Liquid Paraffin as a Rehydrant for Air Dried Buccal Smear

 

Dr. Hannah.R1, Dr. Pratibha Ramani2, Dr. M.P. Brundha3, Dr. Herald. J. Sherlin4, Dr. Gheena Ranjith5, Dr. Abilasha Ramasubramanian6, Dr. GifrinaJayaraj7, Dr. K. R. Don8, Dr. Archana.S9

1MDS Postgraduate Student, Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

2Professor and Head of the Department, Department of Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

3Professor, Department of General Pathology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

4Professor, Department of Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

5Reader, Department of Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

6Reader, Department of Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

7Reader, Department of Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

8Senior Lecturer, Department of Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

9Reader, Department of oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospitals, SIMATS, No-24, Moorthy Nagar, Chettiaragaram, Thiruverkadu Post, Chennai -600077.

*Corresponding Author E-mail: rgrace_89@yahoo.co.in, dr_pratibha@rediffmail.com, drherald888@rediffmail.com, gheena_ranjith@yahoo.co.in, abilasha.ramasubramanian@gmail.com, gifrinaj@gmail.com, drkrdon@gmail.com, drarch.s@gmail.com

 

ABSTRACT:

Aim: To assess the efficacy of liquid paraffin as a rehydrant for air dried buccal smear based on the nuclear and cytoplasmic details. Background: Buccal smear is useful for diagnosing Malignancy, Fungal infection, Viral infection and Vesicullobullous dermatoses. The routine practice is to wetfixthe smear and send them to the laboratory for staining and evaluation by a cytopathologist. But drying of smears is inevitable, especially when the aspirate is less and when there is improper fixation. This can cause drying artifacts. An alternative method for overcoming this problem is intentional air drying followed by rehydration. Many rehydrants have been experimented upon. The most common rehydrant being saline. The present study is done to check the efficacy of liquid paraffin as a rehydrant. Material and methods: 2 sets of 20 buccal smears were collected from the patients. One set air dried for 24 hours and the other wet fixed. Conventional pap staining was then carried out. The slides were examined by two observers for preservation of Nuclear and cytoplasmic details based on the semi quantitative scoring system. Results: Excellent nuclear details were seen in 45% of air dried smear compared to 25%in routine wet fixation. The cytoplasmic details of air dried smear and routine wet fixed smear was the same. Conclusion: Liquid paraffin as a rehydrant has shown promising results and the nuclear details were found to marginally better than in routine wet fixed smear. Air dried buccal smears can be used in routine practice especially in rural areas where there is limited access to laboratories and for mass screening.

 

KEYWORDS: Buccal smear, Rehydration, Liquid Paraffin.

 


 

 

 

INTRODUCTION:

A buccal smear is a noninvasive adjunct to routine biopsy, where a sample of cells is collected from the inside of the mouth. It provides useful information for detection of Precancerous lesion, Barr bodies, Organisms like Candida etc. Two types of smears are commonly used for cytological examination wet alcohol-fixed smears and air-dried smears. Air dried smears are commonly used in cervical cytology. An important process in preparation of air dried smears is rehydration.

 

Papanicolaou stain is generally regarded as the best stain for assessment of chromatin pattern in cytologic smears and ensures maximum resemblance with the corresponding cells in tissue sections. However, a prerequisite is immediate fixation, usually in Isopropyl alcohol. Air drying has deleterious effects on the nuclear and cytoplasmic morphology and staining intensity[1].

 

To counteract the artifacts various rehydrants were introduced in the late 90’s to counteract the artifacts formed due to air drying. Lencioni et al used sequential tap-water and acetic acid alcohol solutions[2]. Bonime recommended the use of 50% aqueous glycerin. Nieburgs' method of rehydrating with hydroxypropyl methylcellulose ether in water ether alcohol required prior coating with hydroxypropyl methylcellulose ether. Rehydration techniques, however, have not made a strong impact in exfoliative cytologic examination because of availability of the much simpler spray fixatives[3].

 

In this study we have used Liquid paraffin as a rehydrant. Medicinal liquid paraffin, also known as paraffinum liquidum, is a highly refined mineral oil. Used in Skin care products, as Laxative and as tissue preservative in pathology labs. M.S Israel et al used liquid paraffin as a tissue preservative for pathology specimens. All the major organs in the body were well preserved in it. It has a refractive index comparable to glycerol [4]. This property of liquid paraffin was taken it into account to test its ability to rehydrate the air dried smears. Apart from this Liquid paraffin is also easily available and is inexpensive.

 

MATERIAL AND METHODS:

This study was done on buccal smear collected from 20 patients with no apparent oral lesion. They were selected at random.

 

Two sets of buccal smears were collected from each patient; the smears in the first set were fixed in Isopropyl alcohol for 20 minutes and the second set were air dried for 24 hours, rehydrated with commercially available Liquid paraffin for 10 minutes and then fixed in isopropyl alcohol for 20 minutes. Both the smears were stained with conventional Papanicolaou stain. After fixation the smears were hydrated in running tap water for 2-3minutes. Then is stained in Harris hematoxylin for 3-5minutes followed by washing in tap water. The slide is then dipped in 1% acid alcohol once, for the process of differentiation. Bluing is then done by washing the slides in tap water for 2 minutes. Followed by 95% alcohol for 2 minutes and then left in Eosin Azure-50 for 3 minutes. It is then left in 95% alcohol for 2 minutes twice. The slides are then stained using Orange G-6 for 3 minutes and followed by 95% alcohol for 2 minutes twice. The slides are then air dried and mounted using DPX. The total time taken was 26 minutes. The slides were then labeled in order to facilitate double blinding of both the observer and the analyzer. The slides were examined for preservation of Nuclear and cytoplasmic details based on the semi quantitative scoring system by the observers.

 

Scoring criteria:

For nuclear details, score 1 was given for poor nuclear details, 2 for satisfactory details and 3 for excellent details. Similarly for cytoplasmic details, score 1 was given for poor cytoplasmic details, 2 for satisfactory details and 3 for excellent details.

 

RESULTS:

Table:1 Nuclear details

Score

Air dried Smear

(no of slides)

Wet fixed Smear

(no of slides)

0

2

2

1

3

1

2

6

12

3

9

5

Note: 1. Poor details;   2. Satisfactory details;    3. Excellent details

 

Table:2 Cytoplasmic details

Score

Air dried smear

(no of slides)

Wet fixed Smear

(no of slides)

0

1

1

1

8

8

2

10

10

3

1

1

Note: 1. Poor details;   2. Satisfactory details;     3. Excellent details

 

 

Figure:1 Comparison of Nuclear Details between wet fixed and air dried smears

 

Figure:2 Comparison of Cytoplasmic Details between wet fixed and air dried smears

 

A total of 20 air dried smears and 20 wet fixed smears were analysed. Excellent nuclear details score of 3 was given to 9 of the Airdried smears in comparison to 5 of the wet fixed smear. So excellent nuclear details were seen in 45% of air dried smear compared to 25% in routine wet fixation(Table1, figure1). But it was not statistically significant. The cytoplasmic details of air dried smear and routine wet fixed smear was the same (Table 2,Figure 2).

 

DISCUSSION:

Papanicolaou stain the best stain for the assessment of chromatin pattern in cytologic smears, ensures maximum resemblance with the corresponding cells in tissue sections. A prerequisite for good staining is immediate fixation, usually in Isopropyl alcohol. In exfoliative cytologic some degree of drying is inevitable, particularly at the edges of the smears, where the diagnostic cells are often concentrated. This drying artifact is noticeable if the amount of aspirate is small and relatively dry.

 

Air drying of the smear has deleterious effects on the nuclear and cytoplasmic morphology and staining intensity. The artifacts which arise due to airdrying polychromatic staining, cellular and nuclear enlargement [1]. john K.C Chan et al noticed that air dried smears stained with H and E or Papshain showed large nucleus which is poorly stained, smudged and the chromatin pattern could not be assessed[3]. These artifacts can render the smear nonsatisfactory or lead to misdiagnosis and a repeat smear is preferred[5,6].

 

To counteract these artifacts various rehydrants were experimented upon in the late 90’s. Sequential tap water, acetic acid solution, aqueous glycerin, hydroxypropyl methyl cellulose ether in water ether alcohol and saline but none of these rehydrants have made a strong impact on exfoliative cytology[3,2].Therefore, it is highly desirable to develop methods to rehydrate these dried-up cells. In this study we have used Liquid paraffin as a rehydrant.

 

The results obtained were that excellent nuclear details were seen in 45% of the air dried smear in comparison with 25% of wet fixed smear. The nuclear details were comparable or better than that of wet fixed smear. The nuclear details were preserved even after air drying the smear for 24 hours and rehydrating it for ten minutes. John K.C Chan in his study using hypotonic Saline showed that nuclear wrinkling was seen in the cells when immersed for more than half an hour and the same was seen in a study by Zare-Mirzaie[3,7]. But there was no wrinkling noticeable even after immersing in liquid paraffin for more than half an hour and the nucleus were crisper, the chromatin pattern clearer, and nucleoli more conspicuous.

 

The cytoplasmic details of air dried smear was similar to the wet fixed smear. Studies done with tap water, aqueous glycerin, and hypotonic showed lysis of a proportion of nucleated cells[3]. There was no evidence of lysis of cells even after immersion for long duration in liquid paraffin.

 

The other advantages this method offered over other rehydrating techniques and conventional wet fixation have been enlisted. The smears could be spread more thinly and leisurely; smears made hastily for fear of air drying are usually not very satisfactory. The problem of air drying in the edges of the smear can be avoided. When a wet smear is placed in isopropyl alcohol, the larger particles or thicker portions of the smear may fall off. If the smear has been fully air dried, the cells adhere better to the slide and do not fall off as easily on rehydration and fixation. The background was very clear. The air dried smear could be easily transported as there was no fear of spillage of alcohol as in wet fixation.

 

The disadvantage was that liquid parafiin when compared to saline is a little expensive. But normal saline is easily available and is cost effective. The disadvantages of wet fixed smears like floating of the cells off the slide, folding of cells and increased background stains were counteracted by air dried smears.

 

Based on the results of this study we recommend airdrying of buccal smear for 24 hours and rehydration with liquid paraffin for 10 minutes followed by fixation in alcohol. Liquid paraffin can be used as an efficient rehydrant for air dried buccal smears.

 

 

 

CONCLUSION:

liquid paraffin as a rehydrant has shown promising results and the nuclear details were found to marginally better than in routine wet fixed smear. Air dried buccal smears can be used in routine practice especially in rural areas where there is limited access to laboratories.

 

REFERENCES:

1.       Hoda R.S., Hoda S.A. Artifacts, contaminants and incidental findings. In: Fundamentals of Pap Test Cytology. Humana Press 2007; 179-186.

2.       Lencioni LJ, Staffieri JJ, Cardinnet LJ. Vaginal and urinary sediment smear staining technique without previous fixation; adapted to Papanicolaou’s and Shorr’s staining methods. J Lab Clin Med.1954; 44(4): 595-9.

3.       Chan JKC, Kung ITM. Rehydration of airdried smears with normal saline: application in fine-needle aspiration cytologic examination. Am J Clin Pathol 1988; 89(1):30-4.

4.       M. S. Israel, L.F. Young. Use of liquid paraffin in the preservation of pathological specimens. J Clin Path.1978; 31(5):499-500.

5.       Crothers, Barbara and Henry, Michael and Firat, Pinar and Hamper, Ulrike. Nondiagnostic/Unsatisfactory: The Bethesda System for Reporting Thyroid Cytopathology. 2010.

6.       Ahmed HG, Tom MA. The consequence of delayed fixation on subsequent preservation of urine cells. Oman Medical Journal. 2011; 26(1):14-8.

7.       Zare –Mirzaie, K. Kalili-Alam and M. Abolhasani. Rehydration of air-dried cervical smears: An alternative to routine wet fixation. Acta Medica Iranica 2007; 45(5):365-68.

 

 

 

 

 

Received on 21.11.2018         Modified on 28.12.2018

Accepted on 14.01.2019      © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(3): 1197-1200.

DOI: 10.5958/0974-360X.2019.00199.9