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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 6  |  Page : 964-967

Spritzer: For diagnostic cytopathology


Department of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Udaipur, Rajasthan, India

Date of Web Publication13-Dec-2017

Correspondence Address:
Dr. Rashmi Metgud
Department of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Debari, Udaipur, Rajasthan - 313 024
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.174548

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 > Abstract 

Background: Exfoliative cytology is an easy, economical, noninvasive, and feasible method for early detection and screening program of any premalignant or malignant lesion. In case of routine cytological procedure, classical Papanicolaou (PAP) stain is widely used while Romanowsky stains are sparingly used. Leishman-Giemsa (LG) cocktail, being a easier, cost effective staining technique, has not been used in exfoliative cytology. Therefore, this pilot study was carried out to compare and contrast the role of LG stain in routine cytological procedure which is very cost-effective, less time-consuming and requires less infrastructural support.
Aim and Objectives: To study the diagnostic efficiency of LG cocktail in comparison with PAP stain and Feulgen stain in mucosal cells for evaluating cellular changes of oral squamous cell carcinoma (OSCC).
Materials and Methods: Three cytological smears were prepared from 10 healthy controls and 10 patients clinically diagnosed with OSCC, and they were stained with LG cocktail stain, PAP stain, and Feulgen stain. The stained smears were evaluated for cytologic diagnosis and the staining characteristics such as nuclear and cytoplasmic details were recorded as per criteria by Sujathan et al.
Statistical Analysis: The data were statistically evaluated with analysis of variance test using SPSS 15 software for windows.
Results: The results from the cases diagnosed as OSCC by PAP and LG cocktail were almost identical and superior to Feulgen stain both in diagnostic ability and in staining characteristics.
Conclusion: The one-step LG cocktail is easy, very cost-effective, less time-consuming with less infrastructural support as compared to PAP stain; however, it warrants further evaluation for screening of oral cancer as a potential aid.

Keywords: Cytology, Feulgen stain, Leishman-Giemsa stain, oral squamous cell carcinoma, Papanicolaou stain, Romanowsky


How to cite this article:
Metgud R, Surbhi, Naik S, Patel S. Spritzer: For diagnostic cytopathology. J Can Res Ther 2017;13:964-7

How to cite this URL:
Metgud R, Surbhi, Naik S, Patel S. Spritzer: For diagnostic cytopathology. J Can Res Ther [serial online] 2017 [cited 2018 Oct 24];13:964-7. Available from: http://www.cancerjournal.net/text.asp?2017/13/6/964/174548


 > Introduction Top


Oral squamous cell carcinoma (OSCC) is one of the most formidable health problems in terms of morbidity and mortality. In the past three decades, higher incidences have been observed in Southeast Asian countries, with almost no significant improvement in mortality from these cancers.[1] Despite improvements in the management of diagnosed cases, the need for rapid testing and diagnosis of diseases has posed great challenges to us.

Exfoliative cytology is an easy, economical, noninvasive, and feasible method for early detection and screening program of any premalignant and malignant lesion, etc. In case of routine cytological procedure, classical Papanicolaou (PAP) stain is widely used while Romanowsky stains are sparingly used.[2] PAP stain has the benefit of staining cells from various layers differentially. However, the cellular details are poorly demonstrated, the procedure is time-consuming, expensive and is also associated with drying artifact.[3] Romanowsky stains are universally employed for routine staining of blood films with the remarkable property of making subtle distinctions in shades of staining granules differentially.[4] Feulgen stain, a DNA-specific stain, demonstrates the nuclear chromatin well, although the procedure is time-consuming, requires multistep staining technique and also not cost-effective.[5]

Leishman-Giemsa (LG) cocktail, being relatively a new staining technique, is easy, cost-effective, less time-consuming, and one-step technique. Therefore, it can be applied for the screening of oral cancer through cytological examination. Hence, the present study was planned to evaluate the diagnostic efficiency of LG cocktail in comparison with PAP stain and Feulgen stain in mucosal cells for evaluating cellular changes of OSCC.


 > Materials and Methods Top


The study group comprised 10 healthy controls and 10 patients clinically diagnosed as OSCC.

The LG cocktail was prepared by filtering a unit volume of Giemsa stain (Fisher Scientific, India) and then mixing it with an equal volume of distilled water to prepare Giemsa working solution. Equal volume of Leishman's stain (Leishman's stain, Fisher Scientific, India) filtered and mixed with an equal volume of Giemsa working solution in the ratio (1:1) to prepare the LG cocktail. The cocktail was used and stored just like Leishman's stain.[2]

An informed consent was taken from the patients, and they were asked to rinse the mouth then scrape cytology was performed with a sterile cytobrush and three smears were prepared from controls (buccal mucosa) and patients in the test group. From each group, two smears were ether alcohol-fixed and stained with PAP (Rapid PAP, Bio Lab Diagnostic, India) and Feulgen stain. One air-dried smear was stained with LG cocktail. Standard staining procedures were used for Feulgen [5] stain.

LG cocktail staining procedure was as follows: The air-dried smears were flooded with the LG cocktail and then left for 1 min. An equal volume of tap water was added and left for 5 min with gentle blowing. The slides were washed in tap water, dried, cleared, and mounted.

The stained smears were evaluated for cytologic diagnosis and the staining characteristics such as nuclear and cytoplasmic details as per criteria by Sujathan et al.

Cytoplasmic details were evaluated by single observer based on transparency and nature of cell membrane [6] and scored as:

  • 0 - not preserved
  • 1 - nontransparent with intact cell membrane
  • 2 - nontransparent masking nuclear details
  • 3 - transparent, intact cell membrane without masking nuclear details.


Nuclear detail were assessed based on the nature of the chromatin, vesicularity, membrane integrity [6] and scored as:

  • 0 - poor preservation
  • 1 - smudgy
  • 2 - fair preservation but chromatin granularity not appreciable
  • 3 - excellent preservation with crisp chromatin.


The data were statistically evaluated by analysis of variance test using SPSS 15 (Windows 2007.) software.


 > Results Top


In the control group, a statistically significant difference was observed when the cytoplasmic staining was compared for Feulgen versus PAP, P ≤ 0.01 and LG Cocktail versus Feulgen, P ≤ 0.01. However, no significant difference was observed between cytoplasmic staining with PAP and LG cocktail stains (PAP vs. LG cocktail, P = 0.46) when compared with each other in the control group [Table 1]. The nuclear staining results observed for PAP, Feulgen, and LG cocktail stains were statistically similar (Feulgen vs. PAP, P ≤ 0.01; PAP vs. LG, P = 0.83; LG Cocktail vs. Feulgen, P ≤0.01) [Table 2].
Table 1: Comparison of cytoplasmic details in Leishman-Giemsa, Papanicolaou, and Feulgen stained smears of healthy controls

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Table 2: Comparison of nuclear details in Leishman-Giemsa, Papanicolaou, and Feulgen stained smears of healthy controls

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In the OSCC group, statistically significant difference was observed when the cytoplasmic staining was compared for (Feulgen vs. PAP, P < 0.01 and LG Cocktail vs. Feulgen, P <0.01). No statistically significant difference was observed between cytoplasmic staining with PAP and LG cocktail stains (PAP vs. LG cocktail, P = 0.23) [Table 3]. Statistically similar results observed in the nuclear staining between the three stains (Feulgen vs. PAP, P ≤ 0.01; PAP vs. LG cocktail, P = 0.58; and LG Cocktail vs. Feulgen, P ≤ 0.01) [Table 4].
Table 3: Comparison of cytoplasmic details in Leishman-Giemsa, Papanicolaou, and Feulgen stained smears of oral squamous cell carcinoma patients

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Table 4: Comparison of nuclear details in Leishman-Giemsa, Papanicolaou, and Feulgen stained smears of oral squamous cell carcinoma patients

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 > Discussion Top


With the increasing incidence of oral cancer in the developing countries such as India and other Southeast Asian countries, the role of screening and diagnosing is becoming more vital and has posed challenges to the clinicians.[2] To develop a faster and more economical alternative, LG cocktail was formulated by Garbyal et al., 2006,[7] and very few studies have been done postulating that LG cocktail, which is a Romanowsky stain, is rapid and useful in enhancing pleomorphism and distinguishing the intracellular from extracellular material.[2],[7],[8]

Leishman stain, a good nuclear stain, gives an intense staining of extracellular ground substance, under stained individual cells and three-dimensional clumps. When Giemsa stain, a good cytoplasmic stain, is mixed with Leishman's stain, the LG cocktail provides a moderate metachromasia to the ground substance and brilliantly stained cellular components.[2],[7]

In the present study, it was observed in the OSCC patients that the cytoplasmic and extracellular substance staining quality of the LG cocktail was equal to or better than that of PAP stain. In fact, the nuclear metachromasia and chromatin granularity were much more intense in the LG cocktail than the PAP-stained smears [Figure 1] and [Figure 2]. Feulgen-stained smears showed a more intense metachromasia when compared to LG cocktail, but sometimes obscured cellular details were observed [Figure 3]. Similar staining characteristics were observed in the healthy controls with all the three stains [Figure 4], [Figure 5], [Figure 6].
Figure 1: Microphotograph of Leishman-Giemsa cocktail stained smear showing clear cellular details with an enlarged nucleus and crisp, granular chromatin in oral squamous cell carcinoma

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Figure 2: Microphotograph of Papanicolaou-stained smear showing differential staining of cytoplasm in oral squamous cell carcinoma

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Figure 3: Microphotograph of Feulgen-stained smear showing inadequate cellular details in oral squamous cell carcinoma

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Figure 4: Microphotograph of Leishman-Giemsa cocktail stained smear showing clear cellular details with a prominent nucleus and crisp, granular chromatin in healthy controls

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Figure 5: Microphotograph of Papanicolaou-stained smear showing differential staining of cytoplasm in healthy controls

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Figure 6: Microphotograph of Feulgen-stained smear showing inadequate cellular details in healthy controls

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In a study on smears from fine needle aspirates by Garbyal et al., 2006,[7] cytoplasmic staining was found to be good with LG cocktail and excellent with May-Grünwald-Giemsa (MGG) stain. According to Mitra et al., 2011,[8] both PAP stain and LG cocktail produced the same result in the cytological finding on smears from oral lesions. Belgaumi and Shetty, 2013,[2] observed LG cocktail to be comparable with PAP stain but superior to MGG stain on cytology smears of OSCC.

The overall observations of the present study reflect that LG cocktail is comparable to PAP stain, which is in accordance with the study by Garbyal et al., Mitra et al., and Belgaumi and Shetty and superior to Feulgen stain both in staining characteristics and diagnostic ability.

The recent global trend is toward the reduction in health care costs, especially for developing countries, such as India. This necessitates a faster, easy, and more economical staining technique to be utilized in screening of oral cancer. The time required for staining with PAP stain, i.e., for fixation and staining is about 45 min. The staining procedure requires multiple steps, large volumes of alcohol, and expensive stains.[5] Though rapid PAP kit is available for faster turnaround time of approximately 5 min, still it requires multiple steps and is very expensive.[9] The fixing and staining procedure for Feulgen stain takes about 2 h,[5] and the cost is higher than the LG cocktail. However, the LG cocktail staining procedure of air-dried smears requires no additional fixation and can be completed in <10 min, with the least expenditure, unlike PAP and Feulgen stain.


 > Conclusion Top


The LG cocktail staining technique was found to give results comparable to the PAP stain and superior to Feulgen stain. LG cocktail is an easy, cost-effective, and one-step technique; however, it warrants further study in its potential application in early detection of oral cancer, especially in mass screening of oral cancer. There are also some limitations in this study; for example, small sample size, subjectivity in scoring the sensitivity and specificity of the LG cocktail staining technique which needs further evaluation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

1.
George J, Narang RS, Rao NN. Stromal response in different histological grades of oral squamous cell carcinoma: A histochemical study. Indian J Dent Res 2012;23:842.  Back to cited text no. 1
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2.
Belgaumi U, Shetty P. Leishman Giemsa cocktail as a new, potentially useful cytological technique comparable to Papanicolaou staining for oral cancer diagnosis. J Cytol 2013;30:18-22.  Back to cited text no. 2
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3.
Culling CF, Allison RT, Barr WT. Cellular Pathology Technique. 4th ed. London: Butterworth; 1985. p. 491.  Back to cited text no. 3
    
4.
Fasakin KA, Okogun GR, Omisakin CT, Adeyemi AA, Esan AJ. Modified Leishman stain: The mystery unfolds. Br J Med Med Res 2014;4:4591-606.  Back to cited text no. 4
    
5.
Bancroft J, Gamble M. Theory and Practice of Histological Techniques. 5th ed. Edinburg: Churchill Livingstone; 2002. p. 105-20.  Back to cited text no. 5
    
6.
Sujathan K, Pillai RK, Kannan S, Chandralekha B, Mathew A, Nair KM. Cytodiagnosis of serous effusions: A combined approach to morphological features in Papanicolaou and May-Grunwald Geimsa stain and a modified cell block preparation. J Cytol 2000;17:89-95.  Back to cited text no. 6
    
7.
Garbyal RS, Agarwal N, Kumar P. Leishman-Giemsa cocktail: An effective Romanowsky stain for air-dried cytologic smears. Acta Cytol 2006;50:403-6.  Back to cited text no. 7
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8.
Mitra S, Bose S, Mukherjee G. Comparative studies on the Leishman Giemsa stains and Papanicolau stains for cytological diagnosis of oral lesion. Sci Cult 2011;77:139-40.  Back to cited text no. 8
    
9.
Dighe 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.  Back to cited text no. 9
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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