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Abstract
Background: The May-Grünwald Giemsa Stain is one of the preferred Romanwsky stains in studying cell morphology of air-dried smears with respect to cellular and nuclear size details and metachromatic extracellular ground with an approximate staining time of 20–30 min. A reduction in staining time and possible application of an ultrafast stain for rapid onsite evaluation (ROSE) of cytological material is the need of the hour. With the application of the new modified ultrafast Giemsa (MUFG) technique, rapid staining can be achieved, thereby helping in triaging of samples and, most importantly, providing an early preliminary diagnosis. Aims: The aim is to assess the quality index of the MUFG technique in FNAC of various organs in comparison with the standard MGG stain. Materials and Methods: A total of 61 FNAC cases were studied by random sampling. Two smears were prepared for each case and stained by both. Scores were given based on five parameters, and the quality index was calculated. Statistical Analysis: Results were analyzed using mean, median, standard deviation, “t” paired test, “P” value, and M-diff for statistical significance. Results: The quality index of MUFG smears was comparable to the standard MGG stain in salivary gland, breast, and thyroid aspirates and low in lymph node and soft tissue aspirates. MUFG is a rapid cost-effective stain which can be applied in the setting of ROSE for a preliminary diagnosis. Conclusion: MUFG is a reliable alternative and rapid technique for cytology diagnosis.
Keywords: Alternative stain, MGG, MUFG, rapid
How to cite this article: Introduction 
Fine-needle aspiration cytology (FNAC) is one of the most effective, and low-cost preliminary diagnostic procedures used in medical practice with high sensitivity and specificity. Quick diagnosis of FNAC plays an important role in efficient clinical practice and can be made possible by the application of rapid onsite evaluation (ROSE) of cytological material.[1],[2],[3],[4] The need for minimal turnaround time for assessing FNA smears has encouraged new innovations and modifications in staining techniques that require less staining time with unequivocal cell morphology.[1],[5],[6],[7],[8]
The purpose of any biologic staining is to ease the perception of structures by increasing the contrast between them. The stains that are routinely used to assess FNA materials basically involve alcohol-fixed wet smears and air-dried smears. The routinely used stains to examine the fixed smears are Hematoxylin eosin stain and Papanicolaou stain, which are employed to study chiefly the crisp nuclear details. The air-dried smears are stained with any of the Romanowsky stains like the May-Grünwald Giemsa (MGG) stain, Wright Giemsa, Diff Quick or toluidine blue to study the cytoplasmic details along with cellular and nuclear size details and metachromatic extracellular ground substances. MGG is the most preferred air dried stain to study the cytological material and it is useful for studying in detail the cell morphology and takes approximately 20–30 min staining time. The reduction in staining time can be made possible by the application of the modified ultrafast Giemsa (MUFG) technique and thereby can be of use in the evaluation of critical cytology samples in intensive care unit (ICU) settings and ROSE in determining the adequacy of material, rendering a preliminary diagnosis, thereby providing an early diagnosis and hence in fine-tuning and triaging of the cytological samples. The objective of the study is to evaluate the quality of staining of air-dried cytology smears by the MUFG stain and compare its quality with that of the standard MGG stain.
Aims and objectives
The objectives of the prospective study are to assess the feasibility and applicability of MUFG stain in fine-needle aspiration smears of various organs and study its role in ROSE for assessing adequacy of sampling and rendering a preliminary diagnosis and also to assess and compare the quality of MUFG with standard MGG.
Materials and Methods The prospective study was carried out in the cytopathology laboratory of a tertiary care teaching hospital in Southern India for a period of three months. Informed consent was taken from the patient prior to the FNAC procedure. Fine-needle aspiration was carried out from various organs as an outpatient procedure referred from different clinical departments for diagnostic purposes. Cases with inadequate cytological material were excluded from the study. Extra air-dried smears were made from the FNAC material after obtaining the required smears for routine reporting, which includes both fixed and air-dried smears.
Two sets of air-dried smears were prepared from each of the 61 cytology samples. One set was stained with standard MGG diluted with 1:10 buffer and stained for 30 min. The other set was stained with four to five drops of undiluted, adequately ripened and filtered Giemsa stock solution and covered with a cover slip (22 × 40 mm) for uniform spreading of stain and preventing oxidation of stain. The stain was left for 3 min at room temperature. The cover slip was removed carefully and the slide was washed gently with buffered water. The cover slip was also gently washed with tap water to be re-used for mounting again. The slides and coverslip were air dried and then mounted with the same dried cover slip.
Both the sets of slides of all 61 cases were labelled and numbered.
The MUFG slides were studied by the study pathologist at the time of the FNAC procedure in assessing the adequacy of cellularity and ROSE to give a provisional diagnosis and later in assessing the quality of stain with standard MGG. The quality of the standard MGG and MUFG stains were compared by a scoring system based on five parameters which includes background details, overall staining, cellular morphology, nuclear staining with chromatin details, cytoplasmic, and stromal details – [Table 1]. The maximum score possible for a single case, taking into account all the four parameters, is 12. The quality index for each set of slides was calculated by the ratio of the actual score obtained and the maximum score possible.
The overall scores obtained for MUFG were compared with the overall scores for the standard MGG in each organ separately. Results were analyzed using mean, median, standard deviation, “t” paired test, “P” value for statistical significance.
Results With the application of rapid MUFG stain, comment on the adequacy of cellularity was possible in all the cases. The foremost advantage was in identifying cases with critically low cellularity with a resultant reflex immediate repeat procedure, thereby helping in preventing inadequate sampling during the FNAC procedure. An attempt at rendering a provisional diagnosis was done in all cases, categorizing them as non-neoplastic, neoplastic (benign), and malignant, with no significant discordance between the provisional and final diagnosis for all cases. The MUFG slides were also studied during the final interpretation of the slides along with the standard MGG smears. However, the present study was not aimed to render the final diagnosis by interpreting the MUFG smears in isolation but to study its quality and usefulness in ROSE settings.
The quality of paired slides of MGG and MUFG smears of each individual case from the total of 61 cases was reviewed in detail by the investigating pathologist and scoring was given. FNACs of lymph node (31) were the commonest followed by breast (11), thyroid (9), salivary gland (5), and soft tissues (5). The scores and quality indices of MGG and MUFG stain of different cytological aspirates are summarized in [Table 2]. The MGG and MUFG quality index is near comparable in salivary gland (0.98 vs 0.95) and thyroid (0.972 vs 0.935) aspirates than that of lymphnode (0.975 vs 0.89), breast (0.931 vs 0.84), and soft tissue aspirates (1.00 vs 0.8). On further analysis with a parametric test (paired t-test) of comparison of mean ± SD in different organs [Table 3], no statistical difference in staining patterns of the standard MGG with MUFG was observed in thyroid (11.66 ± 1.0 vs11.22 ± 1.44; P = 0.454), salivary glands (11.8 ± 0.45 vs 11.4 ± 0.58; P = 0.25), and breast aspirates (11.18 ± 0.8 vs 10.18 ± 1.77; P = 0.13). However, the staining quality of MUFG was significantly compromised with respect to lymph node (11.69 ± 0.66 vs 10.28 ± 2.06; P = 0.005) and soft tissue aspirates (12.00 ± 0 vs 10.6 ± 1.34; P = 0.04) in comparison with the standard MGG.
Table 3: Comparison of standard MGG with MUFG stain with respect to staining pattern scores in various organs by paired t-testAspiration from the breast masses, both neoplastic and benign, were of equal quality with regard to both stains (p > 0.5). Moreover, the myoepithelial cells were better delineated in MUFG preparations. [Figure 1].
Figure 1: MUFG-stained smears smears of breast fibroadenoma showing better delineation of myoepithelial cells (a-c; 10×, 40×)) in comparison with standard MGG (d and e; 40×)Smears from salivary glands and thyroid lesions were also of equal quality, with comparable quality index scores (p-value > 0.5). The extracellular chondromyxoid matrix material in cases of pleomorphic adenoma was equally well appreciable with the MUFG-stained smears [Figure 2].
Figure 2: Good staining of metachromatic matrix in salivary gland pleomorphic adenoma (a, 4×) along with overall better nuclear and cytoplasmic details (b and c; 10×, 40×) with comparable MGG smears (d and e; 4×, 40×)The nuclear features of papillary carcinoma thyroid with nuclear grooves and intranuclear pseudoinclusions were crisp and evidently detectable in MUFG smears. [Figure 3]a and [Figure 3]b The aspirates from Hashimoto's thyriditis showed well-stained lymphoid cells with follicular lysis [Figure 3]c and [Figure 3]d.
Figure 3: Thyroid aspirates showing crisp nuclear details with intranuclear inclusions of papillary carcinoma thyroid (a and b; 10×, 40×). Aspirates from Hashimoto's thyroiditis showing well-stained lymphoid cells with follicular lysis (c and d; 10×, 40×)The distinct malignant cytological and nuclear features with coarse chromatin, prominent nucleoli, and dense cytoplasm of squamous cell carcinoma metastatic to lymph node [Figure 4]a and [Figure 4]b was appreciated and one case of haematological malignancy in lymph node with crisp cell morphology of blasts was diagnosed aptly with the application of MUFG technique in a rapid time, favoring early diagnosis and triaging of samples [Figure 4]c, [Figure 4]d and [Figure 4]e. The tinctorial properties of metachromatic extracellular basement membrane and matrix material in salivary gland pleomorphic adenoma [Figure 2] and one case of soft tissue schwannoma were highlighted by the rapid technique, facilitating correct cytological diagnosis.
Figure 4: MUFG-stained cellular aspirates from metastatic squamous cell carcinoma showing malignant nuclear features with coarse chromatin, dense cytoplasm with necrotic background (a and b; 10×, 40×) and high-quality index of MUFG-stained smears of a highly cellular lymph nodal swelling with hematologic malignancy showing distinct nuclear and cytoplasmic details with nucleoli (c-e; 4×, 10×, 40×)The main disadvantage of MUFG smears was the non-uniformity in staining and was found to be suboptimal and inconsistent with haemorrhagic, necrotic smears, and also in richly cellular smears with cellular overlapping with uneven thick spreading. There were areas of patchy under staining with poor cell morphology combined with areas of better staining in the rarefied areas with a resultant reduction in overall uniform staining pattern. In some cases, the background Red Blood Cells (RBCs) were heavily stained, leading to the obscuration of certain subtle details.
Discussion The Giemsa stain, developed by Gustav Giemsa, was primarily designed for the demonstration of malarial parasites and subsequently used for the identification of Treponema pallidum.[9],[10] The Giemsa stain was translated to cytopathology from microbiology because of the high-quality staining of chromatin and nuclear membrane of all cells, the metachromatic staining property of some cellular components like the cytoplasmic granules and different qualities of cytoplasmic staining depending on the cell type.[11]
One of the initial modifications of Giemsa stain was the MGG stain which also belongs to the Romanowsky group of stains described as early as 1900[12],[13] and was the standard staining technique in hematology samples and subsequently became a dominant and routine stain in diagnostic cytopathology; primarily, the air-dried smears of fluids, FNA smears, and lymph node touch preparations.
Though the Pap stain and the routine H and E stains of wet preparations yield better results on thick smears with crisp nuclear details, the Romanowsky group of stains provide a complementary enhanced elucidation of cytoplasmic components with subtle details on the background, stromal and intercellular matrix appreciation. MGG has now become the routine cytological technique; however, the overall fine quality is seen to be deeply influenced by several factors that include uniform spreading of smears without thick smearing, adequate air drying, pH of the buffer solutions, timing of stain contact with the smears, variation of chemical composition between the batches of preparation of stock solution, and instability of stock solution due to exposure to light and air. The desirable goal of “Standardisation of MGG” is taken as a key quality assurance of the accuracy of a cytopathology laboratory and aims at a quality check that needs to be regularly controlled at every single step of this sensitive technique. Any deviation from the optimal quality has to be identified and addressed promptly.
The DiffQuik stain is a modified Romanowsky method intended as a rapid and a very simple staining of air-dried smears that might be prepared in a physician's office laboratory, which involves three steps, including fixation and staining steps, with preparation of buffer solutions as well.[14]
The Diff-Quik stain has proved to be reliable and effective for rapid assessment. It particularly highlights metachromatic material such as mucins, colloid and ground substance of mesenchymal lesions, as well as lymphoglandular bodies.
With the increasing trend of short turn around time (TAT), there is an increasing trend in the application of rapid staining techniques utilizing Romanowsky-type stains (primarily Diff-Quik stain), rapid Papanicolaou stain, or rapid hematoxylin-eosin (H&E) and reflect the personal and institutional choices of the users.[15] Many researchers have developed modifications to Giemsa's method to achieve better results, with the simplification of the technique and utilizing the value of methylene blue as the primary dye.
The present technique of MUFG was developed to attempt immediate interpretation of FNA smears without tedious processing and preparatory procedures involving buffer preparations and pH maintenance as compared with the abovementioned Romanowsky stains, including the MGG and DiffQuik. The sheer advantage of the MUFG technique is that it involves direct staining with filtered, adequately ripened Giemsa stock solution with a stain contact time of only 3 min.
The MUFG stain has the technical advantages, including lack of immediate fixation obviating the need for alcohol as a preservative in resource poor primary care settings. As a result, there is less cellular loss than with the alcohol-fixed Papanicolaou stain. The other best advantage is that the staining procedure is simple and rapid. Morphlogical advantages include the large size of cells because of lack of shrinkage from immediate fixation and easier identification of cytoplasmic granularity (including neuroendocrine granules) and intranuclear inclusions. Metachromatic materials, such as mucin, colloid, matrix, mesenchymal or stromal fragments, and microorganisms are also better visualized. Hematological disorders can be better evaluated with Romanowskys stain. Lymphoglandular bodies (cytoplasmic cell fragments), a feature of lymphoid lesions, are readily appreciated in MUFG-stained material and are a helpful feature in the work of lymph node masses [Figure 5]. The nuclear chromatin details were also of comparable quality and aided in the diagnosis of many malignant lesions as well [Figure 4].
Figure 5: MUFG stain showing background lymphoglandular bodies in case of lymphoma (40×)The choice of which stain to be used in rapid assessment is made by cytopathologists, which easily allows them to make a reliable diagnosis and is also true when choosing a stain for immediate interpretation. With the current trend of rapid turnaround time reporting of lab samples, we strongly believe that immediate initial interpretation of FNA specimens should be performed and we favor using MUFG stain as it is only a single-step stain, far cheaper and rapid with comparable transparency; thereby can be used in rapid assessment that complements the traditional Papanicolaou stain and has advantages in demonstrating specific cytologic features of benign and malignant lesions.
ROSE of cytological samples obtained by clinicians or radiologists can be stained by MUFG stain and examined microscopically, with an immediate provisional diagnosis. There can be a rapid correlation with clinical and radiology features as imaging films are available and an immediate diagnosis can be made possible. This rapid assessment can be comparable to frozen section in surgical pathology and can give answers to many questions relating to the adequacy of a sample, inflammatory or neoplastic; if neoplastic, benign or malignant; whether material is sufficient for further ancillary techniques, thereby hinting at additional aspirates in the same go, preventing re-aspiration procedures at a later date.[16] Utilization of immediate interpretation decreases the false-negative rate of FNA biopsy and increases the sensitivity and specificity of the procedure. With the rapid assessment, additional samples can be taken for further sophisticated modalities like IHC, cytologenetics/molecular studies, and electron microscopic evaluation.[17]
The study also attempted in a possible cost reduction by reusing the same cover slip which was used for uniform spreading of stain for mounting; by careful removal followed by immediate and thorough washing with gently flowing running tap water. There was a faint minimal blue hue to the used coverslips, which did not hinder cytological evaluation, as well as occasional damage to cover slips, requiring new ones. Therefore, making it a practical cost effective, distinctly advantageous perfect stain in primary care settings is difficult owing to the high cost of cover slips. Neither preparation of working solution nor variation of staining with buffer is required and provides good nuclear and cytoplasmic details as the cells appear large with crisp morphological features. Air drying removes the artifactual changes seen in wet-fixed smears due to poor fixation.
The most important drawback of the MUFG stain is that it requires an even thin spread of aspirated material. Preparation of adequate uniform spread slides is of supreme importance for microscopic analysis. It is not uncommon to observe overly thick sections that are excessively stained and/or poorly differentiated and thus unacceptable for observation. The overall staining quality was markedly affected by thick and hemorrhagic smears. Hemorrhagic smears showed heavy background stain with poor delineation of cell morphology. There was variation in overall staining, with some well-stained areas and under-stained areas within the same slides, leading to a reduction in quality index, especially in smears from soft tissue and lymph nodes, as they tend to have thick and hemorrhagic smears. The stained smears did not show any fading changes for up to three months, and hence they can be stored and retrieved.
This is a pilot study primarily aimed at assessing the quality of MUFG rapid stain,[18] and there are no similar studies published in the literature to the best of our knowledge. The study needs further validation by similar experience from other institutes and to further study the usefulness of sole interpretation of MUFG smears in rendering a final diagnosis.
In conclusion, the MUFG stain is a simple and rapid staining method in terms of applicability in resource poor settings where feasibility of alcohol for fixation and preparation of pH-balanced buffer and working solutions is far from reach, associated with its benefits of rapid assessment for an immediate yet reliable preliminary diagnosis. With the rapid development of advanced modalities, we attempted to appreciate the usefulness and simplicity of this ancient stain to contribute a great deal to cytopathology practice. However, a note of caution and a conservative attitude should be exercised by the pathologist in communicating with the clinicians in giving a preliminary diagnosis.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
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Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/joc.joc_43_22
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