Journal of Cytology
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Year : 2014  |  Volume : 31  |  Issue : 1  |  Page : 11-14
Utility of squash smear cytology in fiber-optic bronchoscopic biopsies

1 Department of Oncopathology, Malabar Cancer Centre, Thalassery, Kannur, India
2 Department of Pathology, Medical College Calicut, Calicut, Kerala, India

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Date of Web Publication15-Apr-2014


Background: Fiber-optic bronchoscopic biopsies yield very small bits of tissue, leading to high false negativity in lung cancer diagnosis, after paraffin embedding.
Aim: The aim of the present study is to assess the diagnostic efficacy of squash smear cytology of fiber-optic bronchoscopic biopsies and to compare this with standard paraffin embedded sections and sputum cytology.
Materials and Methods: A total of 100 suspected cases of lung cancer were subjected to fiber-optic bronchoscopic biopsies. Multiple biopsies from each were divided into two portions. One portion was processed routinely and paraffin sections made. Squash smears were made from the other and stained by Papanicolaou method. The diagnostic efficiency of two methods was compared. A positive diagnosis of cancer by any of the diagnostic modalities initially or during 6 months follow-up was taken as the gold standard.
Results: Out of 100 cases, 91 cases proved to be cancer. The pick-up rate was 0.77 for squash cytology, 0.55 for tissue sections, and 0.31 for sputum cytology. The pick-up was higher for endo-bronchial tumors by all methods. The agreement between squash cytology and tissue sections was 100% for small cell carcinoma and adenocarcinoma and 88% for squamous cell carcinoma.
Conclusion: Squash smear cytology has better pick-up rate than paraffin embedding in fiber-optic bronchoscopic biopsies and should be the preferred method when only one or few bits are available.

Keywords: Fiber-optic bronchoscope; lung cancer; squash smear cytology

How to cite this article:
Nayanar SK, Puthiyaveettil AK, Bhasurangan KC. Utility of squash smear cytology in fiber-optic bronchoscopic biopsies. J Cytol 2014;31:11-4

How to cite this URL:
Nayanar SK, Puthiyaveettil AK, Bhasurangan KC. Utility of squash smear cytology in fiber-optic bronchoscopic biopsies. J Cytol [serial online] 2014 [cited 2022 Dec 6];31:11-4. Available from:

   Introduction Top

Lung cancer remains the most common and the most lethal cancer in the world. The survival from lung cancer is poor and has not changed much during the last 25 years. Rapid advances in imaging modalities like the spiral computerized tomography scan have led to the early detection of lesions. The flexible fiber-optic bronchoscope (FOB) has added yet another means for the pulmonologist to negotiate smaller and smaller airways and different types of endo-bronchial ultrasound and navigation systems have led to improved diagnostic yield and lung cancer staging capabilities. [1] However, this has also created more of a challenge to the pathologist who has to deal with smaller and smaller biopsies. Biopsy specimens are generally small and average about 300 malignant cells in aggregate biopsies. [2] Coghlin et al. [3] found that on an average tumor represented only 33% of the biopsied tissue in an endo-bronchial biopsy sample. In addition, not every biopsy contained tumor. In fact, fewer than half the cases (48%) in the study contained tumor in all biopsy specimens.

Under the circumstances, inadvertent loss of such tiny biopsy material is a real risk. Imprint or "touch smears" could be a solution to this problem, but again, many times the sample is too small for both imprint cytology and histology. Sometimes bleeding which is an inherent risk of FOB biopsies may prevent further attempts, again limiting the amount of material obtained.

Squash smear biopsies have been used extensively in the diagnosis of central nervous system tumors. Its role has increased with the development of stereotactic biopsies, where the amount of tissue available is small. [4] We thought that squash smear cytology of the FOB biopsy samples could be a feasible alternative wherein, even minute samples can be wholly examined and loss during transit or processing avoided. It has could have the advantage of being more time-saving and cost-effective than routine processing for histology.

The objective of this study was to assess the diagnostic efficacy of squash smear cytology of FOB biopsies and to compare it with standard paraffin embedded sections and sputum cytology.

   Materials and Methods Top


A total of 100 consecutive patients who satisfied the inclusion and exclusion criteria during the 18 months period at a tertiary care hospital were included in the study.

Inclusion criteria

Patients who underwent FOB for suspected lung cancer and in whom biopsies were taken. The suspicion of lung cancer was based on the clinical or radiological grounds.

Exclusion criteria

Cases in which at least two bits of biopsy tissue was not available.

Sample size

A minimum sample size of 83 was estimated with the following assumptions; alpha error 0.05; expected sensitivities for two groups (sputum and squash) 0.5 and 0.7, power (1-β) 0.8. [5]


Squash smear was made from one bit wherein the tissue fragment was taken on a clean glass slide and spread using a disposable needle or another glass slide and immediately fixed in 95% ethyl alcohol for a minimum of 15-20 min before being sent to the cytology laboratory. They were then stained by Papanicolaou method.

The other tissue bit was fixed in 10% buffered formalin and conveyed to the histopathology laboratory for routine processing, paraffin embedding, sectioning and hematoxylin and eosin staining. If indicated, deeper serial sections were cut from the block. The reports of the cytology smears and the histology sections were made independently, blind to the diagnosis of each other.

Bronchial brushings were taken for every case and stained by Papanicolaou method. Three consecutive samples of sputum were also assessed for malignant cells, in available cases.

The cases which turned out to be negative for malignancy with squash smear, sections or sputum cytology were further investigated with fine needle aspiration (FNA) of enlarged peripheral lymph nodes, if present or by direct computed tomography scan guided percutaneous FNA of the lesion. Patients negative for all tests were followed up for a minimum of 6 months.

   Results Top

100 patients who underwent FOB were the subjects of the study. Of these 91 were diagnosed to have cancer by one of the diagnostic modalities and follow-up. Nine were found to have no malignancy. Eighty of the 91 patients could be diagnosed by squash, section or sputum, either alone or in combination. Squash alone was positive in 18, section alone in two and sputum in five. Eleven cases not picked up by above three methods in the first instance were diagnosed on follow-up and by procedures like transthoracic FNA.

[Table 1] gives the histological breakup of the lesions. In cases where both section and squash were positive, the tumor subtype by histological section was taken as the final diagnosis. The agreement between squash cytology and histology regarding the subtype was 100% for small cell carcinoma and adenocarcinoma and 88% for squamous cell carcinoma.
Table 1: Histological types of tumor

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The squash smears provided good morphological detail of cells like conventional cytology material. In addition architectural features could be appreciated in some cases. [Figure 1] shows the squash smear appearance of different lesions.
Figure 1: (a) Reactive bronchial epithelial cells. Squash smear (Pap, ×400). (b) Adenocarcinoma. Squash smear (Pap, ×400). (c) Squamous cell carcinoma. Squash smear (Pap, ×400). (d) Small cell carcinoma. Squash smear (Pap, ×400)

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The pick-up rates of the different modalities viz., squash smear, histology and sputum cytology are given in [Table 2]. The pick-up for intrabronchial lesions by squash, histology and sputum cytology were 0.91, 0.74, and 0.39, respectively and for the extra-bronchial lesions 0.58, 0.28, and 0.28, respectively.
Table 2: Pick-up rate of lung cancer by different diagnostic modalities

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   Discussion Top

The diagnosis of lung cancer by cytologic methods is of historic interest as it was demonstrated very early that malignancy could be diagnosed by examining exfoliated cells. The first major series of patients in which the examination of sputum led to the detection of lung cancer was by Hamplen 1919, 13 of 25 cases were examined. [6] Pulmonary cytology again enjoyed a rapid development in the 1970s and 1980s when FNA was validated as an alternative to open lung biopsy. The major methods to obtain cellular material for the diagnosis of lung cancer are:

  1. Sputum collection, the oldest and the most fundamental which depends on spontaneous exfoliation of cells.
  2. Bronchoscopic techniques of bronchial brushing, bronchial washing and bronchoalveolar lavage.
  3. FNA techniques performed trans-bronchoscopically or through chest wall under radiographic guidance.

No collection method is necessarily superior to the others. The choice of the cell collection technique is shaped by factors such as the personal preference of the physician, the status of the patient, the location of the lesion and the differential diagnosis. Endobronchial or transbronchial biopsies, bronchial brushings and bronchial washings, bronchoalveolar lavage along with modifications like endobronchial or trans-bronchial needle aspiration are the principle techniques of specimen retrieval. Forceps biopsy has the advantage of providing histological samples with preservation of architecture of bronchial wall and the lung parenchyma. Yet, even for centrally located tumors the maximum diagnostic yield may not be achieved until the fourth forceps biopsy and as many as ten biopsies may be necessary to maximize the diagnostic yield for peripheral carcinomas. [7] According to one study, diagnostic yield of endobronchial needle aspiration is higher than that of brush biopsy and increased the diagnostic yield when the specimen was inadequate because of crush artefact, necrosis, or tissue resistance. [8]

FOB biopsies sometimes yield very tiny bits of tissue which are difficult to handle and are often lost during processing due to the smallness of its size. This can result in a high false negativity rates in the diagnosis of lung cancer. The squash smear technique was originally developed for rapid diagnosis of brain biopsies. It allows superb preservation of nuclear and cytoplasmic details. The main advantages in addition to its simplicity and rapidity are that the whole specimen, however little it is, can be subjected to examination in a couple of glass slides. In contrast, multiple and complete sectioning of the tissue block is a time consuming and costlier alternative. Papanicolaou stained squash smears can be screened at the earliest within an hour or so.

The disadvantages attributable to the squash smears are the crush artifacts due to excessive pressure during smearing and the drying artifacts due to delay in fixation, both of which can be overcome with experience. There is inevitably some variation in thickness in individual smears and thick areas always appear densely cellular no matter what the appearances would be in corresponding paraffin sections of uniform thickness.

Conventional cytologic methods like bronchial brushing and washing are well known to be efficient methods for lung cancer diagnosis. Jones et al. [9] compared the diagnostic efficiency of wash and brush cytology to histology and found that cytology alone diagnosed 17.7% of case and that an overall yield of 89.3% was obtained using a combination of all three tests. They concluded that all three tests should be performed routinely in cases of suspected malignancy.

In a review of pathologic findings in cases of primary lung cancer in a span of decade, it was found that the total number of trans-bronchial FNA and endobronchial forceps biopsy specimens had increased and the total number of sputum samples had declined in the later period when compared to the earlier. The collective positivity of bronchoscopic cytology specimens was significantly higher than simultaneous forceps endobronchial biopsy during both periods, but bronchoscopic histology and cytology were complementary. [10]

In the current study, the tissue which was usually examined only by paraffin sectioning was also subjected to an alternative method namely, squash smear cytology. The pick-up rate of this technique is found to be better, i.e., 0.77 compared to 0.55 for tissue sections. The pick-up of both methods are understandably better for intraluminal lesions, reaching 0.91 in squash smears compared to 0.58 for squash of extraluminal lesions. In the current study, in cases where both squash smears and histology were positive, the agreement between the two was high. It was 100% for small cell carcinoma and adenocarcinoma and slightly less at 88% for squamous cell carcinoma.

A limitation of our study design is that when the biopsy material is divided into 2 halves the problem of non-representation of a particular bit may occur leading to fallacious results. The mitigating factor is that the possible non-representation is likely to be distributed equally between squash cytology and histology and hence would not affect the final result.

What is to be done if only one or two small bits are all that is obtained during the FOB procedure? Such a situation is not rare because the procedure may be interrupted by events such as bleeding, thereby limiting the amount of material obtained. In such instances, we feel it is more beneficial and easier to squash smear majority of the material for Papanicolaou staining, rather than process it for paraffin embedding. This is justified by the findings of this study, namely better pick-up rate of the former. Moreover, the study also demonstrates that the discrimination of subtypes by squash cytology is nearly as good as that obtained by tissue sections. The smaller portion may be preserved for immunohistochemistry, which is now required frequently for further typing of non-small cell lung cancer.

   References Top

1.Dhillon SS, Dexter EU. Advances in bronchoscopy for lung cancer. J Carcinog 2012;11:19.  Back to cited text no. 1
[PUBMED]  Medknow Journal  
2.Ofiara LM, Navasakulpong A, Ezer N, Gonzalez AV. The importance of a satisfactory biopsy for the diagnosis of lung cancer in the era of personalized treatment. Curr Oncol 2012;19:S16-23.  Back to cited text no. 2
3.Coghlin CL, Smith LJ, Bakar S, Stewart KN, Devereux GS, Nicolson MC, et al. Quantitative analysis of tumor in bronchial biopsy specimens. J Thorac Oncol 2010;5:448-52.  Back to cited text no. 3
4.Roessler K, Dietrich W, Kitz K. High diagnostic accuracy of cytologic smears of central nervous system tumors. A 15-year experience based on 4,172 patients. Acta Cytol 2002;46:667-74.  Back to cited text no. 4
5.Jones SR, Carley S, Harrison M. An introduction to power and sample size estimation. Emerg Med J 2003;20:453-8.  Back to cited text no. 5
6.Linder J. Lung cancer cytology. Something old, something new. Am J Clin Pathol 2000;114:169-71.  Back to cited text no. 6
7.Popp W, Rauscher H, Ritschka L, Redtenbacher S, Zwick H, Dutz W. Diagnostic sensitivity of different techniques in the diagnosis of lung tumors with the flexible fiberoptic bronchoscope. Comparison of brush biopsy, imprint cytology of forceps biopsy, and histology of forceps biopsy. Cancer 1991;67:72-5.  Back to cited text no. 7
8.Bilaçeroðlu S, Günel O, Caðirici U, Perim K. Comparison of endobronchial needle aspiration with forceps and brush biopsies in the diagnosis of endobronchial lung cancer. Monaldi Arch Chest Dis 1997;52:13-7.  Back to cited text no. 8
9.Jones AM, Hanson IM, Armstrong GR, O′Driscoll BR. Value and accuracy of cytology in addition to histology in the diagnosis of lung cancer at flexible bronchoscopy. Respir Med 2001;95:374-8.  Back to cited text no. 9
10.Steffee CH, Segletes LA, Geisinger KR. Changing cytologic and histologic utilization patterns in the diagnosis of 515 primary lung malignancies. Cancer 1997;81:105-15.  Back to cited text no. 10

Correspondence Address:
Sangeetha Keloth Nayanar
Sri Sakthi, Talap, Kannur - 670 002, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9371.130624

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  [Figure 1]

  [Table 1], [Table 2]

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