Sample Turnaround Times for Three Histoprocessing Methods

Introduction

Turnaround time for any pathological laboratory is very important which depends upon the preparation and diagnosis of the histopathologic material. The rapidity advantages the clinician to treat acutely ill patients and influence the work practice of the pathologist. With the advent of modernization tissue processing is modified from the point of tissue removal to embedding for instant histopathological diagnosis by various techniques or methods.

After the surgical removal, the tissue undergoes preparatory protocol for preparation of sections which usually involves impregnation with a suitable supporting medium. The stages of tissue processing include fixation, dehydration, clearing, impregnation and embedding for designated durations of time to ensure completion of the procedure. Culling 1974, Bancroft & Gamble 2002

The reproducibility and relatively low expense attached to the most commonly employed method continues to recommend it as a valuable tool after nearly 100 years of existence. But with the demand of faster or early reporting, newer techniques like rapid manual and microwave processing are getting introduced. Each of them is unique with their own set of advantages and disadvantages.

The conventional tissue processing is reliable and cost effective but time consumption, reagent toxicity and delay in providing diagnosis are the major disadvantages. The rapid manual tissue processing has major disadvantages like the use of noxious chemicals, greater degree of tissue distortion and shrinkage which led to exploration for new short processing schedules. The microwave tissue processing eliminates the use of noxious chemicals, causes lesser distortion of tissue and has shorter processing time but the cost involved in instrumentation is very high. (Panja et al. 2007)

Microwaves were invented by Percy Spencer in 1945 which work on the principle of producing heat by oscillating or exciting polar molecules. The microwave irradiation forces dipolar molecules of proteins to rotate through 180° at the rate of 2.45 billion cycles per second (Srinivasan, Sedmak & Jewell 2002, Bancroft & Gamble 2002). These excited molecules due to kinetics cause collision with adjacent molecules resulting in transfer of rotational energy. This friction causes production of heat within the material itself leading to accelerated diffusion of processing fluids hence faster processing is possible.

The advantages associated with microwave processing led to the production of commercially available microwaves specifically designed for tissue processing, however, the cost involved in these is very high (Leong 2004, Rohr et al. 2001). Domestic microwaves are readily available, affordable and had been used for tissue processing with good results earlier by some authors.

Thus, the aim of the present study was to compare and analyse the efficacy of three histoprocessing methods and to determine the impact on turnaround times of tissue processing by these three methods.

Materials and method

In the present study, 60 specimens were selected randomly. The soft tissue specimen fixed in 10% NBF for 24h were included in the study and hard tissues like cartilage, bone and tooth were not included in the study. The gross features of the specimen were recorded and tissues were cut into three pieces of approximately same size to be processed by three methods. The sections obtained after processing were subsequently stained with H&E by routine and microwave staining method.

The stained slides in each group processed by three techniques were randomly numbered for a blind study and circulated among four observers referred as O1-O4. The observers graded each parameter on the format given in appendix D by following specific criteria as given in appendix C.

Methodology

Microwave tissue processing

The microwave oven was calibrated as the microwave energy is non uniform within the chamber. Thus, hot and cold spots were detected in the chamber with the use of thermal paper sheet instead of the use of extra water load as suggested by various authors. The cold spot provided the most consistent results every time.

The technique was self-standardized by trial and error method in the LG domestic Microwave (Model no. MS-285SD). The microwave was operated at the maximum output power of 40% (approximately 360 W) with rotating tray and ring removed. The cut piece from a fixed tissue sample was placed in a plastic tissue cassette and water washed in running tap water so that tissue was free of formalin. The tissue was irradiated in 200ml of 100% methanol and 200ml of 100% isopropyl alcohol for dehydration at cold spot for 2 cycles of 10 minutes each respectively in the microwave. After dehydration tissue was impregnated in 200ml of molten paraffin wax for 2 cycles at cold spot of 10 min each and was embedded in paraffin wax.

Conventional Tissue Processing

The cut piece from a fixed tissue sample was placed in a metal tissue cassette and water washed in running tap water so that tissue should be free of formalin. The tissue was dehydrated in 70% alcohol (one change), 90% alcohol (one change) and 100% alcohol three changes of 1 h each respectively. After dehydration tissue was cleared in two changes of xylene of 1 h each. Finally, tissue was impregnated in 2 changes of molten paraffin wax for 1 h each and was embedded.

Rapid Manual Tissue Processing

The cut piece from a fixed tissue sample was water washed in running tap water so that tissue should be free of formalin, after that tissue was wrapped in filter paper and dehydrated in 95% alcohol, 100% alcohol for 20 min on a stir plate. The dehydrated tissue was cleared in xylene for 20 min on a stir plate 20 min on a stir plate. Tissue was impregnated in 2 changes of molten paraffin wax of 1h each and was then embedded.

The microwave processed tissue were stained as given in table no. ____. Conventional and rapid manual processed tissues were stained as given in table no.___________.

Statistical Analysis

The values obtained from different observers after assessment of sections processed by the three techniques were subjected to statistical analysis by Kruskal Wallis Test. One way ANOVA (Analysis of variance) was used for comparing mean shrinkage in tissues processed by the three histoprocessing methods. The P value <0.05 was considered to be significant.

Results

All observers were assumed to be reliable as the Cronbach’s reliability test was statistically significant. Complete concordance was found amongst all pathologists in most of the cases. Hence, observer 1 was randomly selected for further analysis.

The histopathological evaluation of the epithelium, fibrous tissues and glandular tissue revealed that the nuclear cytoplasmic contrast was good and cellular outline was distinct in tissues processed by microwave assisted technique followed by conventional processing and rapid manual processing techniques. The stroma was good with distinct cellular outline. The secretory products can be easily appreciated and the RBCs, inflammatory cells were intact. The results were statistically non-significant as observed by Kruskal Wallis test.

The colour intensity of the tissues graded by four observers revealed that the microwave sections were crisper and there was a good contrast between the hematoxyphilic and eosinophillic areas. Though some slides were not visualised up to the mark, all the three histoprocessing methods were comparable to each other.

One way Analysis of Variance (ANOVA) revealed a significant value on comparison of the tissue shrinkage processed by the three techniques. The dimensions of the tissues were recorded before dehydration and paraffin embedding. The mean percentage of shrinkage in rapid manual technique was significantly higher as compared to the other two techniques whereas statistically non-significant value was obtained on comparing conventional and microwave method of tissue processing.

Discussion

Microwaves are electromagnetic waves^{(Microwave Processing Techniques for Microscopy)} which causes oscillation and excitation of polar molecules which are usually dipolar molecules of proteins in tissues. The excited molecules cause collision with adjacent molecules due to kinetics producing friction and causes production of heat within the material itself. The heat produced enhances the rate of diffusion of fluids to permeate into the tissues. The rise in temperature decreases the viscosity of processing fluids that facilitates diffusion. Therefore it is theoretically possible to fasten the tissue fixation and processing. This has resulted in a substantial reduction in the basic steps of histoprocessing, thereby reducing turnaround time and providing same day diagnosis.

The applications of microwaves are extensive which includes tissue fixation, stabilization of large specimens, tissue processing for light and electron microscopy, histochemical and immunohistochemical staining.

Microwave tissue processing technique was introduced by Boon and Kok in 1985 (Leong 2004) but the potential application of microwave energy was first recognized by Mayers in 1970 who successfully fixed tissue with a microwave generator (Kok, Visser & Boon 1988). Boon et al. (1986) reported that it was possible to produce significant acceleration of tissue processing by using microwave radiation. Visinoni et al. (1998) first described the tissue processor that completed the processing in 30-120 min, thus reducing the processing time from 24 h to just 1-2 h providing early reporting and easy patient management.

Thus, the aim of the present study was to compare the cytoplasmic and nuclear details as well as staining characteristics of tissue sections processed by conventional, rapid manual and microwave techniques.

The noxious chemicals used in conventional tissue processing were replaced in microwave tissue processing. In the microwave processing in contrast to conventional tissue processing, isopropyl alcohol was replaced by methanol as dehydrating agent and xylene by isopropyl alcohol as intermediate agent. Molten paraffin wax remained the impregnating and embedding medium for both the techniques. The reagent selection was in consonance with Babu, Malathi and Mangesh (2011) who also used methanol, isopropyl alcohol and molten paraffin wax for microwave tissue processing.

Microwave radiation produced when enter the chamber it is reflected by the chamber walls until these gets absorbed by the material placed inside the chamber (Wong 2011). However, the spreading is not even throughout the chamber leading to formation of hot and cold spot zones. (Kok, Boon & Smid 1993, Thostenson & Chow 1999, Rutgers 2013).

Hence, hot & cold spots should be detected to achieve consistent results. Various authors have described methods for detection of hot and cold spots.

Microwave processing was self-standardized by trial and error method in which the hot and cold spots were detected by using a damp thermal paper Kok, Boon and Smid (1993). All the procedures in the microwave were carried out in the cold spot zones as suggested by Sharp and Paperiello (1971), Benard (1974), Rangell and Keller (2000) in their respective studies.

Microwavable plastic tissue cassettes were used for microwave tissue processing which are cheap and reusable as metallic utensils are contraindicated in the microwave because the electric fields of the waves produced by microwave magnetron are completely reflected at the same frequency by metals which can lead to sparking. (Vollmer 2004).

In the present study, the staining protocol for microwave was followed as given by Babu, Malathi and Mangesh (2011) which included the stains used to be accelerated in the microwave. Kayser and Bubenzer (1990) used domestic microwave oven for acceleration of the various stains which also included H&E stain. Valle (1986), Moorlag, Boon and Kok (1987) and Mathai et al. (2008) modified various special stain protocols for microwave and concluded that microwave did not produce any deleterious effects on staining.

In our study, the three pieces of tissue processed by three techniques sectioned by a soft tissue microtome and stained as per their respective protocols were evaluated. We adopted the criterion for evaluation of tissue sections given by Kango and Deshmukh (2011).

The overall quality of the tissue sections processed by microwave and manual methods was comparable. The microwave processed sections had same or similar cytoplasmic and nuclear details with good erythrocyte integrity and lymphocytic appearance than the manual methods. Similar results are given by Mathai et al. (2008), Morales et al. (2002), Bhuvanamha et al. (2013), Panja et al. (2007), Boon et al. (1986), Kok et al. (1988).

We also observed that the stroma in some cases was slightly more condensed focally in microwave processed tissue sections which is similar to the findings reported by Boon, Kok and Ouwerkerk-Noordam (1986) This lead to the erroneous categorization of these cases as indistinct in studies by Kango and Deshmukh (2011). Since our criterion was adopted from the above mentioned study we also placed focal condensation of stroma as indistinct. In contrast Kok, Visser and Boon (1988) refuted the importance of focal condensation of stroma in diagnostic pathology.

The colour intensity of the tissues graded by four observers revealed that the microwave sections were crisper and there was a good contrast between the hematoxyphilic and eosinophillic areas. The microwave processed tissues showed an increased reaction to H&E. The sections stained were slightly more eosinophilic as compared to the manual techniques. Similar findings are reported by Hopwood et al. (1984), Boon et al. (1986), Chaudhari, Chattopadhyay and Dutta (2000), Leong and Price (2004), Panja et al. (2007), Mathai et al. (2008), Babu, Malathi and Mangesh (2011). Hopwood et al. (1984) suggested that this eosinophilia could be easily corrected by altering the stain composition or staining time in eosin. In contrast Leong & Price (2004) observed that eosinophilia of the cytoplasm was advantageous as it produced good nuclear cytoplasmic contrast and enhancement of the cellular features.

The dysplastic features i.e. hyperchromatism, pleomorphism of tumor cells and mitotic figures were easily appreciable in the microwave processed tissue sections of malignancy. There was also an easy appreciation of the giant cells in the tissues of Central Giant cell Granuloma and tubercular lymphadenitis processed by microwave processing technique.

Rapid processing of histopathologic material is becoming increasingly desirable for intraoperative consultations and timely diagnosis. We found positive impact on turnaround time in microwave method as the time taken for block preparation from fixed tissue was 1h as compared to conventional method (9h) and rapid manual method (3h).

In some cases, proper diagnosis could not be reached as the size of the tissue was small and the sample was not representative of the site. Similar difficulties were also encountered by Suri et al. (2006), Kango and Deshmukh (2011) in their respective studies.

As assessed in our study, the effects of the three methods of histoprocessing on cytoplasmic and nuclear details of epithelial, fibrous and glandular tissue showed no statistically significant variation. The microwave technique was comparable or slightly better than the manual methods.

Conclusion

The applications and versatility of microwave processing methods are unattainable with conventional procedures. The method reported herein reproducibly yields similar histologic quality to that provided by conventional processing. It has many advantages including feasibility, safety and elimination of noxious chemicals that might be used for improvement in the practice of the histopathology laboratory, permitting the preparation of diagnostic material within a day. Domestic microwaves are easily available and cost effective but have certain notable disadvantages like uneven heating and inability to record and maintain temperature within the chamber. Further exploration in the field is required for development of cost effective microwave histoprocessors for histopathology which provide similar histologic material for rapid diagnosis and reporting.

References