Journal of Cytology

: 2014  |  Volume : 31  |  Issue : 3  |  Page : 154--157

Efficacy of rapid, economical, acetic acid, Papanicolaou stain in cervical smears as an alternative to conventional Papanicolaou stain

Shabnam Izhar1, Rupinder Kaur2, Kanwal Masih2,  
1 Department of Pathology, Rohilkhand Medical College and Hospital, Bareilly, Uttar Pradesh, India
2 Department of Pathology, Christian Medical College and Hospital, Ludhiana, Punjab, India

Correspondence Address:
Shabnam Izhar
Akash Puram Colony, Lala Begum Jagatpur, Bareilly, Uttar Pradesh


Background: Papanicolaou (Pap) stain has been used over the years for cervical cytology screening. However; it utilizes a considerable amount of alcohol which is expensive and difficult to procure. In one of the modifications, ethyl alcohol is replaced by 1% acetic acid and is termed as rapid, economical, acetic acid Papanicolaou (REAP) stain. It is cost effective, easily available and provides a suitable and rapid staining alternative. Aim: This study was undertaken to assess the efficacy of REAP stain as an alternative method to conventional Pap stain. Materials and Methods: This study was done over a period of 18 months in a tertiary care hospital. Two sets of cervical smears were prepared of which one was stained with conventional Pap stain, and other was stained with REAP stain. The smears were examined for cytomorphological parameters and were evaluated using a modification of parameters given by Ng et al. Results: A total of 737 smears were examined in duplicate. Most of the conventional Pap smears showed excellent preservation (91.6%) with very few showing optimal (7.6%) and sub-optimal staining (0.8%). In contrast to this excellent preservation was seen in just 33.6% of the REAP stained smears with majority showing optimal and sub-optimal preservation (46.5% and 20% respectively). The P value was statistically significant (<0.0001) depicting inferior staining quality of REAP stain. Conclusion: Rapid, economical, acetic acid Papanicolaou stain undoubtly is a simple, fast and cost effective stain which can be adopted mainly in resource limited settings, but cannot be utilized for research purpose in a tertiary care setup due to poor preservation of the staining quality.

How to cite this article:
Izhar S, Kaur R, Masih K. Efficacy of rapid, economical, acetic acid, Papanicolaou stain in cervical smears as an alternative to conventional Papanicolaou stain.J Cytol 2014;31:154-157

How to cite this URL:
Izhar S, Kaur R, Masih K. Efficacy of rapid, economical, acetic acid, Papanicolaou stain in cervical smears as an alternative to conventional Papanicolaou stain. J Cytol [serial online] 2014 [cited 2022 Aug 9 ];31:154-157
Available from:

Full Text


Papanicolaou (Pap) staining was first described by Papanicolaou in 1943 [1] is widely used as a screening test in spite of being time consuming and requiring a large amount of alcohol. [2]

The original Pap staining of cervical smears has undergone modifications for various reasons, either for decreasing turnaround time or for finding alternative to alcohol to be cost effective. [3],[4] In India, it has relevance because a laboratory needs a license from the government for supply of alcohol. The quota of supply is limited and at times irregular.

Dighe et al. [5] used 1% acetic acid replacing alcohol in most of the steps. Acetic acid acts as a mild dehydrating agent, is cheaper and easily available. This modified method was referred to as rapid, economical, acetic acid Papanicolaou (REAP) stain. In their study, they compared the cytoplasmic and nuclear features of the cells with those with conventional Pap stain. The differentiation and transparency of the cytoplasm of REAP stain were optimal in 90.5% of the cases, and the nuclear details and chromatin pattern were clear and crisp in 96% of the cases. Similar observation was made by another study done by Biswas et al. [6]

Since only two studies had been done on REAP stain, this study was undertaken to compare the conventional Pap stain with REAP stain for staining cervical smears.

 Materials and Methods

The present study was conducted in the cytology section of Department of Pathology, Tertiary Care Hospital for a period of 18 months.

The cervical smears were taken in the gynecology out-patient department as a part for routine screening as well as for various indications. Two slides prepared from each patient, were encoded and were immediately wet fixed in 95% ethanol. Thereafter one of the smear was stained by the standard Pap stain and the other slide was stained by REAP stain.

In REAP stain, acetic acid was used instead of alcohol in the protocol of conventional Pap staining.

1% acetic acid-10 dips.Harris hematoxylin, preheated 60°C-10 dips.Tap water-10 dips.1% acetic acid-10 dips.OG-6-10 dips.1%acetic acid-10 dips.EA-50-10 dips.1% acetic acid-10 dips.Methanol-10 dips.Xylene-10 dips.

The original labels were replaced by another set of identification code that was unknown to two of the three pathologists screening the slides to avoid bias. All the cases were screened for cytomorphological parameters. The smears were then numerically evaluated by three different observers using the modification of parameters given by Ng et al. [7] in four categories.

Excellent cytological preservation with sharp nuclear and cytological features - 4.Optimal preservation of nuclear and cytological features - 3.Sub-optimal preservation of nuclear and cytological features - just able for assessment - 2.Very poor cytological preservation of nuclear and cytoplasmic features unsuitable for assessment - 1.

In the cases where there was the difference in the scores a common consensus was taken. The results were then analyzed using an independent t-test considering significance at the level of P < 0.05.


A total of 737 cases were included in the study. Majority of the patients that is 218 (29.6%) were in the fifth decade. Out of the total, 41.4% came for routine checkup while remaining 58.6% came with various other complaints, e.g., history of discharge per vaginum, menstrual irregularities and bleeding per vaginum.

On examination majority of them (77.5%) had a normal cervix. Only a few had cervical erosion (11.7%), chronic cervicitis (5.9%) and cervix suspicious of carcinoma (2.8%).

The cytological study of cervical smears showed inflammatory changes in approximately one-third of the cases (34.2%) whereas it was normal in 14.4%.

Premalignant changes in the form of dyskaryosis were observed in 1.3% of the cases. Many cell groups in these smears displayed high nuclear/cytoplasmic ratio, irregular nuclear membranes, coarse chromatin with chromatin clumping in few and scant to moderate amount of cytoplasm.

Frank malignancy (squamous cell carcinoma) was detected in only two cases. On examination cervix of one of the patient showed cervical growth whereas other patient presented with bleeding per vaginum.

Cytological study of the smears were done and numerically evaluated using the modification of parameters given by Ng et al. [7]

Out of the total 737 conventional pap smears, 675 (91.6%) showed excellent cytomorphology with a score of 4 [Figure 1]. Optimal preservation of nuclear and cytoplasmic features was seen in 56 (7.6%) cases. Six cases (0.8%) showed sub-optimal preservation. The average score of 3.90 was observed with this stain [Table 1].{Table 1}{Figure 1}

In REAP stained smears, excellent nuclear and cytoplasmic details were seen in 247 (33.6%) cases only. This was followed by a score of 3 in 342 cases (46.5%) with optimal cytomorphology whereas a score of 2 was seen in 148 cases (20.0%) [Figure 2]. The average score was 3.13 [Table 1].{Figure 2}

There were no cases of poor preservation (score 1) in any of the Pap or REAP stained smears.

Excellent nuclear and cytoplasmic features were noted in all of the 12 (100%) cases of dyskaryotic and malignant cervical smears by conventional Pap method whereas the morphology was excellent in only 50% of the cases by REAP method. In rest of 50% of the REAP smears, the preservation of nuclear and cytoplasmic features were optimal.

None of the smears (PAP or REAP) showed a score of 1 where the preservation of nuclear and cytoplasmic details was compromised.

The average score was 4.0 in PAP and 3.5 in REAP for dyskaryotic smears.

The P < 0.0001 hence significant depicting PAP stained smear to be superior to than those stained with REAP.


The Pap test has been used extensively for the early detection of cancerous, precancerous as well as inflammatory lesions of cervix worldwide. Increase turnaround time, high cost and difficulty in availability of alcohol has led to various modifications of Pap stain in different laboratories.

Ultrafast [3] and rapid Pap [4] staining methods are less time consuming but require a large quantity of alcohol. There are certain methods in which methanol is used as a substitute for ethyl alcohol. However, the problem of usage of alcohol which is expensive has not been completely resolved so far. Dighe et al. [5] in 2006 used acetic acid as an alternative to alcohol to overcome the problem of alcohol and also to reduce the duration in REAP stain. Later on Biswas et al. [6] undertook the study using acetic acid to limit the use of ethyl alcohol.

Optimal preservation in cervical smears stained with REAP stain was seen in the majority of the cases in the study done by Dighe et al. [5] and Biswas et al. [6] (90.5% and 90% respectively) whereas our study showed optimal preservation in just 33.6% of REAP stained smears. Sub-optimal preservation was seen in 20% which was much higher than the study done by Dighe et al. [5] (9.5%) and Biswas et al. [6] (9.0%).

An additional observation made in our study was that, the morphology of the endocervical cells when present singly were better stained as compared to when in clusters in REAP, which could be attributed to poor penetration of the stain in overlapping clusters of cells.

Our study also revealed lysis of the red blood cells in the hemorrhagic smears with no change in squamous or glandular cell morphology due to the presence of acetic acid in REAP stain facilitating in the interpretation of cervical smears.


Rapid, economical, acetic acid Papanicolaou stain is a simple, cost-effective and fast staining technique for cervical smears with less turnaround time and can be used in the resource limited setting where alcohol availability is limited, and cost factor is an issue. It cannot be utilized for routine staining in a tertiary setup or for research purposes due to poor preservation of the staining quality. Slides fades when kept for more than 6 months.


1Papanicolaou GN. A new procedure for staining vaginal smears. Science 1942;95:438-9.
2Bales CE. Laboratory techniques. In: Koss LG, editor. Koss Diagnostic Cytology and Its Histopathological Bases. 5 th ed., Vol. II. Philadelphia: Lippincott Williams & Wilkins; 2006. p. 1592-01.
3Yang GC, Alvarez II. Ultrafast Papanicolaou stain. An alternative preparation for fine needle aspiration cytology. Acta Cytol 1995;39:55-60.
4Sato M, Taniguchi E, Kagiya T, Nunobiki O, Yang Q, Nakamura M, et al. A modified rapid Papanicolaou stain for imprint smears. Acta Cytol 2004;48:461-2.
5Dighe SB, Ajit D, Pathuthara S, Chinoy R. Papanicolaou stain: Is it economical to switch to rapid, economical, acetic acid, Papanicolaou stain? Acta Cytol 2006;50:643-6.
6Biswas RR, Paral CC, Dey R, Biswas SC. Rapid, economic, acetic acid, Papanicolaou stain (REAP) is it suitable alternative to standard PAP stain? Al Ameen J Med Sci 2008;1:99-103.
7Ng WF, Choi FB, Cheung LL, Wu C, Leung CF, Ng CS. Rehydration of air-dried smears with normal saline. Application in fluid cytology. Acta Cytol 1994;38:56-64.