Rapid, Economic, Acetic acid, Papanicolaou (REAP) staining technique analogous to the routine Papanicolaou staining (PAP) technique

Introduction

George Papanicolaou created the PAP dye, a vivid color examination of the cell tinge method, in the year 1942. Dr. Papanicolaou afterward updated his research in the years 1954 and 1960.¹ As G.N. Papanicolaou initially studied the PAP stain, he went through numerous alterations, and was later modified by him in the year 1960.¹ The Mallory aniline blue method, the Masson trichrome method, and the short methods can all be found in the history of the Papanicolaou staining method.² In human medicine, Pap is the gold-standard staining technique for FNAC or exfoliate cells, which are used to diagnose neoplastic disorders.³

SB Dighe (2005) published the REAP method.⁴ To compare the excellence of REAP blotching of spatter to conventional PAP staining using upcoming guidelines: Solar & protoplasmic blotching intensity, staining time, vanished success, lengthy duration of shade maintenance.⁵

Papanicolaou stain has been modified to increase staining quality and/or to shorten staining time.⁶ PAP stains are accustomed to detecting and differentiating units from smears obtained in different physical fluids,⁷ gynecological smears,⁸ and FNAC⁹ smears from different organs. Various types of body fluids and needle biopsies are used to stain the specimens; the samples include gynecological Papanicolaou spatter, froth, grazing, cleaning, discharge, BAL, ascitic fluidic, pericardial fluidic, CSFs,¹⁰ malignant ascites, pleural effusion, joint effusion, semen discharge,¹¹ FNAC,⁸ neoplasm affecting specimens, and matter containing loose cells. An application of spot-on tissue slides is connected with loose cell inspection. Papanicolaou spots are gold standard cells of the cervix covering up. It produces a metachronous, clear spotting response by distinct solar & cellular characteristics.

Conventional Papanicolaou Staining Procedure: Shift glass slides immediately out of the alcohol aether adhesive to 80% alcohol watery, then via 70% & 50% alcohol to D/W. For 4 minutes, ‘stain’ with ‘Harris hematoxylin’. Drain with D/W for a few seconds. Six times, bath through 0.25% Hydrochloric Acid in 50% eth-an-ol (20-60 sec). 6 minutes in flowing tap water Run through 50%, 70%, 80%, and 95% alcohol after rinsing with distilled water. 2 minutes staining in OG-6. Rinse in two 95% alcohol changes. For 2 minutes, stain in EA 36/EA 50. Rinse three times in 95% alcohol. Remove water from the abs. alc-oh-ol, then in similar pieces xylol and abs. alc; clear in dimethylbenzene and cover with DPx.¹²

REAP P-A-P Staining Method: 1% ACID ACETIC ten soaks. Harris Haematoxylin 10 dips. [Previously heat to 60°C] Ten soaks in running H2O. ethanoic acid 1%, 10 dips for OG-6. 1%acetic acids ten soaks. Eosin azure-50 ten dips. 1.0% acetic - acid Meth-an-ol ten dips dissolvent ten dips perform mop up with every process. Install in DPx. In REAP, at every stage of alc-oh-ol in standard Papanicolaou was changed with 1%ethanoic acid.¹³

Phosphotungstic acid is applied to counterstains to modify the pH and aid maximize the color potency. This Eosin Azure contrast stain comprises Bis-marck-brown and phos-pho-tungstic acid, both of which settled down away from emulsion when combined, limiting usable lifespan concerning blending.¹⁶

This dye must produce very clear units, allowing often thick samples of merged units to be analyzed. Cellular nuclei should be frangible and blue-black in chroma, with well-defined histone designs. Cellular cytosol colors blue to green, while protein keratin gets – orange.¹⁷

The benefits of REAP stain include: superior to normal Pap staining, good nucleus/cytosol stain improved color clarity, and low value.¹⁸ Acetic acid substitutes expensive eth-an-ol (25% of the complete price -of normal Papanicolaou stain), allowing for lasting duration color retention (>1 FY without dwindling), The technique is rapid, and the stain-ing of transitional cells such as RedBloodC/WhiteBloodC/Bacteria is safely stored, the nucleus/histones/karyon features = transparent/bracing, the cyno-philia/raised eosinophils within smears¹⁹ is equivalent to PAP stain, which breakdown RED blood Cells without alteration in tubular-cells shape.⁹ Due to its low value, it’s a viable option for regular PAP smear seen within a lump of cervix carcinoma identified in underdeveloped nations. The downside though REAP stain comprises discoloration observable within the smudge coat, especially observed cellular clumping, because of its low color accumulation.²⁰ Some scientists believe that when slides are stored for more than 6 months, REAP stain provides lower color standards regarding improper storage.²¹ Also, stains – are best employed in resource-constrained contexts in which there is a price issue, rather than in exploration/triennial settings.¹⁵

Ashok Kumar Deshpande et al.⁴ conducted a study comparing Papanicolaou stain (PAP) with Rapid Economic Acetic acid Papanicolaou Stain [REAP] at the cellular level of the cervix.¹² This research comprised 200 samples from persons of various ages. The study found that PAP Stain is substantially more time-consuming and costly than REAP Stain- lump scanning of cervical carcinoma. When juxtapose with PAP stain, these smears’ scenarios stained with REAP = highly transparent and free of detritus.

Abhilasha Asthana et al. (2014) conducted research comparing the regular Papanicolaou staining procedure to the fast, economical, acetic acid, Papanicolaou (REAP) approach. This study covers a total of 100 samples from two separate sites, each with 50 sample sets. When compared to standard PAP stain, the REAP approach yields good colorful slides thus less expensive and takes less set-up considering lump scanning of mouth carcinoma.¹⁸

Ranu Roy Biswas et al. (2008) investigated Rapid Economic, Acetic Acid, Papanicolaou Stain {REAP} — “Is an acceptable substitute for traditional PAP Stain. There are 220 PAP smears from 110 participants in the research. According to the findings of the study, REAP stain, when compared to standard Papanicolaou stain, provides an appropriate, good, and speedy option for cytological screening at a low cost. Overall, absolute alcohol consumption is low. The stain preservation in the REAP process is also good. In our nation with a high prevalence of the illness, it may be regarded as a good alternative to traditional PAP stain for cell screening programs.¹³

Kalyani Raju et al. conducted research comparing convectional PAP stain with rapid PAP stain. The study covers 50 samples in all. According to the study, - the superiority of REAP ‘stain’ is as follows: prevailing over regular “Pap stain”, marvelous nuclear/protoplasm coloring more advance color-magnitude/clarity, worthwhile just like ethan-oic-acid replaces costly ethanol {25% -complete cost’s general quality Pap stain}, prolonged color safeguard [> one annual aside from vanishing], clever concept, varnishing of karyon/histones/nucleole characteristics – transparent comparable to Pap stain. Because of its low cost, it is a viable substituent for regular PAP smears under cervix carcinoma lump scanning in developed nations.²²

Rationale

FNAC is a technique whereby cells are obtained from a lesion using a thin bore needle and smears are made for cytological diagnosis.

The benefits of REAP stain over standard Pap stain include superior nuclear and cytoplasmic staining, improved color intensity and transparency, and lower cost. Acetic acid replaces expensive ethanol, which accounts for 25% of the cost of standard Pap stain, and provides long-term color preservation (more than a year without fading), a quick procedure, less time required, staining of non-epithelial cells like RBC/WBC/bacteria that are well preserved, nuclear/chromatin/nucleolus details that are clear/crisp, comparable cynophilia/eosinophilia of the cells.

Methods

The following study is a cross-sectional investigative study that will be conducted in the Cytopathological division of the Dept. of Pathology, Jawaharlal Nehru Medical College (JNMC), Sawangi (Meghe), Wardha, in collaboration with the Acharya Vinoba Bhave Rural Hospital (AVBRH), Sawangi (Meghe), Wardha, from 2021 to 2023. The institutional Ethical Committee approval has been obtained from Datta Meghe Institute of Medical Sciences (DMIMS (DU)/IEC/2022/1205). This investigation will take place after obtaining the patients’ written informed consent.

Results

The present study explores whether the qualitative and quantitative analysis of stained smears by REAP Stain is much better than conventional PAP Stain for nucleus and cytoplasm staining.

Discussion

Cervical cancer develops progressively over a certain amount of time.²³ Most accidents take place in less developed countries where there are no reliable testing campaigns available. Some of the risk factors include acquired immunodeficiency syndrome, smoking, and human papillomavirus exposure. Treatment-related extended morbidity is common, however, most women with early-stage tumors can be cured. According to the outcomes of randomized clinical research, chemotherapy,²⁴ and radiation therapy is considered a standard of care for cancer-developed patients and especially for women with locally advanced cancer, whereas its applicability to women in less developed nations is largely not tested.²⁵

In 2006, Dighe SB et al., conducted research on the development of PAP stain and compared traditional PAP stain to REAP stain. The study evaluated the nuclear and cytoplasmic characteristics of both stains. The study concludes that REAP stain’s cytoplasm differentiation and transparency were ideal, and the nuclear features and chromatin pattern were visible.¹⁵

In 2008, Ranu Roy Biswas et al., the question of whether Rapid Economic, Acetic Acid, Papanicolaou Stain (REAP), a suitable substitute for traditional PAP Stain, has been examined. 110 participants provided 220 PAP smears for the study. According to the study’s findings, REAP stain offers an appropriate, excellent, quick, and affordable alternative to traditional Papanicolaou for cytological screening.²⁶ Overall, very little alcohol is consumed. The REAP technique also does a superb job of stain preservation. In our nation, where the disease is quite prevalent, it might be thought of as a good substitute for the traditional PAP stain for cells screening program.¹³

In 2014, Abhilasha Asthana et al., compared the conventional Papanicolaou staining method with the quick, inexpensive acetic acid, Papanicolaou (REAP) method in a study. This study uses a total of 100 samples from two different sites, with 50 sets of samples per site. The study concluded that REAP method, as opposed to traditional PAP Stain, generates superior stained smears that are cost-effective and require less time for mass screening of oral cancer.¹⁸

In 2014, Shabnam Izhar et al. studied the effectiveness of a quick, affordable, acetic acid Papanicolaou stain as an alternative to traditional Papanicolaou stain in cervical smears. There are 737 smears in the study. According to the study’s findings, traditional PAP stain does a better job of color retention than REAP PAP stain, reduces turnaround time, and provides unmistakable cell shape due to superior staining quality.¹⁵

In 2015, Ashok Kumar Deshpande et al., Papanicolaou stain (PAP) and Rapid Economic Acetic acid Papanicolaou Stain (REAP) were compared in a study on cervical cytology. In total, 200 samples from persons of various ages were used in this investigation. The study found that PAP Stain required more time and money than REAP Stain for routine cervical cancer screening. When compared to PAP stain, the background of smears stained by REAP was incredibly clean and debris-free.²⁷

In 2016, Bhagat P. et al. undertook research on the effectiveness of a modified, quick, affordable, acetic acid-based Papanicolaou stain. 102 liquid base cytology samples are included in Ref. 28. The investigation concludes that standard PAP staining produced good cytoplasmic staining. The study endorsed the adoption of the REAP approach as a practical substitute.²⁹

In 2016, Kalyani Raju et al. studied the differences between the conventional PAP stain and the rapid PAP stain. The study uses 50 samples in total.³⁰ The study came to the conclusion that REAP stain has several advantages over standard Pap stain, including superior nuclear/cytoplasmic staining with better color intensity/transparency, cost-effectiveness due to the substitution of acetic acid for expensive ethanol (which accounts for 25% of the cost of standard Pap stain), long-term color preservation (more than a year without fading), a quick procedure, and staining of nuclear/chromatin/nucleolus details that are clear and comparable It is a good substitute for the traditional pap smear in developing nations’ widespread cervical cancer screening programs due to its low cost.³¹

In 2018, Suresh Amin et al., conducted research on PAP Stain for mass screening of micronuclei in buccal smears. Identification and counting of micronuclei are made possible by the stained smear backdrop.³²

In 2020, Garima Goel et al. completed research on Papanicolaou Stain, a cheap, quick, and suitable alternative to traditional PAP Stain for cervical smear staining. 100 smears from 50 female patients³³ are included in the study. In comparison to traditional PAP stain, the study finds that REAP is a quick, affordable, and good approach for both cytoplasmic and nuclear staining with minimal alcohol use.³⁴

In 2022, Karthika Panneerselvam et al. performed research on Papanicolaou, Acetic acid, and Rapid Staining techniques versus traditional staining techniques in the healthy oral mucosa. Eight patients and 160 slides were used in the investigation. The study’s findings show that even though the REAP staining technique had shorter staining times and costs, conventional staining methods were still preferred over them.²⁹