PAPANICOLAOU TECHNIQUES 



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PAPANICOLAOU TECHNIQUES 



during the normal estrous or menstrual 

 cycle, as well as changes due to various 

 hormonal factors. For his studies in 

 both guinea pigs and the human, he 

 used alcohol-ether fixed smears which 

 were stained first with hematoxylin 

 and then counterstained with aqueous 

 solutions of eosin and water blue (see 

 staining technique I). This method 

 was a simple one technically and re- 

 sulted in good differentiation between 

 basophilia and acidophilia. It is still 

 considered useful in hormonal studies. 



During the second period, modifica- 

 tions in technique were investigated 

 which would be better adapted to the 

 diagnosis of malignant neoplasms. In 

 thick smears where cells are found in 

 compact clusters, overljang one an- 

 other, or mixed with blood or mucus, 

 a light, transparent, cytoplasmic stain 

 is necessary to give a clearer view of 

 individual cells and their nuclei. 

 Papanicolaou found that this could be 

 achieved by using 95% alcohol as the 

 solvent for the counterstains. Stains 

 006 and EA36 (formulae 2 & 3 follow- 

 ing the staining procedures) were intro- 

 duced in 1942; EA 65 (formula 4) is a 

 later modification of EA36. When 

 used in combination with a good nuclear 

 stain, these counterstains are particu- 

 larly well adapted to cancer diagnosis 

 but are also excellent for endocrine 

 studies because of their delicate differ- 

 ential staining qualities. They can 

 also be used in staining tissue which 

 is advantageous in the correlation of 

 smears and sections. (For use of alco- 

 holic counterstains, see staining tech- 

 niques II, III, IV, V.) 



Female Genital Tract. Vaginal, endo- 

 cervical and endometrial aspiration 

 smears; cervical swab and spatula 

 smears. 



Equipment: For vaginal smears: a 

 glass pipette, five inches in length and 

 one quarter inch in diameter, slightly 

 curved about two inches from its blunt 

 tip, and equipped with a tightly fitting 

 strong rubber bulb. The pipette should 

 be made of hard pyrex glass to with- 

 stand sterilization. For endocervical 

 and endometrial aspiration smears: a 

 Becton-Dickenson laryngeal cannula 

 equipped with a strong rubber bulb. 

 For cervical smears: a non-absorbent 

 cotton swab or the wooden spatula 

 designed by Ayre (Avre, J. E., Am. J. 

 Obst. & Gynec, 1947, 53, 609). For 

 all types of smears: a jar of fixing solu- 

 tion, i.e. equal parts of 95% ethyl alco- 

 hol and ether, and clean slides. When 

 more than one slide is placed in a bottle 

 of fixative, a paper clip is attached to 

 one end of each slide in order to prevent 



contact between the smeared surfaces. 

 The slides are marked in advanced 

 with the patient's name or case num- 

 ber, type of smear and date. This can 

 be done either with a diamond pencil, 

 or by clipping a small piece of paper or 

 card to the slide. In the latter case, 

 the label should be written with lead 

 pencil, not ink. 



Preparation of smears: Smears should 

 not be prepared after a douche, bath, 

 or use of a lubricant. They should be 

 prepared prior to the pelvic examina- 

 tion, if possible. 



Vaginal smears: The dry pipette, 

 with bulb compressed, is introduced 

 into the posterior fornix of the vagina 

 and the bulb released. The pipette is 

 rotated carefully during aspiration to 

 obtain samples from different parts of 

 the vagina. The fluid thus obtained 

 in the pipette is expelled on to a slide, 

 and spread in a uniform smear with 

 the side of the pipette. The slide is 

 immersed immediately, before the 

 smear dries, in the alcohol-ether fixative 

 and allowed to remain for at least 15 

 minutes for thorough fixation of cells 

 before staining. Smears may be left 

 in alcohol-ether for a week or even 

 longer without harmful effect. 



Endocervical or endometrial smears: 

 After a speculum is in place, endocervi- 

 cal or endometrial secretion is aspirated 

 by means of the laryngeal cannula, 

 expelled on to a slide, spread uniformly 

 with the cannula, and the slide im- 

 mersed immediately in alcohol-ether. 



Cervical swab smears: Secretion ob- 

 tained by means of a swab or spatula 

 from the external os, or from any suspi- 

 cious appearing lesion of the cervix, 

 is quickly spread on a slide and fixed 

 in alcohol-ether. 



The vaginal smear, because it is 

 composed chiefly of squamous cells 

 which reflect hormonal activity is the 

 best type for hormonal evaluation, and 

 for a study of the menstrual cycle. 

 For the latter, smears may be prepared 

 daily throughout the complete cycle 

 by the patient. Slides, either dated 

 or numbered serially in advance, with 

 clips attached, a pipette and jar of 

 fixative are given to the patient and 

 she is instructed how to prepare them. 



For diagnosis of malignancy, a vagi- 

 nal smear gives a more complete overall 

 picture of the cytology of the genital 

 tract since cells from all parts accumu- 

 late in the vaginal pool. Endocervical 

 smears reveal the presence of a cervical 

 carcinoma, endometrial smears are 

 better for diagnosis of fundal, and occa- 

 sionally, tubal carcinomas. Cervical 

 swab or spatula smears give informa- 



