Tissue fixation and processing have been the backbone and gold standard for histopathological diagnosis. However, conventional processing of histopathological tissue is time-consuming and takes approximately 14-16 hours which results in delay in the disease diagnosis and treatment.1-3In this eraemphasis islaid down on reducingthe turnaround time. Several newer techniques like frozen section and automated processing have beenattempted to give a faster diagnosis, but are not cost-effective and most importantly theyare mainly limited to intraoperative consultation.4 The use of microwaves in tissue fixation and processing has revolutionizedtissue processingtechniques, by providing faster processing of the histopathological sections, with its morphology comparable with conventional processing technique.3,5,6 Microwave is a form of non-ionizing radiation in the range of 300 MHz to 300 GHz that produces alternating electromagnetic fields resulting in the generation of instantaneous heat proportional to energy flux which continues until radiation stops.7-8 This property helps in uniform tissue warming in a shorter time with faster penetration of the chemicals inside the tissue, resulting in rapid processing for making paraffin blocks, thereby helping in issuing emergency reports by the same day.3 In the last 30 years, the use of microwaves in tissue processing has been studied in depth, showing that with this technique, reliable results on paraffin sections can be achieved which is comparable to the sections from the conventional histology technique.1,3,9,10Furthermore, the tissue processed through the microwave technique can also be used for immunohistochemistry.1-4,10-18 The present study was done to compare the quality of the tissue sections obtained both from conventional and microwave-assisted techniques for tissue processing and also to find its usefulness and applicability in our setting.

This was a prospective comparative study analysis for the microwave and conventional technique of tissue processing, conducted in the Histopathology section of the Department of Pathology for 1 year and 7 months. The Institutional Research and Ethics Committee approved the study via. letter No. BFUHS/2K15p-TH/8909. The study included two hundred randomly selected paired tissue samples of varied specimens from different organs received for histopathological examination. Small biopsy specimens including the tru-cut needle biopsy, endoscopic biopsies, small skin, bony biopsies and biopsies not received in formalin were excluded from the study. Sample Selection A total of 400 samples comprising 200 paired tissue samples were randomly taken from different organs which included the gastrointestinal tract, soft tissues, salivary gland, nose, synovium, tonsils, vertebral disc, thyroid, ovary, uterus, brain, lung, placenta, spleen, pancreas, kidney, breast, lipoma, skin, lymph node, liver, prostate, penis, and urinary bladder. Representative slices of standard size (15mm x 10mm x 2mm) were taken from the specimens and divided into two halves. One of the slices was taken for conventional routine processing (Group A) while the other was taken for microwave processing (Group B) in the domestic microwave. Conventional Tissue Processing (Group A):The cassettes with the tissue slice (Group A) were put in the Leica automated histokinette. The conventional tissue processing protocol is shown in Table 1. No. of beakers Solution Time Function Beaker no. 1 10% buffered formalin 1 hour Fixative Beaker no. 2 10% buffered formalin 1 hour Fixative Beaker no. 3 50% ethyl alcohol 1 hour Dehydration Beaker no. 4 60% ethyl alcohol 1 hour Dehydration Beaker no. 5 70% ethyl alcohol 1 hour Dehydration Beaker no. 6 80% ethyl alcohol 1 hour Dehydration Beaker no. 7 100% ethyl alcohol 1 hour Dehydration Beaker no. 8 100% ethyl alcohol 1 hour Dehydration Beaker no. 9 Xylene 1 ½ hour Clearing Beaker no. 10 Xylene 1 ½ hour Clearing Wax bath Paraffin wax (62 ºC) 1 ½ hour Impregnation Wax bath Paraffin wax (62 ºC) 1 ½ hour Impregnation Wax bath Paraffin wax (62 ºC) 1 ½ hour Impregnation Total time 15 ½ hours Table 1 Protocol for conventional tissue processing. Microwave Tissue Processing (Group B): A pilot study on 96 randomly selected tissue specimens using a domestic microwave(Samsung Domestic Microwave, Voltage-230 V, AC, 50 Hz (frequency), power input-1300-2800W, Power output-100-900 W (6 LEVELS), Frequency 2450 MHz). Glass lid-covered borosil beakers were used for reagents. During the impregnation step, an additional glass beaker filled with plain tap water was kept. The processing of the tissues was based on the protocol given by Suri et al11with slight modifications in timing and change of reagents. A final protocol (Table2)for microwave-assisted tissue processing was adopted based on using various combinations and permutations (12 cycles) in the processing techniques and time duration.For the final procedure, all the reagents in microwave processing were replaced by fresh ones after 6 cycles or after 48 sections (whichever was earlier). Table2 showsthe modified protocol for microwave tissue processing Beakers Solution Volume Power Time Function Beaker no. 1 10 % buffered formalin 200 ml 300 watts 1 min Fixation of tissue piece. Beaker no. 2 70% ethyl alcohol 200 ml 300 watts 4 min Dehydration. Beaker no. 3 90% ethyl alcohol 200 ml 300 watts 4 min Dehydration. Beaker no. 4 100% ethyl alcohol 200 ml 300 watts 3 min Dehydration. Beaker no. 5 100% ethyl alcohol 200 ml 300 watts 3 min Dehydration. Beaker no. 6 Xylene 200 ml 300 watts 3 min Clearing. Beaker no. 7 Xylene 200 ml 300 watts 3 min Clearing. Beaker no. 8 Paraffin wax (pre-heated at 62 ºC) 200 ml 600 watts 8 min Impregnation. Total time 29 min Once the tissue was processed by both techniques, the remaining procedure for both sets of tissue remained the same which was embedding, tissue sectioning(3-5µm thickness), staining [Haematoxylin and eosin stain] and mounting [dibutyl phthalate xylene (DPX)]. Following this, the slides prepared fromboth microwave and conventional techniques were blindedwith a sticker and was given a fictitious numberby the first author (SD). The slides were evaluated by two authors. Evaluation of sections: The paired slides were evaluated under a microscope by both observers independently using the modification of the criteria used byTripathi et al19as shown in Table3. The scoring (4- Excellent / 3- Good/ 2-Fair /1-Poor) of the slides was done for each of the parameters- cellular outlines, cytoplasmic outlines, nuclear details, the appearance of inflammatory cells, interface of epithelium and stromal/ mesenchymal tissue and stromal/ mesenchymal tissue. The scores were then added and a total score was obtained. The minimum score that could be given was 6 and a maximum score was 24. According to the total score, the grade Excellent/ Good/ Fair/ Poor was assigned for every slide against the fictitious number by both observers and recorded separately. The mean of the total score given by the observers was deduced. The decoding of the slides was done at the end and recorded. Parameters POOR FAIR GOOD EXCELLENT Cellular Outline 1 2 3 4 Cytoplasmic Details 1 2 3 4 Nuclear Details 1 2 3 4 Appearance of inflammatory cells 1 2 3 4 Interface of epithelium and stromal/ mesenchymal tissue 1 2 3 4 Stromal/ mesenchymal tissue 1 2 3 4 Total Score Table3 Parameters used for evaluation of slides. Total Score GRADE 20-24 EXCELLENT 15-19 GOOD 10-14 FAIR 6-9 POOR Statistical Analysis: The final results were subjected to statistical analysis using student’s t-test, Pearson chi-square test and kappa agreement. RESULTS The age of the patients included in this study group ranged from 45 days of life to 88 years. The mean age was 39 years. There wasa total of 109 females and 91 males. The male-to-female ratio was 1:1.2. Out of 200 paired tissues processed by both techniques there were 62 (31%) malignant cases, 85 (42.5%) benign cases and 53 (26.5%) inflammatory cases. There was a significant reduction (p<0.05) in each of the basic steps of histoprocessing by microwave technique which overall decreased the turnaround time. (Table4) Table4 Comparison of time taken by microwave and conventional tissue processing with p-value. PROCEDURE MICROWAVE CONVENTIONAL p-VALUE Fixation 1 minute 120 minutes < 0.0001 Dehydration 14 minutes 360 minutes < 0.0001 Clearing 6 minutes 180 minutes < 0.0001 Impregnation 8 minutes 270 minutes < 0.0001 TOTAL 29 minutes 930 minutes < 0.0001 Parameters evaluated by the observers: The slides were evaluated for each parameter by two of the pathologists and the mean of the total score given by both observers was taken. The sections from tissues processed using both methods were comparable and equally good. The p-value of each parameter comprising microwave and conventional processing is given in Table5. Table5 Parameters evaluated, mean scores of microwave and conventional tissue processing with p-value and Alpha (α) value. Parameters Microwave Processing Conventional Processing p-value Alpha Value Cellular Outlines 3.97 3.57 0.19 0.715 Cytoplasmic details 3.93 3.95 0.15 0.705 Nuclear details 3.90 3.95 0.05 0.598 Appearance of Inflammatory cells 3.89 3.90 0.07 0.540 Interface of epithelium and stromal/ mesenchymal tissue 3.61 3.80 0.002 0.340 Stromal/ mesenchymal tissue 2.89 2.99 0.07 0.082 Overall 0.5 The reliability test (α value) was done to evaluate the interobserver variation. The interobserver variation was found to be minimal as shown in Table5. Tissues processed by both microwaveand conventional techniquehad comparable morphology(78.4% and 80.8%, respectively) with the majority graded as having excellent morphology(Fig. 1-7)(Table6). Table6 Grades obtained for the tissues processed by microwave and conventional technique. GRADE MICROWAVE PROCESSING CONVENTIONAL PROCESSING P-value No. of samples Percentage No. of samples Percentage EXCELLENT 157 78.4% 161 80.8% 0.9743 GOOD 39 19.3% 36 18% 0.920 FAIR 4 2.3% 3 1.2% 0.745 POOR 0 0 0 0 0 TOTAL 200 100% 200 100% The volume of reagents such as ethyl alcohol, xylene and paraffin wax consumption were less compared to reagent consumption in conventional tissue processing. After each step of processing in the microwave, the volume of the reagents decreased and this was overcome by covering the beaker with a glass plate to avoid excessive evaporation of the reagents while processing. The tissues grossly became firm, hard and pale after microwave processing however, it did not show any gross shrinkage. There was no loss of tissue in most of the specimens processed by microwave technique. Mild microscopic tissue shrinkage was seen in the microwave-processed tissues (Fig. 1)when compared to conventionally processed tissues,however, this did not hinder the observers from making a pathological diagnosis. CONVENTIONAL PROCESSED MICROWAVE PROCESSED Figure 1. Photomicrographs showing adenomyosis, uterus, showing endometrial glands with evident glandular shrinkage. (100x). Various sections from the intestines, endometrium and thyroid processed by microwave technique showed that the glands appeared slightly smaller in size when compared with the conventionally processed sections from the respective tissues (Fig. 1,2,3,4). No erythrocytolysis was seen in any of the sections processed by microwave technique. CONVENTIONALPROCESSED MICROWAVE PROCESSED Figure 2. Photomicrograph showing well differentiated carcinoma, colon. (100x). CONVENTIONALPROCESSED MICROWAVE PROCESSED Figure 3. Photomicrographs showing variable sized thyroid follicles, multinodular goiter, thyroid (100x). CONVENTIONALPROCESSED MICROWAVE PROCESSED Figure 4. Photomicrographs showing fibrocystic disease of breast. (100x). Smaller biopsies like prostatic chips, gall bladder strips, endometrium, and nasal mucosa tissue were morphologically better processed in microwave processing. Fatty tissues like lipoma and breast with adipose tissue showed fair preservation of cellular and nuclear details in the study.(Fig. 4,5). CONVENTIONALPROCESSED MICROWAVE PROCESSED Figure 5. Photomicrographs showing lipoma, back. (100x) Hyperchromatism and pleomorphism of the malignant cells were maintained both in microwave and conventional techniques.(Fig. 6,7). CONVENTIONALPROCESSED MICROWAVE PROCESSED Figure 6 Photomicrographs showing IDC, breast. (100x). Inset showing malignant ductal cells (400x). CONVENTIONALPROCESSED MICROWAVE PROCESSED Figure 7 Photomicrographs showing spindle cell sarcoma. (100x). Arrows showing mitosis. (SARCOMA)

Tissue diagnosis remains the gold standard and plays a consequential role for clinicians, aiding in determining the treatment plan. This,however, is largely dependent upon good sample preparation and staining, which inturn requires proper diffusion of fluids and dyes within and out of the tissue, crucial to tissue staining.Conventional procedures take almost 14-16 hours to process, thereby delaying giving the diagnosis.20Procedures like warming up the essential fluids before processing or use of automated tissue processors have had a positive impact on the same, especially in regards to good staining as well as reducing the time.Microwave tissue processing provides an alternative to histopathologists as it gives comparable results with the additional advantage of reduced turn-around time with the provision of issuing the report on the same day,especially in cases where the management of the patient’s diseases solely depends upon the diagnosis made by the pathologist. Studies have been done in the past demonstrating the application of microwaves in histological procedures including fixation, staining for light and electron microscopy, preservation of cryostat sections and immunohistochemistry.11,15-18 The present study compared both microwave and conventional processing done of 200 paired randomly selected samples from various tissues.Varied types of tissue specimens (soft tissues, breast, thyroid, gall bladder, intestine, and miscellaneous) were processed by both techniques. Various studies showed the use of one or two particular types of tissues for comparing both processes. Gastrointestinal and female genital tract specimens formed the majority in the study done by Devi et al20 whereas only soft tissues were used by the Kango &Deshmukh13 study. Chaudhari et al 22 used smaller biopsies to assess the morphology andused them to compare histochemical as well as immunohistochemistry. The total time taken by microwave processing in our study was 29 minutes as compared to 15½ hours of conventional tissue processing. Shortening of tissue processing time was observed by other studies.3,4,10-13,19-23This improved workload distribution and reporting of the case on the same day, thereby enhancing the pathologist’s role in the management of the patients. Borosilglasswares covered with a glassplate were used for the reagents used during microwave processing. No burnt areas or hot spots were observed in tissues due to overheating during microwave processing in our study which was in agreement with the study done by Mathai et al.12 In addition to this, a separate 1000 ml borosil glass beaker containing normal tap water was kept with the 1000 ml borosil glass beaker containing paraffin wax during the impregnation step. A similar procedure was also followed by Suri et al.11 A maximum of 8 sections were processed at a time in the present study since microwave processing was ineffective while processing a larger number of tissues. The reagents were changed every 6 cycles either/ or processing 48 tissues in total. A similar problem of ineffective processing with large samples was noticed in a study done by Kok et al7and Mathai et al.12 Different permutations and combinations concerning container, fluids as well as wax were used for microwave tissue processing by various authors. Tissues processed by both the techniques in our study were stained with Haematoxylin and Eosin (H&E) stain and were mounted with DPX. They were then blinded and given a fictitious number, which was evaluated by the two observers for cellular outlines, cytoplasmic details, nuclear details, the appearance of inflammatory cells, interface of epithelium and stromal/ mesenchymal tissue and stromal/mesenchymal tissue. The scores given were graded as per Table 2. A similar pattern was followed in a study done by Babu et al 10, Patil et al20 and Devi et al.21The details showing comparative analysis by various authors in comparison to per study are given in Table 7. Table: 7Comparison between the microscopic findings of microwave processing (MW) and conventional tissue processing (CP) in various studies Study Cellular outline Cytoplasmic details Nuclear details Inflammatory cell infiltrate RBC morphology Mayer 1970 MW=CP MW=CP MW=CP - Lysed Chaudhari et al, 2000 MW=CP MW=CP MW=CP - - Rohr et al, 2001 MW=CP MW=CP MW=CP - - Suri et al, 2006 MW=CP MW=CP MW=CP - - Panja et al, 2007 MW=CP MW=CP MWCP MW>CP MW>CP MW>CP - Kango et al, 2011 MW>CP MW>CP MW>CP MW>CP Preserved Devi et al, 2013 MW>CP MW>CP MW>CP - - Tripathi et al. 2013 MW=CP MW=CP MWCP MW>CP MW>CP - - Jain et al, 2015 MW=CP MW=CP MW=CP - - Present Study MW=CP MW=CP MW