was refrigerated for 24 hr. Fol-lowing low-speed centrifugation, the glutaraldehyde was

removed and the cells washed twice with 10 cc phos-phate bufTered saline and stored under refrigeration.The suspension of cells was diluted with 50 cc of distilledwater and the cells placed on a hydrated (previouslyalcohol expanded) Gelman filter.

The second specimen aliquot was transferred to a filterholder containing an alcohol expanded filter. Mucosolwas removed by washing the cells with water throughthe filter while applying low vacuum. The cells werethen step dehydrated to 95% ethanol a nd fixed by allow-ing them to stan d in 95% ethanol for 15 min. Finally,cells were step hydrated, and the filter was kept moistwith water.

The Papanicolaou stai ning procedure for this study isa modification of the routine dip staining protocol used

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PAPANICOLAOU STAINING POSTFLOW SPECIME NS 103

by the Cytopathology Laboratory at the University ofRochester Medical Center. Both specimen aliquots werehandled in a simi lar mann er for Papanicolaou staining.

Harris Alum hematoxylin (Harleco 638) staining so-

lution (filtered two times through Whatman2

filterpaper) was added to cover the filter and remained incontact with the cells for 4 min. The hematoxylin wasremoved by washing with water, and the filter floodedwith 0.025% (aq.) hydrochloric acid solut ion, an d subse-quently washed wi th water. The filter was then floodedwith 0.01% (aq.) ammonium hydroxide solution and im-mediately washed with water. Following step dehydra-tion to 95% ethanol, Orange G OG-6,Harleco 7052X)sta in was added to the filter and allowed to sta nd for 1.5min, before washing with 95% ethanol. Eosin (EA-65,Harleco 7054X) st ain was added to the filter and al-lowed to st and for 3.5 min. The sample was the n washedwith 95% ethanol, 99% ethanol, and finally xylene(Fisher X-5). The xylene acted to clear th e filter, mak-ing it transparent. The filter was trimmed to 38 mmdiameter (inside diameter of th e filter funnel) and placedcell-side-up on a 75 x 50 mm glass slide (Fisher 12-550Cf in Euki tt mounting media 0. Kindler, West Ger-many), and covered using a 45 x 50 mm coverslip (Fisher

12-545H). No cells were observed on the portion of thefilter th at h ad been removed.

Cell Recovery

To document cell recovery ra te s of the reported proto-col, ten additional gynecologic samples were divided intotwo equal aliquots following syringing. One specimenaliquot was ethanol fixed and Papanicolaou stainedwhile remaining in th e filtration ap paratus. The otherspecimen aliquot was ethanol fixed on a filter, removedfrom the apparatus, and conventionally dip stained ac-cording to the Papanicolaou technique. Following stain-ing, both specimen aliquots were mounted on glass slidesand coverslipped. Cells i n 50 random fields (as viewedat 2 0 0 ~ ) ere counted on each slide, to evaluate cellloss.

RESULTS ND DISCUSSIONSamples tha t were Papanicolaou stained following A 0

poststain fixation were compared with alcohol fixed Pa-panicolaou-stained samples. Cells from both aliquotswere assessed for overall s tain ing quality, cytoplasmican d nuclear detail, an d general eas e of diagnosis. Nu.clear morphology and cytoplasmic detail was very crisp ,and nuclear hyperchromasia was evident in abnormalcells. Eosinophilic staining was evident in superficialsquamous cells, and cyanophilic staining was noted in

intermediate squamous and squamous metaplastic cells.Polymorphonuclear leukocytes were characteristicallyhyperchromatically stained, and Doderlein bacillis wereclearly discernible in several squamous cells. Nuclear

and cytoplasmic Papanicolaou st ain ing was slightly re-duced in t he previously AO-stained glutaraldehyde-fixedcells. Despite the possible exclusion of some binding ofPapanicolaou stains by the AO, glutaraldehyde bufferpoststain fixation protocol, sufficient sta in was bound toallow visualization of cytoplasmic and nuclear morphol-ogy, and the stain permitted accurate cytologic evalua-tion. Figure 1 llustrat es the stainin g quality present innormal and abnormal cells. These cells were previouslystained with A 0 fixed in glutaraldehyde, an d subse-quently Papanicolaou stained.

Papanicolaou staining of cells with the filter main-tained in the filtration apparatus greatly increased cellrecovery as compared to conventional dip staining. Anaverage 13-fold increase in number of cells was ob-served. Cell loss appeared to be randomly distributedover all cell types (leukocytes, normal epithelial cells,and abnormal epithelial cells).

Thi s protocol is applicable in sit uati ons where limitednumbers of cells are collected in large fluid volumes,and where cell loss and specimen cross contaminationmust be minimal. Evaluation of the postflow aliquotsfrom the X-Y-Z multidimensional slit-scan flow sytem isonly one application. This techn ique should prove usefulin other areas in which high sample recovery and ade-quate Papanicolaou sta ining ar e required.

LITERATURE CITED1. Cambier JL, Kay DB, Wheeless LL: muitidime nsional flow sys-

tem. J Histochem Cytochem 27:321, 1979.2. Cambier MA, Wheeless LL, Patte n SF: A new post-staining fixation

tcchniqu e for Acridine Ora nge. A&a Cytol (Baltimore) 21:477, 1977.3 Cambier MA, Wheeless LL, Patten SF: post-staining fixation

technique for Acridine Orange: Quantitative aspects. Anal Qua ntCytol 1:57, 1979.

4. Frost JL, Gill GW, Hankins AG, LaCorte FJ, Miller RA, HollanderDH: Cytology filter preparations: Factors affecting their quality forstudy of circ ulat ing cancer cells in th e blood. Acta Cytol (Baltimore)11:363, 967.

5. Gelm an Sciences: Diagnostic Cytology by Membrane Filter. Appli-cation Bu lletin 100R, 1982.

6. Lopez PA, Cambier MA, Wheeless LL: Syringing as a method ofcell dispersal. 11. Effects on abnormal cells. Anal Quant Cytol3:235,1981.

7. Mead JS, Horan PK, Wheeless LL: Syrin ging as a mcthod of celldispersal. I. Effects on intermediate and superficial squamous cclls.Acta Cytol (Baltimore) 22:86, 1978.

8. Wheeless LL, Patten SF, erka n TK, Brooks CL, Gorman KM, LeshSR, Lopez PA, Wood J C S Multidimensional slit-scan prescreeningsystem: Preliminary results of a single blind clinical study. Cyto-metry 5:1, 984.