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Table of Contents   
ORIGINAL RESEARCH  
Year : 2011  |  Volume : 22  |  Issue : 1  |  Page : 50-55
A comparative study on microwave and routine tissue processing


1 Department of Oral Pathology, Aditya Dental College, Beed, Maharashtra, India
2 Department of Oral Pathology, Sri Ramachandra Dental College, Porur, Chennai, India
3 Department of Oral Pathology, S.R.M. Dental College, Ramapuram, Chennai, India

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Date of Submission29-Sep-2009
Date of Decision23-Apr-2010
Date of Acceptance01-Sep-2010
Date of Web Publication25-Apr-2011
 

   Abstract 

Aim and Objective: The present study was aimed at assessing the rapid microwave-assisted tissue processing and staining to determine if it can replace standard formalin-fixed and paraffin-embedded processing and staining technique.
Materials and Methods: The study group consisted of 15 oral mucosal biopsies. The specimens were fixed in 10% formalin for 24 hours to ensure adequate fixation and their gross features were recorded, photographed, and then the specimens were cut into equal halves to be processed by both conventional and microwave histoprocessing methods and then subsequently stained with H and E by microwave and conventional methods. The stained slides in each group processed by both microwave and routine methods were randomly numbered for a blind study and circulated among six observers. All the observers were asked to grade each parameter into Excellent/Good/Average/Poor in a data sheet comprising a total of six parameters. These gradings were given a numerical value of 4, 3, 2 and 1, respectively. The parameters included in the data sheet were cellular clarity, cytoplasmic details, nuclear details, color intensity, interface of epithelium and connective tissue. Wilcoxon-matched pairs signed rank test (non-parametric) was used to calculate the test of significance (P value).
Results: The total processing time involved in microwave was 42 minutes and 270 minutes for the conventional method. H and E staining in microwave took 33 minutes and 40 minutes for conventional method.
Conclusions: The individual scores by different observers regarding the various parameters included in the study were statistically insignificant, the overall quality of microwave-processed and microwave-stained slides appeared slightly better than conventionally processed and stained slides.

Keywords: Comparative study, microwave tissue processing, turn-around time

How to cite this article:
Babu T M, Malathi N, Magesh K T. A comparative study on microwave and routine tissue processing. Indian J Dent Res 2011;22:50-5

How to cite this URL:
Babu T M, Malathi N, Magesh K T. A comparative study on microwave and routine tissue processing. Indian J Dent Res [serial online] 2011 [cited 2017 Jan 20];22:50-5. Available from: http://www.ijdr.in/text.asp?2011/22/1/50/79975
Rapid processing of histopathologic material is becoming increasingly desirable to fulfill the needs of clinicians treating acutely ill patients. Turn-around time has been an important issue for many years and has become increasingly important in this age of managed care and commitments to overall reduction of costs for health care services. Initially, efforts were directed toward reducing specimen processing time for intraoperative consultations and rush specimens when a clinical requirement existed for a rapid diagnosis and initiation of emergency therapy based on the histopathology findings. The development of the frozen section technique allowed a rapid processing time for intraoperative histopathologic evaluation, but this technique is associated with difficulty in preparing and cutting certain types of specimens (fatty tissues) as well as certain morphologic changes that make histopathologic interpretation more difficult. These limitations have inhibited the use of frozen sections from becoming the predominant tissue processing method for routine specimens. [1]

Modifications of routine processing techniques have allowed the processing of small biopsy specimens through paraffin with production of H and E stained slides in approximately 4 hours. While this represents a considerable time saving procedure in contrast with the overnight procedures routinely used, it is relatively labor intensive and applicable predominantly to small biopsy specimens. Hence, its use has been restricted largely to the processing of small biopsy specimens from patients for whom rapid histopathologic diagnosis is required to initiate life-saving antimicrobial, immunosuppressant or chemotherapeutic intervention. The demands of these methods have not permitted their wide acceptance for routine tissue preparation for H and E slides. [1]

During the last 30 years, microwave-assisted tissue processing has been studied. The technique has achieved increasing acceptance in the last decade. In 1993, Leong reviewed microwave techniques described for diagnostic laboratories. Since then, the increased popularity of microwave-assisted tissue processing has led to the production of commercially available microwave ovens specifically designed to ensure uniform rapid tissue processing under precisely controlled specimen temperatures. These machines also precisely control on-off cycling of the heating. Such commercial units have facilitated tissue processing and diagnosis on the same day on which the specimen is obtained. [1],[2]

While a number of authors have reviewed the techniques and results of microwave-facilitated tissue fixation and processing, we are unaware of any previous studies comparing the quality of microwave processing and routine processing from matched specimens procured from the workload of an ordinary surgical pathology laboratory, using a commercially available microwave oven. The purpose of the present study was to document the usefulness of microwave-assisted tissue processing and to determine whether it can replace standard formalin fixation and paraffin-embedded overnight processing as the new routine technique for tissue preparation. Changing the standard technique for tissue fixation and preparation from the currently used overnight processing to same-day microwave tissue preparation could substantially reduce the turnaround times, permitting same-day diagnosis that would facilitate patient diagnosis and management on a one-day basis. This improvement in turnaround time could reduce the costs associated with diagnosis and increase the rapidity with which neoplastic diseases are diagnosed and therapy is initiated. [1]


   Materials and Methods Top


The study group consisted of 15 oral mucosal biopsies received in the Department of Oral Pathology, Sri Ramachandra Dental College, Chennai, and the specimens were fixed in 10% formalin for 24 hours to ensure adequate fixation. Their gross features, namely, weight, color, consistency, and dimensions were recorded. The specimens were then subsequently photographed and cut into equal halves to be processed by both conventional and microwave histoprocessing methods and subsequently stained with H and E by microwave and conventional methods. The stained slides in each group processed by both microwave and routine methods were randomly numbered for a blind study and circulated among six observers. All the observers were asked to grade each parameter into Excellent/Good/Average/Poor in a data sheet comprising a total of six parameters. These gradings were given a numerical value of 4, 3, 2 and 1, respectively and the observers were referred to as O1, O2, O3, O4, O5 and O6, respectively. The parameters included in the data sheet were cellular clarity, cytoplasmic details, nuclear details, color intensity, interface of epithelium and connective tissue. Wilcoxon-matched pairs signed rank test (nonparametric) was used to calculate the test of significance (P value).

Microwave processing

Samsung Microwave Oven (model 183ST) with a maximum output of 850 W was used. Two microwave-resistant glass beakers of 200 ml each, 100% methyl alcohol as dehydrating agent (Qualigens, Product no. 32407), 100% isopropyl alcohol as intermedium (Qualigens, Mumbai, India Product no. 26897), paraffin wax (Qualigens, Product no. 19215), thermometer to measure temperature, plastic tissue cassettes and hand towel to handle the utensils used in microwave oven were used. Only one tissue was processed at a time.

The adequately fixed sample was water washed for 5 minutes to remove excess formalin from the tissue. The tissue was transferred to a beaker containing 200 ml of 100% methanol for dehydration and the microwave was set at 300 W for two 7-minute cycles. The tissue was transferred to a beaker containing 100% isopropyl alcohol with the same microwave settings. The specimen was then transferred to a beaker containing 200 ml of molten paraffin wax for wax impregnation and the tissue was embedded in molten paraffin wax immediately. The tissue block thus obtained was sectioned, dewaxed and then stained with H and E using microwave oven. The time gap between the irradiation cycles was almost immediate and as soon as the first cycle was completed, the second cycle was started.

Microwave hematoxylin and eosin staining

The ribbon sections obtained were dewaxed in xylene, with two changes of 10 minutes each, and hydrated in running tap water for 5 minutes. Fifty milliliters of hematoxylin stain was taken in a small  Petri dish More Details and the slides containing tissue sections were placed in the Petri dish and the microwave oven was set at 300 W for 30 seconds. The slides were immediately transferred to running tap water for blueing. The sections were differentiated in acid alcohol (one to two dips) and then they were water washed in running tap water for 5 minutes. Fifty milliliters of eosin stain was taken into a small Petri dish and the sections were transferred to it. Microwave was set at 300 W for 30 seconds. The sections were water washed in running tap water for 2-3 minutes to remove excess stain. They were dehydrated in absolute alcohol (two changes). The slides were air dried and mounted.

Routine tissue processing

Fixed tissue sample was transferred to running tap water to remove excess formalin. Tissue sample was transferred to graded alcohol for dehydration: 70% alcohol - one change for 30 minutes; 90% alcohol - one change for 30 minutes; 100% alcohol - three changes of 1 hour each. Xylene was used as clearing agent with two changes of 30 minutes each. Tissue sample was then transferred to molten paraffin wax - 2 changes of 1 hour each, and finally the specimen was embedded in molten paraffin wax. [3],[4]

Hematoxylin and eosin staining

Embedded tissue was sectioned and dewaxed in xylene - two changes of 10 minutes each. The tissue sections were hydrated in running tap water for 5 minutes. Slides were then transferred to a coplin jar containing hematoxylin stain solution for 5-7 minutes and then placed in running tap water for 2-3 minutes for blueing. Sections were differentiated in acid alcohols (one to two dips). They were water washed for 5 minutes and then transferred to a coplin jar containing eosin stain solution for 30 seconds. They were water washed to remove excess stain and later were dehydrated in absolute alcohol - two changes of one dip each, and were air dried and mounted. [3],[4]

The stained slides in each group, processed by both microwave and routine methods, were randomly numbered for a blind study and circulated among six observers. Each observer was asked to grade each parameter into Excellent/Good/Average/Poor in a data sheet comprising a total of six parameters. These gradings were given a numerical value of 4, 3, 2 and 1, respectively, and the observers were referred to as O1, O2, O3, O4, O5 and O6, respectively. The parameters included in a data sheet were cellular clarity, cytoplasmic details, nuclear details, color intensity, interface of epithelium and connective tissue. Wilcoxon-matched pairs signed ranks test (nonparametric) was used to calculate the test of significance (P value).


   Results Top


The slides were produced in 75 minutes as compared to 310 minutes in routine processing [Table 1], [Table 2] and [Table 3].
Table 1: Time comparison between routine and microwave tissue processing

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Table 2: H and E staining: Routine and microwave methods

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Table 3: Total time taken for processing and staining

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The parameters in our study namely cellular clarity, cytoplasmic details, nuclear details and color intensity, interface of epithelium and connective tissue, fibrous tissue appeared slightly better in microwave method than routine method [Table 4], [Table 5], [Table 6], [Table 7], [Table 8] and [Table 9], [Figure 1], [Figure 2] and [Figure 3].
Figure 1: Color intensity and interface of epithelium and connective tissue

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Figure 2: Comparison of fibrous tissue

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Figure 3: Cellular, nuclear and cytoplasmic details

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Table 4: Cellular clarity

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Table 5: Cytoplasmic details

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Table 6: Nuclear details

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Table 7: Color intensity

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Table 8: Interface of epithelium and connective tissue

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Table 9: Fibrous tissue

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We experienced a 51% of shrinkage in microwave method as compared to 47% in routine method after alcohol steps. Tissues appeared much firmer in microwave method than in routine method as it is attributed to heat generated by microwave oven.

The average temperature rise (ΔT) recorded by thermometer after dehydration step was 53.25°C (maximum 87°C and range 50-57°C) and after intermedium step was 54.75°C (maximum 87°C and range 50-57°C) [Table 10].
Table 10: Temperature rise ÄT = ÄT2 - ÄT1 (°C)

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   Discussion Top


The practice of histopathology during the last five decades has been enriched by advances in our knowledge of morphologic expression of disease and by new technologies such as immunohistochemical and molecular assays. Handling of tissue samples from surgical removal to the preparation of H and E stained slides, however, has remained impervious to scientific advances. In particular, formalin fixation, followed by currently used conventional processing methods has been the standard for almost 100 years. [5]

A 1-day minimum delay in the preparation of diagnostic cases, toxicity of reagents used, and degradation of nucleic acids, are a few of the important shortcomings associated with that practice. The introduction of microwave technology into the histology laboratory provides a way to overcome many of these problems. [5]

The method reported herein reproducibly yields histologic material of similar or superior quality to that provided by time-honored conventional processing. It has many advantages including expediency, safety, potential for preservation of molecular integrity of specimens that might be used in subsequent studies, and improvement in the workflow of laboratory, permitting the preparation of diagnostic material during the day time. [5]

Microwaves are an integral part of our daily lives, and applications in the pathology laboratory are an inevitable outcome. This form of non-ionizing radiation produces alternating electromagnetic fields that result in the rotation of polar molecules such as water and the polar side chains of proteins through 180° at the rate of 2.45 billion cycles per second. The molecular kinetics so induced result in the generation of instantaneous heat that is proportional to the energy flux and continues until the radiation ceases. In addition to most biologic materials, inorganic molecules with an asymmetric electrical charge also can be rotated by this electromagnetic flux. [6]

We used a domestic microwave oven (Samsung 183 ST) with a maximum output of 850 W. Histoprocessing was carried out with a power output of 300 W and a relatively short cycle time of 7 minutes each and this setting was adopted by us, as this had not shown any detrimental effects on final microscopic view of the specimen.

Metals and metallic utensils are contraindicated for use in microwave ovens due to total internal reflection of microwaves, leading to sparkling. [7],[8] Hence plastic cassettes in place of metal cassettes during tissue processing were used. These cassettes are relatively cheap and can be reused.

Here dimensions of specimen in terms of volume were considered, and reduction in volumes was referred to as shrinkage. A 4% excess shrinkage as compared to routine processing in this study was experienced. A little excess in shrinkage noted here could be due to the heat generated by microwave oven. But the volume changes after paraffin wax impregnation could not be measured, as stated by Baker, who measured a net shrinkage of 33% in volume after paraffin wax impregnation. [9],[10]

Tissues appeared much firmer after alcohol steps and pale in color in microwave method as compared to routine method.

Microwaves can penetrate to a maximum depth of 2 cm and hence they may require a few additional cycles of dehydration in larger tissues. [7] This problem has been in overcome in this study, as all specimens selected were less than 2 cm in thickness.

Microwave irradiation has several advantages over routine methods from the perspective of the laboratory personnel, also with certain environmental advantages. It eliminates the need for xylene in tissue processing and may reduce or eliminate the need for formalin, as determined by the laboratory. [11]

From the perspective of the final product, microwave irradiation substantially shortens the time from specimen reception to diagnosis. In the present study, the slides were produced in 75 minutes as compared to 310 minutes in routine processing [Table 1], [Table 2] and [Table 3]. This allowed same-day tissue processing and diagnosis of small biopsy specimens without compromising the overall quality of histological section. This is in agreement with Ralph Rohr et al, where histological slides in 2-3 hours were produced using microwave irradiation. [7],[11],[12],[13]

Two sets of slides comprising 15 in each group, processed by both microwave and routine methods, among six observers were circulated. Their compiled data were taken into account.

Cellular clarity, cytoplasmic details, nuclear details and color intensity were slightly better in microwave method than in routine method [Table 4], [Table 5], [Table 6] and [Table 7], [Figure 1] and [Figure 3]. This is consistent with the studies of Kok and Boon. [7],[13],[14] This is attributed to uniform distribution of heat and effective dehydration. All the observers were in agreement with this finding.

Interface of epithelium and connective tissue has been a special area of interest for a pathologist in ruling out invasion and in many other immune-mediated disorders. [15],[16] It appeared better in microwave method than in routine method and is supported by five out of the six observers. There were not enough studies highlighting this aspect using a microwave method of tissue processing [Table 8], [Figure 1].

Fibrous tissues have a special interest in pathological diagnoses and often are the cause of difficulty in sectioning on a microtome. They often require additional or extended dehydration times which was not a problem in our study with our microwave setting. These tissues appeared better in microwave method than in routinely prepared slides. Majority of the observers were in agreement with this finding [Table 9], [Figure 2].

The overall quality of microwave-processed and microwave-stained slides appeared slightly better than routinely processed and routinely stained slides.


   Conclusions Top


The microscopic results were excellent even for adipose tissue and fibrous tissue, which have been a considerable problem to a histotechnologist, often requiring additional dehydration times and additional time on ice before sectioning on a microtome.

Staining time was considerably reduced from 40 minutes in routine method to 33 minutes in microwave method for regular H and E slides.

In our study it was found that microwave processing considerably shortens the time for permanent histological sections without a demonstrable decrease in section quality or interpretation. The overall quality of microscopic tissues of routine and microwave processing methods was similar and it was not possible to distinguish between the two techniques.

Hence, from our study, we would like to conclude that microwave technique of tissue processing can be adopted in the regular histopathology laboratory on a regular basis, and considering the considerable shortening of time period, microwave technique can replace the well-established routine tissue processing.

We strongly believe in a famous quote "A stitch in time saves nine" and hence we have made an attempt toward faster, reliable, cost-effective diagnosis and timely institution of treatment for better health care.

 
   References Top

1.Rohr LR, Layfield LJ, Wallin D, Hardy D. A comparison of routine and rapid tissue processing in a surgical pathology laboratory. Am J Clin Pathol 2001;115:703-8.  Back to cited text no. 1
[PUBMED]  [FULLTEXT]  
2.Kok LP, Boon ME, Suurmeijer AJ. Major improvement in microscopic-image quality of cryostat sections. Am J Clin Pathol 1987;88:620-3.  Back to cited text no. 2
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3.Culling′s CF, Allison RT, Barr WT. Cullings Histological Technique Processing. 4th ed. London : Butterworths and Co-publishers; 1985. p. 51-77.  Back to cited text no. 3
    
4.Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. 5 th ed. Missouri: Harcourt Publishers; 2002.  Back to cited text no. 4
    
5.Morales AR, Nassiri M, Kanhoush R, Vincek V, Nadji M. Experience with an automated microwave assisted rapid tissue processing method. Am J Clin Pathol 2004;121:528-36.  Back to cited text no. 5
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6.Leong AS. Microwaves and turnaround times in histoprocessing: Is this a new era in histotechnology? Am J Clin Pathol 2004;121:460-2.  Back to cited text no. 6
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7.Kok LP, Boon ME. Microwave Cookbook for Microscopists. Art and Science of Visualization. 3 rd revised ed. Leiden: Coulomb Press Leyden; 1992. p. 9.  Back to cited text no. 7
    
8.Microwave processing technique for microscopy. Available from : http://www.ebsciences.com/papers/microwaves.htm [last accessed on 2009 Nov 24].  Back to cited text no. 8
    
9.Kayser K, Stute H, Lubcke J, Wazinski U. Rapid microwave fixation: A comparative morphometric study. Histochem J 1988;20:222-5.  Back to cited text no. 9
    
10.Bancroft JD, Gamble M. Theory and Practice of Histological Techniques, Chapter no. 6. 5 th ed. London: Harcourt Publishers; 2002. p. 85-108.  Back to cited text no. 10
    
11.Bostwick DG, Al Annouf N, Choi C. Establishment of the formalin free surgical pathology laboratory. Arch Pathol Lab Med 1994;118:298-302.  Back to cited text no. 11
    
12.Slap S. Application of microwave technology to histology. In: Bancroft JD, Gamble M, editors. Theory and Practice of Histological Techniques. 5 th ed. London: Harcourt Publishers; 2002. p. 416.  Back to cited text no. 12
    
13.Boon ME, Kok LP, Ouwerkerk-Noordam E. Microwave-stimulated diffusion for fast processing of tissue: Reduced dehydrating, clearing and impregnating times. Histopathology 1986;10:303-9.   Back to cited text no. 13
    
14.Kok LP, Visser PE, Boon ME. Histoprocessing with the microwave oven: An update. Histochem J 1988;20:323-8.  Back to cited text no. 14
    
15.Neville BW, Damm DD, Allen CM, Bouquot JE. Oral and Maxillofacial Pathology. 2 nd ed. Missouri: Elsevier Publications; 2002.  Back to cited text no. 15
    
16.Regezi JA, Scuibba JJ, Jordan RC. Ulcerative Conditions. Oral Pathology. Clinical Pathological Correlations. 3rd ed. Philadelphia: W. D. Saunders Company; 1999. p. 69-80.  Back to cited text no. 16
    

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Correspondence Address:
T Mahesh Babu
Department of Oral Pathology, Aditya Dental College, Beed, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.79975

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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]

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