Year : 2021 Month : January Volume : 8 Issue : 3 Page : 162-167.
Aiswarya K.P.1, Priya V.S.2, Deepthi Raj M.L.3, Indira K.4
1 Department of Health Services, Government of Kerala, India.
2, 3 Department of Pathology, Government Medical College, Thiruvananthapuram, Kerala, India.
4 Department of MLT, Government Medical College, Thiruvananthapuram, Kerala, India.
Dr. Priya V.S.,
Saras, T.C 18/1809 (1),
Plavila, Thirumala P.O.,
Thiruvananthapuram - 695006,
Email : firstname.lastname@example.org
The importance of cytology in the early diagnosis of neoplastic as well as non-neoplastic lesions has been widely accepted. But the diagnosis of bone lesions by fine needle aspiration is non diagnostic most of the time due to insufficient material. In such situations a bone biopsy becomes necessary, even though an early histopathological diagnosis is not possible due to the long process of decalcification. A new technique called “Sediment cytology” has been described which involves the study of smears prepared from sediment of the fixative containing biopsy specimen.1 It is otherwise called ‘Biopsy Sediment cytology’ as it is prepared from fixatives of the biopsy specimen. This technique utilises exfoliated malignant cells which lose their cohesiveness.2 Malignant tumours are highly vascular, friable and easily sheds its cells in the fixative medium. Cells can be exfoliated from the cut surface of the specimen. So, this fixative is rich in cytology material and can be used in the preliminary diagnosis of different lesions especially malignant lesions. Sediment cytology shows importance of the fixative fluids of biopsy specimens which are usually discarded after specimen processing. Analysis of the fixative sediment for cytological interpretation with relevant clinical and radiological findings allows an early diagnosis.
As the bone lesions are inaccessible for cytological technique, sediment cytology can be used as complimentary test along with histopathology. Sediment cytology does not require long duration of decalcification procedures as in histopathological examination and can provide rapid and early diagnosis which helps in effective treatment plan.3 It can be used in the evaluation of variety of lesions in bone, cervix, oral cavity, urinary bladder and breast. Our present study was done to evaluate the role of sediment cytology of the bone biopsy specimen fixative in the early diagnosis of bone lesions. We also aimed to find the correlation between cytological and histopathological diagnoses.
This is a cross sectional study – diagnostic test evaluation. The study population included specimens of bone biopsy received at the histopathology laboratory, Department of Pathology of a tertiary health care centre in South India. The study period was for 6 months from April 12, 2019 to October 12, 2019 and commenced after institutional ethical committee approval (HEC NO: 05/04/2019 / MCT dated 12/4/19.
All bone biopsy specimens received in the histopathology laboratory regardless of age and sex of the patient were used in the study.
Specimen Processing Methods
Bone biopsy specimens received in the histopathology department in 10 % neutral buffered formalin as fixative were studied. The material for the sediment cytology was taken from the formalin in the specimen containing bottles. The received bottles were agitated in order to obtain exfoliated cells from the specimen surface. The specimen was then transferred to another container with fresh fixative. The fixative in the original container was allowed to settle down for about 1 hour. In cases of large specimens, they were bisected and kept in the original container for settling of cells. Most of the supernatant from the original container were pipetted off or discarded according to the volume of the fixative, leaving about 2 - 5 ml of the fixative containing the sediment in it. This fluid was agitated and mixed thoroughly, then transferred to falcon tubes and were centrifuged at 3000 rpm for 5 minutes.3 After centrifugation the supernatant was discarded, and sediment was washed twice with normal saline. Smears were prepared from the sediment on albuminised glass slide using pick and smear method.4 Pick and smear method was used when the sediment contains blood tinged and solid particles. The sediment was completely decanted on an albuminised glass slide, another clean glass slide was placed over the first one, overlapping horizontal strokes were made and the material with particles were spread evenly over the slide. Albumin coated slides were preferred as it ensures the adherence of cells. The prepared smears were immediately placed in a Coplin jar with 95 % ethyl alcohol fixative.
Papanicolaou staining method was used for staining the smears prepared from sediments. This staining method used Harris haematoxylin as nuclear stain and Orange G and EA 36 as cytoplasmic stain. Harris haematoxylin stain was prepared by dissolving 1 g haematoxylin in the alcohol (10 ml) & alum (20 g) in hot water. These two solutions were mixed together and heated to boiling. Mercuric oxide (20 g) was added immediately & then cooled rapidly by plunging the flask in to cold water. The solution so prepared was ready for immediate use. Glacial acetic acid added after cooling gives more precise nuclear staining. To remove excess haematoxylin from unwanted sites the smears were differentiated in 0.5 % HCl solution which was made by mixing 700 ml distilled water with 60 ml 1N HCl. After differentiation a step called bluing was done in alkaline solution containing lithium carbonate (1.5 g) mixed with 100 ml of water. Bluing was followed by cytoplasmic staining in Orange G solution (Orange G (2.5 g) powder mixed with 25ml distilled water and 475 ml of absolute ethanol) and subsequently in EA 36 (light green 1 g, distilled water 15 ml, absolute ethanol 235 ml, eosin 1 g, 200 mg phosphotungstic acid and 1 % phloxine).
Papanicolaou Staining Procedure5
Cytology smears from the sediment were evaluated along with proper clinical, radiological and gross findings and categorised according to the cytological features. The malignant lesions showed adequate or increased cellularity, high nuclear-cytoplasmic ratio, cellular pleomorphism, aniso nucleosis and hyperchromatic nuclei whereas the benign lesion showed less cellularity and benign microscopic features.7 The cytology smears were divided as malignant, benign, inflammatory and inconclusive. Benign category included benign tumours as well as non-neoplastic lesions. The cytological diagnosis was then compared with histopathological diagnosis which was taken as gold standard.
Sensitivity, specificity, positive predictive value and negative predictive value for benign, malignant and inflammatory bone lesions in sediment cytology were calculated
The current study included 50 bone lesions. The cytological analysis of bone biopsy sediment was done after correlation with radiological findings and finally compared with histopathological diagnosis. Out of 50 samples of bone lesions taken for the study, majority of the cases belonged to the age group 40 - 60 (56 %) followed by the age group 20 - 40 (24 %). The least number of cases were obtained from the age group 80 - 100 (2 %). Both males and females constituted 50 % each of the total cases selected. Out of 50 samples of bone biopsy samples taken for the study, the lesions were broadly divided into 3 categories; malignant, benign, and inflammatory. The histopathological diagnosis labelled 20 lesions as malignant (40 %), 13 lesions as benign (26 %) and 17 lesions as inflammatory (34 %). Of the 20 malignant lesions, there were 2 cases of chondrosarcoma, 11 cases of metastases, 1 case of myopericytoma, 4 cases of plasmacytoma, 1 case of Hodgkin’s lymphoma and 1 case of Langerhans cell histiocytosis. Of the 13 benign lesions, there were 6 cases of giant cell tumour, 1 case of osteochondroma and 6 cases of avascular necrosis. The inflammatory cases were 17 in number which included lesions like synovitis, osteoarthritis and acute / chronic inflammation. The proportion of cases according to histopathology diagnosis is depicted in Table 1.
In sediment cytology method, lesions included in the current study were broadly divided into 4 categories; benign, malignant, inflammatory, and inconclusive. Sediment cytology labelled 16 lesions as malignant (32 %), 11 lesions as benign (22 %), 16 lesions as inflammatory (32 %) and 7 cases were labelled inconclusive (14 %). Sediment cytology was able to correctly diagnose 16 out of 20 malignant lesions, 11 out of 13 benign lesions and 16 out of 17 inflammatory lesions. 7 cases were labelled as inconclusive. Most of the inconclusive cases showed only red blood cells (RBCs), necrotic material and some eosinophilic material in smears. These inconclusive cases were histopathologically diagnosed as chondrosarcoma, metastasis, myopericytoma, Langerhans cell histiocytosis, osteochondroma, avascular necrosis and inflammatory lesion. The proportion of cases according to sediment cytology is depicted in Figure 2.
Out of the 10 cases of metastasis, 7 cases showed metastasis from adenocarcinoma which were diagnosed as adenocarcinoma histopathologically also. Some of the smears showed better architectural pattern as cell clusters, tissue fragments and glandular pattern. 1 case was metastasis from squamous cell carcinoma and showed scattered atypical squamoid cells (Figure 1). Histopathology of that was well differentiated squamous cell carcinoma. Two smears showed pleomorphic spindle cells which was diagnosed as metastasis from malignant spindle cell neoplasm in histopathology. Histopathological correlation was obtained in 10 out of 11 cases of metastasis. 1 case was inconclusive due to inadequate cellularity and that was metastasis from adenocarcinoma histopathologically.
Sediment smears of 4 out of 4 cases of plasmacytoma showed many plasma cells with occasional binucleate forms. Radiology of these cases showed lytic lesions. Plasmacytoma showed 100 % correlation with histopathological diagnosis. Among the 2 cases of chondrosarcoma, 1 case showed chondroid cells with atypia and eosinophilic background material in the smears. A possibility of chondroid neoplasm with atypia could be made out. Other case showed RBCs only. Histopathological correlation was noticed in 1 out of 2 cases. Smears from giant cell tumours showed few multinucleated giant cells, histiocytes, and spindle cells (Figure 2a). Possibility of giant cell tumour was considered after radiology correlation. 100 % histopathological correlation was obtained in giant cell tumours. In case of lymphoma smears showed many lymphocytes and occasional large cells with nucleoli and it was diagnosed as Hodgkin’s lymphoma histopathologically. The smears in cases of avascular necrosis showed necrotic fragments, necrotic material and RBCs. 5 out of 6 cases showed histopathological correlation. Osteochondroma could not be diagnosed from the sediment smears.
Inflammatory conditions showed moderate cellularity compared with malignant lesions. Most of the smears from synovitis showed lymphocytes and plasma cells. Smears from osteoarthritis showed clusters of rounded cells with moderate cytoplasm and round vesicular nucleus which were taken as proliferated synovial cells, along with inflammatory cells & eosinophilic material, all of that favoured a degenerative arthritic change. 16 out of 17 inflammatory lesions showed correlation with histopathological diagnosis except one case of degenerative synovitis, smears of which showed inadequate cellularity.
Putting all these figures together, 43 out of 50 bone lesions were diagnosed from sediment cytology. False negative cases were due to inadequate cellularity and they constituted chondrosarcoma, metastasis, myopericytoma, Langerhans cell histiocytosis, Osteochondroma, avascular necrosis and inflammatory lesion. There were no false positive results. Overall diagnostic accuracy was 86 %. Cytological diagnosis of sediment smears with histopathological correlation which was taken as gold standard is depicted in Table 3.
Sediment cytology of malignant bone lesions showed highest specificity of 100 % and moderate sensitivity of 80 %. Benign bone lesions also showed 100 % specificity and a moderate sensitivity of 84.6 %. (Table 4)
Sediment cytology is one of the cytological techniques in which the sediment of the biopsy specimen fixative has been used for cytological examination. It has been suggested as a complimentary test in the preliminary and rapid diagnosis of different lesions of bone,8 breast, cervix,9 ovary10 oesophagus, stomach11 urinary bladder12 and lungs13. There are very few published reports indicating the utility and advantage of sediment cytology exclusively for bones. Sediment cytology can suggest an early diagnosis as compared to histopathology which needs long decalcification process.
Advantages of sediment cytology are, it is rapid, simple, cost effective and does not require additional instruments. But it has disadvantage like obscuring of cell morphology by blood.
Another advantage is the use of fixative in the biopsy specimen for the preparation of smears which are usually discarded.3 Usually these fixatives in the specimen containers are considered as non-productive and discarded after tissue processing though they are rich source for cytological material. In aspiration cytology of bone lesions, getting a representative sample is difficult. Thus, sediment cytology can be considered as a better option for early diagnosis of biopsy specimen.
Albumin coated slides were used for the purpose of adherence of cells. Common cytology staining technique like Papanicolaou was used as it was long lasting and gave better cytomorphological details as well as matrix.
The current study showed sediments from the malignant bone lesions were adequate and the smears were cellular when compared with benign lesions. This finding was concordant with studies conducted by Shah et al, Valiathan et al and Nayak et al.14 Smears had well preserved cell morphology in most of the cases to suggest a diagnosis after correlating with clinical and radiological findings. Malignant lesions showed increased cellularity due to the loss of cohesiveness which leads to increased exfoliation of tumour cells and were picked in sediment smears.
Some of the smears showed cell cluster or tissue fragments which gave better idea about architectural pattern of the lesion especially in metastatic carcinoma. As in FNAC, cases of plasmacytoma showed many mature plasma cells with eccentric nucleus and perinuclear hof and binucleated plasma cells.15 In chondrosarcoma atypical chondroid cells were noted in a background of eosinophilic material.16 Giant cell tumours showed multinucleated giant cells and mononuclear spindle cells.17 In all these cases correlation with clinical and radiological findings were helpful. All the cases of plasmacytoma had multiple lytic lesions. Serum electrophoresis of 2 cases showed M band, elevated serum calcium level and low haemoglobin as in the study conducted by Jagtap et al.18 Eccentric expansile lytic lesions were noticed in giant cell tumours. Both chondrosarcoma cases were presented as pathological fracture and showed lytic lesion with calcification
Valiathan et al. studied one hundred cases out of which a total of 33 malignant bone lesions were diagnosed with the help of sediment cytology. They were able to correctly diagnose 26 lesions and got an accuracy of 79 %. They had one false positive case. One of the difficulties they found was differentiating giant cell tumour from chondroblastoma as both have similar clinical and radiological features.8 They were also unable to suggest two cases of low-grade chondrosarcoma as malignant by this method.
In the study by Nagalotimath et al. which included 15 cases of bone lesions, all the benign and malignant lesions were correctly identified giving an accuracy of 100 %. A total of 500 lesions from different sites were evaluated by this technique. There were 193 true positives, 340 true negatives, 1 false positive and 6 false negative cases, giving a sensitivity of 98.4 %, specificity of 99.4 %, and overall diagnostic accuracy of 97.5 %.19
Shah et al studied 65 bone lesions, out of which 58 lesions were correctly diagnosed.3 The study diagnosed 29 lesions as malignant, 26 as benign lesions, 3 inflammatory and 7 inconclusive. The diagnostic accuracy was 89.2 %. The findings in the current study were almost similar to these studies.
In the current study, out of 20 malignant lesions 16 were positively identified and out of 13 benign lesions 11 were correctly diagnosed. Out of 17 inflammatory lesions, 16 lesions were correctly diagnosed. Seven cases were labelled as inconclusive as they had insufficient cellularity to suggest a diagnosis. The current study analyses the sensitivity and specificity of the cytology technique. When compared with final histopathology, sediment cytology was having 100 % specificity in diagnosing malignant, benign and inflammatory lesions. But the sensitivity of the cytological diagnosis was only 80 %, 84.6 % and 94 % respectively. Overall diagnostic accuracy achieved was 86 % and in the malignant lesions it was 80 %.
As compared with normal epithelial cells, cells of malignant and benign lesions show more exfoliation. This property can be utilised in sediment cytology. In most of the situations FNAC from the bone lesions are non-diagnostic due to insufficient material obtained because of the structural difficulties.
Sediment cytology gives early diagnosis, however, benign lesions yielded less cellular specimens and were more difficult to cytologically diagnose. Examination of the gross specimen and correlation with radiological findings before arriving at a cytological diagnosis of the biopsy sediments in bone lesions was absolutely helpful. Even though these studies show that sediment cytology can be suggested as a preliminary diagnostic test in bone lesions especially when suspecting malignant conditions, histopathological appearance of a lesion is considered to be the final arbiter of its diagnosis. This technique can be utilised in the centers where the frozen section facilities are unavailable.
Even though histopathological diagnosis remains the gold standard, biopsy sediment cytology can be considered as a rapid and reliable preliminary diagnostic test in bone lesions especially in malignant conditions so that it helps in better treatment plan without delay. The basic advantage of sediment cytology of bone lesions is that it does not require long decalcification process as in histopathology. Sediment cytology has several other advantages like being a simple, cost effective, rapid tool which does not require additional instruments. A provisional diagnosis can be made in benign, malignant or inflammatory lesions. This method has been employed for the evaluation of various diseases of breast and cervix, oesophagus and stomach, bladder, lungs and bones. The current study showed that the cell yield in the sediments was more and that of smears was adequately cellular in most of the cases to suggest a diagnosis as compared with histopathology.
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Financial or other competing interests: None.
Copyright © 2021 Aiswarya K.P. et al. This is an open access article distributed under Creative Commons Attribution License [Attribution 4.0 International (CC BY 4.0)]
Aiswarya KP, Priya VS, Raj MLD, et al. Role of sediment cytology in the early diagnosis of bone lesions and its comparison with histopathological findings. J Evid Based Med Healthc 2021;8(03):162-167. DOI: 10.18410/jebmh/2021/31