Leishman Giemsa cocktail as a new, potentially useful cytological technique comparable to Papanicolaou staining for oral cancer diagnosis.

BACKGROUND: Papanicolaou staining is commonly used for staining exfoliative cytology smears with Romanowsky stains being used sparingly. Leishman Giemsa (LG) cocktail, being a relatively new staining technique, has not been used in exfoliative cytology. This easy, cost-effective and one-step technique warrants further study because of its potential application in screening of oral cancer. AIM: To study and evaluate the diagnostic efficiency and reliability of Leishman Giemsa (LG) cocktail in comparison with Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stains in exfoliated cells for the detection of oral squamous cell carcinoma.
MATERIALS AND METHODS: Three smears were prepared from each 100 controls (buccal mucosa) and 100 patients, clinically diagnosed with oral squamous cell carcinoma and stained with Pap, MGG and LG cocktail stains. The slides were evaluated for the staining characteristics of nucleus and cytoplasm. The diagnostic efficiency of each stain was evaluated by comparing the cytologic diagnosis of each stain with the histopathological diagnosis. Finally, the diagnostic reliability was evaluated by comparing the three stains with each other and the histologic diagnosis. STATISTICAL ANALYSIS: The data were statistically evaluated with Friedman test, Wilcoxon sign rank test and McNemar chi square test using SPSS15 software.
RESULTS: The results from the histologically confirmed cases of squamous cell carcinoma and the number of cases diagnosed by Pap and LG cocktail were almost identical and both were superior to MGG. The P value obtained for the confirmed cases of squamous cell carcinoma in comparison for Pap vs MGG was 0.001, MGG vs LG cocktail was 0.001 and LG cocktail vs Pap was 0.157. Hence, no statistical significant difference was observed between the diagnostic ability of Pap and LG cocktail stains.
CONCLUSION: LG cocktail is an easy, cost-effective and one-step technique comparable to Pap staining; however, it warrants further study in its potential application in screening of oral cancer.

Introduction

Oral cancer is the most common malignancy worldwide. Despite improvements in the management of diagnosed cases, delay in diagnosis undoubtedly increases the morbidity and mortality resulting from it.[1] Its burden on the economy for providing healthcare is substantial and with the increasing incidence of oral cancer in developing countries like India and the other South-East-Asian countries, the role of screening is becoming more vital.[2]

Exfoliative cytology is a valuable aid for screening of malignant and potentially malignant oral lesions.[3] It is an easy, economical, non-invasive and feasible method for detection of malignancies.[45] The most commonly followed technique for staining exfoliative cytology smears is the Papanicolaou (Pap) technique. It has the benefit of staining cells from various layers differentially. But, the procedure is time consuming with multiple steps and is also expensive.[67] Romanowsky stains are universally employed for staining blood films with very satisfactory results due to their remarkable property of making subtle distinctions in shades of staining, and staining of granules differentially. Many laboratories use May-Grünwald Giemsa (MGG) (Romanowsky type stain) staining method for cytological diagnosis of specimens in addition to Pap. However, some of the disadvantages of MGG include tendency to precipitate, high background staining, preparation of fresh solution every day and technique sensitivity.[78]

Leishman Giemsa (LG) cocktail, being a relatively new staining technique, has not been used in exfoliative cytology. This easy, cost-effective and one-step technique warrants further study because of its potential application in screening of oral cancer. Hence, this study was planned to evaluate and compare routinely used stains like Pap, MGG with a relatively new LG cocktail staining technique, in exfoliated cells of oral malignancy.

Materials and Methods

The study group comprised of 100 healthy controls and 100 patients clinically diagnosed as having oral squamous cell carcinoma.

Inclusion criteria for control group were as follows: Controls selected were between 18 to 30 years with no history of habits (tobacco smoking, betel chewing and alcohol consumption) and no mucosal pathology.

Inclusion criteria for test group were as follows: Patients clinically diagnosed as having oral squamous cell carcinoma and were ready to undergo a biopsy procedure.

Exclusion criteria for test group were as follows: Cases where the biopsy procedure was not carried out for any reason, cases where smears were inadequate (less than 50 cells) and cases that were not diagnosed histologically to be squamous cell carcinoma.

The LG cocktail was prepared by filtering a unit volume of Giemsa (Giemsa's solution for microscopy, Merck, India) and mixing it with an equal volume of distilled water to prepare Giemsa working solution. Equal volume of Leishman's stain (Leishman's stain, Span Diagnostics, India) was filtered and mixed with an equal volume of Giemsa working solution (1:1) to prepare the LG cocktail.[9] The cocktail was used and stored just like Leishman's stain.

An informed consent was taken. The patients were asked to rinse the mouth, scrape cytology was performed with a sterile metallic cement spatula and three smears were prepared from controls (buccal mucosa) and patients in the test group. From each group, one smear was ether alcohol-fixed and stained with Pap (Orange G, EA 36, Merck, India) and two slides air-dried. One air-dried smear was stained with MGG (May Grünwald's solution modified for microscopy, Merck, India) after fixation with methanol and the second air-dried smear was stained with LG cocktail stains. Standard staining procedures were used for Pap[10-12] and MGG[68] stains.

LG cocktail staining procedure was as follows: The air-dried smears were flooded with the LG cocktail and left for 1 minute. An equal volume of buffer (pH-6.8) was added and left for 5 minutes with gentle blowing. The slides were washed in tap water, dried, cleared and mounted.

The slides were evaluated for the staining characteristics like nuclear and cytoplasmic detail. Each stained slide was evaluated for 50 well-stained cells and scored according to the scoring criteria as per Sujathan et al.[13]

Cytoplasmic details were evaluated based on transparency and nature of cell membrane[13] and scored as:

0 - not preserved,

1+ - non-transparent with intact cell membrane,

2+ - non transparent masking nuclear details,

3+ - transparent, intact cell membrane without masking nuclear details.

Nuclear detail was assessed based on the nature of the chromatin, vesicularity, membrane integrity[13] and scored as:

0 - poor preservation,

1+ - smudgy,

2+ - fair preservation but chromatin granularity not appreciable,

3+ - excellent preservation with crisp chromatin.

The stained slides were also evaluated for cytologic diagnosis, by a single examiner without the knowledge of clinicopathological details of the case. The diagnostic ability of each stain was evaluated by comparing the cytologic diagnosis with the histopathological diagnosis. Finally, the diagnostic reliability was evaluated by comparing the three stains with each other and the histologic diagnosis.

The data were statistically evaluated with Friedman test, Wilcoxon sign rank test and Mcnemar chi square test using SPSS15 software.

Results

In the control group, a statistically significant difference was observed when the cytoplasmic staining was compared for Pap vs MGG, P = 0.0001 and MGG vs LG cocktail, P = 0.0001. But, no significant difference was observed between cytoplasmic staining with Pap and LG cocktail stains (Pap vs LG cocktail, P = 0.144) when compared with each other in the control group. The nuclear staining result observed for Pap, MGG and LG cocktail stains was statistically similar (Pap vs MGG - P = 0.0001, Pap vs LG = 0.001 and MGG vs LG cocktail).

In the test group, statistically significant difference was observed between the cytoplasmic staining of Pap and LG cocktail stains when compared to MGG (Pap vs MGG, P = 0.0001 and MGG vs LG cocktail, P = 0.0001). No statistically significant difference was observed between cytoplasmic staining with Pap and LG cocktail stains (Pap vs LG cocktail, P = 0.705) when compared with each other in the test group [Table 1]. Statistically similar results observed in the nuclear staining between the three stains (Pap vs MGG, P = 0.0001, Pap vs LG cocktail, P = 0.001 and MGG vs LG cocktail, P = 0.0001) [Table 1].

Table 1

Cytoplasmic and nuclear staining for Papanicolaou, MGG and LG stains in the test group

The diagnostic reliability was evaluated for Pap, MGG and LG cocktail stains comparing the cytologic diagnosis with the histopathology. The smears obtained in the test group, after evaluating the cytoplasmic and nuclear details, had been given a cytologic diagnosis of no malignancy when the cytologic changes ranged from normal to atypical, indeterminate when the cytologic changes were intermediate and positive of malignancy when suggestive and frankly positive for squamous cell carcinoma.[14]

When the smears were compared with the histopathology reports, it was found that 91 of the 100 clinically suspected cases were diagnosed as squamous cell carcinoma, seven as epithelial dysplasia and in two patients the tissue received did not show features suggestive of epithelial dysplasia or carcinoma and were excluded from data analysis.

Of the 91 confirmed cases of squamous cell carcinoma, the number of cases diagnosed by Pap and LG cocktail were almost identical and both were superior to MGG. The P value obtained for the confirmed cases of squamous cell carcinoma on comparison of Pap vs MGG was 0.001, MGG vs LG cocktail was 0.001 and LG cocktail vs Pap was 0.157. Hence, no statistically significant difference was observed between the diagnostic ability of Pap and LG cocktail stains.

Discussion

Leishman stain, a good nuclear stain, when used alone, gives an intense staining of extracellular ground substance, under stained individual cells and 3-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.[9]

It was observed that the cytoplasmic staining in both control and test group was better appreciated with Pap and LG cocktail stains when compared to MGG (P = 0.0001). Pap stain was better than LG cocktail, but the difference was statistically insignificant. For nuclear staining, it was observed that LG cocktail gave comparatively better results followed by Pap, and MGG, in both, control and test group.

In a study on smears from fine needle aspirates by Gabryal et al.,[9] cytoplasmic staining was found to be good with LG cocktail and excellent with MGG. But in the present study, it was observed that LG cocktail stain was better than MGG for cytoplasmic staining. The results for cytoplasmic staining for Pap and MGG were similar to a study by Idris and Hussain.[15] However, in a study by Sujathan et al.,[13] comparing Pap and MGG stains, MGG was a better cytoplasmic stain and Pap a better nuclear stain, and they suggested the use of both stains to increase efficacy. Though the nuclear transparency of Pap was absent in LG cocktail, the chromatin granularity and vesicularity was better appreciated in air-dried LG cocktail stained smears. This is in accordance with Gabryal et al.[9] Additionally, the nuclear enlargement and variation in nuclear size is exaggerated in air-dried smears which is helpful in cytological diagnosis. If the background staining is too intense, it may also prevent adequate visualisation of cell clusters.[13] In the present study too, MGG-stained smears showed a more intense metachromasia when compared to LG cocktail and sometimes obscured cellular detail [Figure 1].

Figure 1

Smear showing metachromasia of ground substance (MGG, ×400)

Finally, the cytologic diagnosis of Pap, MGG and LG cocktail-stained smears was compared with the histopathology reports. It was observed that no statistically significant difference was found between the diagnostic ability of Pap and LG cocktail stains, while MGG stain gave slightly inferior results. The overall observations of the present study were that the LG cocktail is comparable to Pap stain, which is in accordance with the study by Gabryal et al.[9] and Mitra et al.[16] and superior to MGG stain both in staining characteristics [Figure 2 a-c] and diagnostic ability. The sensitivity of LG cocktail was 95%, which was higher than Pap and MGG stains, and the specificity was 88.23% [Table 2].

Figure 2

(a) Smear showing differential staining of cytoplasm (Pap, ×400); (b) Smear showing inadequate cellular details (MGG, ×400); (c) Smear showing clear cellular details with an enlarged nucleus and crisp, granular chromatin (LG cocktail, ×400)

Table 2

Comparison of diagnostic efficacy of Pap, MGG and LG stains

For a stain to be utilised in a mass screening programme, in addition to good staining characteristics, the technique must be easy, rapid and economical. The time required for staining with Pap stain, i.e., for fixation and staining is about 45 minutes. The staining procedure requires multiple steps, large volumes of alcohol and expensive stains.[10-12] The fixing and staining procedure for MGG takes about 45 minutes[68] and the cost is higher than the LG cocktail. However, the LG cocktail staining procedure of air-dried smears require no additional fixation as in MGG stain and can be completed in less than 10 minutes, with the least expenditure. Some disadvantages of MGG stain include tendency to precipitate, high background staining and preparation of fresh solution every day. Moreover, the staining technique is designed for staining a number of slides and not individual slides.[78] Though Rapid Pap kit is available for faster turnaround time of approximately 5 minutes, it still requires multiple steps and is very expensive when compared to the above stains.[17] Therefore, in addition to good staining characteristics, other features that go in favor of the LG cocktail is the ease of staining technique, the time required for staining and the cost factor.

However, with a few limitations like evaluation by a single examiner, subjectivity in scoring the sensitivity and specificity of the LG cocktail staining technique need to be further evaluated. Within the limitations of the study, the LG cocktail staining technique was found to give results comparable to the Pap stain. Keeping in mind the added advantages of a single step procedure, cost effectiveness and speed of the technique, the study supports the idea of utilising this method for early detection of oral cancer, especially in mass screening programmes.