CHANGES IN BUCCAL CELLS THE ANAEMIAS - jcp.bmj.com · Occasional nuclei, however, are...

J. clin. Path. (1959), 12, 222.

CHANGES IN BUCCAL CELLS IN THE ANAEMIASBY

M. M. BODDINGTONFrom the Department of Pathology, Churchill Hospital, Headington, Oxford

(RECEIVED FOR PUBLICATION DECEMBER 19, 1958)

Morphological abnormalities of the haemo-poietic cells in pernicious anaemia have been wellknown for many years, but it is only recently thatattention has been drawn to abnormalities in theepithelial cells. Graham and Rheault (1954)studied the buccal epithelial cells in gastricwashings and showed that there was generalenlargement of the cytoplasm and of the nucleus,the presence of some cells with much enlargednuclei, and an increase in the number of cells withtwo or more nuclei. The nucleus may be hyper-chromatic, with unevenly distributed chromatinand a prominent nucleolus, and have an irregularmembrane.

Massey and Klayman (1955) and Rubin (1955,1956) confirmed these findings, and, with Gardner(1956), found that these changes are also presentin megaloblastic anaemia not due to vitamin B12deficiency; however, they are not completelyspecific for megaloblastic anaemia. Changes invarious other epithelia from cases with megalo-blastic anaemia have been reported by severalauthors (Boddington and Spriggs, 1959).

G6rz-Kardaszewicz (1956), Boen (1957), andFarrant (1958) measured abnormal cells from themouth in cases of pernicious anaemia andconfirmed that the nucleus in the buccal cellsis abnormally enlarged but this is reversible withtreatment.

Scarcely any mention has been made of thecondition of the tongue in the cases so farexamined, and it therefore seems highly rel-vant todiscover whether the cytological changes in buccalcells are dependent upon atrophy of the papillaeor on the megaloblastic anaemia. The presentstudy has been made with this end in view.

Material and MethodsInitially tongue scrapings taken with a glass slide

were used for this study, but specimens of saliva laterproved more satisfactory. In some cases both typesof specimen were examined (Boddington and Spriggs,1959). Comparison of the average diameters of thecytoplasm and of the nucleus in specimens taken bythe two methods in the same patient showed little

variation in the general pattern. In all cases thenuclear diameter in saliva was greater than in thetongue smear and in the majority the difference was asignificant one.

Saliva also made possible a direct comparison withthe buccal cells seen in gastric washings. In 10 casesthe average diameters in saliva showed closeagreement with those in gastric washings taken at thesame time. The tendency was towards smaller cellsand nuclei in the gastric washings, but not in allcases.

In this paper only the results obtained from salivaare reported. Specimens were taken from 59 patientsof whom 18 were diagnosed as pernicious anaemiaby the usual criteria, 11 as other megaloblasticanaemias (due to pregnancy, five cases, steatorrhoea,two cases, total gastrectomy, one case, nutritionaldeficiency, one case, carcinomatosis, one case, andphenobarbitone treatment for epilepsy, one case).Twenty had iron-deficiency anaemia, and 10 cases hadabnormal tongues not associated with anaemia.Twenty-three patients had papillated tongues and inthe rest the papillae were atrophied to a greater orlesser degree. Specimens from 10 people of differentage groups were used as controls.

Specimens were collected by asking the patient tospit several times into a container. The saliva wascentrifuged and two films made from the depositeither with a wire loop or by spreading between thetwo slides; these were immediately " wet-fixed " inether-alcohol or ethyl alcohol containing 3% aceticacid for staining by the Papanicolaou technique.The slides were examined through the 1/ 12 oil-

immersion objective, and the mean diameter of 100cells and their nuclei in each case was determined bymeasuring both the long and the short diameters withan eyepiece micrometer. Some selection was necessaryto avoid those cells with folded cytoplasm, unusuallydistorted nuclei, and those in tight collections. Asfar as possible the measurable cells were consecutive.The mean diameter of these measurements wascalculated for each case and this is the "meandiameter" referred to below. In addition, percentagesof abnormally enlarged nuclei (greater than 14 A indiameter) and of binucleate cells were calculated fromcounts of 1,000 cells.

ResultsMemurement of Diameters.-These are set out

in the Table and Fig. 4.

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CHANGES IN BUCCAL CELLS IN THE ANAEMIAS

TABLEAVERAGE DIAMETERS OF BUCCAL SQUAMOUS CELLS

Average AverageCytoplasmic Nuclear

Disease Group Cases Diameter andStandard StandardError (,4) Error (p)

Tongue papillated:Normal control .. .. 10 50-11±1 17 8-64±0-17Pernicious anaemia .. 5 51-65±1-17 8-66±0-08Other megaloblastic anaem-

ias 4 51-48±A137 8 93±0-21All megaloblastic anaemias 9 51-57±0-87 8:78±0-08Iron-deficiency anaemia 10 46-46±0-94 8 94± 0.11

Other cases with abnormaltongues.4 43-86±2-62 8-83±0-28

Tongue depapillated:Pernicious anaemia 13 49-32- 1-49 9-92±0-27Other megaloblastic anaem-

ias . .

All megaloblastic anaemiasIron-deficiency anaemia ..

Other cases with abnormaltongues

72010

6

47 33±2-14 10-28±0-15148 77 1-122 10 10±0 1948-55± 1-33 9-41 ±0-16'44-96 1-27 9-39 '0-16

..h

4.

-__c- _Fgr i......... *.;*.:0.

. .. i.g . :Normal Buccal Cells.-Buccal squamous cells

from a normal healthy person have a cytoplasmicdiameter from 20 / to 80 ,u, with a mean of about50 ji. The nucleus is small and oval with a fineregular chromatin pattern (except when pyknotic)and a diameter varying from 5 ,t to 13 /i, with amean of about 8.6 /u. Occasional binucleate cellsare present. The cytoplasm frequently containskerato-hyaline granules and often there is aperinuclear halo. The appearance of these cells isshown in Fig. 1.

Cells from Cases of Megaloblastic Anaemia.-Many of the buccal cells from cases of megalo-blastic anaemia have a normal appearance apartfrom the slight increase in nuclear diameter.

FIG. 1.-Normal buccal squamous cells. The lower one is binucleateand the other contains kerato-hyaline granules. x 400.

FIG. 2.-Buccal cells from a case of pernicious anaemia with a smoothtongue. All the nuclei are larger than normal and the two largestcells have " giant" nuclei, i.e., greater than 14 ,z in diameter.x 400.

Occasional nuclei, however, are hyperchromaticwith prominent nucleoli and a serrated nuclearmembrane, but are not usually much enlarged.The greatly enlarged (giant) nuclei are commonlyvesicular and round with a regular nuclear berder(Fig. 2). The cytoplasmic diameter in these cellsis rarely very large and is commonly even smallerthan normal.Measurements of cytoplasmic diameters showed

close agreement with those of the normal series.The cases with normal tongues tended to haveslightly larger diameters than normal, while in thegroup with depapillated tongues they were rathersmaller (see Table). In only one case was thereany considerable increase in size. In six casesexamined 10 to 15 days after treatment withvitamin B12 there was a decrease in diameter infive, while in the sixth case the tongue remainedsmooth in spite of treatment.The nuclear diameters, on the other hand, were

essentially normal in the cases with papillatedtongues, but those with smooth tongueshad much larger nuclei; the differencebetween the averages for this group (see Table)and that of the controls was highly significant(P= <0.001).One patient with a normal tongue showed a

high mean diameter, whereas two of them withsmooth tongues had diameters within normallimits (Fig. 4). Five of the six patients examinedafter treatment showed the expected reversiontowards normal; two had subnormal diametersafter 10 and 15 days respectively.

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M. M. BODDINGTON

Cells from Cases of Iron-deficiency Anaemia.-Changes in the buccal cells in iron-deficiencyanaemia have not so far been reported. Thisstudy has shown that the cytoplasmic diametersin the 20 cases examined tended to be smallerthan normal especially in 10 cases with papillatedtongues (see Table). The difference between theaverage for this group of 10 and that of thenormal controls is a significant one at the 5%level.The meain nuclear diameters of the 10 cases with

normal tongues were within normal limitsalthough in two cases there was some enlargement.On the other hand, most of the cases with atrophyof the papillae showed nuclear enlargement (Fig.4). In some cases the diameters were comparableto those seen in megaloblastic anaemia (Fig. 3)and the average for this group showed a statistic-ally significant increase over normal (P= <0.05).

Cases with Abnormal Tongues not Associatedwith Anaemia.-The 10 cases with abnormal orsmooth tongues in the absence of anaemia inwhich buccal cells were measured all had normalhaemoglobin values. (Six had completelydepapillated tongues, two had thrush, one hadleukoplakia of the tongue, and one had a painfultongue normal in appearance of unknownaetiology.)

In all but one of this miscellaneous group therewas a smaller cytoplasmic diameter than normaland both the groups (see Table) showed asignificantly smaller average diameter at the 5%level.The nuclear diameters were within normal

limits in three of the cases without epithelialatrophy of the tongue, and in two of those witha depapillated tongue (Fig. 4). The group withoutatrophy showed an average diameter which wasessentially normal, whilst the group with smoothtongues (see Table) had a diameter which wassignificantly higher than normal (P=<0.05).

Percentages of Nuclear Abnormalities.-Fig. 5illustrates these in the patients and controls.The most readily observed changes and those

which are most easily measured are the greatlyenlarged (giant) nuclei and the binucleate cells.These appeared to be more numerous in megalo-blastic anaemia than in other conditions, and inorder to verify this supposition the percentagesof nuclei greater than 14 ,t in mean diameter andthe cells with two or more nuclei in counts of1,000 cells were found for each case. Fig. 5shows the mean values for each group.

FIG. 3.-Buccal cells from a case of iron-deficiency anaemia with asmooth tongue. The largest cell has a " giant " nucleus whichis also hyperchromatic with three prominent nucleoli and anirregular border. x 400.

Nuclei Greater than 14 jt in Diameter.-Nonuclei of this size were seen in specimens fromnormal persons."Giant" nuclei were found in all cases of

megaloblastic anaemia with smooth tongues. Twocases with near normal diameters showed quitefrequent giant nuclei and small percentages werealso found in five of the cases with papillatedtongues, four of them having normal meandiameters.None of the cases with iron-deficiency anaemia

having normal tongues had abnormally largenuclei, but all but two of the group with smoothtongues showed them (Fig. 3).Of the 10 cases without anaemia five with

increased nuclear diameters also showed " giant"nuclei.

" Giant" nuclei, therefore, were present in themajority of cases with smooth tongues and in halfof the cases of megaloblastic anaemia with normaltongues. Whilst many of the cases of megalo-blastic anaemia had a high percentage of largenuclei (over 5%) and the mean values (Fig. 5)show a striking difference from the means of theother groups, there is some overlap between thegroups and no specific diagnostic value can beattached to the " giant " nuclei alone.

Multinucleation.-Cells with more than twonuclei were rarely seen. Graham and Rheault(1954) and Gorz-Kardaszewicz (1956) havereported cells with five and nine nuclei respec-tively, although the photomicrographs of these

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Normal controls

Pernicious anaemia

Other megaloblasticanaemias

Iron-deficiencyanaemia

Other cases withabnormal tongues

Series

Normal control

Perniciousanaemia

Other megaloblasticanaemias

Iron-deficiencyanaemia

Other cases withabnormal tongues

Papillated tongue 0

Depapillated tongue *

0 000 O0D 0

0 0Q0l 0 0 "

a 00? me

*a*04 * 0

OOo0 0* 0a *0

0 @ 0 -

0

. a -. *- . A . I f .

7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 1 1.5Mean diameter (u)

FIG. 4.-The mean nuclear diameters for each case examined.

Conditionof Tongue

Normal

PapillatedDepapillated




tIIIIIIIM ~ ~ IIIIIIIIIIT17

Nuclei greater than14 p in diameterin

Binucleate cells M. . I . a

0 1 2 3 4 5 6 7Mean percentage of cells

FIG. 5.-The mean percentage values of nuclei greater than 14 , in diameter and of binucleate cells in eachgroup of cases.

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M. M. BODDINGTON

suggest that they may in fact have been cellclusters. An acceptable example has beenpublished by Massey and Klayman (1955).The percentages of binucleate cells calculated

from counts of 1,000 cells again showed them tobe more numerous in cases with megaloblasticanaemia although not necessarily correlated withthe " giant" nuclei (Fig. 5). There was still someoverlap between the normals and the other groups.

Other Abnormalities.-Nuclear abnormalitiessuch as hyperchromatism with unevenlydistributed chromatin and irregular nuclearmembrane are qualitative, and interpretation willvary from one observer to another. Suchabnormalities may be more frequent in cases withmegaloblastic anaemia, but are not always presentand do not appear to be specific.

ConclusionsGraham and Rheault's (1954) initial observation,

later confirmed by Boen (1957), of the enlarge-ment of the cytoplasmic diameters in buccalsquamous cells in cases with pernicious anaemiawas not confirmed in this investigation.The cells in pernicious anaemia were not much

changed in size from those in normal persons andtended to be smaller in those cases with atrophyof the lingual epithelium. This tendency towardsa smaller cytoplasmic size was more apparent incases with iron-deficiency anaemia and certainother diseases. In addition the cytoplasmicdiameter decreased still further in perniciousanaemia after treatment.We have confirmed that in many cases of

megaloblastic anaemia the buccal cell nucleus isabnormally large and that there are other nuclearabnormalities. In the majority of cases, however,these abnormalities were associated with atrophyof the lingual epithelium and were also present incases with certain other diseases where a smoothtongue was present. Farrant (1958) hasdescribed one such case without anaemia, andlingual atrophy may account for the six caseswhich did not have megaloblastic anaemiadescribed by Massey and Klayman (1955).

Cases in our series of megaloblastic anaemiawith normal tongues did not show nuclearenlargement, and it was not invariable in the caseswith smooth tongues. Boen (1957) has alsodescribed three cases of pernicious anaemia withnormal nuclear diameters. In such cases theremay be other nuclear abnormalities, also non-specific, but usually much more marked inmegaloblastic anaemia.

Gardner (1956) described nuclear fragmentationand nuclear haloes as additional abnormalities intropical sprue. Nuclear fragments in the cytoplasmwere never seen in our preparations, and Gardnermay have been observing kerato-hyaline granules.Percentages of cells containing these vary con-siderably in each specimen, and we have found nocorrelation with the disease or the state of thetongue (thus confirming Gorz-Kardaszewicz,1956). Perinuclear haloes are not specific formegaloblastic anaemia and are a normal featureof buccal cells (Ziskin and Moulton, 1948). Certaincases did show extreme examples of this pheno-menon, and our case of tropical sprue was one ofthem.

Whilst concluding that percentage values ofsuch nuclear abnormalities as gigantism andbinucleation are not an adequate diagnostic testfor megaloblastic anaemia in all cases, these maybe high enough to assist in the diagnosis. Boen,Molhuysen, and Steenbergen (1958) have demon-strated such an instance.

It is evident from our series that the abnormalenlargement of the buccal cell nucleus is notdirectly correlated with the smoothness of thetongue. This is illustrated by the finding of thisabnormal change in cells from cheek scrapings(Gorz-Kardaszewicz, 1956; Farrant, 1958). itseems that these abnormalities appear at a stagein the deficiency state when the tongue also (inmost cases) becomes clinically abnormal. Noattempt was made to classify the degree of lingualatrophy, but it is of interest that most of thecases in the "smooth" group with normal ornear-normal nuclear diameters did not havecompletely depapillated tongues. A method ofindexing the degree of depapillation, such as thatof DiPalma (1946), would possibly give a closercorrelation.

It appears, therefore, that in megaloblasticanaemia the abnormality in cellular metabolism,whose nature is still not fully understood, at thesame time causes the megaloblastic transformationin the red cell precursors, the gigantism of thegranular leucocytes, and the nuclear abnormalitiesof the epithelial cells (of which the buccal cellsare the most strikingly affected). The enlargementof the nucleus of the buccal squamous cell maybe produced by other metabolic defects such asiron-deficiency, but the present methods ofexamination cannot distinguish between thedifferent causes.

SummaryMean cytoplasmic and nuclear diameters of

salivary squamous cells were measured in 69

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persons. Eighteen of these had perniciousanaemia, 11 had other megaloblastic anaemias,20 had iron-deficiency anaemia, 10 had abnormaltongues not associated with anaemia, and 10 werenormal controls. Thirty-six of the patients hadsmooth tongues.The cytoplasmic diameters of buccal cells from

cases of pernicious anaemia were not enlargedbut tended to be smaller than in normal healthypersons. In other megaloblastic anaemias, iron-deficiency anaemia, and in some other diseasesnot associated with anaemia but having abnormaltongues, the cytoplasmic diameter also appearedto be abnormally small.The nuclei of buccal cells showed a distinctly

enlarged diameter in those cases of megaloblasticanaemia in which there was lingual atrophy.Cases of iron-deficiency anaemia also sometimesshowed this nuclear enlargement in the presence

of a smooth tongue, and a high nuclear diameteris by no means specific for any particular cause

of lingual atrophy. However, the highest figureswere found in pernicious anaemia.

Specific nuclear abnormalities such as " giant"nuclei and binucleation were more common inmegaloblastic anaemia than in other diseasegroups with smooth tongues. A small percentageof abnormal nuclei could be seen in cases ofmegaloblastic anaemia with normal tongues.These abnormalities are not a specific diagnostictest for the megaloblastic anaemias but may assistdiagnosis when they are present to a markeddegree.

My thanks are due to the medical staff of theUnited Oxford Hospitals for providing me with

specimens and details of patients under their care;to Mrs. D. Jackson for her assistance with thephotography; and to Mr. N. T. J. Bailey for his adviceon the statistics.

I am also grateful to Dr. A. H. T. Robb-Smith forcorrecting the manuscript and in particular to DrA. I. Spriggs for all the advice and help given duringthis investigation.The work was carried out while receiving a grant

from the British Empire Cancer Campaign.

REFERENCESBoddington, M. M., and Spriggs, A. I. ( 1959). J. clin. Path., 12, 228.Boen, S. T. (1957). Acta med. scand., 159, 425.- Molhuysen, J. A., and Steenbergen, J. (1958). Lancet, 2, 294.DiPalma, J. R. (1946). Arch. intern. Med., 78, 405.Farrant, P. C. (1958). Lancet, 1, 830.Gardner, F. H. (1956). J. Lab. clin. Med., 47, 529.G6rz-Kardaszewicz, S. (1956). Pat. pol., 7, 373.*Graham, R. M., and Rheault, M. H. (1954). J. Lab. clin. Med., 43,

235.Massey, B. W., and Klayman, M. I. (1955). Amer. J. med. Sci., 230,

506.Rubin, C. E. (1955). Gastroenterology, 29, 563.-(1956). Ann. N.Y. Acad. Sci., 63, 1377.Ziskin, D. E., and Moulton, R. (1948). J. clin. Endocr., 8 , 146.

*As it is only available in Polish the following summary of thepublication by G6rz-Kardaszewicz (1956) is given.From 10 to 15 cheek scrapings at three- to four-day intervals

were taken from 1 cm. below the right parotid duct in each patient,and examined in wet-fixed films stained with haematoxylin andeosin. Percentages of abnormal cells were calculated in each of 32patents with anaemia (22 pernicious anaemia) and in 10 normalmales and 10 normal females. The abnormalities counted were largecells with normal nuclei, large nuclei in normal cells, multinucleatecells, cells containing cytoplasmic granules, and anucleate cells.The results were tabulated and showed about 10% of abnormal

cells in pernicious anaemia. Cells with a total diameter above 91 Paccounted for 4% of this increase, those with nuclei above 16 iin diameter 2%, and binucleate cells 4%. The abnormal enlargementpersisted in a smaller percentage of cells in 16 cases after treatment.The number of cells with granules and anucleate cells was notabnormally high.G6rz-Kardaszewicz postulates that the same factor controls both

the changes in the epithelium and in the marrow because gigantismand multinucleation can be found in cells obtained from allaccessible regions of the body, but no data for this hypothesis aregiven.

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