Office gynecologic cytology

Office gynecologic cytology An evaluation

ROBERT Denver,

A.

of the Riva-Turner

MUNSICK,

staining

M.D.,

and screening

method

PH.D.

Colorado

CYTOLOGIC STUDY of the female reproductive tract has achieved great precision and deserved popularity in the diagnosis of gynecologic malignancies. Contemporary cytologists generally employ modifications of the original method of Papanicolaou and Trautl for the staining, screening, and final determination of criteria of malignancy. Nevertheless, various investigators have sought to circumvent disadvantages inherent in the Papanicolaou technique. Some of these impediments are: (1) necessity for slide fixation, (2) prolonged, tedious staining techniques, (3) relatively long delays between smear preparation and final cytologic screening, (4) long training periods for cytotechnicians, (5) relatively slow screening rates, even among experienced cytologists, and (6) a temporal and spatial schism created by departmentalization between the physician and the cytologist. Friedman” first used fluorescence microscopy for the cytologic detection of malignancy. Bertalanffy3 then studied fluorescence technique in detail and found that acridine orange provided good differential fluorescent staining of nuclear deoxyribonucleic acid (DNA) and cytoplasmic ribonucleic acid (RNA) with ultraviolet irradiation. Bertalanffy3 has continued to use the acridine orange method, screening for increased cytoplasmic RNA, which fluoresces red. His

results have been quite acceptable and have recently been extensively reviewed. Some investigators have endorsed this method.4l 5 Others, however, have had variable success with it and generally find that the method is too imprecise for clinical application.+1° Recently, Riva and Turner11-12 have devised a new acridine orange staining technique which requires only 10 seconds and in which the red fluorescence of RNA has been markedly suppressed. Their screening is done with relatively economical equipment and is dependent entirely upon nuclear morphology and fluorescence. Recently, Kawasaki and Wilson13 and Batt** have confirmed the value of the Riva-Turner method for the detection of cervical cancer. The Riva-Turner method appeared to encompass so many favorable features that an independent appraisal of its value appeared desirable. This investigation was, therefore, undertaken in order to determine whether or not a clinician with no cytologic experience prior to a brief training course could use this method and obtain results suitable for office practice or clinical teaching. As the results will show, this study reaffirms the reliability and practicality of the method. Materials and methods All equipment was that recommended by Riva and Turner.ll Slides were obtained from April through September, 1963, from patients having routine or indicated Papanicolaou smears performed in the obstetric and gynecologic clinics of the University of Colorado Medical Center. Papanicolaou smears were obtained first

From the University of Colorado Medical Center, Department of Obstetrics and Gynecology. This investigation was supported grant-in-aid from the Millheim Foundation for Cancer Research, Denver, Colorado.

by a

411

with the use of separate slides for the cervis and the posterior vaginal pool. Smears wert’ prepared by medical students, residents, and attending staff physicians with cotton tipped applicators. The slides were immediately placed in a fixative and smears for acridine orange examination were then obtained. In this case cervical and vaginal smears were placed separately on one slide and were allowed to dry in the air at ambient temperatures. A brief clinical history was provided to the Papanicolaou cytologists and to the author. Papanicolaou smears were stained, mounted, and screened by the Exfoliative Cytology Laboratory of the Department of Pathology, University of Colorado Medical Center, and final interpretations of all atypical smears were provided by a pathologist experienced in clinical cytolocgy. Acridine orange smears were interpreted exclusively by the author. Staining and screening were performed a few minutes to a few weeks after the smears were obtained. The 10 second staining method of Riva and Turner was used.ll Screening was performed by examining first the vaginal end of the smear under low power magnification 1x100) for hormonal effect, inflammatory and atypical cells, and pathogens. The remainder of the slide was screened quickly by the Riva-Turner method, observing for a “break in pattern,” and for cell nuclei brighter or larger than the predominant type. Abnormal cells were then examined and interpreted under high-power magnification (x450). Smears without significant inflammation or atypia are usually screened in this manner in 30 to 60 seconds. The author’s only experience in the field of cytology had been acquired during a total of 7 days with Riva and Turner. Thus, with only one week of training, and with no other cytologic experience, the study was started. The acridine orange smears were classified according to the scheme of Papanicolaou: Class I, normal; Class II, atypical; Class III, suspicious of malignancy; Class IV, highly suspicious of malignancy; and Class V, with criteria cytolo,+c of malignancy.15 The

cytology laboratory IISC’S a similar classification, but dots not 11s~ the Class V category. In tabulating the acridine orange results, the Class V smears were, thcreforc,: incorporated with those of (:lass IV. Only those Papanicolaou and acridine orange smears obtained within 72 hours of one another were considered comparable and applicable to the study (over 90 per cent were obtained at the same clinic visit). Histologic diagnoses wrre obtained from all patients with persistently suspicious Papanicolaou smears regardless of the acridine orange result. If the acridine orange result with variable was repeatedly suspicious Papanicolaou reports, conical cervical biopsy and uterine curettage were, likewise, performed. Thus, from the total of 1,093 patients, 26 were subjected to diagnostic biopsy procedures. In a few patients with (Xass III acridine orange smears and persistently negative Papanicolaou smears, no biopsy was “false performed. They were considered positives” in the acridine orange series. Results The results have been compared in several ways. In analyzing only the initial smears of the 1,093 patients examined, Table I shows that there was a 0.1 per cent false negative rate in Class I acridine orange reports and a 0.09 per cent rate in the Papanicolaou group. One and seven tenths per cent of the Class II smears were false negative by a&dine orange versus 24 per cent by Papanicolaou. By acridine orange 25 per cent of Class III and 86 per cent of combined Classes IV and V had malignancies. By Papanicolaou 75 per cent of Class III and 100 per cent of Class IV smears were positive. When these data are divided into negative (Classes I and II) and suspicious (Classes III and IV) groups: the results are as follows: By acridine orange 0.19 per cent of negative smears were false negative and 58 per cent of suspicious smears were positive. By Papanicolaou, there was a 0.46 per cent false negative and an 86 per cent positive rate among suspicious smears. When the results of all comparable smears,

Volume !24 Number 3

OfFice cytology

including repeated smears, are considered, the results for both methods are also seen in Table I. False negative smears were found in 0.1 per cent of Class I and 3.1 per cent of Class II acridine orange smears and in 0.37 per cent of Class I and 33 per cent of Class II Papanicolaou smears. The malignancy rate was 37 per cent in Class III and 88 per cent in Classes IV and V of acridine orange smears and was 64 per cent in Class III and 100 per cent in Class IV Papanismears. Combined negative and colaou suspicious results for all comparable smears, including repeated smears, are as follows: By acridine orange there was a 0.28 per cent false negative rate and by Papanicolaou a 1.0 per cent false negative rate. Sixty per cent of the acridine orange suspicious smears and 82 per cent of the Papanicolaou suspicious smears were shown later to have been derived from patients with cancer. The acridine orange data, reclassified into negative (Classes I and II), suspicious (Classes III and IV), and with criteria of malignancy (Class V) are tabulated in Table II, both for initial smear and for all smear data. For initial smears, 0.19 per cent of negative, 35 per cent of suspicious, and 100 per cent of smears with criteria of malig-

Table I. Comparison

of acridine orange by classification, method,

of malignancy

Initial Acridine

I II III IVandV

1,007 60 12 14

Table II. Incidence

No. 1 1 3 12

0.1 1.7 25 86

of malignancy

Smears

Negative Suspicious With criteria malignancy Totals

Several facts became apparent to us when we reviewed our data. Our Papanicolaou laboratory is far more conservative than most in its classification of slides, for approximately one quarter of the patients with Class II smears were later found to have malignancy and more than half of the Class III smears were from patients with proved cancer. The author’s results with the Riva-Turner 10 second acridine orange staining method were quite rewarding. They revealed fewer false negative smears (Classes I and II) than in the Papanicolaou series and positive smears in suspicious categories which are in approximate accordance with other series. The author concludes, therefore, that the Riva-Turner method offers clinicians a reliable screening device for cervical carcinoma. Proper training in this method is mandatory, however, for, as stipulated by its originators, “screening for atypia by increased nuclear fluorescence is such a radical

and Papanicolaou smears, showing and in initial and repeated smears All

0.09 24 75 100

in acridine

1,009 65 19 16

orange

9

smears

I;;$-1

Papanicolaou

$;1 2 7 14

No. 0.1 3.1 37 88

smears by another

%

Smears

/“““:I

1,071 21 11 6

MT& 4 7 7 6

0.4 33 64 100

classification All

1

incidence

orange

smears

1 Malignancies

2

1,093

No.

1 4 6 6

1,067 17 of

A&dine

~~4+?i~-

1,062 17 8 6

Initial Class

Comment

Papanicolaou

-~“:“:/M& No.

Class

nancy were proved to have malignancies. For all smears, including repeated ones, the percentages were 0.28 per cent, 46 per cent, and 100 per cent, respectively.

smears

orange

413

smears

) Malignancies

1

s/o

9

0.19 35 100

1,074 26 9

3 12 9

0.28 46 100

17

1.6

1,109

22

2

414

Munsick

departure from usual microscopy that indocrination in this method is critical if acceptable results are to be obtained.“ll The Kiva-Turner method has other advantages and some disadvantages which defy statistical analysis. Advantages over the Papaniocolaou method include: no fixation, rapid screening, minimal rapid staining, laboratory space and equipment, rapid teaching, reliable diagnosis of trichomonads and yeast, and finally, economy. Disadvantages are: loss of cells from slides with repeated examinations, relatively rapid fading of the dye, nonvisualization of cytoplasmic details, slightly poorer nuclear detail, and the very remote possibility of cornea1 or retinal burns secondary to ultraviolet irradiation. Although we do not currently employ the Riva-Turner acridine orange method for official readings of cervicovaginal smears, we do find it rewarding that our resident staff physicians and medical students have familiarized themselves with the basic concepts of gynecologic cytodiagnosis and that they employ acridine orange in cases where: (1) an immediate evaluation for malignancy is desirable, (2) hormonal evaluations may aid in a patient’s management, and (3) confirmation or primary diagnosis of the tricho-

monal or monilial etiology of a vaginitis is desirable and a wet-drop preparation is nondiagnostic. The immediate availability of a staining method by which hormonal effects, vaginitis diagnosis, and malignancy detection are combined has enthused students and teachers alike, and will contribute, we hope, to a much greater awareness among physicians and students of the importance and nature of cytodiagnosis. For office cytodiagnosis the method is highly satisfactory for it combines economy, speed, reliability, versatility, and simplicity. Summary The 10 second acridine orange staining method of Riva and Turner has been compared with the classical Papanicolaou method for the cytodiagnosis of gynecologic cancer. The results indicate that a clinician with 2 weeks of special training in the method can obtain results with sufficient accuracy for incorporation of this method into the oflice laboratory. Other advantages of the method are also discussed. The author wishes to express his thanks to H. L. Riva and T. R. Turner for the time which they have sacrificed in teaching their method.

REFERENCES

Papanicolaou, G. N., and Traut, H. R.: Diagnosis of uterine cancer by the vaginal 1943, Commonwealth smear, New York, Fund. 2. Friedman, H. P.: AM. J. OBST. & GYNEC. 59: 1.

852, 3.

10. 11. 12.

1950.

Bertalanffy,

9. Rockey,

F. D.: Ann.

New

York

Acad.

SC.

93: 715, 1962. Marks, R., and Goodwin, A. M.: Brit. J. Cancer 16: 390, 1962. 5. Frampton, J.: ’ J. Obst. & Gynaec. Brit. Comm. 70: 561, 1963. 6. Liu, W.: Arch. Path. 71: 282, 1961. J. C., and Ackerman, M. R.: Obst. 7. Holland, & Gynec. 17: 38, 1961. 8. Betts, A., Achenbach, R. R., and Friedell, G. H.: Arch. Path. 73: 250, 1962.

13.

4.

14. 15.

T. J. F.: J. Obst. & Gynaec. Brit. Comm. 70: 571, 1963. Stevenson, J.: Acta Cytol. 8: 224. 1964. Riva, H. L., and Turner, T. R.: Obst. & Gynec. 20: 451, 1962. Riva, H. L., and Turner, T. R.: AM. J. OBST. & GYNEC. 85: 713, 1963. Kawasaki, D. M., and Wilson, J. H.: Obst. & Gvnec. ‘22: 636. 1963. Batt,‘R. E.: Obst: & Gynec. 24: 248, 1964. Papanicolaou, G. N.: Atlas of exfoliative cytology, Cambridge, Massachusetts, 1954, Harvard University Press, pp. 20-21. Bernalillo County Indian Hospital 2211 Lomas Blvd., N. E. Albuquerque, New Mexico 87106