The irrigation smear

CURRENT

OPINION

The irrigation A comparative

trial

smear of vaginal

smears

in the detection

0.

N.

A.

London,

HUSAIN,

irrigation

of cervical

M.D.,

pipette

and spatula

cancer

M.C.PATH.

England

A survey of 2,350 women, comparing doctor/nurse-collected scrape smears and self-collected cytopipette smears, has shown around an 85 per cent detection rate for cancer with the cytopipette, though a less eficient rate for dysplasia. As the purpose of the “do-it-herself” pipette is to alert to disease by a relatively cheap method, it is felt that the instrument is worthy of consideration and development as there is little doubt that the principle is sound and effective, though the design of pipette and its contents may not yet be ideal. The test in no way replaces a scrape smear with clinical examination where this is practicable.

S I N c E H . J . D A v I s51 6 described his vaginal irrigation pipette in 1962, there have been numerous attempts at reproducing his results to compare them with other methods of cell collection. Some have been simple surveys using the cytopipette only11 5, 7, ’ and some comparative tests with scrape smears.2-4, lo, I23 l3 Opinions vary as to its value and reliability, and the present survey was undertaken for the Ministry of Health in order to investigate the validity and acceptance. Design

and

purpose

of

to make on this. We endeavored to compare quality of cell sample from the cytopipette with that of the scrape smear, each in the way they would be collected in routine practice. Material

trial

Evaluation of this technique can be divided into two aspects: (a) population acceptance and (b) quality of technical result. The present study does not set out to test the former attribute, though we have some observations From the Regional Group Laboratories, Hospital.

and

methods

An English make of irrigation pipette (Vann Bros., London), a little smaller than the American and Scandinavian instrument (Skandialab A/S, Copenhagen, Denmark), was used throughout this trial. It has a single squeeze-out volume of 2.0 to 2.5 ml., and return of 1.5 to 2.0 ml. by in vitro test. The pipette was filled to the tip with an irrigation solution made up of 20 per cent alcohol in normal saline with a few thymol crystals to prevent bacterial growth. No dye was added, as this caused romplaints of staining clothes. The pipette traveled in a thin polythene sleeve within a cardboard tube with end caps,

Cytology Centre, St. Stephen’s 138

Volume Number

106 1

and this was tied into a linen postal bag with a stamped and addressed tab attached. A printed form was enclosed with the pipette explaining the idea of the test with an invitation to join the trial. This stated that the test was to be taken one week after the end of the next menstrual period, this being the simplest way of aiming at the midcycle phase, and not less than 2 weeks after the scrape test being performed, in order to allow regeneration of epithelium. If pregnant, the woman was asked not to do the test: if simply amenorrheic or menopausal, then she should perform the test 2 weeks following the scrape smear. Intercourse, douches, and contraceptive caps or cream were to be avoided for 48 hours prior to the test. On the reverse side of the form were printed instructions of how to perform the test. By half sitting on the edge of the toilet seat, the woman could insert the pipette into the vagina up to the neck of the bulb, and by squeezing hard once only, then moving the tip about before releasing the pressure on the bulb, the fluid was permitted to return to the bulb now admixed with cells and mucus. At the bottom of this page the woman was asked which test she preferred, and whether she had any criticism of the cytopipette test. Selection of women included in the trial These were women attending Local Health Authority and Family Planning Association clinics, patients of a few selected general practitioners, and some hospital clinic patients. Almost all were within the childbearing age but a few early menopausal cases were included, No pregnant women were allowed to take the test, though a number of postpartum patients were included. At the clinic or hospital, after the scrape smear had been taken, the doctor and nurse invited the woman to accept a cytopipette, explaining its purpose and method of use. If the patient declined or the doctor considered her unlikely or unsuitable to per-

Irrigation

smear

139

form the test due to poor cooperation or for various gynecologic reasons, she was not given a pipette. The latter group, in general, constituted about 3 to 5 per cent of women and varied from clinic to clinic. The series was fortified by the addition of cytopipette material from patients having scrape smears in general or clinic practice, and here a nurse or doctor would collect the sample at the next attendance, again usually after an interval of about 2 weeks from the scrape smear. laboratory

procedures

The time interval between test collection and receipt of pipette in the laboratory was as follows: 1 day = 30 per cent, 2 days = 33 per cent, 3 days = 18 per cent, 4 days = 9.0 per cent, 5 days = 6.0 per cent, 6 and more days = 4 per cent. The pipettes were labeled, shaken, and then centrifuged gently at 1,200 r.p.m. for 2 minutes. Most of the supernatant was decanted and replaced by fixative solution containing Carbowax. The pipettes then stood for half an hour and were spun again, this time leaving behind as much fluid as there were cells. These were mixed together, and a drop or two of this thick cell suspension was transferred to a slide and spread by the ridged tip of the pipette. The smears were allowed to dry completely and then were stained by the traditional Papanicolaou technique. One full smear was screened over an area of 4.5 x 2.4 cm. which contains an average of 50,000 cells. Screening was by cytotechnicians specially trained in irrigation smear scanning. In essence, this is more sensitive than for scrape smears. Thus, into Papanicolaou Grade III would fall any smear containing mildly dyskaryotic superficial or intermediate squames, a finding in scrape smears that might reflect an inflammatory dysplasia which would not qualify for more than Papanicolaou classsification of Grade II (inflammatory) : otherwise, we employed ‘Papanicolaou classification throughout, apart from grouping Grades IV and V together as malignant for both scrape and irrigation smears.

140

Husain

Amer.

In practice, the scrape smears were read first and reported out before the cytopipettes arrived. The pipettes were processed in batches each day, and were screened blind; that is, the technicians had no knowledge of, or access to, the results of the scrape smear, which had often been read from one to 3 months before. All “pick-ups” from each series (scrape and pipette) were routinely submitted for scrutiny by the author in the normal way. Once graded III or above, the matching smear scrape and pipette were then reviewed to see if they were truly negative. Any alteration of grades on review are mentioned in the text or charts. Results Around 2,750 women were issued pipettes and 2,350 women returned pipettes under the conditions of the trial. About 5 per cent of women either refused or were deemed unsuitable for the test. Of the kits issued, between 10 and 15 per cent failed to return the pipette. This varied from clinic to clinic. The reason given on inquiry ranged from: “I forgot”; “I couldn’t be bothered”; “I’d had a test anyway”; “It got lost”; to one who said: “Little Johnny used it as a water pistol” The two questions asked on the instruction form were answered as follows: (a) “Which test do you prefer?“: Prefer cytopipette, 68 per cent; prefer scrape test, 24 per cent; no preference, 8 per cent. (b) In spite of the majority favoring the pipette, there were a number of varied criticisms in answer to that specific question. These are listed in Table I. Table I. Criticisms by patients test (per 1,000 women)

of cytopipette

Doubt of reliability of test Bulb too difficult to squeeze Bulb did not regain shape Stinging sensation Pipette painful or difficult to insert C&i& of position of test (easier on chair) Uncomfortable or sore Dribbling after test “Made me nervous”

55 40 31 31 9 5 4 3

2

January J. Obstet.

1, 1970 Gynec.

Other comments that came up singly included : “Can’t see what I’m doing”; “Easy if y-ou know angle of vagina”; “More private, less loss of modesty”; “Too easy for selfsatisfaction”; “Distasteful and embarrassing”; “It’s marvelous”; and indicate various aspects of the principle of self-collection. One reply must be quoted in full to illustrate that sometimes our English on the instruction forms was not welI-chosen. Dear Sir or Madam, You state in the form

that I must

refrain from intercourse at least 48 hours prior to the test. . . . As I never read that part and didn’t know whether prior meant before or after, I had already taken the test after intercourse.

Such comments demonstrate a failure to communicate to the very sector of population we most wish to approach. A new instruction sheet, this time more simply and clearly Lvorded and moreover illustrated will, TV~ hope, help to overcome this problem. laboratory aspects Processing time. The processing of the pipettes consumed material time in preparing the smears, and a study of this produced the following figures: Processing

time for

1 pipette

=

16

=

25

=

40

minutes.

Processing minutes Processing minutes

time

for 6 pipettes = 4 minutes each. time for 12 pipettes = 3% minutes each.

Twelve was the maximum processed at any one time, but up to 32 could be accommodated in a large centrifuge. Processing from here on was the same as for the scrape smears. Criticism of technique by laboratory staff. There is, of course, an additional task in processing the pipettes, but the only serious

criticism was leveled at handling the contaminated pipettes. This could be overcome by immersion in detergent receipt and wiping cIean, plastic gloves.

or antiseptic on or by the use of

Volume Number

Irrigation

IG6 1

Screening time. A check was made of the time taken for screening scrape and pipette smears. The results in Table II were based on the average by 3 screeners, each scanning 300 smears. We confirm the claim by Davis that one could “speed screen” the thin and monocellular cytopipette smear, though only about 25 per cent faster than the scrape smear in the truly negative cases. With the doubtful and positive cases there was a wide variation of screening time. Here conclusive evidence of cancer might be found within a few fields using either technique, or may require prolonged search. Again, a marginally faster screen occurred with the thinner cytopipette smear. When seeking for Davis’ own criteria of A, B, Z-Bas, and C cells, a summary of M-hich is given below, the screening time was increased to cover that of the scrape smear, but this may well be improved with experience. For those who are not familiar with this cell classification, a short description is appended. A cells:

Active nucleus-at least 100 per cent increase in the quantity of dark-staining chromatin material over the staining of the normal intermediate cell nuclei in the same preparation. Chromatin structure

must

be

evident.

Big nucleus-a nucleus which is more than 12 ,u. in diameter. Minor degrees of hyperchromasia may or may not be present. 2-Bas cells: A substantially rounded parabasal cell with nuclear diameter occu-

B cells:

Table

C cells:

Quantitative

(min.)

Negative smear (Papanicolaou Grade I and II) Doubtful and positive smears (Papanicolaou Grade III, IV, and V)

pying half OP more of the cytoplasmic diameter provided the overall smear pattern is not atrophic. Against an atrophic background only parabasal cells exceeding A cells in degree of hyperchromasia or B cells in nuclear diameter were considered sufficiently identifiable as aberrant for the P-Bus category. Curious nucleus-borderline degrees of hyperchromasia, roughness of chromatin pattern or abnormal clearing. analysis

of

Table III. Papanicolaou classification

4

2-10

3

2-8

smears

grading

Cytopipette smear

/ z?;,L

141

A series of charts depict the analysis in this project. In Table III it will be noted that there is close similarity in Grades I and II and in fact Grade III, but there was double the incidence of unreliable smears in the cytopipette series. By unreliable we mean poor quality of preparation or a cell content of less than 50,000 per smear. Quantity and quality analysis, Tables IV and V, give an indication of proportion of readable and reliable smears. It will be obvious that though 2 per cent showed no macroscopic cellular deposit, there were some cells present, and most were of readable quality. It was found that the equivalent cells in the cytopipette smears were a shade smaller on average than those on the scrape smears, confirming an observation which has been made by Reagan and Lin.12 Whether this

II Cytopipette

smear

No. Grade 0 (unsatisfactory) Grade I Grade II Grade III Grades IV and V

127 1,833

Scrape

% 5.40

34

78.01 14.00 1.45

---- 27

1.14

329

2.350

100.00

smear No. ) % 54 1,915 305 31 45 2.350

2.32 81.48 12.97 1.32

1.91 100.00

142

Husain Arne~.

Table IV. Quantity macroscopic* 0 (less than + (l-2 mm.)

of cell deposit

1 mm.)

= = = =

+ (2-3 mm.) ++ +++

(3-5 mm.) (5 mm. +)

Table delay 2% 22.6 37.2 23.4

=

Delay

VII.

(days) I- 8 8-12 1 Z-20 20 f

9.8

Quality

1

January J. Ohstet.

deterioration

Same

(%)

(

I, 1970 Gynrc.

due

Worse

90 66 44 12

to

(70) 10 ?‘? 56 82

100.0 *Rough measurements of depth of cell button in conical end by bulb. Note: Even a ? cell deposit would be sufficient to make one adequate smear, and a 0 will often contain SOme cells.

Table VIII. Detection of Trichomonas and Monilia infection (analysis of 1,000 cases) Scraje

Table V. Quality (microscopic) *

of cell appearances

Trichomonas Monilia

Bad

=

Poor but readable Fair Good

= 16.1% = 49.8% =

appearance of quality and preservation.

Table VI. Comparison preparation

Collector Nurse

Patient

Unsatisfactory 0 5.2 7.4

in quality

3.3

1.4

1.8

0.9

0.9%

33.2% 100.0%

“Final microscopic by the staining quality

1 Cytopipette

of cells

as judged

of smear

Poor t

Fair ++

Good +++

Total

11.3 15.6

50.9 51

32.6 26

100% 100%

was due to the osmotic action of the saline solution or the condition of drying in the presence of Carbowax is difficult to ascertain and is the subject of a further study. Quality of patient-versus-nurse collection of pipette material A comparison between patient and nurse collection was carried out in relation to both quality and quantity of specimens and is given in terms of Grade 0 or unsatisfactory smear. The unsatisfactory rates are not greatly different in the two methods of collection. Looking at the small positive group of cases, it is interesting to note that the ability to detect disease from specimens collected by patient or nurse/doctor was also closely

similar. The “miss rate” or failure to detect in the so-called “positive series” with the cytopipette smears was 9/36 by nurses compared with 1 l/36 by patient. Deterioration due to delay in processing There was no appreciable difference in the quality of cells examined immediately on receipt (i.e., 24 hours after collection) and little after a 5 to 8 day delay in processing. There is, however, a distinct but small deterioration in the first 24 hours, and in the nature of things this cannot be avoided. In the second week approximately half the specimens showed loss of quality and this was much increased by the end of the third week, as seen in Table VII. It is obvious that a simple saline solution is not ideal, and the new solutions used now appear to be better. Infections Table VIII shows the superiority of the scrape smear in detecting Trichomonas and Monilia infection, a finding shared by most other authors. It must be realised that at least a fortnight separated the two tests and elimination of infection by clinicians or patient could have taken place. Hormonal state An attempt at assessing the correct of the cycle was made by the hormonal

phase pat-

Volume Number

106 1

Irrigation

tern in 100 smears in the proliferative phase and 100 smears in the secretory phase. Table IX shows the result. In our hands, and using the 20 per cent alcoholic saline solution, the irrigation smear showed poor correlation with hormonal state. It is obvious that to make use of the cytopipette for hormonal evaluation, a better solution will have to be used. Screening

for

malignancy

Table IX

Table X. Comparative

51 18

42

28 3

24 5

29

detection

rates by disease* Detection

method Cytopipette

Scrape

Lesion

143

subsequent course of all these cases over 2 years has been uneventful. The positive cytopipette or scrape smears were followed up either by repeat scrape smears and/or biopsy, and the following list gives the composition of the diagnosis. All the malignancies and dysplasias, and most of the infections have had histological confirmation. The unknown or unprovens are still in need of elucidation, and here at least 3 out of the 10 are probably carcinoma in situ from their smear appearances. Most of the cases involved in the survey have been followed for more than one year and no further malignant disease has emerged in this group. Table X demonstrates the proportions of cases. It will be apparent that the more malignant the disease, the more reliable appears to be the cytopipette in detection. The low detection rate in dysplasia probably depicts the low exfoliation rate in this disease. A further exercise into the volume of evidence for detection in the “positive series” was to count the number of dyskaryotic or malignant cells present within a 3 x 2 cm. frame produced by a rubber stamp on the cover slip. This was still well short of the total cell content of the smear-perhaps about two-thirds in most instances-and the number of (a) malignant cells, (b) markedly dyskaryotic cells, and (c) mild to moderate dyskaryotic cells (as often seen also in inflammation )were recorded. A further scan of those cytopipette smears without malignant or dyskaryotic cells was made to detect

All “positive” or “pick-up” smears were marked, Papanicolaou graded, and reported on by the screeners, then referred to the author for final decision. Each cytopipette smear was given a grading independent of its matching scrape smear. A total of 86 “pick-up” smears, either scrape or cytopipette, resulted from the screening. Of these, 6 did not, to my mind, warrant grading as Papanicolaou Grade III and over, and were rejected from the series. In none of these cases did the matching smear demonstrate alerting signs, and the

Proliferative pattern Secretory pattern Mixed hormonal pattern Cytolytic pattern

smear

Total

No.

%

No.

11 (+l)

By %

D&s

criteria

Invasive squamous cancer Adenocarcinoma

14 7

14

100

6

86

7

100

-

Carcinoma

32

31

97

27

84

-

17

17

100

10

58

(71%)

3

1

in situ

Dysplasia Infection Unknown *Figures

10 in parentheses

are those

resulting

8 from

the use of Davis’

criteria.

(+2)

33

3

80

7 (+I)

78

100 70

(86%)

(80%)

144

Husain

Amer.

Janua1-y J. Obstet.

1, 1970 Gynec.

BCBAI

00.0

1 iumber of

8

Cells

8

per Bmear

$0: OAAA l

0.0000 cooooooo

808

OAOOA

l

m

)88=

m0mm OOoOAo

1

A

Cells

iBAB Cell0

CYTO

PETTE

B

NllIb%?r of

00

Cells

8.

per Smear

70

I Dyey11 I CIS ~8qUM Adenc

A &A

Mild 1Dymkaryod -r-

ia

1

LIB Ca. L.

3

80

. 00

SO

om

8

oom

l

AA 100 0.0.. 0000000

0

40

0

30

. 0 OAA 0

0

l 20.0 80 1080 DO.

(> (1

H0.A

NIL

Cti.

ii-

311s

B BA8 Cella

-

Mild I )yekaryoeis

l

00 00

Marked Dyekaryoels

Malignant

Fig. 1. Cytologic evidence of malignancy. the cells of Davis’ criteria (A cells, B cells, 2-Bas cells, and C cells). A chart, Fig. 1, was prepared plotting the extent to which the graded diagnostic criteria were present against the amount of that particular type of cell. In certain cases of mixed abnormal cell content such as the presence of 10 malignant cells together with 50 dyskaryotic cells, this places the case on the borders of malignancy and dyskaryosis at approximately 30 on the vertical scale. The rough position of the spot does, however, denote

the degree of reliability of diagnosis by quality and quantity. Compared with this, the scrape smears lay much more to the top right hand corner of the chart. Comment

It will be seen that the sensitivity of the cytopipette to detect malignancy varies from 78 to 100 per cent according to type of malignancy, an average of 88 per cent (or 91 per cent using Davis’ criteria). On the other hand, in the state of dys-

Volume Number

106 1

plasia, as with the polyglot unknown group where exfoliation is perhaps not quite so prolific, the detection rate drops to around 70 per cent. The use of this technique would indeed be questionable if the finer or more sensitive criteria of “pick-up” are not used as suggested by Davis. His own criteria, however, are to some degree nonspecific, and a rescreen of part of our negative material produced an “atypical rate” of around 4 per cent, which is obviously too high. We prefer to utilize the traditional criteria of mild and marked dyskaryosis, and scored sensitively we reach almost the same target, but with an over-all atypical rate of just under 2 per cent. If, however, the instrument is used as an “alerting test” where the “pick-up” group is merely given a good scrape smear and no serious objection clinical examination, would be forthcoming if the acceptance rate for the test is higher than for the scrape smear and the use of resources much lower. The quality rate of the few smears collected by nurse and doctor compared with those collected by patient proved to be closely similar, a finding in keeping with those of MacGregor, Frazer, and Mannlo who had an unsatisfactory rate of 16 per cent when patient, and 14 per cent when doctor performed the test; and also Reagan and Linla with rates of doctor (9.2 per cent) and patient (10.0 per cent). On the other hand, Anderson and Gunn2 showed an improved quality of use in the hands of the professional worker, with 6.4 per cent for doctor, compared with 15.5 per cent unsatisfactory rate for patient collection. In this series, however, the patients were composed largely of lower socioeconomic colored population. In this trial a one-test-only policy was carried out, and so a number of pipette samples containing too few cells were not repeated-as would normally be the case. The criteria that an irrigation smear should contain between 30 and 50 cells in a lowpowered field is essential and would necessitate about 3.5 to 4 per cent repeats in this series, though in 3 cases “alerting” dyskary-

Irrigation

smear

145

otic cells were present in below optimum cellularity smears. This compares with 5.4 per cent unsatisfactory smears due mainly to scanty material in a series by Koch and and around 3 per cent in Davis Stakeman,” and Jones’ hands7 One of the greatest attributes of the pipette is its low cost. In this project the whole pipette, postal tube, sack and return postage cost 2/6d. each. This compared favorably with the average cost of clinic attendance of 25/- to 40/- per person. Offset against this is the extra laboratory load of mass processing of the pipette, which, we calctdate, adds a further 6d. to each test. There are many things to say about the pipette and its improvement. A larger pipette is a distinct advantage and a new one is on the market. We are using this in the next trial. A more bland fluid with better preserving qualities is also necessary, and this, too, exists in the new model. Processing can be much simplified, and we have already altered our schedule, using the new solution DS 411 incorporated in the Davis pipette. There is no need to use a Carbowax solution in the processing schedule as preservation and fixation are sound with the new solution, and smears can be made direct onto a slide. Moreover, simple sedimentation for some hours seems sufficient to concentrate the cells, and centrifugation is only necessary if the cellular sample is scanty. Thus, if the pipette is enlarged to increase the volume of irrigating solution, and more descriptive and illustrated instructions given to the patient, a simpler procedure adopted in laboratory processing and greater skill achieved in scanning, the pipette should provide a technique nearly equal to that of the scrape smear for what could bc a fraction of the cost. As prophylactic early screening measures for preclinica1 disease processes increase in number, the most crucial factor will be one of cost and use of resources. It may well be necessary to screen those of higher risk, for example, the older woman, much more frequently if screening is to be

146

January 1, 1970 Amer. J. Obstet. Gynec.

Husain

effective; would

then

the

cost

of

clinic

attendance

be prohibitive.

Moreover, nique

as a principle,

has

could

much

be used

of test

for

the

wider

pipette

implications

chemical

and

techas

other

it

forms

collection.

These

arguments

development instrument.

of

appear such

a

to justify potentially

further useful

Our thanks go to the Ministry of Health who sponsored this project, and to Dr. J. M. G. Wilson for his advice and encouragement: also to the gynaecologists, Mr. Frank Denny, Mr. J. K. Morrison, Mr. D. W. S. Gordon, and Mr. A. H.

REFERENCES

1.

Anderson, A. F., and Clark, F. R.: Lancet 1: 479, 1966. 2. Anderson, W. A. D., and Gunn, S. A.: Cancer 20: 1587, 1967. 3. Anderson, W. A. D., and Gunn, S. A.: Acta Cytol. 10: 149, 1966. K.: Acta 3a. Anderson, G. H., and Krakauer, Cytol. 10: 418, 1966. 4. Bredahl, E., Koch, F., and Stakemann, G.: Acta Cytol. 9: 189, 1965. 5. Davis, H. J.: AMER. J. OBSTET. GYNEC. 84: 1017, 1962. 6. Davis, H. J.: Acta Cytol. 6: 459, 1962. 7. Davis, H. J., and Jones, H. W., Jr.: AMER. J. OBSTET. GYNEC. 96: 605, 1966.

Milne, who kindly permitted their patients to be investigated. We are grateful to the Medical Officers of Health and their colleagues in the following Boroughs: Kensington and Chelsea (Dr. J. H. Weir), Richmond upon Thames (Dr. A. M. Nelson), Hammersmith (Dr. A. D. C. S. Cameron), and Wandsworth, (Dr. J. Tudor Lewis); to the doctors and nurses of the Family Planning Association Clinics in those areas, and to Dr. K. Huntington (general practitioner) who cooperated so fully in the trial: also to Sister K. Grant, and to cytoscreeners R. Puckett, D. Robinson, Y. Harris, and J. Wells, who really did all the work.

8.

Koch, F.: The Population Screening for Cervical Carcinoma in the Borough of Frederiksberg 1962-1963, Copenhagen, 1966, Einar Munkspaard Forlag. 9. Kbch, F., an; Stakemann, G.: Danish Med. Bull. 11: 209, 1964. 10. MacGregor, J. E., Fraser, M. E., and Mann, E. M. F.: Lancet 1: 252, 1966. 10a Mattingly, R. F.: Second International Congress of Exfoliative Cytology, Paris, 1965. 11. Muskett. T. M.. Carter. A. K.. and Dodrre. 0. G.: &it. Med. J. 2:’ 341, 1966. - ’ 12. Reagan, J. W., and Lin, F.: Acta Cytol. 11: 374, 1967. 13. Richart, R. M., and Vaillant, H. W.: J. A. M. A. 192: 199, 1965.