Tumor Cell Representation by an Improvised Technique of Fine-Needle Aspiration Specimen Acquisition and Cell Block Preparation: Our Experience in Lung Cancer Cases in a Peripheral Center of Eastern India.

BACKGROUND: Being a minimally invasive diagnostic technique, Fine-Needle Aspiration Cytology (FNAC) has become the first-line test and corresponding aspirated material has become the target specimen for diagnosis and ancillary tests in lung carcinoma. Although the role of Cell Blocks (CBs) in diagnosis and in ancillary testing is well recognized in literature, limited attention has been paid to specimen procurement and triage in the preparation of CBs. In the present scenario, CBs are not consistently optimal because of its low cellularity. AIMS: This study is aimed to describe an improvised technique of specimen acquisition and cell block preparation in CT-guided FNACs of lung carcinoma cases in a resource-constrained center and to assess its efficacy for optimal representation of cellularity, morphology, and architecture.
MATERIALS AND METHODS: Total 85 lung carcinoma cases undergoing CT-guided FNAC in our center from February 2017 to January 2018 were included in this study. 4 to 5 direct smears and subsequent CBs were made from material obtained by single pass. Cellularity of smears and corresponding cell blocks were assessed and categorized according to a scoring system (score 1 to 3 for number of cells <50, 50-100, >100, respectively). Preserved architecture and morphology were also assessed in smears and CBs.
RESULTS: The evaluated samples showed a cellularity score 3 in 65.4%CBs and score 2 in 24.7% CBs. Overall, 90.1% cell blocks had acceptable cellularity. Cell morphology was preserved in all CBs of acceptable cellularity, except for two adenocarcinoma, one squamous cell carcinoma, and one small cell carcinoma blocks. Cellular architecture was also preserved in all CBs of acceptable cellularity.
CONCLUSIONS: This simple improvised technique of CB preparation optimized its cellularity, morphology, and architectural preservation, even after adequate cellular FNA smears. Copyright:

INTRODUCTION

Lung cancer is still the leading cause of cancer-related mortality.[1] Now, in the era of specific targeted therapy, appropriate diagnosis including the morphological, immunohistochemical, and molecular characterization of the malignant neoplasm has become very important and challenging. Historical separation of small cell carcinoma and nonsmall cell carcinoma (NSCLC) is no longer adequate as different molecular profiles and subsequent different therapeutic strategies for different subtypes of NSCLC are increasingly appreciated.[23] The specific targeted therapies require molecular pathological analysis after the standard cytomorphological or histomorphological evaluation including immunochemistry, mandating the availability of satisfactory tumor cells in the specimen for those tests.

As a recently published guideline of American College of Chest Physicians clearly stated that minimal invasive diagnostic technique should be used as first-line test, the target specimen is shifting from histological to cytological.[4] Cytological specimens compared to biopsy specimens are usually less contaminated by nonneoplastic cells and are, therefore, favorable for sensitive molecular genetic analysis. Conventional cytological smears have the advantage of intactness of cell nuclei, thus providing high DNA quality for molecular analysis. But, the drawback is that the procurement of material by micro-dissection results in limited tumor cells and problems in archiving the specimen.[5] In this context, the utility of cell block study as an integral part of cytology is appreciated more now than ever before, largely owing to the significant role they play in ancillary testing particularly molecular diagnosis. As most of the ancillary testing platforms are developed on formalin-fixed paraffin-embedded (FFPE) tissue, cell block preparation processed in this way generally does not require additional validation.[6] The cell blocks can be archived for future diagnostic and research purpose and at the time of tumor relapse particularly when re-biopsy is not feasible.

Several articles describe that availability of at least 50–100 tumor cells are required and 100 analyzable tumor cells are generally sufficient for effective evaluation and ancillary studies, provided they are contaminated with nonneoplastic cells as few as possible.[57] However, the cell blocks are not consistently optimal with low cellularity being the leading cause of dissatisfaction. Cell loss and reduction of DNA material with each additional section from cell blocks add to the problem. Too often greater attention is focussed on the diagnosis and not on the procurement and triage which have been shown to significantly affect cellularity.[8] Additional dedicated passes for optimal specimen collection and triage (judicious allocation among smears and cell blocks) are crucial to increase the likelihood of enough sample for ancillary testing. Direct smears having enough cell content but the failure of molecular testing in cell blocks signify that the overall specimen cellularity was not suboptimal. Judicious allocation to the smear and redirection of some content to cell block could have improved the yield. Development of protocol for specimen collection and triage are, therefore, very important for optimization of cell blocks. In an earlier published study, our group has already described a simple and cost-effective method for CB preparation for optimal tumor cell representation.[9]

This present study is aimed to describe the improvised technique of specimen acquisition and cell block preparation in CT-guided transthoracic FNAC of lung carcinoma cases in resource-constrained center of India. The efficacy of this improvised technique is assessed for optimal representation of cellularity, morphology, and architecture in cell blocks of different histological types of lung cancer.

MATERIALS AND METHODS

A study was performed with CT-guided FNA of 85 lung carcinoma cases from the period of February 2017 to January 2018. Materials from lung lesions were aspirated for conventional smears and cell block simultaneously. Material was obtained by inserting 21 gauze spinal needle attached to a 10-cc syringe. After localizing the lung lesion with the help of CT scan, the needle was passed into the lesion. The stellate was withdrawn when CT finding confirmed that the anticipated target was reached. The syringe fitted with a FNAC gun was then attached to the needle hub. Mild negative pressure was created and maintained in the syringe and the needle was moved quickly back and forth in a cutting motion within the lesion to chisel and suck the material within the syringe. The needle was withdrawn as soon as blood was seen to cross the needle hub. Needle was detached from the syringe and was kept separately.

The material present in the syringe was then used very quickly to make conventional smears in 4-5 slides of which one was kept for alcohol fixation and others allowed for air drying. The remaining material was ejected into a glass slide and allowed to clot. Just after that, the material present in the needle hub was scooped out by pin head and added to the remaining material in the glass slide. Any remaining material, if present in the needle, was also collected by introducing the stellate. The fragments of these clotted materials were collected in centrifuge tube containing 10% neutral buffered formalin. The test tube was centrifuged at 3000 rpm for 10 min. The deposited fragments were, thus, packed and formalin fixed. They were allowed to dry and subsequently taken out by inverting the tube. The material was then wrapped in filter paper or Surgipath Bio-wrap (Leica) and kept in cassette, processed routinely and formalin-fixed paraffin-embedded (FFPE) cell blocks were prepared and stained with H and E stain. The air-dried and alcohol-fixed smears were also stained with May–Grunwald–Geimsa (MGG) and Papanicolaou (PAP) stains.

Here in this study, FNAC smears showing a clear cut architectural pattern of acini or papillae and cytomorphology showing prominent nucleoli and preservation of cytoplasmic mucin were included in adenocarcinoma type.

Some architectural patterns showing clusters or small sheets with ragged border, sharply angular projections of cytoplasm, and window in between the cells with or without recognizable intercellular bridges and cytomorphology showing cytoplasmic orangophilia (PAP) and bluish tinge or pale (MGG) cytoplasm were considered as squamous cell carcinoma.

Clusters or sheets of small to medium cells with presence of rosette were included as maintained architecture in small cell carcinoma and characteristic cell morphology described in standard textbooks were considered as intactness of cell morphology in small cell carcinoma.

Cluster of large cells forming organoid pattern, ill-formed papillae, questionable acini, or rosette were considered as features of architecture in PDNSCLC (poorly differentiated nonsmall cell lung carcinoma) although those needed to be further confirmed and categorized by immunohistochemistry (IHC).

As per requirement criteria available in literature,[57] cellularity of the smears and cell blocks (i.e. number of tumor cells excluding other contaminating cells like stromal cells and inflammatory cells) were categorized into those having unacceptable cellularity score 1 (<50 cells) and acceptable cellularity score 2 (50–100 cells) and score 3 (>100 cells). Adequate preservation of morphology and architecture (as described above) was scored as 1 and no recognizable morphology or architecture was scored as 0.

The smears and CBs were examined according to above mentioned cytologic features and scoring system. The overall results of smears and CBs having different scores were tabulated [Tables 1-3].

Table 1

Cellularity score among Cell Block and FNA smears

MORPHOLOGY	CELLULARITYFNA SMEAR (%)	SCORE 3CELL BLOCK (%)	CELLULARITYFNA SMEAR (%)	SCORE 2CELL BLOCK (%)	CELLULARITYFNA SMEAR (%)	SCORE 1CELL BLOCK (%)
1. ADENO CA	41 (97.6%)	28 (68.3)	00 (0%)	9 (21.9%)	01 (2.4%)	04 (9.7%)
2. SQUAMOUS CA	09 (81.8%)	05 (50%)	01 (9.1%)	4 (40%)	01 (9.1%)	1 (10%)
3. SMALL CELL CA	11 (91.7%)	06 (60%)	00	03 (30%)	01 (8.3%)	01 (10%)
4. P.D. NSCC	16 (88.8%)	12 (66.7%	02 (11.1%)	4 (22.2%)	00	2 (11.1%)
5. NSCC with adeno + squamous ftrs	02 (100%)	02 (100%)	00	00	00	00
TOTAL	79 (92.9%)	53 (65.4)	3 (3.5%)	20 (24.7)	03 (3.5%)	8 (9.9%)

Table 3

Architecture scoring among Cell Block and FNA smears

TUMOR TYPE	SMEAR SCORE 1(PERCENTAGE)	CELL BLOCK SCORE 1(PERCENTAGE)	SMEAR SCORE 0(PERCENTAGE)	CELL BLOCK SCORE 0(PERCENTAGE)
ADENOCARCINOMA	42 (100%)	39 (95.1%)	0 (00%)	2 (4.9%)
SQUAMOUS CELL CARCINOMA	5 (45.5%)	7 (70%)	6 (54.5%)	3 (30%)
SMALL CELL CA	10 (83.3%)	9 (90%)	2 (16.7%)	1 (10%)
PD NSCC	6 (33.3%)	9 (50%)	12 (66.7%)	9 (50%)
NSCC with adeno and squamous features	2 (100%)	2 (100%)	0 (00%)	0 (00%)
TOTAL	65 (76.7%)	66 (81.5%)	20 (23.5%)	15 (18.5%)

RESULTS

Total 85 cases were included in this study. FNA smears were prepared from all these cases. Subsequent cell block preparations were possible in 81 cases after the acquisition of material by single pass.

Age range of this study population was from 30 to 82 years with overall mean age being 57.9 years. The youngest patient was 30 years male. Adenocarcinoma cases had the lowest mean age of 57.9 years while squamous carcinoma cases had the highest mean age of 62.5 years.

Male: female ratio was 4.6:1. The number of cases categorized into histologic subtypes depending on impression made from FNAC smears was as follows: Adenocarcinoma–42, Squamous cell carcinoma–11, Small cell carcinoma–12, PDNSCLC–18, and nonsmall cell carcinoma with squamous cell and adenocarcinoma pattern–2.

Cell block preparation was not possible in one case each of adenocarcinoma and squamous cell carcinoma and 2 cases of small cell carcinoma.

From the FNAC smears, it was seen that out of 42 cases of adenocarcinoma, 41 (97.6%) cases had cellularity score 3 and 1 (2.4%) case had cellularity score 1. Corresponding cell block preparations had score of 3 in 28 (68.3%) cases and score 2 in 9 (21.9%) cases. Hence, a total of 37 (90.2% of cell blocks) cases had acceptable cellularity [Table 1].

11 cases of squamous cell carcinoma smears revealed cellularity score 3 in 9 (81.8%) cases and 1 (9.1%) each had cellularity score 2 and 1, respectively. Of the cell block preparations, 5 (50% of the cell blocks) had score 3 and 4 (40% of the cell blocks) had score 2. Hence, total 9 (90% of blocks) cases had acceptable cellularity.

Out of 12 cases of small cell carcinoma, 11 cases (91.7%) had FNAC smear cellularity score 3 and remaining 1 case had cellularity score 1. Corresponding cell block showed cellularity score of 3 in 6 (60% of cell blocks) cases and score 2 in 3 (30% of the cell blocks). Hence, in 9 (90% of blocks) cases, cell blocks had acceptable cellularity.

In the case of PDNSCLC, cell block was possible in all 18 cases. Of those, 16 (88.8%) cases had FNAC smear cellularity score 3 and 2 (11.1% of cell block) cases had score 2. Corresponding cell block showed score 3 in 12 (66.7% of cell blocks) cases and score 2 in 4 (22.2% of blocks) cases. Hence, out of 18 cell blocks, 16 (88.8% cell blocks) cases had acceptable cellularity.

Cell block preparation was possible from material obtained by single pass in NSCLC with squamous and adenocarcinoma pattern. They had cellularity score of 3 in both FNAC smears and corresponding cell blocks.

In total 81 cases, 4 to 5 conventional smears and subsequent CB preparation were possible from material obtained by single pass. Out of these 81 cases, cellularity score 3 was found in 76 (93.8% of smears) and score 2 was found in 2 (2.5% of smears). In total, 78 (96.3%) smears and 73 (90.12%) CBs had acceptable cellularity adequate for ancillary investigations.

Considering the cell morphology [Table 2], it was seen that all 42 (100%) cases of adenocarcinoma [Figure 1a-d] had intact cell morphology in smears and 35 (85.4% of cell blocks) cases had intact cell morphology in cell blocks.

Table 2

Morphology score among Cell Block and FNA smears

TUMOR TYPE	SMEAR SCORE 1(PERCENTAGE)	CELL BLOCK SCORE 1(PERCENTAGE)	SMEAR SCORE 0(PERCENTAGE)	CELL BLOCK SCORE 0(PERCENTAGE)
ADENOCARCINOMA	42 (100%)	35 (85.4%)	0 (00%)	6 (14.6%)
SQUAMOUS CELL CARCINOMA	9 (81.8%)	7 (70%)	2 (18.2%)	3 (30%)
SMALL CELL CA	11 (91.7%)	9 (90%)	1 (8.3%)	1 (10%)
PD NSCLC	18 (100%)	17 (94.4%)	0 (00%)	1 (5.6%)
NSCLC with adeno and squamous ftrs	2 (100%)	2 (100%)	0 (00%)	0 (00%)
TOTAL	82 (96.5%)	70 (86.4%)	3 (3.5%)	11 (13.6%)

Figure 1

Composite image showing cellularity, intact cell morphology, and different architectural patterns of lung adenocarcinoma in cell block preparation. (a) Acinar pattern, (b) papillary pattern, (c) clusters with intracellular mucin, and (d) cribriform pattern. [Figure 1a-1c: H and E stain, 400 × magnification; Figure 1d: H and E stain, 100 × magnification]

In squamous cell carcinoma [Figure 2a and b], out of 11 cases, intact morphology was seen in 9 (81.8%) of the smears and 7 (70% of blocks) cell block cases.

Figure 2

(a and b): Composite image showing cellularity and intact cell morphology of squamous cell carcinoma in cell block preparation. (a) Sheets of cells with keratin pearl formation. (b) Cluster of malignant cells with intercellular bridges. (c) Microphotograph showing small cell carcinoma. (d) Microphotograph showing nonsmall cell carcinoma with high cellularity and intact cell morphology. [Figure 2a-2c: H and E stain, 400 × magnification; Figure 2d: H and E stain, 100 × magnification]

11 out of 12 cases (91.7%) of small cell carcinoma [Figure 2c] had intact cell morphology in smears and 9 cases (90% of blocks) had intact morphology in cell block, while in PDNSCLC [Figure 2d], all 18 smears (100%) had intact cell morphology and 17 (94.4%) had intact cell morphology in cell block. Thus, overall 82 (96.5%) of smear and 70 (86.4% of blocks) cases had intact cell morphology.

Considering the architecture [Table 3], the number of cases having intact architecture were 42 (100% of smears) in adenocarcinoma, 5 (45.5% of smears) in squamous cell carcinoma, 10 (83.3%) in small cell carcinoma, 6 (33%) in PDNSCLC, and 100% in adeno-squamous cell carcinoma, respectively. Intact architecture found in cell blocks were 39 (95% of blocks) in adenocarcinoma, 7 (70% of blocks) in squamous cell carcinoma, 9 (90% of blocks) in small cell carcinoma, 9 (50% of blocks) in PDNSCLC, and 2 cases of adeno-squamous cell carcinoma. Thus, overall 66 out of 81 (81.5% cell blocks) cell blocks had intact architecture, whereas 76.5% of smears had intact architecture.

DISCUSSION

CT-guided FNAC of pulmonary masses provides a least invasive, simple, easy, and reliable method for reaching rapid tissue diagnosis with minimal complication.[10] The use of cell block has been widely advocated in the diagnostic workup of patient with masses amenable to FNA since they provide diagnostic architectural information which compliments FNA smears.[11] Although there are some disadvantages like reduced morphological quality, as compared to conventional cytology, some studies have reported cell blocks to be helpful for better demonstration of the architectural pattern.[12] Small tissue fragments appear as “mini biopsies” which are useful for diagnosis, pattern recognition, subclassification, and identification of certain features[1314] When adequately cellular, cell blocks serve as a source of multiple additional sections that are valuable for ancillary studies such as special staining, immunostaining, ultra-structural analysis, and molecular testing.[15] Extensive analytical studies by Dacunha et al. on impact of preanalytic variables including specimen type upon EGFR mutation testing of lung carcinoma found overall similarity between smears and cell blocks.[16] Moreover recommendation of expert consensus opinion for cell blocks over smear for EGFR mutation and ALK rearrangement evaluation in lung cancers makes the role of cell block very important.[17] Material procured from only single pass was included in this study to reflect that cellularity found in the cell blocks after getting sufficiently cellular smear is an attempt to standardize the efficiency of the technique and procedure. There are different methods of CB preparation described in the literature,[1819] where materials were collected in nonfixative solution to overcome negative interference of formalin fixative in clot formation.[20] Clot formation of aspirated material in our study was achieved by natural mechanism of clotting cascade following tissue injury. Contact with glass slide might have some additional role as glass material is a known thrombogenic substance. Addition of any other substance as done in other methods to congeal the cell pellets was not done here. Allowing the material to clot first and its subsequent transfer into formalin fixative were expected to have minimal interference on clot formation in our study.

The incidence of adenocarcinoma was the highest among the 85 cases in the present study. This is in agreement with the study done by Tan et al. but contrasts others Indian studies like that of Saha et al.[1021]

In the current study of the FNA smears, cellularity score 3 was found in 79 cases and score 2 in 3 cases. Hence, 82 smears had cellularity of 50 cells or more. Only 3 cases had cellularity <50 in FNA smears. The corresponding cell blocks prepared showed score 3 cellularity in 53 blocks and score 2 cellularity in 20 blocks. Hence, 73 blocks (90.1% of cell blocks) out of 81 blocks showed cellularity which can be considered acceptable for ancillary investigations. Preservation of cell morphology was seen in 86.4% of cell blocks, whereas that of smears was 96.5%. A similar type of morphologic preservation (87% and 95%, respectively) was in a study conducted by Abdulhameed et al.[22] Architecture preservation seen in the current study was 81.5% of cell blocks and 76.7% of smears, whereas their study showed those to be 100% and 72%, respectively. It has been observed that in all cell blocks of acceptable cellularity, architecture was preserved. Cell morphology was also preserved except for only 2 cell blocks. This reflects that the intactness of architecture and cell morphologyin FFPE traditional cytology preparation platform best approximates FFPE blocks containing biopsy tissue.

In adenocarcinoma cases, out of 41 cell blocks, only 4 (9.7%) blocks had cellularity score 1 (<50 cells). Of these 4 blocks, smear cellularity score was 1 in only 1 case. The presence of abundant mucin in aspirate could be a cause of low cellularity. Additional dedicated pass for cell block preparation, if possible, could be of help in this case. Smear cellularity score 3 was found in 3 cases of which 2 cases had blood and necro-inflammatory component, respectively. One case in which block preparation was not possible had smear cellularity score 3. Better allocation of material would be expected to make optimal cell blocks in these cases where the smear cellularity score was 3.

One case each of squamous cell carcinoma and small cell carcinoma had cell block cellularity score of 1 and smear cellularity score 1. This was attributed to the fact that the aspirated material was necrotic in both. In one case of squamous cell carcinoma and two cases of small cell carcinoma, cell block preparation was not possible. Necrotic material causing needle blockage had led to inadequacy of material and thereby causing failure of cellblock preparation. Additional dedicated pass, if possible, could be of help in these cases having necrotic aspirate and low smear cellularity.

The complications encountered during this study were pneumothorax and emphysematous bulla in 3 (3.5%) cases having peripherally located lesion. Hemoptysis was seen in 4 (4.7%) cases having central or peri-hilar lesion. Transient pleuritic chest pain was seen in 7 (8.2%) cases. All the complications were managed conservatively except for one case of pneumothorax which needed chest drainage. Study conducted by Singh Ningthoujam et al. had similarly low rate of complications.[23] Kocijancic et al. had higher (27.2%) pneumothorax with higher (45%) rate of chest tube insertion.[24] This low rate of complications in our study might be due to use of fine (21G) needle, brief rapid chiselling movements of needles, single pass only, and necessary precautionary steps including exclusion of unfit patients.

In this study, we have put forward a technique where material blown out from syringe was used for preparing 4 to 5 conventional smears first. The residual material from syringe, needle hub, and needle hollow were exclusively reserved for CB preparation. When CBs were made following these steps, more than 90% of blocks represented adequate tumor cells for diagnosis and further ancillary studies even with single pass material. Additional dedicated pass in compliant patients would definitely increase the cellularity of CBs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.