|Year : 2021 | Volume
| Issue : 5 | Page : 566-570
Microwave-based rapid tissue processing technique: A novel aid in histopathologic laboratory
Pallavi Mishra, Alokenath Bandyopadhyay, Harish Kumar, Kailash Chandra Dash, Lipsa Bhuyan, Abikshyeet Panda
Department of Oral Pathology and Microbiology, Kalinga Institute of Dental Sciences, Bhubaneswar, Odisha, India
|Date of Submission||23-Sep-2020|
|Date of Decision||10-Oct-2020|
|Date of Acceptance||18-Nov-2020|
|Date of Web Publication||05-Jun-2021|
Department of Oral Pathology and Microbiology, Kalinga Institute of Dental Sciences, Kalinga Institute of Industrial Technology Deemed to be University, Bhubaneswar, Odisha
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Conventional tissue processing takes a lot of time, but microwave processing method though rapid, its quality of tissue texture and staining properties is still questionable. Therefore, we conducted this study to find out the reliability of histological sections in the microwave method. Aim and Objectives: The aim is to assess whether microwave tissue processing, which is a rapid technique, is comparable to conventional tissue processing in terms of quality and reliability of histological sections. Materials and Methods: A total of 80 formalin-fixed tissue samples were taken and were divided into two pieces. One subjected to conventional tissue processing and the other subjected to microwave tissue processing. The slides were coded and evaluated by three independent observers using various parameters. All the data were subjected to Chi-square test. Results: Both the process did not differ in their efficiency in overall nuclear details, overall cytoplasmic detail, occurrence of artifact and tissue architecture, nuclear staining, cytoplasmic staining, and red blood cell staining. Although the other observers did not find any statistical significance, according to observer 3, conventional processing proved to have marginally significantly better epithelial connective tissue interface than microwave processing (P = 0.047). Conclusion: The microwave method of tissue processing is an economical procedure which reduces the obnoxious chemicals as well as the turnaround time.
Keywords: Comparative study, microwave, tissue processing, turnaround time
|How to cite this article:|
Mishra P, Bandyopadhyay A, Kumar H, Dash KC, Bhuyan L, Panda A. Microwave-based rapid tissue processing technique: A novel aid in histopathologic laboratory. J Pharm Bioall Sci 2021;13, Suppl S1:566-70
|How to cite this URL:|
Mishra P, Bandyopadhyay A, Kumar H, Dash KC, Bhuyan L, Panda A. Microwave-based rapid tissue processing technique: A novel aid in histopathologic laboratory. J Pharm Bioall Sci [serial online] 2021 [cited 2021 Oct 27];13, Suppl S1:566-70. Available from: https://www.jpbsonline.org/text.asp?2021/13/5/566/317531
| Introduction|| |
The most important part in pathology is to be able to do a histo-diagnosis of all biopsies received. Since a century, pathologists have been experimenting to find the ideal method for processing of the biopsied tissue. Most pathologists use the usual conventional procedures for processing, which has evolved from Conventional 2–3 days to rapid manual processing of a day or overnight. This leads to delay in Diagnosis. Initially, efforts were directed toward reducing specimen processing time for emergency diagnosis of the specimen during the surgical procedure. During the past 30 years, microwave-assisted tissue processing has been studied. In 1993, Leong reviewed microwave techniques described for diagnostic laboratories., The recently used microwave technique for tissue processing helps in reducing the total processing time, eliminates the noxious chemicals like xylene and shows a lesser degree of denaturation of nucleic acids. Also by using a domestic microwave, the processing remains cost effective also. Even though advantages are shown by microwave technology, it has not yet taken over the conventional processing and most laboratories still continue the age-old technique of conventional processing. This study was conducted to further compare the conventional and microwave tissue processing for the regular histopathological assessment.
| Materials and Methods|| |
The present study was conducted in the Department of Oral and Maxillofacial Pathology. Institutional ethics committee clearance was obtained. The tissue specimens sent to the histopathology laboratory constituted the study group. A total of 80 tissue samples were taken based on the inclusion and exclusion criteria devised for the study. The tissue with a minimum dimension of 0.8 cm × 0.8 cm or greater and with a thickness of 0.5 cm or above were taken as samples. Tissues not meeting the minimum size criteria were not included in the study.
The formalin-fixed tissue samples were taken up and sectioned into two pieces such that one was sent for routine, conventional tissue processing and the other was used for microwave tissue processing. In conventional tissue processing, each tissue was subjected to dehydration by various grades of alcohol, clearing with Xylene, and impregnated with paraffin wax before being embedded to prepare tissue blocks.
In microwave tissue processing, each tissue was placed in a 200 ml glass beaker filled with 100 ml of 100% Isopropyl alcohol. The microwave was kept on 100 watts for 20 min for tissue < 1 cm × 1 cm size and for tissue larger than 1 cm × 1 cm for 30 min. Wax impregnation was done on 100 watts for 20 min, then the tissue was removed and embedded in molten paraffin wax block and left to cool for 30 min.
Sections of 4 μ were prepared from both the blocks by Leica semiautomatic rotary microtome. Hematoxylin and Eosin staining procedure was the same for both the tissues [Figure 1]. One hundred and sixty slides were coded to blind it and evaluated by three independent oral pathologists. Observations were recorded as per the pre-designed questionnaire as per the protocol. The quality of staining was evaluated based on the parameters such as (i) nuclear staining, (ii) cytoplasmic staining, (iii) epithelial connective tissue interface, and (iv) red blood cell (RBC) integrity. The tissue quality was evaluated based on the parameters such as (i) nuclear details, (ii) cytoplasmic details, (iii) occurrence of artifacts, and (iv) overall tissue architecture.
|Figure 1: Conventional processing (a) versus microwave processing (b) of an incisional biopsy specimen of unicystic ameloblastoma at × 100|
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The results obtained were subjected to a Chi-square test. All the statistical methods were carried out through SPSS Inc. SPSS software, version 16.0 Chicago for windows.
| Results|| |
On comparing conventional and microwave tissue processing, there was no significant difference in overall nuclear details (P = 0.411, 0.393, and 0.6), overall cytoplasmic details (P = 0.635, 0.814, and 0.314) and occurrence of the artifacts (P = 0.850, 0.784, and 0.06) as observer by the three observers. However, observer 3 found significantly better (P = 0.004) tissue architecture in conventional processing than microwave processing. Observer 1 and 2 did not find any statistically significant difference in tissue architecture between both the methods [Table 1].
The statistical analysis of data yielded no significant difference among the observers in evaluation of overall nuclear staining (P= 0.540, 0.347, and 0.194), overall cytoplasmic staining (p = 0.633, 0.216, and 0.360) and RBC integrity (P= 0.801, 0.534, and 0.124). Observer 3 found on ventional processing to be significantly better then microwave processing (P = 0.047) whereas observer 1 and 2 did not find any statistically significant difference between both the methods [Table 2].
The diagnostic ability of the tissues was evaluated based on the observations of the three independent observers. Irrespective of the processing method utilized, all the observers unanimously agreed that all the slides were diagnosable [Figure 1].
| Discussion|| |
Hitherto, not much advancement has been made in the laboratory aspect of the histopathology. This study compared the efficacy of the two different methods of tissue processing qualitatively by three independent observers based on (i) tissue quality, (ii) quality of staining, and (iii) diagnostic ability.
When the nuclear detail in the epithelium was evaluated, one observer found a statistically significant difference between both the processing methods, but the other two observers failed to elicit any difference between both the procedures. When the nuclear details in connective tissue were evaluated, the observations of all three observers led to a conclusion that none of the processing methods were superior rather, they are more or less equal in efficiency. This finding was in agreement with the findings of Panja et al. and Boon et al. Panja et al. Chaudhari and Chattopadhyay, and Morales et al., who found out that nuclear morphology was the same in both the procedures. Similarly when Mathai et al. compared the nuclear size shape and quality in conventional and microwave processed tissues did not find any significant difference. Whereas Babu et al. and Kango and Deshmukh found microwave processing to be better than conventional processing when nuclear detail is concerned.
When the cytoplasmic details in the epithelium, connective tissue, or the overall tissue were evaluated, none of the three observers found any statistical difference between the conventional and microwave processing which was in accordance to various studies.,,,,,
Babu et al. noticed more shrinkage in microwave processed tissues whereas Panja et al. reported less shrinkage.. Kango and Deshmukh reported uneven staining as an artifact in conventional processing. However, in our study, both the processes of tissue processing that is conventional processing and microwave processing did not show any difference based on the observations given by the observers in the occurrence of artifacts. This was against the findings of Shashidara et al. who reported more number of artifacts like section folds, heat artifacts in case of microwave processing.
Kok et al., Morales et al., and Mathai et al. found that the tissue architecture was same in both the processing. Kango and Deshmukh found the tissue architecture to be well maintained with no shrinkage or spongy pattern. In our study, one observer found the tissue architecture after conventional processing to be better than microwave tissue processing. However, the other two observers did not find any such differences in both the procedures. Hence, it is difficult to draw any conclusion about the superiority of any of the processing techniques.
Babu et al. reported better color intensity in microwave tissue processing. Various authors concluded that there is no significant variation in the staining quality of tissues in either of the processes.,,,,,,,,
When nuclear and cytoplasmic staining of was evaluated, none of the observers found any significant difference in the outcome of conventional or microwave processing in the epithelium or connective tissue. Mathai et al. and Kango and Deshmukh reported moderate cytoplasm eosinophilia in microwave processed tissues. According to Leong et al. this cytoplasmic eosinophilia ensures better nuclear cytoplasmic contrast.
Babu et al. found the microwave tissue processing to be better in terms of reproducing the epithelial, connective tissue interface. In our study, only one observer found the conventional processing to be better than microwave processing in terms of quality of staining of epithelial, connective tissue interface. However, the other two observers did not find any difference between the procedures.
None of the observers in our study found any differences in integrity of RBCs both the tissue processing procedures. Mathai et al., Kango and Deshmukh, Boon et al. and Tripathi et al. found the RBCs to be intact without any lysis in microwave assisted tissue fixation methods. Whereas Hopwood et al., Mayers, Leong et al., Bernard found RBCs to be lysed after microwave processing.
In our study, when the diagnostic ability of the tissue processed by microwave was taken into consideration, all the observers unanimously found the microwave processed tissue to be diagnosable. Many previous studies have highlighted the distinct advantages of microwave in various aspects of tissue processing.,,,,,
| Conclusion|| |
Our study demonstrated no substantial difference in overall quality or diagnosability of tissue sections prepared by microwave or the conventional method. This study leads us to believe that routinely received, formalin-fixed tissue maybe processed rapidly by microwave irradiation without compromising the quality of the histologic sections. It also proves that the microwave method of tissue processing is the optimal method for substantially reducing the turnaround time and permitting the histopathology laboratories to consistently provide same-day diagnosis for a variety of types of tissue biopsy specimens. The usage of microwave tissue processing is very good for economization of operational costs, to meet the challenges of shortage of ancillary staff, improved employee productivity and to ensure a cost-effective operation.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Leong AS. Microwave techniques for diagnostic laboratories. Scanning 1993;15:88-98.
Morales AR, Nassiri M, Kanhoush R, Vincek V, Nadji M. Experience with an automated microwave-assisted rapid tissue processing method: Validation of histologic quality and impact on the timeliness of diagnostic surgical pathology. Am J Clin Pathol 2004;121:528-36.
Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. 5th
ed. London: Churchill Livingstone; 2006.
Panja P, Sriram G, Saraswathi TR. Comparison of three different methods of tissue processing. J Oral Maxillofac Pathol 2007;11:15-7. [Full text]
Boon ME, Kok LP, Noordam OE. Microwave-stimulated diffusion for fast processing of tissue: Reduced dehydrating, clearing, and impregnating times. Histopathology 1986;10:303-9.
Chaudhari K, Chattopadhyay AD. Microwave technique in histopathology and its comparison with the conventional technique. Indian J Pathol Microbiol 2000;43:387-94.
] [Full text]
Morales AR, Essenfeld H, Essenfeld E, Duboue MC, Vincek NM. Continuous-specimen-flow, high-throughput, 1-hour tissue processing. A system for rapid diagnostic tissue preparation. Arch Pathol Lab Med 2002;126:583-90.
Mathai AM, Naik R, Pai MR, Rai SB. Microwave histoprocessing versus conventional histoprocessing. Indian J Pathol Microbiol 2008;51:12-6.
] [Full text]
Babu TM, Malathi N, Magesh KT. A comparative study on microwave and routine tissue processing. Indian J Dent Res 2011;22:50-5.
] [Full text]
Kango PG, Deshmukh RS. Microwave processing: A boon for oral pathologists. J Oral Maxillofac Pathol 2011;15:6-13.
] [Full text]
Shashidara R, Sridhara SU, Praveen B. Kitchen microwave-assisted accelerated method for fixation and processing of oral mucosal biopsies: A pilot study. World J Dent 2011;2:17-21.
Kok LP, Visser PE, Boon ME. Histoprocessing with the microwave oven: An update. Histochem J 1988;20:323-8.
Hafajee ZA, Leong AS. Ultra-rapid microwave-stimulated tissue processing with a modified protocol incorporating microwave fixation. Pathology 2004;36:325-9.
Leong AS, Duncis CG. A method of rapid fixation of large biopsy specimens using microwave irradiation. Pathology 1986;18:222-5.
Rohr LR, Layfield LJ, Wallin D, Hardy D. A comparison of routine and rapid microwave tissue processing in a surgical pathology laboratory. Quality of histologic sections and advantages of microwave processing. Am J Clin Pathol 2001;115:703-8.
Leong AS, Daymon ME, Milios J. Microwave irradiation as a form of fixation for light and electron microscopy. J Pathol 1985;146:313-21.
Boon ME, Gerrits PO, Moorlag HE, Nieuwenhuis P, Kok LP. Formaldehyde fixation and microwave irradiation. Histochemical J 1988;20:313-22.
Tripathi M, Bansal R, Gupta M, Bharat V. Comparison of routine fixation of tissues with rapid tissue fixation. J Clin Diagn Res 2013;7:2768-73.
Hopwood D, Coghill G, Ramsay J, Milne G, Kerr M. Microwave fixation: Its potential for routine techniques, histochemistry, immunocytochemistry and electron microscopy. Histochem J 1984;16:1171-91.
Mayers CP. Histological fixation by microwave heating. J Clin Pathol 1970;23:273-5.
Bernard GR. Microwave irradiation as a generator of heat for histological fixation. Stain Technol 1974;49:215-24.
Kovacs L, Szende B, Elek G, Lapis K, Horvath O, Hiszek I, et al
. Working experience with a new vacuum-accelerated microwave histoprocessor. J Pathol 1996;180:106-10.
Kok LP, Boon ME. Microwaves for microscopy. J Microsc 1990;158:291-322.
McLay AL, Anderson JD, McMeekin W. Microwave polymerisation of epoxy resin: Rapid processing technique in ultrastructural pathology. J Clin Pathol 1987;40:350-2.
Giberson RT, Demaree RS Jr., Nordhausen RW. Four-hour processing of clinical/diagnostic specimens for electron microscopy using microwave technique. J Vet Diagn Invest 1997;9:61-7.
[Table 1], [Table 2]