Microwave-Based Rapid Tissue Processing Technique: A Novel Aid in Histopathologic Laboratory.

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. Copyright:

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.[12] 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.[3] 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

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].

Table 1

Evaluation of tissue quality by three independent observers

Evaluation of tissue quality: Nuclear details overall by conventional and microwave procedure
Parameters	Observer number	Observations	Conventional	Microwave	Total	χ2, p
Nuclear details	Observer 1	Poor	3 (3.8)	4 (5.0)	7 (5.0)	1.778, 0.411
		Average	55 (68.8)	61 (76.2)	116 (72.5)
		Good	22 (27.5)	15 (18.8)	37 (23.1)
	Observer 2	Poor	2 (2.5)	3 (3.8)	5 (3.1)	1.87, 0.393
		Average	47 (58.8)	54 (67.5)	101 (63.1)
		Good	31 (38.8)	23 (28.8)	54 (33.8)
	Observer 3	Poor	9 (11.2)	12 (15.0)	5 (3.1)	0.832, 0.66
		Average	39 (48.8)	34 (42.5)	101 (63.1)
		Good	32 (40.0)	34 (42.5)	54 (33.8)
Cytoplasmic details	Observer 1	Poor	4 (5.0)	5 (6.2)	9 (5.6)	0.908, 0.635
		Average	63978.8)	66 (82.5)	129 (80.6)
		Good	13 (16.2)	9 (11.2)	229 (13.8)
	Observer 2	Poor	2 (2.5)	1 (1.2)	3 (1.9)	0.41, 0.814
		Average	67 (83.8)	69 (86.2)	136 (85.0)
		Good	11 (13.8)	10 (12.5)	21 (13.1)
	Observer 3	Poor	16 (20)	19 (23.8)	35 (21.9)	2.151, 0.341
		Average	40 (50)	45 (56.2)	85 (53.1)
		Good	24 (30)	16 (20.0)	40 (25.0)
Occurrence of artefacts	Observer 1	Poor	17 (21.2)	16 (20.0)	33 (20.6)	0.325, 0.85
		Average	57 (71.2)	56 (70.0)	113 (70.6)
		Good	6 (7.5)	8 (10.0)	14 (8.8)
	Observer 2	Poor	9 (11.2)	8 (10.0)	17 (10.6)	0.487, 0.784
		Average	49 (61.2)	46 (57.5)	95 (59.4)
		Good	22 (27.5)	26 (32.5)	48 (30.0)
	Observer 3	Poor	10 (12.5)	17 (21.2)	27 (16.9)	5.641, 0.06
		Average	48 (60.0)	52 (65.0)	100 (62.5)
		Good	22 (27.5)	11 (13.8)	33 (20.6)
Tissue architecture	Observer 1	Poor	12 (15.0)	21 (26.2)	33 (20.6)	3.54, 0.17
		Average	52 (65.0)	48 (60.0)	100 (62.5)
		Good	16 (20.0)	11 (13.8)	27 (16.9)
	Observer 2	Poor	22 (27.5)	27 (33.8)	49 (30.6)	1.078, 0.583
		Average	36 (45)	30 (37.5)	66 (41.2)
		Good	22 (27.5)	23 (28.8)	45 (28.1)
	Observer 3	Poor	12 (15.0)	10 (12.5)	22 (13.8)	11.207, 0.004
		Average	34 (42.5)	54 (67.5)	88 (55.0)
		Good	34 (42.5)	16 (20.0)	50 (31.2)

Chi-square test (P≤0.05)

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].

Table 2

Evaluation of staining quality by three-independent observers

Evaluation of quality of staining: Conventional and microwave procedure
Parameters	Observer number	Observations	Conventional	Microwave	Total	χ2, P
Nuclear staining	Observer 1	Poor	3 (3.8)	6 (5.0)	9 (5.6)	1.231, 0.540
		Average	56 (70.0)	56 (70.0)	112 (70.0)
		Good	21 (26.2)	18 (22.5)	39 (24.4)
	Observer 2	Poor	1 (1.2)	4 (5.0)	5 (3.1)	2.117, 0.347
		Average	51 (63.8)	52 (65.0)	103 (64.4)
		Good	28 (35.0)	24 (30.0)	52 (32.5)
	Observer 3	Poor	4 (5.0)	4 (5.0)	8 (5.0)	3.28, 0.194
		Average	26 (32.5)	37 (46.2)	63 (39.4)
		Good	50 (62.5)	39 (48.8)	89 (55.6)
Cytoplasmic staining	Observer 1	Poor	8 (10.0)	6 (7.5)	14 (8.8)	0.914, 0.633
		Average	60 (75.0)	65 (81.2)	125 (78.1)
		Good	12 (15.0)	9 (11.2)	21 (13.1)
	Observer 2	Poor	0 (0.0)	3 (3.8)	3 (1.9)	3.062, 0.216
		Average	74 (92.5)	71 (88.8)	145 (90.6)
		Good	6 (7.5)	6 (7.5)	12 (7.5)
	Observer 3	Poor	3 (3.8)	4 (5.0)	7 (4.4)	2.043, 0.360
		Average	35 (43.8)	43 (53.8)	78 (48.8)
		Good	42 (52.5)	33 (41.2)	75 (46.9)
Epithelia connective tissue interface	Observer 1	Poor	7 (10.9)	9 (14.1)	16 (12.5)	0.607, 0.738
		Average	27 (42.2)	29 (45.3)	56 (43.8)
		Good	30 (46.9)	26 (40.6)	56 (43.8)
	Observer 2	Poor	3 (4.7)	6 (9.4)	9 (7.0)	1.097, 0.578
		Average	13 (20.3)	13 (20.3)	26 (20.3)
		Good	48 (75.0)	45 (70.3)	93 (72.7)
	Observer 3	Poor	7 (10.9)	9 (14.1)	16 (12.5)	6.129, 0.047
		Average	27 (42.2)	29 (45.3)	56 (43.8)
		Good	30 (46.9)	26 (40.6)	56 (43.8)
RBC integrity	Observer 1	Poor	38 (47.5)	34 (42.5)	72 (45.0)	0.444, 0.801
		Average	34 (42.5)	38 (47.5)	72 (45.0)
		Good	8 (10.0)	8 (10.0)	16 (10.0)
	Observer 2	Poor	36 (45.0)	43 (53.8)	79 (49.4)	1.254, 0.534
		Average	34 (42.5)	28 (35.0)	62 (38.8)
		Good	10 (12.5)	9 (11.2)	19 (11.9)
	Observer 3	Poor	11 (13.8)	18 (22.5)	29 (18.1)	4.178, 0.124
		Average	37 (46.2)	41 (51.2)	78 (48.8)
		Good	32 (40.0)	21 (26.2)	53 (33.1)

Chi-square test (P<0.05). RBC: Red blood cell

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.[4] and Boon et al.[5] Panja et al.[4] Chaudhari and Chattopadhyay,[6] and Morales et al.,[7] who found out that nuclear morphology was the same in both the procedures. Similarly when Mathai et al.[8] compared the nuclear size shape and quality in conventional and microwave processed tissues did not find any significant difference. Whereas Babu et al.[9] and Kango and Deshmukh[10] 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.[5678910]

Babu et al.[9] noticed more shrinkage in microwave processed tissues whereas Panja et al.[4] reported less shrinkage.. Kango and Deshmukh[10] 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.[11] who reported more number of artifacts like section folds, heat artifacts in case of microwave processing.

Kok et al.,[12] Morales et al.,[7] and Mathai et al.[8] found that the tissue architecture was same in both the processing. Kango and Deshmukh[10] 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.[9] 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.[45671012131415]

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.[8] and Kango and Deshmukh[10] reported moderate cytoplasm eosinophilia in microwave processed tissues. According to Leong et al.[16] this cytoplasmic eosinophilia ensures better nuclear cytoplasmic contrast.

Babu et al.[9] 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.,[8] Kango and Deshmukh,[10] Boon et al.[17] and Tripathi et al.[18] found the RBCs to be intact without any lysis in microwave assisted tissue fixation methods. Whereas Hopwood et al.,[19] Mayers,[20] Leong et al.,[16] Bernard[21] 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.[5822232425]

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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.