|Year : 2014 | Volume
| Issue : 4 | Page : 458-462
Role of microwaves in rapid processing of tissue for histopathology
Harsh Kumar, Punita Kalkal, Archana Buch, Shirish S Chandanwale, Sunita Bamanikar, Atul Jain
Department of Pathology, Padmashree Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharashtra, India
|Date of Web Publication||25-Jun-2014|
B-603, Gold Coast, Ivory Estates, Someshwarwadi, Pune - 411 008, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim: The primary aim of the following study is to study the utility of microwave stimulated rapid processing of tissues for histopathology and the secondary aim is to compare the quality of the final product obtained after microwave stimulated rapid tissue processing with the conventional method. Materials and Methods: During the 3 years period of study, 150 cases were studied which were randomly selected from the tissues sent for histopathology. Tissues were processed by conventional method and by microwave assisted tissue processing. Results: Slides obtained by microwave and conventional tissue processing were statistically analyzed by using Chi-square test. The results obtained showed that the cellular and nuclear features of microwave processed tissues were comparable to conventionally processed tissue. Microwave assisted tissue processing reduced the total time for preparing tissue blocks to about an hour. Conclusion: Microwave stimulated processing provides an attractive alternative over traditional conventional processing.
Keywords: Conventional, microwave, tissue processing
|How to cite this article:|
Kumar H, Kalkal P, Buch A, Chandanwale SS, Bamanikar S, Jain A. Role of microwaves in rapid processing of tissue for histopathology. Med J DY Patil Univ 2014;7:458-62
|How to cite this URL:|
Kumar H, Kalkal P, Buch A, Chandanwale SS, Bamanikar S, Jain A. Role of microwaves in rapid processing of tissue for histopathology. Med J DY Patil Univ [serial online] 2014 [cited 2021 Sep 29];7:458-62. Available from: https://www.mjdrdypu.org/text.asp?2014/7/4/458/135267
| Introduction|| |
Microwaves are electromagnetic waves, discovered by Percy Spencer in 1945. Microwave have frequencies between 300 MHz and 300 GHz.  Microwave ovens used in histopathology operate at 2.45 GHz, corresponding to a wavelength in a vacuum of 12.2 cm. 
By using a microwave oven, heat is generated from within the tissues which warms the tissue block uniformly in a short time.  This results in faster penetration of tissue processing chemicals inside tissues resulting in rapid processing for making paraffin blocks. Therefore, microwaves have a great potential to shorten the processing time of tissues and hence their utility in rapid processing of tissues is being increasingly utilized. This study is intended to evaluate the role of the microwave oven for rapid processing of tissues for histopathology and to define a protocol for this process for use in the laboratory.
| Materials and Methods|| |
The present study was carried out in the Department of Pathology, of a tertiary care hospital and research center. This prospective study was conducted from July 2011 to September 2013. This study was carried out to compare the overall quality of tissue sections prepared by microwave stimulated tissue processing versus conventional processing and to ascertain if microwaves significantly accelerate histoprocessing, so that the turnaround time for histopathology slide preparation and report generation were significantly reduced.
Materials used were as follows:
- Microwave oven:
Bajaj microwave oven, model no: 2003 ETB, serial no: G041MW0710061677, voltage-230 V, AC, 50 Hz, power input-1200 W (microwave), power output-800 W, Frequency 2450 MHz
- Glassware used:
Two beakers 250 ml each, two beakers 100 ml each
- Reagents used:
10% formal saline as a fixative, 80% ethyl alcohol, 100% ethyl alcohol, chloroform, paraffin wax
- Other materials:
Temperature probe (industrial mercury thermometer), oven for melting paraffin wax, water bath, rotary microtome, plastic cassettes, tissue processing cassettes, stain hematoxylin and eosin (H and E), mounting media, glass slides, cover slips, compound microscope
A total of 150 specimens were randomly selected from the tissues sent for histopathology studies to the Department of Pathology. Only soft-tissues without bony component of size 1 cm × 1 cm × 0.5 cm were processed. After fixation in 10% formal saline, each tissue was divided into two equal halves. One bit was processed by the conventional method, which was labeled as sample A and the other half by using the microwave assisted tissue processing protocol labeled as sample B.
The cassettes with the tissue (sample A) were initially kept in water for an hour to remove excess formalin before being put in the auto processor. All processing was performed at room temperature, except for impregnation and embedding. The conventional tissue processing protocol is depicted in [Table 1].
Microwave Tissue Processing
The samples B from the fixed tissue were subjected to microwave stimulated processing. The protocol for processing of tissue specimens was based on the study by Kango and Deshmukh.  The processing protocol of Kango and Deshmukh had been suitably modified in a pilot study in our department through which we achieved desirable results. The protocol adopted by us is shown in [Table 2].
All the reagents were directly heated in the microwave except paraffin. Paraffin was melted on the stove separately and then placed in the microwave. Whereas operating microwave for tissue processing, the microwave oven was set at 40% power. Recording the temperature of the reagents at the interval was done manually using an industrial mercury thermometer.
Studying of Sections
The paired (H and E) slides were then evaluated by the professor of our department who was unaware of the processing method used for the slides. The slides were evaluated using criteria are listed in [Table 3] and [Table 4].
|Table 3: Criteria observed for analyzing tissue integrity and overall cell and stromal architecture|
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Besides the cellular and nuclear features, the tissue architecture was also analyzed, for fragmentation, shrinkage and loss of integrity of epithelium and connective tissue, if any. After evaluation, the final results were subjected to statistical analysis.
| Results|| |
Following achievement of satisfactory tissue processing, histopathological evaluation for cellular and nuclear morphology was done on tissues processed in parallel by both conventional and microwave tissue processing methods. [Table 5] shows the evaluation of cellular and nuclear morphology of tissues processed by both conventional and microwave techniques. When the cellular and nuclear morphology was distinct, Grade 1 was given and where morphology was indistinct Grade 0 was given. Chi-square test was applied for statistical evaluation. On evaluation for cellular morphology (P = 0.472) and for nuclear morphology (P = 0.552). It was found out that there were no significant differences in the products obtained by the two protocols under evaluation. This finding was in consonance with a study done by Kango and Deshmukh. 
|Table 5: Histopathological evaluation for cellular and nuclear morphology (n = 150)|
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| Discussion|| |
In histoprocessing for light microscopy, tissue is processed by three basic steps, i.e., dehydration, then clearing, which is followed by impregnation in paraffin wax. These steps are based on diffusion of chemicals and the aim of the exercise is to replace one reagent by another. Tissue contains water molecules and this does not allow embedding media to enter the tissue. In histoprocessing dehydration replace water in the tissue by alcohol (or a substitute). Alcohol permits penetration by tissue processing chemical which will be used subsequently. During clearing, there is an exchange of alcohol by a reagent miscible with paraffin wax (or its substitute) as chloroform or xylene. Finally, impregnation is the process in which the clearing agent is replaced by paraffin wax (or its substitute). Routine processing of tissues will take anything from 4 h to an overnight cycle of 12 h. 
For decades instrumentation used in tissue processing remained relatively unchanged. A recent addition in the list of techniques involved for rapid processing of tissues is the use of microwaves, which has revolutionized histotechniques.
Microwaves are the electromagnetic wave that can penetrate various types of material. Their penetration depth is dependent on the electric conductivity of the medium. Upon penetration into tissues, the energy is absorbed by the molecules. In the oscillating electric fields produced by microwaves irradiation, the dipolar molecules like water are forced to vibrate. Some of the acquired rotational energy is transferred to the random motion upon collision with other molecules. This induced kinetic movement produces instantaneous heat. This heat production increases the diffusion of the reagents and thereby decreases the tissue processing time. Unlike conventional heating, the heating in the microwave is from within (internal heating) and its effect occurs throughout the material being irradiated. ,,
In histoprocessing, diffusion is a key factor. The formula which governs the rate of diffusion is < X2 > = 2Dt, where "X" stands for net distance covered by a particle in solution in a certain direction; "t" is the time period during which diffusion occurs; "D" is the diffusion constant for the substance; < > stands for the average value. The formula states that the average squared distance covered by a particle in solution is proportional to the diffusion time. This indicates that the thickness of the biopsies should be less, the length and breadth of the tissue does not matter. 
In the initial part of the study, the tissues were processed using the technique advocated by Kango and Deshmukh.  Unfortunately, they did not show a desirable quality of H and E staining. Certain tissues had processed well, whereas some had drawbacks caused by improper dehydration and clearing. In view of this, a modified tissue processing protocol was followed shown in [Table 2]. In this protocol, the exposure of tissues to dehydration was increased. By this modification, the tissues were properly dehydrated and sections of good quality were obtained. As we wanted to shorten the time of tissue processing, we also tried further modification by decreasing time in the "clearing" step. We found that the results were good. We also tried decreasing the duration of impregnation and to our surprise we observed that there was no compromise with the quality of the tissue sections obtained. In our study, the processing time excluding fixation and section cutting/staining was about an hour. The same was also observed in studies as done by Rohr et al.,  Morales et al.,  Panja et al.,  Leong  and others.
Boon et al.,  Kango and Deshmukh.  in their study found that the epithelium showed good nuclear and cytoplasm contrast when processed by microwave technique. Similarly, in our study we also observed epithelium with good nuclear and cytoplasm contrast when processed by microwave assisted tissue processing [Figure 1]a and b. Focal condensation of connective tissue was occasionally seen, but this does not affect interpretations for diagnosis. 
|Figure 1: Gallbladder epithelium showing good nuclear and cytoplasmic detail. (a) Conventional tissue processing (H and E, ×100), (b) microwave assisted tissue processing (H and E, ×100)|
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In this study tissue sections processed by microwave assisted tissue processing showed similar tissue architecture, stroma, secretory products when compared with the conventionally processed technique [Figure 2]a and b. No tissue shrinkage, nuclear pyknosis or loss of cytoplasmic details were observed in the present study. These findings were also observed by Chaudhari et al.,  Morales et al. , and Kango and Deshmukh. 
|Figure 2: Appendix showing well preserved tissue architecture, stroma and mucin. (a) Conventional tissue processing (H and E, ×100), (b) microwave assisted tissue processing (H and E, ×100)|
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Using microwaves, Mayers,  Bernard,  Hopwood et al.,  Leong et al.  observed red cell lysis in tissue sections. However, this problem was not found in our study. Red cells [Figure 3]a and b and inflammatory cells like lymphocytes [Figure 4]a and b were well-preserved in the tissue processed by microwave technique. Similar findings were also seen in the study conducted by Kango and Deshmukh. 
|Figure 3: Section from an inflamed appendix showing preserved red blood cells in the lumen of blood vessel present in stroma (arrow). (a) Conventional tissue processing (H and E, ×100), (b) microwave assisted tissue processing (H and E, ×100)|
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|Figure 4: Section shows well preserved lymphocytes in the stroma (arrow). (a) Conventional tissue processing (H and E, ×400), (b) microwave assisted tissue processing (H and E, ×400)|
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In case of tumor tissues processed by us using microwave assisted tissue protocol, all the features of malignancy such as pleomorphic nuclei, prominent nucleoli, hyperchromatism, anisonucleosis, mitotic figures [Figure 5]a and b were comparable to sections from the conventionally processed tissue. A study done by Hopwood et al.,  Mathai et al.,  Kango and Deshmukh.  stated that the tissue diagnosis of malignancy, can be given satisfactorily by reporting on microwave processed tissues.
|Figure 5: Malignant melanoma showing well defined pleomorphic nuclei and prominent nucleoli. (a) Conventional tissue processing (H and E, ×100), (b) microwave assisted tissue processing (H and E, ×100)|
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| Conclusion|| |
Based on the above study it can be concluded that microwaves are a form of electromagnetic waves. They induce heat when applied to tissue. This enhances the diffusion of chemicals into tissues. This property of microwaves has been utilized for rapid processing of tissues for histopathological study. It was observed that microwave assisted tissue processing yields paraffin sections of similar or superior quality to that are produced by conventional tissue processing. By this innovative method, pathologists can now offer an early final diagnosis which eventually results in a more efficient and better management of patients. Since the only equipment required for this method in histopathology is a microwave oven, the technique is considered highly suitable for hospital laboratories as well as research laboratories where histological materials are routinely processed.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]