|Year : 2021 | Volume
| Issue : 1 | Page : 6-11
Diagnostic utility of cell block technique as an adjunct to cytological smears in evaluation of thyroid aspirates on fine needle aspiration cytology
Usha Kiran Raina1, Jyotsna Suri2, Subash Bhardwaj2, Bhavna Sahni3
1 Directorate of Health services, Jammu, Jammu and Kashmir, India
2 Department of Pathology, Government Medical College, Jammu, Jammu and Kashmir, India
3 Department of Community Medicine, Government Medical College, Jammu, Jammu and Kashmir, India
|Date of Submission||18-Jun-2020|
|Date of Decision||25-Aug-2020|
|Date of Acceptance||23-Sep-2020|
|Date of Web Publication||27-Jan-2021|
Department of Community Medicine, Government Medical College, Jammu - 180 001, Jammu and Kashmir
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Direct smears and cell blocks prepared from residual tissue fluids complement each other for establishing a conclusive cytopathologic diagnosis in thyroid lesions. Aim of the Study: Evaluation of the diagnostic utility of fine-needle aspiration cytology (FNAC) alone and in combination with cell blocks in the diagnosis of thyroid lesions. Materials and Methods: A hospital-based prospective longitudinal study was conducted in which 100 thyroid smears were categorized as neoplastic, nonneoplastic and inadequate using FNAC and cell block technique. All specimens were classified using point scoring system modified by Mair et al. Direct smears were compared with cell-block specimens on variables of cellularity, background blood and clot, cellular degeneration and retention of appropriate architecture using Fisher's exact and unpaired t-test. Results: Sixty-two percent of cell block specimens showed a moderate amount of background material (P = 0.01), display of excellent architecture was also significantly more in cell block (20%) technique than direct smears (9%), and degree of cellular degeneration was minimal in significantly more cell blocks (26%) than direct smears (12%), thereby making diagnosis easy as opposed to FNAC which yielded more cellular material than cell blocks (P = 0.02). Four cases showing hemorrhagic material on direct smears were diagnosed on cell block as one case each of colloid goiter and chronic lymphocytic thyroiditis and two cases of adenomatous goiter and two cases showing hemorrhagic material on cell block were diagnosed with chronic lymphocytic thyroiditis on direct smears. Conclusion: FNAC and cell block complement each other and combining both techniques improves the diagnostic yield and facilitates accurate diagnosis. It is advisable to perform cell-block for each case of FNAC of thyroid lesions.
Keywords: Cell block, cytology, fine-needle aspiration, malignancy, thyroid lesions
|How to cite this article:|
Raina UK, Suri J, Bhardwaj S, Sahni B. Diagnostic utility of cell block technique as an adjunct to cytological smears in evaluation of thyroid aspirates on fine needle aspiration cytology. J Nat Sc Biol Med 2021;12:6-11
|How to cite this URL:|
Raina UK, Suri J, Bhardwaj S, Sahni B. Diagnostic utility of cell block technique as an adjunct to cytological smears in evaluation of thyroid aspirates on fine needle aspiration cytology. J Nat Sc Biol Med [serial online] 2021 [cited 2021 May 12];12:6-11. Available from: http://www.jnsbm.org/text.asp?2021/12/1/6/307848
| Introduction|| |
It is estimated that 42 million people in India suffer from thyroid diseases. Neck swelling is a common clinical presentation of a thyroid problem. These swellings may be diffuse or nodular. Solitary nodules are a cause of concern for the clinician due to the possibility of the nodule being malignant.
To avoid misdiagnosis and hence unnecessary surgery in patients with benign thyroid nodules, it is essential to distinguish between benign and malignant lesions.
George N Papanicolaou (PAP) introduced cytology as a tool to detect cancer and precancerous lesions in 1928. The accuracy of the cytologic examination from any site depends greatly on the quality of collection, preparation, staining, and interpretation of the material. Inadequacy in any of these steps will adversely affect the quality of diagnostic cytology. Studies have shown that the cytological examination of specimens by means of smears, no matter how carefully prepared leaves behind a large residue that is not further investigated, but that might contain valuable diagnostic material. The residual material can be evaluated in a simple and expedient fashion by treating it as cell block embedded in paraffin and examined besides routine smears.
The use of cell block for processing cytology fluids was first reported in 1895. This technique presents with some advantages and disadvantages. The advantages being that the histological pattern of the diseases can be appreciated, which is not usually seen in smear preparations and multiple sections can be taken that can be subjected to routine or special staining and immunohistochemistry. Morphological details can also be obtained with cell block method, which includes preservation of the architectural pattern like cell ball and papillae and three-dimensional clusters, excellent nuclear and cytoplasmic details, and individual cell characteristics. On the other hand, fragments of tissue can easily be interpreted in a biopsy-like fashion. During the microscopic examination as well, cell block technique poses less difficulty for observation as lesser dispersal of the cells occurs with this technique. Furthermore, sections prepared from the cell block can be stored for a longer period of time for retrospective studies as well. Cellblock technique has some shortcomings, the most obvious of which is that the technique is time-consuming when compared to conventional smear preparation. It is possible that during the process of centrifugation, material may be lost and may prove too less to come to a conclusive diagnosis. In the process of centrifugation, sometimes artifacts can occur in the cellblock that may be manifested as rosettes or pseudoacini that may be a source of misdiagnosis.
Despite the increased use of fine needle aspiration cytology (FNAC) and immunocytochemistry in the diagnosis of solid tumors of thyroid, only limited study has been done to assess the added contribution of cell block, although the value of cell block has been acknowledged., Several studies have shown that cell block increases the diagnostic accuracy and efficacy of thyroid fine needle aspirate, results in slide reduction and obviates repeat fine-needle aspirate.,
The present study was based on the premise that a large amount of material remains in the needle hub after the preparation of direct smears, which is generally discarded. To evaluate this residual material in a simple and expedient fashion, we employed cell block technique in addition to routine direct smears being done for aspirates of the thyroid gland on FNA with a presumption that this will help us to assess the utility of cell block technique in addition to direct smears on FNA thyroid and find out if the accuracy of diagnosis improves by using these techniques in combination over either of the tests used alone.
| Materials and Methods|| |
The present observational prospective study was conducted over a period of 1-year from November 1, 2016 to October 31, 2017, after obtaining approval from the Institutional Ethics Committee, GMC Jammu through letter no: IEC/Thesis/Research/T12B/C/2016/294 dated October 7, 2016. All patients referred to the cytology section of the department of pathology for FNAC thyroid were included in the sample, ensuring confidentiality. Written consent was sought, and FNAC was done. Aspirated material was subjected to direct smears as well as cell block study, and subsequent microscopic examination of the same was done. Cases yielding inadequate material for cell block, patients not ready to give consent for the FNAC thyroid, and patients with bleeding disorders or on anticoagulation therapy were excluded.
FNAC was performed under aseptic precautions using 25–27 G needle attached to 20 ml syringe with a Cameco syringe holder.
Direct smear on glass slide
After procuring the specimen onto the surface of previously labeled clean glass slides, tongue-shaped smears were prepared on the middle third of the slide. Usually, four slides were prepared, two slides were instantly inserted in Coplin jar for wet fixation: (PAP) Staining (Morse, 2002). Other two were air-dried for May Grunwald Giemsa Staining. This could vary depending on the amount of material aspirated.
Preparation of cell block
Following smear preparations, the needles and syringes used to obtain fine-needle aspirates were rinsed in 10 mL of 50% ethanol in a specimen container. Any residual clot or tissue in the hub of needles was removed carefully in the laboratory with the aid of another needle and rinsed in 50% ethanol. The entire material was centrifuged in a 10-mL disposable centrifuge tube at 4,000 rpm for 6 min to create 1 or more cell pellets (1 pellet in most cases). The supernatant fluid was decanted, and the deposit fixed in freshly prepared Nathan alcohol formalin substitute consisting of 9 parts of 100% ethanol and 1 part of 40% formaldehyde. The fixed cell pellets, at the end of 45 min' fixation, were recentrifuged at 4000 rpm for 6 min. These pellets should detach themselves or can be removed easily with a disposable Pasteur pipette following centrifugation. The cell pellets were wrapped in filter paper, placed in a cassette, and stored in 80% ethanol until ready for processing in the automatic tissue processor using a 13-hour processing schedule as follows: 80% ethanol with 1change (2.5 h); 95% ethanol (1 h); 100% ethanol, 4 times (1 h each); 1:1 ethanol/xylene (1 h); xylene, 3 times (1 h each). The cell blocks were embedded in paraffin and sectioned at 3-μm thickness. The cytomorphology of FNA smears was compared with histopathology of cell blocks in all cases.
On these slides, all the specimens were classified using point scoring system modified by Mair et al. Specimens with insufficient cellularity i.e., <5–6 groups of follicular epithelial cells with 10 or more cells per group on conventional smears and cell block negative for follicular cell were considered as “unsatisfactory” according to the adequacy assessment protocol is taken from Goellner et al. To ensure quality control in this study, 10% of slides were examined by an independent consultant pathologist who was blinded to the previous results.
Data management and statistical analysis
In this study, results are tabulated as proportions and percentages. Independent variables included cell block and conventional smear preparation. Cellularity, quality of the background, degree of cellular degeneration, architectural or cellular arrangement, and cytodiagnosis were the dependent variables compared by Fischer's exact test and unpaired t-test in smears and cell blocks using web-based software Open Epi, Version 3. 03, developed by Emory University in Atlanta, Georgia. Two-tailed P < 0.05 were considered statistically significant.
| Results|| |
A total of 100 patients were enrolled in the study. Most patients were in their 4th decade of life (24%); however, very few were <20 years (7%) and >70 years (5%). The mean age of the patients was 42.75 ± 15.75 years, with a range of 12–75 years. The sample consisted of main females (88%), with male-to-female ratio of 1:7.33. Among 100 cases, Diffuse thyroid swelling was present in 65%, Solitary nodule in 32% cases of which right side thyroid swelling was present in 21% and left side in 11% cases, and multinodular swelling was seen in 3% patients. Most patients reported duration of thyroid swelling ranging from 1 to 12 months (45%). The mean duration of thyroid swelling was 4.43 years, with a range of 3 days to 40 years. Blood mixed aspirate was present in 55% patients, followed by colloid aspirated in 42%, and brown color fluid, straw color fluid, yellowish fluid was aspirated in 1% patients each.
[Table 1] reveals that 88% of lesions were benign, 8% were malignant, and 4% inadequate on conventional smears in comparison to cellblock, which showed 90% to be benign, 8% malignant, and 2% inadequate. Colloid goiter was the most common nonneoplastic lesion observed in both direct smear and cell block technique [Figure 1]a, followed by chronic lymphocytic thyroiditis [Figure 1]b. Papillary Carcinoma was the most common type of the neoplasm observed with both the techniques [Figure 1]c. However, the combination of the cell block and direct smears improved a gain in diagnosis, as four cases showing merely hemorrhagic material on conventional direct smears were diagnosed on cell block as one case of colloid goiter, one case of chronic lymphocytic thyroiditis, and two cases of adenomatous goiter and two cases showing hemorrhagic material on cell block were diagnosed as Chronic lymphocytic thyroiditis on direct smears. Thus, cell block bridges the gap between cytology and histopathology.
|Figure 1:(a) Colliod goitre: Variable sized thyroid follicles (blue arrow) filled with colloid (cell block, (H and E, ×400). (b) Chronic lymphocytic thyroiditis: Lymphoid cells (blue arrow) infiltration in the thyroid follicular (black arrow) cells (cell block H and E, ×100). (c) Papillary carcinoma of thyroid: Section shows multiple papillae (black arrow) with fibro vascular core (blue arrow) (cell block H and E, ×100)|
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|Table 1: Categorization of patients according to direct smears and cell block diagnoses|
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When the comparison of the diagnostic quality of background, blood and clot was carried out as shown in [Table 2], Score 0 and Score 2 were comparable in both techniques. However, two thirds (62%) of cell block specimens showed a moderate amount of background material making diagnosis possible, which was significantly more than in direct smears.
|Table 2: Comparison of background blood and clot in smears and cell blocks|
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It is evident from [Table 3] that cellularity was significantly more in direct smears (25%) when compared with cell block (12%), making the diagnosis by smears simpler while it was minimal in more cell block specimens (41%) than direct smears (22%), reiterating the superiority of direct smears in case of cellularity, difference being statistically significant.
|Table 3: Comparison of cellularity on conventional smears and modified cell block|
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As observed in [Table 4], the display of excellent architecture was significantly more in cell block (20%) technique than direct smears (9%). Moderate retention was comparable in both techniques, while 35% direct smears were nondiagnostic, which was significantly more than cell blocks (15%).
|Table 4: Comparison of retention of appropriate architecture on direct smears and cell block|
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[Table 5] shows that the degree of cellular degeneration was minimal in significantly more cell blocks (26%) than direct smears (12%), thereby making diagnosis easy. SCORE 1 was found to be comparable in both techniques, while marked cellular degeneration was observed in almost double the number of direct smears than cell blocks, but this difference was not statistically significant.
|Table 5: Comparison of degree of cellular degeneration and trauma present in smears and cell blocks|
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As observed in [Table 6], significantly better results were yielded for cell block as compared to conventional smears in parameters of retention of appropriate architecture (P = 0.0003) and cellular degeneration (P = 0.006). However, significantly better results were yielded for conventional smears as compared to the cell block in parameters of cellularity (P = 0.001). Mean scores of the cell block and conventional smears for background blood or clot were comparable and there was no statistical significance (P = 0.91).
|Table 6: Comparison of mean scores for direct smears and cell blocks using mair et al graded scoring|
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Thus, cell block technique, when used as an adjunct to FNAC, not only increased the overall diagnostic accuracy but also helped to demonstrate better architectural patterns which could be of great help in approaching the correct diagnosis in doubtful cases, leading to a more refined cytological diagnosis complementary to smears and histology.
| Discussion|| |
Thyroid nodules are a significant clinical problem in the general population, but majority are benign and do not require surgery. The prevalence of thyroid carcinomas is approximate 4%–8%. In agreement with the literature, we also found that 8% of the cases in our study were malignant.
The present study was conducted on 100 patients. The age of patients ranged from 12 to 75 years with a mean of 42.75 years. These variables are slightly higher as compared with the study done by Madakshira et al. who found that the age range was 22–58 years with a mean age of 38.7 years but comparable with study by Hegazy et al., where the age range was 15–65 years, with a median age of 40 years., In our study, majority of patients (24%) were in their fourth decade of life, followed by 18% of patients in their third decade. In most studies, it was reported that a larger proportion of patients were in their third decade of life.
Eighty-eight percent of patients in our study were females with a male-to-female ratio of 1:7.8. This finding was in agreement with another study that reported the male to female ratio of 1: 6.5. Female preponderance was also seen in a study where out of 59 patients, 49 were females and 10 were males.
Out of 100 cases in our study, diffuse thyroid swelling was present in 65%, solitary nodule in 32% cases of which right side thyroid swelling was seen in 21% and left side in 11% cases. Multinodular swelling was seen in 3% of patients. Ahmed et al. also reported that solitary nodule was the predominant presentation in 53.33% cases, followed by multinodular in 32.22% cases and diffuse thyroid swelling in 14.44% cases.
Blood mixed material was aspirated in 55% patients, followed by colloid aspirate in 42%, brown color fluid, straw color fluid, yellowish fluid aspirated in 1% of the patients each. In our knowledge, no such data is available in the current literature despite exhaustive parsing of the published scientific work.
The present study assessed smears of cell block and conventional smears using a modified scoring system by Mair et al. As per the scoring system, significantly better results were yielded for cell block as compared to conventional smears in parameters of retention of the appropriate architecture. This agrees with other studies which concluded that cell block enabled a better interpretation of morphology of the papillae or follicles along with the study of nuclear features.,,
Significantly better results were yielded on cell block as compared to conventional smears in relation to cellular degeneration. It was achieved because samples were fixed immediately after collection. This result was in agreement with Nathan et al. Another study, however, showed a contrary result of marked cellular degeneration on Cellblock as compared to conventional smears. In their study, material for cell block was aspirated after 3–4 passes for aspirations for the conventional FNA, and this may have contributed to a more traumatized and poorly preserved specimen.
In our study, we witnessed better cellularity with direct smears as direct smears were prepared earlier from the aspirate and then the remaining material in the needle hub and syringe was used for cell block preparation. Similar findings were seen in a study by Sanchez and Selvaghi. Moreover, no dedicated passes were done for modified cellblock preparation, limiting the cellularity.
In the present study, the cytological findings were categorized as neoplastic, non-neoplastic, and not suitable for diagnosis. We found that the majority of cases were benign, i.e., 88% on direct smears and 90% on cell block. This is in agreement with another study which documented that majority (70%) of thyroid FNA are reported as benign. Ahmed et al. also observed nonneoplastic lesions in 83.33% cases on FNAC, of which colloid goiter was most common as in the present study, and the neoplastic diagnosis was made in 14.44% cases. However, in our study, four cases remained un diagnosed on conventional direct smears. This inconclusive diagnosis on FNAC could be due to poor spreading, air-drying artifact, and the presence of thick tissue fragments despite aspiration of adequate material as suggested by Sanchez and Selvaghi. Thus, the combination of the cell block with direct smears improved a 4% gain in diagnosis. This result was in concord with observations made by Rajib et al., who found that by adding cell block technique, 12.5% inadequate direct smears could yield a diagnosis, increasing the percentage of adequate cases to 95.7%. De-Lima et al., also suggested the same finding.
When mean scores for direct smears and cell Blocks were compared using Mair et al. graded scoring, significantly better results were yielded for cell block as compared to conventional smears in parameters of retention of appropriate architecture (P = 0.0003). This is in tune with other studies by Brown et al. and Kulkarni et al. who concluded that cell block preserves architectural patterns with excellent nuclear and cytoplasmic details., Similar findings were seen in another study, which established that the cellblock enabled a better interpretation of morphology of the papillae or follicles along with the study of nuclear features.
Significantly better results were seen on cell block as compared to conventional smears in relation to cellular degeneration (P = 0.006). Minimal cellular degeneration was seen in the majority of cases in both methods, although cell block technique was better than direct smear in preserving cellular integrity. It was achieved because samples were fixed immediately after collection. This result is in agreement with Nithyananda et al. Another study however, showed a contrary result of marked cellular degeneration on Cellblock as compared to conventional smears where the material for cell block was aspirated after 3-4 passes and this may have contributed to a more traumatized and poorly preserved specimen.
In our study, we witnessed better cellularity with direct smears as compared to the cell blocks (P = 0.001). It was because direct smears were prepared earlier from the aspirate, and then the remaining material in the needle hub and syringe was used for cell block preparation. Similar findings were seen in a study by Sanchez and Selvaghi.
No dedicated passes were done for modified cell block preparation, thus limiting the effectiveness of the method.
| Conclusion|| |
The combined use of cell block and direct smear improved testing as we achieved 100% diagnosis. We, therefore, conclude that cell block technique, when used as an adjuvant to routine smear examination, increases diagnostic yield because of better preservation of the architectural pattern, particularly in cases where there is a diagnostic dilemma. Hence, it is advisable to perform cell-block for all cases of FNAC of thyroid lesions to arrive at a definitive diagnosis, thereby increasing the validity of both the techniques.
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Conflicts of interest
There are no conflicts of interest.
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